Lipophilic peptide nucleic acids containing a 1,3-diyne function: Synthesis, characterization and production of derived polydiacetylene liposomes

Article abstract of DOI:10.1016/j.tet.2005.07.021

Adenine-, cytosine- and thymine-containing peptide nucleic acid (PNA) monomers have been synthesized in which either diacetylenic or stearoyl moieties are attached to the N-or C-terminus; the diacetylenic group is embedded within a long hydrocarbon chain.

Full text of DOI:10.1016/j.tet.2005.07.021

Tetrahedron 61 (2005) 8875–8887
Lipophilic peptide nucleic acids containing a 1,3-diyne function:
synthesis, characterization and production of derived
polydiacetylene liposomes
*
Nicola M. Howarth, W. Edward Lindsell, Euan Murray and Peter N. Preston
*
Chemistry, School of Engineering and Physical Sciences, William H. Perkin Building, Heriot-Watt University, Riccarton,
Edinburgh EH14 4AS, UK
Received 15 April 2005; revised 22 June 2005; accepted 7 July 2005
Available online 28 July 2005
Abstract—Adenine-, cytosine- and thymine-containing peptide nucleic acid (PNA) monomers have been synthesized in which either
diacetylenic or stearoyl moieties are attached to the N-or C-terminus; the diacetylenic group is embedded within a long hydrocarbon chain. A
range of analogous lipophilic functionalized PNA oligomers have been prepared using either solid phase synthesis or a post-synthetic
solution phase procedure following cleavage of the PNA oligomer from the solid support. Selected functionalized PNA monomers and
oligomers have been incorporated into liposomal polydiacetylenes and characterized by UV–vis absorption spectroscopy. Preliminary
investigations show that blue PDA-liposomes containing thymine-based PNAs can be formed and that production of liposomes with other
PNA systems are viable.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
employed to detect the enzymic activities of phospho-
lipases,9 the interaction of peptides with cell membranes,10
and antibody–epitope interactions.11 It is notable from the
work cited above6–11 that the recognition element in these
sensors can be either covalently linked to the reporter (PDA)
fragment through copolymerization7 or embedded within
the lipid bilayer by physical interactions.9
Polydiacetylenes (PDAs) [2, Scheme 1] can be prepared
from conjugated diynes 1 by solid state polymerization
through thermal treatment or by UV- and g-irradiation.1
Many PDAs exhibit chromic effects that can be induced by
heat,2 solvent effects3 and pH changes;4,5 such effects have
also been observed through binding processes designed to
mimic molecular recognition events at cell surfaces. For
example, Charych et al. prepared a thin film PDA-assembly
incorporating a sialic acid head group, which afforded a
quantifiable colorimetric response on exposure to influenza
virus.6 Later, they prepared a PDA virus biosensor more
efficiently through preliminary incorporation of the sialic
acid-labelled diacetylene into liposomes,7 and related
assemblies have been used to detect cholera toxin.8 More
recently, selectively labelled PDA liposomes have been
We are using this type of approach to design nucleic acid
biosensors in which the PDA head group or the embedded,
labelled lipid possesses DNA-intercalating or related bind-
ing properties. In earlier work, we synthesized lipophilic
acridine-labelled diacetylenes, thence PDAs,12 and more
recently described, in preliminary communication, the
preparation and polymerization of lipophilic peptide nucleic
acids (PNAs; cf. 3 Scheme 2) derived form stearic acid and
pentacosa-10,12-diynoic acid.13 A notable feature of PNAs
314 is that they bind with higher affinity to single-stranded
DNA (ssDNA) and RNA than their natural oligonucleotide
counterparts.15
Scheme 1.
Keywords: Nucleic acid biosensors; PNA; Polydiacetylenes; Liposomes.
*
Corresponding authors. Tel.: C44 131 451 8026; fax: C44 131 451 3180
(N.M.H); tel.: C44 131 451 8035; fax: C44 131 451 3180 (P.N.P);
e-mail addresses: N.M.Howarth@hw.ac.uk; P.N.Preston@hw.ac.uk
Scheme 2. BZthyminyl, cytosinyl, adeninyl, guaninyl.
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.07.021

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N. M. Howarth et al. / Tetrahedron 61 (2005) 8875–8887
In this paper, we provide full experimental details of our
earlier13 and subsequent investigations in this area.
The objectives of this study were as follows: (1) synthesis of
PNA ‘monomer’ model compounds with diacetylenic or
stearoyl moieties at the N- or C-terminus; (2) solid-phase
synthesis of PNA oligomers incorporating diacetylenic and
(separately) stearoyl groups; (3) production of novel PNA-
containing PDA liposomes.
2. Results and discussion
2.1. C-Terminal diacetylenic derivatives
Whilst the primary objective was the synthesis of PNA
oligomers attached to diacetylenic units, it was important to
prepare model PNA ‘monomers’ to evaluate their suscepti-
bility to topochemical polymerization. C-Terminal diacetyl-
enic derivatives were prepared by two routes: the protected
thymine ‘monomer’ 416,17 was hydrolyzed18 to afford 5 in
74% yield (Scheme 3).
Scheme 4. Reagents and conditions: (i) [(CH3)3COCO]2O, DCM, Et2O, rt;
(ii) CH3(CH2)11C4(CH2)8CO2N(COCH2)2,12 DCM, rt; (iii) 6 M aq HCl,
DCM, rt.
wasteful use of the diacetylenic reagent (87% yield for 14/
15) but an unavoidable byproduct 13 is formed in the first
step (12/13C14).
2.2. N-Terminal diacetylenic and stearoyl derivatives
PNA model compounds in this category were prepared by
two routes. The diyne 11 (Scheme 3) was prepared in 50%
yield from 414,15 by first removing the Boc protecting group
with hydrochloric acid, followed by treatment with N-
succinimidyl-10,12-pentacosadiynoate.12 We subsequently
achieved a higher yield in the coupling step (90%) through
use of 10,12-pentacosadiynoyl fluoride.13
In the alternative approach, the N-terminal diacetylenic
derivatives 24–26 and N-terminal stearoyl derivative 27
were prepared from N-(2-aminoethyl)glycine methyl ester
dihydrochloride 17 (Scheme 5). Treatment of 17 with either
N-succinimidyl-10,12-pentacosadiynoate12 or N-succin-
imidyl stearoate in the presence of N,N-diisopropylethyl-
amine (DIPEA) afforded 18 and 19, respectively, in 65 and
60% yield after purification. Compound 18 was then coupled
to thyminylacetic acid,17 N4-benzyloxycarbonylcytosinyl-
acetic acid18 and N6-benzyloxycarbonyladeninylacetic acid
using either 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyl-
uronium hexafluoro-phosphate (HBTU) or 2-(7-azabenzo-
triazol-1-yl)-1,1,3,3,-tetramethyluronium hexafluorophos-
phate (HATU) as the activating agent in the presence of
DIPEA. After work-up and purification by flash chromato-
graphy, 20, 21 and 22 were isolated in 63, 71 and 80% yields,
respectively. Similar conditions were applied to the coupling
of 19 to thyminyl acetic acid to obtain 23 (86% yield). Finally,
ester hydrolysis using lithium hydroxide afforded the desired
compounds 24–27 in yields ranging from 84–97%.
Scheme 3. BocZ(CH3)3COCO; TZthyminyl. Reagents and conditions: (i)
1 M aq LiOH, THF, rt; (ii) (a) 1.1 equiv EDC, 1.1 equiv HOBt, DMF, 0 8C-
rt (b) 0.94 equiv CH3(CH2)11C4(CH2)8CONH(CH2CH2O)2CH2CH2NH2
9 or 0.67 equiv CH3(CH2)11C4(CH2)9NH2 10, 1 equiv DIPEA, DMF, 0 8C-
rt; (iii) 6 M aq HCl, DCM, rt; (iv) 1 equiv CH3(CH2)11C4(CH2)8-
CO2N(COCH2)2,12 DMF, rt, or 1 equiv CH3(CH2)11C4(CH2)8COF,13 2:1
DMF:DCM, rt, 3 equiv DIPEA.
12
Compound 5 was then coupled using N-hydroxybenzotria-
zole (HOBt) and 1-(3-dimethylaminopropyl)-3-ethylcarbo-
diimide (EDC) to (separately) N-(80-amino-30,60-
dioxaoctyl)-10,12-pentacosadiynamide 912 and 1-amino-
10,12-pentacosadiyne 10; compounds 6 and 7 (Scheme 3)
were isolated in 66 and 38% yields, respectively, after
purification, with deprotection (6 M HCl) of the former
affording the hydrochloride salt 8 in high yield.
Surprisingly, attempts to deprotect compounds 25 and 26
were unsuccessful using standard procedures19 [e.g., 6 M aq
HCl or 10% aq KOH] and an alternative route20 was used to
produce a fully deprotected adenine-containing PNA-
monomer (Scheme 6). Thus, ethyl adenin-9-yl acetate18
was di-Boc protected (28a/28b) and the product was
hydrolyzed (28b/28c); a standard coupling procedure
An inefficient step in the sequence described above is the
low yield (38%) obtained in converting the relatively
expensive N-succinimidyl-10,12-pentacosadiynoate into
9.12 Therefore, an alternative route was also used in this
work (12/14/15/16; Scheme 4), which provided a less

N. M. Howarth et al. / Tetrahedron 61 (2005) 8875–8887
8877
(28c/28d) afforded the diacetylenic derivative, which was
hydrolyzed (6 M aq HCl) in almost quantitative yield to give
the desired, deprotected PNA monomer 28e. A character-
istic feature of the N-terminal diacetylenic derived PNA
monomers (24–26, 28e) is their gradual change on exposure
to light from colourless to pale blue in the solid state as a
result of topochemical polymerization.
2.3. Synthesis of N-stearoyl and diacetylenic PNA
oligomers
All PNA oligomers were assembled manually in a stepwise
fashion on a suitably derivatized solid support employing
the solid-phase protocol described by Christensen et al.21
and successfully utilized previously.22 Oligomers 29 and
31–36 (Scheme 7) were constructed on an N1-Boc-N5-(2-
chloro-Z)-L-lysine-derivatized 4-methyl-benzhydrylamine
(MBHA) resin whereas, for oligomers 30 and 37–40
(Scheme 7), an N-Boc-L-aspartic acid 4-benzyl ester
MBHA-derivatized resin was used. The Boc-protected
thymine monomer 5 (Scheme 3) was coupled using
HBTU as the activating agent in the presence of a slight
excess of DIPEA. All the couplings were monitored using
the Kaiser test23 and in all cases only single couplings were
required. At the end of the syntheses, the oligomers were
cleaved from the solid support using trifluoromethane-
sulfonic acid (TFMSA) under identical conditions to those
employed by Christensen et al.21 The crude oligomers were
purified using RP-HPLC and their identities were verified
by MALDI-TOF mass spectrometry.
Scheme 5. AZZN6-benzyloxycarbonyladeninyl; CZZN4-benzyloxycarbo-
nylcytosinyl; TZthyminyl. Reagents and conditions: (i) 1 equiv CH3
(CH2)11C4(CH2)8CO2N(COCH2)212 or 1 equiv CH3(CH2)16CO2N(COCH2)2,
3 equiv DIPEA, DMF, rt; (ii) 1.2 equiv BCH2CO2H,17,18 1.2 equiv HBTU
or HATU, 3 equiv DIPEA, DMF, rt; (iii) 1 M aq LiOH, THF, rt.
Scheme 7. T0Zthyminyl-PNA monomer unit; Lys and AspZlysine and
aspartic acid units, respectively, CH3(CH2)16CO, Ac, CH3(CH2)11C4
(CH2)8CO, H-PEG and CH3(CH2)11C4(CH2)8CO-PEGZacyl group
attached either to the N-terminal amino group of the PNA oligomer or to
the side-chain amino group of C-terminal lysine unit; H-PEGZH-
NH(CH2CH2O)2CH2CO; NH2ZC-terminal amide.
The stearoyl group was incorporated at the N-terminus of
oligomers 29 and 30 (Scheme 7) in the final coupling step
once the PNA T010-mer had been assembled on the solid
support. Initially, for the preparation of 29, attempts to
couple stearic acid to the PNA oligomer involved using
HBTU in the presence of DIPEA. However, in this case, it
was found necessary to use a 1:1 (v/v) mixture of
DCM:DMF as the coupling solvent for solubility reasons.
This coupling step was repeated twice in an attempt to
ensure complete reaction but, unfortunately, subsequent
analysis by RP-HPLC of the crude product obtained, after
cleavage and precipitation, showed the presence of almost
Scheme 6. Reagents and conditions: (i) [(CH3)3COCO]2O, DMAP, THF,
rt; (ii) 1 M aq LiOH, THF, rt; (iii) 18, DIPEA, HBTU, DMF, rt; (iv) 6 M aq
HCl, DCM, 40 8C.

