2-Methylwyosine, a nucleoside with restricted anti conformation in the East region enforced by nucleobase moiety modification: Synthesis and conformational analysis by NMR and molecular dynamics

Article abstract of DOI:10.1080/15257770.2012.724133

We synthesized a new 2-methyl derivative of wyosine using a multistep procedure starting from guanosine. We examined different synthetic paths and optimized the conditions for each step. Based on MD calculations and analysis of the ³ J HH and J C1′H1′ of the ribose moiety, we discovered that the sugar part adopted conformation specific for the East region rarely occurring in solution. This unusual conformational preference is probably due to steric repulsions between the methyl group at position 2 and the 5′-CH₂OH group. We observed that N-glycosidic bond stability weakened 14-fold upon the introduction of the methyl group in position 2 compared with wyosine.

Full text of DOI:10.1080/15257770.2012.724133

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2-Methylwyosine, a Nucleoside With
Restricted Anti Conformation in the
East Region Enforced by Nucleobase
Moiety Modification: Synthesis and
Conformational Analysis by NMR and
Molecular Dynamics
Daniel Baranowski a , Bozenna Golankiewicz a , Wojciech Folkman a
& Mariusz Popenda b
a Laboratory of Nucleoside Chemistry, Institute of Bioorganic
Chemistry PAS, Poznan, Poland
b Laboratory of Structural Chemistry of Nucleic Acids, Institute of
Bioorganic Chemistry PAS, Poznan, Poland
Version of record first published: 15 Oct 2012.
To cite this article: Daniel Baranowski, Bozenna Golankiewicz, Wojciech Folkman & Mariusz Popenda
(2012): 2-Methylwyosine, a Nucleoside With Restricted Anti Conformation in the East Region Enforced
by Nucleobase Moiety Modification: Synthesis and Conformational Analysis by NMR and Molecular
Dynamics, Nucleosides, Nucleotides and Nucleic Acids, 31:10, 707-719
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Nucleosides, Nucleotides and Nucleic Acids, 31:707–719, 2012
Copyright ꢀC Institute of Bioorganic Chemistry PAS
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2012.724133
2-METHYLWYOSINE, A NUCLEOSIDE WITH RESTRICTED ANTI
CONFORMATION IN THE EAST REGION ENFORCED
BY NUCLEOBASE MOIETY MODIFICATION: SYNTHESIS
AND CONFORMATIONAL ANALYSIS BY NMR
AND MOLECULAR DYNAMICS
Daniel Baranowski,1 Bozenna Golankiewicz,1 Wojciech Folkman,1
and Mariusz Popenda2
1Laboratory of Nucleoside Chemistry, Institute of Bioorganic Chemistry PAS, Poznan, Poland
2Laboratory of Structural Chemistry of Nucleic Acids, Institute of Bioorganic Chemistry PAS,
Poznan, Poland
GRAPHICAL ABSTRACT
We synthesized a new 2-methyl derivative of wyosine using a multistep procedure starting from
2
guanosine. We examined different synthetic paths and optimized the conditions for each step. Based
3
ꢁ
ꢁ
on MD calculations and analysis of the JHH and JC1 H1 of the ribose moiety, we discovered that
the sugar part adopted conformation specific for the East region rarely occurring in solution. This
unusual conformational preference is probably due to steric repulsions between the methyl group at
position 2 and the 5ꢁ-CH2OH group. We observed that N-glycosidic bond stability weakened 14-fold
upon the introduction of the methyl group in position 2 compared with wyosine.
Keywords Modified purine nucleosides; restricted conformation; glycosidic bond sta-
bility
INTRODUCTION
For over 40 years wyosine and its derivatives, called also Y nucleosides,
have been the subject of considerable research on their chemical synthesis
Received 13 February 2012; accepted 21 August 2012.
