Self-assembly of N2-modified guanosine derivatives: Formation of discrete G-octamers

Article abstract of DOI:10.1002/chem.200701411

In the presence of Na⁺ ions, two N²-modified guanosine derivatives, N²-(4-n-butylphenyl)-2′, 3′5′-O-triacetylguanosine (G1) and N²-(4-pyrenylphenyl)- 2′,3′,5′-O-triacetylguanosine (G2), are found to self-associa

Full text of DOI:10.1002/chem.200701411

DOI: 10.1002/chem.200701411
Self-Assembly of N2-Modified Guanosine Derivatives:
Formationof Discrete G-Octamers
Sanela Martic´, Xiangyang Liu, Suning Wang,* and Gang Wu*[a]
Abstract: In the presence of Na+ ions,
two N2-modified guanosine derivatives,
N2-(4-n-butylphenyl)-2’,3’,5’-O-triace-
tylguanosine (G1) and N2-(4-pyrenyl-
phenyl)-2’,3’,5’-O-triacetylguanosine
(G2), are found to self-associate into
discrete octamers that contain two G-
quartets and a central ion. In each oc-
tamer, all eight guanosine molecules
are in a syn conformation and the two
G-quartets are stacked in a tail-to-tail
fashion. On the basis of NMR spectro-
scopic evidence, we hypothesize that
the p–p-stacking interaction between
the N2-side arms (phenyl in G1 and
pyrenyl in G2) can considerably stabi-
lize the octamer structure. For G1, we
have used NMR spectroscopic satura-
tion-transfer experiments to monitor
the kinetic ligand exchange process be-
tween monomers and octamers in
CD3CN. The results show that the acti-
vation energy (Ea) of the ligand ex-
change process is 31 ꢀ5 kJmolꢁ1. An
Eyring analysis of the saturation trans-
fer data yields the enthalpy and entro-
py of activation for the transition state:
Keywords: guanosine · NMR spec-
troscopy · saturation transfer · self-
assembly · supramolecular chemis-
try
DH° =29 ꢀ5 kJmolꢁ1
and
DS° =
ꢁ151 ꢀ10 Jmolꢁ1 Kꢁ1. These results
are consistent with an associative
mechanism for ligand exchange.
Introduction
tamers, dodecamers, hexadecamers, and even longer molec-
ular cylinders depends on p stacking between bases, the
sugar conformation, the glycosyl torsion angle (syn and
anti), the type of templating cations (sometimes anions as
well), protecting groups and other substitutions on the base
that might provide additional attraction or hindrance. In
general, guanosine-based derivatives with well-defined struc-
ture, stability and tuneable electronic, photonic, and mag-
netic properties are highly desirable.
For guanosine and 2’-deoxyguanosine derivatives, there
are several potential points for modification that would not
interfere with G-quartet formation: 2’, 3’, and 5’ positions of
the ribose and C8, N2, and N3 of the guanine base. To date,
only C8 and N2 base modification has been utilized for G-
quartet formation.[7–17] In many cases, base modification in-
troduces new properties and flexibilities that might not be
possible for the unmodified guanine. For example, Sessler
and co-workers[6] reported that a C8-modified guanosine nu-
cleoside forms a G-quartet in the absence of templating
metal ions, that is, it forms an “empty” G-quartet. Gottarel-
li, Spada, and co-workers[8,9] found that 8-oxogunosines self-
assemble into helical architectures. Kaucher and Davis[16] re-
ported that N2,C8-disubstituted guanosine derivatives can
form G-quartets. We recently demonstrated that a N2-modi-
fied guanosine derivate can form discrete G-octamers.[17] It
is anticipated that base modification can be used as a new
handle in the design of new self-assembled guanosine struc-
Molecular self-assembly via non-covalent interactions such
as hydrogen bonding can lead to stable supramolecular
structures. Hydrogen bonding between complementary
DNA bases is one remarkable example of molecular self-as-
sembly in nature. Among nucleobases, guanine exhibits the
most versatile hydrogen-bonding capability;this results in a
variety of self-assembled structures, such as dimers, trimers,
G-ribbons and G-quartets.[1–3] In the past decade, the G-
quartet structural motif has attracted considerable attention
because of its biological implications.[4] In the meantime, ap-
plications of guanosine-based molecules in materials science
and nanotechnology have also appeared.[5,6]
There are many factors that contribute to the overall
structure of a G-quartet-based supramolecular entity. At the
core of the G-quartet motif, non-covalent interactions, such
as hydrogen-bonding and cation–dipole interactions, are the
most important ones. Further stacking of G-quartets into oc-
´
[a] S. Martic, Dr. X. Liu, Prof. Dr. S. Wang, Prof. Dr. G. Wu
Department of Chemistry, Queenꢀs University
90 Bader Lane, Kingston, Ontario K7L 3N6 (Canada)
Fax : (+1)613-533-6669
E-mail: suning.wang@chem.queensu.ca
gang.wu@chem.queensu.ca
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
1196
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Chem. Eur. J. 2008, 14, 1196 – 1204

FULL PAPER
tures. We report in this study the self-assembly of two N2-
modified guanosine nucleosides, N2-(4-n-butylphenyl)-
2’,3’,5’-O-triacetylguanosine (G1) and N2-(4-pyrenylphenyl)-
2’,3’,5’-O-triacetylguanosine (G2). A brief account on the
ZnII complex and its cross coupling to p-diiodobenzene in
the presence of [PdACHTRE(UNG PPh3)4] at 08C to give p-pyrenyliodo-
phenyl (1) in 59% yield. Compound 2 was synthesized
through an Ullmann condensation by using CuI as the cata-
lyst and Cs2CO3 as the base at 1408C. Protection of com-
pound 2 was achieved by using an acetylation reaction with
acetic anhydride in the presence of triethylamine and N-di-
methylaminopyridine (DMAP) as a catalyst to give G2 (see
Scheme 1).
