| Glycobiology | Pages |
Inhibition of L- and P-selectin by a rationally synthesized novel core 2-like branched structure containing GalNAc-Lewisx and Neu5Ac[alpha]2-3Gal[beta]1-3GalNAc sequences
Introduction
Results And Discussion
Materials and methods
Acknowledgments
References
Inhibition of L- and P-selectin by a rationally synthesized novel core 2-like branched structure containing GalNAc-Lewisx and Neu5Ac[alpha]2-3Gal[beta]1-3GalNAc sequences
The selectins interact in important normal and pathological situations with certain sialylated, fucosylated glycoconjugate ligands containing sialyl Lewisx (Neu5Ac[alpha]2-3Gal[beta]1-4(Fuc[alpha]1-3)GlcNAc). Much effort has gone into the synthesis of sialylated and sulfated Lewisx analogs as competitive ligands for the selectins. Since the natural selectin ligands GlyCAM-1 and PSGL-1 carry sialyl Lewisx as part of a branched Core 2 O-linked structure, we recently synthesized Gal[beta]1-4(Fuc[alpha]1-3)GlcNAc[beta]1-6(SE-3Gal[beta]1-3)GalNAc1[alpha]OMe and found it to be a moderately superior ligand for L and P-selectin (Koenig et al., Glycobiology 7, 79-93, 1997). Other studies have shown that sulfate esters can replace sialic acid in some selectin ligands (Yeun et al., Biochemistry, 31, 9126-9131, 1992; Imai et al., Nature, 361, 555, 1993). Based upon these observations, we hypothesized that Neu5Ac[alpha]2-3Gal[beta]1-3GalNAc might have the capability of interacting with L- and P-selectin. To examine this hypothesis, we synthesized Gal[beta]1-4(Fuc[alpha]1-3)GlcNAc[beta]1-6(Neu5Ac[alpha]2-3Gal[beta]1-3)-GalNAc[alpha]1-OB, which was found to be 2- to 3-fold better than sialyl Lex for P and L selectin, respectively. We also report the synthesis of an unusual structure GalNAc[beta]1-4(Fuc[alpha]1- 3)GlcNAc[beta]1-OMe (GalNAc-Lewisx-O-methyl glycoside), which also proved to be a better inhibitor of L- and P-selectin than sialyl Lewisx-OMe. Combining this with our knowledge of Core 2 branched structures, we have synthesized a molecule that is 5- to 6-fold better at inhibiting L- and P-selectin than sialyl Lewisx-OMe,
By contrast to unbranched structures, substitution of a sulfate ester group for a sialic acid residue in such a molecule resulted in a considerable loss of inhibition ability. Thus, the combination of a sialic acid residue on the primary ([beta]1-3) arm, and a modified Lex unit on the branched ([beta]1-6) arm on an O-linked Core 2 structure generated a monovalent synthetic oliogosaccharide inhibitor superior to SLex for both L- and P-selectin.
Key words: Core 2 branched structures/selectins/selectin inhibitors
Introduction
The selectins are mammalian lectins that mediate the early steps of recruitment of leukocytes from the bloodstream in a variety of normal and pathological situations (see Bevilacqua and Nelson, 1993; Rosen and Bertozzi, 1994; Varki 1994, 1997; Lasky, 1995; Lowe and Ward, 1997; McEver and Cummings, 1997, for reviews). All three members of this family of adhesion molecules (L-, P-, and E selectin) have amino terminal C-type lectin domains that originally predicted their ability to bind carbohydrates. The role of sialic acids in ligand recognition by the selectins was also evident from early studies. A landmark event in this field occurred when several groups independently reported that the selectins recognize sialylated fucosylated ligands containing the sialyl Lex motif (SLex) Neu5Ac[alpha]2-3Gal[beta]1-4 (Fuc[alpha]1-3)GlcNAc[beta]1- (structure 1) (Phillips et al., 1990; Walz, 1990; Brandley, 1990). Later it was shown that sialyl Lea type structures can also bind with selectins (Berg et al., 1991). Also, Grinell et al., (1994) demonstrated that an uncommon sequence GalNAc[beta]1-4(Fuc[alpha]1-3)GlcNAc (GalNAc-Lex) occurred as a terminal structure on certain recombinant glycoproteins, and that it was a better inhibitor than SLex for E-selectin.
It has also been well documented that the sulfate group can replace sialic acid in some instances as ligands for selectins (Yuen et al., 1992; Imai et al., 1993). One of us (K.M.) was the first to synthesize 3-O-sulfoLex SE-3Gal[beta]1-4(Fuc[alpha]1-3)GlcNAc (Chandrasekaran et al., 1991, 1992; Matta, 1997). Synthetic sulfo Lex derivative SE-3Gal[beta]1-4(Fuc[alpha]1-3)GlcNAc[beta]1-OMe 2 is recognized by all three selectins (Koenig et al., 1997). These findings stimulated interest in the chemical and enzymatic synthesis of SLex 1 and its isomer SLea, Neu5Ac[alpha]2- 3Gal[beta]1-3(Fuc[alpha]1-4)GlcNAc[beta]1-OMe (structure 3), their sulfated analogs and other modified structures related to these motifs (for example, Nicolaou et al., 1991, 1993; Tyrrel et al., 1991; Maaheimo et al., 1995; Bamford et al., 1996; Dupre et al., 1996; Marron et al., 1996; Sanders et al., 1996; Sprengard et al., 1996; Tsuboi et al., 1996; Woltering et al., 1996).
Most of the efforts towards the procurement of such small molecule inhibitory ligands for selectins have been centered on the synthesis of mimics of SLex type structures. Of note, the affinity of selectins for all these synthetic analogs appear to be much poorer than those of the natural glycoconjugate ligands such as GlyCAM-1, CD34 (Baumheiter et al., 1993; Hemmerich 1994, 1995), P-selectin glycoprotein ligand-1 (PSGL-I) (Moore et al., 1994; Wilkins et al., 1996), and mucosal cell adhesion molecule (MadCAM-I) (Berg et al., 1993), all of which are mucin type glycoproteins carrying large numbers of O-linked chains which include sialylated fucosylated sequences (Krall, 1997).
The complex chains of O-linked glycoproteins consist of three distinct regions: core, backbone, and nonreducing terminus. The core structures are unique to O-linked glycoproteins, while the backbone and nonreducing terminus can be found in glycopeptides and also as part of N-linked glycoproteins. Among the known core structures (Schachter, 1986; Varki and Marth, 1995) in which GalNAc is [alpha]-linked to Ser/Thr, Core 2, GlcNAc[beta](1-6)[Gal[beta](1-3)]GalNAc[alpha]1-O Ser/Thr, appears to be the most prominent among these six structures, and our synthetic selectin inhibitor
 |
27 |
Structural studies on the carbohydrate moieties of these ligands are only available for PSGL-1 and GlyCAM-1. Surprisingly, PSGL-1 is not heavily fucosylated, and the majority of O-glycans are disialylated or neutral forms of core 2 structures (Wilkins et al., 1996). The following Core 2 branched SLex containing structure has been reported as a significant component of both PSGL-1 and GlyCAM-1 (Hemmerich et al., 1995):
 |
4 |
However, according to Hemmerich (1995), sulfate was reported to be present at the C-6 position of galactose, or 6 position of GlcNAc of the SLex moiety linked to the C-6 position of GalNAc (structures 5 and 6). We therefore recently synthesized Neu5Ac[alpha]2-3(SE-6Gal[beta]1-4(Fuc[alpha]1-3)GlcNAc[beta]1-OMe (structure 7) (Jain et al., 1994). However, our data indicate that this is neither a superior nor a specific ligand for L-selectin (Koenig et al., 1997). The only obvious difference was that recognition by E-selectin was lost upon sulfation. The isomeric compound Neu5Ac[alpha]2-3Gal[beta]1-4(Fuc[alpha]1-3)SE-6GlcNAc[beta]1-3Gal was synthesized by Scudder et al. (1994) and found to be only moderately superior to SLex as an inhibitor of L-selectin.
