RESUMO
RNA represents a potential target for new antiviral therapies, which are urgently needed to address public health threats such as the human immunodeficiency virus (HIV). We showed previously that the interaction between the viral Tat protein and the HIV-1 trans-activation response (TAR) RNA was blocked by TB-CP-6.9a. This cyclic peptide was derived from a TAR-binding loop that emerged during lab evolution of a TAR-binding protein (TBP) family. Here we synthesized and characterized a next-generation, cyclic-peptide library based on the TBP scaffold. We sought to identify conserved RNA-binding interactions and the influence of cyclization linkers on RNA binding and antiviral activity. A diverse group of cyclization linkers, encompassing disulfide bonds to bicyclic aromatic staples, was used to restrain the cyclic peptide geometry. Thermodynamic profiling revealed specific arginine-rich sequences with low to submicromolar affinity driven by enthalpic and entropic contributions. The best compounds exhibited no appreciable off-target binding to related molecules, such as BIV TAR and human 7SK RNAs. A specific arginine-to-lysine change in the highest affinity cyclic peptide reduced TAR binding by tenfold, suggesting that TBP-derived cyclic peptides use an arginine-fork motif to recognize the TAR major groove while differentiating the mode of binding from other TAR-targeting molecules. Finally, we showed that HIV infectivity in cell culture was reduced in the presence of cyclic peptides constrained by methylene or naphthalene-based linkers. Our findings provide insight into the molecular determinants required for HIV-1 TAR recognition and antiviral activity. These findings are broadly relevant to the development of antivirals that target RNA molecules.
Assuntos
Antivirais/química , HIV-1/química , Peptídeos Cíclicos/química , RNA Viral/química , Células HEK293 , Infecções por HIV/tratamento farmacológico , Infecções por HIV/genética , Infecções por HIV/metabolismo , HIV-1/genética , HIV-1/metabolismo , Humanos , Ligação Proteica , RNA Viral/genética , RNA Viral/metabolismoRESUMO
The proteasome, the primary protease for ubiquitin-dependent proteolysis in eukaryotes, is usually found as a mixture of 30S, 26S, and 20S complexes. These complexes have common catalytic sites, which makes it challenging to determine their distinctive roles in intracellular proteolysis. Here, we chemically synthesize a panel of homogenous ubiquitinated proteins, and use them to compare 20S and 26S proteasomes with respect to substrate selection and peptide-product generation. We show that 20S proteasomes can degrade the ubiquitin tag along with the conjugated substrate. Ubiquitin remnants on branched peptide products identified by LC-MS/MS, and flexibility in the 20S gate observed by cryo-EM, reflect the ability of the 20S proteasome to proteolyze an isopeptide-linked ubiquitin-conjugate. Peptidomics identifies proteasome-trapped ubiquitin-derived peptides and peptides of potential 20S substrates in Hi20S cells, hypoxic cells, and human failing-heart. Moreover, elevated levels of 20S proteasomes appear to contribute to cell survival under stress associated with damaged proteins.
Assuntos
Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Hipóxia Celular , Sobrevivência Celular , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Humanos , Peptídeos/química , Peptídeos/metabolismo , Conformação Proteica , Proteólise , Especificidade por Substrato , Ubiquitina/química , Proteínas Ubiquitinadas/química , Proteínas Ubiquitinadas/metabolismo , UbiquitinaçãoRESUMO
RNA-protein interfaces control key replication events during the HIV-1 life cycle. The viral trans-activator of transcription (Tat) protein uses an archetypal arginine-rich motif (ARM) to recruit the host positive transcription elongation factor b (pTEFb) complex onto the viral trans-activation response (TAR) RNA, leading to activation of HIV transcription. Efforts to block this interaction have stimulated production of biologics designed to disrupt this essential RNA-protein interface. Here, we present four co-crystal structures of lab-evolved TAR-binding proteins (TBPs) in complex with HIV-1 TAR. Our results reveal that high-affinity binding requires a distinct sequence and spacing of arginines within a specific ß2-ß3 hairpin loop that arose during selection. Although loops with as many as five arginines were analyzed, only three arginines could bind simultaneously with major-groove guanines. Amino acids that promote backbone interactions within the ß2-ß3 loop were also observed to be important for high-affinity interactions. Based on structural and affinity analyses, we designed two cyclic peptide mimics of the TAR-binding ß2-ß3 loop sequences present in two high-affinity TBPs (KD values of 4.2 ± 0.3 and 3.0 ± 0.3 nm). Our efforts yielded low-molecular weight compounds that bind TAR with low micromolar affinity (KD values ranging from 3.6 to 22 µm). Significantly, one cyclic compound within this series blocked binding of the Tat-ARM peptide to TAR in solution assays, whereas its linear counterpart did not. Overall, this work provides insight into protein-mediated TAR recognition and lays the ground for the development of cyclic peptide inhibitors of a vital HIV-1 RNA-protein interaction.
