Mt-tRNA components: Synthesis of (2-Thio)uridines modified with blocked glycine/taurine moieties at C-5,1

Article abstract of DOI:10.1080/15257770.2013.838261

In this paper, we discuss the usefulness of reductive amination of 5-formyl-2′,3′-O-isopropylidene(-2-thio)uridine with glycine or taurine esters in the presence of sodium triacetoxyborohydride (NaBH(OAc) ₃) for the synthesis of the native mitochondrial (mt) tRNA components 5-carboxymethylaminomethyl(-2-thio)uridine (cmnm⁵(s²)U) and 5-taurinomethyl(-2-thio)uridine (τm⁵(s²)U) with a blocked amino acid function. 2-(Trimethylsilyl)ethyl and 2-(p-nitrophenyl)ethyl esters of glycine and 2-(2,4,5-trifluorophenyl)ethyl ester of taurine were selected as protection of carboxylic and sulfonic acid residues, respectively. The first synthesis of 5-formyl-2′,3′-O-isopropylidene-2-thiouridine is also reported.

Full text of DOI:10.1080/15257770.2013.838261

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mt-tRNA Components: Synthesis of (2-
Thio)Uridines Modified with Blocked
Glycine/Taurine Moieties at C-5,1
Grazyna Leszczynska a , Piotr Leonczak a , Agnieszka Dziergowska a
& Andrzej Malkiewicz a
a Institute of Organic Chemistry , Lodz University of Technology ,
Lodz , Poland
Published online: 18 Oct 2013.
To cite this article: Grazyna Leszczynska , Piotr Leonczak , Agnieszka Dziergowska & Andrzej
Malkiewicz (2013) mt-tRNA Components: Synthesis of (2-Thio)Uridines Modified with Blocked
Glycine/Taurine Moieties at C-5,1, Nucleosides, Nucleotides and Nucleic Acids, 32:11, 599-616, DOI:
10.1080/15257770.2013.838261
To link to this article: http://dx.doi.org/10.1080/15257770.2013.838261
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Nucleosides, Nucleotides and Nucleic Acids, 32:599–616, 2013
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2013.838261
mt-tRNA COMPONENTS: SYNTHESIS OF (2-THIO)URIDINES
MODIFIED WITH BLOCKED GLYCINE/TAURINE MOIETIES AT C-5,1
Grazyna Leszczynska, Piotr Leonczak, Agnieszka Dziergowska, and Andrzej
Malkiewicz
Institute of Organic Chemistry, Lodz University of Technology, Lodz, Poland
In this paper, we discuss the usefulness of reductive amination of 5-formyl-2ꢁ,3ꢁ-O-
2
isopropylidene(-2-thio)uridine with glycine or taurine esters in the presence of sodium triacetoxy-
borohydride (NaBH(OAc)3) for the synthesis of the native mitochondrial (mt) tRNA components
5-carboxymethylaminomethyl(-2-thio)uridine (cmnm5(s2)U) and 5-taurinomethyl(-2-thio)uridine
(τm5(s2)U) with a blocked amino acid function. 2-(Trimethylsilyl)ethyl and 2-(p-nitrophenyl)ethyl
esters of glycine and 2-(2,4,5-trifluorophenyl)ethyl ester of taurine were selected as protection of car-
boxylic and sulfonic acid residues, respectively. The first synthesis of 5-formyl-2ꢁ,3ꢁ-O-isopropylidene-
2-thiouridine is also reported.
Keywords Modified nucleosides; reductive amination; 5-formyluridine (f5U); 5-formyl-
2-thiouridine (f5s2U); 5-carboxymethylaminomethyl(-2-thio)uridine (cmnm5(s2)U); 5-
taurinomethyl(-2-thio)uridine (τm5(s2)U)
INTRODUCTION
5-Carboxymethylaminomethyluridine (cmnm5U 1, Figure 1) and its 2-
thioanalogue (cmnm5s2U 2, Figure 1) have been found in the wobble po-
sition (the first anticodon letter) of Saccharomyces cerevisiae mitochondrial
(mt) tRNAs specific for Leu and Lys, respectively.[1, 2] A structurally sim-
ilar pair of wobble uridines, 5-taurinomethyluridine (τm5U 3, Figure 1)
and 5-taurinomethyl-2-thiouridine (τm5s2U 4, Figure 1), have been isolated
from mammalian mt-tRNAs specific for Leu(UUR) and Lys, respectively.[1, 3]
Recently, taurine-modified nucleosides have been also identified in the mt-
tRNALeu,Lys of a squid, Loligo bleekeri.[4]
Received 10 July 2013; accepted 22 August 2013.
This study was supported by grant 1306/B/H03/2011/40 from the National Science Centre, Poland
(for G.L).
Address correspondence to Grazyna Leszczynska, Institute of Organic Chemistry, Lodz University
of Technology, Zeromskiego 116, 90-924 Lodz, Poland. E-mail: grazyna.leszczynska@p.lodz.pl
599

600
Grazyna Leszczynska et al.
FIGURE 1 Chemical structures of 5-carboxymethylaminomethyluridine (cmnm5U, 1), 5-
carboxymethylaminomethyl-2-thiouridine (cmnm5s2U, 2), 5-taurinomethyluridine (τm5U, 3),
and 5-taurinomethyl-2-thiouridine (τm5s2U, 4).
Modified nucleosides 1–4 are crucial structural elements of mt-tRNA
that determines the biopolymer decoding capacity.[2, 4, 5] Site-specific mu-
tations in yeast mt-DNA induce the loss of modifications 1 and 2 leading
to a significant failure of mitochondrial protein biosynthesis.[6, 7] A similar
mechanism has been proposed to explain the absence of modifications 3
and 4 in human mt-tRNA specific for Leu(UUR) and Lys, respectively, and
this phenomenon results in decoding disorders responsible for the incur-
able mitochondrial diseases MELAS and MERRF.[8, 9] Recently, yeast cells
have been proposed as a useful model for studies of molecular and cel-
lular effects related to the above-mentioned mitochondrial pathologies.[7]
For a long time, modified tRNA fragments have served as key components
of oligomer synthesis by the semienzymatic “RNA fragment recombination”
methodology.[5, 10] Using this method, 3ꢁ,5ꢁ-O-bisphosphates of cmnm5U and
τm5U have been inserted into position 34 of Escherichia coli tRNALeu(UUR)
and the same technology has been applied in constructing an analogue of
the human mt-tRNALeu(UUR) anticodon arm domain modified with τm5U
at the wobble position.[5] The incorporation of the hypermodified nucle-
osides 1–4 into oligoribonucleotide sequences by phosphoramidite chem-
istry on solid support requires appropriate protection of their amino acid
residues. The variously substituted phenols have been tested for the pro-
tection of the sulfonic acid residue of nucleosides 3,4, and the synthesis of
respective, fully blocked 3ꢁ-O-phosphoramidites of τm5U and τm5s2U has
also been reported.[11] Their application to the synthesis of modified RNAs
was, however, limited to the dimer level, and final oligomers, in particular
those modified with τm5s2U, were contaminated with considerable amounts
of side products.[12]
In the literature, only one paper describes the chemical synthesis of
RNA anticodon triplets modified with cmnm5U and cmnm5s2U (a triester
approach in solution).[13]

