Synthesis of oligoribonucleotides containing pyrimidine 2′-O-[(hydroxyalkoxy)methyl]ribonucleosides

Article abstract of DOI:10.1135/cccc20060804

A simple and efficient method for the preparation of pyrimidine 2′-O-[(2-hydroxyethoxy)methyl]ribonucleosides and 2′-O-[(3- hydroxypropoxy)methyl]ribonucleosides has been developed. These modified nucleosides were incorporated into oligoribonucleotides, which were shown to form stable RNA/RNA duplexes.

Full text of DOI:10.1135/cccc20060804

804
Bobkov, Brilliantov, Mikhailov, Rozenski, Van Aerschot, Herdewijn:
SYNTHESIS OF OLIGORIBONUCLEOTIDES CONTAINING PYRIMIDINE
2′-O-[(HYDROXYALKOXY)METHYL]RIBONUCLEOSIDES
Georgii V. BOBKOVa1, Kirill V. BRILLIANTOVa2, Sergey N. MIKHAILOVa3,*,
b2
b3
Jef ROZENSKIb1, Arthur VAN AERSCHOT and Piet HERDEWIJN
a
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov str. 32,
Moscow 119991, Russia; e-mail: georgbk@eimb.ru, room312@eimb.ru,
1
2
3
smikh@eimb.ru
b
Rega Institute, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven,
Belgium; e-mail: jef.rozenski@rega.kuleuven.be, arthur.vanaerschot@rega.kuleuven.be,
1
2
3
piet.herdewijn@rega.kuleuven.ac.be
Received February 14, 2006
Accepted April 6, 2006
This paper is dedicated to Professor Antonín Holý on the occasion of his 70th birthday.
A
sim ple an d efficien t m eth od for th e preparation of pyrim idin e 2′-O-[(2-h ydroxy-
eth oxy)m eth yl]ribon ucleosides an d 2′-O-[(3-h ydroxypropoxy)m eth yl]ribon ucleosides h as
been developed. Th ese m odified n ucleosides were in corporated in to oligoribon ucleotides,
wh ich were sh own to form stable RNA/RNA duplexes.
Key w o r d s: Ribon u cleosid es; O ligon u cleot id es; 2′-O-Mod ified n ucleosides; An tisen se
agen ts; RNA duplexes; Th erm al den aturation ; siRNA.
Modified oligon ucleotides are widely used in m olecular biology an d m edici-
n al ch em istry. An tisen se agen ts are valuable tools to in h ibit th e expression
of a target gen e in a sequen ce specific m an n er, an d m ay be used for fun c-
tion al gen om ics, target validation an d th eurapeutic purposes1. Th e im pact
of th e research in th is field in creased due to th e discovery of RNA in terfer-
en ce2,3
.
Num erous attem pts h ave been m ade to im prove th e properties of n atural
oligon ucleotides4,5. It is believed th at th e 2′-m odification s in sugar m oiety
are th e m ost un iversal an d prom isin g6–9. A typical strategy for th e prepara-
tion of such oligon ucleotides is th e syn th esis of a m odified n ucleoside fol-
lowed by con version to th e correspon din g ph osph oram idite suitable for th e
autom ated oligon ucleotide syn th esis. Most of th ese m odification s were
ach ieved via alkylation reaction s of partially protected ribon ucleosides. Th e
h eterocyclic bases sh ould be protected in order to avoid th eir alkylation .
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 6, pp. 804–819
© 2006 Institute of Organic Chemistry and Biochemistry
doi:10.1135/cccc20060804

Synthesis of Oligoribonucleotides
805
For every n ucleoside, th e specific blockin g groups are used10, wh ich in m ost
cases are n ot com patible with stan dard autom ated oligon ucleotide syn th e-
sis. For th e preparation of 2′-O-m odified n ucleosides we h ave recen tly de-
veloped an oth er type of ch em istry based on O-glycosylation reaction s,
wh ich h as several im portan t advan tages, such as in creased yields an d sim -
plification 11,12. A sim ple an d effective m eth od h as been recen tly devel-
oped13–16 for th e preparation of 2′-O-(β-D-ribofuran osyl)n ucleosides startin g
from readily available 3′,5′-O-blocked N-acylribon ucleosides an d 1-O-acetyl-
2,3,5-tri-O-ben zoyl-β-D-ribofuran ose preactivated with tin tetrach loride in
1,2-dich loroeth an e at 0 °C. Th e con den sation proceeds by stereospecific
form ation of th e β-D-glycosidic bon d an d gives a h igh yield. Several oth er
fully acylated m on osacch aride derivatives of D- an d L-arabin ofuran oses,
D-eryth rofuran ose an d D-ribopyran ose h ave been successfully used in th e
sam e reaction 17
.
RESULTS AND DISCUSSION
Synthesis of the Monomeric Nucleosides
Here we presen t our recen t results on th e preparation of 2′-O-h ydroxy-
alkoxym eth ylribon ucleosides an d th eir in corporation in to oligoribo-
n ucleotides. Th is sch em e is n early th e sam e as for th e preparation of
disacch aride n ucleosides an d th eir in corporation in to oligon ucleotides17.
18
Th e startin g com poun ds 1 an d 2 were easily prepared from 1,3-dioxolan e
an d 1,3-dioxan e, respectively, an d were con den sed with 3′,5′-O-blocked
19
ribon ucleosides 3a, 3b in th e presen ce of tin tetrach loride in 1,2-dich loro-
eth an e at –12 °C for 20 m in (Sch em e 1). Th e yields (85–87%) of products
4a, 4b an d 5a, 5b were even h igh er th an in th e case of preparation of th e
above m en tion ed disacch aride n ucleosides13. Th e silyl group was deblocked
to yield partially protected 6a, 6b an d 7a, 7b an d subsequen t deblockin g
with am m on ia in m eth an ol gave free 2′-O-substituted n ucleosides 8a, 8b
an d 9a, 9b in h igh overall yields. Nucleosides 6a, 6b an d 7a, 7b were con -
verted usin g stan dard procedures to th e correspon din g m eth oxytrityl deriv-
atives 10a, 10b an d 11a, 11b an d th eir ph osph oram idites 12a, 12b an d
13a, 13b.
Th e structure of th e com poun ds was supported by NMR spectroscopy an d
m ass sp ect ro m et ry. Th e ch em ical sh ift s were assign ed u sin g d o u ble
reson an ce tech n iques an d COSY experim en ts. Several con clusion s were
drawn from th e 1H NMR spectral an alysis. In n ucleosides 3a, 3b–5a, 5b
with 1,1,3,3-tetraisopropyldisiloxan e-1,3-diyl group th e couplin g con stan ts
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 6, pp. 804–819

806
Bobkov, Brilliantov, Mikhailov, Rozenski, Van Aerschot, Herdewijn:
O
O
O
O
AcO
AcO
O
1
B
O
OAc
(ii)
(i)
Si
O
+
O
O
OAc
Si
O
OH
2
3
B
B
O
HO
(iv)
(iii)
Si
O
O
O
Si
O
HO
O
O
O
O
OAc
OAc
4
6
(v)
B
B'
MMTrO
HO
O
O
(vii)
RO
HO
O
O
O
O
OAc
OH
10 R = H
8
(vi)
HO
12 R = -P(OCH2CH2CN)(N-iPr)
B
B
O
(iv)
(iii)
Si
O
O
O
Si
O
O
O
OAc
HO
O
O
OAc
5
7
(v)
B'
B
HO
MMTrO
O
O
(vii)
OAc
HO
O
O
OH
RO
O
O
9
11 R = H
13 R = -P(OCH2CH2CN)(N-iPr)
(vi)
a B = B' = Ura
b B = CytBz, B' = Cyt
(i) Ac2O, AcOH, H2SO4; (ii) SnCl2/ClCH2CH2Cl, –12 oC; (iii) Bu4NF/THF; (iv) NH3/MeOH;
(v) MMTrCl/Py; (vi) iPrNPCl(OCH2CH2CN); (vii) oligonucleotide sunthesis
SCHEME 1
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 6, pp. 804–819