Address correspondence to Daniel Baranowski, Laboratory of Nucleoside Chemistry, Institute of
Bioorganic Chemistry PAS, Z. Noskowskiego 12/14, Poznan, 61-704, Poland. E-mail: danibari@ibch.
poznan.pl
707

708
D. Baranowski et al.
and biological functions.[1–3] These fluorescent tricyclic nucleosides exist ex-
clusively in position 37, 3ꢁ-adjacent to the anticodon loop of tRNAPhe. They
constitute a considerable part of post-transcriptionally modified nucleoside
families isolated from various tRNA in Eukaryal and Archeal.[4] The struc-
ture of the Y nucleoside family contains the tricyclic aromatic base moiety,
that is, 4,9-dihydro-4,6-dimethyl-9-oxo-imidazo[1,2-a]purine, which is substi-
tuted with β-ribofuranose at position 3 and an acyclic chain at position 7.
The tricyclic base part of Y nucleosides enhances its stacking interactions
with adjacent bases (A36 or A38), engendering restriction of conforma-
tional flexibility of the anticodon.[5] Recent research on tRNAPhe lacking
the wyosine-like modification has shown the enhancement of retroviral ribo-
somal frameshifting,[6–9] thereby influencing the replication of human im-
munodeficiency virus (HIV) RNA.[10] Thus, the presence of the Y nucleoside
at position 37 of tRNAPhe seems to be crucial for translational reading-frame
maintenance[11] by stabilizing the codon–anticodon interactions in the de-
coding site of the ribosome.[12] Currently, it has been proven that the wyosine
biosynthetic pathway consists of multistep enzymatic reactions starting from
guanosine.[13,14] Moreover, the crystal structure of TYW1, the enzyme that
catalyzes the second step to form the tricyclic ring, was determined.[15,16]
Our previous study revealed that wyosine in solution is constrained in the
anti conformation due to intramolecular crowding of the N3-(β-D-ribose)
moiety and the N4-methyl substituent.[17,18] The ribose moiety of wyosine is
in a normal state of dynamic equilibrium with the ratio between N and S
states of approximately 1:1.[18] In the literature on wyosine derivatives substi-
tuted at position 2, data is rather limited without any attempts to determine
conformational preference in solution or glycosidic bond stability.[19,20]
We have observed that the introduction of the methyl group at posi-
tion 2 of the wyosine molecule constrains the ribose moiety of the formed
compound 9 in an unusual East conformation in solution. Moreover, we de-
veloped a simple and easy synthetic procedure for 9 starting from guanosine
and also evaluated t0.5 for the glycosidic bond acid hydrolysis reaction for
the first time.
RESULTS AND DISCUSSION
Synthesis of the title compound 9 (Scheme 1) was conducted ac-
cording to the procedure developed for wyosine,[21–23] using 1,N2-
isopropenoguanosine substituted at the position 8 as an intermediate. We
found that there were two possible ways to obtain 9. The first approach was
the methylation of 2 at position 8 with AlMe3 catalyzed by PdCl2/PPh3 in
tetrahydrofuran (THF) to give 3,[24] followed by alkylation-cyclization with
sodium hydride/bromoacetone in dimethyl sulfoxide (DMSO).[25] In the
second approach, 2 reacted first with sodium hydride/bromoacetone and

2-Methylwyosine, a Nucleoside with Restricted Anti Conformation
709
SCHEME 1 Synthesis of 4,9-Dihydro-9-oxo-2,4,6-trimethyl-3-(β-D-ribofuranosyl) imidazo[1,2-a]purine
(2-methylwyosine, 9). Reagents and conditions: (a) Br2/H2O (93%); (b) NaH, BrCH2COCH3/DMSO
then NH4OH concd (84% for 2→6, 67% for 3→4); (c) HMDS, (NH4)2SO4 then AlMe3, PdCl2,
PPh3/THF then NH4Cl/MeOH (56% for 2→3, 50% for 6→4; d) Ac2O/Py (89% for 6→7, 68% for
4→5); (e) AlMe3, PdCl2, PPh3/THF (56% for 7→5); f) CH2N2, ZnI2/Et2O then 1M NH4HCO3 (22%
for 5→8) (g) NH3/MeOH anh (81% for 8→9).
the resulting 6 was then transformed into 4 using the same reaction con-
ditions as above mentioned for the 2→3 conversion. The second approach
appeared to be more convenient because it simplified the purification of the
product and improved the yield (42% versus 37% for 2→6→4 and 2→3→4,
respectively). We also checked the 7→5 conversion but because of low yield
and the presence of additional C2 methylated products containing deacety-
lated hydroxyl group in ribose, we abandoned further attempts on this route.
