Scalable and sustainable DMF-free solid-phase synthesis of liraglutideby 1-tert-butyl-3-ethylcarbodiimide-mediated couplings and catch-and-release acylation and purification strategies

Scalable and sustainable DMF-free solid-phase synthesis of liraglutideby 1-tert-butyl-3-ethylcarbodiimide-mediated couplings and catch-and-release acylation and purification strategies | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Scalable and sustainable DMF-free solid-phase synthesis of liraglutideby 1-tert-butyl-3-ethylcarbodiimide-mediated couplings and catch-and-release acylation and purification strategies Lorenzo Pacini, Manoj Kumar Muthyala, Robert Zitterbart, Oleg Marder, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5738025/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The growing need for sustainable practices in pharmaceutical manufacturing has stimulated advancements in peptide synthesis. This study focuses on applying green chemistry principles to the synthesis of the Glucagon-Like Peptide-1 analog liraglutide, using novel and sustainable solid-phase synthetic strategies. By adopting the safer coupling reagent 1-tert-butyl-3-ethylcarbodiimide (T-Bec®) in combination with eco-friendly binary solvents like dimethyl sulfoxide and butyl acetate, we demonstrated that it is possible to significantly reduce the environmental impact while maintaining high efficiency and quality of the synthesis. T-Bec® minimizes hazardous byproducts, such as hydrogen cyanide, and enhances solvent compatibility, achieving crude purities and yields comparable to conventional syntheses. Two synthetic strategies were developed for liraglutide production. The first strategy based on a “direct synthesis”, incorporating a lipidated lysine building block into the peptide sequence, achieving 86% HPLC purity after catch-and-release purification. The second strategy based on “catch-lipidation-and-release” approach, allowed to obtain the peptide precursor without the lipid moiety, which was later linked during a controlled lipidation step. This latter strategy yielded purities exceeding 90% and reduced reliance on preparative HPLC. These findings highlight the effectiveness of T-Bec® and green solvent systems to optimize scalable and sustainable SPPS processes. These methods improve resource efficiency and reduce environmental impact, to allow a viable pathway to produce therapeutic peptide ingredients like liraglutide. This work underscores the potential of green chemistry to align pharmaceutical innovation with environmental responsibility. Organic Chemistry Solid-phase peptide synthesis GLP-1 agonist catch-and-release purification green binary solvent mixtures T-Bec® coupling reagent Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The rising focus on sustainable practices in chemical synthesis has led to significant advancements in green chemistry, especially within pharmaceutical production, where reducing hazardous waste and minimizing environmental impact are critical objectives (Kar et al. 2022 ). Peptide-based therapeutics, increasingly vital in treating a wide array of conditions, from metabolic disorders to autoimmune diseases (Selmi et al. 2011 ; Sharma et al. 2023 ), are one area where environmentally friendly approaches are especially valuable (Ferrazzano et al. 2022 ). However, solid-phase peptide synthesis (SPPS), a key method for producing peptides, has traditionally relied on toxic solvents, high reagent consumption, and other practices that contribute to environmental and safety concerns. Integrating green chemistry principles into SPPS is therefore essential to develop more sustainable, less hazardous methodologies for peptide production (Martin et al. 2020 ). One promising strategy for green SPPS involves optimized coupling reagents for amide bond formation (Yang et al. 2023 ). Among these, N,N’-diisopropylcarbodiimide (DIC) is commonly employed but is known for its associated safety risks, including toxicity, side reactions, and undesired by-products (Pawlas and Rasmussen 2023 ). 1-tert-Butyl-3-ethylcarbodiimide (T-Bec®), a novel and safer coupling reagent, offers a compelling alternative (Pawlas et al. 2023 ). T-Bec® reduces the health hazards and environmental impact typically associated with traditional reagents, while maintaining high efficiency and low epimerization rates in peptide synthesis (Fantoni et al. 2024 ). Importantly, compatibility of T-Bec® with green binary solvent mixtures, such as combinations of dimethyl sulfoxide (DMSO) and tert-butyl acetate (BuOAc), further supports its use in sustainable SPPS protocols (Ferrazzano et al. 2019 ; Al Musaimi et al. 2020 ; Wegner et al. 2021 ; Pacini et al. 2024 ). These green solvents provide a more environmentally friendly medium for synthesis compared to traditional toxic solvents such as dimethylformamide (DMF) or dichloromethane (DCM), making T-Bec® and green solvent systems an ideal pairing for advancing green peptide chemistry. Last but not least, while DIC reacts with OxymaPure® at a 1:1 ratio, yielding the byproduct oxadiazole and releasing HCN, T-Bec® reacts with OxymaPure® under the same conditions, forming oxadiazine only, what makes T-Bec® a more health and safety friendly reagent (Manne et al. 2021 ). The development and application of greener SPPS methodologies are especially relevant in production of Glucagon-Like Peptide-1 (GLP-1) agonists, which are prominent in treating diabetes and obesity (Staby et al. 2020 ; Liu et al. 2020 ; Hach et al. 2024 ). GLP-1-based drugs have revolutionized the therapeutic landscape in metabolic health, with agonists like liraglutide achieving blockbuster status due to their efficacy, long lasting action, and favorable safety profiles. However, as demand for these peptides rises, the need for scalable, sustainable synthetic routes that align with environmental responsibility goals is dramatically increasing. Designing green synthetic strategies for GLP-1 agonists would meet this market demand while reducing the ecological footprint of their production (Barredo-Vacchelli et al. 2024 ). Herein, two synthetic strategies, involving the use of PurePep EasyClean (PEC) catch & release technology, are described (Zitterbart et al. 2021 ). In both cases, we successfully achieved levels of liraglutide purity that makes further purification through chromatography much easier. In summary, this work highlights the importance of T-Bec® as a safer, more sustainable coupling reagent, its compatibility with green solvent systems within the production of liraglutide, and the broader role of green SPPS approaches in producing key therapeutic peptide ingredients. Additionally, it has been demonstrated that the use of heating together with T-Bec®/Oxyma Pure accelerates SPPS and further enhances reaction rates, contributing to the efficiency of green peptide synthesis. By promoting eco-friendly methods for the synthesis of GLP-1 agonists, decreasing the need of time-and-solvent-consuming preparative HPLC, we can meet the growing demand for these drugs while setting a new standard for environmental responsibility in pharmaceutical manufacturing. Materials and methods Fmoc-amino acids, Oxyma Pure and Fmoc-Gly-2-Chlorotrityl resin were provided directly by Gyros Protein Technologies (Tucson, AZ, USA). N,N'-Diisopropylcarbodiimide was purchased from Carbosolution Chemicals GmbH (St. Ingbert, Germany). Dimethylformamide, ethyl acetate, propyl acetate, butyl acetate, dimethyl sulfoxide, acetic anhydride, pyridine, anisole, and triisopropylsilane were obtained from Carl Roth GmbH (Karlsruhe, Germany). Piperidine, trifluoroacetic acid, and Rink amide AM resin (0.64 mmol/g), Fmoc-Lys(Palm-Glu-OtBu)-OH, Palm-L-Glu(OSu)-OtBu were purchased from Iris Biotech GmbH (Marktredwitz, Germany). Fmoc-Gly-Tentagel S resins (0.17 mmol/g) was acquired from Rapp Polymere (Tuebingen, Germany). 