Synthesis and antibacterial activity of 5′-tetrachlorophthalimido and 5′-azido 5′-deoxyribonucleosides

Article abstract of DOI:10.1080/15257770.2016.1250906

Reported is an efficient synthesis of adenyl and uridyl 5′-tetrachlorophthalimido-5′-deoxyribonucleosides, and guanylyl 5′-azido-5′-deoxyribonucleosides, which are useful in solid-phase synthesis of phosphoramidate and ribonucleic guanidine oligonucleotides. Replacement of 5′-hydroxyl with tetrachlorophthalimido group was performed via Mitsunobu reaction for adenosine and uridine. An alternative method was applied for guanosine which replaced the 5′-hydroxyl with an azido group. The resulting compounds were converted to 5′-amino-5′-deoxyribonucleosides for oligonucleotide synthesis. Synthetic intermediates were tested as antimicrobials against six bacterial strains. All analogs containing the 2′,3′-O-isopropylidine protecting group demonstrated antibacterial activity against Neisseria meningitidis, and among those analogs with 5′-tetrachlorophthalimido and 5′-azido demonstrated increased antibacterial effect.

Full text of DOI:10.1080/15257770.2016.1250906

Nucleosides, Nucleotides and Nucleic Acids
ISSN: 1525-7770 (Print) 1532-2335 (Online) Journal homepage: http://www.tandfonline.com/loi/lncn20
Synthesis and Antibacterial Activity of 5′-
tetrachlorophthalimido and 5′-azido 5′-
deoxyribonucleosides
Robert Van Ostrand, Casey Jacobsen, Alicia Delahunty, Carley Stringer, Ryan
Noorbehesht, Haidi Ahmed & Ahmed M. Awad
To cite this article: Robert Van Ostrand, Casey Jacobsen, Alicia Delahunty, Carley Stringer, Ryan
Noorbehesht, Haidi Ahmed & Ahmed M. Awad (2017): Synthesis and Antibacterial Activity of
5′-tetrachlorophthalimido and 5′-azido 5′-deoxyribonucleosides, Nucleosides, Nucleotides and
Nucleic Acids, DOI: 10.1080/15257770.2016.1250906
To link to this article: http://dx.doi.org/10.1080/15257770.2016.1250906
Published online: 03 Jan 2017.
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Date: 05 January 2017, At: 02:31

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
, VOL. , NO. , –
http://dx.doi.org/./..
Synthesis and Antibacterial Activity of
ꢀ-tetrachlorophthalimido and ꢀ-azido
ꢀ-deoxyribonucleosides
Robert Van Ostrand , Casey Jacobsen, Alicia Delahunty, Carley Stringer,
Ryan Noorbehesht, Haidi Ahmed, and Ahmed M. Awad
Chemistry Program, California State University Channel Islands, Camarillo, CA, USA
ABSTRACT
ARTICLE HISTORY
Received  May 
Accepted  October 
Reported is an efficient synthesis of adenyl and uridyl 5ꢀ-
tetrachlorophthalimido-5ꢀ-deoxyribonucleosides, and guanylyl
5ꢀ-azido-5ꢀ-deoxyribonucleosides, which are useful in solid-phase
synthesis of phosphoramidate and ribonucleic guanidine
oligonucleotides. Replacement of 5ꢀ-hydroxyl with tetra-
chlorophthalimido group was performed via Mitsunobu reaction
for adenosine and uridine. An alternative method was applied
for guanosine which replaced the 5ꢀ-hydroxyl with an azido
group. The resulting compounds were converted to 5ꢀ-amino-
5ꢀ-deoxyribonucleosides for oligonucleotide synthesis. Synthetic
intermediates were tested as antimicrobials against six bac-
terial strains. All analogs containing the 2ꢀ,3ꢀ-O-isopropylidine
protecting group demonstrated antibacterial activity against
Neisseria meningitidis, and among those analogs with 5ꢀ-
tetrachlorophthalimido and 5ꢀ-azido demonstrated increased
antibacterial effect.
KEYWORDS
ꢀ-Amino nucleosides;
ꢀ,ꢀ-O-isopropylidene
nucleosides; modified
nucleoside; mitsunobu
reaction; ribonucleic
guanidine; antibacterial
activity of nucleoside
analogs
Introduction
Many applications of modified nucleosides include incorporation into therapeu-
tic oligonucleotides and usage as potent antibacterial and antiviral agents.[1–3]
Azido and amino nucleosides, in particular, received considerable attention with
new applications in chemistry, biology, and medicine.[4,5] Azidothymidine (AZT,
Figure 1A), for example, is an antiretroviral nucleoside analog used for treatment
of HIV.[6] Problems associated with AZT, such as immunodeficiency, lymphoma,
muscle atrophy, and dementia, motivate synthetic chemists to develop alternative
azido nucleosides that could provide the same benefits with less or no toxic effects.
Azidoguanosine (AZG, Figure 1B) is another nucleoside drug which is a potent and
selective inhibitor of HIV with a resistance profile superior to AZT.[7] A gemcitabine
analog (Figure 1C) containing a 5ꢀ-amino modification has demonstrated inhibition
CONTACT Ahmed M. Awad
ahmed.awad@csuci.edu
Islands, One University Drive, Camarillo, CA , USA.
Chemistry Program, California State University Channel
©  Taylor & Francis Group, LLC

2
R. V. OSTRAND ET AL.
Figure . Modified nucleosides (A) ꢀ-azidothymidine (AZT); (B) ꢀ-azido-ꢀ,ꢀ-dideoxyguanosine
(AZG); (C) Gemcitabine analog; (D) ꢀ-arylamino nucleoside.
of RRM1.[8] A 5ꢀ-arylamino-nucleoside (Figure 1D) exhibited significant inhibi-
tion of HIV-1 reverse transcriptase.[9] Moreover, therapeutic oligonucleotides which
exhibit unique characteristics, such as sequence specific binding affinity, have been
developed through chemical modification of the nucleoside sugar, nitrogenous base,
and the phosphate backbone.[10–13] Among these, phosphoramidate and ribonu-
cleic guanidine (RNG) oligonucleotides (Figure 2) have demonstrated capability as
therapeutic nucleic acids that utilize 5ꢀ-amino-5ꢀ-deoxyribonucleoside monomers
as building block components.[14–16]
Several experiments highlighted the potential of RNG to improve binding affin-
ity, cellular uptake, and nuclease resistance. The positively charged guanidinium
linkage within RNG is capable of overcoming electrostatic repulsion from native
DNA/RNA.[17] The wide pH range through which the guanidinium group remains
protonated allows binding to readily occur through both hydrogen bonding and
charge pairing.[18] The positive charge facilitates interaction with softer anions such
as phosphates and sulfates.[19] Prior work in vitro demonstrated pentameric uridyl
RNG forms remarkably stable complexes with pentameric adenyl DNA, and no
structures with DNA consisting of non-complementary bases were detected.[20]
Figure . (A) Pꢀ→Nꢀ phosphoramidate RNA; (B) Ribonucleic guanidine (RNG).