1H NOESY NMR spectra: Figure 1 shows the spectral re-
1
gions of the H NOESY NMR spectra of G1 and G2 self-as-
semblies that were prepared in the presence of Na+ ions.
The observed interbase NOE cross peaks between H8 and
N2H protons are characteristic features that indicate G-quar-
tet formation. In addition, strong NOE cross peaks are ob-
served between H8 and H1’ for both G1 and G2 self-assem-
blies, which suggests that all G1 and G2 molecules are ex-
clusively in a syn conformation. The observed NOE cross
peaks between Hortho/Hmeta and H2’/H3’ protons further sup-
port a syn conformation of the glycosidic bond so that the
N2-subtituent is approximately above the ribose ring. The
crystal structure of the G1 monomer also exhibits a syn con-
formation with a glycosyl torsion angle cCN (O4’-C1’-N9-C4)
self-assembly of G1 has been reported.[17] Here we report a
detailed examination of the self-assembled structures that
are formed by G1 and G2, and of the kinetic ligand ex-
change between G1 monomers and aggregates in solution.
Results and Discussion
Synthesis: The N2-functional-
ized molecule, N2-(4-n-butyl-
phenyl)-2’,3’,5’-O-triacetylgua-
nosine (G1) was synthesized
by using a previously reported
method.[18] The synthetic route
to N2-(4-pyrenylphenyl)-2’,3’,5’-
O-triacetylguanosine) G2 is
shown in Scheme 1. Typically,
ꢁ
Pd-catalyzed C N bond cou-
pling reactions are performed
by using halogenated nucleo-
sides or aryl halides;however,
such methodology requires
protection of the hydroxyl
groups of the ribose moiety as
well as the protection and sub-
sequent deprotection of the O6
site.[19] Our procedure is based
on the well-known Ullmann
condensation reactions, which
ꢁ
are commonly used for the C
N coupling of aryl halides with
aryl amines,[20,21] but have not
been attempted with nucleo-
sides previously. The starting
material, p-pyrenyliodophenyl
(1) was synthesized by lithiat-
ing 1-bromopyrene first, then
by transmetalation to form a
Scheme 1. Synthetic route to G2.
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1197

G. Wu, S. Wang et al.
served solvent-dependence effect. The 23Na NMR spectrum
of the G1 self-assembly in CDCl3 exhibits a single peak at
d=ꢁ17.8 ppm;this is in agreement with a G-octamer struc-
ture that contains a central Na+ ion.[23–25] The ESI-MS spec-
tra also suggest the presence of G1 octamers. It is interesting
to note that neither G1 dodecamers nor G1 hexadecamers
were observed in the ESI-MS spectra, which is different
from previous observations that have been made for other
guanosine derivatives, for which polymeric aggregates are
always likely to form in the presence of Na+ or K+
ions.[17,26,27] In addition, individual G1 or G2 tetramers were
not observed either in the solution or gas phase. Perhaps,
the particular stacking scheme between the two all-syn G-
quartets in each G1 octamer and the presence of acetyl pro-
tecting groups impose steric hindrance that prevents the
stacking of two G1 ocamers together. As will be discussed
later, our models provide indirect evidence that supports
this argument. The circular dichroism (CD) spectrum of G1
in CH3CN exhibits a nearly degenerate negative exciton
couplet that is centered at 290 nm;this is similar to that re-
ported for a G-octamer by Davis and co-workers.[27] This
CD spectral feature is indicative of the twisted stacking of
two chiral G-quartets.[28,29]
DOSY NMR spectroscopy: As mentioned in the previous
section, NOESY data provide unambiguous evidence that
G1 and G2 self-assemblies are based on the G-quartet struc-
tural unit. On the other hand, the ESI-MS data support the
presence of G octamers rather than dodecamers and hexa-
decamers in the gas phase. However, none of these tech-
niques can answer the question as to the exact size of molec-
ular aggregates in solution. To determine the precise size of
molecular self-assemblies from G1 and G2 in solution, we
used an NMR technique known as diffusion-ordered spec-
troscopy (DOSY).[30,31] In general, a DOSY spectroscopic
experiment measures the translational diffusion coefficient
(D) of a molecule, which can then be used to infer the exact
size of the molecule or molecular aggregate in solution. Re-
cently, DOSY or diffusion NMR spectroscopy has been ex-
tensively used in supramolecular chemistry.[27,32–40] Experi-
mental results of translational diffusion constants for G1
and G2 in various solvents are given in Table 1. We have
also measured the value of D for the unmodified 2’,3’,5’-O-
triacetylguanosine (TAG) in DMSO. Because TAG exists as
Figure 1. Regions of the NOESY spectra of A) G1 in CDCl3 and B) G2
in CD2Cl2.