1-tert-Butyl-3-ethylcarbodiimide (T-Bec®) was provided by Luxembourg Bio Technologies (Ness Ziona, Israel). Solubility Tests - After calibrating a 15 mL plastic tube, 1 mmol of each coupling reagent was weighed, and 1 mL of the tested solvent was added. The mixture was stirred at room temperature for 30 minutes. If the reagent remained insoluble, the solvent volume was gradually increased to check solubility at concentrations of 0.5 M, 0.25 M, and 0.1 M. Solid-Phase Peptide Synthesis - All syntheses were performed using the PurePep® Chorus (Tucson, AZ, USA) peptide synthesizer in parallel synthesis mode, with induction heating and at room temperature. For 100 µmol scale syntheses, 303 mg of Fmoc-Gly-2-Chlorotrityl resin (0.33 mmol/g loading), 417 mg of Fmoc-Gly-Tentagel S resin (0.24 mmol/g loading), and 588 mg of Tentagel S RAM resin (0.17 mmol/g loading) were weighed. Fmoc-amino acids were dissolved in the tested mixture or in DMF using an ultrasonic bath at 0.3M concentration. DIC or T-Bec® and Oxyma Pure were dissolved in the tested mixture or in DMF at 1M concentration. The solvents were freshly prepared and transferred to the appropriate bottles corresponding to separate lines on the machine. After synthesis, the loaded resins were washed with isopropyl alcohol and dried under vacuum. Before starting each synthesis with different solvents, the instrument was fully calibrated to ensure that the correct volume of solvent was delivered. Test Cleavage - A small amount (2–4 mg) of dry loaded resin was transferred into a 500 µL plastic tube, and 100 µL TFA, 5 µL TIS, and 5 µL H 2 O were added. After 1.5 hours of mechanical mixing, the suspension was filtered and cold diethyl ether (-20°C) was added to the peptide TFA solution. The precipitated peptide was isolated by centrifugation and dried under vacuum. The crude peptides were dissolved in water/ACN 1:1 and analyzed by RP-UPLC coupled with ESI-MS. PurePep EasyClean (PEC) lipidation – The first step of this process is the coupling of the reductively, traceless cleavable linker PEC-RC+, TFA cleavage resistant, that allows further attachment on activated agarose beads, on the N-terminal amino function of the to-be-purified peptide still on resin that was conducted at r.t. for 18 hours under mechanical shaking using 4 equivalents of the linker, 6 equivalents of Oxyma pure, and 6 equivalents of DiPEA in DMSO/EtOAc 1:9 (v/v) mixture. After monitoring with LC-MS, the linker-precursor peptide was cleaved from the resin, and all protecting groups on the amino acid side chains were removed. The linker-peptide was solubilized in TFA/H 2 O 9:1 (v/v) and cooled at 0°C. Pyridine was added dropwise, the solution was diluted with ACN and added to polymethacrylate (PMA) activated beads. In the case of activated Agarose beads, the linker-peptide was solubilized with DMSO and added directly on the beads. The mixture was shaken for 5 hours at r.t. and, after immobilization, the beads were washed with 3×H 2 O. Unreacted aldehyde functions were blocked on beads using a solution of 1% (w/v) NH 2 OMe in 0.1 M aqueous citric acid buffer (pH 4.5) for 5 minutes. The loaded beads (agarose or PMA) were washed with DMSO/butyl acetate 3:7 (v/v). A solution of 4 equivalents of Palm-L-Glu(OSu)-OtBu, 4 equivalents of coupling reagent (HOAt or Oxyma Pure) and 6 equivalents of base (DiPEA or Pyridine) were added to the loaded beads after 5 minutes of pre-activation. The formation of the new amide bond occurred in 5 hours. PurePep EasyClean (PEC) purification – After immobilization (and acylation, in the case of the second strategy), the beads were washed with 3×0.9 M guanidinium chloride (GdmCl) in DMSO and with 3×0.1 M NaCI in H 2 O/EtOH 3:7 (v/v). The linker on the peptide was reduced using a solution of 0.3 M dithiothreitol (DTT) in 0.6 M aqueous NaHCO 3 (pH 8) for 15 minutes at r.t. Then, the beads were washed with H 2 O and ACN. The final release consisted in treatment with TFA/H 2 O 95:5 (v/v) for 45 minutes at r.t. for PMA beads and TFA/H 2 O 40:60 (v/v) for 1 hour at r.t. followed by precipitation in cold diethyl ether and lyophilization. Result and discussion Green solvents binary mixtures and T-Bec® - The first part of our study involved the test of a safer coupling reagent such as T-Bec® in combination with binary mixtures previously studied. T-Bec® is a non-symmetric carbodiimide as showed in Fig. 1 . In the very enlightening work from Pawlas et al. (Pawlas and Rasmussen 2023 ), it was demonstrated that the coupling system T-Bec®/Oxyma Pure, during the formation of a new amide bond, releases a marked lower quantity of HCN deriving from Oxyma Pure in comparison with the standard coupling system DIC/Oxyma Pure even if the performances, in terms of coupling reaction rate are essentially the same. Another important feature is that the T-Bec® derived urea, 1-tert-butyl-3-ethyl urea (TBEU), is more soluble in DMF and in many other solvent systems. We investigated the coupling system T-Bec®/Oxyma Pure to synthesize, at 100µmol scale and at 90°C, three model peptides using the green binary mixture butyl acetate/DMSO, propyl acetate/DMSO or anisole/DMSO for coupling and Fmoc removal reactions, and ethyl acetate/DMSO for washings. The three model peptides are ACP(65–74)-NH 2 (H-VQAAIDYING-NH 2 ), a peptide commonly employed to assess the efficiency of synthetic protocols, polyALA (H-AAAAAAAAAAK-NH 2 ) a well-known self-assembling peptide even on resin, and AlaSTD (H-AKADEVSLHKWYG-NH 2 ) a standard peptide used as a reference non-challenging sequence (Pacini et al. 2024 ). The sequences were produced with the PurePep® Chorus automated synthesizer (Gyros Protein Technologies, Tucson, AZ, USA), employing mechanical shaking and nitrogen bubbling for mixing. The exact protocol, outlining volumes and durations, is provided in Table S1 and S2. Table 1 Crude HPLC Purities of peptides synthesized using T-Bec®/Oxyma Pure in green solvents binary mixtures DMF Oxyma Pure T-Bec® 90°C, 2 min BuOAc DMSO Oxyma Pure T-Bec® 90°C, 2 min Anisole DMSO Oxyma Pure T-Bec® 90°C, 2 min PrOAc DMSO Oxyma Pure T-Bec® 90°C, 2 min Peptide HPLC Purity (% A/A) HPLC Purity (% A/A) HPLC Purity (% A/A) HPLC Purity (% A/A) ACP-NH 2 VQAAIDYING-NH 2 89.1 85.4 78.6 76.8 PolyALA-NH 2 AAAAAAAK-NH 2 77.4 76.4 79.9 64.6 GTPstd-NH 2 * AKADEVSLHKWYG-NH 2 89.6 93.3 83.9 77.4 The data in Table 1 show that the crude purities obtained from the three peptide syntheses in green binary solvent mixtures and DMF are fairly comparable. There is no single solvent mixture that consistently outperforms the others. For instance, the crude purity of ACP-NH 2 is higher when synthesized with DMF, but this trend does not hold for all sequences. The purity of PolyALA is higher when anisole/DMSO mixture was used, while GTPstd achieves better purity with a BuOAc/DMSO mixture. On average, the purities of the three sequences are comparable when using DMF and BuOAc/DMSO. At this stage, we decided to investigate the use of T-Bec® in association with BuOAc/DMSO mixture also for the synthesis of the linear precursor of liraglutide at high temperature (90°C) without double couplings and use of pseudo prolines. The synthesis yielded an over 70 A/A% purity and 93% yield demonstrating the full comparability with DIC/Oxyma Pure synthesis in BuOAc/DMSO mixture used for the second strategy. Green synthesis approaches for liraglutide production - In the second part of this work, we focused on the synthetic strategy of liraglutide, a blockbuster drug included in the class of GLP-1 agonists. This study presents two synthetic strategies that utilize green binary solvent mixtures. The first strategy involves the synthesis of the complete liraglutide sequence, incorporating the building block Lys(Palm-Glu-OtBu) directly in the sequence via green SPPS followed by catch-and-release PEC purification as reported in Fig. 3 . In contrast, the second strategy involves the synthesis of the linear sequence of liraglutide without the lipid moiety attached to the lysine residue. This approach also employs green binary solvent mixture for a green solid-phase peptide synthesis. Following the completion of the SPPS, cleavage from the resin and complete removal of side-chain protecting groups in the resulting peptide undergoes catch-and-release purification (PEC). After the “catch” step, lipidation occurs directly on the agarose solid phase. This process involves the free primary amine on the side chain of the lysine residue, which is the only reactive amine present in the peptide. This strategy allows for precise control over the lipidation process, thereby optimizing the final yield and purity. The “direct synthesis” strategy – In this pathway, the entire peptide was synthesized using solid-phase peptide synthesis (SPPS) on a 100 µmol scale, utilizing the PurePep Chorus instrument by Gyros Protein Technology (Tucson, AZ, USA). In line with green chemistry principles applied to peptide synthesis, and based on our previous work (Pacini et al. 2024 ), we selected environmentally friendly binary solvent mixtures for the synthesis of liraglutide. Specifically, the EtOAc/DMSO mixture (8:2, v/v) was used as the primary solvent for washings. For amide bond formation and Fmoc deprotection, BuOAc/DMSO mixtures (ratios of 1:1 and 7:3, respectively) were employed due to their varying polarity. Additionally, these mixtures were designed for use at elevated temperatures, as their boiling points exceed 100°C, potentially enhancing yield, purity, and reducing synthesis time. However, for this study, all reactions were conducted at room temperature to facilitate scale-up on the PurePep Sonata + instrument by Gyros Protein Technology (Tucson, AZ, USA), which lacks a heating system. For coupling, we used