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
3
Proposing novel and more efficient synthetic methods to develop amino and
azido nucleosides is crucial in nucleic acids chemistry. The precedent synthetic
method for 5ꢀ-azido-5ꢀ-deoxyribonucleosides involves nucleophilic displacement of
5ꢀ-O-sulfonate esters with azide, which can then be reduced to the 5ꢀ-amino deriva-
tive.[21,22] In this work, we describe convenient and efficient methods to synthesize
adenyl and uridyl 5ꢀ-tetrachlorophthalimido-5ꢀ-deoxyribonucleosides, and guany-
lyl 5ꢀ-azido-5ꢀ-deoxyribonucleosides. Subsequent preparation of the corresponding
5ꢀ-amino-5ꢀ-deoxyribonucleoside analogs is also reported. The target compounds
may serve as precursors to synthesize the building blocks for automated solid-phase
synthesis of P3ꢀ→N5ꢀ phosphoramidate RNA or the 3ꢀ-terminal monomers in RNG
oligonucleotides. In addition, adenyl, uridyl, and guanylyl synthetic intermediates
were tested for antibacterial activity against three gram positive and three gram neg-
ative strains of bacteria. Six of the tested compounds demonstrated significant inhi-
bition of the growth of bacteria Neisseria meningitidis.
Results and discussion
Synthesis
Preparation of monomers for oligonucleotide synthesis first required protec-
tion of the nitrogenous base for adenosine and guanosine, which was accom-
plished by using well-established protocols to produce N6-benzoyladenosine and
N2-isobutyrylguanosine.[23] Protection of the nucleoside 2ꢀ,3ꢀ-hydroxyls was then
achieved using 2,2-dimethoxypropane for adenosine, uridine, and guanosine
analogs, utilizing the isopropylidene protecting group to produce compounds
1, 7, and 13, respectively. 5ꢀ-hydroxyl group of the nucleosides was then con-
verted to 5ꢀ-tetrachlorophthalimido in high yield for adenosine and uridine
analogs 2 (91%) and 8 (78%) via Mitsunobu reaction (Scheme 1) using 3,4,5,6-
tetrachlorophthalimide.[24] This reaction did not produce the desired guanylyl ana-
log, perhaps due to the necessary protection of the O6-position of guanine.[25] The
guanylyl nucleoside 5ꢀ-hydroxyl was instead converted to 5ꢀ-azido according to a
previously published method to produce 14 (Scheme 2) in 80% yield.[26]
The 2ꢀ,3ꢀ-O-isopropylidene protecting group was then removed by stirring in
75% TFA to reveal the free 2ꢀ,3ꢀ-hydroxyls on compounds 3, 9, and 15 at yields
of 94%, 91%, and 82%, respectively. Next, TBDMS was selectively added to the
2ꢀ-hydroxyl, leaving the 3ꢀ-hydroxyl exposed for its linker on analogs 4, 10, and 16.
This reaction was performed with TBDMSCl and imidazole in anhydrous pyridine
for the uridyl analog, while TBDMSCl, AgNO3, and pyridine in anhydrous DMF
provided higher reaction yields for adenyl and guanylyl analogs. In this silylation
step, the undesired 3ꢀ-O-TBDMS nucleoside derivatives were separated from the
desired 2ꢀ-O-TBDMS nucleoside analogs by silica gel flash column chromatog-
raphy. The nitrogen-containing groups at the nucleoside 5ꢀ-position on adenyl
and uridyl analogs were then converted to 5ꢀ-amino using ethylenediamine via
modified Gabriel synthesis.[27] A catalytic reduction of the azido guanylyl analog

4
R. V. OSTRAND ET AL.
Scheme . Synthesis of ꢀ-tetrachlorophthalimido-ꢀ-deoxyribonucleosides: (a) ,-dimethoxypro
pane, p-toluenesulfonic acid, acetone,  reflux; (b) Mitsunobu reaction, ,,,-
h
tetrachlorophthalimide, PPh, THF,  h reflux; (c) % aq TFA; (d) TBDMSCl, AgNO, pyridine,
DMF or TBDMSCl, imidazole, pyridine; (e) TBDMSCl, AgNO, pyridine, THF (f) % aq TFA, °C.
was performed using 10% Pd/C. The formed 5ꢀ-amino group was then protected
with MMTr, which is useful during oligonucleotide synthesis cycles, resulting in
compounds 5, 11, and 17 (Scheme 3). The last step involved the addition of the 3ꢀ-
O-succinyl linker, required to facilitate the attachment to controlled-pore glass solid
support. This was performed using succinic anhydride, leading to the formation of
2ꢀ-O-(tert-butyldimethylsilyl)-5ꢀ-(4-monomethoxytritylamino)-3ꢀ-O-succinyl-5ꢀ-
deoxyribonucleosides 6, 12, and 18 in respective yields of 79%, 84%, and 76%.
An alternate protective strategy was developed to synthesize compounds 4 and
10, which involved silyl protection of the 2ꢀ,5ꢀ-hydroxyls to produce compounds
19 and 21 (Scheme 1). The 5ꢀ-O-TBDMS group was then removed via selective
deprotection by stirring in 90% TFA at 0°C to produce 20 or 22 (93%, 91%),
which allowed high yield conversion to 4 or 10 (85%, 79%) via Mitsunobu reaction.
This was another convenient method providing 2ꢀ-O-(tert-butyldimethylsilyl)-5ꢀ-
tetrachlorophthalimido-5ꢀ-deoxyribonucleosides in high yield, and further demon-
strated the versatility of the herein reported Mitsunobu reaction in which no
necessity for the protection of the 3ꢀ-hydroxyl was required. However, it is worth

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
5
Scheme . Synthesis of ꢀ-azido-ꢀ-deoxyguanosine: (a) ,-dimethoxypropane, p-toluenesulfonic
acid, acetone,  h reflux; (b) DMF, PPh, CBr, NaN, °C; (c) % aq TFA; (d) TBDMSCl, AgNO, pyri-
dine, DMF.
mentioning that the Mitsunobu reaction failed when applied directly on N6-
benzoyladenosine with both 2ꢀ and 3ꢀ-hydroxyls remain unprotected, even when the
THF solvent was replaced by DMF to overcome the low solubility of the unprotected
analog.
Antibacterial activity
With an increasing number of antibiotic resistant pathogens, and a shorten-
ing supply of new antibiotics, it is critical to design new, effective antibacterial
Scheme . Oligonucleotide monomer preparation: (a) (i) ethylenediamine, THF or Pd/C, H, CHCl;
(ii) MMTrCl, EtN, CHCl; (b) succinic anhydride, EtN, CHCl.

