Synthesis of pyrrolidine-based oxy-peptide nucleic acids carrying four types of nucleobases and their transport into cytoplasm

Article abstract of DOI:10.1016/j.tet.2010.10.056

We synthesized 16 pyrrolidine-based oxy-peptide nucleic acid (POPNA) monomers carrying four different nucleobases onto four different stereoisomers of pyrrolidine rings. Using these monomers, we prepared POPNA oligomers, which formed sequence-specific hyb

Full text of DOI:10.1016/j.tet.2010.10.056

Tetrahedron 66 (2010) 9659e9666
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis of pyrrolidine-based oxy-peptide nucleic acids carrying four types of
nucleobases and their transport into cytoplasm
*
Mizuki Kitamatsu , Akiko Takahashi, Takashi Ohtsuki, Masahiko Sisido
Department of Bioscience and Biotechnology, Okayama University, 3-1-1 Tsushimanaka, Kita-ku, Okayama 700-0082, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 August 2010
Received in revised form 20 October 2010
Accepted 20 October 2010
Available online 26 October 2010
We synthesized 16 pyrrolidine-based oxy-peptide nucleic acid (POPNA) monomers carrying four dif-
ferent nucleobases onto four different stereoisomers of pyrrolidine rings. Using these monomers, we
prepared POPNA oligomers, which formed sequence-specific hybrids with DNAs. The oligomer config-
urations influenced the hybrid stability. The oligomers were not taken into CHO cells. However, they
could enter the cell cytoplasm when mixed with the influenza virus hemagglutinin peptideearginine
heptamer conjugate.
Keywords:
Peptide nucleic acid
Ó 2010 Elsevier Ltd. All rights reserved.
Solid-phase peptide synthesis
Cell-penetrating peptide
Confocal laser scanning microscopy
1. Introduction
structure of DNA by introducing pyrrolidine rings in the backbone;
An example is pyrrolidine-based oxy-PNAs (POPNAs; Fig. 1D).5,6
Nielsen-type peptide nucleic acids (PNAs; Fig. 1A) are DNA
surrogates that form stable duplexes and triplexes with DNAs
(Fig. 1B) and RNAs.1 The non-ionic backbone of PNAs leads to stable
hybrids with the nucleic acids, but at the same time, the neutral
backbone results in low water solubility of the PNAs owing to the
formation of aggregates in aqueous solutions. This undesirable
property limits the use of PNAs in medicinal and other applications.
One way of overcoming this drawback is to conjugate PNAs with
cationic peptides such as cell-penetrating peptides (CPPs).2 How-
ever, the PNAeCPP conjugates cause non-specific interactions with
nucleic acids consisting of anionic backbones.
The pyrrolidine ring possesses two chiral centers and there are
four stereoisomers (cis- -, trans- -, cis- -, andtrans- -configurations).
We have previously shown that among the adenine homo-oligomers
L
L
D
D
To overcome these drawbacks, we synthesized new peptide
nucleic acids by introducing ether linkages in the main chain of
peptide nucleic acids; an example is oxy-PNA (OPNA; Fig. 1C).3
Introduction of ether linkages in the PNA backbones can improve
the water solubility of the PNAs.3a,4 OPNAs are also not expected to
non-specifically interact with nucleic acids because of the non-
ionic backbone of the OPNAs. The OPNA oligomers can be suc-
cessfully hybridized with the complementary DNAs, which is in-
dicated by very sharp melting curves. However, because OPNAs do
not form stable hybrids with RNAs, we designed and synthesized
other types of oxy-PNAs that whose structures resembled the
* Corresponding author. Tel./fax: þ81 86 251 8219; e-mail address: kitamatu@
cc.okayama-u.ac.jp (M. Kitamatsu).
Fig. 1. Chemical structures of PNA, DNA, OPNA, and POPNA oligomers.
0040-4020/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2010.10.056

9660
M. Kitamatsu et al. / Tetrahedron 66 (2010) 9659e9666
of the four different configurations, trans-L-POPNA and cis-L-POPNA
form the most stable hybrids with the complementary RNA and DNA,
respectively.5d In thisstudy, we synthesized 16 POPNA monomersof 4
stereoisomers on the pyrrolidine ring with 4 different nucleobases
(adenine (A), thymine (T), guanine(G), and cytosine (C)). The coupling
of these monomers resulted in the synthesis of POPNA oligomers that
contained sequences of the four nucleobases. The coupling was per-
formed by Fmoc-based solid-phase peptide synthesis (SPPS). The
formation of hybrids of these POPNA oligomers with the DNA se-
quences was examined by studying the melting curves. We also in-
vestigated the cellular uptake and endosomal release of the POPNA
oligomers. The cellular uptake of POPNA oligomers was achieved by
mixing the POPNA oligomers with HA2 (an N-terminal 23-mer pep-
tide of the influenza virus hemagglutinin protein)7 conjugated with
a CPP consisting of an arginine heptamer.8
2. Results and discussion
2.1. Synthesis of POPNA monomers and their oligomers
Sixteen POPNA monomers were synthesized from two com-
pounds, 1 (the trans-
starting materials, 1 and the cis-
hydroxyproline and cis- -hydroxyproline in six steps according to
the literature procedure, respectively.5b cis-
-2 and trans- -2 were
synthesized in two steps through the inversion of the free hydroxyl
groupdin the fourth position of the pyrrolidine ring of trans- -1
and cis- -1dby formylation and subsequent treatment with
aqueous ammonia in methanol. The synthetic routes from com-
pound 2 to the trans- -POPNA monomers with A, T, G, and C
nucleobases are shown in Scheme 1.
For preparing the trans- -POPNA T monomer, first, compound 3
L
-1, see Scheme 1) and the cis-
D
-1. These
D
-1, were synthesized from trans-
L-
D
L
D
L
D
L
L
was synthesized by a direct substitution of the hydroxyl group of 2
with N3-benzoylthymine9 under Mitsunobu conditions.5,10 Next, 3
was treated with 30% HBr/AcOH to remove the tert-butylox-
ycarbonyl (Boc), tert-butyl ester (t-Bu), and benzoyl (Bz) protecting
groups. The resulting free amine was then protected with 9-fluo-
renylmethyl succinimidyl carbonate (FmoceOSu) to give the trans-
L
-POPNA T monomer 4 in an overall yield of 85% from 2. For the
Scheme 1. Synthetic routes of the trans-L-POPNA monomers 4, 7, 9, and 14: (i) HCOOH,
DEAD, Ph3P, THF/toluene, rt, overnight; (ii) 25% NH3 aq, MeOH, rt, 1 h; (iii) N3-ben-
zoylthymine, DEAD, Ph3P, THF/toluene, rt, overnight; (iv) 30% HBr/AcOH, rt, 30 min; (v)
FmoceOSu, NaHCO3, H2O/MeCN (1/1, v/v), rt, overnight; (vi) MseCl, TEA, DCM, rt, 3 h;
(vii) N6-benzoyladenine, K2CO3, 18-crown-6, DMF, 65 ꢀC, overnight; (viii) N4-Cbz-cy-
tosine, K2CO3, 18-crown-6, DMF, 65 ꢀC, overnight; (ix) TFA, rt, 30 min; (x) 2-NH2e6-Cl-
Purine, K2CO3, 18-crown-6, DMF, 65 ꢀC, overnight; (xi) 2-nitrophenol, DABCO, TEA, 1,2-
dichloroethane, rt, overnight; (xii) i-PrCOCl, pyridine, rt, overnight; (xiii) 2-nitro-
benzaldoxime, 1,1,3,3-tetramethylguanidine, MeCN, rt, overnight.
