Synthesis and properties of chiral peptide nucleic acids with a N-aminoethyl-D-proline backbone

Article abstract of DOI:10.1016/S0960-894X(00)00507-2

A synthon of D-proline substituted at the 4-position by thymine and at N by a flexible aminoethyl linker, has been used to prepare a novel chiral peptide nucleic acid (cPNA) with (2R,4R) stereochemistry using solid phase methodology. The homothymine decamer cPNA binds to complementary polyadenylic acid to form a 2:1 hybrid with high affinity and specificity according to UV and CD studies, whereas no binding to the corresponding polydeoxyadenylic acid was observed. (C) 2000 Published by Elsevier Science Ltd.

Full text of DOI:10.1016/S0960-894X(00)00507-2

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2541±2545
Synthesis and Properties of Chiral Peptide Nucleic Acids with a
N-Aminoethyl-D-proline Backbone
Tirayut Vilaivan,a Chanchai Khongdeesameor,a Pongchai Harnyuttanakorn,b
Martin S. Westwellc and Gordon Lowec,*
aOrganic Synthesis Research Unit, Department of Chemistry, Faculty of Science, Chulalongkorn University, Phayathai Road, Patumwan,
Bangkok 10330, Thailand
bDepartment of Biology, Faculty of Science, Chulalongkorn University, Phayathai Road, Patumwan, Bangkok 10330, Thailand
cDyson Perrins Laboratory, University of Oxford, South Parks Road, Oxford, OX1 3QY, UK
Received 30 July 2000; revised 4 September 2000; accepted 5 September 2000
AbstractÐA synthon of d-proline substituted at the 4-position by thymine and at N by a ¯exible aminoethyl linker, has been used
to prepare a novel chiral peptide nucleic acid (cPNA) with (2R,4R) stereochemistry using solid phase methodology. The homo-
thymine decamer cPNA binds to complementary polyadenylic acid to form a 2:1 hybrid with high anity and speci®city according
to UV and CD studies, whereas no binding to the corresponding polydeoxyadenylic acid was observed. # 2000 Published by
Elsevier Science Ltd.
Since the discovery by Nielsen and co-workers in
1991,1 4 peptide nucleic acids (PNA) (1)5 7 have attrac-
ted much attention due to the exceptionally high anity
and speci®city towards complementary oligoribo-
nucleotides and oligodeoxyribonucleotides. In addition
they are stable towards nucleases and proteases and can
be easily synthesized via solid phase techniques. Such a
combination of properties, not found in natural oligo-
nucleotides, make PNA potentially useful as a tool in
antisense technology8 as well as other areas of research.
The stronger binding properties of PNA compared to
natural oligonucleotides were attributed to the lack of
electrostatic repulsion between the uncharged PNA
backbone and the negatively charged sugar phosphate
backbone of the oligonucleotides. The three-dimen-
sional structure of hybrids between PNA and oligo-
nucleotides were studied by X-ray crystallography9 and
NMR spectroscopy10 which revealed that the con-
formation of the oligonucleotides in such hybrids are
quite di€erent from A or B-DNA conformations.
Modi®cation to PNA could provide a system which
might exhibit superior properties such as water solubi-
lity and the ability to penetrate cell membranes, without
loss of binding anity and speci®city. Many such mod-
i®cations have been made in order to achieve these
properties with varying degrees of success. Recent
examples include: introduction of substituents into (1)
to create chirality,11 introduction of positively-charged
groups onto the PNA backbone,12 restricting the move-
ment of the backbone or the side chain of PNA, e.g. by
replacing the amide bond with a double bond,13 or
by making the backbone or side chain part of a ring
system.14 22
We have recently reported a synthesis of chiral PNA (2)
based on a rigid glycyl-d-proline skeleton with the
nucleobases attached to the 4-position on the proline
ring.20 22 These chiral PNAs were shown to interact
strongly with complementary DNA and RNA.23 This
chiral PNA and its hybrid with oligonucleotides, how-
ever, has poor solubility in aqueous media, making
biological studies dicult. Attempts to improve the
solubility of PNAs have so far met with variable
success.24 26 Here we report the synthesis and binding
studies of the deoxy-analogue of (2), i.e. the glycine
being replaced by an aminoethyl linker (3).
