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Glc-PC a New Type of Glucosidic Phospholipid.

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Glc-PC, a New Type of Glucosidic Phospholipid**
Michael Mickeleit, Thomas Wieder, Klaus Buchner,
Christoph Geilen, Johann Mulzer,* and
Werner Reutter*
Dcdic,ctierlto ProfL.ssor Richard R. Sclmzidi
ow t l i ~o i ~ i ~ t i s i oofr ~his 60th hirtlduy
Phospholipids such as phosphatidyl choline (PC) are fundamental structural components of biological membranes. In addition to their structural role. they are also important in cellular
signal transduction['' and as regulators of enzyme activity.['] It
has been known for some time that 2-0-alkyl and 2-0-acyl
derivatives of 24ysophosphatidylcholine display both cytotoxic
and growth-promoting a ~ t i v i t i e s ,and
~ ~ ]this has led to the development of dermatological agents. for instance for the treatment
of skin metastases.[4]Until now, it was not possible to suppress
the cytotoxic effects of these compounds without decreasing
their ability to inhibit cell proliferation. With the synthesis of a
new type of glyceroglycophospholipid (Glc-PC 1, Scheme I ) ,
we have now found a solution to this problem. The starting
material. (R)-isopropyliderieglyceraldehyde@),['I was converted into the x./&unsaturated ester 3 to better differentiate between the quasi-enantiotopic end groups. The primary advantage of this unusual "protective group" is that it minimizes steric
hindrance in the later 2-glycosidation and can be removed under
extremely mild conditions (ozonolysis'Ieduction 1 without the
danger of a 1,3-acyl migration. After acidic hydrolysis of the
acetonide ring in 3 to give the diol4. ester 5 was xynthesized by
a highly chemoselective monoacylation of the primary OH func~ 9: 1)
tion and then converted into a mixture of anomera 7 ( s c : =
by treatment with glucosylfluoride 6.['l
Before the phosphate could be introduced as the final glycerol
substituent, the olefinic unit generated early in the synthesis WAS
oxidized to produce the aldehyde 8, and this was reduced to the
alcohol 9. Selective reduction of 8 without endangering the
stearoyl ester was achieved with zinc borohydride in ether as the
reducing agent.['I The alcohol was esterified with phosphorylchloride. and the dichloro ester was treated immediately with
''I
choline tosylate to produce the phosphocholine
Since the stearoyl group is labile under hydrogenolytic conditions, it was not entirely surprising that removal of the protective benzyl group presented considerable problems. After careful optimization, however. the reaction gave good yields
(Scheme 1). Compound 1 was obtained in gram quantities in
eight reaction steps with an overall yield of 2 2 % (for spectroscopicdata see Table 1 ) . It should be possible to introduce many
different acyl and glycosyl residues.
The biological test of cytotoxicity was performed in a serumfree cell culture system. Figure 1 B shows that Glc-PC 1 is non-
0
0
\
2
3
0
OH
4: R = H
0
d
----t
*
f
Scheme 1 . Synthesiz of 1 .md 10. a) Triethylphosphonoacetate.NaH. THF. -20 'C (84%); b) AcOH:'H,O (60:40) (81 %); c) CH,(CH,),,COCI, DMAP. py. 0 C (75%):
d ) 7.3.4.6-tetra-O-benryl-/i-u-glucopyranosylfluoride
(6). AgCIO,. SnCI,. 4 A mol. sieves, Et,O, - 15 C (89%): e) 1 . 0,. CH,CI,. -78'C; 2. Ph,P (XY%,): f) Zn(BH,),.
Et,O. 0 C (75")"): g) 1. POCI,, Et,N, CHCI,, 0 C . 2 . choline tosyldte. py. O T (67%); h) H, ( 2 atm.). Pd,'C ( 5 % ) . CH,OH, I 2 d (81 %). DMAP = 4-(dimethylamino)pyridine. py = pyridine.
