Synthesis of new peptide nucleic acid monomer with glycylglycine backbone

Article abstract of DOI:10.1002/jhet.5570430445

New thymine peptide nucleic acid (PNA) monomer with glycylglycine backbone was prepared. This involved a key step of the coupling between iodinated serine (3) and 3-benzoylthymine.

Full text of DOI:10.1002/jhet.5570430445

Jul-Aug 2006
SynthesisꢀofꢀNewꢀPeptideꢀNucleicꢀAcidꢀMonomer withꢀꢀ
ꢁꢁꢁꢁꢀ
Glycylglycine Backbone
Tetsuo Yamasaki [a]*, Mohamed Abdel-Aziz [b], Takashi Iwashita [b],
Akiko Watanabe [a], Masanori Sakamoto [a] and Masami Otsuka [b]
[a] School of Pharmaceutical Sciences, Kyusyu University of Health and Welfare, 1714-1 Yoshino-cho, Nobeoka
City, Miyazaki 882-8508, Japan, [b] Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, 5-
1 Oe-Honmachi, Kumamoto 862-0973, Japan.
Received October 5, 2005
Bz
N
O
O
N
OH
I
O
2 steps
3 steps
FmocHN
N
H
CO2H
BocHN
CO2Bn
BocHN
CO2Bn
1
3
6
New thymine peptide nucleic acid (PNA) monomer with glycylglycine backbone was prepared. This
involved a key step of the coupling between iodinated serine (3) and 3-benzoylthymine.
J. Heterocyclic Chem., 43, 1111 (2006).
Introduction.
B: Nucleobase
NH
O
NH
B
Aminoethylglycine peptide nucleic acid (aegPNA) is an
analogue with the deoxyribose phosphate replaced by a
polyamide backbone [1-3]. In spite of such a dramatic
structural change, aegPNA shows a high affinity to DNA
and RNA in a sequence-specific fashion. As polyamide
based nucleic acid analogues have the great potential as
tools in many biological applications, development of
novel analogues of PNA became an attractive area of
research soon after aegPNA was reported by Nielsen and
co-workers [4-8].
We previously reported on the binding activity to DNA
and RNA with aegPNAs including PNA monomers with
glycylalanine backbone (isogaPNA monomers) [9].
IsogaPNA is 2',5'-isoDNA mimic chiral peptide nucleic
acid that is one of interesting antisense compounds having
the preferential binding activity to RNA [10]. However,
unfortunately the incorporation of isogaPNA monomers
to aegPNAs resulted in decrease of hybridization
properties to DNA and RNA. The decreased Tm values
compared to unmodified aegPNAs indicate that the
glycylalanine backbone of isogaPNA may not be optimal
in length to hybridize with DNA and RNA. In this
context, we examined the preparation of PNA monomer
with glycylglycine backbone (isoggPNA monomer), in
which length is different from that of isogaPNA, in order
to investigate the hybridization property of isoggPNA
with DNA and RNA (Figure 1).
HN
O
O
B
O
B
O
B
HN
N
O
HN
O
O
P
O
O
n
n
n
O
n
O
isogaPNA
isoggPNA
aegPNA
2',5'-isoDNA
Figure 1. Structures of aegPNA, 2',5'-isoPNA, isogaPNA and isoggPNA.
thymine [11] under the Mitsunobu conditions by the
same method as that for the coupling between hydroxy-
methylalanine and 3-benzoylthymine in the preparation
of isogaPNA monomers resulted in failure (the starting
material 3-benzoylthymine was recovered). The next
attempt of the coupling with tosylated compound (2)
[12] and 3-benzoylthymine under basic conditions did
not proceed either (tosylation of BocSer methyl ester
underwent facile ꢀ elimination at the tosylated position
to give methyl acrylate compound). To this end,
compound (2) was converted to the corresponding
iodide (3), that was further reacted with 3-benzoyl-
thymine, affording compounds (4a) and (4b) in 68 %
and 11 % yields successfully after flash silica gel
chromatography and subsequent recrystallyzation.
Removal of the Boc group of 4a followed by the
coupling with FmocGly in the presence of TBTU,
finally reductive cleavage of benzyl ester of 5 by the
treatment with 10% Pd-C and H2 gas gave thymine
isoggPNA monomer (6) (Scheme 1).
