Preparation and Application of an Inexpensive α-Formylglycine Building Block Compatible with Fmoc Solid-Phase Peptide Synthesis

α-Formylglycine (fGly) is a rare residue located in the active site of sulfatases and serves as a precursor to pharmaceutically relevant motifs. The installation of fGly motifs into peptides is currently challenging due to degradation under the acidic and nucleophile-rich conditions accompanying resin cleavage during solid-phase peptide synthesis. We report the synthesis of acid- and nucleophile-tolerant α-formylglycine building blocks from vitamin C and use them to prepare callyaerin A, a macrocyclic peptide containing an fGly-derived motif.


General considerations
Anhydrous solvents were dried over a PureSolv MD 7 Solvent Purification System. Anhydrous solvents were used in reactions unless otherwise stated, or where aqueous/organic cosolvent mixtures were employed. GPR-grade solvents were used for flash chromatography purposes. Solution-phase synthetic reactions were carried out using oven-dried glassware. All concentrations were performed in vacuo unless otherwise stated. Thin layer chromatography was carried out on Merck silica gel 60 F254 precoated aluminium foil sheets and these were visualized using UV light (254 nm) and/or PPh3 (10% in DCM) and/or ninhydrin (1.5% ninhydrin, 3% AcOH in n-butanol), and/or H2SO4 (5% H2SO4 in MeOH). Unless otherwise indicated, flash column chromatography was performed on Supelco® silica gel (particle size 35-75 µm, pore diameter 60 Å, 220-440 mesh) and the solvent system used is recorded in parentheses.
Reagents (including peptide coupling reagents) were purchased from Sigma-Aldrich and used as supplied, unless otherwise indicated. Fmoc-protected amino acids were purchased from Fluorochem.
Optical rotations were measured using a Bellingham and Stanley ADP 450 Automatic Digital Peltier Controlled Polarimeter equipped with a 589 nm LED and a path length of 1 dm. [α]D 20 values reported in units of 10 -1 deg cm 2 g -1 .
Small-molecule high resolution mass spectrometry (HRMS) data were obtained at RT on a Bruker Daltonics microTOF mass spectrometer coupled to an Agilent 1200 series LC system at The University York Centre of Excellence in Mass Spectrometry (CoEMS). Nominal and exact m/z values are reported in Daltons.
High Performance Liquid Chromatography-Electrospray Ionization Mass Spectrometry (LC-MS) of peptides was performed using a Dionex UltiMate® 3000 Ci Rapid Separation LC system equipped with an UltiMate® 3000 photodiode array detector probing at 210-400 nm, coupled to a HCT ultra ETD II (Bruker Daltonics) ion trap spectrometer, using an Accucore C18 column (150 × 2.1 mm, 2.6 µm particle size. The LC-MS apparatus was controlled using Chromeleon® 6.80 SR12 software (ThermoScientific), esquireControl version 6.2, Build 62.24 software (Bruker Daltonics), and Bruker compass HyStar 3.2-SR2, HyStar version 3.2, Build 44 software (Bruker Daltonics) at CoEMS. Water (solvent A) and acetonitrile (solvent B), both containing 0.1% formic acid, were used as the mobile phase at a flow rate of 0.3 mL min -1 . LC traces were measured via UV absorption between 210-400 nm. Two LC gradients were used. Gradient A and Gradient B. These gradients were programmed as shown below:  Synthesis and characterisation of small molecule probes 5,6-O-isopropylidene-L-ascorbic acid 1 1 was synthesised using an adapted literature procedure. [1] To a solution of L-ascorbic acid (20 g, 114 mmol) in acetone (80 mL) was added a catalytic amount of acetyl chloride (2.1 mL, 30 mmol, 0.26 equiv) at rt. After stirring for 3.5 h at rt, the mixture was placed in the refrigerator overnight. The solid was then filtered off and washed with cold acetone to yield 1 as a white solid (26.5 g, 85%).

