(1S,2R/1R,2S)-aminocyclohexyl glycyl thymine PNA: Synthesis, monomer crystal structures, and DNA/RNA hybridization studies

Article abstract of DOI:10.1021/ol034933m

(Matrix Presented) The synthesis of ethyl cis-(1s,2R/1R,2S)-2.aminocyclohex-1-yI-N-(thymin-1-yl-acetyl) glycinate (10a and 10b) via enzymatic resolution of the key racemic intermediate trans-2-azido cyclohexanols 3 is reported. The crystal structures of 10 show equatorial disposition of the tertiary amide group, with the torsion angle β in the range 60-70°. The PNA oligomers incorporating these show differential effects in hybridizing with complementary DNA and RNA.

Full text of DOI:10.1021/ol034933m

ORGANIC
LETTERS
2003
Vol. 5, No. 17
3013-3016
(1S,2R/1R,2S)-Aminocyclohexyl Glycyl
Thymine PNA: Synthesis, Monomer
Crystal Structures, and DNA/RNA
Hybridization Studies
T. Govindaraju,† Rajesh G. Gonnade,‡ Mohan M. Bhadbhade,‡
,†
,†
Vaijayanti A. Kumar,* and Krishna N. Ganesh*
DiVision of Organic Chemistry (Synthesis) and Centre for Materials Characterisation
(X-ray Facility), National Chemical Laboratory, Pune 411008, India
kng@ems.ncl.res.in; Vakumar@dalton.ncl.res.in
Received May 28, 2003
ABSTRACT
The synthesis of ethyl cis-(1S,2R/1R,2S)-2-aminocyclohex-1-yl-N-(thymin-1-yl-acetyl) glycinate (10a and 10b) via enzymatic resolution of the
key racemic intermediate trans-2-azido cyclohexanols 3 is reported. The crystal structures of 10 show equatorial disposition of the tertiary
amide group, with the torsion angle â in the range 60−70°. The PNA oligomers incorporating these show differential effects in hybridizing with
complementary DNA and RNA.
Peptide nucleic acids (PNA, I) are acyclic, achiral DNA
mimics, in which nucleobases are attached via an acetyl
linker to a pseudopeptide backbone composed of N-(2-
aminoethyl) glycyl units.1 PNA hybridizes through Watson-
Crick hydrogen bonding to complementary oligonucleotides.
PNA:DNA and PNA:RNA duplexes exhibit thermal stability
higher than that of the corresponding DNA:DNA and DNA:
RNA duplexes.2 PNAs are of great interest in medicinal
chemistry, with potential for the design of gene-targeted
drugs.3 Several rationale modifications have been reported
in order to understand the structure-activity relationship.1b
The objectives of these modifications are to overcome the
ambiguity in binding orientations of cDNA/RNA, to restrict
conformational flexibility in backbone and impart features
for selective RNA and DNA binding.4 The approaches
include introduction of chirality in the achiral PNA backbone
to influence the orientation of cDNA binding and designing
of cyclic analogues to preorganize the PNA structure, an
attribute that could entropically drive the duplex formation.4
One such earlier modification was to constrain the flex-
ibility in the aminoethyl segment of the PNA monomer by
introducing a cyclohexyl ring as in II.5 The trans-(1S,2S)-
cyclohexyl PNA II hybridizes with the complementary DNA
similarly to unmodified PNA, while trans-(1R,2R)-cyclo-
hexyl PNA lacked such a property.5 Thermodynamic data
† Division of Organic Chemistry (Synthesis).
‡ Centre for Materials Characterisation.
(1) (a) Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. Science
1991, 254, 1497-1500. (b) Ganesh, K. N.; Nielsen, P. E. Curr. Org. Chem.
2000, 4, 1931-1943.
(2) (a) Egholm, M.; Buchardt, O.; Christensen, L.; Behrens, C.; Freier,
S. M.; Driver, D. A.; Berg, R. H.; Kim, S. K.; Norden, B.; Nielsen, P. E.
Nature 1993, 365, 566-568. (b) Jensen, K. K.; Qrum, H.; Neilsen, P. E.;
Norden, B. Biochemistry 1997, 36, 5072-5077.
