J. Am. Chem. SOC.1994,116, 3922-3931
3922
The Significance of Electrostatic Effects in Phospho-Ester Hydrolysis? Philip Tole and Carmay Lim' Contribution from the Departments of Molecular and Medical Genetics, Chemistry, and Biochemistry, University of Toronto, 1 King's College Circle, Toronto, Ontario M5S 1 A8, Canada Received August 25, 1993. Revised Manuscript Received December 6, 1993'
Abstract: The detailed activation free energy profiles for alkaline hydrolysis of the methyl aminoethylenephosphonate (MNP) in vacuum and the changes upon solvation have been computed. The calculations predict that alkaline hydrolysis of M N P in solution proceeds via (OH)- attack opposite the ring oxygen instead of the nitrogen, in agreement with the concept of apicophilicity. The M N P + (OH)- solution reaction is predicted to yield a mixture of products, viz., inversion of configuration production from P-0 endocyclic cleavage and retention of configuration products from P-0 exocyclic cleavage and P-N endocyclic cleavage. Intramolecular charge-charge interactions between the hydroxyl and/or amide protons with neighboring atoms influence the position of the ring nitrogen relative to the ring oxygen in a TBP and facilitate endo- and exocyclic cleavage. There is no evidence of transition-state stereoelectronic effects controlling the M N P (OH)- reaction pathway.
+
1. Introduction
Scheme 1
Phosphate esters and anhydrides dominate the chemistry of life processes and are of great chemical, technical, and biological importance.' In particular, due to the significance of cyclic fivemembered phospho-esters as intermediates in the hydrolysis of ribonucleic acids,2 there have been numerous experimental studies3v4on the hydrolysis of this type of ester. From thesestudies a set of guidelines have been developed for predicting the product distribution, hydrolysis rates, and stereochemistry for associative nucleophilic substitution reactions at phosphorus. The pioneering studies by Westheimer and co-workers3,sfjhave provided indirect evidence for the formation of a trigonal-bipyramidal (TBP) pentacovalent intermediate under certain reaction conditions as well as a set of rules governing the dynamics of TBPintermediate~.~ Pseudorotation of a TBP intermediate can proceed via a Berry mechanism, as illustrated in Scheme 1. Recently, a detailed theoretical study of the basic hydrolysis of the methyl ethylenephosphate (MEP) has established a number of novel mechanistic principles for phospho-ester reactivity in the absences.9 and presence of enzymes.10 The calculations reveal a reaction, where hydroxyl new mechanism for the MEP (OH)ion attack is concerted with pseudorotation to form a TBP intermediate which undergoes ring opening faster than isomerization and subsequent exocyclic cleavage of the methoxy group. Thus, the calculations predict endocyclic cleavage products with retention of configuration in solution. A key finding was that the rate-determining step in solution was governed by the formation of a long-range distorted TBP transition state rather than endo-
+
t This work was supported by the Protein Engineering Network Centers of Excellence of Canada. 0 Abstract published in Aduance ACS Abstracts, March 15, 1994. (1) Westheimer, F. H. Science 1987, 237, 1173-1 178. (2) Fersht, A. Enzyme Structure and Mechanism; W. H. Freeman: New York, 1985. (3) Westheimer, F. H. Acc. Chem. Res. 1968, 1, 70-78. (4) Thatcher, G. R. J.; Kluger, R. Adu. Phys. Org. Chem. 1989, 25, 99. ( 5 ) Haake, P. C.; Westheimer, F. H. J. Am. Chem. SOC.1961,83,11021109. (6) Kluger, R.; Covitz, F.; Dennis, E.; Williams, L. D.; Westheimer, F. H. J. Am. Chem. SOC.1969, 91, 6066-6072. (7) Luckenbach, R. Dynamic Stereochemistry of Pentaco-ordinated Phosphorus and Related Elements; Georg Thieme Publishers: Stuttgart, 1973. (8) Lim, C.; Tole, P. J. Phys. Chem. 1992, 96, 5217-5219. (9) Tole, P.; Lim, C. In The Anomeric Effect and Associated Srereoelectronic Effects;Thatcher, G. R., Ed.; ACS Symposium Series 539; American Chemical Society: Washington, DC, 1993. (10) Lim, C.; Tole, P. J. Am. Chem. SOC.1992, 114, 7245-7252.
