The use of Sonogashira coupling for the synthesis of modified uracil peptide nucleic acid

Article abstract of DOI:10.1016/S0040-4039(02)00024-2

Palladium-catalyzed Sonogashira coupling has been shown to be compatible with PNA monomers as illustrated by the reaction of 5-iodouracil peptide nucleic acid monomer (^IU-PNA) with several terminal alkynes. These reactions have been performed in the solution phase and with ^IU-PNA linked to an insoluble polymer support. The results presented herein show that while the isolated yields from the solution phase chemistry are modest (38-53%), the yields of the resin-bound coupling reactions are essentially quantitative, at the monomer level. A selection of alkynes was used to install various additional functionality on the uracil nucleobase. Examples of a hydroxyl, protected thiol and protected amino group are given. Further, an example of derivatization of a resin-bound oligomer with a single ^IU insert is given.

Full text of DOI:10.1016/S0040-4039(02)00024-2

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 1381–1386
The use of Sonogashira coupling for the synthesis of modified
uracil peptide nucleic acid
Robert H. E. Hudson,* Ge Li and Joseph Tse
Department of Chemistry, The University of Western Ontario, London, Ontario, Canada N6A 5B7
Received 26 November 2001; revised 14 December 2001; accepted 18 December 2001
Abstract—Palladium-catalyzed Sonogashira coupling has been shown to be compatible with PNA monomers as illustrated by the
reaction of 5-iodouracil peptide nucleic acid monomer (IU-PNA) with several terminal alkynes. These reactions have been
I
performed in the solution phase and with U-PNA linked to an insoluble polymer support. The results presented herein show that
while the isolated yields from the solution phase chemistry are modest (38–53%), the yields of the resin-bound coupling reactions
are essentially quantitative, at the monomer level. A selection of alkynes was used to install various additional functionality on
the uracil nucleobase. Examples of a hydroxyl, protected thiol and protected amino group are given. Further, an example of
I
derivatization of a resin-bound oligomer with a single U insert is given. © 2002 Elsevier Science Ltd. All rights reserved.
Peptide nucleic acid (PNA) is an oligonucleotide analog
composed of nucleobase derivatives attached to a
polyamide backbone. Although PNA is structurally
quite different than DNA, the geometric constraint of
placing the pendant nucleobases along the polymer
backbone is the same ‘6+3’ bond arrangement found in
natural DNA, as shown below.1 It has been determined
that PNA may form sequence-specific complexes with
either single-stranded DNA or RNA.2 In addition,
PNA is able to bind double-stranded DNA by a strand-
displacement mechanism1,3 or by the more usual triple-
helix formation dependent on the sequence context.4,5
tics. For such applications, the oligomer is commonly
derivatized with a reporter group such as a radioisotope
or a fluorophore. It may also be important to use
labeled PNA to track cellular compartmentalization or
localization in studies that involve antisense-type or
ribonucleoprotein inhibition. To date, the radiolabeling
of PNA has been done via an appended peptide or
oligonucleotide.6,7 These methods have the potential
drawback that the reporter group comprises a substan-
tial portion of the molecule and may modify or govern
some of its properties and may be susceptible to degra-
dation by proteases or nucleases. Labeling of PNA with
smaller reporter groups has generally been done
through the N-terminus or C-terminus that has been
limited to only one or two labels thus far.8 We report
herein the development of a method for introducing
substituents to uracil peptide nucleic acid monomer
that may potentially be used to label or polylabel PNA.
