Analytical Sciences Advance Publication by J-STAGE Received November 30, 2020; Accepted January 15, 2021; Published online on January 22, 2021 DOI: 10.2116/analsci.20SCN06

NOTE Special issues for Analytical Biomaterials

Preparation of Spherical Containing Self-Immolative Poly(carbamate) Core

Shione FUKUMOTO,* Mami KAWADE,* Kazunori KIMURA,* Yoshitsugu AKIYAMA,** † and Akihiko KIKUCHI*

* Department of and Technology, Tokyo University of Science, 6-3-1

Niijuku, Katsushika, Tokyo 125-8585, Japan

** Faculty of Industrial Science and Technology, Tokyo University of Science, 6-3-1

Niijuku, Katsushika, Tokyo 125-8585, Japan

† To whom correspondence should be addressed.

E-mail: [email protected]

1 Abstract

We prepared a novel spherical nucleic acid, containing a core structure of self-immolative poly(carbamate) (PC), with aminobenzyl alcohol as a repeating unit, by conjugating an end-activated PC derivative with an amine-terminated oligoDNA on a solid support for PC-oligoDNA. Dynamic light-scattering measurements revealed a hydrodynamic diameter of 107 nm with a narrow size distribution. A fluorescent monomer with aminobenzyl alcohol is available for PC-oligoDNA synthesis for enhancement of fluorescence emission by domino-like disassembly of PC in response to various external stimuli.

Keywords: spherical nucleic acids, self-immolative polymer, poly(carbamate), DNA conjugation

2 Introduction

Spherical nucleic acids (SNAs) consisting of a nanostructure core densely modified with a brush structure of oligomeric nucleic acids (oligoDNAs) have been widely investigated for decades for various bioanalytical applications, such as sensing1, imaging2,3, and diagnostics4,5. Of interest, the negatively charged oligoDNAs on these nanostructures leads to improved colloidal stability of the nanoparticles in physiological solutions and enhances resistance to nuclease degradation, as well as enhances the ability to bind to complementary oligoDNAs.6 Thus, SNAs may have several bioanalytical applications that differ from biosensing approaches in which homogeneous DNA probes are used.

For example, a dense single-stranded (ss) DNA shell surrounding a gold core (ssDNA-GNP) has been previously used as a sensing platform in the form of NanoFlares that .7-9 Fluorescein (FAM)-labeled-ssDNA (FAM-ssDNA; 10 nucleotide [nt]) hybridizes to a portion of the complementary sequences in ssDNA-GNP

(23 nt). Since the hybridized gold nanoparticles and FAM in ssDNA-GNP are close to each other, the emission of FAM is quenched effectively. Furthermore, when an available mRNA target binds to the mRNA recognition site in ssDNA-GNP, the

FAM-ssDNA is replaced by mRNA with a full-match 18- sequence to ssDNA-GNP, thereby releasing FAM-ssDNA by DNA strand replacement and increasing the intensity of fluorescence emission. In addition, a hydrophobic camptothecin-conjugated ssDNA (CPT-ssDNA) self-assembles in aqueous solutions to form a CPT-cored SNA.10 This micellar nanostructure can respond to external stimuli because it has three CPT molecules connected to a phenol-based, self-immolative linkage with the 2-nitrobenzyl group. The 2-nitrobenzyl group is sensitive to UV light

(365 nm), which induces core degradation and release of CPT and ssDNA. Thus, owing

3 to these advantages, nanoparticles with a dense layer of DNA are promising materials for bioanalytical applications.

In a recent study, Mirkin and co-workers11 reported the use of ssDNA conjugated with biodegradable poly(lactic-co-glycolic acid) as a core material in the liquid-phase reaction for degradable SNA strategies. However, the selection of degradation stimuli and control of degradation kinetics are limited due to random ester hydrolysis on the polymer backbone. Here, we present a design facilitating the efficient conjugation of ssDNA with a self-immolative poly(carbamate) (PC) derivative on the solid support for constructing a new class of SNA-based nanoparticles (Fig. 1). The novel nanoparticles have a PC derivative core (PC-oligoDNA) with a potent ability to control the release of ssDNA through a self-immolative chain fragmentation of PC, with

4-aminobenzyl alcohol as a repeating unit, in response to several external triggers.

Experimental

Reagents and chemicals

All reagents and solvents used in this study were reagent grade and were used without further purification. 4-Aminobenzyl alcohol, phenyl chloroformate, and methylamine solution (33 wt%) in absolute ethanol were purchased from Sigma-Aldrich

(MO, USA). Hexane and methanol were purchased from Kanto Chemical Co., Inc.

