A set of nonpolar thymidine nucleoside analogues with gradually increasing size

Article abstract of DOI:10.1021/ol048487u

(Chemical Equation Presented) We describe a series of nonpolar nucleoside analogues having similar shapes and gradually increasing size. The structure of the nucleobase thymine was mimicked with toluene derivatives, replacing 02/04 with hydrogen, fluorine, chlorine, bromine, and iodine. Glycosidic bonds were formed by reactions of lithiated 2,4-dihalotoluenes with a deoxyribonolactone derivative. Structural analysis by NMR showed similar conformations across the series. The compounds are useful for study of the biological recognition of nucleotides and nucleic acids.

Full text of DOI:10.1021/ol048487u

ORGANIC
LETTERS
2004
Vol. 6, No. 22
3949-3952
A Set of Nonpolar Thymidine
Nucleoside Analogues with Gradually
Increasing Size
Tae Woo Kim and Eric T. Kool*
Department of Chemistry, Stanford UniVersity, Stanford, California 94305-5080
kool@stanford.edu
Received July 30, 2004
ABSTRACT
We describe a series of nonpolar nucleoside analogues having similar shapes and gradually increasing size. The structure of the nucleobase
thymine was mimicked with toluene derivatives, replacing O2/O4 with hydrogen, fluorine, chlorine, bromine, and iodine. Glycosidic bonds
were formed by reactions of lithiated 2,4-dihalotoluenes with a deoxyribonolactone derivative. Structural analysis by NMR showed similar
conformations across the series. The compounds are useful for study of the biological recognition of nucleotides and nucleic acids.
The biological effects of nucleosides and nucleotides range
widely, from their incorporation into DNA (and their
resulting interactions with many DNA-recognizing proteins
and enzymes) to their involvement in many other aspects of
cellular metabolism. Understanding the recognition of nucleo-
tides by enzymes is important not only because of these
biological roles but also because such enzymes are often
medicinal targets. In an effort to separate steric and electronic
effects in nucleotide recognition, we have designed nonpolar
nucleoside isosteres,1 which maintain the size and shape of
natural nucleosides as closely as possible but lack polar
hydrogen bonding groups such as the ones responsible for
Watson-Crick hydrogen bonds. Such compounds have been
useful in underlining the importance of hydrogen bonding
in some biochemical activities2 and in revealing that some
biological processes such as DNA replication can fare
surprisingly well without hydrogen bonding groups.3
deformability.4 Here we describe the preparation and struc-
tures of such a series of compounds. We took thymidine as
a starting point and designed a set of compounds with smaller
(the dihydrogen case, 1) or increasingly larger substituents
(dihalogen deoxyribosides 2-5). Computer models suggested
that progression from hydrogen and moving down the
halogen series would produce bond lengths at the 2,4-
positions (corresponding to oxygens of thymidine, 6) ranging
from 1.1 to 2.0 Å (Figure 1). Group radii also would increase
gradually along the series as well.
We adopted a generalized strategy for preparing the first
four of the five C-glycosides. Our approach involved
(2) (a) Maki, A. S.; Kim, T. W.; Kool, E. T. Biochemistry 2004, 43,
1102. (b) Maki, A. S.; Brownewell, F. F.; Liu, D.; Kool, E. T. Nucleic
Acids Res. 2003, 31, 1059. (c) Lan, T.; McLaughlin, L. W. Biochemistry
2001, 40, 968. (d) Woods, K. K.; Lan, T.; McLaughlin, L. W.; Williams,
L. D. Nucleic Acids Res. 2003 31, 1536. (e) Francis, A. W.; Helquist, S.
A.; Kool, E. T.; David, S. S. J. Am. Chem. Soc. 2003, 125, 16235. (f)
Schofield, M. J.; Brownewell, F. E.; Nayak, S.; Du, C.; Kool, E. T.; Hsieh,
P. J. Biol. Chem. 2001, 276, 45505. (g) Drotschmann, K.; Yang, W.;
Brownewell, F. E.; Kool, E. T.; Kunkel, T. A. J. Biol. Chem. 2001, 276,
46625. (h) Begley, T. J.; Haas, B. J.; Morales, J. C.; Kool, E. T.;
Cunningham, R. P. DNA Repair 2002, 2, 107. (i) Washington, M. T.;
Helquist, S. A.; Kool, E. T.; Prakash, L.; Prakash, S. Mol. Cell. Biol. 2003,
23, 5107. (j) Rausch, J. W.; Qu, J.; Yi-Brunozzi, H. Y.; Kool, E. T.; Le
Grice S. F. J. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 11279. (k) Sun, L.;
Zhang, K.; Zhou, L.; Hohler, P.; Kool, E. T.; Yuan, X. F.; Wang, Z.; Taylor,
J.-S. Biochemistry 2003, 42, 9431.
To probe steric effects in a systematic way, it would be
desirable to have a set of molecules of gradually increasing
size, to act as “molecular rulers” for active site size and
(1) (a) Schweitzer, B. A.; Kool, E. T. J. Org. Chem. 1994, 59, 7238. (b)
Kool, E. T.; Morales, J. C.; Guckian, K. M. Angew. Chem., Int. Ed. 2000,
39, 990. (c) Guckian, K. M.; Schweitzer, B. A.; Ren R. X.-F.; Sheils C. J.;
Tahmassebi, D. C.; Kool, E. T. J. Am. Chem. Soc. 2000, 122, 2213.
10.1021/ol048487u CCC: $27.50
© 2004 American Chemical Society
Published on Web 09/29/2004

