Nitrocefin

The substrate is amici11in for penicillinases and cephalothin for Cephases unless otherwise iadicated. The substrate is nitrocefin. 

The minimum effective concentration was determined by a spot test using nitrocefin Ceph, cephalosporinase Pen, penicillinase. For abbreviations, see footnote to Table 6.1. 

The 3-chloromethyl cephalosporin 41 has been used for the preparation of nitrocefin 42 via Finkelstein and Wittig reactions <05JOC367>. The bicyclic P-lactam 43, a simple C3 homologue of sulbactam, has been prepared and evaluated as an improved inhibitor of class C P-lactamases <05JOC4510>. 

Mechanistic studies of the reaction catalyzed by the metallo-(3-lactamase from Bacteroides fragilis have been performed using nitrocefin (Scheme 25) as a substrate.127,131,132 Several theoretical studies pertinent to possible steps in the mechanistic pathway have also been reported.133-139 Issues stemming from these studies that remain unresolved include whether the nucleophilic hydroxide is bridging or terminal, and the chemical nature of an intermediate that is detected during the hydrolysis of nitrocefin. 

Scheme 26 Proposed mechanism for nitrocefin hydrolysis catalyzed by [Zn2(BPAN) ( i-0H)( a-02PPh2)](C104)2.

Proposed mechanism and rate law for pH-dependence of nitrocefin hydrolysis. 

Additional kinetic studies performed under conditions wherein less water was present (1 9 DMSO acetone containing 0.28 M H20) yielded =(8.9 0.3)x 103M-1. The difference between the equilibrium binding constant K for the nitrocefin hydrolysis reaction catalyzed by [Zn2(BPAN)( i-0H)( i-02PPh2)](C104)2 under... 

Variable temperature kinetic studies for the hydrolysis of nitrocefin catalyzed by [Zn2L3(p-0H)(N03)2] in acetone DMSO yielded AH% = 88.4 + 5.3 kJlVT1 and A= —45.3 + 16.0 JM 1K 1. These values are similar to those found for nitrocefin hydrolysis catalyzed by [Zn2(BPAN)(ii-0H)( i-02PPh2)](C104)2 in 1 9 DMSO ac-etone (Akfi = 96.1 5.2kJM 1 and AS = —22.4+12 JM 1K 1) and are consistent with intramolecular attack by the bridging hydroxide on the coordinated P-lactam substrate in the rate-determining step.140... 

Using pyridinium triflate, the deprotonated amide intermediate was estimated to have pKa = 4.20. This low value is consistent with Zn(II) ion stabilization of the deprotonated amide. The formation of this deprotonated amide intermediate occurs in wet DMSO with a microscopic rate constant that corresponds to the intramolecular hydrolysis of coordinated nitrocefin catalyzed by [Zn2L3( i-0H)(N03)2]. Activation parameters for this reaction (A= 77.6 + 8.0kJM-1 and AS = —62.7 + 20.0 JM"1 K-1) are within error of those found for the same reaction in acetone DMSO... 

Fig. 24 Proposed structure of spectroscopically observable deprotonated intermediate in the hydrolysis of nitrocefin catalyzed by [Zn2L3(g-0H)(N03)2]-...

Hydrolysis of nitrocefin catalyzed by [([12]aneN4)Zn(H20)](N03)2 (Fig. 5)" proceeds via a pathway that does not involve substrate coordination to the zinc center. Specifically, as the zinc center in the hydroxide species [([12]aneN4)Zn(0H)]N03 is coordinatively saturated, the reaction proceeds via a bimolecular pathway (Fig. 26). 

An important conclusion from these comparative studies is that mononuclear Zn(II) complexes can be as efficient for nitrocefin hydrolysis as binuclear systems, thus providing evidence that the second zinc center (Zn2) in metallo- 3-lactamases is not required for nucleophile activation or catalytic activity. 

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