Linear free energy relationships and kinetic isotope effects reveal the chemistry of the Ado 2′-OH group

Article abstract of DOI:10.1016/j.tetlet.2007.01.139

Using kinetic isotope effects (KIE) and Hammett correlations, we show that the main role of the adenosine 2′-OH group on deprotonation by the non nucleophilic base DBU during external acyl group transfer is to generate enhanced electron density on the attacking nucleophile through ionization. The small primary KIEs (1.2 and 1.6) and the large Hammett reaction constants (+2.25 and +3.19) obtained for the ethanolysis of 2′/3′-O-p-substituted benzoyl 5′-O-trityl adenosines and 2′-deoxyadenosines are consistent with an A_N + D_N reaction mechanism. The implications of our results are discussed in terms of chemical contributions of the 2′-OH group in the ribosome catalysis of peptide bond formation.

Full text of DOI:10.1016/j.tetlet.2007.01.139

Tetrahedron Letters 48 (2007) 2381–2384
Linear free energy relationships and kinetic isotope effects
reveal the chemistry of the Ado 20-OH group
Mohamed M. Changalov* and Dimiter D. Petkov
Laboratory of BioCatalysis, Institute of Organic Chemistry, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
Received 5 September 2006; revised 15 January 2007; accepted 24 January 2007
Available online 30 January 2007
Abstract—Using kinetic isotope effects (KIE) and Hammett correlations, we show that the main role of the adenosine 20-OH group
on deprotonation by the non nucleophilic base DBU during external acyl group transfer is to generate enhanced electron density on
the attacking nucleophile through ionization. The small primary KIEs (1.2 and 1.6) and the large Hammett reaction constants
(+2.25 and +3.19) obtained for the ethanolysis of 20/30-O-p-substituted benzoyl 50-O-trityl adenosines and 20-deoxyadenosines
are consistent with an AN + DN reaction mechanism. The implications of our results are discussed in terms of chemical contri-
butions of the 20-OH group in the ribosome catalysis of peptide bond formation.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Ribosome catalyzes peptide bond formation between
aminoacyl-tRNA (aa-tRNA)1 bound to the A site of
the ribosome and peptidyl-tRNA at the P-site. The pep-
tide bond is formed as a result of nucleophilic attack by
the a-amino group of aminoacyl-tRNA on the ester car-
bonyl group of peptidyl-tRNA. The first step is deproto-
the intermediate structure. In principle, ribosome can
catalyze peptide bond formation via several mecha-
nisms, such as proper positioning of the peptidyl and
aminoacyl substrates in the active site, general acid–base
catalysis during deprotonation and protonation, or elec-
trostatic stabilization of the transition states.1,4–9 Sub-
strate assisted catalysis,10 ribosome being an entropy
trap11 in which the increase of DS* is a primary thermo-
dynamic motivation for the catalysis and 20-OH acting
as a ‘proton shuttle’ catalyst12 have also been proposed.
Here, we reveal in detail the reaction mechanism and the
role of the 20-OH group as a vicinal mediator in the pro-
ton transfer for a model transesterification reaction. For
the ethanolysis of 20/30-O-p-substituted benzoyl 50-O-tri-
tyl adenosines and 20-deoxyadenosines (Scheme 1), we
obtained Hammett plots with two different reaction con-
stants, qA = 3.19 and qdA = 2.25, respectively. These
values imply that the reaction for the two types of sub-
strates proceeds through a similar AN + DN mechanism
but with different transition state structures. The latter is
strongly supported by the primary kinetic isotope effects
(KIE) of 1.2 and 1.6 observed for the ethanolysis of the
two types of substrates.
þ
nation of the a-NH3 group to create the nucleophilic
NH2 group. Subsequent nucleophilic attack of the a-
NH2 group on the electrophilic carbonyl group leads
to the formation of the zwitterionic tetrahedral interme-
diate, which by deprotonation forms a negatively
charged tetrahedral intermediate. The breakdown of
the tetrahedral intermediate is initiated by donating a
proton back to the leaving oxygen to form the products.
