Complex self-assembly of pyrimido[4,5-d]pyrimidine nucleoside supramolecular structures

Abstract

Supramolecular self-assembly is not only one of the chemical roots of biological structure but is also drawing attention in different industrial fields. Here we study the mechanism of the formation of a complex flower-shaped supramolecular structure of pyrimido[4,5-d]pyrimidine nucleosides by dynamic light scattering, scanning electron microscopy, differential scanning calorimetry, nuclear magnetic resonance and X-ray analysis. Upon removing the hydroxyl group of sugars, different flower-shaped superstructures can be produced. These works demonstrate that complex self-assembly can indeed be attained through hierarchical non-covalent interactions of single molecules. Furthermore, chimerical structures built from molecular recognition by these monomers indicate their potential in other fields if combined with other chemical entities.

ARTICLE

Received 14 Oct 2013 | Accepted 13 Dec 2013 | Published 24 Jan 2014

Hang Zhao1,2,*, Xiurong Guo2,*, Shiliang He2,*, Xin Zeng1, Xinglong Zhou2, Chaoliang Zhang1, Jing Hu1, Xiaohua Wu2, Zhihua Xing2, Liangyin Chu1, Yang He2 & Qianming Chen1

Supramolecular self-assembly is not only one of the chemical roots of biological structure but is also drawing attention in different industrial elds. Here we study the mechanism of the formation of a complex ower-shaped supramolecular structure of pyrimido[4,5-d]pyrimidine nucleosides by dynamic light scattering, scanning electron microscopy, differential scanning calorimetry, nuclear magnetic resonance and X-ray analysis. Upon removing the hydroxyl group of sugars, different ower-shaped superstructures can be produced. These works demonstrate that complex self-assembly can indeed be attained through hierarchical non-covalent interactions of single molecules. Furthermore, chimerical structures built from molecular recognition by these monomers indicate their potential in other elds if combined with other chemical entities.

DOI: 10.1038/ncomms4108 OPEN

Complex self-assembly of pyrimido[4,5-d]pyrimidine nucleoside supramolecular structures

1 State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, No. 14, Section 3, Renminnan Road, Chengdu,Sichuan 610041, China. 2 Laboratory of Ethnopharmacology, Institute for Nanobiomedical Technology and Membrane Biology, Regenerative Medicine Research Center, West China Hospital, West China Medical School, Sichuan University, Chengdu, Sichuan 610041, China. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to Y.H. (email: mailto:[email protected]

Web End [email protected] ) or to Q.C. (email: mailto:[email protected]

Web End [email protected] ).

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Biological structures developed by living organisms have their chemical roots in molecular (covalent) and supra-molecular (non-covalent) morphogenesis and are cur

rently at the frontiers of physics, chemistry, biology and other disciplines with high hopes to generate novel entities and properties16. Some micro- and nano-structures with plant-like morphologies have been reported713. Recently, highly branched ower-shaped structures have attracted attention due to their potential in the elds of magnetic materials, catalysis, optoelectronic devices, solar cells and medicine1419. Previously, the ower-shaped structures were mainly synthesized by covalent inorganic species. Later, a fullerene derivative was reported to be the rst ower-shaped organic supramolecular micro-structure synthesized through the combination of the pp interaction of C60 and the

van der Waals interaction of aliphatic side chains20,21. Certain ower-shaped assemblies of benzothiophene derivatives also showed superhydrophobic properties due to this special morphology22,23. Very recently, a protein-inorganic hybrid nanoower has been reported with the advantages of enhancing enzymatic activity and stability24.

Another type of lifes central molecules, nucleic acids (DNA/RNA), have been exploited at polymeric level, with advantages of high-delity molecular recognitions and programmable synthesis, to assemble nanowires, lattices and 3D octahedron superstructures, and have found many applications (not only as genetic materials) in nanotechnology2531. Nucleosides, with specic basebase interactions, have also been used to construct certain supramolecular structures at monomeric level, such as G-quartets, columnar nanotubes and nanoparticles3234. However, using them to construct structures with complex shapes is a relatively unexplored area. Varieties of nucleobase moieties other than canonical purines and pyrimidines have been developed to construct novel super-structures through diverse base pair patterns3539. For instance, the Janus-type guaninecytosine base derivatives have been reported to form rosette nanotubes (RNTs)35,38. Inspired by these works, we designed and synthesized a series of Janus-type pyrimido[4,5-d]pyrimidine nucleosides combining all of the four chemical letters of the genetic alphabet, tridentate guaninecytosine and bidentate adeninethymine (uracil) nucleosides40,41. In the attempt to build nanostructures with these nucleosides through their self-complementary two-faced base pair motifs, serendipitously and excitingly, we found that compound 1 (J-AT) can form a complicated and aesthetically appealing ower-shaped superstructure in solution, which was the rst report of ower-shaped morphology formed by nucleoside derivatives42,43. Regrettably, there was no insight into the formation process and ner molecular architectures of these assemblies obtained at that time. Herein we reveal its formation mechanism by time-elapsed SEM, DLS, DSC and VT-NMR. The inner atomic-level molecular interactions are revealed by single-crystal X-ray analysis. In addition, different ower-shaped species are constructed and the recognition property of this ower-shaped structure is investigated. Previously, due to directional restrictions, H-bonds mainly directed the formation of spatially regular micro- or nano-structures in the case of conformational rigid small molecules. In the current case, because of the intrinsic three-dimensional structure and exible conformation of nucleo-sides, complex ower-shaped supramolecular morphologies have been constructed and it epitomizes that the reproducing and tailoring of complex structures through hierarchical non-covalent interactions with relatively singular molecules are achievable only by ne tuning the structural parameters.

