Molecular and supramolecular chemistry of rosette nanotubes

Abstract

A pyrimido[4,5-d]pyrimidine featuring the hydrogen bond donors and acceptors of both guanine (G) and cytosine (C) in the appropriate geometry undergoes hierarchical self-assembly into nanotubular architectures. Specifically, in solution this heterocycle self-organizes into cyclic hexamers through hydrogen bonding interactions, which then further π–π stack into rosette nanotubes (RNTs). The present work reviews the synthetic strategies used to tune the structure of this class of heterocyclic molecules and ultimately tailor the physical properties of the resulting RNTs for potential applications in nanomedicine, catalysis, and renewable energy.

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Rachel L. Beingessner

Dr Beingessner studied Biochemistry (BSc) and Organic Chemistry (MSc) at the University of Waterloo (Canada). After obtaining her PhD at the University of Ottawa (Canada) in 2007, she joined the National Institute for Nanotechnology (Canada) for one year of postdoctoral training in the area of Supramolecular Nanomaterials, prior to transitioning to a staff research position. In 2015, she accepted a new position at the Massachusetts Institute of Technology where she currently works as a Research Scientist.

Yiwen Fan

Ms Yiwen Fan is a Chemical Engineering PhD candidate in the Supramolecular Nanomaterials Laboratory at Northeastern University, Boston, MA, USA. She received a B.S. in Chemistry from Wuhan University, China. Her research interests mainly include synthesis and self-organization processes of rosette nanotubes for biomedical applications.

Hicham Fenniri

Dr Fenniri received all his degrees from the Université de Strasbourg, France. After postdoctoral training at the Scripps Research Institute, he moved to Purdue University, where he initiated his independent academic career, and established the Purdue Laboratory for Chemical Nanotechnology (1999). In 2003, He joined the National Research Council and the University of Alberta (Edmonton, AB, Canada) as Professor of Chemistry (2003–2013). Dr Fenniri is currently Professor of Chemical and Biomedical Engineering at Northeastern University, Boston, MA, USA. Dr Fenniri's contributions appeared in over 220 publications, 20 patents and patent applications, and over 450 contributed national and international conference papers. Dr Fenniri has also lectured extensively around the globe and he has been an invited professor at several institutes and universities.

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1. Introduction

Supramolecular 1D nanostructures have far reaching applications in chemistry, biology and materials science,^1,2 owing in part to the ability to correlate properties and function with the nature of the individual building blocks. Among 1D nanostructures are cylindrical architectures generated from the stacking of homo-modular or hetero-modular rosettes formed through hydrogen bonding acceptor (A)–donor (D) interactions.^3,4 Many examples of different sizes of rosettes including trimeric,^5–8 tetrameric,^9–23 pentameric,^24–27 and hexameric^28–44 have been showcased in the literature, a few of which have demonstrated further stacking into cylindrical architectures such as nanowires or nanofibers. An elegant example of this hierarchical assembly process was described by Jonkheijm et al. ⁴⁵ They demonstrated the self-assembly of oligo-(p-phenylenevinylene) functionalized diamino triazines into hexameric rosettes that further assembled into cylindrical aggregates. Height measurements of the stacks obtained from AFM images were consistent with small-angle neutron scattering data as well as diameter measurements of the hexameric rosettes observed in scanning tunneling microscopy images.⁴⁵ Another interesting example, in this case, a photoresponsive system, was reported by Yagai and coworkers.⁴⁶ Hexameric rosettes comprised of functionalized cyanurate and melamine featuring photoswitchable azobenzene sidechains, were shown to stack into columnar aggregates and form elongated fibrous assemblies over time. The dissociation and reformation of the columnar aggregates could be controlled by inducing the trans–cis isomerization of the peripheral azobenzene substituents under UV-irradiation.⁴⁶

In this review, the molecular and supramolecular chemistry of the pyrimido[4,5-d]pyrimidine shown in Fig. 1a and its analogues are described.⁴⁷ This heterocycle, termed the G∧C motif herein, was first synthesized by the Lehn group in 1996 (with alkyl or aryl substituents)⁴⁸ to feature a self-complementary triad of the ADD hydrogen bonding arrays of guanine (G) and DAA hydrogen bonding arrays of cytosine (C). In water, this motif undergoes an entropically driven,^49–51 self-organization process to form hexameric rosettes maintained by 18 hydrogen bonds (Fig. 1b). Since the redundant NH group in the cytosine ring is functionalized, the molecule is less prone to pyrimidine–hydroxypyrimidine tautomerism, thereby effectively locking the DAA array for rosette formation, regardless of the solvent (aqueous or organic) it is dissolved in. Once these rosettes are generated, they then organize through π-stacking, van der Waals interactions and solvophobic effects to form discrete tubular architectures called rosette nanotubes (RNTs) that have an inner channel diameter of ca. 1.1 nm (Fig. 1c).^49,50

Fig. 1 (a) Bicyclic G∧C motif, where “A” and “D” denote a hydrogen bond acceptor and hydrogen bond donor, respectively. (b) Six G∧C motifs organize in solution to form a hexameric rosette. (c) Stacking of the rosettes generates the RNT. Representative SEM (d), TEM (e) and AFM (f) images of the RNTs.

