Bioconjugate Chem. 2002, 13, 143−149
143
TECHNICAL NOTES Synthesis of Novel Glycolipids That Bind HIV-1 Gp120 Rachel Y. LaBell, Neil E. Jacobsen, Jacquelyn Gervay-Hague, and David F. O’Brien* The University of Arizona, Department of Chemistry, Tucson. Arizona 85721 . Received July 24, 2001
As part of a research effort to design and prepare high affinity ligands for the galactosyl ceramide (GalCer) binding site on the HIV cell surface glycoprotein, gp120, several GalCer analogues have been prepared and characterized. The molecular design of analogues permits independent variations of the carbohydrate, the length of a hydrophilic spacer between the ligand and the lipid, and the composition of the hydrophobic lipid chains. Five different galactosyl analogues were synthesized having hydrophilic spacers of tri-, tetra-, and penta-ethylene glycol separating the carbohydrate from the lipid region which has either oleoyl or stearoyl lipid chains. The synthetic design allows for a convergent synthesis of the three components of the glycolipid conjugate. The structural characterization includes the proton and carbon chemical shifts, which were assigned after analysis of 1D and 2D NMR spectra.
INTRODUCTION
HIV-1 can gain entry into human T-cells by the initial binding of an associated surface glycoprotein (gp120) to the critical domain receptor (CD4) on the T-cell membrane surface. Subsequently, a cascade of events occur leading to HIV-1 replication which ultimately causes impairment of the immune system as HIV infection progresses to AIDS. However, an alternative receptor has been discovered that mediates the infection of CD4 negative cells (1-3). This galactosyl ceramide (GalCer) receptor is present on the surface of many types of cells including colorectal cells and vaginal epithelial cells (4). GalCer (Figure 1) is a relatively simple glycosphingolipid consisting of a single galactose unit bound via a β-linkage to ceramide. In biological membranes, the galactose portion is displayed very close to the membrane surface. Several laboratories have shown that the length of the spacer group between an antigen and its anchoring lipid can affect the efficiency of binding to antibodies. For example, Ringsdorf et al. found that extending fluorescein away from the membrane surface with a tetraethylene glycol spacer increased the binding of fluorescein to its antibody (5). The use of a hydrophilic linker as a spacer group in glycolipids has been reported to enhance the specific interaction of carbohydrates with receptor proteins (6, 7). Therefore, we elected to design GalCer analogues with variable length hydrophilic linkers to investigate the effect of spacer group length on the binding of galactosyl lipids with gp120. The β configuration of galactose is believed to be necessary for effective binding of GalCer to gp120 (8). One problem with GalCer is the susceptibility of the anomeric linkage to cleavage by glycosidases. The replacement of the anomeric oxygen with carbon stabilizes the anomeric linkage and is reported to have little effect on the binding of galactose to gp120 (9, 10). The third variable considered in the glycolipid is the nature of the lipid chains. There seems to be conflicting
evidence in the literature about whether variation in the lipid chains can influence the binding of GalCer analogues to gp120. Silberger and Bhat provided evidence that variation of the GalCer fatty acid chain length from 16 to 24 carbons showed little effect on binding efficiency when 125I-labeled gp120 was incubated with GalCer doped liposomes (8). Other authors reported that varying the GalCer fatty acid chain length from 1 to 14 has a distinct effect on the binding of GalCer analogues to rgp120 in an ELISA-type assay (9). Rico-Lattes has reported that a seven-carbon fatty acid chain showed no activity while a ten-carbon chain showed weak activity in binding to SPC3 peptide which mimics the V3 loop of gp120 (11). This literature provides evidence that a sufficiently long hydrophobic lipid chain is necessary to effectively anchor the lipid into the membrane, and there is little additional effect on binding