Pseudo-Wang Handle for the Preparation of Fully Protected Peptides

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Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX

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Pseudo-Wang Handle for the Preparation of Fully Protected Peptides. Synthesis of Liraglutide by Fragment Condensation Daniel Carbajo,†,‡ Peter Fransen,† Ayman El-Faham,§,∥ Miriam Royo,†,‡ and Fernando Albericio*,†,‡,§,⊥,#

Org. Lett. Downloaded from pubs.acs.org by UNIV OF TEXAS AT DALLAS on 03/14/19. For personal use only.

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CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine, University of Barcelona, 08028 Barcelona, Spain ‡ Institute of Advanced Chemistry of Catalonia (IQAC−CSIC), Spanish National Research Council (CSIC), 08034 Barcelona, Spain § Department of Chemistry, College of Science, King Saud University, 2455, Riyadh 11451, Saudi Arabia ∥ Department of Chemistry, Faculty of Science, Alexandria University, 426, Alexandria 21321, Egypt ⊥ Department of Organic Chemistry, University of Barcelona, 08028 Barcelona, Spain # School of Chemistry and Physics, University of KwaZulu-Natal, Durban 4001, South Africa S Supporting Information *

ABSTRACT: A handle for the protection of the C-terminus of peptides after cleaving with low concentration of trifluoroacetic acid (2−4%) is reported. The handle prevents polymerization reactions in the convergent condensation of peptidic fragments. Moreover, it is traceless, being removed during the final deprotection step of the peptide synthesis. This cheap and convenient handle is easily attached to the solid support, causing no disturbance to peptide elongation and thus proving to be useful in the convergent synthesis of long peptides.

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One of the limitations of this hybrid strategy is the preparation of the C-terminal fragment, whose carboxylic acid should be masked or protected. For the synthesis of Cterminal amide peptides, this C-terminal fragment can be prepared using Sieber resin, which allows the release of the amide peptide with 4% trifluoroacetic acid (TFA).14 However, no direct and convenient strategy allowing the SPS of the Cprotected fragment for C-terminal acid peptides is currently available (see next paragraph for an indirect method previously developed by our laboratory). To date, either the free Cterminal acid is protected in solution in the form of chlorotrityl ester15 or the synthesis is started with the second amino acid of the sequence, and then the properly protected first amino acid is coupled in solution.16 Chlorotrityl esters do not show high stability and can be hydrolyzed during the subsequent fragment incorporation. The second approach is complex as it requires an additional coupling involving a peptide susceptible to racemization and extra purification steps. In our research into a suitable way to conveniently synthesize protected/masked C-carboxylic peptides, several years ago our group developed a procedure based on the use of a linker that forms diketopiperazine (DKP) during the release of the protected peptide from the resin.17 The DKP, which

ince Merrifield introduced solid-phase synthesis (SPS) for small-medium sized peptides,1 the number of new strategies that have expanded his original idea has grown exponentially.2 The use of better resins and linkers,3 coupling reagent cocktails,4 improved protocols,5 and concourse of microwave6 is now allowing the routine synthesis of peptides of up to 30 residues in length, mostly for research purposes.7 However, the preparation of peptides containing “difficult sequences”, with a larger number of residues and on a large scale, such as active pharmaceutical ingredients (APIs), is still a challenge. A convergent approach in which peptide fragments are assembled in solution emerges as the method of choice for the syntheses of these peptides. This also opens the door to a wide range of post-translational modifications and sequence variations.8 Native chemical ligation (NCL)9 is a unique approach to such syntheses, although alternatives, such as Staudinger reaction,10 click chemistry,11 or the use of hydrazide peptides, have also been used for assembly purposes.12 All these strategies involve unprotected peptides, which is thus the main limitation to the scale up. Alternatively, the target peptide can be prepared by solution assembly of the protected peptides, which can be prepared in solid phase. The paradigm of this hybrid (solid-phase/solution) strategy has been the industrial synthesis of T-20. The protected peptides are conveniently prepared using the 2-chlorotrityl chloride (2CTC) resin.13 © XXXX American Chemical Society