a system consisting of diisopropylcarbodiimide (DIC) and Oxyma Pure (5 equivalents each), along with 5 equivalents of Fmoc-protected amino acids. The reaction was performed for one hour at room temperature, mixing by both nitrogen bubbling and mechanical shaking. Instead, the coupling reaction with the building block Fmoc-Lys(Palm-Glu-OtBu)-OH required 2 equivalents of the building block, along with 2 equivalents each of DIC and Oxyma Pure. From the 8th cycle onwards, the reaction was repeated twice to achieve a conversion rate as close as possible to 100%. Fmoc removal was performed in two 5-minute treatments using the appropriate solution, except for the second amino acid in the sequence, where a shorter treatment (5 + 3 minutes) was applied to prevent the formation of diketopiperazines, which could significantly reduce the overall yield, as previously demonstrated for another GLP-1 agonist product (Wang et al. 2022 ). In order to monitor and prevent the formation of diketopiperazines (DKP), we simultaneously synthesized the sequences using two different types of resin functionalization, i.e., Wang and 2-chlorotrityl resin, expecting differences between the two resins due to the steric hindrance of the trityl group on trityl resin that should prevent the formation of diketopiperazine better than Wang resin. We were unable to detect any DKP during the Fmoc removal of the second amino acid on both types of resin. The rapid treatment (5 + 3 minutes) effectively deprotected the N-terminal of the second amino acid without resulting in the formation of DKPs (data not shown). Another important reaction is acetylation after each coupling step, which ensures that only the N-terminal of the desired sequence remains free at the end of the synthesis. After the synthesis was completed on the instrument, a test cleavage was performed, and the HPLC analysis showed that the purity of the peptide was 45% A/A (Supporting information). At this stage, the loaded resins were divided into two aliquots: one half (made of halves of 2-chlorotrityl resin and Wang resins combined in one batch) was cleaved and purified by preparative HPLC, while the others separately underwent PEC catch-and-release purification. For the latter, with only the N-terminal of the desired sequence accessible on the resin, the PEC linker was coupled using 4 equivalents of linker, 6 equivalents of Oxyma Pure, and 6 equivalents of DiPEA. This reaction proceeded in 16 hours at room temperature in a green binary mixture of BuOAc and DMSO (7:3, v/v), with reaction progress monitored by the chloranil test. After the cleavage step, we evaluated the yield based on the isolated crude product mass. Both syntheses on the two types of resin yielded approximately 75% (isolated mass/calculated mass) (data available in supporting information). The two crudes deriving from the two resin types were combined in one batch and PEC purified obtaining a final product with an HPLC purity of 86.05 A/A% (Fig. 5 ). The Catch-Lipidation-And-Release Strategy – The peptide was assembled on Fmoc-Gly- 2-Chlorotrityl resin using standard Fmoc-protected amino acids, following the same green mixtures described in the previous strategy. However, this time Fmoc-Lys(Boc)-OH was used instead of the more expensive building block Fmoc-Lys(Palm-Glu-OtBu)-OH. As a result, at the end of the SPPS, we obtained a liraglutide precursor without the fatty acid plus Glu as linker moiety on the peptide. After PEC linker coupling and cleavage, we obtained a 54 A/A% HPLC pure crude (yield 71%). At this stage, we immobilized 5 µmol of PEC-linker-liraglutide precursor on both agarose and PMA aldehyde-functionalized beads for acylation as a proof of concept. Following immobilization, the first acylation attempt used 4 equivalents of the building block Palm-L-Glu(OSu)-OtBu along with 4 equivalents of Oxyma Pure, dissolved in DMF. After 5 minutes of pre-activation at room temperature, the solution was added to the peptide-loaded beads (agarose and PMA). We hypothesized that the primary amine on lysine would be more reactive than histidine and more prone to nucleophilic attack on the OSu-activated carboxylic group of the building block. The reaction was monitored after 2 and 4 hours drawing a small sample of beads. The product was cleaved after 4 hours from the beads when it yielded a conversion rates of 95% for agarose beads (poor HPLC purity) and 91% for PMA beads (HPLC purity 63 A/A%) (Table 2 ). Table 2 In-Process-Control of Acylation study on liraglutide precursor Bead type conversion (%) Purity (A/A%) Check 2h Agarose 87 69.5 PMA 55 43.8 Final release 4h Agarose 95 75.7 PMA 91 63.3 We observed that the mild final release of liraglutide (40% v/v TFA in water) from agarose beads was neither long nor acidic enough to remove the tBu group from the carboxylic function on the α-carbon of glutamic acid (Fig. 6 ). After precipitation of the product, we resolved the issue of partial tBu deprotection with a treatment of TFA/TIS/H₂O (95:2.5:2.5 v/v/v) at room temperature for 1 hour. This treatment resulted in completely deprotected liraglutide (final purity 72 A/A%). This first encouraging result motivated us to explore further coupling systems by adding a base and testing also HOAt and as solvent the green binary mixture BuOAc/DMSO 7:3 v/v used also for SPPS. We investigated 3 different coupling systems (c.s.) still at 5 µmol, reported in Table 3 , both on agarose and PMA beads. Table 3 Three Coupling Systems evaluated for the optimization of acylation. c.s. 1 c.s. 2 c.s. 3 BB-OSu 4 equiv 20 µmol OxymaPure HOAt OxymaPure 4 equiv 20 µmol DiPEA DiPEA Pyridine 6 equiv 30 µmol in 150 µL BuOAc/DMSO In all these coupling systems, we added 6 equivalents of base to compensate for the minimal basicity of DMF, substituted with BuOAc/DMSO, that provides the optimal environment for the coupling reaction. The best result (79 A/A%, conversion 99%) after 5 hours of reaction, was achieved with coupling system 2 (HOAt and DIPEA) on agarose beads. Based on this result, we decided to scale up the reaction by a factor of 10. 50 µmol were treated with coupling system 2 in BuOAc/DMSO for 5 hours at r.t. obtaining, after the release from agarose beads and a further treatment with TFA/TIS/H 2 O, 90 A/A%, HPLC crude purity and a conversion over 99% (Fig. 7 ). Conclusions This study highlights the successful integration of green chemistry principles into solid-phase peptide synthesis (SPPS), offering a practical and environmentally friendly alternative for producing therapeutic peptide ingredients. By using 1-tert-butyl-3-ethylcarbodiimide (T-Bec®) as a safer coupling reagent in combination with eco-friendly solvent systems like dimethyl sulfoxide (DMSO) and butyl acetate (BuOAc), we reduced the environmental footprint and safety risks commonly associated with traditional synthesis methods. These approaches maintained high efficiency and delivered high-quality peptide products, demonstrating that sustainability and performance can go hand in hand. We explored two innovative strategies for synthesizing liraglutide, a key GLP-1 agonist used to treat diabetes and obesity. The “direct synthesis” approach incorporated a lipidated lysine building block directly into the peptide sequence, achieving excellent purity after catch-and-release purification. The “catch-lipidation-and-release” strategy took a different route, attaching the lipid moiety in a controlled, post-synthesis step, which allowed for better precision and higher final yields. Both methods proved to be scalable and significantly reduced the need for extensive preparative HPLC, saving time and resources. This work demonstrates that adopting greener methods in peptide synthesis is not only possible but practical for meeting the pharmaceutical industry growing demand for sustainability. Our results show that using T-Bec® and green solvents can deliver the same high-quality outcomes as conventional methods while being more friendly to the environment. Additionally, the catch-and-release purification technique enhanced efficiency, making these methods suitable for large-scale applications. Looking ahead, this research sets the stage for further innovation in sustainable pharmaceutical manufacturing. As the need for peptide-based therapeutics like liraglutide continues to grow, these methods offer a reliable, eco-conscious way to meet that demand. By aligning cutting-edge science with environmental responsibility, this study underscores the potential to redefine how we produce life-saving medicines, benefiting both people and the planet. Declarations Manoj Kumar Muthyala and Robert Zitterbart are employers of Gyros Protein Technologies Inc. and declare competing financial interests: the PurePep Chorus synthesizer and the PurePep EasyClean technology described in the manuscript can be purchased from Gyros Protein Technologies. Oleg Marder is an employer of Luxembourg Bio Technologies and declare competing financial interests: T-Bec® described in the manuscript can be purchased from Luxembourg Bio Technologies. Acknowledgements: Lorenzo Pacini acknowledges MUR and EU-FSE for the financial support of the PhD fellowship PON Research and Innovation 2014–2020 (D.M. 1061/2021) XXXVII Cycle in Chemical Sciences: “Greening peptide chemistry, a necessary step to the future”. The project was funded in part by EUniWell: 5th seed funding call for the TTPep project and 3rd Well-Being Research Incubator Call for the project “Sustainable and green upscaling processes for peptide(-based) in vitro diagnostics and cosmeceuticals”. 