Potassium-3,4-O-isopropylidene-L-threonate 2
To a stirred solution of potassium carbonate (59.2 g, 428 mmol) in water (227 mL) was added 1 (39.7 g, 184 mmol). The resultant solution was cooled to 0 °C and 30% aqueous H2O2 was added dropwise (47.9 mL, 469 mmol), taking care to make sure that the temperature of the reaction solution was kept below 10 °C. The resulting solution was then stirred at rt for 24 h, after which time it was concentrated in vacuo to yield a white solid. To the white solid was added EtOH (500 mL), and the resulting suspension refluxed with stirring for 30 minutes. Solids were removed by filtration while the EtOH remained hot using a sintered funnel and the eluate collected. The removed solids were again refluxed in EtOH (500 mL) for 30 minutes, removed via filtration and the eluate collected a further four times. The eluates were then combined and concentrated in vacuo to yield potassium-3,4-O-isopropylidene-L-threonate 2 as a white solid in quantitative yield (39.4 g).

(Methyl (S)-2-azido-2-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)acetate 4 (via 3′)
3 (26.2 g, 137 mmol) was dissolved in anhydrous DCM (250 mL) and pyridine (26 mL, 321 mmol) under N2 and then cooled to -15 °C. Trifluoromethanesulfonic anhydride (50 g, 177 mmol) was then added dropwise over 30 minutes at -15 °C with vigorous stirring. The reaction solution was then allowed to warm to rt and stirred for a further 1 h. The reaction mixture was then transferred to a separating funnel, washed with brine (125 mL), saturated NaHCO3 (125 mL), 1 M HCl (125 mL), saturated NaHCO3 (125 mL) and brine (125 mL). The organic layer was then dried over MgSO4 and concentrated in vacuo to yield a brown oil. The oil was dissolved in ethyl acetate and hexane was then added until a brown tar separated. The supernatant was then decanted, leaving behind the tar. Concentration of the supernatant via slow evaporation under a stream of nitrogen gas yielded triflate 3′ as off-white needle like crystals which were isolated via filtration and dried in vacuo (27.4 g, 82.2 mmol, 60%). 3′ (27.4 g, 82.2 mmol) and sodium azide (8.30 g, 128 mmol) were then placed in a round bottom flask and 175 mL of a 3:1 v/v mixture of acetone to water was added. The resultant solution was stirred vigorously overnight at rt in the dark, during which time the solution turned a dark brown colour. The reaction solution was then transferred to a separating funnel and ethyl acetate (250 mL) and water (250 mL) added. The organic extraction was subsequently washed with water (125 mL) and brine (125 mL). The organic layer was then dried over MgSO4 and concentrated in vacuo to yield a dark orange oil. This oil was purified via flash column chromatography (hexane → 80% EtOAc in hexane) to yield 4 as a colourless oil (17.0 g, 78.8 mmol, 96% from 3', 58% from 3). Fractions containing 4 were identified by treating TLC plates with PPh3 stain followed by ninhydrin stain.
Method 1: To 4 (6.00 g, 27.9 mmol) dissolved in MeOH (100 mL) was added a heaped spatula of 10% palladium on activated carbon. The system was then placed under 1 atm of H2. The resultant mixture was stirred at rt until azide reduction was complete by TLC (approx. 2 h). Azides and amines were visualised on the TLC plates using PPh3 and ninhydrin stains. The Pd/C was then removed via filtration and 9-fluorenylmethyl N-succinimidyl carbonate (11.3 g, 33.5 mmol) was added to the eluate to yield a suspension. DCM (100 mL) was then added to aid solvation. The reaction solution was then stirred overnight at rt, concentrated in vacuo, resuspended in EtOAc (200 mL) and washed with water (100 mL) and brine (100 mL). The organic extract was then dried over MgSO4 and concentrated in vacuo to yield a yellow oil that was purified via flash column chromatography (hexane → EtOAc) to yield 5 (4.43 g, 10.8 mmol, 39%) as an off-colourless oil that solidified into an off-white solid.

