Synthesis and biodistribution of a short nonionic oligonucleotide analogue in mouse with a potential to mimic peptides

Article abstract of DOI:10.1021/jm970538o

A nonionic RNA analogue of the sequence r(U(SO₂)G(SO₂)A(SO₂)C) has been synthesized where each bridging phosphate diester is replaced by a dimethylene sulfone unit (rSNA). The rSNA was synthesized in solution from 3',5'-bi

Full text of DOI:10.1021/jm970538o

276
J . Med. Chem. 1998, 41, 276-283
Articles
Syn th esis a n d Biod istr ibu tion of a Sh or t Non ion ic Oligon u cleotid e An a logu e in
Mou se w ith a P oten tia l to Mim ic P ep tid es
Bernd Eschgfa¨ller,†,‡ Marcel Ko¨nig,† Franziska Boess,§ Urs A. Boelsterli,§ and Steven A. Benner*,‡
Department of Chemistry, Swiss Federal Institute of Technology, CH-8092 Zurich, Switzerland, Department of Chemistry,
University of Florida, Gainesville, Florida 32611, and Institute of Toxicology, Swiss Federal Institute of Technology and
University of Zurich, CH-8603 Schwerzenbach, Switzerland
Received August 11, 1997
A nonionic RNA analogue of the sequence r(USO GSO ASO C) has been synthesized where each
2
2
bridging phosphate diester is replaced by a dimethylene2sulfone unit (rSNA). The rSNA was
synthesized in solution from 3′,5′-bishomo-â-ribonucleoside derivatives as building blocks. Full
experimental procedures are provided, and the product and all synthetic intermediates are
fully characterized. The tetramer is nonionic but highly dipolar due to multiple hydrogen
bonding opportunities. It is freely soluble in water only at higher pH’s, permitting it to be
radiolabeled by exchange of the acidic protons R to the sulfones with tritiated water. The
tritiated molecule was administered intravenously into the tail vein (2.6 mg/kg) of mice, and
its distribution was monitored over 48 h. The rSNA was widely distributed in the biological
tissues, including the brain, and excreted in both the feces and the urine. The accumulation
of radioactivity was significantly higher in liver and kidney than in other tissues. Radiolabel
was recovered from the urine, analyzed by HPLC, and shown to be intact oligonucleotide sulfone.
This is the first bioavailability study on a short nonionic oligonucleotide analogue, a class of
molecules with potential biomedical applications.
In tr od u ction
“SNA”) with Ki ≈ 20 pM, 100-fold lower than the Km
for the corresponding DNA substrate.9 The nonstand-
ard sulfone linkage is known to bend the DNA duplex
in the same way that the recognition site is bent prior
to cleavage,9 making the compound an analogue of the
high-energy intermediate in the enzymatic reaction.
These and other results suggest that the dimethylene
sulfone linker might be useful in creating tight binding,
low molecular weight (1000-1500 Da) oligonucleotide
analogues that serve as inhibitors for restriction en-
zymes, transcription factors, and other proteins that
bend and unwind specific DNA sequences. As the
dimethylene sulfone linkage is uncharged, nuclease
resistant, and extremely stable to chemical degradation,
it meets many other criteria desired for a biologically
active agent as well.
The potential for analogues of oligonucleotides to
exert biological activity has become increasingly ap-
preciated in the past decade, complementing a much
longer history where pharmaceutical research focused
on nucleosides and their analogues.1,2 Much of the drive
to develop oligonucleotide analogues has come from the
desire to obtain “antisense” compounds, long oligonucle-
otides (16-30-mers) that bind to complementary mes-
senger RNA molecules following Watson-Crick base
pairing rules (for reviews see refs 3-7). Alterations in
the structure of natural oligonucleotides have been
sought to improve stability, bioavailability, and hybrid-
ization potential.
Often overlooked in this work is the potential value
of short (4-8 nucleotides) oligonucleotide analogues as
ligands for transcription factors, restriction enzymes, or
other biologically important proteins that recognize
short oligonucleotide sequences. Here, replacement of
anionic phosphates in the oligonucleotide backbone by
nonionic linkers is known to permit the analogue to
fold,8 often to give a conformation preferentially bound
by the protein.
SNAs will have biological value in vivo only if they
are bioavailable. While bioavailability in a number of
longer oligonucleotide analogues has been studied by a
variety of workers,10-15 we were surprised to find that
virtually no work had been done to examine the bio-
availability of short (4-8 nucleotide) oligonucleotide
analogues that might have value in applications unre-
lated to antisense. As noticed elsewhere, nonionic
oligonucleotide analogues are curious variants of natu-
ral oligonucleotides.8 As with DNA and RNA, they
collect a large number of hydrogen bond donor and
acceptor groups, which have the propensity to form
“structure” (bends, folds, turns, aggregates, and pre-
cipitates). Unlike with DNA and RNA, however, the
tendency to form structure is unopposed by the Cou-
For example, Bla¨ttler et al. recently found that
substitution of a dimethylene sulfone unit for a phos-
phate in the hexanucleotide recognition sequence of the
EcoRV restriction endonuclease yielded an inhibitor (an
† Swiss Federal Institute of Technology.
‡ University of Florida.
§ Swiss Federal Institute of Technology and University of Zurich.
S0022-2623(97)00538-4 CCC: $15.00 © 1998 American Chemical Society
Published on Web 01/08/1998

Short Nonionic Oligonucleotide Analogue Synthesis
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 3 277
Sch em e 1a
a
(a) Cs2CO3/THF; (b) Oxone/NaOAc/THF/MeOH/H2O.
lombic repulsion. These differences make the bioavail-
ability of SNAs especially interesting. Therefore, we
describe the synthesis, biodistribution, and clearance of
a tetramer sulfone oligoribonucleotide with the sequence
USO GSO ASO C, an SNA containing one of each of the
four standard nucleobases and unable to form a self-
duplex by Watson-Crick rules, as a model for a short
oligonucleotide analogue.
Dimer 7 had to be deprotected and functionalized at
the 3′′-end to generate a thiol group and 8 at the 6′-end
to give a bromide (Scheme 2). The benzoyl and acetyl
groups of 7 were hydrolyzed with 0.2 M LiOH in THF/
MeOH (1:1) to triol 9 (82%). Compound 9 was converted
by a Mitsunobu reaction with thioacetic acid in THF to
the thioacetate 10 in 76% yield.22 Deprotection of 10
with ammonia in degassed MeOH generated the thiol
11 in quantitative yield. Dimer 8 was desilylated with
HF in pyridine to give 12 (92%). Reaction with CBr4/
PPh3 in acetonitrile yielded bromide 13 (70%).23,24
Coupling of the functionalized dimers 11 and 13 with
Cs2CO3 in THF resulted in tetramer 14 in 82% yield
(Scheme 3). Only traces of disulfide were detected.
