Synthesis of chiral peptide nucleic acids using Fmoc chemistry

Article abstract of DOI:10.1016/S0040-4020(01)00789-X

A Fmoc-based synthesis of chiral PNAs is described. Chiral monomer backbones were efficiently prepared by reductive amination of N-Fmoc-protected L,D-alaninals with glycine esters and the subsequent acylation of free amines with thymine-1-ylacetic acid. T

Full text of DOI:10.1016/S0040-4020(01)00789-X

TETRAHEDRON
Pergamon
Tetrahedron 57 32001) 8107±8113
Synthesis of chiral peptide nucleic acids using Fmoc chemistry
Yun Wu and Jie-Cheng Xup
Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 354 Feng Lin Road, Shanghai 200032, People's Republic of China
Received 15 May 2001; revised 4 July 2001; accepted 26 July 2001
AbstractÐA Fmoc-based synthesis of chiral PNAs is described. Chiral monomer backbones were ef®ciently prepared by reductive
amination of N-Fmoc-protected l,d-alaninals with glycine esters and the subsequent acylation of free amines with thymine-1-ylacetic
acid. The dimer derivative of l-amino acid was prepared in solution. Finally, a chiral decamer was obtained by a solid phase strategy
using a succinyl-linked support. q 2001 Elsevier Science Ltd. All rights reserved.
1. Introduction
chiral PNA monomers from N-Fmoc protected l or
d-alanine and the subsequent incorporation into a chiral
dimer using solution phase techniques and a base-cleavage
solid phase strategy for the preparation of chiral PNA are
described in detail.
Peptide nucleic acids 3PNAs) are DNA mimics in which
nucleic acid bases are incorporated to a pseudopeptide back-
bone.1±3 PNAs recognize their complementary oligonucleo-
tides with a remarkably high speci®city and af®nity, and are
resistant to nucleases and proteases.4±7 These properties of
PNAs have special attention in the development of gene
therapeutics and bio-molecular tools. The original PNA
backbone consists of N-32-aminoethyl)glycine units, and
nucleobases are attached to the glycine nitrogen through
methylene carbonyl linkers. Introduction of chirality into
the backbone has attracted much attention.8±10 Such a
modi®cation might serve as a model for the introduction
of active groups at various positions along the backbone
and leads to the improvement of such parameters as binding
af®nity, speci®city and cell permeability. For instance,
Lenzi et al. have prepared a PNA mimic involving amino-
butyric acid and glycine.11They demonstrated that this
chiral peptidic DNA exhibited good self-recognition similar
to that of DNA±DNA duplexes.
2. Results and discussion
2.1. Monomer synthesis
The chiral monomer backbones can be prepared by
reductive amination of the glycine ester with N-Fmoc
amino aldehydes. This procedure gave N-Fmoc protected
chiral backbones in good purity and yield. First, N-Fmoc
l,d-alanines were transformed into their corresponding
N,O-dimethylhydroxyl-amide derivatives12 31a and 1b) via
mixed anhydrides using isobutylchloroformate 3IBCF).
Reduction of the amides was performed with ®ve equiva-
lents of hydride 31.25 equiv. of LiAlH4) to produce the
corresponding aldehydes 2a, 2b within 10±20 min at 08C.
After crystallization, the aldehydes were reacted with
glycine ester and the resulting imines were ef®ciently
transformed to amines using two different reductants. The
intermediate imines from the ethyl ester of glycine were
treated with 10% Pd/C, while the imines obtained from
the benzyl ester of glycine were treated with NaBH3CN.
N-Fmoc protected l,d chiral monomer backbones 3a±d
were obtained in 70±85% yield, respectively, after puri®ca-
tion by ¯ash chromatography 3Scheme 2). All of these
amines were shelf-stable solids which could be stored for
more than two weeks. A more attractive method involved
transforming the crude materials into the corresponding
stable hydrochloride salts 3c, 3d by treating with an equiva-
lent of dry ethereal HCl 31.5 M) at 2208C, which can be
stored for several months. The optical purities of 3a and 3b
reached 99.3 and 96% by chiral HPLC measurement, which
were similar to the N-Fmoc alanine.
To synthesize chiral PNAs, different methods have been
developed, amongst which a Boc protecting group strategy
is widely used to realize the oligomerization.12±14 The
repeated use of TFA and the harsh HF or TFMSA treatment
mean this strategy will be incompatible with many types of
modi®ed PNAs, especially with the PNA±DNA chimera
due to the sensitivity of DNA to strong acids. In search of
an alternative strategy which may provide a feasible route to
the synthesis of PNAs with special groups and oligo-
nucleotides, we developed a simpli®ed and ef®cient pro-
cedure to synthesize N-Fmoc protected chiral PNAs
3Scheme 1). Herein, the synthesis of the thymine containing
Keywords: N-Fmoc; glycine; oligomerization.
