Synthesis of PNA monomers and dimers by ugi four-component reaction

Article abstract of DOI:10.1055/s-2003-39391

Peptide nucleic acids (PNA) have received great attention as tools in molecular biology and biotechnology, diagnostic agents, and potential antisense drugs. Many derivatives and analogues of PNA were designed and prepared to improve their physico-chemical

Full text of DOI:10.1055/s-2003-39391

PAPER
1171
Synthesis of PNA Monomers and Dimers by Ugi Four-Component Reaction
Synthesis of
M
i
onome
n
ric and Dim
g
Nuclei
X
c
A
cids u,* Ting Zhang, Wenhao Wang, Xiaomin Zou, Xin Zhang, Yiqiu Fu
Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100083,
P. R. China
Fax +861062015584; E-mail: pingxu@bjmu.edu.cn
Received 7 January 2003; revised 3 March 2003
PNA-oligomers are synthesized in most cases by homoge-
Abstract: Peptide nucleic acids (PNA) have received great atten-
neous or solid-phase peptide synthesis from N-protected
tion as tools in molecular biology and biotechnology, diagnostic
monomers (Figure 2).5 The synthesis of PNA monomers
has been described with different strategies using differ-
agents, and potential antisense drugs. Many derivatives and ana-
logues of PNA were designed and prepared to improve their physi-
co-chemical and biological properties. In most cases, PNA ent protecting groups for the amino function (Boc, Fmoc,
oligomers were synthesized by peptide synthesis from N-protected
or Mmt) and for the nucleobases. The commonly used
monomers. In this work, a new method to obtain PNA monomers
methods for the preparation of PNA monomers require
and PNA chain prolongation by Ugi four-component condensation
several steps to generate the N-(2-aminoethyl)glycine unit
reaction was tested by synthesizing PNA monomers and dimers.
followed by N-acylation of the glycine derivative by a
This method differs from classical peptide chemistry in that no prior
carboxymethylated nucleobase.6 Several quite different
preparation of PNA monomers is needed as they are built up along
research reports demonstrated the usefulness of Ugi four-
component condensation (Ugi-4CC) reaction in the syn-
thesis of PNA monomers.7 Herein we describe a new
method to obtain PNA monomer and PNA chain prolon-
gation by progressional Ugi-4CC reactions, which differs
from classical peptide chemistry in that prior preparation
of PNA monomers is not needed as they are built up along
with the extended chain.
with the extension of chain. The synthesis of the key component
isocyanide is also presented.
Key words: peptide nucleic acids (PNA), condensation, isocya-
nide, Ugi four-component reaction
Peptide nucleic acids (PNA) are oligonucleotide ana-
logues in which the sugar-phosphate backbone has been
replaced by a polyamide chain linked to nucleobases
(Figure 1). Invented more than 10 years ago,1 they have
received great attention due to their several favorable
properties, including resistance to nuclease and protease
digestion, stability in serum and cell extracts, and their
high affinity for RNA and single- and double-stranded Figure 2 Structure of N-protected PNA monomer; Pg = protecting
DNA targets.2 For these reasons, they have become very
group
popular, as tools in molecular biology and biotechnology,
as diagnostic agents, and as potential antisense drugs.
However, there are some limitations for the use of PNAs
in diagnostic and research applications, for example, their
low solubility in water and their tendency towards self-ag-
gregation.3 Many derivatives and analogues of PNA were
designed and prepared to improve their physico-chemical
and biological properties.4 A systematic investigation of
structure-function relationships using combinatorial
chemical strategies is still an attractive research area.
The multi-component condensation reactions, especially
Ugi-4CC reaction, is continuously used as a powerful tool
in combinatorial chemistry.8 In this work, PNA oligomer
to be synthesized was designed by a repeated protocol of
two steps (an Ugi reaction and a deprotection) in one turn
(Scheme 1). In the first cycle, a nucleobase-acetic acid 1,
an amine 2, an oxo compound (aldehyde or ketone, 3), and
an N-protected aminoethyl isocyanide 4 were used as
starting materials in the Ugi-4CC reaction to generate the
amino-protected PNA monomer 5 in a one-pot process,
followed by deprotection of the amino group. Then the
deprotected product 6 reacts as the amine component with
the other three components in the second cycle of Ugi re-
action to yield the amino-protected PNA dimer 7. By re-
peating the Ugi reaction and amino-deprotection, the PNA
chain can be extended. This strategy can be applied in the
synthesis of a number of structural derivatives and ana-
logues of PNA by employment of different reactants, 1, 2,
3 and 4. More expediently, the four components may be
selected as four types of building blocks to construct de-
rivatized PNA libraries by solid- or solution-phase syn-
thesis following this strategy.
Figure 1 Structure of PNA; B = nucleobase
Synthesis 2003, No. 8, Print: 05 06 2003.
Art Id.1437-210X,E;2003,0,08,1171,1176,ftx,en;F00403SS.pdf.
© Georg Thieme Verlag Stuttgart · New York

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P. Xu et al.
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Scheme 1 Synthetic scheme of PNA monomer and chain extension; B = nucleobase, Pg = protecting group
For the purpose of examining the feasibility of the syn- not easy to do because the two amino groups were often
thetic route, thymin-1-ylacetic acid (1a), [N4-(benzyloxy- amidated at the same time. But the mono-protected (N-
carbonyl)cytosine-1-yl]acetic acid (1b), 3-methyl- Boc)-ethylenediamine 9 was obtained conveniently with
butylamine (2a), formaldehyde (3a), 3-methylbutanal di-tert-butyl dicarbonate in moderate yield (70%) by fol-
(3b), and (N-tert-butyloxycarbonyl)aminoethyl isocya- lowing the procedure described.9 The formylation of 9
nide (4a) were selected first to generate PNA monomers was carried out by using HCO2Et, HCO2C(=O)Me, DCC/
and dimers in solution phase following the above strategy. HCO2H, respectively; and all gave the anticipated formyl-
Among the four components of the Ugi reaction, 2 and 3 ated product 10. But DCC/HCO2H gave the best results.
are commercially available, and 1a and 1b can be pre- The reaction was over in 4 hours at room temperature with
pared conveniently (Scheme 2).5
high yield (92%), and no byproduct was detected. The re-
actants should be added in the following order: first the
amine 9, then formic acid, and finally a solution of DCC
in anhydrous CH2Cl2.
Isocyanide 4a has to be prepared just before use because
of its instability. The synthetic route to 4a is shown in
Scheme 3. The mono-amidation of ethylenediamine was
Scheme 2 Synthetic route to thymine and cytosine acetic acids
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Synthesis of PNA Monomers and Dimers by Ugi Four-Component Reaction
1173
reacted with each other in the reaction. The structures of
5a–c were confirmed by their FAB-MS, 1H NMR and IR
spectra, and elemental analyses (see experimental sec-
tion). The quantities and qualities of Ugi products were af-
fected by reaction temperature and time. At room
temperature, the reaction was slow and sometimes had to
be prolonged for two days (48 h). When the reaction tem-
perature was increased to 80 °C (refluxing in isopro-
panol), the reaction speeded up greatly. But when the
reactions were carried out at higher temperature, the num-
Scheme 3 Synthetic route to N-Boc-aminoethyl isocyanide
The preparation of isocyanides from their formamide pre- ber and amount of byproducts increased gradually (moni-
cursors has been reviewed.10 The POCl3/Et3N method was tored by TLC). Considering the purity of the products,
used in this work to transform 10 to (N-Boc)-aminoethyl carrying out the reaction at room temperature was better.
isocyanide 4a. During the reaction, hydrogen chloride At this temperature most reactions can be completed with-
was produced, which may deprotect the acid labile Boc in 24–48 hours.