Assuntos
Arginina/química , Repetição Terminal Longa de HIV/genética , HIV-1/metabolismo , Peptídeos Cíclicos/química , RNA Viral/metabolismo , Proteína de Ligação a TATA-Box/química , Sequência de Aminoácidos , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Cinética , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Peptídeos Cíclicos/metabolismo , Ligação Proteica , RNA Viral/química , Proteína de Ligação a TATA-Box/genética , Proteína de Ligação a TATA-Box/metabolismo , TermodinâmicaRESUMO
The Sonic Hedgehog (SHH) pathway plays a key role in cancer. Alterations of SHH canonical signaling, causally linked to tumor progression, have become rational targets for cancer therapy. However, Smoothened (SMO) inhibitors have failed to show clinical benefit in patients with cancers displaying SHH autocrine/paracrine expression. We reported earlier that the SHH receptor Patched (PTCH) is a dependence receptor that triggers apoptosis in the absence of SHH through a pathway that differs from the canonical one, thus generating a state of dependence on SHH for survival. Here, we propose a dual function for SHH: its binding to PTCH not only activates the SHH canonical pathway but also blocks PTCH-induced apoptosis. Eighty percent, 64%, and 8% of human colon, pancreatic, and lung cancer cells, respectively, overexpressed SHH at transcriptional and protein levels. In addition, SHH-overexpressing cells expressed all the effectors of the PTCH-induced apoptotic pathway. Although the canonical pathway remained unchanged, autocrine SHH interference in colon, pancreatic, and lung cell lines triggered cell death through PTCH proapoptotic signaling. In vivo, SHH interference in colon cancer cell lines decreased primary tumor growth and metastasis. Therefore, the antitumor effect associated to SHH deprivation, usually thought to be a consequence of the inactivation of the canonical SHH pathway, is, at least in part, because of the engagement of PTCH proapoptotic activity. Together, these data strongly suggest that therapeutic strategies based on the disruption of SHH/PTCH interaction in SHH-overexpressing cancers should be explored. SIGNIFICANCE: Sonic Hedgehog-overexpressing tumors express PTCH-induced cell death effectors, suggesting that this death signaling could be activated as an antitumor strategy.
Assuntos
Apoptose/fisiologia , Proteínas Hedgehog/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Receptores Patched/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Embrião de Galinha , Xenoenxertos , Humanos , Camundongos , Transdução de Sinais/fisiologia , Peixe-ZebraRESUMO
A promising approach in cancer therapy is to find ligands that directly bind ubiquitin (Ub) chains. However, finding molecules capable of tightly and specifically binding Ub chains is challenging given the range of Ub polymer lengths and linkages and their subtle structural differences. Here, we use total chemical synthesis of proteins to generate highly homogeneous Ub chains for screening against trillion-member macrocyclic peptide libraries (RaPID system). De novo cyclic peptides were found that can bind tightly and specifically to K48-linked Ub chains, confirmed by NMR studies. These cyclic peptides protected K48-linked Ub chains from deubiquitinating enzymes and prevented proteasomal degradation of Ub-tagged proteins. The cyclic peptides could enter cells, inhibit growth and induce programmed cell death, opening new opportunities for therapeutic intervention. This highly synthetic approach, with both protein target generation and cyclic peptide discovery performed in vitro, will make other elaborate post-translationally modified targets accessible for drug discovery.
