Acetyl group for proper protection of β-sugar-amino acids used in SPPS

The synthesis of d-glucosamine-1-carboxylic acid based β-sugar amino acids (β-SAAs) is typically performed in nine consecutive steps via an inefficient OAc → Br → CN conversion protocol with low overall yield. Here, we present the improved and more efficient synthesis of both Fmoc-GlcAPC-OH and Fmoc-GlcAPC(Ac)-OH, β-SAAs consisting of only 4–5 synthetic steps. Their active ester and amide bond formation with glycine methyl ester (H-Gly-OMe) was completed and monitored by 1H NMR. The stability of the pyranoid OHs protecting the acetyl groups was investigated under three different Fmoc cleavage conditions and was found to be satisfactory even at high piperidine concentration (e.g. 40%). We designed a SPPS protocol using Fmoc-GlcAPC(Ac)-OH to produce model peptides Gly-β-SAA-Gly as well as Gly-β-SAA-β-SAA-Gly with high coupling efficiency. The products were deacetylated using the Zemplén method, which allows the hydrophilicity of a building block and/or chimera to be fine-tuned, even after the polypeptide chain has already been synthesized. Supplementary Information The online version contains supplementary material available at 10.1007/s00726-023-03278-1.

The most efficient and widely used method for the synthesis of oligopeptides and chimeras is solid-phase peptide synthesis (SPPS), which has recently been adopted for β-SAAs.(Goldschmidt Gőz et al. 2019, 2021;Nagy et al. 2019;Farkas et al. 2021;Duong et al. 2021) However, both the active ester formation and the coupling reaction conditions require the protection of the nucleophilic -OH groups, similar to the case of Ser and Thr residues.Acetylation is one of the simplest and easiest ways of providing -OH protection in carbohydrate chemistry and has already been used in solution phase peptide synthesis by Suhara et al. (2006).They obtained the N-Boc and O-acetyl protected sugar amino acid (β-SAA) from d-glucosamine‧HCl after completing a ninestep reaction sequence (Scheme 1).From the monomer, they synthesized new dimer, trimer and tetramer 'carbopeptoids' using conventional solution phase peptide synthesis (Suhara et al. 2006).Since our intention was to use the β-SAA moiety in SPPS, we needed to thoroughly investigate the stability of the -OAc-protected β-SAAs under the conditions of the peptide coupling and Fmoc deprotection steps, as well as when the crude product was removed from the resin.
Although a large number of coupling reagents and active esters have been used in the past (Valeur and Bradley 2009;El-Faham and Albericio 2011), HOBt/DIC (Hydroxybenzotriazole/N,N-Diisopropylcarbodiimide) are now the most widely used reagents (Albericio et al. 1998).The PyBOP/DIEA (Benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate/N,N-Diisopropylethylamine) reagent pair, which has been shown to minimize the risk of racemization, is also used to construct more challenging sequences.Recently, we have investigated and optimized different coupling reagents to generate GXXG model peptides (Goldschmidt Gőz et al. 2019;Nagy et al. 2019), where X was either a pyranoid (e.g.Fmoc-GlcAPU(Me)-OH) or a furanoid (e.g.Fmoc-RibAFU(ip)-OH) β-SAAs.To optimize both products formation and coupling as a function of time, their active ester formation was monitored by 1 H NMR. For both furanoid and pyranoid β-SAAs, PyBOP/DIEA was found to be the best coupling reagent.Using this method, active esters are formed rapidly (within 20 min with high conversion: > 99%) and remain stable for at least 6-24 h, significantly longer than the duration of a typical coupling cycle (3 h).
Here, we demonstrate a shorter and scalable synthetic route for the preparation of the novel β-SAA coupling component Fmoc-GlcAPC-OH (1) and its per-O-acetylated derivative Fmoc-GlcAPC(Ac)-OH (2).Although O-acetyl protection is common in carbohydrate chemistry, and commonly used in the synthesis of glycopeptides and related compounds (Bermejo et al 2020), it is infrequently used in conventional SPPS.Therefore, in response to this synthetic challenge, we have fine-tuned protocols in which the reaction is monitored by NMR.Active ester formation and coupling with H-Gly-OMe was followed by time-resolved 1 H NMR. Focusing on the different model peptides introduced earlier, both Ac-GXG-NH 2 and Ac-GXXG-NH 2 were prepared, where X represents the β-SAA.We show here, that the acetyl groups are highly stable under a variety of Fmoc and resin cleavage conditions, resulting in the fully protected oligopeptides.Furthermore, the O-acetyl groups can be subsequently removed by the standard Zemplén deacetylation method, widely used in carbohydrate chemistry, resulting in an unprotected and even more polar β-SAA-containing chimera.

