N,N′-bis-(2-(cyano)ethoxycarbonyl)-2-methyl-2-thiopseudourea: A guanylating reagent for synthesis of 2′-O-[2-(guanidinium)ethyl]-modified oligonucleotides

Article abstract of DOI:10.1080/15257770601112705

A guanylating reagent, N,N′-bis-(2-(cyano)ethoxycarbonyl)-2- thiopseudourea, was synthesized and used for synthesis of 2′-O-[2- (guanidinium)ethyl] (2′-O-GE) modified oligonucleotides. A convenient deprotection method for the 2′-O-GE oligonucleotides was

Full text of DOI:10.1080/15257770601112705

Nucleosides, Nucleotides, and Nucleic Acids, 26:149–159, 2007
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770601112705
N,Nꢀ-BIS-(2-(CYANO)ETHOXYCARBONYL)-2-METHYL-2-
THIOPSEUDOUREA: A GUANYLATING REAGENT FOR SYNTHESIS
OF 2ꢀ-O-[2-(GUANIDINIUM)ETHYL]-MODIFIED OLIGONUCLEOTIDES
Thazha P. Prakash, Ask Pu¨ schl, and Muthiah Manoharan
Department of
2
Medicinal Chemistry, Isis Pharmaceuticals Inc., Carlsbad, California, USA
A guanylating reagent, N,Nꢁ-bis-(2-(cyano)ethoxycarbonyl)-2-thiopseudourea, was synthesized
2
and used for synthesis of 2ꢁ-O-[2-(guanidinium)ethyl] (2ꢁ-O-GE) modified oligonucleotides. A
convenient deprotection method for the 2ꢁ-O-GE oligonucleotides was developed.
Numerous nucleic acid analogues have been synthesized and their
biochemical properties analyzed for therapeutic as well as diagnostic
applications.[1,2] In order to improve upon the activity of nucleic acid-based
drugs, several chemistries have been developed.[2–4] Among these, the most
promising resulted from the chemical modification at the 2ꢁ-position of the
oligonucleotide.[2,3] The 2ꢁ-position is attractive for chemical modification
because it offers enhanced binding affinity to a complementary RNA strand
and imparts nuclease resistance.[3] For example, the 2ꢁ-O-aminopropyl (2ꢁ-
O-AP, Figure 1) provides extremely high nuclease resistance and good
hybridization properties.[5] The high nuclease resistance of the 2ꢁ-O-AP
oligonucleotides has been attributed to their cationic nature. The pKa of
the primary amino group is around 9 and, thus, the group is protonated
under physiological conditions. This is termed the charge effect.
Another interesting highly basic, cationic group is the guanidinium, with
a pKa of 12.5. The guanidinium group plays an important role in funda-
mental organic and biological processes such as catalysis and protein-nucleic
acid interactions. The guanidinium group contains three nitrogen atoms in
a plane, remains protonated over a wide pH range, and can form up to five
hydrogen bonds when present within the arginine side chain.[6] Recently,
efficient postsynthesis methods of guanidination of the phosphate linkage
and the 2ꢁ-position of nucleic acid were reported.[7] Cationic oligonu-
cleotides containing a guanidinium backbone show increased affinity to
Received 5 June 2006; accepted 26 September 2006.
Address correspondence to Muthiah Manoharan, Department of Medicinal Chemistry, Isis Phar-
maceuticals Inc., Carlsbad, CA 92008. E-mail: mmanoharan@alnylam. com
149