The 20-OH group might be involved in proton transfer,
acting either as a general-base to extract a proton from
the amine or as a general acid to provide a proton to the
30-OH group. A recent study presented crystal structures
of A-site and P-site substrates bound simultaneously, as
well as an analogue of the tetrahedral intermediate that
includes the A76 20-OH providing the most complete
picture of the active site to date.2,3 According to these
structures, the 20-OH hydrogen bonds with the a-NH2
group in the substrate structure. The 20-OH is most cer-
tainly involved in aligning the nucleophile prior to reac-
tion; however, it is also noted that the 20-OH is the only
functional group positioned to act as a general-base in
External transesterification is known to be catalyzed by
the ribosome,13,14 in which the P-site excludes water un-
til the moment when release factors interact with a stop
codon.2 To mimic the environment of this site we stud-
ied the ethanolysis of the substrates in the aprotic polar
organic solvent acetonitrile using the strong, bulky
organic base 1,8-diazabicyclo[5.4.0]-undec-7-en (DBU)
*
Corresponding author. Tel.: +359 2 9606 161; fax: +359 2 8700
225; e-mail: mohamed@orgchm.bas.bg
0040-4039/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2007.01.139

2382
M. M. Changalov, D. D. Petkov / Tetrahedron Letters 48 (2007) 2381–2384
BH+
R
-
Ade
BH+
R
O
-
Ade
O
O
BH+
R
O
-
adenosines
1a-6a
Ade
O
O
O
O
O
O
δ−
O
H
H
OH
δ+
OEt
A
O
Ade
OEt
TrO
R
Et
O
*
*
T1A
T2A
?
TIA
O
O
(O)H
Ade
+ EtOH + DBU
TrO
B
O
BH+
R
-
Ade
O
deoxy-
adenosines
O
δ−
Products
O
O
O
1b-5b
δ+
OEt
B
O
H
R
δ+
Et
*
TIdA
T1dA
N
B =
= DBU
R=Ph(X), X= NO2, 1a, 1b; COMe, 2a, 2b; Cl, 3a, 3b; H, 4a, 4b; Me, 5a, 5b; MeO, 6a
N
Scheme 1. Proposed AN + DN mechanism of the ethanolysis of 20/30-O-p-X-substituted benzoyl 50-O-trityl adenosines (1a–6a), A and 20-
deoxyadenosines (1b–5b), B, pKa of DBU in acetonitrile is 23.9 pKa units.25
as basic catalyst. kobs constants (Table 1) were obtained
from the pseudo-first order decrease of the substrate
concentration in HPLC chromatograms. Normal kinetic
isotope effects, kH/kD, were obtained for the ethanolysis
of both types of substrates when EtOH was substituted
with EtOD. The pseudo-first order rate constant ratio
kobsX/kobsH for the DBU-catalyzed ethanolysis of the
20/30-O-p-X-substituted benzoyl 50-O-trityl adenosines
and the 20-deoxyadenosine derivatives were determined
and used to construct the Hammett plots shown in Fig-
ure 1. They fit the plots (R = 0.993 and 0.999, respec-
tively) with a slope qA = 3.19 for the adenosine series
and qdA = 2.25 for the deoxyadenosine series.
from the Hammett plots obtained and the pKa values
that the transesterification of both the adenosine and
deoxyadenosine substrates proceeds through a similar
stepwise AN + DN mechanism. It is unlikely that the
transesterification of these substrates would proceed
through a concerted ANDN mechanism. There are many
reports of tetrahedral adducts between acyl functions
and nucleophiles,15,16 indicating that a stepwise addi-
tion–elimination AE (AN + DN) mechanism is involved
in the transfer of an acyl group between strong nucleo-
philes. There is substantial evidence from both substitu-
ent effect17 and isotope effect18 studies that the carbonyl
group transfer reactions proceed through a concerted
ANDN mechanism when the nucleophile and the leaving
group are only weakly basic. Acyl functions with very
good leaving groups and possessing features stabilizing
an acylium ion favour stepwise displacement reactions
with a DN + AN mechanism19 whereas poor leaving
groups confer an AN + DN mechanism.20 Since 20/30-
O-p-substituted benzoyl 50-O-trityl adenosines 1a–6a
are mixtures of 30 and 20-isoforms (see Supplementary
data) one could suppose that they compete during the
ethanolysis. However, it has long been known that acyl
migration in the 20,30-cis-diol system of nucleosides
takes place very rapidly.21 The respective half times of
hydrolysis and equilibration of 20(30)-O-acetyluridine
in 0.1 M phosphate buffer at 20 ꢁC are ca. 30 days and
These values show that there is a substantial change in
the charge of the reaction centre from the ground state
to the transition state at the rate-determining step. The
large positive reaction constants (qA = 3.19 and
qdA = 2.25) and the high pKas for both the nucleophile
(pKa = 16) and the leaving group (pKa ꢀ 12.5) unambig-
uously demonstrate that the transesterification of the 20/
30-O-p-X-substituted benzoyl 50-O-trityl adenosines and
20-deoxyadenosines proceeds through tetrahedral addi-
tion intermediates TIA and TIdA shown in Scheme 1.
