Mixed-sequence pyrrolidine-amide oligonucleotide mimics: Boc(Z) synthesis and DNA/RNA binding properties

Article abstract of DOI:10.1039/b613386j

Pyrrolidine-amide oligonucleotide mimics (POMs) exhibit promising properties for potential applications, including in vivo DNA and RNA targeting, diagnostics and bioanalysis. Before POMs can be evaluated in these applications it is first necessary to synt

Full text of DOI:10.1039/b613386j

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Mixed-sequence pyrrolidine-amide oligonucleotide mimics: Boc(Z) synthesis
and DNA/RNA binding properties†
Roberta J. Worthington, Adam P. O’Rourke, Jordi Morral, T. H. Samuel Tan and Jason Micklefield*
Received 14th September 2006, Accepted 9th November 2006
First published as an Advance Article on the web 1st December 2006
DOI: 10.1039/b613386j
Pyrrolidine-amide oligonucleotide mimics (POMs) exhibit promising properties for potential
applications, including in vivo DNA and RNA targeting, diagnostics and bioanalysis. Before POMs can
be evaluated in these applications it is first necessary to synthesise and establish the properties of fully
modified oligomers, with biologically relevant mixed sequences. Accordingly, Boc-Z-protected
thyminyl, adeninyl and cytosinyl POM monomers were prepared and used in the first successful solid
phase synthesis of a mixed sequence POM, Lys-TCACAACTT-NH2. UV thermal denaturation studies
revealed that the POM oligomer is capable of hybridising with sequence selectivity to both
complementary parallel and antiparallel RNA and DNA strands. Whilst the duplex melting
temperatures (Tm) were higher than the corresponding duplexes formed with isosequential PNA, DNA
and RNA oligomers the rates of association/dissociation of the mixed sequence POM with
DNA/RNA targets were noticeably slower.
Introduction
Pyrrolidine-amide oligonucleotide mimics (POMs) 1 (Fig. 1) are
stereochemical and conformational mimics of natural nucleic
acids.1–5 Owing to protonation of the pyrrolidine ring, POMs
are positively charged, which aids solubility and could increase
affinity for target DNA and RNA. Previously we have shown
that a series of thyminyl and adeninyl POM oligomers can form
stable complexes with complementary DNA and RNA and in
some cases possess a noticeable kinetic selectivity for RNA over
DNA.1–5 A number of related pyrrolidine-containing oligonu-
cleotide mimics, or peptide nucleic acid analogues, have similarly
been developed.6–16 Several of these pyrrolidine-containing mimics
also exhibit promising nucleic acid recognition and other phys-
iochemical properties, which might be useful for a wide range
of applications including in vivo DNA and RNA targeting,17–19
diagnostics and bioanalysis,20,21 or programmed assembly of nano-
structures.22 Despite this, most of the results compiled so far,1–12,14,15
Fig. 1 Structure of pyrrolidine-amide oligonucleotide mimics (POMs) 1.
The Fmoc approach, described in the preceding paper,1 used monomers
with the exception of a few studies,13,16 are based on the properties
of chimeric oligomers containing a mix of PNA and pyrrolidine
containing Fmoc-backbone amino protecting groups, which are cleavable
with piperidine and standard acyl-protected bases (C, A and G), which are
cleaved with ammonia. A polystyrene resin functionalised with the Rink
amide linker allows oligomers to be cleaved by trifluoroacetic acid (TFA).
monomer units or fully modified pyrrolidinyl-homopolymers
(e.g. poly-T oligomers), which are not useful in real, practical
applications.17–22 Therefore, in order to realise the potential of
In this paper, the Boc-Z strategy is employed which utilises monomers
POM and the other pyrrolidine-containing nucleic acid mimics it
is neccessary to systematically evaluate the properties of a variety
of fully modified mixed sequence oligomers, which would be more
relevant for biological and analytical purposes.17–22
with Boc-backbone amino protecting groups, which are cleavable with
TFA, along with benzyloxycarbonyl(Z)-protected bases (C, A and G)
that are stable to TFA, but can be removed with trifluoromethanesulfonic
acid (TFMSA). A polystyrene resin functionalised with methylbenzhydry-
lamine (MBHA) groups is used as the solid support, in this case, allowing
oligomers to be cleaved with TFMSA, but not TFA.
School of Chemistry and Manchester Interdisciplinary Biocentre, The
University of Manchester, 131 Princess Street, Manchester, UK M1 7DN.
E-mail: jason.micklefield@manchester.ac.uk
† Electronic supplementary information (ESI) available: additional data,
general experimental methods for monomer and solid phase synthesis and
characterisation of compounds not presented in the main experimental
section, and methods employed in thermal denaturation experiments. See
DOI: 10.1039/b613386j
In the preceding paper1 we described the synthesis of Fmoc-
protected POM monomers, with standard acyl-protected nucle-
obases (Fig. 1). Using these monomers we were able to prepare
short homopolymers, using standard Fmoc-peptide chemistry.
However, the Fmoc approach failed to deliver longer mixed-
sequence POM oligomers, despite the fact that similar chemistry
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had been used for the efficient synthesis of PNA.23,24 Prior to
the development of the Fmoc strategy for PNA synthesis, Boc-
solid phase chemistry, with benzyloxycarbonyl (Z)-nucleobase
protecting groups was most widely used.24–26 The Boc-Z strategy
has a number of advantages over Fmoc chemistry,27 including
the stability of the protecting groups to the coupling conditions,
which reduces the occurrence of multiple couplings. Also, Boc
deprotection with trifluoroacetic acid (TFA) is fast and TFA can
help prevent aggregation of the growing oligomers.27 Additionally,
the resultingN-terminalamino groupremains protonated after de-
protection, which can reduce side reactions that involve cyclisation
of terminal free amino groups onto adjacent amide linkages. Such
intramolecular cyclisation reactions can result in six-membered
lactam side products in both PNA23,27 and POM3 synthesis, which
are equivalent to diketopiperazine formation occurring during
standard peptide synthesis. In this paper we demonstrate the first
successful synthesis of longer mixed-sequence POMs, using the
Boc-Z strategy, which has enabled the DNA and RNA hybridisa-
tion properties of a more relevant POM sequence to be evaluated.
phine and Boc-ON30 to afford Boc-protected amine 11 in 51%
yield. The cis-alcohol 11 was then inverted, as before, to give
trans-alcohol 13 via the formyl ester 12. The cis-alcohol 11 was
also inverted with concomitant tosylation to give 14, with methyl
p-toluenesulfonate under Mitsunobu’s conditions.31 Introduction
of N3-benzoylthymine into the pyrrolidine ring of the trans-
alcohol 13 under standard Mitsunobu conditions afforded the N3-
benzoylthymine derivative 8, in 70% yield, which was hydrolysed to
the required thyminyl Boc-acid 10. The advantage of this approach
is that potentially all the nucleobases could be introduced in the
penultimate step, via the trans-alcohol 13 or the tosylate 14 leading
to a more convergent synthesis of the required POM monomers.
With this in mind N6-Z-adenine was used to displace the tosylate
of 14 in the presence of K2CO3 and 18-crown-6 resulting in
the adeninyl derivative 15 in 40% yield. The regiochemistry
of the product 15 was established through comparison of 13C-
NMR chemical shifts with known N7- and N9-alkylated adeninyl
derivatives from the literature.32,33 During this transformation the
N6-Z-protecting group was lost, but was subsequently replaced
by treating 15 with freshly prepared 1-(benzyloxycarbonyl)-3-
ethylimidazolium tetrafluoroborate (‘Rapoport’s reagent’)34 to
afford 16 in 50% yield. Saponification and neutralization as before
revealed the Boc-Z adeninyl acid 17 in 75% yield.
Results and discussion
Synthesis of the Boc-Z-protected POM monomers
Initially the synthesis of the required cytosinyl monomer 25
was investigated via the trans-alcohol 5. However, attempts to
introduce N4-Z-cytosine into the pyrrolidine 5 by Mitsunobu
chemistry gave none of the desired N1-adduct and instead gave
the O2-adduct 19 in a very low yield. Displacement of the tosylate
18 with N4-Z-cytosine also gave exclusively the O2-adduct 19 in
67% yield. Conversely, treatment of tosylate 18 with N4-[p-(tert-
butyl)benzoyl]cytosine gave the desired N1-adduct 20 in a modest
32% yield, along with a minor amount of the O2-adduct 21.
As before, the regiochemistry was assigned through comparison
of 13C-NMR chemical shifts with known compounds,32,35 and
in the case of the N1-adduct 20 the structure and the relative
stereochemistry was ultimately confirmed by X-ray crystallogra-
phy (Fig. 2).‡ The regiochemical outcome of these reactions is
probably a reflection of the different electronic properties of the
N4-protecting groups. For example, the more electron withdrawing
p-(tert-butyl)benzoyl protecting group presumably decreases the
delocalisation of the lone pair of electrons from the N4-amino
group onto the O2-atom of the carbonyl group and, as a result,
predominately N1-alkylation would be favoured. To complete the
synthesis, the azide 20 was reduced to the amine with in situ Boc-
protection, using trimethylphosphine and Boc-ON30 to give 22 in
63% yield. Subsequently it was found that 22 could be prepared
more efficiently via substituting the tosylate of 14 with N4-[p-
(tert-butyl)benzoyl]cytosine in 46% yield. Removal of the N4-[p-
(tert-butyl)benzoyl]cytosine protecting group of 22 with sodium
methoxide in methanol resulted in the cytosine derivative 23 which
was treated with freshly prepared ‘Rapoport’s reagent’34 to give
the Z-protected cytosinyl monomer 24 in 92% yield over the two
In order to investigate the alternative Boc-Z approach for the
synthesis of POM mixed sequences, the appropriately protected
POM monomers were first prepared (Scheme 1). Accordingly the
amine HCl salt 2, used in the synthesis of the Fmoc-protected
POM monomers,1 was alkylated with methyl bromoacetate to
give methyl ester 3. The methyl ester, as opposed to the tert-butyl
ester used previously,1 allows for subsequent deprotection by mild
basic hydrolysis to give the required acid functionality without
jeopardising the Boc- and Z-protecting groups. The cis-alcohol
3 was transformed to the trans-formyl ester 4 using Mitsunobu’s
conditions. Cleavage of the formyl ester of 4 to give trans-alcohol
5 was achieved with potassium carbonate in anhydrous methanol.
