A Fmoc protection reagent: Fmoc-Amox

Most peptide synthesis is achieved by solid-phase peptide synthesis using Fmoc or Boc protection methods, however when Fmoc-OSu is used, due to the formation of dipeptide (or tripeptide) and β-alaninamide impurities, in the amino acid The introduction of unprotected N in Fmoc appears to be challenging. Here we introduce an efficient method for the synthesis of Fmoc-Gly-OH based on the oxime derivative Fmoc-Amox without any side reactions. Fmoc-Amox is inexpensive, and Amox can be easily removed after the reaction, thereby providing pure Fmoc-Gly-OH without any harmful impurities or contaminants (mainly dipeptides or Amox itself), which can be obtained from high-efficiency liquid Phase chromatography and NMR were demonstrated.

In 1963, Merrifield described a new concept of chemical synthesis. The active ingredients (API) of some drugs on the market are TIDES (oligonucleotide and peptide therapeutic agents), which contain as many as 30-40 monomers. The bodies were produced using a solid-phase method first described by Merrifield. Although the solid-phase peptide synthesis (SPPS) method was initially questioned to some extent by his European colleagues, it is now widely used in synthetic research and production.

As can be seen from the nomenclature, all amino acids have at least two functional groups: a carboxylic acid and an amino group. If the activity of the C-terminal carboxylic acid of the amino acid is covered by an insoluble polymer group, the amino group is temporarily protected, and then participates in the reaction step by step and continuously, including removing the protecting group of the amino group and then coupling with the next N-protected amino acid couplet. However, in the case of trifunctional amino acids, the side chains are protected by permanent (or semi-permanent) protecting groups. In the early years, Merrifield used benzyl (Bn) for long-term protection and start-butoxy carbonyl (Boc) as a temporary protection group, but both Boc and Bn can be decomposed under acidic conditions: trifluoroacetic acid (TFA) de-Boc, Strong acids such as HF or trifluoromethanesulfonic acid (TFMSA) hydrolyzes Bn. In the 1970s, Carpino, who also proposed the Boc protecting group, revolutionized the field of peptide chemistry by proposing fluorenylmethyloxycarbonyl (Fmoc) as a group for temporarily protecting the amino group, that is, the N protecting group can be removed with a base, It is thus orthogonal to the Boc group. In addition, Sheppard and Atherton in Europe, together with Chang and Meienhofer in the United States, developed the Fmoc/t-Bu method at the same time, and the reaction was treated with TFA solution to release the peptide. The implementation of this method marks the "civilianization" of peptide synthesis, because the use of the Boc/Bn method requires trained chemists and special equipment, while through the Fmoc/t-Bu method, other biological laboratories can also synthesize peptides. Furthermore, this method has enabled the production of kilogram-scale peptides.

The first commercial Fmoc-amino acids were synthesized by the Schotten-Baumann method: the amino acid was reacted with Fmoc-Cl under basic conditions (Fig. 1A). In the early 1980s, the Ashish group and Bachem and Goodman pointed out that most commercial Fmoc-amino acids contain dipeptides and tripeptides. These impurities come from the high reactivity of Fmoc-Cl, which can also react with the carboxyl group of the amino acid to be protected. Anhydride, which in turn can react with the amine terminus of another amino acid molecule, resulting in the formation of a dipeptide. The mechanism is shown in Figure 1B.

Figure 1 Fmoc protection of amino acids
A. FMOC protection mechanism
B. Mechanism of Protected Dipeptide Formation During Amino Acid Protection

Considering that even a small proportion of impurities can lead to loss of yield and purity, the parties jointly concluded that Fmoc-Cl should be avoided. Because these side reactions are related to the mass of the leaving group, Ashish's group suggested using Fmoc-N3, also mentioned by Carpino and Han in their paper. The Ashish group proposed to synthesize Fmoc-N3 with Fmoc-Cl and sodium azide. To avoid the danger of Fmoc-N3 preparation and storage, it is used now. The Fmoc-amino acid synthesized by this method has a higher purity. Verlander et al. suggested the use of Fmoc-OSU by screening different leaving groups, while Bolin et al. suggested the use of silylating reagents to protect the carboxyl group. Many years later, Barlos et al proposed a method for preparing Trt-amino acids from Trt-Cl to avoid the formation of Trt ester impurities. Later, Suresh et al. proposed the preparation of Fmoc-amino acids from Fmoc-Cl in the presence of activated zinc powder, which allowed for neutral conditions.

