Expanding the Scope of the Cleavable N-(Methoxy)oxazolidine Linker for the Synthesis of Oligonucleotide Conjugates

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 conditions, 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 phosphorothioates 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.


Introduction
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][2][3][4][5][6][7][8][9]. Despite the great potential of ONs as drugs, they suffer from poor pharmacokinetic properties [10]. Backbone modifications such as phosphorothioate and 2 -O-substitutions improve the stability and increase the plasma circulation time of Ons [10,11], but cell/tissue-specific extrahepatic delivery has remained a challenge [8,12]. For targeted delivery, antibodies [13][14][15], aptamers [16,17], nanoparticles [18], extracellular vesicles [19], carbohydrates [20][21][22], cholesterol [23,24] m and other small molecules [25][26][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][30][31], which may make the overall 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][33][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 linkers are hydrazones [35,36], which are cleaved in slightly acidic conditions perceived to that in endosomes and lysosomes, and disulfides [14,16], which are cleaved in a mildly reducible environment in cytosol. Hence, the former linker chemistry may be suitable for lease of ON therapeutics in biological mediums.
Recently, the reversible formation of N-(methoxy)oxazolidine ( Figure 1) w loyed in conjugation between 2′-deoxy-2′-(N-methoxyamino)uridine (U NOMe , a)-modified ONs and Gly-H-modified peptide aldehydes [37]. The U NOMe -ONs a de aldehydes were both synthesized by automated assembly using appropriate ed solid supports. After cleavage, deprotection, and purification, the U NOMe -ONs eptide aldehydes were mixed in slightly acidic conditions to yield conjugates in ble yields. The conjugates were stable during RP HPLC purification and lyophi ut showed an acid-dependent hydrolytic cleavage. ONs were released from Glyied peptide aldehydes with a half-life (t0.5) of 5.8, 42, and 220 h at pH 4, 5, and 6 vely (37 °C) (cf. Table 1 entries 1-3), and only 11% was released after two weeks ation at pH 7 (37 °C). It was additionally shown by small molecular models that f the N-(methoxy)oxazolidine hydrolysis could be adjusted using structurally d ldehydes.
igure 1. Formation of N-(methoxy)oxazolidines between 2′-deoxy-2′-(N-methoxyamino) d small molecule aldehydes (R = cf. Table 2).  mildly reducible environment in cytosol. Hence, the former linker chemistry may be suitable for 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 2′-deoxy-2′-(N-methoxyamino)uridine (U NOMe , Scheme 1a)-modified ONs and Gly-H-modified peptide aldehydes [37]. The U NOMe -ONs and peptide aldehydes were both synthesized by automated assembly using appropriately modified solid supports. After cleavage, deprotection, and purification, the U NOMe -ONs and the peptide aldehydes 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.  Table 2).

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. 2 -deoxy-2 -(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 Supplementary Materials). The reaction stalled at equilibrium (K = 2.82 ± 0.43 × 10 3 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.  [37]. b Cf. R in Figure 1. c According to pseudo first-order rate law. d Acquired by mixing 1 (5 mM) and aldehyde (5 mM) at pH 4.

Synthesis of β-Ala-H-Modified Biomolecules
Two peptide aldehydes and one PNA aldehyde were synthesized by following a published protocol [48,49]. Fmoc-β-Ala-H was bound to an amino-modified ChemMatrix resin via N-(Boc)oxazolidine to obtain solid support 3. On this support, SpyTag-(AEEA) 2 -β-Ala-H (P1), retro inverso THR-β-Ala-H (P2), and GluR3 antisense PNA-β-Ala-H (PNA1) were 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. supporting information for more details); precipitated in cold ether; and dissolved in aq. 0.01% TFA to yield P1, P2, and PNA1, which were purified by RP HPLC (cf. supporting information).
One β-Ala-H-containing trivalent GalNAc cluster, with a good potential for liver targeting via asialoglycoprotein receptor [51], was additionally synthesized starting from branching unit 4 [52] consisting of three alkynyl and one aromatic aldehyde group (Scheme 2b). First, the aldehyde moiety was oxidized by Jones' condition using Cr 3 O. The resulting carboxylic acid (5) was coupled to the diethoxy acetal of β-Ala-H using BOP/DIPEA activation to yield an amide (6). Then, the alkynyl groups of the core were coupled with (3-azidopropyl)-2acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galactopyranoside using Cu(I) catalyzed 1,3-dipolar cycloaddition (i.e., click reaction). Finally, the acetal protection of 7 was removed by aq 0.01% TFA to expose the β-Ala-H functionality. The final product 8 was prepared in an 18% yield after four steps. It may be worth mentioning that in our previous study aryl aldehydes (cf. 4) reacted only barely with 1, and the β-Ala-H-extension (cf. 8) is crucial to gain an efficient conjugation.