Introduction of N3-benzoylthymine onto the pyrrolidine ring of 5,
was carried out under Mitsunobu conditions, to give the (2R,4R)
protected thymine derivative 6 in a yield of 75%, which depends
upon the presence of the additive sodium benzoate.28,29 Azide
reduction and isolation of the resulting amine was not possible
under the conditions used previously in the synthesis of Fmoc
monomers,1 due to the facile formation of a bicyclic lactam 7,
resulting from intramolecular attack of the amine on the methyl
ester. It was therefore necessary to carry out azide reduction with
in situ Boc protection. This was achieved, by the hydrogenation of
azide 6 over 10% Pd-C in the presence of di-tert-butyldicarbonate
(Boc-anhydride) to afford protected thyminyl derivative 8, along
with the de-benzoylated thyminyl derivative 9, in a combined yield
of 65%. Alternatively it was subsequently found that the azide 6
could be reduced more efficiently using trimethylphosphine in the
presence of 2-(tert-butoxycarbonyloxyimino)-2-phenylacetonitrile
(Boc-ON)30 to give 8 as the major product in 81% yield. Saponifi-
cation of both 8 and 9, followed by neutralization of the reaction
mixture with HCl resulted in Boc thyminyl acid 10 in 73 and 87%
yields from 8 and 9 respectively.
‡ Crystal data for compound 20: C23H29N7O4, CH4O, H2O, M = 517.59,
˚
orthorhombic, a = 7.7564(9), b = 13.3948(9), c = 25.589(3) A, V =
3
−1
˚
2658.6(5) A , T = 150(2) K, space group P212121, Z = 4, l = 0.095 mm
,
reflections collected 7393, independent reflections 3846 [R(int) = 0.1129],
In addition to this route, the possibility of introducing the
nucleobase, after azide reduction and Boc protection, was also
investigated. Accordingly, azide 3 was treated with trimethylphos-
2
R1 = 0.0956 [I > 2r(I)], wR = 0.3014 (all data). CCDC reference number
624250. For crystallographic data in CIF or other electronic format see
DOI: 10.1039/b613386j.
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Scheme 1 Reagents and conditions: a) DIEA, BrCH2CO2CH3, CH2Cl2, 0 ◦C → rt, 18 h; b) HCO2H, PPh3, DIAD, THF, −30 ◦C → rt, 18 h; c) NaOCH3
in CH3OH or K2CO3 in CH3OH, rt, 2 h; d) N3-benzoylthymine, PPh3, DIAD, PhCO2Na, THF, rt, 18 h; e) 10% Pd-C, H2, Boc anhydride, EtOAc, rt, 18 h
or PMe3, 2-(tert-butoxycarbonyloxyimino)-2-phenylacetonitrile (Boc-ON); f) 1M aq NaOH, THF, rt 4 h, then 0.1M HCl; g) N3-benzoylthymine, PPh3,
DIAD, THF, rt, 18 h; h) PMe3, Boc-ON, THF, −20 ◦C → rt, 1 h; i) CH3OTs, PPh3, DIAD, THF, −20 ◦C → rt, 20 h; j) N6-benzyloxycarbonyladenine,
K2CO3, 18-crown-6, DMF, 80 ◦C, 4 h; k) 1-(benzyloxycarbonyl)-3-ethylimidazolium tetrafluoroborate (‘Rapoport’s reagent’), CH2Cl2, rt, 18 h; l)
◦
N4-benzyloxycarbonylcytosine, K2CO3, 18-crown-6, DMF, 75 C, 16 h; m) N4-benzyloxycarbonylcytosine, PPh3, DIAD, THF, −25 ◦C → rt, 18 h;
◦
n) tosyl chloride, pyridine, 0 C → rt, 18 h; o) N4-[p-(tert-butyl)benzoyl]cytosine, K2CO3, 18-crown-6, DMF, 75 ◦C, 16 h; p) NaOCH3, CH3OH,
t
rt, 6 h. Abbreviations: BuBz = p-(tert-butyl)benzoyl, Bz = benzoyl, Boc = tert-butoxycarbonyl, DIAD = diisopropyl azodicarboxylate, DIEA =
diisopropylethylamine, Ts = tosyl.
steps. Saponification of 24 then revealed Boc-Z cytosinyl acid 25
in 90% yield. Despite this, we later found that Boc-Z cytosinyl
monomer 24 could be prepared directly from the trans-alcohol
13 which was coupled with N4-benzyloxycarbonylcytosine under
Mitsunobu’s conditions to give the N1-cytosinyl product 24 in 29%
yield, with the regiochemistry again confirmed by 13C NMR.32,35 It
is interesting to note that the regioselectivity in this case is opposite
to that observed in the reaction between the azido trans-alcohol
5 and N4-benzyloxycarbonylcytosine, which gave the O2-adduct
19. This may be due to participation of the Boc amino group
of 13, which could potentially form an H-bond with the more
electronegative O2-atom of the cytosinyl carbonyl group, in the
Fig. 2 X-Ray crystal structure of the cytosinyl POM free amine 20 which
transition state, which may direct nucleophilic attack via the N1-
is shown to exhibit a C5ꢀ-exo-type conformation with a pseudorotational
phase angle (P = −27◦) and backbone torsional angles (c = 63◦, d = 82◦
atom of the ambident nucleophile.
The availability of the X-ray crystal structure of the cytosinyl
POM monomer 20 (Fig. 2) gave the opportunity to consider the
conformation of the POM pyrrolidine ring as the free amine.
and e = 152◦) that correspond more closely with the torsional angles
of ribose in typical A-type RNA duplexes, rather than B-type DNA
duplexes.39
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Owing to nearest neighbour interactions36,37 it is not anticipated
that every pyrrolidine ring in a POM oligomer will be protonated
simultaneously at physiological pH. It is therefore useful to
consider the conformation of the POM monomer units in both
the protonated and the free amine forms. Analysis of the structure
of 20 reveals an overall C5ꢀ-exo conformat◦ion which is described by
a pseudorotation phase angle (P) of −27 .38,39 This conformation
is equivalent to a nucleotide C2ꢀ-exo conformation which most
closely matches the conformation of ribose in A-type RNA
duplexes. The backbone torsion angles (c = 63◦, d = 82◦ and
e = 152◦) are also close to the corresponding angles found in A-
type RNA duplexes.39 This suggests that the protonation state
of the pyrrolidine ring in the POM oligomers will have little
effect on the conformation of oligomers, and both states possess
the prerequisite conformation for hybridisation with RNA and
DNA. The fact that our earlier studies1–5 reveal that changes in
pH do affect UV thermal denaturation temperature (Tm) values
for complexes with DNA and RNA is therefore most likely due to
electrostatic as opposed to conformational effects.
and is superior to the modest average coupling efficiency of ca. 95%
which was obtained for the same synthesis of Lys-POM(T)5-Lys
26 using the Fmoc protocol.
Boc-solid phase synthesis of mixed-sequence POMs
The synthesis of Lys-TCACAACTT-NH2 was achieved following
a similar protocol to that used in the synthesis of the thyminyl
pentamer. However, it was necessary to use 5 equiv. of the POM
Boc amino acid for each coupling (along with 4.9 equiv. HBTU,
and 5.5 equiv. DIEA) and a monomer concentration of 0.1 M. In
addition, it was shown to be advantageous to employ double
couplings when extending from N-terminal adeninyl residues.
The POM mixed sequence was synthesised in a yield of 61% as
determined by analytical C18 HPLC (see ESI†), equating to an
average coupling efficiency of 95.2%. These coupling efficiencies
are respectable compared with the efficiencies typically resulting
from peptide or PNA synthesis. The POM-PNA chimera Lys-
TC*AC*AAC*TT-NH2 (where C* corresponds to a PNA cy-
tosinyl monomer) was synthesised via the same method. The yield
for the synthesis of the chimera was determined by analytical C18
HPLC as 68.7%, corresponding to an average coupling efficiency
of 96.3%. A number of additional biologically relevant mixed-
sequence POM oligomers have now been successfully synthesised
using this approach, which will be published separately.
Boc-solid phase synthesis of Lys-POM(T)5-Lys
In order to establish if Boc-solid phase chemistry is more efficient
than the Fmoc approach for the synthesis of POMs, the previously
prepared Lys-POM(T)5-Lys 26 1 (see ESI†) was re-synthesised
from monomer 10 by the standard Boc-PNA synthetic protocol.26
The pentamer was prepared on methylbenzhydrylamine low
loading (MBHA LL) functionalised resin which was adjusted at
the first coupling of N-a-Boc-N-e-2-chloro-Z-L-lysine to give a
loading of 0.12 mmol g−1 (ca. 20% of the maximum loading of the
resin). POM Boc-T amino acid 10 (2 equiv.) was then preactivated
with 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexa-
fluorophosphate (HBTU) (1.9 equiv.) and diisopropylethylamine
(DIEA) (2.2 equiv.) prior to subsequent coupling reactions, which
proceeded for 2 h and were monitored by the Kaiser test. Acetyl
capping ofany unreacted oligomer was carried outfollowedbyBoc
deprotection and repeated coupling. Cleavage from the resin was
effected by the ‘low-high’ TFMSA method26 to give Lys-POM(T)5-
Lys 26 in a yield of 95.8% as determined by analytical C18 HPLC
(see ESI†). This equates to an average coupling efficiency of 99.2%
Nucleic acid binding properties of POM Lys-TCACAACTT-NH2
In order to investigate the RNA and DNA hybridisation
properties of the mixed-sequence POM, UV thermal denatu-
ration/renaturation experiments were carried out with POM
Lys-TCACAACTT-NH2 and complementary RNA and DNA
oligonucleotides. Initial experiments were carried out under close
to physiological conditions using a 10 mM K2HPO4 buffer
solution, adjusted to a salt concentration of 0.12 M K+ and pH 7.0.
