Synthesis and Evaluation of (1S,2R/1R,2S)-Aminocyclohexylglycyl PNAs As Conformationally Preorganized PNA Analogues for DNA/RNA Recognition

Article abstract of DOI:10.1021/jo035747x

Conformationally constrained cis-aminocyclohexylglycyl PNAs have been designed on the basis of stereospecific imposition of 1,2-cis-cyclohexyl moieties on the aminoethyl segment of aminoethylglycyl PNA (aegPNA). The introduction of the cis-cyclohexyl ring

Full text of DOI:10.1021/jo035747x

Syn th esis a n d Eva lu a tion of (1S,2R/1R,2S)-Am in ocycloh exylglycyl
P NAs a s Con for m a tion a lly P r eor ga n ized P NA An a logu es for
DNA/RNA Recogn ition
T. Govindaraju, Vaijayanti A. Kumar,* and Krishna N. Ganesh*
Division of Organic Chemistry (Synthesis), National Chemical Laboratory, Pune 411008, India
vakumar@dalton.ncl.res.in
Received November 28, 2003
Conformationally constrained cis-aminocyclohexylglycyl PNAs have been designed on the basis of
stereospecific imposition of 1,2-cis-cyclohexyl moieties on the aminoethyl segment of aminoethyl-
glycyl PNA (aegPNA). The introduction of the cis-cyclohexyl ring may allow the restriction of the
torsion angle â in the ethylenediamine segment to 60-70° that is prevalent in PNA2:DNA and
PNA:RNA complexes. The synthesis of the optically pure monomers (10a and 10b) is achieved by
stereoselective enzymatic hydrolysis of an intermediate ester 2. The chiral PNA oligomers were
synthesized with (1S,2R/1R,2S)-aminocyclohexylglycyl thymine monomers in the center and
N-terminus of aegPNA. Differential gel shift retardation with one or more units of modified monomer
units was observed as a result of hybridization of PNA sequences with complementary DNA
sequences. Hybridization studies with complementary DNA and RNA sequences using UV-Tm
measurements indicate that PNA with (1S,2R)-cyclohexyl stereochemistry enhances selective
binding with RNA over DNA as compared to control aegPNA and PNA with the other (1R,2S)
isomer.
In tr od u ction
Peptide nucleic acids (PNA II) are DNA/RNA (I)
mimics in which the natural nucleobases are attached
to an uncharged, pseudopeptide backbone composed of
N-(2-aminoethyl)glycine units via methylene carbonyl
linkers (Figure 1).1 PNA hybridizes to complementary
DNA/RNA sequences via duplex formation for mixed
sequences (W-C base pairing) and triplex formation for
homopyrimidine or homopurine sequences involving both
W-C and Hoogsteen base pairing. The complexes of PNA
with DNA/RNA have consistently shown higher thermal
stabilities as compared to the corresponding DNA-DNA
and DNA-RNA complexes.2 Due to these exceptional
properties and their resistance to proteases and nu-
cleases, PNAs are ideal lead candidates for development
as new therapeutic agents in medicinal chemistry via
antigene or antisense approaches3 and also find useful
applications in molecular biology and diagnostics.4
The current limitations in PNA properties are its poor
water solubility and lack of cell permeability coupled with
ambiguity in DNA/RNA recognition arising from its
F IGURE 1. Structures of I DNA(RNA), II PNA, and III chiral
cis-(1S,2R)- and (1R,2S)-aminocyclohexyl PNAs.
equally facile binding in parallel/antiparallel fashion with
the complementary nucleic acid sequences. These limita-
tions are now being systematically addressed with modi-
fied PNA analogues.1b Major improvements in DNA/RNA
recognition selectivity were anticipated by pre-organiza-
tion of PNA structure via conformational constraint
strategies.5 The approaches include introduction of chiral-
ity in the achiral PNA backbone to influence the orienta-
tion of cDNA/RNA binding1b and designing cyclic ana-
logues that preorganize the PNA structure for attaining
hybridization competent conformation and hence entropic-
ally drive the complex formation.5 Most of the cyclic PNA
analogues have been constructed by insertion of a five-
membered pyrrolidine ring in to the backbone.6 In one
of the earliest approaches, the six-membered trans-
(1S,2S/1R,2R)-cyclohexyl ring was imposed on the PNA
backbone,7 which exhibited destabilization of the derived
(1) (a) Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. Science
1991, 254, 1497. (b) Ganesh, K. N.; Nielsen, P. E. Curr. Org. Chem.
2000, 4, 1931-43.4.
(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, 566-68. (b) J ensen, K. K.; Qrum, H.; Nielsen,
P. E.; Norden, B. Biochemistry 1997, 37, 5072-50.
(3) (a) Bennett, C. F. In Applied Antisense Oligonucleotide Technol-
ogy; Stein, C. A., Craig, A. M., Eds.; Wiley-Liss, Inc.: New York, 1998.
(b) Braasch, D. A.; Corey, D. R. Biochemistry 2002, 41, 4503-4510.
(b) Uhlmann, E.; Peyman, A. Chem. Rev. 1990, 90, 366-374. (c)
Micklefield, J . Curr. Med. Chem. 2001, 8, 1157-1179.
(4) Nielsen, P. E.; Egholm, M. In Peptide nucleic acids (PNA).
Protocols and Apllications; Nielsen, P. E., Egholm, M., Eds.; Horizon
Scientific: Norfolk, CT, 1999.
(5) Kumar, V. A. Eur. J . Org. Chem. 2002, 2021-2032.
10.1021/jo035747x CCC: $27.50 © 2004 American Chemical Society
Published on Web 02/24/2004
1858
J . Org. Chem. 2004, 69, 1858-1865

Synthesis of (1S,2R/ 1R,2S)-Aminocyclohexylglycyl PNAs
SCHEME 1 a
a
Reagents and conditions: (i) NaN3, NH4Cl, aq ethanol, reflux, 18 h; (ii) n-butyric anhydride, dry pyridine, DMAP (cat.), rt, 16 h; (iii)
Pseudomonas cepacia (lipase), phosphate buffer, pH 7.2, 2.5 h; (iv) NaOMe in MeOH.
DNA complexes. In this analogue, the trans-(1,2-diaxial)
disposition of the substituents caused the torsion angle
â to be ∼180°. This geometry is far from the conformation
required for PNA strand in PNA2:DNA triplexes (â
∼73°)8a and PNA:DNA duplex (â ∼141°)8b as inferred
from the reported X-ray structures or PNA:RNA (â
∼65-70°)8c duplex computed from NMR studies.8 Re-
cently, two approaches have emerged that involve the
tuning of the torsion angle â in PNA backbone by
superimposition of cis-(1R,2S/1S,2R)-cyclohexyl9 and trans-
(1S,2S)-cyclopentyl ring systems.10 In this paper, we
elaborate on the synthesis and DNA/RNA hybridization
studies on cis-(1S,2R/1R,2S)-cyclohexyl PNAs that we
reported in a recent communication.9 The homothymine
oligomer incorporated with this modification shows ster-
eochemistry-dependent, sequence-specific triplex forma-
tion with complementary DNA and RNA sequences.
