trans-5-aminopipecolyl-aegPNA chimera: Design, synthesis, and study of binding preferences with DNA/RNA in duplex/triplex mode

Article abstract of DOI:10.1021/jo0506884

The design and synthesis of novel chiral PNA monomer based on trans-5-aminopipecolic acid is reported. The trans diequatorial disposition of the 1,4 ring substituents in six-membered 5-aminopipecolic acid derivative could be favorable over trans 1,3 axial

Full text of DOI:10.1021/jo0506884

trans-5-Aminopipecolyl-aegPNA Chimera:
Design, Synthesis, and Study of Binding
Preferences with DNA/RNA in Duplex/
Triplex Mode
Pallavi S. Lonkar and Vaijayanti A. Kumar*
Division of Organic Chemistry (Synthesis), National
Chemical Laboratory, Pune 411008, India
FIGURE 1. aegPNA and its constrained analogues.
va.kumar@ncl.res.in
Received April 7, 2005
was thus synthesized by inserting a methylene group in
a 2-aminoethyl segment of I (Figure 1). It was found from
UV-Tm studies that the PNA:DNA duplexes or triplexes
incorporating a single apgPNA monomer were less stable
compared to unmodified aegPNA. This decrease in stabil-
ity for the chimeric apg-aegPNA was ascribed to the
increase in the degree of conformational freedom in the
backbone and consequent large entropy loss during
complex formation. Conversely, a favorable structural
preorganization of PNA may lead to the desired selective
complex formation with the target DNA or RNA, provided
the enthalpic contributions are conserved. The structural
preorganization approach using additional conforma-
tional constraint has been especially successful in the
case of DNA analogues such as locked nucleic acids
(LNAs),6 conformationally frozen hexitol,7 cyclohexene,8
and altritol nucleic acids.9 The stability is conferred
because of the RNA-like structures either locked or frozen
in 2′-endo sugar ring conformations. As the structural
preferences of PNA in PNA/DNA or PNA/RNA complexes
are not yet as well deciphered as those of DNA-DNA/
RNA complexes,10 there is intense activity in this field
to obtain an optimal PNA analogue. Our quest to find
correct conformational and configurational preferences
for optimal DNA/RNA recognition has previously resulted
in very interesting structural modifications11 of PNA that
include uncharged 4-aminoprolyl (Figure 1, III) and
4-aminopipecolyl (Figure 1, IV) chiral PNA derivatives.
These involved introduction of a methylene bridge be-
tween the R′ carbon atom of the glycyl moiety and the
â/γ carbon atom of the aminoethyl/aminopropyl segment
of the parent aeg/apgPNA to generate five-/six-mem-
bered pyrrolidine/piperidine rings. The comparative UV-
Tm studies after incorporation of these two modifications
The design and synthesis of novel chiral PNA monomer
based on trans-5-aminopipecolic acid is reported. The trans
diequatorial disposition of the 1,4 ring substituents in six-
membered 5-aminopipecolic acid derivative could be favor-
able over trans 1,3 axial-equatorial disposition in 4-amino-
pipecolic acid of PNA. Studies on the synthesis of trans-4/
5-aminopipecolyl PNA-eagPNA chimeras and their binding
preferences to DNA/RNA in duplex/triplex modes are de-
scribed.
Peptide nucleic acids, PNAs, are nucleic acid mimics
with a pseudopeptide backbone composed of N-(2-ami-
noethyl)glycine (aeg) units (Figure 1, I) to which the
nucleobases are attached through methylenecarbonyl
linkers.1,2 The unique open-chain chemical structure of
aegPNA has much potential for refinement considering
several available modes for providing charge, chirality,
and structural preorganization via chemical bridges
between aminoethyl, glycyl, and the nucleobase linker
segments of the monomer unit. Most efforts in this
direction have been aimed at furthering the potential of
PNAs as therapeutic agents with additional requisite
properties such as improved water solubility, cellular
uptake,3 and discrimination between parallel (p) and
antiparallel (ap) binding modes.4 In an earlier study, to
understand the structural obligations of aegPNA, struc-
tural variations were realized by extending the backbone
in either 2-aminoethyl, glycyl, or linker segments of
aegPNA.5 Aminopropylglycyl PNA apgPNA(Figure 1, II)
(5) Hyrup, B.; Egholm, M.; Nielsen, P. E.; Wittung, P.; Norden, B.;
Buchardt, O. J. Am. Chem. Soc. 1994, 116, 7964-7970.