Under these conditions POM Lys-TCACAACTT-NH2 hybridises
with complementary antiparallel RNA 5ꢀ-AAGUUGUGA-3ꢀ
exhibiting a melting temperature (Tm) of 46 ◦C with a significant
hyperchromic shift of 24% (Fig. 3 and Table 1). Antiparallel, in this
case, is defined as the N-terminal (Lys-capped) end of the POM
Table 1 Tm values for POM Lys-TCACAACTT-NH2 vs. complementary nucleic acids
Tm/◦Ca (% hypochromicb/hyperchromic shiftc)
POM Lys-TCACAACTT-NH2
DNA 5ꢀ-TCACAACTT-3ꢀ
PNA Lys-TCACAACTT-NH2
Heating
Cooling
25a (25)b
26 (29)
23 (10)
20 (12)
Heating
Heatingc
25a (9)
n.t.e
Antiparallel RNA
5ꢀ-AAGUUGUGA-3ꢀ
Parallel RNA
46a (24)c
47d (30)
44 (29)
47d (39)
43 (15)
42d (24)
45 (16)
45d (42)
38a (15)c
24a (12)c
35a (8)c
n.t.
5ꢀ-AGUGUUGAA-3ꢀ
Antiparallel DNA
5ꢀ-AAGTTGTGA-3ꢀ
Parallel DNA
32 (14)
n.t.
5ꢀ-AGTGTTGAA-3
a Melting experiments were carried out at a concentration of 42 lM (in bases) of each strand in 10 mM K2HPO4, 0.12 M K+, pH 7.0 (total volume
1.0 cm3). UV absorbance (A260) was recorded with heating at 5 ◦C min−1 from 15 to 93 ◦C, cooling at 0.2 ◦C min−1 to 15 ◦C and heating at 0.2 ◦C min−1
to 93 ◦C. The Tm was determined from the first derivative of the slow heating and cooling curve. b Hypochromic shifts are indicated in parentheses and
were calculated as follows: [A(93 ◦C) − A(15 ◦C)] × 100/A(93 ◦C). c Hyperchromic shifts are indicated in parentheses and were calculated as for the
hypochromic shifts. d POM Lys-TCACAACTT-NH2 was incubated with the oligonucleotide for 12 h before being subjected to slow thermal denaturation
(0.2 ◦C min−1). e n.t. = no transition observed
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To explore this apparently slow hybridisation event, the POM
Lys-TCACAACTT-NH2 and antiparallel RNA target were
incubated for 12 h at room temperature, prior to melting at
◦
0.2 C min−1. Whilst the Tm extracted from this melting curve
was similar the hyperchromic shift was noticeably larger (30%)
(Fig. 3), compared with the standard heating/cooling ramps
initially employed. The larger hyperchromic shift is most likely
due to the extended incubation time (12 h), allowing a greater
portion of POM and RNA strands to anneal and is also indicative
of slow hybridisation. In addition to this a series of UV thermal
denaturation and renaturation curves were generated for the POM
and antiparallel RNA at decreasing rates of heating and cooling
from 1.0 to 0.1 ◦C min−1 (see ESI†). From this it can been seen that
whilst the Tm values extracted from the melting curves decrease
from 58 to 43 ◦C, whilst the Tm extracted from the cooling curves
increase from 20 to 26 ◦C, as the cooling rate is changed from 1.0 to
0.1 ◦C min−1. By extrapolation, this suggests that the true thermo-
dynamic Tm would lie within the range 30–40 ◦C (Fig. 4). Unex-
pectedly, we also found that POM Lys-TCACAACTT-NH2 can
hybridise to complementary parallel RNA 5ꢀ-AGUGUUGAA-3ꢀ
with a denaturation Tm of 44 ◦C under the same conditions.
Again, significant hysteresis is evident, indicating slow rates of
association/dissociation (see Table 1 and ESI† for more data).
Fig. 3 UV thermal denaturation curves and first derivatives for POM
Lys-TCACAACTT-NH2 vs. antiparallel RNA. (A) (i) shows the slow
◦
cooling (renaturation) curve (0.2 C min−1), (ii) the slow heating (denat-
◦
uration) cu◦rve (0.2 C min−1) and (iii) the slow heating (denaturation)
curve (0.2 C min−1) obtained immediately after the POM and RNA
were incubated at room temperature for 12 h is shown in grey. Control
experiments show that the fluctuations in the slow heating curves (curve ii)
are entirely due to the sample and not the instrumentation and therefore
remain uncorrected. These fluctuations may be due to slow, incomplete
hybridisation given that the same sample gives a very smooth melting
curve following incubation for 12h (curve iii). (B) (i) The corresponding
first derivatives obtained from slow cooling under standard conditions, (ii)
slow heating under standard conditions and (iii) slow heating following
incubation for 12 h at room temperature shown in grey.
Fig. 4 Thermal denaturation (ꢀ) and renaturation (᭹) transition tem-
peratures (Tm) for POM Lys-TCACAACTT-NH2 vs. antiparallel RNA
as a function of rates of heating (ꢀ) and cooling (᭹), as extracted from
the first derivatives shown in the ESI† (Fig. S6B). As the rates of heating
and cooling are reduced the Tm values extracted from renaturation and
denaturation approach one another, allowing the true thermodynamic Tm
to be extrapolated to be within the range of ca. 30–40 ◦C.
hybridising with the 3ꢀ-end of the RNA target. In comparison, the
isosequential DNA:RNA heteroduplex melts with a Tm of 25 ◦C
whilst the PNA Lys-TCACAACTT-NH2:RNA heteroduplex ex-
hibits a Tm of 38 ◦C. Noticeable hysteresis was observed between
the cooling and heating curves for these UV melting experiments
with POM Lys-TCACAACTT-NH2 and the antiparallel RNA
(Fig. 3). As a consequence, the Tm extracted from the denaturation
(cooling curve) was 25 ◦C, which is considerably lower than the Tm
extracted from the melting curve (DTm = −21 ◦C). This indicates
that the rate of heating/cooling employed (0.2 ◦C min−1) is faster
than the rate of association/dissociation of the POM and RNA,
such that a true equilibrium is not attained. In contrast, the isose-
quential DNA:RNA and PNA:RNA UV thermal denaturation
and renaturation shows little or no hysteresis, under the same
conditions.
Preliminary UV thermal denaturation experiments also show
that POM Lys-TCACAACTT-NH2 hybridises with complemen-
tary antiparallel DNA 5ꢀ-AAGTTGTGA-3ꢀ with a Tm (heating)
of 43 ◦C (Table 1) under close to physiological conditions (0.12 M
K+ and pH 7.0). In comparison the isosequential DNA duplex
has a Tm of 32 ◦C, whereas the PNA Lys-T◦CACAACTT-NH2
antiparallel DNA duplex exhibits a Tm of 35 C under the same
conditions. Hysteresis is again evident between the heating and
cooling curves for POM Lys-TCACAACTT-NH2 with antiparal-
◦
lel DNA (DTm = −20 C); similarly, the denaturation Tm values
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decrease, whilst the renaturation Tm values increase as the rate
of cooling is progressively decreased. In addition to this it was
also evident that POM Lys-TCACAACTT-NH2 hybridises with
complementary parallel DNA 5ꢀ-AGUGUUGAA-3ꢀ exhibiting a
similar Tm of 45 ◦C with noticeable hysteresis. Finally we explored
the sequence selectivity of DNA hybridisation with POM Lys-
TCACAACTT-NH2 using a number of mismatched antiparallel
DNA oligomers. For example, the UV thermal denaturation curve
for the hybridisation of POM Lys-TCACAACTT-NH2 to DNA 5ꢀ-
AAGTTCTGA-3ꢀ (containing a single CC mismatch underlined)
resulted in a noncooperative (near linear) hyperchromic shift from
which it was not possible to define a transition melting point (Tm)
(Fig. 5). UV thermal denaturation experiments for POM Lys-
TCACAACTT-NH2 and a double mismatch antiparallel DNA
sequence, 5ꢀ-AAGGTATGA-3ꢀ again showed a noncooperative
transition, from which a Tm could not be determined.
structures, resulting in a more random coil in the single-stranded
state, which might facilitate faster hybridisation. Despite this, the
POM/PNA chimera did not show any evidence of hybridisation
with complementary parallel and antiparallel RNA or DNA in
UV thermal denaturation/renaturation experiments. Whilst this is
not informative for rationalising the slow rates of hybridisation of
POM with DNA or RNA, it does demonstrate how the behaviour
of a chimera cannot be used to understand the properties of
uniformly modified oligomers per se. For example, many studies
with PNA analogues, including pyrrolidine-modified PNAs, are
based on the effects of inserting one or more new modified
monomers within a PNA strand resulting in chimeras.15 Typically,
if the Tm of the chimera is higher than the standard unmodified
PNA the modification is viewed as potentially useful, whilst
modifications that reduce the Tm are discarded.15 Clearly, the
results presented here show the obvious danger of basing any
conclusions on the chimeric approach. Indeed, in the absence
of any other information about PNA, if we were to follow the
logic described above, we would conclude that PNA is not a
potentially useful nucleic acid mimic, because insertion of PNA
monomers into a POM abolishes its ability to hybridise with DNA
and RNA. Moreover, the results presented here and earlier1–5 also
show that whilst studying the DNA and RNA properties of fully
modified homopolymers is better than using chimeras, it too can
lead to conclusions which are not necessarily general. Indeed,
it is well known that DNA homopolymers, for example, behave
differently to random, mixed-sequence nucleic acids.40,41 Thus if
any nucleic acid mimic is to be considered for potential DNA and
RNA hybridisation-based applications, a variety of fully modified
mixed sequences must necessarily be evaluated. To this end we are
currently investigating the biophysical properties of other mixed-
sequence POMs, which will be the subject of future publications.
