Synthesis of photolabile o-nitroveratryloxycarbonyl (NVOC) protected peptide nucleic acid monomers

Article abstract of DOI:10.1016/j.tet.2005.06.002

The chemical synthesis of peptide nucleic acid (PNA) monomers was accomplished using various combinations of the o-nitroveratryloxycarbonyl (NVOC) group (N-aminoethylglycine backbone) and base labile acyl-type nucleobase protecting groups (anisoyl for ade

Full text of DOI:10.1016/j.tet.2005.06.002

Tetrahedron 61 (2005) 7967–7973
Synthesis of photolabile o-nitroveratryloxycarbonyl (NVOC)
protected peptide nucleic acid monomers
a
a
a
b,
Zheng-Chun Liu,a,b, Dong-Sik Shin, Kyu-Teak Lee, Bong-Hyun Jun, Yong-Kweon Kim
*
*
and Yoon-Sik Leea,
*
aSchool of Chemical Engineering, Seoul National University, Kwanak-Gu, Seoul 151-742, South Korea
bSchool of Electrical Engineering and Computer Science, Seoul National University, Kwanak-Gu, Seoul 151-742, South Korea
Received 25 April 2005; revised 3 June 2005; accepted 6 June 2005
Available online 28 June 2005
Abstract—The chemical synthesis of peptide nucleic acid (PNA) monomers was accomplished using various combinations of the
o-nitroveratryloxycarbonyl (NVOC) group (N-aminoethylglycine backbone) and base labile acyl-type nucleobase protecting groups (anisoyl
for adenine and cytosine; isobutyryl for guanine), thus offering a photolithographic solid-phase PNA synthetic strategy compatible with
photolithographic oligonucleotide synthesis conditions and allowing the in situ synthesis of PNA microarrays in an essentially neutral
medium, by avoiding the use of the commonly used deprotection reagents such as trifluoroacetic acid or piperidine. Convenient methods
were also explored to prepare 1-(carboxymethyl)-4-N-(4-methoxybenzoyl)cytosine and 9-(carboxymethyl)-2-N-(isobutyryl)guanine with
good yields.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
peptide nucleic acids as arrayed probe molecules could offer
an alternative to DNA microarrays affording superior
performance.7–11
Peptide nucleic acids (PNAs) are DNA analogs in which
the normal phosphodiester backbone is replaced by
2-aminoethyl glycine linkages.1 PNAs recognize their
complementary DNAs, RNAs or PNAs obeying the
Watson–Crick base-pairing rules with a remarkably high
specificity and affinity, and are resistant to nucleases and
proteases. Accordingly, since their invention, there have
been many reports on PNAs in such diverse fields as
chemistry, biochemistry, biotechnology, and medicine.2–5
More important is that surface-attached PNAs have been
shown to retain the unique and efficient hybridization
properties, which have been reported in solution studies of
regular PNAs.6 PNA recognition layers thus offer significant
advantages over their DNA counterparts in the realm of
sequence-specific DNA biosensors. These advantages
include significantly higher sensitivity and specificity
(including greater discrimination against single-base mis-
matches), faster hybridization at room and elevated
temperatures, minimal dependence on ionic strength, and
the use of shorter probes. Accordingly, the utilization of
In fact, PNA-arrays have come a long way since their initial
discovery a few years ago.12 As with DNA arrays, there are
two approaches that can be used for the preparation of PNA
arrays. In the first approach pre-fabricated PNA molecules
are spotted onto an appropriate support medium. This
method can produce PNA arrays of high quality if HPLC-
purified molecules are used. Ole Brandt et al.13 successfully
employed a modified spotting method to prepare PNA
microarrays for the hybridization of unlabelled DNA
samples. However, PNA chemistry is not widely used and
is therefore rather expensive. Additionally, the scale of the
standard synthesis is about 2 mmol, which is much more
than is needed for microarray production, thus increasing
the cost. Also, the standard HPLC purification of many
oligomers would be cost-intensive and time-consuming.
As an alternative to the spotting method, PNAs can be
synthesized in situ in a parallel manner on an appropriate
substrate.12,14–17 This procedure permits the simultaneous
synthesis of many different molecules and has the advantage
of consuming only very small amounts of the reagents per
oligomer. Using this technique, PNA macroarrays have
been developed on porous support media and silicon or glass
slides using fluorenylmethoxycarbonyl (FMOC) chemistry.
Porous membranes, however, have several inherent
Keywords: PNA monomers; o-Nitroveratryloxycarbonyl; Photo-
lithography.
*
Corresponding authors. Tel.: C82 2 880 7241; fax: C82 2 871 5974
(Y.-K.K.); tel.: C82 2 880 7080; fax: C82 2 876 9625 (Y.-S.L.);
e-mail addresses: liuzhengchunseu@126.com; yongkkim@chollian.net;
yslee@snu.ac.kr
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.06.002

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Z.-C. Liu et al. / Tetrahedron 61 (2005) 7967–7973
Scheme 1. Synthesis of tert-butyl N-[2-N-(4,5-dimethoxy-2-nitrobenzyloxy carbonyl)aminoethyl] glycinate. Reagents and conditions: (a) tert-butyl
bromoacetate, dichloromethane; (b) o-nitroveratryloxycarbonyl chloride, DIEA.
disadvantages that are detrimental to their routine appli-
cation. They lack mechanical stability, for example, which
makes them difficult to handle. In addition, they are rather
inflexible with respect to the mode of labeling and detection.