Corresponding author. Tel.: 186-216-416-3300; fax: 186-216-416-
6263; e-mail: xjcheng@pub.sioc.ac.cn
p
0040±4020/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020301)00789-X

8108
Y. Wu, J.-C. Xu / Tetrahedron 57 02001) 8107±8113
Scheme 1.
Scheme 2. Reagents and conditions: 3i) iBuO±CO±Cl, NMM, THF; HCl´HNMe3OMe), NEt3; 2208C. 3ii) LiAlH4, THF, 08C. 3iii) 3a). HCl´NH2CH2COOEt,
KOAc, 10% Pd/C, MeOH; room temperature. 3b). Tos´NH2CH2COOBn, HOAc, NaBH3CN, MeOH, 08C.
The acylation of free amines with thymin-1-yl acetic acid
can be carried out by conventional methods using BBC-Cl15
as the coupling reagent. 4a±4d were obtained in good to
excellent yields 385±95%), though the other uronium or
phosphonium reagents were ineffective for this coupling.
As shown in Scheme 3, removal of the benzyl ester from
4c and 4d was performed by hydrogenation with 10% Pd/C
and the ethyl group of 4a and 4b was cleaved with NaOH
35 M, 10 equiv.), since ethyl and methyl esters are more
sensitive to aqueous NaOH than the Fmoc group at low
temperature within a short period of time 35±10 min).16
The Fmoc protected chiral PNA monomers were obtained
after puri®cation by ¯ash chromatography.
2.2. Solution phase oligomerization
Dimerization of N-Fmoc protected l-dipeptides was then
investigated. The Fmoc group of 4c was removed easily
using a 50% solution of N,N-diethylamine in DCM to
give monomer 6, which was coupled with 5c using FEP as
the coupling reagent. As expected, FEP17 gave higher
coupling yields than uronium salt 3TBTU) and phos-
phonium salt 3BOP) and afforded dimer 7 in 70% yield.
Only one product is obtained indicating that no
racemization occurred during the coupling. Finally, hydro-
genation of 7 with 10% Pd/C in ethanol gave 8 in 85% yield
3Scheme 4).
Scheme 3. Reagents and conditions: 3i) Thymin-1-yl acetic acid, BBC-Cl, DIPEA, DMF/DCM; rt. 3ii) 3a). RˆBn: 10% Pd/C, EtOH; room temperature.
3b). RˆEt:NaOH 32.5 M), H2O±THF, 08C, within 5±10 min.

Y. Wu, J.-C. Xu / Tetrahedron 57 02001) 8107±8113
8109
Scheme 4. Reagents and conditions: 3i) 50% HNEt2 in DCM, 30 min. 3ii) 5a, FEP, DIPEA, 2108C. 3iii) 10% Pd/C, EtOH; room temperature.
2.3. Solid phase oligomerization
amino acid 5b was conducted in the presence of AOP, HOAt
and DIPEA in anhydrous DMF/DCM. For this, the
reagents are pre-mixed in the delivery syringe, and after a
short preactivation period delivered to the reaction vessel.
After 2 h of coupling, unreacted amino functions were
capped using a mixture of 5% acetic anhydride in DMF,
which is used as a capping reagent in standard Fmoc
strategy SPPS.
Solid phase peptide synthesis using Boc methodology
requires exposure of the peptide resin to strong acids for
each cycle. These conditions are incompatible with an
orthorgonal strategy and will be unsuitable for the synthesis
of phosphono-PNAs because of their acid sensitivity. In
1982, Apsimon et al. developed a new solid support to
synthesis b-aminoalcohol, which is cleavable under base
treatment.18,19 We introduced this support with 5-amino-
pentasuccinate linker into our Fmoc based peptide
synthesis. Thus, 5-aminopentan-1-ol 9 was selectively
protected using Fmoc-OSu to give 10. The resultant alcohol
was succinylated using succinic anhydride and DMAP in
DCM to give 11, which was then coupled to BHA resin
using AOP/DIPEA in DCM 3Scheme 5).
The ef®ciency of reaction is judged by measuring the
absorbance of the dibenzofulvene±piperidine adduct 3j264
18,000 dm3 mol21 cm21), liberated during deprotection.
Finally, the PNA is cleaved from the solid support and
deprotected with 0.5N NaOH/H2O/THF solution, followed
by 50% aqueous acetic acid for the mixture. After de-salting
by gel ®ltration, the chiral PNA was puri®ed by RP-HPLC
using a C18 column and a linear gradient acetonitrile 3from
20 to 23%) in water with 0.05% TFA. The identity and
purity of the oligomer was determined by ESI-MS 3Fig.
