Design and stereoselective synthesis of four peptide nucleic acid monomers with cyclic structures in backbone

Article abstract of DOI:10.1002/jhet.627

Four isomers of the monomer of peptide nucleic acid (PNA) were derived from (2S,4R)-4-hydroxyproline; they had different stereochemistries at the C ² and C⁴ positions in the pyrrolidine ring. These different backbone conformations corresponding to four different stereochemistries were realized through a combination of inversions at the C² and the C⁴ positions in pyrrolidine ring. The obtained backbone frameworks were reacted with N-benzoyl thymine to give the corresponding PNA monomers. Spectroscopic comparison of the resultant monomers confirmed their stereochemistries.

Full text of DOI:10.1002/jhet.627

1132
Design and Stereoselective Synthesis of Four Peptide Nucleic
Acid Monomers with Cyclic Structures in Backbone
Vol 48
Akiko Watanabe,a* Naotoshi Kiyota,b Tetsuo Yamasaki,a Kazuhiro Tanda,a
Tatsunori Miyagoe,a Masanori Sakamoto,a and Masami Otsukab
aSchool of Pharmaceutical Sciences, Kyushu University of Health and Welfare,
Nobeoka, Miyazaki 882-8508, Japan
bFaculty of Medical and Pharmaceutical Sciences, Kumamoto University, Kumamoto,
Kumamoto 862-0973, Japan
*E-mail: akiwata@phoenix.ac.jp
Received June 3, 2010
DOI 10.1002/jhet.627
Published online 7 June 2011 in Wiley Online Library (wileyonlinelibrary.com).
Four isomers of the monomer of peptide nucleic acid (PNA) were derived from (2S,4R)-4-hydroxy-
proline; they had different stereochemistries at the C2 and C4 positions in the pyrrolidine ring. These
different backbone conformations corresponding to four different stereochemistries were realized
through a combination of inversions at the C2 and the C4 positions in pyrrolidine ring. The obtained
backbone frameworks were reacted with N-benzoyl thymine to give the corresponding PNA monomers.
Spectroscopic comparison of the resultant monomers confirmed their stereochemistries.
J. Heterocyclic Chem., 48, 1132 (2011).
INTRODUCTION
designed PNA backbone molecules on the basis of three
concepts: (1) introduction of a ring structure in the back-
bone, (2) introduction of ether bonding, and (3) derivation
of four isomers from four different stereochemistries at
the C2 and C4 positions in pyrrolidine ring (Fig. 1).
The objective of this study is to develop a novel pep-
tide nucleic acid (PNA) with a peptide backbone [1], spe-
cifically for the purpose of site-specific application for
nucleotide-molecule recognition. In recent years, siRNAs
are being commonly used as anti-sense/anti-gene mole-
cules in the anti-sense method, one of the gene expression
control methods. However, siRNAs have unsatisfactory
site-selectivity, particularly, in the stable hairpin-loop
region; further, they are toxic at high concentrations [2].
As an alternate to siRNAs, oligonucleotide analogs bear-
ing amide structure, for example, PNAs, peptide ribonu-
cleic acids, or their derivatives, are also used as not only
anti-sense/anti-gene molecules but also as molecular
detection tools because of their functional attributions
such as tolerance of RNAase, cell uptake stimulation, low
toxicity, and (importantly) site-specificity [3–5]. Nucleic
acid analogs bearing peptide structure are thus worth
investigating for their functional availability in anti-sense/
anti-gene applications. We have reported on several PNA
monomers having chain or ring structures [1,6,7]. In this
study, we selected (2S,4R)-4-hydroxyproline, a commer-
cially available amino acid, as the starting material. We
First, the synthetic strategy to obtain the (2S,4R)-
backbone was established as follows: Trans-4-hydroxy-
L-proline 1a was introduced esterification by using
SOCl2/EtOH in the yield of 80% and N-Boc protection
with (Boc)2O in quantitative yield [8]. The obtained 3a
[9] was reduced with LiBH4 [8] to give 4a [10] in 71%,
and the resulting primary alcohol group was protected by
TBDMS group in 55% [11]. 5a [12] was obtained and
treated with tert-butyl bromoacetate [13] in 80% followed
by desilylation by TBAF [13] in 97% and O-tosylation to
give (2S,4R)-backbone 8a in 75% (Scheme 1).
Second, the synthetic strategy to obtain the (2R,4R)-
backbone is as follows: Inversion at the C2 position of
1a was carried out by treatment with acetic acid and ace-
tic anhydride to give 1b in 74% [14]. Procedures similar
to that described previously gave corresponding products
at the steps of esterification, N-Boc protection [8], LiBH4
reduction [8], O-TBDMS protection [11], treatment
C
V
2011 HeteroCorporation

September 2011 Design and Stereoselective Synthesis of Four Peptide Nucleic Acid Monomers
with Cyclic Structures in Backbone
1133
Scheme 1
Figure 1. Retrosynthetic pathway of PNA backbone with ring
structure.
with tert-butyl bromoacetate [13], desilylation [13], and
O-tosylation yields of 73%, quantitative yield, 83%, 81%,
70%, 77%, and 76%, respectively (Scheme 2).
Scheme 2
Third, the synthetic strategy to obtain the (2S,4S)-back-
bone was as follows: Inversion at the C4 position of 3a
was accomplished by treatment with DEAD [15] by Mit-
sunobu condition to give 9c in 97%. Again, procedures
similar to that described previously gave corresponding
products at the steps of LiBH4 reduction [8], O-TBDMS
protection [11], treatment with tert-butyl bromoacetate
[13], desilylation [13], and O-tosylation in the yields of
quantitative yield, 83%, 62%, 87%, and 82%, respec-
tively (Scheme 3).
Fourth, the synthetic strategy to obtain the (2R,4S)-
backbone was as follows: Inversion at the C4 position of
3b was carried out by treatment with DEAD [15] by
Mitsunobu condition to give 9d in quantitative yield
after inversion at C2 position of 1a [14]. Procedures
similar to that described previously gave corresponding
products at the steps of LiBH4 reduction [8], O-TBDMS
protection [11], treatment with tert-butyl bromoacetate
[13], desilylation [13], and O-tosylation in the yields of
quantitative yield, 68%, 82%, 95%, and 84%, respec-
tively (Scheme 4).
The four backbone conformations (8) were reacted with
N-benzoyl nucleic-acid bases in the presence of K2CO3 and
crownether to give PNA monomers (10, 11) (Scheme 5).
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

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A. Watanabe, N. Kiyota, T. Yamasaki, K. Tanda, T. Miyagoe, M. Sakamoto, and M. Otsuka
Vol 48
Scheme 3
Scheme 5
By deprotection with trifluoroacetic acid and protec-
tion with Fmoc-Opfp, the thymine monomers were con-
verted to N-Fmoc derivatives (12a–d), which can be
applied to solid-phase oligomer synthesis (Scheme 6).
Comparison of the specific rotations of 12a–d con-
firmed that the relationships between 12a and 12d, and
also between 12b and 12c, are enantiomeric (Fig. 2).
In summary, we have successfully synthesized novel
PNA monomers with four different stereochemistries of
substituents in the backbone with a ring structure. The
synthesis of oligomers by solid-phase method and their
evaluation as anti-sense/anti-gene oligomers are cur-
rently under study by our group.
In the coupling reactions of 8 with N-benzoyladenine,
the desired products (11a–d) and their regioisomers
(11a0–d0) were obtained in the approximately ratio of
2:1 to 3:1.
Scheme 4
EXPERIMENTAL
All the melting points were determined on a Yanagimoto
micromelting point apparatus and are uncorrected. 1H- and
13C-NMR spectra were taken with a JEOL JNM-AL 300 and a
JEOL JNM-AL 400. Chemical shifts were determined using
tetramethylsilane as an internal standard. Mass spectra were
Scheme 6
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

September 2011 Design and Stereoselective Synthesis of Four Peptide Nucleic Acid Monomers
with Cyclic Structures in Backbone
1135
(5-CH2), 57.4 (2-CH) 61.9 (Et CH2), 68.2 (4-CH), 168.5
(C¼¼O); FAB-MS (m/z): 168 (Mþ þ 1).
