Synthesis and properties of O-β-D-ribofuranosyl-(1″ → 2′)-guanosine-5″-O-phosphate and its derivatives

Article abstract of DOI:10.1002/hlca.200390050

The efficient synthesis of O-β-D-ribofuranosyl-(1″ → 2′)-guanosine-5″-O-phosphate and O-β-D-ribofuranosyl-(1″ → 2′)-adenosine-5″-O-phosphate, minor tRNA components, have been developed, and their conformational properties were examined by NMR spectroscopy.

Full text of DOI:10.1002/hlca.200390050

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Helvetica Chimica Acta Vol. 86 (2003)
Synthesis and Properties of O-b-d-ribofuranosyl-(1'' ! 2')-guanosine-5''-
O-phosphate and Its Derivatives
by Ekaterina V.Efimtseva a), Alexandra A.Shelkunova a), Sergey N.Mikhailov a), Koen Nauwelaertsb),
Jef Rozenskib), Eveline Lescrinierb), and Piet Herdewijn*b)
a) Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov str. 32, Moscow,
119991 Russia (Phone: ‡7-095-1359704; fax: ‡7-095-1351405; e-mail: smikh@eimb.ru)
b) Rega Institute, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven
(Phone: ‡32-16-337387, fax: ‡32-16337340, e-mail: Piet.Herdewijn@rega.kuleuven.ac.be)
Dedicated to Professor Wolfgang Pfleiderer on the occasion of his 75th birthday
The efficient synthesis of O-b-d-ribofuranosyl-(1'' ! 2')-guanosine-5''-O-phosphate and O-b-d-ribofura-
nosyl-(1'' ! 2')-adenosine-5''-O-phosphate, minor tRNA components, have been developed, and their
conformational properties were examined by NMR spectroscopy.
1.Introduction. Nucleic acids consist of over 100 modified nucleosides, in addition
to eight major ribo- and deoxyribonucleosides. Although a few modifications have been
found in DNA, 81 modified nucleosides were found in tRNAs and 15 were found in
other RNAs [1][2]. Most of the minor ribonucleosides have modified heterocyclic
bases. Only two types of sugar-modified nucleosides were found in RNA, namely 2'-O-
Me and 2'-O-b-d-ribofuranosyl derivatives. The last nucleotides O-b-d-ribofuranosyl-
(1'' ! 2')-guanosine (and adenosine)-5''-O-phosphate (Grp and Arp) were isolated
from initiator tRNAs of yeasts [3 5]. It was shown that this modification controlled
the discrimination between their initiator vs. elongator function [4]. It should be
mentioned that the charged components are usually located in tRNA loops, but Grp
and Arp are located in position 64 of the stem region of T loop [6]. X-Ray analysis
demonstrated that the additional phosphorylated ribofuranose moiety is located in the
minor groove so that the 5''-phosphoryl group interacts with the 2-amino group of the
neighboring guanosine residue [7]. The same position of the additional ribose moiety
was found in solution of the self-complementary decaribonucleotide
5'r(GCGA*AUUCGC)-3' containing 2'-O-b-d-ribofuranosyl-adenosine by NMR
spectroscopy [8]. It was also shown that this modification has no effect on the thermal
stability of the duplex [8].
2.Results and Discussion.
We have developed a general method for the
preparation of disaccharide nucleosides [9 13]. Recently, 2'-O-b-d-ribofuranosyl-
adenosine was prepared by condensation of N6,3',5'-O-protected adenosine 1b with an
excess of 1-O-acetyl-2,3,5-tri-O-benzoyl-b-d-ribofuranose in the presence of SnCl4 and
then converted into O-b-d-ribofuranosyl-(1'' ! 2')-adenosine-5''-O-phosphate (7b) in
overall yield of 9% [14][15] (Scheme). The selective blocking of five OH groups in

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505
Scheme
a) SnCl4/ClCH2CH2Cl, 08. b) 0.1m K2CO3 in MeOH. c) bis[2-(4-nitrophenyl)ethyl]phosphate/TPSCl/1-methyl-
1H-imidazole/Py. d) Bu4NF/THF. e) DBU/Py. f) NH3/MeOH.
2'-O-b-d-ribofuranosyl-nucleosides is an essential strategy in the preparation of the
title compound. Previously, the per-O-benzoylated disaccharide nucleoside was fully
deprotected, tritylated at the 5''-position, acetylated, and detritylated, and the 5''-OH
group (as in 4) of the additional ribose residue was further phosphorylated [15]. This
protocol is rather lengthy, but it can be shortened and simplified, when differentiatively
blocked carbohydrates are utilized for O-glycosylation. Here, we present our results on
the preparation of O-b-d-ribofuranosyl-(1'' ! 2')-guanosine-5''-O-phosphate (7a). To
compare both methods and to develop efficient preparation of suitable synthons for
oligonucleotide synthesis, the corresponding adenosine derivative was also prepared.
Our attempts to use 1,2,3-tri-O-benzoyl-d-ribofuranose and its 5-O-monomethox-
ytrityl derivative [16] resulted in a complex mixture with the formation of the desired
disaccharide product in very low yield. The same results were obtained with 1,2,3-tri-O-

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acetyl-5-O-bis[2-(4-nitrophenyl)ethyl]phosphorylyl-d-ribofuranose [17]. It should be
mentioned that this synthon was used for the preparation of 5'-nucleotides under
conditions developed by Vorbruggen [18] and for glycosylation of secondary OH
groups in 2'-deoxynucleosides [19]. Therefore, we investigated the use of another
synthon 2 for the glycosylation reaction of the hindered secondary OH group in
nucleoside 1, i.e., the use of a 5-O-phenoxyacetyl group, which is 300 times less stable
under alkaline conditions than the Bz group [20][21].