ResultsStability of the ower-shaped superstructure. Upon construction of supramolecular structures with pyrimido[4,5-d]pyrimidine nucleosides, we found that compound 1 (J-AT) formed ower-shaped superstructures with uniform diameters of B40 mm as observed by SEM and DLS (Fig. 1, Supplementary

Fig. 1), which is drastically different from the microsphere-shaped structures formed by the 20-deoxyribonucleoside 2 under the exact same conditions. Such ower-shaped superstructures were stable up to 60 days, and no pronounced changes in sizes and morphologies were found during the whole period, indicating that neither collapse nor aggregation occurred. The inuence of pH on the stability of the ower-shaped superstructure was checked (Supplementary Fig. 2). The microower was stable over a wide pH, ranging from 3 to 11. Below pH 3 microspheres and smooth membranous structure were produced, and above pH 11 brous structures and nano-particles were produced.

Like other self-assembly systems, the formation of such ower-shaped structure was concentration dependent (Supplementary Fig. 3). When the concentrations of compound 1 were above 0.1 mg ml 1 up to the saturated 0.5 mg ml 1, the ower-shaped superstructures were observed. However, at concentrations below 0.1 mg ml 1 only microspheres were observed, which could be taken as the threshold of the phase transition between two states just like the critical micelle concentration (CMC) is needed for the surfactant to assemble a micelle44. The formation process of the microspheres in solution at lower concentrations was monitored by temperature-variant DLS measurements and the dependence of the distribution of hydrodynamic radius of the particles on temperatures indicated that the microspheres observed under SEM were not the result of solvent evaporation (Supplementary Fig. 4).

Formation process of the ower-shaped superstructure. To investigate the formation mechanism of the ower-shaped superstructure, time-elapsing experiments were carried out. From the samples taken at different time intervals between 0 min and 14 h, it can be seen that the ower-shaped superstructure was growing from the microspherical structure (Fig. 2ag). Only complete microowers were observed from all the samples taken after 14 h (Fig. 2h,i). These phenomena suggested that the formation of the microowers also happened in the solution, not as a mere result of surface deposition; otherwise, the same shapes should be observed at different time intervals. The time-dependent SEM images also suggested the following mechanism for the formation of the ower-shaped superstructure. In the rst stage a spherical nucleation was formed (indicated by low-concentration DLS and SEM); in the second stage the petals radially protruded from the surface and grew into the nal fully shaped ower, with the gradual fading of the spherical bud (Fig. 2f).

The energetics of this phase transition was also characterized by DSC, in order to determine the thermodynamic parameters of non-covalent interactions. The overall thermal effects detected were very weak due to the relatively low concentration of the solution; thus, a high-precision microcalorimeter was used. The DSC melting prole of compound 1 (0.4 mg ml 1) in water (Supplementary Fig. 5) also indicated two-phase transitions in accordance with the visual evidences of SEM. The thermodynamic information was obtained as: (Tm 1 50.8 C,

DHcal 1 1,259.4 kJ mol 1, DG25

1 132.2 kJ mol 1

and Tm 2 63.6 C, DHcal

2 93.3 kJ mol 1, DG25

2

79.7 kJ mol 1). The DG25C of the low-temperature transition is much larger than that of the high-temperature transition. To investigate the non-covalent interactions that direct the formation

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4108 ARTICLE

D

A

NH2

O

NH2

O

5

6

78

4

A D

N

NH

N

NH

A T

N

N

O 1

2

3

N

N

O

HO

HO

O

O

4

5

1

2

3

OH

OH

OH

1 2

Figure 1 | SEM images and molecular structures of compounds 1 and 2. (a) Flower-shaped superstructure self-assembled by 1 in water (0.2 mg ml 1) and its molecular structure with the systematic numbering. (b) Microsphere superstructure self-assembled by 2 in water (0.2 mg ml 1) and its molecular structure showing the two-faced hydrogen bond acceptordonor motif of adenine and thymine: the arrows of A at A represent the hydrogen acceptors and the arrows at D represent the hydrogen donors. Scale bars, (a) 10 mm; (b) 5 mm.

of such ower-shaped superstructure, the hydrogen bonds were rst investigated by the variable-temperature 1H NMR (VTNMR) in DMSO (d6). For the base moiety, the chemical shift of

N11HA involved in the intramolecular H-bond remained unchanged, but the chemical shifts of N11HB and N3H involved in intermolecular H-bonds displayed signicant changes upon varying temperature (Supplementary Fig. 6). For the sugar residue, the H-bonds resulting from hydroxyl groups in solution state were also observed: for instance, the chemical shift for 20-OH of the sugar moiety moving from d 5.33 (363 K) to d5.58 p.p.m. (293 K), with Dd 0.25 p.p.m. (Supplementary Fig. 7).