While the Lehn group was unable to conclusively demonstrate the formation of hexameric rosettes or RNTs in solution,⁴⁸ a bicyclic analogue maintaining the same hydrogen bonding array was demonstrated by Mascal and coworkers⁵² to form the predicted supramacrocycle in the solid state. Further hierarchical self-assembly of the supramacrocycles into RNTs was not observed however. More recently, high-field solid-state NMR spectroscopy experiments using an ultra high-field magnet field strength of 21.1 T and ultra-fast magic angle spinning (MAS) (60 kHz) conclusively established the hydrogen bonding network within ¹⁵N-labeled RNTs.⁵³ Specifically, 2D ¹H–¹H double quantum correlation experiments, which probe through-space dipolar interactions, elucidated the spatial arrangement of the protons on the G∧C motifs, while ¹H–¹⁵N and ¹⁵N–¹⁵N 2D correlation spectra, both through-bond and through-space, provided clear evidence for the hydrogen bonding pattern of the self-assembled rosette structure shown in Fig. 1b. The ¹H MAS NMR spectrum also revealed two types of water molecules per rosette with a total integration of seven. This is consistent with modeling calculations which predicted a ring-like structure of six water molecules located in between adjacent rosettes, all in contact with the carbonyl group of the G∧C motif exposed on the RNT channel surface (Fig. 2). An additional water molecule confined to the center of the channel, forms a loose chain along the length of the RNTs.⁵⁴

Fig. 2 (a) Top view of the RNT channel showing the wetting monolayer in the channel. (b) Arrangement of water molecules bridging three stacked rosettes. Adapted with permission from T. Yamazaki, H. Fenniri and A. Kovalenko, ChemPhysChem, 2010, 11, 361–367. Copyright (2010) John Wiley and Sons.

A notable feature of the G∧C motif is the functional groups that are chemically bound to the exocyclic nitrogen atom on the G-face (e.g. CH₃ in Fig. 1a) and the endocyclic nitrogen atom on the C-face (e.g. CH₂CH₂-L-lysine in Fig. 1a) of the hydrophobic core. These functional groups modify the properties of the RNTs including their solubility profile, since they are expressed on the outer periphery. Although RNTs are supramolecular structures assembled through non-covalent forces, they are exceptionally tolerant to a wide range of functional groups and maintain excellent control over the self-organization process. They are stable over a broad range of pH values and temperatures, yet undergo dissociation upon application of high heat (>85 °C), high dilution or treatment with a Brønsted acid.⁴⁹

RNTs have been extensively characterized not only by NMR spectroscopy,⁵³ but also by circular dichroism, UV-Vis, dynamic light scattering and fluorescence spectroscopies and by microscopy techniques.^49,51,55 Fig. 1d–f shows examples of scanning electron microscopy (SEM), transmission electron microscopy (TEM) and atomic force microscopy (AFM) images of these supramolecular assemblies. Although the inner channel diameter of the RNTs generated from this bicyclic molecule is fixed at ca. 1.1 nm, their length can be tuned from a few nanometers up to several millimeters by increasing the sample concentration, heating the solution to higher temperatures and permitting longer solution aging times.⁵⁰ Their outer diameter alternatively, is largely dictated by the functional groups attached to the motif. Further hierarchical organization of the RNTs can lead to bundles,⁴⁹ sheets,⁵⁰ and superhelices⁵¹ in water, as well as prolate nanospheroids in organic solvents.⁵⁶

Studies over the past 15 years have highlighted the potential of the RNTs as novel biocompatible^57–59 nanomaterials for drug delivery,^60–65 siRNA delivery,⁶⁶ bone,^67–78 cartilage,^79–81 myocardial^82,83 and endothelial tissue engineering^84,85 as well as for sustainable energy generation⁸⁶ and catalysis applications.^87,88 While details pertaining to these applications are described elsewhere, herein a detailed account of the synthesis and functionalization of the G∧C motif and its analogues are provided. The development of this chemistry has been critical for enabling the physical and chemical properties of these noncovalent architectures to be tuned in a highly controlled manner for such diverse utility.

