S. Vilà, E. Badosa, E. Montesinos, L. Feliu, M. Planas
FULL PAPER
(CH2Cl2). A solution of Fmoc-Lys-OH (500 mg, 1.40 mmol) in 1,4-
dioxane/2% aqueous Na2CO3 (1:1, 1.69 mL) was then added drop-
wise and the resulting mixture was stirred for 24 h. The pH was
maintained at 8–9 by adding 10% aqueous Na2CO3. The reaction
was monitored by TLC (EtOAc/MeOH/NH3, 5:2:1). Once the reac-
tion was finished, H2O (40 mL) was added and the resulting sus-
pension was washed with tert-butyl methyl ether (3ϫ 20 mL). The
aqueous phase was acidified to pH 2–3 with 3 n HCl and extracted
with EtOAc (3ϫ 20 mL). The organic fractions were combined,
washed with an aqueous HCl solution (pH = 2; 2ϫ 40 mL) and
dried with anhydrous MgSO4. Evaporation of the organic solvent
under reduced pressure yielded Fmoc-Lys(pNZ)-OH as a yellow
oil, which solidified to a white solid upon addition of diethyl ether
Experimental Section
General: Manual peptide syntheses were performed in polypropyl-
ene syringes fitted with a polyethylene porous disk. Solvents and
soluble reagents were removed in vacuo. Commercially available
reagents were used throughout without purification. Solvents were
purified and dried by passing them through an activated alumina
purification system (MBraun SPS-800) or by conventional di-
stillation techniques. Flash chromatography purifications were per-
formed on silica gel 60 (230–400 mesh, Merck).
All compounds were analysed under standard analytical HPLC
conditions with a Dionex liquid chromatography instrument. De-
tection was performed at 220 nm. Analysis was carried out with a
Kromasil 100 C18 (40 mmϫ 4.6 mm, 3.5 μm) column using a 2–
100% B linear gradient over 7 min at a flow rate of 1 mL/min.
Solvent A was 0.1% aqueous TFA and solvent B was 0.1% TFA
in CH3CN.
(309 mg, 42% yield). Rf = 0.5 (AcOH/MeOH/NH3, 5:2:1); [α]1D9
=
–10 (c = 1%, DMF); tR = 8.48 min. 1H NMR (300 MHz,
[D6]DMSO): δ = 1.29–1.40 [m, 4 H, CH2(δ), CH2(γ)], 1.56–1.71
[m, 2 H, CH2(β)], 2.99 [q, J = 6.3 Hz, 2 H, CH2(ε)], 3.87–3.94 [m,
1 H, CH(α)], 4.19–4.29 [m, 3 H, CH2(Fmoc), CH(Fmoc)], 5.15 (s, 2 H,
OCH2), 7.31 (t, J = 7.5 Hz, 2 H, 2 CHarom.), 7.39–7.44 (m, 3 H, 1
NH, 2 CHarom.), 7.59 (d, J = 9.0 Hz, 2 H, 2 CHarom.), 7.63 (d, J =
7.8 Hz, 1 H, NH), 7.72 (d, J = 7.5 Hz, 2 H, 2 CHarom.), 7.89 (d, J
= 7.2 Hz, 2 H, 2 CHarom.), 8.23 (d, J = 9.0 Hz, 2 H, 2 CHarom.),
12.55 (br. s, 1 H, COOH) ppm. 13C NMR (100 MHz, [D6]DMSO):
δ = 23.90 [CH2(γ)], 29.87, 31.34 [CH2(β), CH2(δ)], 41.90 [CH2(ε)],
47.60 [CH(Fmoc)], 54.73 [CH(α)], 64.90 (OCH2), 66.55 (OCH2),
121.10 (2 CHarom.), 124.10 (2 CHarom.), 126.25 (2 CHarom.), 128.04
(2 CHarom.), 128.61 (2 CHarom.), 129.04 (2 CHarom.), 141.67 (1
ESI-MS analyses were performed with an Esquire 6000 ESI ion
Trap LC/MS (Bruker Daltonics) instrument equipped with an elec-
trospray ion source (University of Girona). The instrument was
operated in the positive ESI(+) ion mode. Samples (5 μL) were in-
troduced into the mass spectrometer ion source directly through an
HPLC auto-sampler. The mobile phase (80:20 CH3CN/H2O at a
flow rate of 100 μL/min) was delivered by a 1100 Series HPLC
pump (Agilent). Nitrogen was employed as both the drying and
nebulizing gas. HRMS were recorded under conditions of ESI with
a Bruker MicroTof-Q IITM instrument using a hybrid quadrupole
time-of-flight mass spectrometer (University of Girona). Samples
were introduced into the mass spectrometer ion source by direct
infusion through a syringe pump with external calibration using
sodium formate. The instrument was operated in the positive
ESI(+) ion mode.