6
R. V. OSTRAND ET AL.
agents, and to find new applications of existing compounds that act as antibac-
terial agents. Modified nucleosides resemble naturally occurring RNA analogs,
and therefore may interfere with numerous biological processes, so it is not sur-
prising that many nucleoside-based analogs, including 5ꢀ-amino derivatives, have
demonstrated antimicrobial activity.[28] Considering this, intermediates containing
5ꢀ-tetrachlorophthalimido, 5ꢀ-azido, and respective precursors made suitable candi-
dates for antibacterial screening. Synthetic intermediates were tested for antibacte-
rial activity against six strains of bacteria, including three gram positive and three
gram negative strains. Bacteria tested includes Staphylococcus aureus, Escherichia
coli, Bacillus cereus, Enterococcus faecalis, Proteus vulgaris, and Neisseria meningi-
tidis. Disc diffusion was implemented to determine percent inhibition and to obtain
quantitative susceptibility data. Known antibacterial agents ampicillin, kanamycin,
and erythromycin were used as positive controls, and DMSO was used as a nega-
tive control. No antibacterial activity could be observed with five strains of bacteria.
However, a total of six compounds, two of each nucleobase, demonstrated inhibition
against Neisseria meningitidis serogroup B. Neisseria meningitidis is a gram negative
diplococcus that colonizes the nasopharyngeal mucosa and inhabits approximately
10% of the population on a continual basis, but the bacteria can cause devastat-
ing diseases with high fatality rates and is prone to avoid detection by the immune
system.[29]
Percent inhibition results revealed an observable trend. One of six bacteria,
Neisseria meningitidis, was inhibited by synthetic intermediates 1, 2, 7, 8, 13, and
14, as seen in Table 1. All six compounds that demonstrated inhibition against
N. meningitidis possess the 2ꢀ,3ꢀ-O-isopropylidene protecting group. Adenyl, uridyl,
and guanylyl 2ꢀ,3ꢀ-O-isopropylidene-nucleosides 1, 7, and 13, each possessing
Table . Antibacterial activity.
Percent inhibition of selected bacterial strains
S. aureus
(G+)
B. cereus
(G+)
E. faecalis
(G+)
P. vulgaris
(G−)
E. coli
(G−)
N. meningitidis
(G−)
Compound
DMSO (Negative Control)

















































































































N-Benzoyladenosine
N-Isobutyrylguanosine

















Ampicillin ( μg)
Kanamycin ( μg)
Erythromycin ( μg)












NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
7
Table . Quantitative susceptibility determination.
Observed inhibition by varied concentrations of compounds
Mass loaded Volume pipetted
on disc (μg)
onto disc (μL)
Compound 2 Compound 8 Compound 14
Ampicillin
Kanamycin







Inhibition
Inhibition
Inhibition
No
Inhibition
Inhibition
No inhibition
No inhibition
Inhibition
Inhibition
Inhibition
Inhibition
Inhibition
Inhibition
Inhibition
Inhibition
Inhibition
Inhibition
Inhibition

No inhibition
inhibition
a 5ꢀ-hydroxyl, demonstrated inhibition against N. meningitidis. The presence
of a nitrogen-containing group at the 5ꢀ-position in addition to the 2ꢀ,3ꢀ-O-
isopropylidene protecting group increased antibacterial activity against N. menin-
gitidis for all three tested nucleobases in compounds 2, 8, and 14. Analogs with
a 5ꢀ-amino or 5ꢀ-azido derivative modification, but lacking the 2ꢀ,3ꢀ-O- isopropy-
lidene protecting group (3, 9, and 15), failed to inhibit N. meningitidis at tested
quantities. This indicates the 2ꢀ,3ꢀ-O- isopropylidene protecting group was critical
for bacterial inhibition against Neisseria meningitidis. Nucleosides possessing 5ꢀ-
tetrachlorophthalimido and 2ꢀ-O-TBDMS (4 and 10) did not exhibit antibacterial
activity, reinforcing the importance of the presence of the 2ꢀ,3ꢀ-O-isopropylidene
protecting group to achieve antibacterial activity against N. meningitidis. Enzymes
that utilize nucleoside substrates, such as adenosine deaminase, tolerate some steric
hindrance of modified substrates as seen in prior work with 5ꢀ-substituted-2ꢀ,3ꢀ-O-
isopropylidene adenosine.[30] One potential target of inhibition is the rhamnosy-
lation enzyme EarP, which is crucial for cell viability and has been identified as a
promising target for antibacterial drug design specific to Neisseria meningitidis.[31]
Comparable antibiotic susceptibility results have been previously reported using
both disc diffusion and the agar dilution method commonly used to determine min-
imum inhibitory concentration.[32] Compounds 2, 8, and 14 were tested against
Neisseria meningitidis using disc diffusion to determine the minimum amount of
compound necessary to prevent growth of the bacteria. The working amounts of
known antibiotics ampicillin and kanamycin for disc diffusion tests are 10 μg and
30 μg, respectively.[33] As shown in Table 2, compounds 2 and 14 inhibited the
growth of N. meningitidis using as little as 64 μg, and compound 8 inhibited N.
meningitidis using 128 μg.
These results could be useful in future drug design of nucleoside antimicrobial
agents, by utilizing the 2ꢀ,3ꢀ-O-isopropylidene group in combination with nitrogen-
containing or other possible effective substituents located at the 5ꢀ-position. The iso-
propylidene group should also be investigated for use with other organic molecules
being examined as antibiotics, as it may provide additional efficacy against Neisseria
meningitidis and other bacteria.
Conclusion
This work provides a convenient and high yield synthesis for adenyl, uridyl,
and guanylyl 5ꢀ-amino-5ꢀ-deoxynucleosides which may be used as precursors

8
R. V. OSTRAND ET AL.
to synthesize the building blocks required for the assembly of phosphorami-
date or ribonucleic guanidine oligonucleotides. Also, antimicrobial activity against
Neisseria meningitidis was exhibited by six intermediates containing the 2ꢀ,3ꢀ-
O-isopropylidene group. The antibacterial activity of 2ꢀ,3ꢀ-O-isopropyldiene-
nucleosides was improved when a nitrogen-containing group replaced the
nucleoside 5ꢀ-hydroxyl, as demonstrated by the reported 5ꢀ-tetrachlorophthalmido-
5ꢀ-deoxyribonucleosides and 5ꢀ-azido-5ꢀ-deoxyribonucleosides.
Experimental
General procedures
1H-NMR spectra were recorded on a 500 MHz Varian instrument, using CDCl3
or DMSO-d6 as solvents. Chemical shifts were reported in δ ppm and coupling
constants (J) are given in Hz. Thin-layer chromatography (TLC) was carried out
on Silica Gel 60 F254 pre-coated plates (EMD Millipore, Merck KGaA, Darmstadt,
Germany) and visualization of the products was performed under UV light. Silica
gel used for flash column chromatography was Scientific Silica Gel (particle size 60–
200, Acros Organics, Geel, Belgium). ESI mass spectra were recorded on QSTAR
Pulsar Quadrupole/time-of-flight mass spectrometer with Turbo Ion Spray Ioniza-
tion Source (Applied Biosystems, Foster City, CA, USA). Reactions were performed
using anhydrous solvents under N2 atmosphere.
Synthesis
General procedure for 2ꢀ,3ꢀ-O-isopropylidenenucleosides. To a suspension of
N6-benzoyladenosine, uridine, or N2-isobutyrylguanosine (15.70 mmol) in anhy-
drous acetone (100 mL), p-toluenesulfonic acid monohydrate (0.30 g, 1.57 mmol)
and 2,2-dimethyoxypropane (17.3 mL, 141.19 mmol) were added. After 24 h reflux,
the mixture was filtered to remove any unreacted starting material and the filtrate
was concentrated under vacuum. The residue was dissolved in EtOAc, washed with
5% NaHCO3, brine, and was dried over sodium sulfate anhydrous. The crude mix-
ture was washed with anhydrous diethyl ether and filtered to afford pure compound
1, 7, or 13, respectively.
N6-Benzoyl-2ꢀ,3ꢀ-O-isopropylideneadenosine (1). 5.75 g, yield 89%. 1HNMR
(500 MHz, DMSO-d6): 11.19 s, 1H (NH); 8.76 s, 1H (H-8); 8.66 s, 1H (H-2); 8.03
d, 2H, J = 7.4 (H-Bz); 7.63 t, 1H, J = 7.4 (H-Bz); 7.53 t, 2H, J = 7.8 (H-Bz); 6.26
d, 1H, J = 2.9 (H-1ꢀ); 5.43 dd, 1H, J = 2.7, 6.1 (H-2ꢀ); 5.12 t, 1H, J = 5.2 (OH-5ꢀ);
4.99 dd, 1H, J = 2.5, 6.4 (H-3ꢀ); 4.26 m, 1H (H-4ꢀ); 3.58 – 3.52 m, 2H (H-5ꢀ); 1.55 s,
3H (CH3); 1.33 s, 3H (CH3). 13C NMR (500 MHz, DMSO-d6): 166.1, 152.3, 152.2,
150.9, 143.6, 133.8, 132.9, 128.9, 128.9, 126.2, 113.5, 90.3, 87.3, 84.0, 81.9, 62.0, 27.5,
25.6.
2ꢀ,3ꢀ-O-Isopropylideneuridine (7). 3.53 g, yield 79%. 1H NMR (500 MHz, DMSO-
d6): 11.35 s, 1H (NH); 7.77 d, 1H, J = 8.3 (H-6); 5.81 d, 1H, J = 2.9 (H-1ꢀ); 5.61 d,

