C-terminal Hydrazide Research Articles

Oxidation and Phenolysis of Peptide/Protein C-Terminal Hydrazides Afford Salicylaldehyde Ester Surrogates for Chemical Protein Synthesis.

With the growing popularity of serine/threonine ligation (STL) and cysteine/penicillamine ligation (CPL) in chemical protein synthesis, facile and general approaches for the preparation of peptide salicylaldehyde (SAL) esters are urgently needed, especially those viable for obtaining expressed protein SAL esters. Herein, we report the access of SAL ester surrogates from peptide hydrazides (obtained either synthetically or recombinantly) via nitrite oxidation and phenolysis by 3-(1,3-dithian-2-yl)-4-hydroxybenzoic acid (SAL(-COOH)PDT). The resulting peptide SAL(-COOH)PDT esters can be activated to afford the reactive peptide SAL(-COOH) esters for subsequent STL/CPL. While being operationally simple for both synthetic peptides and expressed proteins, the current strategy facilitates convergent protein synthesis and combined application of STL with NCL. The generality of the strategy is showcased by the N-terminal ubiquitination of the growth arrest and DNA damage-inducible protein (Gadd45a), the efficient synthesis of ubiquitin-like protein 5 (UBL-5) via a combined N-to-C NCL-STL strategy, and the C-to-N semisynthesis of a myoglobin (Mb) variant.

A Shelf Stable Fmoc Hydrazine Resin for the Synthesis of Peptide Hydrazides.

C-terminal hydrazides are an important class of synthetic peptides with an ever expanding scope of applications, but their widespread application for chemical protein synthesis has been hampered due to the lack of stable resin linkers for synthesis of longer and more challenging peptide hydrazide fragments. We present a practical method for the regeneration, loading, and storage of trityl-chloride resins for the production of hydrazide containing peptides, leveraging 9-fluorenylmethyl carbazate. We show that these resins are extremely stable under several common resin storage conditions. The application of these resins to solid phase peptide synthesis (SPPS) is demonstrated through the synthesis of the 40-mer GLP-1R agonist peptide "P5". These studies support the broad utility of Fmoc-NHNH-Trt resins for SPPS of C-terminal hydrazide peptides.

Asparaginyl Endopeptidase-Mediated Protein C-Terminal Hydrazinolysis for the Synthesis of Bioconjugates.

Asparaginyl endopeptidases (AEPs) are cysteinyl enzymes naturally catalyzing the hydrolysis and transpeptidation reactions at Asx-Xaa bonds. These reactions go by a common acyl-enzyme thioester intermediate, which is either attacked by water (for a protease-AEP) or by a peptidic amine nucleophile (for a ligase-AEP) to form the respective hydrolysis or aminolysis product. Herein, we show that hydrazine and hydroxylamine, two α-effect nucleophiles, are capable of resolving the thioester intermediate to yield peptide and protein products containing a C-terminal hydrazide and hydroxamic acid functionality, respectively. The hydrazinolysis reaction exhibits very high efficiency and can be completed in minutes at a low enzyme-to-substrate ratio. We further show the utility of the so-formed asparaginyl hydrazide in native chemical ligation and hydrazone conjugation. Using an EGFR-targeting affibody as a model protein, we have showcased our methodology in the preparation of a number of protein ligation or conjugation products, which are decorated with various functional moieties. The ZEGFR affibody-doxorubicin conjugate shows high selective binding and cytotoxicity toward the EGFR-positive A431 cells. Our results demonstrate the advantages of AEP-mediated protein hydrazinolysis as a simple and straightforward strategy for the precision manufacturing of protein bioconjugates.