[*] Prof. Dr. J. Mulrer, Dr. M. Mickeleit
lnstitut f i r Organische Chemie der Universitat
Maric-Curie-Strasse 11. D-60439 Frankfurt a m Main (Germany)
Telrf2i.i: In1 code +(69) 798-29464
Prof. Dr W. Reutter. Dr. T. Wieder. Dr C. Geilen
Institut f i r Molekularbiologie und Biochemie der Freien Universitit
Ai-niiiiallee 27. D-14195 Berlin-Dahlem (Germany)
Tclefax: I i i t code +(30) 838-2141
Dr. K Buchner
lnatitut fur- Bivchemie der Freien Universitht Berlm (Germany)
[**I
This iresearch was supported by the Fonds der Chemischen lndostne a n d the
Sonnenfeld-Stiftung.
toxic up to a concentration of 10 pmo1L-I (cell viability
>95 %). The protected intermediate 10 (Bn-PC) showed only
low toxicity even at 1000 pmolL-' (Fig. 1 A ) . and it inhibited
cell proliferation only at relatively high concentrations, namely,
greater than 100 pmolL-'; this effect is probably due to unspecific cell damage (Fig. 1 A). In nontoxic concentrations, 1
inhibits the proliferation of HaCaT cells; thus. at 10 pmol L - '
cell proliferation was inhibited by 80% (Fig. 1 B).
Since. as a group, various isoenzymes of protein kinase C
(PKC) play an essential role in the control of cell proliferation,[12] we tested the effect of 1 on this enzyme. As already
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shown for 2-lysophosphatidylcholine,~'31 the P K C activity at
low concentrations of 1 was increased by up to 70%. At higher
10: colorless oil; [XI;" = +53.7 ( c = 1 35 in CHCI,); ' H N M R (500 MHz. CDCI,):
concentrations of 1 this activation decreased (Fig. 2). In con6 = 0 . 8 8 (t. J = 6 . 2 5 H z . 3 H ) . 1 . 1 X 1.30 (m, 28H). 1.53 (mc. 2 H ) . 2.23 (t.
trast to 2-lysophosphatidylcholine, however, even high, toxic
J = 6.25 Hr. 2 H ) . 3.18 (s. 9H). 3.44-3.54 (m, 2 H ) . 3.56 (dd, J = 1 0 . 0 . 3.75 Hz,
1 H ) . 3.61b3.64 (ine. 1 H ) . 3.64-3.6Y (dd. J=lO.OHz. 1H). 3.71-3.75 (dd.
concentrations of 1 d o not inhibit PKC activity in vitro. ComJ = 1 0 . 0 , 3 . 7 5 H z . lH),3.X9-3.94(dd.J=10.0H~,1H),3.96-403(m,3H),4.09- pound 1 also had no effect on basal activity.[14'
Table 1 Selected physical properties m d spectroscopic data of 10 and 1.
4.14 (m. 1 H). 4 14--4.21 (m. 3 H ) . 4.30 (dd. J =10.0. 3.75 Hz. 1 H), 4.42-4.93 (m.
XH). 5.21 (d. J = 3.75 Hz.1 H). 7.12-7.40 (m. 20H): IR (KBr): V = 2925, 2853.
1731. 1456, 1453. 1240, 1091. 1069cm-'; MS (FAB'): m / r ( X ) : 1047 (0.7). 524
(3.2). 224 (4.X).91 (100)
I : colorless amorphous solid; M.p. 185 C. [a]$"' +61.3 ( c = l in CH,OH);
' H NMR(5OO MHz. CD,OD): b = 0.90(t.J = 6.25 Hz. 3 H ) , 1.23-1.36(m. 28 H).
1.60 (mL. 2H). 2.36 (t. J = 6.25 Hz, 2H). 3.23 (s. YH), 3.30 (me. 1 H). 3.32 (dd.
180
1
-
T
T
J=8.75Hz,1H).3.38(dd.J=10.0.3.75Hz,IH).3.59-3.65(m,3H),3.67-3.78
(m. 2H). 3.97-4.04 (m. 2 H ) , 4.08 (mc, 1 H), 4.21-4.33 (m, 4 H ) , 5.04 (d.
J = 3.75 Hz. 1 H); IR (KBr. PI): = 3387, 2922, 1794. 1645, 1467. 1229, 1086,
1053 c m - l ; MS (FAB+): !n/; ( O h ) : 686 ( 8 . 5 ) . 524 (4.9).420 (4.6), 241 (5.2). 224
(19.1), 184 (74.0). 166 (20.7). 86 (54 9)
.I
80
60
40
20
-
-
0
4
50
25
i
0
I
.
