Pyrophosphate ionization

The biosynthetic cyclization reactions leading to the various diterpene skeleta are summarized in Figure 3.16 (for a review see MacMillan and Beale (1999)). In addition to the well-established mode of cyclization driven by pyrophosphate ionization (sometimes referred to as Type T), a second mode ( Type IT), driven by protonation of double bonds. 

The biosynthesis of casbene (43), a phytoalexin of castor bean, is an example of direct pyrophosphate ionization-driven cyclization of GGPP. Casbene synthase has been cloned and overexpressed in E. colt (Huang et al. 1998) the enzyme catalyses formation of a 14-membered ring with the distal double bond acting as nucleophile, followed by a cyclopropanation as shown in Figure 3.16. 

The pentagon stabilization has been found in a biochemical phenomenon [80], The hydrogen on the thiazolium ring 9 (Scheme 7) is easily ionized to afford the corresponding carbene 10, a key catalyst in enzymatic reactions for which thiamine (vitamin B-1,11) pyrophosphate is the cofactor. The pentagon stability is expected to contribute to this unusual deprotonation. A lone pair generated on the carbon atom in 10 can similarly delocalize through the vicinal C-N and C-S a bonds in a cyclic manner. 

The subsamples were split and sent to different laboratories to be subjected to ten commonly-used and proprietary leach/digestion techniques (a) aqua regia partial digestion method at Acme Analytical Laboratories (b) sodium pyrophosphate and cold hydroxylamine leaches at ALS Chemex (c) enzyme and TerraSol leach methods at Skyline Labs (d) Bioleach and soil gas hydrocarbon analyses at Activation Laboratories (e) Mobile metal ion (MMI) extraction at SGS Minerals (f) 4-acid near-total and sodium peroxide sinter total digestions (under the uses contract) at SGS Minerals and (g) de-ionized water leach at the USGS laboratories. 

Decarboxylation of an a-keto acid like pyruvate is a difficult reaction for the same reason as are the ketol condensations (see fig. 12.33) Both kinds of reactions require the participation of an intermediate in which the carbonyl carbon carries a negative charge. In all such reactions that occur in metabolism, the intermediate is stabilized by prior condensation of the carbonyl group with thiamine pyrophosphate. In figure 13.5 thiamine pyrophosphate and its hydroxyethyl derivative are written in the doubly ionized ylid form rather than the neutral form because this is the form that actually participates in the reaction even though it is present in much smaller amounts. 

In the first step of the conversion catalyzed by pyruvate decarboxylase, a carbon atom from thiamine pyrophosphate adds to the carbonyl carbon of pyruvate. Decarboxylation produces the key reactive intermediate, hydroxyethyl thiamine pyrophosphate (HETPP). As shown in figure 13.5, the ionized ylid form of HETPP is resonance-stabilized by the existence of a form without charge separation. The next enzyme, dihydrolipoyltransacetylase, catalyzes the transfer of the two-carbon moiety to lipoic acid. A nucleophilic attack by HETPP on the sulfur atom attached to carbon 8 of oxidized lipoic acid displaces the electrons of the disulfide bond to the sulfur atom attached to carbon 6. The sulfur then picks up a proton from the environment as shown in figure 13.5. This simple displacement reaction is also an oxidation-reduction reaction, in which the attacking carbon atom is oxidized from the aldehyde level in HETPP to the carboxyl level in the lipoic acid derivative. The oxidized (disulfide) form of lipoic acid is converted to the reduced (mer-capto) form. The fact that the two-carbon moiety has become an acyl group is shown more clearly after dissocia-... 

Several groups +have examined the influence of divalent cations, such as Mg2 and Mn2, in catalyzing the solvolysis of allylic pyrophosphates such as geranyl pyrophosphate (58-60). The results strongly suggest that the role of the metal ion in enzymatic transformations of allylic pyrophosphates is to neutralize the negative charge of the pyrophosphate moiety and thus assist in the ionization of the substrate to produce the allylic cation. 

Consideration of chemical models, now largely in hindsight, allows broad outlines of a cyclization scheme to be delineated. Reaction of geranyl pyrophosphate is initiated by ionization which is assisted by low pH and divalent metal ion. Conversion of the geranyl to the linalyl system precedes cyclization to the monocyclic intermediate by the established stereochemical course. The overall process occurs stepwise via a series of... 

The pinene cyclases convert the anomalous linalyl enantiomer to abnormal levels of acyclic (e.g. myrcene) and monocyclic (e.g. limonene) terpenes, these aberrant products perhaps arising via ionization of the tertiary substrate in the transoid or other partially extended (exo) form (see below). In any event, for all "normal" cyclizations examined thus far, the configuration of the cyclizlng linalyl Intermediate has been confirmed to be that which would be expected on the basis of an anti-endo conformation. Scattered attempts at intercalating the cyclization cascade with analogs of proposed cyclic intermediates (e.g. a-terpinyl and 2-pinyl pyrophosphate) have been unsuccessful (20,35,36). 

Thiamine pyrophosphate (TPP) ionizes to form a carbanion. (2) The carbanion of TPP attacks the ketose substrate. (3) Cleavage of a carbon-carbon bond frees the aldose product and leaves a two-carbon fragment joined to TPP. (4) This activated glycoaldehyde intermediate attacks the aldose substrate to form a new carbon-carbon bond. (5) The ketose product is released, freeing the TPP for the next reaction cycle. 

Thiamine is absorbed in the intestine by both active transport mechanisms and passive diffusion. The active form of the cofactor, thiamine pyrophosphate (thiamine diphosphate, TPP), is synthesized by an enzymatic transfer of a pyrophosphate group from ATP to thiamine (Figure 15-1). The resulting TPP has a reactive carbon on the thiazole ring that is easily ionized to form a carbanion, which can undergo nucleophilic addition reactions. 

Reed found that crystalline prenyltransferase could solvolyze the allylic substrates. This reaction required inorganic pyrophosphate and had a velocity of about 2% of the normal reaction rate [6]. Examination of the allylic product, either dimethylallyl alcohol or geraniol, revealed that C-1 had inverted and the csu-binol oxygen had come from water. Since the normal reaction involves inversion of C-1 and scission of C-O bond, the solvolysis seemed to be mimicking the normal reaction, with H2O replacing the organic portion of isopentenyl pyrophosphate in the catalytic site. This indicates that ionization of the allylic pyrophosphate is the first event, followed by condensation to form a new bond, then a hydrogen elimination from C-2 of the former isopentenyl moiety. Thus, there is an ionization-condensation-elimination sequence of events. 

Pyruvate dehydrogenases (PDH) are multienzyme complexes responsible for the conversion of pyruvate of acetyl-CoA. The decarboxylation step requires thiamin pyrophosphate (TP). The mechanism involves ionization of thiamine to produce an yielde which adds to the carbonyl of pyruvate forming a covalent adduct. This adduct has properties which permit CO2, to leave, an event that could not occur in pyruvic acid. 

Thiamine pyrophosphate (13) is the co-factor for a number of enzymes that can be described as stabilizing hypothetical acyl anion intermediates. For instance, it is the coenzyme for the enzyme carboxylase that catalyses the conversion of pyruvic acid to acetaldehyde. We had early shown that this mechanism involves a thiazolium anion (14) whose second resonance form (15) is a carbene. Ionization of the C-2 proton of the thiazolium ring generates this species that can add nucleophilically to carbonyl groups such as that in pyruvic acid, forming an intermediate whose decarboxylation generates a stabilized anion. 

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