Tunable pH-sensitive 2-carboxybenzyl phosphoramidate cleavable linkers

Highlights

• The pK_a of the benzoate in 2-carboxybenzyl phosphoramidates controlled hydrolysis.

• The rate of amine-release was inversely correlated with Hammett sigma values.

• The mechanism of hydrolysis proceeds through a transient cyclic acyl-phosphate.

Abstract

We previously described a pH-sensitive phosphoramidate linker scaffold that can be tuned to release amine-containing drugs at various pH values. In these previous studies it was determined that the tunability of this linker was dependent upon the proximity of an acidic group (e.g., carboxylic acid or pyridinium). In this study, we confirmed that the tunability of pH-triggered amine-release was also dependent upon the pK_a of the proximal acidic group. A series of 2-carboxybenzyl phosphoramidates was prepared in which the pK_a of the proximal benzoic acid was predictably attenuated by substituents on the benzoate ring consistent with their σ-values.

Introduction

Cleavable linker technology has been actively pursued for the past two decades [1], [2], [3]. Cleavable linkers have found use in the context of solid phase synthesis of small-molecule libraries and as strategy for asymmetric synthesis [4], [5]. More recently, the fields of chemical biology and biochemistry have employed cleavable linkers in the development of antibody drug conjugates [6], [7], [8], [9], [10] or triggered release of condition-specific turn-on dyes [11]. Despite the diversity of applications for cleavable linkers, the common requirement is highly predictable and controlled release of a desired compound from the linker scaffold under specific conditions.

We recently reported on two generations of phosphoramidate scaffolds (Fig. 1) that can be triggered to release amine-bearing drugs or self-immolating spacers at varying rates based upon the proximity of a mildly acidic group (e.g., carboxylic acid or pyridinium) [12], [13]. Due to the tunable nature of these scaffolds, we envisioned that these cleavable linker scaffolds can be tailored to the specific pH requirements of controlled release applications. Applications for such tunable pH-programmable cleavable scaffolds include antibody or small-molecule drug conjugates, drug-eluting stents, prodrugs, turn-on dyes for intracellular trafficking, or protecting groups in the chemical synthesis [1], [2], [3], [14].

It is known that the hydrolysis of phosphoramidate PN bonds is pH dependent [15], [16], [17], [18] and influenced by the presence of a proximal carboxylate [19]. We demonstrated that the proximity of a carboxylic acid led to greater susceptibility of the PN bond to hydrolysis at mild pH conditions, proceeding through two possible mechanisms [12], [13]. We have hypothesized that in addition to proximity, the pKa of the neighboring acidic group, and its ability to protonate the leaving amine, could influence the hydrolytic susceptibility of the PN bond. The focus of this study was aimed at attenuating the hydrolytic stability of the phosphoramidate scaffold through substituent effects [20], [21] on a proximal benzoic acid (Fig. 2). We hypothesized that under mildly acidic conditions (pH = 5.5), in which the the carboxylic acid is more fully protonated from electron-donating substituents (σ < 0) present, the rates for hydrolysis of the PN bond would be greater.

The substituted 2-carboxybenzyl phosphoramidates were prepared as outlined in Scheme 1. Phthalates 6a–6g were saponified and converted to the corresponding esters 5a-5g. Fluorenlymethyl ester-protected phosphites 3a–3g were generated from 4a–4g with diphenyl phosphite. Routine Atherton-Todd conditions [12], [13] provided the protected 2-carboxybenzyl phosphoramidates 2a–2g. Global deprotection with LiOH yielded the desired substituted 2-carboxybenzyl phosphoramidates 1a–1g.