For the preparation of 2, readily available in three steps from d-ribose, 1,2,3-tri-O-
benzoyl-d-ribofuranose [16] was chosen as a starting compound. Anomers of 2, which
may be easily separated on silica-gel column, were used for the next glycosylation step
under standard conditions [9 13]. The O-glycosylation reaction proceed stereospecifi-
cally with the formation of b-anomers 3a,b, in which the coupling constants (J(1',2')) in
the additional ribofuranose moiety are less than 0.5 Hz. Selective deblocking of the
phenoxyacetyl group was achieved with 0.1m K2CO3 in MeOH. The overall yields for
the preparation of 4a,b were 46 38%, higher then the earlier multistep conversion
mentioned above (26%) [15]. For the protection of the additional phosphate residue,
the 2-(4-nitrophenyl)ethyl (npe) group [22][23] was chosen. Phosphorylation with
bis[2-(4-nitrophenyl)ethyl]phosphate in the presence of 2,4,6-triisopropylbenzenesul-
fonyl chloride (TPSCl) and 1-methyl-1H-imidazole gave phosphotriesters 5a,b in good
yields. After desilylation [15], 6a,b were converted via its dimethoxytrityl derivatives to
the corresponding phosphoramidites. Their incorporation into oligonucleotides will be
published shortly. Subsequent cleavage of the npe groups in triester 6a with 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU) [22][23] in dry pyridine followed by deacyla-
tion with NH3 in MeOH gave free monophosphate 7a in high overall yield (18%
starting from 1a). The structures of the compounds were supported by NMR
spectroscopy and mass spectrometry. The chemical shifts were assigned by double-
resonance techniques and COSY experiments. It should be mentioned that the
1H-NMR spectra of the compouds obtained are rather complicated due to the presence
of two ribofuranose residues and diastereotopic protons of the npe (CH2)2 group in
phosphates 5a,b and 6a,b. Several conclusions were drawn from the 1H-NMR analysis.
In disaccharides nucleosides 3a,b and 4a,b with the 1,1,3,3-tetraisopropyldisiloxane-1,3-
diyl group, the coupling constants J(1',2') of both ribose and nucleoside moieties are
less than 0.5 Hz.
The removal of the silyl group and introduction of a bulky substituent at the 5'-O-
position of the additional ribofuranosyl residue resulted in significant conformational
changes. The presence of bis[2-(4-nitrophenyl)ethyl]phosphoryl groups in disacchar-
ides 5a,b and 6a,b increases the coupling constant J(1',2') in the ribofuranosyl residue
up to 1.9 Hz. In 13C-NMR spectra of these derivatives, the vicinal coupling constants
J(C(4),P) are larger than the geminal J(C(5),P) ones [24].
The conformation of a five-membered ring can be completely described by two
independent parameters: phase angle and puckering amplitude. Since the puckering
amplitude always remains within a narrow range and the pseudorotational angle can
adopt values ranging from 0 to 360 degrees, the different conformations are mostly
visualized by using the pseudorotational wheel.
It is widely accepted that furanose rings, which are flexible entities, will show a high-
speed equilibrium between two low-energy conformations in solution. One of those

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507
conformations is situated in the northern hemisphere of the pseudorotational wheel
(the so called N conformer), and the other in the southern hemisphere (the S
conformer) (Fig. 1). A complete description of this system now requires five
independent parameters: four parameters describing the conformers and one
parameter describing the fraction of one of the conformers.
Fig. 1. N and S conformations of a ribose
The two conformers and their relative abundance can be resolved by NMR
3
spectroscopy, through measurement of the J(H,H) coupling constants. Because the
equilibrium, as mentioned above, can be considered as fast on the NMR time-scale,
average coupling constants and average chemical shifts will be observed. Assuming the
two-state model described above, it is possible to find the conformations and their
3
populations that best fit the experimentally observed J(H,H) coupling constants
[25][26].
A 2D-TOCSY experiment [27][28] was performed for the assignment of all signals
(Table 1). The presence of a phosphate group was confirmed by 31P-NMR spectroscopy,
and its correct attachment to the molecule was evident from a characteristic downfield
shift of HaÀC(5') and HbÀC(5') of the ribose moiety and their couplings with the P-
atom (J(H,P)). The correct glycosidic linkage of C(1') of the ribose moiety with C(2')
of the nucleoside moiety could be confirmed by a NOE contact between HÀC(1') of
the ribose and HÀC(2') of the nucleoside.
A high-resolution 1D 1H-NMR spectrum was used to measure the coupling
3
constants. The J(H,H) coupling constants of both ribose moieties, determined from
spectra recorded at 292 K, are presented in Table 2. Analysis of these data with
Pseurot 6.2[29] generated the results shown in Table 3.
It was shown that the additional ribose moiety of 7a will appear predominantly in
its N conformation, which we could expect because of stereoelectronic effects due to
the 2'-OH group [30]. The ribose ring of the nucleoside shows a remarkably high
percentage of S conformer (58%), where the large group that is attached to C(2')
occurs in an energetically favorable equatorial-like position. This high percentage of
the S conformer can also be explained by the syn-orientation about the N-glycosidic
bond, which is preferred by guanosine nucleosides, and which drives the equilibrium
towards the S conformer due to steric and stereoelectronic effects and intramolecular
H-bond formation [31].
To compare the structural characteristics of Guo2'Rib5'p (7a) with Ade2'Rib5'p
(7b), described earlier by Rodionov et al. [15], we performed a Pseurot 6.2analysis

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Table 1. Measured Chemical Shifts [ppm] in the Ribose and Nucleoside Moieties of 7a and 7b
Grp (7a)
Nucleoside moiety
Arp (7b)
Nucleoside moiety
Nucleus
Ribose moiety
Ribose moiety
HÀC(1')
HÀC(2')
HÀC(3')
HÀC(4')
HaÀC(5')
HbÀC(5')
HÀC(8)
HÀC(2)
5.02( d)
6.01 (d)
5.02( d)
6.25 (d)
4.93 (dd)
4.63 (dd)
4.32( ddd)
3.90 (dd)
3.87 (dd)
8.42( s)
4.11 (dd)
4.18 (dd)
3.94 (ddd)
3.76 (ddd)
3.56 (ddd)
4.72( dd)
4.48 (dd)
4.10 (ddd)
3.81 (dd)
3.73 (dd)
7.93 (s)
4.20 (dd)
4.24 (dd)
3.99 (ddd)
3.73 (ddd)
3.54 (ddd)
8.28 (s)
C(1')
C(2')
C(3')
C(4')
C(5')
C(2)
C(4)
C(5)
C(6)
C(8)
108.50 (s)
75.28 (s)
88.01 (s)
80.40 (s)
70.13 (s)
85.63 (s)
61.90 (s)
154.58 (s)
152.03 (s)
117.39 (s)
159.80 (s)
139.61 (s)
108.20 (s)
75.20 (s)
88.40 (s)
80.20 (s)
70.50 (s)
86.70 (s)
62.68 (s)
156.54 (s)
149.15 (s)
119.99 (s)
153.31 (s)
142.6 (s)
71.80 (s)
71.50 (s)
82.89 (d)a)
65.88 (d)b)
82.67 (d)c)
65.70 (d)d)
P
1.98
1.93
a) J(C(4'),P) ˆ 6.4 Hz. b) J(C(5'),P) ˆ 5.1 Hz. c) J(C(4'),P) ˆ 6.7 Hz. d) J(C(5'),P) ˆ 5.0 Hz.