I
'
1
.
1
.
I
' I .
I
'
I
.
1
'
1
\
1
0 L
.
T
'
100
' """'I
'"'"1
1
10
"""'I
Compound 1 is therefore a noncytotoxic inhibitor of cell proliferation, whose inhibitory action is not mediated by protein
kinase C. Further 2-glycosidic derivatives must now be synthesized to determine whether this unusual property is due to the
glucoside residue.
' "T
1000 10000
Bn-PC c o n c e n t r a t i o n [pMI
Experimental Procedure
150
Cell culture: HaCaT cells [15] were maintained in liquid culture medium [16]. Confluent cells were released from the plate with 0.1 % trypsin/0.02 % EDTA solution,
then passaged. For the duration of the experiment. the cells were kept in serum-free
keratinocyte growth medium. Compound 10 was first dissolved in DMSO
( 8 5 mmol L - I ) then added to keratinocyte growth medium to obtain the required
concentration. Compound 1 was dissolved directly in keratinocyte growth medium.
125
100
75
50
25
0
0
1
10
100
1000 1 0 0 0 0
Glc-PC c o n c e n t r a t i o n [ p ~ l
Fig. 1 . Influence of 1 and 10 on the viability (m) and proliferation ( 0 ) of HaCaT
cells. Confluent HaCaT cells were treated for 3 h with different concentrations of
Bn-PC 10 (A) or Glc-PC 1 (B). The controls contained DMSO (A) or no further
additions (B). After incubation, cell viability was determined as described in the
Experimental Procedure (m). Values A refer to the activity [%] relative to the control
experiment. HaCaT cells (20000 cellscm-2) were treated for 24 h with different
concentrations of 10 (A) or 1 (B). The controls contained DMSO (A) or no further
additions (B). After incubation the cell count was determined by the crystal violet
method ( 0 ) . Values A refer to the activity [%I relative to the control experiment,
2668
f> VCH VerlugsgeseIlsdia/r mhH. 0.69451 Weinhein?, 1995
Cytotoxicity test: The cytotoxicity of 10 and 1 against HaCaT cells was determined
by the trypan blue exclusion method. Confluently grown HaCaT cells were incubated for 3 h with different concentrations of the two synthesized lipids. The media
were removed by aspiration, and the cells were washed with phosphate-buffered
saline (PBS). Trypan blue solution (0.1% in PBS) was then added, and the blue
(dead) and colorless (live) cells counted under a diavert microscope. At least 200
cells were counted for each determination. As an additional control of the cytotoxicity of 10 and 1, the alkaline phosphatase activity of the treated HaCaT cells was
determined [17].
Measurement of cell proliferation: The influence of 10 and I on the proliferation of
HaCaT cells was determined by the crystal violet method [18]. HaCaT cells were
sown at a density of about 20000 cellscm-z. The cells were allowed to adhere then
incubated for 24 h with different concentrations of 10 and 1. The medium was
removed by aspiration and the cells washed with PBS. Finally. cell numbers were
determined after staining with 0.1 YOcrystal violet solution 1191.
Measurement of protein kinase C activity: Protein kindse C was purified from
bovine brain [20]. Enzyme activity was determined in a reaction mixture (total
volume 50 pL) containing 20 mM triethanolamine (pH 7.4), 4 mM magnesium acetate. 0.1 mM CaCI,. SOmM mercaptoethanol, 0.1 g L - ' histone 111 S, 5 0 g L - l
phosphatidylserine, 5 g L - I diolein, and 20 mM ATP supplement with [y3*P]ATP
(total radioactivity 2 x 10' cpm). Phosphatidylserine, diolein. and I were dissolved
in methanol/chloroform ( 1 : 1). Before addition to the reaction mixture, the solvent
was removed under nitrogen and the residue suspended in a suitable volume of
20 mM triethanolamine by ultrasonic treatment. The reaction was started by addition of enzyme (10-20 ng). After 2 min incubation at 3 0 T , an aliquot (40 pL) of
+
o57o-0~3319513423-2ri688 10.00 .25/0
Angew. C k m . In[. Ed. Engl. 1995. 34, N o . 23/24
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the reaction imxtui-cw:ts transferred to a Whatman P83 filter. The filter was washed
three time\ i n 0.5"; phosphoric acid. and the radioactivity determined by measurement of the Cei-enkov radiation in water [21. 221.
