Experimental and Computational Evidence for a Loose Transition State in Phosphoroimidazolide Hydrolysis

Article abstract of DOI:10.1021/jacs.6b00784

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 reactions.

Full text of DOI:10.1021/jacs.6b00784

Communication
pubs.acs.org/JACS
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
fission to the leaving group both occur in the transition state
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
isotopologue 1b (Figure 1) at pH 4.0 using the competitive
6
11,12
guide optimization of nonenzymatic RNA replication,
method by mass spectrometry (Figure S2).
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
N
involves extensive scissile bond fission, indicating an A DN
mechanism with a loose transition state (Supporting Text 1).
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
KIE is consistent with the phosphate remaining deprotonated
at the rate-limiting step (Supporting Text 2).
Received: January 22, 2016
©
XXXX American Chemical Society
A
DOI: 10.1021/jacs.6b00784
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
Scheme 2. Summary of Calculated A D Mechanisms;
N
N
Calculated P−N Bond Lengths Are Labeled in Å
Figure 1. Isotopologues and their kinetic isotope effects. (a) The
reaction scheme for hydrolysis of isotopologues. (b) KIE measurement
for 1a vs 1b. (c) KIE measurement for 1a vs 1c. The KIE was
determined by measuring R , the enrichment of the heavy to light
S
A D mechanism (sum of axial bond order greater than 1,
isotopologues of the substrate ImpG, over the reaction course. R is
N
N
S
9
Table S3).
In the loose A D mechanism, the hydrolysis proceeds via a
normalized by the initial ratio R . L is the integrated intensity of the
0
light isotopologue, and it is normalized by the initial intensity L . The
N
N
0
solid and dashed lines in (b) and (c) are fits using KIEs of the mean
and the limits of 95% confidence interval (n = 8).
rate-determining transition structure (2, Figure 2) with
To obtain structural insight into the transition state, we
complemented these KIE measurements with quantum
mechanical calculations. The validity of these computational
models was judged by comparing the theoretical KIEs
1
5
calculated using the Bigeleisen−Mayer equation with the
experimental results. Although this powerful approach has
repeatedly provided critical transition-structure insights of
1
6−18
important reactions,
reactions is limited
its application in phosphoryl transfer
1
9,20
due to the difficulties in modeling the
21
20,21
strong phosphate-solvent interactions. Recent advances
overcome this technical difficulty by the microsolvation
approach, in which an explicit model of the solute and a few
key solvent molecules is embedded in an implicit solvent
model. This method calculates isotope effects to high accuracy
Figure 2. Representative transition structure 2 and intermediate 3 of
the loose A D mechanism. Key distances are labeled in Å. Gibbs free
N
N
energies are relative to reactants. In the intermediate, the leaving group
is separated infinitely from 3. Water molecules, except for the
nucleophile, are shown in cyan.
20,21
at an affordable computational cost.
Our computational model of the hydrolysis reaction retains
all the key elements of phosphoroimidazolium hydrolysis
extensive scissile bond fission. In contrast to the planar
metaphosphate-like phosphoryl group in the p-nitrophenol
(Scheme 1). Using the microsolvation approach, we built a
20
series of models with up to eight explicit water molecules that
occupied most hydrogen-bonding sites of the reactants. These
microsolvated structures provide a more comprehensive and
accurate sampling of the experimental transition-state ensem-
ble, which includes various transition structures with different
solvation shells. The structures were optimized at the B3LYP/
phosphate (pNPP) dianion, the phosphoryl group in 2
deviates from a planar structure by ∼10° (Figure S3 and Table
S2). Depending on the local solvent structure, 2 can lead to a
hydrolyzed product either via a concomitant proton transfer
(Scheme 2, Pathway B), or via a zwitterionic intermediate (3,
Figure 2, and Scheme 2, Pathway A). The oxonium ion 3 has
elongated O−P and O−H bonds, and a structurally similar
2
2−24
6
-31+G(d)/PCM level of theory,
which has been
20
successful in elucidating mechanisms of phosphate ester
intermediate has been reported in pNPP dianion hydrolysis.
1
9,25,26
hydrolysis.
Additional calculations performed at different
Figures S5 and S6 show representative intrinsic reaction
coordinates for both pathways of the loose mechanism. In the
following discussion, we consider these two pathways
collectively because their rate-limiting transition structures are
similar (Table S2) and 3 is expected to be short-lived due to its
levels of theory confirmed the nature of the stationary points
and yielded similar geometries (Figures S3 and S4) and
calculated KIEs (Table S1), suggesting that the results are
independent of the theoretical level. All calculated transition
27
structures belong to the A DN mechanism (Scheme 2). We
low pK (ca. −4).
