Chemoselective N-Deacetylation of Protected Nucleosides and Nucleotides Promoted by Schwartz's Reagent

Article abstract of DOI:10.1080/15257770.2015.1075552

Protection and deprotection strategies involving the N-acetyl group are widely utilized in nucleoside and nucleotide chemistry. Herein, we present a mild and selective N-deacetylation methodology, applicable to purine and pyrimidine nucleosides, by means of Schwartz's reagent, compatible with most of the common protecting groups used in nucleoside chemistry.

Full text of DOI:10.1080/15257770.2015.1075552

Nucleosides, Nucleotides and Nucleic Acids
ISSN: 1525-7770 (Print) 1532-2335 (Online) Journal homepage: http://www.tandfonline.com/loi/lncn20
Chemoselective N-Deacetylation of Protected
Nucleosides and Nucleotides Promoted by
Schwartz's Reagent
Valentina Ferrari, Michaela Serpi, Christopher McGuigan & Fabrizio Pertusati
To cite this article: Valentina Ferrari, Michaela Serpi, Christopher McGuigan & Fabrizio
Pertusati (2015): Chemoselective N-Deacetylation of Protected Nucleosides and Nucleotides
Promoted by Schwartz's Reagent, Nucleosides, Nucleotides and Nucleic Acids, DOI:
10.1080/15257770.2015.1075552
To link to this article: http://dx.doi.org/10.1080/15257770.2015.1075552
© 2014 Christopher L. Brown, Elisabeth
Criscuolo Urbinati, Weimin Zhang,
Shannon B. Brown, and Michelle McComb-
Kobza. Published with license by Taylor
& Francis© Valentina Ferrari, Michaela
Published online: 22 Oct 2015.
Serpi, Christopher McGuigan, and Fabrizio
Pertusati
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Date: 23 October 2015, At: 06:24

Nucleosides, Nucleotides and Nucleic Acids, 00:1–16, 2015
Published with license by Taylor & Francis
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2015.1075552
CHEMOSELECTIVE N-DEACETYLATION OF PROTECTED
NUCLEOSIDES AND NUCLEOTIDES PROMOTED BY
SCHWARTZ’S REAGENT
Valentina Ferrari, Michaela Serpi, Christopher McGuigan,
and Fabrizio Pertusati
School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Cardiff, UK
Protection and deprotection strategies involving the N-acetyl group are widely utilized in nucleo-
2
side and nucleotide chemistry. Herein, we present a mild and selective N-deacetylation methodology,
applicable to purine and pyrimidine nucleosides, by means of Schwartz’s reagent, compatible with
most of the common protecting groups used in nucleoside chemistry.
Keywords Chemoselective; N -deacetylation; Schwartz reagent; nucleoside; prodrugs;
protecting groups
1. INTRODUCTION
The chemistry of metallocenes rapidly expanded in the 1970s with
organozircocene emerging as one of the most useful classes of tran-
sition metal derivatives for use in organic synthesis.[1] One important
reagent belonging to this class of organometallic compounds is the
chlorobis(cyclopentadienyl)hydrido-zirconium 1, also called zirconocene
hydrochloride but better known as Schwartz’s reagent.[2] Wailes and
Weigold[3] were the first to prepare 1 and, subsequently, Schwartz[2a] and
many other scientists exploited its potential in the functionalization of or-
ganic compounds.[4] Selected examples of the utility of this reagent in
organic synthesis are depicted in Figure 1. Schwartz’s reagent is most of-
ten used in the hydrozirconation of triple and double bonds (Figure 1,
©
Valentina Ferrari, Michaela Serpi, Christopher McGuigan, and Fabrizio Pertusati
This is an Open Access article. Non-commercial re-use, distribution, and reproduction in any
medium, provided the original work is properly attributed, cited, and is not altered, transformed, or built
upon in any way, is permitted. The moral rights of the named author(s) have been asserted.
Received 12 March 2015; accepted 20 July 2015.
Address correspondence to Fabrizio Pertusati, School of Pharmacy and Pharmaceutical Sciences,
Cardiff University, Redwood Building, King Edwards VII Avenue, CF10 3NB, Cardiff, Wales, UK. E-mail:
pertusatif1@cf.ac.uk
Color versions of one or more of the figures in the article can be found online at
www.tandfonline.com/lncn.
1

2
V. Ferrari et al.
FIGURE 1 Selected organic transformations involving the Schwartz’s reagent.
Reaction a).[5] However, its use in this reaction is sometimes hampered by
the functional group compatibility of the process that is limited by the ox-
ophilic, hard Lewis-acid character of the Schwartz reagent.[2b]
This characteristic has been therefore exploited in other useful synthetic
organic transformations such as the chemoselective reduction of tertiary
amides to aldehydes (Figure 1, Reaction b),[6] and the reduction of sec-
ondary amides to N -substituted imines (Figure 1, Reaction c).[7] The latter
transformation, when performed with most other metal hydride reagents, re-
sults either in over-reduction of the imine to give the corresponding amine
or in the reductive cleavage of the amide. Interestingly, 1 has also been
employed as a reducing agent for phosphine oxides to the corresponding
phosphines (Figure 1, Reaction d).[4d] Recently, Bhat and co-workers re-
ported a very mild and useful deacetylation procedure of acetamides involv-
ing reagent 1.[8] The methodology proved to be very efficient for aromatic,
heteroaromatic, and aliphatic amides and moreover no epimerization was
observed during the N -deacetylation of chiral acetamides.
The amine functionality is present in many compounds of biological in-
terest,[9] for instance natural products and pharmaceuticals. Nucleoside and
nucleotide chemistry frequently involves protecting procedures at the amine
and hydroxyl groups and benefit of selective deprotection methods, in or-
der to obtain the target compound efficiently without any side reactions. N -
Acetyl protection is one of the most widely used in nucleoside and nucleotide
chemistry. However, the requirement of strong base or acid and/or high
reaction temperatures[10] during the deprotection step (N -deacetylation)
is often not compatible with some nucleosidic substrates and especially
their prodrugs. Harsh conditions are also likely to yield fully deprotected