Table 2. Measured Coupling Constants [Hz] in the Ribose and Nucleoside Moieties of 7a and 7b
Grp (7a)
Nucleoside moiety
Arp (7b)
Nucleoside moiety
Ribose moiety
Ribose moiety
J(HÀC(1'),HÀC(2'))
J(HÀC(2'),HÀC(3'))
J(HÀC(3'),HÀC(4'))
J(HÀC(4'),HaÀC(5'))
J(HÀC(4'),HbÀC(5'))
J(HaÀC(5'),HbÀC(5'))
J(P,HaÀC(5'))
1.5
4.6
6.7
4.3
5.7
5.4
5.0
4.8
2.7
4.3
1.4
4.6
6.6
4.3
5.8
6.1
5.2
3.4
2.6
3.5
À 12.9
À 12.1
À 12.8
À 11.6
6.4
6.3
J(P,HbÀC(5'))
7.0
7.0
of the 3J(H,H) couplings of the latter compound. Results in Table 2 show that
the substitution of the adenine by a guanine drives the conformational equilibrium
of the nucleoside moiety towards the N conformer. A comparison of the ribose
moieties in both compounds showed only minor differences. It seems that the
substitution of the adenine by a guanine has no effects on the conformation of the
ribose moiety.
To probe the O- and N-glycosidic-bond populations, NOESY spectra were recorded
with 150, 300, and 500 ms mixing times respectively. No relevant data could be
obtained, most likely due to fast rotation around these bonds in a fast equilibrium

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Table 3. Ring Conformers of the Ribose and Nucleoside Moieties of 7a and 7b
Guo2'Rib5'p (7a)
Ribose moiety Nucleoside moiety
Ade2'Rib5'p (7b)
Ribose moiety
Nucleoside moiety
a
PN
)
2.8
25
4.6
27
Nb)
3T2
3E
3T2
3E
c
XN
)
0.820.42
178
2T3
0.18
0.051
0.80
0.36
a
PS
)
154
2E
0.58
0.095
177
2T3
0.20
0.054
156
2E
0.64
0.094
Sb)
c
XS
)
RMSDd)
a) Phase angle of pseudorotation [degrees]. b) Type of conformer. c) Molar fraction of the conformer [%].
d) Root Mean Square Deviation. The puckering amplitude was kept 38 degrees in all cases.
between several low-energy conformations in solution. This was confirmed by a Monte
Carlo conformational search, performed with Macromodel 5.0, which showed the
existence of two equally populated, low-energy conformations about the N-glycosidic
bond, consistent with the syn- and anti-conformation of the guanine moiety (Fig. 2).
About the O-glycosidic bond, three low-energy conformations were seen. (‡ g: 30%,
À g: 26%, t: 44%). The most-populated conformation (trans) was also observed in
the solution structure of an RNA dodecamer duplex containing 9-[2-O-(b-d-
ribofuranosyl]adenine [8]. To obtain more experimental information on the relative
populations of these bond conformations, experiments on a 13C-labeled sample will be
necessary.
Fig. 2. Low-energyconformations around the O- and N-glycosidic bonds . Points represent conformations within
20 kJ of
a
local minimum. N-Glycosidic angle: C(4)ÀN(9)ÀC(1')ÀO(4'); O-glycosidic angle:
C(2')ÀC(1')ÀO(1')ÀC(2').

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3.Conclusions.
The efficient synthesis of O-b-d-ribofuranosyl-(1'' ! 2')guano-
sine-5''-O-phosphate and O-b-d-ribofuranosyl-(1'' ! 2')-adenosine-5''-O-phosphate,
minor tRNA components, has been developed, and their conformational properties
were examined by NMR spectroscopy. These molecules are rather flexible and show
free rotation around the O- and N-glycosidic bonds. The ribose moiety of the
nucleoside shows a high percentage of S-type conformation.
Experimental Part
General. Column chromatography (CC): silica gel (0.06 0.20 mm). TLC: Kieselgel 260 F (Merck);
eluents: CH2Cl2 (A); CH2Cl2/MeOH 98 :2( B); CH2Cl2/MeOH 95 :5 (C); detection by UV light. NMR Spectra:
Bruker AMX-400 and Varian Unity500 NMR spectrometers; at 300 K; chemical shifts d in ppm relative to the
solvent signals (1H and 13C) and relative external reference (H4PO3 (capil.); 31P); coupling constants J in Hz; the
signals were assigned by double-resonance techniques and COSY experiments. MS and exact mass measure-
ments: quadrupole/orthogonal-acceleration time-of-flight tandem mass spectrometer (Q-Tof-2, Micromass,
Manchester, UK) equipped with a standard electrospray ionization (ESI) interface.
1,2,3-Tri-O-benzoyl-5-O-(phenoxyacetyl)-d-ribofuranose (2). Phenoxyacetic anhydride (2.08 g, 7.25 mmol)
was added to a soln. of 1,2,3-tri-O-benzoyl-d-ribofuranose [12] (2.58 g, 5.58 mmol) in pyridine/1,2-dichloro-
ethane 1:4 (25 ml). The mixture was kept for 1 h at 208, then MeOH (1 ml) was added, and, after storage for
30 min at 208, diluted with CH2Cl2, washed with 10% aq. soln. of NaHCO3 (30 ml) and H2O (30 ml), dried
(Na2SO4), evaporated in vacuo, and co-evaporated with toluene (2 Â 10 ml). The residue was purified by flash
chromatography on silica gel to give 2.83 g (85%) of 2 as a mixture of anomers 1:1. Oil. This mixture was
separated by CC (100 g) with 0.2% MeOH/CH2Cl2.
