Pyruvoyl, a novel amino protecting group on the solid phase peptide synthesis and the peptide condensation reaction

In one of the peptide condensation methods termed thioester method, an amino protecting group is required in the lysine side chain. In this study, to investigate the efficiency of the pyruvoyl group as an amino protecting group, we synthesized N^α-fluorenylmethoxycarbonyl (Fmoc)-N^ε-pyruvoyl-lysine and introduced it into peptides and glycopeptides by the ordinary Fmoc-based solid phase peptide synthesis. The pyruvoyl peptide could be condensed with a peptide thioester by the thioester method, and this protecting group was easily removed by o-phenylenediamine treatment without significant side reactions.     

Fluorogenic probes for detecting deacylase and demethylase activity towards post-translationally-modified lysine residues

Reversible enzymatic post-translational modification of the ε-amino groups of lysine residues (e.g. N-acylation reactions) plays an important role in regulating the cellular activities of numerous proteins. This study describes how enzyme catalyzed N-deprotection of lysine residues of non-fluorescent peptide-coumarin probes can be used to generate N-deprotected peptides that undergo spontaneous O- to N-ester transfer reactions (uncatalyzed) to generate a highly fluorescent N-carbamoyl peptide. This enables detection of enzyme catalyzed N-deacetylation, N-demalonylation, N-desuccinylation and N-demethylation reactions activities towards the N-modified lysine residues of these probes using simple ‘turn on’ fluorescent assays.

We developed “turn-on” fluorescent probes that detect enzymatic lysine deacylation and demethylation critical for epigenetic and other cellular phenomena, using intramolecular O- to N-ester transfer reactions.     

Selective Deletion of the Internal Lysine Residue from the Peptide Sequence by Collisional Activation

The gas-phase peptide ion fragmentation chemistry is always the center of attraction in proteomics to analyze the amino acid sequence of peptides and proteins. In this work, we describe the formation of an anomalous fragment ion, which corresponds to the selective deletion of the internal lysine residue from a series of lysine containing peptides upon collisional activation in the ion trap. We detected several water-loss fragment ions and the maximum number of water molecules lost from a particular fragment ion was equal to the number of lysine residues in that fragment. As a consequence of this water-loss phenomenon, internal lysine residues were found to be deleted from the peptide ion. The N,N-dimethylation of all the amine functional groups of the peptide stopped the internal lysine deletion reaction, but selective N-terminal ??-amino acetylation had no effect on this process indicating involvement of the side chains of the lysine residues. The detailed mechanism of the lysine deletion was investigated by multistage CID of the modified and unmodified peptides, by isotope labeling and by energy resolved CID studies. The results suggest that the lysine deletion might occur through a unimolecular multistep mechanism involving a seven-membered cyclic imine intermediate formed by the loss of water from a lysine residue in the protonated peptide. This intermediate subsequently undergoes degradation reaction to deplete the interior imine ring from the peptide backbone leading to the deletion of an internal lysine residue.     

Peptide thioester preparation based on an N-S acyl shift reaction mediated by a thiol ligation auxiliary

Formation of peptide thioesters, based on an N to S acyl shift mediated by an auxiliary, N-4,5-dimethoxy-2-mercaptobenzyl (Dmmb) group, under acidic conditions, is described. The protected peptide was assembled on a hydroxymethylphenylacetamidomethyl resin via an N-Dmmb-amino acid residue according to standard Fmoc solid-phase peptide synthesis following treatment with trifluoroacetic acid. The peptide α-thioester was released from the resin by reaction with 2-mercaptoethanesulfonic acid in the presence of N,N-diisopropylethylamine.     

An N-protection free ligation of the peptide thioester and the peptide with an N-alkoxy- or N-aryloxyamino group at its N-terminus

Peptides with an N-alkoxy or N-aryloxy amino acid at their N-terminus were synthesized and successfully ligated with a peptide thioester by silver ion activation under a slightly acidic condition without requiring protection of the side chain amino groups. The N-methoxy group was easily cleaved by the SmI₂ reduction in CH₃OH aq. to obtain the desired peptide with a native peptide bond. This method was successfully applied to the synthesis of the human atrial natriuretic peptide showing the efficiency of the novel ligation.     

A new protecting group for tryptophan in solid-phase peptide synthesis which protects against acid-catalyzed side reactions and facilitates purification by HPLC

The indole nucleus of Z-Trp-OBzl is modified by acylation of the indole nitrogen using Boc-N-methyl butyric acid followed by catalytic hydrogenation and introduction of the Fmoc group. The resulting derivative, Fmoc-Trp(Boc-Nmbu)-OH, is incorporated into peptide chains via solid-phase peptide synthesis (SPPS). After assembly of the peptide chain, the Boc group is cleaved by treatment with TFA. The peptide is isolated with the tryptophan residue modified with a cationic 4-(N-methylamino) butanoyl group, which improves the solubility of the peptide during HPLC purification. On treatment of the purified peptide at pH 9.5, the Nmbu group undergoes an intramolecular cyclization reaction; this results in the fully deprotected peptide and N-methylpyrrolidone.     

Aqueous synthesis of sialylglycopeptide-grafted glycopolymers with high affinity for the lectin and the influenza virus hemagglutinin

Glycopolymers with pendant complex-type sialyl N-glycans containing heptapeptides, that is, sialylglycopeptides (SGPs), were synthesized using a water soluble polymer backbone bearing N-hydroxysulfosuccinimidyl esters by post-polymerization modification in water. Although SGP has three amino groups on the peptide chain, the substitution reaction occurs preferentially at the N-terminus α-amino group in the lysine residue onto the polymer side chain because the reactivity of such α-amino group is higher than that of the ε-amino group in the lysine residue under mild acidic aqueous condition. The resulting SGP-grafted glycopolymers exhibited strong interaction with the lectin Sambucus sieboldiana agglutinin and the human influenza A virus hemagglutinin, with higher binding associate constant values than those of free saccharide according to quartz crystal microbalance analysis. © 2020 Wiley Periodicals, Inc. J. Polym. Sci. 2020 , 58, 548–556     

A carbamoyl-protective group for tyrosine that facilitates purification of hydrophobic synthetic peptides

The Boc-N-methyl-N-[2-(methylamino)ethyl]carbamoyl group (Boc-Nmec) is reported as a new side chain-protective group for tyrosine in Fmoc solid-phase peptide synthesis. Tyrosine is incorporated into the peptide as Fmoc-Tyr(Boc-Nmec)-OH by standard coupling methods. During the cleavage of the peptide from the resin with TFA the Boc group is simultaneously cleaved while the cationic N-methyl-N-[2-(methylamino)ethyl]carbamoyl group remains attached to the tyrosine residue, thereby increasing the solubility of the peptide. After purification of the peptide, the Nmec protective group can be cleaved under neutral or mild alkaline conditions via an intramolecular cyclization reaction.     

Collision-Induced Dissociation of Diazirine-Labeled Peptide Ions. Evidence for Brønsted-Acid Assisted Elimination of Nitrogen

Gas-phase dissociations were investigated for several peptide ions containing the Gly-Leu* N-terminal motif where Leu* was a modified norleucine residue containing the photolabile diazirine ring. Collisional activation of gas-phase peptide cations resulted in facile N₂ elimination that competed with backbone dissociations. A free lysine ammonium group can act as a Brønsted acid to facilitate N₂ elimination. This dissociation was accompanied by insertion of a lysine proton in the side chain of the photoleucine residue, as established by deuterium labeling and gas-phase sequencing of the products. Electron structure calculations were used to provide structures and energies of reactants, intermediates, and transition states for Gly-Leu*-Gly-Gly-Lys amide ions that were combined with RRKM calculations of unimolecular rate constants. The calculations indicated that Brønsted acid-catalyzed eliminations were kinetically preferred over direct loss of N₂ from the diazirine ring. Mechanisms are proposed to explain the proton-initiated reactions and discuss the reaction products. The non-catalyzed diazirine ring cleavage and N₂ loss is proposed as a thermometer dissociation for peptide ion dissociations.

N-trifluoroacyl lysine derivatives in the synthesis of L-Lysyl-L-glutamic acid

Conditions were developed for simultaneous preparation of N ^å-trifluoroacetyl-L-lysine and N ^α,N ^å-bis(trifluoroacetyl)-L-lysine at overall conversion of initial lysine monohydrochloride up to 82%. By reaction of dimethyl L-glutamate with N ^α,N ^å-bis(trifluoroacetyl)-L-lysyl chloride in the presence of triethylamine or with N ^α-carboxyanhydride of N ^å-trifluoroacetyl-L-lysine with subsequent removing protecting groups in the formed dipeptides by treating with water-ethanol solution of sodium hydroxide we obtained L-lysyl-L-glutamic acid. Physicochemical characteristics of samples obtained coincided with characteristics of L-lysyl-L-glutamic acid described in the literature thus suggesting that no racemization occurred either at the stage of peptide bond formation or at deprotection.     

