Expanding the scope of the cleavable n-(Methoxy)oxazolidine linker for the synthesis of oligonucleotide conjugates

Article abstract of DOI:10.3390/molecules26020490

Oligonucleotides modified by a 2^′-deoxy-2^′-(N-methoxyamino) ribonucleotide react readily with aldehydes in slightly acidic conditions to yield the corresponding N-(methoxy)oxazolidine-linked oligonucleotide-conjugates. The reaction i

Full text of DOI:10.3390/molecules26020490

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molecules
Communication
Expanding the Scope of the Cleavable N-(methoxy)oxazolidine
Linker for the Synthesis of Oligonucleotide Conjugates
Aapo Aho, Antti Äärelä, Heidi Korhonen and Pasi Virta *
Department of Chemistry, University of Turku, 20014 Turku, Finland; aamaah@utu.fi (A.A.);
ankaaar@utu.fi (A.Ä.); hejoli@utu.fi (H.K.)
* Correspondence: pamavi@utu.fi; Tel.: +358-503-285-719
0
0
Abstract: Oligonucleotides modified by a 2 -deoxy-2 -(N-methoxyamino) ribonucleotide react readily
with aldehydes in slightly acidic conditions to yield the corresponding N-(methoxy)oxazolidine-
linked oligonucleotide-conjugates. The reaction is reversible and dynamic in slightly acidic con-
ditions, while the products are virtually stable above pH 7, where the reaction is in a “switched
off-state”. Small molecular examinations have demonstrated that aldehyde constituents affect the
cleavage rate of the N-(methoxy)oxazolidine-linkage. This can be utilized to adjust the stability of this
pH-responsive cleavable linker for drug delivery applications. In the present study, Fmoc-β-Ala-H
was immobilized to a serine-modified ChemMatrix resin and used for the automated assembly of two
peptidealdehydes and one aldehyde-modified peptide nucleic acid (PNA). In addition, a triantennary
N-acetyl-D-galactosamine-cluster with a β-Ala-H unit has been synthesized. These aldehydes were
conjugated via N-(methoxy)oxazolidine-linkage to therapeutically relevant oligonucleotide phospho-
rothioates and one DNA-aptamer in 19–47% isolated yields. The cleavage rates of the conjugates
were studied in slightly acidic conditions. In addition to the diverse set of conjugates synthesized,
these experiments and a comparison to published data demonstrate that the simple conversion of
Gly-H to β-Ala-H residue resulted in a faster cleavage of the N-(methoxy)oxazolidine-linker at pH 5,
being comparable (T0.5 ca 7 h) to hydrazone-based structures.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Aho, A.; Äärelä, A.;
Korhonen, H.; Virta, P. Expanding
the Scope of the Cleavable
N-(methoxy)oxazolidine Linker for
the Synthesis of Oligonucleotide
Conjugates. Molecules 2021, 26, 490.
https://doi.org/10.3390/
Keywords: oligonucleotide conjugates; cleavable linker; N-(methoxy)oxazolidine
molecules26020490
1. Introduction
Academic Editor: Nicola Borbone
Received: 22 December 2020
Accepted: 11 January 2021
Published: 18 January 2021
Oligonucleotide (ON) therapeutics, such as antisense oligonucleotides (ASO) and
small interfering RNAs (siRNAs), can be applied for the modulation of gene expression in
a wide range of disorders [1–9]. Despite the great potential of ONs as drugs, they suffer
from poor pharmacokinetic properties [10]. Backbone modifications such as phospho-
rothioate and 20-O-substitutions improve the stability and increase the plasma circulation
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
time of Ons [10
lenge [ 12]. For targeted delivery, antibodies [13
extracellular vesicles [19], carbohydrates [20 22], cholesterol [23
molecules [25 27] have been utilized. However, almost without exception these strategies
lead to the endosomal entrapment of ONs [15 18 28]. Endosomal escape may be facilitated
by other structural modifications or conjugate groups [29 31], which may make the overall
,
11], but cell/tissue-specific extrahepatic delivery has remained a chal-
15], aptamers [16 17], nanoparticles [18],
24] m and other small
8,
–
,
–
,
–
,
,
–
synthesis complex. In the synthesis of these biomolecular hybrids, in which even the
bis-conjugation of ONs is needed, orthogonal ligation chemistries play a central role. It
is beneficial if the conjugation itself creates a linker that is cleavable [32–34]. The linker
should also provide efficient conjugation, be stable in physiological conditions, and release
the therapeutic ON cargo in appropriate intracellular compartments. Examples of such
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Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
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4.0/).
linkers are hydrazones [35
to that in endosomes and lysosomes, and disulfides [14
reducible environment in cytosol. Hence, the former linker chemistry may be suitable for
,
36], which are cleaved in slightly acidic conditions perceived
,16], which are cleaved in a mildly
Molecules 2021, 26, 490. https://doi.org/10.3390/molecules26020490
https://www.mdpi.com/journal/molecules

