Dde-protected PNA monomers, orthogonal to Fmoc, for the synthesis of PNA-peptide conjugates

Article abstract of DOI:10.1016/j.tet.2005.06.003

Peptide nucleic acids have become, arguably, one of the most interesting of DNA mimics. Herein the efficient solution phase synthesis of four novel 1-(4,4-dimethyl-2,6-dioxacyclohexylidene)ethyl/4-methoxytrityl (Dde/Mmt) protected PNA monomers is reported

Full text of DOI:10.1016/j.tet.2005.06.003

Tetrahedron 61 (2005) 8295–8305
Dde-protected PNA monomers, orthogonal to Fmoc, for the
synthesis of PNA–peptide conjugates
*
´ ´ ´
Laurent Bialy, Juan Jose Dıaz-Mochon, Edgar Specker, Lise Keinicke and Mark Bradley
School of Chemistry, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, UK
Received 1 April 2005; revised 17 May 2005; accepted 2 June 2005
Available online 7 July 2005
Abstract—Peptide nucleic acids have become, arguably, one of the most interesting of DNA mimics. Herein the efficient solution phase
synthesis of four novel 1-(4,4-dimethyl-2,6-dioxacyclohexylidene)ethyl/4-methoxytrityl (Dde/Mmt) protected PNA monomers is reported
which were then used to synthesise PNA–peptide conjugates through a mild Dde deprotection strategy, which was fully orthogonal to Fmoc
chemistry, allowing at will Fmoc peptide and Dde–PNA synthesis.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
hybridises efficiently to complementary DNA and RNA
sequences according standard base pairing rules (Fig. 1);
indeed, due to the lack of electrostatic repulsion in PNA/
DNA complexes present in DNA/DNA or DNA/RNA
double strands, the binding affinity and selectivity of PNA
towards DNA, RNA and PNA is higher than for its DNA
counterparts under physiological conditions. Moreover,
PNA is resistant to biological degradation by nucleases or
proteases, properties which make PNA an ideal tool in a
Peptide Nucleic Acids (PNAs) were first reported in 1991 as
a DNA mimic1 and since this time, a vast number of studies
have been reported covering their synthesis, properties and
potential applications.2 In PNAs, the sugar–phosphate
backbone present in DNA has been replaced by a repeating
polyamide chain of N-(2-aminoethyl)glycine monomer
units, yet despite this major modification PNA still
Figure 1. General structure of a PNA/DNA duplex.
Keywords: PNA; Dde; Fmoc; Orthogonality; Conjugate; Peptide.
*
Corresponding author. Tel.: C44 131 6504280; e-mail: mark.bradley@ed.ac.uk
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.06.003

8296
L. Bialy et al. / Tetrahedron 61 (2005) 8295–8305
Figure 2. General structure of the four PNA building blocks.
number of different areas of research such as gene therapy,
SNP analysis and mRNA profiling.3 A further advantage of
PNA over other DNA mimetics stems from its pseudo-
peptidic structure which allows PNA synthesis to be carried
out by straightforward and traditional solid phase peptide
chemistry based methods (SPPS). However PNA does suffer
from a number of drawbacks and disadvantages, such as
issues relating to its cellular delivery and uptake, the
inability to enzymatically extend PNA primers and the
complexities of PNA synthesis, which to date has made
concurrent peptide/PNA synthesis and PNA/DNA synthesis
very inefficient. However, the conjugation of PNA to
peptides can be used to enable the properties of the PNA to
be highly modulated, for example, by allowing membrane
permeability which then opens up a host of opportunities for
new applications of PNA and better exploitation of its full
potential. Therefore, methods to allow the efficient
preparation of peptide–PNA conjugates in which the
peptide and PNA strands can be prepared at will would
have huge potential.4
2. Results and discussion
In order to carry out solid phase PNA oligomers synthesis,
PNA monomeric building blocks protected on the amino
group of the N-(2-aminoethyl)glycine backbone as well as
the nucleobases (Fig. 2), had to be prepared.
It was desired that the PNA and the peptide moieties should
be elongated in a truly orthogonal fashion, thus imposing no
restrictions on the design of these conjugates, but protecting
groups on the nucleobases (PG1) and the amino acids should
be cleaved together under the final acidic cleavage
conditions. The use of an acid labile linker such as the
Rink amide linker9 would also enable deprotection to be
performed during the final cleavage of the conjugate from
the solid-support. For this purpose, the mono-methoxytrityl
(Mmt) group was chosen for the nucleobase protecting
group (PG1). The correct choice of PG2-which had to be
orthogonal to the Fmoc group and the acid-labile side-chain
protecting groups on peptides proved more challenging, as
none of the PNA monomers reported to date met these
criteria.
To allow the construction of peptide–PNA conjugates,
monomeric building blocks protected with two orthogonal
protecting groups (one for the N terminus and one for the
nucleobase) are required, and during the past decade PNA
monomers with a range of compatible protecting groups
have been developed with Fmoc/Bhoc5 and Boc/Cbz6
being the most frequently used but other combinations
such as Fmoc/Mmt7 or Fmoc/Cbz8 have also been used.
While these monomers are useful for the synthesis of
standard PNA oligomers, their utility in the synthesis of
PNA–peptide conjugates is limited as they do not allow
orthogonal synthesis with commercially available amino
acid monomers (the most frequently used being the
N-terminal Fmoc protected amino acids with acid labile
side-chain protecting groups).
As a possible solution, the allyloxycarbonyl (Aloc) group,
which can be cleaved by palladium catalysts under
essentially neutral conditions,10 was examined. Although
the Aloc group can be deprotected without affecting the
Fmoc group, the use of Aloc-protected PNA monomers,
synthesized in our group, proved to be unsuccessful in chain
extension with the expected products obtained in very low
yield and purities. Moreover, the length of the PNA
oligomers was severely affected as during Aloc deprotec-
tion, Pd(0) species become trapped within the solid supports
(generating black beads) causing premature and random
Aloc deprotection. Thus synthesis on solid support using
Aloc protected monomers in a coupling–deprotection
strategy was fundamentally flawed.
In the quest for alternative N-terminal protecting groups for
PNA monomers which would allow the desired degree of
orthogonality, 1-(4,4-dimethyl-2,6-dioxacyclohexylidene)
ethyl (Dde) was identified, as a protecting group which
has been used for primary amines and one that is classically
removed with hydrazine.
Hence, after unsuccessful attempts to use Aloc-PNA
monomers for the oligomerization process, another protect-
ing group was identified, 1-(4,4-dimethyl-2,6-dioxacyclo-
hexylidene)ethyl (Dde). While hydrazine, the usual reagent
to deprotect the Dde group, also deprotects the Fmoc group,
we reasoned that the Dde group should be deprotected by
hydroxylamine (mixture of NH2OH$HCl and imidazole)
under slightly acidic conditions which would be fully
orthogonal to the Fmoc group.11 The principle of the
orthogonality being based on the different mechanisms
involved for both deprotections. While Fmoc protected
amines are deprotected through an E1cb elimination
generating dibenzofulvene, the Dde group is cleaved by
Here, we describe the synthesis of novel Dde/Mmt PNA
monomers and the development of new deprotection con-
ditions for the Dde group which were robust and fully
orthogonal to other commonly used protecting groups in
peptide chemistry, such as Fmoc, Boc, Mmt, t-Bu, MeO, etc.
Wefurtherdemonstratetheapplicationofthesemonomerstoa
highly versatile synthesis of various PNA–peptide conjugates.

L. Bialy et al. / Tetrahedron 61 (2005) 8295–8305
8297
Scheme 1. Possible Dde deprotection mechanism using NH2OH$HCl/imidazole.
nucleophiles through a transenamination or Michael type
mechanism followed by ring closure yielding an isoxazole
II (Scheme 1, Fig. 3).
yield.12 Following esterification of 3, Dde protection was
carried out using Dde-OH previously prepared from
dimedone and acetic acid,13 yielding the PNA backbone
ethyl 2-N-Dde-aminoethylglycinate 5 in an overall yield of
40% (Scheme 2).
2.3. Derivatization of the nucleobases
The nucleobases were derivatized through alkylation and
protection of the exocyclic amino group, if necessary, with
the monomethoxytritryl group (Mmt) following a procedure
reported by Breihpol.7
Thymine 6 was thus alkylated following the procedure by
Buchardt,6 by treatment of commercially available thymine
with methyl bromoacetate using K2CO3 as base, followed
by saponification of the resulting compound with NaOH
yielding thymin-1-yl acetic acid 7. Cytosine, was first
protected with Mmt-Cl in pyridine and then alkylated using
a similar procedure as described for 7 (Scheme 3).
Figure 3. Novel Dde/Mmt PNA monomers. *Thymidine monomers do not
require nucleobase protection.
2.1. Synthesis of Dde/Mmt PNA monomers
The synthesis of the PNA monomers containing the four
nucleobases was carried out in solution. Retro-synthetically,
the monomers can be disconnected into two synthons, a
backbone and the derivatised nucleobase and this allows
the straightforward synthesis of all four desired PNA
monomers.
2.2. Synthesis of the Dde-protected backbone
The synthesis of the Dde-protected backbone 5 is depicted
in Scheme 2. Thus, chloroacetic acid was alkylated with an
excess of ethylendiamine 1, which following precipitation
with DMSO gave 2-aminoethylglycine 3 in quantitative
Scheme 3. Pyrimidine nucleobases derivatizion: (a) 1 equiv K2CO3, DMF
under N2, 16 h; (b) 10% NaOH (aq), reflux, 10 min; (c) 1.5 equiv Mmt-Cl,
1 equiv NEM, Pyr, 40 8C, overnight; (d) 1 equiv K2CO3, 1 equiv methyl
2-bromoacetate, DMF, under N2, 16 h.
In order to obtain selective N9 over N7 alkylation, purine
nucleobases were first alkylated followed by exocyclic
amino protection. Following deprotonation of adenine with
sodium hydride, alkylation was carried out using ethyl
bromoacetate to give 12. Amino protection using Mmt
chloride in pyridine and subsequent saponification yielded
[N6-(Mmt)-adenin-9yl]-acetic acid 13 (Scheme 4).
Scheme 2. Dde backbone synthesis: (a) room temperature, 16 h;
(b) 10 equiv SO2Cl, MeOH, reflux, 16 h.; (c) 2 equiv DIPEA, 1 equiv
Dde-OH; CH2Cl2/EtOH (1/1), room temperature, 16 h.

8298
L. Bialy et al. / Tetrahedron 61 (2005) 8295–8305
Scheme 4. Purine nucleobases derivatization: (a) 1.1 equiv NaH, 1 equiv methyl 2-bromoacetate, DMF, under N2, 4 h; (b) 1.5 equiv Mmt-Cl, 1 equiv NEM,
CH2Cl2, 40 8C, 3 h, then 25 8C, 16 h; (c) 1 N aqueous NaOH, reflux, 2 h; (d) 1.5 equiv Mmt-Cl, 1 equiv DIPEA, THF, overnight; (e) 10% NaOH (aq.), reflux,
2 h.
2-Amino-6-chloropurine 14 was used as precursor of the
guanine nucleobase. This was due to the poor solubility of
guanine, the lack of reactivity of its exocyclic amino group
and the difficulty in obtaining selectively the N9 alkyl
derivatives. Alkylation and Mmt protection were carried out
as for adenine, however dry CH2Cl2 instead of pyridine was
used in the Mmt protection step. The use of pyridine as
solvent led to the formation of the 6-pyridinium derivative
as a side product. In the last step, basic hydrolysis of the
ester moiety as well as the C-6 chlorine substituent provided
[N2-(Mmt)-guanin-9-yl]-acetic acid 16 (Scheme 4).
MeOH/H2O, NaOH in MeOH/H2O, KOH in H2O, Na2CO3
in MeOH/H2O, K2CO3 in MeOH/H2O), a 1 M solution of
cesium carbonate in MeOH/H2O (1:1) gave the best results
(19a–d). The lack of Dde deprotection during saponification
is a major advantage compared to the preparation of
Fmoc protected PNA monomers, where an extra Fmoc
re-protection step is often required following ester
hydrolysis.7 Another major advantage of the synthesis of
Dde-protected monomers is that no chromatographic
separations were needed, with only extraction and precipi-
tation procedures used. The final products were obtained as
pure materials (as determined by HPLC and NMR),
allowing multi-gram quantities of the monomers to be
2.4. Monomer synthesis
Two steps remained for the synthesis of the monomers:
coupling of the protected backbone 5 and the derivatised
nucleobases 7, 10, 13, and 16, and hydrolysis of the ester
moiety. Amide formation with the secondary amine moiety
in 5 proved to be sluggish. Protected nucleobases-acetic
acid derivatives, 10, 13 and 17 (mainly purine), are known
to be difficult substrates for amide formation and therefore
various different coupling reagents were screened.
Couplings of pyrimidine nucleobase derivatives 7 and 10 to
the backbone 5 were achieved using different coupling
reagents such as 1-propylphosphonic acid cyclic,14 PyBroP
and DCC/DhbtOH6 with similar efficiencies. Esters 18T and
18C(Mmt) were obtained with average yields of 60%.
PyBroP proved to be the most efficient coupling agent
(according to HPLC analysis) with complete conversion how-
ever, with this coupling reagent, the purification process was
challenging, especially on a large scale. In factremoval oftris-
(pyrrolidin-1-yl)-phosphoramide was best performed after
ester hydrolysis by repeated precipitation (see Section 4).
Scheme 5. Dde protected PNA monomers: (a) T-CH2COOH, 50%
1-propylphosphonic acid cyclic in DMF, NEM, 16 h (18T); C(Mmt)CH2-
COOH or A(Mmt)CH2COOH or G(Mmt)CH2COOH, PyBroP, DIPEA,
DMF, 2.5–15 h (18C(Mmt)-18A(Mmt)–18G(Mmt)); (b) 1 M Cs2CO3
MeOH/H2O 1/1, 1.5 h. NBZNucleobases.
The second step involved basic hydrolysis of the ester
group. Among the conditions tested (LiOH in THF, LiOH in

L. Bialy et al. / Tetrahedron 61 (2005) 8295–8305
8299
easily synthesized following our protocols, indeed we have
prepared up to 5 g of monomers using this synthetic route
(Scheme 5).
2.5. PNA–peptide conjugates
The Dde PNA monomers open up new opportunities for
the flexible synthesis of PNA conjugates. In a previous
communication, our group reported the orthogonality
between Dde and Fmoc protecting groups.11 Furthermore
the Dde deprotection protocol was completely compatible
with the Mmt protecting group and the nucleobases. To
demonstrate this robustness we embarked on the synthesis
of various different conjugates.
Scheme 7. Synthesis of a 13 mer PNA oligo using Dde protected PNA
monomers: (a) Dde-PNA-OH (5.5 equiv), PyBOP (5 equiv 0.1 M), NEM
(11 equiv) in DMF, 3 h; (b) NH2OH$HCl/imidazole in NMP/DMF; repeat
(a) and (b) as necessary; (c) excess 20% piperidine in DMF; (d) Fmoc-Ahx-
OH (5.5 equiv), PyBOP (5 equiv, 0.1 M), DIPEA (11 equiv) in DMF, 3 h;
(e) excess TFA/TIS/CH2Cl2 (90/5/5), 1 h.
mediate 24 was cleaved with a mixture of TFA/TIS/CH2Cl2
(90/5/5) for 1 h. After precipitation from TFA–diethyl ether,
the purity and identity of the crude material was determined
by HPLC and MALDI-TOF, where no loss of the Fmoc
group could be detected. Thus, the new monomers were
demonstrated to be valid building blocks for PNA
oligomerization.