trans- -POPNA A monomer, the hydroxyl group of 2 was first
L
reacted with methanesulfonylchloride (MseCl) to give the mesy-
late 5 in 90% yield. Next, 5 was alkylated with N6-benzoyladenine to
give compound 6 in 55% yield.11 Removal of Boc, t-Bu, and Bz
protecting groups on 6 with 30% HBr/AcOH and treatment with
FmoceOSu gave the trans-
of 48% from 2. In the case of the trans-
L-POPNA A monomer 7 in an overall yield
L
-POPNA C monomer, first,
N4-benzyloxycarbonylcytosine (N4-Cbz-cytosine)12 was introduced
to 5 to give compound 8 in 40% yield. Next, Boc and t-Bu protecting
groups of 8 were removed with TFA to avoid deprotection of the Cbz
group. The resulting free compound was treated with FmoceOSu to
nucleobases) were also synthesized from corresponding starting
materials and characterized in a similar manner.
give the trans-
2. In the case of the trans-
L
-POPNA C monomer 9 in an overall yield of 29% from
trans-
were synthesized by SPPS according to previous procedures.5 The
sequences of trans- -POPNA oligomers were H-TGGTGCGAA-Lys-NH2
(trans- -POPNA9), Fam-Sp2-TGGTGCGAATTC-Lys-NH2(Fam-trans-L-
POPNA12, where Sp2 indicates a linker consisting of ethylene glycol
units and Fam represents 5(6)-carboxy-fluorescein used as a fluores-
L-POPNA oligomers containing four types of nucleobases
L-POPNA G monomer, first, alkylation of 2-
amino-6-chloropurine with 5 gave compound 10 in 42% yield.
Conversion of the 6-chloro group on 10 with 2-nitrophenoxy gave
compound 11 in 86% yield. Next, the N2-amino group on 11 was
protected with an isobutyryl group to avoid undesirable acylation
in the peptide-coupling step, to give compound 12 in 93% yield.
Subsequently, removal of the 2-nitrophenoxy group on 12 with
1,1,3,3-tetramethylguanidine gave compound 13 in 65% yield.13 Fi-
nally, removal of Boc and t-Bu protecting groups with 30% HBr/
L
L
cent label), Fam-Sp2-TGGTGCCTC-Sp2-(Arg)7-NH2(Fam-trans-
L-POP
NA9 -R7), and Fam-Sp2-CAGTTAGGGTTAG-Gly-NH2(Fam-trans-
L-
POPNA13). Lys, Arg, and Gly indicate lysine, arginine, and glycine,
respectively. The N-terminals and C-terminals of these oligomers are
primary amines and primary amides, respectively. The crude oligo-
mers were purified by preparative HPLC, and the purified oligomers
AcOH and treatment with FmoceOSu gave the trans-
L-POPNA G
monomer 14 in an overall yield of 22% from 2.
All the final products (4, 7, 9, and 14) were characterized by 1H
were identified by MALDI-TOF mass (trans-L-POPNA9, calculated
NMR, 13C NMR, HRMS, and RP-HPLC (see Supplementary data).
[MþH]þ¼2635.13, observed [MþH]þ¼2634.26; Fam-trans-
L-
Other POPNA monomers of different configurations (cis-
L
-POPNA,
POPNA12, calculated [MþH]þ¼3918.57, observed [MþH]þ
trans- -POPNA, and cis- -POPNA monomers with A, T, G, and C
D
D
¼3917.95; Fam-trans-
L
-POPNA9-R7, calculated [MþH]þ¼4174.91,

M. Kitamatsu et al. / Tetrahedron 66 (2010) 9659e9666
9661
observed [MþH]þ¼4175.50; Fam-trans-
L
-POPNA13, calculated
Table 1
Melting temperatures (Tm) of hybrids of 13-mer POPNAs (N0-CAGTTAGGGTTAG-C0)
with DNAs (50-CTAACCXTAACTG-30, X¼A, T, C, and G)
[MþH]þ¼4163.63, observed [MþH]þ¼4163.26).14
POPNA
DNA (50-CTAACCXTAACTG-30)a
2.2. Hybrid formation of POPNA oligomers with DNAs
Antiparallel orientation
Parallel orientation
C
Melting curves of trans-L-POPNA9 in equimolar mixtures with
X¼C (full-matched)
A
G
T
DNA (50-TTCGXACCA-30, X¼A, T, G, and C) are shown in Fig. 2. In the
case of the completely complementary DNA (X¼C), the sigmoidal
melting curve was observed having a melting temperature (Tm) of
trans-
trans-
L
32.9
41.2
33.6
32.5
29.8
37.8
30.2
28.9
30.2
37.4
28.6
27.2
29.0
35.7
27.8
26.3
28.6 (4.3)b
39.9 (1.4)
27.3 (6.3)
30.8 (1.6)
D
cis-
cis-
L
22.2 ꢀC. The Tm’s of the hybrids of trans-
L-POPNA9 with DNA se-
D
a
quences having a single-mismatched, where X¼A, T, and G, were
Measurement conditions of melting curves of these mixtures are the same as
21.4, 21.0, and 20.3 ꢀC, respectively. These results indicate that
those of Fig. 1.
b
Values in parentheses show a difference of Tm (antiparallel orientation) for Tm
trans-L-POPNA9, having a sequence that contains the four types of
nucleobases, forms moderate sequence-specific hybrids with DNAs.
(parallel orientation).
oligomers, trans-D-POPNA13 formed the most stable hybrid with
the fully complementary DNA (Tm¼41.2 ꢀC). trans-
L-POPNA13, cis-L-
POPNA13, and cis-D-POPNA13 showed lower hybrid stabilities with
the fully complementary DNA than trans-D-POPNA13. All POPNA
oligomers formed moderate sequence-specific hybrids with DNAs.
Successful hybridizations of all the stereoisomers of POPNA are
explained in terms of their flexible main chains that contain ether
linkages. However, the higher stability of the hybrid between trans-
D-POPNA and DNA, than those of the other oligomers/DNA hybrids,
is a consequence of its restricted main chain, which contains pyr-
rolidine rings. Moreover, hybrids of POPNA oligomers with DNAs
are preferentially formed with antiparallel orientations, especially
trans-L-POPNA13 and cis-L
-POPNA13 (32.9 ꢀC and 33.6 ꢀC for anti-
parallel orientation and 28.6 ꢀC and 27.3 ꢀC for parallel orientation,
respectively). This result also suggests that configurations of POPNA
have a tendency to form a single, stable hybrid with DNA.
2.3. Transport of POPNA oligomers into cytoplasm
Fig. 2. Temperature dependences of absorption intensities at 260 nm for equimolar
mixtures of trans-L-POPNA9 (N0-TGGTGCGAA-C0)/DNA (50-TTCGXACCA-30, X¼A, T, G,
and C) in 100 mM NaCl, 10 mM NaH2PO4, and 0.1 mM EDTA, with pH 7.0. [trans-L-
We examined the internalization of the POPNA oligomer into
Chinese hamster ovary (CHO) cells with confocal laser scanning
microscopy. First, the CHO cells were cultured in the presence of
POPNA9]¼[DNA]¼2.5
mM. The melting curves were recorded by heating the solution at
0.5 ꢀC/0.5 min. The observed absorbance has been normalized at 60 ꢀC.
10
served with the CHO cells. Unfortunately, the POPNA oligomer did
not internalize into the cells by themselves. In the case of the trans-
POPNA oligomer conjugated with a CPP (Fam-trans- -POPNA9-R7),
the fluorescence fromFamwas observed inside the cells, as shown in
Fig. 3. However, this image indicates that the fluorescence is mostly
confined in small vesicular compartments. These endosomes were
observed even after 6 h of incubation, without a decrease in
mM Fam-trans-L-POPNA12. However, no fluorescence was ob-
To examine the effect on the configuration of POPNAs when
hybridized with DNAs, we synthesized 13-mer POPNA oligomers of
L-
different configurations (trans-
L-POPNA13, cis-L-POPNA13, trans-D-
L
POPNA13, and cis- -POPNA13; with
D
a
sequence of H-CAGT-
TAGGGTTAG-NH2) and we estimated the melting temperatures of
the oligomers/DNAs hybrids from their respective melting curves.