N
a
further classified these transition structures into a loose A D
A close inspection of various microsolvated transition
structures (Figures S7 and S8) revealed that additional explicit
N
N
(sum of axial bond order less than 1, Table S2) and a tight
B
DOI: 10.1021/jacs.6b00784
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
solvent molecules stabilized 2 (Table S4) and shortened its P−
N bond distance (Table S2). The energies and geometries
showed significant variation as the number of explicit solvent
molecules increased from one to three, but seemed to converge
with more than three solvent molecules. Importantly, the
calculated 15k and k from these transition structures both
agreed well with the experimental measurement (Tables 1 and
S5).
18
Table 1. Experimental and Calculated KIEs of ImpG
Hydrolysis
loose (Pathway A)
tight
a
15k
18k
15k
18k
no. of H O
2
0
1
2
3
4
5
6
7
8
0.999
0.999
0.999
0.999
0.999
1.000
1.000
0.999
0.999
0.995
1.001
1.000
1.002
0.999
0.999
0.998
0.999
0.997
1.027
1.025
1.021
1.021
1.021
1.021
1.022
1.023
1.001
1.002
1.002
1.002
1.002
1.002
1.001
1.001
Figure 3. Dependence of the hydrolysis rates on the leaving group pKa
for phosphoroimidazolides. The red line is the least-squares fit to the
data. Error bars show one standard deviation (n = 5).
15k
1.019(2)
18k
1.002(3)
19
diesters as well as the loose yet metaphosphate-like transition
b
observed
20
state of the pNPP dianion. This difference is likely due to the
weaker P−N bond, a fundamental difference between
phosphoroimidazolides and phosphate esters.
a
Number of solvent molecules in the calculation except for the
nucleophile. Limits of the 95% confidence interval in the last digit (n
b
=
8) are shown in parentheses.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.6b00784.
■
*
S
The transition structures of the tight A D mechanism have
N
N
a distorted trigonal bipyramidal phosphorus center (Figures S4
and S9) and they show little scissile bond fission (Table S3)
with predicted 15k close to 1 (Table 1). Transition structures
Materials and methods, supporting text, Figures S1−S13,
Tables S1−S7, and energies and full geometries of all
calculated structures (PDF)
obtained by progressive microsolvation (Figure S9) showed
little geometric (Figure S10 and Table S3) and energetic
variation (Table S6). All calculated reaction barriers of the tight
mechanism were much higher than those of the loose
mechanism (Figure S11) as well as the experimental activation
energy (Figure S12). Critically, although the calculated 18k for
the tight mechanism fell in the range of the experimental
AUTHOR INFORMATION
Corresponding Author
*szostak@molbio.mgh.harvard.edu
■
Notes
1
5
results, the calculated k did not (Table 1). These results
The authors declare no competing financial interest.
disfavor the tight A D mechanism.
N
N
The extensive calculated P−N bond fission predicts that
leaving group ability should have a strong influence on the
hydrolysis rate. A linear correlation between the leaving group
ACKNOWLEDGMENTS
■
J.W.S. is an Investigator of the Howard Hughes Medical
Institute. L.L. is a Life Sciences Research Foundation Fellow.
This work was supported in part by a grant (290363) from the
Simons Foundation to J.W.S. The authors thank Szostak lab
members Dr. A. C. Fahrenbach, Dr. N. P. Kamat, Dr. A. Pal, J.
Tam, and T. Walton for assistance with the synthesis and Dr.
Eugene Kwan (Harvard University) for helpful discussions.
Computations were run on the Odyssey cluster supported by
the FAS Division of Science, Research Computing Group, at
Harvard University.
pK and the logarithm of the hydrolysis rate was found with a
a
series of phosphoroimidazolides (Figures 3 and S13). The large
negative slope (Brønsted value, or β ) of −1.49 supports the
lg
loose A D mechanism, and is comparable to the values of
N
N
27
−
1.23 of pNPP dianions and −1.1 of phosphorylated
pyridines. Interestingly, although 2-MeImpG hydrolyzes 21-
28
fold more slowly than 1a, it condenses about 10-fold faster in
29
nonenzymatic primer extension. The precise role of the 2-
methyl group in stimulating primer extension is still unclear,
and will be an important focus of future studies.
