Design and synthesis of TH19P01-Camptothecin based hybrid peptides inducing effective anticancer responses on sortilin positive cancer cells.

Recently, the sortilin receptor (SORT1) was found to be preferentially over-expressed on the surface of many cancer cells, which makes SORT1 a novel anticancer target. The SORT1 binding proprietary peptide TH19P01 could achieve the SORT1-mediated cancer cell binding and subsequent internalization. Inspired by the peptide-drug conjugate (PDC) strategy, the TH19P01-camptothecin (CPT) conjugates were designed, efficiently synthesized, and evaluated for their anticancer potential in this study. The water solubility, in vitro anticancer activity, time-kill kinetics, cellular uptake, anti-migration activity, and hemolysis effects were systematically estimated. Besides, in order to monitor the release of CPT from conjugates in real-time, the CPT/Dnp-based "turn on" hybrid peptide was designed, which indicted that CPT could be sustainably released from the hybrid peptide in both human serum and cancer cellular environments. Strikingly, compared with free CPT, the water solubility, cellular uptake, and selectivity towards cancer cells of hybrid peptide LYJ-2 have all been significantly enhanced. Moreover, unlike free CPT or TH19P01, LYJ-2 exhibited selective anti-proliferative and anti-migration effects against SORT1-positive MDA-MB-231 cells. Collectively, this study not only established efficient strategies to improve the solubility and anticancer potential of chemotherapeutic agent CPT, but also provided important references for the future development of TH19P01 based PDCs targeting SORT1.

Approaches to the Full and Partial Chemical Synthesis of Proteins.

Histones are proteins which help to organize DNA. The way in which they function is complex and is partially controlled by post-translational modifications (PTMs). Histone proteins from numerous organisms can be recombinantly produced in bacteria, but many bacterial strains are incapable of installing the variety of PTMs that histones possess. An alternative method of producing histones, which can be used to introduce PTMs, is native chemical ligation (NCL). This chapter provides a general NCL protocol which can be used to produce synthetic, post-translationally modified, histone proteins. The focus is on the NCL procedure itself and not on producing the modified histone protein fragments as there are many different ways in which these can be synthesized, depending on the modification(s) required. The same NCL protocol is also applicable for expressed protein ligation (EPL) with only small modifications to the purification procedure potentially required.

Synthesis and Semi-Synthesis of Alpha-Synuclein: Insight into the Chemical Complexity of Synucleinopathies.

The chemical rules governing protein folding have intrigued generations of researchers for decades. With the advent of artificial intelligence (AI), prediction of protein structure has improved tremendously. However, there is still a level of analysis that is only possible through wet laboratory experiments, especially in respect to the investigation of the pathological effect of mutations and posttranslational modifications (PTMs) on proteins of interest. This requires the availability of pure peptides and proteins in sufficient quantities for biophysical, biochemical, and functional studies. In this context, chemical protein synthesis and semi-synthesis are powerful tools in protein research, which help to enlighten the role of protein modification in the physiology and pathology of proteins. A protein of high interest in the field of biomedicine is alpha-synuclein (aSyn), a protein deeply associated with several devastating neurodegenerative disorders such as Parkinson's disease (PD), dementia with Lewy bodies (DLB), or multiple systems atrophy (MSA). Here, we describe several methods and pathways to synthesize native or modified aSyn, and discuss how these approaches enable us to address pathological mechanisms that may open novel perspectives for therapeutic intervention.

Synthesis and Biological Studies of New Temporin A Analogs Containing Unnatural Amino Acids in Position 7.

(1) Background: Antimicrobial resistance is growing at an extreme pace and has proven to be an urgent topic, for research into alternative treatments. Such a prospective possibility is hidden in antimicrobial peptides because of their low to no toxicity, effectiveness at low concentrations, and most importantly their ability to be used for multiple treatments. This work was focused on the study of the effect of the modification in position 7 of Temporin A on its biological activity; (2) Methods: The targeted peptides were synthesized using Fmoc/Ot-Bu SPPS. The antibacterial activity of the analogs was determined using the broth microdilution method and disk-diffusion method. In vitro tests were performed to determine the cytotoxicity, phototoxicity, and antiproliferative activity of the peptide analogs on a panel of tumor and normal cell lines; (3) Results: All analogs except DTCit showed good antibacterial activity, with DTDab having the best activity according to the disk-diffusion method. However, DTCit had an acceptable cytotoxicity, combined with good selectivity against the test MCF-7 cell line; (4) Conclusions: The obtained results revealed the importance of the basicity and length of the side chain at position 7 in the Temporin A sequence for both tested activities.