(4) (a) Kumar, V. A. Eur. J. Org. Chem. 2002, 2021-32. (b) D’Costa,
M.; Kumar, V. A.; Ganesh, K. N. J. Org. Chem. 2003 (in press).
(5) Lagriffoule, P.; Witteng, P.; Ericksson, M.; Jensen, K. K.; Norden,
B.; Buchardt, O.; Nielsen, P. E. Chem. Eur. J. 1997, 3, 912-919.
(3) Braasch, D. A.; Corey, D. R. Biochemistry 2002, 41, 4503-4510.
10.1021/ol034933m CCC: $25.00 © 2003 American Chemical Society
Published on Web 07/29/2003

indicated that the DNA binding of the (1S,2S) cyclohexyl
PNA is accompanied by a decrease in the entropy loss and
a reduced gain in enthalpy. Molecular Dynamics simulations
performed on model structures of (1S,2S)- and (1R,2R)-
cyclohexyl PNAs showed the torsion angle â (N-CH2-
CH2-NHCO) to be close to 180°, corresponding to a trans-
(1,2-diaxial) disposition5 (Figure 1). An analysis of
-63° and +66°, which is close to the desired value
supporting the postulate of initial design. To our knowledge,
this is the first report of a crystal structure of any backbone-
constrained PNA monomer.
Synthesis of (1S,2R/1R,2S)-Aminocyclohexyl Glycyl
PNA Thymine Ethyl Ester Monomers (10a/10b). The
synthesis of title compounds was achieved starting from (+)-
and (-)-trans-2-azido cyclohexanols 3a and 3b. These were
obtained by enzymatic hydrolysis of racemic 2-azido cyclo-
hexanoate 2 using lipases, well-known in the literature.7 The
butyrate ester of racemic trans-2-azido cyclohexanols was
synthesized by oxirane ring opening of the commercially
available meso-epoxide 1 with sodium azide8 followed by
esterification with butyric anhydride (Scheme 1). The racemic
Scheme 1a
Figure 1. PNA analogues and chiral cyclohexyl PNA.
PNA:DNA and PNA:RNA duplexes by solution NMR
studies and crystal structure have revealed distinctive dif-
ferences in the PNA conformations between the two du-
plexes.6 The most notable difference is in the torsion angle
â; in a PNA:RNA duplex it is in the range of 60-70°,
whereas in a PNA:DNA duplex, it is approximately 140°.
This suggests that the trans diaxial geometry of the (1S,2S/
1R,2R)-cyclohexyl PNA II is far from the required PNA
conformation and not suitable for facile hybridization with
either DNA or RNA targets.
a Reagents and conditions: (i) NaN3, NH4Cl, 80% aq ethanol,
reflux, 18 h, 89%; (ii) n-butyric anhydride, dry pyridine, DMAP
(cat.), rt, 16 h, 91%; (iii) Pseudomonas cepacia lipase, phosphate
buffer, pH 7.2, 2.5 h; (iv) column chromatography; (v) NaOMe in
MeOH, rt, 0.5 h.
