Synthesis and reactivity of nucleoside sulfilimines

Article abstract of DOI:10.1080/10426500490257113

Treatment of 3′- and 5′-protected deoxycytidine and deoxyadenosine with bis-[α,α-bis(trifluoromethyl)benzyloxy] diphenylsulfur resulted in the formation of the corresponding 4-N- and 6-N-diphenylsulfilimine derivatives in good yields. The 4-N- and the 6-N-dimethylsulfilimines of these nucleosides were obtained using DMSO / trifluoroacetic anhydride. These dimethyl- and diphenylsulfilimine derivatives were found to have characteristic UV absorbances at 284-296 nm. Stability tests of the nucleoside dimethylsulfilimine and diphenylsulfilimine under the conditions commonly used in oligonuclotide chemistry by the H-phosphonate approach were executed, and possible applications of these compounds have been discussed.

Full text of DOI:10.1080/10426500490257113

Phosphorus, Sulfur, and Silicon, 179:153–171, 2004
ꢀC
Copyright Taylor & Francis Inc.
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426500490257113
SYNTHESIS AND REACTIVITY OF NUCLEOSIDE
SULFILIMINES
A. Slaitas and E. Yeheskiely
Division of Organic and Bioorganic Chemistry,
Scheele Laboratory, Karolinska Institute,
Stockholm, Sweden
(Received May 8, 2003; accepted July 26, 2003)
Treatment of 3ꢁ- and 5ꢁ-protected deoxycytidine and deoxyadenosine
with bis-[α,α-bis(trifluoromethyl)benzyloxy]diphenylsulfur resulted in
the formation of the corresponding 4-N- and 6-N-diphenylsulfilimine
derivatives in good yields. The 4-N- and the 6-N-dimethylsulfilimines
of these nucleosides were obtained using DMSO/trifluoroacetic anhy-
dride. These dimethyl- and diphenylsulfilimine derivatives were found
to have characteristic UV absorbances at 284-296 nm. Stability tests
of the nucleoside dimethylsulfilimine and diphenylsulfilimine under
the conditions commonly used in oligonuclotide chemistry by the H-
phosphonate approach were executed, and possible applications of these
compounds have been discussed.
Keywords: H-phosphonate; nucleoside; nucleotide labeling; sulfilimine;
synthesis
In the course of our research directed towards investigating the poten-
tial use of sulfuranes as peptide-coupling agents,1 amino acid sulfilim-
ines were isolated. The sulfilimines were formed during the condensa-
tion step, when bis-[α,α-bis(trifluoromethyl)-benzyloxy]diphenylsulfur
(BTBDS) was added to the amino component 1, prior to treatment
with the carboxyl residue (the major product was diphenylsulfilimine
2, Scheme 1) or when BTBDS was introduced to a mixture comprising
of both the amino and carboxyl constituents. As a part of our on-going
research focused on the development of synthetic methods for the prepa-
ration of modified PNAs,2–5 peptides,6 and nucleopeptides,7–12 later to
be tested for their biological activity, we were interested in preparing
We thank the Swedish Natural Science Research Council for support.
Address correspondence to E. Yeheskiely, Division of Organic and Bioorganic
Chemistry, Scheele Laboratory MBB, Karolinska Institute, S-17177 Stockholm, Sweden.
153

154
A. Slaitas and E. Yeheskiely
nucleoside sulfilimines and studying their properties and evaluating
their possible use in nucleic acid chemistry.
SCHEME 1 Formation of a sulfilimine from an amine and BTBDS.
RESULTS AND DISCUSSION
In order to explore the properties of an aromatic as well as aliphatic
nucleoside sulfilimines, we embarked on the preparation of the
diphenyl- and the dimethylsulfilimine derivatives of suitably-protected
deoxycytidine and deoxyadenosine. Our intention is to use the infor-
mation obtained from investigating these compounds as a basis for a
broader study on the properties of nucleoside sulfilimines. For the syn-
thesis of the nucleoside diphenylsulfilimine, BTBDS was the reagent of
choice, while for making the nucleoside dimethylsulfilimines a protocol
employing dimethylsulfoxide/trifluoroacetic anhydride was followed.13
This method relies on the activation of dimethylsulfoxide (DMSO) fol-
lowed by addition of an amine. The sulfoxide can be activated by a va-
riety of reagents such as P2O5, BF3, SO14 or trifluoroacetic anhydride
(Tfa2O). For the synthesis of dimethyls3ulfilimines 4a and 4b, Tfa2O
was chosen, since it was reported to perform better than P2O5, BF3 and
SO3.13
SCHEME 2 Reagents: (i) NH3/MeOH; (ii) Et3N × 3 HF/THF; (iii) Ac2O/Py.
Hence, treatment of 3ꢁ,5ꢁ-O-protected deoxynucleosides 3a15 and
3b16 with bis-[α,α-bis(trifluoromethyl)benzyloxy]diphenylsulfur in
CH2Cl2 resulted in the formation of diphenyl-sulfilimines 4a and 4b

Synthesis and Reactivity of Nucleoside Sulfilimines
155
SCHEME 3 Reagents: (i) NH3/MeOH; (ii) Et3N × 3 HF/THF; (iii) Ac2O/Py.
in 82% and 88% yield, respectively (Scheme 3). It should be mentioned,
that protection of the free hydroxyl functions of the nucleosides dur-
ing the BTBDS and the DMSO/trifluoroacetic anhydride reactions is
mandatory, since BTBDS can cause elimination of alcohols to produce
alkenes,17,18 and the DMSO/trifluoroacetic anhydride mixture can bring
about their oxidation.19,20 Next, compound 4c was obtained by am-
monolysis of diacetylated 4a in anhydrous NH3/MeOH. Fluoride-ion
assisted cleavage of the t-butyldimethylsilyl (TBDMS) groups from 4b
resulted in desilylated compound 4d, which was further acetylated with
acetic anhydride in pyridine to give, after work-up and purification, 4e
(92%).
Nucleoside dimethylsulfilimines 5a, 5b, and 5c were prepared
from 3a, 3b, and 3c16 in 70%, 62% and 63% yield, respectively
(Scheme 3) by the reactions of the properly bis-O-protected nucleosides
with DMSO/trifluoroacetic anhydride in CH2Cl2. Dimethylsulfilimine
derivative 5a was further ammonolyzed as mentioned above for bis-
acetylated 4a, while 5f was prepared by removal of the TBDMS group
from 5b with Et3N × 3 HF for 18 h (70% isolated yield), followed by
acetylation with acetic anhydride in pyridine, which proceeded in quan-
titative yield. The dimethylsulfilimine group was completely intact dur-
ing the desilylation and the acetylation reactions.
While engaged in the synthesis of 4a–e and 5a–f, we found that
the sulfilimines have characteristic UV absorbance maxima at 284-296
nm. At this wavelength range, the nucleobases have an absorbance
minimum.21 This UV absorbance simplified purification and analysis of
the above-mentioned compounds, since it enabled a fast and clear iden-
tification of sulfilimine-containing compounds in the reaction mixtures.
In the next stage after the preparation of the completely protected
nucleoside sulfilimines, we focused our attention on testing their stabil-
ity during oligonucleotide synthetic conditions for the H-phosphonate

156
A. Slaitas and E. Yeheskiely
approach. In the course of their preparation, it became apparent that
dimethylsulfilimines 5a–f are less stable than their diphenyl counter-
parts 4a–e. Consequently, a successful application of the former deriva-
tives in the assembly of dimer 8 will indicate that diphenylsulfilimine
will also survive this procedure. Therefore, deoxycytidine dimethylsul-
filimine 5e was selected as starting material in the preparation of din-
ucleotide 8 as outlined in Scheme 4.
SCHEME 4 Reagents: (i) MMT-Cl/Py (ii) H3PO3/Py/PivCl (iii) 3ꢁ-O-Lev-dABz
,
PivCl/Py/MeCN or OXP (iv) I2/Py/H2O.
First, tritylation of compound 5e with monomethoxytrityl chloride
(MMT-Cl) in pyridine afforded compound 6 (49%). Then, phospho-
nylation in pyridine using phosphorous acid and pivaloyl chloride
(PivCl) led, after hydrolysis, to the formation of H-phosphonate mo-
noester 7 (56% isolated yield). Next, condensation of monomer 7 with 6-
N-benzoyl-2ꢁ-deoxy-3ꢁ-O-levulinoyladenosine (3ꢁ-O-Lev-dABz 22
under
)
the mediation of bis-(2-oxo-3-oxazolidinyl)phosphoryl chloride (OXP)23
afforded, after oxidation with iodine/water and purification by column
chromatography, dimer 8 (87%). In addition, dinucleotide 8 was also
synthesized by treating phosphonate 7 and 3ꢁ-O-Lev-dABz in acetoni-
trile and pyridine with pivaloyl chloride, which is the most com-
mon coupling agent in oligonucleotide synthesis by the H-phosphonate
method.24 After 15 min, oxidation with iodine in pyridine/water for
5 min afforded phosphate diester-containing dimer 8. There was a com-
plete conversion of starting material 7 into 8 as judged by 31P NMR
spectroscopy and HPLC analysis of crude compound 8 (using dimer 8
previously prepared from the OXP reaction as a reference).
Stimulated by the outcome of the phosphonylation and oxida-
tion steps, which are two of the most crucial points in solid-phase

Synthesis and Reactivity of Nucleoside Sulfilimines
157
oligonucleotide synthesis, we then explored the stability of the
dimethylsulfilimine group under conditions for another essential step,
i.e. detritylation.24 This is a repeated mild acidolysis of the di- or
monomethoxytrityl groups that serve as temporary protection of the
5ꢁ-hydroxy functions of the nucleosides (at the extension point of the
oligonucleotide chain). In addition, the iodine oxidation24 was further
investigated in more detail. Moreover, although a capping25 (acetyla-
tion) protocol is not regularly used in the H-phosphonate methodology,
acetylation is often performed in order to protect the hydroxyl functions
of the nucleosides and is also standard in the amidite approach. It was
therefore included in the stability studies. The results of these tests are
summarized in Table I.
The data in Table I show that the dimethylsulfilimine function of nu-
cleosides 5a and 5f was not stable in 2% dichloroacetic acid (DCA) in 1,2-
dichloroethane (DCE). Some degradation was observed already after
30 min for both compounds 5a and 5f. This implies that the dimethyl-
sulfilimine group on deoxyadenosine and deoxycytidine may not survive
the repeated detritylation steps. Nonetheless, it may be stable during
several detritylations, necessary for the preparation of a short oligonu-
cleotide fragment. In contrast with the observed lability under acidic
conditions of compound 5a, the dimethylsulfilimine of derivative 5c was
not degraded when exposed to the conditions required for the oxida-
tion of the phosphonate diester function into a phosphate diester24,26 or
to the capping solution (Ac2O/sym-collidine/DMAP/MeCN) commonly
used in some solid-phase nucleic acid synthesis protocols.27 On the
other hand, when cytidine dimethylsulfilimine 5b was dissolved in
the above mentioned iodine solution, it was intact for the first 5 min.
However, slightly longer treatment led to cleavage of the sulfilimine
TABLE I Stability of Adenosine and Cytidine Dimethylsulfiliminesa
2% DCA/DCE
0.01 M Iodineb
Capping mixturec
Time
(min)
dA 5a
dC 5f
dA 5c
dC 5b
dA 5c
dC 5b
5
15
30
60
120
99
99
92
90
83
∼100
97
∼100
∼100
∼100
99
∼100
90
∼100
∼100
∼100
∼100
∼100
90
90
79
76
72
96
94
91
88
81
77
99
aThe values given in the table correspond to the percentage of the intact sulfilimine
as determined by straight-phase HPLC (mean error 3%).
bAs a solution in collidine/water/MeCN.
cPrepared by mixing equal volumes of Capping A and B solutions, containing Ac2O/
collidine/MeCN 2:3:5 (v/v/v) and 6% 4-(N,N-dimethylamino)pyridine (DMAP) in MeCN
(w/v), respectively.

158
A. Slaitas and E. Yeheskiely
(10% within the 15 min) as indicated in Table I. Moreover, exposure
of 5b to the capping mixture proved to have a deleterious effect on
the sulfilimine integrity. In this case, 10% of nucleoside 5b was af-
fected within 5 min. The information summarized in Table I sug-
gests that only properly protected dimethylsulfilimine derivatives of
deoxyadenosine may endure oligonucleotide synthesis protocols that
require repeated oxidation steps or oxidation times longer than 5
min. This is also correct for methods that endorse repeated capping
step.
Next, the diphenylsulfilimino group in compounds 4a, 4b, and 4e
was subjected to the same conditions as described in Table I for the
dimethylsulfilimine function. The figures obtained from these stabil-
ity tests are given in Table II, and they indicate that, in contrast to
their dimethyl counterparts, compounds 4a and 4b were stable during
treatment with 2% DCA in DCE for 120 min. Prolonged exposure (24 h)
to this mixture showed that the deoxycytidine derivative 4d remained
unaffected, while Ac2dA = SPh2 (4a) was less stable and was partially
degraded (9%).
The diphenylsulfilimine moiety was also found to be much more sta-
ble in the oxidation mixture of I2/collidine in aqueous acetonitrile. After
2 h, the deoxycytidine derivative 4b was still completely intact in con-
trast with 4a which was partially cleaved (6%) as depicted in Table II.
Furthermore, no cleavage of the diphenylsulfilimine function in 4a and
4b was observed in a mixture of Ac2O/collidine/DMAP in MeCN for at
least 2 h.
The stability of the diphenylsulifilimino group towards the conditions
listed in Table II suggests that it will endure the assembly of longer
TABLE II Stability of Adenosine and Cytidine Diphenylsulfiliminesa
2% DCA/DCE
0.01 M Iodineb
Capping mixturec
Time
(min)
Ac2dA-
SPh2 4a
Ac2dC-
SPh2 4e
Ac2dA-
SPh2 4a
Si2dC-
SPh2 4b
Ac2dA-
SPh2 4a
Si2dC-
SPh2 4b
5
15
30
60
120
∼100
∼100
∼100
∼100
∼100
∼100
∼100
∼100
∼100
99
∼100
∼100
∼100
99
∼100
∼100
∼100
∼100
∼100
∼100
∼100
∼100
∼100
∼100
∼100
∼100
∼100
∼100
∼100
94
aThe values given in the table correspond to the percentage of the intact sulfilimine
as determined by straight-phase HPLC (mean error 3%).
bAs a solution in collidine/water/MeCN.
cPrepared by mixing equal volumes of Capping A and B solutions, containing
Ac2O/collidine/MeCN 2:3:5 (v/v/v) and 6% DMAP in MeCN (w/v), respectively.

Synthesis and Reactivity of Nucleoside Sulfilimines
159
nucleic acid fragments on a solid support using the H-phosphonate ap-
proach. A successful application of nucleoside diphenylsulfilimines in
the phosphoroamidite chemistry is also very likely, provided they would
survive the repeated phosphonylation step.
In the following stage, the stability of dA = SMe2,dC = SMe2, dA =
SPh2 and dC = SPh2 (5d, 5e, 4c and 4d, respectively) in an aqueous en-
vironment and during ammonolysis, which is the final step in oligonu-
cleotide synthesis,24 employed to remove the protecting groups from the
nucleobases and detach the oligonucleotide fragment from the solid-
support, was examined. Exposure to 33% aq. NH3 for 16 h at 21◦C
showed that 4c, 4d, and 5d were un-degraded while 5e lost 12% of the
dimethylsulfilimine group. Upon treatment with 33% aq. NH3 at ele-
vated temperature, the diphenylsulfilimine derivatives 4c and 4d re-
mained unaffected, but compounds 5d and 5e were almost completely
converted into the corresponding deoxyadenosine and deoxycytidine,
respectively. In anhydrous NH3 in methanol, at 21◦C, all four nucleo-
side sulfilimines 5d, 5e, 4c, and 4d were found to be stable for at least
2 h.
The increased stability of the diphenylsulfilimines 4c and 4d, com-
pared to the dimethyl- 5d and 5e, is also evident in water and 50 mM
TRIS buffer (pH 9.0).
The diphenylsulfilimines were stable in aqueous solutions at 21◦C
for 16 hours, while the dimethyl analogues were substantially degraded
under the same conditions. In H2O at elevated temperature however,
only dA = SPh2 (4c) was stable, while compound dC=SPh2 (4d) lost 37%
of the sulfilimino group, and derivatives 5d and 5e were detected only
in trace amounts.
The outcome of the stability tests in aqueous solutions is consistent
with the results of the tests reported above which show the increased
stability of the diphenylsulfilimine function compared to the dimethyl-
sulfilimine. This outcome leads to the conclusion that the diphenyl-
sulfilimine group would not be cleaved at the final stage of nucleic acids
synthesis, i.e. the deprotection of the nucleobases and detachment from
the solid-support which is brought about by ammonolysis either at am-
bient temperature or at 60◦C.
This stability to ammonolysis and to the above-mentioned conditions
depicted in Table II permits retaining the diphenylsulfilimine group
during and after oligonucleotide synthesis, a fact that can make them
useful for the introduction of different functionalities into the nucleic
acid fragment which can be accomplished by substitution and/or exten-
sion of the sulfilimine aromatic ring(s) accordingly.
Finally, since it was reported that 2-mercaptoethanol (β-ME) could
cause cleavage of sulfilimines in the presence of acid,28 we were curious

160
A. Slaitas and E. Yeheskiely
TABLE III Stability of 3ꢁ,5ꢁ-Unprotected Nucleoside Sulfilimines in Aqueous
Solutionsa
50 mM TRIS
H2O,
H2O, 33% aq. NH3, 33% aq. NH3,
(pH 9.0), 21◦C, 21◦C, 60◦C,
21◦C,
16 h
60◦C,
16 h
No.
Compound
16 h
16 h
16 h
1
2
3
4
dA = SMe2 5d
dC = SMe2 5e
dA = SPh2 4c
dC = SPh2 4d
90
59
96
77
0
3
98
64
98
88
1
7
∼100
∼100
∼100
∼100
99
99
∼100
98
aThe values given in the table correspond to the percentage of the intact sulfilimine
as determined by reversed- phase HPLC (mean error 3%).
to determine the effect of β-ME solution on 4a, 4b, 5b, and 5c. We found
that the dimethylsulfilimines 5b and 5c underwent a rapid conversion
(within 5 min) into their corresponding starting materials 3b and 3c,
while the diphenylsulfilimines were much more stable. After 2 h, the
deoxycytidine derivative 4b was still completely intact in contrast with
4a, which was partially cleaved (10%).
CONCLUSIONS
Nucleoside diphenyl- and dimethylsulfilimines were synthesized in
good yields. The stability tests clearly show that the nucleoside
diphenylsulfilimines are significantly more stable than those with
the dimethylsulfilimines group. These tests suggest that nucleoside
dimethylsulfilimines are not suitable for the solid-phase synthesis of
long oligonucleotide fragments by the H-phosphonate approach. How-
ever, they may be used in the synthesis of short fragments, such as
dimers, and possibly trimers and tetramers, either in solution or on a
solid-support with reasonably good yields.
Unlike the dimethylsulfilimine nucleosides, it should be possible to
use the diphenylsulfilimine derivatives in the solid-support assembly of
long oligonucleotides. Incorporating one or several nucleoside diphenyl-
sulfilimine can be advantageous due to the fact that they are unaf-
fected by ammonolysis and that the sulfilimine moiety absorbs at 290–
300 nm. Thus, they could be used as UV tags for oligonucleotides via pin-
pointing their location in the presence of unlabeled fragments. Another
possible advantage is that by modifying one or both the phenyl rings
accordingly, diarylsulfilimine-containing nucleotides could be used to
introduce functionalities, such as, for example an intercalator, at a
predetermined position in a nucleic acid fragment.

Synthesis and Reactivity of Nucleoside Sulfilimines
161
EXPERIMENTAL
General Remarks: Solvents were purchased from Merck Eurolab. Dry
solvents (except THF) were obtained by treatment with the appropriate
˚
˚
molecular sieves (3 A for MeCN and methanol, 4A for DMSO, CH2Cl2,
1,2-dichloroethane, and pyridine). The THF was distilled over Na/K-
benzophenone ketyl.
2ꢁ-Deoxynucleosides were purchased from Pharma-Waldof GmbH
(Germany). sym-collidine (2,4,6-trimethylpyridine), BTBDS, H3PO3
and Piv-Cl were purchased from Aldrich; 2-mercaptoethanol from
Sigma; MMT-Cl, OXP, TBDMS-Cl, and Tfa2O from Fluka; Ac2O, DCA,
and Et3N × 3 HF from Lancaster, DMAP from Novabiochem, and io-
dine from May and Baker Ltd. All reagents were used as supplied,
except Piv-Cl and DCA which were distilled through a short Vigreux
column.
Flash chromatography was performed on Merck Silica gel 60 (40–
63 µm).
TLC was performed using analytical Merck silica plates with
F254 indicator and visualized under 254 nm UV or by staining
with H2SO4/MeOH. The following mobile phase systems were used:
CHCl3/MeOH 9:1 (v/v)—System A, CHCl3/MeOH 4:1 (v/v)—System B,
ethyl acetate/iPrOH 9:1—System C.
Straight-phase HPLC was performed on a Jasco HPLC system
equipped with Jasco Diode Array detector (MD-910). The columns were
as follows: Analytical (5 µm, ThermoHypersil, 250 × 4.6 mm) or prepar-
ative silica column (5 µm, Hypersil, 150 × 21.2 mm). The HPLC exper-
iments were executed at ambient temperature. The mobile phase con-
sists of 2% MeOH and 0.2% Et3N in CH2Cl2 with a flow rate of 1 mL/min
and observed at 285 nm.
The RP HPLC was performed on a C18 column (5 µm, Jupiter, 250 ×
4.6 mm) at 30◦C. Buffer A—50 mm TRIS (pH 9.0), buffer B—70% MeCN
in water, gradient 0–60% B in 60 min.
UV absorbance spectra were recorded on Jasco Diode Array detector
(wavelength range 220–400 nm, acquisition time 800 ms).
Nominal mass spectra were recorded on a Micromass LCT mass spec-
trometer (ESI-TOF). High-resolution mass spectral (HRMS) measure-
ments were obtained in positive or negative ion mode using leucine
enkephalin as an internal mass standard.
NMR spectra were recorded on a Bruker Avance DRX 400 spectrome-
ter at 400.13 and 100.61 MHz for 1H and 13C, respectively. The chemical
shifts are reported in ppm using residual solvent signal as an internal
reference.

162
A. Slaitas and E. Yeheskiely
3ꢀ,5ꢀ-di-O-Acetyl-deoxyadenosine Diphenylsulfilimine
(4a)
A solution of 3ꢁ,5ꢁ-di-O-acetyl-deoxyadenosine (0.31 g, 0.93 mmol) in
dry CH2Cl2 (10 mL) was added under a stream of nitrogen and stirring
to a solution of BTBDS (0.75 g, 1.11 mmol) in dry CH2Cl2 (1.0 mL).
After 1 hour, the reaction mixture was transferred into a separation
funnel containing CH2Cl2 (20 mL) and 0.25 M phosphate buffer (20 mL,
pH 7.0). The organic phase was washed with water and brine (10 mL
each) and then was dried (Na2SO4) and concentrated under reduced
pressure yielding colored oily substance. Purification by flash column
chromatography (silica) using 2% methanol and 0.1% Et3N in CH2Cl2
afforded pure compound 4a.
Yield 0.39 g (82%).
1H NMR (CDCl3): 8.23 (1H, s), 7.97 (1H, s), 7.92-7.87 (4H, m), 7.51-
7.44 (6H, m), 6.44 (1H, dd, J = 5.8, 8.3), 5.42 (1H, dt, J = 2.2, 6.2), 4.41
(1H, dd, J = 5.3, 12.9), 4.36-4.32 (2H, m), 2.96-2.89 (1H, m), 2.59 (1H,
ddd, J = 2.3, 5.8, 14.1), 2.12 (3H, s), 2.10 (3H, s).
13C NMR (CDCl3): 170.90, 170.76, 162.31, 152.79, 149.53, 137.53,
132.21, 130.10, 128.15, 125.32, 84.69, 82.72, 75.15, 64.30, 37.95, 21.37,
21.23.
HRMS calculated for C26H26N5O5S m/z: 520.1655. Found: 520.1673.
HPLC: tR 3.4 min (2% MeOH and 0.2% Et3N in CH2Cl2).
λmax (2% MeOH and 0.2% Et3N in CH2Cl2) 296 nm.
3ꢀ,5ꢀ -bis-O-TBDMS-deoxycytidine Diphenylsulfilimine
(4b)
Compound 4b was synthesized and purified as described above for com-
pound 4a using 3ꢁ,5ꢁ-bis-O-TBDMS-deoxycytidine (0.66 g, 1.45 mmol)
and BTBDS (1.11 g, 1.65 mmol).
Yield 0.82 g (88 %).
1H NMR (CDCl3): 7.86 (1H, d, J = 7.5), 7.74-7.69 (4H, m), 7.49-7.43
(6H, m), 6.33 (1H, dd, J = 5.4, 6.1), 6.02 (1H, d, J = 7.5), 4.39 (1H, dt,
J= 5.7, 5.5), 3.91 (1H, dd, J = 2.6, 11.3), 3.85-3.83 (1H, m), 3.76 (1H,
dd, J = 2.1, 11.3), 2.38-2.32 (1H, m), 2.11-2.05 (1H, m), 0.92 (9H, s),
0.86 (9H, s), 0.10 (6H, d, J = 2.3), 0.04 (6H, s).
13C NMR (CDCl3): 172.24, 155.89, 139.67, 136.74, 136.65, 132.29,
132.20, 130.19, 130.15, 128.56, 128.31, 100.04, 87.33, 85.83, 77.71,
62.35, 42.46, 26.34, 26.16, 18.77, 18.39, −4.16, −4.51, −5.08, −5.14.
HRMS calculated for C33H50N3O4SSi2 m/z: 640.3061. Found:
640.3065.
HPLC: tR 3.3 min (2% MeOH and 0.2% Et3N in CH2Cl2).
λmax (2% MeOH and 0.2% Et3N in CH2Cl2) 297 nm.

Synthesis and Reactivity of Nucleoside Sulfilimines
163
Deoxyadenosine Diphenylsulfilimine (4c)
Compound 4a (142 mg, 0.28 mmol) was dissolved in saturated
NH3/MeOH (12 mL). After 2 h, when TLC analysis (System A) indi-
cated that the starting material was consumed and the solvent and
ammonia were removed under reduced pressure. The remaining solid
was dissolved in methanol, precipitated from diethyl ether, filtered, and
washed with diethyl ether. Chromatographically pure compound 4c was
then obtained and used without additional purification.
Yield 72.6 mg (60%).
1H NMR (CD3CN): 8.02 (1H, s), 7.96 (1H, s), 7.93-7.88 (4H, m), 7.62-
7.55 (6H, m), 6.33 (1H, dd, J = 5.6, 9.0), 5.81 (1H, bs), 4.57 (1H, bd,
J = 5.4), 4.05 (1H, dd, J = 2.3, 3.7), 3.78 (1H, dd, J = 2.2, 12.6), 3.71-
3.64 (1H, m), 3.42 (1H, bs), 2.88 (1H, ddd, J = 5.4, 9.1, 14.5), 2.26 (1H,
ddd, J = 1.5, 5.7, 13.3).
13C NMR (CD3CN): 162.32, 151.23, 148.69, 140.10, 137.70, 137.60,
132.52, 132.46, 130.39, 127.78, 127.69, 89.64, 87.35, 72.92, 67.08, 63.36,
40.46.
HRMS calculated for C22H22N5O3S m/z: 436.1443: Found: 436.1441.
RP-HPLC tR 26.8 min.
λmax (MeOH): 294.4 nm (ε = 21800).
Deoxycytidine Diphenylsulfilimine (4d)
Compound 4d can also be prepared as described above for compound
4c using 99 mg (0.20 mmol) of 3ꢁ,5ꢁ-di-O-acetyl-deoxycytidine diphenyl-
sulfilimine (4e) and 10 mL saturated NH3 in methanol.
Yield 51 mg (62%).
1H NMR (CD3CN): 7.84-7.81 (4H, m), 7.65 (1H, d, J = 7.4), 7.59-7.54
(6H, m), 6.10 (1H, t, J = 6.7), 6.02 (1H, dd, J = 0.3, 7.5), 4.34 (1H, bd,
J = 5.1), 3.84 (1H, q, J = 3.5), 3.71-3.68 (1H, m), 3.66 (1H, m), 3.51 (1H,
bs), 3.40 (1H, bs), 2.18 (1H, dd, J = 1.4, 2.8), 2.16 (1H, t, J = 2.1).
13C NMR (CD3CN): 171.89, 155.28, 140.81, 137.11, 137.05, 132.54,
132.50, 130.38, 127.85, 127.78, 99.69, 87.68, 87.19, 71.47, 67.08, 62.27,
40.36.
HRMS calculated for C21H22N3O4S m/z: 412.1331. Found: 412.1347.
RP-HPLC tR 25.2 min.
λmax (MeOH): 293.7 nm (ε = 17500).
3ꢀ,5ꢀ-di-O-Acetyl-deoxycytidine Diphenylsulfilimine (4e)
Compound 4b (0.34 g, 0.53 mmol) was dissolved in dry THF (12 mL),
and Et3N × 3 HF (0.87 mL, 5.33 mmol) was added. The reaction was

164
A. Slaitas and E. Yeheskiely
followed by TLC (System A), and, upon completion, the solvent was
removed under reduced pressure. The remaining oily residue contain-
ing deoxycytidine diphenylsulfilimine(4d) was co-evaporated with dry
pyridine (2 × 3 mL), re-dissolved in pyridine (3 mL) and Ac2O (0.16 mL,
1.6 mmol) was then added with stirring. After completion, the reaction
was quenched with methanol (1 mL), and the solvent removed at re-
duced pressure. The residue was dissolved in ethyl acetate (20 mL),
extracted with saturated aqueous NaHCO3 (2 × 10 mL), water (10 mL)
and brine (10 mL) and then was dried (Na2SO4) and concentrated.
Compound 4e was purified by short column chromatography using 2%
methanol in chloroform.
Yield 0.24 g (92%).
1H NMR (CDCl3): 7.75-7.71 (4H, m), 7.53-7.46 (6H, m), 7.44 (1H, d,
J = 7.5), 6.39 (1H, dd, J = 5.6, 8.3), 6.08 (1H, d, J = 7.5), 5.19 (1H, dt,
2.2, 6.7), 4.37-4.29 (2H, m), 4.24-4.22 (1H, m), 2.57 (1H, ddd, J = 2.1,
5.6, 14.2), 2.10 (3H, s), 2.09 (3H, s), 2.06 (1H, dd, 6.2, 7.9).
13C NMR (CDCl3): 172.20, 170.94, 170.83, 155.62, 138.12, 136.48,
136.43, 132.40, 132.33, 130.25, 130.22, 128.49, 128.32, 126.29, 100.90,
86.33, 82.21, 74.90, 64.48, 38.64, 21.39, 21.24.
HRMS calculated for C25H26N3O6S m/z: 496.1542. Found: 496.1526.
HPLC: tR 3.1 min (2% MeOH and 0.2% Et3N in CH2Cl2).
λmax (2% MeOH and 0.2% Et3N in CH2Cl2) 296 nm.
3ꢀ,5ꢀ-di-O-Acetyl-deoxyadenosine Dimethylsulfilimine
(5a)
Anhydrous DMSO (0.114 mL, 1.61 mmol) in CH2Cl2 (1.8 mL) was mixed
in an oven-dried flask equipped with a thermometer and a magnetic
stirring bar. The reaction flask was cooled to –65◦C, and neat triflu-
oroacetic anhydride (0.125 mL, 0.90 mmol) was then added drop-wise
(the temperature of the reaction mixture should not exceed –55◦C). Dur-
ing the addition of Tfa2O, a white precipitate was formed (sulfonium
reagent).
Meanwhile, 3ꢁ,5ꢁ-di-O-acetyl-deoxyadenosine (0.30 g, 0.90 mmol) was
dissolved in a mixture of dry CH2Cl2 and DMSO (1.8 mL and 0.26 mL,
respectively). This solution was slowly added to the pre-formed sulfo-
nium reagent (temperature kept below –45◦C). After addition, the re-
action was continued for another 60 min., cooled down to –60◦C, where
upon Et3N (0.28 mL) was added. Finally, the reaction mixture was al-
lowed to warm up to 0◦C and transferred into a separation funnel con-
taining CH2Cl2 (20 mL) and 0.25 M phosphate buffer (10 mL, pH 7).
The organic phase was further washed with water (10 mL) and brine
(10 mL) and was then dried (Na2SO4) and evaporated.

Synthesis and Reactivity of Nucleoside Sulfilimines
165
Compound 5a was purified by flash column chromatography as fol-
lows, the column was first eluted with 10% iPrOH in ethyl acetate (elu-
tion of by-products) and then with 10% MeOH in CH2Cl2 containing
0.1% Et3N (elution of the product).
Yield 0.25 g (70%).
1H NMR (CDCl3): 8.26 (1H, s), 7.91 (1H, s), 6.43 (1H, dd, J = 5.9,
8.1), 5.43 (1H, dt, J = 2.3, 6.3), 4.44-4.40 (1H, m), 4.36-4.31 (1H, m),
2.96-2.88 (1H, m), 2.86 (6H, s), 2.60 (1H, ddd, J = 3.1, 6.6, 14.8), 2.13
(3H, s), 2.11 (3H, s).
13C NMR (CDCl3): 170.93, 170.78, 162.35, 152.85, 149.16, 137.25,
124.84, 84.68, 82.70, 75.12, 64.29, 37.94, 33.60, 21.38, 21.24.
HRMS calculated for C16H22N5O5S m/z: 396.1342. Found: 396.1349.
HPLC: tR 4.4 min (2% MeOH and 0.2% Et3N in CH2Cl2).
λmax (2% MeOH and 0.2% Et3N in CH2Cl2) 289 nm.
3ꢀ,5ꢀ-bis-O-TBDMS-deoxycytidine Dimethylsulfilimine
(5b)
Compound 5b was prepared as described above for compound 5a using
3ꢁ,5ꢁ-bis-O-TBDMS-deoxycytidine (1.00 g, 2.2 mmol), DMSO (0.28 mL,
3.95 mmol), and Tfa2O (0.30 mL, 2.2 mmol).
Yield 0.70 g (62%).
1H NMR (CDCl3): 7.80 (1H, d, J = 7.5), 6.34 (1H, dd, J = 5.5, 6.1),
5.78 (1H, d, J = 7.5), 4.39 (1H, dt, J = 5.5, 5.9), 3.90 (1H, dd, J = 2.5,
11.3), 3.84-3.83 (1H, m), 3.75 (1H, dd, J = 1.9, 11.3), 2.77 (3H, s), 2.76
(3H, s), 2.38-2.32 (1H, m), 2.10-2.04 (1H, m), 0.91 (9H, s), 0.87 (9H, s),
0.08 (6H, s), 0.04 (6H, s).
13C NMR (CDCl3): 171.79, 156.02, 139.48, 99.54, 87.33, 85.73, 70.66,
62.34, 42.45, 32.97, 32.92, 26.31, 26.16, 18.75, 18.40, −4.16, −4.50,
−5.12, −5.17.
HRMS calculated for C23H46N3O4SSi2 m/z: 516.2748. Found:
516.2750.
HPLC: tR 3.3 min (2% MeOH and 0.2% Et3N in CH2Cl2).
λmax (2% MeOH and 0.2% Et3N in CH2Cl2) 292 nm.
3ꢀ,5ꢀ-bis-O-TBDMS-deoxyadenosine Dimethylsulfilimine
(5c)
Compound 5c was prepared as described above for compound 5a us-
ing 3ꢁ,5ꢁ-bis-O-TBDMS-deoxyadenosine (0.30 g, 0.63 mmol), DMSO
(0.16 mL, 2.25 mmol), and Tfa2O (0.174 mL, 1.25 mmol).
Yield 0.21 g (63%).
1H NMR (CDCl3): 8.28 (1H, s), 8.07 (1H, s), 6.45 (1H, t, J = 6.4), 4.62-
4.58 (1H, m), 4.00 (1H, dt, J = 3.3, 3.4), 3.87 (1H, dd, J = 4.2, 11.1),

166
A. Slaitas and E. Yeheskiely
3.78 (1H, dd, J = 3.2, 11.1), 2.87 (6H, s), 2.62-2.55 (1H, m), 2.44-2.38
(1H, m), 0.93 (9H, s), 0.92 (9H, s), 0.10 (6H, s), 0.08 (6H, s).
13C NMR (CDCl3): 152.38, 149.06, 137.95, 124.69, 88.06, 84.39, 72.24,
63.21, 41.78, 33.77, 26.42, 26.18, 18.86, 18.42, −4.27, −4.41, −4.95,
−5.07.
HRMS calculated for C24H46N5O3SSi2 m/z: 540.2860. Found:
540.2863.
HPLC: tR 4.7 min (2% MeOH and 0.2% Et3N in CH2Cl2).
λmax (2% MeOH and 0.2% Et3N in CH2Cl2) 292 nm.
Deoxyadenosine Dimethylsulfilimine (5d)
Compound 5d was prepared as described above for compound 4c start-
ing from 64.2 mg of compound 5a and 10 mL of saturated ammonia in
methanol.
Yield 39.2 mg (60%).
1H NMR (CD3CN): 8.07 (1H, s), 7.83 (1H, s), 6.29 (1H, dd, J = 5.7,
9.2), 6.13 (1H, d, J= 9.7), 4.57 (1H, d, J= 5.1), 4.06 (1H, dd, J= 2.1, 3.5),
3.79 (1H, dd, J = 0.8, 12.8), 3.67 (1H, dt, J = 1.9, 12.4), 3.47 (1H, bs),
2.88 (1H, ddd, J = 5.3, 9.2, 14.5), 2.79 (6H, s), 2.25 (1H, dt, J = 1.4, 5.7).
13C NMR (CD3CN): 162.65, 151.47, 148.15, 139.38, 125.33, 89.70,
87.44, 72.99, 63.43, 40.48, 32.31, 32.27.
HRMS calculated for C12H18N5O3S m/z: 312.1130. Found: 312.1132.
RP-HPLC tR 13.3 min.
Deoxycytidine Dimethylsulfilimine (5e)
To a solution of compound 6b (0.60 g, 1.17 mmol) in dry THF (25 mL)
was added in one portion neat Et3N × 3 HF (1.9 mL, 11.7 mmol). The
reaction was followed by TLC (system A) and, upon completion (16 h),
the mixture was concentrated at reduced pressure. The residue was
quenched with 2 M TEAB buffer (3 mL, pH 7.5) and lyophilized. Chro-
matographically pure compound 5e was obtained as a white powder by
trituration from acetone, and was used without additional purification.
Yield 0.24 g (70%).
1H NMR (CD3OD): 7.84 (1H, d, J = 7.5), 6.30 (1H, t, J = 6.8), 5.91
(1H, d, J = 7.5), 4.38 (1H, dt, J = 3.3, 6.2), 3.93 (1H, q, J = 3.6), 3.76
(2H, dq, J = 3.4, 12.0), 2.82 (6H, s), 2.32 (1H, ddd, J = 3.6, 6.1, 13.5),
2.19-2.10 (1H, m).
13C NMR (CD3OD): 139.51, 100.21, 87.69, 86.30, 71.20, 61.91, 40.81,
31.01, 30.94.
HRMS calculated for C11H18N3O4S m/z: 288.1018. Found: 288.1017.
RP-HPLC tR 9.6 min.

Synthesis and Reactivity of Nucleoside Sulfilimines
167
3ꢀ,5ꢀ-di-O-Acetyl-deoxycytidine Dimethylsulfilimine (5f)
To a suspension of compound 5e (55.8 mg, 0.19 mmol) in dry pyridine
(1 mL) was added acetic anhydride (47 µL, 0.49 mmol). After completion
(determined by TLC analysis in System A), the reaction mixture was
quenched with methanol (1 mL) and concentrated, and the remaining
solid was dissolved in CH2Cl2 (2 mL). Purification on a silica column,
which was eluted with 2% MeOH in CH2Cl2, gave pure compound 5f as
a white foam.
Yield 71.7 mg (99.5%).
1H NMR (CDCl3): 7.39 (1H, d, J = 7.5), 6.43 (1H, dd, J = 5.6, 8.4),
5.85 (1H, d, J = 7.5), 5.22-5.19 (1H, m), 4.37-4.29 (2H, m), 4.25-4.23
(1H, m), 2.78 (6H, s), 2.58 (1H, ddd, J = 2.0, 5.6, 14.2), 2.11 (3H, s), 2.10
(3H, s), 2.06 (1H, dd, J = 6.1, 7.8).
13C NMR (CDCl3): 171.71, 170.94, 155.75, 100.45, 86.19, 82.19, 74.91,
64.47, 46.17, 38.58, 32.89, 32.82, 21.40, 21.24, 9.04.
HRMS calculated for C15H22N3O6S m/z: 372.1229. Found: 372.1226.
HPLC: tR 4.7 min (2% MeOH and 0.2% Et3N in CH2Cl2).
λmax (2% MeOH and 0.2% Et3N in CH2Cl2) 289 nm.
5ꢀ-O-Monomethoxytrityl-deoxycytidine
Dimethylsulfilimine (6)
To a suspension of compound 5e (80 mg, 0.28 mmol) in dry pyridine
(2 mL) was added in one portion monomethoxytrityl chloride (112 mg,
0.35 mmol). The completion of the reaction was determined by TLC
(System A). The reaction mixture was quenched with methanol (1 mL)
and concentrated, and the remaining solid was dissolved in CH2Cl2
(10 mL). The organic layer was washed with sat. aq. NaHCO3 (5 mL) and
brine (5 mL) and then was dried (Na2SO4). Purification was performed
by flash column chromatography (silica) using first EtOAc/iPrOH 9:1
containing 0.1% Et3N as eluent, then CH2Cl2/MeOH 9:1 + 0.1% Et3N.
This gave pure compound 6 as a white foam.
Yield 76 mg (49%).
1H NMR (CDCl3): 7.64 (1H, d, J = 7.5), 7.46-7.43 (4H, m), 7.35-
7.23 (8H, m), 6.88-6.84 (2H, m), 6.44 (1H, t, J = 6.2), 5.66 (1H, d,
J = 7.5), 4.52-4.49 (1H, m), 4.05 (1H, q, J = 3.9), 3.81 (3H, s), 3.47-
3.41 (2H, m), 2.77 (3H, s), 2.77 (3H, s), 2.59-2.52 (1H, m), 2.24-2.18
(1H, m).
13C NMR (CDCl3): 171.73, 159.06, 156.08, 144.51, 144.35, 139.19,
135.47, 130.81, 128.79, 128.36, 127.50, 113.63, 99.94, 87.38, 85.84,
85.72, 72.19, 63.81, 55.66, 42.00, 32.89, 32.78, 30.11.
HRMS calculated for C31H34N3O5S m/z: 560.2219. Found: 560.2216.

168
A. Slaitas and E. Yeheskiely
5ꢀ-O-Monomethoxytrityldeoxycytidine
Dimethylsulfilimine 3ꢀ-H-phosphonate (7)
Phosphorous acid (370 mg, 4.5 mmol) was co-evaporated with dry pyri-
dine (3 × 3 mL) and then re-dissolved in dry pyridine (2.7 mL). To this
solution was added pivaloyl chloride (0.28 mL, 2.3 mmol), resulting in
the formation of the pyrophosphorous acid reagent.
The reagent (0.27 mL) was then added to the solution of compound 6
(50.6 mg, 0.09 mmol) in pyridine (0.27 mL). The reaction was followed by
TLC (System B), and upon, completion, was quenched with 1 M TEAB
buffer (1 mL, pH 7.5). Next, CH2Cl2 was added (15 mL) and the organic
mixture was washed with 2 M TEAB (3 mL, pH 7.5). Drying (Na2SO4)
and evaporation of the organic phase gave crude compound 7, which
was purified by straight-phase HPLC, using 8% methanol in CH2Cl2
with 0.2% Et3N as eluent.
Yield 36.5 mg (56%).
1H NMR (CDCl3): 7.52 (1H, d, J = 7.4), 7.40-7.37 (2H, m), 7.25-7.16
(8H, m), 6.86 (1H, d, J = 620.5), 6.80 (2H, d, J = 8.9), 6.41 (1H, t, J =
6.8), 5.55 (1H, d, J = 7.5), 4.86 (1H, bs), 4.20 (1H, bs), 3.75 (3H, s), 3.33
(2H, bs), 3.00 (6H, q, 7.3), 2.72 (3H, s), 2.71 (3H, s), 2.16-2.10 (1H, m),
1.30 (9H, t, J = 7.3).
13C NMR (CDCl3): 171.53, 158.98, 156.45, 144.25, 138.98, 135.37,
130.83, 128.83, 128.31, 127.43, 113.61, 100.50, 87.31, 85.97, 85.21,
74.39, 64.00, 55.61, 45.90, 40.68, 32.88, 32.78, 27.97, 9.28.
31P NMR (CDCl3): 4.98 (d, J = 612.4).
HRMS calculated for C31H35N3O7PS m/z: 624.1933. Found:
624.1941.
HPLC: tR 12.0 min (8% MeOH and 0.2% Et3N in CH2Cl2).
λmax (2% MeOH and 0.2% Et3N in CH2Cl2) 290 nm.
Dinucleotide 8 (Using OXP as a Coupling Agent)
Compound 7 (12.8 mg, 18 µmol) and 6-N-benzoyl-3ꢁ-O-levulinoyl-
deoxyadenosine (12.3 mg, 27 µmol) were dried by co-evaporation with
dry pyridine (3 × 1 mL) and then were dissolved in pyridine (1 mL).
To this solution was added OXP (9.2 mg, 36 µmol) under stirring. After
16 hours, a 0.01 M solution of iodine in pyridine/water 98:2 (0.36 mL)
was added, and oxidation was continued for 5 min. The reaction mix-
ture was diluted with CH2Cl2 (15 mL) and transferred to a separation
funnel containing 10% aq. Na2S2O3 (5 mL) and 1 M TEAB (pH 7.5,
5 mL). Drying (Na2SO4) and evaporation of the organic phase gave
crude compound 8, which was purified by straight-phase HPLC, using
10% methanol in CH2Cl2 with 0.2% Et3N as eluent.

Synthesis and Reactivity of Nucleoside Sulfilimines
169
Yield 18.4 mg (87%).
1H NMR (CDCl3) selected signals: 8.85 (1H, s), 8.80 (1H, s), 8.01
(2H, d, J = 7.4), 7.59-7.56 (2H, m), 7.50-7.46 (2H, m), 7.40-7.38 (4H, m),
7.28-7.23 (5H, m), 7.19-7.15 (2H, m), 6.81-6.78 (2H, m), 6.63 (1H, dd,
J = 5.8, 8.8), 6.42 (1H, t, J = 5.7), 5.61 (3H, s), 5.53 (1H, d, J = 7.6),
5.49 (1H, d, J = 5.4), 5.07-5.04 (1H, m), 4.96-4.91 (1H, m), 4.27-4.24
(2H, m), 4.09-4.07 (2H, m), 3.42-3.33 (2H, m), 2.20 (3H, s).
13C NMR (CDCl3) selected signals: 172.47, 171.58, 258.96, 156.05,
152.94, 152.29, 149.85, 144.49, 144.22, 142.71, 139.27, 135.36, 134.16,
132.99, 130.88, 129.20, 128.89, 128.86, 128.38, 128.28, 127.39, 123.58,
113.55, 99.99, 87.34, 85.87, 85.22, 84.92, 84.31, 63.79, 55.61, 53.36,
46.18, 38.23, 32.82, 30.24, 30.24, 28.37, 9.04, 8.51.
31P NMR (CDCl3): δ -0.42.
HRMS calculated for C53H54N8O13PS m/z: 1073.3269. Found:
1073.3311.
HPLC: tR 4.1 min (10% MeOH and 0.2% Et3N in CH2Cl2).
Dinucleotide 8 (Using Pivaloyl Chloride as a Coupling
Agent)
Dinucleotide 8 was also prepared by treating a solution of phosphonate
7 (3.2 mg, 4.4 µmol) and 6-N-benzoyl-3ꢁ-O-levulinoyldeoxyadenosine
(2.2 mg, 4.9 µmol) in acetonitrile/pyridine (4/1 v/v, 150 µL) with pival-
oyl chloride (1.1 µL). After 15 min., a solution of iodine in pyridine/water
(98/2 v/v, 88 µL, 0.01 M) was added, and oxidation was continued for
5 min. Reaction work-up was performed as described above. The re-
maining solid was dissolved in CDCl3 and its 31P NMR spectrum was
recorded.
31P NMR (CDCl3):- 0.42 as the only product).
An aliquot was injected into HPLC (10% MeOH/CH2Cl2 + 0.2%
Et3N)—tR 4.1 min.
Stability Tests
2% DCA Test—General Procedure
Compound 5a (1.61 mg) was dissolved in 2% (v/v) DCA in DCE (407 µL).
Aliquots (10 µL) were taken at 0, 5, 15, 30, 60, and 120 min and
quenched with the HPLC mobile phase (490 µL). From this solution,
20 µL were injected into the Straight-Phase HPLC and analyzed. The
mobile phase consists of 2% MeOH and 0.2% Et3N in CH2Cl2 with a
flow rate of 1 mL/min and observed at 285 nm. The peak area of com-
pound 5a at 0 min was assigned to 100% (response standard), and the
subsequent areas (5–120 min) were compared to the standard.

170
A. Slaitas and E. Yeheskiely
Capping Test—General Procedure
Compound 5c (2.57 mg) was dissolved in Capping solution A [6% DMAP
in MeCN (w/v), 238 µL], and an equal volume of Capping solution B
[Ac2O:collidine:MeCN 2:3:5 (v/v/v)] was added. The analysis was per-
formed by HPLC as described above.
Iodine Test—General Procedure
Compound 5c (1.00 mg) was dissolved in an iodine solution (463 µL of
0.01 M) This solution was prepared by dissolving 0.103 g I2 in 26 mL
dry MeCN and then adding 2.4 mL collidine and 12 mL water. Aliquots
(10 µL) were taken at 0, 5, 15, 30, 60, and 120 min, and quenched with
the HPLC mobile phase (490 µL) which contained 0.1% dimethyl phos-
phite (to remove the free iodine). The Straight-Phase HPLC analysis
was performed as described above.
Aqueous Buffers and Ammonia Tests—General Procedure
Compound 4c (1.13 mg) was dissolved in TRIS buffer (pH 9.0, 260 µL
50 mM). After 16 h, an aliquot (5 µL) was withdrawn and quenched into
the HPLC mobile phase (50 µL). In the ammonia tests, the aliquots
were first centrifuged under reduced pressure to ensure the removal
of volatile substances, i.e. ammonia and methanol, and then were re-
dissolved in the mobile phase (55 µL). Aliquots (5 µL) were analyzed
by reversed-Phase HPLC.
REFERENCES
[1] A. Slaitas and E. Yeheskiely, J. Pept. Res., 60, 283 (2002).
[2] A. Slaitas and E. Yeheskiely, Eur. J. Org. Chem., 14, 2391 (2002).
[3] A. C. van der Laan, R. Stro¨mberg, J. H. van Boom, E. Kuyl-Yeheskiely, V. A. Efimov,
and O. G. Chakhmakhcheva, Tetrahedron Lett., 37, 7857 (1996).
[4] A. C. van der Laan, P. Havenaar, R. S. Oosting, E. Kuyl-Yeheskiely, E. Uhlmann,
and J. H. van Boom, Bioorg. Med. Chem. Lett., 8, 663 (1998).
[5] J. C. Verheijen, B. A. L. M. Deiman, E. Yeheskiely, G. A. van der Marel, and J. H.
van Boom, Angew. Chem. Int. Ed., 39, 369 (2000).
[6] J. C. H. M. Wijkmans, W. Ottenheim, E. Kuyl-Yeheskiely, G. A. van der Marel, W.
Bloemhoff, and J. H. van Boom, Synthesis, 1, 97 (1995).
[7] D. Filippov, E. Kuyl-Yeheskiely, G. A. van der Marel, G. I. Tesser, and J. H. van
Boom, Tetrahedron Lett., 39, 3597 (1998).
[8] D. Filippov, N. J. Meeuwenoord, E. Kuyl-Yeheskiely, J. C. H. M. Wijkmans, G. A.
van der Marel, and J. H. van Boom, Innovation Perspect. Solid Phase Synth. Comb.
Libr., Collect. Pap., Int. Symp., 4th (Meeting Date 1995), 367 (1996).
[9] E. Kuyl-Yeheskiely, C. M. Dreef-Tromp, A. Geluk, G. A. van der Marel, and J. H.
van Boom, Nucleic Acids Res., 17, 2897 (1989).
[10] E. Kuyl-Yeheskiely, P. A. M. van der Klein, G. M. Visser, G. A. van der Marel, and
J. H. van Boom, Recl. Trav. Chim. Pays-Bas, 105, 69 (1986).

Synthesis and Reactivity of Nucleoside Sulfilimines
171
[11] E. Kuyl-Yeheskiely, C. M. Tromp, A. W. M. Lefeber, G. A. van der Marel, and J. H.
van Boom, Tetrahedron, 44, 6515 (1988).
[12] E. Kuyl-Yeheskiely, C. M. Tromp, A. H. Schaeffer, G. A. van der Marel, and J. H.
van Boom, Nucleic Acids Res., 15, 1807 (1987).
[13] A. K. Sharma, T. Ku, A. D: Dawson, and D. Swern, J. Org. Chem., 40, 2758 (1975).
[14] T. E. Varkey, G. F. Whitfield, and D. Swern, J. Org. Chem., 39, 3365 (1974).
[15] V. Nair and S. D. Chamberlain, Synthesis, 5, 401 (1984).
[16] K. K. Ogilvie, Can. J. Chem., 51, 3799 (1973).
[17] R. J. Arhart and J. C. Martin, J. Am. Chem. Soc., 94, 5003 (1972).
[18] L. J. Kaplan and J. C. Martin, J. Am. Chem. Soc., 95, 793 (1973).
[19] A. J. Mancuso and D. Swern, Synthesis, 165 (1981).
[20] A. Momotake, J. Mito, K. Yamaguchi, H. Togo, and M. Yokoyama, J. Org. Chem., 63,
7207 (1998).
[21] G. M Blackburn and M. J. Gait, Nucleic Acids in Chemistry and Biology, 2nd Edn.
1996, Oxford University Press, New York, ISBN 0-19-963533-1. p. 22.
[22] C. B. Reese and Q. Song, J. Chem. Soc., Perkin 1, 11, 1477 (1999).
[23] R. Stro¨mberg and J. Stawinski, Nucleic Acids Res., Sym, Ser., 18, 185 (1987).
[24] P. J. Garegg, I. Lindh, T. Regberg, J. Stawinski, R. Stro¨mberg, and C. Henrichson,
Tetrahedron Lett., 27, 4051 (1986).
[25] H. Koester, J. Biernat, J. McManus, A. Wolter, A. Stumpe, C. K. Narang, and N. D.
Sinha, Tetrahedron, 40, 103 (1984).
[26] P. J. Garegg, I. Lindh, T. Regberg, J. Stawinski, C. Henrichson, and R. Stro¨mberg,
Tetrahedron Lett., 27, 4055 (1986).
[27] M. J. Damha and K. K. Ogilvie, Protocols for Oligonucleotides and Analogs (Editor
S. Agrawal, Methods in Molecular Biology, Vol. 20), ISBN 0-89603-247-7, p. 85.
[28] P. R. Young and K. J. Reid, J. Org. Chem., 52, 2695 (1987).