750
A. Anastasiadis, F. Separovic and J. White
NMR δ 174.2, CO2H; 150.05, CO; 85.87, C(CH3)3; 34.8, CCO; 27.7,
column eluted with dichloromethane/methanol/water (800 : 100 : 8) to
afford a major fraction of [6-(aminocaproyl)-N-biotinyl(D10)(amino-
caproyl)]gramicidin (gAXDXB) (8) (6.3 mg, 2.69 × 10–3 mmol, 39%),
RF 0.35. MALDI MS 2346. 1H NMR (CD3OD) δ 0.3–1.8, m, 80H; 2.05,
m, 4H; 2.05–2.20, m, 8H; 2.78, d, 1H; 2.90, dd, 1H; 3.0–3.7, m, 19H;
3.88, dd, 2H; 3.90, s, 2H; 3.9–4.8, m, 16H; 6.8–7.6, m, 20H; 8.15, s, 1H.
(CH3)3; 20.9, 19.4, 18.2, CD2.
[6-[N-(tert-Butoxycarbonylamino)](D10)caproyl]gramicidin (6)
gA was isolated from the natural mixture gD using a flash silica column
eluted with dichloromethane/methanol/water (800 : 60 : 4), RF 0.6
(modified procedure).[12,15] TLC plates were developed in
dichloromethane/methanol/water/acetic acid (400 : 60 : 4 : 4) and
visualized with the tryptophan reagent, N,N-dimethylamino-
benzaldehyde.[16] To the purified gA (22 mg, 0.02 mmol) was coupled
6-[N-(tert-butoxycarbonylamino](D10)-6-caproic acid (19.6 mg,
0.07 mmol) and DMAP (3 mg, 0.02 mmol). Dry dichloromethane
(5 mL) and DCC (13 mg, 0.06 mmol) were added.[15] The solvent was
evaporated, then the crude product was dissolved in methanol and
passed down a Sephadex LH20 column. The eluent was dried and then
Acknowledgments
We gratefully acknowledge Dr Chris Burns and Doreen Bali,
Ambri Pty Ltd, for their helpful discussions.
References
[1] B. A. Cornell, V. L. B. Braacch-Maksvytis, L. G. King, P. D. J.
Osman, B. Raguse, L. Wieczorek, R. Pace, Nature 1997, 387,
580.
further purified on
dichloromethane/methanol/water (800 : 60 : 6) to afford
a
flash silica column and eluted with
major
a
fraction of [6-[N-(tert-butoxycarbonylamino)](D10)caproyl]gramicidin
(6) (15 mg, 7.07 × 10–3 mmol, 36%), RF 0.44. 1H NMR (CD3OD) δ 0.3–
1.8, m, 72H (note: a sharp singlet at 1.5 from BOC methyl 1H was
difficult to integrate due to overlap of aliphatic proton resonances from
gA); 2.05–2.40, m, 9H; 3.0–3.7, m, 16H; 3.8–4.8, m, 14H; 6.8–7.6, m,
20H; 8.15, s, 1H.
[2] T. I. Rokitskaya, Y. N. Antonenko, E. A. Kotova, A. J.
Anastasiadis, F. Separovic, Biochemistry 2000, 39, 13053.
[3] T. C. B. Vogt, J. A. Killian, R. A. Demel, B. De Kruijff, Biochim.
Biophys. Acta 1991, 1069, 157.
[4] T. C. B. Vogt, J. A. Killian, B. De Kruijff, Biochemistry 1994, 33,
2063.
[5] Y. Cheng, W. Liu, S. Chen, Synthesis 1980, 3, 223.
[6] J. March, Advanced Organic Chemistry 1968, pp. 213–216
(McGraw Hill: New York).
(N-6-Amino(D10)caproyl)gramicidin (gAXD) (7)
[6-[N-(tert-Butoxycarbonylamino)](D10)caproyl]gramicidin (6) was
deprotected using trifluoroacetic acid (3 mL) and the solution was
evaporated to dryness and dried under high vacuum to yield
(N-6-amino(D10)caproyl)gramicidin (gAXD) (7). 1H NMR (CD3OD) δ
0.3–1.8, m, 72H; 2.05–2.40, m, 9H; 3.0–3.7, m, 16H; 3.8–4.8, m, 14H;
6.8–7.6, m, 20H; 8.15, s, 1H.
[7] L. Semon, Org. Synth. 1941, 1, 318.
[8] J. C. Eck, C. S. Marvel, Org. Synth. 1943, 2, 76.
[9] J. C. Eck, C. S. Marvel, Org. Synth. 1943, 2, 371.
[10] J. C. Eck, Org. Synth. 1960, 4, 39.
[11] I. Masumi, H. Daijiro, K. Takashi, Tetrahedron Lett. 1975, 49,
4393.
[12] W. R Veatch, E. Fossel, E. R. Blout, Biochemistry 1974, 13, 5249.
[13] F. Separovic, S. Barker, M. Delahunty, R. Smith, Biochim.
Biophys. Acta 1999, 1416, 48.
[14] F. Separovic, T. Gehrmann, T. Milne, B. A. Cornell, S. Y. Lin, R.
Smith, Biophys. J. 1994, 67, 1495.
[15] D. Bali, L. King, S. Kim, Bioconjugate Chem. unpublished
results.
[16] J. P. Greenstein, M. Winitz, Chemistry of the Amino Acids 1961,
Vol. 3, p. 2323 (John Wiley: New York).
[6-(Aminocaproyl)-N-Biotinyl(D10)(aminocaproyl)]gramicidin
(gAXDXB) (8)
To gAXD (7) (15 mg, 7.07 × 10–3 mmol) was added dichloromethane/
methanol (2 : 1) (2 mL) and biotinamidocaproate-N-hydroxysucci-
nimide (8.4 mg, 0.02 mmol). The reaction mixture was stirred overnight
at room temperature. The solvent was evaporated, and then the crude
product was dissolved in methanol and passed down a Sephadex LH20
column. The eluent was dried and then further purified on a flash silica