RNA-selective cross-pairing of backbone-extended pyrrolidine-amide oligonucleotide mimics (bePOMs)

Article abstract of DOI:10.1039/b714580m

Pyrrolidine-amide oligonucleotide mimics (POMs) can cross-pair strongly with complementary parallel and antiparallel DNA and RNA targets in a sequence-specific fashion. As a result POMs have significant potential for applications including in vivo gene silencing, diagnostics and bioanalysis. To further modulate the DNA- and RNA-recognition properties and fine-tune the physiochemical properties of POMs for nucleic acid targeting, backbone-extended pyrrolidine-amide oligonucleotide mimics (bePOM I and II) were introduced. The bePOMs differ from the original POMs through the insertion of an additional methylene group into the backbone units, which increases the flexibility of the oligomers. bePOM I and II oligomers were synthesised using solid-phase peptide chemistry. Interestingly, UV thermal denaturation and circular dichroism studies reveals bePOM I and II can hybridise with complementary RNA, but not DNA. The Royal Society of Chemistry 2008.

Full text of DOI:10.1039/b714580m

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
RNA-selective cross-pairing of backbone-extended pyrrolidine-amide
oligonucleotide mimics (bePOMs)†
Roberta J. Worthington, Neil M. Bell, Raymond Wong and Jason Micklefield*
Received 20th September 2007, Accepted 8th October 2007
First published as an Advance Article on the web 1st November 2007
DOI: 10.1039/b714580m
Pyrrolidine-amide oligonucleotide mimics (POMs) can cross-pair strongly with complementary parallel
and antiparallel DNA and RNA targets in a sequence-specific fashion. As a result POMs have
significant potential for applications including in vivo gene silencing, diagnostics and bioanalysis. To
further modulate the DNA- and RNA-recognition properties and fine-tune the physiochemical
properties of POMs for nucleic acid targeting, backbone-extended pyrrolidine-amide oligonucleotide
mimics (bePOM I and II) were introduced. The bePOMs differ from the original POMs through the
insertion of an additional methylene group into the backbone units, which increases the flexibility of the
oligomers. bePOM I and II oligomers were synthesised using solid-phase peptide chemistry.
Interestingly, UV thermal denaturation and circular dichroism studies reveals bePOM I and II can
hybridise with complementary RNA, but not DNA.
Introduction
Nucleic acid mimics that can selectively hybridise with comple-
mentary DNA and RNA can be used to down regulate gene
expression in vivo, which is particularly valuable for functional
genomics.1 In addition, nucleic acid mimics have been employed
as bioanalytical tools or diagnostic agents,2 as well as building
blocks in the programmed assembly of nanostructures.3 Moreover,
a study of the properties of nucleic acid mimics can also provide
a valuable alternative insight into the structure, recognition
Fig. 1 Pyrrolidine-amide oligonucleotide mimics (POM) and back-
properties, function and origins of the natural genetic material.4
bone-extended POMs (bePOM I and II). The protonated pyrrolidine
ꢀ
N1 -substituent prefers the less sterically demanding trans-configuration
Previously we introduced the pyrrolidine-amide oligonucleotide
mimics (POMs) 3 (Fig. 1). Notably it was shown that fully modified
short POM homopolymers5 and longer mixed sequences6 are ca-
pable of cross-pairing with both complementary DNA and RNA,
exhibiting UV transition melting temperatures (Tm) that on the
whole are higher than isosequential peptide nucleic acids (PNAs).7
Interestingly, mixed sequence POMs (e.g. Lys–TCACAACTT–
NH2)6 cross-pair strongly with parallel and antiparallel DNA
as well as RNA, but with rates of association/dissociation that
are noticeably slower than those typically observed with short
oligonucleotides or PNA. One possible reason for this could
be the rigidity of the POM backbone compared to other more
flexible mimics such as PNA. Indeed, nucleic acid mimics with
more rigid backbone structure could favour formation of stable
secondary structures in the single-stranded state, which are not
optimal for hybridisation.8 As a consequence, the conformational
reorganisation of the backbone into a structure that enables base
pairing to take place may be slow and rate-limiting. In light of this
it was decided to investigate the effects of increasing the flexibility
of the POM backbone, by introducing an additional methylene
group into the backbone. This leads to the backbone extended
and POMs are thus stereochemically equivalent to natural nucleic acids.5,6
POMs (bePOM I (1) and II (2), Fig. 1), which both possess
repeating 7-atom linkages and differ only in the relative position
of the amide linkage. Previous studies have established that it is
not necessary to match the six-atom linkage of the backbone of
native nucleic acids in order to retain base pairing. In fact, modified
nucleic acids with units containing five-9 and seven-atom10 linkages
are also capable of cross-pairing with complementary DNA
and RNA. Accordingly, oligomers of bePOM I (1) and II (2)
were synthesised, using solid-phase peptide chemistry and their
hybridisation properties explored using UV thermal denaturation
experiments and CD spectroscopy.
Results and discussion
Synthesis of backbone-extended POM monomers
It was envisaged that the synthesis of bePOM oligomers would
be accomplished using Boc-Z solid-phase peptide synthesis pro-
tocols, similar to those developed previously for the original
fully modified mixed sequence POM.6 In order to test this the
bePOM I thymine monomer was first prepared from the ethyl ester
hydrochloride salt 4 (Scheme 1).11 Protection of the pyrrolidine
nitrogen of 4 with a benzyloxycarbonyl group gave 5 in 91% yield
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: HPLC traces, UV
melting curves, and CD spectra. See DOI: 10.1039/b714580m
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and subsequent tert-butyldimethylsilyl (TBDMS)-protection of
the secondary alcohol provided pyrrolidine 6 in 85% yield. This
allowed reduction of the ethyl ester of 6 with LiBH4 resulting in
the primary alcohol 7 in 70% yield, which was then transformed
to the mesylate 8. The mesylate was not isolated but treated
with sodium cyanide to give nitrile 9 in a yield of 68% over
the two steps. Reduction of nitrile 9 was then achieved with
NaBH4 in the presence of CoCl2·6H20.12 The resulting primary
amine 10 was then Boc-protected using di-tert-butyldicarbonate
to give Boc-amine 11 in 62% overall yield. Deprotection of the
pyrrolidine nitrogen by hydrogenation over 10% Pd–C afforded
amine 12, which was alkylated with methyl bromoacetate to afford
methyl ester 13 in 65% yield. Following TBDMS-deprotection
with tetrabutylammoniumfluoride (TBAF), it was necessary to
invert the stereochemistry of the C4 alcohol of 14 in order
to obtain the desired (2S,4R) configuration of the bePOM I
monomer. Accordingly (4R)-alcohol 14 was transformed to the
(4S)-formyl ester 15 under Mitsunobu conditions13 in 70% yield.
Cleavage of the formyl ester with sodium methoxide in anhydrous
methanol gave the (4S)-alcohol 16 in 90% yield. Thymine was then
introduced onto the pyrrolidine ring as N3-benzoylthimine14 to
ensure the desired N1-alkylation is obtained. This was achieved
using another Mitsunobu reaction to form the N1-thyminyl
derivative 17 in a yield of 67%. Treatment with aqueous sodium
hydroxide in THF, followed by neutralisation provided the bePOM
I thyminyl acid 18 in 71% yield.
The synthesis of the bePOM II thymine monomer is achieved
via a conjugate addition between the reported5d amine HCl salt
19 and methyl acrylate, which gave methyl ester 20 in 85%
yield (Scheme 2). Azide-reduction with trimethylphosphine under
Staudinger conditions, and in situ Boc-protection of the resulting
amine with 2-(tert-butoxycarbonyloxyimino)-2-phenylacetonitrile
(Boc-ON)15 afforded the Boc-amine 21 in 89% yield. The C4-OH
group of the pyrrolidine ring was again inverted via the (4S)-formyl
ester 22, which was formed in 65% yield then cleaved with sodium
methoxide to give (4S)-alcohol 23 in 82% yield. Introduction of
N3-benzoylthimine under Mitsunobu conditions, similarly gave
the thyminyl derivative 24 in 64% yield and saponification and
neutralisation as before provided the bePOM II thyminyl acid 25
in 69% yield.
Scheme 2 Synthesis of bePOM II thyminyl monomer: (a) methyl
acrylate DIEA, CH2Cl2, 0 ◦C for 30 min then rt for 18 h; (b) PMe3,
THF, rt, 1.5 h, then 2-(tert-butoxycarbonyloxyimino)-2-phenylacetonitrile
(Boc-ON), −20 ◦C, 15 min, then rt for 1 h; (c) HCO2H, PPh3, DIAD, THF,
−20 ◦C → rt, 18 h; (d) NaOCH3, CH3OH, rt, 2 h; (e) N3-benzoylthymine,
PPh3, DIAD, THF, 0 ◦C → rt, 18 h; (f) 1M NaOH (aq), THF, rt for 3 h,
then 0.1 M HCl (aq).