Fig. 5 Thermal denaturation curves for POM Lys-TCACAACTT-NH2
vs. antiparallel DNA (5ꢀ-AAGTTGTGA-3ꢀ) and antiparallel DNA con-
taining a single CC-mismatch underlined (5ꢀ-AAGTTCTGA-3ꢀ). Strands
were incubated for 12 h at room temperature prior to slow melting
(0.2 ◦C min−1).
Conclusion
Boc-solid phase chemistry, with benzyloxycarbonyl (Z)-
nucleobase protecting groups, has been used in the early synthesis
of PNA oligomers. To investigate whether this approach can be
applied to the synthesis of pyrrolidine-amide oligonucleotide mim-
ics, Boc-Z-protected thyminyl, adeninyl and cytosinyl monomers
have been prepared. Solid phase synthesis of POM oligomers using
the Boc-Z approach proved much more efficient than the Fmoc
approach which had been investigated earlier.1 Indeed, the Boc-Z
chemistry enabled the synthesis of the first longer mixed-sequence
POM Lys-TCACAACTT-NH2 to be completed, in an overall
yield of 61%, with an average coupling efficiency of 95%. UV
thermal denaturation studies with this more biologically relevant
POM oligomer and complementary DNA and RNA targets were
then performed. This revealed that the POM Lys-TCACAACTT-
NH2 is capable of hybridising with sequence specificity to both
complementary parallel and antiparallel RNA and DNA strands.
The hysteresis between the heating (denaturation) and cooling (re-
naturation) curves in these UV melting experiments indicated that
the rates of association/dissociation of the mixed-sequence POM
with DNA/RNA targets are slow compared with hybridisation of
isosequential PNA, DNA and RNA oligomers. Interestingly, the
introduction of PNA monomers into the POM mixed-sequence
strand, to form a chimera, completely abolishes the hybridisation
with DNA and RNA. Currently, several more mixed-sequence
Overall, these preliminary results indicate that the mixed-
sequence POM is able to hybridise sequence-specifically to both
complementary antiparallel and parallel DNA and RNA targets
to form duplexes with roughly equal stability. In all cases there is
hysteresis between heating and cooling curves, which is indicative
of slow rates of association and dissociation. This is in contrast
to our earlier studies,1–5 where we found that homopolymers
hybridise slowly with DNA, but faster with RNA. Whilst there
are many possible reasons for the slow rates of hybridisation of
POM, compared with PNA or natural nucleic acids, one possible
cause could be the increased rigidity in the POM backbone.
This may result in the formation of a stable secondary structure
in the single-stranded POMs, which have conformations that
do not facilitate hybridisation. In this case the rate-determining
step in binding to DNA and RNA may be the conformational
reorganisation of the POM backbone into a more linear structure
which enables base pairing to take place. To explore this possibility
we prepared a POM/PNA chimera Lys-TC*AC*AAC*TT-NH2,
which contains three PNA cytosinyl units (C*). It is expected
that the PNA monomers would be more flexible than the POM
monomers and might therefore disrupt the formation of secondary
254 | Org. Biomol. Chem., 2007, 5, 249–259
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POMs, including structural variants, which are designed for duplex
and triplex formation, are being evaluated in order to assess the
generality of these findings.
HCO2H (0.36 mL, 9.54 mmol). The solution was cooled to −20 ◦C
and DIAD (1.90 mL, 9.65 mmol) was added. The reaction mixture
was allowed to warm to room temperature and stirred under N2
for 16 h. Solvent was removed under reduced pressure and flash
chromatography (2 : 1 hexane–EtOAc) afforded formyl ester 12
(1.71 g, 74%) as a pale yellow oil. Rf 0.2 (2 : 1 hexane–EtOAc);
[a]D25 +63.0◦ (c = 1.0, CH3OH); mmax(KBr;)/cm−1: 3397 (NH), 1719
(CO); 1H NMR (400 MHz, CDCl3): d 1.44 (9H, s, C(CH3)3), 1.97–
2.04 (2H, m, HaHb3), 2.67 (1H, dd, J 10.8, 3.1 Hz, Ha5), 3.03–3.12
(2H, m, H2, Ha6), 3.35 (1H, d, J 17.2 Hz, Ha7), 3.37–3.41 (1H, m,
Hb6), 3.54 (1H,d, J 17.2 Hz, Hb7), 3.67 (1H, dd, J 10.8, 6.0 Hz Hb5),
3.72 (3H, s, OCH3), 5.09, (1H, br s, NH), 5.26 (1H, br s, H4), 8.00
(1H, s, CHO); 13C NMR (100.6 MHz, CDCl3): d 28.3 (C(CH3)3),
34. 9 (C3), 40.5 (C6), 51.7 (O-CH3), 53.8 (C7), 59.7 (C5), 61.0
Experimental
(2R,4R)-2-Azidomethyl-4-hydroxy-N-(methoxycarbonylmethyl)-
pyrrolidine (3)
To a stirred suspension of pyrrolidine HCl salt 2 1 (11.5 g,
64.1 mmol) in anhydrous CH2Cl2 (80 mL), under N2, was added
diisopropylethylamine (DIEA) (25.7 mL, 147.5 mmol) at 0 ◦C
until dissolution. Methyl bromoacetate (7.72 mL, 83.3 mmol) was
added dropwise at 0 ◦C and the reaction mixture stirred for a
further 30 min at this temperature followed by 18 h at room temper-
ature. Solvent was subsequently removed under reduced pressure.
Purification by column chromatography (gradient elution using
3 : 1 hexane–EtOAc → 1 : 1 hexane–EtOAc,) gave methyl ester 3
(10.9 g, 79%) as a pale yellow oil. Rf 0.37 (3 : 1 EtOAc–hexane); [a]D25
+36.5◦ (c = 1.1, CHCl3); mmax (KBr)/cm−1: 3386 (OH), 2955 and
2858 (CH), 2101 (N3), 1740 and 1204 (CO); 1H NMR (400 MHz,
CDCl3): d 1.67–1.74 (1H, m, Ha3), 2.32–2.42 (1H, m, Hb3), 2.67
(1H, d, J 8.2 Hz, OH), 2.95 (1H, dd, J 9.7, 3.9 Hz, Ha5), 3.09 (1H,
dd, J 9.7, 0.7 Hz, Hb5), 3.14–3.20 (1H, m, H2), 3.37 (1H, dd, J
12.3, 4.3 Hz, Ha6), 3.49 (1H, dd, J 12.3, 4.3 Hz, Hb6), 3.52 (1H, d,
J 17.4 Hz, Ha7), 3.60 (1H, d, J 17.4 Hz, Hb7), 3.71 (3H, s, OCH3),
4.23–4.30 (1H, m, H4); 13C NMR (75.5 MHz, CDCl3): d 38.6
(C3), 51.5 (CO2CH3), 52.5 (C7), 54.7 (C6), 59.9 (C2), 62.0 (C5),
70.6 (C4), 171.3 (CO2CH3); m/z (ES): 215.1 ([M + H]+, 100%),
237.1 ([M + Na]+, 60%); HRMS m/z (ES): 215.1138 ([M + H]+,
C8H15N4O3 requires m/z, 215.1144).
t
(C2), 72.5 (C4), 79.2 (C(CH3)3), 156.2 (CO2 Bu), 160.5 (CHO),
171.3 (CO2Me); m/z (ES): 399 ([M + Na]+ 100%), 317 ([M + H]+
20%); HRMS m/z (ES): 317.1706 ([M + H]+, C13H25O5N2 requires
m/z, 317.1707).
(2R,4S)-2-[(tert-Butoxycarbonyl)aminomethyl]-4-hydroxy-
N1-(methoxycarbonylmethyl)-pyrrolidine (13)
To a solution of formyl ester 12 (750 mg, 2.37 mmol) in anhydrous
CH3OH (5 mL) was added anhydrous sodium methoxide (20 mg,
0.37 mmol). The reaction mixture was stirred under N2 at room
temperature for 1.5 h. Solvent was removed under reduced
pressure and column chromatography (EtOAc) afforded alcohol
13 (597 mg, 87%) as a pale yellow oil. Rf 0.2 (EtOAc); [a]2D5 +49.6◦
(c = 2.0, CH3OH); mmax(KBr)/cm−1: 3405br (OH), 1735 and 1688
(CO); 1H NMR (300 MHz, CDCl3): d 1.43 (9H, s, C(CH3)3), 1.78
(1H, ddd, J 13.2, 9.7, 5.2 Hz Ha3), 1.91 (1H, dd, J 13.2, 6.4 Hz,
Hb3), 2.66 (1H, d, J 11.1 Hz, Ha5), 3.01 (1H, ddd, J 13.6, 4.2, 3.6,
Hz, Ha6), 3.20–3.36 (2H, m, H2 and Hb6), 3.46 (1H, d, J 11.1 Hz,
Hb5), 3.49 (1H, d, J 17.9 Hz, Ha7), 3.56 (1H, d, J 17.9 Hz, Hb7),
(2R,4R)-2-[(tert-Butoxycarbonyl)aminomethyl]-4-hydroxy-N1-
(methoxycarbonylmethyl)-pyrrolidine (11)
3.71 (3H, s, OCH3), 4.27 (1H, br s, H4), 4.96, (1H, br s, OH); 13
C
To a solution of azide 3 (110.3 mg, 0.52 mmol) in THF (1.5 mL)
was added PMe3 (1 M solution in THF, 0.60 mL, 0.60 mmol) fol-
lowed by 2-(tert-butoxycarbonyloxyimino)-2-phenylacetonitrile
(Boc-ON) (155 mg, 0.63 mmol). The reaction mixture was stirred
at room temperature for 1 h. Solvent was removed under reduced
pressure and the crude product was purified by flash chromatog-
raphy (EtOAc) to afford Boc-protected amine ◦11 (52.6 mg, 53%)
as a pale yellow oil. Rf 0.2 (EtOAc); [a]2D5 + 64.7 (c = 2, CH3OH);
NMR (75.5 MHz, CDCl3): d 28.4 (C(CH3)3), 29.7 (C3), 39.0 (C6),
51.8 (CO2CH3), 52.5 (C7), 60.1 (C2), 62.0 (C5), 70.9 (C4) 79.2
t
(C(CH3)3), 156.3 (CO2 Bu), 172.9 (CO2CH3); m/z (ES): 289 ([M +
H]+ 50%); HRMS m/z (ES): 289.1768 ([M + H]+, C13H25O5N2
required m/z, 289.1763).
