Supplementary Material
Distinctin A chain containing free cysteine residue.
The identical text procedure for the A chain
containing a Cys(Scm) residue was followed, up to the point where crude peptide was dissolved in 0.1%
aqueous TFA (20 mL). Next, 0.1 M phosphate buffer (5 mL, pH 7.5) was added, and the overall pH was
adjusted to the range of 7.0-7.5 with 1.0 N NaOH. Solid DTT (38 mg, 0.25 mmol) was then added,
dissolving quickly, and the desired reduction was carried out under stirring for 15 min. Neat TFA was
added to lower the pH into the range of 2-3, the solution was filtered, and RP-HPLC purification
followed. Yield: 13.3 mg (21%), Tr = 42 min., purity by RP-HPLC: 92%, deconvoluted ESI-MS:
calculated 2525.4, found 2525.3.
Distinctin B chain containing Cys(Scm). To convert Cys(Acm) to Cys(Scm) on-resin, the peptide-resin
(220 mg, 25 mol) was first swollen by extensive washing in CH2Cl2. After the peptide-resin was
suspended in CH2Cl2 (4 mL), neat methoxycarbonylsulfenyl chloride (3.0 L, 33 mol) was added, and
the mixture was shaken for 3 h. The peptide-resin was then washed with CH2Cl2 (3 x 5 mL) and dried.
The peptide was cleaved from the resin with freshly prepared reagent K (5 mL) under argon for 2 h,
precipitated with ether (80 mL), and centrifuged to collect a pellet which was washed twice with ether.
The crude peptide was dissolved in 0.1% aqueous TFA (20 mL), filtered, and purified by RP-HPLC.
Yield: 5.0 mg (7%), Tr = 43 min, purity by RP-HPLC: 98%, deconvoluted ESI-MS: calculated 3041.7,
found 3041.6.
1
0.35
0.3
0.3
0.25
0.25
Abs. (220 nm)
Abs. (220 nm)
0.35
0.2
0.15
0.1
0.2
0.15
0.1
0.05
0.05
0
0
0
10
20
30
40
0
Minutes
10
20
30
40
Minutes
Figure S1. Analytical RP-HPLC, Left panel: Purified A chain with free Cys. Right panel: Purified
Cys(Scm)-containing B chain. Gradient, 10-50% B over 40 min.
Distinctin (5) by directed disulfide formation. The above-described two chains were combined, on a 1.6
mol scale, following the same general procedure described in the main text. Yield: 3.2 mg (37%), Tr =
43 min., purity by RP-HPLC: 95%, deconvoluted ESI-MS: calculated 5478.6, found 5478.0
0.16
0.14
Abs. (220 nm)
0.12
0.1
0.08
0.06
0.04
0.02
0
0
10
20
30
40
Minutes
2
0.6
0.7
0.5
0.6
0.5
0.4
Abs. (220 nm)
Abs. (220 nm)
Figure S2. RP-HPLC of Purified distinctin. Gradient 10-50% B over 40 min.
0.3
0.2
0.4
0.3
0.2
0.1
0.1
0
0
0
10
20
30
40
0
Minutes
Figure S3.
10
20
30
40
Minutes
Analytical RP-HPLC, Left panel: Purified Cys(Scm)-containing chain A. Right panel:
Purified chain B containing
15
N,
13
C-G4,
15
N-L5,
15
N-Q15, 15N-L16 isotopic labels. Gradient 10-50% B
over 40 min.
3