New thiazane and thiazolidine PNA monomers: Synthesis, incorporation into PNAs and hybridization studies

Article abstract of DOI:10.1016/S0960-894X(02)00076-8

New constrained PNA monomers containing a substituted thiazolidine or a thiazane ring were synthesized and incorporated in the center of a 9-mer homothymine PNA. The PNA/DNA hybrids stability was studied by UV-melting experiments which showed that the presence of the modified unit destabilizes the PNA/DNA triplexes.

Full text of DOI:10.1016/S0960-894X(02)00076-8

Bioorganic & Medicinal Chemistry Letters 12 (2002) 1047–1050
New Thiazane and Thiazolidine PNA Monomers:
Synthesis, Incorporation into PNAs and Hybridization Studies
Sarah Bregant, Fabienne Burlina* and Gerard Chassaing
´
Laboratoire de Chimie Organique Biologique, Universite P. et M. Curie, CNRS-UMR 7613, 4 place Jussieu,
75252 Paris Cedex 05, France
Received 20 December 2001; accepted 28January 2002
Abstract—New constrained PNA monomers containing a substituted thiazolidine or a thiazane ring were synthesized and incor-
porated in the center of a 9-mer homothymine PNA. The PNA/DNA hybrids stability was studied by UV-melting experiments
which showed that the presence of the modified unit destabilizes the PNA/DNA triplexes. # 2002 Elsevier Science Ltd. All rights
reserved.
Since their introduction in 1991 by Nielsen et al.,1 pep-
tide nucleic acids (PNAs) have reenergized the area of
antisense and antigene strategies and have found appli-
cations as tools in molecular biology and genetic diag-
nostics. PNAs are DNA mimics in which the
nucleobases are attached via a methylene carbonyl lin-
ker to a polyamide backbone composed of N-(2-amino-
ethyl)-glycine (aeg) units (Fig. 1). These polymers are
resistant to cellular enzymes and bind to DNA and
RNA with higher sequence specificity and affinity than
non-modified oligonucleotides.2
inversion of configuration of the C-4 center. The pre-
sence of the gem-dimethyl at position C-5 of the thia-
zolidine ring is expected to reduce the g and d
fluctuation domains. The racemic syn thiazane U4 unit
in the chair conformation leads to g and d values of
180ꢀ. In contrast, for the racemic anti thiazane U4 unit,
four (g, d) sets are expected considering the two chair
conformations of both enantiomers (180ꢀ, ꢁ60ꢀ) or
ꢀ
(ꢁ60ꢀ, 18 0). We report here the synthesis of the new
units U3 and U4, their incorporation in a PNA and
the hybridization studies with DNA. We have also
Many chemical modifications have been proposed to
improve the PNAs hybridization properties. Some of
these imply the rigidification of the PNA backbone to
favor the structure observed in the PNA/DNA or the
PNA/RNA complexes.3 We have recently reported the
synthesis of the constrained units U1 and U2 containing
a thiazolidine ring which restrains the fluctuation
domain of the g and d torsion angles (Fig. 1, U1: g and
d=120ꢀ ꢁ30, U2: g=ꢂ120ꢀ ꢁ30, d=120ꢀ ꢁ30). The
presence of these units in the center of a PNA was
shown to destabilize the PNA/DNA triplex.4 In order to
further explore the influence of g and d values on the
PNA hybridization properties we have designed the new
units U3 and U4 (Fig. 1). The anti and syn U3 units give
g and d angles having opposite signs compared to the
one obtained for U1 and U2, respectively, due to the
Figure 1. Structure of the different PNA units (T=thymine,
Boc=t-butyloxycarbonyl). The atom numbering and the torsion angle
nomenclature used here are indicated (g: C-20–C-2–N–C-4; d: C-2–N–
C-4–C-40).
*Corresponding author. Tel.: +33-1442-73115; fax: +33-1442-73843;
e-mail: burlina@ccr.jussieu.fr
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00076-8

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S. Bregant et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1047–1050
Scheme 1. (i) EtOH, H2O; (ii) BTSA, CH2Cl2, ClCH2COCl, pyridine; (iii) CH2N2, Et2O; (iv) thymine, K2CO3, DMF; (v) LiOH, dioxane/H2O; (vi)
TFA/CH2Cl2; (vii) Et3N, CH2Cl2.
examined the influence of the thiazolidine and thiazane
sulfur oxidation on the PNA/DNA hybrid stability.
stereospecific assignment of the methyl groups of com-
pound 4a could not be deduced from the heteronuclear
13C–1H couplings since the two 13C methyl groups sig-
nals exhibited the same multiplicity (qqt). However,
NOE experiments were in agreement with an anti rela-
tionship between H-2 and H-4 for bicycle 4a (the methyl
group which gave a strong NOE with H-4 gave none
with H-2). Thus the anti stereochemistry was assigned to
3a. Compounds 3a and 3b were separately reacted with
thymine in the presence of potassium carbonate in
dimethylformamide at room temperature to give in each
case a single coupling compound in 60% yield. The last
step toward the synthesis of the PNA units involved the
saponification of the methyl ester function by treatment
with lithium hydroxide in a mixture of water/dioxane
(2:3). The syn unit U3 was formed quantitatively.9 In
contrast, the saponification of the anti compound led to
a mixture of two diastereomers having the expected
mass and another side product which prevented the
isolation of the anti compound in a sufficient scale to
allow incorporation into a PNA.
The PNA unit U3 was prepared following the synthetic
route outlined in Scheme 1. Condensation of N-Boc-
glycine aldehyde 15 with d-penicillamine was performed
at room temperature in a mixture of ethanol/water (1:1)
to give the thiazolidine-4-carboxylic acid 2. After sol-
vent removal and drying over P2O5 for 48h, it was
directly isolated without purification. Thiazolidine 2
was treated with N,O-bis(trimethylsilyl)acetamide
(BTSA) to ensure its solubility in dichloromethane and
reacted with chloroacetyl chloride in the presence of
pyridine at ꢂ78 ꢀC. Treatment with diazomethane in
ether gave a mixture of two diastereomers 3a and 3b
which were separated by flash chromatography and
found to be present in a 60:40 ratio (51% yield starting
from 1).6 The respective stereochemistry of 3a and 3b
could not be determined by nuclear Overhauser effect
(NOE) experiments. Indeed, no NOE was observed
between protons H-2 and H-4. Furthermore, the com-
1
plexity of the H NMR spectra, due to peaks overlap
and exchange magnetization transfer between the cis/
trans amide bond isomers, prevented any clear inter-
pretation of the other NOE observed. To circumvent
the problem associated with the amide bond rotation,
3a and 3b were respectively converted into the bicyclic
compounds 4a (73%) and 4b (80%) by Boc removal and
triethylamine treatment of the resulting free amino
compound.7 In both cases, a single constrained product
was formed. For both compounds 4a and 4b, no NOE
was observed between protons H-2 and H-4. Thus, the
determination of the stereochemistry of 4a and 4b was
based on the analysis of the heteronuclear 13C–1H cou-
plings and NOE involving the methyl groups. In the 13C
spectrum of compound 4b recorded without proton
decoupling, the two non-equivalent methyl groups
exhibited different multiplicities: quadruplet of quintu-
plet (qqt) for the deshielded signal (CH3a), quadruplet
The racemic U4 unit was synthesized starting from
homocysteine thiolactone chlorohydrate (Scheme 2).
After ring opening in the presence of sodium hydroxide
and condensation with N-Boc-glycine aldehyde 1, the
thiazane 5 was obtained as a single diastereomer (45%
yield, two steps). This compound was treated succes-
sively with chloroacetyl chloride in the presence of
BTSA and diazomethane to give the chloro derivative 6
as a single diastereomer (62%, two steps).6,10 The ana-
1
lysis of the coupling constants in its H NMR spectra
showed that the thiazane ring adopts a twist conforma-
tion and the interpretation of the NOE experiments
indicated the syn stereochemical relationship between
of quadruplet for the shielded one (CH3b). This indi-
3
cated that the coupling constant JH-4,CH b
is close to 0
3
Hz showing the anti relationship between H-4 and
CH3b (torsion angle H-4–C-4–C-5–CH3b close to 90ꢀ
according to Karplus curves).8 Furthermore, a NOE
was observed between CH3a and H-2 revealing their syn
relationship. From these data, the syn relationship
between H-2 and H-4 was assigned for compound 4b
and by deduction for compound 3b. In contrast, the
Scheme 2. (i) NaOH, H2O; (ii) N-Boc-glycine aldehyde, EtOH, H2O;
(iii) BTSA, CH2Cl2, ClCH2COCl, pyridine; (iv) CH2N2, Et2O; (v)
thymine, K2CO3, DMF; (vi) LiOH, dioxane/H2O.

S. Bregant et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1047–1050
1049
the C-2 and C-4 substituents. Thymine was alkylated
by thiazane 6 and the resulting coupling compound was
subjected to a saponification to give the syn unit U4 in
good yield.11 The corresponding anti unit could not be
obtained by this synthetic route.
a thiazolidine motif in the PNA probably improves its
solubility, as the thiazane also does. The PNAs were
hybridized to the complementary DNA sequence dA10
or the mismatched sequence dA4TA4 and the thermal
stabilities of the PNA/DNA hybrids were estimated by
UV-melting experiments (Table 1).15 A 2:1 ratio of
PNA/DNA was used as homopyrimidine PNAs form
PNA2/DNA triplexes2 and we observed previously that
the incorporation of a single thiazolidine unit in the
PNA does not prevent the triplex formation.4 Well-
defined heating (Fig. 2) and cooling curves were
obtained for all the modified PNA/DNA hybrids indi-
cating that the presence of the modified unit does not
prevent the cooperative dissociation and association of
the complexes. However, the thermal stabilities of the
modified complexes were significantly reduced com-
pared to that of the reference triplex PNA ref/dA10
(Table 1). The anti unit U1 appeared to be better
accommodated in the triplex than the different syn units
which led to similar Tm values (17 ꢀC reduction of the
Tm with PNA 1, 24–25 ꢀC reduction with PNAs 2 to 4).
The thiazane ring of the synthetic intermediate 6 was
shown by NMR studies to adopt a twist conformation.
This unexpected result prompted us to perform mole-
cular mechanic calculations on the syn racemic U4 unit.
The twist conformation was found to be the most stable
leading to the (g, d) torsion angle sets (ꢂ90ꢀ ꢁ10,
130ꢀ ꢁ10) and (90ꢀ ꢁ10, ꢂ130ꢀ ꢁ10). These angle values
are close to those obtained with the syn thiazolidine
units which could explain the similar Tm values
observed for PNA 2 to 4. The sulfur oxidation of the
modified units did not improve the thermal stabilities of
the hybrids. However, for PNA 1 and to a lesser extent
PNA 2, the hysteresis between the heating and cooling
curves was reduced (Tm–Tas=2 ꢀC for PNA 1ox instead
of 9 ꢀC for PNA 1 or 7.5 ꢀC for PNA ref) suggesting that
the triplex formation is accelerated. Finally, hybrid-
ization of the modified PNAs to the mismatched DNA
sequence resulted in Tm reductions ranging from 9 to
15 ꢀC (12 ꢀC for PNA ref). As a conclusion, the presence
of the modified units was shown to destabilize the PNA/
DNA hybrids but maintain a base-pairing selectivity.
Furthermore, it was observed that incorporation of the
thiazolidine or thiazane units reduces PNA aggregation.
The constrained units U1 to U4 were introduced in the
center of a 9-mer homothymine aegPNA to give PNA 1
to 4 (Table 1). The reference PNA containing only aegT
units (Fig. 1) was also synthesized. Lysine was intro-
duced at the C-terminus of the PNAs to reduce their
self-aggregation.1 PNAs were assembled as described
previously4 on a 5 mmol scale applying with minor
modifications the in situ neutralization protocol for Boc
solid phase peptide chemistry.12 Cleavage of the PNAs
from the resin was performed by standard HF proce-
dure and the PNAs were purified by reverse phase
HPLC and characterized by MALDI-TOF mass spec-
troscopy.13 Sulfur mono-oxidation of the constrained
units was performed directly on the purified polymers
(PNA 1 to 4) by treatment with sodium periodate (10
equiv) in water at room temperature (48h) to give PNA
1ox to 4ox (Table 1).13 Although a single peak was
always observed during preparative or analytical
HPLC, the presence of the two sulfoxide epimers is not
excluded.
It was observed that unlike the reference PNA the
modified PNAs do not exhibit any tendency to self-
aggregation during storage or manipulation. As pre-
viously reported for peptide sequences,14 the presence of
Table 1. Melting (Tm) and half re-association (Tas) temperatures of
the PNA/DNA complexesa
PNAb
Tm PNA/dA10
Tas PNA/dA10
Tm PNA/dA4TA4
ref
1
66
49
58.5
40
54
39
1ox
2
2ox
3
3ox
4
4ox
4846
42
41
41
39
42
41
39
33
36.5
25
22
29
31
27
27
29
25
30
31
aTemperatures are given in ꢀC with estimated deviations of ꢁ1 ꢀC.
bPNAs sequences: PNA ref=H-(aegT)9-Lys-NH2, PNA n=H-
(aegT)4-Un-(aegT)4-Lys-NH2, Un being one of the modified units pre-
sented in Figure 1. The corresponding oxidized PNAs are named PNA
nox.
References and Notes
1. Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O.
Science 1991, 254, 1497.
2. Reviews: (a) Nielsen, P. E. Acc. Chem. Res. 1999, 32, 624.
(b) Ray, A.; Norden, B. FASEB 2000, 14, 1041.
3. (a) Review: Ganesh, K. N.; Nielsen, P. E. Current Org.
Chem. 2000, 4, 931. (b) Puschl, A.; Boesen, T.; Tedeschi, T.;
Dahl, O.; Nielsen, P. E. J. Chem. Soc., Perkin Trans. 1 2001,
2757 and refs 3 and 4 cited therein. (c) Meena; Kumar, V. A.;
Ganesh, K. N. Nucleosides, Nucleotides & Nucleic Acids 2001,
20, 1193. (d) Lonkar, P. S.; Kumar, V. A.; Ganesh, K. N.
Nucleosides, Nucleotides & Nucleic Acids 2001, 20, 1197. (e) Li,
Y.; Jin, T.; Liu, K. Nucleosides, Nucleotides & Nucleic Acids
2001, 20, 1705. (f) Slaitas, A.; Yeheskiely, E. Nucleosides,
Nucleotides & Nucleic Acids 2001, 20, 1377.
4. Bregant, S.; Burlina, F.; Vaissermann, J.; Chassaing, G.
Eur. J. Org. Chem. 2001, 3285.
Figure 2. UV-melting curves of the PNA ox/DNA complexes.

1050
S. Bregant et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1047–1050
5. Dueholm, K. L.; Egholm, M.; Buchardt, O. Organic Pre-
parations and Procedures Int. 1993, 25, 457.
TCH2CO, CO Boc), 142.2, 142.2 (CH T), 108.2, 108.0 (CCH3
T), 78.2, 77.4 (C(CH3)3), 72.8, 72.0 (C-4), 63.3, 61.8 (C-2),
52.7, 51.4 (C(CH3)2) 49.0, 48.3 (T-CH2), 46.7, 42.9 (NHCH2),
32.7, 32.7, 25.7, 25.7 (C(CH3)2), 28.3, 28.2 (C(CH3)3), 11.9,
11.9 (CH3 T).