 |
|
| 5 | 6 |
One explanation of the lack of high affinity of 6 and 6[prime]-sulfo Lewisx for L-selectin (compared to native ligand of GLYCAM-1) could be that the whole carbohydrate moiety represented in 5 and 6 is involved in interaction with L-selectin. In other words, the Neu5Ac[alpha]2-3Gal[beta]1-3GalNAc- moiety of the Core 2 mucin structure might exert an appreciable impact on binding of L-selectin along with sulfated sialyl Lewisx linked at C-6 position in structures 5 and 6. Based on these data, we decided to examine the role of the Neu5Ac[alpha]2-3Gal[beta]1-3GalNAc sequence by synthesizing Core 2 branched structures. For example, we synthesized structure 34:
 |
We have now synthesized GalNAcLex-[beta]-OMe and studied its inhibitory properties towards all three selectins. We have combined this with our knowledge of the properties of the sialylated Core 2 structures to rationally design a better inhibitor 27 for L- and P-selectin.
Table I.
| IC50 values (±SD) (µM) against SLex | ||||
| Compound | R | E-Selectin | P-Selectin | L-Selectin |
| 1 | Neu5Ac[alpha]2-3Gal[beta] | 550 ± 40 | 500 ± 25 | 600 ± 30 |
| 18 | GalNAc[beta] | 500 ± 120 | 400 ± 100 | 300 ± 100 |
Table II.
| IC50 values (±SD) (µM) against SLex | ||||
| Compound | R | E-Selectin | P-Selectin | L-Selectin |
| 24 | OH | >500 | 400 ± 60 | 300 ± 80 |
| 27 | Neu5Ac | >500 | 85 ± 55 | 105 ± 75 |
| 31 | 3-SE | >500 | >500 | 500 ± 70 |
Table III.
| IC50 values (±SD) (µM) against SLex | ||||
| E | L | P | ||
| 8 | R = H, R[prime] = SE | 600 ± 100 | 300 ± 50 | 270 ± 50 |
| 32 | R = SE, R[prime] = Neu5Ac | 590 ± 90 | 500 ± 10 | 585 ± 25 |
| 33 | R = Neu5Ac, R[prime] = SE | 200 ± 40 | 620 ± 90 | 450 ± 70 |
| 34 | R = H, R[prime] = Neu5Ac, [alpha]-benzyl analog GalNAc |
500 ± 50 | 200 ± 50 | 300 ± 55 |
Results and discussion
A series of branched structures have been examined for inhibition of these three selectins. Synthetic strategies for the synthesis of oligosaccharides represented by structures 32 and 33 (Table III) have been described by Huang et al. (unpublished observations), whereas synthesis of 34 will be published elsewhere. In the present studies, under Materials and methods we have described the synthesis of 18 (Table I), 24, 27 and 31 (Table II). Figure
Figure 1. Key glycosyl donors involved in the synthesis of target compounds. Figure 2. Inhibitory properties of Compounds 24, 27, and 31 against E-, L-, and P-selectin against immobilized SLex. Compounds 24, 27, and 31 were tested for their ability to inhibit the binding of each of the recombinant selectins to immobilized SLex in ELISA inhibition experiments, as described under Materials and methods. The IC50 values from such studies and the structures of the compounds 24, 27, and 31 are represented in Table III. Scheme I represents the synthesis of 18 and key glycosyl donor 20 for the synthesis of GalNAc Lex linked compounds. Regioselective acylation of methyl 3-O-benzyl-2-deoxy-2- phthalimido-[beta]-d-glucopyranoside 12 (Alais and David, 1990) with acetyl chloride in pyridine afforded the 6-O-acetyl derivative (13) in 79% yield. Glycosylation (catalyzed by N-iodosuccinimide-triflic acid (Veeneman et al., 1990) of 13 with donor 11 gave (14) in 52% yield. Hydrogenolytic cleavage of the benzyl group of 14 in glacial acetic acid and in the presence of 10% palladium-on-carbon furnished the partially protected disaccharide (15). [alpha]-l-Fucosylation of 15 with the tri-O-benzyl thiophenyl donor (16) in the presence of N-iodosuccinimide-triflic acid (Scheme I) afforded the fully protected trisaccharide derivative (17) in 68% yield. Compound 17 was converted, in 76% yield, into the diphthaloyl peracetate (19) by hydrogenolysis (glacial acetic acid-10% Pd-C), followed by acetolysis (acetic anhydride-acetic-acid-sulfuric acid). Compound 19 was, in turn, converted (49% yield) into the key glycosyl donor (20) by treatment with thiophenol and boron-trifluoride ethearate. A portion of 17 was converted to free trisaccharide GalNAc[beta]1-4(Fuc[alpha]1-3)GlcNAc[beta]1-OMe 18 in three steps; removal of O-benzyl group by hydrogenolysis, and phthalamido removal followed by N-acetylation. The enzymatic synthesis of 18 has been reported (Bergwerff et al., 1993). Scheme I. (a) Acetyl chloride-pyridine-CH2Cl2, -30°C, 2 h; (b) 10% Pd-C, acetic acid; (c) NIS-triflic acid; CH2Cl2-ether (1:1, v/v), 0°C; (d) acetic acid- H2SO4-acetic anhydride, 5°C, 16 h; (e) BF3-ethereate-thiophenol, CH2Cl2, 5 h, 30°C; (f) hydrazine hydrate ethanol (4:1, v/v), 100°C, 16 h; (g) methanol triethylamine-acetic anhydride (4:2:1, v/v). Selective deacylation of methyl O-(2,3,4-tri-O-acetyl-6-O- trimethylacetyl-[beta]-d-galactopyranosyl)-(1-3)-2-acetamido-2- deoxy-4,6-O-(4-methoxybenzylidene)-[alpha]-d-galactopyranoside (21) in 1:1 dichloromethane-methanol (0.5 M NaOMe, pH ~11), followed by chloroacetylation and cleavage of the 4-methoxybenzylidene acetal with 70% aqueous acetic acid afforded compound 22 (unpublished observations) (Scheme II).