Synthesis and anticancer properties of novel dolastatin 10 analogs featuring five-membered heterocyclic rings with a linkable group at the C-terminus.

Dolastatin 10 (Dol-10), a natural marine-source pentapeptide, is a powerful antimitotic agent regarded as one of the most potent anticancer compounds found to date. Dol-10 however, lacks chemical conjugation capabilities, which restricts the feasibility of its application in targeted drug therapy. This limitation has spurred the prospect that chemical structure of the parent molecule might allow conjugation of the derivatives to drug carriers such as antibodies. By first employing docking studies, we designed and prepared a series of novel Dol-10 analogs with a modified C-terminus, preserving high potency of the parent compound while enhancing conjugation capability. The modifications involved the introduction of a methyleneamine functionality at position 4 of the 1,3-thiazole ring, along with the substitution of the thiazole ring with a 1,2,3-triazole moiety, furnished with methylenehydroxy, carboxy, methyleneamine, and N(Me)-methyleneamine tethering functionalities at position 4. Among the synthesized pentapeptides, DA-1 exhibited the highest potency in prostate cancer (PC-3) cells, eliciting apoptosis (IC50 0.2 ± 0.1 nm) and cell cycle arrest at the mitotic stage after at least 6 days of culture. This delayed response suggests the accumulation of cellular stress or significant physiological alterations that profoundly impact the cell cycle. We believe that these novel Dol-10 derivates represent a new and straightforward route for the development of C-terminus modified Dol-10-based microtubule inhibitors, thereby advancing targeted anticancer therapy.

1,3,5-Triazine as Branching Connector for the Construction of Novel Antimicrobial Peptide Dendrimers: Synthesis and Biological Characterization.

Peptides displaying antimicrobial properties are being regarded as useful tools to evade and combat antimicrobial resistance, a major public health challenge. Here we have addressed dendrimers, attractive molecules in pharmaceutical innovation and development displaying broad biological activity. Triazine-based dendrimers were fully synthesized in the solid phase, and their antimicrobial activity and some insights into their mechanisms of action were explored. Triazine is present in a large number of compounds with highly diverse biological targets with broad biological activities and could be an excellent branching unit to accommodate peptides. Our results show that the novel peptide dendrimers synthesized have remarkable antimicrobial activity against Gram-negative bacteria (E. coli and P. aeruginosa) and suggest that they may be useful in neutralizing the effect of efflux machinery on resistance.

Synthesis and structural optimization of oncolytic peptide LTX-315.

Oncolytic peptides represented potential novel candidates for anticancer treatments especially drug-resistant cancer cell lines. One of the most promising and extensively studied is LTX-315, which is considered as the first in class oncolytic peptide and has entered phase I/II clinical trials. Nevertheless, the shortcomings including poor proteolytic stability, moderate anticancer durability and high synthesis costs may hinder the widespread clinical applications of LTX-315. In order to reduce the synthesis costs, as well as develop derivatives possessing both high protease-stability and durable anticancer efficiency, twenty LTX-315-based derived-peptides were designed and efficiently synthesized. Especially, through solid-phase S-alkylation, as well as the optimized peptide cleavage condition, the derived peptides could be prepared with drastically reduced synthesis cost. The in vitro anticancer efficiency, serum stability, anticancer durability, anti-migration activity, and hemolysis effect were systematically investigated. It was found that derived peptide MS-13 exhibited comparable anticancer efficiency and durability to those of LTX-315. Strikingly, the D-type peptide MS-20, which is the enantiomer of MS-13, was demonstrated to possess significantly high proteolytic stability and sustained anticancer durability. In general, the cost-effective synthesis and stability-guided structural optimizations were conducted on LTX-315, affording the highly hydrolysis resistant MS-20 which possessed durable anticancer activity. Meanwhile, this study also provided a reliable reference for the future optimization of anticancer peptides through the solid-phase S-alkylation and L-type to D-type amino acid substitutions.

An Introductory Guide to Protease Sensitive Linker Design Using Matrix Metalloproteinase 13 as an Example.

