Quintets of uracil and thymine: a novel structure of nucleobase self-assembly studied by electrospray ionization mass spectrometry

Abstract

ESI-MS and molecular dynamic calculations reveal that in the presence of K⁺, Rb⁺ and Cs⁺, uracil, thymine and their homologues form self-assembled quintet structures that are stabilized by hydrogen bonding and ion dipole interactions.

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The self-assembly of nucleobases is of great interest in both biochemistry and supramolecular chemistry. A good structural model of self-assembly can simplify a complex system and stimulate further research activities in many related areas. Such an excellent example is G-quartet, which has drawn tremendous attention over the past 40 years.^1,2 Recently, a synthetic nucleoside isoguanosine (isoG) was found to form pentameric self-assembly in the presence of Cs⁺ in CDCl₃.³ Later, Chaput and Switzer proposed that the relative orientation of the hydrogen-bond donor and acceptor groups on G and isoG may have determined that G favours to form planar cyclic tetramers while isoG prefers to form planar pentamers.⁴ Although the proposed design scheme is general and may be extended to more complex structures, such pentameric or “quintet” structural models have not yet been applied to a common natural nucleobase. In fact, uracil (U) and thymine (T) were observed to specifically form pentameric clusters in the presence of K⁺, Rb⁺ and Cs⁺ by ESI-MS.^5,6 More recently, we reported that [T₅ + K]⁺ could also be directly produced from ambient solid thymine sample mixed with potassium salt by desorption electrospray ionization (DESI).⁷ However, no structural information was available for these magic number cluster ions. Here, we provide evidence to show that these particularly stable nucleobase pentameric ions can be well accounted for by a quintet structural model (see Fig. 1).

Fig. 1 (a) Structure of uracil; (b) quintet structure of the magic number pentameric clusters of uracil and its homologues.

Fig. 1(b) shows the proposed quintet structure of the pentameric cluster ion [B₅ + M]⁺, where B is a base molecule, e.g.uracil (U), thymine (T) or a alkyl-substituted homologue of U such as 5-ethyluracil (5EU), 6-methyluracil (6MU), or 5,6-dimethyluracil (DiU), and M is K⁺, Rb⁺ or Cs⁺. Five of the base molecules form a planar cyclic pentamervia dual self-complementary hydrogen bonds, and the central cation is coordinated by five carbonyl oxygen atoms. Such a model allows one to focus on the factors that are important to the self-assembly, i.e.hydrogen-bonding, size of the central ion, and the effect of the exo-cyclic alkyl groups in the formation and stabilization of this structure. All of these have been supported by the following experimental and theoretical results.

Uracil and its C5 and or C6 alkyl-substituted homologues form magic number pentameric cluster ion [B₅ + M]⁺ in ESI-MS (see Fig. 2(a)–(e)). It is noted here that any two (e.g. denoted here by ¹B and ²B) of these molecules can also form a series of pentameric cluster ions of mixed ligands [¹B_n²B_5−n + M]⁺ (n = 0∼5), which will be further studied in our laboratory. However, in the spectrum of 1-methyluracil (Fig. 2(f)), only monomer, dimer and trimer ions can be observed, which is consistent with the ESI behaviour of uridine.⁸3-Methyluracil and 1,3-dimethyluracil exhibited very similar behaviour with 1-methyluracil (data not shown). These results suggest that N1–H and N3–H of uracil contribute significantly to the stability of the pentameric ion, likely due to their participation in the formation of the hydrogen-bonds. In contrast, alkyl substitutions of uracil on either C5 or C6 or on both improve the stability of the pentameric ions. These substitutions increase the dipole moment and polarizability of the ligands,⁹ which may lead to stronger binding between the ligands and the central cation. The later results also support the proposed quintet structure in which C5 and/or C6 alkyl substitutions do not interrupt the hydrogen-bonding since they are located outside the hydrogen-bonding rings (see Fig. 1).

Fig. 2 ESI mass spectra of 1.0 × 10⁻⁴ mol L⁻¹ KCl and 3.0 × 10⁻⁴ mol L⁻¹ (a) uracil (U); (b) thymine (T); (c) 6-methyluracil (6MU); (d) 5-ethyluracil (5EU); (e) 5,6-dimethyluracil (DiU); (f) 1-methyluracil (1MU).

