Regioselective Mitsunobu Reaction of Partially Protected Uridine

Article abstract of DOI:10.1080/15257770.2016.1188943

Mitsunobu reaction of partially acylated uridine proceeds with high regioselectivity for intramolecular S_N2 anhydro linkage closuring. Under the reaction conditions, an isomeric mixture of diacyl uridine derivatives with either free 2′- or 3′-hydroxyl group was transformed into a single cyclonucleosidic product, 2,2′-anhydro-3′,5′-di-O-acyluridine. This paper presents a possible mechanism of the reactions, the explanation of observed phenomenon based on semiempirical and density functional theory (DFT) calculations and possible utility of this synthetic pathway.

Full text of DOI:10.1080/15257770.2016.1188943

Nucleosides, Nucleotides and Nucleic Acids
ISSN: 1525-7770 (Print) 1532-2335 (Online) Journal homepage: http://www.tandfonline.com/loi/lncn20
Regioselective Mitsunobu Reaction of Partially
Protected Uridine
Maurycy Szlenkier, Karol Kamel & Jerzy Boryski
To cite this article: Maurycy Szlenkier, Karol Kamel & Jerzy Boryski (2016): Regioselective
Mitsunobu Reaction of Partially Protected Uridine, Nucleosides, Nucleotides and Nucleic Acids,
DOI: 10.1080/15257770.2016.1188943
To link to this article: http://dx.doi.org/10.1080/15257770.2016.1188943
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Date: 29 June 2016, At: 08:10

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
, VOL. , NO. , –
http://dx.doi.org/./..
Regioselective Mitsunobu Reaction of Partially Protected
Uridine
Maurycy Szlenkier, Karol Kamel, and Jerzy Boryski
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland
ABSTRACT
ARTICLE HISTORY
Received  August 
Accepted  May 
Mitsunobu reaction of partially acylated uridine proceeds with
high regioselectivity for intramolecular SN2 anhydro linkage clo-
suring. Under the reaction conditions, an isomeric mixture of dia-
cyl uridine derivatives with either free 2ꢀ- or 3ꢀ-hydroxyl group was
transformed into a single cyclonucleosidic product, 2,2ꢀ-anhydro-
3ꢀ,5ꢀ-di-O-acyluridine. This paper presents a possible mechanism
of the reactions, the explanation of observed phenomenon based
onsemiempiricalanddensityfunctionaltheory(DFT)calculations
and possible utility of this synthetic pathway.
KEYWORDS
nucleoside synthesis;
modified nucleosides;
Mitsunobu reaction; partial
acylation; calculations
Introduction
Functionalization of the 2ꢀ,3ꢀ-cis-diole system in ribonucleosides is still a challenging
and attractive topic, because nucleosides modified in positions C-2ꢀ and C-3ꢀ of the
sugar moiety are extremely important in the search of new therapeutic agents, act-
ing as DNA/RNA elongation terminators or antisense oligonucleotides. One of the
possible ways to differentiate between the 2ꢀ- and 3ꢀ-hydroxyl groups is the applica-
tion of anhydro nucleosides. Although they have been known for many years, their
potential does not seem to be fully explored. The synthesis of 2,2ꢀ-anhydrouridine
was reported, for the first time, by Brown et al. in 1950’s,[1] and since then the
preparation methods and synthetic usefulness of the anhydro nucleosides have been
extensively studied. Because of their susceptibility to SN2 substitution with a vari-
ety of nucleophiles, the anhydro synthons can be applied in the synthesis of nucle-
oside analogs with a modified sugar portion, e.g., 3ꢀ-azido-2ꢀ,3ꢀ-dideoxythymidine
(AZT)[2] or 2ꢀ-O-methyluridine.[3] Furthermore, the anhydro nucleosides can read-
ily be transformed into xylofuranosyl and arabinofuranosyl derivatives through
simple hydrolysis of an appropriate (2,3ꢀ or 2,2ꢀ, respectively) anhydro linkage under
acidic[1,4] or basic conditions.[5–7]
A number of synthetic methods for the preparation of anhydro nucleosides have
been reported. In the ribonucleosides series, the method involving the application
of diphenyl carbonate, originally developed by Hampton and Nichol as a result
CONTACT Maurycy Szlenkier
Sciences, Poznan, Poland.
mszlenk@ibch.poznan.pl
Institute of Bioorganic Chemistry, Polish Academy of
©  Taylor & Francis Group, LLC

