Scientifica Acta 1, No. 1, 65 – 69 (2007)
Medicinal Chemistry and Pharmaceutical Technology
Chemo-enzymatic synthesis of nucleosides and nucleotides by
immobilized and stabilized enzymes
Teodora Bavaro
Dipartimento di Chimica Farmaceutica, Università di Pavia, Viale Taramelli 12, 27100 Pavia Italy
Innovate Biotechnology srl, Rivalta Scrivia, Tortona, AL, Italy
[email protected]
Modified nucleosides and nucleotides are widely used as antiviral and anticancer drugs. These compounds
are routinely synthesized by complex chemical procedures, involving several protection/deprotection reactions that affect the overall yield, the reaction time and the process costs. Alternatively, nucleosides and
nucleotides can be prepared via enzymatic processes. The use of immobilized and stabilized enzymes on
solid support enables the exploitation of the inherent selectivity of enzymes with the advantages to avoid the
risk of protein contaminants, to re-use the biocatalysts, and to improve their performances (e.g. stability) in
a wide range of experimental conditions.
The main goal of this research was the preparation of 5’-deoxynucleosides, such as doxifluridine and
capecitabine, antineoplastic drugs, and 5’-mononucleotides, such as UMP, CMP, AMP and GMP, used as
baby food additives.
1 Introduction
Natural nucleosides and their modified derivatives are commonly used as antiviral and antitumour agents
[1]. Their therapeutic activity is due to their ability to act as antimetabolites in the RNA and DNA synthesis
[2,3]. Nucleosides have traditionally been prepared by various chemical methods [4]. Most of them employ
natural nucleosides as starting compounds or are based on a convergent approach via condensation of the
carbohydrate precursor and the heterocyclic base. Protection and deprotection steps and glycosyl activation
are required for the control of the configuration at the anomeric center, and the regioselective glycoside
formation at one of nucleophilic groups in the purine or pyrimidine base. As a result, chemical schemes of
nucleoside analogues preparation are time-consuming and often plagued by low overall yields, low purity
of the final product, high process costs [5].
Enzymatic syntheses have been shown to be an advantageous alternative to chemical methods [6]. The
use of enzymes as catalysts in pharmaceutical chemistry is quite new but it is becoming very popular also
because of the recent attention for “sustainable chemistry”. In fact, enzymes efficiency carries out faster
processes, less waste and a reduced use of organic solvents. Besides, the inherent enzymes selectivity as
well as their ability to work in mild conditions, can favour the obtainment of pure products by avoiding
tedious synthetic and purification steps in comparison with the classical chemical approach. Enzymatic
processes can be very competitive in terms of costs and technology, allowing high quality products to be
obtained. The availability of enzymatic “libraries”, the efficiency of molecular cloning and protein expression platforms, and all the technologies that can improve an enzyme’s selectivity, specificity and stability,
are contribuiting to the diffusion of biocatalysis in the manufacturing of Active Pharmaceutical Ingredients
(APIs). However, despite several efforts and the potential advantages of enzyme catalysts, nucleosides are
still preferably prepared by procedures not involving enzymes. This reflects the relative inefficiency of
current enzymatic methods indicating the need for improvement in the enzymatic approach to nucleoside
synthesis. In particular, the instability of the protein in non physiological conditions (temperature, pH,
ionic strength, cosolvents), the difficult recovery of the catalyst from the reaction medium and the high
production costs may represent important limitations. These problems may be overcome by using immobilized enzymes. The immobilized enzyme can be separated from the reaction medium by filtration and is
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Scientifica Acta 1, No. 1 (2007)
Base1
HO
O
enzymatic
transglycosylation HO
+ Base2
a
OH
OH
Base1
O
OH
Base2 chemical
protection
OH
b
GPO
O
enzymatic
Base2 regioselective
deprotection
Base2
HO
c
OPG OPG
O
OPG OPG
d
chemical
functionalization
Base2
Base2
2-
O3PO
CH3 O
O
OH
OH
OH
OH
Fig. 1: Chemo-enzymatic synthesis of nucleosides and nucleotides: general approach.
