Synthesis of novel phenoxy-linked diazirine glycero- and sphingolipid probes via trialkylaryltin intermediates

Article abstract of DOI:10.1016/S0040-4039(00)01166-7

Two photoaffinity lipid probes, phosphatidylcholine analog 1 and ceramide analog 2, were synthesized via trialkylaryltin intermediates 10b,b' using a Stille coupling strategy. The latter are stable for months at -20°C. Iododestannylation afforded lipid probes 1 and 2. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)01166-7

Tetrahedron Letters 41 (2000) 6737±6741
Synthesis of novel phenoxy-linked diazirine glycero- and
sphingolipid probes via trialkylaryltin intermediates
Guoqing Li and Robert Bittman*
Department of Chemistry and Biochemistry, Queens College of The City University of New York, Flushing,
New York 11367-1597, USA
Received 17 June 2000; revised 11 July 2000; accepted 12 July 2000
Abstract
Two photoanity lipid probes, phosphatidylcholine analog 1 and ceramide analog 2, were synthesized
via trialkylaryltin intermediates 10b,b0 using a Stille coupling strategy. The latter are stable for months at
^20ꢀC. Iododestannylation a€orded lipid probes 1 and 2. # 2000 Elsevier Science Ltd. All rights reserved.
Photoanity labeling is a well-established method for identifying ligand-binding domains in
macromolecules.1 Many investigators consider carbene precursors as the reagents of choice.2
Diazirines3 and tri¯uoromethylphenyl diazirines (which were introduced by Brunner in 1980)4 are
convenient carbene-generating probes.1d,5 7 Since peptide mapping generally employs radioligands
(e.g. 125I and 131I)1c to trace the photoadducts, the commercially available hydrophobic photolabel,
3-tri¯uoromethyl-3-m-125iodophenyl-3H-diazirine (125I-TID), is a widely used tool.8 Incorporation
of the 125I-TID label into speci®c ligands permits characterization of putative membrane receptors
and intracellular target proteins.1c,d Although radioiodination has been achieved by aryldiazonium8
and arylthallium chemistry,9 and by aryltin±iodine exchange,10 the chemical yields are very low.
As part of our studies to identify the putative glycerophospholipid binding site of phospholipid
scramblase,11 we required photoanity probes of phosphatidylcholine (PC) (and other
phospholipids available by transphosphatidylation).12 We also sought to photolabel the viral E1
spike glycoprotein of the Semliki Forest virus13 with a photoactivatable analog of ceramide.14
Accordingly, two new lipophilic probes (1,2) were synthesized in which a tri¯uorophenoxy
diazirine functionality is located near the glycerol backbone (1) or sphingoid base (2). Use of a
phenoxy group instead of a phenyl group as the linker in PC15 increases the lmax to ꢁ375 nm
(" 284 M^1 cm^1). Stille coupling16 of hexabutyldistannane with aryl iodide 9b,b0 a€orded
arylstannane intermediate 10b,b0. The short half-times of 125I (60 days) and 131I (8.1 days)17
* Corresponding author. Tel: 718-997-3279; fax: 718-997-3349; e-mail: robert_bittman@qc.edu
0040-4039/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)01166-7