Fmoc-Lys(Dde)-OH 21 was used as core of these conjugates
(Scheme 6)15 which was coupled to commercially available
Rink amino PEGA resin, using PyBOP, to give 22. Three
possible strategies are realizable for the construction of
PNA–peptide conjugates: (a) building up first the PNA arm
(using Dde monomers 19), and then the peptide arm (using
Fmoc monomers); (b) or vice versa; (c) or alternating PNA
elongation and peptide elongation steps.
Traditionally, peptides have been conjugated to PNA using
either traditional solid phase chemistry by coupling amino
acids followed by PNA monomers or vice versa16–18 using
disulfide linkage19 or ligation through disulfide bonds.20
However, the method reported here allows a much more
flexible strategy which involves elongation of either PNA or
peptide parts at any stage of the synthesis (see above). To
demonstrate this flexibility, a conjugate was synthesised by
alternating PNA coupling and amino acids coupling steps
(Scheme 8). PNA monomer couplings were performed as
for compound 24 and amino acid couplings were carried out
using PyBOP/DIPEA in DMF with HOBt for 3 h. Fmoc
deprotection was carried out using 20% piperidine in DMF
for two cycles of 6 min. No Dde cleavage was observed
under these conditions.
The synthesis of the PNA oligo arm was firstly investigated.
Initially three different resin types were tested: PS, Tentagel
and PEGA but in terms of Dde deprotection, PEGA showed
the fastest cleavage kinetics (complete deprotection after
1 h); while the other resins required longer times (up to 3 h).11
PEGA was thus considered most suited for the purpose of
PNA synthesis with Dde PNA monomers and after selective
Dde deprotection with a solution of NH2OH$HCl/imidazol
(1/0.75 equiv) in NMP/DMF (5/1) for one hour resin 23
(as the salt) was obtained.
A 13mer PNA oligo containing the four natural nucleobases
was synthesised starting from 23 (Scheme 7) with PNA
monomer couplings achieved with PyBOP/NEM in DMF
using 5.5 equiv of monomer (0.11 M) for 3 h while Dde
deprotection was achieved using the protocol described
above. After elaboration of the PNA arm, Fmoc deprotec-
tion was carried using 20% piperidine in DMF and after
coupling of Fmoc–aminohexanoic acid the PNA inter-
Once the construct 25 was prepared the peptide arm was
capped using butyric acid and the conjugate was labelled
with 5(6)-carboxyfluorescein on the PNA arm, to yield 26.
Following cleavage from the resin with TFA/TIS/CH2Cl2
Scheme 6. Fmoc-Lys-Rink-PEGA 23 synthesis: (a) Dde-OH (2 equiv, 26 mM), TFA (0.1 equiv) in EtOH, reflux, 2 days;15 (b) 21 (5.5 equiv), PyBOP (5 equiv,
0.1 M), amino-Rink-PEGA resin, DIPEA (11 equiv) in DMF, 3 h; (c) 20% NH2OH.HCl/imidazole (1/0.75 equiv) in NMP/DMF (5/1).

8300
L. Bialy et al. / Tetrahedron 61 (2005) 8295–8305
Scheme 8. Synthesis of 26 using alternative PNA and amino acid couplings: (a) Dde-PNA-OH (5.5 equiv), PyBOP (5 equiv), NEM (11 equiv) in DMF, 3 h; (b)
20% piperidine in DMF; (c) Fmoc-AA-OH or butyric acid (last step) (5.5 equiv), PyBOP (5 equiv, 0.08 M), DIPEA (16 equiv), HOBt (5.5 equiv) in DMF, 3 h;
(d) NH2OH.HCl/imidazole; repeat a–d as necessary; (e) 5(6)-carboxyfluorescein (22 equiv), PyBOP (22 equiv, 0.08 M), NEM (44 equiv), in DMF, 15 h; (f)
TFA/TIS/CH2Cl2 (90/5/5), 1 h. FAMZ5(6)-carboxyfluorescein.
(90/5/5) and precipitation, HPLC and MALDI-TOF analysis
was undertaken to give the data shown in Figure 4.
which require alternative coupling steps for amino acid and
PNA monomers. Further possible applications beyond the
synthesis of PNA–peptide conjugates include the synthesis
of PNA molecular beacons (i.e., the complementary PNA
arm constructed with Fmoc protected PNA monomers).
However, it should be noted that PNA poly-T sequences
(more than four Ts in a row) cannot be synthesized with this
method, due to the strong interactions of polythymidine
chains within the beads.
3. Conclusion
The solution phase synthesis of a new class of PNA
monomers and their application to PNA oligomerization is
presented. These monomers present an alternative to Fmoc
protected PNA monomers, and the Dde monomers show
distinct advantages, especially in the context of PNA–
peptide conjugates giving the possibility of a more flexible
strategy to obtain PNA–peptide conjugates, such as the
possibility of a truly orthogonal synthesis of PNA and
peptide when using commercially available Fmoc amino
acids. This strategy is routinely used in our group for the
synthesis of split and mix PNA encoded peptide libraries
4. Experimental
4.1. General
NMR spectra were recorded using a Bruker AC300
spectrometer operating at 300 MHz for H and 75 MHz
1
Figure 4. HPLC (acetonitrile gradient is shown, lZ220 nm) and MALDI-TOF of crude compound 26.

L. Bialy et al. / Tetrahedron 61 (2005) 8295–8305
8301
for 13C and a Bruker AC400 spectrometer operating at
400 MHz for 1H and 100 MHz for 13C. All coupling
constants (J values) were measured in Hz. ES mass spectra
were recorded using a VG Platform Quadrupole Electro-
spray Ionisation mass spectrometer. Reverse phase analyti-
cal HPLC (RP HPLC) was performed using a Hewlett
Packard HP1100 Chemstation, using a Phenomenex C18
prodigy 5 mm (150 mm!3.0 mm i.d.) column. The
following methods were used: solvent AZH2O/CH3CN/
TFA 90/10/0.1 to solvent BZH2O/CH3CN/TFA 10/90/0.1,
solvent CZH2O/CH3CN/TFA 100/0/0.1, solvent DZH2O/
CH3CN/TFA 0/100/0.1.
2-bromoacetate (3.43 mL, 31.3 mmol) was added dropwise.
The reaction was stirred at room temperature for 16 h before
evaporating the solvent in vacuo. The resulting oil was
precipitated with H2O and removed by filtration, and
washed with H2O to give a white solid which was dried
over P2O5 in vacuo. RfZ0.32 (EtOAc); HPLC (method 2)
RtZ3.43; mpO200 8C min; HRMS (ESC): m/z calcd for
1
C27H25NaN3O4 [MCNaC] 478.1737, found 478.1741; H
NMR (300 MHz, d6-DMSO): dZ12.8 (br s, 1H, COOH),
3
8.4 (br s, 1H, NH), 7.45 (d, J(H,H)Z7.7 Hz, 1H, CHCyt
)
3
7.4–7.1 (m, 12H, CHMmt), 6.81 (d, J(H,H)Z8.8 Hz, 2H,
CHMmt), 6.18 (d, 3J(H,H)Z7.7 Hz, 1H, CHCyt), 4.25 (s, 2H,
CH2), 3.70 (s, 3H, H3CO–Ar), 3.60 (s, 3H, COOCH3) ppm;
13C NMR (100 MHz, CDCl3): dZ168.4 (COOH), 166.0
(]CCyt), 158.8 (COCH3), 155.7 (CO), 145.2 (CHMmt),
144.2 (CMmt), 135.9 (CMmt), 130.0 (CHCyt), 129.0 (CHMmt),
128.6 (CHMmt), 127.8 (CHMmt), 113.6 (CHMmt), 94.9
(CHCyt), 70.6 (CMmt), 55.2 (OCH3), 52.6 (COOCH3), 50.1
(CH2).