Compound 4 was transformed into the triacetate derivative 5 by treatment
with acetic anhydride in pyridine and subsequent selective removal of the
N-acetyl group with Py:MeOH:H2O. Selective methylation at position N4 of
5 by means of Simmons–Smith reagent afforded 8 in 22% yield.[21–23] Simi-
larly to the methylation reaction of 1,N2-isopropenoguanosine triacetate and
its 2ꢁ-deoxyribose derivative,[23] we noted the formation of 1-methylwyeine
substituted with a methyl group at position 2 of 10, a base which was a prod-
uct of a subsequent deribosylation reaction. Further attempts to reduce the
yield of this byproduct failed. Deacetylation of 8 with anhydrous methanolic
ammonia resulted in 81% of crystalline 9.

710
D. Baranowski et al.
3
TABLE 1 J HH and 1J CH coupling constantsa in the ribose ring of wyosine (W) and 2-methylwyosine 9
Coupled nuclei
W [Hz]
9 [Hz]
Coupled nuclei
W [Hz]
9 [Hz]
H1ꢁ-H2ꢁ
4.7
4.8
4.5
3.4
3.4
7.3
6.6
4.2
3.7
4.5
C1ꢁ–H1ꢁ
168.3
149.0
148.2
147.6
140.3
160.3
146.7
150.1
147.6
140.1
H2ꢁ–H3ꢁꢁ
C2ꢁ–H2ꢁ
H3ꢁ–H4
C3ꢁ–H3ꢁ
H4ꢁ–H5ꢁ b
H4ꢁ–H5ꢁꢁ b
C4ꢁ–H4ꢁ
C5ꢁ–H5ꢁ/H5ꢁꢁ
aIn Hz; DMSO-d6 solvent (TMS); 21◦C. bH5ꢁ has been arbitrary assigned to be in lower field.
We examined conformational alterations induced by the presence of an
additional bulky substituent at position 2 of the base using nuclear magnetic
1
resonance (NMR) spectroscopy and molecular dynamics calculations. H
and 13C NMR spectra of 2-methylwyosine revealed substantial differences in
terms of chemical shifts and coupling constants in the carbohydrate range
when compared with those of wyosine (Table 1). Particularly conspicuous
was the change of the 3JH2 ,H3 , which is known to be relatively insensitive to
different proportions of N and S forms in solution.[26]
ꢁ
ꢁ
According to the concept of pseudorotation[27] the conformation of the
furanose ring can be determined in terms of the phase angle of pseudorota-
tion (P), the degree of pucker (φm) and the conformational population. The
computer program PSEUROT[28] (6.0) was used to calculate the best fit of
all conformational parameters (P and φm for both I and II conformers and
corresponding mole fractions) to three experimental coupling constants
3
3
(3J H1 H2 , J H2 H3 , J H3 H4 ). Wyosine showed the typical rapid exchange be-
tween north and south conformers localized in the most preferred sectors
centered around P = 18◦ (C3ꢁ-endo) and P = 162◦ (C2ꢁ-endo). However, 2-
methylwyosine, adopts conformations characterized by a phase angle (P) of
90◦–126◦, a range of values specific for East region (Figure 1). The trend of
the ribose moiety of 9 to adopt the unusual East conformation in solution
was also demonstrated by molecular dynamics calculations. As illustrated by
the bar graphs in Figure 1 both approaches clearly show substantial confor-
mational dissimilarity between the ribose part of W and 9. There are some
discrepancies in the range of values for phase angle (P = 90◦–126◦) calcu-
lated for 9 from PSEUROT and (P = 72◦–126◦) from molecular dynamics
simulation. However, these differences are likely to be expected when limita-
tions and some arbitrary parameters of both methods are taken into account.
Additional nuclear magnetic resonance evidence for the rare β-D-
ribose conformation of 9 in solution was gained from the 2D-NOESY
spectra (Figure 2). Out of NOE interactions observed for wyosine and 2-
methylwyosine (Table 2), those between H1ꢁ and H4ꢁ proved to be crucial.