(2R,4S)-2-[(tert-Butoxycarbonyl)aminomethyl]-4-(toluene-4-
sulfonyloxy)-N1-(methoxycarbonylmethyl)-pyrrolidine (14)
1
mmax(KBr)/cm−1: 3381 br (OH), 1743 and 1692 (CO); H NMR
(400 MHz, CDCl3): d 1.42 (9H, s, C(CH3)3), 1.66 (1H, dd, J 14.1,
5.4 Hz, Ha3ꢀ), 2.28 (1H, ddd, J 14.1, 9.1, 6.5, Hz, Hb3ꢀ), 2.72 (1H,
dd, J 9.6, 3.7 Hz, Ha5ꢀ), 2.89–2.91 (1H, m, H2ꢀ), 3.06–3.13 (2H, m,
Ha6ꢀ and Hb5ꢀ), 3.28–3.33 (1H, m, Hb6ꢀ), 3.31 (1H, d, J 17.2 Hz,
Ha7ꢀ), 3.50 (1H, d, J 17.2 Hz, Hb7ꢀ), 3.69 (3H, s, OCH3), 4.26 (1H,
br s, H4), 5.44 (1H, br s, NH); 13C NMR (100.6 MHz, CDCl3): d
28.4 (C(CH3)3), 38.0 (C3ꢀ), 41.4 (C6ꢀ), 51.7 (CO2CH3), 53.0 (C7ꢀ),
To a solution of alcohol 11 (1.50 g, 5.22 mmol) in anhydrous THF
(50 mL) at −20 ◦C was added DIAD (1.24 mL, 6.30 mmol) and
methyl p-toluenesulfonate (0.94 mL, 6.23 mmol) followed by PPh3
(1.64 g, 6.25 mmol) portionwise under N2. The reaction mixture
was allowed to warm to room temperature and stirred under N2 for
20 h. Solvent was removed under reduced pressure and the crude
product purified by flash chromatography (1 : 1 Et2O–CH3Ph)
to afford p-toluenesulfonate 14 (1.35 g, 59%) as a pale yellow
t
61.1 (C2ꢀ), 62.5 (C5ꢀ), 70.1 (C4ꢀ), 79.1 (C(CH3)3), 156.6 (CO2 Bu),
171.6 (CO2CH3); m/z (ES): 289 ([M + H]+ 100%); HRMS m/z
(ES): 289.1756 ([M + H]+, C13H25O5N2 requires m/z, 289.1758).
oil. Rf 0.4 (1 : 1 Et2O–CH3Ph); [a]25 +24.4◦ (c = 2.0, CH3OH);
D
mmax(KBr)/cm−1: 3400 (NH), 1750 and 1712 (CO), 1361 and 1186
1
(SO2O); H NMR (400 MHz, CDCl3): d 1.41 (9H, s, C(CH3)3),
(2R,4S)-2-[(tert-Butoxycarbonyl)aminomethyl]-4-formyloxy-
N-(methoxycarbonylmethyl)-pyrrolidine (12)
1.84 (1H, ddd, J 15.1, 14.7, 7.8 Hz, Ha3ꢀ), 1.97 (1H, dd, J 14.7,
5.1 Hz, Hb3ꢀ), 2.45 (3H, s, tosyl CH3), 2.75 (1H, dd, J 11.3, 3.7 Hz,
Ha5ꢀ), 2.97 (1H, ddd, J 14.0, 3.6, 3.3 Hz, Ha6ꢀ), 3.01–3.06 (1H,
m, H2ꢀ), 3.22–3.36 (1H, m, Hb6ꢀ), 3.29 (1H, d, J 17.0 Hz, Ha7ꢀ),
To a solution of alcohol 11 (2.11g, 7.32 mmol) in anhydrous THF
(35 mL) was added PPh3 (2.49 g, 9.49 mmol) and anhydrous
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3.48 (1H, d, J 17.0 Hz, Hb7ꢀ), 3.51 (1H, dd, J 11.3, 5.7 Hz, Hb5ꢀ),
3.70 (3H, s, OCH3), 4.87 (1H, br s, H4ꢀ), 5.05 (1H, br s, NH),
7.34 (2H, d, J 8.2 Hz, tosyl aromatic), 7.76 (2H, d, J 8.2 Hz, tosyl
aromatic); 13C NMR (100.6 MHz, CDCl3): d 21.7 (tosyl CH3), 28.3
(C(CH3)3), 35.1 (C3ꢀ), 40.5 (C6ꢀ), 51.8 (CO2CH3), 54.2 (C7ꢀ), 59.9
(C5ꢀ), 61.3 (C2ꢀ), 79.3 (C(CH3)3), 79.6 (C4ꢀ), 127.7 (tosyl aromatic
CH), 129.9 (tosyl aromatic CH), 133.5 (tosyl p-C), 144.9 (tosyl
Hz, Hb3ꢀ), 2.51–2.59 (2H, m, Ha5ꢀand H2ꢀ), 2.70 (1H, d, J 14.5 Hz,
Ha6ꢀ), 3.08 (1H, dd, J 14.5, 3.0 Hz, Ha7ꢀ), 3.26 (1H, d, J 11.5 Hz,
Hb5ꢀ), 3.33 (1H, d, J 14.5 Hz, Hb6), 3.45 (1H, d, J 14.5 Hz, Hb7ꢀ),
4.83–4.95 (1H, m, H4ꢀ), 8.30 (1H, s, H6);13C NMR (75.4 MHz,
CDCl3): d 13.2 (thymine CH3), 29.1 (C(CH3)3), 37.1 (C3ꢀ), 40.4
(C6ꢀ), 54.0 (C4ꢀ), 59.5 (C7ꢀ), 60.5 (C5ꢀ), 65.3 (C2ꢀ), 80.1 (C(CH3)3),
t
111.8 (C5), 141.5 (C6), 154.1 (C2), 159.5 (CO2 Bu), 168.0 (C4),
179.5, (CO2H); m/z (ES): 405 ([M + Na]+, 20%), 383.1 ([M + H]+,
t
ipso-C), 156.2 (CO2 Bu), 171.4 (CO2CH3); m/z (ES): 443 ([M +
H]+ 100%); HRMS m/z (ES): 443.1857 ([M + H]+, C20H31O7N2S
requires m/z, 443.1852).
45), 327.1 ([M − Bu]+, 100); HRMS m/z (ES): 383.1922 ([M +
t
H]+, C17H27N4O6 require m/z, 383.1931).
(2ꢀR,4ꢀR)-2ꢀ-[(tert-Butoxycarbonyl)aminomethyl]-4ꢀ-(N3-benzoyl-
thymin-1-yl)-N1ꢀ-(methoxycarbonylmethyl)-pyrrolidine (8)
(2ꢀR,4ꢀR)-2ꢀ-[(tert-Butoxycarbonyl)aminomethyl]-4ꢀ-(adenin-9-yl)-
N1ꢀ-(methoxycarbonylmethyl)-pyrrolidine (15)
To a solution of alcohol 13 (574 mg, 1.99 mmol) in anhydrous THF
(7.5 mL) was added N3-benzoylthymine42 (555 mg, 2.41 mmol),
and PPh3 (628 mg, 2.39 mmol) under N2. The mixture was cooled
to −20 ◦C and DIAD (0.54 mL, 2.74 mmol) was added dropwise.