Melting temperatures of these hybrids are listed in Table 1. Of these
Fig. 3. Confocal microscopy images of CHO cells cultured for 2 h at 37 ꢀC in the presence of 1.0
mM Fam-trans-L-POPNA9-R7. The left image shows the green fluorescence image from
Fam-fluorescence. The right image is a superimposed image of the phase contrast and the fluorescence images.

9662
M. Kitamatsu et al. / Tetrahedron 66 (2010) 9659e9666
fluorescence. These results suggest that the POPNA oligomer con-
jugated with a CPP internalized inside CHO cells by an endocytosis
mechanism and that they remained inside the endosomes.
To release the POPNA oligomer from the endosomes, we syn-
thesized an HA2 (Tmr-HA2-R7, a peptide that disrupts endosomes)7
labeled with a 5(6)-tetramethylrhodamine fluorophore at the N-
terminal and conjugated with R7 at the C-terminal. The CHO cells
inside the cytoplasm of CHO cells when mixed with a HA2eCPP
conjugate. The antisense effect of the four types of stereoisomers of
the POPNA oligomers, in combination with the HA2eCPP conjugate
is now under investigation.
4. Experimental
4.1. General
were incubated with the mixture of Fam-trans-
HA2-R7. The fluorescence from the CHO cells, after incubation with
Fam-trans- -POPNA13, in the absence and presence of Tmr-HA2-R7
was measured. Incubation with only Fam-trans- -POPNA13 showed
no internalization of the oligomer. On the other hand, Fam-trans-
L-POPNA13 and Tmr-
L
Diethyl azodicarboxylate (DEAD), triphenylphosphine (Ph3P),
methanesulfonylchloride (MseCl), 25% aqueous ammonia, triethyl-
amine (TEA), N6-benzoyladenine, 18-crown-6, 2-amino-6-chlor-
opurine, 2-nitrophenol, 2-nitrobenzaldoxime, K2CO3, NaHCO3,
KHSO4, MgSO4, pyridine, and other solvents were purchased from
L
L-
POPNA13 was successfully internalized inside CHO cells in the
presence of Tmr-HA2-R7 (Fig. 4), and more importantly, both the red
Fig. 4. Confocal microscopy images of CHO cells cultured for 1 h at 37 ꢀC in the presence of Fam-trans-L-POPNA13 and Tmr-HA2-R7 (Tmr-Sp2-GLFEAIEGFIENGWEGMIDGWYG-Sp2-
RRRRRRR-NH2). [Fam-trans-L-POPNA13]¼[Tmr-HA2-R7]¼10
M.15 The top left image shows the phase contrast image of the optical section at the middle of cells. The top right
image shows the green fluorescence image from Fam groups. The bottom right image shows the red fluorescence image from Tmr groups.
m
(Tmr) and green (Fam) fluorescence were detected throughout the
whole cell. This indicates that the POPNA oligomers were taken up
into the cells with the Tmr-HA2-R7 and released into the cytoplasm
with the disruption of the endosomes by the HA2 peptide within 1 h.
Wako Chemicals (Tokyo, Japan). Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH,
Fmoc-Arg(Pbf)-OH, 30% hydrogen bromide in acetic acid (HBr/AcOH),
trifluoroacetic acid (TFA) and 9-fluorenylmethyl succinimidyl car-
bonate (FmoceOSu) were purchased from Watanabe Chemicals
(Hiroshima, Japan). Formic acid (HCOOH) and Fmoc-NH-PEG-COOH
(Sp2) were purchased from Merck (NJ, USA). 1,4-Diazabicyclo[2.2.2]
octane (DABCO), isobutyrylchloride (i-PrCOCl), 1,1,3,3-tetrame-
thylguanidine, 6-[fluorescein-5(6)-carboxamido]hexanoic acid N-
hydroxysuccinimide ester (Fam), and 6-carboxy-tetramethylrhod-
amine N-succinimidyl ester (Tmr) were purchased from Sigma-
eAldrich (MO, USA). DNA oligomers were purchased from Invitrogen
Japan (Tokyo, Japan). These reagents were used without purification.
3. Conclusions
In summary, we synthesized 16 POPNA monomers with four
types of nucleobases on four stereoisomers of the pyrrolidine rings.
POPNA oligomers of a mixed sequence of four types of nucleobases
were also synthesized. These POPNA oligomers were successfully
hybridized with DNAs. The configurations of POPNA oligomers af-
fect the hybridization. The POPNA oligomers were readily taken up
Distilled water was used throughout the synthesis. 1H NMR and 13
C

M. Kitamatsu et al. / Tetrahedron 66 (2010) 9659e9666
9663
NMR spectra were recorded on a Varian Mercury 300 spectrometer.
High-resolution mass spectra were measured on Nihon Denshi MS
700. MALDI-TOF mass spectra were measured on Applied Biosystems
Voyager DE-Pro, and hybridization behavior was observed on a JASCO
V-560 UVevis spectrophotometer equipped with a temperature
controller. Fluorescent images from confocal laser scanning micros-
copy were observed on Olympus FLUOVIEW FV-1000.
64%. 1H NMR (300 MHz, CDCl3):
d
1.9 (m, 3H, thymine CH3), 2.1e2.7
(m, 2H, C30H2), 2.9e4.8 (m, 6H, CaeCH2eOeCH2eCO, Fmoc CH, and
C20H), 4.2 (m, 2H, FmocCH2), 5.0e5.3 (m, 2H, C50H2), 5.6 (m,1H, C40H),
7.2e7.8 (m, 9H, Fmoc aromatic ring and C6H). 13C NMR (75 MHz,
CDCl3):
d
12.3 (thymine CH3), 34.2 (C30), 47.2 (Fmoc CH), 49.6 (C50),
51.5 (C40), 55.7 (C20), 66.7 (Fmoc CH2), 67.9 (OeCH2eCO), 71.8
(CaeCH2eO), 111.4 (C5), 119.9, 125.0, 127.1, 127.7, 141.2, and 143.8
(Fmoc aromatic ring),137.2 (C6),151.2 (C2),154.2 (Fmoc amide),164.2
(C4), 172.5 (COOH). HRMS (found/calculated): 506.1914/506.1849
[MþH]þ. IR (film, CHCl3, cmꢁ1): 3066, 2959, 2930, 2901, 1697, 1522,
Key intermediates for POPNA monomers, 1 (trans-
L-configura-
tion), cis- -configurated 1(2), cis- -configurated 1, and trans-D-
L
D
configurated 1 were synthesized according to procedures that were
previously reported.5b
1508, 1474, 1452, 1424, 1339, 1273, 1138. Synthesis of trans-
uration of 4 and cis-
aforementioned protocols and these spectroscopic data were identi-
cal with 4 and cis- -configuration of 4, respectively. trans- -configu-
D-config-
D-configuration of 4 was performed according to
4.2. Synthesis of POPNA T monomers
L
D
4.2.1. (2S,4R)-4-(3-Benzoyl-5-methyl-2,4-dioxo-3,4-dihydro-2H-pyr-
imidin-1-yl)-2-tert-butoxycarbonylmethoxymethyl-pyrrolidine-1-
carboxylic acid tert-butyl ester (3). Compound 2 (3.51 g,10.6 mmol),
Ph3P (4.17 g,15.9 mmol), and N3-benzoylthymine (2.39 g,10.6 mmol)
were added to tetrahydrofuran (THF)(10 mL). To this mixture was
added DEAD(2.49 mL,15.9 mmol) underargonatmosphere atꢁ15 ꢀC
and then stirred at room temperature overnight. The resultant mix-
ture was evaporated to dryness and the residue was washed several
times with 50/50 ethyl acetate (AcOEt)/hexane (Hex) mixture and
dried in vacuo. The residue was chromatographed on silica gel with
40/60 AcOEt/Hex mixture as eluting solvent. The title compound 3
was obtained as white powder. Yield: 17%. Rf (AcOEt/Hex¼5/5, v/v):
ration of 4: yield: 36%. HRMS (found/calculated): 506.1931/506.1849
[MþH]þ. cis-
D
-configuration of 4: yield: 40%. HRMS (found/calcu-
lated): 506.1946/506.1849 [MþH]þ.