(6) Petersen, M.; Wengel, J. Trends Biotechnol. 2003, 21, 74-81.
(7) Lescrinier, E.; Esnouf, R.; Schraml, J.; Busson, R.; Heus, H. A.;
Hilbers, C. W.; Herdewijn, P. Chem. Biol. 2000, 7, 719-731.
(8) Wang, J.; Verbeure, B.; Luyten, I.; Lescrinier, E.; Froeyen, M.;
Hendrix, C.; Rosemeyer, H.; Seela, F.; Aerschot, A. V.; Herdewijn, P.
J. Am. Chem. Soc. 2000, 122, 8595-8602.
(9) Kozlov, I. A.; Zielinski, M.; Allart, B.; Kerremans, L.; Aerschot,
A. V.; Busson, R.; Herdewijn, P.; Orgel, L. E. Chem. Eur. J. 2000, 6,
151-155.
(10) (a) Betts, l.; Josey, J. A.; Veal, J. M.; Jorden, 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) Kumar, V. A. Eur. J. Org. Chem. 2002, 2021-2032. (b)
Govindaraju, T.; Kumar V. A.; Ganesh, K. N. Chem. Commun. 2004,
860-861. (c) Shirude, P. S.; Kumar V. A.; Ganesh, K. N. Terahedron
Lett. 2004, 45, 3085-3088. (d) Govindaraju, T.; Gonnade, R. G.;
Bhadbhade, M. M.; Kumar V. A.; Ganesh, K. N. Org. Lett. 2003, 5,
3013-3016. (e) Kumar V. A.; Ganesh, K. N. Acc. Chem. Res. 2005, 38,
404-412.
(1) Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. Science
1991, 254, 1497-1501.
(2) 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.
(3) Koppelhus, U.; Nielsen, P. E. Adv. Drug Delivery Rev. 2003, 55,
267-280.
(4) Uhlmann, E.; Peyman, A.; Breipohl, G.; Will, W. D. Angew.
Chem., Int. Ed. 1998, 37, 2796-2823.
10.1021/jo0506884 CCC: $30.25 © 2005 American Chemical Society
Published on Web 07/27/2005
6956
J. Org. Chem. 2005, 70, 6956-6959

SCHEME 1a
in a triplex forming aegPNAT10 sequence showed that
both (2S,4R) five-12 and (2S,4S) six-membered13 trans
ring conformations caused detrimental effects on the
stability of PNA2:DNA and thus were incapable of adopt-
ing the structural bias required for PNA2:DNA triplex
formation.
The primary knowledge of the stability of trans con-
formations in six-membered rings suggested that a trans-
5-aminopipecolyl monomer (Figure 1,V) could be a better
choice because of the stable, favored diequatorial disposi-
tion of the backbone. In comparison, the trans axial-
equatorial disposition of substituents in either III or IV
(Figure 1) may not be favored. The rationally evolved
structure V arises from the insertion of an ethylene bridge
between R′ carbon atom of the glycyl moiety and the â
carbon atom of the aminoethyl segment of aegPNA I. Our
recent studies on the conformationally frozen six-
membered PNA analogue, a positively charged chimeric
piperidine-aegPNAT8, favored sequence-specific triplex
formation with target oligomers.14
In this note, we present our results on the synthesis
and hybridization properties of the designed trans-
(2S,5R)-5-aminopipecolyl thyminyl PNA monomer, its
incorporation into triplex- and duplex-forming oligomers,
and their DNA and RNA binding studies in comparison
with the trans-4-aminopipecolyl PNA analogue reported
earlier.13
Synthesis of 5-Aminopipecolyl PNA Monomer.