Similar procedure was carried on the 38.4 g (0.23 mol) of
(2S,4S)-4-hydroxyproline hydrochloride 1b to give (2R,4R)-4-
hydroxyproline ethyl ester 2b [16] as colorless needles (33 g,
0.169 mol, 73%); 1H-NMR (DMSO-d6) d: 1.21 (3H, t, J ¼
8.26, Et CH3), 2.0–2.15 (1H, m, 3-CHH), 2.2–2.35 (1H, m,
3-CHH), 3.15–3.25 (2H, m, 5-CH2), 4.18 (2H, q, J ¼ 18,
Et CH2), 4.34 (1H, br-s, 4-CH), 4.44 (1H, dd, J ¼ 3.6, 3.6,
2-CH); 13C-NMR (DMSO-d6) d: 13.7 (Et CH3), 36.9 (3-CH2),
52.8 (5-CH2), 57.4 (2-CH), 62.1 (Et CH2), 67.9 (4-CH), 167.9
(C¼¼O).
General procedure for introduction of N-Boc protection
in 4-hydroxyproline ethyl esters 2. To the mixture of
150 mL of sat. NaHCO3 aq., 150 mL of dioxane, and 10 g
(50 mmol) of hydrochloride salt of (2S,4R)-4-hydroxyproline
ethyl ester (2a) was added 12.6 mL (55 mmol) of (Boc)2O at
0ꢀC and was stirred at room temperature for overnight [8].
Resulted colorless precipitation was removed by suction filtra-
tion and filtrate was concentrated in vacuo. To the resulted res-
idue was added water and was extracted with CH2Cl2. Organic
layer was dried over MgSO4. Solvent was evaporated in vacuo
to give (2S,4R)-4-hydroxyproline-1,2-dicarboxylic acid 1-tert-
butyl ester 2-ethyl ester 3a [9a] as colorless oil (13 g,
50 mmol, quant.); 1H-NMR (CDCl3) d: 1.28 (3H, t, J ¼ 6.9,
Et CH3), 1.41, 1.45 (9H, 2 ꢁ s, Boc CH3 rotamer), 1.96–2.11
(1H, m, 3-CHH), 2.19–2.38 (1H, m, 3-CHH), 3.38–3.68
(2H, m, 5-CH2), 4.08–4.26 (2H, m, Et CH2), 4.37 (1H, t, J ¼
8.4, 2-CH), 4.45 (1H, br-s, 4-CH); 13C-NMR (CDCl3) d: 13.9,
14.0 (Et CH3 rotamer), 28.1, 28.2 (Boc CH3 rotamer), 38.2,
38.9 (3-CH3 rotamer), 54.5 (5-CH2), 57.6, 57.9 (2-CH
rotamer), 60.9 (Et CH2), 68.9, 69.6 (3-CH2) rotamer), 80.0
(Boc C), 154.0, 154.4 (Boc C¼¼O rotamer), 172.9, 173.1
(C¼¼O rotamer); FAB-MS (m/z): 260 (Mþ þ 1).
Figure 2. Comparison of specific rotation of four PNA monomers
12a–d with N-benzoylthymine as base.
obtained from a JEOL JNS-DX303HF Mass Spectrometer and
a JEOL GC-MateII. Optical rotation was measured on a
JASCO DIP-1000 KUY digital polarimeter. All column chro-
matography was performed using silica gel (Kanto Kagaku,
63-210, 60N and Fuji Silysia Chemical, PSQ 100B).
(2S,4S)-4-Hydroxyproline hydrochloride (1b). To the
stirred mixture of 128 mL (1.36 mol) of acetic anhydride and
360 mL (6.24 mol) of acetic acid at 50ꢀC was added 29.6 g
(0.2244 mol) of (2S,4R)-4-hydroxyproline (1a) and was stirred
at reflux temperature [14]. After for 5.5 h, solvent was distilled
in vacuo. To the resulted residue was added 2 mol/L HCl (400
mL) and was stirred at reflux temperature. After 3 h, activated
carbon was added to the reaction mixture and was filtrated,
then solvent was distilled in vacuo. Resulted crystalline residue
was collected by suction filtration and recrystallized from etha-
nol to give (2S,4S)-4-hydroxyproline hydrochloride 1b as col-
Similar procedure was carried on the hydrochloride salt of
(2R,4R)-4-hydroxyproline ethyl ester 2b to give (2R,4R)-4-
hydroxyproline-1,2-dicarboxylic acid 1-tert-butyl ester 2-ethyl
ester 3b [9b] as colorless oil (13 g, 0.05 mol, quant.);
1H-NMR (CDCl3) d: 1.25–1.35 (3H, m, Et CH3), 1.43, 1.46
(9H, 2 ꢁ s, Boc CH3 rotamer), 2.0–2.15 (1H, m, 3-CHH),
2.25–2.4 (1H, m, 3-CHH), 3.4–3.75 (2H, m, 5-CH2), 4.23 (2H,
q, J ¼ 21.47, Et CH2), 4.2–4.4 (2H, m, 2-CH and 4-CH);
13C-NMR (CDCl3) d: 13.9, 14.1 (Et CH3 rotamer), 28.2, 28.3
(Boc CH3 rotamer), 37.7, 38.5 (3-CH3 rotamer), 55.3, 55.8
(5-CH2 rotamer), 80.3 (Boc C), 153.7 (Boc C¼¼O), 174.8 (C¼¼O).
General procedure for LiBH4 reduction of 2-ethoxycar-
bonyl group in 2-ethyl esters 3 and 9. To the suspension of
3.9 g (177 mmol) of LiBH4 in 30 mL of dry THF was added
solution of 13 g (50 mmol) of (2S,4R)-4-hydroxyproline-1,2-
dicarboxylic acid 1-tert-butyl ester 2-ethyl ester (3a) in 50 mL
of dry THF dropwise at 0ꢀC under N2 and was stirred at room
temperature for overnight [8]. Fifty millilitres of water was
added very carefully followed by addition of 25 mL of 6M
HCl on ice-salt bath. Obtained mixture was extracted with
ethyl acetate. Organic layer was dried over MgSO4. Solvent
was evaporated in vacuo. Resulted residue was purified by
short column on silica-gel (ethyl acetate) to give (2S,4R)-4-
hydroxyprolinol-1-carboxylic acid tert-butyl ester 4a [10a] as
1
orless needles (28 g, 0.167 mol, 75%, >99%de by H-NMR);
1H-NMR (DMSO-d6) d: 2.0–2.15 (1H, m, 3-CHH), 2.2–2.35
(1H, m, 3-CHH), 3.15 (2H, s, 5-CH2), 4.25–4.4 (2H, m, 2-CH
and 4-CH), 5.4 (1H, br, NH), 8.7 (1H, br, OH), 10.5 (1H, br,
CO2H); 1H-NMR (D2O) d: 2.2–2.3 (1H, m, 3-CHH), 2.39
(1H, dtd, J ¼ 4.2, 8.1, 4.2, 3-CHH), 3.32 (2H, ddd, J ¼ 12.3,
3.6, 3.6, 5-CH2), 4.4–4.5 (1H, m, 4-CH), 4.49 (1H, dd, J ¼
3.3, 3.3, 2-CH); 13C-NMR (DMSO-d6) d: 37.2 (3-CH2), 52.9
(5-CH2), 57.5 (2-CH), 68.2 (4-CH), 170.6 (C¼¼O).
General procedure for ethyl esterification of C2-carboxyl
group in 4-hydroxyprolines 1. To the suspension of 30.2 g
(0.23 mol) of (2S,4R)-4-hydroxyproline 1a in 250 mL of dry
ethanol was added 40 mL (0.46 mol) of thionyl chloride
slowly and was stirred for overnight at room temperature.
