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T. Sugiyama et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 3337–3341
Fig. 2. Switching from Boc-based PNA monomers to Fmoc-based monomers.
large scale. Since 2 was isolated as a HCl salt, it was stable and
could be stored for years at À20 °C.
The synthesis of Fmoc thiouracil monomer 11 commenced with
the preparation of reagent 5 for protection of the thiocarbonyl
group of 2-thiouracil (Scheme 2). The reduction of 2-methy-
lanisaldehyde 3 with NaBH4 gave alcohol 4, which was converted
to bromide 5 by a reaction with PBr3. Since bromide 5 was not very
stable, it was immediately used in the next step. The treatment of
2-thiouracil 6 with bromide 5 in a mixture of EtOH-H2O gave S-
alkylated derivative 7 in 88% yield. The alkylation of 7 with ethyl
bromoacetate furnished a mixture of the N1- and O-alkylated
regioisomers 8a and 8b in favor of the desired 8a. Column chro-
matography provided 8a and 8b in 39% and 18% yields, respec-
tively. This moderate yield of 8a was acceptable because the
precursor 7 was recovered in 38% yield and was reusable.
The alkylation of compound 7 may yield an N1-, N3-, or O-alky-
lated isomer (Fig. 3). The structure of 8a was established as an N1-
alkylated isomer through comparisons of its 1H NMR and 13C NMR
Fig. 1. (a) Chemical structures of A-T, D-T, D-sU, and A-sU paired nucleobases. (b)
Double-duplex invasion by pcPNA.
monomers for pcPNA, the thiocarbonyl group of sU is protected
with the MPM (p-methoxybenzyl) group and the exocyclic amino
group of D is masked with the Cbz group. To remove these protect-
ing groups from the nucleobases,harsh reagents such as trifluo-
romethanesulfonic acid (TFMSA) or HF are required. The main
advantages of Fmoc chemistry are that after the completion of syn-
thesis, PNA oligomers can be cleaved from the resin and fully
deprotected under relatively mild acidic conditions (TFA) and that
commercial peptide synthesizers typically employ Fmoc chem-
istry. Therefore, we selected the 4-methoxy-2-methybenzyl group
(MMPM) to protect the thiocarbonyl group of the sU monomer for
the following reasons: (1) Due to the electron-donating nature of
the newly introduced methyl group, MMPM may be more sensitive
to TFA than MPM and may be cleaved by a treatment with TFA; (2)
MMPM may exhibit sufficient stability under the reaction condi-
tions used for sU monomer synthesis; (3) since the 2-methyl group
of MMPM is relatively small, its steric congestion may not affect
the synthesis of the sU monomer and may not reduce coupling effi-
ciency in the solid-phase synthesis of PNA oligomers. Regarding
the D monomer, we decided to use the Boc group for amino protec-
tion. Here we report the development of new PNA monomers for
pcPNA synthesis that are fully compatible with standard Fmoc
chemistry (Fig. 2).
spectra in DMSO-d6 with those of the known structure.5 The
22
regiochemistry of 8a was confirmed by NOE experiments. The
presence of an NOE effect of the methylene protons of the –CH2-
COOEt fragment (d 4.8 ppm) on the irradiation of the pyrimidine
H-6 (d 7.7 ppm) was indicative of an N1-alkylated isomer. Addi-
tional evidence was obtained from the HMBC experiment for 8a
in which methylene protons were coupled to C2 and C6 carbons
(red arrows in Fig. 3).
The regiochemical assignment of 8b was performed as follows.
Although compound 7 may be alkylated at the N3 position, this
possibility was excluded by comparisons with the reported NMR
data of the S- and N3-dialkylated isomers of thiouracil.16,17 Fur-
thermore, the HMBC experiment for 8b showed that the methylene
protons of the –CH2-COOEt fragment (d 5.0 ppm) were not coupled
to C2 carbon. Since this coupling is expected for an N3-isomer, the
possibility of alkylation at the N3 position was again discounted.
Therefore, the remaining possibility was an O-alkyl isomer. The
13C chemical shift of the methylene carbon of 8b (d 62.7 ppm) in
DMSO-d6 was shifted downfield from that of the analogous carbon
of 8a (d 52.4 ppm),22 which indicated that the carbon atom of the
methylene linker (-CH2-) directly bonded to an oxygen atom. In
addition, the 1H NMR and 13C NMR spectra of 8b in CDCl3 were
consistent with those reported for the S- and O-dialkylated iso-
mers.16–18 Thus, 8b was an O-alkylated isomer.
We selected benzyl N-[2-(Fmoc)aminoethyl]glycinate 212 as the
backbone unit for the synthesis of Fmoc-protected pcPNA mono-
mers. The synthesis of 2 is outlined in Scheme 1. This scheme
avoids the formation of 2-oxopiperazine, which has been occasion-
ally observed in the synthesis of PNA backbones.13,14 Compound 1
was prepared according to the procedure of Thomson et al.15 1 was
then converted into benzyl ester 2 by a treatment with p-toluene-
sulfonic acid and the subsequent addition of excess benzyl alcohol
in refluxing toluene. This one pot process proceeded efficiently and
the requisite backbone 2 was obtained in excellent yield and on a
The ethyl ester 8a was saponified with LiOH to afford carboxylic
acid 9 in 89% yield. The coupling of 9 with PNA backbone 2 using
HBTU provided 10 in 94% yield. The benzyl ester group of 10 was
selectively cleaved by an alkaline treatment (20 eq. NaOH in
Sugiyama, Toru
