A. LEGGIO ET AL.
(eluent: petroleum ether/Et2O 50 : 50). The organic layer was
separated, and the aqueous phase was extracted with three
additional portions of DCM (3× 10 ml). The combined organic
extracts were washed once with 1 N HCl and once with brine. The
resulting organic extracts were finally dried (Na2SO4), filtered, and
evaporated to dryness under reduced pressure conditions to
give a crude reaction product. The subsequent chromatographic
purification (diethyl ether/petroleum ether) afforded the corre-
sponding N-Nosyl-dipeptides 8a–b in 72–75% yields.
monitoring the removal of the 9-fluorenylmethanesulfonyl
protecting group by TLC (diethyl ether/petroleum ether, 60 : 40 v/v).
After evaporation of the solvent under reduced pressure,
aqueous 1 N HCl was added and the aqueous solution was
extracted with AcOEt (3 × 10 ml) in order to remove the
dibenzofulvene-amine adduct. The aqueous phase was made
basic (pH 8) with a saturated aqueous solution of NaHCO3. The
basic liquors, containing the N-deprotected product 6c, were then
treated with a solution of N-Fmoc-L-alanine chloride (9) (0.17 g,
0.52 mmol) in dry DCM (20 ml). The reaction mixture was stirred
at room temperature for about 2 h monitoring the formation of
the dipeptide by TLC (petroleum ether/Et2O 50 : 50). The organic
layer was separated, and the aqueous phase was extracted with
three additional portions of DCM (3 × 10 ml). The combined
organic extracts were washed once with aqueous 1 N HCl and
once with brine. The resulting organic layers were finally dried
(Na2SO4), filtered, and evaporated to dryness under reduced
pressure conditions to give a crude reaction product. The subse-
quent chromatographic purification (diethyl ether/petroleum
ether) afforded the corresponding N-Fmoc-dipeptide 10 (0.16 g)
N-Nosyl-L-phenylalanyl-N-methyl-L-alanine methyl ester (8a)
The product was prepared following the general procedure using
4a (0.075 g, 0.21 mmol) in DCM and diethylamine (0.22 ml). The
reaction was stirred at room temperature for 90 min. The basic
liquors, containing the deprotected N-methyl-α-amino acid
methyl ester 6a, were then treated with a solution of N-Nosyl-L-
phenylalanine (7) (0.077 g, 0.21 mmol) in dry DCM (10 ml). The
reaction was stirred at room temperature for further 90 min. The
subsequent work up afforded 68mg of the title compound 8a in
72% yield as pale yellow oil; 1H NMR (300 MHz, CDCl3) δ 1.26
(d, J = 7.2Hz, 3H, CHCH3), 2.82 (s, 3H, NCH3), 2.90 (dd, J = 14.1 and
6.9 Hz, 1H, CH2Ph), 3.05 (dd, J = 14.1 and 5.7 Hz, 1H, CH2Ph), 3.70
(s, 3H, OCH3), 4.52 (m, 1H, CHCH2Ph), 5.01 (q, J = 7.2Hz, 1H, CHCH3),
6.25 (d, J= 9.6 Hz, 1H, NH), 7.09–7.27 (m, 5H, C6H5CH2), 7.80
(d, J = 9.0Hz, 2H, ArH-Ns), 8.19 (d, J = 9.0 Hz, 2H, ArH-Ns); 13C NMR
(75 MHz, CDCl3) δ 14.03, 31.19, 39.30, 52.38, 52.67, 54.90, 124.03,
127.21, 128.06, 128.58, 129.51, 135.27, 146.08, 149.76, 170.89,
171.32; ESI-QTOF-MS: 450.1355 (M+ H)+, 472.1187 (M + Na)+.
1
as a colourless oil in 72% yield; H NMR (300 MHz, CDCl3) δ 0.86
(d, J = 6.8 Hz, 3 H, CHCH3), 1.02 (d, J = 6.8 Hz, 3 H, CHCH3), 1.35
(d, J = 6.8 Hz, 3 H, CH3), 2.22 (m, 1 H, CH(CH3)2), 3.02 (s, 3 H,
NCH3), 3.69 (s, 3 H, OCH3), 4.22 (m, 1 H, CHFmoc), 4.32–4.38
(m, 2 H, CH2Fmoc), 4.70 (m, 1 H, CHCH3), 4.86 (d, J = 9.8 Hz, 1 H,
CHCH(CH3)2), 5.86 (d, J = 7.8 Hz, 1 H, NH), 7.21–7.82 (m, 8 H ArH);
anal. calcd for C25H30N2O5: C, 68.47; H, 6.90; N, 6.39, found:
C, 68.43; H, 6.93; N, 6.41.
