Synthesis of the four diastereoisomers of 3-thymine-1-(tbutoxycarbonyl)aminocyclopentane-1-carboxylic acid

Article abstract of DOI:10.1016/S0040-4039(02)02696-5

Synthetic routes to all four diastereoisomers of 3-thymine-1-(^tbutoxycarbonyl)aminocyclopentane-1-carboxylic acid have been developed starting from the commercially available (S)-dimethyl malate. The key step in the synthesis involves dialkylat

Full text of DOI:10.1016/S0040-4039(02)02696-5

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 695–698
Synthesis of the four diastereoisomers of
3-thymine-1-(tbutoxycarbonyl)aminocyclopentane-1-carboxylic
acid
Nicola M. Howarth,a,* Laurence P. G. Wakelinb and David M. Walkera
aChemistry, School of Engineering & Physical Sciences, William H. Perkin Building, Heriot-Watt University, Riccarton,
Edinburgh EH14 4AS, UK
bSchool of Medical Sciences, University of New South Wales, Sydney 2052, Australia
Received 1 November 2002; accepted 22 November 2002
Abstract—Synthetic routes to all four diastereoisomers of 3-thymine-1-(tbutoxycarbonyl)aminocyclopentane-1-carboxylic acid
have been developed starting from the commercially available (S)-dimethyl malate. The key step in the synthesis involves
dialkylation of N-(diphenylmethylene)glycine ethyl ester with 1,4-diiodo-2(S)-trityloxybutane. © 2003 Elsevier Science Ltd. All
rights reserved.
Selective manipulation of gene expression will make a
major impact on the therapy of human disease and
much research is now focused on the design and synthe-
sis of ligands that bind to DNA in a sequence specific
manner so as to inhibit transcription. Nielsen et al.1,2
have developed polyamide nucleic acid (PNA) mimics
of DNA in which the entire deoxyribose–phosphate
backbone has been exchanged with a structurally
homomorphous uncharged polyamide backbone com-
posed of N-(2-aminoethyl)glycine units (Fig. 1). PNAs
bind to both single-stranded DNA (ssDNA) and RNA
with high affinity and sequence specificity and a num-
ber of oligonucleotide-dependent enzymatic functions
have been inhibited on forming PNA/ssDNA or PNA/
Figure 1. Comparison of PNA, a-PNA and a-cycloPNA structures. B=adenine; cytosine; guanine; thymine.
Keywords: PNA; homoserine analogues; cyclic a,a-disubstituted amino acids.
* Corresponding author. Tel.: (+44)-(0)131-451 8026; fax: (+44)-(0)131-451 3180; e-mail: n.m.howarth@hw.ac.uk
0040-4039/03/$ - see front matter © 2003 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)02696-5