thus available for the re-use. Depending on the immobilization technique, the properties of the biocatalyst
such as stability and selectivity, may be significantly affected [7, 8]. Indeed, the design of a tailor-made
immobilization plays a pivotal role to take full advantage from this technology. With a view to preparative
(industrial) applications, it is mandatory to simplify the scale-up process; the availability of an immobilized and stabilized enzyme meets this need both for the easy handling of such a catalyst and its operational
stability for repeated use.
In order to develop cost-effective and environmentally friendly chemical syntheses at industrial scale,
it is essential to integrate biocatalysis and modern chemical research and development to deliver manufacturing routes with fewer synthetic steps, reduced waste streams, and improved overall synthetic efficiency
in yields, regio- and stereoselectivities, process robustness, and safety.
The importance of this strategy is supported by few examples where a second-generation and chemoenzymatic process was developed to replace an existing and less efficient chemical route [9].
2 Synthesis of nucleosides and nucleotides
This project combines techniques of traditional synthesis with a biotechnological approach with the aim
to develop novel chemo-enzymatic processes affording high-value nucleoside derivatives at lower cost
and with a better impurity profile. On this basis, our chemo-enzymatic approach (Scheme 1) utilizes
immobilized and stabilized enzymes in critical synthetic steps.
The first step (a) is an enzyme-catalyzed reaction involving nucleoside phosphorylases which catalyze
the reversible phosphorolysis of the starting nucleoside leading to a sugar-1-phosphate with the release
of the nucleobase; the presence of another nucleobase results in the formation of a new nucleoside. This
nucleoside is then fully protected by a easy and cheap chemical reaction (usually by acetylation, step
b) and again, subjected to an enzymatic reaction (step c), that is the regioselective deprotection of the
desired sugar hydroxyl (5’, in this case). The resulting intermediate is then properly reacted to give the
5’-deoxynucleoside or the 5’-monophosphate (d).
2.1 Enzymatic transglycosylation
The first step of the process concerned the transglycosylation reaction catalyzed by nucleoside phosphorylases (NPs) in fully aqueous medium. Specifically, NPs are able to catalyze the N-glycosidic bond
formation. NPs catalyze the reversible phosphorolysis of a nucleoside leading to a sugar-1-phosphate
with the release of the nucleobase; the presence of another nucleobase results in the formation of a new
nucleoside. The transglycosylation can be bi-enzymatic, coupling different enzymes according to the structure of the donor nucleoside and of the acceptor base, as previously reported for the synthesis of purine
2’-deoxynucleosides catalyzed by immobilized uridine phosphorylase (UP, E.C. 2.4.2.3) and purine nucleoside phosphorylase (PNP, E.C. 2.4.2.1) from Bacillus subtilis [10]. Here, we used UP [11] as a biocatalyst
to develop a mono-enzymatic process.
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Scientifica Acta 1, No. 1 (2007)
67
Products %
Entry
Enzyme
Substrate
Conversion % Time (h)
a (5’) b (3’) Others
1
CRL
PFL
PCL
PPL
uridine
98
100
95
64
24
19
29
24
85
93
73
45
--2
1
13
7
20
18
2
CRL
PFL
PCL
PPL
arabinosyluridine
97
6
72
8
24
24
24
24
89
2
66
6
3
1
1
1
5
3
5
1
3
CRL
PFL
PCL
PPL
2’-deoxyuridine
95
73
100
27
4
24
24
24
24
32
6
4
65
27
59
23
6
14
35
--
4
CRL
PFL
PCL
PPL
5-fluorouridine
94
97
95
22
24
2
24
24
63
94
51
10
-----
31
3
44
12
5
CRL
PFL
PCL
PPL
adenosine
99
99
99
25
5.5
24
24
24
59
89
64
20
-----
40
10
35
5
6
CRL
PFL
PCL
PPL
cytidine
97
39
61
43
98
72
98
48
70
20
39
35
-----
27
19
22
8
Fig. 2: Enzymatic screening. Immobilized lipases: Candida rugosa CRL, Pseudomonas fluorescens PFL,
Pseudomonas cepacia PCL and Porcine pancreas PPL.
