Preparation and determination of optical purity of γ-lysine modified peptide nucleic acid analogues

Article abstract of DOI:10.1007/s12272-012-0315-4

Peptide nucleic acids (PNAs) are DNA analogues in which the nucleic acid backbone is replaced by a pseudopeptide backbone and nucleobases are attached to the backbone by methylene carbonyl linkers. γ-Carbon modification of the PNA structure allows monomers, and subsequently oligomers, with improved properties to be obtained. In this study, we report the convenient synthesis of γ-lysine-modified PNA monomers for pyrimidine bases (thymine and cytosine) with high optical purity (> 99.5%) and direct enantiomer separation of γ-lysine-modified PNA analogs, using chiral HPLC to determine the optical purity.

Full text of DOI:10.1007/s12272-012-0315-4

Arch Pharm Res Vol 35, No 3, 517-522, 2012
DOI 10.1007/s12272-012-0315-4
Preparation and Determination of Optical Purity of γ-Lysine
Modified Peptide Nucleic Acid Analogues
Hu Huang1, Goon Ho Joe2, Sung Rok Choi2, Su Nam Kim2,Yong Tae Kim2, Hwang Siek Pak2, Sung Kee Kim2,
1
3
4,
1,
*
*
Joon Hee Hong , Hyo-Kyung Han , Jong Seong Kang ,and Wonjae Lee
1College of Pharmacy, Chosun University, Gwangju 501-759, Korea, Panagene Incorporated, Daejeon 301-510, Korea,
2
3College of Pharmacy, Dongguk University, Seoul 100-715, Korea, and College of Pharmacy, Chungnam National Uni-
4
versity, Daejeon 305-764, Korea
(Received July 8, 2011/Revised August 17, 2011/Accepted August 29, 2011)
Peptide nucleic acids (PNAs) are DNA analogues in which the nucleic acid backbone is
replaced by a pseudopeptide backbone and nucleobases are attached to the backbone by meth-
ylene carbonyl linkers.
sequently oligomers, with improved properties to be obtained. In this study, we report the
convenient synthesis of lysine-modified PNA monomers for pyrimidine bases (thymine and
γ-Carbon modification of the PNA structure allows monomers, and sub-
γ
-
cytosine) with high optical purity (> 99.5%) and direct enantiomer separation of
ified PNA analogs, using chiral HPLC to determine the optical purity.
γ-lysine-mod-
Key words: Peptide nucleic acid, γ-Lysine modified PNA, Optical purity
permeability to cells, increased stability of PNA-DNA
duplexes, and opportunities for further functionaliza-
INTRODUCTION
Peptide nucleic acids (PNAs) are neutral DNA mimics tion (Englund and Appella, 2005; de Koning et al.,
that bind to complementary DNA or RNA sequences 2006; Dragulescu-Andrasi et al., 2008; Kleiner et al.,
with high affinity and sequence specificity (Nilsen et 2008; Sahu et al., 2009; Totsingan et al., 2009). There-
al., 1991). Recently, a number of modifications to the fore, the preparation of chiral PNA monomers with
basic PNA monomeric units have been reported in high optical purity for PNA oligomers, which afford
order to improve the binding stability to the comple- high binding affinity to the complementary nucleic
mentary nucleic acid and the specificity of complexa- acids, has become an important research area. To that
tion (Summerton and Weller, 1997; Koshkin et al., end, indirect analytical methods using HPLC, GC and
1998; Obika et al., 1998; Falkieicz et al., 1999; Hickman 19F-NMR to determine the optical purity of PNA mon-
et al., 2000; Sforza et al., 2003; Noguchi and Asanuma, omers and/or oligomers have been developed and applied
2007; Dose and Seitz, 2008; Ishizuka et al., 2008). (Szokan et al., 1988; Corradini et al., 1999; Sforza et al.,
Among them, the most promising modification is the 2003; Sahu et al., 2009). However, all these indirect
introduction of the stereogenic center at the
atom of the -aminoethylglycine unit (Englund and hydrolysis and consequently have the potential com-
Appella, 2005; de Koning et al., 2006; Dragulescu- plication of racemization during analyte preparation.