Figure 1. Structures of the thymidine analogues, designed to have gradually increasing steric demand. (A) Space-filling models of the
analogues with methyl groups at the point of attachment to deoxyribose, with calculated electrostatic potentials mapped on the van der
Waals surfaces (electrostatic scale: -50 to 30). (B) PM3-calculated bond lengths for the 2,4-substituents, which range in size from H to I
(Spartan ‘02, Wavefunction, Inc., Ivine, CA). Calculated thymine bond lengths are shown for comparison. Also shown are corresponding
bond lengths from crystal structures of three of the compounds (2, 3, 6). Calculated bond lengths are shown with methyl replacing the
deoxyribose. Crystal structure bond lengths shown are given for the free nucleoside.
reactions of selectively lithiated dihalotoluenes, or toluene
itself in the case of 1, with the known deoxyribonolactone
derivative utilized by Woski.5 In practice, the lithiated arenes
reacted moderately well with the lactone, yielding the
C-glycosides with yields ranging from 29% (for 4) to 35%
(for 5). The acidic H3 between two fluorides of 2a hampered
the lithiation at the bromide, so we replaced the lithiation
step with Grignard for 2b. In the four coupling reactions
the desired â-isomer was the major product; the minor
R-isomers were obtained in yields less than 10%. The desired
compounds were easily separated by silica gel column, and
the â-orientation was confirmed by NOE measurements
involving the 2′-protons and their vicinal neighbors (Sup-
porting Information). Deprotection of the siloxane 3′,5′-
protecting group was carried out smoothly with tetrabuty-
lammonium fluoride, giving the first four compounds of the
series in yields of 90-97%. The 2,4-substitutions were
confirmed (relative to other possible isomers) by HMBC and
NOE measurements (Supporting Information), and mass
spectra confirmed that the 2,4-halogens in 2-4 remained
intact.
The diiodide 5 required a different approach and was
constructed from the dibromonucleoside 4. To avoid the
problem of selective lithiation in the presence of two other
iodo groups, we prepared this final compound (the largest
of the series) by replacing the bromo groups of deoxynucleo-
side 4 with iodines using the copper-catalyzed strategy
described by Buchwald.6 Although the procedure had not