2. Synthesis of the G∧C motif

Molecules featuring the pyrimido[4,5-d]pyrimidine core are typically synthesized using one of two strategies. The first involves an annulation reaction of a uracil derivative such as shown in Table 1 and the second involves a cyclization reaction of a pyrimidine synthon as illustrated with a few examples in Table 2. Since the G∧C motif requires the ADD–DAA hydrogen bonding array and flexibility in terms of the functional groups expressed, a synthetic route more closely resembling the strategy in Table 2 was implemented. In particular, the G∧C motif 1 was viewed retrosynthetically as the annulated product of the trisubstituted pyrimidine 2 and fragment 3 (Fig. 3). Regioselective nucleophilic aromatic substitution reactions (S_NAr) at positions 2, 4 and 6 on 4 in turn, and conversion of the aldehyde to the nitrile, provides entry into pyrimidine 2.

Table 1 Examples of strategies used to synthesize the pyrimido[4,5-d]pyrimidine core structure starting from uracil or a derivative thereof

Entry #	Synthon 1	Synthon 2	Pyrimido[4,5-d]pyrimidine core	Yield (%)	Reference
1		(i) POCl3/DMF, (ii) Y = S, O, NH (thermal)		52–92	89 and 90
2		Formamide (thermal)		64	91
3		ArCHO, urea, HOAc (microwave)		76–85	92
4		I2 (cat) (thermal)		50–73	93
5		ArCHO, urea, CAN (cat) (thermal)		90–98	94
6		(i) NH2NH2, n-BuOH, (ii) NaNO2, AcOH (thermal)		31–54	95

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Table 2 Examples of strategies used to synthesize the pyrimido[4,5-d]pyrimidine core structure via cyclization of pyrimidine synthons

Entry #	Synthon 1	Synthon 2	Pyrimido[4,5-d]pyrimidine core	Yield (%)	Reference
1		(i) PhCOCl/KOtBu, (ii) NaOH/H2O2		50–76 (2^nd step)	96
2		(i) Cl3CCONCO, (ii) NaOMe		65	97
3		PCl5, R^1 = COPh (thermal)		64	98
4		Urea (thermal)		78	99

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Fig. 3 Retrosynthetic analysis of G∧C motif 1.

Based on this retrosynthesis and the previous work by the Lehn group,⁴⁸ a scalable, nine step synthetic strategy for the G∧C motif was described in 2001 and later optimized, as shown in Fig. 4a.⁴⁹ Commercially available barbituric acid (5) is first converted to 2,4,6-trichloropyrimidine-5-carbaldehyde (4) in the presence of phosphoryl chloride in DMF. Regioselective S_NAr at C4 with allylamine (2 equiv., −78 °C), followed by a second regioselective S_NAr reaction at C2 with MeNH₂ (2 equiv., 0 °C – rt) provides the disubstituted pyrimidine 7. While these conditions work well for alkylamines, in 2008 it was demonstrated that the direct introduction of aniline nucleophiles into C2 (and also C4) of 4 is best accomplished using a phase transfer catalysis strategy (Fig. 4b).¹⁰⁰ Specifically, under biphasic conditions (organic solvent/H₂O) and in the presence of a weak base (KHCO₃) and tetrabutylammonium iodide (TBAI), 14a–c can be prepared without the formation of troublesome side reactions (e.g. disubstitution and imine formation) that often occur when using aniline nucleophiles for these types of substitutions.¹⁰¹ The second S_NAr reaction of 14a–c with MeNH₂, also under phase transfer conditions, generates the disubstituted products 15a–c in good yield.

Fig. 4 (a) Synthesis of the allyl-functionalized G∧C motif. (b) Phase transfer catalysis strategy for the S_NAr reaction of aniline nucleophiles with pyrimidine 4.

Following the formation of 7 (Fig. 4a), a third S_NAr reaction at position 6 using benzyl alkoxide under refluxing conditions provides the trisubstituted product 8, which is then selectively Boc-protected on the methylamine group to generate 9. Conversion of the aldehyde to the nitrile is next accomplished using hydroxylamine hydrochloride under basic conditions, followed by addition of trifluoroacetic anhydride (TFAA) and triethylamine (TEA). A ring closure reaction of 11 using trichloroacetyl isocyanate and ammonia, then global deprotection under acidic conditions, furnishes G∧C motif 13 (29% overall yield) functionalized with an allyl group on the C-ring and a methyl group on the exocyclic nitrogen atom of the G-ring.

3. Functionalization strategies of the RNTs

3.1. Functionalizing the protected G∧C motif

As mentioned previously, a key feature of the RNTs is their highly tunable surface properties, achieved by attaching functional groups to the G∧C motif that have distinct physical and biochemical characteristics. The significance of the surface tunability was first demonstrated in 2001.⁴⁹ Circular dichroism spectroscopy revealed that RNTs expressing covalently attached L-lysine had the opposite supramolecular chirality to those expressing D-lysine as shown in Fig. 5. In 2002, amino acids that are electrostatically bound to crown ether anchor points on the RNT surface were also shown to dictate the chiroptical properties of these nanomaterials.¹⁰² This further led to the observation of solvent-induced chirality inversion of RNTs generated from a single chiral G∧C motif,¹⁰³ as well as structure-dependent chirality inversion and pH-controlled chiroptical switching.¹⁰⁴

Fig. 5 Comparison of the CD spectra of solutions containing D-lysine (open diamonds) or L-lysine functionalized G∧C motifs. Reprinted with permission from H. Fenniri, P. Mathivanan, K. L. Vidale, D. M. Sherman, K. Hallenga, K. V. Wood and J. G. Stowell, J. Am. Chem. Soc., 2001, 123, 3854–3855. Copyright (2001) American Chemical Society.

The expression of different surface functional groups is a critical aspect to the potential applications of these nanomaterials in nanomedicine,^57–85 catalysis^87,88 and sustainable energy generation.⁸⁶ It has been shown for example, that RNTs mimic the natural nanoscale structure of the extracellular matrices in bone and cartilage tissues.^67–81 This innate feature along with their surface functionalization with bioactive amino acids and peptides (e.g. bone morphogenetic proteins, RGD and KRSR cell adhesive peptides), enables the RNTs to create a favorable surface environment on implant materials (e.g. Ti) for enhanced cellular adhesion and function. These smart nanomaterials may thus resolve the problems associated with low biocompatibility, poor long-term stability, slow rate of osseointegration and low healing response, that result from the poor structural and functional connections between the bone and the implant surfaces.¹⁰⁵

Given the significance of functionalizing the G∧C motif, considerable effort has focused on developing functionalization methods that proceed both early and late in the synthesis of this heterocycle. As depicted in Fig. 6 (and previously in Fig. 4), installation of NH₂R¹ and NH₂R² via consecutive S_NAr reactions at position 4 and 2 on 2,4,6-trichloropyrimidine-5-carbaldehyde (4), installs the R² and R¹ groups early in the synthetic scheme. This strategy is most amenable for acid and base stable groups such as the alkyl moieties 19a–f (Fig. 6a).¹⁰⁶ In the case of aromatic groups installed via the same manner (Fig. 6b), these can be further elaborated using a Suzuki cross coupling reaction prior to deprotection, to generate electronically and structurally unique aromatic motifs such as 22.¹⁰⁰ Generally, the functionalization of the G∧C motif with such groups renders the corresponding RNTs soluble in non-aqueous solvents for potential applications as discotic liquid crystals, channels, nanowires and LB films.

Fig. 6 (a) Installation of R¹ and R² groups early in the synthetic scheme through consecutive S_NAr reactions at C4 and C2 of 4. (b) Example of using the Suzuki cross-coupling reaction to further elaborate the G∧C motif prior to deprotection.

An alternative functionalization strategy that is often utilized, occurs late in the synthetic scheme of the G∧C motif and involves a reductive amination reaction. Namely, compound 12 is Boc-protected and the allyl group of 23 is transformed to the corresponding aldehyde 24 via an oxidative cleavage reaction under the conditions shown in Fig. 7a. This aldehyde can be prepared on large scale (100 g) and stored for long periods (>1 year) at 4 °C without degradation. Following a reductive amination of 24 with NH₂R and deprotection under acidic conditions, the functionalized G∧C motif 25 is obtained.

Fig. 7 (a) Reductive amination strategy for the functionalization of the G∧C motif. Comparison of the (b) G∧C motif (left), rosette (middle) and RNT (right) with the (c) twin G∧C-motif (left), twin rosette (middle) and twin RNT (right). Adapted with permission from J. G. Moralez, J. Raez, T. Yamazaki, R. K. Motkuri, A. Kovalenko and H. Fenniri, J. Am. Chem. Soc., 2005, 127, 8307–8309. Copyright (2005) American Chemical Society.

A twin version of this motif has also been developed as shown in Fig. 7c, that is constructed using a double reductive amination reaction.⁵¹ This twin motif was specifically designed to accommodate molecules such as peptides that could negatively impact the RNT stability as a result of their charge density and steric bulk. In general, RNTs formed from the twin G∧C motif have an association free energy approximately twice that of a single motif when identically functionalized and of equal length. This is due to (a) the reduction of functional group density and net charge, (b) the thermostability gained as a result of pre-organization, increased amphiphilic character and greater number of hydrogen bonds per module (12 instead of 6), (c) rosettes that are maintained by 36 hydrogen bonds instead of 18 and (d) the decreased surface steric congestion and electrostatic repulsion.⁵¹