Andrasi et al., 2008; Sahu et al., 2009). Compared to In this paper, we report the preparation of enantio-
unmodified normal PNAs, the modified PNAs have pure lysine-modified fluorenylmethoxycarbonyl (Fmoc)
γ-carbon analytical methods require chiral derivatization or
N
γ-
γ-
several advantages such as improved solubility, better PNA monomers and the convenient direct enantiomer
separation for Fmoc-protected PNA intermediates and
monomers without any derivatization for determina-
tion of the optical purity. This is the first report of
direct enantiomer separation of γ-lysine-modified Fmoc-
protected PNA intermediates and monomers by chiral
HPLC.
*These authors contributed equally to this work.
Correspondence to: Wonjae Lee, College of Pharmacy, Chosun
University, Gwangju 501-759, Korea
Tel: 82-62-230-6376, Fax: 82-62-222-5414
E-mail: wlee@chosun.ac.kr
517

518
H. Huang et al.
45.0 mmol) was added to the residue and dissolved in
methanol (200 mL). The reaction mixture was cooled
to 0oC in an ice bath and NaBH3CN (2.83 g, 45.0
mmol), CH3CO2H (2.57 mL, 45.0 mmol) and DIEPA
MATERIALS AND METHODS
Chemicals and general experimental proce-
dures
Boc: tert-butoxycarbonyl; DCC: N,N-dicyclohexyl- (7.84 mL, 45.0 mmol) were added to the stirred
carbodiimide; DhbtOH: 3-hydroxy-1,2,3-benzotriazin- solution. The reaction was then allowed to stir for 2 h
4(3H)-one; DIPEA: diisopropylethylamine; DMF: N,N
dimethylformamide; DMSO: dimethyl sulfoxide; Fmoc: was dissolved in ethyl acetate (150 mL) and washed
fluoren-9-yl-methoxycarbonyl; HBTU: -benzotriazol- with saturated KHSO4, saturated NaHCO3 and brine.
-
at 0oC. After the solvent was evaporated, the residue
O
1-yl-N,N,N’,N’-tetramethyluronium hexafluorophos- The organic layer was dried over magnesium sulfate,
phate; THF: tetrahydrofuran. All starting materials, filtered, and evaporated to obtain an oil. The oil was
reagents and dry solvents were purchased from the purified by flash chromatography (ethyl acetate-
commercial suppliers (Sigma Aldrich and Fluka) and hexane, 1:1). The product was a pale yellow oil. Yield:
were used without further purification. Flash-column 16.5 g (2 steps, 68%).
chromatography was performed on Merck Kieselgel 1H-NMR (600 MH, DMSO-d6):
60 (230-400 mesh). Thin-layer chromatography (TLC) 7.65 (d, 2H, Fmoc-
was performed on Merck Kieselgel 60 TLC plates. 2H, Fmoc- ), 7.03 (d, 1H, Fmoc-NH), 6.71 (t, 1H, Boc-
NMR spectra were recorded on a Varian Inova 600
), 4.15-4.28 (m, 3H, Fmoc-CH2-O + Fmoc-C ), 4.02
MHz spectrometer. Chemical shifts (
values) are in (q, 2H, CO2-CH2), 3.21-3.42 (m, 3H, N-C -CH2-N +
δ
7.83 (d, 2H, Fmoc-
H),
H
), 7.36 (t, 2H, Fmoc- ), 7.27 (t,
H
H
NH
H
δ
H
ppm relative to DMSO-d6 (2.50 ppm for proton and N-CH2-CO), 2.83-2.85 (m, 2H, N-CH-CH2-N), 2.45-
76.9 ppm for carbon). Electrospray mass spectra (ESI- 2.47 (m, 2H, Boc-NH-CH2), 1.30-1.52 (m, 6H, Boc-NH-
MS) were obtained using LTQ mass spectrometer and CH2-CH2-CH2-CH2), 1.43 (s, 9H, tert-butyl-CH3), 1.17
nominal mass measurement. Chromatography was (t, 3H, CO2-CH2-CH3). 13C-NMR (150 MHz, DMSO-
performed at room temperature using an HPLC Breeze d6):
system consisting of a Waters model 1525 binary pump, 127.61, 125.90, 120.77, 77.91, 65.75, 60.53, 53.31,
a Rheodyne model 7125 injector with a 20 L loop, 51.34, 50.92, 47.49, 32.59, 30.14, 28.94, 28.87, 23.50,
and a dual absorbance detector (Waters 2487 detector). 14.78, 14.73. ESI-MS: found 540 (MH+), calcd 540; for
Chiralpak IB column (250 mm L 4.6 mm I.D., 10 C30H41N3O6.
