RESUMO
The p53 tumor suppressor protein is the most well studied as a regulator of transcription in the nucleus, where it exists primarily as a tetramer. However, there are other oligomeric states of p53 that are relevant to its regulation and activities. In unstressed cells, p53 is normally held in check by MDM2 that targets p53 for transcriptional repression, proteasomal degradation, and cytoplasmic localization. Here we discovered a hydrophobic region within the MDM2 N-terminal domain that binds exclusively to the dimeric form of the p53 C-terminal domain in vitro. In cell-based assays, MDM2 exhibits superior binding to, hyperdegradation of, and increased nuclear exclusion of dimeric p53 when compared with tetrameric wild-type p53. Correspondingly, impairing the hydrophobicity of the newly identified N-terminal MDM2 region leads to p53 stabilization. Interestingly, we found that dimeric mutant p53 is partially unfolded and is a target for ubiquitin-independent degradation by the 20S proteasome. Finally, forcing certain tumor-derived mutant forms of p53 into dimer configuration results in hyperdegradation of mutant p53 and inhibition of p53-mediated cancer cell migration. Gaining insight into different oligomeric forms of p53 may provide novel approaches to cancer therapy.
Assuntos
Neoplasias/fisiopatologia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular Tumoral , Citoplasma/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Mutação , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Domínios Proteicos , Multimerização Proteica/genética , Proteólise , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genéticaRESUMO
Protein aggregation correlates with many human diseases. Protein aggregates differ in structure and shape. Strategies to develop effective aggregation inhibitors that reach the clinic failed so far. Here, we developed a family of peptides targeting early aggregation stages for both amorphous and fibrillar aggregates of proteins unrelated in sequence and structure. They act on dynamic precursors before mechanistic differentiation takes place. Using peptide arrays, we first identified peptides inhibiting the amorphous aggregation of a molten globular, aggregation-prone mutant of the Axin tumor suppressor. Optimization revealed that the peptides activity did not depend on their sequences but rather on their molecular determinants: a composition of 20-30% flexible, 30-40% aliphatic and 20-30% aromatic residues, a hydrophobicity/hydrophilicity ratio close to 1, and an even distribution of residues of different nature throughout the sequence. The peptides also suppressed fibrillation of Tau, a disordered protein that forms amyloids in Alzheimer's disease, and slowed down that of Huntingtin Exon1, an amyloidogenic protein in Huntington's disease, both entirely unrelated to Axin. Our compounds thus target early aggregation stages of different aggregation mechanisms, inhibiting both amorphous and amyloid aggregation. Such cross-mechanistic, multi-targeting aggregation inhibitors may be lead compounds for developing drug candidates against various protein aggregation diseases.
RESUMO
Kinases are responsible for regulating cellular and physiological processes, and abnormal kinase activity is associated with various diseases. Therefore, kinases are being used as biomarkers for disease and developing methods for their sensing is highly important. Usually more than one kinase is involved in phosphorylating a target protein. However, kinase detection methods usually detect the activity of only one specific kinase. Here we describe an electrochemical kinase sensing tool for the selective detection of two kinases using the same target peptide. We demonstrate the sensing of kinases ERK2 and PKCδ. This is based on a single sensing element, a peptide that contains two distinct phosphorylation sites of these two kinases. Reversibility experiments with alkaline phosphatase and reaction with the electrochemically active ferrocene-labeled ATP showed that the mechanism of sensing is by detecting the enzymatic phosphorylation. Our approach can be further utilized to develop devices for the detection of multiple kinases and can be expanded to other types of enzymes involved in disease.
