Investigations into the synthesis o f the lipid components o f the LPD vector were
initiated with the synthesis of DOTMA. DOTMA is not commercially available,
although it is available in premixed liposome formulations, for example Lipofectin®,’^^ conversely, DOPE is commercially available. Our initial aim was to establish the influence and importance o f DOTMA and DOPE on the transfection process and evaluate the effect o f the DOTMA counter ion.
A direct route to DOTMA has been reported by Feigner et. al. which involved
the dietherification o f 3-dimethylamino-1,2-propanediol 42, using activated octadec-9- en-l-ol (oleyl alcohol) as the tosylate, and subsequent quaternisation with
chloromethane to yield DOTMA (see Scheme 1.3.1).^^ This methodology was
originally investigated, with the intention o f optimising the synthetic route, to provide a reliable route with analogue synthesis in mind. Interestingly, Feigner and co-workers
used oleyl alcohol which is commercially available in a cis.trans ratio of approximately:
85:15.'”
2.1.1 Synthesis o f Activated Octadec-Ç-en-l-ol Derivatives
A variety of activated (Z)-octadec-9-enyl derivatives were synthesised to explore
the synthesis o f 43. (Scheme 2.1.1) The reported synthesis of DOTMA utilised octadec-
9-en-l-ol 109 activated as the tosylate.^"^ Tosylation o f octadec-9-en-l-ol to give the p-
toluenesulfonyl derivative 110, was readily achieved in a 73% yield, using p-
toluenesulfonyl chloride and triethylamine.^"* The preparation of the bromoalkane 111, was achieved in 95% yield, using triphenylphosphine and carbon tetrabromide."^^'^^^ The methanesulfonic acid derivative 112 was prepared in 85% yield using methanesulfonyl chloride and triethylamine.*^'^
2 DOTMA Lipid Analogues___________________________________________ Results and Discussion 112 MsCI, TEA, DCM, r.t. TsCI, TEA, DCM, r.t. 110 - ________________ R-OH R -O ^ CBr., PPh,, DCM. r.t. R -B r 111 R =
Scheme 2.1.1 Activated oleyl alcohol derivatives.
2,L2 Synthesis o f 3-Dimethylamino-l,2-Propanediol Derivatives
The synthesis o f the dialkylated compound 43 was initially attempted following the conditions reported by F e i g n e r H o w e v e r , when using the tosylate 110 and
potassium tert-h\xXox\&Q with the diol 42 at reflux in anhydrous xylenes, no
dietherification products were detected by either NMR spectroscopy or MS analysis
(Scheme 2.1.2 & Table 2.1.1). The use o f dry toluene as a solvent was explored and the reaction time was increased fi*om 3 hours to 24 hours, but again no dietherification product was detected (Entry 2). The use o f alkyl halides in éthérification procedures during lipid production have been reported previously.^^ The bromo activated derivative 111 was therefore used with potassium rerr-butoxide as base, but this failed to generate the desired ether (Entry 3). In general the use o f bromoalkanes could also lead to the formation o f A^-alkylation products, although here this was not observed. The use of potassium /err-butoxide was questioned due to its apparent poor solubility in either toluene or xylenes, despite Feigner and co-workers’ report, and hence the base
employed was changed. When using sodium hydride in
tetrahydrofiiran/dimethylformamide (5:1), the desired derivative 43 was successfully formed in a low yield o f 25% (Entry 4). The use o f DMF or THF has previously been reported for the successful production of long chain ethers during lipid synthesis.^* In an attempt to optimise the reaction conditions, the preparation o f 43 was repeated using DMF as solvent and heating to 80 °C, but the yield decreased to 14% (Entry 5). Substitution o f the tosylate derivative 110, for the mesylate derivative 112, enhanced the yield to 35% (Entry 6). The yield of 43 was increased further (45%) by the addition of 4 equivalents o f the mesylate (Entry 7).
2 DOTMA Lipid Analogues Results and Discussion RX OH See Table 2.1.1 OR 42 43
Scheme 2.1.2 Alkylation o f 3-dimethylamino-propan-1,2-diol 42.
E N T R Y R X (e q ) Base (eq) Solvent D uration (h) Y ield (% )
1 TsO K ‘BuO xylene 3 0
2 TsO K 'BuO toluene 24 0
3 Br K 'BuO Toluene 24 0
4 TsO NaH THF/DMF 72 25
5 TsO NaH DMF 24 14
6 MsO (3eq) NaH (3 eq) THF 72 35
7 MsO (4eq) NaH (3eq) THF 72 45
Table 2.1.1 Alkylation of 3-dimethylamino-propan-l,2-diol 42.
With the dietherified derivative 43 in hand, this was then successfully converted to the quaternary amine 113 by treating with an excess o f iodomethane in 83% yield. 'H NMR spectroscopy and ESMS analyses were consistent with product formation and the overall synthesis of 113 is summarised in Scheme 2.1.3.
/N
JC U
MsOR (113), NaH, THF Reflux, 72h, 45% 42 43 Iodomethane, r.t, 6h 83% 113S ch em e 2.1.3 Summary of the synthesis of DOTMA 1 113.
Notably, throughout the synthesis these series o f compounds, the amine and quaternary salt were readily detected using electrospray mass spectrometry (ESMS) or
2 DOTMA Lipid Analogues Results and Discussion
E N T R Y Product 3 = or 4 = 8h (NCfla) (ppm ) 5c (NCH3) (ppm ) m /z (+ES)
1 43 3 ° 2.29 46.72 620.7 (MH+)
2 113 4 ° 3.52 55.64 634.6 ({M-I}^)
Table 2.1.2 Spectroscopic properties of compounds 43 and 113.
The chloride salt o f DOTMA 7 was obtained by treating the iodo derivative 113 with an Amberlite® IRA-400 (Cl) ion exchange resin, to obtain 7 in 76% yield (Scheme 2.1.4). The exchange of the counterion was confirmed by elemental analysis. A comparison o f the spectral data cannot be made with the DOTMA synthesised by Feigner and co-workers, since none was published.^^ Direct quaternisation of 43 with chloromethane rather than iodomethane was not attempted due to the high pressures and temperatures required.
113
Amberlite IRA-400 (01) Ion Exchange Resin
Scheme 2.1.4 Synthesis of DOTMA 7.
2 .1 3 Biological Data fo r DOTMA Salts
Transfection studies focused on two main aspects: firstly the influence of the differing counter-ions. In some systems, counterion effects have been reported and whether this was an important consideration needed to be established in our system.^^’’ Secondly, the requirements for the co-lipid DOPE needed to be assessed.
Testing was carried out following the methodology o f Hart and co-workers. Human airway epithelial (HAEo) cell lines were investigated for transfection with the standard and modified LPD vector formulations, throughout this thesis. Standard LPD vector formulations comprised o f Peptide 2 ([K]i6GACRRETAWACG), DNA plasmid