Janus-type at nucleosides: Synthesis, solid and solution state structures

Article abstract of DOI:10.1039/c1ob05577a

Novel Janus-type nucleoside analogues (1a-d) were synthesized. Their pyrimido[4,5-d]pyrimidine base moiety has one face with a bidentate Watson-Crick donor-acceptor (DA) H-bond array of adenine and the other face with an acceptor-donor (AD) H-bond array o

Full text of DOI:10.1039/c1ob05577a

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Janus-type AT nucleosides: synthesis, solid and solution state structures†
Mei-Ying Pan,a Wen Hang,a Xiao-Jun Zhao,a Hang Zhao,a Peng-Chi Deng,b Zhi-Hua Xing,a Yong Qinga and
Yang He*a
Received 12th April 2011, Accepted 17th May 2011
DOI: 10.1039/c1ob05577a
Novel Janus-type nucleoside analogues (1a–d) were synthesized. Their pyrimido[4,5-d]pyrimidine base
moiety has one face with a bidentate Watson–Crick donor–acceptor (DA) H-bond array of adenine and
the other face with an acceptor–donor (AD) H-bond array of thymine. These nucleosides may
self-associate through the self-complementary base pair. Indeed, in the solid state, compound 6d
displayed a honeycomb-like supramolecular structure with tetrameric membered cavities formed
through the combination of reverse Watson–Crick base pairs and aromatic stacking, in which the
solvent molecules were accommodated. The result of temperature-dependent CD studies showed that
the free nucleosides can form higher order chiral structures in aqueous solution.
a new symmetrical carbocyclic system.21 Previously, Janus-type
guanosine-adenine base (J-GA) was synthesized to recognize C
Introduction
and U simultaneously.22 Later, the self-complementary Janus-
type ganosine-cytosine base (J-GC) deriveatives were synthesized
and reported by Lehn and coworkers23 to form a particular
six-membered rosette structure. Mascal et al.24 also reported a
pyridopyrimidine derivative with hydrogen bonding codes of both
guanine and cytosine and confirmed its rosette structure by a
crystal state study. Recently, guanosine-cytosine derivatives with
amino acids or the crown ethers attached to the nitrogen atom
of the cytosine ring were found to form rosette helix nanotubes
in solution state.25–29 Perrin and coworkers30 reported another
self-complementary diaminopurine-thymine/uracil base (J-AT)
system, which is also a tridentate system with alkyl groups attached
to the nitrogen atom of the thymine ring. Through the crystal
studies, they were found to form ribbon-like structures.
DNA is one of the most tailorable and versatile molecules from
the “bottom-up” strategy point of view in the nanotechnology
field due to its unique structural motif and self-recognition
properties.1–4 The power of DNA as a molecular tool is also
enhanced by automated synthetic methods and by the PCR
technique to amplify programmed DNA sequences from micro-
scopic to macroscopic quantities. In addition, nature provides
a complete toolbox of highly specific enzymes that enable the
processing of the DNA material with atomic precision and
accuracy. By modifying the base pairing patterns, except for the
classical right-handed double-helix with Watson–Crick base pairs,
polymorphic structural features5–7 can be obtained which expands
the application of DNA in the field of nanotechnology to great
extent. For example, a cyclic trimeric8 supramolecular structure
based on guanosine-cytidine dinucleoside has been reported.
Tetrameric5,7,9–12 structures based on G-quartet have been used
to construct nanowires,13–15 biosensors,16 ion pair receptors12 and
transmembrane ion transporters.10,14,17 isoG cyclic pentamers with
selective cation binding properties were also reported.18–20
Apart from the purine and pyrimidine derivatives, other het-
erocyclic systems can be employed as well to construct special
supramolecular structures based on different base pair motifs.
For example, Janus-type nucleobases were used as building blocks
to form some unique supramolecular structures. Janus molecules
(from the Roman god Janus) were first proposed to describe
aLaboratory of Ethnopharmacology, Institute for Nanobiomedical Technol-
ogy and Membrane Biology, Regenerative Medicine Research Center, West
China Hospital, West China Medical School, Sichuan University, Chengu,
610041, China. E-mail: heyangqx@yahoo.com.cn; Tel: +86 2885164077
Scheme 1 Molecular structures and atom numbering of bidentate J-AT
nucleosides and tridentate J-GC nucleoside. The arrows of A represent
the hydrogen acceptors; the arrows of D represent the hydrogen donors.
R = D-b-ribose (1a), R = L-b-ribose (1b), R = D-b-2¢-deoxyribose (1c), R =
D-a-2¢-deoxyribose (1d).
bAnalytical & Testing Center, Sichuan University, Chengdu, 610064, China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c1ob05577a
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In order to employ this special base pairing of dual-hydrogen-
pattern systems in the field of DNA (or RNA), the correspond-
ing nucleosides are needed. However, there are few antecedent
examples in which the Janus-type base attaching to a ribose or 2¢-
deoxyribose have been synthesized and investigated. Therefore, J-
GC nucleoside has been synthesized in our laboratory which shows
a preliminary antiviral activity.31 Also it shows a similar nanotube-
shaped structure in solution like alkylated Janus-type GC deriva-
tives and the results will be reported elsewhere. Accordingly, we
would like to expand this tridentate J-GC nucleoside system
to a bidentate J-AT nucleoside system. For GC pyrimido[4,5-
d]pyrimidine system, only the Watson–Crick base pair motif is
feasible to maintain the tridentate H-bond array, which naturally
leads to the formation of rosette structure just as reported.23,32
Different from that, in the case of the AT system except for
Watson–Crick base pairing (Scheme 2), the reverse Watson–
Crick base pairing is also possible (Scheme 2). In the purine and
pyrimidine system, the reverse Watson–Crick AT base pair will
lead to the formation of parallel-stranded DNA which has been
proven experimentally.33–35 The energy calculation also showed a
small difference between the parallel-stranded and the normal
antiparallel-stranded duplex structures.35,36 Consequently, for the
AT pyrimido[4,5-d]pyrimidine system, if the Watson–Crick base
pair is adopted a rosette structure will be formed; on the other
hand if the reverse Watson–Crick base pair is adopted an infinite
linear tape structure will be formed (Scheme 2). Therefore, it is very
interesting to investigate which base pairing motif will dominate in
these Janus-type AT nucleosides. For this purpose, we synthesized
the Janus-type AT nucleosides 1a–d (Scheme 1) which have never
been reported before and studied their structures both in solid
state and solution state.