been previously described for multiple bromo groups, we
applied it with modest success in this case, obtaining the
diiodide in 22% yield. PM3 calculations of the aryl substit-
uents of this series were used to estimate bond lengths of
the base mimics. Of particular interest are the bond lengths
of the varied 2,4-substituents, which vary over a 1.0 Å range
(Figure 1). Maps of the electrostatic charges over the van
der Waals surfaces are also shown for comparison (Figure
1). Overall, the new dichloro, dibromo, and diiodo com-
(3) (a) Moran, S.; Ren, R. X.-F.; Kool, E. T. Proc. Natl. Acad. Sci. U.S.A.
1997, 94, 10506. (b) Moran, S.; Ren, R. X.-F.; Rumney, S.; Kool E. T. J.
Am. Chem. Soc. 1997, 119, 2056. (c) Matray, T. J.; Kool, E. T. Nature
1999, 399, 704. (d) Delaney, J. C.; Henderson, P. T.; Helquist, S. A.;
Morales, J. C.; Essigmann, J. M.; Kool, E. T. Proc. Natl. Acad. Sci. U.S.A.
2003, 100, 4469. (e) McMinn, D. L.; Ogawa, A. K.; Wu, Y.; Liu, J.; Schultz,
P. G.; Romesberg, F. E. J. Am. Chem. Soc. 1999, 121, 11585. (f) Ogawa,
A. K.; Wu, Y.; McMinn, D. L.; Liu, J.; Schultz, P. G.; Romesberg, F. E.
J. Am. Chem. Soc. 2000, 122, 3274. (g) Wu, Y.; Ogawa, A. K.; Berger,
M.; McMinn, D. L.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc.
2000, 122, 76215. (h) Yu, C.; Henry, A. A.; Romesberg, F. E.; Schultz, P.
G. Angew. Chem., Int. Ed. 2002, 41, 3841. (i) Henry, A. A.; Yu, C.;
Romesberg, F. E. J. Am. Chem. Soc. 2003, 125, 9638. (j) Henry, A. A.;
Olsen, A. G.; Matsuda, S.; Yu, C.; Geierstanger, B. H.; Romesberg, F. E.
J. Am. Chem. Soc. 2004, 126, 6923. (k) Ohtsuki, T.; Kimoto, M.; Ishikawa,
M.; Mitsui, T.; Hirao, I.; Yokoyama, S. Proc. Natl. Acad. Sci. U.S.A. 2001,
98, 4922. (l) Mitsui, T.; Kitamura, A.; Kimoto, M.; To, T.; Sato, A.; Hirao,
I.; Yokoyama, S. J. Am. Chem. Soc. 2003, 125, 5298.
(4) (a) Cummins, L. L.; Owens, S. R.; Risen, L. M.; Lesnik, E. A.; Freier,
S. M.; McGee, D.; Guinosso, C. J.; Cook, P. D. Nucleic Acids Res. 1995,
23, 2019. (b) Hou Y.-M.; Zhang, X.; Holland J. A.; Davis, D. R. Nucleic
Acids Res. 2001 29, 976.
(6) (a) Klapars, A.; Buchwald, S. L. J. Am. Chem. Soc. 2002, 124, 14844.
(b) Klapars, A.; Huang, X.; Buchwald, S. L. J. Am. Chem. Soc. 2002, 124,
7421.
(5) Wichai, U.; Woski, S. A. Org. Lett. 1999, 1, 1173.
3950
Org. Lett., Vol. 6, No. 22, 2004