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Molecules 2021, 26, 490
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the conjugation of cell/tissue targeting vehicles, whereas the latter may be suitable for the
conjugation of endosomal escaping moieties. Expanding the chemistry of reversible linkers
is important to find efficient orthogonal conjugation strategies and the targeted release of
ON therapeutics in biological mediums.
Recently, the reversible formation of N-(methoxy)oxazolidine (Figure 1) was employed in
conjugation between 20-deoxy-20-(N-methoxyamino)uridine (UNOMe
, Scheme 1a)-modified
ONs and Gly-H-modified peptide aldehydes [37]. The UNOMe-ONs and peptide aldehydes
were both synthesized by automated assembly using appropriately modified solid supports.
After cleavage, deprotection, and purification, the UNOMe-ONs and the peptide aldehy-
des were mixed in slightly acidic conditions to yield conjugates in reasonable yields. The
conjugates were stable during RP HPLC purification and lyophilization, but showed an acid-
dependent hydrolytic cleavage. ONs were released from Gly-H-modified peptide aldehydes
with a half-life (t0.5) of 5.8, 42, and 220 h at pH 4, 5, and 6, respectively (37 ◦C) (◦cf. Table 1
entries 1–3), and only 11% was released after two weeks of incubation at pH 7 (37 C). It was
additionally shown by small molecular models that the rate of the N-(methoxy)oxazolidine
hydrolysis could be adjusted using structurally different aldehydes.
Figure 1. Formation of N-(methoxy)oxazolidines between 20-deoxy-20-(N-methoxyamino) uridine
and small molecule aldehydes (R = cf. Table 2).
Scheme 1.
solid support. (
bound on an amino-modified ChemMatrix resin via N-Boc-oxazolidine.
(
A
) Automated synthesis of UNOMe -elongated oligonucleotides (ON1
,
ON2
,
ON3, and ON4) using UNOMe
-Ala-H
B
) Automated synthesis of -Ala-H-modified peptides (P1 and P2) and PNA (PNA1) using Fmoc-β
β

