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Chain-Substituted Lipids as Substrates for Phospholipase A2.

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[I] W Kaminsky, K. Kiilper, H. H. Brintzinger, E R. W. P. Wild, Angew.
Chem. 97 (1985) 507; Angew. Chem. Int. Ed. Engl. 24 (1985) 507.
[2] J. A. Ewen, J1 Am. Chem. Soc. 106 (1984) 6355.
131 W. Kaminsky, Angew. Mukromol. Chem. 1451144 (1986) 149.
[4] P. Pino, Adv. Polym. Sci. 4 (1966) 393.
[S] P. Pino, P. Cioni, J. Wei, 1 Am. Chem. Soc. 109 (1987) 6189.
161 P. Pino, P. Cioni, M. Galimberti, J. Wei, N. Piccolrovazzi in W. Kaminsky,
H . Sinn, (Eds.): Transition Metals and OrgunomeraNics us Cuta&srs for
Olefin Polymerrzarion,Springer. Berlin 1988, p. 269.
[7] A. Schafer, E. Karl, L. Zsolnai, G. Huttner, H. H. Brintzinger, J Orgunom a . Chem. 328 (1987) 87.
[8] W. Kaminsky, H. Hahnsen, Dtsch. Pat. Appl., P 32403836, procedure for
the preparation of oligomeric alumoxanes (1982). Hoechst AG; Chem.
Abslr. 101 (1984) 73242~.
[9] R. J. Abraham, P. Loftus: Proton and Carbon-f3 NMRSpectroscopy, Heyden&Son, London 1978, p. 18.
1101 a) W. A. Konig, Nachr. Chem. Tech. Lab. 37 (1989) 471; b) W. A. Konig,
S. Lutz, G. Wenz, Angew. Chem. 100 (1988) 989; Angew. Chem. Int. Ed.
Engl. 27 (1988) 979.
[ l l ] D. E. Dorman, M. Jautelat, J. D. Roberts, 1 Org. Chem. 34 (1971) 2757.
[12] L. P. Lindeman, J. Q. M a m s , Anal. Chem. 43 (1971) 1245.
1131 K. K. Mayer, C. Djerassi, Orx. Muss Specfrom. 5 (1971) 817.
(1 mg Crotalus adamanteusf”’ in 30 pL of 5 mM CaCI,). After 24 hours of magnetic stirring and TLC monitoring, the
reactions were quenched with 5 mL of water, after which the
water layer was separated, washed with ether, and lyophoIized. Reaction yields were determined by one of two methods: (1) Integrated ‘H NMR signals of the a-methylene and
choline methyl groups were compared. (2) Lysophosphatidylcholine was isolated by column chromatography (silica;
8:2 CH,OH/CHCI, as eluant).
As seen in Table 1, a yield of 97% was obtained with the
parent DSPC 1. On the other hand, n-propyl and n-butyl
Table 1. Yields and rates of phospholipase-A,-catalyzed hydrolyses of 1 and its
derivatives.
Substituent
By Fredric M . Menger” and M . G. Wood, Jr.
Recent monolayer,[’] calorimetric,f21 mass spectromet’I 3C NMR,f6Iand transport [’I studies
r i ~ , [biochemical,[4.
~]
of new lipid systems required the synthesis of “lysophospholipid” intermediates via phospholipase-A,-catalyzed hydrolyses [Eq. (a)]. In the course of our work, we found that the
’
CH,OCOR
I
CHOCOR
phospholipase A,
I
CH,OCOR
I
CHOH
I
CH,-
CH,-
RCOX
CH,OCOR
I
CHOCOR‘
I
(a)
C H , e
phospholipase gave no isolable products with phospholipids
bearing certain alkyl groups on the first half of their C,,
chains. This observation inspired further investigation owing
to the possibility that the chain-substituted lipids, like related short-chain lecithins,[81inhibit normal phospholipase-A,
activity. Developing such inhibitors is a major goal of current chemopharmacological researchfg- 1 3 ] because phospholipase A, catalyzes the release of arachidonic acid (a
precursor for prostaglandins, thromboxanes, and leukotrienes). Hydrolysis-resistant phospholipids would also enable construction of drug-carrying liposomes that are better
able to withstand metabolic degradation.
Table 1 lists the synthetic distearoylphosphatidylcholine
(DSPC, 1) derivatives examined with phospholipase A,.
CH,-OCOC, 7H35
I
CH-OCOC, ,HS5
I
B
CH,-OP(O)OCH,CH,N(CH,),
1
I
Oe
In a typical preparative run,f141lipid (40 pmol in 3.3 mL of
1 :9 methanol/ether) was mixed with crude snake venom
[*] Prof. E M. Menger, M. G. Wood, Jr.