As for the in situ synthesis on glass or silicon slides, only a
few nanoliters of activated monomer are consumed per
oligomer. However, the spotting device needs to be highly
accurate, since the monomers have to be delivered to
exactly the same slide coordinates many times over.
Otherwise, a large percentage of truncated sequences will
be created. Therefore, the in situ synthesis of high-density
PNA microarrays will only be possible once appropriately
adapted hardware or PNA chemistry becomes available, a
problem which will most likely be addressed using
photolithography. The combinatorial photolithographic
process was first described by Fodor et al. for peptide array
synthesis18 and then successfully applied to the synthesis of
DNA microarrays.19,20 If we examine this issue from a
different viewpoint, PNA chemistry is basically peptide
chemistry combined with the simplicity of DNA chemistry.
In other words, if appropriate PNA monomers can be
synthesized, light-directed PNA array synthesis will become
possible. Herein, we describe the efficient synthesis of PNA
monomers, using o-nitroveratryloxycarbonyl (NVOC) (back-
bone)/acyl (anisoyl for cytosine and adenine; isobutyryl for
guanine) protecting-group combinations, which are compa-
tible with photolithographic solid phase PNA synthesis,
DNA synthesis and the assembly of DNA–PNA chimeras.
assembly of the protected N-(2-aminoethyl)glycine back-
bone and protected nucleobase-substituted acetic acid
structural units.5 In the case of light-directed PNA synthesis,
photolabile protecting groups are needed which are stable to
the different chemical steps involved in usual peptide
coupling chemistry, but which are photolytically cleaved
with high efficiency by irradiation at a wavelength which
does not damage the basic nucleosides structure. Hence, we
chose o-nitroveratryloxycarbonyl (NVOCZ4,5-dimethoxy-
2-nitrobenzyloxycarbonyl) as the photolabile protecting
group, because of its established use in peptide synthesis
and its o-nitrobenzyl moiety which is generally removed by
irradiation at wavelengths O300 nm without damaging the
nucleic acids.18,21,22 The key intermediate in our synthetic
route to the PNA building blocks is tert-butyl N-[2-N-(4,5-
dimethoxy-2-nitrobenzyloxycarbonyl)aminoethyl]glycinate
(1), which was synthesized according to the route outlined
in Scheme 1. tert-Butyl N-(2-aminoethyl)glycinate was
obtained according to the method previously reported in the
literature.23 The selective reaction of the primary amine of
tert-butyl N-(2-aminoethyl)glycinate with o-nitroveratryl-
oxycarbonyl chloride gave the oily product 1 after
purification with flash chromatography.
The nucleobases have exocyclic amino functions, which can
be protected by base-labile acyl protecting groups.
Protection at these sites not only blocks any undesirable
side reactions, but also increases the solubility of the
monomers, as well as their compatibility with the mild
conditions used for DNA synthesis.24–28 1-N-Carboxy-
methylthymine was synthesized according to the procedure
of Kosynkina et al.29 9-(Carboxymethyl)-6-N-(4-methoxy-
benzoyl)adenine was synthesized as described by Uhlmann
2. Results and discussion
The chemical synthesis of PNA monomers relies on the
Scheme 2. Synthesis of acyl-protected carboxymethyl nucleobases. Reagents and conditions: (a) anisoyl chloride, pyridine; (b) methyl bromoacetate, NaH,
DMF; (c) NaOH, H2O/dioxane; (d) isobutyric acid anhydride, DMF; (e) tert-butyl bromoacetate, NaH, DMF; (f) TFA, triethylsilane dichloromethane.

Z.-C. Liu et al. / Tetrahedron 61 (2005) 7967–7973
7969
et al.24,25 1-(Carboxymethyl)-4-N-(4-methoxybenzoyl)cyto-
3. Experimental
´
sine (4) was synthesized as described by Timar and
Uhlmann et al. with a modification designed to improve
the yield.25,27 As shown in Scheme 2, cytosine was acylated
with anisoyl chloride in pyridine to give 2 with high yield.
Compound 2 was then converted to its sodium salt using
NaH in DMF and alkylated using methyl bromoacetate. The
resulting methyl ester 3 was saponified using NaOH in a
dioxane–water mixture. The desired product 4 was isolated
by pH dependent precipitation with KHSO4 solution. The
synthesis of 9-(carboxymethyl)-2-N-(isobutyryl)guanine
was relatively difficult, because the substitution of guanine
is notorious for giving N9/N7 regioisomers requiring a
labor-consuming separation process. Fortunately, it was
found that lowering the temperature favored the formation
of the desired N9-regioisomer.25,27 2-N-Isobutyrylguanine
was synthesized according to the procedure of Jenny et al.30
This was then alkylated using NaH/tert-butyl bromoacetate
at low temperature to give the desired N9-alkylated
derivative 5 with good yield along with other regio-
isomers,27 which could be separated through simple
recrystallization from ethyl acetate. Removal of the tert-
butyl ester using TFA in dichloromethane in the presence of
triethylsilane gave acid 6 in quantitative yield.
3.1. General
The following abbreviations are employed: N,N-dimethyl-
formamide (DMF); o-nitroveratryloxycarbonyl (NVOC);
4-methoxybenzoyl (An); isobutyryl (iBu); trifluoroacetic
acid (TFA); N,N-diisopropylethylamine0 (DIEA); triethyl-
amine (TEA); O-benzotriazol-l-yl-N,N,N ,N0-tetramethylur-
onium
hexafluorophosphate
(BOP
reagent);
1-hydroxybenzotri-azole (HOBT). All reagents were
obtained from commercial suppliers and used without
further purification unless otherwise stated. Reactions
involving air- and/or moisture-sensitive reagents were
conducted under an atmosphere of dry nitrogen. Flash
chromatography was performed using Merck silica gel 60
(230–400 mesh ASTM). Melting points were obtained with
l
electrothermal 9300 apparatus without correction. H and
13C NMR spectra were obtained in the solvents indicated at
300 and 75 MHz, respectively. Chemical shifts are reported
in parts per million downfield relative to the internal
standard, tetramethylsilane. Coupling constants are reported
in hertz (Hz). Spectral patterns are designated as s, singlet;
d, doublet; t, triplet; q, quartet; m, multiplet; and br, broad.
Mass spectra were recorded using fast atom bombardment
(FAB). Elemental analyses were recorded using Perkin–
Elmer CHN analyzer model 2400.
The general synthetic routes toward the monomers are
outlined in Scheme 3. The carboxymethylated nucleobases
were coupled to the tert-butyl N-[2-N-(4,5-dimethoxy-2-
nitrobenzyloxycarbonyl)aminoethyl]glycinate backbone
unit 1 using several standard methods. The simplest and
most effective reagents were found to be propylphosphonic
anhydride for the pyrimidines and BOP reagent/HOBT for
the purines. As shown in Scheme 3, coupling of the base
acetic acid to the backbone was followed by removal of the
tert-butyl ester under standard conditions.
3.1.1. tert-Butyl N-[2-N-(4,5-dimethoxy-2-nitrobenzyl-
oxycarbonyl)aminoethyl]glycinate (1). To a solution of
tert-butyl N-(2-aminoethyl)glycinate23 (1.75 g, 10.0 m mol)
in dichloromethane (100 ml) was added DIEA (1.69 ml,
9.5 mmol) with mechanical stirring. A solution of 4,5-di-
methoxy-2-nitrobenzylchloroformate (2.62 g, 9.5 mmol) in
dichloromethane (10 ml) was added dropwise at 0 8C over a
period of 2 h. The resulting solution was allowed to warm to
room temperature and stirring was continued for another 2 h
at this temperature. The reaction mixture was then washed
sequentially with 1 M aq. HC1, sat. aq. NaHCO3 (3!20 ml)
and brine. The organic layer was dried over Na2SO4 and
concentrated in vacuo. Purification by flash chromatography
eluting with 10% methanol in ethyl acetate gave 1 as a green
oily product with a yield of 1.51 g (38%). RfZ0.37 (ethyl
In conclusion, in this study we described the synthetic route
to photolabile NVOC protected PNA monomers. Further
studies relating to the use of the above monomers in the
preparation of PNA oligomers using the Micro Array Mirror
system developed in our laboratory31–33 and our quest to
synthesize the PNA backbones in high yield are in progress
and will be reported on in due course.
Scheme 3. Synthesis of NVOC-protected PNA monomers. Reagents and conditions: (a) B–CH2–COOHCpropylphosphonic anhydride for 7a and 7b; B–CH2–
COOHCBOP reagent, HOBT, DIEA for 7c and 7d; (b) TFA, dichloromethane.

7970
Z.-C. Liu et al. / Tetrahedron 61 (2005) 7967–7973
acetate/methanol, 9:1); MS (FAB): 414.1872
(C18H27N3O8CH requires 414.1876); H NMR (CDCl3) d
8.6 Hz, 2H), 4.47 (s, 2H), 3.85(s, 3H). These values were
well matched with those of a previous report.27
1
7.71 (s, 1H), 7.04 (s, 1H), 5.52 (s, 2H), 3.99 (s, 3H), 3.96 (s,
3H), 3.32 (t, JZ5.5 Hz, 2H), 3.28 (s, 2H), 2.78 (t, JZ
5.6 Hz, 2H), 1.47 (s, 9H); 13C NMR (CDCl3) d 171.6, 155.9,
153.3, 147.7, 139.4, 128.3, 109.8, 107.9, 81.2, 63.2, 56.2,
51.0, 48.4, 40.6, 27.9.