Resulted precipitation was collected by suction filtration and
recrystallized from ethanol to give hydrochloride salt of
(2S,4R)-4-hydroxyproline ethyl ester 2a [16] as colorless nee-
dles (36.1 g, 0.185 mol, 80%); 1H-NMR (DMSO-d6) d: 1.22
(3H, t, J ¼ 6.9, Et CH3), 1.96–2.11 (1H, m, 3-CHH), 2.11–
2.26 (1H, m, 3-CHH), 3.04 (2H, d, J ¼ 11.7, 5-CHH), 3.37
(2H, dd, J ¼ 11.7, 4.5, 5-CHH), 4.18 (2H, q, J ¼ 6.9,
Et CH2), 4.32–4.45 (2H, m, 4-CH and 2-CH), 5.63 (br, OH);
13C-NMR (DMSO-d6) d: 13.8 (Et CH3), 37.0 (3-CH2), 52.8
1
colorless oil (7.9 g, 38 mmol, 71%); H-NMR (CDCl3) d: 1.46
(9H, s, Boc CH3), 1.71 (1H, br-s, 3-CHH), 2.04 (1H, br-s,
3-CHH), 3.30–3.79 (4H, m, 5-CH2 and C2-CH2), 3.94–4.19
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

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A. Watanabe, N. Kiyota, T. Yamasaki, K. Tanda, T. Miyagoe, M. Sakamoto, and M. Otsuka
Vol 48
General procedure of C4-O-protection of 5. To the mix-
ture of 3.64 g (11 mmol) of (2S,4R)-2-(tert-butyldimethyl-sila-
nyloxymethyl)-4-hydroxy-pyrrolidine-1-carboxylic acid tert-
butyl ester 5a in benzene/50% NaOH aq. (32 mL/10 mL) was
added 1.13 g (3.3 mmol) of Bu4NHSO4 followed by 3.3 mL
(22 mmol) of tBuOCOCH2Br at 10ꢀC and was stirred at about
20ꢀC for overnight [13]. Reaction mixture was extracted with
ethyl acetate. Obtained organic layer was washed with 10% cit-
ric acid and brine, dried over MgSO4. Solvent was evaporated
in vacuo and resulted residue was purified on silica-gel column
chromatography (eluent; hexane:ethyl acetate ¼ 6:1) to give
(2S,4R)-4-tert-butoxycarbonylmethoxy-2-(tert-butyldimethylsi-
lanyloxymethyl)-pyrrolidine-1-carboxylic acid tert-butyl ester
(1H, m, 2-CH), 4.34 (1H, br-s, 4-CH), 5.28 (br, OH);
13C-NMR (CDCl3) d: 28.3 (Boc CH3), 37.3 (3-CH2), 55.6
(5-CH2), 58.6 (2-CH), 66.5 (C2-CH2), 68.8 (4-CH), 80.4 (Boc
C), 156.9 (Boc C¼¼O); FAB-MS (m/z): 218 (Mþ þ 1).
Similar procedure was carried on the (2R,4R)-4-hydroxypro-
line-1,2-dicarboxylic acid 1-tert-butyl ester 2-ethyl ester 3c,
7.9 g (28 mmol) of (2S,4S)-4-formyloxypyrrolidine-1,2-dicar-
boxylic acid 1-tert-butyl ester 2-ethyl ester 9a, and 8.6 g (30
mmol) of (2R,4S)-4-formyloxypyrrolidine-1,2-dicarboxylic acid
1-tert-butyl ester 2-ethyl ester 9d to give (2R,4R)-4-hydroxy-
prolinol-1-carboxylic acid tert-butyl ester 4b [10a], (2S,4S)-4-
hydroxyprolinol-1-carboxylic acid tert-butyl ester 4c [10a],
and (2R,4S)-4-hydroxyprolinol-1-carboxylic acid tert-butyl
ester 4d [10b] as colorless needles (8.7 g, 41 mmol, 83%), as
colorless needles (5.8 g, 28 mmol, quant.), and as colorless oil
(6.53 g, 30 mol, quant.), respectively.
24
6a as yellow oil (4.03 g, 9.1 mol, 82%); [a]D ꢂ26.43ꢀ
(c 1.0368, CHCl3); 1H-NMR (CDCl3) d: 0.00 (6H, s, Si-Me),
0.85 (9H, s, TBDMS Me), 1.43, 1.45 (18H, 2 ꢁ s, Boc Me
and t-BuOCO Me), 1.94–2.23 (2H, m, 3-CH2), 3.32–3.72 (4H,
m, 5-CH2 and C2-CH2), 3.81–4.04 (1H, m, 2-CH), 3.92 (2H, s,
C4-OCH2), 4.11–4.26 (1H, m, 4-CH); 13C-NMR (CDCl3) d:
ꢂ5.6 (Si-Me), 18.1 (TBDMS C), 25.8 (TBDMS Me), 27.9,
28.0 (Boc Me rotamer), 28.4 (t-BuOCO Me), 33.7, 35.0
(3-CH2 rotamer), 51.5, 52.3 (5-CH2 rotamer), 57.5 (2-CH),
63.4, 64.2 (C2-CH2 rotamer), 66.9 (C4-OCH2), 77.5, 78.1
(4-CH rotamer), 79.1, 79.4 (Boc C rotamer), 81.6 (t-BuOCO
CO); FAB-MS (m/z): 446 (Mþ þ 1) [Calcd. For C22H44NO6Si:
M þ H. 446.294 Found: (M þ H)þ 446.293 (FAB)].
1
4b: H-NMR (CDCl3) d: 1.46 (9H, s, Boc CH3), 1.79–2.00
(1H, m, 3-CHH), 2.23–2.38 (1H, m, 3-CHH), 3.34–3.64 (3H,
m, 5-CH2 and C2-CHH), 3.87–4.08 (2H, m, 2-CH and C2-
CHH), 4.28 (1H, br-s, 4-CH); 13C-NMR (CDCl3) d: 28.3 (Boc
CH3), 37.2, 37.9 (3-CH2 rotamer), 56.6 (5-CH2), 58.2, 58.3 (2-
CH rotamer), 63.3, 63.9 (C2-CH2 rotamer), 68.7, 69.5 (4-CH
rotamer), 79.9 (Boc C), 154.7, 155.5 (Boc C¼¼O rotamer);
FAB-MS (m/z): 218 (Mþ þ 1). 4c ¼ ent-4b. 4d ¼ ent-4a.
General procedure of C2-CH2-O-TBDMS protection of
4. To the mixture of 4.4 g (20 mmol) of (2S,4R)-4-hydroxy-
prolinol-1-carboxylic acid tert-butyl ester (4a) and 1.5 g
(22 mmol) of imidazole in 80 mL of dry CH2Cl2 was added
3.36 g (22 mmol)of TBDMSCl on ice-salt bath and was stirred
at room temperature for overnight [11]. After removal of pre-
cipitation by suction filtration, solvent was evaporated
in vacuo. Obtained residue was purified on silica-gel column
chromatography (eluent; hexane:ethyl acetate ¼ 4:1) to give
(2S,4R)-2-(tert-butyldimethylsilanyloxymethyl)-4-hydroxy-pyr-
rolidine-1-carboxylic acid tert-butyl ester 5a as colorless oil
(3.64 g, 11 mmol, 55%) [12]; 1H-NMR (CDCl3) d: 0.00
(6H, s, TBDMS Si-Me), 0.84 (9H, s, TBDMS Me), 1.43 (9H, s,
Boc Me), 1.85–2.02 (1H, m, 3-CHH), 2.08–2.23 (1H, m,
3-CHH), 3.28–3.54 (4H, m, 5-CH2 and C2-CH2), 3.84–4.03
(1H, m, 2-CH), 4.41 (1H, br-s, 4-CH); 13C-NMR (CDCl3) d:
ꢂ5.5 (TBDMS Si Me), 18.1 (TBDMS C), 25.8 (TBDMS Me),
28.5 (Boc Me), 36.6, 36.8 (3-CH2 rotamer), 55.1, 55.4 (5-CH2
rotamer), 57.4 (2-CH), 63.0, 64.0 (C2-CH2 rotamer), 69.5, 70.2
(4-CH rotamer), 79.4 (Boc C), 154.6 (Boc C¼¼O).
Similar procedure was carried on the 5.58 g (16.3 mmol) of
5b, 5.52 g (16.7 mmol) of 5c, and 4.52 g (13.6 mmol) of 5d to
give 6b (5.09 g, 11.4 mmol, 70%), 6c (4.59 g, 10.3 mmol, 62%),
and 6d (4.8 g, 10.8 mmol, 79%), respectively, as yellow oil.
23
6b: [a]D þ22.06ꢀ (c 1.032, CHCl3); 1H-NMR (CDCl3) d:
0.00 (6H, s, Si-Me), 0.84 (9H, s, TBDMS Me), 1.23, 1.41
(18H, 2 ꢁ s, Boc Me and t-BuOCO Me), 1.83–2.04 (1H, m,
3-CHH), 2.11–2.16 (1H, m, 3-CHH), 3.25–3.35 (1H, m,
5-CHH), 3.43–3.62 (2H, m, 5-CHH and C2-CHH), 3.62–3.76
(1H, m, C2-CHH), 3.62–3.79 (1H, m, 2-CH), 3.89 (2H, s,
C4-OCH2), 4.00–4.09 (1H, m, 4-CH); 13C-NMR (CDCl3) d:
ꢂ5.4 (Si-Me), 14.1 (TBDMS C), 25.8 (TBDMS Me), 27.9,
28.0 (Boc Me rotamer), 28.4 (t-BuOCO Me), 33.7, 35.0
(3-CH2 rotamer), 51.5, 52.3 (5-CH2 rotamer), 57.5, 57.6 (2-CH
rotamer), 62.9, 63.8 (C2-CH2 rotamer), 66.8 (C4-OCH2), 77.6,
78.4 (4-CH rotamer), 79.1, 79.4 (Boc C rotamer), 81.5
(t-BuOCO CO); FAB-MS (m/z): 446 (Mþ þ 1) [Calcd. For
C22H44NO6Si: M þ H. 446.294 Found: (M þ H)þ 446.295
24
(FAB)]. 6c ¼ ent-6b; [a]D ꢂ21.76ꢀ (c 1.0232, CHCl3). 6d ¼
Similar procedure was carried on the 4b, 4c, and 3.3 g (15
mmol) of 4d to give 5b (5.38 g, 16.3 mol, 81%), 5c (5.52 g,
16.7 mmol, 83%), and 5d (3.38 g, 10.2 mmol, 68%), respec-
tively, as colorless oil [12].
24
ent-6a; [a]D þ25.78ꢀ (c 0.9997, CHCl3).
General procedure of removal of C2-CH2-O-TBDMS
protecting group in 6. To the solution of 4.03 g (9.1 mmol)
of (2S,4R)-4-tert-butoxycarbonylmethoxy-2-(tert-butyldimethyl-
silanyloxymethyl)-4-hydroxy-pyrrolidine-1-carboxylic acid tert-
butyl ester 6a in 20 mL of dry THF was added 22.8 mL (22.8
mmol) of 1 mol/L TBAF solution in THF and was stirred at
room temperature for overnight [13]. After evaporation of the
solvent, to the resulted residue was added water and brine then
was extracted with ethyl acetate. Organic layer was dried over
MgSO4. Solvent was evaporated in vacuo and resulted residue
was purified on silica-gel column chromatography (eluent; hexa-
ne:ethyl acetate ¼ 1:1) to give (2S,4R)-4-tert -butoxycarbonyl-
methoxy-2-hydroxymethyl-4-hydroxy-pyrrolidine-1-carboxylic acid
tert-butyl ester 7a (2.91g, 8.8 mmol, 97%) as pale yellow oil;
1
5b: H-NMR (CDCl3) d: 0.00 (6H, s, TBDMS Si-Me), 0.80
(9H, s, TBDMS Me), 1.34, 1.35 (9H, 2 ꢁs, Boc Me rotamer),
1.72–1.86 (1H, m, 3-CHH), 2.16–2.32 (1H, m, 3-CHH), 3.26–
3.50 (3H, m, 5-CH2 and C2-CHH), 3.76–3.94 (1H, m, 2-CH),
4.03–4.16 (1H, m, 4-CH, 4.16–4.25 (1H, dd, J ¼ 5.1,
2.4, C2-CHH)); 13C-NMR (CDCl3) d: ꢂ5.8 (TBDMS Si Me),
18.3 (TBDMS C), 25.8 (TBDMS Me), 28.3, 28.4 (Boc
Me rotamer), 37.7, 38.4 (3-CH2 rotamer), 57.1, 57.4 (5-CH2
rotamer), 57.9, 58.0 (2-CH rotamer), 64.0, 64.8 (C2-CH2
rotamer), 68.9, 69.7 (4-CH rotamer), 79.2, 79.5 (Boc C rotamer),
154.6 (Boc C¼¼O); FAB-MS (m/z): 332 (Mþ þ 1). 5c ¼ ent-5b.
5d ¼ ent-5a.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

September 2011 Design and Stereoselective Synthesis of Four Peptide Nucleic Acid Monomers
with Cyclic Structures in Backbone
1137
23
1
[a]D ꢂ21.40ꢀ (c 1.0324, CHCl3); H-NMR (CDCl3) d: 1.47,
1.48 (18H, 2 ꢁ s, Boc Me and t-BuOCO Me), 2.00–2.26 (2H,
m, 3-CH2), 3.30–3.87 (4H, m, 5-CH2 and C2-CH2), 3.96 (2H,
s, C4-OCH2), 4.00–4.21 (2H, m, 2-CH and 4-CH), 4.91 (br,
OH); 13C-NMR (CDCl3) d: 28.1, 28.4 (Boc Me and t-BuOCO
Me), 34.3 (3-CH2), 53.0 (5-CH2), 59.0 (2-CH), 66.5, 67.0
(C2-CH2 and C4-OCH2), 77.2 (4-CH), 80.5 (Boc C), 81.8
(t-BuOCO C), 156.8 (Boc CO), 169.4 (t-BuOCO C¼¼O); FAB-
MS (m/z): 332 (Mþ þ 1) [Calcd. For: C16H29NO6 M. 331.200
Found: Mþ 331.200 (FAB)].
Ts Me), 3.38–3.55 (2H, m, 5-CH2), 3.84 (2H, s, C4-OCH2),
4.04 (1H, br-s, 4-CH), 4.11 (2H, br-s, C2-CH2) , 4.17–4.4.26
(1H, m, 2-CH), 7.33 (2H, d, J ¼ 7.8, Ph 3,5-CH), 7.79 (2H, d,
J ¼ 8.4, Ph 2,6-CH); 13C-NMR (CDCl3 d: 21.5 (Ts Me), 28.0,
28.2 (Boc Me and t-BuOCO Me), 31.7, 33.3 (3-CH2 rotamer),
51.9, 52.9 (5-CH2 rotamer), 54.8, 55.0 (2-CH rotamer), 66.4
(C4-OCH2), 69.5, 70.1 (C2-CH2 rotamer), 77.6, 78.4 (4-CH
rotamer), 79.9, 80.2 (Boc C rotamer), 81.7 (t-BuOCO C),
127.9 (Ph 2,6-CH), 129.7 (Ph 3,5-CH), 133.1 (Ph 1-C), 144.6
(Ph 4-C), 153.9, 154.2 (Boc C¼¼O rotamer), 169.1 (t-BuOCO
Similar procedure was carried on the 5.09 g (11.4 mmol) of 6b,
4.59 g (10.3 mmol) of 6c, and 4.8 g (10.8 mmol) of 6d to give 7b
(3.23 g, 9.8 mmol, 86%), 7c (2.96 g, 8.9 mmol, 87%), and 7d
(3.38 g, 10.2 mmol, 95%), respectively, as pale yellow oil.
C¼¼O); FAB-MS (m/z): 486 (Mþ
þ
1) [Calcd. For
C23H36NO8S: M þ H. 486.216 Found: (M þ H)þ 486.220
22
(FAB)]. 8c ¼ ent-8b; [a]D ꢂ2.39ꢀ (c 1.016, CHCl3). 8d ¼
22
ent-8a; [a]D þ15.91ꢀ (c 0.986, CHCl3).
23
1
General procedure for Mitsunobu reaction at C4 position
in 3. To the mixture of 13 g (50 mmol) of (2S,4R)-4-hydroxy-
proline-1,2-dicarboxylic acid 1-tert-butyl ester 2-ethyl ester
(3a), 14.6 g (56 mmol) of triphenylphosphine, 2.1 mL (55
mmol) of formic acid, and 270 mL of dry THF was added 8.7
mL (56 mmol) of DEAD on ice-salt bath and was stirred at
room temperature for overnight [15]. After evaporation of sol-
vent in vacuo, ether and hexane were added to the residue.
