values. As reported earlier,13 simple
for the neomycin B and 1,3-diaminopropane samples are the average of four
independent runs.
their individual pK
a
oligoamines such as diethylenetriamine and triethylene-
tetramine are essentially as effective as 1,3-diaminopropane in
hydrolyzing ApA. This suggests that the number of amino
groups may not be the major factor distinguishing neomycin B
from 1,3-diaminopropane. Since the observed rate is a combina-
tion of individual hydrolysis rates mediated by various pairs of
amino groups, it is likely that some pairs are much more active
than others. This may be due to their spatial positioning and
1
For general references discussing RNA hydrolysis, see J. Chin, Acc.
Chem. Res., 1991, 24, 145; R. Breslow, E. Anslyn and D.-L. Huang,
Tetrahedron, 1991, 47, 2365; J. Vincent, M. Crowder and B. Averill,
TIBS, 1992, 17, 105; E. Anslyn and D. Perreult, Angew. Chem., Int. Ed.
Engl., 1997, 36, 430 and references cited therein.
2
D. Magda, R. A. Miller, J. L. Sessler and B. L. Iverson, J. Am. Chem.
Soc., 1994, 116, 7439; K. Matsumura, M. Endo and M. Komiyama,
J. Chem. Soc., Chem. Commun., 1994, 2019; J. Hall, D. H u¨ sken, U.
Pieles, H. E. Moser and R. H a¨ ner, Chem. Biol., 1994, 1, 185; J. K.
Bashkin, E. I. Frolova and U. Sampath, J. Am. Chem. Soc., 1994, 116,
a
individual pK values, as well as the lower conformational
freedom of neomycin B when compared to simple unstructured
diamines. Since cyclohexane-1,3-diamine has been found to be
as effective as 1,3-diaminopropane in hydrolyzing ApA,13 we
5
981; J. Hall, D. H u¨ sken and R. H a¨ ner, Nucleic Acids Res., 1996, 24,
3522; D. Magda, S. Crofts, A. Lin, D. Miles, M. Wright and J. L.
Sessler, J. Am. Chem. Soc., 1997, 119, 2293; D. Magda, M. Wright, S.
Crofts, A. Lin and J. L. Sessler, J. Am. Chem. Soc., 1997, 119, 6947.
M. Endo, Y. Azuma, Y. Saga, A. Kuzuya, G. Kawai and M. Komiyama,
J. Org. Chem., 1997, 62, 846.
favor the modulation of individual pK
source for the rate enhancement observed with neomycin B.
While the amines in 1,3-diaminopropane are rather basic, (pK
.1 and 9.8),13 certain pairs in neomycin B are less basic and
display a wider difference in their pK . For example, the pK
a
values as the likely
3
4
5
a
8
Phe
For the hydrolytic cleavage of tRNA by metal-free bleomycin, see
a
a
16
M. V. Keck and S. M. Hecht, Biochemistry, 1995, 34, 12 029.
U. von Ahsen, J. Davies and R. Schroeder, Nature, 1991, 353, 368; U.
von Ahsen, J. Davies and R. Schroeder, J. Mol. Biol., 1992, 226, 935; J.
Davies, U. von Ahsen and R. Schroeder, The RNA World, ed. R. F.
Gesteland and J. F. Atkins, Cold Spring Harbor Laboratory Press, New
York, 1993, pp. 185–204.
1
3
values reported for N and N are 8.04 and 5.74, respectively.
This may lead to a higher population of a monocationic form at
a given pH compared to 1,3-diaminopropane, and therefore to a
faster hydrolysis.
Neomycin B has been shown to bind to various RNA
sequences5
–8
6 T. K. Stage, K. J. Hertel and O. C. Uhlenbeck, RNA, 1995, 1, 95.
and now to hydrolyze an RNA dinucleoside
7
H.-Y. Mei, A. A. Galan, N. S. Halim, D. P. Mack, D. W. Moreland,
K. B. Sanders, H. N. Troung and A. W. Czarnik, Bioorg. Med. Chem.
Lett., 1995, 5, 2755.
phosphate. These observations provide new leads for the design
of small molecules that combine RNA recognition with
hydrolysis. Such molecules may become useful chemical
8
9
M. L. Zapp, S. Stern and M. R. Green, Cell, 1993, 74, 969; G. Werstuck,
M. L. Zapp and M. R. Green, Chem. Biol., 1996, 3, 129.
M. Hendrix, P. B. Alper, E. S. Priestley and C.-H. Wong, Angew. Chem.,
Int. Ed. Engl., 1997, 36, 95.
