S.N. Khattab et al. / Tetrahedron 68 (2012) 3056e3062
3061
4H, 2CH2). 13C NMR (C2D6SO):
109.79, 127.44, 157.73.
d
24.14, 25.59, 26.19, 43.66, 48.03,
46.64, 47.75, 66.56, 66.77, 72.47, 107.01, 120.49, 125.28, 127.60,
128.48, 141.56, 142.68, 151.41, 155.67. A sample for exact mass de-
termination was prepared, dissolving in H2O/CH3CN and diluting in
H2O/CH3CN/1%TFA: m/z¼406.14105 [MþH]þ, 428.12304 [MþNa]þ,
811.27424 [2MþH]þ.
4.2.4. N-Hydroxy-2-morpholino-2-oxoacetimidoyl cyanide (18).33 The
product was obtained as white crystals, mp 193e194 ꢁC (lit.
155e160)1 in yield 79% (from ethyl cyanoacetic ester). 1H NMR
(C3D6O):
d
3.66e3.75 (m, 8H, 4CH2), 13.12 (s, 1H, OH, D2O ex-
4.4. Synthesis of N-(9-fluorenylmethyloxycarbonyl)glycine
(Fmoc-Gly-OH)
changeable). 13C NMR (C2D6SO):
127.31, 158.23.
d
43.13, 47.62, 66.22, 66.51, 109.79,
A solution of the Fmoc-OX derivatives (20 mmol) in 100 mL
acetone was added dropwise to a stirred solution of Glycine
(20 mmol) and NaHCO3 (50 mmol) in 100 mL water. After stirring
overnight the reaction mixture was concentrated under reduced
pressure and then extracted with CH2Cl2 (50 mL) to remove the
unreacted Fmoc-OX derivatives. The reaction mixture was cooled
and acidified with 10% HCl to congo red litmus paper to give a white
solid, which was filtered and washed with water several times,
dried to give a white solid, with mp¼175e178 ꢁC (mp of an au-
thentic commercial sample of 99.8% purity¼177e178 ꢁC;
literature14¼174e176 ꢁC (Table 2). The purity of Fmoc-Gly-OH was
4.3. General method for preparation of Fmoc-oxime
derivatives (6e9)
A
solution of (9H-fluoren-9-yl)methyl carbonochloridate
(10 mmol) in 30 mL CH2Cl2 was added slowly to a solution of
(10 mmol) of oxime (15e18) and sodium carbonate (20 mmol) in
20 mL of H2O with stirring at 0 ꢁC. The resulting clear mixture was
stirred at 0 ꢁC for 30 min and then at room temperature for 2 h.
After dilution with CH2Cl2 (50 mL), the organic phase was collected
and washed with water and saturated aqueous NaCl (30 mL), dried
over anhydrous MgSO4. Filtered and the solvent was removed with
a rotary evaporator, the residue was recrystallized from CH2Cl2/
hexane to give Fmoc-oxime derivatives.
determined by injection of 5
Gly-OH in acetonitrile onto HPLC using the following conditions:
Waters SunFire C18 column (2.5
ml of a sample prepared from Fmoc-
mm, 4.6ꢂ75 mm); linear gradient
10e100% of 0.036% TFA in CH3CN/0.045%TFA in H2O over 8 min;
flow¼1.0 ml/min; PDA detection at 254 nm tR¼Fmoc-Gly-OH¼6.2;
tR¼Fmoc-Gly-Gly-OH¼5.6 min and coinjection with an authentic
sample of Fmoc-Gly-OH and Fmoc-Gly-Gly-OH.
4.3.1. N-(((9H-fluoren-9-yl)methoxy)carbonyloxy)-2-amino-2-
oxoacetimidoyl cyanide (6). The product was obtained as white
crystals, mp 152e153 ꢁC in yield 91%. 1H NMR (C2D6SO):
d 4.46 (t,
1H, CH, J¼6.4 Hz), 4.83 (d, 2H, CH2, J¼6.4 Hz), 7.36 (t, 2H, AreH,
J¼7.6 Hz), 7.44e7.47 (m, 3H, AreHþNH), 7.73 (d, 2H, AreH,
4.5. Synthesis of N-(9-fluorenylmethyloxycarbonyl)alanine
(Fmoc-Ala-OH)
J¼7.2 Hz), 7.89e7.91 (m, 3H, AreHþNH). 13C NMR:
d
46.72, 71.68,
107.19, 120.40, 125.26, 127.51, 128.26, 134.07, 141.56, 143.26, 151.08,
157.82. A sample for exact mass determination was prepared, dis-
solving in H2O/CH3CN 1:1 and diluting in H2O/CH3CN/1%TFA: m/
z¼336.09841 [MþH]þ, 358.08030 [MþNa]þ, 693.17150 [2MþNa]þ.
A solution of the Fmoc-OX derivatives (20 mmol) in 100 mL
acetone was added dropwise to a stirred solution of Alanine
(20 mmol) and NaHCO3 (50 mmol) in 100 mL water. After stirring
overnight the reaction mixture was concentrated under reduced
pressure and then extracted with CH2Cl2 (50 mL) to remove the
unreacted Fmoc-OX derivatives. The reaction mixture was cooled
and acidified with 10% HCl to congo red litmus paper to give a white
solid, which was filtered and washed with water several times,
dried to give a white solid, mp 137e142 ꢁC (authentic commercial
sample mp 147e153 ꢁC (Table 2). The purity of Fmoc-Ala-OH was
4.3.2. N-(((9H-fluoren-9-yl)methoxy)carbonyloxy)-2-(ethylamino)-
2-oxoacetimidoyl cyanide (7). The product was obtained as white
crystals, mp 160e161 ꢁC in yield 84%. 1H NMR (CDCl3):
d 1.24 (t, 3H,
CH3, J¼7.2 Hz), 3.42e3.49 (m, 2H, CH2), 4.35 (t, 1H, CH, J¼7.6 Hz),
4.63 (d, 2H, CH2, J¼7.6 Hz), 7.35 (t, 2H, AreH, J¼7.6 Hz), 7.44 (t, 2H,
AreH, J¼7.6 Hz), 7.61 (d, 2H, AreH, J¼6.8 Hz), 7.79 (d, 2H, AreH,
J¼6.8 Hz). 13C NMR:
d
14.64, 35.46, 46.60, 72.64, 106.54, 120.51,
determined by injection of 5
Ala-OH in acetonitrile onto HPLC using the following conditions:
Waters SunFire C18 column (2.5
ml of a sample prepared from Fmoc-
125.28, 127.63, 128.52, 133.48, 141.57, 142.61, 151.41, 155.53. A
sample for exact mass determination was prepared, dissolving in
H2O/CH3CN and diluting in H2O/CH3CN/1%TFA: m/z¼364.12973
[MþH]þ, 386.11162 [MþNa]þ, 727.2516 [2MþH]þ, 749.23387
[2MþNa]þ.
mm, 4.6ꢂ75 mm); linear gradient
10e100% of 0.036% TFA in CH3CN/0.045%TFA in H2O over 8 min;
flow¼1.0 ml/min; PDA detection at 254 nm tR¼Fmoc-Ala-
OH¼6.6 min; tR¼Fmoc-Ala-Ala-OH¼6.2 min and coinjection with
an authentic sample of Fmoc-Ala-OH and Fmoc-Ala-Ala-OH (mp:
188e189 ꢁC).
4.3.3. N-(((9H-fluoren-9-yl)methoxy)carbonyloxy)-2-oxo-2-(piper-
idin-1-yl)acetimidoyl cyanide (8). The product was obtained as
waxy product in yield 79%. 1H NMR (CDCl3):
d
1.66e1.70 (m, 6H,
Acknowledgements
3CH2), 3.58, 3.67 (2t, 4H, 2CH2, J¼5.6 Hz), 4.34 (t, 1H, CH, J¼7.6 Hz),
4.59 (d, 2H, CH2, J¼7.6 Hz), 7.32e7.37 (m, 2H, AreH), 7.44 (t, 2H,
AreH, J¼7.6 Hz), 7.61 (d, 2H, AreH, J¼6.8 Hz), 7.79 (d, 2H, AreH,
The work in the laboratory of the authors has been partially fi-
ꢀ
nanced by the Secretaría de Estado de Cooperacion Internacional
J¼6.8 Hz). 13C NMR:
d
24.33, 25.48, 26.47, 44.44, 46.65, 48.59, 72.33,
(AECI), the CICYT (CTQ2009-07758), the Generalitat de Catalunya
(2009SGR 1024), the Institute for Research in Biomedicine Barce-
lona (IRB Barcelona), and the Barcelona Science Park. RSF thanks
107.17, 120.46, 125.33, 127.59, 128.44, 133.06, 141.55, 142.78, 151.57,
155.31. A sample for exact mass determination was prepared, dis-
solving in H2O/CH3CN and diluting in H2O/CH3CN/1%TFA: m/
z¼404.16274 [MþH]þ, 426.14243 [MþNa]þ, 829.29634 [2MþNa]þ.
ꢀ
the Ministerio de Educacion y Ciencia for a FPU PhD fellowship.
Yoav Luxembourg (Luxembourg Biotechnologies) is thanked for his
support.
4.3.4. N-(((9H-fluoren-9-yl)methoxy)carbonyloxy)-2-morpholino-2-
oxoacetimidoyl cyanide (9). The product was obtained as white
Supplementary data
crystals, mp 135e136 ꢁC in yield 88%. 1H NMR (CDCl3):
d 3.71e3.79
(m, 8H, 4CH2), 4.34 (t, 1H, CH, J¼7.2 Hz), 4.62 (d, 2H, CH2, J¼7.2 Hz),
1H and 13C NMR spectra of compounds 6e9 and 15e18. Sup-
plementary data related to this article can be found online at
7.35 (t, 2H, AreH, J¼6.4 Hz), 7.43 (t, 2H, AreH, J¼7.6 Hz), 7.62 (d, 2H,
AreH, J¼7.2 Hz), 7.79 (d, 2H, AreH, J¼7.2 Hz). 13C NMR:
d 43.71,