SYNTHESIS OF 2-DEOXY-β-D-RIBONUCLEOSIDES AND2,3-DIDEOXY.β-D-PENTOFURANOSIDES ON IMMOBILIZED BACTERIAL CELLS

Article abstract of DOI:10.1135/cccc19942303

Alginate gel-entrapped cells of auxotrophic thymine-dependent strain of E. coli catalyze the transfer of 2-deoxy-D-ribofuranosyl moiety of 2'-deoxyuridine to purine and pyrimidine bases as well as their aza and deaza analogs.All experiments invariably gave β-anomers; in most cases, the reaction was regiospecific, affording N⁹-isomers in the purine and N¹-isomers in the pyrimidine series.Also a 2,3-dideoxynucleoside can serve as donor of the glycosyl moiety.The acceptor activity of purine bases depends only little on substitution, the only condition being the presence of N⁷-nitrogen atom.On the other hand, in the pyrimidine series the activity is limited to only a narrow choice of mostly short 5-alkyl and 5-halogeno uracil derivatives.Heterocyclic bases containing amino groups are deaminated; this can be avoided by conversion of the base to the corresponding N-dimethylaminomethylene derivative which is then ammonolyzed.The method was verified by isolation of 9-(2-deoxy-β-D-ribofuranosyl) derivatives of adenine, guanine, 2-chloroadenine, 6-methylpurine, 8-azaadenine, 8-azaguanine, 1-deazaadenine, 3-deazaadenine, 1-(2-deoxy-β-D-ribofuranosyl) derivatives of 5-ethyluracil, 5-fluorouracil, and 9-(2,3-deoxy-β-D-pentofuranosyl)hypoxanthine, 9-(2,3-deoxy-β-D-pentofuranosyl)-6-methylpurine, and other nucleosides.

Full text of DOI:10.1135/cccc19942303

Biotechnological Preparation of 2’-Deoxyribonucleosides
2303
SYNTHESIS OF 2-DEOXY-β-D-RIBONUCLEOSIDES AND
2,3-DIDEOXY-β-D-PENTOFURANOSIDES ON IMMOBILIZED BACTERIAL
CELLS*
Ivan VOTRUBA, Antonin HOLY**, Hana DVORAKOVA, Jaroslav GUNTER,
Dana HOCKOVA, Hubert HREBABECKY, Tomas CIHLAR and Milena MASOJIDKOVA
Institute of Organic Chemistry and Biochemistry,
Academy of Sciences of the Czech Republic, 166 10 Prague 6, The Czech Republic
Received January 14, 1994
Accepted April 20, 1994
Alginate gel-entrapped cells of auxotrophic thymine-dependent strain of E. coli catalyze the transfer
of 2-deoxy-D-ribofuranosyl moiety of 2′-deoxyuridine to purine and pyrimidine bases as well as their
aza and deaza analogs. All experiments invariably gave β-anomers; in most cases, the reaction was
regiospecific, affording N9-isomers in the purine and N1-isomers in the pyrimidine series. Also a 2,3-
dideoxynucleoside can serve as donor of the glycosyl moiety. The acceptor activity of purine bases
depends only little on substitution, the only condition being the presence of N7-nitrogen atom. On the
other hand, in the pyrimidine series the activity is limited to only a narrow choice of mostly short
5-alkyl and 5-halogeno uracil derivatives. Heterocyclic bases containing amino groups are deami-
nated; this can be avoided by conversion of the base to the corresponding N-dimethylaminomethylene
derivative which is then ammonolyzed. The method was verified by isolation of 9-(2-deoxy-β-D-ribo-
furanosyl) derivatives of adenine, guanine, 2-chloroadenine, 6-methylpurine, 8-azaadenine, 8-aza-
guanine, 1-deazaadenine, 3-deazaadenine, 1-(2-deoxy-β-D-ribofuranosyl) derivatives of 5-ethyluracil,
5-fluorouracil, and 9-(2,3-dideoxy-β-D-pentofuranosyl)hypoxanthine, 9-(2,3-dideoxy-β-D-pentofurano-
syl)-6-methylpurine, and other nucleosides.
2′-Deoxy-β-D-ribonucleosides of pyrimidine and purine bases are very important pre-
cursors in the biosynthesis of deoxyribonucleic acids. Natural deoxyribonucleosides are
of importance as starting compounds for synthesis of oligodeoxyribonucleotides in
gene manipulations or development of diagnostic probes. Many deoxynucleosides
derived from chemically modified heterocyclic bases of both series are also significant
registered or potential drugs (particularly antivirals and cancerostatics) or diagnostic
tools.
* The results were presented in part at the VII. Symposium on the Chemistry of Nucleic Acid
Components, Bechyne 1987.
**The author to whom correspondence should be addressed.
Collect. Czech. Chem. Commun. (Vol. 59) (1994)

2304
Votruba et al.:
Natural 2′-deoxyribonucleosides are usually prepared by enzymatic degradation of
DNA or its precursors (nucleotides). Their chemical synthesis is of no advantage: of the
four deoxyribonucleosides only thymidine and 2′-deoxycytidine can be easily prepared
in bulk by conversion of the corresponding ribonucleosides1. Equally complicated is
the synthesis of 2′-deoxyribonucleosides containing modified heterocyclic bases. For
the chemical synthesis of this type of compounds many methods are available that
consist in deoxyribosylation of suitable heterocyclic bases, transformation of the corre-
sponding ribonucleosides, or modification of the heterocyclic system in preformed
2′-deoxyribonucleosides. Total synthesis by glycosylation is difficult because of limited
accessibility and low stability of a suitable blocked derivative of 2-deoxy-D-ribose;
recently, there appeared an alternative in deoxyribosylation by the phase-transfer
method (see e.g. ref.2 and references therein) but not all heterocyclic systems withstand
the drastic conditions used in this reaction. However, the main problem with glycosida-
tion techniques is the formation of mixtures of α- and β-anomers which in most cases
are separable only with difficulty. It appears that (at least in some cases) the stereospe-
cificity of this reaction is kinetically controlled and thus may be to a certain degree
influenced3 by suitable choice of conditions; nevertheless, the whole approach is still
far from perfection.
For the transformation ribo → 2-deoxyribo, the corresponding ribonucleoside has to
be synthesized first. Although in these cases it is not difficult to obtain uniform
β-anomers4, further conversion is possible without greater problems only in the pyri-
midine series where one can utilize the easy formation of 2,2′-anhydronucleosides5.
With purine derivatives, such attempts were feasible only when performed on a small
scale because they require expensive procedures (use of triflates, silyl protecting groups
etc., see e.g. ref.6).
Therefore, the enzyme-catalyzed transglycosylation of some synthetically easily ac-
cessible 2′-deoxyribonucleosides appears to be of advantage. As the most suitable we
may consider thymidine or 2′-deoxyuridine, accessible by partial synthesis from uridine
or even by total synthesis from D-arabinose7. The mentioned enzymatic method should
be characterized by mild (physiological) conditions, the usually expected regiospeci-
ficity as well as stereospecificity of the transfer reaction, and simple scale-up ability.
The enzymatically catalyzed synthesis of deoxyribonucleosides has been described
several times8–11 making use of whole (procaryotic) cells, cell homogenates or enzyme
preparations of various purity. Enzymes, catalyzing such transfer reactions, can be
either nucleoside N-transdeoxyribosylases (e.g. from Lactobacillus sp.) or nucleoside
phosphorylases. Whole cells or unpurified extracts contain also other enzymes metabo-
lizing the bases as well as deoxynucleosides; any improvement is possible only at the
expenses of effort exerted on purification of the given enzyme, lowering thus the effec-
tivity of the way chosen.
Collect. Czech. Chem. Commun. (Vol. 59) (1994)

Biotechnological Preparation of 2’-Deoxyribonucleosides
2305
We described recently a very effective and simple preparation of 2′-deoxy-β-D-ribo-
nucleosides of heterocyclic bases, using immobilized cells of an auxotrophic thymine-
dependent strain of E. coli12. The method consists in the transfer of 2-deoxy-
ribofuranoside moiety from 2′-deoxyuridine (or other suitable donor) to the hetero-
cyclic base:
dUrd
+
base
BdRf
+
uracil
As indicated by preliminary experiments, the reaction is stereospecific and purine and
pyrimidine bases and their analogs afford “natural” isomers (i.e. the N9-isomers from
the purine and the N1-isomers from the pyrimidine bases), whereas some imidazole
derivatives give rise to both the possible regioisomers13.
The mentioned catalyst contains immobilized bacterial cells harvested in the postlo-
garithmic growth phase and in comparison with the mentioned enzymatically catalyzed
reactions offers the following advantages: (i) because of high immobilization capacity
of the system chosen, it is possible to achieve a high content of the biomass (and high
specific enzymatic activity) in the catalyst; (ii) not only the auxotroph used has high
transdeoxyribosylation activity but also in the immobilized cells the undesired enzy-
matic side activities (phosphorylating, phosphorolytic, hydrolytic) are suppressed (ex-
cept deamination); (iii) thanks to these properties, the syntheses can be performed in a
dilute aqueous buffer and at relatively high concentration, to a considerable extent com-
parable with the methods of organic synthesis of these compounds.
We therefore decided to investigate in detail the applicability of this method to the
preparation of 2-deoxy-β-D-ribofuranosides of modified heterocyclic bases. Special at-
tention we focused to such bases whose deoxyribonucleosides already are of practical
importance (e.g. in medicine), as well as to the investigation of regiospecificity of the
transfer reaction in cases of bases for which we may expect an ambiguity of the transfer
site. In the latter context, we were interested particularly in the deaza and aza analogs
of purine bases. By isolation and characterization of their deoxyribonucleosides, of
which some have not hitherto been prepared, we intend to illustrate the practical appli-
cability of the method.
In the course of the last years, nucleosides modified on the sugar component received
an extraordinary attention in connection with the quest for new antivirals (particularly
against HIV) and cytostatics. Especially 3-azido-2,3-dideoxynucleosides (Retrovir),
2,3-dideoxyinosine (Didanosin), 2,3-dideoxycytidine (Zalcitabine) and other modified
sugar nucleosides of natural bases are very important. It has been found that 2,3-
dideoxynucleosides can be prepared in low yield by transglycosylation, catalyzed either
with nucleoside-N-transdeoxyribosylase from Lactobacillus sp. or with nucleoside
phosphorylase, i.e. with salvage pathway enzymes14,15. Since generally only sparse in-
Collect. Czech. Chem. Commun. (Vol. 59) (1994)

2306
Votruba et al.:
formation exists about the donor specificity of transdeoxyribosylation reactions, we
intended to extend our method to sugar-modified nucleosides.
RESULTS AND DISCUSSION
Basic Parameters
In the preliminary phase of our studies we have found that one of the best acceptors in
the transdeoxyribosylation reaction is 6-methylpurine. Moreover, the exceptional
properties of this base enabled a good HPLC and TLC separation of all the components
of the reaction mixture to be made. Therefore, much of basic information on the course
of the studied processes was obtained using this base as acceptor.
The first key problem is the stability of the catalyst and reproducibility of the proce-
dures. The catalyst is prepared by dropping a suspension of bacterial cells in an alginate
sol into a calcium chloride solution. We have verified that the catalyst is stable enough
to afford practically the same conversion of model mixture 2′-deoxyuridine–6-methyl-
purine after storage for 1 week at +4 °C. In a series of experiments with the mentioned
mixture, in which the catalyst was repeatedly used in 16-h cycles, the conversion
practically did not change even after ninefold use of the same catalyst and amounted to
98.7 ± 0.8%. We also studied how significant is the amount of the compound withheld
in the catalyst beads which swell considerably during the incubation. Upon disintegra-
tion of the beads and analysis of their content we found no significant presence of
substrate, product or (which is more important) their possible metabolites (nucleotides).
The found pH optimum was 5.8, the conversion being not very different in the region
pH 5 – 6 (Fig. 1). Acetate as well as phosphate buffer can be employed, however, after
some time the phosphate buffer dissolved the catalyst beads. Therefore, we performed
all the studies in an ammonium acetate buffer pH 5.8.
FIG. 1
Catalytic activity–pH profile of 2-deoxyriboside
synthesis (Ca2+-alginate entrapped E. coli SPT−
cells). c, Concentration of 6-methylpurine 2-deoxy-
riboside formed
Collect. Czech. Chem. Commun. (Vol. 59) (1994)