Method 2:
To 4 (1.00 g, 4.64 mmol) dissolved in THF (47.5 mL) was added water (2.5 mL). PPh3 (2.44 g, 9.28 mmol) was then added and the resultant solution stirred for 2 h at rt. 9-fluorenylmethyl Nsuccinimidyl carbonate (1.9 g, 5.57 mmol), NaHCO3 (0.585 g, 6.96 mmol) and water (2.5 mL) were then added and the reaction solution was stirred overnight at rt. After this time the solution was concentrated in vacuo, resuspended in EtOAc (200 mL) and washed with water (100 mL) and brine (100 mL). The organic extract was then dried over MgSO4 and concentrated in vacuo to yield a crude residue. This residue was dissolved in the minimum volume of hot EtOAc and was then cooled using an ice-bath, prompting the precipitation of PPh3O. The PPh3O was removed via filtration and the eluate was purified via flash column chromatography (hexane → EtOAc) to yield 5 (0.998 g, 2.43 mmol, 52%) as an off-colourless oil that solidified into an off-white solid.

(2S)-[(4R)-2,2-dimethyl-1,3-dioxolan-4-yl]({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)acetic acid 6
CaCl2 (25.0 g, 226 mmol) was dissolved with stirring in a 7:3 v/v 2-propanol:water mixture (290 mL). 13.2 mL of 1 M NaOH was then added and the resultant cloudy mixture was cooled to 0 °C. 5 (3.64 g, 8.84 mmol) was dissolved in the minimum volume of DCM and the CaCl2/NaOH solution was then added in one portion. The reaction solution was then stirred rt until TLC showed the reaction to have reached completion (approx. 4 hours). The reaction solution was then transferred into a separating funnel and DCM (250 mL) and brine (250 mL) were added. The aqueous layer was acidified to pH 5 via the dropwise addition of 1 M HCl and the biphasic system shaken to extract the product into the organic layer. The aqueous layer was then further extracted with DCM (2 × 100 mL). All organic extracts were then combined and dried over MgSO4 before being concentrated in vacuo. This yielded a pale green oil which was purified via flash column chromatography (25% EtOAc in hexane + 0.5% AcOH → EtOAc + 0.5%) to yield 6 (2.95 g, 7.43 mmol, 84%) as a colourless oil that solidified into a white solid.  Test peptide 7 7 was synthesised using Fmoc solid-phase peptide synthesis (SPPS). H-Gly-2-ClTrt resin (40 mg, 0.79 mmol/g, 0.032 mmol, styrene+1% divinylbenzene copolymer matrix, 200-400 mesh, Novabiochem) was weighed out into an SPPS cartridge fitted with a PTFE stopcock, swollen in DMF for 30 minutes and then filtered. For each amino acid coupling during the SPPS of 7 the following method was used: DIPEA (58 µL, 0.35 mmols, 11 eq) was added to a solution of Fmoc-protected amino acid (0.16 mmols, 5 eq) and HCTU (64 mg, 0.16 mmols, 5 eq) dissolved in the minimum volume of DMF. The resultant solution was then immediately added to the resin. The reaction mixture was gently agitated by rotation for 1 h and the resin was filtered off and washed with DMF (3 x 2 minutes with rotation). A solution of 20 % piperidine in DMF was added to the resin and the mixture gently agitated by rotation for 2 minutes. The resin was filtered off and this piperidine treatment process was repeated a further four more times. The resin (now bearing deprotected amine functionality) was then washed using DMF (5 x 2 minutes with rotation) prior to coupling on the next amino acid.
After coupling on the final glycine residue and washing with DMF (5 x 2 minutes with rotation), the resin was washed with DCM (3 x 2 minutes with rotation) and MeOH (3 x 2 minutes with rotation). The resin was dried on a vacuum manifold and further dried on a high vacuum line overnight. 5 mL of a cleavage cocktail solution (95:2.5:2.5 TFA:H2O:triisopropylsilane) was then added to the resin and the mixture gently agitated by rotation for 1 h. The reaction mixture was drained into cold Et2O (incubated at -20 °C prior to use) and centrifuged at 4000 rpm at 4 °C until pelleted (ca 5-10 minutes). The supernatant was then carefully decanted. The pellet was subsequently resuspended in cold Et2O and the centrifugation and supernatant decantation process repeated a further three more times. The precipitated peptide pellet was then dissolved in 20% AcOH (aq) and lyophilised to obtain 7 as a fluffy solid (23 mg, 95%) for use without further purification.    Figure 1 of the main paper). The UV trace was recorded at 210-400 nm. The peak at ca. 10 s corresponds to injection.

