|
|
| 课题组研究方向 |
自由基和激发态是高反应活性的瞬态物种，是影响多种化学、生物过程的关键活泼中间体。我们致力于发展和运用时间分辨激光光谱技术，尤其是时间分辨红外光谱方法，以能源、环境、材料和生命科学中的一些基础科学问题为背景，探测一系列重要的自由基及激发态分子的反应动力学过程，结合量子化学计算，深入阐述反应微观机理。研究包括有机分子、DNA生物分子、以及纳米金表界面分子等体系中各种复杂的物理化学动态过程，涉及化学动力学、分子光化学、分子光生物、激光光谱等领域中的前沿基础。近期代表性工作：
|
| |
| 1. Fluorescence Quenching Dynamics of 2‑Amino-7-methyl-1,8-naphthyridine in Abasic-Site-Containing DNA Duplexes for Nucleobase Recognition |
Dramatic fluorescence quenching of small heterocyclic ligands trapped in the abasic site (AP) of DNA has been implemented as an unprecedented strategy recognizing single-base mutations in sequence analysis of cancer genes. However, the key mechanisms governing selective nucleobase recognition remain to be disentangled. Herein, we perform fluorescence quenching dynamics studies for 2-amino-7-methyl1,8-naphthyridine (AMND) in well-designed AP-containing DNA single/double strands. The primary mechanism is discovered, showing that AMND only targets cytosine to form a pseudo-base pair, and therefore, fluorescence quenching of AMND arises through the DNA-mediated electron transfer (ET) between excited state AMND* and flanking nucleobases, most favorably with flanking guanines. Subtle dynamic conformational variations induced by different flanking nucleobases are revealed and found to modulate efficiencies of electron transfer and fluorescence quenching. These findings provide critical mechanistic insights for guiding the design of photoinduced electron transfer (PET)-based fluorescent ligands as sensitive singlebase recognition reporters.
|
| |
| 2. Reactivity and DNA Damage by Independently Generated 2′-
Deoxycytidin‑N4‑yl Radical |
Oxidative stress produces a variety of radicals in DNA, including pyrimidine nucleobase radicals. The nitrogencentered DNA radical 2′-deoxycytidin-N4-yl radical (dC·) plays a role in DNA damage mediated by one electron oxidants, such as HOCl and ionizing radiation. However, the reactivity of dC· is not well understood. To reduce this knowledge gap, we photochemically generated dC· from a nitrophenyl oxime nucleoside and within chemically synthesized oligonucleotides from the same precursor. dC· formation is confirmed by transient UV-absorption spectroscopy in laser flash photolysis (LFP) experiments. LFP and duplex DNA cleavage experiments indicate that dC· oxidizes dG. Transient formation of the dG radical cation (dG+•) is observed in LFP experiments. Oxidation of the opposing dG in DNA results in hole transfer when the opposing dG is part of a dGGG sequence. The sequence dependence is attributed to a competition between rapid proton transfer from dG+• to the opposing dC anion formed and hole transfer. Enhanced hole transfer when less acidic O6-methyl-2′-deoxyguanosine is opposite dC· supports this proposal. dC· produces tandem lesions in sequences containing thymidine at the 5′-position by abstracting a hydrogen atom from the thymine methyl group. The corresponding thymidine peroxyl radical completes tandem lesion formation by reacting with the 5′-adjacent nucleotide. As dC· is reduced to dC, its role in the process is traceless and is only detectable because of the ability to independently generate it from a stable precursor. These experiments reveal that dC· oxidizes neighboring nucleotides, resulting in deleterious tandem lesions and hole transfer in appropriate sequences.
|
| |
| 3. Independent Generation and Time-Resolved Detection of 2’-Deoxyguanosin-N2-yl Radicals |
Guanine radicals are important reactive intermediates in DNA damage. Hydroxyl radical (HOC) has long been believed to react with 2’-deoxyguanosine (dG) generating 2’-deoxyguanosin-N1-yl radical (dG(N1-H)C) via addition to the nucleobase p-system and subsequent dehydration. This basic tenet was challenged by an alternative mechanism, in which the major reaction of HOC with dG was proposed to involve hydrogen atom abstraction from the N2-amine. The 2’deoxyguanosin-N2-yl radical (dG(N2-H)C) formed was proposed to rapidly tautomerize to dG(N1-H)C. We report the first independent generation of dG(N2-H)C in high yield via photolysis of 1. dG(N2-H)C is directly observed upon nanosecond laser flash photolysis (LFP) of 1. The absorption spectrum of dG(N2-H)C is corroborated by DFT studies, and anti- and syn-dG(N2-H)C are resolved for the first time. The LFP experiments showed no evidence for tautomerization of dG(N2-H)C to dG(N1-H)C within hundreds of microseconds. This observation suggests that the generation of dG(N1-H)C via dG(N2-H)C following hydrogen atom abstraction from dG is unlikely to be a major pathway when HOC reacts with dG.
|
| |
| 4. Degradation of Cytosine Radical Cations in 2′-Deoxycytidine and in i-Motif DNA: Hydrogen-Bonding Guided Pathways. |
Radical cations of nucleobases are key intermediates causing genome mutation, among which cytosine C•+ is of growing importance because the ensuing cytosine oxidation causes GC → AT transversions in DNA replication. Although the chemistry and biology of steady-state C oxidation products have been characterized, time-resolved study of initial degradation pathways of C•+ is still at the preliminary stage. Herein, we choose i-motif, a unique C-quadruplex structure composed of hemiprotonated base pairs C(H)+:C, to examine C•+ degradation in a DNA surrounding without interference of G bases. Comprehensive time-resolved spectroscopy were performed to track C•+ dynamics in i-motif and in free base dC. The competing pathways of deprotonation (1.4 × 107 s–1), tautomerization (8.8 × 104 s–1), and hydration (5.3 × 103 s–1) are differentiated, and their rate constants are determined for the first time, underlining the strong reactivity of C•+. Distinct pathway is observed in i-motif compared with dC, showing the prominent features of C•+ hydration forming C(5OH)• and C(6OH)•. By further experiments of pH-dependence, comparison with single strand, and with Ag+ mediated i-motif, the mechanisms of C•+ degradation in i-motif are disclosed. The hydrogen-bonding within C(H)+:C plays a significant role in guiding the reaction flux, by blocking the tautomerization of C(−H)• and reversing the equilibrium from C(−H)• to C•+. The C radicals in i-motif thus retain more cation character, and are mainly subject to hydration leading to lesion products that can induce disruption of i-motif structure and affect its critical roles in gene-regulation.
|
| |
| 5. Preferential Binding of π-Ligand Porphyrin Targeting 5′-5′ Stacking Interface of Human Telomeric RNA G-Quadruplex Dimer. |
Human telomeric RNA (TERRA) containing thousands of G-rich repeats has the propensity to form parallel-stranded G-quadruplexes. The emerging crucial roles of TERRA G-quadruplexes in RNA biology fuel increasing attention for studying anticancer ligand binding with such structures, which, however, remains scarce. Here we utilized multiple steady-state and time-resolved spectroscopy analyses in conjunction with NMR methods and investigated thoroughly the binding behavior of TMPyP4 to a TERRA G-quadruplex dimer formed by the 10-nucleotide sequence r(GGGUUAGGGU). It is clearly identified that TMPyP4 intercalates into the 5′-5′ stacking interface of two G-quadruplex blocks with a binding stoichiometry of 1:1 and binding constant of 1.92 × 106 M–1. This is consistent with the unique TERRA structural features of the enlarged π–π stacking plane of the A·(G·G·G·G)·A hexad at 5′-ends of each G-quadruplex block. The preferential binding of π-ligand porphyrin to the 5′-5′ stacking interface of the native TERRA G-quadruplex dimer is first ascertained by the combination of dynamics and structural characterization.
|
| |
| 6. Monitoring the Structure-Dependent Reaction Pathways of Guanine Radical Cations in Triplex DNA: Deprotonation Versus Hydration. |
Exposure of DNA to one-electron oxidants leads initially to the formation of guanine radical cations (G•+), which may degrade by deprotonation or hydration and ultimately cause strand breaks or 8-oxoG lesions. As the structure is dramatically changed by binding of the third strand in the major groove of the target duplex, it makes the triplex an interesting DNA structure to be examined and compared with the duplex on the G•+ degradation pathways. Here, we report for the first time the time-resolved spectroscopy study on the G•+ reaction dynamics in triplex DNA together with the Fourier transform infrared characterization of steady-state products, from which structural effects on the reactivity of G•+ are unraveled. For an antiparallel triplex-containing GGC motif, G•+ mainly suffers from fast deprotonation (9.8 ± 0.2) × 106 s–1, featuring release of both N1–H and N2–H of G in the third strand directly into bulk water. The much faster and distinct deprotonation behavior compared to the duplex should be related to long-resident water spines in the third strand. The G•+ hydration product 8-oxoG is negligible for an antiparallel triplex; instead, the 5-HOO–(G–H) hydroperoxide formed after G•+ deprotonation is identified by its vibrational marker band. In contrast, in a parallel triplex (C+GC), the deprotonation of G•+ occurs slowly (6.0 ± 0.3) × 105 s–1 with the release of N1–H, while G•+ hydration becomes the major pathway with yields of 8-oxoG larger than in the duplex. The increased positive charge brought by the third strand makes the G radical in the parallel triplex sustain more cation character and prone for hydration. These results indicate that non-B DNA (triplex) plays an important role in DNA damage formation and provide mechanistic insights to rationalize why triplex structures might become hot spots for mutagenesis.
|
| |
| 7. Photochemical reaction dynamics studies of nucleic acids. |
Nucleic acids are the most important biomolecules, which function to express, store and transmit genetic information. The major radiation damages to the living things are essentially relative to the physical and chemical reactions of nucleic acids. Therefore, interests in photochemical reaction dynamics of nucleic acids have been intensified in recent years with the development of powerful experimental and computational techniques, boosting a challenging and promising subfield of chemical reaction dynamics interdisciplinary with molecular biology. In this context, we have made great efforts in developing the transient spectroscopic methods, particularly time-resolved infrared, to investigate the excited state and free radical reaction dynamics involved in the photochemical reactions of nucleic acid bases and DNA secondary structures (duplex, quadruplex etc.). By capturing the fast chemical events of these key short-lived transient species and combining quantum chemical calculations, we revealed the reaction mechanisms of [2+2] photocycloaddition, spore photoproduct (SP) photolesion, electron transfer, proton transfer, photosensitization, ROS oxidation, and photocleavage that are closely associated with the crosslink or oxidative DNA damages. The key roles of non-adiabatic surface intersections, excited state electronic structure property, conformation of secondary structure, base-pairing, π-stacking, and hydrogen-bonding in affecting the photochemical reaction pathways are elucidated. Our results provided important chemical insights to understand the DNA damage at the molecular and quantum state specific levels. This review mainly summarizes the recent progress made by our groups and the related work in literatures.
|
| |
| 8. Porphyrin Bound to i‐Motifs: Intercalation versus External Groove Binding. |
G‐rich and C‐rich DNA can fold into the tetrastranded helical structures G quadruplex or C quadruplex (i‐motif), which are considered to be specific drug targets for cancer therapy. A large number of small molecules (so‐called ligands), which can bind and modulate the stability of G quadruplex structures, have been widely examined. Much less is known, however, about the ligand binding interactions with the C quadruplex (i‐motif). By combining steady‐state measurements (UV/Vis, fluorescence, and induced circular dichroism (ICD)) with time‐resolved laser flash photolysis spectroscopy, we have studied the binding interactions of cationic porphyrin (5,10,15,20‐tetrakis(N‐methylpyridinium‐4‐yl)‐21 H,23 H‐porphyrin, abbreviated as TMPyP4) with i‐motifs (C3TA2)3C3T and (C4A4C4)2. The intercalation binding mode through π–π stacking of the porphyrin macrocycle and the C:C+ hemiprotonated base pair has been identified for the first time. The coexistent binding modes of intercalation (≈80 %) versus external major‐groove binding (≈20 %) have been determined quantitatively, thereby allowing a fuller understanding of the porphyrin–i‐motif interactions. The ionic strength was found to play an important role in affecting affects the binding modes, with the progressive increase in the ionic strength resulting in the gradual decrease in the intercalation percentage and an increase in the groove‐binding percentage. Furthermore, an extended study of the porphyrin derivative with four bulky side‐arm substituents (T4) suggests a complete prohibition of the intercalation mode owing to large steric hindrance, thereby providing a novel groove‐binding ligand with site selectivity. These results provide in‐depth mechanistic insights to better understand the ligand interactions with i‐motifs and guidance for related applications in anticancer drug design.
