Spin-labelled sulfur containing neoglycolipids

Biochimie (1992) 74, 57-62 © Soci6t~ franqaise de biochimie et biologie mol~culaire / Elsevier, Paris

57

S p i n . l a b e l l e d sulfur c o n t a i n i n g n e o g | y c o l i p i d s JMJ Tronchet l, M Zs~ly l, A Ricca l, F Barbalat-Rey ~, M Geoffroy 2 IDepartment of Pharmaceutical Chemistry, 2Department of Physical Chemistry Sciences ll, University of Geneva, CH-1211 Geneva 4, Switzerland (Received 22 July 1991; accepted 21 October 1991)

Summary - - A neoglycolipid of structure ~-D-Glcp-S-(CH2)~N(OH)(CH2)4-O-cholest-5-en-3~-yl has been prepared in fair overall yield by reduction of the nitrone obtained by condensation of ~-D-GIcp-S-(CH2)3NHOH and OCH-(CH,)3-O-cholest-.~-en-3~-yi. This synthetic procedure is very flexible, allowing a large range of lengths for the spacer arm, different positions for the NOlt group along the spacer ann chain and the replacement of the sulfur by other bio-isosteric groups. The new neoglycolipid spontaneeusly oxidized to the corresponding nitroxide free radical whose EPR spectrum gave information on its conformational equilibrium ~hich was further studied by molecular mechanics. EPR spectroscopy I conformational analysis / spacer arms I spin-labelling / glycolipids Introduction The usefulness of paramagnetic probes (spin-labelled molecules) for the monitoring of biological events is well established [ 1]. Their detection by EPR spectroscopy is specific and the sensitivity of the method high. Most workers in this area have been using stable nitroxide free radicals in the form of a preformed spin-labelled reagent which was fixed onto a biological molecule via a simple condensation reaction. In this 'classical spin-labelling' the stability of the radical is obtained through extensive branching of the or-carbon atoms (fig l a), at the expense of the structural information obtainable from hyperfine couplings with hydrogen nuclei. Another consequence of this branching is the creation of large structural diff,,'rences between the spin-labeled and the native molecules. In these conditions, the only useful information provided by such 'classical spin-labelling' concerns the freedom of motion (rotational correlation time) of the labelled molecule. For these reasons, we developed [6] 'non-classical spin-labelling' techniques consisting of the synthesis o f deoxy-N-hydroxyamino analogs of sugars and nucleosides. These hydroxylamine derivatives differ Abbreviations: EPR, electron paramagnetic resonance; MP, melting point; TLC, thin layer chromatography; MS, mass spectrum; TMS, tetramethylsilane; DPPH, diphenylpicrylhydrazyl; Anal, elementary analysis; chol, cholesteryl; DMSO, dimethylsulfoxide

from natural compounds only in the replacement of an oxy group (-O-) with the structurally close Nhydroxyimino(-N(OH)-) group. They oxidize spontaneously to the corresponding nitroxide free radicals (fig l b - d ) whose stationary concentration is suffic|ent to give rise to usable ESR spectra. We describe, as an example, the synthesis of one such non-classically labelled neoglycolipid.

Materials and m e t h o d s General MP (uncorrected) were determined under microscope with a Mettler FP52 MP apparatus. TLCs were performed on silica gel HF~4 Merck with detection by UV light and a solution of thymol (0.5%), and sulfuric acid (5%) in EtOH. Column chromatography was conducted on silica gel 60 (0.063-0.2 mm) Merck. Silica gel 60 (0.040-0.200 mm) Merck was used for flash column chromatography. IR spectra were re~:orded with a FT-IR Nicolet 20 SXB spectrometer. UV spectra were measured on a Kontron Uvicon 810 spectrophotometer. NMR spectra were recorded at 20°C on a Braker WP 200 SY spectrometer (tH 200 MHz; chemical shifts in ppm from TMS; 8 units; b broad; s singlet...). The mass spectra were recorded on Finningan 4023 or VG 70-70E spectrometers. Optical rotations were measured with a Schmidt-Haensch polarimeter. EPR spectra were recorded on a Varian E-9 spectrometer (X band, 100 KHz modulation) equipped with a variable temperature device. The g values were measured by using a DPPH sample and the magnetic field was calibrated with an NMR marker. All the hyperfine coupling constants were checked by simulating the corresponding EPR spectra with an IBM compatible desk

58 computer using a program developed in this laboratory (Bartalat-Rey, Lichtle, unpublished results). The reaction scheme is represented in figure 2.

