Gobbo et al.
NMM (0.05 mL, 0.30 mmol). The reaction mixture was stirred 1 h
at 0 ꢀC, then at room temperature for 2–3 days, by keeping the pH
to 8. The solvent was removed in vacuo, the residue was dissolved
in EtOAc, and repeatedly washed with 0.5 M citric acid, water, 5%
NaHCO3, and water. The organic phase was dried over Na2SO4 and
evaporated to dryness in vacuo. The crude product was purified by
flash chromatography (8% MeOH in DCM), and the desired peptide
was isolated in 40% yield.
treatments with 30% 1,1,1,3,3,3-hexafluoropropan-2-ol (HFIP) in
DCM for 30 min. The filtrates were combined and evaporated to
dryness providing the crude protected peptide. Removal of the side-
chain-protecting groups was achieved by treatment with reagent K
(TFA⁄ phenol ⁄ thioanisole ⁄ water ⁄ 1,2-ethanedithiol, 82:5:5:5:2.5 by
vol) for 90 min. The cleavage mixture was concentrated under
reduced pressure. The product was precipitated by addition of
diethyl ether and lyophilized from a mixture of acetonitrile in 5%
aqueous acetic acid. The crude peptide (70 mg, 70% purity based
on HPLC) was purified by semi-preparative HPLC to afford the
desired product with a 95% purity in 43% yield. HPLC: (C18 gradient
20
M. p. 149–151 ꢀC; Rf 0.60 (I); Rf 0.85 (II); Rf 0.10 (III); ½aꢀD )29.8
(c = 0.5, MeOH); HPLC: (C18 gradient A) Rt 32.5 min, (C4 gradient B)
Rt 25.1 min. electrospray ionization mass spectrometry (ESI-MS)
(m ⁄ z): calcd for C88H132N16O21 1748.94; found 875.48 [M + 2H]2+. IR
20
A) Rt 18.24 min; ½aꢀD )15.0 (c = 0.1, MeOH); IR (KBr): 3418, 1658,
1538 cm)1; ESI-MS (m ⁄ z): calcd for C72H118N18O19 1538.85; found
770.42 [M+2H]2+, 781.90 [M+H+Na]2+, and 789.39 [M+H+K]2+. 1H-
NMR (600 MHz, CD3OH): d 10.42 (d, 1H, e-NH Trp1); 8.25 (s and d,
2H, NH Gln6 and Aib8); 8.19–8.17 (m, 2H, NH Leu12 and Trp1); 8.15
(s, 1H, NH Aib10) 8.02 (s and d, 2H, NH Aib13 and Ala5); 7.89 (s,
1H, NH Aib4); 7.87 (d, 1H, NH Ala11); 7.86 (d, 2H, NH Ala7 and
Ser9); 7.60–7.59 (m, 2H, 2 NH Val2 and Lol); 7.57 (s, 1H, NH Aib3);
7.52–7.50 (m, 3H, NH and dNH Gln14, H-4 Trp1); 7.42 (s, 1H, dNH
Gln6); 7.33–7.32 (d, 1H, H-7 Trp1); 7.22 (d, 1H, H-2 Trp1); 7.10–7.08
(t, 1H, H-6 Trp1); 7.00–6.97 (t, 1H, H-5 Trp1); 6.82 (s, 1H, dNH
Gln14); 6.72 (s, 1H, dNH Gln6); 5.35 (m, 1H, OH Lol); 5.19–5.14 (t,
1H, OH Ser9); 4.51–4.48 (m, 1H, a-CH Trp1); 4.20–4.15 (m, 1H, a-CH
Gln14); 4.18–3.98 (m, 8H, a-CH Ala5, a-CH Ala7, a-CH Gln6, a-CH
Ala11, a-CH Leu12, a-CH Lol15, a-CH and b-CH Ser9); 3.57–3.54 (m,
3H, a-CH Val2 and b2-CH2 Lol); 3.26–3.24 (m, 2H, b-CH2 Trp1);
2.59–2.46 (m, 3H, c-CH2 Gln6 and c-CH Gln14); 2.43–2.37 (m, 1H, c-
CH Gln14); 2.33–2.25 (m, 2H, b-CH Gln6 and b-CH Gln14); 2.21–2.18
(m, 1H, b-CH Gln6); 2.16–2.10 (m, 1H, b-CH Gln14); 2.04 (s, 3H, CH3
Ac); 1.94–1.85 (m, 3H, b-CH Val2, b-CH and c-CH Leu12); 1.79–1.72
(m, 1H, c-CH Lol); 1.65–1.60 (m, 2H, b-CH Leu12 and b1-CH Lol);
1.58 (s, 3H, b-CH3 Aib10); 1.57 (s, 3H, b-CH3 Aib8); 1.55 (s, 3H, b-
CH3 Aib13); 1.52–1.51 (m, 15H, b-CH3 Aib4, Ala7, Aib8, Ala11 and
Aib13); 1.50–1.496 (d, 3H, b-CH3 Ala5); 1.49 (s, 3H, b-CH3 Aib10);
1.48 (s, 3H, b-CH3 Aib4); 1.43 (s, 3H, b-CH3 Aib3); 1.42 (s, 3H, b-
CH3 Aib3); 1.37–1.31 (m, 1H, b1-CH Lol); 0.94–0.89 (m, 15H, 2 d-
CH3 Leu12, 2 d-CH3 Lol and c-CH3 Val2); 0.72–0.70 (d, 3H, c-CH3
Val2).
