SHORT PAPER
Synthesis of 6-N-Biotinylaminohexyl Isopropyl Phosphorofluoridate
409
o
in a fridge (4 C) for 4 h. The precipitate was filtered and washed
repeatedly with Et2O to give 398 mg (85%) of product; [a]D20 +60.4
(c = 0.98, CH3OH).
6-N-Biotinylaminohexyl Isopropyl Phosphorofluoridate (2)
The H-phosphonate 5 (57 mg, 0.13 mmol) was dried by coevapora-
tion with pyridine (2 ¥ 2 mL) and finally dissolved in pyridine (2
mL). To the stirred solution Et3N (72mL, 0.52 mmol, 4 equiv.) was
added followed by triethylamine trihydrofluoride (21 mL 0.13
mmol, 1 equiv.) and, finaly, I2 (49 mg, 1.19 mmol, 1.5 equiv.). The
reaction was stirred for 10 min and then diluted with CH2Cl2 (30
mL). The solution was washed successively with 1M Na2S2O3 (30
mL), sat. aq NaHCO3 (30 mL) and concentrated. Toluene was evap-
orated from the residue to remove residual pyridine. The crude
product was purified by FCC eluting with propan-2-olÐEtOAc (2:3)
1H NMR [(CD3)2SO]: d = 1.20Ð1.50 (14H, m, 7 ¥ CH2), 2.04 (2H,
t, J = 6.8, CH2CO), 2.56 (1H, d, JH ,H = 12.5, Hb), 2.83 (1H, dd,
a
b
JH ,Hc = 4.7, Ha) 3.02 (3H, m, CH2N and He), 3.47 (2H, m, CH2O),
a
4.13 (1H, m, Hd), 4.32 (1H, m, Hc), 4.37 (1H, t, J = 5.5, OH), 6.39
(1H, s, NH), 6.46 (1H, s, NH), 7.76 (1H, t, J = 5.0, CH2NH).
EIMS: m/z = 343.2 [M]+ (C16H29N3O3S requires M, 343.19).
Anal. Calcd for C16H29N3O3S: C, 55.95; H, 8.51; N, 12.23. Found:
C, 55.65; H, 8.60; N, 12.12.
21
to give white waxy solid (57 mg, 96%); [a]D + 21.7 (c = 0.9,
CHCl3).
1H NMR (CDCl3): d = 1.2Ð1.8 (20H, m, 7 ¥ CH2 and 2 ¥ CH3), 2.24
6-N-Biotinylaminohexyl Hydrogenphosphonate (4)
(2H, t, J = 7.0, CH2CO), 2.78 (1H, d, JH ,Hb = 12.8, Hb), 2.96 (1H,
a
The biotin derivative 3 (100 mg, 0.29 mmol) was dissolved in DMF
(5 mL) and pyridine (0.06 mL, 0.74 mmol) was added. To this
stirred solution, salicyl chlorophosphite (76 mg, 0.38 mmol, 1.3
equiv.) in DMF (5 mL) was added dropwise over 10 min. After a
further 10 min, 1M aq TEAB (triethylammonium bicarbonate, pH
8, 0.5 mL) was added and the solution stirred for 15 min. Et2O (55
mL) was added to the mixture, which was kept at 4 ûC overnight.
The precipitate was filtered and washed with Et2O (3 ¥ 30 mL) and
CH2Cl2 (3 ¥ 30 mL) to give 111 mg (94%) of product; [a]D23 + 44
(c = 1, CH3OH).
dd, JH ,Hc = 4.5, Ha), 3.24 (3H, m, CH2N and He), 4.20 (2H, dt, JH,P
a
= 7.4, CH2OP), 4.37 (1H, dd, JH ,Hd = 8.0 and JH ,He = 4.5, Hd), 4.57
(1H, dd, Hc), 4.82 [1H, dsep, JH,H = JP,H = 6.3, POCH(CH3)2], 6.25
(3H, bs, NH).
c
d
31P NMR (CDCl3): d = Ð9.8 (JP,F = 978).
19F NMR (CDCl3): d = Ð78.4.
ESMS(+): m/z = 506.0 [M + K]+, 490.0 [M + Na]+, 468.1 [M + H]+,
426.0 [M Ð iPr + 2H]+ (C19H35FN3O5PS requires M, 467.20).
1H NMR (CD3OD): d = 1.20Ð1.70 (14H, m, 7 ¥ CH2), 2.28 (2H, t,
Anal. Calcd for C19H35FN3O5PS.1/2H2O: C, 47.89; H, 7.61; N, 8.82.
Found: C, 48.10; H, 7.56; N, 8.48.
J = 7.2, CH2CO), 2.74 (1H, d, JH ,Hb = 12.9, Hb), 2.97 (1H, dd, JH ,H
a
a c
= 4.6, Ha), 3.21 (2H, t, J = 6.7, CH2N), 3.25 (1H, m, He), 4.05 (2H,
c
d e
dt, JP,H = 8.3, JH,H = 6.4, CH2OP), 4.34 (1H, dd, JH ,Hd = 7.9, JH ,H
= 4.6, Hd), 4.53 (1H, dd, Hc), 6.80 (1H, d, JP,H = 678, PH).