2
M. SZLENKIER ET AL.
of an attempted synthesis of uridine 2ꢀ,3ꢀ-cyclic carbonate, offers the best results
in the formation of 2,2ꢀ-anhydro linkage.[8] The method is simple, efficient (90%
yield on kilogram scale), and does not require any protection of the sugar por-
tion.[3] In turn, the intramolecular cyclization reaction of 2ꢀ(3ꢀ)-tosyl,[1] -mesyl,[5,6]
or -trifluoromethanesulfonyl[6] substrates has commonly been applied for synthe-
sis of the anhydro nucleosides in the both ribo and 2ꢀ-deoxyribo series. However,
the latter method requires the use of protecting groups for the preparation of sub-
strates. Its usefulness can be exemplified here by the synthesis of 2,3ꢀ-anhydro-2ꢀ-C-
methyluridine[5] or 2ꢀ,3ꢀ-bis-modified derivatives of uridine.[6]
The third common approach to anhydro nucleosides involves a tandem Mit-
sunobu reaction[9,10] and it is mainly applied for the 2ꢀ-deoxy series.[2] The Mit-
sunobu reaction is widely used as a versatile regioselective method for the dehy-
drative condensation of pronucleophile with primary or secondary alcohols, and
requires the combination of a reducing phosphine reagent together with an oxidiz-
ing azo reagent. The general mechanism of Mitsunobu reaction is well examined.[10]
In this article, we would like to present an interesting regioselectivity, discovered
during anhydrouridine synthesis under Mitsunobu conditions. We would like to
propose a probable mechanism of this reaction, based on current state of knowl-
edge, and the explanation of the observed regioselectivity, supported by molecular
modeling, which has not been presented yet and gives rise to a better understanding
of widely utilized synthesis of 2,2ꢀ-anhydrouridine with diphenyl carbonate.
Results and discussion
Mitsunobu reaction
Recently, 4-N,2ꢀ,5ꢀ-O,O-tripivaloylcytidine has successfully been obtained in crys-
talline form in good yields.[11] In the case of uridine, a similar, partial acylation
results in a mixture of diacylated 3ꢀ-hydroxyl and 2ꢀ-hydroxyl isomers.[12] In pre-
sented experiment, a mixture of two dipivaloylated isomers was obtained (2a, 3a;
Scheme 1) in a ratio of 4:1, respectively. The structure of the two isomers in a mixture
was elucidated, and characteristic 1H NMR signals were assigned (Figure 1) with the
COSY method supported with HSQC and HMBC techniques. Such a mixture was
presented to Mitsunobu reaction. Both isomers could give anhydrocyclic derivatives
and, because of substantial structure differentiation between 2,2ꢀ-anhydro-3ꢀ,5ꢀ-
di-O-pivaloyluridine and 2,3ꢀ-anhydro-2ꢀ,5ꢀ-di-O-pivaloyluridine, their separation
could be easy. However, Mitsunobu reaction performed on the isomeric mixture
gave 2,2ꢀ-anhydro-3ꢀ,5ꢀ-di-O-pivaloyluridine (4a; Scheme 1; overall yield from uri-
dine 56%), as a single product, which could readily be isolated as a pure precipitate
from ether. Such a regioselectivity of Mitsunobu reaction has never been reported
in the literature, what prompted us to further examine the nature of observed phe-
nomenon. To prove its structure, the product (4a) was treated with NH3/MeOH,
causing cleavage of pivaloyl groups, and then subjected to 2 D NMR spectral anal-
ysis, which indicated that the observed compound was 2,2ꢀ-anhydrouridine (5).

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
3
Scheme . Reagents and conditions: (i)  eq. PivCl(a)/BzCl(b), Py, t < °C; (ii) DEAD, PhP, DMF, r t (4a
% from uridine, 4b % from uridine); (iii) NH/MeOH, rt (%); (iv) DEAD, PhP, °C (%).
The procedure was repeated with benzoyl protective group giving analogous
result—the mixture of two dibenzoyl precursors 2b and 3b in a ratio 2:1 and only
one product 2,2ꢀ-anhydro-3ꢀ,5ꢀ-di-O-benzoyluridine (Scheme 1), although with a
better yield (68% from uridine). It suggests that Mitsunobu reaction leading to
Figure . Characteristic H NMR signals in the isomeric mixture of 2a and 3a.

4
M. SZLENKIER ET AL.
Scheme . The proposed mechanism of regioselective formation of 4a–b.
3ꢀ,5ꢀ-bis-protected 2,2ꢀ-anhydrouridine may be of general character for acyl pro-
tective groups suitable for partial protection of nucleosides.[12] Furthermore, in
order to establish the regioselectivity of anhydro linkage formation, in the case of
2ꢀ,3ꢀ-O,O-unprotected substrate, we conducted the Mitsunobu reaction with 5ꢀ-O-
(4,4ꢀ-dimethoxytrityl)uridine (6). As a result, we obtained a single product, 2,2ꢀ-
anhydro-5ꢀ-O-(4,4ꢀ-dimethoxytrityl)uridine (7; 23%, Scheme 1). One can observe
the influence of bulkiness of the DMTr group in position O-5ꢀ on the yield of 2,2ꢀ-
anhydrouridine formation and the demand for energy. This result may indicate that
the presented Mitsunobu reaction regioselectivity is not driven by the labile acyl
protective group, but rather occurs despite its presence within a cis-diole system.
The labile character of acyl protective groups, under the conditions of Mitsunobu
reaction, is implied by the ratio of diacylated precursors, in favor of O2ꢀ,O5ꢀ ones,
however this does not interrupt the formation of only 2,2ꢀ-anhydro product in both
situations.
The probable mechanism of the regioselective formation of 4a–b can be depicted
(Scheme 2) analogously to the silyl migration in diol system reported recently.[13]
At the first stage, the proton from heterocyclic base residue is detached by 8 gen-
erating ion pairs (9 and 10a–b; 9 and 11a–b; Scheme 2), because pKa values of
hydroxyl groups in positions 2ꢀ and 3ꢀ are higher (pKaOH2 = 12.7, pKaOH3 = 13)
ꢀ
ꢀ

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
5
than amide group in uracil (pKaU = 9.7). The compound 8 can act as basic cata-
lyst. In the presence of 8, compounds 2a–b and 3a–b are isomerizing, and anions
10a–b and 11a–b can isomerize as well, while the amount of 11a–b is depleting
because of the formation of oxyphosphonium ion 13a–b. An alternative oxyphos-
phonium ion 14a–b can be formed as well. The observed product 4a–b is formed
from 13a–b, in intramolecular SN2 substitution. This is a crucial step because it is
practically irreversible. The regioselectivity of the reaction could be caused by differ-
ent factors—steric hindrance, electron density on the centers of nucleophilic attack,
and geometrical feasibility of closing the anhydro bridge. To estimate the factor
magnitudes, we have performed semiempirical and density functional theory (DFT)
calculations.
Further reactions
We tested 2,2ꢀ-anhydro-3ꢀ,5ꢀ-di-O-pivaloyluridine (4a) for acidic hydrolysis and for
substitutions with three different nucleophiles (Scheme 3). As it was summarized
by Piccirilli et al.,[14] some nucleophiles substitute to a position C-2ꢀ of a sugar
moiety giving a ribose like product and some attack a position C-2 of nucleobase
what results in C-2-substituted 1-(β-D-arabinofuranosyl)uracil derivatives. First
substitution to 4a was performed with magnesium methoxide in absolute methanol
and resulted with a good yield (84%), but it was followed by full deprotection of
hydroxyl groups giving 2ꢀ-methoxyuridine (15). The synthesis of 2ꢀ-methoxyuridine
from 2,2ꢀ-anhydrouridine was presented inter alia by Roy and Tang[3] with 92%
yield. Second reaction of 4a was with lithium azide and gave a C-2ꢀ-substituted
product with yield 72% (16), while reaction of 5 with the same nucleophile gave
an expected product with yield 68% (17). Third nucleophile was generated from
4-methylthiophenol with catalytic amount of potassium phthalimide in DMF. In
the reaction with 4a it is substituted to the C-2ꢀ position giving product 18 with
quantitative yield, however the analogous reaction with 5 occurred equally good
(TLC), although standard purification gave some losses (19). The conducted sub-
stitutions indicate that 4a does not have any significant advantage over unprotected
5 in nucleophilic substitutions. The acidic hydrolysis of 4a occurred correctly with
retention of protective groups giving a desired product 1-(3ꢀ,5ꢀ-di-O-pivaloyl-β-D-
arabinofuranosyl)uracil (20) with yield 74%. Isolation and purification of 20 did
not encounter any difficulties, because the migration of pivaloic group within 2ꢀ,3ꢀ-
trans-diole system could not take place. The configuration of sugar moiety was con-
firmed by NOESY. Finally, we wanted to answer an obvious question if straight acy-
lation of 2,2ꢀ-anhydrouridine (5) is a better way to obtain compounds like 4a or 4b.
Shen et al.[15] synthesized 4b by simple benzoylation of 5 with 3 eq. of benzoyl chlo-
ride in room temperature with yield >88%. In the same manner, we have conducted
pivaloylation of 5 obtaining 4a with yield 87%. Assuming 90% efficacy in obtaining
5 with diphenyl carbonate,[3] yield from uridine would be 78% for 4a and >79%
for 4b.