N-Deacetylation of Nucleosides and Nucleotides
3
products, an outcome often undesired. Zinc bromide[11] and hydrazine hy-
drate[12] were initially reported as selective and mild N -deacetylating agents
for nucleosides, although their compatibility with other protecting groups
was not explored broadly. Our goal was the identification of a selective N -
deacetylation methodology involving mild reaction conditions that could be
applied to a broad spectrum of differently protected nucleosides. Therefore,
we became immediately interested in Bhat’s methodology[8] and in its pos-
sible application in nucleoside chemistry. To the best of our knowledge, no
attempt to use Schwartz’s reagent in the N -deacetylation of nucleosides and
their prodrugs has been previously reported. Herein, we would like to report
our efforts directed at deacetylation of N -acetyl purine and pyrimidine nucle-
oside analogues bearing a variety of protecting groups (PGs) at the hydroxyl
moiety, commonly used in nucleoside chemistry, such as O-acetyl, O-tert-
butyloxycarbonyl, O-tert-butyldimethylsilyl, O-tetrahydropyranyl, O-benzoyl,
and O-isopropylidene groups, with the aim of evaluating the ability of reagent
1 to selectively remove the acetyl group from the amino moiety, leaving the
other PGs untouched.
2. RESULTS AND DISCUSSION
Our investigation began with the preparation, according to literature
procedures, of differently substituted N-acetyl-2ꢀ-deoxycytidine (2a-c) and
N-acetylcytidine (2d-g), as indicated in Table 1. With all the O-protected
nucleosides in hand, we proceeded to test the deacetylation reaction. First,
compound 2a was subjected to the action of three equivalents of reagent 1 in
a THF solution, under a nitrogen atmosphere, for 30 minutes (Table 1, Entry
1). Pleasingly, we were able to isolate after aqueous work-up and column
chromatography compound 3a in 57% yield, where only the N -acetyl group
was removed. Delighted by this result, we directed our efforts towards other
protecting groups. Application of these conditions to 2b, once again, led
to the selective removal of the acetyl group from the amino moiety without
affecting the silicon protected hydroxyl groups and yielding 3b in 49% yield
(Table 1, Entry 2).
Similar results were obtained testing the compatibility of Schwartz’s
reagent with other protecting groups such as tetrahydropyranyl, tertbutoxy-
carbonyl, benzoyl, and isopropylidene (Table 1, Entries 3–7). In all exam-
ples, the O-protecting groups were unaffected by this procedure, and only
the acetyl group on the nucleobase was removed, affording the desired com-
pounds in moderate to good yields (39–70%), along with the recovery of
unreacted starting material. Attempts to increase the yield of the desired
N -deacetylated nucleoside either by increasing the equivalents of 1 (up to
six) or by adding it portionwise over a period of three hours, proved to
be unsuccessful and some amount of starting material was always recovered

4
V. Ferrari et al.
TABLE 1 Deacetylation reaction of differentially protected N -acetyl-2ꢀ-deoxycytidines and
N -acetylcytidines, promoted by Schwartz reagent
Recovered SM
Entry Cpd R1
R2
R3
1 (eq) Temp (◦C) Time (h) Prod. Yield (%)
(%)
1
2
3
4
5
6
7
8
9
2a
2b
2c
2d
H
H
H
Ac
Ac
3
3
3
3
3
3
3
6
3
rt
rt
rt
rt
rt
rt
rt
rt
70
0.5
0.5
3
0.5
3
3
3
3
12
3a
3b
3c
3d
3e
3f
3g
3a
3a
57
49
68
39
70
53
55
55
48
40
33
20
55
23
41
38
42
39
TBDMS TBDMS
THP
Boc
Ac
THP
Boc
Ac
Bz
H
OH
2e OAc
2f OBz
2g CMe2
2a
2a
Bz
H
H
Ac
Ac
Ac
Ac
(Table 1, Entry 8). Incrementing the temperature up to 70◦C did not en-
hance the yield (Table 1, Entry 9). Subsequently, we decided to broaden the
scope of the methodology by testing it on examples of other pyrimidine and
purine nucleoside derivatives bearing different PGs (Table 2). Lamivudine
was acetylated on both oxygen and amine groups and the resulting N,O-
diacetylated compound 2h was then submitted to the deacetylating protocol
(Table 2, Entry 1). As expected, the acetyl group was removed only from the
nitrogen on the nucleobase, leaving the O-acetyl group untouched.
N -acetyl-O-trityl-lamivudine 2i was found to be a poor substrate for this
protocol (Table 2, Entry 2). The desired product 3i was obtained only in
25% isolated yield, most probably due to the partial trityl group instability
to the reaction conditions. No starting material was isolated in this case.
When the protocol was tested on purine nucleoside analogues, reagent
1 proved efficient in removing the acetyl group selectively from the amine
group on both the guanine ring of peracetylated acyclovir 2j and the adenine
ring of N -acetyl-5ꢀ-O-acetyl-2ꢀ,3ꢀ-O-isopropylidene adenosine 2k, affording 3j
and 3k respectively in 47% and 76% isolated yields (Table 2, Entries 3,4).
Finally, N,N -diacetyl-2ꢀ,3ꢀ-isopropylidene adenosine 2l was submitted to
the optimized protocol. In this case Schwartz’s reagent was able to remove
both N -acetyl groups, affording compound 3k in 53% yield, with only traces
of mono-N -acetylated derivative 2k.