Boc-Z solid-phase synthesis of POM, bePOM I and bePOM II
Lys–(T)8–NH2 oligomers
POM Lys–(T)8–NH2 27, bePOM I Lys–(T)8–NH2 28 and bePOM
II Lys–(T)8–NH2 29 were synthesised following the Boc-Z POM
synthetic protocol.6,16 The octamers were prepared on methylben-
zhydrylamine (MBHA LL)-functionalised resin, adjusted at the
first coupling to give a loading of 0.12 mmol.g−1. Unreacted amino
groups were capped with acetic anhydride. In the case of POM
Lys–(T)8–NH2 27 subsequent couplings employed four equivalents
of Boc-protected POM thyminyl acid 26,6 preactivated with 2-
(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylguanidinium hexaflu-
orophosphate (HBTU) (3.8 equiv.) and diisopropylethylamine
(DIEA) (4.4 equiv.). Coupling reactions proceeded for 2 h and
were monitored by the Kaiser test.17 Capping of any unreacted
oligomer was carried out by reaction with acetic anhydride
again in the presence of DMF and collidine. Boc-deprotection
with trifluoroacetic acid (TFA) and m-cresol as a scavenger was
followed by repeated coupling, capping and deprotection steps. In
the case of bePOM I Lys–(T)8–NH2 28 and bePOM II Lys–(T)8–
NH2 29 five equivalents of thyminyl acid 18 or 25 were preactivated
with HBTU (4.75 equiv.) and DIEA (5.5 equiv.) prior to the second
Scheme 1 Synthesis of bePOM I thyminyl monomer: (a) benzylchlo-
roformate, Et3N, 1 : 1 water–1,4-dioxane, 50 ◦C for 2 h then rt for 18 h;
(b) TBDMS–Cl, imidazole, DIEA, DMF, rt, 18 h; (c) LiBH4, THF, 0 ◦C →
rt, 18 h; (d) MsCl, DIEA, CH2Cl2, 0 ◦C → rt, 3 h; (e) NaCN, DMF, 75 ◦C,
20 h; (f) NaBH4, CoCl2·6H2O, CH3OH, rt, 4 h; (g) Boc anhydride, Et3N,
1 : 1 water–1,4-dioxane, rt, 18 h; (h) 10% Pd–C, CH3OH, H2, rt, 18 h;
(i) BrCH2CO2CH3, DIEA, CH2Cl2, 0 ◦C → rt, 18 h; (j) TBAF, THF, rt,
4 h; (k) HCO2H, PPh3, DIAD, THF,–20 ◦C → rt, 18 h; (l) NaOCH3,
CH3OH, rt, 5 h; (m) N3-benzoylthymine, PPh3, DIAD, THF,–20 ◦C → rt,
18 h; (n) 1 M NaOH (aq), THF, rt for 18 h, then 0.1 M HCl (aq).
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and subsequent coupling reactions. Cleavage of the oligomers from
the resin was carried out by the “low–high” TFMSA method16 and
the crude oligomers were analysed by analytical C18 HPLC and
MALDI-TOF mass spectrometry (Fig. 2). POM Lys–(T)8–NH2
27 was synthesised in a yield of 52%, as determined by analytical
C18 HPLC, this corresponds to an average coupling efficiency of
93.0%. The yield for the synthesis of bePOM I Lys–(T)8–NH2 28
was 86%, equating to an average coupling efficiency of 98.3%. The
yield for the synthesis of bePOM II Lys–(T)8–NH2 29 was 60%,
equating to an average coupling efficiency of 94.5%. Oligomers
were purified by semi-preparative C18 HPLC and the purity of
oligomers was estimated to be higher than 95% based on analytical
C18 HPLC.
Nucleic acid-binding properties of bePOM I and II: UV thermal
denaturation and renaturation experiments
The POM and bePOM oligomers were next subjected to UV
thermal denaturation/renaturation experiments. In line with
earlier observations,5 the prototype POM Lys–(T)8–NH2 27 hy-
bridises strongly with both RNA and DNA, exhibiting transition
melting temperatures (Tm heating) of 41.5 and 36.4 ◦C with
r(CGCA8CGC) and d(CGCA8CGC), respectively, under close to
physiological conditions (Table 1). With longer homopolymers
poly(rA) and poly(dA), higher Tm were observed, which are
accompanied by more pronounced hysteresis, indicative of slow
rates of association/dissociation. Interestingly the extent of this
hysteresis, is similar for both poly(rA) and poly(dA), suggesting
that the kinetic selectivity for RNA over DNA observed previously
with thyminyl POM pentamers,5 is not evident with the octameric
POM 27.
The oligomer with the type-I extended-backbone bePOM I
Lys–(T)8–NH2 28 hybridises with r(CGCA8CGC) with a Tm
(heating) of 36.7 ◦C (Fig. 3) and exhibits slight hysteresis with
Tm (cooling) of 32.5 ◦C. Incubation at room temperature for
24 h prior to denaturation has little effect on Tm or hyperchromic
shift (Table 1). Under identical conditions bePOM I Lys–(T)8–
NH2 28 shows no evidence of cooperative melting transitions with
d(CGCA8CGC), poly(dA) or poly(rA) and there is no significant
Fig. 2 (A) MALDI-MS of crude POM Lys–(T8)–NH2 27 showing
m/z 2260.1 ([M + H]+ 100%, C111H154N51O19 requires m/z, 2260.2);
(B) MALDI-MS of crude POM bePOM I Lys–(T8)–NH2 28 showing
m/z 2372.1 ([M + H]+ 100%, C119H160N51O19 requires m/z, 2372.2); (C)
MALDI-MS of crude POM bePOM I Lys–(T8)–NH2 29 showing m/z
2372.2 ([M + H]+ 100%, C119H160N51O19 requires m/z, 2372.2).
hyperchromic shifts. For the type-II backbone-extended POM
(29) apparent hybridisation with r(CGCA8CGC) is observed
Table 1 UV thermal denaturation/renaturation transition temperatures (Tm) for POM 27, bePOM I 28 and bePOM II 29 vs. complementary nucleic
acids
POM Lys–(T)8–NH2 27
bePOM I Lys–(T)8–NH2 28
bePOM II Lys–(T)8–NH2 29
Tm/◦Ca (hyperchromicb and
hypochromicc shifts (%))
Heating
Cooling
Heating
Cooling
Heating
Cooling
r(CGCA8CGC)
d(CGCA8CGC)
Poly(rA)
41.5a (12.6)b
43.8d (13.4)
36.4a (12.3)
41.0d (20.5)
52.4a (22.0)
54.0d (24.7)
53.4a (10.0)
53.4d (15.8)
37.9a (12.2)c
35.6a (10.6)
38.2a (21.5)
41.4a (12.1)
36.7a (9.1)b
34.8d (13.4)
n.t.e (4.0)
32.5a (7.7)c
n.t.e (1.0)
n.t. (14.5)
n.t. (11.0)
31.2 and 48.8a (13.5)b
n.t.de (16.2)
n.t.e (5.4)
38.6a (7.7)b
n.t.e (3.0)
33.3a (8.8)b
n.t.e (8.3)
n.t.de (12.5)
n.t.e (17.1)
n.t.de (17.6)
n.t.e (12.7)
n.t.de (12.9)
n.t.de (12.7)
44.4a (9.2)b
48.2d (13.6)
n.t.e (9.4)
Poly(dA)
n.t.de (17.7)
a Experiments were carried out with 84 lM total conc. in bases, in a 1 : 1 ratio of strands for d(CGCA8CGC) and r(CGCA8CGC) and 1 : 1 ratio of bases
for poly(rA) and poly(dA), and 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 23 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 1st derivative of the slow
heating and cooling curve. b Hyperchromic and are indicated in parentheses and were calculated as follows: [Abs 93 ◦C − Abs 15 ◦C] × 100/Abs 93 ◦C.
c Hypochromic shifts are indicated in parentheses and were calculated as follows: [Abs 93 ◦C − Abs 15 ◦C] × 100/Abs 93 ◦C. d Samples were incubated
with nucleic acid for 24 h before being subjected to slow thermal denaturation (0.2 ◦C min−1). e n.t. = no transition evident. Note that no transitions are
evident in control experiments where POMs are subjected to UV thermal denaturation in the absence of complementary nucleic acid targets.
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prolonged period of time. These experiments indicate that both
the type-I and type-II backbone-extended POMs (28 and 29)
can hybridise with complementary RNA, but not DNA. This
apparent cross-pairing selectivity of bePOM I and II for RNA
over DNA was further investigated using circular dichroism (CD)
spectroscopy.
Circular dichroism experiments
Initially the CD spectra of POM Lys–(T)8–NH2 27, bePOM I
Lys–(T)8–NH2 28 and bePOM II Lys–(T)8–NH2 29 single strands
were recorded (Fig. 4). In the case of prototype, POM Lys–(T)8–
NH2 27 shows a strong negative bands at 215 and 285 nm and
a strong positive band at 260 nm. Interestingly, the sign of the
bands for the single stranded POM 27 are opposite to those
typically observed in the CDs of short RNA and DNA strands
(see ESI†).18 This might suggest that POM 27 is preorganised into
a base-stacked conformation with a left-handed helical sense that
is opposite to that typically observed with right-handed helical
DNA and RNA. The bePOM I and II oligomers (28 and 29) have
CD spectra that exhibit bands of lower intensity than the original
POM 27. This is indicative of bePOM I and II possessing less
structurally ordered single strands than the more rigid POM 27,
which is presumably due to the extra methylene group increasing
the intrinsic flexibility of the backbone. Also notable is the fact
that bePOM II 29 possesses bands, which are opposite in sign to
Fig.
3 UV thermal denaturation curves and first derivatives for
POM Lys–(T8)–NH2 27, bePOM I Lys–(T8)–NH2 28 and bePOM II
Lys–(T8)–NH2 29 vs. r(CGCA8CGC) at 7.6 lM (total conc. in strands, 1 :
1 ratio of strands) and 10 mM K2HPO4, 0.12 M K+, pH 7.0 (total volume
1.0 cm3): (A) slow heating (denaturation) curves for POM 27 (i), bePOM
I 28 (ii) and bePOM II 39 (iii) vs. r(CGCA8CGC); (B) the corresponding
first derivatives for POM 27 (i), bePOM I 28 (ii) and bePOM II 39 (iii) vs.
r(CGCA8CGC).
(Fig. 3), with two distinct transitions in the denaturation curve,
at 31.2 ◦C and 48.8 ◦C, possibly indicative of transitions from
triplex to duplex to single strands. However attempts to define
stoichiometry of binding through Job plots were inconclusive. It
was previously noted that the prototype POM can hybridise with
DNA and RNA in a parallel or antiparallel fashion.6 Therefore
the presence of a mixture of parallel and antiparallel complexes
of differing thermodynamic stability, may also account for the
observed double transition. Clearly this issue is best resolved
using mixed-sequence oligomers, with defined orientations and
modes of hybridisation. Cross-pairing between bePOM II 29 and
poly(rA) was also apparent. In this case, notable hysteresis was
observed with Tm of 44.4 and 33.3 ◦C (DTm = 11.1 ◦C) for
the denaturation and renaturation curves, respectively. On the
other hand, bePOM II 29 shows no evidence of thermal denat-
uration/renaturation d(CGCA8CGC) or poly(dA) even after the
complementary strands are incubated at room temperature for a
Fig. 4 CD spectra of POM 27, bePOM I 28 and bePOM II 29 vs.
r(CGCA8CGC) at 7.6 lM (total conc. in strands, 1 : 1 ratio of strands)
and 10 mM K2HPO4, 0.12 M K+, pH 7.0 (total volume 1.0 cm3):
(A) single strands (i) POM 27, (ii) bePOM I 28, (iii) bePOM II 29,
(iv) r(CGCA8CGC); (B) bePOM I 28 vs. r(CGCA8CGC), (i) calculated,
(ii) acquired; (C) bePOM II 29 vs. r(CGCA8CGC), (i) calculated,
(ii) acquired.
