Synthesis and polymerisation of lipophilic peptide nucleic acids derived from stearic acid and pentacosa-10,12-diynoic acid

Article abstract of DOI:10.1016/j.tetlet.2003.09.047

The adenine, cytosine and thymine peptide nucleic acid (PNA) monomers and PNA T₁₀ oligomers bearing either a diacetylenic or stearoyl moiety at the N- or C-terminus have been successfully prepared. The resulting thymine monomeric and T₁₀

Full text of DOI:10.1016/j.tetlet.2003.09.047

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 8089–8092
Synthesis and polymerisation of lipophilic peptide nucleic acids
derived from stearic acid and pentacosa-10,12-diynoic acid
Nicola M. Howarth,* W. Edward Lindsell, Euan Murray and Peter N. Preston*
Chemistry, School of Engineering & Physical Sciences, William H. Perkin Building, Heriot-Watt University, Riccarton,
Edinburgh EH14 4AS, UK
Received 30 July 2003; revised 27 August 2003; accepted 5 September 2003
Abstract—The adenine, cytosine and thymine peptide nucleic acid (PNA) monomers and PNA T10 oligomers bearing either a
diacetylenic or stearoyl moiety at the N- or C-terminus have been successfully prepared. The resulting thymine monomeric and
T10-mer derivatives have been subsequently incorporated into polydiacetylene-containing liposomes.
© 2003 Elsevier Ltd. All rights reserved.
Polydiacetylenes (PDAs) can be prepared by topochem-
ical polymerisation of conjugated 1,3-diynes by thermal
treatment or by UV- and g-irradiation (Scheme 1);1
they can also be formed in thin films2 or as liposomes.3
An important feature of PDAs is manifested in colour
changes that can be induced thermally, by pH changes
and through solvent variations.4 Blue to red transitions
also result from binding events in which lipophilic
biospecific receptors are embedded in a host PDA
matrix; such assemblies can be generated either by
co-polymerisation of appropriately functionalised
diacetylenes (e.g. carbohydrate-mediated recognition of
influenza virus3) or by the use of lipid/PDA mixed
vesicles (e.g. elucidation of peptide-membrane
interactions5).
ing properties. Previously, we have reported the prepa-
ration of lipophilic acridine-labelled diacetylenes6 and
PDA liposomes thereof.7 Here, we report the synthesis
of peptide nucleic acids (PNAs) and model PNA
monomer analogues in the 1,3-diyne series. PNAs are
DNA mimics in which the entire deoxyribose–phos-
phate backbone has been exchanged with a structurally
homomorphous uncharged polyamide backbone com-
posed of N-(2-aminoethyl)glycine units (Scheme 2).8 An
important feature of PNAs is that they bind with higher
affinity and sequence specificity to both single-stranded
DNA (ssDNA) and RNA than their natural oligonucle-
otide counterparts.9
The objectives of the present work 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 (sps) of PNA oligomers
incorporating diacetylenic and (separately) stearoyl
groups.
We envisage that nucleic acid biosensors should be
accessible using PDA matrixes in which the receptor is
a group with suitable intercalating or other DNA-bind-
3. Production of new PDA liposomes.
Scheme 1. Preparation of PDAs from conjugated 1,3-diynes.
Keywords: nucleic acid biosensors; PNA; polydiacetylenes; liposomes.
* Corresponding authors. Tel.: +(44)-(0)131-451 8026; fax: +(44)-
(0)131-451 3180 (N.M.H.); Tel.: +(44)-(0)131-451 8035; fax: +(44)-
(0)131-451 3180 (P.N.P.); e-mail: n.m.howarth@hw.ac.uk;
p.n.preston@hw.ac.uk
Scheme 2. Structure of PNA. B=adenine; cytosine; guanine;
thymine.