The reaction mixture was allowed to warm to room temperature
and stirred for 18 h. Solvent was removed under reduced pressure
and flash chromatography (1 : 1 hexane–EtOAc, Rf 0.3) afforded
thyminyl derivative 8 (681 mg, 68%) as a white foam. Mp 75–
To a solution of p-toluenesulfonate 14 (270 mg, 0.61 mmol) in an-
hydrous DMF (4.5 mL) was added N6-benzyloxycarbonyladenine
(280 mg, 1.04 mmol), K2CO3 (144 mg, 1.04 mmol) and 18-crown-
6 (81 mg, 0.31 mmol). The reaction mixture was stirred at 80 ◦C
under N2 for 4 h. H2O (250 mL) was added to the reaction mixture
and the mixture was extracted with EtOAc (5 × 250 mL). The
organic fractions were combined and dried over MgSO4, filtered
and then evaporated under reduced pressure. The crude product
was purified by flash chromatography (3% CH3OH in CHCl3) to
afford adeninyl derivative 15 (111 mg, 45%) as a white foam. Rf
◦
77 C (hexane/EtOAc); [a]2D5 −25.07 (c = 0.5, CH3OH); mmax
(NaCl)/cm−1: 3376 (NH), 2978 (CH), 1745, 1698 and 1653 (CO);
kmax(CH3OH)/nm 253.0; 1H NMR (300 MHz, CDCl3): d 1.29 (s,
9 H, C(CH3)3), 1.70 (1H, m, Ha3ꢀ), 1.89 (3H, s, thymine CH3), 2.43
(1H, ddd, J 15.0, 8.0, 8.0 Hz, Hb3ꢀ), 2.55–2.75 (2H, m, Ha5ꢀ and
H2ꢀ), 2.99 (1H d, J 14.5 Hz, Ha6ꢀ), 3.08 (1H, d, J 17.5 Hz, Ha7ꢀ),
3.19 (1H, d, J 11.0 Hz, Hb5ꢀ), 3.33 (1H, dd, J 14.5, 8.5 Hz, Hb6ꢀ),
3.49 (1H, d, J 17.5 Hz, Hb7ꢀ), 3.63 (3H, s, OCH3), 4.93 (1H, m,
H4ꢀ), 7.34 (2H, t, J 7.5 Hz, Bz CH), 7.49 (2H, t, J 7.5 Hz, Bz
CH), 7.76 (1H, d, J 8.0 Hz, Bz CH), 8.02 (1H, s, H6); 13C NMR
(75.4 MHz, CDCl3): d 13.2 (thymine CH3), 28.7 (C(CH3)3), 36.4
(C3ꢀ), 40.3 (C6ꢀ), 52.3 (OCH3), 52.4 (C4ꢀ), 53.2 (C7ꢀ), 59.5 (C5ꢀ),
63.2 (C2ꢀ), 80.0 (C(CH3)3), 111.5 (C5), 129.5 (Bz CH), 130.8 (Bz
CH), 132.1 (Bz C), 135.3 (Bz CH), 138.2 (C6), 150.4 (C2), 156.7
◦
0.1 (3% CH3OH in CHCl3); mp 52–54 C (CHCl3); [a]25 +65.9◦
D
(c = 2.0, CH3OH); mmax(KBr)/cm−1: 3327 and 3183 (NH), 1743
1
and 1696 (CO); kmax(CH3OH)/nm: 261.0; H NMR (400 MHz,
CDCl3): d 1.31 (9H, s, C(CH3)3), 1.92 (1H, ddd, J 14.3, 7.0, 2.5 Hz,
Ha3ꢀ), 2.63 (1H, ddd, J 14.3, 8.6, 8.4 Hz, Hb3ꢀ), 2.94–2.98 (1H, m,
H2ꢀ), 3.02 (1H, dd, J 10.4, 6.3 Hz, Ha5ꢀ), 3.11 (1H, d, J 14.4 Hz,
Ha6), 3.30 (1H, d, J 17.4 Hz, Ha7ꢀ), 3.36 (1H, dd, J 14.4, 8.3 Hz,
Hb6ꢀ), 3.54 (1H, d, J 10.4 Hz, Hb5ꢀ), 3.67 (1H, d, J 17.4 Hz,
Hb7ꢀ), 3.72 (3H, s, OCH3), 5.06 (1H, br s, H4), 5.34 (1H, br s,
NH), 6.22 (2H, br s, NH2), 8.29 (1H, s, H2), 8.41 (1H, s, H8);
13C NMR (100.6 MHz, CDCl3): d 28.2 (C(CH3)3), 36.1 (C3ꢀ), 40.4
(C6ꢀ), 51.7 (CO2CH3), 51.8 (C2ꢀ), 52.8 (C7ꢀ), 59.4 (C5ꢀ), 62.0 (C4ꢀ),
79.3 (C(CH3)3), 119.4 (C5), 139.4 (C8), 149.5 (C4), 152.4 (C2),
t
(CO2 Bu), 163.2 (C4), 169.6 (Bz CO), 171.7 (CO2CH3); m/z (ES):
501.1 ([M + H]+, 80%); HRMS m/z (ES): 501.2344 ([M + H]+,
C25H33N4O7 requires m/z, 501.2349).
155.4 (C6), 156.2 (CO2 Bu), 171.2 (CO2CH3); m/z (ES): 406 ([M +
t
H]+ 100%); HRMS m/z (ES): 406.2194 ([M + H]+, C18H28O4N7
requires m/z, 406.2197).
(2ꢀR,4ꢀR)-2ꢀ-(tert-Butoxycarbonylaminomethyl)-4ꢀ-(thymin-1-yl)-
pyrrolidine-1ꢀ-yl-acetic acid (10)
(2ꢀR,4ꢀR)-2ꢀ-(tert-Butoxycarbonylaminomethyl)-4ꢀ-(N6-benzyloxy-
carbonyladenin-9-yl)-N1ꢀ-(methoxycarbonylmethyl)-pyrrolidine (16)
To a solution of benzoyl-protected derivative 8 (650 mg,
1.30 mmol) in THF (5 mL) was added 1 M aqueous NaOH
(3.25 mL, 3.25 mmol). The mixture was stirred at room tempera-
ture for 3 h then a second portion of 1 M aqueous NaOH (3.25 mL,
3.25 mmol) was added to the reaction mixture which was stirred
for a further 3 h. THF was removed under a stream of nitrogen
and the pH of the remaining aqueous solution was adjusted to
7 by addition of 0.1 M aqueous HCl. Water was removed under
reduced pressure and the resulting white residue was submitted
to column chromatography (EtOAc–CH3OH 7 : 3) followed by
To
a freshly prepared solution of 1-(benzyloxycarbonyl)-
3-ethylimidazolium tetrafluoroborate (‘Rapoport’s reagent’)34
(1.49 g, 4.7 mmol) in anhydrous CH2Cl2 (6 mL) was added adenine
derivative 15 (200 mg, 0.49 mmol) and the reaction mixture
stirred at room temperature under N2 for 18 h. Saturated aqueous
NaHCO3 (10 mL) was then added to the reaction mixture and
the aqueous solution was extracted with CH2Cl2 (4 × 25 mL).
The combined organic extracts were dried over MgSO4, filtered
and evaporated under reduced pressure. The crude product was
purified by flash chromatography (2% CH3OH in CHCl3) to afford
the benzyloxycarbonyl-protected product 16 (158 mg, 60%) as a
white foam. Rf 0.1 (2% CH3OH in CHCl3); mp 58–60 ◦C (CHCl3);
[a]2D5 +58.0◦ (c = 1.0, CH3OH); mmax(KBr)/cm−1: 3373 (NH), 1747
R
reversed-phase chromatography (BondElutꢀ C18, H2O–CH3CN
9 : 1◦) to afford acid 10 (361 mg, 73%) as a white solid. Mp 185–
186 C (H2O); [a]25 +38.9◦ (c = 0.5, CH3OH); mmax(KBr)/cm−1:
D
1
3406 (NH), 2979 (CH), 1693 (CO); kmax(CH3OH)/nm: 271.0; H
=
NMR (300 MHz, CDCl3): d 1.36 (9H, s, C(CH3)3), 1.53 (1H, m,
Ha3ꢀ), 1.97 (3H, s, thymine CH3), 2.44 (1H, ddd, J 14.0, 9.0, 8.5,
and 1708 (CO), 1610 (aromatic C C); kmax(CH3OH)/nm: 271.0;
1H NMR (400 MHz, CDCl3): d 1.25 (9H, s, C(CH3)3); 1.86 (1H,
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dd, J 16.7, 7.2 Hz, Ha3ꢀ), 2.61 (1H, ddd, J 16.7, 14.3, 8.6 Hz, Hb3ꢀ),
2.87–2.93 (1H, m, H2ꢀ), 2.97–3.05 (2H, m, Ha5ꢀ and Ha6ꢀ), 3.26
(1H, d, J 17.4 Hz, Ha7ꢀ), 3.33 (1H, dd, J 15.3, 8.5 Hz, Hb6ꢀ), 3.47
(1H, d, J 10.5 Hz, Hb5ꢀ), 3.60 (1H, d, J 17.4 Hz, Hb7ꢀ), 3.69 (3H, s,
OCH3), 4.94 (1H, br s, H4ꢀ), 5.09 (1H, br s, NH), 5.24 (2H, s, benzyl
CH2), 7.28–7.34 (3H, m, benzyl aromatic), 7.39 (2H, d, J 6.6 Hz,
benzyl aromatic), 8.02 (1H, s, H2), 8.68 (1H, s, H8); 13C NMR
(100.6 MHz, CDCl3): d 28.1 (C(CH3)3), 36.0 (C3ꢀ), 40.5 (C7ꢀ),
41.7 (C2ꢀ), 51.9 (C6ꢀ), 52.7 (CO2CH3), 59.2 (C5ꢀ), 61.9 (C4ꢀ), 67.5
(benzyl CH2), 79.3 (C(CH3)3), 121.9 (C5), 128.4 (benzyl aromatic
CH), 129.1 (benzyl CH), 129.2 (benzyl CH), 135.5 (benzyl ipso-
with EtOAc (4 × 10 mL) and the combined organic extracts were
dried over MgSO4, and then evaporated under reduced pressure.
Purification by column chromatography (5% CH3OH in EtOAc)
afforded pro◦duct 22 (68 mg, 46%) as a white foam Rf 0.20 (EtOAc);
mp 99–101 C (from CHCl3, decomp.); [a]25 −36.2◦ (c = 0.89,
D
CHCl3); mmax (KBr)/cm−1: 3269 (NH), 1749 and 1699 (CO); kmax
1
(CHCl3)/nm 266; H NMR (400 MHz, CDCl3): d 1.31 (9H, s,
Bz-C(CH3)3), 1.35 and 1.38 (9H, 2 × s, Boc C(CH3)3), 2.35–2.48
(1H, m, Ha3ꢀ), 2.48–2.92 (3H, m, Ha5ꢀ, Hb3ꢀ and H2ꢀ), 2.95–3.25
(2H, m, Ha7ꢀ and Ha6ꢀ), 3.28–3.93 (6H, m, OCH3, Hb7ꢀ, Hb6ꢀ and
Hb5ꢀ), 4.78 and 5.13 (1H, 2 × br s, H4ꢀ rotamers), 7.24–7.53 (3H,
m, H5 and m-Bz H rotamers), 7.61 and 7.72 (1H, 2 × br s, BocNH
rotamers), 7.83–7.97 (2H, m, o-Bz H rotamers), 8.64 (1H, d, J
6.9 Hz, H6), 11.29 (1H, br s, BzNH); 13C NMR (100.6 MHz,
CDCl3): d 28.0 and 28.2 (Bz C(CH3)3 rotamers), 31.0 and 31.1 (Boc
C(CH3)3 rotamers), 34.8 (Bz C(CH3)3), 35.0 (C3ꢀ), 40.0 (C6ꢀ), 51.6
and 51.9 (CO2CH3 rotamers), 53.3 (C7ꢀ), 55.3 (C4ꢀ), 56.5 (C5ꢀ),
62.3 (C2ꢀ), 79.3 and 79.4 (Boc C(CH3)3 rotamers), 96.8 (C5), 124.8
and 125.8 (Bz CH rotamers), 126.9 (Bz CH), 127.3 and 127.7
(Bz CH rotamers), 128.3 and 128.6 (Bz CH rotamers), 130.0 and
131.1 (Bz p-C rotamers), 147.4 (C6), 155.0 (Boc CO), 155.8 and
156.2 (C2 rotamers), 156.8 and 157.0 (Bz ipso-C rotamers), 162.9
(C4), 167.9 (Bz CO), 171.0 (CO2CH3); m/z (ES): 542.3 ([M +
H]+, 100%); HRMS m/z (ES): 542.2977 ([M + H]+, C28H40N5O6
requires m/z, 542.2979).
t
C), 141.8 (C8), 149.3 (C4), 151.1 (C6), 152.4 (C2), 156.1 (CO2 Bu),
156.2 (CO2Bn), 171.1 (CO2CH3); m/z (ES): 540 ([M + H]+ 100%);
HRMS m/z (ES): 540.2567 ([M + H]+, C26H34O6N7 requires m/z,
540.2565).
