Fast Solid-Phase Synthesis of Chiral Peptide Nucleic Acids
FULL PAPER
4.22 (s, 1 H, α-H), 4.4Ϫ4.6 (m, 5 H, CH2CH Fmoc ϩ OCH2 allyl),
5.03 (s broad, 2 H, NH Boc ϩ NH lysine side chain), 5.20 (s, 2 H,
CH2 2-Cl-Z-lysine), 5.1Ϫ5.3 (m, 2 H, CH2 allyl), 5.8Ϫ5.9 (m, 1 H,
CH allyl), 7.2Ϫ7.4 (m, 8 H, CH aromatic 2-Cl-Z-lysine ϩ CH aro-
and high enantiomeric excess. Different synthetic pathways
towards a -lysine-based submonomer to be used with the
Boc strategy have been discussed, outlining optimal proced-
ures. However, the route via a methyl ester is preferable
since it combines fairly high yield with optimal optical pur-
ity.
Solid-phase syntheses of different chiral PNAs were per-
formed by the submonomeric approach yielding chiral
PNAs with high optical purity and high yield. The method
3
matic Fmoc), 7.62 (d, JH,H ϭ 7.4, 2 H, CH aromatic Fmoc), 7.75
3
(d, JH,H ϭ 7.3, 2 H, CH aromatic Fmoc) ppm. 13C NMR
(300 MHz, CDCl3, 25 °C): δ ϭ 23.4, 28.3, 28.6, 29.2, 39.5, 40.6,
46.5, 47.3, 60.4, 63.7, 65.8, 66.8, 79.2, 118.6, 119.8, 124.6, 126.7,
126.9, 127.6, 129.2, 129.4, 129.6, 131.6, 134.2, 134.3, 141.3, 143.8,
156.0, 156.1, 156.2, 170.9 ppm. MS-ESI (MeOH): calcd. for
appears to be excellent when only one chiral center is in- C39H47ClN3O8 [MHϩ]: m/z ϭ 720.3, found 720.0; calcd. for
C39H46ClNaN3O8 [MNaϩ]: m/z ϭ 742.3, found 742.0. ee (chiral
volved; in the syntheses of ‘‘chiral box’’ PNAs, where more
GC-MS according to the method reported in ref.[25]): 99.1%.
adjacent bulky chiral centers are involved, this method al-
lowed for the synthesis of chiral PNAs with the highest op-
tical purity ever obtained.
The results presented here open the possibility to obtain
NЈ-Boc-Aminoethyl-Nε-2-chloro-Z-Nα-Fmoc-
D-lysine (3) by Allyl
Ester Deprotection: NЈ-Boc-Aminoethyl-Nε-2-chloro-Z-NαϪFmoc-
-Lysine allyl ester (2) (0.37 g, 0.51 mmol) was dissolved in THF
highly optically pure chiral PNAs with a reasonable facility, (15 mL) at room temperature together with [Pd (PPh3)4] (0.06 g,
0.051 mmol). After stirring for 10 min, morpholine (0.44 mL,
5.1 mmol) was added to the mixture. After 5 min, the reaction was
quenched with a 1 potassium hydrogen sulfate solution (60 mL).
The organic layer was separated and washed with 0.1 potassium
hydrogen sulfate (3 times). The combined organic phases were dried
with magnesium sulfate, filtered and the solvents evaporated under
vacuum. The residue was purified by flash chromatography (ace-
tonitrile/methanol, gradient elution from CH3CN 100% to
CH3CN/CH3OH 50:50); the product was obtained as a white solid.
Yield: 0.27 g (80%). ee (chiral GC-MS according to the method
reported in ref.[25]): 98.9%.
improving the abilities of PNAs in the specific recognition
of nucleic acids, and yielding more pure compounds for
structural studies (X-ray crystallography, NMR spectro-
scopy) of the PNA-DNA complexes.
Experimental Section
General: Boc: tert-butoxycarbonyl; BTSA: bis(trimethylsilyl-
acetamide); DCC: N,NЈ-dicyclohexylcarbodiimide; DCM: di-
chloromethane; DCU: N,NЈ-dicyclohexylurea; DHBtOH: 3-hy-
droxy-1,2,3-benzotriazin-4(3H)-one; DIPEA: diisopropylethyl-
amine; DMF: N,N-dimethylformamide; DMSO: dimethyl sulfox-
ide; Fmoc: fluoren-9-yl-methoxycarbonyl; HATU: O-(7-azabenzo-
NЈ-Boc-Aminoethyl-Nε-2-chloro-Z-Nα-Fmoc-
D-lysine (3) by Zwitter-
ion Protection: Bis(trimethylsilyl)acetamide (BTSA) (0.3 mL,
1.2 mmol) and DIPEA (0.16 mL, 0.9 mmol) were added to NЈ-Boc-
aminoethyl-Nε-(2-chloro-Z)--Lysine (4) (0.27 g, 0.6 mmol) sus-
pended in DCM (6 mL), with exclusion of water by a CaCl2 drying
tube. When the solution was nearly clear (10Ϫ15 min were usually
required), Fmoc-Cl (0.31 g, 1.2 mmol) was added at 0 °C. After
10 min, the mixture was heated to room temperature and then
stirred for 2 h. Methanol (2.4 mL) was carefully added and the mix-
ture was stirred for an additional 15 min, diluted with DCM
(20 mL), washed successively with a saturated KHSO4 solution and
brine, dried with magnesium sulfate and the solvents were evapor-
ated. The residue was purified by flash chromatography (dichloro-
methane/methanol, 95:5). The product was obtained as a white
foam. Yield: 0.29 g (60%). ee (chiral GC-MS according to the
method reported in ref.[25]): 99.1%. Melting point 90Ϫ92 °C. 1H
triazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate;
HBTU: O-benzotriazol-1-yl-N,N,NЈ,NЈ-tetramethyluronium hexa-
fluorophosphate; MBHA-PS: methylbenzhydrylamine-polystyrene:
Moz: 4-methoxybenzyloxycarbonyl; NMP: N-methylpyrrolidone;
TFA: trifluoroacetic acid; TFFH: fluoro-N,N,NЈ-tetramethylforma-
midinium hexafluorophosphate; TFMSA: trifluoromethanesul-
fonic acid; THF: tetrahydrofuran; Z: benzyloxycarbonyl. All sol-
vents and starting materials were used as commercially available.
The PNA sequences are given from N to C.
Carboxymethyl Nucleobases: The (carboxymethyl)thymine and the
Z-protected (carboxymethyl)adenine and (carboxymethyl)cytosine
were synthesized as reported in ref.[30]
NЈ-Boc-Aminoethyl-Nε-2-chloro-Z-
D-lysine Allyl Ester (1): The pro-
NMR (300 MHz, CDCl3, 25 °C):
δ ϭ 1.1Ϫ1.5 (m, 6 H,
CH2CH2CH2 lysine side chain), 1.32 [s, 9 H, (CH3)3 Boc], 2.9Ϫ3.1
(m, 6 H, CH2 lysine side chain ϩ CH2CH2 aminoethyl group),
4.1Ϫ4.4 (m, 4 H, α-H ϩ CH2CH Fmoc), 5.13 (s, 2 H, CH2 2-
Cl-Z-lysine), 7.1Ϫ7.3 (m, 8 H, CH aromatic 2-Cl-Z-lysine ϩ CH
aromatic Fmoc), 7.4Ϫ7.5 (m, 2 H, CH aromatic Fmoc), 7.67 (d,
3JH,H ϭ 7.3, 2 H, CH aromatic Fmoc) ppm. 13C NMR (300 MHz,
CDCl3, 25 °C): δ ϭ 23.6, 28.3, 29.3, 29.7, 39.4, 40.7, 46.1, 47.2,
61.4, 63.6, 67.3, 79.4, 119.8, 124.8, 127.1, 127.6, 126.7, 129.1, 129.3,
129.6, 133.4, 134.5, 141.3, 143.9, 156.4, 156.5, 156.6 ppm. FT-IR
tected backbone was synthesized according to a literature proced-
ure.[23]
NЈ-Boc-Aminoethyl-Nε-2-chloro-Z-Nα-Fmoc-
D-lysine Allyl Ester (2):
NЈ-Boc-Aminoethyl-Nε-2-chloro-Z--Lysine allyl ester (1) (0.56 g,
1.12 mmol) was dissolved in DCM together with DIPEA (0.29 mL,
1.67 mmol) and the solution was cooled to 0 °C. Fmoc-Cl (0.58 g,
2.24 mmol) was added and the mixture was stirred at room temper-
ature for 2 h. The reaction was quenched with 1 potassium hy-
drogen sulfate (60 mL). The organic layer was separated and the
aqueous phase was washed with DCM (3 ϫ 60 mL). The combined
organic phases were dried with magnesium sulfate, filtered and the
solvents evaporated under vacuum. The residue was purified by
flash chromatography (ethyl acetate/hexane, 1:2). The product was
(KBr): ν˜ ϭ 3422 (s), 2932 (m), 1700 (s), 1521 (w), 1251 (m) cmϪ1
.
MS (ESI, CH3OH): calcd. for C36H42ClN3NaO8 [MNaϩ]: m/z ϭ
702.3, found 702.0.
NЈ-Boc-Aminoethyl-Nε-2-chloro-Z-
D-lysine (4) by Allyl Ester Depro-
1
a colourless oil. Yield: 0.73 g (97%). H NMR (300 MHz, CDCl3,
tection: NЈ-Boc-Aminoethyl-Nε-2-chloro-Z--Lysine allyl ester (1)
25 °C): δ ϭ 1.2Ϫ1.6 (m, 6 H, CH2CH2CH2 lysine side chain), 1.40
(0.15 g, 0.30 mmol) was dissolved in THF (15 mL) at room temper-
[s, 9 H, (CH3)3 Boc], 2.8Ϫ3.0 (m, 2 H, CH2 aminoethyl group), ature together with [Pd (PPh3)4] (0.035 g, 0.03 mmol). After stirring
3.0Ϫ3.4 (m, 4 H, CH2 aminoethyl group ϩ CH2 lysine side chain),
for 10 min, morpholine (0.38 mL, 4.3 mmol) was added to the mix-
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Eur. J. Org. Chem. 2003, 1056Ϫ1063