Synthesis of Oligoribonucleotides
807
J1′,2′ of n ucleoside m oieties are lower th an 0.5 Hz. Most of th e ch em ical
sh ifts an d couplin g con stan ts m ay be calculated directly from NMR spectra.
In som e cases com parison with th e publish ed spectra of disacch aride n u-
cleosides13 an d double reson an ce, 1H-13C correlation an d COSY spectra
were used for th e assign m en t. It sh ould be m en tion ed th at som e of th e
1H NMR spectra of th e obtain ed com poun ds are rath er com plicated due to
th e overlap of sign als of th e ribofuran ose residue an d th e diastereotopic
proton s of CH2OCH2CH2O an d CH2OCH2CH2CH2O groups. Th e acetal
group appeared as two doublets aroun d 5 ppm . As in th e case of
disacch aride n ucleosides13 an d 2′-O-m eth yln ucleosides20 in corporation of
th e 2′-O-substituen t results in a low-field sh ift (2.6–3.1 ppm ) of C-2′ in
13C NMR spectra.
Incorporation Studies
Coun tless m odification s h ave been in troduced in to n ucleosides in search
for im proved bin din g to com plem en tary stran ds, but n ot m an y with
form acetal sugar derivatized n ucleoside an alogues h ave been in corporated
in to oligon ucleotides as a perm an en t m odification 7,21. Th e O-(alkoxy-
m eth yl) groupin g h as largely been used as an an ch or for th e developm en t
of sugar h ydroxyl protectin g groups like in (silylm eth oxy)m eth yl22 or
2-(silyleth oxy)m eth yl eth ers23, or (2-m eth oxyeth oxy)m eth yl eth ers24.
Fully protected an alogues are m an datory for oligon ucleotide syn th esis,
an d stan dard reaction s lead to th e required am idites useful for in corpora-
tion in to oligon ucleotides. Hereto, ph osph itylation was carried out on th e
m on om eth oxytritylated derivatives (10a, 10b, 11a an d 11b, respectively)
in dich lorom eth an e usin g fresh ly distilled eth yldiisopropylam in e an d
2-cyan oeth yl diisopropylph osph oram idoch loridite un der argon to afford
12a, 12b an d 13a, 13b in h igh yield. With th ese ph osph oram idites, several
oligoribon ucleotides with on e or m ore m odification s were assem bled, usin g
com m ercial TOM am idites for th e n atural n ucleosides. Th e 2′-O-[(triiso-
propylsilyl)oxy]m eth yl (TOM) protectin g group is kn own for its h igh cou-
plin g efficien cy alon g with fast, sim ple deprotection 25,26. Trityl yields were
excellen t, for th e sh orter oligos reach in g 100% for full-len gth assem bly,
even with m ultiple in corporation s of th e m odified buildin g blocks. All
oligos described h ere were assem bled on a LCAA-CPG support loaded with
a dim eth ylform am idin e-protected guan osin e. For deprotection of th e m odi-
fied RNAs a m eth ylam in e cocktail in water–eth an ol was used, followed by a
1 M TBAF treatm en t. All oligos were purified by ion exch an ge ch rom atogra-
ph y, desalted by gel filtration an d an alysed by m ass spectrom etry (Table I
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 6, pp. 804–819

808
Bobkov, Brilliantov, Mikhailov, Rozenski, Van Aerschot, Herdewijn:
an d Fig. 1). On ly th e m ain part of th e product con tain in g peak was col-
lected, affordin g in gen eral 20 to 30 OD260 startin g from a 1 µm ol loaded
support. In addition several siRNAs com prisin g on e or m ore of th e n ew
m odification s h ave been prepared an d are curren tly un der evaluation .
As an exam ple, th e m ass spectral an alysis of m odified oligon ucleotide
(en try 14 in Table I) carryin g 5 m odified n ucleosides is sh own in th e ac-
com pan yin g Fig. 1, with th e top pan el sh owin g th e UV trace as well as th e
m ass ion coun t (base peak in ten sity). In th e UV trace a sligh t bum p is n o-
ticed, but th is is gen erated by th e buffer, an d th e ion coun t is very low. Th e
m ass spectrum at th e elutin g tim e of th e oligon ucleotide is sh own in pan el
b, with th e decon voluted spectrum with th e expected m ass as th e in set.
Altogeth er, it sh ows th e iden tity an d purity of th e oligon ucleotides obtain ed.
Th e effect of th e n ew m odification s was verified on n on am ers by m eltin g
experim en ts studies, in an alogy with our previous work on differen t h exitol
TABLE I
Molecular weigh t m easurem en ts an d m eltin g experim en ts for oligoribon ucleotides con tain -
in g 2′-O-[(h ydroxyalkoxy)m eth yl]ribon ucleosides in 0.1 M NaCl, 0.1 m M EDTA an d 20 m M
KH2PO4 at pH 7.5 (Tm , °C)
Com plem en tary
oligoribon ucleotide oligoribon ucleotide
3′-CGACACAGC-5′ 3′-CGU GAG UGC-5′
Com plem en tary
MS
calcd.
M+
MS
foun d
M+
Startin g
syn th on
No.
Oligoribon ucleotide
Tm , °C
Tm , °C
1
2
5′-GCUGUGUCG-3′
5′-GCUGUOMeGUCG-3′
5′-GCUGUGUCG-3′
5′-GCUGUGUCG-3′
5′-GCACUCACG-3′
5′-GCACUOMeCACG-3′
5′-GCACUCACG-3′
5′-GCACUCACG-3′
5′-GCUGUGUCG-3′
5′-GCUGUGUCG-3′
5′-GCACUCACG-3′
5′-GCACUCACG-3′
5′-GCUGUGUCG-3′
5′-GCUGUGUCG-3′
55.4
57.2
54.8
54.7
3
12a
13a
2920.4 2920.5
2934.4 2934.5
4
5
56.9
57.9
55.1
55.4
6
7
12b
2886.5 2886.5
2900.5 2900.3
2994.5 2994.5
3022.5 3022.5
2960.5 2960.5
2988.5 2988.4
3354.6 3354.7
3424.7 3424.5
8
13b
9
12a
54.6
54.2
10
11
12
13
14
13a
12b
54.1
54.2
13b
12a/12b
13a/13b
53.1
51.4
U: 2′-O-[(h ydroxyalkoxy)m eth yl]uridin e residue.
C: 2′-O-[(h ydroxyalkoxy)m eth yl]cytidin e residue.
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 6, pp. 804–819

Synthesis of Oligoribonucleotides
809
n ucleoside con gen ers27. In con trast for in corporation of a 2′-O-m eth yl-
ribon ucleoside in such n on am ers, wh ich on average in creased th e m eltin g
tem perature (Tm ) by 1.5 °C per m odification (from 1 to 2 °C depen din g on
th e sequen ce con text; com pare en tries 1 an d 2, 5 an d 6 in Table I), th e lon -
ger ch ain s in troduced with th e n ew con gen ers 8a, 8b or 9a, 9b sligh tly
destabilize th e RNA duplex. Wh ile 2′-alkyl m oieties (in ribo con figuration )
in gen eral give con siderable destabilisation of a duplex, m ost of th e studied
2′-alkoxy substituen ts do stabilise th e duplex7. Like in th e presen t study,
th e on ly form acetal fun ction in corporated in to th e duplex as a 2′-O-(ethoxy-
m eth yl)ribon u cleosid e7, or a 2′-O-(m eth oxym eth yl)ribon u cleosid e21
,
h owever, proved disadvan tageous. Th is largely con trasts with th e
well-kn own MOE m odification (2-m eth oxyeth yl)ribon ucleosides7,28. It was
reported th at 2′-(2-m eth oxyeth yl) substituen ts are con form ation ally
preorgan ized for th e duplex state in an A-type duplex as for an om eric an d
gauch e effects an d electrostatic in teraction s between th e backbon e an d
a
b
FIG. 1
HPLC-MS an alysis of m odified oligon ucleotide (en try 14 in Table I). Pan el a: Overlaid UV
ch rom atogram at 260 n m (UV260) an d base peak in ten sity (BPI) ch rom atogram . Pan el b: Mass
spectrum taken at 4.0 m in with decon voluted spectrum in in set
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 6, pp. 804–819

