Synthesis of cholesterol-polyamine carbamates: PKa studies and condensation of calf thymus DNA

Article abstract of DOI:10.1039/a803284j

Novel cholesterol-polyamine carbamates have been prepared and their pK_as determined potentiometrically for conjugates substituted with up to five amino functional groups and the binding affinity for calf thymus DNA has also been determined; these polyamine carbamates are models for lipoplex formation with respect to gene delivery (lipofection), a key first step in gene therapy.

Full text of DOI:10.1039/a803284j

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Synthesis of cholesterol-polyamine carbamates: pKa studies and condensation
of calf thymus DNA
Andrew J. Geall,a Richard J. Taylor,b Mark E. Earll,b Michael A. W. Eatonb and Ian S. Blagbrougha*†
a Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath, UK BA2 7AY
b Celltech Therapeutics Ltd., Bath Road, Slough, UK SL1 4EN
H
Novel cholesterol-polyamine carbamates have been pre-
N
NH2
H2N
N
H
pared and their pKas determined potentiometrically for
conjugates substituted with up to five amino functional
groups and the binding affinity for calf thymus DNA has
also been determined; these polyamine carbamates are
models for lipoplex formation with respect to gene delivery
(lipofection), a key first step in gene therapy.
Spermine (3.4.3) 1
H
R
R
N
N
NR1
RN
H
N
R
2 R = H, R1 = H
3 R = H, R1 = COCF3
4 R = Boc, R1 = COCF3
5 R = Boc, R1 = H
Polyamines such as tetra-amine spermine 1 are widely distrib-
uted in nature and display a variety of important biological
activities.1,2 Polyamines affect DNA replication and translation,
protein synthesis, membrane stabilization and the activity of
certain kinases and topoisomerases. Stabilization of specific
DNA conformations and charge neutralisation of intracellular
polyanions (e.g. DNA and RNA) may be among the most
important physiological roles of polyamines. Their binding has
a profound effect on DNA structure, causing transitions from B
to both A and Z forms, and at higher concentrations conforma-
tional changes, e.g. aggregation and condensation.2,3 Condensa-
tion is caused by alleviation of charge repulsion between
neighbouring phosphates on the DNA helix allowing collapse
into a more compact structure.3 Polyamine mediated condensa-
tion is a rapidly expanding area of research for non-viral vectors
in gene therapy (lipoplexes in lipofection).4
O
H2N
N
H
N
H
N
H
O
n
m
n
6 m = 2, n = 1
7 m = 1, n = 1
8 m = 0, n = 1
9 m = 1, n = 0
O
H
N
H
N
H2N
N
H
N
H
O
The application of synthetic lipo-polyamines constitutes a
safer and more efficient gene transfer strategy which, unlike the
use of viral vectors, does not elicit immune responses.5 Within
the prerequisites for delivery of DNA across an intact
cytoplasmic membrane are condensation and masking the
negative charge of the phosphate backbone; polyammonium
ions are therefore suitable for use as gene delivery systems.6,7
Covalent attachment of a lipid moiety,8 such as an aliphatic
chain2,7,9 or a steroid,6,10,11 further enhances polyamine-
mediated DNA condensation.12 The mechanism by which these
compounds cause lipofection is poorly understood. Knowledge
about the pKas, especially accurate prediction along a poly-
amine (polyammonium) chain, will allow protonation states at
physiological pH to be determined and therefore DNA binding
affinities can be predicted with greater confidence. Such
physicochemical properties are important in the design of
lipoplexes for efficient lipofection.