efficiency. The second issue concerning the lipid chains is the presence of unsaturation. GalCer fatty acid is saturated to the extent that it exists in microdomains in biological membranes (12). Lipids with an unsaturation in the chains tend to not form domains in biological membranes because they do not pack together well and are more fluidlike. Lipids with saturated chains tend to form domains because the lipid chains pack efficiently and are more solidlike. Whether GalCer analogues that do not form domains will bind to gp120 has not been systematically investigated. Therefore, the design allows for the synthesis of both saturated and unsaturated analogues that will allow evaluation of this variable. In an effort to design GalCer analogues that could compete with the binding of gp120 to GalCer, a versatile synthetic scheme was developed. The analogue design permits the independent variation of three components that could increase binding: carbohydrate head group, hydrophilic spacer group, and lipid tails. The synthesis of these GalCer analogues is convergent with respect to each component. The hydrophilic spacer is derived from commercially available oligo-ethylene glycols by dif-
10.1021/bc015533r CCC: $22.00 © 2002 American Chemical Society Published on Web 12/08/2001
144 Bioconjugate Chem., Vol. 13, No. 1, 2002
Figure 1. Structure of galactosyl ceramide. Scheme 1
Scheme 2
ferentiation of the end groups. The carbohydrate head group is derived from β-D-galactose pentaacetate by conversion into the C-glycoside using trimethylsilyl cyanide (TMSCN). The lipid chains can be easily obtained by reaction of any long chain alcohol with maleic anhydride to form the maleic diester. Variation of one or more of these components has the potential to affect the binding affinity of the analogue to gp120. RESULTS
The synthesis started with a commercially available spacer, 2-(2-chloroethoxy)ethoxyethanol (1a). The corresponding longer spacers were derived from tetraethylene glycol and pentaethylene glycol by monochlorination with triphenylphosphine and carbon tetrachloride (Scheme 1) (16). The statistical mixture of dichloride, monochloride, and diol were separated by silica gel chromatography. In each spacer the remaining alcohol underwent Jones oxidation to give the corresponding monochlorocarboxylic acids 2a,b,c (15). The synthesis of each spacer is completed by conversion of the ω-chloro carboxylic acid to the ω-thiol by reaction with thiourea, sodium hydroxide, and water 3a,b,c (15). The literature procedure was modified by addition of 1 equiv of sodium iodide to form the iodide in-situ and improve yields up to 96%. The galactose portion was synthesized from β-D-galactose pentaacetate by displacing the anomeric acetate using TMSCN in the presence of BF3‚OEt2 4 (Scheme 2) (17). Only the β anomer is formed because of participation of the C-2 acetate (17). The peracetylated C-galactose was
reduced with lithium aluminum hydride in THF to remove all the acetates and convert the cyano group to methylene amine 5 (18). There was some acyl migration from the C-2 to the primary amine during the reaction followed by reduction of the amide to give 18% ethylamine as a byproduct. Two different lipid chains were used to synthesize the five Gal-Cer analogues. The unsaturated series of glycolipids were made using oleoyl alcohol. These analogues were synthesized in order to investigate the effect of nondomain-forming glycolipids on binding affinity. The saturated series was made using stearoyl alcohol. Saturated glycolipids have the propensity to form domains when mixed with dioleoyl phosphatidylcholine (DOPC). The unsaturated lipid was prepared (Scheme 3) from maleic anhydride and oleoyl alcohol with a catalytic amount of p-toluenesulfonic acid in chloroform to give the desired unsaturated diester. The conjugated double bond of the maleic ester provides a site for nucleophilic addition of the ω-thiol carboxylic acid 3a,b,c. The product is a lipid-spacer conjugate with a carboxylic acid end group 7a,b,c (19). The last step of the synthesis is the formation of an amide bond between the carboxylic acid of the lipid 7a,b,c and the C-galactoside 6. The acid was combined with pentafluorophenyl trifluoroacetate to form the activated pentafluoro ester followed by addition of 6 (20). Low yields are a result of minimal solubility of the deprotected carbohydrate in DMF. The unsaturated series of glycolipid molecules are named according to the carbohy-