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N. M. Howarth et al. / Tetrahedron 61 (2005) 8875–8887
equal amounts of the acetyl-capped failure sequence 31
(Scheme 7) [R.T.Z17 min] and the desired oligomer 29
[R.T.Z29 min]. Therefore, alternative strategies for incor-
porating the stearoyl moiety were sought. Our earlier
success in the use of 10,12-pentacosadiynoyl fluoride for the
preparation of model N-terminal PNA compounds led us
next to investigate the use of stearoyl fluoride in the solid
phase synthesis of 29.13 Thus, following N-terminal
deprotection of the PNA T010-mer, the solid-supported
oligomer was treated with a solution of stearoyl fluoride
and DIPEA in 1:1 (v/v) DCM:DMF. Once again, this
coupling procedure was repeated twice more before the
oligomer was cleaved from the support. This time subse-
quent analysis by RP-HPLC of the crude product revealed
the desired oligomer 29 to be the principal component, with
the capped failure sequence 31 accounting for !12% of the
material. This strategy for addition of the stearoyl group to
the N-terminus of a PNA-oligomer was also successfully
utilized for preparation of the analogous oligomer 30.
molecules.24 Amphiphilic diacetylenes with polar head-
groups and extended hydrocarbon chains form liposomes in
aqueous solutions with one set of head-groups exposed at
the surface and the other enclosing an interior hydration
sphere;25 in many cases the ordered structure permits
topochemical polymerization to produce stable PDA-
liposomes. Such PDA-liposomes have been described for
diacetylene lipids with, inter alia, carboxylic acid,7,25,26
ester,25 alcohol,25 amine,25 hydrazyl27 and carbohydrate
terminal groups6,25 and vary in colour from deep blue to
red, corresponding to decreasing effective p-conjugation
lengths of the ene–yne backbone. Moreover, the incorpor-
ation of suitable receptor elements at the surface of such
PDA-liposomes, either by covalent linking to the PDA or
by physical embedding in the matrix, can produce sensors
in which interactions with specific substrates induce
changes in polymer conformation with concomitant colour
changes.7–11,25,26,28,29
In this work, PDA-liposomes were prepared by a procedure
similar to that described by Charych and co-workers,9,28,29
involving sonication of an aqueous suspension of the amphi-
philic molecules, filtration, refrigeration and subsequent
photo-polymerization of the diacetylene arrays. As pre-
viously described,7,25 10,12-pentacosadiynoic acid 41
(Scheme 7) formed deep blue solutions of PDA-liposome
and this compound can be employed as a matrix for lipo-
somes of mixed composition.9 The model, mono-thymine
diacetylene 24 with an acid head-group, formed deep purple
solutions of liposome under similar synthetic conditions to
those applied to 41; the related ester 20 also turned purple
after irradiation but much precipitation of the product
occurred leaving a very dilute solution of the PDA. The
visible spectrum of PDA-liposome poly-41 shows typical
bands of the blue form, with lmax ca. 630 and 580 nm
(cf. Ref. 25); similar visible absorption bands were observed
for the products from 24 and 20 (dilute solution), but an
additional band with lmax in the region 510–540 nm
indicates the presence of some red form, contributing to
the overall purple colours (see Table 1). Addition of 2 M aq
NaOH to aqueous solutions of liposomes derived from 24,
as for 41, caused a colour change to red and a shift in the
main absorption band to ca. 530 nm (cf. Refs. 26, 30). A
band at lmax 271 nm (in poly-24) or 263 nm (in poly-20),
not observed in poly-41, may be assigned to absorption by
the thymine group. PDA-liposomes were subsequently
Preliminary investigations into the preparation of the related
N-terminal diacetylene-functionalized PNA oligomers
employing a similar approach to that described above,
using 10,12-pentacosadiynoyl fluoride in the final coupling
step, proved to be unsuccessful.13 The reasons for this are as
yet unclear but it appears that the diacetylene function may
polymerize under the conditions required to cleave the
oligomer from the solid support. Therefore, in order to
overcome this limitation, a post-synthetic solution-phase
modification method was developed. Thus, following
construction of the appropriate PNA oligomer on the solid
support, the N-terminus was acetyl capped and the oligomer
cleaved from the resin to afford 31 and 34, respectively
(Scheme 7). The lysine amino side-chains of the oligomers
31 and 34 were subsequently acylated by treatment of a
solution of the appropriate oligomer in 1:1 (v/v) DMF:
pyridine with the required acyl fluoride in the presence of
DIPEA to give 32, 33, 35 or 36, respectively.
This post-synthetic solution-phase modification approach
was also used successfully for the preparation of two N-
terminal diacetylenic-functionalized PNA oligomers [38
and 40 (Scheme 7)] needed for completion of our liposome
studies. Thus, following construction of the parent PNA-
oligomers 37 and 39 (Scheme 7), respectively, the oli-
gomers were cleaved from the solid support and treated with
10,12-pentacosadiynoyl fluoride13 in the presence of
DIPEA. Although, in both cases, these couplings failed to
go to completion, analysis of the crude products by RP-
HPLC showed that both 38 and 40 had been formed in ca.
50% yield. Furthermore, since there was a large difference
in retention times between the diacetylene-functionalized
PNA oligomers 38 and 40 and the parent PNA oligomers 37
and 39, respectively, it was relatively easy to separate out
the desired products.
Table 1. PDA-liposomes prepared in this work
Liposome
lmax (nm)
poly-41
poly-24
poly-20
632, 586, 235
624, 576, 518, 271
623, 536, 263, (220)a
630, 582, 254
copoly-41 (95%)Ccopoly-24 (5%)}
poly-41 (95%)Cpoly-20 (5%)}
{poly-41Cadded 27 (5%)}
{poly-41Cadded 23 (5%)}
copoly-41 (95%)Ccopoly-36 (5%)}
poly-41 (95%)Cpoly-38 (5%)}
{poly-41Cadded 30 (5%)}
{poly-41C added 35 (5%)}
631, 584, 230b
631, 587, 254
640, 591, 240b
634, 586, 259
2.4. Formation of liposomes and polymerization of
diacetylenes
623, 578, 240b
633, 587, 260
625, 579, 363, 256, (222)a
Liposomes (or, in more general terminology, vesicles)
comprise discrete aggregates of amphiphilic molecular
bilayers, formed by naturally occurring phospholipids but
also by self-assembly of synthetic surfactants and related
Variations in lmax of up to 10 nm are observed for samples from different
preparations.
a Dilute solution.
b Mainly matrix, poly-41.