Received: March 5, 2019

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DOI: 10.1021/acs.orglett.9b00813 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters blocks the C-carboxylic end, is later removed during global deprotection. Despite the success of the linker in the convergent synthesis of small-medium size peptides, its use is limited because the DKP formation reaction is relatively slow, thereby reducing the final yield of the synthesis. Furthermore, the cleavage requires long treatments with piperidine in either THF or DMF, which should be removed before further coupling (see the Supporting Information for a detailed comparison between this previous work and the one described herein). With the aim to develop a simpler handle, here we focused on 4-hydroxymethylphenol, which is the base of the Wang resin, a solid support that is widely used in SPS of peptides. When the first amino acid forms a p-alkoxy benzyl ester with the Wang resin, a high amount of TFA is required to release the peptide from the support. Given the presence of the phenol function of 4-hydroxymethylphenol, we turned our attention to the multidetachable concept developed by Merrifield and coworkers.18 Thus, we envisioned that the introduction of 4hydroxymethylphenol into a more labile resin such 2-CTC would result in a suitable multidetachable support for our purposes. Once the protected peptide is build up, treatment with a reduced concentration of TFA (only 2−4%) renders a peptide with intact protecting groups. More importantly, 4hydroxymethylphenol (ψ-WangHANDLE) is still attached to the C-terminus, thereby leaving a phenol group that is not reactive toward coupling reagents involving hindered carboxylic groups, such as the incoming protected fragment in a fragment condensation approach (Figures 1 and 2).

Figure 2. General scheme of ψ-WangHANDLE functionality. A 2% TFA cleavage renders the C-terminal protected peptide, while 100% TFA cleavage leads to free unprotected peptide.

Figure 3. Sequence of liraglutide.

ligation site. Another issue to be considered is the optimum step at which to introduce the palmitoyl-γ-Glu moiety, because, once attached to the peptidic backbone, the long aliphatic chain can hinder amino acid elongation. Therefore, the step at which this moiety is introduced was considered to be of critical importance and various options were explored in this regard. First, the synthesis of the liraglutide linear sequence lacking the branched unit was attempted in order to prove the validity of our strategy (Figure 4). The protected fragment 1 [Boc-His(9−22)-Gly-OH] was manually synthesized on 2-CTC resin using Fmoc-amino acids, except the last residue, which was introduced as Boc-His(Boc)-OH and using DIC/OxymaPure as coupling reagent in N,N-dimethylformamide (DMF). Ninhydrin tests were performed to determine whether the reaction had reached completion, with recouplings done when required. The synthesis was accomplished without significant issues and the protected Fragment 1 was obtained in good yield (75%) and purity (80%) after cleavage [2% TFA in dichloromethane (DCM)] and precipitation. The synthesis of the protected fragment 2 started with the incorporation of the ψ-WangHANDLE (4-hydroxymethylphenol) to CTC resin using 0.9 equiv of the former in the presence of freshly distilled DIEA (2 equiv). Unreacted Cl in the CTC

Figure 1. Design of ψ-WangHANDLE for the cleavage from resin of protected peptides in mild conditions.

In order to evaluate and use this handle for the synthesis of a long peptide of industrial interest, we focused on the synthesis of liraglutide. Liraglutide is a glucagon-like peptide (GLP)-1 (fragment 8−38) analogue used for glycemic control in type 2 diabetes19 and for the treatment of obesity.20 It is currently a blockbuster sold under the name of Victoza.21 Liraglutide is a long peptide with a linear sequence of 31 amino acids that is branched at the Lys27 side chain with a palmitoyl-γ-Glu moiety (Figure 3). The stepwise synthesis of liraglutide using either 2-CTC or Wang resin rendered acceptable crude products for research purposes. However, they were unsuitable for scaling up in a production campaign (results not shown). On the other hand, liraglutide is particularly amenable to synthesis via a convergent approach because it contains 31 residues and a Gly is strategically placed in the middle of the sequence, thus dividing the peptide into two fragments of 15 and 16 residues, a length considered to be the general limit for a convergent approach. The presence of Gly, which is poorly hindered and immune to the racemization of the free amino acid, is an ideal B