of 75.78.[17] Our observation falls into the general trend that
N2 and C8-modified guanosine derivatives usually prefer to
adopt a syn conformation.[7,17,22] Also seen in Figure 1 are
the strong NOE cross peaks between Hortho and N2H protons
for both G1 and G2, which are due to intrabase interactions.
1
The fact that a single set of H NMR signals for each octa-
mer are observed suggests that the G1 and G2 octamers are
D4-symmetric.
The self-assembly process of
both G1 and G2 appears to be
Table 1. Translational diffusion coefficients (in units of 10ꢁ10 m2 s) determined for TAG monomer, G1 mono-
mer, G2 monomer, G1 octamer, and G2 octamer in different solvents. All NMR diffusion measurements were
performed at 298 K.
rather sensitive to the solvent.
For example, G1 exists as mon-
omers in CD2Cl2, as a mono-
TAG[a]
Dmonomer
G1
D8
G2
mer–aggregate
mixture
in
Solvent viscosity (h)[c]
Dmonomer
D8/Dmonomer
Dmonomer
D8
D8/Dmonomer
CD3CN, and exclusively as ag-
gregates in CDCl3. G2 exists as
monomers in CDCl3, CD3CN,
and [D6]acetone, and as aggre-
gates only in CD2Cl2. At this
time we do not have a satisfac-
tory explanation for the ob-
DMSO (2.001)
CDCl3 (0.542)
CD3CN (0.345)
CD2Cl2 (0.432)
CD3COCD3 (0.307)
1.44
–
-
–
–
–
–
-
–
–
–
–
–
0.51
0.49
5.32[b]
8.35[b]
6.67[b]
9.38[b]
6.06
9.28
–
3.09
4.76
–
0.51
0.51
–
8.14[b]
11.2
4.17
–
–
–
5.56[b]
[a] TAG=2’,3’,5’-O-triacetylguanosine [b] Calculated by using the relationship between solvent viscosity and
D. [c] Solvent viscosity data at 298 K in units of cP.
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Self-Assembly of Guanosine Derivatives
FULL PAPER
monomers in DMSO, the experimental D value for TAG
was used as an independent reference for G monomers. For
easy comparison, we have calculated the expected D values
for TAG in other solvents by using the inverse dependence
between D and the solvent viscosity (h). As seen from
Table 1, the value of D for G1 monomers in CDCl3, which
was prepared in the absence of Na+ ions is 6.06 ꢀ0.05”
10ꢁ10 m2 s. This value is approximately 14% larger than that
of TAG in the same solvent, 5.32”10ꢁ10 m2 s. This suggests
that the N2 modification of G1 also plays a role in the over-
all shape of the molecule compared to that of the unmodi-
fied guanosine. For G1 and G2 monomers in other solvents,
the values of D are generally larger than the corresponding
values of TAG by approximately 20%. When Na+ ions are
present in the solution, G1 self-associates into aggregates
and has a much smaller D value of 3.09”10ꢁ10 m2 s. The
ratio between the two D values for G1 monomers and G1
aggregates suggests that G1 molecules form octamers in
CDCl3.[22] As seen from Table 1, the values of D for G2 ag-
gregates also suggest the formation of G2 octamers.
The most interesting case is that of G1 in CD3CN. The
1H NMR spectrum of G1 in CD3CN that was prepared in
the presence of Na+ ions generally exhibits two sets of
NMR signals. Furthermore, the two sets of signals exhibit
quite different D values: 4.76 and 9.28”10ꢁ10 m2 s. Once
again, the diffusion data immediately suggest that G1 exists
as a mixture of monomers and octamers in CD3CN. This sit-
uation is best illustrated in the 2D representation of the
DOSY data that is shown in Figure 2. In a sense, DOSY
spectroscopy is a type of NMR chromatography that sepa-
rates molecular species according to their translational mo-
bility. Another interesting observation is that the NOE cross
peaks that are due to G1 monomers and G1 octamers have
opposite signs;this is illustrated in Figure 2, and can be un-
derstood on the basis that G1 monomers and G1 octamers
have very different rotational correlation times (tC). It is
well known that for small molecules in which tC is short rel-
ative to 1/w0 (w0 is the angular Larmor frequency of the nu-
cleus under observation), the NOE cross peaks exhibit an
opposite sign to the diagonal peaks. On the other hand, for
large molecules or molecular aggregates with a long tC
value relative to 1/w0, NOE cross peaks have the same sign
as the diagonal peaks. In the present case, because G1 oc-
tamers (ca. 4300 Da) are much larger than G1 monomers
(540 Da), they have quite different values of tC;this gives
rise to NOE cross peaks with opposite signs. This observa-
tion, in turn, is in agreement with the diffusion NMR spec-
troscopic results. We have also found that the amount of G1
octamers relative to that of G1 monomers increases with a
decrease of temperature, this indicates that the octamer for-
mation is an exothermic process. Our dilution experiments
of G1 in CD3CN also suggest that the octamer formation is
favored at higher concentrations at 298 K, while a concen-
tration effect is negligible at 283 K. G2 exists exclusively as
octamers in CD2Cl2 and as monomers in [D6]acetone. We
did not find any solvent in which G2 exists as a mixture of
monomers and octamers.
Figure 2. A) 2D representation of the DOSY data for G1 in CD3CN.
B) A spectral region of the NOESY spectrum of G1 in CD3CN. The
NOE cross peaks in blue and red have the same and opposite signs as
the diagonal peaks, respectively.
Molecular structures of G1 and G2 octamers: Because crys-
tallization of G1 and G2 octamers were unsuccessful, the
model building in this section is based primarily on NMR
1
spectroscopic evidence. As mentioned earlier, the H NMR
spectroscopic data suggest that G1 and G2 form D4-sym-
metric octamers, each of which contains two all-syn G-quar-
tets. As shown in Figure 3, the molecular structure of the
G1 monomer[17] exhibits a syn conformation with a glycosyl
torsion angle of 75.78. Another important structural feature
of the G1 monomer is that the five-membered ribose ring is
2
puckered in the E (2’-endo) conformation. We have there-
fore built a G octamer model by assuming a similar glycosyl
torsion angle and sugar conformation. Because each G-quar-
tet has two faces, head and tail, as illustrated in Figure 3,
there are still two possible ways to form a D4-symmetric oc-
tamer, that is, either head-to-head or tail-to-tail. Our model
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1199

G. Wu, S. Wang et al.
phenyl (G1) and pyrenyl (G2)
aromatic rings from the two
different G-quartets. Indeed,
as seen from Figure 4, the ob-
1
served H NMR chemical shifts
for the phenyl protons (Hortho
and Hmeta) for the G1 octamer
are considerably more shielded
than those in the G1 monomer.
In CDCl3, such changes
amount to Dd=0.34 and
0.18 ppm for Hortho and Hmeta
,
respectively. In CD3CN, we
also found Dd=0.15 and
0.20 ppm for Hortho and Hmeta
respectively. For G2, significant
1H NMR
chemical shift
,
changes (ca. Ddꢂ0.5 ppm)
were observed for H7 and H2
of the pyrenyl group. These
chemical shift changes are
comparable to those that were
observed for the H8 protons
(Dd=0.42 ppm for G1 and
Dd=0.45 ppm for G2), which
are due to the p–p stacking be-
tween the bases. Other protons
of the pyrenyl group, even in
the absence of a complete as-
signment, also show small
chemical shift changes. All
these observations are consis-
tent with the formation of p–p
stacking. Interestingly, the
phenyl protons in the G2 octa-
mer exhibit little chemical shift
change compared to those in
the G2 monomer. Inspection
of the molecular model reveals
that the phenyl rings in the G2
octamer are more perpendicu-
lar to the guanine base plane
than those in the G1 octamer.
Figure 3. A) Crystal structure of the G1 monomer. B) Illustration of the head and tail faces of a G-quartet.
Top views (D and F) and side views (C and E) of the G1 and G2 octamers.
suggests that the tail-to-tail stacking is most likely to occur
because of the arrangement of the three acetyl groups. In
particular, as seen in Figure 3, the 2’- and 3’-O-acetyl groups
are on the head side of the G-quartet and 5’-O-acetyl group
is on the tail side. As a result, the head face is more crowd-
ed than the tail side. Furthermore, because of the additional
methylene group that is linked to the 5’-O-acetyl group, it is
more flexible than the 2’- and 3’-O-acetyl groups that are di-
rectly attached to the ribose ring. A tail-to-tail G-octamer
formation can also provide an explanation for the fact that
further stacking between octamers has not been observed
for G1 and G2.
In particular, the C2-N2-Cipso-Cortho torsion angle in the G2
octamer is 788, whereas it is only 418 in the G1 octamer.