1). These oligomers showed an ability to form adducts
with alkali metal ions especially potassium. The principal
mass peak at 2946.2 Da corresponds to the potassium salt of
the desired PNA oligomer 3C125H173N41O41: Mˆ2906) and
The chiral decathymine PNA with d con®guration was
successfully prepared by the successive coupling of the
corresponding monomers. The PNA was synthesized on a
40 mmmol scale. First, deprotection of the Fmoc group was
realized with 20% piperidine in DMF. Then the coupling of
the resultant amine with 2.5 equiv. of the Fmoc protected
Scheme 5. Reagents and conditions: 3i) Fmoc-OSu, DIPEA, DCM. 3ii) Succinic anhydride, DMPA, DCM/Py 33:1), 08C, room temperature. 3iii) BHA resin,
AOP, DIPEA, DMF/DCM, 2108C.

8110
Y. Wu, J.-C. Xu / Tetrahedron 57 02001) 8107±8113
Figure 1. ESI mass spectrum of the pure oligomer.
the other one at 2929.1 Da to the sodium salt of the oligomer
3i.e. [M1Na]1).
cooled to 2208C. Then, isobutylchloroformate 32.6 mL,
20 mmol) was added in one portion under stirring and left
for several minutes. Triethylamine 33.06 mL, 22 mmol) was
added followed by a solution of N,O-dimethyl-hydroxyl-
amine hydrochloride 31.94 g, 20 mmol) in 50 mL of dry
DMF, which had been chilled to the same temperature.
After stirring for 30 min at 2208C, the reaction mixture
was allowed to warm to room temperature. The precipitates
were removed by ®ltration, the ®ltrate was diluted with
200 mL of water and 20 mL of 1N HCl. After evaporation
in vacuo, the solution was adjusted to pH 8 with 2N K2CO3
and then cooled in freezer overnight. The precipitates were
collected by ®ltration, brie¯y washed with deionized water,
and dried in vaccum at 408C for 3 h.
3. Conclusions
A Fmoc based strategy was successfully applied to the
synthesis of chiral PNAs. In this synthesis, chiral monomers
were prepared using a reductive amination method. Subse-
quent connection of the backbones with nucleobases was
accomplished using BBC-Cl as the coupling reagent, and
the dimerization was carried out in solution using FEP as
coupling reagent. A modi®ed amino linker resin was then
used as the support to accomplish the Fmoc-based solid
phase synthesis of a chiral PNA under mild base conditions.
Compound 1a was obtained as crystalline solid, yield 92%,
H
20
mp: 120±1218C 3dec.), [a]D ˆ12.1 3c 0.68 CHCl3).20 1
4. Experimental
NMR 390 MHz, CDCl3) d 7.85±7.2 38H, m, Fmoc-
aromatic-CH), 5.6 31H, m, NH), 4.75±4.20 34H, m, Fmoc-
aliphatic-CH and ±CH2 and ±CHMe), 3.75 33H, s, OCH3),
3.2 33H, s, NCH3), 1.35 33H, d, Jˆ5.4 Hz, ±CHCH3).
Melting points were taken on digital melting point apparatus
and uncorrected. Infrared spectra were recorded on a
Shimadzu IR-440 spectrometer. Mass spectra were recorded
1
on HP 5989A and VG QUATTRO mass spectrometers. H
Compound 1b was obtained as crystalline solid, yield 91%,
20
20
mp: 124±1258C 3dec.), [a]D ˆ22.33 3c 1 CHCl3). 1H
NMR spectra were recorded on Bruker AM 300 3300 MHz)
and Bruker DRX-400 3400 MHz) using TMS as internal
standard. Combustion analysis for elemental composition
was carried out on an Italy MOD 1106 analyzer. Optical
rotations were determined using a Perkin±Elmer 241 MC
polarimeter. Flash column chromatography was performed
with 300±400 mesh silica gel, and analytical thin layer
chromatography was performed on pre-coated silica gel
plates 360F-254) with the systems 3v/v) indicated. Solvents
and reagents were puri®ed by standard methods as
necessary. Amino acids were l-con®guration if not
otherwise stated. HOAt, AOP, BOP, TBTU, DIPEA,
thymine, N,O-dimethylhydroxylamine hydrochloride,
LiAlH4, BHA resin were purchased from Aldrich Chemical
Co, WI, and used without puri®cation.
NMR 390 MHz, CDCl3) d 8.0±7.3 38H, m, Fmoc-
aromatic-CH), 5.70 31H, m, NH), 5.0±4.3 34H, m, Fmoc-
aliphatic-CH and-CH2 and ±CHMe), 3.80 33H, s, ±OCH3),
3.20 33H, s, NCH3), 1.30 33H, d, Jˆ5.4 Hz, ±CH3).