Resulted colorless precipitation was removed by suction filtra-
tion and solvent was evaporated in vacuo. Obtained residue
was purified on silica-gel column chromatography (eluent;
CH2Cl2:methanol ¼ 40:1) to give (2S,4S)-4-formyloxypyrroli-
dine-1,2-dicarboxylic acid 1-tert-butyl ester 2-ethyl ester 9c as
7b: [a]D þ22.33ꢀ (c 1.0206, CHCl3); H-NMR (CDCl3) d:
1.47, 1.48 (18H, 2 ꢁ s, Boc Me and t-BuOCO Me), 1.80–1.95
(1H, m, 3-CHH), 2.11–2.31 (1H, m, 3-CHH) , 3.40–3.88 (4H,
m, 5-CH2 and C2-CH2), 3.97 (2H, m, C4-OCH2), 3.88–4.20 (2H,
m, 2-CH and 4-CH), 4.43 (br, OH); 13C-NMR (CDCl3) d: 27.9,
28.3 (Boc Me and t-BuOCO Me), 34.0, 34.7 (3-CH2 rotamer),
52.5 (5-CH2), 58.0, 59.1 (2-CH rotamer), 66.8 (C4-OCH2), 67.0
(C2-CH2), 77.8 (4-CH), 80.2 (Boc C), 81.8 (t-BuOCO C), 156.3
(Boc CO), 169.2 (t-BuOCO C¼¼O); FAB-MS (m/z): 332 (Mþ þ
1) [Calcd. For: C16H29NO6 M. 331.200 Found: Mþ 331.202
23
(FAB)]. 7c ¼ ent-7b; [a]D ꢂ23.67ꢀ (c 1.0445, CHCl3). 7d ¼
24
ent-7a; [a]D þ21.61ꢀ (c 1.033, CHCl3).
General procedure of C2-CH2-O-tosyl protection of 7. To
the solution of 2.9 g (8.8 mmol) of 7a in 30 mL of dry pyri-
dine was added 2 g (10.6 mmol) of tosyl chloride in three por-
tions every 20 min at 0ꢀC and was stirred at room temperature
for overnight [15]. Pyridine was evaporated by azeotropy with
toluene. A total of 10% citric acid aq. was added to the resi-
due and was extracted with ethyl acetate. Organic layer was
washed with brine, dried over MgSO4, and was evaporated
in vacuo. Obtained residue was purified on silica-gel column
chromatography (eluent; hexane:ethyl acetate ¼ 2:1) to give
(2S,4R)-4-tert-butoxycarbonylmethoxy-2-(4-tosyloxymethyl)-4-
hydroxy-pyrrolidine-1-carboxylic acid tert-butyl ester 8a (3.22
25
colorless needles (12.3 g, 43 mmol, 86%); [a]D ꢂ55.92ꢀ
1
(c 1.0004, CHCl3); H-NMR (CDCl3) d: 1.17–1.34 (3H, m, Et
CH3), 1.43, 1.48 (9H, 2 ꢁ s, Boc CH3 rotamer), 2.28, 2.33
(1H, 2 ꢁ s, 3-CHH), 2.42–2.62 (1H, m, 3-CHH), 3.50–3.68
(1H, m, 5-CHH), 3.72–3.87 (1H, m, 5-CHH), 4.11–4.30 (2H,
m, Et CH2), 4.34–4.55 (1H, m, 2-CH), 5.38 (1H, br-s, 4-CH),
7.97 (1H, s, CHO); 13C-NMR (CDCl3) d: 14.0, 14.1 (Et CH3
rotamer), 28.1, 28.2 (Boc CH3 rotamer), 35.3, 36.3 (3-CH2
rotamer), 51.7, 52.0 (2-CH rotamer), 61.6 (Et CH2), 71.5, 72.2
(4-CH rotamer), 80.3 (Boc C), 153.4, 153.9 (Boc C¼¼O
rotamer), 160.0, 160.1 (CHO rotamer), 172.0, 172.3 (C¼¼O
21
rotamer); FAB-MS (m/z): 288 (Mþ
þ
1) [Calcd. For
g, 6.6 mmol, 75%) as colorless oil; [a]D ꢂ17.97ꢀ (c 1.0124,
1
C13H21NO6: M. 287.137 Found: (M þ H)þ 287.135 (FAB)].
CHCl3); H-NMR (CDCl3) d: 1.38 (9H, d, J ¼ 15.6, t-BuOCO
Me), 1.48 (9H, s, Boc Me), 1.94–2.28 (2H, m, 3-CH2), 2.44
(3H, s, Ts Me), 3.21–3.75 (2H, m, 5-CH2), 3.94 (2H, s, C4-
OCH2), 4.00–4.25 (2H, m, 2-CH and 4-CH), 4.00–4.4.42 (2H,
m, C2-CH2), 7.35 (2H, br-s, Ph 3,5-CH), 7.76 (2H, d, J ¼ 7.8,
Ph 2,6-CH); 13C-NMR (CDCl3 d: 20.9, 21.5 (Ts Me rotamer),
28.0, 28.2 (Boc Me and t-BuOCO Me), 33.4, 34.9 (3-CH2
rotamer), 51.3, 52.1 (5-CH2 rotamer), 54.6 (2-CH), 66.8 (C4-
OCH2), 70.1, 70.3 (C2-CH2 rotamer), 77.0, 77.4 (4-CH
rotamer), 79.8, 80.1 (Boc C rotamer), 81.7 (t-BuOCO C), 127.8
(Ph 2,6-CH), 129.8 (Ph 3,5-CH), 132.7 (Ph 1-C), 144.5 (Ph
4-C), 154.2 (Boc C¼¼O), 169.2 (t-BuOCO C¼¼O); FAB-MS
(m/z): 486 (Mþ þ 1) [Calcd. For C23H36NO8S: M þ H. 486.216
Found: (M þ H)þ 486.215 (FAB)].
Similar procedure was carried on the 3b to give 9d as color-
25
less oil (12.3 g, 43 mol, 86%); [a]D þ43.28ꢀ (c 1.0146,
1
CHCl3); H-NMR (CDCl3) d: 1.29 (3H, t, J ¼ 13.8, Et CH3),
1.43, 1.46 (9H, 2 ꢁ s, Boc CH3 rotamer), 2.15–2.30 (1H, m,
3-CHH), 2.30–2.50 (1H, m, 3-CHH), 3.72 (2H, br,-s, 5-CH2),
4.19 (2H, q, J ¼ 19.2, Et CH2), 4.3–4.45 (1H, m, 2-CH), 5.41
(1H, br-s, 4-CH), 8.03 (1H, s, CHO); 13C-NMR (CDCl3) d:
13.9, 14.0 (Et CH3 rotamer), 28.1, 28.2 (Boc CH3 rotamer),
35.3, 36.3 (3-CH2 rotamer), 51.7, 52.0 (2-CH rotamer), 61.6
(Et CH2), 71.5, 72.2 (4-CH rotamer), 80.3 (Boc C), 153.4,
153.9 (Boc C¼¼O rotamer), 160.0, 160.1 (CHO rotamer),
172.0, 172.3 (C¼¼O rotamer); FAB-MS (m/z): 288 (Mþ þ 1)
[Calcd. For C13H21NO6: M. 287.137 Found: Mþ 287.139
(FAB)].
Similar procedure was carried on the 2.9 g (8.8 mmol) of
7b, 2.96 g (8.9 mmol) of 7c, and 3.38 g (10.2 mmol) of 7d to
give 8b, 8c, and 8d as colorless needles (3.26 g, 6.7 mmol,
76%), as colorless oil (3.54 g, 7.3 mmol, 82%), and as color-
less needles (4.15 g, 8.5 mmol, 84%), respectively.