group. In fact the deprotected compound was indeed de-
The protecting group on the main chain amino of PNA
tected in our experiment. Also the isocyanide 4a was
monomers 5 need to be cleaved before the extension of the
found to be unstable. Hence the procedure was improved
chain. The typical 50% CF3CO2H–CH2Cl2 solution was
to finish the reaction in shorter time (treatment with
used first for N-Boc deprotection of 5b. The reaction was
completed easily within 1 hour to obtain the trifluoroace-
tate product 6a (Scheme 4). When 6a was reacted with
thymine acetic acid, 3-methylbutanal and (N-Boc)-amino-
POCl3 for 30 min instead of 1 h, then with K2CO3 for 30
min instead of 1 h) to reduce the unwanted deprotection.
Additionally, removal of the gaseous hydrogen chloride
evolved during the reaction under vacuum was also help-
ethyl isocyanide 4a in a second Ugi cycle to generate the
ful. Then, the reaction mixture was worked up immediate-
corresponding PNA dimer, the product was not the antic-
ly to decrease the decomposition of isocyanide product.
ipated compound 7b (Mw = 832.14), but a new com-
Isocyanide 4a was purified chromatographically and its
pound 7a (Mw = 762.00, Scheme 4). The structure of 7a
1
structure was determined by H NMR spectrum and the
was confirmed by its mass spectrum (M+ – Boc = 662). Its
characteristic IR absorbance of isocyanide group at 2120
formation can be explained as follows: in the Ugi reaction,
the trifluoroacetate anion, which was carried in the reac-
cm–1.
Thymin-1-ylacetic acid (1a), [N4-(benzyloxycarbonyl)cy- tion mixture by compound 6a instead of thymine acetate
tosine-1-yl]acetic acid (1b), 3-methylbutylamine (2a), anion, reacts with the amine, aldehyde, and isocyanide to
formaldehyde (3a), 3-methylbutanal (3b), and (N-Boc)- yield the Ugi product 7a. This hypothesis was proved by
aminoethyl isocyanide (4a) were used to build up three the fact that the thymine acetic acid was recovered as ex-
PNA monomers 5a–c (Table 1) by Ugi reaction (cf. pected. Anion resin was used to try to remove the trifluo-
roacetate anion, but to no avail. Finally HCl–DMF was
Scheme 1). Equimolar amounts of the four components
Table 1 PNA Monomers Prepared
PNA monomers
Components
1
2
3
4
5a
HCHO
5b
5c
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P. Xu et al.
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Scheme 4 Formation of compound 7a; T = thymine
used to deprotect the Boc group, and the hydrochloride cient to cause the self-aggregation of PNA. Considering
product 6b (Scheme 5) is transformed to the correspond- the solubility, the reaction was carried out in DMF solu-
ing PNA dimer successfully. Moreover, the PNA mono- tion. The aldehyde is a potential diverse component in Ugi
mer 5c, which has a Cbz-protected cytosine on the side- reaction and substituted aldehydes will be more meaning-
chain, was also transformed to main chain deprotected ful than formaldehyde. So in the second Ugi cycle 3-me-
product 6c by HCl–DMF (Scheme 5). Meanwhile the side thylbutanal instead of formaldehyde was used to test the
chain protecting group Cbz was unaffected. This made it method.
possible that the amino groups on the main chain and side-
In conclusion, a novel PNA synthesis strategy was de-
chain can be protected with Boc and Cbz, respectively,
signed based on Ugi four-component condensation reac-
and deprotected orthogonally.