0040-4039/$ - see front matter © 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2003.09.047

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N. M. Howarth et al. / Tetrahedron Letters 44 (2003) 8089–8092
1. Synthesis of PNA monomer model compounds12
aminoethyl)glycine methyl ester dihydrochloride 616
(Scheme 4). Treatment of 6 with either N-succin-
imidyl-10,12-pentacosadiynoate6 or N-succinimidyl
stearoate in the presence of N,N-diisopropylethyl-
amine afforded 7 and 8, respectively, in 65 and 60%
yields after purification. Compound 7 was then cou-
pled to thyminyl acetic acid,14 N4-benzyloxycarbonyl
cytosinyl acetic acid15 and N6-benzyloxycarbonyl
adeninyl acetic acid15 using either HBTU or HATU
as the activating agent in the presence of N,N-diiso-
propylethylamine. After work-up and purification by
flash chromatography, 9, 10 and 11 were ioslated in
63, 71 and 80% yields. Similar conditions were
applied in the coupling of 8 to thyminyl acetic acid
to obtain 12 (86% yield). Finally, ester hydrolysis
using lithium hydroxide afforded the desired com-
pounds 13, 14, 15 and 16 in yields ranging from 84
to 97%.
1.1. C-Terminal diacetylenic derivatives
The two C-terminal diacetylenic derivatives, 3 and 4,
were successfully prepared from the protected thymine
PNA monomer 113,14 as outlined in Scheme 3. Ester
hydroysis using the procedure reported by Dueholm
et al.15 afforded 2 in a 74% yield. Compound 2 was
then converted in situ into its corresponding N-
hydroxybenzotriazole (HOBt)- activated ester using
HOBt and EDC. Subsequent addition of either N-(8-
amino-3,6-dioxaoctyl)-10,12-pentacosadiynamide7 or 1-
amino-10,12-pentacosadiyne10 and N,N-diisopropyl-
ethylamine gave the desired compounds 3 and 4,
respectively, in 66 and 38% yields after purification.
1.2. N-Terminal diacetylenic and stearoyl derivatives
Two routes to the N-terminal PNA monomer model
compounds have been employed. The N-terminal
diacetylenic derivative 5 was prepared from the pro-
2. Synthesis of N-stearoyl and diacetylenic PNA
oligomers
tected thymine PNA monomer
1
(Scheme 3).
The PNA oligomer of sequence CH3(CH2)16CO-
Removal of the Boc protecting group was accom-
plished using aqueous hydrochloric acid. Treatment of
the isolated crude hydrochloride salt with N-succin-
imidyl-10,12-pentacosadiynoate6 in the presence of
N,N-diisopropylethylamine gave compound 5 in 50%
yield after purification. We have found more recently
that 5 can be obtained in a higher yield (90%) if
10,12-pentacosadiynoic acid fluoride11 is used in the
last step rather than N-succinimidyl-10,12-penta-
cosadiynoate.
(T)10Lys-NH2, 17, has been prepared by sps17,18 using
the N1-Boc-N5-(2-chloro-Z)-
L-lysine-derivatised 4-
methylbenzhydrylamine (MBHA) resin. The Boc-pro-
tected thymine monomer 2 (Scheme 3) was coupled
using HBTU as the activating agent in the presence
of a slight excess of N,N-diisopropylethylamine. All
the couplings were monitored using the Kaiser test19
and in all cases only a single coupling was required.
The stearoyl group was incorporated at the N-termi-
nus of the oligomer in the final coupling step once
the PNA T10-mer had been assembled on the solid
support. This was achieved using 3.33 equiv. of
stearoyl fluoride20 and 10 equiv. of N,N-diisopropyl-
ethylamine. At the end of the synthesis, the oligomer
The N-terminal diacetylenic derivatives 13–15 and N-
terminal stearoyl analogue 16 have been synthesised
using an alternative strategy starting from N-(2-
Scheme 3. Boc=(CH3)3COCO; T=thyminyl. Reagents and conditions: i. 1 M (aq.) LiOH, THF, rt; ii. (a) 1.1 equiv. EDC, 1.1
7
equiv. HOBt, DMF, 0°C–rt (b) 0.94 equiv. CH3(CH2)11C4(CH2)8CONH(CH2CH2O)2CH2NH2
CH3(CH2)11C4(CH2)9NH2,10
equiv. DIPEA, DMF, 0°C–rt; iii. (aq.) HCl, DCM, rt; iv.