4.3. Synthesis of POPNA A monomers
4.3.1. (2S,4S)-2-tert-Butoxycarbonylmethoxymethyl-4-methane-sul-
fonyloxy-pyrrolidine-1-carboxylic acid tert-butyl ester (5). MseCl
(2.5 g, 21.6 mmol) and TEA (2.2 g, 21.6 mmol) were added to a so-
lution of 2 (5.5 g, 16.6 mmol) in dichloromethane (DCM, 17 mL)
under ice cooling, and the mixture was stirred for 3 h at room
temperature. The mixture was evaporated to dryness. The residue
was dissolved in water (50 mL) and extracted three times with
AcOEt (each 50 mL). The AcOEt layer was washed with water
(50 mL) and brine (50 mL), then, dried over MgSO4. Filtration fol-
lowed by solvent evaporation gave crude viscous oil. The crude oil
was chromatographed on silica gel with 30/70 AcOEt/Hex mixture
as eluting solvent. The title compound 5 was obtained as a colorless
0.41.1H NMR (300 MHz, CDCl3):
d
1.5 (sþs,18H, Boc CH3 and tert-butyl
CH3), 1.96 (s, 3H, thymine CH3), 2.2e2.6 (m, 2H, C30H2), 3.4e4.3 (m,
7H, C50H2, CaeCH2eOeCH2eCO and C20H), 5.2 (m, 1H, C40H), 7.1 (s,
1H, C6H), 7.4e7.9 (m, 5H, phenyl). 13C NMR (75 MHz, CDCl3):
d 14.3
(thymine CH3), 28.1 and 28.4 (Boc CH3 and tert-butyl CH3), 32.4 (C30),
49.6 (C50), 55.8 (C20), 62.3 (C40), 68.9 (OeCH2eCO), 72.4 (CaeCH2eO),
80.5and 81.7(BocCandtert-butylC),111.5(C5),129.1,130.4,131.5, and
134.9 (phenyl), 136.3 (C6), 149.8 (C2), 154.5 (Boc amide), 161 (C4),
168.9 (tert-butyl ester and benzoyl amide).
viscous oil. Yield: 93%. 1H NMR (300 MHz, CDCl3):
d
1.5 (sþs, 18H,
Boc CH3 and tert-butyl CH3), 2.2e2.6 (m, 2H, C30H2), 3.1 (s, 3H,
mesyl CH3), 3.4e4.2 (m, 7H, C20H, C50H2 and CaeCH2eOeCH2eCO),
5.2 (m, 1H, C40H). 13C NMR (75 MHz, CDCl3):
d 28.1 and 28.4 (Boc
4.2.2. (2S,4R)-2-Carboxymethoxymethyl-4-(5-methyl-2,4-dioxo-3,4-
dihydro-2H-pyrimidin-1-yl)-pyrrolidine-1-carboxylic acid 9H-fluo-
CH3 and tert-butyl CH3), 34.8 (C30), 38.7 (mesyl CH3), 53.1 (C50), 55.5
(C20), 68.8 (OeCH2eCO), 71.4 (CaeCH2eO), 79.1 (C40), 80.3 and 81.5
(Boc C and tert-butyl C), 154.0 (Boc amide), 169.5 (tert-butyl ester).
ren-9-ylmethyl ester (4, trans- -POPNA T monomer). Compound 3
L
(1.00 g, 1.83 mmol) was treated with 30% HBr/AcOH (10 mL) for
30 min at room temperature. The resultant mixture was evaporated
to dryness. The residue was dissolved in 5% aqueous NaHCO3 (10 mL)
to adjust the pH to 8. A solution of FmoceOSu (0.71 g, 2.20 mmol) in
acetonitrile (MeCN, 10 mL) was added to the aqueous solution with
stirring under ice cooling. The reaction mixture was stirred at room
temperature overnight and evaporated to dryness. The residue was
dissolved in water and the aqueous layer was washed three times
with diethyl ether (Et2O, each 30 mL), acidified to pH 7 with 5%
aqueous KHSO4, and extracted three times with AcOEt. The AcOEt
layer was washed with water and brine, then, dried over MgSO4.
Filtration followed by solvent evaporation gave the title compound 4.
Compound 4 was obtained as a white powder. Compound 4 was
purified by preparatory RP-HPLC (C18 column) before using to SPPS.
4.3.2. (2S,4R)-4-(6-Benzoylamino-purin-9-yl)-2-tert-butox-
ycarbonylmethoxymethyl-pyrrolidine-1-carboxylic acid tert-butyl
ester (6). Compound 5 (2.5 g, 6.1 mmol), N6-benzoyladeninne (2.9 g,
12 mmol), 18-crown-6-ether (1.0 g, 3.8 mmol), and K2CO3 (1.6 g,
12 mmol) were added to N,N0-dimethylformamide (DMF,10 mL). The
mixture was stirred at 60 ꢀC overnight. The mixture was evaporated
to dryness. The residue was dissolved in water (50 mL) and extracted
three times with AcOEt (each 50 mL). The AcOEt layer was washed
with water (50 mL) and brine (50 mL), then, dried over MgSO4. Fil-
tration followed by solvent evaporation gave crude yellow powder.
The crude compound was chromatographed on silica gel with 90/10
AcOEt/Hex mixture as eluting solvent. The title compound 6 was
obtained as a white powder. Yield: 41%. Rf (AcOEt/Hex¼7/3 (v/v)):
Yield: 60%.1H NMR (300 MHz, CDCl3):
d
1.7 and 1.8 (sþs, 3H, thymine
0.13.1H NMR (300 MHz, CDCl3):
d
1.44 and 1.48 (sþs,18H, Boc CH3 and
CH3), 2.0e2.4 (m, 2H, C30H2), 3.0e4.2 (m, 7H, C50H2,
CaeCH2eOeCH2eCO, and Fmoc CH), 4.3 (m, 2H, Fmoc CH2), 4.4e4.7
(m, 1H, C20H), 5.0e5.3 (m, 1H, C40H), 7.3e7.9 (m, 8H, Fmoc aromatic
tert-butyl CH3), 2.6e2.8 (m, 2H, C30H2), 3.6e4.4 (m, 7H, C20H, C50H2
and CaeCH2eOeCH2eCO), 5.42 (m, 1H, C40H), 7.4e8.0 (m, 5H, phe-
nyl), 8.2 (m,1H, benzoyl amide), 8.77 (s,1H, C2H), 9.23(s,1H, C8H).13C
ring), 7.6 (s,1H, C6H).13C NMR (75 MHz, CDCl3):
d
12.5 (thymine CH3),
NMR (75 MHz, CDCl3): d 28.1 and 28.4 (Boc CH3 and tert-butyl CH3),
32.4and 33.4 (C30), 47.2(FmocCH), 49.6 (C50), 53.0and54.0(C40), 55.9
and 56.4 (C20), 66.6 (Fmoc CH2), 68.1 (OeCH2eCO), 71.8 (CaeCH2eO),
111.9 (C5), 119.9, 124.9, 127.0, 127.7, 141.3, and 143.8 (Fmoc aromatic
ring), 136.6 (C6), 151.3 (C2), 154.5 (Fmoc amide), 164.2 (C4), 173.6
(COOH). HRMS (found/calculated): 506.1953/506.1849 [MþH]þ. IR
(film, CHCl3, cmꢁ1): 3067, 2957, 2895, 1697, 1522, 1508, 1474, 1451,
33.6 (C30), 51 (C50), 53.7 (C40), 55.9 (C20), 69.0 (OeCH2eCO), 71.9
(CaeCH2eO), 80.3 and 81.7 (Boc C and tert-butyl C), 123 (C5), 127.8,
128.8, 132.7, and 133.6 (phenyl), 129 (C6),141.3 (C8), 149.5 (C2), 152.4
(C4),153.9(Bocamide),164.7(benzoylamide),169.4(tert-butylester).
