Synthesis and binding affinity of a chiral PNA analogue

Article abstract of DOI:10.1081/NCN-100105906

The synthesis of a chiral peptide nucleic acid (PNA), which is composed of N-aminoethyl-cis-4-nucleobase-L-proline units, was described. The chiral PNA monomers containing all four nucleobases (A, T, C and G) were steroselectively prepared. The x-ray diff

Full text of DOI:10.1081/NCN-100105906

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SYNTHESIS AND BINDING AFFINITY OF A CHIRAL PNA
ANALOGUE
Ying Li a , Tao Jin a & Keliang Liu b
a Beijing Institute of Pharmacology & Toxicology, 27 Taiping Road, Beijing, 100850, China
b Beijing Institute of Pharmacology & Toxicology, 27 Taiping Road, Beijing, 100850, China
Published online: 21 Aug 2006.
To cite this article: Ying Li , Tao Jin & Keliang Liu (2001) SYNTHESIS AND BINDING AFFINITY OF A CHIRAL PNA ANALOGUE,
Nucleosides, Nucleotides and Nucleic Acids, 20:9, 1705-1721, DOI: 10.1081/NCN-100105906
To link to this article: http://dx.doi.org/10.1081/NCN-100105906
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(9), 170571721 (2001)
SYNTHESIS AND BINDING AFFINITY
OF A CHIRAL PNA ANALOGUE
Ying Li, Tao Jin, and Keliang Liu*
Beijing Institute of Pharmacology & Toxicology,
27 Taiping Road, Beijing 100850, China
ABSTRACT
The synthesis of a chiral peptide nucleic acid (PNA), which is composed of
N-aminoethyl-cis-4-nucleobase-L-proline units, was described. The chiral
PNA monomers containing all four nucleobases (A, T, C and G) were
steroselectively prepared. The x-ray diffraction data from a single crystal
confirmed the configuration of a key intermediate. Binding activity of the
oligomers with their complementary DNA targets was also investigated.
Synthetic molecules that can bind specifically to a chosen target in a
gene sequence are of great interest in medicinal and biotechnological fields.
They have shown promise for the development of gene therapeutic agents,
diagnostic devices for genetic analysis and as molecular tools for nucleic acid
manipulations1. PNA2 is a nucleic acid analogue in which the entire deoxy-
ribose-phosphate backbone in DNA has been replaced by a completely dif-
ferent backbone composed of N-(2-aminoethyl) glycine units (Fig. 1). PNA
has shown advantages377 superior to other DNA analogues. In recent years,
many applications of PNA have been exploited, especially in a biotechno-
logical context8710. However, as far as developing a drug is concerned, PNA
still has some limitations. One is its poor solubility in aqueous media11.
Another drawback is that a PNA oligomer can bind to a nucleic acid target in
both parallel and antiparallel orientations12, which may lead to lower binding
specificity. It is suggested that this kind of orientation independence could be
a consequence of the achiral nature of PNA.
*Corresponding author.
1705
Copyright # 2001 by Marcel Dekker, Inc.
www.dekker.com

1706
LI, JIN, AND LIU
Figure 1. Structure of DNA, PNA and the chiral PNA.
Our research is focused on the design and synthesis a series of chiral
PNAs with a partially cyclic backbone to mimic natural nucleic acids. The
pyrrolidine ring in proline is a suitable unit for mimicking the ribose moiety
in DNA, therefore, chiral PNA is designed to be composed of alternated N-
aminoethyl-cis-4-nucleobase-L-proline units (Fig. 1). This chiral PNA not
only has a similar dimension and rigidity as natural nucleic acids, but has
a tertiary amino structure in each unit, which could be expected to obtain a
good solubility and a high affinity for negatively charged nature nucleic acids.
As we were working on this project, D’Costa et al.13 reported a homo-
thymine PNA having the same backbone, but their results showed some
differences from ours. In this paper, we described the detailed synthesis of the
chiral PNA monomers containing all four natural bases (A, C, T and G) and
their oligomerization. Three of the monomers (C, A, G) have not been
reported yet. We also studied the crystal structure of compound 5 to confirm
its configuration. The data showed that no racemization occurred during the
synthesis. The hybridization properties of the chiral PNA oligomers with
complementary DNA targets were also discussed.
RESULTS AND DISCUSSION
Since the chiral PNA had a polyamide backbone, the oligomers were
assembled by solid phase peptide synthesis (SPPS) with different Boc pro-
tected monomers.

