Synthesis of Functional Building Blocks for Type III-B Rotaxane Dendrimer

Second-generation type III-B rotaxane dendrons, equipped with succinimide and acetylene functional groups, were synthesized successfully and characterized by NMR spectroscopy and mass spectrometry. A cell viability study of a dendron with a normal cell line of L929 fibroblast cells revealed no obvious cytotoxicity at a range of 5 to 100 μM. The nontoxic properties of the sophisticated rotaxane dendron building blocks provided a choice of bio-compatible macromolecular machines that could be potentially developed into polymeric materials.


Introduction
Rotaxanes are a unique class of molecules that consist of one or multiple macrocycles that encircle one or more dumbbell-shaped threads. Such an entanglement between the macrocycles and threads in space through a non-covalent interaction is known as a mechanical bond [1], where these components cannot be separated until the chemical bond breaks or distorts within the atom of the components. Rotaxanes have been widely applied in molecular switching [2,3], molecular pumps [4][5][6], molecular motors [7], molecular machines [8] and organocatalysis [9,10]. Dendrimers are another class of highly ordered, hyperbranched macromolecules, composed of a repeating dendron unit with an exact molecular weight [11]. Typically, the molecular weight of dendrimers is higher than that of most drug molecules but smaller than that of linear polymers, in which such a molecular weight range can provide ample space for drug encapsulations in dendrimers' near-spherical, three-dimensional morphology [12][13][14]. The fusion of rotaxane and dendrimers gives another, new class of mechanically interlocked macromolecules known as "Rotaxane Dendrimers". The concept of rotaxane dendrimers was first proposed by Lee and Kim [15] in 2003, and was further elaborated on by Stoddart [1], Leung [16,17] and Yang [18]. Basically, there are three types of rotaxane dendrimers (I, II and III), while each type can be further subdivided into small categories (A, B and C). Type I and type II are the most abundant reported examples because of their structural simplicity and friendly preparation [19][20][21][22][23]. In the case of type III, it is defined as dendritic polyrotaxane, which means that the rotaxane mechanically bonds in a branch-like manner, similar to a dendrimer. Type III-A rotaxane dendrimers are defined as the mechanical bonds branching via the thread [24,25]. On the other hand, type III-B rotaxane dendrimers are defined as the mechanical bonds branching through the macrocycles [26]. Our group has reported a series of pure organic, metal-free higher-generation type III-B [26][27][28] and type III-C [29] rotaxane dendrimers, and demonstrated that such sophisticated macromolecules can act as drug carriers by encapsulating an anti-cancer drug (chlorambucil) [27] as well as actively releasing the drug from the carrier upon the acid-base switching mechanism (macromolecular machines). We also showed that such rotaxane dendrimers were non-inflammatory as well as non-toxic in mice bodies, and could be characterized by the mass spectrometric imaging technique [30].
The current synthetic method of producing higher-generation type III-B rotaxane dendrimers is based on the versatile copper-catalyzed azide-alkyne cycloaddition (CuAAC) [26][27][28] by using the building blocks of a relatively unstable pseudorotaxane instead of using a stable, pre-formed rotaxane. Pseudorotaxanes require relatively strong supramolecular interactions to maintain their interlocking structure between the thread and the macrocycle, subject to dissociation in harsh conditions. In order to expand the scope of synthetic methods for producing type III rotaxane dendrimers, we herein revisited the preparation of type III-B rotaxane second-generation (G2) dendrons with a N-hydroxysuccinimide (NHS) or an acetylene functional group with a molecular weight of approximately 5000 g/mol. Such functional dendrons can be the dendrimer building blocks, anchoring to other core materials such as nanoparticles [31,32], functional surfaces [33] or biomolecules through biorthogonal click chemistry or nucleophilic reactions [34]. It is necessary to assess the normal cell viability of such building blocks of rotaxane dendrimers before employing them as potential drug carriers [27,29] for cancer treatments [35][36][37][38][39], mainly based on enhancing the drug's stability, lesion site targeting and prolonged drug release.

