Supporting information: Experimental Synthesis Procedures and Characterization Data
General.
Starting materials and solvents were purchased from commercial suppliers (Alfa Aesar,
CHEMsolute, Iris Biotech, Sigma Aldrich, VWR, AppliChem, Fluka, ABCR, LabScan and Merck)
and used without further purification. TLC was performed on Merck TLC Silica gel 60 F254
aluminium sheets (5 × 7.5 cm); plates were developed with 5% H2SO4 in MeOH followed by
heating. Vacuum liquid chromatography (VLC) was performed with Merck Silica gel 60 H (90 %
particle size < 55 µm). 1H and 13C nuclear magnetic resonance (NMR) spectra were acquired by
using a 600 MHz MHz Bruker Avance III HD equipped with a cryogenically cooled 5 mm dual
probe optimized for 13C or a 400 MHz Bruker Avance III spectrometer. Samples were dissolved in
dimethylsulfoxide-d6 or D2O (Cambridge Isotope Laboratories) and analyzed at 300 K. Coupling
constants (J values) are given in Hertz (Hz); multiplicities are reported as follows: singlet (s),
doublet (d), triplet (t), quartet (q), and multiplet (m). Water used during analytical and preparative
HPLC was filtered through a 0.22 μm membrane filter. Analytical HPLC was used to determine
purity and was carried out on a Phenomenex Luna C18 (2) (3 μm) column (150 mm × 4.6 mm)
using binary mixtures of eluent A (water−MeCN−TFA 95:5:0.1) and eluent B (water−MeCN−TFA
5:95:0.1) for elution with a flow rate of 0.8 mL min-1; Fmoc-protected building block: a linear
gradient of 0−80% B during 5 min followed by a linear rise to 100% B during 25 min; peptides and
peptidomimetics 2-11: a linear gradient of 10−60% B during 30 min; peptidomimetic 12: a linerar
gradient of 20-80% B during 30 min. UV-detection: building block at λ = 267 nm and
peptidomimetics at λ = 220 nm or 280 nm. All compounds had a purity of at least 95%. Retention
times (tR) are given for each compound. Preparative HPLC was performed by using a Luna C18 (2)
(5 μm) column (250 mm × 21.2 mm) on an Agilent 1100 LC system with a multiple-wavelength
UV detector. Elution was performed with linear gradients of 10−25% B to 20-100% during 20 min
at a flow rate of 20 mL/min; peptidomimetics were detected at λ = 220 nm or 280 nm. LC-HRMS
was performed with a Phenomenex Luna C18 (2) (3 μm) column (150 mm × 4.6 mm) using binary
mixtures of eluent C (water−MeCN−formic acid 95:5:0.1) and eluent D (water−MeCN−formic acid
5:95:0.1); elution was performed with a linear gradient of 10−60% D during 30 min at a flow rate of
0.5 mL/min. HRMS spectra were obtained by using a Bruker MicrOTOF-Q II Quadropol MS
detector. The analyses were performed as ESI-MS (m/z): [M + nH]n+ for peptidomimetics.
Synthesis of β-(2,5,7-tri-tert-butylindol-3-yl)alanine (H-Tbt-OH). H-Trp-OH (10.0 g) was
dissolved in TFA (245 mL), and then tert-BuOH (47 mL; 10 equiv) was added. The solution was
stirred for 4 days in a sealed flask. The solvent was removed by evaporation under reduced
pressure. The dark oily residue was redissolved in toluene, and then the solvent was removed; this
was repeated until most TFA was removed. Water (50 mL) was added and a gum-like precipitate
formed. Then pH was adjusted to 7 with aq. satd. NaHCO3 (150 mL). The water was decanted off
and the solid dissolved in EtOH (70 mL) and heated to approximately 70 °C. Water (70 mL) was
slowly added under vigorous stirring. Upon cooling to rt the product precipitated as white crystals
(2.23 g; 12.4%). The mother liquor was concentrated in vacuo and crystallized in the same way to
give an additional crop of crystals (1.80 g; 10.0%).
1
H NMR (400 MHz, DMSO-d6) : 8.70 (1H, br s), 7.44 (1H, br s, Indole-H), 7.00 (1H, br s, Indole-
H) 3.52 (1 H, dd, J = 14.8 and 5.1 Hz, HA-β), 3.43 (1H, dd, J = 8.6 and 5.1 Hz, H-α), 2.98 (1 H, dd,
J = 14.8 and 8.6 Hz), 1.47 (9 H, s, tBu), 1.45 (9 H, s, tBu), 1.32 (9 H, s, tBu). 13C NMR (150 MHz,
DMSO-d6) : 170.12, 142.69, 140.67, 131.60, 130.02, 129.82, 115.17, 112.26, 105.19, 56.46,
34.44, 34.25, 33.25, 32.01 (3C), 30.89 (3C), 30.26 (3C), 27.24.