1
b-Isomer of 2. Oil. Rf (CH2Cl2/MeOH 99.5 :0.5) 0.7. H-NMR (CDCl3): 8.09 6.75 (m, 20 arom. H); 6.65
(s, HÀC(1)); 5.93 (m, HÀC(2), HÀC(3)); 4.76 (ddd, J(4,3) ˆ 6.3, J(4,5a) ˆ 3.7, J(4,5b) ˆ 4.8, HÀC(4)); 4.65
(dd, J(5a,5b) ˆ À12.1, HaÀC(5)); 4.53 (d, J ˆ À16.0, 1 H, PhOCH2); 4.42( dd, HbÀC(5)); 4.40 (d, 1 H,
PhOCH2). 13C-NMR (CDCl3): 168.54, 165.50, 165.14, 164.68 (CˆO); 157.61 (Ph); 133.94, 133.76, 129.96,
129.84, 129.57, 129.02, 128.72, 128.63, 128.57 (Bz); 121.80, 114.67 (Ph); 98.97 (C(1)); 79.73 (C(4)); 75.0 (C(2));
71.26 (C(3)); 64.83 (C(5)); 64.19 (CH2).
a-Isomer of 2. Oil. Rf (CH2Cl2/MeOH 99.5 :0.5) 0.56. 1H-NMR (CDCl3): 8.08 6.95 (m, 20 arom. H); 6.75
(d, J(1,2) ˆ 4.3, HÀC(1)); 5.68 (dd, J(3,2) ˆ 6.6, J(3,4) ˆ 2.2, HÀC(3)); 5.42( dd, HÀC(2)); 4.76 (m, HÀC(4),
CH2); 4.64 (dd, J(5a,4) ˆ 2.9, J(5a,5b) ˆ À12.1, HaÀC(5)); 4.53 (dd, J(5b,4) ˆ 3.3, HbÀC(5)). 13C-NMR
(CDCl3): 168.84, 165.81, 165.17, 164.96 (CˆO); 157.70 (Ph); 133.66, 133.54, 129.96, 129.87, 129.75, 129.20,
128.44 (Bz, Ph); 121.92, 114.54 (Ph); 94.88 (C(1)); 82.40 (C(4)); 71.36 (C(2)); 70.69 (C(3)); 65.04 (C(5)); 64.13
(CH2).
9-[2-O-[2,3-di-O-benzoyl-3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-5-O-(phenoxyacetyl)-b-d-ribo-
furanosyl]-b-d-ribofuranosyl]-N2-isobutyryl guanine (3a). To a cooled soln. (08) of 2 (1.35 g, 2.26 mmol) under
N2 in 1,2-dichloroethane (25 ml), SnCl4 (0.32ml, 2.71 mmol) was added, and the soln. was kept at 0 8 for 10 min.
After addition of N2-isobutyryl-9-[3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-b-d-ribofuranosyl]guanine
(1a; 1.16 g, 1.95 mmol), the resulting soln. was kept at 08 for 16 h. The mixture was diluted with CH2Cl2 (25 ml),
10% aq. soln. of NaHCO3 (30 ml) was added, and the suspension was stirred at 208 for 20 min. The suspension
was filtered through Hyflo Super Cel, the org. layer was separated, washed with H2O (20 ml), dried (Na2SO4),
and evaporated to dryness. The residue was purified by CC (silica gel (50 g)). The column was washed with
system A, and then elution with 1% MeOH/CH2Cl2 gave 1.41 g (68%) of 3a. Foam. Rf (C) 0.62. 1H-NMR
(CDCl3): 12.19 (br. s, NH); 9.64 (br. s, NH); 8.12( s, HÀC(8)); 7.99 7.86 (m, 4 H, Bz); 7.62 7.21 ( m, 8 H, Bz,
Ph); 6.95 (t, J ˆ 7.3, 1 H, Ph); 6.82( d, J ˆ 8.1, 2H, Ph); 5.98 ( s, HÀC(1') (Guo)); 5.93 (dd, J(3',2') ˆ 5.3,
J(3',4') ˆ 5.9, HÀC(3')(Rib)); 5.85 (s, HÀC(1')(Rib)); 5.78 (d, HÀC(2')(Rib)); 4.93 (dd, J(5'a,4') ˆ 4.7,
J(5'a,5'b) ˆ À11.5, HaÀC(5')(Rib)); 4.88 (d, J ˆ 16.5, 1 H, PhOCH2); 4.79 (d, 1 H, PhOCH2); 4.61
(m, HÀC(2'), HÀC(3')(Guo)); 4.42( dd, J(5'b,4') ˆ 3.4, HbÀC(5')(Rib)); 4.29 (m, HaÀC(5')(Guo),
HÀC(4')(Rib)); 4.19 (dd, J(4',3') ˆ 9.3, J(4',5'b) ˆ 2.5, HÀC(4')(Guo)); 4.03 (dd, J(5'b,5'a) ˆ À13.7,
HbÀC(5')(Guo)); 2.61 (sept., J ˆ 6.8, CH (i-Bu)); 1.17 0.93 (m, 34 H, i-Bu, i-Pr). 13C-NMR (CDCl3): 179.43,
171.01, 165.40, 164.92(C ˆO); 157.36 (Ph); 155.20 (C(6)); 148.47 (C(2)); 147.26 (C(4)); 135.84 (C(8)); 133.59,
129.72, 129.68, 129.04, 128.85, 128.50, 128.46 (Bz, Ph); 122.14 (Ph); 121.18 (C(5)); 114.35 (Ph); 105.39
(C(1')(Rib)); 87.55 (C(1')(Guo)); 81.27 (C(4')(Guo)); 79.46 (C(4')(Rib)); 79.40 (C(2')(Guo)); 75.91

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(C(2')(Rib)); 72.12 (C(3')(Rib)); 69.64 (C(3')(Guo)); 65.18 (C(5')(Rib)); 64.75 (CH2); 59.55 (C(5')(Guo));
36.03 (CH (i-Bu)); 18.85 (Me (i-Bu)); 17.47, 17.34, 17.29, 17.25, 17.13, 16.98, 16.70, 13.39, 13.09, 12.87, 12.61 (i-Pr).
‡
ESI-MS (pos.): 1070.4235 ([C53H67N5O15Si2 ‡ H] ; calc. 1070.4250).