The chiral pipecolyl PNA thymine monomer trans-(5R)-
(N-Boc-amino)-N-(thymin-1-ylacetyl)-(2S)-pipecolic acid
7 was synthesized from cis-N-benzyloxycarbonyl-(5S)-
hydroxy-(2S)-pipecolic acid methyl ester 1, which in turn
was synthesized from L-glutamic acid15 (Scheme 1). The
5-hydroxy group was converted to mesylate 2 by reaction
with MsCl in dry pyridine followed by SN2 displacement
of (5S)-O-mesyl group by treatment with sodium azide
to yield (5R)-azide 3. The azide group was selectively
reduced to the 5-amino derivative by hydrogenation in
the presence of Ra/Ni as a catalyst. The resulting free
amine was protected in situ as Boc derivative 4. Depro-
tection of the ring nitrogen by hydrogenation over 10%
Pd-C and subsequent acylation with chloroacetyl chlo-
ride gave compound 5. N-1 alkylation of thymine was
effected by reaction with 5 in the presence of K2CO3 in
DMF to give N-1 thyminylmethylcarbonyl derivative 6.
The methyl ester in 6 was hydrolyzed using sodium
hydroxide in aqueous methanol to give free acid 7, the
required monomer block for building the oligomers. The
structural integrity of the PNA monomer was confirmed
by NMR and mass spectroscopic analysis.
a Reagents: (i) MsCl, Py, 70%. (ii) NaN3, DMF, 90%. (iii) H2,
Ra/Ni, (Boc)2O 90%. (iv) (a) Pd-C/H2; (b) ClCH2COCl 60%. (v)
Thymine, K2CO3, DMF, 73%. (vi) 1 M NaOH, H2O-MeOH, 90%.
duction of conformationally constrained modified units
at predefined positions was therefore undertaken to study
their effect on the conformational organization of PNA
that could determine the binding preferences. Introduc-
tion of the chiral unit at C/N-termini in sequences 8-11
could preferentially induce helicity in PNA oligomers.17
The propagation of such induced structural bias may be
enhanced by the introduction of a second chiral unit as
in sequences 12 and 13.18 Introduction of the chiral units
at adjacent positions or synthesis of homogeneous back-
bone-modified PNAs was not realized, as the rigidity of
the backbone in such PNAs reported earlier did not allow
binding to the complementary nucleic acids.12,19 The
mixed base oligomers 17-19 were synthesized to study
the effect of modification in duplex binding mode. The
synthesis of PNA oligomers comprising standard aegPNA
monomers, 4-aminopipecolyl PNA monomer,13 and 7 was
carried out using standard solid-phase synthesis proto-
cols.20 The oligomers after the synthesis were cleaved
from the support using trifluoroacetic acid-trifluo-
romethanesulfonic acid21 to yield the PNA oligomers
carrying â-alanine at their carboxy termini (Table 1 and
Table 2). The purity of oligomers was rechecked by RP-
HPLC on a C18 column and characterized by MALDI-
TOF mass spectrometry (Supporting Information). The
PNA sequences used here are either homopyrimidine
sequences 8-14 that are known to form PNA2:DNA
triplexes or duplex forming mixed purine-pyrimidine
sequences (17-19). A Job plot22 confirmed a 2:1 stoichi-
ometry of binding between the PNA strand 13 and DNA
15 (Supporting Information). All DNA/RNA binding
studies were performed with 1:2 stoichiometry of DNA
and homothyminyl pipecolyl-aegPNA. Mixed base se-
quence complex formation studies were performed using
1:1 PNA:DNA/RNA stoichiometry.
Synthesis of aegPNA and Pipecolyl-aegPNA Chi-
mera. The aegPNA oligomers are known to bind DNA/
RNA sequences with high specificity and affinity.1,2
Incorporation of modified chiral PNA monomer units
derived from chiral amino acids in the aegPNA backbone
seem to induce conformational preferences that assist the
binding of such chimeric PNAs to DNA/RNA.11,16 Intro-
(16) (a) Puschl, A.; Boesen, T.; Tedeschi, T.; Dahl, O.; Nielsen, P. E.
J. Chem. Soc., Perkin Trans. 1 2001, 2757-2763. (b) Jordan, S.;
Schwemler, C.; Kosch, W.; Kretschmer, A.; Stropp, U.; Schwenner, E.;
Mielke, B. Bioorg. Med. Chem. Lett. 1997, 7, 687-692. (c) Lagriffoule,
P.; Wittung, P.; Eriksson, M.; Jensen, K. K.; Norden, B.; Buchardt,
O.; Nielsen, P. E. Chem. Eur. J. 1997, 3, 912-919.
(17) Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. Biocon-
jugate Chem. 1994, 5, 3-7.
(18) Shirude, P. S.; Kumar, V. A.; Ganesh, K. N. Tetrahedron 2004,
60, 9485-9491.
(19) Lowe,G.;Vilaivan,T.J.Chem.Soc.,PerkinTrans.11997,547-554.
(20) Nielsen, P. E.; Egholm, M. Peptide Nucleic Acids: Protocols and
Applications; Horizon Scientific Press: Norfolk, England, 1999.
(21) Fields, G. B.; Fields, C. G. J. Am. Chem. Soc. 1991, 113, 4202-
4207.
(22) (a) Job, P. Ann. Chim. 1928, 9, 113-203. (b) Cantor, C. R.;
Schimmel, P. R. Biophys. Chem. 1980, Part III, 624pp.
(12) Gangamani, B. P.; Kumar, V. A.; Ganesh, K. N. Tetrahedron
1999, 57, 177-192
(13) Lonkar, P. S.; Kumar, V. A.; Ganesh, K. N. Nucleosides
Nucleotides and Nucleic Acids 2001, 20, 1197-1200.
(14) Lonkar, P. S.; Kumar, V. A. Bioorg. Med. Chem. Lett. 2004, 14,
2147-2149.
(15) Adams, D, R.; Bailey, P. D.; Collier, I. D.; Heffernan, J. D.;
Stokes, S. Chem. Commun. 1996, 349-350.
J. Org. Chem, Vol. 70, No. 17, 2005 6957

TABLE 1. UV-Tm Measurements of PNA2:DNA
Triplexesa
PNA sequences
Tm (°C)
H-(T)9 t-â-ala-OH
H-t (T)9-â-ala-OH
H-(T)3t (T)5 tâ-ala-OH
H-(T)10-â-ala-OH
(2S,4S)13
(2S,5R) 9
(2S,4S) 10
(2S,5R) 11
(2S,4S) 12
(2S,5R) 13
14
8
55.9
61
56
68
34
78
67
a Buffer: 10 mM sodium cacodylate, 100 mM NaCl, 0.01 mM
EDTA, pH 7.3. Melting experiments were performed with a
PNA:DNA strand concentration of 1 µM each. DNA-15 5′-
GCAAAAAAAAAACG-3′ complementary to PNA 8-14. DNA-16 5′-
GCAAAATAAAAACG-3′ T represents aegPNA units and t 4/5-
aminopipecolyl T ) mismatched base in DNA The values reported
here are the average of three independent experiments and are
accurate to (0.5 °C.
FIGURE 3. UV-melting profile of (A) PNA:DNA (a) 19:20,
(b) 17:20, (c) 19:21, (d) 17:21, (e) 18:21, (f) 18:20 and (B) PNA:
RNA (g) 19:22, (h) 18:22, (i) 17:22.
in the oligomer were detrimental to the PNA2-DNA
complex stability.