Synthesis of Fmoc-Tbt-OH.
H-Tbt-OH (13; 2.23 g) was dissolved in MeCN–H2O (5:1; 115 mL) under vigorous stirring and
heating. Then Et3N (1.7 mL ; 2 equiv) was added. When a clear solution was obtained, Fmoc-Cl
(1.54 g; 1 equiv) in MeCN (4 mL) was added while cooling on an ice-bath. The reaction was stirred
for 30 min. The solvent was removed in vacuo and the residue dissolved in EtOAc (90 mL), which
was washed with aq. 1M HCl (70 mL) and brine (70 mL). Drying over Na2SO4 and removal of
solvent in vacuo yielded the crude product (3.53 g). Along with 1.41 g of another similar batch this
was dissolved in CH2Cl2 (20 mL) and loaded onto a VLC column (h × d: 8 × 7 cm; silicagel 60H
presaturated with heptane). Gradient elution: heptane (600 mL), heptane–EtOAc 20:1 + 0.1%
HOAc (525 mL), heptane–EtOAc 10:1 + 0.1% HOAc (550 mL), heptane–EtOAc 8:1 + 0.1% HOAc
(450 mL), heptane–EtOAc 6:1 + 0.1% HOAc (700 mL), heptane–EtOAc 4:1 + 0.1% HOAc (500
mL). Fractions containing the desired product were concd to give 14 (4.27 g) as a white solid.
Analyt. HPLC: tR = 26.0 min (0%  80%  100% B); TLC (heptane-EtOAc 3:1 + 0.5% HOAc):
Rf = 0.13.
1
H NMR (600 MHz, DMSO-d6) : 12.60 (1H, br s, COOH), 8.70 (1H, s), 7.88 (1H, d, J = 7.5 Hz),
7.87 (1H, d, J = 7.7 Hz), 7.78 (1 H, d, J = 7.5 Hz), 7.68 (1H, d, J = 7.5 Hz), 7.64 (1 H, d, J = 7.5
Hz), 7.47 (1 H, d, J = 1.5 Hz), 7.41 (1 H, t, J = 7.5 Hz), 7.39 (1 H, d, J = 7.5 Hz), 7.30 (1 H, br t, J =
7.5 Hz), 7.23 (1 H, br t, J = 7.5 Hz), 7.00 (1 H, d, 1.5 Hz), 4.24–4.13 (4 H, m, H-α, Fmoc-CH,
Fmoc-CH2), 3.39 (1 H, dd, J = 14.8 and 5.8 Hz, HA-β), 3.15 (1 H, dd, J = 14.8 and 8.3 Hz, HB-β),
1.47 (9 H, s, tBu), 1.44 (9 H, s, tBu), 1.31 (9 H, s, tBu).
C NMR (150 MHz, DMSO-d6) : 173.69, 155.99, 143.78, 143.69, 142.33, 140.65 (2C), 140.47,
13
131.52, 130.22, 129.69, 127.61, 127.57, 127.02 (2C), 125.30 (2C), 120.05 (2C), 115.22, 112.48,
105.58, 65.73, 56.13, 46.56, 34.36, 34.23, 33.21, 31.95 (3C), 30.74 (3C), 30.27 (3C), 26.95.
Manual peptide synthesis.
Tetrapeptides and peptidomimetics were synthesized by manual standard Fmoc SPPS on a Rink
Amide solid support (H-Rink Amide ChemMatrix or Fmoc-Rink Amide AM resin; loadings: 0.520.70 mmol/g) in teflon reaction vessels (10 mL) fitted with polypropylene filters. Vessels were
agitated at 320 shakes min-1. Between each step the resin was washed successively with DMF,
MeOH, DCM (3 × 3 mL, each for 3 min). Upon swelling in DMF and initial Fmoc deprotection
sequential SPPS was perfomed. Couplings (0.1 mmol scale) with a given building block (5 equiv)
was performed with PyBOP (5 equiv) and DIPEA (10 equiv) in 3 mL DMF for 2 hours at rt. Due to
scarcety of Fmoc-Tbt-OH couplings with this building block was performed with 2 equiv for 12
hours at rt. After loading, capping with Ac2O:DIPEA:NMP (1:2:3; 3 mL) was performed for 10
min. Deprotection was carried out by treatment with 20% piperidine–DMF (3 mL; 2 × 10 min). All
compounds were cleaved by using TFA:TIS:H2O (95:2.5:2.5; 3 mL) for 2 hours. After cleavage the
crude peptides were concentrated with MeOH and toluene (3 ×) followed by precipitation with
Et2O. After drying, peptides were purified by preparative RP-HPLC and characterized by analytical
RP-HPLC, HRMS, and 1H NMR.