Biotechnological Preparation of 2’-Deoxyribonucleosides
2307
We compared the donor ability of two pyrimidine deoxyribonucleosides, 2′-deoxy-
uridine and thymidine. It appeared that for the transfer of the sugar moiety to 6-methyl-
purine, after 6 h of incubation, the donor activity of thymidine was somewhat lower
(by 28%) than of 2′-deoxyuridine (Fig. 2). In most experiments we employed 2′-deoxy-
uridine because of its good synthetic accessibility. In the system 2′-deoxyuridine –
6-methylpurine, the conversion of the heterocyclic base was proportional to the donor
concentration only at lower donor/acceptor values, being practically the same for
values higher than 2 (data not shown). For this reason we systematically performed our
experiments with a twofold excess of the donor relative to the base.
Acceptor Activity of Heterocyclic Bases
In the discussion of our results we first of all compare the conversion of the heterocy-
clic bases into the corresponding nucleosides irrespectively of their isomeric or
anomeric purity. This approach is an approximative one because it considers neither the
possible differences between the UV-characteristics of the base and the nucleosides
formed from it nor the possible distortion of the results due to low solubility of the base
in the reaction medium. Consequently, we cannot consider the found conversion to be
an absolute measure of the acceptor activity of the base and the numbers in Tables I
and II are only of semiquantitative value. Of an equally little objectivity would be the
evaluation by preparative experiments: the determination of conversion from the filtrate
before separation of the mixture involves possible neglecting the undissolved base
whereas values obtained after the separation relate to the isolated yield but not to the
primary conversion of the heterocyclic base.
Another evaluating parameter that, according to our experience invariably coincided
with the base conversion, was the concurrent conversion of 2′-deoxyuridine to uracil. In
cases where no deoxyribonucleoside was formed from the base, we did not observe any
uracil formation from 2′-deoxyuridine.
FIG. 2
Comparison of donor efficiency of 2′-deoxy-
uridine and thymidine (catalyzed by Ca2+-alginate
entrapped E. coli SPT− cells, 5 beads/ml). c, Con-
centration of 6-methylpurine 2-deoxyriboside
formed. 1 74 mM 2′-Deoxyuridine, 2 74 mM Thy-
midine. Concentration of 6-methylpurine (acceptor)
was 37 mmol/l
Collect. Czech. Chem. Commun. (Vol. 59) (1994)

2308
Votruba et al.:
TABLE I
Acceptor activity of purine bases in the reaction with 2′-deoxyuridine
Compound
Base
Conversiona, %
I
Adenine
63b
67
72
46
ND
55
63
0
II
2-Methyladenine
III
2-Methylthioadenine
2,6-Diaminopurine
IV
V
2-Chloroadenine
VI
6-Methylpurine
VII
6-Chloropurine
VIII
IX
6-Mercaptopurine
6-Methylthiopurine
N6-Methyladenine
N6-Dimethyladenine
N6-Furfuryladenine
63
64
70
90
33
39
ND
37
75
64
39
21
11
68c
68
45
4
X
XI
XII
XIII
XIV
2,6-Dichloropurine
Hypoxanthine
XV
Guanine
N2-Acetylguanine
N6-Dimethylaminomethyleneadenine
2-Chloro-N6-dimethylaminomethyleneadenine
N2-Dimethylaminomethyleneguanine
8-Aminoadenine
XVI
XVII
XVIII
XIX
XX
XXI
4-Aminopyrazolo[3,4-d]pyrimidine
XXII
XXIII
XXIV
XXV
XXVI
XXVII
XXVIII
XXIX
XXX
XXXI
XXXII
XXXIII
XXXIV
XXXV
XXXVI
XXXVII
XXXVIII
8-Azaadenine
N6-Dimethylaminomethylene-8-azaadenine
8-Aza-2,6-diaminopurine
8-Azahypoxanthine
8-Azaguanine
N6-Dimethylaminomethylene-8-azaguanine
ND
68
88
93
73
43
0
1-Deazapurine
1-Deazaadenine
1-Deaza-6-nitropurine
3-Deazapurine
6-Chloro-3-deazapurine
6-Azido-3-deazapurine
3-Deazaadenine
58
22
traces
47
26
30
3-Deaza-2,6-diaminopurine
3-Deazaguanine
2-Azaadenine
2-Azahypoxanthine
Collect. Czech. Chem. Commun. (Vol. 59) (1994)

Biotechnological Preparation of 2’-Deoxyribonucleosides
2309
TABLE I
(Continued)
Compound
Base
Conversion, %
XL
1-Deazapurine-N3-oxide
3-Deazapurine-N1-oxide
0
XLI
0
XLII
XLIII
XLIV
XLV
XLVI
4-Aminopyrrolo[2,3-d]pyrimidine
0
4-Amino-2-mercaptopyrrolo-[2,3-d]pyrimidine
4-Amino-2-methylthiopyrrolo[2,3-d]pyrimidine
2-Amino-4-hydroxypyrrolo[2,3-d]pyrimidine
N1,N6-Etheno-2-azaadenine
0
0
ND
ND
a
Prior to analysis, dimethylaminomethylene and acetyl derivatives were subjected to ammonolysis in
b
12.5% aqueous ammonia at room temperature overnight; ND not determined; product 2′-deoxy-
inosine; mixture of 2′-deoxy-8-azaadenosine and 2′-deoxy-8-azainosine.
c
With purine bases and their analogs (Table I), as acceptors may serve derivatives of
purine, 1-deazapurine, (imidazo[4,5-b]pyridine), 3-deazapurine (imidazo[4,5-c]py-
ridine), 8-azapurine (1,2,3-triazolo[4,5-d]pyrimidine) and 2-azapurine (imidazo[4,5-d]-
1,2,3-triazine), the only condition being the presence of a nitrogen atom in position 7 of
the purine system: derivatives of 7-deazapurine (pyrrolo[2,3-d]pyrimidine) (XLIIa – XLVa)*
and pyrazolo[3,4-d]pyrimidine (XXI) are all inactive. Such a strict condition is not un-
usual even for other enzymes metabolizing purine derivatives. Another structural fea-
ture, clearly hindering interaction of the purine base with the enzyme, is the presence
of a group affecting the π-electron distribution of the heteroaromatic system in the
base: N-oxides of 1-deaza- and 3-deazapurine derivatives XLa and XLIa do not act as
acceptors in the transglycosylation reaction.
Other substituents on the purine nucleous can affect the yields but have no principal
influence. The position 6 in the parent purine system or its aza or deaza analogs can
evidently be occupied by an atom of hydrogen or halogen, or an azido, nitro, alkyl,
amino, alkyl(aryl)amino or dialkylamino group; the presence of a lactam–lactim tau-
tomerism is detrimental to the acceptor activity and replacement of the oxo group in the
position 6 of hypoxanthine by a thioxo group (VIIIa) resulted in complete failure, the
only one observed in this series. However, S-alkylation (compound IXa) restores again
the activity. Substitution in position 2 of the purine moiety with a halogen atom, or an
alkyl or amino group has no principal effect on the susceptibility of the base toward the
* In the formulae, a denotes heterocyclic base, b 2-deoxynucleoside, c 2,3-dideoxynucleoside.
Collect. Czech. Chem. Commun. (Vol. 59) (1994)

2310
Votruba et al.:
Collect. Czech. Chem. Commun. (Vol. 59) (1994)

Biotechnological Preparation of 2’-Deoxyribonucleosides
2311
glycosyl transfer. Although information in this respect is not exhaustive, it seems that
the effect of this substitution is rather negative, similarly to the case of introduction of
an amino group into position C-8 of adenine (compound XXa).
Evaluating the practical applicability of this method in the series of purine deriva-
tives, we encounter two kinds of problems: (i) with adenine derivatives, the transglyco-
sylation reaction may be considerably complicated by residual activity of adenosine
aminohydrolase in the immobilized cells which in some cases leads to subsequent for-
mation of the corresponding hypoxanthine derivatives and, which is no less important,
(ii) the reaction is often limited by the very low solubility of the purine bases. The latter
difficulty can be positively influenced by a technical modification (dimethyl sulfoxide
can be added as cosolvent; up to final concentration 8% in the incubation mixture it
presents no difficulties); however, we developed a procedure that simultaneously elimi-
nates also the above-mentioned subsequent deamination: the amino group of adenine
and guanine derivatives and their aza analogs was converted into an amidine group (the
so called dimethylaminomethylene derivative) by reaction with dimethylformamide
dineopentyl acetal (the corresponding dimethyl acetal might N-methylate the base). In
all the cases studied (XVII – XIX, XXIII, XXVII), this group was sufficiently stable
under the experimental conditions and could be easily removed by ammonolysis di-
rectly in the incubation mixture. Of course, it is possible, and sometimes because of
Collect. Czech. Chem. Commun. (Vol. 59) (1994)

2312
Votruba et al.:
different chromatographic parameters advantageous, to isolate first the 2′-deoxynucleo-
side N-dimethylaminomethylene derivative and then to subject it to ammonolysis.
The practical applicability of the transdeoxyribosylation technique was demonstrated
by the preparation of 2′-deoxyadenosine (Ib), 2′-deoxyguanosine (XVb), 2′-deoxyino-
sine (XIVb), 1-deaza-2′-deoxyadenosine (XXIXb), and 9-(2-deoxy-β-D-ribofuranosyl)
derivatives of 1-deazapurine (XXVIIIb), 6-azido-3-deazapurine (XXXIIIb) and 1-deaza-
6-nitropurine (XXXb). Using UV and NMR spectra16, we ascribed the 9-(2-deoxy-β-D-
ribofuranosyl) structure to all these compounds and thus the transfer reaction proceeds
under formation of the “natural” N9-isomers with both the purine and azapurine sys-
tems. The 1-deazaadenine derivative XXIXb was assigned structure by using the charac-
Collect. Czech. Chem. Commun. (Vol. 59) (1994)

Biotechnological Preparation of 2’-Deoxyribonucleosides
2313
teristic spectrum of N9-derivatives, the 6-nitro derivative XXXb was identified by com-
parison with the published 1H NMR spectrum17. The structure of 3-deazapurine deriva-
tive XXXIb was established by comparison with the known N9-substituted nucleoside
analogs16. On the preparative scale we performed the synthesis of 2-chloro-2′-deoxy-
adenosine18 (Vb), an important drug against hairy cell leukemia, and 9-(2-deoxy-β-D-ri-
bofuranosyl)-6-methylpurine (VIb), a significant diagnostic of myco- plasma infection
of eucaryotic cells19.
We paid particular attention to 8-azapurine derivatives where the N8-isomer was in-
variably the principal product of alkylation reactions20,21. Isolation of the product aris-
TABLE II
Acceptor activity of pyrimidine bases in reaction with 2′-deoxyuridine
Compound
Base
Conversion, %
XLIX
L
Thymine
67
35
0
5-Ethyluracil
LI
5-Propyluracil
LII
5-(2-Propyl)uracil
5-(3-Butenyl)uracil
5-Cyclopropyluracil
5-Cyclobutyluracil
5-Fluorouracil
0
LIII
0
LIV
39
0
LV
LVI
64
57
0
LVII
LVIII
LIX
5-Iodouracil
5-Cyanouracil
5-Nitrouracil
0
LX
5-Carboxyuracil
5-Acetyluracil
0
LXI
31
0
LXII
LXIII
LXIV
LXV
LXVI
LXVII
LXVIII
LXIX
LXX
LXXI
LXXII
6-Methyluracil
2-Pyrimidone
0
4-Methyl-2-pyrimidone
4-Methoxy-2-pyrimidone
Cytosine
0
0
0
N4-Dimethylaminomethylenecytosine
22
0
6-Azauracil
2-Thiouracil
0
2-Thiocytosine
0
2-Pyridone
0
4,6-Dihydroxypyrimidine
0
Incubation mixture contained: 0.85 mmol base, 1.7 mmol 2′-deoxyuridine in 50 ml 50 mM am-
monium acetate, pH 5.8 and 150 catalyst beads (diameter 5 mm); incubation time 6 h at 37 °C
Collect. Czech. Chem. Commun. (Vol. 59) (1994)