Method 1 (flow rate 0.5 mL/min). 100% A to 100% B over
10 min, followed by 100% B for 5 min. Method 2 (flow rate
1 mL/min) 100% A H2O to 100% B over 3 min followed by
100% B for 1 min. Method 3 (flow rate 0.5 mL/min); 100%
C over 5 min, then 100% C to 40% C/60% D over 10 min
then to 100% D followed by 100% D over 4 min).
Retention time (Rt). Thin layer chromatography (TLC) was
performed using Alugram SIL G/UV/254 precoated plates.
Visualisation was achieved by UV radiation.
Methyl [N4-(4-methoxytrityl)-cytosin-1-yl]-acetate7,21
(31.1 g, 67 mmol) was suspended in 2 N aqueous NaOH
(200 mL) and the mixture was stirred for 2 h at reflux. The
reaction was cooled to room temperature, washed with
CH2Cl2 (3!50 mL) and acidified with 2 N aqueous HCl
until pH 2. The precipitated was collected by filtration,
washed with small portions of water until the filtrate was
neutral (pHZ7) and dried in vacuo for 12 h to give acid 10
(11.4 g, 66% over two steps) as a white solid.; RfZHPLC
(method 2); RtZ3.33 min; mp 210 8C; HRMS (ESC) m/z
calcd for C26H24N3O4 [MCHC] 442.1762; found
PyBOP (benzotriazole-1-yl-oxy-tris-pyrrolidinio-phos-
phonium hexafluorophosphate) and PyBroP (bromo-tris-
pyrrolidinio-phosphonium hexafluorophophate) were
purchased from Novabiochem (Merck Bioscience, UK).
PEGA resin (acrylolated O,O-bis(aminoproplyl)polyethyl-
ene glycol resin) 0.4 mmol/g 150–300 mm was purchased
from Polymer Labs (UK), DIPEA (N,N-diisopropyl-N-
ethylamine), NEM (N-ethylmorpholine).
1
442.1770; H NMR (400 MHz, d6-DMSO): 8.31 (s, 1H,
3
NHMmt), 7.44 (d, J(H,H)Z6.5 Hz, 1H, CHCyt), 7.27–7.16
(m, 12H, CHMmt), 6.82 (d, 3J(H,H)Z5.5 Hz, 2H,
4.1.1. Methyl N-{2-[1-(4,4-dimethyl-2,6-dioxo-cyclohexyl-
iden)-ethylamino]-ethyl}-glycinate (5). To a stirred solu-
tion of diamine salt 412 (41.6 g, 203 mmol) and DIPEA
(71 mL, 406 mmol) in CH2Cl2/MeOH (1/1 500 mL) was
added Dde-OH (37 g, 203 mmol). The solution was stirred
at room temperature for 16 h before evaporating the solvent
in vacuo. The crude material was taken up in EtOAc
(400 mL) and extracted with 1 M KHSO4 (4!100 mL),
which was brought to pH 9 with NaHCO3 before extraction
with further EtOAc (4!100 mL). The organic phases were
combined, washed with brine (1!100 mL), dried over
MgSO4 and concentrated in vacuo to give ester 5 as a brown
oil. (34.2 g, 57%). RfZ0.34 (EtOAc); HPLC (method 2)
RtZ2.5 min; mp 37 8C; HRMS (ESC): m/z calcd for
C15H24N2O4 [MCHC] 297.1808, found 297.1807; 1H
NMR (400 MHz, CDCl3): dZ13.42 (s, 1H, NH-Dde),
3.66 (s, 3H, OCH3), 3.43–3.48 (m, 4H, CH2), 2.87 (t,
3J(H,H)Z6.2 Hz, 2H, CH2), 2.50 (s, 3H, CCH3), 2.29 (s,
4H, CH2-Dde), 1.96 (s, 1H, NH), 0.95 (s, 6H, (CH3-
Dde) ppm; 13C NMR (100 MHz, CDCl3): dZ198.1 (CO-
Dde), 173.4 (CCH3), 172.7 (COOCH3), 107.9 (Dde-C]C),
52.8 (CH2-Dde), 51.8 (OCH3), 50.2 (NHCH2CO), 47.6
(DdeNHCH2CH2NH), 44.0 (DdeNHCH2CH2NH), 30.0
((CH2)2C(CH3)2), 28.2 (CH3-Dde), 18.1 (C–CH3).
3
CHCOCH3), 6.17 (d, J(H,H)Z5.8 Hz, 1H, CHCyt), 4.24
(s, 2H, CH2), 3.72 (s, 3H, H3CO–Ar) ppm; 13C NMR
(100 MHz, d6-DMSO): 169.9 (COOH), 164.1 (CCyt), 158.6
(C–OCH3), 157.5 (CO), 144.9 (CHMmt), 144.2 (CMmt),
135.1 (CMmt), 129.9 (CHCyt), 128.6 (CHCyt), 127.4 (CHCyt),
126.1 (CHMmt), 112.8 (CHMmt), 95.5 (CHCyt), 69.7
(C(Ar)3), 55.0 (O–CH3), 49.7 (CH2COOH) ppm.
4.1.3. [N6-(4-Methoxytrityl)-adenin-9-yl]-acetic acid
(13). A mixture of ester 126 (7.21 g, 32.3 mmol), NEM
(4.1 mL, 32.3 mmol) and 4-monomethoxytrityl chloride
(15.0 g, 48.5 mmol) in pyridine (100 mL) and dichloro-
methane (100 mL) was heated at 40 8C for 3 h, then at 25 8C
for 16 h. After evaporation of the solvent in vacuo, the
residue was re-dissolved in EtOAc (400 mL), washed with
1 N aqueous KHSO4 (100 mL), 10% aqueous NaHCO3
(100 mL), brine (100 mL) and dried over MgSO4. After
evaporation of the solvent in vacuo, the residue was washed
with petroleum ether to give a yellowish solid which was
refluxed for 2 h in 1 N aqueous NaOH (200 mL) and stirred
at 25 8C for 2 h. The solution was cooled to 0 8C. Following
addition of 1 N aqueous KHSO4 (250 mL), the mixture was
stirred at 0 8C for 30 min, giving rise to the precipitation of
acid 13. After filtration, the residue was washed with small
portions of water until the filtrate was neutral (pHZ7) and
dried in vacuo for 12 h to give acid 13 (14.8 g, 78% over two
steps) as a white solid.
4.1.2. [N4-(4-Methoxytrityl)-cytosin-1-yl]-acetic acid
(10). NaH (1.04 g, 60% in mineral oil, 26 mmol) was
added to a stirred suspension of pyrimidine 97 (10 g,
26 mmol) in DMF (100 mL). After stirring for 1 h at room
temperature the mixture was cooled to 0 8C and methyl
RfZ0.28 (CH2Cl2/MeOH/HOAc 100/10/1); HPLC Rt

8302
L. Bialy et al. / Tetrahedron 61 (2005) 8295–8305
(method 1)Z8.79 min; mp 111–120 8C; HRMS (ESC): m/z
calcd for C27H24N5O3 [MCHC] 466.1874, found 466.1871;
1H (300 MHz, d6-DMSO): dZ8.15 (s, 1H, CHpur), 7.88 (s,
1H, CHpur), 7.30–7.20 (m, 12H, CHMmt), 6.85 (d, 2H,
3J(H,H)Z8.9 Hz, CHCOCH3), 4.80 (s, 2H, CH2COO) 3.72
(s, 3H, H3CO–Ar) ppm; 13C NMR (75 MHz, d6-DMSO):
dZ169.2 (CO), 157.7 (C), 153.3 (C), 151.0 (CHPur), 148.9
(C), 145.2 (C), 142.3 (CHPur), 137.1 (C), 129.8 (CHMmt),
128.4 (CHMmt), 127.7 (CHMmt), 126.5 (CHMmt), 119.9 (C),
113.0 (CHMmt), 69.9 (C(Ar)3), 55.0 (CH3O), 45.0 (CH2).
0 8C, and the reaction was stirred under N2 at 25 8C for 2 h.
The solvent was removed in vacuo, the residue was
re-dissolved in CH2Cl2 (300 mL), washed with 1 M
KHSO4 (100 mL), 10% aqueous NaHCO3 (100 mL) and
brine (100 mL). The organic phase was dried over MgSO4
and concentrated in vacuo to give ester 18A(Mmt) as a
brown solid which was used in the next step without
purification. An analytical sample was obtained by
chromatography.
RfZ0.62 (CH2Cl2/MeOH 5/1); HPLC (method 1) RtZ
9.60 min; mp 125–135 8C; HRMS (ESC): m/z calcd for
C42H46N7O6 [MCHC] 744.3504, found 744.3510; 1H
(300 MHz, d6-DMSO) two rotamers: dZ13.28 and 13.17
(s, 1H, Dde-NH), 8.09 and 8.08 (s, 1H, CHpur), 7.88 (s, 1H,
CHpur), 7.31–7.22 (m, 12H, CHMmt), 6.85 (d, 2H, 3J(H,H)Z
8.5 Hz, CHMmt), 5.26 and 5.09 (s, 2H, CH2COO), 4.52 and
4.13 (s, 2H, CH2CO), 3.79 (m, 2H, CH2N), 3.74 (s, 3H,
CH3O), 3.72 (s, 3H, CH3O), 3.57 (m, 2H, CH2N), 2.57 and
2.46 (s, 3H, CH3C), 2.30 and 2.27 (s, 4H, CH2-Dde), 0.94 (s,
6H, CH3-Dde) ppm; 13C NMR (75 MHz, d6-DMSO) two
rotamers: dZ196.6 and 196.4 (CO), 173.2 (CO), 169.8 (CO),
169.3 (CO), 167.7 and 167.1 (C), 157.7 (C), 153.4 (C), 151.1
(CH), 149.0 (C), 145.2 (C), 142.3 and 142.1 (CH), 137.1 (C),
129.8 (CH), 128.4 (CH), 127.7 (CH), 126.5 (CH), 119.7 (C),
113.0 (CH), 107.4 and 107.2 (C), 69.9 (C(Ar)3), 55.0 (CH3),
52.4 (CH2), 51.8 (CH3), 49.0 and 48.1 (CH2), 45.8 (CH2),
43.7 (CH2), 41.0 (CH2), 29.7 (C), 27.8 (CH3), 17.3 and 17.1
(CH3) ppm.
4.1.4. Methyl N2-(4-methoxytrityl)-amino-6-chloro-
purin-9-yl acetate (16). To a solution of ester 157 (5 g,
20 mmol) and 4-methoxytrityl chloride (9.58 g, 30 mmol)
in dry THF (135 mL) was added DIPEA (3.6 mL, 20 mmol)
and the mixture was stirred under a N2 atmosphere for 12 h.
Following evaporation of the solvent, the mixture was
taken up in Et2O (150 mL) and washed with 1 M KHSO4
(3!25 mL); 10% aqueous NaHCO3 (3!25 mL) and
brine (1!25 mL). The organic layer was dried over
Na2SO4 and concentrated in vacuo to give oil which was
triturated with petroleum ether 40–60 8C to give ester 16 as
a yellowish solid (6.2 g, 60%). RfZ0.5 (EtOAc); HPLC
(method 1) RtZ10.00 min; mp 55 8C; MS (ESC): m/z
calcd for C28H25ClN5O3 [MCHC] 514.16 found 514.2
1
[35Cl]; 516.2 [37Cl] H NMR (400 MHz, CDCl3): dZ7.67
(s, 1H, CHpur), 7.35–7.24 (m, 12H, CHMmt), 6.81 (d, 2H,
3J(H,H)Z8.6 Hz, CHCOCH3), 6.65 (s, 1H, NH-Mmt), 4.37
(s, 2H, CH2), 3.81 (s, 3H, H3CO–Ar), 3.67 (s, 3H,
COOCH3) ppm; 13C NMR (100 MHz, CDCl3): dZ166.8
(CO), 158.2 (C), 157.6 (C), 152.7 (C), 150.5 (C), 145.0 (C),
141.5 (CHpur), 137.1 (C), 130.2 (CHMmt); 128.9 (CHMmt),
127.6 (CHMmt), 126.5 (CHMmt), 124.0 (C), 112.9 (CHMmt),
70.8 (C(Ar)3), 55.3 (H3CO–Ar), 52.9 (COOCH3), 43.8
(CH2) ppm.
4
4.1.7. Methyl N-{2-[N0 -(4-methoxytrityl)-cytosin-1-yl]-
acetyl}-N-{2-[1-(4,4-dimethyl-2,6-dioxo-cyclohexyliden)
ethylamino]ethyl}-glycinate (18C(Mmt)). This compound
was synthesized in analogy to ester 18A(Mmt).
RfZ0.21 (EtOAc); HPLC (method 2) RtZ3.63 min; mp
135 8C; HRMS (ESC): m/z calcd for C41H46N5O7 [MC
HC] 720.3397, found 720.3402; 1H NMR (400 MHz,
CDCl3): two rotamers dZ13.50 and 13.37 (br s, 1H, Dde-
4.1.5. [N2-(4-Methoxytrityl)-guanin-9-yl]-acetic acid
(17). Ester 16 (6.2 g, 12 mmol) was suspended in 2 N
aqueous NaOH (150 mL) and the mixture was stirred for 4 h
at reflux. The reaction was cooled to room temperature and
acidified with 2 N aqueous HCl (pHZ2). The precipitated
was collected by filtration, washed with water and dried, in
vacuo overnight giving a solid which was washed with
diisopropyl ether to give acid 17 (4.2 g, 72%) as a yellowish
solid.; RfZ0.12 (CH2Cl2/MeOH 7/3); HPLC (method 1)
RtZ7.50 min; mp O200 8C; HRMS (ESC) calcd for
C27H24N5O4 [MCHC] 482.1828; found 482.1831; 1H
NMR (400 MHz, d6-DMSO): dZ11.18 (s, 1H, NHpur),
8.06 (s, 1H, NH-Mmt), 8.04 (s, 1H, CHpur), 7.27–7.16 (m,
NH), 7.23–7.05 (m, 12 H, CHMmt,
1H CHCyt), 6.76 (d,
3
3J(H,H)Z9.0 Hz, 2H, CHMmt), 4.99 (d, J(H,H)Z7.8 Hz,
1H, CHCyt), 4.53 and 4.36 (s, 2H, CH2COO), 4.27 and 4.02
(s, 2H, CH2CO), 3.72–3.63 (m, 3H, CH3O, 4H, CH2N), 3.52
(CH3O), 2.51 and 2.47 (s, 3H, CH3C), 2.25 and 2.24 (s, 4H,
CH2-Dde), 0.95 and 0.93 (s, 6H, CH3-Dde); 13C NMR
(100 MHz, CDCl3) two rotamers: 199.0 and 198.6 (CO),
175.7 (CNH), 170.8 and 170.4 (CO), 168.8 and 168.0 (CO),
163.8 (C), 159.8 (C), 153.0 (CO), 148.2 and 147.2 (CH),
145.1 (C), 136.7 (C), 130.2 (CH), 129.6 (CH), 129.8 (CH),
128.8 (CH), 128.6 (CH), 114.7 and 114.2 (CH), 95.9 (CH),
71.8 (C(Ar)3), 56.3 (H3C), 53.9 and 53.5 (CH2), 51.9 and
51.3 (CH2), 49.9 and 49.5 (CH2), 47.4 and 47.3 (CH2), 42.6
and 41.8 (CH2), 31.0 (C), 29.2 (CH3), 18.9 and 18.7 (CH3).