It is worthwhile mentioning that H-bonds from both base moiety and sugar residues displayed biphasic melting transitions as well (the melting curves of N11HB and 20-OH are given as examples in Supplementary Fig. 8) and the reversibility was conrmed by the overlapping of the VT-NMR melting curves between heating and cooling processes. As the difference of molecular structure between compound 1 and 2 lies only on the 20-OH group, we supposed that the 20-OH group plays a key role in the direction of the formation of the ower-shaped supramolecular structures. However, the specic connectivities of these groups could not be solved by this method. To decipher the inner workings of this superstructure at atomic level, we therefore tried to get the single crystal of compound 1 from water for X-ray analysis.

Single-crystal structure of compound 1. The preparation of the single crystal was carried out in a much slower process by cooling the saturated aqueous solution of 1 from 100 to 30 C in steps of 0.5 C/h in an incubator. Colourless needle single crystals suitable for X-ray diffraction were obtained in 1 week. This was the very rst example of a crystalline free pyrimido[4,5-d]pyrimidine nucleoside and unusual structural features were found. Inspection of the single crystal of 1 disclosed the

co-existence of two conformers in equal amounts (Supplementary Data 1). These two conformers, A (Fig. 3a) and B (Fig. 3b), were different in all the three interacted fundamental structure parameters for nucleosides. We used the torsion angle w(O40-C10-N8-

C9) to indicate the rotational position of the nucleobase relative to the sugar ring (syn/anti conformation) for pyrimido[4,5-d]pyrimidine nucleosides. The torsion angles w of conformers

A and B were measured to be 151.9(9) and 171.8(1),

respectively. Therefore, conformer A adopted an anti conformation (Supplementary Fig. 9a) and conformer B adopted an unusual high-anti conformation with the almost eclipsing of O4B0-C1B0 and N8B-C7B bonds (Supplementary Fig. 9b). The high-anti conformation was very scarce for other nucleosides except for azanucleosides and formycin45. In the current case, B conformer appeared to be stabilized through an unprecedently reported intermolecular H-bond between 20-OH and N1 of base moiety as mentioned above (Fig. 3b).

Sugar puckering (dened by pseudorotation phase angle, P) is the driving force to dominate the whole conformations of nucleosides and even of DNA/RNA. Among two principal puckering modes (C30-endo, North or C20-endo, South), ribonucleosides invariably adopt C30-endo (North) puckering. Here, we found, conformer A adopted a typical N-type conformation with a twist of C30-endo (3T4, P 20.7(2), tm 34.1(7)), but

conformer B adopted an S-type conformation with a twist of C30-exo (3T4, P 206.0(3), tm 32.5(5)), which resulted from

the unusual intramolecular H-bond between 20-OH and N1 mentioned above.

The orientation of the 50-hydroxyl group relative to the furanose, dened by the torsion angle g (O50C50C40O40), is also a crucial structural parameter. Conformer A was in ap (gauche, trans) range with the O5A0-OH at equatorial position and pointing into the sugar ring (g 68.6(5)). Conformer B was

in sc (gauche, gauche) range with the O5B0-OH at axial

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a b c

d e f

g h i

Figure 2 | SEM images showing the formation process of the ower-shaped superstructure. (ag) Intermediates of incomplete microowers were observed within 14 h, which was in a quite similar manner mimicking the natural owers blooming from buds. (h,i) only the fully unfolded microowers were observed after 14 h (h, backside; i, frontside). Scale bars, (ac) 5 mm; (di) 10 mm.

position and pointing outside the sugar ring (g 69.1(3)).

Consequently, the distance between 50-OH and 30-OH of conformer B is more stretched than that for conformer A (Fig. 3a,b).

For the normal bidentate adeninethymine base pairs, not only WatsonCrick but also reverse WatsonCrick pattern is possible, and the latter will lead to the formation of a parallel-stranded duplex DNA46,47. The current J-AT system is endowed with the same options (Fig. 3c), but only the reverse WatsonCrick base pairs were found in the single crystal (Fig. 3d). Strikingly, these base pairs existed invariably between conformers A and B through the faces of adenine and thymine (Fig. 3d, N3B-H3B N6A, N11A-H11AO13B, N3A-H3AN6B, N11B-H11BO13A), which extended to form a linear ribbon structure with an alternating arrangement of conformers A and B with ribose residues located on both the sides. In addition, an intramolecular hydrogen bond between N11-H and O12 was found in both conformers, which was a general characteristic for the J-AT system.

At the 3D supramolecular level, a novel multilayered structure with complicated hydrogen bond networks was formed (Fig. 4ad). The layers can be clearly seen from Fig. 4a, but adjacent base pair layers are not stacked (Fig. 4b and Supplementary Fig. 10). To display the complexity clearly, here we present the whole hydrogen bond interactions in three

categories: AA, BB and AB interactions across different layers with the participation of some water molecules. The AA conformer interactions are displayed in Fig. 4c: one water molecule links together three A conformers from three neighboring layers with four hydrogen bonds and a direct intermolecular hydrogen bond of O5A0H5A0O13A tethers the sugar residue and base moiety of two A conformers from adjacent layers together. The BB conformer interactions are displayed in Fig. 4d: one water molecule acts as a bridge joining two B-conformers from the upper and lower layers with bifurcated hydrogen bonds and a direct intermolecular hydrogen bond of C1B0-H1B0O13B connects the sugar residue and base moiety of two B conformers between the middle and the lower layer. The interactions of conformers AB are more complicated and can be seen from Fig. 4b: The 30-OH of one conformer A is connected to the 20-OH of one conformer B at the same layer through a water molecule. The 20-OH of one conformer A is directly connected to the 20-OH of one conformer B (O2A0H2A0O2B0). Another water molecule (O15) holds two conformers A and one conformer B together. There is another direct hydrogen bond holding conformers A and B together from the adjacent layers (O3A0H3A0O3B0, N11AHAO5B0). Totally, there are nine intermolecular hydrogen bonds acting as the repeating unit connecting conformer A and B together.