Electrophoretic gel shift assay is presented to show
differential retardation as a result of hybridization of
PNA sequences carrying one or more units of modified
monomer with the complementary DNA sequences. Hy-
bridization study with complementary DNA and RNA
sequences using UV-Tm measurements to study the
DNA/ RNA binding selectivity and comparative mismatch
tolerance is also reported.
ture.11 The butyrate ester of racemic trans-2-azidocyclo-
hexanol was synthesized by the oxirane ring opening of
the commercially available meso-epoxide 1 with sodium
azide12 followed by esterification with butyric anhydride
(Scheme 1). The racemic butyrate ester 2 was resolved
by enzymatic enantioselective hydrolysis using the lipase
from Pseudomonas cepacia (Amano-PS) in sodium phos-
phate buffer which proceeded with 40% conversion,
followed by chromatography, to obtain the optically pure
(1R,2R)-azido alcohol 3a . The earlier reported procedure
for this resolution step employed the lipase from Candida
cylindracea but was found to be less effective as compared
to Amano-PS. After a comparative resolution of racemic
butyrate 2 with both the enzymes, we found that the P.
cepacia lipase (Amano-PS) gave a better enantiomeric
purity of the azido alcohol than the lipase from C.
cylindracea under identical reaction conditions and in
less reaction time. Further enzymatic treatment of the
mixture of minor (1R,2R) and major (1S,2S) butyrate
esters 2 for the second time followed by column purifica-
tion and subsequent methanolysis of the 2 (1S,2S) 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 confirmed by comparing with
known values for the optical rotations reported in the
literature11 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 catalyst13
and in situ t-Boc protection of the resulting amine
function yielded the Boc-protected amino alcohol (1R,2R)-4
(Scheme 2). Other hydrogenation catalysts such as Raney
Ni and 10% Pd/C were also effective at catalyzing the
reduction of azide, but the product obtained required
extensive purification due to the resulting side products
and consequently lower yields. The alcohol 4 was 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.
This was confirmed from the crystal structures of mesy-
late (1R,2R)-5 and azide (1S,2R)-6 (Supporting Informa-
tion). The azide (1S,2R)-6 was hydrogenated using Adam’s
catalyst to give the amine (1S,2R)-7, which without
further purification was alkylated with ethyl bromoac-
Resu lts a n d Discu ssion
Syn th esis of cis-(1S,2R)- a n d (1R,2S)-Am in ocyclo-
h exylth ym in e Mon om er s a n d th e P NA Oligom er s.
The synthesis of the title compounds was achieved
starting from a mixture of (+)- and (-)-trans-2-azidocy-
clohexanoate 2 using lipases well-known in the litera-
(6) (a) Gangamani, B. P.; Kumar, V. A.; Ganesh, K. N. Tetrahedron
1999, 55, 177-192. (b) D’Costa, M.; Kumar, V. A.; Ganesh, K. N. Org.
Lett. 1999, 1, 1513-1516. (c) Vilaivan, T.; Khongdeesameor, C.;
Harnyauttanokam, P.; Westwell, M. S.; Lowe, G. Biorg. Med. Chem.
Lett. 2000, 10, 2541-2545. (d) Vilaivan, T.; Khongdeesameor, C.;
Harnyauttanokam, P.; Westwell, M. S.; Lowe, G. Tetrahedron. Lett.
2001, 42, 5533-5536. (e) Hickman, D. T.; King, P. M.; Cooper, M. A.;
Slater, J . M.; Micklefield, J . Chem. Commun. 2000, 2251-2252.
(7) Lagriffoule, P.; Witteng, P.; Ericksson, M.; J ensen, K. K.; Norden,
B.; Buchardt, O.; Nielsen, P. E. Chem. Eur. J . 1997, 3, 912-919.
(8) (a) Betts, L.; J osey, J . A.; Veal, M.; J ordan, S. R. Science 1995,
270, 1838-1841. (b) Ericksson, M.; Nielsen, P. E. Nat. Struct. Biol.
1996, 3, 410-413. (c) Brown, S. C.; Thomson, S. A.; Veal, J . M.; Davis,
D. J . Science 1994, 265, 777-780.
(11) (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.
(12) Swift, G.; Swern, D. J . Org. Chem. 1967, 32, 511-517.
(13) Schaus, S. E.; Larrow, J . F.; J acobsen, E. N. J . Org. Chem. 1997,
62, 4197-4199.
(9) Govindaraju, T.; Gonnade, R. G.; Bhadbhade, M. M.; Kumar, V.
A.; Ganesh, K. N. Org. Lett. 2003, 17, 3013-3016.
(10) Myers, M. C.; Witschi, M. A.; Larionova, N. V.; Franck, J . M.;
Haynes, R. D.; Hara, T.; Grajkowski, a.; Appella, D. H. Org. Lett. 2003,
15, 2695-2698.
J . Org. Chem, Vol. 69, No. 6, 2004 1859

Govindaraju et al.
SCHEME 2 a
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, dioxane/H2O (1:1), 0 °C, 30 min; (viii) thymine, K2CO3 dry DMF, 60-65°C, 3.5 h; (ix) 0.5 M LiOH, aq THF, 0.5 h.
etate in the presence of KF-Celite. This resulted in the
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
using 0.5 M LiOH in aqueous THF gave the monomer
11a . All attempts to couple thymine acetic acid and the
amine 8 using coupling reagents such as DCC, HBTU,
and TBTU were unsuccessful. This may be attributed to
the presence of the bulky tert-butyloxycarbonyl (Boc)
group in the adjacent position. This is seen in the crystal
structures of the final monomer esters 10a and 10b
where the thymine base attached to the main backbone
via methylene carbonyl linker is away from both Boc
group and the glycine in chain (Supporting Information).
All new compounds were characterized by 1H and 13C
NMR and mass spectral data. The synthesis of the other
enantiomer (1R,2S)-11b monomer was accomplished
starting from (1S,2S)-3b following same steps as de-
scribed above.
The enantiomeric purity of alcohols (1R,2R)-3a and
(1S,2S)-3b was confirmed from 13C NMR studies of their
acetyl derivatives utilizing the chiral chemical shift
reagent, (+)-tris[3-(heptafluoropropylhydroxymethylene)-
d-camphorato]europium (III) [Eu(hfc)3] (7.7 mol %)14
(Supporting Information). The torsion angles â as de-
duced from the X-ray crystal structure9 structures for 10a
(1S,2R) and 10b (1R,2S) are -63° and +66°, respectively,
which are closer in magnitude to that found in PNA2:
DNA and PNA:RNA complexes, with difference in their
relative signs as expected for the enantiomeric pairs. In
solution, the tertiary amide bond in PNA is known to
exist as a rotameric mixture.8 In the present structures,
the amide bond is trans with carbonyl pointing toward
the C-terminus, similar to that previously observed by
us for cyanuryl monomer15 and to that seen in the
recently reported crystal structure of the D-lysine-based
chiral PNA-DNA duplex.16 The crystal structure data
also show axial-equatorial dispositions for the cis-
disubstituted (1S,2R/1R,2S)-cyclohexyl PNA monomers,
with the bulky substituent carrying the nucleobase
directed into an equatorial position.