2314
Votruba et al.:
ing from N6-dimethylaminomethylene-8-azaadenine (XXIIIa) has shown that also in
this case the reaction afforded exclusively the N9-isomer XXIIIb. Analogously, we pre-
pared 8-aza-2′-deoxyguanosine (XXVIb) by reaction of N6-dimethylaminomethylene-8-
azaguanine (XXVIIa) followed by ammonolysis. In neither case we detected any other
isomers. Surprisingly, the reaction of 8-aza-2,6-diaminopurine (XXIVa) gave the
8-isomer XLVIII as the only product with no other isomers being detected. We have no
interpretation for this unexpected reversal of regiospecificity. Proof of all the structures
1
was convincingly given by UV spectra characteristic of these isomers, by H NMR
spectra, and particularly by 13C NMR spectra.
The isomer structure of 2′-deoxyribosides of 8-azaadenine (XXIIb), 8-aza-2,6-
diaminopurine (XXIVb) and 8-azaguanine (XXVIb) was determined by 13C NMR
(proton-decoupled) spectra. The N9-structure of compounds XXIIb and XXVIb was con-
firmed by the characteristic chemical shifts of the carbon atoms of the base as well as
the sugar part. These data (see Experimental) are in accord with the literature
values22,23. On the other hand, in the spectrum of derivative XXIVb we observed a
marked downfield shift of the signal due to C-4 of the base (8.4 ppm) and of the
anomeric C-1′ carbon signal (8.9 ppm) compared with the N9-substituted derivatives of
this base, other signals being affected only negligibly. These changes are characteristic
of a substitution of 8-aza-2,6-diaminopurine in N8-position.
The regiospecific ambiguity was encountered in the case of 2-azapurine derivatives:
from 2-azahypoxanthine (XXXVIIIa) we obtained the N7- and N9-isomers XXXVIIIb and
XXXIXb in equimolar ratio, the tricyclic derivative N1,N6-etheno-2-azaadenine XLVIa
afforded a mixture of N9-isomer XLVIb and N7-isomer XLVIIb, in which the former
predominated (9 : 2). It is, however, worth notice that NMR analysis did not prove
glycosylation of the triazine ring of the 2-azapurine system although alkylations into
this position proceed easily. For the structural assignment we used the same charac-
teristic shifts of the carbon atoms of the base24: whereas for compound XXXVIIIb the
chemical shifts correspond to N9-substitution, the spectrum of compound XXXIXb ex-
hibits a characteristic downfield shift of the C-4 (9.4 ppm), C-8 (1.3 ppm) and C-1′ (1.5
ppm) signals, and an upfield shift of the C-5 signal (10 ppm). According to our experi-
ence, these changes are characteristic of the N7-isomer. The structures of compounds
XLVIb and XLVIIb were assigned analogously.
The applications of the transglycosylation reaction to the purine and analogous systems
are thus wide and in majority of cases lead to the expected N9-isomers. A substantially
different situation was found in the pyrimidine series. As seen from Table II, the scope
of modifications is generally very limited. From the failure of the acceptor activity of
2-pyrimidinone (LXIIIa), its 4-methyl derivative LXIVa, and 4-methoxy-2-pyrimidin-
one (LXVa) it is evident that for the acceptor activity a complete uracil or cytosine
system is irreplaceable. Since with cytosine the results are distorted by residual cytosine
aminohydrolase activity of the immobilized cells (the transdeoxyribosylation with
Collect. Czech. Chem. Commun. (Vol. 59) (1994)

Biotechnological Preparation of 2’-Deoxyribonucleosides
2315
2′-deoxyuridine of the formed uracil is not discernible), also in this case we success-
fully made use of N4-dimethylaminomethylene derivative LXVIIa. Of the whole studied
group of derivatives, only a limited number of 5-alkyluracils are transglycosylation
acceptors: the reaction was realized with thymine and its homolog 5-ethyluracil (La)
but not with the propyl and 2-propyl uracil derivatives LIa and LIIa and the higher
Collect. Czech. Chem. Commun. (Vol. 59) (1994)

2316
Votruba et al.:
alkyl derivative LIII. Only the perhaps sterically less demanding 5-cyclopropyluracil
(LIVa) proved to be good substrate, contrary to its homolog, 5-cyclobutyluracil (LVa).
Whereas the reaction was rendered impossible by some electronegative substituents in
position C-5 (cyano, nitro or carboxy groups), with other electronegative substituents
(fluorine or iodine atoms) it proceeded normally. Also 5-acetyluracil (LXIa) reacted as
acceptor of the deoxyribosyl moiety. A substitution of the uracil ring in position 6 is
not acceptable (compound LXIIa) and the same holds for the oxo–thioxo change in
position C-2 (compounds LXIXa and LXXa). In contrast to the purine series, replace-
ment of −CH= grouping with −N= is not acceptable (LXVIII). We cannot make any
general conclusions; obviously neither the apparent pK of the base nor the bulk of the
substituent in position 5 are the decisive factors. It is clear, however, that the reaction,
if proceeds, affords the N1-regioisomer. Such considerable limitation of structural vari-
ability indicates that the catalysis may be due to another enzyme than that in the trans-
deoxyribosylation of purine bases. Also in the pyrimidine series we illustrated the
potentialities of the method by preparing biologically active nucleosides: 5-ethyl-2′-de-
oxyuridine (Lb, antiherpetic Aedurid) and 5-fluoro-2′-deoxyuridine (LVIb, cytostatic
Floxuridine).
It is important to stress that the transdeoxyribosylation activities operating in immo-
bilized cells need not be necessarily all activities of this type in the corresponding live
bacterial cell. As an evident case we can mention the discrepancy between the re-
peatedly confirmed absence of acceptor activity of 2-pyrimidone (LXIIIa) in the trans-
glycosylation catalyzed by immobilized cells and the high conversion of this base into
its 2-deoxy-β-D-ribofuranoside LXIIIb which we observed in the live cells of E. coli in
vivo as well as in their cell-free extract in vitro25.
Donor Activity of Uracil and Thymine Nucleosides Modified in the Sugar Moiety
The donor activity was studied on two acceptor models: 6-methylpurine, mentioned
above, and hypoxanthine. The results in Table III prove that the hydrogen atom in
position 2 of deoxyribose cannot be replaced by hydroxy, alkoxy or amino groups or by
a halogen atom in the ribo configuration. Also a hydroxyl group in arabo configuration
in uracil arabinoside (LXXIX) has a prohibitive effect whereas an atom of fluorine in
this configuration (compound LXXXI) is to a certain degree acceptable. The hydroxy
group itself in position 2 of 2-deoxy-D-ribose is not indispensable, although the donor
activity, if present in compounds without this group, is reduced by several orders of
magnitude: in spite of this, we succeeded in performing the transfer of “2,3-dideoxy-
ribofuranosyl” moiety to 6-methylpurine under formation of hitherto undescribed nu-
cleoside VIc, as well as to hypoxanthine from dideoxyuridine and dideoxythymidine
(LXXVII) (Fig. 3) (the isolated 2′,3′-dideoxyinosine (XIVc) is the antiviral Didanosin,
used in treatment of AIDS). Also 3′-fluoro-2′,3′-dideoxyuridine (LXXVIII) proved to be
a weakly active donor, whereas 3′-azido-2′,3′-dideoxythymidine (LXXXII) is inactive.
Collect. Czech. Chem. Commun. (Vol. 59) (1994)

Biotechnological Preparation of 2’-Deoxyribonucleosides
2317
TABLE III
Donor activity of sugar-modified pyrimidine nucleosides
Compound
Donor
Activity
LXXIII
LXXIV
LXXV
2′-Deoxyuridine
+
−
−
−
+
2′-Amino-2′-deoxyuridine
2′-Chloro-2′-deoxyuridine
2′-O-Methyluridine
LXXVI
LXXVII
LXXVIII
LXXIX
LXXX
2′,3′-Dideoxyuridine
a
1-(2,3-Dideoxy-3-fluoro-D-threo-pentofuranosyl)uracil
1-(D-Arabinofuranosyl)uracil
±
−
−
+
−
1-(2-Deoxy-2-L-erythro-pentofuranosyl)uracil
1-(2-Deoxy-2-fluoro-D-arabinofuranosyl)uracil
3′-Azido-2′,3′-dideoxythymidine
LXXXI
LXXXII
a
Traces of product
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2318
Votruba et al.:
In this respect, the 5′-hydroxy functionality seems to be of key importance and its
relative position to the heterocyclic base system is decisive: 1-(2-deoxy-α-L-erythro-
pentofuranosyl)uracil (LXXX), in which the configuration at C-4′ is opposite to that in
2′-deoxyuridine, is not a transglycosylation donor. We can thus conclude that, provided
long reaction time, the transfer, catalyzed with immobilized cells described in this
work, can be used also for the preparation of 2,3-dideoxynucleosides, particularly in the
purine series. Continuation of this investigation, isolation of the possible modified com-
pounds that might be accessible by this method, as well as utilization of the transglyco-
sylation reaction for other types of nucleoside donors (hexofuranosides etc.) reaches
already beyond the scope of this paper.
The described method may also be used for the preparation of isotopically labelled
deoxyribonucleosides; the label can be present in the heterocyclic base and/or in the
sugar component. In such cases, the mild conditions and easy isolation on the micro
scale, as well as the possible recovery of both reaction components are of advantage.
EXPERIMENTAL
Unless stated otherwise, solutions were evaporated at 40 °C/2 kPa and compounds were dried at 13
Pa over phosphorus pentoxide. Melting points were determined on a Kofler block and are uncor-
rected. Thin-layer chromatography was performed on Silufol UV254 sheets in the solvent systems: S1,
chloroform–methanol (3 : 1), S2, chloroform–methanol (4 : 1), S3, chloroform–methanol (7 : 3), S4,
water–ethanol–acetone–ethyl acetate (1 : 2 : 2 : 12), S5, water–ethanol–acetone–ethyl acetate (1 : 1 : 1 : 4).
Preparative chromatography on loose layers was carried out on 40 × 17 × 0.4 cm plates of silica gel
containing a UV indicator (Kavalier, Votice, The Czech Republic). Paper chromatography was per-
formed on a paper Whatman No. 1 in system S6, 2-propanol–concentrated aqueous ammonia–water
(7 : 1 : 2), paper electrophoresis on a paper Whatman No. 3 MM at 40 V/cm for 1 h in 0.05 M
triethylammonium hydrogen carbonate pH 7.5. The electrophoretical mobilities are referenced to
uridine 3′-phosphate. Reversed-phase liquid chromatography was carried out on Separon SGX columns
(150 × 3.3 mm) in 50 mM potassium dihydrogen phosphate pH 5.5, linear gradient of methanol (H1,
10 – 25% methanol during 20 min; H2, 10 – 45% methanol during 20 min; H3, 5 – 20% methanol
FIG. 3
Time course of 2′,3′-dideoxyribonucleoside syn-
thesis catalyzed by Ca2+-alginate entrapped E. coli
SPT− cells. c, concentration of 2′,3′-dideoxyribo-
nucleoside formed. 1 18.5 mM Hypoxanthine,
37 mM 2′,3′-dideoxyuridine and 10 beads/ml. 2
18.5 mM Hypoxanthine, 37 mM 2′,3′-dideoxyuridine
and 5 beads/ml. 3 18.5 mM 6-Methylpurine, 37 mM
2′,3′-dideoxyuridine and 10 beads/ml. 4 18.5 mM
Hypoxanthine, 37 mM 2′,3′-dideoxythymidine and
5 beads/ml
Collect. Czech. Chem. Commun. (Vol. 59) (1994)