Thioether 14 was oxidized with Oxone to sulfone 15 in
84% yield. 15 was fully deprotected in one step with 2
M NaOH/MeOH/THF to give 16 in 87% yield (Scheme
3).
La belin g of Oligon u cleotid e An a logu es. The
protons on the CH2 groups bonded to sulfur were
exchanged with tritium derived from tritiated water
(Scheme 3). Following a procedure developed using
deuterium as a label, purified oligomer (2.0 mg) was
suspended in tritiated water (1 mL, specific activity, 25
mCi/g ) 925 MBq/g, DuPont-NEN) and treated with
triethylamine (1 M). The oligomer dissolved immedi-
ately after the addition of the base. The solution was
shaken for 48 h at room temperature. The sample was
lyophilized in a SpeedVac, resuspended in deionized
water (1 mL), and incubated with shaking at room
temperature (30 min) to remove rapidly exchangeable
protons followed by lyophilization. This cycle was
repeated two more times. The rate of loss of the label
was estimated by incubating a deuterated GC sulfone-
2
2
2
Ch em istr y
Syn th esis of Oligon u cleotid e An a logu es. The
functionalized monomers 1-4 were synthesized from
diacetone D-glucose as previously described (Scheme
1).8,16,17 Hydrolysis of the thioacetates 1 and 3 with
ammonia at 0 °C yielded the corresponding thiols, but
generated especially with 1 large amounts of disulfide.
Therefore 1 and 3 were deprotected in situ to thiolate
via acetyl migration and were coupled with Cs2CO3 in
THF with bromides 2 and 4 to yield thioethers USG (5a ,
70%) and ASC (6a , 74%).8,18,19 The acetate group
migrated hereby to the neighboring 2′-OH group. Ap-
proximately 10% of the thioethers were isolated with a
2′-OH group, presumably because of an intermolecular
acetyl migration (5b, 8% and 6b, 8%). These byproducts
were used together with the acetylated thioethers in the
further reaction steps.
Thioethers were immediately oxidized with excess
Oxone in methanol/water to give the sulfones 7 in
quantitative and 8 in 85% yield (Scheme 1).20 Earlier
experiments in our laboratory showed that the oxidation
at a later stage of the synthesis resulted in lower
yields.21 TLC showed the fast reaction to the more polar
sulfoxide intermediates followed by a slower oxidation
to the sulfones.

278 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 3
Eschgfa¨ller et al.
Sch em e 2a
a
(a) 0.2 M LiOH/THF/MeOH; (b) DIAD/PPh3/AcSH/THF; (c) NH3/MeOH; (d) HF/pyridine; (e) PPh3/CBr4/CH3CN.
Sch em e 3a
a
(a) Cs2CO3/THF; (b) Oxone/THF/MeOH/H2O; (c) 2 N NaOH/THF/MeOH; (d) 1 M Et3N/T2O. Note that tritium T is present as a tracer.
dimer dissolved in PBS buffer at pH 7.4 at 37 °C for 48
h. As analyzed by 1H NMR, a loss of 5-10% of the label
was detected. This should not interfere with the results
of the bioavailability study, because the exchanged
tritium should predominately appear in the form of
tritiated water which is easily removed by lyophiliza-
tion.
arise from the deprotonation of the U and G bases,
which have pKas of ca. 9.2. It appears to be easier to
manipulate nonionic oligonucleotide analogues under
alkaline conditions, and this may provide a general
strategy for nonionic oligonucleotide analogues as long
as the linker is stable under alkaline conditions. This
may also be valuable in synthesis and solution-phase
manipulation of peptide nucleic acids (PNAs).25
The improved solubility of 16 in water under alkaline
conditions was valuable in preparing its tritiated de-
rivative 17. The dimethylene sulfone unit appears to
have nearly optimal acid-base properties. It is suf-
ficiently acidic to permit incorporation of tritium from
water under relatively mild conditions. At pH 7.4,
however, the SO2-C-H bond is sufficiently stable to
allow properly controlled bioavailability studies to be
done.
Resu lts a n d Discu ssion
The tetrasulfone USO GSO ASO C 16 is poorly soluble
2
2
in water at neutral pH (10 µ2M), but moderately soluble
in DMSO. In water containing 1 M triethylamine, the
compound was freely soluble. These results contrast
markedly with the behavior of natural DNA, whose
repeating anionic character renders it freely soluble in
water under all conditions.8 The enhanced solubility
of 16 in water under alkaline conditions is presumed to

Short Nonionic Oligonucleotide Analogue Synthesis
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 3 279
Ta ble 1. Total Fecal and Urinary Excretion of Dimethylene
Sulfone-Bridged Oligonucleotide after Single iv Injection (%
Recovered Dose)
time interval
(h)
urinary excretion
(%)
fecal excretion
(%)
mouse
0-3
1
2
2.7
17.3
0.1
0-8
3
4
5
27.8
20.3
33.4
7.1
18.6
2.5
0-24
6
7
23.0
20.6
49.9
19.6
The radioactivity was recovered both in the urine and
in the feces, indicating that the oligosulfone was ex-
creted via the kidney and also cleared via biliary
excretion. Over the entire time course, the main
pathway of excretion of the dimethylene sulfone oligo-
nucleotide was via the feces. Approximately 9% of the
administered dose was found in the feces 8 h after the
injection, while 26% was found in the urine. After 24
h, however, approximately 35% of the label was found
in the feces, while further secretion into the urine was
not observed (Table 1). The large variance in radioac-
tivity found in excreted samples is accounted for by the
irregularity of the excretion phenomenon.
To establish the chemical identity of the species
carrying tritium in the urine, the urine samples were
filtered through SepPak C18 cartridges to remove salt,
concentrated by lyophilization, and analyzed by HPLC.
The label was present predominantly in one peak that
eluted between 24 and 26 min. Authentic tritiated
sulfone injected on the same column showed the same
retention time, suggesting that the sulfone excreted in
the urine had not undergone chemical degradation.
Studies from other laboratories with longer oligo-
nucleotides and their analogues report that these mol-
ecules achieve widespread biodistribution in several
animal systems.9,10,12-14 The biodistribution of the
rUSO GSO ASO C oligosulfone differs from that of other
oligonucleotide analogues in several respects. First,
excretion was slower with the oligosulfone than ob-
served in a test methylphosphonate 12 nucleobases in
length. The methylphosphonates are also nonionic
oligonucleotide analogues. In contrast, excretion of the
oligosulfone was more rapid than several phosphoro-
thioates 20-40 nucleotides in length. These results
show, therefore, no simple dependence of pharmacody-
namics either on size or polarity.