An important feature in many molecular biological
techniques is the recognition of nucleic acid sequences
by oligonucleotides. Established techniques such as
FISH and quantitative PCR are beginning to make use
of PNA due to its improved hybridization characteris-
* Corresponding author. Fax: 519 661 3022; e-mail: robert.hudson@uwo.ca
0040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)00024-2

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R. H. E. Hudson et al. / Tetrahedron Letters 43 (2002) 1381–1386
For this work, we started with uracil. Under conditions
similar to Robins’,18 5-iodouracil was easily and reli-
ably prepared in high yield using iodine monochloride
in methanol from which the product crystallizes
(Scheme 1). The choice of solvent is crucial for the
success of this reaction, other common solvents for this
type of chemistry such as acetonitrile result in an
emulsion from which the product is isolated only with
difficulty and in lower yield. A one-step alkylation is
then accomplished using chloroacetic acid in aqueous
alkali. The moderate yield may be due to the instability
of the iodouracil in aqueous base19 or dialkylation,20
but it is rapid, convenient and is as efficient as the
traditional approach of alkylation with ethyl bromoac-
etate followed by hydrolysis.21 The (5-iodouracil-1-
yl)acetic acid was then condensed with ethyl
(N-Boc-aminoethyl)glycinate22 under standard condi-
tions to give cross-coupling substrate 2.23
The route is based on well-established coupling of
terminal alkynes with aryl iodides and has been
employed previously for the synthesis of modified
nucleosides.9,10 Incorporation of a label to the nucleo-
base avoids the introduction of a stereocenter, as is the
case for labeling along the polyamide backbone, for
instance by use of an amino acid other than glycine.
Importantly, derivatization at the C5 position of uracil
does not modify the Watson–Crick base pairing face of
the nucleobase and a propynyl-substituent at this posi-
tion may stabilize duplex formation, as is seen for
DNA.11
As with base modified nucleosides, we envision a rich
area of PNA chemistry to be accessed by developing
this strategy. For instance, internal derivatization of an
oligomer is a route to branched or dendrimeric struc-
tures,12 which have not yet been reported for PNA. An
internal thiol could be used to form bis-PNA type
structures from homo- or hetero-oxidative crosslinking
with a second thiol labeled PNA oligomer. PNA func-
tionalized with a thiol group may be used to form an
intramolecular crosslink resulting in bent PNA, or to
bend PNA:NA hybrid duplexes.13 The hydroxypropy-
nyl or aminopropynyl PNAs that are possible by exten-
sion of this method may have improved solubility14 or
other interesting properties, especially the cationic
aminopropynyl derivative.15 Internal labeling of PNA
also leaves both the C- and N-terminus available for
other forms of derivatization, most notably the possi-
bility of intramolecular cyclization to form cyclic
bisPNA molecules.16 The incorporation of labels inter-
nal to the oligomer or at the terminal residues or in
conjunction with appended labels on the C-or N-termi-
nus may be used to construct new types of PNA-based
molecular beacons.17 Internal labeling of PNA
oligomers also gives rise to the possibility of placing
labels with well-defined spacing along the polymer
backbone. Finally, internal modification of PNA
oligomers could conceivably modulate their antisense/
antigene efficacy and the potency of interruption of
nucleic acid processing enzymes based on steric grounds
and merits investigation.
The cross-coupling with all of the alkynes was accom-
plished under standard conditions with only a variation
in the reaction times.24 The alkynes, conditions and
yields are illustrated in Table 1. Alkyne a, propargyl
alcohol, is compatible with the reaction conditions and
the hydroxyl group need not be protected. It is envis-
aged that this alkyne may be used to prepare a hydrox-
ylated PNA oligo that may have improved solubility
characteristics. 2-Propynylmercaptan was masked as
the p-methoxybenzylthioether (alkyne b), a standard
protecting group compatible with a graded acidolysis
deprotection scheme.25 Incorporation of a thiol group
into a PNA represents a unique chemically addressable
group and may be used as a site for conjugation to
thiol-reactive probes or dyes. Alkynes c and d are both
derivatives of propargylamine in which an electroactive
reporter group26 or photolabile protecting group were
used, respectively. The coupling reactions (Scheme 1)
and subsequent hydrolysis proceeded with chemical
yields given in Table 1. The modest overall chemical
yields are due in part to difficulties encountered in the
isolation and chromatographic purification of these
compounds. However, NMR and HPLC analysis of the
purified products indicated that they were homoge-
neous and they gave satisfactory spectroscopic
characterization.27–30
Scheme 1. General scheme for the synthesis of 5-derivatized uracil peptide nucleic acid monomers. Reagents and conditions (i) ICl,
MeOH, 50°C, 4 h, 98%; (ii) ClCH2CO2H, aq. KOH, D, 1 h, 62%; (iii) DCC, HOBt, BOCNH(CH2)2NHCH2CO2Et, 1:1
DCM:DMF, 73%; (iv) R%CH2CCH, Pd(PPh3)4, CuI, NEt3, DMF 3–16 h; (v) 1 M NaOH/THF, 45 min.

