Fast Solution-Phase and Liquid-Phase Peptide Syntheses (SolPSS and LPPS) Mediated by Biomimetic Cyclic Propylphosphonic Anhydride (T3P®) (original) (raw)
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Aqueous solid-phase peptide synthesis (ASPPS): A novel concept of peptide synthesis
2020
In the frame of the present work, an efficient and sustainable aqueous solid-phase peptide synthesis was developed based on a novel sulfonated derivative of fluorenyl protecting group. The viability of this approach and its application range was demonstrated upon the assembly of 22 biologically active peptides. To make access to aqueous peptide synthesis, coupling efficency was assessed in water-based systems applying respective Nα-Smoc amino acids and using different activation approaches. In our hands, several water-compatible activating additives were found appropriate, with EDC-Cl, Oxima and HOPO being the most efficient ones. Our experiments showed that although coupling of amino acids in water gave reasonable yields and purity of peptides, the addition of organic co-solvents enhanced coupling performance significantly. Additional studies on enantiomeric composition showed no increased racemization levels during the ASPPS process. Ionic properties of the Smoc protecting group g...
Green Chemistry, 2023
In peptide synthesis, the issues related to poor sustainability, long reaction times and high process mass intensity (PMI) are necessary to promote actions aimed at redefining procedural aspects projected towards more sustainable synthetic processes. Herein, we report a fast, widely applicable and green solution-phase peptide synthesis (GSolPPS) via a continuous protocol using propylphosphonic anhydride T3P® as the coupling reagent and N-benzyloxycarbonyl-protecting group (Z), which is easily removed by hydrogenation. Because N,N-dimethylformamide (DMF) replacement was a priority, the iterative process was performed in EtOAc, pushing further on overall sustainability. The efficiency of the synthetic protocol in terms of conversion, racemization and reaction times allowed extending the scope of the work to the synthesis of the standard peptide Leu-enkephalin as a proof of concept. Among the various explored procedures, the one-pot protocol (A cont plus), avoiding work-ups, intermediate purification and any dispersion effect, allowed the achievement of PMI = 30 for each deprotection/coupling sequence necessary to introduce a single amino acid in the iterative process, without considering the possibility of solvent and base recovery. This value is the lowest reported for an oligopeptide synthesis protocol to date.
ACS Sustainable Chemistry & Engineering, 2019
mixture (2 × 3 mL each). A solution of Fmoc-Leu-OH (3 equiv), N,N′-diisopropylcarbodiimide (DIC) (3 equiv), and Oxyma Pure (3 equiv) in the proper mixture, preactivated for 5 min, was charged onto the resin and stirred for 1 h. After the peptide coupling, the resin was washed with DMF, DCM and DMF or mixture, iPrOH, and mixture (2 × 3 mL each). Then, 20% piperidine in DMF or selected mixture was charged on the resin (2 × 3 mL × 15 min). The resin was washed and ready for the subsequent couplings, deprotections, and washings, as reported before, to obtain the pentapeptide. The peptide was cleaved from the resin with trifluoroacetic acid (TFA)/H 2 O/ triisopropylsilane (TIS) (95:2.5:2.5) solution for 2 h at room temperature. The crude was directly analyzed by HPLC-MS. Solid-Phase Synthesis of H-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol (Linear Octreotide) in 70:30 Anisole/Dimethyl Carbonate (Method 8). The synthesis was performed in a glass syringe, attached at the bottom to a vacuum source to remove excess of reagents and solvents. The resin (H-Thr(tBu)-ol-2CT-PS 0.6 mmol/g, 500 mg) was washed with 3 mL of Mix C3, 3 mL of iPrOH, and 3 mL of Mix C3. A preactivated solution of Fmoc-Cys(Trt)-OH (3 equiv), DIC (3 equiv), and Oxyma Pure (3 equiv) in Mix C3 (3.3 mL) was charged onto the resin and stirred for 1 h. After the peptide coupling, the resin was washed with 3 mL of Mix C3, 3 mL of iPrOH, and 3 mL of Mix C3. Fmoc removal was performed by adding 