150
T. P. Prakash et al.
FIGURE 1 2ꢁ-O-(2-(guanidinium)ethyl) (2ꢁ-O-GE) and 2ꢁ-O-(3-aminopropyl) (2ꢁ-O-AP) RNA.
target RNA.[8] Nucleic acids with guanidinium functionality at 5-position
of pyrimidine stabilize a triplex structure with a DNA duplex.[9]
A suitable protecting strategy for the guanidinium group that is com-
patible with solid-phase DNA synthesis protocols is essential for the syn-
thesis of 2ꢁ-O-GE modified oligonucleotides. Unfortunately, conventional
guanidinium protecting groups such as benzyloxycarbonyl (Cbz) or tert-
butyloxycarbonyl (Boc)[10] are not compatible with solid-phase DNA syn-
thesis. Here we report a guanylating reagent with a base labile protect-
ing group and its use in synthesis of 2ꢁ-O-(2-guanidinium)ethyl modified
oligonucleotides. We recently reported the stabilizing effect of 2ꢁ-O-(2-
guanidinium)ethyl-modified oligonucleotides on duplex and triplex nucleic
acid structure.[11] The full account of synthetic strategy used for the synthe-
sis of 2ꢁ-O-GE-modified oligonucleotides and their biophysical properties are
described in this report.
We previously reported the use of an amino protecting group com-
patible with oligonucleotide synthesis, the N -(2-(cyano)ethoxycarbonyl)
group (CEOC).[12] Here we report use of the CEOC protecting group
in the synthesis of 2ꢁ-O-GE-modified oligonucleotides. The guanylat-
ing reagent was prepared by treatment of CEOE-succinimide 1[12]
with S-methylisothiourea hemisulfate to yield N,Nꢁ-bis-CEOC-2-methyl-2-
thiopseudourea 2 (Scheme 1).
SCHEME 1 a(a) S-methylisothiourea hemisulfate, CH2Cl2, NaHCO3, rt.

N,Nꢁ-Bis-(2-(cyano)ethoxycarbonyl)-2-methyl-2-thiopseudourea
151
SCHEME
2
aDMT
=
4,4ꢁ-dimethoxytrityl; (a) 2, anhydrous DMF, triethylamine, rt; (b)
N ,N -diisopropylammonium tetrazolide, (2-cyanoethyl)-N ,N ,N ꢁ,N ꢁ-tetraisopropylphosphorodiami-dite,
CH3CN, rt; (c) succinic anhydride, pyridine, CH2Cl2, DMAP, rt; (d) 2-(1H-benzotriazole-1-yl)-1,1,3,3-
tetramethyluroniumtetrafluoroborate (TBTU), DMF, amino alkyl controlled pore glass (CPG), rt.
Scheme 2 shows the synthesis of the CEOC-protected 2ꢁ-O-GE-5-
methyluridine-3ꢁ-phosphoramidite 5 and solid support 7. The 2ꢁ-O-[2-
(amino)ethyl]-5ꢁ-O-(4,4ꢁ-dimethoxytrityl)-5-methyluridine 3 was synthesized
using previously reported procedures.[12] Compound 3 was treated with
guanylating reagent 2 and triethylamine in DMF at room temperature
to yield 4 (66%) with a protected guanidinium functionality. Compound
4 was converted into 3ꢁ-phosphoramidite 5 using standard procdures.[5]
Compound 4 was converted into the 3ꢁ-O-succinyl derivative 6 and loaded
onto amino alkyl controlled pore glass (CPG) according to standard syn-
thetic procedures[13] to yield solid support 7 (32.8 µmol g−1). Oligonu-
cleotides shown in the Table 1 were synthesized using phosphoramidite
5 and solid support 7, and the standard phosphoramidites and solid
supports for incorporation of adenine (A), thymine (T), guanine (G),
and cytocine (C) residues. Oxidation of the internucleosidic phosphite
groups was carried out using 1-S-(+)-(10-camphorsulfonyl)oxaziridine[14]
or tert-butylhydroperoxide/acetonitrile/water (10:87:3). The step-wise
coupling efficiency of the modified phosphoramidites was more than
97%.
When the oligonucleotides modified with CEOC-protected 2ꢁ-O-(2-
guanidinium)ethyl were heated with aqueous ammonia at 55◦C, one major
additional product along with expected oligonucleotide was observed in the
electrospray mass spectrometry (ES-MS) analysis. The additional product
had a molecular weight of 68 mass units per modification greater than
molecular weight of the expected oligonucleotide, suggesting the formation