Assuntos
Sistema de Sinalização das MAP Quinases , Peptídeos , Fosforilação , Peptídeos/metabolismoRESUMO
Coiled-coil domains (CCDs) play key roles in regulating both healthy cellular processes and the pathogenesis of various diseases by controlling protein self-association and protein-protein interactions. Here, we probe the mechanism of oligomerization of a peptide representing the CCD of the STIL protein, a tetrameric multi-domain protein that is over-expressed in several cancers and associated with metastatic spread. STIL tetramerization is mediated both by an intrinsically disordered domain (STIL400-700) and a structured CCD (STIL CCD718-749). Disrupting STIL oligomerization via the CCD inhibits its activity in vivo. We describe a comprehensive biophysical and structural characterization of the concentration-dependent oligomerization of STIL CCD peptide. We combine analytical ultracentrifugation, fluorescence and circular dichroism spectroscopy to probe the STIL CCD peptide assembly in solution and determine dissociation constants of both the dimerization, (KD = 8 ± 2 µM) and tetramerization (KD = 68 ± 2 µM) of the WT STIL CCD peptide. The higher-order oligomers result in increased thermal stability and cooperativity of association. We suggest that this complex oligomerization mechanism regulates the activated levels of STIL in the cell and during centriole duplication. In addition, we present X-ray crystal structures for the CCD containing destabilising (L736E) and stabilising (Q729L) mutations, which reveal dimeric and tetrameric antiparallel coiled-coil structures, respectively. Overall, this study offers a basis for understanding the structural molecular biology of the STIL protein, and how it might be targeted to discover anti-cancer reagents.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular , Fenômenos Biofísicos , Dicroísmo Circular , Dimerização , Peptídeos/química , Domínios Proteicos , Proteínas , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/químicaRESUMO
One of the most important properties of intrinsically disordered proteins is their ability to undergo liquid-liquid phase separation and form droplets. The Adenomatous Polyposis Coli (APC) protein is an IDP that plays a key role in Wnt signaling and mutations in Apc initiate cancer. APC forms droplets via its 20R domains and self-association domain (ASAD) and in the context of Axin. However, the mechanism involved is unknown. Here, we used peptides to study the molecular mechanism and regulation of APC droplet formation. We found that a peptide derived from the ASAD of APC-formed droplets. Peptide array screening showed that the ASAD bound other APC peptides corresponding to the 20R3 and 20R5 domains. We discovered that the 20R3/5 peptides also formed droplets by themselves and mapped specific residues within 20R3/5 that are necessary for droplet formation. When incubated together, the ASAD and 20R3/5 did not form droplets. Thus, the interaction of the ASAD with 20R3 and 20R5 may regulate the droplet formation as a means of regulating different cellular functions. Phosphorylation of 20R3 or 20R5 at specific residues prevented droplet formation of 20R3/5. Our results reveal that phosphorylation and the ability to undergo liquid-liquid phase separation, which are both important properties of intrinsically disordered proteins, are related to each other in APC. Phosphorylation inhibited the liquid-liquid phase separation of APC, acting as an 'on-off' switch for droplet formation. Phosphorylation may thus be a common mechanism regulating LLPS in intrinsically disordered proteins.
Assuntos
Proteínas Intrinsicamente Desordenadas , Proteínas Intrinsicamente Desordenadas/química , Mutação , Fosforilação , Domínios Proteicos , Polipose Adenomatosa do ColoRESUMO
The role kinases play in regulating cellular processes makes them potential biomarkers for detecting the onset and prognosis of various diseases, including many types of cancer. Current kinase biosensors, including electrochemical and radiometric methods, rely on sensing the ATP-dependant enzymatic phosphorylation reaction. Here we introduce a new type of interaction-based electrochemical kinase biosensor that does not require any chemical labelling or modification. The basis for sensing is the interactions between the catalytic site of the kinase and the phosphorylation site of its substrate rather than the phosphorylation reaction. We demonstrated this concept with the ERK2 kinase and its substrate protein HDGF, which is involved in lung cancer. A peptide monolayer derived from the HDGF phosphorylation site was adsorbed onto a gold electrode and was used to sense ERK2 without ATP. The sensitivity of the assay was down to 10â nM of ERK2, corresponding with the range of its cellular concentrations. Surface chemistry analysis confirmed that ERK2 was bound to the HDGF peptide monolayer. This increased the permeability of redox-active species through the monolayer and resulted in ERK2 electrochemical sensing. Since our detection approach is based on protein-protein interactions and not on the enzymatic reaction, it can be further utilized for more selective detection of different types of enzymes.
