Kinetic Analysis of Ester Aminolysis Catalyzed by Nucleosides in a Nonpolar Medium. Evidence of Bifunctional Catalysis

Article abstract of DOI:10.1021/jo971614y

Kinetic analysis of ester aminolysis in benzene catalyzed by 2′,3′,5′-O-tris(tert-butyldimethylsilyl)-protected nucleosides and 2-pyridone is reported. The catalytic rate constant k_3′ was determined for protected nucleosides A, C, G, U, and pseudouridine (Ψ). The relatively high value associated with C and 2-pyridone is indicative of bifunctional catalysis occurring through stabilization of the aminolysis transition state. The implications of this finding on the possible role C plays in biological catalysis during protein synthesis is hypothesized. ? Abstract published in Advance ACS Abstracts, December 1, 1997.

Full text of DOI:10.1021/jo971614y

J . Org. Chem. 1997, 62, 9295-9297
9295
Kin etic An a lysis of Ester Am in olysis Ca ta lyzed by Nu cleosid es in a
Non p ola r Med iu m . Evid en ce of Bifu n ction a l Ca ta lysis
Christian Melander and David A. Horne*
Department of Chemistry, Columbia University, New York, New York 10027
Received August 29, 1997X
Kinetic analysis of ester aminolysis in benzene catalyzed by 2′,3′,5′-O-tris(tert-butyldimethylsilyl)-
protected nucleosides and 2-pyridone is reported. The catalytic rate constant k3′ was determined
for protected nucleosides A, C, G, U, and pseudouridine (Ψ). The relatively high value associated
with C and 2-pyridone is indicative of bifunctional catalysis occurring through stabilization of the
aminolysis transition state. The implications of this finding on the possible role C plays in biological
catalysis during protein synthesis is hypothesized.
An initial report from our laboratory demonstrated
that functional groups of TBS-protected ribonucleoside
bases are capable of catalyzing amide bond formation in
a nonpolar medium (Figure 1).1 This bond is the seminal
bond formed during protein synthesis, and its formation
is catalyzed by the ribosome.2 A relatively large rate
F igu r e 1.
enhancement was observed for cytidine in comparison
with the other ribonucleosides, A, G, U, and pseudouri-
dine (Ψ) (Figure 2). Although there exists a plethora of
RNA-catalyzed processes including the recent ribozyme-
catalyzed aminolysis reaction,3 the catalytic role which
functional groups of nucleoside bases play remains
obscure due to the complex nature of the polynucleotide
structure. Herein, we report a kinetic description of ester
aminolysis catalyzed by nucleosides. The data indicates
C catalyzes aminolysis through a bifunctional mechanism
that involves simultaneous donation and acceptance of
protons in the aminolysis transition state.
Previous studies have shown4-6 that under pseudo-
first-order conditions and in the presence of nonnucleo-
side catalysts, aminolysis of esters in aprotic solvents
proceeds according to the rate expression given in eq 1.
F igu r e 2.
Ta ble 1. Su m m a r y of Ca ta lysis
catalyst
[catalyst] (mM)
[amine] (M)
102k3′ (M-2 s-1
)
k1obs ) k2[amine] + k3[amine]2 +
k3′[amine][catalyst] (1)
C
0.220-1.83
0.209-1.83
0.412-1.83
0.915-1.83
0.915-1.83
0.915-1.83
0.0303-0.101
0.0303-0.101
0.0303-0.101
0.0405-0.101
0.0405-0.101
0.0405-0.101
730 ( 50
610 ( 70
175 ( 5
40.0 ( 0.4
40.0 ( 2.8
26.0 ( 1.6
2-pyridone
G
Ψ
A
U
The k2 term is not detectable at room temperature7 but
has been observed at higher temperatures.8 The k3 term
has been shown to be relatively consistent, regardless of
the catalyst type or concentration, and this was found to
be true in the case of nucleoside catalysis (data not
shown). For the case of nucleosides, a plot of k1obs/[amine]
vs [amine] was linear from which k3 and k3′ were
determined. Table 1 lists the values for k3′ of ribonucleo-
sides as well as the well-known bifunctional catalyst
2-pyridone9 for comparison.
as well as ribonucleosides, dimerization in a nonpolar
medium is an unavoidable phenomenon. Since the
catalytically active species is believed to manifest itself
in its monomeric form,9 obtaining true kinetic parameters
requires modification of the kinetic equation to account
for catalyst association. Nucleosides are known to self-
dimerize weakly in nonpolar solvents,10 but the extent
of dimerization under the present conditions is unknown.
Both C and 2-pyridone were assumed to self-dimerize to
noncatalytically active species, and their dimerization
and rate constants were calculated according to method
of Watson.8 Equation 2 shows the relationship between
the dimerization constant and various rate terms. K is
the dimerization constant. k2obs′ is (k1obs/[amine]) - (k3-
[amine]) and k3obs′ ) k2obs′/T where T is the stoichiometric
concentration of catalyst. Plots of 1/k3obs′ vs k2obs′ for C
and 2-pyridone were linear from which the catalytic rate
In determining an accurate value for the catalytic rate
constant k3′, the correct choice of catalyst concentration
is complicated by catalyst associations. For 2-pyridone,
X Abstract published in Advance ACS Abstracts, December 1, 1997.
(1) Melander, C.; Horne, D. A. J . Org. Chem. 1996, 61, 8344.
(2) Noller, H. F.; Hoffarth, V.; Zimniak, L. Science 1992, 256, 1416.
(3) Lohse, P. A.; Szostak, J . W. Nature 1996, 381, 442.
(4) Shawali, A.; Biechler, S. S. J . Am. Chem. Soc. 1967, 89, 3020.
(5) Anderson, H.; Su, C.-W.; Watson, J . W. J . Am. Chem. Soc. 1969,
91, 482.
(6) Menger, F. M.; Smith, J . H. J . Am. Chem. Soc. 1969, 91, 5346.
(7) Menger, F. M. J . Am. Chem. Soc. 1966, 88, 3081.
(8) Su, C.-W.; Watson, J . W. J . Am. Chem. Soc. 1974, 96, 1854.
(9) Rony, P. R. J . Am. Chem. Soc. 1969, 91, 6090.
(10) (a) Kyogoku, Y.; Lord, R. C.; Rich, A. Science 1966, 154, 520.
(b) Kyogoku, Y.; Lord, R. C.; Rich, A. J . Am. Chem. Soc. 1967, 89, 496.
S0022-3263(97)01614-9 CCC: $14.00 © 1997 American Chemical Society