17
probes for RNA structure and folding. We are currently
exploring these possibilities with larger and well-defined RNA
structures.
This work was supported by the Universitywide AIDS
Research Program, University of California, grant No. R96-SD-
10 H. Wang and Y. Tor, J. Am. Chem. Soc., 1997, 119, 8734; H. Wang and
Y. Tor, Bioorg. Med. Chem. Lett., 1997, 7, 1951; H. Wang and Y. Tor,
Angew. Chem., in the press.
067, and by the Hellman Faculty Fellowship (Y. T.).
1
1
1 T. Hermann and E. Westhof, J. Mol. Biol., in the press.
2 K. Yoshinari, K. Yamazaki and M. Komiyama, J. Am. Chem. Soc.,
1991, 113, 5899. See also: K. N. Dalby, A. J. Kirby and F. Hollfelder,
Pure Appl. Chem., 1994, 66, 687.
Footnotes and References
*
†
E-mail: ytor@ucsd.edu
All samples contained 50 mm Tris and 0.1 mm ApA. The pH was adjusted
1
1
1
3 M. Komiyama and K. Yoshinari, J. Org. Chem., 1997, 62, 2155.
4 B. Barbier and A. Brack, J. Am. Chem. Soc., 1992, 114, 3511.
5 H. Dugas, Bioorganic Chemistry, A Chemical Approach to Enzyme
Action, 2nd edn., Springer Verlag, New York, 1989, pp. 123–137.
6 R. E. Botto and B. Coxon, J. Am. Chem. Soc., 1983, 105, 1021. See also:
D. E. Dorman, J. W. Paschal and K. E. Merkel, J. Am. Chem. Soc., 1976,
to 8.0 at room temperature (21 °C) with highly pure HCl (low trace metals:
iron < 1 ppb, lead < 0.01 ppb, lutetium < 0.005 ppb, zinc < 0.02 ppb). At
5
‡
2
0 °C, there is a drop of approximately 0.5 pH unit.
1
HPLC analysis was performed using a C18 reversed-phase column with
5 mm sodium phosphate buffer, pH 3.5, containing 2% MeCN as the initial
9
8, 6885; L. Szil a´ gyi, Z. Sz. Pusztahelyi, S. Jakab and I. Kov a´ cs,
21
eluent, at a flow-rate of 1 ml min . Gradient elution up to 50% MeCN was
used to optimize separation.
Carbohydr. Res., 1993, 247, 99.
1
7 C. P. H. Vary and J. N. Vournakis, Proc. Natl. Acad. Sci. USA, 1984, 81,
§
The chromatograms were monitored at 260 nm and the resolved peaks
were integrated and corrected for any volume changes against a peak of
A-deoxyadenosine used as an internal standard. Plotting ln(1 2 A/Atot) vs.
6
5
978; J. M. Kean, S. A. White and D. E. Draper, Biochemistry 1985, 24,
062; C. Ehresmann, F. Baudin, M. Mougel, P. Romby, J.-P. Ebel and
2
B. Ehresmann, Nucleic Acids Res., 1987, 15, 9109; G. Knapp, Methods
Enzymol., 1989, 180, 192; D. S. Sigman and C.-H. Chen, Annu. Rev.
Biochem., 1990, 59, 207; D. S. Sigman, A. Mazumder and D. M. Perrin,
Chem. Rev., 1993, 93, 2295.
t (min), where A = integration for adenosine, and Atot = sum of integrals
for all species, gives a straight line with a slope of 2k. The linear correlation
coefficient was > 0.99 for samples with substantial hydrolysis (e.g.
neomycin B and 1,3-diaminopropane) and somewhat lower for control
samples with very little hydrolysis, as expected. The rate constants reported
Received in Corvallis, OR, USA, 15th August 1997; 7/06023H
148
Chem. Commun., 1998