N6-Benzoyl-9-{2-O-[2,3-di-O-benzoyl-3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-5-O-(phenoxyace-
tyl)-b-d-ribofuranosyl]-b-d-ribofuranosyl}adenine (3b). As described for 3a, with 2 (840 mg, 1.41 mmol), SnCl4
(0.2ml, 1.71 mmol) and N6-benzoyl-9-[3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl]-b-d-ribofuranosyl)ade-
nine (1b; 720 mg, 1.17 mmol): 765 mg (60%) of 3b. Foam. Rf (C) 0.59. 1H-NMR (CDCl3): 9.10 (br. s, NH), 8.76
(s, HÀC(8)); 8.30 (s, HÀC(2)); 8.04 7.85 (m, 6 H, Bz); 7.61 7.18 (m, 11 H, Bz, Ph); 6.88 (m, 3 H, Ph); 6.02
(s, HÀC(1')(Ado)); 5.80 (m, HÀC(1'), HÀC(2'), HÀC(3')(Rib)); 4.98 (dd, J(3',2') ˆ 4.8, J(3',4') ˆ 8.8,
HÀC(3')(Ado)); 4.79 4.56 (m, HÀC(2')(Ado), HÀC(4'), CH2(5')(Rib), CH2); 4.26 3.98 (m, HÀC(4'),
CH2(5')(Ado)); 1.08 0.97 (m, 28 H (i-Pr)). 13C-NMR (CDCl3): 168.78, 165.44, 165.05, 164.62(C ˆO); 157.67
(Ph); 152.82 (C(2)); 150.93 (C(6)); 149.48 (C(4)); 142.01 (C(8)); 133.54, 129.75, 129.51, 128.87, 128.50, 128.44,
127.87 (Bz, Ph); 123.56 (C(5)); 121.74, 114.60 (Ph); 105.80 (C(1')(Rib)); 88.75 (C(1')(Ado)); 81.40
(C(4')(Ado)); 79.67 (C(4')(Rib)); 78.88 (C(2')(Ado)); 75.51 (C(2')(Rib)); 72.27 (C(3')(Rib)); 69.81
(C(3')(Ado)); 65.47 (C(5')(Rib)); 65.13 (CH2); 59.73 (C(5')(Ado)); 17.30, 17.15, 16.97, 16.76, 16.66, 13.20,
‡
12.78, 12.69, 12.45 (i-Pr). ESI-MS (pos.): 1088.4103 ([C56H65N5O14Si2 ‡ H] ; calc. 1088.4144).
9-[2-O-(2,3-Di-O-benzoyl-b-d-ribofuranosyl)-3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-b-d-ribo-
furanosyl]-N2-isobutyryl guanine (4a). A soln. of 3a (1.41 g, 1.32mmol) in 0.1 m K2CO3 in MeOH (13.2ml) was
kept for 10 min at 208, and then 10% AcOH in MeOH was added to adjust pH 7.0. The resulting soln. was
concentrated in vacuo to dryness, and the residue was partitioned between AcOEt (100 ml) and H2O (30 ml),
the org. layer was washed with H2O (30 ml), dried (Na2SO4), and evaporated to dryness. The residue was
applied to CC (silica gel (30 g)). The column was washed with system A and then eluted with system B to give
840 mg (68%) of 4a Foam. Rf (C) 0.5. 1H-NMR (CDCl3): 12.10 (br. s, NH); 9.96 (br. s, NH); 8.05 (s, HÀC(8));
7.99 7.89 (m, 4 H, Bz); 7.61 7.33 (m, 6 H, Bz); 5.91 (s, HÀC(1')(Guo)); 5.90 (dd, J(3',2') ˆ 4.9, J(3',4') ˆ 7.6,
HÀC(3')(Rib)); 5.87 (s, HÀC(1')(Rib)); 5.84 (d, HÀC(2')(Rib)); 4.59 (ddd, J(4',5'a) ˆ 2.1, J(4',5'b) ˆ 4.4,
HÀC(4')(Rib)); 4.53 (dd, J(3',2') ˆ 4.1, J(3',4') ˆ 9.5, HÀC(3')(Guo)); 4.47 (d, (Guo)); 4.33 (dd, J(5'a,4') ˆ 1.0,
J(5'a,5'b) ˆ À13.7, HaÀC(5')(Guo)); 4.24 (ddd, J(4',5'b) ˆ 2.4, HÀC(4')(Guo)); 4.16 (dd, J(5'a,5'b) ˆ À12.5,
HaÀC(5')(Rib)); 4.04 (dd, HbÀC(5')(Guo)); 3.97 (dd, HbÀC(5')(Rib)); 2.62 (sept., J ˆ 6.9, CH (i-Bu)); 1.28
(d, Me (i-Bu)); 1.27 (d, Me (i-Bu)); 1.11 0.93 (m, 28 H, i-Pr). 13C-NMR (CDCl3): 178.64, 165.46, 164.90
(CˆO); 155.23 (C(6)); 148.58 (C(2)); 146.41 (C(4)); 135.72 (C(8)); 133.47, 133.50, 129.69, 129.15, 128.96, 128.48,
128.40 (Bz); 121.15 (C(5)); 104.55 (C(1')(Rib)); 88.73 (C(1')(Guo)); 82.23 (C(4')(Rib)); 81.43 (C(4')(Guo));
77.59 (C(2')(Guo)); 75.86 (C(2')(Rib)); 70.17 (C(3')(Rib)); 69.58 (C(3')(Guo)); 61.47 (C(5')(Rib)); 59.34
(C(5')(Guo)); 36.74 (CH (i-Bu)); 18.83, 18.78 (Me (i-Bu)); 17.44, 17.27, 17.18, 16.98, 16.72, 13.38, 12.89, 12.69 (i-
‡
Pr). ESI-MS (pos.): 936.3907 ([C45H61N5O13Si2 ‡ H] ; calc. 936.3882).