As shown in Figure 2, the NOE for H1ꢁ and H4ꢁ in 2-methylwyosine is much
stronger than for H1ꢁ and H2ꢁ, contrary to wyosine itself. This is evidence for
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ

2-Methylwyosine, a Nucleoside with Restricted Anti Conformation
711
FIGURE 1 (a) PSEUROT program and (b) MD calculations percentage of pseudorotation phase angles
of ribose rings of wyosine (left) and 2-methylwyosine (right).
FIGURE 2 Selected region of the 2D-NOESY spectrum of the wyosine/2-methylwyosine mixture (ap-
prox. 1:1) showing cross-peaks between H1ꢁ and H2ꢁ, H4ꢁ and N4CH3.

712
D. Baranowski et al.
TABLE 2 1D NOE enhancements for wyosine and 2-methylwyosine after H1ꢁ frequency irradiation
(DMSO-d6 21◦C)
Nucleus observed
wyosine
2-methylwyosine
2ꢁOH
3ꢁOH
H2ꢁ
1.1%
0.6%
1.6%
10.7%
1.2%
1.1%
0.4%
1.5%
9.3%
2.6%
N4CH3
H4ꢁ
a shorter distance between H1ꢁ-H4ꢁ than between H1ꢁ-H2ꢁ that is character-
istic of an East conformation as demonstrated in Figure 3.[29] The above was
also confirmed by 1D NOE experiments.
The East conformational preference of the sugar moiety of 2-
methylwyosine was also indicated by its one-bond 13C–1H spin-coupling con-
stant 1J C1 ,H1 , significantly smaller than that those of wyosine (Table 1). This
result is consistent with the corresponding J CH value for β-D-ribose itself,
which has been computed as a function of ring conformation.[26]
Orientation of the base plane of 9 toward the ribose moiety is anti, the
same as in wyosine,[18] which is confirmed by the strong NOE signal between
H1ꢁ and N4CH3 (Table 2).
ꢁ
ꢁ
Inspection of t0.5 for the glycosidic bond hydrolysis reaction of 9 has re-
vealed approximately a 14-fold decrease of its value compared with wyosine
which is generally known for its lability[30,31] (Table 3). Such increased sus-
ceptibility of glycosidic bond to fission may be attributed to an unfavorable
orientation between the lone pair of O4ꢁ of ribose and N3 of the base result-
ing from the adoption of rare East conformation by the ribose moiety.[32]
FIGUREꢁ3 Structures of nucleosides adopting North (C3ꢁ-endoꢁC2ꢁ-exo), South (C2ꢁ-endo C3ꢁ-exo), and
ꢁ
ꢁ
ꢁ
˚
East (O4 -endo) ribose conformation and distances (A) H1 –H2 and H1 –H4 characteristic of particular
structure. Calculations for ribofuranose rings of sugar pucker amplitude 40◦ were conducted by using
DISCOVER program.

2-Methylwyosine, a Nucleoside with Restricted Anti Conformation
713
TABLE 3 t0.5 for glycosidic bond hydrolysis in HCl at t = 37◦C determined by means of Vierordt
method
Compound
0.01 N
0.001 N
Wyosine
2-Methylwyosine (9)
375 s
27 s
4350 s
300 s
To best of our knowledge, 2-methylwyosine is the first example of a
nucleoside having the ribose moiety in the rare East conformation O4ꢁ-endo-
C1ꢁ-exo in solution imposed not by any bond but by non-bonded interactions,
induced by nucleobase modification. In our opinion, this unique conformer
of ribose in 9 probably resulted from steric repulsions between the methyl
group at position 2 and the 5ꢁ-CH2OH group. The east conformation is re-
sponsible for a definite decrease in the stability of the glycosidic bond due
to electrostatic repulsion between unfavorably oriented lone pairs of O4ꢁ
and N3 leading to a weakening strength of the anomeric effect. This con-
clusion corroborates the hypothesis that the anomeric effect is responsible
for the N⇔S pseudorotational equilibrium of pentofuranose moiety and gly-
cosidic bond stability.[33] Recently, such an unexpected furanose conforma-
tion in solution was found for 2ꢁ-deoxy-2ꢁ-fluoro-5-methylarabinouridine[34]
and oligonucleotide duplexes containing 2ꢁ-deoxy-2ꢁ-fluoroarabinofuranosyl
residues.[35,36] The other example is D-mannose forming a weak complex
3
with platinum(IV).[37] The characteristic large value for J H2 ,H3 , (5.9 Hz)
which was displayed by the complex was also observed for the ribose moiety
of 9 (6.6 Hz).
