52.7, 50.0, 48.6, 47.0, 45.7, 44.1, 43.1, 40.3, 37.7, 36.7, 35.5, 33.6,
(CO, 7 signals detected whereas 8 are expected, probably two
signals overlap), 155.4, 138.3, 136.4, 133.6, 130.9, 130.2, 129.6,
128.0, 125.2, 121.2 (all ArC), 81.0, 79.6 (Cq), 64.5, 58.0, 57.5,
54.4, 53.2, 47.6, 44.1, 41.0, 38.1, 38.0, 36.3, 30.8, 29.2, 28.7, 28.2,
25.9, 23.5, 21.7, 18.6, −1.5 (CH, CH2 and CH3; 20 signals detected
whereas 21 are expected. Possibly two signals overlap or a signal
is obscured by the MeOH signal); m/z (FAB+; NBA)1064.5789
([M + H]+, C53H82N9O12Si requires 1064.5852).
33.2, 33.1, 32.5, 32.5, 32.1, 31.4, 29.8, 27.6, 27.5, 21.1, 20.7, 20.5,
20.5, 20.4 (d, JC, P 2.6), 18.8, 17.7, 17.3, 16.6, 16.3, −1.3 (Si-(CH3)3);
P (202.5 MHz; CD3OD)1.24; m/z (MeOH; ESI-pos.): 1710.4
([M + H]+, C79H115CoN17O18PSi requires 1709.9), 855.0 [M + 2H]2+,
866.7 [M + Na + H]2+.
Vitamin B12-b-acid conjugate 16
To a suspension of 15 (19 mg; 11.1 mol) in CH2Cl2 (4 ml) at 0 °C
under an atmosphere of N2 was added CF3COOH (1 ml), result-
ing in a clear red solution. The mixture was stirred for 4 h at 0 °C
followed by evaporation of the solvent in vacuo. Purification by
preparative HPLC (RP8 30 × 100 mm column; acetate buffer; gra-
dient: 1.0% per min, starting from buffer a), yielded 11 mg (64%)
of a red solid.
Enkephalin conjugate 13
Compound 12 (42 mg; 0.04 mmol) was dissolved in 3 ml of a mix-
ture of CH2Cl2/TFA (1/1 v/v). After the mixture was stirred at RT
for 2 h, it was concentrated to dryness in vacuo. Purification by pre-
parative HPLC (C18ec-column; TFA buffer) afforded 29 mg (88%)
of the trifluoroacetate salt of 13 as a colourless solid.
H (500.25 MHz; CD3OD) 8.91 (1H, s, N2CHHis), 7.54 (1H, s,
CHHis), 7.27 (4H, m, 4 × ArCHPhe), 7.21 (1H, m,ArCHPhe), 7.10 (2H,
pseudo-d, 2 × ArCHTyr), 6.76 (2H, pseudo-d, 2 × ArCHTyr), 4.53
(1H, m, CH), 4.45 (1H, m, CH), 4.21 (3H, m, CH + N-CH2),
4.08 (1H, m, CH), 3.93 (2H, m, CH2-Gly), 3.85 (3H, s, OCH3),
3.79 (2H, m, CH2-Gly), 3.15 (4H, overlapping m, CH2 + NH-
CH2), 2.99 (2H, m, CH2), 2.07 (2H, m, N-CH2-CH2), 1.67 (1H, m,
CHLeu), 1.58 (2H, m, CH2), 0.91 (3H, d, J 6.2, Leu-CH3), 0.89 (3H,
d, J 6.2, Leu-CH3); C (125.8 MHz; CD3OD) 175.3, 174.3, 172.5,
172.1, 170.9, 169.6 (6 × CO), 163.0 (q, JC, F 35, CF3COOH),
158.5, 138.3, 137.5, 131.8, 130.4, 129.3, 129.8, 128.2, 126.2,
122.7, 117.0 (All ArC), 57.4, 56.3, 54.3, 54.3, 53.2, 47.8, 43.8,
43.7, 41.2, 38.7, 37.9, 36.5, 30.8, 27.9, 26.9, 24.2, 21.9; m/z (FAB+;
NBA) = 764.4128 ([M + H]+, C38H54N9O8 requires764.4095).