E )- and (Z )-3-N-hydro.~yiminopropyl 2,3,4,6-tetra-O-acetyl- 1 thio-lJ-D-glucopyranoside (2) To a solution of 1 [7] (2.5 g, 5.95 retool) in EtOH (30 ml), pyridine (2 ml) and N-hydroxyammonium chloride (0.8 g, 11.5 retool) were added. After 14 h stirring at room temperature, the reaction mixture was concentrated, extracted with CH.,CI., (100 ml), and the organic phase washed (H,O), dried (Na_,SO4) and submitted to a column chromatograghy (CH2CI,J acetone 4:1) which gave 2 (2.4 g0 92.8%) as a 4:5 E/Z mixture. "~'~ MP 109.5-110.5 O C (ether), [otis38 ° (c 1.84, CHCI.0, RF 0.52 and 0.63 (CH.,CI.4acetone 4:1), X~Km~3459 (N-OH), 1747 (C = O), and 1714 (C = N) cm-t; ~.Etott 206 nm (e 757.7). IH-NMR mLtx (CDCI3): 7.83 (bs, = N-OH E), 7.48 (t, Jcll. cH, = 6.0 Hz, = CH E), 7.45 (bs, = N-OH Z), 6.82 (t, JcH. cn. = 5.0 Hz = CH Z), 5.25 (t, I H, J.,..~ = 9 Hz, H-3), 5.10 (t, IH, J3.~ = 9 Hz, H-4), 5.05 (t, IH, J~.: - 10 Hz, H-2), 4.52 (d, I H, H-I), 4.20 (m, 2H, J.~.~ = 5 Hz, J~. ~b= 2.5 Hz, J.~.,..~b = 12.5 Hz, Ha, b-6), 3.75 (m, IH, H-5), 3.0-2.5 (m, 4 H, SCH,, CH,-CH=), and 2.15-1.93 (4s, 12 H, 40Ac). MS: m/z (%) 109 (77), 127 (29), 182 (13), 139 (12), 97 (13), 81 (ll), 195 (10), 331 (12, M .÷ -S(CHgaCH = NOH), 376 (0.3, M .+ -OAc), and 271 (4, M .+ -OAc-S(CH2)2. NOH). Anal calculated for Ct~H2sNOt0S (435.45): C, 46.89; H; 5.79; N, 3.22; S, 7.36. Found: C, 46.84; H, 5.62; N, 3.18; S, 7.25. 3-N-Hydroas'aminopropyl 2 ,3,4,6-tetra-O-acetyi- l -thio-lJ-Dglucopyranoside (3) To a solution of 2 (1.0 g; 2.3 mmoi) in MeOH (20 mi), NaBH3CN (!.6 g, 24.6 mmoi) was added and the pH kept at 2-3 by continuous addition of 6 N HCi in MeOH. After the pH

,i 0

/I~

/ - ........ 0

,/ o,0,y

OMe

AeO ~ OAe

•

/.o

N I H

Me

a

O

OBz

b

O 0

OH

e

d

N

_~0

~. 0

Fig 1. Examples of spin-labelled sugar derivatives, a. Classically' spin-labolled blocked O-glucoside [2]. b, [3]. ¢. [4]. d. [5] non classically spin-labelled sugar and nucleoside analogs.