m) was purchased from Daicel Chemical Company.
δ 172.82, 156.64, 156.22, 144.62, 141.39, 128.16,
µ
×
µ
The chromatographic conditions were methanol-ethanol-
Fmoc-L-Lys(Boc)-PNA-thymine-OEt monomer
hexane = 5:15:80 (v/v/v) for Fmoc-L-Lys(Boc)-PNA (5T)
backbone (
15-20:80-85:0.1 (v/v/v) for
omers as the mobile phase at 1.0 mL/min of flow rate DhbtOH (0.76 g, 4.68 mmol) and DCC (0.97 g, 4.68
4
) and alcohol-hexane-trifluoroacetic acid =
Carboxymethylthymine (0.86 g, 4.68 mmol) was dis-
γ-
lysine-modified PNA mon- solved in dry DMF (20 mL) at 0oC, together with
with UV detection at 254 nm.
mmol). The solution was stirred for 1.5 h at 0oC, then
for 30 min at room temperature. The N,N dicyclohexy-
-
lurea was then filtered and the Fmoc-L-Lys(Boc)-PNA
backbone (1.25 g, 2.34 mmol) in DMF (20 mL) was
added to the mixture. The solution was stirred for 2 h
Procedure for the preparation (from 2 to 6T
and 6C )
Fmoc-L-Lys(Boc)-N,O-dimethyl (2)
This compound was prepared according to the previ- and the DMF was evaporated. The residue was dissolv-
ously reported methods (Kleiner et al., 2008; Totsingan ed in ethyl acetate and washed with saturated KHSO4
et al., 2009).
(twice) and saturated NaHCO3 (twice). The organic
layer was dried over magnesium sulfate and filtered.
After the solvent was removed, the residue was purified
Fmoc-L-Lys(Boc)-PNA backbone (4)
To a solution of Fmoc-L-Lys(Boc)-N,O-dimethyl (23.0 by solidification with diethyl ether (50 mL) to afford
g, 45.0 mmol) in dry THF (150 mL) was added dropwise 1.42 g (yield: 86%) of 5T as a pale yellow solid.
2 M solution of LAH in THF (22.5 mL, 45 mmol) at
78oC. The reaction mixture was stirred at 78oC 11.25 (s, 1H, imide-N
until HPLC showed no starting amide. The reaction 2H, Fmoc- ), 7.06-7.38 (m, 6H, Fmoc-
mixture was quenched with H2O (50 mL) and ethyl + Fmoc-N 6.73 (m, 1H, Boc-N ), 3.89-4.71 (m, 9H,
acetate (50 mL) mixture with stirring below
10oC. thymine-CH2-CO, N-CH2-CO2H + Fmoc-CH-CH2-O +
Extracted ethyl acetate mixture was evaporated below CO2CH2 + Fmoc-C -CH2), 2.84-3.65 (m, 5H, Fmoc-
room temperature. Glycine ethyl ester HCl (6.28 g, NH-CH-CH2-N, Fmoc-NH-C -CH2-N + Boc-NH-CH2),
1H-NMR (600 MHz, DMSO-d6):
δ
major rotamer
), 7.84 (d, 2H, Fmoc- ), 7.63 (m,
+ thymine-H
−
−
H
H
H
H
H),
H
−
H
H

Synthesis of
γ-Lysine Modified Peptide Nucleic Acids
519
2.04-2.15 (m, 2H, Boc-NH-CH2-CH2), 1.64 (s, 3H, thy- + Fmoc-CH-CH2-O), 2.82-3.65 (m, 5H, Fmoc-NH-C
mine-CH3), 1.35 (s, 9H, tert-butyl-CH3), 1.42-1.54 (m, CH2 + Fmoc-NH-CH-CH2-N + Boc-NH-CH2), 1.64 (s,
4H, Boc-NH-CH2-CH2-CH2), 1.42 (t, 3H, CO2CH2CH3); 3H, thymine-CH3), 1.06-1.43 (m, 6H, BocNH-CH2-CH2
H-
-
minor rotamer 1.68 (s, 3H, thymine-CH3), 1.33 (s, 9H,
C
H2-CH2), 1.31 (s, 9H, tert-butyl-CH3); minor rotamer
tert-butyl-CH3). 13C-NMR (150 MHz, DMSO-d6):
δ
11.23 (s, 1H, imide-NH), 1.67 (s, 3H, thymine-CH3), 1.31
169.83, 169.40, 168.48, 168.24, 165.02, 156.74, 156.60, (s, 9H, tert-butyl-CH3). 13C-NMR (150 MHz, DMSO-
156.23, 151.60, 144.65, 144.51, 144.45, 142.49, 141.41, d6);
δ 171.29, 170.91, 168.42, 168.02, 165.03, 156.75,
128.33, 127.75, 125.70, 120.72, 108.82, 108.78, 77.97, 156.62, 156.23, 151.62, 144.67, 144.53, 144.44, 142.67,
65.95, 65.58, 61.11, 52.17, 50.37, 48.84, 48.43, 47.46, 141.41, 128.23, 127.75, 125.89, 125.78, 120.81, 120.72,
31.87, 29.96, 28.94, 28.88, 23.49, 14.59, 12.56. ESI- 108.78, 77.98, 65.97, 65.58, 52.05, 48.54, 48.37, 47.46,
+
MS: found 723 (MNH4 ); 728 (MNa+), calcd 723; 728, 31.87, 29.95, 28.94, 28.88, 23.51, 15.79, 12.55. ESI-
for C37H47N5O9.
MS: found 695 (MNH4+); 700 (MNa+), calcd 695; 700,
for C35H343N5O9.
Fmoc-L-Lys(Boc)-PNA-cytosine(Bhoc)-OEt mon-
omer (5C)
Fmoc-L-Lys(Boc)-PNA-cytosine(Bhoc)-OH mon-
1H-NMR (600 MHz, DMSO-d6):
δ
major rotamer 10.94 omer (6C)
), 6.70-7.84 (m, 22H, Fmoc-
+ Bhoc- 1H-NMR (600 MHz, DMSO-d6):
+ Fmoc-N + Boc-N ), 3.90-4.84 (m, 7.87 (m, 22H, Fmoc- + Bhoc-
+ Fmoc-C -CH2 + Fmoc-CH-CH2-O + + Boc-N ), 3.85-4.85 (m, 10H, Bhoc-C
N-CH2-CO + CO-CH2-cytosine + CO2CH2), 2.68-3.64
(m, 5H, Fmoc-NH-CH-CH2-N, Fmoc-NH-C -CH2-N + cytosine + CO2CH2), 2.82-3.67 (m, 5H, Fmoc-NH-CH-
Boc-NH-CH2), 1.31 (s, 9H, tert-butyl-CH3), 1.18 (t, 3H, H2-N, Fmoc-NH-C -CH2-N + Boc-NH-CH2), 1.31 (s,
CO2CH2CH3); minor rotamer 10.92 (s, 1H, Bhoc-N ), 9H, tert-butyl-CH3); minor rotamer 10.92 (s, 1H, Bhoc-
1.33 (s, 9H, tert-butyl-CH3). 13C-NMR (150 MHz, DMSO- ), 1.33 (s, 9H, tert-butyl-CH3). 13C-NMR (150 MHz,
d6); 169.86, 169.40, 168.38, 168.11, 163.70, 156.76, DMSO-d6); 173.45, 171.31, 170.92, 170.37, 168.33,
(s, 1H, Bhoc-N
+ cytosine-
10H, Bhoc-C
H
H
H
H
δ major rotamer 6.70-
H
H
H
H
H
H
+ cytosine-
H
+ Fmoc-
+ Fmoc-
N
H
H
H
CH-CH2 + Fmoc-CH-CH2-O + N-CH2-CO + CO-CH2-
H
C
H
H
N
H
δ
δ
156.61, 156.24, 155.62, 152.99, 151.41, 144.68, 144.55, 167.95, 163.70, 163.65, 156.78, 156.64, 156.25, 155.59,
144.51, 141.41, 141.06, 129.26, 128.54, 127.65, 127.15, 153.05, 151.47, 144.70, 144.56, 144.50, 143.28, 141.41,
127.03, 125.79, 120.74, 94.46, 78.07, 77.97, 65.95, 141.06, 129.86, 129.26, 129.22, 129.17, 128.54, 128.32,
61.80, 61.12, 50.46, 48.90, 47.46, 46.61, 31.87, 29.95, 127.67, 127.15, 127.02, 126.53, 125.82, 120.80, 120.71,
28.95, 28.88, 23.51. ESI-MS: found 901 (MH+); 923 94.48, 78.08, 77.99, 66.00, 65.59, 52.17, 51.55, 50.20,
(MNa+), calcd; 901; 923, for C50H56N6O10.
47.46, 46.61, 32.11, 31.89, 29.97, 28.95, 28.88, 25.90,
23.54, 15.95, 15.79. ESI-MS: found 873 (MH+); 895
Fmoc-L-Lys(Boc)-PNA-thymine-OH monomer (MNa+), calcd 873; 895, for C48H52N6O10.
(6T)
All characterization data of D-(4), D-(5T), D-(5C), D-
Fmoc-L-Lys(Boc)-thymine-OEt (1.09 g, 1.55 mmol)
(
6T) and D 6C) matched ( ), (5T), (5C), (6T) and (6C),
-
(
4
was dissolved in dry THF (10 mL) at 0oC. To the stirred respectively.
solution was added 1 M NaOH solution (4.7 mL, 4.67
mmol) and then stirred for 1 h at room temperature.
The reaction mixture was adjusted pH to 9 by adding
1 N HCl solution; Fmoc-OSu (0.63 g, 1.86 mmol) was
RESULTS AND DISCUSSION
The chiral
γ-lysine-modified PNA monomers for
then added to the solution and stirred for 1 h at room pyrimidine nucleobase [thymine (T) and cytosine (C)]
temperature. The reaction mixture was adjusted to were prepared as outlined in Scheme 1, by modifying
pH = 3 with 1 N HCl at 0oC, extracted with ethyl the previously reported methods (Englund and Appella,
acetate (10 mL), washed with saturated NaHCO3 solu- 2005; de Koning et al., 2006; Dragulescu-Andrasi et al.,
tion (twice) and dried over magnesium sulfate. The 2008; Kleiner et al., 2008; Sahu et al., 2009; Totsingan
organic layer was filtered, evaporated, and purified by et al., 2009). The commercially available Fmoc-L-lys
solidification with diethyl ether (50 mL) to afford 0.93 (Boc)-OH (
g (yield: 89%) of 6T as a white solid. diprotected L-lysine (
1H-NMR (600 MHz, DMSO-d6):
major rotamer 11.25 L-Lys(Boc)-N-methoxy-
(s, 1H, imide-N ), 7.84 (d, 2H, Fmoc- ), 7.63 (t, 2H, methoxy- -methylamine hydrochloride using HBTU
Fmoc- ), 7.08-7.38 (m, 6H, Fmoc- + thymine-H + activation in DMF. The amide ( ) was then reduced
Fmoc-N ), 6.71-6.75 (m, 1H, Boc-N ), 3.83-4.70 (m, with LiAlH4 in THF at low temperature and the sub-
7H, thymine-CH2-CO, N-CH2-CO2H + Fmoc-C -CH2 sequent hydrolysis gave Fmoc-L-Lys(Boc)-H ( ). The
1
) was selected as a starting material. The
) was transformed into Fmoc-
-methyl amide ( ) with
1
N
δ
2
N-
H
H
N
H
H
2
H
H
H
3

520
H. Huang et al.
Scheme 1. Synthesis of γ-L-lysine-modified PNA monomers
crude aldehyde (3) was immediately used for reduc- mobile phases of ethanol-hexane-trifluoroacetic acid =
tive amination with ethyl glycine hydrochloride and 20:80:0.1 (v/v/v) and methanol-ethanol-hexane-tri-
NaBH3CN in the presence of acetic acid and DIEPA in fluoroacetic acid = 5:10:85:0.1 (v/v/v/v) were used on
methanol to make the
backbone ( ). After purification of the chiral PNA :<0.50) and (6C) (L:D = >99.50:<0.50) but also D
backbone by column chromatography, the pure back- (L:D = 0.41:99.59) and D 6C) (L:D = 0.06:99.94) analytes
γ
-L-Lys(Boc)-PNA monomer ester Chiralpak IB, respectively. Not only (6T) (L:D = >99.50
4
-(6T)
-
(
bone was converted into Fmoc-L-Lys(Boc)-PNA-thy- showed extremely high optical purity, which indicates
mine-OEt monomer (5T) by reaction with thymine almost no racemization during all PNA monomer syn-
acetic acid using the DCC/DhbtOH coupling method thesis procedures, including the final basic hydrolysis
in DMF. After hydrolysis with 1 M NaOH in THF (de- step. This is the first report of direct enantiomer
protection of Fmoc occurs during hydrolysis, so the separation of
subsequent re-protection in situ was performed by intermediates and monomers by chiral HPLC. Other
γ-lysine-modified Fmoc-protected PNA
using Fmoc-OSu), the desired γ-L-Lys(Boc)-PNA-thy- research groups have used indirect analytical methods
mine-OH monomer (6T) was obtained as a white solid using HPLC and 19F-NMR to determine the diastereo-
by solidification, in which a further purification process meric excess after chiral derivatization (Sforza et al.,
was not performed. For the other pyrimidine base, 2003; Sahu et al., 2009). Typical chromatograms demon-
cytosine-derived
was synthesized by the same procedure described in PNA-thymine-OH (6T) are presented in Fig. 1.
Scheme 1. Racemization can potentially occur during the
Also, Fmoc-D-Lys(Boc)-PNA-thymine-OH [D 6T)] and reduction step ( ) in the preparation of -lysine-
Fmoc-D-Lys(Boc)-PNA-cytosine(Bhoc)-OH [D-(6C)] were modified Fmoc-protected PNA monomers, since it is
synthesized from Fmoc-D-Lys(Boc)-OH [D )] as a well-known that -electron withdrawing group-sub-
starting material. Applying the modified direct enanti- stituted -amino aldehydes are thermally unstable
γ
-lysine PNA analogue monomer (6C
)
strating the optical purity of Fmoc-D and L-Lys(Boc)-
-(
2→3
γ
-(1
N
α
omer separation method developed in our group (Jin and easy to racemize (Jurczak and Golebiowski, 1989;
et al., 2006, 2007), we determined the optical purities Gryko et al., 2003; Soto-Cairoli et al., 2008). Therefore,
of Fmoc-L-Lys(Boc)-PNA-thymine-OH (6T) and Fmoc- we examined the degree of racemization under various
L-Lys(Boc)-PNA-cytosine(Bhoc)-OH (6C) as well as D- conditions at the reduction step (
6T) and D-(6C). In the chromatographic conditions of important step for the optical purity of PNA-mono-
thymine- and cytosine-derived -lysine PNA analogs, mers. Because Fmoc-lysinal ( ) was not isolated and
2→3), which is an
(
γ
3

Synthesis of
γ-Lysine Modified Peptide Nucleic Acids
521
Fig. 1. Determination of optical purity and typical chromatograms of enantiomer separation of racemic Fmoc-Lys(Boc)-PNA-
thymine-OH (left), Fmoc-D-Lys(Boc)-PNA-thymine-OH (D-6T) (middle, L:D = 0.41:99.59) and Fmoc-L-Lys(Boc)-PNA-thymine-
OH (6T) (right, L:D = >99.50 : <0.50) on Chiralpak IB; Mobile phase: ethanol-hexane-trifluoroacetic acid = 20:80:0.1 (v/v/v);
Flow rate = 1 mL/min; UV 254 nm.
immediately reacted with glycine ethyl ester in situ
the degree of racemization in the reduction was ex- amide to form a more stable complex with the
amined at the next step of the reductive-aminated in- group of the Weinreb amide without cleavage of pro-
termediate ( ) by using our direct enantiomer separa- tection group (Nahm and Weinreb, 1981). Therefore,
tion method (Jin et al., 2006, 2007). As shown in Table Fmoc-deprotection could be minimized at the quench-
I, we found that the quenching temperature was more ing step and the aldehyde compound ( ) could be
,
at low temperature it might migrate to the Weinreb
β-methoxy
4
3
important than the complexation temperature. Ra- obtained in higher yield at low temperature. Typical
cemization did not occur at the complexation step chromatograms of determination of the optical purity
below 0oC but occurred at the quenching step above of Fmoc-L-Lys(Boc)-PNA-backbone (
4
) obtained after
,O-dimethyl ( ) and the
) are
0oC, due to formation of a stable aluminum-complex reduction of Fmoc-L-Lys(Boc)-
N
2
with a Weinreb amide. We also observed that the degree subsequent amination of Fmoc-L-Lys(Boc)-H (
3
of racemization increased very slightly as the quench- presented in Fig. 2.
ing temperature increased. However, the yields were
dependent on the complexation temperature rather 99.5%) Fmoc-L and D-Lys(Boc)-PNA-thymine-OH and
than the quenching temperature. Presumably, the alu- Fmoc-L and D-Lys(Boc)-PNA-cytosine(Bhoc)-OH as
minum ion of LiAlH4 could bind to the Weinreb amide lysine-modified PNA pyrimidine base (thymine and
) or Fmoc-carbamate or Boc-carbamate moiety, but cytosine) monomers starting from the commercially
In conclusion, we synthesized the enantiopure (>
γ-
(2
Table I. Determination of optical purity and yield of the
reduction of Fmoc-L-Lys(Boc)-N,O-dimethyl (2) under
various reaction conditions
Temperature (oC)
Exp.
No.
Optical
purity* (L:D)
Yield**
(%)
Complexation Quenching
1
2
3
4
5
6
−
−
−
78
78
10
0
0
0
−
10
0
99.96:0.04
99.95:0.05
99.95:0.05
99.95:0.05
99.73:0.27
99.20:0.80
68.0
62.2
59.4
46.1
44.1
42.0
−
−
10
10
0
r.t.
*The optical purity of Fmoc-L-Lys(Boc)-PNA backbone (
4)
obtained after reduction of Fmoc-L-Lys(Boc)- ,O-dimethyl
N
Fig. 2. Determination of optical purity and typical chroma-
tograms of enantiomer separation of racemic Fmoc-Lys(Boc)-
PNA backbone (left) and Fmoc-L-Lys(Boc)-PNA backbone
(right, L:D = 99.96:0.04) on Chiralpak IB; Mobile phase:
methanol-ethanol-hexane = 5:15:80 (v/v/v); Flow rate = 1
mL/min; UV 254 nm.
(
2
) and the subsequent amination of Fmoc-L-Lys(Boc)-H
(
3) was determined on Chiralpak IB [Mobile phase: metha-
nol-ethanol-hexane = 5:15:80 (v/v/v); Flow rate 1 mL/min;
UV 254 nm].
**Isolation yield obtained after column chromatography of
Fmoc-L-Lys(Boc)-PNA backbone (4).

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