4.3.3. (2S,4R)-4-(6-Benzoylamino-purin-9-yl)-2-carboxymethox-
1419,1356, 1339, 1267, 1140. Synthesis of cis-L-configuration of 4 was
ymethyl-pyrrolidine-1-carboxylic acid 9H-fluoren-9-ylmethyl ester
also performed in a similar manner to that described above. Yield:
(7, trans- -POPNA A monomer). Compound 6 (1.4 g, 2.5 mmol) was
L

9664
M. Kitamatsu et al. / Tetrahedron 66 (2010) 9659e9666
treated with 30% HBr/AcOH (10 mL) for 30 min at room temper-
ature. The resultant mixture was evaporated to dryness. The res-
idue was dissolved in 5% aqueous NaHCO3 (50 mL) to adjust the
pH to 8. A solution of FmoceOSu (0.94 g, 2.8 mmol) in MeCN
(50 mL) was added to the aqueous solution with stirring under ice
cooling. The reaction mixture was stirred at room temperature
overnight and evaporated to dryness. The residue was dissolved in
water and the aqueous layer was washed three times with Et2O
(each 30 mL), acidified to pH 7 with 5% aqueous KHSO4, and the
resultant precipitate was collected by filtration. The residue was
washed several times with water and dried in vacuo. Compound 7
was obtained as a white powder. Compound 7 was purified by
preparatory RP-HPLC (C18 column) before using to SPPS. Yield:
(C6), 152.2 (Cbz ester), 153.9 (Boc amide), 155.4 (C2), 161.7 (C4),
169.3 (tert-butyl ester).
4.4.2. (2S,4R)-4-(4-Benzyloxycarbonylamino-2-oxo-2H-pyrimidin-1-
yl)-2-carboxymethoxymethyl-pyrrolidine-1-carboxylic acid 9H-fluo-
ren-9-ylmethyl ester (9, trans- -POPNA C monomer). Compound 8
L
(0.50 g, 0.89 mmol) was treated with TFA (10 mL) for 30 min at room
temperature. The resultant mixture was evaporated to dryness. The
residue was dissolved in 5% aqueous NaHCO3 (50 mL) to adjust the
pH to 8. A solution of FmoceOSu (0.33 g, 0.98 mmol) in MeCN
(50 mL) was added to the aqueous solution with stirring under ice
cooling. The reaction mixture was stirred at room temperature
overnight and evaporated to dryness. The residue was dissolved in
water and the aqueous layer was washed three times with Et2O
(each 30 mL), acidified to pH 3 with 5% aqueous KHSO4, and
extracted three times with AcOEt (each 50 mL). The AcOEt layer was
washed with water (50 mL) and brine (50 mL) and dried over
MgSO4. Filtration followed by solvent evaporation gave the title
compound 9. Compound 9 was obtained as a white powder. Com-
pound 9 was purified by preparatory RP-HPLC (C18 column) before
77%. 1H NMR (300 MHz, CDCl3): 2.4e3.2 (m, 3H, C30H2 and C20H),
d
3.5e4.8 (m, 9H, Fmoc CH, C50H2, CaeCH2eOeCH2eCO and Fmoc
CH2), 5.48 and 5.67 (mþm, 1H, C40H, rotamer), 7.2e8.2 (m, 13H,
Fmoc aromatic ring and phenyl), 8.19 and 8.29 (sþs, 1H, C8H,
rotamer), 8.72 and 8.80 (sþs, 1H, C2H, rotamer), 10.52 (br, 1H,
COOH). 13C NMR (75 MHz, CDCl3):
d
33.6 (C30), 47.2 (Fmoc CH),
50.5 (C50), 54.2 (C40), 56.0 and 56.6 (C20), 67.4 (Fmoc CH2), 68.0
(OeCH2eCO), 71.8 (CaeCH2eO), 119.9, 124.9, 127.0, 127.7, 141.2,
and 143.7 (Fmoc aromatic ring), 127.2, 128.4, 132.8, and 133.0
(phenyl), 128.6 (C5), 142.4 (C8), 149.6 (C4), 151.5 (C6), 152.2 (C2),
154.2 (Fmoc amide), 165.4 (benzoyl amide), 173.5 (COOH). HRMS
(found/calculated): 619.2282/619.2227 [MþH]þ. IR (film, CHCl3,
cmꢁ1): 3069, 2895, 1701, 1611, 1518, 1456, 1420, 1339, 1242, 1138.
using to SPPS. Yield: 54%. 1H NMR (300 MHz, CDCl3):
d 2.1e3.2 (m,
3H, C30H2 and C20H), 3.4e4.8 (m, 9H, Fmoc CH, C50H2,
CaeCH2eOeCH2eCO, and Fmoc CH2), 5.22 (s, 2H, Cbz CH2), 5.30 (m,
1H, C40H), 7.2e7.8 (m, 15H, C6H, Fmoc aromatic ring, phenyl, and
C5H), 9.25 (br,1H, COOH). 13C NMR (75 MHz, CDCl3):
d 32.3 and 33.4
(C30), 47.2 (Fmoc CH), 50.2 (C50), 56.2 (C40), 57.1 (C20), 66.4 (Cbz CH2),
67.2 (Fmoc CH2), 67.8 (OeCH2eCO), 71.4 (CaeCH2eO), 96.0 (C5),
119.9,124.8,127.1,127.7,141.2, and 143.7 (Fmoc aromatic ring),124.4,
128.1, 128.5, and 135.0 (phenyl), 145.5 (C6), 152.8 (Cbz ester), 154.3
(Fmoc amide), 154.9 (C2), 162.2 (C4), 172.8 (COOH). HRMS (found/
Synthesis of cis-
L
-configuration of 7 was also performed in an
identical manner to that described above. Yield: 89%. 1H NMR
(300 MHz, CDCl3):
d
2.3e3.2 (m, 3H, C30H2 and C20H), 3.2e5.4 (m,
10H, Fmoc CH, C50H2, CaeCH2eOeCH2eCO, Fmoc CH2 and C40H),
7.2e7.8 (m, 13H, Fmoc aromatic ring and phenyl), 7.96 (br, 1H,
C8H), 8.76 and 8.86 (brþbr, 1H, C2H, rotamer), 10.85 (br, 1H,
calculated): 625.2314/625.2220 [MþH]þ. Synthesis of cis-
L-config-
uration of 9 was also performed in an identical manner to that de-
COOH). 13C NMR (75 MHz, CDCl3):
d
32.9 (C30), 47.2 (Fmoc CH), 51.5
scribed above. Yield: 10% at two steps. 1H NMR (300 MHz, CDCl3):
(C50), 52 (C40), 55.8 (C20), 67.2 (Fmoc CH2), 67.9 (OeCH2eCO), 71.6
(CaeCH2eO), 119.9, 124.7, 127.1, 127.7, 141.3, and 143.8 (Fmoc ar-
omatic ring), 128.5 (C5), 127.4, 128.2, 132.7, and 133.1 (phenyl),
142.5 (C8), 149.2 (C4), 151 (C6), 151.9 (C2), 154.2 (Fmoc amide),
165.5 (benzoyl amide), 172.9 (COOH). HRMS (found/calculated):
619.2332/619.2227 [MþH]þ. IR (film, CHCl3, cmꢁ1): 3069, 2895,
1699, 1611, 1522, 1452, 1420, 1338, 1246, 1140. Syntheses of trans-
d
1.9e3.2 (m, 4H, C30H2 and C50H2), 3.4e4.8 (m, 8H,
CaeCH2eOeCH2eCO, Fmoc CH, Fmoc CH2 and C20H), 5.2e5.5 (m, 3H,
C40H and Cbz CH2), 7.1e8.8 (m,15H, C6H, Fmoc aromatic ring, phenyl,
and C5H).13C NMR (75 MHz, CDCl3): 33.2 (C30), 47.4 (Fmoc CH), 51.5
d
(C50), 52 (C40), 55.8 (C20), 67.1 (Cbz CH2), 68.0 (Fmoc CH2), 68.4
(OeCH2eCO), 72.8 (CaeCH2eO), 96.3 (C5), 120.0, 124.8, 126.9, 127.1,
141.3, and 143.8 (Fmoc aromatic ring), 124, 127.8, 128.3, and 134.8
(phenyl),148.6 (C6), 153.0 (Cbz ester), 154.0 (Fmoc amide), 154 (C2),
162.0 (C4), 175.9 (COOH). HRMS (found/calculated): 625.2305/
625.2220 [MþH]þ. IR (film, CHCl3, cmꢁ1): 1751, 1697, 1624, 1558,
1508, 1452,1429,1400,1354,1253,1140, 1124,1071,1005. Syntheses
D
-configuration of 7 and cis-
according to aforementioned protocols and these spectroscopic
data were identical with 7 and cis- -configuration of 7, re-
D
-configuration of 7 were performed
L
spectively. trans- -configuration of 7: yield: 41%. HRMS (found/
D
calculated): 619.2332/619.2227 [MþH]þ. cis-
D
-configuration of 7:
of trans-
formed according to aforementioned protocols and these spectro-
scopic data were identical with 9 and cis- -configuration of 9,
D
-configuration of 9 and cis- -configuration of 9 were per-
D
Yield: 9%. HRMS (found/calculated): 619.2312/619.2227 [MþH]þ.
