Use of an Asparaginyl Endopeptidase for Chemo-enzymatic Peptide and Protein Labeling (original) (raw)
Related papers
2019
Asparaginyl endopeptides (AEP) are recognized for their catalytic efficiency, presenting as ideal tools for protein bioconjugation. However, the peptide ligation catalyzed by AEP is reversible. In an attempt to obtain high reaction yields, thiodepsipeptides have been used as substrates but found to be highly unstable, and labeling is only limited to the N-terminus. To maximize the potential use of AEP, here we developed a novel chemo-enzymatic sequence for protein bioconjugation at both the N- and C-termini. In this system, an alternative recognition sequence, Asn-Cys-Leu, was used. Upon ligation, the reaction yields Cys-Leu as leaving group, and its reactive 1,2-aminothiol functionality was quenched by an effective and affordable electrophile, 2-formyl phenylboronic acid (FPBA), to yield a non-reactive cyclic byproduct. In the presence of FPBA our model reaction proceeds with ~95% yield using only 1.2 equivalent of substrate, whereas the yield remains at ~50% in the absence of this...
Bioconjugate chemistry, 2018
Peptides with an N-terminal cysteine residue allow site-specific modification of proteins and peptides and chemical synthesis of proteins. They have been widely used to develop new strategies for imaging, drug discovery, diagnostics, and chip technologies. Here we present a method to produce recombinant peptides with an N-terminal cysteine residue as a convenient alternative to chemical synthesis. The method is based on the release of the desired peptide from a recombinant fusion protein by mild acid hydrolysis of an Asp-Cys sequence. To test the general validity of the method we prepared four fusion proteins bearing three different peptides (20-37 amino acid long) at the C-terminus of a ketosteroid isomerase-derived and two Onconase-derived carriers for the production of toxic peptides in E. coli. The chosen peptides were (C)GKY20, an antimicrobial peptide from the C-terminus of human thrombin, (C)ApoB, an antimicrobial peptide from an inner region of human Apolipoprotein B, and (C...
Direct Production of Proteins with N-Terminal Cysteine for Site-Specific Conjugation
Bioconjugate Chemistry, 2004
Proteins with N-terminal cysteine can undergo native chemical ligation and are useful for site-specific N-terminal labeling or protein semisynthesis. Recombinant production of these has usually been by site-specific cleavage of a precursor fusion protein at an internal cysteine residue. Here we describe a simpler route to producing these proteins. Overexpression in E. coli of several proteins containing cysteine as the second amino acid residue yielded products in which the intiating methionine residue had been completely cleaved by endogenous methionine aminopeptidase. While secondary modification of the terminal cysteine was a complicating factor, conditions were identified to eliminate or minimize this problem. Recombinant proteins produced in this way were suitable for site-specific modification of the amino terminus via native chemical ligation technology, as demonstrated by conjugation of a thioester-containing derivative of fluorescein to one such protein. The ability to directly produce proteins with N-terminal cysteine should simplify the application of native chemical ligation technology to recombinant proteins and make the technique more amenable to researchers with limited expertise in protein chemistry.
Natural Occurring and Engineered Enzymes for Peptide Ligation and Cyclization
Frontiers in Chemistry, 2019
The renaissance of peptides as prospective therapeutics has fostered the development of novel strategies for their synthesis and modification. In this context, besides the development of new chemical peptide ligation approaches, especially the use of enzymes as a versatile tool has gained increased attention. Nowadays, due to their inherent properties such as excellent regio-and chemoselectivity, enzymes represent invaluable instruments in both academic and industrial laboratories. This mini-review focuses on natural-and engineered peptide ligases that can form a new peptide (amide) bond between the C-terminal carboxy and N-terminal amino group of a peptide and/or protein. The pro's and cons of several enzyme classes such as Sortases, Asparaginyl Endoproteases, Trypsin related enzymes and as a central focus subtilisin-derived variants are summarized. Most recent developments with regards to ligation and cyclization are highlighted.
Analytical Biochemistry, 2011
Protease inhibitors represent a major class of drugs, even though a large number of proteases remain unexplored. Consequently, a great interest lies in the identification of highly sensitive substrates useful for both the characterization and the validation of these enzyme targets and for the design of inhibitors as potential therapeutic agents through high-throughput screening (HTS). With this aim, a synthetic substrate library, in which the highly fluorescent (L)-pyrenylalanine residue (Pya) is efficiently quenched by its proximity with the p-nitro-(L)-phenylalanine (Nop) moiety, was designed. The cleavage between Pya and Nop leads to a highly fluorescent metabolite providing the required sensitivity. This library, characterized by a water-soluble primary sequence Ac-SGK-Pya-(X)(n)(-)Nop-GGK-NH(2), X being a mixture of 10 natural amino acids (A, I, L, K, F, W, E, Q, T, P) and n varying from 0 to 3, was validated using enzymes belonging to the four main types of hydrolases: serine-, metallo-, cystein-, and aspartyl-proteases. The selectivity of substrates belonging to this library was evidenced by characterizing specific substrates for the isoenzymes NEP-1 and NEP-2. This library easily synthesized is of great interest for the identification and development of selective and specific substrates for still uncharacterized endoproteases.
Angewandte Chemie International Edition, 2002
Proteins with N-terminal cysteines are useful in a wide range of biotechnological applications ranging from protein semi-synthesis to site-specific N-terminal labeling. Peptides and proteins with N-terminal cysteines undergo native chemical ligation and expressed protein ligation reactions with thioesters to form native peptide bonds. These reactions have been used to extend the size of proteins that can be synthesized chemically and to incorporate synthetic peptides with modifications and labels into expressed proteins. [3±8] In addition, proteins with N-terminal cysteines also react chemoselectively with aldehydes to form thiazolidines, and this reaction has been utilized to label and immobilize peptides and proteins. [9±12] Here we present a novel method ( ) to produce proteins with N-terminal cysteines by Scheme 5. A new approach to the synthesis of Milnacipran (11). ethyl formate to give 13 (73 % yield of isolated product), a known Milnacipran precursor. Aromatic and benzyl CH groups are more acidic than those of cyclopropanes and cubanes, so it is not surprising that BuMgDA stoichiometrically metalates an amide-activated CH group appropriately positioned thereupon. What if the CH group is less acidic? Cyclobutane CH is kinetically 2500 times more weakly acidic than cyclopropane CH. Under conditions as harsh as most base/solvent combinations can withstand (e.g. LiTMP/THF or LiTMP Hg(TMP) 2 /THF at 08 C (TMP 2,2,6,6-tetramethylpiperidine) or LDA/THF at reflux), there is no observed metalation of 14. However, under the conditions of our preliminary trials, reaction of 14 with excess BuMgDA in THF at reflux for 5 h gave about 20 % conversion to the cis-b-metalated compound 15, identified by formation of ester 16 (17 % yield of isolated product, Scheme 6). Although still to be optimized, this b deprotonation/metalation of a cyclobutane amide is extraordinary and unprecedented. Scheme 6. Deprotonation/magnesiation of an amide-activated cyclobutane using BuMgDA.
Efficient site-specific labeling of proteins via cysteines
Bioconjugate chemistry, 2008
Methods for chemical modifications of proteins have been crucial for the advancement of proteomics. In particular, site-specific covalent labeling of proteins with fluorophores and other moieties has permitted the development of a multitude of assays for proteome analysis. A common approach for such a modification is solvent-accessible cysteine labeling using thiol-reactive dyes. Cysteine is very attractive for site-specific conjugation due to its relative rarity throughout the proteome and the ease of its introduction into a specific site ...
Chemical Science, 2018
C-Terminal cysteine peptide acids are difficult to access without epimerization of the cysteine astereocenter. Diversification of the C-terminus after solid-phase peptide synthesis poses an even greater challenge because of the proclivity of the cysteine a-stereocenter to undergo deprotonation upon activation of the C-terminal carboxylic acid. We present herein two general strategies to access Cterminal cysteine peptide derivatives without detectable epimerization, diketopiperazine formation, or piperidinylalanine side products. C-Terminal cysteine peptides, including prenylated and farnesylated peptides, 1 disulde linked peptide toxins, 2 and insulinotropic peptides, 3,4 comprise an important but synthetically challenging class of biologically active peptides. Many of these peptides are modied at the C-terminus. C-terminal modications such as esters and amides can be critical to maintaining a peptide's active conformation, 5 in vivo activity, and pharmacokinetics; 6 therefore, the ability to vary the peptide structure in this location is crucial to drug development efforts. 7 Although several methods have been reported for C-terminal functionalization aer solid-phase peptide synthesis (SPPS) is complete, 8 these approaches either result in epimerization when applied to C-terminal Cys peptides 9 or the applicability of the method to Cterminal Cys peptides is not addressed. 10,11 While activation of the C-terminal carboxylic acid can induce epimerization via oxazolone formation in most amino acids, 12 cysteine is also prone to epimerization via direct deprotonation during its attachment to the resin 13 and upon prolonged or repeated exposure to base (i.e., during peptide elongation via Fmoc SPPS). 14 Therefore, even the preparation of simple carboxylic acids or carboxamides of C-terminal cysteine peptides can be fraught with contamination by epimerized products, 1f,g,13a,15 reducing the overall yield and complicating the purication of the target peptides. A method for the epimerization-free synthesis and subsequent C-terminal modication of C-terminal Cys peptides would be highly impactful. In this work, we report the rst mild and convenient method for the epimerization-free diversication of peptides bearing a Cterminal cysteine. 16 Carboxylic acids, primary and secondary amides, and esters are accessed without epimerization or formation of diketopiperazine and piperidinyl-alanine side products. 17 We apply this strategy to the total synthesis of the nicotinic acetylcholine receptor (nAChR) antagonist a-conotoxin ImI. 18 Additionally, we include an alternate strategy employing Ndeprotected cysteine derivatives as nucleophiles, and we demonstrate its utility via the synthesis of the insect pheromone a-factor. 1 In the context of our ongoing efforts toward the synthesis of disulde-linked aand m-conotoxins, 19,20 we were concerned about possible epimerization of the C-terminal cysteine during the SPPS. We recently reported a strategy for C-terminal functionalization of non-cysteine peptides involving activation of the methyl-diaminobenzoyl (MeDbz) linker (1 / 2) 21 followed by nucleophilic cleavage of the N-acyl urea (MeNbz) group 22 to yield various protected (3) or unprotected (4) peptides (Scheme 1). 23 If this approach were to prove mild enough to enable Scheme 1 Our strategy for C-terminal functionalization of non-Cys terminated peptides.