*Corresponding author. Tel.: +44-1865-275649; fax: +44-1865-
275674; e-mail: gordon.lowe@chem.ox.ac.uk
0960-894X/00/$ - see front matter # 2000 Published by Elsevier Science Ltd.
PII: S0960-894X(00)00507-2

2542
T. Vilaivan et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2541±2545
Replacing the glycine carbonyl group in the glycyl-
proline backbone of cPNA with a methylene group
(`aminoethylprolyl cPNA') should create a more con-
formationally ¯exible backbone while the conformation
of the side chain is still restricted. Increasing conforma-
tional ¯exibility of the backbone might decrease the
binding anity due to the increased entropy loss upon
hybridization, but it should allow the cPNA to adopt a
wider range of conformations than the cPNA (2).
Combination of the two factors may decrease or increase
the binding anity of the resulting cPNA to its com-
plementary oligonucleotide, depending on how close the
conformation of the cPNA in the hybrid is to that in its
native state. The basic proline-nitrogen atom should be at
least partially protonated under physiological conditions
and should attract the negatively charged phosphate
group of DNA so providing further stabilization of the
hybrid formed with natural DNA. This idea has been
exploited in Nielsen's type PNA by replacing the
methylenecarbonyl linker which joined the base and the
backbone to an ethylene linker12 so that the a-nitrogen
atom became basic. Unfortunately, the resulting modi®ed
PNA had lower anity towards DNA presumably due
to the increased ¯exibility of the linker. Very recently a
system related to (3), with di€erent stereochemistry
around the proline ring, has been described.27
standard HBTU/DIEA activation protocol with capping
at the end of each cycle. Quantitative monitoring of the
coupling eciency, by measurement of the absorbance
of dibenzofulvene-piperidine adduct at 264 nm, after the
deprotection cycle revealed the average coupling yield of
approximately 92%. Using the Fmoc-ON puri®cation
strategy,31 it was possible to isolate the Fmoc-(3a)
together with its partially debenzoylated product with-
out diculty using reverse phase HPLC. Subsequent
treatment with 20% aqueous piperidine followed by
HPLC gave the fully deprotected (3a). ESI mass spec-
trometric analysis revealed a single peak corresponding
to the expected product (Mr=2787.27, calcd for
Mr=2788.06). In contrast to the analogous glycylpro-
line PNA (2) with the same T10 sequence, the product is
freely soluble in aqueous solvents and a concentration
exceeding 5 mg/mL could be achieved (Scheme 1).
Interaction between the decathymine PNA oligomer
(3a) with poly(rA), poly(dA) and (dA)10 was investi-
gated by Tm measurement. Melting was observed only
when the nucleic acid component is poly(rA) with a
single transition at Tm=53 ꢀC (150 mM NaCl). A typi-
cal melting curve is shown in Figure 1 and the Tm values
are as shown in Table 1. Control experiments with
poly(rU) and poly(rI) showed no observable melting
A synthetic route towards the N-aminoethylproline
synthon carrying suitable protecting groups (7a and 7b)
for solid phase peptide synthesis was therefore devel-
oped. N-p-Nitrobenzenesulfonyl (`Nosyl') aziridine,
obtained by treatment of N-nosylethanolamine with
Ph3P/DEAD in THF at low temperature, was employed as
the electrophilic N-aminoethylating agent.28 Protected
cis-4-thyminyl-d-proline (4) was synthesized as described
previously.20 22 The Boc group was selectively removed
in the presence of diphenylmethyl (Dpm) ester using p-
toluenesulfonic acid in acetonitrile under conditions
previously reported. Nucleophilic ring opening of N-
nosylaziridine by this free amine proceeded eciently to
give the desired N-aminoethylproline derivative (5).
Reaction of (5) with Boc2O was achieved in the presence
of 4-dimethylaminopyridine (DMAP) to give the N-Boc
derivative. Treatment with thiophenol in the presence of
potassium carbonate in DMF at room temperature gave
the N-Boc derivative (6a) as an amorphous solid [50%
yield from (4)].29 Deprotection of the carboxyl group by
catalytic transfer hydrogenolysis (cyclohexene, Pd±C)
gave the free N-Boc amino acid (7a) in quantitative yield.29
.
curve, suggesting that the interaction is speci®c for A T
base-pairing. UV titration between (3a) and poly(rA)
revealed a 2:1 stoichiometry of T:A, indicating the for-
mation of a triple helical complex, probably via Wat-
. .
son±Crick and Hoogsteen-type T A T pairing similar to
those observed with other homopyrimidine PNAs.1 4
The interaction of cPNA and poly(A) was further stu-
died by CD spectroscopy. While single stranded
poly(rA) exhibited a characteristic CD signal in the
region of 200±300 nm, single-stranded cPNA (3a)
The Boc protecting group in (6a) was converted to the
Fmoc group in order to take advantage of the milder
conditions of peptide synthesis employing Fmoc pro-
tection strategy. When (6a) was treated with p-toluene-
sulfonic acid in acetonitrile followed by 9-
¯uorenylmethyl chloroformate (Fmoc-Cl) in the presence
of DIEA, the Fmoc derivative (6b) was obtained in 80%
yield.30 The carboxyl protecting group was removed by
HCl/dioxane to give the free acid (7b) (71%) as its
hydrochloride salt with the N3-benzoyl protecting group
of thymine intact.30 The Fmoc protected monomer (7b)
was oligomerized on Novasyn TGR resin, previously
loaded with Fmoc-Lys(Boc) on a 5 mmol scale using
Scheme 1. Synthesis of the monomer for aminoethylproline PNA.
Reagents and conditions: (i) 2.5 equiv pTsOH/MeCN; (ii) N-nosyl-
aziridine/DIEA/MeCN, rt; (iii) Boc2O/Et3N/DMAP in CH2Cl2; (iv)
PhSH/K2CO3 in DMF, rt; (v) cyclohexene, cat Pd/C in MeOH, re¯ux;
(vi) FmocCl/DIEA; (vii) 4 M HCl/dioxane.

T. Vilaivan et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2541±2545
2543
Figure 1. Melting curve of (3a) and poly(rA), poly(rU) and poly(dA). Conditions: 10 mM sodium phosphate bu€er pH 7.0; 150 mM NaCl; 1.0 mM
(3a); ratio of T:A=1:1; 20% hypochromicity.
exhibited very weak CD spectra in this region. Any
change in CD signal of the mixture between (3a) and
poly(rA) would therefore indicate the formation of new
species. As expected, addition of (3a) to poly(rA)
resulted in a strong induced CD signal with a minima at
275 nm (Fig. 2a) whereas no signi®cant change was
observed when (3a) was added to poly(dA) under the
same conditions (Fig. 2b). The shape of the CD curve is,
however, signi®cantly di€erent from those of conven-
tional DNA-DNA and DNA-RNA complexes thus
possibly indicating a new type of helical structure. By
following the CD signal as a function of amount of (3a)
added, the stoichiometry of binding of PNA:RNA was
found to be 2:1, consistent with the result obtained from
UV titration. Gel electrophoresis experiments between
(3a) and ¯uorescent labelled (dA)10 also con®rmed the
lack of binding between (3a) and (dA)10.
Although the hybrid between PNA oligomer (3a) and
poly(rA) exhibited lower Tm value compared to Niel-
sen's type PNA (1a) or glycylproline cPNA (2a), the Tm
is still remarkably high compared to the natural oligo-
deoxyribonucleotide (dT)10 (Tm <20 ꢀC under compar-
able conditions). The lowered Tm compared to the
glycylproline cPNA (2a) emphasizes the importance of
restricting the conformation. The electrostatic interac-
tion between the protonated form of the proline nitro-
gen atom and the negatively charged phosphate group
of the nucleic acid may be a factor that provides a
positive contribution to the binding. This is supported
by the fact that no binding of (3a) to poly(rA) could be
observed at high salt concentration (1.0 M NaCl). The
selective recognition of polyribonucleotide in preference
to polydeoxyribonucleotide is, however, of great interest
with regard to its use in antisense research. Further-
more, the results are in sharp contrast to recent work27
on related aminoethylproline PNA systems but with
di€erent stereochemistry around the proline ring [i.e.