The fact that the label excreted in the urine was found
predominantly in the form of the oligosulfone shows that
relatively little of the compound was degraded in the
body. This is consistent with the expected stability of
the sulfone linker.
Interestingly, the amount of oligosulfone found in the
brain is much higher than that found in studies using
other oligonucleotide analogues. Pardridge et al. re-
ported that the uptake of an 18-mer PNA by the brain
was negligible following intravenous administration;
uptake was increased by at least 28-fold when the PNA
was bound to the OX26-SA vector. The brain uptake
with the vector-mediated PNA was 0.1% of the injected
dose per gram of brain at 60 min after an iv injection.26
In contrast, 0.15% of the injected dose of oligosulfone
was found per gram of brain 1 h after injection.
F igu r e 1. Measured radioactivity in the different tissues after
a single iv injection of 2700 dpm/g of tissue. Three mice were
sacrificed per time interval and the radioactivity was deter-
mined via liquid scintillation counting.
Nevertheless, in the bioavailability studies, a small
but significant amount of the label was found in lyo-
philizable fractions, presumably as water. The slow
exchange of tritium to water was expected in light of
control experiments that showed that a small amount
of label was lost in vitro under physiological conditions.
The amount of released label, never more than 7% of
the total label introduced in the bioavailability studies,
uniformly distributed from all tissues, was determined
by lyophilization. The data reported in Figure 1 reflect
correction for the tritiated water.
The tritiated sulfone was widely distributed in all
tissues examined (Figure 1). The concentration of label
was significantly higher in liver and kidney than in all
other tissues. Substantial amount of oligosulfone was
found in the brain. The concentration of SNA in the
liver was similar to that in the kidney, 3 times that in
spleen and lungs, 4 times that in serum, and 20 times
that in brain. After 8 h, the oligosulfone concentration
in all tissues was higher than in the serum with the
exception of brain tissue (Figure 1). The uptake of the
orally administered tritiated sulfone was also tested.
Low radioactivity was found in all examined tissues, and
up to 95% of the administered dose was excreted within
24 h, suggesting poor gastrointestinal absorption of the
oligonucleotide (data not shown).
2
2
2

280 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 3
Eschgfa¨ller et al.
at 50 °C for 2 h. 2-Propanol (0.5 mL) and 30% H2O2 (0.2 mL)
were added to decolorize the samples, the mixture was heated
to 50 °C (1 h) to destroy excess peroxide, and the mixture was
then suspended in Hionic-Fluor (13 mL) scintillation cocktail.
The plasma and urine samples were suspended directly in
Hionic-Fluor scintillation cocktail (13 mL/g of sample).
The radioactivity was measured with low level counting in
a Liquid Scintillation Analyzer Tri-Cab 2250CA (Low Level)
(Canberra Packard, Meriden, CT). The counts per minute
were converted to disintegrations per minute by standard
quench corrections. The concentration of the sulfone oligo-
nucleotide in the sample was calculated from the specific
activity of the tetramer. The background activity of the control
mice were subtracted from the data reported in Figure 1.
For HPLC-analysis (Waters, Milford, MA) the urine samples
were prepurified with SepPak C18 cartridges (Waters, Milford,
MA).
Con clu sion
The sulfone linkage, mimicking the natural phos-
phodiester linkage, was originally examined because of
the remarkable pharmacodynamic properties displayed
by simple sulfoxides and sulfones, which distribute
rapidly in tissues.27,28 However, rSNAs have the pro-
pensity to aggregate in solution, which may hinder such
distribution. These studies show that an rSNA with a
mass of 1200 Da is distributed rapidly and widely in
mice, including the brain.29,30 Oligonucleotide ana-
logues of this size have the potential to be a new class
of biologically active substances due to their ability to
interact with and inhibit proteins that bind and catalyze
transformation on DNA.9
USG Dim er s 5a a n d 5b. Uridine thioester 1 (126.0 mg,
0.222 mmol) and guanidine bromide 2 (176.9 mg, 0.221 mmol)
were dried on HV at room temperature for 24 h, Cs2CO3 (287.6
mg, 0.883 mmol) was added, and the mixture was dried on
HV at room temperature for 15 h. THF (7.2 mL) was added,
and the fine white suspension was stirred for 8 h at room
temperature. The solvent was rotary evaporated and the
residue chromatographed on silica gel (33 g) with CH2Cl2 and
MeOH (1%, 100 mL; 2%, 500 mL; 10%, 200 mL) as eluent to
give the 2′-OAc dimer 5a (200.6 mg, 70%) and the 2′-OH dimer
5b (21.5 mg, 8%). The dimers were contaminated with small
amounts of side products resulting from the thioester com-
pound 1. The side products formed during this type of reaction
are discussed elsewhere.8
Exp er im en ta l Section
Gen er a l P r oced u r e. All reactions were run under a
positive pressure of dry argon. Tetrahydrofuran was distilled
from sodium; dimethylformamide, pyridine, acetonitrile, and
methanol were purchased from Fluka (puriss, stored over
molecular sieves). Reagents were purchased from Fluka with
the highest commercial quality and used without further
purification. Acetate buffer was made from 3 M AcOH and 1
M NaOAc in deionized water. E. Merck silica gel (60, 0.040-
0.063 mm) was used for flash chromatography. NMR spectra
were obtained from Bruker AMX 500, Varian EM-390, or
Varian XL-300. All spectra were recorded using tetrameth-
ylsilane as an internal standard. Mass spectra (MS) were
obtained from a VG ZAB2-SEQ (FAB, 3-nitrobenzyl alcohol
(NOBA) matrix) or a Bruker Reflex (MALDI-TOF, 2-[(4-
hydroxyphenyl)azo]benzoic acid (HABA) matrix) spectrometer.
Biologica l Meth od s. All experiments were approved by
the institutional supervisory board and performed according
to Swiss Federal regulations. Male Zur:ICR mice (average
weight, 30 g) obtained from the Institute of Laboratory Animal
Sciences, University of Zurich, were used for all experiments.