Received: June 9, 1995 [Z 8079 IE]
German version. Aii,quw. Chon. 1995. 107. 2879- 2881
Keywords: glyceraldehyde . glycosidations . phospholipids . proliferation inhibitors . protein kinase C
[ I ] D. E Vance. B i o d i ~ w i .Cell Bid. 1990. 68. 1151-1165: J. H. Exton. J. B i d
Chem 1990, 26.7. 1-4: S. H . Zeisel, FASEE J. 1993. 7, 551 - 557.
[I] P. c'. Clioy. D E Vance. J. Biol. U i m . 1978,253,5163-5167; K.Oishi. R. L.
Raynor. P. A Charp. J. F. Kuo. ihid. 1988. 263(14). 6865-6871: C. C. Geilen.
T. Wicder. W. Reutter. ihid. 1992.267.6719-6724: M. Mosior, R. M. Epand,
Bior lwnijs/r>. 1993.32,66-75: C. C. Geilen, A. Haase. T. Wieder. D . Arndt, R
Zeisig. W. Reuttei-. J Lipid Rcs. 1994. 35. 625-632.
[3J H. IJ Weltneii. 0. Westphal. Liebig., hii. C h ~ n i .1967. 709. 240-243; R
Aiidreeseii. M Modolell. H U. Weltzien. H. Eibl, H. H. Common. G. W. Lor,
P Ci Mundrr-. <'uiiwi Rcr 1978. 38. 3894-3899; W. E. Berdel. W R. E.
B'iuaert. V. F i n k . J. Rastettei-. P. G . Munder. Aiiticuncer Re., 1981. 1. 345-352;
C. C Geilen. R Haase, K. Buchner. T. Wieder. F. Hucho. W. Reutter. Eur. J
Coii<<,r1991. 27, 1650 1653.
141 C Unger. H Eihl. H W. von Heyden. M. Peukert, H. Sindermann. G . A.
N2Ipel. O l l k O / r J ~ ~ l1990.
<'
13. 56
[SJ J. Mulier. A Angei-mann, li,rrulich~~n
Lcrr. 1983. 24. 2843-2846: J. Jurczak.
S. Pikul. T. Biiuer. h d . 1986,4-7,447-488; J. Mann, N . K. Partlett. A.Thomas.
J. C'IWIII.Rc,\ 1987. 369.
[6] T. Mukaiyaiii,i. Y. Muiai. S. Shoda. Clieiri. Lctr 1981. 431 -432.
[7] Ci. H Pomcr. S H. Homes. Tcwulidron Lerr. 1985, 26, 5-8.
[ X I The use or heiizyl protective groups is essential. The acetates. which are commoiil) used iii wgar chemistry. give on15 siiiall yields.
[9] B. C Ranu. S i . i ~ l e r r 1993. 885-892: W J. Gender. F. A. Johnson, A. D. B.
Sloan. J A m C ' l i w i Soc. 1960, 82, 6074--6079.
[I01 G Hirth. H Sai-okz W. Bannwarrh. R. Barner. Huh. Chun. A < f u1983. 66.
1210 i m
'tage. thc anomers produced i n the glycosidation can he separated by
MeOH:CH,CI,. 4 : l ) . The final product is the pure cr-anomer.
[I?] M . 1. Clemens, I . Trnyner. J. Menaya. J. Cell Sci. 1992, 103. 881-887: Y.
Wishiruka. S ( i o i ~ r1992, 2.78. 607-614.