Scheme II. (a) NIS-triflic acid; CH2Cl2, -65°C; (b) thiourea-lutidine, ethanol- CH2Cl2 (1:1, v/v), 6 h, 80°C; (c) hydrazine hydrate-methanol (4:1, v/v); (d) methanol-triethylamine-acetic anhydride (4:2:1, v/v); (e) MeOH-MeONa. N-Iodosuccinimide-triflic acid glycosylation of compound (22) with glycosyl donor 20, followed by removal of the chloroacetyl groups in the [beta]-galactopyranosyl residue afforded the partially-protected pentasaccharide intermediate (23) in 35% yield (based on 22). The 1H NMR spectrum of 23 was in conformity with the overall structure expected. Compound 23 is the key intermediate for obtaining the desired inhibitors 24 and 27. Thus, for the production of compound 24 the partially-protected 23 was subjected to complete removal of the blocking groups in three successive steps (see Materials and methods), whereas for obtaining the sialylated compound 27, the same intermediate 23 was allowed to react with known (Marra and Sinay, 1989) sialyl donor (25) to give, in 47% yield, the hexasaccharide derivative (26) (Scheme III). The [alpha]-configuration for the sialic acid residue was confirmed by the 1H NMR of 26 which exhibited a double doublet at [delta] 2.67 (J = 4.6 Hz), attributable to H-3e of this residue. The conversion of 26 into the target compound (27) was performed in four successive steps: (1) lithium iodide-pyridine (Nicolaou et al., 1992) (methyl ester to free acid), (2) methanol-hydrazine hydrate (removal of the phthalimido group), (3) acetic anhydride-methanol-dichloromethane (N-acetylation), and (4) methanolic sodium methoxide (O-deacetylation). The 1H and 13C NMR spectra of 27 were in accord with the structure assigned. A similar glycosylation of compound (Jain and Matta, 1994) (28) with 20 gave, in 76% yield, the pentasaccharide derivative (29), the chloroacetyl group of which was removed to give, in 71% yield, intermediate (30) (Scheme IV). For the production of the target compound (31), intermediate 30 was treated with five molar equivalents of sulfur trioxide-pyridine complex in N,N-dimethylformamide at 0°C (Scheme IV), followed by customary removal of the protecting groups. Column chromatographic purification on silica gel then furnished the desired compound 31 in 37% yield. The structure and purity of our synthetic compounds was established by TLC, NMR, and FAB mass spectroscopy. Inhibition studies To ascertain the importance of GalNAc in the GalNAcLex structure, we synthesized the two compounds shown in Table 1, and compared their inhibitory properties against all three selectins. As shown in the table, the presence of a [beta]-linked GalNAc residue at the 4 position of GlcNAc(Fuc[alpha]1-3) was clearly superior to having the [alpha]2-3sialylated Gal residue (Neu5Ac2-3Gal) of SLex, structure 1. These data confirm the previous report for E-selectin (Grinnell et al., 1994), and extend them to show that GalNAcLex is an even better inhibitor than SLex of L- and P-selectin. Based upon this finding and the rationale discussed in the introduction, we felt that Neu5Ac[alpha]2-3Gal[beta]1-3GalNAc sequence in Core 2 structure ligands could combine with GalNAcLex to obtain even better binding to L- and P-selectin. We have therefore synthesized an oligosaccharide containing both of these sequences. The results of inhibition studies shown in Table II support the value of this approach. It can be seen that putting the GalNAc-Lex into a Core 2 branched structure made no difference when compared to GalNAc-Lex. However, adding a sialic acid residue to the Gal[beta]1-3GalNAc sequence of this structure potentiated the inhibitory properties to the point where it is ~6-fold better as an inhibitor of L- and P-selectin. The improvement in binding to E-selectin was not as great. Interestingly, a sulfate ester at the 3-position of Gal[beta]1-3GalNAc could not substitute for the sialic acid residue. These data suggest that L- and P-selectin are recognizing a specific clustered motif involving components of both branches of the Core 2 structure. Our interest in the synthesis of branched ligands containing Neu5Ac[alpha]2-Gal[beta]1-3GalNAc[alpha]- arm has been also due to the following findings. We recently identified a 3-O-sulfotransferase from ovarian, breast, and other tissues capable of incorporating sulfate esters at C-3 of Gal in the Gal[beta]1-3GalNAc sequence (Kuhns et al., 1995; Chandrasekaran et al., 1996, 1997). Recently, reports of a native oligosaccharide carrying SE-3Gal[beta]1-3GalNAc have appeared (Chance and Mawhinney, 1996; Florea et al., 1997). We synthesized a branched derivative Core 2 structure 8, containing a 3-O-sulfate ester group instead of sialic acid moiety and found it to be inhibitory for L- and P- selectin (Koenig et al., 1997). This encouraged us to synthesize 34 having the Neu5Ac[alpha]2-3Gal[beta]1-3GalNAc sequence. We also synthesized structure 9 wherein the sulfate ester is located at C-6 of Gal in the Gal[beta]1-3GalNAc moiety In Table III, we have given the inhibition data for compounds 8, 32-34 and sialyl Lewisx. The carbohydrate sequence represented by structure 32 has been reported to be part of respiratory mucin (Lo-Guidice et al., 1994). Compound 32 was found to behave similar to sialyl Lewisx. Compound 33 was 2-fold better than sialyl Lewisx against E-selectin and slightly better against P-selectin. Comparative inhibition data of 8, 32, and 33 suggest that in core 2 structures, sialyl Lewisx arm is a preferred site for interacting with E-selectin. However, we have observed an interesting phenomena for L- and P-selectin ligands containing sulfate at the C-3 position of galactose on one arm and sialic acid at C-3 of galactose in the other arm of Core 2 structure. When sulfate is present at C-3 of Gal in Gal[beta]1-3GalNAc the presence of sialic acid at C-3 of Gal in the Lex moiety further linked to C-6 of GalNAc (as in compound 33) has a negative impact toward binding, as our previous studies showed that Gal[beta]1-4(Fuc[alpha]1-3)GlcNAc[beta]1-6(SE-3Gal[beta]1-3)GalNAc[alpha]1-OMe 8 is a 2-fold better ligand than sialyl Lewisx for L- and P-selectin (Koenig et al., 1997). It is also surprising that when sialic acid is linked to C-3 of Gal in Gal[beta]1-3GalNAc, the presence of a 3-O-sulfo Lex moiety on C-6 of GalNAc (as in compound 32) also has a negative impact. Our compound Gal[beta]1-4(Fuc[alpha]1-3)GlcNAc[beta]1-6(Neu5Ac[alpha]2- 3Gal[beta]1-3)GalNAc[alpha]1-OBn 34 (R.K.Jain and K.L.Matta, unpublished observations) is almost 3-fold better than sialyl Lewisx for L-selectin. It is apparent that in an approach for the construction of a library of analogs of the Gal[beta]1-4(Fuc[alpha]1-3)GlcNAc[beta]1-6(Gal[beta]1-3)GalNAc[alpha]1- sequence if sialic acid (or replacement of sialic acid by a carboxy alkyl group) (Allanson, 1993) is kept at the C-3 position of galactose in one arm it will be advisable to introduce sulfate at a position other than C-3 of galactose in the other arm. Thus, it is not surprising to see a sulfate ester group located on C-6 of galactose or GlcNAc, as shown in 5 and 6 which represent portions of GlyCAM-1. Once again, our recent inhibition studies of these selectins with branched Core 2 structures warrant mention