Acknowledgement
31P NMR (CD3OD): d = 6.93 (JP,H = 678).
31P NMR (CD3OD + Et3N): d = 4.88 (JP,H = 611).
ESMS(Ð): m/z = 406.2 [M Ð H]Ð (C16H30N3O5PS requires M,
407.20).
This work and A.P.H. were supported by a Wellcome Trust Grant
048564/Z/96/Z. The research of A.V.N. was supported by an Inter-
national Research Scholar's award from the Howard Hughes Medi-
cal Institute.
Anal. Calcd for C16H30N3O5PS: C, 47.16; H, 7.42; N, 10.31. Found:
C, 46.90; H, 7.60; N, 10.21.
References
(1) Cohen, J. A.; Oosterbaan, R. A.; Barends, F. Meth. Enzymol.
1967, 11, 686.
(2) Hermanson, G. T. Bioconjugate Techniques; Academic Press:
6-N-Biotinylaminohexyl Isopropyl Hydrogenphosphonate (5)
H-phosphosphonate 4 (50 mg, 0.12 mmol) was dissolved in a mix-
ture of DMF (2 mL), propan-2-ol (18 mL, 0.24 mmol, 2 equiv.) and
Et3N (67 mL, 0.48 mmol, 4 equiv.). Pivaloyl chloride (44 mL, 0.36
mmol, 3 equiv.) was added to the solution and the mixture was
stirred for 20 min. The reaction was quenched with 1M aq. TEAB
(1 mL) and the solution stirred for 15 min, and concentrated. A so-
lution of the residue in CH2Cl2 (20 mL) was washed with H2O (20
mL) and concentrated. The crude product was purified by FCC elut-
ing with a gradient of CH3OH in CH2Cl2 (2Æ15%) containing 1%
Et3N to give a white waxy solid (40 mg, 71%); [a]D23 +30.7 (c = 1,
CHCl3).
San Diego, 1996; p 570.
(3) McCormick, D. B. J. Hetrocycl. Chem. 1973, 10, 235.
(4) McCormick, D. B.; Roth, J. A. Anal. Biochem. 1970, 34, 226.
(5) Marugg, J. E.; Tromp, M.; Kuyl-Yeheskiely. E.; van der
Marel, G. A.; van Boom, J. H. Tetrahedron Lett. 1986, 27,
2661.
(6) Westerduin, P.; Veeneman, G. H.; van der Marel, G. A.; van
Boom, J. H.Tetrahedron Lett. 1986, 27, 6271.
(7) Stawinski, J.; Thelin, M. J. Chem. Soc. Perkin Trans. 2 1990,
849.
1H NMR (CDCl3): d = 1.2Ð1.7 (20H, m, 7 ¥ CH2 and 2 ¥ CH3), 2.18
(8) Lindh, I.; Stawinski, J. J. Org. Chem. 1989, 54, 1338.
(9) Dabkowski, W.; Cramer, F.; Michalski, J. J. Chem Soc., Per-
kin Trans. 1 1992, 1447.
(2H, t, J = 7.1, CH2CO), 2.71 (1H, d, JH ,H
a
b
= 12.8, Hb), 2.87 (1H,
dd, JHa,Hc = 4.0, Ha), 3.17 (3H, m, CH2N and He), 4.02 (2H, dt, JH,H
c
d
d e
= 6.3, JH,P = 8.0, CH2OP), 4.28 (1H, dd, JH ,H = 7.4, JH ,H = 4.3,
Hd), 4.30 (1H, m, Hc), 4.68 [1H, dsep, JH,H = 6.2, JH,P = 8.2,
POCH(CH3)2], 6.57 (2H, m, NH), 6.64 (1H, s, NH), 7.10 (1H, d,
JH,P = 691, PH).
(10) Dabkowski, W.; Michalski, J.; Wasiak, J.; Cramer, F. J. Chem
Soc., Perkin Trans. 1 1994, 817.
(11) Bollmark, M.; Zain, R.; Stavinski, J. Tetrahedron Lett. 1996,
37, 3537.
(12) Dabkowski, W.; Tworowska, I. Chem. Lett. 1995, 727.
(13) DelMar, E. G.; Largman, C.; Brodrick, J. W.; Geokas, M. C.
Anal. Biochem. 1979, 99, 316.
31P NMR (CDCl3): d = 6.34.
ESMS(+): m/z = 450.0 [M + H]+, 408.0 [M Ð iPr + 2H]+, 390.0 [M
Ð OiPr]+ (C19H36N3O5PS requires M, 449.21).
Anal. Calcd for C19H36N3O5PS: C, 50.76; H, 8.07; N, 9.35. Found:
C, 50.33; H, 8.52; N, 9.25.
Synthesis 1999, No. No. 3, 407–409 ISSN 0039-7881 © Thieme Stuttgart · New York