6
M. SZLENKIER ET AL.
Scheme . Further reactions of 4a and 5: Reagents and conditions: (a) Mg(MeO)/MeOHabs, reflux;
(b)/(e) LiN, DMF, °C; (c)/(f) p-Thiocresol, PPI, DMF, °C; (d) N HClaq/DMF, °C; (g) PivCl, Py, rt.
Calculations
Because of the size of the molecules, only carbon backbones of two anhydrouridines
were calculated with the DFT (B3LYP/6-31++G(d,p)) method. Elimination of
neighboring side groups enabled the estimation of energetic cost of anhydro ring
closure only. The calculated total energy figures are −644.863 hartree for 2ꢀ-anhydro
backbone (2ꢀAB) and −644.852 hartree for 3ꢀ-anhydro backbone (3ꢀAB), which
gives 6.9 kcal/mol difference in favor of 2ꢀAB (Figure 2).

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
7
Figure . Optimized structures of two possible anhydrouridine backbones.
To find a rationale for the calculated energies, the analysis of intrinsic structural
strains was made. Generally, sugar residue of 3ꢀAB was more compressed and the
˚
ꢀ
ꢀ
ꢀ
ꢀ
lengths of diagonals of a furanose backbone total dC3 –C1 = 2.272 A and dC4 –C2
=
ꢀ
ꢀ
˚
˚
ꢀ
ꢀ
ꢀ
ꢀ
2.377 A for 3 AB, whereas, for 2 AB, they total dC3 –C1 = 2.428 A and dC4 -C2
=
˚
2.392 A. In the models of THF, constructed on the basis of crystallographic bond
longitudes and the maximum and minimum size of the bond angles,[16] the diago-
ꢀ
˚
˚
nals ranged from 2.370 A to 2.426 A. In the case of the 3 AB model, one diagonal
was smaller and out of the range, while both diagonals were smaller than in the 2ꢀAB
model. The calculations might suggest that the geometric strains were the cause of
the energy difference. However, comparison of the bond lengths of 2ꢀAB and 3ꢀAB,
computed with the DFT method, with literature data,[16] and the bond angles with
ideal values, did not reveal which deformation had a crucial role.
To estimate the electron effect on the described reaction, two simplified sys-
tems were taken into consideration. To compare which hydroxyl group is more
susceptible to interaction with phosphonium intermediate (9), two structures of
2ꢀ-fluorouridine and 3ꢀ-fluorouridine were calculated with the DFT (B3LYP/6-
31G(d,p)) method. The substitution with fluoride was done to eliminate the con-
tribution of undesired 2ꢀ,3ꢀ OH…O hydrogen bonds. The analysis of partial charges
ꢀ
ꢀ
gave δ = −0.31 e and δ = −0.33 e, which had a small but counteracting effect.
O2
O3
Therefore, this factor was not responsible for the discussed regioselectivity. To esti-
mate which center of nucleophilic attack was more preferable for the anhydro bridge
closuring, uridine 2ꢀ,3ꢀ-cyclic carbonate was optimized with the DFT (B3LYP/6–
31G(d,p)) method. Uridine carbonate is an intermediate product of the reaction
of uridine with diphenylcarbonate and sodium hydrogen carbonate, which only
leads to 2,2ꢀ-anhydrouridine formation. The global minimum on potential energy
surface for 2ꢀ,3ꢀ-cyclic carbonate corresponded to a structure with hydrogen bond
between 5ꢀ-OH and O-2 (Figure 3). The partial charges located in C-2ꢀ and C-3ꢀ
within the calculated structure were equal (+0.05 e). For the 2ꢀ-fluorouridine and
3ꢀ-fluorouridine, the comparison of partial charges on carbons bonded to hydroxyl