0002-7863/94/1516-3922$04.50/0
d or exocyclic cleavage of the pseudorotated TBP intermediate. Another new finding was that ring strain, as manifested by the deviation of the ring 0-P-0 angle relative to its acyclic counterpart, was relieved in the long-range transition state but was retained in the TBP intermediate. To further establish the factors governing nucleophilic (OH)addition to the phosphorus center, a thorough investigation of the basic hydrolysisof the methyl aminoethylenephosphonate (MNP, Figure 1) has been carried out. As the phosphorus center is chiral in MNP, the stereospecificity of the M N P (OH)- reaction can be clearly followed. The stereochemical outcome of the various possible pathways for the alkaline hydrolysis of M N P is summarized in Scheme 2. The gas-phase activation free energy profiles (see Method section) for the M N P + (OH)-reaction were calculated using a b initio molecular orbital methods" at the MP2/6-3 l+G*//HF/3-21 +G* level.I2 Solvent effects on the gas-phase activation free energy profiles were estimated using continuum dielectric methodsI3J4to compute the solvation free energies of the reactants, transition states, intermediates, and products. In section 2, the quantum mechanical method and the continuum dielectric method are outlined briefly. Section 3 presents the activation free energy profiles for the M N P (OH)and how they are affected by solvation. The important factors that control the reactivity of M N P with (OH)-are discussed in section 4, and the results are summarized in section 5. The present work demonstrates that there is an underlying set of unifying
+
+
~~
~~
(11) Hehre, W. J.; Radom, L.; Schleyer, P.v. R.; Pople, J. A. Ab Initio Molecular Orbital Theory; John Wiley and Sons: New York, 1986. (12) This denotes a single-point energy calculation on the geometry fully optimized at the Hartree-Fock (HF) level with the 3-21+G* basis set using second-orderMoller-Plesset perturbation theory (MP2) with a 6-3 1+G* basis
set. (13) Gilson, M. K.; Honig, B. H. Proteins: Struct., Funcf.,Genet. 1988, 4, 7-18. (14) Lim, C.; Bashford, D.; Karplus, M. J. Phys. Chem. 1991,95, 56105620.
0 1994 American Chemical Society
J . Am. Chem. SOC..Val. 116, No. 9. 1994
Elecfrosroric Effecrs in Phospho- Esfer Hydrolysis
d \
3923
*.1";.
8
6%
112
)2=4b4 2 I
0 1 II
11
12
Figure 1. HF/3-21+G* ball and stick geometry of methyl aminoethylenephosphonate energy minims
Scheme 2 +
CHELP (charges from electrostatic potentials)." A natural bond order (NBO)" population analysis was performed. Vibrational frequencies were analyzed in terms of the internal coordinates using the program MOLVIB.19 Fromthefrequenciesandgeometries,thevibratianalenergy ( E d andentropy (Sdwerecalculatedaccording tostandardstatistical mechanical formulas." The rotational (E,*,) and translational [E,") energy were treated classically. The electronic correlation energy was estimated by second-order Mdler-Plesset perturbation theory (MPZ) with the 6-31+G* basis for a single p i n t corresponding to the HF/321+G* optimized geometry. Addition of the energetic and entropic corrections to the MP2/6-31+G*//HF/3-21+G* 12activationenergies gave the gas-phaes free energy barriers. The solution free energy barrier. ha:,., from A to B was calculated from a thermodynamic cycle.
