Synthesis of oligonucleotides containing thiazole and thiazole N-oxide nucleobases

Article abstract of DOI:10.1021/ol017003g

(equation presented) The thiazole C-nucleoside analogue was synthesized by the Hantzsch cyclization method to form the thiazole ring and was then converted to the thiazole N-oxide C-nucleoside analogue by peracid oxidation of the heterocycle nitrogen. Inc

Full text of DOI:10.1021/ol017003g

ORGANIC
LETTERS
2002
Vol. 4, No. 6
877-880
Synthesis of Oligonucleotides
Containing Thiazole and Thiazole
N-Oxide Nucleobases
Tod J. Miller, Hannah D. Farquar, Arsham Sheybani, Camille E. Tallini,
Andrea S. Saurage, Frank R. Fronczek, and Robert P. Hammer*
Department of Chemistry, Louisiana State UniVersity, Baton Rouge, Louisiana 70803
rphammer@lsu.edu
Received November 6, 2001
ABSTRACT
The thiazole C-nucleoside analogue was synthesized by the Hantzsch cyclization method to form the thiazole ring and was then converted to
the thiazole N-oxide C-nucleoside analogue by peracid oxidation of the heterocycle nitrogen. Incorporation of the thiazole and thiazole N-oxide
phosphoramidites into DNA was successful though significant deoxygenation of the N-oxide occurred during DNA assembly. The mechanism
proposed for the reduction of the thiazole N-oxide to thiazole involves the formation of an N-oxide phosphite ester.
Many naturally occurring and synthetic C-nucleosides show
biological activity and are synthetic targets of interest for
their structural and biochemical properties as well as
antitumor and antiviral activity.1,2 More recently, the syn-
thesis of a variety of C-nucleosides has been undertaken to
explore the biophysical and biochemical properties of DNA.
These studies have been aimed at exploring the role that
hydrogen-bonding, base stacking, and dipolar interactions
have on both duplex stability (thermodynamic properties)
and the templating ability (enzymatic properties) of oligo-
nucleotides.3
A relatively unexplored area of oligonucleotide duplex
structure and stability lies in the design of nucleobase
analogues that contain both a pronounced directional dipole
and the capability to form specific hydrogen bonds with one
of the four canonical bases in DNA. One functional group
that would introduce a pronounced directional dipole, as well
as potential for hydrogen-bonding specificity, is the aromatic
N-oxides. Such an analogue would provide a means to both
examine dipole-dipole interactions and provide a means of
introducing functionality capable of forming specific and
strong hydrogen bonds. In addition to the unconventional
base-pairing selectivity introduced, such nucleoside analogues
containing specific hydrogen-bonding capabilities could play
an important role in deciphering the importance of DNA
polymerase contacts with nucleosides being replicated.
Crystallographic studies have shown that the N3 of purines
and O2 of pyrimidines make H-bonding contacts with the
active site of a number of DNA polymerases.4-6 Pyrimidine
nucleotide triphosphate analogues lacking O2 of the base
were not incorporated by Klenow fragment or by Taq DNA
polymerase and at higher concentrations actually inhibit
polymerase activity.7 We envisioned that nucleoside ana-
(4) Steitz, T. A. J. Biol. Chem. 1999, 274, 17395-17398.
(5) Kiefer, J. R.; Mao, C.; Braman, J. C.; Beese, L. S. Nature 1998,
391, 304-307.
(6) Eom, S. H.; Wang, J. M.; Steitz, T. A. Nature 1996, 382, 278-281.
(7) Guo, M. J.; Hildbrand, S.; Leumann, C. J.; McLaughlin, L. W.;
Waring, M. J. Nucleic Acids Res. 1998, 26, 1863-1869.
(1) Daves, G. D., Jr.; Cheng, C. C. Prog. Med. Chem. 1976, 13, 303-
349.
(2) Hacksell, U.; Daves, G. D., Jr. Prog. Med. Chem. 1985, 22, 1-65.
(3) Loakes, D. Nucleic Acids Res. 2001, 29, 2437-2447.
10.1021/ol017003g CCC: $22.00 © 2002 American Chemical Society
Published on Web 02/21/2002