The modified cis-(1S,2R/1R,2S)-aminocyclohexylgly-
cylthymine monomers 11a and 11b were incorporated
into PNA oligomers using Boc chemistry on L-lysine-
derivatized (4-methylbenzhydryl)amine (MBHA) resin as
reported before.9 Various homothymine PNA decamers
(13-16, Table 1) incorporating one or two cis-(1S,2R/
1R,2S)-aminocyclohexylglycylthymine PNA monomers in
different positions were synthesized. The oligomers were
cleaved from the resin using TFMSA procedure17 followed
by RP HPLC purification and characterized by mass
spectrometry (MALDI-TOF).
UV-Tm St u d ies of P NA-DNA/R NA Com p lexes.
The hybridization of modified PNAs with complementary
DNA I sequences were studied by temperature-depend-
ent UV absorbance experiments. The stoichiometry for
(15) Sanjayan, G. J .; Pedireddi, V. R.; Ganesh, K. N. Org. Lett. 2000,
2, 2825-2828.
(16) Menchise, V.; Simone, G. D.; Tedeschi, T, Corradini, R.; Sforza,
S.; Marchelli, R.; Capasso, D.; Saviano, M.; Pedone, C. Proc. Natl. Acad.
Sci U.S.A. 2003, 100, 12021-12026.
(17) Christensen, L.; Fitzpatrick, R.; Gildea, B.; Petersen, K. H.;
Hansen, H. F.; Koch, T.; Egholm, M.; Buchardt, O.; Nielsen, P. E.;
Coull, J .; Berg, R. H. J . Peptide Sci. 1995, 3, 175.
(14) Gogte, V. N.; Pandit, V. S.; Natu, A. A.; Nanda, R. K.; Sastry,
M. K. Org. Magn. Reson. 1984, 22, 624.
1860 J . Org. Chem., Vol. 69, No. 6, 2004

Synthesis of (1S,2R/ 1R,2S)-Aminocyclohexylglycyl PNAs
TABLE 1. UV-Meltin g Tem p er a tu r es (Tm Va lu es)a of P NA2/DNA Com p lexes
PNA
DNA Ib
DNA IIb
poly rA
poly dA
72.4
12, H-TTTTTTTTTT-Lys-NH2
13, H-TTTTTSRTTTTT-Lys-NH2
14, H-TTTTTRSTTTTT-Lys-NH2
15, H-TSRTTTTSRTTTTT-Lys-NH2
16, H-TRSTTTTRSTTTTT-Lys-NH2
69.2
41.1
45.0
34.4
37.5
58.0 (-11.2)c
26.1 (-15.0)
29.1 (-16.1)
23.3 (-11.0)
25.7 (-12.0)
>80.0
77.2
70.7
64.4
58.6
61.5 (-15.7)d
63.5 (-7.2)
53.7 (-10.7)
53.5 (-5.1)
a
Tm ) melting temperature (measured in the buffer 10 mM sodium phosphate, 100 mM NaCl, 0.1 mM EDTA, pH ) 7.0), PNA2:DNA
complexes. b I ) 5′d(CGCA10CGC); II 5′d(CGCA4CA5CGC). b Values in parentheses indicate the difference in Tm as a result of the mismatch
in DNA sequence. c Values in parentheses indicate difference in binding with RNA over DNA. The values reported here are the average
of three independent experiments and are accurate to (0.5 °C.
of the modified oligomers with poly rA exhibited de-
creased Tm, indicating destabilization as compared to the
unmodified PNA 12, but the magnitudes were relatively
small for either poly-rA or poly dA (∆Tm ∼7° per
modification) than observed for the complexes with DNA
oligonucleotides. The most interesting observation is that
the stereochemistry of the monomeric units is able to
direct the DNA verses RNA binding selectivity; thus, the
(1S,2R)-PNAs 13 and 15 with one and two modifications
show better binding to poly-rA compared to the corre-
sponding (1R,2S)-PNAs 14 and 16. The situation is
reverse for binding with DNAsthe (1R,2S)-PNAs 14 and
16 bind DNA better compared to (1S,2R)-PNAs 13 and
15. In general, all PNA oligomers showed a slightly lower
binding with poly dA than with poly-rA. The observed
transitions are sharp for modified PNA complexes with
poly rA and poly dA as compared to complexes of
F IGURE 2. UV absorbance (at 260 nm) of mixtures of PNA
14 and the complementary DNA I in the relative molar ratios
of 0:100, 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20,
90:10, and 100:0 (buffer, 10 mM sodium phosphate pH 7.0,
100 mM NaCl, 0.1 mM EDTA).
unmodified PNA 12. Introduction of the second modified
unit at the N-terminus actually reduced the overall
stability of complexes both with dA10, poly dA, and poly-
rA as well as the differential RNA over DNA binding
selectivity.
The thermal stabilities of PNA complexes with DNA
II [d(A4CA5] containing a single mismatch at the middle
modification site were also measured. The ∆Tm for control
PNA 12 was -11° while that for modified PNAs 13 and
14 were -15° when a single modified unit is present.
With the second modified unit this difference also reduced
to -11° with a lowering of Tm. These results indicated
that the cyclohexyl modified PNAs retain the ability of
PNAs of sequence specific recognition and binding to
cDNA allowing discrimination against mismatch se-
quences.
PNA:DNA complexation as established by UV absorbance
mixing data at 260 nm (J ob plot)18 was a 2:1 ratio (Fig-
ure 2).
The thermal stabilities (Tm) of PNA2:DNA triplexes
(Table 1, Figure 3) were obtained for different PNA
modifications with both complementary DNA I and DNA
II having a single mismatch. The corresponding com-
plexes with poly rA and poly dA were also studied for
relative compatibility of the imposed stereochemistry
with ribo- and deoxyribonucleic acids. In general, the
PNA2:DNA triplexes were found to be less stable for
PNAs containing enantiomeric (1S,2R)- or (1R,2S)-modi-
fied cyclohexyl units, as compared to the unmodified
PNA. The PNA oligomers 13 and 14 having a modifica-
tion in the center of the sequence destabilized the
complexes with DNA I (∆Tm ) -28 and -24 °C, respec-
tively) in comparison to the control PNA 12. In the case
of PNA oligomers 15 and 16 having two modified units
(at N-terminal and center), a further decrease in Tm (∆Tm
) -35 and -32 °C) was observed. PNAs 14 and 16
modified with (1R,2S) isomer were slightly better binding
with DNA I than the PNAs 13 and 15 with enantiomeric
(1S,2R) isomer. The comparative binding study was also
conducted for complexation with poly-rA. For appropriate
comparison of DNA and RNA complex stabilities, UV
experiments were done with poly dA, which exhibited a
higher Tm than the DNA oligonucleotide I. The complexes
CD Sp ectr oscop ic Stu d ies of P NA:DNA Com -
p lexes. PNA is nonchiral and does not show any signifi-
cant CD signatures. However, PNA:DNA complexes
exhibit characteristic CD signatures due to chirality
induced by DNA component. It is known that formation
of PNA triplexes is accompanied by the appearance of
positive CD bands at 258 and 285 nm that are not present
in DNA. The 258 nm band is slightly sharper and of
higher intensity than 285 nm. The presence of chiral
centers in cyclohexyl PNAs should further influence the
CD patterns of the derived PNA:DNA triplexes. Figure
4 shows the CD spectra of individual PNAs 13-15, DNA
I, and the PNA:DNA complexes. The single-stranded
cyclohexyl PNAs (13-15) show weak CD bands in the
250-290 nm region that are absent in unmodified PNA
12 (Supporting Information). The CD spectra of com-
plexes of PNA 13, PNA 14, PNA 15, and PNA 16 with
d(A10) are nearly identical (Figure 4A,B) and are similar
to the CD spectrum of the unmodified (PNA12)2-d(A10)
(18) (a) J ob, P. Ann. Chim. 1928, 9, 113-203. (b) Cantor, C. R.;
Schimmel, P. R. Biophys. Chem. Part III 1980, 624.