here. Interestingly, our synthetic compound 9 having SE-6Gal[beta]1-3GalNAc is found to be a ligand for both L and P selectin. It is striking that 6[prime]-sulfo Lex or 6-sulfo Lex type structures without a sialic acid moiety do not act as inhibitors of selectin, whereas the presence of a 6-O-sulfo group on the galactose moiety of Gal[beta]1-3GalNAc showed inhibitory effects. It should be noted that the SE-6Gal[beta]1-3GalNAc sequence has been reported to be a part of various O-linked mucin glycoproteins (Chance et al., 1996). Also, the Neu5Ac[alpha]2-3(SE-6Gal)[beta]1-3GalNAc sequence has been suggested to comprise a part of O-linked glycoprotein (Mawhinney et al., 1992). Thus, it is quite possible that earlier mentioned naturally occurring selectin ligands such as CD34 or other unidentified sialo mucin type ligands (Kraal and Mebius, 1997) may contain 6[prime]-sulfo Gal in the Gal[beta]1-3GalNAc chain contained in a core 2 structure in the form of SE-6Gal[beta]1-3GalNAc or SE-6(Neu5Ac[alpha]2-3)Gal[beta]1-3GalNAc[alpha] sequences. In fact, the present studies demonstrating the ability of Neu5Ac[alpha]2- 3Gal[beta]1-3GalNAc to bind to selectins, combined with our previous finding of inhibitory impact of SE-6Gal[beta]1-3GalNAc, enhances our interest in the preparation of branched core 2 structure with SE-6(Neu5Ac[alpha]2-3)Gal[beta]1-3GalNAc having sialyl Lex or Lex moiety O-linked at C-6 position of GalNAc. It is not clear at this time why an oligosaccharide containing GalNAc[beta]1-4(Fuc[alpha]1-3)GlcNAc[beta]1-OMe sequence, which lacks a negative charge, appears to be a better ligand for E-selectin. However, Grinnell et al. (1994) reported SLex and GalNAc[beta]1-4(Fuc[alpha]1-3)GlcNAc appear to be structurally similar through modeling studies. We became interested in the synthesis of oligosaccharides containing GalNAc[beta]1-4(Fuc[alpha]1-3)GlcNAc determinant partly because GalNAc as a starting material is cheaper than sialic acid. Moreover, the chemical synthesis of an oligosaccharide containing the GalNAc[beta]1-4(Fuc[alpha]1-3)GlcNAc determinant is not as time consuming as an oligosaccharide with sialyl Lewis x moiety. GalNAc[beta]1-4(Fuc[alpha]1-3)GlcNAc[beta]- sequence has been found to be a part of various glycoproteins, especially N-linked glycoproteins (Dell et al., 1995; Manzella et al., 1996). The disaccharide GalNAc[beta]1-4GlcNAc[beta]- is part of certain O-linked glycoproteins (Hirano et al., 1993), some containing fucose (Siciliano, 1994). The sequence GalNAc[beta]1-4(Fuc[alpha]1-3)GlcNAc has been reported to be a part of oligosaccharides isolated from sea squirt H antigen, an O-linked glycoprotein (Ohata et al., 1991). The sequence shown in structure 24 has been reported recently (Strecker et al., 1994). Thus, our present studies provide a new avenue toward the synthesis of GalNAc-Lex containing branched ligands for the inhibition of L- and P-selectin that is considerably better than SLex. Furthermore, we have clearly demonstrated that the Neu5Ac[alpha]2-3Gal[beta]1-3GalNAc sequence of the core 2 structure contributes to the binding of L- and P- selectin.
Scheme III. (a) NIS-triflic acid, propionitrile, -65°C, 3 h; (b) LiI-pyridine, 120°C, 6 h; (c) methanol-hydrazine hydrate (4:1, v/v), 80°C, 16 h; (d) methanol-CH2Cl2 (1:1, v/v), acetic anhydride, 0°C, 1 h; (e) MeOH- MeONa, 48 h.
8
9
Materials And Methods
Materials
Sources of several chemicals used are indicated below. Most of the other materials used were obtained from the Sigma Chemical Co. The following materials were obtained from the sources indicated
All other chemicals were of reagent grade or better, from commercial sources. The recombinant L-selectin Ig-fusion chimeric protein was prepared as described (Norgard et al., 1993), and the E- and P-selectin Ig-fusion chimeric constructs were produced using the pcDM8 vectors (Nelson et al., 1993).
ELISA inhibition assays
ELISA inhibition assays were done as previously reported (Koenig et al., 1997). Sterile polystyrene 96 well ELISA plates (no. 25801, Corning) were coated with 200 ng of polyacrylamide-SLex (no. 18205PA, Syntesome) by overnight incubation at 4°C in 100 µl of 50 mM sodium carbonate/bicarbonate buffer, pH 9.5. Plates were then blocked with 200 µl per well of assay buffer: 20 mM Hepes (no. 16926, U.S. Biochemical), 125 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, 1% protease-free BSA (no. 82-045-1, Pentex, Miles, Inc.), pH 7.45 (osmolality 290 milliosmoles, determined with a Vapor Pressure Osmometer, model 5500XR, Wescor, Inc.) for a minimum of 2 h at 4°C. During the blocking step, the selectin chimeras were separately preincubated at 4°C with a secondary antibody, peroxidase-conjugated goat anti-human IgG (No. 109-035-098, Jackson Immunoresearch Laboratories, Inc.) in assay buffer for ~1 h. Final selectin-Rg concentration was 20 nM, and the optimal secondary antibody dilution was determined to be 1:1000 for the particular serum used. Potential inhibitors were serially diluted in assay buffer, at 2× the final required concentration. The selectin-Rg/secondary antibody stock was aliquoted into tubes containing an equivalent volume of inhibitor solution; buffer alone for the positive control, or buffer with 10 mM Na2 EDTA, pH 7.5, for the negative control (giving a final concentration of 5 mM EDTA). These tubes were preincubated at 4°C for 30 min, and then added to ELISA plates, in duplicates, at final well volume of 100 µl. After 4 h of plate incubation at 4°C, plates were washed three times with 200 µl per well of assay buffer at 4°C, followed by development with 150 µl per well of OPD solution at room temperature: 0.002 mg o-phenylenediamine dihydrochloride (OPD)/ml in 50 mM sodium citrate, 50 mM disodium phosphate buffer, pH 5.2 containing 1 µl/ml 30% H2O2. Using a timer, each well was sequentially quenched with 40 µl of 4 M H2SO4 after a fixed time of peroxidase reaction. Softmax software and a microplate reader (Molecular Devices, Inc.) determined and recorded absorbance at 492 nM. Prior to curve fitting, the data was changed into percentages for comparative purposes, using the formula: ([average of duplicates) - (negative control)]/[(positive control) - (negative control)] × 100) again with the Softmax software.
General methods
Optical rotations were measured at ~25°C with a Perkin-Elmer 241 Polarimeter. Thin layer chromatography (TLC) was conducted on glass plates precoated with 0.25 mm layers of silica gel 60F-254 (Analtech GHLF uniplates). The compounds were located by exposure UV light or by spraying with 5% H2SO4 in ethanol and charring, or by both techniques. The silica gel used for column chromatography was Baker Analyzed (60-200 mesh). NMR spectra were recorded at ~25°C, 1H-spectra with a Varian EM-390 at 90 MHz and with a Bruker AM-400 at 400 MHz, and the 13C-spectra with a Bruker AM-400 at 100.6 MHz. All chemical shifts are referenced to tetramethylsilane. Solutions in organic solvents were generally dried with anhydrous sodium sulfate. Dichloromethane, N,N-dimethylformamide, 1,2-dichloroethane, benzene, and 2,2-dimethoxypropane were kept dried over 4 Å molecular sieves. Elemental analyses were performed by Robertson Laboratory, Madison, New Jersey, USA.