8
M. SZLENKIER ET AL.
Figure . Optimized structure of uridine ꢀ,ꢀ-cyclic carbonate.
groups gave a similar result: for C-3ꢀ in 2ꢀ-fluorouridine δ = +0.03 e, for C-2ꢀ in
ꢀ
C3
3ꢀ-fluorouridine δ = +0.02 e, and ꢀδ
= 0.01 e. Consequently, the electron
ꢀ
ꢀ
ꢀ
C2
C3 /C2
density effect did not play any significant role in the observed regioselectivity.
Uridine carbonate is a relatively simple structure, which allowed to calculate the
energy barrier of anhydro bridge closuring, using the PM7 semiempirical method.
The structure corresponding to the lowest local minimum without hydrogen bond
was used as a starting point. The energy of a substrate was set at 0 kcal/mol, as the ref-
erence value. The calculated energy of the transition state for 2ꢀ-anhydro bridge clo-
suring (TS2ꢀ) was lower by 8.5 kcal/mol than that of 3ꢀ-anhydro bridge (TS3ꢀ), which
is consistent with previous conclusions. Schematic illustration of potential energy
surface for both, theoretically possible, reaction directions is presented in Figure 4.
Furthermore, the example of regioselective reaction of uridine with diphenyl car-
bonate indicated that steric hindrance of side groups and large phosphonium inter-
mediate was not a contributing factor in the presented Mitsunobu reaction either.
However, the optimized structure of uridine carbonate displayed another factor that
turned out to be relevant. The distances between the C2ꢀ-O2 and C3ꢀ-O2 atoms were
˚
˚
2.970 A and 3.680 A, respectively. For the intramolecular reaction, it is a significant
determinant that controls the reaction kinetically, which explains the difference in
the calculated transition states energy values. It is clear that the sugar puckering of
uridine carbonate is unique, and the example has only an illustrative value. However,
this effect should hold true meaning for the presented Mitsunobu reaction.
Summary
Our study presents the regioselective 2,2ꢀ-anhydro linkage formation in partially
acylated uridine by the Mitsunobu reaction. The molecular modeling helped us to

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
9
Figure . Schematic representation (energy vs. reaction coordinate) of two alternative anhydro link-
age formation.
comprehend the observed regioselectivity and suggested that geometrical feasibility
of locking the anhydro bridge preferably to the C-2ꢀ position, than to the C-3ꢀ is a
leading factor, responsible for different energy values of transition states and prod-
ucts. The effect seems to apply to all methods of generating 2,2ꢀ-anhydronucleosides,
where both C-3ꢀ and C-2ꢀ are available. The observed regioselectivity is interesting,
however, it has not escaped our notice, that presented synthetic solution probably
will not find an application since an 2,2ꢀ-anhydrouridine can be easily obtained with
the Hampton and Nichol’s method with high yield, unprotected 2,2ꢀ-anhydrouridine
(5) undergoes substitutions equally good to 2,2ꢀ-anhydro-3ꢀ,5ꢀ-di-O-pivaloyluridine
(4a) and straight acylation of 5 leads to 4a and 4b with better yields. Acidic hydrol-
ysis of 4a allowed to obtain 1-(3ꢀ,5ꢀ-di-O-pivaloyl-β-D-arabinofuranosyl)uracil.
Experimental section
Calculations
Conformational analyses were made using the HyperChem 8.0 package[17] by
molecular mechanics in the AMBER force field. Geometry optimization calcu-
lations were performed, using the NWChem 6.1 package,[18] employing DFT
(B3LYP/6-31++G(d,p)) and DFT (B3LYP/6-31G(d,p)) levels of theory. The IR
spectra for all of the resulting structures were calculated to check if the obtained
structures meet their corresponding energy minima. The Mulliken and Lowdin
analyses were carried out in the GAMESS software[19] at the DFT (B3LYP/6-
31G(d,p)) level of theory, in order to calculate partial charges and assess bond

