Communication
Experimental and Computational Evidence for a Loose Transition
State in Phosphoroimidazolide Hydrolysis
Li Li, Victor S. Lelyveld, Noam Prywes, and Jack W. Szostak*
Howard Hughes Medical Institute, Department of Molecular Biology and Center for Computational and Integrative Biology,
Massachusetts General Hospital, Boston, Massachusetts 02114, United States
*
S Supporting Information
A good model system for such a mechanistic study is the
hydrolysis reaction (Scheme 1). Earlier work by Kanavarioti et
al. on the hydrolysis pH−rate profile suggested that from pH 4
to 10 hydrolysis occurs via the attack of water on a
phosphoroimidazolium zwitterion with a deprotonated phos-
ABSTRACT: Phosphoroimidazolides play a critical role
in several enzymatic phosphoryl transfer reactions and
have been studied extensively as activated monomers for
nonenzymatic nucleic acid replication, but the detailed
mechanisms of these phosphoryl transfer reactions remain
elusive. Some aspects of the mechanism can be deduced by
studying the hydrolysis reaction, a simpler system that is
amenable to a thorough mechanistic treatment. Here we
characterize the transition state of phosphoroimidazolide
hydrolysis by kinetic isotope effect (KIE) and linear free
energy relationship (LFER) measurements, and theoretical
calculations. The KIE and LFER observations are best
explained by calculated loose transition structures with
extensive scissile bond cleavage. These three-dimensional
models of the transition state provide the basis for future
mechanistic investigations of phosphoroimidazolide reac-
tions.
8
phate and a protonated leaving group. To obtain insight into
the transition state structures, additional experimental and
computational tools are necessary. These include kinetic
isotope effect (KIE) and linear free energy relationships
(
LFER) that probe changes in bonding and charge distribution
in the transition state, respectively, as well as quantum
mechanical calculations that provide three-dimensional tran-
sition structures to better interpret these experimental results.
Phosphoryl transfer reactions may proceed via three
mechanisms: an S 1-like stepwise mechanism with a
N
metaphosphate intermediate (D + A per IUPAC nomencla-
N
N
ture), a stepwise AN + DN mechanism with a phosphorane
intermediate, and an S 2-like concerted A D mechanism in
N
N
N
which the bond formation to the nucleophile and the bond
2
hosphate esters confer a stable backbone upon nucleic
(Figure S1). The transition state of the A D mechanism is
N
N
1
P
acids and are ubiquitous in biochemical reactions.
Phosphoroimidazolides, being much more labile, have been
mainly found as enzymatic intermediates, such as those formed
loose if it has less axial bonding than the ground state, or tight if
9
it has more. Extensive KIE and LFER studies have shown that
phosphate esters hydrolyze via either the A D or A + D
N
N
N
N
2
3
mechanisms. Furthermore, as the alkylation state of the
phosphate increases from monoesters to triesters, the transition
by acid phosphatase and the RtcB RNA ligase. Their
reactivity has been exploited in laboratory efforts to synthesize
2
,9,10
4
state tightens with less scissile bond fission.
self-replicating model protocells, in which nucleoside 5′-
To characterize the extent of scissile bond fission in the
transition state of phosphoroimidazolium hydrolysis, we
phosphoroimidazolides are used as activated substrates to
5
chemically copy RNA templates (Scheme 1). Resolving the
determined the leaving-group KIE by hydrolyzing guanosine-
transition structures of these phosphoryl transfer reactions will
advance our understanding of enzymatic reactions and may
15
5
′-phosphoroimidazolide (ImpG) 1a and its N-labeled
6
11,12
guide optimization of nonenzymatic RNA replication,
KIE for the hydrolysis of 1a relative to 1b, 15k, was 1.019 ±
.002 from eight independent measurements (Figure 1). Its
The observed
potentially enabling the replication of a broader range of
7
0
template sequences inside model protocells.
large amplitude suggests that the hydrolytic rate-limiting step
Scheme 1. Model Systems for the Study of Nonenzymatic
RNA Replication
We next measured the KIE for hydrolysis of phosphoro-
18
imidazolides 1a relative to 1c, or k. The observed KIE is 1.002
±
0.003 (Figure 1). Since the 18O isotope effect for phosphate
13,14
protonation is inverse, between 0.98 and 0.99,
the observed
Received: January 22, 2016
©
XXXX American Chemical Society
A
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX