Syntheses of (2′)3′-15N/-Amino-(2′)3′- deoxyguanosine and determination of Their pKa values by15N NMR spectroscopy

Article abstract of DOI:10.1021/ol071129h

2′-Amino-2′-deoxyguanosine and 3′-amino-3′- deoxyguanosine are valuable probes for investigating the metal ion interactions at the active site of the group I ribozyme. However, these experiments require a thorough understanding of the protonation state of

Full text of DOI:10.1021/ol071129h

ORGANIC
LETTERS
2007
Vol. 9, No. 16
3057-3060
Syntheses of
(2 )3 ′)3′-deoxyguanosine
-15N-Amino-(2
′
′
and Determination of Their pKa Values
15
by N NMR Spectroscopy
Qing Dai,† Charles R. Lea,‡ Jun Lu,§ and Joseph A. Piccirilli*,†,‡,§
Department of Biochemistry & Molecular Biology, Department of Chemistry, and
Howard Hughes Medical Institute, The UniVersity of Chicago, 929 East 57th Street,
MC 1028, Chicago, Illinois 60637
jpicciri@uchicago.edu
Received May 15, 2007
ABSTRACT
2
′
-Amino-2
of the group I ribozyme. However, these experiments require a thorough understanding of the protonation state of the amino group at a
specific pH. Here, we describe the first syntheses of 2 -deoxyadenosine, 2 -deoxyguanosine, and 3
15N-amino-2 15N-amino-2 15N-amino-3
deoxyguanosine. The 15N-enriched nucleus allows convenient and accurate determination of the amine pKa by 15N NMR.
′-deoxyguanosine and 3′-amino-3′-deoxyguanosine are valuable probes for investigating the metal ion interactions at the active site
′
-
′
′
-
′
′
-
′-
2′-Amino-2′-deoxynucleosides and 3′-amino-3′-deoxynucleo-
sides (herein referred as 2′(3′)-amino-2′(3′)-deoxynucleo-
sides) and oligonucleotides containing them have received
much attention in recent years as potential therapeutic
agents,1 chemogenetic agents,2 and diagnostic and biochemi-
cal probes. For example, 3′-amino-3′-deoxyadenosine deriva-
tives inhibit replication of HIV-1 and serve as tailored
agonists for activation of an engineered adenosine receptor.3
As components of antisense oligonucleotides or small
interfering RNAs,4 2′(3′)-amino-2′(3′)-deoxynucleosides have
the potential to improve drug efficacy by imparting resistance
to chemical and enzymatic degradation.5 In addition to their
therapeutic potential, the distinct physiochemical properties
of 2′(3′)-amino-2′(3′)-deoxynucleosides render them espe-
cially powerful tools for exploring RNA structure, function,
and dynamics,6 particularly in defining the roles and environ-
ments of specific 2′- or 3′-hydroxyl groups within structured
and catalytic RNA molecules.5,7
(3) Gao, Z.-G.; Duong, H., T.; Sonina, T.; Kim, S.-K.; Van Rompaey,
P.; Van Calenbergh, S.; Mamedova, L.; Kim, H. O.; Kim, M. J.; Kim, A.
Y.; Liang, B. T.; Jeong, L. S.; Jacobson, K. A. J. Med. Chem. 2006, 49,
2689.
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S. Biochim. Biophys. Acta 1999, 1489, 53. (c) Estibeiro, P.; Godfray, J.
Trends Neurosci. 2001, 24, S56.
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Nucleic Acids Res. 1994, 22, 20. (b) McGee, D. P. C.; Vaughn-Settle, A.;
Vargeese, C.; Zhai, Y. J. Org. Chem. 1996, 61, 781.
† Department of Biochemistry & Molecular Biology.
‡ Department of Chemistry.
§ Howard Hughes Medical Institute.
(1) (a) Ikehara, M.; Maruyama, T.; Miki, H. Tetrahedron Lett. 1976,
17, 4485. (b) Bobek, M. Carbohydr. Res. 1979, 70, 263. (c) Ranganathan,
R. Tetrahedron Lett. 1977, 18, 1291. (d) Santiago, F. S.; Khachigian, L.
M. J. Mol. Med. 2001, 79, 695.
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10.1021/ol071129h CCC: $37.00
© 2007 American Chemical Society
Published on Web 07/13/2007