Materials and Methods
General. All chemicals, reagents and solvents were purchased without further purification from Sigma-Aldrich, St. Louis, MO, USA, unless otherwise stated. Deuterated solvents were purchased from Cambridge Isotope Laboratories, Tewksbury, MA, USA.
Followed by the introduction of acetylene with propargylamine (Figure 2), the G2 rotaxane dendron-acetylene 5-3H·3PF 6 was successfully synthesized with a 50% yield from the G2 rotaxane dendron-OSu 4-3H·3PF 6 . This reaction provided a clean and efficient functional group interconversion for the high-molecular-weight dendrons. The molecular structures of the two functional G2 rotaxane dendrons 4-3H·3PF 6 and 5-3H·3PF 6 were confirmed by 1 H NMR spectroscopy. The observation ( Figure 3A) of the proton signals (2.84 ppm) of the succinimide (H a ) in the core of the G2 rotaxane dendron-OSu 4-3H·3PF 6 revealed the successful introduction of the NHS group of the G2 dendron ( Figures S3 and S4), compared to the spectra (Figures S1 and S2) of 1-H·PF 6 . On the other hand ( Figure 3B), the disappearance of the succinimide proton (H a ) and the appearance of the acetylene proton (H b ) (2.19 ppm) and the propargyl proton (H c ) (4.13 ppm) in the core of the G2 rotaxane dendron-acetylene 5-3H·3PF 6 (Figures S5 and S6) indicated the successful introduction of a propylene group to the G2 rotaxane dendron by a traditional acyl substitution reaction.  For characterization of the G2 rotaxane dendron-OSu 4-3H·3PF 6 , the electrospray ionization mass spectrum (ESI-MS) revealed ( Figure 4) the most abundant molecular ion signal (m/z 1504.1218) as [M-3PF 6 +4H 2 O] 3+ , losing its three counter-anion PF 6 -, as the detectable cation species. The expanded molecular ion signal ( Figure S7) revealed that the peak spacings were approximately 0.3333, which demonstrated a +3 charged ion. Furthermore, the patterns of the peaks were basically the same between the experimental result and the theoretical analysis. Another molecular ion signal (m/z 1515.1113) can be attributed as [M-3PF 6 +6H 2 O] 3+ . Both ions revealed a dendron with a H 2 O adduct in the gas phase. For characterization of the G2 rotaxane dendron-acetylene 5-3H·3PF 6 , the ESI-MS revealed ( Figure 5) the most abundant molecular ion signal (m/z 1483.7431) as [M-3PF 6 ] 3+ , losing its three counter-anion PF 6 -, as the detectable cation species. Another molecular ion signal (m/z 1495.4098) can be attributed as the molecular ion [M-3PF 6 +2H 2 O] 3+ . Overall, water adducts to two dendrons in the gas phase were observed through mass spectrometric analyses. On the other hand, an in vitro study of the new dendron was performed with L929 mouse fibroblast cells to shed light on cytotoxicity towards cells for extracellular matrix and collagen biosynthesis. To our delight, the tested dendron had no obvious cytotoxicity in L929 cells, even at the highest concentration of 100 µM ( Figure 6).

Conclusions
In conclusion, novel type III-B second-generation rotaxane dendrons equipped with succinimide and acetylene functional groups were successfully synthesized and characterized. The new dendron compound did not reveal cytotoxicity with L929 fibroblast cells, even at a high concentration of 100 µM of the normal cells. The nontoxic nature as well as their sophisticated chemical structures would be essential criteria for developing next-generation macromolecular machines for potential biomedical use.

Institutional Review Board Statement:
The study was conducted according to the guidelines approved by The Hong Kong Baptist University and The Chinese University of Hong Kong.

Data Availability Statement:
The data presented in this study are available on request from the corresponding authors.