810
Bobkov, Brilliantov, Mikhailov, Rozenski, Van Aerschot, Herdewijn:
substituen t, an d th is con trasts with a 2′-O-(eth oxym eth yl) group wh ich
leads to sligh tly reduced RNA affin ity of th e correspon din g oligon ucleo-
tides29. Th e presen t results sh ow th at in corporation of th e extra h ydroxyl
group in th e side ch ain does n ot con tribute to or com pen sate th e overall ef-
fect. Th e destabilizin g effect of th e (3-h ydroxypropoxym eth yl) group is
sligh tly larger in com parison with th e (2-h ydroxyeth oxym eth yl) group,
wh ich is n oticed best after in troducin g five m odification s in th e n on am er
sequen ce (en tries 13 an d 14 in Table I). It n eeds to be stressed, h owever,
th at stable duplexes are still obtain ed, with about 0.5 °C destabilisation per
m odification .
CONCLUSIONS
Th e gen eral m eth od for th e preparation of 2′-O-β-D-ribofuran osyln ucleo-
sides was foun d to be applicable to th e syn th esis of pyrim idin e
2′-O-[(h ydroxyalkoxy)m eth yl]ribon ucleosides. Th e 2′-O-substituen t was
foun d to be stable durin g oligon ucleotide syn th esis. Addition al work on th e
preparation of oth er 2′-O-fun ction alized n ucleosides an d oligon ucleotides
bearin g am in o groups is in progress an d will be publish ed sh ortly.
EXPERIMENTAL
Colum n ch rom atograph y (CC) was perform ed on silica gel Kieselgel 60 (0.063–0.200 m m ,
Merck). TLC was carried out on Kieselgel 260 F (0.040–0.063 m m , Merck) with detection by
UV an d th e followin g solven t system s (v/v): A CH2Cl2–EtOH, 95:5; B CH2Cl2–EtOH, 9:1; C
iPrOH–con cen trated NH4OH–H2O, 7:1:2; D h exan e–aceton e–NEt3, 49:49:2. NMR spectra
were recorded usin g Bruker AMX 400 an d Varian Un ity 500 spectrom eters at 300 K. Ch em i-
cal sh ifts δ in ppm were m easured relative to th e solven t sign als (1H an d 13C) or H3PO4 as
relative extern al referen ce (31P). Th e couplin g con stan ts (J) are given in Hz. Th e sign als were
assign ed usin g double reson an ce tech n iques an d COSY experim en ts. Th e UV spectra were re-
corded on a Cary-300 UV/VIS spectroph otom eter (Varian ). Mass spectrom etry an d exact
m ass m easurem en ts of n ucleoside in term ediates were perform ed on a quadrupole/orth o-
gon al-acceleration tim e-of-fligh t tan dem m ass spectrom eter (Q-Tof-2, Microm ass, Man ch es-
ter, U.K.) equipped with a stan dard electrospray ion ization (ESI) in terface. ESI spectra for th e
m odified oligon ucleotides were obtain ed by couplin g th e Q-Tof-2 to a capillary HPLC
(CapLC, Waters, Milford, MA, U.S.A.). Masses were obtain ed by decon volution of th e spectra
usin g th e MaxEn t 1 algorith m (MassLyn x 3.4, Microm ass, Man ch ester, U.K.). Ch rom atogra-
ph y: C18 colum n 0.5 m m × 15 m m (PepMap, LC Packin gs), m obile ph ase: 0.05
M
trieth ylam in e with 1,1,1,3,3,3-h exafluoropropan -2-ol (pH 7.5), aceton itrile as th e organ ic
ph ase. Flow rate: 12 µl/m in . Th e gradien t started at 2% of organ ic ph ase an d in creased 2%
per m in ute over 15 m in . Mass spectra were acquired every 2 s in n egative ion ization m ode
applyin g 2850 V on th e electrospray capillary. Con e voltage 35 V, collision cell voltage 10 V.
TOM-am idites an d th e ph osph orylation reagen t were purch ased from Glen Research .
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 6, pp. 804–819

Synthesis of Oligoribonucleotides
811
2-(Hydroxym eth oxy)eth an -1-ol Diacetate (1)18
1H NMR (CDCl3): 5.25 s, 2 H (OCH2O); 4.18 t, 2 H, J = 4.8 (OCH2CH2OAc); 3.80 t, 2 H
(OCH2CH2OAc); 2.06 s, 3 H (Ac); 2.04 s, 3 H (Ac). 13C NMR (CDCl3): 170.87, 170.46 (C=O);
89.16 (OCH2O); 68.24 (OCH2CH2OAc); 63.25 (OCH2CH2OAc); 21.00, 20.86 (Me).
3-(Hydroxym eth oxy)propan -1-ol Diacetate (2)18
1H NMR (CDCl3): 5.17 s, 2 H (OCH2O); 4.07 t, 2 H, J = 6.4 (OCH2CH2CH2OAc); 3.63 t, 2 H,
J = 6.4 (OCH2CH2CH2OAc); 2.01 s, 3 H (Ac); 1.97 s, 3 H (Ac); 1.83 p, 2 H (OCH2CH2CH2OAc).
13C NMR (CDCl3): 170.98, 170.56 (C=O); 89.26 (OCH2O); 66.99 (OCH2CH2CH2OAc); 61.28
(OCH2CH2CH2OAc); 28.97 (OCH2CH2CH2OAc); 21.01, 20.91 (Me).
3′,5′-(1,1,3,3-Tetraisopropyldisiloxan e-1,3-diyl)uridin e (3a)19
RF 0.33 (A). 1H NMR (CDCl3): 8.52 brs, 1 H (NH); 7.66 d, 1 H, J(6,5) = 8.1 (H-6); 5.72 s, 1 H
(H-1′), 5.67 dd, 1 H, J(5,NH) = 1.8 (H-5); 4.38 dd, 1 H, J(3′,2′) = 4.7, J(3′,4′) = 8.7 (H-3′); 4.19
dd, 1 H, J(5′a,4′) = 2.2, J(5′a,5′b) = –13.1 (H-5′a); 4.17 d, 1 H (H-2′); 4.09 ddd, 1 H, J(4′,5′b) =
2.8 (H-4′); 4.01 dd, 1 H (H-5′b); 1.09–1.02 m , 28 H (iPr). 13C NMR (CDCl3): 163.43 (C-4);
150.21 (C-2); 140.15 (C-6); 102.13 (C-5); 91.24 (C-1′); 82.14 (C-4′); 75.32 (C-2′); 69.26 (C-3′);
60.58 (C-5′); 17.58, 17.47, 17.40, 17.24, 17.15, 16.97, 13.54, 13.11, 12.72 (iPr).
N4-Ben zoyl-3′,5′-(1,1,3,3-tetraisopropyldisiloxan e-1,3-diyl)cytidin e (3b)19
RF 0.34 (A). 1H NMR (CDCl3): 8.85 brs, 1 H (NH); 8.22 d, 1 H, J(6,5) = 7.5 (H-6); 7.92–7.50 m ,
6 H (Bz, H-5); 5.84 s, 1 H (H-1′); 4.35 dd, 1 H, J(3′,2′) = 4.6, J(3′,4′) = 8.9 (H-3′); 4.28 dd, 1 H,
J(5′a,4′) = 1.8, J(5′a,5′b) = –13.4 (H-5′a); 4.26 d, 1 H (H-2′); 4.21 ddd, 1 H, J(4′,5′b) = 2.8
(H-4′); 4.01 dd, 1 H (H-5′b); 1.11–1.00 m , 28 H (iPr). 13C NMR: 165.78 (C=O); 162.63 (C-4);
154.33 (C-2); 144.78 (C-6); 133.29, 129.13, 127.82 (Ph ); 96.52 (C-5); 91.99 (C-1′); 82.25
(C-4′); 75.36 (C-2′); 68.89 (C-3′); 60.32 (C-5′); 17.57, 17.52, 17.42, 17.14, 16.97, 13.54, 13.09,
12.69 (iPr).
1-{2-O-[(2-Acetoxyeth oxy)m eth yl]-3,5-O-(1,1,3,3-tetraisopropyldisiloxan e-1,3-diyl)-
β-D-ribofuran osyl}uracil (4a)
To a cool solution (–15 °C) un der n itrogen of n ucleoside 3a (0.973 g, 2.0 m m ol) an d
2-(h ydroxym eth oxy)eth an -1-ol diacetate (1; 0.705 g, 4.0 m m ol) in 1,2-dich loroeth an e
(15 m l) tin tetrach loride (0.36 m l, 3 m m ol) was added an d th e solution was kept at –12 °C
for 20 m in . A 10% aqueous solution of sodium h ydrogen carbon ate (10 m l) an d m eth ylen e
ch loride (20 m l) were added an d th e suspen sion was stirred at 0 °C for 20 m in . Th e suspen -
sion was filtered th rough Hyflo Super Cel, organ ic layer was separated, wash ed with water
(20 m l), dried over an h ydrous Na2SO4 an d evaporated to dryn ess. Th e residue was purified
by colum n ch rom atograph y on silica gel (30 g). Th e colum n was wash ed with m eth ylen e
ch loride (200 m l), m eth ylen e ch loride–eth an ol 99:1 (200 m l) an d th en eluted with m eth yl-
en e ch loride–eth an ol 98:2 to give 4a as a foam . Yield 1.04 g (86%). RF 0.35 (A). 1H NMR
(CDCl3): 9.72 brs, 1 H (NH); 7.88 d, 1 H, J(6,5) = 8.1 (H-6); 5.74 s, 1 H (H-1′), 5.65 d, 1 H
(H-5); 5.02 d, 1 H, J = –6.8 (OCHHO); 4.94 d, 1 H (OCHHO); 4.22 d, 1 H, J(5′a,5′b) = –13.7
(H-5′a); 4.20–4.13 m , 4 H (H-2′, H-3′, OCH2CH2OAc); 4.12 dd, 1 H, J(4′,3′) = 8.8, J(4′,5′b) =
1.8 (H-4′); 3.96 dd, 1 H (H-5′b); 3.88 m , 2 H (OCH2CH2OAc); 2.05 s, 3 H (Ac); 1.05–0.99 m ,
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 6, pp. 804–819

