Synthesis of a C-linked glycosylated thymine-based PNA monomer and its incorporation into a PNA oligomer

Article abstract of DOI:10.1039/b607463d

Backbone modification of peptide nucleic acids (PNAs) by glycosylation has been shown to enhance selective biodistribution and cellular targeting of PNA oligomers based on sugar and cell surface lectin interactions. Here we report the synthesis of a new backbone-glycosylated thymine-based PNA monomer (T ^gal). The sugar residue was attached to the backbone of PNA via a stable carbon-carbon linkage between the sugar and the PNA monomers. Also, incorporation of the modified monomer into a PNA decamer (H-Ala ^gal-G-G-G-T^gal-C-A-G-C-T^gal-T-Lys-NH ₂) was successfully performed. Melting temperature (UV-T _m) of the modified PNA against the complementary DNA was only slightly lower than unmodified PNA. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b607463d

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis of a C-linked glycosylated thymine-based PNA monomer and its
incorporation into a PNA oligomer
Ramin Hamzavi,a Christoph Meyera and Nils Metzler-Nolte*a,b
Received 25th May 2006, Accepted 12th July 2006
First published as an Advance Article on the web 17th August 2006
DOI: 10.1039/b607463d
Backbone modification of peptide nucleic acids (PNAs) by glycosylation has been shown to enhance
selective biodistribution and cellular targeting of PNA oligomers based on sugar and cell surface lectin
interactions. Here we report the synthesis of a new backbone-glycosylated thymine-based PNA
monomer (Tgal). The sugar residue was attached to the backbone of PNA via a stable carbon–carbon
linkage between the sugar and the PNA monomers. Also, incorporation of the modified monomer into
a PNA decamer (H-Alagal-G-G-G-Tgal-C-A-G-C-Tgal-T-Lys-NH2) was successfully performed. Melting
temperature (UV-Tm) of the modified PNA against the complementary DNA was only slightly lower
than unmodified PNA.
the amino acid and the anomeric carbon of the sugar. Synthetic
Introduction
C-glycosyl amino acids and C-glycosylated PNA derivatives are
Peptide nucleic acid (PNA) is a DNA analog in which the
very interesting molecules because of the chemical and enzymatic
deoxyribose phosphate backbone of DNA is replaced with an
stability of the carbon–carbon linkage compared to ester- or
ethylenediamine glycine backbone that has very similar spatial
requirements.1,2 PNA can bind to DNA in a sequence-specific
amide-linked derivatives. We now report the synthesis of a new
class of C-glycosylated PNA monomers in which a C-bound
manner with a higher affinity compared to double-stranded DNA
glycosylated building block is integrated into a PNA monomer.
We prepared a C-galactosylated alanine derivative (Alagal) that
was used as a building block replacing glycine in the PNA
backbone. The new synthetic route described herein requires fewer
chemical synthesis steps and has a higher yield compared to the
previously reported synthesis of a C-glycosylated PNA monomer.9
Moreover, UV melting experiments with complementary DNA
indicate similar stability for oligomers containing the new C-
glycosylated PNA monomers compared to unmodified PNA
oligomers.