CH3(CH2)11C4(CH2)8CO2N(COCH2)2,6 DMF, rt, or 1 equiv. CH3(CH2)11C4(CH2)8COF,11 2:1 DMF:DCM, rt, 3 equiv. DIPEA.
or 0.67 equiv.
1
6
M
1
equiv.

N. M. Howarth et al. / Tetrahedron Letters 44 (2003) 8089–8092
8091
†
17 CH3(CH2)16CO-(T)10-LysNH2
was cleaved from the solid support using tri-
fluoromethanesulfonic acid (TFMSA) under identical
conditions to those employed by Christensen et al.17
The crude oligomer was purified using RP-HPLC
(R.T.=28 min; buffer A=0.1% TFA in water; buffer
B=0.1% TFA in acetonitrile; flow rate=1 mL/min;
umax=260 nm). The identity of the oligomer was ver-
ified by MALDI-TOF mass spectrometry which
showed a principal peak at 3096.2 Da, corresponding
to the Na+ adduct of the desired oligomer.
18 CH3(CH2)16CO-(T)10-AspNH2
19 CH3(CH2)11C4(CH2)8CO-(T)10-LysNH2
20 AcAsp-(T)10-LysNH2
21 AcAsp-(T)10-Lys(CO(CH2)16CH3)NH2
22 AcAsp-(T)10-Lys(CO(CH2)8C4(CH2)11CH3)NH2
Similarly prepared21 was 18, [M+Na]+=3082.1 Da,
but the final coupling step on the solid support to
prepare the related diacetylene derivative 19 failed.
However,
a
post-synthetic modification method
involving acylation of the lysine side chain was used
successfully to attach a diacetylenic group; thus,
capped PNA 20 in 1:1 DMF/pyridine was allowed to
react with the appropriate acyl fluoride (10 equiv.)
with DIPEA (30 equiv.) in 1:1 DCM:DMF to afford
21, [M+Na]+=3252.5 Da, and 22, [M+Na]+=3342.7
Da, both in 67% yield.
3. Formation of liposomes
Liposomes of selected lipophilic products, including
derivatives with diacetylene groups as a component,
were prepared in deionised water22 using a 25 KHz
probe sonicator at 80°C, followed by filtration (0.8
mm nylon filter) and cooling to 4°C. Photo-polymeri-
sation of the N2-purged solutions with a UV lamp
(254 nm) afforded deep blue solutions of PDA-lipo-
somes. The following examples, A–E, were stable to
precipitation at room temperature.
A poly-CH3(CH2)11C4(CH2)8CO2H 23 (100%)
umax 626, 582, 229 nm
B poly-13 (100%)
umax 623, 574, 522, 269 nm
C poly-{23 (95%)+13 (5%)} umax 627, 583, 254 nm
D poly-{23 (95%)+22 (5%)} umax 624, 580, 259 nm
E poly-23 (95%)+16 (5%) umax 631, 587, 254 nm
Polymerised carboxylic acid 23 forms a liposome A
with an electronic spectrum typical of PDAs (e.g. see
Ref. 3). The thyminyl-containing PDA-liposome B
includes an absorption band, 269 nm, assignable to
the pyrimidine ring (the higher energy band <229 nm,
observed in A, being obscured). Liposomes formed
from mixtures of 23 (95%) and a thymine-containing
lipid, 13, 16 or 22 (5%), show spectral evidence for
incorporation of the latter component both in C and
D, with polymerisable 1,3-diyne functions in the
minor constituent, and also in E, with only an unre-
active stearoyl tail in 16. Thus, the formation of
PDA-liposomes containing PNA head-groups with
potential for biosensor applications has been achieved
(Scheme 5).