10
M. SZLENKIER ET AL.
order in the optimized structures. Semiempirical calculations were performed using
the HyperChem 8.0 package and MOPAC2012 package,[20] employing the PM7
method.
Chemistry
All reagents were purchased from commercial suppliers and were used without
further purification. 5ꢀ-O-(4,4ꢀ-dimethoxytrityl)uridine (6) was obtained according
to Ref.[21] Melting points were determined on a Laboratory Devices Mel-Temp
II micromelting points apparatus (Laboratory Devices, Holliston, MA, USA) and
remain uncorrected. The NMR spectra were recorded on a Bruker Avance 400 MHz
spectrometer in DMSO-d6 with tetramethylsilane as an internal standard: chemical
shifts are reported in δ-values (ppm) and coupling constants are given in Hz.
1
For assigning the NMR signals to specific atom positions COSY, H–13C HSQC
1
and H–13C HMBC techniques were used. To establish sugar moiety structure of
1-(3ꢀ,5ꢀ-di-O-pivaloyl-β-D-arabinofuranosyl)uracil (20) NOESY technique was
employed. Mass spectra (MS) were measured on a Bruker microTOF-Q spectrom-
eter. The RP-HPLC analysis was performed on Waters 1525 Binary HPLC with
Waters 2487 UV Detector on LiChrospher RP18–5 Endcapped C18 (5.0 µm) 4.6 mm
× 250 mm column from Supelco Analytical using solvent system A—0.1 M triethy-
lammonium acetate and B—A/acetonitrile 1:4 (v/v) and gradient B in A 60%–100%
within 5 min, held 100% B for 8 min and then reversed gradient B in A 100%–60%
within 5 min with a flow rate of 1 mL/min at 25°C. For compound 19, the RP-HPLC
analysis was made using gradient B in A 5%–90% within 25 min. Thin-layer chro-
matography (TLC) was conducted on the Merck silica gel 60 F254 plates, using the
following solvent systems (measured by volume): C—dichloromethane-methanol
(95:5), D—dichloromethane-methanol (9:1), E—dichloromethane-acetone (2:1),
F—dichloromethane-methanol (4:1), G—dichloromethane-methanol (98:2), H—
dichloromethane-acetone (4:1), I—dichloromethane-methanol (99:1). For prepar-
ative column chromatography, the Merck silica gel 60 (0.040–0.063 mm) was used
along with the solvent systems mentioned above.
ꢀ,ꢀ-Di-O-pivaloyluridine and ꢀ,ꢀ-di-O-pivaloyluridine (2a,3a)
Uridine (4.4913 g, 18.39 mmol) was added to dry pyridine (80 mL). The solution
was concentrated to ca. 70 mL and cooled down to 0°C. Then, pivaloyl chloride
(4.52 mL, 36.78 mmol) was added in nine portions at a temperature below 5°C, and
the solution was maintained at 4°C for 18 hours. The solvent was coevaporated with
toluene under diminished pressure. The resulting oil was dissolved in methylene
chloride (80 mL) and washed with cold, saturated solution of NaHCO3 (200 mL).
The organic layer was separated, dried over Na2SO4, concentrated to thick oil and
dried in vacuum. The product (6.9778 g) was analyzed with the NMR spectroscopy
(1H NMR and COSY), showing the presence of two isomeric compounds (2a,3a) in
ratio 4:1, respectively.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
11
ꢀ,ꢀ-Di-O-benzoyluridine and ꢀ,ꢀ-di-O-benzoyluridine (2b,3b)
Uridine (1 g, 4.09 mmol) was added to dry pyridine (30 mL). The solution was con-
centrated to ca. 25 mL and cooled down to 0°C. Then, benzoyl chloride (0.950 mL,
8.18 mmol) was added in five portions at a temperature below 5°C, and the solution
was maintained at 4°C for 18 hours. The solvent was coevaporated with toluene
under diminished pressure. The resulting oil was dissolved in methylene chloride
(80 mL) and washed with cold, saturated solution of NaHCO3 (200 mL). The organic
layer was separated, dried over Na2SO4, concentrated to thick oil, and dried in vac-
uum. The product (1.5728 g) was analyzed with the NMR spectroscopy (1H NMR
and COSY), showing the presence of two isomeric compounds (2b,3b) in ratio 2:1,
respectively.
,ꢀ-Anhydro-ꢀ,ꢀ-di-O-pivaloyluridine (4a)
The obtained mixture of 2a and 3a and Ph3P (5.3314 g, 20.33 mmol) was dissolved
in dry DMF (100 mL) and concentrated to ca. 90 mL. Then, the DEAD (3.5363 g,
3.2 mL, 20.3 mmol) was added and the solution was stirred at room temperature for
21 hours. The solvent was evaporated under diminished pressure, and the residue
was suspended in diethyl ether (80 mL). The white precipitate was collected and
dried in vacuum yielding 4.074 g of the product (10.33 mmol, 56% from uridine).
The product was 99% pure by RP-HPLC; m.p. 223–224°C; Rf 0.42 (D); 1H NMR: δ
7.89 (d, J = 7.6 Hz, 1H, H6), 6.40 (d, J = 5.2 Hz, 1H, H1ꢀ), 5.91 (d, J = 7.6 Hz, 1H,
H5), 5.49 (d, J = 5.6 Hz, 1H, H2ꢀ), 5.32 (d, J = 2.4 Hz, 1H, H3ꢀ), 4.46 (m, 1H, H4ꢀ),
4.05 (dd, J = 4.8, 12.4 Hz, 1H, H5ꢀa), 3.94 (dd, J = 6.8, 12.4 Hz, 1H, H5ꢀb), 1.19 (s,
13
=
9H, (CH3)3), 1.08 (s, 9H, (CH3)3); C NMR: δ 177.3, 177.0 (2 × C O), 171.2 (C4),
159.8 (C2), 137.1 (C6), 109.4 (C5), 90.1 (C1ꢀ), 86.5 (C2ꢀ), 82.5 (C4ꢀ), 76.4 (C3ꢀ), 62.4
(C5ꢀ), 38.6 (2 × C(Me)3), 27.1 ((CH3)3), 27.0 ((CH3)3). Mass (ESI-MS) m/z calcd
for [C19H26N2O7 + H]+ 395.1813, found 395.1801, err 3.0 ppm.
,ꢀ-Anhydro-ꢀ,ꢀ-di-O-pivaloyluridine (4a from 5)
Dried 5 (2.1500 g, 9.51 mmol) was dissolved in dry pyridine (35 mL), concentrated
to ca. 32 mL and cooled down in ice bath. Then, the pivaloyl chloride was added in
portions (3.4401 g, 28.53 mmol, 3.5104 mL). The mixture was allowed to warm up
to rt and stirred overnight. The solvent was coevaporated with toluene, the residual
solid dissolved in methylene chloride and extracted with saturated aqueous solu-
tion of NaHCO3. Organic layer was dried over Na2SO4, concentrated, and the prod-
uct was precipitated with diethyl ether. The crude product was contaminated with
a compound of smaller Rf—probably product of monoacylation. Therefore, it was
subsequently purified by silica gel column chromatography in solvent system G giv-
ing 3.2698 g (8.29 mmol, 87%). The product was 96% pure by RP-HPLC, m.p. 223–
224°C, NMR and MS data were consisted with those of 4a.
,ꢀ-Anhydro-ꢀ,ꢀ-di-O-benzoyluridine (4b)
The obtained mixture of 2b and 3b and Ph3P (1.5029 g, 5.73 mmol) was dissolved
in dry DMF (25 mL) and concentrated to ca. 20 mL. Then, the DEAD (0.9982 g,