(dsDNA) itself. Several in vitro and in vivo studies have already
shown an antisense effect of PNAs that are targeted to specific
mRNA regions.3–6 PNA has a high stability towards cellular
nucleases and proteases. This, together with the apparent lack
of general toxicity and non-specific protein binding makes PNA
an attractive agent in the development of gene targeted drugs.7
The success in developing PNA as a gene targeted drug also
depends on its pharmacokinetic behaviour. Results from in vivo
studies of McMahon et al. indicate that unmodified PNAs are
rather poorly taken up and rapidly eliminated by the kidney
after intravenous injection in rats. Most of the PNA was excreted
unchanged in the urine after 24 hours.8 Due to this, PNA modi-
fications that lead to a more effective and specific biodistribution
have to be invented. It has been shown that pharmacokinetic
behaviour of PNA can be controlled by modification of PNA with
various glycosyl substituents (backbone glycosylation). Effective
liver targeting of glycosylated PNA has been reported due to
galactose and N-acetylgalactosamine interactions with a sialo-
glycoprotein receptor (hepatic lectin) of liver cells.9
Experimental procedures
Reagents and solvents were obtained from commercial sources and
used without further purification. PNA monomers were obtained
from Applied Biosystems, DNA oligomers (HPLC purified qual-
ity, checked in-house for purity and correct mass) were purchased
from IBA. Mass spectra were recorded on a Mat 8200 instrument
in FAB mode (positive ions, glycerol or 3-nitrobenzyl alcohol ma-
trix), ESI mass spectra on a Finnigan TSQ 700 and MALDI-TOF
mass spectra on a Bruker Biflex instrument. NMR spectra were
obtained on a Bruker AM 360 instrument. Flash chromatography
was carried out using Silica Gel 60 (Merck particle size 0.040–
0.063 mm). HPL chromatograms were measured on a customized
Varian Prostar instrument on reverse-phase Dynamax Microsorb
60-8 C18 columns. A linear gradient composed of A (0.1% TFA in
water) and B (0.1% TFA in acetonitrile) was used for analytical
and preparative HPLC. Analytical: Time 0–2 min 5% B. Time
30 min 50% B. Time 33 min 70% B. Time 40 min 5% B. Time
45 min 5% B (250 × 4.6 mm, 1 mL min−1). Preparative: Time
0–10 min 5% B. Time 40 min 60% B. Time 45 min 5% B (250 ×
10.0 mm, 5 mL min−1).
Glycopeptide synthesis strategies have been introduced as
models in such work either for monomer or for oligomer synthesis
of glycosylated PNAs. Glycosylation of peptides is accomplished
by direct attachment of a sugar to an amino acid through a
linker. The suitably protected monomer is then incorporated into
the peptide chain as a building block in solid phase peptide
synthesis (SPPS). The attachment usually links the a position of
aInstitute for Pharmacy and Molecular Biotechnology, University of Heidel-
berg, Im Neuenheimer Feld 364, 69120, Heidelberg, Germany
bDepartment of Chemistry, University of Bochum, Universita¨tsstrasse 150,
44801, Bochum, Germany. E-mail: nils.metzler-nolte@ruhr-uni-bochum.de;
Fax: +49 (0)234 32 14378
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N-(Boc)-3-(2,3,4,6-tetra-O-acetyl-galactopyranose-1-yl)-2-
aminopropionic acid benzyl ester (1)
169.7, 169.8, 169.9, 170.9, 173.9, 174.0; HRMS (M + H)+, calc.
(found) for C41H47N2O13: 775.3078 (775.3071).
Boc-protected (a,b-dehydroalanine) benzyl ester (2.5 g, 6.8 mmol)
and 2,3,4,5-tetra-O-acetyl-galactosyl bromide (2.0 g, 7.2 mmol)
were twice co-evaporated with toluene, dissolved in dry toluene
(20 mL) and heated to 60 ◦C under nitrogen. nBu3SnH (2.1 g,
11.7 mmol) and AIBN (150 mg, 0.9 mmol) in dry toluene
(12 mL) were added. The reaction mixture was stirred for 1 hour.