Results and discussion
Chemical synthesis
There are two major approaches for the synthesis of nucleosides.
The first approach employs the corresponding base derivatives
reacting with an appropriate sugar component (convergent gly-
cosylation reaction). The second one involves the construction
of a purine or pyrimidine system from a simple N-glycosylated
precursor (linear method). We intended to adopt the direct
Scheme 2 Base pair motifs (R represents sugar moieties).
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glycosylation reaction through Vorbrueggen method37 using cor-
responding silylated Janus-type AT base moiety. To prepare the
nucleobase 4, we adopted the reported procedure employing N-
bis(methylthio)methylenecyanamide as starting material.38 How-
ever, during the synthesis we could not obtain the base 4 in larger
scale as stated in the reference. The initial precipitate obtained in
the reaction was not the final product we desired and probably
with the structure of 3 according to its molecular weight of
152.03 from the mass spectra. So, after washing it with methanol,
this intermediate was treated further with formamide, then pure
compound 4 was obtained as yellowish powder precipitated from
methanol (Scheme 3). Next, for silylation, base 4 and HMDS were
refluxed with catalytic amount of TMSCl. The completion of the
silylation of 4 was indicated by clarification of the reaction mixture
and the excess of HMDS was removed by evaporation. The
silylated base moiety 5 was used immediately in the glycosylation
step due to its sensitivity to moisture. The glycosylation was
accomplished by treating the compound 5 with 1-O-acetyl-2,3,5-
tri-O-benzoyl-b-D-ribofuranose at the presence of Lewis acid
catalyst SnCl4. The reaction leads to the formation of main product
6a (Scheme 4). Its structure was studied with X-ray analysis
(Fig. 1a) in which we found the sugar moiety attached to the N8
atom of adenine ring. The reason of regioselectivity in coupling
of heterocyclic base and sugar may be due to steric hindrance
of the thymine carbonyl group. To remove the protecting group,
compound 6a was treated with 0.5 M sodium methoxide. The
structure of obtained compound 1a was confirmed with 1H and
13 C HMQC and HMBC NMR spectroscopy. A similar procedure
was used for synthesis of J-AT L-ribonucleoside (Scheme 4).
The silylated base 5 was treated with 1,2,3,5-O-tetra-acetyl-b-L-
ribofuranose employing catalyst SnCl4 followed by deprotection,
the corresponding L-nucleoside 1b was afforded. In the case of L-
nucleoside 1b, the same structure determination procedure using
NMR techniques was employed as in the case of D-ribonucleoside
1a. Compound 1b was assigned to be the L-b-nucleoside which
indicated that the sugar ring was connected to the adenine ring.
For the synthesis of 2¢-deoxyribonucleosides, the same silylated
base 5 was used as the acceptor and 1-a-chloro-3,5-di-O-toluoyl-
2-deoxy-D-ribofuranose was used as donor. In the case of riboside,
because of the steric hinderance of the 2¢-acyloxy group, only b
anomer nucleoside was formed. However, in the case of deoxyri-
bonucleoside, without the neighboring-group participation, usu-
ally 1 : 1 mixtures of a : b anomers will be formed when employing
the Vorbrueggen’s glycolsylation conditions.37 It was reported that
CuI in CHCl3 can facilitate the b-configuration selectivity with a
electrical push-pull process for the normal purine and pyrimidine
system.39 However, this method did not give satisfactory result in
this pyrimido[4,5-d]pyrimidine system. We had tried the reaction
Scheme 3 Synthesis of J-AT nucleobase: (i) DMSO, methyl cyanoacetate, K2CO3, 4 h, 10% HCl, 95%. (ii) 10% NaOH, r.t., 2 h, 70 ◦C, 10 min, 93%. (iii)
Formamide, 180 ◦C, 4 h, 80%. (iv) Formamide, 180 ◦C, 4 h, 80%.
Scheme 4 Synthesis of J-AT D- and L-ribonucleosides: (i) HMDS, TMSCl, reflux. (ii) Dry acetonitrile, SnCl4, 45%. (iii) Dry acetonitrile, SnCl4 50%. (iv)
0.5 M MeONa, reflux, 80%. (v) 0.1 M MeONa, reflux, 93%.
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Fig. 1 A perspective view of compounds 6a (a) and 6d (b) showing the atom-numbering and N8 atom on the adenine ring is connected to C1¢-atom of
sugar residues.
of silylated base 5 by treating with 1-a-chloro-3,5-di-O-toluoyl-
2-deoxy-D-ribofuranose and with CuI as catalyst, however both
anomeric isomers 6c and 6d were obtained in equal amounts and
the total yield was poor. Later on, when using SnCl4 as catalyst,
the yield of the products was poor as well. Therefore, we finally
chose Lewis acid trimethylsilyl triflate as catalyst which provided
better total yield than either CuI or SnCl4. However, due to the
similar polarity of compound 6c and 6d, we failed to separate them
by flash column chromatograph using different eluent systems.
Even the protecting groups on sugars were removed, it was also
not feasible to separate a and b isomers of the free nucleosides.