ꢁ
ꢁ
The occurrence of the rare East conformation of the ribose part
in a biologically important system has been recently proved for adeno-
sine complexed with human adenosine kinase by the X-ray analysis.[38]
On the contrary, the ribose ring of adenosine complexed with adeno-
sine kinase from Toxoplasma gondii has displayed more common North
conformation.[39]
In conclusion, 2-methylwyosine is an example of anti-fixed nucleoside
where ribose adopts a rare conformation in the East region enforced by
substitution of base moiety. Unfortunately, prospective application of 9 as
a substrate for oligonucleotide synthesis is seriously limited because of ex-
ceptional lability of the glycosidic bond. These results inspired us to con-
tinue our research work to obtain more stable derivatives of wyosine sub-
stituted at the position 2. The strategy for synthesis of oligomers contain-
ing hypermodified nucleosides making allowance for their lability has been
developed with the aim of investigation of formation of a stable homod-
imeric “kissing complex” during the replication of Moloney murine leukemia
virus.[40,41]

714
D. Baranowski et al.
EXPERIMENTAL
General
Nuclear magnetic resonance spectra were recorded on a Varian Unity
300FT spectrometer at 25◦C with tetramethylsilane as an internal standard.
Chemical shifts (δ) are reported in parts per million (ppm), and signals are
reported as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet),
or br s (broad singlet). Mass spectra were recorded on an Intectra AMD 604
spectrometer wit LSIMS method. Elemental analyses were performed on a
Perkin–Elmer 240CHN analyzer. The rate of glycosidic bond was measured
on a Perkin–Elmer Lamda EZ 201 spectrometer using Vierodt method in wa-
ter. Thin layer chromatography (TLC) was conducted on Merck precoated
silica gel F254 plates (thickness 0.25mm). For a preparative short column
chromatography Merck TLC silica gel type 60H was used.
8-Bromoguanosine (2)
Compound 2 was prepared according to the literature method[42] to
1
yield 93%. H NMR (300 MHz DMSO-d6) δ 10.82 (brs, 1H, N1–H), 6.51
(brs, 2H, NH2), 5.68 (d, 1H, H1ꢁ), 5.46, 5.10 (d, d, 1H, 1H, 2ꢁ–OH, 3ꢁ–OH),
5.01 (dd, 1H, H2ꢁ), 4.92 (1H, t, 5ꢁ–OH), 4.15–4.11 (1H, m, H3ꢁ), 3.87–3.82
(1H, m, H4ꢁ), 3.68–3.61 (1H, m, H5ꢁ), 3.54–3.46 (1H, m, H5ꢁꢁ).
2-Bromo-3,9-dihydro-6-methyl-9-oxo-3-(β-D-ribo-furanosyl)-5H-imidazo[1,2-a]
Purine (6)
Compound 6 was obtained by the reaction of 2 with sodium hydride and
bromoacetone according to the literature method.[25] The dark red solution
was concentrated, poured into diethylether:acetone 3:1 and left for 24 h. The
off-white precipitate was crystallized from methanol to give 6 (84%). 1H NMR
(300 MHz DMSO-d6) δ 12.57 (brs, 1H, H5), 7.39 (d, 1H, H7), 5.78 (d, 1H,
H1ꢁ), 5.49 (d, 1H, 2ꢁ–OH), 5.19 (d, 1H, 3ꢁ–OH), 5.14 (dd, 1H, H2ꢁ), 4.97 (dd,
1H, 5ꢁ–OH), 4.22 (q, 1H, H3ꢁ), 3.91 (q, 1H, H4ꢁ), 3.74-3.67 (m, 1H, H5ꢁ),
3.59–3.51 (m, 1H, H5ꢁꢁ), 2.26 (d, 3H, CH3). 13C NMR (75 MHz DMSO-d6) δ
150.2 (C9), 149.9 (C3a), 144.8 (C4a), 125.6 (C6), 122.6 (C2), 116.2 (C9a),
103.5 (C7), 90.3 (C1ꢁ), 85.9 (C4ꢁ), 70.4 (C2ꢁ), 70.3 (C3ꢁ), 62.0 (C5ꢁ), 10.3
(CH3).
