Scalable synthesis and coupling of quaternary α-arylated amino acids: α-aryl substituents are tolerated in α-helical peptides†

Quaternary amino acids are important tools for the modification and stabilisation of peptide secondary structures. Here we describe a practical and scalable synthesis applicable to quaternary alpha-arylated amino acids (Q4As), and the development of solid-phase synthesis conditions for their incorporation into peptides. Monomeric and dimeric α-helical peptides are synthesised with varying degrees of Q4A substitution and their structures examined using biophysical methods. Both enantiomers of the Q4As are tolerated in folded monomeric and oligomeric α-helical peptides, with the (R)-enantiomer slightly more so than the (S).

The reaction mixture was allowed to warm to room temperature and left to stir for 2 h. The mixture was cooled to 0 °C and pyridine (2.70 mL, 32.9 mmol, 2.0 eq.) was added dropwise. The reaction was allowed to warm to room temperature and left to stir for a further 16 h before quenching with HCl (1 M, aq.). The mixture was concentrated in vacuo, diluted in EtOAc and washed with HCl (3×, 1 M, aq.) and brine, dried over MgSO4, filtered and concentrated in vacuo. The crude product was purified by automated FCC to give the title compound (5.31 g, 12.8 mmol, 78 %, >99:1 dr) as a white solid. Data in agreement with reported values. 3

Peptide Purification
Crude peptides were purified by reverse-phase HPLC on a JASCO HPLC system equipped with a Phenomenex Luna C18 column (5 μm particle size; 100 Å pore size; 150×10 mm). A gradient of water (0.1 % TFA, buffer A) and acetonitrile (0.1 % TFA, buffer B) between 20 and 80 % or 40 and 100 % buffer B over 30 min at a flow rate of 3 mL min -1 with absorbance recorded at 220 and 280 nm was typically used. The fractions collected from HPLC were analysed by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) on a Bruker ultraFlexXtreme II MALDI-TOF mass spectrometer operating in positive-ion reflector mode. Peptides were co-crystallised on a ground-steel target plate using α-cyano-4-hydroxycinnamic acid as the matrix. Peptide purity was confirmed by reverse-phase analytical HPLC on a JASCO chromatography system fitted with a Phenomenex® Kinetex C18 (5 μM particle size; 100 x 4.5 mm) column.

Peptide Concentration Determination
Peptide concentrations were determined in H2O by UV-vis absorption at 280 nm on a Nanodrop 2000 (Thermo Scientific) using the extinction coefficient for tryptophan: ε280 = 5690 dm 3 mol -1 cm -1 . Any contribution to this from the arylated amino acids were deemed negligible.

Circular Dichroism Spectroscopy
Circular dichroism (CD) spectra were measured at 5 °C as the average of eight scans from 260-190 nm using a JASCO 810 spectropolarimeter fitted with a Peltier temperature controller, a 1 mm pathlength quartz cuvette (Starna), a scanning speed of 100 nm min -1 , and a bandwidth of 1 nm. Peptides were prepared at 50 µM concentration (150 µL

Analytical Ultracentrifugation
Sedimentation equilibrium analytical ultracentrifugation (AUC) experiments were conducted at 20 °C in a Beckman XL-A Analytical Ultracentrifuge using an An-60 Ti rotor and Epon or aluminium 2-channel centrepieces fitted with quartz windows. Solutions were prepared at 75 μM (but 37.5 μM for peptide 15) in PBS to a volume of 100 μL for Epon centrepieces and 110 μL for aluminium centrepieces. The reference channel contained 110 μL of PBS for Epon centrepieces and 120 for aluminium centrepieces.
The samples were centrifuged from 44-60 krpm in increments of 4 krpm. The absorbance was measured across the cell at a radial distance of 5.