Herein we report the design and synthesis of polyamine
carbamates of cholesterol (at position 3), using our orthogonal
protection stragegy for efficient syntheses of unsymmetrical
polyamine amides.13 Six compounds have been made using
polyamines: 1,12-diamino-4,9-diazadodecane 1 (spermine,
3.4.3), 1,11-diamino-4,8-diazaundecane (thermine, norsper-
mine, 3.3.3), 1,10-diamino-4,7-diazadecane (3.2.3), 1,9-diam-
ino-3,7-diazanonane (2.3.2), tetraethylenepentamine 2 (2.2.2.2)
and pentaethylenehexamine (2.2.2.2.2) affording 6–11 re-
spectively. Our protocol for the synthesis of carbamate 10 is
outlined.‡ The pKa values of these compounds were then
measured using a Sirius PCA101 automated pKa titrator, in 0.15
m KCl ionic strength adjusted water; values obtained for
spermine (3.4.3) 1 are comparable with literature values
determined both potentiometrically and spectroscopi-
cally.14–16
n
10 n = 1
11 n = 2
The DNA binding affinities for this series of polyamine
conjugates 6–11 were determined using calf thymus DNA (6
mg, [DNA base-pair] = 3.0 mm),§ and a fluorescence quenching
assay based upon exclusion of ethidium bromide which is
effectively present in excess (1.3 mm).11,12 The binding
affinities are critically compared as both the charge ratio4 and
concentration at which 50% of the ethidium bromide fluores-
cence was quenched (in 20 mm NaCl, see Table 1). These data
give support to our hypothesis that binding is a function of
charge and that the regiochemical distribution of such charges is
also significant for DNA affinity; conjugates 3.4.3 6, 3.3.3 7,
3.2.3 8 and 2.3.2 9 show this trend. Likewise, polyethylene
imine conjugates 10 and 11 were (respectively) weaker with
only 2.0 charges at pH 7.4 and stronger wtih 2.3 charges
distributed along the polyamine. Carbamates 7 and 11 have
comparable pKas across the first three protonation sites, but
their structural differences are reflected in their DNA binding
affinities (Table 1). These subtle differences in DNA conden-
sation as a function of charge distribution are clearly important
for lipoplex formation when compared with the higher charge
on unconjugated spermine (3.8 at pH 7.4).
In a recent, comprehensive paper on the role of charge in
polyamine analogue recognition, Bergeron et al. demonstrated
that small structural alterations resulted in substantial differ-
ences in biological activities.15 pKas are a function of the inter-
amine distance as well as their substituents. It is important to
recognise that any charge is shared across several of the basic
Chem. Commun., 1998
1403

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Table 1 Polyamine pKas and ethidium bromide exclusion data
then cleaved by increasing the pH to 11 with conc. aq. ammonia, stirring
(25 °C, 15 h) to afford, after flash chromatography over silica gel (CH2Cl2–
MeOH–conc. aq. NH4OH 200:10:1 to 150:10:1 v/v/v), tetra-Boc
protected polyamine 5 (19%). Reaction of the free primary amine of 5 with
3-cholesteryl chloroformate (1.2 equiv., 3.0 equiv. TEA, CH2Cl2, 0 °C for
10 min then to 25 °C for 12 h) afforded, after purification over silica gel
(EtOAc–hexane 8:2 to 6:4 v/v) fully protected carbamate 10 (81%).
Deprotection (CH2Cl2–TFA 10:90 v/v, 0 °C, 2 h) and purification by RP-
HPLC over ABZ + Plus (5 mm, Supelcosil) (MeCN–0.1% aq. TFA 1:1 v/v,
l = 220 nm) afforded the polytrifluoroacetate salt of polyamine carbamate
10 (50%) HR-FABMS (+ve ion in m-NBA) [Found 602.5380 (M + 1).
Polyamine Measured pKas Net chargea Charge ratiob Conc./mmc
1
10.9 ± 0.01
10.1 ± 0.01
8.9 ± 0.01
8.1 ± 0.01
10.1 ± 0.06
8.6 ± 0.06
7.3 ± 0.05
10.7 ± 0.04
8.8 ± 0.02
7.2 ± 0.02
10.0 ± 0.02
8.0 ± 0.02
5.5 ± 0.02
9.3 ± 0.01
7.6 ± 0.01
5.7 ± 0.01
9.9 ± 0.20
8.4 ± 0.20
6.3 ± 0.21
3.9 ± 0.21
10.2 ± 0.10
8.6 ± 0.08
7.2 ± 0.09
4.4 ± 0.09
2.5 ± 0.28
3.8
> 4.0
> 17.0
6
2.4
2.3
1.8
1.6
2.0
0.62
0.76
0.80
0.88
0.92
1.3
1.6
1.7
2.4
2.7
7
C36H68N5O2 requires 602.5380].