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
9
1H, J = 8.3 (H-5); 5.06 t, 1H, J = 5.4 (OH-5ꢀ); 4.88 dd, 1H, J = 2.7, 6.1 (H-2ꢀ); 4.72
dd, 1H, J = 3.4, 6.4 (H-3ꢀ); 4.07 q, 1H, J = 5.4 (H-4ꢀ); 3.57–3.52 m, 2H (H-5ꢀ); 1.47 s,
3H (CH3); 1.27 s, 3H (CH3). 13C NMR (500 MHz, DMSO-d6): 163.6, 150.8, 142.4,
113.4, 102.2, 91.6, 87.0, 84.1, 80.9, 61.7, 27.5, 25.6.
N2-Isobutyryl-2ꢀ,3ꢀ-O-isopropylideneguanosine (13). 5.37 g, yield 87%. 1H NMR
(500 MHz, CDCl3): 7.86 s, 1H (H-8); 5.84 d, 1H, J = 3.8 (H-1ꢀ); 5.13 dd, 1H, J =
4.0, 6.2 (H-2ꢀ); 5.01 dd, 1H, J = 2.3, 6.2 (H-3ꢀ); 4.40 q, 1H, J = 2.5 (H-4ꢀ); 3.93 –
3.76 m, 2H (H-5ꢀ); 2.71 m, 1H (CH); 1.58 s, 3H (CH3); 1.34 s, 3H (CH3); 1.24, 6H,
J = 6.9 (CH(CH3)2). 13C NMR (500 MHz, DMSO-d6): 180.6, 155.2, 148.6, 138.5,
120.8, 113.6, 89.2, 87.4, 84.2, 81.5, 62.0, 35.2, 27.5, 25.7, 24.7, 19.3, 19.3.
General procedure for 5ꢀ-tetrachlorophthalimido-2ꢀ,3ꢀ-O-isopropylidene-5ꢀ-
deoxyribonucleosides. A suspension was made of 1 or 7 (3.10 mmol), PPh3 (1.02 g,
3.88 mmol), and 3,4,5,6-tetrachlorophthalimide (1.06 g, 3.72 mmol) in anhydrous
THF (50 mL). Diisopropyl azodicarboxylate (0.73 mL, 3.72 mmol) was added
dropwise over 15 min, and the mixture was refluxed for 15 h. The solvent was con-
centrated under vacuum, and the crude mixture was purified by silica gel column
chromatography to produce 2 or 8, respectively.
N6-Benzoyl-5ꢀ-tetrachlorophthalimido-2ꢀ,3ꢀ-O-isopropylidene-5ꢀ-deoxyadenosine
(2). 1.91 g, yield 91%, purified using 1:1 ratio of hexanes to EtOAc. HRMS (ESI)
1
m/z Calcd for C28H20N6O6Cl4 (M + Na)+ 699.0096. Found 699.0084. HNMR
(500 MHz, CDCl3): 9.04 s, 1H (NH); 8.73 s, 1H (H-8); 8.07 s, 1H (H-2); 8.03 d,
2H, J = 7.3 (H-Bz); 7.69 – 7.44 m, 3H (H-Bz); 6.12 d, 1H, J = 1.4 (H-1ꢀ); 5.58 dd,
1H, J = 1.7, 6.1 (H-2ꢀ); 5.26 dd, 1H, J = 3.4, 6.4 (H-3ꢀ); 4.58 m, 1H (H-4ꢀ); 4.06
– 3.95 m, 2H (H-5ꢀ); 1.59 s, 3H (CH3); 1.39 s, 3H (CH3). 13C NMR (500 MHz,
DMSO-d6): 166.0, 163.7, 152.0, 151.9, 151.0, 144.2, 138.8, 133.8, 133.6, 132.9, 132.8,
132.5, 132.5, 129.0, 128.9, 128.6, 128.4, 126.1, 114.0, 89.4, 84.4, 84.0, 82.1, 55.4, 27.5,
25.8.
5ꢀ-Tetrachlorophthalimido-2ꢀ,3ꢀ-O-isopropylidene-5ꢀ-deoxyuridine (8). 1.33 g,
yield 78%, purified using 1:1 ratio of hexanes to EtOAc. HRMS (ESI) m/z Calcd
1
for C20H15Cl4N3O7 (M + Na)+ 573.9509, found 573.9485. H NMR (500 MHz,
DMSO-d6): 11.34 d, 1H, J = 2.0 (NH); 7.71 d, 1H, J = 8.3 (H-6); 5.67 d, 1H, J = 1.0
(H-1ꢀ); 5.59 dd, 1H, J = 2.2, 8.1 (H-5); 5.12 dd, 1H, J = 1.5, 6.3 (H-2ꢀ); 4.86 dd, 1H,
J = 4.4, 6.4 (H-3ꢀ); 4.20 dd, 1H, J = 2.0, 4.5 (H-4ꢀ); 3.97–3.85 m, 2H (H-5ꢀ); 1.45 s,
3H (CH3); 1.27 s, 3H (CH3). 13C NMR (500 MHz, CDCl3): 164.0, 163.3, 150.7,
144.0, 140.0, 129.5, 127.4, 114.1, 103.0, 97.3, 86.1, 84.6, 82.5, 60.4, 40.3, 26.9, 25.2,
14.2.
General procedure for 5ꢀ-tetrachlorophthalimido-5ꢀ-deoxyribonucleosides. To com-
pound 2 or 8 (1.59 mmol) in methylene chloride (5 mL), aqueous trifluoroacetic
acid (75%, 20 mL) was added, and the mixture was stirred at room temperature
for 2 hours. The solvent was evaporated under reduced pressure, and the residue
was filtered and washed with anhydrous diethyl ether to produce compound 3 or 9,
respectively.
N6-Benzoyl-5ꢀ-tetrachlorophthalimido-5ꢀ-deoxyadenosine (3). 0.95 g, yield 94%.
HRMS (ESI) m/z Calcd for C25H16N6O6Cl4 (M + Na)+ 658.9783, found 658.9767.