Such an arrangement of the phenyl ring relative to the gua-
nine plane was also predicted by quantum chemical calcula-
tions at the B3LYP/6–311+G(d) level for G2 and from the
crystal structure for the G1 monomer. Consequently, there
is very little p–p stacking between the two phenyl rings in
the G2 octamer. This is entirely consistent with the observa-
tion shown in Figure 4, in which the 1H NMR signals for
Hortho and Hmeta show little variations between G2 monomers
1
and G2 octamers. On the other hand, significant H NMR
spectroscopic chemical shift changes are observed for Hortho
and Hmeta protons between the G1 monomer and G1 octa-
mer as mentioned earlier.
The molecular models of the G1 and G2 octamers also
suggest possible p–p-stacking interactions between the
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Self-Assembly of Guanosine Derivatives
FULL PAPER
to examine ligand exchange
between the free (monomers)
and bound (octamers) states;
see Scheme 2. However, varia-
1
ble temperature H NMR spec-
tra of G1 do not exhibit any
significant line broadening,
which indicates that the ligand
exchange rate is much slower
than the NMR chemical shift
time scale. For this reason, we
employed a saturation transfer
NMR spectroscopic technique
to measure ligand exchange
rates.[41,42] Typically, saturation
transfer NMR experiments are
performed by selectively satu-
rating one NMR signal (sig-
nal A) from the G1 monomer
(or the octamer) and then
monitoring the time evolution
of the signal (signal B) from
the G1 octamer (or the mono-
mer). According to Forsen and
Hoffman,[41,42] the time evolu-
Figure 4. Regions of 1H NMR spectra for G1 in CDCl3 (monomer and octamer) and G2 in CD2Cl2 (octamer)
and DMSO (monomer). * marks the solvent peak.
Another piece of evidence that suggests that there is p–p
stacking between the pyrenyl rings in the G2 octamer comes
from the NOESY data. In the NOESY spectrum that is
shown in Figure 5, cross peaks are observed between H7 and
H2 of the pyrenyl group. As shown in Figure 5, the distance
between H2 and H7 within the same pyrenyl ring is approxi-
mately 8.027 Š. This distance is generally too long to gener-
ate any NOE effect. On the other hand, the G2 octamer
model suggests that the distance between H2 and H7 from
two different G-quartets (interquartet) is about 2.926 Š.
This is a reasonable short contact for producing the ob-
served NOE cross peaks. We have also measured the fluo-
rescence spectra for G2 in CH2Cl2. However, we did not ob-
serve a pyrene excimer peak. Presumably at the low concen-
trations that are suitable for the optical measurement, very
little G2 octamers are present.
In a G-octamer, the main forces to hold two G-quartets
together are the ion–carbonyl interaction and the p–p stack-
ing between the guanine bases. It is plausible that the addi-
tional p–p stacking between the N2 side-arms in both G1
and G2 octamers further stabilizes these octamer structures.
It would be interesting to design new N2-modified guanosine
derivatives in which the p–p stacking between the N2 groups
can be optimized. It might also be possible that such a p–p
stacking between N2 groups would provide a strong enough
attraction to hold the two G-quartets so that the central
cation becomes unnecessary, this would give rise to an
“empty” G-octamer. Experiments to search for such an
empty G-octamer are underway in our laboratory.
Ligand exchange between G1 monomers and octamers: As
mentioned earlier, G1 exists as a mixture of monomers and
octamers in CD3CN. This provides an excellent opportunity
Figure 5. A) Part of the G2 octamer model that shows the stacking be-
tween two pyrenyl groups. B) Expansion of the NOESY spectrum of G2
octamers in CD2Cl2.
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1201

G. Wu, S. Wang et al.
Table 2. Saturation transfer NMR experimental results for G1 kinetic
ligand exchange in CD3CN.
Saturation of octamer signals
Saturation of monomer signals
T
[K]
T1A [s]
tA [s]
k=1/tA
T1A [s]
tA [s]
k=1/tA
[sꢁ1
]
[sꢁ1
]
283
298
313
1.39
(H8)
2.01
(H1)
2.05
(H8)
2.4ꢀ0.5 0.4
1.7ꢀ0.2 0.6
0.8ꢀ0.1 1.3
1.18
(H8)
1.59
(H1)
1.61
(H8)
3.0ꢀ0.5 0.3
1.6ꢀ0.2 0.6
0.7ꢀ0.1 1.4
Scheme 2. Ligand exchange between G1 monomers and octamers.
tion of signal A under the conditions of complete saturation
of signal B can be written as:
uration transfer NMR experimental results for G1 in
CD3CN at different temperatures.
ꢀ
ꢁ
1
1
A
¼
e
t þ
ꢁ
tA þ T1A
The ligand exchange rates are in the order of a few sꢁ1.