Scheme 4. AZ=N6-Benzyloxycarbonyladeninyl; CZ=N4-
benzyloxycarbonylcytosinyl; T=thyminyl. Reagents and con-
ditions: i. 1 equiv. CH3(CH2)11C4(CH2)8CO2N(COCH2)2 or
7
† We employ the following nomenclature: CH3(CH2)16CO,
CH3(CH2)11C4(CH2)8CO and Ac denote the acyl group attached to
the N-terminal; T the thyminyl PNA monomer unit; Lys and Asp
refer to lysine and aspartic acid, respectively, and NH2 indicates a
C-terminal amide.
1
equiv. CH3(CH2)16CO2N(COCH2)2, 3 equiv. DIPEA,
DMF, rt; ii. 1.2 equiv. R%CH2CO2H,14,15 1.2 equiv. HBTU
or HATU, 3 equiv. DIPEA, DMF, rt; iii. 1 M (aq.) LiOH,
THF, rt.

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N. M. Howarth et al. / Tetrahedron Letters 44 (2003) 8089–8092
Freier, S. M.; Driver, D. A.; Berg, R. H.; Kim, S. K.;
Norden, B.; Nielsen, P. E. Nature 1993, 365, 566.
10. Synthesis and spectroscopic data for CH3(CH2)11C4-
(CH2)9NH2: 10,12-Pentacosadiynoyl amide7 and excess
Red-Al® were stirred in toluene, 0–20°C, 17 h; quenching
with NaOH aq. and extraction into ethyl acetate afforded
the product as
a
colourless solid, 72%. 1H NMR
(CDCl3): l ppm 0.80 (t, J=6.2 Hz, 3H, CH3), 1.2–1.7 (m,
34H, CH2), 2.18 (t, J=6.6 Hz, 4H, C4CH2), 2.8 (br s, 2H,
NH2), 3.55 (t, J=6.4 Hz, 2H, NCH2); 13C{1H} NMR
(CDCl3): l ppm 14.1, 19.1, 22.6, 25.6, 28.2, 28.8, 29.0,
29.3, 29.4, 29.6, 31.8, 32.7, 63.0.
11. Synthesis and spectroscopic data for CH3(CH2)11C4-
(CH2)8COF: 10,12-Pentacosadiynoic acid, 2 equiv. cyan-
uric fluoride and 1 equiv. pyridine were heated at reflux
in DCM, 3 h; aq./DCM work-up gave the product as a
colourless, oily solid, 99%. 1H NMR (CDCl3): l ppm
0.85 (t, J=6.4 Hz, 3H, CH3), 1.2–1.7 (m, 30H, CH2), 1.7
(m, 2H, CH2), 2.25 (t, J=6.7 Hz, 4H, C4CH2), 2.50 (dt,
J=0.9, 4.9 Hz, 2H, CH2); 13C{1H} NMR (CDCl3): l
ppm 14.1, 19.1 (×2), 22.7, 28.2, 28.3, 28.6, 28.7, 28.8,
28.9, 29.1, 29.3, 29.5, 29.6 (×3), 31.9 65.2, 65.4, 77.2, 77.6,
163.5 {d, 1J(13C–19F)=361 Hz}; 19F NMR (CDCl3): l
ppm 44.9. IR (Nujol) cm−1: 1836 (wCO). HRMS (EI):
m/z 394.3480 [M+NH4]+, C25H45NOF calcd 394.3485.
12. Satisfactory analytical and spectroscopic data were
obtained for all new compounds.
Scheme 5. Formation of the mixed assembly derived from
polymerisable diacetylenes 10,12-pentacosadiynoic acid (95%)
and 22 (5%).
Acknowledgements
13. Finn, P. J.; Gibson, N. J.; Fallon, R.; Hamilton, A.;
Brown, T. Nucleic Acid Res. 1996, 24, 3357.
14. Kosynkina, L.; Wang, W.; Liang, T. C. Tetrahedron Lett.
1994, 35, 5173.