UP was immobilized by a first strong adsorption of the enzyme on a flexible ionic support obtained
by derivatization of the epoxide resin Sepabeads EC-EP with high molecular weight polyethyleneamine.
Successively, the adsorbed enzyme was cross-linked with 20% oxidized dextran affording, after reduction,
the covalent immobilization of the protein [12].
A number of nucleosides and pyrimidine bases have been screened as possible sugar donors and sugar
acceptors, respectively, for the transglycosylation reactions [11].
For the synthesis of doxifluridine, we used 5’-deoxyuridine as sugar donor (Scheme 3).
2.2 Regioselective enzymatic hydrolysis of peracetylated nucleosides
Lipases (glycerol ester hydrolases, E.C.3.1.1.3) catalyze the hydrolysis/synthesis of a wide range of soluble
or insoluble carboxylic acid ester and amides. These enzymes are one of the most useful biocatalysts due
to their efficiency, easy work up and stability in organic solvents [13].
For the second step, we performed a screening of several lipases with the aim to identify the best catalysts in terms of activity and regioselectivity for the hydrolysis of peracetylated nucleosides, prepared by
standard procedures, starting from the nucleosides obtained by transglycosylation. Lipases from different
biological sources were immobilized on a hydrophobic support (Octyl-Sepharose) as previously reported
[13] (Figure 3).
The exclusive hydrolysis at the primary position (C-5’) was observed by using CRL, PFL, PCL with
the substrates 1, 2, 4, 5 and 6 (Figures 2–3). These results remark the influence of the sugar on the
enzyme regioselectivity; in fact, in the hydrolysis of 2’-deoxyuridine, CRL and PCL were more selective
for the C-3’ position. PPL showed a poor activity toward all the tested substrates. When the enzyme
was poorly regioselective, the hydrolysis afforded a mixture of partially deprotected nucleosides that were
isolated, purified, fully characterized and successively used as analytical standards [14]. This screening
allowed to find catalysts able to regioselectively deprotect the C-5’ position of the sugar for the successive
functionalization. The regioselectivity for C-3’, however, can be interesting for the obtainment of 3’functionalized nucleosides as well.
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Scientifica Acta 1, No. 1 (2007)
B
B
AcO
B
AcO
HO
LIPASE
O
O
H
R
H
H
OAc R1
H
R
H
H
OAc R1
1-6
1a-6a
1 R=H
2 R=OAc
3 R=H
4 R=H
5 R=H
6 R=H
R1=OAc
R1=H
R1=H
R1=OAc
R1=OAc
R1=OAc
B=uracil
B=uracil
B=uracil
B=5-fluorouracil
B=adenine
B=cytosine
O
+
H
R
H
H
OH R1
1b-3b
1a, 1b
2a, 2b
3a, 3b
4a
5a
6a
R=H
R1=OAc
R=OAc R1=H
R=H
R1=H
R=H
R1=OAc
R=H
R1=OAc
R=H
R1=OAc
Fig. 3: Enzymatic hydrolysis. Reaction conditions: immobilized lipase, 10% CH3 CN in KH2 PO4 buffer pH 7.