Bioconjugate Chem., Vol. 13, No. 1, 2002 145
Figure 2. Numbering scheme for NMR assignments. Scheme 3
drate, Gal; the length of the ethylene glycol spacer: 3, 4, or 5; and the nature of the lipid chain: oleoyl (unsat) or stearoyl (sat). Hence the unsaturated series is referred to as Gal-3-Unsat, Gal-4-Unsat, and Gal-5-Unsat. The saturated series (Scheme 3) was prepared by the same reaction sequence except that the starting alcohol was stearoyl alcohol and the resulting maleic ester has saturated lipid chains 9. The spacer-lipid conjugate 10a,b and the final glycolipids 11a,b were formed in the same manner as the unsaturated series to give Gal-3Sat and Gal-4-Sat. NMR chemical shift assignments were made for Gal3-Sat, a representative glycolipid analogue (Figure 2). 2DNMR data (HMQC, HMBC, and COSY) was acquired and analyzed to assign all protons and carbons. NMR chemical shift assignments (Table 1) were obtained by analysis
of the HMBC data (Figure 4). The G4 (equatorial) proton signal has small coupling constants and thus can be located in the HSQC spectrum (Figure 3) as a sharp, positive cross-peak. G7 and G6 diastereotopic pairs can also be identified in the HSQC, with the G7 carbon 20 ppm upfield of the G6 carbon since it is bonded to amide nitrogen instead of oxygen. Clear HMBC connectivities allow assignment of the entire C-glycoside, with confirmation from the DQF-COSY (data not shown). An HMBC cross-peak from one of the G7 protons to the S1 (carbonyl) carbon verifies the spacer attachment through the amide linkage, and HMBC and COSY connections lead to assignment of the entire spacer. One of the S6 protons shows an HMBC connection through the thioether linkage to the L2 carbon, connecting the spacer to the lipid moiety. The more downfield of the lipid carbonyl
146 Bioconjugate Chem., Vol. 13, No. 1, 2002 Table 1. Chemical Shift Assignments for Gal-3-Sat in CDCl3/CD3OD (1:1) positiona δ(1H) multb G1 G2 G3 G4 G5 G6a G6b G7a G7b S1 S2 S3 S4 S5 S6a S6b L1 L2 L3a L3b L4 L5a L5b L5a′ L5b′ L6 L6′ L7 L7′
J(Hz)
3.15 3.39 3.40 3.80 3.41 3.69 3.60 3.63 3.40
ddd m m dd m dd d d m
9.4,6.4,2.9
3.94 3.61 3.59 3.62 2.87 2.79
s m m m dtc dt
JAB 15.7
13.6(JAB),6.4 13.7(JAB),6.4
3.65 2.87 2.6
dd dd dd
10.5,5.2 16.9,10.4 16.9,5.3
4.06 4.05 3.998 3.992 1.54 1.58 1.24 1.28
dt dt dt dt m m m m
10.7(JAB),6.6 10.7(JAB),6.6 10.9(JAB),6.7 10.9(JAB),6.7
L8-L19/ 1.20 L8′-L19′
m
L20/L20′ 1.19 L21/L21′ 1.22 L22/L22′ 0.77
m m t
2.7,1.0 11.6,6.9 11.7 13.9
7.0
δ(13C) 79.70 69.85 75.49 70.47 79.78 62.75 62.75 41.14 41.14 172.56 71.07 71.67 70.95 71.26 31.96 31.96 173.09 42.91 37.49 37.49 171.92 66.12 66.12 66.54 66.54 29.41 29.38 26.70 26.70 30.07(2)d 30.16(2) 30.35(1) 30.37(1) 30.40(2) 30.46(2) 30.49(14) 32.75 23.45 14.51
HMBC carbons G2,G3 G1,G3 G3,G5 G1,G4,G6 G5 G4,G5 G1,G2 G1,G2,S1 S1,S3 S4 S3 S4 S5 S5,L2 L1,L3,S6 L1,L2 L1,L2,L4 L4,L6,L7/7′ L4,L6,L7/7′ L1,L6′,L7/7′ L1,L6′,L7/7′ L5,L7 L5′,L7′
Figure 3. Portion of the edited 2D Gradient-Selected HSQC spectrum of Gal-3-Sat in CD3OD/CHCl3 (1:1). Data were acquired in TPPI mode with 512 complex pairs in t2, 750 increments of t1, and 8 scans per t1 increment. A skewed, 45°shifted sinebell was applied in both dimensions with zero-filling to a final real matrix of 2048 (F2) × 1024 (F1) data points. Single contours (open) are shown for negative (CH2) cross-peaks with many contours (filled) for positive (CH3, CH) cross-peaks.
L5′
L20/L20′ L20/L20′,L21/L21′
a See Figure 2 for numbering system. Lower-case a and b refer to diastereotopic protons in methylene groups which are not stereospecifically assigned. b d ) doublet, t ) triplet, dd ) doublet of doublets, dt ) doublet of triplets, ddd ) double double doublet, m ) multiplet. c Doublet coupling listed first for all dt. d Estimated number of carbons shown in parentheses.