C
arom.), 141.69 (1 Carom.), 144.77 (1 Carom.), 144.80 (1 Carom.),
146.31 (1 Carom.), 147.84 (1 Carom.), 156.73 (NHCOO), 157.15
(NHCOO), 174.99 (COOH) ppm. MS (ESI): m/z = 548.1 [M +
H]+.
Synthesis of Fmoc-Glu(NH-CH2-CϵCH)-OtBu:[10a] Propargyl-
amine (110 μL, 1.56 mmol), Oxyma (404 mg, 2.84 mmol), N,NЈ-di-
isopropylethylamine (DIEA; 720 μL, 4.22 mmol) and 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDC; 320 mg, 3.12 mmol)
were added to a solution of Fmoc-Glu-OtBu (600 mg, 1.42 mmol)
in dry THF (70 mL) under N2. The reaction mixture was stirred at
room temperature under N2 and monitored by HPLC. After 24 h,
further EDC (160 mg), Oxyma (101 mg) and DIEA (125 μL) were
added and the mixture was stirred for an additional 24 h. The reac-
tion was stopped by addition of EtOH (2 mL). Removal of the
solvents under vacuum gave a residue that was dissolved in EtOAc
(50 mL), extracted with H2SO4 (0.5 m, 4ϫ 50 mL), washed with
distilled H2O (2ϫ 50 mL), dried with anhydrous MgSO4 and con-
centrated. The crude product was purified by column chromatog-
raphy (hexane/EtOAc, 2:1) to give Fmoc-Glu(NH-CH2-CϵCH)-
OtBu as a yellow oil (639 mg, 98% yield). Rf = 0.81 (hexane/
1H and 13C NMR spectra were recorded with a Bruker 300 or
400 MHz NMR spectrometer. Chemical shifts are reported as δ
values (ppm) directly referenced to the solvent signal.