J . Org. Chem, Vol. 69, No. 6, 2004 1861

Govindaraju et al.
F IGURE 3. First derivative UV-Tm Plots of PNA2:DNA I complexes (A) for the (1S,2R)-PNA, (a) PNA 12, (b) PNA 13, (d) PNA
15 and (B) (1R,2S) modification, (a) PNA 12, (c) PNA 14, (e) PNA 16.
F IGURE 4. (A) CD spectra of PNA 13 (SS), PNA 14 (SS), and complexes (PNA 13)2:DNA I and (PNA 14)2:DNA I. (B) CD spectra
of PNA 15 (SS), PNA 16 (SS), and complexes (PNA 15)2:DNA I and (PNA 16)2:DNA I (10 mM sodium phosphate buffer, pH ) 7,
100 mM NaCl, 0.1 mM EDTA, 20 °C).
triplex.19 All PNA:DNA triplexes show the expected
positive bands at 258 and 285 nm, with intensity of the
former higher than that of latter. Interestingly the
relative intensity ratios of 258/285 nm bands are different
for the cyclohexyl PNA:DNA triplexes as compared to the
control PNA:DNA triplex. The 285 nm band increased
in intensity with the degree of modification, irrespective
of the nature of the modification (RS/SR). These results
suggest that the cyclohexyl modifications significantly
alter the base stacking in the modified PNA:DNA com-
plexes. The systematic changes in the CD spectral fea-
tures upon variable stoichiometric mixing of PNA and
DNA components can be used to generate a J ob plot,
which also indicated a binding ratio of 2:1 for the
cyclohexyl PNA:DNA complexes, confirming the forma-
tion of triplexes (Supporting Information).
Gel Sh ift Assa y a n d Com p etition Bin d in g Exp er i-
m en ts. Electrophoretic gel shift assay20 was used to
establish the binding of different PNAs to the comple-
mentary DNA I. The PNAs modified with one or two
units of SR/RS cyclohexyl units and the control PNA 12
were individually treated with oligonucleotide I (see
Figure 5 legend for experimental conditions), and the
complexations were monitored by nondenaturing gel
electrophoresis at 10 °C. The spots were visualized on a
fluorescent TLC background, and the results are shown
in Figure 5. The formation of PNA2:DNA complexes was
accompanied by the disappearance of the single strand
DNA I and appearance of a lower migrating band due to
PNA:DNA complexes. The single modified PNA2:DNA
complexes migrated about the same as the unmodified
PNA:DNA complex but lower than that of DNA I (Figure
5A, lanes 6-8), while the doubly modified PNA:DNA
complexes showed more retardation (Figure 5A, about
the same as the unmodified PNA:DNA complex but lower
than that of DNA I (Figure 5A, lanes 6-8), while the
doubly modified PNA:DNA complexes showed more
retardation (Figure 5A, lanes 1 and 2). The different
mobility retardation shifts of cyclohexyl PNA:DNA com-
plexes may arise from the increased molecular weight
upon formation of the complexes, a rigid geometry
imposed by the cyclohexyl ring on the derived complexes
or from an enhanced hydrophobicity due to cyclohexyl
rings. Under the electrophoretic conditions employed, the
single stranded PNAs that carry a net positive charge
did not move out of the well (Figure 5A, lanes 3 and 5).
(20) Sambrook, J .; Fritsch, E. F.; Maniatis, T. In Molecular Clon-
ing: A Laboratory Manual, 2; Cold Spring Harbor Laboratory Press:
Cold Spring Harbor, NY, 1989.
(19) Kim, S. K.; Nielsen, P. E.; Egholm, M.; Buchardt, O.; Berg, R.
H. J . Am. Chem. Soc. 1993, 115, 6477.
1862 J . Org. Chem., Vol. 69, No. 6, 2004

Synthesis of (1S,2R/ 1R,2S)-Aminocyclohexylglycyl PNAs
F IGURE 5. PNA/DNA complexation. (A) Gel shift assay: lane 1, PNA 15 + DNA I; lane 2, PNA 16 + DNA I; lane 3, ssPNA-12
(aeg-T10); lane 4, DNA I; lane 5, ssPNA 13; lane 6, PNA 13 + DNA I; lane 7, PNA 14 + DNA I; lane 8, PNA 12 (aeg-T10) + DNA
I. (B) Competition binding experiments: lane 1, DNA I + DNA III + PNA 15; lane 2, DNA I + DNA III + PNA 16; lane 3, ssDNA
I; lane 4, DNA duplex (I + III); lane 5, ssDNA III; lane 6, DNA I + DNA III + PNA 13; lane 3, ssDNA I; lane 4, DNA duplex (I
+ III); lane 5, ssDNA III; lane 6, DNA I + DNA III + PNA 13; lane 7, DNA I + DNA III + PNA 14; lane 8, DNA I + DNA III
+ PNA 12; DNA I ) CGCA10CGC (d A10), DNA III ) GCGT10GCG (dT10). 0.5X TBE pH 8.0.
The results of competition binding experiments carried
out by adding PNAs to the DNA duplex (I/III) followed
by annealing and gel electrophoresis is shown in Figure
5B. Due to a higher binding affinity of PNA:DNA com-
plexes compared with DNA:DNA complexes, the added
PNA binds to the complementary DNA I in the DNA
duplex, releasing the DNA strand III. This is clearly seen
in the gel electrophoresis in which the DNA duplex band
(Figure 5B, lane 4) is converted into a PNA:DNA complex
seen as a retarded band and the released DNA strand
(Figure 5B, lane 6-8) seen as a faster moving band. The
results indicate that the modified PNAs are as good as
the unmodified PNA in successfully competing for bind-
ing with the complementary DNA strand. The systematic
changes in the CD spectral features upon variable
stoichiometric mixing of PNA and DNA components can
be used to generate a J ob’s plot which also indicated a
binding ratio of 2:1 for the cyclohexyl PNA:DNA com-
plexes, confirming the formation of triplexes. An isobestic
point seen at 262 nm in the CD spectra (Supporting
Information) clearly confirmed the CD changes to arise
as a consequence of complex formation.
in mixed sequences that form PNA:DNA/RNA duplexes
and explore other ring structures to further improve the
binding affinity via entropic gains and impart efficient
DNA/RNA selectivity.