N-Deacetylation of Nucleosides and Nucleotides
5
TABLE 2 N-deacetylation of protected pyrimidine and purine nucleosides, promoted by Schwartz’s
reagent
Entry
Compound
Product
Yield (%)
56
1
2
25
3
47
4
76
(Continued on next page)

6
V. Ferrari et al.
TABLE 2 N-deacetylation of protected pyrimidine and purine nucleosides, promoted by Schwartz’s
reagent (Continued)
Entry
Compound
Product
Yield (%)
5
53
2.1. In Situ Generation of the Schwartz’s Reagent
and Yield Improvement
At the same time we were conducting our investigations, Snieckus et al.[6c]
reported a practical method to generate Schwartz’s reagent in situ using
Li(Ot-Bu)3AlH, demonstrating the advantage of this procedure both in re-
ducing tertiary amides to aldehydes and in hydrozirconation reactions, in
comparison to the use of the commercial Schwartz’s reagent. We, therefore,
decided to assess this efficient procedure in the deacetylation of nucleo-
side 2g in order to be able to compare the in situ methods with the use of
the preformed 1 (Scheme 1). Therefore, to a THF solution of 2g and 1.5
equivalents of Cp2ZrCl2 in THF, solid Li(Ot-Bu)3AlH was added in one por-
tion and the mixture was stirred for 40 minutes. Aqueous workup, followed
by column chromatography, afforded compound 3g in 80% isolated yield,
demonstrating a yield improvement from the previous 55%, possibly due to
the partial air, light, and moisture sensitivity of 1, which are reported to be
responsible for the reduction of its effectiveness with time. [13]
2.2. Schwartz’s Reagent N-Deacetylation Applied
to Nucleoside Phosphoroamidate
Nucleoside analogues (NAs) are an important class of molecules account-
ing for half of all antiviral drugs currently on the market and a number of an-
ticancer agents that are widely used.[14] NAs mostly require phosphorylation
to be active. Unfortunately, many nucleoside analogues are not phosphory-
lated effectively in vivo, and thus their therapeutic potential is often quite
limited. Using various approaches, the ionizable phosphate group can be
masked by derivatization, generating prodrugs with increased biological ac-
tivity.[15] Among different nucleoside monophosphate prodrug strategies is

N-Deacetylation of Nucleosides and Nucleotides
7
SCHEME 1 N -deacetylation of N -acetyl-2ꢀ,3ꢀ-O-isopropylidene cytidine 2 g with in situ generated
Schwartz’s reagent 1.
the phosphoroamidate prodrug approach (ProTide), consisting of an amino
acid ester promoiety linked via P–N bond to a nucleoside aryl phosphate.[16]
Several leading pharmaceutical companies have applied this technology for
antiviral and anticancer treatments. Gilead has just launched on the market
its anti-HCV ProTide, Sofosbuvir (PSI-7977),[17] whereas Nucana Biomed has
taken to trial a gemcitabine ProTide (NUC-1031), for patients with advanced
solid tumours.[18] Considering the importance that the ProTide technology
is assuming in the antiviral and anticancer scenarios, we were interested in
checking if Schwartz’s reagent is compatible with the phosphoramidate mo-
tif and whether this protocol can be used at later stage in the synthesis of
this class of prodrugs.
SCHEME 2 Synthesis and deacetylation of N -acetyl-2ꢀ,3ꢀ-O-isopropylidene- cytidine-5ꢀ-O-[napthyl
(cyclohexyl-L-alanyl)] phosphate (5) promoted by Schwartz reagent.
Phosphoroamidate 5 was synthesized from 2g and (2S)-cyclohexyl 2-
((chloro(naphthalen-1-yloxy)phosphoryl)amino)propanoate 4 in 40% yield
(Scheme 2), according to a previously described method.[16] Phosphoroami-
date 5 was then treated with three equivalents of the Schwartz’s reagent,
yielding the corresponding deacetylated prodrug 6 in 65% isolated yield.

8
V. Ferrari et al.
3. CONCLUSIONS
In conclusion, we have demonstrated that the general and mild method
for the deacetylation of N -acetyl group developed by Bhat et al. can be
successfully applied to purine and pyrimidine nucleosides, with diverse O-
protection. Moreover, we have shown that Schwartz’s reagent is compatible
with the phosphoramidate moiety and can be used for N -deacetylation reac-
tion in the later stages of the synthesis of this class of nucleoside monophos-
phate prodrugs. Lastly, we have proved that modification of the reported
procedure[6c] involving in situ generation of Schwartz’s reagent leads to
yield improvement and is promising of similar results on other nucleoside
substrates.
4. EXPERIMENTAL SECTION
General information. All anhydrous solvents were purchased from Sigma-
Aldrich. All commercially available reagents were used without further pu-
rification. Schwartz reagent was purchased from Sigma-Aldrich or prepared
in situ as described and all nucleosides used as starting materials were
purchased from Carbosynth. All reactions were performed under an Ar-
1
gon atmosphere, unless otherwise stated. H NMR (500 MHz), 13C NMR
(125 MHz), and 31P NMR (202 MHz) spectra were recorded on a Bruker
Avance 500 MHz spectrometer at 25ºC. Chemical shifts (δ) are quoted in
1
parts per million (ppm) relative to internal CD3OD (δ 3.34 H NMR, δ
49.86 13C NMR) and CDCl3 (δ 7.26 1H NMR, δ 77.36 13C NMR) or external
85% H3PO4 (δ 0.00 31P NMR). Coupling constants (J ) are given in Hertz.
The following abbreviations are used in the assignment of NMR signals:
singlet (s), doublet (d), triplet (t), quartet (q), quintet (qn), multiplet (m),
broad singlet (bs), doublet of doublet (dd), and doublet of triplet (dt). High
and low-resolution mass spectrometry analyses were performed on a Bruker
microTof-LC system.
Standard procedure for the N-deacetylation reaction.[8] To a stirred solution
of N -acetyl nucleoside (100 mg) in anhydrous THF (2 mL), Schwartz’s
reagent (3–6 eq.) is added at room temperature and the reaction mixture is
stirred for 0.5–3 hours. Water is then added to quench the reaction. The re-
sulting solution is extracted with CH2Cl2 (2 × 5 mL). The combined organic
layer is washed with brine solution and dried over anhydrous Na2SO4, fil-
tered and evaporated to afford the crude product, which is purified by silica
gel column chromatography to finally afford pure deacetylated nucleoside.
N-Deacetylation reaction via in situ generation of Schwartz’s.[6c] To a solution
of N -acetyl nucleoside (100 mg) and Cp2ZrCl2 (1.5 equiv) in THF (2 mL)
at rt, solid LiAlH(OtBu)3 (1.5 equiv) was rapidly added. The resulting so-
lution was stirred at rt for 40 minutes and the reaction was monitored by
TLC analysis. Water is then added to quench the reaction. The resulting

