Development of fructose-1,6-bisphosphate aldolase enzyme peptide mimics as biocatalysts in direct asymmetric aldol reactions

This study describes the design and synthesis of mimetic peptides modelled on the catalytic active site of the fructose-1,6-bisphosphate aldolase (FBPA) enzyme. The synthesized peptides consisting of the turn motifs and catalytic site amino acids of FBPA enzyme were evaluated for catalytic activity in direct asymmetric aldol reactions of ketones and aldehydes. The influence of substrate scope, catalyst loading and solvents including water, on the reaction were also investigated. Nuclear magnetic resonance (NMR) and circular dichroism (CD) were used to determine the secondary structure of the peptides to provide an understanding of the structure–activity relationship. The peptides showed catalytic activity and the aldol products were obtained in low yields (up to 44%), but excellent enantioselectivity (up to 93%) and moderate diastereoselectivity (65 : 35).


General information and materials
All solvents and reagents were obtained from commercial sources. Solvents for column chromatography (ethyl acetate and hexanes) were purchased from Protea Chemicals (South Africa) and distilled before use to remove non-volatile components. Aldol reactions were monitored using thin layer chromatography (TLC) plates (0.2 mm silica gel 60 with fluorescent indicator UV254) were obtained from Sigma-Aldrich and further visualization was done by staining with potassium permanganate (KMnO4) solution followed by heating. Purification of aldol products was conducted on Merk normal silica gel (particle size 0.063-0.200 mm) and flash silica gel (particle size 0.040-0.063). 1 H NMR and 13 C NMR spectra were recorded on either a Bruker AVANCE 300 MHz, Bruker AVANCE 400 MHz or on a Bruker AVANCE III 500 MHz spectrometer. 2D NMR spectra which include COSY, 13C-HSQC, TOCSY, NOESY and ROESY experiments, were recorded at 293, 300, and 308 K on a 500 MHz NMR Bruker III 500 MHz spectrometer. All 2D spectra were recorded at the phase sensitive mode using time proportional phase increment (TPPI). The residual water peak of DMSO-d6 was suppressed by a presaturation pulse of 2 s duration. The first NOESY experiments were recorded with mixing time of 150, 200 and 250 ms; ROESY spectrum were recorded with mixing times of 100, 150, 200, 250 and 300 ms; while TOCSY was recorded with mixing times of 48, 64, 70, 80 and 100 ms; each increment was the sum of 32 scans with a relaxation delay of 2.0 s; 2048 data point were collected per experiment.
The enantiomeric excess (ee) was determined by chiral high performance liquid chromatography (HPLC) analysis on a Dionex HPLC Ultimate 3000 instrument (CHROMELEON version 6.80 software); coupled to a pump and photodiode array detector. A Lux 5µ cellulose-2 column was used for the analysis with hexane and isopropyl alcohol (IPA) as the mobile phase. CD spectra were recorded on a JASCO J-18 spectropolarimeter between the range of 190 to 250 nm in the specified solvents (water and phosphate buffer), with 10 scans at 20°C. Analytical LC-MS analysis was carried out on an Ultra High Performance Liquid Chromatography (Thermo Scientific Ultimate 3000, RS diode array detectors)-High Resolution Mass Spectrometer (Bruker Compact quadruple time of-flight) coupled to Diode Array (215 and 254 nm).

Synthesis of peptides catalysts.
Peptide were synthesized on an automated Protein Technologies, Inc PS-3TM peptide synthesizer and manually following the general Fmoc solid phase synthesis.

2.1.1-Swelling, activation and coupling of the first amino acid
The Fmoc-rink amide resin, 600 mg, (0, 160 mmol/g) was swelled in DMF (10.0 mL) for 20 minutes in a 70 mL glass reaction vessel with fritted filters. The DMF was removed by suction and a 20% piperidine solution in DMF (5 mL) was added and mixed by bubbling for 5 minutes with inert nitrogen gas (N2). The piperidine solution was then filtered out and the resin was washed with DMF (5×5 mL) for 30 sec second per each wash. The first amino acid cysteine (1.20 mmol, 0.2 M), and coupling reagent HBTU (1.14 mmol, 0.19 M) were dissolved in a solution of 1.0 M DIPEA in DMF (6 mL) and added into the reaction vessel. An additional 6 mL of DMF was also added. The reaction mixture was allowed to react for 45 minutes while a gentle flow of N 2 was bubbled into the reaction. For double coupling, the resin was washed with (3×5 mL) DMF, and the coupling was repeated using the same mixture for 60 minutes. The resin was then washed with (3×5 mL) DMF, and (2×5mL) DCM.
After the first coupling, Fmoc protecting group was removed using (2×10 mL) 20% piperidine solution and the resin was washed with DMF (5×5 mL) before the second amino acid was coupled. A solution containing 6 mL of 1.0 M DIPEA, (1.14 mmol, 0.19 M) HBTU, and (1.02 mmol, 0.2 M) arginine was added to the resin. The reaction mixture was mixed gently by bubbling nitrogen gas for 45 minutes. The remaining amino acids in the sequence were also coupled using the same procedure until the full peptide was synthesized.