(2-thio)uridines Modified with Blocked Glycine/Taurine
601
Reductive amination of 5-formyl-2ꢁ-deoxyuridine (f5dU) has found wide
application in the synthesis of 5-alkylamino derivatives of pyrimidine nucle-
osides[14] and nucleic acid conjugates.[15] To date, no reports have been
published concerning the utilization of 5-formyluridine (f5U) for reduc-
tive amination with amino acids or the synthesis of 5-formyl-2-thiouridine
(f5s2U).
In this paper, we present a practical approach for synthesis of 5-
methyleneaminoacid(-2-thio)uridines utilizing a two-step procedure of re-
ductive amination of 5-formyl(-2-thio)uridine with glycine and taurine es-
ters in the presence of sodium triacetoxyborohydride (NaBH(OAc)3) as a
reducing agent. Based on literature data, alternative procedures that uti-
lize 5-chloromethyl(-2-thio)uridine[16] or a quaternary ammonium salt of
5-pyrrolidinomethyluridine[17] as the starting material have also been exper-
imentally examined.
RESULTS AND DISCUSSION
Two groups, 2-(p-nitrophenyl)ethyl (NPE, 8a, Scheme 1) and 2-
(trimethylsilyl)ethyl (TMSE, 8b, Scheme 1), were tested for the pro-
tection of the glycine carboxyl function. The usefulness of the above-
mentioned protecting groups was previously verified by incorporation of
N6-threonylcarbamoyladenosine (t6A) and its 2-methylthio analogue
(ms2t6A) into the anticodon arm sequence of tRNA by phosphoramidite
chemistry.[18] N -Boc glycine esters 7a and 7b (Scheme 1) were obtained by
condensation of the N -Boc-protected glycine 5 with 2-(p-nitrophenyl)ethyl
or 2-(trimethylsilyl)ethyl alcohol, (6a) or (6b), respectively, in the presence
of DCC. We found that both masking groups in compounds 7a and 7b were
stable in 8 M ethanolic ammonia and easily removed under standard condi-
tions for their deprotection during oligoribonucleotide synthesis, 10% DBU
in MeCN, 40 min, 40◦C for NPE ester and 1 M TBAF in NMP for TMSE
ester.[18] The stability of the 2-(trimethylsilyl)ethyl ester protection in 8 M
ethanolic ammonia offers a simple way for simultaneous deblocking of alka-
line labile protecting groups, e.g., 2-cyanoethyl, -tac (or -pac) and cleavage
oligomer from CPG-support without risk of amide formation. Subsequent
removal of TMSE group can be performed together with TBDMS protection
of sugar residues. In contrary, the 2-(p-nitrophenyl)ethyl group is removed
by DBU in β-elimination process before deprotection of base-labile groups
SCHEME 1 Reagents and conditions: (i) DCC, py, MeCN, 12 h, 0◦C → rt; (ii) 4 M HCl/dioxane, 1.5 h, rt.

602
Grazyna Leszczynska et al.
SCHEME 2 Reagents and conditions: (i) Boc2O, Bu4NOH, H2O, acetone, 12 h, rt; (ii) triphosgene,
DMF(cat.), CH2Cl2, 30 min, rt; (iii) Et3N, CH2Cl2, 2 h, 0◦C → rt; (iv) 4 M HCl/dioxane, 1 h, rt; (v)
TMSCl, 2,2-dimethoxypropane, MeOH, 24 h, rt; (vi) NaBH4, THF, 15 min, 65◦C; (vii) MeOH, 2.5 h,
reflux.
as well as cleavage oligomer from the solid support. This strategy also elim-
inates the risk of aminolysis. Despite numerous attempts we were not able
to protect the taurine acidic function with the TMSE group, while the NPE
blockage was found too labile to be used in oligoribonucleotide synthesis.
The list of known masking groups for the protection of the sulfonic acid
residue is short, in particular for taurine-modified nucleosides applied in
oligomer syntheses via phosphoramidite chemistry.[11, 12]
We synthesized several 2-arylethyl esters of N -Boc taurine (p-
trifluoromethyl-, 2,4-difluoro-, 2,4,5-trifluoro-, 2,4-dichloro-, p-fluoro-,
p-nitrophenylethyl esters) by conversion of N -Boc taurine tetrabuty-
lammonium salt[19] (10) (Scheme 2) to sulfonyl chloride 11,[20] which
was subsequently reacted with an appropriate alcohol using a modified
procedure of sulfonyl chloride esterification.[21] To synthesize alcohols,
commercially available variously substituted phenylacetic acids were almost
quantitatively converted to methyl esters,[22] which were then reduced with
NaBH4/MeOH/THF to desired products.[23] Nevertheless, only N -Boc
taurine 2-(2,4,5-trifluorophenyl)ethyl ester (12) revealed the stability
under alkaline conditions, e.g., triethylamine, isopropyldiethylamine,
necessary for the synthesis of suitable monomer unit. We found that the
2-(2,4,5-trifluorophenyl)ethyl ester protection can be easily removed by
the β-elimination process under relatively mild conditions (10% DBU in
MeCN, 40 min, 40◦C).
N -Boc glycine and taurine esters, 7a, 7b, and 12, respectively, were trans-
formed to hydrochlorides 8a–8c with very good yields by treatment with 4 M
hydrochloric acid in dioxane (Schemes 1 and 2).
In the literature, 5-chloromethyl-2ꢁ,3ꢁ-O-isopropylidene(-2-thio)uridine
or quaternary ammonium salt of 2ꢁ,3ꢁ-O-isopropylidene-5-pyrroli-
dinomethyl(-2-thio)uridine were reacted with methyl, ethyl or tert-
butyl glycine esters to give products of nucleophilic substitution at the
activated 5-methylene group, albeit with moderate yields.[16, 17] Using
these procedures, we tried to synthesize cmnm5U, τm5U, and their

(2-thio)uridines Modified with Blocked Glycine/Taurine
603
SCHEME 3 Reagents and conditions: (i) Et3N, DMF or MeCN, rt; (ii) TFAA, py, 1 h, 0◦C → rt.
2-thio analogues protected with NPE/TMSE (for glycine residue) and
2-(2,4,5-trifluorophenyl)ethyl (for taurine residue; Scheme 3). The result-
ing nucleosides 19a–19c/20a–20c underwent partial degradation under
conditions of silica gel column chromatography. Thus, the crude products
19a–19c/20a–20c were treated with trifluoroacetic anhydride (TFAA)
in pyridine to obtain amides 21a–21c/22a–22c, which are stable during
silica gel column purification. The trifluoroacetyl group has been used
as standard protection of the aliphatic, secondary amine function in the
synthesis of RNA fragments modified with uridines containing such a
group.[16, 24] The final yields of the isolated products 21a–21c/22a–22c
(Scheme 3) were poor (15–25%; experimental data not shown).
As an alternative, we tested reductive amination of 5-formyl(-2-
thio)uridine with amino acid esters in the presence of NaBH(OAc)3
(Scheme 4). We found that oxidation of 5-hydroxymethyl-2ꢁ,3ꢁ-O-
isopropylideneuridine (23) to 5-formyl-2ꢁ,3ꢁ-O-isopropylideneuridine (25)
with activated MnO2 in acetone/CH2Cl2 (4:1, v/v) solution at elevated tem-
perature (55◦C, 2–7 h) gives better yield than the previously reported proce-
dure[25] (55% versus 37%). Surprisingly, the same protocol may be used for
the oxidation of 5-hydroxymethyl-2ꢁ,3ꢁ-O-isopropylidene-2-thiouridine (24)
to the appropriate 5-formyl-2-thiouridine 26 without the formation of side-
products of oxidation/oxidative desulphurization of the 2-thiocarbonyl func-
tion.[26]
The 5-formyluridine 25 and its 2-thio analogue 26 were subjected to
reductive amination with amino acid esters 8a–8c (Scheme 4) according to a
two-step procedure that involves the formation of imines 27a–27c/28a–28c
and subsequently their reduction with NaBH(OAc)3. The crude secondary
amines 19a–19c and their 2-thio analogues 20a–20c were acylated with TFAA
in pyridine to respective amides 21a-21c/22a-22c. According to NMR data,
resulting uridines exist as a mixture of rotamers about the –NC(O)CF3
amide bond. Glycine derivatives 21a and 21b were isolated in 66% and
81% yields, respectively (the yields calculated refer to aldehydes 25 and
26, respectively). For the 2-thio analogues 22a and 22b, the yields were

