Synthesis of carbocyclic nucleotides as potential substrates for thymidylate kinase

Article abstract of DOI:10.1081/NCN-200060251

Enantiomerically pure carbocyclic 2′-deoxy-3′-azidothymidine monophosphate (AZTMP) and carba-2′deoxy-3′-thiocyanatothymidine monophosphate were synthesized to study their behavior toward their phosphorylation by thymidylate kinase. The nucleotides were sy

Full text of DOI:10.1081/NCN-200060251

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SYNTHESIS OF CARBOCYCLIC NUCLEOTIDES AS
POTENTIAL SUBSTRATES FOR THYMIDYLATE KINASE
O. R. Ludek a & C. Meier a
a Institute of Organic Chemistry, University of Hamburg, Hamburg, Germany
Published online: 15 Nov 2011.
To cite this article: O. R. Ludek & C. Meier (2005): SYNTHESIS OF CARBOCYCLIC NUCLEOTIDES AS POTENTIAL SUBSTRATES FOR
THYMIDYLATE KINASE, Nucleosides, Nucleotides and Nucleic Acids, 24:5-7, 683-686
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Nucleosides, Nucleotides, and Nucleic Acids, 24 (5–7):683–686, (2005)
Copyright D Taylor & Francis, Inc.
ISSN: 1525-7770 print/ 1532-2335 online
DOI: 10.1081/NCN-200060251
SYNTHESIS OF CARBOCYCLIC NUCLEOTIDES AS POTENTIAL
SUBSTRATES FOR THYMIDYLATE KINASE
5
O. R. Ludek and C. Meier
Institute of Organic Chemistry, University of Hamburg,
Hamburg, Germany
5
Enantiomerically pure carbocyclic 2’-deoxy-3’-azidothymidine monophosphate (AZTMP) and carba-
2’deoxy-3’-thiocyanatothymidine monophosphate were synthesized to study their behavior toward their
phosphorylation by thymidylate kinase. The nucleotides were synthesized starting from the parent
nucleosides by an alkaline hydrolysis of the corresponding cycloSal-phosphate triesters.
Keywords Carbocyclic Nucleosides, Mitsunobu Reaction, cycloSal-Pronucleotides,
Nucleotides
INTRODUCTION
Nucleoside analogues used in antiviral or antitumor therapies need to be
activated intracellularly by an enzymatic phosphorylation cascade in order to be
biologically active. Unfortunately, some of the well-established active nucleosides
are only poor substrates for these activating enzymes. For example, d4T is a bad
substrate in the first phosphorylating step, catalyzed by thymidine kinase, whereas
AZT is only phosphorylated inefficiently from the monophosphate to the
diphosphate by thymidylate kinase.[1] In the case of d4T, this kinetic bottleneck
can be successfully circumvented by using an established pronucleotide approach,
e.g., the cycloSal-concept (thymidine kinase bypass).[2] So far, this strategy could not
be applied to the intracellular activation of AZT.
The reason for the slow conversion from AZTMP to AZTDP is a structural
change of the enzyme thymidylate kinase while binding the unnatural substrate
AZT-monophosphate. The bulky 3’-azido-group of AZTMP causes a shift in the p-
loop region, leading to an almost complete breakdown of the catalytic mechanism,
responsible for the phosphoryl group transfer.[3]
Address correspondence to C. Meier, Institute of Organic Chemistry, University of Hamburg, Martin-Luther-
King-Platz 6, Hamburg D-20146, Germany.
683
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684
O. R. Ludek and C. Meier
FIGURE 1 Phosphorylation cascade of antivirally active nucleosides.
RESULTS
In our group, we are interested in investigating the structural requirements of
nucleotides phosphorylation efficiently by thymidylate kinase. In a first attempt we
replaced the azido group in AZTMP by isosteric substituents, e.g., the thiocyanato-,
the allyl-, and propargyl group, and we increased the flexibility of the nucleotide by
using acyclic analogues of thymidine.[4] Surprisingly, none of the prepared nucle-
otides were substrates for TmpK. Apparently, the acyclic nucleotides were too
flexible to bind to the active site of the kinase.
For the synthesis of carbocyclic 3’-azido-2’-deoxythymidine monophosphate
(carba-AZTMP) and carba-3’-thiocyanato-2’deoxythymidine monophosphate, we
started from enantiomerically pure carbocyclic thymidine 1, which was prepared
by a condensation reaction of the corresponding protected cyclopentanol with the
protected nucleobase under Mitsunobu conditions.[5] As reported before, besides
FIGURE 2 Variable parameters for the SAR of nucleotides towards thymidylate kinase.