However, for a long time, the most commonly used method for the synthesis of Fmoc-amino acids was Fmoc-OSu (or NHS), which was also questioned when Hlebowicz et al. Bachem Europe's research shows that Fmoc-AA-OH prepared using Fmoc-OSu contains Fmoc-β-Ala-OH and Fmoc-β-Ala-AA-OH, and these two impurities pass through Lossen after OSu attacks a carbonyl group. formed by rearrangement (Figure 2).

Figure 2 The formation mechanism of β-alaninamide through Lossen rearrangement

These findings have reinvigorated research enthusiasm for the development of new reagents or methods for the safe introduction of Fmoc groups. Table 1 lists the different Fmoc derivatives used to protect amino acids and their results for the preparation of Fmoc-Gly-OH. Although this reaction can be used for the protection of all amino acids, Gly is more obvious because of its low steric hindrance. Conducive to high-efficiency polymerization. These Fmoc-introduced derivatives should have two key features: (i) no special high reactivity, avoiding the formation of oligopeptides; (ii) the leaving group is usually a hydroxyl compound substituted by an amino acid, which is relatively stable during processing. Easier to remove. Therefore, the Ashish group first proposed Fmoc-2-mercaptobenzimidazole, which synthesized Fmoc derivatives with fewer oligopeptides (Table 1, #4). However, the by-product 2-MBT released in the reaction has poor solubility and needs to be completely removed by washing with organic solvents, while Fmoc-amino acids also have certain solubility in organic solvents, which is not conducive to the final yield. A similar problem was encountered by Verlander et al., when using (poly)chlorophenyl derivatives, organic solvent alcohols contaminate the final product, resulting in low overall yields (4-30%).

Derivatives of other succinimides, such as phthalimide, norbornenyl (which is a free radical derived from norbornene), and the corresponding spiro analogs, six-membered derivatives, etc., were also carried out analyzed but did not have a significant advantage (Table 1, #5-7). Simultaneously, the formation of β-alanine was detected when norbornene groups were combined with EDC for solid-phase peptide synthesis (SPPS) in water. To overcome the problem of intercalation of β-alaninamide residues caused by the use of succinimide derivatives, Najera et al. proposed a reagent in the form of a polymer, and the final β-alaninamide contaminant was immobilized on the polymer support, However, this method limits the use for preparing small amounts of protected amino acids.

Other reactive substances commonly used with carbodiimide coupling compounds such as pentafluorophenyl (Pfp) or benzotriazole (Bt), although these reagents are relatively expensive (Pfp) or explosive (Bt) (Table 1, #8, 9), but the yields are high, and some laboratories have even investigated Fmoc-triazine derivatives (Table 1, #10) as a way to synthesize Fmoc-amino acids. For the method of introducing Fmoc using a pure aqueous solution, Fmoc-phenyl dimethyl sulfonium methyl sulfate (Fmoc-ODsp) has been used, but the formation of the dipeptide was not investigated (Table 1, #11).

To overcome one of the greatest challenges in the synthesis of peptide building blocks, namely the undesired formation of dipeptides and tripeptides during amino acid protection, the approach of a large number of Fmoc leaving groups has been intensively investigated. Although a small number of reagents have had decent results, almost none of them have the potential to be used in industrial production. In this regard, based on the structure of the commonly used carbodiimide additive oxime, the Fmoc leaving group derivative Fmoc-Amox was proposed. Fmoc-Amox has been used to protect H-Gly-OH, which is very prone to side reactions, with a high yield (93%), and there is no by-product in the form of Fmoc-dipeptide at all, which can be seen from HPLC and NMR analysis Get confirmed. In addition, Amox derivatives may be used in the future to introduce other protecting groups such as pNZ, Alloc, and Boc.

The Ashish group believes that Oxyma is a better additive than carbodiimide. Oxyma has strong reactivity and is more and more widely used in the industrial production of peptides. The group also investigated other less reactive oxime derivatives to introduce the Fmoc group, from the first screen (Table 1, #12-16), including 2-hydroxypyridine N-oxide (HOPO), derived from Oxyma (Table 1, #12) gave a higher content of dipeptide due to its high reactivity, followed by HOPO (Table 1, #16). Cyanopyridinium oxime is also a good additive (Table 1, #15), but it is expensive and difficult to remove from the reaction. The dipeptide formation in the second screen was generally less (Table 1, #17-20), so the cyanoamide derivative (Amox) (Table 1, #20) was chosen as an alternative to HOSu to introduce Fmoc. Fmoc-Amox is affordable and can be easily removed after the reaction, unlike the case of MBT, Amox has a solubility of 0.9 M in water, which ensures that it does not contaminate the final Fmoc-amino acid (Table 1, #4 ).