The animals were kept in Macrolone cages with wood shavings
as bedding under controlled environmental conditions. They
were fed with Nafag 890 rodent pellets (Nafag, Gossau,
Switzerland) and water ad libitum for 1 week prior to the
study. The oligosulfone was dissolved in a H2O/DMSO solution
(1:1, vol/vol). Doses were injected intravenously into the tail
vein during anesthesia with Ketamine (Ketaminol, Veteri-
naria, Zurich, Switzerland, 150 mg/kg, ip) or administered by
oral gavage in 50 µL of a solution to 2.6 mg/kg of body weight
(2700 dpm/g). In toxicity tests, DMSO displayed an LD50 for
mice of 3.8-11.0 mg/kg,31,32 significantly above the approxi-
mately 0.9 mg/kg injected in these studies. Accordingly, no
mice were lost due to toxic response, nor was toxicity detected
in inspection of organs from sacrificed mice. After 1, 3, 8, 24,
and 48 h, three test animals were sacrificed with carbon
dioxide. Due to the limited amount of rSNA, we were only
able to choose five time intervals. Vehicle control mice
receiving no oligosulfone injection were also sacrificed. Blood
and tissue samples (liver, kidney, spleen, lungs, and brain) of
each animal were collected. Each tissue was trimmed of
extraneous fat or connective tissue and weighed. Samples of
blood were centrifuged to separate plasma, and the organs
were homogenized (in deionized water, 3 mL/g wet weight).
The urine and feces of the animals were collected in metabo-
lism cages up to 24 h. Samples were stored at -20 °C until
analysis.
Compound 5a : FAB MS (3-NOBA) m/z 1289.4 (M + H+, 35);
1231.4 (6); 919.3 ((M-GNPE, i-Bu)+, 13); 371.1 (GNPE, i-Bu+H+, 17);
197.1 (21); 105.0 (Bz+, 100). Compound 5b: FAB MS (3-
NOBA) m/z 1247.4 (M + H+, 33); 877.3 ((M - GNPE,i-Bu)+, 21);
371.1 (GNPE,i-Bu+H+, 35); 197.1 (21); 105.0 (Bz+, 100).
ASC Dim er s 6a a n d 6b. 3 (232.1 mg, 0.334 mmol), 4 (215.5
mg, 0.333 mmol), and Cs2CO3 (433.2 mg, 1.33 mmol) were
dried as described for the preparation of 5a and 5b above. THF
(10 mL) was added, and the resulting fine white suspension
was heated to 50 °C and vigorously stirred for 2 h at 50 °C.
The suspension was cooled to room temperature, acetate buffer
(0.83 mL), CH2Cl2, and 9/10 saturated brine were added, the
organic layer was separated, and the aqueous layer was three
times reextracted with CH2Cl2. The combined organic layers
were concentrated in vacuo, and the residue was chromato-
graphed on silica gel (40 g) with CH2Cl2 and MeOH (1%, 700
mL; 2%, 400 mL; 10%, 200 mL) as eluent. The dimers 6a
(311.7 mg, 74%) and 6b (33.6 mg, 8%) were obtained slightly
contaminated with side products resulting from 3 (see above).
Compound 6a : FAB MS (3-NOBA) m/z 1289.4 (M + H+, 10);
240.1 (ABz + H+, 23); 197.1 (13); 136.0 (59); 105.0 (Bz+, 100).
Compound 6b: FAB MS (3-NOBA) m/z 1219.6 (M + H+, 11);
240.1 (ABz + H+, 36); 197.1 (13); 136.0 (58); 105.0 (Bz+, 100).
USO2G Dim er s 7a a n d 7b. Thioether 5 (130.2 mg, 0.101
mmol) was dissolved in a mixture of THF (2.8 mL) and MeOH
(17.8 mL). A freshly prepared solution of Oxone (2KHSO5‚
KHSO4‚K2SO4) (250.9 mg, 0.408 mmol) and NaOAc (110.3 mg,
1.34 mmol) in water (3.6 mL) was added under vigorous
stirring. The resulting white suspension was stirred for 2 h
at room temperature. Saturated aqueous Na2S2O3 and CH2Cl2
were added resulting in a slightly cloudy solution. The organic
layer was separated, and the aqueous layer was reextracted
three times with CH2Cl2. The residue was filtered through
silica gel (40 g, CH2Cl2/MeOH, 92:8) to give 7 in quantitative
yield. An analytical sample was prepared by chromatography
on silica gel with CH2Cl2/MeOH (98:2) as eluent. The following
are data for 7a . The 1H NMR signals were assigned by 1H/
1H-COSY. 1H NMR (500 MHz, CDCl3): δ (ppm) 1.03 (s, 9H,
CH3-tBu); 1.14 (d, J ) 6.9 Hz, 3H, CH3-iBu); 1.16 (d, J ) 6.8
Hz, 3H, CH3-iBu); 1.73 (m, 1H, H-5′-U); 1.88 (m, 1H, H-5′-U);
2.07 (s, 3H, COCH3); 2.52 (m, 2H, H-5′-G); 2.63 (m, 1H, CH-
iBu); 2.84 (dd, J ) 3.3 Hz, J ) 14.0 Hz, 1H, H-3′′-U); 2.92 (m,
1H, H-3′-U); 3.20 (m, 1H, H-6′-G); 3.24 (dd, J ) 3.5 Hz, J )
All samples were lyophilized twice to remove tritiated water.
The homogenized tissue was digested with Soluene 350 (1 mL/
g, Canberra Packard, Meriden, CT) in a glass scintillation vial.