604
Grazyna Leszczynska et al.
SCHEME 4 Reagents and conditions: (i) activated MnO2, acetone : CH2Cl2 (4:1, v/v), 2–7 h, 55◦C, (ii)
Et3N, CH2Cl2/DMF, 1–1.5 h, rt; (iii) NaBH(OAc)3, 40 min–2 h, rt; (iv) TFAA, py, 1 h, 0◦C → rt; (v) 25%
aq. AcOH, 1 h, 90◦C.
slightly lower, 56% and 78%, respectively. However, reductive amination
of aldehydes 25 and 26 with taurine 2-(2,4,5-trifluorophenyl)ethyl ester 8c
gave a notably lower yield (∼35%). The significant decrease in the yield
of reductive amination involving taurine ester 8c may be explained as a
result of unfavorable equilibrium position of the reversible reaction of Schiff
bases formation (27c, 28c) and/or their less susceptibility to reduction with
sodium triacetoxyborohydride. In this specific case, a Michael-type addition
of phenyl vinylsulfate with the 5-aminomethyl(-2-thio)uridine derivative is
the most effective methodology and should be considered the method of
choice.[11]
It is worth to notice that presented strategy of the reductive amination
allowed to increase the yield of C-N-C linkage formation by 40%–60% in the
case of glycine esters and 20% in the case of taurine ester in comparison
with methods described in the literature.[16, 17]
The acetal protection of the sugar moieties in compounds 21a–21c and
22a–22c was removed with 25% acetic acid (90◦C, 1h) and the title com-
pounds 29a–29c and 30a–30c were isolated with 80% yield.
In conclusion, we report facile synthesis of (2-thio)uridines substituted
at C-5,1 with protected glycine or taurine residues (29a–29c/30a–30c) as
useful intermediates in the chemical synthesis of oligomers modified with

(2-thio)uridines Modified with Blocked Glycine/Taurine
605
5-carboxymethylaminomethyl(-2-thio)uridine (1,2) and 5-taurinomethyl(-2-
thio)uridine (3,4).
EXPERIMENTAL
General Procedure for the Synthesis of Hydrochlorides of Glycine
Esters 8a and 8b
N -Boc glycine 5 (1.1 g, 6.0 mmol, 1.0 equiv) was dissolved in anhydrous
MeCN (6.0 mL); alcohol 6a/6b (6.6 mmol, 1.1 equiv) and anhydrous pyri-
dine (1.5 mL) were added. The mixture was cooled in an ice bath and DCC
(1.4 g, 6.6 mmol, 1.1 equiv) was added. The cooling bath was removed and
the mixture was stirred overnight at room temperature (rt). The reaction
was quenched by 1 M aqueous solution of oxalic acid (1.1 mL) and stirred
at rt for 30 min. The precipitated DCU was filtered and washed with EtOAc.
The filtrate was extracted with 1 M aqueous solution of NaHCO3 (3 mL),
water (3 mL), and then dried over anhydrous MgSO4 and evaporated under
reduced pressure. The residue was co-evaporated with toluene to give N -Boc
glycine ester 7a/7b. The crude compound 7a/7b (5.0 mmol) was treated
with 4 M solution of HCl/dioxane (50 mL) and stirred at rt for 1.5 h. The
mixture was concentrated under reduced pressure and precipitated with
hexane affording hydrochloride 8a/8b.
Hydrochloride of glycine 2-(p-nitrophenyl)ethyl ester (8a)
Compound 7a was isolated in 95% yield. TLC Rf = 0.57 (CHCl3/MeOH
1
98:2, v/v); H NMR (250 MHz, CD3OD) δ 1.45 (s, 9H), 3.08 (t, 2H, J =
6.50 Hz), 3.88 (bs, 2H), 4.41 (t, 2H, J = 6. 50 Hz), 4.98 (bs, 1H), 7.37–7.41
(m, 2H), 8.16–8.20 (m, 2H). Compound 8a was obtained in 95% yield as a
white powder. TLC Rf = 0.15 (CHCl3/MeOH 98:2 v/v); 1H NMR (250 MHz,
CD3OD) δ 3.15 (t, 2H, J = 6.50 Hz), 3.81 (s, 2H), 4.52 (t, 2H, J = 6.50 Hz),
7.52 (d, 2H, J = 8.75 Hz), 8.17 (d, 2H, J = 8.75 Hz); 13C NMR (62.9 MHz,
CD3OD) δ 34.10, 39.58, 65.51, 123.24, 129.78, 145.72, 146.92, 167.07; HRMS
m/z calcd for C10H13N2O4 [M+H]+ 225.0875, found 225.0873.
Hydrochloride of glycine 2-trimethylsilylethyl ester (8b)
Compound 7b was isolated in 85% yield. TLC Rf = 0.66 (CHCl3/MeOH
98:2, v/v); 1H NMR (250 MHz, CD3OD) δ 0.05 (s, 9H), 1.04–1.11 (m, 2H),
1.32 (s, 9H,), 3.79 (s, 2H), 4.30–4.37 (m, 2H). Compound 8b was obtained
in 95% yield as a white powder. TLC Rf = 0.25 (CHCl3/MeOH 98:2 v/v);
1H NMR (250 MHz, CD3OD) δ 0.06 (s, 9H,), 1.04–1.11 (m, 2H), 3.80 (s,
2H), 4.30–4.37 (m, 2H); 13C NMR (62.9 MHz, CD3OD) δ –2.94, 16.86, 39.78,
64.34, 167.15; HRMS m/z calcd for C7H18NO2Si [M+H]+ 176.1107, found
176.1111.