Their formation is rate-determining since the nucleofuge
for both substrates is less basic and is a better leaving
group than the attacking nucleophile. There is no doubt
Table 1. Kinetic data and kinetic isotope effects at 25 ꢁC for the ethanolysis of 20/30-O-p-substituted benzoyl 50-O-trityl adenosines (1a–6a) and 30-O-
p-substituted benzoyl 50-O-trityl 20-deoxyadenosines (1b–5b) in acetonitrile
p-X
Adenosine derivatives kobs (minꢁ1
)
KIE
Deoxyadenosine derivatives kobs (minꢁ1
)
KIE
r
EtOH
EtOD
EtOH
EtOD
NO2 (1)
COMe (2)
Cl (3)
H (4)
Me (5)
27.92 1.12
1.54 0.06
0.42 0.02
0.06 0.002
0.03 0.002
0.007 0.0004
22.75 0.68
1.28 0.05
0.35 8 · 10ꢁ3
0.05 1 · 10ꢁ3
0.025 1 · 10ꢁ3
0.0057 3 · 10ꢁ4
1.2
1.2
1.2
1.2
1.2
1.2
0.052 0.002
0.009 0.003 ꢁ5
0.0025 7 · 10
0.0007 2 · 10ꢁ5
0.0003 1 · 10ꢁ5
—
0.0324 9.7 · 10ꢁ4
0.0060 1.5 · 10ꢁ5
0.0016 4.2 · 10ꢁ5
0.0004 1.6 · 10ꢁ5
0.0002 6 · 10ꢁ6
—
1.6
1.5
1.6
1.6
1.5
—
0.78
0.50
0.23
0.0
ꢁ0.17
MeO (6)
ꢁ0.27

M. M. Changalov, D. D. Petkov / Tetrahedron Letters 48 (2007) 2381–2384
2383
of 1.2 for the adenosine substrates is in agreement with
the larger reaction constant of 3.19 since the first shows
significant movement of the ethanol OHꢂproton towards
the 20-oxyanion at the transition state T1A and the latter
advanced bond formation between the attacking nucleo-
phile (EtOꢁ anion) and the carbonyl carbon at that rate-
determining transition state (Scheme 1 A). The greater
kinetic isotope effect of 1.6 for the deoxyadenosine sub-
strates reveals a more symmetric transition state T1ꢂdA
(Scheme 1 B) in which the total change in the zero-point
energies is greater than that in the Tꢂ1A state and an in-
creased kinetic isotope effect is observed for the trans-
esterification of the deoxyadenosine substrates. It is in
good agreement with the reaction constꢂant of 2.25 show-
ing a more planar transition state T1dA in which the
bond order between the nucleophile and the carbonyl
group has a lower extent than that in the transition state
Tꢂ1A. The good agreement between the reaction constants
and the kinetic isotope effects proves that the proton
transfer and the nucleophilic attack are concerted for
the ethanolysis of both kinds of substrates. Tetrahedral
intermediate TIA breakdown is likely to proceed
through transition state Tꢂ2A in which departure of the
leaving group is facilitated by the 20-OH as the latter
donates a proton back to the leaving oxygen, making it
a better leaving group. Adenosine is a better leaving
group than deoxyadenosine; however, we did not ob-
serve a break in the Hammett plot for the ethanolysis
of the deoxyadenosine substrates. This shows that there
is no change in the rate-determining step for the transe-
sterification of the deoxyadenosines 1b–5b, and it
follows a similar AN + DN mechanism. These studies
clearly show that the role of the deprotonated 20-OH
group in the transesterification is to enhance the nucleo-
philicity of the attacking nucleophile generating en-
hanced electron density on it either by ionization or by
general-base catalysis. It can be seen from the kinetic
data (Table 1) that the amount of anchimeric assistance
by the vicinal OH group is enormous, up to 600 fold.