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Chem Sci 12:2389–2396. https://doi.org/10.1039/D0SC06285E Additional Declarations The authors declare potential competing interests as follows: Manoj Kumar Muthyala and Robert Zitterbart are employers of Gyros Protein Technologies Inc. and declare competing financial interests: the PurePep Chorus synthesizer and the PurePep EasyClean technology described in the manuscript can be purchased from Gyros Protein Technologies. Oleg Marder is an employer of Luxembourg Bio Technologies and declare competing financial interests: T-Bec® described in the manuscript can be purchased from Luxembourg Bio Technologies. Supplementary Files PAPINIAMSupportingPREPRINT.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5738025","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":395984742,"identity":"e8112e0c-8251-4e35-9018-8444fc079794","order_by":0,"name":"Lorenzo Pacini","email":"","orcid":"https://orcid.org/0000-0003-0737-2213","institution":"University of Florence","correspondingAuthor":false,"prefix":"","firstName":"Lorenzo","middleName":"","lastName":"Pacini","suffix":""},{"id":395984743,"identity":"eb4e3ba8-d12b-4293-9398-0f105d1ca0b5","order_by":1,"name":"Manoj Kumar Muthyala","email":"","orcid":"https://orcid.org/0009-0008-7145-8402","institution":"Gyros Protein Technologies Inc","correspondingAuthor":false,"prefix":"","firstName":"Manoj","middleName":"Kumar","lastName":"Muthyala","suffix":""},{"id":395984744,"identity":"108cf9f2-6691-4f18-9bfd-b9507ba54ef3","order_by":2,"name":"Robert Zitterbart","email":"","orcid":"https://orcid.org/0000-0003-1729-6111","institution":"Gyros Protein Technologies Inc","correspondingAuthor":false,"prefix":"","firstName":"Robert","middleName":"","lastName":"Zitterbart","suffix":""},{"id":395984745,"identity":"23e1d301-8f65-4776-8990-2212c4e34344","order_by":3,"name":"Oleg Marder","email":"","orcid":"","institution":"Luxembourg Bio Technologies","correspondingAuthor":false,"prefix":"","firstName":"Oleg","middleName":"","lastName":"Marder","suffix":""},{"id":395984746,"identity":"cf557ccc-6e5f-4b0b-a89a-1da8c7826b16","order_by":4,"name":"Paolo Rovero","email":"","orcid":"https://orcid.org/0000-0001-9577-5228","institution":"University of Florence","correspondingAuthor":false,"prefix":"","firstName":"Paolo","middleName":"","lastName":"Rovero","suffix":""},{"id":395984747,"identity":"1a1e6da5-33a6-4d3f-989b-a81238f6acf7","order_by":5,"name":"Anna Maria Papini","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAUlEQVRIie2PsUoDQRBAFwLajLl2ZY/kFyYsXBPhfmUhYDUnBzYWFldpldQH/swcC9oc1gsJllYWQcEmQdxTEhWTO0uLfcXOzLJvZlaIQOAfA58BBUQ+8AVubv6iHBdeqX3S6WxB9kf91eUX0c20enaXD3FUHFUveb6Itcsq5lzE6R5FLu4nim7PQXJ/okp8hMSdGea2xRyhogMDggEVoPUKoV22KENHekVvBoYMetUouiRsnYKOEpVdGUCG5GMKyg5l5JVxNjMwsv3TcekVWT81igTg3crALzanV5MO7qZ2nq9tGl2TXvL6JD0s9v1/Qw9E71spu943/FQCgUAgsOUdFm1UKLnznIQAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-2947-7107","institution":"University of Florence","correspondingAuthor":true,"prefix":"","firstName":"Anna","middleName":"Maria","lastName":"Papini","suffix":""}],"badges":[],"createdAt":"2024-12-30 22:34:30","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":true,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-5738025/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5738025/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":72733177,"identity":"966d9fdd-e49c-4adb-8b9b-de8f5711c9d2","added_by":"auto","created_at":"2025-01-01 07:23:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":37400,"visible":true,"origin":"","legend":"\u003cp\u003eStructure of T-Bec®\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-5738025/v1/3fcdff34e1a49d17dfb70a0e.png"},{"id":72733179,"identity":"bd6082d8-b5d6-45ad-ac0f-0ca68f5f9226","added_by":"auto","created_at":"2025-01-01 07:23:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":550762,"visible":true,"origin":"","legend":"\u003cp\u003eRP-UHPLC traces of the precursor of liraglutide synthesized using BuOAc/DMSO and T-Bec®/Oxyma Pure as coupling system. C4 column Kromasil 300-5-C4 (300 Å, 5 μm, 4.6 × 150 mm); temperature 35 °C; flow, 1 mL/min; eluent, 0.1% (v/v) TFA in H\u003csub\u003e2\u003c/sub\u003eO (A) and 0.1% (v/v) TFA in CH\u003csub\u003e3\u003c/sub\u003eCN (B); λ, 215 nm, gradient, 5−95% B in 20 min. Rt = 8.95 min: precursor liraglutide 70.05% purity.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-5738025/v1/6782df3854fae911b4838c2b.png"},{"id":72733187,"identity":"baf72f30-f082-4a43-8105-207bd038e1f0","added_by":"auto","created_at":"2025-01-01 07:23:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3924579,"visible":true,"origin":"","legend":"\u003cp\u003e\"Direct synthesis\" of liraglutide\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-5738025/v1/a1882fc740afe1198dabf76a.png"},{"id":72733291,"identity":"4f617a19-847e-4ba1-a160-a6ec003a3425","added_by":"auto","created_at":"2025-01-01 07:31:54","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4433680,"visible":true,"origin":"","legend":"\u003cp\u003e\"Catch-lipidation-and-release\" strategy for the synthesis of liraglutide\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-5738025/v1/89883c0b5daeb7003a800b6f.png"},{"id":72733197,"identity":"77a50adb-10c3-46ff-8e2b-3b2cd159b398","added_by":"auto","created_at":"2025-01-01 07:23:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":527807,"visible":true,"origin":"","legend":"\u003cp\u003eRP-UHPLC traces of PEC purified liraglutide synthesized using BuOAc/DMSO, direct synthesis strategy. C18 column Waters Acquity BEH (130 Å, 1.7 μm, 2.1 × 50 mm); temperature 65 °C; flow, 0.5 mL/min; eluent, 0.1% (v/v) TFA in H\u003csub\u003e2\u003c/sub\u003eO (A) and 0.1% (v/v) TFA in CH\u003csub\u003e3\u003c/sub\u003eCN (B); λ, 215 nm, gradient, 10−90% B in 5 min. Rt = 2.79 min: liraglutide 86.05% purity.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-5738025/v1/70b38b22a0fb9976c63de282.png"},{"id":72733190,"identity":"b415ff9e-d859-48c9-a7c5-c5ff93f4d8c9","added_by":"auto","created_at":"2025-01-01 07:23:55","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":840269,"visible":true,"origin":"","legend":"\u003cp\u003eStructure of Palm-L-Glu(OSu)-OtBu\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-5738025/v1/95ba1dabb508eba2cd07f0ab.png"},{"id":72733186,"identity":"9ebe3dc9-f17a-4e50-aa74-10e044b8ee82","added_by":"auto","created_at":"2025-01-01 07:23:54","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":475632,"visible":true,"origin":"","legend":"\u003cp\u003eRP-UHPLC traces of PEC purified liraglutide synthesized using BuOAc/DMSO, Acylation strategy. C18 column Waters Acquity BEH (130 Å, 1.7 μm, 2.1 × 50 mm); temperature 65 °C; flow, 0.5 mL/min; eluent, 0.1% (v/v) TFA in H\u003csub\u003e2\u003c/sub\u003eO (A) and 0.1% (v/v) TFA in CH\u003csub\u003e3\u003c/sub\u003eCN (B); λ, 215 nm, gradient, 10−90% B in 5 min. Rt = 2.81 min: liraglutide 90.5% purity.\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-5738025/v1/b410ad276c1cb73f5dd108b3.png"},{"id":72733802,"identity":"33087864-c234-4b93-89c8-b90a57a05d60","added_by":"auto","created_at":"2025-01-01 07:48:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7882393,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5738025/v1/72ab6913-d8ee-479d-bfa1-248df5a5c1b4.pdf"},{"id":72733182,"identity":"c17068ff-5d09-4b30-bdac-051b1a65e640","added_by":"auto","created_at":"2025-01-01 07:23:54","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1818442,"visible":true,"origin":"","legend":"","description":"","filename":"PAPINIAMSupportingPREPRINT.docx","url":"https://assets-eu.researchsquare.com/files/rs-5738025/v1/2490c0503d819fb00d650d37.docx"}],"financialInterests":"The authors declare potential competing interests as follows: Manoj Kumar Muthyala and Robert Zitterbart are employers of Gyros Protein Technologies Inc. and declare competing financial interests: the PurePep Chorus synthesizer and the PurePep EasyClean technology described in the manuscript can be purchased from Gyros Protein Technologies.