Evaluation of the Trifluoromethanosulfonic Acid/Trifluoroacetic Acid/Thioanisole Cleavage Procedure for Application in Solid-Phase Peptide Synthesis.

It is well known that cationic antimicrobial peptides (cAMPs) are potential microbicidal agents for the increasing problem of antimicrobial resistance. However, the physicochemical properties of each peptide need to be optimized for clinical use. To evaluate the effects of dimerization on the structure and biological activity of the antimicrobial peptide Ctx-Ha, we have synthesized the monomeric and three dimeric (Lys-branched) forms of the Ctx-Ha peptide by solid-phase peptide synthesis using a combination of 9-fluorenylmethyloxycarbonyl (Fmoc) and t-butoxycarbonyl (Boc) chemical approaches. The antimicrobial activity assay showed that dimerization decreases the ability of the peptide to inhibit growth of bacteria or fungi; however, the dimeric analogs displayed a higher level of bactericidal activity. In addition, a dramatic increase (50 times) in hemolytic activity was achieved with these analogs. Permeabilization studies showed that the rate of carboxyfluorescein release was higher for the dimeric peptides than for the monomeric peptide, especially in vesicles that contained sphingomyelin. Despite different biological activities, the secondary structure and pore diameter were not significantly altered by dimerization. In contrast to the case for other dimeric cAMPs, we have shown that dimerization selectively decreases the antimicrobial activity of this peptide and increases the hemolytic activity. The results also show that the interaction between dimeric peptides and the cell wall could be responsible for the decrease of the antimicrobial activity of these peptides.

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“…The cleavage of BHA resin was performed with 73.5% TFA, 12.5% thioanisole, 9% trifluoromethanesulfonic acid, and 5% m-cresol (22).…”

Section: Chemicals and Microorganismsmentioning

confidence: 99%

Effects of Dimerization on the Structure and Biological Activity of Antimicrobial Peptide Ctx-Ha

Lorenzón

Cespedes

Vicente

et al. 2012

Antimicrob Agents Chemother

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“…Our proposal was based on the stability of model peptide-resin linkages toward existing acid cleavage procedures, 20,21) as well as on the degree to which premature peptide chains are removed during trifluoroacetic acid (TFA)/Boc removal of peptide-resins. The results of other studies 22,23) have indicated the need for caution in selecting the type of resin to be used in the synthesis of peptide sequences containing C-terminal residue in either the a-carboxamide or the a-carboxyl function. In addition, appropriate final cleavage experiments, which are typically carried out either in anhydrous hydrogen fluoride 24) or in a trifluoromethanesulfonic acid/TFA/thioanisole cocktail, 25) should be performed, since a significant decrease in the amount of cleaved peptide can occur in this step.…”

mentioning

confidence: 99%

Comparative Investigation of the Cleavage Step in the Synthesis of Model Peptide Resins: Implications for N.ALPHA.-9-Fluorenylmethyloxycarbonyl-Solid Phase Peptide Synthesis

Jubilut

Cilli

Crusca

et al. 2007

CHEMICAL & PHARMACEUTICAL BULLETIN

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Since its inception, 1) the solid-phase peptide synthesis (SPPS) methodology has been systematically improved as the result of a wide variety of experimental investigations. These efforts have ranged from optimizing the coupling reaction itself (through the use of efficient acylating reagents, microwave irradiation and variations in temperature) [2][3][4][5] to broadening our knowledge of the complex peptide-resin solvation process. [6][7][8][9] Predictably, methods such as nuclear magnetic resonance 10,11) and Fourier transform infrared spectroscopy 12,13) have also been tested in attempts to further improve SPPS. In our case, [14][15][16][17] we pioneered the application of the electron paramagnetic resonance (EPR) technique, which is based on the use of a previously developed amino acid-type marker. 18,19) All of these efforts have led to ongoing improvement of the SPPS method. Intriguingly, little attention has yet been given to the possibility that incomplete cleavage occurs or that there is premature removal of peptide chains from the solid support. Within this context, we previously proposed some rules for the selection of resins used in the N a -tertbutyloxycarbonyl (Boc) chemistry. Our proposal was based on the stability of model peptide-resin linkages toward existing acid cleavage procedures, 20,21) as well as on the degree to which premature peptide chains are removed during trifluoroacetic acid (TFA)/Boc removal of peptide-resins. The results of other studies 22,23) have indicated the need for caution in selecting the type of resin to be used in the synthesis of peptide sequences containing C-terminal residue in either the a-carboxamide or the a-carboxyl function. In addition, appropriate final cleavage experiments, which are typically carried out either in anhydrous hydrogen fluoride 24) or in a trifluoromethanesulfonic acid/TFA/thioanisole cocktail, 25) should be performed, since a significant decrease in the amount of cleaved peptide can occur in this step. The type of the C-terminal residue and the length of the peptide sequence seem to affect the overall cleavage yield (which, surprisingly, can be as high as 30%) as a consequence of incomplete final chain removal accompanied by premature loss from the resin during TFA removal of peptide-resins.The results of a comparison between benzhydrylamine-resin (BHAR) 26) and methylbenzhydrylamine-resin (MBHAR), 27) both used for the synthesis of a-carboxamide peptides in the Boc-chemistry, led us to conclude that the latter is the resin of choice mainly when the resin-bound amino acid is of the hydrophobic type. However, in the presence of a hydrophilic residue at the C-terminal position, the difference between the two aminated resins in terms of their efficiency depends on the length of the peptide. When the peptide sequence is longer, BHAR produces higher yields than does MBHAR. This is a consequence of the greater stability of the peptideresin linkage of the former toward successive TFA treatments in each synthesis cycle. 20,21) In the present study, w...