The mixture was gently swirled and heated to 50 °C in a water
bath overnight. Hionic-Fluor scintillation cocktail (13 mL,
Canberra Packard, Meriden, CT) was added and swirled until
a clear solution appeared. The dried feces samples were
pulverized, 20 mg rehydrated with deionized water (0.1 mL)
in a scintillation vial, and digested with Soluene 350 (1 mL)

Short Nonionic Oligonucleotide Analogue Synthesis
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 3 281
12.4 Hz, 1H, H-3′′-U); 3.29 (t, J ) 6 Hz, 2H, CH2-NPE); 3.47
(m, 1H, H-6′ G); 3.79 (m, 2H, H-6′-U); 3.93 (dt, J ) 2.2 Hz, J
) 9.6 Hz, 1H, H-4′-U); 4.18 (m, 1H, H-3′-G); 4.55 (m, 1H, H-4′-
G); 4.64 (dd, J ) 6.5 Hz, J ) 11.6 Hz, 1H, H-3′′-G); 4.76 (d, J
) 6.2 Hz, 1H, H-3′′-G); 4.78 (t, J ) 6.6 Hz, 2H, CH2-NPE);
5.21 (d, J ) 1.7 Hz, 1H, H-1′-U); 5.40 (dd, J ) 1.7 Hz, J ) 6.5
Hz, 1H, H-2′-U); 5.62 (dd, J ) 2.3 Hz, J ) 8.0 Hz, 1H, H-5-U);
5.99 (d, J ) 1.6 Hz, 1H, H-1′-G); 6.01 (dd, J ) 1.6 Hz, J ) 6.2
Hz, 1H, H-2′-G); 7.01 (d, J ) 8.2 Hz, 1H, H-6-U); 7.34-7.55
(m, 14H, p-Ph, p-Bz, o-NPE); 7.58-7.63 (m, 4H, o-Ph); 7.91
(s, 1H, H-8-G); 7.99-8.06 (m, 4H, o-Bz); 8.13 (d, J ) 8.8 Hz,
2H, m-NPE); 8.89 (br, 1H, NH). FAB MS (3-NOBA): m/z
thioacetic acid (6.7 µL, 70.4 µmol) was added. The slightly
yellow solution was stirred for 1.5 h at 0 °C, the solvents were
rotary evaporated, and the residue was chromatographed on
silica gel (11.2 g, CH2Cl2/MeOH, 97:3; 95:5; 90:10) to yield
thioester 10 (40.1 mg, 76%) as a white foam. 1H NMR (500
MHz, CDCl3): δ (ppm) 1.03 (s, 9H, CH3-tBu); 1.26 (d, J ) 6.9
Hz, 6 H, CH3-iBu); 1.86 (m, 1H, H-5′-U); 2.02 (m, 1H, H-5′-U);
2.14 (m, 1H, H-3′-U); 2.30 (s, 3H, SAc); 2.35 (m, 2H, H-5′-G);
2.49 (m, 1H, H-3′-G); 2.73 (m, 1H, CH-iBu); 2.97 (m, 2H, 1 ×
H-3′′-U, 1 × H-3′′-G); 3.28 (m, 2H, H-6′-U); 3.40 (dt, J ) 4.9
Hz, J ) 12.8 Hz, 1H, H-6′-G); 3.68 (dt, J ) 3.4 Hz, J ) 12.7
Hz, 1H, H-6′-G); 3.90 (t, J ) 6.1 Hz, 2H, CH2-NPE); 3.92 (m,
1H, H-4′-G); 4.29 (t, J ) 9.9 Hz, 1H, H-4′-G); 4.42 (d, J ) 3.5
Hz, 1H, H-2′-U); 4.53 (d, J ) 4.9 Hz, 1H, H-2′-G); 4.75 (m, 2H,
CH2-NPE); 5.52 (d, J ) 1.8 Hz, 1H, H-1′-U); 5.59 (d, J ) 8.1
Hz, 1H, H-5-U); 5.62 (s, 1H, H-1′-G); 6.35 (br, 1H, OH-2′); 7.32
(d, J ) 8.2 Hz, 1H, H-6-U); 7.35-7.44 (m, 6H, p-Ph); 7.53 (d,
J ) 8.7 Hz, 2H, o-NPE); 7.64-7.66 (m, 4H, o-Ph); 8.02 (s, 1H,
H-8-G); 8.14 (d, J ) 8.7 Hz, 2H, m-NPE); 8.29 (br, 1H, NH).
FAB MS (3-NOBA): m/z 1129.3 (M + H+, 25); 743.2 (9); 371.1
(GNPE,i-Bu + H+, 100); 221.1 (28); 76.9 (Ph+, 36).
i
1321.2 (M + H+, 13); 1263.5 ((M - Bu)+, 3); 951.1 ((M -
GNPE,i-Bu)+, 3); 371.8 (GNPE,i-Bu + H+, 8); 307. 0 (32); 105.0 (Bz+,
91); 76.9 (Ph+, 54).
ASO2C Dim er s 8a a n d 8b. Following the procedure used
for the preparation of 7 above, AC-thioether 6 (310.4 mg, 0.246
mmol) was dissolved in THF (27.2 mL)/MeOH (42.7 mL), and
a mixture of Oxone (604.8 mg, 0.984 mmol) and NaOAc (266.5
mg, 3.25 mmol) in water (3.6 mL) was added. After 2 h,
saturated aqueous Na2S2O3 and CH2Cl2 were added, and the
product was extracted as above. Chromatography on silica
gel (40 g, CH2Cl2/MeOH, 98.5:1.5; 98:2; 95:5) gave 8 (269.0 mg,
USO2G Th iol 11. Thioacetate 10 (40.1 mg, 35.5 µmol) was
dissolved in Ar-saturated MeOH (3.6 mL) at 0 °C. Ammonia
was bubbled through the solution for 15 min. The lines were
removed, and the solution was stirred a further 105 min at 0
°C before carefully removing the solvent and NH3 by evapora-
tion at ca. 1 °C with the exclusion of air contact. The white
solid was dried on HV for 12 h.
1
85%) as a white foam. The following are data for 8a . The H
NMR signals were assigned by 1H/1H-COSY. 1H NMR (500
MHz, CDCl3): δ (ppm) 1.05 (s, 9H, CH3-tBu); 1.97 (m, 1H, H-5′-
A); 2.04 (m, 1H, H-5′-A); 2.18 (s, 3H, COCH3); 2.46 (m, 1H,
H-5′-C); 2.57 (m, 1H, H-5′-C); 3.09 (d, J ) 12.4 Hz, 1H, H-3′′-
A); 3.46 (m, 4H, 1 × H-3′′-A, 2 × H-6′-C, H-3′-C); 3.62 (m, 1H,
H-3′-A); 3.86 (m, 2H, H-6′-A); 4.25 (t, J ) 8.8 Hz, 1H, H-4′-A);
4.47 (t, J ) 6.7 Hz, 1H, H-4′-C); 4.53 (dd, J ) 7.0 Hz, J ) 11.4
Hz, 1H, H-3′′-C); 4.72 (dd, J ) 6.4 Hz, J ) 11.5 Hz, 1H, H-3′′-
C); 5.45 (d, J ) 1.7 Hz, 1H, H-1′-C); 5.88 (d, J ) 5.6 Hz, 1H,
H-2′-A); 5.96 (d, J ) 1.6 Hz, 1H, H-1′-A); 6.97 (dd, J ) 1.6 Hz,
J ) 6.2 Hz, 1H, H-2′-C); 7.33-7.60 (m, 20H, p-Ph, p-Bz, H-5-
C, H-6-C); 7.62-7.68 (m, 4H, o-Ph); 7.77 (d, J ) 7.6 Hz, 2H,
o-NBz-A); 7.91 (d, J ) 7.4 Hz, 2H, o-NBz-C); 7.96-8.00 (m,
4H, o-OBz); 8.23 (s, 1H, H-8-A); 8.71 (s, 1H, H-2-A); 8.92 (br,
1H, NH); 9.32 (br, 1H, NH). FAB MS (3-NOBA): m/z 1293.5
(M + H+, 18); 240.0 (ABz + H+, 9); 105.0 (Bz+, 100); 76.9 (Ph+,
28).