§ Using the literature average weight per nucleotide of 330 Da.4
8
1 For selected reviews on polyamines, see B. Ganem, Acc. Chem. Res.,
1982, 15, 290; R. J. Bergeron, Acc. Chem. Res., 1986, 19, 105;
I. S. Blagbrough, S. Carrington and A. J. Geall, Pharm. Sci., 1997, 3,
223 and refs. cited therein.
2 J.-P. Behr, Tetrahedron Lett., 1986, 27, 5861; J.-P. Behr, Acc. Chem.
Res., 1993, 26, 274; N. Schmid and J.-P. Behr, Tetrahedron Lett., 1995,
36, 1447 and refs. cited therein.
3 S. C. Tam and R. J. P. Williams, Struct. Bonding, 1985, 63, 103;
E. Rowatt and R. J. P. Williams, J. Inorg. Biochem., 1992, 46, 87;
K. D. Stewart and T. A. Gray, J. Phys. Org. Chem., 1992, 5, 461;
V. A. Bloomfield, Curr. Opin. Struct. Biol., 1996, 6, 334.
4 P. L. Felgner, Y. Barenholz, J. P. Behr, S. H. Cheng, P. Cullis, L. Huang,
J. A. Jessee, L. Seymour, F. Szoka, A. R. Thierry, E. Wagner and G. Wu,
Hum. Gene Ther., 1997, 8, 511.
5 R. G. Crystal, Science, 1995, 270, 404; P. L. Felgner, Sci. Am., 1997,
276, 86; C. O’Driscoll, Chem. Brit., 1997, 33, 66; R. I. Mahato,
A. Rolland and E. Tomlinson, Pharm. Res., 1997, 14, 853; I. M. Verma
and N. Somia, Nature, 1997, 389, 239.
9
10
11
2.3
0.66
1.3
a Net positive charge calculated from the Henderson–Hasselbach equation
at pH 7.4. b Charge ratio4 at which 50% exclusion of ethidium bromide is
c
effected using calf thymus DNA at pH 7.4. Concentration of polyamine
6 E. R. Lee, J. Marshall, C. S. Siegel, C. Jiang, N. S. Yew, M. R. Nichols,
J. B. Nietupski, R. J. Ziegler, M. B. Lane, K. X. Wang, N. C. Wan,
R. K. Scheule, D. J. Harris, A. E. Smith and S. H. Cheng, Hum. Gene
Ther., 1996, 7, 1701; R. G. Cooper, C. J. Etheridge, L. Stewart,
J. Marshall, S. Rudginsky, S. H. Cheng and A. D. Miller, Chem. Eur. J.,
1988, 4, 137.
conjugate at which 50% exclusion of ethidium bromide (1.3 mm) is effected
using calf thymus DNA (3.0 mm) at pH 7.4.
centres and that it cannot be attributed to a single point. Even
when the first charge is introduced principally on the primary
amine, it is also distributed on to the secondary amines. This has
been shown using unsymmetrical triamine, spermidine.17
The four methylene central spacer found in spermine 1 has
also been shown to be important for binding affinity, confirming
that both the number of positive charges and their distribution
has a profound effect on the polyamine’s ability to induce DNA
conformational changes.18 The measured pKas for polyamines
containing aminopropyl16 and aminoethyl10 units and Trans-
fectam (DOGS)7,19 add further weight to this hypothesis. These
results will be of use in gene therapy studies and should find
ready application in the design of lipoplexes with particular
reference to spermidine and spermine class alkaloids. This
evaluation of pKa data, the number and regiochemical distribu-
tion of charges along the polyamine backbone, may lead to a
clearer understanding of lipoplex modes of action.
