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| Abstract: | |
| Lipases
[E.C.3.1.1.3] are ubiquitous in nature where their role in vivo is the
enzymatic hydrolysis of triglycerides. In these transformations lipases
display a high degree of regio- and enantioselectivity, properties which
are of great interest for the use of these enzymes in vitro. The experimental
determination of these selectivities is somewhat problematic due to the
fact that the primary products of hydrolysis are notoriously instable due
to rapid acyl group migrations especially under protic conditions and at
elevated temperatures. These are also substrates of lipases and therefore
their optical purity can be strongly conversion dependent. Thus, one prerequisite
for the successful determination of the enantioselectivities are extremly
small conversions (<2-6%) and a rapid derivatisation of the primary
reaction products prior to analysis. Moreover, the method is only of analytical
value, the resulting products are not suitable for further synthetic use
such as for the preparation of phospholipids. In view of this situation
we felt that isosteric mimics of natural lipids would be an attractive
alternative for a system and medium independent determination of these
important enzyme parameters, as well as for the synthesis of novel carba-analogues
of lipids and phospholipids. The systematic replacement of sp3-oxygens
by sp3-carbon atoms in triglycerides leads to such mimics with minimal
deviation of bond angles and bond distances. Their behaviour should be
identically - with the exception of hydrolytic cleavage in the modified
position - towards biological systems. In order to test this hypothesis
synthetic routes for the synthesis of carba- analogues of triglycerides
were developed. Carba-analogues of tricaprylin 11, trilaurin 25 and tripalmitin
26 with modification of the sn-2 position were prepared starting from the
corresponding fatty acid chlorides as well as racemic carba-analogues of
triacetin 39 and trilaurin 40 with modification of the sn-1(3) position.
The synthesis of triglycerides modified in the sn-1,2(2,3) positions -
like the triacetin analogues (?)-56 and (S)-56 - were realised via Diels-Alder
reaction of 2,3-dimethylbutadiene with the acrylic acid esters of D-pantolactone
42 as the key reaction step. This cycloaddition with 42 in presence of
catalytic amounts of titaniumtetrachloride gives the cyclohexene derivative
50 with very high diastereoselectivity. Reduction with LiAlH4 to the alcohol
(S)-53 (? 99% ee) proceeds without racemisation and acetylation followed
by oxidative cleavage of the C=C-double bound gives the triacetin-analogue
(S)-56. One triglyceride-analogue modified in the sn-1,3-positions 64 was
synthesized starting from levulinic acid ethyl ester. This general approach
also allows the synthesis of novel carba-analogous phospholipids of high
enantiomeric purity with a) modification of the sn-2 position such as (S)-79
and b) modification of the sn-1,2(2,3) positions like (S)-82. Such analogues
of phospholipids could have interesting biological activities, e.g. as
inhibitors of phospholipase A2. Using the above carba-analogues of triglycerides
their activities and enantioselectivities towards 44 commercially available
lipases was studied in comparison with the corresponding triglycerides.
The results - obtained with tricaprylin and its carba-analogue 11 (modification
in the sn-2 position) - showed considerable differences between these two
substrates towards the studied lipases both regarding their specific activities
and enantioselectivities.
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