N6-Benzoyl-9-[2-O-(2,3-di-O-benzoyl-b-d-ribofuranosyl)-3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-
b-d-ribofuranosyl]adenine (4b). As described for 4a, with 3b (544 mg, 0.5 mmol): 300 mg (63%) of 4b. Foam. Rf
(C) 0.42. 1H-NMR (CDCl3): 9.18 (br. s, NH); 8.76 (s, HÀC(8)); 8.47 (s, HÀC(2)); 8.01 7.85 (m, 6 H, Bz); 7.57
7.26 ( m, 9 H, Bz); 6.2 0 (s, HÀC(1')(Ado)); 6.03 (dd, J(3',2') ˆ 4.7, J(3',4') ˆ 7.8, H ÀC(3')(Rib)); 5.79
(d, HÀC(2')(Rib)); 5.57 (s, HÀC(1')(Rib)); 4.49 (m, HÀC(2'), HÀC(3')(Ado), HÀC(4')(Rib)); 4.29
(d, J(5'a,5'b) ˆ À13.5, HaÀC(5')(Ado)); 4.25 (dd, J(3',4') ˆ 9.0, J(4',5'b) ˆ 2.5, HÀC(4')(Ado)); 4.08
(dd, J(5'a,4') ˆ 1.5, J(5'a,5'b) ˆ À12.5, HaÀC(5')(Rib)); 4.01 (dd, HbÀC(5')(Ado)); 3.79 (dd, J(5'b,4') ˆ 3.1,
HbÀC(5')(Rib)); 1.05 0.87 (m, 28 H (i-Pr)). 13C-NMR (CDCl3): 165.53, 165.05, 164.68 (CˆO); 152.39 (C(2));
150.05 (C(6)); 149.84 (C(4)); 140.68 (C(8)); 133.66, 133.27, 132.88, 129.72, 129.42, 128.90, 128.44, 128.35, 127.93
(Bz); 123.98 (C(5)); 104.65 (C(1')(Rib)); 89.08 (C(1')(Ado)); 82.65 (C(4')(Rib)); 81.74 (C(4')(Ado)); 77.24
(C(2')(Ado)); 75.70 (C(2')(Rib)); 69.99 (C(3')(Rib)); 69.08 (C(3')(Ado)); 60.31 (C(5')(Rib)); 59.37
(C(5')(Ado)); 17.27, 17.18, 16.88, 16.72, 16.66, 13.26, 12.72, 12.54 (i-Pr). ESI-MS (pos.): 954.3787
‡
([C48H59N5O12Si2 ‡ H] ; calc. 954.3777).
9-{2-O-[2,3-Di-O-benzoyl-5-O-bis[2-(4-nitrophenyl)ethyl]phosphorylyl-b-d-ribofuranosyl]-3,5-O-(1,1,3,3-
tetraisopropyldisiloxane-1,3-diyl)-b-d-ribofuranosyl]-N-isobutyryl guanine (5a). A mixture of 4a (1.17 g,
1.25 mmol) and a 0.5m soln. of the triethylammonium salt of bis[2-(4-nitrophenyl)ethyl] phosphate (5 ml,
2.5 mmol) was co-evaporated with dry pyridine (3 Â 5 ml) and finally dissolved in pyridine (5 ml). Then, 1-
methyl-1H-imidazole (1 ml, 12.5 mmol) and TPSCl (1.51 g, 5 mmol) were added. The mixture was stirred for 4 h
at 208, and then concentrated in vacuo. The residue was dissolved in CHCl3 (100 ml) and washed with 0.06m
phosphate buffer (pH 7.0, 2 Â 30 ml). The org. layer was dried (Na2SO4), evaporated in vacuo, co-evaporated
with toluene (2 Â 10 ml), and purified by CC (silica gel (30 g)). The column was washed with Et2O (300 ml) and
eluted with system B to give 1.12g (68%) of 5a. Foam. Rf (C) 0.65. 1H-NMR (CDCl3): 12.36 (br. s, NH); 10.86

512
Helvetica Chimica Acta Vol. 86 (2003)
(br. s, NH); 8.14 7.83 (m, 9 H, HÀC(8), Bz, Ph); 7.60 7.27 (m, 10 H, Bz, Ph); 5.86 (m, HÀC(1')(Guo),
HÀC(3')(Rib)); 5.81 (d, J(1',2') ˆ 1.5, HÀC(1')(Rib)); 5.68 (d, J(2',3') ˆ 5.4, HÀC(2')(Rib)); 4.63
(dd, J(3',2') ˆ 4.2, J(3',4') ˆ 9.3, HÀC(3')(Guo)); 4.47 (d, HÀC(2')(Guo)); 4.44 4.36 (m, HaÀC(5')(Rib),
CH2O); 4.28 4.16 (m, HÀC(4'), HbÀC(5')(Rib), HaÀC(5')(Guo), CH2O); 4.09 (dd, J(4',5'b) ˆ 2.3,
HÀC(4')(Guo)); 4.01 (dd, J(5'b,5'a) ˆ À13.6, HbÀC(5')(Guo)); 3.12( m, CH2); 3.01 (m, CH2); 2.83
(sept., J ˆ 6.8, CH (i-Bu)); 1.24 0.93 (m, 34 H, i-Bu, i-Pr). 13C-NMR (CDCl3): 180.08, 165.32, 164.87 (CˆO);
155.58 (C(6)); 148.84 (C(2)); 147.54 (C(4)); 144.38 (Ph); 135.90 (C(8)); 133.74, 133.62, 129.78, 129.67, 129.58,
128.79, 128.50, 128.46, 124.00, 123.79, 123.74 (Bz, Ph); 121.28 (C(5)); 105.67 (C(1')(Rib)); 87.09 (C(1')(Guo));
81.23 (C(4')(Guo)); 80.73 (d, J(C,P) ˆ 9.8, C(4')(Rib)); 79.50 (C(2')(Guo)); 75.86 (C(2')(Rib)); 72.35
(C(3')(Rib)); 69.45 (C(3')(Guo)); 68.04 (d, J(C,P) ˆ 4.6, CH2O); 67.77 (d, J(C,P) ˆ 5.5, CH2O); 67.20
(d, J(C,P) ˆ 3.9, C(5')(Rib)); 59.55 (C(5')(Guo)); 36.18 (CH2); 35.46 (CH (i-Bu)); 19.08, 19.02(Me (i-Bu));
17.44, 17.31, 17.26, 17.12, 16.95, 16.78, 13.35, 13.10, 12.82, 12.61 (i-Pr). ESI-MS (pos.): 1314.4503
‡
([C61H76N7O20PSi2 ‡ H] ; calc. 1314.4499).