General procedure for glycosidation
Using 1-thiophenyl glycoside as glycosyl donor, a solution of the acceptors (1.0-1.2 mmol) and donor (1.0-1.5 mmol) and N-iodosuccinimide (2.5-3.0 mmol) in dichloromethane (20 ml, for preparation of compound 14, 23,and 29) or 1:1 dichloromethane-ether (30 ml, for compound 17), propionitrile (15 ml, for compound 26) was stirred for 0.5 h with 4 Å molecular sieves (2 g) under an argon atmosphere at 0°C (compound 14), or -40°C (compound 26) or -65°C (compound 23 and 29). Then a dilute solution of trifluoromethanesulfonic acid (triflic acid, 0.2 ml in 10 ml dichloromethane or propionitrile) was added dropwise. Stirring was continued at the same temperature for an additional hour, and the acid was neutralized with aqueous sodium bicarbonate solution. The mixture was filtered (Celite bed), and the solids thoroughly washed with water, saturated sodium bicarbonate solution, 10% sodium thiosulfate solution, dried and concentrated under diminished pressure.
Phenyl 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-1-thio-[alpha]/[beta]-d- galactopyranoside (11)
To a stirred solution of 10 (6.4 g, 13.4 mmol) in dichloromethane (70 ml) was added thiophenol (4.0 ml, 36.4 mmol) and BF3 ethereate (4.0 ml, 28.4 mmol). Stirring was continued for 4 h at room temperature. The reaction mixture was then washed with aqueous sodium bicarbonate solution, water, dried, and concentrated. The residue was purified on a column of silica gel with a solvent gradient consisting of hexane-ethyl acetate 3:2 -> 1:1 (v/v) to afford 11 (6.1 g, 84%); [[alpha]]D +28° (c 1.0, CHCl3); 1H NMR (CD2Cl2): [delta] 7.88-7.26 (m, 9 H, arom.), 5.79 (dd, J = 9.1 Hz and 10.1 Hz, 1 H, H-3), 5.70 (d, J = 10.5 Hz, 0.8 H, H-1), 5.49 (d, J = 3.5 Hz, 1 H, H-4), 2.2, 2.06, and 1.98 (each s, 3 × OAc-[alpha]), 2.18, 2.04, and 1.81 (each s, 3 × OAc-[beta]).
Anal Calc. for C26H25NO9S: C, 59.19; H, 4.78; N, 2.66. Found: C, 59.21; H, 4.91; N, 2.54.
Methyl 6-O-acetyl-3-O-benzyl-2-deoxy-2-phthalimido-[beta]-d- glucopyranoside (13)
To a cold (-30°C), stirred solution of methyl 3-O-benzyl-2-deoxy-2-phthalimido-[beta]-d-glucopyranoside 12 (4.8 g, 11.6 mmol) in pyridine (50 ml) was added, dropwise, a solution of acetyl chloride (0.97 ml, 12.4 mmol) in pyridine-dichloromethane (1:2, 15 ml). Stirring was continued for 2 h at the same temperature, and then the mixture was kept overnight at 5-6°C. It was then cooled to O°C and methanol (5 ml) was added to decompose excess reagent. The solvents were removed under diminished pressure and the residue dissolved in dichloromethane. The organic layer was successively washed with 10% aqueous hydrochloric acid, water, saturated sodium bicarbonate solution, dried, and concentrated. The crude product was purified on a column of silica gel with a solvent gradient consisting of 40-50% ethyl acetate in hexane to give 13 (4.2 g, 79%); [[alpha]]D +23° (c 1.0, CHCl3); 1H NMR (CD2Cl2): [delta] 7.73-6.98 (m, 9 H, arom.), 5.04 (d, J = 8.3 Hz, 1 H, H-1), 4.04 (dd, J = 8.6 Hz, H-6), 3.37 (s, 3 H-OMe), 2.14 (s, 3 H, OAc).
Anal Calc. for C24H25NO8: C, 63.29; H, 5.53; N, 3.08. Found: C, 63.31; H, 5.60; N, 3.01.
Methyl O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-[beta]-d-galacto-pyranosyl)-(1
->4)-6-O-acetyl-3-O-benzyl-2-deoxy-2-phthalimido- [beta]-d-glucopyranoside (14)
Glycosidation of 13 (4.0 g, 8.6 mmol) with 11 (6.0 g, 11.1 mmol) followed by silica gel column chromatography (solvent gradient consisting of hexane-ethyl acetate 3:2 -> 1:1) afforded 14 (6.1 g, 52%); [[alpha]]D +10° (c 1.0, CHCl3); 1H NMR (CD2Cl2): [delta] 7.90-6.92 (m, 13 H, arom.), 5.79 (dd, 1 H, H-3[prime]), 5.48 (d, J = 8.7 Hz, 1 H, H-1), 5.44 (d, J = 3.1 Hz, 1 H, H-4[prime]), 4.91 (dd, 1 H, H-2[prime]), 4.46 (d, J = 8.0 Hz, 1 H, H-1[prime]), 3.27 (s, 3 H, OMe), 2.10, 2.03, 2.01, and 1.81 (each s, 12 H, 4 × OAc).
Anal Calc. for C44H44N2O17: C, 60.54; H, 5.08; N, 3.21. Found: C, 60.35; H, 5.11; N, 3.16.
Methyl O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-[beta]-d-galactopyranosyl)-(1
->4)-6-O-acetyl-2-deoxy-2-phthalimido-[beta]-d- glucopyranoside (15)
A mixture of compound 14 (1.0 g) and 10% Pd-C (1.0 g) in glacial acetic acid (20 ml) was shaken at ~345 kPa. The suspension was then filtered (Celite bed), the solids were thoroughly washed with glacial acetic acid, and the combined filtrate and washings were concentrated under reduced pressure. The crude product was applied to a column of silica gel and eluted with hexane-ethyl acetate 2:3 -> 1:4 (v/v). The fractions corresponding to 15 were pooled and concentrated to give an amorphous solid (0.55 g, 62%); [[alpha]]D -11° (c 1.5, CHCl3); 1H NMR (CD2Cl2): [delta] 7.89-7.74 (m, 8 H, arom.), 5.79 (dd, 1 H, H-3[prime]), 5.06 (d, J = 9.1 Hz, 1 H, H-1), 4.54 (dd, 1 H, H-2[prime]), 3.34 (s, 3 H, OMe), 2.17, 1.91, 1.82 and 1.81 (each s, 12 H, 4 × OAc).
Anal Calc. for C37H38N2O17: C, 56.77; H, 4.89; N, 3.58. Found: C, 56.82; H, 4.91; N, 3.49.
Methyl O-(2,3,4-tri-O-acetyl-2-deoxy-2-phthalimido-[beta]-d-galactopyranosyl)-(1
->4)-O-[2,3,4-tri-O-benzyl-[alpha]-l-fucopyranosyl)-(1->3)-O]-6-O-acetyl-2-deoxy-2-phthalimido-[beta]-d-glucopyranoside (17)
Glycosidation of 17 (3.9 g, 5.0 mmol) with 16 (10.8 g, 20.0 mmol) in dichloromethane-ether (1:1, 100 ml) and purification of the crude product mixture by silica gel column chromatography [solvent gradient consisting of hexane-ethyl acetate 3:2 -> 1:1 (v/v) furnished compound 18 (3.0 g, 68%); [[alpha]]D +3° (c 1.5, CHCl3); 1H NMR (CD2Cl2): [delta] 7.88-6.99 (m, 23 H, arom.), 5.79 (dd, 1 H, H-3[prime]), 5.31 (d, J = 4.1 Hz, 1 H, H-1[prime][prime]), 4.89 (d, J = 8.6 Hz, 1 H, H-1), 4.82 (d, J = 10.0 Hz, 1 H, H-1[prime]), 3.27 (s, 3 H, OMe), 2.07, 2.03, 2.02, and 1.78 (each s, 12 H, 4 × OAc), 1.30 (d, J = 6.5, Hz, 3 H, CMe).
