ORIGINAL ARTICLES
Fig. 2: Calcium mobilization studies in HEK293mNPSR cells. Left panel: agonist effects of NPS, [Ipv5]NPS A and B. Right panel: antagonist effects of [Ipv5]NPS B and
[tBu-D-Gly5]NPS vs NPS. Data are the mean sem of 4 separate experiments performed in duplicate.
including the synthesis of novel non natural Val derivatives.
The incorporation of Ipv in position 5 of NPS was success-
fully performed using classical solid phase peptide synthesis
procedures and the diastereomeric mixture of [L-Ipv5]NPS and
[D-Ipv5]NPS obtained was successfully separated in preparative
HPLC. In calcium mobilization assays [Ipv5]NPS A behaves as
a NPSR partial agonist while [Ipv5]NPS B as a pure antagonist
and both peptides displayed low potency at the mouse NPSR.
Our results indicated that the unnatural amino acid described
in this study could be used as a novel building block for com-
binatorial libraries of peptides, as well as for structure-activity
relationship studies of bioactive peptides.
CH(CH3)2). 13C NMR (100 MHz, CDCl3): ␦ 168.7, 60.8, 58.6, 39.1, 34.5,
20.9, 17.3, 14.2. MS (ESI): [MH]+ = 245.
3.1.4. 2-(1,1,2-Trimethyl-propyl)-malonic acid monoethyl ester (3)
To a stirred solution of 2 (521 mg, 2.135 mmol) in 15 mL of ethanol, a
1N solution of NaOH (5.5 mL, 7.9 mmol) was added dropwise. The reac-
tion mixture was stirred at room temperature for 10 h, monitored by TLC
(EtOAc/Light Petroleum/AcOH, 1/5/0.3) and mass spectrometry until to the
appearing of diacid molecular weight. The solvent was evaporated to dry-
ness and the aqueous layer extracted twice with ethyl acetate (10 mL each)
to remove the di-ester derivative. Then aqueous layer was acidified with 1N
HCl and extracted 3 times with ethyl acetate (20 mL each) to recover the
desired monoacid, Yield 401 mg (87%), based on the consumed di-ester,
enough pure to be used in the next reaction without further purification.1H
NMR (400 MHz, CDCl3): ␦ 4.27-4.16 (2H, q, J = 7.2 Hz, OCH2CH3), 3.56
(1H, s, EtOOC-CH-COOH), 1.85-1.78 (1H, m, CH(CH3)2), 1.32-1.25 (3H,
t, J = 7.2 Hz, OCH2CH3), 1.05 (3H, s, CH3), 1.00 (3H, s, CH3), 0.92-0.90
(3H, d, J = 3.6 Hz, CH(CH3)2), 0.88-0.87 (3H, d, J = 3.6 Hz, CH(CH3)2).
13C NMR (100 MHz, CDCl3): ␦ 171.7, 170.8, 61.7, 58.1, 40.1, 34.4, 21.1,
20.9, 17.4, 17.3, 14.1. MS (ESI): [MH]+ = 217.
3. Experimental
3.1. Synthesis
3.1.1. General procedures
HPLC grade solvents were purchased from Sigma Aldrich (Steinheim,
Germany). The purity of the tested compounds has been assessed by RP-
HPLC. All compounds showed > 95% purity. One-dimensional and two
dimensional NMR spectra were recorded on a VARIAN 400 MHz instru-
ment. Chemical shifts are given in ppm (␦) relative to TMS and coupling
constants are in Hz. MS analyses were performed on a ESI-Micromass ZMD
2000. Optical rotation data were recorded on a Perkin-Elmer polarime-
ter 241. Flash chromatography was carried out on a silica gel (Merck,
230–400 Mesh). Silica gel (Polygram SIL G/UV254) was used for thin
layer chromatography.
3.1.5. 2-(9H-Fluoren-9-yl-methoxycarbonylamino)-3,3,4-trimethyl-
pentanoic acid ethyl ester (4)
To a stirred solution of 3 (113 mg, 0.52 mmol) in dry toluene, triethylamine
(0.18 mL, 1.3 mmol) and diphenylphosphoryl azide (0.17 mL, 0.78 mmol)
were added. The reaction mixture was heated at reflux for 2 h. After cooling
at room temperature, fluorenyl-methanol (204 mg, 1.04 mmol) was added
and the reaction was heated again at reflux overnight. After evaporation
of the solvent, the crude material was purified by flash chromatography
(EtOAc/Light petroleum, 0.5/9.5) to give compound 4, Yield 99.96 mg
(47%). 1H NMR (400 MHz, CDCl3): ␦ 7.78-7.74 (4H, d, J = 8 Hz, Ar Fmoc),
7.61-7.57(4H, d, J = 8 Hz, ArFmoc), 5.34(2H, d, J = 8 Hz, CH-CH2-O), 4.41
(1H, s, EtOOC-CH-NH), 4.20 (2H, q, J = 7.6 Hz, CH3-CH2-O), 4.17 (1H,
m, (Ar)2-CH-CH2-O), 1.58 (1H, m, CH3-CH-CH3), 1.28 (3H, t, J = 7.6 Hz,
CH3-CH2-O), 0.93 (6H, s, (CH3)2-C), 0.89 (6H, m, (CH3)2-CH).