696
N. M. Howarth et al. / Tetrahedron Letters 44 (2003) 695–698
RNA complexes.2 Homopyrimidine PNAs have been
found to invade double-stranded DNA (dsDNA) by
displacing the non-complementary strand to form
The four diastereoisomers of 3-thymine-1-Boc-
aminocyclopentane-1-carboxylic acid acid (10, 11, 14
and 15 (Scheme 2)) required for construction of a-
cycloPNA oligomers have been prepared as outlined in
Schemes 1 and 2. Their synthesis starts from the com-
mercially available (S)-dimethyl malate (1), which was
recently employed by Ma et al. in the stereoselective
synthesis of (1S,3R)-1-aminocyclopentane-1,3-dicar-
boxylic acid.12 The first step involved protection of the
hydroxyl group of 1 with a trityl group by treatment
with trityl chloride in the presence of DBU to afford 2
in a 99% yield (Scheme 1).13 The diester 2 was then
reduced using lithium borohydride in the presence of a
catalytic amount of B-methoxy-9-BBN to give the diol
3 in a quantitative yield after purification (Scheme 1).14
Mesylation of both hydroxyl groups, followed by heat-
ing the resulting dimesylate with sodium iodide in
acetone, gave the more stable 1,4-diiodide product, 4,
in an overall yield of 84% (Scheme 1). Dialkylation of
N-(diphenylmethylene)glycine ethyl ester with 4 was
accomplished using 2.2 equiv. of lithium hexamethyldis-
ilazide to yield the cyclised compound 5 as an insepara-
ble mixture of two isomers (Scheme 1).15 The ratio of
(PNA)2/DNA triplexes and
a
displaced strand
analogous to a P-loop.2 Homopurine PNAs also invade
dsDNA but fail to form triplexes and their invasion
complexes are less stable.2 Strand invasion is of great
importance because, in principle, it provides a general
solution to the molecular recognition problem since
duplex formation is governed by the universal Watson–
Crick hydrogen bonding scheme. Unfortunately, simple
mixed purine–pyrimidine PNAs do not invade dsDNA,
in general, although there are a few exceptions.3–5 Thus,
there is the need to explore further PNA analogues for
the purpose of expanding the strand invasion alphabet.
Recently, we have embarked on the development of
true peptide mimics of DNA and have reported the
design and synthesis of one such analogue,
(Fig. 1).6 Unfortunately, despite molecular models indi-
cating structural complementarity, -a-PNA oligomers
L
-a-PNA
L
fail to hybridise to appropriate ssDNA targets.7 We
believe that the most likely explanation for this
observed lack of hybridisation is side chain flexibility.
That such effects may be significant is highlighted by
the results of Nielsen et al. who found that reducing the
side chain methylene carbonyl in PNA dramatically
destabilised both PNA:DNA heteroduplexes and
triplexes.8 The issue of side chain flexibility has also
been raised for acyclic DNA analogues9,10 and for novel
PNAs based on d-amino acids.11 Thus, we have insti-
gated a research programme to examine side chain
1
cis:trans products was estimated to be 2:1 from the H
NMR of the crude material. Concomitant deprotection
of the hydroxyl and amino functions under acidic con-
ditions and reprotection of the amino moiety with a
Boc group gave the two key alcohols, 6 and 7, in 55%
yield over the two steps (Scheme 1). Separation of the
two isomers was achieved at this stage using flash
chromatography and the stereochemistry of the minor
product (19%) was assigned as (1R,3S) (i.e. 7) from
NOE experiments.
restricted
the proposed oligomers for study.
L
-a-PNAs and a-cycloPNA (Fig. 1) is one of
Scheme 1. Reagents and conditions: i. 1.2 equiv. Ph3CCl, 1.4 equiv. DBU, DCM, rt; ii. 1.5 equiv. LiBH4, 0.1 equiv.
B-methoxy-9-BBN, THF, rt; iii. 2.2 equiv. MsCl, 3.3 equiv. Et3N, DCM, rt; iv. 5 equiv. NaI, acetone, reflux; v. 1 equiv.
Ph2CNCH2CO2Et, 2.2 equiv. LiHMDS, THF, −78°C; vi. (a) 2 M (aq.) HCl, rt (b) 1.1 equiv. (Boc)2O, 2 equiv. Na2CO3,
CHCl3:H2O, reflux.