R. H. E. Hudson et al. / Tetrahedron Letters 43 (2002) 1381–1386
Table 1. Alkyne derivatives used for coupling to 5-iodouracil PNA monomer
1383
Next, we investigated the conversion of the iodouracil
PNA whilst attached to solid support. We choose to
attach to the 5-iodouracil PNA monomer to HMBA-AM
resin,31 a linker that is cleaved under basic conditions to
yield C-terminal acids.32 The products in the cleavage
mixture could then be directly compared to the com-
pounds synthesized by solution methods. Fig. 1 illus-
trates comparison of the crude product mixtures taken
directly from resin treated with CsOH, to the standard
RP-HPLC (Fig. 2, panel a: crude oligomer; panel b:
purified oligomer). A portion of the same resin was then
treated to the usual coupling conditions with propargyl
alcohol (PA) as the alkyne component. Panel c and d are
the chromatograms for the crude and purified H2N-lys-
TPAUTCCTT-lys-CO2H36 (6). Although it is difficult to
gauge the chemical yield of cross-coupling reaction it is
evident that it proceeds to give the major product. Both
of these oligomers were examined for their capacity to
bind to complementary DNA. Under the given ionic
conditions (10 mM PO43−, 100 mM NaCl, 0.1 mM
EDTA, pH 7) the DNA:DNA duplex did not give a
measurable Tm (<10°C) and the unmodified PNA:DNA
duplex melted at 47.5°C. Examination of the Tm for both
the 5-iodouracil-containing PNA oligomer (Tm=46.0°C)
and the propargyl alcohol modified uracil PNA oligomer
(Tm=47.0°C) indicate that these substitutions are well
tolerated.
I
compounds. Panel a illustrates that U-PNA can be
loaded onto the resin and directly cleaved without
modification as shown by coinjection with authentic
material. Panels b–d are for the final products whose
structures are shown inset. Of note, each of these
examples shows the complete absence of IU-PNA
monomer indicating complete conversion to the cross-
coupled product.
It was clear that the coupling conditions were effective
for derivatization of a PNA monomer on the support,33
but arguably a more useful approach is the derivatization
in the context of an oligomer. For this experiment, a
7-mer PNA of the sequence H2N-lys-TIUTCCTT-lys-
CO2H34 (5) containing a single IU-PNA insert was
In summary, we have demonstrated that both solution-
phase and solid-phase Sonogashira coupling is a feasible
route to 5-derivatized uracil-PNA residues. As expected,
this reaction is tolerant to a variety of alkynes. Impor-
tantly, the cross-coupling reaction also works in the
context of an oligomer. We are currently examining the
scope and utility of this method for the synthesis of
nucleobase-modified PNA.
prepared by manual solid-phase peptide synthesis.35
A
portion of the IU-PNA containing oligomer was cleaved
under standard TFMSA conditions and analyzed by

1384
R. H. E. Hudson et al. / Tetrahedron Letters 43 (2002) 1381–1386
Figure 1. RP HPLC analysis of the solid-phase derivatization of 5-iodouracil PNA monomer. (i) solution: authentic sample prepared
by solution-phase methods, (ii) polymer: crude mixture direct from cleavage of polymer-supported reaction, (iii) coinjection:
approximately an equimolar mixture of (i) and (ii). Panel a: 5-iodouracil PNA monomer. Coupling with alkyne component - Panel
,
b: 4a; Panel c; 4b; Panel d: 4c. Conditions: reversed-phase HPLC (C18, Rainin Microsorb-MV, 100 A) and a gradient composed
of A (aqueous H3PO4, pH 2.00) and B (acetonitrile) at a flow rate 1.00 mL/min. Gradient: initial conditions - 90% A, 10% B for
one minute then a linear gradient to 35% A, 65% B over 9 min followed by a linear gradient to 100% B over the next 3 min.