The solvent was evaporated and the residue was dissolved in
acetonitrile (100 mL) and extracted with n-pentane (3 × 50 mL) in
order to remove tin compounds. After evaporation of the solvents,
the residue was dissolved in ethyl acetate and extracted with
saturated aq. NaHCO3. Volatiles were removed and the residue
was purified by flash chromatography eluting with n-hexane–ethyl
acetate (2 : 1 v/v). Compound 1 was obtained as a white solid
(yield = 69.5%). MS (FAB) m/z 609 (M + H)+. 1H NMR (CDCl3):
d 1.4 (s, 9H, Boc), 2.0–2.2 (4s, 12H, sugar acetyls), 4.1 (m, 3H, H-5
and H-6), 4.2 (dd, 1H, J = 11.0, 5.5 Hz, H-1), 4.4 (m, 2H, b-
CH2), 4.5 (m, 1H, a-CH2), 5.1 (dd, 1H, J = 11.0, 3 Hz, H-3), 5.2
(overlapping, 3H, H-2 and -CH2 of benzyl), 5.4 (t, 1H, J = 3 Hz,
N-(2-Fmoc-aminoethyl)-N-(thymine-1-ylacetyl)-3-(2,3,4,6-tetra-
O-acetyl-galactopyranose-1-yl)-2-aminopropionic acid benzyl
ester (4)
Thymine-1-ylacetic acid (550 mg, 3 mmol), DhbtOH (520 mg,
3.2 mmol) and DCC (1.0 g, 5 mmol) were dissolved in DMF
(20 mL) and stirred for 20 minutes. Compound 3 (1.2 g, 1.5 mmol)
was added and the reaction mixture was stirred overnight. The
volatiles were removed under vacuum and the remaining solid was
dissolved in ethyl acetate (150 mL). Insoluble DCU was filtered
off and the filtrate was extracted with sat. aq. NaHCO3 (2 ×
50 mL) and brine (50 mL). After drying over MgSO4 the organic
phase was evaporated to dryness. The residue was purified by
flash chromatography eluting with ethyl acetate. Compound 4 was
obtained as a white solid. (yield = 64%), MS (FAB) m/z 941 (M +
H)+. 1H NMR (CDCl3): d 1.8 (m, 2H), 1.9 (s, 3H, thymine-CH3),
2.0–2.4 (4s, 12H, sugar acetyls), 2.7 (m, 1H), 3.4 (m, 2H), 3.6 (m,
1H), 3.9 (m, 1H), 4.0 (dd, 1H, J = 11.0, 5.5 Hz, H-1), 4.1–4.2 (m,
3H), 4.3–4.5 (m, 4H), 4.6 (m, 1H), 5.2 (overlapping, 4H, H-2, H-3
and -CH2 of benzyl), 5.4 (t, 1H, J = 3 Hz, H-4), 5.7 (br. s, 1H,
NHFmoc), 6.8 (s, 1H, thymine aromatic), 7.3–7.7 (m, 13H, benzyl
and Fmoc), 9.0 (br. s, 1H, NHthymine); 13C NMR (CDCl3): d 12.4,
20.5, 20.6, 20.7, 20.8, 24.9, 25.6, 33.9, 39.4, 47.2, 58.9, 61.2, 61.3,
66.6, 67.2, 67.3, 67.4, 67.6, 67.7, 67.8, 110.5, 120.0, 120.1, 125.0,
127.0, 127.1, 127.2, 127.7, 127.9, 128.4, 128.6, 128.7, 128.8, 128.9,
130.9, 141.1, 141.3, 164.1, 166.5, 166.6, 167.2, 167.7, 169.7, 169.8,
170.0; HRMS (M + H)+, calc. (found) for C48H5N4O16: 941.3457
(941.3465).
H-4), 5.5 (s, 1H, -NH-Fmoc), 7.4 (m, 5H, benzyl aromatics); 13
C
NMR (CDCl3): d 20.6, 20.7, 20.8, 20.9, 28.2, 28.3, 28.4, 42.7,
50.6, 62.0, 66.7, 67.1, 67.3, 67.7, 68.3, 71.5, 74.9, 80.3, 128.3,
128.5, 128.6, 128.7, 128.8; Elemental analysis for C29H39NO13·0.5
H2O: calc. C 56.30, H 6.52, N 2.26; found C 56.47, H 6.51,
N 2.21%.
N-(2-Fmoc-aminoethyl)-3-(2,3,4,6-tetra-O-acetyl-
galactopyranose-1-yl)-2-aminopropionic acid benzyl ester (3)
Compound 1 (3.5 g, 5.75 mmol) was added to a mixture of
water–TES–TFA (1 + 1 + 28 mL) at 0 ◦C and stirred for
1 hour. Toluene (50 mL) was added, and volatiles were removed
under vacuum. The remaining solid was dissolved in ethyl acetate
(100 mL) and extracted with saturated aq. NaHCO3 (2 × 50 mL)
and brine. After drying the organic phase with Na2SO4, all
volatiles were removed under vacuum and 2-(2,3,4,6-tetra-O-
acetyl-galactopyranosyl)alanine benzyl ester (2) was obtained as
a white solid (yield = 90%). MS (FAB) m/z 509 (M + H)+.
