The tea-bag protocol for comparison of Fmoc removal reagents in solid-phase peptide synthesis

Guzmán, Fanny; Gauna, Adriana; Luna, Omar; Román, Tanya; Álvarez, Claudio; Alberício, Fernando; Cárdenas, Constanza

doi:10.1007/s00726-020-02883-8

Cited by 23 publications

(12 citation statements)

References 12 publications

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“…This protocol has demonstrated to be quite useful when a large number of sequences need to be synthesized. Although it can be used for the preparation of totally unrelated sequences, it is particularly convenient in the case of protein epitope mapping [ 18 , 19 ], in the case of Ala scan for the identification of key residues in a peptide [ 20 , 21 ], to test different strategies or reagents [ 10 ], in the synthesis of antigen epitopes for use in diagnosis (antibody generation) [ 22 , 23 ], in the search of new compounds [ 24 , 25 ], and in the development of peptide-based vaccines, among other applications.…”

Section: Discussionmentioning

confidence: 99%

“…In this way, our groups have been working on the “tea bag” for many years, using the fluorenylmethoxycarbonyl (Fmoc)/tert-butyl (tBu) protection scheme in contrast to Houghten, who utilized the less friendly tert-butyloxycarbonyl (Boc)/benzyl (Bzl) scheme. In addition, we have analyzed new Fmoc removal reagents with advantages regarding toxicity and handling [ 10 ]. Thus, during that time, we have optimized all the parameters of this strategy, and herein, we describe a comprehensive protocol detailing all of the steps.…”

Section: Introductionmentioning

confidence: 99%

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Tea Bags for Fmoc Solid-Phase Peptide Synthesis: An Example of Circular Economy

et al. 2021

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Peptide synthesis is an area with a wide field of application, from biomedicine to nanotechnology, that offers the option of simultaneously synthesizing a large number of sequences for the purpose of preliminary screening, which is a powerful tool. Nevertheless, standard protocols generate large volumes of solvent waste. Here, we present a protocol for the multiple Fmoc solid-phase peptide synthesis in tea bags, where reagent recycling steps are included. Fifty-two peptides with wide amino acid composition and seven to twenty amino acid residues in length were synthesized in less than three weeks. A clustering analysis was performed, grouping the peptides by physicochemical features. Although a relationship between the overall yield and the physicochemical features of the sequences was not established, the process showed good performance despite sequence diversity. The recycling system allowed to reduce N, N-dimethylformamide usage by 25–30% and reduce the deprotection reagent usage by 50%. This protocol has been optimized for the simultaneous synthesis of a large number of peptide sequences. Additionally, a reagent recycling system was included in the procedure, which turns the process into a framework of circular economy, without affecting the quality of the products obtained.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Tea Bags for Fmoc Solid-Phase Peptide Synthesis: An Example of Circular Economy

et al. 2021

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“…Peptide synthesis was performed according to the Fmoc/tBu standard strategy using a “tea-bag” protocol ( 18 ). Peptides were characterized by high-performance liquid chromatography (HPLC) in a JASCO system (JASCO Corp., Tokyo, Japan), and molecular mass was determined by electrospray-mass spectrometry (ESI–MS) in an LCMS-2020 ESI–MS equipment (Shimadzu Corp., Kyoto, Japan).…”

Section: Methodsmentioning

confidence: 99%

Functional Profile of CD8+ T-Cells in Response to HLA-A*02:01-Restricted Mutated Epitopes Derived from the Gag Protein of Circulating HIV-1 Strains from Medellín, Colombia