tion by a repeated protocol. Three PNA monomers and
In the synthesis of PNA dimer by a second cycle Ugi re- two dimers were prepared in solution phase to test the
action (Scheme 5), the hydrochloride 6 needed to be treat- method. Most of the reactants are commercially available
ed with an alkaline agent to liberate the amino group or prepared easily. The synthesis of the key component in
before the other three components were added. Pyridine the Ugi reaction, (N-Boc)-aminoethyl isocyanide, is re-
was found to be good for this purpose. Its basicity was ported in detail. This strategy can be applied in the synthe-
strong enough to neutralize the chloride ion, but insuffi- sis of PNA analogues, and the four components may be
Scheme 5 Synthesis of PNA dimer
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Synthesis of PNA Monomers and Dimers by Ugi Four-Component Reaction
1175
MS (FAB): m/z = 304 (M + 1).
selected as four types of building blocks to construct de-
rivatized PNA libraries. But this solution-phase method is
not satisfactory to build up large scale libraries and long
chain PNA oligomers because of the difficulty of purifica-
tion and the low yields. The solid-phase procedure is be-
ing studied to improve the method.
(N-tert-Butyloxycarbonyl)ethylenediamine (9)
A solution of (Boc)2O (2.18 g, 10 mmol) in anhyd THF (10 mL) was
added dropwise to a solution of ethylenediamine (2.1 mL, 30 mmol)
in anhyd THF (10 mL) cooled in an ice-bath, and the mixture was
stirred for 30 min. After stirring for a further 24 h at r.t., the solvent
was removed by evaporation under reduced pressure. The residue
was partitioned between CH2Cl2 (20 mL) and H2O (5 mL) and the
aqueous layer was washed with CH2Cl2 (2 × 5 mL). The CH2Cl2 so-
lutions were combined and washed with 20% aq NaCl (2 × 5 mL),
dried (MgSO4), and evaporated to yield 2.4 g of a colorless oil. This
crude materiel was purified by column chromatography on silica gel
(CH2Cl2–EtOH–aq NH3, 15:15:1) to give 9; yield: 1.2 g (75%); col-
orless oil.
Unless otherwise mentioned, all the reagents were obtained com-
mercially and used without further purification. Petroleum ether
used had a boiling point range of 60–90 °C. Melting points were
1
taken with an X-4 apparatus and are uncorrected. IR (KBr), H
NMR, MS and elemental analyses data were taken with PE-983,
VXR-300S, VG-ZAB-HS, Carloerba-1106 instruments, respective-
ly.
IR (film): 3308, 3066, 2923, 1702, 1636, 1547, 1415, 1332, 1237,
1034, 696 cm–1.
Thymin-1-ylacetic Acid (1a)
1H NMR (CDCl3): = 5.24 (s, 1 H, CONH, exchangeable with
D2O), 3.18 (t, 2 H, J = 5.85 Hz, NCH2), 2.82 (t, 2 H, J = 5.85 Hz,
NCH2), 2.34 (d, 2 H, J = 13.8 Hz, NH2, exchangeable with D2O),
1.38 [s, 9 H, C(CH3)3].
To a stirred solution of thymine (5.00 g, 0.04 mol) and K2CO3 (5.48
g, 0.04 mol) in anhyd DMF (120 mL) was added dropwise ethyl
bromoacetate (4.35 mL, 0.04 mol) at r.t. under N2, and the mixture
was stirred for 20 h. After filtration, the filtrate was evaporated to
dryness under reduced pressure. The residual solid was mixed thor-
oughly with H2O (37.5 mL) and aq HCl (4 mol/L, 1.6 mL) at 0 °C,
and the mixture was stirred for 30 min, and filtered to give ethyl
thymin-1-ylacetate. This solid was washed with H2O (3 × 20 mL),
then mixed with H2O (40 mL) and aq NaOH (1 mol/L, 20 mL), and
boiled for 10 min. After cooling to 0 °C, aq HCl (4 mol/L, 13.5 mL)
was added and the mixture was stirred for 30 min. After filtration,
the filter cake was washed with H2O (3 × 20 mL), and dried; yield:
4.53 g (65%); white powder; mp 260–262 °C (Lit.5 mp not report-
ed).