3.1.5. 2-N-(Isobutyryl)-9-(tert-butyloxycarbonylmethyl)
guanine (5). To a suspension of 2-N-isobutyrylguanine30
(6.63 g, 30.0 mmol) in anhydrous DMF (250 ml) was added
NaH (0.72 g, 30.0 mmol) at 0 8C. The mixture was stirred
for 1 h and then tert-butyl bromoacetate (6.44 g,
33.0 mmol) was added dropwise over a period of 30 min
at 0 8C. The reaction was stopped after 2 h by the addition of
a small amount of solid CO2 and methanol (5 ml). The
reaction mixture was evaporated in vacuo and the residue
was treated with dichloromethane and water. The water
phase was re-extracted with dichloromethane. The dichloro-
methane fractions were combined and concentrated in
vacuo to give the crude product. The resulting crude product
was recrystallized from ethyl acetate to give the desired
N9-isomer product 5 with a yield of 6.46 g (64%). MpZ
204–205 8C (decomp.); RfZ0.13 (dichloromethane/metha-
nol, 9.5:0.5); 1H NMR (CDCl3) d 12.06 (s, 1H), 8.87 (s, 1H),
7.88 (s, 1H), 4.72 (s, 2H), 2.76 (m, 1H); 1.28 (d, JZ6.8 Hz,
6H). Anal. Calcd for C15H21N5O4: C, 53.72; H, 6.31; N,
20.88. Found: C, 53.88; H, 6.47; N, 20.75. These values
were well matched with those of a previous report.27
3.1.2. 4-N-(4-Methoxybenzoyl)cytosine (2). To a suspen-
sion of cytosine (2.5 g, 22.5 mmol) in pyridine (100 ml) was
added 4-methoxybenzoyl chloride (5.76 g, 33.8 mmol) at
room temperature. The reaction mixture was stirred in an
oil-bath at 80 8C. The cystosine rapidly dissolved and then
the product precipitated from the solution. After 2 h, the
precipitate was filtered off, washed with methanol and dried
in vacuo to afford 2 as a white powder with a yield of 4.5 g
(81%). 1H NMR (d6-DMSO) d 8.00 (d, JZ8.9 Hz, 2H), 7.84
(d, JZ7.0 Hz, 1H), 7.22 (s, br, 1H), 7.02 (d, JZ8.9 Hz, 2H),
´
3.84(s, 3H).ThiscompoundwaspreviouslyobtainedbyTimar
et al., but without detailed NMR characterization.27
3.1.3. 4-N-(4-Methoxybenzoyl)-1-(methoxycarbonyl-
methyl)cytosine (3). To a suspension of 2 (3.68 g,
15.0 mmol) in dry DMF (300 ml) was added NaH (0.36 g;
15.0 mmol) at 0 8C and the mixture was stirred for 1 h.
Methyl bromoacetate (2.32 g; 15.2 mmol, in 5 ml of
dichloromethane) was added dropwise at 0 8C using a
syringe with stirring over a period of 30 min. The resulting
solution was then allowed to warm to room temperature.
Stirring was continued for 1.5 h at room temperature, and
methanol (10 ml) was then added. The solvent was removed
in vacuo, and the residue was partitioned between
dichloromethane and water. The organic phase was washed
with water, dried (Na2SO4), filtered and evaporated in
vacuo. The resulting crude product was recrystallized from
methanol (200 ml) to give 3 as a white needle-like
crystalline solid with a yield of 4.1 g (86%). MpZ218–
220 8C; MS (FAB): 318.1098 (C15H15N3O5CH requires
318.1098); RfZ0.56 (ethyl acetate/methanol, 9:1); 1H NMR
(d6-DMSO) d 11.12 (s, 1H), 8.08 (d, JZ7.3 Hz, 1H), 8.00
(d, JZ9.0 Hz, 2H), 7.32 (d, JZ7.0 Hz, 1H), 7.02 (d, JZ
9.0 Hz, 2H), 4.66 (s, 2H), 3.77 (s, 3H), 3.54 (s, 3H); 13C
NMR (d6-DMSO) d 168.5, 166.5, 164.0, 162.9, 155.3,
150.4, 130.7, 125.1, 113.7, 96.2, 55.5, 52.3, 50.7. Anal.
Calcd for C15H15N3O5: C, 56.78; H, 4.76; N, 13.24. Found:
C, 56.58; H, 4.82; N, 13.12.
3.1.6. 9-(Carboxymethyl)-2-N-(isobutyryl)guanine (6).
To a suspension of 5 (2.0 g, 6 mmol) in anhydrous
dichloromethane (10 ml) was added triethylsilane (4.8 ml,
30 mmol). The mixture was cooled to 0 8C and TFA (15 ml)
was then added over a period of 5 min. After 30 min the
reaction was allowed to warm to room temperature. Upon
completion of the reaction (traced by TLC), the mixture was
concentrated in vacuo to give a foam which was stirred with
diethyl ether, filtered, washed with diethyl ether, and dried
in vacuo to afford the desired product 6 with a yield of
1.67 g (100%). RfZ0.44 (n-butanol/acetic acid/H2O, 3:1:1);
1H NMR (d6-DMSO) d 12.07 (s, 1H), 11.69 (s, 1H), 7.94 (s,
1H), 4.87 (s, 2H), 2.75 (q, 1H), 1.11 (d, JZ6.8 Hz, 6H). The
NMR values were well matched with those of a previous
report.24
3.1.7. tert-Butyl N-[2-N-(4,5-dimethoxy-2-nitrobenzyloxy-
carbonyl)aminoethyl]-N-[(thymin-l-yl)acetyl]glycinate
(7a). To a solution of 1 (4.13 g, 10.0 mmol) in anhydrous
DMF (50 ml) was added the acid 1-N-carboxymethyl-
thymine29 (1.84 g, 10.0 mmol), and the mixture was stirred
until most of the acid was dissolved. Propylphosphonic
anhydride (50% solution in ethyl acetate) (10.5 ml;
10.0 mmol) and DIEA (3.44 ml; 20 mmol) were added at
room temperature. The mixture was stirred at room
temperature for 2 h and then poured into a stirred mixture
of ice water (200 ml) and sat. NaHCO3 solution (15 ml).