812
Bobkov, Brilliantov, Mikhailov, Rozenski, Van Aerschot, Herdewijn:
28 H (iPr). 13C NMR (CDCl3): 171.08 (C=O, Ac); 163.70 (C-4); 150.14 (C-2); 139.34 (C-6);
101.77 (C-5); 94.60 (OCH2O); 89.40 (C-1′); 81.92 (C-4′); 78.40 (C-2′); 68.19 (C-3′); 65.91
(OCH2CH2OAc); 63.60 (OCH2CH2OAc); 59.50 (C-5′); 20.99 (Me); 17.59, 17.50, 17.42, 17.33,
17.22, 17.07, 16.93, 13.51, 13.23, 13.00, 12.75 (iPr). LSI-MS: (C26H46N2O10Si2 + H+)
603.2764, calculated 603.2769.
1-{2-O-[(2-Acetoxyeth oxy)m eth yl]-3,5-O-(1,1,3,3-tetraisopropyldisiloxan e-1,3-diyl)-
β-D-ribofuran osyl}-N4-ben zoylcytosin e (4b)
An alogous con den sation of diacetate 1 (0.705 g, 4.0 m m ol) in th e presen ce of tin tetra-
ch loride (0.36 m l, 3 m m ol) with n ucleoside 3b (1.180 g, 2.0 m m ol) in 1,2-dich loroeth an e (15
m l) at –12 °C for 20 m in gave 4b as a foam . Yield 1.235 g (87%). RF 0.37 (A). 1H NMR
(CDCl3): 8.75 brs, 1 H (NH), 8.34 d, 1 H, J(6,5) = 7.5 (H-6); 7.90–7.50 m , 6 H (Bz, H-5); 5.84 s,
1 H (H-1′), 5.14 d, 1 H, J = –6.7 (OCHHO); 4.99 d, 1 H (OCHHO), 4.29 d, 1 H, J(5′a,5′b) =
–13.7 (H-5′a); 4.24–4.18 m , 5 H (H-2′, H-3′, H-4′, OCH2CH2OAc); 4.01 d, 1 H (H-5′b); 3.92 m ,
2 H (OCH2CH2OAc); 2.07 s, 3 H (Ac); 1.11–0.92 m , 28 H (iPr). 13C NMR (CDCl3): 171.13
(C=O, Ac); 167.40 (C=O, Bz); 162.55 (C-4); 154.30 (C-2); 144.36 (C-6); 133.30, 129.19,
127.67 (Ph ); 96.22 (C-5); 94.74 (OCH2O); 90.45 (C-1′); 82.07 (C-4′); 78.25 (C-2′); 67.96
(C-3′); 65.95 (OCH2OCH2CH2OAc); 63.73 (OCH2OCH2CH2OAc); 59.53 (C-5′); 21.06 (Me);
17.58, 17.42, 17.14, 16.98, 13.52, 13.04, 12.77 (iPr). LSI-MS: (C33H51N3O10Si2 + H+)
706.3186, calculated 706.3190.
1-{2-O-[(3-Acetoxypropoxy)m eth yl]-3,5-O-(1,1,3,3-tetraisopropyldisiloxan e-1,3-diyl)-
β-D-ribofuran osyl}uracil (5a)
An alogous con den sation of diacetate 2 (0.761 g, 4.0 m m ol) in th e presen ce of tin tetra-
ch loride (0.36 m l, 3 m m ol) with n ucleoside 3a (0.973 g, 2.0 m m ol) in 1,2-dich loroeth an e
(15 m l) at –12 °C for 20 m in gave 5a as a foam . Yield 1.065 g (86%). RF 0.35 (A). 1H NMR
(CDCl3): 9.68 brs, 1 H (NH); 7.87 d, 1 H, J(6,5) = 8.1 (H-6); 5.74 s, 1 H (H-1′); 5.65 d, 1 H
(H-5); 4.98 d, 1 H, J = –6.5 (OCHHO); 4.89 d, 1 H (OCHHO); 4.25 d, 1 H, J(5′a,5′b) = –13.4
(H-5′a); 4.30–4.10 m , 5 H (H-2′, H-3′, H-4′, OCH2CH2CH2OAc); 3.95 dd, 1 H, J(5′b,4′) = 2.2
(H-5′b); 3.71 t, 2 H, J = 6.4 (OCH2CH2CH2OAc); 2.02 s, 3 H (Ac); 1.90 p, 2 H, J = 6.4
(OCH2CH2CH2OAc); 1.08–0.99 m , 28 H (iPr). 13C NMR (CDCl3): 171.16 (C=O); 163.71 (C-4);
150.06 (C-2); 139.41 (C-6); 101.70 (C-5); 94.64 (OCH2O); 89.40 (C-1′); 81.92 (C-4′); 78.21
(C-2′); 68.17 (C-3′); 64.96 (OCH2CH2CH2OAc); 61.87 (OCH2CH2CH2OAc); 59.51 (C-5′);
28.89 (OCH2CH2CH2OAc); 21.02 (Me); 17.58, 17.50, 17.41, 17.32, 17.2, 17.06, 16.93, 13.54,
13.22, 12.99, 12.71 (iPr). LSI-MS: (C27H48N2O10Si2 + H+) 617.2926, calculated 617.2925.
1-{2-O-[(2-Acetoxypropoxy)m eth yl]-3,5-O-(1,1,3,3-tetraisopropyldisiloxan e-1,3-diyl)-
β-D-ribofuran osyl}-N4-ben zoylcytosin e (5b)
An alogous con den sation of diacetate
2 (0.761 g, 4.0 m m ol) in th e presen ce of tin
tetrach loride (0.36 m l, 3 m m ol) with n ucleoside 3b (1.180 g, 2.0 m m ol) in 1,2-dich loroeth an e
(15 m l) at –1 2 °C fo r 2 0 m in gave 5 b as a fo am . RF 0 .3 7 (A). Yield 1 .2 2 0 g (8 5 % ).
1 H N MR (CDCl3): 9.15 brs, 1 H (NH); 8.39 d, 1 H, J(6,5) = 7.5 (H-6); 7.94–7.52 m , 6 H (Bz,
H-5); 5.85 s, 1 H (H-1′); 5.09 d, 1 H, J = –6.5 (OCHHO); 4.96 d, 1 H (OCHHO); 4.31 d, 1 H,
J(5′a,5′b) = –12.8 (H-5′a); 4.27–4.17 m , 5 H (H-2′, H-3′, H-4′, OCH2CH2CH2OAc); 4.02 d, 1 H
(H-5′b); 3.77 m ,
2 H (OCH2CH2CH2OAc); 2.07 s, 3 H (Ac); 1.94 p, 2 H, J = 6.3
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 6, pp. 804–819