606
Grazyna Leszczynska et al.
Hydrochloride of taurine 2-(2,4,5-trifluorophenyl)ethyl ester (8c)
Taurine 9 (1.25 g, 10 mmol, 1 equiv) was dissolved in water (10 mL), and
then 40% aqueous solution of n-Bu4NOH (6.5 mL, 10 mmol, 1 equiv) was
added. Next, Boc2O (2.18 g, 10 mmol, 1 equiv) in acetone (30 mL) was added
dropwise. The mixture was stirred overnight at rt. Acetone was evaporated
under reduced pressure and the aqueous phase was extracted with CH2Cl2
(3 × 12 mL). Organic layers were combined and dried over MgSO4. The
solvent was evaporated affording 4.4 g (95%) of tetrabutylammonium salt of
N -Boc taurine (10) as a yellowish oil. Rf = 0.6 (CHCl3/MeOH 9:1, v/v); 1H
NMR (250 MHz, CDCl3) δ 1.00 (t, 12H, J = 7.50 Hz), 1.36–1.51 (m, 17H),
1.58–1.71 (m, 8H), 2.92–2.97 (m, 2H), 3.25–3.32 (m, 8H), 3.52–3.57 (m,
2H); 13C NMR (62.9 MHz, CDCl3) δ 13.4, 19.49, 23.75, 28.26, 36.83, 50.51,
58.44, 78.17, 155.88. The crude material 10 (2 g, 4.29 mmol, 1.0 equiv) was
dissolved in anhydrous CH2Cl2 (15 mL), DMF (0.032 mL, 0.43 mmol, 0.43
equiv), and then triphosgene (0.51 g, 1.72 mmol, 0.4 equiv) was added. The
mixture was stirred at rt for 30 min and then concentrated under reduced
pressure, dissolved in EtOAc/hexane (1:1, v/v, 20 mL) and filtered over a
small amount of silica gel with EtOAc/hexane (1:1, v/v) as eluent. Evap-
oration under reduced pressure afforded 0.85 g (81%) of N -Boc taurine
chloride 11 as a white powder. Rf = 0.53 (EtOAc/hexane 1:1, v/v); 1H NMR
(250 MHz, CDCl3) δ 1.46 (s, 9H), 2.99−3.04 (m, 2H), 3.07–3.14 (m, 2H),
4.34 (bs, 1H). 2-(2,4,5-Trifluorophenyl)ethanol 15 (0.54 g, 3.07 mmol, 1.0
equiv) and triethylamine (0.47 mL, 3.38 mmol, 1.1 equiv) were dissolved
in CH2Cl2 (1.2 mL) and cooled in an ice bath. N -Boc taurine chloride 11
(1.12 g, 4.61 mmol, 1.5 equiv) in CH2Cl2 (8 mL) was then added drop-
wise. After the addition, the cooling bath was removed and the mixture was
stirred at rt for 2 h. Then, the mixture was diluted with CH2Cl2 (8 mL),
washed with saturated aqueous NH4Cl (12.5 mL), H2O (12.5 mL) and brine
(12.5 mL), and then dried and evaporated. The crude product was puri-
fied by column chromatography using hexane/EtOAc (1:1, v/v) as eluent to
give 0.92 g (78%) of N -Boc taurine 2-(2,4,5-trifluorophenyl)ethyl ester (12).
Rf = 0.72 (CHCl3/MeOH 98:2, v/v), 0.84 (EtOAc/hexane 1:1,
v/v); 1H NMR (250 MHz, CDCl3) δ 1.44 (s, 9H), 3.05 (t, 2H,
J = 7.50 Hz), 3.27 (t, 2H, J = 7.50 Hz), 3.56 (q, 2H, J = 7.50 Hz), 4.40 (t, 2H,
J = 7.50 Hz), 5.05 (bs, 1H), 6.89–6.99 (m, 1H), 7.05–7.15 (m, 1H); 13C NMR
(62.9 MHz, CDCl3) δ 26.47, 26.98, 33.50, 48.45, 66.34, 78.33, 103.49–104.28
(m), 116.89–117.28 (m), 153.78. Compound 12 (0.9 g, 2.35 mmol) was
treated with a solution of 4 M HCl/dioxane (47 mL) and stirred at rt for
1 h. The mixture was concentrated under reduced pressure, washed with
petroleum ether (20 mL) and filtered, affording 0.68 g (91%) of hydrochlo-
ride of taurine 2-(2,4,5-trifluorophenyl)ethyl ester (8c) as a white powder.
1H NMR (250 MHz, CD3OD) δ 3.02 (bs, 2H), 3.28 (bs, 2H), 3.58 (bs, 2H),
4.43 (bs, 2H), 7.00–7.10 (m, 1H), 7.17–7.30 (m, 1H); 13C NMR (62.9 MHz,

(2-thio)uridines Modified with Blocked Glycine/Taurine
607
CD3OD) δ 29.27; 35.52, 47.21, 71.16, 106.48 (dd, J = 21.30 Hz, J = 28.90 Hz),
120.20 (dd, J = 5.30 Hz, J = 19.60 Hz), 121.71 (dt, J = 4.80 Hz, J = 17.90 Hz),
148.25 (dd, J = 12.20 Hz, J = 242.30 Hz), 150.54 (dt, J = 13.30 Hz, J =
248.60 Hz), 157.51 (dd, J = 9.70 Hz, J = 244.40 Hz); HRMS m/z calcd for
C10H13NO3F3S [M+H]+ 284.0568, found 284.0566.
2-(2,4,5-trifluorophenyl)ethanol (15)
To a suspension of 2,4,5-trifluorophenylacetic acid (13, 5 g, 26.3 mmol,
1 equiv) in anhydrous methanol (11 mL), 2,2-dimethoxypropane (42 mL)
and TMS-Cl (0.33 mL, 2.6 mmol, 0.1 equiv) were added. After stirring at rt
for 24 h, the mixture was concentrated under reduced pressure and then
purified by gravity column chromatography on silica gel using CHCl3 as
eluent. 5.26 g (98%) of 2,4,5-trifluorophenylacetic acid methyl ester (14)
was obtained as a transparent liquid. TLC Rf = 0.68 (hexane/AcOEt, 3:1
1
v/v); H NMR (250 MHz, CDCl3) δ 3.62 (s, 2H), 3.72 (s, 3H), 6.88–6.99
(m, 1H), 7.07–7.17 (m, 1H). Compound 14 (5.0 g, 24.5 mmol, 1 equiv) was
dissolved in THF (88 mL) and sodium borohydride powder (5.5 g, 0.15 mol,
6 equiv) was added. The suspension was stirred at 65◦C for 15 min and then
methanol (88 mL) was added dropwise. The mixture was refluxed for 2.5 h.
After cooling to rt, the reaction was quenched by 2 N aq. HCl (4.9 mL). The
mixture was concentrated under reduced pressure and extracted with EtOAc
(3 × 100 mL). The combined organic layers were dried over anhydrous
MgSO4, filtered, and concentrated under reduced pressure. The residue
was purified by gravity column chromatography (CHCl3), affording 3.7 g
(86%) of 2-(2,4,5-trifluorophenyl)ethanol (15) as a brownish viscous liquid.
1
TLC Rf = 0.38 (hexane/AcOEt, 2:1 v/v); H NMR (250 MHz, CDCl3) δ
2.85 (t, J = 7.50 Hz, 2H), 3.85 (t, J = 7.50 Hz, 2H), 6.85–6.96 (m, 1H),
7.04–7.14 (m, 1H); 13C NMR (176 MHz, CDCl3) δ 31.75, 61.99, 105.34 (dd,
J = 20.60 Hz, J = 28.30 Hz), 118.74 (dd, J = 6.20 Hz, J = 19.00 Hz), 121.93
(dt, J = 4.80 Hz, J = 18.70 Hz), 146.63 (ddd, J = 3.20 Hz, J = 11.80 Hz,
J = 244.30 Hz), 148.70 (dt, J = 13.70 Hz, J = 249.20 Hz), 156.11 (ddd, J =
2.50 Hz, J = 8.90 Hz, J = 243.90 Hz).
General Procedure for the Synthesis of 5-formyl-2ꢀ,3ꢀ-O-
isopropylidene(-2-thio)uridine (25/26)
Nucleoside 23[27]/24[28] (9.5 mmol, 1 equiv) was dissolved in
acetone/CH2Cl2 (4:1, v/v; 80 mL), then activated MnO2 (14.8 g, 0.17 mol,
17.5 equiv) was added. After being stirred for 2 h (23)/7 h (24) at 55◦C,
the mixture was cooled to rt, and MnO2 was centrifuged. The solution was
transferred to a fresh tube and the residual MnO2 was resuspended in a
mixture of acetone/CH2Cl2 (1:1, v/v; 3 × 40 mL), shaken vigorously, and