Synthesis of Fmoc-Lys-OH: Fmoc-Lys(Boc)-OH (2.0 g, 4.40 mmol)
was dissolved in a solution of TFA/CH2Cl2 (1:1; 32 mL) and stirred
for 2 h at room temperature. Following TFA evaporation and di-
ethyl ether extraction, Fmoc-Lys-OH was obtained as a white pow-
der (1.54 g, 98% yield). Rf = 0.5 (CHCl3/MeOH/AcOH, 5:3:1); tR
= 7.16 min. IR (neat): ν = 3066.47 (ϵCH, s), 1670.64 (C=O, s),
˜
1519.64 (C=C), 1449.63 (δ CH2), 1181.57 (δ C–H ip), 789.19 (γ
CH2), 738.60 (δ NH opp) cm–1. 1H NMR (400 MHz,
[D6]DMSO): δ = 1.34–1.43 [m, 2 H, CH2(γ)], 1.49–1.74 [m, 4 H,
CH2(β), CH2(δ)], 2.73–2.78 [m, 2 H, CH2(ε)], 3.90–3.96 [m, 1 H,
CH(α)], 4.21–4.26 [m,
1 H, CH(Fmoc)], 4.28–4.34 [m, 2 H,
EtOAc, 1:5); tR = 8.89 min. IR (neat): ν = 3305.99 (ϵCH, s),
˜
CH2(Fmoc)], 7.33 (td, J = 0.7, 7.2 Hz, 2 H, 2 CHarom.), 7.42 (t, J =
7.2 Hz, 2 H, 2 CHarom.), 7.63 (d, J = 7.2 Hz, 1 H, 1 CHarom.), 7.71–
7.74 (m, 1 H, 1 CHarom.), 7.79 (br., 1 H, CONH), 7.89 (d, J =
7.2 Hz, 2 H, 2 CHarom.) ppm. 13C NMR (100 MHz, [D6]DMSO):
δ = 22.57 [CH2(γ)], 26.51 [CH2(β)], 30.17 [CH2(δ)], 38.59 [CH2(ε)],
46.66 [CH(Fmoc)], 53.61 [CH(α)], 65.58 [CH2(Fmoc)], 120.13 (1
CHarom.), 120.14 (1 CHarom.), 125.26 (2 CHarom.), 127.07 (1
CHarom.), 127.08 (1 CHarom.), 127.65 (2 CHarom.), 140.73 (1 Carom.),
140.76 (1 Carom.), 143.79 (1 Carom.), 143.81 (1 Carom.), 156.20
(CONH), 173.85 (COOH) ppm. MS (ESI): m/z = 369.1 [M + H]+.
2980.46 (CϵC, s), 1725.79, 1689.73 (C=O, s), 1637.08 (δ NH2),
1083.99 (C–O, s), 738.99 (δ NH opp), 646.23 (δ ϵCH) cm–1. 1H
NMR (400 MHz, CDCl3): δ = 1.51 [s, 9 H, C(CH3)3], 1.90–1.97
[m, 2 H, 1 CH2(β), 1 CH2(γ)], 2.23–2.30 [m, 3 H, 1 CH2(β), 1
CH2(γ), 1 CϵCH], 4.07–4.09 (m, 2 H, NCH2), 4.24–4.27 [m, 2 H,
1 CH(Fmoc), 1 CH(α)], 4.42–4.51 (m, 2 H, OCH2), 5.59 (d, J =
7.6 Hz, 1 H, NHCOO), 6.32 (br., 1 H, CONH), 7.36 (t, J = 7.6 Hz,
2 H, 2 CHarom.), 7.45 (t, J = 7.6 Hz, 2 H, 2 CHarom.), 7.63 (d, J =
7.6 Hz, 2 H, 2 CHarom.), 7.81 (d, J = 7.6 Hz, 2 H, 2 CHarom.) ppm.
13C NMR (100 MHz, CDCl3): δ = 27.92 (CH3), 29.06, 29.19, 32.23
Synthesis of Fmoc-Lys(pNZ)-OH:[14] p-Nitrobenzyl chloroformate [CH2(β), CH2(γ), CH2N], 47.15 [CH(Fmoc)], 53.80 [CH(α)], 66.93
(301 mg, 1.35 mmol) was dissolved in 1,4-dioxane (590 μL) and a
solution of NaN3 (106 mg, 1.62 mmol) in H2O (420 μL) was added.
The resulting emulsion was stirred for 2 h and the formation of p-
nitrobenzyl azidoformate (pNZ-N3) was monitored by TLC
(OCH2), 71.52 (ϵCH), 79.41 (Cϵ), 82.63 [C(CH3)3], 119.94 (1
CHarom.), 119.97 (1 CHarom.), 125.01 (1 CHarom.), 125.06 (1
CHarom.), 127.04 (2 CHarom.), 127.70 (2 CHarom.), 141.25 (1 Carom.),
141.29 (1 Carom.), 143.57 (1 Carom.), 143.81 (1 Carom.), 156.42
1124
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Eur. J. Org. Chem. 2015, 1117–1129