Skin secretory amphibian antimicrobial peptides are the part of their immune defense. The present work is devoted to the study
of the influence of “water environment stress” and additional bacterial impact on the composition of the skin secretion of
the Common frog (Rana temporaria) by means of high-performance liquid chromatography (HPLC) and matrix assisted laser desorption/ionization (MALDI) mass spectrometry.
It was shown that the contact of the amphibian species with Micrococcus luteus and Staphylococcus aureus stimulates the release of antimicrobial peptides, maintains the high bradykinin and related peptides levels in the skin secretion
and influences the processing of the latter ones. The possibilities of mass spectrometric profiling by using HPLC and MALDI
were demonstrated. This feature allows the detection of potentially bioactive peptides for their future direct testing, as
has been shown for temporin M and brevinin 1Tb in the present study. 相似文献
Amphibian skin secretion represents a cerain scientific interest as a source of biologically active natural peptides. In the
present research skin peptidome of wide-spread European frog Rana lessonae (Camerano, 1882) was studied for the first time ever. Peptide sequencing was accomplished with Fourier transform ion cyclotron
resonance mass spectrometer in collision-induced and electron capture dissociation modes. A portion of amphibian peptides
contains intramolecular C-terminal disulfide cycle which obstructs mass spectrometric sequencing. Two methods were utilized
to overcome this difficulty: reduction with dithiotreithol followed by thiol group alkylation and oxidation into sulfonic
acid groups with performic acid. Integrated approach employed in the present study allowed the identification of 49 peptides
(of 6 to 37 amino acid residues), including 19 novel species. 相似文献
Summary. Secreted peptides from diverse sources have been found to contain a d-amino acid. From the sequence of cloned mRNAs coding for the precursors of such peptides it could be deduced that in all cases tested so far the d-amino acid in the final product is derived from the corresponding l-amino acid present in the primary product of translation. Enzymes catalyzing such an l- to d-isomerization in peptide linkage have been isolated from the venom of a spider and the skin secretions of frogs. Even though
these are completely different proteins, the reaction mechanism is the same, namely a de-protonation/re-protonation of the
α-carbon of an amino acid with concomitant inversion of the chirality. Sequences potentially coding for homologues of the
frog enzyme are present in the genome of different vertebrate species. 相似文献
Cancer has always been one of the most common malignant diseases in the world. Therefore, there is an urgent need to find potent agents with selective antitumor activity against cancer cells. It has been reported that antimicrobial peptides (AMPs) can selectively target tumor cells. In this study, we focused on the anti-tumor activity and mechanism of Brevivin-1RL1, a cationic α-helical AMP isolated from frog Rana limnocharis skin secretions. We found that Brevivin-1RL1 preferentially inhibits tumor cells rather than non-tumor cells with slight hemolytic activity. Cell viability assay demonstrated the intermolecular disulfide bridge contributes to the inhibitory activity of the peptide as the antitumor activity was abolished when the disulfide bridge reduced. Further mechanism studies revealed that both necrosis and apoptosis are involved in Brevivin-1RL1 mediated tumor cells death. Moreover, Brevivin-1RL1 induced extrinsic and mitochondria intrinsic apoptosis is caspases dependent, as the pan-caspase inhibitor z-VAD-FMK rescued Brevinin-1RL1 induced tumor cell proliferative inhibition. Immunohistology staining showed Brevivin-1RL1 mainly aggregated on the surface of the tumor cells. These results together suggested that Brevivin-1RL1 preferentially converges on the cancer cells to trigger necrosis and caspase-dependent apoptosis and Brevivin-1RL1 could be considered as a pharmacological candidate for further development as anti-cancer agent. 相似文献
Five natural peptides isolated from ranid skin secretions of European frog species of Rana ridibunda and Rana arvalis (molecular masses 3516, 2674, 2636, 1874, and 1810 Da) were studied by MALDI-TOF/TOF to compare two procedures of disulfide bond cleavage: (1) performic oxidation and (2) reduction/carboxamidomethylation. The processes are relevant for the elucidation of the amino acid sequence inside the seven-member cystine ring at the C-terminus. The results clearly demonstrated that oxidation of the disulfide bond led to notably higher abundances of b- and y-ions, corresponding to the C-terminal peptide bonds, than reduction/carboxamidomethylation. This conclusion is true for all five peptides studied. Besides that, the oxidation procedure is simpler than carboxamidomethylation, as it is a one-step process with no purification required. The oxidation is more reproducible. The results were similar each time the peptide was subjected to the process. It was successfully applied to all five peptides while reduction/carboxamidomethylation failed in the case of brevinin-1Ra, despite all variations of reaction conditions. 相似文献