N. M. Howarth et al. / Tetrahedron 61 (2005) 8875–8887
8879
produced from two component mixtures of 41 with 24 and,
separately, 20, to investigate the ability of forming
copolymeric structures: at 5% (by weight) 24, the resulting
deep blue liposome was similar in appearance to the matrix
poly-41 but exhibited a strong UV absorption at lmax
254 nm, supporting the incorporation of the mono-thymine
units into this product; however, with 20 (5%), the resulting
blue liposome showed a UV band lmax ca. 230 nm, similar
to that of the matrix alone, providing no evidence of incor-
poration of the thymine unit. PDA-liposome formation was
also carried out on mixtures of matrix material 41 with
varying proportions of the non-polymerizable N-stearoyl
thymine-monomers 27 and, separately, 23; mixtures
containing the acid 27 (5%) showed evidence, from the
UV spectrum, for the inclusion of the thymine-containing
component into the resulting blue liposome, but not in
the blue product of similar mixtures containing 23 with an
ester-head group. These results indicate that thymine-
containing, carboxylic acid lipids can be successfully
doped into liposomes derived from 10,12-pentacosadiynoic
acid as the matrix, either through covalent bonding within
the polymer chain (e.g., with 24) or by secondary
interactions with the structure (e.g., with 27).
the method of Still et al.32 and was carried out using silica
gel 60 (200–400 mesh). Analytical HPLC was performed on
˚
a Phenomenex Atlantis 300 A C18-reverse phase column
using a Gilson HPLC system. Buffer A was 0.5% TFA in
water and buffer B was 0.5% TFA in acetonitrile. Analysis
and purification of oligomers was carried out by applying
the following elution protocol: 0% buffer B for 10 min at a
flow rate of 1.0 mL/min followed by a linear gradient from
0 to 100% buffer B over 30 min. The eluents were moni-
tored at 260 nm. In all cases, unmodified PNA oligomers
had retention times between 17 and 18 min whereas
stearoyl- or diacetylene-functionalized PNA oligomers
had longer retention times between 29 and 33 min. Melting
points were determined using an electrothermal instrument
and are uncorrected. IR spectra were recorded on Perkin-
Elmer FTIR 1600 or 1430 instruments and calibrated
1
against polystyrene. H and 13C{1H} NMR spectra were
recorded on Bruker AC 200 or DPX 400 spectrometers;
chemical shifts (ppm) are reported with respect to SiMe4 as
reference (positive shifts to high frequency/low field). UV–
vis spectra were recorded on a Shimadzu UV-160
spectrophotometer. Mass spectra were measured on an
upgraded VG MS9 instrument; high resolution mass spectra
(HRMS) were also recorded at the EPSRC National Mass
Spectrometer Service Centre, University of Wales,
Swansea. MALDI-TOF spectra were recorded on an
Amersham Biosciences instrument using the matrix 3,5-
dimethoxy-4-hydroxycinnamic acid (sinapinic acid).
Experimental errors of the MALDI-TOF peaks are
estimated to be G0.1%. Elemental analyses were performed
at Heriot-Watt University.
The formation of liposomes from two component mixtures
of acid-terminated, T10-oligomeric PNAs, 30, 35, 36 or 38
(5%) and the diacetylene matrix 41 (95%) was also
investigated. Dark blue solutions of liposomes were
obtained from samples containing diacetylene-PNA-con-
taining lipids 36, 38 or the stearoyl-PNA lipid 30; these
solutions were relatively stable, although some precipitation
was observed after several days at rt; UV–vis spectra were
typical of PDA-lipsomes but only samples containing 30 or
36 as the minor component showed a UV absorption at lmax
ca. 260 nm assignable to incorporated pyrimidine (Table 1).
The mixture of 35 (5%) and 41 (95%) gave only a dilute,
light blue solution of liposome and much precipitation
occurred during the preparation.
3.1.1. 1-Amino-10,12-pentacosadiyne 10. This compound
was prepared by the following sequence:
10,12-Pentacosadiynoyl chloride—Thionyl chloride (1.8 g,
15 mmol) was added dropwise to 10,12-pentacosadiynoic
acid (1.0 g, 26.7 mmol) in THF (4 mL). The mixture was
heated under reflux for 60 min until no more HCl gas
evolved. The solvent was evaporated under reduced pres-
sure to yield 10,12-pentacosadiynoyl chloride as an orange
oil, which was used without further purification. dH (CDCl3)
0.90 (t, JZ6.2 Hz, 3H, CH3), 1.2–1.8 (m, 32H, CH2), 2.25
(t, JZ6.4 Hz, 4H, CH2C^C), 2.85 (t, 2H, JZ7.2 Hz,
CH2COCl). dC (CDCl3) 14.0 (CH3), 19.1, 22.6, 24.9, 26.0,
28.1, 28.3, 28.6, 28.7, 28.8, 29.0. 29.2, 29.4, 29.5, 31.8, 47.0
These results prove that blue PDA-liposomes containing
thymine-based PNAs can be prepared and indicate that
production of liposomes with other PNA systems are viable.
With the propensity of tailor-made PNAs to hybridize
strongly with complementary ss-DNA or RNA, such PNA-
containing PDA-liposomes have potential to act as
biosensors for specific nucleic acids. Further studies in
this area are in progress.
(CH2), 65.0, 65.3, 77.1 (C^C), 173.7 (CO). nmax (Nujol, cmK1
)
2924, 2854 (CH str), 1803 (CO).
3. Experimental
3.1. General
10,12-Pentacosadiynoyl amide—10,12-Pentacosadiynoyl
chloride (0.58 g, 15 mmol) was added to cold aqueous
ammonia (sp. gr. 0.88, 20 mL) and the mixture was stirred
for 3 h. The product was then extracted with DCM (5!
100 mL). The organic extract was dried (MgSO4) and the
solvent evaporated under reduced pressure to yield a yellow
solid, which was purified by flash column chromatography
on silica gel using CH2Cl2/MeOH (97:3) as eluent to yield
10,12-pentacosadiynoyl amide as a colourless solid (0.34 g,
62%), mp 100–102 8C. Found: C, 80.30; H, 11.85; N,
3.82%. C25H43NO requires C, 80.37; H, 11.60; N, 3.75%. dH
(CDCl3) 0.85 (t, 3H, JZ6.3 Hz, CH3), 1.2–1.7 (m, 32H,
CH2), 2.20–2.30 (m, 6H, CH2C^C and CH2CO), 5.40 (br s,
2H, NH2). dC (CDCl3) 14.1 (CH3), 19.1, 22.6, 25.4, 28.2,
All starting materials were purchased either from Avocado
Research, Lancaster Research or Sigma-Aldrich Chemical
companies. Anhydrous solvents were dried according to the
procedures described by Casey and Leonard.31 HPLC grade
N,N-dimethylformamide (DMF) was used as supplied.
Analytical TLC was carried out on aluminium plates pre-
coated with silica gel 60 F254 (Merck). Products were
visualized either by UV light or by staining the plate with a
7% phosphomolybdic acid solution in methanol (w/v)
followed by heating. Column chromatography refers to

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N. M. Howarth et al. / Tetrahedron 61 (2005) 8875–8887
28.3, 28.6, 28.8, 29.0, 29.3, 29.4, 29.5, 31.8, 35.8 (CH2),
65.1, 77.4 (C^C), 175.8 (CO). nmax (Nujol, cmK1) 3402
and 3188 (NH str), 2923, 2853 (CH str), 1646 (CO). HRMS
(ES) m/z (MCH)C374.3425 (C25H44NO requires
374.3423).
77.4 (C^C), 77.6 and 79.8 (C–O), 110.8 (thymine C-5),
141.2 (thymine C-6), 151.4 (CO), 156.2 (CONH), 164.4
(CO), 167.6 and 168.1 (CON), 168.2 and 168.6 (CONH),
173.6 (CONH). nmax (thin film, cmK1) 3320, 3066 (NH str),
2926, 2854 (CH str), 1678 (CO). HRMS (ES) m/z (MC
H)C871.5906 (C47H79N6O9 requires 871.5909).
1-Amino-10,12-pentacosadiyne—To a suspension of 10,12-
pentacosadiynoyl amide (0.20 g, 0.54 mmol) in diethyl
ether (20 mL) was added LiAlH4 (0.20 g, 5.4 mmol) at 0 8C.
The mixture was stirred for 17 h, after which time wet
diethyl ether was added slowly until effervescence ceased.
The mixture was filtered, the filtrate was washed with water
and then dried (MgSO4). The solvent was evaporated under
reduced pressure to yield the title compound 10 as a
colourless solid (0.13 g, 69%), which turned blue in ambient
light, mp 60–62 8C. dH (CDCl3) 0.80 (t, 3H, JZ6.4 Hz,
CH3), 1.2–1.5 (m, 36H, CH2), 2.18 (t, 4H, JZ6.8 Hz,
CH2C^C), 2.6 (br s, 2H, NH2). dC (CDCl3) 14.1 (CH3),
19.2, 22.7, 28.3 (!2), 28.8 (!2), 29.0, 29.1, 29.3, 29.4 (!2),
29.5, 29.6 (!3), 31.9, 33.6, 42.1 (CH2), 65.2, 65.3, 77.2,
77.5 (C^C). nmax (KBr, cmK1) 3352 (NH str) 2954, 2917,
2850 (CH str), 1644, 1595, 1471. HRMS (ES) m/z (MC
H)C360.3626 (C25H46N requires 360.3625).
3.1.3. (Amino-10,12-pentacosadiynoyl)-N-(2-t-butoxy-
carbonylaminoethyl)-N-(thymin-1-ylacetyl)glycinate 7.
To N-(2-t-butoxycarbonylaminoethyl)-N-(thymin-1-ylacetyl)
glycine 5 (0.057 g, 0.14 mmol) in DMF (10 mL) was added
DIPEA (73 ml, 0.42 mmol) and 1-amino-10,12-pentacosa-
diyne (0.050 g, 0.14 mmol), followed by HBTU (0.053 g,
0.14 mmol). The mixture was stirred for 17 h at rt, after
which the solvent was evaporated under reduced pressure.
The residue was dissolved in DCM (25 mL) and the solution
was washed successively with 1 M aq NaHCO3 (3!25 mL),
1 M aq KHSO4 (2!25 mL), water (25 mL) and finally brine
(25 mL). The organic phase was then dried (MgSO4) and the
solvent evaporated under reduced pressure to yield a crude
product, which was purified by flash column chromato-
graphy on silica gel using CH2Cl2/MeOH (97:3) as eluent to
yield the title compound 7 as a glassy solid (0.047 g, 45%).
dH (CDCl3) (two rotational isomers in a 2:1 ratio were
observed) 0.85 (t, 3H, JZ6.4 Hz, CH2CH3), 1.2–1.5 (m,
34H, CH2) 1.40 (s, 9H, (CH3)3), 1.90 (s, 3H, thymine-CH3),
2.20 (t, 4H, JZ6.7 Hz, CH2C^C), 3.1–3.5 (m, unresolved,
4H, NHCH2CH2N), 3.85 (major) and 4.18 (minor) (s, 2H,
NCH2CONH), 4.42 (minor) and 4.50 (major) (s, 2H,
NCH2CON), 5.72 (t, 1H, NH,), 6.35 (t, 1H, NH) 6.90
(major) and 7.0 (minor) (s, 1H, thymine-H), 8.90 (br s, 1H,
NH). dC (CDCl3) (two rotational isomers in a 2:1 ratio were
observed) 12.3 (!2) (thymine-CH3), 14.1 (CH2CH3), 19.2
(!2), 22.7, 26.8, 26.9, 28.3 (!2) (CH2), 28.4 (!2)
((CH3)3), 28.8 (!2), 29.0, 29.1, 29.2, 29.3, 29.4, 29.5, 29.6
(!3), 29.7, 31.2, 38.8, 39.8, 40.0, 48.3, 48.7, 49.1, 51.4,
51.9 (CH2), 65.2, 65.3, 77.2, 77.4 77.5, 77.6 (C^C), 77.6
and 79.9 (C–O), 110.7 and 110.8 (thymine), 141.1 and 141.2
(C6), 151.1 and 151.2 (CO), 156.2 (CONH), 164.2 and
164.3 (CO), 167.6 and 167.7 (CON), 168.1 and 168.4
(CONH). nmax (thin film, cmK1) 3348, 3306, 3164 (NH str),
2926, 2854 (CH str), 1693, 1658 (CO). HRMS (ES) m/z
(MCNH4)C743.5436 (C41H71N606 requires 743.5430).
3.1.2. N-[N0-(2-t-Butoxycarbonylaminoethyl)-N0-(thymin-
1-ylacetyl)-N0-(2-(2-(2-methylamidoethoxy)ethoxy)ethyl)]-
10,12-pentacosadiynamide 6. To N-(2-t-butoxycarbonyl-
aminoethyl)-N-(thymin-1-ylacetyl)glycine
5
(0.66 g,
1.72 mmol) in DMF (50 mL) was added EDC (0.36 g,
1.88 mmol) and HOBt (0.25 g, 1.88 mmol) at 0 8C. The
mixture was stirred for 1 h at 0 8C, then at rt for 30 min and
finally cooled again to 0 8C. To this solution was added a
cooled solution of the amide 912 (0.82 g, 1.63 mmol) and
DIPEA (0.3 mL, 1.88 mmol) in DMF (50 mL). The mixture
was stirred for 17 h at rt, after which the solvent was
evaporated under reduced pressure. The residue was
dissolved in DCM (50 mL) and the solution was washed
successively with saturated sodium bicarbonate solution
(3!25 mL), citric acid solution (2!25 mL), water (25 mL)
and finally brine (25 mL). The organic phase was dried
(MgSO4) and the solvent was evaporated under reduced
pressure to leave a crude glassy solid, which was further
purified by column chromatography on silica gel with
CH2Cl2/MeOH (19:1) as eluent to yield the title compound
6 as a glassy solid (0.92 g, 66%). dH (CDCl3) (two rotational
isomers in a 2:1 ratio were observed) 0.90 (t, 3H, JZ6.4 Hz,
CH2CH3), 1.2–1.7 (m, 32H, CH2), 1.40 (s, 9H, (CH3)3), 1.90
(s, 3H, thymine-CH3), 2.20 (t, 2H, JZ8.0 Hz, CH2CH2-
CONH), 2.25 (t, 4H, JZ6.7 Hz, CH2C^C), 3.25 (minor)
and 3.35 (major) (t, 2H, JZ5.0 Hz, CH2), 3.45 (t, 4H, JZ
4.9 Hz, CH2), 3.55 (m, unresolved, 2H, CH2), 3.60 (m,
unresolved, 4H, CH2), 3.65 (m, unresolved, 4H, CH2), 4.00
(major) and 4.10 (minor) (s, 2H, NCH2CONH), 4.50 (minor)
and 4.55 (major) (s, 2H, NCH2CON), 5.35 (minor) and 5.90
(major) (t, 1H, BocNH), 6.45 (minor) and 6.60 (major) (t,
1H, NH), 6.95 (major) and 7.10 (minor) (s, 1H, thymine-H),
7.15 (major) and 7.50 (minor) (t, 1H, NH). dC (CDCl3) (two
rotational isomers in a 2:1 ratio were observed) 12.2 and
12.3 (thymine-CH3), 14.1 (CH2CH3), 19.1 (!2), 22.6, 25.7,
28.3 (!2) (CH2), 28.4 (!2) ((CH3)3), 28.8, 28.8, 28.9,
29.0, 29.2, 29.2, 29.3, 29.4, 29.6, 31.9, 36.5, 36.5, 38.7,
38.9, 39.1, 39.3, 39.4, 45.9, 48.4, 48.6, 48.9, 50.8, 65.2 and
65.3 (C^C), 69.4, 69.6, 69.8, 69.9, 70.0 (CH2), 77.3 and
3.1.4. N-[N0-(Aminoethyl)-N0-(thymin-1-ylacetyl)-N0-(2-
(2-(2-methylamidoethoxy)ethoxy)ethyl)]-10,12-pentaco-
sadiynamide hydrochloride 8. Six molar (6 M) aq HCl
(5 mL) was added to compound 6 (0.067 g, 0.077 mmol) in
DCM (5 mL) and the mixture was stirred at rt for 2 h. The
product was evaporated under reduced pressure to afford the
title compound 8 as a glassy solid (0.055 g, 89%). dH (d6-
DMSO) (two rotational isomers in a 2:1 ratio were
observed) 0.82 (t, 3H, JZ6.4 Hz, CH2CH3), 1.2–1.5 (m,
32H, CH2) 1.72 (s, 3H, thymine-CH3), 2.05 (t, 2H, JZ7.3 Hz,
CH2CH2CONH), 2.25 (t, 4H, JZ6.4 Hz, CH2C^C), 3.2–3.7
(m unresolved, 16H, CH2), 3.95 and 4.10 (s, 2H, NCH2-
CONH), 4.45 and 4.75 (s, 2H, NCH2CON), 7.42 (s, 1H,
thymine-H), 7.80 (t, 1H, CH2CH2CONH), 7.98 and 8.25
(br s, 3H, NH3), 8.35 and 8.48 (t, 1H, NCH2CONH), 11.30
and 11.32 (s, 1H, thymine-NH). dC (DMSO) (two rotational
isomers in a 2:1 ratio were observed) 11.9 (!2) (thymine-
CH3), 13.9 (CH2CH3), 18.2, 18.3, 22.1, 25.2, 27.7 (!2),
28.1, 28.2, 28.4, 28.6 (!2), 28.7, 28.8, 28.9, 29.0, 31.3,