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those observed with POM 27, suggesting that the single strands
possess opposite helical sense.
characterised by MALDI mass spectrometry and analytical RP-
HPLC. The DNA- and RNA-hybridisation properties of bePOMs
POM I and II thyminyl octamers (28 and 29) were then compared
with the prototype POM oligomer, using UV thermal denaturation
and renaturation experiments and CD spectroscopy. This showed
that bePOM I thyminyl octamer 28 binds to r(CGCA8CGC) with
a slightly lower Tm (heating) than that of the prototype POM,
but shows no evidence of hybridisation with d(CGCA8CGC).
The bePOM II thyminyl octamer 29 similarly exhibits hybridi-
sation with RNA, but not DNA. Hybridisation of bePOM II
with r(CGCA8CGC) is accompanied by two transitions in the
denaturation curve. This could be due to triplex formation or the
formation of a mixture of parallel and antiparallel complexes of
differing thermodynamic stability. Circular dichroism experiments
also show additional evidence of complex formation between the
bePOM I and II oligomers with RNA but not DNA. These find-
ings are consistent with the earlier observation that a backbone-
extended nucleic acid mimic containing (2ꢀS,4ꢀS)-pyrrolidine units,
which is the enantiomer of the bePOM II presented here, is also
capable of selective cross-pairing with RNA.10d Currently, the
synthesis of longer mixed-sequence bePOMs is under way in order
to fully investigate the recognition of more biologically relevant
nucleic acid sequences.6
The CD spectra for the equimolar complexes of oligomers (27,
28 and 29) with r(CGCA8CGC) and d(CGCA8CGC) were next
compared against the calculated CD spectra resulting from the
average of the CD spectra obtained for the corresponding separate
single strands. For POM 27 with RNA and DNA (CGCA8CGC)
significant difference in both the wavelength and intensity of
the CD bands is observed between the calculated and observed
spectra (Fig. 5). Notably, the overall CD spectra of the complex
between POM 27 and r(CGCA8CGC) closely resembles that of
typical A-type RNA helices.18 This suggests that the RNA strand
has greater influence over the final conformation of the POM–
RNA complex. In the case of bePOM I and II (28 and 29) with
r(CGCA8CGC) a noticeable increase in band intensity is evident
for the observed CD compared with the calculated CD, and again
the CD spectra closely resemble those observed for A-type helical
RNA (Fig. 4). In contrast, there are essentially no differences
between theobservedandcalculated CDs ofequimolar mixtures of
bePOM I and II (28 and 29) with d(CGCA8CGC) (see ESI†). This
fully supports the earlier observations, showing that whilst bePOM
I and II can cross-pair with RNA, no hybridisation is evident with
isosequential DNA. Of course the interpretation of the CD spectra
is only qualitative and NMR or X-ray crystallography are required
for more detailed structural and conformational analysis.
Experimental
NMR spectra were recorded on a Bruker DPX 300 operating at
300 MHz (1H) and 75.5 MHz (13C) or a Bruker DPX 400 operating
1
at 400 MHz (1H) and 100.6 MHz (13C). Chemical shifts in H
and 13C NMR spectra are expressed in ppm relative to tetram-
ethylsilane and were internally referenced to the residual solvent
1
signal. Chemical shift assignments for H and 13C spectra were
assisted with COSY, DEPT, HMQC and HMBC experiments. The
splitting patterns for NMR spectra are designated as follows: s
(singlet), d (doublet), t (triplet), q (quartet), dd (doublet of
doublets), ddd (doublet of doublet of doublets), m (multiplet) and
br (broad). Mass spectra were obtained using electrospray (ES)
on a MassLynx orthogonal accelerated-TOF mass spectrometer
with samples introduced from a Waters 7240 sample injector.
MALDI mass spectra were obtained on a Micromass TOF Spec
2e or a Shimadzu AXIMI-CF+ using a-cyano-4-hydroxycinnamic
acid as matrix. Infrared spectra were recorded on a Nicolet
Nexus 670 FT-IR spectrometer with samples prepared as a thin
film on KBr discs or as Nujol mulls. UV measurements were
carried out on a Varian Cary 400 spectrometer with cell-transport
accessories with samples. Molar extinction coefficients (e) were
calculated from Beer–Lambert law from a sample solution ◦of
known concentration. Optical rotations were measured at 25 C
with an Optical Activity AA-1000 polarimeter. Melting points
were determined with an electrothermal capillary apparatus and
are uncorrected. X-Ray crystallographic analysis was collected on
a Nonius jCCD diffractometer. Thin layer chromatography was
performed on Fluka silica gel (60 F254) coated on aluminium plates.
TLC plates were visualised by UV (254 nm) and/or developed
using potassium permanganate, vanillin or Ehrlichs reagent and
ninhydrin. Flash column chromatography was performed on silica
gel LC 60A purchased from Fluorochem Ltd. Chemicals were
purchased from Aldrich Chemical Company, Acros Organics
and Lancaster Synthesis Ltd and were used without further
Fig. 5 CD spectra for POM Lys–(T)8–NH2 27 vs. d(CGCA8CGC) and
r(CGCA8CGC) 7.6 lM (total conc. in strands, 1 : 1 ratio of strands) and
10 mM K2HPO4, 0.12 M K+, pH 7.0 (total volume 1.0 cm3). (A) CD
spectra of acquired and calculated for POM 27 vs. d(CGCA8CGC);
(B) CD spectra of acquired and calculated for POM 27 vs. r(CGCA8CGC).
Conclusion
Boc-protected thyminyl monomers were prepared and used for
the solid-phase synthesis of backbone-extended pyrrolidine-amide
oligonucleotide mimics. The synthetic bePOMs POM I and II
thyminyl octamers (28 and 29) were purified by RP-HPLC and
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purification unless otherwise noted. Solvents were purified and
dried where necessary; THF was distilled from sodium with
benzophenone as indicator under nitrogen. Dichloromethane was
distilled from CaH2. DMF, 1,4-dioxane, DIEA and pyridine
were purchased anhydrous from Aldrich Chemical Company or
Acros Organics and used without further purification. Deionised
water was used throughout. For reactions requiring anhydrous
conditions, glassware was flame dried under vacuum and cooled
under a positive pressure of nitrogen.
NMR (400 MHz, CDCl3) d 0.00 and 0.01 (6H, 2 × s Si(CH3)2,
rotamers), 0.81 (9H, s, SiC(CH3)3), 1.11 and 1.22 (2 × t, J 7.1 Hz,
CH2CH3, rotamers), 2.07–2.15 (1H, m, Ha3), 2.22–2.31 (1H, m,
Hb3), 3.36 and 3.40 (1H, 2 × dd, J 11.3 Hz, 2.6 Ha5, rotamers),
3.62 and 3.66 (1H, 2 × dd, J 11.3, 5.3 Hz, Hb5, rotamers), 4.03
and 4.13 (1H, 2 × q, J 7.1 Hz, CH2CH3, rotamers), 4.04 and
4.14 (1H, 2 × q, J 7.1 Hz, CH2CH3, rotamers), 4.31–4.35 (1H,
m, H4), 4.36 and 4.42 (1H, 2 × dd, J 8.8, 3.7 Hz, H2 rotamers),
5.05 and 5.09 (1H, d, J 12.4 Hz, benzyl CH2, rotamers), 5.13
and 5.15 (1H, d, J 12.4 Hz, benzyl CH2, rotamers), 7.23–7.36
(5H, m, benzyl aromatic); 13C NMR (100.6 MHz, CDCl3) d −5.5
and −5.4 (Si(CH3)2 rotamers), 13.6 and 13.7 (CH2CH3, rotamers),
17.4 (SiC(CH3)3), 25.2 (SiC(CH3)3), 38.3 and 39.2 (C3, rotamers),
54.3 and 54.7 (C5, rotamers), 57.3 and 57.6 (C2 rotamers), 60.5
(CH2CH3), 66.5 (benzyl CH2), 69.4 and 70.2 (C4 rotamers), 127.3,
127.4, 127.5 and 127.5, (benzyl aromatic, rotamers), 127.9 and
128.0 (benzyl aromatic, rotamers), 136.2 and 136.3 (benzyl ipso-
C, rotamers), 154.0 and 154.3 (CO2Bn rotamers); m/z (ES) 446
([M + K]+ 100%), 408 ([M + H]+ 40%); HRMS m/z (ES), 446.1768
calculated for C19H31O4NSiNa 446.1760.
(2R,4R)-2-Ethoxycarbonyl-4-hydroxy-N-
(benzyloxycarbonyl)pyrrolidine (5)
To a solution of pyrrolidine hydrochloride salt 4 (61.9 g,
0.316 mmol) in 1 : 1 water–1,4-dioxane (465 mL), was added
triethylamine (110 mL, 0.783 mmol), followed by benzylchloro-
formate (66.4 mL, 0.472 mmol) dropwise. The reaction mixture
was stirred at 50 ◦C for 2h then at room temperature for 18 h.