H-Arg-Trp-Arg-Phe-NH2 (2)
Analyt. HPLC: tR = 14.61 min (purity: 98.9% at 220 nm). HRMS calcd for C32H46N12O4 [M + 2H]2+
350.7061, found 350.7064; ΔM = 0.9 ppm. 1H NMR (600 MHz; D2O) δ: 7.67 (1 H, d, J = 8.0 Hz),
7.54 (1 H, d, J = 8.2 Hz), 7.39 (2 H, m), 7.33–7.18 (6 H, m), 4.72 (1 H, t, J = 8.0 Hz, H-), 4.44 (1
H, dd, J = 8.7 and 6.5 Hz, H-), 4.14 (1 H, br t, J = 7.5 Hz, H-), 3.98 (1 H, t, J = 6.5 Hz, H-),
3.86 (1 H, m, H-), 3.25 (2 H, m), 3.18 (2 H, t, J = 7.0 Hz), 3.10 (1 H, dd, J = 13.9 and 5.9 Hz),
3.05 (2 H, t, J = 7.0 Hz), 2.85 (1 H, dd, J = 13.9 and 8.8 Hz), 1.85 (2 H, m), 1.58 (2 H, m), 1.48 (2
H, m), 1.29 (2 H, m).
H-Arg-[p-phenyl-Phe]-Arg-Phe-NH2 (3)
Analyt. HPLC: tR = 19.66 min (purity: 98.8% at 220 nm). HRMS calcd for C36H49N11O4 [M + 2H]2+
332.1959, found 332.1963; ΔM = 1.2 ppm. 1H NMR (400 MHz; D2O) δ: 7.65 (2 H, d, J = 7.3 Hz),
7.56 (2 H, d, J = 8.2 Hz), 7.48 (2 H, t, J = 7.5 Hz), 7.38 (1 H, m), 7.31–7.20 (5 H, m), 7.08 (2 H, d, J
= 7.0 Hz), 4.60 (1 H, dd, J = 8.6 and 7.4 Hz, H-), 4.28 (1 H, dd, J = 8.5 and 6.5 Hz, H-), 4.14 (1
H, t, J = 7.0 Hz, H-), 3.97 (1 H, t, J = 7.0 Hz, H-), 3.15–3.08 (3 H, m), 3.03-2.94 (4 H, m), 2.79
(1 H, dd, J = 13.8 and 8.6 Hz), 1.89–1.76 (2 H, m), 1.62–1.46 (4 H, m), 1.38–1.18 (2 H, m).
H-Arg-Cha-Arg-Phe-NH2 (4)
Analyt. HPLC: tR = 16.21 (purity: 98.9% at 220 nm). HRMS calcd for C30H51N11O4 [M + 2H]2+
315.7136, found 315.7139; ΔM = 1.0 ppm. 1H NMR (400 MHz; D2O) δ: 7.35–7.21 (5 H, m, Ph),
4.57 (1 H, dd, J = 9.0 and 6.0 Hz, H-), 4.32 (1 H, dd, J = 9.0 and 6.0 Hz, H-), 4.20 (1 H, t, J = 7.0
Hz, H-), 3.98 (1 H, t, J = 6.5 Hz, H-), 3.18–3.07 (5 H, m), 2.95 (1H, dd, J = 14.0 and 9.0 Hz),
1.90–1.82 (2 H, m), 1.71–1.30 (12 H, m), 1.30–1.05 (4 H, m), 0.97–0.80 (3 H, m).
H-Arg-[L-2-Aoc]-Arg-Phe-NH2 (5)
Analyt. HPLC: tR = 17.87 (purity: 99.7% at 220 nm). HRMS calcd for C29H51N11O4 [M + 2H]2+
309.7133, found 309.7136; ΔM = 1.0 ppm. 1H NMR (400 MHz; D2O) δ: 7.34–7.21 (5 H, m, Ph),
4.56 (1 H, dd, J = 9.0 and 6.0 Hz, H-), 4.18–4.24 (2 H, m, H-), 3.99 (1H, t, J = 7.0 Hz, H-),
3.16 (2 H, t, J = 7.0 Hz), 3.18–3.07 (3 H, m), 2.95 (1 H, dd, J = 14.0 and 8.8 Hz), 1.85 (2 H, m)
1.71–1.34 (8 H, m), 1.29–1.18 (8 H, m), 0.83–0.78 (3 H, m).