Anal Calc. for C64H66N2O21: C, 64.10; H, 5.55; N, 2.34. Found: C, 64.31; H, 5.51; N2, 2.16.
Methyl O-(2-acetamido-2-deoxy-[beta]-d-galactopyranosyl)- (1->4)-O-[[alpha]-l-fucopyranosyl)-(1->3)-O]-2-acetamido-2-deoxy-[beta]-d-glucopyranoside (18)
A mixture of 17 (0.3 g) and 10% Pd-C (0.8 g) in glacial acetic acid (20 ml) was shaken under hydrogen at ~345 kPa for 16 h at room temperature. After processing as described for the preparation of 16 (from 12) and followed by phthalamido removal with hydrazine hydrate-ethanol (1:4; v/v) at 100°C for 16 h and N-acetylation with methanol-triethylamine-acetic anhydride (4:2:1, v/v) afforded 18. After purification over a silica gel column with CHCl3-MeOH-water (13:6:1->5:4:1) as the eluent, 18 (0.07 g, 51%); [[alpha]]D -88° (c 0.5, H2O); 1H NMR (D2O): [delta] 5.12 (d, J = 3.9 Hz, 1H, H-1[prime][prime]), 4.55 (d, J = 8.4 Hz, 1H, H-1), 4.49 (d, J = 7.8 Hz, 1H, H-1[prime]), 3.52 (s, 3H, OMe), 2.09 and 2.05 (each s, 6 H, 2×NAc), 1.28 (d, J = 6.6 Hz, 3 H, CMe); 13C-NMR: GalNAc-[beta]-(1->4) residue: 100.69 (C-1), 51.34 (C-2), 70.97 (C-3), 66.50 (C-4), 72.36 (C-5), 60.40 (C-6), 21.15 (NAc); Fuc-[alpha]-(1->3) residue: 97.41 (C-1), 66.68 (C-2), 68.16 (C-3), 69.79 (C-4), 65.90 (C-5), 14.33 (C-6); GlcNAc-[beta]-OMe residue: 99.72 (C-1), 54.38 (C-2), 74.44 (C-3), 73.81 (C-4), 73.70 (C-5), 59.01 (C-6), 56.09 (OMe), 21.18 (NAc). ES-MS: m/z = 583.22 [M-1]- (584.58).
Anal Calc. for C23H40N2O15: C, 47.25; H, 6.90; N, 4.79. Found: C, 47.09; H, 6.85; N, 4.67.
O-(3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-[beta]-d-galactopyranosyl)-(1->4)-O-[(2,3,4-tri-O-acetyl-[alpha]-l-fucopyranosyl)- (1->3)-O]-1,6-di-O-acetyl-2-deoxy-2-phthalimido-d-glucopyranose (19)
A solution of compound 17 (6.0 g) in glacial acetic acid (60 ml) was treated with 10% Pd-C (4.0 g) and the mixture was shaken for 16 h at room temperature under hydrogen (~345 kPa). The suspension was then filtered (Celite bed) and the solids were thoroughly washed with methanol. The filtrate and washings were combined and concentrated and the residue was directly utilized in the next step. A solution of this residue in acetic acid (80 ml) and acetic anhydride (96 ml) containing conc. H2SO4 (8.4 ml) was stirred for 16 h at 5°C. The mixture was then diluted with dichloromethane (700 ml), and successively washed with water, saturated aqueous sodium bicarbonate solution, water, dried, evaporated to dryness, and redissolved in dichloromethane. Addition of ether-hexane caused the precipitation of 19 as an amorphous solid (4.0 g, 76%); [[alpha]]D -63° (c 1.0, CHCl3); 1H NMR (CD2Cl2): [delta] 7.92-7.78 (m, 8 H, arom.), 6.00 (d, J = 8.5 Hz, 0.6 H, H1[beta]), 5.94 (d, J = 3.2 Hz, 0.4 H, H-1[alpha]), 5.82 (dd, 1H, H-3[prime]) 5.51 (d, J = 3.8 Hz, 1 H, H-4[prime][prime]), 5.48 (d, J = 3.6 Hz, 1 H, H-4[prime]), 5.40 (d, J = 4.6 Hz, 1 H, H-1[prime][prime]), 4.83 (d, J = 10.6 Hz, 1 H, H-1[prime]) 2.22-1.76 (cluster of s, 24 H, 8 × OAc), 1.41 (d, J = 6.7 Hz, 1.8 H, CMe-[beta]), 1.36 (d, J = 6.5 Hz, 1.2 H, CMe-[alpha]).
Anal Calc. for C50H54N2O25: C, 55.48; H, 5.03; N, 2.59. Found: C, 55.29; H, 5.11; N, 2.58.
Phenyl O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-[beta]-d-galactopyranosyl)-(1->4)-O-[(2,3,4-tri-O-acetyl-[alpha]-l-fucopyranosyl)-(1
->3)-O]-6-O-acetyl-2-deoxy-2-phthalimido-1-thio- [alpha]/[beta]-d-glucopyranoside (20)
To a stirred solution of 19 (2.0 6, 1.8 mmol) in dichloromethane (40 ml) was added thiophenol (2.0 ml, 18 mmol) and BF3-ethereate (0.8 ml, 5.6 mmol). Stirring was continued for 5 h at room temperature. The reaction mixture was washed with aqueous sodium bicarbonate solution, water, dried, and concentrated. The residue was purified on a column of silica gel with a solvent gradient consisting of hexane-ethyl acetate 1:1 -> 1:4 to afford 20 (1.1 g, 49%); [[alpha]]D -72° (c 1.1, CHCl3); 1H NMR (CD2Cl2): [delta] 7.91-7.17 (m, 13 H, arom.), 5.84 (dd, 1 H, H-3[prime]), 5.51 (d, J = 3.8 Hz, 1 H, H-4[prime][prime]), 5.41 (d, 2.8 Hz, 1H, H-4[prime]) 5.36 (d, J = 8.4 Hz, 1 H, and H-1), 5.34 (d, J = 4.0 Hz, 1 H, H-1[prime][prime]), 5.28 (d, J = 10.5 Hz, 1 H, H-1[prime]), 5.19 (dd, 1 H, H-2[prime]), 2.20, 2.12, 2.11, 2.08, 2.07, 1.93, and 1.80 (each s, 21 H, 7 × OAc), and 1.35 (d, J = 6.7 Hz. 3 H, CMe).
Anal Calc. for C54H56N2O23S: C, 57.24; H, 4.98; N, 2.47. Found: C, 57.37; H, 5.01; N, 2.29.