Using this reductive amination approach, G∧C and/or twin G∧C motifs expressing many different groups have been prepared for both water-soluble and organic-soluble RNTs, including those which express a variety of alkyl and aromatic groups¹⁰⁶ (including porphyrin⁸⁶) and amino acids.¹⁰⁷ The expression of different types of peptides including the tubular renal imaging mercaptoacetyl triglycine (MAG₃) ligand,^108,109 poly-lysine,¹¹⁰ and bone morphogenetic peptides¹¹¹ have also been demonstrated. To further modulate the RNTs' properties, two or three of these G∧C motifs with different functional groups can be co-assembled together to obtain multi-functionalized RNTs.⁶²

In addition to these functionalization approaches, a novel homoallyl protecting group shown in Fig. 8 (compound 26) has also been developed, that once selectively removed in the presence of the Boc and Bn groups, enables direct nitrogen alkylation with an electrophile.¹¹² The initial deprotection of 26 involves an oxidative cleavage of the homoallyl group to the corresponding aldehyde 27. Following addition of a primary amine (RNH₂) in 1,2-dichloroethane, imine 28 is generated. After stirring at rt for 24 h, the free NH group of 31, formed via the loss of imine 30 and water, is then available for alkylation as demonstrated by the addition of carbohydrate 32 to produce 33.

Fig. 8 Homoallyl protecting group strategy for the G∧C motif.

3.2. Modifying the functional groups of the G∧C motif post-deprotection

While Section 3.1 described the various strategies developed over the years to functionalize a protected version of the G∧C motif, it has also been shown that functional groups attached to the deprotected heterocycle in its self-assembled state can be further modified. In 2012 for example, an aqueous basic (Na₂CO₃) solution of self-assembled 34a (or 34b) with amine groups on the RNT surface were successfully polymerized using adipoyl chloride in CH₂Cl₂ to create a more thermostable polymeric material (Fig. 9).¹¹³ Along with confirming that the polymerization process had indeed occurred using ¹H NMR, FTIR, DLS and DSC studies, SEM and TEM images of the washed film of 35a revealed a parallel tape-like formation in which the RNTs were aligned along their main axis. This suggested that polymerization using this nylon-6,6 process likely occured along the length of the RNTs as well as between adjacent nanotubes.

Fig. 9 (a) Synthesis of RNT polymers 35a and 35b. (b–d) TEM images of 35a (1 mg mL⁻¹ in MeCN). The parallel tape-like formation of the RNTs is visible in image (e). Scale bars in nm. Adapted with permission from B.-L. Deng, R. L. Beingessner, R. S. Johnson, N. K. Girdhar, C. Danumah, T. Yamazaki and H. Fenniri, Macromolecules, 2012, 45, 7157–7162. Copyright (2012) American Chemical Society.

In 2010, the one-pot nucleation, growth, morphogenesis and passivation of 1.4 nm Au nanoparticles (NPs) on the surface of lysine-functionalized twin RNTs generated from 36 (Fig. 10) was also described.⁸⁷ The lysine side-chains were used to coordinate negatively charged tetrachloroaurate. Upon reduction with hydrazine, evenly distributed nucleation sites on the surface of the RNTs led to the formation of nearly monodisperse Au NPs with a size of 1.4–1.5 nm. These results were further extended in 2011, with the electroless synthesis of 1.4 nm Pd as well as Pt NPs on the RNT surface.⁸⁸ Such RNT/metal-based NP hybrid materials offer exciting opportunities for applications in catalysis, nanoelectronics and nanophotonics.

Fig. 10 (a) Twin G∧C motif (b) TEM image showing the monodisperse Au NP/RNT composite on a carbon-coated grid. Scale bar 20 nm. (c) Self-assembled RNT generated from (a) with nucleated Au NPs (gold spheres). Adapted with permission from R. Chhabra, J. G. Moralez, J. Raez, T. Yamazaki, J.-Y. Cho, A. J. Myles, A. Kovalenko and H. Fenniri, J. Am. Chem. Soc., 2010, 132, 32–33. Copyright (2010) American Chemical Society.

4. Extending the G∧C core

4.1. Synthesis of xG∧C and xK1

Along with developing the aforementioned chemistry to functionalize the surface of the RNTs, efforts have also focused on synthesizing extended versions of the bicyclic motif shown in Fig. 11 (called K1); namely the tricycle in both the water (called xK1)⁵⁵ and organic (called xG∧C)¹¹⁴ soluble forms, as well as the tetracycle (called xxG∧C).¹¹⁵

Fig. 11 Bicyclic (left), tricyclic (middle) and tetracyclic (right) G∧C motifs and their corresponding self-assembly into RNTs with inner channel diameters of 1.1 nm, 1.4 nm and 1.7 nm, respectively.

These extended heterocycles feature the same G and C hydrogen bonding arrays as the bicycle, but are separated by one or two pyridine rings. They were specifically designed to self-assemble into RNTs that have increased inner channel diameters of ca. 1.4 nm and ca. 1.7 nm, respectively. By also having larger outer diameters, these RNTs would enable more sterically demanding moieties to be expressed on the outer surface relative to the bicyclic counterpart. Moreover, functionalization at position A on the pyridine ring(s) provides opportunities to display the respective R groups within the inner RNT channel.

