Comprehensive Peptide Cyclization Examination Yields Optimized APP Scaffolds with Improved Affinity toward Mint2.

Peptides targeting disease-relevant protein-protein interactions are an attractive class of therapeutics covering the otherwise undruggable space between small molecules and therapeutic proteins. However, peptides generally suffer from poor metabolic stability and low membrane permeability. Hence, peptide cyclization has become a valuable approach to develop linear peptide motifs into metabolically stable and potentially cell-permeable cyclic leads. Furthermore, cyclization of side chains, also known as "stapling", can stabilize particular secondary peptide structures. Here, we demonstrate that a comprehensive examination of cyclization strategies in terms of position, chemistry, and length is a prerequisite for the selection of optimal cyclic peptide scaffolds. Our systematic approach identifies cyclic APP dodecamer peptides targeting the phosphotyrosine binding domain of Mint2 with substantially improved affinity. We show that especially all-hydrocarbon stapling provides improved metabolic stability, a significantly stabilized secondary structure and membrane permeability.

Integrated and High-Throughput Approach for Sensitive Analysis of Tyrosine Phosphoproteome.

Tyrosine phosphorylation (pTyr) regulates various signaling pathways under normal and cancerous states. Due to their low abundance and transient and dynamic natures, systematic profiling of pTyr sites is challenging. Antibody and engineered binding domain-based approaches have been well applied to pTyr peptide enrichment. However, traditional methods have the disadvantage of a long sample preparation process, which makes them unsuitable for processing limited amount of samples, especially in a high-throughput manner. In this study we developed a 96-well microplate-based approach to integrate all the sample preparation steps starting from cell culture to MS-compatible pTyr peptide enrichment in three consecutive 96-well microplates. By assembling an engineered SH2 domain onto a microplate, nonspecific adsorption of phosphopeptides is greatly reduced, which allows us to remove the Ti-IMAC purification and three C18 desalting steps (after digestion, pTyr enrichment, and Ti-IMAC purification) and, therefore, greatly simplifies the entire pTyr peptide enrichment workflow, especially when processing a large number of samples. Starting with 96-well microplate-cultured, pervanadate-stimulated cells, our approach could enrich 21% more pTyr sites than the traditional serial pTyr enrichment approach and showed good sensitivity and reproducibility in the range of 200 ng to 200 µg peptides. Importantly, we applied this approach to profile tyrosine kinase inhibitor-mediated EGFR signaling pathway and could well differentiate the distinct response of different pTyr sites. Collectively, the integrated 96-well microplate-based approach is valuable for profiling pTyr sites from limited biological samples and in a high-throughput manner.

A Formylglycine-Peptide for the Site-Directed Identification of Phosphotyrosine-Mimetic Fragments.

Discovery of protein-binding fragments for precisely defined binding sites is an unmet challenge to date. Herein, formylglycine is investigated as a molecular probe for the sensitive detection of fragments binding to a spatially defined protein site . Formylglycine peptide 3 was derived from a phosphotyrosine-containing peptide substrate of protein tyrosine phosphatase PTP1B by replacing the phosphorylated amino acid with the reactive electrophile. Fragment ligation with formylglycine occurred in situ in aqueous physiological buffer. Structures and kinetics were validated by NMR spectroscopy. Screening and hit validation revealed fluorinated and non-fluorinated hit fragments being able to replace the native phosphotyrosine residue. The formylglycine probe identified low-affinity fragments with high spatial resolution as substantiated by molecular modelling. The best fragment hit, 4-amino-phenyl-acetic acid, was converted into a cellularly active, nanomolar inhibitor of the protein tyrosine phosphatase SHP2.

Blockade of phosphotyrosine pathways suggesting SH2 superbinder as a novel therapy for pulmonary fibrosis.