6738
preclude installation of the radioiodide at the outset of the synthetic route and storage of radio-
iodinated TID derivatives for signi®cant periods. Stannyl derivative 1b can be stored under an
inert atmosphere for months in the dark and cold without signi®cant decomposition. Iodode-
stannylation is a convenient means for introducing a radioiodine atom into photoreactive ligands
1 and 2.
4-Methoxytri¯uoroacetophenone (3) was prepared from 4-bromoanisole and N-tri-
¯uoroacetylpiperidine as previously reported.18 Halogenation of aryl tri¯uoroketone 3 (2 equiv.
of HgO, conc. H2SO4, CCl4, re¯ux) provided bromide 4a and iodide 4b in high yield (Scheme 1).
O-Demethylation was carried out with 3 equiv. of LiCl in DMF19 at re¯ux for 2 h; the electron-
withdrawing nature of the 4-tri¯uoroacetyl group facilitates the demethylation, and phenol 5a,b
was obtained in high yield.
Scheme 1.
The synthesis of trialkylstannylphenoxy-containing diazirinyl acids is outlined in Scheme 2.
The linker group between the phenoxy moiety and the lipid contained either one or three
methylene groups; we used `b' to denote the n=1 series and b0 to denote the n=3 series. Coupling
of phenols 5a,b with ethyl bromoacetate or 4-bromobutyrate (Li2CO3/DMF, overnight, at 50±60ꢀC
for n=1 and 80±90ꢀC for n=3) a€orded esters 6 in ꢁ80% yield. (The esters were subsequently
hydrolyzed to acids 11a,b and coupled to the lipid target as shown in Scheme 3.21) The diazir-
idines 8 were prepared by the following reaction sequence: (a) formation of a mixture of E/Z
.
oximes (NH2OH HCl in dry EtOH/py); (b) O-tosylation of the oximes (p-TsCl, Et3N, CH2Cl2,
0ꢀC); (c) reaction of O-tosyl oxime 7 with liq. NH3 in a pressure tube (^78ꢀC to rt). With n=1 in
the linker, the ethyl esters were converted into amides 8a,b; however, with 7b0 (n=3), the ethyl
ester group survived, giving 8b0. Oxidation of diaziridines 8a,b,b0 using iodine and excess
triethylamine a€orded the corresponding diazirines 9. The formation of tin compounds 10b,b0
from aryl halides is the key step in our synthesis. The aryl bromide 9a was subjected to Stille
coupling with hexaalkylditin and various Pd catalysts without success. However, aryl iodides
9b,b0 were more reactive than 9a, and the desired products 10b and 10b0 were formed with
catalytic Pd(MeCN)2Cl2 in HMPA in 50 and 45% yield, respectively.

6739
Scheme 2.
Scheme 3.
Acylation of lysophosphatidylcholine (lyso-PC) with the acyl chloride or anhydride derived
from acid 11b (n=1) was unsuccessful, probably because of steric hindrance. Reaction of the less
hindered (n=3) arylstannyl acid 11b0 and iodoaryl acid 12b0, via their anhydrides generated in
situ, with lyso-PC at rt in a water bath a€orded PC analogs 1b0 (28%) and 1 (70%),20 respectively
(Scheme 3), after chromatography (elution with CHCl3; then with CHCl3:MeOH, 10:1; ®nally
with CHCl3:MeOH:H2O, 65:25:4). The lower yield and longer reaction time (4 days) for 1b0 may
arise from the steric e€ect of the tri-n-butyltin group. It is important to note that the stannyl
derivative 1b0 is stable for at least 2 months at ^20ꢀC. The trialkylstannyl group is eciently
replaced by iodo as discussed below.