Solvent was removed under reduced pressure, water (300 mL) was
added and the product extracted with Et2O (4 × 500 mL). The
organic fractions were combined and dried over MgSO4, MgSO4
was removed by filtration and solvent was removed under reduced
pressure. The product was purified by flash chromatography (3 :
1 EtOAc–hexanes, Rf 0.6) to afford benzyloxycarbonyl-protected
product 5 (84.4 g, 91%) as a colourless oil. [a]D + 19.5◦ (c =
(2R,4R)-2-Hydroxymethyl-4-[(tert-butyl)dimethylsilyloxy]-N-
(benzyloxycarbonyl)pyrrolidine (7)
To a solution of ethyl ester 7 (99.0 g, 0.243 mmol) in anhydrous
1
1, CHCl3); mmax(KBr)/cm−1 3428 br (OH), 1754, 1712, (CO); H
◦
THF at 0 C under nitrogen was added LiBH4 portionwise. The
NMR (400 MHz, CDCl3) d 1.14 and 1.31 (3H, 2 × t, J 7.1 Hz,
CH2CH3 rotamers), 2.12 (1H, dd, J 14.1 Hz, 6.7 Hz, Ha3), 2.29–
2.40 (1H, m, Hb3), 3.59 and 3.63 (1H, 2 × dd, J 11.9 Hz, 4.1 Hz,
Ha5 rotamers), 3.73 and 3.78 (1H, 2 × d, J 11.9 Hz, Hb5 rotamers),
4.09 and 4.26 (2H, 2 × q, J 7.1 Hz, CH2CH3 rotamers), 4.36–4.43
(2H, m, H2 and H4), 5.06–5.21 (2H, m, benzyl CH2), 7.32–7.36
(5H, m, benzyl aromatic); 13C NMR (75.5 MHz, CDCl3) d 13.4
and 13.5 (CH2CH3 rotamers), 37.3 and 38.1 (C3 rotamers), 54.4
and 54.7 (C5 rotamers), 57.3 and 57.6 (C4 rotamers), 60.8 and
60.9 (CH2CH3 rotamers), 66.5 and 66.6 (benzyl CH2 rotamers),
68.8 and 69.7 (C2 rotamers), 127.1, 127.2, 127.4, 127.8 and
127.9 (benzyl aromatic CH), 135.8 and 136.0, (benzyl ipso-C
rotamers), 153.9 and 154.4 (CO2Bn rotamers), 172.7 and 172.8
(CO2Et rotamers); m/z (ES) 332 ([M+K]+ 70%); HRMS m/z (ES)
332.0895, calculated for C15H19O5NK 332.0895.
reaction mixture was allowed to warm to room temperature and
stirred under nitrogen for 18 h. The reaction mixture was cooled to
−10 ◦C and quenched by dropwise addition of 1 : 1 water–sat. aq.
K2CO3 (200 mL), this mixture was stirred at room temperature
for 24 h. Water (200 mL) was added and the product extracted
with EtOAc (4 × 500 mL). The organic fractions were combined
and dried over MgSO4, MgSO4 was removed by filtration and
solvent was removed under reduced pressure. The product was
purified by flash chromatography (2 : 1 hexanes–EtOAc, Rf 0.3)
to afford alcohol 7 (62.2 g, 70%) as a colourless oil. [a]D + 10.7◦
(c = 1, CHCl3); mmax(KBr)/cm−1 3428 br (OH), 1762, 1708 (CO);
1H NMR (400 MHz, CDCl3) d 0.08 and 0.12 (6H, 2 × s, Si(CH3)2
rotamers), 0.88 and 0.89 (9H, 2 × s, SiC(CH3)3 rotamers), 1.70
and 1.91 (1H, 2 × d, J 13.7 Hz, Ha3 rotamers), 2.10–2.31 (1H, m,
Hb3), 3.45 and 3.37 (1H, 2 × d, J 11.7 Hz, Ha5 rotamers), 3.60–
3.68 (1H, m, Hb5), 3.78–3.88 (2H, m, Ha6 and Hb6), 4.06–4.14
(1H, m, H2), 4.34 and 4.40 (1H, 2 × br s, H4 rotamers), 5.09–5,19
(2H, m, benzyl CH2), 7.29–7.37 (5H, m, benzyl aromatic); 13C
NMR (75.5 MHz, CDCl3) d −5.3 (Si(CH3)2), 17.6 (SiC(CH3)3),
25.2 and 25.4 (SiC(CH3)3 rotamers), 37.5 and 38.1 (C3 rotamers),
55.3 and 56.1 (C5 rotamers), 59.5 (C2), 66.1 (benzyl CH2), 66.7
and 66.8 (C6 rotamers), 70.0 and 70.2 (C4 rotamers), 126.5, 126.8,
127.6, 127.7, 128.0 and 128.2 (benzyl aromatic CH, rotamers),
136.2 (benzyl ipso-C) 154.6 and 156.2 (CO2Bn rotamers); m/z
(ES) 388 ([M + Na]+ 50%); HRMS m/z (ES) 388.1915, calculated
for C19H31O4NSiNa 388.1915.
(2R,4R)-2-Ethoxycarbonyl-4-[(tert-butyl)dimethylsilyloxy]-N-
(benzyloxycarbonyl)pyrrolidine (6)
To a solution of alcohol 5 (84.0 g, 0.286 mmol) in anhydrous DMF
(110 mL) was added tert-butyldimethylsilylchloride (TBDMS–Cl)
(64.3 g, 0.427 mmol), imidazole (38.1 g, 0.56 mmol) and DIEA
(50 mL, 0.303 mmol) under nitrogen. The reaction mixture was
stirred at room temperature under nitrogen for 18 h. The solvent
volume was reduced in vacuo and water (200 mL) was added.
The product was extracted with CH2Cl2 (4 × 300 mL) and the
organic fractions were combined and dried over MgSO4, MgSO4
was removed by filtration and solvent was removed under reduced
pressure. The product was purified by flash chromatography (3 :
1 hexanes–EtOAc, Rf 0.4) to afford TBDMS-protected alcohol
6 (99.6 g, 85%) as a colourless oil. Found: C 61.59; H 8.43, N
3.31, Calculated for C19H31O4NSi; C 61.88, H 8.16, N 3.44%; [a]D
(2S,4R)-2-Cyanomethyl-4-[(tert-butyl)dimethylsilyloxy]-N-
(benzyloxycarbonyl)pyrrolidine (9)
To a solution of alcohol 7 (5.25 g, 14.36 mmol) in anhydrous
+ 44.9◦ (c = 1, CHCl3); mmax(KBr)/cm−1 1750, 1708 (CO); H
CH2Cl2 (20 mL) at 0 ◦C, under nitrogen was added DIEA (3.8 mL,
1
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23.0 mmol) followed by methanesulfonyl chloride (1.3 mL,
17.0 mmol) dropwise. The reaction mixture was allowed to warm
to room temperature and stirred under nitrogen for 3 h. The
reaction was quenched by addition of sat. NaHCO3 (aq) (25 mL).
Water (25 mL) was added and the crude product was extracted with
CH2Cl2 (4 × 100 mL). The organic fractions were combined and
dried over MgSO4, MgSO4 was removed by filtration and solvent
was removed under reduced pressure. The crude methanesulfonate
8 was dried under reduced pressure and dissolved in anhydrous
DMF (40 mL). NaCN (3.55 g, 72.4 mmol) was added under
(NH), 1710, 1685 (CO); 1H NMR (400 MHz, CDCl3) d 0.05 (6H, s,
Si(CH3)2), 0.86 (9H, s SiC(CH3)3), 1.43 (9H, s, C(CH3)3), 1.72–1.82
(2H, m, Ha3 and Ha6), 1.95–2.17 (2H, m, Hb3 and Hb6), 2.92–3.00
(1H, m, Ha8), 3.28 (1H, d, J 11.7 Hz, Ha5), 3.34–3.40 (1H, m,
Hb8), 3.67 (1H, dd, J 11.7 Hz, 5.2 Hz, Hb5 rotamers), 3.93 and
4.06 (1H, 2 × d, J 7.0 Hz, H2 rotamers), 4.36 (1H. br s, H4), 5.07–
5.17 (2H, m, benzyl CH2), 7.34–7.38 (5H, m, benzyl aromatic); 13
C
NMR (100.6 MHz, CDCl3) d −5.0 (Si(CH3)2), 17.8 (SiC(CH3)3,
25.6 (SiC(CH3)3, 28.4 (C(CH3)3), 35.4 (C6), 37.5 (C8), 39.6 and
39.7 (C3 rotamers), 54.6 and 54.8 (C2 rotamers), 55.3 (C5), 66.7
and 67.0 (benzyl CH2 rotamers), 70.5 and 71.3 (C4 rotamers), 78.6
and 78.9 (C(CH3)3 rotamers), 127.7, 127.9, 128.1, 128.4 and 128.6,
(benzyl aromatic CH rotamers), 136.4 and 136.7 (benzyl ipso-C ro-
◦
nitrogen and the suspension stirred at 75 C, under nitrogen for
20 h. Water (150 mL) was added to the reaction mixture and the
product extracted with Et2O (4 × 250 mL). The aqueous layer
was drained into a solution of NaOCl. The organic fractions were
combined, washed with water and dried over MgSO4, MgSO4 was
removed by filtration and solvent was removed under reduced
pressure. Flash chromatography (hexanes–EtOAc 2 : 1 Rf 0.7)
afforded nitrile 9 (3.65 g, 68%) as a pale yellow oil. [a]D + 21.3◦
(c = 2, CHCl3); mmax(KBr)/cm−1 2253 (CN), 1707 (CO); 1H NMR
(400 MHz, CDCl3) d 0.07 and 0.10 (6H, 2 × s, Si(CH3)2 rotamers),
0.89 (9H, s, SiC(CH3)3), 2.04–2.09 (1H, m, Ha3), 2.13–2.20 (1H,
m, Hb3), 2.85–3.10 (2H, m, Ha6Hb6), 3.39 and 3.34 (1H, 2 × d, J
11.6 Hz, Ha5 rotamers), 3.55 and 3.60 (1H, 2 × dd, J 11.6, 4.5 Hz,
Hb5 rotamers), 4.16–4.21 (1H, m, H2), 4.41 (1H, br s, H4), 5.08–
5.18 (2H, m, benzyl CH2), 7.36–7.37 (5H, m, benzyl aromatic);
13C NMR (75.5 MHz, CDCl3) d −5.1 and −5.0 (Si(CH3)2
rotamers), 17.8 (SiC(CH3)3, 22.3 and 23.2 (C6 rotamers), 25.6
(SiC(CH3)3, 38.2 and 39.0 (C3, rotamers), 53.5 and 54.0 (C2
rotamers), 55.6 and 56.2 (C5 rotamers), 67.0 and 67.3 (benzyl
CH2 rotamers), 70.4 and 71.1 (C4 rotamers), 117.9 and 118.0
(CN rotamers), 127.8, 128.0, 128.1, 128.2, 128.4 and 128.6 (benzyl
aromatic CH rotamers), 136.0 and 136.3 (benzyl ipso-C rotamers),
154.2 and 154.6 (CO2Bn rotamers); m/z (ES) 375 ([M + H]+
100%); HRMS m/z (ES) 375.2100, calculated for C20H31O3N2Si
375.2098.
tamers), 155.6 (CO2Bn), 156.1 (CO2 Bu); m/z (ES) 479 ([M + H]+
t
100%); HRMS m/z (ES) 479.2929, calculated for C25H43O5N2Si
479.2936.