608
Grazyna Leszczynska et al.
centrifuged. The combined solutions were filtered through Celit. The fil-
trate was concentrated under reduced pressure and the solid residue was
purified by column chromatography using MeOH in CHCl3.
5-formyl-2ꢀ,3ꢀ-O-isopropylideneuridine (25)
Compound 25 was isolated in 55% yield using 6% MeOH in CHCl3 as
eluent. TLC Rf = 0.45 (CHCl3/MeOH, 9:1 v/v); 1H NMR (700 MHz, DMSO-
d6) δ 1.29 (s, 3H), 1.48 (s, 3H), 3.56–3.64 (m, 2H), 4.23–4.25 (m, 1H), 4.75
(dd, 1H, J = 6.25 Hz, J = 3.00 Hz), 4.94 (dd, 1H, J = 6.25 Hz, J = 2.25 Hz),
5.16 (t, 1H, J = 5.00 Hz), 5.85 (d, 1H, J = 2.25 Hz), 8.61 (s, 1H), 9.76 (s, 1H),
11.80 (s, 1H); 13C NMR (176 MHz, DMSO-d6) δ 24.61, 26.44, 60.63, 80.11,
84.14, 87.28, 92.55, 109.96, 112.11, 147.73, 149.13, 161.21, 185.68; HRMS
(FAB+) calcd for C13H17N2O7 [M+H]+ 313.1036, found 313.1042.
5-formyl-2ꢀ,3ꢀ-O-isopropylidene-2-thiouridine (26)
Compound 26 was isolated in 65% yield using 2% MeOH in CHCl3 as
eluent. TLC Rf = 0.50 (CHCl3/MeOH, 9:1 v/v); 1H NMR (250 MHz, DMSO-
d6) δ 1.34 (s, 3H), 1.56 (s, 3H), 3.70 (dd, 1H, J = 3.50 Hz, J = 11.90 Hz),
3.77 (dd, 1H, J = 2.80 Hz, J = 11.90 Hz), 4.34–4.35 (m,1H), 4.83 (dd, 1H,
J = 3.50 Hz, J = 6.30 Hz), 4.92 (dd, 1H, J = 2.10 Hz, J = 6.30 Hz), 6.74 (d,
1H, J = 2.10 Hz), 8.79 (s, 1H), 9.83 (s, 1H); 13C NMR (62.9 MHz, DMSO-d6) δ
25.69, 27.45, 60.86, 79.78, 85.64, 87.79, 94.71, 113.28, 114.42, 146.87, 158.55,
177.33, 186.71; HRMS (FAB+) calcd for C13H17N2O6S [M+H]+ 329.0807,
found 329.0802.
General Procedure for the Synthesis of Compounds
21a-21c/22a-22c
Nucleoside 25/26 (4.2 mmol, 1.0 equiv) was suspended in CH2Cl2
(22 mL) and a few drops of DMF was added to complete substrate disso-
lution. Then, hydrochloride of amino acid ester 8a-8c (5.0 mmol, 1.2 equiv)
and Et3N (702 μL, 5.0 mmol, 1.2 equiv) were added and the mixture was
stirred at rt for 1–1.5 h. TLC analysis (CHCl3/MeOH 95:5 or 90:10, v/v)
showed full conversion of aldehyde 25/26 into suitable imine 27a-27c/28a-
28c. The reaction mixture was treated with NaBH(OAc)3 (1.1 g, 5.0 mmol,
1.2 equiv) and stirring continued for another 40 min for compounds 27a,
27c, 28a or for 2 h for 27b, 28b, 28c. The reaction was quenched with 5%
aq. NaHCO3 (8 mL) and then extracted with CH2Cl2 (3 × 30 mL). The
combined organic layers were dried over MgSO4, filtered, and concentrated
under reduced pressure. The crude material 19a-19c/20a-20c (3.5 mmol,
1 equiv) was co-evaporated with anhydrous pyridine, dissolved in the same
solvent (59 mL), cooled in ice bath and trifluoroacetic anhydride (2.5 mL,
17.5 mmol, 5.0 equiv) was added dropwise. The reaction mixture was stirred