1
(KBr): 3328, 1740, 1660, 1538 cm)1. H-NMR (400 MHz, CD3OH): d
10.415 (d, 1H, e-NH Trp1); 8.25 (s, 1H, NH Aib10); 8.18–8.16 (m, 1H,
NH Trp1); 8.10–8.08 (d, 1H, NH Glu(OMe)6]; 8.04 (s and d, 2H, NH
Aib8 and Ser(Bzl)9); 7.98–7.96 (m, 2H, NH Ala5 and Leu12); 7.87–
7.86 (d, 1H, NH Ala11); 7.81 (s, 1H, NH Aib4); 7.79–7.78 (d, 1H, NH
Ala7); 7.73 (s, 1H, NH Aib13); 7.63–7.61 (d, 1H, NH Glu(OMe)14);
7.59–7.57 (d, 1H, NH Val2); 7.52–7.50 (m, 2H, NH Aib14, H-4 Trp1);
7.49–7.47 (d, 1H, NH Lol); 7.36–7.24 (m, 11H, H-7 Trp1, 2 · 5CH
arom Bzl); 7.22 (d, 1H, H-2 Trp1); 7.11–7.07 (t, 1H, H-6 Trp1); 7.01–
6.97 (t, 1H, H-5 Trp1); 5.19 (s, 2H, CH2-Ph Ser(Bzl)9); 4.52 (s, 2H,
CH2-Ph Lol-Bzl); 4.50–4.49 (m, 1H, a-CH Trp1); 4.24–3.19 (m, 1H, a-
CH Glu(OMe)14); 4.18–4.12 (m, 3H, a-CH Lol, a-CH and b-CH
Ser(Bzl)9); 4.11–3.94 (m, 5H, a-CH Ala5, a-CH Glu(OMe)6, a-CH Ala7,
a-CH Ala11 and a-CH Leu12); 3.89–3.88 (d, 1H, b-CH Ser(Bzl)9); 3.62
or
or
(s, 3H, CH3 Glu(OMe)6
14); 3.56 (s, 3H, CH3 Glu(OMe)6
14);
3.49–3.41 (m, 3H, a-CH Val2 and b2-CH2 Lol); 3.26–3.24 (m, 2H, b-
CH2 Trp1); 2.65–2.40 (m, 4H, c-CH2 Glu(OMe)6 and c-CH2
Glu(OMe)14); 2.35–2.22 (m, 2H, b-CH Glu(OMe)6 and b-CH
Glu(OMe)14); 2.15–2.06 (m, 2H, b-CH Glu(OMe)6 and b-CH
Glu(OMe)14); 2.04 (s, 3H, CH3 Ac); 1.94–1.85 (m, 3H, b-CH Val2, b-
CH and c-CH Leu12); 1.81–1.71 (m, 1H, c-CH Lol); 1.65–1.60 (m, 2H,
b-CH Leu12 and b1-CH Lol); 1.58 (s, 6H, b-CH3 Aib8 and Aib10); 1.53
(s, 3H, b-CH3 Aib13); 1.52–1.49 (m, 18H, b-CH3 Aib4, Ala7, Aib8,
Aib10, Ala11 and Aib13); 1.46 (s, 3H, b-CH3 Aib4); 1.42 (s, 6H, b-CH3
Ala5 and b-CH3 Aib3); 1.37–1.39 (bs, 4H, b-CH3 Aib3 and b1-CH
Lol); 1.02–1.01 (m, 1H); 0.91–0.87 (m, 15H, 2 d-CH3 Leu12, 2 d-CH3
Lol and c-CH3 Val2); 0.72–0.70 (d, 3H, c-CH3 Val2).
FTIR absorption
Solid-phase peptide synthesis (SPPS)
The solution FTIR spectra were recorded at 293 K using a Perkin-
Elmer model 1720X FTIR spectrophotometer, nitrogen flushed,
equipped with a sample-shuttle device, at 2 cm)1 nominal resolu-
tion, averaging 100 scans. Solvent (baseline) spectra were recorded
under the same conditions. For spectral elaboration, the software
SPECTRACALC provided by Galactic (Salem, MA, USA) was employed.
Cells with path lengths of 1.0 and 10 mm (with CaF2 windows)
were used. Spectrograde deuterated chloroform (99.8%, d2) was
purchased from Merck.
Tylopeptin B was synthesized in a 0.05 mmol scale, using an
Advanced Chemtech (Louisville, KY, USA) 348X peptide synthesizer,
starting from H-L-Lol-2-chlorotrityl resin. The trityl (Trt) group was
used for masking the Gln side chain, and Boc and tert-butyl groups
were used to protect the Trp and Ser side chains, respectively. 9-
Fluorenylmethoxycarbonyl (Fmoc) deprotection was achieved with
20% piperidine in DMF (5 + 15 min). Couplings were performed in
the presence of HATU ⁄ diisopropylethylamine (DIPEA) (reaction time
45–60 min or 120 min for Aib on Aib), using an excess of 4 equiva-
lents of the carboxyl component. A single coupling protocol was
used to acylate Lol, Leu12, Ala7, Gln(Trt)6, and Val2; in all other
cases, the coupling step was repeated. N-terminal acetylation of
the peptide-resin was performed by a repeated treatment with ace-
tic anhydride (0.75 mmol) and DIPEA (0.2 mmol) in DMF for 30 min.
The 1,2-aminoalcohol peptide was cleaved from the resin upon 3–4
Nuclear magnetic resonance
Nuclear magnetic resonance experiments were carried out on a
Bruker Avance model DMX-600 spectrometer. The peptide concen-
tration was 6.6 mM in CD3OH. The alcohol –OH signal was sup-
pressed by presaturation during the relaxation delay. All
172
Chem Biol Drug Des 2010; 75: 169–181