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4108 ARTICLE

a

b

N11B

N11A

C5B

O12B

C4B

C5A

N6A

C7A

N8A

C10A

C9A C4A

C2A

N1A

O13A

N3A

N6B

C7B

C10B

O12A

N3B

C9B

C5B

O5B

N8B

C5A

O5A

C3A

N1B

O4A

O4B

C1A

C2A

O2A

C4B

C3B

O3B

C1B

O13B

C2B

C4A

O3A

C2B

O2B

c

H

O

H

H

O

H

H

N

H

O NH2

R

N H

O

N H

O

O

N

N

T

N

R

N

N

N

HN

N

A N

N

N

N

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T A

H

A

T

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A

A N

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H

N

T

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N

N

R

R

O

R

O

H

N

1

R = ribose

H

Watson-Crick base pair Reverse Watson-Crick base

d

O2B

N1B

C2B

N1B

O12A

O12A

N11A

N11A

N8A

N6B

N11B

N3B

N6B

N6A

N3A

N11B

O12B

O13B

O12B

O13A

O13A

Figure 3 | Structures of two monomeric conformers and the base pair motifs between them. (a) Conformer A adopts an anti conformation withan N-type (30-endo) sugar puckering and 50-OH at ap position. (b) Conformer B adopts high-anti conformation with an S-type (30-exo) sugar puckering and 50-OH at sc position. The intramolecular H-bond between 20-OH and N1B is shown in green colour. (c) Two possible base pair motifs of compound 1. (d) A detailed view of the reverse WatsonCrick base pairs in the solid state of 1. The repeated hydrogen bonds unit connecting conformers A and B together in the whole assembly is highlighted in green colour. Atoms are coded as follows: red, oxygen; blue, nitrogen; gray, carbon; white, hydrogen.

Therefore, for compound 1, a very complicated hydrogen bond network (20 hydrogen bonds in total from both base and sugar parts) is repeated innitely in the whole assembly to form the 3D multilayered supramolecular structure in single-crystal state. Obviously, both base moieties and the sugar residues are intrinsic deterministic factors (through proper conformation) to direct the three-dimensional superstructure shape. Meanwhile, the dried

ower-shaped powders obtained from the SEM samples were also analysed by XRPD48. The pattern of XRPD displayed close similarity with the pattern simulated from the single-crystal data (Supplementary Fig. 11), suggesting that the ower-shaped solution aggregate on fast cooling adopted the same interactions. The phenomenon that compound 2 (lacking the 20-OH group compared with compound 1) could only form the

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a b

z

x x

N11A

O5B

O13A

O15

O3B

O2B

O3B

O2A

N1A

N1B

O16

O3A

c

b

d

c

d

O15

O16

O13A

N1A

O13B

O5A

O16

O2B

O5B

O15

O13A

N13B

C1B

O13A

N1A

O5B

O16

N1A

N16

O2B

O5A

C1B

O15

O15

O5B

O13A

N1A

O2B

O2B

O15

Figure 4 | Complicated hydrogen bond networks of compound 1. (a) The overall multilayered supramolecular structure of compound 1. (b) The interactions between conformers A and B (viewed through z direction of a). The base moieties of adjacent layers are not stacked and the covalent bonds of different layers are displayed in yellow or purple colour, respectively. (c) The interactions between conformer A and A (viewed through x direction of a).

(d) The interactions between conformer B and B (viewed through x direction of a). The total repeated 20 hydrogen bonds unit crossing different layers of the whole assembly is highlighted in green colour. Atoms are coded as follows: red, oxygen; blue, nitrogen; gray, carbon; white, hydrogen.

microsphere suggested that the 20-OH group might play a key role in this anisotropic growth process. Its importance was also revealed here by the multiple participations in the whole H-bond network. To further conrm this suppose, we designed and synthesized the 50-deoxyrbonucleoside (3) and 30-deoxyribonucleoside (4); both conserved the 20-OH group but one -OH group was removed like the case of 2, to see whether or not they could still form the ower-shaped superstructure in solution state.