(Tokyo, Japan). Dimethylsulfoxide-d6 (99.9 atom%D) and 4-phenyl-1-butanol were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Tetrahydrofuran

(super dehydrated, THF), sodium hydrogen carbonate (NaHCO3), ethyl acetate (EtOAc), ammonium chloride (NH4Cl), dibutyltin dilaurate (DBTL), dimethylsulfoxide (super dehydrated, DMSO), N,N’-carbonyldiimidazole (CDI), N,N-dimethylformamide (super dehydrated, DMF), 40% methylamine solution, and dialysis membrane (molecular

4 weight cut-off: 1,000) were purchased from FUJIFILM Wako Pure Chemical Corp.

(Osaka, Japan). OligoDNA-modified Controlled Pore Glass (CPG) resin

(CPG-17nt-NH2, 5’H2N-ATGCCCATACTGTTGAG-CPG-3’) was purchased from

Tsukuba Oligo Service Co., Ltd. (Ibaraki, Japan). Thin layer chromatography (TLC) measurements were carried out using Merck 60 F254 silica gel plates (Merck Millipore,

MA, USA); compounds were visualized with a standard handheld short-wave UV lamp

(254 nm). 1H NMR spectra were obtained using 400 MHz Bruker Digital NMR

AVANCE NEO 400 system (Bruker Japan, Kanagawa, Japan), and chemical shifts were reported in parts per million (ppm ) relative to the solvent (DMSO-d6: 2.49 ppm).

Dynamic light scattering (DLS) measurements were carried out using a Malvern

Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK).

Synthesis of Poly(carbamate) derivatives possessing a carbonylimidazole moiety at the polymer end (PC-CI)

Hydroxy-terminated PC derivatives (PC-OH, Supporting information and Fig.

2-(a))12 (60 mg，15 mol) and CDI (25 mg，0.15 mmol) were dissolved in dry DMF (1.0 mL) under nitrogen atmosphere. The reaction mixture was stirred at 20-25°C for 3 h, then concentrated under diminished pressure. The residue was purified by reprecipitation from methanol, filtered, and dried under diminished pressure to give the

1 PC-CI as an yellow powder (18 mg, yield; 30%). H NMR (DMSO-d6);  (ppm) 9.80

(s), 9.67 (s, 1H), 8.27 (s, 1H), 7.18-7.49 (aminobenzyl chain), 5.37 (s, 2H), 5.07 (s),

4.10 (t, 2H), 2.63 (t, 2H), 1.65 (m, 2H), and 1.26 (m, 2H).

Preparation of PC-oligoDNA conjugates and in situ construction of DNA-covered nanostructure

5 PC-CI (4.3 mg, 1.0 mol) was dissolved in dry DMSO (50 L). To this solution, 0.1 mol of oligoDNA (17 bases) on a CPG resin (CPG-17nt-NH2) was added.

The reaction mixture was maintained at 20-25°C for 24 h to allow the coupling reaction of oligoDNA with PC to proceed. The supernatant containing excess PC-CI was removed by washing three times with dry DMSO (500 L). The CPG resin with

PC-oligoDNA was cleaved from the solid support and deprotection of oligoDNA was carried out by suspension in 1.0 mL solution of ethanolic methylamine/aqueous methylamine (1:1 in volume ratio) at 20-25°C for overnight, followed by filtration to remove the CPG resin. The filtrate was dialyzed against pure water using a dialysis membrane with a molecular weight cutoff of 1,000 kDa (Spectrum Laboratories) at

20-25°C for 24 h to offer DNA-covered nanostructures.

Results and Discussion

PC derivatives, known as self-immolative polymers, consist of 4-aminobenzyl alcohol derivatives as the repeating unit. They can release several monomers with different substituents in response to various external stimuli, including acid-base, redox, light, and enzymatic activity.13 To obtain a conjugation of PC derivatives with ssDNA

(PC-oligoDNA), PC derivatives were synthesized according to previously established methods12 (Fig. S1, Supporting Information). Briefly, the phenyl carbamate of aminobenzyl alcohol was polymerized by the addition of catalytic DBTL in anhydrous

DMSO (110°C, 15 min), followed by capping the polymer terminal through the addition of 4-phenyl-1-butanol, leading to a 68% yield of PC derivatives possessing a phenylbutyl moiety and a hydroxy group at both the terminals (PC-OH).