7 J.-P. Behr, B. Demeneix, J.-P. Loeffler and J. Perez-Mutul, Proc. Natl.
Acad. Sci. USA, 1989, 86, 6982.
8 I. S. Blagbrough, S. Taylor, M. L. Carpenter, V. Novoselskiy,
T. Shamma and I. S. Haworth, Chem. Commun., 1998, 929.
9 G. Byk, C. Dubertret, V. Escriou, M. Frederic, G. Jaslin, R. Rangara,
B. Pitard, J. Crouzet, P. Wils, B. Schwartz and D. Scherman, J. Med.
Chem., 1998, 41, 224.
10 J. K. Guy–Caffey, V. Bodepudi, J. S. Bishop, K. Jayaraman and
N. Chaudhary, J. Biol. Chem., 1995, 270, 31391; D. Moradpour,
J. I. Schauer, V. R. Zurawski, Jr., J. R. Wands and R. H. Boutin,
Biochem. Biophys. Res. Commun., 1996, 221, 82; S. Walker, M. J. Sofia,
R. Kakarla, N. A. Kogan, L. Wierichs, C. B. Longley, K. Bruker,
H. R. Axelrod, S. Midha, S. Babu and D. Kahne, Proc. Natl. Acad. Sci.
USA, 1996, 93, 1585.
11 H.-P. Hsieh, J. G. Muller and C. J. Burrows, J. Am. Chem. Soc., 1994,
116, 12077.
12 A. J. Geall and I. S. Blagbrough, Tetrahedron Lett., 1998, 39, 443.
13 I. S. Blagbrough and A. J. Geall, Tetrahedron Lett., 1998, 39, 439.
14 G. Anderegg and P. Bla¨uenstein, Helv. Chim. Acta, 1982, 65, 162.
15 R. J. Bergeron, J. S. McManis, W. R. Weimar, K. M. Schreier, F. Gao,
Q. Wu, J. Ortiz-Ocasio, G. R. Luchetta, C. Porter and J. R. T. Vinson,
J. Med. Chem., 1995, 38, 2278.
16 Y. Takeda, K. Samejima, K. Nagano, M. Watanabe, H. Sugeta and
Y. Kyogoku, Eur. J. Biochem., 1983, 130, 383; D. Aikens, S. Bunce,
F. Onasch, R. Parker, III, C. Hurwitz and S. Clemans, Biophys. Chem.,
1993, 17, 67.
17 M. M. Kimberly and J. H. Goldstein, Anal. Chem., 1981, 53, 789; D. A.
Aikens, S. C. Bunce, O. F. Onasch, H. M. Schwartz and C. Hurwitz,
J. Chem. Soc., Chem. Commun., 1983, 42.
18 H. S. Basu, H. C. A. Schwietert, B. G. Feuerstein and L. J. Marton,
Biochem. J., 1990, 269, 329.
We thank the EPSRC and Celltech Therapeutics Ltd, for a
CASE studentship (to A. J. G.). We acknowledge some
preliminary experimental work of Ms Dima Al-Hadithi (Uni-
versity of Bath) and useful discussions with Dr Ian S. Haworth
(University of Southern California). I. S. B. and I. S. H. are
recipients of a NATO grant (CRG 970290).
Notes and References
† E-mail: prsisb@bath.ac.uk
‡ An important first step is the ready purification of technical grade ( ~ 80%)
2.2.2.2-pentamine 2 by selective protection of one primary amino functional
group by reaction with ethyl trifluoroacetate (1.0 equiv., MeOH, 278 °C for
1 h then to 0 °C over 1 h) to form trifluoroacetamide 3. Immediately, in this
solution, the remaining four amino functional groups were Boc protected
with di-tert-butyl dicarbonate (5 equiv., 0–25 °C over 1 h then 14 h) to
afford fully protected polyamine 4. The trifluoroacetyl protecting group was
19 J.-S. Remy, C. Sirlin, P. Vierling and J.-P. Behr, Bioconjugate Chem.,
1994, 5, 647.
Received in Glasgow, UK, 30th April 1998; 8/03284J
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Chem. Commun., 1998