carbons was assigned to L1 due to its proximity to the thioether. HMBC connectivities across the ester linkages in the lipid portion allow unambiguous assignment of the L5 and L5′ (differing in both 1H and 13C shifts) and the L6 and L6′ (differing in 1H shifts) positions. Approximately eight carbons from the L8-L19/L8′-L19′ portion of the lipid chains were resolved in the 1D 13C spectrum but could not be assigned (Table 1). DISCUSSION
Binding of the unsaturated GalCer analogues to gp120 has already been assessed by total internal reflection fluorescence spectroscopy (TIRF) (21). Fluorescently tagged recombinant gp120 was allowed to bind to the monolayer surface containing GalCer analogues and an equilibrium measurement of protein bound to the monolayer was accomplished using TIRF. The binding studies investigated the effect of varying the spacer length on binding affinity of GalCer analogues to gp120. The analogues bind cooperatively to gp120 when doped at 5 mol % in a monolayer of DOPC. The binding curves were extrapolated to obtain binding affinity constants (Ka) of 2 × 106. The protein coverage was determined and highest protein coverage was achieved with a spacer group of triethylene glycol. Therefore, a critical spacer arm length has been determined for these GalCer analogues (21). A compara-
Figure 4. Two portions of the phase-sensitive 2D GradientSelective HMBC spectrum of Gal-3-Sat in CD3OD/CHCl3 (1:1). Data were acquired in echo-antiecho mode with 2048 complex pairs in t2, 356 increments of t1, and 16 scans per t1 increment using a TANGO sequence and a gradient to dephase 13C-bound proton signals before the start of the HMBC (Kover, K. E., personal communication). A 90°-shifted sinebell was applied in both dimensions with zero-filling to a final real matrix of 2048 (F2) × 1024 (F1) data points. Single contours (open) are shown for negative cross-peaks with many contours (filled) for positive cross-peaks. Circles indicated one-bond correlations.
tive study of the saturated and unsaturated GalCer analogues will be reported elsewhere. This research has led to the development of a versatile synthesis of glycolipids that conjugate a carbohydrate, spacer group, and a lipid. These analogues should improve attempts to design and synthesize new HIV-1 therapies using membrane-tethered receptors for gp120.
Bioconjugate Chem., Vol. 13, No. 1, 2002 147 EXPERIMENTAL SECTION
General Procedures. All materials were obtained from commercial sources and used without additional purification, unless otherwise noted. 1D 1H and 13C NMR spectra were recorded on Varian Gemini-200, Varian Unity-300 MHz, Bruker DRX-500 or Bruker DRX-600 spectrometers. 1D 1H spectra were referenced to internal tetramethylsilane (0 ppm) and 1D 13C spectra are referenced to the solvent 13C signal, (CDCl3 at 77.0 ppm and CD3OD at 49.0 ppm). NMR spectra for Gal-3-Sat (1H, 13C, HSQC (13), HMBC (13), and DQF-COSY (14) were acquired on a Bruker DRX-600 spectrometer with a 5 mm Bruker TXI triple-resonance three-axis gradient probe at 25 °C. The sample (30 mg) was dissolved in 0.6 mL of CDCl3/CD3OD (1:1). The spectral widths were 4.6 ppm (1H) and 200 ppm (13C) for the 2D spectra. Data were processed and plotted using Felix2000 (Molecular Simulations, San Diego, CA). All 2D spectra were referenced using specific signals in the 1D spectra, which were referenced to internal tetramethylsilane (0 ppm) for 1H and CD3OD (49.15 ppm) for 13C. (2-{2-[2-Chloroethoxy]ethoxy}ethoxy)acetic Acid (2b). A mixture of (2-{2-[2-chloroethoxy]ethoxy}ethoxy)ethanol (3.00 g, 14.2 mmol) in acetone (60 mL) was stirred at room temperature as 6 mL of a 2.67 M Jones reagent (15) was added dropwise over 15 min. The reaction was stirred for an additional 30 min followed by addition of 3 drops of 2-propanol. Water (40 mL) was added to the mixture followed by removal of the acetone in vacuo. Saturated NaCl solution (20 mL) was added, and the aqueous mixture was extracted with CHCl3 (7 × 30 mL). The organic layers were combined and dried with anhydrous MgSO4. The product was visualized with bromocresol green on a silica gel TLC plate after elution with 30:5:3 CHCl3/EtOH/HOAc, with an Rf value of 0.5. The solvent was removed in vacuo, and the product was purified by silica gel column chromatography in 30:5:3 CHCl3/EtOH/HOAc to yield 2.63 g of product in 82% yield. 1H NMR (300 MHz, CDCl3) δ 3.50-3.65 (m, 12H), 4.15 (s, 2H), 10.63 (bs, 1H). 13C NMR (75 MHz, CDCl3) δ 42.5, 68.0, 70.1, 70.2, 70.7, 71.0, 173.8. High-resolution mass spectrum (FAB+) calcd for C8H15O5Cl (MH)+: 227.0686. Found: 227.0680. (2-{2-[2-(2-Mercaptoethoxy)ethoxy]ethoxy}ethoxy)acetic Acid (3c). (2-{2-[2-(2-Chloroethoxy)ethoxy]ethoxy}ethoxy)acetic acid (2.60 g, 9.63 mmol) was combined with thiourea (2.93 g, 38.5 mmol) and sodium iodide (1.45 g, 9.63 mmol) in 40 mL of water, and the reaction mixture was heated at refluxed for 4 h. After addition of 52 mL of 2.5 M, heating was continued for another 4 h. The reaction was cooled to room temperature, and the pH was adjusted to 1 with concentrated HCl. The aqueous mixture was extracted with CHCl3 (7 × 40 mL), and the organic layers were combined and dried with anhydrous MgSO4. The product was visualized with bromocresol green on a silica gel TLC plate after elution with 3:2 CHCl3/EtOH, with an Rf value of 0.30. The solvent was removed in vacuo, and the resulting oil was purified on neutral alumina column chromatograpy using a gradient starting with 6:1 CHCl3/EtOH and ending with 60:40:5 CHCl3/EtOH/HOAc to yield 2.43 g of a colorless oil with a 94% yield. 1H NMR (300 MHz, CDCl3) δ 1.55 (t, J ) 8.6 Hz, 1H), 2.64 (q, J ) 7.3 Hz, 2H), 3.53-3.73 (m, 14H), 4.14 (s, 2H). 13C NMR (75 MHz, CDCl3) δ 24.0, 68.3, 70.0, 70.3, 70.4, 70.9, 72.7, 174.2. High-resolution mass spectrum (FAB+) calcd for C10H21O6S (MH)+: 269.1059. Found: 269.1067. 2-[2-(2-Carboxymethyoxyethoxy)ethylsulfanyl]succinic Acid dioctadec-9-enyl Ester (7a). Butene-
dioic acid dioctadec-9-enyl ester (cis) (0.660 g, 1.07 mmol) was dissolved in 40 mL of 2-propanol at 80 °C under an Ar atmosphere. In a separate vial, [2-(2-mercaptoethoxy)ethoxy]acetic acid (3a) (0.193 g, 1.07 mmol) was titrated to pH of 8 with 15% NaOH. The basic solution was added to the reaction mixture followed by a catalytic amount of piperidine (2 drops). The reaction was heated at reflux for 1 h. Solvent was removed in vacuo, and the residue was taken up in water (20 mL). The pH was adjusted to 4 with 10% HCl, and the resulting solution was extracted with ethyl acetate (4 × 80 mL). The organic layers were combined and dried with anhydrous MgSO4, and the solvent was removed in vacuo. The product was visualized by phosphomolybdic acid on a silica gel TLC plate after elution with 9:1 CHCl3/MeOH, giving an Rf of 0.25. The product was purified by silica gel column chromatography using pure CHCl3 to elute the starting ester, followed by 9:1 CHCl3/MeOH to elute the product. A colorless oil (0.50 g) was obtained in 59% yield. 