4.1.2. N-[+9H-Fluoren-9-ylmethoxy)carbonyl]-l or d-
alaninal +2a or 2b). A solution of 1a or 1b 35.31 g,
15 mmol) in 80 mL of dry THF was cooled to 08C, and
then LiAlH4 30.69 g, 18.8 mmol) was added. The reaction
was followed by TLC mixture until completion. A solution
of KHSO4 35.44 g, 40 mmol) was added and the reaction
mixture was extracted with ethyl acetate 33£50 mL). The
combined organic extracts were washed with 1N HCl
32£50 mL), NaHCO3 32£50 mL) and brine 32£50 mL),
dried and evaporated to give a white solid. Pure product
was obtained by recrystallization from petroleum±ethyl
acetate.
4.1. Synthesis of compounds
4.1.1.
N-[+9H-Fluoren-9-ylmethoxy)carbonyl]-l
or
Compound 2a, white solid, yield 95%, mp:137±1388C 3lit.21
d-alanyl-N-methoxy-N-methylamide +1a or 1b). N-Fmoc-
l or d-alanine 36.224 g, 20 mmol) and N-methylmorpholine
322 mL, 20 mmol) were dissolved in 120 mL of dry THF and
mp:1458C), mp:147±1488C 3lit.21 mp:1458C), [a]D ˆ43.4
20
3c 1 CHCl3) [lit.21 [a]Dˆ10 3c 1 CHCl3)]. 1H NMR

Y. Wu, J.-C. Xu / Tetrahedron 57 02001) 8107±8113
8111
390 MHz, CDCl3) d 9.5 31H, s, CHO), 7.85±7.20 38H, m,
Fmoc-aromatic-CH), 5.40 31H, m, NH), 4.40±4.10 34H, m,
Fmoc-aliphatic-CH and ±CH2 and ±CHMe), 1.30 33H, d,
Jˆ5.4 Hz, ±CH3).
methanol 340 mL) was treated with 2a or 2b. The suspended
solution was stirred at 08C for 30 min, to which acetic acid
30.75 mL) and NaCNBH3 30.37 g, 5.9 mmol) were added
sequentially and left for 2 h, the solvent was removed
under reduced pressure to give a colorless oil. The oil was
dissolved in 200 mL of ethyl acetate and 80 mL of 5%
NaHCO3. The aqueous layer was separated and extracted
with ethyl acetate 33£100 mL), the combined organic layer
was washed successively with 5% NaHCO3 33£80 mL) and
brine 33£80 mL) and dried with Na2SO4. The solvent was
evaporated and the residue was treated with dry ethereal
HCl 31.5 M) and precipitated by dry hexane 380 mL).
The resultant solid hydrochloride salt was collected by
®ltration. The pure product was given as white solid after
recrystallization from methanol±ether±hexane.
Compound 2b, white solid, yield 93%, mp:136±1378C 3lit.21
mp:1458C), [a]D ˆ244.1 3c 1.08 CHCl3) [lit.21 [a]Dˆ211
20
1
3c 1 CHCl3)]. H NMR 390 MHz, CDCl3) d 9.45 31H, s,
CHO), 7.85±7.15 38H, m, Fmoc-aromatic-CH), 5.40 31H,
m, NH), 4.40±4.15 34H, m, Fmoc-aliphatic-CH and ±CH2
and ±CHMe), 1.25 33H, d, Jˆ5.4 Hz, ±CH3).
4.2. Reductive amination of aldehydes and glycine ester
4.2.1. Ethyl N-+2-Fmoc-amino-l or d-methylethyl)glyci-
nate +3a or 3b). A solution of N-Fmoc-alaninal 2a or 2b
34.25 g, 14.4 mmol) in methanol 3100 mL) was added to a
solution of glycine ethyl ester hydrochloride 32.008 g,
Compound 3c, white solid, yield 70%, mp: 180±1818C
3dec.). [Found: C, 66.71; H, 6.02; N, 5.75.
C27H28N2O4´HCl´1/4H2O requires C, 66.80; H, 6.12; N,
5.77%]; nmax3KBr): 3316 3NH), 1746, 1690 3CvO), 759
14.4 mmol)
and
anhydrous
CH3COOK
32.83 g,
28.9 mmol) in methanol 3150 mL). The mixture was cooled
to 08C under nitrogen for 10 min and 10% Pd/C 346 mg) was
added with vigorous stirring. The reaction mixture was
hydrogenated at atmospheric pressure and room temperature
until hydrogen uptake had ceased. The catalyst was ®ltered
off and solvent was evaporated under reduced pressure. The
residue was suspended in water 325 mL) and the pH was
adjusted to 8 by addition of 1N NaOH. The aqueous phase
was extracted with DCM 35£25 mL), the organic layer was
washed with NaHCO3 32£25 mL), brine 32£25 mL), and
dried 3Na2SO4). After evaporation of the solvent, the crude
product was puri®ed by ¯ash chromatography.