Synthesis of Oligoribonucleotides
813
(OCH2CH2CH2OAc); 1.11–0.98 m , 28 H (iPr). 13C NMR (CDCl3): 171.19 (C=O, Ac); 166.80
(C=O, Bz); 162.48 (C-4); 154.50 (C-2); 144.50 (C-6); 133.31, 129.15, 127.75 (Ph ); 96.23 (C-5);
94.77 (OCH2O); 90.46 (C-1′); 82.08 (C-4′); 77.94 (C-2′); 67.92 (C-3′); 64.95
(OCH2CH2CH2OAc);
61.95
(OCH2OCH2CH2CH2OAc);
59.54
(C-5′);
28.92
(OCH2OCH2CH2CH2OAc); 21.07 (Me); 17.60, 17.42, 17.14, 16.98, 13.52, 13.23, 13.02 (iPr).
LSI-MS: (C34H53N3O10Si2 + H+) 720.3341, calculated 720.3347.
1-{2-O-[(2-Acetoxyeth oxy)m eth yl]-β-D-ribofuran osyl}uracil (6a)
Nucleoside 4a (0.602 g, 1 m m ol) was dissolved in 0.5 M tetrabutylam m on ium fluoride
trih ydrate in tetrah ydrofuran (5 m l), kept at 20 °C for 10 m in , evaporated to dryn ess, evapo-
rated with ch loroform (10 m l) an d applied on to a colum n with silica gel (20 g). Th e colum n
was wash ed with m eth ylen e ch loride (100 m l), m eth ylen e ch loride–eth an ol 98:2 (200 m l),
m eth ylen e ch loride–eth an ol 96:4 (200 m l), an d th en eluted with m eth ylen e ch loride–
eth an ol 94:6 to give 6a as a foam . Yield 0.330 g (92%). RF 0.22 (B). 1H NMR (CDCl3): 8.69
brs, 1 H (NH); 7.74 d, 1 H, J(6,5) = 8.1 (H-6); 5.82 d, 1 H, J(1′,2′) = 4.5 (H-1′); 5.72 d, 1 H
(H-5); 4.91 d, 1 H, J = –6.7 (OCHHO); 4.87 d, 1 H (OCHHO); 4.42 dd, 1 H, J(2′,3′) = 5.0
(H-2′); 4.37 t, 1 H, J(3′,4′) = 5.0 (H-3′); 4.23 m , 2 H (OCH2CH2OAc); 4.10 dt, 1 H, J(4′,5′a) =
1.9, J(4′,5′b) = 1.9 (H-4′); 3.98 dd, 1 H, J(5′a,5′b) = –12.2 (H-5′a); 3.86 dd, 1 H (H-5′b); 3.82
m , 2 H (OCH2CH2OAc); 2.08 s, 3 H (Ac). 13C NMR (CDCl3): 171.10 (C=O); 164.00 (C-4);
150.31 (C-2); 141.68 (C-6); 102.64 (C-5); 96.01 (OCH2O); 90.29 (C-1′); 85.19 (C-4′); 80.30
(C-2′); 69.58 (C-3′); 66.98 (OCH2CH2OAc); 63.38 (OCH2CH2OAc); 61.62 (C-5′); 20.15 (Me).
LSI-MS: (C14H20N2O9 + H+) 361.1241, calculated 361.1246.
1-{2-O-[(2-Acetoxyeth oxy)m eth yl]-β-D-ribofuran osyl}-N4-ben zoylcytosin e (6b)
An alogous desilylation of 4b (706 m g, 1 m m ol) yielded 6b as a foam (407 m g, 88%). RF 0.25
(B). 1H NMR (CDCl3): 9.24 brs, 1 H (NH); 8.46 d, 1 H, J(6,5) = 7.5 (H-6); 7.91–7.47 m , 6 H
(Bz, H-5); 5.86 d, 1 H, J(1′,2′) = 2.2 (H-1′); 5.03 d, 1 H, J = –6.4 (OCHHO); 4.92 d, 1 H
(OCHHO); 4.44 dd, 1 H, J(2′,3′) = 4.5 (H-2′); 4.35 dd, 1 H, J(3′,4′) = 7.2 (H-3′); 4.19 m , 2 H
(OCH2CH2OAc); 4.13 ddd, 1 H, J(4′,5′a) = 1.9, J(4′,5′b) = 1.6 (H-4′); 4.06 dd, 1 H, J(5′a,5′b) =
–12.5 (H-5′a); 3.90 dd, 1 H (H-5′b); 3.86–3.75 m , 2 H (OCH2CH2OAc); 2.03 s, 3 H (Ac).
13C NMR (CDCl3): 171.21 (C=O, Ac); 167.62 (C=O, Bz); 162.61 (C-4); 154.53 (C-2); 146.44
(C-6); 133.25, 132.80, 128.95, 127.79 (Ph ); 96.91 (C-5); 95.65 (OCH2O); 91.54 (C-1′); 84.91
(C-4′); 80.13 (C-2′); 68.24 (C-3′); 66.66 (OCH2OCH2CH2OAc); 63.35 (OCH2OCH2CH2OAc);
60.48 (C-5′); 20.80 (Me). LSI-MS: (C21H25N3O9 + H+) 464.1661, calculated 464.1668.
1-{2-O-[(3-Acetoxypropoxy)m eth yl]-β-D-ribofuran osyl}uracil (7a)
An alogous desilylation of 5a (617 m g, 1 m m ol) yielded 7a as a foam (337 m g, 90%). RF 0.22
(B). 1H NMR (CDCl3): 9.34 brs, 1 H (NH); 7.89 d, 1 H, J(6,5) = 8.0 (H-6); 5.86 d, 1 H, J(1′,2′) =
3.1 (H-1′); 5.72 d, 1 H (H-5); 4.86 d, 1 H, J = –6.5 (OCHHO); 4.82 d, 1 H (OCHHO); 4.35 m ,
2 H (H-2′, H-3′); 4.15–4.08 m , 3 H (H-4′, OCH2CH2CH2OAc); 3.95 dd, 1 H, J(5′a,4′) = 2.2,
J(5′a,5′b)
= –12.3 (H-5′a); 3.86 dd, 1 H, J(5′b,4′) = 2.1 (H-5′b); 3.69–3.58 m , 2 H
(OCH2CH2CH2OAc); 2.08 s, 3 H (Ac); 1.86 p, 2 H, J = 6.3 (OCH2CH2CH2OAc). 13C NMR
(CDCl3): 171.25 (C=O); 163.48 (C-4); 150.51 (C-2); 141.57 (C-6); 102.49 (C-5); 95.61
(OCH2O); 89.67 (C-1′); 85.10 (C-4′); 79.78 (C-2′); 69.34 (C-3′); 65.50 (OCH2CH2CH2OAc);
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 6, pp. 804–819

814
Bobkov, Brilliantov, Mikhailov, Rozenski, Van Aerschot, Herdewijn:
61.58 (OCH2CH2CH2OAc); 59.29 (C-5′); 28.99 (OCH2CH2CH2OAc); 21.04 (Me). LSI-MS:
(C15H22N2O9 + H+) 375.1406, calculated 375.1403.
1-{2-O-[(3-Acetoxypropoxy)m eth yl]-β-D-ribofuran osyl}-N4-ben zoylcytosin e (7b)
An alogous desilylation of 5b (720 m g, 1 m m ol) yielded 7b as a foam (423 m g, 89%). RF 0.28
(B). 1H NMR (CDCl3): 9.17 brs, 1 H (NH); 8.45 d, 1 H, J(6,5) = 7.5 (H-6); 7.90–7.46 m , 6 H
(Bz, H-5); 5.87 d, 1 H, J(1′,2′) = 2.2 (H-1′); 4.99 d, 1 H, J = –6.5 (OCHHO); 4.88 d, 1 H
(OCHHO); 4.45 dd, 1 H, J(2′,3′) = 5.0 (H-2′); 4.36 dd, 1 H, J(3′,4′) = 6.5 (H-3′); 4.14–4.05 m ,
4 H (H-4′, H-5′a, OCH2CH2CH2OAc); 3.91 d, 1 H, J(5′a,5′b) = –12.1 (H-5′b); 3.71–3.56 m , 2 H
(OCH2CH2CH2OAc); 2.02 s, 3 H (Ac); 1.86 p, 2 H, J = 6.3 (OCH2CH2CH2OAc). 13C NMR
(CDCl3): 171.50 (C=O, Ac); 167.40 (C=O, Bz); 162.65 (C-4); 154.70 (C-2); 146.60 (C-6);
133.41, 133.01, 129.13, 127.91 (Ph ); 97.02 (C-5); 95.67 (OCH2O); 91.69 (C-1′); 85.17 (C-4′);
79.99 (C-2′); 68.52 (C-3′); 65.52 (OCH2OCH2CH2CH2OAc); 61.53 (OCH2OCH2CH2CH2OAc);
60.79 (C-5′); 29.01 (OCH2OCH2CH2CH2OAc); 21.04 (Me). LSI-MS: (C22H27N3O9 + H+)
478.1823, calculated 478.1825.
1-{2-O-[(2-Hydroxyeth oxy)m eth yl]-β-D-ribofuran osyl}uracil (8a)
A solution of n ucleoside 6a (360 m g, 1.0 m m ol) in 5 M am m on ia in m eth an ol (10 m l) was
kept at 20 °C for 3 days an d th en con cen trated to dryn ess in vacuo. Th e residue was
partion ed between m eth ylen e ch loride (10 m l) an d water (20 m l), an d th e water layer was
wash ed with m eth ylen e ch loride (2 × 10 m l). Th e aqueous layer was con cen trated to dry-
n ess, th e residue was dissolved in 5 m l of m eth an ol, th e solution evaporated to dryn ess in
vacuo to give 8a as a h ygroscopic foam (300 m g, 94%). RF 0.70 (C). UV (pH 1–7): λm ax 262
n m (ε 9600); (pH 13): λm ax 261 n m (ε 6900). 1H NMR (DMSO-d6): 11.38 brs, 1 H (NH); 7.91 d,
1 H, J(6,5) = 8.1 (H-6); 5.89 d, 1 H, J(1′,2′) = 5.3 (H-1′); 5.64 d, 1 H (H-5); 5.25 brs, 2 H
(OH-3′, OH-5′); 4.71 brs, 3 H (OCH2O, OH); 4.16 t, 1 H, J(2′,3′) = 5.3 (H-2′); 4.11 dd, 1 H,
J(3′,4′) = 3.3 (H-3′); 3.88 dt, 1 H, J(4′,5′a) = 3.0, J(4′,5′b) = 3.0 (H-4′); 3.63 dd, 1 H, J(5′a,5′b) =
–12.2 (H-5′a); 3.56 dd, 1 H (H-5′b); 3.51–3.42 m , 4 H (OCH2CH2OH). 13C NMR (DMSO-d6):
163.19 (C-4); 150.73 (C-2); 140.43 (C-6); 101.92 (C-5); 94.27 (OCH2O); 86.11 (C-1′); 85.29
(C-4′); 78.24 (C-2′); 69.48 (OCH2CH2OH); 68.75 (C-3′); 60.72 (C-5′); 60.06 (OCH2CH2OH).
LSI-MS: (C12H18N2O8 + H+) 319.1147, calculated 319.1141.
1-{2-O-[(2-Hydroxyeth oxy)m eth yl]-β-D-ribofuran osyl}cytosin e (8b)
An alogous deacylation of 6b (463 m g, 1.0 m m ol), after evaporation with m eth an ol yielded
8b as a h ygroscopic foam (295 m g, 93%). RF 0.60 (C). UV (pH 7–13): λm ax 271 n m (ε 8400);
(pH 1): λm ax 279 n m (ε 13100). 1H NMR (D2O): 7.96 d, 1 H, J(6,5) = 7.7 (H-6); 6.19 d, 1 H
(H-5); 6.15 d, 1 H, J(1′,2′) = 4.9 (H-1′); 5.02 d, 1 H, J = –7.2 (OCHHO); 4.98 d, 1 H (OCHHO);
4.49 dd, 1 H, J(2′,3′) = 5.4 (H-2′); 4.43 t, 1 H, J(3′,4′) = 5.4 (H-3′); 4.25 ddd, 1 H, J(4′,5′a) =
2.8, J(4′,5′b) = 4.4 (H-4′); 4.02 dd, 1 H, J(5′a,5′b) = –12.8 (H-5′a); 3.93 dd, 1 H (H-5′b);
3.83–3.72 m , 4 H (OCH2CH2OH). 13C NMR (DMSO-d6): 165.59 (C-4); 155.20 (C-2); 141.18
(C-6); 93.99 (C-5); 93.98 (OCH2O); 87.52 (C-1′); 84.33 (C-4′); 78.50 (C-2′); 69.43
(OCH2OCH2CH2OH); 68.31 (C-3′); 60.36 (C-5′); 60.13 (OCH2OCH2CH2OH). LSI-MS:
(C12H19N3O7 + H+) 318.1304, calculated 318.1301.
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 6, pp. 804–819

