Accessing human selenoproteins through chemical protein synthesis

The human body contains 25 selenoproteins, but challenges in their preparations have prevented biological characterizations thus far. Here we report the first total chemical syntheses of two human selenoproteins, selenoprotein M (SELM) and selenoprotein W (SELW).

Linear gradients of ACN (with 0.1 % TFA, buffer B) in water (with 0.1 % TFA, Buffer A) were used for all systems to elute bound peptides. The flow rates were 1 mL/min (analytical, column heated at 30 o C), 10 mL/min (semi-preparative), and 20 mL/min (C18 preparative).

Electrospray Ionization Mass Spectrometry (ESI-MS). ESI-MS was performed on
LCQ Fleet Ion Trap mass spectrometer (Thermo Scientific). Peptide masses were calculated from the experimental mass to charge (m/z) ratios from the observed multiply charged species of a peptide. Deconvolution of the experimental MS data was performed with the help of MagTran v1.03 software.

Circular Dichroism (CD).
The secondary structure content of the synthetic human SELM and SELW were compared to the commercially available E. coli Trx ( Figure S3) using far-UV CD spectroscopy (200 to 260 nm). Spectra were recorded on J-810 spectropolarimeter (Jasco), using a quartz cuvette with a path length of 0.1 cm, and obtained by averaging 5 wavelength scans in 1 or 0.5 nm steps, with a signal averaging time of 2 s and a bandwidth of 1 nm. Each purified protein was dissolved separately in folding buffer as described bellow. [Θ] centrifuged at 5000 rpm for 5 min, and concentration was determined by UV-Vis spectrophotometer (using theoretical Ɛ 280 nm = 5,180 M -1 ·cm -1 ).  Figure S3) was carried out on a C18 analytical column (ACQUITY UPLC C18 column, 1.7 μm, 130 Å, 2.1 × 100 mm), using a gradient of 5% B over 3 min then 5-70% B over 7 min).

Alloc protection:
The Fmoc-Dbz-resin was washed with DMF and DCM, and 0.35 M Allylchloroformate and DIEA (1 equiv to resin loading) in DCM were added to the resin and mixed for 24 h at room temperature. 14 Standard Fmoc-SPPS was followed and finally, Boc-Ala-OH was manually doubly coupled (3.0 equiv Boc-Ala-OH, 3.0 equiv HATU and 2.9 equiv DIEA).

Alloc deprotection:
The Alloc protected peptide-resin was washed and swollen in DCM for 30 min and briefly sparged with Ar. The PhSiH 3 (20 equiv) and Pd(PPh 3 ) 4 (0.35 equiv) in 5 mL DCM was added and mixed for 3 h. 14    The detailed synthesis of the two peptide segments as well as the ligation reaction are described below.