was stabilized at 3 without further addition of HCI, the reaction mixture was stirred for another 2 h, concentrated and the residue dissolved into Titrisol pH 3 citrateB-ICl buffer (25 ml). The aqueous phase washed with CH2C12 (10 ml) was brought to pH 8-8.5 and re-extracted with CH2Cl2 (3 x 50 ml). The collected organic phase, dlied (Na,SO4) gave, after column chromatography (Et,O/MeOH) 10:1) 3 (0.55 g, 54.7%) as a syrup. [ot]~ -28" (c 1.2, CHCI3); DK~ 3477 (NH), 3285 (OH), 2943 (CH), and 1749.6 (C = O) cm-I; ~.EmtOH203 nm (~ 1004). IH-NMR (CDCI3): 5.20 (t, IH, ./.,.3 = ./3.4 = 9 Hz, H-3), 5.10 (t, 1 H, J4. 5 = 9 Hz, H-4), 5.05 (t, 1 H, Ji.2 = 9.5 Hz, H-2), 4.20 (m, 2 H, Js.~ = 5 Hz, Js.6b = 2.5 Hz, J~.6b = 12 Hz, Ha, b-6), 3.70 (m, 3 H, H-5, NH, OH), 3.03 (t, 2 H, Jcm. CH, = 7 Hz, NCH2), 2.75 (m, 2 H, SCHD, 2.15-1.95 (ss, 12 H, 4 0 A c ) , and 1.88 (q, 2 H, -CH2-). MS: m/z (%) 109 (100), 169 (96), 45 (67), 127 (41), 73 (26), 97 (22), 331 (7, M .+ -S(CH.,hNHOH), 271 (3), 289 (2), and 438 (0.5, M .+ + !). Anal calculated for Ct~H.,TNOIoS (43.7): C, 46.68; H, 6.22: N, 3.20; S, 7.33. Found: C, 46.49: H, 6.13; N, 3.331 S, 7.50.

3-N-Hydro.~yaminopropyl l-thio-~-o-glucopyranoside (4) A solution of 3 (0.5 g, I. 15 retool) in 0.02 M NaOMe in MeOH (10 ml) was kept at room temperature for 5 h, concentrated, then submitted to a flash chromatography (CH2Ch./MeOH 3:2) to yield 4 (0.2% 87.1%) as a foam. RF 0.10 (CH,ChJMeOH 3:2), [ot]~s -38°; ~K~ 3600-2900 (OH), 2856 and 2832 (CH), and 1636 (NH) cm -t. tH-NMR (spectrum in DMSO-d6 coupling constants were measured in presence of D,O and CF.~COOH): 7.10 (bs, i H, N-OH), 5.70-4.0 (5 overlapping bs, 4 OH, NH), 4.25 (d, I H, Jr.,. = 9.5 Hz, H-l), 3.67 (m, 1 H, ~. 6b = 2 Hz-, Hb-6), 3.40 (m, 1 H, Js. ~ = 5 Hz, J~. 6b = 12 HZ, Ha-6), 3.30-2.86 (m, 4 H, H-2,3A,5), 2.75 (t, 2 H, SCH2), 2.65 (t, 2 H, NCH,), and 1.67 (q, 2 H, -CH.,-). MS: ml,. (%) 45 (100), 83 (86, -(CH2h-NHOH), 90 (52), 85 (49), 119 (33), 106 (10, S(CH.,hNHOH), 140 (19), 154 (10), 163 (3,8 glucopyranosyl), ! 95 ( 1.4, glucopyranosyl-S-), and 269 (0.12, M-+). Anal calculated for CgHtgNO6S (269.32); C, 40.14; H, 7.1 !; N, 5.20; S, 11.91. Found: C, 40.26; H, 7.23; N, 5.01: S, 12.06. 4-( C holest-5-en-3 ~-ylo.~:y)-butanol (6) 3-O-Tosylcholest-5-en-3[3-ol [8] (10 g, 18.2 retool) was refluxed for 2 h with a solution of butyleneglycoi (20 ml) in dioxane (40 ml). The reaction mixture was brought to room temperature, concentrated, diluted with water (200 ml), then extracted with chloroform (4 x 50 ml). The collected organic phases, dried (MgSO4), concentrated, gave after column chromatography (hexane/ether 7:3) 6 (6 g, 72%). MP 75-100°C (ether/hexane), R~ 0.08 (hexane/ether 7:3), [0t]~ -31 ° (c 1.49, CHCID; ~Km~ 3256 (OH), 2924.6 (CH), and 1461 (C-O-C) cm-L IH-NMR (CDCI3): 5.35 (m, ! H, HC=), 3.67 (t, 2 H, OCH2), 3.53 (t, 2 H, OCH~.), 3.20 (tt, I H, J2~.3 = J4a..~ = ! 1 Hz, J.~b. 3 = J4b..~ = 4.5 Hz, H-3 chol) 2.50 (bs, I H, OH), and 2.45--0.70 (m, 47H, chol skeleton + CH,CH,). MS: m/z (%) 55 (100), 81 (50), 73 (48, HO(CH2)4-), 95 (40), 145 (33), 107 (30), 121 (28), 247 (19), 368 (18), 353 (4), and 329 (2). Anal calculated for C3tH540., (458.77): C, 81.16, 11.86. Found: C, 80.96; H, I 1.96. 4-(Cholest-5-en-3[J-ylo.~y)-butanal (7) A mixture of pyridinium chlorochromate (1.2 g, 5.57 mmol), powered 4 A molecular sieves (!.2 g), sodium acetate (0.4 g, 4.89 mmol) and CH2CI2 (50 mi) was stirred at room temperature for 20 min, then a solution of 5 (0.5 g, 1.09 mmol) in CH2CI, (5 ml) was added. After 3 h stirring at room temperature, the reaction was finished (TLC), EhO (200 ml) was added and the reaction mixture filtered and concentrated, was submitted to a column chromatography (hexane/ether 7:3) which

59 O~

OAe NHaOH9811,~-~ AeOo M ~ S ~ ' c H = N

Aeo~S%~"CH=O 1

O&:

2

OAe

l I (CH~)8 l X

A~O~

6 X.. CH~OH

7jsT~

~OH

•

~0k~

OH

r

"I---T

±~± ~ ~"

''~ 0A:

.A,

v

OAc A , ~ ~ J

11

4 R-H

AI

oe

~1~~

10

,o

OR 3 RuAc ~8"/~t

7 X=CHO _ ~

OH

9 0.~

,~hos

Sw/.~H

_o~

(4a---

"~OH

~''~ v

OH

Fig 2. Synthetic scheme leading to a spin-labelled neoglycolipid. yielded 7 (0.41 g, 82.4%). MP 68-73°C (hexane), Rv 0.40 (hexane/ether 7:3), [ot]~ -33 ° (c 1.77, CHCI~); ~gm~ 2952, 2925, 2851 (CH), 1732 (C ffi O), and 1461, 1377 (C-O-C) cm-L ~H-NMR (CDCI~); 9.78 (t, 1 H, Jc. ca, ffi 1.5 Hz, CHO), 5.33 (m, 1 H, HCffi), 3.48 (t, 2 H, OCH2),'3. ro (tt, 1 H, J2a 3 - J ~ 3 11 Hz, J2b 3 = J~b ~ = 4.5 HZ, H-3 chol), 2.53 (dr, 2 H,'CHzCH ffi O), and 2'.45--0.55 (m, chol skeleton + CH2). MS: m/z (%) 71 (100), 81 (44), 57 (41), 145 (38), 95 (35), 368 (24), 353 (3), 329 (2), 387 (0.5), and 418 (0.4). Anal calculated for C~Hs202 (456.76): C, 81.25; H, 11.48. Found: C, 81.40; H, 11.33.

4.Aza.8-( cholest.5.en-3 fl.yloxy )oct-4-enyi 2 3,4,6 -tetra-Oacetyl-l-thio-fl-D-glucopyranoside N.oxyde (8) A solution of 3 (0.44 g, 1.01 retool) and 7 (0.46 g, 1.01 retool), in a 5:1 EtOH/pyridine mixture (12 ml) was stirred 6 h at room temperature, concentrated and the residue submitted to a flash chromatography (Et20/MeOH 95:5) to give 8 (0.50 g, 56.7%) as a foam, Rv 0.13 (Et20/MeOH 95:5), [¢z]~ -30.5 ° (c 1.18, CHCI3); ZEtOH max 202 (e 6289), and 235 mn (~ 9852); ~ger msx 2949--2830 (CH), 1753 (C = O), 1612 (C = N), 1437, 1376 and 1240 (C-O-C) cm-L ~H-NMR (CDCI3): 6.80 (t, 1 H, ./ca. ca, = 6.0 Hz, N = CH), 5.34 (m, 1 H, C = CH), 5.22 (t, 1 H, ./2.3 = ./3 ~ = 9 Hz, H-3), 5.05 (t, 1 H, ./4 s = 9 Hz, H-4), 5.00 (t, 1 H, H'-2), 4.50 (d, 1 H, J~ 2 = 9.5 Hz, H-l), 4.18 (m, 2 H, J ~ s = 4.5 Hz, Jeb.~ = 2.5 Hz, J ~ 6b = 12 Hz, Ha, b-6), 3.86 ( t , 2 H, OCH2), 3.70 (m, 1 H, H-5), 3.50 (t, 2 H, N, NCH2), 3.13 (tt,