[**I
Department of Chemistry, Emory University
Atlanta, GA 30322 (USA)
This work was supported by the National Institutes of Health
1218
0 VCH
Yer[agsgesellschafimbH, 0-6940 Weinheim, 1989
Location [a]
Yield
[%I
~
Chain-Substituted Lipids as Substrates
for Phospholipase A,**
Chain(s)
Methyl
Methyl
Methyl
Methyl
Methyl
Methyl
Methyl
Ethyl
Ethyl
Ethyl
Ethyl
Ethyl
n-Propyl
n-Propyl
n-Propyl
n-Propyl
n-Butyl
n-Butyl
n-Butyl
n-Butyl
n-Butyl
n-Butyl
n-Butyl
Phenyl
Phenyl
~
1.2
2
1.2
2
1.2
2
12
1.2
1.2
2
12
2
1.2
2
1.2
2
1,2
2
1.2
2
1,2
2
1-2
12
12
4
4
6
6
8
8
12
4
6
6
8
8
6
6
8
8
4
4
6
6
8
8
12
8
12
[b]
97*
95*
87
90
92
91*
100
85’
5
7
15
14
15
0
0
0
0
0
0
0
0
0
0
34*
-
Relative
initial rate v , , ~ [c]
~,
100
12
20
23
48
45
100
100
3
3
13
5
18
4
14
11
29
<0.3
3
10.3
9
2
9
42
2
50
~
[a] Numbering system counts the cdrbonyl carbon of the fatty acid as C-1.
Either both chains (“1.2”) or only the second chain (“2”) bears a substituent at
the indicated location. [b] The percentages marked with a n asterisk reflect the
isolated yields after column chromatograpy. All other yields were determined
in duplicate by ‘H N M R spectroscopy. [c] Rates were measured using 5.9 mM
lipid substrate. Reaction conditions were different from those used to determine
yields (see text). V,,, (I) = 420 pmol mg-’ min-’.
branching at positions 4,6, and 8 gave 0 % yields regardless
of whether both chains (“1,2”) or only the second chain
(“2”) was substituted. An n-butyl at a more distal locus
(C-12) did not result in 0 % yield but instead reduced it to
34%. Methyl groups exerted little effect on the yield (85100%) even when they were situated close to the hydrolytic
site. Yields tumbled to 5-15% with ethyl groups on carbons 4, 6, and 8. In summary, phospholipid hydrolyses are
impaired by alkyl substituents, particularly large ones, located in the first half of the chains.
Initial rates of phospholipase-A,-catalyzed hydrolyses
(Table 1) were all measured under identical conditions:
6.0 mL of an aqueous mixture composed of 5.9 mM lipid,
20 mM Triton X-100, and 10 mM CaCI,, bath-sonicated at
65 “C to produce a mixed-micellar system.112.161Subsequent
hydrolysis, initiated with 0.2 units of purified phospholipase
from Crotaius adamanteus venomf171(1 67 units per mg), was
measured with a Radiometer pH-stat at 40.0 “C by titrating
at pH = 8.0 under N, with 2 or 5 mM KOH.
0S70-0X33~X9j0909-12rR$02.S0/0
Angew. Chem. Int. Ed. Engl. 28 (1989) Nr. 9
Relative rates Y , , in~ Table
~ ~
1 parallel roughly the reaction
yields (an exact correspondence not being expected since
conditions for obtaining the two parameters were entirely
different). The following conclusions can be drawn from the
Y
~ data:
,
~ ( 1 ) ~ The
~ v , . values
~ ~ ~ increase steadily as the methyl
groups are shifted away from the carbonyl group (e.g.,
v,, = 12, 23, 45, and 100 for bis-methylation of carbons 4,
6, 8, and 12). (2) Methyl groups on both chains produce
about a twofold lower vi, rel than identically positioned
methyl groups on chain-2 alone. Thus, substitution need not
occur on the labile fatty acid to affect the rate. (3) Ethyl and
n-propyl groups reduce vi, rel from 3- to 30-fold depending on
the proximity to the hydrolytic site. Interestingly, a single
n-propyl group on C-8 of chain-2 gave a 0 % yield with an
ether solvent system but only a threefold rate decrease with
the lipid incorporated into aqueous micelles. (4) n-Butyl
groups induce dramatic rate decreases: DSPC bearing two
n-butyl groups on C-4 hydrolyzes more slowly than DSPC 1
by a factor of 390 (a value that could be only approximated
owing to the extreme sluggishness of the former reaction).
When the butyl groups were moved to C-12, the inhibition
almost disappeared. As seen from Table 1, the effect of
phenyl groups is similar to that of n-butyl groups.
Conventional enzyme kinetics revealed details about the
n-butyl group inhibition. Thus, micellar 1 gave K,,, and V,,,
values of 3.8 mM and 420 pmol mg-I min-' (in satisfactory
agreement with related studies on egg phosphatidylchoThe 4,4'-dibutyl derivative was shown to behave as
a competitive inhibitor with Ki = 2.0 mM (Fig. 1). n-Butyla-
[I] F. M. Menger. M. G. Wood. Jr.. S. D. Richardson, Q.Zhou. A. R. Elrington. M. J. Sherrod, J. Am. Chem. SOC.110 (1988) 6797.
[2] F. M. Menger. M. G. Wood, Jr., Q . 2. Zhou. H. P. Hopkins. J. Fumero. J.
,4177.Chem. Soc. 110 (1988) 6804.