Proteases play a crucial role, not only in physiological, but also in pathological processes, such as cancer, inflammation, arthritis, Alzheimer's, and infections, to name but a few. Their ability to cleave peptides can be harnessed for a broad range of biotechnological purposes. To do this efficiently, it is essential to find an amino acid sequence that meets the necessary requirements, including interdependent factors like specificity, selectivity, cleavage kinetics, or synthetic accessibility. Cleavage sequences from natural substrates of the protease may not be optimal in terms of specificity and selectivity, which is why these frequently require arduous and sometimes unsuccessful optimization such as by iterative exchange of single amino acids. Hence, here we describe the systematic design of protease sensitive linkers (PSLs)─peptide sequences specifically cleaved by a target protease─guided by the mass spectrometry based determination of target protease specific cleavage sites from a proteome-based peptide library. It includes a procedure for identifying bespoke PSL sequences, their optimization, synthesis, and validation and introduces a program that can indicate potential cleavage sites by hundreds of enzymes in any arbitrary amino acid sequence. Thereby, we provide an introduction to PSL design, illustrated by the example of matrix metalloproteinase 13 (MMP13). This introduction can serve as a guide and help to greatly accelerate the development and use of protease-sensitive linkers in diverse applications.

Macromolecular tool box to elucidate CLAVATA3/Embryo Surrounding Region-Related-RLK binding, signaling and downstream effects.

Plant peptides communicate by binding to a large family of receptor-like kinases (RLKs) and they share a conserved binding mechanism, which may account for their promiscuous interaction with several RLKs. In order to understand the in vivo binding specificity of CLAVATA3/Embryo Surrounding Region-Related peptide family, we have developed a novel set of CLAVATA 3 (CLV3) based peptide tools. After carefully evaluating the CLE peptide binding characteristics, using solid phase synthesis process, we have modified the CLV3 peptide and attached a fluorophore and a photoactivable side group. We observed that the labeled CLV3 shows binding specificity within CLAVATA1 clade of RLKs while avoiding the distantly-related PEP RECEPTOR clade, thus resolving the contradictory results obtained previously by many in vitro methods. Furthermore, we observed that the RLK-bound CLV3 undergoes clathrin-mediated endocytosis and gets trafficked to vacuole via ARA7-labeled endosomes. Additionally, modifying CLV3 for light-controlled activation enabled spatial and temporal control over CLE signalling. Hence, our CLV3 macromolecular toolbox can be used to study rapid cell specific down-stream effects. Given the conserved binding properties, in the future our toolbox can also be used as a template to modify other CLE peptides.

Facile Peptide Macrocyclization and Multifunctionalization via Cyclen Installation.

Cyclen-peptide bioconjugates are usually prepared in multiple steps that require individual preparation and purification of the cyclic peptide and hydrophilic cyclen derivatives. An efficient strategy is discovered for peptide cyclization and functionalization toward lanthanide probe via three components intermolecular crosslinking on solid-phase peptide synthesis with high conversion yield. Multifunctionality can be conferred by introducing different modular parts or/and metal ions on the cyclen-embedded cyclopeptide. As a proof-of-concept, a luminescent Eu3+ complex and a Gd3+-based contrasting agent for in vitro optical imaging and in vivo magnetic resonance imaging, respectively, are demonstrated through utilizing this preparation of cyclen-embedded cyclic arginylglycylaspartic acid (RGD) peptide.

Chemical and Chemoenzymatic Synthesis of Peptide and Protein Therapeutics Conjugated with Human N-Glycans.

Glycosylation is one of the most ubiquitous post-translational modifications. It affects the structure and function of peptides/proteins and consequently has a significant impact on various biological events. However, the structural complexity and heterogeneity of glycopeptides/proteins caused by the diversity of glycan structures and glycosylation sites complicates the detailed elucidation of glycan function and hampers their clinical applications. To address these challenges, chemical and/or enzyme-assisted synthesis methods have been developed to realize glycopeptides/proteins with well-defined glycan morphologies. In particular, N-glycans are expected to be useful for improving the solubility, in vivo half-life and aggregation of bioactive peptides/proteins that have had limited clinical applications so far due to their short duration of action in the blood and unsuitable physicochemical properties. Chemical glycosylation performed in a post-synthetic procedure can be used to facilitate the development of glycopeptide/protein analogues or mimetics that are superior to the original molecules in terms of physicochemical and pharmacokinetic properties. N-glycans are used to modify targets because they are highly biodegradable and biocompatible and have structures that already exist in the human body. On the practical side, from a quality control perspective, close attention should be paid to their structural homogeneity when they are to be applied to pharmaceuticals.

d-type peptides based fluorescent probes for "turn on" sensing of heparin.