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Molecules 2021, 26, 490
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◦
Table 1. Hydrolysis rates of the UNOMe conjugates at 37 C.
pH b
Entry
Conjugate a
t0.5 (h)
ON Released at Equilibrium (%)
1 c
2 c
3 c
4
5
6
7
8
9
C1*
C1*
C1*
C1
C1
C1
C2
C3
C4
4
5
6
4
5
6
5
5
5
5.80 ± 0.52
41.7 ± 2.3
222 ± 20
95.2 ± 2.1
95.8 ± 1.6
quant.
91.5 ± 5.1
89.7 ± 2.9
95.7 ± 3.4
59.2 ± 0.6
94.8 ± 2.4
63.9 ± 2.5
1.53 ± 0.25
7.17 ± 0.77
37.3 ± 3.3
4.41 ± 0.17
10.2 ± 0.81
7.40 ± 1.03
10 µM initial concentration. 100 mM NaOAc/AcOH. c Previously published data [36].
a
b
In this study, the scope of the N-(methoxy)oxazolidine ligation was expanded by the
synthesis of a more diverse set of conjugates. UNOMe-extended ONs, consisting of three
therapeutically relevant ONs, ISE-AR-V7 [38], Nusinersen [39], IONIS-DGAT2RX
and one DNA-aptamer, TfRA3 [42], were synthesized and conjugated to four different
-Ala-H-containing biomolecules: SpyTag [43], D-retro inverso THR [44 46], an antisense
[40,41],
β
–
PNA [47], and a trivalent N-acetyl galactosamine (GalNAc) cluster (cf. further description
of these biomolecules below). The ligation products could be obtained in 19–47% isolated
yields. The hydrolysis rate of the conjugates (i.e., reverse ligation) was studied at pH 4, 5, 6,
and 7.4. The N-(methoxy)oxazolidine linker, bound to the β-Ala-H residue, clearly cleaved
faster in acidic conditions (pH 4–6) than the previously studied Gly-H-based conjugates [37].
The cleavage was comparable to most hydrazone linkers, and the conjugates maintained
their hydrolytic stability in physiological conditions (pH 7.4).
2. Results
2.1. Small Molecular Model Study
Prior to real conjugation experiments (described below), the N-(methoxy)oxazolidine
formation with a
β
-Ala-H residue was studied using small-molecule models. 20-deoxy-20-
(N-methoxyamino)uridine (1, 5 mM) and N-Bz-β-Ala-H (5 mM) were mixed in buffered
aqueous solution (pH 4) at room temperature and the progress of the reaction was followed
by RP HPLC. As expected, two N-(methoxy)oxazolidine ligation products (R/S isomers)
were formed (cf. RP HPLC profile of the reaction and characterization of the products in Sup-
plementary Materials). The reaction stalled at equilibrium (K = 2.82 ± 0.43 × 103 L mol−1),
yielding a 75% conversion of 1 to the ligation products. The hydrolysis rate of the obtained
N-(methoxy)oxazolidine was determined at pH 4, 5, and 6 by following the degradation
of the major ligation product. As expected, the hydrolysis rate was pH-dependent, with
half-lives of 5.3, 29, and 310 h at pH 4, 5, and 6, respectively, being ca. three-fold faster than
the hydrolysis of N-Bz-Gly-H ligation product (Figure 1 and Table 2). Despite the modest
rate enhancement of the hydrolysis, this model reaction was well-behaving and promising,
considering the conjugation of ONs with β-Ala-H-containing biomolecules.
0
0
Table 2. Formation and decay of N-(methoxy)oxazolidines between 2 -deoxy-2 -(N-methoxyamino)
uridine and small molecular aldehydes.
Equilibrium Constant K
Equilibrium Yield
(%) d
R b
Entry
pH
t0.5 Decay (h) c
c
(l mol−1
)
1 a
2 a
3 a
4
5
6
BzNHCH2
4
5
6
4
5
6
16.1 ± 0.7
75.5 ± 16.3
n/a
4958 ± 50
82
“
“
BzNHCH2CH2
5.28 ± 0.58
28.5 ± 2.2
310 ± 34
2398 ± 425
75
“
“
a
b
c
d
Previously published data [37]. Cf. R in Figure 1. According to pseudo first-order rate law. Acquired by
mixing 1 (5 mM) and aldehyde (5 mM) at pH 4.