N. M. Howarth et al. / Tetrahedron 61 (2005) 8875–8887
8881
35.3, 36.8, 36.9, 38.4, 38.8, 40.8, 45.1, 45.3, 47.7, 48.0,
49.2, 49.9 (CH2), 65.3 (C^C), 68.8, 69.1, 69.5, 69.6 (CH2),
77.9 (!2) (C^C), 108.0 (!2) (C5), 142.1 (!2) (C6),
151.0 and 151.1 (CO), 164.3 and 164.4 (CO), 167.4 and
168.3 (CON), 168.6, 169.5, 172.2 (CONH). nmax (thin film,
cmK1) 3305 (NH str), 2920, 2850 (CH str), 1672, 1554,
1468. HRMS (ES) m/z (MKCl)C, 771.5378 (C42H71N6O7
requires 771.5379).
3.1.7. N-(2-(2-(2-Aminoethoxy)ethoxy)ethyl)-10,12-penta-
cosadiynamide hydrochloride 16. N-(2-(2-(2-t-Butoxy-
carbonylaminoethoxy)ethoxy)ethyl)-10,12-pentacosadiyn-
amide 15 (0.16 g, 0.26 mmol) was dissolved in DCM (2 mL)
and 6 M aq HCl (2 mL) was added. The mixture was stirred
for 30 min and then the solvents were evaporated under
reduced pressure to yield the title compound 16 (0.14 g) in
quantitative yield. This compound was used in situ without
further purification. dH (d6-DMSO) 0.83 (t, 3H, JZ6.0 Hz,
CH3), 1.2–1.7 (m, 32H, CH2), 2.15 (t, 2H, JZ7.2 Hz,
CH2CO), 2.25 (t, 4H, JZ6.3 Hz, CH2C^C), 2.95 (q, 2H,
JZ5.4 Hz, CH2), 3.20 (q, 2H, JZ5.6 Hz, CH2), 3.40 (t, 2H,
JZ5.4 Hz, CH2), 3.55 (s, 4H, CH2O), 3.60 (t, 2H, JZ
5.2 Hz, CH2), 7.90 (t, 1H, NH), 8.05 (br s, NH).
3.1.5. 2-(2-(2-t-Butoxycarbonylaminoethoxy)ethoxy)-
ethylamine 14 and bis-1,2-(2-t-butoxycarbonylamino-
ethoxy)ethane 13. To a stirred solution of 1,8-diamino-
3,6-dioxaoctane (5.0 g, 33.2 mmol) in DCM (40 mL) was
added a solution of di-t-butyl dicarbonate (3.6 g, 16.9 mmol)
in diethyl ether (30 mL) over 30 min. Stirring was continued
for an additional 2 h, after which the solvent was evaporated
under reduced pressure to yield a crude yellow oil. This oil
was subjected to separation by flash column chromato-
graphy on silica gel using DCM/MeOH (19:1–17:3) as eluent
to yield the two title compounds 14 and 13 as clear oils.
(These compounds were not purified to analytical standard).
3.1.8. (Aminoethyl)-N-(thymin-1-ylacetyl)glycine ethyl
ester hydrochloride. N-(2-t-Butoxycarbonylaminoethyl)-
N-(thymin-1-ylacetyl)-glycine ethyl ester 414,15 (0.55 g,
1.33 mmol) was dissolved in ethyl acetate (25 mL) and
6 M aq HCl (25 mL) was added. The mixture was stirred for
30 min after which time the solvent was evaporated under
reduced pressure to yield the title compound as a glassy
solid. This was then used in situ without further purification.
Compound 14 (1.89 g, 45%, RfZ0.12 [DCM/MeOH 95:5]).
dH (CDCl3) 1.40 (s, 9H, CH3), 1.60 (s, 2H, NH2), 2.83 (t,
2H, JZ5.2 Hz, CH2NH2), 3.25 (apparent q, 2H, JZ5.2 Hz,
BocNHCH2CH2), 3.49 (m, unresolved, 4H, CH2), 3.58 (s,
4H, OCH2CH2O), 5.15 (br s, 1H, BocNH). dC (CDCl3) 28.3
(CH3), 40.2 (CH2), 41.6 (CH2), 70.1 (CH2), 73.2 (CH2), 79.0
(C–O), 155.9 (CO).
3.1.9. N-(2-Pentacosa-10,12-diynoylaminoethyl)-N-
(thymin-1-ylacetyl)glycine ethyl ester 11. Method A. To
a solution of N-succinimdyl-10,12-pentacosadiynoate12
(0.63 g, 1.34 mmol) in DMF (10 mL) was added (amino-
ethyl)-N-(thymin-1-ylacetyl)glycine ethyl ester hydrochlor-
ide (0.45 g, 1.30 mmol) and DIPEA (0.70 mL, 3.9 mmol) in
DMF (10 mL) at 0 8C. The mixture was stirred for 17 h at rt,
then the solvent was evaporated under reduced pressure.
The resulting residue was purified by flash column
chromatography on silica gel with DCM/MeOH (19:1) as
eluent to yield the title compound 11 as a white foam
(0.40 g, 50%).
Compound 13 (1.07 g, 37%, RfZ0.23 [DCM/MeOH 95:5]).
dH (CDCl3) 1.42 (s, 18H, CH3), 3.30 (apparent q, 4H, JZ
5.0 Hz, BocNHCH2), 3.51 (t, 4H, JZ5.3 Hz, BocNHCH2-
CH2), 3.54 (s, 4H, OCH2CH2O), 5.03 (br s, 2H, BocNH). dC
(CDCl3) 28.3 (CH3), 40.3 (CH2), 70.2 (CH2), (CH2), 79.2
(C–O), 155.9 (C]O).
Method B. To a solution of (aminoethyl)-N-(thymin-1-
ylacetyl)glycine ethyl ester hydrochloride (0.064 g,
0.16 mmol) in DMF (10 mL) was added DIPEA (85 ml,
0.49 mmol) followed by a solution of 10,12-pentacosadiy-
noyl fluoride13 (0.061 g, 0.16 mmol) in DCM (5 mL). The
mixture was stirred at rt for 17 h, then worked up as above
(Method A) to yield the title compound 11 as a foam
(0.096 g, 90%). dH (CDCl3) (two rotational isomers in a 2:1
ratio were observed) 0.88 (t, 3H, JZ6.8 Hz, CH2CH2CH3),
1.2–1.7 (m, 35H, 16!CH2 and 1!CO2CH2CH3), 1.90 (s,
3H, thymine-CH3), 2.15 and 2.30 (t, 2H, JZ7.3 Hz,
CH2CONH), 2.22 (t, 4H, JZ7.2 Hz, CH2C^C), 3.40
(minor) and 3.44 (major) (apparent q, 2H, JZ5.6 Hz,
NHCH2CH2) 3.55 (t, 2H, JZ5.7 Hz, NHCH2CH2), 4.05 and
4.15 (s, 2H, CH2CONH), 4.21 (major) and 4.28 (minor) (q,
2H, JZ7.2 Hz, CH2CH3), 4.41 (minor) and 4.55 (major) (s,
2H, CH2CON), 6.45 (minor) and 6.90 (major) (t, 1H, NH),
6.95 (major) and 7.05 (minor) (s, 1H, thymine-H), 9.38
(major) and 9.42 (minor) (s, 1H, thymine-NH). dC (CDCl3)
(two rotational isomers in a 2:1 ratio were observed) 12.3
(thymine-CH3), 14.1 (!2) (CH2CH3), 18.4, 19.2, 19.2,
22.7, 25.5, 25.5, 25.6, 28.3, 28.4, 28.4, 28.7, 28.8, 28.4,
28.9, 28.9, 29.0, 29.1, 29.2, 29.2, 29.3, 29.3, 29.3, 29.3,
49.5, 29.6, 29.6, 29.7, 29.7, 29.7, 32.0, 36.3, 36.4, 37.6,
37.7, 48.1, 48.3, 48.4, 48.4, 48.9, 50.2, 61.8, 62.4 (CH2),
65.2, 65.3, 77.2, 77.5, 77.6, 77.7 (C^C), 110.8 and 110.9
3.1.6. N-(2-(2-(2-t-Butoxycarbonylaminoethoxy)ethoxy)
ethyl)-10,12-pentacosadiynamide 15. A solution of 2-(2-
(2-t-butoxycarbonylaminoethoxy)ethoxy)ethylamine 14
(0.67 g, 2.70 mmol) and N-succinimidyl-10,12-pentacosa-
diynoate12 (0.10 g, 0.21 mmol) in DCM (50 mL) was stirred
at rt for 17 h. The mixture was then washed with water
(50 mL) and the solvent was evaporated from the organic
fraction to yield a solid, which was subjected to flash
column chromatography on silica gel using CH2Cl2/MeOH
(19:1) as eluent. The title compound 15 (1.10 g, 87%) was
isolated as a colourless solid, mp 52–54 8C, which turned
blue in ambient light. dH (CDCl3) 0.80 (t, 3H, JZ6.2 Hz,
CH2CH3), 1.20–1.60 (m, 32H, CH2), 1.40 (s, 9H, t-Bu), 2.10
(t, 2H, JZ7.9 Hz, CH2CO), 2.15 (t, 4H, JZ6.7 Hz,
CH2C^C), 3.25 (apparent q, 2H, JZ5.0 Hz, BocNHCH2-
CH2), 3.40 (m, 2H, CH2), 3.50 (t, 4H, JZ5.3 Hz, CH2), 3.60
(s, 4H, OCH2), 5.0 (br s, 1H, NH), 6.0 (br s, 1H, NH). dC
(CDCl3) 14.0 (CH2CH3), 19.1, 22.6, 25.6 (CH2), 28.3
(C(CH3)3), 28.7, 28.8, 28.9, 29.0, 29.1, 29.4, 29.5, 31.8,
36.6, 39.0, 40.2 (CH2), 65.1 (!2) (C^C and CH2), 70.1
(!2) (CH2), 77.5 (C^C), 79.3 (C–O), 155.9 (BocCONH),
173.1 (CONHCH2). nmax (KBr, cmK1) 3352, 3310, 3082
(NH str), 2920, 2848 (CH str), 1685 (CO), 1644 (CO),
1553, 1540, 1466. HRMS (ES) m/z (MCH)C605.4896
(C36H65N205 requires 605.4893).

