3/1/AnDn mechanism

Other phosphoryl transfer mechanisms are an associative, two-step mechanism (An + Dn) and a concerted mechanism (ANDN) with no intermediate. The AN+DN mechanism is an addition-elimination pathway in which a stable pentacoordinate intermediate, called a phosphorane, is formed. This mechanism occurs in some reactions of phosphate triesters and diesters, and has been speculated to occur in enzymatic reactions of monoesters. In the concerted ANDN mechanism, bond formation to the nucleophile and bond fission to the leaving group both occur in the transition state. This transition state could be loose or tight, depending upon the synchronicity between nucleophilic attack and leaving group departure. The concerted mechanism of Fig. 2 is drawn to indicate a loose transition state, typical of phosphate monoester reactions. 

Some authors have referred to highly dissociative AnDn reactions as being SnI-like . If the transition state is bimolecular, with both the nucleophile and the leaving group in the reaction coordinate, then the reaction has an AnDn mechanism, regardless of the extents of bond loss or formation at the transition state. In this chapter, we will refer to highly dissociative A Dn transition states as being oxocarbenium ion-like rather than SNl-like . 

One striking observation about the reactions catalyzed by RTA and pertussis toxin is that the enzymes appear to use opposite catalytic strategies. The analogous non-enzymatic reactions, hydrolysis of adenosine 5 -monophosphate (AMP) and NAD, both have highly dissociative AnD mechanisms with oxocarbenium ionlike transition states. In the ADP-ribosylation of protein Gid by pertussis toxin, the AnDn mechanism is more synchronous, with more nucleophile participation and lower oxocarbenium ion character than the non-enzymatic reaction. " In contrast, the RTA-catalyzed depurination of RNA" and DNA substrates proceeds through stepwise Dj,j An mechanisms where the enzyme stabilizes an oxocarbenium ion to the point that it becomes a discrete intermediate. This is illustrated with a hypothetical free energy surface for each reaction (Fig. 14). 

Glucopyranosyl fluorides. The larger primary KIEs for 9a and 9/8 (Table 3) are consistent with an AnDn mechanism.The authors used BOVA and the previously solved TS stmcture for the enzyme-catalyzed hydrolysis of 9a as a... 

The experimental KlEs for acid-catalyzed AMP hydrolysis indicate an AnDn mechanism. The 9- N KIE = 1.030 is less than the semi-classical limit of 1.044 because the increase in bond orders within the adenine ring compensate for the loss of the Cl -N9 bond. Protonation at N7 in the acid-catalyzed reaction makes the incipient anionic leaving group neutral, and helps to transfer rr-bonding electrons into the bonds with N9. 

TS analysis was performed for two PNP-catalyzed reactions, arsenolysis of inosine using arsenate as a phosphate analogue, and a pre-steady state TS analysis of PNP-catalyzed inosine hydrolysis. The ribosyl ring KIEs (Table 7) were typical of those observed previously for other reactions of A -ribosides, indicating an AnDn mechanism for hydrolysis and either an AnDn or Dn An for arsenolysis. The... 

TS analysis of inosine arsenolysis. TS analysis was performed on the arsenolysis of inosine under steady state conditions. The primary, KIE was near the lower limit (1.025-1.029) calculated for an AnDn mechanism with adenosine and could indicate either an AnDn or Dn An mechanism. The large a- and j8-secondary KIEs indicated a transition state with high oxocarbenium ion character. Analysis of X-ray crystal structures of PNP demonstrates that there is not enough space in the enzyme active site between the leaving group purine and the phosphate nucleophile to allow formation of a stable oxocarbenium ion (see Section 3). Thus, the most likely mechanism for arsenolysis is an extremely dissociative AnDn mechanism. The primary, 9- N KIE = 1.010 was lower than expected, as discussed below. 

TS analysis of inosine hydrolysis. Experimental KIEs were determined for inosine hydrolysis under pre-steady state conditions because of the exceptionally slow dissociation of hypoxanthine (estimated K = 1.3 pM), which is rate-limiting under steady state conditions. Hypoxanthine binds to the other two PNP subunits with much lower affinity. KIEs in the ribosyl ring were typical of an AnDn mechanism. When the reaction was run in 20% methanol, the product ratio was 85 15 1-methylribose ribose. This is close to the ratio expected based on the relative nucleophilicities of MeOH and water, indicating that there is significant participation of the nucleophile in the reaction coordinate. As with the arsenolysis, the primary, 9- N KIE of 1.000 was anomalous. These KIEs were initially rationalized as indicating an internal equilibrium formation of ribose and hypoxanthine however, this is inconsistent with what is now known about the observable KIEs for stepwise reactions. 

The second major mechanistic criterion besides kinetics is the configuration of the products racemization is expected for a Dn + An mechanism, inversion of configuration for an AnDn mechanism. For obvious reasons, this criterion cannot be applied to systems in which the alkanediazonium ion is formed in a protonation equilibrium from a diazoalkane because that leads to racemates (see, for a classical case, Streitwieser and Schaeffer, 1957 b). 

Based on Brosch and Kirmse s result (1991) that from optically active [l- H]butylamine completely inverted [l- H]butan-l-ol was obtained, we added this type of bimolecular displacement to White s Scheme 7-17 (pathway AnDn to 7.38). As shown later (Sect. 7.6), it is not, however, unambiguous to assume that this reaction indeed follows an AnDn mechanism. 

Scheme 10 Hypothetical stepwise An + Dn and concerted AnDn mechanisms for La " "2( OCH3)2-catalyzed cleavage of 34 and 35. Adapted from J. Am. Chem. Soc 2009 131 13738-13748. 

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