L
4.4. Synthesis of POPNA C monomers
respectively. trans-D-configurationof 9:yield: 5%at twosteps. HRMS
(found/calculated): 625.2323/625.2220 [MþH]þ. cis-
D-configura-
4.4.1. (2S,4R)-4-(4-Benzyloxycarbonylamino-2-oxo-2H-pyrimidin-1-
yl)-2-tert-butoxycarbonylmethoxymethyl-pyrrolidine-1-carboxylic
acid tert-butyl ester (8). Compound 5 (2.00 g, 4.88 mmol), N4-Cbz-
cytosine (2.99 g, 12.2 mmol), 18-crown-6 (3.22 g, 12.2 mmol), and
K2CO3 (1.68 g, 12.2 mmol) were added to DMF (50 mL) and stirred
at 65 ꢀC overnight. The mixture was filtered and evaporated dry-
ness. The crude oil was chromatographed on silica gel with 70/30
AcOEt/Hex mixture as eluting solvent. The title compound 8 was
obtained as a colorless viscous oil. Yield: 40%. 1H NMR (300 MHz,
tion of 9: yield: 33% HRMS (found/calculated): 625.2326/625.2220
[MþH]þ. IR (film, CHCl3, cmꢁ1): 1748, 1701, 1654, 1628, 1558, 1521,
1450, 1420, 1352, 1331, 1142, 1103, 1069, 993, 974.
4.5. Synthesis of POPNA G monomers
4.5.1. (2S,4R)-4-(2-Amino-6-chloro-purin-9-yl)-2-tert-butox-
ycarbonylmethoxymethyl-pyrrolidine-1-carboxylic acid tert-butyl
ester (10). Compound 5 (5.0 g, 12.4 mmol), 2-amino-6-chloropurin
(6.3 g, 37.1 mmol), 18-crown-6-ether (9.8 g, 37.1 mmol), and K2CO3
(5.1 g, 37.1 mmol) were added to DMF (50 mL) and stirred at 65 ꢀC
overnight. The mixture was filtered and evaporated to dryness. The
mixture was added to AcOEt and the insoluble residue was re-
moved. The crude oil was chromatographed on silica gel with 70/30
AcOEt/Hex mixture as the eluting solvent. The title compound 10
was obtained as a colorless viscous oil. Yield: 36%. 1H NMR
CDCl3):
d
1.44 and 1.47 (sþs, 18H, Boc CH3 and tert-butyl CH3), 2.35
and 2.55 (mþm, 2H, C30H2), 3.4e4.3 (m, 7H, C50H2,
CaeCH2eOeCH2eCO, and C20H), 5.21 (s, 2H, Cbz CH2), 5.28 (m, 1H,
C40H), 7.1e7.9 (m, 8H, C6H, phenyl, C5H, and Cbz ester). 13C NMR
(75 MHz, CDCl3): d 28.1 and 28.4 (Boc CH3 and tert-butyl CH3), 32.3
and 34.2 (C30), 50 (C50), 51.1 (C40), 55.6 (C20), 67.9 (Cbz CH2), 68.9
(OeCH2eCO), 71.5 and 72.2 (CaeCH2eO), 80.2 and 81.6 (Boc C and
tert-butyl C), 95.2 (C5), 128.3, 128.6, 131, and 134.9 (phenyl), 145.0
(300 MHz, CDCl3):
d
1.45 and 1.46 (sþs, 18H, Boc CH3 and tert-butyl

M. Kitamatsu et al. / Tetrahedron 66 (2010) 9659e9666
9665
CH3), 2.61 (t, 2H, C30H2), 3.5e4.3 (m, 7H, C20H, C50H2 and
CaeCH2eOeCH2eCO), 5.17 (m, 1H, C40H), 5.29 (br, 2H, NH2), 7.75 (s,
and brine (100 mL), and it was then dried over MgSO4. Filtration
followed by solvent evaporation gave a crude viscous oil. The crude
oil was chromatographed on silica gel with 95/5 AcOEt/MeOH
mixture as the eluting solvent. The title compound 13 was obtained
1H, C8H). 13C NMR (75 MHz, CDCl3):
d 28.1 and 28.4 (Boc CH3 and
tert-butyl CH3), 33.4 and 34.6 (C30), 50.5 and 51.2 (C50), 52.8 and
53.5 (C40), 55.9 (C20), 68.9 (OeCH2eCO), 71.9 (CaeCH2eO), 80.3 and
81.7 (Boc C and tert-butyl C), 125.5 (C5), 140.5 (C8), 151.4 (C2), 153.5
(Boc amide), 158.9 (C4), 162.5 (C6), 169.3 (tert-butyl ester).
as a white powder. Yield: 50%. 1H NMR (300 MHz, CDCl3):
d 1.22 and
1.24 (d, 6H, iso-butyl CH3), 1.42 and 1.45 (sþs, 18H, Boc CH3 and tert-
butyl CH3), 2.4e2.8 (m, 3H, C30H2 and iso-butyl CH), 3.5e4.3 (m, 7H,
C20H, C50H2 and CaeCH2eOeCH2eCO), 5.02 (m, 1H, C40H), 7.61 (s,
1H, C8H), 9.37 (br,1H, iso-butyl amide),12.08 (br, 1H, C1H). 13C NMR
4.5.2. (2S,4R)-4-[2-Amino-6-(2-nitrophenoxy)-purin-9-yl]-2-tert-
butoxycarbonylmethoxymethyl-pyrrolidine-1-carboxylic acid tert-
butyl ester (11). DABCO (0.46 g, 4.15 mmol) and TEA (1.26 g,
12.45 mmol) were added to a solution of 10 (2.00 g, 4.15 mmol) in
1,2-dichloroethane (20 mL) and the mixture was stirred at room
temperature overnight. The mixture was evaporated to dryness. The
residue was dissolved in water (100 mL) and extracted three times
with DCM (each 50 mL). The DCM layer was washed with water
(100 mL) and brine (100 mL), and it was then dried over MgSO4.
Filtration followed by solvent evaporation gave crude viscous oil.