Without further purification, compound 2 (2.0 g, 3.9 mmol)
and Fmoc-aminoacetaldehyde (850 mg, 3.0 mmol) were dissolved
in methanol (5 mL) and stirred for 10 minutes. Acetic acid
(250 ll, 4 mmol) and NaBH3CN (250 mg, 3.0 mmol) were added
sequentially. The reaction mixture was stirred for 2 h. All volatiles
were removed under vacuum and the residue was dissolved in ethyl
acetate (100 mL) and extracted with sat. aq. NaHCO3 (2 × 50 mL).
The organic phase, after drying with MgSO4, was removed under
vacuum and the residue was purified by flash chromatography
eluting with ethyl acetate–n-hexane (3 : 2 v/v). Compound 3 was
obtained as a white solid. (yield = 38.5%). MS (FAB) m/z 775 (M
+ H)+;1H NMR (CDCl3): d 1.8 (m, 2H), 2.0–2.2 (4s, 12H, sugar
acetyls), 2.6 (m, 1H), 2.8 (m, 1H), 3.4 (m, 2H), 4.1 (m, 1H), 4.2 (m,
2H), 4.3 (m, 2H), 4.5 (m, 4H), 5.2 (overlapping, 4H, H-2 and H-3
and -CH2 of benzyl), 5.4 (s, 1H, H-4), 5.5 (broad s, 1H, -NHFmoc),
7.2–7.8 (m, 13H, benzyl and Fmoc). 13C NMR (CDCl3): d 20.6,
20.7, 20.8, 20.9, 29.2, 40.8, 47.2, 47.6, 57.9, 60.6, 66.2, 66.6, 66.8,
66.9, 67.7, 68.3, 68.4, 69.4, 119.9, 125.1, 125.2, 127.0, 127.6, 128.3,
128.5, 128.6, 128.8, 130.9, 135.5, 141.3, 144.0, 156.1, 156.5, 156.9,
N-(2-Fmoc-aminoethyl)-N-(thymine-1-ylacetyl)-3-(2,3,4,6-tetra-
O-acetyl-galactopyranose-1-yl)-2-aminopropionic acid (5)
Compound 4 (900 mg, 0.96 mmol) was dissolved in methanol
(20 mL) and a catalytic amount of palladium on charcoal
(Pd/C) was added. The reaction mixture was hydrogenated under
atmospheric pressure until TLC (ethyl acetate–n-hexane, 2 : 1
v/v) showed the absence of all starting material (approximately
2 h). The catalyst was filtered off with Celite and the filtrate was
evaporated to dryness. The residue was triturated with diethyl ether
until compound 5 precipitated as a white powder. (yield = 61.5%),
MS (FAB) m/z 629 (M + H)+. 1H NMR (CD3OD) resolved signals:
d 1.9 (s, 3H, thymine-CH3), 2.0–2.2 (4s, 12H, sugar acetyls), 2.6
(m, 1H), 3.5 (m, 2H), 3.7 (m, 1H), 4.1 (m, 2H), 4.3 (m, 3H), 4.5 (m,
2H), 4.7 (m, 2H), 5.3 (overlapping s, 2H, H-2 and H-3), 5.4 (s, 1H,
H-4), 7.3 (s, 1Hthymine), 7.4–7.9 (m, 8H, Fmoc); 13C NMR (CDCl3):
d 12.2, 20.5, 20.6, 20.8, 40.5, 62.6, 67.7, 69.0, 69.2, 110.9, 120.8,
120.9, 126.1, 126.3, 127.9, 128.1, 128.3, 128.8, 142.5, 142.6, 143.7,
143.8, 145.3, 171.3, 171.4, 171.5, 171.6, 171.8, 172.4; Elemental
analysis for C41H46N4O16·3H2O: calc. C 54.42, H 5.79, N 6.19;
found C 54.77, H 5.83, N 6.66%.