*H
\
where ha;, is the gas-phase free energy barrier and the M , ' s arc solvation free energies; that is A&
Retention
Retention
Retention
Retention
=
+
-W A )
(2.1)
The Helmholtz solvation free energyzo was estimated by solving Poisson's equation using the finitedifferencemethod.".14 Thecalculations employed a 71 X 71 X 71 lattice centered on the phosphorus with a grid principles, which on one hand reinstates some of the concepts spacing of 0.25 A. The low-dielectric region, which was assigned a proposed by Westheimer and ~ o - w o r k e r s 3 , ~ a nond t h e o t h e r hand dielectric constant of 2. was defined as the region inaccessible to contact i n t r d u c e s n e w key concepts that governthereactivityofphosphoby a 1.4-A sphcre rolling over a surface defined by the effective solule esters. radii. The latter were taken from the CHARMM version 22l' van der Waalsradii. TheHF/3-21+G*geometriesandCHELP"panialcharges. 2. Method which were assumed to be the same in vacuum and solution. wereemployed. We briefly outline the methods here and rely on previous ~ o r k ~ ~ ' 4 ' ~The diffcrence between the electrostatic potential calculated in ~olvent and vacuum, characterized by a dielectric constant of 80 and I, to provide the details. The a b initio calculations were performed using respectively, yielded the solvation free energy. the program Gaussian 92.16 The hydroxyl oxygen to phosphorus distance waschosen as the reactioncoordinate forthe approach of (OH)-to MNP. 3. Results and the phosphorusto departingatom distancewas chosen as the reaction coordinate for e n d o and exwyclic cleavage of the TBP intermediates. T h e following nomenclature for t h e pentacovalent complexes Each geometry was fully optimized at the HF'/3-21+G* level, and the illustrated in Figures 2 a n d 4 is adopted here, consistent with resulting wave function was used to calculate the electrostatic potential. ( 1 7 ) Chidun. L F , Frmcl. M U J Compui Chem 1987 8. R94 which was fitted to an atomic point charge model using the program (In)Reed. A E .Curtis%.L A , Weinhold. F Chem Rer 19RR.88 R99 (I5)Tale. P.; Lim. C. J . Phys. Chem. 1993.97.6212. I191 Kuc7era. J Kuciera K P r o m m MOI V I R Hanard Unncrrilv (16) Frisch, M. J.: Trucks, G. W.; Head-Gordon. M.; Gill, P. M. W.: Ca&b;idgc, 1988. Wong. M. W.; Foreman. J. 6.; Johnson, 6. G.:Schlegcl, H. E.: Robb. M.: (20) Chan, S. L.: Lim. C. Bridging continuum dielectric theory. thermRcploglc, E. S.; Gomperts, R.; Andres. J. L.: Raghavachari. K.: Binklcy. J. dynamicrandstatirtical mechanics. J . Chem. Phys.,rubmitted for publication. S.:Gonzalcr. C.; Martin. R. L.: Fox. D.J.: Defrccr. D.J.; Baker. J.:Stcwart. (21) MacKercll. A. D.. Jr.: Wiorkiewicr-Kuezera. 1.: Karolus. M. AllJ. J. P.; Pople. J. A. Gourrion 92. Revision A, Gaussian Inc.: Pittsburgh, PA hydrogen empiricalparame1cri;atian of nucleic acids fir malecdlar modeling. 15213. 1992. minimizations 2nd dynamics simulations. Manuscript in preparation.
3924 J . Am. Chem. SOC.,Yo/. 116, No. 9, 1994
Tole and Lim
J . Am. Chem. Soc., Vol. 116, No. 9, 1994 3925
Electrostatic Effects in Phospho-Ester Hydrolysis Table 1. Energies. Entropies, and Solvation Free Energies
E T R V(kcal/mol) ~
ST&' (cal mol-' K-I)
AA/ (kcal/mol)
MNP (OH)I"L TSaaL 1-N TS-N (MHAEP)-
-809.398 -809.424 -809.409 -809.474 -809.464 -809.510
E H( a~4 69 35 01 54 04 26
-814.890 -814.911 -814.906 -814.973 -814.961 -815.010
25 36 89 31 15 76
97.168 98.611 97.935 100.107 98.613 100.499
132.171 110.540 98.653 92.900 94.923 100.174
-113.17 -82.08 -78.24 -65.92 -67.58 -66.42
TS-H 1"X TS-X
-809.456 -809.467 -809.457 -809.474
87 05 92 41
-814.959 -814.970 -814.963 -814.976
24 09 09 29
98.780 99.927 98.585 97.795
92.397 93.922 95.454 137.917
-68.89 -68.02 -64.96 -83.42
-809.458 -809.443 -809.464 -809.460 -809.467 -809.438
81 13 21 21 90 84
-814.957 -814.944 -814.965 -814.960 -814.965 -814.937
16 14 39 91 51 84
98.206 98.392 99.882 99.159 99.861 98.109
99.918 95.836 93.687 91.308 94.505 95.225
-67.64 -67.87 -68.92 -69.41 -70.23 -72.25
-809.439 -809.458 -809.437 -809.434 -809.443
53 03 95 53 91
-814.944 87 -814.962 57 -8 14.944 42 -814.942 37 -814.952 46
99.01 1 99.875 97.806 98.244 98.144
90.705 93.716 96.976 94.574 92.659
-70.42 -65.96 -67.19 -67.91 -62.72
+
(AEP)I'eL TPCL
I= TSaCM IaCN TSaCN TSP
IQX TS"X TSQH TSQN
E,,t
+ MeOH
E M P(au) ~~
H F energies for fully optimized 3-21+G* geometries. Single-point MP2/6-31+G* energy calculation at 3-21+G* geometry. ETRV= EmM+ in cal mol-' K-*,Solvation free energies in kcal/mol. + Evibin kcal/mol. STRV = S,t st,
+
+
b
Figure 3. Gas-phase activation free energy profile for (OH)- attack opposite the ring nitrogen of M N P and the change in profile upon solvation. The circles correspond to species on the exocyclic cleavage pathway.