Facile solid phase synthesis of N-cycloguanidinyl-formyl peptides

A novel strategy of solid phase synthesis of N-cycloguanidinyl-formyl peptides has been established and investigated which involved coupling orthogonal protected diaminoacid with resin bound peptide, α-amino group deprotection, guanidinylation of α-amino group by bis-Cbz-1H-pyrazole-1-carboxamidine followed by cleavage and cyclization in solution, and finally removing Cbz by palladium catalyzed hydrogenation. Through this method, cycloguanidine could be introduced to either N-terminus or sidechain of designated peptides. The reaction conditions were facile, straightforward, and totally adaptive to common solid phase peptide synthesis strategy.     

Synthesis and ring‐opening (co)polymerization of L‐lysine N‐carboxyanhydrides containing labile side‐chain protective groups

This contribution describes the synthesis and ring‐opening (co)polymerization of several L ‐lysine N‐carboxyanhydrides (NCAs) that contain labile protective groups at the ?‐NH₂ position. Four of the following L ‐lysine NCAs were investigated: N^?‐trifluoroacetyl‐L ‐lysine N‐carboxyanhydride, N^?‐(tert‐butoxycarbonyl)‐L ‐lysine N‐carboxyanhydride, N^?‐(9‐fluorenylmethoxycarbonyl)‐L ‐lysine N‐carboxyanhydride, and N^?‐(6‐nitroveratryloxycarbonyl)‐L ‐lysine N‐carboxyanhydride. In contrast to the harsh conditions that are required for acidolysis of benzyl carbamate moieties, which are usually used to protect the ?‐NH₂ position of L ‐lysine during NCA polymerization, the protective groups of the L ‐lysine NCAs presented here can be removed under mildly acidic or basic conditions or by photolysis. As a consequence, these monomers may allow access to novel peptide hybrid materials that cannot be prepared from ?‐benzyloxycarbonyl‐L ‐lysine N‐carboxyanhydride (Z‐Lys NCA) because of side reactions that accompany the removal of the Z groups. By copolymerization of these L ‐lysine NCAs with labile protective groups, either with each other or with γ‐benzyl‐L ‐glutamate N‐carboxyanhydride or Z‐Lys NCA, orthogonally side‐chain‐protected copolypeptides with number‐average degrees of polymerization ≤20 were obtained. Such copolypeptides, which contain different side‐chain protective groups that can be removed independently, are interesting for the synthesis of complex polypeptide architectures or can be used as scaffolds for the preparation of synthetic antigens or protein mimetics. © 2003 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 41: 1167–1187, 2003     

A Tamao–Fleming oxidation route to dipeptides bearing N,O-acetal functionality

Tamao–Fleming oxidation of the N-dimethylphenylsilylmethyl group linked to the nitrogen of a peptide bond enables access to dipeptide N,O-acetal functionality. The N-silylmethyl functionality serves as a latent form of the N,O-acetal which is revealed after peptide bond construction.     

Facile synthesis of N-(benzyl, methyl)-lysine as a building block for site-specifically lysine monomethylated peptides

Facile, mild and efficient one-pot preparation of N^α-Fmoc-N^ε-(benzyl, methyl)-lysine, a building block for monomethylated peptide synthesis, was described. This building block was proved to be efficient for the synthesis of site-specifically monomethylated peptide. Benzyl group, which was incorporated by reductive benzylation and removed via catalytic hydrogenolysis, served as an excellent protecting group.     

Hyperbranched poly(L-lysine) modified with histidine residues via lysine terminal amino groups: Synthesis and structure

A method for preparing hyperbranched poly(L-lysine) via the polymerization of N^?-carbobenzoxy-L-lysine-N-carboxyanhydride has been developed in order to regulate the molecular mass and size of this polymer and to modify amino groups of its N-terminal lysine residues. This method includes the reductive removal of an N^?-carbobenzoxy group by hydrogen over activated palladium in the presence of a chain termination agent, which is the activated ether of N^α-tert-butyloxycarbonylhistidine. The structure of the polymers has been studied by capillary electrophoresis, circular dichroism, and molecular hydrodynamics.     

Synthesis of polyglutamine conjugates of lysine dendrimers in solution and polymer gel

The synthesis of polyglutamine conjugates of lysine dendrimers, in which carboxyl groups of polyglutamine chains are linked via one point to the amino groups of N-terminal lysine fragments of dendrimers, is studied. Lysine dendrimers of third, fourth, and fifth generations are preliminarily prepared via the solid-phase synthesis on a polymer support, crosslinked p-methylbenzohydrylaminopolystyrene. The first approach to the synthesis of polyglutamine conjugates of dendrimers consisted in that, after the synthesis of lysine dendrimers, their removal from the polymer support, their full unblocking, and their purification, γ-benzyl glutamate N-carboxyadhydride is involved in solution polymerization at the amino groups of lysine of the outer sphere of dendrimers. The second approach includes the polymerization of γ-benzyl glutamate N-carboxyadhydride on the amino groups of N-terminal lysine residues of lysine dendrimers performed before dendrimer removal from the polymer support. The structural study of star-shaped conjugate of lysine dendrimers makes it possible to for the first time estimate the similarities and differences in these two approaches to the synthesis of polyglutamine conjugates of lysine dendrimers.     