Probably A 76 20-OH group has a similar role in peptide
bond formation, enhancing nucleophilic poweþr of the
attacking nucleophile. The pKa of the a-NH3 group
in aa-tRNA is estimated to be around 8 and it is likely
that the proton to be accepted by a water molecule24 fol-
lowed by subsequent nucleophilic attack of the a-NH2
group is facilitated by the 20-OH, the latter is supported
by the recently presented crystal structures.2,3 Taking
into account the high pKa of the activated a-NH2 group
and that of the depeptidylated tRNA A76 20/30-OH as a
leaving group, we can speculate that the peptide bond
formation could proceed through a similar AN + DN
mechanism. It involves the formation of a highly asso-
ciative transition state in which the proton transfer to
the A76 20-OH and the nucleophilic attack of the a-
NH2 group are concerted. An additional tetrahedral
intermediate is formed as its structure resembles
strongly that of the transition state. We propose a mech-
anism of peptide bond formation quite similar to that of
the transesterification of 20/30-O-p-substituted benzoyl
50-O-trityl adenosines 1a–6a depicted in Scheme 1 A.
Although the proposed mechanism for peptidyl transfer-
ase reaction is certainly an oversimplification, it never-
theless provides a description of the transition state
4
3
2
1
0
p-NO
3
2
2
ρ
= 3.19
A
p-COMe
p-Cl
1
p-NO
2
H
p-COMe
p-Me
p-MeO
0
p-Cl
H
ρ
= 2.25
-1
dA
p-Me
-0.4
0.0
0.4
0.8
σ
Figure 1. Hammett plots of the ethanolysis of 20/30-O-p-X-substituted
benzoyl 50-O-trityl adenosines (d) and 20-deoxyadenosines (s).
7.5 s, respectively, their ratio being 350,000. The latter
implies that there is no competition between 30 and
20-isoforms during the external acyl group transfer
(ethanolysis).
Qualitatively, the fundamental mechanism of enhancing
the nucleophilic power of any given oxygen atom is to
enhance the electron density of the nucleophilic lone
electron pair. This leads to three possible molecular
mechanisms for generating enhanced electron density
in the reactant state of the nucleophile22 by altering
the coordination and/or bonding of the nucleophile:
(1) desolvation or more precisely the stripping away of
H-bond donors to the nucleophilic lone pair, (2) coordi-
nation of the alcohol proton, that is, H-bonding to a
general-base and (3) ionization. In addition to these
three mechanisms, an electrical field generated at the
enzyme active site can polarize the bond to the nucleo-
phile enhancing its nucleophilicity.
In our model reaction, we observed normal primary
kinetic isotope effects for the ethanolysis of both adeno-
sines 1a–6a and deoxyadenosine 1b–5b substrates of
1.2 and 1.6, respectively. The normal kinetic isotope
effects are indicative of a proton ‘in flight’ at the transi-
tion state of the rate-determining step; however, the ob-
served kinetic isotope effects for the ethanolysis of 20/30-
O-p-substituted benzoyl 50-O-trityl adenosines (1a–6a) are
well below 2 (Table 1), which is the generally accepted
threshold for distinguishing general-base catalysis. More
likely, the reason in this case for the generated enhanced
electron density on EtOH is ionization by the DBU-
deprotonated 20-OH group; however, general-base catal-
ysis could not be entirely excluded, as the pKa of the 20-
OH group complies well with Jencks’ libido rule23 and
can act as a general-base. The 20-oxyanion is obviously
more efficient in increasing the nucleophilic power of
EtOH than the external nonnucleophilic base DBU.
The generated enhanced electron density of the 20-oxy-
anion results in a greater bond order between the nucleo-
phile and the electrophilic carbonyl group, which leads
to the formation of a full negative charge on the car-
bonyl oxygen, as indicated by the large Hammett reac-
tion constant of 3.19. The small kinetic isotope effect

2384
M. M. Changalov, D. D. Petkov / Tetrahedron Letters 48 (2007) 2381–2384
6. Maden, B. E.; Monro, R. E. Eur. J. Biochem. 1968, 6, 309–
316.
7. Moore, P. B.; Steitz, T. A. Ann. Rev. Biochem. 2003, 72,
813–850.
and the reaction path of peptide bond formation which
will not be altered greatly by the specificities of the
EtOH and the a-NH2 group as O and N nucleophiles.
8. Nierhaus, K. H.; Schulze, H.; Cooperman, B. S. Biochem.
Int. 1980, 1, 185–192.
This study shows that the main role of the deprotonated
20-OH group in our model transesterification reaction is
to enhance the nucleophilic power of the attacking
nucleophile. The Hammett correlations and kinetic iso-
tope effects support a stepwise AN + DN reaction mech-
anism in which attack of the nucleophile is rate-
determining. Arguments show that a similar AN + DN
mechanism (Scheme 1 A) probably operates for the pep-
tide bond formation.
9. Katunin, V. I.; Muth, G. W.; Strobel, S. A.; Wintermeyer,
W.; Rodnina, M. V. Mol. Cell 2002, 10, 339–346.