The mixture was shaken vigorously for several minutes,
which led to the precipitation of fine pale yellow solids. The
solids were collected by filtration, stirred with water
(15 ml), filtered, washed with cold water, and dried in a
vacuum. Purification by flash chromatography eluting with
10% methanol in CHCl3 gave 7a in the form of pale green
solids with a yield of 5.68 g (98%). RfZ0.50 (ethyl acetate/
methanol, 9:1); MS (FAB): 580.2251 (C25H33N5O11CH
requires 580.2254); 1H NMR (CDCl3) (two rotamers) d 8.00
(s, 1H), 7.97 (s, 1H), 7.70 (s, 1H), 7.04 and 7.02 (rotamer,
2!s, 1H), 7.00 (s, 1H), 5.49 (s, 2H), 4.52 and 4.39 (rotamer,
3.1.4. 1-(Carboxymethyl)-4-N-(4-methoxybenzoyl)cyto-
sine (4). To a solution of 3 (3.62 g, 24 mmol) in a mixture
of dioxane (50 ml) and water (25 ml) was added a 2 M aq.
NaOH solution dropwise with stirring at room temperature
(pH 11–12). When hydrolysis of the methyl ester was
complete (traced by TLC), the pH of the reaction solution
was adjusted to 3 using 2 M KHSO4 solution. The
precipitate which separated out was filtered off. This crude
product was dissolved in aq. NaHCO3 solution and
reprecipitated by adding 2 M KHSO4 solution. The product
was filtered off, washed with a small amount of water and
dried in vacuo to give the product as a white solid with a
yield of 3.31 g (95%). RfZ0.52 (n-butanol/acetic acid/H2O,
3:1:1); 1H NMR (d6-DMSO) d 8.09 (d, JZ7.3 Hz, 1H), 8.02
(d, JZ8.8 Hz, 2H), 7.31 (d, JZ7.5Hz, 1H), 7.03 (d, JZ

Z.-C. Liu et al. / Tetrahedron 61 (2005) 7967–7973
7971
2!s, 2H), 4.09 and 3.99 (rotamer, 2!s, 2H), 3.98 (s, 3H),
3.96 (s, 3H), 3.57 (t, JZ6.0 Hz, 2H), 3.41 (t, JZ6.8 Hz,
2H), 1.92 and 1.91 (rotamer, 2!s, 3H), 1.50 and 1.46
(rotamer, 2!s, 9H); 13C NMR (CDCl3) d 168.8, 168.5,
168.1, 167.3, 164.1, 156.2, 156.1, 153.5, 153.4, 151.1,
151.0, 148.4, 148.0, 141.2, 140.9, 140.1, 139.7, 128.1,
127.1, 111.4, 110.8, 110.5, 110.2, 108.2, 108.1, 83.7, 82.7,
64.1, 63.6, 56.5, 56.4, 51.1, 49.6, 48.7, 47.8, 39.2, 39.1,
28.0, 27.9, 12.3.
139.7, 139.5, 129.8, 128.4, 127.6, 125.0, 114.1, 114.0,
110.8, 110.0, 108.0, 107.9, 96.6, 83.5, 82.4, 63.9, 63.4, 56.6,
56.5, 56.3, 56.2, 55.5, 51.4, 49.7, 49.4, 48.8, 48.6, 39.1,
39.0, 27.9.
3.1.10. N-[2-N-(4,5-Dimethoxy-2-nitrobenzyloxycarbo-
nyl)aminoethyl]-N-{[4-N-(4-methoxybenzoyl)-cytosin-1-
yl]acetyl}glycine (8b). To a suspension of 7b (6.98 g,
10 mmol) in dichloromethane (30 ml) was added TFA
(30 ml). A clear solution was formed which was stirred at
room temperature for 3 h (traced by TLC). The reaction
mixture was dried in vacuo and residual TFA was removed
by co-evaporation twice with toluene. The residue was
triturated with diethyl ether and filtered. Purification by
recrystallization from acetone gave 8b as pale green solids
with a yield of 5.91 g (92%). MpZ204 8C (decomp.); RfZ
0.69 (n-butanol/acetic acid/H2O, 3:1:1); MS (FAB):
3.1.8. N-[2-N-(4,5-Dimethoxy-2-nitrobenzyloxycarbo-
nyl)aminoethyl]-N-[(thymin-1-yl)acetyl]glycine (8a). To
a suspension of 7a (10 mmol) in dichloromethane (30 ml)
was added TFA (45 ml). A clear solution was formed which
was stirred at room temperature for 2 h (traced by TLC).