1
and 739 3Fmoc and Ph) cm21; H NMR 3300 Hz, CDCl3)
d 9.80 31H, m, NH´HCl), 7.73±7.27 313H, m, phenyl- and
Fmoc-aromatic-CH), 6.54 31H, s, FmocNH), 5.11 32H, s,
CH2Ph), 4.63±4.12 34H, m, Fmoc-aliphatic-CH and ±CH2
and ±CHMe), 3.96 32H, s, HNCH2CO), 3.42±3.21 32H, m,
CHCH2NH), 1.26 33H, m, CHCH3); ESIMS m/z: 445
[M1H]1, 100%, 178 [Fmoc]1, 42%, 91 [Bn]1, 35%.
4.2.3. Acylation of the pseudodipeptide with thymin-1-
ylacetic acid. Amine 3a±3c or 3d 310 mmol), thymin-1-
ylacetic acid 32.02 g, 11 mmol) and BBC-Cl 33.66 g,
11 mmol) were suspended in 10 mL of dry DMF and
5 mL of dry DCM at 08C and stirred for a few minutes,
then DIPEA 33.84 mL, 22 mmol) was added at the same
temperature. The mixture was allowed to warm to room
temperature with stirring overnight, the solvent was evapo-
rated and the residue was partitioned between water
350 mL) and ethyl acetate 350 mL), the aqueous phase was
extracted with ethyl acetate 34£50 mL). The combined
organic extract was washed with 1N HCl 32£50 mL),
NaHCO3 32£50 mL) and brine 32£50 mL), and then dried
with Na2SO4. The solvent was evaporated to dryness and the
crude products were puri®ed by ¯ash chromatography
3hexane±ethyl acetate, 1:2) to give the desired products.
Compound 3a, white solid, yield 85%, mp: 85.5±878C
16
3dec.), [a]D ˆ5.55 3c 0.88 CHCl3). [Found: C, 68.92; H,
6.69; N, 7.24%. C22H26N2O4 requires C, 69.09; H, 6.85; N,
7.32%]; nmax3KBr): 3323 3NH), 1737, 1686 3CvO), 759,
1
739 3Fmoc) cm21; H NMR 3300 MHz, CDCl3) d 7.77±
7.26 38H, m, Fmoc-aromatic-CH), 5.21 31H, s, FmocNH),
4.39 32H, d, Jˆ6.6 Hz, Fmoc-aliphatic-CH2), 4.25±4.02
33H, m, Fmoc-aliphatic-CH, CH2Me), 3.81 31H, m,
±CHMe), 3.41 32H, d, Jˆ7.2 Hz, HNCH2CO2Et), 2.62
32H, d, Jˆ1.1 Hz, CHCH2NH), 1.97 31H, s, CH2NHCH2),
1.26 33H, t, Jˆ6.0 Hz, CH2CH3), 1.18 33H, d, Jˆ5.3 Hz, CH
CH3); ESIMS m/z: 383.3 [M1H]1, 100%, 765 [2M1H]1,
8%, 178 [Fmoc]1, 44%; eeˆ99.3%.
Compound 4a, white solid, yield 95%, mp: 179±1808C.
[Found: C, 63.20; H, 6.17; N, 10.16%. C29H32N4O7 requires
C, 63.49; H, 5.88; N, 10.41%]; nmax3KBr): 1682 3CvO),
Compound 3b, white solid, yield 85%, mp: 86±878C 3dec.),
16
[a]D ˆ25.96 3cˆ0.869 in CHCl3). [Found: C, 68.91; H,
1
760 and 739 3Fmoc) cm21; HNMR 3400 MHz, d6-DMSO)
6.65; N, 7.21%. C22H26N2O4 requires C, 69.09; H, 6.85; N,
7.32%]; nmax3KBr): 3321 3NH), 1739, 1685 3CvO), 759,
two isomers d 11.28 and 11.27 31H, 2£s, thymine-NH33)),
7.90±7.31 38H, m, Fmoc aromatic-CH), 7.25 31H, s,
thymine-CH36)), 4.76±3.0 38H, m, CHMe, Fmoc aliphatic-
CH and ±CH2, NCH2CO, CH2CO, CHCH2N and CH2Me),
1.73 and 1.70 33H, 2£s, thymine-CH3), 1.20 33H, 3£t,
Jˆ7.0 Hz, CH2Me), 1.05 33H, 2£d, Jˆ6.3 Hz, CH3); ESI-
MS m/z: 549.4 [M1H]1, 100%, 571.4 [M1Na±H]1, 90%,
587.4 [M1K±H]1, 20%.