A facile method for vancomycin C-terminus functionalization and derivatization through hydrazide

Over 60-year clinical use of vancomycin led to the emergence of vancomycin-resistant bacteria and threatened our health. To combat vancomycin-resistant strains, numerous vancomycin analogues were developed, such as Telavancin, Oritavancin and Dalbavancin. Extra structures embedded on C-terminus has been proved to be an effective strategy to promote antibacterial activity of vancomycin against vancomycin-resistant strains. Here, we reported a facile strategy, inspired by native chemical ligation, for vancomycin C-terminus functionalization and derivatization. The introduction of C-terminal hydrazide on vancomycin not only provided us an accessible method for C-terminus functionalization through carbonyl azide and thioester, also acted as an efficient site for vancomycin structure modifications. Based on hydrazide-vancomycin, we effectively conjugated cysteine and cysteine containing peptides onto vancomycin C-terminus, and two fluorescent FITC-vancomycin were prepared through Cys-Maleimide conjugation. Meanwhile, we introduced lipophilic structures onto vancomycin C-terminus via the hydrazide moiety. The obtained vancomycin derivatives were evaluated against both Gram-positive and negative bacteria strains.

The spinal anti-allodynic effects of endomorphin analogs with C-terminal hydrazide modification in neuropathic pain model

The present study was undertaken to further investigate the spinal anti-allodynic effects of endomorphins (EMs) and their C-terminal hydrazide modified analogs EM-1-NHNH2 and EM-2-NHNH2 in the spared nerve injury (SNI) model of neuropathic pain in mice. Our results demonstrated that intrathecal (i.t.) administration of endomorphin-1 (EM-1), endomorphin-2 (EM-2), EM-1-NHNH2 and EM-2-NHNH2 produced potent anti-allodynic effects ipsilaterally in neuropathic pain model. Judging from the area under the curve (AUC) values, these two analogs exhibited higher antinociception than their parent peptides. Moreover, they also displayed significant antinociceptive effects in the contralateral paw administered intrathecally. Interestingly, EM-1 and its analog EM-1-NHNH2 displayed their antinociception probably by μ2-opioid receptor subtype since the μ1-opioid receptor antagonist naloxonazine didn’t significantly block the anti-allodynia of EM-1 and EM-1-NHNH2, which implied a same opioid mechanism. However, the anti-allodynia induced by EM-2, but not EM-2-NHNH2 was significantly reduced by both μ1-opioid antagonist, naloxonazine and κ-antagonist, nor-binaltorphamine (nor-BNI), indicating multiple opioid receptors were involved in the anti-allodynic effects of EM-2. Most importantly, EM-1-NHNH2 decreased the antinociceptive tolerance, and EM-2-NHNH2 displayed non-tolerance-forming antinociception. Therefore, C-terminal amide to hydrazide conversion changed the spinal antinociceptive profiles of EMs in neuropathic pain. The present investigation is of great value in the development of novel opioid therapeutics against neuropathic pain.

Strategies for the highly efficient synthesis of erythropoietin N-glycopeptide hydrazides.

A convergent synthesis for erythropoietin (EPO) 1-28 N-glycopeptide hydrazides was developed. In this approach, EPO 1-28 peptides were synthesized on the solid phase and converted to C-terminal hydrazides after cleavage from the resin. After selective deprotection of the Asp24 side chain, the desired glycosylamine was coupled by pseudoproline-assisted Lansbury aspartylation. Although the initial yields of the EPO 1-28 glycopeptides were satisfactory, they could be markedly improved by increasing the purity of the peptide using a reversed-phase high-performance liquid chromatography (RP-HPLC) purification of the protected peptide.

Cover Feature: Efficient Generation of Hydrazides in Proteins by RadA Split Intein (ChemBioChem 3/2020)

Hydrazides at proteins’ C termini are much desired because of their straightforward use for bioconjugations and chemical modifications. However, traditional preparation methods often lack the necessary biocompatibility. In their full paper, J. Liu, S. Rozovsky, et al. on page 346 in Issue 3, 2020 (DOI: 10.1002/cbic.201900160) show that combining thiols and hydrazine under mild conditions results in the high-yield generation of such C-terminal hydrazides. The addition of thiols not only accelerates the formation of the transient thioester intermediate, and the subsequent hydrazinolysis that promotes hydrazide formation at proteins’ C terminus, but also alleviates the problem of destructive protein aggregation. Employing split inteins to trigger hydrazinolysis provides an additional level of control of this now biocompatible generation method.