152
T. P. Prakash et al.
TABLE 1 2ꢁ-O-GE modified oligonucleotides
Number
Sequence
8
9
5ꢁ-d(T∗CC AGG T∗GT∗ CCG CAT∗ C)-3ꢁ
5ꢁ-d(CTC GTA CT∗T∗ T∗T∗C CGG TCC)-3ꢁ
5ꢁ-d(T∗T∗T∗ T∗T∗C TCT CTC TCT)-3ꢁ
5ꢁ-d (T∗TT∗ TT∗C TCT CTC TCT)-3ꢁ
5ꢁ-d(TT∗T TT∗C TCT∗ CTC T∗CT)-3ꢁ
5ꢁ-d(TTT TTT TTT TTT TTT TTT T∗)-3ꢁ
5ꢁ-d(TTT TTT TTT TTT TTT TT∗T T∗)-3ꢁ
5ꢁ-d(TTT TTT TTT TTT TTT T∗T∗T∗ T∗)-3ꢁ
10
11
12
13
14
15
All oligonucleotides were phosphodiesters;
T∗ indicates 2ꢁ-O-[2-(guanidinium)ethyl]-5-methyluridine.
of a triazine derivative. One possible mechanism for the formation of a
triazine derivative is the nucleophilic attack of the ammonia at one of
the carbonyls bearing the 2-cyanoethoxy group, followed by intramolecular
cyclization to form 16 (Figure 2).
FIGURE 2 Possible mechanism of formation of triazine derivative during deprotection of oligonu-
cleotides with a CEOC-protected guanidinium group at the 2ꢁ-position with aqueous ammonia.

N,Nꢁ-Bis-(2-(cyano)ethoxycarbonyl)-2-methyl-2-thiopseudourea
153
FIGURE 3 HPLC profile of oligonucleotide 17 after deprotection with aqueous ammonia. HPLC
conditions: Altech SAX 4.6 × 250 mm column; mobile phase A = 10% acetonitrile, mobile phase B
= 10% acetonitrile in 2 M ammonium acetate; gradient 0–100% B in 40 minutes; flow rate 1 mL min-1;
detection at 260 nm.
In order to confirm this mechanism, we synthesized a pentamer oligonu-
cleotide 17, 5ꢁ-d(GAT∗CT)-3ꢁ, where T∗ is 2ꢁ-O-GE-5-methyl-uridine, using
phosphoramidite 5 to introduce the 2ꢁ-O-GE residue. The solid support
carrying the oligonucleotide was heated with aqueous ammonia at 55◦C for
6 hours. The solid support was filtered and the filtrate was concentrated.
The residue was dissolved in water and analyzed by HPLC (Figure 3) on a
SAX column. The two products formed were separated and analyzed by ES-
MS. The fraction from Peak A in Figure 3 gave a mass corresponding to the
molecular weight of oligonucleotide 17 (Peak A: Calcd. MS 1579.31, found
1579.95). However, the Peak B fraction gave a mass 69 units greater than
expected for oligonucleotide 17 (Peak B: Calcd. MS 1648.4, found 1648.9).
This molecular weight corresponds to the triazine-modified oligonucleotide
18 (Figure 4), presumably generated during the ammonium hydroxide-
mediated deprotection by the mechanism shown in Figure 2. Deprotection
with ammonium hydroxide at room temperature also resulted in the
formation of triazine derivative.
Removal of the 2-cyanoethoxycarbonyl group from modified oligonu-
cleotides with 50% piperidine[15] prior to heating with aqueous ammonia

154
T. P. Prakash et al.
FIGURE 4 2ꢁ-Triazine modified oligonucleotide 18 formed when solid support bearing oligonucleotide
17 was heated with agueous ammonia at 55◦C.
prevented the formation of the triazine derivative as shown by ES-MS analy-
sis. Piperidine is a base, but is a poor nucleophile. Hence, it may initiate the
elimination of acrylonitrile, resulting in the formation of 2ꢁ-O-GE modified
oligonucleotides while suppressing the competing nucleophilic reaction.
A two-step deprotection condition was developed for the synthesis of
2ꢁ-O-GE oligonucleotides with a CEOC-protected guanidinium group. The
oligonucleotides on solid support were treated with 50% piperidine in water
and kept at room temperature for 24 hours to remove the CEOC protecting
groups from the guanidinium groups, and the cyanoethyl group from the
internucleoside phosphates and simultaneously release the oligonucleotides
from solid supports. The solid supports were removed by filtration and
the filtrates were concentrated to dryness. The oligonucleotides were then
heated with aqueous ammonia (28–30 wt%) at 55◦C for 6 hours to complete
deprotection of the protecting groups from the exocyclic amino groups of
the bases. The oligonucleotides (Table 1) were purified by reverse-phase
HPLC and characterized by ES-MS (Table 2) and purity was assessed using
HPLC (Table 2) and capillary gel electrophoresis.
Our previous work showed that the 2ꢁ-O-GE modification imparted
stabilization of 2◦C per modification to a duplex with complementary RNA,
relative to an unmodified DNA of the same sequence, when modifications
were dispersed as in oligonucleotide 8.[11] Consecutive modifications, as in
sequence 9, destabilized the duplex with RNA slightly relative to unmodified
DNA. This behavior also was observed for oligonucleotides bearing 2ꢁ-
O-aminopropyl or homologous groups.[5] The loss of stability observed
for duplexes between RNA and oligonucleotides with consecutive 2ꢁ-O-GE
or other cationic modifications is most likely due to repulsions between
positively charged moieties in the minor groove. Hybridization with the
complementary DNA led to duplexes less stable than those formed with the
complementary RNA.[11]
We also evaluated the affinity of 2ꢁ-O-GE-modified oligonucleotides for
a double-stranded DNA target.[11] An increase in the midpoint of the
dissociation of the third strand (T) of 4.1◦C per modification was observed