Synthesis of H-GlcAPC-OH derivatives
The synthesis of several H-GlcAPC-OH derivatives has already been described (Suhara et al. (loc cit;von Roeden et al. 1996), but the method published by Suhara et al. is the most widely accepted and used, leading to the N-Boc and O-Ac protected β-SAA: Boc-GlcAPC(Ac)-OH (3).However, we found that this route-which consists of 9 steps-is too long and that it could be shortened and optimized.Furthermore, we wanted to prepare building blocks suitable for SPPS (Goldschmidt Gőz et al. 2018;Nagy et al. 2017) and also for flow peptide chemistry (Farkas et al. 2021;Goldschmidt Gőz et al. 2021).Thus, in order to be compatible with Fmoc chemistry, the targeted β-SAA must be protected by Fmoc and Ac groups, respectively.
In the original Suhara synthesis, the key intermediate 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-dglucopyranosyl-1-carbonitrile (5) was obtained in four steps and the final product 3 was obtained in five consecutive steps.The chain elongation leading to the nitrile derivative (5), made by OAc → Br → CN exchange, are the key steps of this synthesis.Subsequently, 5 was hydrolyzed in three steps to give the fully unprotected d-glucosamine-1-carboxylic acid (H-GlcAPC-OH, 8), which was then acetylated and N-protected by Boc to give Boc-GlcAPC(Ac)-OH (3), a molecule finally suitable for synthesis using solution-state protocols.This building block was used to complete the homooligomer synthesis with BOP/DIEA, a stepwise synthesis in solution, resulting in the dimer, trimer, etc. up to the hexamer (Suhara et al. 2006).The O-acetyl protection was removed after the final step using the Zemplén deacetylation method.Based on NMR and CD spectroscopy data, only the hexamer is long enough to adopt a 14-helix structure (Suhara et al. 2006).The -GlcAPC-building block can form oligomers by using different coupling agents (e.g.BOP/DIEA or HOBt/EDC‧HCl).However, the synthesis of the β-SAA building block is tedious in this way, and these synthons are only suitable for peptide synthesis in the solution state.
Our method is more efficient than that of Suhara et al. who obtain their final compound, Boc-GlcAPC(Ac)-OH, in 9 steps with an overall yield of 26%.We achieved our target molecules Fmoc-GlcAPC-OH (1) and obtained Fmoc-GlcAPC(Ac)-OH (2) in four and five steps, with overall yields of 36% and 29%, respectively.The process is also more environmentally friendly by reducing the number of synthetic steps.The key step, chain elongation, is performed in a single step.After their formation, both 1 and 2 can be precipitated with 2 M HCl.Molecules 5 and 2 were both successfully recrystallized from ethanol, eliminating column chromatography and providing a greener synthetic route.

Active ester formation
We have previously investigated several coupling reagents and conditions to obtain the best method for β-SAAs coupling (Nagy et al. 2019).Before proceeding with SPPS, we performed 1 H NMR measurements to determine if the PyBOP/DIEA coupling reagent pair was suitable and to determine the optimal coupling conditions.We monitored the active ester formation of the β-SAAs (Fmoc-GlcAPC-OH and Fmoc-GlcAPC(Ac)-OH) as a function of time (Goldschmidt Gőz et al. 2019;Nagy et al. 2019).In DMF (Fig. 1a), the hydroxybenztriazol moiety of PyBOP transforms into the active ester moiety of both Fmoc-GlcAPC-OBt (11) and Fmoc-GlcAPC(Ac)-OBt (12), whose formation was indicated by the characteristic chemical shift changes of the aromatic H D with respect to the parent H D' (Fig. 1b).The formation of the active ester was monitored for both β-SAA derivatives and we found that the formation of Fmoc-GlcAPC-OBt (11) requires ~ 1 h, resulting in an almost quantitative (96%) conversion (Fig. 1c and SFigure 1).We have also found that the active ester remains stable in solution for up to 24 h at room temperature.The reaction with Fmoc-GlcAPC(Ac)-OH (2) was slower (~ 3 h), but again the conversion was almost quantitative (~ 99%) and the product was stable even after 24 h.Note how useful the NMR monitoring of the reaction is for these β-SAAs, we were able to determine that the activation is slower than expected (1-3 h), but in return the active ester formations are almost quantitative.