General procedure of coupling reaction of trans-8 with
N-benzoyl nucleic acid. To the solution of 730 mg (1.5
mmol) of 8a in 7.5 mL of dry DMF was added 860 mg (2.25
mmol) of N-benzoylthymine, 200 mg (0.75 mmol) of 18-
crown-6 and 1.04 g (7.5 mmol) of K2CO3 and was stirred at
room temperature for 10 days [15]. Water was added to the
reaction mixture and was extracted with ethyl acetate. Organic
20
8b: [a]D þ1.46ꢀ (c 1.0386, CHCl3); 1H-NMR (CDCl3) d:
1.40, 1.48 (18H, 2 ꢁ s, t-BuOCO Me amd Boc Me), 1.91–
2.09 (1H, m, 3-CHH), 2.09–2.28 (1H, m, 3-CHH), 2.44 (3H, s,
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

1138
A. Watanabe, N. Kiyota, T. Yamasaki, K. Tanda, T. Miyagoe, M. Sakamoto, and M. Otsuka
Vol 48
layer was dried over MgSO4 and solvent was evaporated
in vacuo. Resulted residue was purified on silica-gel column
chromatography to give (2S,4R)-2-(N3-benzoylthymine-1-
ylmethyl)-4-tert-butoxycarbonyl-methyoxypyrrolidine-1-carbox-
ylic acid tert-butyl ester 10a (533 mg, 0.98 mmol, 65%) as
[Calcd. For C28H36N6O6: M. 552.270 Found: Mþ 552.269
20
(FAB)]. 11d0 ¼ ent-11a0; [a]D þ96.14ꢀ (c 1.045, CHCl3).
General procedure of coupling reaction of cis-8 with N-
benzoyl nucleic acid. To the solution of 730 mg (1.5 mmol)
of 8b in 7.5 mL of dry DMF was added 860 mg (2.25 mmol)
of N-benzoylthymine, 200 mg (0.75 mmol) of 18-crown-6, and
1.04 g (7.5 mmol) of K2CO3 and was stirred at 50ꢀC for 40
hrs [15]. Water was added to the reaction mixture and was
extracted with ethyl acetate. Organic layer was dried over
MgSO4 and solvent was evaporated in vacuo. Resulted residue
was purified on silica-gel column chromatography to give
(2R,4R)-2-(N3-benzoylthymine-1-ylmethyl)-4-tert-butoxycarbonyl-
methyoxypyrrolidine-1-carboxylic acid tert-butyl ester 10b
colorless solid; [a]D þ3.27ꢀ (c 1.026, CHCl3); 1H-NMR
23
(CDCl3) d: 1.46, 1.48 (18H, 2 ꢁ s, Boc Me and t-BuOCO
Me), 1.90 (3H, s, thymine Me), 1.80–2.03 (1H, m, 3-CHH),
2.07–2.27 (1H, m, 3-CHH), 3.36–3.73 (2H, m, 5-CH2), 3.91,
3.92 (2H, 2 ꢁ s, C4-OCH2), 3.79–4.23 (2H, m, C2-CH2), 4.03–
4.23 (1H, m, 4-CH), 4.23–4.40 (1H, m, 2-CH), 7.11 (1H, s,
thymine CH), 7.40–7.54 (2H, m, Ph 2,6-CH), 7.57–7.69 (1H,
m, Ph 4-CH), 7.99–8.13 (2H, m, Ph 3,5-CH); 13C-NMR (CDCl3)
d: 12.1 (thymine Me), 27.9, 28.8 (Boc Me and t-BuOCO Me),
34.4 (3-CH), 51.2 (C2-CH2), 51.9 (5-CH2), 54.7 (2-CH), 66.6
(C4-OCH2), 77.3 (4-CH), 80.1 (Boc C), 81.7 (t-BuOCO C),
110.0 (thymine 5-C), 128.9 (Ph 2,6-CH), 130.5 (Ph 3,5-CH),
131.6 (Ph 1-C), 134.8 (Ph 4-CH), 140.7 (thymine CH), 150.2
(thymine 2-C¼¼O), 155.1 (Boc C¼¼O), 163.1 (Bz CO), 169.1
(thymine 4-C¼¼O), 169.3 (t-BuOCO C¼¼O); FAB-MS (m/z):
544 (Mþ þ 1) [Calcd. For C28H37N3O8: M. 543.258 Found:
Mþ 543.257 (FAB)].
21
(133 mg, 0.24 mmol, 16%) as colorless solid; [a]D ꢂ95.24ꢀ
(c 1.036, CHCl3); 1H-NMR (CDCl3) d: 1.44, 1.49 (18H, 2 ꢁ
s, Boc Me and t-BuOCO Me), 1.88 (3H, s, thymine Me),
1.38–2.10 (2H, m, 3-CH2), 3.40–3.63 (2H, m, 5-CH2), 3.73–
4.25 (5H, m, C4-OCH2, C2-CH2 and 4-CH), 4.44 (1H, br-s, 2-
CH), 7.31 (1H, s, thymine CH), 7.38–7.54 (2H, m, Ph 3,5-
CH), 7.54–7.67 (1H, m, Ph 4-CH), 8.26 (2H, d, J ¼ 7.8, Ph
3,5-CH); 13C-NMR (CDCl3) d: 12.0 (thymine Me), 27.9, 28.2
(Boc Me and t-BuOCO Me), 33.7 (3-CH2), 52.7 (5-CH2), 52.9
(C2-CH2), 54.0 (2-CH), 66.8 (C4-OCH2), 79.6 (4-CH), 81.7
(Boc C and t-BuOCO C), 108.9 (thymine 5-C), 128.6 (Ph 2,6-
CH), 130.8 (Ph 3,5-CH), 131.9 (Ph 1-C), 134.4 (Ph 4-CH),
141.5 (thymine CH), 150.1 (thymine 2-C¼¼O), 154.9 (Boc
C¼¼O), 163.6 (Bz C¼¼O), 169.5 (t-BuOCO C¼¼O)169.7, (thy-
mine 4-C¼¼O); FAB-MS (m/z): 544 (Mþ þ 1) [Calcd. For
C28H37N3O8: M. 543.258 Found: Mþ 543.259 (FAB)].
Similar procedure was carried on the 8d with N-benzoylthy-
mine to give 10d (518 mg, 0.95 mmol, 63%) as colorless
23
solid; ¼ ent-10a; [a]D ꢂ3.16ꢀ (c 1.0104, CHCl3).
Similar procedure was also carried on the 8a and 8d with N-
benzoyladenine to give 11a (457 mg, 0.83 mmol, 55%) and 11d
(458 mg, 0.83 mmol, 55%), respectively, as colorless solid.
Simultaneously, N7-coupled regioisomers, 11a0 (127 mg, 15%)
and 11d0 (126 mg, 15%), were also isolated, respectively.
17
1
Similar procedure was carried on the 8c with N-benzoylthy-
mine to give 10c (106 mg, 0.2 mmol, 13%) as colorless solid;
11a: [a]D ꢂ65.12ꢀ (c 1.021, CHCl3); H-NMR (CDCl3) d:
1.44, 1.49 (18H, 2 ꢁ s, Boc Me and t-BuOCO Me), 1.81–1.94
(1H, m, 3-CHH), 2.11–2.26 (1H, m, 3-CHH), 3.00–3.23 (2H,
m, 5-CH2), 3.72–3.96 (1H, m, 2-CH), 3.86 (2H, s, C4-OCH2),
4.26–4.42 (1H, m, 2-CH), 4.42–4.81 (2H, m, C2-CH2), 7.42–
7.53 (2H, m, Ph 3,5-CH), 7.53–7.64 (1H, m, Ph 4-CH), 7.95
(1H, s, adenine 8-CH), 8.04 (2H, d, J ¼ 7.5, Ph 2,6-CH), 8.74
(1H, s, adenosine 2-CH); 13C-NMR (CDCl3) d: 27.9, 28.2
(Boc Me and t-BuOCO Me), 34.3 (3-CH2), 44.5 (C2-CH2),
52.4 (5-CH2), 56.1 (2-CH), 66.4 (C4-OCH2), 76.8 (4-CH), 80.2
(Boc C), 81.6 (t-BuOCO C), 122.4 (adenine 5-C), 127.8 (Ph
2,6-CH), 128.5 (Ph 3,5-CH), 132.4 (Ph 4-CH), 133.5 (Ph 1-C),
141.7 (adenine 6-C), 143.6 (adenine 8-CH), 149.4 (adenine 4-
C), 152.4 (adenine 2-C), 154.8 (Boc C¼¼O), 164.7 (Bz C¼¼O),
168.9 (t-BuOCO C¼¼O); FAB-MS (m/z): 553 (Mþ þ 1)
[Calcd. For C28H36N6O6: M. 552.270 Found: Mþ 552.267
22
¼ ent-10b ; [a]D þ103.91ꢀ (c 1.0352, CHCl3).