\nOleg Marder is an employer of Luxembourg Bio Technologies and declare competing financial interests: T-Bec® described in the manuscript can be purchased from Luxembourg Bio Technologies.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eScalable and sustainable DMF-free solid-phase synthesis of liraglutideby 1-tert-butyl-3-ethylcarbodiimide-mediated couplings and catch-and-release acylation and purification strategies\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe rising focus on sustainable practices in chemical synthesis has led to significant advancements in green chemistry, especially within pharmaceutical production, where reducing hazardous waste and minimizing environmental impact are critical objectives (Kar et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Peptide-based therapeutics, increasingly vital in treating a wide array of conditions, from metabolic disorders to autoimmune diseases (Selmi et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Sharma et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), are one area where environmentally friendly approaches are especially valuable (Ferrazzano et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, solid-phase peptide synthesis (SPPS), a key method for producing peptides, has traditionally relied on toxic solvents, high reagent consumption, and other practices that contribute to environmental and safety concerns. Integrating green chemistry principles into SPPS is therefore essential to develop more sustainable, less hazardous methodologies for peptide production (Martin et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOne promising strategy for green SPPS involves optimized coupling reagents for amide bond formation (Yang et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Among these, N,N\u0026rsquo;-diisopropylcarbodiimide (DIC) is commonly employed but is known for its associated safety risks, including toxicity, side reactions, and undesired by-products (Pawlas and Rasmussen \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). 1-tert-Butyl-3-ethylcarbodiimide (T-Bec\u0026reg;), a novel and safer coupling reagent, offers a compelling alternative (Pawlas et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). T-Bec\u0026reg; reduces the health hazards and environmental impact typically associated with traditional reagents, while maintaining high efficiency and low epimerization rates in peptide synthesis (Fantoni et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Importantly, compatibility of T-Bec\u0026reg; with green binary solvent mixtures, such as combinations of dimethyl sulfoxide (DMSO) and tert-butyl acetate (BuOAc), further supports its use in sustainable SPPS protocols (Ferrazzano et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Al Musaimi et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Wegner et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Pacini et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). These green solvents provide a more environmentally friendly medium for synthesis compared to traditional toxic solvents such as dimethylformamide (DMF) or dichloromethane (DCM), making T-Bec\u0026reg; and green solvent systems an ideal pairing for advancing green peptide chemistry. Last but not least, while DIC reacts with OxymaPure\u0026reg; at a 1:1 ratio, yielding the byproduct oxadiazole and releasing HCN, T-Bec\u0026reg; reacts with OxymaPure\u0026reg; under the same conditions, forming oxadiazine only, what makes T-Bec\u0026reg; a more health and safety friendly reagent (Manne et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe development and application of greener SPPS methodologies are especially relevant in production of Glucagon-Like Peptide-1 (GLP-1) agonists, which are prominent in treating diabetes and obesity (Staby et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Hach et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). GLP-1-based drugs have revolutionized the therapeutic landscape in metabolic health, with agonists like liraglutide achieving blockbuster status due to their efficacy, long lasting action, and favorable safety profiles. However, as demand for these peptides rises, the need for scalable, sustainable synthetic routes that align with environmental responsibility goals is dramatically increasing. Designing green synthetic strategies for GLP-1 agonists would meet this market demand while reducing the ecological footprint of their production (Barredo-Vacchelli et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Herein, two synthetic strategies, involving the use of PurePep EasyClean (PEC) catch \u0026amp; release technology, are described (Zitterbart et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In both cases, we successfully achieved levels of liraglutide purity that makes further purification through chromatography much easier.\u003c/p\u003e \u003cp\u003eIn summary, this work highlights the importance of T-Bec\u0026reg; as a safer, more sustainable coupling reagent, its compatibility with green solvent systems within the production of liraglutide, and the broader role of green SPPS approaches in producing key therapeutic peptide ingredients. Additionally, it has been demonstrated that the use of heating together with T-Bec\u0026reg;/Oxyma Pure accelerates SPPS and further enhances reaction rates, contributing to the efficiency of green peptide synthesis. By promoting eco-friendly methods for the synthesis of GLP-1 agonists, decreasing the need of time-and-solvent-consuming preparative HPLC, we can meet the growing demand for these drugs while setting a new standard for environmental responsibility in pharmaceutical manufacturing.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eFmoc-amino acids, Oxyma Pure and Fmoc-Gly-2-Chlorotrityl resin were provided directly by Gyros Protein Technologies (Tucson, AZ, USA). N,N'-Diisopropylcarbodiimide was purchased from Carbosolution Chemicals GmbH (St. Ingbert, Germany). Dimethylformamide, ethyl acetate, propyl acetate, butyl acetate, dimethyl sulfoxide, acetic anhydride, pyridine, anisole, and triisopropylsilane were obtained from Carl Roth GmbH (Karlsruhe, Germany). Piperidine, trifluoroacetic acid, and Rink amide AM resin (0.64 mmol/g), Fmoc-Lys(Palm-Glu-OtBu)-OH, Palm-L-Glu(OSu)-OtBu were purchased from Iris Biotech GmbH (Marktredwitz, Germany). Fmoc-Gly-Tentagel S resins (0.17 mmol/g) was acquired from Rapp Polymere (Tuebingen, Germany). 1-tert-Butyl-3-ethylcarbodiimide (T-Bec®) was provided by Luxembourg Bio Technologies (Ness Ziona, Israel).\u003c/p\u003e \u003cp\u003eSolubility Tests - After calibrating a 15 mL plastic tube, 1 mmol of each coupling reagent was weighed, and 1 mL of the tested solvent was added. The mixture was stirred at room temperature for 30 minutes. If the reagent remained insoluble, the solvent volume was gradually increased to check solubility at concentrations of 0.5 M, 0.25 M, and 0.1 M.\u003c/p\u003e \u003cp\u003eSolid-Phase Peptide Synthesis - All syntheses were performed using the PurePep® Chorus (Tucson, AZ, USA) peptide synthesizer in parallel synthesis mode, with induction heating and at room temperature. For 100 µmol scale syntheses, 303 mg of Fmoc-Gly-2-Chlorotrityl resin (0.33 mmol/g loading), 417 mg of Fmoc-Gly-Tentagel S resin (0.24 mmol/g loading), and 588 mg of Tentagel S RAM resin (0.17 mmol/g loading) were weighed. Fmoc-amino acids were dissolved in the tested mixture or in DMF using an ultrasonic bath at 0.3M concentration. DIC or T-Bec® and Oxyma Pure were dissolved in the tested mixture or in DMF at 1M concentration. The solvents were freshly prepared and transferred to the appropriate bottles corresponding to separate lines on the machine. After synthesis, the loaded resins were washed with isopropyl alcohol and dried under vacuum. Before starting each synthesis with different solvents, the instrument was fully calibrated to ensure that the correct volume of solvent was delivered.\u003c/p\u003e \u003cp\u003eTest Cleavage - A small amount (2–4 mg) of dry loaded resin was transferred into a 500 µL plastic tube, and 100 µL TFA, 5 µL TIS, and 5 µL H\u003csub\u003e2\u003c/sub\u003eO were added. After 1.5 hours of mechanical mixing, the suspension was filtered and cold diethyl ether (-20°C) was added to the peptide TFA solution. The precipitated peptide was isolated by centrifugation and dried under vacuum. The crude peptides were dissolved in water/ACN 1:1 and analyzed by RP-UPLC coupled with ESI-MS.\u003c/p\u003e \u003cp\u003ePurePep EasyClean (PEC) lipidation – The first step of this process is the coupling of the reductively, traceless cleavable linker PEC-RC+, TFA cleavage resistant, that allows further attachment on activated agarose beads, on the N-terminal amino function of the to-be-purified peptide still on resin that was conducted at r.t. for 18 hours under mechanical shaking using 4 equivalents of the linker, 6 equivalents of Oxyma pure, and 6 equivalents of DiPEA in DMSO/EtOAc 1:9 (v/v) mixture. After monitoring with LC-MS, the linker-precursor peptide was cleaved from the resin, and all protecting groups on the amino acid side chains were removed. The linker-peptide was solubilized in TFA/H\u003csub\u003e2\u003c/sub\u003eO 9:1 (v/v) and cooled at 0°C. Pyridine was added dropwise, the solution was diluted with ACN and added to polymethacrylate (PMA) activated beads. In the case of activated Agarose beads, the linker-peptide was solubilized with DMSO and added directly on the beads. The mixture was shaken for 5 hours at r.t. and, after immobilization, the beads were washed with 3×H\u003csub\u003e2\u003c/sub\u003eO. Unreacted aldehyde functions were blocked on beads using a solution of 1% (w/v) NH\u003csub\u003e2\u003c/sub\u003eOMe in 0.1 M aqueous citric acid buffer (pH 4.5) for 5 minutes. The loaded beads (agarose or PMA) were washed with DMSO/butyl acetate 3:7 (v/v). A solution of 4 equivalents of Palm-L-Glu(OSu)-OtBu, 4 equivalents of coupling reagent (HOAt or Oxyma Pure) and 6 equivalents of base (DiPEA or Pyridine) were added to the loaded beads after 5 minutes of pre-activation. The formation of the new amide bond occurred in 5 hours.\u003c/p\u003e \u003cp\u003ePurePep EasyClean (PEC) purification – After immobilization (and acylation, in the case of the second strategy), the beads were washed with 3×0.9 M guanidinium chloride (GdmCl) in DMSO and with 3×0.1 M NaCI in H\u003csub\u003e2\u003c/sub\u003eO/EtOH 3:7 (v/v). The linker on the peptide was reduced using a solution of 0.3 M dithiothreitol (DTT) in 0.6 M aqueous NaHCO\u003csub\u003e3\u003c/sub\u003e (pH 8) for 15 minutes at r.t. Then, the beads were washed with H\u003csub\u003e2\u003c/sub\u003eO and ACN. The final release consisted in treatment with TFA/H\u003csub\u003e2\u003c/sub\u003eO 95:5 (v/v) for 45 minutes at r.t. for PMA beads and TFA/H\u003csub\u003e2\u003c/sub\u003eO 40:60 (v/v) for 1 hour at r.t. followed by precipitation in cold diethyl ether and lyophilization.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Result and discussion","content":"\u003cp\u003e \u003cb\u003eGreen solvents binary mixtures and T-Bec®\u003c/b\u003e - The first part of our study involved the test of a safer coupling reagent such as T-Bec® in combination with binary mixtures previously studied. T-Bec® is a non-symmetric carbodiimide as showed in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. In the very enlightening work from Pawlas et al. (Pawlas and Rasmussen \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), it was demonstrated that the coupling system T-Bec®/Oxyma Pure, during the formation of a new amide bond, releases a marked lower quantity of HCN deriving from Oxyma Pure in comparison with the standard coupling system DIC/Oxyma Pure even if the performances, in terms of coupling reaction rate are essentially the same. Another important feature is that the T-Bec® derived urea, 1-tert-butyl-3-ethyl urea (TBEU), is more soluble in DMF and in many other solvent systems.\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003cp\u003eWe investigated the coupling system T-Bec®/Oxyma Pure to synthesize, at 100µmol scale and at 90°C, three model peptides using the green binary mixture butyl acetate/DMSO, propyl acetate/DMSO or anisole/DMSO for coupling and Fmoc removal reactions, and ethyl acetate/DMSO for washings.\u003c/p\u003e\u003cp\u003eThe three model peptides are ACP(65–74)-NH\u003csub\u003e2\u003c/sub\u003e (H-VQAAIDYING-NH\u003csub\u003e2\u003c/sub\u003e), a peptide commonly employed to assess the efficiency of synthetic protocols, polyALA (H-AAAAAAAAAAK-NH\u003csub\u003e2\u003c/sub\u003e) a well-known self-assembling peptide even on resin, and AlaSTD (H-AKADEVSLHKWYG-NH\u003csub\u003e2\u003c/sub\u003e) a standard peptide used as a reference non-challenging sequence (Pacini et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe sequences were produced with the PurePep® Chorus automated synthesizer (Gyros Protein Technologies, Tucson, AZ, USA), employing mechanical shaking and nitrogen bubbling for mixing. The exact protocol, outlining volumes and durations, is provided in Table S1 and S2.\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCrude HPLC Purities of peptides synthesized using T-Bec®/Oxyma Pure in green solvents binary mixtures\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDMF\u003c/p\u003e \u003cp\u003eOxyma\u003c/p\u003e \u003cp\u003ePure\u003c/p\u003e \u003cp\u003eT-Bec®\u003c/p\u003e \u003cp\u003e90°C, 2 min\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBuOAc\u003c/p\u003e \u003cp\u003eDMSO\u003c/p\u003e \u003cp\u003eOxyma\u003c/p\u003e \u003cp\u003ePure\u003c/p\u003e \u003cp\u003eT-Bec®\u003c/p\u003e \u003cp\u003e90°C, 2 min\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAnisole\u003c/p\u003e \u003cp\u003eDMSO\u003c/p\u003e \u003cp\u003eOxyma\u003c/p\u003e \u003cp\u003ePure\u003c/p\u003e \u003cp\u003eT-Bec®\u003c/p\u003e \u003cp\u003e90°C, 2 min\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePrOAc\u003c/p\u003e \u003cp\u003eDMSO\u003c/p\u003e \u003cp\u003eOxyma\u003c/p\u003e \u003cp\u003ePure\u003c/p\u003e \u003cp\u003eT-Bec®\u003c/p\u003e \u003cp\u003e90°C, 2 min\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePeptide\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHPLC Purity\u003c/p\u003e \u003cp\u003e(% A/A)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHPLC Purity\u003c/p\u003e \u003cp\u003e(% A/A)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHPLC Purity\u003c/p\u003e \u003cp\u003e(% A/A)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHPLC Purity\u003c/p\u003e \u003cp\u003e(% A/A)\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eACP-NH\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003cp\u003eVQAAIDYING-NH\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e89.1\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e85.4\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e78.6\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e76.8\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePolyALA-NH\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003cp\u003eAAAAAAAK-NH\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e77.4\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e76.4\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e79.9\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e64.6\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGTPstd-NH\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e \u003csup\u003e\u003cb\u003e*\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eAKADEVSLHKWYG-NH\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e89.6\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e93.3\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e83.9\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e77.4\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe data in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e show that the crude purities obtained from the three peptide syntheses in green binary solvent mixtures and DMF are fairly comparable. There is no single solvent mixture that consistently outperforms the others. For instance, the crude purity of ACP-NH\u003csub\u003e2\u003c/sub\u003e is higher when synthesized with DMF, but this trend does not hold for all sequences. The purity of PolyALA is higher when anisole/DMSO mixture was used, while GTPstd achieves better purity with a BuOAc/DMSO mixture. On average, the purities of the three sequences are comparable when using DMF and BuOAc/DMSO.\u003c/p\u003e\u003cp\u003eAt this stage, we decided to investigate the use of T-Bec® in association with BuOAc/DMSO mixture also for the synthesis of the linear precursor of liraglutide at high temperature (90°C) without double couplings and use of pseudo prolines. The synthesis yielded an over 70 A/A% purity and 93% yield demonstrating the full comparability with DIC/Oxyma Pure synthesis in BuOAc/DMSO mixture used for the second strategy.\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003cp\u003e \u003cb\u003eGreen synthesis approaches for liraglutide production\u003c/b\u003e - In the second part of this work, we focused on the synthetic strategy of liraglutide, a blockbuster drug included in the class of GLP-1 agonists. This study presents two synthetic strategies that utilize green binary solvent mixtures. The first strategy involves the synthesis of the complete liraglutide sequence, incorporating the building block Lys(Palm-Glu-OtBu) directly in the sequence \u003cem\u003evia\u003c/em\u003e green SPPS followed by catch-and-release PEC purification as reported in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003cp\u003eIn contrast, the second strategy involves the synthesis of the linear sequence of liraglutide without the lipid moiety attached to the lysine residue. This approach also employs green binary solvent mixture for a green solid-phase peptide synthesis. Following the completion of the SPPS, cleavage from the resin and complete removal of side-chain protecting groups in the resulting peptide undergoes catch-and-release purification (PEC).