MS (FAB): m/z = 161 (M + 1).
(N-tert-Butyloxycarbonyl)-N -formylethylenediamine (10)
Anhyd formic acid (0.075 mL, 2 mmol) was added to a solution of
9 (320 mg, 2 mmol) in anhyd CH2Cl2 (5 mL) cooled in an ice-bath,
followed by addition of the solution of DCC (412 mg, 2 mmol) in
anhyd CH2Cl2 (5 mL). The mixture was stirred for 3–4 h at r.t., and
the solid dicyclohexylurea was filtered. The filtrate was washed
with sat. aq NaHCO3 (2 mL), dried (MgSO4), and evaporated; yield:
340 mg (90%); white solid; mp 61–63 °C.
1H NMR (DMSO-d6): = 13.09 (s, 1 H, CO2H), 11.33 (s, 1 H,
CONH), 7.49 (s, 1 H, C=CH), 4.37 (s, 2 H, CH2), 1.75 (s, 3 H, CH3).
IR (KBr): 3324, 2927, 1668, 1530, 1271, 1247, 1168 cm–1.
1H NMR (CDCl3): = 8.13 (s, 1 H, HCO), 6.67 (s, 1 H, CONH),
5.14 (s, 1 H, CONH), 3.34 (m, 2 H, NCH2), 3.23 (m, 2 H, NCH2),
1.38 [s, 9 H, C(CH3)3].
[N4-(Benzyloxycarbonyl)cytosine-1-yl]acetic Acid (1b)
Benzyl chloroformate (52 mL, 0.36 mol) was added dropwise slow-
ly to a suspension of cytosine (20.0 g, 0.18 mol) in anhyd pyridine
(1000 mL) at 0 °C under N2. The mixture was stirred for 20 h and
evaporated under reduced pressure to remove the solvent. The solid
residue was mixed thoroughly with H2O (200 mL) and the pH was
adjusted to 1 with concd HCl. The white solid was filtered off,
washed with H2O, and dried. The crude product was recrystallized
from EtOH (500 mL) to give N4-(benzyloxycarbonyl)cytosine.
MS (FAB): m/z = 189 (M + 1).
Anal. Calcd for C8H16N2O3 (188.26): C, 51.04; H, 8.58; N, 14.88.
Found: C, 51.12; H, 8.50; N, 14.80.
(N-tert-Butyloxycarbonyl)aminoethyl Isocyanide (4a)
To a solution of 10 (1.64 g, 8.7 mmol) in anhyd CH2Cl2 (13 mL) was
added Et3N (3.6 mL, 26.2 mmol) and to the mixture cooled in an
ice-bath was added dropwise POCl3 (0.82 mL, 8.7 mmol) and
stirred for 30 min. A solution of K2CO3 (1.21g, 8.7 mmol) in H2O
(5.7 mL) was added to the mixture and stirred for another 30 min.
The aqueous layer was extracted with CH2Cl2 (2 × 5 mL). The or-
ganic layer and the CH2Cl2 solutions were combined and washed
with H2O (2 × 5 mL), dried (MgSO4), and evaporated under reduced
pressure to yield a dark-brown cream. This crude materiel was pu-
rified by column chromatography on silica gel (petroleum ether–
EtOAc, 3:1); yield: 1.0 g (68%); pale yellow solid; mp 46–47 °C.
N4-(Benzyloxycarbonyl)cytosine
Yield: 14.7 g (33%); white crystals; mp 270 °C (dec) (Lit.5 mp >250
°C).
To a stirred suspension of the above prepared N4-(benzyloxycarbo-
nyl)cytosine (20.3 g, 83 mmol), and K2CO3 (11.4 g, 83 mmol) in an-
hyd DMF (230 mL) was added dropwise ethyl bromoacetate (8.9
mL, 83 mmol) at r.t. under N2, and the mixture was stirred for 24 h.