Our interest in 2′(3′)-amino-2′(3′)-deoxyguanosine origi-
nates from its value in mechanistic investigations of the group
I ribozyme, which efficiently catalyzes nucleotidyl transfer
between an oligonucleotide substrate and guanosine.8 Previ-
ous metal ion rescue experiments9 and crystal structures10
have implicated metal ions in the catalytic mechanisms of
exon-ligation and intron-excision. However, whether three
metal ions (Figure 1A) or two metal ions (Figure 1B)
Defining the interactions between 3′-amino-3′-deoxy-
guanosine and the ribozyme requires knowledge of the amine
pKa, as protonation may compete with metal ion coordina-
tions and influence binding of the guanosine analogue to the
ribozyme. Eckstein et al. have used 13C NMR to determine
the ribofuranosyl amine pKa of 2′-amino-2′-deoxycytidine
in a dinucleotide.12 The corresponding pKa value for 2′-
amino-2′-deoxyguanosine has not been determined by NMR
but was inferred from biochemical experiments.11 We wanted
to use 15N NMR to determine the pKa values of 2′(3′)-amino-
2′(3′)-deoxyguanosine, which requires the installation of a
15N-enriched amino group at the 2′(3′)-R position of gua-
nosine. The low natural abundance of 15N (0.36%) allows
facile chemical shift assignment of 15N-enriched (98%)
nuclei, which yield one singlet in the 15N NMR spectrum.
Moreover, the ionization state of the amine influences the
15N chemical shift, allowing a sensitive and convenient way
to determine the pKa of the amino group.13 Herein, we
describe the first syntheses of 2′-15N-amino-2′-deoxyadeno-
sine, 2′-15N-amino-2′-deoxyguanosine, and 3′-15N-amino-3′-
deoxyguanosine and determination of their pKa values using
15N NMR.
To synthesize 2′(3′)-15N-amino-2′(3′)-deoxyguanosine, we
considered strategies that introduce the 15N atom into the
C-2′- or C-3′ position of guanosine using the commercially
available 15N-labeled reagents. Some of these include 15NH3,
PhCH215NH2, and phthalimide-15N potassium salt. Consider-
ing the nucleophilicity of these 15N-labeled reagents, the most
straightforward approach would involve SN2 displacement
reactions with the 2′- or 3′-â-triflate derivative of guanosine.
Iodide or bromide has been reported to displace the 2′-â-
triflate from the 3′,5′-O-disilyl protected guanosine derivative
(1b) to give the corresponding 2′-R-halo-2′-deoxyguanosine
derivatives.14 We recently reported that t-butylthiol15 reacts
with 1b smoothly to generate a 2′-t-butylthio-2′-deoxygua-
nosine derivative. Direct SN2 displacement reaction of 1b
with amines, however, has not been reported.
We first tried to introduce the 2′-amino group directly by
treating 1b with ammonia but obtained no desired product
under various conditions.16 The 2′-â-triflate derivatives of
adenosine, cytosine and uridine react with phthalimide to
afford the corresponding 2′-R-phthalimido analogues.17 How-
ever, we observed no reaction for guanosine analogue 1b
under similar conditions. Based on the previous work
showing that the 2′-â-triflate derivative of adenosine (1a)
reacts with methylamine and dimethylamine,18 we attempted
Figure 1. Models of the transition state for both step 1 and step
2 of the group I intron RNA splicing reaction with (A) three
catalytic metals and (B) two catalytic metals. In both models, there
is a metal ion, MA or M1, which coordinates with the 3′-hydroxyl
group of the U-1 residue. However, in the three-metal ion model,
two distinct metal ions, MB and MC, coordinate with the 3′- and
2′-hydroxyl groups of guanosine, respectively, whereas in the two-
metal ion model, M2 coordinates with both the 3′- and 2′-hydroxyl
groups.10
contribute to transition-state interactions remains unclear. The
crystal structure of the Azoarcus intron suggests that the 3′-
oxygen of the guanosine cofactor interacts with M2 in the
ground state (Figure 1B). The guanosine analogue, 3′-amino-
3′-deoxyguanosine, provides a probe to test this interaction
via metal ion rescue experiments in the same manner that
studies using 2′-amino-2′-deoxyguanosine have established
the interaction between MC and the guanosine 2′-hydroxyl
group as shown in Figure 1A.11
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Weeks, K. M. J. Am. Chem. Soc. 2000, 122, 216. (f) Gordon, P. M.;
Sontheimer, E. J.; Piccirilli, J. A. Biochemistry 2000, 39, 12939. (g) Gordon,
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B.; Jones, B.; Cosstick, R.; Cech, T. R. Nature 1997, 388, 805. (e) Piccirilli,
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3058
Org. Lett., Vol. 9, No. 16, 2007