logues containing an N-oxide functionality, in a structural
position analogous to the O2 of the pyrimidine base, would
provide critical data pertaining to the importance of this
contact in DNA polymerase catalysis. We have chosen
thiazole as our heterocyclic base; it has been previously
proven to be stable to the conditions required for oligonucleo-
tide synthesis8 and is readily oxidized to the N-oxide.9
Numerous reports have provided synthetic details for the
preparation of the thiazole ring in tiazofurin and related
analogues.10-12 The thiazole ring was constructed from
ribofuranosylcyanide using the classical Hantzsch method,13
but with modifications to allow the preparation of 2-(2′-
deoxy-â-D-ribofuranosyl)thiazole and its N-oxide. Reaction
of chlorosugar 1 with excess sodium cyanide in dry DME
provided the nitrile 2 in a 3.2:1 â:R mixture of anomers in
83% yield. As previously reported separation of these
anomers proved difficult.10 Quantitative conversion of 2 to
the thioamide by treatment with hydrogen sulfide and a
catalytic amount of DMAP allowed ready purification of the
â-anomer to provide compound 3. The thioamide 3 was
refluxed with 50% aqueous chloroacetaldehyde in 2:1 THF:
ethanol,14 but the reaction was low-yielding (28%) and
chromatographic separation of the thiazole nucleoside (4)
from side-products proved difficult. However, reaction of 3
with bromoacetaldehyde diethyl acetal and a catalytic amount
of 4M HCl/dioxane in refluxing acetone15 provided com-
pound 4 in improved yield (70%), with ready purification
from side-products.
The structure and absolute configuration of 4, 5, and 6
were confirmed by X-ray crystallography (Figure 1). Struc-
For formation of the N-oxide (5), compound 4 was treated
with 2 equivalents of mCPBA9 in THF (72 h) to provide the
N-oxide 5 in 48% yield. Longer reaction times provided little
additional product, although unreacted thiazole 4 was readily
recycled. However a more efficient method using 1.1
equivalents each of trifluoroacetic anhydride (TFAA) and
hydrogen peroxide urea complex (UHP) was utilized,16 which
provided a much higher yield (97%) after only 3 h at room
temperature. Removal of the hydroxyl protecting groups with
NH4OH proved destructive to the thiazole ring of 4.
Therefore, the esters of 4 and 5 were dissolved in 50 mM
sodium methoxide/methanol to provide the free nucleoside
analogues 6 and 7 in 74% and 83% yields, respectively.
(8) (a) Gutierrez, A. J.; Terhorst, T. J.; Matteucci, M. D.; Froehler, B.
C. J. Am. Chem. Soc. 1994, 116, 5540-5544. (b) Hammer, R. P.; Cameron,
M. A.; Sapp, A. L. Synthesis and Thermodynamic Properties of Nucleic
Acids containing Non-Hydrogen-Bonding Nucleoside Analogues, T225; 35th
National Organic Symposium, Trinity University, San Antonio, Texas, June
22-26, 1997.
(9) Begtrup, M.; Hansen, L. B. L. Acta Chem. Scand. 1992, 46, 372-
383.
(10) Srivastava, P. C.; Pickering, M. V.; Allen, L. B.; Streeter, D. G.;
Campbell, M. T.; Witkowski, J. T.; Sidwell, R. W.; Robins, R. K. J. Med.
Chem. 1977, 20, 256-262.
(11) Dalley, N. K.; Petrie, C. R., III; Revankar, G. R.; Robins, R. K.
Nucleosides Nucleotides 1985, 4, 651-659.
Figure 1. X-ray crystallography ORTEP diagrams of (A) 2-(3′,5′-
O-p-toluoyl-2′-deoxy-â-D-ribofuranosyl)thiazole, (B) 2-(3′,5′-O-p-
toluoyl-2′-deoxy-â-D-ribofuranosyl)thiazole-N-oxide, and (C) 2-(2′-
deoxy-â-D-ribofuranosyl)thiazole.
(12) Ramasamy, K. S.; Lau, J. Y. N. Nucleosides Nucleotides Nucleic
Acids 2001, 20, 1329-1331.
(13) Vernin, G. In Thiazole and Its DeriVatiVes; Metzger, J. V., Ed.;
John Wiley & Sons: New York, 1979; Vol. 34, pp 165-335.
(14) Wigerinck, P.; Snoeck, R.; Claes, P.; Declercq, E.; Herdewijn, P.
J. Med. Chem. 1991, 34, 1767-1772.
(15) Baldwin, J. J.; Christy, M. E.; Denny, G. H.; Habecker, C. N.;
Freedman, M. B.; Lyle, P. A.; Ponticello, G. S.; Varga, S. L.; Gross, D.
M.; Sweet, C. S. J. Med. Chem. 1986, 29, 1065-1080.
(16) Caron, S.; Do, N. M.; Sieser, J. E. Tetrahedron Lett. 2000, 41, 2299-
2302.
tural analysis indicates a C2′ endo-sugar conformation
(P)∼180). The thiazole ring is planar with the sulfur
positioned over the oxygen of the furanose ring. The dihedral
878
Org. Lett., Vol. 4, No. 6, 2002

