Rapid building block-economic synthesis of long, multi-O-GalNAcylated MUC5AC tandem repeat peptides

The study of mucin function requires access to highly O-glycosylated peptides with multiple tandem repeats. Solid-phase synthesis would be a suitable method, however, the central problem in the synthesis of mucin glycopeptides is the need to use precious and potentially vulnerable glycoamino acid building blocks in excess. In this article, we report the development of a method based on SPPS and native chemical ligation/desulfurization chemistry that allows the rapid, reliable, and glyco-economical synthesis of long multi-O-GalNAcylated peptides. To facilitate access to the glycosyl donor required for the preparation of Fmoc-Ser/Thr(αAc3GalNAc)-OH we used an easily scalable azidophenylselenylation of galactal instead of azidonitration. The problem of low yield when coupling glycoamino acids in small excess was solved by carrying out the reactions in 2-MeTHF instead of DMF and using DIC/Oxyma. Remarkably, quantitative coupling was achieved within 10 minutes using only 1.5 equivalents of glycoamino acid. The method does not require (microwave) heating, thus avoiding side reactions such as acetyl transfer to the N-terminal amino acid. This method also improved the difficult coupling of glycoamino acid to the hydrazine-resin and furnished peptides carrying 10 GalNAc units in high purities (>95%) of crude products. Combined with a one-pot method involving native chemical ligation at a glycoamino acid junction and superfast desulfurization, the method yielded highly pure MUC5AC glycopeptides comprising 10 octapeptide tandem repeats with 20 α-O-linked GalNAc residues within a week.


1.General Information
Commercially available compounds were used without further purification.Solvents for peptide synthesis were dried by using MBraun Solvent Purification System SPS 800.Dry solvents for glycoamino acid synthesis were obtained from Thermo Scientific (Schwerte, Germany).Molecular sieves 4Å were activated prior to reaction overnight at 250°C in vacuo.
NMR-spectra were recorded on a Bruker Avance II 300 MHz Spectrometer and referenced to the residual protonated solvent signal or/and tetramethylsilane.
High resolution ESI-MS spectra were recorded on a Waters H-class instrument equipped with a quaternary solvent manager, a Waters sample manager-FTN, a Waters PDA detector and a Waters column manager with an Acquity UPLC protein BEH C18 column (1.7 μm, 2.1 mm x 50 mm).Samples were eluted with a flow rate of 0.3 mL/min at 40 °C.The following gradient was used: A: 0.01% FA in H2O; B: 0.01% FA in MeCN.5% B: 0-0.5 min; 5 to 95% B: 0.5-3.5 min; 95% B: 3.5-4 min.Mass analysis was conducted with a Waters XEVO G2-XS QTof analyzer and the recorded data was subsequently analyzed using the provided built-in software.
Stirring was continued for an hour, then the zinc dust was filtered off and washed with CH 2 Cl 2 .The combined organic solutions were washed with sat.aq.NaHCO 3 (4x300 mL), the organic phase was dried by filtration through Na 2 SO 4 and concentrated in vacuo.The residue was purified by column chromatography [cyclohexene: EtOAc (2:3)], giving 6.77 g (47%) of glycothreonine tert-butyl ester 6t; white solid.All spectral characteristics were completely identical to the compound 6t obtained by method A.
d NMR-sample was taken to assess α/β-ratio of crude (page S10).e In case of Fmoc-Ser(Ac 3 GalN 3 )-OtBu, it is crucial to separate α-from β-anomer as they cannot be separated in the next step.The column was performed twice in this case.f NMR-sample was taken to assess α/β-ratio of crude (page S11).g The column was performed twice in this case.The resin (~0.17 mmol/g) was transferred into a syringe equipped with a filter frit and allowed to swell in anhydrous CH 2 Cl 2 (10 min).Hydrolysed trityl residues were reactivated by addition of SOCl 2 in anhydrous CH 2 Cl 2 (10% v/v) for 2x30 min.The resin was washed (5x anhydrous CH 2 Cl 2 , 5xDIPEA in anhydrous CH 2 Cl 2 (5% v/v), 5x anhydrous CH 2 Cl 2 ).

