Supporting Information for
Towards intrinsically colored peptides: the synthesis and investigation of the
spectral properties of methylated azatryptophans in Trp-cage mutants
B. P. Noichl,1 P. M. Durkin, 1 N. Budisa.* 1
1
Department of Chemistry, Berlin Institute of Technology, Müller-Breslau-Straße 10, 10623 Berlin.
Peptide-Synthesis
TC
Sequence: H-Asn-Leu-Tyr-Ile-Gln-Trp-Leu-Lys-Asp-Gly-Gly-Pro-Ser-Ser-Gly-Arg-Pro-Pro-Pro-Ser-OH
For the purification of the Trp-cage-wild-type was used the following gradient of system A in system B:
Time
0.00 Min
1.00 Min
2.00 Min
9.00 Min
11.00 Min
13.00 Min
System B
10%
10%
25%
30%
90%
90%
Retention time (tR): 5.45 Min
ESI-MS: m/z = 724.04 (M+3H3+), 1085.56 (M+2H2+); calculated for (M+H+): 2169.1087.
calculated for (M+2H2+): 1085.5597
calculated for (M+3H3+): 724.0422
Isolated yield: 10 mg.
Figure 1: LC-MS Run of Trp-Cage at the LTQ Orbitrap XL
TC-W66
Sequence: H-Asn-Leu-Tyr-Ile-Gln-6-Leu-Lys-Asp-Gly-Gly-Pro-Ser-Ser-Gly-Arg-Pro-Pro-Pro-Ser-NH2
The Trp-Cage mini protein was synthesized to the amino acid leucine on the automated peptide
synthesizer with the procedure described above. The non-canonical amino acid 1,7DM7AW at position 6 and
the following amino acids were coupled manually. For the purification by HPLC was used the following gradient
of system A in system B:
Time
0.00 Min
1.00 Min
2.00 Min
9.00 Min
11.00 Min
13.00 Min
System B
10%
10%
25%
30%
90%
90%
Retention time (tR): 5.04 Min
ESI-MS: m/z = 733.71 (M+3H3+), 1100.07 (M+2H2+); calculated for (M+H+): 2199.1624
calculated for (M+2H2+): 1100.0849
calculated for (M+3H3+): 733.7257
Isolated yield: 5 mg.
Figure 2: LC-MS Run of Trp-Cage- W6_1,7DM7AW at the LTQ Orbitrap XL
TC-W65
Sequence: H-Asn-Leu-Tyr-Ile-Gln-5-Leu-Lys-Asp-Gly-Gly-Pro-Ser-Ser-Gly-Arg-Pro-Pro-Pro-Ser-OH
The Trp-Cage mini protein was synthesized to the amino acid leucine on the automated peptide
synthesizer with the procedure described above. The non-canonical amino acid 1M7AW at position 6 and the
following amino acids were coupled by hand. For the purification by HPLC was used the following gradient of
system A in system B:
Time
0.00 Min
1.00 Min
2.00 Min
12.00 Min
14.00 Min
17.00 Min
System B
10%
10%
25%
30%
90%
90%
Retention time (tR): 4.94 Min
ESI-MS: m/z = 729.05 (M+3H3+),1093.07 (M+2H2+); calculated for (M+H+): 2185.1230
calculated for (M+2H2+): 1093.0651
calculated for (M+3H3+): 729.0458
Isolated yield: 2 mg.
Figure 3: LC-MS Run of Trp-Cage- W6_1M7AW at the LTQ Orbitrap XL
Supplementary Data
(S)-3-(2-(((Benzyloxy)carbonyl)amino)-2-carboxyethyl)-1,7-dimethyl-1H-pyrrolo[2,3b]pyridin-7-ium iodide
The crude starting material of 15 (200 mg, 0.85 mmol, 1.0 eq.) was added to a solution of saturated NaHCO 3(aq)
solution/THF (9 mL, 1:1) at 0 °C. Benzyl chloroformate (174 mg, 1.02 mmol, 1.2 eq.) was added and the mixture
was stirred for 2 hours at 0 °C. The precipitate was filtered and concentrated under reduced pressure.
Purification on reversed phase HPLC afforded the desired product as a yellow solid (40 mg).
HPLC-conditions: Gradient from 35 % to 45 % acetonitrile in 15 minutes. Retention time of the Cbz-1,7dimethyl-7-azatryptophan: τR = 3.84 minutes. Retention time of the Cbz-Val-OH: τR = 11.22 Minutes. ESI-MS:
m/z = 368.16 (M+); calculated for (C20H22N3O4+): 368.16; 1H NMR (400 MHz, CD3CN) δ: 8.55 (1H, d, J = 8.0 Hz),
8.12 (1H, d, J = 6.0 Hz), 7.29-7.42 (7H, m), 5.97 (1H, d, J = 7.0 Hz), 4.97 - 5.08 (2H, m), 4.46-4.56 (4H, m), 4.14
(3H, s), 3.18-3.41 (2H, m); 13C NMR (101 MHz, CD3CN) δ: 173.2, 156.8, 140.4, 140.0, 138.1, 137.3, 135.8, 130.2,
129.5, 129.1, 128.9, 116.6, 112.9, 67.2, 55.4, 45.6, 38.3, 27.3.
This coumpound was included for completeness, it is described in the body text but is not present in any
of the schemes.
(S)-3-(2-Amino-2-carboxyethyl)-1,7-dimethyl-1H-pyrrolo[2,3-b]pyridin-7-ium chloride
The Z-protected amino acid (50 mg, 0.14 mmol) was dissolved in water (4 mL) and 6 N HCl (4 mL) was added.
The reaction was stirred for 27 hours at 75°C. The reaction mixture was cooled to room temperature and the
solvent was evaporated under reduced pressure. The desired product was obtained as a yellow solid in 96 %
yield (35 mg, 0.13 mmol).
ESI-MS: m/z = 234.12 (M+H+); calculated for (C12H16N3O2+H+): 234.12; 1H NMR (400 MHz, D2O) δ: 8.56 (1H, d,
J=8.0 Hz), 8.24 (1H, d, J=6.0 Hz), 7.42-7.48 (1H, m), 4.61 (3H, s), 4.21-4.23 (1H, m), 4.20 (3H, s), 3.43 (2H, d,
J=6.0 Hz); 13C NMR (101 MHz, D2O) δ: 172.7, 139.7, 139.3, 135.9, 135.3, 127.7, 115.9, 108.6, 53.5, 44.4, 37.2,
24.9.
This coumpound was included for completeness, it is described in the body text but is not present in any
of the schemes.