The crude oil was chromatographed on silica gel with 70/30 AcOEt/
Hex mixture as the eluting solvent. The title compound 11 was
obtained as a yellow crystal. Yield: 78%. 1H NMR (300 MHz, CDCl3):
(75 MHz, CDCl3): d 18.9 and 19.0 (iso-butyl CH3), 28.1 and 28.4 (Boc
CH3 and tert-butyl CH3), 34.0 (C30), 36.3 (iso-butyl CH), 50.9 (C50),
52.1 (C40), 56.0 (C20), 68.9 (OeCH2eCO), 71.9 (CaeCH2eO), 80.3 and
81.8 (Boc C and tert-butyl C), 121.6 (C5), 137.2 (C8), 147.3 and 148.1
(C2 and C6), 154.3 (Boc amide), 155.7 (C4), 169.6 (tert-butyl ester),
178.8 (iso-butyl amide).
4.5.5. (2S,4R)-2-Carboxymethoxymethyl-4-(2-isobutyrylamino-6-
oxo-1,6-dihydro-purin-9-yl)-pyrrolidine-1-carboxylic acid 9H-fluo-
ren-9-ylmethyl ester (14, trans- -POPNA G monomer). Compound 13
L
(0.9 g, 1.6 mmol) was treated with 30% HBr/AcOH (10 mL) for
30 min at room temperature. The resultant mixture was evaporated
to dryness. The residue was dissolved in 5% aqueous NaHCO3 to
adjust the pH to 8 (30 mL). A solution of FmoceOSu (0.6 g,
1.8 mmol) in MeCN (30 mL) was added to the aqueous solution
with stirring under ice cooling. The reaction mixture was stirred at
room temperature overnight and evaporated to dryness. The resi-
due was dissolved in water and the aqueous layer was washed three
times with Et2O (each 30 mL) and acidified to pH 7 with 5% aqueous
KHSO4; the resultant precipitate was collected by filtration. The
residue was washed several times with water and dried in vacuo.
Compound 14 was obtained as a white powder. Compound 14 was
purified by preparatory RP-HPLC (C18 column) before using to
d
1.45 and 1.47 (sþs, 18H, Boc CH3 and tert-butyl CH3), 2.62 (m, 2H,
C30H2), 3.5e4.4 (m, 7H, C20H, C50H2 and CaeCH2eOeCH2eCO), 4.84
(br, 2H, NH2), 5.16 (m, 1H, C40H), 7.41, 7.67, and 8.09 (tþtþd, 4H,
phenyl), 7.69 (s, 1H, C8H). 13C NMR (75 MHz, CDCl3):
d 28.1 and 28.4
(Boc CH3 and tert-butyl CH3), 33.4 and 34.7 (C30), 51.3 (C50), 52.6 and
53.2 (C40), 55.9 (C20), 69.0 (O-CH2-CO), 71.9 (Ca-CH2-O), 80.2 and 81.6
(Boc C and tert-butyl C), 115.6 (C5), 125.3, 125.5, 126.0, 134.5, 142.5,
and 145.5 (phenyl), 138.9 (C8), 155.2 and 158.5 (C2 and C6), 154.1
(Boc amide), 159.1 (C4), 169.4 (tert-butyl ester).
4.5.3. (2S,4R)-2-tert-Butoxycarbonylmethoxymethyl-4-[2-iso-
butyrylamino-6-(2-nitrophenoxy)-purin-9-yl]-pyrrolidine-1-carbox-
ylic acid tert-butyl ester (12). Compound 11 (1.9 g, 3.2 mmol) was
dissolved in pyridine and i-PrCOCl (0.42 g, 3.9 mmol) was added to
the solution of 11 in pyridine (20 mL) under ice cooling and the
mixture was stirred at room temperature overnight. The mixture
was then evaporated to dryness. The residue was added to water
(100 mL) and extracted three times with AcOEt (each 100 mL). The
AcOEt layer was washed with 5% aqueous citric acid (100 mL) and
water (100 mL), and it was then dried over MgSO4. Filtration fol-
lowed by solvent evaporation gave crude viscous oil. The crude oil
was chromatographed on silica gel with 70/30 AcOEt/Hex mixture
as the eluting solvent. The title compound 12 was obtained as
SPPS. Yield: 40%. 1H NMR (300 MHz, CDCl3):
d 1.20 and 1.22 (d, 6H,
iso-butyl CH3), 2.2e2.8 (m, 3H, iso-butyl CH and C30H2), 3.1 (m, 1H,
C20H), 3.3e4.7 (m, 9H, Fmoc CH, C50H2, CaeCH2eOeCH2eCO, and
Fmoc CH2), 5.0e5.3 (m, 1H, C40H), 7.1e7.9 (m, 9H, Fmoc aromatic
ring and C8H), 8.67 (d, 1H, iso-butyl amide), 10.0e10.3 (br, 1H,
COOH), 12.32 (br, 1H, C1H). 13C NMR (75 MHz, CDCl3):
d 19.0 (iso-
butyl CH3), 33.7 (C30), 36.1 (iso-butyl CH), 47.0 (Fmoc CH), 51.0 (C50),
52.7 (C40), 56.7 (C20), 67.4 (Fmoc CH2), 68 (OeCH2eCO), 71.6
(CaeCH2eO), 119.9, 124.8, 126.9, 127.7, 141.1, and 143.6 (Fmoc aro-
matic ring), 124 (C5), 138.4 (C8), 147.9 and 148.4 (C2 and C6), 154.3
and 154.7 (Fmoc amide), 155.5 (C4), 172.7 (COOH), 179.7 (iso-butyl
amide). HRMS (found/calculated): 601.2391/601.2332 [MþH]þ.
yellow foam. Yield: quant. 1H NMR (300 MHz, CDCl3):
d
1.03 and
Synthesis of cis-L-configuration of 14 was also performed in
1.05 (d, 6H, iso-butyl CH3), 1.46 and 1.48 (sþs, 18H, Boc CH3 and tert-
butyl CH3), 2.6e2.8 (m, 2H, C30H2), 3.16 (m, 1H, iso-butyl CH),
3.6e4.3 (m, 7H, C20H, C50H2 and CaeCH2eOeCH2eCO), 5.32 (m, 1H,
C40H), 7.4e8.2 (mþmþd, 5H, phenyl and C8H), 7.92 (s, 1H, iso-butyl
a manner identical to that described above. Yield: 96%. Rf (AcOEt/
MeOH¼9/1 (v/v)): 0.38. 1H NMR (300 MHz, CDCl3):
d 1.19 and 1.21
(d, 6H, iso-butyl CH3), 2.3e3.0 (m, 3H, iso-butyl CH and C30H2), 3.1
(m, 1H, C20H), 3.4e4.9 (m, 10H, Fmoc CH, C50H2,
CaeCH2eOeCH2eCO, Fmoc CH2 and C40H), 7.2e8.0 (m, 9H, Fmoc
aromatic ring and C8H), 8.50 (d, 1H, iso-butyl amide), 9.6e9.9 (br,
amide). 13C NMR (75 MHz, CDCl3):
d 18.8 (iso-butyl CH3), 28.1 and
28.4 (Boc CH3 and tert-butyl CH3), 33.5 (C30), 34.2 (iso-butyl CH),
51.3 (C50), 53.8 (C40), 55.9 (C20), 68.9 (OeCH2eCO), 71.9 and 72.5
(CaeCH2eO), 80.3 and 81.7 (Boc C and tert-butyl C), 118.0 (C5),
125.5, 125.8, 126.6, 134.8, 142.2, and 145.5 (phenyl), 141.3 (C8), 151.2
(C2), 154.2 (C6), 154 (Boc amide), 159.3 (C4), 169.3 (tert-butyl ester),
177.3 (iso-butyl amide).
1H, COOH), 12.19 (br, 1H, C1H). 13C NMR (75 MHz, CDCl3):
d 18.9
(iso-butyl CH3), 32.5 (C30), 36.0 (iso-butyl CH), 47.1 (Fmoc CH), 50.6
(C50), 52.4 (C40), 56.2 (C20), 66.6 (Fmoc CH2), 68.0 (OeCH2eCO), 70.8
(CaeCH2eO), 119.9, 124.9, 127.1, 127.7, 141.2, and 143.7 (Fmoc aro-
matic ring),124.5 (C5),138.5 (C8),147.7 and 148.6 (C2 and C6),154.2
(Fmoc amide), 155.4 (C4), 172.4 (COOH), 179.6 (iso-butyl amide).