Exp er im en ta l Section
En zym a tic Resolu tion of Ra cem ic a lcoh ols (1R/S,2R/
S) 3 w ith Lip a se P . cepa cia . To a solution of lipase Amano-
PS (500 mg) in phosphate buffer (150 mL, pH ) 7.2) was added
racemic butyrate (1R/ S,2R/ S)-2 (10 g). The mixture was
stirred vigorously for 2.5 h, which accomplished 40% conver-
sion for alcohol (-) (1R,2R)-3a . The enzyme was separated by
filtration, and the filtrate was extracted into DCM. The solvent
was evaporated, and the column chromatographic separation
of the residue afforded chirally pure alcohol (1R,2R)-3a and
the optically enriched butyrate (1S,2S)-2. The later was again
subjected to enzyme hydrolysis as described to yield the
optically pure butyrate (1S,2S)-2 after purification. From this
the alcohol (1S,2S)-3b was obtained by methanolysis with
catalytic amount of NaOMe in MeOH in 30% overall yield. The
enantiomeric purity was confirmed by comparison with known
values of the optical rotations of alcohols (1R,2R)-3a and
(1S,2S)-3b from the literature [for alcohol (1R,2R)-3a : [R]20
D
) -69.33 (lit.8 [R]20 -66.9, c 1.5, CH2Cl2); alcohol (1S,2S)-
D
3b: [R]20 ) +68.7 (lit.8 [R]20 +66.3, c 1.6, CH2Cl2)].
D
D
Con clu sion
(1R,2R)-2-(N-ter t-Bu t yloxyca r b on yla m in o)cycloh ex-
a n ol [(1R,2R)-4]. To a solution of (1R,2R)-2-azidocyclohexanol
(5.6 g, 39.7 mmol) in dry ethyl acetate (10 mL) placed in a
hydrogenation flask were added di-tert-butyl dicarbonate (10.4
g, 47.6 mmol) and Adams catalyst (2 mol %). The mixture was
hydrogenated in Parr apparatus (rt, 35-40 psi 3 h). The
catalyst was filtered, solvent in the filtrate was evaporated
under reduced pressure, and the residue was purified by
column chromatography (EtOAc/petroleum ether) to afford a
white solid of the alcohol (1R,2R)-4: yield (7.9 g, 92.6%); mp
On the basis of a rational design, we have synthesized
the (1S,2R)- and (1R,2S)-aminocyclohexylglycyl PNA
monomers, in which the torsion angle â is restricted to
60-70° by virtue of a cis axial-equitorial disposition of
the backbone substutuents. The designed monomers were
built into PNA oligomers by solid-phase synthesis using
Boc protection strategy. The UV melting temperatures
of the modified oligomers with the complemetary/
mismatched DNA and RNA were measured. The imposed
stereochemistry of the PNA oligomer having the (1S,2R)
isomer preferred to bind RNA and the oligomer having
the (1R,2S) isomer showed higher affinity toward DNA
in homothymine oligomers, leading to a stereodiscrimi-
nation in recognition of DNA and RNA. The gel shift
experiments indicated that the modifications retain the
competitive binding attributes of PNA toward DNA
duplex. The preorganization of the PNA backbone by
chemical modifications such that the torsion angle â is
restricted in a range found by NMR and crystal structure
data may lead to the induction of substantial selectivity
(discrimination) in DNA/RNA recognition. On the basis
of this design, work is in progress to examine the effects
111 °C; [R]20 +5.0 (c 0.6, CH2Cl2); 1H NMR (CHCl3-d, 500
D
MHz) δH 1.04-1.34 (4H, m), 1.42 (9H, s), 1.61-1.73 (2H, m),
1.89-2.06 (2H, m) 3.2-3.36 (2H, m) 2.8-3.5 (1H, bd), 4.0-
5.0 (1H, bd); 13C NMR (CHCl3-d, 500 MHz) δC 23.9, 24.6, 28.3,
31.7, 34.0, 56.5, 75.2,79.9, 160.0; MS (FAB+) 216 [M + 1] (50),
116 (30) [M + 1 - Boc]. Anal. Calcd for C11H21NO3: C, 61.39;
H, 9.76; N, 6.51. Found: C, 61.17; H, 9.71; N, 6.33;
(1S,2S)-2-(N-ter t-b u t yloxyca r b on yla m in o)cycloh ex-
a n ol [(1S,2S)- 4]. This compound was prepared from the azido
alcohol (1S,2S)-3b using a procedure similar to the one
described for the alcohol (1R,2R)-4: mp 111 °C; [R]20 -6.6 (c
D
0.6, CH2Cl2); 1H NMR (CHCl3-d, 500 MHz) δH 1.04-1.34 (4H,
m), 1.42 (9H, s), 1.61-1.73 (2H, m), 1.89-2.06 (2H, m), 3.2-
3.36 (2H, m), 2.8-3.5 (1H, bd), 4.0-5.0 (1H, bd); 13C NMR
(CHCl3-d, 500 MHz) δC 23.9, 24.6, 28.3, 31.7, 34.0, 56.5, 75.2,
79.9, 60.0; MS (FAB+) 216 [M + 1] (50), 116 (30) [M + 1 -
J . Org. Chem, Vol. 69, No. 6, 2004 1863

Govindaraju et al.
Boc]. Anal. Calcd for C11H21NO3: C, 61.39; H, 9.76; N, 6.51.
Found C, 61.56; H, 10.01; N, 6.40.
(1R,2S)-2-(N-ter t-Bu tyloxyca r bon yla m in o)-1-a m in ocy-
cloh exa n e [(1R,2S)-7]. The amine (1R,2S)-7 was obtained on
hydrogenation of the azide (1R,2S)-6 following the same pro-
cedure as described for the synthesis of the amine (1S,2R)-7.
(1R,2R)-2-(N-ter t-Bu tyloxycar bon ylam in o)cycloh exan e-
1-m eth yl Su lfon a te [(1R,2R)-5]. To a stirred solution of
alcohol (1R,2R)-4 (6.0 g, 27.9 mmol) and DMAP (0.05 g) in dry
pyridine (100 mL) at 0 °C under nitrogen was added meth-
anesulfonyl chloride (4.16 g, 36.3 mmol) over a period of 20
min. The mixture was stirred for 1 h and then refrigerated
overnight. Pyridine was evaporated under reduced pressure,
and the residue was purified by column chromatography
(EtOAc/petroleum ether) affording mesylate (1R,2R)-5 as a
N-[(2R)-ter t-Bu tyloxyca r bon yla m in ocycloh ex-(1S)-yl]-
glycin e Eth yl Ester [(1S,2R)]-8. To a stirred mixture of
amine (1S,2R)-7 (3.4 g, 15.9 mmol) and freshly prepare
KF-Celite (5.52 g, 47.66 mmol) in dry acetonitrile (150 mL)
was added ethyl bromoacetate (2.38 g, 14.3 mmol) dropwise
for 30 min at rt under nitrogen atmosphere. After 3.5 h, the
Celite was filtered off and the solvent in the filtrate was
evaporated under reduced pressure which on column chro-
matographic purification (EtOAc) afforded the ethyl ester
white crystalline solid: yield (8.0 g, 97.9%); mp 121 °C; [R]20
D
-10.67 (c 1.5, CH2Cl2): 1H NMR (CHCl3-d, 500 MHz); δH 1.15-
1.34 (3H, m), 1.41 (9H, s), 1.52-1.86 (3H, m), 1.92-2.09 (1H,
m), 2.1-2.2 (1H, m), 3.0 (3H, s), 3.45-3.65 (1H, bd), 4.3-4.5
(1H, bd), 4.6-4.8 (1H, bd); 13C NMR (CHCl3-d, 500 MHz) δC
23.5, 23.7, 28.2, 31.6, 32.0, 38.3, 51.8, 79.3, 82.4, 155.2; MS
(FAB+) 294 [M + 1] (5), 194 (85) [M + 1 - Boc]. Anal. Calcd
for C12H23NO5S: C, 49.0; H, 7.80; N, 4.77; S, 10.92. Found: C,
49.30; H, 7.86; N, 4.50; S, 10.55.
