Development of Protecting Groups for Prodrug-Type Oligonucleotide Medicines

Abstract

In recent years, nucleic acid-based drug therapeutics have gained considerable attention for their potential in the treatment of various diseases. However, their therapeutic value is greatly hindered by the challenge of delivering them into cells. One possible strategy to improve cellular uptake is the use of “prodrug-type oligonucleotide medicine” in which negatively charged phosphodiester moieties are masked by bio-labile protecting groups. In this review, we describe our recent studies related to bio-labile protecting groups for phosphodiester moieties in the development of prodrug-type oligonucleotide medicines.

1. Introduction

Oligonucleotide-based drug therapeutics could become effective treatment options against human diseases such as cancer and viral infections.^1,2) However, the delivery of oligonucleotide drugs to their target sites of action remains a major challenge. Due to their structural aspects, oligonucleotides have poor cellular membrane permeability and are unstable against nucleases in biological fluids. To improve the cellular membrane permeability of oligonucleotide-based drugs, good drug-delivery systems (DDSs) should be developed. To date, the typical DDSs of oligonucleotide drugs are: (i) using nanoparticles as carriers, (ii) conjugating small molecules and/or polymers to improve the cellular membrane permeability of oligonucleotides; and (iii) developing prodrug-type oligonucleotide medicines. In this review, we have focused on (iii), the development of prodrug-type oligonucleotide medicines; (i) and (ii), which have been discussed in depth elsewhere,^3–7) are not in the focus.

The prodrug approach to oligonucleotides is based on neutralizing the negative charges of phosphodiesters using bio-labile protecting groups for improving cellular membrane permeability. After prodrug-type oligonucleotide medicines have internalized within cells, their protecting groups are deprotected with “internal triggers” such as esterases, a reductive environment, etc. (Chart 1).

Chart 1. Expected Action of Oligonucleotide Prodrugs

Pioneering work on prodrugs for oligonucleotides was done in the 1990s. S-Acylthioethyl groups, as esterase labile protecting groups, were originally reported as nucleotide prodrugs⁸⁾ and later applied to the development of oligonucleotide prodrugs.^9–11) The main advantages of the prodrug-type oligonucleotide strategy include: (i) elimination of the need for transfection reagents and carriers to introduce oligonucleotides into cells; (ii) the general resistance of protected oligonucleotides to nucleases in biological fluids; and (iii) the ability to apply synthetic chemical techniques to develop protecting groups with different deprotection triggers.^12–36) Recently, the prodrug approach has been expanded from using antisense oligonucleotides to small interfering-RNA (siRNA) prodrugs, with application to biological studies. Furthermore, protecting groups have been linked to ligand molecules to achieve tissue-selective delivery.³¹⁾ However, the development of protecting groups for oligonucleotide prodrugs is not easy. The protecting group must be stable during the synthetic cycle of oligonucleotide and deprotection steps, and the protecting groups must be stable in biological fluids, yet they must be capable of being quickly deprotected at the target site.

In this review, we describe our recent studies on the design and synthesis of bio-labile protecting groups for phosphodiester moieties which could be applicable to prodrug-type oligonucleotide medicines.

2. Bio-labile Protecting Groups for Prodrug-Type Oligonucleotide Medicines

Outlines of the bio-labile protecting groups in the previous studies, along with our method, are described in Chart 2.

Chart 2. Structures of Bio-labile Protecting Groups for Phosphodiester Moieties, and the Expected Deprotection Pathway

In Chart 2(a), previously mentioned S-acylthioethyl groups are described. The protecting groups on the backbones of oligonucleotides are cleaved by esterase to generate free thiol functions which attack to the α-carbon near phosphorus atoms to provide naked oligonucleotides.^10,11,31,37) The preparation of these oligonucleotides requires non canonical synthetic conditions.

Nitrofuranylmethyl and nitrothienylmethyl groups for phosphodiester moieties in oligonucleotides are cleaved via the nitro reduction-elimination process triggered by nitroreductase²⁵⁾ (Chart 2b). Experiments were done with oligothymidylates; oligonucleotides with mixed sequences bearing these protecting groups have not been reported. It is presumed that the heteroarylmethyl structure was probably unstable in DNA synthetic processes.

Therefore, we investigated new protecting groups which have been found to be stable in standard DNA synthetic cycles, in order to make protected oligonucleotide preparations easier and more easily reproducible. The general design of our bio-labile protecting groups is described in Chart 2c. A typical protecting group is composed of a propyl linker connected to a benzene ring that has a heteroatom as a nucleophile. The heteroatom is protected with various trigger sensitive moieties which can respond to specific cellular triggers. The cleavage of trigger-sensitive moieties by triggers in cells gave nucleophiles such as hydroxyl, amino and mercapto groups; the intramolecular attack of these groups to the carbon near phosphortriester gave native oligonucleotides.