The separation of them was achieved by introducing DMT group
to 5¢-OH group of the nucleosides later on. The compounds
7c and 7d with DMT attached can be separated by column
chromatography. Subsequently, the DMT group was removed by
treating 7c and 7d with 2.5% DCA methylene chloride solution
(2.5 mL DCA was added in 10 mL methylene chloride), and
the free nucleosides 1c and 1d were obtained as white powders
(Scheme 5). Considering the laborious process of such operations,
we tried to find another convenient method. After numerous
attempts with different solvents, finally we found 6c can precipitate
as a white powder from ethyl acetate while 6d will be remained
in solution. Subsequently, 1c and 1d were obtained by removing
the toluoyl group accordingly. The regioselectivity and anomeric
configuration of 6d were proven by X-ray analysis (Fig. 1b). It was
identified as the D-a-deoxyribonucleoside and the sugar moiety
was also attached to the N8 atom of the adenine ring (Fig. 1b).
For compound 6c, we failed to obtain a good quality single crystal
1
for X-ray diffraction studies. Therefore, we employed the H-13C
HMQC and 1H-13C HMBC NMR spectroscopy to determine the
configuration of compound 1c to be D-b-2¢-deoxyribonucleoside.
Solid state structure
The single crystals of compounds 6a and 6d were obtained and
some interesting structural differences in their solid state can be
seen from the X-ray diffraction studies. The structure and atom
numbering are shown in Fig. 1. Compound 6a crystallized slowly
from methanol while compound 6d crystallized from methylene
chloride/ethanol solution after the failure of crystallization from
methanol. The detailed structural parameters of 6a have been
published elsewhere.40 The nucleobase ring of 6d is nearly planar
˚
just as 6a. The deviations of ring atoms are less than 0.02 A for each
pyrimidine ring and the dihedral angle between the two rings is
2.03(8)◦. The biggest deviation of exocyclic groups from the rings
˚
is -0.084(6) A (O12). The orientation of the nucleobase relative to
the sugar ring (anti/syn conformation) in normal N9-glycosylated
purine nucleoside system is defined by the torsion angle c (O4¢–
C1¢–N9–C4).41 When the pyrimidine ring of the purine is located
outside the sugar plane, the conformation is defined as anti; on
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Scheme 5 Synthesis of J-AT 2¢-deoxy-ribonucleosides: (i) Dry 1,2-dichloroethane, trimethylsilyl triflate, 34%. (ii) 0.2 M MeONa, reflux, 80%. (iii) Dry
pyridine, DMT-Cl, triethylamine, DMAP, overnight at r.t., 81%. (iv) 2.5% of DCA in CH2Cl2 r.t., 50%.
the other hand, when it is located above the sugar plane, the
conformation is defined as syn. In the case of 6d, the anti/syn
conformation is also defined by the torsion angle c (O4¢–C1¢–
N8–C9) and the value is 173.6 (3)◦, with the thymine ring located
outside the sugar ring. Therefore, 6d adopts an anti conformation
which is consistent with that of 6a. However, there is a difference in
the sugar puckering mode between 6a and 6d. 6a adopts an N-type
(3¢-endo) conformation with a twist of C3-endo (3T4) P = 24.5 (2)◦,
tm = 38.3 (2)◦, a typical sugar puckering mode for ribonuleosides.
6d adopts an N-type (2¢-endo) conformation, with an asymmetrical
twist of (2T1), P = 159.1 (2)◦, tm = 30.9 (2)◦ which is a typical sugar
puckering mode for 2¢-deoxynucleosides.
The most striking difference between the two compounds was
displayed in their hydrogen bond patterns. For compound 6a,
each nucleoside molecule contains an intramolecular H-bond
formed between the bifurcated H11A atom and O12 atom and
six intermolecular hydrogen bonds (Fig. 2a). The intermolecular
hydrogen bonds N3–H3 ◊ ◊ ◊ N6 (formed directly between the
thymine and adenine faces), N11–H11A ◊ ◊ ◊ O7¢ together with the
bridged methanol act as the repeated units connecting adjacent
nucleosides head to head in an anti-parallel way to form an infinite
one dimension tape with a nearly perpendicular dihedral angle
between the adjacent nucleobase planes. For compound 6d, each
nucleoside molecule contains an intramolecular hydrogen bond
Fig. 2 A detailed view of the H-bonds on compounds 6a (a) and 6d (b). H-bonds are shown in dashed lines. Atoms are coded as follows: red, oxygen;
blue, nitrogen; gray, carbon. H atoms and some groups which are not involved in H-bonds have been omitted for clarity.
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which is the same as in 6a and five intermolecular hydrogen bonds.
The intermolecular hydrogen bonds are formed between the faces
of adenine (A) and thymine (T): (A) N11–H11A ◊ ◊ ◊ O13 (T)
and (A) N3–H3 ◊ ◊ ◊ N6 (T). They connected adjacent nucleoside
molecules to form an infinite wavy tape in typical reverse Watson–
Crick base pair pattern. The distance between the hydrogen bond
of A (H11B) and T (O13) is 0.211 nm and the distance between
another hydrogen bond of A (N6) and T (H3) is 0.194 nm, which
were just located in the reported hydrogen bonds range of 0.19–
0.22 nm for the reverse Watson–Crick AT base pairs.34
The supramolecular structure for 6a and 6d was also different. In
case of 6a, the one dimensional tapes were arranged in parallel to
form an infinite two-dimensional pleated sheet structure through
the aromatic stacking between the nucleobase plane and the
benzene ring of the benzoyl protecting group on furanoribose
5¢-OH with a distance of 0.35 nm (Fig. 3a1). Comparing to the
pleated sheet structure of 6a, a quite different structure was formed
by 6d. The neighboring two tapes were associated together through
two types of aromatic stacking (Fig. 3b1 and 3b2). One such
stacking mode is a sandwich shape with the 3¢-benzoyl groups
located at the bottom, the nucleobase plane of the reverse Watson–
Crick base pair from the same tape inserted into the middle and
the 5¢-benzoyl groups from adjacent tape located on the top of
the base pair (Fig. 3b1). The other stacking is also in a sandwich
shape but with the base pair on the top, 3¢-benzoyl groups in
the middle, and the 5¢-benzoyl groups of next nucleoside from
adjacent tape located below the 3¢-benzoyl group (Fig. 3b2). The
distance of the aromatic stacking is 0.34 nm, exactly the same as
the base pair stack distance in the DNA duplexes. As a whole, both
aromatic stacking and the reverse Watson–Crick base pair tethered
four 6d molecules together to form a rectangular cavity with two
water molecules and two ethanol molecules located inside. This
cavity was repeated to form an infinite honeycomb-like network
structure.