In terms of Fmoc-dipeptide formation, H-Gly-OH proved to be the best reagent for evaluating the performance of Fmoc-Amox because of its low steric hindrance favoring a high ratio of oligomerization. Using 1 gram and 40 grams of two parallel experiments to prepare Fmoc-Gly-OH results are close, and the reaction process is described as follows: the acetone solution of Fmoc-Amox is slowly added to the stirred H-Gly-OH and sodium carbonate aqueous solution, through continuous separation Sodium carbonate, was added batch-wise to maintain the pH of the reaction mixture at 9-10. The reaction was monitored by TLC as well as pH stability (a drop in pH as a sign of reaction). After the reaction was complete (4 hours), the solvent was removed and the remaining aqueous layer was washed with DCM, followed by the addition of 1N HCl (until pH<2), resulting in an off-white precipitate, which was then filtered and treated with ethyl acetate and n-hexane (93%) After recrystallization, the purity of Fmoc-Gly-OH detected by HPLC was very good. Since Fmoc-Gly-Gly-OH prepared by solid-phase technology will be eluted together with Fmoc-Gly-OH, it indicates that the formed dipeptide impurity is difficult to remove, as shown in Figure 3, it is not observed in the chromatogram Traces of dipeptides (Figure 3). In addition, 1H NMR did not show any contamination of Amox (Fig. 4), which clearly shows that Amox has a great advantage in the synthesis of Fmoc amino acids.

Figure 3 (A) HPLC chromatograms of Fmoc-Gly-OH and Fmoc-Gly-Gly-OH eluting together
(B) HPLC chromatogram of Fmoc-Gly-OH

Figure 4 Comparison of 1H and 13C NMR of Fmoc-Gly-OH, Fmoc-Amox and Amox

Motivated by these results, Fmoc-Phe-OH and Fmoc-Val-OH (10 g mini-trials each) were prepared using the method described above (Figures 5 and 6). The purity of the final product was confirmed by HPLC and NMR, showing no trace formation of dipeptide. The importance of the solvent during the work-up was also investigated. The resulting product was dissolved in DCM and extracted using distilled water. DCM work-up helped to remove traces of Amox, resulting in a pure product as confirmed by NMR (Fig. 7).

Figure 5 (I) A. HPLC chromatogram of Fmoc-Phe-OH and Fmoc-Phe-Phe-OH eluting together
B. HPLC chromatogram of Fmoc-Phe-OH
(II) 1H and 13C NMR comparison of Fmoc-Phe-OH, Fmoc-Amox and Amox

Figure 6 (I) A. HPLC chromatogram of Fmoc-Val-OH and Fmoc-Val-Val-OH eluting together
B. HPLC chromatogram of Fmoc-Val-OH
(II) 1H and 13C NMR comparison of Fmoc-Val-OH, Fmoc-Amox and Amox

Figure 7 Comparison of the post-treatments of Fmoc-Phe-OH extracted with ethyl acetate and DCM

These results clearly show that Fmoc-Amox has great advantages in the synthesis of Fmoc-amino acids which are most prone to side reactions.

Example of synthesis of Fmoc-amino acids:

1. Add glycine (131.34mmol) and sodium carbonate (106mmol) to purified water, and add Fmoc-Amox (119.4mmol) acetone solution dropwise to ensure that the pH of the reaction solution is always kept at 9-10. After TLC detected that the Fmoc-Amox reaction was complete, the reaction solution was concentrated to remove acetone and then extracted with dichloromethane to remove impurities. The aqueous phase was acidified with 1N HCl and a large amount of white solid precipitated. After filtration, the filter cake was washed three times with purified water. The collected solid was recrystallized with ethyl acetate/n-hexane to obtain high-purity Fmoc-glycine.

2. Add phenylalanine or valine (33mmol) and sodium carbonate (75mmol) to purified water, and add Fmoc-Amox (30mmol) acetone solution dropwise to ensure that the pH of the reaction solution is always kept at 9-10. After TLC detected that the Fmoc-Amox reaction was complete, the reaction solution was concentrated to remove acetone and then extracted with dichloromethane to remove impurities. The aqueous phase was acidified with 1N HCl and a large amount of white solid precipitated. After filtration, the filter cake was washed three times with purified water. The collected solid was recrystallized with ethyl acetate/n-hexane to obtain highly pure Fmoc-phenylalanine or Fmoc-valine.

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