Figure 2. HPLC analysis of the solid-phase derivatization of 5-iodouracil within a 7-mer PNA. Panel a, b: oligomer-H2N-lys-
TIUTCCTT-lys-CO2H (5), crude and purified, respectively. Panel c, d: oligomer-H2N-lys-TPAUTCCTT-lys-CO2H (6), crude and
purified, respectively. Note: different elution conditions were used, thus retention times, shown inset, cannot be reliably compared.
,
Analytical RP HPLC (C18, Rainin Microsorb-MV, 100 A, solvent A: 0.1% TFA in water; solvent B: 0.1% TFA in acetonitrile)
conditions for the analysis of oligomer 5: 1 min. 100% A, linear gradient over 45 min to 30% B followed by a gradient to 100%
B over the next 2 min. Analysis of 6: 1 minute 100% A, linear gradient over 30 min to 35% B followed by a gradient to 100%
B over the next 2 min, at 50°C.

R. H. E. Hudson et al. / Tetrahedron Letters 43 (2002) 1381–1386
1385
Acknowledgements
18. Robins, M. J.; Barr, P. J.; Giziewicz, J. Can. J. Chem.
1982, 60, 554–557.
19. We have not attempted to identify any by-products.
Ueda, T. In Chemistry of Nucleosides and Nucleotides;
Townsend, L. B., Ed.; Plenum Press: New York, USA,
1988; Vol. 1, pp. 33–37.
20. Dallaire, C.; Arya, P. Tetrahedron Lett. 1998, 39, 5129–
5132.
21. In our hands, the alkylation of 5-iodouracil with ethyl
bromoacetate and K2CO3 in DMF and subsequent
hydrolysis gave the free acid in approximately 60% yield
(isolated).
We gratefully acknowledge the Natural Sciences and
Engineering Research Council of Canada (NSERC),
the Canadian Foundation for Innovation and the Uni-
versity of Western Ontario for financial support of this
project. The Faculty of Graduate Studies is thanked for
a Special University Scholarship (G.L., J.T.) as well as
NSERC and the Ontario Graduate Scholarship Pro-
grams (J.T.) Thanks to Dr. C. Kirby (UWO) for assis-
tance with NMR spectroscopic studies and the CFI for
infrastructure support. Professor M. J. Damha of
McGill University and Antisar Hlil of the McGill Uni-
versity Mass Spectrometry Facility are thanked for
assistance with these studies. We also thank an anony-
mous referee for valuable comments.
22. (a) Ethyl (N-Boc-aminoethyl)glycinate was prepared by
the alkylation of ethyl bromoacetate with 2-N-tert-
butoxycarbonylaminoethylamine as reported: Meltzer, P.
C.; Liang, A. Y.; Matsuidaira, P. J. Org. Chem. 1995, 60,
4305–4308; (b) Preparation of 2-N-tert-butoxycarbonyl-
aminoethylamine followed the method of: Krapcho, A.
P.; Kuell, C. S. Synth. Commun. 1990, 20, 2559–2564.
23. Ethyl N-(2-Boc-aminoethyl)-N-(5-iodouracil-1-yl) glyci-
nate (2): 1H NMR (400 MHz, (CD3)2CO, major (ma.)
and minor (mi.) rotamer signals) l 7.96 (ma.) and 7.94
(mi.) (s, 1H), 6.25 (ma.) and 5.97 (mi.) (s, 1H), 4.86 (ma.)
and 4.68 (mi.) (s, 2H), 4.37 (mi.) and 4.12 (ma.) (s, 2H),
4.12 (q, 2H, J=7.7 Hz), 3.58 (ma.) and 3.50 (mi.) (t, 2H,
J=6.0 Hz), 3.36 (ma. and 3.21 (mi.) (m, 2H), 1.41 (ma.)
and 1.39 (mi.) (s, 9H), 1.22 (t, 3H, J=7.0 Hz). HR-MS
(FAB) calcd for C17H25IN4O7, 524.0768, found: 523.9984.