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“…In recent years, advances like the removal of Fmoc using less nucleophilic bases, introduction of a thioester after peptide assembly, and on‐resin conversion of peptide acids into peptide thioesters have alleviated the problems associated with the generation of a thioester using Fmoc chemistry . However generating a thioester using Boc chemistry is still considered an effective approach and here we report a technology that complements the existing methods in NCL peptide construction …”

Section: Introductionmentioning

confidence: 99%

“…Here we investigate a technique that further enhances the replacement of HF with trifluoromethanesulfonic acid (TFMSA), by specifically focusing on its compatibility with the standard Boc chemistry used to produce thioesters, which are an essential requirement for NCL. The replacement of HF with TFMSA is an idea that has produced several protocols as early as the 90s, however here we have optimized the conditions for Huwentoxin‐I (HwTx‐I), a 33 amino acid spider toxin. TFMSA is a strong acid that is comparatively less hazardous than HF.…”

Section: Introductionmentioning

confidence: 99%

t‐boc synthesis of huwentoxin‐i through native chemical ligation incorporating a trifluoromethanesulfonic acid cleavage strategy

et al. 2016

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Tert-butyloxycarbonyl (t-Boc) based Native Chemical Ligation (NCL) techniques commonly employ hydrogen fluoride (HF) to create the thioester fragment required for the ligation process. Our research aimed to assess the replacement of HF with Trifluoromethanesulfonic acid (TFMSA). Here we examined a 33 amino acid test peptide, Huwentoxin-I (HwTx-I) as a novel candidate for our TFMSA cleavage protocol. Structurally HwTx-I has an X-Cys16-Cys17-X sequence mid-region, which makes it an ideal candidate for NCL. Experiments determined that the best yields (16.8%) obtained for 50 mg of a thioester support resin were achieved with a TFMSA volume of 100 μL with a 0.5 hour incubation on ice, followed by 2.0 hours at room temperature. RP-HPLC/UV and mass spectra indicated the appropriate parent mass and retention of the cleaved HwTx-I N-terminal thioester fragment (Ala1-Cys16), which was used in preparation for NCL. The resulting chemically ligated HwTx-I was oxidized/folded, purified, and then assessed for pharmacological target selectivity. Native-like HwTx-I produced by this method yielded an EC50 value of 340.5 ± 26.8 nM for Nav1.2 and an EC50 value of 504.1 ± 81.3 nM for Nav1.3, this being similar to previous literature results using native material. This paper represents the first NCL based synthesis of this potent sodium channel blocker. Our illustrated approach removes potential restrictions in the advancement of NCL as a common peptide laboratory technique with minimal investment, and removes the hazards associated with HF usage.

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Evaluation of the Trifluoromethanosulfonic Acid/Trifluoroacetic Acid/Thioanisole Cleavage Procedure for Application in Solid-Phase Peptide Synthesis.

Cited by 33 publications

References 31 publications

Effects of Dimerization on the Structure and Biological Activity of Antimicrobial Peptide Ctx-Ha

Effects of Dimerization on the Structure and Biological Activity of Antimicrobial Peptide Ctx-Ha

Comparative Investigation of the Cleavage Step in the Synthesis of Model Peptide Resins: Implications for N.ALPHA.-9-Fluorenylmethyloxycarbonyl-Solid Phase Peptide Synthesis

t‐boc synthesis of huwentoxin‐i through native chemical ligation incorporating a trifluoromethanesulfonic acid cleavage strategy

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