O
O
HN
5'
HO
O
O
chemical
protection
N
OH OH
1
a
AcO
HN
O
O
enzymatic
regioselective
deprotection
(PFL)
N
OAc OAc
2
b
HO
O
HN
O
O
N
OAc OAc
3
chemical
deoxygenation
and
deprotection
c, d
O
HN
CH3O
O
O
F
HN
O N
H
enzymatic
5 transglycosylation
O
F
HN
(UP)
N
e
uracil
CH3O
O
N
OH OH
6, doxifluridine
OH OH
4
a: Ac2O/CH3CN/DMAP
b: KH2PO4 buffer 25 mM, pH 7.0, 10% CH3CN, enzyme: immobilized Pseudomonas fluorescens lipase (PFL)
c: 1) MeSO2-Cl 2) Br- N+Bu4 3) (Bu)3SnH, AIBN
d: MeOH/NaOMe
e: KH2PO4 buffer 10 mM, pH 7.5, enzyme: immobilized uridine phosphorylase from Bacillus subtilis (UP)
Fig. 4: The results obtained for capecitabine has been disclosed in an Italian Patent Application [16].
2.3 Doxifluridine
The synthesis of doxifluridine (6) consists of the following steps (Figure 4): by treatment of uridine (1)
with acetic anhydride, the peracetylated derivative 2 is easily obtained and successively subjected to the
regioselective enzymatic hydrolysis to give 3. From the extensive screening above reported (Table 1),
the lipase with the highest regioselectivity for the C-5’ position of this substrate was PFL. Compound 3
was deoxygenated [15] and deprotected affording 5’-deoxyuridine (4). The enzymatic transglycosylation
between 4 and 5-fluorouracil (5) gave doxifluridine (6) in >95% purity.
The chemo-enzymatic synthesis of doxifluridine remarks the versatility of the proposed approach (Figure 1). In fact, according to the substrate specificity of the enzyme, the enzymatic transglycosylation could
be performed as the last step starting from 5’-deoxyuridine.
2.4 5’-Mononucleotides
The phosphorylation of nucleosides can be performed by phosphorus oxycloride (POCl3 ) in trialkyl phosphates (TAP) and in the presence of water to give to nucleoside 5’-monosphosphates [17]. Our target compounds, uridine and cytidine 5’-monophosphate (UMP and CMP), were obtained by a chemo-enzymatic
process starting from the acetylated of uridine and cytidine (7, 8). Once selected the best lipase source
(PFL and CRL, respectively), the biocatalyst was used for the hydrolysis of 7 and 8, to give 9 and 10,
respectively, having only a free hydroxyl group in C-5’ position [18]. These intermediates were used for
the synthesis of UMP and CMP according to the chemical procedure previously reported [17] (11, 12,
Figure 5).
According to the results obtained, the use of biological catalysts has shown considerable advantages over
chemical synthesis, such as stereo- and regioselectivity and reduction of the preparation and purification
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Scientifica Acta 1, No. 1 (2007)
AcO
Base
O
OAc OAc
7, 8
enzymatic
regioselective
deprotection
a
Uridine = 93%
Cytidine = 70%
69
HO
Base
O
OAc OAc
2-
phosphorylation
b
70%
9, 10
O3PO
Base
O
OH
OH
11, UMP (5'-monophosphate uridine)
12, CMP (5'-monophosphate cytidine)
Base= uracil, cytosine
a: KH2PO4 buffer 25 mM, pH 7, 10% CH3CN, enzyme: immobilized PFL or CRL
b: (C2H5O)3PO/POCl3
Fig. 5
steps. This chemo-enzymatic process can be considered, indeed, an alternative strategy to the traditional
synthesis of nucleosides and nucleotides.
Acknowledgements I thank Dr. Silvia Rocchietti (Innovate Biotechnology srl, Rivalta Scrivia, Tortona, AL, Italy)
for her precious collaboration and my tutor Prof. Massimo Pregnolato. A special thank to Prof. Marco Terreni and Dr.
Daniela Ubiali for their kind collaboration.
We thank the subproject BIOCAT REGINS 2E0006R within the INTERREG IIIC Regional Framework Operation
(RFO) for the fellowship to T.B. for the period March-October 2006.
We thank Sovvenzione Globale INGENIO POR Ob. 3 F.S.E. 2000-2006 for the fellowship to T.B. (2007).
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