ASO2C Alcoh ol 12. Dimer 8 (253.3 mg, 0.196 mmol) was
dissolved in a solution of HF (1.25 mmol) in pyridine. The
reaction mixture was vigorously stirred for 3.5 h, methoxy-
trimethylsilane (0.34 mL, 2.48 mmol) was added, and the
mixture was stirred for 10 min. The solvent was removed,
and the residue was chromatographed on silica gel (40 g, CH2-
Cl2/MeOH, 95:5) to yield 12 (190.7 mg, 92%). 1H NMR (500
MHz, CDCl3): δ (ppm) 2.04 (m, 2H, H-5′-A); 2.16 (s, 3H,
COCH3); 2.57 (m, 2H, H-5′-C); 3.36 (dd, J ) 11.7 Hz, J ) 14.4
Hz, 1H, H-3′′-A); 3.94 (m, 1H, H-6′-C); 3.54 (dd, J ) 2.2 Hz, J
) 14.6 Hz, 1H, H-3′′-A); 3.68 (m, 2H, H-3′-A/C); 3.85 (m, 2H,
H-6′-A); 4.04 (br, 1H, OH); 4.23 (m, 1H, H-4′-A); 4.47 (m, 1H,
H-4′-C); 4.53 (dd J ) 7.2 Hz, J ) 11.5 Hz, 1H, H-3′′-C); 4.75
(dd, J ) 6.4 Hz, J ) 11.5 Hz, 1H, H-3′′-C); 5.38 (d, J ) 1.7 Hz,
1H, H-1′-C); 5.93 (dd, J ) 1.9 Hz, J ) 6.4 Hz, 1H, H-2′-A);
6.00 (s, 1H, H-1′-A); 6.06 (d, J ) 5.5 Hz, 1H, H-2′-C); 7.37 (m,
2H, m-NBz-A); 7.43 (m, 6H, m-Bz); 7.50 (d, J ) 7.5 Hz, 1H,
H-5-C); 7.52-7.61 (m, 4H, p-Bz); 7.71 (d, J ) 7.4 Hz, 1H, H-6-
C); 7.77 (d, J ) 7.7 Hz, 2H, o-NBz-A); 7.89 (d, J ) 7.8 Hz, 2H,
o-NBz-C); 7.99 (m, 4H, p-OBz); 8.18 (s, 1H, H-8-A); 8.61 (s,
1H, H-2-A); 8.95 (br, 1H, NH); 9.11 (br, 1H, NH). FAB MS
(3-NOBA): m/z 1055.3 (M + H+, 11); 601.2 ((M - ABz - CBz)+,
22); 240.1 (ABz + H+, 22); 105.0 (Bz+, 100).
ASO2C Br om id e 13. Alcohol 12 (86.7 mg, 82.2 µmol) and
PPh3 (43.4 mg, 165.1 µmol) were dissolved in CH3CN (3.0 mL).
CBr4 (49.2 mg, 148.3 µmol), dissolved in CH3CN (1.0 mL), was
added at room temperature. After 1 h, the solution was poured
into a mixture of saturated sodium bicarbonate, ice, and CH2-
Cl2. The organic layer was separated, and the aqueous layer
was reextracted four times with CH2Cl2. The organic layers
were combined, the solvent was removed, and the residue was
chromatographed on silica gel (20 g, CH2Cl2/MeOH, 98:1.5; 98:
2; 96:4; 90:10) to yield 13 (64.5 mg, 70%). 1H NMR (400 MHz,
CDCl3): δ (ppm) 2.18 (s, 3H, COCH3); 2.34 (m, 1H, H-5′-A);
2.43 (m, 1H, H-5′-A); 2.46 (m, 1H, H-5′-C); 2.58 (m, 1H, H-5′-
C); 3.17 (dd, J ) 3.2 Hz, J ) 14.3 Hz, 1H, H-3′′-A); 3.37-3.52,
3.58 (m, 6H, H-3′-A, 4 × H-6′-A/C, 1 × H-3′′-A); 3.82 (m, 1H,
H-3′-C); 4.30 (dt, J ) 2.1 Hz, J ) 9.2 Hz, 1H, H-4′-A); 4.46 (m,
1H, H-4′-C); 4.54 (dd, J ) 7.0 Hz, J ) 11.7 Hz, 1H, H-3′′-C);
4.72 (dd, J ) 6.4 Hz, J ) 11.5 Hz, 1H, H-3′′-C); 5.44 (d, J )
2.0 Hz, 1H, H-1′-C); 5.93 (d, J ) 5.7 Hz, 1H, H-2′-A); 5.99 (dd,
J ) 1.8 Hz, J ) 6.2 Hz, 1H, H-2′-C); 6.06 (s, 1H, H-1′-A); 7.33-
7.49 (m, 8H, m-Bz); 7.51-7.61 (m, 5H, p-Bz, H-5-C); 7.64 (d,
J ) 7.4 Hz, 1H, H-6-C); 7.86 (d, J ) 7.7 Hz, 2H, o-NBz-A);
7.91 (d, J ) 7.3 Hz, 2H, o-NBz-C); 7.98 (m, 4H, p-OBz); 8.29
(s, 1H, H-8-A); 8.72 (s, 1H, H-2-A); 8.95 (br, 1H, NH); 9.20 (br,
USO2G Tr iol 9. Dimer 7 (136.3 mg, 103.1 µmol) was
dissolved in a mixture of THF (1.1 mL) and MeOH (1.2 mL)
and was cooled to 0 °C. A solution of 0.2 M LiOH (1.70 mL,
340.0 µmol) was added dropwise. The solution was stirred for
2 h at 0 °C before acetate buffer (163 µL) was added slowly.
Then, CH2Cl2 was added and the organic layer was separated.
The aqueous layer was reextracted five times with CH2Cl2.
The combined organic layers were concentrated in vacuo.