6. General procedure for the acylation reaction: To the car-
boxylic acid derivative suspended in dry CH2Cl2, BTSA (1
equiv) was added dropwise under argon. The reaction mixture
was stirred at rt until complete dissolution then cooled to
ꢂ78 ꢀC, pyridine (1.5 equiv) and chloroacetyl chloride (1
equiv) were added successively. After 30 min, CH2Cl2 was
added. The organic layer was washed twice with a 10% citric
acid aqueous solution and brine, dried over MgSO4 and con-
centrated. The resulting residue was dissolved in ether before
careful addition of freshly prepared diazomethane. When the
starting material was consumed, argon was bubbled through
the solution in order to remove diazomethane excess. The
reaction mixture was concentrated under vacuum and the
residue was purified by flash chromatography on silica gel
(EtOAc/cyclohexane).
7. Bicyclic compounds 4a: 1H NMR (400 MHz, CDCl3) d 5.40
(dd, 1H, 3J=10.2, 4.0 Hz, H-2), 4.74 (s, 1H, H-4), 3.75 (s, 3H,
OCH3), 3.66, 3.49 (2d, 2H, 2J=18.2 Hz, NHCH2CO), 3.43
(dd, 1H, 2J=13.4 Hz, 3J=4.0 Hz, NHCHH), (dd, 1H,
2J=13.4 Hz, 3J=10.2 Hz, NHCHH), 1.60, 1.45 (2s, 6H,
C(CH3)2). 13C NMR (100.61 MHz, CDCl3) d 169.0, 169.0
(COCH3, NCOCH2), 71.0 (C-4), 62.3 (C-2), 52.4 (OCH3), 51.5
(C(CH3)2), 48.5 (NHCH2CO), 48.4 (NHCH2), 30.1, 26.6
(C(CH3)2). 4b: 1H NMR (400 MHz, CDCl3) d 5.02 (dd, 1H,
3J=10.1, 3.0 Hz, H-2), 4.35 (s, 1H, H-4), 3.72 (s, 3H, OCH3),
3.61, 3.52 (2d, 2H, 2J=17.8Hz, NHC H2CO), 3.43 (dd, 1H,
2J=12.7 Hz, 3J=3.0 Hz, NHCHH), (dd, 1H, 2J=12.7 Hz,
3J=10.1 Hz, NHCHH), 1.59, 1.37 (2s, 6H, C(CH3)2). 13C NMR
(100.61MHz, CDCl3) d 169.4, 167.4 (COCH3, NCOCH2), 71.8
(C-4), 62.0 (C-2), 52.2 (OCH3), 51.0 (C(CH3)2), 48.1
(NHCH2CO), 47.3 (NHCH2), 31.5, 23.8(C( CH3)2).
1
10. Thiazane 6: H NMR (400 MHz, CDCl3) d 5.70 (m, 1H,
3
3
NHBoc), 5.25 (t, 1H, J=6.2 Hz, H-4), 4.84 (dd, 1H, J=9.8,
5.5 Hz, H-2), 4.29, 4.11 (2d, 2H, 2J=13.3 Hz, ClCH2), 3.72 (s,
3H, CH3O), 3.53, 3.19 (2m, 2H, NHCH2), 2.81 (m, 1H,
3J=9.9, 3.3 Hz, J=13.5 Hz, H-6), 2.51 (m, 1H, J=13.5 Hz,
H-6), 2.42 (m, 1H, H-5), 2.20 (m, 1H, H-5), 1.40 (s, 9H, CH3
Boc). 13C NMR (100.61 MHz, CDCl3) ꢀ 172.2 (COOCH3),
167.1 (ClCH2CO), 155.9 (CO Boc), 79.9 (C(CH3)3), 53.4 (C-2),
52.8(CH 3O), 51.3 (C-4), 45.7 (NHCH2), 41.3 (ClCH2), 28.2
(C(CH3)3), 26.8(C-5), 20.3 (C-6).