2-Bromo-3,9-dihydro-6-methyl-9-oxo-3-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)-
5H-imidazo[1,2-a]Purine (7)
Compound 7 was obtained by reacting 6 with acetic anhydride in pyri-
dine according to the literature method.[23] The product was isolated by
chromatography on a silica gel column with CH2Cl2–EtOH (98:2) as eluent
1
to obtain 7 (89%). H NMR (300 MHz DMSO-d6) δ 12.61 (brs, 1H, H5),
7.41 (d, 1H, H7), 6.01 (d, 1H, H1ꢁ), 6.20 (dd, 1H, H2ꢁ), 5.68 (t, 1H, H3ꢁ),

2-Methylwyosine, a Nucleoside with Restricted Anti Conformation
715
4.47–4.42 (dd, 1H, H5ꢁ), 4.39-4.34 (m, 1H, H4ꢁ), 4.25-4.19 (dd 1H, H5ꢁꢁ),
2.26 (d, 3H, CH3), 2.13, 2.07, 1.96 (3s, 9H, 3CH3 of acetyl).
8-Methylguanosine (3)
Compound 3 was synthesized according to the literature procedure de-
scribed at.[24] Crude product was chromatographed on a silica gel column
1
with CHCl3-MeOH (2:1) as eluant to obtain 3 (56%). H NMR (300 MHz
DMSO-d6) δ 10.64 (brs, 1H, N1–H), 6.34 (brs, 2H, NH2), 5.66 (d, 1H, H1ꢁ),
5.33, 5.13–5.08 (d, m, 1H, 2H, 2ꢁ–OH, 3ꢁ–OH i 5ꢁ–OH), 4.68 (dd, 1H, H2ꢁ),
4.10–4.06 (1H, m, H3ꢁ), 3.84 (1H, q, H4ꢁ), 3.67–3.60 (1H, m, H5ꢁ), 3.56–3.49
(1H, m, H5ꢁꢁ), 2.40 (3H, s, CH3)
3,9-Dihydro-2,6-dimethyl-9-oxo-3-(β-D-ribofuranosyl)-5H-imidazo [1,2-a]
Purine (4)
Variant A. Compound 4 was obtained by the reaction of 3 with bro-
moacetone and sodium hydride according to literature method.[25] The
dark red solutions was concentrated, poured into diethylether: acetone 3:1
and left for 12 h. The oily residue was decanted then dissolved in MeOH
and chromatographed on a silica gel column with CHCl3-MeOH (9:1) as
eluant to obtain 4 (67%).
Variant B. Compound 4 was synthesized by the reacting 6 with AlMe3
according to described procedure.[24] Crude product was chromatographed
on a silica gel column with CHCl3–EtOH (85:15) as eluent to obtain 4.
(50%) 1H NMR (300 MHz DMSO-d6) δ 12.33 (brs, 1H, N5–H), 7.34 (d, 1H,
H7), 5.75 (d, 1H, H1ꢁ), 5.37 (d, 1H, 2ꢁ–OH), 5.16 (d, 1H, 3ꢁ–OH), 5.15 (m,
1H, 5ꢁ–OH) 4.85 (q, 1H, H2ꢁ), 4.18–4,14 (m, 1H, H3ꢁ), 3.91 (q, 1H, H4ꢁ),
3.73–3.66 (m, 1H, H5ꢁ), 3.61–3.53 (m, 1H, H5ꢁꢁ), 2.47 (d, 3H, CH3), 2.25 (d,
3H, CH3). 13C NMR (75 MHz DMSO-d6) δ 150.5 (C9), 150.3 (C3a), 146.1
(C2), 144.6 (C4a), 126.1 (C6), 114.1 (C9a), 103.2 (C7), 88.0 (C1ꢁ), 85.5
(C4ꢁ), 70.9 (C2ꢁ), 70.4 (C3ꢁ), 61.8 (C5ꢁ), 14.6 (CH3), 10.4 (CH3).