C (CD3OD; 125.8 MHz): 181.7, 180.3, 177.7, 177.7, 176.7,
175.7, 175.6, 175.5, 175.5, 174.8, 174.8, 174.3, 167.3, 167.0, 143.6,
138.8, 138.4, 138.2, 135.7, 133.9, 131.6, 118.9, 118.0, 112.7, 108.8,
105.3, 95.8, 88.1, 86.5, 83.8 (d, JC, P 5.6), 76.5, 75.6, 73.7 (d, JC, P
6.1), 70.9, 62.8, 60.5, 57.6, 57.1, 55.3, 55.2, 52.8, 52.7, 50.0, 46.9,
45.7, 44.0, 43.1, 40.3, 37.7, 36.7, 35.5, 33.6, 33.6, 33.1, 32.5, 32.5,
32.0, 29.8, 27.5, 27.5, 21.1, 20.7, 20.5, 20.3, 20.1, 17.7, 17.2, 16.5,
16.2; P (202.5 MHz; CD3OD)1.24; m/z (ESI-pos.; MeOH): 1565.2
([M + H]+, C73H104CoN17O16P requires 1565.6), 1587.2 [M + Na]+,
783.4 [M + 2H]2+, 794.1 [M + Na + H]2+.
Re complex 17
To a solution of Re(His)(CO)3 (178 mg; 0.4 mmol) and Boc-NH-
CH2CH2CH2Br (100 mg; 0.4 mmol) in DMF (4 ml) was added
Cs2CO3 (140 mg; 0.4 mmol). The resulting suspension was heated
at 40 °C for 16 h. After the solvent was removed in vacuo, the resi-
due was taken up in CH2Cl2 (25 ml) and H2O (20 ml). The phases
were separated and the aqueous solution was extracted with CH2Cl2
(25 ml). The CH2Cl2 were combined and washed with brine (2 ×
20 ml) and dried (MgSO4). Yield: 230 mg (97%) of a colourless
solid.
Found: C, 35.4; H, 3.9; N, 9.8. Calcd. for C17H23N4O7Re: C,
35.1; H, 4.0; N, 9.6%; max (KBr)/cm−1 3436m NH, 2020s, 1894s,
CO; C (500.25 MHz; CD3CN): 7.94 (1H, s, N2CHHis), 6.92 (1H,
s, CHHis), 5.45 (1H, br, NH-Boc), 4.61 (1H, m, CH) 3.96 (2H, t,
J 7.0, N-CH2), 3.88 (1H, m, Re-NH), 3.83 (1H, m, Re-NH), 3.20
(1H, m, CH), 2.99 (3H, m, CH + Boc-NH-CH2), 1.87 (2H, m,
CH2-CH2-CH2), 1.40 (9H, s, OC(CH3)3). C (75.47 MHz; CD3CN):
199.5 (CO), 197.9 (2 × CO), 182.8 (His-CO2), 163.8 (Boc-CO),
142.5, 135.8, 119.7 (ArCHis), 79.5 (Cq-Boc), 52.6, 46.2, 38.0, 31.7,
28.8 (3 × CH2 + C + C) 28.6 (Boc-CH3); m/z (ESI-pos.; MeOH)
483 [M − Boc + 2H]+, 527 [M − C4H9 + 2H]+, 582 [M + H]+, 605
[M + Na]+, 1165 [2M + H]+, 1187 [2M + Na]+.
Vitamin B12-b-acid; cyanocobalamin b-monocarboxylic acid
(14)
Vitamin B12 (1.88 g, 1.39 mmol) was hydrolyzed in HCl 0.1 M
(190 ml) as described in the literature.25–27 The purification was
modified in the following way: the Dowex column allowed, after
desalting by phenol extractions, the isolation of three fractions,
one containing exclusively d-acid, a second one consisting of a
mixture of b-acid and d-acid, and a third fraction consisting of a
mixture of b-acid and e-acid. The mixture of b-acid and d-acid was
separated by preparative HPLC (column: Waters XTerra Prep RP8,
5 m, 30 × 100 mm; buffer a: acetate buffer; gradient: 0.5% min−1
starting from 100% buffer a). The mixture of b-acid and e-acid was
separated analogously but using the Tris buffer as buffer a. Cyano-
cobalamin-b-acid was isolated in a yield of 280 mg (15%).