The end-group analysis and degree of polymerization (DP) of the obtained PC-OH were determined from 1H NMR spectrum as shown in Fig. 2(a). The signals of the main

6 chain, the end of the phenylbutyl moiety, and the terminated benzyl alcohol moiety were assignable to the obtained PC-OH. In addition, the DP of PC-OH is determined from the intensity ratio of the aminobenzyl backbone signals (7.18-7.49 ppm) with methylene proton peaks of benzyl alcohol (4.42 ppm) at the terminals was 26. These results are consistent with those of a previous study.12

The hydroxy-terminated PC-OH was then activated with N,N’-carbonyldiimidazole

(CDI) in DMF (20-25°C, 3 h) to form an end-functionalized PC possessing a carbonylimidazole (CI) moiety (PC-CI) with a 30% yield. Fig. 2 shows the 1H NMR spectra before and after reaction with CDI. As shown in Fig. 2(b), signals of the methylene (4.4 ppm) protons at the end of the benzyl alcohol moiety had completely shifted to 5.3 ppm, whereas new peaks appeared at 8.2 ppm owing to the CI moiety. The conversion of the hydroxy group to the CI moiety was confirmed to be 76%, based on the peak ratio of the polymer chain to the CI moiety in the 1H NMR spectrum. This conversion efficiency was optimized by the insertion of a condensation step under diminished pressure after the reaction to increase the activated efficiency (Table S1,

Supporting Information). These results clearly indicated the successful conversion of the hydroxy group to the CI moiety.

To obtain the PC-oligoDNA conjugate, PC-CI was treated with amine-terminated 17 nucleotide (nt) oligoDNA on a solid support in DMSO (20-25°C, 24 h). This was followed by deprotection and cleavage from the solid support by treatment with

CH3NH2 in aqueous ethanol (20-25°C, 12 h) to obtain PC-oligoDNA for use without isolation. Subsequently, in situ self-assembled nanoparticles were developed using dialysis against water overnight. Fig. 3 shows the size distribution of the nanoparticles in solution after dialysis. DLS measurements revealed that the hydrodynamic diameter and polydispersity index (PDI) were 107 nm and 0.09 with good reproducibility. The

7 size of the particles as well as other parameters such as aggregation number per particle would be significantly influenced by depending on the length of DNA and PC segment.

In addition, the size distribution in DLS measurement showed a similar hydrodynamic diameter (= 107 nm) and PDI (= 0.09) after 1day incubation at 20-25 °C (Fig. S2,

Supporting Information). Furthermore, the resulting nanostructures retained good colloidal stability without visible changes such as precipitation or cloudiness in aqueous solution at 4°C for at least 2 weeks. These results demonstrate the formation of a nanostructure covered with a dense DNA shell in an aqueous solution.

In summary, we successfully established SNA nanoparticles, containing PC derivatives with a repeating unit of 4-aminobenzyl alcohol as the core material, using a solid-based PC-oligoDNA conjugate and in situ self-assembly. DLS measurements revealed unimodal nanostructures with a hydrodynamic diameter of 107 nm. The resulting nanostructures could achieve the release of fluorescent functional molecules

(monomers) in the core and oligoDNA formed the shell by domino-like disassembly of

PC in response to various stimuli12,13 through incorporation of several triggers (instead of the phenylbutyl moiety) in PC-oligoDNA. Furthermore, the high-density layer of the

DNA phase in the nanostructures could be used as a nucleic acid sensor.14-16

Experiments regarding the physicochemical properties of light-triggered SNA resulting from the conjugation of PC derivatives possessing a light-responsive nitrobenzyl group at the terminal are underway and will be published elsewhere. These nanoparticles, therefore, have potential for application as a new analytical biomaterial.

Acknowledgements This work was supported by Japan Society for the Promotion of Science

KAKENHI (grant number: JP17K05938).

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10 Figure Captions

Fig. 1 Schematic diagram of the preparation of self-assembled nanoparticles comprised of a dense DNA shell surrounding a self-immolative PC derivative core.

1 Fig. 2 H-NMR spectra of (a) PC-OH, and (b) PC-CI in DMSO-d6 at 25°C.

Fig. 3 DLS size distribution of nanoparticles composed of PC-oligoDNA conjugates at

25°C.

11 Fig. 1

12 Fig. 2

13 Fig. 3

14 Graphical Index

15