previous work.15 The letters I and TS denote intermediate and transition state, respectively. The superscripts a and e denote an axial and equatorial position in a TBP, respectively; the first superscript refers to the position of the (OH)- nucleophile and the second to the N H group location in the TBP. The nonsuperscript letter after I or TS describes the nature of the TBP complex: L for long-ranged ion-dipole complexes, P for pseudorotation, H or M for rotation of the hydroxyl or methoxy group about the P-O(H/Me) bond, N for endocyclic cleavage, and X for exocyclic cleavage. Note that the pseudorotation transition state TSP in Figure 4 was found to be a tetragonal-
pyramidal structure with an apical ligand and four basal ligands (see section 3.3.1). Replacing one of the ring oxygens in MEP with an N H group breaks the symmetry of the ring so that (OH)- can attack opposite a ring oxygen or nitrogen in MNP. The results for (OH)attack opposite MNP(N) will first be discussed, followed by the results for attack opposite M N P ( 0 ) . Figures 2 and 4 illustrate the gasphase free energy profiles, and Figures 3 and 5 show how they are altered in solution. The thermodynamic data for the figures are collected in Table 1. The structural information for each of the species is provided as supplementary material in three tables. Unless stated otherwise the values of the atomic charges were obtained from the CHELP program.1' In the following discussion a frequency for a P-O/P-N bond means that it contains the maximal percentage contribution of a P-O/P-N stretch, but it does not necessary correspond to a pure (100%) P-O/P-N stretch. 3.1. MNP. In vacuum, M N P exists in a gauche and a trans conformation, characterized by a CMe-0-P-01 dihedral of 29 and 176', respectively; the -gauche conformation was found to be unstable. Like MEP, the trans conformer with the methyl group over the ring is less stable than the gauche conformer by 3 kcal/mol at the HF/3-21+G* level (see Figure 1). The energetic and structural data for the lowest energy conformer are employed in the tables and ensuing discussion. The ring N-P-0 angle (94.3') is similar to the ring 0-P-0 angle (95.1 ') in MEP. This suggests that the ethylene bridge and its relative rigidity controls the ring angle about the P atom in a five-membered ring. Replacing a ring oxygen by an N H group does not lead to significant changes in geometry between MEP and M N P except that the P-N bond length is 0.04 A longer than the P-Oring bond (1.60A). The ring forces the nitrogen intoa rather flat umbrellalike conformation with the amide proton in the plane of the ring and the single lone pair of electrons out of the ring plane. This and the reduced charge density of a face containing N H (rather than 0)are important distinctions between M N P and MEP that result in different reactivity characteristics. 3.2. (OH)-Attackopposite MNP(N). 3.2.1. Activation Free Energy Profile in Vacuum for (OH)- MNP(N). The gasphase activation free energy profile for (OH)- attack opposite the ring nitrogen (Figure 2) is similar to that for (OH)- attack opposite a ring oxygen in MEP (see Figure 1 in Tole and Lim ( 1993)15). In the ensuing discussion, the structural and thermodynamic results will be presented and compared to the corresponding data for the MEP + (OH)- pathway.IS
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3926 J. Am. Chem. SOC.,Vol. 116. No. 9, 1994
Tole and Lim
z w v
*'
J . Am. Chem. SOC.,Vol. 116, No. 9, 1994 3927
Electrostatic Effects in Phospho-Ester Hydrolysis
Table 2. Two-Electron Stabilization Energies from NBO Analysis
S t a i ~ p o i n b a l o n s d a n ~ Y
species TSaaL
TS% MNPC0I-I- 1%
1
TS?