Background: Idiopathic pulmonary fibrosis (IPF) is a progressive and irreversible fibrotic disease with high mortality. Currently, pirfenidone and nintedanib are the only approved drugs for IPF by the U.S. Food and Drug Administration (FDA), but their efficacy is limited. The activation of multiple phosphotyrosine (pY) mediated signaling pathways underlying the pathological mechanism of IPF has been explored. A Src homology-2 (SH2) superbinder, which contains mutations of three amino acids (AAs) of natural SH2 domain has been shown to be able to block phosphotyrosine (pY) pathway. Therefore, we aimed to introduce SH2 superbinder into the treatment of IPF. Methods: We analyzed the database of IPF patients and examined pY levels in lung tissues from IPF patients. In primary lung fibroblasts obtained from IPF patient as well as bleomycin (BLM) treated mice, the cell proliferation, migration and differentiation associated with pY were investigated and the anti-fibrotic effect of SH2 superbinder was also tested. In vivo, we further verified the safety and effectiveness of SH2 superbinder in multiple BLM mice models. We also compared the anti-fibrotic effect and side-effect of SH2 superbinder and nintedanib in vivo. Results: The data showed that the cytokines and growth factors pathways which directly correlated to pY levels were significantly enriched in IPF. High pY levels were found to induce abnormal proliferation, migration and differentiation of lung fibroblasts. SH2 superbinder blocked pY-mediated signaling pathways and suppress pulmonary fibrosis by targeting high pY levels in fibroblasts. SH2 superbinder had better therapeutic effect and less side-effect compare to nintedanib in vivo. Conclusions: SH2 superbinder had significant anti-fibrotic effects both in vitro and in vivo, which could be used as a promising therapy for IPF.

Comprehensive binary interaction mapping of τ phosphotyrosine sites with SH2 domains in the human genome: Implications for the rational design of self-inhibitory phosphopeptides to target τ hyperphosphorylation signaling in Alzheimer's Disease.

Human microtubule-associated protein Tau (τ) is abundant in the axons of neurons where it stabilizes microtubule bundles; abnormally hyperphosphorylated τ is a hallmark of Alzheimer's disease (AD) and related tauopathies. The hyperphosphorylation events can be recognized by phosphotyrosine-recognition domain SH2 (Src homology 2) to elicit downstream τ signaling in AD pathology. In this study, a comprehensive binary interaction map (CBIM) of all the 6 τ phosphotyrosine sites with 120 SH2 domains in the human genome was systematically created at structural level using computational analyses and binding assays, from which we were able to identify those of strong and moderate binding pairs of sites to domains. It is found that the SH2-recognition specificity of different τ phosphotyrosine sites has been evolutionally optimized to become roughly orthogonal to each other, and thus these site phosphorylations would regulate different but probably partially overlapped biological functions in τ signaling. Some SH2 groups such as SRC, RIN, PLCG, SOCS and SH2D were revealed to have effective binding potency as compared to others; they could be regarded as potential τ-associated proteins to transduce the downstream signaling. We further determined the systematic binding affinities of 6 τ-phosphopeptides to the 11 SH2 domains in SRC group, from which the FYN-τ18 and YES-τ29 pairs were identified as strong binders. Subsequently, rational molecular design was performed on τ18 and τ29 to derive a number of τ-phosphopeptide mutants with increased affinity; they are self-inhibitory candidates to competitively target τ hyperphosphorylation events in AD. In addition, it is revealed that the primary anchor pY0 and secondary anchor X+3 of τ-phosphopeptides play an important role in SRC-group SH2 recognition, which confer stability and specificity to the SH2-phosphopeptide binding, respectively.

Engineered SH2 Domains for Targeted Phosphoproteomics.

A comprehensive analysis of the phosphoproteome is essential for understanding molecular mechanisms of human diseases. However, current tools used to enrich phosphotyrosine (pTyr) are limited in their applicability and scope. Here, we engineered new superbinder Src-Homology 2 (SH2) domains that enrich diverse sets of pTyr-peptides. We used phage display to select a Fes-SH2 domain variant (superFes; sFes1) with high affinity for pTyr and solved its structure bound to a pTyr-peptide. We performed systematic structure-function analyses of the superbinding mechanisms of sFes1 and superSrc-SH2 (sSrc1), another SH2 superbinder. We grafted the superbinder motifs from sFes1 and sSrc1 into 17 additional SH2 domains and confirmed increased binding affinity for specific pTyr-peptides. Using mass spectrometry (MS), we demonstrated that SH2 superbinders have distinct specificity profiles and superior capabilities to enrich pTyr-peptides. Finally, using combinations of SH2 superbinders as affinity purification (AP) tools we showed that unique subsets of pTyr-peptides can be enriched with unparalleled depth and coverage.

Pentafluorophosphato-Phenylalanines: Amphiphilic Phosphotyrosine Mimetics Displaying Fluorine-Specific Protein Interactions.

Phosphotyrosine residues are essential functional switches in health and disease. Thus, phosphotyrosine biomimetics are crucial for the development of chemical tools and drug molecules. We report here the discovery and investigation of pentafluorophosphato amino acids as novel phosphotyrosine biomimetics. A mild acidic pentafluorination protocol was developed and two PF5 -amino acids were prepared and employed in peptide synthesis. Their structures, reactivities, and fluorine-specific interactions were studied by NMR and IR spectroscopy, X-ray diffraction, and in bioactivity assays. The mono-anionic PF5 motif displayed an amphiphilic character binding to hydrophobic surfaces, to water molecules, and to protein-binding sites, exploiting charge and H-F-bonding interactions. The novel motifs bind 25- to 30-fold stronger to the phosphotyrosine binding site of the protein tyrosine phosphatase PTP1B than the best current biomimetics, as rationalized by computational methods, including molecular dynamics simulations.

A Tyrosine Phosphoproteome Analysis Approach Enabled by Selective Dephosphorylation with Protein Tyrosine Phosphatase.

Protein tyrosine phosphorylation (pTyr) plays a prominent role in signal transduction and regulation in all eukaryotic cells. In conventional immunoaffinity purification (IP) methods, phosphotyrosine peptides are isolated from the digest of cellular protein extracts with a phosphotyrosine-specific antibody and are identified by tandem mass spectrometry. However, low sensitivity, poor reproducibility, and high cost are universal concerns for IP approaches. In this study, we presented an antibody-free approach to identify phosphotyrosine peptides by using protein tyrosine phosphatase (PTP). It was found that most of the PTPs including PTP1B, TCPTP, and SHP1 can efficiently and selectively dephosphorylate phosphotyrosine peptides. We then designed a workflow by combining two Ti4+-IMAC-based phosphopeptide enrichment steps with PTP-catalyzed dephosphorylation for tyrosine phosphoproteomics analysis. This workflow was first validated by selective detection of phosphotyrosine peptides from semicomplex samples and then applied to analyze the tyrosine phosphoproteome of Jurkat T cells. Around 1000 putative former phosphotyrosine peptides were identified from less than 500 µg of cell lysate. The tyrosine phosphosites on the majority of these peptides could be unambiguously determined for over 70% of them possessing only one tyrosine residue. It was also found that the tyrosine sites identified by this method were highly complementary to those identified by the SH2 superbinder-based method. Therefore, the combination of Ti4+-IMAC enrichment with PTP dephosphorylation provides an alternative and cost-effective approach for tyrosine phosphoproteomics analysis.

Peptide probes containing a non-hydrolyzable phosphotyrosine-mimetic residue for enrichment of protein tyrosine phosphatases.

We developed peptide probes containing a non-hydrolyzable phosphotyrosine mimetic, 4-[difluoro(phosphono)methyl]-L-phenylalanine (F2 Pmp) for the enrichment of protein tyrosine phosphatases (PTPs). We found that different F2 Pmp probes can enrich different PTPs, depending on the probe sequence. Furthermore, proteins containing a Src homology 2 (SH2) domain were enriched together. Importantly, probes containing phosphotyrosine instead of F2 Pmp failed to enrich PTPs due to dephosphorylation during the pulldown step. This enrichment approach using peptides containing F2 Pmp could be a generic tool for tyrosine phosphatome analysis without the use of antibodies.

Enzymatically Forming Intranuclear Peptide Assemblies for Selectively Killing Human Induced Pluripotent Stem Cells.

Tumorigenic risk of undifferentiated human induced pluripotent stem cells (iPSCs), being a major obstacle for clinical application of iPSCs, requires novel approaches for selectively eliminating undifferentiated iPSCs. Here, we show that an l-phosphopentapeptide, upon the dephosphorylation catalyzed by alkaline phosphatase (ALP) overexpressed by iPSCs, rapidly forms intranuclear peptide assemblies made of α-helices to selectively kill iPSCs. The phosphopentapeptide, consisting of four l-leucine residues and a C-terminal l-phosphotyrosine, self-assembles to form micelles/nanoparticles, which transform into peptide nanofibers/nanoribbons after enzymatic dephosphorylation removes the phosphate group from the l-phosphotyrosine. The concentration of ALP and incubation time dictates the morphology of the peptide assemblies. Circular dichroism and FTIR indicate that the l-pentapeptide in the assemblies contains a mixture of an α-helix and aggregated strands. Incubating the l-phosphopentapeptide with human iPSCs results in rapid killing of the iPSCs (=<2 h) due to the significant accumulation of the peptide assemblies in the nuclei of iPSCs. The phosphopentapeptide is innocuous to normal cells (e.g., HEK293 and hematopoietic progenitor cell (HPC)) because normal cells hardly overexpress ALP. Inhibiting ALP, mutating the l-phosphotyrosine from the C-terminal to the middle of the phosphopentapeptides, or replacing l-leucine to d-leucine in the phosphopentapeptide abolishes the intranuclear assemblies of the pentapeptides. Treating the l-phosphopentapeptide with cell lysate of normal cells (e.g., HS-5) confirms the proteolysis of the l-pentapeptide. This work, as the first case of intranuclear assemblies of peptides, not only illustrates the application of enzymatic noncovalent synthesis for selectively targeting nuclei of cells but also may lead to a new way to eliminate other pathological cells that express a high level of certain enzymes.

