1644 Bioconjugate Chem., Vol. 21, No. 9, 2010
Wang et al.
ate buffer (0.1 M, pH 8.0; 4 mL) while stirring. After 4 h at
room temperature, the reaction mixture was acidified by HCl
solution (1 N) to assist in the formation of white precipitate. The
white solid was washed with cold HCl solution, dried over ether,
5 (0.25 g, 1.14 mmol) in DMF (3 mL) dropwise while stirring.
After overnight reaction at room temperature, the mixture was
filtered, evaporated in vacuo, and loaded to a silica column for
further purification. The products were separated by eluting with
mobile phases of 5-20% MeOH in DCM to afford 6 (0.45 g;
93.8%) as a yellowish solid. 1H NMR (400 MHz) (CD3OD) δ:
4.53 (t, 1H, CHN), 4.34 (t, 1H, CHN), 3.25 (dd, 1H, CHS),
3.20 (t, 2H, CH2NH), 3.08 (t, 2H, CH2NH), 2.96 (d, 1H, CHHS),
2.73 (d, 1H, CHHS), 2.22 (t, 4H, CH2CO), 1.38-1.80 (m, 29H).
13C NMR (100.67 MHz) (DMSO) δ: 176.02, 175.97, 166.05,
158.50, 79.50, 63.39, 61.63, 57.01, 41.24, 41.04, 40.24, 36.99,
36.82, 30.90, 30.35, 30.13, 29.78, 29.49, 28.80, 27.56, 26.92,
26.74. HRMS (ESI) calculated for C27H49N5O5S, [M+Na]+
578.3352 (calcd.), 578.3349 (found).
1
and air-dried to afford 3 (0.30 g; 80.5%). H NMR (400 MHz)
(DMSO) δ: 7.78 (s, 1H, CONH), 6.46 (s, 1H, CONH), 6.38 (s,
1H, CONH), 4.32 (t, 1H, CHN), 4.14 (t, 1H, CHN), 3.11 (dd,
1H, CHS), 3.02 (t, 2H, CH2NH), 2.84 (d, 1H, CHHS), 2.59 (d,
1H, CHHS), 2.18 (t, 2H, CH2COOH), 2.05 (t, 2H, CH2CO),
1.23-1.68 (m, 12H). 13C NMR (100.67 MHz) (DMSO) δ:
174.45, 171.79, 162.70, 61.03, 59.18, 55.43, 35.21, 33.60, 28.90,
28.02, 25.97, 25.33, 24.22. HRMS (ESI) calculated for
C16H27N3O4S, [M+Na]+ 380.1620 (calcd.), 380.1617 (found).
6-[5-(2-Oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pentanoy-
lamino]-hexanoic Acid 2,5-Dioxo-pyrrolidin-1-yl Ester (4). Syn-
thesis of 4 with high purity was achieved following the method
of Wilchek and Bayer with critical changes in workup proce-
dures (28). 6-[5-(2-Oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-
pentanoylamino]-hexanoic acid (3; 0.31 g, 0.87 mmol) was
completely dissolved in DMF (20 mL) with gentle warming.
After cooling the 3 solution to room temperature without
reprecipitation, DCC (0.34 g, 1.65 mmol) and pyridine (0.07
mL, 0.83 mmol) were added to the solution while stirring for 5
min, followed by the addition of NHS (0.16 g, 1.46 mmol) with
continuous stirring for 18 h. The reaction product was filtered
and concentrated under reduced pressure. The remaining solid
was redissolved in 2-propanol (40 mL) with gentle heating,
cooled down to room temperature, and reprecipitated at 4 °C
overnight. The product was scraped out of the glassware, washed
with cold 2-propanol, and air-dried to obtain 4 (0.22 g; 56.8%)
as a white solid. 1H NMR (400 MHz) (DMSO) δ: 7.77 (s, 1H,
CONH), 6.45 (s, 1H, CONH), 6.38 (s, 1H, CONH), 4.31 (t,
1H, CHN), 4.15 (t, 1H, CHN), 3.11 (dd, 1H, CHS), 3.03 (t,
2H, CH2NH), 2.83 (s, 4H, CH2 of NHS), 2.79 (d, 1H, CHHS),
2.67 (t, 2H, CH2COOH), 2.60 (d, 1H, CHHS), 2.06 (t, 2H,
CH2CO), 1.04-1.75 (m, 12H). 13C NMR (100.67 MHz)
(DMSO) δ: 171.90, 170.36, 169.03, 162.79, 61.13, 59.27, 55.53,
38.14, 35.32, 30.24, 28.74, 28.32, 28.14, 25.54, 25.41, 24.05.
HRMS (ESI) calculated for C20H30N4O6S, [M+Na]+ 477.1784
(calcd.), 477.1784 (found).
6-[5-(2-Oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pentanoy-
lamino]-hexanoic Acid (6-amino-hexyl)-amide, TFA Salt (7). (6-
{6-[5-(2-Oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pentanoy-
lamino]-hexanoylamino}-hexyl)-carbamic acid tert-butyl ester
(6; 0.23 g, 0.39 mmol) was dissolved in trifluoroacetic acid
(TFA) and stirred at room temperature for 45 min. The product
was concentrated under reduced pressure, washed by ether, and
again concentrated under reduced pressure to obtain oil-like 7
(0.22 g; 100%) as TFA salts. 1H NMR (400 MHz) (CD3OD) δ:
4.58 (t, 1H, CHN), 4.35 (t, 1H, CHN), 3.25 (dd, 1H, CHS),
3.21 (t, 4H, CH2NH), 2.96 (d, 1H, CHHS), 2.95 (t, 2H,
CH2NH3+), 2.75 (d, 1H, CHHS), 1.22-1.80 (m, 20H). 13C NMR
(100.67 MHz) (DMSO) δ: 176.67, 166.14, 63.38, 61.63, 57.03,
41.05, 40.64, 40.19, 40.01, 36.98, 36.81, 30.17, 29.78, 29.50,
28.50, 27.53, 27.33, 26.96, 26.72, 26.27. HRMS (ESI) calculated
for C22H41N5O3S, [M+H]+ 456.3008 (calcd.), 456.3009 (found).