1H NMR (200 MHz, CDCl3) δ 0.85 (t, J ) 6.8 Hz, 6H), 1.25 (m, 44H), 1.58 (t, J ) 7.6 Hz, 4H), 1.98 (q, J ) 5.8 Hz, 8H), 2.57-3.03 (m, 4H), 3.59-3.76 (m, 6H), 4.04 (t, J ) 7.0 Hz, 2H), 4.10-4.13 (m, 2H), 5.46 (t, J ) 5.2 Hz, 4H). 13C NMR (50 MHz, CDCl3) δ 14.1, 22.6, 25.8, 27.2, 28.5, 29.2, 29.3, 29.4, 29.5, 29.7, 30.8, 31.9, 36.5, 41.7, 65.2, 65.6, 70.0, 70.4, 127.9, 129.9, 170.6, 171.7. Electrospray mass spectrum calcd. for C46H84O8S (M)-: 796.4. Found: 795.5. 2-{2-[2-(2-Carboxymethoxyethoxy)ethoxy]ethylsulfanyl}succinic Acid dioctadec-9-enyl Ester (7b). Prepared in a silimar manner to (7a). 1H NMR (300 MHz, CDCl3) δ 0.85 (t, J ) 7.48 Hz, 6H), 1.17-1.35 (m, 44H), 1.53-1.67 (m, 4H), 1.94-2.02 (m, 8H), 2.55-2.69 (m, 1H), 2.74-2.95 (m, 2H), 3.58-3.71 (m, 12H), 3.994.13 (m, 6H), 5.31 (t, J ) 5.28 Hz, 4H). 13C NMR (75 MHz, CDCl3) δ 14.0, 21.6, 21.7, 22.6, 25.7, 27.1, 28.4, 29.1, 29.3, 29.4, 29.6, 30.7, 31.8, 36.4, 36.7, 41.7, 65.0, 65.5, 68.4, 68.9, 70.0, 70.2, 129.6, 129.8, 170.6, 171.0, 171.6. Electrospray mass spectrum cald for C48H88O9S (M)-: 840.0. Found: 839.3. 2-(2-{2-[2-(2-Carboxymethoxyethoxy)ethoxy]ethoxy}ethylsulfanyl)succinic Acid Dioctadec-9enyl Ester (7c). Prepared in a silimar manner to (7a). 1H NMR (300 MHz, CDCl ) δ 0.86 (t, J ) 7.0 Hz, 6H), 3 1.23-1.30 (m, 44H), 1.51-1.65 (m, 6H), 1.86-2.03 (m, 8H), 2.65 (dd, J ) 16.8, 4.4 Hz, 1H), 2.73-3.35 (m, 6H), 3.58-3.70 (m, 10H), 3.96-4.14 (m, 6H), 5.32 (t, J ) 5.3 Hz, 4H). 13C NMR (75 MHz, CDCl3) δ 14.1, 22.6, 25.7, 25.8, 27.1, 28.5, 29.2, 29.3, 29.4, 29.5, 29.7, 30.9, 31.8, 36.5, 41.6, 65.1, 65.5, 70.0, 70.2, 129.7, 129.9, 170.6, 171.6. Electrospray mass spectrum calcd. for C50H92O10S (M)-: 884.0. Found: 883.1. 2-[2-(2-Carboxymethoxyethoxy)ethylsulfanyl]succinic Acid Dioctadecyl Ester (10a). Prepared in a silimar manner to (7a). 1H NMR (300 MHz, CDCl3) δ 0.84 (t, J ) 6.4 Hz, 6H), 1.21 (m, 60H), 1.58 (p, J ) 11.8, 5.9 Hz, 4H), 2.64 (dd, J ) 17.0, 11.0 Hz, 1H), 2.74-2.98 (m, 4H), 3.61-3.73 (m, 6H), 4.01-4.13 (m, 6H). 13C NMR (75 MHz, CDCl3) δ 14.1, 22.7, 25.7, 25.8, 28.5, 29.2, 29.3, 29.5, 29.6, 29.7, 30.8, 31.9, 36.5, 41.7, 62.3, 65.2, 65.6, 68.6, 70.1, 70.4, 71.0, 76.6, 78.9, 170.7, 171.7. Highresolution mass spectrum (FAB+) calcd for C46H89O8S (MH)+: 801.6278. Found: 801.6289. 2-{2-[2-(2-Carboxymethoxyethoxy)ethoxy]ethylsulfanyl}succinic Acid Dioctadecyl Ester (10b). Prepared in a silimar manner to (7a). 1H NMR (300 MHz, CDCl3) δ 0.88 (t, J ) 7.6 Hz, 6H), 1.25 (m, 60H), 1.62 (p, J ) 12.7, 6.8 Hz, 4H), 2.68 (dd, J ) 17.1, 11.7 Hz, 1H), 2.77-3.01 (m, 4H), 3.61-3.78 (m, 10H), 4.06 (t, J ) 7.3 Hz, 2H), 4.10-4.17 (m, 4H). 13C NMR (75 MHz, CDCl3)
148 Bioconjugate Chem., Vol. 13, No. 1, 2002