Assuntos
Lisina/química , Peptídeos Cíclicos/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Ubiquitinas/metabolismo , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Enzimas Desubiquitinantes , Células HeLa , Humanos , Estrutura Molecular , Peptídeos Cíclicos/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Ligação Proteica , Bibliotecas de Moléculas Pequenas/farmacologia , Ubiquitinas/síntese química , Ubiquitinas/químicaRESUMO
One of the enigmas in the ubiquitin (Ub) field is the requirement for a poly-Ub chain as a proteasomal targeting signal. The canonical chain appears to be longer than the distance between the two Ub-binding proteasomal receptors. Furthermore, genetic manipulation has shown that one receptor subunit is sufficient, which suggests that a single Ub can serve as a degradation signal. To shed light on this mystery, we chemically synthesized tetra-Ub, di-Ub (K48-based), and mono-Ub adducts of HA-α-globin, where the distal or proximal Ub moieties were tagged differentially with either Myc or Flag. When incubated in a crude cell extract, the distal Ub moiety in the tetra-Ub adduct was mostly removed by deubiquitinating enzymes (DUBs) and reconjugated to other substrates in the extract. In contrast, the proximal moiety was most likely degraded with the substrate. The efficacy of degradation was proportionate to the chain length; while tetra-Ub globin was an efficient substrate, with mono-Ub globin, we observed rapid removal of the Ub moiety with almost no degradation of the free globin. Taken together, these findings suggest that the proximal moieties are necessary for securing the association of the substrate with the proteasome along the proteolytic process, whereas the distal moieties are important in protecting the proximal moieties from premature deubiquitination. Interestingly, when the same experiment was carried out using purified 26S proteasome, mono- and tetra-Ub globin were similarly degraded, highlighting the roles of the entire repertoire of cellular DUBs in regulating the degradation of proteasomal substrates.
RESUMO
Ester-linked ubiquitinated proteins have been reported by several groups to be involved in ubiquitin signalling. However, due to the lack of the suitable tools to homogeneously produce such conjugates, their exact physiological roles and biochemical behavior remain enigmatic. Here, we report for the first time on the development of a novel synthetic strategy based on total chemical synthesis of proteins to construct ubiquitinated proteins, where ubiquitin is linked to the substrate via an ester bond. In this study, we prepared ester- and isopeptide-linked ubiquitinated α-globin and examined their relative behaviors with various deubiquitinases. We found that deubiquitinases are able to cleave the ester linkage with different efficiency relative to the isopeptide-linked substrate. These results may indicate that ester-linked ubiquitinated proteins are natural substrates for deubiquitinases.
RESUMO
We report a general and novel semisynthetic strategy for the preparation of ubiquitinated protein-activity-based probes on the basis of sequential dehydroalanine formation on expressed proteins. We applied this approach to construct a physiologically and therapeutically relevant ubiquitinated α-globin probe, which was used for the enrichment and proteomic identification of α-globin-modulating deubiquitinases. We found USP15 as a potential deubiquitinase for the modulation of α-globin, an excess of which aggravates ß-thalassemia symptoms. This development opens new opportunities for activity-based-probe design to shed light on the important aspects underlying ubiquitination and deubiquitination in health and disease.
