We have developed a novel pH-sensitive linker based on a phosphoramidate scaffold that can be tuned to release amine-containing drug molecules at various pH values. The pH-triggered phosphoramidate-based linkers are responsive to pH alone and do not require intracellular enzymatic action to initiate drug release. Key to the pH-triggered amine release from these linkers is a proximal acidic group (e.g., pyridinium or carboxylic acid) to promote the hydrolysis of the phosphoramidate P-N bond, presumably through an intramolecular general-acid type mechanism. Phosphoramidate hydrolysis is largely governed by the pKa of the leaving amine (e.g., primary, secondary, aniline). However, the proximity of the neighboring pyridine group attenuates the stability of the P-N bond to hydrolysis, thus allowing for control over the release of an amine from the phosphoramidate center. Based on the model scaffolds examined, phosphoramidate-based linkers could be selected for particular properties for controlled-release applications such as amine type, stability under physiological conditions, or release rates at various pH values such as intracellular endosomal conditions. The tunability of the phosphoramidate scaffold is expected to find broad applicability in various controlled drug-release applications such as antibody or small-molecule drug conjugates, drug-eluting stents, prodrug activation, as well as intracellular trafficking studies in which pH changes can trigger the release of turn-on dyes.

We developed a second generation of tunable pH-sensitive linkers based on our phosphoramidate scaffold to release amine-containing drugs under acidic conditions. The pH-triggered phosphoramidate-based linkers are responsive to pH and do not require intracellular enzymatic action to initiate drug release. On the basis of the model scaffolds examined, phosphoramidate-based linkers were selected for particular properties for controlled release applications such as amine type, stability under physiological conditions, or release rates at various pH values such as intracellular endosomal conditions. Key to the pH-triggered amine release from these linker is a proximal carboxylic acid to promote hydrolysis of the phosphoramidate P-N bond, presumably through an intramolecular general acid-type mechanism. Phosphoramidate hydrolysis is largely governed by the pKa of the leaving amine. However, the proximity of the neighboring carboxylic acid attenuates the stability of the P-N bond to hydrolysis, thus allowing for control over the release of an amine from the phosphoramidate center. In addition, we observed that the Thorpe-Ingold effect and rigidification of the scaffold could further enhance the rate of release. Esterification of the neighboring carboxylic acid was found to protect the scaffold from rapid release at low pH. This latter observation is particularly noteworthy as it suggests that the phosphoramidate-based drug-conjugate scaffold can be protected as an ester prodrug for oral administration. While the tunability phosphoramidate linkers is attractive for applications in intracellular trafficking studies in which pH changes can trigger release of turn-on dyes, antibody drug conjugates, small-molecule drug conjugates, and drug eluting stents (DES), the promise of oral delivery of drug conjugates is expected to have broad impact in controlled release applications.

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Using a linearly swept chirp function to modulate a Bradbury-Nielsen (BN) ion gate and application of a common signal processing technique (cross-correlation), we outline a method for obtaining high resolution IMS-MS spectra with ion gate duty cycles approaching 50%. Correlation IMS (CIMS) offers advantages over current multiplexing approaches in IMS-MS, which include the Hadamard and Fourier transforms, by minimizing transform artifacts while maintaining high ion throughput. Although cross-correlation techniques have been utilized previously in the field of IMS, to the best of our knowledge, this approach has not been utilized to obtain spectrum that resembles traditional IMS spectrum with resolving powers approaching the theoretical limit. This new approach relies on a linear sweep, which is a swept frequency signal, commonly utilized in different applications because of its compatibility with the fast Fourier transform (FFT). However, unlike spectra derived from Fourier transformation, CIMS yields data sampling rates that are not dependent upon terminal frequency and takes advantage of several factors unique to IMS operation; the non-linear response of ions at relatively low gate pulse widths, fluctuations in intensity, and peak profiles resembling the input gate pulse vector observed especially noted at low gating frequencies.