C (125.80 MHz; CD3OD): 181.8, 180.2, 177.6, 176.8, 176.7,
175.7, 175.6, 175.5, 174.7, 174.4, 174.3, 167.2, 166.9, 143.5, 138.3,
135.7, 134.0, 131.5, 118.3, 112.6, 108.8, 105.2, 95.7, 88.0, 86.5,
83.7 (d, JC, P 5.7), 76.4, 75.5, 73.7 (d, JC, P 5.8), 62.8, 62.1, 61.1, 60.5,
57.6, 56.9, 55.1, 52.6, 49.9, 46.8, 43.9, 43.3, 40.2, 35.6, 35.4, 33.5,
33.1, 33.0, 32.5, 32.4, 29.7, 27.5, 27.1, 21.0, 20.6, 20.4, 20.3 (d, JC, P
2.7), 20.0, 17.7, 17.2, 16.6, 16.2. P (202.5 MHz; CD3OD)1.24.
Biotin conjugate 18
To a solution of complex 17 (98 mg; 0.17 mmol) in CH2Cl2 (4 ml)
at 0 °C was added TFA (2 ml). The mixture was stirred for 1 h at
0 °C, followed by removal of the solvent in vacuo. In a separate
flask D-(+)-biotin (41 mg; 0.17 mmol) was dissolved in a 4/1 (v/v)
DMF/NEt2 solution. This mixture was added to the Re complex and
TBTU (55 mg; 0.17 mmol) was added. After it had been stirred at
RT for 45 min, the mixture was evaporated to dryness in vacuo. The
sticky residue was washed with EtOAc (2 × 50 ml) and H2O (2 ×
10 ml to remove some side-products. The residue was dissolved in
a minimum amount of MeOH and subsequently purified by column
chromatography (EtOH). Yield: 47 mg (40%) of a white colourless
solid.
Vitamin B12-b-acid conjugate 15
Compound 7 (44 mg; 0.07 mmol) was dissolved in DMF (1.5 ml)
and Et2N (1.5 ml). The mixture was stirred for 1 h at RT. Subse-
quently the mixture was evaporated to dryness in vacuo. In another
flask, 14 (20.0 mg, 14.8 mol) was dissolved in 4.5 ml of a DMSO/
DMF (1/5 v/v) mixture. This mixture was transferred to the flask
containing the deprotected histidine derivative. Subsequently NEt3
(1.0 ml) and TBTU (32.1 mg; 0.1 mmol) were added. The mixture
was stirred for 45 min at RT, after which period it was concentrated
to dryness in vacuo. Purification by preparative HPLC (C8-column;
acetate buffer; gradient: 2.0% per min, starting from buffer a) af-
forded 16 mg (67%) of a red solid.
C (125.80 MHz; CD3OD) 181.7, 180.3, 177.8, 177.7, 176.8,
175.8, 175.7, 175.5, 174.8, 174.8, 174.4, 174.2, 167.3, 167.1, 158.8,
143.7, 138.6, 138.5, 138.4, 135.8, 133.9, 131.7, 118.6, 118.0, 116.9,
112.7, 108.9, 105.3, 95.8, 88.1, 86.6, 83.9 (d, JC, P 5.9), 76.5, 75.7,
73.7 (d, JC, P 5.9), 70.9, 64.4, 62.9, 60.5, 57.7, 57.2, 55.8, 55.3, 52.9,
max (KBr)/cm−1 3309w NH, 2019vs CO, 1884vs br, CO, 1687s,
1636s, CO; H (500.25 MHz; CD3OD) 8.09 (1H, s, N2CHHis),
6.92 (1H, s, CHHis), 5.73 (1H, m, Re-NH), 5.09 (1H, Re-NH), 4.48
(1H, m), 4.29 (1H, m), 4.02 (3H, overlapping m, CH + N-CH2),
3.17 (5H, overlapping m, CH2 + CH-biotin + CH2-biotin), 2.90
2
(1H, dd, J 5.0, J 12.8, S-CH), 2.71 (1H, d, J 12.8 Hz, S-CH),
2.20 (2H, J 7.3 Hz, biotin-CH2), 1.94 (2H, m, N-CH2-CH2),
2 6 0 2
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 5 9 3 – 2 6 0 3