Assuntos
Técnicas Biossensoriais , Domínio Catalítico , Ouro , Peptídeos , FosforilaçãoRESUMO
Invited for the cover of this issue is Assaf Friedler, Shlomo Yitzchaik and co-workers at the Hebrew University of Jerusalem and the Academia Sinica. The image depicts a new approach for electrochemical kinase sensing that does not rely on phosphorylation. The kinase binds a peptide layer, which undergoes rearrangement, resulting in the permeation of redox-active species through the layer and electrochemical sensing. Read the full text of the article at 10.1002/chem.202104227.
Assuntos
Peptídeos , Domínio Catalítico , HumanosRESUMO
Protein aggregation is involved in a variety of diseases, including neurodegenerative diseases and cancer. The cellular environment is crowded by a plethora of cosolutes comprising small molecules and biomacromolecules at high concentrations, which may influence the aggregation of proteins in vivo. To account for the effect of cosolutes on cancer-related protein aggregation, we studied their effect on the aggregation of the cancer-related L106R mutant of the Axin protein. Axin is a key player in the Wnt signaling pathway, and the L106R mutation in its RGS domain results in a native molten globule that tends to form native-like aggregates. This results in uncontrolled activation of the Wnt signaling pathway, leading to cancer. We monitored the aggregation process of Axin RGS L106R in vitro in the presence of a wide ensemble of cosolutes including polyols, amino acids, betaine, and polyethylene glycol crowders. Except myo-inositol, all polyols decreased RGS L106R aggregation, with carbohydrates exerting the strongest inhibition. Conversely, betaine and polyethylene glycols enhanced aggregation. These results are consistent with the reported effects of osmolytes and crowders on the stability of molten globular proteins and with both amorphous and amyloid aggregation mechanisms. We suggest a model of Axin L106R aggregation in vivo, whereby molecularly small osmolytes keep the protein as a free soluble molecule but the increased crowding of the bound state by macromolecules induces its aggregation at the nanoscale. To our knowledge, this is the first systematic study on the effect of osmolytes and crowders on a process of native-like aggregation involved in pathology, as it sheds light on the contribution of cosolutes to the onset of cancer as a protein misfolding disease and on the relevance of aggregation in the molecular etiology of cancer.
Assuntos
Neoplasias , Polietilenoglicóis , Amiloide , Proteína Axina/genética , Mutação , Neoplasias/genética , Transdução de SinaisRESUMO
We describe a molecular characterization of the interaction between the cancer-related proteins WWOX and p73. This interaction is mediated by the first of two WW domains (WW1) of WWOX and a PPXY-motif-containing region in p73. While phosphorylation of Tyr33 of WWOX and association with p73 are known to affect apoptotic activity, the quantitative effect of phosphorylation on this specific interaction is determined here for the first time. Using ITC and fluorescence anisotropy, we measured the binding affinity between WWOX domains and a p73 derived peptide, and showed that this interaction is regulated by Tyr phosphorylation of WW1. Chemical synthesis of the phosphorylated domains of WWOX revealed that the binding affinity of WWOX to p73 is decreased when WWOX is phosphorylated. This result suggests a fine-tuning of binding affinity in a differential, ligand-specific manner: the decrease in binding affinity of WWOX to p73 can free both partners to form new interactions.
Assuntos
Proteína Tumoral p73/metabolismo , Oxidorredutase com Domínios WW/metabolismo , Motivos de Aminoácidos , Calorimetria , Polarização de Fluorescência , Humanos , Modelos Moleculares , Fosforilação , Ligação Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteína Tumoral p73/química , Oxidorredutase com Domínios WW/química , Oxidorredutase com Domínios WW/genéticaRESUMO
Intrinsically disordered regions in proteins (IDRs) mediate many disease-related protein-protein interactions. However, the unfolded character and continuous conformational changes of IDRs make them difficult to target for therapeutic purposes. Here, we show that a designed peptide based on the disordered p53 linker domain can be used to target a partner IDR from the anti-apoptotic iASPP protein, promoting apoptosis of cancer cells. The p53 linker forms a hairpin-like structure with its two termini in close proximity. We designed a peptide derived from the disordered termini without the hairpin, designated as p53 LinkTer. The LinkTer peptide binds the disordered RT loop of iASPP with the same affinity as the parent p53 linker peptide, and inhibits the p53-iASPP interaction in vitro. The LinkTer peptide shows increased stability to proteolysis, penetrates cancer cells, causes nuclei shrinkage, and compromises the viability of cells. We conclude that a designed peptide comprising only the IDR from a peptide sequence can serve as an improved inhibitor since it binds its target protein without the need for pre-folding, paving the way for therapeutic targeting of IDRs.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos/metabolismo , Proteínas Repressoras/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sequência de Aminoácidos , Apoptose , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos/química , Ligação Proteica , Proteínas Repressoras/química , Proteína Supressora de Tumor p53/químicaRESUMO
Invited for the cover of this issue is the group of Assaf Friedler at the Hebrew University of Jerusalem. The image depicts the protein-protein interactions reported in this work. Read the full text of the article at 10.1002/chem.202000465.