Construction of ower-shaped superstructures. 50-Deoxyribonucleoside (3) was synthesized from the sugar donor of 1, 2, 3-tri-O-acetyl-5-deoxy-b-D-ribofuranose. The initial attempts to synthesize 30-deoxyribonucleoside (4) through selective deoxygenation of 1 failed due to the poor solubility of free J-AT nucleoside in various organic solvents. Consequently, 4 was obtained via Vorbrueggen glycosylation procedures with SnCl4 as the catalyst and 1,2-di-O-acetyl-5-O-benzoyl-3-deoxy-b-D-ribofuranose as the sugar donor (Supplementary Figs 1217). As we anticipated, both compound 3 and 4 can form ower-shaped superstructures, which conrmed the key role of the 20-OH group: once it exists in the molecule, the ower shape retains. Of course, the different molecular structures also led to different overall shapes of the owers: spiral form for 3 and ball ower for 4, but both retained the petals as shown in Fig. 5. Fortunately, the single crystal of 4 was obtained in aqueous solution by a slowly cooling procedure (Supplementary Data 2). The differences of inner workings between 1 and 4 can be seen clearly in the single-crystal state. There were also two conformers (anti and high-anti) for compound 4 in equal amounts, which were connected together by the reverse WatsonCrick base pairs

(Supplementary Figs 18,19) as well. However, owing to lack of the 30-OH group, the sugar puckering of both conformers of 4 adopted N-type modes without the intramolecular hydrogen bond of 20-OH-N1. Instead, the high-anti conformation of conformer B was locked through the intermolecular hydrogen bonds with the participation of 20-OH, in the context of the whole assembly. In general, the whole supramolecular assembly was formed by spatial repeating of 13 hydrogen bonds involving both base and sugar parts (Fig. 5c, Supplementary Fig. 20). The four hydrogen bonds formed around 20-OH (O2A0H2A0O5B0,

O2A0N11AH11A, O2B0H2B0O5A0, O2B0N11BH11B) clearly indicated its key roles in directing the whole assembly. It is worth mentioning that water molecules were totally expelled from the whole assembly of 4. These differences might suggest that the ower formed by 4 was more compact than 1.

Molecular recognition by compound 1. The base moiety of J-AT nucleoside derivatives has one face of adenine H-bond array and the other face of thymine array. In addition to the self-complementarity, J-AT, in principle, can be competitively base paired with either adenosine or uridine/thymidine. The inuences of the complementary or non-complementary natural ribonucleosides on supramolecular shapes were investigated by adding them into the aqueous solution of 1 (0.2 mg ml 1) and comparing the new structures with the corresponding structures of individual normal nucleosides (Supplementary Fig. 21). Initially, we supposed that both adenosine and uridine could interrupt the microower formation of 1 because the elongation of the linear ribbon structure will be terminated by J-AT-A or J-AT-U base pairs. Indeed, the ower-shaped structure of 1 completely disappeared in the case of J-AT A (1:1) mixture, with the

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4108 ARTICLE

NH2

O

NH2

O

N

NH

N

NH

N

N

O

N

N

O

CH3

HO

O

O

OH

OH

OH

3 4

O12A N11A

N3A

N11A

N6B

N11B

N3B

O13B

N11A

N6A

O12B

O13A

O2B

O5B

O5A

O2A

N1B O13B

Figure 5 | SEM images of compounds 34 and the single-crystal structure of compound 4. (a) Spiral ower-shaped superstructure formed by 3 in water(0.2 mg ml 1). (b) Ball ower-shaped superstructure formed by 4 in water (0.2 mg ml 1). (c) The single-crystal structure of 4. The repeated unit of 13 intra and intermolecular hydrogen bonds is highlighted in green colour. Atoms are coded as follows: red, oxygen; blue, nitrogen; gray, carbon; white, hydrogen. Scale bars, (a,b) 10 mm.

formation of microspheres (Fig. 6a), which was completely different from either the J-AT microower or the adenosines ber-shaped structure. This meant the J-AT and adenosine

were indeed hybridized. Contrary to our anticipation, compound 1 did not pair with uridine easily. When 1 equiv. U was added, the microower remained intact (Fig. 6b). When 5 equiv.

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a

b

c

d

e

f

g

h

i

Figure 6 | Chimeric morphologies formed by mixing J-AT with other nucleosides. (a) J-AT:Adenosine (1:1 mole ratio). (b) J-AT:Uridine (1:1 mole ratio). (c) J-AT:Uridine (1:5 mole ratio). (d) J-AT:Cytidine (1:1 mole ratio). (e) J-AT:Cytidine (1:5 mole ratio). (f) J-AT:Guanosine (1:1 mole ratio). (g) J-AT:Guanosine (1:5 mole ratio). (h) J-AT:compound 2 (1:1 mole ratio). (i) J-AT:J-TA (1:1 mole ratio). Scale bars, (a,gi), 5 mm; (bf), 10 mm.

U was added, an amazing chimerical structure with petals protruding from the smooth membrane formed by uridine was observed. This chimerical morphology indicated the partial base pairs between 1 and uridine (Fig. 6c). The different base pair properties between 1 and adenosine or uridine can be explained by the rotation barrier of the glycosyl bond (Supplementary Fig. 22). Current results suggested the anti conformation was predominant in solution as well, which is consistent with the single-crystal structures (anti and high-anti conformers).

1 or 5 equiv. of cytidine was not able to interrupt the ower-shaped superstructure of 1, in accordance with that cytosine pairs with neither the adenine face nor the thymine face of J-AT under such conditions (Fig. 6d,e). In addition, this result supported that the base pairs were the main force in bringing the molecules together; otherwise the cytidine could be incorporated if the interactions between riboses were the main forces. Guanosine showed a moderate inuence on the microower superstructure. The addition of 1 equiv. of guanosine did not change the overall shape of the microower (Fig. 6f) with the co-existence of a tape-shaped structure from guanosine itself. By further increasing guanosine up to 5 equiv., the ower disappeared and transformed to a microsphere like in the case of adding adenosine (Fig. 6g). This phenomenon was practically in accordance with the

well-documented G-A and G-T/G-U mismatches due to the guanines multiple hydrogen-bonding sites.

