C O M M U N I C A T I O N S
Table 1. Comparison of â-Furanosyl Ribonucleoside Yields for
It is instructive to consider that, at neutral pH, the bases that
form glycosides under the present conditions, namely adenine,
hypoxanthine, and pyrimidinone, all have a reacting system that
consists of an amidine moiety (the imidazole portion of the purine)
or a vinylogous amidine moiety. This feature allows a ring nitrogen
atom that carries an in-plane electron pair to act as a nucleophile
while the second nitrogen atom, which carries an in-plane hydrogen
atom, can simultaneously or subsequently lose this hydrogen as a
proton. Cytosine, in its canonical keto-amino tautomer, also has
such a structure, but the lone pair is on a nitrogen atom that is
sterically hindered, being flanked by an NH2 and a carbonyl group,
apparently preventing reaction.
The pKa of N1-H of uracil is about 9.5, so that there is less
than 1% of the anion at neutral pH. However, Kimura et al. have
shown that placement of the uracil base near a cationic species
can selectively reduce the pKa of N1-H by more than 2 pH units.8
Thus, coordination of uracil within a supramolecular assembly, such
as in a coaxial stack with cationic intercalators,4b might sufficiently
lower the N1-H pKa to allow glycosylation.
Three Bases under Selected Reaction Conditionsa
reaction
zebularine
adenosine
inosine
Orgel reaction conditions
pH 2.1
pH 6.3
12%
2%
<1%
<1%
1%
<1%
3%
1%
<1%
a Reaction conditions are described in Figure 2. Yields are absolute and
neglect side products, which we estimate at <20%. The maximum variability
in the measured yields was 45%. Previously reported nucleoside yields were
calculated BRSM.2 See SI for additional details.
Table 2. The B3LYP/6-31G(d) Predicted Activation Energies (in
kcal/mol) for Glycosidic Bond Formation between Ribose and
2-Pyrimidinone or Uracila
reactants
activation energy
ribose, pyrimidinone
ribose, uracil
ribose, protonated-pyrimidinone
ribose, protonated-pyrimidinone, Mg2+
ribose, uracil, Mg2+
52.7
50.8
34.3
20.2
39.9
If 2-pyrimidinone nucleosides existed in the first RNA-like
polymers, they could have been replaced by a post glycosylation
base modification or by evolution. Conversion to uridine could have
occurred by the addition of water across the N3-C4 bond,9 followed
by hydride transfer of the C-4 hydrogen to an acceptor (e.g.,
glyoxylate). The latter process would have been driven by the
superior stability of uridine against glycosidic bond hydrolysis (SI)
and greater functionality of the uracil substituents for base pairing.10
In any case, we suggest that the studies presented here indicate
what might have been the essential features of pyrimidine (or
pyrimidine-like) bases in early life.
a See SI for pathway structures and associated energies, and for
calculation methodology.
that, under the same reaction conditions, the most easily formed
nucleosides will also be those for which the glycosidic bond is most
easily broken, and we observe a positive correlation between the
propensity for a particular nucleoside to degrade and its formation
yield (SI).
We have used high-level calculations to model the zebularine
formation reaction to understand why 2-pyrimidinone readily forms
a glycosidic bond with ribose. Protonated 2-pyrimidinone has a
pKa of 2.24.5 Thus, most of the base will be protonated under the
acidic reaction condition during the actual reaction, although this
does not mean that the base must be the proton donor in an acid-
catalyzed reaction. In the presence of Mg2+ at pH 6.3, less than
0.1% of the base will be protonated.
Acknowledgment. This research was supported by the NASA
Exobiology Program (Grant NNG04GJ32G) (N.V.H.) and NSF
Crest Grant No. 9805465 (J.S.U.). This work is dedicated to the
memory of James P. Collins.