Synthesis of Oligoribonucleotides
815
1-{2-O-[(2-Hydroxypropoxy)m eth yl]-β-D-ribofuran osyl}uracil (9a)
A solution of n ucleoside 7a (374 m g, 1.0 m m ol) in 5 M am m on ia in m eth an ol (10 m l) was
kept at 20 °C for 3 days an d th en con cen trated to dryn ess in vacuo. Th e residue was
partion ed between m eth ylen e ch loride (10 m l) an d water (20 m l), an d th e water layer was
wash ed with m eth ylen e ch loride (2 × 10 m l). Th e aqueous layer was con cen trated to dry-
n ess, aceton e (5 m l) was added to th e residue an d, after 20 h at 20 °C, th e precipitate was
filtered off, wash ed with eth er an d dried to give 9a as a powder (267 m g, 80%). RF 0.70 (C).
UV (pH 1–7): λm ax 262 n m (ε 9700); (pH 13): λm ax 261 n m (ε 6800). 1H NMR (DMSO-d6):
11.21 brs, 1 H (NH); 7.89 d, 1 H, J(6,5) = 8.1 (H-6); 5.91 d, 1 H, J(1′,2′) = 5.6 (H-1′); 5.64 d, 1 H
(H-5); 5.06 d, 1 H, J(OH,3′) = 5.3 (OH-3′); 5.04 dd, 1 H, J(OH,5′a) = 4.9, J(OH,5′b) = 5.9
(OH-5′); 4.69 d, 1 H, J = –6.7 (OCHHO); 4.67 d, 1 H (OCHHO); 4.27 t, 1 H, J(OH,H) = 5.2
(OCH2CH2CH2OH); 4.15 dd, 1 H, J(2′,3′) = 5.0 (H-2′); 4.12 ddd, 1 H, J(3′,4′) = 3.6 (H-3′); 3.89
ddd, 1 H, J(4′,5′a) = 3.4, J(4′,5′b) = 2.7 (H-4′); 3.64 ddd, 1 H, J(5′a,5′b) = –11.9 (H-5′a); 3.56 ddd,
1 H (H-5′b); 3.60–3.37 m , 4 H (OCH2CH2CH2OH); 1.60 p, 2 H, J = 6.5 (OCH2CH2CH2OH).
13C NMR (DMSO-d6): 162.82 (C-4); 150.48 (C-2); 140.32 (C-6); 101.79 (C-5); 93.89 (OCH2O);
86.07 (C-1′); 85.27 (C-4′); 77.78 (C-2′); 68.63 (C-3′); 64.63 (OCH2CH2CH2OH); 60.70 (C-5′);
57.65 (OCH2CH2CH2OH); 32.42 (OCH2CH2CH2OH). LSI-MS: (C13H20N2O8 + H+) 333.1291,
calculated 333.1297.
1-{2-O-[(2-Hydroxypropoxy)m eth yl]-β-D-ribofuran osyl}cytosin e (9b)
An alogous deacylation of 7b (477 m g, 1 m m ol) as in th e case of 6a, after evaporation with
m eth an ol, yielded 9b as a h ygroscopic foam (301 m g, 91%). RF 0.60 (C). UV (pH 7–13): λm ax
271 n m (ε 8500); (pH 1): λm ax 279 n m (ε 13200). 1H NMR (D2O): 7.98 d, 1 H, J(6,5) = 7.7
(H-6); 6.24 d, 1 H (H-5); 6.20 d, 1 H, J(1′,2′) = 5.3 (H-1′); 5.00 d, 1 H, J = –7.0 (OCHHO);
4.97 d, 1 H (OCHHO); 4.51 dd, 1 H, J(2′,3′) = 5.6 (H-2′); 4.47 dd, 1 H, J(3′,4′) = 4.7 (H-3′);
4.30 ddd, 1 H, J(4′,5′a) = 3.1, J(4′,5′b) = 4.4 (H-4′); 4.04 dd, 1 H, J(5′a,5′b) = –12.6 (H-5′a);
3.96 dd,
1 H (H-5′b); 3.79–3.72 m , 4 H (OCH2CH2CH2OH); 1.89 p, 2 H, J = 6.6
(OCH2CH2CH2OH). 13C NMR (DMSO-d6): 165.59 (C-4); 155.22 (C-2); 141.19 (C-6); 94.03
(C-5); 93.66 (OCH2O); 87.50 (C-1′); 84.42 (C-4′); 78.04 (C-2′); 68.31 (C-3′); 64.44
(OCH2OCH2CH2CH2OH);
60.43
(C-5′);
57.72
(OCH2OCH2CH2CH2OH);
32.54
(OCH2OCH2CH2CH2OH). LSI-MS: (C13H21N3O7 + H+) 332.1451, calculated 332.1457.
1-{2-O-[(2-Acetoxyeth oxy)m eth yl]-5-O-(4-m eth oxytrityl)-β-D-ribofuran osyl}uracil (10a)
Nucleoside 6a (0.676 g, 1.87 m m ol) was dried by evaporation with pyridin e (2 × 10 m l). Th e
residue was dissolved in dry pyridin e (15 m l), m on om eth oxytrityl ch loride (0.648 g, 2.10
m m ol) was added an d th e resultin g solution was kept in th e dark at 20 °C for 16 h . Th en
MeOH (0.5 m l) was added an d after 30 m in th e m ixture was con cen trated alm ost to dryn ess
in vacuo. Th e residue was dissolved in m eth ylen e ch loride (50 m l), wash ed with 10% aque-
ous solution of sodium h ydrogen carbon ate (20 m l) an d water (2 × 20 m l). Th e organ ic layer
was dried over an h ydrous Na2SO4, evaporated in vacuo, evaporated with toluen e (2 × 10 m l)
an d purified by colum n ch rom atograph y on silica gel (30 g). Th e colum n was wash ed with
m eth ylen e ch loride (200 m l), m eth ylen e ch loride–eth an ol 99:1 (200 m l) an d th en eluted
with m eth ylen e ch loride–eth an ol 97:3 to give 10a as a foam . Yield 0.832 g (70%). RF 0.33
(A). 1H NMR (DMSO-d6): 11.41 brs, 1 H (NH); 7.75 d, 1 H, J(6,5) = 8.1 (H-6); 7.40–7.22 m , 12 H
(Ph ); 6.91 d, 1 H, J = 8.8 (Ph OMe); 5.85 d, 1 H, J(1′,2′) = 3.4 (H-1′); 5.85 d, 1 H, J(OH,3′) =
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816
Bobkov, Brilliantov, Mikhailov, Rozenski, Van Aerschot, Herdewijn:
5.9 (OH-3′); 5.31 dd, 1 H, J(5,NH) = 2.2 (H-5); 4.79 d, 1 H, J = –7.2 (OCHHO); 4.77 d, 1 H
(OCHHO); 4.27–4.24 m , 2 H (H-2′, H-3′); 4.12–3.97 m , 2 H (OCH2CH2OAc); 4.09 m , 1 H
(H-4′); 3.74 s, 3 H (OMe); 3.69 m , 2 H (OCH2CH2OAc); 3.29 dd, 1 H, J(5′a,4′) = 4.4, J(5′a,5′b) =
–10.5 (H-5′a); 3.21 dd, 1 H, J(5′b,4′) = 2.2 (H-5′b); 1.97 s, 1 H (Ac). 13C NMR (DMSO-d6):
170.17 (C=O); 162.76 (C-4); 158.22 (Ph OMe); 150.26 (C-2); 143.79, 143.53 (Ph ); 140.36
(C-6); 134.35, 129.87, 127.71, 127.70, 126.97 (Ph ); 113.31 (Ph OMe); 101.32 (C5); 94.10
(OCH2O); 87.13 (C-1′); 86.10 (Ph 3C); 82.90 (C-4′); 77.43 (C-2′); 68.38 (C-3′); 65.18
(OCH2CH2OAc); 63.09 (OCH2CH2OAc); 61.58 (C-5′); 54.87 (OMe); 20.59 (Me, Ac).
1-{2-O-[(2-Acetoxyeth oxy)m eth yl]-5-O-(4-m eth oxytrityl)-β-D-ribofuran osyl}-
N4-ben zoylcytosin e (10b)
An alogous tritylation of n ucleoside 6b (463 m g, 1 m m ol) yielded 10b as a foam (589 m g,
80%). RF 0.33 (A). 1H NMR (DMSO-d6): 11.24 brs, 1 H (NH); 8.32 d, 1 H, J(6,5) = 7.5 (H-6);
8.02–7.28 m , 17 H (Bz, Tr); 7.16 brd, 1 H (H-5); 6.94 d, 2 H, J = 8.1 (Ph OMe); 5.89 d, 1 H,
J(1′,2′) = 1.6 (H-1′); 5.35 brs, 1 H (OH); 4.97 d, 1 H, J = –6.7 (OCHHO); 4.87 d, 1 H
(OCHHO); 4.36 dd, 1 H, J(3′,2′) = 5.0, J(3′,4′) = 8.1 (H-3′); 4.21 dd, 1 H (H-2′); 4.15 t, 2 H, J =
4.7 (OCH2CH2OAc); 4.09 ddd, 1 H, J(4′,5a′) = 3.4, J(4′,5b′) = 2.7 (H-4′); 3.79 m , 2 H
(OCH2CH2OAc); 3.76 s, 3 H (OMe); 3.42 dd, 1 H, J(5′a,5′b) = –11.2 (H-5′a); 3.35 dd, 1 H
(H-5′b); 1.99 s, 3 H (Ac). 13C NMR (DMSO-d6): 170.24 (C=O, Ac); 167.65 (C=O, Bz); 163.11
(C-4); 158.27 (Ph OMe); 154.73 (C-2); 144.43 (C-6); 143.96, 143.68, 134.83, 132.65, 129.94,
128.37, 128.09, 127.79 (Ph ); 113.30 (Ph OMe); 96.12 (C-5); 93.69 (OCH2O); 89.30 (C-1′);
86.26 (Ph 3C); 82.02 (C-4′); 78.31 (C-2′); 67.86 (C-3′); 65.20 (OCH2OCH2CH2OAc); 63.09