TABLE 2. UV-Tm (°C) Studies of PNA:DNA/RNA
Duplexesa
PNA oligomers 13 and 12 with DNA 16 having a single
mismatch at the site complementary to the pipecolyl unit
caused a decrease in the Tm values of the corresponding
triplexes as compared to the fully complementary triplex
by 9 and 4 °C, respectively. In comparison, the control
PNA2 14:DNA 16 showed a linear increase in absorbance
with increasing temperature with no sigmoidal transi-
tion.
DNA
RNA
PNA sequences
ap 20 p 21 ap 22
H-GTA GATCACT-â-ala-OH 17
47
48
43
46
37
40
43
38
32
H-GTAGAt (2S,4S)CACT-â-ala-OH 18
H-GTAGAt (2S,5R)CACT-â-ala-OH 19
a T, C, G, and A represent aegPNA unit and t represents
pipecolyl PNA units, DNA (ap) 20 5′-AGT GAT CTA C-3′, DNA
(p) 21 5′-CAT CTA GTG A-3′, RNA (ap) 22 5′- AGT GAT CTA C-3′.
Pipecolyl-aegPNA:DNA/RNA Duplexes. It was con-
sidered necessary to examine the effect of the pipecolyl
PNA backbone chirality on the binding orientation in
duplexes with mixed purine/pyrimidine sequences. Re-
cently, there is also considerable interest in designing
compounds that would show bias for binding either to
DNA or RNA.11,23 Thus, the 4/5-aminopipecolyl PNA units
were introduced in the mixed purine-pyrimidine se-
quence (17-19). All the pipecolyl-aegPNA oligomers
formed more stable duplexes in the ap mode with
complementary DNA. This trend is also observed with
aegPNAs tethered with lysine2 at the C-terminus. A
single (2S,4S) pipecolyl unit destabilized the parallel
duplex by 11 °C, whereas the antiparallel duplex is
marginally stabilized (∆Tm ) +1 °C). Destabilization was
observed for the complexes having 5-aminopipecolyl unit
in the p mode as well as the ap mode (Table 2, Figure 3
A). Both 4-aminopipecolyl (2S,4S) and 5-aminopipecolyl
(2S,5R) modified oligomers 18 and 19 hybridized with
RNA 22 (Figure 3 B) but with a decrease in Tm compared
to control PNA (∆Tm ) -5 and -11 °C). Thus, trans-4-
aminopipecolyl PNA monomer IV that destabilized PNA:
RNA and PNA2:DNA triplexes effected significant p/ap
orientation selectivity for PNA:DNA duplex formation.
The 1,4 trans diequatorial disposition of the substitu-
ents in the monomeric form of the trans-5-amino pipecolyl
derivative could additively effect the conformational bias
that stabilized triplex with PNA 13. The 1,3 trans axial-
equatorial substitution in the 4-amino pipecolyl deriva-
tive resulted in additive destabilization of the triplex with
PNA 12. The duplex formation with either DNA or RNA
suffered a set back by introduction of a single modified
FIGURE 2. UV-melting profile of PNA2:DNA complexes (a)
9:15, (b) 11:15, (c) 13:15, (d) 14:15.