The reaction mixture was dried in a vacuum and residual
TFA was removed by co-evaporation twice with toluene.
The residue was triturated with diethyl ether and filtered.
Purification by recrystallization from acetone gave 8a as
pale green solids with a yield of 5.68 g (93%). MpZ182 8C
(decomp.); RfZ0.52 (n-butanol/acetic acid/H2O, 3:1:1); MS
(FAB): 524.1631 (C21H25N5O11CH requires 524.1628); 1H
NMR (d6-DMSO) (two rotamers) d 11.28 and 11.27
(rotamer, 2!s, 1H), 8.0 (br, 1H), 7.92 (s, 1H), 7.70 (s,
1H), 7.30 and 7.26 (rotamer, 2!s, 1H), 7.20 (s, 1H), 5.35
and 5.33 (rotamer, 2!s, 2H), 4.65 and 4.47 (rotamer, 2!s,
2H), 4.18 and 3.99 (rotamer, 2!s, 2H), 3.90 (s, 3H), 3.87
(s, 3H), 3.46–2.99 (br, CH2CH2O), 1.73 (s, 3H); 13C NMR
(d6-DMSO) d 171.1, 170.7, 167.9, 167.4, 164.6, 156.1,
155.9, 153.5, 153.4, 151.1, 147.9, 147.8, 142.2, 142.1,
139.4, 139.3, 128.0, 127.6, 110.9, 110.5, 108.4, 108.3,
108.1, 62.8, 62.6, 56.3, 56.1, 49.4, 48.7, 47.9, 47.1, 38.8,
38.1, 12.0. Anal. Calcd for C21H25N5O11: C, 48.19; H, 4.81;
N, 13.38. Found: C, 48.29; H, 4.78; N, 13.24.
1
665.1991 (C28H30N6O12CH requires 643.1999); H NMR
(d6-DMSO) (two rotamers) d 8.00(d, JZ8.8 Hz, 2H), 7.93
and 7.60 (rotamer, 2!d, JZ7.7, 5.5 Hz, 1H), 7.69 and 7.55
(rotamer, 2!s, 2H), 7.43 and 7.27 (rotamer, 2!d, JZ5.9,
6.9 Hz, 1H), 7.21 and 7.17 (rotamer, 2!s, 2H), 7.01(d, JZ
9.0 Hz, 2H), 5.35 and 5.32 (rotamer 2!s, 2H), 4.86 and
4.68 (rotamer s, 2H), 4.27 and 4.01 (rotamer s, 2H) 3.89 and
3.88 (rotamer s, 3H), 3.85 and 3.84 (rotamer s, 3H), 3.83
(s, 3H), 3.48–3.13 (br, CH2CH2O); 13C NMR (d6-DMSO) d
170.9, 170.5, 167.6, 167.1, 163.5, 162.8, 156.0, 155.8,
155.2, 153.4, 153.3, 150.9, 147.7, 147.6, 139.3, 139.2,
130.6, 127.9, 127.6, 125.2, 113.7, 110.8, 110.4, 108.0, 95.8,
62.7, 62.5, 56.2, 56.0, 55.5, 50.9, 49.6, 49.1, 47.7, 46.9,
38.7, 38.0. Anal. Calcd for C28H30N6O12: C, 52.34; H, 4.71;
N, 13.08. Found: C, 52.46; H, 4.77; N, 13.05.
3.1.11. tert-Butyl N-[2-N-(4,5-dimethoxy-2-nitrobenzyl-
oxycarbonyl)aminoethyl]-N-{[6-N-(4-methoxybenzoyl)
adenin-9-yl]acetyl}glycinate (7c). To a solution of 1
(4.13 g, 10 mmol) in anhydrous DMF (50 ml) were added
9-N-(carbonylmethyl)-6-N-(4-methoxybenzoyl)adenine24,25
(3.27 g, 10 mmol), BOP reagent (6.62 g, 15 mmol), HOBT
(2.02 g, 15 mmol) and DIEA (4.30 ml, 26 mmol). After
stirring at room temperature for 5 h (traced by TLC), the
mixture was concentrated in vacuo and the residue was
partitioned between ethyl acetate (100 ml) and half
saturated brine (100 ml), followed by extraction of the
aqueous phase with ethyl acetate (3!100 ml). The
combined organic extract was washed with 1 M aq. HC1
(2!50ml), sat. aq. NaHCO3 (2!50 ml), brine (1!50 ml),
and then dried (Na2SO4) and concentrated in vacuo.