These values are comparable to those that were reported by
Davis and co-workers for isoguanosines.[43] We also per-
formed saturation transfer experiments for different concen-
trations, and the results suggest that ligand exchange rates
decrease considerably at low concentrations. This is consis-
tent with an associative bimolecular mechanism for the
ligand exchange process.[44] An Arrhenius analysis of the
data that is shown in Table 2 yields an activation energy
(Ea) of 31 ꢀ5 kJmolꢁ1 for the kinetic ligand exchange pro-
cess. We have also applied an Eyring analysis for the satura-
tion transfer data, and have obtained the enthalpy of activa-
tion and the entropy of activation for the transition state:
DH° =29 ꢀ5 kJmolꢁ1 and DS° =ꢁ151 ꢀ10 Jmolꢁ1 Kꢁ1.
Again, a large and negative value of DS° is in agreement
with the associative mechanism for ligand exchange.
Mz ðtÞ
T1A
T1A þ tA
tA
A
MO
T1A þ tA
in which T1A is the spin–lattice relaxation time constant of
signal A and tA is the lifetime of molecules in state A. The
ligand exchange process between G1 monomers and octam-
ers is depicted in Figure 6. Because it is possible to find
1H NMR spectral regions in which signals for both mono-
mers and octamers are well resolved, we performed the sat-
uration transfer experiments by saturating signals from mon-
omers and octamers separately. Table 2 summarizes the sat-
Conclusion
We have shown that two N2-modified guanosine derivatives
form discrete G-octamers that contain all-syn guanosine
molecules. The stacking between the two G-quartets is most
likely in a tail-to-tail fashion. NMR spectroscopic results
strongly suggest the formation of p–p stacking between the
phenyl and pyrenyl groups in the G1 and G2 octamers, re-
spectively. The kinetic ligand exchange between G1 mono-
mers and G1 octamers is generally slow, and is in the order
of a few sꢁ1 between 283 and 313 K. We have determined
the enthalpy and entropy of activation for the transition
state:
DH° =29 ꢀ5 kJmolꢁ1
and
DS° =ꢁ151 ꢀ
10 Jmolꢁ1 Kꢁ1. These data suggest an associative mechanism
for the ligand exchange process. This study provides new in-
sights into the self-assembly of N2-modified guanosine com-
pounds in organic solvents. It is quite clear that N2-modifica-
tion is a viable approach in the design of new G-quartet-
based materials. Although this study deals with guanosine
nucleosides, it is possible that the same strategy might be
applicable to nucleotides (DNA and RNA) because the exo-
ꢁ
cyclic amino N H bond is pointing into the groove region.
Figure 6. Saturation transfer NMR experiments for the G1 ligand ex-
change in CD3CN. A) Saturation of the NMR signal from octamers and
We believe that residue-specific N2 modification should be
exploited as a new handle for fine-tuning G-quadruplex
structure and function.
*
B) saturation of the NMR signal from monomers.
283K; & 298K;
*
313K.
1202
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ꢁ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 1196 – 1204

Self-Assembly of Guanosine Derivatives
FULL PAPER
5.82 Hz, 1H;H 1’), 5.45 (t, J=5.40 Hz, 1H;H 3’), 4.31 (m, J=3.43, 4.02 Hz,
1H;H 4’), 4.28 (m, J=4.6, 6.1, 10.1 Hz, 1H;H 5’’), 4.15 (m, J=4.8, 6.1,
10.2 Hz, 1H;H 5’), 2.06 (s, 3H;CH ), 2.02 (s, 3H;CH ), 1.86 ppm (s, 3H;
Experimental Section
3
3
Synthesis details: All starting materials and reagents were purchased
from Aldrich Chemical Company and were used without further purifica-
tion. Thin-layer chromatography was carried out by using silica gel 60
plates, and the column chromatography was performed by using silica gel
of particle size 60–200 mm and C-18 silica gel for reversed-phase chroma-
tography, all of which were purchased from Silicycle. G1 was synthesized
by using a previously reported procedure.[17]
CH3); 13C NMR (400 MHz, [D6]DMSO, 258C): d=171.7, 170.2, 170.1,
157.3, 150.5, 150.2, 138.8, 137.6, 135.7, 131.8, 131.6 (2C), 131.2, 130.7,
128.4 (2C), 128.3 (2C), 128.2 (2C), 127.2, 126.1, 125.8, 125.7 (2C), 125.4,
125.0, 124.9, 121.1 (2C), 87.3 (C1), 79.9 (C4), 72.6 (C2), 70.8 (C3), 63.6
(C5), 21.3 (CH3), 21.0 (CH3), 20.8 ppm (CH3);HRMS (ESI +): m/z: calcd
for C38H31N5O8: 686.2508;found: 686.22760 [ M+H]+.
