Pentacovalent intermediate

There has been a series of papers on the deoxygenation of 2-nitrophenyl phenyl ethers and sulphides. Of particular interest has been the role of pentacovalent intermediates (47) in these reactions. These are certainly not the primary intermediates in the reactions but are of great importance in determining the course of the reaction. Their genesis is thought to be that shown in Scheme 3.57... 

Although the detailed catalytic mechanisms of these phosphatases have not been elucidated, an accepted general mechanism is that the two metal ions are cooperatively working by interacting directly with the scissible phosphate and stabilizing the pentacovalent intermediate (33, 45). Moreover, one zinc(II) ion generates the attacking OH ion. 

Since (28) possesses a negative charge at an apical position, it should be unstable relative to (29). Rapid pseudorotation to (29) should then compete with decomposition of (28) to yield products arising from exocyclic cleavage. This explanation requires, however, a pentacovalent intermediate and that the rate of pseudorotation of (28) be increased by a minimal 103 relative to (26) to accommodate the experimental findings. By implication then the above base-catalyzed hydrolysis also is thought to involve the pentacovalent species. 

In the case of the monobenzyl ester the experiments with hydroxylamine do not require intermediates analogous to the above. This is in accord with the decreased stability of a monoanionic pentacovalent intermediate whose zwitterion (45)... 

A rate enhancement of about 107 is observed over the slow spontaneous hydrolysis of the corresponding di-3-nitrophenyl phosphate. The specific displacement of the exocyclic phenolate may be rationalized in terms, of a pentacovalent intermediate (56)... 

Whereas the phosphetanium (Sect. 2.2) derivatives undergo base-catalyzed hydrolysis with essentially complete retention of configuration, the reaction with the related thietanium salts proceeds with complete inversion of configuration and can be described simply as a direct nucleophilic substitution 35. As far as the author is aware, no exchange evidence of the type er countered in the acid-catalyzed hydrolysis of ethylene phosphate, has been found for the cyclic sulfate esters. Consequently despite the geometrical similarities between the cyclic sulfates and phosphates and the related hybridization of the central atoms, the situation in which a finite pentacovalent intermediate sulfur species exists has not been delineated (restricting the discussion to esters). 

The chemical reactivities of groups in the apical and equatorial positions of pentacovalent intermediates are different.664 In particular, elimination of a nucleophilic group to form a tetrahedral phosphate is easier from an apical position than from an equatorial position. For the in-line displacement of Eq. 12-27 elimination of RO should be easy. However an adjacent attack would leave - OR in an equatorial position. Before it could be eliminated, the intermediate would probably have to undergo a permutational rearrangement by which -OR would be transferred from an equatorial to an apical position. 

The structures of the native enzyme and its complexes with several inhibitors have since been obtained at higher resolution in other laboratories, to afford a more complete description of the enzyme - substrate interactions.191 Particularly noteworthy are the lysine residues 7,41, and 66. That these are an important part of the catalytic machinery has been deduced from their conservation in evolution (they have been found in all homologous ribonucleases that have been sequenced), and from their loss of activity when they are acetylated. Lys-41 is particularly important. The lysine side chains are very mobile in the free enzyme, but their mobilities are much decreased on the binding of nucleotide substrate analogues. Lys-41 interacts directly with the phosphate moiety and is thought to stabilize the pentacovalent intermediate. Another residue that has been con-... 

In an associative mechanism, there is first the addition of the nucleophile to give a pentacovalent intermediate, followed by the elimination of the leaving group. This mechanism is subdivided further. There is an in-line mechanism, in which the attacking nucleophile enters opposite the leaving group (equation 8.32),... 

The chemistry and stereochemistry of the reactions were extensively discussed in Chapter 8, sections El and E3. There is an in-line mechanism that generates a pentacovalent intermediate or transition state, with the attacking nucleophile and leaving group occupying the apical positions of the trigonal bipyramid. 

The various proposals have each evolved with time, and the issues are numerous and intertwined. There is general agreement that the phosphorus is attacked by an oxygen atom made more nucleophilic catalyti-cally. A catalytically stabilized pentacovalent intermediate (or activated state) is accepted. Specific catalytic protonation of the leaving group is involved. There is also agreement that at least two of the three residues His 12, His 119, and Lys 41 are involved crucially in the mechanism. 

In Fig. 20, B, R, B2, and R2 are the positions of the base and ribose components of the dinucleotide or independent pyrimidine and purine nucleotides, respectively. The phosphate position pi can be occupied by the 3, 5"-diester (5" refers to the 5 position of R2 in a diester) or the 3 - and 5 -nucleotides, respectively. In the protein crystal a sulfate ion occupied this position in variable degree depending on the pH. Histidine 119 can be in any one of four or more positions depending on various factors. The second base might be in position B2 when it is a pyrimidine. The phosphate of a cyclic substrate or pentacovalent intermediate may be at p,. The position labeled H20 is the position of an isolated peak on the electron density map which is interpreted to be a water molecule, Wi, present in the protein and in the complexes. 

Attack opposite 05" would allow immediate departure of 05" without pseudorotation or protonation of X or Y. Protonation or juxtaposed charges could facilitate attack by making P more positive and thus reduce the energy of the activated complex. Either would also likely stabilize the pentacovalent intermediate ES and might trap it for a finite time. Witzel (519) has emphasized both of these points without reference to pseudorotation. Protonation of X or Y coupled with pseudorotation could allow 03 to leave forming the 2, 5"-diester, but such isomerization does not occur. 

In the in-line push-pull mechanisms of Rabin and Roberts, the highest energy transition state may be either the pentacovalent intermediate or the alkoxide (hydroxide) state with 02 or 05" deprotonated but not bonded to P. Incipient deprotonation of 02 in an activated state is equivalent. Protonation of X or Y or nearby positive charge could stabilize the pentacovalent intermediate. Removal of either could facilitate formation of the alkoxide in the breakdown of the intermediate. Restoration of the initial state of the enzyme is required in this mechanism and could be rate limiting. In the adjacent (pseudorotation) models of Witzel, Hammes, Usher, or Wang protonation of X or Y would be required to allow one of the two pseudomers to exist. In step 1 this requirement (and thus perhaps a rate limiting process) applies to the attack by 02. Deprotonation would force or facilitate reversal or pseudorotation to... 

The ks curve for C > p hydrolysis is not symmetrical, and a secondary acid pathway has been proposed to explain part of the activity at pH 4.0 (499). Perhaps the protonation of both histidines produces enough polarization of the phosphate to permit direct attack by water or stabilizes the transitional state in the formation of the pentacovalent intermediate. With excess protons present, protonation of the leaving group would be easy. If histidine is not involved as a base, the ks/Km curve should also be distorted according to this view. This description would not seem to fit their model. In any case it would be interesting to see if this acid pathway is different from the normal pathway with respect to in-line vs. adjacent mechanisms. An alternate proposal is that the protonation of the acetate-tris buffer was distorting the curve. Another factor to consider is the chloride behavior as a competitive inhibitor, but this would not affect fcs if it is strictly competitive. 

West (91), amplifying on these results, argued that since the solvolysis is bimolecular it must proceed either through a normal Sn2 bimolecular displacement or involve a rather stable pentacovalent intermediate. Both mechanisms. West believes, must involve a 5-coordinate transition state, and therefore may really be thought of as equivalent. West found that silacyclopentane was 13 times as reactive as diethylmethylsilane and 200 times as reactive as silacydohexane (which could be construed as evidence for I-strain in silacyclopentane). Since this order of reactivity is the same as that found in carbocyclic compounds, it was concluded that similar considerations of energy and entropy of reaction are encountered, a possibility that had also been advanced by Price. 

The formation of both isomers after the fluorodephenylation seems to occur not only in the interaction with sulphuric acid, but also with alcohol or moisture. The authors postulated a mechanism similar to the racemization of 1-naphthylphenylmethylfluorosilane, which included a pentacovalent intermediate state ... 

Figure 12. Proposed metal-ion-stabilized pentacovalent intermediates...

A plausible rationale for the above Mg2+ ion catalysis may be electrostatic facilitation of nucleophilic attack to form a pentacovalent intermediate as depicted in the structures of Figure 12. This rationale already has been proposed by Steffens et al. for the metal ion catalysis in the intramolecular displacement reaction of phenolate ion by car-boxylate (17). 

What we found is that all metal ions catalyze P—O fission. Selective P—O fission by amines was increased from 80% to 100% in the presence of Mg2+ ion, which also enhanced the rate. Exclusive P—O fission also occurred in the attack by the oxyanion of PCA in the presence of Zn2+ ion. A plausible rationale is that such a path, which involves metal ion assistance in a pentacovalent intermediate as illustrated in Figure 12a, is energetically much more favorable than that of Sn2 displacement on sulfur. Conversely, if an enzyme that catalyzes the reaction of phosphosulfate is metal ion dependent, the reaction probably involves P—O fission, as suggested by Roy (4). 

Now the alkoxide anion can attack the positively charged phosphorus atom. This is a good reaction in two ways. First, there is the obvious neutralization of charge and, second, the P-O bond is very strong. This reaction, which we have drawn as an S>j2 reaction at phosphorus, really goes through a pentacovalent intermediate shown to the right, but you will usually see it drawn in a concerted fashion. r -i... 

Evidence that the 1Sn2 reaction at silicon does indeed go through a pentacovalent intermediate comes from the silicon analogue of the migration step in hydroboration-oxidation. Treatment of reactive organosilanes (that is, those with at least one heteroatom—F, OR, NR2—attached to silicon to encourage nucleophilic attack of hydroperoxide at silicon)... 

The actual substrates are Mg2+ complexes of ADP and ATP, as in all knovm phosphoryl transfer reactions with these nucleotides. A terminal oxygen atom of ADP attacks the phosphorus atom of P to form a pentacovalent intermediate, which then dissociates into ATP and H2O (Figure 18.28). The attacking oxygen atom of ADP and the departing oxygen atom of P occupy the apices of a trigonal bipyramid. 

Figure 18.28. ATP Synthesis Mechanism. One of the oxygen atoms of ADP attacks the phosphorus atom of Pj to form a pentacovalent intermediate, which then forms ATP and releases a molecule of H2O.

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