to use PhCH215NH2 for the synthesis of 2′-15N-benzylamino-
2′-deoxyadenosine and 2′-15N-benzylamino-2′-deoxygua-
nosine, anticipating that the benzyl group could be subse-
quently removed by catalytic transfer hydrogenation19 (Scheme
1).
react similarly with15N-benzylamine to give the correspond-
ing 3′-15N-benzylamino-3′-deoxyguanosine derivative. How-
ever, we had difficulty in preparing the 3′-â-triflate of
guanosine due to poor selectivity during protection of the
2′- and 5′-hydroxyl groups.24 As an alternative, we prepared
the corresponding 3′-â-bromo-3′-deoxyguanosine derivative
(5, Scheme 2) from 5′-O-TBDPS-guanosine.25 However, we
Scheme 1. Syntheses of 2′-15N-Amino-2′-deoxypurine
Nucleosides
Scheme 2. Synthesis of 3′-15N-Amino-3′-deoxyguanosine
Reaction of 1a20 with 15N-labeled benzylamine in THF in
the presence of triethylamine21 at 75 °C for 2 days gave 2a
in 65% yield. Hydrogenolysis with formic acid and Pd/C in
methanol for 6 h removed the benzyl protecting group to
afford 3a in 76% yield. Removal of the 3′,5′-silyl protecting
group with TBAF generated product 4a. It was purified by
reversed-phase column chromatography but the tetra-butyl-
ammonium salt could not be removed completely (as
found that treatment of 5 with benzylamine formed the 2′,3′-
epoxide derivative exclusively. Probably, benzylamine first
removes the 2′-acetyl group, releasing the 2′-hydroxyl group,
which then attacks the C-3′ from the R-face.
1
indicated by H NMR). To circumvent this problem, we
Subsequent attempts to synthesize 3′-15N-amino-3′-deoxy-
guanosine were carried out by modifying Zhang’s recent
synthesis of 3′-amino-3′-deoxyguanosine.25 The key steps of
their synthesis include the intramolecular SN2 reaction of 9a
(Scheme 2), followed by ring opening under basic conditions
to introduce the 3′-benzylamino group. The intermediate 9a
was prepared by treating 3′-bromoguanosine derivative 6
with benzyl isocyanate. Unfortunately, 15N-benzyl isocyanate
is not commercially available and preparing it from expensive
15N-benzylamine seemed impractical. To use 15N-benzyl-
amine directly, we adopted a two-step strategy to introduce
treated 3a with ammonium fluoride (NH4F) in methanol22
and obtained 4a in 87% yield, which showed no contamina-
tion of NH4F (as revealed by the absence of a 19F NMR
signal) after purification by reversed-phase chromatography.
We synthesized 2′-15N-amino-2′-deoxyguanosine (4b)
using the same approach. Although 1b behaved differently
from 1a in its reaction with phthalimide, we found that 1b
reacted smoothly with 15N-benzylamine in DMF23 at 75 °C
to generate 2b in 55% yield. Direct displacement of the 2′-
â-triflate from guanosine by an amine to generate the
corresponding 2′-R-amino-2′-deoxyguanosine analogue has
not been reported previously. We expect that other primary
amines may react similarly. Removal of benzyl (82%) and
silyl protecting groups (84%) in the same manner as
described for the adenosine derivative afforded 2′-15N-amino-
2′-deoxyguanosine 4b (Scheme 1).
a
15N-labeled benzylaminocarbonyl group at the 2′-R-C
position of 6. Reaction of 6 with 4-nitrophenyl chloro-
formate26 first gave an intermediate, which then reacted with
15N-benzylamine to give the 2′-O-15N-benzylaminocarbonyl-
guanosine analogue. We found that the reaction of 6 with
4-nitrophenyl chloroformate gave different major products
depending upon the solvent. In dichloromethane, the reaction
gave chloroformate 7 as the major product, while in pyridine,
carbonate 8 formed as the major product (Scheme 2). Both
7 and 8 reacted readily with 15N-benzylamine in dichloro-
methane in the presence of triethylamine to give the key
Encouraged by the successful introduction of the 2′-15N-
benzylamino group into adenosine and guanosine, we
reasoned that the 3′-â-triflate derivative of guanosine would
(19) Yagi, H.; Thakker, D. R.; Lehr, R. E.; Jerina, D. M. J. Org. Chem.
1979, 44, 3443.
(20) Jiang, C.; Suhadolnik, R. J.; Baker, D. C. Nucleosides Nucleotides
1988, 7, 271.
(21) Considering the cost of 15N-labeled benzylamine, we added triethyl-
amine as base instead of using an excess amount of 15N-labeled benzylamine.
(22) Zhang, W.; Robins, R. J. Tetrahedron Lett. 1992, 33, 1177.
(23) When THF was used as solvent, 2a failed to give desired product
2b.
(24) (a) Robins, M. J.; Hawrelak, S. D.; Hernandez, A. E.; Wnuk, S. F.
Nucleosides Nucleotides 1992, 11, 821. (b) Kim, B.-T.; Kim, S.-K.; Lee,
S.-J.; Hwang, K.-J. Bull. Kor. Chem. Soc. 2004, 25, 243.
(25) Zhang, L.; Cui, Z.; Zhang, B. HelV. Chim. Acta 2003, 86, 703.
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Org. Lett., Vol. 9, No. 16, 2007
3059