(1S,2R)-8 as a colorless oil: yield (3.63 g, 76.6%); [R]20 -9.15
D
(c 1.42, CH2Cl2); 1H NMR (CHCl3-d, 200 MHz) δH 1.24 (3H, t),
1.41 (s), 1.6-1.85 (2H, m), 2.0-2.35 (2H, m), 2.35-2.7 (3H,
m), 2.75-3.52 (5H, m), 3.67 (1H, bd), 4.0-4.5 (2H, m), 4.9-
5.75 (1H, bd); 13C NMR (CHCl3-d, 200 MHz) δC 13.9, 28.1,
33.74, 37.7, 40.7, 49.1, 60.2, 61.3, 61.6, 69.2, 78.7, 156.3, 172.4;
MS (FAB+) 301 [M + 1] (100), 201 (12) [M + 1 - Boc].
(1S,2S)-2-(N-ter t-Bu tyloxyca r bon yla m in o)cycloh exa n -
1-m et h yl Su lfon a t e [(1S,2S)-5]. The compound (1S,2S)-5
was prepared from the alcohol (1S,2S)-4 using a procedure
N-[(2S)-ter t-Bu tyloxyca r bon yla m in ocycloh ex-(1R)-yl]-
glycin e Eth yl Ester [(1R,2S)]-8. A procedure similar to the
one described for the ethyl ester (1S,2R)-8 afforded (1R,2S)-8
similar to the one described for the mesylate (1R,2R)-5: mp
starting from the amine (1R,2S)-7: [R]20 +10.56 (c 1.42,
D
1
121 °C; [R]20 +13.33 (c 1.5, CH2Cl2); H NMR (CHCl3-d, 500
CH2Cl2); 1H NMR (CHCl3-d, 200 MHz) δH 1.24 (3H, t), 1.41
(s), 1.6-1.85 (2H, m), 2.0-2.35 (2H, m), 2.35-2.7 (3H, m),
2.75-3.52 (5H, m), 3.67 (1H, bd), 4.0-4.5 (2H, m), 4.9-5.75
(1H, bd); 13C NMR (CHCl3-d, 200 MHz) δC 13.9, 28.1, 33.7,
37.7, 40.7, 49.1, 60.2, 61.3, 61, 69.2, 78.7, 156.3, 172.4; MS
(FAB+) 301 [M + 1] (100), 201 (12) [M + 1 - Boc].
D
MHz) δH 1.15-1.34 (3H, m), 1.41 (9H, s), 1.52-1.86 (3H, m),
1.92-2.09 (1H, m), 2.1-2.2 (1H, m), 3.0 (3H, s), 3.45-3.65 (1H,
bd), 4.3-4.5 (1H, bd), 4.6-4.8 (1H, bd); 13C NMR (CHCl3-d,
500 MHz) δC 23.5, 23.7, 28.2, 31.6, 32.0, 38.3, 51.8, 79.3, 82.4,
155.2; MS (FAB+) 294 [M + 1] (5), 194 (85) [M + 1 - Boc].
Anal. Calcd for C12H23NO5S: C, 49.0; H, 7.80; N, 4.77; S, 10.92.
Found: C, 49.21; H, 7.81; N, 4.56; S, 10.67.
N-[(2R)-ter t-Bu tyloxyca r bon yla m in ocycloh ex-(1S)-yl]-
N-(ch lor oa cet yl)glycin e E t h yl E st er [(1S,2R)-9. To a
stirred solution of the amine (1R,2S)-8 (3.6 g, 12 mmol) in 10%
Na2CO3 (90 mL) and 1,4-dioxane (90 mL) cooled to 0 °C was
added chloroacetyl chloride (6.78 g, 60 mmol) in two additions.
After 30 min, the dioxane was removed under reduced pressure
and the residue was extracted into EtOAc (2 × 100 mL) and
dried over Na2SO4. The solvent was evaporated under reduced
pressure, and the residue was purified by column chromatog-
raphy (MeOH/ CH2Cl2) affording chloro compound (1S,2R)-9
as a white solid: yield (3.3 g, 73%); mp 65-68 °C; [R]20D -89.54
(c 2.2, CH2Cl2); 1H NMR (CHCl3-d, 200 MHz) δH 1.0-2.0 (20H,
m), 3.65-4.5 (8H, m), 4.7-5.2 (1H, bd); 13C NMR (CHCl3-d,
200 MHz) δC 13.9, 19.8, 23.7, 25.2, 28.2, 30.9, 41.4, 45.8, 49.0,
57.0, 61.0, 79.5, 155.5, 167.0, 169.0; MS LCMS 377 [M + 1],
277 [M + 1 - Boc].
(1S,2R)-2-(N-ter t-Bu tyloxyca r bon yla m in o)-1-a zid ocy-
cloh exa n e [(1S,2R)-6]. A stirred mixture of the mesylate
(1R,2R)-5 (8.0 g, 33.33 mmol) and NaN3 (17.3 g, 0.26 mol) in
DMF (80 mL) under nitrogen was heated at 65-70 °C for 12
h. After cooling, the solvent was evaporated under reduced
pressure and the residue was purified by column chromatog-
raphy (EtOAc/petroleum ether) to afford a white solid of azide
(1S,2R)-6: yield (5.7 g, 87%); mp 69-70 °C; IR, ν (cm-1) (KBr)
3359, 2954, 2104, 1720, 1681, 1367, 1319, 1166 cm-1; [R]20
D
1
+102 (c 1.5, CH2Cl2); H NMR (CHCl3-d, 500 MHz) δH 1.21-
1.35 (1H, m), 1.35-1.51 (12H), 1.52-1.64 (2H, m), 1.65-1.77
(1H, m), 1.89-2.01 (1H, m), 3.5-3.65 (1H, bd), 3.8-4.0 (1H,
bd), 4.56-4.8 (1H, bd); 13C NMR (CHCl3-d, 500 MHz) δC 19.6,
24.2, 27.5, 28.2, 28.6, 51.0, 61.5, 79.4, 154.9; MS (FAB+) 241
(35), [M + 1], 141 (15) [M + 1 - Boc]. Anal. Calcd for
N-[(2S)-ter t-Bu t yloxyca r b on yla m in oa m in ocycloh ex-
(1R)-yl]-N-(ch lor oa cetyl)glycin e Eth yl Ester [(1R,2S)-9.