Biotechnological Preparation of 2’-Deoxyribonucleosides
2319
during 15 min) or in 50 mM triethylammonium hydrogen carbonate pH 7.5 alone (H4) or with 6%
of acetonitrile (H5). NMR spectra were measured on a spectrometer Varian UNITY-200 (1H at 200
MHz, 13C at 50.3 MHz) or Varian UNITY-500 (1H at 500 MHz and 13C at 125.7 MHz) in
CD3SOCD3. Chemical shifts of protons were referenced to tetramethylsilane as internal standard
whereas shifts of carbon atoms to the solvent signal and then calculated according to the relationship
δ(CD3SOCD3) = 39.7 ppm. In addition to the usual proton-decoupled 13C NMR spectra we measured
also J-modulated spectra (“attached proton test pulse sequence”) or proton-coupled spectra, enabling
carbon signals of C and CH2 to be distinguished from those of CH and CH3. UV absorption spectra
were measured on a Beckmann DU-65 spectrometer; the wavelengths of extrema are given in nm.
Mass spectra were taken on a ZAB-EQ spectrometer (VG Analytical) using EI (electron energy 70 eV)
and FAB (ionization by Xe, accelerating voltage 8 kV) techniques. Spectrophotometric determination
of content of compounds was done in aqueous solutions (pH 2) and at wavelength of the absorption
maximum, using published extinction coefficient values.
Materials
Thymidine, 2′-deoxyadenosine, 2′-deoxyguanosine, 2′-deoxyinosine, hypoxanthine and 5-fluorouracil
were obtained from Calbiochem, adenine, guanine and thymine from Fluka. 2′-Deoxy-5-fluoro-
uridine, 8-azaadenine, 8-aza-2,6-diaminopurine, 8-azaguanine and 2,6-dichloropurine were Sigma
products. 2′-Deoxyuridine1, dimethylformamide dineopentyl acetal26 and 6-methylpurine27 were pre-
pared by published procedures. The pyrimidine bases were kindly given by Dr M. Ledvina of this
Institute, other purine base analogs were prepared by published procedures. Bacterial cells were im-
mobilized using alginate Protanal LF 10/60 (Protan, Norway).
Preparation of the Biocatalyst
Cultures of Escherichia coli SPT− (ref.28) were grown at 37 °C in a synthetic medium29 supplemented
with thymine (1 µg/ml) until the cell density has reached 8 . 108 cells/ml. The harvested cells were
exhaustively washed by centrifugation (6 000 g, 35 min; 3 times) in the presence of 0.9% KCl. The
resulting sediment of bacterial cells was suspended in 0.9% KCl (1 g of wet cells in 4 ml) and mixed
with a water solution of alginate (1 g of the wet biomass in 4 ml of 0.9% KCl with 6 ml of 4.76%
alginate). The ionotropic size-controlled gelation to entrap bacterial cells was achieved by dropping
E. coli SPT−-alginate suspension to 2% CaCl2 (the precipitation bath)30. The resulting calcium-algi-
nate beads (5 mm diameter) were then stirred in the precipitation bath for 4 h, washed 4 times with
an excess of 0.9% KCl and stored in the same medium at 4 °C until used.
Stability of the Biocatalyst
The incubation mixture contained 6-methylpurine (158 mg, 1.18 mmol) and 2′-deoxyuridine (537 mg,
2.35 mmol) in 30 ml of 0.05 M ammonium acetate pH 5.8. After addition of the catalyst (90 beads,
370 mg of biomass), the mixture was incubated in a Dubnoff′s incubator at 37 °C for 16 h. After
decanting the supernatant, fresh solution of the same composition was added and the incubation pro-
cedure was repeated nine times (total). The samples were analyzed by HPLC on an SGX C18 column
(150 × 3.3 mm); isocratic elution with 0.01 M potassium dihydrogen phosphate containing 13.5% of
methanol. Molar absorption coefficients of 6-methylpurine and its deoxyriboside were taken as
identical. The conversion in the last mixture achieved 99.6%, the average in all experiments was
98.7 ± 0.76%.
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2320
Votruba et al.:
Synthesis of 2′-Deoxyribonucleosides
A) Analytical procedure. The incubation mixture contained the corresponding heterocyclic base
(0.37 mmol) and 2′-deoxyuridine (0.74 mmol) in 50 mM ammonium acetate pH 5.7 (10 ml); 30 to 50
beads of the biocatalyst were added and the mixture was incubated for 16 h at 37 °C. The reaction
was followed by HPLC (reversed phase Tessek-SeparonTM SCX C18, 7 µm, 15 cm × 3.3 mm) in an
appropriate buffered solvent system. The data are given in Tables I and II.
B) Preparative procedure. Unless stated otherwise, the amount of reagents and solvents was
scaled up 5 times with respect to procedure A, the reaction conditions and analytical procedure being
identical. The supernatant was decanted, the catalyst washed by the same buffer as above (10 ml) and
the combined supernatants were mebrane-filtered (nitrocellulose filters, 0.45 µm). The filtrate was
further processed as indicated in specific cases.
Synthesis of 2′,3′-Dideoxyribonucleosides
The biocatalyst (50 to 100 beads) was added to a solution of the appropriate purine base (0.185
mmol) and 2′,3′-dideoxyuridine or 2′,3′-dideoxythymidine (0.37 mmol) in 50 mM ammonium acetate,
pH 5.7 (10 ml). The reaction course (at 37 °C) was followed by HPLC.
2-Chloroadenine (Va)
A mixture of 2,6-dichloropurine (XIIIa, 1.5 g, 7.9 mmol) and 30% methanolic ammonia (60 ml) was
heated at 100 °C for 12 h in an autoclave and then evaporated. The residue was dissolved in water
and deionized on a column of Dowex 50 X 8 (H+ form, 50 ml). The ion exchanger was washed with
water to loss of UV absorption, suspended in dilute (1 : 1) aqueous ammonia (250 ml), stirred for 2 h,
filtered, washed with water, and the filtrate was evaporated in vacuo. The dry residue was codistilled
with ethanol (3 × 50 ml), mixed with acetone and filtered; yield 0.90 g (67%) of chromatographically
homogeneous product Va, not melting up to 260 °C, RF 0.42 (S4). For C5H4ClN5 (169.6) calculated:
35.42% C, 2.38% H, 20.91% Cl, 41.30% N; found: 35.28% C, 2.44% H, 21.11% Cl, 41.06% N.
2-Chloro-N6-dimethylaminomethyleneadenine (XVIIIa)
A mixture of compound Va (2.0 g, 11.8 mmol), dimethylformamide (30 ml) and dimethylformamide
dineopentyl acetal (6 ml) was stirred at 80 °C to dissolution (calcium chloride protecting tube) and
the stirring was continued at room temperature overnight. The solvent was evaporated at 40 °C/2 kPa
to dryness, the residue was codistilled with toluene (2 × 25 ml) under the same conditions, and mixed
with ethanol (20 ml). Ether (300 ml) was added, the suspension was filtered, washed with ether and
dried in vacuo. Yield 2.0 g (75%) of compound XVIIIa, not melting up to 280 °C. For C8H9ClN6
(224.7) calculated: 42.76% C, 4.04% H, 15.78% Cl, 37.41% N; found: 42.56% C, 4.24% H, 15.42% Cl,
37.38% N.
N2-Dimethylaminomethyleneguanine (XIXa)
A mixture of guanine (3 g, 20 mmol), dimethylformamide (90 ml) and dimethylformamide dineopen-
tyl acetal (15 ml) was stirred at 110 °C (calcium chloride protecting tube) to dissolution (2 h) and
then evaporated at 60 °C/13 Pa. The residue was codistilled with toluene and ethanol (50 ml) was
added together with small pieces of solid CO2 (about 10 g). After standing at room temperature for
30 min, the mixture was evaporated in vacuo and the residue was recrystallized from water; yield 3.5 g
(85%) of compound XIXa not melting up to 280 °C. For C8H10N6O (206.2) calculated: 46.60% C,
Collect. Czech. Chem. Commun. (Vol. 59) (1994)

Biotechnological Preparation of 2’-Deoxyribonucleosides
2321
1
4.89% H, 40.76% N; found: 46.54% C, 4.95% H, 41.02% N. H NMR spectrum: 12.70 brs, 1 H and
11.20 brs, 1 H (NH); 8.55 s, 1 H (H-8); 7.85 s, 1 H (N=CH); 3.00 s, 3 H and 3.10 s, 3 H (2 × CH3).
8-Aza-N6-dimethylaminomethyleneadenine (XXIIIa)
A mixture of 8-azaadenine (XXIIa, 5 g, 36.8 mmol), dimethylformamide (50 ml) and dimethylform-
amide dineopentyl acetal (25 ml) was stirred at 100 °C (calcium chloride tube) for 16 h and then
evaporated at 40 °C/2 kPa. The dry residue was codistilled with toluene (2 × 25 ml) under the same
conditions and then mixed with ethanol (100 ml) under simultaneous addition of solid CO2 to main-
tain neutrality. After 30 min, the product was filtered, washed with ethanol and ether, and dried to
give 5.6 g (79%) of compound XXIIIa, not melting up to 260 °C. The product was homogeneous
according to HPLC in H5 (k 0.80). For C7H9N7 (191.2) calculated: 43.97% C, 4.74% H, 51.28% N;
found: 44.17% C, 4.46% H, 51.80% N.
2′-Deoxyadenosine (Ib)
The catalyst (100 beads) was added to a solution of N6-dimethylaminomethyleneadenine (XVIIa, 76.4 mg,
0.4 mmol) and 2′-deoxyuridine (182.4 mg, 0.8 mmol) in 0.05 M ammonium acetate pH 5.8 (10 ml)
and the mixture was incubated in a Dubnoff′s incubator at 37 °C for 18 h. After decanting, the cata-
lyst was washed with water (10 ml), the combined portions were membrane-filtered (vide supra) and
the filtrate was mixed with the same volume of concentrated aqueous ammonia. The mixture was
heated rapidly to the boil, set aside for 30 min and concentrated in vacuo to about 10 ml. The con-
centrate was filtered through a small layer of Celite and applied onto a column of octadecylsilica gel
(30 µm, 100 ml). After elution with water (2 ml/min) which washed out 2′-deoxyuridine and uracil,
the column was further eluted with 10% aqueous methanol. The fractions containing 2′-deoxyadeno-
sine (TLC, S3) were evaporated and the product was crystallized from ethanol with addition of ether;
yield 44 mg of compound Ib (44% related to the base). The product was identical (HPLC) with an
authentic material.
9-(2-Deoxy-β-D-ribofuranosyl)-2,6-diaminopurine (IVb)
The catalyst (250 beads) was added to a solution of 2′-deoxyuridine (0.925 g, 4.05 mmol) and 2,6-
diaminopurine (IVa, 0.255 g, 1.7 mmol) in 0.05 M ammonium acetate buffer pH 5.8 (50 ml) and the
mixture was incubated in a Dubnoff′s incubator at 37 °C for 7 h. The mixture was decanted and the
catalyst washed with water. After membrane-filtration, the mixture was separated by preparative thin-
layer chromatography in system S5. The product-containing fraction was evaporated and the residue
crystallized from ethanol–ether to give 0.20 g (41%) of 2′-deoxynucleoside IVb, m.p. 147 °C, RF 0.33
(S5). For C10H14N6O3 . H2O (284.1) calculated: 42.26% C, 5.67% H, 29.56% N; found: 42.68% C,
1
5.38% H, 29.58% N. H NMR spectrum: 7.92 s, 1 H (H-8); 6.77 brs, 2 H and 5.76 brs, 2 H (2 × NH2);
6.17 dd, 1 H, J(1′,2a′) = 8.1, J(1′,2b′) = 6.0 (H-1′); 5.28 brs, 1 H and 5.25 brs, 1 H (5′-OH and
3′-OH); 4.35 brdt, 1 H, ΣJ = 10.8 (H-3′); 3.84 td, 1 H, ΣJ = 10.5 (H-4′); 3.59 dd, 1 H, J(5a′,4′) = 4.1,
J(gem) = 12.0 (H-5a′); 3.51 dd, 1 H, J(5b′,4′) = 3.9, J(gem) = 12.0 (H-5b′); 2.58 ddd, 1 H, J(2a′,3′) = 5.6,
J(2a′,1′) = 8.1, J(gem) = 13.2 (H-2a′); 2.18 ddd, 1 H, J(2b′,3′) = 2.7, J(2b′,1′) = 6.0, J(gem) = 13.2
(H-2b′). Mass spectrum, m/z: 267.2 (M + H). UV spectrum, λmax (ε), pH 2: 290.0 (9 800), 253.0 (11 000);
pH 7: 281.0 (12 000), 255.0 (9 300); pH 12: 280.0 (10 700), 256.0 (9 800).
Collect. Czech. Chem. Commun. (Vol. 59) (1994)