3,9-Dihydro-2,6-dimethyl-9-oxo-3-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)-5H-
imidazo [1,2-a]Purine (5)
Variant A. Compound 5 was obtained by reacting 4 with acetic anhydride
in pyridine according to literature method.[23] Product was isolated by chro-
matography on a silica gel column with CHCl3–EtOH (97:3) as eluent to
obtain 5 (68%).
Variant B. A solution of 7 (0.53 g, 1 mmol) in THF (6 mL), PdC12
(0.018 g, 0.1 mmol), PPh3 (0.053 g, 0.2 mmol) and AlMe3 (1 mL, 2 mmol)
was refluxed for 24 h under Ar. The solvent was then removed and the
resulting off-white foam was chromatographed on a silica gel column with
CHCl3–EtOH (97:3) as eluent to obtain 5 (100 mg, 21%). 1H NMR (300 MHz
DMSO-d6) δ 12.40 (brs, 1H, H5), 7.35 (d, 1H, H7), 6.04 (d, 1H, H1ꢁ), 6.01

716
D. Baranowski et al.
(dd, 1H, H2ꢁ), 5.67 (t, 1H, H3ꢁ), 4.45–4.40 (dd, 1H, H5ꢁ), 4.36–4.31 (m,
1H, H4ꢁ), 4.28–4.22 (dd 1H, H5ꢁꢁ), 2.46 (d, 3H, CH3), 2.26 (d, 3H, CH3),
2.14, 2.06, 1.97 (3s, 9H, 3xCH3 of acetyl) 13C NMR (75 MHz DMSO-d6) δ
170.05, 169.58, 169.51 (3xC O) 150.5 (C9), 150.1 (C3a), 145.7 (C2), 144.9
(C4a), 126.2 (C6), 114.0 (C9a), 103.4 (C7), 86.2 (C1ꢁ), 78.8 (C4ꢁ), 71.3 (C2ꢁ),
69.9 (C3ꢁ), 62.8 (C5ꢁ), 20.4, 20.3, 20.3 (3xCH3 of acetyl), 14.2 (2CH3), 10.5
(6CH3)
4,9-Dihydro-9-oxo-2,4,6-trimethyl-3-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)
imidazo[1,2-a]Purine (8)
Compound 8 was obtained according to the literature method with mod-
ifications.[13] To a stirring solution of zinc iodide (2.4 g, 7.5 mmol) in diethyl
ether (30 mL) a saturated solution of diazomethane in diethyl ether (30 mL)
was added dropwise. The obtained suspension was quickly treated with so-
lution of 5 (0.23 g, 0.5 mmol) in CH2Cl2 (10 mL) and stirred for 30 min
at room temperature. After this time 1M aqueous ammonium bicarbonate
solution (30 mL) was added and left stirred for 20 min. Preciptitate was
filtered off and washed with CH2Cl2 (2 × 10 mL). Aqueous and organic
phases of the filtrate were separated, the former was extracted with CH2Cl2
(3×20 mL). Combined organic phases were evaporated, and the obtained
foam was chromatographed on a silica gel column with CHCl3–iPrOH (95:5)
as eluent to obtain 6 (52 mg, 22%). 1H NMR (300 MHz DMSO-d6) δ 7.38 (d,
1H, H7), 6.43 (d, 1H, H1ꢁ), 5.52 (t, 1H, H2ꢁ), 5.42 (dd 1H, H3ꢁ), 4.44–4.40
(m, 1H, H4ꢁ), 4.39 (s, 1H, H5ꢁ), 4.38 (s, 1H, H5ꢁꢁ), 4.06 (d, 3H, 4CH3), 2.57
(d, 3H, 2CH3), 2.23 (d, 3H, 6CH3), 2.12, 2.07, 2.06 (3s, 9H, 3xCH3 of acetyl).