1
Scheme 1
Table 1. Nucleoside Conformations for 1-5 Determined by H
NMR Measurements in D2O
coupling constants
H1′-H2′ H1′-H2′′ H2′-H3′ H2′′-H3′ H3′-H4′ H2′-H2′′
1
10.68
5.49
5.80
5.62
5.76
5.73
5.92
5.79
6.01
6.08
5.75
1.68
2.24
2.44
2.18
2.47
2.33
13.71
13.58
13.70
13.76
13.59
2b 10.37
∼1.0
3
4
5
10.26
10.23
10.14
1.85
1.95
1.53
summed J valuesa
∑ 2′
∑ 1′
∑ 2′′
∑ 3′
1
2b
16.17
16.17
15.88
15.99
15.87
30.31
29.74
29.97
30.07
29.48
20.88
20.88
21.17
21.47
20.85
9.84
9.73
3
10.04
10.50
9.61
4
5
a ∑1′ ) J1′2′ + J1′2′′; ∑2′ ) J1′2′ + J2′3′ + J2′2′′; ∑2′ ) J1′2′′ +
J2′′3′ + J2′2′′; ∑3′ ) J2′3′ + J2′′3′ + J3′4′. b Data for 2 from ref 7a.
a C2′-endo sugar conformation, falling into the “S” family
of conformers, and an anti glycosidic orientation is present.
Comparison of this structure to that of the difluorotoluene
analogue7a shows a similar structure and conformation
(Figure 2B). The C-Cl bond lengths in the crystal structure
are 1.74 and 1.75 Å, which is, of course, substantially longer
than the corresponding C-F bonds in the difluorotoluene
deoxyriboside crystal structure (1.34 Å) (Figure 1B).7a
Overall, then, the dichloro analogue adopts a structure nearly
the same as the reported crystal structure of thymidine.7b
pounds appear to have quite low polarity. The surface
potentials vary by a relatively small amount, but they suggest
that the three largest cases have negative charge density at
the center of the ring similar to that of the dihydrogen case,
with a small increase for the largest case (the diiodide). Only
the difluoro case has less negative charge density, presumably
because of the high electronegativity of fluorine. Overall,
the shapes are similar to that of the parent thymine.
We were able to obtain crystals of the dichlorotoluene
deoxyribose derivative from chloroform, which afforded a
solid-state X-ray structure (Figure 2A). The structure shows
Because crystal packing forces may alter conformations
in relatively flexible five-membered rings, the structures of
these isosteres in solution were of greater interest than the
solid-state structures. We carried out an analysis of ring
coupling constants to assign deoxyribose conformations for
the series of five compounds.8 The data are shown in Table
1. All five compounds are quite similar, and all are assigned
as S-type sugars. Thus, the conformations fall into a relatively
narrow range, and this range is not greatly different from
the conformational preference of thymidine itself (which is
reported as 70% S).7a We conclude that varying bond lengths
(at the 2-position on the benzene ring in particular) does not
markedly affect the ring conformation.
We also examined bond rotameric preferences for the
glycosidic bonds, which was evaluated by NOE experiments
focused on the C-1′ proton. A syn-glycoside generally shows
large NOE enhancements at the 6-position of the “nucleo-
base” due to the proximity of these two protons. However,
natural thymidine shows only a small enhancement due to
its relatively strong anti orientational preference, and our
Figure 2. Solid-state X-ray crystal structure of dichlorotoluene
deoxyglycoside 3. (A) ORTEP drawing of 3, showing â-anomeric
configuration, C-3′-exo (S type) conformation of deoxyribose, and
anti glycosidic orientation. (B) Comparison of dihedral angles in
the solid-state structures of 3, 2, and thymidine (6). Data for 2 are
from ref 7a, and data for thymidine (6) are from ref 7b.
(7) (a) Guckian, K. M.; Kool, E. T. Angew. Chem., Int. Ed. 1997, 36,
2825. (b) Chechlov, A. N. J. Struct. Chem. 1995, 36, 155.
(8) (a) Wijemenga, S. S.; Mooren, M. M. W.; Hilbers, C. W. In NMR of
Macromolecules: A Practical Approach; Roberts, G. K., Ed.; Oxford
University, 1993; p 258. (b) Rinkel, L. J.; Altona, C. J. Biomol. Struct.
Dyn. 1987, 4, 621.
Org. Lett., Vol. 6, No. 22, 2004
3951

experiments showed similarly small NOEs to the C6-protons
of the four dihalo compounds in this series.
Acknowledgment. This work was supported by the
National Institutes of Health (EB002059 and GM072705).
T.W.K. acknowledges a KOSEF postdoctoral fellowship.
Overall, the results show that this new series of five
compounds is readily prepared and that all five adopt
conformations very close to that of thymidine. Thus, we
expect these nucleoside analogues to be broadly useful as
steric probes of enzyme active sites that normally process
thymidine nucleosides or nucleotides. In addition, when
incorporated into DNA they may well be useful as steric
probes of protein-DNA recognition in general. Experiments
directed along these lines are underway.
Supporting Information Available: Experimental details
of nucleoside synthesis and characterization, details of NMR
structural methods, and details of the crystal structure of
nucleoside 3 (CIF). This material is available free of charge
via the Internet at http://pubs.acs.org.
OL048487U
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Org. Lett., Vol. 6, No. 22, 2004