2 × 3 mL of 20% piperidine in Mix C3 on the resin, shaking it for 10 min each. After the deprotection, the resin was washed with 4 × 3 mL of Mix C3. The resin was ready for the subsequent couplings, deprotections, and washings, as reported before, to obtain the decapeptide. The final washings were performed with Mix C3 (4 × 3 mL) and iPrOH (3 × 2 mL). The peptide was cleaved from the resin with TFA/TIS/1-dodecanthiol (9 mL/0.7 mL/0.6 mL) solution for 4 h at room temperature. The solution was recovered by filtration. Diisopropyl ether (37 mL) was added dropwise at 0−5°C to the acidic solution until precipitation of peptide was achieved. The resulting mixture was stirred for 1.5 h at 0−5°C. The precipitate was filtered, washed with diisopropyl ether and petroleum ether, and dried under vacuum, affording an off-white solid. The crude was analyzed by HPLC-MS. For the synthesis with method 1, as a substitution for Mix C3 and iPrOH, DMF and DCM were used. Cyclization of Linear Octreotide. Crude trifluoroacetate linear Octreotide (1 g of a raw synthetic product containing 82.3% or 88.0%
The Journal of Organic Chemistry, 1993
A study of the utilization of tert-butyloxycarbonyl-protected amino acid N-carboxyanhydrides (Boc-NCAs) in solid-phase peptide synthesis revealed that coupling rates were favored in solvents with a high dielectric constant such as DMF. Tertiary amines such as DIEA are not required for efficient coupling in DMF. However, the use of 1 equiv of DIEA in DMF in the synthesis of a pentapeptide resulted in a cleaner crude product as compared with that obtained in the absence of DIEA. The rate of BwNCA coupling was comparable to that found for (benzotriazol-l-yloxy)tris(dimethy~o)phosphonium hexafluorophosphate (BOP) coupling or 2 4 1H-benzotriazol-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate (HBTU) coupling as judged by kinetic analysig of coupling of BocIleNCA or BocValNCA to an Ala-PAM-resin or an ne-Ala-PAM-resin. Coupling df Boc-L-ValNCA to a Phe-PAM resin or of Boc-L-PheNCA to an Ala-PAM resin resulted in 0.2%4.25% formation of D-Val-Phe and D-Phe-Ala, respectively, indicating that racemization during UNCA coupling is comparable to that found using BOP or HBTU.
SLAS Technology
The development of peptide-based pharmaceutics is a hot topic in the pharmaceutical industry and in basic research. However, from the research and development perspective there is an unmet need for new, alternative, solid-phase peptide synthesizers that are highly efficient, automated, robust, able to synthetize peptides in parallel, inexpensive (to obtain and operate), have potential to be scaled up, and even comply with the principles of green chemistry. Moreover, a peptide synthesizer of this type could also fill the gap in university research, and therefore speed the advancement of peptide-based pharmaceutical options. This paper presents a Tecan add-on peptide synthesizer (TaPSy), which has operational flexibility (coupling time: 15-30 min), can handle all manual synthesis methods, and is economical (solvent use: 34.5 mL/cycle, while handling 0.49 mmol scale/reactor, even with ≤ 3 equivalents of activated amino acid derivatives). Moreover, it can carry out parallel synthesis of up to 12 different peptides (0.49 mmol scale in each). TaPSy uses no heating or high pressure, while it is still resistant to external influences (operating conditions: atmospheric pressure, room temperature 20-40 ˚C, including high [ > 70%] relative humidity). The system's solvent can also be switched from DMF to a green and biorenewable solvent,-valerolactone (GVL), without further adjustment. The designed TaPSy system can produce peptides with high purity (> 70%), even with the green GVL solvent alternative. In this paper we demonstrate the optimization path of a newly developed peptide synthesizer in the context of coupling reagents, reaction time and reagent equivalents applying for a synthesis of a model peptide. We compare the results by analytical characteristics (purity of raw material, crude yield, yield) and calculated overall cost of the syntheses of one mg of crude peptide using a specified set of reaction conditions.