Next, the effect of incorporating J-AT analogues with different sugar residues was also investigated. The SEM experiments showed that chimerical supramolecular structures consisted of microowers and microspheres were formed in the mixed solution containing equal amounts of compounds 1 and 2 (Fig. 6h). Another chimerical structure combining microowers with interwoven nanobundles was also observed in the mixed solution containing equal amounts of 1 and J-TA nucleoside; the latter formed nanobundles by itself (Fig. 6i, Supplementary Fig. 23). Therefore, the specic molecular recognition property of J-AT nucleoside was retained, indicated by the different hybridized shapes, which is benecial for further functionalizing of this structure through specic non-covalent interactions.

DiscussionPreviously, during our research of some Janus-type nucleoside analogues concerning their antiviral and antitumor activities, in parallel we found that compound 1 could form a beautiful ower-shaped superstructure in different solutions. However, there was no thorough insight into the dynamic formation process and the molecular architecture of this special nucleoside self-assembly

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with high resolution at that time. Considering the scientic and practical signicance of complex natural mimic-shaped chemical structures, we intended to investigate this ower-shaped nucleo-side superstructure thoroughly, including its formation mechanism, the inner interactions at the atomic level, and how the modied structures and its molecular recognition properties inuence the superstructures. In general, the experimental results from DLS, NMR and SEM suggested a two-staged mechanism (from microsphere to a complete ower-shaped state) for the anisotropic growth of the ower-shaped superstructure. In addition, the DSC provided the thermodynamic parameters for such biphasic process. Therefore, to form such complex morphology, rst the non-specic H-bonds hold fast the individual molecules together to form the nucleation and grow into a microsphere, and then once they are held closer a reorganizing process follows. In order to form specic base pairs, the correct conformation around the glycosyl bond was required (high-anti conformation in this case), which further inuenced the sugar puckering. Consequently, all the hydroxyl groups were rearranged again in a xed spatial direction to form an energy-favourable complicated H-bond network, which led to the ne tuning of the whole system into a branched ower-shaped superstructure. The atomic-level interactions of the single crystal consisting of complicated hydrogen bond-mediated networks have been revealed by X-ray analysis, which was very rarely reported for related topics45. This information was fundamental to understanding all the forces and connectivities of such complicated superstructures. An added value is that this structure is a nice example of a H-bonded system in aqueous environment, which is rather difcult to form due to the competition of water molecules49,50. We also carried out XRPD experiments, which bridged the structures between the powders prepared from the ower-shaped solution state and that prepared from the single-crystal state by comparing the experimental pattern with the calculated pattern. The results suggested the ower-shaped solution aggregate on fast cooling adopted the same interactions as the single-crystal state.

The reproduction of similar ower-shaped morphologies by related compounds 3 and 4 by modifying the hydroxyl groups proved the viabilities of using this new type of compounds to build complex superstructures with their unique inner working and meanwhile conrmed our doubts about the relationship between the single molecular structural parameter and the shapes of the nal supramolecular assemblies. This information is also very helpful for us to construct or functionalize such complex-shaped structure in the future. The most powerful property of nucleosides is their unique base pair recognition, which is the base of DNA/RNA replications and transcriptions29,30. These recognition properties were also employed in the current case to build rather interesting hybrid morphologies, which might expand its usages greatly.

In summary, the two-staged formation process of a complicated ower-shaped supramolecular structure was revealed by various techniques. The sophisticated hydrogen bond networks have been revealed by X-ray analysis. Such complex-shaped superstructures can be constructed and expanded to related chemical entities by modifying certain functional groups as well. Therefore, nucleosides can also play an important role, with the combination of the rich chemistry of both heterocycles and sugars and its 3D conformational exibility, to fabricate more complex chemical architectures in the supramolecular self-assembly area.

Methods

General. All chemicals were commercially available. The solvents and reagents were analytic pure. Solvents 1,2-dichloroethane and acetonitrile were puried by distilling from P2O5. Thin-layer chromatography (TLC) was performed on an

aluminium sheet covered with silica gel 60 F254 (0.2 mm, Merck, Germany). Flash column chromatography (FC) was carried out with silica gel 60 (Haiyang chemical company, PR China) at 0.4 bar. NMR spectra were recorded on a AV II (Bruker, Germany) spectrometer at 400 and 600 MHz; the d values in p.p.m. are relative to

Me4Si as internal standard; High-resolution mass spectra were measured with a mass analyser (Q-TOF, Bruker, Germany). The UV absorption spectra were recorded on a DU-800 spectrophotometer (Beckman, US), lmax in nm, E in dm3 mol 1 cm 1; the XRPD spectra were measured on XPert Pro MPD. The DLS spectra were recorded on JL-6000 and Nano-ZS (Malvern); differential scanning calorimetry (DSC) measurements were carried out using a VP-DSC micro-calorimeter (Microcal, Northampton, MA).