(2S,4R)-2-[2-(tert-Butoxycarbonylamino)ethyl]-4-[(tert-butyl)-
dimethylsilyloxy]-N-(methoxycarbonylmethyl)pyrrolidine (13)
A solution of 11 (12.84 g, 26.8 mmol) in anhydrous CH3OH
(350 mL) was degassed with nitrogen before being added to
10% palladium on carbon (1.50 g) under a nitrogen atmosphere.
Hydrogen was bubbled through the reaction mixture for 5 minutes
and the reaction mixture was then stirred under a hydrogen
atmosphere for 18 h. Palladium on carbon was removed by
filtration and solvent was removed under reduced pressure. The
crude amine 12 was dried and then dissolved in anhydrous
CH2Cl2 (40 mL) under nitrogen. The solution was cooled to
0
◦C and DIEA (9.8 mL, 59.1 mmol) was added followed by
dropwise addition of methyl bromoacetate (4.9 mL, 53.1 mmol).
The reaction mixture was allowed to warm to room temperature
and stirred under nitrogen for 18 h. Solvent was removed under
reduced pressure and flash chromatography (4 : 1 hexanes–EtOAc,
Rf 0.5) afforded methyl ester 13 (7.21 g, 65%) as a pale yellow oil.
[a]D −11.5◦ (c = 1, CHCl3); mmax(KBr)/cm−1 3360 (NH), 1751,
1
1710, 1688 (CO); H NMR (400 MHz, CDCl3) d 0.03 (6H, s,
(2S,4R)-2-[2-(tert-Butoxycarbonylamino)ethyl]-4-[(tert-
Si(CH3)2), 0.86 (9H, s, SiC(CH3)3), 1.42 (9H, s, C(CH3)3), 1.55–
1.75 (3H, m, Ha3, Ha6 and Hb6), 2.23 (1H, ddd, J 13,4, 7.3, 6.1 Hz,
Hb3), 2.63 (1H, dd, J 9.7, 5.8 Hz, Ha5), 2.75–2.81 (1H, m, H2),
3.06 (1H, dd, J 9.7 1.6, Hz, Hb5), 3.14–3.27 (3H, m, Ha7, Ha8 and
Hb8), 3.55 (1H, m, J 16.7 Hz, Hb7), 3.70 (3H, s, OCH3), 4.30–
4.35 (1H, m, H4), 5.30 (1H, br s, NH); 13C NMR (100.6 MHz,
CDCl3) d −4.8 (Si(CH3)2), 18.1 (SiC(CH3)3, 25.8 (SiC(CH3)3, 28.4
(C(CH3)3), 32.2 (C6), 37.3 (C8), 40.3 (C3), 51.5 (OCH3), 53.7 (C7),
butyl)dimethylsilyloxy]-N-(benzyloxycarbonyl)pyrrolidine (11)
To a solution of nitrile 9 (230 mg, 0.66 mmol) in CH3OH (4 mL) at
room temperature was added CoCl2·6H2O (315 mg, 1.32 mmol),
followed by portionwise addition of NaBH4 (250 mg, 6.60 mmol).
The solution was stirred at room temperature for 4 h. EtOAc
(20 mL) was added to the reaction mixture and the resulting black
precipitate removed by filtration. Water (20 mL) was added to the
filtrate and the crude amine extracted with EtOAc (4 × 100 mL).
The organic fractions were combined and dried over MgSO4,
MgSO4 was removed by filtration and solvent was removed under
reduced pressure. The crude amine 10 was dried under reduced
pressure and dissolved in 1 : 1 H2O–1,4-dioxane (0.9 mL). To this
solution was added triethylamine (200 lL, 1.43 mmol) and di-
tert-butyl-dicarbonate (Boc anhydride) (220 mg, 1.01 mmol). The
reaction mixture was stirred at room temperature for 18 h and the
product was extracted with Et2O (5 × 100 mL). The organic frac-
tions were combined and dried over MgSO4. MgSO4 was removed
by filtration and solvent was removed under reduced pressure.
Purification by flash chromatography (3 : 1 hexanes–EtOAc, Rf
0.7) afforded the Boc-protected product 11 (195 mg, 62%) as a
colourless oil. [a]D −16.6◦ (c = 1, CHCl3); mmax(KBr)/cm−1 3357
t
60.5 (C2), 62.3 (C5), 70.5 (C4), 78.7 (C(CH3)3), 156.0 (CO2 Bu),
171.2 (CO2CH3); m/z (ES) 417 ([M + H]+ 100%); HRMS m/z
(ES) 417.2787, calculated for C20H41O5N2Si 417.2779.
(2S,4R)-2-[2-(tert-Butoxycarbonylamino)ethyl]-4-hydroxy-N-
(methoxycarbonylmethyl)pyrrolidine (14)
To a solution of 13 (6.51 g, 15.64 mmol) in anhydrous THF (65 mL)
was added tetrabutylammonium fluoride (TBAF) (14.00 g,
44.37 mmol) under nitrogen. The reaction mixture was stirred at
room temperature for 4 h. Solvent was removed under reduced
pressure and flash chromatography (EtOAc, Rf 0.2) afforded
alcohol 14 (3.88 g, 82%) as a pale yellow oil. [a]D + 28.5 (c =
1
1, CHCl3); mmax(KBr)/cm−1 3360 br (OH), 1740, 1684 (CO); H
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NMR (400 MHz, CDCl3) d 1.42 (9H, s, (C(CH3)3), 1.56–1.66 (2H,
m, Ha3 and Ha6), 1.74–1.79 (1H, m, Hb6), 2.30–2.37 (1H, m, Hb3),
2.54 (1H, dd, J 9.8 4.4 Hz, Ha5), 2.61–2.64 (1H, m, H2), 3.10–3.18
(4H, m, Hb5, Ha7, Ha8 and Hb8), 3.56 (1H, d, J 16.8 Hz, Hb7),
3.70 (3H, s, OCH3), 4.25 (1H, br s, H4); 13C NMR (100.6 MHz,
CDCl3) d 28.3 (C(CH3)3), 32.8 (C6), 37.3 (C8), 40.2 (C3), 51.6
(OCH3), 53.4 (C7), 60.5 (C2), 62.6 (C5), 69.6 (C4), 78.9 (C(CH3)3),
5.2 Hz, Hb5), 3.55 (1H, d, J 17.6 Hz, Hb7), 3.69 (3H, s, OCH3),
4.26–4.28 (1H, m, H4); 13C NMR (100.6 MHz, CDCl3) d 28.2
(C(CH3)3), 32.7 (C6), 37.3 (C8), 40.5 (C3), 51.6 (OCH3), 53.2 (C7),
t
59.1 (C2), 61.8 (C5), 69.9 (C4), 78.8 (C(CH3)3), 155.9 (CO2 Bu),
172.2 (CO2CH3); m/z (ES) 303 ([M + H]+ 100%); HRMS m/z
(ES) 303.1914, calculated for C14H27O5N2 303.1914.
156.0 (CO2 Bu), 171.4 (CO2CH3); m/z (ES) 325 ([M + Na]+ 100%),
t
(2ꢀS,4ꢀR)-2-[2-(tert-Butoxycarbonylamino)ethyl]-4-(N3-
benzoylthymin-1-yl)-N-(methoxycarbonylmethyl)pyrrolidine (17)
303 ([M + H]+ 85%); HRMS m/z (ES) 303.1913, calculated for
C14H27O5N2 303.1914.
To a solution of alcohol 17 (400 mg, 1.32 mmol) in anhydrous THF
(50 mL) under nitrogen, was added N3-benzoylthymine (370 mg,
1.61 mmol) and triphenylphosphine (420 mg, 1.60 mmol). The
(2S,4S)-2-[2-(tert-Butoxycarbonylamino)ethyl]-4-formyloxy-N-
(methoxycarbonylmethyl)pyrrolidine (15)
◦
mixture was cooled to −20 C and DIAD (360 lL, 1.83 mmol)
To a solution of alcohol 14 (3.20 g, 10.59 mmol) in anhydrous THF
(50 mL) under nitrogen, was added triphenylphosphine (3.60 g,
13.73 mmol). The solution was cooled to −20 ◦C and anhydrous
formic acid (560 lL, 13.78 mmol) was added followed by dropwise
addition of DIAD (2.75 mL, 13.97 mmol). The reaction mixture
was allowed to warm to room temperature and stirred under
nitrogen for 18 h. Triphenylphosphine (1.80 g, 6.87 mmol) was
added and the reaction mixture was cooled to −20 ◦C, anhydrous
formic acid (260 lL, 6.89 mmol) was added followed by dropwise
addition of DIAD (1.38 mL, 6.99 mmol). The reaction mixture was
allowed to warm to room temperature and stirred under nitrogen
for 3 h. Solvent was removed under reduced pressure and flash
chromatography (1 : 1 hexanes–EtOAc Rf 0.3) afforded formyl
ester 15 (2.45 g, 70%) as a pale yellow oil. [a]D−10.2◦ (c = 1,
was added dropwise. The reaction mixture was allowed to warm to
room temperature and stirred under nitrogen for 18 h. Solvent was
removed under reduced pressure and flash chromatography (1 : 1
hexanes–EtOAc Rf 0.4) afforded thyminyl derivative 17 (453 mg,
67%) as a white foam. Found: C 60.28; H 6.76, N 10.36, Calculated
for C26H34O7N4; C 60.69, H 6.66, N 10.89%; [a]D−49.3◦ (c =
0.5, CHCl3); mmax(KBr)/cm−1 3373 (NH), 1746, 1698, 1652 (CO);
1
kmax (CH3OH)/nm 252 (e/dm3mol−1 cm−1 1.6 × 104); H NMR
(400 MHz, CDCl3) d 1.42 (9H, s, C(CH3)3), 1.48–1.55 (1H, m,
Ha3ꢀ), 1.60 (1H, dd, J 13.9, 7.2 Hz, Ha6ꢀ), 1.83–1.91 (1H, m, Hb6ꢀ),
1.99 (3H, s, thymine CH3), 2.56–2.66 (3H, m, H2ꢀ, Hb3ꢀ and Ha5ꢀ),
2.99 (1H, d, J 17.2 Hz, Ha7ꢀ), 3.09–3.18 (2H, m, HaHb8ꢀ), 3.33
(1H, d, J 11.1 Hz, Hb5ꢀ), 3.69 (1H, d, J 17.2 Hz, Hb7ꢀ), 3.74
(3H, s, O–CH3), 4.85 (1H, br s, NH), 5.00–5.04 (1H, m, H4ꢀ), 7.47
(2H, t, J 7.5 Hz, Bz meta-H), 7.62 (1H, t, J 7.5 Hz, Bz para-H),
7.90 (2H, d, J 7.5 Hz, Bz ortho-H), 8.09 (1H, s, H6); 13C NMR
(100.6 MHz, CDCl3) d 12.7 (thymine CH3), 28.3 (C(CH3) 3), 32.6
(C6ꢀ), 37.2 (C8ꢀ), 38.7 (C3ꢀ), 51.8 (O-CH3), 51.9 (C4ꢀ), 52.7 (C7ꢀ),
58.7 (C5ꢀ), 60.7 (C2ꢀ), 79.3 (C(CH3)3), 111.3 (C5), 129.0 (Bz meta-
C), 130.3 (Bz ortho-C), 131.6 (Bz ipso-C), 134.8 (Bz para-C), 137.7
1
CHCl3); mmax(KBr)/cm−1 3336 (NH), 1739, 1720, 1685 (CO); H
NMR (400 MHz, CDCl3) d 1.43 (9H, s, (C(CH3)3), 1.50–1.59 (1H,
m, Ha6), 1.75–1.82 (1H, m, Hb6), 1.92 (1H, ddd, J 13.6, 6.7, 2.1 Hz,
Ha3), 2.04 (1H, ddd, J 13.6, 6.4, 2.2 Hz, Hb3), 2.54 (1H, dd, J 11.1,
3.6 Hz, Ha5), 2.92–2.98 (1H, m, H2), 3.09–3.19 (2H, m, Ha8 and
Hb8), 3.22 (1H, d, J 16.7 Hz, Ha7), 3.60 (1H, d, J 16.7 Hz, Hb7),
3.69 (1H, dd, J 11.1, 6.3 Hz, Hb5), 3.72 (3H, s, OCH3), 4.95 (1H,
br s, NH), 5.26–5.31 (1H, m, H4), 8.00 (1H, s, OCHO); 13C NMR
(100.6 MHz, CDCl3) d 28.4 (C(CH3)3), 32.4 (C6), 37.3 (C8), 37.4
(C3), 51.8 (OCH3), 54.0 (C7), 59.5 (C5), 59.9 (C2), 72.5 (C4), 79.1
t
(C6), 149.9 (C2), 155.8 (CO2 Bu), 162.8 (C4), 169.2 (Bz CO), 170.9
(CO2CH3); m/z (ES) 537 ([M + Na]+ 100%), 515 ([M + H]+ 60%);
HRMS m/z (ES) 515.2514, calculated for C26H35O7N4 515.2500.
t
(C(CH3)3), 155.9 (CO2 Bu), 160.5 (OCHO), 171.0 (CO2CH3); m/z
(2ꢀS,4ꢀR)-2-[2-(tert-Butoxycarbonylamino)ethyl]-4-(thymin-1-
yl)pyrrolidine-1-yl-acetic acid (18)
(ES) 331 ([M + H]+ 100%), 353 ([M + Na+] 40%); HRMS m/z
(ES) 331.1864, calculated for C15H27O6N2 331.1864.
To a solution of methyl ester 17 (400 mg, 0.78 mmol) in THF
(4 mL) was added 1 M aqueous NaOH (2.4 mL, 2.4 mmol)
and the reaction mixture was stirred at room temperature for
18 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 (7 : 3 EtOAc–CH3OH Rf 0.2) followed by reversed
phase chromatography (BondElut C18, H2O–CH3CN 9 : 1), the
product was lyophilised to afford acid 18 (220 mg, 71%) as a white
powder. [a]D + 5.2◦ (c = 1, CH3OH); mmax(KBr)/cm−1 3353 br (OH),
1720, 1680, 1651 (CO); kmax (CH3OH)/nm 267 (e/dm3 mol-1 cm-1
1.27 × 104); 1HNMR(400MHz, CD3OD) d 1.35 (9H, s, (C(CH3)3),
1.72–1.77 (1H, m, Ha6ꢀ), 1.80 (3H, s, thymine CH3), 1.98–2.08 (2H,
m, Ha3ꢀ and Hb6ꢀ), 2.74–2.81 (1H, m, Hb3ꢀ), 3.03–3.08 (2H, m, Ha8ꢀ
and Hb8ꢀ), 3.28–3.38 (2H, m, H2ꢀ and Ha5ꢀ), 3.43 (1H, d, J 16.2 Hz,
Ha7), 3.74 (1H, d, J 16.2 Hz, Hb7ꢀ), 3.91 (1H, d, J 12.6 Hz, Hb5ꢀ),
(2S,4S)-2-[2-(tert-Butoxycarbonylamino)ethyl]-4-hydroxy-N-
(methoxycarbonylmethyl)pyrrolidine (16)
To a solution of formyl ester 15 (2.21 g, 6.68 mmol) in anhydrous
CH3OH (15 mL) under nitrogen, was added anhydrous sodium
methoxide (90 mg, 1.67 mmol). The reaction mixture was stirred
at room temperature for 2 h. Anhydrous sodium methoxide
(45 mg, 0.84 mmol) was added and the reaction mixture stirred
for a further 3 h. Solvent was removed under reduced pressure
and flash chromatography (EtOAc Rf 0.2) afforded 4S alcohol
16 (1.82 g, 90%) as a pale yellow oil. [a]D + 31.4◦ (c = 1,
CHCl3); mmax(KBr)/cm−1 3362 br (OH), 1744, 1690 (CO); 1H NMR
(400 MHz, CDCl3) d 1.39 (1H, s, (C(CH3)3), 1.44–1.51 (1H, m,
Ha6), 1.63–1.75 (2H, m, Ha3 and Hb6), 1.93 (1H, dd, J 13.0,
6.1 Hz, Hb3), 2.53 (1H, d, J 10.8 Hz, Ha5), 3.01–3.13 (3H, m, H2,
Ha8 and Hb8), 3.35 (1H, d, J 17.6 Hz, Ha7), 3.48 (1H, dd, J 10.8,
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4.68–4.75 (1H, m, H4ꢀ), 7.47 (1H, s, H6); 13C NMR (100.6 MHz,
CD3OD) d 12.4 (thymine CH3), 28.8 (C(CH3) 3), 31.9 (C6ꢀ), 36.6
(C3ꢀ), 38.2 (C8ꢀ), 56.2 (C7ꢀ), 58.5 (C4ꢀ), 60.2 (C5ꢀ), 66.5 (C2ꢀ), 80.3
173.0 (CO2CH3); m/z (ES) 325 ([M + Na]+ 100%), 303 ([M + H]+
90%; HRMS m/z (ES) 325.1725, calculated for C14H26N2O5Na
325.1734.
t
(C(CH3)3), 111.6 (C5), 142.7 (C6), 153.2 (C2), 158.5 (CO2 Bu),
166.5 (C4), 171.2 (CO2H); m/z (ES) 397 ([M + H]+ 100%), 419
([M + Na]+ 50%); HRMS m/z (ES) 397.2094, calculated for
C18H29O6N4 397.2082.
(2R,4S)-2-[(tert-Butoxycarbonyl)aminomethyl]-4-formyloxy-N-
(methylpropanoate)pyrrolidine (22)
To a solution of alcohol 21 (2.01 g, 6.68 mmol) in THF (30 mL),
was added triphenylphosphine (2.25 g, 8.3 mmol) and anhydrous
formic acid (0.32 mL, 8.48 mmol) under nitrogen. The solution
was cooled to −20 ◦C and DIAD (1.7 mL, 8.57 mmol) was
added dropwise. The reaction mixture was allowed to warm to
room temperature and stirred under nitrogen for 18 h. Solvent
was removed under reduced pressure and the crude product was
purified by flash chromatography (5 : 1 hexanes–EtOAc, Rf 0.5
EtOAc) to afford the formyl ester 22 (1.44 g, 65%) as a pale
yellow oil. [a]D +57.7 (c = 1, CHCl3); mmax(BaF)/cm−1 2976 (CHO),
1723 (CO); 1H NMR (400 MHz, CDCl3) d 1.40 (9H, s, C(CH3)3),
1.82–1.96 (2H, m, Ha3, Hb3), 2.23 (1H, dd, J 10.6, 3.7 Hz, Ha5),
2.41–2.46 (3H, m, Ha8, Ha7, Hb7), 2.82 (1H, br s, H2), 3.08–
3.15 (2H, m, Hb8, Hb5), 3.31–3.37 (1H, m, Ha6), 3.56 (1H, dd, J
10.6, 6.1 Hz Hb6), 3.67 (3H, s, OCH3), 5.04, 5.06 (1H, 2 × s, NH
rotomers), 5.14–5.19 (1H, m, H4), 7.96 (1H, s, CHO); 13C NMR
(100.6 MHz; CDCl3) d 28.3 (C(CH3)3), 33.5 (C7), 34.5 (C3), 39.9
(C6), 48.7 (C8), 51.7 (OCH3), 59.0 (C5), 61.2 (C2), 72.3 (C4), 78.9
(2R,4R)-2-(Azidomethyl)-4-hydroxy-N-
(methylpropanoate)pyrrolidine (20)
To a suspension of the azide hydrochloric salt 19 (4.17 g,
23.34 mmol) in anhydrous CH2Cl2, was added DIEA (1 mL,
8.22 g, 63.57 mmol) at 0 ◦C under nitrogen and the suspension
stirred until dissolution. To the solution was added methyl acrylate
(4.25 mL, 4.06 g, 47.19 mmol) dropwise, and the reaction mixture
was stirred at 0 ◦C for 30 min. The reaction mixture was allowed
to warm to room temperature and stirred for a further 18 h under
nitrogen. Solvent was removed under reduced pressure and the
crude product purified by flash chromatography (1 : 1 hexanes–
EtOAc, Rf 0.2 EtOAc) to afford methyl ester 20 (4.52 g, 85%)
as a pale yellow oil. [a]D +57.7 (c = 1, CHCl3); mmax(BaF)/cm−1
3401 br (OH), 2104 (N3), 1727 (CO); 1H NMR (400 MHz; CDCl3)
d 1.65 (1H, dd, J 14.3, 4.6 Hz, Ha3), 2.20–2.27 (1H, m, Hb3),
2.30 (1H dd, J 9.8, 4.0 Hz Ha5), 2.45–2.57 (3H, m, Ha7, Hb7
and Ha8), 2.65–2.71 (1H, m, H2), 2.83 (1H, s, OH), 3.05–3.17
(2H, m, Hb5 and Hb8), 3.29 (1H, dd, J 12.4, 4.4 Hz, Ha6), 3.46
(1H, dd, J 12.4, 3.2 Hz, Hb6), 3.65 (3H, s, OCH3) 4.17 (1H, s,
H4); 13C NMR (100.6 MHz; CDCl3) d 33.5 (C7), 38.1 (C3), 48.1
(C8), 51.6 (OCH3), 53.8 (C6), 61.8 (C5), 61.9 (C2), 70.1 (C4),
172.6 (CO2CH3); m/z (ES) 251 ([M + Na]+ 100%), 229 ([M + H]+
55%); HRMS m/z (ES) 251.1118, calculated for C9H16N5O2
251.1115.
t
(C(CH3)3), 156.3 (CO2 Bu), 160.5 (CHO), 172.8 (CO2CH3); m/z
(ES) 353 ([M + Na]+ 100%), 331 ([M + H]+ 40%); HRMS m/z
(ES) 353.1690, calculated for C15H26N2O6Na 330.1783.
(2R,4S)-2-[(tert-Butoxycarbonyl)aminomethyl]-4-hydroxy-N-
(methylpropanoate)pyrrolidine (23)
To a solution of formyl ester 22 (1.39 g, 4.19 mmol) in anhydrous
CH3OH (15 mL) was added sodium methoxide (34 mg, 0.62 mmol)
under nitrogen at room temperature. The reaction mixture was
stirred until no starting material was left (ca. 2 h), as shown by
TLC. Solvent was removed under reduced pressure and the crude
product was purified by flash chromatography (1 : 1 hexane–
EtOAc, Rf 0.1 EtOAc) to afford the S-alcohol 23 (1.03 g, 82%)
as a pale yellow oil. [a]D +111.7 (c = 1, CHCl3); mmax(KBr)/cm−1
3389 br (OH) 1737 and 1688 (CO); 1H NMR (400 MHz, CDCl3)
d 1.40 (9H, s, C(CH3)3), 1.70–1.75 (1H, m, Ha3), 1.78–1.90 (1H,
m, Hb3), 2.20 (1H, dd, J 9.85, 4.67 Hz, Ha5), 2.40–2.51 (3H, m,
Ha8, Ha7 and Hb7), 2.86 (1H, br s, H2), 3.07–3.13 (2H, m, Hb5
and Hb8), 3.25–3.30 (1H, m, Ha6), 3.40 (1H, dd, J 9.72, 5.81 Hz,
Hb6), 3.66 (3H, s, OCH3), 4.30 (1H, br s, H4), 5.06 (1H, d, J 6.57,
NH); 13C NMR (100.6 MHz; CDCl3) d 28.3 (C(CH3)3), 33.7 (C7),
37.9 (C3), 40.4 (C6), 49.1 (C8), 51.7 (OCH3), 61.3 (C2), 61.7 (C5),
(2R,4R)-2-[(tert-Butoxycarbonyl)aminomethyl]-4-hydroxy-N-
(methylpropanoate)pyrrolidine (21)
To a solution of the methyl ester azide 20 (522 mg, 2.28 mmol) in
THF (10 mL) was added a 1 M solution of trimethylphosphine
in THF (3.43 mL, 3.43 mmol) and water (42 lL, 2.33 mmol).
The solution was stirred until all the starting material had been
consumed, as determined by TLC (ca. 1.5 h). The solution
was cooled to −20 ◦C and 2-(tert-butoxycarbonyloxyimino)-2-
phenylacetonitrile (Boc-ON) (1.41 g, 5.74 mmol) was added, the
reaction mixture was stirred at −20 ◦C for 15 minutes then allowed
to warmed to room temperature and stirred for a further hour.
Solvent was removed under reduced pressure and the crude
product was purified by flash chromatography (1 : 1 hexanes–
EtOAc, Rf 0.1 EtOAc) to afford the Boc-protected product 21
(619 mg, 89%) as a pale yellow oil. [a]D +63.2 (c = 1, CHCl3);
t
69.6 (C4), 78.9 (C(CH3)3), 156.4 (CO2 Bu), 173.0 (CO2CH3); m/z
(ES) 325 ([M + Na]+ 100%), 303 ([M + H]+ 75%); HRMS m/z
1
mmax(BaF)/cm−1 3389 br (OH) 1742 and 1692 (CO); H NMR
(ES) 303.1916, calculated for C14H27N2O5 303.1914.
(400 MHz, CDCl3) d 1.42 (9H, s, C(CH3)3), 1.60 (1H, dd, J 14.3,
5.9 Hz, Ha3), 2.16–2.26 (1H, m, Hb3), 2.28 (1H, dd, J 9.8, 4.1 Hz,
Ha5), 2.37–2.43 (1H, m, Ha8), 2.46–2.53 (2H, m, Ha7 and Hb7),
2.59 (1H, br s, OH), 3.07–3.17 (3H, m, Hb5, H2 and Ha6), 3.32–
3.37 (1H, m, Hb6), 3.69 (3H, s, OCH3), 4.20 (1H, t, J 4.4 Hz,
H4), 5.20 (1H, br s, NH); 13C NMR (100.6 MHz; CDCl3) d 28.4
(C(CH3)3), 33.5 (C7), 38.0 (C3), 40.9 (C6), 48.2 (C8), 51.7 (OCH3),
(2ꢀR,4ꢀR)-2-[(tert-Butoxycarbonyl)aminomethyl]-4-(N3-
benzoylthymin-1-yl)-N-(methylpropanoate)pyrrolidine (24)
To the S-alcohol 23 (475 mg, 1.57 mmol) in anhydrous THF
(20 mL) under nitrogen was added triphenylphosphine (550 mg,
2.04 mmol) and N3-benzoylthymine (462 mg, 2.00 mmol). The
t
61.9 (C2), 62.0 (C5), 69.9 (C4), 79.1 (C(CH3)3), 156.5 (CO2 Bu),
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◦
suspension was cooled to 0 C and DIAD (462 lL, 2.00 mmol)
POM oligomer synthesised. All other chemicals used in solid-
phase work were obtained at the highest purity grade from
Aldrich Chemical Company or Acros Organics and were used
without further purification. Reagents used for the Kaiser test
were prepared according to literature.17
was added dropwise and the reaction mixture was stirred for 5 min.
The reaction mixture was allowed to warm to room temperature
and stirred for 18 h under nitrogen. Solvent was removed under
reduced pressure and the crude product was purified by flash
chromatography (1 : 1 hexane–EtOAc, Rf 0.1 EtOAc) to afford
the thyminyl derivative 24 (516 mg, 64%) as a white foam. [a]D
+68.1 (c = 1, CHCl3); mmax(KBr)/cm−1 1746, 1699 and 1652 (CO);
1H NMR (400 MHz; CDCl3) d 1.46 (9H, s, C(CH3)3), 1.63–1.70
(1H, m, Ha3ꢀ), 2.00 (3H, s, thymine CH3), 2.23–2.30 (1H, m, Ha8ꢀ),
2.52–2.60 (5H, m, Ha7ꢀ, Hb7ꢀ, Hb3ꢀ, Ha5ꢀ and H2ꢀ), 3.19–3.32 (3H,
m, Ha6ꢀ, Hb8ꢀ and Hb5ꢀ), 3.59 (1H, dd, J 14.2, 9.6 Hz, Hb6ꢀ),
3.77 (3H, s, OCH3), 5.14 (1H, br s, H4ꢀ), 5.37 (1H, d, J 7.1 Hz,
NH), 7.47 (2H, t, J 7.6 Hz, Bz meta-H), 7.62 (1H, t, J 7.6 Hz,
Bz para-H), 7.79 (1H, s, H6), 7.89 (2H, d, J 7.6 Hz, Bz ortho-
H); 13C NMR (100.6 MHz; CDCl3) d 12.7 (thymine CH3), 28.3
(C(CH3)3), 33.2 (C7ꢀ), 35.5 (C3ꢀ), 39.0 (C6ꢀ), 47.2 (C8ꢀ), 51.0 (C4ꢀ),
51.8 (OCH3), 59.0 (C5ꢀ), 63.2 (C2ꢀ), 79.4 (C(CH3)3), 111.0 (C5),
129.1 (Bz ortho-C), 130.4 (Bz meta-C), 131.5 (Bz ipso-C), 134.9
General procedure for solid-phase synthesis
Into a 1 mL solid-phase synthesis vessel was weighed MBHA
resin (5 equiv.). Washing of the resin was carried out 3 times
with DMF (all washings use 1 mL per 25 lmol resin loading,
in all cases performed with rotation of vessel for 30 s each time,
after which solvent was removed through a Buchner flask under
reduced pressure) and 3 times with CH2Cl2. The resin was swelled
in CH2Cl2. Washing of the resin was carried out 3 times with DMF
for 30 s each time, once with 5% piperidine–DMF for 4 minutes
and 3 times with DMF–CH2Cl2 (1 : 1). In a separate small vial,
Boc-POM–(T)–OH (1 equiv.), HBTU (0.95 equiv.) and DIEA
(1.1 equiv.) in DMF–pyridine (3 : 1) (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. The coupling reagent was removed and the
resin washed 2 times with DMF for 30 s each time. The resin
was treated with freshly prepared acetic anhydride–collidine–
DMF (1 : 1 : 8) (1 mL per 25 lmol) with agitation for 15 min.