REFERENCES
1) Westheimer, F. H. Science 1987, 235, 1173.
2) Cleland, W. W.; Hengge, A. C. Chem. Rev. 2006, 106, 3252.
(3) Chakravarty, A. K.; Subbotin, R.; Chait, B. T.; Shuman, S. Proc.
Natl. Acad. Sci. U. S. A. 2012, 109, 6072.
■
In conclusion, our quantum mechanical calculations provide
a three-dimensional transition-state model that unifies the
(
(
experimental KIEs and β of phosphoroimidazolide hydrolysis.
lg
At the transition state, there is significant P−N bond fission
without forming a metaphosphate-like phosphoryl group. This
is distinct from the much tighter transition state of phosphate
(4) Blain, J. C.; Szostak, J. W. Annu. Rev. Biochem. 2014, 83, 615.
(5) Inoue, T.; Orgel, L. E. J. Am. Chem. Soc. 1981, 103, 7666.
C
DOI: 10.1021/jacs.6b00784
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
(
(
(
6) Szostak, J. W. J. Syst. Chem. 2012, 3, 2.
7) Adamala, K.; Szostak, J. W. Science 2013, 342, 1098.
8) Kanavarioti, A.; Bernasconi, C. F.; Doodokyan, D. L.; Alberas, D.
J. J. Am. Chem. Soc. 1989, 111, 7247.
9) Lassila, J. K.; Zalatan, J. G.; Herschlag, D. Annu. Rev. Biochem.
011, 80, 669.
(
2
(
10) Hengge, A. C. Acc. Chem. Res. 2002, 35, 105.
(
11) Berti, P. J.; Blanke, S. R.; Schramm, V. L. J. Am. Chem. Soc. 1997,
1
(
19, 12079.
12) Harris, M. E.; Dai, Q.; Gu, H.; Kellerman, D. L.; Piccirilli, J. A.;
Anderson, V. E. J. Am. Chem. Soc. 2010, 132, 11613.
13) Knight, W. B.; Weiss, P. M.; Cleland, W. W. J. Am. Chem. Soc.
986, 108, 2759.
14) Hengge, A. C.; Edens, W. A.; Elsing, H. J. Am. Chem. Soc. 1994,
16, 5045.
(
1
(
1
(
15) Bigeleisen, J.; Mayer, M. G. J. Chem. Phys. 1947, 15, 261.
(
16) Houk, K. N.; Gustafson, S. M.; Black, A. K. J. Am. Chem. Soc.
1
992, 114, 8565.
17) Storer, J. W.; Raimondi, L.; Houk, K. N. J. Am. Chem. Soc. 1994,
16, 9675.
18) Ussing, B. R.; Singleton, D. A. J. Am. Chem. Soc. 2005, 127,
888.
19) Wong, K.-Y.; Gu, H.; Zhang, S.; Piccirilli, J. A.; Harris, M. E.;
York, D. M. Angew. Chem., Int. Ed. 2012, 51, 647.
20) Duarte, F.; Åqvist, J.; Williams, N. H.; Kamerlin, S. C. J. Am.
Chem. Soc. 2015, 137, 1081.
21) Kolmodin, K.; Luzhkov, V. B.; Åqvist, J. J. Am. Chem. Soc. 2002,
24, 10130.
(
1
(
2
(
(
(
1
(
22) Becke, A. D. J. Chem. Phys. 1993, 98, 5648.
(
23) Lee, C.; Yang, W.; Parr, R. G. Phys. Rev. B: Condens. Matter
Mater. Phys. 1988, 37, 785.
24) Tomasi, J.; Mennucci, B.; Cammi, R. Chem. Rev. 2005, 105,
999.
25) Ribeiro, A. J. M.; Ramos, M. J.; Fernandes, P. A. J. Chem. Theory
Comput. 2010, 6, 2281.
26) Lopez, X.; Schaefer, M.; Dejaegere, A.; Karplus, M. J. Am. Chem.
Soc. 2002, 124, 5010.
(
2
(
(
(
(
(
27) Kirby, A. J.; Varvoglis, A. G. J. Am. Chem. Soc. 1967, 89, 415.
28) Jameson, G. W.; Lawlor, J. M. J. Chem. Soc. B 1970, 53.
29) Wu, T.; Orgel, L. E. J. Am. Chem. Soc. 1992, 114, 5496.
D
DOI: 10.1021/jacs.6b00784
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX