Exocyclic bond cleavage, hydrolysis

A pathway (Scheme I) (8.9) for the hydrolysis of oligoglycosides by lysozyme that differs from the previously accepted mechanism (Scheme II) (3,10-12) is described in this section. The alternative pathway, suggested by results of a 55-ps MD simulation of the lysozyme (GlcNAc)e complex (1), is consistent with the available experimental data and with stereoelectronic considerations. Experimental data have demonstrated that Glu 35 and Asp 52 are essential, as shown by recent site-directed mutagenesis results (13.) which corroborate chemical modification studies (3.14 and references cited therein), and that the reaction proceeds with retention of configuration at Ci Q and references cited therein). A fundamental feature of the alternative pathway is that an endocyclic bond is broken in the initial step, in contrast to the exocyclic bond cleavage in the accepted mechanism. 

The Ciamician-Dennstedt reaction can be thought of as the complement to the Reimer-Tiemann reaction (Scheme 8.3.2). The first step of both reactions is cyclopropanation of one of the carbon-carbon double bonds of a pyrrole with a dichlorocarbene, resulting in intermediate 3. The Ciamician-Dennstedt reaction results from cleavage of the internal C-C bond and elimination of chloride (path a), while the Reimer-Tiemann reaction results from cleavage of the exocyclic bond, and subsequent hydrolysis of the dichloromethyl moiety to furnish aldehyde 5 (path b). 

Details of the hydrolytic process are somewhat more complicated because the acid-catalyzed hydrolysis proceeds via the initial protonation of an alkoxy oxygen followed by bond cleavage. Because the protonation can involve the exocyclic or endocyclic alkoxy group, two different sets of initial products are possible. However, in both cases the ultimate degradation products remain the same. These two possible reaction paths are shown on page 130. 

The implications of the above observations may be important, especially if similar trends are observed in pyranose anomers. For example, with respect to the mechanism of acid-catalyzed hydrolysis of pyranosides, endocyclic C-0 bond cleavage (preceeded by 05 protonation) may be assisted in P-anomers in which the Cl-01 bond is equatorial, since the 04-Cl bond may already be extended in these anomers. By a similar argument, exocyclic C-0 scission (preceeded by 01 protonation) may be assisted in the hydrolysis of a-pyranosides in which the Cl-01 is axial and extended, thus resembling the transition state. Post and Karplus have recently suggested that enzyme-catalyzed glycoside hydrolysis of P-pyranosides may indeed take place by ring oxygen protonation, followed by endocyclic C-0 bond scission. 

Large rate enhancements (> 10 -10 ) are observed for hydrolysis of [24a,b,c] relative to acyclic analogues [25a,b], consistent with breakdown of the initial TBP [27] via endocyclic P—N bond cleavage (Scheme 11). However, the cyclic phenyl ester also undergoes hydrolysis by exocyclic P—O... 

Mulliez and Wolf (1986) have studied the aminolysis and alcoholysis of similar five-membered species [29] to those studied by Kluger et al. (1987). Initial exocyclic cleavage is observed in solvolysis of the phosphoramidate diester [29a], whereas the phosphonamidate [29b] undergoes alcoholysis and hydrolysis with endocyclic P—N bond cleavage. These reactivity patterns are most easily accommodated by a mechanistic scheme in which initial attack is apical to nitrogen. 

Since the transition state structures of exocyclic C-O bond cleavage resemble the oxocarbenium ion intermediates, favorable interactions that stabilize the charged intermediates would facilitate hydrolysis [16]. The p5U anoside 5 bearing axial substituents at the C3 and C4 carbons reacted at the highest rate via an... 

The acid-catalyzed hydrolysis of methyl 5-thioxylopyranosides 2 occurs via fast protonation of the C-O bond followed by slow exocyclic C-O bond cleavage. The study of the different types of kinetic isotope effects indicates that a thiacarbenium ion is formed in the process. A similar study of the hydrolysis of xylopyranosides 1 has confirmed the mechanism generally accepted for the reaction and has demonstrated the intermediacy of oxacarbenium ions as intermediates. 

The initial step in the alternative hydrolysis mechanism is protonation of the ring 0i by Glu 35 (Scheme I). Cleavage of the endocyclic C1-O5 bond forms the acyclic oxocarbonium ion intermediate, which is stabilized by Asp 52. Attack by water, cleavage of the C1-O4 bond, and ring closure then lead to the observed products. Existing experimental data on lysozyme hydrolysis are consistent with Scheme I (see references in Post and Karplus ( )). Moreover, distortion of the ring in site D is not required and the antiperiplanar orientation of an exocyclic O4 lone pair orbital relative to the cleaved C1-O5 bond found in the simulation (see section on "Enhancement of a Substrate Conformation Optimum for Catalysis") is in accord with stereoelectronic requirements (1 ). ... 

Because a knowledge of all degradation products is important for regulatory approval, studies aimed at establishing the exact hydrolysis path were carried out [50, 51]. As already described in Sect. 4.2.3, the acid-catalyzed hydrolysis of the cyclic ortho ester bond can proceed via two paths, depending on whether cleavage occurs at the exocyclic alkoxy or endocyclic alkoxy group. 

Fig. 14 Trigonal bipyramidal intermediates (a) for RNase catalysed cleavage of the P—0(5 ) bond of an oligonucleotide (b) for exocyclic cleavage in the hydrolysis of MEP. Equatorial bridging oxygen nonbonding electrons are shown as sp hybrids.

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