Following the same line of examining the role of hydrogen-bonding, we also studied ESI behaviour of thio-substituted uracil analogues including 2-thiouracil (2SU) and 4-thiouracil (4SU). It is expected from the structural model (Fig. 1) that thio-substitution can eliminate or at least reduce the contribution of hydrogen-bonding in the stabilization of the pentameric cluster ions. Indeed, no magic number pentameric cluster ions were observed for both of the thio-substituted analogue compounds. In the ESI spectrum of 2SU, even pentameric cluster ions could not be observed. Interestingly, however, ESI spectra of 4SU exhibited a minor peak of the pentameric cluster ion [4SU₅ + M]⁺ though its relative intensity was only about 20–30% of the tetrameric cluster ion depending on the cation M. These results suggest again that the hydrogen-bonds play key roles in the stabilization of the quintet and its inner hydrogen-bonding (O2⋯H–N3) has a larger effect than its outer one (O4⋯H–N1), which is consistent with the proposed structural model.

Collision induced dissociation (CID) experiments may also suggest the nature and strengths of the interaction among the components of the clusters.¹⁰ With gradual increase of the collision energy, the pentameric cluster ion [B₅ + M]⁺ undergoes sequential loss of single unit B, indicating that there is only non-covalent bonding among these units. Furthermore, as Fig. 3 shows, the dissociation energy (DE)^10,11 for the loss of a unit B at 50% of the parent ion intensity decreases with the association number of the examined clusters except for the pentamer. It is expected that the binding strength between a unit B and the cation M will be weakened when there are more Bs simply aggregating around the cation. The unusual stability of the pentameric cluster implies that the interactions between Bs in the pentamer play decisive roles, and such additional stabilizing effects correspond well with the dual complementary hydrogen bonds which can only establish between Bs in the proposed quintet structure for uracil-like molecules with properly oriented donor and acceptor atoms.⁴

Fig. 3 Dissociation profiles of various clusters of uracil with Cs⁺. The collision energy was in percentage of the maximum “tickling” voltage, and the relative abundance of the precursor ion was expressed in fraction of the total ion current.

The cation size is also an important factor that determines the formation and stability of the quintet. ESI experiments by varying the central cation showed that U, T and other homologues of U could not form pentameric clusters with Li⁺ and Na⁺. A subtler comparison among K⁺, Rb⁺, Cs⁺ was also studied using ESI-MS by mixing a base (e.g. T) with a pair of the alkali metal ions (1 : 1, mol/mol) (see Fig. S1 in ESI).† As we expected, two pentameric clusters each with a different central cation were observed in one spectrum. Their signal intensities reflect the relative capability of the central cation in stabilizing the quintet. The stabilizing order was Cs⁺≈ Rb⁺ > K⁺, implying that a larger cation was favored for the quintet structure. Na⁺ or Li⁺ may be too small to form stable interactions with all the surrounding carbonyl oxygen atoms if it was the central cation of the quintet.

The above estimates are further supported by DFT-based quantum chemical calculations¹² on both [U₅ + K]⁺ and [U₅ + Na]⁺. The optimized stable structure of [U₅ + K]⁺ (see Fig. S2 in ESI)† is formed by uracils intimately surrounding the central cation in a C₅ symmetry with dual self-complementary hydrogen bonds between two neighbouring molecules, just like what we proposed in Fig. 1. Interestingly, the structure is non-planar, which is similar to the calculated quintet structure of iso-guanine.¹³ In contrast, the sodium ion is not in the center of the deliberately optimized structure of [U₅ + Na]⁺ (see Fig. S3 in ESI).† The metal ion may only significantly interact with the carbonyl oxygen of one uracil, which makes the structure much less stable, in consistent with the fact that magic number cluster ion [U₅ + Na]⁺ could not be observed by ESI-MS.⁵

In conclusion, a quintet structure for the pentameric magic number clusters of uracil and thymine, as well as for other C5 and or C6 alkyl substituted uracil homologues, observed by ESI-MS was proposed, which was supported by a series of experiments and calculations. The quintet structure formed by uracil resembles the isoG pentamer model¹⁴ and may be the simplest one according to the prediction of Chaput and Switzer.⁴ It is expected that with proper derivatization on either C5 and or C6 of uracil, more complex and interesting molecules may self-assemble into highly ordered quintet structures in the presence of cations of suitable size. The uracil quintet and related motifs are expected to find potential applications in diverse areas of chemistry, such as formation of self-assembled ionophores, analytical sensors, and other functional materials. The methods described in the paper for investigating non-covalent interactions by electrospray ionization mass spectrometry are also applicable to other interesting assemblies in supramolecular chemistry and complexes of biological and pharmaceutical importance.

This work was supported by the National Natural Science Foundation of China (grant no. 20575006 and 20727002).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: [DETAILS]. See DOI: 10.1039/b903857d

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