Solution state structure
For free nucleosides 1a–d, without aromatic stacking of the
protecting groups, in principle only the base pairs can be used
for self-assembly. In order to explore if they can, in fact, form
the regular structures like the tridentate-hydrogen-bonds J-GC
system, we investigated their self-assemble property in solution
state. Earlier, the variable temperature 1HNMR (VT NMR)
spectrometry has been used to investigate the self-assembly of the
J-GC base derivatives in toluene.23 Therefore, we employed this
VT NMR technique to study the hydrogen bonds formation of
compounds 1a and 1d in DMSO. As can be seen from Fig. 4, for
compound 1a, the chemical shift of the hydrogen atom of imino
group on the thymine ring (N3H) moves from d 10.80 (293 K) to
d 10.57 ppm (333 K) with Dd = 0.23 ppm; and the chemical shift
for N11H atom of the amino group on the adenine ring moving
from d 8.94 (293 K) to d 8.75 ppm (333 K) with Dd = 0.19 ppm.
These results indicate that these hydrogen atoms participating in
the intermolecular hydrogen bonds formation. Another hydrogen
atom from the amino group on the adenine ring at d 8.96 ppm
was assigned to be the intramolecular hydrogen bond since its
chemical shift remains unchanged. For compound 1d, similar
chemical shift changes were observed, with Dd = 0.24 ppm (from
293 K to 333 K) for N3H and Dd = 0.20 ppm (from 293 K to
Fig. 3 The pleated supramolecular structure formed by one dimensional
tapes in compounds 6a (a) and the honeycomb-like structure formed by one
dimensional tapes in compound 6d (b). The aromatic stacking interaction
between the tapes in 6a (a1). Two types of p-stacking interactions between
the tapes in 6d (b1), (b2). With the exception of the green p-stacking planes,
all atoms are coded as follows: red, oxygen; blue, nitrogen; gray, carbon.
The H atoms were omitted for clarity.
Fig. 4 The variable temperature 1HNMR spectra of compounds 1a (a) and 1d (b).
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Fig. 5 (a) Temperature-dependent CD spectra of unbuffered aqueous solution for compound 1d (3.69 ¥ 10-4 mol L-1). The arrow indicates the trend of
temperature increasing: 5.0 ◦C, 15.0 ◦C, 25.0 ◦C, 35.0 ◦C, 45.0 ◦C, 50.0 ◦C, 55.0 ◦C, 60.0 ◦C, 65.0 ◦C, 70.0 ◦C, 75.0 ◦C, 80.0 ◦C, 85.0 ◦C; temp. ramp:
1 ◦C min-1. Wavelength start: 360 nm, wavelength end: 220 nm, wavelength step: 1 nm. (b) Temperature dependence of 1d at 284 nm. Tm: 67.2 ◦C.
333 K) for N11H. Another N11H atom (d 8.88 ppm) in amino
group on the adenine ring participating in the intramolecular
hydrogen bond remained unchanged upon the increasing of the
temperature. Therefore, the VT NMR studies indicated the self-
assembly structure of compounds 1a and 1d in DMSO were
formed through hydrogen bonds. Next, in order to study whether
some higher order structures will be formed by compounds 1a–d
in aqueous solution like the J-GC derivatives, the temperature-
dependent CD spectra were performed.
was also formed by the bidentate J-AT deoxyribonuleosides in
solution state. In the case of J-AT ribonucleosides, their CD
spectra also displayed positive and negative Cotton effects and
the D-isomer (1a) and L-isomer (1b) gave a spatial mirror image
(Fig. 7) due to their enantiomeric character of the sugar residues.
On the contrary, their CD peaks of maximum and minimum
were not vanished as the temperature increasing from 5 ◦C to
◦
85 C. This phenomenon indicates that the structures of 1a and
1b formed in aqueous solution may be more stable than that
of compounds 1c and 1d, the Tm values are higher than 85 ◦C;
or the structure of ribonucleosides 1a and 1b formed in water
is very different to that of the deoxyribonucleosides 1c and 1d.
To solve this problem other techniques are needed, the suitable
conditions for further X-ray structure determination are under
investigations.
In the case of 2¢-deoxyribonucleosides 1c, 1d, CD spectra
displayed the positive and negative Cotton effect. With the tem-
perature changes, a temperature dependent melting phenomenon
was observed. The maximum (253 nm) and minimum (284 nm)
of CD spectra for compound 1d (Fig. 5) disappeared completely
◦
upon the temperature rising to 85 C. By plotting the CD signal
at 284 nm against the temperature, a sigmoid melting curve was
obtained with the Tm value to be 67.2 ◦C. For compound 1c,
a similar phenomenon was observed which gave a Tm value of
64 ◦C (Fig. 6). The phenomenon in CD spectra is similar to that
of the J-GC derivatives indicating a higher order chiral structure
Conclusion
We synthesized a series of novel Janus-type AT ribonucleo-
sides and 2¢-deoxyribonucleosides by Vorbrueggen glycosylation
Fig. 6 (a) Temperature-dependent CD spectra of unbuffered aqueous solution for compound 1c (3.39 ¥ 10-4 mol L-1), The arrow indicates the trend of
temperature increasing: 5.0 ◦C, 15.0 ◦C, 25.0 ◦C, 35.0 ◦C, 45.0 ◦C, 50.0 ◦C, 55.0 ◦C, 60.0 ◦C, 65.0 ◦C, 70.0 ◦C, 75.0 ◦C, 80.0 ◦C, 85.0 ◦C; temp. ramp:
1 ◦C min-1. Wavelength start: 360 nm, wavelength end: 220 nm, wavelength step: 1 nm. (b) Temperature dependence of 1c at 256 nm. Tm: 64 ◦C.