1
739 3Fmoc) cm21; H NMR 3300 MHz, CDCl3) d 7.76±
7.29 38H, m, Fmoc-aromatic-CH), 5.21 31H, s, FmocNH),
4.39 32H, d, Jˆ6.5 Hz, Fmoc-aliphatic-CH2), 4.25±4.16
33H, m, Fmoc-aliphatic-CH, CH2Me), 3.83 31H, m,
CHMe), 3.43 32H, d, Jˆ7.6 Hz, HNCH2CO2Et), 2.70 32H,
d, Jˆ3.6 Hz, CHCH2NH), 2.25 31H, s, CH2NHCH2), 1.28
33H, t, Jˆ7.1 Hz, CH2CH3), 1.19 33H, d, Jˆ5.7 Hz,
CHCH3); EIMS m/z: 383 [M1H]1, 2%, 178 [Fmoc]1,
85%, 165 [M-Fmoc-CO2]1, 100%; eeˆ96%.
Compound 4b, white solid, yield 94%, mp 179±1808C.
[Found: C, 63.05; H, 6.19; N, 10.16%. C29H32N4O7´
1/4H2O requires C, 62.98; H, 5.92; N, 10.13%]; nmax3KBr):
1743 and 1682 3CvO), 760 and 739 3Fmoc) cm21; 1H NMR
3400 MHz, d6-DMSO) two isomers d 11.28 an 11.27 31H,
4.2.2. Benzyl N-+2-Fmoc-amino-l or d-methylethyl)gly-
cinate +3c or 3d). A pre-cooled 10% solution of benzyl
glycinate toluene-p-sulfonate 33.71 g, 11 mmol) in

8112
Y. Wu, J.-C. Xu / Tetrahedron 57 02001) 8107±8113
2£s, thymine-NH33)), 7.90±7.18 38H, m, Fmoc aromatic-
CH), 7.25 31H, s, thymine-CH36)), 4.70 31H, q, Jˆ7.7 Hz,
CHMe), 4.47±4.0 37H, m, Fmoc aliphatic-CH and ±CH2,
CH2Me and CH2CO), 4.09±3.00 34H, m, CHCH2N and
NCH2CO), 1.73 and 1.71 33H, 2£s, thymine-CH3), 1.20
33H, 2£t, Jˆ7.0 Hz, CH2Me), 1.05 33H, 2£d, Jˆ6.3 Hz,
CH3); ESI-MS m/z: 549.4 [M1H]1, 21%, 571.4 [M1Na±
H]1, 100%, 587.4 [M1K±H]1, 28%.
3Fmoc) cm21; 1H NMR 3300 MHz, d6-DMSO) two isomers
d 11.28 and 11.24 31H, 2£s, thymine-NH33)), 7.90±7.25
39H, m, Fmoc-aromatic-CH and T-CH36)), 4.71 31H, m,
±CHMe), 4.46±3.58 39H, m, Fmoc-aliphatic-CH and
±CH2 and ±CH2NCH2 and CH2CO and H2O), 1.72 and
1.71 33H, 2£s, thymine-CH3), 1.05 33H, 2£d, Jˆ6.4 Hz,
CHCH3); ESIMS m/z: 521 [M1H]1, 10%, 543 [M1Na]1,
100%, 1064 [2M1Na]1, 75%, 1584 [3M1Na]1, 15%.
Compound 4c, white solid, yield 85%, mp: 204±2058C
Compound 5b, white solid, yield 80%, mp: 202±2038C;
20
16
3dec.), [a]D ˆ19.0 3c 0.88 in DMF). [Found: C, 63.18;
[a]D ˆ210.02 3c 1.08 DMF), HPLC 99.3%. [Found: C,
H, 5.31; N, 8.93%. C34H34N4O7´2H2O requires C, 63.15;
H, 5.92; N, 8.66%]; nmax3KBr): 1713, 1712 3CvO);
60.77; H, 5.63; N, 10.00%. C27H28N4O7´H2O requires C,
60.22; H, 5.61; N, 10.40%]; nmax3KBr): 3316 3OH), 1688
756,737 and 697 3Fmoc and -Ph) cm21
;
1H NMR
3CvO), 760, 738 3Fmoc) cm21 1H NMR 3300 MHz,
;
3300 MHz, d6-DMSO) two isomers d 11.30 and 11.28
31H, 2£s, thymine-NH33)), 7.89±7.29 313H, m, phenyl
and Fmoc-aromatic-CH), 7.23 and 7.17 31H, 2£s,
thymine-CH36)), 5.20 and 5.12 32H, 2£s, CH2Ph), 4.71
31H, m, CHMe), 4.49±3.00 39H, m, Fmoc-aliphatic-CH
and ±CH2 and ±CH2CO and CH2NCH2), 1.73 and 1.71
33H, 2£s, thymine-CH3), 1.05 33H, 2£d., Jˆ6.3 Hz,
CHCH3); ESIMS m/z: 633 [M1Na]1, 100%, 1233
[2M1Na]1, 55%, 1855 [3M1Na]1, 34%.
d6-DMSO) two isomers d 11.32 and 11.28 31H, 2£s,
thymine-NH33)), 7.91±7.23 39H, m, Fmoc-aromatic-CH
and T-CH36)), 4.69 31H, m, ±CHMe), 4.53±3.04 39H, m,
Fmoc-aliphatic-CH and ±CH2 and CH2CO and CH2NCH2
and H2O), 1.71 and 1.72 33H, 2£s, thymine-CH3), 1.10 33H,
m, CHCH3); ESIMS m/z: 521 [M1H]1, 65%, 543
[M1Na]1, 25%, 1041 [2M1H]1, 100%, 1063
[2M1Na2H]1, 50%.