(2S,4S) and (2R,4S)], in which both stereoisomers were
reported to bind strongly with complementary oligo-
deoxyribonucleotides according to Tm studies. At pre-
sent we do not have a satisfactory model to explain the
di€erence observed, but clearly stereochemistry of the
proline ring must play an important role in determining
the speci®city.
Table 1. Comparison of Tm of hybrids between PNA and oligonu-
cleotidesa
Hybrids
Tm (ꢀC)
Stoichiometry (PNA:DNA/RNA)
(1a):poly(dA)
(2a):poly(dA)
(3a):poly(dA)
(3a):poly(rA)
(3a):poly(rU)
(3a):poly(rI)
73
70
2:1
1:1
Ð
2:1
Ð
Ðb
53
Ðb
Ðb
Ð
In conclusion, we have demonstrated that a novel cPNA
carrying a hydrophilic N-aminoethylproline backbone
can be readily synthesized using standard peptide
chemistry. The cPNA is readily soluble in aqueous
(1a): H-[Aeg(T)]10-LysNH2; (1b): H-[Gly-d-Pro(cis-T)]10-LysNH2
aConditions: see Fig. 1.
bNo melting above 20 ꢀC was observed.

2544
T. Vilaivan et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2541±2545
Freier, S. M.; Driver, D. A.; Berg, R. H.; Kim, S. K.; Norden,
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13. Cantin, M.; Schutz, R.; Leumann, C. J. Tetrahedron Lett.
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17. Gangamani, B. P.; Kumar, V. A.; Ganesh, K. N. Tetra-
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Stropp, U.; Schwenner, E.; Mielke, B. Bioorg. Med. Chem.
Lett. 1997, 7, 687.
Figure 2. (a) CD spectra of mixture of poly(rA) and (3a) at di€erent
equivalents of T; inset: plot of e260 versus equivalents of T; (b) CD
spectra of mixture of poly(dA) and (3a) at di€erent equivalents of T.
Conditions 10 mM sodium phosphate bu€er pH 7.0. The concentra-
tion of poly(rA) and poly(dA) was 10 mM based on AMP.
20. Lowe, G.; Vilaivan, T. J. Chem. Soc., Perkin Trans. 1
1997, 539.
21. Lowe, G.; Vilaivan, T. J. Chem. Soc., Perkin Trans. 1
1997, 547.
22. Lowe, G.; Vilaivan, T. J. Chem. Soc., Perkin Trans. 1
1997, 555.
23. Lowe, G.; Vilaivan, T.; Westwell, M. S. Bioorg. Chem.
1997, 25, 321.
solvents and exhibit strong interaction with oligo-
ribonucleotides but not with oligodeoxyribonucleotides.
Such high selectivity suggests it has potential as an
antisense agent where selective targeting of mRNA
would be bene®cial.
24. Vilaivan, T.; Khongdeesameor, C.; Wiriyawaree, W.; Man-
sawat, W.; Westwell, M. S.; Lowe, G. submitted for publication.
25. Altmann, K. H.; Husken, D.; Cuenoud, B.; Garcia-Eche-
verria, C. Bioorg. Med. Chem. Lett. 2000, 10, 929.
26. Kuwahura, M.; Arimisu, M.; Sisido, M. J. Am. Chem.
Soc. 1999, 121, 256.
Acknowledgements
We gratefully acknowledge the National Science and
Technology Development Agency (Central Oce Pro-
ject RDE-CO grant number: CO-B-06-22-09-001, ®nancial
year 1997), the Department of Chemistry, Chula-
longkorn University (TV) and Lincoln College, Oxford
(MSW) for ®nancial support. Local Graduate Scholar-
ship from NSTDA (Thailand) (to C.K.) is gratefully
acknowledged. We also would like to thank Chula-
longkorn Research Equipment Centre (Bangkok) for
microanalysis services and Dr. R. T. Aplin (Dyson Per-
rins Laboratory, Oxford) for the ESI-MS spectra.