N,Nꢁ-Bis-(2-(cyano)ethoxycarbonyl)-2-methyl-2-thiopseudourea
155
TABLE 2 ES-MS and HPLC analysis of oligonucleotides with 2ꢁ-O-GE
modification
ES-MS
HPLC Retention
Number
Calcd
Found
Time, mina
8
9
5238.22
5797.49
4931.41
4729.73
4830.80
5818.47
5920.59
6122.93
5238.21
5797.15
4931.46
4729.47
4830.29
5818.62
5920.21
6122.60
18.69
18.06
21.93
19.11
16.07
19.46
19.32
18.21
10
11
12
13
14
15
aConditions were Waters C-4 3.9 × 300 mm column; mobile phase A was
50 mM triethylammonium acetate; pH 7; mobile phase B was acetonitrile;
gradient was 5% to 60% B in 55 minutes; flow rate 1.5 mL min−1; detection
at λ = 260.
for oligonucleotide 10 with sequential modification as compared to an
unmodified DNA third strand. The observed enhancement in T was 0.6◦C
per modification higher than observed with the 2ꢁ-O-aminoethyl-modified
oligonucleotide.[11,16] When the modifications were dispersed as in oligonu-
cleotides 11 and 12, the T was increased relative to unmodified DNA, but
was considerably less than that of the oligonucleotide with consecutive
modifications.[11]
The stability of 2ꢁ-O-GE oligonucleotides against nucleases also was
evaluated.[11] A phosphodiester oligonucleotide with a single 2ꢁ-O-GE
residue at the 3ꢁ end (Table 1, 13) was almost completely degraded after 4
hours in the presence of snake venom phosphodiesterase. Oligonucleotides
with two or four 3ꢁ terminal 2ꢁ-O-GE residues (Table 1, 14–15) were stable at
>95% full length after 24 hours in the presence of nuclease.
Previous work indicated that 2ꢁ-O-GE-modified oligonucleotides showed
high affinity to complementary RNA and double-stranded DNA and ex-
ceptional nuclease resistance.[11] These properties make the 2ꢁ-O-GE mod-
ification of interest for many nucleic acid-based applications, including
antisense, RNA interference (RNAi), decoy, and aptamer therapeutic
and diagnostic technologies. Initial synthetic routes to 2ꢁ-O-GE-modified
oligonucleotides were not compatible with solid-phase oligonucleotide syn-
thesis protocols. We have developed a guanylating reagent with a CEOC
protecting group that is compatible with solid-phase synthesis. A two-step
deprotection protocol was required to avoid formation of a triazine side
product. This guanylating reagent will make synthesis of 2ꢁ-O-GE-containing
oligonucleotides practical and will hopefully lead to further investigation of
this promising modification.