(2-thio)uridines Modified with Blocked Glycine/Taurine
609
at 0◦C for 1 h, quenched with 5% aq. NaHCO3 (25 mL) and extracted
with CH2Cl2 (3 × 50 mL). The combined organic layers were dried over
MgSO4, filtered, and concentrated under reduced pressure. The residual
pyridine was removed by co-evaporation with anhydrous toluene, and the
resulting foam was purified by column chromatography affording product
21a-21c/22a-22c as rotamers about –NC(O)CF3 amide bond (two chemi-
1
cal shifts were observed for some of the H and 13C NMR resonances; the
secondary shifts in 13C NMR spectra are given in parentheses). The yields
calculated for 21a-21c/22a-22c refer to substrate 25 or 26.
2ꢀ,3ꢀ-O-isopropylidene-N-[(1-β-D-ribofuranosyl-1H-pyrimidin-5-
yl)methyl]-N-trifluoroacetylglycine 2-(p-nitrophenyl)ethyl ester
(21a)
Compound 21a was purified by column chromatography with CHCl3 as
eluent to obtain a white foam in 66% yield. TLC Rf = 0.52 (CHCl3/MeOH
95:5, v/v); 1H NMR (250 MHz, CDCl3) δ 1.36 (s, 3H), 1.58 (s, 3H), 3.08 (t,
2H, J = 6.50 Hz), 3.80 (dd, 1H, J = 2.50 Hz, J = 12.00 Hz), 4.00 (dd, 1H, J =
2.00 Hz, J = 12.00 Hz), 4.09 (d, 1H, J = 14.25 Hz), 4.24 (d, 1H, J = 14.25 Hz),
4.40–4.63 (m, 5H), 4.77–4.94 (m, 2H), 5.80 (d, 0.9H, J = 2.75 Hz), 5.87 (d,
0.1H, J = 3.00 Hz), 7.38 (d, 2H, J = 8.75 Hz), 7.80 (s, 0.1H), 8.01 (s, 0.9H),
8.19 (d, 2H, J = 8.75 Hz), 8.79 (s, 0.9H), 8.89 (s, 0.1H); 13C NMR (176 MHz,
CDCl3) δ 24.94 (25.11), 26.93 (27.04), 34.58, 46.50, 50.45 (49.12), 62.57,
65.00, 80.69 (80.16), 85.11 (84.36), 87.33 (86.27), 94.59 (93.24), 107.44
(107.70), 113.71, 114.53 (q, J = 104.01 Hz), 123.72, 129.53, 143.19, 144.65,
146.92, 149.37, 157.92 (q, J = 36.43 Hz), 163.93, 167.93; HRMS calcd for
C25H26N4O11F3 [M-H]− 615.1550, found 615.1559.
2ꢀ,3ꢀ-O-isopropylidene-N-[(1-β-D-ribofuranosyl-1H-pyrimidin-5-
yl)methyl]-N-trifluoroacetylglycine 2-(trimethylsilyl)ethyl ester
(21b)
Compound 21b was purified by column chromatography using 2%
MeOH in CHCl3 as eluent to obtain a white foam in 81% yield. TLC Rf
= 0.46 (CHCl3/MeOH 95:5, v/v); 1H NMR (250 MHz; CDCl3): δ 0.04–0.06
(m, 9H), 0.98–1.05 (m, 2H), 1.36 (s, 3H), 1.58 (s, 3H), 3.80 (dd, 1H, J =
2.75 Hz, J = 12.25 Hz), 3.99 (dd, 1H, J = 2.00 Hz, J = 12.25 Hz), 4.09–4.35
(m, 4H), 4.44–4.62 (m, 3H), 4.86 (dd, 1H, J = 2.50 Hz, J = 6.00 Hz),
4.92 (dd, 1H, J = 1.75 Hz, J = 6.00 Hz), 5.79 (d, 0.9H, J = 2.50 Hz),
5.91 (d, 0.1H, J = 2.75 Hz), 7.85 (s, 0.1H), 8.01 (s, 0.9H), 8.71 (s, 0.9H),
8.80 (s, 0.1H); 13C NMR (176 MHz, CDCl3): δ -1.82 (-1.76), 17.14 (17.07),
24.91 (25.07), 26.89 (27.02), 46.49 (44.59), 50.62 (49.28), 62.46 (62.17),
64.27 (63.76), 80.63 (80.12), 84.93 (84.31), 87.29 (86.25), 94.55 (92.84),
107.68 (107.83), 113.66 (114.12), 115.68 (q, J = 287.76 Hz), 143.18, 149.73

610
Grazyna Leszczynska et al.
(149.64), 157.90 (q, J = 36.78 Hz), 163.75 (162.65), 168.21 (167.73); HRMS
calcd for C22H33N3O9F3Si [M+H]+ 568.1938, found 568.1937.
2ꢀ,3ꢀ-O-isopropylidene-N-[(1-β-D-ribofuranosyl-1H-pyrimidin-5-
yl)methyl]-N-trifluoroacetyltaurine 2-(2,4,5-trifluorophenyl)ethyl
ester (21c)
Compound 21c was purified by column chromatography using 2% of
MeOH in CHCl3 as eluent to obtain a white foam in 36% yield. TLC Rf =
0.42 (CHCl3/MeOH 95:5, v/v); 1H NMR (700 MHz, CDCl3) δ 1.35 (s, 3H),
1.58 (s, 3H); 3.03–3.08 (m, 2H), 3.41–3.57 (m, 2H), 3.75–3.83 (m, 1H),
3.91–3.98 (m, 1H), 4.01–4.24 (m, 3H), 4.35–4.47 (m, 4H), 4.77 (dd, 0.27H,
J = 2.80 Hz, J = 5.60 Hz), 4.85 (dd, 0.7H, J = 2.80 Hz, J = 6.30 Hz), 4.87 (dd,
0.3H, J = 2.80 Hz, J = 5.60 Hz), 4.91 (dd, 0.7H, J = 2.80 Hz, J = 6.30 Hz),
5.80 (d, 0.7H, J = 2.80 Hz), 5.93 (d, 0.3H, J = 2.80 Hz), 6.92–6.96 (m, 1H),
7.07–7.14 (m, 1H), 7.78 (s, 0.3H), 8.04 (s, 0.7H); 13C NMR (176 MHz, CDCl3)
δ 25.13 (25.28), 27.12 (27.23), 28.72 (29.68), 43.67 (42.04), 44.91 (45.01),
46.66 (48.67), 62.67 (62.48), 68.56 (68.83), 80.79 (80.34), 85.08 (84.85),
87.38 (86.53), 93.09 (94.63), 105.53–105.68 (m), 107.11 (107.73), 113.98
(114.32), 115.90 (q, J = 287.16 Hz), 118.87–119.01 (m), 119.43–119.58 (m),
144.33, 146.74 (ddd, J = 3.70 Hz, J = 12.80 Hz, J = 245.21 Hz), 149.29 (dt,
J = 13.52 Hz, J = 251.00 Hz), 149.89 (149.65), 156.16 (ddd, J = 2.10 Hz,
J = 8.90 Hz, J = 244.50 Hz), 158.0 (q, J = 36.16 Hz), 163.54 (162.35);
HRMS calcd for C25H26N3O10F6S [M-H]− 674.1243, found 674.1250.
2ꢀ,3ꢀ-O-isopropylidene-N-[(1-β-D-ribofuranosyl-1H-2-
thiopyrimidin-5-yl)methyl]-N-trifluoroacetylglycine
2-(p-nitrophenyl)ethyl ester (22a)
Compound 22a was purified by column chromatography with CHCl3 to
give a light yellow foam in 56% yield. TLC Rf = 0.64 (CHCl3/MeOH 95:5,
v/v); 1H NMR (250 MHz, CDCl3) δ 1.37 (s, 3H), 1.61 (s, 3H), 3.07 (t, 2H, J =
5.00 Hz), 3.84–4.26 (m, 4H), 4.41–4.67 (m, 5H), 4.80 (dd, 1H, J = 2.25 Hz,
J = 6.00 Hz), 4.92 (dd, 1H, J = 2.50 Hz, J = 6.00 Hz), 6.65 (d, 0.9H, J =
2.25 Hz), 6.87 (d, 0.1H, J = 2.25 Hz), 7.38 (d, 2H, J = 8.50 Hz), 8.12 (d, 2H J
= 8.50 Hz), 8.33 (s, 1H); 13C NMR (176 MHz, CDCl3) δ 25.33 (25.55), 27.15
(27.35), 34.77 (34.67), 46.66, 50.67 (49.30), 62.25 (61.75), 65.24 (65.30),
80.16 (79.89), 86.10 (85.64), 87.57 (86.31), 96.01 (94.01), 112.05 (112.57),
113.89 (114.57), 115.79 (q, J = 288.00 Hz), 123.90, 129.75, 142.91, 144.87,
147.15, 158.10 (q, J = 37.00 Hz), 160.83, 168.09 (167.95), 175.06 (174.97);
HRMS calcd for C25H26N4O10F3S [M-H]− 631.1322, found 631.1316.