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cuvettes, lmax in nm, e in dm3 mol-1 cm-1. CD spectra were recorded
on circular dichroism spectrometer (Aviv Biomedical, Inc.). Proper
size single crystals of 6a and 6d were stabilized into a tiny glass tube
including mother liquor with epoxy resin to minimize solvent loss.
Crystal Data were collected on CAD4SDP-44 M/H instrument
(Xcalibur, Eos, Oxford Diffraction Ltd.).
5-Amino-8-(2,3,5-tri-O-benzoyl-b-D-furanosyl)
pyrimido[4,5-d]pyrimidine-2,4(3H,8H)-dione (6a)
Compound 4 (600 mg, 3.3 mmol) was suspended in Hexamethyl-
disilazane (HMDS) (30 mL) and stirred at 140 ◦C for about
3 min, then trimethylsiyl chloride (TMSCl) (600 mL, 4.7 mmol) was
added, the reaction was stirred at reflux for 18 h until the mixture
was clear, then the solution was evaporated to remove HMDS,
the silylated base 5 was obtained which was immediately used
in next step without further purification. Dry 1,2-dichloroethane
(20 mL) was added in the pot containing silylated base 5 and
stirred. 1-O-acetyl-2,3,5-tri-O-benzoyl-b-D-ribofuranose (630 mg,
1.6 mmol) dissolved in dry acetonitrile (20 mL) was added in this
solution above. SnCl4 (550 mL, 4.2 mmol) was added as catalyst
at 0 ◦C. After the mist vanished, the reaction stirred at room
temperature. 0.5 h later, saturated NaHCO3 aqueous solution
Fig. 7 CD spectra of unbuffered aqueous solution for compound 1a
(3.37 ¥ 10-4 mol L-1) and compound 1b (3.47 ¥ 10-4 mol L-1). Temp. start:
5
◦C, end: 85 ◦C, temp. step: 10 ◦C, temp. ramp: 1 ◦C min-1; wavelength
start: 360 nm, wavelength end: 220 nm, wavelength step: 1 nm.
reaction using silylated AT pyrimido[4,5-d]pyrimidine base moi-
eties and the corresponding sugar donors. In the solid state,
depending on the variation of sugar residues and solvent en-
vironment different supramolecular structures will be formed.
Especially, with the combination of reverse Watson–Crick base
pairs and the aromatic stacking from sugar hydroxyl protecting
groups, compound 6d can form a honeycomb-like supramolecular
network structure with rectangle cavities containing two water
molecules and two ethanol molecules inside. From the NMR
studies, the hydrogen bonds were maintained in DMSO for both
free ribonuleosides and 2¢-deoxyribonuleosides. According to the
CD spectra, the nucleoside monomers can form a higher order
chiral suprastructure in aqueous solution. If these nucleoside
monomers are incorporated into the oligonucleotides, with the
help of phosphodiester and sugar backbone and the base pair
stacking, whether some interesting structures will be formed at
the DNA/RNA level is a very interesting topic to be explored.
The relevant work is under investigation and will be reported in
due time.
◦
(40 mL) was added at 0 C to quench the reaction; CH2Cl2 (3 ¥
40 mL) was used to extract the organic phase, after dried with
anhydrous Na2SO4, the organic phase was evaporated. The residue
was applied to F.C. (silica gel, column 4 ¥ 8 cm, elution with
CH2Cl2 to CH2Cl2 : methanol 98 : 2) and afforded 6a (940 mg,
45%): UV (MeOH): 225 (95658), 251 (27263), 273 (15395), 278
(14355); dH (600 MHz; d6-DMSO): 4.73–4.75 (2H, t, J = 6 Hz, 5¢-
H2), 4.82–4.84 (1H, t, J = 6 Hz, 4¢-H), 6.12–6.13 (1H, d, J = 6 Hz,
3¢-H), 6.17–6.20 (1H, t, J1 = 12 Hz, J2 = 6 Hz, 2¢-H), 6.51 (1H, s,
1¢-H), 7.40–7.99 (15H, m, H-arom), 8.66 (1H, s, CH), 9.07 (1H, s,
NHa) 9.14 (1H, s, NHb), 10.87 (1H, s, NH). dC (600 MHz; d6-
DMSO): 64.41, 71.12, 74.68, 80.25, 87.59, 129.00, 129.05, 129.13,
129.20, 129.62, 129.73, 129.78, 129.89, 133.98, 134.27, 134.37,
153.06, 156.72, 157.70, 162.03, 164.99, 165.13, 165.31. HRMS
(ESI+) m/z: Calc. for C32H25N5O9: 646.1550 [M+Na]+. Found
646.1549 [M+Na]+.
5-Amino-8-(b-D-furanosyl) pyrimido[4,5-d]pyrimidine-
2,4(3H,8H)-dione (1a)
Experimental
Compound 6a (80 mg, 0.13 mmol) was suspended in solution of
0.5 M NaOMe/MeOH (10 mL), the reaction continued at reflux
for 20 min, the mixture solution turned to be gel attached to
the wall of the glass pot. After cooling to room temperature, the
gel was neutralized with diluted acetic acid to about pH 6.5 and
filtered; the precipitate obtained was washed with methanol (2 ¥
10 mL) and water (1 ¥ 10 mL). After vacuum drying, 1a was
obtained (32 mg, 80%): UV (MeOH): 251 (25943), 277 (5681); dH
(600 MHz; d6-DMSO): 3.60–3.80 (2H, m, 5¢-H2), 3.94–3.97 (1H,
m, 4¢-H), 4.08–4.17 (2H, m, 3¢-H, 2¢-H), 5.08–5.10 (1H, d, J =
12 Hz, 5¢-OH), 5.27–5.29 (1H, t, J = 6 Hz, 3¢-OH), 5.55–5.56 (1H,
d, J = 6 Hz, 2¢-OH), 6.17–6.18 (1H, d, J = 6 Hz, 1¢-H), 8.88 (1H, s,
CH), 8.92 (1H, s, NHa), 8.98 (1H, s, NHb), 10.77 (1H, s, NH). dC
(600 MHz; d6-DMSO): 60.04, 69.04, 74.98, 85.06, 87.44, 90.26,
151.88, 157.00, 158.18, 162.00, 165.44. HR-MS (ESI-) m/z: Calc.
for C11H13N5O6: 310.0788 [M - H]-. Found 310.0787 [M - H]-.
General
All chemicals were bought from Sigma–Aldrich. The solvent and
reagent are analytic pure. Solvents 1,2-dichloroethane and acetoni-
trile have been purified by distilling from phosphorus pentoxide.
Pyridine is treated with calcium hydride in a similar way. Thin-layer
chromatography (TLC) is performed on aluminium sheet covered
with silica gel 60 F254 (0.2 mm, Merck, Germany). Flash column
chromatography (FC): silica gel 60 (Haiyang chemical company,
P. R. China) at 0.4 bar. The new compounds were characterized by
NMR, high-resolution ESI Mass spectra and UV-spectra. NMR
spectra were recorded on a AV II (Bruker, Germany) spectrometer
at 400 MHz and 600 MHz. ESI mass spectra were performed
on a mass spectrometer (Q-TOF-premier, Waters company, US),
the UV/vis absorption spectra were collected on a DU-800
spectrophotometer (Beckman, US) using 1 cm path length quartz
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5-Amino-8-(2,3,5-tri-O-benzoyl-b-L-furanosyl)pyrimido[4,5-
d]pyrimidine-2,4(3H,8H)-dione (6b)
(2 mL, 15.8 mmol) was added as catalyst, the reaction was stirred at
reflux until the mixture solution became clear, and then evaporated
to remove HMDS, the silylated base was obtained which was used
for next step without further purification. The silylated base 5
was dissolved in dry 1,2-dichloroethane (60 mL), to which 1-a-
chloro-3,5-di-O-toluoyl-2-deoxy-D-ribofuranose (3 g 7.7 mmol)
dissolved in dry acetonitrile (60 mL) was added, trimethylsilyl
triflate (TMSOTf) (1 mL, 5.6 mmol) was added at 0 ◦C, the
reaction continued at room temperature for about 1.5 h. Saturated
NaHCO3 aqueous solution (120 mL) was added to quench the
reaction at 0 ◦C, CH2Cl2 (3 ¥ 120 mL) was used to extracted
organic phase. The organic phase was combined and dried with
anhydrous Na2SO4, then evaporated; the residues were applied to
FC (silica gel, column 4 ¥ 10 cm, elution with CH2Cl2 to CH2Cl2 :
methanol 99 : 1 to 98 : 2), afforded a mixture of 6c and 6d as light
yellow powder (2 g, 34%), and the mixture powder was dissolved
in ethyl acetate, 6c was separated as a precipitate, the supernate
was evaporated to furnish the compound 6d (at a ratio of 6c:6d ª
2 : 1).
Compound 6c: UV (MeOH) 247.0 (25867), 274.0 (4481). dH
(600 MHz; d6-DMSO): 2.37, 2.40 (6H, 2 s, 2CH3), 2.72–2.74 (1H,
t, J = 6 Hz, 2¢-Ha), 2.816–2.827 (1H, t, J = 4.2 Hz; 2¢-Hb), 4.66–
4.70 (3H, m, 4¢-H, 5¢-H2), 5.62–5.64 (1H, t, J = 6 Hz, 3¢-H), 6.53–
6.55 (1H, t, J = 6 Hz; 1¢-H), 7.30–7.94 (8H, m, H-arom), 8.57
(1H, s, CH), 8.970–8.974 (1H, d, J = 2.4 Hz, NHa), 9.01 (1H, s,
NHb), 10.83 (1H, s, NH); dC (600 MHz; d6-DMSO): 21.65, 21.69,
38.62, 64.69, 75.21, 83.07, 87.36, 87.48, 126.89, 129.72, 129.82,
129.93, 144.41, 144.57, 151.02, 156.96, 157.73, 162.01, 165.34,
165.75, 165.93. HR-MS (ESI+): Calc. for [C27H25N5O7]: 553.1573
[M+Na]+; Found 554.1623 [M+Na]+.
Compound 4 (4.2 g, 23.4 mmol) was suspended in HMDS
◦
(300 mL) and stirred at 140 C for about 3 min, TMSCl (4 mL,
31.6 mmol) was added as catalyst, and the reaction was refluxed
with exclusion of humidity for 7 days until it became clear. Then,
the solution was evaporated to remove HMDS. Compound 5 was
afforded, which was used for the next step immediately without
further purification. The silylated base 5 was dissolved in dry
1, 2-dichloroethane (200 mL) and stirred at room temperature.
To which 1,2,3,4-tetra-O-acetyl-b-L-ribofuranose (3.8 g 12 mmol)
suspended in dry acetonitrile (200 mL) was added. SnCl4 (4 mL,
30.3 mmol) was added as catalyst at 0 ◦C. After the mist vanished,
the reaction mixture was stirred at room temperature for 1.5 h.
