Summary:
The purpose of the current study was to synthesize 2΄-deoxy-β-
D-ribofuranosyl-5-hydroxy-5-methylhydantoin 1.1b, one of the main oxidation
products of thymidine in DNA. In the first part, the synthetic efforts towards
the open-chain N-(2΄-deoxy-β-D-ribofuranosyl)-N΄-pyruvoyl-urea 1b. In the
second part, the studies towards the generation of the target molecule
(compound 1.1b) from the ozonolysis reaction of protected thymidines, are
described.
In the first part, two synthetic methods were applied: Under the first method
(Scheme 10, p. 24), the reaction of β-D-ribofuranosyl isocyanate 3a with amides
was studied. It was found that 3a was a particularly unstable substance with
the isocyanate group behaving as a good leaving group, leading to N-pyruvoyl-β-
D-ribofuranosylamine 10 after reaction with a low reactivity amide such as
pyruvamide (4). Pyruvamide proved a poor nucleophile in reactions with
isocyanates, such as 18. Similar reactivity was exhibited by the nitrogen of
(S)-lactamide, as it was shown when the protected lactamide 15 was left to
react with 18 under similar conditions.
Under the second synthetic method (Scheme 31, p. 43), the isocyanate of
protected lactamide 14 reacted with 2,3,4,6-tetra-O-acetyl-β-
D-glucopyranosylamine 19. From the two products obtained, product 23
corresponds to the first successful preparation of the desired precursor which
may be converted in two steps, namely deprotection of the silyl group and
oxidation of the free hydroxyl, to the final target compound.
In the second part, the products of the ozonolysis reaction of protected
thymidines were characterized. Specifically, the precursor compounds 27a, 27b
and 27c were characterized for the first time. These structures have been
postulated in the literature1 but have not been characterized to date. The
precursor decomposition products were also isolated and characterized and more
specifically, the N-formylurea 28a-c, the Ν-formamide 29a-c and the hydantoin
2.1a-c. In the case of the tribenzylated thymidine, hydantoin 33 was isolated
directly from the ozonolysis reaction which led to a proposed mechanism. The
open-chain pyruvoyl-urea structure 2a-c was not observed indicating the
relative stability of the closed ring of the 5-hydroxy-5-methyl-hydantoin.
Our results are in agreement with the studies from J. Cadet and coworkers1,
with the only exception that Ν-(2-deoxy-β-D-ribofuranosyl)-Ν-acetyl-urea
(Scheme 7, p. 18) was not observed. It is also important that, in agreement
with the above work, 2-deoxy-β-D-ribofuranosyl-urea was not observed although
it has been mentioned as aproduct in a more recent study by von Sonntag &
coworkers.2 This fact provides useful hints in order to draw a suitable
mechanism for the reaction. Finally, through the present study, a method for
the stereoselective preparation of (5R)-Ν-(2΄-deoxy-3΄,5΄-di-Ο-
tert-butyldimethylsilyl-β-D-ribofuranosyl)-5-hydroxy-5-methylhydantoin has been
developed by utilizing the steric hindrance of a TBDMS group at the 5’-postion.
Keywords:
ozonolysis, thymine, thymidine, oxidation damage, de novo synthesis