Coupling with the active esters
The coupling of the two active β-SAAs esters, Fmoc-GlcAPC-OBt (11) and Fmoc-GlcAPC(Ac)-OBt ( 12), was investigated with glycine, the simplest and most flexible proteinogenic amino acid residue.The C-terminal of Gly was protected as a methyl ester: H-Gly-OMe.Slow amide bond formation was observed for both Fmoc-GlcAPC-OBt (11) and Fmoc-GlcAPC(Ac)-OBt (12) in the presence of 2 equivalents of H-Gly-OMe and 6 equivalents of DIEA: even after 24 h the conversion was only 63% and 17%, respectively.(Fig. 2 and SFigure 2).Although this pilot reaction was too slow and the conversion was insufficient for direct implementation for SPPS, it should also be remembered that these initial experiments were carried out in an NMR tube without any stirring.In the following, we will show that the same coupling reaction proceeds more rapidly and more effectively when the reaction conditions are optimized for SPPS.

Stability of the acetyl groups in chimera containing the -GlcAPC(Ac)-subunit
As our intention was to develop Fmoc chemistry suitable β-SAAs using acetylated derivatives, e.g.Fmoc-GlcAPC(Ac)-Gly-OMe, the question arises to what extent an acetyl protecting group resists cleavage conditions.Therefore, we investigated the stability of this group under conventional Fmoc cleavage conditions and beyond.The N-phthaloyl-protected model compound Phth-GlcAPC(Ac)-NH 2 (13) was used, as the Phth-group is an inexpensive and simple amide protecting group (Fig. 3).Acetyl groups were found to be stable under conventionally accepted Fmoc cleavage conditions at room temperature (rt), such as 20% piperidine in DMF, or its equivalent 2% piperidine with 2% Scheme 2 Improved synthesis of H-GlcAPC-OH based β-sugar amino acids (1, 2).Reaction conditions: a) MeCN, TMSCN, BF 3 ‧OEt 2 , rt, 18 h, 50% , b) i) 12% NaOH in H 2 O, reflux (18 h), ii) 2 M HCl, reflux (18 h), c) Fmoc-OSu, MeOH, dioxane, rt (48 h) and d) Ac 2 O, Pyridine, rt (18 h) DBU in DMF (Fig. 3c-d and SFigure 3-5).Furthermore, even under the harshest conditions of flow chemistry (Farkas et al. 2021), 40% piperidine in DMF, the O-Ac group remains intact at 25 °C.In conclusion, acetyl groups seem to be suitable to protect -OH groups of β-SAAs moieties in SPPS, at least if (i) the chimera synthesis is not too long, (ii) the sequence is not too long and (iii) the sugar amino acid is not too close to the C-terminus of the sequence.

Coupling on solid phase
Our end products (1, 2) are promising candidates for coupling to SPPS.Having both the O-acetylated Fmoc-GlcAPC(Ac)-OH (2) and its "free" derivative Fmoc-GlcAPC-OH (1) in hand, SPPS was investigated for both.Note that the free β-SAA 1 has a total of 3 free -OH groups as nucleophiles, which could cause miscoupling, especially that of the primer -OH of the C-6 position.Armed with the previously established NMR data on active ester formation and coupling data, SPPS was used to generate the Ac-GXXG-NH 2 model chimera (X = β-SAA: -GlcAPC-and -GlcAPC(Ac)-).Using this model peptide, we investigated all 3 different conditions of amide bond formation, namely those of α-β, β-β and β-α amide formation.For Fmoc-Gly-OH coupling, DIC/HOBt was used (1 h coupling time), whereas for β-SAAs PyBOP/ DIEA was used (3 h).For Fmoc cleavage, 2% piperidine and 2% DBU in DMF protocol was used.When using Fmoc-GlcAPC-OH, a building block with free -OH groups, we found low coupling efficiencies (5-25% in Table 1).Furthermore, raw material analysis showed that the desired model On the other hand, the per-O-acetylated Fmoc-GlcAPC(Ac)-OH was successfully incorporated into the Ac-GXXG-NH 2 model chimera: both coupling efficiencies (84-91%) and yields (69-90%) are acceptable (Table 1).We have previously shown that all protecting groups used for Fmoc/tBu SPPS protocols can be successfully removed during the final cleavage step using an acid-reduced cleavage mixture containing 50% TFA (Goldschmidt Gőz et al. 2021;Duong et al. 2021).Using the same mixture in this case, we found that the crude model chimera Ac-GXXG-NH 2 was isolated with all O-acetyl groups successfully retained.Furthermore, even when using the 95% TFA mixture, the O-acetyl protecting groups remain intact as confirmed by MS (SFigure 15).Thus, both the milder and the harsher TFA conditions allow to obtain the desired peracetylated chimera.