Purification by flash chromatography eluting with 5%
methanol in dichloromethane gave 7c in the form of pale
green solids with a yield of 6.29 g (87%). RfZ0.31 (ethyl
acetate/methanol, 9:1); MS (FAB): 723.2735
3.1.9. tert-Butyl N-[2-N-(4,5-dimethoxy-2-nitrobenzyl-
oxycarbonyl)aminoethyl]-N-{[4-N-(4-methoxybenzoyl)-
cytosin-1-yl]acetyl}glycinate (7b). To a solution of 1
(4.13 g, 10.0 mmol) in ethyl acetate (50 ml) were added
the acid 4 (3.33 g, 11.0 mmol) and TEA (2.77 ml;
20.0 mmol). Propylphosphonic anhydride (50% solution in
ethyl acetate) (9.55 ml; 15.0 mmol) was added at room
temperature. The mixture was adjusted to pH 8 by the
addition of TEA and stirred for a further 1 h, after which the
mixture was evaporated in vacuo. The residue was taken up
in dichloromethane (100 ml), and the resulting solution was
washed with water (3!20 ml). The organic phase was dried
(Na2SO4), filtered and evaporated in vacuo. Purification by
flash chromatography eluting with 5% methanol in
dichloromethane gave 7b in the form of pale green solids
with a yield of 6.09 g (87%). RfZ0.50 (ethyl acetate/
methanol, 9:1); MS (FAB): 699.2619 (C32H38N6O12CH
requires 699.2625); 1H NMR (CDCl3) (two rotamers) d 7.85
(d, JZ8.4 Hz, 2H), 7.71 (s, 1H), 7.69(s, 1H), 7.61 and 7.58
(rotamer, 2!s, 1H), 7.10 and 7.04 (rotamer, 2!s, 1H),
6.98(d, JZ8.8 Hz, 2H), 5.51 and 5.50 (rotamer, 2!s, 2H),
4.75 and 4.46 (rotamer, 2!s, 2H), 4.24 and 4.02 (rotamer,
2!s, 2H), 3.99 and 3.98 (rotamer, 2!s, 3H), 3.95 and 3.92
(rotamer, 2!s, 3H), 3.89 (s, 3H), 3.66 and 3.60 (rotamer,
2!t, JZ6.0, 6.3 Hz, 2H), 3.48 and 3.41 (rotamer, 2!t, JZ
6.0, 6.1 Hz, 2H), 1.51 and 1.45 (rotamer, 2!s, 9H); 13C
NMR (CDCl3) d 168.6, 168.5, 168.0, 167.1, 163.4, 162.9,
156.2, 156.1, 155.6, 153.6, 153.5, 150.2, 148.1, 147.8,
1
(C33H38N8O11CH requires 723.2738); H NMR (CDCl3)
(two rotamers) d 8.90(s, 1H), 8.71 and 8.69 (rotamer, 2!s,
1H), 8.14 and 8.13 (rotamer, 2!s, 1H), 7.98 (d, JZ8.6 Hz,
2H), 7.69 and 7.67 (rotamer, 2!s, 1H), 7.02 and 7.01
(rotamer, 2!s, 1H), 6.98 (d, JZ2.4 Hz, 2H), 5.51 and 5.47
(rotamer, 2!s, 2H), 5.16 and 5.02 (rotamer, 2!s, 2H), 4.22
and 3.98 (rotamer, 2!s, 2H), 3.95 (s, 3H), 3.93 (s, 3H), 3.90
(s, 3H), 3.70 and 3.60 (rotamer, 2!t, JZ6.0, 6.1 Hz, 2H),
3.50 and 3.40 (rotamer, 2!t, JZ5.5, 5.7 Hz, 2H), 1.47 and
1.45 (rotamer, 2!s, 6H). 13C NMR (CDCl3) d 168.6, 168.2,
167.1, 166.4, 164.2, 164.1, 163.2, 163.1, 156.3, 156.1,

7972
Z.-C. Liu et al. / Tetrahedron 61 (2005) 7967–7973
153.5, 153.4, 152.4, 152.3, 151.9, 151.8, 149.5, 149.4,
148.2, 147.9, 144.2, 144.0, 139.9, 139.5, 130.0, 129.9,
128.0, 127.1, 125.8, 125.7, 122.0, 121.9, 114.0, 113.9,
110.95, 110.8, 108.2, 108.0, 83.9, 82.7, 64.1, 63.6, 56.4,
56.3, 55.5, 51.0, 50.7, 48.8, 48.6, 39.2, 38.9, 34.6, 34.5,
27.9, 27.8.
148.1, 140.6, 140.1, 139.9, 127.6, 127.5, 119.4, 119.3,
111.9, 110.5, 108.2, 108.1, 82.8, 82.6, 65.0, 64.0, 56.5, 56.4,
49.6, 49.5, 48.6, 48.3, 39.5, 39.3, 35.1, 34.9, 30.0, 29.7,
28.0, 27.9, 18.9, 18.8.