|
| |
| 9. Capturing the radical ion-pair intermediate in DNA guanine oxidation. |
Although the radical ion pair has been frequently invoked as a key intermediate in DNA oxidative damage reactions and photoinduced electron transfer processes, the unambiguous detection and characterization of this species remain formidable and unresolved due to its extremely unstable nature and low concentration. We use the strategy that, at cryogenic temperatures, the transient species could be sufficiently stabilized to be detectable spectroscopically. By coupling the two techniques (the cryogenic stabilization and the time-resolved laser flash photolysis spectroscopy) together, we are able to capture the ion-pair transient G+Cl- in the chlorine radical–initiated DNA guanine (G) oxidation reaction, and provide direct evidence to ascertain the intricate type of addition/charge separation mechanism underlying guanine oxidation. The unique spectral signature of the radical ion-pair G+-Cl- is identified, revealing a markedly intense absorption feature peaking at 570 nm that is distinctive from G+• alone. Moreover, the ion-pair spectrum is found to be highly sensitive to the protonation equilibria within guanine-cytosine base pair (G:C), which splits into two resolved bands at 480 and 610 nm as the acidic proton transfers along the central hydrogen bond from G+• to C. We thus use this exquisite sensitivity to track the intrabase-pair proton transfer dynamics in the double-stranded DNA oligonucleotides, which is of critical importance for the description of the proton-coupled charge transfer mechanisms in DNA.
|
| |
| 10. Direct Observation of Guanine Radical Cation Deprotonation in G-Quadruplex DNA |
Although numerous studies have been devoted to the charge transfer through double-stranded DNA (dsDNA), one of the major problems that hinder their potential applications in molecular electronics is the fast deprotonation of guanine cation (G+•) to form a neutral radical that can cause the termination of hole transfer. It is thus of critical importance to explore other DNA structures, among which G-quadruplexes are an emerging topic. By nanosecond laser flash photolysis, we report here the direct observation and findings of the unusual deprotonation behavior (loss of amino proton N2–H instead of imino proton N1–H) and slower (1–2 orders of magnitude) deprotonation rate of G+• within G-quadruplexes, compared to the case in the free base dG or dsDNA. Four G-quadruplexes AG3(T2AG3)3, (G4T4G4)2, (TG4T)4, and G2T2G2TGTG2T2G2 (TBA) are measured systematically to examine the relationship of deprotonation with the hydrogen-bonding surroundings. Combined with in depth kinetic isotope experiments and pKa analysis, mechanistic insights have been further achieved, showing that it should be the non-hydrogen-bonded free proton to be released during deprotonation in G-quadruplexes, which is the N2–H exposed to solvent for G bases in G-quartets or the free N1–H for G base in the loop. The slower N2–H deprotonation rate can thus ensure less interruption of the hole transfer. The unique deprotonation features observed here for G-quadruplexes open possibilities for their interesting applications as molecular electronic devices, while the elucidated mechanisms can provide illuminations for the rational design of G-quadruplex structures toward such applications and enrich the fundamental understandings of DNA radical chemistry.