4.2.5. Dimer N-Fmoc-l-T-T-OBn +7). Compound 4a
30.31 g, 0.5 mmol) was suspended in dry DCM 32 mL)
and then N,N-diethylamine 31 mL) was added at room
temperature. After reacting for 30 min, the solvent was
evaporated in vacuo to give the crude amine-free chiral
monomer 6. It was dissolved in dry DMF 32 mL) and stored
at 2108C and directly used in the next step of coulping
without puri®cation. l-monomer 5a 30.260 g, 0.5 mmol),
FEP 30.14 g, 0.55 mmol) and DIPEA 3360 mL, 1.25 mmol)
were dissolved in dry DMF 32 mL) and dry DCM 32 mL),
and then cooled to 2108C in an ice±salt bath. The DMF
solution of 6 was added to this stirred mixture at low
temperature. The reaction was continued for 2 h at the
same temperature, and an additional 3 h at room tempera-
ture under stirring. The mixture was concentrated in vacuo
and the residue was puri®ed by ¯ash chromatography 3ethyl
acetate±methanol±water 25:1:0.1) to give the dimer as a
white foam 30.25 g, 56%). [Found: C, 61.55; H, 5.73; N,
12.26%. C46H50N8O11´1/2H2O requires C, 61.39; H, 5.71; N,
12.45%]; nmax3KBr): 1681 3CvO); 759, 739 3Fmoc and
Compound 4d, white solid, yield 88%, mp: 204±2058C
3dec.). [Found: C, 66.75; H, 5.78; N, 9.08%. C34H34N4O7
requires C, 66.87; H, 5.61; N, 9.17%]; nmax3KBr): 1732,
1712 and 1687 3CvO); 756,737 and 698 3Fmoc and
1
-Ph) cm21; H NMR 3300 MHz, d6-DMSO) two isomers d
11.13 31H, brm, thymine-NH33)), 7.90±7.18 313H, m,
phenyl and Fmoc-aromatic-CH), 7.09 and 7.08 31H, 2£s,
thymine-CH36)), 5.20 and 5.12 32H, 2£s, CH2Ph), 4.72
31H, q, Jˆ7.1 Hz, CHMe), 4.50±4.19 35H, m, Fmoc-
aliphatic-CH and ±CH2 and ±CH2CO), 4.12±3.05 34H, m,
CH2NCH2), 1.73 and 1.71 33H, 2£s, thymine-CH3), 1.05
33H, 2£d, Jˆ6.3 Hz, CHCH3); ESIMS m/z: 611 [M1H]1,
100%, 1221 [2M1H]1, 75%.
4.2.4. N-+2-Fmoc-Amino-2-l or d-methylethyl)-N-
+thymin-1-ylacetyl)glycine +5a or 5b). 3a) 5 M Sodium
hydroxide 32 mL) was added to a stirred solution of 4a or
4b 30.549 g, 1 mmol) in THF 32 mL) at 08C. The completion
of reaction was monitored by TLC 3within 5±10 min).
Water 310 mL) was added and the pH was adjusted to 7±8
by the addition of 1N HCl. The aqueous phase was extracted
with CHCl3 33£10 mL), the pH was adjusted to 2 and
extracted with ethyl acetate 38£20 mL). The organic layer
was dried over Na2SO4 and evaporated to give the crude
product. After crystallization or puri®cation by ¯ash chro-
matography 3ethyl acetate±methanol±water 5:1:0.2), the
desired product was obtained.3b) 4c or 4d 30.305 g,
0.5 mmol) was suspended in 200 mL of ethanol. After
re¯uxing for 20 min, the solution was cooled to room
temperature and 10% Pd/C 350 mg) was added under nitro-
gen and hydrogenated until the substrate disappeared 3by
TLC). The reaction mixture was ®ltered and the solvent
was evaporated. The crude product was puri®ed by ¯ash
chromatography 3ethyl acetate±methanol±water 5:1:0.2).