24. A typical procedure for the cross-coupling reaction and
subsequent purification follows: To a solution of com-
pound ethyl N-(2-Boc-aminoethyl)-N-(5-iodouracil-1-yl)
glycinate 2 (1 equiv.), alkyne (3 equiv.), dry triethylamine
(2 equiv.) in 5 mL of dry DMF were added tetra-
kis(triphenylphosphine) palladium (0) (0.1 equiv.) and
copper(I) iodide (0.3 equiv.). The resulting mixture was
stirred at room temperature for the indicated time and
the reaction was quenched by the addition of 7 mL of
cold water. The crude product was isolated by extraction
into EtOAc (3×10 mL). The combined organic layers
were washed with water (2×10 mL), (brine 10 mL) and
then dried over Na2SO4. The desired product was isolated
by silica gel column chromatography (typically 5–10%
MeOH in DCM). The alkynes used were: propragyl
alcohol (Aldrich, used as received); methyl 4-[(2-propy-
nylthio)methyl] phenyl ether, 1H NMR (400 MHz,
CDCl3) l 7.27 (d, 1H, J=8.8 Hz), 6.88 (d, 1H, J=8.4
Hz), 3.83 (s, 2H), 3.76 (s, 3H), 3.14 (d , 2H, J=2.8 Hz),
2.74 (t, 1H, J=2.4 Hz), HRMS calcd for C11H12OS:
192.0609, found: 192.0615; 3-(ferrocenyl carbonyl)amino-
1-propyne, 1H NMR (200 MHz, CDCl3) l 5.80 (br s,
1H), 4.70 (s, 2H), 4.38 (s, 2H), 4.23 (s, 5H), 4.17 (s, 2H),
2.27 (t, 1H, J=2.0 Hz) HRMS calcd for C14H13ONFe:
267.0346, found: 267.0347; 4, 5-dimethoxy-2-nitrobenzyl
2-propynylcarbamate, 1H NMR (400 MHz, CDCl3)
l7.65 (s, 1H), 6.92 (s, 1H), 5.47 (s, 2H), 5.05 (b, 1H), 3.96
(q, 2H, J=1.6 Hz), 3.92 (s, 3H), 3.89 (s, 3H), 2.20 (t, 1H,
J=2.4 Hz). MS (EI) m/z 294 (M+).
References
1. Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O.
Science 1991, 254, 1497–1500.
2. Egholm, M.; Buchardt, O.; Christensen, L.; Behrens, C.;
Freier, S. M.; Driver, D. A.; Berg, R. H.; Kim, S. K.;
Norden, B.; Nielsen, P. E. Nature 1993, 365, 566–568.
3. Nielsen, P. E.; Egholm, M.; Buchardt, O. J. Mol. Recog-
nit. 1994, 7, 165–170.
4. Wittung, P.; Nielsen, P. E.; Norden, B. Biochemistry
1997, 36, 7973–7979.
5. The chemistry and properties of PNA has been reviewed
recently and appears in Peptide Nucleic Acids Protocols
and Applications; Nielsen, P. E., Egholm, M., Eds.; Hori-
zon Scientific Press: Wymnodham, England, 1999.
6. Kozlov, I. A.; Nielsen, P. E.; Orgel, L. E. Bioconjugate
Chem. 1998, 9, 415–417.
7. Uhlmann, E. Biol. Chem. 1998, 379, 1045–1052.
8. A recent example: Hess, A.; Metzler-Nolte, N. Chem.
Commun. 1999, 885–886 and Ref. 5.
9. Sonogashira, K.; Tohda, Y.; Hagihara, N. Tetrahedron
Lett. 1975, 4467–4470.
10. Hobbs, F. W., Jr. J. Org. Chem. 1989, 54, 3420–3422.
11. Froehler, B. C.; Wadwani, S.; Trehorst, T. J.; Gerrard, S.
R. Tetrahedron Lett. 1992, 33, 5307–5310.
12. Comb-type branched nucleics have been synthesized by
branching from the nucleobase, for example: Horn, T.;
Urdea, M. S. Nucleic Acids Res. 1989, 17, 6959–6967.
13. Glick, G. D. Biopolymers 1988, 48, 83–96 and references
cited therein.
14. An experimental observation is that the C-5 (3-hydroxy-
propynyl) derivative PNA monomer is a deliquescent
solid, unlike thymine PNA monomer and is easily dis-
solved in water.
15. (a) Strauss, J. K.; Roberts, C.; Nelson, M. G.; Switzer,
C.; Maher, L. J., III. Proc. Natl. Acad. Sci. USA 1996,
93, 9515–9520; (b) Dande, P.; Liang, G.; Chen, F.-X.;
Roberts, C.; Nelson, M. G.; Hashimoto, H.; Switzer, C.;
Gold, B. Biochemistry 1997, 36, 6024–6032.
25. Barany, G.; Merrifield, R. B. In The Peptides Analysis,
Synthesis and Biology; Gross, E.; Meienhofer, J., Eds.;
Academic Press: New York, USA, 1980; Vol. 2, pp.
233–240.
26. A recent report highlighting an unexpected modification
to a uridine base under cross-coupling conditions with
16. Cyclic oligonucleotides have been extensively studied and
reviewed by: Kool, E. J. Chem. Rev. 1997, 1473–1487.
17. Tyagi, S.; Kramer, F. R. Nature Biotech. 1996, 14, 303–
308.

1386
R. H. E. Hudson et al. / Tetrahedron Letters 43 (2002) 1381–1386
ferrocenylacetylene (Yu, C. J. et al. J. Am. Chem. Soc.
2000, 122, 6767–6768.) was not found to occur with
alkyne c, nor a, b and d.
(mi.) and 4.08 (ma.) (s, 2H), 4.08 (s, 2H), 3.98 (s, 2H),
3.97 (s, 6H), 3.59 (ma.) and 3.54 (mi.) (m, 2H), 3.29 (ma.)
and 3.24 (mi.) (m, 2H), 1.41 (s, 9H). MS (MALDI TOF)
calcd for C28H34N6O13 662.602, found: 662.6.
27. N-(2-Boc-aminoethyl)-N-{[5-(3-hydroxy-1-propynyl)-
uracil-1-yl]acetyl} glycine (4a).
31. HMBA-AM resin (HMBA-AM: p-hydroxymethylbenzoic
acid aminomethyl Merrifield resin) was obtained from
NovaBiochem (La Jolla, CA, USA). Conditions used for
1H NMR (400 MHz, basic D2O, major (ma.) and minor
(mi.) rotamer signals): l 7.78 (ma.) and 7.77 (mi.) (s, 1H),
4.75 (ma.) and 4.59 (mi.) (s, 2H), 4.32 (s, 2H), 3.93 (ma.)
and 3.86 (mi.) (s, 2H), 3.63 (mi.) and 3.40 (ma.) (m, 2H),
3.27 (mi.) and 3.14 (ma.) (m, 2H), 1.34 (s, 9H). MS
(MALDI TOF) calcd for C18H24N4O3 424.405, found:
424.5.
I
loading U-PNA: HMBA-AM resin (421 mg, 0.43 mmol)
was swollen with 2 mL of dry DMF for 30 min. After
draining the solvent, a solution containing compound 2a
(645 mg, 1.30 mmol), HBTU (411 mg, 1.08 mmol),
DIPEA (153 mg, 1.30 mmol) and catalytic amount of
DMAP dissolved in 2 mL of dry DMF was added to the
resin. The resulting mixture was stirred at room tempera-
ture for 24 h. The excess reagents were drained off and
the resin was washed alternating with 3×2 mL of DCM,
3×2 mL of MeOH.
28. N-(2-Boc-aminoethyl)-N-[5-{3-[(4-methoxybenzyl)thio]-1-
propynyl}uracil-1-yl]acetyl glycine (4b).
1H NMR (400 MHz, (CD3)2CO) l 10.25 (s, 1H), 7.64
(ma.) and 7.63 (mi.) (s, 1H), 7.21 (d, 2H, J=8.8 Hz), 6.75
(d, 2H, J=8.8 Hz), 6.15 (ma.) and 5.88 (mi.) (br s, 1H),
4.73 (ma.) and 4.58 (mi.) (s, 2H), 4.25 (mi.) and 4.03
(ma.) (s, 2H), 3.76 (s, 2H), 3.65 (s, 3H), 3.47 (ma.) and
3.40 (mi.) (t, 2H, J=6.4 Hz), 3.26 (ma.) and 3.11 (mi.)
(m, 2H), 3.17 (s, 2H), 1.29 (ma.) and 1.27 (mi.) (s, 9H).
MS (MALDI TOF) calcd for C26H32N4O8S 560.620,
found: 562.4.
32. Cleavage was effected by a brief treatment of the resin
(50 mg) with 1 mL of a solution of the composition: 1
mL of 50% aqueous CsOH solution, 1 mL MeOH, 10 mL
water and 11 mL THF.
I
33. 50 mg of the U-PNA derivatized HMBA-AM resin was
swollen with 0.5 mL of dry DMF for 30 min. After
draining the solvent, a solution of copper(I) iodide (0.2
equiv.) and dry triethylamine (2 equiv.), alkyne (3 equiv.),
tetrakis(triphenylphosphine) palladium (0.1 equiv.) in 0.5
mL of dry DMF were added in that order. The mixture
was then agitated for 16 h after which the excess reagents
were washed off with 3×1 mL of DCM, 3×1 mL of
MeOH alternatively. The resin was then dried in air.
34. MALDI-TOF, C86H119N34O29I , calcd: 2220 amu, found:
2224 amu.
35. Boc-lys 2-Cl-Z PAM resin is available from Nova-
Biochem. The oligomerization chemistry follows that of:
Koch, T. In Peptide Nucleic Acids: Protocols and Applica-
tions; Nielsen, P. E.; Egholm, M., Eds., Horizon Scien-
tific Press: UK, 1999; Chapter 2.1.
29. N-(2-Boc-aminoethyl)-N-{5-[3-(ferrocenecarbonyl)amino-
1-propynyl]uracil-1-yl}-acetyl glycine (4c).
1H NMR (400 MHz, (CD3)2CO) l 10.32 (s, 1H), 7.74
(ma.) and 7.71 (mi.) (s, 1H), 7.56 (br s, 1H), 6.28 (ma.)
and 6.00 (mi.) (br s, 1H), 4.84 (s, 2H), 4.84 (ma.) and 4.74
(mi.) (s, 2H), 4.35 (d, 2H, J=1.8 Hz), 4.44 (mi.) and 4.22
(ma.) (s, 2H), 4.27 (d, 2H, J=2.3 Hz), 4.21 (s, 5H), 4.15
(ma.) and 4.27 (mi.) (s, 2H), 3.58 (ma.) and 3.51 (mi.) (t,
2H, J=5.8 Hz), 3.38 (ma.) and 3.22 (mi.) (m, 2H), 1.42
(ma.) and 1.40 (mi.) (s, 9H). MS (MALDI TOF) calcd
for C29H33FeN5O8 635.446, found: 635.3.
30. N-(2-Boc-aminoethyl)-N-{5-[3-([(4,5-dimethoxy-2-nitro-
benzyl)oxycarbonyl]amino)-1-propynyl]-uracil-1-yl}acetyl
glycine (4d).
1H NMR (400 MHz, (CD3)2CO) l 10.39 (s, 1H), 7.78 (s,
1H), 7.74 (s, 1H), 7.22 (s, 1H), 7.12 (br s, 1H), 6.28 (br s,
1H), 5.48 (s, 2H), 4.88 (ma.) and 4.73 (mi.) (s, 2H), 4.39
36. MALDI-TOF, C89H122N34O30 , calcd: 2148 amu, found:
2189 amu (K+ adduct).