N-Nosyl-L-phenylalanyl-N-methyl-D-alanine methyl ester (8b)
Results and Discussion
The product was prepared following the general procedure using
4b (0.11g, 0.31 mmol) in DCM and diethylamine (0.32 ml). The
reaction was stirred at room temperature for 2 h. The basic liquors,
containing the deprotected N-methyl-α-amino acid methyl ester
6b, were then treated with a solution of N-Nosyl-L-phenylalanine
(7) (0.11 g, 0.31 mmol) in dry DCM (15 ml). The reaction was stirred
at room temperature for 2 h. The subsequent work up afforded
104 mg of the title compound 8b in 75% yield as pale yellow oil;
1H NMR (300MHz, CDCl3) δ 1.15 (d, J = 7.5Hz, 3H, CHCH3), 2.51
(s, 3H, NCH3), 2.90–3.08 (m, 2H, CH2Ph), 3.61 (s, 3H, OCH3), 4.55
(m, 1H, CHCH2Ph), 4.90 (q, J= 7.5 Hz, 1H, CHCH3), 5.90 (d,
J = 9.0Hz, 1H, NH), 7.11–7.29 (m, 5H, C6H5CH2), 7.91 (d, J = 8.7Hz,
2H, ArH-Ns), 8,27 (d, J =8.7 Hz, 2H, ArH-Ns); 13C NMR (75 MHz, CDCl3)
δ 14.35, 31.05, 40.87, 52.23, 52.31, 54.48, 124.40, 127.53, 128.07,
128.66, 129.56, 134.93, 145.52, 149.79, 170.59, 171.25; ESI-QTOF-
MS: 450.1356 (M + H)+, 472.1177 (M+ Na)+.
The development of our methodology started synthesising
N-Fms-α-amino acid methyl esters. To this aim, the protecting
reagent (9H-fluoren-9-yl)methanesulfonyl chloride (Fms-Cl) (1)
(Scheme 1) was prepared starting from (9H-fluoren-9-yl)methanol
1
[25]. The molecular structure of (1) was assigned by H and 13C
NMR spectroscopy. The obtained spectroscopic data were
consistent with those reported [25].
N-9-fluorenylmethanesulfonyl-α-amino acid methyl esters (3a–f)
were prepared by treating lipophilic α-amino acid methyl esters
(2a–f) with FmsCl (1) and N,N-diisopropylethylamine (DIPEA) in
DCM at room temperature (Scheme 1).
After about 2 h, the reaction afforded N-9-fluorenylmethane-
sulfonyl-α-amino acid methyl esters (3a–f) in very good yields
(Table 1) and high purity as verified by TLC and elemental analysis.
1H NMR and 13C NMR analyses of 3a–f fully confirmed the
molecular structure of N-9-fluorenylmethanesulfonyl-α-amino acid
methyl esters.
Removal of 9-Fluorenylmethanesulfonyl Protecting Group
and Synthesis of N-Fmoc-L-Alanine-N-Methyl-L-Valine Methyl
Ester (10)
The protection reaction was subsequently tested with one side
chain functionalized amino acid, L-tyrosine methyl ester protected
on side chain with the benzyl group (2 g). The corresponding
N-Fms-protected derivative 3g was obtained in 72% yields
Diethylamine (0.54 ml) was added to a solution of 4c (0.20 g,
0.52 mmol) in dry DCM. The resulting mixture was stirred for
about 2 h at room temperature under nitrogen atmosphere
1
(Scheme 1 and Table 1) and high purity as confirmed by H and
13C NMR spectroscopy.
Scheme 1. Synthesis of N-9-fluorenylmethanesulfonyl-α-amino acid methyl esters 3a–g.
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Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.
J. Pept. Sci. 2015; 21: 644–650