An Improved Procedure for N- to C-Directed (Inverse) Solid-Phase Peptide Synthesis
Journal of Combinatorial Chemistry, 2000
A method for solid-phase peptide synthesis in the N-to C-direction that delivers good coupling yields and a low degree of epimerization is reported. The optimized method involves the coupling, without preactivation, of the resin-bound C-terminal amino acid with excess amounts of amino acid tri-tert-butoxysilyl (Sil) esters, using HATU as coupling reagent and 2,4,6-trimethylpyridine (TMP, collidine) as a base. For the amino acids investigated, the degree of epimerization was typically 5%, except for Ser(t-Bu) which was more easily epimerized (ca. 20%). Five tripeptides (AA 1 -AA 2 -AA 3 ) with different properties were used as representative model peptides in the development of the synthetic method: Asp-Leu-Glu, Leu-Ala-Phe, Glu-Asp-Val, Asp-Ser-Ile, and Asp-D-Glu-Leu. The study used different combinations of HATU and TBTU as activating agents, N,N-diisopropylethylamine (DIEA) and TMP as bases, DMF and dichloromethane as solvents, and cupric chloride as an epimerization suppressant. The epimerization of AA 2 in the coupling of AA 3 was further reduced in the presence of cupric chloride. However, the use of this reagent also resulted in a decrease in loading onto the resin and significant cleavage between AA 1 and AA 2 . Experiments indicated that the observed suppressing effect of cupric chloride on epimerization in the present system merely seemed to be a result of a base-induced cleavage of the oxazolone system, the key intermediate in the epimerization process. Consequently, the cleavages were most pronounced in slow couplings. An improved synthesis of fully characterized amino acid tri-tert-butoxysilyl (Sil) ester hydrochloride building blocks is presented. The amino acid Sil esters were found to be stable as hydrochlorides but not as free bases. Although only a few peptides have been used in this study, we believe that the facile procedure devised herein should provide an attractive alternative for the solid-phase synthesis of short (six residues or less) C-terminally modified peptides, e.g., in library format.
Journal of the American Chemical Society, 1998
Peptide targets for synthesis are often desired with C-terminal end groups other than the more usual acid and amide functionalities. Relatively few routes exist for synthesis of C-terminal-modified peptidessincluding cyclic peptidessby either solution or solid-phase methods, and known routes are often limited in terms of ease and generality. We describe here a novel Backbone Amide Linker (BAL) approach, whereby the growing peptide is anchored through a backbone nitrogen, thus allowing considerable flexibility in management of the termini. Initial efforts on BAL have adapted the chemistry of the tris(alkoxy)benzylamide system exploited previously with PAL anchors. Aldehyde precursors to PAL, e.g. 5-(4-formyl-3,5-dimethoxyphenoxy)valeric acid, were reductively coupled to the R-amine of the prospective C-terminal amino acid, which was blocked as a tert-butyl, allyl, or methyl ester, or to the appropriately protected C-terminal-modified amino acid derivative. These reductive aminations were carried out either in solution or on the solid phase, and occurred without racemization. The secondary amine intermediates resulting from solution amination were converted to the 9-fluorenylmethoxycarbonyl (Fmoc)-protected preformed handle derivatives, which were then attached to poly-(ethylene glycol)-polystyrene (PEG-PS) graft or copoly(styrene-1% divinylbenzene) (PS) supports and used to assemble peptides by standard Fmoc solid-phase chemistry. Alternatively, BAL anchors formed by onresin reductive amination were applied directly. Conditions were optimized to achieve near-quantitative acylation at the difficult step to introduce the penultimate residue, and a side reaction involving diketopiperazine formation under some circumstances was prevented by a modified protocol for N R-protection of the second residue/ introduction of the third residue. Examples are provided for the syntheses in high yields and purities of representative peptide acids, alcohols, N,N-dialkylamides, aldehydes, esters, and head-to-tail cyclic peptides. These methodologies avoid postsynthetic solution-phase transformations and are ripe for further extension.
Application of carboxylic-phospholanic mixed anhydrides to fragment coupling in peptide synthesis
International Journal of Peptide and Protein Research, 1992
The application of 1-0x0-1-chlorophospholane as a novel reagent for the in situ activation of peptide fragments for use in peptide bond forming reactions, either in liquid or solid phase, has been examined. 24.1 MHz 31P NMR spectroscopy has been employed to follow the formation, stability and reactivity of the intermediate phospholanic-carboxylic mixed anhydride
Fundamentals of Modern Peptide Synthesis
ChemInform, 2006
The purpose of this article is to delineate strategic considerations and provide practical procedures to enable non-experts to synthesize peptides with a reasonable chance of success. This article is not encyclopedic but rather devoted to the Fmoc/tBu approach of solid phase peptide synthesis (SPPS), which is now the most commonly used methodology for the production of peptides. The principles of SPPS with a review of linkers and supports currently employed are presented. Basic concepts for the different steps of SPPS such as anchoring, deprotection, coupling reaction and cleavage are all discussed along with the possible problem of aggregation and side-reactions. Essential protocols for the synthesis of fully deprotected peptides are presented including resin handling, coupling, capping, Fmoc-deprotection, final cleavage and disulfide bridge formation. Index Entries: Solid phase peptide synthesis (SPPS); resin; Fmoc SPPS; coupling reagents; protecting groups; anchoring; side reaction.