12
M. SZLENKIER ET AL.
0.9 mL, 5.73 mmol) was added and the solution was stirred at room temperature for
21 hours. The solvent was evaporated under diminished pressure, and the residue
was suspended in diethyl ether (30 mL). The white precipitate was collected and
dried in vacuum yielding 1.2137 g of the product (2.79 mmol, 68% from uridine).
The product was 97% pure by RP-HPLC; m.p. 263–264°C; Rf 0.42 (D); 1H NMR: δ
8.05 (d, J = 7.2 Hz, 2H, Ph), 7.92 (d, J = 7.2 Hz, 1H, H6), 7.90 (d, J = 7.2 Hz, 2H,
Ph), 7.72 (t, J = 7.2 Hz, 1H, Ph), 7.66 (t, J = 7.2 Hz, 1H, Ph), 7.58 (t, J = 7.6 Hz, 2 H,
Ph), 7.50 (t, J = 7.6 Hz, 2 H, Ph), 6.49 (d, J = 5.6 Hz, 1H, H1ꢀ), 5.89 (d, J = 7.6 Hz,
1H, H5), 5.77 (d, J = 5.6 Hz, 1H, H2ꢀ), 5.71 (d, J = 2.8 Hz, 1H, H3ꢀ), 4.88 (m, 1H,
H4ꢀ), 4.36 (m, 2 H, H5ꢀa,b); 13C NMR: δ 170.7 (C4), 165.2 (C O), 164.9 (C O),
159.4 (C2), 136.7 (C6), 134.0 (Ph), 133.6 (Ph), 129.6 (Ph), 129.2 (Ph), 129.0 (Ph),
128.8 (Ph), 128.7 (Ph), 128.6 (Ph), 109.0 (C5), 89.9 (C1ꢀ), 86.1 (C2ꢀ), 82.2 (C4ꢀ), 77.2
(C3ꢀ), 63.1 (C5ꢀ). Mass (ESI-MS) m/z calcd. for [C23H18N2O7+H]+ 435.1187, found
435.1184, err. −0.6 ppm.
=
=
,ꢀ-Anhydrouridine (5)
4a (100 mg, 0.25 mmol) was dissolved in 2 mL of MeOH saturated with NH3. The
solution was stirred for 3 hours at 0°C, the solvent was evaporated, and the product
was purified on previously washed silica gel column in solvent system D. The col-
umn was washed with CH2Cl2:MeOH 1:1 v/v. The fractions containing the prod-
uct were collected and evaporated under diminished pressure. The product was
crystallized from DMF and flushed with MeOH resulting in 52.5 mg (0.23 mmol,
yield 92%) of 5. The product was 99% pure by RP-HPLC; m.p. 242–243°C; Rf 0.27
1
(F); H NMR: δ 7.83 (d, J = 7.2 Hz, 1H, H6), 6.30 (d, J = 5.6 Hz, 1H, H1ꢀ), 5.87
(s, 1H, 3ꢀOH), 5.84 (d, J = 7.6 Hz, 1H, H5), 5.20 (d, J = 5.6 Hz, 1H, H2ꢀ), 4.97
(s, 1H, 5ꢀOH), 4.38 (s, 1H, H3ꢀ), 4.07 (t, J = 4.8 Hz, 1H, H4ꢀ), 3.28 (dd, J = 4.8,
11.6 Hz, 1H, H5ꢀa), 3.19 (dd, J = 5.6, 11.2 Hz, 1H, H5ꢀb); 13C NMR: δ 171.2 (C4),
159.8 (C2), 136.8 (C6), 108.6 (C5), 90.0 (C1ꢀ), 89.2 (C2ꢀ), 88.7 (C4ꢀ), 74.7 (C3ꢀ), 60.8
(C5ꢀ); Mass (ESI-MS) m/z calcd. for [C9H10N2O5 + H]+ 227.0662, found 227.0666,
err −1.7 ppm.
,ꢀ-Anhydro-ꢀ-O-(,ꢀ-dimethoxytrityl)uridine (7)
ꢀ
Dried O5 -(4,4ꢀ-dimethoxytrityl)uridine (305.0 mg, 0.56 mmol) and Ph3P
(204.6 mg, 0.78 mmol) were dissolved in dry DMF (12 mL) and concentrated
to ca. 10 mL. Then, the DEAD (135.9 mg, 123 μL, 0.78 mmol) was added and
stirred for 21 hours. The solvent was evaporated under diminished pressure and the
mixture was purified on a silica gel column in solvent system C. The fractions con-
taining the product were collected, evaporated, precipitated from toluene, flushed
with hexane, and dried in vacuum resulting in 70.9 mg of 7 (0.13 mmol, yield 23%).
The product was 98% pure by RP-HPLC; m.p. 129.5–130°C; Rf 0.34 (D); 1H NMR:
δ 7.93 (d, J = 7.6 Hz, 1H, H6), 7.27 (m, 4H, Ph), 7.21 (m, 1H, Ph), 7.14 (dd, J = 2.8,
8.8 Hz, 4H, Ph), 6.83 (dd, J = 6.0, 8.8 Hz, 4H, Ph), 6.33 (d, J = 5.6 Hz, 1H, H1ꢀ),
5.98 (d, J = 4.8 Hz, 1H, 3ꢀOH), 5.88 (d, J = 7.2 Hz, 1H, H5), 5.21 (d, J = 5.6 Hz,