δ 14.1, 22.7, 25.8, 28.5, 29.2, 29.4, 29.5, 29.6, 29.7, 31.0, 31.9, 36.6, 41.7, 65.2, 65.6, 70.1, 70.5, 70.6, 170.7, 171.7. Electrospray mass spectrum cald for C48H92O9S (M)-: 845.3. Found: 845.0. Gal-3-Unsat (8a). 2-[2-(2-Carboxymethyoxyethoxy)ethylsulfanyl]succinic acid dioctadec-9-enyl ester (7a) (165 mg, 0.21 mmol) was dissolved in 1.0 mL of DMF followed by addition of triethylamine (117 µL, 0.84 mmol) and pentafluorophenyltrifluoroacetate (55 µL, 0.32 mmol) via a Hamilton syringe. The reaction mixture was stirred at room temperature for 2 h to allow formation of the activated pentafluorophenyl ester. The C-glycoside amine (5) (40 mg, 0.21 mmol) was added, and the reaction was allowed to stir overnight. A solution of 10% NaHCO3 (10 mL) was added directly to the reaction mixture, and then the aqueous layer was extracted with CHCl3 (4 × 10 mL). The organic extracts were combined and dried with anhydrous MgSO4, and the product was visualized with phosphomolybdic acid on a silica gel TLC plate. The Rf was 0.3 when eluted with 9:1 CHCl3/MeOH. The product was purified by silica gel chromatography using a gradient solvent system starting with pure CHCl3 followed by 99:1, 95:1, and 9:1 CHCl3/MeOH. The product (32 mg) was obtained as a colorless, viscous liquid in 17% yield. 1 H NMR (600 MHz, CD3OD) δ 0.80 (t, J ) 6.9 Hz, 6H), 1.19 (m, 44H), 1.51-1.57 (m, 4H), 1.94 (m, 8H), 2.65 (d, J ) 16.9 Hz, 1H), 2.74-2.88 (m, 3H), 3.21 (m, 8H), 3.30 (q, J ) 6.9 Hz, 1H), 3.36 (d, J ) 5.9 Hz, 2H), 3.39 (t, J ) 6.0 Hz, 1H), 3.55-3.68 (m, 10 H), 3.76 (bs, 1H), 3.92 (s, 2H), 3.98 (q, J ) 6.9 Hz, 2H), 4.04 (dt, J ) 2.6, 6.5 Hz, 2H), 5.25 (t, J ) 4.6 Hz, 4H). 13C NMR (150 MHz, CD3OD) δ 14.1, 22.6, 25.8, 27.2, 28.5, 29.2, 29.3, 29.4, 29.5, 29.7, 31.0, 31.9, 36.5, 39.7, 41.7, 50.8, 63.2, 65.3, 69.9, 70.0, 70.1, 70.2, 70.7, 76.4, 76.6, 76.8, 77.8, 78.8, 129.7, 129.9, 170.7, 171.6, 172.4. High-resolution mass spectrum (FAB+) calcd for C53H97NO12S (MH)+: 972.6810. Found: 972.6815. Gal-4-Unsat (8b). Prepared in a silimar manner to (8a). 1H NMR (600 MHz, CD3OD/CDCl3) δ 0.86 (t, J ) 6.7 Hz, 6H), 1.19-1.34 (m, 44H), 1.50-1.65 (m, 4H), 1.99 (q, J ) 12.6, 5.9 Hz, 8H), 2.66 (dd, J ) 17.0, 5.5 Hz, 1H), 2.82 (q, J ) 6.9 Hz, 1H), 2.88-2.98 (m, 3H), 3.27 (q, J ) 8.8 Hz, 1H), 3.37-3.43 (m, 4H), 3.45-3.52 (m, 6H), 3.583.70 (m, 10H), 3.73-3.76 (m, 1H), 3.78-3.84 (m, 1H), 3.86-3.91 (m, 1H), 4.02-4.07 (m, 4H), 4.11 (t, J ) 6.9 Hz, 2H), 5.33 (p, J ) 3.3, 6.9 Hz, 4H. Electrospray ionization mass spectrum (ESI+) calcd for C55H101NO13S (MH)+: 1016.34. Found: 1016.3. Gal-5-Unsat (8c). Prepared in a silimar manner to (8a). 1H NMR (600 MHz, CD3OD/CDCl3) δ 0.86 (t, J ) 7.1 Hz, 6H), 1.19-1.34 (m, 44H), 1.52-1.65 (m, 4H), 1.99 (q, J ) 12.4, 5.9 Hz, 8H), 2.66 (dd, J ) 17.0, 5.4 Hz, 1H), 2.82 (q, J ) 6.7 Hz, 1H), 2.88-2.98 (m, 3H), 3.23-3.31 (m, 3H), 3.47-3.52 (m, 3H), 3.59-3.70 (m, 14H), 3.783.84 (m, 2H), 3.85-3.90 (m, 2H), 3.95-4.06 (m, 7H), 4.11 (t, J ) 7.1 Hz, 2H), 5.33 (p, J ) 3.1, 6.3 Hz, 4H). Mass spectrum (FAB+) calcd for C57H105NO14S (MH)+: 1060.7. Found: 1061.2. Gal-3-Sat (11a). Prepared in a silimar manner to (8a). 1H and 13C assignments: see Table 1. High-resolution mass spectrum (FAB+) calcd for C53H101NO12S (MH)+: 976.7123. Found: 976.7156. Gal-4-Sat (11b). Prepared in a silimar manner to (8a). 1H NMR (300 MHz, CDCl ) δ 0.87 (t, J ) 7.0 Hz, 6H), 3 1.25 (m, 60H), 1.61 (p, J ) 12.0, 5.0 Hz, 4H), 2.67 (dd, J ) 16.4, 6.1 Hz, 1H), 2.77-3.01 (m, 4H), 3.25-3.37 (m, 2H), 3.48-3.55 (m, 2H), 3.62-3.72 (m, 14H), 3.85 (t, J ) 6.5 Hz, 2H), 4.03-4.15 (m, 10H). 13C NMR (75 MHz, CDCl3) δ 14.1, 22.7, 25.7, 25.8, 28.5, 29.2, 29.3, 29.5, 29.7,
30.8, 31.9, 36.5, 39.7, 41.7, 65.2, 65.7, 67.5, 70.1, 70.2, 70.5, 70.9, 77.4, 170.7, 171.6. Low resolution mass spectrum (FAB+) calcd for C55H105NO13S (MH)+: 1020.7. Found: 1021.3. ACKNOWLEDGMENT