N-Deacetylation of Nucleosides and Nucleotides
9
solution is extracted with CH2Cl2 (2 × 5 mL). The combined organic layer
is washed with brine solution and dried over anhydrous Na2SO4, filtered
and evaporated to afford the crude product, which is purified by silica gel
column chromatography to finally afford pure deacetylated nucleoside.
3ꢀ,5ꢀ-O-Bis(acetyl)-2ꢀ-deoxycytidine (3a). Prepared from 2a[19a,b] accord-
ing to standard procedure for the N -deacetylation reaction. The crude com-
pound is purified by flash chromatography on silica gel gradient elution of
CH2Cl2/MeOH from 98/2 to 95/5 to give 3a[19c] as a white solid (0.050 g,
57% yield). 1H NMR (500 MHz) CDCl3 δH 2.08 (3H, s, OCOCH3), 2.09 (3H,
s, OCOCH3), 2.19–2.12 (1H, m, H-2ꢀa), 2.58 (1H, dd, J = 4.0, 14.0 Hz, H-
2ꢀb), 4.24–4.26 (1H, m, H-4ꢀ), 4.32 (2H, d, J = 12.5 Hz, H-5ꢀ), 5.18–5.19 (1H,
m, H-3ꢀ), 6.11 (1H, d, J = 6.5 Hz, H-5), 6.22 (1H, t J = 6.5 Hz, H-1ꢀ), 7.57
(1H, d, J = 6.5 Hz, H-6). 13C NMR (125 MHz) CDCl3 δC 20.90, 20.98 (CH3),
38.24 (CH2-2ꢀ), 63.82 (CH2-5ꢀ), 74.12 (C-3ꢀ), 82.63 (C-4ꢀ), 88.18 (C-1ꢀ), 95.87
(C-5), 140.21 (C-6), 170.32 (C-O), 155.15 (C-2), 164.90 (C-4); MS (ES+) m/z
+
(312) [M+1]; HRMS (ES+) m/z found 312.1192 [calcd for C13H18N3O6
(M+H)+ 312.1190].
3ꢀ,5ꢀ-O- Bis(tert-butyldimethylsilyl)-2ꢀ-deoxycytidine (3b). Prepared from
2b[20a,b] according to N -deacetylation reaction. The crude compound is puri-
fied by flash chromatography on silica gel gradient elution of CH2Cl2/MeOH
from 98/2 to 95/5 to give 3b[20c] as a white solid (0.044 g, 49%). H NMR
1
(500 MHz) CDCl3 δH 0.12 (6H, s, 2 × CH3), 0.15 (6H, s, 2 × CH3), 0.91 (9H,
s, C(CH3)3), 0.98 (9H, s, C(CH3)3), 2.09–2.13 (1H, m, H-2ꢀa), 2.35–2.40
(1H, m, H-2ꢀb), 3.83 (1H, dd, J = 2.5, 11.5 Hz, H-5ꢀa), 3.92 (1H, dd, J =
3.0, 11.0 Hz, H-5ꢀb), 3.93–3.96 (1H, m, H-4ꢀ), 4.47–4.49 (1H, m, H-3ꢀ), 5.89
(1H, d, J = 7.5 Hz, H-5), 6.24 (1H, t J = 6.0 Hz, H-1ꢀ), 7.97 (1H, d, J =
7.5 Hz, H-6); 13C NMR (125 MHz) CDCl3 δC –4.92, –4.58 (SiCH3), 17.93,
18.94 (C(CH3)3) 26.04, 26.22 (C(CH3)3), 42.13 (C-2ꢀ), 62.04 (C-5ꢀ), 70.44
(C-3ꢀ), 85.80 (C-1ꢀ), 87.25 (C-4ꢀ), 94.26 (C-5), 141 (C-6), 155.94 (C-2), 165.87
(C-4); MS (ES+) m/z 456 [M+1]; HRMS (ES+) m/z found 456.2710 [calcd
for C21H42N3O4Si2+ (M+H)+ 456.2708].
3ꢀ,5ꢀ-O-Bis(tetrahydropyranyl)-2ꢀ-deoxycytidine (3c). Prepared from
2c[21] according to standard procedure for the N -deacetylation reaction.
The crude compound is purified by flash chromatography on silica gel gra-
dient elution of CH2Cl2/MeOH from 98/2 to 95/5 to give 3c as a white
1
solid (0.061 mg, 68%). H NMR (500 MHz) CD3OD δH 1.44–1.48 (8H, m,
CH2 Py), 1.61–1.76 (4H, m, CH2 Py), 2.34–2.50 (1H, m, H-2ꢀb), 1.95–2.12
(1H, m, H-2ꢀa), 3.43–3.47 (2H, m, CH2O Py), 3.51 (0.25H, dd, J = 3.0,
11.0 Hz, H-5ꢀb), 3.53 (0.25H, dd, J = 3.0, 11.5 Hz, H-5ꢀb), 3.54–3.61 (0.5H,
dt J = 3.9, 16.5 Hz, H-5ꢀb), 3.71–3.82 (2H, m, CH2O Py), 3.80–3.86 (0.5,
m, H-5ꢀa), 3.90–3.95 (0.5H, m, H-5ꢀa), 4.07–4.11 (0.5H, m, H-4ꢀ), 4.13–4.15
(0.25H, m, H-4ꢀ), 4.17–4.19 (0.25H, m, H-4ꢀ), 4.29–4.31(0.25H, m, H-3ꢀ),
4.34–4.36 (0.5H, m H-3ꢀ), 4.40–4.41 (0.25H, m, H-3ꢀ), 4.55–4.67 (2H, m,