Similar procedure was also carried on the 8b and 8c with N-
benzoyladenine to give 11b (310 mg, 0.56 mmol, 37%) and
11c (348 mg, 0.63 mmol, 42%), respectively, as colorless
solid. Simultaneously, N7-coupled regioisomers, 11b0 (134 mg,
16%) and 11c0 (159 mg, 19%), were also isolated, respectively.
17
11b: [a]D ꢂ26.28ꢀ (c 1.000, CHCl3); 1H-NMR (CDCl3) d:
1.45, 1.49 (18H, 2 ꢁ s, Boc Me and t-BuOCO Me), 1.91–2.17
(2H, m, 3-CH2), 3.47–3.72 (2H, m, 5-CH2), 4.06 (2H, s, C4-
OCH2), 4.09–4.23 (1H, m, 4-CH), 4.28–4.57 (1H, m, 2-CH),
4.66 (2H, d, J ¼ 6.3, C2-CH2), 7.40–7.55 (2H, m, Ph 2,6-CH),
7.55–7.62 (1H, m, Ph 4-CH), 7.96–8.09 (2H, m, Ph 3,5-CH),
8.11(1H, s, adenine 8-CH), 8.76 (1H, s, adenine 2-CH), 9.57
(br, NH); 13C-NMR (CDCl3) d: 27.9, 28.1 (Boc Me and
t-BuOCO Me), 33.0, 34.4 (3-CH2 rotamer), 45.8, 46.8
(C2-CH2 rotamer), 52.0, 52.7 (5-CH2 rotamer), 56.0, 56.4 (2-
CH rotamer), 66.7 (C4-OCH2), 78.2, 78.5 (4-CH rotamer),
80.0, 80.1 (Boc C rotamer), 81.7 (t-BuOCO C), 122.8 (adenine
5-C), 127.7 (Ph 2,6-CH), 128.5 (Ph 3,5-CH), 132.4 (Ph 4-CH),
133.6 (Ph 1-C), 141.7 (adenine 6-C), 143.8 (adenine 8-CH),
149.2 (adenine 4-C), 152.2 (adenine 2-C), 154.3 (Boc C¼¼O),
164.7 (Bz C¼¼O), 169.0 (t-BuOCO C¼¼O); FAB-MS (m/z):
553 (Mþ þ 1) [Calcd. For C28H36N6O6: M þ H. 552.270
17
(FAB)]. 11d ¼ ent-11a; [a]D þ65.40ꢀ (c 1.012, CHCl3).
19
11a0; [a]D ꢂ96.10ꢀ (c 1.045, CHCl3); 1H-NMR (CDCl3) d:
1.41, 1.48 (18H, 2 ꢁ s, Boc Me and t-BuOCO Me), 1.87–2.34
(2H, m, 3-CH2), 3.11–3.26 (2H, m, 5-CH2), 3.70–3.92 (1H, m,
2-CH), 3.76 (2H, s, C4-OCH2), 4.38–4.60 (1H, m, 2-CH),
4.94–5.38 (2H, m, C2-CH2), 7.36–7.6 (3H, m, Ph 3,4-5-CH),
8.04 (1H, s, adenine 8-CH), 8.20 (2H, d, J ¼ 7.5, Ph 2,6-CH),
8.44 (1H, s, adenosine 2-CH); 13C-NMR (CDCl3) d: 27.8, 28.1
(Boc Me and t-BuOCO Me), 33.4 (3-CH2), 48.1 (C2-CH2),
52.2 (5-CH2), 56.5 (2-CH), 66.4 (C4-OCH2), 77.0 (4-CH), 80.3
(Boc C), 81.5 (t-BuOCO C), 115.4 (adenine 5-C), 128.0 (Ph
2,6-CH), 128.9 (Ph 3,5-CH), 132.0 (Ph 4-CH), 137.0 (Ph 1-C),
142.0 (adenine 8-C), 147.0 (adenine 2-CH), 150.2 (adenine 6-
C), 154.9 (Boc C¼¼O), 157.8 (adenine 4-C), 168.9 (Bz C¼¼O),
168.9 (t-BuOCO C¼¼O); FAB-MS (m/z): 553 (Mþ þ 1)
17
Found: Mþ 552.271 (FAB)]. 11c ¼ ent-11b; [a]D þ26.38ꢀ
21
1
(c 1.018, CHCl3). 11b0: [a]D ꢂ53.51ꢀ (c 1.025, CHCl3); H-
NMR (CDCl3) d: 1.36, 1.47 (18H, 2 ꢁ s, Boc Me and
t-BuOCO Me), 1.92–2.38 (2H, m, 3-CH2), 3.43–3.85 (2H, m,
5-CH2), 3.97, 4.05 (2H, 2 ꢁ s, C4-OCH2), 4.11–4.34(1H, m,
4-CH), 4.81 (2H, br-s, C2-CH2), 5.00 (1H, br-s, 2-CH), 7.28–
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

September 2011 Design and Stereoselective Synthesis of Four Peptide Nucleic Acid Monomers
with Cyclic Structures in Backbone
1139
7.57 (3H, m, Ph 3,4-5-CH), 8.06–8.30 (2H, m, Ph 2,6-CH),
8.36(1H, br-s, adenine 8-CH), 8.44 (1H, s, adenosine 2-CH);
13C-NMR (CDCl3) d: 27.2, 27.7 (Boc Me and t-BuOCO Me),
35.0, 35.3 (3-CH2 rotamer), 50.6, 51.2 (C2-CH2 rotamer), 52.0
(5-CH2), 56.2, 57.2 (2-CH rotamer), 66.6 (C4-OCH2), 78.2,
78.8 (4-CH rotamer), 79.5 (Boc C), 81.5 (t-BuOCO C), 114.7,
115.6 (adenine 5-C rotamer), 127.6 (Ph 2,6-CH), 128.8 (Ph
3,5-CH), 131.4 (Ph 4-CH), 137.1, 137.5 (Ph 1-C rotamer),
141.5, 141.9 (adenine 8-CH rotamer), 146.7, 147.2 (adenine
2-CH rotamer), 149.2, 149.8 (adenine 6-C rotamer), 154.2
(Boc C¼¼O), 157.2, 158.1 (adenine 4-C rotamer), 168.9
(t-BuOCO C¼¼O), 170.6 (Bz C¼¼O); FAB-MS (m/z): 553 (Mþ
þ 1) [Calcd. For C28H36N6O6: M þ H. 552.270 Found: Mþ
4-CH and Fmoc CH), 7.76 (2H, d, J ¼ 7.5, Fmoc CH), 8.07
(2H, d, J ¼ 5.1, Ph 3,5-CH); 13C-NMR (CDCl3) d: 12.1 (thy-
mine Me), 33.5 (3-CH), 47.1 (Fmoc 9-CH), 51.6 (5-CH2), 52.4
(C2-CH2), 56.0 (2-CH), 67.0 (C4-OCH2), 67.6 (Fmoc CH2),
79.5 (4-CH), 110.4 (thymine 5-C), 120.0, 124.8, 127.1, 127.8
(Fmoc CH), 128.4, 129.0 (Ph 2.6-CH rotamer), 130.1, 130.7,
(Ph 3,5-CH rotamer), 131.6 (Ph 1-C), 133.8, 134.8 (Ph 4-CH
rotamer), 140.9 (thymine CH), 141.2 (Fmoc 4a,4b-C), 143.6,
143.7 (Fmoc 8a,9a-C), 150.3 (thymine 2-C¼¼O), 155.4 (Fmoc
C¼¼O), 163.4 (Bz C¼¼O), . 169.3 (thymine 4-C¼¼O), 171.4
(CO2H); FAB-MS (m/z): 610 (Mþ
þ
1) [Calcd. For
C34H31N3O8: M. 609.211 Found: Mþ 609.212 (FAB)]. 12c ¼
23
ent-12b; [a]D þ61.30ꢀ (c 1.015, CHCl3). 12d ¼ ent-12a;
23
[a]D þ19.98ꢀ (c 1.005, CHCl3).