1 H, Jchol2a,3 -- Jchol4a.3 ----11 Hz, Jchol2 b . 3 = Jchol4b.3 - - 4.5 Hz, H-3 chol), 2.90--0.65 (m, chol skeleton), 2.55 (q, 2 H, =CH-CH2), 2.23 (t, 2 H, SCH2), 2.00, 2.03, 2.05, and 2.07 (4 s, 4 0 A c ) . MS: m/z (%) 169 (100), 109 (82), 55 (58), 81 (50), 97 (45), 368 (44), 145 (4"), 247 (20), 508 (15), 528 (15), 801 (1), 862 (0.5, M .+ -CH3), 877 (0.4 M .+ + 1). Anal calculated for C4sH77NOnS (876.21): C, 65.80; H, 8.86; N, 1.60; S, 3.66. Found: C, 65.56; H, 8.94; N, 1.72; S, 3.78.

4-Aza-8-( cholest-5-en-3 fl-yloxy )-4-hydroxy-octyl 2 ~3,4 ,6-tetraO-ace~. l-l-thio-fl-D-glycopyranoside (9) 8 (0.7 g, 0.8 mmol) was reduced in MeOH (20 ml) using NaBH3CN (0.2 g, 2.71 mmol) at pH 3 as described for the preparation of 3. After completion of the reaction (TLC), the reaction mixture was neutralized, concentrated, extracted with CH2C12 (50 ml), and the organic phase, washed (H20, 10 ml), dried (MgSO4) was submitted to a flash column chromatography (ether/petroleum ether 4:1) to afford 9 (0.63 g, 89.5%) as a white foam. R~ 0.25 (ether/petroleum ether 4:1), [¢x]21-32° (c 1.1, CHCI3); ~,~m~a 205 nm (~ 5141), and 230 (¢ 895); ~K~ 3472 (OH), 2945, 2903 and 2850 (CH), 1756 (C -- O), and 1375 (C-O-C) cm-i. IH-NMR (CDCI3): 5.35 (m, 1 H, C - CH), 5.20 (t, 1 H, J2.3 --"J3.4 " 9 Hz, H-3), 5.10-5.00 (m, 3 H, J4 5 " 9 Hz, H-2,4, NOH), 4.50 (d, 1 H, Jr.2 -- 9.5 Hz, H-l), 4.20'(m, 2 H, J ~ s -- 5 Hz, J~. s --- 2.5 Hz, Js,. 6b --'--12 HZ, Ha, b--6), 3.70 (m, 1 H, H-5), 3.50 it, 2 H, OCH2), 3.15 (tt, 1 H, Jcnol2~.3 -Jchol 4a, 3 ----"11 Hz, Jchol 2b,3 --=Jchol 4b, 3 -'~ 4.5 Hz, H-3 chol), 2.70 (m, 4 H, 2 NCH2), 2.45--0.65 (m, chol skeleton with 4 0 A c , SCH2, and 3 C H 2 of the spacer ann). MS: m/z (%) 84 (100), 169 (75), 109 (72), 128 (67), 144 (67), 368 (65), 55 (52), 492 (40), 530 (20), and 386 (18). Anal calculated for C4sH79NOnS (878.23): C, 65.65; H, 9.07: N, 1.59; S, 3.65. Found. C, 65.51; H, 9.06; N, 1.82; S, 3.91. 