9296 J . Org. Chem., Vol. 62, No. 26, 1997
1/k3obs′ ) 1/k3′ + 2Kk2obs′/(k3′)2
Melander and Horne
(2)
constant k3′ and the dimerization constant K were
calculated (Figure 3). Table 2 summarizes these results.
The catalytic rate and dimerization constants for C and
2-pyridone are similar in magnitude. The values of k3′
and K obtained for 2-pyridone under the present condi-
tions are comparable to the previously reported values
for 2-pyridone in chlorobenzene (k3′ ) 10 ( 1 M-2 s-1, K
) 200 M-1).8
In an aprotic medium, breakdown of the tetrahedral
intermediate has been shown to be rate determining.11
The third-order nature of the rate expression is indicative
of catalysis occurring, in part, by acceptance of the
ammonium proton by catalyst in the transition state.
This enables expulsion of the phenolate anion rather than
the more energetic amide anion from the tetrahedral
intermediate. The extent of proton transfer required for
the catalysis is estimated between 10 and 30% of full
proton transfer.8 The observed rate enhancement for C
is consistent with the proposal that C manifests its
catalytic effect through a bifunctional mechanism (Figure
4). Stabilization of the aminolysis transition state by the
amidinone functionality occurs through simultaneous
donation and acceptance of protons. The other catalyti-
cally active nucleosides, however, show only modest rate
enhancements, and this method proved unreliable for
determining their dimerization constants. Therefore,
without experimental insight into the extent of dimer-
ization, the catalytic rate constants for A, U, G, and Ψ
were determined by simply plotting k1obs/[amine] vs
[amine] and dividing by the stoichiometric amount of
nucleoside used.12
F igu r e 3. Determination of K and k3′.
F igu r e 4.
As evidenced by the data, C and 2-pyridone are
superior catalysts compared to the other nucleosides and
are comparable in their effects. A recent study on the
mutarotation of tetramethylglucose catalyzed by nucleo-
sides, however, revealed that C was less effective in
catalyzing this process than 2-pyridone.13 This finding
suggests that C may be an inherently inferior catalyst
than 2-pyridone in catalyzing processes that strictly
involve bifunctional/tautomeric catalysis. This is par-
ticularly true when considering that similar dimerization
constants were found for C and 2-pyridone in the present
study. The transition state in Figure 4 depicts C
participating in three hydrogen bonds in catalyzing the
aminolysis reaction. This arrangement is reminescent
of the hydrogen-bonding arrangement seen in a Watson-
Crick G-C base pair. Although 2-pyridone may be a
fundamentally better bifunctional catalyst than C, the
added stabilization by C in delivering a third hydrogen
bond in the aminolysis reaction may account for the
similarity seen in the rate constants for these two
catalysts. The involvement of three hydrogen bonds may
also account for the fact that C is a significantly better
catalyst than the other nucleosides which do not possess
the requisite donor-acceptor combination.
Ta ble 2. Ra te a n d Dim er iza tion Con sta n ts
catalyst
102k3′ (M-2 s-1
)
K (M-1
)
C
830 ( 60
790 ( 90
110
190
2-pyridone
rate enhancements seen by the other nucleosides indicate
some form of general base catalysis. Finally, one intrigu-
ing speculation focuses on the possible role for C75 of the
universally conserved CCA tail of tRNAs in peptidyl
transfer reactions. While C74 of tRNA has been shown
to be involved in base pairing with a G residue of
ribosomal RNA, no such function has been found for
C75.14 It is known, however, that this nucleoside is
required for efficient ribosome-mediated protein synthe-
sis. Given the close proximity of C75 to the peptidyl
transferase center, the possibility of C75 participating
in protein synthesis through some type of bifunctional
catalysis is an intriguing possibility that has not been
previously examined.
Exp er im en ta l Section
Chemicals were purchased from common vendors and
purified prior to use. n-Butylamine was distilled over KOH
and stored under argon. Benzene was distilled over Na/
benzophenone and stored over activated 4 Å molecular sieves
under argon, 2-pyridone was sublimed twice, and p-nitrophen-
yl acetate was recrystallized from hexanes. 2′,3′,5′-O-Tris(tert-
butyldimethylsilyl)nucleosides were prepared as previously
described.15
In conclusion, the nucleoside C manifests its catalytic
potential via a bifunctional mechanism, while the modest
(11) (a) Menger, F. M.; Smith, J . H. Tetrahedron Lett. 1970, 4163.
(b) Menger, F. M.; Smith, J . H. J . Am. Chem. Soc. 1972, 94, 3824.
(12) While the catalytic effects of A, G, U, and Ψ are small, we do
not interpret this result as a consequence of nucleoside self-complex-
ation since the extent of such associations in pure solvents under the
concentrations used would be small.10 This is particularly true in the
presence of 0.1 M amine. In fact, the determination of k3′ for C without
incorporating dimerization gave a value of 7.3 M-2 s-1 which is only
slightly less than the value of 8.3 M-2 s-1 reported in Table 1.
(13) Melander, C.; Horne, D. A. Tetrahedron Lett. 1997, 38, 713.
(14) Samaha, R. R.; Green, R.; Noller, H. F. Nature 1995, 377, 309.
(15) Ogilvie, K. K.; Schifman, A. L.; Penney, C. L. Can. J . Chem.
1979, 57, 2230 and references therein.