8882
N. M. Howarth et al. / Tetrahedron 61 (2005) 8875–8887
(thymine-C5), 141.0 and 141.2 (thymine-C6), 151.3 (CO),
164.3 and 164.3 (CO), 167.4 and 168.2 (CON), 169.4 and
169.6 (CONH), 174.1 and 174.5 (CO2Et). nmax (thin film,
cmK1) 3319 (NH str), 2924, 2852 (CH str), 1738, 1709,
1689, 1666, 1650, 1548, 1468. HRMS (ES) m/z (MC
NH4)C686.4862 (C38H64N5O6 requires 686.4857).
adjusted to pH 8.5 with solid NaHCO3. The aqueous phase
was then extracted with DCM (3!100 mL), the combined
organic extract was dried (MgSO4) and the solvent was
evaporated under reduced pressure. The crude residue was
then purified by flash column chromatography [silica gel,
CH2Cl2/MeOH 19:1 as eluent] to yield the title compound
19 as a colourless solid (1.66 g, 60%). Found: C, 69.09; H,
11.81; N, 6.99%. C23H46N2O3 requires C, 69.30; H, 11.63;
N, 7.03%. dH (CDCl3) 0.86 (t, 3H, JZ6.4 Hz, CH2CH3),
1.18–1.70 (m, 30H, CH2), 2.17 (t, 2H, JZ7.5 Hz, CH2-
CONH), 2.75 (t, 2H, JZ5.8 Hz, CH2NHCH2), 3.35 (appar-
ent q, 2H, JZ5.5 Hz, CONHCH2), 3.40 (s, 2H, CH2CO2Me),
2.72 (s, 3H, OCH3), 6.10 (br s, 1H, NH). [1!NH not
observed] dC (CDCl3) 14.0 (CH3), 22.6, 25.7, 29.3, 29.4,
29.6, 30.8, 31.8, 36.7, 388, 48.3, 50.1 (CH2), 51.8 (CH3O),
172.9, 173.3 (CO). HRMS (ES) m/z (MCH)C399.3585
(C23H47N2O3 requires 399.3586).
3.1.10. N-(2-Pentacosa-10,12-diynoylaminoethyl)glycine
methyl ester 18. To N-succinimdyl-10,12-pentacosadiynoate
(1.28 g, 2.71 mmol) in DMF was added N-(2-aminoethyl)-
glycine methyl ester dihydrochloride (0.57 g, 2.77 mmol)
and DIPEA (1.43 mL, 10.3 mmol) in DMF. The mixture
was stirred at rt for 17 h. The solvent was evaporated under
reduced pressure and the pink-white residual solid was
partitioned with 1 M aq KHSO4 (150 mL) and ethyl acetate
(150 mL). Subsequently, the pH of the aqueous phase was
adjusted to 8.5 using solid NaHCO3 and a saturated aqueous
solution of NaHCO3. The mixture was then extracted with
DCM (3!150 mL), the combined organic fractions were
dried (MgSO4) and the solvent evaporated under reduced
pressure to afford the title compound 18 (0.76 g, 65%) as a
colourless solid, mp 59–63 8C, which turned blue in ambient
light. dH (CDCl3) 0.88 (t, 3H, JZ6.5 Hz, CH2CH3), 1.2–1.7
(m, 32H, CH2), 2.15 (t, 2H, JZ7.5 Hz, CH2CH2CO), 2.23
(t, 4H, JZ6.8 Hz, CH2C^C), 2.75 (t, 2H, JZ5.7 Hz,
CH2CH2NHCH2), 3.35 (q, 2H, JZ5.6 Hz, CONHCH2),
3.42 (s, 2H, NHCH2CO), 3.72 (s, 3H, OCH3), 6.20 (br s, 1H,
NH). [1!NH not observed] dC (CDCl3) 14.0 (CH2CH3),
19.1, 22.6, 25.0, 25.6, 28.3, 28.8, 28.8, 29.0, 29.1, 29.2,
29.4, 29.5, 31.8, 26.6, 38.7, 48.3, 49.8 (CH2), 51.9 (OCH3),
3.1.13. N-(2-Pentacosa-10,12-diynoylaminoethyl)-N-
(thymin-1-ylacetyl)glycine methyl ester 20. To a solution
of N-(2-pentacosa-10,12-diynoylaminoethyl)glycine methyl
ester 18 (1.00 g, 2.05 mmol) and triethylamine (0.84 mL,
6.03 mmol) in DMF (25 mL) was added thymin-1-yl acetic
acid (0.38 g, 2.06 mmol) followed by HATU (0.78 g,
2.05 mmol). The mixture was stirred at rt for 17 h then the
solvent was evaporated under reduced pressure. The residue
was dissolved in DCM (25 mL) and the solution was washed
successively with 1 M aq NaHCO3, 1 M aq KHSO4, water,
brine and finally dried (MgSO4). The solvent was evapor-
ated under reduced pressure to leave a white foam, which
was purified by flash column chromatography [silica gel,
CH2Cl2/MeOH 17:3 as eluent] to yield the title compound
20 (0.80 g, 63%) as a white foam, mp 59–63 8C, which
turned purple in ambient light. Found: C, 67.92; H, 9.07; N,
8.42%. C37H58N4O6 requires C, 67.86; H, 8.93; N, 8.56%.
dH (CDCl3) (two rotational isomers in a 2:1 ratio were
observed) 0.88 (t, 3H, JZ6.6 Hz, CH2CH3), 1.20–1.65 (m,
32H, CH2), 1.88 (major) and 2.00 (minor) (s, 3H, thymine-
CH3), 2.15 (minor) and 2.25 (major) (t, 2H, JZ7.5 Hz,
CH2CONH), 2.22 (t, 4H, JZ6.0 Hz, CH2C^C), 3.40
(minor) and 3.45 (major) (m, unresolved, 2H, NHCH2CH2),
3.45 (minor) and 3.55 (major) (m, unresolved, 2H, NHCH2-
CH2), 3.75 (major) and 3.80 (minor) (s, 3H, OCH3), 4.05
(major) and 4.20 (minor) (s, 2H, CH2CO2Me), 4.35 (minor)
and 4.50 (major) (s, 2H, CH2CON), 6.15 (minor) and 6.77
(major) (t, 1H, JZ5.8 Hz, CONH), 6.95 (major) and 6.99
(minor) (s, 1H, thymine-H). [1!NH not observed] dC
(CDCl3) (two rotational isomers in a 2:1 ratio were observed)
12.3 (thymine-CH3), 14.1 (CH2CH3), 19.2, 19.2, 22.7, 25.5,
28.3, 28.3, 28.8, 28.8, 28.9, 29.1, 29.2, 29.2, 29.3, 29.3,
29.4, 29.6, 29.6, 29.6, 31.9, 36.3, 36.5, 37.5, 37.6, 47.9,
48.2, 48.3, 48.4, 48.7, 50.0 (CH2), 52.5 and 52.9 (OCH3),
65.2, 65.3, 77.2, 77.4, 77.6, 77.6 (C^C), 110.8 and 110.9
(thymine-C5), 140.8 and 141.0 (thymine-C6), 151.1 (CO),
164.0 and 164.0 (CO), 167.4 and 168.1 (CON), 169.7 and
170.0 (CONH), 173.9 and 174.4 (CO2Me). nmax (thin film,
cmK1) 3321, 3191, 3060 (NH str), 2926, 2854 (CH str),
1746, 1672, 1548, 1530, 1467 (CO). HRMS (ES) m/z (MC
H)C655.4435 (C37H59N4O6 requires 655.4435).
64.1, 77.4 (C^C), 172.7, 173.4 (CO). nmax (KBr, cmK1
)
3296, 3089 (NH str), 2920, 2048 (CH str), 1742, 1640 (CO
str), 1558, 1465. HRMS (ES) m/z (MCH)C489.4049
(C30H53N2O3 requires 489.4056).
3.1.11. N-Succinimidyl octadecanoate. N-Hydroxysuccin-
imide (0.76 g, 6.56 mmol), followed by EDC (1.25 g,
6.56 mmol) were added to a solution of stearic acid
(1.87 g, 6.56 mmol) in DCM (50 mL) and the mixture was
stirred at rt for 21 h. The solvent was evaporated under
reduced pressure. The residue was dissolved in DCM
(100 mL) and the solution washed with water (100 mL); the
aqueous phase was then back-extracted with DCM (2!
100 mL). The combined organic phase was dried (MgSO4)
and the solvent evaporated under reduced pressure to yield
the title compound as a colourless solid (2.45 g, 98%). This
compound was characterized by NMR spectroscopy but was
not purified to analytical standard. It was used without
further purification. dH (CDCl3) 0.87 (t, 3H, JZ6.4 Hz,
CH3), 1.10–1.80 (m, 30H, CH2), 2.33 (t, 2H, JZ7.5 Hz,
CH2COO), 2.82 (s, 4H, COCH2CH2CO). dC (CDCl3) 14.0
(CH3), 22.6, 24.5, 25.5, 28.7, 28.9, 29.3, 29.3, 30.8, 31.8,
33.7 (CH2), 168.6, 169.1 (CO).
3.1.12. N-(-2-Octadecanoylaminoethyl)glycine methyl
ester 19. To a solution of N-succinimidyl octadecanoate
(2.65 g, 6.95 mmol) in DCM (100 mL) was added a solution
of N-(2-aminoethyl)glycine methyl ester dihydrochloride
(1.42 g, 6.95 mmol) and DIPEA (2.9 mL, 20.85 mmol). The
mixture was stirred at rt for 20 h then the solvent was
evaporated under reduced pressure. The residue was
partitioned between ethyl acetate (100 mL) and 1 M aq
KHSO4 (100 mL). The aqueous phase was separated and
3.1.14. N-(2-Pentacosa-10,12-diynoylaminoethyl)-N-(N4-
(benzyloxycarbonyl)cytosine-1-ylacetyl)glycine methyl
ester 21. (N4-(Benzyloxycarbonyl)cytosin-1-yl)acetic