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 administrated drugs. However, nanoparticle-based delivery systems may be additionally needed in this approach. The GalNac cluster 8 could increase the liver targeting of ON3 (C3). P1 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 U NOMe oligonucleotides ON1, ON2, ON3, and ON4 were mixed with an excess of the corresponding β-Ala-H conjugate groups P1, P2, PNA1, and 8 (Scheme 2a) and incubated in AcOH/DMSO (1:3, v/v), 2 M LiCl at 55 • C (reaction specific parameters in Table 3). After 1 h of incubation, the reaction mixtures were neutralized using dilute aq. NaOH and subjected as such to RP HPLC. As seen in the RP HPLC profiles of the crude product mixtures (Figure 2), a good conversion of the products was obtained with a moderate excess of the aldehyde constituents (P1, P2, PNA1, and 8, 2-8 equiv.). The product fractions were lyophilized to give the conjugates C1, C2, C3 Ac , and C4 in 19-47% yields (Table 3). Conjugate C3 Ac was deacetylated by soaking the conjugate in concentrated aq. ammonia (3 h at rt), and, without further purification, lyophilized to give C3. ministrated drugs. However, nanoparticle-based delivery systems may be additionally needed in this approach. The GalNac cluster 8 could increase the liver targeting of ON3 (C3). P1 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 U NOMe oligonucleotides ON1, ON2, ON3, and ON4 were mixed with an excess of the corresponding β-Ala-H conjugate groups P1, P2, PNA1, and 8 (Scheme 2a) and incubated in AcOH/DMSO (1:3, v/v), 2 M LiCl at 55 °C (reaction specific parameters in Table 3). After 1 h of incubation, the reaction mixtures were neutralized using dilute aq. NaOH and subjected as such to RP HPLC. As seen in the RP HPLC profiles of the crude product mixtures (Figure 2), a good conversion of the products was obtained with a moderate excess of the aldehyde constituents (P1, P2, PNA1, and 8, 2-8 equiv.). The product fractions were lyophilized to give the conjugates C1, C2, C3 Ac , and C4 in 19-47% yields (Table 3). Conjugate C3 Ac was deacetylated by soaking the conjugate in concentrated aq. ammonia (3 h at rt), and, without further purification, lyophilized to give C3.

Hydrolysis of the Ligations Products
The hydrolysis rates of the conjugates (C1-C4) were studied by incubating them (10 µM) in aq. buffers at 37 °C and monitoring the release of the ONs by RP HPLC (Figure 3 and 4, Table 1). As predicted by the small-molecule models, ON1 released from β-Ala-Hderived conjugate C1 5-6 times faster (at pH 5, t0.5 = 7.17 ± 0.77 h) than from Gly-H-derived conjugate C1* (at pH 5, t0.5 = 41.7 ± 2.3). Indeed, as illustrated in Figure 3, C1 requires approximately one pH unit less acidic environment than C1* to reach the same rate of hydrolysis in the range of pH 4-6. Similarly, at pH 5 conjugates C2, C3, and C4 were all hydrolyzed within t0.5 of 4.0-11.1 h (entries 7, 8, 9 in Table 1). Interestingly, there was variation in the hydrolysis rates and also in the equilibrium yields. The reaction is most likely affected by the macromolecular interactions (e.g., by electrostatic interactions between the ONs and positively charged peptides), and not only by the closest environment of the reaction center. All the conjugates were virtually stable at pH 7.4 after three days of incubation ( Figure 4).