We have modeled glycosylation involving a closed sugar in
analogy with other glycosylation and deglycosylation reactions,6
although an alternative route may also exist with the open-chain
sugar.7 The stationary structures obtained at the B3LYP/6-31G(d)
level reveal that protonated 2-pyrimidinone forms a complex with
ribose that includes a strong H-bond between the N1 proton of
2-pyrimidinone and the O1′ hydroxyl group of ribose. (Direct proton
transfer from the solvent to the ribose was not studied.) This proton
is also transferred to the O1′ hydroxyl group during cleavage of
the C1′-OOH bond.
Supporting Information Available: Sample preparation and data
analysis details, NMR spectra, computational details, including energy
state diagrams. This material is available free of charge via the Internet
References
(1) Zubay, G.; Mui, T. Origins Life EVol. Biosphere 2001, 31, 87-102.
(2) Fuller, W. D.; Sanchez, R. A.; Orgel, L. E. J. Mol. EVol. 1972, 1, 249-
257.
The reaction mechanism for zebularine formation in the presence
of Mg2+ was also investigated at the B3LYP/6-31G(d) level. An
initial complex was formed between ribose and protonated 2-py-
rimidinone with the assistance of the Mg2+ cation. In a transition-
state structure the breaking of the C1′-OOH bond is facilitated by
the Mg2+ cation and a bond is formed between the leaving HO-
group and the Mg2+ cation. The activation energy for this reaction
step is calculated to be 20.2 kcal/mol (see Table 2).
(3) (a) Ingar, A.-A.; Luke, R. W. A.; Hayter, B. R.; Sutherland, J. D. Chem.
Biol. Chem. 2003, 4, 504-507.
(4) (a) Benner, S.; Burgstaller, P.; Battersby, T.; Jurczyk, S. In The RNA
World; Gesteland, R. F., Atkins, J. F., Eds.; CSHL Press: Cold Spring
Harbor, New York, 1999; pp 163-181. (b) Hud, N. V.; Anet, F. A. L. J.
Theor. Biol. 2000, 205, 543-562. (c) Kolb, V. M.; Dworkin, J. P.; Miller,
S. L. J. Mol. EVol. 1994, 38, 549-557. (d) Mittapalli, G. K.; Osornio, Y.
M.; Guerrero, M. A.; Reddy, K. R.; Krishnamurthy, R.; Eschenmoser, A.
Angew. Chem., Int. Ed. 2007, 46, 2478-2484. (e) Mittapalli, G. K.; Reddy,
K. R.; Xiong, H.; Munoz, O.; Han, B.; De Riccardis, F.; Krishnamurthy,
R.; Eschenmoser, A. Angew. Chem., Int. Ed. 2007, 46, 2470-2477.
Comparison of results obtained from all calculations performed
provides the following conclusions: (1) Acid catalyzes glycosidic
bond formation by facilitating cleavage of the C1′-OOH bond, and
the acidic proton can be delivered by the protonated base; (2) Mg2+
facilitates the reaction between ribose and protonated 2-pyrimidi-
none by both lowering the energy of the transition states and by
holding ribose and the protonated base in close proximity and in a
relative orientation that is compatible with the transition states and
the nucleoside product.
(5) Brown, D. J. Nature 1950, 165, 1010.
(6) (a) Horenstein, N. A. AdV. Phys. Org. Chem. 2006, 41, 275-314. (b)
Berti, P. J.; McCann, J. A. B. Chem. ReV. 2006, 106, 506-555.
(7) Dworkin, J. P.; Miller, S. L. Carbohydr. Res. 2000, 39, 359-365.
(8) Kimura, E.; Kitamura, H.; Koike, T.; Shiro, M. J. Am. Chem. Soc. 1997,
119, 10909-10919.
(9) Frick, L.; Yang, C.; Marquez, V. E.; Wolfenden, R. Biochemistry 1989,
28, 9423-9430.
(10) Gildea, B.; McLaughlin, L. W. Nucleic Acids Res. 1989, 17, 2261-2281.
JA072781A
9
J. AM. CHEM. SOC. VOL. 129, NO. 31, 2007 9557