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2.2. Synthesis of 20-deoxy0-20-(N-methoxyamino)uridine-Modified Oligonucleotides
Four oligonucleotides elongated by UNOMe (AON-ISE-AR-V7-UNOMe
ON1), Nusinersen-
UNOMe UNOMe ON3), and TfRA3-UNOMe
ON2), IONIS-DGAT2RX ON4) (Scheme 1a) were
next synthesized using previously prepared solid support 37], commercial phosphoramidite
(
(
-
(
(
2
[
building blocks, and automated chain assembly. AON-ISE-AR-V7 suppresses prostate tumor
cell survival by the inhibition of androgen receptor variant 7 mRNA synthesis [38], Nusinersen
is an approved drug used for treating spinal muscular athropy [39], and IONIS-DGAT2RX ASO
reduces DGAT2 enzyme production and is a potential treatment for nonalcoholic steatohep-
atitis [40
vehicle to traverse the blood brain barrier (BBB) (i.e., the role of this ON in the cargo-delivery
vehicle construct C4 is inverse compared to that of conjugates C1 C3, cf below). After chain
,41]. TfRA3 is a transferring receptor-binding aptamer [41] that can act as a delivery
–
elongation, cleavage with concentrated aqueous ammonia followed by RP HPLC purification
gave the desired oligonucleotides in 29–40% yields (cf. supporting information).
2.3. Synthesis of β-Ala-H-Modified Biomolecules
Two peptide aldehydes and one PNA aldehyde were synthesized by following a pub-
lished protocol [48
via N-(Boc)oxazolidine to obtain solid support
H (P1), retro inverso THR- -Ala-H (P2), and GluR3 antisense PNA-
,
49]. Fmoc-
β
-Ala-H was bound to an amino-modified ChemMatrix resin
. On this support, SpyTag-(AEEA)2- -Ala-
-Ala-H (PNA1) were
3
β
β
β
synthesized using automated Fmoc-chemistry (Scheme 1b). SpyTag peptide binds through
irreversible isopeptide bond to a SpyCatcher protein domain [43]. This autocatalytic
process has been utilized, e.g., for the preparation of antibody-ON-conjugates [50], but
immunogenicity issues should be resolved prior to drug delivery applications. THR and
its peptidase-resistant retro inverso version [44] binds to the transferrin receptor, and it has
been applied to deliver RNA nanoparticles through the blood brain barrier [45,46]. GluR3
antisense PNA has been shown to reduce the glutamate excitotoxicity associated with
amyotrophic lateral sclerosis (ALS) by reducing GluR3 protein expression [47]. After chain
elongation, the peptides/PNA were cleaved from the resin using a TFA cocktail (cf. support-
ing information for more details); precipitated in cold ether; and dissolved in aq. 0.01% TFA
to yield P1
One
ing via asialoglycoprotein receptor [51], was additionally synthesized starting from branching
unit 52] consisting of three alkynyl and one aromatic aldehyde group (Scheme 2b). First,
the aldehyde moiety was oxidized by Jones’ condition using Cr3O. The resulting carboxylic
acid ( ) was coupled to the diethoxy acetal of -Ala-H using BOP/DIPEA activation to yield
an amide ( ). Then, the alkynyl groups of the core were coupled with (3-azidopropyl)-2-
acetamido-3,4,6-tri-O-acetyl-2-deoxy- -D-galactopyranoside using Cu(I) catalyzed 1,3-dipolar
cycloaddition (i.e., click reaction). Finally, the acetal protection of was removed by aq 0.01%
TFA to expose the -Ala-H functionality. The final product was prepared in an 18% yield
after four steps. It may be worth mentioning that in our previous study aryl aldehydes
(cf. ) reacted only barely with , and the -Ala-H-extension (cf. ) is crucial to gain an
efficient conjugation.
,
P2, and PNA1, which were purified by RP HPLC (cf. supporting information).
β-Ala-H-containing trivalent GalNAc cluster, with a good potential for liver target-
4
[
5
β
6
β
7
β
8
4
1
β
8
2.4. Synthesis of Oligonucleotide Conjugates C1-C4 Using N-(methoxy)oxazolidine Ligation
The rationale of the synthesized conjugates C1 C4 below is based on the therapeutic
-
relevance of the ASOs (cf. above) and the reported delivery potential of the corresponding
conjugate groups to target tissues: P2 and ON4 could potentially enhance the CNS targeting
of ON2 and PNA1 (i.e., C2 and C4) and increase their potential as intravenously adminis-
trated drugs. However, nanoparticle-based delivery systems may be additionally needed
in this approach. The GalNac cluster
can readily be extended to an antibody construct specific to prostate membrane antigen
(PMSA) and, in this way, improve the targeting of ON1
C1). The UNOMe oligonucleotides
ON1 ON2 ON3, and ON4 were mixed with an excess of the corresponding -Ala-H
conjugate groups P1 P2 PNA1, and (Scheme 2a) and incubated in AcOH/DMSO (1:3,
8 could increase the liver targeting of ON3 (C3). P1
(
,
,
β
,
,
8