Assuntos
Peptídeos , Modelos Biológicos , Peptídeos/química , Domínios e Motivos de Interação entre ProteínasRESUMO
Unraveling the role of post-translational modification (PTM) patterns is one of the most urgent and unresolved issues facing the scientific community. Attempts to crack the phosphorylation bio-barcode led to significant findings, which suggest that many proteins cannot be regarded as a single entity but exist as several forms which differ in their phosphorylation patterns and their functions. While protein regions that do not contain PTMs can be rather simply mimicked using peptide libraries, heavily phosphorylated regions are much harder to study using the same tools. The differences between the syntheses of simple mono-, di- and tri-phosphopeptides and the synthesis of multiphosphopeptides are dramatic. While simple phosphopeptides can be synthesized using almost standard SPPS strategies, the synthesis of multiphosphopeptides is to date a major synthetic challenge. Synthesis of multiphosphopeptides requires the insertion of several phosphate groups simultaneously or sequentially into various positions on the peptide in the presence of many other potential modification sites. These groups are bulky, unstable and cannot be easily introduced when in close proximity. Moreover, since the same protein region can possess many alternative multiphosphorylation patterns, libraries comprising a large number of peptides with different degrees and positions of phosphorylation are essential. Many strategies have been developed to provide routes to enable the preparation of multiphosphopeptides. These methods are essentially different from the methods used for the preparation of simple phosphopeptides. In this review, we specifically emphasize the challenges and importance of synthesizing multiphosphopeptides and their libraries. The historical perspective and state of the art strategies are described. We demonstrate here how the different synthetic approaches attempt to address the special problems associated with the synthesis of multiphosphopeptides. The advantages and disadvantages of each strategy are discussed in order to provide a roadmap for the synthesis of such libraries. An overview of the existing strategies and some comments regarding future directions are provided. Applications of multiphosphopeptide libraries as tools to study the effect of phosphorylation patterns on the biological function of proteins are also described.
Assuntos
Peptídeos/síntese química , Peptídeos/metabolismo , Biblioteca de Peptídeos , Peptídeos/química , Fosforilação , Processamento de Proteína Pós-TraducionalRESUMO
[This corrects the article DOI: 10.1371/journal.ppat.1000977.].
RESUMO
Protein phosphorylation barcodes, clusters of several phosphorylation sites within a short unfolded region, control many cellular processes. Existing biochemical methods used to study the roles of these barcodes suffer from low selectivity and provide only qualitative data. Chemically synthesized multiphosphopeptide libraries are selective and specific, but their synthesis is extremely difficult using the current peptide synthesis methods. Here we describe a new microwave assisted approach for synthesizing a library of multiphosphopeptides, using the C-terminus of rhodopsin as a proof of concept. Our approach utilizes multiple protocols for synthesizing libraries of multiphosphopeptides instead of the inefficient single protocol methods currently used. Using our approach we demonstrated the synthesis with up to seven phosphorylated amino acids, sometimes next to each other, an accomplishment that was impractical before. Synthesizing the Rhodopsin derived multiphosphopeptide library enabled dissecting the precise phosphorylation barcode required for the recruitment, activation and modulation of the conformation of Arrestin. Since phosphorylation barcodes modulate the activity of hundreds of GPCRs, synthesizing libraries of multiphosphopeptides is the method of choice for studying their molecular mechanisms of action. Our approach provides an invaluable tool for evaluating how protein phosphorylation barcodes regulate their activity.