Results and Discussion.
The synthesis of thyminylmethylglycine (4) is the key
step for the preparation of thymine isoggPNA monomer
(6). The coupling reaction of L-serine with 3-benzoyl-
In summary, we have achieved the synthesis of novel
thymine isoggPNA monomer with the different length of
backbone from that of isoga PNA monomers. It's

1112
T. Yamasaki, M. Abdel-Aziz, T. Iwashita, A. Watanabe, M. Sakamoto and M. Otsuka
Vol 43
These compounds were prepared according to the literature
methods [11].
Scheme 1
Tosylated compound (2): 1H-NMR (300 MHz, CDCl3) ꢀ:
1.34(s, 9H, CMe3), 2.35(s, 3H, Me), 4.17-4.26(m, 1H, CHH),
4.30-4.37(m, 1H, CHH), 4.44-4.51(m, 1H, CH), 5.02(d, J=12.0
Hz, 1H, PhCHH), 5.10((d, J=12.0 Hz, 1H, PhCHH), 5.24(d,
J=7.6 Hz, 1H, NH), 7.10-7.36(m, 7H, Ph and arom), 7.64(d,
J=8.3 Hz, 2H, arom).
DEAD, Ph3P,
3-benzoylthymine
DMF
OH
4a
BocHN
CO2Bn
1
Iodide compound (3): 1H-NMR (300 MHz, DMSO-d6) ꢀ:
1.37(s, 9H, CMe3), 3.29-3.38(m, 1H, CHH), 3.47-3.55(m, 1H,
CHH), 3.47-3.55(m, 1H, CH), 5.13(s, 2H, CH2), 7.35(s, 5H,
Ph), 7.45(d, J=8.1 Hz, 1H, NH).
TosCl, pyridine,
-10oC
I
OTos
NaI, acetone
BocHN
CO2Bn
BocHN
CO2Bn
3-(3-Benzoyl-5-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-
yl)-2-tert-butoxycarbonylaminopropionic acid benzyl ester (4a)
and 2-tert-Butoxycarbonylamino-3-(5-methyl-2,4-dioxo-3,4-
dihydro-2H- pyrimidin-1-yl)propionic acid benzyl ester (4b).
2
3
3-benzoylthymine,
K2CO3, DMF
3-benzoylthymine,
K2CO3, DMF
To a solution of iodide compound (3) (4.06 g, 10 mmol) and
3-benzoylthymine (4.60 g, 20 mmol) in DMF (30 mL) was
added anhydrous K2CO3 (2.76 g, 20 mmol) at rt. After stirring
for 12 h, the solvent was evaporated to dryness. The resulting
residue was dissolved in ethyl acetate (200 mL), washed with
water (200 mL), and then dried (MgSO4). After concentration of
the solution, the crude products were purified by flash
chromatography on silica gel with ethyl acetate:hexane (1:2) to
give 4a (68 %) and 4b (11%). Analytical samples were further
purified by recrystalization from ethyl acetate - hexane.
4a
Bz
N
H
O
O
N
O
O
+
N
N
BocHN
CO2Bn
BocHN
CO2Bn
4a
4b
(i) conc. HCl, dioxane
(ii) FmocGly, TBTU, HOBt, DMF
25
1
4a: mp 133-134ºC; [ꢁ]D -3.2º (c 0.1, methanol); H-NMR
(300 MHz, CDCl3) ꢀ: 1.43(s, 9H, CMe3), 1.90(s, 3H, Me), 3.70-
3.85(m, 1H, CHH), 4.34-4.42(m, 1H, CHH), 4.59-4.70(m, 1H,
CH), 5.07(d, J=12.0Hz, 1H, PhCHH), 5.19(d, J=12.0 Hz, 1H,
PhCHH), 5.47(d, J=6.6 Hz, 1H, NH), 7.02(s, 1H, CH-6 of
Thymine), 7.33(s, 5H, Ph), 7.48(dd, J=7.8 and 7.8, 2H, PhC=O),
7.63(dd, J=7.8 and 7.8 Hz, 1H, PhC=O), 8.04(d, J=7.8 Hz, 2H,
PhC=O); 13C-NMR(75 MHz, CDCl3) ꢀ: 12.30(Me), 28.21(Me),
50.47(CH2), 52.26(CH), 68.08(CH2), 80.68(C), 110.41(C),
128.53(CH),128.63(CH), 129.01(CH), 130.68(CH), 131.59(C),
134.68(C), 134.89(CH), 140.46(CH), 150.06(C), 155.22(C),
163.04(C), 168.86(C), 169.39(C); FAB-MS m/z: 508 (M++1);
Anal. Calcd for C27H29N3O7: C, 63.89; H, 5.76; N, 8.28. Found:
C, 63.98; H, 5.73; N, 8.30.
4b: mp 191-192ºC; [ꢁ]D 1.0º (c 0.1, methanol); H-NMR
(300 MHz, CDCl3) ꢀ: 1.41(s, 9H, CMe3), 1.85(s, 3H, Me),
4.02(dd, J=14.2 and 6.8 Hz, 1H, CHH), 4.20(dd, J=14.2 and 5.6
Hz, 1H, CHH), 4.52(dd, J=6.8 and 5.6 Hz, 1H, CH), 5.16(d,
J=12.0Hz, 1H, PhCHH), 5.22(d, J=12.0 Hz, 1H, PhCHH),
5.46(br, 1H, NH), 6.92(s, 1H, CH-6 of Thymine), 7.34(s, 5H,
Ph), 8.25(brs, 1H, NH); 13C-NMR(75 MHz, CDCl3) ꢀ:
11.95(Me), 27.98(Me), 48.12(CH2), 51.70(CH), 66.38(CH2),
78.72(C), 108.03(C), 127.89(CH),128.11(CH), 128.39(CH),
135.58(C), 141.80(CH), 150.87(C), 155.26(C), 164.20(C),
169.76(C); FAB-MS m/z: 404 (M++1); Anal. Calcd for
C27H29N3O7: C, 59.54; H, 6.25; N, 10.42. Found: C, 59.65; H,
5.95; N, 10.23.
Bz
N
Bz
N
O
N
O
O
O
H2,
10 % Pd-C,
MeOH
N
O
O
FmocHN
FmocHN
N
CO2H
N
CO2Bn
H
H
5
6
Synthesis of 3-(3-Benzoyl-5-methyl-2,4-dioxo-3,4-dihydro-2H- pyrimidin-1-yl)-
2-[2-(9H-fluoren-9-ylmethoxycarbonylamino)acetyl-amino]propionic acid (6).
incorporation into aegPNA oligomers and binding studies
with DNA and RNA are currently under progress.
23
1
EXPERIMENTAL
All the melting points were determined on a Yanagimoto
micromelting point apparatus and are uncorrected. H and 13C
1
NMR and IR spectra were taken with a JEOL JNM-AL 300 and
a JASCO JIR-6500W. Chemical shifts were determined using
tetramethylsilane as an internal standard. Mass spectra were
obtained from a JEOL JNS-DX303HF Mass Spectrometer.
Optical rotation was measured on a JASCO DIP-1000 KUY
digital polarimeter. All flash column chromatography was
performed using flash-grade silica gel (Kanto Kagaku, 40-100,
60N).
3-(3-Benzoyl-5-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-
yl)-2-[2-(9H-fluoren-9-ylmethoxycarbonylamino)acetylamino]
propionic acid benzyl ester (5).
Benzyl 2(S)-(tert-butoxycarbonylamino)-3-p-toluenesulfonyl-
propionate (2) and Benzyl 2(R)-(tert-butoxycarbonylamino)-3-
iodopropionate (3).
To a solution of compound (4a) (0.51 g, 1 mmol) in 1,4-
dioxane (10 mL) was added conc. HCl (8 mL). The reaction

Jul-Aug 2006
SynthesisꢀofꢀNewꢀPeptideꢀNucleicꢀAcidꢀMonomerꢀwith Glycylglycine Backbone
1113
mixture was stirred at rt till the spot of 4a disappeared on TLC.
The solution was neutralized with saturated NaHCO3, extracted
with CH2Cl2 (3 x 100 mL), and then dried (MgSO4). The organic
solvent was evaporated to get the crude deprotected compound of
Boc as an oil. After drying under reduced pressure for 24 h, the oil
compound was used in the subsequent coupling without
purification. Diisopropylethylamine (0.35 mL, 2 mmol) was added
to a stirred solution of the crude oily compound, FmocGly (0.30 g,
1 mmol), HOBt (0.15 g, 1 mmol), and TBTU (0.32 g, 1 mmol) in
DMF (6 ml) under argon at rt. After stirring for 1.5 h, the solvent
was evaporated under reduced pressure. The residue was dissolved
in CH2Cl2 (100 mL) and the solution was washed consecutively
with H2O (80 mL), 4% aq. NaHCO3 (3 x 80 mL), 5% aq. KHSO4
(3 x 80 mL), H2O (2 x 80 mL), dried (MgSO4), and the organic
solvent was evaporated under reduced pressure. The residue was
purified by flash chromatography on silica gel with ethyl
acetate:n-hexane (3:1) and then recrystallization from ethyl
alcohol-diethyl ether to give 5 (74% from 4a). mp 194-195ºC;
was purified by flash chromatography on silica gel (ethyl acetate-
methyl alcohol gradient) to give 6 (87%) as a white solid. Analytical
sample was purified by recrystallization from CH2Cl2 - n-hexane.
mp 183-184ºC; [ꢀ]D25 -9.4º (c 0.1, methanol); 1H-NMR (300 MHz,
CDCl3) ꢁ: 1.78(s, 3H, Me), 3.55-3.70(m, 3H, CH2 and CHH), 4.18-
4.28(m, 3H, CH2 and CHH), 4.37-4.45(m, 2H, CH x 2), 7.25-
7.41(m, 4H, arom), 7.27(s, 1H, CH-6 of Thymine), 7.42-7.56(m,
3H, Ph), 7.61-7.75(m, 3H, Ph and NH), 7.81(br, 1H, NH), 7.87(d,
J=7.5 Hz, 2H, arom), 8.04(d, J=7.5 Hz, 2H, arom); 13C-NMR (75
MHz, DMSO-d6) ꢁ: 11.74(Me), 43.67(CH2), 46.60(CH),
51.27(CH2), 52.28(CH), 65.80(CH2), 107.63(C), 120.09(CH),
125.21(CH), 127.09(CH), 127.61(CH), 129.24(CH), 130.56(CH),
131.32(C), 135.13(CH), 140.69(C), 142.78(CH), 143.80(C),
149.45(C), 156.47(C), 163.03(C), 168.94(C), 170.01(C), 171.12(C);
FAB-MS m/z: 596 (M++1); Anal. Calcd for C32H28N4O8: C, 64.42;
H, 4.73; N, 9.39. Found: C, 64.44; H, 4.88; N, 9.12.
REFERENCES
25
1
[ꢀ]D 5.0º (c 0.1, methanol); H-NMR (300 MHz, CDCl3) ꢁ:
1.87(s, 3H, Me), 3.78-3.85(m, 2H, CH2), 4.13-4.28(m, 2H, CH2),
4.39(d, J=6.9 Hz, 2H, CH2), 4.82-4.91(m, 1H, CH), 5.08(d, J=12.0
Hz, 1H, PhCHH), 5.17(d, J=12.0 Hz, 1H, PhCHH), 5.30-5.38(m,
1H, CH), 7.02(d, J=5.2 Hz, 1H, NH), 7.28-7.48(m, 7H, arom),
7.33(s, 5H, Ph), 7.52-7.62(m, 5H, arom and NH), 7.76(d, J=7.4
Hz, 2H, arom), 7.93(d, J=7.4 Hz, 2H, arom); 13C-NMR (75 MHz,
CDCl3) ꢁ: 12.24(Me), 44.54(CH2), 47.04(CH), 49.51(CH2),
52.11(CH), 67.33(CH2), 68.28(CH2), 111.37(C), 120.00(CH),
125.05(CH), 127.08(CH), 127.76(CH), 128.63(CH), 128.72(CH),
128.81(CH), 129.10(CH), 130.60(CH), 131.44(C), 134.58(C),
135.07(CH), 140.21(CH), 141.29(C), 143.67(C), 150.52(C),
156.67(C), 162.88(C), 168.77(C), 169.49(C); FAB-MS m/z: 687
(M++1); Anal. Calcd for C39H34N4O8: C, 68.21; H, 4.99; N, 8.16.
Found: C, 67.92; H, 4.95; N, 8.18.
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