15. Dueholm, K. L.; Egholm, M.; Behrens, C.; Christensen,
L.; Hansen, H. F.; Vulpius, T.; Petersen, K. H.; Berg, R.
H.; Nielsen, P. E.; Buchardt, O. J. Org. Chem. 1994, 59,
5767.
We thank Heriot-Watt University for support (to
E.M.), Dr. A. S. F. Boyd for NMR spectra, and the
National Mass Spectrometry Service Centre, Swansea,
for HRMS data.
16. Heimer, E. P.; Gallo-Torres, H. E.; Felix, A. M.; Ahmad,
M.; Lambros, T. J.; Scheidl, F.; Meinenhofer, J. Int. J.
Pept. Protein Res. 1984, 23, 203.
References
17. Christensen, L.; Fitzpatrick, R.; Gildea, B.; Petersen, K.
H.; Hansen, H. F.; Koch, T.; Egholm, M.; Buchardt, O.;
Nielsen, P. E.; Coull, J.; Berg, R. J. Pept. Sci. 1995, 3,
175.
1. (a) Wegner, G.; Z. Naturforsch. B 1969, 24, 284; Macro-
mol. Chem. 1972, 154, 35; Pure Appl. Chem. 1977, 49,
443; (b) Polydiacetylenes; Bloor, D.; Chance, R. R., Eds.;
Martinus Nijhoff: The Netherlands, 1985.
18. Howarth, N. M.; Wakelin, L. P. G. J. Org. Chem. 1997,
62, 5441.
19. Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I.
2. Charych, D. H.; Nagy, J. O.; Spevack, W.; Bednarski, M.
D. Science 1993, 261, 585.
3. Reichert, A.; Nagy, J. O.; Spevack, W.; Charych, D. J.
Anal. Biochem. 1970, 84, 595.
Am. Chem. Soc. 1995, 117, 829.
20. Synthesis and spectroscopic data for CH3(CH2)16COF:
Preparation: cf. Ref. 11, product obtained as a colourless,
4. Jenkins, I. H.; Kar, A. J.; Lindsell, W. E.; Murray, C.;
Preston, P. N.; Wang, C.; Wherrett, B. S. Macromolecules
1996, 29, 6365 and references cited therein.
5. Kolusheva, S.; Shahal, T.; Jelinek, R. Biochemistry 2000,
39, 15851.
1
oily solid, 90%. H NMR (CDCl3) l ppm: 0.85 (t, J=6.4
Hz, 3H, CH3), 1.2–1.4 (m, 28H, CH2), 1.65 (m, 2H,
CH2), 2.50 (dt, J=1.1, 4.9 Hz, 2H, CH2); 13C{1H} NMR
(CDCl3) l ppm: 14.1, 22.7, 23.9, 28.7, 29.1, 29.3, 29.4,
29.5, 29.6, 29.7, 31.9 (×2), 32.4, 163.6 (d, 1J(13C–19F)=
361 Hz); 19F NMR (CDCl3): l ppm 44.9. IR (Nujol)
cm−1: 1846 (wCO).
6. Lindsell, W. E.; Murray, C.; Preston, P. N.; Woodman,
T. A. J. Tetrahedron 2000, 56, 1233.
7. Murray, C. Ph.D. Thesis, Heriot-Watt University, Edin-
burgh, 1999.
21. Accurate, principal peak MALDI-TOF mass spectral
data were obtained for all new PNAs.
22. Charych, D.; Cheng, Q.; Reichert, A.; Kuziemko, G.;
Stroh, M.; Nagy, J. O.; Spevak, W.; Stevens, R. C. Chem.
Biol. 1996, 3, 113.
8. (a) Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O.
Science 1991, 54, 1497; (b) Uhlmann, E.; Peyman, A.;
Breipohl, G.; Will, D. W. Angew. Chem., Int. Ed. Engl.
1998, 37, 2796.
9. Egholm, M.; Buchardt, O.; Christensen, L.; Behrens, C.;