3.1.2. 2-Isopropylidene-malonic acid diethyl ester (1)
To a stirred solution of diethyl malonate (20 mL, 131.87 mmol), acetone
(14.5 mL 197.81 mmol) and acetic anhydride (15.57 mL, 164.84 mmol)
anhydrous zinc dichloride (2.68 g, 19.78 mmol) was added. The reaction
mixture was heated at reflux for 24 h and checked by TLC using EtOAc/light
petroleum 0.5/9.5 as eluent. After cooling at room temperature, toluene
(50 mL) was added and the organic phase was washed twice with water
(20 mL each), the organic phase was concentrated in vacuo and the crude
product purified by flash chromatography (EtOAc/Light petroleum 0.5/9.5)
to yield compound 1, Yield 12.63 g (48%). 1H NMR (400 MHz, CDCl3): ␦
4.27-4.17 (4H, q, J = 7 Hz, OCH2CH3), 2.05 (6H, s, 2CH3), 1.31-1.24 (6H,
t, J = 7 Hz, 2(OCH2CH3)). MS (ESI): [MH]+ = 201.
3.1.6. 2-(9H-Fluoren-9-yl methoxycarbonylamino)-
3,3,4-trimethyl-pentanoic acid (5)
Compound 4 (75 mg, 0.183 mmol) was dissolved in glacial acetic acid
(2 mL) and HCl 37% (2 mL); reaction mixture was than heated at reflux
over night and monitored by TLC (EtOAc/Light petroleum/AcOH, 1/1/0.3)
successively, reaction mixture was evaporated in vacuo and the crude residue
was dissolved in water (5 mL) and extracted three times with EtOAc (15 mL
each). The organic phases were dried over Na2SO4 and evaporated to dry-
ness to obtain compound 5 in 95% yield as a pure compound. 1H NMR
(400 MHz, CDCl3): ␦ 7.78-7.74 (4H, d, J = 8 Hz, Ar Fmoc), 7.61-7.57 (4H,
d, J = 8 Hz, Ar Fmoc), 5.34 (2H, d, J = 8 Hz, Fmoc-CH2-O), 4.41 (1H, s,
HOOC-CH-NH), 4.17 (1H, m, (Ar)2-CH-CH2-O), 1.58 (1H, m, CH3-CH-
CH3), 0.93 (6H, s, (CH3)2-C), 0.89 (6H, m, (CH3)2-CH). HR-MS (ESI):
[MH]+ = 382.20171, calc: 382.20183.
3.1.3. 2-(1,1,2-Trimethyl-propyl)-malonic acid diethyl ester (2)
In a two neck round bottom flask, under argon atmosphere, diethyl ether
(20 mL), magnesium (82.4 mg, 3.4 mmol) and isopropylbromide (0.38 mL,
4.06 mmol) were added. When all the metallic magnesium was consumed,
the solution was cooled at -5 ◦C and a catalytically amount of copper (I) chlo-
ride (4.5 mg, 0.045 mmol) was added. After 15 min compound 1 (451.5 mg,
2.26 mmol) dissolved in diethyl ether were added. After 4 h, 10 mL of
10% sulphuric acid were added and the organic phase was separated, dried
over sodium sulphate and purified by flash chromatography (EtOAc/ light
petroleum 0.5/9.5) to give compound 2, Yield 60.66 mg (11%). 1H NMR
(400 MHz, CDCl3): ␦ 4.19-4.11 (4H, q, J = 7.2 Hz, OCH2CH3,), 3.50 (1H,
s, CH(COOEt)2), 1.85-1.79 (1H, m, CH), 1.28-1.21 (6H, t, J = 7.2 Hz,
2(OCH2CH3)), 1.04 (6H, s, -C(CH3)2), 0.87-0.84 (6H, d, J = 6.8 Hz, -
3.1.7. General procedures for the solid phase peptide synthesis and
purification
Fmoc-Ser(tBu)-4-benzyloxybenzyl alcohol resin (Fmoc-Ser(tBu)-Wang
resin) (0.62 mmol/g, 0.2 g) was treated with 20% piperine/N,
N-dimethylformamide (DMF) and linked with Fmoc-Lys(Boc)-OH
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Pharmazie 69 (2014)