Spatiotemporal Control over Chemical Assembly in Living Cells by Integration of Acid-Catalyzed Hydrolysis and Enzymatic Reactions.

Spatiotemporal control of chemical assembly in living cells remains challenging. We have now developed an efficient and general platform to precisely control the formation of assemblies in living cells. We introduced an O-[bis(dimethylamino)phosphono]tyrosine protection strategy in the self-assembly motif as the Trojan horse, whereby the programmed precursors resist hydrolysis by phosphatases on and inside cells because the unmasking of the enzymatic cleavage site occurs selectively in the acidic environment of lysosomes. After demonstrating the multistage self-assembly processes in vitro by liquid chromatography/mass spectrometry (LC-MS), cryogenic electron microscopy (Cryo-EM), and circular dichroism (CD), we investigated the formation of site-specific self-assembly in living cells using confocal laser scanning microscopy (CLSM), LC-MS, and biological electron microscopy (Bio-EM). Controlling chemical assembly in living systems spatiotemporally may have applications in supramolecular chemistry, materials science, synthetic biology, and chemical biology.

Resurgence of phosphotyrosine binding domains: Structural and functional properties essential for understanding disease pathogenesis.

BACKGROUND: Phosphotyrosine Binding (PTB) Domains, usually found on scaffold proteins, are pervasive in many cellular signaling pathways. These domains are the second-largest family of phosphotyrosine recognition domains and since their initial discovery, dozens of PTB domains have been structurally determined. SCOPE OF REVIEW: Due to its signature sequence flexibility, PTB domains can bind to a large variety of ligands including phospholipids. PTB peptide binding is divided into classical binding (canonical NPXY motifs) and non-classical binding (all other motifs). The first atypical PTB domain was discovered in cerebral cavernous malformation 2 (CCM2) protein, while only one third in size of the typical PTB domain, it remains functionally equivalent. MAJOR CONCLUSIONS: PTB domains are involved in numerous signaling processes including embryogenesis, neurogenesis, and angiogenesis, while dysfunction is linked to major disorders including diabetes, hypercholesterolemia, Alzheimer's disease, and strokes. PTB domains may also be essential in infectious processes, currently responsible for the global pandemic in which viral cellular entry is suspected to be mediated through PTB and NPXY interactions. GENERAL SIGNIFICANCE: We summarize the structural and functional updates in the PTB domain over the last 20 years in hopes of resurging interest and further analyzing the importance of this versatile domain.

The intramolecular allostery of GRB2 governing its interaction with SOS1 is modulated by phosphotyrosine ligands.

Growth factor receptor-bound protein 2 (GRB2) is a trivalent adaptor protein and a key element in signal transduction. It interacts via its flanking nSH3 and cSH3 domains with the proline-rich domain (PRD) of the RAS activator SOS1 and via its central SH2 domain with phosphorylated tyrosine residues of receptor tyrosine kinases (RTKs; e.g. HER2). The elucidation of structural organization and mechanistic insights into GRB2 interactions, however, remain challenging due to their inherent flexibility. This study represents an important advance in our mechanistic understanding of how GRB2 links RTKs to SOS1. Accordingly, it can be proposed that (1) HER2 pYP-bound SH2 potentiates GRB2 SH3 domain interactions with SOS1 (an allosteric mechanism); (2) the SH2 domain blocks cSH3, enabling nSH3 to bind SOS1 first before cSH3 follows (an avidity-based mechanism); and (3) the allosteric behavior of cSH3 to other domains appears to be unidirectional, although there is an allosteric effect between the SH2 and SH3 domains.

Late Stage Phosphotyrosine Mimetic Functionalization of Peptides Employing Metallaphotoredox Catalysis.