Then the acetonitrile was removed by evaporating, the reaction
mixture was diluted with (3 ¥ 200 mL) CH2Cl2 to extract the
organic phase, and then cold saturated NaHCO3 aqueous solution
(200 mL) was added to wash the organic phase. The combined
organic phase was dried with anhydrous Na2SO4 and evaporated,
the residue was applied to FC (silica gel, column 5 ¥ 10 cm, elution
with CH2Cl2 to CH2Cl2 : methanol 98 : 2), furnishing compound
6b (5.8 g, 50%): UV (MeOH): 251 (16690), 231 (11163), 278 (4319).
dH (600 MHz; d6-DMSO): 2.05–2.08 (9H, t, 3OAc), 4.32–4.40 (3H,
m, 4¢-H, 5¢-H2), 5.53–5.56 (1H, t, J1 = 8 Hz, J2 = 4 Hz, 3¢-H), 5.64–
5.67 (1H, q, J = 4 Hz, 2¢-H), 6.27–6.28 (1H, d, J = 4 Hz, 1¢-H),
8.56 (s, 1H, CH), 9.06–9.07 (1H, d, J = 4 Hz, NHa), 9.15–9.16 (1H,
d, J = 4 Hz, NHb), 10.85 (s, 1H, NH). dC (600 MHz; d6-DMSO):
20.74, 20.77, 20.98, 63.42, 70.15, 73.39, 80.36, 87.41, 89.88, 152.58,
156.70, 157.69, 161.93, 165.28, 169.74, 169.86, 170.51. MS (ESI-)
m/z: Calc. for C17H19N5O9: 436.1104 [M - H]-. Found: 436.1102
[M - H]-.
Compound 6d: UV (MeOH) 226.0 (139822), 203.0 (135088),
272.0 (22378), 278.0 (20111), dH (600 MHz; d6-DMSO): 2.36, 2.40
(6H, 2 s, 2CH3), 2.60–2.63 (1H, d, J = 18 Hz, 2¢-Ha), 2.97–3.00
(1H, q, J = 6 Hz, 2¢-Hb), 4.50–4.51 (2H, d, J = 6 Hz, 5¢-H2), 5.28–
5.30 (1H, t, J = 6 Hz, 4¢-H), 5.57–5.58 (1H, d, J = 6 Hz, 3¢-H),
6.47–6.48 (1H, d, J = 6 Hz, 1¢-H), 7.25–7.94 (8H, m, arom-H),
8.65 (1H, s, CH), 8.89–8.90 (1H, d, J = 6 Hz, NHa), 8.947–8.953
(1H, d, J = 3.6 Hz, NHb), 10.78 (1H, s, NH); dC (600 MHz; d6-
DMSO): 21.63, 21.67, 64.37, 75.18, 85.48, 87.66, 89.63, 126.79,
127.00, 129.62, 129.77, 129.82, 129.92, 144.39, 144.47, 150.98,
156.98, 157.84, 162.24, 165.37, 165.92. HR-MS (ESI+): Calc. for
[C27H25N5O7]: 553.1573 [M+Na]+; Found 554.1623 [M+Na]+.
5-Amino-8-(b-L-furanosyl)pyrimido[4,5-d]pyrimidine-2,4(3H,8H)-
dione (1b)
Compound 6b (2 g, 4.5 mmol) was suspended in 0.2 M
NaOMe/MeOH (50 mL), the mixture reaction was stirred at
room temperature until the compound 6b disappeared monitored
by TLC. Then the reaction solution was neutralized with diluted
acetic acid, filtered and the precipitate was washed with methanol
(3 ¥ 50 mL), after the vacuum drying, giving compound 1b (1.3 g,
93%): UV (MeOH) 226 (47903), 251 (22074), 272 (9568). dH
(600 MHz; d6-DMSO): 3.60–3.63 (1H, m, 5¢-Ha), 3.76–3.79 (1H,
m, 5¢-Hb), 3.94–3.96 (1H, m, 4¢-H), 4.08–4.09 (1H, d, J = 6 Hz,
3¢-H), 4.14–4.15 (1H, d, J = 6 Hz, 2¢-H), 5.10–5.11 (1H, d, J =
6 Hz, 5¢-OH), 5.28–5.30 (t, J = 6 Hz, 1H, 3¢-OH), 5.57–5.58 (1H,
d, J = 6 Hz, 2¢-OH), 6.15–6.16 (1H, d, J = 2 Hz, 1¢-H), 8.87 (1H, s,
CH), 8.92 (1H, s, NHa), 8.97 (1H, s, NHb), 10.79 (s, 1H, NH). dC
(600 MHz; d6-DMSO): 59.98, 68.97, 74.96, 85.01, 87.45, 90.27,
151.86, 157.06, 158.16, 161.98, 165.43. MS (ESI-) m/z: Calc. for
C11H13N5O6: 310.0787 [M - H]-. Found: 310.0793 [M - H]-.