Methyl O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-[beta]-d-galactopyranosyl-(1->4)-O-[(2,3,4-tri-O-acetyl-[alpha]-l-fucopyranosyl)-(1
->3)-O]-)6-O-acetyl-2-deoxy-2-phthalimido-[beta]-d-glucopyranosyl)-(1->6)-O-[(6-O-trimethylacetyl-[beta]-d-galactopyranosyl)-(1->3)
-O]-2-acetamido-2-deoxy-[alpha]-d-galactopyranoside (23) and Methyl O-(2-acetamido-2-deoxy-[beta]-d-galactopyranosyl)-(1->4)-O-[([alpha]-l-fucopyranosyl)-(1->3)-O]-(2-acetamido-2- deoxy-[beta]-d-glucopyranosyl)-(1->6)-O-[([beta]-d-galactopyranosyl)- (1->3)-O]-2-acetamido-2-deoxy-[alpha]-d-galactopyranoside (24)
Glycosidation of 22 (0.9 g, 1.26 mmol) with 20 (1.0 g, 0.88 mmol) followed by processing in the usual manner gave a crude product mixture which was directly employed in the next step without further purification. A solution of the crude product in 1:1 ethanol-dichloromethane (30 ml) containing thiourea (2.8 g, 37.8 mmol) and lutidine (2.0 ml, 18.72 mmol) was stirred for 6 h at 80°C. The solvents were evaporated under reduced pressure and the residue redissolved in dichloromethane. The organic layer was washed with water, dried, and concentrated under diminished pressure. The residue was purified on a column of silica gel by elution with a solvent gradient consisting of 10-15% MeOH in dichloromethane to give 23 (0.44 g, 37%; based on 22); [[alpha]]D -40° (c 0.5, CHCl3); 1H NMR (CD2Cl2): [delta] 7.92-7.77 (m, 8 H, arom.), 5.83 (dd, 1 H, H-3[prime][prime][prime]), 5.76 (d, J = 9.6 Hz, 1 H, H-1[prime][prime]), 5.51 (d, J = 3.5 Hz, 1H, H-4[prime][prime][prime][prime]), 5.41 (d, J = 2.9 Hz, 1 H, H-4[prime][prime][prime]), 5.38 (d, J = 8.6 Hz, 1 H, H-1[prime][prime][prime]), 5.22 (d, J = 3.3 Hz, 1 H, H-1[prime][prime][prime][prime]), 5.20 (d, J = 2.9 Hz, 1 H, H-1), 2.83 (s, 3 H, OMe), 2.20-1.81, (cluster of s, 24 H, 7 × OAc and NAc), 1.36 (d, J = 6.4 Hz, 3 H, CMe), and 1.14 (s, 9 H, CMe3).
Anal Calc. for C68H85N3O35: C, 54.29; H, 5.69; N, 2.79. Found: C, 54.03; H, 5.69; N, 2.79.
A portion of compound 23 was treated with hydrazine hydrate in methanol to cleave the phthalimido group, followed by N-acetylation (MeOH-Et3N-Ac2O) chromatographed and finally O-deacetylation in furnish in 66% yield, amorphous 24; [[alpha]]D -12° (c 1.0, H2O); 1H NMR (D2O): [delta] 5.11 (d, J = 4.0 Hz, 1 H, H-1[prime][prime][prime][prime]), 4.74 (d, J = 3.8 Hz, 1 H, H-1), 4.52 (d, J = 8.3 Hz, 1 H, H-1[prime][prime]), 4.46 (d, J = 7.8 Hz, 1 H, H-1[prime][prime][prime]), 4.44 (d, J = 7.0 Hz, 1 H, H-1[prime]), 3.35 (s, 3 H, OMe), 2.04, 2.00 and 1.99 (each s, 9 H, 3 × NAc), and 1.26 (d, J = 6.6 Hz, 3 H, CMe); 13C NMR: GalNAc-[beta]-(1->4) residue: 100.36 (C-1), 51.44 (C-2), 69.79 (C-3), 66.76 (C-4), 73.71 (C-5), 60.00 (C-6), 21.23 (NAc); Fuc-[alpha]-(1->3): 97.49 (C-1), 67.97 (C-2), 68.24 (C-3), 69.67 (C-4), 65.94 (C-5), 14.40 (C-6); GlcNAc-[beta]-(1->6) residue: 99.76 (C-1), 53.97 (C-2), 74.45 (C-3), 74.00 (C-4), 72.43 (C-5), 59.06 (C-6), 21.30 (NAc); Gal-[beta]-(1->3) residue: 103.68 (C-1), 69.03 (C-2), 71.06 (C-3), 67.63 (C-4), 73.88 (C-5), 60.48 (C-6); GalNAc-[alpha]-OMe residue: 97.24 (C-1), 47.56 (C-2), 76.11 (C-3), 66.41 (C-4), 71.58 (C-5), 68.19 (C-6), 54.60 (OMe), 21.06 (NAc). ES-MS: m/z = 948.39 [M-1]-.
Anal. Calc. for C37H63N3O25.H2O: C, 45.91; H, 6.77; N, 4.34. Found: C, 46.08; H, 6.63; N, 4.29.Methyl O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-[beta]-d-galactopyranosyl)-(1->4)-O-[(2,3,4-tri-O-acetyl-[alpha]-l-fucopyranosyl)-(1->3)
-O]-6-O-acetyl-2-deoxy-2-phthalimido-[beta]-d-glucopyranosyl)-(1->6)-O-[methyl-(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5
-dideoxy-d-glycero-[alpha]-d-galacto-2-nonulopyranosylonate-(2->3)
-O-(6-O-trimethylacetyl-[beta]-d-galactopyranosyl-(1->3)-O]-2-acetamido-2-deoxy-[alpha]-d-galactopyranoside (26)
Compound 23 (0.2 g, 0.13 mmol) was treated with donor 25 (0.6 g, 1.1 mmol) in propionitrile (15 ml) at -65°C for 3 h. The reaction mixture was then processed as described above, and the crude product subjected to column chromatography on silica gel with 10% MeOH in dichloromethane as the eluent to give 26 (0.12 g, 46%); [[alpha]]D -28° (c 0.5, CHCl3); 1H NMR (CD2Cl2): [delta] 7.88-7.76 (m, 8 H, arom.), 5.48 (d, J = 9.2 Hz; 1 H, NH), 5.84 (dd, 1 H, H-3[prime][prime][prime]), 5.49 (d, J = 3.3 Hz, 1H, H-4[prime][prime][prime][prime]), 5.40 (d, J = 3.0 Hz, 1 H, H-4[prime][prime][prime]), 5.37 (d, J = 8.6 Hz, 1 H, H-1[prime][prime]), 5.28 (d, J = 3.3 Hz, 1 H, H-1[prime][prime][prime][prime]), 5.18 (d, J = 3.3 Hz, 1 H, H-1), 5.14 (d, J = 9.4 Hz, 1 H, H-1[prime][prime][prime]), 3.78 (s, 3 H, OMe), 2.79 (s, 3 H, OMe), 2.67 (dd, J = 4.6 Hz, H-3e[prime][prime][prime][prime][prime]), 2.18-1.77, (cluster of s, 39 H, 12 × OAc and NHAc), 1.34 (d, J = 6.5 Hz, 3 H, CMe) and 1.15 (s, 9 H, Cme3).
Anal Calc. for C88H112N4O47: C, 53.44; H, 5.71; N, 2.83. Found: C, 53.09; H, 5.89; N, 2.93.
