HF‐Free Boc Synthesis of Peptide Thioesters for Ligation and Cyclization

Abstract We have developed a convenient method for the direct synthesis of peptide thioesters, versatile intermediates for peptide ligation and cyclic peptide synthesis. The technology uses a modified Boc SPPS strategy that avoids the use of anhydrous HF. Boc in situ neutralization protocols are used in combination with Merrifield hydroxymethyl resin and TFA/TMSBr cleavage. Avoiding HF extends the scope of Boc SPPS to post‐translational modifications that are compatible with the milder cleavage conditions, demonstrated here with the synthesis of the phosphorylated protein CHK2. Peptide thioesters give easy, direct, access to cyclic peptides, illustrated by the synthesis of cyclorasin, a KRAS inhibitor.


Preparation of the mercaptopropionic acid leucine (MPAL) resin:
Resin loading was adapted from Hackeng et al. [S2] and performed on small batches typically: hydroxymethyl resin (0.250 g, 0.316 mmol, Merck Chemicals) was swollen in dry CH 2 Cl 2 for at least 2 h and derivatised as follows. DIC (0.2 g, 1.58 mmol, 5 eq.) in CH 2 Cl 2 (2 mL) was added to a suspension of Boc-Leu-OH (0.80 g, 3.15 mmol, 10 eq.) in CH 2 Cl 2 (2 mL) over 10 min at 0 °C followed by addition of DMAP (0.038 g, 0.315 mmol, 1 eq.). The resulting suspension was warmed to room temperature and stirred for 45 min to allow complete formation of the symmetric anhydride which was then added to the swollen hydroxymethyl Merrifield resin. The resin was left overnight and thoroughly washed with CH 2 Cl 2 and DMF. The Boc-group was removed using neat TFA (2 x 1 min) and washed with DMF. DIEA was added until fuming ceased to neutralize any remaining TFA. 3-Tritylmercaptopropionic acid (0.44 g, 1.26 mmol, 4 eq.) was activated with HCTU (0.52 g, 1.26 mmol, 4 eq.) and DIEA (0.12 g, 0.95 mmol, 3 eq.) for 3 min and added to the resin. For peptide elongation of the MPAL resin, the trityl group was removed with treatment with a TFA mixture (2.5 % TES, 2.5 % H 2 O in TFA, 3 x 2 min until all yellow color had disappeared). The resulting MPAL resin was used for polypeptide chain assembly by the implementation of in-situ neutralization SPPS protocols.

Preparation of the trityl-mercaptophenylacetic acid (Trt-MPAA):
The title compound was adapted from Dang et al. [S3] briefly, trityl chloride (1.0 g, 3.59 mmol) was added to a suspension of 4-mercaptophenylacetic acid (0.559 g, 3.32 mmol) in CH 2 Cl 2 (10 mL), stirred for 3 h, and the reaction monitored by TLC to completion. The reaction was quenched with the addition of H 2 O (10 mL) and extracted with EtOAc (1 x 40 mL). The organic phase was washed with brine (1 x 30 mL), dried (Na 2 SO 4 ) and evaporated. Silica Chromatography (

In-situ neutralisation SPPS protocols:
Boc-SPPS in situ neutralisation protocols were adapted from Schnolzer et al. [S5] The Boc-amino acid (1.26 mmol, 4 eq.) and HCTU (1.197 mmol, 3.8 eq.) were dissolved in DMF (2.5 mL). DIEA (1.89 mmol, 6 eq.) was added, the coupling mixture was shaken for 3 min and added to a reaction vessel containing the MPAL resin. The reaction was left for 30 min followed by a DMF flow-wash (2 x 30 s).
Coupling of the remaining amino acids was accomplished as follows: After removal of the Boc-group using neat TFA (2 x 1 min), the resin was flow-washed vigorously with DMF (3 x 10 mL) for no more than 30 s under concurrent draining via vacuum. The next amino acid was then activated as previously described and added to the resin to couple for 30 min.

TFA/TMSBr Cleavage:
After coupling of the last amino acid, the resin was washed with DMF and CH 2 Cl 2 and dried in vacuo. Cleavage from the resin and total deprotection was accomplished with TFA/TMSBr/Thioanisole/EDT (1/0.05/0.05/0.025) (typically 5 mL per 0.1 g of peptide-resin). The TMSBr bottle was pressurised with Argon, and TMSBr was transferred via syringe and canula to the reagent vessel. The volatiles were sparged under a stream of N 2 , the peptide was precipitated from Et 2 O (Na-dried, 4 °C). HPLC Purification of the crude peptide was performed on a C18 column (see General Methods) the fractions were collected and lyophilized to give the purified peptide as a white powder.