(2-thio)uridines Modified with Blocked Glycine/Taurine
611
2ꢀ,3ꢀ-O-isopropylidene-N-[(1-β-D-ribofuranosyl-1H-2-
thiopyrimidin-5-yl)methyl]-N-trifluoroacetylglycine
2-(trimethylsilyl)ethyl ester (22b)
Compound 22b was purified by column chromatography using 10%
MeOH in CHCl3 to obtain a light yellow foam in 78% yield. TLC Rf =
0.58 (CHCl3/MeOH 95:5, v/v); 1H NMR (250 MHz, CDCl3) δ 0.04 (s, 9H),
0.96–1.05 (m, 2H), 1.38 (s, 3H), 1.62 (s, 3H), 3.86 (dd, 1H, J = 2.25 Hz,
J = 12.25 Hz), 4.05–4.11 (m, 1H), 4.20–4.30 (m, 4H), 4.42–4.50 (m, 2H),
4.52–4.62 (m, 1H), 4.81 (dd, 1H, J = 6.00 Hz, J = 2.50 Hz), 4.91 (dd, 1H,
J = 6.00 Hz, J = 2.50 Hz), 6.65 (d, 0.9H, J = 2.50 Hz), 6.89 (d, 0.1H, J =
2.75 Hz), 8.16 (s, 0.1H), 8.33 (s, 0.9H), 9.56 (bs, 1H); 13C NMR (176 MHz,
CDCl3) δ -1.56, 17.40, 25.31 (25.54), 27.16 (27.35), 46.73, 50.88, 62.28, 64.63
(64.71), 80.23 (79.18), 86.13 (85.61), 87.62 (86.32), 96.19, 112.21 (112.66),
113.83 (114.57), 115.87 (q, J = 286.88 Hz), 142.70, 158.31 (q, J = 36.00 Hz),
160.22, 168.27, 168.68, 175.03; HRMS calcd for C22H33N3O8F3SiS 584.1709,
found 584.1709.
2ꢀ,3ꢀ-O-isopropylidene-N-[(1-β-D-ribofuranosyl-1H-2-
thiopyrimidin-5-yl)methyl]-N-trifluoroacetyltaurine
2-(2,4,5-trifluorophenyl)ethyl ester (22c)
Compound 22c was purified by column chromatography using 2%
MeOH in CHCl3 as eluent to obtain a light yellow foam in 32% yield. TLC
Rf = 0.66 (CHCl3/MeOH 95:5, v/v); 1H NMR (700 MHz, CDCl3) δ 1.61 (s,
3H), 1.85 (s, 3H), 3.15–3.20 (m, 2H), 3.50–4.21 (m, 4H), 4.00–4.58 (m, 7H),
4.74 (dd, 0.4H, J = 2.80 Hz, J = 5.60 Hz), 4.86 (dd, 0.6H, J = 2.80 Hz, J =
6.30), 4.92 (dd, 0.4H, J = 2.80 Hz, J = 5.60 Hz), 4.98 (dd, 0.6H, J = 2.80 Hz,
J = 6.30 Hz), 6.80 (d, 0.6H, J = 2.80 Hz), 6.95 (d, 0.4H, J = 2.80 Hz),
7.00–7.03 (m, 1H), 7.16–7.21 (m, 1H), 8.22 (s, 0.4H); 8.41 (s, 0.6H); 13C
NMR (176 MHz, CDCl3) δ 25.36 (25.53), 27.18 (27.35), 28.71 (29.69), 42.53
(43.58), 44.69 (45.32), 48.59 (46.59), 62.10 (61.70), 68.62 (68.92), 79.99
(79.19), 85.82 (85.70), 87.24 (86.47), 95.44 (94.07), 105.56–105.87 (m),
111.71 (112.68), 114.06 (114.43), 115.92 (q, J = 287.00 Hz), 118.83–119.01
(m), 138.91, 143.48, 145.97–150.09 (m), 155.38–155.55 (m), 156.77–156.96
(m), 158.01 (q, J = 36.00 Hz), 160.65 (159.28), 175.72 (175.10); HRMS
calcd for C25H27N3O9F6NaS2 [M+Na]+ 714.0991, found 714.0985.
General Procedure for the Synthesis of Compounds
29a-29c/30a-30c
Nucleoside 21a-21c/22a-22c (1 mmol) was dissolved in 25% aq. acetic
acid (20 mL). After being stirred for 1 h at 90◦C the mixture was cooled
to rt and then concentrated under reduced pressure. The solid residue was

612
Grazyna Leszczynska et al.
co-evaporated with anhydrous toluene and purified by column chromatogra-
phy using MeOH in CHCl3. The resulting product 29a-29c/30a-30c exist as
rotamers about –NC(O)CF3 amide bond (two chemical shifts were observed
1
for some of the H and 13C NMR resonances; the secondary shifts in 13C
NMR spectra are given in parentheses).
N-[(1-β-D-ribofuranosyl-1H-pyrimidin-5-yl)methyl]-N-
trifluoroacetylglycine 2-(p-nitrophenyl)ethyl ester
(29a)
Compound 29a was purified by column chromatography using 10%
MeOH in CHCl3 as eluent to obtain a white foam in 85% yield. TLC Rf =
0.33 (CHCl3/MeOH 90:10, v/v); 1H NMR (700 MHz, C6D5N) δ 2.97 (t, 1H, J
= 7.00 Hz), 3.03 (t, 1H, J = 6.30 Hz), 4.23–4.52 (m, 4H), 4.61–4.99 (m, 9H),
6.84 (d, 0.5H, J = 4.20 Hz), 6.86 (d, 0.5H, J = 2.80 Hz), 7.13 (bs, 1H), 7.36 (d,
1H, J = 8.40 Hz), 7.40 (d, 1H, J = 8.40 Hz), 8.17 (d, 1H, J = 8.40 Hz), 8.21 (d,
1H, J = 8.40 Hz), 8.86 (s, 0.5H), 8.97 (s, 0.5H); 13C NMR (176 MHz, C6D5N)
δ 34.48 (34.45), 46.42 (45.57), 48.89 (50.12), 60.99 (61.60), 64.93 (65.21),
71.09 (70.59), 76.39 (76.02), 86.22 (86.01), 90.30, 108.31 (107.99), 116.45
(q, J = 288.82 Hz) [116.65 (q, J = 288.80 Hz)], 129.95, 130.00, 140.08,
142.26, 145.80 (145.88), 146.97 (146.91), 151.45 (151.49), 156.95–157.78
(m), (164.06) 164.72, (168.19) 168.79; HRMS calcd for C22H23N4O11F3Na
[M+Na]+ 599.1213, found 599.1215.
N-[(1-β-D-ribofuranosyl-1H-pyrimidin-5-yl)methyl]-N-
trifluoroacetylglycine 2-(trimethylsilyl)ethyl Ester
(29b)
Compound 29b was purified by column chromatography using 6%
MeOH in CHCl3 as eluent to obtain a white foam in 81% yield. TLC Rf
1
= 0.38 (CHCl3/MeOH 90:10, v/v); H NMR (250 MHz, CDCl3) δ 0,03 (s,
2H), 0.04 (s, 7H), 0.97–1.04 (m, 2H), 3.47 (bs, 1H), 3.64–4.55 (m, 12H),
4.80 (bs, 1H), 5.79 (d, 0.8H, J = 3.75 Hz), 5.84 (bs, 0.2H), 8.03 (s, 0.2H),
8.17 (s, 0.8H), 9.91 (bs, 0.9H); 13C NMR (176 MHz, CDCl3) δ -0.66, 18.28
(19.27), 47.36 (45.99), 51.56 (50.17), 62.61 (62.13), 65.54 (65.47), 70.84
(71.49), 75.96 (76.16), 86.36 (85.95), 91.95 (91.23), 109.69, 116.89 (q, J
= 287.80 Hz), 143.51, 152.08 (151.95), 158.87 (q, J = 36.08 Hz), 164.97
(164.06), 169.57 (169.03); HRMS calcd for C19H28N3O9F3NaSi [M+Na]+
550.1445, found 550.1446.

