9490
P. S. Shirude et al. / Tetrahedron 60 (2004) 9485–9491
25
4.1.3. 5-(R)-Hydroxy-N-benzyloxycarbonyl-2-(R)-pipe-
colic acid methyl ester. 1H NMR (CDCl3) d: 7.35 (s,
5H), 5.15 (s, 2H), 4.89–4.77 (m, 1H), 4.27–4.21 (m, 1H),
3.73–3.65 (m, 4H), 2.85–2.71 (m, 1H), 2.48–2.26 (m, 2H)
[a]
Mobs: 514.
ZC10.0 (c 0.5, CH3OH). MS: (ESI) Mcalc: 514.58,
D 589
4.1.10. 1-(N-Boc-aminoethyl)-5-(S)-(N3-benzoylthymin-
1-yl)-2-(R)-pipecolic acid methyl ester. 1H NMR
(CDCl3) d: 7.90–7.89 (m, 2H), 7.68–7.45 (m, 4H) 5.24 (br
s, 1H), 3.75 (s, 1H), 3.72 (s, 3H), 3.46–3.22 (m, 3H), 3.00–
2.68 (m, 3H) 2.16–2.00 (m, 1H), 2.00 (s, 3H), 1.96–1.67 (m,
4H), 1.43 (s, 9H): 13C NMR (CDCl3) d: 175.55, 169.36,
163.50, 156.27, 150.57, 142.84, 135.13, 131.91, 130.62,
129.39, 109.76, 79.82, 66.87, 64.97, 56.26, 52.50, 51.51,
1.98–1.73 (m, 2H): [a]D25
ZC15.38 (c 0.26, CH3OH).
589
4.1.4. 5-(S)-Hydroxy-2-(S)-pipecolic acid methyl ester
(7). The N-deprotection of methyl ester 6 (2.20 g, 7.8 mmol)
was done under hydrogenation over Pd–C (10%) to obtain 7
(1.10 g, 92%). 1H NMR (CDCl3) d: 3.83–3.81 (m, 1H), 3.74
(s, 3H), 3.40–3.33 (t, JZ6.6 Hz, 1H), 3.08–3.00 (dd, JZ2.4,
12 Hz, 1H) 2.88–2.80 (dd, JZ2, 12.2 Hz, 1H), 1.90–1.83
(m, 3H), 1.74–1.61 (m, 1H).
39.99, 29.67, 28.66, 28.02, 12.53: [a]D25
0.25, CH3OH). MS: (ESI) Mcalc: 514.58, Mobs: 515.
ZK12.0 (c
589
4.1.11. 1-(N-Boc-aminoethyl)-5-(R/S)-(thymin-1-yl)-2-
(S/R)-pipecolic acid (10). To a solution of the methyl
ester 9 (0.36 g, 0.7 mmol) in methanol (2 mL), was added
aqueous 2 M NaOH (2 mL). The reaction mixture was
further stirred overnight followed by neutralization with
Dowex-50 HC resin, which was then filtered off. The filtrate
was concentrated under vacuum and the residue was taken
up in water. This was washed with ethyl acetate before
concentrating it to dryness to obtain the product 10 (0.25 g,
95%) as white solid foam.
4.1.5. 1-(N-Boc-aminoethyl)-5-(S/R)-hydroxy-2-(S/R)-
pipecolic acid methyl ester (8). To a cooled solution of
5-(S/R)-hydroxy-2-(S/R)-pipecolic acid methyl ester 7
(1.0 g, 6.3 mmol), DIPEA (2.7 mL, 15.7 mmol), DMAP
(0.15 g, 1.3 mmol) in dry DMF/acetonitrile (1:1) (10 mL)
was added with stirring for 15 min. N-Boc aminoethyl
mesylate (1.50 g, 6.3 mmol) in DMF (3 mL) was then added
and the reaction mixture was heated to 50 8C for 20 h.
Evaporation of the solvent followed by column chromato-
graphy gave thick brown oil of 8 (0.7 g, 37%).
4.1.12. 1-(N-Boc-aminoethyl)-5-(R)-(thymin-1-yl)-2-(S)-
pipecolic acid. H NMR (D2O) d: 7.50 (s, 1H) 4.52–4.44
(m, 1H), 4.21–3.94 (m, 3H) 3.50–3.45 (m, 4H), 2.34–2.24
(m, 4H), 1.89 (s, 3H), 1.45 (s, 9H): [a] ZC32.0 (c 0.1,
CH3OH). MS: (ESI) Mcalc: 396.45, Mobs: 396.