Two representatives of a new type of β‐amino acids, carrying two functionalized side chains, one in the 2‐ and one in the 3‐position, have been prepared stereoselectively: a β‐Ser derivative with an additional CH2OH group in the 2‐position (for β‐peptides with better water solubility; Scheme 2) and a β‐HCys derivative with an additional CH2SBn group in the 2‐position (for disulfide formation and metal complexation with the derived β‐peptides; Scheme 3). Also, a simple method for the preparation of α‐methylidene‐β‐amino acids is presented (see Boc‐2‐methylidene‐β‐HLeu‐OH, 8 in Scheme 3). The two amino acids with two serine or two cysteine side chains are incorporated into a β‐hexa‐ and two β‐heptapeptides ( 18 and 23/24 , resp.), which carry up to four CH2OH groups. Disulfide formation with the β‐peptides carrying two CH2SH groups generates very stable 1,2‐dithiane rings in the centre of the β‐heptapeptides, and a cyclohexane analog was also prepared (cf. 27 in Scheme 6). The CD spectra in H2O clearly indicate the presence of 314‐helical structures of those β‐peptides ( 18 , 23 , 24 , 27b ) having the `right' configurations at all stereogenic centers (Fig. 2). NMR Measurements (Tables 1 and 2, and Fig. 4) in aqueous solution of one of the new β‐peptides ( 24 ) are interpreted on the assumption that the predominant secondary structure is the 314‐helix, a conformation that has been found to be typical for β‐peptides in MeOH or pyridine solution, according to our previous NMR investigations. 相似文献
Simple and fast identification of disulfide linkages in insulin is demonstrated with a peptic digest using the Route 66 method.
This is accomplished by collisional activation of singly and doubly charged cationic Na+ and Ca2+ complexes generated using electrospray ionization mass spectrometry (ESI-MS). Collisional activation of doubly charged metal
complexes of peptides with intermolecular disulfide linkages yields two sets of singly charged paired products separated by
66 mass units resulting from selective S-C bond cleavages. Highly selective elimination of 66 mass units, which corresponds
to the molecular weight of hydrogen disulfide (H2S2), is observed from singly charged metal complexes of peptides with disulfide linkages. The mechanism proposed for these processes
is initiated by formation of a metal-stabilized enolate at Cys, followed by cleavage of the S-C bond. Further activation of
the products yields sequence information that facilitates locating the position of the disulfide linkages in the peptic digest
fragments. For example, the doubly charged Ca2+ complex of the peptic digest product GIVEQCCASVCSL/FVNQHLCGSHL yields paired products separated by 66 mass units resulting
from selective S-C bond cleavages at an intermolecular disulfide linkage under low-energy collision-induced dissociation.
Further activation of the product comprising the A chain reveals the presence of a second disulfide bridge, an intramolecular
linkage. Experimental and theoretical studies of the disulfide linked model peptides provide mechanistic details for the selective
cleavage of the S-C bond. 相似文献
In this study, we systematically investigated gas-phase fragmentation behavior of [M + nH + OH]n•+ ions formed from peptides containing intra-molecular disulfide bond. Backbone fragmentation and radical initiated neutral
losses were observed as the two competing processes upon low energy collision-induced dissociation (CID). Their relative contribution
was found to be affected by the charge state (n) of [M + nH + OH]n•+ ions and the means for activation, i.e., beam-type CID or ion trap CID. Radical initiated neutral losses were promoted in
ion-trap CID and for lower charge states where mobile protons were limited. Beam-type CID and dissociation of higher charge
states of [M + nH + OH]n•+ ions generally gave abundant backbone fragmentation, which was highly desirable for characterizing peptides containing disulfide
bonds. The amount of sequence information obtained from CID of [M + nH + OH]n•+ ions was compared with that from CID of disulfide bond reduced peptides. For the 11 peptides studied herein, similar extent
of sequence information was obtained from these two methods. 相似文献
Mass spectrometric de novo sequencing of skin secretion peptides from genus Rana is complicated because of C-terminal disulfide cycles present in their structure. Brevinin-1E and brevinin-2Ec from the skin
secretion of the Marsh Frog R. ridibunda were used for a comparative study of six N-phenylmaleimide derivatives as new alkylating agents for cysteine thiol moieties.
The paper describes the synthesis and confirmation of the structures of the obtained compounds. A procedure was developed
for modifying thiol groups with the proposed reagents. Alkylation efficiency and the effect on the peak intensity in matrix-assisted
laser desorption/ionization (MALDI) spectra were investigated. The best results were obtained for 2,4- and 2,5-dimethylphenylmaleimides.