From a synthetic standpoint, the tricyclic motifs xK1 and xG∧C as well as the tetracyclic motif xxG∧C, may be viewed as derivatives of the pyrido[2,3-d]pyrimidine scaffold (Fig. 12). In the case of xxG∧C, the core structure is a juxtaposition of two pyrido[2,3-d]pyrimidine molecules. Similar to pyrimido[4,5-d]pyrimidines, numerous strategies have been developed to synthesize these molecules that mainly involve cyclization of the functionalized pyridine scaffold to generate the pyrimidine ring (strategy 1, Fig. 12)^116–129 or cyclization of the functionalized pyrimidine scaffold to generate the pyridine ring (strategy 2, Fig. 12).^130–146

Fig. 12 General synthetic strategies to prepare pyrido[2,3-d]pyrimidines.

On the basis of the known strategies for synthesizing pyrido[2,3-d]pyrimidines and the previous work on the G∧C pyrimido[4,5-d]pyrimidine scaffold, the tricyclic constructs xK1 and xG∧C were prepared using three key regioselective S_NAr reactions at C2, C4, and C7 on the trichloro substituted pyrido[2,3-d]pyrimidine 37 (Fig. 13a).^55,114 Installation of the third ring following a similar route described for the bicyclic G∧C motif (Fig. 4) provided access to the desired heterocycles. While a protected alcohol at C4 of 38 was necessary for installing the carbonyl group on the G-face of both xK1 and xG∧C, the appendage of different C2 and C7 functionalized amines, conferred distinct chemical and physical properties on the resulting self-assembled xK1 and xG∧C RNTs, including their solubilities.

Fig. 13 (a) General synthetic strategy for the formation of the tricyclic modules xG∧C and xK1. (b) Three-step synthesis of intermediate 42.

The synthesis of the trichloro substituted pyrido[2,3-d]pyrimidine 37 was initiated using the type 2 strategy described in Fig. 12 (i.e. cyclization of the pyrimidine ring). Beginning from 2,4,6-trichloropyrimidine-5-carbaldehyde (4), a condensation reaction was performed with malononitrile in the presence of β-alanine to provide 41 in 50% yield (Fig. 13b).^55,114 Under thermal conditions (180 °C), cyclization of 41 generated the desired product 37 in 75% yield. Although 37 has three electrophilic sites, C4 is the most reactive towards nucleophiles and a regioselective S_NAr reaction using benzyl alcohol in the presence of Et₃N at −40 °C, offered 42 in 70% yield.

For the synthesis of xG∧C, intermediate 42 was first treated with excess allylamine in CH₂Cl₂, followed by heating to 50 °C in a sealed tube to provide 43 in 89% yield (Fig. 14a). The allylamine at C2 of 43 was then selectively Boc-protected and the resulting compound 44 was treated with trichloroacetyl isocyanate. Total consumption of 44 was observed by TLC within 5 h, after which 7 M NH₃ in MeOH was added to yield 45 in 90% over the two steps. Global deprotection under acidic conditions provided xG∧C as the hydrochloride salt. Subsequent self-assembly studies revealed that xG∧C was only sparingly soluble in DMF (0.6 mM with heat) and moderately soluble in DMSO (1.2 mM without heat; 15 mM with heat). While SEM confirmed the presence of high aspect ratio RNTs in DMF, TEM and AFM revealed that their outer diameter was 3.6 ± 0.2 nm and 3.6 ± 0.3 nm respectively, in agreement with the modeling calculations of 3.5 nm (Fig. 14b–e).¹¹⁴

Fig. 14 (a) Synthesis of xG∧C. Tapping mode atomic force microscopy (b) height and (c) amplitude images of xG∧C (0.6 mM in DMF) on mica and transmission electron micrographs of xG∧C in a (d) 0.3 mM in DMF and (e) 1.1 mM solution in DMF. Reproduced with permission from ref. 114 from the Royal Society of Chemistry.

Unlike xG∧C which features two allyl groups, the water-soluble version xK1 was prepared as an extended analogue of K1 (Fig. 11). As a result, the synthesis of this heterocycle required the installation of a methylamine group at C2 and an allylamine group at C7 on pyrido[2,3-d]pyrimidine 42 (Fig. 15), that could be used as a handle later on for installing the L-lysine functional group.⁵⁵ Although previous work by Broom and coworkers¹⁴⁷ suggested that C7 of 42 may be more reactive than C2 toward S_NAr, this was in fact not the case. Treatment of 42 with methylamine provided the desired C2-substituted product 46, thereby revealing the importance of the electron withdrawing ability of the adjoining deactivated pyridyl ring and the adjacent pyrimidine ring nitrogens on the substitution pattern.