H-Arg-Tbt-Arg-Phe-NH2 (6)
Analyt. HPLC: tR = 27.94 min (purity: 99.7% at 220 nm). HRMS calcd for C44H70N12O4 [M +
2H]2+ 277.8623, found 277.8629; ΔM = 2.2 ppm. 1H NMR (400 MHz; D2O) δ: 7.43–7.27 (7 H, m),
4.66 (1 H, dd, J = 9.7 and 6.2 Hz, H-), 4.35 (1 H, t, J = 7.5 Hz, H-), 4.21 (1 H, t, J = 6.3 Hz, H), 4.04 (1 H, t, J = 6.5 Hz, H-), 3.41 (1 H, dd, J = 14.5 and 10.2 Hz), 3.32 (1 H, dd, J = 14.5 Hz
and 6.2 Hz), 3.25 (2 H, t, J = 6.9 Hz), 3.08 (2 H, t, J = 7.1 Hz), 3.06 (1 H, obsc.), 2.94 (1H, dd, J =
13.8 and 8.0 Hz), 2.00–1.94 (2 H, m), 1.76–1.62 (3 H, m), 1.61–1.30 (3H, obsc. by tBu signals),
1.51 (9 H, s, tBu), 1.46 (9 H, s, tBu), 1.41 (9 H, s, tBu).
H-Arg-Tbt-Arg-NH2 (7)
Analyt. HPLC: tR = 23.85 min (purity: 99.9% at 220 nm). HRMS calcd for C35H61N11O3 [M + 2H]2+
314.7244, found 314.7248; ΔM = 1.3 ppm. 1H NMR (400 MHz; D2O) δ: 7.40 (1 H, s, indole-H),
7.29 (1 H, s, indole-H), 4.59 (1 H, dd, J = 10.4 and 5.5 Hz, H-), 4.13 (1 H, t, J = 6.3 Hz, H-),
4.08 (1 H, dd, J = 7.9 and 4.9 Hz, H-α), 3.52–3.40 (2 H, m), 3.29 (2 H, t, J = 7.0 Hz), 3.11 (2 H, t, J
= 6.8 Hz), 2.06–1.93 (2 H, m), 1.78-1.62 (3 H, m), 1.55-1.35 (3 H, obsc. by tBu signals), 1.52 (9 H,
s, tBu), 1.51 (9 H, s, tBu), 1.39 (9 H, s, tBu).
Ac-Arg-Tbt-Arg-Phe-NH2 (8)
Analyt. HPLC: tR = 30.37 min (purity: 99.8% at 220 nm). HRMS calcd for C46H72N12O5 [M + 2H]2+
437.2954, found 437.2955; ΔM = 0.2 ppm. 1H NMR (400 MHz; D2O) δ: 7.33–7.27 (3 H, m), 7.22–
7.19 (4 H, m) 4.63 (1 H, t, J = 8.0 Hz, H-), 4.34 (1 , dd, J = 9.0 and 6.2 Hz, H-), 4.18–4.12 (1 H,
m, H-), 4.04 (1 H, t, J = 7.0 Hz, H-), 3.40 (1 H, dd, J = 14.8 and 8.0 Hz), 3.15 (1 H, dd, J = 14.8
and 7.8 Hz), 3.09 (1 H, dd, J = 14.0 and 6.2 Hz), 3.02 (2 H, t, J = 7.0 Hz), 2.96 (2 H, m), 2.87 (1 H,
dd, J = 14.0 and 7.7 Hz), 1.88 (3 H, s, Ac), 1.62-1.44 (4 H, m), 1.42 (9 H, s, tBu), 1.40 (9 H, s, tBu),
1.32 (9 H, s, tBu), 1.36–1.24 (4 H, obsc. by tBu signal).