10
V. Ferrari et al.
CHO Py), 5.77–5.80 (1H, m, H-5), 6.11–6.16 (1H, m, H-1ꢀ), 7.88 (0.25H,
d, J = 7.5 Hz, H-6), 7.89 (0.25H, d, J = 7.5 Hz, H-6), 7.94 (0.25H, d, J =
7.5 Hz, H-6), 7.97 (0.25H, d, J = 7.0 Hz, H-6); 13C NMR (125 MHz) CD3OD
δC 20.47, 20.50, 20.68, 20.77 (CH2 Py), 26.51, 26.55 (CH2 Py), 31.69, 31.75,
31.94, 32.01 (CH2 Py), 39.54, 39.71, 40.82, 40.98 (C-2ꢀ), 63.69, 63.73, 63.75,
63.92 (CH2O), 68.30, 68.42, 68.49, 68.80 (C-5ꢀ), 77.39, 77.82, 78.25 (C-3ꢀ),
85.41, 85.47, 85.97, 85.18 (C-4ꢀ), 87.65, 87.83, 87.87, 87.97 (C-1ꢀ), 95.80,
95.90 (C-5),99.29, 99.36, 99.79, 99.88, 100.14, 100.33, 100.97,101.03 (CH
Py), 142.38, 142.81 (C-6), 158.17 (C-2), 167.63 (C-4); MS (ES+) m/z = 396
[M+1]. HRMS (ES+) m/z found 396.2127 [calcd for C19H30N3O6+ (M+H)+
396.2129].
5ꢀ,3ꢀ-O-Bis-(tert-butoxycarbonyl)-cytidine (3d). Prepared from 2d[22] ac-
cording to standard procedure for the N -deacetylation reaction. The crude
compound is purified by flash chromatography on silica gel gradient elution
of CH2Cl2/MeOH from 98/2 to 95/5 to give 3d as a white solid (0.035 g,
1
39% yield). H NMR (500 MHz) CD3OD δH 1.47 (9H, s, C(CH3)3), 1.50
(9H, s, C(CH3)3), 3.77 (1H, dd, J = 3.0, 12.5 Hz, H-5ꢀ), 3.88 (1H, dd, J
= 2.5, 12.5 Hz, H-5ꢀ), 4.22–4.20 (1H, m, H-4ꢀ), 5.27 (1H, dd, J = 5.5 Hz,
J = 5.5 Hz, H-3ꢀ), 5.32 (1H, dd, J = 4.0, 4.0 Hz, H-2ꢀ), 5.90 (1H, d, J =
7.5 Hz, H-5), 6.10 (1H, d, J = 4.5 Hz, H-1ꢀ), 8.0 (1H, d, J = 7.5 Hz, H-6);
13C NMR (125 MHz) CD3OD δC 27.96, 28.02 (C(CH3)3), 31.70 (C(CH3)3),
61.96 (C-5ꢀ), 74.17 (C-3ꢀ), 77.25 (C-2ꢀ), 83.93 (C-4ꢀ), 89.94 (C-1ꢀ), 96.64 (C-
5), 143.07 (C-6), 153.61, 153.89 (COC(CH3)3), 158.01(C-2), 167.56 (C-4);
MS (ES+) m/z = 444 [M+1]. HRMS (ES+) m/z found 444.1973 [calcd for
C19H30N3O9+ (M+H)+ 444.1977].
Cytidine 2ꢀ, 3ꢀ, 5ꢀ-triacetate (3e). Prepared from 2e[19a,10b] according
to standard procedure for the N -deacetylation reaction. The crude com-
pound is purified by flash chromatography on silica gel gradient elution of
CH2Cl2/MeOH from 98/2 to 95/5 to give 3e[10b] as a white solid (0.063 g,
1
70% yield). H NMR (500 MHz) CD3OD δH 2.10 (3H, s, COCH3), 2.11
(3H, s, COCH3), 2.12 (3H, s, COCH3), 4.34–4.40 (3H, m, H4ꢀ, H5ꢀ), 5.41
(dd, J = 5.0, 5.9 Hz, 1H, H3ꢀ), 5.48 (dd, J = 4.6, 6.0 Hz, 1H, H3ꢀ), 5.96
(1H, d, J = 7.5 Hz, H5), 5.98 (1H, d, J = 4.6 Hz, H1ꢀ), 7.69 (1H, d, J
= 7.5 Hz, H6); 13C NMR (125 MHz) CD3OD δC 20.40 (COCH3), 20.45
(COCH3), 20.69 (COCH3), 64.32 (C5ꢀ), 71.68 (C3ꢀ), 74.69 (C2ꢀ), 80.99
(C4ꢀ), 91.09 (C1ꢀ), 96.76 (C5), 143.23 (C6), 157.91 (C4), 167.82 (C2),
171.35 (COCH3), 171.39 (COCH3), 172.19 (COCH3); MS (ES+) m/z (370)
[M+1]. HRMS (ES+) m/z found 370.1243 [calcd for C15H20N3O8+ (M+H)+
370.1245].
Cytidine 2ꢀ, 3ꢀ, 5ꢀ-tribenzoate (3f). Prepared from 2f[23a,b] according
to standard procedure for the N -deacetylation reaction. The crude com-
pound is purified by flash chromatography on silica gel gradient elution of
CH2Cl2/MeOH from 98/2 to 96/4 to give 3f[23c] as a white solid (0.049 g,

N-Deacetylation of Nucleosides and Nucleotides
11
53% yield). 1H NMR (500 MHz) CD3Cl δH 2.12 (3H, s, COCH3), 4.63 (1H,
dd, J = 3.9, 12.2 Hz, H5ꢀ), 4.72–4.68 (1H, m, H4ꢀ), 4.76 (1H, dd, J = 2.8,
12.2 Hz, H5ꢀ), 5.74–5.78 (1H, m, H2ꢀ), 5.79–5.84 (1H, m, H3ꢀ), 6.31 (1H,
d, J = 4.2 Hz, H1ꢀ), 7.31–7.24 (5H, m, Ph, H5), 7.37–7.42 (2H, m, Ph),
7.43–7.49 (2H, m, Ph), 7.50–7.55 (1H, m, Ph), 7.81–7.89 (5H, m, Ph, H6),
7.98–8.08 (2H, m, Ph), 9.58 (1H, br s, NHCOCH3). 13C NMR (125 MHz)
CD3Cl δc 63.92 (C5ꢀ), 71.20 (C3ꢀ), 74.45 (C2ꢀ), 80.15 (C4ꢀ), 89.25 (C1ꢀ),
95.45 (C5), 128.46 (CH Ar), 128.49 (CH-Ar), 128.68 (CH-Ar), 128.75 (C-
Ar), 129.40 (C-Ar), 129.71 (CH-Ar), 129.85 (CH-Ar), 129.97 (CH-Ar), 133.49
(CH-Ar), 133.61 (CH-Ar), 133.62 (CH-Ar), 141.36 (C6), 155.28 (C5), 165.33
(COPh), 165.37 (COPh), 165.39 (C4), 166.14 (COPh); MS (ES+) m/z (556)
[M+1]; HRMS (ES+) m/z found 556.1711 [calcd for C30H26N3O8+ (M+H)+
556.1714].
2ꢀ, 3ꢀ-O-Isopropylidene-cytidine (3g). Prepared from 2g[24a,b] according
to standard procedure for the N -deacetylation reaction. The crude com-
pound is purified by flash chromatography on silica gel gradient elution of
CH2Cl2/MeOH from 98/2 to 95/5 to give 3g[24c] as a white solid (0.043g,
49% yield). Prepared according to the in situ Schwartz’s reagent generation
procedure for the deacetylation reaction. The crude compound is purified
by flash chromatography on silica gel gradient elution of CH2Cl2/MeOH
1
from 98/2 to 95/5 to give 3g as a white solid (0.070 g, 80% yield). H
NMR (500 MHz) CD3OD δH 1.25 (3H, s, CH3), 1.45 (3H, s, CH3), 3.62
(1H, dd, J = 4.5, 12.0 Hz, H-5ꢀa), 3.69 (1H, dd, J = 3.5, 12.0 Hz, H-5ꢀb),
4.11–4.13 (1H, m, H-4ꢀ), 4.72–4.74 (1H, m, H-3ꢀ), 4.78–4.80 (1H, m, H-2ꢀ),
5.75 (1H, s, H-1ꢀ), 5.81 (1H, d, J = 7.5 Hz, H-5), 7.73 (1H, d, J = 7.5Hz, H-
6); 13C NMR (125 MHz) CD3OD δC 25.55 (CCH3)2), 27.55 (CCH3)2), 63.19
(C5ꢀ), 82.25 (C3ꢀ), 86.40 (C2ꢀ), 88.69 (C4ꢀ), 95.45 (C1ꢀ), 96.20 (C5), 115.03
(CCH3)2), 144.42 (C6), 154.90 (C2), 167.82 (C4).MS (ESI+) m/z = (284)
[M+1].HRMS (ES+) m/z found 284.1243 [calcd for C12H18N3O5+ (M+H)+
284.1241].
O-Acetyl-lamivudine (3h). Prepared from 2h[19a,25] according to standard
procedure for the N -deacetylation reaction. The crude compound is purified
by flash chromatography on silica gel gradient elution of CH2Cl2/MeOH
1
from 98/2 to 95/5 to give 3h[25] as a white solid (0.048 g, 56% yield). H
NMR (500 MHz) CD3OD δH 1.90 (3H, s, COCH3), 2.95 (1H, dd, J = 3.0,
12.5 Hz, H-4ꢀ), 3.35 (1H, dd, J = 5.5, 12.5 Hz, H-4ꢀ), 4.20 (1H, dd, J = 3.0,
12.5 Hz, CH2OH), 4.35 (1H, dd, J = 5.0, 12.5 Hz, CH2OH), 5.20 (1H, dd, J =
3.5, 5.0 Hz, H-2ꢀ), 5.70 (1H, d, J = 7.5 Hz, H-5), 6.10 (1H, dd, J = 3.5, 5.5 Hz,
H-5ꢀ), 7.68 (1H, d, J = 7.5 Hz, H-6); 13C NMR (125 MHz) CD3OD δC 20.62
(CH3), 38.15 (C-4ꢀ), 65.64 (CH2OH), 84.44 (C-2ꢀ), 89.11 (C-5ꢀ), 96.03 (C-6),
142.49 (C-5), 157.83 (C-2), 167.72 (C-4), 172.08 (COCH3); MS (ESI+) m/z
= 272 [M+1]. HRMS (ES+) m/z found 272.0701 [calcd for C10H14N3O4S+
(M+H)+ 272.0700].

12
V. Ferrari et al.
O-Trityl-lamivudine (3i). Prepared from 2i[26] according to standard pro-
cedure for the N -deacetylation reaction. The crude compound is purified
by flash chromatography on silica gel gradient elution of CH2Cl2/MeOH
from 98/2 to 95/5 to give 3i as a white solid (0.023 g, 25% yield). 1H NMR
(500 MHz) CDCl3 δH 3.09 (1H, dd, J = 2.5, 12.5 Hz, H-4ꢀ), 3.44 (1H, dd, J =
5.5, 12.5 Hz, H-4ꢀ), 3.49 (2H, d, J = 3.5 Hz, CH2OH), 5.19 (1H, t, J = 3.5 Hz,
H-2ꢀ), 5.38 (1H, d, J = 7.0 Hz, H-5), 6.27 (1H, dd, J = 3.0, 5.5 Hz, H-5ꢀ),
7.26–7.15 (10H, m, Ph), 7.39–7.37 (5H, m, Ph), 7.93 (1H, d, J = 7.0 Hz,
H-6); 13C NMR (125 MHz) CDCl3 δC 36.72 (C-4ꢀ), 61.31 (CH2OH), 83.90
(C-2ꢀ), 84.75 (C-5ꢀ), 84.88 (C-Ph), 91.12 (C-5), 124.39, 125.59, 126.23 (CH-
Ph), 139.06 (C-6), 140.08 (C-Ph), 153.01 (C-2), 163.16 (C-4); MS (ESI+) m/z
= 472 [M+1]. HRMS (ES+) m/z found 472.1685 [calcd for C27H26N3O3S+
(M+H)+ 472.1689].
O-Acetyl-acyclovir (3j). Prepared from 2j[19a,27a] according to standard
procedure for the N -deacetylation reaction. The crude compound is purified
by flash chromatography on silica gel gradient elution of CH2Cl2/MeOH
1
from 98/2 to 95/5 to give 3j[27b] as a white solid (0.041 g, 47%). H NMR
(500 MHz) (CD3)2SO δH 1.95 (3H, s, COCH3), 3.65 (2H, dd, J = 7.0, 7.5 Hz,
CH2O), 4.10 (2H, dd, J = 5.0, 6.0 Hz, CH2O), 6.55 (2H, s, NCH2O), 7.80
(1H, s, H-8), 10.65 (1H, brs, NH); 13C NMR (125 MHz) (CD3)2SO δC 20.53
(CH3), 62.70 (NCH2), 66.48 (CH2O), 71.80 (NCH2O), 116.45 (C-5), 137.62
(C-8), 151.39 (C-4), 153.92 (C-2), 156.71(C-6), 170.22 (COCH3); MS (ESI+)
m/z = 268 [M+1]. HRMS (ES+) m/z found 268.1044 [calcd for C10H14N5O4
+
(M+H)+ 268.1040].
5ꢀ-O-Acetyl-2ꢀ,3ꢀ-O-isopropylidene-adenosine (3k). Prepared from
2k[19a,28] or 2l[19a] according to standard procedure for the N -deacetylation
reaction. The crude compound is purified by flash chromatography on
silica gel gradient elution of CH2Cl2/MeOH from 98/2 to 95/5 to give 3k
1
as a white solid (0.068 g, 76% from 2k; 0.034 g, 53% from 2l). H NMR
(500 MHz) CD3Cl δH 1.33 (3H, s, CH3).1.55 (3H, s, CH3), 1.92 (3H, s,
COCH3), 4.15 (1H, dd, J = 6.2 Hz, 11.7 Hz, H5ꢀ), 4.29 (1H, dd, J = 4.4 Hz,
11.7 Hz, H5ꢀ), 4.43–4.38 (1H, m, H4ꢀ), 4.99 (1H, dd, J = 3.4 Hz, 6.3 Hz,
H3ꢀ), 5.41 (1H, dd, J = 2.0 Hz, 6.3 Hz, H2ꢀ), 5.94 (2H, br s, NH2), 6.04 (1H,
d, J = 2.0 Hz, H1ꢀ), 7.82 (1H, s, H8), 8.28 (1H, s, H2); 13C NMR (125 MHz)
CDCl3 δC 20.68 (COCH3), 25.41 (CCH3), 27.14 (CCH3), 64.09 (C5ꢀ), 81.73
(C4ꢀ), 84.23 (C3ꢀ), 85.04 (C2ꢀ), 91.06 (C1ꢀ), 114.59 (C5), 120.35 (CCH3),
139.71 (C8), 149.27 (C4), 153.22 (C2), 155.71 (C6), 170.41 (COCH3).
MS (ES+) m/z (350) [M+1]; HRMS (ES+) m/z found 350.1451 [calcd for
C15H20N5O5+ (M+H)+ 350.1459].
N-acetyl-2ꢀ,3ꢀ-O-isopropylidene-5ꢀ-O-[napthyl (cyclohexyl-L-alanyl)] pho-
sphate cytidine (5). To a solution of 2g[24a,b] (0.5 g, 1.53 mmoL) and
(2S)-cyclohexyl 2-((chloro(naphthalen-1-yloxy)phosphoryl)amino)propa-
noate (4[16,18a], 1.02g, 3.073 mmoL) in anhydrous THF (10mL), 1M tBuMgCl
(3.08 mL, 3.073 mmoL) is added dropwise and the reaction mixture is stirred

