PMC

Substituted phosphates and related compounds important for biology and mechanism. Phosphate monoesters and diesters form crucial biological compounds. Phosphoanhydrides contain one or more anhydride linkages between phosphate groups. Triesters, thio-substituted compounds, and phosphorylated pyridines have contributed significantly to mechanistic understanding. The protonation states shown are the dominant forms at pH 7–8.

Charge distribution on the phosphoryl oxygen atoms in the transition state. Based on a simple arrow-pushing scheme, one might expect the charge on the nonbridging oxygen atoms to decrease in a loose transition state (a) and increase in a tight transition state (b) (the transition-state charges depicted are the extreme limits). However, the charge distribution for a metaphosphate-like species in a loose transition state is unknown. Three different resonance forms can be depicted with more or less charge on the nonbridging oxygen atoms (c).

Phosphate monoesters and proposed hydrolysis reaction mechanisms. (a) Hydrolysis of phosphate monoester dianion. (b) At low pH, phosphate monoesters become protonated to form monoanions (). (c,d ) The rapid hydrolysis of these phosphate monoester monoanions led early researchers to propose that phosphate ester hydrolysis reactions proceeded through a metaphosphate intermediate. There is now strong evidence against the intermediacy of metaphosphate. (e) Reaction coordinate diagram. A transition state (‡) represents a local maximum along a reaction coordinate, whereas an intermediate exists in an energy well.

Interpreting observed linear free energy relationships (LFERs). (a) Observed β_LG LFERs. In gray: substituted phosphate monoester dianions reacting with water [diamonds indicate benzoyl leaving groups (); circles () and square () are phenolate leaving groups]. Points are indicated for phenyl phosphate and p-nitrophenyl phosphate. In red: hydrolysis reactions of phosphorylated pyridines [squares () and circles ()]. In blue: morpholine nucleophile reacting with phosphorylated pyridines (). (b) Observed β_NUC LFERs. In gray: amine nucleophiles reacting with p-nitrophenyl phosphate (). In red: oxygen nucleophiles with phosphorylated 4-methyl pyridine (). In blue: amine nucleophiles with phosphorylated 3-methoxypyridine (). (c) Pyridine nucleophiles and leaving groups do not have transferrable protons and show the same trends as for oxygen nucleophiles (panels a and b).

Changes in structure can alter the nature of the transition state. (a) Along the reaction pathway, the transition state is at a maximum in free energy. When the equilibrium between reactants and products changes, the transition state moves toward the species that has increased in energy. (b) Perpendicular to the reaction pathway, the transition state is at a minimum in free energy. When the equilibrium between the phosphorane and metaphosphate species changes, the transition state moves toward the species that has decreased in energy. (c) A two-dimensional reaction coordinate diagram for symmetric phosphoryl-transfer reactions with alkyl nucleophiles and leaving groups, with approximate predicted changes in transition-state structure as ester substituents are added to the phosphoryl group and the energy of the phosphorane corner decreases. (d) Similar approximate predictions for symmetric reactions with p-nitrophenolate nucleophiles and leaving groups. Whereas monoester reactions show little variability in transition state, triesters show considerable variability (as discussed further in ).

Substituent effects can provide information about charge development in the transition state. (a) In the complete hydrolysis reaction of phenyl phosphate, the P-O bond to the leaving-group oxygen is fully broken, and negative charge develops on the phenolate oxygen. In the transition state, the P-O bond is partially broken, and negative charge partially accumulates on the phenolate oxygen. (b) Adding an electron-withdrawing p-nitro group distributes electron density away from the leaving-group oxygen in the transition state, making the partial cleavage of the P-O bond more favorable and thereby increasing the reaction rate. (c,d ) Adding a p-nitro group also withdraws electron density in the free phenolate, shifting the overall equilibrium toward products and reducing the affinity for a proton. This results in a reduced pK_a value. (e) Hypothetical linear free energy relationships (LFERs) for a series of leaving groups of differing pK_a values.

Kinetic isotope effects (KIEs) for nonenzymatic phosphoryl-transfer reactions with p-nitrophenyl leaving groups (, , –) (includes one unpublished value from E.A. Tanifum & A.C. Hengge). Complete data tables and corresponding references are presented in the . The reactions shown are for the fully ionized species (i.e., phosphate monoester dianions and sulfate monoester monoanions, rather than the corresponding protonated forms). (a) Leaving-group KIEs (¹⁸k_bridge) for monoester hydrolysis are large, suggesting extensive bond cleavage to the leaving group in the transition state. Values of ¹⁸k_bridge are significantly smaller for diesters and triesters. Phosphorothioate and sulfate esters exhibit similar trends in their leaving-group KIEs. (b) The nonbridging oxygen atom KIE (¹⁸k_nonbridge, per oxygen atom) for phosphate monoester hydrolysis is close to unity, whereas that for triester hydrolysis is large and normal. Values for diester hydrolysis are more variable. Phosphorothioate esters are not included because the presence of sulfur at the nonbridging position significantly perturbs ¹⁸k_nonbridge (see ), and sulfate esters are omitted because ¹⁸k_nonbridge is available for only a single sulfate monoester.

Kinetic isotope effects (KIEs) report on changes in bonding in the transition state. (a) Sites of isotopic substitution in the phosphate monoester p-nitrophenyl phosphate (pNPP) are shown in color. KIEs can be measured for multiple sites within a molecule, including the bridging oxygen at the position of bond cleavage (red ) and the nonbridging oxygen atoms of the phosphoryl group (blue). The presence of a nitrogen atom (green) in pNPP allows KIEs to be measured with the remote label method (), as it is technically easier to measure ¹⁵N/¹⁴N than ¹⁸O/¹⁶O ratios. Recently, however, some KIEs have been determined by direct measurement of ¹⁸O/¹⁶O ratios (, , ). (b) Heavy-isotope substitution at the position of bond cleavage (red, bridging oxygen atom) slows the reaction because the difference in zero-point vibrational energy is larger in the ground state than in the transition state, leading to a larger activation barrier for the heavy-isotope-substituted molecule (, ). The broader vibrational potential well at the transition state reflects the weaker state of the partially broken bond.

Mechanistic possibilities in phosphoryl-transfer reactions. (a) An elimination-addition reaction through a metaphosphate intermediate (D_N + A_N), an addition-elimination reaction through a phosphorane intermediate (A_N + D_N), and a concerted reaction pathway with simultaneous bond formation and bond cleavage (A_ND_N). The parentheses give the IUPAC nomenclature (). (b) A range of possible transition states for a concerted pathway. (c) A two-dimensional reaction coordinate diagram, also known as a More O'Ferrall-Jencks diagram (, ). The diagram represents a range of possible concerted reaction pathways passing through loose, synchronous, or tight transition states. Bond breaking and bond formation proceed along the x- and y-axes, respectively, and the energy axis is perpendicular to the page. The transition state is located at a maximum along the path from reactants to products, but a minimum in the direction perpendicular to the reaction pathway. The two-dimensional reaction coordinate depiction emphasizes that there is a continuum of reaction pathways, and the transition state can occur at any point along the pathway. For simplicity, the diagram above depicts symmetrical transition states halfway along each reaction pathway. Reactions proceeding through intermediates would have additional energy wells in the lower right corner for a metaphosphate intermediate and in the upper left corner for a phosphorane intermediate.