DOI: 10.1021/acs.orglett.9b00813 Org. Lett. XXXX, XXX, XXX−XXX

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crude products, with some product decomposition.22 When PyBOP was used as coupling reagent, the reaction was much slower than that achieved previously, but nonetheless provided clearer crude products. Thus, PyBOP was selected as the coupling reagent of choice. The apparent pH of the coupling cocktail was a key factor, which should be kept between 7.5 and 8.5. A lower pH prevented the reaction from occurring, while a higher pH led to dirtier crude products with significant side products. Fully coupled peptide was deprotected with TFA [TFA-H2O-TIS (95:2.5:2.5)], providing crude peptide 7 with excellent yields. Once the validity of the ψ-WangHANDLE strategy had been confirmed, we proceeded with the synthesis of liraglutide, taking care when introducing the branching moiety. Three approaches were tested (Figure 5): (A) in solution, after

Figure 4. Fragment strategy for the preparation of liraglutide, using ψWangHANDLE. Palm introduction is omitted here.

resin was capped with MeOH. The first amino acid, Fmoc-GlyOH, was introduced into the ψ-WangHANDLE-CTC resin using DIC/DMAP in DMF. The rest of the couplings were carried out in DIC/OxymaPure, as for the other fragment. Interestingly, after the introduction of 11 amino acids, even fully piperidine treated peptide resins were ninhydrin-negative. Therefore, the progress of the amino acid elongation had to be carefully monitored by HPLC analysis of the peptide obtained after a mini-cleavage (approximately 5 mg of resin) following each coupling. The Fmoc of the last residue [Gln(Trt)] was removed. The final cleavage to obtain the protected fragment 2, in which the C-carboxylic group was protected as 4hydroxybenzyl ester, required a slightly higher concentration of TFA (2−4%), which is compatible with the protecting groups and the ψ-WangHANDLE, than that usually required for the preparation of free acid peptides using CTC-resin (1−2%). The protected fragment 2 was obtained with high yield and was found to be soluble only in DCM and other organic solvents, thereby hindering its analysis by HPLC. The purity of the peptide was checked after global deprotection [TFA-H2OTIS (95:2.5:2.5)] of 5. This procedure provided fragment 2, which showed excellent purity (over 90%). Having achieved both fragments with excellent yield and purity, we studied fragment condensation. A key point was the total removal of piperidine traces in the protected fragment 2, as the presence of this compound greatly favored the formation of piperidides of the protected fragment 1. Piperidine was removed by repetitive washings of the peptide resin with DMF, MeOH, and DCM (5 × 1 min) and finally with DCM containing 0.01% of TFA. After peptide cleavage, the solution was precipitated over H2O, and finally the precipitate was washed several times with H2O to ensure a complete removal of piperidine. The fragment coupling was first assayed using DMF as a solvent; however, fragment solubility was unsatisfactory, so DMSO was tested as coupling solvent. The use of the uronium/aminium salts, COMU and HCTU, provided dirty

Figure 5. Scheme of Palm-moiety incorporation possibilities.

condensation of both fragments; (B) in solution, after isolation of the protected fragment 2; and (C) in solid phase, before cleavage the protected fragment 2 from the resin.. We considered that the presence of such a long aliphatic chain might hamper the effectiveness of fragment condensation, so we explored the addition of Palm-Glu(OSu)-OtBu in the last step of the synthesis, after condensation of the two fragments (A, Figure 5). Despite the presence of the unprotected final α-amino from His, we expected the Lys εamino group to be significantly more reactive, so acylation could be selectively performed in the desired position. Nevertheless, despite our efforts, double acylation was consistently significant, which forced us to discard the strategy (A) (Figure S4). Alternatively, we explored the possibility of reprotecting the α-amino with the Boc group after fragment condensation and global deprotection. In this case, the εamino group of Lys27 was protected with the allyloxycarbonyl (Alloc) group during the synthesis and this group was removed after the reprotection with Boc and before the incorporation of the branching moiety. The reprotection with Boc led to the main product with a double Boc incorporation, probably in the His residue (Figure S5). Interestingly, this extra protecting group did not hamper the introduction of the Pal-Glu-OtBu moiety after Alloc removal in a slow reaction that required 72 h to reach completion (Figure S6) and using Palm-Glu(OSu)OtBu/DIEA (2/4 equiv). Having demonstrated that Palm-Glu-OtBu could be introduced into the final chain, we expected a similar behavior C