10
R. V. OSTRAND ET AL.
HNMR (500 MHz, DMSO-d6): 8.72 s, 1H (H-8); 8.61 s, 1H (H-2); 8.04 d, 2H, J =
7.3 (H-Bz); 7.63 t, 1H, J = 7.3 (H-Bz); 7.54 t, 2H, J = 7.6 (H-Bz); 6.01 d, 1H, J =
5.3 (H-1ꢀ); 4.83 t, 1H, J = 5.2 (H-2ꢀ); 4.30 t, 1H, J = 4.4 (H-3ꢀ); 4.19 m, 1H (H-
4ꢀ); 4.01 – 3.90 m, 2H (H-5ꢀ). 13C NMR (500 MHz, DMSO-d6): 163.8, 152.5, 151.9,
150.8, 144.1, 138.8, 133.8, 132.9, 132.5, 132.0, 132.0, 129.3, 129.2, 129.0, 128.9, 128.6,
128.6, 126.2, 88.4, 81.8, 73.1, 71.9.
5ꢀ-Tetrachlorophthalimido-5ꢀ-deoxyuridine (9). 0.74 g, yield 91%. HRMS (ESI)
1
m/z Calcd for C17H11N3O7Cl4 (M + Na)+ 531.9249, found 531.9240. H NMR
(500 MHz, DMSO-d6): 11.33 d, 1H, J = 1.9 (NH); 7.73 d, 1H, J = 8.3 (H-6); 5.67
d, 1H, J = 5.4 (H-1ꢀ); 5.63 dd, 1H, J = 2.2, 8.1 (H-5); 4.18 t, 1H, J = 5.2 (H-2ꢀ);
4.01–3.95 m, 2H (H-3ꢀ & H-4ꢀ); 3.89 – 3.82 m, 2H (H-5ꢀ). 13C NMR (500 MHz,
DMSO-d6): 163.7, 163.5, 151.0, 142.0, 138.7, 128.6, 128.5, 102.3, 89.6, 80.9, 72.7,
71.5.
N6-Benzoyl-2ꢀ-O-(tert-butyldimethylsilyl)-5ꢀ-tetrachlorophthalimido-5ꢀ-
deoxyadenosine (4). To a solution of 3 (0.40 g, 0.63 mmol) in anhydrous DMF
(20 mL), anhydrous pyridine (0.25 mL, 3.15 mmol) and AgNO3 (0.19 g, 1.13 mmol)
were added. The mixture was stirred for 10 min, and tert-butyldimethylsilyl
chloride (0.17 g, 1.13 mmol) was added and the mixture was stirred at room
temperature for 48 h. The mixture was filtered through celite, washed with ethanol,
and the solvent was removed under reduced pressure. The residue was dissolved
in EtOAc, washed with H2O, brine, and dried over sodium sulfate anhydrous.
The solvent was removed under vacuum and the crude mixture was purified
using silica gel column chromatography (1.5:1 ratio hexanes to EtOAc) to obtain
compound 4 (0.29 g, 61%). HRMS (ESI) m/z Calcd for C31H30N6O6SiCl4 (M +
1
Na)+ 773.0648, found 773.0638. HNMR (500 MHz, CDCl3): 9.13 s, 1H (NH);
8.63 s, 1H (H-8); 8.21 s, 1H (H-2); 8.02 d, 2H, J = 7.4 (H-Bz); 7.60 t, 1H, J =
7.4 (H-Bz); 7.52 t, 2H, J = 7.6 (H-Bz); 5.90 d, 1H, J = 3.4 (H-1ꢀ); 5.04 t, 1H, J
= 4.2 (H-2ꢀ); 4.42 – 4.35 m, 2H (H-3ꢀ, H-4ꢀ); 4.21 – 4.03 m, 2H (H-5ꢀ); 0.86 s,
9H (SiC(CH3)3); 0.04 s, 3H (SiCH3); −0.06 s, 3H (SiCH3). 13C NMR (500 MHz,
DMSO-d6): 170.8, 165.9, 163.8, 152.4, 151.9, 150.9, 144.0, 138.8, 133.8, 132.9, 128.9,
128.9, 128.6, 128.6, 126.2, 88.7, 82.0, 74.9, 71.8, 60.2, 26.0, 21.2, 18.2, 14.5, −4.36,
−4.85.
2ꢀ-O-(tert-Butyldimethylsilyl)-5ꢀ-tetrachlorophthalimido-5ꢀ-deoxyuridine (10). To
a mixture of 9 (0.33 g, 0.64 mmol) and imidazole (0.061 g, 0.90 mmol) in anhy-
drous pyridine (10 mL), TBDMSCl (0.135 g, 0.90 mmol) was added, and the mix-
ture was stirred under N2 overnight. The solvent was evaporated under vacuum,
and the residue dissolved in EtOAc and washed with H2O, brine, then was dried
over sodium sulfate anhydrous. The crude was purified using silica gel column chro-
matography (1.75:1 ratio hexanes to EtOAc) to afford compound 10 (0.30 g, 76%).
HRMS (ESI) m/z Calcd for C23H25N3O7SiCl4 (M + Na)+ 648.01, found 648.01. 1H
NMR (500 MHz, DMSO-d6): 11.36 s, 1H (NH); 7.78 d, 1H, J = 7.8 (H-6); 5.68 d,
1H, J = 4.9 (H-1ꢀ); 5.66 d, 1H, J = 7.4 (H-5); 5.20 d, 1H, J = 5.9 (OH-3ꢀ); 4.33 t, 1H,
J = 5.2 (H-2ꢀ); 4.02–4.01 m, 1H (H-3ꢀ); 3.92–3.89 m, 3H (H-4ꢀ & H-5ꢀ); 0.81 s, 9H