|
| |
| 11. Fine-Tuning of β-Substitution to Modulate the Lowest Triplet Excited States: A Bioinspired Approach to Design Phosphorescent Metalloporphyrinoids |
Learning nature’s approach to modulate photophysical properties of NIR porphyrinoids by fine-tuning β-substituents including the number and position, in a manner similar to naturally occurring chlorophylls, has the potential to circumvent the disadvantages of traditional “extended π-conjugation” strategy such as stability, molecular size, solubility, and undesirable π–π stacking. Here we show that such subtle structural changes in Pt(II) or Pd(II) cis/trans-porphodilactones (termed by cis/trans-Pt/Pd) influence photophysical properties of the lowest triplet excited states including phosphorescence, Stokes shifts, and even photosensitization ability in triplet–triplet annihilation reactions with rubrene. Prominently, the overall upconversion capability (η, η = ε·ΦUC) of Pd or Pt trans-complex is 104 times higher than that of cis-analogue. Nanosecond time-resolved infrared (TR-IR) spectroscopy experiments showed larger frequency shift of ν(C═O) bands (ca. 10 cm–1) of cis-complexes than those of trans-complexes in the triplet excited states. These spectral features, combining with TD-DFT calculations, suggest the strong electronic coupling between the lactone moieties and the main porphyrin chromophores and thus the importance of precisely positioning β-substituents by mimicking chlorophylls, as an alternative to “extended π-conjugation”, in designing NIR active porphyrinoids.
|
| |
| 12. Explicit Differentiation of G-Quadruplex/Ligand Interactions: Triplet Excited States as Sensitive Reporters |
We report a new transient spectral method utilizing triplet excited state as sensitive reporters to monitor and differentiate the multiplex G-quadruplex/ligand interactions in a single assay, which is a difficult task and usually requires a combination of several techniques. From a systematic study on the interactions of porphyrin (TMPyP4) with each telomeric G-quadruplex: AG3(T2AG3)3, G2T2G2TGTG2T2G2, (G4T4G4)2, and (TG4T)4, it is convincingly shown that the ligand triplet decay lifetimes are sensitive to the local bound microenvironment within G-quadruplexes, from which the coexisting binding modes of end-stacking, intercalation, and sandwich are distinguished and their respective contribution are determined. The complete scenario of mixed interaction modes is thus revealed, shedding light on the past controversial issues. Additional control experiments demonstrate the sensitivity of this triplet reporter method, which can even capture the binding behavior change as the G-quadruplex structures are adjusted by Na+ or K+.
|
| |
| 13. Photophysical and Photochemical Properties of 4-Thiouracil: Time-Resolved IR Spectroscopy and DFT Studies |
Intensified research interests are posed with the thionucleobase 4-thiouracil (4-TU), due to its important biological function as site-specific photoprobe to detect RNA structures and nucleic acid–nucleic acid contacts. By means of time-resolved IR spectroscopy and density functional theory (DFT) studies, we have examined the unique photophysical and photochemical properties of 4-TU. It is shown that 4-TU absorbs UVA light and results in the triplet formation with a high quantum yield (0.9). Under N2-saturated anaerobic conditions, the reactive triplet undergoes mainly cross-linking, leading to the (5–4)/(6–4) pyrimidine–pyrimidone product. In the presence of O2 under aerobic conditions, the triplet 4-TU acts as an energy donor to produce singlet oxygen 1O2 by triplet–triplet energy transfer. The highly reactive oxygen species 1O2 then reacts readily with 4-TU, leading to the products of uracil (U) with a yield of 0.2 and uracil-6-sulfonate (USO3) that is fluorescent at ∼390 nm. The product formation pathways and product distribution are well rationalized by the joint B3LYP/6-311+G(d,p) calculations. From dynamics and mechanistic point of views, these results enable a further understanding for 4-TU acting as reactive precursors for photochemical reactions relevant to 1O2, which has profound implications for photo cross-linking, DNA photodamage, as well as photodynamic therapy studies.