NMR spectroscopic experiments: All routine 1H and 13C NMR spectra
were recorded on Bruker Avance-500 and Avance-600 spectrometers. For
G1 octamers in CDCl3, the source of Na+ ions was either NaClO4 or
sodium picrate. For G1 octamers in CD3CN and G2 octamers in CD2Cl2,
no additional Na+ ions were added to the samples because Na+ ions had
apparently been extracted into the samples during the compound synthe-
sis and NMR sample preparation. Although at that time, the exact
source of Na+ and the nature of counter ions were uncertain, the pres-
ence of the Na+ ions in these samples was unambiguously proved by so-
lution 23Na NMR and cryptand extraction experiments.
p-Pyrenyliodophenyl (1): A 1.6m hexane solution of nBuLi (1.75 mL,
2.79 mmol) was added to a stirred solution of bromopyrene (0.71 g,
2.54 mmol) in THF (100 mL)at ꢁ788C. After 1 h at this temperature,
ZnCl2 (0.41 g, 3.04 mmol) was added and stirring was continued for 0.5 h
at 08C. Diiodobenzene (1.81 g, 5.4 mmol) and [PdACHTRE(UNG PPh3)4] (0.22 g,
9 mol%) were added to the mixture, and the reaction was allowed to
warm up to room temperature, and was stirred overnight under N2. The
solution was partitioned by using ethyl acetate (100 mL) and water
(100 mL). The aqueous layer was further extracted with dichloromethane
(3”40 mL), and the combined organic fractions were dried over anhy-
drous MgSO4 and concentrated under vacuum. The product was purified
by using column chromatography (hexane/ethyl acetate 5:1) and was iso-
Pulse field gradient (PEG) NMR spectroscopy: Diffusion experiments
were carried out with Bruker Avance-600 MHz spectrometer by using
the pulse sequence of longitudinal-eddy-current delay (LED) with bipo-
lar-gradient pulses. The 1H 90o and 180o pulse widths were 10 and 20 ms,
respectively. The pulse-filed gradient duration was varied from 4–15 ms,
and the variable gradient (G) was changed from 6 to 350 mT/m. The dif-
fusion period was varied from 50 to 90 ms. A total of 16 transients were
collected for each of the 32 increment steps with a recycling delay 12 s.
The eddy-current delay was set to 5 ms. Diffusion coefficients were ob-
tained by integration of the desired peaks to a single exponential decay
curve by using “Simfit Bruker XWINNMR” software. Calibration of the
field gradient strength was achieved by measuring the value of transla-
tional diffusion coefficient (D) for the residual 1H signal in D2O
lated as
a
white solid. Yield: 0.610 g (59.4%). 1H NMR (400 MHz,
CDCl3, 258C): d=8.24–7.94 (m, J=3.6, 7.8, 9.2 Hz, 9H), 7.91 (d, J=
8.3 Hz, 2H, Hortho), 7.39 ppm (d, J=8.3 Hz, 2H, Hmeta); 13C NMR
(400 MHz, CDCl3, 258C): d=140.7, 137.5, 136.4, 132.5, 131.5, 131.2,
130.9, 130.8, 128.3, 128.1, 127.7, 127.6, 127.4, 127.3, 126.1, 125.8, 125.3,
125.0, 124.9, 124.8, 124.6, 93.1 ppm;HRMS (EI +): m/z: calcd for
C22H13I: 404.0062;found: 404.0046 [ M]+.
N2-(4-Pyrenylphenyl)guanosine (2): A mixture of p-pyrenyliodophenyl
(0.199 g, 0.49 mmol), guanosine (0.209 g, 0.73 mmol), cesium carbonate
(0.192 g, 0.59 mmol), copper iodide (0.014 g, 0.07 mmol, 15%), and
DMSO (5 mL) in small sealable 25 mL vial was degassed with N2 for
10 min, then the vial was sealed, and the reaction was carried out at
1408C for 24 h in a preheated oil bath. Water (10 mL) was added to reac-
tion mixture, and the solution was neutralized to pHꢂ7 by using aq.
HCl. Further addition of water (30 mL) led to the precipitation of the
product as a beige solid. The solid was washed with water to remove un-
reacted guanosine, then it was purified by using reversed-phase silica
(CH2Cl2, then MeOH/H2O 4:1) to obtain N2-(4-pyrenylphenyl) guanosine
2
(99.99%, H atom), D=1.91”10ꢁ9 m2 s.
NOESY spectroscopy: All NOESY spectra at 298 K were acquired by
using a mixing time of 0.4 s and a total of 64 transients with a recycling
delay of 10 s. The NOESY experiment at 218 K was acquired by using a
mixing time of 0.1 s and a recycling delay of 2 s and a total of 64 transi-
ents.
Saturation transfer experiments: Selective saturation transfer experi-
ments were performed for G1 in CD3CN between 283 and 313 K. The ex-
periments were conducted by using a selective saturation pulse on the
peak of interest. An irradiation at H8 of the G1 monomer or (G1)8 octa-
mer was used at 283 and 313 K, while irradiation at H1’ of the G1 mono-
mer or (G1)8 octamer was performed at 298 K. The spectra were collect-
ed by varying the mixing time from 0 to 14 s for a total of 24 data points.