Classical PNA is very flexible and can attain different
conformations to accommodate binding to both DNA and
RNA. In view of results on trans-(1S,2S/1R,2R)-cyclohexyl
PNAs and PNA-DNA/RNA duplex structural data, it oc-
curred to us that if torsion angle â can be restricted to values
in the range 60-70°, the resulting PNA may bind to RNA
selectively over DNA. This is possible in a cis-1,2-disub-
stituted cyclohexyl system wherein the substituents are in
axial-equitorial disposition. In cyclohexyl PNAs, this cor-
responds to (1S,2R/1R,2S) configuration III, wherein the
torsion angle â would be around 65°. These may be better
suited for incorporation into the PNA backbone to impart
DNA/RNA discriminating properties. In this paper, we
describe a chemoenzymatic synthesis of cis-(1S,2R/1R,2S)-
aminocyclohexyl glycyl PNA thymine monomers for incor-
poration into PNA oligomers and the crystal structure of the
title monomers. The torsion angle â in these corresponds to
butyrate ester 2 was resolved by enzymatic enantioselective
hydrolysis using the lipase from Pseudomonas cepacia
(Amano-PS) in sodium phosphate buffer (pH ) 7.2, 40%
conversion), followed by chromatography, to obtain optically
pure (1R,2R)-azido alcohol 3a. Amano-PS lipase was found
to be much better than the earlier reported lipase from
Candida cylindracea for this resolution step and gave better
enantiomeric purity for the azido alcohol 3 under identical
reaction conditions and in less reaction time. Further enzyme
treatment of the mixture of (1R,2R) (minor) and (1S,2S)
(major) butyrate ester 2 for the second time, column
purification, and subsequent methanolysis of the (1S,2S)-2
ester using NaOMe in methanol gave pure (1S,2S)-3b in 30%
overall yield. The enantiopurity of both (1R,2R)-3a and
(1S,2S)-3b isomers was about 98-99% by comparing with
known values for the optical rotations reported in the
(7) (a) Faber, K.; Honig, H.; Seufer-Wasserthal, P. Tetrahedron Lett.
1988, 29, 1903-1904. (b) Honig, H.; Seufer-Wasserthal, P. J. Chem. Soc.,
Perkin Trans. 1 1989, 2341-2345.
(6) (a) Brown, S. C.; Thomson, S. A.; Veal, J. M.; Davis, D. G. Science
1994, 265, 777-780. (b) Ericksson, M.; Neilsen, P. E. Nat. Struct. Biol.
1996, 3, 410-413.
(8) Swift, G.; Swern, D. J. Org. Chem. 1967, 32, 511-517.
3014
Org. Lett., Vol. 5, No. 17, 2003

literature7 and by 13C NMR using the chiral chemical shift
reagent Eu(hfc)3.
The reduction of the azide function in (1R,2R)-3a by
hydrogenation using Adam’s catalyst9 and in situ t-Boc
protection of the resulting amine function yielded the Boc-
protected amino alcohol (1R,2R)-4 (Scheme 2), which was
X-ray Crystal Structures of (1S,2R/1R,2S)-Aminocy-
clohexyl PNA Thymine Monomers 10a/10b. X-ray quality
crystals of both (1S,2R)-10a and (1R,2S)-10b were obtained
only from chloroform,10 and the ORTEP diagrams are shown
in Figure 2. The torsion angles for (1S,2R)-10a and (1R,2S)-
Scheme 2a
Figure 2. ORTEP13 view of (a) (1S,2R)- and (b) (1R,2S)-
aminocyclohexyl glycyl PNA thymine monomer ethyl ester 10.
10b are shown in Table 1. The torsion angle â in both
isomers are around 65° in magnitude, but with opposite signs.
Table 1. Torsion Angles of PNA Monomers and PNA-DNA/
PNA-RNA Duplexes
compound
R
â
γ
δ
ø1
ø2
a Reagent and conditions: (i) PtO2, dry EtOAc, H2, 35-40 psi,
rt, 3.5 h; (ii) (Boc)2O; (iii) MsCl, dry pyridine, DMAP, 0-5 °C, 5
h; (iv) NaN3, dry DMF, 72 °C, 18 h; (v) PtO2, MeOH, H2, 35-40
psi, rt, 3.5 h; (vi) BrCH2COOEt, KF-Celite, dry CH3CN, rt, 4 h;
(vii) ClCH2COCl, Na2CO3, dioxan/H2O (1:1), 0 °C, 0.5 h; (viii)
thymine, K2CO3 dry DMF, 65 °C, 3.5 h; (ix) 0.5 M LiOH/aq THF,
rt, 0.5 h.
(1S,2R)-10a
(1R,2S)-10b
PNA-DNAa
PNA-RNAb
128
-129
105
-63
66
141
67
76
-78
78
119
-119
139
1.02
-0.87
-3
-175
174
151
170
79
85
4
-171
a Reference 6b. b Reference 6a.