ethyl tetraisopropylphosphorodiamidite (3 equiv), diisopro-
pylamine (DIPA; 1 equiv) and tetrazole (1 equiv) in dry
CH2Cl2. After a 2 h reaction time, CH3OH was added and
the product purified to provide thiazole phosphoramidite 9
in 89% yield. These conditions were initially used for the
phosphitylation of thiazole N-oxide nucleoside 10 and
resulted in a complex reaction mixture containing significant
amounts of the deoxygenated thiazole amidite 9 as indicated
by TLC. Attempted phosphitylation of the N-oxide 10 with
the more reactive chlorophoramidite reagent also led to a
mixture of products containing mostly starting material as
well as thiazole phorphoramidite 9. Thus a modified protocol
was developed whereby phosphordiamidite reagent is added
portionwise to avoid large excesses. To a solution of DMT-
N-oxide 10 in CH2Cl2 was added DIPA (1.1 equiv), tetrazole
(1.1 equiv), 2-cyanoethyl tetraisopropylphosphorodiamidite
(1.3 equiv) and was reacted for 30 min. A second portion of
2-cyanoethyl tetraisopropylphosphorodiamidite (1.3 equiv)
was added. Following an additional 20 min of reaction,
CH3OH was added to quench the reaction and thiazole
N-oxide phosphoramidite 11 was purified by flash chroma-
tography in 67% yield.
Scheme 1
For oligonucleotide synthesis, each nucleoside phosphor-
amidite was dissolved in dry acetonitrile to a 0.10 M
concentration and used to prepare several self-complementary
oligonucleotides based on the Dickerson dodecamer sequence
as well as oligonucleotides to examine the enzymatic
properties of each analogue. Due to the observed ammonia
lability of thiazole, phenoxyacetyl (Pac), 4-isopropylphenoxy-
acetyl, and acetyl N-protection for dA, dG, and dC phos-
phoramidites, respectively, were used for oligonucleotide
assembly (Glen Research, Sterling, VA). The synthesis was
performed on a 1.0 µmol scale, and all coupling steps except
for the thiazole analogues were >98% by trityl assay (vide
infra). Base and phosphodiester deprotection was accom-
plished using 50 mM potassium carbonate in methanol (200
µL) at room temperature for 3 h. Acetic acid (1.5 equiv) in
water (200 µL) was added, and the resulting solutions were
evaporated to dryness.
angles, ø (O1′-C1′-C2-S1), for the toluoyl protected
thiazole nucleoside, the toluoyl protected thiazole N-oxide
nucleoside, and the free thiazole nucleoside are 21.6°, 23.0°,
and 32.6°, respectively, which is comparable to other crystal
structure data obtained for similar thiazole nucleosides.17
For incorporation into oligonucleotides, nucleoside ana-
logues 6 and 7 were converted to their protected nucleoside
phosphoramidites (Scheme 2). The 5′-hydroxyl of each was
Scheme 2
The deprotected oligonucleotides were analyzed by anion-
exchange HPLC. Figure 2 shows the resulting HPLC profile
for the oligonucleotide sequence 5′-d(CGCXAATTTGCG)-
3′, where X represents a nucleoside having either thiazole
or thiazole N-oxide as heterocyclic base. Analysis of the
products by MALDI spectroscopy indicates that the thiazole
nucleoside is completely stable to the steps used for
oligonucleotide synthesis and deprotection. However, three
product peaks are evident for the thiazole N-oxide oligo-
nucleotide. The mass of the peaks corresponds to an
octanucleotide (Figure 2B, peak III) as well as intact
dodecanucleotides containing either thiazole (Figure 2B, peak
I) or thiazole N-oxide (Figure 2B, peak II). Both the problem
in the synthesis of the thiazole N-oxide phosphoramidite 11
and the deoxygenation of thiazole N-oxide during solid-phase
oligonucleotide synthesis suggest that the activated P(III)
reagents are causing the reduction. A common method for
reduction of aromatic N-oxides is the reaction with phos-
phorus trichloride.18,19 Thus, it appears that for deoxygenation
dimethoxytritylated in anhydrous pyridine using 1.0 equiv
of Et3N and dimethoxytrityl chloride (DMTCl) to provide
DMT ethers 8 and 10 in 70% and 90% yield, respectively.
For complete conversion, compound 7 required treatment
with a second equiv of Et3N and DMTCl. Phosphitylation
of thiazole 8 was accomplished by treatment with 2-cyano-
(17) Goldstein, B. M.; Takusagawa, F.; Berman, H. M.; Srivastava, P.
C.; Robins, R. K. J. Am. Chem. Soc. 1983, 105, 7416-7422.
Org. Lett., Vol. 4, No. 6, 2002
879