(OCH2OCH2CH2OAc); 61.85 (C-5′); 54.98 (OMe); 20.57 (Me, Ac).
1-{2-O-[(3-Acetoxypropoxy)m eth yl]-5-O-(4-m eth oxytrityl)-β-D-ribofuran osyl}uracil (11a)
An alogous tritylation of n ucleoside 7a (375 m g, 1 m m ol) yielded 11a as a foam (498 m g,
77%). RF 0.33 (A). 1H NMR (CDCl3): 8.47 brs, 1 H (NH); 7.94 d, 1 H, J(6,5) = 8.1 (H-6);
7.52–7.34 m , 12 H (Ph ); 6.86 d, 2 H, J = 8.7 (Ph OMe); 5.99 d, 1 H, J(1′,2′) = 2.8 (H-1′); 5.31 dd,
1 H, J(5,NH) = 1.9 (H-5); 4.98 d, 1 H, J = –6.7 (OCHHO); 4.87 d, 1 H (OCHHO); 4.48 dt, 1 H,
J(3′,2′) = 5.6, J(3′,OH) = 7.5, J(3′,4′) = 5.6 (H-3′); 4.29 dd, 1
H (H-2′); 4.16 m , 2 H
(OCH2CH2CH2OAc); 4.05 dt, 1 H, J(4′,5a′) = 2.2, J(4′,5b′) = 2.2 (H-4′); 3.80 s, 3 H (OMe);
3.72 m , 1 H (OCHHCH2CH2OAc); 3.62 m , 1 H (OCHHCH2CH2OAc); 3.54 d, 2 H (H-5′a,
H-5′b); 2.72 d, 1 H (OH-3′); 2.04 d, 3 H (Ac); 1.92 p, 2 H, J = 6.2 (OCH2CH2CH2OAc). 13C NMR
(DMSO-d6): 170.17 (C=O); 162.76 (C-4); 158.22 (Ph OMe); 150.26 (C-2); 143.99, 143.73 (Ph );
140.11 (C-6); 134.55, 129.97, 127.91, 127.80, 126.87 (Ph ); 113.21 (Ph OMe); 101.52 (C-5);
94.00 (OCH2O); 87.03 (C-1′); 86.20 (Ph 3C); 82.80 (C-4′); 77.53 (C-2′); 68.58 (C-3′); 64.18
(OCH2CH2CH2OAc); 62.88 (C-5′); 60.98 (OCH2CH2CH2OAc); 54.97 (OMe); 28.40
(OCH2CH2CH2OAc); 20.49 (Me, Ac).
1-{2-O-[(3-Acetoxypropoxy)m eth yl]-5-O-(4-m eth oxytrityl)-β-D-ribofuran osyl}-
N4-ben zoylcytosin e (11b)
An alogous tritylation of n ucleoside 7b (477 m g, 1 m m ol) yielded 11b as a foam (578 m g,
77%). RF 0.33 (A). 1H NMR (DMSO-d6): 11.23 brs, 1 H (NH); 8.33 d, 1 H, J(6,5) = 7.6 (H-6);
8.02–7.28 m , 17 H (Bz, Tr); 7.15 brd, 1 H (H-5); 6.93 d, 2 H, J = 9.0 (Ph OMe); 5.90 s, 1 H
(H-1′); 5.32 brs, 1 H (OH-3′); 4.93 d, 1 H, J = –6.5 (OCHHO); 4.83 d, 1 H (OCHHO); 4.35 dd,
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Synthesis of Oligoribonucleotides
817
1 H, J(3′,2′) = 5.6, J(3′,4′) = 6.8 (H-3′); 4.21 d, 1 H (H-2′); 4.10 ddd, 1 H, J(4′,5a′) = 3.3,
J(4′,5b′) = 2.7 (H-4′); 4.05 t, 2 H, J = 6.5 (OCH2CH2CH2OAc); 3.76 s, 3 H (OMe); 3.69–3.57
m , 2 H (OCH2CH2CH2OAc); 3.41 dd, 1 H, J(5′a,5′b) = –10.9 (H-5′a); 3.35 dd, 1 H (H-5′b);
1.97 s, 3 H (Ac); 1.82 p, 2 H, J = 6.4 (OCH2CH2CH2OAc). 13C NMR (DMSO-d6): 170.27 (C=O,
Ac); 167.21 (C=O, Bz); 162.84 (C-4); 158.27 (Ph OMe); 154.81 (C-2); 144.54 (C-6); 143.96,
143.68, 134.82, 132.64, 129.95, 128.37, 128.07, 127.94, 127.00 (Ph ); 113.30 (Ph OMe); 96.14
(C-5); 93.74 (OCH2O); 89.16 (C-1′); 86.28 (Ph 3C); 82.09 (C-4′); 78.25 (C-2′); 67.90 (C-3′);
64.07 (OCH2OCH2CH2CH2OAc); 61.90 (C-5′); 61.15 (OCH2OCH2CH2CH2OAc); 54.98 (OMe);
28.42 (OCH2OCH2CH2CH2OAc); 20.59 (Me, Ac).
2′-O-[(2-Acetoxyeth oxy)m eth yl]-3′-O-[(2-cyan oeth yl)(diisopropylam in o)ph osph an yl]-
5′-O-(4-m eth oxytrityl)uridin e (12a)
Th e m eth oxytritylated derivative 10a (450 m g, 0.71 m m ol) was dissolved in 6 m l dich loro-
m eth an e un der argon , an d eth yl(diisopropyl)am in e (371 µl, 2.13 m m ol) an d 2-cyan oeth yl
diisopropylph osph oram idoch loridite (285 µl, 1.28 m m ol) were added an d, after stirrin g th e
solution for 2 h , TLC in dicated com plete reaction . A 10% aqueous solution of sodium
h ydrogen carbon ate (2 m l) was added, th e solution was stirred for 10 m in an d partition ed
between CH2Cl2 (50 m l) an d aqueous NaHCO3 (30 m l). Th e organ ic ph ase was wash ed with
aqueous sodium ch loride (2 × 30 m l) an d th e aqueous ph ases were back extracted with
CH2Cl2 (20 m l). Evaporation of th e organ ics left an oil wh ich was flash -purified on 40 g of
silica gel (h exan e–aceton e–TEA, 64:35:1) to afford th e product as a foam after coevaporation
with dich lorom eth an e. Th e foam was dissolved in 2 m l of dich lorom eth an e an d precipitated
in 150 m l cold (–70 °C) h exan e–diisopropyl eth er (9:1) to afford 373 m g (0.45 m m ol, 63%)
of th e title product as a wh ite powder. RF 0.37 (D). 31P NMR (CDCl3): 151.21, 150.35.
LSI-MS: (C43H53N4O11P1 + H+) 833.3517, calculated 833.3526.
An alogously to th e previous procedure th e followin g am idites were prepared:
2 ′-O-[(3 -Acetoxypropoxy)m eth yl]-3 ′-O-[(2 -cya n oeth yl)(diisopropyla m in o)ph osph a n yl]-
5′-O-(4-methoxytrityl)uridine (13a). Startin g from com poun d 11a (0.62 m m ol, 398 m g), 377 m g
of th e required am idite was obtain ed (0.44 m m ol, 72%). RF 0.41 (D). 31P NMR (CDCl3):
150.43, 150.14. LSI-MS: (C44H55N4O11P1 + H+) 847.3697, calculated 847.3682.
2 ′-O-[(2 -Acetoxyeth oxy)m eth yl]-N4 -ben zoyl-3 ′-O-[(2 -cya n oeth yl)(diisopropyla m in o)-
phosphanyl]-5′-O-(4-methoxytrityl)cytidine (12b). Startin g with com poun d 10b (507 m g, 0.69
m m ol), 523 m g of th e required am idite was obtain ed (0.56 m m ol, 81%). RF 0.43 (D). 31P NMR
(CDCl3): 151.40, 150.13. LSI-MS: (C50H58N5O11P1 + H+) 936.3932, calculated 936.3948.
2 ′-O-[(3 -Acetoxypropoxy)m eth yl]-N4 -ben zoyl-3 ′-O-[(2 -cya n oeth yl)(diisopropyla m in o)-
phosphanyl]-5′-O-(4-methoxytrityl)cytidine (13b). Startin g with com poun d 11b (603 m g, 0.80
m m ol), 458 m g of th e required am idite was obtain ed (0.48 m m ol, 60%). RF 0.40 (D). 31P NMR
(CDCl3): 151.40, 150.13. LSI-MS: (C51H60N5O11P1 + H+) 950.4117, calculated 950.4104.
Syn th esis an d An alysis of Oligon ucleotides
Oligon ucleotide assem bly was perform ed on an Expedite™ DNA syn th esizer (Applied
Biosystem s) usin g th e ph osph oram idite approach . Th e stan dard RNA assem bly protocol was
used with a 10 m in couplin g tim e, usin g 0.07
M of th e n ewly syn th esized un n atural
am idites an d 0.06 M of th e TOM am idites with 0.25 M 5-(eth ylsulfan yl)tetrazole (ETT) as th e
activator. Th e oligom ers were deprotected an d cleaved from th e solid support by treatm en t
with a 1:1 m ixture of 40% aqueous m eth ylam in e an d 8 M m eth ylam in e in eth an ol at 35 °C
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 6, pp. 804–819