N-(Boc)-3-(2,3,4,6-tetra-O-acetyl-galactopyranose-1-yl)-2-
aminopropionic acid (6)
The same procedure for hydrogenation of compound 4 (de-
scribed above) was used for hydrogenation of compound 1
(1.0 g, 1.6 mmol). After removal of the catalyst, the filtrate was
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evaporated and the residue was precipitated from THF–n-hexane
(1 : 1 v/v). The precipitate was filtered off and dried in vacuum
(yield = 92%). MS (FAB) m/z 520 (M + H)+;1H NMR (CD3OD):
d 1.4 (s, 9H, Boc), 2.0–2.2 (4s, 12H, sugar acetyls), 4.2 (m, 2H),
4.3 (m, 3H), 4.4 (m, 1H, a-CH2), 5.1 (m, 1H), 5.3 (m, 2H),
5.4 (overlapping, 2H, H-4 and NHFmoc); 13C NMR (CDCl3): d
20.6, 20.7, 20.8, 20.9, 28.7, 28.8, 28.9, 39.3, 61.4, 62.0, 63.1, 67.4,
70.4, 72.9, 75.9, 80.4, 171.3, 171.7, 172.2; Elemental analysis for
C22H33NO13 calc. C 50.86, H 6.40, N 2.70; found C 50.75 H 6.84
N 2.76%.
Synthesis of PNA oligomer conjugates
50 mg of Tentagel R-RAM resin (capacity = 0.2 mmol g−1)
with preloaded Fmoc-Lys(Boc) amino acid was used as solid
support for the solid phase synthesis. A Teflon syringe with
a frit at the bottom was used as reactor and all reactions
were carried out on a shaker at 600 vibrations per minute.
Fmoc/Bhoc protected PNA monomers (Applied Biosystems) and
galactosylated derivatives (40 lmol, 4-fold access) were activated
in an Eppendorf tube with TBTU (12.2 mg, 38 lmol dissolved
in 270 lL DMF). DIPEA (30 lL, 4.7 eq.) was added and the
acid pre-activations were performed by vibration for 2 minutes (7
minutes for C(Bhoc) monomer). Activated acids were transferred
to the syringe containing the resin and the coupling was allowed to
proceed for 40 minutes (120 minutes for glycosylated derivatives)
under vibration. The resin was washed with DMF and DCM
successively. Success of the coupling reactions was controlled by
the Kaiser test. Piperidine (20%) in DMF (3 + 2 min) was used for
double Fmoc deprotection. Prior to cleavage of the final product
from the resin, acetyl groups at the galactosyl moieties were cleaved
by vibration for 24 hours with 1 mL of a mixture of TEA–
methanol–water (5 : 3 : 2 v/v). The oligomer was cleaved from the
resin with 700 lL of a mixture of TFA–anisol–m-cresol (14 : 5 : 1
v/v) for 2 hours. The crude product was precipitated by draining
the cleavage mixture into ice-cold diethyl ether. The precipitate
was washed with cold diethyl ether (5 × 10 mL), centrifuged each
time and collected and purified by preparative RP-HPLC. The
molecular mass was determined by MALDI-TOF. m/z 3513.7 (M
+ H)+, m/z 3536.6 (M + H + Na)+.
Scheme 1 Synthesis of the C-linked glycosylated PNA monomer. (a)
2,3,4,5-tetra-O-acetyl-galactosyl bromide, nBu3SnH, AIBN, 60 ◦C; (b)
H2O–TES–TFA, 1 : 1 : 28 v/v/v; (c) Fmoc-aminoacetaldehyde, acetic
acid, NaBH3CN, 2 h; (d) thymine-1-ylacetic acid, DhbtOH, DCC; (e) H2,
Pd/C in MeOH, 1 atm.