H-Arg-Tbt-Arg-[hPhe]-NH2 (9)
Analyt. HPLC: tR = 28.67 min (purity: 99.0% at 220 nm). HRMS calcd for C45H72N12O4 [M + 3H]3+
282.5342, found 282.5343; ΔM = 0.4 ppm. 1H NMR (400 MHz; D2O) δ: 7.35–7.30 (3 H, m), 7.27–
7.19 (3 H, m), 7.16 (1 H, d, J = 1.5 Hz), 4.59 (1 H, dd, J = 10.2 and 6.0 Hz, H-), 4.12 (1 H, br t, J
= 6.0 Hz, H-), 3.99 (1 H, t, J = 6.5 Hz, H-), 3.94 (1 H, dd, J =8.3 and 6.2 Hz, H-), 3.38 (1 H,
dd, J = 14.5 and 10.4 Hz, dd), 3.29 (1 H, dd, J = 14.5 and 6.0 Hz), 3.16 (2 H, t, J = 7.0 Hz), 3.02 (2
H, t, J = 7.2 Hz), 2.67–2.53 (2 H, m), 2.04–1.82 (4 H, m), 1.69–1.43 (4 H, m), 1.42–1.29 (2 H, obsc.
by tBu signals), 1.41 (9 H, s, tBu), 1.40 (9 H, s, tBu), 1.31 (9 H, s, tBu).
H-Arg-Tbt-Arg-[β3Phe]-NH2 (10)
Analyt. HPLC: tR = 28.33 min (purity: 95.8% at 220 nm). HRMS calcd for C45H72N12O4 [M + 3H]3+
282.5339, found 282.5339; ΔM = 0.0 ppm. 1H NMR (400 MHz; D2O) δ: 7.27 (1 H, d, J = 1.5 Hz),
7.17–7.02 (5 H, m), 4.50 (1 H, dd, J = 9.0 and 6.7 Hz, H-), 4.12 (1 H, m, H-), 4.00 (1 H, t, J =
6.5 Hz, H-), 3.78 (1 H, t, J = 6.6 Hz, H-), 3.27 (1 H, dd, J = 14.8 and 9.2 Hz), 3.11 (1 H, dd, J =
14.8 and 6.5 Hz), 3.02 (2 H, m), 2.91 (2 H, t, J = 7.0 Hz), 2.59 (1 H, dd, J = 13.8 and 6.8 Hz), 2.49
(1 H, dd, J = 13.8 and 8.0 Hz), 2.26 (1 H, J = 14.8 and 5.0 Hz), 2.08 (1 H, dd, J = 14.8 and 9.2 Hz)
1.74 (2 H, m), 1.50–1.35 (3 H, m), 1.34–1.18 (3 H, obsc. by tBu signals), 1.33 (9 H, s, tBu), 1.29 (9
H, s, tBu), 1.23 (9 H, s, tBu).
H-Arg-Tbt-Arg-[α-NPhe]-NH2 (11)
Analyt. HPLC: tR = 28.23 min (purity: 99.6% at 220 nm). HRMS calcd for C44H70N12O4 [M + 3H]3+
277.8621, found 277.8618; ΔM = 1.1 ppm. 1H NMR (400 MHz; D2O) δ: 7.45–7.28* (4 H, m), 7.25–
7.21* (1 H, m), 7.16–7.08* (2 H, m), 4.68–4.20* (4 H, m), 4.02–3.52* (3 H, m), 3.44-2.95* (6 H, m),
1.95–1.81* (2 H, m), 1.80–1.46* (4 H, m), 1.45–1.28* (2 H, obsc. by tBu signals), 1.44** (9 H, s,
t
Bu), 1.41* (9 H, s, tBu), 1.40*** (9 H, s, tBu), 1.32*** (9H, s, tBu), 1.30** (9 H, s, tBu); * Overlapping
signals from both rotamers; ** Signal from major rotamer; *** Signal from minor rotamer.
H-Arg-Tbt-Arg-Tbt-NH2 (12)
Analyt. HPLC: tR = 29.54 min (purity: 99.9% at 220 nm). HRMS calcd for C58H95N13O4 [M +
3H]3+ 346.9281, found 346.9282; ΔM = 0.3 ppm. 1H NMR (400 MHz; D2O) δ: 7.37 (1 H, br s,
indole-H), 7.29 (1 H, br s, indole-H), 7.09 (2 H, m, indole-H), 4.55 (1 H, dd, J = 9.2 and 6.4 Hz, H), 4.37 (1 H, dd, J = 9.0 and 6.1 Hz, H-α), 4.24 (1 H, t, J = 6.1 Hz, H-), 3.98 (1 H, t, J = 6.4 Hz,
H-), 3.33–3.14 (3 H, m), 3.16 (2 H, t, J = 7.1 Hz), 3.04 (2 H, t, J = 6.9 Hz), 2.96–2.80 (1 H, m),
1.88 (2 H, m), 1.75–1.48 (5 H, m), 1.45–1.20 (1 H, obsc. by tBu signals), 1.37 (9 H, s, tBu), 1.32 (18
H, br s, 2  tBu), 1.28 (9 H, s, tBu), 1.24 (9 H, s, tBu), 1.18 (9 H, s, tBu).