4.Acetoxy-4-aza-8( cholest-5-en-3 fl-yloxy )octyi 2 ,3 ,4 ,6-tetra-Oaceryl- l -thio-fl-D-g lucopyranoside (10) 9 (0.2 g) was acetylated with Ac20 (0.5 ml) and pyridine (2 ml) at room temperature for 16 h. Usual workup followed by a column chromatography (ether/petroleum ether 4:1) afforded 10 (0.2 g, 95.4%) as a syrup. RF 0.43 (ether/petroleum ether 4:1), [¢x]~ -31.5 ° (c 1.07, CHCI3); ~,EmtOH205 nm (¢ 4562); ~gm~ 2947, 2904, 2867 (CH), 1758 (C -- O), and 1375 (C-O-C) cm-I. IH-NMR (CDCI3): 5.35 (m, 1 H, C -- CH), 5.23 (t, 1 H, J2.3-J3.4--9Hz, H-3), 5.10 (t, 1 H , ~ --9Hz, H-4), 5.00 (t, 1 H, H-2), 4.55 (d, 1H, JL2- 10Hz, ,.'51), 4.20 (m, 2H, J6~.5= 5 Hz, J6b.s = 2.5 Hz, Jca eb = 12 Hz, Ha- b-6), 3.72 (m, 1 H, H5), 3.45 (t, 2 H, OCH2), ~. 12 (tt, 1 H, J~hol2a.3 = Jchol4~.3 = 11 Hz, J~hol2~ 3 = Jcho14b 3 = 4.5 HZ, H-3 chol), 2.85 (m, 4 H, 2 NCH2), and 2'.50-0.50 (chol skeleton with 5 0 A c , SCH2, 3 CH2 of the spacer arm). MS: m/z (%) 118 (100), 76 (82), 257 (79), 59 (76), 66 (60), 101 (58), 344 (40), 323 (20), 247 (20), 369 (15), and 539 (2). Anal calculated for CsoSs,NOnS (920.27): C, 65.26; H, 8.87; N, 1.52; S, 3.42. Found: C, 65.01; H, 8.98; N, 1.79; S, 3.27. 4-Aza-8.( cholest-5-en-3 [J-y loxy )-4-hydroxyocty l l -thio- ~-Dglucopyranoside (11) A solution of 9 (0.46 g) in 0.02 M NaOMe in MeOH (20 ml) was left 14 h at room temperature, then neutralized (0.1 M aqueous AcOH) and concentrated. A flash column chromatography (CH2CI2/MeOH 4:1) afforded 11 (0.25 g, 67.2%). MP 139.5-140.2°C (ether), RF 0.46 (CH2CI2]MeOH 4:1), [¢x]~ -31 ° (c 0.61, THF); ~EtOHm,204 nm (£ 4032); ~)germ,~3405, 3280 (OH), 2954, 2931, 2849 (CH), and 1109 (C-O-C ring) cm-~. IH-NMR (DMSO-d6): 7.60 (s, 1 H, N-OH), 5.28 (m, 1 H, --CH), 5.05, 5.00 and 4.90 (3d, 3 H, 3 OH), 4.50 (bs, I H, CH2OH), 4.20 (d, 1 H, J~. 2 = 9.5 Hz, H- 1), 3.68 (m, 1 H, Js. 6b=