SEM experiments. SEM was performed using a high-resolution INSPECT F50. All SEM images were obtained without staining. Samples were prepared by dissolving corresponding compounds in H2O (0.2 mg ml 1). The solutions were heated to 100 C and allowed to cool to room temperature. The samples were taken for SEM experiments at specied time intervals. For the time-elapsing experiments, 100 ml sample solution was taken at different time intervals (0, 20, 40 min, 1, 2, 3, 5, 8, 10, 12, 14, 16, 18, 20, 22 and 24 h) to be observed under SEM.

DSC experiments. Microcalorimetry experiments were performed on VP-DSC microcalorimeter driven by a DSC-run software. Compound 1 was dissolved at a concentration of 0.4 mg ml 1 in water. Solutions were carefully degassed prior to their utilization and their thermal proles were analysed in the 2580 C temperature range at a scan rate of 0.5 C min 1.

Synthetic procedures. All compounds were synthesized from commercially available materials and characterized by 1H, 13C NMR spectroscopy and HRMS.

Crystallographic study of 1 and 4. Single crystals were obtained by slowly cooling the corresponding saturated solution in steps of 0.5 C h 1 from 100 to 30 C.

Proper-sized single crystals of compounds 1 and 4 were stabilized into a tiny glass tube including mother liquor with epoxy resin to minimize solvent loss. Crystal data were collected on Bruker Apex II.

Synthesis of Compound 9. Compound 5 (5-aminopyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione)51 (500 mg, 2.79 mmol) was suspended in hexamethyldisilazane (HMDS, 50 ml). After the mixture being stirred at 140 C for 3 min, trimethylsilyl chloride (TMSCl, 500 ml) was added. The mixture was stirred under reuxing for about 10 h until it turned clear. Then, the solution was evaporated to remove excess HMDS and the silylated base 6 was obtained and immediately used without further purication. At room temperature, to this silylated base residue was added 1,2-dichloroethane:acetonitrile (1:1, 50 ml) followed by adding 1,2,3,-tri-O-acetyl-5-deoxy-b-D-ribofuranose (7). This mixture was cooled to 0 C and SnCl4 (500 ml)

was added as catalyst. After the mist vanished, the reaction was brought to room temperature and the stirring was continued for 1 h. Then, saturated NaHCO3 aqueous solution (20 ml) was added at 0 C to quench the reaction. Next, 30 ml CH2Cl2 was added. The organic phase was collected and dried with anhydrous

Na2SO4. After the evaporation of the CH2Cl2, the residue was applied to FC (CH2Cl2:methanol 99:1) and compound 9 (550 mg, 52%) was obtained as

colourless powder. TLC (CH2Cl2:MeOH, 99:1 v/v): Rf 0.2; UV/Vis: lmax

(MeOH)/nm (e/dm3 mol 1 cm 1): 232 (29,355), 250 (54,418), 276 (14,913), 273 (4,798); 1H NMR(400 MHz, DMSO-d6): d 1.40 (d, J 4.00 Hz, 3H, 50-CH3),

2.07 (d, J 8.80 Hz, 6H, OAc), 4.19 (t, J 4.4 Hz, 1H, 40-H), 5.29 (t, J 4.40 Hz,

1H, 30-H), 5.64 (q, J 2.80 Hz, 1H, 20-H), 6.22 (d, J 2.8 Hz, 1H, 10-H), 8.56

(s, 1H, 7-CH), 9.09 (dd, J 2.40 Hz, J 2.00 Hz, NH2), 10.83 (s, 1H, NH); 13

C NMR (100 MHz, DMSO-d6): d 18.32, 20.74, 20.81, 73.53, 74.09, 78.57, 87.34,90.21, 152.80, 156.73, 157.73, 161.90, 165.31, 169.90, 169.93; HRMS (ESI )

m/z: calcd for: [C15H17N5O7 Na] : 402.1026; found: 402.1028.

Synthesis of Compound 3. Compound 9 (200 mg, 0.52 mmol) was suspended in freshly prepared 15 ml 0.10 M sodium methoxide. The reaction mixture was stirred at r.t. for 20 min. After neutralization with diluted acetic acid, the mixture was ltrated and the residue was washed with MeOH and H2O three times to get the product 3 (140 mg, 91%); UV/Vis: lmax (MeOH)/nm (e/dm3 mol 1 cm 1):233 (16,787), 250 (17,520), 276 (4,833), 273 (4,798); 1H NMR(400 MHz, DMSO-d6): d 1.33 (d, J 4.00 Hz, 3H, 50-CH3), 3.82 (t, J 3.60 Hz, 1H, 40-H), 3.95-3.99

(m, J 4.00 Hz, 30-H), 4.23 (t, J 2.80 Hz, 20-H), 5.13 (s, 1H, 30-OH), 5.52 (s, 1H,

20-OH), 6.13 (d, J 2.40 Hz, 1H, 10-H), 8.40 (s, 1H, 7-CH), 9.02 (s, 2H, NH2), 10.81

(s, 1H, NH); 13C NMR (100 MHz, DMSO-d6): d 18.63, 74.33, 74.62, 80.17, 87.43,90.70, 151.86, 156.95, 158.21, 161.94, 165.43; HRMS (ESI-) m/z: calcd for: [C11H13N5O5-H] : 294.0838; found: 294.0837.