Assuntos
Alanina/análogos & derivados , Enzimas Desubiquitinantes/metabolismo , Sondas Moleculares/metabolismo , alfa-Globinas/metabolismo , Alanina/biossíntese , Alanina/química , Enzimas Desubiquitinantes/química , Humanos , Sondas Moleculares/química , Estrutura Molecular , alfa-Globinas/químicaRESUMO
Small ubiquitin like modifier (SUMO) proteins are known to regulate many important cellular processes such as transcription and apoptosis. Recently, hybrid SUMO-ubiquitin chains containing SUMO-2 linked to Lys63-di-ubiquitin were found to play a major role in DNA repair. Despite some progress in understanding the role of these hybrid chains in DNA repair, there are various fundamental questions remaining to be answered. To further investigate the importance of hybrid SUMO-ubiquitin chains in DNA repair, the homogenous material of these chains, and their unique analogues, are needed in workable quantities. By applying advanced chemical strategies for protein synthesis, we report the first total chemical synthesis of four different SUMO-2-Lys63-linked di-ubiquitin hybrid chains, in which the di-ubiquitin is linked to different lysines in SUMO. In these syntheses, the usefulness of removable solubilizing tags is demonstrated, and two different approaches were examined in terms of reliability and efficiency. In the first approach, a poly-Arg tag was attached to the C-terminus of SUMO via a 3,4-diaminobenzoic acid cleavable linker, whereas in the second we attached the tag via a phenylacetamidomethyl linker, which can be cleaved by PdCl2. The comparison between these different strategies offers guidelines for future scale-up preparation of these analogues and other proteins, which currently use synthetic peptide intermediates that are difficult to handle and purify. The availability of the SUMO-ubiquitin hybrid chains opens up new opportunities for studying the role of these chains in DNA repair and other cellular processes.
RESUMO
Posttranslational modification of proteins by ubiquitin (Ub), i.e., ubiquitination, mediates a variety of cellular processes, including protein homeostasis, cell cycle, DNA repair, and viral infections. Understanding the molecular mechanism of ubiquitination in these events is the basis for unraveling its precise role in health and disease. However, the inherent complexity of Ub signaling due to the high atomic complexity of Ub conjugates, where Ub is attached to other Ub molecules and to protein substrates in various forms, imposes a major challenge for these studies. In this regard, the enzymatic approaches employed for the preparation of important Ub conjugates have severe limitations to deliver them in high homogeneity and in adequate amounts for the desired study. Recent developments in the area of chemical synthesis and semisynthesis of proteins offer great solutions to the enzymatic limitations and enabling the preparation of various Ub conjugates with precise control over the atomic structure. These conjugates significantly contribute to deciphering Ub signaling at the molecular level, and with the synthetic tools in hand, chemical biologists have become key players in efforts toward understanding the complexity of the Ub code. In this Perspective, we highlight the key contributions of these synthetic approaches and how the development of novel Ub-based reagents is greatly assisting in uncovering unknown aspects of Ub signaling. We also discuss future aspirations to address unresolved questions in this exciting area of research.
RESUMO
Various hypotheses have been proposed regarding how chain length, linkage type, position on substrate, and susceptibility to deubiquitinases (DUBs) affect processing of different substrates by proteasome. Here we report a new strategy for the chemical synthesis of ubiquitinated proteins to generate a set of well-defined conjugates bearing an oxime bond between the chain and the substrate. We confirmed that this isopeptide replacement is resistant to DUBs and to shaving by proteasome. Analyzing products generated by proteasomes ranked how chain length governed degradation outcome. Our results support that (1) the cleavage of the proximal isopeptide bond is not a prerequisite for proteasomal degradation, (2) by overcoming trimming at the proteasome, tetraUb is a fundamentally different signal than shorter chains, and (3) the tetra-ubiquitin chain can be degraded with the substrate. Together these results highlight the usefulness of chemistry to dissect the contribution of proteasome-associated DUBs and the complexity of the degradation process.
Assuntos
Enzimas Desubiquitinantes/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Proteínas Ubiquitinadas/metabolismo , Enzimas Desubiquitinantes/química , Humanos , Estrutura Molecular , Complexo de Endopeptidases do Proteassoma/química , Ubiquitina/química , Proteínas Ubiquitinadas/síntese química , Proteínas Ubiquitinadas/químicaRESUMO
We report a strategy for site-specific protein ubiquitination using dehydroalanine (Dha) chemistry for the preparation of ubiquitin conjugates bearing a very close mimic of the native isopeptide bond. Our approach relies on the selective formation of Dha followed by conjugation with hexapeptide bearing a thiol handle derived from the C-terminal of ubiquitin. Subsequently, the resulting synthetic intermediate undergoes native chemical ligation with the complementary part of the ubiquitin polypeptide. It has been proposed that the Michael addition step could result in the formation of a diastereomeric mixture as a result of unselective protonation of the enolate intermediate. It has also been proposed that the chiral protein environment may influence such an addition step. In the protein context these questions remain open and no experimental evidence was provided as to how such a protein environment affects the diastereoselectivity of the addition step. As was previously proposed for the conjugation step on protein bearing Dha, the isopeptide bond formation step in our study resulted in the construction of two protein diastereomers. To assign the ratio of these diastereomers, trypsinization coupled with high-pressure liquid chromatography analysis were performed. Moreover, the obtained peptide diastereomers were compared with identical synthetic peptides having defined stereogenic centers, which enabled the determination of the configuration of the isopeptide mimic in each diastereomer. Our study, which offers a new method for isopeptide bond formation and protein ubiquitination, gives insights into the parameters that affect the stereoselectivity of the addition step to Dha for chemical protein modifications.