60 Table !. ESR data of nitroxide free radical 3', 9' and 11' in diglyme, x represents the line width. Compound

3"

Tem~mture (°C) 10o g

2.0056

9'

80 2.0060

11'

110 2.0057

14.9

14.9

10.6 10.6

10.3 10.3 10.3 10.3

10.3 10.3 10.3 10.3

a.V(O)

0.6 0.6

0.5 0.5 0.5 0.5

0.5 0.5 0.5 0.5

F (G)

1.1

0.8

0.8

as (G) ag (G) a,~ (G)

12.8

these conditions, the 3[$ cholesteryl ether 6 was obtained in 72% yield and easily oxidized to 7 (82%). Condensation of 3 with 7 afforded in fair yield the nitrone 8 which was reduced in almost quantitative yield into the hydroxylamine 9, which could be acetylated to 10 and deacetylated to the free S-glycoside 11, Hydroxylamines 3, 9 and 11 oxidized spontaneously in the air into the corresponding nitroxide free radicals, 3', 9' and 11' respectively, ESR data of which are collected in table I.

11.0

2.5 Hz, J~.b - 12 Hz Hb-6), 3.55-2.90 (m, 12H, Ha-6, H-5, H4, H-3, H-2, 2 NCH2, OCH20CH chol), 2.70.(m, 2 H, SCH2), 2.35--0.5 (m, chol skeleton with 3 CH2 of the spacer ann). MS: m/z (%) 368 (100), 144 (62), 811 (53), 55 (52), 247 (28), 386 (26, chol), 353 (23), 491 (4), 530 (2), and 440 (1,6, cholO(CH2)4). Anal calculated for C4oH~iNO~S (710.08): C, 67.66; H, 10.08; N, 1.97; S, 4.52. Found: C, 67.44; H, 10.09: N, 2.08; S, 4.76.

Results The general strategy used to build the neoglycolipid 11 (fig 2) consisted of the preparation of the hydroxylamine 3 (the glycosyl moiety and half of the spacer arm) on one hand and the modified cholesterol 7 on the other and to condense these two building blocks. The known [7] thioglycoside derivative 1, obtained by conjugate nucleophilic addition of 2,3,4,6-tetra-Oacetyl-l-thio-[$-D-glucose upon acroleine, was oximated to 2 (E:Z mixture) which was reduced [9, 10] to the terminal hydroxylamine 3. Terminal hydroxylamines like 3 oxidize to the corresponding nitroxide free radicals giving usable ESR spectra (table I) but can not be considered good candidates for spin-labelling as the generated radicals are quite unstable and prone to dimerization via the corresponding terminal nitrone, Upon Zemlen deacetylation, 3 led to the free S-glycoside 4. The reaction of cholesteryl tosylate [8] $, with butane- 1,4-diol proceeded as expected [ 11] via a SN1 mechanism with retention of configuration as confirmed by the IH-NMR vicinal couplings of the H3 proton of the 3[$-cholesteryl moiety (two axial-axial (11 Hz) and two axial-equatorial (4.5 Hz) Y values). In

Discussion This synthetic pathway which afforded neoglycolipid 11 in a fair overall yield is extremely general and allows the preparation of spin-labelled neoglycolipids of the general structure Glyc-X-(CH2),-N(OH)(CHz)m OChol where Glyc can represent virtually any sugar moiety, X standing for S, O, or CH2, Chol for cholest-5-en-3[$-yl, and a large range of values being possible for n and m. The only reaction specific of a thioglycoside was in fact the conjugate addition to 1 but many other routes are available to prepare moxoalkyl glycosides. The O-glycoside (X = O, n = 2, 5, and 10, ot and [$) and C-glycoside (n -- 1, ot and [3) analogs of II have been prepared in such a way (Tronchet, Zs61y, unpublished results). Good EPR spectra, ie of l l ' (fig 3), can be obtained by heating to 80-110°C, exposing to UV light or oxidizing with a trace of lead dioxide the substrate hydroxylamine. These last two techniques should be compatible with most biochemical conditions. The well-resolved signals of figure 3 can be explained by the existence of a unique radical species of structure 11' and corresponds to a time-averaged spectrum. The same remark applies to the spectra of 3' and 9'. This situation is generally obtained at these temperatures. The values of g, F, an and aaa (table I) are those expected from this type of nitroxide in these conditions. The hyperfine coupling constants aa~ are indicative of the conformation of methylene groups vicinal to the imino-N-oxyl bridge. When a C-H bond lies in the nitroxide plane, its hyperfine coupling is null and this of the second proton of the same methylene group is close to 20 (3 [12, 13]. When the only conformations to be populated are those in which one of the C-H bond eclipses the nitroxide plane, the sum of two hyperfine coupling constants pertaining to the methylene group is close to 20 G, irrespective of the relative population of the two types of conformers. This situation, which is frequently encountered [3], prevails here. Another useful fact obtained from the number of ax~ hyperfine couplings is the number of y hydrogen atoms. A conformational study of 11' using CHARMM (a trademark for Polygen Corporation) [14] and a set of parameters developed for the nitroxide group [15] has

61

o

temp

110"C

I N~/~/"

S~

0

HO a N = 14.9G 4xa

H

OH ...