CHIRAL PNA ANALOGUE
1707
Scheme 1.
Synthesis of Monomers
Synthesis of the Key Intermediate N-(2-Boc-amino)ethyl-trans-
4-methanesulfonoxyl-L-proline ethyl ester 4
Compound 4 was the key intermediate in our synthetic pathways. It was
prepared as shown in Scheme 1. Alkylation of trans-4-hydroxy-L-proline
ethyl ester 2 with N-Boc-aminoethyl bromide14 gave compound 3 in an
overall yield 68%. Then compound 3 was converted to its mesylate derivative
4 in a quantitative yield according to the method described by Borcherding
et al15.
Monomers Containing Pyrimidine Bases
Alkylation of thymine with compound 4 was achieved by stirring the
mixture of compound 4, thymine, K2CO3 and 18-crown-6 in DMF at 75ꢀC
for 36 hours16. After extraction and purification by flash chromatography,
compound 5 was obtained in a 71% yield. Finally, the ethyl ester was
removed by hydrolysis, giving the thymine monomer 6 in a 79% yield
(Scheme 2). The cis-sterochemistry of compound 5 was confirmed by a
single-crystal x-ray diffraction analysis (Fig. 2).
Alkylation of cytosine with mesylate 4 was carried out under a similar
condition reported by Lewis et al17. Compound 4 reacted with sodium
cytosine in anhydrous DMF to give compound 7 as the major product in a
28% yield (Scheme 2). It was reported16 that direct displacement on an
inactive carbon with cytosine may generate N1-coupled and O2-coupled
isomers. We determined the exact structure of compound 7 by selective
DEPT NMR technique16. Irradiation H-40 of compound 7 led to selective
enhancement of C-6, C-2 and C-20 resonance at 143.5, 157.1 and 65.9 ppm,

1708
LI, JIN, AND LIU
Scheme 2.
respectively (Fig. 3). The result showed that compound 7 was the N1-coupled
isomer.
The exocyclic amino group of the nucleobase should be protected to
prevent chain extension or acetylation on it during the capping procedure in
the oligomer synthesis circles5,18. Benzyloxycarbonyl(Cbz) was used as the
Figure 2. The crystal structure of compound 5 from x-ray diffraction.

CHIRAL PNA ANALOGUE
1709
Figure 3.
protecting group for the exocyclic amino of cytosine. Thus compound 7
reacted with benzyloxycarbonyl chloride (CbzCl) to give compound 8 and
hydrolyzed to give the cytosine monomer 9 (Scheme 2).
Monomers Containing Purine Bases
For the synthesis of the adenine monomer, nucleophilic displacement of
the mesylate 4 with adenine=NaH in DMF as reported in literature15 worked
well, affording compound 10 in a 24% yield. Attempts to protect the exo-
cyclic amino group of compound 10 with CbzCl failed under a variety of
different conditions. We decided to adopt the benzoyl group (Bz), used in
conventional DNA chemistry19 as the protecting group. Compound 10
reacted with 4 eq. excess of benzoyl chloride in pyridine to give compound 11
in a 53% yield. Final removal of the ethyl ester by hydrolysis gave the
adenine monomer 12 in a 75% yield (Scheme 3).

1710
LI, JIN, AND LIU
Scheme 3.
In DNA synthesis, two deprotecting methods19 are available for
removing acyl group (including isobutyryl group, acetyl group and benzoyl
group). One was treatment with concentrated aqueous ammonia at 55ꢀC for 2
hours. Another was treatment with concentrated aqueous ammonia and 30%
methylamine (1:1) at room temperature for 90min. To test which method was
suitable for peptide synthesis, we treated a dipeptide Gly-Trp with the two
methods respectively. The result showed that the dipeptide partially degraded
in concentrated aqueous ammonia at 55ꢀC for 2 hours, however, no degra-
dation or racemization20 was observed in aqueous ammonia and methylamine
at room temperature for 5 hours. This demonstrated that the latter method
was suitable for deprotection in the PNA oligomers synthesis.
Guanine did not react with compound 4 under similar conditions par-
tially because of its poor solubility. We then used N2-isobutyrylguanine (N2-
iBuG) as the starting material. Direct alkylation21 N2-iBuG gave a mixture of
two products, the N9-isomer 13 and N7-isomer 14, in 10% and 20% yields
respectively. Hydrolysis of compound 13 gave the guanine monomer 15 in a
64% yield (Scheme 4).
Synthesis of Oligomers
Four PNA oligomers were assembled manually in a stepwise fashion
using a similar protocol of SPPS described by Dueholm5. Lysine was
incorporated at the C-terminus in order to suppress self-aggregation and
increase solubility in aqueous media5. A 4-methylbenzhydrylamine (MBHA)
resin was used as the polymeric support. Monomers 6, 9, 12 and 15 were
coupled using a 1,3-dicyclohexylcarbodiimide(DCC)-coupling protocol.
Once all the monomers had been linked one by one, the resin was treated
with concentrated aqueous ammonia-methylamine (1:1) at room temperature
for 2 hours to remove the protecting groups. After cleavage from the solid

CHIRAL PNA ANALOGUE
1711
Scheme 4.
support with anhydrous HF, the crude products were purified by sephadex
gel filtration and RP-HPLC on a Kromasil C-18 reverse phase column using
acetonitrile-water containing 0.1% TFA gradient system to give the pure
( >95%, 260nm) PNA-oligomers. All the oligomers were confirmed by TOF-
MS (Table 1). The solubility of these new PNA-oligomers in water is satis-
factory ( >15 mg=ml for (pT10)-lys).
Thermal Transition
The thermal transition curves of the above oligomers were recorded at
260nm over 6764ꢀC range (Fig. 4). Neither p(A12)-lys nor dT10 showed any
Table 1. TOF-MS Data of the Chiral PNA-Oligomers
PNA
MW (Calc.) MW (Found)
p(A12)-lys
p(T10)-lys
p(TATAAATT)-lys
p(ATTCCTTCTTCGGGAA)-lys
3424.8
3424.0
2788.02788.4
2294.5
4415.7
2299.1
4416.6

1712
LI, JIN, AND LIU
Figure 4. Melting curves (normalized) e for single strand dA12; þ for single strand dT12;6
for single strand p(A12)-lys; s for complex dA12=dT12; 7 for complex p(A12)-lys/dT10; n for
complex p(A12)-lys=d(T4GT5).
clear hyperchromicity, indicating that no self-aggregation occurred. The
complex p(A12)-lys=dT10 displayed a well defined single-phased melting
profile (Fig. 4), with 40% hypochromicity and a melting temperature(Tm) of
46ꢀC, higher than that of dA12=dT12 (Fig. 4, Table 2). It suggested that
p(A12)-lys can bind strongly to dT10. The complex p(A12)-lys=d(50-T4GT5-30),
containing one mismatched base pair, exhibited a 13ꢀC decrease in Tm
(Fig. 4, Table 2), in comparison to fully complementary p(A12)-lys=dT10.
This demonstrated that binding of p(A12)-lys with complementary DNA was
sequence specific.
Table 2. Tm Values of Complexes Between the Chiral PNA and DNA*
Hybrid Complex
Tm (ꢀC)
Hypochromicity (%)
dA12=dT12
p(A12)-lys=dT10
p(T10)-lys=dA10
p(TATAAATT)-lys=
d(50-ATATTTAA-3’)
p(TATAAATT)-lys=
d(50-AATTTATA-3’)
p(A12)-lys=d(T4GT5)
28
46
n.d.
n.d.
35.0
40.7
9.0
5.6
n.d.
33
9.0
24.6
n.d.: not detectable.
*Experiment condition: Buffer containing 0.1M NaCl, 0.02 M (CH3)2-
AsO2Na, pH7.0.
Each complex was composed of 1:1 stoichiometry of two component parts.

CHIRAL PNA ANALOGUE
1713
Figure 5. Melting curves. 7 for complex dA10=dT10; 6 for complex dA10=p(T10)-lys; n for
complex dA10=2[p(T10)-lys].
As p(T10)-lys had the same backbone as p(A12)-lys, it was expected to
bind with its DNA target, however, no distinct melting transition step was
detected for p(T10)-lys=dA10 (both 1:1 and 1:2) mixture over a 1797ꢀC range
(Fig. 5). According to the results of D’Coster et al.13, the Tm of p(T10)-
lys=dA10 complex might be too high to be detected. To clarify this, UV-
mixing curves were measured at 260and 280nm, but no clear transition point
at 1:1 or 1:2 (A=T, mole=mole) was observed (Fig. 6). It seemed that p(T10)-
lys did not bind with its complementary DNA target. We also synthesized a
shorter, mix-sequenced oligomer p(TATAAATT)-lys and evaluated its
hybridization properties. No melting step was observed (melting curves not
shown). It demonstrated that thymine on the chiral PNA could not pair with
adenine on DNA.
Figure 6. Mixing curve for dA10=(pT10)-lys m for 260nm data, r for 280nm data total
conc. ¼ 5 mM, measured at 16ꢀC.

1714
LI, JIN, AND LIU
EXPERIMENTAL SECTION
Reagents and solvents were obtained commercially and used without
1
further purification unless indicated. H and 13C NMR spectra were mea-
sured on JNM-GX 400MNMR spectrometers. Chemical shifts were recorded
in ppm relative to tetramethylsilane(TMS). FAB-MS spectra were recorded
on a Zabspec mass spectrometer. Melting points were uncorrected.
Chemical Synthesis
N-(2-Boc-amino)ethyl-trans-4-hydroxy-L-proline ethyl ester (3)
Triethylamine (32 ml, 0.23 mol) was added dropwise to a stirred solution
of compound 1 (24.61 g, 0.11 mol) and compound 2 (19.58 g, 0.10 mol) in
anhydrous DMF (300 ml) at room temperature and the reaction mixture was
left to stir overnight. The mixture was filtered and evaporated to dryness. The
oily residue was dissolved in EtOAc (300 ml) and washed with a saturated
solution of NaHCO3 (100 ml) and brine (50 ml). Then the solution was
acidified to pH 2 with 10% citric acid (aqueous). The water phase was neu-
tralized to pH 7 with NaHCO3 and extracted with EtOAc (36150ml). The
combined extracts were dried over anhydrous MgSO4 and then evaporated to
dryness invacuum. Compound 3 was obtained as yellow syrup (20.61 g, 68%).
1HNMR(CDCl3) d1.25(t, 3H), 1.41(s, 9H), 2.073.6(m, 10H), 4.15(q,
2H), 4.44(m, 1H). 5.26(s, 1H); MS(FAB): m=z 303.2(MþH)þ, 247.1(M-
tBu)þ, 222.9(M-tBuO)þ, 203.1(M-Boc)þ; Anal. Calcd for C14H26N2O5: C,
55.16; H, 8.67; N,9.27. Found: C, 55.16; H, 8.85; N, 9.46.
N-(2-Boc-amino)ethyl-trans-4-methanesulfonyloxy-L-proline ethyl ester (4)
A solution of compound 3 (18.01 g, 60 mmol) and methanesulfonyl
chloride (10.31 g, 90 mmol) in CH2Cl2 (500 ml) was cooled in an ice bath.
Triethylamine (12.12 g, 120mmol) was added dropwise. After the addition
was complete, the reaction mixture was allowed to come to room tempera-
ture. The reaction was judged to be complete as TLC showed the absence of
compound 3 (4:1 EtOAc=petroleum ether). The mixture was washed with
water (100 ml) and 5% aqueous NaHCO3 (100 ml). The aqueous layers were
extracted with CH2Cl2(2650ml). The combined organic layers were dried
over anhydrous Na2SO4 and concentrated to give 4 as yellow syrup (23 g,
100%). This material was used immediately without further purification.
1HNMR(CDCl3) d1.29(t, 3H), 1.45(s, 9H), 2.274.0(m, 10H), 4.15(q,
2H), 5.2475.25(m, 2H); MS(FAB) m=z 381.1(MþH)þ, 323.1(MꢁtBu)þ,
307.1(MꢁtBuO)þ; Anal. Calcd for C15H28N2O7S: C, 47.36; H, 7.42; N, 7.36.
Found: C, 47.11; H, 7.51; N, 7.35.

CHIRAL PNA ANALOGUE
1715
N-(2-Boc-amino)ethyl-cis-4-(thymin-1-yl)-L-proline ethyl ester (5)
A suspension of compound 4 (7.60g, 20mmol), thymine (3.78 g,
30mmol), anhydrous K 2CO3 (5.53 g, 40mmol) and 18-crown-6 (10.56 g,
ꢀ
40mmol) in DMF (250ml) was stirred at 75 C for 36 hours. The solids in the
reaction mixture were filtered and washed thoroughly with EtOAc. The
combined filtrate was concentrated and the residue was purified by flash
chromatography using 7:3 EtOAc=petroleum ether (60790ꢀC) as the eluting
solvent. Compound 5 was obtained as yellow viscous oil (3.34 g, 40%).
Crystallization from EtOAc gave the product as colorless crystals.
m.p.:1417143ꢀC.
1HNMR(CDCl3) d1.28(t, 3H), 1.48(s, 9H), 1.92(s, 3H), 1.873.4(m,
10H), 4.18(q, 2H), 5.22 (br, 1H), 7.92(s, 1H); 9.0(s, 1H); 13CNMR(CDCl3)
d12.61, 14.10, 28.34, 36.66, 38.79, 51.98, 52.63, 58.03, 61.44, 64.80, 79.22,
111.30, 137.80, 151.35, 155.90, 164.02, 173.21; HRMS m=z 411.2234(MþH)þ,
calcd. for C19H30N4O6þH ¼ 411.2238; 311.1711(MꢁBoc)þ, calcd. for
C14H22N4O4þH ¼ 311.1711; Anal. Calcd for C19H30N4O6: C, 55.60; H, 7.37;
N, 13.65. Found: C, 55.78; H, 7.46; N, 13.79.
N-(2-Boc-amino)ethyl-cis-4-(cytosin-1-yl)-L-proline ethyl ester (7)
Sodium hydride (60% disp., 3.00 g, 75 mmol) was added to a vigorously
stirred suspension of cytosine (8.33 g, 75 mmol) in anhydrous DMF (280ml)
at 50ꢀC. After hydrogen production had ceased, compound 4 (9.5 g, 25 mmol)
was added and the reaction mixture was left to stir for a week. The solids in the
mixture were filtered and washed thoroughly with EtOAc. The combined
filtrate was concentrated and the residue was dissolved in EtOAc (100 ml) and
filtered again to remove the undissolved solids. The filtrate was purified by
flash chromatography using 8:2 EtOAc=EtOH as the eluting solvent. Com-
pound 7 was obtained as white powder (4.0g, 27.5%). m.p.: 1427144ꢀC.
1HNMR(CDCl3) d1.29(t, 3H), 1.46(s, 9H), 1.973.4(m, 10H), 4.21(q,
2H), 5.14(br, 1H), 5.28 (m, 1H), 6.18(d, 1H), 8.22(d, 1H); 13CNMR(CDCl3)
d14.85, 29.18, 37.72, 39.75, 53.71, 54.22, 59.28, 61.98, 65.89, 79.86, 95.84,
143.88, 156.75, 156.99, 165.83, 174.08; MS(FAB) m=z 791.5(2MþH)þ,
396.3(MþH)þ, 322.3(MꢁtBuO)þ, 296.3(MꢁBoc)þ; Anal. Calcd for
C18H29N5O5: C, 54.67; H, 7.39; N, 17.71. Found: C, 54.78; H, 7.38; N, 17.84.
N-(2-Boc-amino)ethyl-cis-4-((N4-benzoxycarbonyl)cytosine-1-yl)-L-proline
ethyl ester (8)
A solution of benzoxycarbonyl chloride (3 ml, 17.4 mmol) in anhydrous
pyridine (10ml) was carefully added dropwise to a stirred solution of com-
pound 7 (1.0 g, 2.5 mmol), DMAP (0.08 g, 0.62 mmol) in anhydrous pyridine

1716
LI, JIN, AND LIU
(50ml) at 0 ꢀC. The reaction was allowed to warm slowly to room temperature
before being left to stir overnight. Subsequently, the reaction was poured into
a saturated solution of NaHCO3 (6 ml) and extracted with EtOAc (3650ml).
The combined organic extracts were evaporated to dryness in vacuum. The
residue was purified by flash chromatography using ethyl acetate as the eluting
solvent. Compound 8 was obtained as foam (1.03 g, 77%).
1HNMR(CDCl3) d1.26(t, 3H), 1.43(s, 9H), 1.973.4(m, 10H), 4.18(q,
2H), 5.1075.36 (m, 3H), 7.35(s, 5H); 7.27(d,1H), 8.22(s, 1H); MS(FAB) m=z
530.2(MþH)þ, 472.1(MꢁtBu)þ, 456.1(MꢁtBuO)þ, 430.1(MꢁBoc)þ; Anal.
Calcd. for C26H35N5O7: C, 58.97; H, 6.66; N, 13.22. Found: C, 58.97; H,
6.87; N, 13.11.
N-(2-Boc-amino)ethyl-cis-4-(adenin-9-yl)-L-proline ethyl ester (10)
Sodium hydrid (60%, 1.78 g, and 44.5 mmol) was added to a vigor-
ously stirred suspension of adenine (6.01 g, 44.5 mmol) in anhydrous DMF
(100 ml). The reaction mixture was stirred at 55760ꢀC for 3 hours. Then
compound 4 (5.64 g, 14.8 mmol) was added and the reaction mixture was
left to stir for 2 days. The solids in the mixture were filtered and washed
thoroughly with EtOAc. The filtrate was evaporated to dryness and the
resulting residue was purified by flash chromatography using 8:2
EtOAc=EtOH as the eluting solvent. Compound 10 was afforded as viscous
oil (1.5 g, 24%).
1HNMR(CDCl3) d1.26(t, 3H), 1.43(s, 9H), 1.873.5(m, 10H), 4.19(q,
2H), 5.09(s, 1H), 5.28(m, 1H), 8.29(s, 1H), 8.60(s, 1H); MS(FAB) m=z
420.1(MþH)þ, 364.1(MꢁtBu)þ, 346.1(MꢁtBuO)þ, 320.1(MꢁBoc)þ; Anal.
Calcd for C19H29N7O4: C, 54.40; H, 6.97; N, 23.37. Found: C, 54.35; H, 6.97;
N, 23.20.
N-(2-Boc-amino)ethyl-cis-4-((N6-dibenzoyl)adenin-9-yl)-L-proline
ethyl ester (11)
A solution of benzoyl chloride (1.5 ml, 10mmol) in anhydrous pyridine
(5 ml) was carefully added dropwise to a stirred solution of compound 10
(1.05 g, 2.5 mmol) in anhydrous pyridine (5 ml) at 0ꢀC. The reaction was
allowed to warm slowly to room temperature before being left to stir over-
night. Subsequently, the reaction mixture was poured into a cooled saturated
solution of NaHCO3 (20ml) and extracted with ethyl ether (3 620ml). The
combined organic extracts were evaporated to dryness in vacuum to give a
crude yellow oil which was purified by flash chromatography using 6:4 ethyl
acetate=petroleum ether (60790ꢀC) as the eluting solvent. Compound 11 was
obtained as foam (0.7 g, 53%).

CHIRAL PNA ANALOGUE
1717
1HNMR(CDCl3) d1.20(s, 3H), 1.36(s, 9H), 1.573.5(m, 10H), 4.19(q,
2H), 5.09(s, 1H), 5.32(s, 1H), 7.277.8(m, 10H), 8.55(s, 1H), 8.70(s, 1H);
MS(FAB) m=z 628.1(MþH)þ, 524.0(MꢁBz)þ, 343.9(Bz2A)þ; Anal. Calcd for
C33H37N7O6: C, 63.15; H, 5.94; N, 15.62. Found: C, 62.94; H, 5.90; N, 15.68.
N-(2-Boc-amino)ethyl-cis-4-((N2-isobutyryl)guanin-9-yl)-L-proline
ethyl ester (13)
A mixture of N2-isobutyryl-guanine (2.76 g, 12.5 mmol), K2CO3 (1.73 g,
12.5 mmol), and compound 4 (1.90g, 5.0mmol) in DMF (30ml) was stirred
at 50ꢀC for 3 days. The solvent was removed under reduced pressure and the
residue was purified by flash chromatography with 1:2 acetone=petroleum
ether (30760ꢀC) as the eluting solvent. The more polar fraction were com-
bined and evaporated to give the title compound as foam (0.26 g, 10%).
1HNMR(d6-DMSO) d1.10(s, 3H), 1.14(d, 6H), 1.35(s, 9H), 2.23(m, 1H),
2.074.4(m, 10H), 4.20(q, 2H), 4.92(d, 1H), 8.18(s, 1H); 13CNMR(d6-DMSO)
d13.8, 18.7, 28.1, 34.7, 36.2, 38.7, 51.9, 53.3, 58.3, 60.4, 64.4, 77.5, 119.6,
137.9, 147.7, 148.2, 154.9, 155.5, 172.6, 180.0; MS(FAB) m=z 506.3(MþH)þ,
406.2(MꢁBoc)þ, 222.1(iBuGþH)þ.
General Procedure for Preparing Monomer 6, 9, 12, 15
In each case, a 2 M aqueous solution of sodium hydroxide (3.0eq.) was
added to a stirred solution of ester 5, 8, 11 or 14 (1 eq.) in methanol at room
temperature. The reaction was judged to be complete as TLC showed the
absence of the ester. The reaction mixture was then cooled to 0ꢀC and
acidified to pH5 with 1 M HCl (aqueous).
N-(2-Boc-amino)ethyl-cis-4-(thymine-1-yl)-L-proline (6)
The above procedure was followed using compound 5 (0.41 g, 1 mmol)
as starting material. After acidification, the solution was evaporated to
dryness and the residue was redissolved in ethanol (20ml). The solid material
was removed by filtration and the filtrate was purified by flash chromato-
graphy using 9:1 CH2Cl2=MeOH as the eluting solvent to give the product as
foam (0.30 g, 79%).
1HNMR(CD3OD) d1.43(s, 9H), 1.88(s, 3H), 1.874.0(m, 9H), 4.83(s,
1H), 7.67(s, 1H); MS(FAB) m=z 405.0(MþNa)þ, 383.0(MþH)þ.
N-(2-Boc-amino)ethyl-cis-4-((N4-benzoxycarbonyl)cytosin-1-yl)-L-proline (9)
The above procedure was followed using compound 8 (1.05 g, 2 mmol)
as starting material. After acidification, the solution was evaporated to

1718
LI, JIN, AND LIU
dryness and the residue was redissolved in methanol (5 ml). The solid
material was removed by filtration and the filtrate was purified by flash
chromatography using 9:1 CH2Cl2=MeOH as the eluting solvent to give the
product as foam (0.55 g, 55%).
1HNMR(CD3OD) d1.43(s, 9H), 1.973.4(m, 9H), 5.1075.36(m, 4H),
7.35(s, 5H), 7.27(d, 1H), 8.22(s, 1H); MS(FAB) m=z 1002.8(2MþH)þ,
524.1(MþNa)þ, 502.2(MþH)þ.
N-(2-Boc-amino)ethyl-cis-4-((N6-benzoyl)adenin-9-yl)-L-proline (12)
The above procedure was followed using compound 11 (2.62 g,
4.2 mmol) as starting material. After acidification, the solution was evapo-
rated to dryness and the residue was redissolved in methanol (10ml). The
solid material was removed by filtration and the filtrate was purified by flash
chromatography using 9:1 CH2Cl2=MeOH as the eluting solvent to give the
product as foam (1.56 g, 75%).
1HNMR(CD3OD) d1.37(s, 9H), 2.073.5(m, 9H), 4.91(s, 1H), 5.23(m,
1H), 7.90(s, 1H), 8.20(s, 5H), 8.77(s, 1H); MS(FAB) m=z 518.1(MþNa)þ,
496.1(MþH)þ, 396.1(MꢁBoc)þ.
N-(2-Boc-amino)ethyl-cis-4-((N2-isobutyryl)guanin-9-yl)-L-proline (15)
The above procedure was followed using compound 13 (0.30 g,
0.59 mmol) as starting material. After acidification, the solution was evapo-
rated to dryness and the residue was redissolved in methanol (2 ml). The solid
material was removed by filtration and the filtrate was purified by flash
chromatography using 1:1 CH2Cl2=MeOH as the eluting solvent to give the
product as foam (0.18 g, 64%).
1HNMR(d3-DMSO) d1.14(d, 6H), 1.35(s, 9H), 2.23(m, 1H), 2.074.4(m,
10H), 4.92(d, 1H), 8.18(s, 1H); 13CNMR(d3-DMSO) d18.7, 28.8, 34.7, 37.2,
40.2, 53.4, 54.6, 59.8, 66.0, 80.2, 117.0, 138.6, 152.4, 152.7, 155.2, 158.9,
175.4, 180.0; MS(FAB) m=z 478.2 (MþH)þ.
Oligomerization (General Protocol)
The chiral PNA-oligomers were prepared by standard solid-phase
peptide synthesis. The syntheses were initiated on a MBHA resin
(0.55 mmol=g) from C terminal to N terminal. DCC was used as coupling
reagent. The detailed procedure was as follows: 1) Washing the resin with
CH2Cl2, MeOH, CH2Cl2, 262 min; 2) neutralizing with N,N-diisopropyl-
ethylamine (DIEA)=CH2Cl2 (8%, v=v), 265 min; 3) Washing with CH2Cl2,
MeOH, CH2Cl2, 262 min; 4) Coupling with Boc-lys(2Cl-Cbz)-OH(3 eq.) or

CHIRAL PNA ANALOGUE
PNA monomer(2 eq.),
1719
1-hydroxybenzotriazole(HOBt)(4 eq.)
and
DCC(4 eq.), using DMF as solvent, 478 hours; 5) Washing with CH2Cl2,
MeOH, CH2Cl2, 262 min; 6) Monitoring the coupling using Kaiser test, if
negative, go on with next step; Otherwise capping the unreacted amino group
with Ac2O=DIEA=CH2Cl2 (3:1:30, v =v=v), 1620min, washing with CH 2Cl2,
MeOH, CH2Cl2, 262 min; 7) Deprotecting Boc with TFA=CH2Cl2 (1:1,
v=v), 165 min, 1630min. Steps 177 were repeated until the required
sequence was obtained.
Stepwise Assembly of H-T10-lys-NH2
The synthesis was initiated on 100 mg MBHA resin. After the final
coupling, the resin was dried under vacuum. The oligomer was cleaved from
the support with HF for 1 hour at 0ꢀC. After sufficient washing and lyo-
philisation, the obtained fluffy solid was purified by sephadex gel filtration:
yield 45 mg crude PNA (purity: 44%, according to HPLC at 260nm). A
portion of the sample was purified by RP-HPLC purification to give the pure
oligomer for further use. MS(TOF) m=z 2788.4, (calc.2788.0).
Stepwise Assembly of H-A12-lys-NH2
The synthesis was initiated on 100 mg MBHA resin. After the final
coupling, the resin was treated with concentrated aqueous ammonia-
methylamine (1:1) at room temperature for 2 hours to remove the protecting
group on adenine. After cleavage from the solid support and purification by
sephadex gel filtration, 35 mg crude PNA were obtained (purity: 41%,
260nm). A portion of the sample was purified by RP-HPLC purification to
give the pure oligomer for further use. MS(TOF) m=z 3424.0, (calc.3424.8).
Stepwise Assembly of H-TATAAATT-lys-NH2
The synthesis was initiated on 50mg MBHA resin. After deprotection,
cleavage and sephadex gel filtration, 52 mg crude PNA were obtained (purity:
87%, 260nm). A portion of the sample was purified by RP-HPLC purification
to give the pure oligomer for further use. MS(TOF) m=z 2299.1, (calc.2294.5).
Stepwise Assembly of H-ATT CCT TCT TCG GGA A-lys-NH2
The synthesis was initiated on 100 mg MBHA resin. After deprotection,
cleavage and sephadex gel filtration, 79 mg crude PNA were obtained (purity:
34%, 260nm). A portion of the sample was purified by RP-HPLC purification
to give the pure oligomer for further use. MS(TOF) m=z 4416.6, (calc.4415.7).

1720
LI, JIN, AND LIU
Thermal Transition Measurement
UV-melting curves and wavelength scan were recorded using an UV-260
spectrophotometer. The oligomers concentration was determined by mea-
suring the absorbance at 260nm and assuming the following extinction
coefficients: T ¼ 8.5, A ¼ 13.0, C ¼ 7.5, G ¼ 11.7 ml=mmol.cm. The two com-
plementary strands were dissolved in a buffer containing 0.1 M NaCl, 0.02 M
sodium cacodylate, pH 7.0. The solution was heated to 95ꢀC for 10minutes
and then cooled slowly to 6ꢀC. Absorbance vs. temperature curve was
measured at a heating rate of 0.3ꢀC=min. Tm value was determined as the
temperature of the maximum in the first derivative of dissociation curves.
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1721
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acetic acid), and the optical rotation of the controlled dipeptide is [a]20 ¼ 16.81,
(c ¼ 0.0963, 1 M acetic acid).
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Received September 19, 2000
Accepted March 29, 2001