Chromatography on silica gel (12 g, CH2Cl2/MeOH, 96:4; 95:
5; 90:10) afforded triol 9 (90.5 mg, 82%) as a white foam. 1H
NMR (400 MHz, CDCl3): δ (ppm) 1.03 (s, 9H, CH3-tBu); 1.24
(2d, J ) 6.9 Hz, J ) 6.9 Hz, 6 H, 2 × CH3-iBu); 1.83 (m, 1H,
H-5′-U); 1.99 (m, 1H, H-5′-U); 2.12, 2.28 (m, 2H, H-3′-U/G);
2.42 (m, 2H, H-5′-G); 2.64 (m, 1H, CH-iBu); 2.90 (d, J ) 12.5
Hz, 1H, H-3′′-U); 3.21 (m, 1H, H-3′′-U); 3.26 (t, J ) 6.8 Hz,
2H, CH2-NPE); 3.34 (m, 1H, H-6′ G); 3.55 (br, 1H, OH-3′′-G);
3.64 (dt, J ) 3.4 Hz, J ) 12.6 Hz, 1H, H-6′-G); 3.75 (m, 1H,
H-3′′-G); 3.88 (t, J ) 6.1 Hz, 2H, H-6′-U); 3.97 (m, 1H, H-3′′-
G); 4.25 (m, 2H, H-4′-U, H-2′-U); 4.56 (br, 1H, H-2′-G); 4.72
(m, 1H, H-4′-G); 4.72 (t, J ) 6.7 Hz, 2H, CH2-NPE); 5.56, 5.57
(s, 1H, H-1′-U/G); 5.62 (d, J ) 8.1 Hz, 1H, H-5-U); 5.85 (br,
1H, OH-2′); 6.37 (br, 1H, OH-2′); 7.31 (d, J ) 8.2 Hz, 1H, H-6-
U); 7.34-7.44 (m, 6H, p-Ph); 7.49 (d, J ) 8.7 Hz, 2H, o-NPE);
7.63-7.65 (m, 4H, o-Ph); 8.06 (s, 1H, H-8-G); 8.13 (d, J ) 8.7
Hz, 2H, m-NPE); 8.24 (br, 1H, NH). FAB MS (3-NOBA): m/z
1093.3 (M + Na+, 8); 1071.1 (M + H+, 28); 370.9 (GNPE,i-Bu
+
H+, 100); 76.9 (Ph+, 36).
USO G Th ioa ceta te 10. A solution of PPh3 (24.7 mg, 94.2
2
µmol) in THF (1.0 mL) was cooled to 0 °C. Diisopropyl
azodicarboxylate (DIAD) (13.6 µL, 70.4 µmol) was added, and
the mixture was stirred for 35 min. 9 (50.4 mg, 47.0 µmol)
was dried (48 h, HV at room temperature), dissolved in THF
(0.78 mL), and added to the mixture. Immediately thereafter,

282 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 3
Eschgfa¨ller et al.
1H, NH). FAB MS (3-NOBA): m/z 1117.1, 1119.1 (M + H+,
7/8); 663.0, 665.0 ((M - ABz - CBz)+, 4/4); 240.0 (ABz + H+,
12); 104.9 (Bz+, 68).
Acetate buffer (0.37 mL) was added, yielding a white precipi-
tate. The organic solvents were removed by rotary evapora-
tion, and CH2Cl2/diethyl ether 9:1 and H2O were added to yield
a clear, slightly yellow organic layer and a milky aqueous
layer. The organic layer was separated and the aqueous layer
washed three times with CH2Cl2/diethyl ether (9:1). The white
precipitate was recovered by centrifugation from the aqueous
layer. The white precipitate was suspended in water in an
Eppendorf tube, shaken and the product recovered by precipi-
tation. This procedure was repeated twice. 16 (2.0 mg, 87%)
was recovered as a white solid. Analytical HPLC (Waters RP-
Nova-Pak C18 column, 3.9 × 150 mm, flow 0.9 mL/min; A )
H2O, B ) CH3CN, 0% B for 5 min, gradient 0% to 25% B in 40
min, 25% to 100% B in 15 min, 100% B for 10 min, retention
time of the single peak: 24-26 min) showed a single product.
1H NMR (300 MHz, DMSO-d6): δ (ppm) The spectrum upfield
from 3.70 ppm could not be interpreted; 3.70 (m, 2H, H-3′′);
3.85-4.13 (m, 4H, H-4′); 4.19 (m, 1H, H-2′-Py); 4.27 (m, 1H,
H-2′-Py); 4.57 (m, 1H, H-1′-Pu); 4.72 (m, 1H, H-2′-Pu); 5.50
(br, 1H, OH); 5.66 (m, 2H, H-1′, H-5-U); 5.71 (s, 1H, H-1′); 5.76
(s, d, 2H, H-1′, H-5-C); 5.95 (s, 1H, H-1′-A); 6.01 (br, 1H, OH);
6.13 (br, 1H, OH); 6.22 (br, 1H, OH); 6.53 (br, 2H); 7.15 (br,
2H, NH2); 7.30 (br, 1H, NH); 7.54 (d, 1H, H-6-U); 7.58 (d, 1H,
H-6-C); 7.90 (s, 1H, H-2-A); 8.17 (s, H-8-A); 8.35 (s, 1H, H-8-
A); 10.95 (br, NH). MALDI-TOF MS (HABA-matrix, linear,
positive mode, 7 kV): m/z 1284.4 (M - H+2 Na+); 1262.6 (M +
Na+).
USO GSASO C Tetr a m er 14. Cs2CO3 (37.4 mg, 114.8 µmol),
2
2
dried on HV (2 min at 130 °C, 10 min at room temperature),
was placed under Ar, and THF (2.0 mL) was added. Crude
thiol 11 (ca. 35.5 µmol, based on 100% ammonolysis yield) and
13 (47 mg, 42.0 µmol) dissolved in THF (1.6 mL) were added.