Quantifying Fmoc loading
The resin with N-terminal Fmoc group was swollen in 2 mL of piperidine:DMF solution (1:4, v/v) and placed on a shaker for 5 minutes.The liquid was drained in a 10 mL glass flask, equipped with a sept, and vacuum was applied to ensure all liquid is drained.Treatment was repeated once more.
In a glass cuvette, 990 μL of the same piperidine:DMF solution was added.From the solution containing piperidine-fulvene adduct, 10 μL was withdrawn and added to the glass cuvette.The mixture was shaken for 30 seconds.
UV-absorbance value was measured, and converted to μmol value according to the following formula: The final concentration of all compounds was 50 mM.After preactivation for 3 minutes, liquid was filtered from formed diisopropylurea crystals, and the filtrate was added to H 2 N-NH-TRT resin in a fritted syringe.At indicated time points, liquid was drained, and the resin was washed (5xDMF, 5xCH 2 Cl 2 , 5xDMF).To cap unreacted sites, resin was treated with DMF:Ac 2 O:DiPEA (70:20:10, v/v/v) mixture for 5 min.The yields of reaction were estimated by the UVabsorbance of the piperidine-fulvene adduct (λ = 301 nm, ε = 7800 M -1 cm -1 ).
Coupling: Fmoc-protected amino acids (5 eq.) were transferred to the resin, followed by DIC and Oxyma (5 eq.each).The final concentration of amino acid was 167 mM.Reaction temperature was elevated to 90°C (coupling time = 1 min).The resin was washed with 2x 2 mL of DMF, each washing was performed for 1 minute.
Fmoc removal: The resin was treated with 20% piperidine in DMF for 1 min at 90°C.The resin was washed with 3x 2 mL of DMF, each washing was performed for 1 minute.
Capping: The resin was treated with DMF:Ac 2 O:DiPEA (70:20:10, v/v/v) for 5 min at RT.The resin was washed with 2x 2 mL of DMF, each washing was performed for 1 minute.
TFA cleavage: Prior to TFA cleavage the Fmoc group was removed as described.The resin was washed with CH 2 Cl 2 and dried under vacuum.Then 5 mL of a mixture of TFA:TIS:H 2 O (96:2:2, v/v/v) was added to the resin.After 2 h the cleavage cocktail was collected by filtration, the resin was washed once with 5 mL of TFA and the combined filtrates were concentrated under air flow.
Peptide work-up: To the remaining residue cold Et 2 O (9-fold volume) was added and the suspension was centrifuged (4000 rpm, 5 min).Afterwards the ether phase was decanted.The peptide precipitate was dissolved in Milli-Q water.
5.2 Optimizing coupling of Fmoc-Thr(αAc 3 GalNAc)-OH (7t) in DMF solution (Table 4) Condition 1: Automated coupling was performed with 10 µmol of 8* by using the Biotage Initiator+ Alstra synthesizer.Fmoc-protected glycoamino acid 7t was dissolved in Oxyma/DMF solution (55 mM) and combined with DIC until a final concentration of 50 mM 7t/DIC/Oxyma was obtained.Reaction temperature was elevated to 75°C and coupling was performed for 5 min.The resin was washed (5xDMF, 5xCH 2 Cl 2 ).No capping was performed.Fmoc removal and TFA cleavage were performed as described under 5.1.
Conditions 2-14: Couplings were performed manually with 4 mg (~0.4 µmol) of 8*.The indicated amount of Fmoc-Thr(Ac 3 GalNAc)-OH, HATU, HOAt, and base were dissolved in DMF to achieve a final concentration of 50 mM glycoamino acid.The solution was added to peptide resin 8* in a fritted syringe.At indicated time points, liquid was drained, and the resin was washed (5xDMF, 5xCH 2 Cl 2 , 5xDMF).No capping was performed.Fmoc removal was performed manually by treating the resin with 20 % piperidine in DMF for 10 min.The resin was washed (5xDMF, 5xCH 2 Cl 2 ).TFA cleavage was performed as described under 5.1.The yields of reactions were estimated by integrating peaks in UPLC chromatograms.Glycopeptide 13 (2.49mg, 0.42 μmol) was dissolved in 84 μL of ligation buffer B (6M Gn-HCl, 0.2M Na 2 HPO 4 , pH = 8.50) to reach a concentration of 5 mM.TCEP hydrochloride salt (1.2 mg) was added to reach a concentration of 50 mM.This mixture was combined with the mixture of 11TE.The pH was adjusted to ~7.1 with 1M NaOH solution.After vigorous vortexing, the reaction was allowed to proceed at ambient temperature.Aliquots were withdrawn at indicated time points for UPLC analyses.After completion of reaction, the mixture was forwarded to onepot desulfurization (chapter 8).

Figure S10 .
Figure S10.UPLC analysis of thioesterification of 11 after 90 min and 150 min.At 90 minutes, starting glycopeptide 11 is consumed, but complete conversion to 11TE is not yet achieved because the peptide-pyrazolate intermediate 11Pyr is still present.After 150 minutes, formation of peptide thioester 11TE is completed.
The stirred solution of tri-O-acetyl-galactal 1 (25 g, 91.91 mmol) and Se 2 Ph 2 (17.21 g, 55.15 mmol) in anhydrous CH 2 Cl 2 (384 mL) was cooled to -30 o C, then TMSN 3 (24.2mL,183.82 mmol) and PhI(OAc) (32.56 g, 101.1 mmol) were added sequentially.After PhI(OAc) 2 was dissolved, the temperature was maintained at -20 o C for 16 hours.The mixture was diluted with CH 2 Cl 2 and washed with sat.aq.NaHCO 3 (2x400 mL).The organic phase was dried by filtration through Na 2 SO 4 and concentrated in vacuo.Crystallization from iPrOH, filtration and washing with petroleum ether gave 23.77 g (55%) of selenogalactoside 2 as a white solid. 1 H NMR data are in accord with the literature.1 2.1.Synthesis of galactosyl donors 2 and 5 of the obtained compound 4s (2.37 g) in a mixture of THF, AcOH and Ac 2 O (3:2:1, v/v, 30 mL) activated zinc dust3(2.45g, 37.44 mmol) was added.Stirring was continued for an hour, then the zinc dust was filtered off and washed with CH 2 Cl 2 .The combined organic solutions were washed with sat.aq.NaHCO 3 (3x250 mL), the organic phase was dried by filtration through Na 2 SO 4 and concentrated in vacuo.