818
Bobkov, Brilliantov, Mikhailov, Rozenski, Van Aerschot, Herdewijn:
for 6 h . Th e supern atan t was lyoph ilized an d th e residue was treated with 1 m l of a 1
M
TBAF solution in THF at 55 °C for 10 m in an d at room tem perature for 24 h . Th e m ixture
was n eutralized with 1 m l of a 1 M solution of Tris-HCl, pH 7.4, sligh tly con cen trated an d
desalted on a NAP-25® colum n (Seph adex G25-DNA grade; Ph arm acia). Th e crude product
was an alyzed on a Mon o-Q® HR 5/5 an ion exch an ge colum n , th en purified on a Mon o-Q®
HR 10/10 colum n (Ph arm acia) with th e followin g gradien t system (A = 10 m M NaClO4; B =
600 m M NaClO4, both in aqueous 20 m M Tris-HCl, 15% CH3CN, 0.1 m M EDTA). Th e
low-pressure liquid ch rom atograph y system con sisted of a Merck–Hitach i L 6200 A in telli-
gen t pum p, a Uvicord SII 2138 UV detector (Ph arm acia-LKB) an d a recorder. Th e product-
con tain in g fraction was desalted on a NAP-25® colum n an d lyoph ilized.
Oligon ucleotides were ch aracterized an d th eir purity was ch ecked by HPLC/MS on a capil-
lary ch rom atograph (CapLC, Waters, Milford, MA, U.S.A.). Colum n s of 150 m m × 0.3 m m
len gth (LCPackin gs, San Fran cisco, CA, U.S.A.) were used. Oligon ucleotides were eluted with
an aceton itrile gradien t in 50 m M trieth ylam m on ium adjusted to pH 8.0 with
1,1,1,3,3,3-h exafluoropropan -2-ol. Th e flow rate was 5 µl/m in . Electrospray spectra were ac-
quired on an orth ogon al acceleration /tim e-of-fligh t m ass spectrom eter (Q-Tof-2, Microm ass,
Man ch ester, U.K.) in th e n egative ion m ode. Th e scan tim e used was 2 s. Th e com bin ed
spectra from a ch rom atograph ic peak were decon voluted usin g th e MaxEn t algorith m of th e
software (Masslyn x 3.4, Microm ass, Man ch ester, U.K.). Th eoretical oligon ucleotide m olecular
weigh ts were calculated usin g th e m on oisotopic atom ic weigh ts.
Meltin g Tem peratures
Oligom ers were dissolved in 0.1 M NaCl, 0.02 M potassium ph osph ate, pH 7.5, 0.1 m M EDTA.
Th e con cen tration was determ in ed by m easurin g th e absorban ce in MilliQ water at 260 n m
an d 80 °C, an d assum in g th e m odified n ucleoside an alogues to h ave th e sam e m olar adsorp-
tion coefficien ts per base m oiety in th e den atured state as th e n atural n ucleosides (C* ε =
7100; U* ε = 9600). Th e con cen tration in all experim en ts was 4 µM for each stran d. Meltin g
curves were determ in ed with a Varian Cary 300 BIO spectroph otom eter. Cuvettes were
m ain tain ed at con stan t tem perature by m ean s of water circulation th rough th e cuvette
h older. Th e tem perature of th e solution was m easured with a th erm istor directly im m ersed
in th e cuvette. Tem perature con trol an d data acquisition were perform ed autom atically with
an IBM-com patible com puter usin g Cary Win UV th erm al application software. Followin g
a fast h eatin g an d coolin g cycle to allow proper an n ealin g of both stran ds, th e sam ples were
h eated at a rate of 0.2 °C/m in startin g at 10 °C up to 80 °C an d coolin g again at th e sam e
rate. Meltin g tem peratures were determ in ed by plottin g th e first derivative of th e absorban ce
versus tem perature curve an d are th e average of two run s. Heatin g an d coolin g curves were
m easured two tim es at two wavelen gth s 260 an d 270 n m an d in gen eral sh owed th e sam e
Tm values.
The authors thank V. V. Novikov for NMR measurements and G. Schepers for the synthesis of
oligonucleotides and Tm determination. Financial support of the Russian Foundation for Basic
Research, Programme “Molecular and Cellar Biology”, INTAS and KUL Research Council is
acknowledged.
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Synthesis of Oligoribonucleotides
819
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