\u003c/p\u003e\u003cp\u003eAfter the “catch” step, lipidation occurs directly on the agarose solid phase. This process involves the free primary amine on the side chain of the lysine residue, which is the only reactive amine present in the peptide. This strategy allows for precise control over the lipidation process, thereby optimizing the final yield and purity.\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003cp\u003e \u003cb\u003eThe “direct synthesis” strategy –\u003c/b\u003e In this pathway, the entire peptide was synthesized using solid-phase peptide synthesis (SPPS) on a 100 µmol scale, utilizing the PurePep Chorus instrument by Gyros Protein Technology (Tucson, AZ, USA). In line with green chemistry principles applied to peptide synthesis, and based on our previous work (Pacini et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), we selected environmentally friendly binary solvent mixtures for the synthesis of liraglutide. Specifically, the EtOAc/DMSO mixture (8:2, v/v) was used as the primary solvent for washings. For amide bond formation and Fmoc deprotection, BuOAc/DMSO mixtures (ratios of 1:1 and 7:3, respectively) were employed due to their varying polarity.\u003c/p\u003e\u003cp\u003eAdditionally, these mixtures were designed for use at elevated temperatures, as their boiling points exceed 100°C, potentially enhancing yield, purity, and reducing synthesis time. However, for this study, all reactions were conducted at room temperature to facilitate scale-up on the PurePep Sonata + instrument by Gyros Protein Technology (Tucson, AZ, USA), which lacks a heating system.\u003c/p\u003e\u003cp\u003eFor coupling, we used a system consisting of diisopropylcarbodiimide (DIC) and Oxyma Pure (5 equivalents each), along with 5 equivalents of Fmoc-protected amino acids. The reaction was performed for one hour at room temperature, mixing by both nitrogen bubbling and mechanical shaking. Instead, the coupling reaction with the building block Fmoc-Lys(Palm-Glu-OtBu)-OH required 2 equivalents of the building block, along with 2 equivalents each of DIC and Oxyma Pure. From the 8th cycle onwards, the reaction was repeated twice to achieve a conversion rate as close as possible to 100%. Fmoc removal was performed in two 5-minute treatments using the appropriate solution, except for the second amino acid in the sequence, where a shorter treatment (5 + 3 minutes) was applied to prevent the formation of diketopiperazines, which could significantly reduce the overall yield, as previously demonstrated for another GLP-1 agonist product (Wang et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In order to monitor and prevent the formation of diketopiperazines (DKP), we simultaneously synthesized the sequences using two different types of resin functionalization, i.e., Wang and 2-chlorotrityl resin, expecting differences between the two resins due to the steric hindrance of the trityl group on trityl resin that should prevent the formation of diketopiperazine better than Wang resin. We were unable to detect any DKP during the Fmoc removal of the second amino acid on both types of resin. The rapid treatment (5 + 3 minutes) effectively deprotected the N-terminal of the second amino acid without resulting in the formation of DKPs (data not shown).\u003c/p\u003e\u003cp\u003eAnother important reaction is acetylation after each coupling step, which ensures that only the N-terminal of the desired sequence remains free at the end of the synthesis.\u003c/p\u003e\u003cp\u003eAfter the synthesis was completed on the instrument, a test cleavage was performed, and the HPLC analysis showed that the purity of the peptide was 45% A/A (Supporting information).\u003c/p\u003e\u003cp\u003eAt this stage, the loaded resins were divided into two aliquots: one half (made of halves of 2-chlorotrityl resin and Wang resins combined in one batch) was cleaved and purified by preparative HPLC, while the others separately underwent PEC catch-and-release purification. For the latter, with only the N-terminal of the desired sequence accessible on the resin, the PEC linker was coupled using 4 equivalents of linker, 6 equivalents of Oxyma Pure, and 6 equivalents of DiPEA. This reaction proceeded in 16 hours at room temperature in a green binary mixture of BuOAc and DMSO (7:3, v/v), with reaction progress monitored by the chloranil test. After the cleavage step, we evaluated the yield based on the isolated crude product mass. Both syntheses on the two types of resin yielded approximately 75% (isolated mass/calculated mass) (data available in supporting information).\u003c/p\u003e\u003cp\u003eThe two crudes deriving from the two resin types were combined in one batch and PEC purified obtaining a final product with an HPLC purity of 86.05 A/A% (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003cp\u003e \u003cb\u003eThe Catch-Lipidation-And-Release Strategy –\u003c/b\u003e The peptide was assembled on Fmoc-Gly- 2-Chlorotrityl resin using standard Fmoc-protected amino acids, following the same green mixtures described in the previous strategy. However, this time Fmoc-Lys(Boc)-OH was used instead of the more expensive building block Fmoc-Lys(Palm-Glu-OtBu)-OH. As a result, at the end of the SPPS, we obtained a liraglutide precursor without the fatty acid plus Glu as linker moiety on the peptide. After PEC linker coupling and cleavage, we obtained a 54 A/A% HPLC pure crude (yield 71%).\u003c/p\u003e\u003cp\u003eAt this stage, we immobilized 5 µmol of PEC-linker-liraglutide precursor on both agarose and PMA aldehyde-functionalized beads for acylation as a proof of concept. Following immobilization, the first acylation attempt used 4 equivalents of the building block Palm-L-Glu(OSu)-OtBu along with 4 equivalents of Oxyma Pure, dissolved in DMF. After 5 minutes of pre-activation at room temperature, the solution was added to the peptide-loaded beads (agarose and PMA). We hypothesized that the primary amine on lysine would be more reactive than histidine and more prone to nucleophilic attack on the OSu-activated carboxylic group of the building block.\u003c/p\u003e\u003cp\u003eThe reaction was monitored after 2 and 4 hours drawing a small sample of beads. The product was cleaved after 4 hours from the beads when it yielded a conversion rates of 95% for agarose beads (poor HPLC purity) and 91% for PMA beads (HPLC purity 63 A/A%) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIn-Process-Control of Acylation study on liraglutide precursor\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBead\u003c/p\u003e \u003cp\u003etype\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003econversion\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePurity (A/A%)\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCheck 2h\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAgarose\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e69.5\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePMA\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e55\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e43.8\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eFinal release 4h\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAgarose\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e95\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e75.7\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePMA\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e91\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e63.3\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe observed that the mild final release of liraglutide (40% v/v TFA in water) from agarose beads was neither long nor acidic enough to remove the tBu group from the carboxylic function on the α-carbon of glutamic acid (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003cp\u003eAfter precipitation of the product, we resolved the issue of partial tBu deprotection with a treatment of TFA/TIS/H₂O (95:2.5:2.5 v/v/v) at room temperature for 1 hour. This treatment resulted in completely deprotected liraglutide (final purity 72 A/A%).\u003c/p\u003e\u003cp\u003eThis first encouraging result motivated us to explore further coupling systems by adding a base and testing also HOAt and as solvent the green binary mixture BuOAc/DMSO 7:3 v/v used also for SPPS.