Scheme 3
N-oxide phosphoramidite and therefore the large peak
corresponding to the octanucleotide seen in the HPLC
chromatogram. The tetrazole-activated N-oxide may be
reacting with the phosphorus of other N-oxide phosphor-
amidite molecules, thus decreasing the concentration of the
active phosphorus species.
In summary, we have successfully synthesized nucleosides
bearing thiazole and thiazole N-oxide as the heteroaromatic
base. These nucleosides were incorporated into oligonucleo-
tides both for duplex melting studies and to study the
properties of each nucleoside when transcribed. The thermo-
dynamic and enzymatic behavior of oligonucleotides con-
taining thiazole and thiazole N-oxide analogues are currently
being investigated.
Figure 2. (A) Chromatogram of the Dickerson dodecamer contain-
ing thiazole C-nucleoside (Th); MALDI (M - H)- 3593, (calcd)
3593. (B) Chromatogram of the Dickerson dodecamer containing
thiazole N-oxide C-nucleoside (Ox); I. MALDI (M - H)- 3593,
(calcd) 3593; II. MALDI (M - H)- 3613 (calcd) 3609; III. MALDI
(M - H)- 2426 (calcd) 2422. Conditions: Hydrocell NS 1000
(anion exchange) 150 × 4.6 mm column; solvent A 25 mM CHES
pH 8, 30% MeOH; solvent B 25 mM CHES/1 M (NH4)2SO4 pH 8,
30% MeOH; 0-100% B over 60 min, flow rate ) 1 mL/min.
Acknowledgment. The authors wish to thank Susan
Newman at the School of Veterinary Medicine Genelab for
generous use of facilities. Financial support was provided
by the National Cancer Institute (CA 65930) of the National
Institutes of Health and by an LSU Dr. Charles E. Coates
Scholar Research Grant awarded to H.D.F.
to occur the N-oxide must first be covalently attached to the
P(III) moiety. In Scheme 3 we suggest a mechanism (A)
whereby the thiazole N-oxide, whether resin bound or in
solution, reacts with activated phosphoramidite reagent to
give phosphitylated N-oxide species 12. Once attached to
the oxygen, the phosphorus can donate its electrons to reduce
the thiazole and create a metaphosphate-like intermediate.
We favor this mechanism over direct nucleophilic reduction
by the P(III) reagents (B) as we find the thiazole N-oxide
phosphoramidite to be stable to long-term storage.20 This
explains the decreased coupling efficiency of the thiazole
Supporting Information Available: Experimental details
for Schemes 1 and 2, full characterization of compounds
2-11, X-ray crystallographic information for Figure 1, and
MALDI mass spectra of the oligonucleotides synthesized.
This material is available free of charge via the Internet at
http://pubs.acs.org.
OL017003G
(18) Ochiai, E. In Aromatic Amine Oxides; Elsevier Publishing Com-
pany: New York, 1967; pp 190-196.
(19) Albini, A.; Pietra, S. In Heterocyclic N-oxides; CRC Press: Boca
Raton, FL, 1991; pp 120-123.
(20) Thiazole N-oxide phosphoramidite 11 is stable for 1 year when
stored at -20 °C.
880
Org. Lett., Vol. 4, No. 6, 2002