[3] M . J. I. Mattina, S. D. Richardson, M. G. Wood, Jr.. Q. 2. Zhou, M. J.
Spcctrom. 23 (1988) 292.
Contado. F. M. Menger. L. E. Abbey, Org. MUJS.
(41 Q. Zhou. R. L. Raynor, M . G.Wood. Jr.. F. M. Menger. J F. Kuo. 5iochemrstrj 27 (1988) 7361.
[5] P. A. Charp, Q . Zhou. M. G. Wood. Jr., R.L. Raynor. F. M. Menger. J. F.
Kuo. 510chrmi.srr~
27 (1988) 4607.
[6] E M. Menger. P. Aikens. M. Wood, Jr.. J. Chivn. Soc. C h m . Comnrrm.
1988, 180.
[7] P. Aikens. J. J. Lee, R. Persichetti. unpublished results.
[XI W. G. J. Hol, A n g w . Cl7em. 98 (1986) 765; Angew. C'hrm. I n / . GI Engl. 25
(1986) 767.
[9] N . S. Chandrzkumar, J. Hajdu, J. Org. t h e m . 47 (1982) 2144.
[lo] L. J. Reynolds, B. P. Morgan, G. A. Hite, E. D. Mihelich, E. A. Dennis, J.
Am. them. SO(..1 / 0 (1988) 5172.
[ I l l M. H. Gelb. J. Am. L'hem. SOC./OH (1986) 3146.
[12] W. Yuan, R. J. Berman. M. H.Gelb, J. Am. Chrm. Soc. l O Y (1987) 8071
[13] C . D. De Bose. R. A. Burns, J. M. Donovan, M. F. Roberts. B/ochrini.\tr$
24 (1985) 1298.
1141 R.L. Misiorowski, M. A. Wells, Bio~hcn?i.stryI S (1974) 4921.
[15] Sigma catalogue No. V6X75.
1161 R. A. Deems, E. A. Dennis, Merhorls En:jmo/. 71 (1981) 703.
[17] Sigma catalogue No. PO790 (EC 3.1.1.4).
(181 C. R. Kensil. E. A. Denis, J. Bid. Chrm. 254 (1979) 5843.
[I91 We cannot exclude the possibility that alkylated phosphatidylcholine modifies the micellar system, although this appears unlikely since a large excess
of neutral surfactant, Triton X-100. was employed.
[20] Protein kinase C activation and inhibition by our lipid derivatives [4, 51
must be taken into consideration here.
Phosphonioboratoacetylenes: C, Stabilized
by Donor and Acceptor Molecules
By Hans Jiirgen Bestmann,* Harald Behl,
and Matthias Bremer
Dedicated to Professor Ernst Ruch on the occasion
of'his 70th birthday
Hexaphenylcarbodiphosphorane 1 ['I can be formally regarded as a "complex" consisting of two donor molecules
and an electron-rich, excited carbon atom.['] Such a purely
hypothetical formulation raises the question whether the C,
molecule131can be stabilized by complexation. In principle,
.i,
e
H,P-CGC-BH,
-
15
150
f Is1
Fig. 1. C'ompetitive inhibition of the hydrolysis of 1 by 4,4'-di-n-butyl-DSPC
(pH-stat output: pH = 8.0, 40.0 'C). c = added equivalents of KOH. A :
0.84 mM DSPC 1, no inhibitor. B: 1 + 1.0 mM inhibitor. c: 1 + 3.0 mM inhibitor. D: 5.9 mM 4.4-di-n-butyl-DSPC alone.
tion of the fatty acid chains, therefore, does not impair binding to the active site but instead severely impedes subsequent
hydrolysis." 91
On the basis of our results, chain modification of phospholipids would seem to constitute a promising method of
altering lipid susceptibility to phospholipase A , . The potential for thereby controlling prostaglandin levels, and for designing more stable drug delivery systems, should again be
mentioned .1201
Received. February 20. 1989 [Z 3182 IE]
German version: Angew. Chcm. 101 (1989) 1277
2
1
this should be possible by coordination of a donor molecule
to one C atom and an acceptor molecule to the other C atom.
For our investigations, we chose a phosphane as donor and
a borane as acceptor. Compound 2 was used in ab initio
calculations as the simplest example of this as yet unknown
molecule with betaine structure. The following synthetic
route led to the target molecules 9 (Scheme 1):
Ph,P-CEC-H
C,H,Li 4
THF.
20 C
BR, 6
Ph,P-C-C-Li
3
E1,O'THF.
20 C
>
5
CH,I 8
Ph,P-C'-C-ER,Li
7
Scheme 1. Synthesis of 9,6-9: a, R
0
E t 2 0 THF. 0 C
=
Ph; b, R
=
0
Ph2P-C-C-BR,
I
CH,
9
CH,Ph
[*] Prof. Dr. H. J. Bestmann. DipLChem. H. Behl, DipLChem. M. Bremer
Institut fur Organische Chemie der UniversitHt Erlangen-Nurnberg
Henkestrasse 42, D-8520 Erlangen (FRG)
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