This research was supported by NIH (AI 40359). Scientific discussions with Dr. A. Somogyi and Dr. A. S. Bhat are gratefully acknowledged. LITERATURE CITED (1) Fantini, J., Cook, D. G., Nathanson, N., Spitalnik, S. L., and Gonzalez-Scarano, F. (1993) Infection of colonic epithelial cell lines by type 1 human immunodeficiency virus is associated with cell surface expression of galactosylceramide, a potential alternative gp120 receptor. Proc. Natl. Acad. Sci. U.S.A. 90, 2700-2704. (2) Blanzat, M., Perez, E., Rico-Lattes, I., Prome, D., Prome, J. C., and Lattes, A. (1999) New catanionic glycolipids. 1. Synthesis, characterization, and biological activity of doublechain and gemini catanionic analogues of galactosylceramide (gal.beta.1cer). Langmuir 15, 6163-6169. (3) Cook, D. G., Fantini, J., Spitalnik, S. L., and GonzalezScarano, F. (1994) Application of high-performance thin-layer chromatography to investigate interactions between human immunodeficiency virus type 1 glycoprotein gp 120 and galactosylceramide. Methods Mol. Genet. 4, 86-92. (4) Delezay, O., Yahi, N., and Fantini, J. (1996) Detection of functional galactosylceramide (GalCer) receptors on CD4negative HIV-1 target cells. Perspect. Drug Discovery Des. 5, 192-202. (5) Ahlers, M., Muller, W., Reichert, A., Ringsdorf, H., and Venzmer, J. (1990) Specific Interactions of Proteins with Functional Lipid Monolayers - Ways of Simulating Biomembrane Processes. Angew. Chem., Int. Ed. Engl. 29, 1269-1285. (6) Adachi, K., Yamada, Y., Wada, H., Kameyama, A., Ishida, H., and Kiso, M. (1998) Synthesis of sialyl Lewis X ganglioside analogues containing a variable length spacer between the sugar and lipophilic moieties. J. Carbohydr. Chem. 17, 595607. (7) Sasaki, A., Murahashi, N., Yamada, H., and Morikawa, A. (1994) Syntheses of novel galactosyl ligands for liposomes and their accumulation in the rat liver. Biol. Pharm. Bull. 17, 680-685. (8) Bhat, S., Spitalnik, S. L., Gonzalez-Scarano, F., and Silberberg, D. H. (1991) Galactosyl ceramide or a derivative is an essential component of the neural receptor for human immunodeficiency virus type 1 envelope glycoprotein gp120. Proc. Natl. Acad. Sci. U.S.A. 88, 7131-7134. (9) Bertozzi, C. R., Cook, D. G., Kobertz, W. R., GonzalezScarano, F., and Bednarski, M. D. (1992) Carbon-linked galactosphingolipid analogues bind specifically to HIV-1 gp120. J. Am. Chem. Soc. 114, 10639-41. (10) Bertozzi, C., and Bednarski, M. (1996) Synthesis of Cglycosides; stable mimics of O-glycosidic linkages. Front. Nat. Prod. Res. 1, 316-351. (11) Rico-Lattes, I., Gouzy, M.-F., Andre-Barres, C., Guidetti, B., and Lattes, A. (1998) Synthetic bolaamphiphilic analogues of galactosylceramide (GalCer) potentially binding to the V3 domain of HIV-1 gp 120: key role of their hydrophobicity. New J. Chem. 22, 451-457. (12) Brown, D. A., and London, E. (2000) Structure and Function of Sphingolipid and Cholesterol-rich Memebrane Rafts. J. Biol. Chem. 275, 17221-17224. (13) Willker, W., Leibfritz, D., Kerssebaum, R., and Bermel, W. (1993) Magn. Reson. Chem. 31, 287-292. (14) Piantini, U., Sorensen, O. W., and Ernst, R. R. (1982) J. Am. Chem, Soc. 104, 6800-6801. (15) Frisch, B., Boeckler, C., and Schuber, F. (1996) Synthesis of short polyoxyethylene-based heterobifunctional cross-linking reagents. Application to the coupling of peptides to liposomes. Bioconjugate Chem. 7, 180-186.
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