2
2
11. syn PNA unit U4 (amide bond isomers, 1:1): 1H NMR
(500 MHz, CD3OD) d 7.33, 7.30 (2s, 1H, CH T), 5.68(m,
0.5H, H-2), 5.32 (m, 0.5H, H-4), 5.00–4.55 (m, 3H, H-2, H-4,
T-CH2), 3.80–3.15 (m, 2H, NHCH2), 3.12, 3.04 (2t, 1H,
2J=3J=12.5 Hz, 2J=3J=11.6 Hz, H-6), 2.75–2.47 (m, 2H, H-
6, H-5), 2.23 (m, 1H, H-5), 1.91 (s, 3H, CH3 T), 1.48, 1.43 (2s,
9H, CH3 Boc). 13C NMR (125.73 MHz, CD3OD) d 175.2,
175.0 (COOH), 169.8, 169.5, 167.5, 158.7, 153.5 (CO), 144.1,
143.9 (CH T), 111.6, 111.5 (CCH3 T), 81.4, 80.9 (C(CH3)3),
55.6, 54.9 (C-4), 52.8, 52.0 (C-2), 50.5, 50.4 (T-CH2), 46.3, 44.1
(NHCH2), 29.2 (C(CH3)3), 28.9, 28.4 (C-5), 21.5, 20.8 (C-6),
12.8, 12.7 (CH3 T).
12. Schnolzer, M.; Alewood, P.; Jones, A.; Alewood, D.;
Kent, S. B. H. Int. J. Protein Res. 1992, 40, 180.
13. PNA characterization by MALDI-TOF MS using
a-cyano-4-hydroxycinnamic acid as matrix. [MH]+: PNA ref
2541.2 (calcd 2541.0); PNA 1 2584.9, PNA 2 2585.3 (calcd
2585.0); PNA 1ox 2601.0, PNA 2ox 2600.9 (calcd 2601.0);
PNA 3 2612.9 (calcd 2613.0); PNA 3ox 2629.1 (calcd 2629.0);
PNA 4 2598.8 (calcd 2599.0); PNA 4ox 2615.0 (calcd 2615.0).
14. Wohr, T.; Wahl, F.; Nefzi, A.; Rohwedder, B.; Sato, T.;
Sun, X.; Mutter, M. J. Am. Chem. Soc. 1996, 118, 9218.
15. UV-melting experiments were performed in 10 mM
sodium phosphate buffer (pH=7), 100 mM NaCl with
[PNA]=4 mM and [DNA]=2 mM. The mixture was heated for
5 min at 85 ꢀC, slowly cooled and stored overnight at R.T and
incubated 30 min at 15 ꢀC before Tm measurements (heating
and cooling rates=0.5 ꢀC/min).
8. Karplus, M. J. Chem. Phys. 1959, 30, 11.
9. syn PNA unit U3: 1H NMR (400 MHz, DMSO-d6) d 11.37,
11.31 (2s, 1H, NH T), 7.63, 7.33 (2m, 1H, NHBoc), 7.47, 7.39
(2s, 1H, CH T), 5.44 (dd, 0.5H, 3J=6.2, <1 Hz, H-2), 5.37 (m,
2
0.5H, H-2), 4.80, 4.71 (2d, 2H, J=16.4 Hz, T-CH2), 4.65 (s,
2
0.5H, H-4), 4.58, 4.23 (2d, 2H, J=16.4 Hz, T-CH2), 4.45 (s,
0.5H, H-4), 3.85–3.30 (m, 2H, NHCH2), 1.76, 1.74 (2s, 3H,
CH3 T), 1.50–1.40 (4s, 6H, C(CH3)2), 1.38, 1.35 (2s, 9H, CH3
Boc). 13C NMR (100.61 MHz, DMSO-d6) 170.6, 170.3, 166.4,
166.1, 164.4, 164.4, 156.0, 155.8, 151.0, 151.0 (CO T, COOH,