Pipecolyl-aegPNA2:DNA Triplexes. The normalized
absorbance versus temperature plots derived from the
UV melting data indicated a single transition indicative
of the simultaneous dissociation of the two PNA strands
from the DNA in the DNA:PNA2 complex. The results of
UV-Tm studies are summarized in Table 1 and shown
in Figure 2. The C-terminal 5-aminopipecolyl (2S,5R)
unit in PNA 9 as well as the 4-aminopipecolyl (2S,4S)
unit in PNA 8 destabilized the resulting complex with
DNA (∆Tm ) -6 and -12 °C, respectively) compared to
the unmodified aegPNA 14. The presence of aminopipe-
colyl units at the N-terminal (2S,4S) unit caused a
destabilization of the complex (∆Tm ) -11 °C, PNA 10),
while the (2S,5R) unit had minor effect in altering the
stability of its complex with DNA (∆Tm ) +1 °C PNA
11). Compared to unmodified aegPNA, increasing the
number of aminopipecolyl units to two led to favorable
complex formation (∆Tm ) +11 °C) for the (2S,5R) unit
(PNA 13) but highly unfavored complex formation (∆Tm
) -33 °C) for the (2S,4S) unit (PNA 12). Thus, the
(2S,5R) unit seems to be quite efficient in binding to
complementary DNA in triplex mode. In comparison,
both 4-amino pipecolyl and 4-aminoprolyl PNA units 13
(23) (a) Slaitas, A.; Yeheskiely, E. Eur. J. Org Chem. 2002, 2391-
2399. (b) 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, 5, 2695-2698. (c) Govindaraju T.; Kumar, V. A.; Ganesh, K. N.
J. Org. Chem. 2004, 69, 1858-1865. (d) Samuel, T. H.; David, T.;
Hickman, T.; Morral, J.; Beadham, G.; Micklefield, J. Chem. Comm.
2004, 516-517.
6958 J. Org. Chem., Vol. 70, No. 17, 2005

pressure of 40 psi in a Parr hydrogenation apparatus at which
point TLC indicated complete disappearance of starting material.
The Ra/Ni suspension was removed by filtration through Celite.
The filtrate was concentrated to give a crude product that was
unit of 4/5-amino pipecolyl derivative. although the
4-amino pipecolyl derivative differentiated between p
versus ap orientation of complementary DNA. It is
interesting to note that the constrained apgPNA ana-
logue, 4-aminopipecolyl (2S,4S), in 18 reverts the desta-
bilization caused by the open-chain apgPNA unit in the
same sequence.5 Thus, the structural differences in PNA:
DNA, PNA:RNA, and PNA2:DNA and elegant use of
substitution pattern of six-membered pipecolic acid de-
rivatives directed the complexation of pipecolic aegPNA
with DNA/RNA sequences. These results lead us to
believe that a biased preorganized conformation capable
of tuning the correct internucleobase geometry could be
particularly important when duplex/triplex, p/ap, or
DNA/RNA selectivity is being addressed.
In summary, the data presented in this note point out
the effectiveness of the design that allows aegPNAs to
be biased for binding in a triplex mode and an antipar-
allel duplex mode. The design also enforces superior
binding to DNA over RNA. It is likely that PNA oligomers
composed exclusively or predominantly of pipecolyl PNA
monomers may hybridize better than the chimeric back-
bone oligomer studied here. More data in terms of
sequence and backbone context (number and position of
pipecolyl units) are required to fully evaluate the proper-
ties of 4/5-aminopipecolyl PNA in terms of producing
hybridization-competent conformations. In our recent
work using aminoethylpipecolyl PNA, we reported addi-
tive properties of the monomeric units in the triplex
forming oligomers.11c,18 Further work to fully understand
these features is currently in progress in our laboratory.
1
then purified by column chromatography (yield 0.9 g, 90%). H
NMR CDCl3 δ: 7.3 (s, 5H), 5.15 (s, 2H), 5.0-4.75 (s, 1H), 4.2-4
(m, 1H), 3.7 (s, 4H), 3.35-2.9 (m, 1H), 2.49-1.6 (m, 4H), 1.4 (s,
9H). 13C NMR CDCl3 δ: 171.5, 159.8, 155.2, 136.5, 128.6, 128.1,
127.7, 79.4, 67.6, 52.3, 46.0, 44.2, 28.4, 25.9, 21.1. ESIMS: calcd
for C20H28N2O6 (M+) 392, found 409 (M + 17).