N6-Benzoyl-9-[2-O-(2,3-di-O-benzoyl-5-O-{bis[2-(4-nitrophenyl)ethyl]phosphorylyl}-b-d-ribofuranosyl)-
3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-b-d-ribofuranosyl]adenine (5b). As described for 5a, with 4b
(220 mg, 0.23 mmol): 200 mg (65%) of 5b. Foam. Rf (C) 0.66. 1H-NMR (CDCl3): 9.03 (br. s, NH); 8.72
(s, HÀC(8)); 8.34 (s, HÀC(2)); 8.01 7.83 (m, 10 H, Bz, Ph); 7.61 7.30 (m, 13 H, Bz, Ph); 6.08
(s, HÀC(1')(Ado)); 5.85 (dd, J(3',2') ˆ 4.9, J(3',4') ˆ 6.3, HÀC(3')(Rib)); 5.79 (d, HÀC(2')(Rib)); 5.77
(s, HÀC(1')(Rib)); 5.07 (dd, J(3',2') ˆ 4.6, J(3',4') ˆ 9.0, HÀC(3')(Ado)); 4.84 (d, HÀC(2')(Ado)); 4.50
(m, HÀC(4')(Rib)); 4.30 4.11 (m, HÀC(4'), HaÀC(5')(Ado), CH2(5')(Rib), CH2O); 4.02( dd, J(5'b,4') ˆ
2.7, J(5'b,5'a) ˆ À13.2, HbÀC(5')(Ado)); 3.00 (m, CH2); 2.97 (m, CH2); 1.08 0.95 (m, 28 H, i-Pr). 13C-NMR
(CDCl3): 165.47, 165.02, 164.59 (CˆO); 152.60 (C(2)); 150.96 (C(6)); 149.51 (C(4)); 146.96, 144.68 (Ph); 142.80
(C(8)); 133.63, 132.88, 129.78, 128.93, 128.54, 127.84, 123.71 (Bz, Ph); 123.69 (C(5)); 105.56 (C(1')(Rib)); 88.84
(C(1')(Ado)); 81.40 (C(4')(Ado)); 80.16 (d, J(C,P) ˆ 7.2, C(4')(Rib)); 78.70 (C(2')(Ado)); 75.39 (C(2')(Rib));
71.66 (C(3')(Rib)); 70.05 (C(3')(Ado)); 67.17 (m, C(5')(Rib), CH2O); 59.85 (C(5')(Ado)); 36.07, 36.06 (CH2),
‡
17.15, 16.97, 16.76, 13.17, 12.78, 12.66, 12.48 (i-Pr). ESI-MS (pos.): 1332.4365 ([C64H74N7O19PSi2 ‡ H] ; calc.
1332.4394).
9-[2-O-(2,3-di-O-benzoyl-5-O-{bis[2-(4-nitrophenyl)ethyl]phosphorylyl}-b-d-ribofuranosyl)-b-d-ribofura-
nosyl]-N2-isobutyryl guanine (6a). The protected triester 5a (830 mg, 0.63 mmol) was dissolved in THF (3 ml),
and a soln. of Bu4NF ¥ 3 H2O (554 mg, 1.76 mmol) in THF (2ml) was added. After 15 min at 20 8, H2O (1 ml) and
‡
Dowex-50 (Na form; 1.5 ml) were added, and the mixture was stirred for 30 min at 208. The resin was filtered
off and washed with AcOEt. The filtrate was washed with 0.06m phosphate buffer (pH 7.0; 2 Â 30 ml). The org.
layer was dried (Na2SO4), evaporated to dryness, and subjected to CC (silica gel (30 g)). The column was
washed with system A, then B, and eluted with system C to give 520 mg (77%) of 6a. Foam. Rf (C) 0.25.
1H-NMR (CDCl3): 12.30 (br. s, NH); 10.83 (br. s, NH); 8.56 (s, HÀC(8)); 8.10 7.79 (m, 8 H, Bz, Ph); 7.56 7.26
(m, 10 H, Bz, Ph); 6.14 (d, J(1',2') ˆ 2.9, HÀC(1')(Guo)); 5.82( dd, J(3',2') ˆ 5.1, J(3',4') ˆ 6.1, HÀC(3')(Rib));
5.68 (dd, J(2',1') ˆ 1.2, HÀC(2')(Rib)); 5.62( d, HÀC(1')(Rib)); 4.81 (dd, J(3',2') ˆ 4.6, J(3',4') ˆ 8.0,
HÀC(3')(Guo)); 4.56 (dd, HÀC(2')(Guo)); 4.51 (m, HÀC(4')(Guo)); 4.42( m, CH2O); 4.36
(dddd, J(4',5'a) ˆ 4.4, J(4',5'b) ˆ 3.9, J(4',P) ˆ 1.9, HÀC(4')(Rib)); 4.30 (ddd, J(5'a,5'b) ˆ À11.0, J(5'a,P) ˆ 4.6,
HaÀC(5')(Rib)); 4.16 (m, CH2O); 4.11 (ddd, J(5'b,P) ˆ 4.6, HbÀC(5')(Rib)); 4.06 (m, HaÀC(5')(Guo)); 3.98
(m, HbÀC(5')(Guo)); 3.11 (m, CH2); 2.97 (m, CH2); 2.75 (sept., J ˆ 6.8, CH (i-Bu)); 2.10 (m, HOÀC(3'),
HOÀC(5')(Guo)); 1.19 (d, Me (i-Bu)); 1.17 (d, Me (i-Bu)). 13C-NMR (CDCl3): 180.14, 165.29, 164.83 (CˆO);
155.93 (C(6)); 148.51 (C(2)); 146.97 (C(4)); 144.97, 144.46 (Ph); 135.50 (C(8)); 133.63, 129.84, 129.77, 129.70,
129.63, 128.74, 128.50, 123.99, 123.69, 123.64 (Bz, Ph); 120.47 (C(5)); 106.02 (C(1')(Rib)); 87.14 (C(1')(Guo));
84.36 (C(4')(Guo)); 81.47 (C(2')(Guo)); 79.99 (d, J(C,P) ˆ 9.8, C(4')(Rib)); 75.69 (C(2')(Rib)); 71.24
(C(3')(Rib)); 70.00 (C(3')(Guo)); 68.08 (d, J(C,P) ˆ 3.9, CH2O); 67.56 (d, J(C,P) ˆ 4.9, CH2O); 66.47
(d, J(C,P) ˆ 3.7, C(5')(Rib)); 60.69 (C(5')(Guo)); 36.14, 36.09 (CH2); 35.67 (CH (i-Bu)); 19.01, 18.92(Me (i-
‡
Bu)). ESI-MS (pos.): 1094.2791 ([C49H50N7O19P ‡ Na] ; calc. 1094.2797).