3
12H, CHMmt), 6.81 (d, 2H, J(H,H)Z8.6 Hz, CHCOCH3),
4.28 (s, 2H, CH2), 3.72 (s, 3H, H3CO–Ar) ppm; 13C NMR
(100 MHz, d6-DMSO): dZ168.5 (CO), 168.0 (CO), 157.7
(C), 155.2 (C), 151.6 (C), 149.1 (C), 148.0 (C) 144.6 (C),
138.0 (CHpur), 136.6 (C); 129.8 (CHMmt), 128.3 (CHMmt),
127.4 (CHMmt), 126.4 (CHMmt), 112.8 (CHMmt), 69.9
(C(Ar)3), 55.0 (H3CO–Ar), 43.8 (CH2) ppm.
2
4.1.8. Methyl N-[N0 -(4-methoxytrityl)-guanin-9-yl]-N-
[2-[1-(4,4-dimethyl-2,6-dioxacyclohexylidene) ethyl-
amino]ethyl]glycinate (18G(Mmt)). This compound was
synthesized in analogy to ester 18A(Mmt).
6
4.1.6. Methyl N-{2-[N0 -(4-methoxytrityl)-adenin-9-yl]
acetyl}-N-{2-[1-(4,4-dimethyl-2,6-dioxo-cyclohexyliden)
ethylamino]ethyl}glycinate (18A(Mmt)). To a solution of
acid 13 (5.10 g, 10.9 mmol) and amine 5 (3.38 g,
10.9 mmol) in DMF (25 mL) at 0 8C was added PyBroP
(5.07 g, 10.9 mmol) and DIPEA (3.7 mL, 21.8 mmol) at
18 G(Mmt) was obtained as a brown solid (48%) RfZ0.31
(CH2Cl2/MeOH 5/1); HPLC (method 1) RtZ8.42 min; mp
142–145 8C; HRMS (ESC) m/z calcd for C42H46N7O7
1
760.3459 found 760.3460 [MCHC] H NMR (400 MHz,

L. Bialy et al. / Tetrahedron 61 (2005) 8295–8305
8303
d6-DMSO) two rotamers dZ13.16 and 13.12 (br s, 1H, Dde-
NH), 10.58 and 10.55 (br s, 1H, NHpur), 7.58 and 7.55 (s,
1H, NH-Mmt), 7.44 and 7.42 (s, 1H, CHpur), 7.28–7.12 (m,
12H, CHMmt), 6.83 (d, 3J(H,H)Z8.5 Hz, 2H, CHMmt), 4.43
and 4.24 (s, 2H, CH2COO), 4.10 and 4.05 (s, 2H, CH2CO),
3.72–3.67 (m, 4H, N–CH2CH2-N), 3.66 and 3.61 (s, 3H,
CH3O), 3.43 (s, 3H, CH3O), 2.49 and 2.47 (s, 4H, CH2-
Dde), 2.30 and 2.26 (s, 3H, CH3C), 0.95 and 0.94 (s, 6H,
CH3-Dde); 13C NMR (100 MHz, d6-DMSO): dZ196.4 and
196.2 (CO), 173.0 and 172.7 (CO), 169.4 and 169.2 (CO),
166.9 (CO), 166.3 (C), 157.5 (C), 156.4 (C), 150.8 and
150.7 (C), 149.9 (C), 144.8 (C), 136.7 (CHpur), 129.7 and
129.6 (CH); 128.8 (CH), 127.6 and 127.4 (CH), 126.3
and 126.3 (CH), 116.1 and 116.0 (C), 112.7 (CH), 107.2 (C),
69.7 and 69.6 (C(Ar)3), 54.8 (CH3), 52.2 (CH2), 51.7 (CH3),
48.8 (CH2), 48.1 (CH2), 46.5 (CH2), 40.7 (CH2), 29.6 (C)
27.7 (CH3), 17.2 and 17.0 (CH3) ppm.
(m, 10H, CHMmt, 1H, CHCyt), 7.20 (d, 2H 3J(H,H)Z8.5 Hz,
CHMmt), 6.90 (d, J(H,H)Z9.0 Hz, 2H, CHMmt), 5.30 and
3
5.26 (d, 3J(H,H)Z7.5 Hz, 1H, CHCyt), 4.71 and 4.57 (s, 2H,
CH2COO), 4.26 and 4.12 (s, 2H, CH2CO), 3.90–3.66 (m,
3H, CH3O, 4H, CH2N), 2.56 and 2.53 (s, 3H, CH3C), 2.33
and 2.29 (s, 4H, CH2-Dde), 0.98 and 0.96 (s, 6H, CH3-Dde)
ppm; 13C NMR (100 MHz, CD3OD) two rotamers: 198.9
and 198.6 (CO), 174.8 (CNH), 171.4 and 171.1 (CO), 168.8
and 1678.0 (CO), 163.8 (C), 159.5 (C), 153.5 (CO), 148.8
(CH), 143.6 (C), 135.1 (C) 129.9 (CH), 128.5 (CH), 128.1
(CH), 127. (CH), 113.2 (CH), 95.1 (CH), 71.6 (C(Ar)3), 56.3
(H3C), 53.9 (CH2), 51.2 and 50.9 (CH2), 50.0 (CH2), 49.2
and 49.0 (CH2), 42.9 and 42.7 (CH2), 29.9 (C), 27.4 (CH3),
17.4 and 17.2 (CH3) ppm.
2
4.1.11. N-[N0 -(4-Methoxytrityl)guanin-9-yl]-N-[2-[1-
(4,4-dimethyl-2,6-dioxacyclohexylidene)ethylamino]
ethyl]glycine (19G(Mmt)). To a solution of ester
18G(Mmt) (2.5 g, 3.3 mmol) in methanol (16.5 mL) was
added dropwise a 2 M Cs2CO3 aqueous solution (16.5 mL).
The solution was stirred for 1.30 h. MeOH was evaporated
in vacuo and the remaining solution was diluted with water
(50 mL) washed with CH2Cl2 (3!15 mL). The basic
solution was brought to pH 2 using 1 M KHSO4. The
precipitate was collected by filtration, washed with water
and re-dissolved in a minimum of MeOH. Following
addition of an excess of water, the precipitated solid was
collected and dried in vacuo to give acid 19G(Mmt) (1.4 g,
57%) as a brown solid RfZ0.24 (BuOH/HOAc/H2O 3/1/1);
HPLC (method 1) RtZ7.072 min; mp 175 8C(dc); HRMS
(ESC) calcd for C41H44N5O4 [MCHC]746.3297, found
6
4.1.9. N-{2-[N0 -(4-Methoxytrityl)-adenin-9-yl]-acetyl}-
N-{2-[1-(4,4-dimethyl-2,6-dioxo-cyclohexyliden)-ethyl-
amino]-ethyl}-glycine (19A(Mmt)). Crude ester
18A(Mmt) was suspended in a 1:1 (v/v) mixture of
MeOH and 2 M Cs2CO3 (60 mL) and stirred for 1.5 h.
MeOH was evaporated in vacuo. The remaining solution
was diluted with 200 mL water, washed with EtOAc
(100 mL), acidified with 1 M aqueous KHSO4, extracted
with CH2Cl2 (3!200 mL). The organic extracts were
washed with brine (100 mL) and the solvent was evaporated
in vacuo. The residue was dissolved in a minimum amount
of EtOAc. After addition of an excess of hexane, the
precipitated solid was collected and dried in vacuo. This
precipitate was dissolved in a minimum amount of MeOH.
After addition of excess of water, the precipitated solid was
collected and dried in vacuo to give acid 19A(Mmt) (3.55 g,
45%) as an off-white solid.