27. Costa, M. D.; Kumar, V. A.; Ganesh, K. N. Org. Lett.
1999, 1, 1513.
28. Maligres, P. E.; See, M. M.; Askin, D.; Reider, P. J. Tet-
rahedron Lett. 1997, 38, 5253.
29. N-2-(N-tert-Butoxycarbonylamino)ethyl-cis-4-(N3-benz-
oylthymin-1-yl)-d-proline diphenylmethyl ester (6a) dH (500
t
MHz; CDCl3) 1.43 [9H, s, Bu CH3], 1.87 [3H, s, thymine
CH3], 1.92 [1H, m, 1ÂCH2(30)], 2.52±2.59 [1H, m,
1ÂBocNHCH2CH2N], 2.71±2.80 [2H, m, 1ÂBocNHCH2-
CH2N and 1ÂCH2(50)], 2.85 [1H, ddd, J=19.0, 15.0, 9.5 Hz,
1ÂCH2(30)], 3.03±3.12 [1H, m, 1ÂBocNHCH2CH2N], 3.18±
3.21 and 3.21±3.26 [2H, m, 1ÂBocNHCH2CH2N and
1ÂCH2(50)], 3.40 [1H, dd, J=7.0, 9.5 Hz, CH(2)], 5.09 [1H, br
s, Boc NH], 5.17±5.24 [1H, m, CH(40)], 6.96 [1H, s, CHPh2],
7.24±7.38 [10H, m, Dpm aromatic CH], 7.45 [2H, t, J=8.0 Hz,
benzoyl m-CH], 7.61 [1H, t, J=8.0 Hz, benzoyl p-CH], 7.87
[2H, d, J=8.0 Hz, benzoyl o-CH], 7.96 [1H, s, thymine
C(6)H]; m/z (APCI+) 676 (M+Na+, 18.5%), 653 (M+H+,
59), 597 (M C4H8+, 25), 554 (M Boc+, 9), 475 (26.5), 167
(Ph2CH+, 100); [a]D22 +6.5 (c=1.06, CHCl3).
References and Notes
1. Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O.
Science 1991, 254, 1497.
2. Egholm, M.; Buchardt, O.; Nielsen, P. E.; Berg, R. H. J.
Am. Chem. Soc. 1992, 114, 1895.
3. Egholm, M.; Buchardt, O.; Nielsen, P. E.; Berg, R. H. J.
Am. Chem. Soc. 1992, 114, 9677.
4. Egholm, M.; Buchardt, O.; Christensen, L.; Behrens, C.;
N-2-(N-tert-Butoxycarbonylamino)ethyl-cis-4-(N3-benzoylthy-

T. Vilaivan et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2541±2545
2545
min-1-yl)-d-proline (7a) dH (200 MHz; CDCl3) 1.42 [9H, s, tBu
CH3], 1.95 [3H, s, thymine CH3], 2.05 [1H, m, 1ÂCH2(30)],
2.60±3.50 [m, CH2(50) and 2Âaminoethyl CH2 and CH(20)],
4.65 [1H, br m, OH], 5.18 [1H, m, CH(40)], 5.60 [1H, br m,
NH]; 7.36 [10H, m, Dpm aromatic CH], 7.48 [2H, t, Bz m-
CH], 7.60 [1H, t, Bz p-CH], 7.89 [2H, d, Bz o-CH], 8.01 [1H, s,
thymine C(6)H]; m/z (APCI+) 509 (M+Na+, 38%), 487
(M+H+, 56), 431 (M C4H8+, 45), 309 (100), 155 (82); [a]2D3
+12.2 (c=1.04, CHCl3).