6740
Finally, ipso iododestannylation of 1b0 was carried out on a small scale with unlabeled sodium
iodide in the presence of an oxidizing agent (Scheme 3).22 The goal was to establish the conditions
needed for conversion of 1b0 to product 1 with a minimum of handling and chromatographic
procedures so that the reaction could be subsequently carried out using Na125I just prior to use
with the biological targets.
Acknowledgements
This work was supported by NIH Grant HL-16660.
References
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other intramolecular rearrangements that can occur in carbenes after photolysis.1e
7. Advantages of tri¯uoromethylphenyl diazirines: photoactivation at l >350 nm, avoiding protein-damaging
wavelengths; high chemical stability in subdued ambient lighting toward mild reducing agents, oxidizing agents,
acidic and basic conditions, and elevated temperatures.1
8. Brunner, J.; Semenza, G. Biochemistry 1981, 20, 7174±7182.
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11. Bevers, E. M.; Comfurius, P.; Dekkers, D. W.; Zwaal, R. F. Biochim. Biophys. Acta 1999, 1439, 317±330.
12. For a review of transphosphatidylation, see: Bittman, R. In Lipid Synthesis and Manufacture; Gunstone, F. D.,
Ed.; Marcel Dekker: New York, 1999; pp. 125±144.
13. Sphingolipid speci®city of Semliki Forest virus fusion: e.g. Moesby, L.; Corver, J.; Erukulla, R. K.; Bittman, R.;
Wilschut, J. Biochemistry 1995, 34, 10319±10324.
14. For a previous synthesis of a radioiodinated TID photoanity analog of ceramide, see: Huwiler, A.; Brunner, J.;
Hummel, R.; Vervoordeldonk, M.; Stabel, S.; Van den Bosch, H.; Pfeilschifter, J. Proc. Natl. Acad. Sci. USA
1996, 93, 6959±6963. For a radioiodinated aryl azido analog of ceramide, see: Zegers, M. M. P.; Kok, J. W.;
Hoekstra, D. Biochem. J. 1997, 328, 489±498.
15. [125I-TID]-PC: (a) Ref. 1d. (b) Durrer, P.; Gaudin, Y.; Ruigrok, R. W.; Graf, R.; Brunner, J. J. Biol. Chem. 1995,
270, 17575±17581. (c) Goldmann, W. H.; Niles, J. L.; Arnaout, M. A. Eur. J. Biochem. 1999, 261, 155±162.
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6741
20. Compound 1: 1H NMR (CDCl3): ꢀ 7.52 (d, J=2.0 Hz), 7.15 (dd, J=1.7, J=8.5 Hz), 6.76 (d, J=8.7 Hz), 5.18 (m),
4.38±4.37 (m), 4.26 (m), 4.11±4.01 (m), 3.91 (br s), 3.76 (br s), 3.32 (s), 2.57 (t, J=7.3 Hz), 2.19 (t, J=7.6 Hz), 2.09
(m, 2H), 1.50 (m, 2H), 1.34±1.20 (m), 0.84 (t, J=6.8 Hz). 13C NMR: ꢀ 173.5, 172.4, 158.3, 137.5, 128.3, 122.8,
121.9 (q, J=273 Hz), 111.7, 86.9, 70.9, 67.9, 66.3, 63.4, 62.7, 59.3, 54.3, 34.0, 31.9, 30.6, 29.7±29.1 (m), 27.4 (q,
J=40.5 Hz), 24.8, 24.3, 22.6, 14.1.
21. Ceramide probe 2 was obtained in 83% yield by coupling diazirine acid 12b with d-erythro-sphingosine; Rf 0.7
(CH2Cl2/MeOH); 1H NMR (CDCl3): ꢀ 7.64 (d, J=8.0 Hz), 7.56 (d, J=2.0 Hz), 7.21 (dd, J=8.0, J=2.0 Hz), 6.75
(d, J=8.0 Hz), 5.78 (dt, J=15.0, J=7.0 Hz), 5.53 (dd, J=15.0, J=7.0 Hz), 4.52 (d, J=2.0 Hz), 4.37 (t, J=5.0
Hz), 4.02±3.98 (m), 3.80±3.73 (m), 2.04±2.00 (m), 1.33±1.10 (m), 0.85 (t, J=7.0 Hz). 13C NMR (CDCl3): ꢀ 167.1,
157.0, 156.7, 137.6, 134.7, 128.6, 128.5, 124.3, 121.9 (q, J=270 Hz), 111.9, 86.8, 74.3, 67.8, 62.2, 54.3, 42.3, 32.3,
31.9, 29.7, 29.6, 29.5, 29.3, 29.2, 29.1, 27.4 (q, J=40 Hz), 23.4, 22.7, 14.1.
0
.
22. Addition of freshly prepared ethanolic chloramine±T 3H2O to 1b in EtOH and NaI in aq. NaH2PO4 solution,
then quenching with NaHSO3, a€orded PC probe 1 in 92% yield after ®ltration through an aminopropyl solid-
phase extraction cartridge (elution with CHCl3, then with CHCl3:MeOH 1:1).