Assuntos
Proteínas/síntese química , Proteínas/metabolismo , Sequência de Aminoácidos , Fosforilação , Conformação Proteica , Rodopsina/químicaRESUMO
Intrinsically disordered regions (IDRs) in proteins are highly abundant, but they are still commonly viewed as long stretches of polar, solvent-accessible residues. Here we show that the disordered C-terminal domain (CTD) of HIV-1 Rev has two subregions that carry out two distinct complementary roles of regulating protein oligomerization and contributing to stability. We propose that this takes place through a delicate balance between charged and hydrophobic residues within the IDR. This means that mutations in this region, as well as the known mutations in the structured region of the protein, can affect protein function. We suggest that IDRs in proteins should be divided into subdomains similarly to structured regions, rather than being viewed as long flexible stretches.
Assuntos
HIV-1/química , Proteínas Intrinsicamente Desordenadas/química , Produtos do Gene rev do Vírus da Imunodeficiência Humana/química , Infecções por HIV/virologia , HIV-1/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Proteínas Intrinsicamente Desordenadas/metabolismo , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Estabilidade Proteica , Produtos do Gene rev do Vírus da Imunodeficiência Humana/metabolismoRESUMO
We have recently reported the covalent inhibition of HIV-1 integrase by an N-terminal succinimide-modified lens epithelium-derived growth factor (361-370) peptide. We also showed that this peptide is proteolytically stable. Here, we show that this inhibitor is stored as fibrils that serve as a stock for the inhibitory monomers. The fibrils increase the local concentration of the peptide at the target protein. When the monomers bind integrase, the equilibrium between the fibrils and their monomers shifts towards the formation of peptide monomers. The combination of fibril formation and subsequent proteolytic stability of the peptide may bring to new strategy for developing therapeutic agents. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.
Assuntos
Inibidores de Integrase de HIV/química , Integrase de HIV/química , HIV-1/química , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos/química , Sequência de Aminoácidos , Inibidores de Integrase de HIV/síntese química , HIV-1/enzimologia , Humanos , Microscopia de Força Atômica , Peptídeos/síntese química , Multimerização Proteica , Estabilidade Proteica , Proteólise , Succinimidas/químicaRESUMO
Life requires orchestrated control of cell proliferation, cell maintenance, and cell death. Involved in these decisions are protein complexes that assimilate a variety of inputs that report on the status of the cell and lead to an output response. Among the proteins involved in this response are nutrient-deprivation autophagy factor-1 (NAF-1)- and Bcl-2. NAF-1 is a homodimeric member of the novel Fe-S protein NEET family, which binds two 2Fe-2S clusters. NAF-1 is an important partner for Bcl-2 at the endoplasmic reticulum to functionally antagonize Beclin 1-dependent autophagy [Chang NC, Nguyen M, Germain M, Shore GC (2010) EMBO J 29(3):606-618]. We used an integrated approach involving peptide array, deuterium exchange mass spectrometry (DXMS), and functional studies aided by the power of sufficient constraints from direct coupling analysis (DCA) to determine the dominant docked conformation of the NAF-1-Bcl-2 complex. NAF-1 binds to both the pro- and antiapoptotic regions (BH3 and BH4) of Bcl-2, as demonstrated by a nested protein fragment analysis in a peptide array and DXMS analysis. A combination of the solution studies together with a new application of DCA to the eukaryotic proteins NAF-1 and Bcl-2 provided sufficient constraints at amino acid resolution to predict the interaction surfaces and orientation of the protein-protein interactions involved in the docked structure. The specific integrated approach described in this paper provides the first structural information, to our knowledge, for future targeting of the NAF-1-Bcl-2 complex in the regulation of apoptosis/autophagy in cancer biology.