This design allows for a diverse set of protecting groups for phosphodiester moieties applicable to various oligonucleotide prodrugs. Moreover, substituents on the benzene ring can regulate the speed of the cyclization steps,^38,39) and biocompatibility can be improved by attaching the functional molecules.³³⁾ Importantly, the protecting groups are highly stable in standard DNA synthetic cycles, thus the experiments can be reproduced in many laboratories.

3. Some Examples of Bio-labile Protecting Groups

3.1. Oligonucleotide Prodrugs Bearing Photo-sensitive Protecting Groups

In order to confirm the stability of the protecting group in DNA synthetic cycles, and to detect intermediates in the deprotection pathway, we introduced a photosensitive group to the protecting group (Chart 3). Since the photoreaction was clean, we expect that the reaction pathway can be easily monitored.³³⁾

Chart 3. Expected Deprotection Reaction Pathways of a Protecting Group Using a Photo-labile Moiety, a 2-Nitrobenzyl Group

A synthetic route for the protecting group and an amidite unit (a monomer unit for oligonucleotide synthesis) is shown in Chart 4. An alcohol (3) was synthesized from 2,6-dimethylphenol according to a procedure described previously,⁴⁰⁾ and 3 was coupled with an o-NO₂ benzyl bromide to give the protecting group 4. A phosphoramidite unit (6) was synthesized using a standard synthetic procedure and incorporated into the model oligonucleotides (Chart 4).

Chart 4. Synthesis of a Monomer Unit Having a Photo-labile Protecting Group 6

ODN1 (Chart 4) was synthesized, deprotected, and purified using standard procedures for unmodified oligonucleotide preparation. ODN1 was obtained in good yield. By photo-irradiation of a solution containing ODN1, the 2-nitrobenzyl group was removed to generate a phenolic intermediate which was gradually converted into the “naked” oligothymidylate. These results suggested a proposed deprotection pathway as shown in Chart 3.

3.2. Oligonucleotide Prodrugs Bearing Reduction-Activated Protecting Groups

Hypoxia in locally advanced solid tumors is a characteristic environment caused by an insufficient supply of oxygen due to poorly developed vasculature.⁴¹⁾ Hence, several hypoxia-activated prodrugs using reduction-activated protecting groups have been developed and reported.^42,43) As described above, nitrofuranyl methyl and nitrothienylmethyl groups were used for protecting phosphodiester moieties in oligthymidylates²⁵⁾ (Chart 2b). However, ODNs with mixed sequences bearing these protecting groups have not been successfully synthesized, probably because the protecting groups have inadequate stability for standard DNA synthetic circles. Thus, we prepared a durable protecting group, the 3-(2-nitrophenyl)propyl group, as a reduction-activated protecting group for phosphodiester moieties in prodrug-type oligonucleotides³⁵⁾ (Chart 5).

Chart 5. The Structure of a Prodrug-Type Oligonucleotide Bearing a Reduction-Activated Protecting Group, and Its Expected Deprotection Pathway

Oligonucleotides bearing reduction-activated protecting groups were synthesized using a standard DNA synthetic protocol (Chart 6). We also synthesized an oligonucleotide containing a non-cleavable protecting group as a control (ODN 3).

Chart 6. Synthesis of ODNs Bearing Reduction-Activated Protecting Groups

Oligonucleotides bearing reduction-activated protecting groups were treated with nitroreductase (from Escherichia coli) in the presence of the reduced form of nicotinamide adenine dinucleotide (NADH). The protecting groups were deprotected to give native oligonucleotides.³⁵⁾ Conversely, the non-cleavable substrate (ODN 3) was completely stable under the same condition.

We evaluated the stability of oligonucleotides bearing the protecting group against exonuclease, endonuclease, and 50% human serum. Protected oligonucleotides exhibited much greater stability in 50% human serum than the full phosphodiester ODN.

Next, we evaluated the cellular uptake of the protected ODNs into HeLa cells. ODNs 6–8 were labeled with fluorescein at their 5′-ends.³⁵⁾ HeLa cells were treated with solutions containing the different ODNs (10 µM) for 1 h. The fixed cells were observed by confocal microscopy. The fluorescence intensity of the labeled ODNs in HeLa cells increased as the number of protecting groups increased. The maximum fluorescence intensity was observed for ODN 8. This result suggests that the oligonucleotides bearing reduction-activated protecting groups (hydrophobic groups) can be taken up into cells without the use of transfection reagents.

3.3. Functionalization of Prodrug-Type Oligonucleotides

It has been known that functional groups such as carbohydrates^44,45) and vitamins^46,47) attached to oligonucleotides induce the cellular uptake of oligonucleotides. These functional molecules, attached to the bio-labile protecting groups, can be deprotected in cells, and naked oligonucleotide drugs will then be generated (Chart 7).