(2ꢀR,4ꢀR)-2ꢀ-(tert-Butoxycarbonylaminomethyl)-4ꢀ-(N6-benzyloxy-
carbonyladenin-9-yl)-pyrrolidine-1-yl-acetic acid (17)
To a solution of methyl ester 16 (30.6 mg, 0.057 mmol) in
THF (360 lL) was added a 1 M aqueous solution of NaOH
(62 lL, 0.062 mmol). The reaction mixture was stirred at room
temperature for 4 h. 1 M aqueous NaOH (28 lL, 0.028 mmol)
was added and the reaction mixture stirred for a further 2 h at
room temperature. THF was then removed under a stream of
N2 and the pH of the resulting aqueous solution was adjusted
to 7 by dropwise addition of aqueous HCl (0.02 M). H2O was
then removed under reduced pressure and the crude product
was purified by reversed-phase (C18) column chromatography
(H2O → CH3CN) to afford Boc-protected acid monomer 17
(2ꢀR,4ꢀR)-2ꢀ-(tert-Butoxycarbonylaminomethyl)-4ꢀ-cytosin-1-yl-
N1ꢀ-(methoxycarbonylmethyl)-pyrrolidine (23)
NaOCH3 (35.6 mg, 0.659 mmol) was dissolved in anhydrous
CH3OH under a stream of N2 (3.40 mL) with the aid of sonication.
This solution was cooled to 0 ◦C, before being added to tert-butyl
benzoyl-protected cytosine derivative 22 (340 mg, 0.628 mmol).
The mixture was stirred at room temperature under N2 for 10 h
and solvent was removed under reduced pressure. Purification by
column chromatography (gradient elution: 1 : 1 EtOAc–hexane →
EtOAc → 1:4 CH3OH–EtOAc) gave give cytosine derivative 23
(211 mg, 88%) as a white foam. Rf 0.13 (5% EtOH in CH2Cl2);
[a]2D5 −21.1◦ (c = 0.35, CHCl3); mmax (KBr)/cm−1: 3334, 3210
(NH), 1747, 1700, 1653 (CO); kmax (CH3OH)/nm 277.0; 1H NMR
(300 MHz, CD3OD): d 1.32 (9H, s, C(CH3)3), 1.43–1.56 (1H, m,
Ha3ꢀ), 2.38–2.54 (1H, m, Hb3ꢀ), 2.60–2.73 (2H, m, H2ꢀ and Ha5ꢀ),
2.99–3.15 (3H, m, Ha7ꢀ, Ha6ꢀand Hb6ꢀ), 3.25–3.32 (1H, m, Hb5ꢀ),
3.65 (3H, s, OCH3), 3.74 (1H, d, J 17.4 Hz, Hb7ꢀ), 4.85–4.93 (1H, m,
H4ꢀ), 5.84 (1H, d, J 7.4 Hz, H5), 6.48 (1H, br t, J 5.5 Hz, BocNH),
8.32 (1H, d, J 7.4 Hz, H6); 13C NMR (75 MHz, CD3OD): d 28.8
(C(CH3)3), 37.5 (C3ꢀ), 41.2 (C6ꢀ), 52.3 (CO2CH3), 53.9 (C7ꢀ), 54.9
(C4ꢀ), 59.3 (C5ꢀ), 63.8 (C2ꢀ), 80.0 (C(CH3)3), 96.1 (C5), 145.0 (C6),
158.6 (Boc CO), 158.9 (C2), 167.2 (C4), 173.3 (CO2CH3); m/z
◦
(21.3 mg, 72%) as a white powder. Mp 120–121 C (H2O); [a]D25
+58.8◦ (c = 0.5, CH3OH); mmax(KBr)/cm−1: 3370 (NH), 1746 (CO);
1
kmax(CH3OH)/nm: 269.0; H NMR (400MHz, CD3OD): d 1.13
(9H, s, C(CH3)3), 1.74 (1H, dd, J 14.1, 6.6 Hz, Ha3ꢀ), 2.62 (1H,
ddd, J 14.1, 8.8, 8.4 Hz, Hb3ꢀ), 2.71 (1H, dd, J 8.4, 6.6 Hz, H2ꢀ),
2.79 (1H, dd, J 10.8, 5.7 Hz, Ha5ꢀ), 2.85 (1H, d, J 14.9 Hz, Ha7ꢀ),
3.06 (1H, dd, J 14.5, 2.6 Hz, Ha6ꢀ), 3.35 (1H, br s, Hb6ꢀ), 3.51
(1H, d, J 14.9 Hz, Hb7ꢀ), 3.58 (1H, d, J 10.8 Hz, Hb5ꢀ), 5.08–5.11
(1H, m, H4ꢀ), 5.31 (2H, s, benzyl CH2), 7.31–7.40 (3H, m, benzyl
aromatic), 7.47 (2H, d J 7.1 Hz, benzyl aromatic), 8.57 (1H, s,
H2), 9.06 (1H, s, H8); 13C NMR (100.6 MHz, CD3OD): d 28.5
(C(CH3)3), 37.0 (C3ꢀ), 40.2 (C6ꢀ), 53.7 (C2ꢀ), 58.8 (C7ꢀ), 60.5 (C5ꢀ),
64.5 (C4ꢀ), 68.4 (benzyl CH2), 79.6 (C(CH3)3), 122.9 (C5), 129.3
(benzyl aromatic CH), 129.4 (benzyl aromatic CH), 129.6 (benzyl
aromatic CH), 137.5 (benzyl ipso-C), 144.9 (C8), 150.6 (C4), 152.4
t
(C6), 152.8 (C2), 153.5 (CO2 Bu), 158.8 (CO2Bn), 179.1 (CO2H);
m/z (ES): 524 ([M − H]− 100%); HRMS (ES): 548.2224 ([M +
Na]+, C25H31O6N7Na requires m/z, 548.2228).
(ES): 282.2 ([M − CO2 Bu + H]+, 100%); 382.2 ([M + H]+, 80%);
t
(2ꢀR,4ꢀR)-2ꢀ-(tert-Butoxycarbonylaminomethyl)-4ꢀ-(N4-[para-
(tert-butyl)benzoyl]cytosin-1-yl)-N1ꢀ-(methoxycarbonylmethyl)-
pyrrolidine (22)
402.2 ([M + Na]+, 80%); HRMS m/z (ES): 382.2084 ([M + H]+,
C17H28N5O5 requires m/z, 382.2090).
A suspension of tosylate 14 (120 mg, 0.271 mmol), N4-[p-(tert-
butyl)benzoyl]cytosine (186 mg, 0.67 mmol), K2CO3 (190 mg,
1.38 mmol) and 18-crown-6 (27 mg, 0.102 mmol) in anhydrous
DMF (1.4 mL) was stirred at 75 ◦C under N2 for 18 h. The
solvent was removed under reduced pressure and brine (5 mL)
was added to the resulting brown paste. The mixture was extracted
(2ꢀR,4ꢀR)-2ꢀ-(tert-Butoxycarbonylaminomethyl)-4ꢀ-(N4-benzyloxy-
carbonylcytosin-1-yl)-N-(methoxycarbonylmethyl)-pyrrolidine (24)
Method A. N-(Benzyloxycarbonyl)imidazole (923 mg,
4.56 mmol) was dissolved in anhydrous CH2Cl2 (922 lL) under
a stream of N2. The stirred solution was cooled to 0 ◦C before
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Et3O·BF4 (2.74 mL 1 M solution in anhydrous CH2Cl2, 2.74 mmol)
was added. After 7.5 h stirring under N2 the cytosine derivative
23 (174 mg, 456 lmol) in CH2Cl2 (174 lL) was transferred into
the reaction mixture which was stirred for a further 14.5 h. The
reaction mixture was cooled to 0 ◦C and saturated NaHCO3
(9.2 mL) was added. The mixture was partitioned between
CH2Cl2 (30 mL) and H2O (30 mL) and the organic extracts
were dried over MgSO4 and evaporated under reduced pressure.
Purification by column chromatography (gradient elution: 1 :
1 EtOAc–hexane → EtOAc → 1 : 4 CH3OH–EtOAc) gave
Z-protected cytosine derivative 24 (216 mg, 92%) as a white
foam.
Hb3ꢀ), 2.71–2.81 (2H, m, H2ꢀ and Ha5ꢀ), 2.90 (1H, d, J 15.8 Hz,
Ha7ꢀ), 3.06 (1H, d, J 12.3 Hz, Ha6ꢀ), 3.30 (1H, d, J 14.6 Hz, Hb6ꢀ),
3.46 (1H, d, J 11.4 Hz, Hb5ꢀ), 3.55 (1H, d, J 15.8 Hz, Hb7ꢀ), 4.75–
4.87 (1H, m, H4ꢀ), 5.12 (1H, d, J 12.4 Hz, Cbz CH2), 5.18 (1H, d,
J 12.4 Hz, Cbz CH2), 7.22–7.37 (6H, m, benzyl aromatic CH and
H5), 8.62 (1H, d, J 6.4 Hz, H6); 13C NMR (100.6 MHz, CD3OD):
d 28.7 (C(CH3)3), 36.4 (C3ꢀ), 41.1 (C6ꢀ), 54.9 (C7ꢀ), 58.6 (C4ꢀ),
60.1 (C5ꢀ), 65.5 (C2ꢀ), 68.6 (Cbz CH2), 80.3 (C(CH3)3), 97.4 (C5),
129.4 (Z Ar-CH), 129.5 (Z Ar-CH), 129.6 (m-Z-CH), 137.1 (ipso-
Z-C), 150.2 (C6 cytosine), 154.3 (Z-CO), 158.2 (C2), 158.8 (Boc
CO), 164.3 (C4), 178.5 (CO2H); m/z (ES): 500 ([M − H]−, 100%);
HRMS m/z (ES): 524.2118 ([M + Na]+, C24H32N5O7Na requires
m/z, 524.2121).