II: TFA stability tests
Fmoc-Gly-resin and Fmoc-Gly-MPAL-resin were prepared as previously described, using PBr 3 for the method of attachment to the solid support. Upon completion of the synthesis, the resin was washed with DMF (2 x 30 s continuous flow), CH 2 Cl 2 (2 x 30 s. continuous flow), and dried overnight in vacuo. The dried resins (6.52 mg for Fmoc-Gly-resin and 6.97 mg for Fmoc-Gly-MPAL-resin) were treated with TFA (10 mL) for 8 h. Time points were taken at 10 min, 30 min, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, and the absorbance measured at 301 nm with a Jasco UV-Vis Spectrometer employing glass suprasil cuvettes with a path length of 10 mm. The data was fitted with a non-linear curve using Graph Pad Prism 6.0 to determine half-life stabilities of the measured resins.

Non-thioester Syntheses:
H-PGVGPGVGV-OH (Elastin) (1). The peptide was synthesized on a Merrifield hydroxymethyl resin (0.2 g, 0.2 mmol) using standard in situ neutralisation protocols for Boc SPPS. 0.382 g of resin was obtained upon completion of the synthesis (98 % yield based on expected dry weight). After TFA/TMSBr cleavage and precipitation from Et 2 O (Na-dried, 4 °C) from a portion of 170 mg, the title compound was obtained as a white powder (

IV: Peptide Cyclisations
Cyclic CdYVYNTRSGWRWYT (23). Peptide 18 (4 mg, 1.7 mmol; 1 mM) was dissolved in phosphate buffer (1.7 mL, 200 mM NaH 2 PO 4 , 2 mM EDTA, 6 M Guanidine HCl) with TCEP (0.015 g.mL -1 , 50 mM final concentration) and MPAA (0.5 mg.mL -1 , 3 mM final concentration). The reaction was allowed to incubate at 40 ˚C and monitored by analytical HPLC by taking aliquots (2 µL) of the reaction mixture and diluting with buffer A (8 µL). The samples were injected on an RP-C18 column using a linear gradient of 20-50 % B in 30 min with a flow rate of 1 mL min -1 . After 15 min, the cyclisation was complete by analytical HPLC and diluted with Buffer A. Semi-preparative HPLC (20-50 % B in 30 min with a flow rate of 15 mL min -1 ) yielded the title compound as a white powder (2.64 mg, 75 %). Cyclorasin 9A5 (24). The cyclisation of peptide 17 was performed as previously described by Houghten [S6] without purification of the crude starting material. Crude peptide 17 (0.042 g, 0.028 mmol) was dissolved in a mixture of CH 3 CN:H 2 O (7:1) imidazole (12 mL, final concentration of imidazole 1 M). The reaction was allowed to incubate at 40 ˚C for 2 h and followed by analytical HPLC by taking aliquots (2 µL) of the reaction mixture and diluting with buffer A (8 µL). The samples were injected on an RP-C18 column using a linear gradient of 25-55 % B in 30 min with a flow rate of 1 mL min -1 . After 2 h, the cyclisation was determined complete, the reaction mixture was diluted with Buffer A and lyophilized to dryness. The resulting powder was redissolved in Buffer A and purified on a semi-preparative HPLC column (25-55 % B in 30 min) to give the title compound 24 as a white powder (7.4 mg, 13 % overall yield). Figure S23. Cyclorasin 9A5 cylisation. Above: Time course of cyclisation of the linear H-dAla-Arg-Arg-Arg-dNal-Arg-Phe(4-F)-dNle-Gln-Trp-Thr-MPAA to Cyclorasin 9A5 24. a) hydrolysis of the peptide thioester b) non head-to-tail cyclised product c) MPAA d,e,f) non head-to-tail cyclised product g) starting material h) correctly cyclised product i) artifact from the column. Below: HPLC of pure Cyclorasin 9A5 24: R t 18.1 min (20-50 % B over 30 min, λ = 214 nm); MALDI-TOF of the main peak: m/z = 1586.6 [M+H] + (First isotope), calc.: 1586.9. Figure S24. Microscale thermophoresis data showing the binding affinity of the Cyclorasin 9A5 with wt-K-Ras_GTPγS (A), wt-K-Ras_GDP (C), K-Ras-G12V_ GTPγS (B) and K-Ras-G12V_ GDP (D). The Kd values for each interaction were calculated as an average of three measurements.