156
T. P. Prakash et al.
EXPERIMENTAL
General Procedures
Anhydrous CH3CN, standard phosphoramidites and reagents for
oligonucleotide synthesis were purchased from Glen Research, Inc. (Ster-
ling, VA, USA). All other reagents and anhydrous solvents were purchased
from Sigma-Aldrich (St. Louis, MO, USA) and used without further purifi-
1
cation. H NMR spectra were referenced using internal standard (CH3)4Si
and 31P NMR spectra were referenced using external standard 85% H3PO4.
Microanalyses were performed by Quantitative Technologies Inc. (White-
house, NJ, USA). Mass spectra were recorded by Mass Consortium (San
Diego, CA, USA) and the College of Chemistry, University of California
(Berkeley, CA, USA).
N,Nꢁ-Bis-CEOC-2-methyl-2-thiopseudourea (2). S-methylisothiourea he-
misulfate (5.29 g, 38.0 mmol) was suspended in CH2Cl2 (250 mL) and sat.
NaHCO3 (250 mL). CEOC-O-succinimide 1 (20.2 g, 95.2 mmol) was added
and the reaction mixture was stirred for 2 hours at room temperature.
The organic phase was separated from the aqueous phase. The aqueous
phase was extracted with dichloromethane (2 × 200 mL) and the combined
organic phase was dried (Na2SO4), filtered, and evaporated. The crude
product was purified by flash silica gel column chromatography with ethyl
acetate/dichloromethane (95:5) as eluent to yield 2 (3.78 g, 35%) as a white
1
solid. H NMR (200 MHz, CDCl3) δ 11.80 (brs, 1H), 4.39 (p, J = 6.0 Hz,
4H), 2.80 (t, J = 6.0 Hz, 4H), 2.45 (s, 3H); 13C NMR (75 MHz, CDCl3) δ
173.3, 160.3, 150.7, 117.0, 116.5, 60.9, 60.6, 18.0, 14.7; HRMS (FAB) Calcd
for C10H13N4O4S+ 285.0656, found: 285.0658.
2ꢁ-O-[2-(N,Nꢁ-Bis-CEOC-guanidinium)ethyl]-5ꢁ-O-(4,4ꢁ-dimethoxytrityl)-
5-methyl uridine (4). Compound 3 (1.56 g, 2.58 mmol) was dissolved in
anhydrous DMF (9 mL). To this, guanylating reagent 2 (0.81 g, 2.85 mmol)
and then triethylamine (0.36 ml, 0.2.58 mmol) were added and the reaction
mixture was stirred at room temperature for 4 hours. The reaction was
quenched by addition of 5% aqueous NaHCO3 (40 mL), extracted with
ethyl acetate (2 × 60 mL), and the combined organic phase was dried
over anhydrous Na2SO4, filtered, and evaporated. The crude product was
purified by flash silica gel column chromatography with ethyl acetate as
eluent to yield 4 (1.44 g, 66%). 1H NMR (300 MHz, CDCl3) δ 11.67 (s, 1 H),
9.80 (s, 1 H), 8.59 (t, J = 6.0 Hz, 1H), 7.63 (s, 1H), 7.42–7.21 (m, 9H), 6.84
(d, J = 9 Hz, 4H), 6.01 (d, J = 3 Hz, 1H), 4.47 (brs, J = 6 Hz, 2H), 4.36
(t, J = 6 Hz, 2H), 4.27 (t, J = 6 Hz, 2H) 4.15–3.97 (m, 3H), 3.93–3.84 (m,
1H), 3.78 (s, 6H), 3.73 (m, 2H) 3.57–3.42 (m, 2H), 2.73 (m, 4H), 1.34 (s,
3H); 13C NMR (75 MHz, CDCl3) δ 164.5, 162.9, 158.7, 156.1, 153.0, 150.8,
144.4, 135.5, 135.3, 130.2, 128.1, 127.7, 127.2, 117.5, 116.9, 113.3, 111.1,
87.1, 86.8, 83.5, 82.4, 69.2, 68.9, 62.4, 61.1, 59.9, 55.3, 40.9, 18.1, 17.9, 11.9;
HRMS (FAB) Calcd for C42H45N7O12Na+ 862.3024, found: 862.2991.