Test peptide 8
To a solution of 7 (865 µL, 2.0 mM, 1.3 mg, in HPLC-grade water) was added a solution of NaIO4 (42 µL, 50 mM in HPLC-grade water). The reaction was mixed thoroughly and allowed to sit for 20 minutes at rt, after which time LC-MS showed the complete conversion of 7 into 8. The solution was then loaded onto a solid phase extraction cartridge (Grace Davison Extract Clean, 8 mL reservoir, Fisher Scientific) equilibrated with water/acetonitrile. After initial washing with water, the product was eluted over a gradient of acetonitrile. The product was then diluted with water, and subsequently lyophilised to give 8 as a white fluffy solid.

Linear Callyaerin A precursor 9
Peptide 9 was synthesised via Fmoc SPPS using a CEM Liberty Lite Automated Microwave Peptide Synthesiser, according to the manufacturers standard protocols. Briefly, Fmoc-protected amino acids (5.5 equiv., 0.2 m in DMF) including fGly building block 6 were coupled in the presence of N,N'-diisopropylcarbodiimide (DIC, 15 equiv.) and Oxyma Pure (5 equiv.) under microwave irradiation at a temperature of 90 °C for 2 minutes. Fmoc deprotection was performed using 20% piperidine in DMF at 90 °C for 60 seconds. The synthesis was performed on a 0.1 mmol scale using Rink Amide MBHA resin (C-terminal amide, 0.5 mmol/g, styrene+1% divinylbenzene copolymer matrix, 100-200 mesh, Fluorochem). Prior to cleavage, the resin was washed sequentially washed with DMF (5 x 2 minutes with rotation), DCM (3 x 2 minutes with rotation) and MeOH (3 x 2 minutes with rotation). The resin was then dried on a high vacuum line overnight. 5 mL of a cleavage cocktail solution (95:2.5:2.5 TFA:H2O:triisopropylsilane) was then added to the resin and the mixture gently agitated by rotation for 1 h. The reaction mixture was drained into cold Et2O (incubated at -20 °C prior to use) and centrifuged at 4000 rpm at 4 °C until pelleted (ca 5-10 minutes). The supernatant was then carefully decanted. The pellet was subsequently resuspended in cold Et2O and the centrifugation and supernatant decantation process repeated a further three more times. The precipitated peptide pellet was then dissolved in 20% AcOH (aq) and lyophilised to obtain 9 as a fluffy off-white solid (78 mg, 55%) for use without further purification.