Developing "turn on" fluorescent probes was desirable for the detection of the effective anticoagulant agent heparin in clinical applications. Through combining the aggregation induced emission (AIE) fluorogen tetraphenylethene (TPE) and heparin specific binding peptide AG73, the promising "turn on" fluorescent probe TPE-1 has been developed. Nevertheless, although TPE-1 could achieve the sensitive and selective detection of heparin, the low proteolytic stability and undesirable poor solubility may limit its widespread applications. In this study, seven TPE-1 derived fluorescent probes were rationally designed, efficiently synthesized and evaluated. The stability and water solubility were systematically estimated. Especially, to achieve real-time monitoring of proteolytic stability, the novel Abz/Dnp-based "turn on" probes that employ the internally quenched fluorescent (IQF) mechanism were designed and synthesized. Moreover, the detection ability of synthetic fluorescent probes for heparin were systematically evaluated. Importantly, the performance of d-type peptide fluorescent probe XH-6 indicated that d-type amino acid substitutions could significantly improve the proteolytic stability without compromising its ability of heparin sensing, and attaching solubilizing tag 2-(2-aminoethoxy) ethoxy) acid (AEEA) could greatly enhance the solubility. Collectively, this study not only established practical strategies to improve both the water solubility and proteolytic stability of "turn on" fluorescent probes for heparin sensing, but also provided valuable references for the subsequent development of enzymatic hydrolysis-resistant d-type peptides based fluorescent probes.

Optimization of peptide synthesis time and sustainability using novel eco-friendly binary solvent systems with induction heating on an automated peptide synthesizer.

On December 12th, 2023, the European Commission took regulatory action to amend Annex XVII of REACH, imposing restrictions on the use of N,N-dimethylformamide (DMF) within the EU market owing to its high toxicity. Historically, DMF has been widely considered the gold standard for solid-phase peptide synthesis (SPPS). Being urgent to propose alternative solvents, we tested the suitability of non-hazardous neat and mixed solvents. Notably, binary solvent mixtures containing dimethyl sulfoxide as one of the solvent partners demonstrated high efficacy in solubilizing reagents while maintaining the desired swelling characteristics of common resins. A series of binary solvent mixtures were tested in automated SPPS, both at room temperature and high temperature, employing the PurePep® Chorus synthesizer, which enabled controlled induction heating between 25 and 90°C with oscillation mixing. The performances were assessed in challenging peptide sequences, i.e., ACP (65-74), and in longer and aggregating sequences like SARS-CoV-2 RBM (436-507) and ß-amyloid (1-42). Furthermore, as part of the proposed sustainable approach to minimize the utilization of hazardous solvents, we coupled the novel PurePep EasyClean catch-and-release purification technology. This work, addressing regulatory compliance, emphasizes the crucial role of green chemistry in advancing safer and more environmentally friendly practices in SPPS.

Reflections on a Copenhagen-Minneapolis Axis in Bioorganic Chemistry.

The international peptide community rejoiced when one of its most distinguished members, Morten Meldal of Denmark, shared the 2022 Nobel Prize in Chemistry. In fact, the regiospecific solid-phase "copper(I)-catalyzed 1,3-dipolar cycloaddition of terminal alkynes to azides" (CuACC) reaction-that formed the specific basis for Meldal's recognition-was reported first at the 17th American Peptide Symposium held in San Diego in June 2001. The present perspective outlines intertwining conceptual and experimental threads pursued concurrently in Copenhagen and Minneapolis, sometimes by the same individuals, that provided context for Meldal's breakthrough discovery. Major topics covered include orthogonality in chemistry; the dithiasuccinoyl (Dts) protecting group for amino groups in α-amino acids, carbohydrates, and monomers for peptide nucleic acids (PNA); and poly(ethylene glycol) (PEG)-based solid supports such as PEG-PS, PEGA, and CLEAR [and variations inspired by them] for solid-phase peptide synthesis (SPPS), solid-phase organic synthesis (SPOS), and combinatorial chemistry that can support biological assays in aqueous media.

Development of an oxazole-based cleavable linker for peptides.