Once prepared, the hydrolytic stability of 2-carboxybenzyl phosphoramidates 1a–g was assessed at pH 5.5, and 7.4 by ³¹P NMR [12], [13]. To determine the pH-dependent rates of hydrolysis, the peak areas for each parent phosphoramidate (1a–g), and its respective hydrolytic product and intermediate, were determined over 8 h with respect to an internal standard (triphenylphosphine oxide, TPPO). The observed hydrolysis rates of phosphoramidates 1a–g followed first order kinetics as previously noted [12], [13]. Half-lives and rate constants for 2-carboxybenzyl phosphoramidates 1a–1g, as well as control compounds 13, 16, and 19, at pH 5.5 and 7.4 are summarized in Table 1. Our interest in pH-triggered cleavable linkers is their utility in tumor biomarker-targeted drug conjugates. Therefore, the most relevant conditions for our applications are physiological (pH 7.4) and that of early endosomes (pH 5.5) following the internalization of the biomarker-targeted drug conjugates. However, to more fully illustrate the pH-dependence of the 2-carboxybenzyl phosphoramidate scaffold, the stability of 1c was monitored at pH 5.0, 5.5, 6.0. 6.5, and 7.4 as an example (Fig. 3).

Based on the data in Table 1, electron withdrawing groups (EWGs) led to increased stability of the phosphoramidate scaffold toward hydrolysis of the PN bond, while electron donating groups (EDGs) led to a decrease in stability (Table 1). This trend was inversely correlated to the σ values at both pH 7.4 and pH 5.5, resulting in negative ρ values of −0.84 and −1.02 for pH 7.4 and pH 5.5, respectively (Fig. 4). These results suggest that the pH-triggered release of amines from the phosphoramidate scaffold is also tunable through attenuating the pK_a of the neighboring acidic group.

While the mechanism for the hydrolysis of our previous pH-triggered phosphoramidate linker scaffolds [12], [13] has remained conjectural (Scheme 2), the presence of a transient intermediate in the ³¹P spectra (Fig. 3) supports the 2-step intramolecular substitution mechanism as shown in Scheme 2b. This mechanism would be consistent with the inverse Hammett correlation observed for the phosphoramidates (Fig. 4) whereby electron donating groups promote the nucleophilicity of the benzoyl carboxylate for the substitution at phosphorus, forming the acyl-phosphate intermediate. Because 1c showed the greatest formation of this putative acyl-phosphate intermediate, it was used as a representative example to further elucidate the mechanism of hydrolysis.

The hydrolysis of 1c was monitored by ³¹P and ¹H NMR (Fig. 5) in pD 6.0 buffer (acetic acid‑d₄ : sodium acetate-d₃), and the rate of its hydrolysis in pD 6.0 buffer was found to be similar to that at pH 6.5 [23]. The chemical shift of the transient intermediate P’ (I) was consistent with those of previously reported acyl-phosphates [24]. An expansion of the phenethylamine region in the ¹H NMR spectra (II and V) showed no significant change for the distant β-protons (a) as expected, however, the α-protons (b), which were proximal to the PN bond, rapidly decayed to the α-protons (b’) of released phenethylamine. Expansion of the benzylic region in the ¹H NMR spectra (III and VI) showed that the benzylic protons (c) of 1c rapidly decayed to the benzylic protons (c’) of an acyl-phosphate intermediate before finally decaying to the benzylic protons (c”) of the hydrolyzed product. Combined, the data from the ³¹P and ¹H NMR spectra for the hydrolysis of 1c support the proposed 2-step intramolecular mechanism (Scheme 2B) over a general acid catalyzed mechanism (Scheme 2A).

In summary, we have demonstrated that by attenuating the pK_a of the neighboring acidic group in the 2-carboxybenzyl phosphoramidate scaffold the controlled hydrolytic release of an amine can be predictably tuned. These results are consistent with those reported by Kirby and coworkers [25], [26], [27], [28] who noted the effects of neighboring ionizable groups on hydrolysis of phosphate mono, -di, and -tri esters. Together with our prior findings, we conclude that the rate of hydrolysis of such phosphoramidate scaffolds depends upon three main factors: (1) the pK_a of the departing amine [12], (2) the proximity of the ionizable group [13], and (3) the pK_a of the proximal ionizable group. Combined, these aspects allow for considerable control of this scaffold for the release of amine payloads in the context of cleavable linkers, which can be tuned to meet the specific demands of various controlled-release applications.