Additionally tested iodoacetic acid was shown to be a powerful modifier of thiol groups, while its excess notably increases
the intensities of the peaks of protonated molecules in the MALDI mass spectra of both peptides. 相似文献
Structure–activity relationship studies are a highly time-consuming aspect of peptide-based drug development, particularly in the assembly of disulfide-rich peptides, which often requires multiple synthetic steps and purifications. Therefore, it is vital to develop rapid and efficient chemical methods to readily access the desired peptides. We have developed a photolysis-mediated “one-pot” strategy for regioselective disulfide bond formation. The new pairing system utilises two ortho-nitroveratryl protected cysteines to generate two disulfide bridges in less than one hour in good yield. This strategy was applied to the synthesis of complex disulfide-rich peptides such as Rho-conotoxin ρ-TIA and native human insulin. 相似文献
Disulfide bonds are post-translationnal modifications that can be crucial for the stability and the biological activities of natural peptides. Considering the importance of these disulfide bond-containing peptides, the development of new techniques in order to characterize these modifications is of great interest. For this purpose, collision cross cections (CCS) of a large data set of 118 peptides (displaying various sequences) bearing zero, one, two, or three disulfide bond(s) have been measured in this study at different charge states using ion mobility-mass spectrometry. From an experimental point of view, CCS differences (ΔCCS) between peptides bearing various numbers of disulfide bonds and peptides having no disulfide bonds have been calculated. The ΔCCS calculations have also been applied to peptides bearing two disulfide bonds but different cysteine connectivities (Cys1-Cys2/Cys3-Cys4; Cys1-Cys3/Cys2-Cys4; Cys1-Cys4/Cys2-Cys3). The effect of the replacement of a proton by a potassium adduct on a peptidic structure has also been investigated.
Despite the importance of stapled peptides for drug discovery, only few practical processes to prepare cross‐linked peptides have been described; thus the structural diversity of available staple motifs is currently limited. At the same time, C−H activation has emerged as an efficient approach to functionalize complex molecules. Although there are many reports on the C−H functionalization of amino acids, examples of post‐synthetic peptide C−H modification are rare and comprise almost only C(sp2)−H activation. Herein, we report the development of a palladium‐catalyzed late‐stage C(sp3)−H activation method for peptide stapling, affording an unprecedented hydrocarbon cross‐link. This method was first employed to prepare a library of stapled peptides in solution. The compatibility with various amino acids as well as the influence of the size (i ,i +3 and i ,i +4) and length of the staple were investigated. Finally, a simple solid‐phase procedure was also established. 相似文献
Previously reported studies of the iodine oxidation of S-trityl-cysteine peptides and S-acetamidomethyl-cysteine peptides, leading directly to cystine peptides, have been extended. Detailed investigations have been made of the reactivities of the S-trityl and the S-acetamidomethyl group towards iodine in various solvents. In chloroform, methylene chloride, trifluoroethanol, and hexafluoroisopropyl alcohol the differences in the reaction rates of the two groups have been found to be extremely large, allowing the selective conversion of the tritylthio groups to disulfides in the presence of the S-acetamidomethyl derivatives. In a second group of solvents, consisting of methanol, acetic acid, dioxane, and mixtures of these solvents with water, simultaneous iodine oxidation of S-trityl- and S-acetamidomethyl-cysteine peptides leads to a preferential combination of these two residues, resulting in predominantly asymmetrical cystine derivatives. - The suitability of the two sulfur-protecting groups in the synthesis of cyclic cystine peptides has been assessed. - Possible reaction mechanisms are discussed. - The scope and limitations of iodine oxidation in peptide synthesis have been studied. The applicability of the method has been demonstrated in the preparation of the open-chain asymmetrical cystine peptide 5 , the protected somatostatin derivative 17 , and the A(1–13) segment 19 of human insulin, previously employed in the total synthesis of this hormone. 相似文献
Human insulin‐like peptide‐6 (INSL‐6) belongs to the insulin superfamily and shares the distinctive disulfide bond configuration of human insulin. In this report we present the first chemical synthesis of INSL‐6 utilizing fluorenylmethyloxycarbonyl‐based (Fmoc) solid‐phase peptide chemistry and regioselective disulfide bond construction protocols. Due to the presence of an oxidation‐sensitive tryptophan residue, two new orthogonal synthetic methodologies were developed. The first method involved the identification of an additive to suppress the oxidation of tryptophan during iodine‐mediated S‐acetamidomethyl (Acm) deprotection and the second utilized iodine‐free, sulfoxide‐directed disulfide bond formation. The methodologies presented here offer an efficient synthetic route to INSL‐6 and will further improve synthetic access to other multiple‐disulfide‐containing peptides with oxidation‐sensitive residues. 相似文献