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had been reactive enough to attack the active species, we would have found it attached to fragment 1, perhaps in some unusual polymerization. Nevertheless, the reaction proceeded properly with PyBOP, the crude product was clean, and neither the presence of starting material nor polymerization was detected after TFA treatment for deprotection. We therefore conclude that 4-hydroxymethylphenol was still attached to the peptidic backbone, which prevented any secondary reaction caused by the C-terminal of the peptide and allowed us to perform the condensation reaction and obtain the desired peptide. We have therefore completed the convergent synthesis of a long and difficult peptide with ease, obtaining liraglutide with high yield (condensation yield 74%, total yield 31%) and purity.24 In conclusion, we have described for the first time a multidetachable linker, which combines the Wang handle with a 2-CTC-resin, for a convenient preparation of C-terminal protected peptides amenable to be used in fragment condensation for the synthesis of long peptides. In addition to the application of this linker to other difficult peptides with more complex sequences (cyclic peptides or disulfidecontaining peptides), further studies addressing the compatibility of this linker in solution with other deprotecting conditions (e.g., H2 reduction, heat) are envisioned. The results of these projects will be published in due course.

when attaching it to the corresponding fragment prior to condensation. In this regard, we examined the introduction of the branching moiety during the solid-phase elongation of fragment 2 (C, Figure 5). Taking into account the sequence -Gln(Trt)-Ala-Ala-Lys(X)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly, the Alloc group could be removed after the introduction of the Lys, the two Ala, and the Gln. Alloc removal of Fmocprotected Lys is prone to α-amino deprotection,23 so we skipped it. First, when the Palm-based moiety was introduced after the last amino acid (Gln), incomplete incorporation of the Palm-Glu-OtBu was detected, so this point was discarded. On the other hand, introduction of the Palm side chain in solid phase after the coupling of either Ala residue proceeded easily and smoothly. No significant difference in yield was detected. However, its insertion after the introduction of the second Ala residue was more convenient in a day-to-day procedure. We therefore decided to keep that point as the desired Palm introduction for the final synthetic strategy. The optimized procedures described earlier for the synthesis of liraglutide were followed, with no significant influence of the presence of the Palm-Glu(OtBu) moiety. No differences in behavior were detected even for the final fragment condensation. As before, the crude deprotected peptide was obtained with great purity (68%) and was further purified by semipreparative HPLC to render liraglutide (purity 99%, condensation yield 62%) (Figure 6). As further confirmation of product identity,

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.9b00813.

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Sequence of liraglutide; comparision between DKP17 and pseudo-Wang strategies; materials and synthetic procedures; EM and HPLC spectra (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; [email protected]. ORCID

Ayman El-Faham: 0000-0002-3951-2754 Fernando Albericio: 0000-0002-8946-0462 Author Contributions

The strategy was designed by the all authors, the experiments were carried mainly out by D.C. and also by P.F., and all authors discussed the results and prepared the manuscript. Notes

The authors declare no competing financial interest. Figure 6. (A) HPLC trace of crude unprotected Liraglutide after fragment condensation (strategy C, Figure 5). (B) HPLC trace of final liraglutide. 5−100% H2O/ACN (0.1% TFA in 13 min), 220 nm.

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coelution assays of commercial liraglutide and prepared peptide were performed. No difference between the two molecules was found by HPLC, thereby confirming the synthesized peptide as being liraglutide. If ψ-WangHANDLE had been labile to the cleavage or condensing conditions, some polymerization or secondary reaction through the C-terminal would have been detected during the reaction. Likewise, if the phenol group of the handle

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ACKNOWLEDGMENTS This work was funded in part by the following: Lonza AG (Wisp, Switzerland), the Spanish Ministry of Economy, Industry, and Competitiveness (MINECO) (CTQ201567870-P & RTC-2014-2207-1), CIBER-BBN, the Generalitat de Catalunya (2017 SGR 1439) (Spain), and the International Scientific Partnership Program ISPP at King Saud University (ISPP# 0061) (Saudi Arabia). REFERENCES

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DOI: 10.1021/acs.orglett.9b00813 Org. Lett. XXXX, XXX, XXX−XXX