N. M. Howarth et al. / Tetrahedron 61 (2005) 8875–8887
8883
acid18 (0.061 g, 0.20 mmol) and triethylamine (0.061 g,
0.60 mmol) were added to a solution of N-(2-pentacosa-
10,12-diynoylaminoethyl)glycine methyl ester 18 (0.10 g,
0.20 mmol) in DMF (10 mL) followed by HATU (0.081 g,
0.22 mmol). The solution was stirred for 17 h at rt, then the
solvent was evaporated under reduced pressure. The residue
was dissolved in DCM (20 mL) and the solution was washed
successively with 1 M aq NaHCO3 (2!20 mL), 1 M aq
KHSO4 (2!20 mL), water (10 mL) and finally brine
(10 mL). The organic solution was dried (MgSO4) and the
solvent was evaporated under reduced pressure to yield the
title compound 21 (0.11 g, 71%) as a colourless solid, mp
110 8C. dH (CDCl3) (two rotational isomers in a 2:1 ratio
were observed) 0.85 (t, 3H, JZ6.3 Hz, CH2CH3), 1.2–1.45
(m, 32H, CH2), 2.10 (minor) and 2.25 (major) (t, 2H, JZ
7.5 Hz, CH2CONH), 2.25 (t, 4H, JZ7.3 Hz, CH2C^C),
3.30–3.60 (m unresolved, 4H, NHCH2CH2N), 3.70 (major)
and 3.75 (minor) (s, 3H, OCH3), 4.05 (major) and 4.35
(minor) (s, 2H, CH2CO2Me), 4.50 (minor) and 4.65 (major)
(s, 2H, CH2CON), 5.2O (s, 2H, CH2OCO), 6.15 (minor) and
6.90 (major) (t, 1H, NH), 7.20–7.70 (m unresolved, 7H, 5!
Ar-H, 2!cytosinyl-H). [1!NH not observed] dC (CDCl3)
(two rotational isomers in a 2:1 ratio were observed) 14.1
(CH2CH3), 19.2 (!2), 22.7, 28.3, 28.4, 28.8, 28.9 (!2),
29.1, 29.2, 29.3, 29.5, 29.6 (!3), 31.9, 36.3, 31.2, 28.7,
41.1, 48.2, 48.5, 49.9 (CH2), 52.5 and 52.9 (OCH3), 65.2
and 65.3 (C^C), 67.9, 68.1, 68.2 (CH2), 77.2, 77.5, 77.6,
77.7 (C^C), 95.1 (cytosinyl-C5), 128.3, 128.4, 128.7,
128.8, 130.9, 132.5, 134.9, 149.7, 152.1 (NHCOO), 155.7
(cytosinyl-C6), 162.9 (cytosinyl-CO), 167.0 and 167.7
(CON), 169.8 (CONH), 173.8 and 174.3 (CO2Me). HRMS
(ES) (MCH)C774.4808 (C44H63N5O7 requires 774.4805).
(CH2CH3), 19.1, 19.2, 22.6, 25.4, 25.4, 28.3, 28.3, 28.7,
28.8, 28.9, 29.1, 29.1, 29.2, 29.2, 29.3, 29.4, 29.6, 29.6,
29.6, 29.7, 31.9, 26.5, 36.5, 36.6, 37.4, 37.5, 43.7, 43.9,
48.3, 48.7, 48.9, 50.0 (CH2), 52.6 and 53.1 (OCH3) 65.2 and
65.3 (C^C), 67.7 and 67.8 (CH2), 77.2, 77.4, 77.6 (C^C),
121.3 (adenine-C5), 128.5, 128.5, 128.6 (CH), 135.4, 135.4,
143.8 (adenine-C6), 149.3 (NHCOO), 151.5, 152.7, 152.8,
166.4, 167.2 (CON), 169.5 and 170.2 (CONH), 174.2
(CO2Me). HRMS (ES) (MCH)C798.4911 (C45H64N7O6
requires 798.4918).
3.1.16. N-(2-Octadecanoylaminoethyl)-N-(thymin-1-yl-
acetyl)glycine methyl ester 23. Thymine-1-acetic acid
(0.36 g, 1.95 mmol) was dissolved in DMF (10 mL) and
HBTU (0.6 g, 1.57 mmol) was added. The mixture was
stirred for 15 min and then added to a solution of N-(-2-
octadecanoylaminoethyl)glycine methyl ester 19 (0.63 g,
1.57 mmol) and DIPEA (2.47 mL, 17.8 mmol) in DMF
(10 mL). The mixture was stirred for 19 h, then the solvent
was evaporated under reduced pressure. The residue was
dissolved in DCM and the solution was washed successively
with 1 M aq NaHCO3, 1 M aq KHSO4, water and brine and
was finally dried (MgSO4). The solvent was evaporated
under reduced pressure to leave an orange oil, which was
purified by flash column chromatography [silica gel,
CH2Cl2/methanol 17:3 as eluent] to yield the title compound
23 (0.76 g, 86%) as a colourless solid, mp 123–127 8C.
Found: C, 63.4; H, 9.4; N, 9.8%. C30H52O6N4 requires C,
63.8; H 9.2; N, 9.9%. dH (CDCl3) (two rotational isomers in
a 2:1 ratio were observed) 0.76 (t, 3H, JZ6.4 Hz, CH2CH3),
1.10–1.60 (m, 30H, CH2), 1.88 (s, 3H, thymine-CH3), 2.10
(minor) and 2.20 (major) (t, 2H, JZ7.5 Hz, CH2CONH),
3.40 (minor) and 3.48 (major) (q, 2H, JZ5.8 Hz, NHCH2),
3.55 (t, 2H, JZ4.9 Hz, CH2CH2N), 3.74 (major) and 3.80
(minor) (s, 3H, OCH3), 4.05 (major) and 4.21 (minor) (s,
2H, CH2CO2Me), 4.38 (minor) and 4.55 (major) (s, 2H,
CH2CON), 6.10 (minor) and 6.70 (major) (t, 1H, CONH),
6.95 (major) and 7.03 (minor) (s, 1H, thyminyl C-6–H), 8.84
(minor) and 9.05 (major) (s, 1H, thyminyl-N3–H). dC
(CDCl3) (two rotational isomers in a 2:1 ratio were observed)
12.3 (thyminyl-CH3), 14.1 (CH2CH3), 22.7, 25.6, 29.3,
29.4, 29.5, 29.6, 29.7, 30.8, 31.9, 36.5, 36.6, 37.5, 37.6,
47.9, 48.3, 48.5, 48.8, 50.0 (CH2), 52.6 and 53.0 (OCH3),
110.8 and 110.9 (thyminyl-C5), 140.8 (thyminyl-C6), 150.9
(CO), 163.9 (CO), 167.3 and 168.1 (CON), 170.0 and 170.1
3.1.15. N-(2-Pentacosa-10,12-diynoylaminoethyl)-N-(N6-
(benzyloxycarbonyl)adenin-9-ylacetyl)glycine methyl
ester 22. To a solution of N-(2-pentacosa-10,12-diynoyl-
aminoethyl)glycine methyl ester 18 (0.83 g, 1.71 mmol) in
DMF (25 mL) and triethylamine (0.71 mL, 5.12 mmol) was
added (N6-(benzyloxycarbonyl)adenin-9-yl)acetic acid18
(0.56 g, 1.71 mmol) followed by HATU (0.71 g, 1.88 mmol).
The mixture was stirred at rt for 17 h, then the solvent was
evaporated under reduced pressure. The residue was
dissolved in DCM (25 mL) and the solution was washed
successively with 1 M aq NaHCO3, 1 M aq KHSO4, water
and brine The solution was dried (MgSO4) and the solvent
was evaporated under reduced pressure to leave a white
foam, which was purified by flash column chromatography
[silica gel, CH2Cl2/MeOH 97:3 as eluent] to yield the title
compound 22 as a white foam (1.10 g, 80%). dH (CDCl3)
(two rotational isomers in a 2:1 ratio were observed) 0.85 (t,
3H, JZ6.4 Hz, CH2CH3), 1.20–1.70 (m, 32H, CH2), 2.00
(minor) and 2.26 (major) (t, 2H, JZ7.5 Hz, CH2CONH),
2.16 (t, 4H, JZ7.1 Hz, CH2C^C), 3.40 (minor) and 3.50
(major) (apparent q, 2H, JZ6.0 Hz, NHCH2), 3.55 (minor)
and 3.65 (major) (t, 2H, JZ5.6 Hz, CH2CH2N), 3.68
(major) and 3.76 (minor) (s, 3H, OCH3), 3.99 (major) and
4.23 (minor) (s, 2H, CH2CO2Me), 4.88 (minor) and 5.20
(major) (s, 2H, CH2CON), 5.24 (s, 2H, CH2–O), 5.90
(minor) and 6.67 (major) (t, 1H, JZ6.1 Hz, NHCH2), 7.25–
7.40 (m, 5H, Ar-H), 7.90 (major) and 7.98 (minor) (s, 1H,
adenine C-8–H), 8.55 (br s, 1H, CbzNH), 8.69 (major) and
8.71 (minor) (s, 1H, adenine-C2–H). dC (CDCl3) (two
rotational isomers in a 2:1 ratio were observed) 14.1
(CO2Me), 173.9 and 174.4 (CONH). nmax (KBr, cmK1
)
3293, 3190, 3043 (NH str), 2919, 2850 (CH str), 1741, 1682,
1644 (CO), 1548, 1472, 1432, 1416. HRMS (ES) m/z (MC
H)C565.3958 (C30H53O6N4 requires 565.3959).
3.1.17. N-(2-Pentacosa-10,12-diynoylaminoethyl)-N-
(thymin-1-ylacetyl)glycine 24. N-(2-Pentacosa-10,12-diy-
noylaminoethyl)-N-(thymin-1-ylacetyl)glycine methyl ester
20 (0.15 g, 0.23 mmol) was dissolved in THF (5 mL) and
1 M aq LiOH (3 mL) and the mixture was stirred at rt for
2 h. Water (5 mL) was then added and the aqueous layer
was separated and washed with DCM (2!10 mL) before
being acidified to pH 2.0 with 1 M aq HCl. This acidic
aqueous solution was extracted with DCM (5!25 mL), the
combined organic phase was dried (MgSO4) and the solvent
was evaporated to yield the title compound 24 (0.14 g,
97%).as a white foam. dH (d6-DMSO) (two rotational
isomers in a 2:1 ratio were observed) 0.80 (t, 3H, JZ6.4 Hz,