2322
Votruba et al.:
9-(2-Deoxy-β-D-ribofuranosyl)-2-chloroadenine (Vb)
A solution of 2-chloro-N6-dimethylaminomethyleneadenine (XVIIIa, 178 mg, 0.75 mmol) in a mix-
ture of ethanol and dimethyl sulfoxide (3 : 2, 10 ml), together with the catalyst (160 beads), was
added to a solution of 2′-deoxyuridine (370 mg, 1.5 mmol) in 0.05 M acetate buffer pH 5.8 (40 ml).
The mixture was incubated at 37 °C for 16 h and worked up as described for 9-(2-deoxy-β-D-ribofu-
ranosyl)-6-methylpurine. Crystallization from ethanol with addition of ether afforded 285 mg (67%)
of product Vb, m.p. 232 °C. For C10H12ClN5O3 (285.7) calculated: 42.04% C, 4.23% H, 12.41% Cl,
1
24.51% N; found: 42.20% C, 4.11% H, 12.32% Cl, 24.47% N. H NMR spectrum: 8.36 s, 1 H (H-8);
7.82 brs, 2 H (NH2); 6.26 dd, 1 H, J(1′,2a′) = 7.3, J(1′,2b′) = 6.3 (H-1′); 5.32 br, 1 H (3′-OH); 4.97 brt,
1 H, J = 4.5 (5′-OH); 4.38 m, 1 H, J(3′,2b′) = J(3′,4′) = 3.2, J(3′,2a′) = 5.9 (H-3′); 3.85 brdt, 1 H,
J(3′,4′) = 2.7, J(4′,5a′) = J(4′,5b′) = 4.6 (H-4′); 3.60 dt, 1 H and 3.51 dt, 1 H, J(5′,4′) = J(5′,OH) = 4.5,
J(gem) = 11.7 (2 × H-5′); 2.64 ddd, 1 H, J(2a′,1′) = 7.5, J(2a′, 3′) = 5.9, J(gem) = 13.2 (H-2a′); 2.28 ddd,
1 H, J(2b′,1′) = 6.1, J(2b′,3′) = 3.2, J(gem) = 13.2, (H-2b′). Mass spectrum, m/z: 286 (M+). UV
spectrum, λmax (ε), pH 2: 265 (14 500); pH 12: 265 (14 800).
9-(2-Deoxy-β-D-ribofuranosyl)-6-methylpurine (VIb)
The catalyst (600 beads, vide supra) was added to a solution of 2′-deoxyuridine (4.56 g, 20 mmol)
and 6-methylpurine (VIa, 1.35 g, 10 mmol) in 0.05 M acetate buffer pH 5.7 (200 ml) and the mixture
was incubated in a Dubnoff′s incubator at 37 °C for 7 h. The supernatant was decanted, the catalyst
washed with water (50 ml) and the combined portions were membrane filtered (Millipore, 0.45 µm).
The filtrate was applied onto a column of octadecylsilica gel (30 µm, 190 ml), preequilibrated with
water. The column was washed with water (2 ml/min) and the UV-absorbing fractions (20 ml) were
analyzed by TLC in system S1. Fractions containing 2′-deoxyuridine were combined, taken down in
vacuo and the residue was crystallized from ethanol with addition of ether to give 1.25 g (27%) of
recovered 2′-deoxyuridine, identical with an authentic material. Further elution with 5% aqueous
methanol and subsequent crystallization from ethanol with addition of light petroleum afforded 1.60 g
(64%) of 9-(2-deoxy-β-D-ribofuranosyl)-6-methylpurine (VIb), m.p. 161 °C. For C11H14N4O3 (250.3)
calculated: 52.79% C, 5.64% H, 22.39% N; found: 52.85% C, 5.56% H, 22.14% N. Mass spectrum,
1
m/z: 250 (M+). H NMR spectrum: 8.77 s, 1 H (H-8); 8.71 s, 1 H (H-2); 6.44 dd, 1 H, J(1′,2a′) = 7.2,
J(1′,2b′) = 6.3 (H-1′); 5.34 d, 1 H, J(3′,OH) = 4.0, (3′-OH); 4.99 t, 1 H, J(5′,OH) = 5.6 (5′-OH); 4.43 m,
1 H (H-3′); 3.88 td, 1 H, J(3′,4′) = 3.0, J(4′,5a′) = J(4′,5b′) = 4.5 (H-4′); 3.61 dq, 1 H, J(5a′,4′) = 4.5,
J(5a′,OH) = 5.4, J(gem) = 12.0 (H-5a′); 3.51 dq, 1 H, J(5b′,4) = 4.5, J(5b′,OH) = 5.4, J(gem) = 12.0,
(H-5b′); 2.77 dq, 1 H, J(2a′,1′) = 7.2, J(2a′,3′) = 5.8, J(gem) = 13.3 (H-2a′); 2.71 s, 3 H (6-CH3);
2.32 dq, 1 H, J(2b′,1′) = 6.3, J(2b′,3′) = 3.5, J(gem) = 13.3 (H-2b′). UV spectrum, λmax (ε), pH 2:
265 (7 000); pH 12: 263 (7 900).
9-(2-Deoxy-β-D-ribofuranosyl)-6-methylthiopurine (IXb)
The catalyst (100 beads, vide supra) was added to a solution of 2′-deoxyuridine (0.37 g, 1.6 mmol)
and 6-methylthiopurine (IXa, 0.12 g, 0.7 mmol) in 0.05 M ammonium acetate buffer pH 5.8 (20 ml)
and the mixture was incubated in a Dubnoff′s incubator at 37 °C for 7 h. The supernatant was de-
canted and the catalyst washed with water. After membrane filtration, the mixture was separated by
preparative thin-layer chromatography in system S2. The product fraction was evaporated and the
residue crystallized from ethanol–ether to give 0.19 g (95%) of 2′-deoxynucleoside IXb, m.p. 159 °C,
RF 0.68 (S2). For C11H14N4O3S (282.2) calculated: 46.81% C, 5.00% H, 19.84% N; found: 46.75% C,
1
5.00% H, 20.18% N. H NMR spectrum: 8.73 s, 1 H and 8.66 s, 1 H (H-2 and H-8); 6.43 brt, 1 H,
ΣJ = 13.4 (H-1′); 5.35 d, 1 H, J(3′,OH) = 4.4 (3′-OH); 5.00 t, 1 H, J = 5.6 (5′-OH); 4.44 m, 1 H,
Collect. Czech. Chem. Commun. (Vol. 59) (1994)

Biotechnological Preparation of 2’-Deoxyribonucleosides
2323
(H-3′); 3.89 td, 1 H, J(4′,3′) = 3.2, J(4′,5a′) = J(4′,5b′) = 4.5 (H-4′); 3.62 dt, 1 H, J(5a′,4′) =
J(5a′,OH) = 5.0, J(gem) = 11.7 (H-5a′); 3.52 ddd, 1 H, J(5b′,4′) = 4.6, J(5b′,OH) = 5.9, J(gem) =
11.7 (H-5b′); 2.76 ddd, 1 H, J(2a′,3′) = 5.9, J(2a′,1′) = 7.1, J(gem) = 13.2 (H-2a′); 2.66 s, 3 H (CH3);
2.34 ddd, 1 H, J(2b′,3′) = 3.2, J(2b′,1′) = 6.3, J(gem) = 13.2 (H-2b′). Mass spectrum, m/z: 283.2 (M + H).
UV spectrum, λmax (ε), pH 2: 291.0 (19 100); pH 7: 291.0 (19 100), pH 12: 291.0 (24 000).
9-(2-Deoxy-β-D-ribofuranosyl)-N6-furfuryladenine (XIIb)
The catalyst (250 beads, vide supra) was added to a solution of 2′-deoxyuridine (0.925 g, 4.05 mmol)
and N6-furfuryladenine (XIIa, 0.4 g, 1.85 mmol) in 0.05 M ammonium acetate buffer pH 5.8 (50 ml)
and the mixture was incubated in a Dubnoff′s incubator at 37 °C for 7 h. The supernatant was de-
canted and the catalyst washed with water. After membrane filtration, the mixture was separated by
preparative thin-layer chromatography in system S2. The product fraction was evaporated and the
residue on precipitation from ethanol with ether afforded 0.35 g (56%) of hygroscopic 2′-deoxy-
nucleoside XIIb, RF 0.72 (S2). 1H NMR spectrum: 8.37 s, 1 H and 8.23 s, 1 H (H-8 and H-2);
8.29 br, 1 H (NH); 7.53 brdd, 1 H, J(5″,4″) = 2.0, J(5″,3″) = 1.0 (H-5″); 6.33 dd, 1 H, J(4″,3″) = 3.5,
J(4″,5″) = 2.0 (H-4″); 6.22 brd, 1 H, J(3″,4″) = 3.5, J(3″,5″) = 1.0 (H-3″); 6.35 dd, 1 H, J(1′,2a′) =
7.8, J(1′,2b′) = 6.1 (H-1′); 5.31 d, 1 H, J(3′,OH) = 4.0 (3′-OH); 5.19 dd, 1 H, J = 4.9 and 5.6 (5′-OH);
4.68 br, 2 H (CH2); 4.41 dq, 1 H, ΣJ = 15.4 (H-3′); 3.88 td, 1 H, J(4′,3′) = 2.5, J(4′,5a′) = J(4′,5b′) =
4.2 (H-4′); 3.62 dt, 1 H, J(5a′,4′) = J(5a′,OH) = 4.6, J(gem) = 11.7 (H-5a′); 3.52 ddd, 1 H, J(5b′,4′) =
4.2, J(5b′,OH) = 6.6, J(gem) = 11.7 (H-5b′); 2.73 ddd, 1 H, J(2a′,3′) = 5.9, J(2a′,1′) = 7.8, J(gem) =
13.2 (H-2a′); 2.26 ddd, 1 H, J(2b′,3′) = 2.9, J(2b′,1′) = 6.1, J(gem) = 13.2 (H-2b′). C15H17N5O4
(331.3): mass spectrum, m/z: 332.1 (M + H). UV spectrum, λmax (ε), pH 2: 268.0 (21 700); pH 7:
267.0 (23 400), pH 12: 268.0 (8 200). Spectrophotometrically determined content: >90%.
2′-Deoxyinosine (XIVb)
The incubation mixture contained hypoxanthine (204 mg, 1.5 mmol), 2′-deoxyuridine (684 mg, 3 mmol)
and 400 beads of the catalyst in 0.05 M ammonium acetate buffer pH 5.8 (30 ml). After incubation
at 37 °C for 16 h, the supernatant was decanted, the catalyst washed with water (10 ml) and the
combined portions membrane filtered. After concentration in vacuo to about 10 ml, the mixture was
separated by preparative HPLC, elution with a gradient water–methanol. The product fraction was
evaporated in vacuo and the residue crystallized from ethanol–ether; yield 0.25 g (65%) of product
XIVb, homogeneous according to HPLC, TLC and identical with an authentic sample.
2′-Deoxyguanosine (XVb)
A solution of N2-dimethylaminomethylene-2′-deoxyguanosine (XIXb, 100 mg, 0.3 mmol) in a mixture
of water (5 ml) and concentrated aqueous ammonia (2 ml) was allowed to stand at room temperature
overnight and then taken down in vacuo, affording 2′-deoxyguanosine (XVb) in quantitative yield.
The product was identical with an authentic sample according to paper chromatography in system S6
and to HPLC in system H4.
N2-Dimethylaminomethylene-2′-deoxyguanosine (XIXb)
The title compound was prepared from N2-dimethylaminomethyleneguanine (XIXa, 207 mg, 1.0 mmol)
and 2′-deoxyuridine (456 mg, 2.0 mmol) in 0.05 M ammonium acetate buffer pH 5.8 (20 ml) in the
same manner as described for 2′-deoxyadenosine (incubation for 30 h). After membrane filtration, the
filtrate was applied onto a column of octadecylsilica gel (vide supra). Washing with water separated
Collect. Czech. Chem. Commun. (Vol. 59) (1994)