Fig. 15 (a) Synthesis of xK1.

In order to promote the second S_NAr reaction of 46 with allylamine, it was first necessary to electronically deactivate the ring by protecting the methylamine group with an electron withdrawing Boc substituent. Substitution at C7 with allylamine in the presence of DIPEA then proceeded, albeit in 50% yield. Closure of 47 using trichloroacetyl isocyanate/NH₃ followed by protection of the amine, provided tricycle 49. Oxidative cleavage of alkene 49 under standard conditions, followed by reductive amination of aldehyde 50 with protected L-lysine, furnished compound 51. Finally, deprotection with TFA/thioanisole provided the water-soluble tricyclic motif, xK1 as the TFA salt. Overall, xK1 was prepared from intermediate 42 in 13% yield.⁵⁵

Self-assembly studies revealed that xK1 rapidly organized into RNTs at rt (pH_final = 2.8) in water. This is an advantage over the bicyclic motif K1, which requires heat to induce the entropically driven self-assembly process as a result of having less hydrophobic character. UV-visible, CD and fluorescence spectroscopy experiments further revealed the unique J-aggregate properties of xK1 RNTs. Most notably, the very large recorded molar ellipticity value of 4 × 10⁶ deg M⁻¹ m⁻¹ for a chiral helical stack, indicated strong dipole transitions between adjacent molecules within the RNT. The increased hydrophobic/amphiphilic character of this tricyclic core compared to the bicyclic core K1, presumably results in stronger assemblies in water and promotes stronger and larger π–π interactions that are favorable for establishing optimal interchromophoric distances and geometries for exciton coupling and increased electronic delocalization.⁵⁵

4.2. Synthesis of xxG∧C

Following the synthesis of the tricyclic modules, the construction of the extended tetracycle xxG∧C (Fig. 16) was next explored due to its potential to self-assemble into RNTs with an even larger internal diameter of ca. 1.7 nm.¹¹⁵ As mentioned previously, xxG∧C is a juxtaposition of two pyrido[2,3-d]pyrimidine cores. Regardless of whether the synthesis was approached from the G or the C side, the synthetic strategy required the multifunctional pyrido[2,3-d]pyrimidine intermediate 53 (Fig. 16) to build the third ring using a Friedländer-type condensation. Mixed urea synthesis and cyclization of 54 would then install the final cytosine ring.

Fig. 16 Synthetic strategy of xxG∧C.

As illustrated in Fig. 17, the synthesis was thus initiated by treating 42 with allylamine to provide the C2-substituted product 55 in 64% yield.¹¹⁵ Previous experience with the synthesis of the tricyclic module xK1 (Fig. 15),^55,114 indicated that the electron density of the ring in 55 needed to be reduced in order to facilitate the subsequent S_NAr reaction at C7 with ammonia. Given that an aldehyde at C6 was also required for the eventual Friedländer condensation reaction, the strategy was to first reduce nitrile 55 to the aldehyde (56) and then protect the allylamine with a Boc group to decrease the electron density.

Fig. 17 Synthesis of the trisubstituted pyrido[2,3-d]pyrimidine 60.

Although the initial DIBAL-H reduction of 55 did proceed, only 50% conversion to the desired product 56 was achieved, with the balance being the starting material 55 (Fig. 17). To facilitate the isolation of 56, the mixture was treated with NaBH₄ in MeOH and CHCl₃. While the resulting products 57 and 58 were readily separated by silica gel flash chromatography, the pyridyl ring of 57 was found to be reduced to the dihydropyridine. This could be reoxidized using PDC however, to provide 55 in 72% yield. Reoxidation of 58 using PCC alternatively, provided 56 in 74% yield. The allylamine group of 56 was then protected with Boc₂O to facilitate the S_NAr reaction with ammonia (10 equiv., 2 M in i-PrOH) in THF, but with a maximum yield of ca. 30%.¹¹⁵

During the course of the reaction optimization of the previous Boc protection of 56, it was observed that the yield of 59 decreased as the number of equivalents of DMAP increased. Investigation of the reaction kinetics and product distribution by ¹H NMR in CD₃CN suggested that the addition of 1 equiv. of DMAP at rt resulted in quantitative conversion of 59 to adduct 61 (Fig. 18). This was further confirmed by mass spectrometry. It was therefore postulated that DMAP and other tertiary amines could act as catalysts for the third S_NAr, through the generation of a stable, yet more reactive intermediate species 61. After an extensive examination of various combinations and amounts of tertiary amines (DMAP, 4-methoxypyridine, 4-pyrrolidinopyridine, and DABCO), ammonia solutions as well as reaction conditions, the most favorable results were obtained when 59 was treated with a 0.5 M solution of NH₃ in THF (10 equiv.), DABCO (1.1 equiv.) and DIPEA (1.0 equiv.). After 13 h at rt, 60 was provided, but only in 52% yield, along with the hydrolysis product 62 in 40% yield. It is expected however, that this reaction can be further improved under anhydrous conditions.¹¹⁵

Fig. 18 Optimization of the synthesis of the trisubstituted pyrido[2,3-d]pyrimidine 60.