1
-Ph) cm21; H NMR 3400 MHz, d6-DMSO) more than two
isomers d 7.90±7.31 315H, m, phenyl and Fmoc-aromatic-
CH and T-CH36)), 5.20 and 5.11 32H, 2£s, CH2Ph), 4.68±
4.02 39H, m, CHMe, Fmoc-aliphatic-CH and ±CH2 and
thymine-CH2CO,), 3.8±3.0 38H, m, H2O and ±CH2NCH2),
1.73±1.64 36H, brm, thymine-CH3), 1.18±0.97 36H, 2£m,
CH3); ESIMS m/z: 891.5 [M1H]1, 100%, 913 [M1Na]1,
75%, 669.4 [M2Fmoc2CO21H]1, 20%.
4.2.6. N-Fmoc-l-T-T-OH +8). The synthetic procedure was
similar to that of N-Fmoc-l,d-T-OH, white solid.
n
max3KBr): 3303 3OH), 1681 3CvO), 761, 739
3Fmoc) cm21; H NMR 3400 MHz, d6-DMSO) more than
two isomers d 11.24 31H, brm, COOH), 7.89±7.16 310H,
m, Fmoc aromatic-CH and T-CH36)), 4.82±3.6 313H, m,
CHMe, Fmoc aliphatic-CH and ±CH2 and thymine-
CH2CO, NCH2CO), 3.55±3.0 34H, m, H2O and
CHCH2N), 1.68 36H, brm, T-CH3), 1.23±1.00 36H, 2£m,
CHCH3); ESI-MS: 803 [M12H]1, 100%, 846
[M12Na2H]1, 90%, 867.3 [M13Na22H]1, 45%.
1
Compound 5a, white solid, yield 76%, mp: 197±1988C,
HPLC 99.7%. [Found: C, 56.77; H, 5.52; N, 10.23%.
C27H28N4O7´3H2O requires C, 56.44; H, 5.96; N, 9.75%];
n
max3KBr): 3334 3OH), 1682 3CvO); 761,739

Y. Wu, J.-C. Xu / Tetrahedron 57 02001) 8107±8113
8113
4.2.7. N-+Fluoren-9-ylmethoxycarbonyl)amino-penta-1-
yl succinate +10). Compound 9 30.81 g, 2.5 mmol) was
dissolved in dry DCM 310 mL) and dry pyridine 31 mL).
To this solution was added succinic anhydride 30.25 g,
2.5 mmol) and DMAP 330.4 mg, 0.25 mmol). After stirring
for 3 h at room temperature, an additional portion of
succinic anhydride 325.0 mg, 0.25 mmol) and DMPA
360.8 mg, 0.50 mmol) were added and the solution was
stirred for 6 h at 508C and 16 h at room temperature. The
solution was evaporated in vacuo, the residue was dissolved
in ethyl acetate and washed once with ice cold 5% citric
acid. The organic phase was dried under Na2SO4, ®ltered
and evaporated under reduced pressure. The residue was
puri®ed by ¯ash chromatography 3from DCM±ethyl acetate
2:1 to DCM±methanol 50:1) to give the product 11 as a
white solid 30.59 g, 65%). [Found: C, 67.99; H, 6.40; N,
3.21%. C24H27NO6 requires C, 67.75; H, 6.40%; N,
3.29%]; nmax3KBr): 3352 3OH), 1716, 1689 3CvO), 758,
unreacted amino group using 5% Ac2O in DMF,
2£15 min; 36) washing with DMAc 32£2 mL), EtOH
32£2 mL), DCM 32£2 mL);. Steps 1±6 were repeated
until the desired sequence were obtained. After the ®nal
coupling, the resins were treated with 0.5 M NaOH in
water±THF 31:1) for 3 h at room temperature. Then, the
solution was collected and adjusted to pH 7.0 by aq. 1 M
AcOH. After concentration to dryness, the residue 3ESI-MS
of crude PNA m/z 2906.0) was dissolved in H2O and puri-
®ed on kromasil RP-C18 34.6£250 mm), eluting with a
linear gradient of 20±23% B in A 3A: 0.05% TFA in
water; B: 0.05% TFA in acetontrile) yielding 62% of
oligmer H2N-3T)10-3penta).
Acknowledgements
We thank the National Science Foundation of China and the
State Key Laboratory of Bioorganic and Natural Products
Chemistry for their ®nancial support.
738 3Fmoc) cm21
; d
1H NMR 3300 MHz, CDCl3)
7.78±7.26 38H, m, Fmoc-aromatic-CH), 4.97 31H, s,
FmocNH), 4.47±4.40 32H, m, ±CH2O), 4.23±4.09 33H,
m, Fmoc-aliphatic-CH and ±CH2), 3.17 32H, m, NCH2),
2.64 34H, m, COCH2CH2CO), 1.66±1.37 36H, m,
CH2CH2CH2CH2CH2); ESIMS m/z: 448 [M1Na]1, 100%,
873 [2M1Na]1, 80%.
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succinylamido-BHA resin
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Á
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A
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BHA resin. The resulting suspension was shaken for 3 h at
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Â
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.
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