\u003c/p\u003e\u003cp\u003eWe investigated 3 different coupling systems (c.s.) still at 5 µmol, reported in Table \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, both on agarose and PMA beads.\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThree Coupling Systems evaluated for the optimization of acylation.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ec.s. 1\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ec.s. 2\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ec.s. 3\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eBB-OSu\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4 equiv\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20 µmol\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOxymaPure\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHOAt\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOxymaPure\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4 equiv\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20 µmol\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDiPEA\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDiPEA\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePyridine\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6 equiv\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e30 µmol\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003ein 150 µL BuOAc/DMSO\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn all these coupling systems, we added 6 equivalents of base to compensate for the minimal basicity of DMF, substituted with BuOAc/DMSO, that provides the optimal environment for the coupling reaction. The best result (79 A/A%, conversion 99%) after 5 hours of reaction, was achieved with coupling system 2 (HOAt and DIPEA) on agarose beads. Based on this result, we decided to scale up the reaction by a factor of 10.\u003c/p\u003e\u003cp\u003e50 µmol were treated with coupling system 2 in BuOAc/DMSO for 5 hours at r.t. obtaining, after the release from agarose beads and a further treatment with TFA/TIS/H\u003csub\u003e2\u003c/sub\u003eO, 90 A/A%, HPLC crude purity and a conversion over 99% (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study highlights the successful integration of green chemistry principles into solid-phase peptide synthesis (SPPS), offering a practical and environmentally friendly alternative for producing therapeutic peptide ingredients. By using 1-tert-butyl-3-ethylcarbodiimide (T-Bec\u0026reg;) as a safer coupling reagent in combination with eco-friendly solvent systems like dimethyl sulfoxide (DMSO) and butyl acetate (BuOAc), we reduced the environmental footprint and safety risks commonly associated with traditional synthesis methods. These approaches maintained high efficiency and delivered high-quality peptide products, demonstrating that sustainability and performance can go hand in hand.\u003c/p\u003e \u003cp\u003eWe explored two innovative strategies for synthesizing liraglutide, a key GLP-1 agonist used to treat diabetes and obesity. The \u0026ldquo;direct synthesis\u0026rdquo; approach incorporated a lipidated lysine building block directly into the peptide sequence, achieving excellent purity after catch-and-release purification. The \u0026ldquo;catch-lipidation-and-release\u0026rdquo; strategy took a different route, attaching the lipid moiety in a controlled, post-synthesis step, which allowed for better precision and higher final yields. Both methods proved to be scalable and significantly reduced the need for extensive preparative HPLC, saving time and resources.\u003c/p\u003e \u003cp\u003eThis work demonstrates that adopting greener methods in peptide synthesis is not only possible but practical for meeting the pharmaceutical industry growing demand for sustainability. Our results show that using T-Bec\u0026reg; and green solvents can deliver the same high-quality outcomes as conventional methods while being more friendly to the environment. Additionally, the catch-and-release purification technique enhanced efficiency, making these methods suitable for large-scale applications.\u003c/p\u003e \u003cp\u003eLooking ahead, this research sets the stage for further innovation in sustainable pharmaceutical manufacturing. As the need for peptide-based therapeutics like liraglutide continues to grow, these methods offer a reliable, eco-conscious way to meet that demand. By aligning cutting-edge science with environmental responsibility, this study underscores the potential to redefine how we produce life-saving medicines, benefiting both people and the planet.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eManoj Kumar Muthyala and Robert Zitterbart are employers of Gyros Protein Technologies Inc. and declare competing financial interests: the PurePep Chorus synthesizer and the PurePep EasyClean technology described in the manuscript can be purchased from Gyros Protein Technologies.\u003c/p\u003e \u003cp\u003eOleg Marder is an employer of Luxembourg Bio Technologies and declare competing financial interests: T-Bec\u0026reg; described in the manuscript can be purchased from Luxembourg Bio Technologies.\u003c/p\u003e\u003ch2\u003eAcknowledgements:\u003c/h2\u003e \u003cp\u003eLorenzo Pacini acknowledges MUR and EU-FSE for the financial support of the PhD fellowship PON Research and Innovation 2014\u0026ndash;2020 (D.M. 1061/2021) XXXVII Cycle in Chemical Sciences: \u0026ldquo;Greening peptide chemistry, a necessary step to the future\u0026rdquo;. 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Chem Sci 12:2389\u0026ndash;2396. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/D0SC06285E\u003c/span\u003e\u003cspan address=\"10.1039/D0SC06285E\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"University of Florence","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Solid-phase peptide synthesis, GLP-1 agonist, catch-and-release purification, green binary solvent mixtures, T-Bec® coupling reagent","lastPublishedDoi":"10.21203/rs.3.rs-5738025/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5738025/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe growing need for sustainable practices in pharmaceutical manufacturing has stimulated advancements in peptide synthesis. This study focuses on applying green chemistry principles to the synthesis of the Glucagon-Like Peptide-1 analog liraglutide, using novel and sustainable solid-phase synthetic strategies. By adopting the safer coupling reagent 1-tert-butyl-3-ethylcarbodiimide (T-Bec\u0026reg;) in combination with eco-friendly binary solvents like dimethyl sulfoxide and butyl acetate, we demonstrated that it is possible to significantly reduce the environmental impact while maintaining high efficiency and quality of the synthesis. T-Bec\u0026reg; minimizes hazardous byproducts, such as hydrogen cyanide, and enhances solvent compatibility, achieving crude purities and yields comparable to conventional syntheses. Two synthetic strategies were developed for liraglutide production. The first strategy based on a \u0026ldquo;direct synthesis\u0026rdquo;, incorporating a lipidated lysine building block into the peptide sequence, achieving 86% HPLC purity after catch-and-release purification. The second strategy based on \u0026ldquo;catch-lipidation-and-release\u0026rdquo; approach, allowed to obtain the peptide precursor without the lipid moiety, which was later linked during a controlled lipidation step. This latter strategy yielded purities exceeding 90% and reduced reliance on preparative HPLC.\u003c/p\u003e \u003cp\u003eThese findings highlight the effectiveness of T-Bec\u0026reg; and green solvent systems to optimize scalable and sustainable SPPS processes. These methods improve resource efficiency and reduce environmental impact, to allow a viable pathway to produce therapeutic peptide ingredients like liraglutide. This work underscores the potential of green chemistry to align pharmaceutical innovation with environmental responsibility.\u003c/p\u003e","manuscriptTitle":"Scalable and sustainable DMF-free solid-phase synthesis of liraglutideby 1-tert-butyl-3-ethylcarbodiimide-mediated couplings and catch-and-release acylation and purification strategies","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-01 07:23:50","doi":"10.21203/rs.3.rs-5738025/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"294b0293-8a3a-4ae9-88d5-8358051b2f96","owner":[],"postedDate":"January 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":42183970,"name":"Organic Chemistry"}],"tags":[],"updatedAt":"2025-01-02T01:53:20+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-01 07:23:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5738025","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5738025","identity":"rs-5738025","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}