|
| |
| 14. Formation of Guanine-6-sulfonate from 6-Thioguanine and Singlet Oxygen: A Combined Theoretical and Experimental Study |
As an end metabolism product of the widely used thiopurine drugs, 6-thioguanine (6-TG) absorbs UVA and produces 1O2 by photosensitization. This unusual photochemical property triggers a variety of DNA damage, among which the oxidation of 6-TG itself by 1O2 to the promutagenic product guanine-6-sulfonate (GSO3) represents one of the major forms. It has been suspected that there exists an initial intermediate, GSO, prior to its further oxidation to GSO2 and GSO3, but GSO has never been observed. Using density functional theory, we have explored the energetics and intermediates of 6-TG and 1O2. A new mechanism via GSOOH → GSO2 → GSO4 → GSO3 has been discovered to be the most feasible energetically, whereas the anticipated GSO mechanism is found to encounter an inaccessibly high barrier and thus is prevented. The mechanism through the GSOOH and GSO4 intermediates can be validated further by joint experimental measurements, where the fast rate constant of 4.9 × 109 M–1 s–1 and the reaction stoichiometry of 0.58 supports this low-barrier new mechanism. In addition to the dominant pathway of GSOOH → GSO2 → GSO4 → GSO3, a side pathway with higher barrier, GSOOH → G, has also been located, providing a rationalization for the observed product distributions of GSO2 and GSO3 as major products and G as minor product. From mechanistic and kinetics points of view, the present findings provide new chemical insights to understand the high phototoxicity of 6-TG in DNA and point to methods of using 6-TG as a sensitive fluorescence probe for the quantitative detection of 1O2, which holds particular promise for detecting 1O2 in DNA-related biological surroundings.
|
| |
| 15. Aggregation-Induced Enhancement Effect of Gold Nanoparticles on Triplet Excited State |
Remarkable optical properties are posed with gold nanoparticles (AuNPs) due to the excitation of localized surface plasmon resonances, which makes AuNPs affect strongly both the ground state and the excited state of adjacent organic molecules. Compared with the ground state, the effect of AuNPs on excited state of organic molecules is not always fully understood. Here, we performed transient UV–vis absorption experiments to monitor the triplet excited state formation of three cationic dyes and one anionic dye in the presence of two types of gold nanoparticles: the citrate-stabilized AuNPs and ATP-protected AuNPs. It is found that the three cationic dyes can cause efficient aggregation of citrate-stabilized AuNPs, leading to AuNPs aggregates with varied size, whereas the ATP-protected AuNPs can be sustained in the monodispersed state. By comparing the circumstances of aggregated AuNPs and monodispersed AuNPs, we demonstrate that the enhancement effect on triplet excited state formation results from the aggregation of gold nanoparticles and depends on the aggregation size. These findings reveal the aggregation induced plasmon field interaction of AuNPs with excited state population dynamics and may enable new applications of aggregated metal nanoparticles, where aggregates can serve as stronger plasmonic nanoantennas.
|
| |
| 16. Ultrafast Formation of the Benzoic Acid Triplet upon UV Photolysis and Its Sequential Photodissociation in Solution |
Time-resolved infrared (TR-IR) absorption spectroscopy in both the femtosecond and nanosecond time domain has been applied to examine the photolysis of benzoic acid in acetonitrile solution following either 267 nm or 193 nm excitation. By combining the ultrafast and nanosecond TR-IR measurements, both the excited states and the photofragments have been detected and key mechanistic insights were obtained. We show that the solvent interaction modifies the excited state relaxation pathways and thus the population dynamics, leading to different photolysis behavior in solution from that observed in the gas phase. Vibrational energy transfer to solvents dissipates excitation energy efficiently, suppressing the photodissociation and depopulating the excited S2 or S3 state molecules to the lowest T1 state with a rate of ∼2.5 ps after a delayed onset of ∼3.7 ps. Photolysis of benzoic acid using 267 nm excitation is dominated by the formation of the T1excited state and no photofragments could be detected. The results from TR-IR experiments using higher energy of 193 nm indicate that photodissociation proceeds more rapidly than the vibrational energy transfer to solvents and C–C bond fission becomes the dominant relaxation pathway in these experiments as featured by the prominent observation of the COOH photofragments and negligible yield of the T1excited state. The measured ultrafast formation of T1excited state supports the existence of the surface intersections of S2/S1, S2/T2, and S1/T1/T2, and the large T1 quantum yield of ∼0.65 indicates the importance of the excited state depopulation to triplet manifold as the key factor affecting the photophysical and photochemical behavior of the monomeric benzoic acid.
|
|
|