Method B. To a solution of alcohol 13 (650 mg, 2.25 mmol)
in anhydrous THF (8.5 mL) under N2 was added N4-
benzyloxycarbonylcytosine (670 mg, 2.73 mmol) and PPh3
(715 mg, 2.73 mmol). The reaction mixture was cooled to −20 ◦C
and DIAD (610 lL, 3.10 mmol) was added dropwise. The reaction
mixture was allowed to warm to room temperature and stirred
under N2 for 18 h. Solvent was removed under reduced pressure
and the residue was purified by flash chromatography, as described
above, to give the Z-protected cytosine derivative 24 (340 mg, 29%)
as a white foam. Rf 0.28 (5% EtOH/EtOAc); [a]2D5 −42.7◦ (c = 0.88,
CHCl3); mmax (KBr)/cm−1: 3249, 1622 and 1505 (NH), 2984, 2930
and 2844 (CH), 1747 and 1696 (CO), 1225 and 992 (NCOO); kmax
(CH3OH)/nm 239.0; 1H NMR (300 MHz, CH3OD): d 1.28 (9H, s,
C(CH3)3), 1.47–1.61 (1H, m, Ha3ꢀ), 2.42–2.59 (1H, m, Hb3ꢀ), 2.63–
2.79 (2H, m, Ha5ꢀ and H2ꢀ), 3.04–3.15 (3H, m, Ha6ꢀ, Hb6ꢀ and
Ha7ꢀ), 3.36 (1H, br d, J 11.3 Hz, Hb5ꢀ), 3.67 (3H, s, CO2CH3), 3.77
(1H, d, J 17.4 Hz, Hb7ꢀ), 4.83–4.92 (1H, m, H4ꢀ), 5.17 (2H, AB q,
J 12.4 Hz, Bn CH2), 7.18–7.46 (6H, m, Bn aromatic H and H5),
8.76 (1H, d, J 7.5 Hz, H6); 13C NMR (75 MHz, CD3OD): d 28.7
(C(CH3)3), 37.1 (C3ꢀ), 40.8 (C6ꢀ), 52.3 (CO2CH3), 53.6 (C7ꢀ), 56.1
(C4ꢀ), 58.8 (C5ꢀ), 63.6 (C2ꢀ), 68.5 (Cbz CH2), 80.0 (C(CH3)3), 96.9
(C5), 129.3 (o- and p-Z-CH), 129.6 (m-Z-CH), 137.3 (ipso-Z-C),
148.6 (C6 cytosine), 154.6 (Z-CO), 158.5 (Boc CO), 158.5 (C2),
164.1 (C4), 173.3 (CO2CH3); m/z (ES): 516.2 ([M + H]+, 100%);
HRMS m/z (ES): 516.2450 ([M + H]+, C25H34N5O7 requires m/z,
516.2458).
Solid phase synthesis of POM oligomers
Into a 1 mL solid phase synthesis vessel was weighed MBHA
LL resin (100–200 mesh) (5 equiv. relative to the first monomer
loaded). The resin was then washed three times with DMF and
three times with CH2Cl2 (all washings use 1 mL per 25 lmol resin
loading, with rotation of reaction vessel for 30 s each time unless
stated otherwise). The resin was swelled overnight in CH2Cl2, and
then the solvent was removed by vacuum suction. The resin was
washed three times with DMF, once with 5% piperidine/DMF
(with 4 min agitation) and three times with DMF–CH2Cl2 (1 :
1). In a separate small vial, monomers Boc-POM(T)-OH 10, Boc-
POM(Az)-OH 17, Boc-POM(Cz)-OH 25, Boc-PNA(Cz)-OH or N-
a-Boc-N-e-2-chloro-Z-L-lysine (1 equiv.), HBTU (0.95 equiv.) and
diisopropylethylamine (DIEA) (1.1 equiv.) in DMF–pyridine (3 :
1) (total monomer concentration of 0.1 M) were allowed to activate
for 3 min. The mixture was then added to the resin. Coupling was
allowed to proceed with agitation for 6 h for the first coupling. The
coupling mixture was removed and the resin washed two times
with DMF before a freshly prepared acetic anhydride–collidine–
DMF (1 : 1 : 8, v/v/v) (1 mL per 25 lmol) mixture was added
with agitation for 15 min. The acetylating reagent was removed
by vacuum suction and the resin washed with DMF (three times),
complete reaction was indicated by negative Kaiser test. The resin
was then washed with 5% piperidine/DMF (once for 4 min) and
DMF–CH2Cl2 (1 : 1) (three times).
Deprotection of the resin-bound Boc-protected POM oligomer
was accomplished using TFA–m-cresol (95 : 5, v/v) (1 mL per
25 lmol) four times for 4 min each. The resin was washed with
DMF–CH2Cl2 (1 : 1) (three times) and successful deprotection
was indicated by a positive Kaiser test. The resin was then
washed with pyridine (two times). Subsequent coupling employed
monomers 10, 17, 25 or Boc-PNA(Cz)-OH (5 equiv.), HBTU
(2.38 equiv.), DIEA (2.25 equiv.) and coupling times of 2 h. A
second coupling onto adenine residues using monomer 10, 17,
25 or Boc-PNA(Cz)-OH (2.5 equiv.), HBTU (2.38 equiv.), DIEA
(2.75 equiv.) and coupling times of 1 h was carried out. In the case
of the terminal lysine residue, N-a-Boc-N-e-2-chloro-Z-L-lysine
(6 equiv.), HBTU (5.7 equiv.), and DIEA (6.6 equiv.) were used.
Capping after subsequent couplings was carried out for 5 min.
The coupling–capping–deprotection sequence was repeated until
the desired oligomer was obtained. Deprotection of Z-protected
nucleobases and cleavage of the oligomer from the resin was
achieved using trifluoromethanesulfonic acid (TFMSA) via the
‘low–high TFMSA’ method.26 During ‘low TFMSA’ treatment
(2ꢀR,4ꢀR)-2ꢀ-(tert-Butoxycarbonylaminomethyl)-4ꢀ-(N4-benzyloxy-
carbonylcytosin-1-yl)-N1ꢀ-(methoxycarbonylmethyl)-pyrrolidine-
1-yl-acetic acid (25)
Methyl ester 24 (216 mg, 419 lmol) was dissolved in THF (864 lL)
and 1 M NaOH aq. (440 lL, 440 lmol) was added to the stirred
solution at room temperature. After 6 h, the reaction was cooled
to 0 ◦C and 0.1 M HCl aq. (4.40 mL) was added to neutralize
the mixture. The solvent was evaporated under reduced pressure
and the resulting white powder was purified by silica gel column
chromatography (gradient elution: CHCl3 → 1 : 4 CH3OH–
CHCl3) followed by C18 reversed-phase column chromatography
(Varian BondElut, 10 g cartridge, gradient elution H2O → MeOH)
to give the cytosinyl Boc-acid 25 (190 mg, 90%) as a white powder
powder. Rf 0.29 (30% CH3OH in CHCl3); mp 134 ◦C (from H2O,
decomp.); [a]25 −146.4◦ (c = 0.87, CH3OH); kmax (CH3OH)/nm 239
(e/dm3 mol−1Dcm−1 2.67 × 104); 1H NMR (400 MHz, CD3OD): d
1.24 (9H, s, C(CH3)3), 1.59–1.66 (1H, m, Ha3ꢀ), 2.46–2.56 (1H, m,
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the resin was treated with a solution of (TFA–DMS–m-cresol, 1 :
References
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1 mL per 20 lmol resin loading) each separately cooled to 0 C
before being added to the resin, followed by agitation for 1 h. The
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evaporated and lyophilised to give pure product as a white powder.
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(Phenomenex Gemini 5 l C18, 150 × 4.6 mm) and oligomers were
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Retention time on analytical HPLC was 26 min, using a Phe-
nomenex Gemini 5 l C18 150 × 4.6 mm analytical column. Solvent
A was H2O with 0.1% HCO2H and solvent B was acetonitrile with
0.1% HCO2H. The flow rate was 1 mL min−1 with 100% A for
9 min followed by a gradient from 100% A changing to 90% A
with 10% B over 52 min. m/z MALDI-TOF MS: 2505 ([M +
H]+ 100%, C111H154N51O19 requires m/z, 2505.3), 2527 ([M + Na]+
90%, C111H153N51O19Na requires m/z, 2527.2); 2543 ([M + K]+
70%, C111H153N51O19K requires m/z, 2543.2).
POM-PNA chimera Lys-TC*AC*AAC*TT-NH2
Retention time on analytical HPLC was 24 min, using a Phe-
nomenex Gemini 5 l C18 150 × 4.6 mm analytical column. Solvent
A was H2O with 0.1% HCO2H and solvent B was acetonitrile with
0.1% HCO2H. The flow rate was 1 mL min−1 with 100% A for
9 min followed by a gradient from 100% A changing to 90% A
with 10% B over 52 min. m/z MALDI-TOF MS: 2512 ([M + H]+
100%, C108H148N51O22 requires m/z, 2512.2)
Acknowledgements
This work was funded by the BBSRC (research grant 36/B15998)
and EPSRC (partial PhD studentships to R.J.W. and A.O.). We
also thank Dr Robin Pritchard for X-ray crystallography and the
EPSRC National Mass Spectrometry Centre (University of Wales,
at Swansea).
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