2324
Votruba et al.:
uracil and 2′-deoxyuridine, 5% aqueous methanol eluted N2-dimethylaminomethyleneguanine. Elution
with 10% aqueous methanol afforded the deoxynucleoside. The product fractions were combined, the
solvent evaporated in vacuo and the residue precipitated with ether from methanolic solution. Yield
161 mg (50%) of the title compound XIXb, m.p. 154 °C. For C13H18N6O4 (322.3) calculated: 48.44% C,
5.63% H, 26.08% N; found: 48.27% C, 5.46% H, 26.18% N. Mass spectrum, m/z: 322 (M+).
1H NMR spectrum: 8.62 s, 1 H (H-8); 8.35 s, 1 H (N=CH); 6.52 t, 1 H, J = 6.7 (H-1′); 4.32 m, 1 H
(H-3′); 3.84 m, 1 H (H-4′); 3.55 m, 2 H (2 × H-5′); 3.15 s, 3 H and 3.02 s, 3 H (2 × NCH3).
UV spectrum, λmax (ε), pH 7: 235 (17 300), 290 (23 700).
8-Aza-2′-deoxyadenosine (XXIIb)
The reaction was performed with 8-aza-N6-dimethylaminomethyleneadenine (XXIIIa, 354 mg, 1.85 mmol)
and 2′-deoxyuridine (844 mg, 3.7 mmol) in 0.05 M ammonium acetate buffer pH 5.8 (40 ml) in the
same manner as described for 2′-deoxyadenosine (Ib) (incubation at 37°C for 16 h). After ammono-
lysis, the mixture was evaporated in vacuo and the product was isolated by preparative HPLC in a
gradient methanol–water. The product fraction was evaporated and the residue crystallized from
ethanol–ether; yield 120 mg (26% related to the base) of 8-aza-2′-adenosine (XXIIb), m.p. 196 – 199 °C.
For C9H12N6O3 (252.2) calculated: 42.86% C, 4.80% H, 33.32% N; found: 42.57% C, 4.60% H,
1
33.50% N. H NMR spectrum: 8.30 s, 1 H (H-2); 8.15 brs, 1 H and 8.30 brs, 1 H (NH2); 6.61 t, 1 H,
J(1′,2a′) = J(1′,2b′) = 6.35 (H-1′); 5.38 brs, 1 H (3′-OH); 4.89 brt, 1 H, J = 5.0 (5′-OH); 4.54 m, 1 H, (H-3′);
3.90 brq, 1 H, ΣJ = 14.4, J(3′,4′) = 3.4 (H-4′); 3.57 dd, 1 H, J(5a′,4′) = 5.1, J(gem) = 11.7 (H-5a′); 3.40 dd,
1 H, J(5b′,4′) = 5.9, J(gem) = 11.7 (H-5b′); 3.03 dt, 1 H, J(2a′,1′) = J(2a′,3′) = 6.1, J(gem) = 13.4 (H-2a′);
2.41 ddd, 1 H, J(2b′,3′) = 4.4, J(2b′,1′) = 6.8, J(gem) = 13.4 (H-2b′). 13C NMR spectrum: 157.14
(C-2); 156.42 (C-6); 148.97 (C-4); 124.50 (C-5); 88.66 (C-4′); 85.80 (C-1′); 71.09 (C-3′); 62.25 (C-5′);
38.32 (C-2′). UV spectrum, λmax (ε), pH 2: 278 (10 600).
9-(2-Deoxy-β-D-ribofuranosyl)-8-azaguanine (XXVIb)
The catalyst (250 beads, vide supra) was added to a solution of 2′-deoxyuridine (0.925 g, 4.05 mmol)
and 8-aza-N2-dimethylaminomethyleneguanine (XXVIIa, 0.275 g, 1.3 mmol) in 0.05 M ammonium
acetate buffer pH 5.8 (46 ml) and dimethyl sulfoxide (4 ml) and the mixture was incubated in a
Dubnoff′s incubator at 37 °C for 7 h. The supernatant was decanted and the catalyst washed with
water. The mixture was worked up as described for 2′-deoxyadenosine (Ib). After membrane-filtra-
tion, the mixture was separated by preparative thin-layer chromatography in system S5. The product
fraction was evaporated and the residue crystallized from ethanol–ether to give 0.05 g (15%) of
1
2′-deoxynucleoside XXVIb, not melting up to 280 °C, RF 0.49 (S5). H NMR spectrum: 10.25 br, 1 H
(NH); 6.95 br, 2 H (NH2); 6.29 t, 1 H, J = 6.4 (H-1′); 4.75 t, 1 H, J = 5.5 (5′-OH); 4.46 brpent, 1 H,
ΣJ = 17.6 (H-3′); 3.83 td, 1 H, J(4′, 3′) = 3.9, J(4′,5a′) = J(4′,5b′) = 5.8 (H-4′); 5.32 d, 1 H, J(3′,OH) = 4.4
(3′-OH); 3.50 dt, 1 H, J(5a′,4′) = J(5a′,OH) = 5.4, J(gem) = 11.7 (H-5a′); 3.37 dt, 1 H, J(5b′,4′) =
4.2, J(5b′,OH) = 6.1, J(gem) = 11.7 (H-5b′); 2.89 ddd, 1 H, J(2a′,3′) = J(2a′,1′) = 6.1, J(gem) = 13.2
(H-2a′); 2.32 ddd, 1 H, J(2b′, 3′) = 4.6, J(2b′,1′) = 6.7, J(gem) = 13.2 (H-2b′). 13C NMR spectrum:
155.91 (C-6); 155.86 (C-2); 151.84 (C-4); 124.84 (C-5); 88.47 (C-4′); 84.32 (C-1′); 71.13 (C-3′);
62.42 (C-5′); 38.27 (C-2′). C9H12N6O4 (268.2): mass spectrum, m/z: 269.1 (M + H). UV spectrum,
λmax (ε), pH 2: 255.0 (10 500); pH 7: 255.0 (10 600), pH 12: 279.0 (9 600). Spectrophotometrically
determined content: >90%.
Collect. Czech. Chem. Commun. (Vol. 59) (1994)

Biotechnological Preparation of 2’-Deoxyribonucleosides
9-(2-Deoxy-β-D-ribofuranosyl)-1-deazapurine (XXVIIIb)
2325
The catalyst (135 beads, vide supra) was added to a solution of 2′-deoxyuridine (0.8 g, 3.4 mmol)
and 1-deazapurine (XXVIIIa, 0.2 g, 1.7 mmol) in 0.05 M ammonium acetate buffer pH 5.8 (40 ml)
and the mixture was incubated in a Dubnoff′s incubator at 37 °C for 7 h. The supernatant was de-
canted and the catalyst washed with water. After membrane-filtration, the mixture was separated by
preparative HPLC, elution with a gradient methanol–water. Upon mixing with dry ether, the product
crystallized. Yield 0.20 g (51%) of deoxyribonucleoside XXVIIIb, m.p. 139 – 141 °C. For
C11H13N3O3 (235.2) calculated: 56.17% C, 5.57% H, 17.86% N; found: 55.81% C, 5.39% H, 17.77% N.
1
HPLC (H5, k = 6.54). H NMR spectrum: 8.69 s, 1 H (H-8), 8.38 dd, 1 H, J(2,6) = 1.4, J(2,1) = 4.8
(H-2); 8.13 dd, 1 H, J(6,1) = 8.1, J(6,2) = 1.4 (H-6); 7.33 dd, 1 H, J(1,2) = 4.8, J(1,6) = 8.1 (H-1);
6.54 dd, 1 H, J(1′,2a′) = 7.6, J(1′,2b′) = 6.3 (H-1′); 5.32 d, 1 H, J(OH,3′) = 4.2 (3′-OH); 5.08 t, 1 H,
J(OH,5′) = 5.6 (5′-OH); 4.47 m, 1 H (H-3′); 3.92 dt, 1 H, J(4′,3′) = 2.7, J(4′,5a′) = J(4′,5b′) = 4.5
(H-4′); 3.62 m, 2 H (H-5′); 2.81 ddd, 1 H, J(2a′,3′) = 5.8, J(2a′,1′) = 7.6, J(gem) = 15.4 (H-2a′); 2.32 ddd,
1 H, J(2b′,3′) = 3.2, J(2b′,1′) = 6.3, J(gem) = 15.4 (H-2b′). 13C NMR spectrum: 146.80 (C-4); 144.40
(C-8); 144.39 (C-2); 136.90 (C-5); 128.36 (C-6); 119.20 (C-1); 88.54 (C-4′); 84.65 (C-1′); 71.57 (C-3′);
62.43 (C-5′); 40.08 (C-2′). UV spectrum, λmax (ε), pH 2: 280.9 (8 600), 274.8 (9 600), 235.8 (4 800);
pH 12: 280.8 (8 500), 242.8 (5 200).
9-(2-Deoxy-β-D-ribofuranosyl)-1-deazaadenine (XXIXb)
The catalyst (135 beads, vide supra) was added to a solution of 2′-deoxyuridine (0.8 g, 3.4 mmol)
and 1-deazaadenine (XXIXa, 0.23 g, 1.7 mmol) in 0.05 M ammonium acetate buffer pH 5.8 (40 ml)
and the mixture was incubated in a Dubnoff′s incubator at 37 °C for 7 h. The supernatant was de-
canted and the catalyst washed with water. After membrane-filtration, the mixture was separated by
preparative HPLC, elution with a gradient methanol–water. Crystallization from ethanol–ether–light
petroleum afforded 0.1 g (27%) of product XXIXb, m.p. 224 – 226 °C. For C11H14N4O3 (250.2) cal-
culated: 52.80% C, 5.64% H, 22.38% N; found: 52.57% C, 5.60% H, 22.50% N. HPLC (H5, k = 5.48).
1H NMR spectrum: 8.26 s, 1 H (H-8); 7.78 d, 1 H, J(1,2) = 5.6 (H-2); 6.44 brs, 2 H (NH2); 6.38 d,
1 H, J(2,1) = 5.6 (H-1); 6.38 dd, 1 H, J(1′,2a′) = 8.4, J(1′,2b′) = 5.9 (H-1′); 5.77 dd, 1 H, J = 4.3
and 7.5 (5′-OH); 5.28 d, 1 H, J(OH,3′) = 3.7 (3′-OH); 3.91 brq, 1 H, ΣJ = 9.2 (H-4′); 3.64 dt, 1 H,
ΣJ = 20.0 (H-5a′); 3.53 m, 1 H (H-5b′); 2.79 ddd, 1 H, J(2a′,1′) = 8.4, J(2a′,3′) = 5.5, J(gem) = 13.4
(H-2a′); 2.20 ddd, 1 H, J(2b′,1′) = 5.9, J(2b′,3′) = 2.5, J(gem) = 13.4 (H-2b′). UV spectrum: λmax (ε),
pH 2: 280.6 (17 100), 263.0 (12 400); pH 12: 261.8 (13 800), 275.0 (10 100).
9-(2-Deoxy-β-D-ribofuranosyl)-6-nitro-1-deazapurine (XXXb)
The catalyst (135 beads, vide supra) was added to a solution of 2′-deoxyuridine (0.8 g, 3.4 mmol)
and 6-nitro-1-deazapurine (XXXa, 0.268 g, 1.6 mmol) in 0.05 M ammonium acetate buffer pH 5.8 (40 ml)
and the mixture was incubated in a Dubnoff′s incubator at 37 °C for 7 h. The supernatant was de-
canted and the catalyst washed with water. After membrane-filtration, the mixture was separated by
preparative HPLC, elution with a gradient methanol–water. Standing with dry ether in a refrigerator
afforded 0.13 g (27%) of yellow crystalline product XXXb, m.p. 122 – 124 °C. For C11H12N4O5 . H2O
(298.2) calculated: 44.30% C, 4.73% H, 18.78% N; found: 44.05% C, 4.50% H, 18.81% N. HPLC
1
(H5, k = 9.01). H NMR spectrum: 9.01 s, 1 H (H-8); 8.67 d, 1 H, J(1,2) = 5.4 (H-2); 8.01 d, 1 H,
J(2,1) = 5.4 (H-1); 6.58 t, 1 H, J = 6.6 (H-1′); 5.35 brd, 1 H and 4.97 brt, 1 H, J = 4.0 and 5.0
(3′-OH and 5′-OH); 4.44 m, 1 H (H-3′); 3.91 dt, 1H (H-4′); 3.63 dt, 1 H and 3.53 dt, 1 H (H-5a′ and
H-5b′); 2.78 brpent, 1 H, J(2a′,1′) = 6.8, J(2a′,3′) = 6.2, J(gem) = 13.4 (H-2a′); 2.40 ddd, 1 H, J(2b′,1′)
Collect. Czech. Chem. Commun. (Vol. 59) (1994)