5-Amino-8-(2-deoxy-b-D-erythro-pentofuranosyl)pyrimido[4,5-
d]pyrimidine-2,4(3H,8H)-dione (1c)
Method 1: compound 6c (820 mg, 1.5 mmol) was suspended in
0.2 M NaOMe/MeOH (40 mL), the reaction was stirred at reflux
until 6c had disappeared (monitored by TLC, CH2Cl2 : MeOH
95 : 5), and then the reaction mixture was neutralized with diluted
acetic acid to about pH 6.5, filtered and the precipitate was washed
with methanol (3 ¥ 40 mL), and water (1 ¥ 40 mL), after vacuum
drying, afforded 1c (410 mg, 93%). Method 2: A solution of 7c
(200 mg, 0.3 mmol) in CH2Cl2 (6 mL) was treated with 2.5% DCA
in CH2Cl2 (4 ml). The reaction was stirred at room temperature
for 5min. When compound 7c disappeared (monitored by TLC,
CH2Cl2 : MeOH, 12 : 1), triethylamine was added to neutralize
the reaction mixture. Then the reaction mixture was filtered and
the precipitate was washed with CH2Cl2 (3 ¥ 10 mL) and acetone
5-Amino-8-[2-deoxy-3,5-di-O-(p-toluoyl)-b-D-erythro-pentofuran-
osyl]pyrimido[4,5-d]pyrimidine-2,4(3H,8H)-dione (6c); 5-amino-8-
[2-deoxy-3,5-di-O-(p-toluoyl)-a-D-erythro-pentofuranosyl]pyri-
mido[4,5-d]pyrimidine-2,4(3H, 8H)-dione (6d)
Compound 4 (2 g, 11.2 mmol) was suspended in HMDS (150 mL)
and the mixture was stirred at 140 ◦C for about 3 min. TMSCl
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(3 ¥ 10 mL). After vacuum drying, 1c was given 48 mg (50%): UV
(MeOH) 251(28571), 277(6158). dH (600 MHz; d6-DMSO): 2.18–
2.22 (1H, m, 2¢-Ha), 2.35–2.39 (1H, m, 2¢-Hb), 3.59–3.61 (1H, d,
J = 12 Hz, 5¢-Ha), 3.67–3.69 (1H, d, J = 12 Hz, 5¢-Hb), 3.89–3.91
(1H, t, J = 6 Hz, 4¢-H), 4.27 (1H, s, 3¢-H), 5.20 (1H, s, 5¢-OH), 5.34
(1H, s, 3¢-OH), 6.41–6.43 (1H, t, J = 6 Hz, 1¢-H), 8.77 (1H, s, CH),
8.89–8.93 (2H, 2 s, NH2), 10.76 (1H, s, NH). dC (600 MHz; d6-
DMSO): 41.60, 60.95, 69.99, 86.75, 87.46, 88.59, 151.39, 157.11,
157.78, 162.07, 165.43. HR-MS (ESI-): Calc. for [C27H25N5O4]:
294.0838 [M - H]-, Found 294.0838 [M - H]-.
Compound 7d: UV (MeOH) 230.0 (45654), 250.0 (36857), 273.0
(11634). dH (600 MHz; d6-DMSO): 2.17–2.19 (1H, d, J = 12 Hz,
2¢-Ha), 2.61–2.65 (1H, m, 2¢-Hb), 3.02–3.14 (2H, m, 5¢-H2), 3.74
(6H, s, 2OCH3), 4.23 (1H, s, 4¢-H), 4.56–4.57 (1H, t, J = 6 Hz, 3¢-
H), 5.289–5.293 (1H, d, J = 2.4 Hz; 3¢-OH), 6.45–6.46 (1H, d, J =
6 Hz; 1¢-H), 6.92–7.40 (13H, m, H-arom), 8.47 (1H, s, CH), 8.875–
8.881 (1H, d, J = 3.6 Hz; NHa), 8.90–8.91 (1H, d, J = 6 Hz; NHb),
10.78 (1H, s, NH). dC (600 MHz; d6-DMSO): 40.96, 55.01, 63.77,
70.80, 85.69, 87.00, 88.67, 89.07, 113.26, 126.73, 127.64, 127.91,
129.62, 129.66, 135.31, 135.44, 144.67, 151.14, 156.58, 157.32,
158.09, 161.75, 164.91. HR-MS (ESI-): Calc. for [C32H31N5O7]
597.2223, Found [M - H]- 596.2150.
5-Amino-8-(2-deoxy-a-D-erythro-pentofuranosyl)pyrimido[4,5-
d]pyrimidine-2,4(3H,8H)-dione (1d)
Acknowledgements
Compound 6d (170 mg, 0.3 mmol) was suspended in 0.2 M
NaOMe/MeOH (10 mL), the reaction was stirred at reflux until
6d had disappeared (monitored by TLC, CH2Cl2 : MeOH 95 : 5),
and then the reaction solution was neutralized with diluted acetic
acid to about pH 6.5, filtered and washed with methanol (3 ¥
10 mL) and water (1 ¥ 10 mL). After vacuum drying, 1d was
afforded (80 mg, 90%): UV (MeOH) 225 (49672), 272 (66694),
279.0 (60606), 295 (20771), 323 (16281). dH (600 MHz; d6-DMSO):
2.08–2.11 (1H, d, J = 18 Hz, 2¢-Ha), 2.56–2.60 (1H, m, 2¢-Hb), 3.42–
3.45 (2H, dd, J = 6 Hz 5¢-H2), 4.25–4.26 (1H, d, J = 6 Hz, 4¢-H),
4.39–4.41 (1H, t, J = 6 Hz, 3¢-H), 4.96 (1H, s, 5¢-OH), 5.23 (1H, s,
3¢-OH), 6.34–6.35 (1H, d, J = 6 Hz, 1¢-H), 8.45 (1H, s, CH), 8.87–
8.89 (2H, 2 s, NH2), 10.76 (1H, s, NH). dC (600 MHz; d6-DMSO):
41.23, 62.12, 71.09, 87.49, 89.06, 91.35, 151.66, 157.04, 157.81,
162.25, 165.40. HR-MS (ESI-): Calc. for [C27H25N5O4]: 294.0838
[M - H]-, Found 294.0838 [M - H]-.
We thank the National Natural Science Foundations of China
(document no. 20772087) for the financial support. We also thank
Ming-Hai Tang from National Key Laboratory of Biotherapy for
providing us with high quality mass spectra.
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(8239). dH (600 MHz; d6-DMSO): 2.31–2.45 (2H, m, 2¢-H2), 3.21–
3.28 (2H, m, 5¢-H2), 3.73 (6H, s, 2OCH3), 4.01–4.03 (1H, m, 4¢-H),
4.29–4.31 (1H, t, J = 6 Hz, 3¢-H), 5.40–5.41 (1H, d, J = 6 Hz,
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