New C-terminal hydrazide L-Leucine derivatives as multi-solvent low molecular weight organogelators

Three new low-molecular-weight gelators (LMWGs) based on L-Leucine derivatives bearing different chain lengths (i.e. 9, 12 or 16) were designed and synthesized (designated as 1-3). The N-terminal is carrying the various alkyl chain lengths where the C-terminal is modified with hydrazide moiety. The prepared gelators 1-3 were fully characterized using 1H, 13C NMR, FTIR and mass spectroscopy. The gelation behavior has also been investigated using different organic solvents and oils with determination of critical gelation concentration (CGC). The self-assembly process was investigated by recording FTIR spectra gel states to investigate the driving forces for gelation process. The morphology of the prepared xerogels was studied using SEM.

Advantage of a Narrow Spectrum Host Defense (Antimicrobial) Peptide Over a Broad Spectrum Analog in Preclinical Drug Development.

The APO-type proline-arginine-rich host defense peptides exhibit potent in vitro killing parameters against Enterobacteriaceae but not to other bacteria. Because of the excellent in vivo properties against systemic and local infections, attempts are regularly made to further improve the activity spectrum. A C-terminal hydrazide analog of the Chex1-Arg20 amide (ARV-1502) shows somewhat improved minimal inhibitory concentration against Moraxellaceae. Here we compared the activity of the two peptides as well as an inactive dimeric reverse amide analog in a systemic Acinetobacter baumannii infection. Only the narrow spectrum amide derivative reduced the 6-h blood bacterial burden by >2 log10 units reaching statistical significance (p = 0.03 at 5 mg/kg and 0.031 at 2 mg/kg administered intramuscularly). The hydrazide derivative, probably due to stronger activity on cell membranes, lysed erythrocytes at lower concentrations, and caused toxic effects at lower doses (10 mg/kg vs. 25 mg/kg). In a limited study, the amide induced a >5-fold production of the anti-inflammatory cytokine IL-10 over untreated naïve mice and minor increases in the anti-inflammatory IL-4 and pro-inflammatory cytokines TNF-α and IL-6, in blood. The blood of hydrazide-treated mice exhibited significantly lowered levels of IL-10 and slightly decreased IL-4 and TNF-α. These results suggest that the improved efficacy of the narrow-spectrum amide analog is likely associated with increased anti-inflammatory cytokine production and better stimulation of the immune system. Although blood IL-6 and TNF-α levels are frequently used as markers of potential toxicity in drug development, we did not observe any notable increase in mice receiving the toxic polyamide antibiotic colistin.

Chemical Synthesis of Natural Polyubiquitin Chains through Auxiliary-Mediated Ligation of an Expressed Ubiquitin Isomer.

An efficient method for the assembly of polyUb chains using auxiliary-modified Ub isomers is reported. This strategy takes advantages of auxiliary-mediated native chemical ligation between the distal Ub C-terminal hydrazide and the auxiliary of proximal Ub. Using removable protecting groups, Lys48-linked and Lys6-linked tri-Ub and even a mixed-linkage Lys6, Lys48-linked triUb in multimilligram quantities was made. These results demonstrate that this strategy yields natural polyubiquitin chains of desired length and linkage by using Ub isomer.

Practical Chemical Synthesis of Atypical Ubiquitin Chains by Using an Isopeptide-Linked Ub Isomer.

Chemical ubiquitination is an effective approach for accessing structurally defined, atypical ubiquitin (Ub) chains that are difficult to prepare by other techniques. Herein, we describe a strategy that uses a readily accessible premade isopeptide-linked 76-mer (isoUb), which has an N-terminal Cys and a C-terminal hydrazide, as the key building block to assemble atypical Ub chains in a modular fashion. This method avoids the use of auxiliary-modified Lys and instead employs the canonical and therefore more robust Cys-based native chemical ligation technique. The efficiency and capacity of this isoUb-based strategy is exemplified by the cost-effective synthesis of several linkage- and length-defined atypical Ub chains, including K27-linked tetra-Ub and K11/K48-branched tri-, tetra-, penta-, and hexa-Ubs.