(2-thio)uridines Modified with Blocked Glycine/Taurine
613
N-[(1-β-D-ribofuranosyl-1H-pyrimidin-5-yl)methyl]-N-
trifluoroacetyltaurine 2-(2,4,5-trifluorophenyl)ethyl ester
(29c)
Compound 29c was purified by column chromatography using 8%
MeOH in CHCl3 as eluent to obtain a white foam in 75% yield. TLC Rf = 0.30
1
(CHCl3/MeOH 90:10, v/v); H NMR (700 MHz, CD3OD) δ 3.14–3.18 (m,
2H), 3.54–3.93 (m, 5H), 4.02–4.14 (m, 2H), 4.21–4.26 (m, 2H), 4.32–4.39
(m, 1H), 4.46–4.56 (m, 3H), 5.98 (d, 0.5H, J = 4.90 Hz), 6.00 (d, 0.5H, J
= 4.20 Hz), 7.19–7.24 (m, 1H), 7.37–7.41 (m, 1H), 8.19 (s, 0.5H), 8.25 (s,
0.5H); 13C NMR (176 MHz, CD3OD) δ 27.95, 41.86 (42.52), 43.80 (44.70),
45.73, 60.54 (61.11), 69.05 (68.95), 69.86 (70.07), 74.91 (74.48), 85.08
(85.13), 89.42 (89.45), 104.98–105.29 (m), 107.81 (107.89), 113.74–120.53
(m), 139.67, 142.37, 145.89–149.76 (m), 150.73, 156.83–157.47 (m), 163.45
(164.02); HRMS calcd for C22H23N3O10F6NaS [M+Na]+ 658.0906, found
658.0901.
N-[(1-β-D-ribofuranosyl-1H-2-thiopyrimidin-5-yl)methyl]-N-
trifluoroacetylglycine 2-(p-nitrophenyl)ethyl ester
(30a)
Compound 30a was purified by column chromatography using 5%
MeOH in CHCl3 as eluent to obtain a yellow foam in 80% yield. TLC Rf
= 0.41 (CHCl3/MeOH 90:10, v/v); 1H NMR (700 MHz, C6D5N) δ 2.90–2.99
(m, 2H), 4.16–4.81 (m, 11H), 7.27 (bs, 1H), 7.33 (d, 1H, J = 8.40 Hz),
7.37 (d,1H, J = 8.40 Hz), 8.04 (d, 1H, J = 8.40 Hz), 8.15 (d, 1H, J =
8.40 Hz), 8.90 (s, 0.5H), 9.07 (s, 0.5H); 13C NMR (176 MHz, C6D5N) δ 34.50
(34.46), 46.64 (45.86), 49.15 (50.24), 60.56 (59.95), 65.22 (65.52), 69.48
(69.87), 69.48 (69.87), 76.32 (76.44), 85.66 (85.85), 94.65 (94.73), 112.97
(113.02), 116.52 (q, J = 279.65 Hz) [116.82 (q, J = 288.11 Hz)], 130.14,
130.20, 139.91, 142.06, 146.06 (146.11), 146.86 (146.91), 149.16, 157.40 (q,
J = 31.15 Hz) [157.95 (q, J = 36.12 Hz)], 161.59 (160.96), 168.92 (168.34),
176.58 (176.44); HRMS calcd for C22H23N4O10F3NaS [M+Na]+ 615.0985,
found 615.0978.
N-[(1-β-D-ribofuranosyl-1H-2-thiopyrimidin-5-yl)methyl]-N-
trifluoroacetylglycine 2-(trimethylsilyl)ethyl ester
(30b)
Compound 30b was purified by column chromatography using 5%
MeOH in CHCl3 as eluent to obtain a light yellow foam in 85% yield. TLC
Rf = 0.24 (CHCl3/MeOH 95:5, v/v); 1H NMR (700 MHz, CD3OD) δ 0,05 (s,
3.3H), 0.06 (s, 5.7H), 0.99–1.02 (m, 2H), 3.48–4.27 (m, 9H), 4.39–4.70 (m,
3H), 6.58 (d, 0.4H, J = 2.10 Hz), 6.59 (d, 0.6H, J = 2.10 Hz), 8.38 (s, 0.4H),

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Grazyna Leszczynska et al.
8.46 (s, 0.6H); 13C NMR (176 MHz, CD3OD) δ -1.56, 18.22, 47.31, 51.38, 61.51
(60.82), 65.37 (65.06), 70.28 (69.86), 76.73, 86.02, 95.16, 113.86 (113.76),
115.12–120.02 (m), 143.108 (142.78), 158.48–159.20 (m), 162.40 (161.74),
170.24 (169.68), 177.28 (177.16); HRMS calcd for C19H28N3O8F3NaSiS
[M+Na]+ 566.1216, found 566.1213.
N-[(1-β-D-ribofuranosyl-1H-2-thiopyrimidin-5-yl)methyl]-N-
trifluoroacetyltaurine 2-(2,4,5-trifluorophenyl)ethyl ester
(30c)
Compound 30c was purified by column chromatography using 10%
MeOH in CHCl3 as eluent to obtain a light yellow foam in 80% yield.
1
TLC Rf = 0.46 (CHCl3/MeOH 90:10, v/v); H NMR (700 MHz, CD3OD)
δ 3.14–3.17 (m, 2H), 3.55–3.59 (m, 2H), 3.82–4.16 (m, 5H), 4.19–4.22 (m,
1H), 4.27–4.29 (m, 1H), 4.33–4.58 (m, 4H), 6.66 (d, 0.6H, J = 2.10 Hz),
6.68 (d, 0.4H, J = 2.80 Hz), 7.19–7.24 (m, 1H), 7.36–7.41 (m, 1H), 8.44 (s,
0.6H), 8.52 (s, 0.4H); 13C NMR (176 MHz, CD3OD) δ 27.96, 42.15 (42.67),
44.12, 45.73 (44.95), 59.33 (60.16), 68.43 (68.91), 69.06 (68.95), 75.39
(75.25), 84.58 (84.64), 93.77 (93.72), 104.98–105.27 (m), 112.18 (111.88),
116.02 (q, J = 287.00 Hz), 118.61–120.55 (m), 139.17, 142.04, 145.87–149.84
(m), 155.58–157.57 (m), 160.16 (160.78), 175.72 (175.89); HRMS calcd for
C22H23N3O9F6NaS2 [M+Na]+ 674.0678, found 674.0681.
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