'I
TSP
?X
TS=X
TS'M
1%
TS'N
donor orbital 0'
m
OMC PN
02'
05'
OH TS"N
02'
TWH
OH OH OH
1"X
05' 05'
OH TSCCX
05' 05'
OH
P-05'
TSaCL
0'
I=
OH OMC OMC OMC OMC
P-N N-H P-OH P-OH P-OH P-02' CMC-H P-02' CM'-H P-02' 0-H 0-H P-OMC 0-H P-OMC P-0MC P-N 0-H P-N
TSaCM Solufion
IaCN
02'
OMC
TSaCN
02'
OMS
I
TSP 1-x
d
P-N endocyclic cleavage pathways, respectively.
Long-Range Complexes. An ion-dipole minimum (IaaL) with a tetrahedral phosphorus (averageangleabout P = logo) is found at a P-OH distance of 5.45 8, (5.35 A in MEP). As the reaction coordinate decreases to 2.90 A, a distorted TBP transition state (TSaaL) with average N-P-Y, OH-P-Y, and Y-P-Y ( Y = O', OMc, 03') angles of 101, 78, and 116O, respectively, is obtained. The long-range complexes are structurally analogous to those found on the MEP (OH)- pathway.15 Thus, the axial ring nitrogen has little geometrical influence in the initial stages of the reaction, which is dictated by steric and charge repulsion between the incoming nucleophile and the three negatively charged atoms 01, OMc, and 0 3 ' . However, it has an effect on the IaaL TSaaL gas-phase activation free energy, which is almost double that of the MEP (OH)- pathway. This is because an axial ring nitrogen destabilizes TSaaL relative to the corresponding longrange MEP-(OH)- transition state since oxygen, which forms a more ionicbond with phosphorus as it is more polar than nitrogen, prefers the axial position compared to nitrogen. Evidence for this is provided by the slightly shorter and stronger P-0 axial bond (1.65 A, 803 cm-l)lS in the long-range MEP-.(OH)transition state relative to the P-N axial bond (1.69 A, 788 cm-I) in the TSaaL transition state. Pseudorotation. As in the case of MEP,*J5 an intermediate with the hydroxyl group axial was found to be unstable since it underwent spontaneous pseudorotation to yield 1-N. This is not surprising since the factors that drive pseudorotation in the MEP + (OH)- pathway's are present here, Le., (i) a very favorable free energy gradient to form an 1-N intermediate with the hydroxyl group equatorial (-38 kcal/mol vs -35 kcal/mol for MEP), (ii) attractive HO--O3'and repulsive OH-.O3'/OMe interactions, which causes lengthening of the P-03' bond and widening of the 03'P-OMe angle, and (iii) low-frequency ring torsions and lack of steric hindrance. In addition, the reduced polarity of nitrogen compared to oxygen (resulting in an increased covalency of the P-N bond) favors an equatorial nitrogen in a TBP and, thus, pseudorotation.
+
-
0"W
05'
OH
Figure 5. Gas-phase activation free energy profile for (OH)- attack opposite the ring oxygenof MNP and the changein profile upon solvation. The solid circles and triangles correspond to species on the exocyclic and
+
acceptor orbital P-0" P-OH 0-H N-H P-02' 0-H P-02' P-02' P-05' 0-H N-H P-OS' 0-H N-H
TSCaX
05'
OH OH OH N OH
TScaH TS"N
-
A E t r (kcal/mol) 39.3 C0.05
2.9 1 .o
cos 19.1 co.5 5.2 3.3 2.3 COS
0.7 60.9 co.05
