Toward sustainable solid-phase peptide synthesis strategy – in situ Fmoc removal

ABSTRACT

Solid-phase peptide synthesis (SPPS) is the method of choice for the synthesis of peptides for research and production purposes. Despite having several positive features, it remains a challenge to reduce the amount of solvent waste generated during the synthesis. We proposed a 3-step protocol (in-situ Fmoc removal) where washing after coupling was eliminated. Here, the in-situ Fmoc removal protocol was optimized by adding an extra 4-methylpiperidine (4-MP) treatment to ensure the removal of the Fmoc. Additionally, a second addition of the carbodiimide during the coupling step is performed to form an in situ more active ester of the Fmoc-amino acid. The number of washings has been kept to a minimum of three adding in two of them 1% of OxymaPure to ensure the total removal of the 4-MP. This strategy, which saves up to 60% of solvent, was successfully demonstrated in two important Active Pharmaceutical Ingredients (APIs), angiotensin II and afamelanotide synthesis, with high purity. This strategy will be added to the green toolbox of SPPS making the approach more sustainable.

GRAPHICAL ABSTRACT

KEYWORDS:

• Fluorenylmethoxycarbonyl (Fmoc)
• green chemistry
• in situ coupling deprotection
• peptide
• 4-methylpiperidine
• sustainable chemistry

Introduction

Peptides are an important class of Active Pharmaceutical Ingredients (APIs) (1, 2). They form part of approximately 8% of all drugs approved by the US Food and Drug Administration (FDA) during the last years (3). In most cases, this has been possible thanks to the development of the solid-phase peptide synthesis (SPPS) strategy carried out by Merrifield (4) and refined by many groups over the last few years (5–7). As a result of all these continued methodological works, nowdays it is possible to manufacture peptides such as T-20, Liraglutide, Semaglutide, and Tirzepatide containing more than 30 amino acids, and in some cases bearing side-chains with fatty acids at the multi-Kg scale peptides (8–11).

From a strictly synthetic point of view, these milestones seemed to be unattainable just a few decades ago. However, these days, the manufacturing of these peptides is facing another challenge i.e. the sustainability of the overall process. However, SPPS incorporates several features associated with the 12 Principles of Green Chemistry (12–14), such as (i) ease manipulations (all reactions in the same reactor; no isolation of intermediates; no major cleaning procedures), (ii) good-excellent yields; (iii) ease work-ups (excess of reagents and soluble side-products are removed by filtration and washings), and (iv) ease of scaling-up; the number of protecting groups used and the large number of solvents needed are the main drawbacks in terms of sustainability (13, 14).

While the issue of the protecting groups should be addressed from a multifaced point of view, the large consumption of solvents could be minimized without changing excessively the SPPS rules of the game during the sixty years of this synthetic methodology. A former study from our group has proven the concept of using a 3-step in-situ Fmoc removal approach for SPPS (Figure 1) (15). This was inspired in one of the synthetic programs on the CEM microwave-assisted automatic synthesizer (16), where at the end of the coupling step, the base used for the removal of the Fmoc was added and after the deprotection step washing was carried out before the next coupling step. In a recent study, CEM demonstrated successful peptide synthesis with no washings involved (17). Our idea was to see if a similar program could also be implemented at room temperature. As the main problem was the need for an efficient removal of the base, we proposed to perform the washing with 1% OxymaPure in DMF, taking advantage of the mild acidity of the OxymaPure to remove the piperidine (16). Thus, the number of washing after Fmoc-removal can be minimized.

Figure 1. Comparison between standard 4-step standard SPPS and 3-step in-situ Fmoc removal protocol.

This strategy is associated with two risks: the first one is that the base (piperidine or other secondary amines) could remove first the Fmoc group from the Fmoc-peptidyl resin and the incoming activated amino acid and then the formation of double or even triple hits can take place (Scheme 1, A). On the other hand, as the piperidine (secondary amine) has to deactivate the active ester and remove the Fmoc of the incoming amino acid, a lack of it can be translated in the presence of the Fmoc in the growing peptide chain and result in the formation of deletion peptides.

Scheme 1. Potential drawbacks associated with the 3-step in-situ Fmoc removal.

Using as model Leu-enkephalin-amide (H-Tyr-Gly–Gly-Phe-Leu-NH₂), we have demonstrated that the 3-step in-situ Fmoc removal (coupling → in-situ Fmoc removal → washing) was able to render the model peptide with similar purity to the regular strategy, where extensive washings are introduced after the coupling and Fmoc removal as well (15). Here, the study was further extended by investigating the use of a 3-step in-situ Fmoc removal condition [20% 4-methyl piperidine (4-MP) in DMF] to synthesize more complex peptides. 4-MP is considered a greener base because piperidine is a regulated substance, which is not possible to be acquired in several countries due to its use in the production of phencyclidine, which is a recreational drug. Piperidine is included in the List of Precursors and Chemicals Frequently Used in the Illicit Manufacture of Narcotic Drugs and Psychotropic Substances under International Control issued by the International Narcotics Control Board (INCB) (18). 4-MP kinetically works like piperidine in DMF or other green solvents (19, 20). No trace of racemization was observed during the SPPS. Therefore, 20% 4-MP in DMF was used as an Fmoc removal condition during the peptide synthesis.

Results and discussion

The peptides were synthesized simultaneously through coupling of Fmoc-amino acids followed by in-situ Fmoc removal without washing the previous step. At the end of the synthesis, the peptidyl resin was treated with a standard cleavage cocktail, trifluoroacetic acid (TFA)-triisopropylsilane (TIS)-H₂O, followed by the washing of the residual cleavage mixture with cold diethyl ether to obtain the desired peptides. These peptides were then analysed by HPLC for observation.

SPPS of H-YSSFL-NH₂, 3-step in-situ Fmoc removal protocol using 20% 4-MP

First of all, the Ser², Ser³-Leu-enkephalin-amide (H-Tyr-Ser-Ser-Phe-Leu-NH₂) was prepared to validate the previously demonstrated strategy. The use of the model Leu-enkephalin-amide (H-Tyr-Gly–Gly-Phe-Leu-NH₂) was not an ideal choice, because the Gly gives a double hit due to its poor hindrance (21). The Ser-Ser derivative peptide was assembled on Fmoc-RinkAmide-AM-PS resin (72.5 mg, resin loading 0.69 mmol/g) using the 3-step in-situ protocol, as shown in Figure 2. For all the couplings, 3 eq. each of Fmoc-AA-OH, OxymaPure, and DIC were dissolved in 0.5 mL DMF. After 1 h coupling, 0.12 mL of neat 4-MP, which represents 20% of 4-MP, was directly added to the coupling cocktail present in the peptidyl resin for Fmoc removal. After 10 min, the peptidyl resin was washed twice with 1 mL of 1% OxymaPure in DMF and then with 1 mL DMF. The presence of OxymaPure assures a complete removal of the remaining 4-MP (15).

Figure 2. SPPS of H-YSSFL-NH₂, a 3-step in-situ protocol using 20% 4-MP in DMF.

The HPLC analysis shows the presence of the desired peptide H-YSSFL-NH₂ with high purity, as shown in Figure 3 and further confirmed by LCMS (supplementary information Figure S1). This result is better than the one obtained in our previous work where the synthesis of H-YGGFL-NH₂ was attempted. In that case, extra incorporation of Gly was detected due to the highlight reactivity of Gly that provokes its double insertion (15).

Figure 3. Chromatogram of H-YSSFL-NH₂ synthesized using 3-step in-situ Fmoc removal protocol using 20% 4-MP in DMF. Method used: 5-60% B into A in 15 min.

SPPS of Angiotensin II (H-DRVYHPF-NH2)

The synthesis of Angiotensin II was performed using two protocols as explained below:

(a) SPPS of Angiotensin II (H-DRVYHPF-NH₂)_, 3-step in-situ 20% 4-MP for Fmoc removal

Next, the most relevant peptide, angiotensin II (H-DRVYHPF-NH₂), was assembled using the same protocol followed for H-Tyr-Ser-Ser-Phe-Leu-NH₂ on Fmoc-RinkAmide-AM-PS resin, as shown in Figure 4. The synthesis of the peptide was performed using Fmoc-AA-OH/DIC/OxymaPure (1:1:1; 3 eq), for 1 h, followed by the in-situ addition of neat 4-MP (for Fmoc removal) for 10 min. Asp renders aspartimide formation in the presence of a base (22). However, in this case, Asp is exposed to the base only once (as it is the last residue in the peptide sequence during SPPS). Therefore, only one treatment for Fmoc removal was performed.

Figure 4. SPPS of Angiotensin II, 3-step in-situ protocol using 20% 4-MP in DMF.

Synthesizing Angiotensin II by a 3-step in-situ protocol of 20% 4-MP gave unexpected results. Multiple peaks were observed containing the Fmoc protecting group (supplementary information Figure S2). These results reinforce the idea that the Fmoc removal steps many times is more important for rendering a good-quality peptide than the coupling step by itself.

After this, some changes were made to improve the Fmoc removal, which has been demonstrated to be key for the good quality of the final peptide. As the Fmoc removal step is a more demanding step, an extra treatment with 4-MP has been incorporated without extra washings. Thus, initially, in-situ Fmoc removal was performed for 10 mins and then the reaction cocktail was filtered and 20% 4-MP in DMF was added for an extra 10 mins.

(b) SPPS of Angiotensin II (H-DRVYHPF-NH₂)_, 3-step in-situ double 20% 4-MP in DMF for Fmoc removal

The peptide Angiotensin II was assembled, as shown in Figure 5. Thus, after each coupling, neat 4-MP was added until a proportion of 20% for 10 min and then after filtration, an extra 20% 4-MP in DMF was added for 10 min for Fmoc removal.

Figure 5. SPPS of Angiotensin II, 3-step in situ double Fmoc removal condition.

The HPLC of the synthesis using a 3-step in-situ double Fmoc removal condition gives the peptide with high purity, as shown in Figure 6, and confirmed by LCMS (supplementary information Figure S3). Here, the complexity in Fmoc removal on a difficult peptide sequence using our preceding in-situ condition (20% 4-MP in DMF) was eliminated with double Fmoc removal protocol, but without extra washings. This indicates the importance of the incorporation of in-situ double Fmoc removal conditions for difficult peptides.

Figure 6. Chromatogram of Angiotensin II, In-situ Fmoc removal using 20% 4-MP [In-situ neat 4-MP (10 min) + 20% 4-MP in DMF (10 min)]; Total = 20 min. Method used: 5-95% B into A in 15 min.

SPPS of Afamelanotide (H-SYSLEHFRWGKPV-NH₂)

The synthesis of Afamelanotide was performed using 3 protocols, as explained below:

(a) SPPS of Afamelanotide, 3-step in-situ double 20% 4-MP (in-situ neat 4-MP, 10 min + 20% 4-MP in DMF, 10 min) treatment for Fmoc removal

Following the positive results, Afamelanotide (H-SYSLEHFRWGKPV-NH₂), a thirteen amino acid peptide, was synthesized to test the validity of the double Fmoc removal protocol. The synthesis was performed following a similar protocol, as shown in Figure 5. After the synthesis, the HPLC analysis shows the desired peptide with a good purity, but with an impurity corresponding to the peptide with a Pro deletion (Figure 7). We envisage that this can be due to an inefficient coupling of Fmoc-Pro-OH to the hindered H-Val-Rinkamide-resin. As it is well known, the incorporation of the first amino acids into the resin is jeopardized by the hindrance of the resin itself.

Figure 7. Chromatogram of Afamelanotide, in-situ double Fmoc removal using 20% 4-MP [In-situ neat 4-MP (10 min) + 20% 4-MP in DMF (10 min)]; Total = 20 min. Method used: 5-60% B into A in 15 min.

(b) SPPS of Afamelanotide (H-SYSLEHFRWGKPV-NH₂), 3-step in-situ double 20% 4-MP (in-situ neat 4-MP, 10 min + 20% 4-MP in DMF, 10 min) treatment for Fmoc removal and double addition of DIC

In all couplings performed in SPPS, an active ester of the Fmoc-AA-OH is formed by the coupling reagents and the coupling additives (DIC and OxymaPure in this case). This active ester is not stable and with time, it decomposes giving the initial Fmoc-AA-OH and the coupling additive (OxymaPure), where both are inefficient in terms of coupling. However, if an extra coupling reagent is added, it will be performing again the active ester, which could complete the acylation of the peptide resin. This does not support any extra consumption of solvent. For all the couplings, 3 eq. each of Fmoc-AA-OH/DIC/OxymaPure (1:1:1; 3 eq.) was dissolved in 0.5 mL DMF and left to react for 30 min. After 30 min, 3 eq. more DIC was added to the coupling cocktail with peptidyl resin and left to react for another 30 min. After 1 h, neat 20% 4-MP (0.12 mL for 0.05 mmol resin) was added into the coupling cocktail and left to react for 10 min. After 10 min, the peptidyl resin was filtered and 20% 4-MP in DMF was added for another 10 min (Figure 8). After 1 h 20 mins, the peptidyl resin was filtered and washed only three times [the first two washings with OxymaPure (1%) to ensure the removal of 4-MP] for the next coupling.

Figure 8. SPPS of Afamelanotide, double DIC addition was performed (3 eq. for 30 min + 3 eq. for 30 min = 1 h), followed by in situ double Fmoc removal (in-situ neat 4-MP, 10 min + 20% 4-MP in DMF, 10 min).

HPLC analysis for the revised 3-step double in-situ Fmoc removal and DIC incorporation (Figure 9) showed the desired peptide with excellent purity without Pro deletion, as confirmed by LCMS (supplementary information Figure S4). This indicates the importance of using double DIC addition for the coupling.

Figure 9. Chromatogram of Afamelanotide, double DIC addition was performed (3 eq. for 30 min + 3 eq. for 30 min = 1 h), followed by double in situ double Fmoc removal (in-situ neat 4-MP, 10 min + 20% 4-MP in DMF, 10 min). Total = 20 min. Method used: 5-60% B into A in 15 min.

(c) SPPS of Afamelanotide, a standard 4-step protocol using double DIC addition with washings and double Fmoc removal conditions

To compare, the in situ Fmoc removal protocol (double Fmoc removal treatment and washings after coupling and Fmoc removal steps) a new synthesis of Afamelanotide was carried out using standard conditions, but with double incorporation of DIC.

All the couplings, 3 eq. each of Fmoc-AA-OH/DIC/OxymaPure (1:1:1; 3 eq.) was dissolved in 0.5 mL DMF and left to react for 30 min. After 30 min, 3 eq. more DIC was added to the coupling cocktail with peptidyl resin and left to react for another 30 min. After 1 h, the resin was filtered and washed five times with DMF, followed by double Fmoc removal using 20% 4-MP in DMF (2 × 10 min), as shown in Figure 10. After 20 min, the peptidyl resin was filtered and washed again fine times with DMF for the next coupling step.

Figure 10. SPPS of Afamelanotide, a standard 4-step protocol of double DIC addition (3 eq. for 30 min + 3 eq. for 30 min = 1 h), followed by double Fmoc removal 20% 4-MP [2 x10 min].

HPLC analysis of a 4-step protocol using double DIC addition and double Fmoc removal (Figure 11) shows the presence of the desired peptide with good purity without Pro deletion.

Figure 11. Chromatogram of Afamelanotide synthesized using revised 4-step protocol i.e. double DIC addition was performed (3 eq. for 30 min + 3 eq. for 30 min = 1 h), followed by double in situ double Fmoc removal (in-situ neat 4-MP, 10 min + 20% 4-MP in DMF, 10 min). Total = 20 min. Method used: 5-60% B into A in 15 min

Figure 12 shows the comparison of revised 3-step and 4-step protocols for the synthesis of afamelanotide. The revised 3-step protocol seems to be better in terms of purity compared to the 4-step protocol.

Figure 12. Chromatograms of Afamelanotide, using 3-step in-situ protocol and standard 4-step protocol. Method used: 5-50% B into A in 15 min.

Experimental

General

All reagents and solvents were purchased from commercial suppliers and used without further purification. Fmoc amino acids, Fmoc-RinkAmide-AM-PS resin (72.5 mg, resin loading 0.69 mmol/g), were purchased from Iris Biotech. OxymaPure and DIC were gifted from Luxembourg Biotech. 4-Methylpiperidine (4-MP) was supplied by Sigma-Aldrich. Organic solvents, dimethylformamide (DMF) and HPLC quality acetonitrile (CH₃CN), were purchased from Merck. Milli-Q water was used for RP-HPLC. Analytical HPLC was performed on an Agilent 1100 system using a Phenomenex Aeris^TMC18 (3.6 μm, 4.6 × 150 mm) column, with a flow rate of 1.0 mL/min and UV detection at 220 nm. Chemstation software was used for data processing. Buffer A: 0.1% TFA in H₂O; buffer B: 0.1% TFA in CH₃CN. LC-MS was performed on a Thermo Fisher Scientific UltiMate 3000 UHPLC-ISQTM EC single quadrupole mass spectrometer in positive ion mode using a Phenomenex Aeris^TM C18 (3.6 μm, 4.6 × 150 mm) column. Buffer A: 0.1% formic acid in H₂O; buffer B: 0.1% formic acid in CH₃CN. Method: 5-60% B into A in 15 min.

Peptide synthesis using SPPS

All peptides were assembled manually in plastic syringes fitted with a porous polypropylene disk. The Fmoc/tBu strategy was used for synthesis using DIC/OxymaPure in DMF as a coupling cocktail. Fmoc removal was carried out in situ using 4-methylpiperidine (4-MP) which was added to peptidyl resin without filtering of coupling cocktail after coupling. All the peptides were synthesized using the protocol explained below.

SPPS using in-situ Fmoc removal protocol

Fmoc-Rink-Amide AM-PS resin (72.5 mg, 0.69 mmol/g resin loading) was washed with DMF (2 × 1 mL for 5 min). Deprotection of the Fmoc group was achieved by the treatment of the resin with 20% 4-MP/DMF (1 × 7 min) followed by washing with 1% OxymaPure/DMF (2 × 1 mL) + DMF (1 × 1 mL). The protected Fmoc-amino acids (3.0 eq.) and OxymaPure (3.0 eq.) in 0.5 mL DMF (0.6 M) were preactivated for 1 min with DIC (3.0 eq.) before adding resin. Upon addition and after 1 h of coupling 0.12 mL neat 4-MP was added at rt to the coupling reaction mixture without filtration and washing. After 10 min, the reaction mixture was filtered, and peptidyl resin was washed with 1% OxymaPure/DMF (2 × 1 mL) + DMF (1 × 1 mL). Coupling and deprotection were repeated until the peptide was assembled onto the resin. The peptidyl resin was dried, and the peptide was cleaved from the resin by treating it with TFA/TIS/H₂O (95:2.5:2.5) for 90 min at rt. After that, the peptide was precipitated using chilled diethyl ether. The precipitate was centrifuged to afford the desired peptide, as confirmed by LCMS for purity and mass.

SPPS using double in-situ Fmoc removal protocol

Fmoc-Rink-Amide AM-PS resin (72.5 mg, 0.69 mmol/g resin loading) was washed with DMF (2 × 1 mL for 5 min). Deprotection of the Fmoc group was achieved by treatment of the resin with 20% 4-MP/DMF (1 × 7 min) followed by washing with 1% OxymaPure/DMF (2 × 1 mL) + DMF (1 × 1 mL). The protected Fmoc-amino acids (3.0 eq.) and OxymaPure (3.0 eq.) in 0.5 mL DMF (0.6 M) were preactivated for 1 min with DIC (3.0 eq.) before adding resin. Upon addition and after 1 h of coupling 0.12 mL neat 4-MP was added at rt to the coupling reaction mixture without filtration. After 10 min, the reaction mixture was filtered, and an extra 20% 4-MP in DMF was added for 10 min at rt. The peptidyl resin was then washed with 1% OxymaPure/DMF (2 × 1 mL) + DMF (1 × 1 mL). Coupling and deprotection were repeated until the peptide was assembled onto the resin. The peptidyl resin was dried, and the peptide was cleaved from the resin by treating it with TFA/TIS/H₂O (95:2.5:2.5) for 90 min at rt. After that, the peptide was precipitated using chilled diethyl ether. The precipitate was centrifuged to afford the desired peptide, as confirmed by LCMS for purity and mass.

SPPS using double in-situ Fmoc removal and double DIC addition protocol

Fmoc-Rink-Amide AM-PS resin (72.5 mg, 0.69 mmol/g resin loading) was washed with DMF (2 × 1 mL for 5 min). Deprotection of the Fmoc group was achieved by treatment of the resin with 20% 4-MP/DMF (1 × 7 min) followed by washing with 1% OxymaPure/DMF (2 × 1 mL) + DMF (1 × 1 mL). The protected Fmoc-amino acids (3.0 eq.) and OxymaPure (3.0 eq.) in 0.5 mL DMF (0.6 M) were preactivated for 1 min with DIC (3.0 eq.) before additing resin. The reaction was performed for 30 mins. After 30 mins, 3 eq. more DIC was added to the coupling cocktail with peptidyl resin and left to react for another 30 min. After 1 h of coupling 0.12 mL neat 4-MP was added at rt to the coupling reaction mixture without filtration. After 10 min, the reaction mixture was filtered, and an extra 20% 4-MP in DMF was added for 10 min at rt. The peptidyl resin was then washed with 1% OxymaPure/DMF (2 × 1 mL) + DMF (1 × 1 mL). Coupling and deprotection were repeated until the peptide was assembled onto the resin. The peptidyl resin was dried, and the peptide was cleaved from the resin by treating it with TFA/TIS/H₂O (95:2.5:2.5) for 90 min at rt. After that, the peptide was precipitated using chilled diethyl ether. The precipitate was centrifuged to afford the desired peptide, as confirmed by LCMS for purity and mass.

Conclusion

An optimized protocol has been developed for the synthesis of two peptide-based APIs, angiotensin II and afamelanotide. This protocol is based on two treatments with 4-MP, which is considered greener than piperidine. The first one is in situ in the presence of the coupling cocktail and sthe econd is the addition of 20% 4-MP to the peptidyl resin just after filtration. This implies that no washing is carried out after the coupling step. In addition, the washing after Fmoc-removal has been reduced from five (23) to three, using two washes containing 1% of OxymaPure, which is a mild acid to assure the complete removal of 4-MP. Furthermore, the coupling step has been also optimized by adding at the middle of the coupling a second portion of DIC, to render in situ the active ester of the Fmoc-amino acid. Using this optimized protocol, the HPLC profile afamelanotide is slightly superior to the same peptide obtained with a standard protocol with two separate Fmoc-removal treatments and extensive washings after coupling and Fmoc-removal steps.

This protocol employs five treatments involving solvents: coupling, one Fmoc-removal, and three washings. In contraposition, the standard protocol requires 13 treatments involving solvents, one more Fmoc-removal, and seven washings. This reduction is translated into more than 60% of solvent savings. We envisage that this new protocol will be added to the green toolbox for SPPS and will make this strategy much more sustainable.

Disclosure statement

No potential conflict of interest was reported by the author(s).