10. Weinger, J. S.; Parnell, K. M.; Dorner, S.; Green, R.;
Strobel, S. A. Nat. Struct. Mol. Biol. 2004, 11, 1101–1106.
11. Sievers, A.; Beringer, M.; Rodnina, M. V.; Wolfenden, R.
Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 7897–7901.
12. (a) Changalov, M. M.; Ivanova, G. D.; Rangelov, M. A.;
Acharya, P.; Acharya, S.; Minakawa, N.; Foldesi, A.;
Stoineva, I. B.; Yomtova, V. M.; Russev, C. D.; Matsuda,
A.; Chattopadhyaya, J.; Petkov, D. D. ChemBioChem
2005, 6, 992–996; (b) Trobo, S.; Aqvist, J. Proc. Natl.
Acad. Sci. U.S.A. 2005, 102, 12395–12400.
Acknowledgements
13. Bieling, P.; Beringer, M.; Adio, S.; Rodnina, M. Nature
Struct. Mol. Biol. 2006, 13, 423–428.
14. Scolnick, E.; Milman, G.; Rosman, M.; Caskey, T. Nature
1970, 225, 152–154.
15. McClelland, A. R.; Santry, J. L. Acc. Chem. Res. 1983, 16,
394–399.
We thank the National Research Fund of Bulgaria
for financial assistance (Grant K-901) and Professor
I. Pojarlieff for helpful suggestions and critical reading
of the manuscript.
16. Capon, B.; Ghosh, A. K.; Grieve, A. M. D. Acc. Chem.
Res. 1981, 14, 306.
Supplementary data
17. (a) Ba-Saif, S.; Luthra, A. K.; Williams, A. J. Am. Chem.
Soc. 1987, 109, 6362; (b) Ba-Saif, S.; Luthra, A. K.;
Williams, A. J. Am. Chem. Soc. 1989, 111, 2647; (c) Ba-
Saif, S.; Colthurst, M.; Waring, M. A.; Williams, A. J.
Chem. Soc., Perkin Trans. 2 1991, 1901; (d) Williams, A.
Acc. Chem. Res. 1989, 22, 387; (e) Colthurst, M. J.; Nanni,
M.; Williams, A. J. Chem. Soc., Perkin Trans. 2 1996, 2285.
18. (a) Hengge, A. C. J. Am. Chem. Soc. 1992, 114, 6575; (b)
Hengge, A. C.; Hess, R. A. J. Am. Chem. Soc. 1992, 114,
11256; (c) Cleland, W. W.; Hengge, A. C. FASEB J. 1995,
9, 1585.
Experimental procedures and characterization of the
compounds. Supplementary data associated with this
article can be found, in the online version, at doi:10.1016/
j.tetlet.2007.01.139.
References and notes
19. Williams, A.; Douglas, K. T. Chem. Rev. 1975, 75, 627.
20. Bender, M. L. Chem. Rev. 1960, 60, 53; Johnson, S. L.
Adv. Phys. Org. Chem. 1967, 5, 237.
1. Barta, A.; Halama, I. In Ribosomal RNA and Group I
introns; Green, R., Schroeder, R., Eds.; R.G. Landes
Company: Austin, TX, 1996; pp 35–54.
21. Griffin, B. E.; Jarman, M.; Reese, C. B.; Sultston, J. E.;
Trentham, D. R. Biochemistry 1966, 5, 3638–3649.
22. Anderson, V. E.; Ruszczycky, M. W.; Harris, M. E. Chem.
Rev. 2006, 106, 3236–3251.
23. Jencks, W. P. J. Am. Chem. Soc. 1972, 94, 4731.
24. Wolfenden, R. Biochemistry 1963, 338, 1090–1092.
25. Leffek, K. T.; Pruszynski, P.; Thanapaalasingham, K.
Can. J. Chem. 1989, 67, 590–595.
2. Schmeing, T. M.; Huang, K. S.; Strobel, S. A.; Steitz,
T. A. Nature 2005, 438, 520–524.
3. Schmeing, T. M.; Huang, K. S.; Kitchen, D. E.; Strobel,
S. A.; Steitz, T. A. Mol. Cell 2005, 20, 437–448.
4. Krayevsky, A. A.; Kukhanova, M. K. Prog. Nucleic Acids
Res. Mol. Biol. 1979, 23, 1–51.
5. Lieberman, K. R.; Dahlberg, A. E. Prog. Nucleic Acids
Res. Mol. Biol. 1995, 50, 1–23.