HRMS (found/calculated): 601.2437/601.2332 [MþH]þ. IR (film,
CHCl3, cmꢁ1): 3066, 2901, 1732, 1684, 1608, 1559, 1474, 1452, 1418,
4.5.4. (2S,4R)-2-tert-Butoxycarbonylmethoxymethyl-4-(2-iso-
butyrylamino-6-oxo-1,6-dihydro-purin-9-yl)-pyrrolidine-1-carbox-
ylic acid tert-butyl ester (13). Compound 12 (2.2 g, 3.3 mmol) and 2-
nitrobenzaldoxime (5.5 g, 33 mmol) were dissolved in MeCN
(30 mL), and 1,1,3,3-tetramethylguanidine (3.5 g, 30 mmol) was
added to the solution. The mixture was stirred at room temperature
overnight. The mixture was evaporated to dryness. The residue was
dissolved in water (100 mL) and extracted five times with DCM
(each 100 mL). The DCM layer was washed with water (100 mL)
1356, 1250, 1140, 1103, 1069, 993, 974. Syntheses of the trans-
configuration of 14 and cis- -configuration of 14 were performed
according to aforementioned protocols, and the spectroscopic data
were identical with those of 14 and the cis- -configuration of 14,
D-
D
L
respectively. trans- -configuration of 14: yield: 36%. HRMS (found/
D
calculated): 601.2403/601.2332 [MþH]þ. cis-
D
-configuration of 14:
yield: 50%. HRMS (found/calculated): 601.2432/601.2332 [MþH]þ.

9666
M. Kitamatsu et al. / Tetrahedron 66 (2010) 9659e9666
IR (film, CHCl3, cmꢁ1): 3066, 2901,1683,1607,1559,1474,1451,1418,
1362, 1317, 1250, 1190, 1142, 1031, 930.
M.; Kubo, T.; Matsuzaki, R.; Endoh, T.; Ohtsuki, T.; Sisido, M. Bioorg. Med. Chem.
Lett. 2008, 19, 3410.
3. (a) Kuwahara, M.; Arimitsu, M.; Sisido, M. J. Am. Chem. Soc. 1999, 121, 256; (b)
Kuwahara, M.; Arimitsu, M.; Sisido, M. Tetrahedron 1999, 55, 10067; (c) Ku-
wahara, M.; Arimitsu, M.; Sisido, M. Bull. Chem. Soc. Jpn. 1999, 72, 1547; (d)
Kuwahara, M.; Arimitsu, M.; Shigeyasu, M.; Saeki, N.; Sisido, M. J. Am. Chem. Soc.
2001, 123, 4653.
Acknowledgements
4. Sawa, N.; Wada, T.; Inoue, Y. Tetrahedron 2010, 66, 344.
This work was supported by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Science, Sports and Cul-
ture, Japan (No. 19750143), Okayama Foundation for Science and
Technology, and the Kurata Memorial Hitachi Science and Tech-
nology Foundation.
5. (a) Shigeyasu, M.; Kuwahara, M.; Sisido, M.; Ishikawa, T. Chem. Lett. 2001, 31,
634; (b) Kitamatsu, M.; Shigeyasu, M.; Okada, T.; Sisido, M. Chem. Commun.
2004, 1208; (c) Kitamatsu, M.; Shigeyasu, M.; Sisido, M. Chem. Lett. 2005, 34,
1216; (d) Kitamatsu, M.; Shigeyasu, M.; Saitoh, M.; Sisido, M. Biopolymers (Pept.
Sci.) 2006, 84, 267.
6. The POPNAs also possess high water solubility, as well as the OPNAs. The sol-
ubility of the adenine 12-mer POPNAs in pure water was each 0.60 base M. The
solubility of the adenine 12-mer OPNA and PNA was 0.66 base M and 0.33 base
M under same conditions as described in Ref. 3a, respectively.
7. (a) Wadia, J.; Stan, R. V.; Dowdy, S. F. Nat. Med. 2004, 10, 310; (b) Michiue, H.;
Tomizawa, K.; Wei, F.-Y.; Matsushita, M.; Lu, Y.-F.; Ichikawa, T.; Tamiya, T.; Date,
I.; Matsui, H. J. Biol. Chem. 2005, 280, 8285.
8. (a) Futaki, S.; Suzuki, T.; Ohashi, W.; Yagami, T.; Tanaka, S.; Ueda, K.; Sugiura, Y.
J. Biol. Chem. 2001, 276, 5836; (b) Suzuki, T.; Futaki, S.; Niwa, M.; Tanaka, S.;
Ueda, K.; Sugiura, Y. J. Biol. Chem. 2002, 277, 2437.
9. Cruickshank, K. A.; Jiricny, J.; Reese, C. B. Tetrahedron Lett. 1984, 25, 681.
10. Kitamatsu, M.; Kashiwagi, T,; Matsuzaki, R.; Sisido, M. Chem. Lett. 2006, 35, 300.
11. (a) D’Costa, M.; Kumar, V. A.; Ganesh, K. N. Org. Lett. 1999, 1, 1513; (b) D’Costa,
M.; Kumar, V.; Ganesh, K. N. Org. Lett. 2001, 3, 1281; (c) Sharma, N. K.; Ganesh,
K. N. Chem. Commun. 2003, 2484.
Supplementary data
1H NMR and 13C NMR spectra of 1e14, HPLC chromatograms,
HRMS spectra, and IR spectra of 4, 7, 9, and 14, and melting curves of
mixtures of trans-L-POPNA13,trans-D-POPNA13, cis-L-POPNA13, cis-D-
POPNA13, and DNAs. Supplementary data associated with this article
can be found in the online version, at doi:10.1016/j.tet.2010.10.056.
These data include MOL file and InChIKeys of the most important
compounds described in this article.
12. Dueholm, K. L.; Egholm, M.; Behrens, C.; Christensen, L.; Hansen, H. F.; Vulpius,
T.; Petersen, K. H.; Berg, R. H.; Nielsen, P. E.; Buchardt, O. J. Org. Chem. 1994, 59,
5767.
References and notes
1. (a) Nielsen, P. E. Peptide Nucleic AcidsdProtocols and Applications; Horizon
Bioscience: Norfolk, England, 2004; (b) Ohtsuki, T.; Fujimoto, T.; Kamimukai,
M.; Kumano, C.; Kitamatsu, M.; Sisido, M. J. Biochem. 2008, 144, 415; (c) Endoh,
T.; Kitamatsu, M.; Sisido, M.; Ohtsuki, T. Chem. Lett. 2009, 38, 438.
13. Howarth, N. M.; Wakelin, L. P. G. J. Org. Chem. 1997, 62, 5441.
14. It must be noted that the sequence of Nielsen-type PNA, H-CAGTTAGGGTTAG-
Gly-NH2
, is difficult to synthesize because of three consecutive guanine units in
the sequence. In the synthesis of trans-L-POPNA oligomers, no such restriction
2. (a) Koppelhus, U.; Shiraishi, T.; Zachar, V.; Pankratova, S.; Nielsen, P. E. Bio-
conjugate Chem. 2008, 19, 1526; (b) Bendifallah, N.; Rasmussen, F. W.; Zachar,
V.; Ebbesen, P.; Nielsen, P. E.; Koppelfus, U. Bioconjugate Chem. 2006, 17, 750; (c)
Koppelhus, U.; Nielsen, P. E. Adv. Drug Delivery Rev. 2003, 55, 267; (d) Kitamatsu,
is encountered.
15. The CHO cells cultured for 2 h at 37 ꢀC under the same conditions were sub-
jected to an assay for cell viability by using the Cell Counting Kit-8 (Dojin).
Under the above conditions, no cytotoxicity was detected.