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
11
(SiC(CH3)3); 0.04 s, 3H (SiCH3); 0.00 s, 3H (SiCH3). 13C NMR (500 MHz, DMSO-
d6): 163.8, 163.4, 150.9, 141.6, 138.7, 128.6, 102.3, 89.6, 81.2, 74.7, 71.4, 26.1, 18.3,
−4.3, −4.7.
General procedure for 2ꢀ-O-(tert-Butyldimethylsilyl)-5ꢀ-(4-monomethoxyt
ritylamino)-5ꢀ-deoxyribonucleosides. To a solution of compound 4 or 10 (1.90 mmol)
in anhydrous THF (25 mL), ethylenediamine (0.15 mL, 2.28 mmol) was added
and the mixture was stirred for 2 hours at room temperature. The solvent was
concentrated under vacuum. The crude 5ꢀ-amino nucleoside product was then
dissolved in anhydrous methylene chloride (45 mL), Et3N (1.15 mL, 8.25 mmol)
and MMTrCl (0.77 g, 2.48 mmol) were added, and the mixture was stirred under
N2 at room temperature for 2 h. The mixture was then cooled to 0°C, and quenched
with MeOH (10 mL). The solvent was removed under vacuum, and the residue was
dissolved in methylene chloride, washed with H2O and brine, dried over sodium
sulfate anhydrous. The crude was purified by silica gel column chromatography
pre-washed with 2% Et3N to afford 5 or 11, respectively.
N6-Benzoyl-2ꢀ-O-(tert-butyldimethylsilyl)-5ꢀ-(4-monomethoxytritylamino)-5ꢀ-
deoxyadenosine (5). 1.04 g, yield 72%, purified using 1:1 ratio hexanes to EtOAc.
HRMS (ESI) m/z Calc for C43H48N6O5Si (M + Na)+ 779.3353, found 779.3334.
1HNMR (500 MHz, CDCl3): 9.03 s, 1H (NH); 8.01 d, 2H, J = 7.4 (H-Bz); 7.96 s,
1H (H-8); 7.73 s, 1H (H-2); 7.62 t, 1H, J = 7.6 (H-Bz); 7.55 – 7.17 m, 14H (H-Ar
and H-Bz); 6.79 d, 2H, J = 8.3 (H-Ar); 5.77 d, 1H, J = 7.3 (H-1ꢀ); 5.67 t, 1H, J = 5.9
(H-2ꢀ); 4.72 d, 1H, J = 4.4 (H-3ꢀ); 4.37 s, 1H (H-4ꢀ); 3.85 – 3.81 m, 1H (H-5ꢀ); 3.54
– 3.46 m, 1H (H-5ꢀ); 3.77 s, 3H (MMTr-OCH3); 0.85 s, 9H (SiC(CH3)3); −0.03 s,
3H (SiCH3); −0.27 s, 3H (SiCH3).
2ꢀ-O-(tert-Butyldimethylsilyl)-5ꢀ-(4-monomethoxytritylamino)-5ꢀ-deoxyuridine
(11). 0.89 g, yield 74%, purified using 1:1 ratio hexanes to EtOAc. HRMS (ESI) m/z
Calcd for C35H43N3O6Si (M + Na)+ 652.2819, found 652.2797. 1HNMR (500 MHz,
CDCl3): 7.47 – 7.09 m, 13H (ArH, BzH, and H-6); 6.81 d, 2H, J = 8.8 (ArH); 5.70
d, 1H, J = 3.9 (H-1ꢀ); 5.66 d, 1H, J = 8.3 (H-5); 4.14 – 4.05 m, 4H (H-2ꢀ, H-3ꢀ, H-4ꢀ,
& H-5ꢀ); 3.78 m, 4H (MMTr-OCH3 & 5ꢀ-H); 0.88 s, 9H (SiC(CH3)3); 0.06 s, 3H
(SiCH3); 0.01 s, 3H (SiCH3).
2ꢀ-O-(tert-Butyldimethylsilyl)-N2-isobutyryl-5ꢀ-(4-monomethoxytritylamino)-
5ꢀ-deoxyguanosine (17). Compound 16 (0.40 g, 0.81 mmol) was dissolved in
anhydrous methylene chloride (8 mL), and Pd/C (0.04 g, 0.41 mmol) was added,
and the mixture was stirred under H2 overnight. The resulting precipitate was dis-
solved in methanol, filtered, and concentrated under vacuum. The crude 5ꢀ-amino
nucleoside product was then dissolved in anhydrous methylene chloride (5 mL),
Et3N (1.06 mL, 7.64 mmol) and MMTrCl (0.28 g, 0.92 mmol) were added, and the
mixture was stirred under N2 at room temperature for 2 h. The mixture was then
cooled to 0°C, and quenched with MeOH (5 mL). The solvent was removed under
vacuum, and the residue was dissolved in methylene chloride, washed with H2O
and brine, dried over sodium sulfate anhydrous. The crude was purified by silica
gel column chromatography pre-washed with 2% Et3N using 1:1 ratio hexanes to

12
R. V. OSTRAND ET AL.
EtOAc to afford 17 (0.32 g, 53%). 1H NMR (500 MHz, CDCl3): 11.97 s, 1H (NH);
7.66 s, 1H (H-8); 7.51 – 7.21 m, 12H (Ar-H); 6.83 d, 2H, J = 8.8 (Ar-H); 5.65 d, 1H,
J = 6.4 (H-1ꢀ); 4.68 q, 1H, J = 6.9 (H-2ꢀ); 4.37 q, 1H, J = 2.9 (H-3ꢀ); 4.27 – 4.05 m,
3H (H-4ꢀ & H-5ꢀ); 3.78 s, 3H (OCH3); 2.73 m, 1H (CH); 1.56 s, 6H (CH(CH3)2);
0.89 s, 9H (SiC(CH3)3); 0.05 s, 3H (SiCH3); −0.03 s, 3H (SiCH3).
General procedure for 2ꢀ-O-(tert-Butyldimethylsilyl)-5ꢀ-(4-monomethoxytrity
lamino)-3ꢀ-O-succinyl-5ꢀ-deoxyribonucleosides. To a solution of 5, 11, or 17
(0.25 mmol) in anhydrous methylene chloride (5 mL), Et3N (0.34 mL, 2.45 mmol)
and succinic anhydride (0.08 g, 0.75 mmol) were added and the mixture was
stirred for 24 h at room temperature. The solvent was evaporated under reduced
pressure, and the residue was dissolved in EtOAc and washed with 5% citric acid,
brine, then was dried over sodium sulfate anhydrous. The crude was purified by
silica gel column chromatography pre-washed with 2% Et3N to afford 6, 12, or 18,
respectively.
N6-Benzoyl-2ꢀ-O-(tert-butyldimethylsilyl)-5ꢀ-(4-monomethoxytritylamino)-3ꢀ-O-
succinyl-5ꢀ-deoxyadenosine (6). 0.17 g, yield 79%, purified using 4:1 ratio EtOAc to
MeOH. HRMS (ESI) m/z Calcd for C47H52N6O8Si (M + Na)+ 879.3514, found
1
879.3487. HNMR (500 MHz, DMSO-d6): 11.17 s, 1H (NH); 8.70 s, 1H (NH);
8.01 d, 2H, J = 7.3 (H-Bz); 7.64 – 7.18 m, 17H (H-8, H-2, H-Ar, and H-Bz); 6.85
d, 2H, J = 8.8 (H-Ar); 5.98 d, 1H, J = 7.8 (H-1ꢀ); 5.80 d, 1H, J = 5.4 (H-3ꢀ); 5.58
dd, 1H, J = 2.4, 7.8 (H-2ꢀ); 4.29 s, 1H (H-4ꢀ); 4.07 – 4.04 m, 2H (H-5ꢀ); 3.71 s, 3H
(MMTr-OCH3); 2.60 – 2.54 m, 4H (CO(CH2)2); 0.65 s, 9H (SiC(CH3)3); −0.09 s,
3H (SiCH3); −0.31 s, 3H (SiCH3).
2ꢀ-O-(tert-Butyldimethylsilyl)-5ꢀ-(4-monomethoxytritylamino)-3ꢀ-O-succinyl-5ꢀ-
deoxyuridine (12). 0.15 g, yield 84%, purified using 1:1.5 ratio hexanes to EtOAc.
HRMS (ESI) m/z Calcd for C39H47N3O9Si (M + Na)+ 752.3192, found 752.3118.
1H NMR (500 MHz, DMSO-d6): 11.36 s, 1H (NH); 7.63 d, 1H, J = 7.9 (H-6); 7.38 –
7.14 m, 12H (ArH); 6.82 d, 2H, J = 8.8 (ArH); 5.72 d, 1H, J = 3.9 (H-1ꢀ); 5.59 d, 1H,
J = 8.3 (H-5); 5.24 t, 1H, J = 4.9 (H-3ꢀ); 4.31 t, 1H, J = 5.9 (H-2ꢀ); 4.06 – 3.91 m, 3H
(H-4ꢀ & H-5ꢀ); 3.70 s, 3H (MMTr-OCH3); 2.62 – 2.60 m, 4H (CO(CH2)2); 0.74 s,
9H, (SiC(CH3)3); −0.06 s, 3H (SiCH3); −0.11 s, 3H (SiCH3).
2ꢀ-O-(tert-Butyldimethylsilyl)-N2-isobutyryl-5ꢀ-(4-monomethoxytritylamino)-3ꢀ-
O-succinyl-5ꢀ-deoxyguanosine (18). 0.16 g, yield 76%, purified using 4:1 ratio EtOAc
to MeOH. 1H NMR (500 MHz, CDCl3): 12.10 s, 1H (NH); 7.63 s, 1H (H-8); 7.47 –
7.16 m, 12H (Ar-H); 6.81 d, 2H, J = 8.8 (Ar-H); 5.87 d, 1H, J = 5.4 (H-1ꢀ); 5.62 s,
1H, (H-3ꢀ); 4.27 s, 1H (H-2ꢀ); 4.15 s, 1H (H-4ꢀ); 3.80 – 3.61 m, 2H (H-5ꢀ); 3.77 s, 3H
(OCH3); 2.80 – 2.61 m, 5H (CH & CO(CH2)2); 1.08 d, 3H, J = 6.8 (CH(CH3)2);
0.98 s, 3H, J = 6.8 (CH(CH3)2); 0.82 s, 9H (SiC(CH3)3); −0.05 s, 3H (SiCH3);
−0.12 s, 3H (SiCH3).
5ꢀ-Azido-N2-isobutyryl-2ꢀ,3ꢀ-O-isopropylidene-5ꢀ-deoxyguanosine (14). To
a
stirred solution of compound 13 (1.40 g, 3.56 mmol) in anhydrous DMF (8 mL),
PPh3 (1.52 g, 5.81 mmol) and CBr4 (2.31 g, 6.97 mmol) were added. The mixture
was stirred under N2 at room temperature for 5 min, then excess NaN3 (1.13 g,
17.42 mmol) was added, and the reaction mixture was stirred at 90°C for 24 h.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
13
The reaction was quenched by adding H2O (5 mL). After stirring for 5 min, the
mixture was diluted with EtOAc, washed with 5% sodium bicarbonate, brine,
and the organic layer was dried over sodium sulfate anhydrous. The solvent was
evaporated under vacuum and the crude product was purified by silica gel column
chromatography (1:1 ratio hexanes to EtOAc) to produce 14 (1.19 g, 80%). 1H NMR
(500 MHz, CDCl3): 12.23 s, 1H (NH); 7.81 d, 1H, J = 1.4 (H-8); 5.95 s, 1H (H-1ꢀ);
5.19 m, 1H (H-2ꢀ); 4.95 m, 1H (H-3ꢀ); 4.29 m, 1H (H-4ꢀ); 3.59 – 3.51 m, 2H (H-5ꢀ);
2.71 m, 1H (CH); 1.57 s, 3H (CH3); 1.33, 3H (CH3); 1.25 m, 6H (CH(CH3)2). 13
C
NMR (500 MHz, DMSO-d6): 180.6, 155.2, 148.6, 138.9, 129.3, 121.1, 114.0, 89.0,
85.4, 83.8, 81.8, 52.2, 35.3, 27.4, 25.8, 19.3, 19.3.
5ꢀ-Azido-N2-isobutyryl-5ꢀ-deoxyguanosine (15). To compound 14 (0.67 g,
1.59 mmol) in methylene chloride (4 mL), aqueous trifluoroacetic acid (75%, 20 mL)
was added, and the mixture was stirred at room temperature for 2 hours. The
solvent was evaporated under reduced pressure, and the residue was filtered and
washed with anhydrous diethyl ether to produce compound 15 (0.49 g, 82%). HRMS
(ESI) m/z Calcd for C14H18N8O5 (M + Na)+ 401.1298, found 401.1294. 1H NMR
(500 MHz, DMSO-d6): 12.07 s, 1H (NH); 8.23 s, 1H (H-8); 5.82 d, 1H, J = 5.9 (H-
1ꢀ); 5.60 d, 1H, J = 5.9 (OH-2ꢀ); 5.33 d, 1H, J = 4.9 (OH-3ꢀ); 4.58 q, 1H, J = 5.5
(H-2ꢀ); 4.08 q, 1H, J = 4.6 (H-3ꢀ); 4.00 q, 1H, J = 3.7 (H-4ꢀ); 3.66 – 3.53 m, 2H (H-
5ꢀ); 2.75 m, 1H (CH); 1.11 dd, 6H, J = 1.3, 6.6 (CH(CH3)2). 13C NMR (500 MHz,
DMSO-d6): 158.9, 120.8, 87.6, 83.4, 73.2, 71.4, 65.3, 56.4, 52.3, 35.3, 19.5, 19.4, 15.6.
5ꢀ-Azido-2ꢀ-O-(tert-butyldimethylsilyl)-N2-isobutyryl-5ꢀ-deoxyguanosine (16). To
a solution of 15 (0.20 g, 0.53 mmol) in anhydrous DMF (5 mL), anhydrous pyridine
(0.16 mL, 2.12 mmol) and AgNO3 (0.12 g, 0.69 mmol) were added. The mixture
was stirred for 10 min, and tert-butyldimethylsilyl chloride (0.10 g, 0.69 mmol) was
added and the mixture was stirred at room temperature for 48 h. The mixture was
filtered through celite, washed with ethanol, and the solvent was removed under
reduced pressure. The residue was dissolved in EtOAc, washed with H2O, brine, and
dried over sodium sulfate anhydrous. The solvent was concentrated under vacuum
and the crude mixture was purified using silica gel column chromatography (1:1
ratio hexanes to EtOAc) to obtain compound 16 (0.13 g, 48%).1H NMR (500 MHz,
CDCl3): 11.99 s, 1H (NH); 7.88 s, 1H (H-8); 5.84 s, 1H (H-1ꢀ); 5.29 s, 1H (OH-
3ꢀ); 4.49 s, 1H (H-2ꢀ); 4.41 s, 1H (H-3ꢀ); 4.11 s, 1H (H-4ꢀ); 3.72 – 3.49 m, 2H (H-
5ꢀ); 2.64 m, 1H (CH); 1.28 m, 6H (CH(CH3)2); 0.94 s, 9H (SiC(CH3)3); 0.17 s, 3H
(SiCH3); −0.01 s, 3H (SiCH3).
General procedure for 2ꢀ,5ꢀ-bis-O-(tert-butyldimethylsilyl)-ribonucleosides. N6-
benzoyladenosine or uridine (9.79 mmol), AgNO3 (4.59 g, 29.37 mmol), and anhy-
drous pyridine (5.57 mL, 68.53 mmol) were suspended in anhydrous THF (100 mL)
and stirred for 10 minutes. TBDMSCl (4.42 g, 29.37 mmol) was added, and the sus-
pension was stirred for 48 hours. The reaction was quenched by addition of ethanol
(8 mL), and the mixture was filtered through celite and washed with ethanol. The
filtrate was concentrated under vacuum and the residue was dissolved in EtOAc,
washed with H2O, brine, and dried over sodium sulfate anhydrous. The crude mix-
ture was purified by silica gel column chromatography to afford 19 or 21.