General procedure for cleaving peptides from the resin
The resin bound peptide was washed with (3 ×5 mL) DMF and then (3×5 mL) DCM and dried by suction. A cleavage cocktail (10 mL) containing 94% TFA: 2.5%EDT:2.5%H 2 O:1%TIS was then reacted with the resin bound peptide for 3 hrs. The cleavage solution was filtered off, and the resin was washed with 5 mL of TFA and the filtrated was divided into portions in and poured into 50 ml centrifuged tubes. Cold diethyl ether was added to the filtered solution upon which a white precipitate of the crude peptide was formed. The two samples were centrifuged at 5000 rpm for 10 minutes. The step was repeated 3 times with cold diethyl ether. The obtained white precipitate was then dissolved in 10 mL (60/40; H 2 O/MeCN) for further analysis and purification.

Purification of the peptides
Peptides were purified via an Agilent 1260 Infinity semi-preparative HPLC system with a UV/VIS detector and an automated fraction collector on a Kinetix® 5 µm B-C18, 100 Å (250 ×230 nm) column. A two-buffer system was employed; Buffer A consisted of 0.1% formic acid in H 2 O and buffer B consisted of 0.1% formic acid in CH 3 CN. A flow rate of 20 ml/min or 15 ml/min and UV wavelengths of 215 and 254 nm were utilized A peptide catalyst (0.0064 mmol, 4 mol%) was added to a vial containing 0.75 mL of 3:1 acetone/water and the mixture was stirred for 15 min. An acceptor aldehyde (0.157 mmol, 24 mg) was then added, and the resulting reaction mixture was stirred vigorously at room temperature for 24-72 hours. The reaction was stopped upon completion as indicated by TLC and acetone was evaporated under reduced pressure. The crude product was extracted using (3 ×10 mL) ethyl acetate (EtOAc) and 2 mL water. The combined organic phases were dried over Na 2 SO 4 and concentrated under reduced pressure. The crude product was then purified by flash silica gel column chromatography using ethyl acetate/hexane (1:3). The pure product was then subjected to chiral-phase HPLC analysis to determine the ee.

Aldol reaction between cyclohexanone and aromatic aldehydes (homogenous)
A peptide catalyst (0.0064 mmol, 4 mol%) was added to the ketone (0.45 mL) in water (40µL) and stirred for 15 minutes. An acceptor aldehyde was then added, and the resulting reaction mixture was left to stir for 24-72 hours at room temperature. The reaction was stopped upon completion as indicated by TLC. The reaction was quenched by extraction with (3×10 mL) EtOAc and brine solution (2 mL). The combined organic extract was washed with brine and dried with Na 2 SO 4 , filtered and concentrated in vacuo. Diastereomeric ratio of the crude product was determined by 1H NMR analysis. The crude aldol product was purified by flash silica-gel column chromatography using EtOAc/Hexane (1:3) and the desired aldol product was subjected to chiral-phase HPLC analysis to determine the ee.

Aldol reaction between cyclohexanone and aromatic aldehydes (heterogenous)
A peptide catalyst (0.0064 mmol, 4 mol%) was added to a solution of a ketone (0.450 mL) and 60 µL of water and stirred for 15 minutes. An acceptor aldehyde (0.157 mmol, 24 mg) was then added to reaction mixture which was stirred for 24-72 hours. The reaction was monitored by TLC and the reaction was quenched by extraction with (3×10 mL) EtOAc and (1×2 mL) brine solution. The combined organic layers were dried with Na2SO4, filtered and concentrated in vacuo. The crude product was purified by flash chromatography on silica gel using EtOAc: hexane (1:3). (1×2.0 mL). The combined organic layers were dried over Na 2 SO 4 and concentrated in vacuo. Diastereomeric ratio of the crude product was determined by 1 H NMR analysis. The crude product was purified by flash chromatography on silica-gel with hexane/ethyl acetate (3:1). The pure products were then analyzed by the chiral HPLC and the ee values were determined

Catalyst recyclability for peptide catalyzed aldol reaction
The filtered aqueous layers obtained after extraction of the crude product, were combined and washed with Et 2 O (3 mL) and C 2 H 5 O (4 mL). The peptide was catalyst obtained was after drying and decantation of the resuspended mixture. The recycled peptide was added to a vial containing a 0.75 mL solvent mixture (0.5173 mL solvent and 0.237 mL water) and