Employing enzymes for the regioselective formation of phosphate esters can
eliminate many of these disadvantages thus making these syntheses more efficient.
Additionally, enantioselective transformations via desymmetrization of prochiral or
meso-diols or through racemate resolution is also possible.
In biological systems, phosphate esters are usually synthesized by means of
phosphorylating transferases called kinases, which catalyze the transfer of a phosphate moiety (more rarely a di17 or triphosphate moiety) from an energy-rich
phosphate donor, such as ATP, onto a nucleophile alcohol.
18 Due to the high
price of these phosphate donors, they cannot be employed in stoichiometric
amounts.
19 As with all cofactors in general, ATP cannot be replaced by less
expensive man-made chemical equivalents, which requires efficient in-situ regeneration to render enzymatic phosphorylations more economic. Fortunately, ATP
recycling has become feasible on a molar scale [554–557].
ATP Recycling In living organisms, ATP is regenerated by metabolic processes, but
for biocatalytic transformations performed in vitro using purified enzymes, this does
not occur. The (hypothetical) addition of stoichiometric amounts of these cofactors
would not only be undesirable from a commercial standpoint but also for thermodynamic reasons, because accumulation of the consumed cofactor (most commonly the
corresponding diphosphate, ADP) can tip the equilibrium of the reaction in the reverse
direction. Thus, nucleoside triphosphate cofactors, such as ATP, are used only in
catalytic amounts and are continuously regenerated during the course of the reaction
by an auxiliary system which usually consists of a second kinase enzyme and a
stoichiometric quantity of a cheap high-energy phosphate donor (Scheme 2.75,
Table 2.1). As nucleoside triphosphates are intrinsically unstable in solution, the
triphosphate species is typically recycled a few 100 times [558, 559]. Sophisticated
reaction engineering using a macromolecular ATP-PEG-construct in a membrane
reactor has raised the ATP-cycle number to a solitary record number of
~20,000 mol product/mol ATP [560]. The total turnover numbers (TTN) concerning
the enzyme performance are the range of ~10
6
–10
8 mol of product per mol of enzyme.
The pro’s and con’s of the commonly used ATP-regenerating systems are as
follows:
• The use of the phosphoenol pyruvate (PEP)/pyruvate kinase system is probably the
most useful method for the regeneration of nucleoside triphosphates [561]. PEP is
not only very stable towards spontaneous hydrolysis but it is also a very strong
phosphorylating agent (Table 2.1). Furthermore, nucleosides other than adenosine
phosphates are also accepted by pyruvate kinase. The drawbacks of this system is
the considerable cost of PEP due to its more complex synthesis [562, 563] and the
fact that pyruvate kinase is inhibited by pyruvate at higher concentrations.
• Acetyl phosphate can be easily synthesized from acetic anhydride and phosphoric acid and is therefore much cheaper than PEP [564]; together with acetate
17 Also termed ‘pyro-phosphates’
18 Phosphorylations involving C, N or S are very rare.
19 The retail price for one mole of ATP is about US $4500, bulk prices are about one tenth of that.
2.1 Hydrolytic Reactions
107
eliminate many of these disadvantages thus making these syntheses more efficient.
Additionally, enantioselective transformations via desymmetrization of prochiral or
meso-diols or through racemate resolution is also possible.
In biological systems, phosphate esters are usually synthesized by means of
phosphorylating transferases called kinases, which catalyze the transfer of a phosphate moiety (more rarely a di17 or triphosphate moiety) from an energy-rich
phosphate donor, such as ATP, onto a nucleophile alcohol.
18 Due to the high
price of these phosphate donors, they cannot be employed in stoichiometric
amounts.
19 As with all cofactors in general, ATP cannot be replaced by less
expensive man-made chemical equivalents, which requires efficient in-situ regeneration to render enzymatic phosphorylations more economic. Fortunately, ATP
recycling has become feasible on a molar scale [554–557].
ATP Recycling In living organisms, ATP is regenerated by metabolic processes, but
for biocatalytic transformations performed in vitro using purified enzymes, this does
not occur. The (hypothetical) addition of stoichiometric amounts of these cofactors
would not only be undesirable from a commercial standpoint but also for thermodynamic reasons, because accumulation of the consumed cofactor (most commonly the
corresponding diphosphate, ADP) can tip the equilibrium of the reaction in the reverse
direction. Thus, nucleoside triphosphate cofactors, such as ATP, are used only in
catalytic amounts and are continuously regenerated during the course of the reaction
by an auxiliary system which usually consists of a second kinase enzyme and a
stoichiometric quantity of a cheap high-energy phosphate donor (Scheme 2.75,
Table 2.1). As nucleoside triphosphates are intrinsically unstable in solution, the
triphosphate species is typically recycled a few 100 times [558, 559]. Sophisticated
reaction engineering using a macromolecular ATP-PEG-construct in a membrane
reactor has raised the ATP-cycle number to a solitary record number of
~20,000 mol product/mol ATP [560]. The total turnover numbers (TTN) concerning
the enzyme performance are the range of ~10
6
–10
8 mol of product per mol of enzyme.
The pro’s and con’s of the commonly used ATP-regenerating systems are as
follows:
• The use of the phosphoenol pyruvate (PEP)/pyruvate kinase system is probably the
most useful method for the regeneration of nucleoside triphosphates [561]. PEP is
not only very stable towards spontaneous hydrolysis but it is also a very strong
phosphorylating agent (Table 2.1). Furthermore, nucleosides other than adenosine
phosphates are also accepted by pyruvate kinase. The drawbacks of this system is
the considerable cost of PEP due to its more complex synthesis [562, 563] and the
fact that pyruvate kinase is inhibited by pyruvate at higher concentrations.
• Acetyl phosphate can be easily synthesized from acetic anhydride and phosphoric acid and is therefore much cheaper than PEP [564]; together with acetate
17 Also termed ‘pyro-phosphates’
18 Phosphorylations involving C, N or S are very rare.
19 The retail price for one mole of ATP is about US $4500, bulk prices are about one tenth of that.
2.1 Hydrolytic Reactions
107