The effect of glutamic acid side chain on acidity constant of lysine in beta-sheet: A density functional theory study

In this work, the possibility of proton transfer between side chain of lysine and glutamic acid in peptide of Glu^?-Ala-Lys⁺ was demonstrated using density functional theory (DFT). We have shown that the proton transfer takes place between side chain of glutamic and lysine residues through the hydrogen bond formation. The structures of transition state for proton transfer reaction were detected in gas and solution phases. Our kinetic studies show that the proton transfer reaction rate in gas phase is higher than solution phase. The ionization constant (pK _a) value of lysine residue in peptide was estimated 1.039 which is lower than intrinsic pK _a of lysine amino acid.     

Hyperbranched polylysines: Mechanism of formation

To gain insight into the mechanism treating formation of hyperbranched polylysines through the polymerization of N ^?-carbobenzoxylysine N-carboxyanhydride under conditions of the reductive removal of a N ^?-carbobenzoxy group, hyperbranched polylysine has been synthesized with the use of trifluoroacetic acid as a terminator in the polymerization of N-carboxyanhydride. The structure of the polymers is studied by capillary electrophoresis, low-pressure gel-permeation chromatography, circular dichroism, and enzymatic hydrolysis with trypsin. At the first stage of synthesis, a low-molecular-mass strongly branched core of the polymer is formed. At the second stage, polylysine chains are grafted via one point onto amino groups of N-terminal lysine moieties of the low-molecular-mass core through their carboxyl ends.     

Peptide Scrambling During Collision-Induced Dissociation is Influenced by N-terminal Residue Basicity

‘Bottom up’ proteomic studies typically use tandem mass spectrometry data to infer peptide ion sequence, enabling identification of the protein whence they derive. The majority of such studies employ collision-induced dissociation (CID) to induce fragmentation of the peptide structure giving diagnostic b-, y-, and a- ions. Recently, rearrangement processes that result in scrambling of the original peptide sequence during CID have been reported for these ions. Such processes have the potential to adversely affect ion accounting (and thus scores from automated search algorithms) in tandem mass spectra, and in extreme cases could lead to false peptide identification. Here, analysis of peptide species produced by Lys-N proteolysis of standard proteins is performed and sequences that exhibit such rearrangement processes identified. The effect of increasing the gas-phase basicity of the N-terminal lysine residue through derivatization to homoarginine toward such sequence scrambling is then assessed. The presence of a highly basic homoarginine (or arginine) residue at the N-terminus is found to disfavor/inhibit sequence scrambling with a coincident increase in the formation of b_(n-1)+H₂O product ions. Finally, further analysis of a sequence produced by Lys-C proteolysis provides evidence toward a potential mechanism for the apparent inhibition of sequence scrambling during resonance excitation CID. Graphical Abstract

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Peptide affinity chromatography media that bind N fusion proteins under chaotropic conditions

To design a generic purification platform and to combine the advantages of fusion protein technology and matrix-assisted refolding, a peptide affinity medium was developed that binds inclusion body-derived N^pro fusion proteins under chaotropic conditions. Proteins were expressed in Escherichia coli using an expression system comprising the autoprotease N^pro from Pestivirus, or its engineered mutant called EDDIE, with C-terminally linked target proteins. Upon refolding, the autoprotease became active and cleaved off its fusion partner, forming an authentic N-terminus. Peptide ligands binding to the autoprotease at 4 M urea were screened from a combinatorial peptide library. A group of positive peptides were identified and further refined by mutational analysis. The best binders represent a common motif comprising positively charged and aromatic amino acids, which can be distributed in a random disposition. Mutational analysis showed that exchange of a single amino acid within the peptide ligand caused a total loss of binding activity. Functional affinity media comprising hexa- or octapeptides were synthesized using a 15-atom spacer with terminal sulfhydryl function and site-directed immobilization of peptides derivatized with iodoacetic anhydride. The peptide size was further reduced to dipeptides comprising only one positively charged and one aromatic amino acid. Based on this, affinity media were prepared by immobilization of a poly amino acid comprising lysine or arginine, and tryptophan, phenylalanine, or tyrosine, respectively, in certain ratios. Binding capacities were in the range of 7–15 mg protein mL⁻¹ of medium, as could be shown for several EDDIE fusion proteins. An efficient protocol for autoproteolytic cleavage using an on-column refolding method was implemented.