This compound was prepared from the amine (1R,2S)-8
following the procedure described for the chloro compound
C
11H20N4O2: C, 55.00; H, 8.33; N, 23.33. Found: C, 55.18; H,
8.00; N, 23.14.
(1R,2S)-2-(N-ter t-Bu tyloxyca r bon yla m in o)-1-a zid ocy-
cloh exa n e [(1R,2S)-6]. A procedure similar to the one
described for (1S,2R)-6 was used to prepare (1R,2S)-6 from
(1S,2S)-5: mp 69-70 °C; IR, ν (cm-1) (KBr) 3359, 2954, 2104,
(1S,2R)-9: mp 65-68 °C; [R]20 + 91.36 (c 2.2, CH2Cl2); 1H
D
NMR (CHCl3-d, 200 MHz) δH 1.0-2.0 (20H, m), 3.65-4.5
(8H, m), 4.7-5.2 (1H, bd); 13C NMR (CHCl3-d, 200 MHz) δC
13.9, 19.8, 23.7, 25.2, 28.2, 30.9, 41.4, 45.8, 49.0, 57.0, 61.0,
79.5, 155.5, 167.0, 169.0; MS LCMS 377 [M + 1], 277 [M + 1
- tBoc].
1720, 1681, 1367, 1319, 1166 cm-1; [R]20 -105.33 (c 1.5,
D
CH2Cl2); 1H NMR (CHCl3-d, 500 MHz) δH 1.21-1.35 (1H,
m), 1.35-1.51 (12H), 1.52-1.64 (2H, m), 1.65-1.77 (1H, m),
1.89-2.01 (1H, m), 3.5-3.65 (1H, bd), 3.8-4.0 (1H, bd),
4.56-4.8 (1H, bd); 13C NMR (CHCl3-d, 500 MHz) δC 19.6,
24.2, 27.5, 28.2, 28.6, 51.0, 61.5, 79.4, 154.9; MS (FAB+) 241
(35) [M + 1], 141 (15) [M + 1 - Boc]. Anal. Calcd for
N-[(2R)-ter t-Bu tyloxyca r bon yla m in ocycloh ex-(1S)-yl]-
N-(th ym in -1-a cetyl)glycin e Eth yl Ester [(1S,2R)-10a ]. A
mixture of chloro compound (1S,2R)-9 (1.5 g, 4.0 mmol),
thymine (0.55 g, 4.38 mmol), and anhydrous K2CO3 (0.66 g,
4.8 mmol) in dry DMF (10 mL) under nitrogen was heated
with stirring at 65 °C for 3.5 h. After cooling, the solvent was
removed under reduced pressure to leave a residue, which was
extracted into DCM (2 × 25 mL) and dried over Na2SO4. The
solvent was evaporated, and the crude compound was puri-
fied by column chromatography (MeOH/DCM) to afford a
white solid of thymine monomer ethyl ester (1S,2R)-10a : yield
C
11H20N4O2: C, 55.00; H, 8.33; N, 23.33. Found: C, 55.11; H,
8.07; N, 23.21.
(1S,2R)-2-(N-ter t-Bu tyloxyca r bon yla m in o)-1-a m in ocy-
cloh exa n e [(1S,2R)-7]. To a solution of the azide (1S,2R)-6
(4.0 g, 16.7 mmol) in methanol (5 mL) taken in a hydrogenation
flask was added Adam’s catalyst (2 mol %). The reaction
mixture was hydrogenated in a Parr apparatus for 3 h at rt
and H2 of pressure 35-40 psi. The catalyst was filtered off,
and then solvent was removed under reduced pressure to yield
a residue of the amine (1S,2R)-7 as a colorless oil, yield (3.45
g, 96.9%). This compound was used for the further reaction
without any purification.
(1.3 g, 70.2%); mp 115-119 °C; [R]20 -85.33 (c 1.5, CH2Cl2);
D
1H NMR (CHCl3-d, 500 MHz) δH 1.17-1.8 (17H), 1.82 (3H,
s), 3.5-5.0 (8H, m), 5.0-5.5 (1H, bd), 7.1 (1H, s), 8.3-8.5
(1H, bd); 13C NMR (CHCl3-d, 500 MHz) δC 12.0, 14.1, 19.8,
23.5, 25.0, 28.1, 30.1, 36.6, 45.3, 47.5, 55.6, 61.0,79.4, 111.0,
1864 J . Org. Chem., Vol. 69, No. 6, 2004

Synthesis of (1S,2R/ 1R,2S)-Aminocyclohexylglycyl PNAs
141.1, 151.0, 155.5, 164.4, 167.4, 169.3; MS (FAB+) 467
characterized by MALDI-TOF mass spectrometry. The overall
yields of the raw products were 35-65%. The normal PNAs
were prepared as described in the literature.
[M + 1] (10), 367 (100) [M + 1 - tBoc]. Anal. Calcd for
C
22H34N4O7: C, 56.65; H, 7.29; N, 12.01. Found: C, 56.81; H,
7.31; N, 11.97.
MALDI-TOF: PNA 12, calcd for C116H154N42O42 2806.0,
N-[(2S)-ter t-Bu tyloxyca r bon yla m in ocycloh ex-(1R)-yl]-
N-(th ym in -1-a cetyl)glycin e Eth yl Ester [(1R,2S)-10b]. A
procedure similar to the one described for the monomer
found 2808.0 [M + 2H]+; PNA 13, calcd for C120H160N42O42
2860.0, found 2862.0 [M + 2H]+; PNA 14, calcd for C120H160
-
N
42O42 2860.0, found 2861.0 [M + H]+; PNA 15, calcd for
(1S,2R)-10a afforded (1R,2S)-10b from (1R,2S)-9: mp 115-
C124H166N42O42 2915.0; found 2917.0 [M + 2H]+; PNA 16, calcd
119 °C; [R]20 +86.67 (c 1.5, CH2Cl2); H NMR (CHCl3-d, 500
for C124H166N42O42 2915.0, found 2916.0 [M + H]+.
1
D
MHz) δH 1.17-1.8 (17H), 1.82 (3H, s), 3.5-5.0 (8H, m), 5.0-
5.5 (1H, bd), 7.1 (1H, s), 8.5-8.5 (1H, bd); 13C NMR (CHCl3-d,
500 MHz) δC 12.0, 14.1, 19.8, 23.5, 25.0, 28.1, 30.1, 36.6, 45.3,
47.5, 55.6, 61.0,79.4, 111.0, 141.1, 151.0, 155.5, 164.4, 167.4,
169.3; MS (FAB+) 467 [M + 1] (10), 367 (100) [M + 1 - tBoc].
Anal. Calcd for C22H34N4O7: C, 56.60; H, 7.22; N, 12.23.
Found: C, 56.81; H, 7.31; N, 11.97.