N. M. Howarth et al. / Tetrahedron Letters 44 (2003) 695–698
697
Scheme 2. Reagents and conditions: i. 3 equiv. BrC6H4SO2Cl, 3 equiv. DMAP, 3 equiv. Et3N, CHCl3, rt; ii. 2.2 equiv.
N3-benzoylthymine,16 2 equiv. NaH, DMF, 40°C; iii. 1.5 equiv. NaOEt, EtOH, rt; iv. 2/3 M (aq.) NaOH, dioxane, rt; v. 1.2 equiv.
PPh3, 1.2 equiv. DIAD, 1.2 equiv. MeI, THF, rt.
Acknowledgements
With alcohols 6 and 7 to hand, it was now possible to
make all four diastereoisomers of 3-thymine-1-Boc-
aminocyclopentane-1-carboxylic acid. This was effected
by firstly converting 6 or 7 into their respective brosyl-
ates 8 or 9 (Scheme 2). Preparation of the (1S,3R)- and
(1R,3R)-diastereoisomers of 3-thymine-1-Boc-aminocy-
clopentane-1-carboxylic acid (i.e. 10 and 11) involved
alkylation of N3-benzoylthymine16 with either 8 or 9 in
the presence of sodium hydride, removal of the benzoyl
protecting group from thymine using sodium ethoxide,
and alkaline ester hydrolysis (Scheme 2). Synthesis of
the (1S,3S)- and (1R,3S)-diastereoisomers (i.e. 14 and
15) first required transformation of alcohols 6 or 7 into
their corresponding iodo derivatives 12 or 13 through a
Mitsunobu reaction (Scheme 2).17 Subsequent alkyla-
tion of N3-benzoylthymine16 with either 12 or 13,
removal of the benzoyl protecting group and alkaline
ester hydrolysis afforded 14 and 15 in reasonable yields
after purification.
The authors gratefully acknowledge the Association for
International Cancer Research for financial support.
We also thank the EPSRC Mass Spectrometry Service
Centre for recording all mass spectra and Dr. Alan S.
F. Boyd (Chemistry, School of Engineering & Physical
Sciences, Heriot-Watt University) for performing the
NOE experiments.
References
1. Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O.
Science 1991, 254, 1497–1500.
2. Uhlmann, E.; Peyman, A.; Breipohl, G.; Will, D. W.
Angew. Chem., Int. Ed. 1998, 37, 2796–2823 and refer-
ences cited therein.
3. (a) Boffa, L. C.; Carpaneto, E. M.; Allfrey, V. G. Proc.
Natl. Acad. Sci. USA 1995, 92, 1901–1905; (b) Boffa, L.
C.; Morris, P. L.; Carpaneto, E. M.; Louissaunt, M.;
Allfrey, V. G. J. Biol. Chem. 1996, 271, 13228–13233.
4. (a) Ishihara, T.; Corey, D. R. J. Am. Chem. Soc. 1999,
121, 2012–2020; (b) Zhang, X.; Ishihara, T.; Corey, D. R.
Nucleic Acids Res. 2000, 28, 3332–3338.
In conclusion, we have developed synthetic routes to all
four diastereoisomers of 3-thymine-1-Boc-aminocy-
clopentane-1-carboxylic acid starting from commer-
cially available (S)-dimethyl malate. The key step in the
synthesis involves dialkylation of N-(diphenyl-
methylene)glycine ethyl ester with 1,4-diiodo-2(S)-trityl-
oxybutane. We are currently in the process of incorpo-
rating these monomers into a-cycloPNA oligomers.
5. Nielsen, P. E.; Egholm, M. Bioorg. Med. Chem. 2001, 9,
2429–2434.

698
N. M. Howarth et al. / Tetrahedron Letters 44 (2003) 695–698
6. Howarth, N. M.; Wakelin, L. P. G. J. Org. Chem. 1997,
62, 5441–5450.
mann, K.-H. Bioorg. Med. Chem. Lett. 1997, 7, 1123–
1126.
12. Ma, D.; Ma, J.; Dai, L. Tetrahedron: Asymmetry 1997, 8,
825–827.
7. Howarth, N. M.; Topham, C. M.; Nielsen, P. E.;
Wakelin, L. P. G., unpublished results.
13. Colin-Messager, S.; Girard, J.-P.; Rossi, J.-C. Tetra-
8. Hyrup, B.; Egholm, M.; Buchardt, O.; Nielsen, P. E.
Bioorg. Med. Chem. Lett. 1996, 6, 1083–1088.
9. Schneider, K. C.; Benner, S. A. J. Am. Chem. Soc. 1990,
112, 453–455.
hedron Lett. 1992, 33, 2689–2692.
14. Brown, H. C.; Narasimhan, S. J. Org. Chem. 1984, 49,
3891–3898.
15. Kotha, S.; Kuki, A. Tetrahedron Lett. 1992, 33, 1565–
1568.
16. Cruickshank, K. A.; Jiricny, J.; Reese, C. B. Tetrahedron
Lett. 1984, 25, 681–684.
17. Schumacher, K. K.; Jiang, J.; Joullie´, M. M. Tetrahedron:
Asymmetry 1998, 9, 47–53.
10. (a) Augustyns, K.; Aerschot, A. V.; Schepdael, A. V.;
Urbanke, C.; Herdewijn, P. Nucleic Acids Res. 1991, 19,
2587–2593; (b) Vandendriessche, F.; Augustyns, K.; Aer-
schot, A. V.; Busson, R.; Hoogmartens, J.; Herdewijn, P.
Tetrahedron 1993, 49, 7223–7238.
11. Garc´ıa-Echeverr´ıa, C.; Hu¨sken, D.; Chiesi, C. S.; Alt-