76
A. Friedrich et al. / Tetrahedron Letters 50 (2009) 75–76
highly substrate-specific enzymes. To examine this issue, we
NH2
NH2
1) NaNO2
2) NaN3
chemically synthesized the metabolic biosynthetic intermediate
strictosidine 7a–d from 1a–d (Scheme 3). Photolabeled azidostrict-
osidines 7a–d were incubated with Catharanthus roseus plant cell
culture that produces monoterpene indole alkaloids ajmalicine
(m/z 353) 9, serpentine (m/z 349) 10, and tabersonine (m/z 337)
11 that are derived from strictosidine 7.7 Mass spectrometry anal-
ysis of these C. roseus extracts revealed the formation of new com-
pounds displaying masses consistent with azido analogs of
alkaloids with m/z 353.8 Furthermore, in cultures supplemented
with azidostrictosidines 7c and 7d, compounds with molecular for-
mulae consistent with azido analogs of alkaloids having m/z 3499
and m/z 33710 were also observed. These compounds were not ob-
served in control cultures lacking azidostrictosidine.
Although MS analysis cannot allow us to predict the structure of
these unknown analogs, these studies nevertheless strongly sug-
gest that the biosynthetic enzymes of an alkaloid metabolic path-
way can bind to and turn over azide-labeled precursors.
Biosynthetic intermediates derived from 1a–d and 7a–d may
therefore potentially be used for photoaffinity labeling of enzymes
in this metabolic pathway. In combination with a chemoselective
handle (such as an alkyne installed at the ester of 7)11 that allows
for identification in a crude mixture, these photolabeled com-
pounds could be used to identify desired metabolic enzymes in cell
lysates.
R
acetic acid
R
N
H
N
H
4a R= 4-NH2
4b R= 5-NH2
4c R= 6-NH2
4d R= 7-NH2
1a R= 4-N3, 62%
1b R= 5-N3, 65%
1c R= 6-N3, 57%
1d R= 7-N3, 50%
Scheme 2. Diazotation of aminotryptamines 4a–d to form azidotryptamines 1a–d.
H
O
O
O-Glc
N3
O-Glc
O
NH
H
NH2
O
N
H
O
N3
strictosidine 7
O
secologanin 8
N
H
O
pH 2
tryptamine 1
unknown
enzymes
monoterpene indole alkaloids derived from 7
N3
N3
N
H
N
H
H
Acknowledgments
N
H
N
H
9
10
O
O
[M]+ 349
serpentine
We gratefully acknowledge support from the Koch Fund and
GM074820, as well as support from Landesgraduiertenförderung
Baden-Württemberg and DAAD for fellowship support for A.F.
We thank Elizabeth McCoy for helpful discussion.
[M+H]+ 353
ajmalicine
O
O
O
O
N
H
N3
[M+H]+ 337
tabersonine
Supplementary data
N
H
O
11
O
Supplementary data (experimental protocols and spectroscopic
characterization of compounds 1–5a–d, 7a–d) associated with this
article can be found, in the online version, at doi:10.1016/
Scheme 3. Chemical reaction of tryptamine 1a–d and secologanin
strictosidine 7a–d, which appears to be incorporated into several terpene indole
alkaloids including 9, 10, and 11.
8
to yield
References and notes
and elimination (Henry reaction) to give nitro vinyl indole 6c.
Reduction with lithium aluminum hydride in refluxing THF gave
6-amino-tryptamine 4c. This method could also be used to gener-
ate 5-aminotryptamine 2b and 7-aminotryptamine 2d, though
addition/elimination and reduction of 2a to 4a did not proceed in
good yields through this route (Scheme 1B).5
1. Dorman, G. Top. Curr. Chem. 2001, 211, 169–225.
2. Miles, E. W.; Phillips, R. S. Biochemistry 1985, 24, 4694–4703.
3. Zettl, R.; Schell, J.; Palme, K. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 689–693.
4. Wu, T. Y. H.; Schultz, P. G. Org. Lett. 2002, 4, 4033–4036.
5. An older report of the synthesis of aminotryptamines is described in: Hiremath,
S. P.; Siddappa, S. J. Med. Chem. 1965, 8, 142–143. However, no yields or
spectroscopic characterization were provided in this study.
With aminotryptamines 4a–d in hand, the azide group could be
introduced by diazotation of the corresponding amines. Following
a protocol from Melhardo et al., sodium nitrite and sodium azide in
glacial acetic acid were used to convert the aminotryptamines 4a–
d into the azidotryptamines 1a–d.6 The primary alkyl amine did
not react under these conditions, and all four azidotryptamines
1a–d could be obtained in good yields in one step from 4a–d
(Scheme 2). From the nitroindoles 2a–d, the corresponding azidot-
ryptamines 1a–d could be obtained in overall yields ranging from
15% to 38%.
6. (a) Li, M.; Johnson, M. E. Tetrahedron Lett. 1994, 35, 6255–6258; (b) Melhardo, L.
L.; Leonard, N. J. J. Org. Chem. 1983, 48, 5130–5133.
7. (a) Hamill, J. D.; Parr, A. J.; Rhodes, M. J. C.; Robins, R. J.; Walton, N. J. Bio/
Technology 1987, 5, 800–804; (b) Rijhwani, S. K.; Shanks, J. V. Enzyme Microb.
Technol. 1998, 22, 606–611.
8. Azido analog of [M+H]+ 353 (e.g., 9). Expected [M+H]+ 394.1879. Observed
[M+H]+ after co-culture with: no substrate not observed; 7a 394.1886; 7b
394.1884; 7c 394.1873; 7d 394.1893.
9. Azido analog of [M]+ 349 (e.g., 10). Expected [M]+ 390.1566. Observed [M+H]+
after co-culture with: no substrate not observed; 7a not observed; 7b not
observed; 7c 390.1581; 7d 390.1581.
10. Azido analog of [M+H]+ 337 (e.g. 11). Expected [M+H]+ 378.1930. Observed
[M+H]+ after co-culture with: no substrate not observed; 7a not observed; 7b
not observed; 7c 378.1933; 7d 378.1943.
A concern when using photoaffinity-derivatized substrates to
identify proteins is that the azide group could disrupt binding to
11. Galan, M. C.; McCoy, E.; O’Connor, S. E. Chem. Commun. 2007, 3249–3251.