The conjugate of adenine–cyclen Zn(II) complex: its synthesis and selective recognition abilities for uracil and uridine

A synthetic route to a novel adenine–cyclen conjugate is described. Preliminary results showed that the adenine–cyclen conjugate can bind Zn 2+ rapidly in water and the Zn(II) complex can selectively recognize uracil and uridine. The recognition abilities have been demonstrated directly by UV spectrophotometric titration, NMR titration, and ESI-MS. The apparent 1:2 complex for uracil with the complex 5 was determined by spectrophotometric titration and ESI-MS at pH 8.2 with I = 0.1 (NaNO 3 ).


Introduction
The recognition and selective binding of nucleobases and nucleosides play fundamental roles in all of the three stages of genetic information transfer: replication, transcription, and protein synthesis.][3][4][5] Among the various bases and nucleosides, uracil and uridine have received much attention.Uracil arises in DNA from mis-incorporation during DNA replication (A-U) or as a result of the spontaneous hydrolytic deamination of cytosine (G-U).While the A-U base pair is not directly mutagenic, some transcription factors have significantly reduced binding affinity for A-U compared to A-T base pairs, 6 which can affect gene expression.Thus, although the eukaryotic replicative polymerases have a high fidelity, with mis-incorporation occurring at a rate of approximately 1×10 -7 per base pair per generation, 7 the finite probability presents undesired biological consequences.In humans, spontaneous deamination of cytosine occurs at a rate of approximately 100-500 events per cell per day. 8Failure to repair the G-U base pair leads to a G-C transition mutation to A-C during DNA replication.Consequently the presence of uracil in DNA is detrimental, and its recognition and removal are essential.In general, uracil DNA glycosylase (UDG) is a base-excision repair enzyme that specifically recognizes and removes uracil from double-or single-stranded DNA.So, we need to design artificial receptors to uracil to mimic the biochemical processes.Recently, Kimura reported that cyclen-Zn(II) complexes appended with different side groups can selectively recognize different nucleobases. 9In particular, the Zn(II)-macrocyclic polyamine complexes which appended aromatic sulfonamides [10][11][12] have been applied to the recognition of nucleobases, for example, thymine (dT) and uracil (U), which possess similarly weak acidic protons at the "imide"groups. 13Kalesse reported that the tyrosine-cyclen conjugate could serve as a new tool for the selective cleavage of RNA, with a preference for unpaired uridine. 14ucleobases as supramolecular motifs have the most interesting and intriguing class of molecular architectures. 157][18][19][20][21] Herein, we present a Zn(II) complex of a novel adeninecyclen conjugate which may possibly combine the properties of adenine and cyclen in the hope of increasing the specificity for recognition of uracil and uridine.To the best of our knowledge, this is the first example of an adenine-cyclen conjugate which has a flexible spacer to connect adenine and cyclen.The synthetic route is shown in Scheme 1.

Results and Discussion
ESI-MS studies on the complexation of uracil, uridine with 5. ESI-MS is one of the most promising tools for the characterization of supramolecular complexes. 22In order to determine the     With the addition of uracil to 5, new peaks 12-16 appeared and indicate that the supramolecular recognition of 5 and uracil exists in the mixed system.Furthermore, the initial peaks 1 and 2 of the N(3)-H, N(1)-H in uracil shifted to peak 11, peak 4 to peak 13, and implied that N(3)-H, N(1)-H in uracil attended coordination or formed a hydrogen bond with adenine; peak 5 to peak 10 implied that adenine interacted with uracil; peak 10 to peak 15 implies that Zn 2+ -cyclen moieties of 5 became non-equivalent in the complex of 5/uracil.So, the conjugate of adenine-Zn 2+ -cyclen can interact with uracil through the coordination between Zn 2+ -cyclen and N(3)H in uracil and the Watson-Crick type of binding (Figure 5).

1-[1-(1,4,7,10-Tetraazacyclododecane)] -3-(9-adenine)propane tetrahydrobromide (4).
To a solution of 3 (0.647 g, 1.0 mmol) in dry EtOH (5 mL) at 0 o C, 40% aqueous HBr (1 mL) was added slowly.After being stirred overnight at room temperature, the reaction mixture was concentrated in vacuo below 40 o C to give a solid.This was crystallized from EtOH/24% aqueous HBr to afford 4•4 HBr as a white powder (0.490g, 78%).Mp: 178-179 o C. 1   is the concentration of uracil, ∆A =A f -A b where A f , A b correspond to uracil-bound and uracilunbound form of the tested compound, respectively.The data fitted to the Bensi-Hildebrand Equation (1), wherein the slope is equal to 1/K app ·a and the y-intercept equal to 1/a.K app was determined from the ratio of the slope to the y-intercept.

Conclusions
In conclusion, we have synthesized the conjugate of Zn(II)-cyclen and adenine which could be a highly selective receptor for uracil and uridine in aqueous solution at pH=8.2 with I=0.1 (NaNO 3 ).Furthermore, the formation of the 1:2 complex with uracil indicates that this work can be extended to design receptors for the site-specific binding of nucleic acids.

stoichiometry of the complex formed by uracil and 5 ,
ESI-MS experiments were performed regularly for a mixture of uracil or uridine and 5 (mM) in H 2 O (pH=8.2,I=0.1 (NaNO 3 )).The ESI-MS signals are detected in the positive mode and the spectrum is shown as Figure 1.The experimental mass spectra for the 1:2 ratio of 5:uracil (ESI-MS: m/z =732.4 (M + ), 620.5 (M +uracil)) fit into the theoretical mass distribution spectra of 5⋅2 uracil.Its recognition mechanism between 5 and uracil may be as shown in Figure 3.Under the conditions at pH=8.2, the N(3)H in uracil loses a proton, and coordinates with Zn(II) in the conjugate 5; then adenine in conjugate 5 interacts with the other uracil through the Watson-Crick type of binding.When the pH was adjusted to 8.2, the complex formed by 5 and uridine in 1:1 ratio of 5:uridine (ESI-MS: m/z=655.8(M+ +1), 679.8 (M+Na) + ) (Figure 2) should fit the theoretical mass spectrum with the assembly structure 5⋅uridine.The assembly between 5 and uridine is shown in Figure 4.