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
13
1H, H2ꢀ), 4.30 (m, 1H, H3ꢀ), 4.23 (m, 1H, H4ꢀ), 2.94 (dd, J = 4.4, 10.4 Hz, 1H,
H5ꢀa), 2.81 (dd, J = 7.2, 10.4 Hz, 1H, H5ꢀb); 13C NMR: δ 170.9 (C4), 159.3 (C2),
158.1 (Ph), 158.0 (Ph), 144.6 (Ph), 136.7 (C6), 135.2 (Ph), 135.1 (Ph), 129.5 (Ph),
129.4 (Ph), 127.8 (Ph), 127.4 (Ph), 126.7 (Ph), 113.2 (Ph), 108.87 (C5), 89.7 (C1ꢀ),
88.4 (C2ꢀ), 86.8 (C4ꢀ), 85.4 (C(MeOPh)2Ph), 74.7 (C3ꢀ), 62.8 (C5ꢀ), 55.0 (CH3O),
54.9 (CH3O); Mass (ESI-MS) m/z calcd. for [C30H28N2O7 + H]+ 529.1969, found
529.1989, err −3.7 ppm.
ꢀ-Deoxy-ꢀ-methoxyuridine (15)
Dried 4a (200 mg, 0.51 mmol) was added to freshly prepared solutions of magne-
sium methoxide (88.1 mg, 1.02 mmol) in absolute methanol (6 mL) with catalytic
amount of iodine (6.5 mg, 0.051 mmol). The solution was refluxed for 5 hours. Then
the mixture was cooled to room temperature and pH was neutralized by slow addi-
tion of glacial acetic acid. The solvent was evaporated under diminished pressure
and refluxed with ethanol (5 mL) for 2 hours. The solid magnesium salts were filtered
off and residual solvent was evaporated giving 120.1 mg of a crude product. Final
purification was made by silica gel column chromatography with solvent system C.
Product was obtained with yield 84% giving 111.2 mg (0.43 mmol). The product
was 95% pure by RP-HPLC; m.p. 158–158.5°C; Rf 0.75 (F); 1H NMR: δ 11.38 (s, 1H,
NH), 7.94 (d, J = 7.6 Hz, 1H, H6), 5.85 (d, J = 4.8 Hz, 1H, H1ꢀ), 5.63 (d, J = 8.0 Hz,
1H, H5), 5.31 (bs, 2H, 3ꢀOH, 5ꢀOH), 4.12 (t, J = 4.8 Hz, 1H, H3ꢀ), 3.84 (d, J = 4.4 Hz,
H1, H4ꢀ), 3.78 (t, J = 4.8 Hz, H1, H2ꢀ), 3.63 (dd, J = 2.8, 12.4 Hz, H1, H5ꢀa), 3.56
(dd, J = 3, 12.2 Hz, 1H, H5ꢀb), 3.36 (s, 3H, CH3); 13C NMR: δ 163.25 (C2), 150.6
(C4), 140.34 (C6), 101.78 (C5), 85.96 (C1ꢀ), 85.05 (C4ꢀ), 82.7 (C2ꢀ), 68.12 (C3ꢀ), 60.34
(C5ꢀ), 57.48 (CH3); Mass (ESI-MS) m/z calcd for [C10H13N2O6]− 257.0779, found
257.0776, err. 1.1 ppm.
ꢀ-Azido-ꢀdeoxy-ꢀ,ꢀ-di-O-pivaloyluridine (16)
Dried 4a (200 mg, 0.51 mmol) and LiN3 (49.9 mg, 1.02 mmol) were added to
distilled and dried DMF (6 mL), which was then concentrated to ca. 5 mL. The
solution was stirred in 135°C for 18 hours. Then the solvent was evaporated and
the residual oil was separated by silica gel chromatography with solvent system
E. The fractions with product were collected and the product was recrystallized
from MeOH/H2O giving 160.5 mg (0.37 mmol) of white solid with yield 72%. The
1
product was 95% pure by RP-HPLC; m.p. 115–116°C, Rf 0.74 (D); H NMR: δ
11.50 (s, 1H, NH), 7.43 (d, J = 8.4 Hz, 1H, H6), 5.79 (d, J = 6.0 Hz, 1H, H1ꢀ),
5.72 (d, J = 8.0 Hz, 1H, H5), 5.32 (t, J = 6.0 Hz, H1, H3ꢀ), 4.78 (t, J = 6.0 Hz,
1Hꢀ , H2ꢀ), 4.23 (m, 3 H, H4ꢀ, H5ꢀa, H5ꢀb), 1.22ꢀ (s, 9H, 3ꢀ(CH3)3), 1.15 (s, 9H,
13
ꢀ
=
=
5 (CH3)3); C NMR: δ 177.11 (5 C O), 176.61 (3 C O), 162.90 (C4), 150.25 (C2),
140.81 (C6), 102.44 (C5), 87.60 (C1ꢀ), 79.14 (C4ꢀ), 71.22 (C3ꢀ), 62.71 (C5ꢀ), 62.23
(C2ꢀ), 38.37 (3ꢀC(CH3)3), 38.24 (5ꢀC(CH3)3), 26.76 (5ꢀCH3Piv), 26.62 (3ꢀCH3Piv);
Mass (ESI-MS) m/z calcd. for [C19H27N5O7]− 436.1838, found 436.1859, err. −
4.8 ppm.

14
M. SZLENKIER ET AL.
ꢀ-Azido-ꢀ-deoxyuridine (17)
Dried 5 (200 mg, 0.88 mmol) and LiN3 (86.2 mg, 1.76 mmol) were added to dis-
tilled and dried DMF (6 mL), which was then concentrated to ca. 5 mL. The solu-
tion was stirred in 135°C for 18 hours. Then the solvent was evaporated and the
residual oil was separated by silica gel chromatography with solvent system C. The
fractions with product were collected and the solvent was evaporated. The product
was suspended twice in dichloromethane and dried on an evaporator. Any attempts
to recrystallize failed. The product was a sticky, yellowish solid. After drying in vac-
uum it became more powdery yielding 161.0 mg (0.60 mmol, 68%), however melting
point measurement was uncertain. The product was 95% pure by RP-HPLC; Rf 0.40
(D); 1H NMR: δ 11.41 (s, 1H, NH), 7.86 (d, J = 7.6 Hz, 1H, H6), 5.96 (d, J = 5.2 Hz,
1H, H1ꢀ), 5.88 (d, J = 5.6 Hz, 1H, 3ꢀOH), 5.67 (d, J = 8.0 Hz, 1H, H5), 5.17 (t, J
= 4.8 Hz, 1H, 5ꢀOH), 4.30 (dd, J = 5.2, 10.4 Hz, 1H, H2ꢀ), 4.05 (t, J = 5.2 Hz, 1H,
H3ꢀ), 3.89 (dd, J = 3.2, 7.6 Hz, 1H, H4ꢀ), 3.66 (m, 1H, H5ꢀa), 3.57 (m, 1H, H5ꢀb);
13C NMR: δ 163.0 (C4), 150.4 (C2), 140.0 (C6), 102.0 (C5), 85.6 (C1ꢀ), 85.2 (C4ꢀ),
70.4 (C2ꢀ), 64.6 (C2ꢀ), 60.2 (C5); Mass (ESI-MS) m/z calcd. for [C9H11N5O5+Na]+
292.0652, found 292.0662, err. −3.3 ppm.
ꢀ-Deoxy-ꢀ,ꢀ-di-O-pivaloyl-ꢀ-(-tolylthio)uridine (18)
Dried 4a (200 mg, 0.51 mmol) and 4-methylthiophenol (126.7 mg, 1.02 mmol) were
added to distilled and dried DMF (6 mL) and it was concentrated to ca. 5 mL.
Then a potassium phthalimide was added (9.2 mg, 0.05 mmol) and the solution was
stirred in 120°C for 4 hours. The reaction went quantitative (TLC). The solvent was
evaporated and residual mixture was purified on silica gel column chromatography
with solvent system I and crystallized from MeOH with water. After the purification
product was obtained with yield 97% giving 256.7 mg (0.495 mmol). The product
was 96% pure by RP-HPLC; m.p. 134.5–135°C, Rf 0.69 (H); 1H NMR: δ 11.29 (s, 1H,
NH), 7.39 (d, J = 8.0 Hz, 1H, H6), 7.26 (d, J = 8.0 Hz, 2H, Phor∗), 7.08 (d, J = 8.0 Hz,
2H, Phm∗), 6.11 (d, J = 9.2 Hz, 1H, H1ꢀ), 5.50 (d, J = 8.0 Hz, 1H, H5), 5.40 (d, J =
6.0 Hz, H1, H3ꢀ), 4.22 (m, 4H, H5ꢀa, H5ꢀb, H2ꢀ, H4ꢀ), 2.23 (s, 3 H, CH3), 1.22 (s, 9H,
ꢀ
ꢀ
3ꢀ(CH3)3), 1.13 (s, 9H, 5ꢀ(CH3)3); 13C NMR: δ 177.10 (5 C O), 176.20 (3 C O),
162.39 (C4), 150.31 (C2), 139.51 (C6), 137.81 (Php∗), 132.45 (Phor∗), 129.79 (Phm∗),
128.11 (PhC-S∗), 102.64 (C5), 88.29 (C1ꢀ), 80.47 (C4ꢀ), 73.48 (C3ꢀ), 63.20 (C5ꢀ),
51.44 (C2ꢀ), 38.53 (3ꢀC(CH3)3), 38.22 (5ꢀC(CH3)3), 26.76 (CH3Piv), 20.55 (CH3);
Mass (ESI-MS) m/z calcd. for [C26H34N2O7S + H]+ 519.2159, found 519.2149,
=
=
∗
err. 1.9 ppm. Abbreviations: “m”—meta, “or”—ortho, “p”—para, “C-S”—carbon
bonded to sulfur in 4-tolylthio group.
ꢀ-Deoxy-ꢀ-(-tolylthio)uridine (19)
Dried 5 (200 mg, 0.88 mmol) and 4-methylthiophenol (218.6 mg, 1.76 mmol) was
added to distilled and dried DMF (6 mL) and it was concentrated to ca. 5 mL. Then a
potassium phthalimide was added (16.7 mg, 0.09 mmol) and the solution was stirred
in 120°C for 4 hours. The reaction was quantitative (TLC). The solvent was evapo-
rated and residual mixture was purified on silica gel column chromatography with