Synthesis of Compound 10. Compound 5 (1.00 g, 5.60 mmol) was suspended in hexamethyldisilazane (HMDS) (50 ml) and stirred at 140 C for about 3 min; then trimethylsilyl chloride (TMSCl, 1 ml) was added. The reaction was stirred at reux until the mixture became clear. Next, the solution was evaporated to remove

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HMDS. The silylated base 6 was obtained, which was immediately used in the next step without further purication. Dry 1,2-dichloroethane (45 ml) was added to the pot containing silylated base 6 and stirred. 1,2-di-O-acetyl-5-O-benzoyl-3-deoxy-b-

D-ribofuranose 8 (920 mg, 2.90 mmol) dissolved in dry acetonitrile (45 ml) was added to the above solution. SnCl4 (952 ml) was added as catalyst at 0 C. After the mist vanished, the reaction mixture was stirred at room temperature for about 2 h, and saturated NaHCO3 aqueous solution (60 ml) was added at 0 C to quench the reaction; CH2Cl2 (3 60 ml) was used to extract the organic phase. After drying

with anhydrous Na2SO4, the organic phase was evaporated. The residue was applied to FC (CH2Cl2: methanol 98:2) and afforded compound 10 (50%, 1.23 g).

TLC (CH2Cl2:MeOH, 98:2 v/v): Rf 0.2; UV/Vis: lmax (MeOH)/nm (e/dm3 mol 1

cm 1): 231 (21,016), 251 (25,376), 277 (6,515); 1H NMR (600 MHz, DMSO-d6): d2.15 (s, 3H, CH3), 3.35 (s, 2H, 30-H), 4.10 (d, J 3.40 Hz, 1H,40-H), 4.634.80 (m,

3H, 20-H and 50-H), 6.37 (s, 1H, 10-H), 7.53-8.03 (m, 5H, H-Ar), 8.43 (s, 1H, C7H),9.01 (d, J 7.50 Hz, 2H, NH2), 10.82 (s, 1H, NH). 13C NMR (150 MHz, DMSO-d6):

d 21.14, 55.38, 58.09, 62.77, 78.23, 81.49, 82.22, 87.36, 89.53, 129.28, 129.76, 134.03, 151.06, 156.83, 157.86, 161.86, 165.34, 166.02, 169.77; HRMS (ESI ) m/z: calcd

for [C20H19N5O7 Na] : 464.1182; found 464.1177.

Synthesis of Compound 4. Compound 9 (100 mg, 0.23 mmol) was dissolved in0.5 M NaOMe/MeOH (7 ml), and the solution was reuxed at 75 C for about10 min. After cooling to room temperature, the solution was neutralized with diluted acetic acid to pH 6.5 and precipitate was formed. After ltration the precipitate was washed with methanol (3 2 ml) and water (1 2 ml). Target com

pound 4 was obtained as a white powder by vacuum drying (50 mg, 74%). UV/Vis: lmax (MeOH)/nm (e/dm3 mol 1 cm 1): 233 (14,294), 250 (25,260), 276 (5,787);

1H NMR (600 MHz, DMSO-d6): d 1.71-1.74 (m, 1H, 30-H), 2.03-2.08 (m, 1H, 30-H), 3.60 (d, J 12.50 Hz, 1H,50-H), 3.87-3.90 (m, 1H, 50-H), 4.34 (s, 1H, 40-H),

4.40-4.47 (m, 1H, 20-H), 5.31 (t, J 4.70 Hz, 1H, 50-OH), 5.63 (d, J 3.80 Hz, 1H,

20-OH), 6.04 (s, 1H, 10-H), 8.86 (s, 1H, NH), 8.92 (s, 1H, NH), 8.96 (s, 1H, CH),10.77 (s, 1H, NH). 13C NMR (150 MHz, DMSO-d6): d 32.56, 61.04, 75.76, 82.65,87.55, 93.13, 151.40, 157.05, 158.08, 162.13, 165.45; HRMS (ESI ) m/z: calcd for

[C11H13N5O5 Na] : 318.0815; found 318.0809.

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Acknowledgements

We thank the National Natural Science Foundations of China (document no.: 81321002, 20772087, 81061120531, 30930100), ISTCPC (2012DFA31370) and the Open Foundation (SKLODSCUKF2012-04) from the State Key Laboratory of Oral Diseases Sichuan University for the nancial support. We also thank Xiaoyan Wang and Daibing Luo of the Analysis and Testing Center of Sichuan University for helping us to process variable-temperature NMR and single-crystal analysis.

Author contributions

H.Z., X.G. and S.H. undertook the synthesis of the all compounds, experimental studies, carried out crystallographic works and wrote the paper; X.Z., X.Z., J.H., X.W., Z.X. and

L.C. carried out instrumental analytic works; C.Z. performed the SEM experiments; Y.H. and Q.C. together designed the whole concept and project, directed and supervised the experimental works and wrote the paper.

Additional information

Accession codes: The X-ray crystallographic coordinates for structures reported in this Article have been deposited at the Cambridge Crystallographic Data Centre (CCDC), under deposition number CCDC: 940972 (1) and 946631 (4). These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via http://www.ccdc.cam.ac.uk/data_request/cif

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How to cite this article: Zhao, H. et al. Complex self-assembly of pyrimido[4,5-d] pyrimidine nucleoside supramolecular structures. Nat. Commun. 5:3108 doi: 10.1038/ ncomms4108 (2014).

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