8884
N. M. Howarth et al. / Tetrahedron 61 (2005) 8875–8887
CH2CH3), 1.20–1.45 (m, 32H, CH2), 1.75 (s, 3H, thymine-
CH3), 2.00 (minor) and 2.10 (major) (t, 2H, JZ7.3 Hz,
CH2CONH), 2.25 (t, 4H, JZ6.4 Hz, CH2C^C), 3.15
(minor) and 3.25 (major) (m, unresolved, 2H, NHCH2CH2),
3.25 (minor) and 3.38 (major) (m, unresolved, 2H, NHCH2-
CH2), 3.95 (major) and 4.15 (minor) (s, 2H, CH2CO2H),
4.45 (minor) and 4.65 (major) (s, 2H, CH2CON), 7.25
(minor) and 7.30 (major) (s, 1H, thymine-H), 7.70 (minor)
and 7.85 (major) (t, 1H, JZ5.8 Hz, NHCH2), 11.20 (minor)
and 11.22 (major) (s, 1H, OH). dC (d6-DMSO) (two
rotational isomers in a 2:1 ratio were observed), 11.8 and
11.9 (thymine-CH3), 13.9 (CH3), 18.3, 22.1, 25.0, 25.1,
27.7, 27.7, 28.1, 28.2, 28.3, 28.4, 28.7, 28.7, 28.8, 28.9,
28.9, 31.3, 35.3, 35.4, 36.2, 36.8, 40.7, 46.5, 46.6, 47.4,
47.7, 54.9 (CH2), 65.3, 65.3, 77.9, 77.9 (C^C), 108.0 and
108.1 (thymine-C5), 142.0 and 142.1 (thymine-C6), 151.1
(CO), 164.4 (CO), 167.1 and 167.6 (CON), 170.4 and 170.8
(CONH), 172.2 and 172.7 (CO2H). nmax (KBr, cmK1) 3351,
3169 (NH str), 2923, 2850 (CH str), 1736, 1692, 1660, 1601,
1422. HRMS (ES) m/z (MCH)C641.4278 (C36H57N4O6
requires 641.4278).
CH2CO2H), 5.13 (minor) and 5.34 (major) (s, 2H,
CH2CON), 5.20 (s, 2H, CH2O), 7.30–7.50 (m, 5H, Ar-H),
7.75 (minor) and 8.00 (major) (t, 1H, NH), 8.32 (s, 1H,
adeninyl-C8–H), 8.60 (s, 1H, adeninyl-C2–H), 10.65 (br s,
1H, OH). dC (CDCl3) (two rotational isomers in a 2:1 ratio
were observed) 13.9 (CH3), 18.3, 22.1, 25.0, 25.1, 27.7,
27.7, 28.1, 28.2, 28.3, 28.4, 28.7, 28.7, 28.8, 28.9, 29.0,
31.3, 35.4, 36.1, 36.9, 40.5, 43.9, 44.1, 46.7, 47.5, 49 (CH2),
65.3 and 65.3 (C^C), 66.2 (CH2), 72.2 and 77.9 (C^C),
122.9 (adenine-C5), 127.8, 127.9, 128.4 (CH), 136.4, 145.2
(adenine-C6), 149.3 (NHCOO), 151.4, 152.1, 152.3, 166.5
and 167.0 (CON), 170.3 and 170.8 (CONH), 172.3, 172.8
(CO2H). MS (ES) m/z 784.6 (MCH)C(70%).
3.1.20. N-(2-Octadecanoylaminoethyl)-N-(thymin-1-yl-
acetyl)-glycine 27. N-(2-Octadecanoylaminoethyl)-N-
(thymin-1-ylacetyl)glycine methyl ester 23 (0.56 g,
0.63 mmol) was dissolved in THF (60 mL) and 1 M aq
LiOH (30 mL). The mixture was stirred for 3 h, then the
solution was washed with DCM. The aqueous phase was
separated, acidified with 1 M aq HCl and then extracted
with ethyl acetate (5!50 mL). The ethyl acetate extract was
dried (MgSO4) and the solvent evaporated under reduced
pressure to yield the title compound 27 as a colourless solid
(0.34 g, 97%, mp 145–147 8C). dH (d6-DMSO) (two rota-
tional isomers in a 2:1 ratio were observed) 0.84 (t, 3H, JZ
6.4 Hz, CH2CH3), 1.20–1.50 (m, 30H, CH2), 1.74 (s, 3H,
thymine-CH3), 2.08 (m, unresolved, 2H, CH2CONH), 3.37
(m, 4H, NCH2CH2N), 3.98 (major) and 4.20 (minor) (s, 2H,
CH2CO2H), 4.48 (minor) and 4.64 (major) (s, 2H,
CH2CON), 7.35 (minor) and 7.42 (major) (s, 1H, thymine-
C6–H), 7.65 (minor) and 7.88 (major) (s, 1H, NHCH2),
11.30 (s, 1H, OH). [1!NH not observed] dC (d6-DMSO)
(two rotational isomers in a 2:1 ratio were observed) 12.3
(thymine-CH3), 14.1, 22.7, 25.6, 29.3, 29.4, 29.5, 29.6, 29.7,
30.8, 31.9, 36.5, 36.6, 37.5, 37.6, 47.9, 48.3, 48.5, 48.8
(CH2), 50.0 (CH3), 110.8 and 110.9 (thymine-C5), 150.9
(CO), 163.9 (CO), 167.3 and 168.1 (CON), 170.0 and 170.1
(CONH), 173.9 and 174.4 (CO2H). nmax (KBr, cmK1) 3319,
3066 (NH str), 2918, 2850 (CH str), 1667, 1644 (CO), 1549,
1469, 1415. HRMS (ES) m/z (MCH)C551.3803
(C29H51N4O6 requires 551.3806).
3.1.18. N-(2-Pentacosa-10,12-diynoylaminoethyl)-N-
(N4(benzyloxycarbonyl)cytosin-1-ylacetyl)glycine 25. N-
(2-Pentacosa-10,12-diynoylaminoethyl)-N-(N4-(benzyl-oxy-
carbonyl)cytosin-1-ylacetyl)glycine methyl ester 21 (0.10 g,
0.13 mmol) was dissolved in THF (10 mL) and 1 M aq
LiOH (5 mL) and the mixture was stirred for 3 h. Water
(20 mL) was added and the solution was washed with DCM
(2!20 mL) and then acidified to pH 1.0 using a 0.7 M
solution of citric acid. The solution was extracted with DCM
(6!10 mL), the extract was washed with brine (10 mL) and
then dried (MgSO4). The solvent was evaporated under
reduced pressure to yield the title compound 25 as a
colourless solid (0.094 g, 95%). dH (d6-DMSO)
(two rotational isomers in a 2:1 ratio were observed) 0.85
(t, 3H, JZ7.1 Hz, CH3), 1.2–1.6 (m, 32H, CH2), 2.10 (m
unresolved, 2H, CH2CONH), 2.30 (t, 4H, JZ6.2 Hz,
CH2C^C), 3.4 (m, 4H, NCH2CH2N), 3.95 (major) and
4.2 (minor) (s, 2H, CH2CO2H), 5.15 (s, 2H, CH2Ph), 7.0
(2!d) and 7.9 (2!d) (2H, JZ7.2 Hz, cytosinyl-H), 7.4
(m, 5H, Ph). HRMS (ES) (MCH)C760.4647 (C43H62N5O7
requires 760.4649).
3.1.21. Ethyl (N6-bis(t-butoxycarbonyl)adenin-9-yl)acetate
28b. To a solution of ethyl adenin-9-ylacetate18 28a
(1.00 g, 4.52 mmol) in THF (25 mL) was added DMAP
(1.66 g, 13.59 mmol) followed by di-t-butyl dicarbonate
(2.96 g, 13.56 mmol) and the mixture was stirred at rt for
17 h. The solvent was evaporated and the crude residue was
purified by flash column chromatography [silica gel,
CH2Cl2/MeOH (0–1%) as eluent] to yield the title
compound 28b as a colourless oil (1.19 g, 62%). dH
(CDCl3) 1.28 (t, 3H, JZ7.2 Hz, CH2CH3), 1.44 (s, 9H,
(CH3)3), 4.26 (q, 2H, JZ7.1 Hz, CH2CH3), 5.05 (s, 2H,
CH2CO2Et), 8.14 (s, 1H, H-8), 8.36 (s, 1H, H-2). dC
(CDCl3) 14.1 (CH2CH3), 27.8 ((CH3)3), 44.4, 62.5 (CH2),
83.7 (C–O), 128.3 (C5), 144.9 (C6), 150.3, 150.5, 152.3,
153.4 (CON), 166.7 (CO2Et). nmax (thin film, cmK1) 2981,
2937 (CH str), 1787, 1753 (CO), 1603, 1581, 1508, 1452.
HRMS (ES) (MCH)C422.2032 (C19H28O6N5 requires
422.2034).
3.1.19. N-(2-Pentacosa-10,12-diynoylaminoethyl)-N-(N6-
(benzyloxycarbonyl)adenin-9-ylacetyl)glycine 26. N-(2-
Pentacosa-10,12-diynoylaminoethyl)-N-(N6-(benzyl-oxy-
carbonyl)adenin-9-ylacetyl)glycine methyl ester 22 (0.24 g,
0.30 mmol) was dissolved in THF (5 mL) and 1 M aq LiOH
(3 mL) and the mixture was stirred at rt for 2 h. Water
(5 mL) was then added and the solution was washed with
DCM (2!10 mL) before being acidified to pH 2.0 with 1 M
aq HCl. The aqueous phase was extracted with DCM (5!
10 mL), the extract was dried (MgSO4) and the solvent was
evaporated under reduced pressure to yield the title
compound 26 as a white foam (0.16 g, 69%), which turned
purple in ambient light. dH (CDCl3) (two rotational isomers
in a 2:1 ratio were observed) 0.80 (t, 3H, JZ6.4 Hz, CH3),
1.20–1.50 (m, 32H, CH2), 1.95 (minor) and 2.05 (major)
(t, 2H, JZ7.3 Hz, CH2CONH), 2.25 (t, 4H, JZ6.4 Hz,
CH2C^C), 3.15 (minor) and 3.35 (major) (m, unresolved,
2H, NHCH2), 3.35 (minor) and 3.50 (major) (m, unresolved,
2H, CH2CH2N), 4.00 (major) and 4.32 (minor) (s, 2H,
3.1.22. (N6-Bis(t-butoxycarbonyl)adenin-9-yl)acetic acid