N,Nꢁ-Bis-(2-(cyano)ethoxycarbonyl)-2-methyl-2-thiopseudourea
157
2 ꢁ-O-[2-(N ꢁ,N ꢁ,-bis-CEOC-guanidinium)ethyl]-5ꢁ-O-(4,4ꢁ-dimethoxytrityl)-
5-methyl uridine-3ꢁ-[(2-cyanoethyl)-N,N-diisopropyl]phosphoramidite (5).
Compound 4 (0.78 g, 0.92 mmol) and diisopropylamine tetrazolide (0.16 g,
0.92 mmol) were dried by coevaporation with anhydrous CH3CN.
The residue obtained was re-dissolved in anhydrous CH3CN (6 mL).
2-cyanoethyl-N ,N ,N ꢁ,N ꢁ-tetraisopropylphosphorodiamidite
(0.54
mL,
1.74 mmol) was added and the reaction mixture was stirred at room
temperature under argon for 5 hours. The solvent was evaporated and the
crude product purified by flash silica gel column chromatography with
ethyl acetate/hexane as eluent to give 5 (0.62 g, 64%). 1H NMR (300 MHz,
CDCl3) δ 11.66 (s, 1 H), 8.58 (t, J = 6.0 Hz, 1H), 8.31(s, 1H), 7.65–7.61 (m,
1H), 7.43–7.23 (m, 9H), 6.86–6.81 (m, 4H), 6.04–6.01 (m, 1H), 4.55–4.46
(m, 1H), 4.41–4.28 (m, 5H), 4.22–4.08 (m, 2H), 4.06–3.87 (m, 3H), 3.79
(s, 6H), 3.70–3.47 (m, 6H) 3.37–3.29 (m, 1H), 2.76–2.72 (m, 4H),2.65 (t,
J = 6 Hz, 1H), 2.37 (t, J = 6 Hz, 1H), 1.38–1.36 (m, 3H), 1.17–1.14 (m,
9H), 1.01 (d, J = 6 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 164.5, 164.4,
163.0, 158.9, 156.1, 156.0, 153.0, 150.8, 144.4, 135.4, 135.3, 130.4, 130.3,
128.5, 128.4, 128.1, 127.4, 118.0, 117.6, 117.2, 116.6, 113.4, 111.4, 111.3,
87.5, 87.4, 87.2, 87.1, 83.2, 82.9, 82.1, 70.8, 70.7, 70.6, 69.1, 68.5, 62.4,
62.1, 61.0, 59.9, 58.1, 58.0, 57.8, 55.4, 43.5, 43.4, 43.2, 40.9, 24.9, 24.8, 24.7,
24.6, 20.4, 20.3, 18.2, 18.1, 12.0, 11.9; 31P NMR (80 MHz, CDCl3): δ 150.87
and 150.78; HRMS (FAB) Calcd for C51H63O13N9PCs+ 1172.3259, found:
1172.3203.
5ꢁ-O-(4,4ꢁ-Dimethoxytrityl)-2ꢁ-O-[2-(N,Nꢁ -CEOC-guanidinium)ethyl]-5-
methyluridine-3ꢁ-O-succinate (6). Compound 4 (0.25 g, 0.3 mmol) was
co-evaporated with anhydrous CH3CN. To this, succinic anhydride (0.06 g,
0.6 mmol), DMAP (0.02 g, 0.16 mmol), anhydrous pyridine (0.05 mL, 0.6
mmol) and CH2Cl2 (1 mL) were added and stirred at room temperature
under argon atmosphere for 4 hours. The reaction mixture was diluted
with CH2Cl2 (25 mL) washed with cold 10% aqueous citric acid (20 mL)
and brine (25 mL). The organic phase was dried over anhydrous Na2SO4
and concentrated to yield 6 (0.25 g, 88%) as a foam. Rf (0.25, 5% MeOH
in CH2Cl2); 1H NMR (200 MHz, CDCl3) δ 1.34 (s, 3H), 2.20–2.50 (m, 8H),
3.45 (d, J = 5.4 Hz, 1H), 3.64 (d, J = 11.6 Hz, 1H), 3.72–4.01 (m, 10H),
4.15 (brs, 1H), 4.26–4.38 (m, 6H), 5.40 (d, J = 4.4 Hz, 1H), 5.93 (d, J = 2.1
Hz, 1H), 6.83 (d, J = 7.2 Hz, 4H), 7.24–7.41 (m, 9H), 7.77 (s, 1H), 8.57 (s,
1H), 10.51 (s, 1H); 13C NMR (75 MHz, CDCl3) δ 176.4, 171.7, 165.8, 163.1,
159.0, 156.2, 153.0, 150.4, 144.2, 135.9, 135.2, 130.3, 128.2, 127.4, 117.3,
116.6, 113.5, 110.8, 88.0, 87.3, 81.1, 80.8, 70.0, 69.5, 61.6, 60.9, 60.0, 55.5,
41.1, 28.8, 18.3, 18.1, 11.9; HRMS (FAB) Calcd for C46H50N7O15+ 940.3365,
found: 940.3346.
5ꢁ-O-(4,4ꢁ-dimethoxytrityl)-2ꢁ-O-[2-(N,Nꢁ -CEOC-guanidinium)ethyl]-5-
methyluridine-3ꢁ-O-succinyl-CPG (7). The 3ꢁ-O-succinate 6 (0.23 g, 0.24
mmol) was dried over P2O5 in vacuo at 40◦C overnight. Anhydrous