Assuntos
Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ribonucleoproteínas/metabolismo , Sequência de Aminoácidos , Humanos , Espectrometria de Massas , Modelos Moleculares , Dados de Sequência Molecular , Oligopeptídeos/química , Ligação ProteicaRESUMO
The leading risk factor for gastric cancer in humans is infection by Helicobacter pylori strains that express and translocate the oncoprotein CagA into host epithelial cells. Once inside host cells, CagA interacts with ASPP2, which specifically stimulates p53-mediated apoptosis and reverses its pro-apoptotic function to promote ASPP2-dependent degradation of p53. The X-ray crystal structure of a complex between the N-terminal domain of CagA and a 56-residue fragment of ASPP2, of which 22 residues were resolved, was recently described. Here, we present biochemical and biophysical analyses of the interaction between the additional regions of CagA and ASPP2 potentially involved in this interaction. Using size exclusion chromatography-multiangle laser light scattering, circular dichroism, and nuclear magnetic resonance analyses, we observed that the ASPP2 region spanning residues 331-692, which was not part of the ASPP2 fragment used for crystallization, is intrinsically disordered in its unbound state. By surface plasmon resonance analysis and isothermal titration calorimetry, we found that a portion of this disordered region in ASPP2, residues 448-692, binds to the N-terminal domain of CagA. We also measured the affinity of the complex between the ASPP2 fragment composed of residues 693-918 and inclusive of the fragment used for crystallization and CagA. Additionally, we mapped the binding regions between ASPP2 and CagA using peptide arrays, demonstrating interactions between CagA and numerous peptides distributed throughout the ASPP2 protein sequence. Our results identify previously uncharacterized regions distributed throughout the protein sequence of ASPP2 as determinants of CagA binding, providing mechanistic insight into apoptosis reprogramming by CagA and potential new drug targets for H. pylori-mediated gastric cancer.
Assuntos
Antígenos de Bactérias/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Bactérias/metabolismo , Infecções por Helicobacter/complicações , Helicobacter pylori/metabolismo , Neoplasias Gástricas/microbiologia , Antígenos de Bactérias/química , Proteínas Reguladoras de Apoptose/química , Proteínas de Bactérias/química , Humanos , Modelos Moleculares , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Neoplasias Gástricas/etiologiaRESUMO
An electrochemical biosensor has been developed for ultrasensitive, label-free determination of protein kinase activity. The sensor is composed of a unique peptide monolayer on a gold electrode. It identifies the order change in the monolayer upon phosphorylation, via square wave voltametry (SWV) measurements. Disorder caused by the introduction of the phosphate groups onto the middle of the peptide sequence results in pinhole formation and therefore an increase in the electrochemical signal. The measured sensitivity was 100 nM of kinase and the dynamic range was 100 nM up to 11 µM. Sensitivity was an order of magnitude higher, and the dynamic range wider by two orders of magnitude, as compared to our previously reported impedimetric method, in which the sensitivity was 1 µM, and the dynamic range was 1-20 µM.
Assuntos
Técnicas Biossensoriais/métodos , Ensaios Enzimáticos/métodos , Ouro/química , Proteínas Quinases/química , Eletroquímica , Eletrodos , Limite de DetecçãoRESUMO
We have previously introduced an easy to perform, cost-effective and highly efficient acetylation technique for solid phase synthesis (SPPS). Malonic acid is used as a precursor and the reaction proceeds via a reactive ketene that acetylates the target amine. Here we present a detailed mechanistic study of the malonic acid-mediated acylation. The influence of reaction conditions, peptide sequence and reagents was systematically studied. Our results show that the methodology can be successfully applied to different types of peptides and nonpeptidic molecules irrespective of their structure, sequence, or conformation. Using alkyl, phenyl, and benzyl malonic acid, we synthesized various acyl peptides with almost quantitative yields. The ketenes obtained from the different malonic acid derived precursors were characterized by in situ (1) H-NMR. The reaction proceeded in short reaction times and resulted in excellent yields when using uronium-based coupling agents, DIPEA as a base, DMF/DMSO/NMP as solvents, Rink amide/Wang/Merrifield resins, temperature of 20°C, pH 8-12 and 5 min preactivation at inert atmosphere. The reaction was unaffected by Lewis acids, transition metal ions, surfactants, or salt. DFT studies support the kinetically favorable concerted mechanism for CO2 and ketene formation that leads to the thermodynamically stable acylated products. We conclude that the malonic acid-mediated acylation is a general method applicable to various target molecules.