N-Deacetylation of Nucleosides and Nucleotides
13
at room temperature overnight. After this period, the solvent is removed un-
der reduced pressure. The crude is purified by column chromatography
on silica gel gradient elution of CH2Cl2/MeOH from 98/2 to 95/5. The
compound 5 is recovered as a white solid[16,18a] (0.420, 40% yield). 1H NMR
(500 MHz) CD3OD δH 1.28–1.40 (11H, m, CHCH3, CH3, 2 × CH2-cHex),
1.52–1.58 (4H, m, CH3, CH2-cHex), 1.69–1.78 (4H, m, 2 × CH2-cHex), 2.15,
2.18 (3H, s, COCH3), 3.99–4.02 (1H, m, CHCH3), 4.40–4.52 (3.5H, m, CH-
2ꢀ, H-4ꢀ, H-5ꢀ), 4.68–4.72 (2H, m, H-2ꢀ, H-3ꢀ), 4.90 (0.5H, m, H-3ꢀ), 5.80 (1H,
d, J = 2.5 Hz, H1ꢀ), 7.25 (0.5H, d, J = 7.5 Hz, H-5), 7.35 (0.5H, d, J = 7.5 Hz,
H-6), 7.39–7.42 (1H, m, Naph), 7.47–7.49 (1H, m, Naph), 7.52–7.58 (2H,
m, Naph), 7.70–7.72 (1H, m, Naph), 7.87–7.89 (1.5H, m, Naph, H-5), 7,96
(0.5H, d, J = 7.5 Hz, H-5), 8.08–8.10 (0.5H, m, Naph), 8.13–8.15 (0.5H, m,
Naph); 13C NMR (125 MHz) CD3OD δC 20.45 (d, JCP = 7.5 Hz, CHCH3),
20.62 (d, JCP = 7.5 Hz, CHCH3), 24.57 (COCH3), 24.60, 24.62 (CH2-cHex),
25.42, 25.52, (CH3), 26.38 (CH2-cHex), 27.37, 27.43 (CH3), 32.35, 32.45
(CH2-cHex), 51.89, 51.97 (CHCH3), 68.01, 68.02 (d, JCP = 7.5 Hz, C-5ꢀ),
75.00 (CHO), 82.22, 82.24 (CH3), 86.57 (CH2), 87.51 (d, JCP = 2.5 Hz, C-
4ꢀ), 87.57 (d, JCP = 2.5 Hz, C-4ꢀ), 96.58, 96.65 (C-1ꢀ), 98.02, 98.12 (C-6),
115.06, 115.21 (C-Naph), 116.22 (d, JCP = 3.7 Hz, CH-Naph), 116. 49 (d,
JCP = 3.7 Hz, CH-Naph), 122.60, 122.68, 126.15, 126.53 (CH-Naph), 126.59,
127.60, 127.70, 127.80, 127.85, 127.93, 128.97 (CH-Naph), 136.28 (C-Naph),
147.20, 147.35 (C5), 147.85 (d, JCP = 7.5 Hz, ipsoC-Naph), 148.00 (d, JCP
= 7.5 Hz, ipsoC-Naph), 157.55, 157.59 (C-2), 164.57 (C-4), 172.85, 172.91
(COCH3), 174.32 (d, JCP = 3.7 Hz, CO2), 174.63 (d, JCP = 3.7 Hz, CO2); 31
P
NMR (500 MHz) MeOD δP 3.92, 4.15. MS (ESI+) m/z = 685 [M+1]. HRMS
(ES+) m/z found 685.2631 [calcd for C33H42N4O10P+ (M+H)+ 685.2633].
2ꢀ,3ꢀ-O-isopropylidene-5ꢀ-O-[napthyl (cyclohexyl-L-alanyl)] phosphate
cytidine (6). Prepared from 5[16,18a] according to standard procedure for
the N -deacetylation reaction. The crude compound is purified by flash
chromatography on silica gel gradient elution of CH2Cl2/MeOH from 98/2
1
to 95/5 to give 6 as a white solid (0.061g, 65% yield). H NMR (500 MHz)
CD3OD δH 1.32–1.41 (12H, m, CHCH3, CH3, 3 × CH2-cHex), 1.54 (3H,
s, CH3), 1.55–1.84 (4H, m, CH2-cHexyl), 4.00–4.05 (1H, m, CHCH3),
4.34–4.44 (3H, m, H-4ꢀ, H-5ꢀ), 4.64–4.77 (2H, m, H-2ꢀ, H-3ꢀ), 4.81–4.84 (m,
1H, CHO), 5.74 (0.5H, d, J = 7.5 Hz, H-6), 5.78–5.81 (1.5H, m, H-1ꢀ, H-6),
7.43–7.48 (1.5H, m, CH-Naph.), 7.50–7.53 (1.5H, m, CH-Naph), 7.55–7.59
(2H, m, CH-Naph, H-5), 7.73 (1H, d, J = 5.0 Hz, CH-Naph), 7.90–7.92
(1H, m, CH-Naph), 8.16–8.20 (1H, m, Naph); 13C NMR (125 MHz) CD3OD
δC 20.45 (d, JCP = 7.5 Hz, CHCH3), 20.60 (d, JCP = 7.5 Hz, CH3), 24.61
(CH2-cHex), 25.50, 25.56, (CH3), 26.39 (CH2-cHex), 27.45, 27.47 (CH3),
32.36, 32.45 (CH2-cHex), 51.91, 51.96 (CHCH3), 68.11, 68.13 (d, JCP
=
5.0 Hz, C-5ꢀ), 74.98 (CHO), 82.23, 82.27 (C-3ꢀ), 86.04, 86.09 (C-2ꢀ), 86.63
(d, JCP = 2.5 Hz, C-4ꢀ), 86.70 (d, JCP = 2.5 Hz, C-4ꢀ), 95.65 (C-1ꢀ), 96.24
(C-6), 115.24, 115.31 (C-Naph), 116.31 (d, JCP = 3.7 Hz, CH-Naph), 116.

14
V. Ferrari et al.
48 (d, JCP = 3.7 Hz, CH-Naph), 122.74, 126.08, 126.54, 126.58, 127.55,
127.60, 127.84 127.87, 128.93 (CH-Naph), 136.33 (C-Naph), 144.19 (C-5),
147.85 (d, JCP = 7.5 Hz, ipsoC-Naph), 148.00 (d, JCP = 7.5 Hz, ipsoC-Naph),
157.84 (C-2), 167.84 (C-4), 174.32 (d, JCP = 3.7 Hz, CO2), 174.63 (d, JCP
=
3.7 Hz, CO2); 31P NMR (500 MHz) CD3OD δP 3.96, 4.11; MS (ES+) m/z =
643 [M+1]. HRMS (ES+) m/z found 643.2521 [calcd for C31H40N4O9P+
(M+H)+ 643.2527].
SUPPLEMENTARY MATERIALS
Supplementary file includes experimental procedures for the synthesis
of protected nucleosides and analytical data for all new compounds. Supple-
mentary materials are available for this article. Go to the publisher’s online
edition of Nucleosides, Nucleotides and Nucleic Acids to view the free supple-
mentary files.
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