2326
Votruba et al.:
= 6.4, J(2b′,3′) = 3.8, J(gem) = 13.4 (H-2b′). UV spectrum, λmax (ε), pH 2: 296.9 (5 600); pH 12:
322.4 (4 900).
9-(2-Deoxy-β-D-ribofuranosyl)-6-azido-3-deazapurine (XXXIIIb)
The catalyst (135 beads, vide supra) was added to a solution of 2′-deoxyuridine (0.43 g, 1.88 mmol)
and 6-azido-3-deazapurine (XXXIIIa, 0.150 g, 0.94 mmol) in 0.05 M ammonium acetate buffer pH 5.8
(40 ml) and the mixture was incubated in a Dubnoff′s incubator at 37 °C for 7 h. The supernatant
was decanted and the catalyst washed with water. After membrane-filtration, the mixture was separated
by preparative HPLC, elution with a gradient methanol–water. The product was crystallized from
ethanol–ether–light petroleum; yield 0.09 g (35%) of white crystals of compound XXXIIIb, m.p.
177 – 178 °C. HPLC (H5, k = 6.68). For C11H12N6O3 (276.2) calculated: 47.83% C, 4.38% H, 30.42% N;
1
found: 47.92% C, 4.45% H, 30.56% N. H NMR spectrum: 9.10 d, 1 H, J(2,3) = 7.3 (H-2); 8.87 s,
1 H (H-8); 7.76 d, 1 H, J(3,2) = 7.3 (H-3); 6.84 t, 1 H, J = 6.6 (H-1′); 5.45 d, 1 H and 5.08 t, 1 H,
J = 4.5 and 5.5 (3′-OH and 5′-OH); 4.47 m, 1 H (H-3′); 3.97 q, 1 H, ΣJ = 12.0 (H-4′); 3.69 dt, 1 H and
3.58 dt, 1 H (H-5a′ and H-5b′); 2.76 ddd, 1 H, J(2a′,1′) = 7.2, J(2a′,3′) = 6.0, J(gem) = 13.2 (H-2a′);
2.50 ddd, 1 H, J(2b′,1′) = 6.0, J(2b′,3′) = 3.5, J(gem) = 13.2 (H-2b′). Mass spectrum, m/z: 276 (M + H).
UV spectrum, λmax (ε), pH 2: 263.2 (4 500); pH 12: 263.5 (4 400).
9-(2-Deoxy-β-D-ribofuranosyl)-3-deazaguanine (XXXVIb)
The catalyst (250 beads, vide supra) was added to a solution of 2′-deoxyuridine (0.925 g, 4.05 mmol)
and 3-deazaguanine (XXXVIa, 0.250 g, 1.66 mmol) in 0.05 M ammonium acetate buffer pH 5.8 (50 ml)
and the mixture was incubated in a Dubnoff′s incubator at 37 °C for 7 h. The supernatant was de-
canted and the catalyst washed with water. After membrane-filtration, the mixture was separated by
preparative thin-layer chromatography in system S5. The product fraction was evaporated and the
residue crystallized from ethanol–ether to give 0.06 g (13%) of 2′-deoxynucleoside XXXVIb, m.p.
165 °C (decomp.), k = 1.79 (H4). For C11H14N4O4 . H2O (284.2) calculated: 46.48% C, 5.67% H,
1
19.70% N; found: 46.07% C, 5.26% H, 19.31% N. H NMR spectrum: 10.30 br, 1 H (NH); 7.86 s,
1 H (H-8); 5.94 dd, 1 H, J = 6.1 and 7.3 (H-1′); 5.59 brs, 2 H (NH2); 5.43 s, 1 H (H-3); 5.32 brs, 1 H
and 4.91 brs, 1 H (5′-OH and 3′-OH); 4.37 dt, 1 H, J(3′,2b′) = J(3′,4′) = 3.2, J(3′,2a′) = 6.1 (H-3′);
3.80 td, 1 H, J(4′,3′) = 3.2, J(4′,5a′) = J(4′,5b′) = 4.7 (H-4′); 3.52 dd, 1 H, J(5a′,4′) = 4.7, J(gem) =
11.7 (H-5a′); 3.47 dd, 1 H, J(5b′,4′) = 4.7, J(gem) = 11.7 (H-5b′); 2.44 ddd, 1 H, J(2a′,3′) = 6.1,
J(2a′,1′) = 7.3, J(gem) = 13.2 (H-2a′); 2.21 ddd, 1 H, J(2b′,3′) = 3.2, J(2b′,1′) = 6.1, J(gem) = 13.2
(H-2b′). Mass spectrum, m/z: 267.1 (M + H). UV spectrum, λmax (ε), pH 2: 281.0 (10 800); pH 7:
300.0 (8 200), 270.0 (10 600); pH 12: 276.0 (10 500).
9-(2-Deoxy-β-D-ribofuranosyl)-2-azahypoxanthine (XXXVIIIb) and
7-(2-Deoxy-β-D-ribofuranosyl)-2-azahypoxanthine (XXXIXb)
The catalyst (250 beads, vide supra) was added to a solution of 2′-deoxyuridine (0.925 g, 4.0 mmol)
and 2-azahypoxanthine (XXXVIIIa, 0.255 g, 1.9 mmol) in 0.05 M ammonium acetate buffer pH 5.8
(50 ml) and the mixture was incubated in a Dubnoff′s incubator at 37 °C for 7 h. The supernatant
was decanted and the catalyst washed with water. After membrane-filtration, the mixture was separated
by preparative HPLC, elution with a gradient methanol–water. The product was crystallized from
ethanol–ether; yield 0.38 g (79%) of a crystalline mixture of 7- and 9-isomers XXXVIIIb and XXXIXb
in the ratio 1 : 1 (NMR); repeated separation of this mixture by HPLC under the same conditions
1
afforded samples of both isomers for identification. XXXVIIIb: H NMR spectrum: 8.53 s, 1 H (H-8);
6.47 t, 1 H, J = 6.6 (H-1′); 5.30 br, 2 H (OH); 4.44 dt, 1 H, J(3′,2b′) = J(3′,4′) = 3.5, J(3′,2a′) = 6.1
Collect. Czech. Chem. Commun. (Vol. 59) (1994)

