Introduction
Linkerology®, (1) a technique with increasing impact in the drug discovery arena, is usually based on the use of a linear molecule that binds two moieties of a drug. Examples of these can be found in Antibody-Drug Conjugates (ADCs) for cancer, where the linker connects the cytotoxic drug to the antibody, (2) or in the most recent drugs approved for diabetes type 2 or obesity, where the linker connects the peptide drug with the fatty acid responsible for its binding to albumin to extend the in vivo half-life of the drug (3).
In terms of linkers, those most widely used are based on poly- or oligomers of ethylene glycol. Polyethylene glycol (PEG) has been widely employed in drug discovery because it enhances the circulation of half-life medication (4).
Due to the excellent properties of PEG, among which are solubility in water, low cellular toxicity and protein resistance. PEGylated products have been used for the treatment of a variety of conditions, including melanoma, hepatitis, hematological cancer, and autoimmune diseases (5), to reduce the limitations of peptide-based drugs (6).
Unfortunately, clinical applications of PEGylated therapeutic molecules are hindered mainly because of their non-biodegradable properties, which may also result in cumulative toxic effects upon repetitive administration. Thus, Peginesatide—a peptide drug with a high PEG-content—initially approved by the FDA in 2012 was withdrawn one year later due to several adverse effects (7). Another potential issue associated with the use of PEGs in drug discovery is the challenge of synthesizing monodisperse PEGs/OEGs, as well as their high cost, which often poses an obstacle to mass use. Furthermore, recent studies have shown evidence that pegylated products exhibit immunogenicity, which reduces their efficiency as therapeutic agents (8, 9). Therefore, there is a growing need for alternative linkers for polymer-based conjugation, including, but not limited to, poly(2-oxazolines) (10), polyglycerol (11), zwitterionic polymers (12), OEGylated poly(meth)acrylates (13) and poly(amino acids)/oligomer peptides (14).
Oligomer peptides, defined as peptides with repeated amino acids, are challenging to synthesize, and this difficulty very often extends to their purification. These oligomers are more conveniently prepared by Solid-Phase Peptide Synthesis (SPPS), a technology developed by Merrifield in the second part of the 20th century (15). Even the SPPS of (Ala)10, which contains the simplest chiral proteinogenic amino acid, is used as a reference for the evaluation of new resins, coupling reagents, and also new solvents (16). This is due to the conformation adopted by the growing peptide, which hinders the coupling and deprotection steps. In the synthesis of oligomer peptides, incomplete coupling or inadequate removal of protecting groups at each step results in a single type of deleted peptide. In other words, the repeated sequence amplifies all potential synthetic problems (for instance, a 99% yield for each step in the preparation of a decaoligomer peptide will result in an impurity of approximately 10% of the nonapeptide). In the case of oligomers of secondary amino acids, there is another potential reaction to be controlled, as the secondary amine being acylated can lead to the early removal of the Fmoc group of the incoming amino acid, resulting in a peptide with an extra residue. In terms of purification, the properties of n+1, n, and n-1 oligomers are often remarkably similar and therefore difficult to separate chromatographically as retention times might be identical.
Over the years, poly(amino acid) polymers have been explored as substitutes for PEG (17). Among them, polysarcosine (PSar) shows more “stealth-like” properties in comparison to PEG. PSar comprises repeated units of the endogenous amino acid Sar (N-methyl-Gly), which is a derivative of glycine (Gly). Furthermore, research has shown that biodegradable PSar is more biocompatible than PEG. For instance, coated inorganic particles (quantum dots and gold nanorods) preserved enhanced colloidal stability and allowed longer circulation time in vivo (18-20). Finally, copolymers incorporating PSar designed for peptide or protein encapsulation have been shown to preserve more activity than PEG (21, 22). Therefore, PSar emerges as a promising material for biomedical applications (23-25).
From a synthetic perspective, the preparation of PSar should be more straightforward than that of PEG because the monomer (Sar) is a bifunctional molecule (amine and acid) with excellent and notable differentiated reactivity. On the other hand, ethylene glycol is a symmetrical molecule with two alcohols that are not very reactive in comparison to those of the amino acid (26, 27). This distinction is important because the moieties (linkers) used in the preparation of an active pharmaceutical ingredient (API) must be of excellent purity to fulfill the requirements set by the correspondingmedical regulatory agencies.
SPPS can be carried out using either protected amino acids or small peptides. The presence of Sar is particularly suitable for using small, protected peptides because Sar is not a chiral amino acid and therefore cannot undergo racemization. To address challenges in coupling efficiency and side reactions observed, ETT was employed as a coupling additive. Herein, we describe the synthesis of dodeca oligomers of Sar using Fmoc-Sar-OH, Fmoc-(Sar)2-OH and Fmoc-(Sar)4-OH.
Materials and methods
Fmoc-Sar-OH, Fmoc-(Sar)2-OH, Fmoc-(Sar)4-OH, and TengaGel S RAM were provided by Iris Biotech (Marktredwitz, Germany). ETT, OxymaPure, and DIC were a generous gift from Luxembourg Biotech (New Siona, Israel). The rest of the reagents and solvents were purchased from Millipore-Aldrich and were used without further purification unless otherwise stated. Analytical HPLC was performed on an Agilent 1100 system using Phenomenex C18 column (3 μm, 4.6×50 mm), and Chemstation software was used for data processing over a 5–95% gradient of CH3CN (0.1% TFA) / H2O (0.1% TFA) over 15 min, flow rate: 1.0 mL/min, detection at 220 nm. All mass spectrometry data were obtained from a Thermo Fisher Scientific UltiMate 3000 UHPLC-ISQTM EC single quadrupole mass spectrometer in positive ion mode over a 5–95 % gradient of MeCN (0.1% HCOOH)/H2O (0.1% HCOOH) for 15 min unless otherwise specified.
All peptides were prepared following a standard Fmoc/tBu-based solid-phase synthesis protocol (SPPS). TengaGel S RAM 90 µ (0.24 mmol/g) was used as solid support for the peptides. Initially, the resin was washed using DMF (3 × 1 min). The Fmoc group was removed by treatment of the resin with 25% piperidine/DMF (1 × 1 min and 1 × 10 min), followed by washing with DMF. The protected Fmoc-amino acids (2 equiv.) were incorporated using DIC (3 equiv.) and additives like Oxyma Pure or ETT (2 equiv.) as coupling reagents in DMF, for (2 x 1 h) at rt. This was repeated until the final peptide was achieved. Fmoc from the last coupled amino acid was removed as explained above. After drying the peptidyl resin, cleavage was performed by treatment with TFA/TIS/H2O (95:2.5:2.5) for 1 h at rt. The cleavage mixture was then precipitated with Et2O and centrifuged, and the pellet was re-dissolved in H2O/ACN (1:1) for analysis by HPLC and LCMS.
Results and Discussion
Dodeca oligomers of Sar were prepared using three Sar derivatives, namely Fmoc-Sar-OH, Fmoc-(Sar)2-OH, and Fmoc-(Sar)4-OH. The first synthesis was carried out using Fmoc-Sar-OH, to which DIC and OxymaPure (2 equiv.) were initially added, and then an extra equiv. of DIC was added after 30 min while the reaction was still ongoing. In total, the reaction was left to run for 1 h. Since Fmoc-Sar-OH is considered a hindered amino acid, a double coupling was performed, followed by removal of the Fmoc group using 25% of piperidine in DMF (1 x 1 min and 1 x 10 min). At different steps of the synthesis, specifically after (Sar)4 and (Sar)8, the peptide was cleaved from the resin with trifluoracetic acid (TFA), triisopropylsilane (TIS), and H2O at a ratio of 95:2.5:2.5 for 1 h at room temperature (rt) to monitor the course of the synthetic process.
After the addition of the fourth Sar residue, the Fmoc group was removed and a mini-cleavage was performed. However, the H-(Sar)4-NH2 eluted in the solvent front on the HPLC, making it difficult to analyze. Therefore, we decided to retain the Fmoc group on the peptide to simplify subsequent analysis.
In the synthesis of Fmoc-(Sar)12-NH2 using Fmoc-Sar-OH, a mini-cleavage was performed after the addition of the fourth Fmoc-Sar-OH to monitor the progress of the reaction. The main peak (Figure S1) indicated the correct molecular weight in the LC-MS analysis (Figure S2), but several impurities were also present. Next, four more Sar residues were added, and the resulting Fmoc-(Sar)8-NH2 was identified by LC-MS after another mini-cleavage (Figures S3, S4). In this intermediate, the solvent front showed a significant presence of H-(Sar)3-NH2 (Figures S3, S5) and some Fmoc-(Sar)2-NH2 (Figures S3, S6). Finally, the last four Sar residues were added to produce Fmoc-(Sar)12-NH2, which again was confirmed by LC-MS (Figures S7, S8). The LC-MS and HPLC spectra showed the presence of impurities, including Fmoc-(Sar)6-NH2 (Figures S7, S9) and, again, H-(Sar)3-NH2 (Figures S7, S10). Although the purity of the synthetic peptide containing 12 hindered amino acid repeats was acceptable, it was not sufficient to allow its use as a linker for conjugation.
After the initial preparation of Fmoc-(Sar)12-NH2 using Fmoc-Sar-OH, the synthesis was repeated using the protected dipeptide Fmoc-(Sar)2-OH as a building block. As in the previous case, the progress of the synthesis was analyzed at the stages of Fmoc-(Sar)4-NH2, Fmoc-(Sar)8-NH2, and at the end, Fmoc-(Sar)12-NH2. At the tetramer level, although the main peak was observed (Figures S11, S12), the HPLC analysis revealed more impurities corresponding to Fmoc-(Sar)2-NH2 (Figures S11, S13), with the remaining peaks unrelated to the peptide (see Figure S14). At the octamer level, Fmoc-(Sar)8-NH2 was the main peak (Figures S14, S15), but it was accompanied by Fmoc-(Sar)4-NH2 (Figure S16) and Fmoc-(Sar)2-NH2 (Figure S17). Finally, Fmoc-(Sar)12-NH2 (Figures S18, S19) was obtained, but it was accompanied by a significant impurity of Fmoc-(Sar)6-NH2 (Figure S20).
Finally, the target peptide was synthesized using Fmoc-(Sar)4-OH as the monomer. The quality of Fmoc-(Sar)8-NH2 (Figures S21, S22) was improved compared to the previous synthesis, although it was still accompanied by an impurity of Fmoc-(Sar)4-NH2 (Figures S21, S23). A similar trend was observed in the analysis of Fmoc-(Sar)12-NH2 (Figures S24, S25), which showed enhanced quality compared to the previous synthesis but still contained some impurities of Fmoc-(Sar)8-NH2 (Figures S24, S26).
Our group has recently demonstrated that ethylthio-1-tetrazole (ETT) outperforms OxymaPure, hydroxybenzotriazole (HOBt), and even 1-hydroxy-7-azabenzotriazole (HOAt) as a coupling additive in combination with DIC for hindered amino acids such as Sar (28). In the next set of syntheses, ETT was used instead of OxymaPure, following the same protocol of double coupling with 2 equiv. of Fmoc-Sar-OH, DIC, and ETT each. After 30 min, an additional extra equiv. of DIC was added for a further 30 min (total coupling time of 1 h). Figure 2 shows that ETT is a superior additive than OxymaPure for these kinds of hindered couplings (Figure 2 vs. Figures S1, S3, S7) [the intermediates, Fmoc-(Sar)4-NH2 and Fmoc-(Sar)8-NH2 from Fmoc-Sar-OH, Fig. S27-S32]. However, MS analysis revealed the presence of Fmoc-(Sar)13-NH2 as a double charge mass of 582 m/z (Figure S32).
After observing the positive results from the synthesis of the target peptide Fmoc-(Sar)12-NH2 using DIC/ETT as coupling reagent with Fmoc-Sar-OH as a building block, the dodeca oligomer was prepared again using Fmoc-(Sar)2-OH and Fmoc-(Sar)4-OH as building blocks. Figure 3 shows that, like the synthesis using Fmoc-Sar-OH, the target peptides were obtained with excellent purity. However, in the synthesis of the dodeca oligomer from Fmoc-(Sar)2-OH, MS analysis revealed an additional incorporation of the building block, resulting in Fmoc-(Sar)14-NH2 as a double charge mass of 617 m/z (Figure S34). In contrast to the MS analysis of Fmoc-(Sar)12-NH2 synthesized using Fmoc-(Sar)4-NH2, there were neither deletions nor additional incorporation of the building block (Figure S35).
Conclusion
It has been demonstrated that PSar containing 4, 8, or 12 units of Sar can be efficiently prepared using three distinct building blocks [Fmoc-Sar-OH, Fmoc-(Sar)2-OH, and Fmoc-(Sar)4-OH].
Syntheses were carried out using DIC as the coupling reagent in combination with either OxymaPure or ETT as additives. Although those using OxymaPure yielded the target peptide (Fmoc-(Sar)12-OH) with acceptable purity, this level was insufficient for the intended use of PSar in Linkerology®. In this context, ETT has been shown to outperform OxymaPure, as previously demonstrated by our group for a simple model peptide.
When comparing Fmoc-(Sar)12-OH synthesized with DIC/ETT from three different monomers, the LC-MS analysis revealed that the target peptide obtained from Fmoc-(Sar)4-OH does not show impurities from Fmoc-(Sar)16-OH (indicative for premature Fmoc removal during the acylation step and double incorporation of the monomer) or Fmoc-(Sar)8-OH (suggesting either incomplete Fmoc removal or coupling of the monomer). On the other hand, analysis of Fmoc-(Sar)12-OH from Fmoc-Sar-OH showed a single peak in HPLC. However, the MS analysis indicated the presence of Fmoc-(Sar)13-OH from Fmoc-Sar-OH and Fmoc-(Sar)14-NH2 from Fmoc-(Sar)2-OH.
This extensive study focused on the synthesis of PSar also highlights the importance of the purity of the building blocks and reagents used. The optimal performance of the synthesis carried out with Fmoc-(Sar)4-OH is attributed to the purity of this building block, which is comparable to that of the monomers. The use of a less pure tetramer is unlikely to match the quality of the target peptide synthesized from the monomer.
It is envisaged that the strategy described herein will facilitate the preparation of larger PSar molecules with satisfactory purity for use in Linkerology®.
Figure 1. Chemical structures of polysarcosine and polyethylene glycol highlighting their respective functional groups.
Figure 2. HPLC of Fmoc-(Sar)4-NH2 (I), Fmoc-(Sar)8-NH2 (II), and Fmoc-(Sar)12-NH2 (III), synthesized from Fmoc-Sar-OH using DIC/ETT.
Figure 3. HPLC of Fmoc-(Sar)12-NH2 synthesized from (I) Fmoc-(Sar)2-NH2 and (II) Fmoc-(Sar)4-OH using DIC/ETT.
References and notes
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