The subsequent Friedländer-type condensation to form the third ring was next accomplished by treating 60 with malononitrile in pyridine to provide 63 in 88% yield (Fig. 19). The formation of the final pyrimidine ring was then attempted by treating 63 with trichloroacetyl isocyanate at 0 °C in CH₂Cl₂ for 2 h, followed by the addition of ammonia (2 M in i-PrOH). FTIR-analysis however, revealed the cyclization had not occurred due to the presence of the nitrile IR vibration at 2200 cm⁻¹. Further attempts to induce the ring closure using other bases such as 7 M NH₃ in MeOH, NaH, or t-BuONa in THF under various conditions were also unsuccessful. However, the ring closure reaction could be promoted by converting 63 to 65 in the presence of benzoyl isocyanate in CH₂Cl₂ at 0 °C. Subsequent treatment of 65 with DIPEA in CH₂Cl₂ for 4 days offered the cyclized adduct 66, which was then deprotected with 4 M HCl in dioxane at 70 °C to provide the desired tetracyclic product xxG∧C. Future efforts will focus on exploring the self-assembly of this motif as well as preparing functionalized derivatives.

Fig. 19 Completion of the synthesis of xxG∧C.

5. Conclusion

As described herein, the G∧C hybrid motif is a pyrimido[4,5-d]pyrimidine scaffold featuring the Watson–Crick ADD hydrogen bonding array of guanine and the DAA hydrogen bonding array of cytosine. In solution, this heterocycle self-assembles into hexameric rosettes, which then further π-stack to form RNTs. As a result of the functionalization strategies developed over the past 15 years, these nanomaterials have displayed an array of structural units on their outer surface including amino acids and peptides, alkyl and aromatic groups, sugars, and even metal NPs. In addition to modifying the physical, optical, electronic and chemical properties of the RNTs in this manner, their inner channel diameter has also been tuned by extending the core of the pyrimido[4,5-d]pyrimidine scaffold. This has not only led to J-aggregate properties of the RNTs, but provides future opportunities for inner channel functionalization and encapsulation of host molecules.

While the synthesis and self-assembly of the G∧C motif bearing the pyrimido[4,5-d]pyrimidine core has been the central focus thus far, analogue structures such as the pyrido[4,3-d]pyrimidine scaffold 67 shown in Fig. 20a have also been explored. This motif displays the same ADD–DAA hydrogen bonding pattern as 68 necessary for hexameric rosette formation, but has a carbon atom in lieu of a nitrogen atom at C8.¹⁴⁸ In cyclohexane, 67 (R = C₁₆H₃₃) self-assembles into RNTs having an outer diameter of 4.8 ± 0.3 nm (by TEM), in agreement with the modeling calculations. When functionalized with an n-heptyl (R = C₇H₃₃) chain, the motif crystallizes in DMSO, revealing the characteristic hexameric organization of the motifs in the solid state. RNTs that exhibit fluorescent properties in DMF have also been generated from this same pyrido[4,3-d]pyrimidine scaffold, but which is functionalized at C8.¹⁴⁹ This analogue is beneficial from a synthetic standpoint due to ease of accessibility (6-steps) and ability for late-stage functionalization at the N1 position (Fig. 20a). These studies therefore provide insight into methods in which the large-scale production of the nanotubes could be further streamlined, while maintaining the essential capability of tuning their chemical and physical properties for specific applications. Future studies of these versatile supramolecular assemblies will continue along these lines.

Fig. 20 (a) Structural comparison of the pyrido[4,3-d]pyrimidine 67 and pyrimido[4,5-d]pyrimidine 68. (b) Six-step synthesis of the self-assembling pyrido[4,3-d]pyrimidine molecules 67 (R = C₇H₁₅ or R = C₁₆H₃₃). (d) X-ray crystal structure of 67 (R = C₇H₁₅) showing (left) the hydrogen bonding array, and (right) portion of the stacking motif of the hexamers. Adapted with permission from A. Durmus, G. Gunbas, S. C. Farmer, M. M. Olmstead, M. Mascal, B. Legese, J.-Y. Cho, R. L. Beingessner, T. Yamazaki and H. Fenniri, J. Org. Chem., 2013, 78, 11421–11426. Copyright (2013) American Chemical Society.

Acknowledgements

We acknowledge the support of the National Science Foundation, the National Research Council of Canada, the Natural Science and Engineering Research Council of Canada, the University of Alberta and Northeastern University.

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