4.1.6. 1-(N-Boc-aminoethyl)-5-(S)-hydroxy-2-(S)-pipeco-
lic acid methyl ester. 1H NMR (CDCl3) d: 5.18 (br s, 1H),
3.93–3.82 (m, 1H), 3.73 (s, 3H), 3.54–3.50 (m, 3H), 3.05–
2.99 (m, 1H) 2.75–2.50 (m, 3H), 1.90–1.64 (m, 4H), 1.45 (s,
1
25
D 589
9H): 13C NMR (CDCl3) d: 175.1, 161.2, 79.2, 64.9, 62.8,
25
55.3, 55.1, 51.8, 40.6, 37.2, 29.1, 28.3: [a]
D 589
ZK10.7 (c
4.1.13. 1-(N-Boc-aminoethyl)-5-(S)-(thymin-1-yl)-2-(R)-
pipecolic acid. H NMR (D2O) d: 7.55 (1H) 4.55–4.48
0.56, CH3OH). MS: (ESI) Mcalc: 302.37, Mobs: 302.
1
(m, 1H), 4.26–3.94 (m, 3H) 3.54–3.42 (m, 4H), 2.50–2.32
(m, 3H), 1.81–1.72 (m, 1H), 1.96 (s, 3H), 1.51 (s, 9H): [a]D25
4.1.7. 1-(N-Boc-aminoethyl)-5-(R)-hydroxy-2-(R)-pipe-
colic acid methyl ester. H NMR (CDCl3) d: 5.34 (br s,
1
ZK34.0 (c 0.1, CH3OH). MS: (ESI) Mcalc: 396.45,
589
1H), 3.96–3.93 (m, 1H), 3.75 (s, 3H), 3.41–3.25 (m, 3H),
3.16–3.13 (m, 1H) 2.69–2.63 (m, 3H), 1.96–1.66 (m, 4H),
1.45 (s, 9H): 13C NMR (CDCl3) d: 173.47, 156.26, 79.13,
65.67, 64.90, 55.69, 54.98, 51.55, 40.60, 37.70, and 28.43:
Mobs: 398.
4.2. Solid phase synthesis of the PNA oligomers on the
solid support
[a]2D5
ZC11.9 (c 0.42, CH3OH). MS: (ESI) Mcalc:
589
302.37, Mobs: 302.
The PNAoligomers were synthesizedmanually bysolidphase
peptide synthesis using the Boc-protection strategy and
employing diisopropylcarbodiimide (DIPCDI) or O-(Benzo-
triazol-1-yl)-N,N,N0,N0 tetramethyl-uronium hexafluoro-
phosphate (HBTU) and 1-hydroxy-benzotriazole (HOBt)
as the coupling agents. The solid support used was
Merrifield resin derivatized with b-alanine (0.17 mequiv/g
resin). The synthesis involved repetitive cycles, each
comprising (i) deprotection of the N-protecting Boc-group
using 50% trifluoroacetic acid (TFA) in CH2Cl2, (ii)
neutralization of the TFA salt formed with DIPEA (5%
solution in CH2Cl2, v/v) and (iii) coupling of the free amine
with the free carboxylic acid group of the incoming
monomer (4 equiv) in the presence of DIPCDI and HOBt,
in DMF or NMP as the solvent. The deprotection of the
N-Boc protecting group and the coupling reaction were
monitored by Kaiser’s test.23 The coupling efficiencies were
found to be O98%.
4.1.8. 1-(N-Boc-aminoethyl)-5-(R/S)-(N3-benzoylthymin-
1-yl)-2-(S/R)-pipecolic acid methyl ester (9). To a stirred
solution of 1-(N-Boc-aminoethyl)-5-(S/R)-hydroxy-2-(S/R)-
pipecolic acid methyl ester 8 (0.55 g, 1.8 mmol), N3-benzoyl-
thymine (0.84 g, 3.6 mmol) and triphenyl phosphine (0.95 g,
3.6 mmol) in dry THF (10 mL) at 0 8C, was added dropwise
diethylazodicarboxylate (DIAD, 0.47 mL, 3.6 mmol). After
completion of the reaction as indicated by TLC (24 h), the
solvent was removed in vacuo and residue purified by silica
gel column chromatography to get the pure product 9 (0.3 g,
32% yield).
4.1.9. 1-(N-Boc-aminoethyl)-5-(R)-(N3-benzoylthymin-1-
yl)-2-(S)-pipecolic acid methyl ester. 1H NMR (CDCl3) d:
7.92 (s, 1H), 7.88 (s, 1H), 7.62–7.44 (m, 4H) 5.28 (br s, 1H),
3.74 (s, 1H), 3.72 (s, 3H), 3.48–3.22 (m, 3H), 2.94–2.69 (m,
3H) 2.14–2.00 (m, 1H), 2.00 (s, 3H), 2.00–1.63 (m, 4H),
1.42 (s, 9H): 13C NMR (CDCl3) d: 175.3, 169.0, 163.2,
156.0, 150.3, 142.5, 138.8, 131.6, 130.3, 129.1, 109.5, 79.5,
66.6, 64.7, 56.0, 52.2, 51.2, 39.7, 29.4, 28.4, 27.7, 12.2:
The PNA oligomers were cleaved from the solid support
using TFA-TFMSA to yield oligomers with free carboxy
terminus.10 The resin-bound PNA oligomer (10 mg) was