In the isomer (1S,2R)-10a, â is negative and all other angles
are closer to the values found in PNA:RNA complex both
in sign and magnitude.6 Though in solution, the tertiary amide
bond exists as a rotameric mixture,6 in crystal structures the
amide bond is trans, with the carbonyl pointing toward the
glycyl component.11 The crystal structure data, particularly
with reference to the angle â, suggest that the PNA oligomers
derived from cis-disubstituted (1S,2R)-cyclohexyl PNA
monomers, with axial-equatorial dispositions, are better
preorganized for a favorable interaction with RNA than with
DNA. In the PNA monomer 10 with the backbone imposed
on the cyclohexyl ring, the N-terminus substituent occupies
the axial position, while the more bulky tertiary amide
substituent takes up the equatorial disposition.
PNA T10 oligomers 12 and 13 incorporating the modified
monomers and the unmodified control 14 were synthesized
using Boc chemistry on L-lysine-derivatized MBHA resin5
and characterized by mass spectrometry analysis.12 These
sequences having single modifications are identical to the
reported5 ones in order to make appropriate comparisons.
then converted to corresponding mesylate (1R,2R)-5. The
mesylate was treated with NaN3 in dry DMF to yield the
azide (1S,2R)-6 with inversion of configuration at C1. The
azide (1S,2R)-6 was hydrogenated using Adam’s catalyst to
give the amines (1S,2R)-7, which without further purification
was alkylated with ethyl bromoacetate in the presence of
KF-Celite. This resulted in monosubstituted cis-1,2-diamines
(1S,2R)-8, which on acylation with chloroacetyl chloride gave
the compound (1S,2R)-9. The condensation of (1S,2R)-9 with
thymine in the presence of K2CO3 in DMF yielded the
desired (1S,2R)-aminocyclohexyl (thymin-1-yl-acetyl) glycyl
PNA monomer ethyl ester (1S,2R)-10a, which on hydrolysis
gave the monomer (1S,2R)-11a. The synthesis of other
enantiomer (1R,2S)-11b was accomplished starting from
(1S,2S)-3b following same steps as described above. All
compounds were characterized by 1H,13C NMR spectroscopy
and mass spectrometry analysis.
(9) Schaus, S. E.; Larrow, J. F.; Jacobsen, E. N. J. Org. Chem. 1997,
62, 4197-4199.
Org. Lett., Vol. 5, No. 17, 2003
3015

The Tm values of PNAs 12 and 13, hybridized with
complementary RNA (poly rA) and DNA (poly dA), were
obtained from temperature-dependent UV absorbance data
(Figure 3) and are shown in Table 2. From the data it is
Table 2. Thermal Stability (Tm, °C) of PNA-DNA/RNA
Hybridsa
PNA oligomer
poly rA
poly dA
H-TTTTTSRTTTTT-Lys-NH2 (12)
H-TTTTTRSTTTTT-Lys-NH2 (13)
H-TTTTTTTTTT-Lys-NH2 (14)
77.2
70.7
>80
61.5
63.5
72.4
a Buffer: 10 mM sodium phosphate, pH 7.0, 100 mM NaCl, 0.1 mM
EDTA. The Tm values reported are the average of 3 independent experiments
and are accurate to (0.5 °C, PNA:DNA/RNA (2:1).
transition was also very sharp compared to the broad nature
in other cases. In comparison, the two isomers had no
significant differences in binding with complementary DNA
(∆Tm ) -2 °C). The preferential binding to RNA over DNA
by one of the enantiomers was not observed with the reported
trans isomers,5 although the trans-(1S,2S) was the preferred
geometry for binding to both DNA and RNA. These
preliminary studies suggest that preorganization of backbone
geometry using monomers with appropriate torsion angles
may lead to induction of substantial selectivity in DNA/RNA
binding.
On the basis of a structural rationale, we have synthesized
[(1S,2R)/(1R,2S)]-aminocyclohexyl (thymin-1-yl-acetyl) gly-
cyl PNA monomers 10, followed by their incorporation into
PNA oligomers. Preliminary binding data with cDNA/RNA
have suggested stereochemical discrimination in binding of
the derived PNA oligomers with RNA that is not significant
in DNA. Further studies on the synthesis and properties of
mixed PNA and homochiral oligomers are currently in
progress. The synthetic route described here also provides
an easy access for cis-cyclohexyl 1,2-diamines 7, which are
useful ligands for applications in chiral catalysis14 and
peptidomimetics.