that paper).10 The glycosylated Ala derivative could be obtained
in a higher yield (69.5%). As a radical donor, tetra-O-acetyl-
galactosyl bromide was reacted with a protected dehydroalanine
derivative in the presence of AIBN as radical initiator and
nBu3SnH as a hydrogen donor. This reaction represents the key
step in the preparation of the C-linked glycosylated derivatives
since it cleanly generates the C–C bond between sugar and
PNA. The Boc protecting group of compound 1 was removed
by TFA and a scavenger for the subsequent formation of the
PNA backbone. The deprotection product 2 was used freshly
for the reductive amination step with Fmoc-aminoacetaldehyde9
without further purification. NaBH3CN and acetic acid were
used as reducing agents and glycosylated PNA backbone 3 was
obtained in good yield. The nucleobase thymine was introduced
to the backbone via amide bond formation between thymine-1-
ylacetic acid3 and the secondary amino group of 3. DCC and
DhbtOH were used as coupling reagents to yield compound 4 in
good yield. Finally, the benzyl groups of compounds 1 and 4 were
removed by catalytic hydrogenation using Pd/C at atmospheric
pressure. The glycosylated monomer 5 (Fmoc-T(O-Ac)gal)-OH) and
the glycosylated amino acid 6 (Boc-Ala(O-Ac)gal)-OH) were obtained
as white powders after trituration with ether and did not require
further purification. Derivatives 5 and 6 were incorporated into
a decamer PNA sequence (H-Alagal-G-G-G-Tgal-C-A-G-C-Tgal-T-
Lys-NH2) by Fmoc solid phase chemistry (Scheme 2). A Lys(Boc)-
Fmoc preloaded Tentagel resin with RAM linker was used as the
solid support. The couplings were carried out in a syringe using a
TBTU–DIPEA mixture as coupling reagents. All coupling steps
were monitored by the Kaiser test. By using an optimized synthetic
protocol, capping reactions could be avoided. Acetyl protecting
groups on sugars were removed by a TEA–methanol–water
UV-Tm measurements
The UV-Tm values were measured in a phosphate buffer (10 mM,
pH 7.0, containing 100 mM NaCl) using a DNA decamer with
complementary sequence. Heating and cooling were performed
◦
at 0.5 C min−1. between 5 and 80 ◦C. The mixture was held at
80 ◦C for 5 min to ensure complete dissociation prior to starting
◦
the measurement at 5 C. Temperature profiles were recorded at
260 nm and all profiles showed monophasing transitions from
which the Tm was determined as the maximum of the first
derivative.
Results and discussion
The monomer synthesis is outlined in Scheme 1. Orthogonal
protection groups for the synthesis of C-galactosylated PNA
are Fmoc, acetyl and benzyl for amine, hydroxyl and carboxylic
acid groups, respectively. Initially, compound 1 was synthesized
as described in the literature (analytical data are not given in
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derivatives, which are also studied in our group,13–16 glycosylated
PNA oligomers may enhance the applications of this promising
new class of antisense agents.7,17,18
Abbreviations
ꢀ
AIBN, a,a -azobisisobutyronitrile; Bhoc: benzhydryloxycar-
bonyl; Boc: tert-butyloxycarbonyl; DCC: N,Nꢀ-dicyclohexylcar-
bodiimide; DIPEA: diisopropylethylamine; DCU: N,Nꢀ-dicyclo-
hexylurea; DhbtOH: 3-hydroxy-1,2,3-benzotriazine-4(3H)-one;
ESI: electrospray ionization; FAB: fast atom bombard-
ment; Fmoc: 9-fluorenylmethoxycarbonyl; MALDI-TOF: matrix-
assisted laser desorption ionization time-of-flight; TBTU: 2-(1H-
benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate;
TLC: thin layer chromatography.
Scheme 2 Introduction of the glycosylated derivatives into a PNA
oligomer. See Experimental section for details.
Acknowledgements
mixture prior to cleavage of the final product from the resin. The
crude product was further purified by preparative RP-HPLC and
the molecular mass was confirmed by MALDI-TOF.
The authors are grateful to H. Rudy (MS and elemental analysis)
and T. Zimmermann (NMR) for technical assistance. Financial
support from the Fond der Chemischen Industrie and the Max-
Buchner-Foundation is gratefully acknowledged.