30. N-2-(9-Fluorenylmethoxycarbonylamino)ethyl-cis-4-(N3-
benzoylthymin-1-yl)-d-proline diphenylmethyl ester (6b)
(80%), mp 94±98 ꢀC (Found C, 72.3; H, 5.7; N, 7.4%;
C47H42N4O7 requires C, 72.3; H, 5.5; N, 7.2%); dH (500 MHz;
CDCl3) 1.73 [3H, s, thymine CH3], 1.85±1.93 [1H, m,
1ÂCH2(30)], 2.50±2.59 [1H, m, 1ÂFmoc NHCH2CH2N], 2.65±
2.76 [2H, m, 1ÂCH2(50) and 1ÂFmoc NHCH2CH2N], 2.77±
2.88 [1H, m, 1ÂCH2(30)], 3.02±3.11 [1H, m, 1ÂFmoc
NHCH2CH2N], 3.14±3.24 [1H, m, 1ÂCH2(50)], 3.25±3.33 [1H,
t, 1ÂFmoc NHCH2CH2N], 3.35±3.42 [1H, m, CH(20)], 4.14
[1H, t, J=7.0 Hz, 1ÂFmoc aliphatic CH2], 4.24 [1H, t, J=8.5
Hz, Fmoc aliphatic CH2], 5.14±5.20 [1H, br m, CH(40)], 5.40
[1H, br s, Fmoc NH], 6.92 [1H, s, CHPh2], 7.18±7.30 [10H, m,
Dpm aromatic CH], 7.30±7.36 [4H, m, Fmoc aromatic CH],
7.40 [2H, t, J=7.5 Hz, benzoyl m-CH], 7.52±7.58 [3H, m,
benzoyl p-CH, 2ÂFmoc CH aromatic], 7.71 [2H, d, J=7.5 Hz,
benzoyl o-CH], 7.81±7.87 [3H, m, thymine C(6)H, 2ÂFmoc
aromatic CH]; m/z (APCI+) 775 (M+H+, 98%), 553 (18.5),
1
265 (21), 167 (Ph2CH+, 74), 137 (100); nmax (KBr)/cm
1746s, 1722s, 1697s and 1653s (CˆO); [a]2D3 +7.03 (c=0.92,
CHCl3).
N-2-(9-Fluorenylmethoxycarbonylamino)-ethyl-cis-4-(N3-ben-
zoylthymin-1-yl)-d-proline hydrochloride (7b) (71%) mp 144±
146 ꢀC (Found C, 60.9; H, 5.0; N, 8.2%; C34H33N4O7Cl+2H2O
requires C, 59.9; H, 5.5; N, 8.2%); dH (500 MHz; DMSO-
d6+1 drop D2O) 2.50 [3H, s, thymine CH3], 2.34±2.44 [1H, m,
1ÂCH2(30)], 2.83 [1H, m, 1ÂCH2(30)], 3.12±3.22, 3.26±3.36,
3.37±3.42 and 3.44±3.53 [4H, 4Âm, FmocNHCH2CH2N],
3.55±3.75 [1H, m, 1ÂCH2(50) overlap with D2O peak], 3.93±
4.00 [1H, m, 1ÂCH2(50)], 4.20 [1H, t, J=6.5 Hz, Fmoc ali-
phatic CH], 4.25±4.36 [2H, m, Fmoc aliphatic CH2], 4.46±4.54
[1H, m, CH(20)], 5.27±5.35 [1H, m, CH(40)], 7.24±7.42 [6H, m,
Fmoc aromatic CH], 7.57 [2H, t, J=7.5 Hz, benzoyl m-CH],
7.62±7.68 [3H, m, Fmoc aromatic CH], 7.74 [1H, t, J=7.5 Hz,
benzoyl p-CH], 7.86 [2H, d, J=7.5 Hz, benzoyl o-CH], 7.96
[2H, d, J=7.5, Fmoc aromatic CH], 8.03 [1H, br s, thymine
C(6)H]; m/z (APCI+) 631 (M+Na+, 6%), 609 (M+H+, 8),
1
333 (7), 265 (12), 179 (26), 137 (100); nmax (KBr)/cm 1748s,
1733s, 1694s and 1645s (CˆO); [a]2D3 +13.5 (c=1.00, DMF).
31. Thomson, S. A.; Josey, J. A.; Cadilla, R.; Gaul, M. P.;
Hassman, C. F.; Luzzio, M. J.; Pipe, A. J.; Reed, K. L.;
Ricca, D. J.; Wiethe, R. W.; Noble, S. A. Tetrahedron 1995,
51, 6179.