Sustainable synthesis of N-methylated peptides in a continuous-flow fixed bed reactor
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A rapid, simplified and highly efficient continuous-flow solid-phase peptide synthesis technology is reported for the direct synthesis of mono and multiple N-methylated cyclic alanine and valine peptides. Through an optimization study, we find that only 1.5 equivalents of the amino acids are sufficient for the couplings to maintain excellent conversions. Importantly, the technology is outstandingly sustainable, since three chemical steps are cancelled from the procedure and low amount of solvent is used, compared to traditional technologies. Furthermore, it is also applicable to the coupling of challenging amino acids, since pentavalines were constructed with high yield. The technology was successfully upscaled and peptide cyclization was carried out too.
Keywordspeptides synthesis peptidomimetics continuous-flow SPPS N-methylation
Studying peptides has been the focus of research for many years, because of the large variety of application of these biomolecules in a number of fields, including rational drug development [1, 2, 3, 4], nanotechnology [5, 6, 7, 8], material science [9, 10, 11, 12], etc. However, there are serious limitations in the use of peptides in medicine. Major obstacles are metabolic instability of peptides owing to fast degradation by the proteases and peptidases in the systemic circulation of the human body (short biological half-life) and poor oral bioavailability [13, 14, 15]. Another great drawback of peptides is their poor membrane permeability . One of the synthetic modifications to depress enzymatic cleavage and to insure facile absorption into the systemic blood circulation is the methylation of peptide backbone [17, 18, 19]. Peptides containing N-methyl amino acids appear regularly in nature: in plants, marine sponges and numerous microorganisms, for example Cyclosporine A, with interesting pharmacological profile and therapeutic potential [20, 21]. The importance of N-methylated peptides is warranted by further significant biomedical applications too [22, 23].
Multiple N-methylated peptides are reluctantly applied  possibly due to the cumbersome and often failed couplings of the amino acids onto sterically hindered N-methylated sites  and incalculable conformational changes [26, 27]. N-methylated peptides are mainly synthesized in an indirect way by the on-resin methylation of ordinary α-amino acids. The reason is the relatively high price of Fmoc-protected N-methylated amino acids, which excludes their direct utilization thereof. Nonetheless, the in situ methylation requires three additional time-consuming steps, extra reagents and produces excess amount of waste. Moreover, during Fmoc removal epimerization and formation of diketopiperazine  can occur. Even more problematic is the spontaneous deprotection of the Fmoc-amino acid. This is caused by the secondary amine functional group of the N-terminus resulting in the additional incorporation of amino acids .
Continuous flow (CF) techniques have recently emerged as a productive methodology in modern synthetic chemistry. This is due to the substantial number of advantages against conventional batch procedures, such as faster heat and mass transfer, the efficient mixing of substrates, shorter reaction times and facile scale up [29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46].
In this work a fast and highly economic continuous-flow solid-phase peptide synthesis (CF-SPPS) technique is presented for the preparation of mono and multiple N-methylated oligopeptides. The technology ensures high yields for the desired peptides with the aid of readily available Fmoc-protected N-methylated amino acids applied in 1.5 M excess. Importantly, the method requires a considerably shorter time, while lower amount of waste is produced. Mono and multiple N-methylated oligoalanines and oligovalines were synthesized as model peptides including difficult sequences.
Results and discussion
The optimized CF-SPPS technique might allow accomplishing the fast and powerful synthesis of numerous N-methylated peptides on solid support. As an evidence of the effectiveness of the procedure, mono and multiple N-methylated pentaalanine peptides were prepared. Oligoalanine peptides containing one or more methyl groups can be employed as template structures for designing biologically active compounds.
The synthesized N-methylated oligoalanines and obtained yields
The Fmoc-Ala loaded resin was filled into the PEEK column. The coupling and deprotection steps were performed under the conditions optimized previously. After the synthesis the resin was transferred to a flask and the cleavage was carried out by the utilization of 90% trifluoroacetic acid (TFA) and 10% water. The crude peptides were analyzed by HPLC-MS and the yields are shown in Table 1. Importantly, the yields of the peptides are >90% and no truncated sequences were found in the product. The yields were calculated from resin loading. Outstandingly, no extra amino acid incorporation was detected resulting from the spontaneous deprotection of the activated amino acid. This fact can be explained by the short residence time of the coupling mixture spent on the resin bed compared to ordinary SPPS technologies and by the fast and efficient coupling of the amino acid to the peptide chain. The residence time was measured to be 5.8 min.
The investigated oligovalines and yields of the syntheses
Importantly, our methodology required lower amounts of amino acid for coupling (1.5 equiv.) making the synthesis more cost-effective. Noteworthy, the coupling and deprotection times were 28 min under flow conditions, compared with 274 min in the former strategy. With this CF technique an amount of only 4.2 mL of solvent was used, as compared with the high solvent consumption (66 mL) of the original approach. Importantly, 3 additional chemical steps were avoided. Importantly, the technology is not only faster, but considerably more sustainable.
Easy and effective solution-phase cyclization of peptides can be carried out by means of a dual syringe pump utilizing the pseudo-high dilution peptide cyclization technology [51, 52, 53, 54]. In a typical experiment, a solution of 1 equiv. of linear peptide in 10 mL DMF was transferred into a syringe. To the equal volume of DMF 1 equiv. of HATU was added and poured into another syringe. Both reaction mixtures were infused into a vigorously stirred solution of 3 equiv. of DIEA in 40 mL DMF at a flow rate of 0.015 mL min−1. After completion, the mixture was stirred for 30 min. The reaction mixture was diluted with 50 mL water and lyophilized. The conversion and the purity of the crude cyclic peptide was determined by RP-HPLC-MS analysis. The conversion of the cyclization experiment was quantitative and the RP-HPLC purification provided 68% yield from resin loading for peptide 13. It is a considerably higher value than the published result (26%) .
In summary, a rapid, simplified and highly efficient CF-SPPS technology is reported for the direct synthesis of mono and multiple N-methylated cyclic alanine and valine peptides. Through an optimization study, we find that in general only 1.5 equivalents of the amino acids are sufficient for the couplings to maintain excellent conversions. This is a cheap and time-efficient approach requiring low amounts of solvents and reagents. Furthermore, it is also applicable to the coupling of challenging amino acids. Importantly, the peptides were synthesized directly, avoiding the on-resin methylation of the amino acids, which allows shortening the synthesis by three chemical steps. Mono and multiple N-methylated pentaalanines and pentavalines were prepared as model and difficult sequences respectively. In the case of alanines, the results were satisfactory under the optimized conditions. To obtain excellent crude purities of pentavalines, however, required the use of oxyma pure as coupling agent and 2 equivalents of amino acids. A scaled-up synthesis was also performed and a subsequent cyclization of the peptide utilizing the pseudo-high dilution solution-phase technology was carried out too.
Experimental (materials and methods)
Peptide synthesis: The linear peptide chains were extended on a Tentagel R PHB resin (0.20 mmol g−1). For CF experiments, a modular CF apparatus was assembled, consisting of a cylindrical PEEK column (with internal dimensions of 100 × 4 mm) filled with the amino acid loaded resin (200 mg), an HPLC pump (JASCO PU-880), an HPLC autosampler (JASCO AS-2055 Plus), an HPLC column thermostat (Dionex STH 585), and a backpressure regulator (Upchurch Scientific/IDEX Health & Science P-465 PEEK). A coupling mixture consisting of 1.5 equivalents of Fmoc-protected amino acid and 1.5 equivalents of HATU as coupling reagent dissolved in 1 mL DMF and 3 equivalents of DIEA was mixed by the autosampler. The coupling mixture has been prepared just before the coupling reaction. The coupling reactions were carried out at the optimized reaction conditions, 80 bar pressure, 60 °C temperature, 0.15 mL min−1 flow rate. For Fmoc deprotection 2 mL of 2% DBU 2% piperidine DMF solution has been used. Between two chemical steps for washing DMF has been used for 6 min long with 0.15 mL min−1 flow rate.
We are grateful to the Hungarian Research Foundation (OTKA No. K 115731). The financial support of the GINOP-2.3.2-15-2016-00014 project is acknowledged. Supported by the ÚNKP-16-4-III New National Excellence Program of the Ministry of Human Capacities
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