In order to probe the interaction with complementary DNA,
UV melting temperatures (UV-Tm) were measured.11,12 For com-
parison, a non-glycosylated PNA oligomer with the same sequence
(H-Ala-G-G-G-T-C-A-G-C-T-T-Lys-NH2) was prepared from
commercially available monomers. Tm values were recorded twice
for glycosylated and control PNA. Only a slight decrease of the
References
1 P. E. Nielsen, Pure Appl. Chem., 1998, 70, 105.
2 A. Ray and B. Norden, FASEB J., 2000, 14, 1041.
3 P. E. Nielsen, M. Egholm, R. H. Berg and O. Burchardt, Science, 1991,
254, 1497.
4 G. Wang and X. S. Xu, Cell Res., 2004, 14, 111.
5 H. J. Larsen and P. E. Nielsen, Nucleic Acids Res., 1996, 24, 458.
6 H. Knudsen and P. E. Nielsen, Nucleic Acids Res., 1996, 24, 494.
7 L. Good and P. E. Nielsen, Antisense Nucleic Acid Drug Dev., 1997, 7,
431.
T
m value from (76 0.5) ◦C to (73 0.5) ◦C was observed due to
the sugar modifications.
Conclusions
The synthesis of a C-linked glycosylated thymine-based PNA
monomer and its incorporation into a PNA oligomer has been
achieved in this work. A number of O-, C- and N-glycosylated
PNA monomers were recently reported by Hamzavi et al.9 In this
work, it was shown that biologically and chemically very stable
glycosylated PNA derivatives are obtained in the case of a carbon–
carbon linkage between the sugar and the backbone. The number
of synthetic steps for preparation of the new monomer described
herein are fewer than for the previously reported analogues and the
total yield is higher. By using optimized SPPS methods for PNA
oligomer synthesis, the glycosylated monomer can be incorporated
into a PNA oligomer in combination with a glycosylated alanine
derivative which was attached to the PNA N-terminus. We
observed that incorporation of both derivatives into a PNA
decamer did not hamper binding to complementary DNA. On the
contrary, glycosylated PNA oligomers are more soluble in water
compared to normal PNA oligomers. This constitutes a significant
advantage in biological application. In addition to metal–PNA
8 B. M. McMahon, D. Mays, J. Lipsky, J. A. Stewart, A. Fauq and E.
Richelson, Antisense Nucleic Acid Drug Dev., 2002, 12, 65.
9 R. Hamzavi, F. Dolle, B. Tavitian, O. Dahl and P. E. Nielsen,
Bioconjugate Chem., 2003, 14, 941.
10 H. Kessler, V. Wittmann, M. Ko¨ck and M. Kottenhahn, Angew. Chem.,
1992, 104, 874.
11 A. Holme´n and B. Norde´n, in Peptide Nucleic Acids, ed. P. E. Nielsen
and M. Egholm, Horizon Scientific Press, Wymondham, 1999, pp. 87–
97.
12 T. Ratilainen, A. Holme´n, E. Tuite, G. Haaima, L. Christensen, P. E.
Nielsen and B. Norde´n, Biochemistry, 1998, 37, 12331.
13 J. C. Verheijen, G. A. van der Marel, J. H. van Boom and N. Metzler-
Nolte, Bioconjugate Chem., 2000, 11, 741.
14 A. Maurer, H.-B. Kraatz and N. Metzler-Nolte, Eur. J. Inorg. Chem.,
2005, 3207.
15 R. Hamzavi, T. Happ, K. Weitershaus and N. Metzler-Nolte,
J. Organomet. Chem., 2004, 689, 4745.
16 N. Metzler-Nolte, in Bioorganometallics, ed. G. Jaouen, Wiley-VCH,
Weinheim, 2006, pp. 125–179.
17 N. Metzler-Nolte, in Molekulare Biotechnologie, ed. M. Wink, Wiley-
VCH, Weinheim, 2004, pp. 579–593.
18 A. M. Gewirtz, D. L. Sokol and M. Z. Ratajczak, Blood, 1998, 92, 712.
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