1
(0.061 g, 22.3%) as a white solid. H NMR (400 MHz, [D6]DMSO, 258C):
1
2
d=10.76 (brs, 1H;N H), 9.14 (brs, 1H;N H), 8.4–8.26 (m, 3H), 8.21 (d,
J=9.17 Hz, 4H), 8.11 (s, 1H, H8), 8.06 (t, J=8.01 Hz, 2H), 7.86 (d, J=
8.28 Hz, 2H;H ortho), 7.64 (d, J=8.21 Hz, 2H;H meta), 5.83 (d, J=5.46 Hz,
1H;H 1’), 5.53 (d, J=5.94 Hz;C -OH), 5.21 (d, J=5.12 Hz;C -OH), 5.02
2
3
(t, J=5.16, 5.35 Hz;C 5-OH), 4.55 (q, J=5.33, 5.68 Hz, 1H;H 2’), 4.13 (d,
J=4.33 Hz, 1H;H 3’), 3.90 (d, J=3.52 Hz, 1H;H 4’), 3.55 ppm (m, J=4.06,
6.78, 14.78 Hz, 2H;H 5’, H5’’); 13C NMR (400 MHz, [D6]DMSO, 258C):
d=157.7, 150.9, 150.5, 138.9, 137.9, 132.1 (2C), 131.9 (2C), 131.5, 131.1,
128.8 (2C), 128.7 (2C), 128.5 (2C), 127.6, 126.4, 126.1 (2C), 125.4, 125.3,
125.2, 123.8, 121.4 (2C), 120.2, 86.9 (C1), 79.5 (C4), 72.3 (C2), 70.4 (C3),
63.3 ppm (C5);HRMS (ESI +): m/z: calcd for C32H25N5O5: 560.1934;
found: 560.1943 [M+H]+.
N2-(4-Pyrenylphenyl)-2’,3’,5’-O-triacetylguanosine (G2): Acetic anhydride
(0.094 mL, 0.98 mmol) was added to a suspension of N2-(4-pyrenylphen-
yl) guanosine (0.041 g, 0.07 mmol) and N-dimethylaminopyridine
(0.003 g, 0.02 mmol) in a mixture of acetonitrile (6 mL) and triethylamine
(0.151 mL, 1.08 mmol) at room temperature. After stirring for 1 h, when
all of the starting material had dissolved, MeOH (5 mL) was added to
the mixture, and stirring was continued for an additional 5 min. The solu-
tion was then evaporated to dryness, and the resulting oil was precipitat-
ed out with iPrOH. The solid was isolated by centrifugation and was
washed with diethyl ether. The solid was dissolved in THF and the com-
pound was purified by preparatory TLC (MeOH/CH2Cl2, 1:9 (v/v) and
MeOH/ethyl acetate 1:9 (v/v)). After extensive purification, a white
product (15 mg, 31.8%) was isolated. 1H NMR (400 MHz, [D6]DMSO,
258C): d=10.93 (brs, 1H;N 1H), 9.17 (brs, 1H;N 2H), 8.39–8.03 (m, J=
1
Each H NMR spectrum was acquired by using 32 scans.
Mass spectrometry: ESIMS experiments were performed by using the
positive-ionization mode on QSTA XL MS/MS systems by using the Ana-
lyst QS Method or on Waters Micromass ZQ Spectra were acquired over
a m/z range of 100–10000. MALDI-TOF experiments were performed on
the Voyager AB Applied Biosystems.
Circular dichroism (CD) spectroscopy: CD spectra of G1 and G2 solu-
tions were recorded on a Jasco 715 circular dichroism spectrometer in a
0.1 cm path length cuvette. The wavelength was varied from 190 to
800 nm at 1000 nm per min with 10 overall scans. The concentration of
the G1 solution was 250 mm and that of G2 was 0.53 mm. The equilibrium
CD curves were obtained in the 285–325 K range, and a suitable time
(10 min) was used to achieve the equilibrium before recording CD.
Acknowledgement
3.12, 4.22, 8.27 Hz, 10H;9H pyrene, H ), 7.78 (d, J=8.38 Hz, 2H;H ortho),
7.63 (d, J=8.41 Hz, 2H;H meta), 6.13 (d, J=5.12 Hz, 1H;H 2’), 6.08 (t, J=
This work was supported by NSERC of Canada. S.M. thanks the Prov-
ince of Ontario for an Ontario Graduate Scholarship (2006-07).
8
Chem. Eur. J. 2008, 14, 1196 – 1204
ꢁ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
1203

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Received: September 6, 2007
Published online: November 26, 2007
1204
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Chem. Eur. J. 2008, 14, 1196 – 1204