C-terminal hydrazide modification changes the spinal antinociceptive profiles of endomorphins in mice

Previously, we have demonstrated that endomorphins (EMs) analogs with C-terminal hydrazide modification retained the μ-opioid receptor affinity and selectivity, and exhibited potent antinociception after intracerebroventricular (i.c.v.) administration. In the present study, we extended our studies to evaluate the antinociceptive profiles of EMs and their analogs EM-1-NHNH2, EM-2-NHNH2 given spinally in the radiant heat paw withdrawal test. Following intrathecal (i.t.) administration, EM-1, EM-2, EM-1-NHNH2 and EM-2-NHNH2 dose-dependently increased the latency for paw withdrawal response. EM-1-NHNH2 displayed the highest antinociceptive effects, with the ED50 values being 1.63 nmol, more potent than the parent EM-1 (1.96 nmol), but with no significant difference. By contrast, the analgesic activities of EM-2 and its analog EM-2-NHNH2 were almost equivalent (P>0.05). Naloxone and β-funaltrexamine (β-FNA) almost completely attenuated the antinociceptive effects of EMs and their analogs EM-1-NHNH2, EM-2-NHNH2 (10 nmol, i.t.), indicating the involvement of μ-opioid receptors. Notably, the antinociception of EM-1 was not significantly antagonized by naloxonazine, a selective μ1-opioid receptor antagonist, but partially reversed the effects of EM-2, suggesting that EM-1 and EM-2 may produce antinociception through distinct μ1- and μ2-opioid receptor subtypes. Moreover, naloxonazine didn’t significantly block the antinociceptive effects of EM-1-NHNH2 and EM-2-NHNH2, and nor-BNI, the κ-opioid receptor antagonist, attenuated the analgesic effects of EM-2, but not EM-1, EM-1-NHNH2 or EM-2-NHNH2. These results indicated that C-terminal amide to hydrazide conversion changed the antinociceptive opioid mechanisms of EM-2 but not EM-1 at the spinal level. Herein, the acute antinociceptive tolerance were further determined and compared. EM-1-NHNH2 and EM-2-NHNH2 shifted the dose-response curve rightward by only 2.8 and 1.5-fold as determined by tolerance ratio, whereas EM-1 and EM-2 by 3.4 and 4.6-fold, respectively, indicating substantially reduced antinociceptive tolerance. The present study demonstrated that C-terminal hydrazide modification changes the spinal antinociceptive profiles of EMs.

C-Terminal Modification and Multimerization Increase the Efficacy of a Proline-Rich Antimicrobial Peptide.

Two series of branched tetramers of the proline-rich antimicrobial peptide (PrAMP), Chex1-Arg20, were prepared to improve antibacterial selectivity and potency against a panel of Gram-negative nosocomial pathogens including Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa. First, tetramerization was achieved by dithiomaleimide (DTM) conjugation of two C-terminal-cysteine bearing dimers that also incorporated C-terminal peptide chemical modification. DTM-linked tetrameric peptides containing a C-terminal hydrazide moiety on each dimer exhibited highly potent activities in the minimum inhibitory concentration (MIC) range of 0.49-2.33 μm. A second series of tetrameric analogues with C-terminal hydrazide modification was prepared by using alternative conjugation linkers including trans-1,4-dibromo-2-butene, α,α'-dibromo-p-xylene, or 6-bismaleimidohexane to determine the effect of length on activity. Each displayed potent and broadened activity against Gram-negative nosocomial pathogens, particularly the butene-linked tetrameric hydrazide. Remarkably, the greatest MIC activity is against P. aeruginosa (0.77 μm/8 μg mL-1 ) where the monomer is inactive. None of these peptides showed any cytotoxicity to mammalian cells up to 25 times the MIC. A diffusion NMR study of the tetrameric hydrazides showed that the more active antibacterial analogues were those with a more compact structure having smaller hydrodynamic radii. The results show that C-terminal PrAMP hydrazidation together with its rational tetramerization is an effective means for increasing both diversity and potency of PrAMP action.