Chart 7. Functionalization of Prodrug-Type Oligonucleotides and Their Expected Actions in the Cells

We reported a method for easily preparing oligonucleotides that have functionalized bio-labile protecting groups.⁴⁸⁾ An oligonucleotide with an alkyne-linked bio-labile protecting group was synthesized, then functional groups were attached to the oligonucleotide by copper-catalyzed azide alkyne cycloaddition (CuAAC) reaction (Chart 8).

Chart 8. A Schematic Representation for the Preparation of Functionalized Oligonucleotides

An oligonucleotide with an alkyne-linked reduction-activated protecting group is synthesized, then a coupling reaction between a functional molecule with an azide group and the alkyne group of the oligonucleotide gave a functionalized oligonucleotide.

Copper-catalyzed azide alkyne cycloaddition (CuAAC) to conjugate functional molecules and oligonucleotide on—the solid support have been reported.^49–53) This strategy allowed for the synthesis of a diverse range of oligonucleotide prodrugs from one oligonucleotide precursor: oligonucleotides with alkyne groups.

A synthetic route⁴⁸⁾ of an alkyne-linked reduction-activated protecting group (11) and an amidite unit (12) with a protecting group is shown in Chart 9.

Chart 9. Synthesis of Thymidine Phosphoramidite Bearing an Alkyne-Linked Reduction-Activated Protecting Group

A schematic representation for the synthesis of functionalized oligonucleotides is shown in Chart 10. Using amidite unit 12, an oligonucleotide with protecting groups was synthesized. Before deprotection, oligonucleotide on-support (controlled pore glass, CPG) was conjugated with various functional molecules, such as vitamins and carbohydrates. The functionalized oligonucleotide was treated with conc. NH₄OH for deprotection, then cleaved from the solid support (CPG).

Chart 10. On-Support Conjugation with Functional Molecules Using the CuAAC Reaction

In our experiments, the phosphoramidite unit 12 was quantitatively incorporated into oligonucleotides, and the on-support CuAAC reaction also succeeded quantitatively. Various functional molecules were conjugated in good yield in the same manner.

We investigated the deprotection reaction of functionalized reduction-activated protecting groups in oligonucleotides (Chart 11). Oligonucleotides with functional groups were treated with nitroreductase (from E. coli), and reactions were monitored by HPLC analysis. The functional groups were removed and naked oligonucleotides were detected.

Chart 11. Deprotection of Functionalized Protecting Groups on the Backbones of Oligonucleotide Prodrugs

4. Discussion

In the above paragraphs, protecting groups for phosphodiester groups were described. The protecting groups can be used for preparing prodrug-type DNA oligonucleotides. Also, prodrug-type oligonucleotides with 2′-O-methylribose or 2′-deoxy-2′-fluororibose can be prepared.³¹⁾ However, prodrug-type RNA oligonucleotides cannot be prepared. This is unfortunate, since RNA type oligonucleotides, especially siRNA, have been important candidates for oligonucleotide therapeutics. For preparing prodrug-type RNA oligonucleotides, a good combination of protecting groups for the phosphodiester group and the 2′-hydroxy group should be developed (Chart 12). The protecting groups should be deprotected in the following order: first, the protecting group of the phosphodiester, then that of the 2′-hydroxy group. When the protecting group of the 2′-hydroxy group is deprotected first, the RNA strand will be degraded by reaction of the free hydroxy group to the phosphotriester group. Today, some good bio-labile protecting groups for 2′-hydroxy groups of RNA strands have been reported.⁵⁴⁾ However, the development of effective combinations of protecting groups has not yet been accomplished.

Chart 12. A Schematic Representation of an Expected Deprotection Pathway for Prodrug Type RNA Oligonucleotide

As previously described, as the numbers of protecting groups at the phosphodiester groups increased, cellular membrane permeability of protected oligonucleotides increased.³⁵⁾ However, the solubility of oligonucleotides decreased as the numbers of protecting groups increased. The balance between solubility and cellular membrane permeability may be important in the development of effective oligonucleotide drugs. Alternatively, by adding soluble functions to the protecting groups, the solubility of oligonucleotides could be improved (Chart 13). As described previously, functional groups such as carbohydrates^44,45) and vitamins^46,47) may improve the cellular uptake of oligonucleotides.

Chart 13. A Schematic Representation of Functionalized Protecting Groups

Summary

Our recent studies focused on the development of protecting groups for prodrug-type oligonucleotides. The skeleton of protecting groups is stable in standard DNA synthetic cycles, thus our experiments can be easily reproduced in any laboratories. Also, because there is some space on the benzene ring, some functional groups, such as peptides and sugars, can be connected for improving the solubility and cellular membrane permeability of these prodrug-type oligonucleotides.

Acknowledgment

This work was supported by a Grant-in-Aid for Scientific Research (C) (17K01966) of the Strategic Research Base Development Program for Private Universities (Kanagawa University, 2012–2016) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Conflict of Interest

The authors declare no conflict of interest.

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