N. M. Howarth et al. / Tetrahedron 61 (2005) 8875–8887
8885
28c. A solution of ethyl (N6-bis(t-butoxycarbonyl)adenin-9-
yl)acetate 28b (0.53 g, 1.26 mmol) in THF (15 mL) and 1 M
aq LiOH (10 mL) was stirred at rt for 1 h. The solution was
then partitioned between 1 M aq KHSO4 (15 mL) and ethyl
acetate (15 mL). The aqueous phase was separated and re-
extracted with ethyl acetate (4!15 mL) and the combined
extract was dried (MgSO4). The solvent was evaporated to
yield the title compound 28c (0.36 g, 72%) as a colourless
solid, mp 104–105 8C. dH (d6-DMSO) 1.38 (s, 9H, CH3),
5.15 (s, 2H, CH2), 8.60 (s, 1H, H-8), 8.83 (s, 1H, H-2), 10.10
(br s, 1H, OH). dC (d6-DMSO) 27.4 ((CH3)3), 44.5 (CH2),
83.4 (C–O), 127.4 (C-5), 147.4, 149.0, 150.0, 151.6, 153.3
(CON), 168.9 (CO2H). nmax (KBr, cmK1) 3420 (OH str),
2981, 2936 (CH str), 1790, 1740 (CO), 1652, 1609, 1585,
1511, 1472. HRMS (ES) (MCH)C394.1721 (C17H24O6N5
requires 394.1721).
17 h. The mixture was evaporated under reduced pressure to
leave the title compound 28e as a glassy solid (0.083 g,
99%). dH (d6-DMSO) (two rotational isomers in a 2:1 ratio
were observed) 0.85 (t, 3H, JZ6.5 Hz, CH2CH3), 1.2–1.5
(m, 32H, CH2), 2.00 (minor) and 2.15 (major) (t, 2H, JZ
7.0 Hz, CH2CONH), 2.25 (t, 4H, JZ6.0 Hz, CH2C^C),
3.2–3.6 (m, unresolved, 4H, NHCH2CH2N), 4.00 (major)
and 4.32 (minor) (s, 2H, CH2CO2H), 5.15 (minor) and 5.35
(major) (s, 2H, CH2CON), 7.80 (minor) and 8.05 (major)
(t, 1H, NH), 8.25 (s, 1H, H-8), 8.35 (s, 1H, H-2). dC (d6-
DMSO) (two rotational isomers in a 2:1 ratio were
observed) 13.9 (CH2CH3), 18.2, 22.0, 25.0, 25.1, 27.7
(!2), 28.1, 28.2, 28.3 (!2), 28.6, 28.7, 28.8, 28.9, 29.0,
31.2, 25.3, 36.1, 36.9, 44.1, 46.7, 47.5 (CH2), 65.3 (!2),
77.9 (C^C), 143.7, 147.8, 152.4, 166.3 and 166.7 (CON),
170.2 and 170.7 (CONH), 172.3 and 172.8 (CO2H). nmax
(thin film, cmK1) 3074 (NH str), 2926, 2854 (CH str), 1660,
1605, 1563, 1552, 1524, 1467, 1422. HRMS (ES) (MC
H)C650.4387 (C36H56N7O4 requires 650.4388).
3.1.23. N-(2-Pentacosa-10,12-diynoylaminoethyl)-N-(N6-
(bis-t-butoxycarbonyl)adenin-9-ylacetyl)glycine methyl
ester 28d. To a solution of N-(2-pentacosa-10,12-diynoyl-
aminoethyl)glycine 18 (0.15 g, 0.31 mmol) in DMF
(10 mL) was added DIPEA (0.15 mL, 0.84 mmol), (N6-
bis(t-butoxycarbonyl)adenin-9-yl)acetic acid 28c (0.11 g,
0.28 mmol) and HBTU (0.11 g, 0.28 mmol). The mixture
was stirred at rt for 17 h, then the solvent was evaporated
under reduced pressure. The residue was dissolved in DCM
(50 mL) and this solution was washed successively with
1 M aq NaHCO3 (3!50 mL), 1 M aq KHSO4 (50 mL),
water (50 mL) and finally brine (50 mL). The solution was
dried (MgSO4) and the solvent was evaporated under
reduced pressure to give the crude product, which was
purified by flash column chromatography [silica gel,
CH2Cl2/methanol (97:3) as eluent] to yield the title com-
pound 28d as a glassy solid (0.11 g, 44%). dH (CDCl3) (two
rotational isomers in a 2:1 ratio were observed) 0.85 (t, 3H,
JZ6.5 Hz, CH2CH3), 1.22 (s, 9H, (CH3)3), 1.4–1.7 (m,
32H, CH2), 2.05 (minor) and 2.35 (major) (t, 2H, JZ7.6 Hz,
CH2CONH), 2.22 (t, 4H, JZ6.8 Hz, CH2C^C), 3.35
(minor) and 3.50 (major) (q, 2H, JZ5.6 Hz, NHCH2),
3.55 (minor) and 3.65 (major) (t, 2H, JZ5.5 Hz,
CH2CH2N), 3.73 (major) and 3.84 (minor) (s, 3H, OCH3),
4.07 (major) and 4.30 (minor) (s, 2H, CH2CO2Me), 5.00
(minor) and 5.18 (major) (s, 2H, CH2CON), 5.95 (minor)
and 6.70 (major) (t, 1H, NH), 8.10 (major) and 8.18 (minor)
(s, 1H, H-8), 8.78 (major) and 8.79 (minor) (s, 1H, H-2). dC
(CDCl3) (two rotational isomers in a 2:1 ratio were
observed) 14.1 (CH2CH3), 19.2 (!2), 22.7 (CH2), 27.8
and 28.3 ((CH3)3), 28.3, 28.7, 28.8, 28.9, 29.1, 29.3, 29.4,
29.6 (!3), 31.9, 36.6, 37.6, 44.0, 48.6 (CH2), 48.9, 52.6
(OCH3), 65.2 and 65.3 (C^C), 77.4 and 77.6 (C^C), 83.7
and 83.8 (C–O), 128.3 (purine-C5), 145.7 (purine-C6),
150.3 (!2), 151.9, 153.5 (NCO2), 166.4 (CON), 170.2
(CONH), 174.2 (CO2Me). nmax (thin film, cmK1) 3375 (NH
str), 2927, 2854 (CH str), 1786, 1751 (CO), 1670, 1604,
1581, 1540, 1457, 1415. HRMS (ES) (MCH)C886.5422
(C47H74N7O8 requires 886.5413).
3.2. General solid phase method for attachment of
N-terminal stearoyl group to PNA oligomers.
Synthesis of CH3(CH2)16CO-(T0)10-LysNH2 29 and
CH3(CH2)16CO-(T0)10-AspNH2 30
The N-Boc protected PNA T010-mer was assembled
manually on either a Boc-Lys(2-Cl–Z)- or Boc-Asp(Bz)-
derivatized MBHA resin, respectively, following the
strategy described by Christensen et. al.21 (0.200 g of
resin (dry weight), loading factorZca. 0.15 mmol/g).
Subsequently, the following procedure was performed: (1)
N-Boc deprotection using a solution of TFA:m-cresol (95:5,
v/v), 2!4 min. (2) Washing with DMF:DCM (1:1, v/v),
3!20 s; washing with pyridine, 2!20 s (3) Coupling using
a solution of octadecanoyl fluoride13 (0.029 g, 0.10 mmol)
and DIPEA (0.052 mL, 0.30 mmol) in DMF:DCM (1:1, v/v)
(1 mL). Coupling was allowed to proceed for 20 min at rt.
(4) Washing with DMF:DCM (1:1, v/v), 2!20 s. Steps 3
and 4 were repeated a further two times. After the final
washing, the resins were dried under vacuum and the PNA
oligomers were concomitantly deprotected and cleaved
from the solid support using the TFMSA method reported
by Christensen et al.21 The crude products afforded were
purified by RP-HPLC to give the pure PNA oligomers 29
and 30 as white pellets. The identities of oligomers 29 and
30 were verified by MALDI-TOF mass spectroscopy
(Table 1).
3.3. General method for solution phase coupling of a
stearoyl or diacetylene group to a PNA oligomer.
Synthesis of Ac-(T0)10-Lys{CO(CH2)16CH3}NH2 32,
Ac-(T0)10-Lys{CO(CH2)8C4(CH2)11CH3}NH2 33,
AcAsp-(T0)10-Lys{CO(CH2)16CH3}NH2 35,
AcAsp-(T0)10-Lys{CO(CH2)8C4(CH2)11CH3}NH2 36,
CH3(CH2)11C4(CH2)8CO-(T0)10-AspNH2 38, and
CH3(CH2)11C4(CH2)8CO-PEG-(T0)10-AspNH2 40
3.1.24. N-(2-Pentacosa-10,12-diynoylaminoethyl)-N-
(adenin-9-ylacetyl)-glycine hydrochloride 28e. To a
solution of N-(2-pentacosa-10,12-diynoylaminoethyl)-N-
(N6-bis(t-butoxycarbonyl)adenin-9-ylacetyl)glycine methyl
ester 28d (0.106 g, 0.12 mmol) in DCM (10 mL) was added
6 M aq HCl (10 mL) and the mixture was stirred at 40 8C for
The appropriate parent PNA oligomers {that is 31 for
preparation of 32 and 33, 34 for 35 and 36, 37 for 38 and 39
for 40 (Scheme 7)} were assembled manually according to
the solid phase strategy developed by Christensen et al.21
Following deprotection and cleavage from the resin, the
identities of the crude PNA oligomers afforded were verified

8886
N. M. Howarth et al. / Tetrahedron 61 (2005) 8875–8887
Table 2. MALDI-TOF mass spectral data for oligomers 29–40 (Scheme 7)
Calculated average mass
Found (m/z)
29
30
31
32
33
34
35
36
37
38
39
40
3097.2 [MCNa]C
3084.1 [MCNa]C
2850.8 [MCH]C
3139.2 [MCNa]C
3229.4 [MCNa]C
2965.9 [MCH]C
3254.3 [MCNa]C
3344.4 [MCNa]C
2817.6 [MCNa]C
3174.2 [MCNa]C
2940.8 [MCH]C
3319.4 [MCNa]C
3096.4 [MCNa]C
3082.9 [MCNa]C
2850.3 [MCH]C
3136.5 [MCNa]C
3227.6 [MCNa]C
2963.1 [MCH]C
3252.5 [MCNa]C
3342.5 [MCNa]C
2816.0 [MCNa]C
3174.2 [MCNa]C
2939.2 [MCH]C
3319.2 [MCNa]C
CH3(CH2)16CO-(T0)10-LysNH2
CH3(CH2)16CO-(T0)10-AspNH2
Ac-(T0)10-LysNH2
Ac-(T0)10-Lys{CO(CH2)16CH3}NH2
Ac-(T0)10-Lys{CO(CH2)8C4(CH2)11CH3}NH2
AcAsp-(T0)10-LysNH2
AcAsp-(T0)10-Lys{CO(CH2)16CH3}NH2
AcAsp-(T0)10-Lys{CO(CH2)8C4(CH2)11CH3}NH2
H-(T0)10-AspNH2
CH3(CH2)11C4(CH2)8CO-(T0)10-AspNH2
H-PEG-(T0)10-AspNH2
CH3(CH2)11C4(CH2)8CO-PEG-(T0)10-AspNH2
The matrix used was 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid).
by MALDI-TOF mass spectrometry (Table 1). Sub-
sequently, the required PNA oligomer was dissolved in
DMF:pyridine (1:1, v/v) (1 mL/0.01 g) and either octa-
decanoyl fluoride13 or 10,12-pentacosadiynoic acid fluor-
ide13 (10 equiv) was added followed by DIPEA (30 equiv).
The resulting mixture was left to stir at rt for 24 h before the
solvent was removed in vacuo. The remaining residue was
re-dissolved in TFA (1 mL) and the crude product was
precipitated from this solution by addition of diethyl ether
(10 mL). The suspension was centrifuged and the super-
natant was decanted off from the pellet yielded. The pellet
was then re-suspended in fresh diethyl ether (10 mL) and the
centrifugation and decanting processes were repeated. This
step was performed a further 5 times before the pellet was
dried using a gentle stream of dry nitrogen gas. Finally, the
crude product afforded was purified by RP-HPLC to give the
pure PNA oligomers 32, 33, 35, 36, 38 and 40 as white
pellets. The identities of oligomers 32, 33, 35, 36, 38 and 40
were confirmed by MALDI-TOF mass spectroscopy
(Table 2).
the EPSRC Mass Spectrometry Service Centre, Swansea,
UK, for HRMS data.
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