1131 K Oishi. R L. Raynor. P. A. Charp. J. F. Kuo. J. Biol. Clirnr. 1988. 263(14),
A New Dimension in Radical Chain
Group Transfer Reaction
by Photosensitized Electron Transfer (PET)
Reductive Activation of PhSeSiR3**
Ganesh Pandey* and K. S. Sesha Poleshwar Rao
Dedicuted to Professor U. R. Gliutak
on the occasion oj'his 65th birthduy
Despite the drawbacks['] associated with tin hydrides, most
of the recent applications of radical reactions in organic synthesisr2] are based on the use of this reagent. While alternative
approaches involving the silyl radical,[31 which is a better
halophile than its tin radical ~ o u n t e r p a r t ,have
~ ~ ] partially overcome the limitations of the tin reagent, the high cost of its
precursor tris(trimethylsily1)silane (TMSS) and the loss of functionality during the last chain propagation step has limited its
popularity among organic chemists. To circumvent the known
limitations of these two reagents, and the necessity for a better
reagent for radical chain reactions in organic synthesis, we introduce here PhSeSiR,
(R, = Ph,tBu), which under PET
(photosensitized electron transfer) reductive reaction conditions
initiates radical chain group transfer reactions (Scheme I ) .
1 [ 5 3 6 1
112 HA- + 1/2 A + 1/2
. hv
PhSe-
6x65 6x71
1141 The b a s i a c t n i l y was measured without addition of phosphatidylserin;
diolein
[I51 P. Houknmp. R T. Petrussevska. D. Breitki-eutz, J. Hornung. A. Markham.
W E. Fusenix, .I. Ccll B i d 1988, 106. 761 -771.
[16] Thc qwntancously iminortalized human keratlnocyte cell line HaCaT (1141)
\raa kindly provided by Dr. N. E. Fusenig ( D K F Z Heidelberg. Germany).
1171 J ( i . ('ulvenor. A. W. Harris. T. E. Mandel. A. Whitela%', E. Ferber. J. hiI F I I I I I I I / 1981. l2h. 1974-1977.
1181 R. J Gillie\. N Didier. M. Denton, .4no/. Biochon. 1986. 159, 109-113.
1191 C'. C (ieilcn. R. Haase, K . Buchner. T. Wieder. F. Hucho. W. Reutter. E m J.
( ' a m r r 1991. 27, 1650-1653.
[20] H. Kruger. W. Schrdder, K Buchner. E Hucho. J. Proruin Clwm. 1990. 9.
467 313
[21] Siiicc ilic siihwate is histone. the measured activity is due predominantly to the
"clri\?licitl" iwlorms of protein kinase C (x. /i.
7 ) . The test system consists of
puritied components only and contains n o other enzymes besides PKS. A role
for Glc-PC A S ii prodrug with respect to 2-lysophosphatidylcholine (LPC) can
thus hc ruled out.
1221 M . Liycinagc. D. Frith. E. Livneh, S. Stabels. E i r ~ d i ~ i J.
i r .1992.283, 781 -787.
H+
1
O1
PlrSeSePll
\
Scheme I . Mechanwn of the PET-driven reduction of I and the radical chain
sequences. DMN = 1.5-dimethoxynaphthalene:HIA = ascorbic acid: HA- = ascorbate: A = dehydroascorbic acid. PhSeSiR, = PhSeSiPhgBu.
The potential of I to initiate the radical chain sequences
shown in Scheme 1 was envisaged by considering the expected
dissociation of the radical anion 1'- into the silyl radical, useful
for chain initiation, and PhSeSePh (2). The latter was formed
after the oxidative dimerization of the corresponding PhSeanion and participates in the termination step.[" The generation
of 1'- was envisioned through one-electron PET reductive processes involving 1,5-dimethoxynaphthalene (DMN) as a light
harvesting ( > 300 nm) electron donor and ascorbic acid (H,A)
as co-reductant.[*] The thermodynamic feasibility of electron
I*] Dr. G. Pandey. K. S. S. P. Rao
Division of Organic Chemistry (Synthesis)
National Cheinical Laboratory
Pune 411 008 (India)
Telefax. Int. code +(212) 330233
e-inail: pandey!o ncl.ernet.in
[**I
This work was supported by the Department of Atomic Energy (Dr. K. S.
Krishnan Fellowship to K. S. S. P. R.) and partly by the Department of
Science and Technology, New Delhi. We are grateful to Dr. K. Vijay Mohan
and M. P. Vinod for measuring reduction potentials oi' I and H,A.
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