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
15
solvent system C and crystallized from MeOH with water. After the purification
product was obtained with yield 82% giving 252.3 mg (0.72 mmol). The product
was 99% pure by RP-HPLC; m.p. 209–210°C, Rf 0.55 (D); 1H NMR: δ 11.12 (s, 1H,
NH), 7.55 (d, J = 7.6 Hz, 1H, H6), 7.22 (d, J = 8.0 Hz, 2H, Phor∗), 7.04 (d, J =
8.0 Hz, 2H, Phm∗), 6.16 (d, J = 9.2 Hz, 1H, H1ꢀ), 5.86 (d, J = 5.6 Hz, 1H, 3ꢀOH),
5.44 (d, J = 8.0 Hz, 1H, H5), 5.10 (t, J = 5.2 Hz, 1H, 5ꢀOH), 4.31 (t, J = 5.6 Hz, 1H,
H3ꢀ), 3.91 (t, J = 3.2 Hz, 1H, H4ꢀ), 3.73 (dd, J = 5.2, 9.2 Hz, 1H, H2ꢀ), 3.56 (t, J =
4.4 Hz, 2H, H5ꢀa,b), 2.21 (s, 3 H, CH3); 13C NMR: δ 162.6 (C4), 150.5 (C2), 139.8
(C6), 137.0 (Php∗) 132.1 (Phor∗), 129.6 (Phm∗), 129.5 (PhC-S∗), 102.3 (C5), 87.8 (C1ꢀ),
86.7 (C4ꢀ), 72.4 (C3ꢀ), 61.5 (C5ꢀ), 55.0 (C2ꢀ), 20.5 (CH3); Mass (ESI-MS) m/z calcd.
for [C16H18N2O5S+Na]+ 373.0829, found 373.0840, err. −2.9 ppm. ∗Abbreviations:
“m”—meta, “or”—ortho, “p”—para, “C-S”—carbon bonded to sulfur in 4-tolylthio
group.
-(ꢀ,ꢀ-Di-O-pivaloyl-β-D-arabinofuranosyl)uracil (20)
4a (75.0 mg, 0.19 mmol) was dissolved in DMF (3 mL) and 2 mL of 2N HClaq was
added. The solution was heated to 80°C and after next 2.5 hours the reaction was
neutralized with saturated NaHCO3aq, extracted with methylene chloride and puri-
fied by silica gel column chromatography with solvent system G. Product was crys-
tallized from toluene giving 57.2 mg (0.14 mmol) with yield 74%. The product was
94% pure by RP-HPLC; m.p. 139–140°C, Rf 0.71 (D); 1H NMR: δ 11.37 (s, 1H, NH),
7.51 (d, J = 8.0 Hz, 1H, H6), 6.11 (d, J = 4.8 Hz, 1H, 2ꢀOH), 6.00 (d, J = 4.0 Hz, 1H,
H1ꢀ), 5.59 (d, J = 8.0 Hz, H1, H5), 4.96 (s, 1H, H3ꢀ), 4.37 (m, 1H, H5ꢀa), 4.22 (m,
1H, H5ꢀb), 4.15 (s, 1H, H2ꢀ), 4.11 (m, 1H, H4ꢀ), 1.18 (s, 9H, (CH3)3), 1.16 (s, 9H,
13
=
=
(CH3)3); C NMR: δ 177.2 (C O), 176.5 (C O), 163.1 (C4), 150.3 (C2), 141.7 (C6),
100.2 (C5), 85.6 (C1ꢀ), 79.5 (C4ꢀ), 78.0 (C3ꢀ), 72.4 (C2ꢀ), 62.9 (C5ꢀ), 26.8 (CH3Piv),
26.6 (CH3Piv); Mass (ESI-MS) m/z calcd. for [C19H28N2O8+Na]+ 435.1738, found
435.1725, err. 3.1 ppm.
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