Access to phosphotyrosine (pTyr) mimetics requires multistep syntheses, and therefore late stage incorporation of these mimetics into peptides is not feasible. Here, we develop and employ metallaphotoredox catalysis using 4-halogenated phenylalanine to afford a variety of protected pTyr mimetics in one step. This methodology was shown to be tolerant of common protecting groups and applicable to the late stage pTyr mimetic modification of protected and unprotected peptides, and peptides of biological relevance.

Performance of phospho-L-tyrosine immobilized onto alginate/polyacrylamide-based cryogels: Effect of ligand coupling on human IgG adsorption and Fab fragments separation.

The ortho-phospho-tyrosine (P-Tyr) pseudoaffinity ligand was immobilized via ether linkage onto polyacrylamide-alginate (PAAm-Alg)-epoxy cryogels prepared according to two different approaches in order to explore their performance in the immunoglobulin G (IgG) purification from human serum. In the first approach, the P-Tyr was attached to cryogel prepared by cryocopolymerization of acrylamide and alginate with allyl glycidyl ether (AGE) as functional comonomer, and methylenebisacrylamide and Ca(II) as crosslinkers, obtaining the PAAm-Alg-AGE-P-Tyr. In the second approach, the PAAm-Alg was synthesized under the same conditions, but without AGE, and the P-Tyr was coupled to epichlorohydrin (ECH)-activated cryogel, obtaining the PAAm-Alg-ECH-P-Tyr. Both pseudoaffinity cryogels were characterized by scanning electron microscopy, swelling tests, porosity, ligand density, and flow dynamics. The human IgG differently interacted with the PAAm-Alg-ECH-P-Tyr and PAAm-Alg-AGE-P-Tyr cryogels, depending on the pH and adsorption buffer system used. The selectivity analyzed by electrophoretic profiles was similar for both cryogels, but PAAm-Alg-ECH-P-Tyr achieved higher IgG adsorption capacity (dynamic capacity of 12.62 mg of IgG/mL of cryogel). The IgG purity assayed by ELISA was 95%. The maximum IgG adsorption capacity and dissociation constant of the PAAm-Alg-ECH-P-Tyr, determined by Langmuir isotherm, were found to be 91.75 mg IgG/g of dry cryogel and 4.60 × 10-6 mol/L at pH 6.0 from aqueous solutions. The PAAm-Alg-AGE-P-Tyr showed potential to purify the Fab fragments from papain-digested human IgG solution at pH 7.0. Fab fragments were separated from Fc fragments (but with uncleaved IgG) in eluted fractions (analyzed by the Western blot technique), with yield of 82% and purity of 95% (determined by radial immunodiffusion).

Revisiting the phosphotyrosine binding pocket of Fyn SH2 domain led to the identification of novel SH2 superbinders.

Protein engineering through directed evolution is an effective way to obtain proteins with novel functions with the potential applications as tools for diagnosis or therapeutics. Many natural proteins have undergone directed evolution in vitro in the test tubes in the laboratories worldwide, resulting in the numerous protein variants with novel or enhanced functions. we constructed here an SH2 variant library by randomizing 8 variable residues in its phosphotyrosine (pTyr) binding pocket. Selection of this library by a pTyr peptide led to the identification of SH2 variants with enhanced affinities measured by EC50. Fluorescent polarization was then applied to quantify the binding affinities of the newly identified SH2 variants. As a result, three SH2 variants, named V3, V13 and V24, have comparable binding affinities with the previously identified SH2 triple-mutant superbinder. Biolayer Interferometry assay was employed to disclose the kinetics of the binding of these SH2 superbinders to the phosphotyrosine peptide. The results indicated that all the SH2 superbinders have two-orders increase of the dissociation rate when binding the pTyr peptide while there was no significant change in their associate rates. Intriguingly, though binding the pTyr peptide with comparable affinity with other SH2 superbinders, the V3 does not bind to the sTyr peptide. However, variant V13 and V24 have cross-reactivity with both pTyr and sTyr peptides. The newly identified superbinders could be utilized as tools for the identification of pTyr-containing proteins from tissues under different physiological or pathophysiological conditions and may have the potential in the therapeutics.

SH2 Domain Binding: Diverse FLVRs of Partnership.