Screening of Pre-miRNA-155 Binding Peptides for Apoptosis Inducing Activity Using Peptide Microarrays.

MicroRNA-155, one of the most potent miRNAs that suppress apoptosis in human cancer, is overexpressed in numerous cancers, and it displays oncogenic activity. Peptide microarrays, constructed by immobilizing 185 peptides containing the C-terminal hydrazide onto epoxide-derivatized glass slides, were employed to evaluate peptide binding properties of pre-miRNA-155 and to identify its binding peptides. Two peptides, which were identified based on the results of peptide microarray and in vitro Dicer inhibition studies, were found to inhibit generation of mature miRNA-155 catalyzed by Dicer and to enhance expression of miRNA-155 target genes in cells. In addition, the results of cell experiments indicate that peptide inhibitors promote apoptotic cell death via a caspase-dependent pathway. Finally, observations made in NMR and molecular modeling studies suggest that a peptide inhibitor preferentially binds to the upper bulge and apical stem-loop region of pre-miRNA-155, thereby suppressing Dicer-mediated miRNA-155 processing.

Cysteine Promoted C‐Terminal Hydrazinolysis of Native Peptides and Proteins

Cysteine Promoted C‐Terminal Hydrazinolysis of Native Peptides and Proteins

Analysis of Density‐Dependent Binding of Glycans by Lectins Using Carbohydrate Microarrays

To investigate the density-dependent binding of glycans by lectins using carbohydrate microarrays, a number of C-terminal hydrazide-conjugated neoglycopeptides with various valences and different spatial arrangements of the sugar ligands were prepared on a solid support. The synthetic strategy includes (1) assembly of alkyne-linked peptides possessing C-terminal hydrazide on a solid support, (2) coupling of azide-linked, unprotected sugars to the alkyne-linked peptides on the solid support utilizing click chemistry, and (3) release of the neoglycopeptides from the solid support. By using this synthetic methodology, sixty five neoglycopeptides with a valency ranging from 1 to 4 and different spatial arrangements of the carbohydrate ligands were generated. Carbohydrate microarrays were constructed by immobilizing the prepared neoglycopeptides on epoxide-derivatized glass slides and were used to analyze the density-dependent binding of glycans by lectins. The results of binding property determinations show that lectin binding is highly dependent on the surface glycan density.

Nondestructive, Colorimetric Monitoring of Amines and Thiols on a Solid Support

A new, nondestructive, highly sensitive method for colorimetric monitoring of primary amines, secondary amines, and thiols on a solid support was developed. The resin used in this method is simply regenerated for the repetition of the reaction or an ensuing reaction. By using this new method, several peptides containing secondary amide linkages and C-terminal hydrazide groups were prepared in high purities and yields.

Construction of protein analogs by site-specific condensation of unprotected fragments

The extreme sensitivity to periodate of 1-amino, 2-hydroxy compounds permits the selective conversion of N-terminal serine and threonine to an aldehydic group. We have used this reaction to construct analogues of human granulocyte colony stimulating factor (G-CSF) by allowing such oxidized peptides to react with others that have had a hydrazide derivative attached to the C-terminus by reversed proteolysis. Two recombinant analogues of G-CSF were used as starting materials. Both had only a single lysine residue (at position 62 and 75, respectively) followed immediately by a serine. Digestion of each analogue by the lysine-specific protease from Achromobacter lyticus gave two fragments, one of which could be N-terminally oxidized and the other converted to the C-terminal hydrazide derivative by reversed proteolysis using the same enzyme. After preliminary studies with model peptides, we first reacted the corresponding peptide pairs together and then, in order to eliminate the 64-74 disulfide loop, fragment 1-62 from the first analogue with fragment 76-174 from the second. Reactions are efficient (up to 80% product based on the oxidized fragment) and take place under very mild conditions. The hydrazone bond can easily be stabilized by reduction with NaBH3CN. This method represents a new, reasonably general route for the construction of large protein chimeras of precisely controlled structure.