Figure 3. Derivative UV-temp curves. Poly rA with (a) PNA
12, (b) PNA 13. Poly dA with (c) PNA 14, (d) PNA 12, (e) PNA
13.
seen that the PNA oligomer 12 with cyclohexyl (1S,2R)
geometry is preferred for binding to RNA over the PNA 13
having cis (1R,2S) geometry (∆Tm ) +7°C). The observed
(10) Single crystals of both 10a (1S,2R) and 10b (1R,2S) were obtained
by crystallization from chloroform only. X-ray intensity data were collected
on a Bruker SMART APEX CCD diffractometer at low temperature. Crystal
data for 10(a): C23H33.25Cl4.50N4O7, M ) 637.31, crystal dimensions 0.75
× 0.34 × 0.02 mm3; crystal system orthorhombic, space group P212121, a
) 6.201(4), b ) 22.599(14) c ) 23.835(14) Å, V ) 3340(3) Å3, Z ) 4, Dc
) 1.267 g cm-3, µ(Mo KR) ) 0.436 mm-1, T ) 150(2) K, F(000) ) 1327,
Max and min transmission 0.9913 and 0.7370, 31159 reflections collected,
5864 unique [I > 2σ(I)], S ) 1.164, R value 0.1043, wR2 ) 0.2477 (all
data R ) 0.1197, wR2 ) 0.2567). Data for 10(b): C23.50H35Cl5.25N4 O7, M
) 671.67, crystal dimensions 1.21 × 0.27 × 0.05 mm3, crystal system
orthorhombic, space group P212121, a ) 6.2353(9), b ) 22.617(3) c )
23.988(3) Å, V ) 3382.8(8) Å3, Z ) 4, Dc ) 1.319 g cm-3, µ(Mo KR) )
0.492 mm-1, T ) 150(2) K, F(000) ) 1397, Max and min transmission
0.9773 and 0.5888, 43174 reflections collected, 5932 unique [I > 2σ(I)], S
) 1.046, R value 0.0723, wR2 ) 0.2074 (all data R ) 0.0847, wR2 )
0.2147). All data were corrected for Lorentzian, programs. SHELX-97
(Sheldrick, G. M. SHELX-97 Program for Crystal Structure Solution and
Refinement; University of Gottingen: Germany, 1997) was used for structure
solution and full polarization and absorption effects using Bruker’s SAINT
and SADABS matrix least squares refinement on F2. Hydrogen atoms were
included in the refinement as per the riding model. Both enantiomers contain
one molecule of ordered chloroform with full occupancy and another
molecule of highly disordered chloroform with occupancy less than unity.
The high R values despite data collection at low temperature could be
attributed to the extensive disorder of the solvent molecule in 10a and 10b
and extensive disorder of the end methyl group of the side chain in 10a.
(11) Sanjayan, G. J.; Pedireddi, V. R.; Ganesh, K. N. Org. Lett, 2000,
2, 2825-2828.
Acknowledgment. T.G. thanks CSIR, New Delhi for the
award of a research fellowship. V.A.K. thanks the Depart-
ment of Science and Technology, New Delhi for research
grants.
OL034933M
(12) MALDI-TOF: PNA 12, 2862.0 [M + 2H+], PNA 13, 2861.0 [M
+ H+], and PNA 14, 2808.0 [M + 2H+].
(13) Johnson, C. K, ORTEP II, report ORNL-5138, Oak Ridge National
Laboratory, Tennesse, U.S.A., 1976. (Incorporated in SHELX TL package
from Bruker-axs.
(14) Martinez, L. E.; Leighton, J. L.; Carsten, D. H.; Jacobsen, E. N. J.
Am. Chem. Soc. 1995, 117, 5897-5858.
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Org. Lett., Vol. 5, No. 17, 2003