O. R. Ludek and C. Meier
685
the desired N-1-alkylated product, the undesired O-2-alkylated product is formed.
The ratio of these two products can be fine-tuned by variation of the solvents and
the substituents on the benzoyl group in the N-3-position of the nucleobase. We
found that nonpolar solvents (1,4-dioxane, diethylether) and electron-donating
groups in the benzoyl residue favour the O-2-alkylation, while polar solvents
(acetonitrile) and electron-withdrawing substituents lead predominantly to the
desired N-1-product. We also found a strong dependence of the product ratio on
the electron density on the carbon bearing the hydroxy group. The nucleobase can
react as an ambident nucleophile with a hard (O) and a soft (N) alkylation site. The
less electron density (highfield shift in 13C NMR), the more O-2-alkylated product
can be isolated.
After tritylation of the 5’-hydroxy group in carba-dT 1 with triphenylmethyl-
chloride in pyridine, the protected nucleoside 2 was converted into the 2,3’-anhydro
derivative 3 under Mitsunobu conditions (90% yield). Nucleophilic opening of the
anhydro structure with sodium azide in DMF gave the protected target structure
(82% yield). After detritylation with 2% benzensulfonic acid (BSA) in dichloro-
methane/methanol (7:3) in 79% yield, the desired carbocyclic AZT 5 was obtained.
For the synthesis of the thiocyanato derivative 6, a second portion of the protected
2,3’-anhydro structure 3 was hydrolyzed quantitativly to the corresponding xylo-
nucleoside 4 with sodium hydroxide in ethanol. To our surprise, after activation of
the 3’-hydroxy group by triflic anhydride in dichloromethane and subsequent
nucleophilic displacement by sodium thiocyante in DMF, the protected target
structure could only be obtained in 12% yield.[6] Detritylation led to the desired
3’-thiocyanato derivative 6 in 79% yield.
Finally, the nucleosides were transferred into their monophosphate esters
(nucleotides) by an alkaline hydrolysis of the corresponding cycloSal-phosphate
triesters. The cycloSal pronucleotide system was originally designed for a selective
intracellular delivery of therapeutically active nucleoside monophosphates by a
purely pH-driven mechanism, but it can also be used for the synthesis of nucleotides
in the laboratory. The synthesis of the cycloSal-phosphate triesters was performed
FIGURE 3 Synthesis of 3’-modified carbocyclic nucleosides.

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O. R. Ludek and C. Meier
FIGURE 4 Synthesis of carbocyclic nucleoside 5’-monophosphates.
using acetonitrile/DMF and the chlorophosphite/t-butylhydroperoxide method at
À20°C, as published before.[7] These phosphate triesters were then dissolved in
actetonitrile/water and triethylamine was added. The reaction mixture was stirred
until a complete decomposition of the educt was observed.
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dideoxythymidine (zidovudine). Mol. Pharmacol. 1999, 56, 1354–1361.
2. Meier, C. cycloSal-pronucleotides—design of chemical trojan horses. Mini Rev. Med. Chem. 2002, 2, 219–234.
3. Lavie, A.; Schlichting, I.; Vetter, I.R.; Konrad, M.; Reinstein, J.; Goody, R.S. The bottleneck in AZT activation.
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TmpK—a key enzyme in the metabolism of AZT. Nucleosides Nucleotides Nucleic Acids 2003, 5–8, 821–
823.
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6. Schreiber, S.L.; Ikemoto, N. Synthesis of chemically reactive analogues of AZT and their biological evaluation
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