14
R. V. OSTRAND ET AL.
N6-Benzoyl-2ꢀ,5ꢀ-bis-O-(tert-butyldimethylsilyl)adenosine (19). 3.82 g, yield 65%,
purified using 1.75:1 ratio of hexanes to EtOAc. 1HNMR (500 MHz, CDCl3): 9.30 s,
1H (NH); 8.80 s, 1H (H-8); 8.43 s, 1H (H-2); 8.03 d, 2H, J = 7.8 (H-Bz); 7.58 t, 1H,
J = 7.4 (H-Bz); 7.50 t, 2H, J = 7.9 (H-Bz); 6.18 d, 1H, J = 4.9 (H-1ꢀ); 4.64 t, 1H, J =
4.9 (H-2ꢀ); 4.29 q, 1H, J = 4.4 (H-3ꢀ); 4.22 d, 1H, J = 3.4 (H-4ꢀ); 4.03 – 3.85 m, 2H (H-
5ꢀ); 0.94 s, 9H (SiC(CH3)3); 0.84 s, 9H (SiC(CH3)3); 0.15 s, 3H (SiCH3); 0.13 s, 3H
(SiCH3); −0.05 s, 3H (SiCH3); −0.12 s, 3H (SiCH3). 13C NMR (500 MHz, CDCl3):
164.7, 152.8, 151.6, 149.5, 141.2, 133.8, 132.7, 128.8, 127.9, 122.9, 88.2, 85.3, 71.1,
63.0, 30.9, 26.0, 25.5, 18.5, 17.9, −5.0, −5.3, −5.4, −5.5.
2ꢀ,5ꢀ-Bis-O-(tert-butyldimethylsilyl)uridine (21). 6.66 g, yield 69%, purified using
2:1 ratio of hexanes to EtOAc. 1HNMR (500 MHz, DMSO-d6): 7.79 d, 1H, J = 8.3
(H-6); 5.79 d, 1H, J = 4.9 (H-1ꢀ); 5.57 dd, 1H, J = 7.8, 7.8 (H-5); 5.06 d, 1H, J =
5.3 (OH-3ꢀ); 4.07 t, 1H, J = 4.9 (H-2ꢀ); 3.93 m, 1H (H-3ꢀ); 3.90 m, 1H (H-4ꢀ); 3.85
– 3.72 m, 1H (H-5ꢀ); 0.88 s, 9H (SiC(CH3)3); 0.82 s, 9H (SiC(CH3)3); 0.07 s, 6H
(Si(CH3)2); 0.01 s, 3H (SiCH3); −0.01 s, 3H (SiCH3). 13C NMR (500 MHz, DMSO-
d6): 163.3, 150.9, 140.2, 102.1, 88.1, 85.0, 76.3, 70.0, 63.0, 31.1, 26.2, 26.0, 18.5, 18.3,
−4.4, −4.8, −5.2.
General procedure for ꢀ-O-(tert-butyldimethylsilyl)-ribonucleosides
Compound 19 or 21 (6.37 mmol) was stirred in aqueous trifluoroacetic acid (90%,
25 mL) at 0°C for 2 hours. The solvent was removed under reduced pressure, and
the crude was purified by silica gel column chromatography (EtOAc) to produce 20
or 22.
N6-Benzoyl-2ꢀ-O-(tert-butyldimethylsilyl)adenosine (20). 2.87 g, yield 93%, puri-
fied using EtOAc. HRMS (ESI) m/z Calcd for C23H31N5O5Si (M + Na)+ 508.1992,
found 508.1971. HNMR (500 MHz, DMSO-d6): 11.17 s, 1H (NH); 8.74 s, 2H (H-8,
H-2); 8.03 d, 2H, J = 7.3 (H-Bz); 7.63 t, 1H, J = 7.6 (H-Bz); 7.53 t, 2H, J = 7.9 (H-Bz);
6.06 d, 1H, J = 5.4 (H-1ꢀ); 5.17 t, 1H, J = 5.4 (OH-5ꢀ); 5.12 d, 1H, J = 5.4 (OH-3ꢀ);
4.72 t, 1H, J = 5.2 (H-2ꢀ); 4.18 q, 1H, J = 4.7 (H-3ꢀ); 4.01 m, 1H (H-4ꢀ); 3.76 – 3.58 m,
2H (H-5ꢀ); 0.74 s, 9H (SiC(CH3)3); −0.06 s, 3H (SiCH3); −0.16 s, 3H (SiCH3). 13
C
NMR (500 MHz, DMSO-d6): 166.0, 152.5, 152.1, 150.9, 143.4, 133.8, 132.9, 128.9,
128.9, 126.3, 88.4, 86.3, 76.1, 70.6, 61.5, 60.2, 31.1, 26.0, 18.3, 14.5, −4.4, −4.8.
2ꢀ-O-(tert-Butyldimethylsilyl)uridine (22). 3.06 g, yield 91%. HNMR (500 MHz,
DMSO-d6): 7.93 d, 1H, J = 8.3 (H-6); 5.77 d, 1H, J = 4.9 (H-1ꢀ); 5.63 dd, 1H,
J = 8.3 (H-5), 7.8; 4.12 t, 1H, J = 4.9 (H-2ꢀ); 3.93 t, 1H, J = 4.9 (H-3ꢀ); 3.86 m, 1H
(H-4ꢀ); 3.65–3.54 m, 1H (H-5ꢀ); 0.82 s, 9H (SiC(CH3)3); 0.02 s, 3H (SiCH3); 0.00 s,
3H (SiCH3). 13C NMR (500 MHz, DMSO-d6): 163.5, 151.1, 140.8, 102.2, 88.1, 85.4,
76.1, 70.0, 60.9, 31.1, 26.1, 26.0, 18.3, −4.4, −4.7.
Antibacterial testing
Percent inhibition
Percent inhibition was determined using disc diffusion. DMSO (5 μL) was pipetted
onto a 5 mm sterile filter paper disc, and the wet disc had 1.0 mg of solid com-
pound applied. Test discs, along with control discs, were loaded on sterile Mueller

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
15
Hinton agar plates or 5% sheep’s blood agar plates that had been inoculated with the
bacteria of interest. Plates were then incubated for 24 hours at 37°C, and Neisseria
meningitidis required a 5% CO2 supplement for reliable growth. The diameter of the
zone of inhibition provided by a compound was measured to demonstrate inhibitory
effectiveness. Percent inhibition is found by dividing the measured diameter of a
compound’s zone of inhibition by the total diameter of the test plate (100 mm), and
multiplying the result by 100. DMSO was used as a negative control and did not
show any inhibitory activity. The reported experimental results were compared to
reference antibiotic compounds.
Quantitative susceptibility determination
Disc diffusion was used to resolve minimum susceptibility limits. A known amount
of compound was dissolved in DMSO and diluted so that amounts of 256μg, 128 μg,
64 μg, and 32 μg of compound were delivered in 5 μL of solution onto a 6 mm
sterile test disc. Test discs and control discs containing the compounds of interest,
ampicillin, and kanamycin of varied dilutions were applied to 5% sheep’s blood agar
plates inoculated with Neisseria meningitidis. Plates were then incubated at 37°C
with a 5% CO2 supplement for 24 hours. The lowest concentration at which bacterial
growth was inhibited by a given test disc was then observed.
Acknowledgments
Financial support for this project was provided by the start-up funding from CSU Channel Islands
and the Chemistry Program general funds. We thank project ACCESO at CSUCI for support-
ing student assistants. Many thanks to Catherine Hutchinson for helping with the antibacterial
assay.
Conflicts of interest
The authors declare no conflict of interest.
ORCID
Robert Van Ostrand http://orcid.org/0000-0002-5134-6269
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