Carbamoylmethyl-carbamic Acid tert-Butyl Ester (8a). Gly-
cine (0.45 g, 6.05 mmol) was dissolved in 6 mL of dioxane/
water mixture (dioxane:water ) 4:2) with stirring, followed by
the addition of 1 N NaOH (2 mL). The acquired glycine solution
was immersed in an ice-water bath and mixed with di-tert-
butyl dicarbonate (1.38 g, 6.31 mmol) dropwise while stirring
to initiate the reaction. The reaction was allowed to proceed at
room temperature overnight. The reaction was diluted with water
(2 mL) and EA (10 mL), extracted with 1 N HCl twice, and
extracted with water twice. The final organic phase was dried
over MgSO4 and evaporated under reduced pressure to give oil-
like 8a (0.94 g; 90%). 1H NMR (400 MHz) (CDCl3) δ: 5.42 (s,
1H, CONH), 3.91 (d, 2H, CH2CO), 1.44 (s, 9H, CCH3). 13C
NMR (100.67 MHz) (CDCl3) δ: 177.0, 156.0, 79.6, 35.8, 34.3,
28.2.
(6-Amino-hexyl)-carbamic Acid tert-Butyl Ester (5). 1,6-
Hexanediamine (1.0 g, 8.61 mmol) was completely dissolved
in dichloromethane (DCM; 7 mL) (29). The solution was chilled
to 0 °C, followed by the addition of di-tert-butyl dicarbonate
(0.63 g, 2.87 mmol) with stirring. The reaction was allowed to
proceed at room temperature overnight. The acquired reaction
product was diluted with chloroform (7.5 mL), extracted with
5% Na2CO3 twice, and its organic phase concentrated under
reduced pressure. The obtained solid was dissolved in 1 N HCl
and extracted with ether twice. The pH of the acquired aqueous
phase was adjusted to 10 with NaOH and extracted with ethyl
acetate (EA) five times. The final organic phase was dried over
MgSO4 and concentrated under reduced pressure to give oil-
tert-Butoxycarbonylamino-acetic Acid 2,5-Dioxo-pyrrolidin-1-
yl Ester (8b). Carbamoylmethyl-carbamic acid tert-butyl ester
(8a; 0.61 g, 3.48 mmol) and NHS (0.61 g, 5.34 mmol) were
dissolved in THF (7 mL), followed by dropwise addition of
DCC (1.42 g, 6.9 mmol) in THF (7 mL) with stirring (30). After
overnight reaction at room temperature, the mixture was
quenched by the addition of three drops of glacial acetic acid,
stirred for 1 h, and filtered to remove suspension. The acquired
filtrate was concentrated under reduced pressure, resuspended
in 2-propanol (25 mL) while stirring for 1 h, and filtered to
separate the suspension from its filtrate. The remaining solid
was washed by 2-propanol and dried to afford white-colored
1
like 5 (0.25 g; 39.8%). H NMR (400 MHz) (CDCl3) δ: 4.54
(s, 1H, CONH), 3.11 (t, 2H, CH2NH), 2.68 (t, 2H, CH2NH2),
1.40-1.50 (s, 9H, CH3; m, 4H, CH2), 1.25-1.37 (m, 4H, CH2).
13C NMR (100.67 MHz) (DMSO) δ: 77.00, 41.98, 33.55, 29.95,
28.33, 26.48. HRMS (ESI) calculated for C11H24N2O2, [M+H]+
217.1916 (calcd.), 217.1917 (found).
1
8b (0.62 g; 68.8%). H NMR (400 MHz) (CDCl3) δ: 5.00 (s,
1H, CONH), 4.27 (d, 2H, CH2CO), 2.85 (s, 4H, CNOCH2CH2),
1.43 (s, 9H, CCH3). 13C NMR (100.67 MHz) (CDCl3) δ: 168.6
(CONH), 77.0 (CH2N), 40.2 (CH2CO), 37.4 (CH2CO), 25.5
(CH3CO).
(6-{6-[5-(2-Oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pen-
tanoylamino]-hexanoylamino}-hexyl)-carbamic Acid tert-Butyl Es-
ter (6). 6-[5-(2-Oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-
pentanoylamino]-hexanoic acid 2,5-dioxo-pyrrolidin-1-yl ester
(4; 0.39 g, 0.86 mmol) was completely dissolved in DMF (15
mL) with gentle warming. After cooling the solution to room
temperature without reprecipitation, it was mixed with triethy-
lamine (Et3N; 0.52 g, 5.15 mmol) followed by the addition of
[(6-{6-[5-(2-Oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pen-
tanoylamino]-hexanoylamino}-hexylcarbamoyl)-methyl]-carbam-
ic Acid tert-Butyl Ester (9). tert-Butoxycarbonylamino-acetic acid
2,5-dioxo-pyrrolidin-1-yl ester (8b; 0.18 g, 0.69 mmol) dissolved
in DMF (5 mL) was added slowly to a DMF solution (3 mL)