Fig 3. EPR spectrum of 11' in diglyme. The four a~ small couplings are included in the linewidth but assigned by spectrum simulation. been achieved through a rotation around the two C-N bonds with an increment of 30 ° (fig 4). The two dihedral angles (O-N-C-C) have been designated 0~ (toward the sugar) and 02 (toward the cholesteryl moiety) respectively. For each conformation, the molecule was subjected to a total (except for the two dihedral constraints) relaxation (Adopted Basis Newton-Raphson, 1000 steps). 0m was incremented first, then, for each of its values, a complete rotation of 02 was performed. The two favorable values of 0~ were 60 ° and 300 ° corresponding to conformation where one C-H bond was anti-periplanar to the N-O bond. For each of these values of 0m, one set of three low energy surfaces was found (fig 4a, d), one conformation of each set, c l (fig 4b, CHARMM energy 10.05 kcal/mol) and c2 (fig 4c, CHARMM energy 10.3 kcai/mol) being particularly stable. The six low energy areas corresponded to values of 02 of [(105 + 15) + m 120°] (m = 0-2). The conformations where one C-H bond of each methylene group would be antiperiplanar to N-O (01 = 02 = 60 ° + m 120 °) (m = 0-2), are excluded owing to unfavorable 1,3-cis coplanar interaction. In the two most stable conformations c l and c2, the two zig-zag aikylidene chains are roughly parallel and pointing to opposite directions relative to the nitroxide plane, one (c2) being doubly eclipsed (0~ = 300 °, 02 = 240°), the other, c 1, being eclipsed on the sugar side (01 = 60 °) and stagerred (02 = 90 °) on the cholesteryl side. Only c2 is in accordance with the experimental EPR values (sum of the a~ values for each methylene close to 20 G). For c 1, the expected values would be approximately 20 G for the methylene on the sugar side and 13 G for the other. Starting from c l and c2, a total relaxation was achieved after removal of the dihedral constraints leading to two new

isoenergetic (CHARMM energy 9.6 kcal/mol) conformation c'l and c'2 respectively, differing from the starting conformation by only a few degrees in the dihedral angles. To summarize, molecular mechanics computations (in vacuum) indicated that two conformations were favorable (one eclipsed-eclipsed, the other eclipsedstaggered), both with their alkylidene chains antiparallel, whereas, experimentally, in diglyme solution, only eclipsed-eclipsed conformations were found. In real conditions, c2 should be substantially more stable than c I. On the other hand, the conformational search was somewhat biased by the choice of 01 as the first dihedral to change and the conformation 'symmetrical' of c2 in which the dihedral angles are exchanged (0~ = 240 °, 02 ffi 300 °) should be equally favorable. The reason why the antiparallel eclipsedeclipsed conformers 0~ = 60 °, 02 = 120 ° and 0j = 120 °, 02 = 60 ° seemed not to be favorable is unclear. In any case, a good overall picture of the tridimensional structure of 11' has been assessed. One hydroge, atom of each N-CH2 methylene group lies in the plane of the nitroxide, one being anti-periplanar to NO, the other synperiplanar and the two alkylidene chains are antiparallel so differing at a minimum from a unique fully extended zig-zag chain. The conformation of 11 which can not be experimentally deduced from the experimental coupling constants in NMR should be, owing to the sp 3 hydridization of its nitrogen, still closer to a fully extended zig-zag for its spacer part. In fact, in Otis type of spinlabelling, the situation is very peculiar, the native biological probe (the hydroxylamine) and its paramagnetic derivative (the nitroxide) are different, but both structurally very close to their model (here an aikyl-

62 References

Fig 4. Conformational analysis of 11'. a. Two-dimensional map, energies increasing from deep blues (below 11 kcal/ tool, to red (above 21 kcal/mol, b. Conformation el. c. Conformation c2. d. Three-dimensional map. MOLCAD [16] was used for the drawings of molecules and UNIMAP (a trademark of UNIRAS A/S) for the maps. idene chain). In classical spin-labelling, the biological probe which interacts with the biological partners and the magnetic probe axe a single species but structurally different from the model compound. In a sense, with their own advantages and drawbacks, classical and non-classical spin-labelling techniques are complementary. Conclusion

Neoglycolipids in which a pyranose moiety is connected to cholesterol through an alkylidene spacer have shown some usefulness as host resistance stimulators against bacteria [ 17] and as constituants of liposomes [ 18]. Spin-labelled neoglycolipids as 11' should constitute an improvement over more usual synthetic glycolipids. Acknowledgments This work was supported by the Swiss National Science Foundation (grant ne 20-26460.89).

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