(2S,5R)-N-(tert-Butoxycarbonyl)-N-1-(chloroacetyl)-pipe-
colic Acid Methyl Ester 5. Compound 4 (0.61 g, 1.55 mmol)
was dissolved in methanol, and 10% Pd-C (90 mg) was then
added. This mixture was subjected to hydrogenation at 60 psi
for 8 h. The catalyst was filtered off over Celite, and the filtrate
was evaporated under vacuum to obtain free amine. Free amine
(0.19 g, 0.73 mmol) was taken in 10% Na2CO3 solution in
dioxane:water (1:1) and cooled in an ice bath. Chloroacetyl
chloride (0.16 mL, 1.4 mmol) was added with vigorous stirring.
The pH of the reaction was maintained around 9.0 during
reaction with addition of solid Na2CO3. After 1 h, at which point
TLC indicated complete disappearance of starting material, the
reaction mixture was concentrated to remove the dioxane. The
product was extracted with EtOAc and purified by column
chromatography (yield 0.11 g, 60%). [R]24D -13 (c 0.02, MeOH).
1H NMR CDCl3 δ: 4.9-4.8 (d, 1H), 4.2-3.8 (m, 2H), 3.7 (s, 3H),
3 (s, 1H), 2.4-2 (m, 2H), 1.9-1.51 (m, 3H), 1.4 (s, 10H). 13C NMR
CDCl3 δ: 170.9, 170.4, 167.0, 155.08, 80.34, 79.5, 74.9, 74.6, 52.2,
46.7, 45.3, 44.1, 40.6, 38.6, 28.0, 27.8, 24.8, 20.5. ESIMS: calcd
for C14H23N2O5Cl (M+) 334, found 335 (M + 1).
(2S,5R)-5-(tert-Butyloxycarbonyl)-N-1-(thyminylacetyl)-
pipecolic Acid Methyl Ester 6. Compound 5 (0.15 g, 0.49
mmol) was stirred with anhydrous K2CO3 (0.186 g, 1.34 mmol)
and thymine (0.17 g, 1.34 mmol) in dry DMF at 60 °C for 5 h.
When TLC indicated complete disappearance of starting mate-
rial, DMF was evaporated, and the product was purified by
column chromatography (yield ) 110 mg, 61%). 1H NMR CDCl3
δ: 9.2 (s, 1H), 7.3 (s, 1H), 5.5 (d, 1H), 4.9-4.8 (m, 1H), 4.3-4.2
(m, 1H), 4.1-3.8 (m, 2H), 3.75 (s, 3H), 3.6-3.49 (m, 1H), 2-1.6
(m, 8H), 1.45 (s, 9H). 13C NMR CDCl3 δ: 170.85, 167.13, 164.71,
155.74, 140.94, 52.50, 54.14, 48.18, 46.12, 44.64, 28.33, 25.32,
21, 12.22. ESIMS: calcd for C19H28N4O7 (M+) 424, found 425(M
+ 1), 442 (M + NH4+).
Experimental Section
(2S,5S)-5-O-Methylsulfonyl-N-1 (Benzyloxycarbonyl)-
pipecolic Acid Methyl Ester 2. To a stirred, cooled solution
of compound 1 (1.5 g, 5.1 mmol) in dry pyridine (20 mL) was
added methane sulfonyl chloride (0.7 mL, 6.11 mmol) dropwise.
Stirring was continued for 2 h at which point TLC indicated
complete conversion of starting material. The solvent was
removed under vacuum, and the residue was taken in water and
extracted with ethyl acetate (2 × 15 mL). The organic layers
were pooled, washed with water (2 × 10 mL) followed by brine
(1 × 10 mL), and dried over sodium sulfate. Evaporation of the
organic layer yielded a crude product that was then purified by
(2S,5R)-5-(tert-Butyloxycarbonyl)-N-(thyminylacetyl)-
pipecolic Acid 7. Compound 6 (0.12 g, 0.15 mmol) was
dissolved in 2 N NaOH in methanol/water (1 mL) and stirred
for 20 min. The pH of the solution was adjusted to 2 by addition
of cation-exchange resin, which was then filtered off. The filtrate
was then evaporated, producing the product as a white foam
silica gel column chromatography (yield, 70%). [R]24 -27.5 (c
D
(0.1 g, 92%). [R]24 ) -1.25 (c 0.008, MeOH). 1H NMR D2O δ:
0.004, MeOH). 1H NMR CDCl3 δ: 7.35(s, 5H), 5.2 (d, 2H),
5-4.75(m, 1H), 4.7-4.3 (m, H6), 3.7 (m, 3H), 3.1 (m, 3H), 2.5-
1.4 (m, 5H). 13C NMR CDCl3 δ: 170, 158,137.1 136.0, 128.4,
128.1, 127.8, 67.8, 52.3, 44.9(C6), 38.6, 27.9, 20. ESIMS: calcd
for C16H21NO7 S (M+) 371, found 389 (M + NH4+).