3.1.14. N-[2-N-(4, 5-Dimethoxy-2-nitrobenzyloxycarbo-
nyl)aminoethyl]-N-{[2-N-(isobutyryl)guanin-9-yl]acetyl}-
glycine (8d). To a suspension of 7d (6.74 g, 10 mmol) in
dichloromethane (20 ml) was added TFA (30 ml). A clear
solution was formed which was stirred at room temperature
for 3.5 h (traced by TLC). The reaction mixture was dried in
a vacuum and residual TFA was removed by co-evaporation
twice with toluene. The residue was triturated with diethyl
ether and filtered. Purification by recrystallization from
methanol gave 8d as pale green solids with a yield of 5.57 g
(90%). MpZ227 8C (decomp.); RfZ0.46 (n-butanol/acetic
acid/H2O, 3:1:1) MS (FAB), m/z 619.2110 (C25H30N8O11C
H requires 619.2112), 1H NMR (d6-DMSO) (two rotamers)
d 11.69 and 11.62 (rotamer, 2!s, 1H), 7.81 (s, 1H), 7.67
and 7.64 (rotamer, 2!s, 1H), 7.19 and 7.13 (rotamer, 2!s,
1H), 5.34 and 5.31 (rotamer, 2!s, 2H), 5.11 and 4.94
(rotamer, 2!s, 2H), 4.01 and 3.84 (rotamer, 2!s, 2H), 3.88
and 3.85 (2!s, 6H), 3.51 and 3.34 and 3.17 (3!br, CH2C
H2O), 2.74 (m, 1H), 1.08 (d, JZ6.8 Hz, 6H). 13C NMR
(d6-DMSO) d 180.2, 180.1, 171.1, 170.6, 167.1, 166.5,
156.1, 155.8, 155.0, 154.9, 153.4, 153.3, 149.3, 148.0,
147.9, 147.7, 140.6, 139.5, 139.2, 127.8, 127.3, 119.6,
111.1, 110.5, 108.1, 108.0, 62.8, 62.7, 56.2, 56.1, 49.6, 49.5,
47.1, 46.9, 44.1, 44.0, 34.8, 34.7, 31.0, 30.9, 18.9, 18.8.
Anal. Calcd for C25H30N8O11: C, 48.54; H, 4.89; N, 18.12.
Found: C, 48.58; H, 4.81; N, 18.08.
3.1.12. N-[2-N-(4,5-Dimethoxy-2-nitrobenzyloxycarbo-
nyl)aminoethyl]-N-{[6-N-(4-methoxybenzoyl)adenin-9-
yl]acetyl}glycine (8c). To a suspension of 7c (7.22 g,
10 mmol) in dichloromethane (30 ml) was added TFA
(30 ml). A clear solution was formed which was stirred at
room temperature for 2.5 h (traced by TLC). The reaction
mixture was dried in a vacuum and residual TFA was
removed by co-evaporation twice with toluene. The residue
was triturated with diethyl ether and filtered. Purification by
recrystallization from methanol gave 8c as pale green solids
with a yield of 6.33 g (95%). MpZ200 8C (decomp.); RfZ
0.49 (n-butanol/acetic acid/H2O, 3:1:1); MS (FAB):
1
667.2106 (C29H30N8O11CH requires 667.2112); H NMR
(d6-DMSO) (two rotamers) d 11.0 (s, 1H), 8.65 and 8.62
(rotamer, 2!s, 1H), 8.31 (s, 1H), 8.04 (d, JZ8.8 Hz, 2H),
7.68 (s, 1H), 7.21 and 7.17 (rotamer, 2!s, 1H), 7.06 (d, JZ
8.6 Hz, 2H), 5.39 and 5.35 (rotamer, 2!s, 2H), 5.15 (s, 2H),
3.97–3.84 (m, 11H), 3.41–3.17 (br, CH2CH2O). 13C NMR
(d6-DMSO) d 170.9, 170.5, 167.1, 165.2, 162.7, 156.2,
155.9, 153.4, 153.3, 152.7, 151.5, 150.2, 147.8, 147.7,
145.4, 139.5, 139.3, 130.7, 127.9, 127.5, 125.6, 124.5,
113.7, 110.9, 110.5, 108.1, 62.8, 62.5, 56.2, 56.1, 55.5, 50.0,
49.2, 47.8, 47.0, 44.2, 44.0, 35.7. Anal. Calcd for
C29H30N8O11: C, 52.25; H, 4.54; N, 16.81. Found: C,
52.28; H, 4.59; N, 16.79.
3.1.13. tert-Butyl N-[2-N-(4,5-dimethoxy-2-nitrobenzyl-
oxycarbonyl)aminoethyl]-N-{[2-N-(isobutyryl)guanin-9-
yl]acetyl}glycinate (7d). To a solution of 1 (4.13 g,
10 mmol) and 6 (3.27 g, 10 mmol) in anhydrous DMF
(50 ml) were added BOP reagent (6.62 g, 12.5 mmol),
HOBT (2.02 g, 12.5 mmol) and DIEA (4.30 ml,
15.0 mmol). After stirring at room temperature for 4.5 h
(traced by TLC), the mixture was concentrated in vacuo and
the residue was partitioned between dichloromethane
(150 ml) and half saturated brine (150 ml), and the aqueous
phase was extracted with ethyl acetate (3!100 ml). The
combined organic extract was washed with 1 M aq. HC1
(2!100 ml), sat. aq. NaHCO3 (2!100 ml), brine (1!
100 ml), and then dried (Na2SO4) and concentrated in
vacuo. Purification by flash chromatography eluting with
10% methanol in dichloromethane gave 2d in the form of
pale green solids with a yield of 5.46 g (81%). RfZ0.11
(ethyl acetate/methanol, 9:1); MS (FAB): 675.2736
Acknowledgements
This work was funded by grants from the Brain Korea 21
Program and the Korean Ministry of Science and
Technology, which supports the development of biochip
technology projects (M10415010006-04N1501-00610).
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