Biotechnological Preparation of 2’-Deoxyribonucleosides
2327
(H-3′); 3.92 td, 1 H, ΣJ = 11.2 (H-4′); 3.63 dd, 1 H, J(5a′,4′) = 4.1, J(gem) = 12.0 (H-5a′); 3.54 dd,
1 H, J(5b′,4′) = 4.4, J(gem) = 12.0 (H-5b′); 2.74 ddd, 1 H, J(2a′,1′) = 7.1, J(2a′,3′) = 6.1, J(gem) =
13.2 (H-2a′); 2.38 ddd, 1 H, J(2b′,1′) = 6.3, J(2b′,3′) = 3.7, J(gem) = 13.2, (H-2b′). 13C NMR spec-
trum: 157.50 (C-6); 145.48 (C-4); 141.48 (C-8); 125.37 (C-5); 88.50 (C-4′); 85.08 (C-1′); 70.75 (C-3′);
1
61.71 (C-5′); 40.14 (C-2′). XXXIXb: H NMR spectrum: 8.72 s, 1 H (H-8); 6.58 t, 1 H, J = 6.6 (H-1′);
5.60 br, 2 H (OH); 4.39 dt, 1 H, J(3′,2b′) = J(3′,4′) = 3.4, J(3′,2a′) = 5.9 (H-3′); 3.90 brq, 1 H, ΣJ =
11.2 (H-4′); 3.63 dd, 1 H, J(5a′,4′) = 4.2, J(gem) = 12.0 (H-5a′); 3.56 dd, 1 H, J(5b′,4′) = 4.2, J(gem)
= 12.0 (H-5b′); 2.57 pent, 1 H, J(2a′,1′) = J(2a′,3′) = 6.5, J(gem) = 13.2 (H-2a′); 2.38 ddd, 1 H,
J(2b′,1′) = 6.1, J(2b′,3′) = 3.7, J(gem) = 13.2 (H-2b′). 13C NMR spectrum: 154.90 (C-4); 154.20 (C-6);
142.75 (C-8); 115.37 (C-5); 88.47 (C-4′); 86.64 (C-1′); 70.30 (C-3′); 61.38 (C-5′); 41.81 (C-2′).
9-(2-Deoxy-β-D-ribofuranosyl)-1,N6-etheno-2-azaadenine (XLVIb) and
7-(2-Deoxy-β-D-ribofuranosyl)-1,N6-etheno-2-azaadenine (XLVIIb)
The catalyst (250 beads, vide supra) was added to a solution of 2′-deoxyuridine (0.925 g, 4.0 mmol)
and 1,N6-etheno-2-azaadenine (XLVIa, 0.305 g, 1.9 mmol) in 0.05 M ammonium acetate buffer pH 5.8
(50 ml) and the mixture was incubated in a Dubnoff′s incubator at 37 °C for 7 h. The supernatant
was decanted and the catalyst washed with water. After membrane-filtration, the mixture was separated
by preparative HPLC, elution with a gradient methanol–water. The product was crystallized from
ethanol–ether; yield 0.430 g (82%) of unseparable crystalline mixture of both isomers in the ratio
1
2 : 9 (NMR). XLVIIb: H NMR spectrum: 8.89 s, 1 H (H-8); 8.81 d, 1 H, J(11,10) = 1.7 (H-11); 7.85 d,
1 H, J(10,11) = 1.7 (H-10); 6.70 t, 1 H, J = 6.3 (H-1′); other signals not assigned. 13C NMR spectrum:
145.21 (C-4); 133.39 (C-10); 129.26 (C-6); 118.95 (C-5); 115.45 (C-11); 88.80 (C-4′); 70.46 (C-3′);
1
61.40 (C-5′); 40.98 (C-2′). XLVIb: H NMR spectrum: 8.96 s, 1 H (H-8); 8.76 d, 1 H, J(11,10) = 1.7
(H-11); 7.85 d, 1 H, J(10,11) = 1.7 (H-10); 6.67 t, 1 H, J = 6.3 (H-1′); 5.40 br, 2 H (OH); 4.49 brdt, 1 H,
J(3′,4′) = 3.5, (H-3′); 3.95 brq, 1 H, ΣJ = 12.7 (H-4′); 3.66 dd, 1 H, J(5a′,4′) = 4.6, J(gem) = 11.7
(H-5a′); 3.57 dd, 1 H, J(5b′,4′) = 4.6, J(gem) = 11.7 (H-5b′); 2.84 dt, 1 H, J(2a′,1′) = 6.1, J(2a′,3′) = 6.3,
J(gem) = 13.4 (H-2a′); 2.51 ddd, 1 H, J(2b′,1′) = 6.6, J(2b′,3′) = 4.4, J(gem) = 13.4 (H-2b′). 13C NMR
spectrum: 137.79 (C-4); 133.66 (C-10); 133.27 (C-6); 126.55 (C-5); 115.45 (C-11); 88.46 (C-4′);
85.28 (C-1′); 70.36 (C-3′), 61.38 (C-5′); 40.16 (C-2′).
8-(2-Deoxy-β-D-ribofuranosyl)-8-aza-2,6-diaminopurine (XLVIIIb)
The catalyst (250 beads, vide supra) was added to a solution of 2′-deoxyuridine (0.925 g, 4.05 mmol)
and 8-aza-2,6-diaminopurine (XLVIIIa, 0.280 g, 1.8 mmol) in 0.05 M ammonium acetate buffer pH 5.8
(50 ml) and the mixture was incubated in a Dubnoff′s incubator at 37 °C for 7 h. The supernatant
was decanted and the catalyst washed with water. After membrane-filtration, the mixture was separated
by preparative thin-layer chromatography in system S5. The product fraction was evaporated and the
residue crystallized from ethanol–ether; yield 0.03 g (6%) of 2′-deoxynucleoside XLVIIIb, not melting
1
up to 280 °C, RF 0.31 (S5). H NMR spectrum: 7.70 br, 1 H and 7.50 br, 1 H (NH2); 6.13 brs, 2 H
(NH2); 6.31 dd, 1 H, J(1′,2a′) = 4.6, J(1′,2b′) = 6.8 (H-1′); 5.40 br, 1 H and 4.78 br, 1 H (5′-OH and
3′-OH); 4.49 brq, 1 H, ΣJ = 15.6 (H-3′); 3.87 brq, 1 H, ΣJ = 15.6 (H-4′); 3.52 dd, 1 H, J(5a′,4′) = 5.1,
J(gem) = 11.7 (H-5a′); 3.38 dd, 1 H, J(5b′,4′) = 6.3, J(gem) = 11.7 (H-5b′); 2.80 ddd, 1 H, J(2a′,3′) = 6.3,
J(2a′,1′) = 4.6, J(gem) = 13.4 (H-2a′); 2.38 ddd, 1 H, J(2b′,3′) = 5.1, J(2b′,1′) = 6.8, J(gem) = 13.4
(H-2b′). 13C NMR spectrum: 162.80 (C-2); 159.92 (C-4); 157.06 (C-6); 123.06 (C-5); 93.09 (C-1′);
88.85 (C-4′); 71.05 (C-3′); 62.49 (C-5′); 39.52 (C-2′). C9H13N7O3 (267.2): mass spectrum, m/z: 268.2
(M + H). UV spectrum, λmax (ε), pH 2: 280.0 (8 000), 263.0 (8 800); pH 7: 309.0 (5 000), 259.0 (3 900);
pH 12: 311.0 (5 200), 258.0 (4 100). Spectrophotometrically determined content: >90%.
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Votruba et al.:
Thymidine (XLIXb)
The incubation mixture contained 2′-deoxyuridine (228 mg, 1 mmol), thymine (63 mg, 0.5 mmol),
100 catalyst beads and 50 ml of 0.05 M ammonium acetate pH 5.8. After incubation at 37 °C for 16 h,
the supernatant was decanted, the catalyst washed with water (5 ml) and the combined filtrates were
membrane-filtered. The filtrate was concentrated in vacuo to about 5 ml, filtered through a layer of
Celite and applied onto a column of octadecylsilica gel (180 ml). Elution with water (2 ml/min, fractions
20 ml, followed by TLC in S1) washed out successively uracil, 2′-deoxyuridine (100 mg, 46%) and
thymidine (XLIXb, 70 mg, 61%, after crystallization from ethanol–ether), m.p. 125 °C. The product
was identical with an authentic material (HPLC, TLC (S1) and paper chromatography (S6)).
5-Ethyl-2′-deoxyuridine (Lb)
The incubation mixture contained 2′-deoxyuridine (2.28 g, 10 mmol), 5-ethyluracil (La, 0.70 g, 5 mmol),
about 1 000 beads of the catalyst and 0.05 M ammonium acetate pH 5.8 (500 ml). After 18 h of
incubation at 37 °C, the supernatant was decanted, the catalyst washed with water (50 ml) and the
combined filtrates were membrane-filtered. The filtrate was concentrated in vacuo to about 50 ml,
filtered through a layer of Celite and applied onto a column of octadecylsilica gel (180 ml). Elution
with water (2 ml/min, fractions 20 ml, monitored by TLC in S4) washed out uracil and then
2′-deoxyuridine (1.7 g, 75%). Elution with 5% aqueous methanol afforded 0.87 g (68%) of 5-ethyl-
2′-deoxyuridine (Lb), m.p. 156 °C (ethanol–ether). For C11H16N2O5 (256.2) calculated: 51.56% C,
6.29% H, 10.93% N; found: 52.12% C, 5.72% H, 11.15% N. UV spectrum, λmax (ε), pH 2: 268 (9 600).
2′-Deoxy-5-fluorouridine (LVIb)
The incubation mixture contained 5-fluorouracil (LVIa, 65 mg, 0.5 mmol), thymidine (245 mg, 1 mmol),
100 beads of the catalyst and 0.05 M ammonium acetate pH 5.8 (50 ml). After incubation at 37 °C
for 16 h, the supernatant was decanted, the catalyst washed with water (25 ml) and the combined
filtrates were concentrated in vacuo at 30 °C to about 20 ml. The mixture was filtered through Celite
and the filtrate was made alkaline with ammonia (pH 9). This solution was applied onto a column of
−
Sephadex A-25 (HCO3 form, 200 ml), preequilibrated with 0.01 M triethylammonium hydrogen car-
bonate pH 7.5. The column was washed with the same buffer (2 ml/min) to drop of UV absorption
(thymine and thymidine). Subsequent elution with 0.1 M triethylammonium hydrogen carbonate
afforded a mixture of 5-fluorouracil and the product. After evaporation of solvent in vacuo, the
residue was applied onto a column of octadecylsilica gel (100 ml). Elution with water, evaporation
of the product fractions (monitored by TLC in S1) and crystallization from ethanol with addition of
ether afforded 75 mg (62%) of 2′-deoxy-5-fluorouridine (LVIb), identical with an authentic material
(HPLC, TLC in S4, paper chromatography in S6 and paper electrophoresis (mobility 0.56 related to
uridine-3′-phosphate)), m.p. 150 – 151 °C (reported31 m.p. 150 °C). UV spectrum, λmax, pH 2: 269.
9-(2,3-Dideoxy-β-D-ribofuranosyl)-6-methylpurine (VIc)
The catalyst (420 beads, vide supra) was added to a solution of 2′,3′-dideoxyuridine (0.6 g, 2.8 mmol)
and 6-methylpurine (VIa, 0.2 g, 1.3 mmol) in 0.05 M ammonium acetate buffer pH 5.8 (80 ml) and
the mixture was incubated in a Dubnoff′s incubator at 37 °C for 7 h. The supernatant was decanted
and the catalyst washed with water. After membrane-filtration, the mixture was separated by
preparative thin-layer chromatography in system S2. The product fraction was evaporated and the
residue crystallized from ethanol–ether; yield 0.06 g (20%) of 2′,3′-dideoxynucleoside VIc, m.p. 107 °C,
RF 0.70 (S2). For C11H14N4O2 (234.2) calculated: 56.40% C, 6.03% H, 23.91% N; found: 56.60% C,
1
6.14% H, 23.52% N. H NMR spectrum: 8.77 s, 1 H and 8.74 s, 1 H (H-2 and H-8); 6.34 dd, 1 H,
Collect. Czech. Chem. Commun. (Vol. 59) (1994)

Biotechnological Preparation of 2’-Deoxyribonucleosides
2329
J(1′,2a′) = 6.1, J(1′,2b′) = 3.9 (H-1′); 4.97 t, 1 H, J = 5.5 (5′-OH); 4.14 m, 1 H, (H-4′); 3.63 ddd, 1 H,
J(5a′,4′) = 4.1, J(5a′,OH) = 5.6, J(gem) = 11.7 (H-5a′); 3.51 dt, 1 H, J(5b′,4′) = J(5b′,OH) = 5.0,
J(gem) = 11.7 (H-5b′); 2.71 s, 3 H (CH3); 2.48 m, 2 H (2 × H-2′); 2.01 – 2.14 m, 2 H (2 × H-3′).
Mass spectrum, m/z: 235.1 (M + H). UV spectrum, λmax (ε), pH 2: 263.0 (7 500); pH 7: 261.0 (7 800),
pH 12: 261.0 (8 400).
2′,3′-Dideoxyinosine (XIVc)
The title compound was prepared from 2′,3′-dideoxyuridine and hypoxanthine in the yield of 18%;
m.p. 188 – 189 °C. For C10H12N4O3 (236.2) calculated: 50.84% C, 5.12% H, 23.72% N; found:
1
51.01% C, 5.04% H, 23.88% N. H NMR spectrum: 12.35 brs, 1 H (NH); 8.33 s, 1 H and 8.07 s, 1 H
(H-2 and H-8); 6.20 dd, 1 H, J(1′,2′) = 6.8 (H-1′); 5.00 br, 1 H (5′-OH); 4.10 m, 1 H, J(1′,2′) = 3.4
and 6.8 (H-4′); 3.62 dd, 1 H, J(5a′,4′) = 3.9, J(gem) = 11.7 (H-5a′); 3.51 dd, 1 H, J(5b′,4′) = 4.6, J(gem)
= 11.7 (H-5b′); 2.44 m, 1 H, ΣJ = 38.1 (H-2a′); 2.33 m, 1 H, ΣJ = 28.3 (H-2b′).
The authors are indebted to Mrs M. Matejkova, Mrs H. Miklova, and Mrs B. Novakova for the
excellent technical assistance, and to Dr. J. Kohoutova of this Institute for the mass spectroscopic
measurements. This study was sponsored by the Grant Agency of the Academy of Sciences of the Czech
Republic (Grant No. 45572).
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Translated by M. Tichy.
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