Figure 2. Determination of aminonucleoside pKa values by following 15N NMR chemical shifts versus pH. (A) 2′-15N-amino-2′-
deoxyadenosine, pKa ) 6.2; (B) 2′-15N-amino-2′-deoxyguanosine, pKa ) 6.2; (C) 3′-15N-amino-3′-deoxyguanosine, pKa ) 7.0. Data were
fit according to eq 1.
suggest that the nucleobase exerts little influence on the pKa
of the 2′-amino group, as observed for the 2′-hydroxyl group
pKa in natural RNA.30 The regioisomer 3′-15N-amino-3′-
deoxyguanosine ionizes with pKa ) 7.0 (Figure 2C), higher
than its 2′-counterpart.
intermediate 9b. Cyclization with NaH in DMF gave
intermediate 10 in 87% yield.27 Removal of the protecting
groups25 gave 3′-15N-amino-3′-deoxyguanosine 12.
To determine the pKa values by 15N NMR, we dissolved
each 15N-labeled nucleoside in deionized water and combined
the solutions with an equal volume of buffer.28 We kept the
final concentration of nucleoside in each sample (17 mM)
well below the buffer concentration (250 mM).12 We acquired
15N NMR spectra using 90% formamide in DMSO-d6 as the
reference. In all cases, 15N chemical shifts moved upfield
with increasing pH as shown in Figure 2. The chemical shifts
were plotted against pH and fit to eq 1, which was derived
from the Henderson-Hasselbalch equation,29
In conclusion, we have efficiently synthesized 2′-15N-
amino-2′-deoxyadenosine and 2′-15N-amino-2′-deoxygua-
nosine by SN2 displacement of the 2′-â-triflate analogues of
adenosine and guanosine with 15N-benzylamine. We prepared
3′-15N-amino-3′-deoxy-guanosine using a two-step strategy
to introduce an 15N-labeled benzylaminocarbonyl group at
the 2′-R-C position by first treating 6 with 4-nitrophenyl
chloroformate followed by treatment with 15N-benzylamine.
We determined the pKa values for the ribofuranosyl amines
in these nucleoside analogues by monitoring the chemical
shifts of their 15N NMR spectra as a function of pH. The
pKa value for 3′-amino-3′-deoxyguanosine allows us to
calculate the relative concentrations of the ionized and neutral
species at specific pH values. This fundamental physio-
chemical information will help to define the interaction of
3′-amino-3′-deoxyguanosine with the group I intron.
(pH-pKa)
δ ) δacid + δbase10(pH-pK )/[1 + 10
]
(1)
a
where δ is the 15N chemical shift and δacid and δbase represent
the chemical shift values at the low and high extremes of
pH, respectively. The influence of pH on the measured 15
N
chemical shifts in Figure 2 follows the above equation, where
the pKa value corresponds to the midpoint of the curve. In
all cases, the fits gave very good correlation coefficients (R
> 0.999). Our data indicate that both 2′-15N-amino-2′-
deoxyadenosine and 2′-15N-amino-2′-deoxyguanosine ionize
with pKa ) 6.2 (Figure 2A,B), identical to that obtained for
2′-amino-2′-deoxycytidine by 13C NMR. These observations
Acknowledgment. Q.D. is a Research Assistant Professor
at the University of Chicago, J.L. is a Research Associate,
and J.A.P. is an Investigator of the Howard Hughes Medical
Institute. We thank the following members of the Piccirilli
laboratory for critical comments on the manuscript: J. Ye,
N-S. Li, R. Fong, J. Frederiksen, T. Novak, R. Sengupta, K.
Sundaram, and S. Koo.
(27) In the report from Zhang et al.,25 the 5′-TBDPS silyl protecting
group was also removed in this step. However, under the same reaction
conditions, the protecting group survived in our experiments.
(28) The following buffers were used: sodium acetate, pH 4.5-5.50;
NaMES, pH 5.75-6.50; NaMOPS, pH 6.75-7.50; NaEPPS, pH 7.75-
8.50; NaCHES, pH 9.0-9.5.
(29) (a) Forman-Kay, J. D.; Clore, G. M.; Gronenborn, A. Biochemistry
1992, 31, 3442. (b) Schaller, W.; Rpbertson, A. D. Biochemistry 1995, 34,
4714.
Supporting Information Available: Full experimental
1
and analytical data and the H and 13C NMR spectra for
compounds 2a,b, 3a,b, 4a,b, and 7-12. This material is
available free of charge via the Internet at http://pubs.acs.org.
OL071129H
(30) Li, Y.; Breaker, R. R. J. Am. Chem. Soc. 1999, 121, 5364.
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Org. Lett., Vol. 9, No. 16, 2007