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T. P. Prakash et al.
DMF (0.62 mL) was added, followed by 2-(1H-benzotriazole)-1-yl)-1,1,3,3-
tetramethyluronium tetrafluoroborate (TBTU, 0.08 g, 0.24 mmol) and
N -methylmorpholine (53 µL, 0.48 mmol). The solution was vortexed until
a clear solution was obtained. To this, anhydrous DMF (2.38 mL) and
amino alkyl CPG (1.03 g, 115.2 mmol g−1, particle size 120/200, mean
˚
pore diameter 520 A) were added. The mixture was allowed to shake
for 18 hours at room temperature. The functionalized solid support was
filtered and washed thoroughly with DMF, CH3CN, and Et2O and dried
under reduced pressure overnight. The functionalized sold support (7)
was suspended in capping solution (10% acetic anhydride, 10% pyridine,
10% N -methylimidazole in THF) and was allowed to shake for 2 hours at
room temperature. It was filtered and washed with CH3CN and Et2O. After
drying under reduced pressure, the loading capacity was determined using
standard trityl assay (32.8 µmol g−1).
Synthesis of Oligonucleotides Containing 2ꢁ-O-[2-(Guanidinium)ethyl]
modification. Oligonucleotides with 2ꢁ-O-GE modifications were synthesized
(Table 1) using phosphoramidite building block 5 and functionalized CPG
7. A 0.1 M solution of phosphoramidite 5 in anhydrous CH3CN was used
for the synthesis of the modified oligonucleotides. For incorporation of 5,
phosphoramidite solutions (12 equivalents per coupling) were delivered
in two portions, each followed by a 5-minute coupling wait time. The
standard phosphoramidites and commercial solid supports were used for
the incorporation of A, C, G, and T residues. Oxidation of the inter-
nucleosidic phosphite to the phosphate was carried out using CSO [1-S-
(+)-(10-camphorsulfonyl)oxaziridine] with 4-minute oxidation wait time or
tert-butylhydroperoxide/acetonitrile/water (10:87:3) with 10 min oxidation
wait time. All other steps in the protocol supplied by Millipore (Billerica,
MA, USA) were used without modification. The DMT group at the 5ꢁ-
end of the modified oligonucleotides was retained after the final step of
the synthesis. After completion of the synthesis, solid support bearing the
oligonucleotides was suspended in 50% piperidine in water (2 mL for a
2 µmol scale synthesis) and kept at room temperature for 24 hour to de-
protect the CEOC group from the guanidinium functionality at 2ꢁ-position
as well as cyanoethyl group from the phosphate. Under these conditions
oligonucleotides also were removed from the solid support. The solid
support was filtered and washed with water (2 × 0.5 mL). The combined
washes and the filtrate were evaporated to dryness. To the residue, aqueous
ammonia solution (2 mL for a 2 µmol scale synthesis, 28–30 wt%) was
added and the solution was heated at 55◦C for 6 hours to complete the
deprotection of exocyclic amino protecting groups of the bases. After
evaporation to dryness, the residue was purified by High Performance
Liquid Chromatography (HPLC, Waters (Milford, MA, USA) C-4, 7.8 ×
300 mm column, A = 50 mM triethylammonium acetate, pH = 6.5–7, B =
CH3CN, 5 to 60% B in 55 minutes, flow rate 2.5 mL min−1, detection

N,Nꢁ-Bis-(2-(cyano)ethoxycarbonyl)-2-methyl-2-thiopseudourea
159
λ = 260 nm). Detritylation with aqueous 80% acetic acid followed by
desalting gave the 2ꢁ-modified oligonucleotides (Table 1). A second HPLC
purification, after detritylation, was carried out using the same conditions
described above to increase the purity of the modified oligonucleotide to
>90%. Oligonucleotides were characterized by ES-MS analysis. HPLC and
CGE analysis assessed the purity.
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