The Src homology 2 (SH2) domain has a special role as one of the cornerstone examples of a "modular" domain. The interactions of this domain are very well-conserved, and have long been described as a bidentate, or "two-pronged plug" interaction between the domain and a phosphotyrosine (pTyr) peptide. Recent work has, however, highlighted unusual features of the SH2 domain that illustrate a greater diversity than was previously appreciated. In this review we discuss some of the novel and unusual characteristics across the SH2 family, including unusual peptide binding pockets, multiple pTyr recognition sites, recognition sites for unphosphorylated peptides, and recently identified variability in the conserved FLVR motif.

Targeting Phosphotyrosine in Native Proteins with Conditional, Bispecific Antibody Traps.

Engineering sequence-specific antibodies (Abs) against phosphotyrosine (pY) motifs embedded in folded polypeptides remains highly challenging because of the stringent requirement for simultaneous recognition of the pY motif and the surrounding folded protein epitope. Here, we present a method named phosphotyrosine Targeting by Recombinant Ab Pair, or pY-TRAP, for in vitro engineering of binders for native pY proteins. Specifically, we create the pY protein by unnatural amino acid misincorporation, mutagenize a universal pY-binding Ab to create a first binder B1 for the pY motif on the pY protein, and then select against the B1-pY protein complex for a second binder B2 that recognizes the composite epitope of B1 and the pY-containing protein complex. We applied pY-TRAP to create highly specific binders to folded Ub-pY59, a rarely studied Ub phosphoform exclusively observed in cancerous tissues, and ZAP70-pY248, a kinase phosphoform regulated in feedback signaling pathways in T cells. The pY-TRAPs do not have detectable binding to wild-type proteins or to other pY peptides or proteins tested. This pY-TRAP approach serves as a generalizable method for engineering sequence-specific Ab binders to native pY proteins.

Preparation of a phosphotyrosine-protein standard for use in semiquantitative western blotting with enhanced chemiluminescence.

Protein tyrosine phosphorylation is key to activation of receptor tyrosine kinases (RTK) that drive development of some cancers. One challenge of RTK-targeted therapy is identification of those tumors that express non-mutated but activated RTKs. Phosphotyrosine (pTyr) RTK levels should be more predictive of the latter than expressed total protein. Western blotting (WB) with a pTyr antibody and enhanced chemiluminescence (ECL) detection is sufficiently sensitive to detect pTyr-RTKs in human tumor homogenates. Presentation of results by comparing WB images, however, is wanting. Here we describe the preparation of a new pTyr-protein standard, pTyr-ALK48-SB (pA), derived from a commercial anaplastic lymphoma kinase (ALK) recombinant fragment, and its use to quantify pTyr-epidermal growth factor receptor (pTyr-EGFR) in commercial A431 cell lysates. Linearity of one-dimensional (1D) WB plots of pA band density versus load as well as its lower level of detection (0.1 ng, 2 fmole) were determined for standardized conditions. Adding pA to two lots of A431 cell lysates with high and low pTyr-EGFR allowed normalization and quantification of the latter by expressing results as density ratios for both 1D and 2D WB. This approach is semi-quantitative because unknown RTKs may be outside the linear range of detection. Semiquantitative ratios are an improvement over comparisons of images without a reference standard and facilitate comparisons between samples.

Development and In Vitro Evaluation of Stearic Acid Phosphotyrosine Amide as New Excipient for Zeta Potential Changing Self-Emulsifying Drug Delivery Systems.

PURPOSE: Development of zeta potential changing SEDDS containing newly synthesized derivative stearic acid phosphotyrosine amide. METHODS: Stearoyl chloride was conjugated with phosphotyrosine, which is substrate for the brush border enzyme intestinal alkaline phosphate. The synthesized derivative was implemented in different SEDDS formulations and the zeta potential changing properties and the concluding mucus diffusion abilities were evaluated. RESULTS: Stearic acid phosphotyrosine amide was successfully synthesized and incorporated into SEDDS. A SEDDS formulation containing the new derivative showed a zeta potential of -14 mV before, and + 2 mV after enzymatic cleavage by intestinal alkaline phosphatase. Experiments on a Caco-2 monolayer demonstrated that the phosphate cannot only be cleaved by isolated enzyme, but also by enzyme, which was expressed by cells. The mucus diffusion abilities of the untreated, negatively charged SEDDS were significantly higher compared to the enzymatically cleaved, positively charged SEDDS. CONCLUSION: The developed stearic acid phosphotyrosine represents a promising excipient for zeta potential changing SEDDS. Graphical Abstract.