et al. 2022

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CD8+ T-cells play a crucial role in the control of HIV replication. HIV-specific CD8+ T-cell responses rapidly expand since the acute phase of the infection, and it has been observed that HIV controllers harbor CD8+ T-cells with potent anti-HIV capacity. The development of CD8+ T-cell-based vaccine against HIV-1 has focused on searching for immunodominant epitopes. However, the strong immune pressure of CD8+ T-cells causes the selection of viral variants with mutations in immunodominant epitopes. Since HIV-1 mutations are selected under the context of a specific HLA-I, the circulation of viral variants with these mutations is highly predictable based on the most prevalent HLA-I within a population. We previously demonstrated the adaptation of circulating strains of HIV-1 to the HLA-A*02 molecule by identifying mutations under positive selection located in GC9 and SL9 epitopes derived from the Gag protein. Also, we used an in silico prediction approach and evaluated whether the mutations found had a higher or lower affinity to the HLA-A*02. Although this strategy allowed predicting the interaction between mutated peptides and HLA-I, the functional response of CD8+ T-cells that these peptides induce is unknown. In the present work, peripheral blood mononuclear cells from 12 HIV-1+ HLA-A*02:01+ individuals were stimulated with the mutated and wild-type peptides derived from the GC9 and SL9 epitopes. The functional profile of CD8+ T-cells was evaluated using flow cytometry, and the frequency of subpopulations was determined according to their number of functions and the polyfunctionality index. The results suggest that the quality of the response (polyfunctionality) could be associated with the binding affinity of the peptide to the HLA molecule, and the functional profile of specific CD8+ T-cells to mutated epitopes in individuals under cART is maintained.

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“…Fmoc groups removal was completed with 20% v/v of 4-methyl piperidine (4MP) (Merck KGaA, Darmstadt, Germany) in N,N-dimethylformamide (DMF) (Merck) [62]. Furthermore, the coupling of Rhd B, for its later use in microscopy, was carried out manually using 5:5:5:7.5 equivalents of Rhd B:(2-(1H-benzotriazol-1-yl)-1,1,3,3tetramethyluronium hexafluorophosphate (HBTU) (Iris Biotech GmbH): Oxyme-Pure (Iris Biotech GmbH): N-ethyldiisopropylamine (DIEA) (Merck) [63]. Peptide cleavage was performed with a solution of 92.5% trifluoroacetic acid (TFA)/2.5% triisopropylsilane (TIS)/2.5% diethanethiol (DOT)/2.5% ultrapure water), washed with cold ether and finally purified by C18 extraction columns with acetonitrile/water gradient to a purity higher than 95%.…”

Section: Peptide Synthesis and Characterizationmentioning

confidence: 99%

Immunomodulatory Lectin-like Peptides for Fish Erythrocytes-Targeting as Potential Antiviral Drug Delivery Platforms

Salvador-Mira

Chico

Aróstica

et al. 2021

IJMS

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One of the challenges of science in disease prevention is optimizing drug and vaccine delivery. Until now, many strategies have been employed in this sector, but most are quite complex and labile. To overcome these limitations, great efforts are directed to coupling drugs to carriers, either of natural or synthetic origin. Among the most studied cell carriers are antigen-presenting cells (APCs), however, red blood cells (RBCs) are positioned as attractive carriers in drug delivery due to their abundance and availability in the body. Furthermore, fish RBCs have a nucleus and have been shown to have a strong involvement in modulating the immune response. In this study, we evaluated the binding of three peptides to rainbow trout RBCs, two lectin-like peptides and another derived from Plasmodium falciparum membrane protein, in order to take advantage of this peptide-RBCs binding to generate tools to improve the specificity, efficacy, immunostimulatory effect, and safety of the antiviral therapeutic or prophylactic administration systems currently used.

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The tea-bag protocol for comparison of Fmoc removal reagents in solid-phase peptide synthesis

Cited by 23 publications

References 12 publications

Tea Bags for Fmoc Solid-Phase Peptide Synthesis: An Example of Circular Economy

Tea Bags for Fmoc Solid-Phase Peptide Synthesis: An Example of Circular Economy

Functional Profile of CD8+ T-Cells in Response to HLA-A*02:01-Restricted Mutated Epitopes Derived from the Gag Protein of Circulating HIV-1 Strains from Medellín, Colombia

Immunomodulatory Lectin-like Peptides for Fish Erythrocytes-Targeting as Potential Antiviral Drug Delivery Platforms

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