After filtration, the filtrate was evaporated under reduced pressure
to dryness. The residual solid was mixed thoroughly with H2O (80
mL) and aq HCl (4 mol/L, 3.1 mL) at 0 °C, and the mixture was
stirred for 15 min, and filtered to furnish ethyl N4-(benzyloxycarbo-
nyl)cytosine-1-ylacetate. This solid was washed with H2O (2 × 20
mL), then mixed with H2O (120 mL) and aq NaOH (1 M, 620 mL),
and stirred for 30 min at r.t., cooled to 0 °C, and filtered. Aq HCl (4
mol/L, 35 mL) was added, the precipitated white solid was filtered
and recrystallized from MeOH (1000 mL) to give 1b; yield: 7.76 g
(31%); white crystals; mp 272–274 °C (Lit.5 mp 266–274 °C).
IR (KBr): 2960, 2120, 1630, 1510, 1250 cm–1.
1H NMR (CDCl3): = 4.98 (s, 1 H, CONH), 3.54 (s, 2 H, NCH2),
3.39 (s, 2 H, NCH2), 1.46 [s, 9 H, C(CH3)3].
MS (FAB): m/z = 171 (M + 1).
Anal. Calcd for C8H14N2O2 (170.24): C, 56.44; H, 8.30; N, 16.46.
Found: C, 56.41; H, 8.32; N, 16.43.
PNA Monomer 5a; Typical Procedure
Paraformaldehyde (117 mg, 3.9 mmol), 3-methylbutylamine (0.49
mL, 4.2 mmol), 4a (600 mg, 3.5 mmol) and 1a (718 mg, 4.1 mmol)
were mixed with isopropanol (4.2 mL) at r.t. and stirred for 48 h.
The suspension was filtered, washed thoroughly with cold EtOH to
give 1.18 g of a white solid. This crude product was dissolved in a
1b
1H NMR (DMSO-d6): = 10.80 (s, 1 H, CONH, exchangeable with
D2O), 8.02 (d, 1 H, J = 7.0 Hz, =CH), 7.36 (m, 5 H, C6H5), 7.01 (d,
1 H, J = 7.0 Hz, =CH), 5.20 (s, 2 H, PhCH2), 4.51 (s, 2 H, CH2).
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P. Xu et al.
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small amount of DMF, and purified by column chromatography on
silica gel (eluent: CH2Cl2–EtOH, 30:1); yield: 1.06 g (67%); white
solid; mp 170 °C.
1H NMR (DMSO-d6): = 11.23 (s, 1 H, NH, exchangeable with
D2O), 8.05 (s, 1 H, CONH, exchangeable with D2O), 7.82 (s, 1 H,
CONH), 7.41 (s, 1 H, =CH), 4.22 (s, 2 H, CH2), 3.73 (t, 2 H, J = 6.4
Hz, NCH2), 3.08 (t, 2 H, J = 6.4 Hz, NCH2), 1.74 (s, 3 H, =CCH3),
1.55 (m, 1 H, CH), 1.36 (s, 2 H, CH2), 1.32–1.23 (m, 10 H, 2 CH3,
2 CH2), 0.84 [s, 9 H, C(CH3)3].
at r.t. for 24 h, the condensed product 7b was obtained from the re-
action mixture by column chromatography on silica gel (eluent:
CH2Cl2–EtOH, 30:1); yield: 103 mg (31%); white solid; mp 150–
152 °C.
IR (KBr): 2929, 2858, 1671, 1388, 1255, 1095 cm–1.
MS (FAB): m/z = 833 (M + 1).
Anal. Calcd for C40H65N9O10 (832.14): C, 57.73; H, 7.89; N, 15.15.
Found: C, 57.79; H, 7.93; N, 15.18.
MS (FAB): m/z = 492 (M + K).