Tm Mea su r em en ts. The concentration was calculated on
the basis of absorbance from the molar extinction coefficients
of the corresponding nucleobases. The complexes were pre-
pared in 10 mM sodium phosphate buffer, pH 7.0 containing
NaCl (100 mM), and EDTA (0.1 mM) and were annealed
by keeping the samples at 85 °C for 5 min followed by slow
cooling to room temperature. Absorbance versus tempera-
ture profiles were obtained by monitoring at 260 nm with a
UV-VIS spectrophotometer scanning from 5 to 85 °C at a
ramp rate of 0.2 °C per minute. The data were processed using
Microcal Origin 5.0 and Tm values derived from the derivative
curves.
Gel Mobility Sh ift Assa y. The PNAs (12-16, Table 1)
were individually mixed with DNA (I or III) in 2:1 ratio (PNA
strand, 0.4 mM and DNA I or III, 0.2 mM) in water. The
samples were lyophilized to dryness and re-suspended in
sodium phosphate buffer (10 mM, pH 7.0, 10 µL) containing
EDTA (0.1 mM). The samples were annealed by heating to 85
°C for 5 min followed by slow cooling to rt and refrigeration
at 4 °C overnight. To this, 10 µL of 40% sucrose in TBE buffer
pH 8.0 was added and the sample was loaded on the gel.
Bromophenol blue (BPB) was used as the tracer dye separately
in an adjacent well. Gel electrophoresis was performed on a
15% nondenaturing polyacrylamide gel (acrylamide/bis-acry-
lamide, 29:1) at constant power supply of 200 V and 10 mA,
until the BPB migrated to three-fourth of the gel length.
During electrophoresis the temperature was maintained at 10
°C. The spots were visualized through UV shadowing by
illuminating the gel placed on a fluorescent silica gel plate,
F254 using UV light.
N-[(2R)-ter t-Bu tyloxyca r bon yla m in ocycloh ex-(1S)-yl]-
N-(th ym in -1-a cetyl)glycin e [(1S,2R)-11a ]. To the monomer
ester (1S,2R)-10a (1.0 g, 2.283 mmol) suspended in THF (10
mL) was added a solution of 0.5 M LiOH (10 mL, 5.1 mmol),
and the mixture was stirred for 30 min. The mixture was
washed with EtOAc (2 × 10 mL). The aqueous layer was
acidified to pH 3 and extracted with EtOAc (3 × 50 mL). The
EtOAc layer was dried over sodium sulfate and evaporated
under reduced pressure to afford monomer (1S,2R)-11a as a
white solid: yield 0.92 g (97.8%); mp 172-177 °C; [R]20D -107
(c 1.06, CH2Cl2); 1H NMR (CHCl3-d, 200 MHz) δH 0.5-1.8
(17H, m, tBoc, 4CH2), 1.88 (3H, s, thymine CH3), 3.3-5.5 (7H,
m, N-CH2, acyl CH2, 1-CH, 2-CH, carbamate NH), 7.18 (1H,
s, thymine-H), 10.0-11.0 (2H, bd, thymine NH and -COOH);
13C NMR (DMSO-d6, 200 MHz) δC 12.2, 19.7, 23.1, 25.5, 28.6,
31.0, 47.7, 55.7, 78.1, 108.5, 142.3, 151.4, 156.1, 164.7, 167.7,
174.0. MS (FAB+) 439 [M + 1] (6), 339 (100) [M + 1 - tBoc].
Anal. Calcd for C20H30N4O7: C, 54.78; H, 6.84; N, 12.77.
Found: C, 54.61; H, 7.01; N, 12.61.
N-[(2S)-ter t-Bu tyloxyca r bon yla m in ocycloh ex-(1R)-yl]-
N-(th ym in -1-a cetyl)glycin e [(1R,2S)-11b]. To the monomer
ester (1R,2S)-10b (1.0 g, 2.283 mmol) suspended in THF (10
mL) was added a solution of 0.5 M LiOH (10 mL, 5.1 mmol),
and the mixture was stirred for 30 min. The mixture was
washed with EtOAc (2 × 10 mL). The aqueous layer was
acidified to pH 3 and extracted with EtOAc (3 × 50 mL). The
EtOAc layer was dried over sodium sulfate and evaporated
under reduced pressure to afford monomer (1R,2S)-11a as a
white solid: Yield 0.92 g (97.8%); mp 172-177 °C; [R]20D +110
(c 1.06, CH2Cl2); 1H NMR (CHCl3-d, 200 MHz) δH 0.5-1.8
(17H, m, t-Boc, 4CH2), 1.88 (3H, s, thymine CH3), 3.3-5.5 (7H,
m, N-CH2, acyl CH2, 1-CH, 2-CH, carbamate NH), 7.18 (1H,
s, thymine-H), 10.0-11.0 (2H, bd, thymine NH and -COOH);
13C NMR (DMSO-d6, 200 MHz) δC 12.2, 19.7, 23.1, 25.5, 28.6,
31.0, 47.7, 55.7, 78.1, 108.5, 142.3, 151.4, 156.1, 164.7, 167.7,
174.0; MS (FAB+) 439 [M + 1] (6), 339 (100) [M + 1 - Boc].
Anal. Calcd for C20H30N4O7: C, 54.78; H, 6.84; N, 12.77.
Found: C, 54.67; H, 7.13; N, 12.8.
Ack n ow led gm en t. T.G. thanks CSIR, New Delhi,
for the award of a research fellowship. V.A.K. thanks
Department of Science and Technology, New Delhi, for
a research grant. K.N.G. is an honorary professor at
J awaharlal Nehru Centre for Advanced Scientific re-
search, Bangalore.
Su p p or tin g In for m a tion Ava ila ble: General experimen-
tal procedures; 1H NMR, 13C NMR, and mass spectra of
compounds 4-6 and 8-11. Enantiopurity data for alcohols 3a
and 3b. HPLC profiles, mass spectra (MALDI-TOF), and
characterization data table for PNA 12, PNA 13, PNA 14, PNA
15, and PNA 16. CD spectra of (a) PNA 12, (b) DNA I, and (c)
complex PNA 12: DNA I and CD J ob’s plot. Melting curves of
PNA 12, PNA 13, PNA 14, PNA 15, PNA 16 with d(A10),
d[A5CA4], poly dA, and poly rA, and their corresponding
derivatives. X-ray cryatal structures of (a) monomer esters
10a , 10b, and cyanuryl PNA monomer and (b) compounds 5a ,b
and 6a . This material is available free of charge via the
Internet at http://pubs.acs.org.
Syn th esis of P NA-Oligom er s In cor por atin g cis-(1S,2R)-
a n d (1R,2S)-Am in ocycloh exyl P NA Mon om er s. The modi-
fied PNA monomers were built into PNA oligomers using
standard procedures on an L-lysine-derivatized (4-methylben-
zhydryl)amine (MBHA) resin (initial loading 0.25 mequiv g-1
)
with HBTU/HOBt/DIEA in DMF/DMSO as a coupling reagent.
The PNA oligomers were cleaved from the resin with TFMSA.
The oligomers were purified by RP HPLC (C18 column) and
J O035747X
J . Org. Chem, Vol. 69, No. 6, 2004 1865