1
746.3280; H NMR (400 MHz, d6-DMSO): two rotamers
dZ13.14 and 13.13 (br s, 1H, Dde-NH), 10.50 (br s, 1H,
NHpur), 7.5 and 7.55 (s, 1H, NH-Mmt), 7.46 and 7.44 (s, 1H,
3
CHpur), 7.25–7.13 (m, 12H, CHMmt), 6.83 (d, J(H,H)Z
8.7 Hz, 2H, CHMmt), 4.45 and 4.22 (s, 1H, CH2COO), 3.98
and 3.93 (s, 1H, CH2CO), 3.73–3.70 (m, 4H, NCH2CH2N),
3.41 (s, 3H, CH3O), 2.50 and 2.47 (s, 4H, CH2-Dde), 2.31
RfZ0.24 (CH2Cl2/MeOH/HOAc 100/10/1); HPLC (method
1) RtZ9.01 min; mp 180 8C (dc); HRMS (ESC): m/z calcd
1
for C41H44N7O6 [MCHC] 730.3348, found 730.3346 H
and 2.27 (s, 3H, CH3C), 0.95 and 0.94 (s, 6H, CH3-Dde) 13
C
(300 MHz, d6-DMSO): dZ13.26 and 13.16 (s, 1H, Dde-
NH), 8.09 and 8.08 (s, 1H, CHpur), 7.87 (s, 1H, CHpur),
7.31–7.21 (m, 12H, CHMm), 6.85 (d, 2H, 3J(H,H)Z8.8 Hz,,
CHMmt), 5.24 and 5.07 (s, 2H, CH2COO), 4.40 and 4.03 (s,
2H, CH2CO), 3.77 (s, 2H, CH2N), 3.72 (s, 3H, CH3O), 3.53
(m, 2H, CH2N), 2.56 and 2.46 (s, 3H, CH3CN), 2.29 and
2.26 (s, 4H, CH2CO), 0.93 (s, 6H, CH3) ppm; 13C NMR
(75 MHz, d6-DMSO): dZ196.4 (CO), 173.2 (CO), 170.7
(CO), 170.2 (CO), 167.7 and 166.9 (C), 157.7 (C), 153.4 (C),
151.1 (CH), 149.0(C), 145.2 (C), 142.4 (CH), 137.1(C), 129.8
(CH), 128.5 (CH), 127.7 (CH), 126.5 (CH), 119.7 (C), 113.0
(CH), 112.2 (C), 69.9 (C), 55.0 (CH3), 52.4 (CH2), 49.2
(CH2), 48.0 and 46.6 (CH2), 43.7 (CH2), 40.9 (CH2), 29.7
(C), 27.8 (CH3), 17.3 and 17.1 (CH3).
NMR (100 MHz, d6-DMSO) two rotamers: dZ196.4 and
196.2 (CO), 172.9 and 172.7 (CO), 170.4 and 170.2 (CO),
167.0 (CO), 166.1 (C), 157.5 (C), 156.4 and 156.4 (C),
150.8 and 150.6 (C), 149.7 (C), 144.8 (C), 138.1 and 137.8
(CHpur), 129.7 and 129.6 (CH); 128.3 and 128.3 (CH), 127.4
(CH), 126.3 (CH), 116.1 and 116.0 (C), 112.7 (CH), 107.2
and 107.1 (C), 69.7 and 69.5 (C(Ar)3), 54.8 (CH3), 52.3
(CH2), 49.3 and 49.3 (CH2), 48.1 (CH2), 46.6 and 46.5
(CH2), 42.7 and 42.5 (CH2), 40.7 (CH2), 29.6 (C), 27.7 and
27.7 (CH3), 17.2 and 17.0 (CH3) ppm.
4.1.12.
N-[2-(Thymin-1-yl)-acetyl]-N-{2-[1-(4,4-
dimethyl-2,6-dioxo-cyclohexyliden)-ethylamino]-ethyl}-
glycine (19T). To a solution of amine 5 (4.5 g, 15.1 mmol)
in EtOAc (17 mL) was added propylphosphonic acid cyclic
(50% solution in DMF) (17 mL, 15.7 mmol), acid 7 (2.9 g,
15.7 mmol), and DIPEA (5.57 mL, 32 mmol and the
reaction was stirred for 16 h. Then a mixture of ice-cold
water (650 mL) and saturated NaHCO3 solution (350 mL)
was added and stirred. The mixture was extracted with
EtOAc (100 mL !9). The organic layers were dried over
MgSO4 and concentrated to give a crude product which was
reprecipitated using EtOAc/diisopropyl ether mixture. The
4
4.1.10. N-{2-[N0 -(4-Methoxytrityl)-cytosin-1-yl]-acetyl}-
N-{2-[1-(4,4-Dimethyl-2,6-dioxo-cyclohexyliden)-ethyl-
amino]-ethyl}-glycine (19C(Mmt)). This compound was
synthesized in analogy to acid 19A(Mmt).
RfZ0.37 (BuOH/HOAc/MeOH 3/1/1); HPLC (method 2)
RtZ3.52 min; mp 174 8C; HRMS (ESC): m/z calcd for
C40H43N5O7 [MCNaC] 728.3055, found 728.30341H
NMR (400 MHz, CD3OD)Z two rotamers dZ7.42–7.25

8304
L. Bialy et al. / Tetrahedron 61 (2005) 8295–8305
solid was collected by filtration to give ester 18T (4.5 g,
65%) as a white solid.
(11 equiv). The resulting solution was mixed for 10 sec.
before adding to the resin (1 equiv) pre-swollen in DMF
and the mixture was then shaken for 3 h. Resins were
washed with DMF (3!3 mL), CH2Cl2 (3!3 mL) and
MeOH (3!3 mL),
Crude 18T (4.5 g, 9 mmol) was suspended in a 1:1 (v/v)
mixture of MeOH and 2 M Cs2CO3 (90 mL) and stirred for
1.5 h. The MeOH was evaporated in vacuo, the aqueous
phase was acidified with 4 N HCl (pHZ1), the solvents
were removed in vacuo. The residue was treated with hot
2-propanol and the hot suspension was filtrated. The filtrate
was collected and 2-propanol was removed in vacuo. The
residue was sonicated with water (25 mL) and filtrated to
give acid 19T (3.1 g, 77%) as a white solid.
4.3. General procedure for amino acids couplings
Fmoc amino acids (5.5 equiv), PyBOP (5 equiv) and HOBt
(5 equiv) were dissolved in DMF followed by addition of
DIPEA (11 equiv). The resulting solution was mixed for
10 s. before adding to resins (1 equiv) pre-swollen in DMF
and the mixture was then shaken for 3 h. Resins were
washed with DMF (3!3 mL), CH2Cl2 (3!3 mL) and
MeOH (3!3 mL),
RfZ0.13 (CH2Cl2/MeOH/HOAc 100/10/1); HPLC (method
1) RtZ7.59 min; mp 243–247 8C; HRMS (ESC): m/z calcd
for C21H28NaN4O7 [MCNaC] 471.1850, found 471.1846;
1H (300 MHz, d6-DMSO) two rotamers: dZ13.15/13.10
(m, 1H, Dde-NH), 11.30 and 11.27 (s, 1H, COOH), 7.32 and
4.3.1. Conjugate 24. HPLC (method 3) RtZ13.75 min;
MALDI-TOF: m/z calcd for C21H28NaN4O7 (average mass)
[MCHC] 4327.21 found 4237.45.