We report the development of a new oxazole-based cleavable linker to release peptides from attached cargo. Oxazoles are stable to most reaction conditions, yet they can be rapidly cleaved in the presence of single-electron oxidants like cerium ammonium nitrate (CAN). An oxazole linker could be synthesized and attached to peptides through standard solid-phase peptide coupling reactions. Cleavage of these peptide-oxazole conjugates is demonstrated on a broad scope of peptides containing various natural and unnatural amino acids. These results represent the first example of a peptide-based linker that is cleaved through single-electron oxidation. The oxazole is also demonstrated to be a suitable linker for both the release of a peptide from a conjugated small molecule and the orthogonal release of cargo from a peptide containing multiple cleavable linkers. Oxazole linkers could serve as a promising tool for peptide screening platforms such as peptide-encoded libraries.

Hydrocarbon stapled temporin-L analogue as potential antibacterial and antiendotoxin agents with enhanced protease stability.

Antimicrobial resistance (AMR) is a serious global concern and a huge burden on the healthcare system. Antimicrobial peptides (AMPs) are considered as a solution of AMR due to their membrane-lytic and intracellular mode of action and therefore resistance development against AMPs is less frequent. One such AMPs, temporin-L (TL) is a 13-mer peptide reported as a potent and broad-spectrum antibacterial agent with significant immunomodulatory activity. However, TL is toxic to human erythrocytes at their antibacterial concentrations and therefore various analogues were synthesized with potent antimicrobial activity and lower hemolytic activity. In this work, we have selected a non-toxic engineered analogue of TL (eTL) and performed hydrocarbon stapling of amino acid residues at i to i + 4 positions at different part of sequence. The synthesized peptides were investigated against both the gram-positive and gram-negative bacteria as well as methicillin resistant S. aureus, its MIC was measured in the concentrations range of 0.9-15.2 µM. All analogues were found equal or better antibacterial as compared to parent peptide. Interestingly one analogue eTL [5-9] was found to be non-cytotoxic and stable in presence of the human serum. Mode of action studies revealed membrane depolarizing and disruptive mode of action with live MRSA. Further in vivo studies of antimicrobial against MRSA infection and anti-endotoxin activities in mice model revealed potential activity of the stapled peptide analogue. Overall, this reports on stapled analogue of the AMPs highlights an important strategy for the development of new antibacterial therapeutics against AMR.

An Overview of Peptides and Peptide Pools for Antigen-Specific Stimulation in T-Cell Assays.

The analysis of antigen-specific T-cell responses has become routine in many laboratories. Functional T-cell assays like enzyme-linked-immuno-spot (ELISPOT), which depend on antigen-specific stimulation, increasingly use peptides to represent the antigen of interest. Besides single peptides, mixtures of peptides (peptide pools) are very frequently applied. Such peptide pools may, for example, represent entire proteins (with overlapping peptides covering a protein sequence) or include noncontiguous peptides such as a collection of T-cell-stimulating peptides. The optimum specification of single peptides or peptide pools for T-cell stimulation assays will depend on the purpose of the test, the target T-cell population, the availability of sample, requirements regarding reproducibility, and, last but not least, the available budget, to mention only the most important factors. Because of the way peptides are produced, they will always contain certain amounts of impurities such as peptides with deletions or truncated peptides, and there may be additional by-products of peptide synthesis. Optimized synthesis protocols as well as purification help reduce impurities that might otherwise cause false-positive assay results. However, specific requirements with respect to purity will vary depending on the purpose of an assay. Finally, storage conditions significantly affect the shelf life of peptides, which is relevant especially for longitudinal studies. The present book chapter addresses all of these aspects in detail. It should provide the researcher with all necessary background knowledge for making the right decisions when it comes to choosing, using, and storing peptides for ELISPOT and other T-cell stimulation assays.

Natural carboxyterminal truncation of human CXCL10 attenuates glycosaminoglycan binding, CXCR3A signaling and lymphocyte chemotaxis, while retaining angiostatic activity.