N6-Benzoyl-9-[2-O-(2,3-di-O-benzoyl-5-O-{bis[2-(4-nitrophenyl)ethyl]phosphorylyl}-b-d-ribofuranosyl)-
b-d-ribofuranosyl]adenine (6b). As described for 6a, with 5b (90 mg, 0.068 mmol): 53 mg (72%) of 6b. Foam. Rf
(C) 0.27. 1H-NMR (CDCl3): 9.07 (br. s, NH); 8.77 (s, HÀC(8)); 8.39 (s, HÀC(2')); 8.11 7.83 (m, 10 H, Bz, Ph);
7.61 7.32( m, 13 H, Bz, Ph); 6.17 (d, J(1',2') ˆ 6.9, HÀC(1')(Ado)); 5.61 (dd, J(3',2') ˆ 5.0, J(3',4') ˆ 5.6,
HÀC(3')(Rib)); 5.55 (dd, J(2',1') ˆ 1.9, HÀC(2')(Rib)); 5.17 (d, HÀC(1')(Rib)); 5.15 (dd, J(2',3') ˆ 4.7,
HÀC(2')(Ado)); 4.65 (d, HÀC(3')(Ado)); 4.33 4.18 (m, HÀC(4')(Ado), HÀC(4')(Rib), CH2O); 4.06
(ddd, J(5'a,4') ˆ 3.7, J(5'a,5'b) ˆ À10.9, J(5'a,P) ˆ 5.9, HaÀC(5')(Rib)); 3.98 (br. d, J(5'a,5'b) ˆ À12.8,
HaÀC(5')(Ado)); 3.89 (ddd, J(5'b,4') ˆ 4.9, J(5'a,P) ˆ 5.6, HbÀC(5')(Rib)); 3.78 (br. d, HbÀC(5')(Ado)); 3.07

Helvetica Chimica Acta Vol. 86 (2003)
513
(m, CH2); 3.03 (m, CH2). 13C-NMR (CDCl3): 165.39, 165.33, 164.72(C ˆO); 151.99 (C(2)); 150.65 (C(6));
150.20 (C(4)); 144.56, 144.47 (Ph); 141.67 (C(8)); 133.82, 133.78, 133.41, 132.91, 129.77, 129.66, 128.84, 128.53,
128.44, 127.98 (Bz, Ph); 124.14 (C(5)); 123.72 (Ph); 106.53 (C(1')(Rib)); 89.37 (C(1')(Ado)); 87.15
(C(4')(Ado)); 81.25 (C(2')(Ado)); 80.68 (d, J(C,P) ˆ 7.0, C(4')(Rib)); 75.63 (C(2')(Rib)); 71.76 (C(3')(Rib));
71.31 (C(3')(Ado)); 67.63 (d, J(C,P) ˆ 6.4, CH2OP); 67.51 (d, J(C,P) ˆ 5.7, CH2OP); 66.55 (d, J(C,P) ˆ 5.7,
‡
C(5')(Rib)); 62.99 (C(5')(Ado)); 36.27, 36.20 (CH2). ESI-MS (pos.): 1090.2864 ([C52H48N7O18P ‡ H] ; calc.
1090.2871).
O-b-d-Ribofuranosyl-(1'' ! 2')-guanosine-5''-O-phosphate (7a). A soln. of 6a (86 mg, 0.08 mmol) in 0.5m
DBU in dry pyridine (6.4 ml, 3.2 mmol) was stored for 24 h at 208, neutralized with AcOH (0.182ml, 3.2mmol),
and evaporated. The residue was dissolved in 5m NH3 in MeOH (5 ml) and kept for 3 d at 208 and then
concentrated in vacuo, dissolved in H2O (30 ml), and washed with CH2Cl2 (2 Â 10 ml). The aq. layer was
concentrated in vacuo, diluted with H2O (50 ml), and then applied to a column of DEAE-cellulose (200 ml,
HCOyÀ3 form). The column was washed with H2O (500 ml) and a 0.05m soln. of NH4HCO3, and eluted with a
0.1m soln. of NH4HCO3. The UV-absorbing fractions were combined, evaporated in vacuo, and co-evaporated
‡
with H2O (5 Â 10 ml). The residue was dissolved in H2O (2ml), applied to a column of Dowex 50 (Na form)
(2ml), eluted with H 2O, and freeze-dried: 0.061 mmol (76%) of 7a was obtained as its Na salt. Rf (i-PrOH/
NH4OH/H2O 7:1:2) 0.16. UV (pH 1): lmax 257 (11900). UV (pH 7): lmax 252 (13300). UV (pH 13): lmax 260
‡
(11000). ESI-MS (pos.): 496.1068 ([C15H22N5O12P ‡ H] ; calc. 496.1080).
NMR Spectra. Samples were prepared with 3 mg of 7a and 3 mg of 7b, resp., dissolved in 250 ml of D2O. The
pD was adjusted to 7.2. Spectra were recorded with a Varian 500 Unity spectrometer at 292 K. Chemical shifts
were measured relative to the H2O signal. The coupling constants were measured in Hz. The signals were
assigned by double-resonance techniques and TOCSY experiments [28][29]. The TOCSY consisted of 2048
datapoints in t2 and 256 increments in t1. The data were apodized with a shifted sine-bell square function in both
dimensions and processed to a 2K Â 1K matrix.
Analysis of the couplings was performed with Pseurot. 6.2[92]. This program uses the
Altona
Sundaralingam formalism [25][26] to describe the ring puckering and a generalized Karplus equation
developed by Diez et al. in collaboration with Donders et al. [32] to describe the relation between coupling and
dihedral angle. RMS was minimized by a Newton Raphson minimization.
NOESY Spectra [33] were recorded with 150, 300, and 500 ms mixing times, resp. The NOESY consisted of
2048 datapoints in t2 and 256 increments in t1. The data were apodized with a shifted sine-bell square function in
both dimensions and processed to a 2K Â 1K matrix.
Conformational Search. A 3D structure was created with Insight II and exported as a pdb file. A Monte
Carlo conformational search was performed with Macromodel 5.0, generating 5000 random structures by
changing dihedral angles and by ring opening and closure. These 5000 structures were minimized with the
Amber Forcefield [34] and a Newton Raphson minimization.
Authors thank KUL Research Council, RFBR, and NATO for financial support, and Chantal Biernaux for
editorial help. K. N. is a research associate of the Fund of Scientific Research, Flanders.
REFERENCES
[1] P. A. Limbach, P. F. Crain, J. A. McCloskey, Nucleic Acids Res. 1994, 22, 2183.
[2] J. Rozenski, P. F. Crain, J. A. McCloskey, Nucleic Acids Res. 1999, 27, 196.
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Received October 17, 2002