D
7.29 (s, 1H), 5.13 (bs, 1H), 4.9-4.78 (m, 1H), 4.64 (s, 1H), 4.61-4.3
(m, 1H), 3.85-3.73 (m, 1H), 3.70-3.4 (m, 2H), 2.46-1.88 (m, 2H),
1.82 (s, 3H), 1.79-1.65 (m, 2H), 1.38 (s, 9H). 13C NMR D2O δ:
170.62,169.99,157.43,153.63,143,47.75,47.28,46.5,29.53,27.34,
11.10. ESIMS: calcd for C18H26N4O7 (M+) 410, found 410 (M+).
(2S,5R)-5-Azido-N-1 (Benzyloxycarbonyl)pipecolic Acid
Methyl Ester 3. To the compound 2 (0.74 g, 1.99 mmol) in dry
DMF was added NaN3 (0.76 g, 11.6 mmol). The mixture was
stirred at 60 °C for 12 h at which point TLC indicated the
complete conversion of starting material. DMF was removed
under vacuum; water (10 mL) was added to the mixture, and
the mixture was extracted with EtOAc (3 × 15 mL). Organic
layers were pooled, washed with water (1 × 10 mL) and brine
(1 × 10 mL), and concentrated to give a crude product that was
then purified by silica gel column chromatography (yield 0.56
g, 90%). [R]24D -20 (c 0.008, MeOH). 1H NMR CDCl3 δ: 7.35 (s,
5H), 5.2 (s, 2H), 5.1-4.8 (m, 1H), 4.35-4.1 (m, 1H), 3.9-3.6 (m,
4H), 3.49-3.2 (m, 1H), 2.2-2 (m, 1H), 1.9-1.5 (m, 2H). ESIMS:
calcd for C15H18N4O4 318, found 319 (M + 1).
Acknowledgment. We thank Dr. K. N. Ganesh for
his continued support for this work. P.S.L. thanks CSIR,
New Delhi, for a research fellowship. V.A.K. thanks
Department of Science and Technology for financial
support.
Supporting Information Available: 1H and 13C NMR
spectra of compound 6; ESIMS for compounds 2, 6, and 7;
elemental analysis for compounds 1 and 5; representative
HPLC for oligomers 9, 11, 13, and 17-19; representative
MALDI-TOF spectra for PNAs 8-10, 11, 17, and 18; Job plot
for PNA 13 and DNA 15 showing PNA2:DNA stoichiometry;
representative Tm (first derivative) curves for thermal dena-
turation of the complexes; and gel-shift assay of chimeric
pipecolyl-aegPNA:DNA complexes. This material is available
free of charge via the Internet at http://pubs.acs.org.
JO0506884
(2S,5R)-N-(tert-Butyloxycarbonyl)-N-1-(benzyloxycarb-
onyl) Pipecolic Acid Methyl Ester 4. Compound 3 (0.74 g,
2.32 mmol) was taken in ethyl acetate. To this were added Ra/
Ni (0.5 mL) and di-tert-butyl dicarbonate (0.55 mL, 2.52 mmol).
This mixture was subjected to hydrogenation for 2 h at a
J. Org. Chem, Vol. 70, No. 17, 2005 6959