BACKGROUND: Interferon-γ-inducible protein of 10 kDa (IP-10/CXCL10) is a dual-function CXC chemokine that coordinates chemotaxis of activated T cells and natural killer (NK) cells via interaction with its G protein-coupled receptor (GPCR), CXC chemokine receptor 3 (CXCR3). As a consequence of natural posttranslational modifications, human CXCL10 exhibits a high degree of structural and functional heterogeneity. However, the biological effect of natural posttranslational processing of CXCL10 at the carboxy (C)-terminus has remained partially elusive. We studied CXCL10(1-73), lacking the four endmost C-terminal amino acids, which was previously identified in supernatant of cultured human fibroblasts and keratinocytes. METHODS: Relative levels of CXCL10(1-73) and intact CXCL10(1-77) were determined in synovial fluids of patients with rheumatoid arthritis (RA) through tandem mass spectrometry. The production of CXCL10(1-73) was optimized through Fmoc-based solid phase peptide synthesis (SPPS) and a strategy to efficiently generate human CXCL10 proteoforms was introduced. CXCL10(1-73) was compared to intact CXCL10(1-77) using surface plasmon resonance for glycosaminoglycan (GAG) binding affinity, assays for cell migration, second messenger signaling downstream of CXCR3, and flow cytometry of CHO cells and primary human T lymphocytes and endothelial cells. Leukocyte recruitment in vivo upon intraperitoneal injection of CXCL10(1-73) was also evaluated. RESULTS: Natural CXCL10(1-73) was more abundantly present compared to intact CXCL10(1-77) in synovial fluids of patients with RA. CXCL10(1-73) had diminished affinity for GAG including heparin, heparan sulfate and chondroitin sulfate A. Moreover, CXCL10(1-73) exhibited an attenuated capacity to induce CXCR3A-mediated signaling, as evidenced in calcium mobilization assays and through quantification of phosphorylated extracellular signal-regulated kinase-1/2 (ERK1/2) and protein kinase B/Akt. Furthermore, CXCL10(1-73) incited significantly less primary human T lymphocyte chemotaxis in vitro and peritoneal ingress of CXCR3+ T lymphocytes in mice. In contrast, loss of the four endmost C-terminal residues did not affect the inhibitory properties of CXCL10 on migration, proliferation, wound closure, phosphorylation of ERK1/2, and sprouting of human microvascular endothelial cells. CONCLUSION: Our study shows that the C-terminal residues Lys74-Pro77 of CXCL10 are important for GAG binding, signaling through CXCR3A, T lymphocyte chemotaxis, but dispensable for angiostasis.

Total synthesis of antifungal lipopeptide iturin A analogues and evaluation of their bioactivity against F. graminearum.

The pursuit of novel antifungal agents is imperative to tackle the threat of antifungal resistance, which poses major risks to both human health and to food security. Iturin A is a cyclic lipopeptide, produced by Bacillus sp., with pronounced antifungal properties against several pathogens. Its challenging synthesis, mainly due to the laborious synthesis of the ß-amino fatty acid present in its structure, has hindered the study of its mode of action and the development of more potent analogues. In this work, a facile synthesis of bioactive iturin A analogues containing an alkylated cysteine residue is presented. Two analogues with opposite configurations of the alkylated cysteine residue were synthesized, to evaluate the role of the stereochemistry of the newly introduced amino acid on the bioactivity. Antifungal assays, conducted against F. graminearum, showed that the novel analogues are bioactive and can be used as a synthetic model for the design of new analogues and in structure-activity relationship studies. The assays also highlight the importance of the ß-amino acid in the natural structure and the role of the stereochemistry of the amino fatty acid, as the analogue with the D configuration showed stronger antifungal properties than the one with the L configuration.

Semisynthesis of segmentally isotope-labeled and site-specifically palmitoylated CD44 cytoplasmic tail.

CD44, a ubiquitously expressed transmembrane receptor, plays a crucial role in cell growth, migration, and tumor progression. Dimerization of CD44 is a key event in signal transduction and has emerged as a potential target for anti-tumor therapies. Palmitoylation, a posttranslational modification, disrupts CD44 dimerization and promotes CD44 accumulation in ordered membrane domains. However, the effects of palmitoylation on the structure and dynamics of CD44 at atomic resolution remain poorly understood. Here, we present a semisynthetic approach combining solid-phase peptide synthesis, recombinant expression, and native chemical ligation to investigate the impact of palmitoylation on the cytoplasmic domain (residues 669-742) of CD44 (CD44ct) by NMR spectroscopy. A segmentally isotope-labeled and site-specifically palmitoylated CD44 variant enabled NMR studies, which revealed chemical shift perturbations and indicated local and long-range conformational changes induced by palmitoylation. The long-range effects suggest altered intramolecular interactions and potential modulation of membrane association patterns. Semisynthetic, palmitoylated CD44ct serves as the basis for studying CD44 clustering, conformational changes, and localization within lipid rafts, and could be used to investigate its role as a tumor suppressor and to explore its therapeutic potential.