4
7.30 (d, 1H, J(H,H)Z1.5 Hz, CH), 4.64 and 4.48 (s, 2H,
CH2COO), 4.26 and 3.99 (s, 2H, CH2CO), 3.60–3.70 (m,
2H, CH2N), 3.43–3.57 (m, 2H, CH2N), 2.50 and 2.45 (s, 3H,
CH3C), 2.26 and (s, 4H, CH2-Dde), 1.72 (s, 6H, CH3-Dde),
0.91 and 0.90 (s, 6H, CH3-Dde) ppm; 13C NMR (75 MHz,
CD3COOD): dZ199.7 and 199.5 (CO), 175.8 (CO), 172.7
and 172.5 (CO), 169.2 and 168.5 (CO), 166.2 (C), 151.8
(CO), 142.8 (CH), 110.5 (C), 107.9 (C), 51.6 (CH2), 51.4
(CH2), 49.7 (CH2), 48.4 and 47.7 (CH2), 41.5 and 41.0
(CH2), 29.7 (C), 27.1 (CH3), 17.8 and 17.6 (CH3), 11.1
(CH3).
4.3.2. Conjugate 26. HPLC (method 3) RtZ12.26 min;
MALDI-TOF: m/z calcd for C179H227N72O48 (average
mass) [MCHC] 4155.1 found 4157.4.
Acknowledgements
This research was supported by the BBRSC (EBS). L.B. is
grateful to the DAAD for a scholarship.
4.1.13. Fmoc-Lys(Dde)-Rink amide PEGA resin (22).
Fmoc-Lys(Dde)-OH 2115 (851 mg, 1.6 mmol) and HOBt
(211 mg, 1.6 mmol) were dissolved in DMF (16 mL) and
shaken for 10 min. DIC (0.296 mL, 1.92 mmol) was added
to the mixture and stirred for a further 10 min. The resulting
solution was added to the PEGA Rink amine resin (10 g of
wet resin, loading 0.04 mmol/g) which was pre-swollen in
CH2Cl2 (3!25 mL). The mixture was shaken overnight and
the progress of the reaction was monitored by a qualitative
ninhydrin test. The resin was filtered and washed thoroughly
with DMF (3!20 mL), CH2Cl2 (3!20 mL) and MeOH
(3!20 mL). An analytical sample was taken and cleaved
using TFA/TIS (95:5) to give Fmoc-Lys(Dde)-NH2. HPLC
(method 1) RtZ9.62 min; MS (ESC): m/z calcd for
C31H38N3O5 [MCHC] 532.3, found 532.3.
References and notes
1. Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. Science
1991, 1497–1500.
2. Nielsen, P. E. Peptide Nucleic Acids: Protocols and
Applications; Horizon Biosience: Norfolk, 2004; p 318.
3. (a) Marin, V. L.; Roy, S.; Armitage, B. A. Expert Opin. Biol.
Ther. 2004, 4, 337–348. (b) Xodo, L. E.; Cogoi, S.; Rapozzi,
V. Curr. Pharm. Des. 2004, 7, 805–819. (c) Stender, H.;
Fiandaca, M.; Hyldig-Nielsen, J. J.; Coull, J. J. Microbiol.
Methods 2003, 48, 1–17.
4. (a) Koppelhus, U.; Nielsen, P. E. Adv. Drug Deliv. Rev. 2003,
55, 267–280. (b) Diaz-Mochon, J. J.; Bialy, L.; Watson, J.;
Sanchez-Martin, R. M.; Bradley, M. Chem. Commun. 2005,
26, 3316–3318.
4.1.14. Fmoc-Lys-Rink amide PEGA resin (23). Resin 22
was pre-swollen in DMF (3!25 mL) before adding a 20%
solution of imidazole/hydroxylamine hydrochloride
(1/0.75 equiv) in NMP/DMF (5/1, 25 mL).11 The resulting
mixture was shaken for 1 h and the resin was filtered and
washed thoroughly with DMF (3!20 mL), CH2Cl2 (3!
20 mL) and MeOH (3!20 mL). An analytical sample was
taken and coupled to Fmoc-Gly-OH (see general procedure
for amino acids coupling) before cleavage was carried out
using TFA/TIS (95:5) to give Fmoc-Lys(Fmoc-Gly)-NH2.
HPLC (method 1) RtZ10.49 min; MS (ESC): m/z calcd for
C38H38NaN4O6 [MCNaC] 669.2, found 669.22.
5. These monomers are commercially available through Applied
Biosystems.
6. Dueholm, K. L.; Egholm, M.; Behrens, C.; Christensen, L.;
Hansen, H. F.; Vulpius, T.; Petersen, K. H.; Berg, R. H.;
Nielsen, P. E.; Buchardt, O. J. Org. Chem. 1994, 59,
5767–5773.
7. Breipohl, G.; Knolle, J.; Langner, D.; O’Malley, G.; Uhlmann,
E. Bioorg. Med. Chem. Lett. 1996, 6, 665–670.
8. Thomson, S. A.; Josey, J. A.; Cadilla, R.; Gaul, M. D.;
Hassman, C. F.; Luzzio, M. J.; Pipe, A. J.; Reed, K. L.; Ricca,
D. J.; Wiethe, R. W.; S, A. Noble. Tetrahedron 1995, 51,
6179–6194.
4.2. General procedure for PNA monomer couplings
9. Rink, H. Tetrahedron Lett. 1987, 28, 3787.
10. Royo, M.; Farrera-Sinfreu, J.; Sole, L.; Albericio, F.
Tetrahedron Lett. 2002, 43, 2029–2032.
Dde PNA monomer 19 (5.5 equiv) and PyBOP (5 equiv)
were dissolved in DMF followed by the addition of NEM

L. Bialy et al. / Tetrahedron 61 (2005) 8295–8305
8305
11. Diaz-Mochon, J. J.; Bialy, L.; Bradley, M. Org. Lett. 2004, 6,
1127.
18. Pooga, M.; Soomets, U.; Hallbrink, M.; Valkna, M.; Saar, K.;
Rezaei, K.; Kahl, U.; Hao, J. X.; Xu, X. J.; Wiesenfeld-Hallin,
Z.; Hokfelt, T.; Bartfai, T.; Langel, U. Nat. Biotechnol. 1998,
16, 857–861.
12. Heimer, E. P.; Gallo-Torres, H. E.; Felix, A. M.; Ahmad, M.;
Lambros, T. J.; Scheidl, F.; Meienhofer, J. Int. J. Pep. Pro.
Res. 1984, 23, 203–211.
19. Koppelhus, U.; Awasthi, S. K.; Zachar, V.; Holst, H. U.;
Ebbesen, P. E.; Nielsen, P. E. Antisense Nucleic Acid Drug
Dev. 2002, 12, 51–63.
13. Kellman, B.; Chan, W. C.; Chhabra, S. R.; Bycroft, B. W.
Tetrahedron Lett. 1997, 38, 5391.
14. Breipohl, G.; Will, D. W.; Peyman, A.; Uhlmann, E.
Tetrahedron 1997, 53, 14671.
20. de Koning, M. C.; Filippov, D. V.; Meeuwenoord, N.; Overh,
M.; van der Marel, G. A.; van Boom, J. H. Tetrahedron Lett.
2002, 43, 8173–8176.
15. Chhabra, S. R.; Hothi, B.; Evans, D. J.; White, P. D.; Bycroft,
B. W.; Chan, W. C. Tetrahedron. Lett. 1998, 39, 1603–1606.
16. Good, L.; Awasthi, S. K.; Dryselius, R.; Larsson, O.; Nielsen,
P. E. Nat. Biotechnol. 2001, 19, 360–364.
21. Muaurinsh, Y.; Schram, J.; de Winter, H.; Blaton, N.; Peeters,
O.; Lescrinier, E.; Rozenscki, J.; Van Aerschot, A.; De Clerq,
E.; Busson, R.; Herdewijn, P. J. Org. Chem. 1997, 62,
2861–2871.
17. Cutrona, G.; Carpaneto, E. M.; Ulivi, M.; Roncella, S.; L.t, O.;
Ferrarini, M.; Boffa, L. C. Nat. Biotechnol. 2000, 18, 300–303.