Callyaerin A 10
Test scale: To a solution of 9 (350 µL, 2.0 mM, 1.0 mg, in HPLC-grade water) was added a solution of NaIO4 (17 µL, 50 mM in HPLC-grade water). The reaction was mixed thoroughly and allowed to sit for 20 minutes at rt, after which time LC-MS showed the complete conversion of 9 into 9' and 10. The solution was then loaded onto a solid phase extraction cartridge (Grace Davison Extract Clean, 8 mL reservoir, Fisher Scientific) equilibrated with water/acetonitrile. After initial washing with water, the product was eluted over a gradient of acetonitrile. Fractions containing 10 were identified via LC-MS, diluted with water and lyophilised to give 10 as a white fluffy solid.
Prep scale: To a solution of 9 (4.6 mL, 2.1 mM, 13 mg, in HPLC-grade water) at 0 °C was added a solution of NaIO4 (221 µL, 50 mM in HPLC-grade water) in 5 instalments. The reaction was mixed thoroughly and then incubated for 20 minutes at rt. NaCl (780 mg) was added to reaction solution and the mixture gently agitated until the NaCl dissolved and peptide was observed to have precipitated. MeCN (5 mL) was then added and the resultant biphasic mixture shaken and cooled to -20 °C. The organic layer was separated from the aqueous layer and set aside. A further 4 extractions of the aqueous layer using MeCN were performed in this manner. The organic extractions were then combined, dried over MgSO4 and concentrated in vacuo to yield a crude material containing both uncyclized and cyclized Callyaerin A (9′ and 10). This crude material was re-dissolved in MeCN (50 mL) and MgSO4 (one spatula) and formic acid (50 µL) were then added. The resultant solution was stirred for 1.5 hours at rt, after which time the MgSO4 was removed via filtration and a crude sample of 10 (13 mg) was recovered by concentrating the eluate in vacuo (the addition of toluene to the eluate was used to assist in the removal of residual formic acid during concentration in vacuo).
The crude sample of 10 was further purified via reverse-phase flash chromatography on a Teledyne CombiFlash® NEXTGEN 300+ system using a C18 column (RediSep® Rf Gold C18 Reversed Phase column, 5.5 gram media) that was pre-equilibrated with water. 10 was purified using a linear gradient from water → MeCN over 10 minutes at a flow rate of 13 mL min -1 , holding at 100% MeCN (13 mL min -1 ) for a further 6 minutes after the linear gradient before re-equilibrating the column back into water (see Figure S43). Fractions containing 10 were identified using LC-MS and were then pooled and lyophilised to give 10 (5.5 mg, 42%) as a white fluffy powder.   Figure S46 and Table S1.        Use of an hThr motif as an fGly precursor can circumvent complications associated with the acid catalysed fragmentation of/nucleophilic attack on fGly motif during SPPS resin cleavage, as illustrated here for the acetal protected fGly motif incorporated using the previously reported acetal protected fGly building block. [4] Figure S 52. The oxidation of serine with RuO4 is known to lead to oxidative scission and retroaminal fragmentation pathways. [5] By contrast, during mass spectrometry analysis of the oxidation of 7 to 8 using NaIO4, fragmentation products attributable to oxidative scission/retroaminal fragmentation were not observed, suggesting that the oxidation of the 4hydroxythreonine (hThr) motif to fGly using NaIO4 is mild and selective.

Figure S 53. A)
The cyclisation of 9′ to 10. B) LC-MS chromatogram of a mixture of 9′ and 10 freshly prepared via the treatment of 9 with NaIO4. C) Mass spectrum of mixture of 9′ and 10 freshly prepared via the treatment of 9 with NaIO4. D) LC-MS chromatogram of a sample of 10 prepared via the incubation of a 9′/10 mixture in MeCN + 0.1% formic acid for 1.5 hours. E) Mass spectrum of a crude sample of 10 prepared via the incubation of a 9′/10 mixture in MeCN + 0.1% formic acid for 1.5 hours. Note that the peak at m/z 926.57 corresponds to a b2 fragment ion of 10. The impurity from SPPS is present in 9 and has been carried through into the samples of 9′/10 presented here. While this species ionises efficiently, the UV traces indicate that it is an abundant species in the above samples. This impurity is unaffected by the treatment with NaIO4. LC-MS chromatograms show the BPC+All MS trace and the UV chromatogram shows the absorbance for wavelengths 210-400 nm. LC-MS was conducted using LC gradient B (see Figure S2).