13C NMR (75 MHz DMSO-d6) δ 169.98, 169.43, 169.17 (3xC O, acetyl),
151.03 (C9), 145.08 (C2), 142.62 (C4a), 141.28 (C3a), 137.37 (C6), 116.42
(C9a), 105.76 (C7), 85.73 (C1ꢁ), 78.67 (C4ꢁ), 71.23 (C2ꢁ), 67.54 (C3ꢁ), 62.09
(C5ꢁ), 20.47, 20.26, 20.06 (3xCH3, acetyl), 35.72 (4CH3), 16.29 (2CH3), 14.07
(6CH3).
4,9-Dihydro-9-oxo-2,4,6-trimethyl-3-(β-D-ribofuranosyl)imidazo[1,2-
a]Purine (9)
Compound 6 (50 mg, 0.11 mmol) was dissolved in 10 N anhydrous
methanolic ammonia (5 mL) and kept at room temperature for 1.5 h. The
solution was concentrated to ca 2 mL and left in a cool place. Crystalline ma-
terial that separated out from the methanol was collected by filtration. Yield
31 mg, 81%. 1H NMR (300 MHz DMSO-d6) δ 7.37 (d, 1H, H7), 6.06 (d, 1H,
H1ꢁ), 5.64 (d, 1H, 2ꢁ–OH), 5.31 (d, 1H, 3ꢁ–OH), 5.07 (t, 1H, 5ꢁ–OH), 4.35 (q,
1H, H2ꢁ), 4.08 (m, 4H, H3ꢁ, 4CH3), 3.88 (q, 1H, H4ꢁ), 3.73–3.67 (m, 1H, H5ꢁ),
3.67–3.61 (m, 1H, H5ꢁꢁ), 2.56 (d, 3H, 2CH3), 2.22 (d, 3H, 6CH3). 13C NMR
(75 MHz DMSO-d6) δ 151.01 (C9), 145.15 (C2), 142.63 (C4a), 141.38 (C3a),
137.21 (C6), 113.60 (C9a), 105.61 (C7), 88.30 (C1ꢁ), 85.41 (C4ꢁ), 72.15 (C2ꢁ),

2-Methylwyosine, a Nucleoside with Restricted Anti Conformation
717
68.47 (C3ꢁ), 60.58 (C5ꢁ), 34.55 (4CH3), 16.68 (2CH3), 14.02 (6CH3). MS m/z
calcd for C15H19N5O5 [M+H+] 350.35 found 350.3 [M+Na+] 372.34 found
372.2 MW = 349.35; Anal. calcd for C15H19N5O5×CH3OH Found: C, 50.32;
H, 5.79; N, 18.09 requires: C, 50.39; H, 6.08; N, 18.36
1,2,4,6-Tetramethyl-1,4-dihydro-9H-imidazo[1,2-a]purin-9-one (10)
1H NMR (300 MHz CDCl3) δ 7.37 (d, 1H, H7), 4.02 (s, 3H, 1CH3), 3.92
4
2
6
(s, 3H, CH3), 2.53 (d, 3H, CH3), 2.36 (d, 3H, CH3). 13C NMR (75 MHz
CDCl3) δ 151.71 (C9), 149.68 (C2), 148.19 (C4a), 142.69 (C3a), 138.14 (C6),
106.44 (C9a), 105.37 (C7), 32.00 (1CH3), 30.98 (4CH3), 14.36 (6CH3), 13.31
(2CH3)
MD and PSEUROT Calculations Procedure
In calculations standard parameters for a Karplus equation were applied
(NRIBOSEH). All five values PI, PII, φmaxI, φmaxII, XII were fitted to experi-
mental data (Table 1). Initial values of φmaxI, φmaxII equaled 40◦ and initial
XII was 50%. Moreover, initial values of pseudorotation phase angles PI and
PII changed independently from 0◦ to 351◦ every 9◦. For statistics only results
giving rms values less than 0.1 and φmaxI, φmaxII between 30◦ and 50◦, were
employed. Molecular dynamics calculations were done using CVFF potential
energy force field based on DISCOVER program (MSI package). Wyosine
and 2-methylwyosine molecules were built by means of BIOPOLYMER and
BUILDER modules (MSI). Each of these molecules had have two initial
conformations of the sugar moieties (North or South). After preliminary
energy minimalization, molecular dynamics were done in vacuum at 300◦K
for 100 ps (105 iterations). The last 50 structures (collected every single ps)
from any MD trajectories were minimized and statistically searched.
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