17
552.269 (FAB)]. 11c0 ¼ ent-11b0; [a]D þ53.91ꢀ (c 1.029,
CHCl3).
General procedure of N-Fmoc replacement of 10. To the
solution of 533 mg (0.98 mmol) of 10a in 5 mL of dry
CH2Cl2 was added 2 mL of trifluoroacetic acid and was stirred
at room temperature for 30 min [17]. Solvent was evaporated
in vacuo. To the obtained residue was added 4 mL of water
and 4 mL of dioxane, followed by 478 mg (1.2 mmol) of
REFERNCES AND NOTES
[1] Yamasaki, T.; Abdel-Aziz, M.; Iwashita, T.; Watanabe, A.;
Sakamoto, M.; Otsuka, M. J Heterocycl Chem 2006, 43, 1111.
[2] Nesterova, M.; Cho-Chung, Y. S. Curr Drug Targets 2004,
5, 683.
[3] (a) Grossmann, T. N.; Ro¨glin, L.; Seitz, O. Angew Chem
Int Ed 2008, 47, 7119; (b) Socher, E.; Bethge, L.; Knoll, A.; Jungnick,
N.; Herrmann, A.; Seitz, O. Angew Chem Int Ed 2008, 47, 9555; (c)
Altenbrunn, F.; Seitz, O. Org Biomol Chem 2008, 6, 2493.
[4] (a) Ito, K.; Katada, H.; Shigi, N.; Komiyama, M. Chem
Commun 2009, 6542; (b) Ishizuka, T.; Otani, K.; Sumaoka, J.;
Komiyama, M. Chem Commun 2009, 1225; (c) Miyajima, Y.; Ishi-
zuka, T.; Yamamoto, Y.; Sumaoka, J.; Komiyama, M. J Am Chem
Soc 2009, 131, 2657; (d) Aiba, Y.; Komiyama, M. Org Biomol Chem
2009, 7, 5078.
Fmoc-OPfp after alkalization with 1.2
g (15 mmol) of
NaHCO3 and reaction mixture was stirred at room temperature
for overnight. To the reaction mixture, 10% citric acid aq. was
added and was extracted with ethyl acetate. Organic layer was
dried over MgSO4 and evaporated in vacuo. Obtained residue
was purified on silica-gel column chromatography (eluent;
CH2Cl2:methanol ¼ 30:1) to give (2S,4R)-2-(N3-benzoylthy-
mine-1-ylmethyl)-4-carboxymethoxy-pyrrolidine-1-carbpxylic
acid 9H –fluoren-9-ylmethyl ester 12a (287 mg, 0.47 mmol,
23
48%) as colorless solid; [a]D ꢂ19.44ꢀ (c 1.004, CHCl3);
¨ ¨
[5] Lundin, K. E.; Good, L.; Stromberg, R.; Graslund, A.;
1H-NMR (CDCl3) d: 1.82 (3H, s, thymine Me), 1.92, 2.19
(2H, 2 ꢁ br-s, 3-CH2), 3.32–3.74 (2H, m, 5-CH2), 3.74–4.06
(2H, m, C2-CH2), 3.92, 3.98 (2H, 2 ꢁ s, C4-OCH2), 4.10 (1H,
br-s, 4-CH), 4.15–4.30 (2H, m, Fmoc 9CH and 2-CH), 4.30–
4.50 (2H, m, Fmoc CH2), 7.12 (1H, s, thymine CH), 7.19–7.89
(8H, m, Fmoc CH); 13C-NMR (CDCl3) d: 15.1 (thymine Me),
34.9 (3-CH), 47.0, 47.4 (Fmoc 9-CH rotamer), 50.3 (C2-CH2),
51.5 (5-CH2), 55.6, 56.1 (2-CH rotamer), 65.8 (C4-OCH2),
67.8 (Fmoc CH2), 129.1 (Ph 2.6-CH), 130.2, 130.4 (Ph 3,5-CH
rotamer), 131.4 (Ph 1-C), 135.0 (Ph 4-CH), 139.9, 140.5 (thy-
mine CH rotamer), 141.2 (Fmoc 4a,4b-C), 143.6 (Fmoc 8a,9a-
C), 150.4 (thymine 2-C¼¼O), 155.7 (Fmoc C¼¼O), 163.1 (Bz
C¼¼O), 169.1 (thymine 4-C¼¼O), 173.1 (CO2H); FAB-MS
(m/z): 610 (Mþ þ 1) [Calcd. For C34H31N3O8: M. 609.211
Found: Mþ 609.209 (FAB)].
Smith, C. I. E. Adv Genet 2006, 56, 1.
[6] Yamasaki, T.; Adbel-Aziz, M.; Kiyota, N.; Maruyama, T.;
Otsuka, M. Heterocycles 2003, 60, 1561.
[7] Adbel-Aziz, M.; Yamasaki, T.; Otsuka, M. Bioorg Med
Chem Lett 2003, 13, 1041.
[8] Baker, G. L.; Fritschel, S. J.; Stille, J. K. J Org Chem 1981,
46, 2954.
[9] (a) Taylor, C. M.; Hardre, R.; Edwards, P. J. B. J Org
Chem 2005, 70, 1306; (b) Hickman, D. T.; Tan, T. H. S.; Morral, J.;
King, P. M.; Cooper, M. A.; Micklefield, J. Org Biomol Chem 2003,
1, 3277.
[10] (a) Rajeev, K. G.; Sanjayan, G. J.; Ganesh, K. N. J Org
Chem 1997, 62, 5169; (b) Peterson, M. L.; Vince, R. J Med Chem
1991, 34, 2787.
[11] Nicolau, K. C.; Hwang, C. K.; Duggan, M. E. J Am Chem
Soc 1989, 111, 6682.
Similar procedure was carried on the 312 mg (0.57 mmol)
of 10b, 362 mg (0.67 mmol) of 10c, and 518 mg (0.95 mmol)
of 10d to give 12b (180 mg, 0.3 mmol, 53%), 12c (247 mg,
0.41 mmol, 61%), and 12d (251 mg, 0.41 mmol, 43%), respec-
tively, as colorless solid.
12b: [a]D ꢂ59.87ꢀ (c 1.005, CHCl3); H-NMR (CDCl3) d:
1.81 (3H, s, thymine Me), 1.73–2.19 (2H, m, 3-CH2), 3.39–
3.73 (3H, m, 5-CH2 and C2-CHH), 3.77–3.97 (1H, m,
C2-CHH), 4.04 (2H, br-s, C4-OCH2), 4.10 (1H, s, 4-CH),
4.11–4.30 (2H, m, Fmoc 9-CH and 2-CH), 4.30–4.50 (2H, m,
Fmoc CH2), 7.22 (1H, s, thymine CH), 7.22–7.66 (3H, m, Ph
[12] Aoyagi, Y.; Williams, R. M. Tetrahedron 1998, 54, 13045.
[13] Altmann, K.-H.; Husken, D.; Cuenoud, B.; Garcia-Echever-
ria, C. Bioorg Med Chem Lett 2000, 10, 929–933.
[14] (a) Robinson, D. S.; Greentein, J. P. J Biol Chem 1952,
195, 383.; (b) Funabashi, K.; Jachmann, M.; Kanai, M.; Shibasaki, M.
Angew Chem Int Ed 2003, 42, 5489.
21
1
[15] Lowe, G.; Vilaivan, T. J Chem Soc Perkin Trans 1989, 1, 539.
[16] (a) Mix, H.; Wilcke, F. W.; Langenbeck, W. Chem Berichte
1958, 91, 2066; (b) Baker, G. L.; Fritschel, S. J.; Stille, J. R.; Stille, J.
K. J Org Chem 1981, 46, 2954.
[17] Schon, I.; Kisfaludy, L. Synthesis 1986, 303.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet