Another important phosphate species, which is needed as a cosubstrate for DHAPdependent aldolase reactions, is dihydroxyacetone phosphate (Scheme 2.78, also see
Sect. 2.4.1). Its chemical synthesis using phosphorus oxychloride is hampered by
moderate yields and the generation of side products. Enzymatic phosphorylation,
however, gives significantly enhanced yields of a product which is sufficiently pure so
that it can be used without isolation for the subsequent carboligation step [583, 584].
5-Phospho-D-ribosyl-α-1-pyrophosphate (PRPP) serves as a key intermediate in
the biosynthesis of purine, pyrimidine, and pyridine nucleotides, such as nucleotide
cofactors [ATP, UTP, GTP, CTP, and NAD(P)H]. It was synthesized on a large
scale from D-ribose using two consecutive phosphorylating steps [585] (Scheme
2.79). First, D-ribose was phosphorylated at the prim-hydroxy group using
ribokinase. Subsequently a pyrophosphate moiety was transferred from ATP onto
the anomeric center in the α-position by PRPP synthase. In this latter step, AMP
(rather than ADP) was generated, which required adenylate kinase and pyruvate
kinase for step-wise regeneration of ATP. Phosphoenol pyruvate (PEP) served
as phosphate donor in all phosphorylation steps. The PRPP thus obtained was
subsequently transformed into orotidine monophosphate (O-5-P) via enzymatic
linkage of the nucleobase by orotidine-5
0 -pyrophosphorylase (a transferase),
followed by decarboxylation of O-5-P by orotidine-5
0 -phosphate decarboxylase
(a lyase) yielding UMP in 73% overall yield.
OH
R eq
R ax
R ax
R eq
R eq
R ax
OR
X
6
= phosphate
pyruvate
phosphoenol pyruvate
pyruvate kinase
hexokinase
P
ADP
ATP
O
R eq
R ax
R ax
R eq
R eq
R ax
OR
X
P
Substituents
Variations tolerated
R
H , M e
R eq
H, F, OH, NH 2 , AcNH
R ax
H, F
X
O, S, NH
Scheme 2.77 Regioselective phosphorylation of hexose derivatives by hexokinase
O
O
HO
O
OH
HO
= phosphate
acetate kinase
or
acid phosphatase
acetate
or
phosphate
acetyl phosphate
or
pyrophosphate
POCl 3 60%
glycerol kinase 80%
ADP
ATP
P
P
Scheme 2.78 Phosphorylation of dihydroxyacetone by glycerol kinase
110
2 Biocatalytic Applications
Sect. 2.4.1). Its chemical synthesis using phosphorus oxychloride is hampered by
moderate yields and the generation of side products. Enzymatic phosphorylation,
however, gives significantly enhanced yields of a product which is sufficiently pure so
that it can be used without isolation for the subsequent carboligation step [583, 584].
5-Phospho-D-ribosyl-α-1-pyrophosphate (PRPP) serves as a key intermediate in
the biosynthesis of purine, pyrimidine, and pyridine nucleotides, such as nucleotide
cofactors [ATP, UTP, GTP, CTP, and NAD(P)H]. It was synthesized on a large
scale from D-ribose using two consecutive phosphorylating steps [585] (Scheme
2.79). First, D-ribose was phosphorylated at the prim-hydroxy group using
ribokinase. Subsequently a pyrophosphate moiety was transferred from ATP onto
the anomeric center in the α-position by PRPP synthase. In this latter step, AMP
(rather than ADP) was generated, which required adenylate kinase and pyruvate
kinase for step-wise regeneration of ATP. Phosphoenol pyruvate (PEP) served
as phosphate donor in all phosphorylation steps. The PRPP thus obtained was
subsequently transformed into orotidine monophosphate (O-5-P) via enzymatic
linkage of the nucleobase by orotidine-5
0 -pyrophosphorylase (a transferase),
followed by decarboxylation of O-5-P by orotidine-5
0 -phosphate decarboxylase
(a lyase) yielding UMP in 73% overall yield.
OH
R eq
R ax
R ax
R eq
R eq
R ax
OR
X
6
= phosphate
pyruvate
phosphoenol pyruvate
pyruvate kinase
hexokinase
P
ADP
ATP
O
R eq
R ax
R ax
R eq
R eq
R ax
OR
X
P
Substituents
Variations tolerated
R
H , M e
R eq
H, F, OH, NH 2 , AcNH
R ax
H, F
X
O, S, NH
Scheme 2.77 Regioselective phosphorylation of hexose derivatives by hexokinase
O
O
HO
O
OH
HO
= phosphate
acetate kinase
or
acid phosphatase
acetate
or
phosphate
acetyl phosphate
or
pyrophosphate
POCl 3 60%
glycerol kinase 80%
ADP
ATP
P
P
Scheme 2.78 Phosphorylation of dihydroxyacetone by glycerol kinase
110
2 Biocatalytic Applications