7c
Anal. Calcd for C21H35N5O6 (453.61): C, 55.60; H, 7.79; N, 15.44.
Found: C, 55.70; H, 7.64; N, 15.56.
Following the above typical procedure, 6c (0.37 mmol), 3-methyl-
butanal (0.04 mL, 0.37 mmol), 4a (63 mg, 0.37 mmol) and 1a (130
mg, 0.74 mmol) were reacted togetrher to give 7c; yield: 98 mg
(28%); white solid; mp 157–159 °C.
5b
Following the above typical procedure, 3-methylbutanal (5.0 mL,
47 mmol), 3-methylbutylamine (6.0 mL, 52 mmol), 4a (7.3 g, 43
mmol) and 1a (8.6 g, 49 mmol) were reacted to give 5b; yield: 17.0
g (80%); white solid; mp 161–163 °C.
MS (FAB): m/z = 952 (M + 1).
Anal. Calcd for C47H70N10O11 (951.27): C, 59.34; H, 7.43; N, 14.73.
Found: C, 59.83; H, 7.39; N, 14.78.
IR (KBr): 3346, 2958, 1680, 1395, 1367, 1250 cm–1.
1H NMR (DMSO-d6): = 11.30 (s, 1 H, CONH, exchangeable with
D2O), 8.22 (s, 1 H, CONH), 7.83 (s, 1 H, CONH), 7.39 (s, 1
H, =CH), 4.59 (s, 2 H, CH2), 3.10–2.90 (m, 4 H, CH2CH2), 1.77 (s,
3 H, =CCH3), 1.50–1.30 (m, 5 H, 3 CH, NCH2), 1.37 [s, 9 H,
C(CH3)3], 0.94–0.82 (m, 16 H, 4 CH3, 2 CH2).
Acknowledgment
This work was supported by the National Major Program for Basic
Research Development (973 projects) from the Ministry of Science
and Technology of China (G1998051114).
MS (FAB): m/z = 510 (M + 1).
References
Anal. Calcd for C25H43N5O6 (509.73): C, 58.90; H, 8.52; N, 13.74.
Found: C, 58.95; H, 8.50; N, 13.78.
(1) Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O.
Science 1991, 254, 1497.
5c
(2) (a) Egholm, M.; Buchardt, O.; Christensen, L.; Behrens, C.;
Freier, S. M.; Driver, D.; Berg, R. H.; Kim, S. K.; Norden,
B.; Nielsen, P. E. Nature (London) 1993, 365, 566.
(b) Wittung, P.; Nielsen, P. E.; Buchardt, O.; Egholm, M.;
Nordén, B. Nature (London) 1994, 368, 561. (c) Nielsen, P.
E.; Egholm, M.; Buchardt, O. Gene 1994, 149, 139.
(d) Demidov, V.; Potaman, V. N.; Frank-Kamenetskii, M.
D.; Buchardt, O.; Egholm, M.; Nielsen, P. E. Biochem.
Pharmacol. 1994, 48, 1309.
Following the above typical procedure, 3-methylbutanal (0.5 mL,
4.7 mmol), 3-methylbutylamine (0.55 mL, 4.7 mmol), 4a (0.7 g, 4.1
mmol) and 1b (1.5 g, 4.9 mmol) were reacted together to give 5c;
yield: 1.6 g (62%); white solid; mp 171–173 °C.
MS (FAB): m/z = 666 (M + K – 1).
Anal. Calcd for C32H48N6O7 (628.86): C, 61.11; H, 7.71; N, 13.37.
Found: C, 61.54; H, 7.38; N, 13.41.
(3) Egholm, M.; Buchardt, O.; Nielsen, P. E.; Berg, R. H. J. Am.
Chem. Soc. 1992, 114, 1895.