The acetylating reagent was removed by vacuum suction and
resin washed with DMF (3 times for 30 s each time), complete
reaction was indicated by negative Kaiser test. The resin was
then washed with 5% piperidine–DMF (once for 4 minutes) and
DMF–CH2Cl2 (1 : 1) (3 times for 30 s each time). Deprotection of
the resin-bound Boc-protected POM oligomer was accomplished
using TFA–m-cresol (1 mL per 25 lmol) 4 times for 4 minutes
each time. The resin was washed with DMF–CH2Cl2 (1 : 1)
(3 times for 30 s each time) and deprotection was indicated by
a positive Kaiser test. The resin was then washed with pyridine
(2 times for 30 s each time). Subsequent coupling employed Boc-
POM(T)–OH (5 equiv.), HBTU (4.75 equiv.), DIEA (5.5 equiv.)
and coupling times of 2 h. In the case of lysine, Boc-Lys-(2-Cl-Z)–
OH (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 Cbz-
protected nucleobases and cleavage of the oligomer from the resin
was achieved by the ‘Low–high TFMSA’ method. During ‘low
TFMSA’ the resin was treated with a solution of (TFA–DMS–m-
cresol (1 : 3 : 1)) and a solution of (TFA–TFMSA (9 : 1)) (each
t
(Bz para-C), 137.5 (C6), 149.8 (C2), 156.2 (CO2 Bu), 162.7 (C4),
169.1 (benzamide CO) 173.2 (CO2CH3); m/z (ES) 515 ([M + H]+
100%); HRMS m/z (ES) 515.2504, calculated for C26H35N4O7
515.2500.
(2ꢀR,4ꢀR)-2-(tert-Butoxycarbonylamino-methyl)-4-(thymin-1-
yl)pyrrolidine-1-yl propanoic acid (25)
To a solution of methyl ester 24 (314.7 mg, 0.611 mmol) in THF
(5 mL) was added 1 M aqueous NaOH (1.85 mL, 1.85 mmol). The
reaction mixture was stirred at room temperature for 3 h. THF was
removed under a stream of nitrogen and the aqueous solution was
adjusted to pH 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 Rf 0.2) followed by reversed phase chromatography (BondElut
C18, H2O–CH3CN 9 : 1), the product was lyophilised to afford acid
25 (295 mg, 69%), as a white solid. [a]D + 60.3 (c = 1, CH3OH);
mmax(KBr)/cm−1 1701, 1696, 1685, 1680 and 1675 (CO); 1H NMR
(400 MHz; CD3OD) d 1.31 (9H, s, C(CH3)3), 1.52–1.61 (1H, m,
Ha3ꢀ), 1.84 (3H, s, thymine CH3), 2.32–2.39 (1H, m, Ha8ꢀ), 2.42–
2.54 (3H, m, Ha7ꢀ, Hb3ꢀ and Hb7ꢀ), 2.61–2.70 (2H, m, Ha5ꢀ and H2ꢀ),
3.12–3.21 (1H, m, Ha6ꢀ), 3.25–3.42 (3H, m, Hb8ꢀ, Hb5ꢀand Hb6ꢀ),
4.84 (1H, s, H4ꢀ), 7.76 (1H, s, H6). 13C NMR (100.6 MHz; CD3OD)
d 12.7 (thymine CH3), 28.7 (C(CH3)3), 34.7 (C7ꢀ), 36.6 (C3ꢀ), 40.8
(C6ꢀ), 50.4 (C8ꢀ), 54.6 (C4ꢀ), 58.9 (C5ꢀ), 65.7 (C2ꢀ), 80.3 (C(CH3)3),
t
111.5 (C5), 140.8 (C6), 153.0 (C2), 158.7 (CO2 Bu), 166.5 (C4),
177.6 (CO2H); m/z (ES) 419 ([M + Na]+ 100%), 397 ([M + H]+
10%); HRMS m/z (ES) 419.1909, calculated for C17H26O6N4Na
419.1901.
◦
1 mL per 20 lmol resin loading) each separately cooled to 0 C
before being added to resin and agitated for 1 h. The cleavage
mixture was removed by vacuum suction. ‘High TFMSA’ was
carried by treating the resin with a solution of TFMSA–TFA–
m-cresol (1 : 8 : 1) (1 mL per 10 lmol resin loading) cooled
to 0 ◦C before being added to resin and agitated for 1 h. The
cleavage mixture was removed by vacuum suction. The cleavage
solutions were separately concentrated under a stream of nitrogen
to ∼50 lL and the oligomer was precipitated from the cleavage
mixtures by addition of a ten-fold excess of anhydrous diethyl
ether. The mixture was subject to centrifugation (10 min, 12 000
rpm, 4 ◦C) and the resulting pellet was redissolved in formic acid
POM oligomer synthesis
All experiments were carried out in solid-phase synthesis vessels
purchased from Kinesis and fitted a with porosity-3 frit. Resin
was agitated by rotation of the vessel and reagents were removed
by suction filtration through a Buchner flask. MBHA resin LL
(100–200 mesh) (loading of 0.62 mmol/g), Boc–Lys–(2-Cl–Z)–
OH and HBTU were purchased from Novabiochem. Fresh bottles
of anhydrous solvents from Acros Organics were used for each
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and diluted again with anhydrous diethyl ether. The centrifugation
process was repeated a further three times. After the final time
the pellets were dissolved in water and lyophilised to give crude
POM oligomers as off-white powders. The oligomers were then
purified by semi-preparative reversed-phase HPLC on a C18
column (Phenomenex Gemini 5 l C18, 250 × 10 mm) with a typical
gradient of 0–10% acetonitrile with 0.1% HCO2H–0.1% aqueous
HCO2H. Fractions collected were evaporated and lyophilised to
give pure product as a white powder. Product purity was verified
by analytical reversed-phase HPLC (Phenomenex Gemini 5 l C18,
150 × 4.6 mm) and oligomers were characterised by MALDI-TOF
mass spectrometry.
use. Sterile nuclease, protease and DEPC-free deionised water was
used throughout. All samples were stored at −20 ◦C. Oligonu-
cleotides were purchased from Sigma-Genosys or sigma Proligo.
PNA monomers were purchased from Applied Biosystems. Each
thermal denaturation experiment consists of 3 ramps and an
averaging time of 1 s was used throughout. Data was collected
every 1 ◦C for the first ramp and 0.1 ◦C for subsequent part of the
experiment. Samples were initially heated at a rate of 5 ◦C min−1
to 93 ◦C to dissociate all strands. After 1 min, samples were cooled
at 0.2 C min−◦1 to 15 C and after a holding time of 1 min were
heated at 0.2 C min−1 to 93 ◦C. All Tm values were obtained
from the maxima of first derivative curves calculated from
Varian Thermal software using a filter size of 97 and smoothed
every 0.3 ◦C.
◦
◦
POM Lys–(T)8–NH2 (27)
Retention time on analytical HPLC was 29 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 2259 ([M + H]+
100%, C102H144N35O25 requires m/z, 2259.1).
Circular dichroism experiments
CD spectra were recorded on a JASCO J-715 spectropolarimeter.
The CD spectra of the POM–DNA complexes and the relevant
single strands were recorded in 10 mM potassium phosphate
buffer, 0.12 M KCl at pH 7.0 unless otherwise stated. The CD
spectra were recorded as an accumulation of 10 scans from 320 to
180 nm using a 0.5 cm cell, a resolution of 0.1 nm, band-width of
1.0 nm, sensitivity of 2 m deg, response of 2 seconds and a scan
speed of 50 nm min−1.
bePOM I Lys–(T)8–NH2 (28)
Retention time on analytical HPLC was 33 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 2371 ([M + H]+
100%, C110H160N35O25 requires m/z, 2371.2).
Acknowledgements
This work was funded by the BBSRC (research grant 36/B15998)
and EPSRC (PhD studentships to R. J. W. and N. M. B)
References
bePOM II Lys–(T)8–NH2 (29)
1 (a) J. Micklefield, Curr. Med. Chem., 2001, 8, 1157; (b) H.-J. Hsu, M.-R.
Liang, C.-T. Chen and B.-C. Chung, Nature, 2006, 439, 480; (c) I. A.
Sheptopalov, S. Sinha and J. K. Chen, Nat. Chem. Biol., 2007, 3, 650;
(d) X. Tang, S. Maegawa, E. S. Weinberg and I. J. Dmochowski, J. Am.
Chem. Soc., 2007, 129, 11000.
2 (a) B. Vester and J. Wengel, Biochemistry, 2004, 43, 13233; (b) P. E.
Nielsen, Curr. Opin. Biotechnol., 2001, 12, 16; (c) M. Petersen and J.
Wengel, Trends Biotechnol., 2003, 21, 74.
Retention time on analytical HPLC was 21 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 97% A
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Thermal denaturation experiments
UV melting plots of absorbance versus temperature were measured
at 260 nm on a Varian Cary 400 Scan UV-visible spectropho-
tometer fitted with a 6 × 6 Peltier thermostatable multicell holder
connected to a temperature-controller module. Experiments were
performed in double-beam mode and controlled by an interfaced
Dell OptiPlex GX150 computer. Denaturation experiments were
performed in 10 mm path length 4 mm path width self-masking
semi-micro quartz cells fitted with a Teflon stopper. Concentra-
tions of POM oligomers, oligonucleotides and polynucleotides
were measured spectrophotometrically at 80 ◦C from molar
extinction coefficients of nucleotidyl units calculated from the
literature.
Buffers were prepared as double-concentrated stock solutions
and diluted to the appropriate concentrations during sample
preparation. All appropriate equipment were autoclaved before
102 | Org. Biomol. Chem., 2008, 6, 92–103
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