Nonpolar Ester Aminolysis Catalyzed by Nucleosides
J . Org. Chem., Vol. 62, No. 26, 1997 9297
Kin etics
tion of p-nitrophenylacetate was 9.15 × 10-5 M. k1obs was
determined from the slope of the graph ln[(A∞ - A0)/(At
- A0)]. Determination of k1obs was accomplished with
three different concentrations of n-butylamine and three
All reactions were conducted in benzene and were
monitored by measuring the UV absorption of the re-
leased p-nitrophenylate anion. Reactions were carried
out in a stoppered 1 mL quartz cell at a constant
temperature of 25 °C. The release of p-nitrophenylate
anion was monitored at 345 nm for the reactions contain-
ing the catalysts 2-pyridone and A, while 335 nm was
used for all other measurements. The reaction was
initiated by mixing an appropriate amount of n-butyl-
amine to a mixture of p-nitrophenyl acetate and catalyst.
An automatic sample changer was used for kinetic
measurements, with a reference cell being used to correct
for instrumental drift. Measurements were made peri-
odically. All kinetic runs were performed under pseudo-
first-order conditions under an excess of n-butylamine.
The n-butylamine concentration was varied between
0.0303 and 0.101 M. The catalyst concentration ranged
from 2.09 × 10-4 to 1.83 × 10-3 M, while the concentra-
different concentrations of catalyst. The plot of k1obs
/
[amine] vs [amine] was linear from which k3′ was
determined (except for 2-pyridone and C, see text).
Ack n ow led gm en t. Financial support from the Na-
tional Science Foundation (NSF Young Investigator
Award to D.A.H.) and the American Chemical Society
Petroleum Research Fund is gratefully acknowledged.
Su p p or tin g In for m a tion Ava ila ble: Graphs of k1obs
/
[amine] (k2) vs [amine] for catalysts (5 pages). This material
is contained in libraries on microfiche, immediately follows this
article in the microfilm version of the journal, and can be
ordered from the ACS; see any current masthead page for
ordering information.
J O971614Y