Deprotected PNA Monomer 6b; Typical Procedure
A solution of HCl in EtOAc (3 M, 0.33 mL) was added to a solution
of 5b (200 mg, 0.4 mmol) in DMF (3 mL) and the mixture was
stirred at 80 °C for 3 h. After evaporation, the residual oil was trit-
urated with Et2O (10 mL) to obtain 6b as a solid; yield: 168 mg
(94%); pale yellow solid; mp 160–163 °C.
(4) Ganesh, K. N.; Nielsen, P. E. Curr. Org. Chem. 2000, 4, 931.
(5) Dueholm, K. L.; Egholm, M.; Behrends, C.; Christensen, L.;
Hansen, H. F.; Vulpius, T.; Petersen, K.; Berg, R. H.;
Nielsen, P. E.; Buchardt, O. J. Org. Chem. 1994, 59, 5767.
(6) (a) Hyrup, B.; Egholm, M.; Nielsen, P. E.; Wittung, P.;
Nordén, B.; Buchardt, O. J. Am. Chem. Soc. 1994, 116,
7964. (b) Will, D. W.; Breipohl, G.; Langner, D.; Knolle, J.;
Uhlmann, E. Tetrahedron 1995, 51, 12069.
(7) (a) Gröger, H.; Hatam, M.; Kintscher, J.; Martens, J. Synth.
Commun. 1996, 26, 3383. (b) Dömling, A.; Richter, W.;
Ugi, I. Nucleosides Nucleotides 1997, 16, 1753. (c)Maison,
W.; Schlemminger, I.; Westerhoff, O.; Martens, J. Bioorg.
Med. Chem. Lett. 1999, 9, 581. (d) Maison, W.;
IR (KBr): 2957, 2934, 2872, 1671, 1387, 1253 cm–1.
1H NMR (DMSO-d6): = 11.31 (s, 1 H, CONH), 8.34 (s, 1 H,
+
CONH), 7.72 (m, 3 H, NH3 , exchangeable with D2O), 7.40 (s, 1
H, =CH), 4.58 (s, 2 H, CH2), 3.41–3.22 (m, 4 H, CH2CH2), 1.75 (s,
3 H, CH3), 1.58–1.46 (m, 5 H, 3 CH, NCH2), 0.94–0.82 (m, 16 H, 4
CH3, 2 CH2).
MS (FAB): m/z = 410 (ammonium salt).
Schlemminger, I.; Westerhoff, O.; Martens, J. Bioorg. Med.
Chem. 2000, 8, 1343. (e) Dömling, A. Nucleosides
Nucleotides 1998, 17, 1667. (f) Dömling, A.; Chi, K.-Z.;
Barrère, M. Bioorg. Med. Chem. Lett. 1999, 9, 2871.
(8) (a) Dömling, A. Comb. Chem. High Throughput Screening
1998, 1, 1. (b) Dömling, A.; Ugi, I. Angew. Chem. Int. Ed.
2000, 39, 3168.
(9) Richter, L. S.; Zuckermann, R. N. Bioorg. Med. Chem. Lett.
1995, 5, 1159.
(10) Ugi, I.; Fetzer, U.; Eholzer, U.; Knupfer, H.; Offermann, K.
Angew. Chem., Int. Ed. Engl. 1965, 4, 472.
6c
Following the above typical procedure, 5c (190 mg, 0.3 mmol) af-
forded 6c; yield: 153 mg (90%); pale yellow solid; mp 173–175 °C.
MS (FAB): m/z = 529 (ammonium salt).
PNA Dimer 7b; Typical Procedure
Pyridine (0.032 mL, 0.4 mmol) was added to a solution of 6b (209
mg, 0.4 mmol) in DMF (4 mL), and allowed to stand overnight. 3-
Methylbutanal (0.43 mL, 0.4 mmol), 4a (68 mg, 0.4 mmol) and 1a
(147 mg, 0.8 mmol) were then added to this solution. After stirring
Synthesis 2003, No. 8, 1171–1176 ISSN 1234-567-89 © Thieme Stuttgart · New York