The suspension was vigorously stirred at 50 °C for 2 h. The
reaction was cooled to room temperature and neutralized with
acetate buffer (57 µL). CH2Cl2 and saturated aqueous NaCl/
H2O (9:1) were added, the organic layer was separated, and
the aqueous layer was reextracted five times with CH2Cl2. The
organic layers were combined, and the solvent was removed
by evaporation. Chromatography on silica gel (12 g, CH2Cl2/
MeOH, 97:3; 96:4; 95:5; 90:10) afforded tetramer 14 (62.2 mg,
82%). 1H NMR (500 MHz, DMSO-d6): δ (ppm) 0.94 (s, 9H,
CH3-tBu); 1.06 (d, J ) 7.2 Hz, 6H, 2 × CH3-iBu); 1.78 (m, 1H,
H-5′-U); 1.92 (m, 1H, H-5′-U); 2.10 (m, 2H, H-5′); 2.12 (s, 3H,
COCH3); 2.21, 2.33 (m, 4H, H-5′); 2.46 (m, 1H, H-3′-U); 2.58,
2.66 (m, 2H, 1 × H-3′′-G, H-3′-G); 2.80 (m, 4H, 2 × H-6′-A, 1
× H-3′′-G, CH-iBu); 3.10 (dd, J ) 3.5 Hz, J ) 12.7 Hz, 1H,
H-3′′-U); 3.15 (dd, J ) 6.5 Hz, J ) 9.5 Hz, 1H, H-3′′-A); 3.22
(m, 1H, H-3′); 3.41 (dd, J ) 8.6 Hz, J ) 14.6 Hz, 1H, H-3′′);
3.50, 3.57, 3.74 (m, 8H, partially covered by the DMSO signal,
4 × H-6′-G/C), 2 × H-3′′-A/U, 2 × H-3′-A/C); 3.74 (t, J ) 6.5
Hz, 2H, H-6′-U); 3.95 (t, J ) 10.7 Hz, 1H, H-4′-U); 4.03 (m,
1H, H-4′-G); 4.14 (t, J ) 8.3 Hz, 1H, H-4′-A); 4.23 (m, 1H, H-2′-
U); 4.51, 4.58 (m, 4H, H-4′-C, H-3′′-C, H-2′-G); 4.77 (t, J ) 6.8
Hz, 2H, CH2-NPE); 5.57 (d, J ) 8.1 Hz, 1H, H-5-U); 5.60 (d, J
) 1.7 Hz, 1H, H-1′-C); 5.73 (d, J ) 5.2 Hz, 1H, OH); 5.89 (d,
1H, OH); 5.89 (d, J ) 1.8 Hz, 1H, H-1′-U); 5.93 (dd, J ) 2.0
Hz, J ) 6.8 Hz, 1H, H-2′-A); 5.99 (d, J ) 5.0 Hz, 1H, H-2′-C);
6.01 (d, J ) 2.0 Hz, 1H, H-1′-G); 6.18 (d, J ) 1.2 Hz, 1H, H-1′-
A); 7.37-7.67 (m, 27H, H-6-U, H-5-C, H-6-C, p-Ph, p-Bz,
o-NPE); 7.91 (d, J ) 7.2 Hz, 2H, o-NBz-A); 7.98-8.05 (m, 6H,
o-NBz-C, o-OBz); 8.16 (d, J ) 8.7 Hz, 2H, m-NPE); 8.25 (d, J
) 7.5 Hz, 1H, NH-iBu); 8.38 (s, 1H, H-8-G); 8.67 (s, 1H, H-8-
A); 8.75 (s, 1H, H-2-A); 10.30 (s, 1H, NH); 11.30 (br, 2H, NH).
FAB MS (3-NOBA): m/z 2123.6 (M + H+, 100); 1885.4 ((M -
ABz)+, 4); 1063.3 (12); 766.4 (10); 666.8 (9); 613.1 (30).
Ack n ow led gm en t . We are indebted to the Swiss
National Science Foundation for partial support of this
work, to Dr. I. Allmann at the Institute of Toxicology
for veterinary assistance, and to Klaus Girgenrath at
the Institute for Organic Chemistry for help in radio-
chemical work.
Su p p or tin g In for m a tion Ava ila ble: Spectral assign-
ments for 7a ,b, 8a ,b, 9, 10, and 12-15 (4 pages). Ordering
information is given on any current masthead page.
Refer en ces
USO GSO ASO2C Tetr a m er 15. UGAC-thioether 14 (62.2
2
2
mg, 28.2 µmol) was dissolved in THF (1.9 mL)/MeOH (5.2 mL),
and a mixture of Oxone (71.1 mg, 115.7 µmol) and NaOAc (31.5
mg, 384.0 µmol) in water (0.95 mL) was added. After 1.5 h,
aqueous Na2S2O3 and CH2Cl2 were added, and the product was
extracted as above. Chromatography on silica gel (12 g, CH2-
Cl2/MeOH, 96.5:3.5; 96:4; 95:5; 90:10) yielded 15 (53.1 mg,
84%) as a white foam. 1H NMR (500 MHz, DMSO-d6): δ (ppm)
0.95 (s, 9H, CH3-tBu); 1.07 (d, J ) 7.1 Hz, 6H, 2 × CH3-iBu);
1.78 (m, 1H, H-5′-U); 2.08 (m, 1H, H-5′-U); 2.13 (s, 3H,
COCH3); 2.31, 2.47 (m, 5H, 4 × H-5′, H-3′-U); 2.81 (m, 1H,
CH-iBu); 3.11, 3.30, 3.45, 3.65 (m, 17H, partially covered by
the DMSO signal, CH2-NPE, 6 × H-6′-G/A/C, 6 × H-3′′-U/G/
A, 3 × H-3′-G/A/C); 3.74 (t, J ) 6.3 Hz, 2H, H-6′-U); 3.95 (t, J
) 9.5 Hz, 1H, H-4′-U); 4.03 (t, J ) 8.8 Hz, 1H, H-4′-G); 4.16
(t, J ) 8.3 Hz, 1H, H-4′-A); 4.23 (br, 1H, H-2′-U); 4.51 (m, 2H,
H-4′-A, 1 × H-3′′-C); 4.59 (m, 1H, H-3′′-C); 4.67 (br, 1H, H-2′-
G); 4.77 (t, J ) 6.6 Hz, 2H, CH2-NPE); 5.57 (d, J ) 8.1 Hz,
1H, H-5-U); 5.60 (d, J ) 1.7 Hz, H-1′-C); 5.73 (d, J ) 5.2 Hz,
1H, OH); 5.90 (s, 1H, H-1′-U); 5.93 (d, J ) 8.5 Hz, 1H, H-2′-
A); 6.00 (s, d, 2H, H-2′-C, H-1′-G); 6.15 (d, J ) 4.7 Hz, 1H,
OH); 6.23 (s, 1H, H-1′-A); 7.38-7.66 (m, 27H, H-6-U, H-5-C,
H-6-C, o-, m-, p-Ph, m-, p-Bz, o-NPE); 7.91 (d, J ) 7.2 Hz, 2H,
o-NBz-A); 7.98-8.05 (m, 6H, o-NBz-C, o-OBz); 8.16 (d, J ) 8.7
Hz, 2H, m-NPE); 8.25 (d, J ) 7.5 Hz, 1H, NH-iBu); 8.38 (s,
1H, H-8-G); 8.67 (s, 1H, H-8-A); 8.75 (s, 1H, H-2-A); 10.30 (s,
1H, NH); 11.30 (br, 2H, NH). FAB MS (3-NOBA): m/z 2155.4
(M + H+, 5); 371.1 (GNPE,i-Bu + H+, 16); 289.0 (21); 240.1 (ABz
+ H+, 9); 216.1 (CBz + H+, 5); 105.0 (Bz+, 59); 76.9 (Ph+, 43).
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Dep r otected USO GSO ASO2C 16. Tetramer 15 (4.0 mg, 1.86
2
2
µmol) was dissolved in MeOH (0.5 mL) and THF (0.5 mL).
NaOH (2 M, 0.5 mL) was added at room temperature, and the
yellow solution was stirred for 23 h at room temperature.

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