Discussion
The N-(methoxy)oxazolidine linker was found to be a reliable tool for conjugating U NOMe -extended ONs to a variety of β-Ala-H-containing biomolecules. The rate of hydrol-

Hydrolysis of the Ligations Products
The hydrolysis rates of the conjugates (C1-C4) were studied by incubating them (10 µM) in aq. buffers at 37 • C and monitoring the release of the ONs by RP HPLC (Figures 3 and 4, Table 1). As predicted by the small-molecule models, ON1 released from β-Ala-H-derived conjugate C1 5-6 times faster (at pH 5, t 0.5 = 7.17 ± 0.77 h) than from Gly-H-derived conjugate C1* (at pH 5, t 0.5 = 41.7 ± 2.3). Indeed, as illustrated in Figure 3, C1 requires approximately one pH unit less acidic environment than C1* to reach the same rate of hydrolysis in the range of pH 4-6. Similarly, at pH 5 conjugates C2, C3, and C4 were all hydrolyzed within t 0.5 of 4.0-11.1 h (entries 7, 8, 9 in Table 1). Interestingly, there was variation in the hydrolysis rates and also in the equilibrium yields. The reaction is most likely affected by the macromolecular interactions (e.g., by electrostatic interactions between the ONs and positively charged peptides), and not only by the closest environment of the reaction center. All the conjugates were virtually stable at pH 7.4 after three days of incubation ( Figure 4).

Hydrolysis of the Ligations Products
The hydrolysis rates of the conjugates (C1-C4) were studied by incubating them µM) in aq. buffers at 37 °C and monitoring the release of the ONs by RP HPLC (Figur and 4, Table 1). As predicted by the small-molecule models, ON1 released from β-Ala derived conjugate C1 5-6 times faster (at pH 5, t0.5 = 7.17 ± 0.77 h) than from Gly-H-deriv conjugate C1* (at pH 5, t0.5 = 41.7 ± 2.3). Indeed, as illustrated in Figure 3, C1 requires proximately one pH unit less acidic environment than C1* to reach the same rate of drolysis in the range of pH 4-6. Similarly, at pH 5 conjugates C2, C3, and C4 were hydrolyzed within t0.5 of 4.0-11.1 h (entries 7, 8, 9 in Table 1). Interestingly, there was v iation in the hydrolysis rates and also in the equilibrium yields. The reaction is most lik affected by the macromolecular interactions (e.g., by electrostatic interactions between ONs and positively charged peptides), and not only by the closest environment of reaction center. All the conjugates were virtually stable at pH 7.4 after three days of in bation ( Figure 4).

Discussion
The N-(methoxy)oxazolidine linker was found to be a reliable tool for conjugat U NOMe -extended ONs to a variety of β-Ala-H-containing biomolecules. The rate of hydr H2O/MeCN (1:1, v/v) and subjected them to RP HPLC (Figure 4). The product fractio were collected and lyophilized. Conjugate C3 Ac was dissolved in concentrated aq. amm nia. The mixture was incubated for 3 h in room temperature and evaporated to dryness yield C3. The yields of the isolated conjugates (Table 3) were determined from the U absorbance at 260 nm using the molar absorptivity of the corresponding nucleobases. T authenticity of the products was verified by MS (ESI-TOF).

Discussion
The N-(methoxy)oxazolidine linker was found to be a reliable tool for conjugating U NOMe -extended ONs to a variety of β-Ala-H-containing biomolecules. The rate of hydrolysis of the N-(methoxy)oxazolidine conjugates (t 0.5 = 4.4-10.2 at pH 5) was in the range of the currently used acid-labile linkers that are applied in antibody-drug conjugates. For example, a phenylketone-derived hydrazine linker used in gemtuzumab ozogamicin (Mylotarg) [53] and inotuzumab ozogamicin (Besponsa) [54] has been determined to hydrolyze 97% in 24 h at pH 4.5, which equals t 0.5 = 4.74 h (according to first-order kinetics), and only 6% at pH 7.4 [53]. Obviously, the optimal release profiles of therapeutic ON conjugates may differ greatly from those of small-molecule drug conjugates. That said, the most central result here was that the release profile could be tuned by modifying the Gly-H aldehyde to a slightly less electron-deficient β-Ala-H without losing the convenience of the conjugate synthesis. It may be assumed that the release rate may be further accelerated using similar simple modifications. Furthermore, the N-(methoxy)oxazolidine conjugation was stable in concentrated ammonia. The option of removing the base-labile protecting groups post-conjugation may be useful in the synthesis of more complex conjugates or/and facilitating chromatographic issues.