Assuntos
Alanina/análogos & derivados , Ubiquitinação , Alanina/química , Sequência de Aminoácidos , Sítios de Ligação , Modelos Moleculares , Conformação Proteica , Estereoisomerismo , Especificidade por Substrato , Ubiquitina/química , Ubiquitina/metabolismoRESUMO
Chemical synthesis offers unique opportunities to prepare proteins with precise control of the atomic composition. Thanks to recent breakthroughs in synthetic methods, the preparation of large and complex proteins composed of 200-300 residues has now become possible. With these advances, a unique toolbox has been created to enable chemical biologists to investigate proteins that are difficult or even impossible to achieve otherwise, such as posttranslationally modified proteins and proteins composed of d-amino acids. In this review we describe the latest achievements in constructing protein conjugates of record sizes, such as those that are involved in the ubiquitin system.
Assuntos
Peptídeos/síntese química , Ubiquitina/síntese química , Peptídeos/química , Ubiquitina/químicaRESUMO
The chemical synthesis of a protein from four fragments or more applying native chemical ligation could be achieved stepwise, in one-pot, convergently, or on a solid support. With the increasing demands of applying protein synthesis to highly complex targets, examining these approaches becomes essential to achieve highly efficient synthesis. Different chemical synthetic strategies are compared for the preparation of the H2B protein having different post-translational modifications. The analogues include H2B that is ubiquitinated at Lys34, Lys120, glycosylated at Ser112, and doubly modified with ubiquitin and N-acetylglucosamine. This study demonstrates that the applied convergent strategy for the synthesis of most of these complex targets was better than the one-pot approach in terms of yield and purity. Some guidelines are offered for future synthetic endeavors of similar challenging proteins.
Assuntos
Acetilglucosamina/química , Histonas/síntese química , Ubiquitina/química , Ubiquitinação , Glicosilação , Histonas/química , Conformação MolecularRESUMO
The synthesis and utilization of novel thiostatines (ß-SH-substituted γ-amino acids) in the design of backbone-disulfide-stabilized ß-hairpin mimetics, solution conformations of hybrid ß-hairpins and Cys-disulfide-stabilized α-peptide analogue, their thiol exchange, and proteolytic stability are investigated. The results suggest that thiostatines can be used to design proteolytically stable water-soluble ß-hairpin mimetics without deviating from overall ß-hairpin conformation.
Assuntos
Aminoácidos/síntese química , Dissulfetos/química , Cininogênios/síntese química , Peptídeos/química , Aminoácidos/química , Cisteína/química , Ligação de Hidrogênio , Cininogênios/química , Estrutura Molecular , Conformação Proteica , Estrutura Secundária de ProteínaRESUMO
HBTU is a standard coupling agent commonly used for the activation of free carboxylic acids during the solution and solid phase peptide synthesis. 1-Hydroxybenzotriazole (HOBt) plays a significant role in reducing the racemization during peptide synthesis; hence it is regularly used as a coupling additive. Here, we are reporting the mild and facile conjugate addition of HOBt to E-vinylogous γ-amino acids mediated by the HBTU. The reaction is moderately diastereoselective and novel ß-benzotriazole N-oxide (ß-BtO) substituted γ-amino acids were isolated in moderate to good yields. The single crystal analysis of methyl esters of major (anti) and minor (syn) conjugate addition products infers the formation of exclusively N-alkylated benzotriazole N-oxides instead of O-alkylation of HOBt. In addition, we showed the utilization of ß-BtO substituted γ-amino acids in peptide synthesis and studied their conformations in single crystals.
Assuntos
Aminoácidos/síntese química , Peptídeos/síntese química , Triazóis/química , Aminoácidos/química , Carbodi-Imidas/química , Cristalografia por Raios X , Modelos Moleculares , Óxidos/química , Peptídeos/química , Técnicas de Síntese em Fase Sólida , EstereoisomerismoRESUMO
Thioacids are recently gaining momentum due to their versatile reactivity. The reactivity of thioacids has been widely explored in the selective amide/peptide bond formation. Thioacids are generally synthesized from the reaction between activated carboxylic acids such as acid chlorides, active esters, etc., and Na2S, H2S, or NaSH. We sought to investigate whether the versatile reactivity of the thioacids can be tuned for the conversion of carboxylic acids into corresponding thioacids in the presence of NaSH. Herein, we report that thioacetic acid- and NaSH-mediated synthesis of N-protected amino thioacids from the corresponding N-protected amino acids, oxidative dimerization of thioacids, crystal conformations of thioacid oxidative dimers, and the utility of thioacids and oxidative dimers in peptide synthesis. Our results suggest that peptides can be synthesized without using standard coupling agents.
Assuntos
Aminoácidos/química , Peptídeos/síntese química , Compostos de Sulfidrila/síntese química , Compostos de Enxofre/química , Estrutura Molecular , Oxirredução , Peptídeos/química , Compostos de Sulfidrila/químicaRESUMO
N-Acylated amines are ubiquitous in nature. Selective N-acylation at neutral conditions remains a key area of interest. Here we are reporting the copper sulfate-mediated highly selective, mild, and rapid N-acylation of various aliphatic and aromatic amines using thioacids in methanol at neutral conditions. All N-acylated products of primary and secondary amines were isolated in good to excellent yields. This method is found to be highly selective for the amines and not sensitive to other functional groups such as phenols, alcohols, and thiols. The simple workup, high yields, and high selectivity of this reaction can be an attractive alternative to those of the existing acyl halide- and acid anhydride-mediated N-acylation reactions.
Assuntos
Ácidos/química , Aminas/síntese química , Compostos de Sulfidrila/química , Acilação , Aminas/química , Estrutura MolecularRESUMO
The high diastereoselectivity in the Michael addition of nitromethane to α,ß-unsaturated γ-amino esters, crystal conformations of ß-nitromethane substituted γ-amino acids and peptides are studied. Results suggest that the N-Boc protected amide NH, conformations of α,ß-unsaturated γ-amino esters and alkyl side chains play a crucial role in dictating the high diastereoselectivity of nitromethane addition to E-vinylogous amino esters. Investigation of the crystal conformations of both α,ß-unsaturated γ-amino esters and the Michael addition products suggests that an H-C(γ)-C(ß)=C(α) eclipsed conformer of the unsaturated amino ester leads to the major (anti) product compared to that of an N-C(γ)-C(ß)=C(α) eclipsed conformer. The major diastereomers were separated and subjected to the peptide synthesis. The single crystal analysis of the dipeptide containing ß-nitromethane substituted γ-amino acids reveals a helical type of folded conformation with an isolated H-bond involving a nine-atom pseudocycle.
Assuntos
Aminoácidos/química , Metano/análogos & derivados , Nitroparafinas/química , Peptídeos/química , Aminoácidos/síntese química , Cristalografia por Raios X , Ésteres/síntese química , Ésteres/química , Metano/síntese química , Metano/química , Modelos Moleculares , Nitroparafinas/síntese química , Peptídeos/síntese química , EstereoisomerismoRESUMO
A novel, ultrafast, mild and scalable amide bond formation strategy in methanol using simple thioacids and amines is described. The mechanism suggests that the coupling reactions are initially mediated by CuSO(4)·5H(2)O and subsequently catalyzed by in situ generated copper sulfide. The pure peptides were isolated in satisfactory yields in less than 5 minutes.