Deacetylation
To further investigate the stability of the O-acetyl protecting groups, Ac-GXG-NH 2 was synthesized and studied, as partial deacetylation is easy to detect on this simple chimera (SFigure 9).The final cleavage was carried out at 40 °C with 95% TFA and we found that the fully O-acetylated chimera is intact.Neither the MS spectrum nor the HPLC chromatogram show any sign of decomposition (SFigure 10 and 14).
To obtain the fully unprotected chimera, the Zemplén deacetylation method, a reaction widely used in carbohydrate chemistry to remove acetyl protection, was performed  on Ac-GXG-NH 2 .As the deacetylation was performed in MeOH, which induces the collapse of the S RAM Tentagel ® resin, the reaction was performed after the final cleavage.
The purified per-O-acetyl Ac-GXG-NH 2 was dissolved in MeOH and a few drops of 2 M NaOMe/MeOH solution were added.After 1 h at room temperature, the reaction was stopped by adding TFA to the mixture.The result was a fully -OH deprotected Ac-GXG-NH 2 chimeric peptide (SFigure 13 and 17).In summary, the O-acetyl groups were successfully removed by the Zemplén deacetylation method, whereas the conventional 50% or 95% TFA final cleavage mixture did not interfere with the O-acetyl groups.

Conclusion
In the present work, new β-sugar amino acids, namely Fmoc-GlcAPC-OH (1) and Fmoc-GlcAPC(Ac)-OH (2), suitable for SPPS were synthesized in only four and five synthetic steps.Using NMR measurements, we found that the corresponding active esters are formed with PyBOP/ DIEA in 1-3 h and when coupled with an α-amino acid, H-Gly-OMe, they form a β-α-amide bond.The stability of the O-acetyl protecting groups was confirmed by 1 H NMR under Fmoc cleavage conditions using Phth-GlcAPC(Ac)-NH 2 (13) as a model.Both Ac-GXG-NH 2 and Ac-GXXG-NH 2 chimeras with O-acetyl protected β-SAA (2) were successfully synthesized in SPPS: both coupling efficiencies and overall yields were found to be sufficient.The acetyl groups were removed by the Zemplén deacetylation method after final resin cleavage.In conclusion, it is possible to prepare chimeric peptides containing β-α-, α-β-and/or β-β-amide linkages using the new and efficiently synthesized β-SAA, Fmoc-GlcAPC(Ac)-OH (2).In addition, this biocompatible synthetic product is even more versatile and tunable, because the O-Ac groups of the β-SAA can be retained or removed at the end of the synthesis.

General information
Analytical data for all compounds (HPLC chromatograms, NMR and MS spectra) can be found in Supporting information, in the online version.
Reagents, materials and solvents were purchased from Sigma-Aldrich, Merck, Reanal, VWR and Iris Biotech.Moisture-sensitive solvents were dried on molecular sieve (3 Å), while acetonitrile was distilled from CaH 2 .Solvents and reagents for MS measurements were purchased from VWR. TLC was performed on silica gel 60 F254, 230 mesh (E.Merck) and spots were detected by UV light (254 nm), charring with 5% H 2 SO 4 solution.
NMR experiments were performed at 298 K on Bruker Avance DRX 500 MHz spectrometer equipped with TXI probe with z-gradient, operating at 500.13 MHz for 1 H and 125.76 MHz for 13 C.The sample concentrations ranged from 0.1 M to 0.2 M. Spectra were recorded in DMF-d 7 using the solvent residual peak as the 1 H internal reference.Spectra evaluation was done with TopSpin 4.1.1software.
The mass spectrometry measurements were performed using a Bruker Amazon SL ion trap mass spectrometer equipped with an electrospray ion source.

Zemplén deacetylation
The purified Ac-Gly-GlcAPC(Ac)-Gly-NH 2 (20 mg) was dissolved in MeOH (2 ml) and 2 M NaOMe/MeOH (1 ml) solution was added.After 1 h at room temperature, the reaction was stopped with the addition of TFA (1 ml).The acidic solution was concentrated to get white solids (10 mg).

Fig. 1
Fig. 1 Monitoring of active ester formation using 1 H NMR spectroscopy.Both Fmoc-GlcAPC-OH (1) and Fmoc-GlcAPC(Ac)-OH (2) form the corresponding active esters, which then, retain their stability even after 24 h.a) The reaction of Fmoc-GlcAPC(Ac)-OH (2) with PyBOP/DIEA in DMF-d 7 gives the active ester 12. b) The active ester formation was monitored by time-resolved 1 H NMR spectra: chemical shift changes of selected aromatic protons were moni-