Methyl O-(2-acetamido-2-deoxy-[beta]-d-galactopyranosyl-(1->4)- O-[[alpha]-l-fucopyranosyl)-(1->3)-O]-(2-acetamido-2-deoxy-[beta]-d- glucopyranosyl)-(1->6)-O-[(5-acetamido-3,5-dideoxy-d-glycero-[alpha]-d-galacto-2-nonulopyranosylonic acid)-(2->3)-O-([beta]-d- galactopyranosyl)-(1->3)-O]-2-acetamido-2-deoxy-[alpha]-d-galactopyranoside (27)
A solution of 26 (0.1 g, 0.05 mmol) and lithium iodide (0.3 g, 2.2 mmol) in pyridine (10 ml) was stirred for 6 h at ~120°C. The solvent was then removed under diminished pressure and the residue was passed through a small column of silica gel by elution with 20-30% methanol in dichloromethane to give the protected free acid derivative. This compound was taken in methanol-hydrazine hydrate (4:1, 20 ml) and heated at ~80°C for 16 h. After evaporation to dryness, the residue was redissolved in methanol-dichloromethane (1:1, 20 ml) and treated with acetic anhydride (6 ml) for 1 h at 0°C. The mixture was then evaporated to dryness and the residue so obtained was deacetylated by stirring in methanolic sodium methoxide (0.05 N; 20 ml) for 2 days at room temperature. The crude product was purified by column chromatography on silica gel by using chloroform/methanol/water 13:6:1 and 4:5:1 (v/v/v) as the eluent, to give the target compound 27 (0.015 g, 24%); [[alpha]]D -8° (c 0.15, H2O); 1H NMR (D2O): [delta] 5.11 (d, J = 3.9 Hz, 1 H, H-1[prime][prime][prime][prime]), 4.76 (d, J = 3.9 Hz; 1 H, H-1), 4.52 (d, J = 8.2 Hz, 1 H, H-1[prime][prime]), 4.51 (d, J = 7.7 Hz, 1 H, H-1[prime][prime][prime]), 4.46 (d, J = 7.0 Hz, 1 H, H-1[prime]), 3.34 (s, 3 H, OMe), 2.75 (dd, J3[prime][prime][prime][prime][prime][prime]e,44[prime][prime][prime][prime][prime] = 4.6 Hz, 1 H, H-3[prime][prime][prime][prime][prime]e), 2.04, 2.03, 2.01 and 1.99 (each s, 12 H, 4 × NHAc), 1.81 (t, J3[prime][prime][prime][prime][prime][prime]a,44[prime][prime][prime][prime][prime] = J3[prime][prime][prime][prime][prime]a, 3[prime][prime][prime][prime][prime]e = 12.1 Hz, 1 H, H-3[prime][prime][prime][prime][prime]a), and 1.26 (d, J = 6.5 Hz, 3 H, CMe); 13C NMR; D2O; GalNAc-[beta]-(1->4) residue: 100.36 (C-1), 51.43 (C-2), 69.79 (C-3), 66.77 (C-4), 73.72 (C-5), 60.00 (C-6), 21.23 (Nac); Fuc-[alpha]-(1->3) residue: 97.49 (C-1), 67.83 (C-2), 68.24 (C-3), 69.12 (C-4), 65.94 (C-5), 14.41 (C-6); GlcNAc-[beta]-(1->6) residue: 99.76 (C-1), 53.97 (C-2), 74.44 (C-3), 73.87 (C-4), 72.44 (C-5), 59.05 (C-6), 21.30 (NAc); Gal-[beta]-(1->3) residue: 103.46 (C-1), 68.22 (C-2), 76.22 (C-3), 66.42 (C-4), 73.81 (C-5), 60.49 (C-6); NeuAc-[alpha]-(2->3) residue: 174.05 (C-1), 98.75 (C-2), 38.82 (C-3), 67.16 (C-4), 50.73 (C-5), 71.85 (C-6), 67.40 (C-7), 70.86 (C-8), 61.59 (C-9), 21.12 (NAc); GalNAc-[alpha]-OMe residue: 97.21 (C-1), 47.46 (C-2), 74.71 (C-3), 66.42 (C-4), 71.07 (C-5), 68.09 (C-6), 54.62 (OMe), 21.09 (NAc). ES-MS: m/z = 1239.8 [M-1]-.
Anal Calc. for C48H80N4O33.1.5 H2O: C, 45.46; H, 6.60; N, 4.42. Found: C, 45.37; H, 6.62; N, 4.40.
Methyl O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-[beta]-d-galactopyranosyl)-(1->4)-O-[(2,3,4-tri-O-acetyl-[alpha]-l-fucopyranosyl)-(1->
3)-O]-(6-O-acetyl-2-deoxy-2-phthalimido-[beta]-d-glucopyranosyl)-(1->6)-O-[(2,4,6-tri-O-acetyl-3-O-chloroacetyl-[beta]
-d-galactopyranosyl-(1->3)-O]-2-acetamido-2-deoxy-[alpha]-d-galactopyranoside (29)
Compound 28 (0.92 g, 1.5 mmol) was treated with 20 (1.5 g, 1.3 mmol) as described in the general glycosidation methods. After the customary processing, the crude product was purified by silica gel column chromatography with a solvent gradient consisting of 20-25% acetone in dichloromethane to give 29 (1.0 g, 74%); [[alpha]]D -36° (c 1.0, CHCl3); 1H NMR (CD2Cl2): [delta] 7.90-7.78 (m, 8 H, arom.), 5.82 (dd, 1 H, H-3[prime][prime][prime]), 5.50 (d, J = 3.5 Hz, 1 H, H-4[prime][prime][prime][prime]), 5.40 (d, J = 3.4 Hz, 1 H, H-4[prime][prime][prime]) 5.38 (d, J = 7.9 Hz, 1 H, H-1[prime]), 5.21 (d, J = 3.5 Hz, 1 H, H-1[prime][prime][prime]), 5.19 (d, J = 3.2 Hz, 1 H, H-1), 5.14 (d, J = 8.2 Hz, 1 H, H-1[prime][prime][prime]), 4.11-4.07 (bs, 2 H, CH2Cl), 2.83 (s, 3 H, OMe), 2.19-1.80 (cluster of s, 33 H, 10 × OAc and NHAc), 1.35 (d, J = 6.8 Hz, 3 H, CMe).
Anal Calc. for C71H84N3O38Cl: C, 52.55; H, 5.22; N, 2.59. Found: C, 52.31; H, 5.16; N, 2.38.
Methyl O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-[beta]-d-galactopyranosyl)-(1->4)-O-[(2,3,4-tri-O-acetyl-[alpha]-l-fucopyranosyl)-(1->3)
-O]-(6-O-acetyl-2-deoxy-2-phthalimido-[beta]-d-glucopyranosyl)-(1->6)-O-[(2,4,6-tri-O-acetyl-[beta]-d-galactopyranosyl)-(1->3)-O]
-2-acetamido-2-deoxy-[alpha]-d-galactopyranoside (30)
Compound 29 (0.96 g, 0.58 mmol) was de-O-chloroacetylated in a manner analogous to that described for the preparation of 14. After customary processing, silica gel column chromatographic purification (5-10% MeOH in dichloromethane), gave 30 (0.65 g, 71%); [[alpha]]D -40° (c 1.0, CHCl3); 1H NMR (CD2Cl2): [delta] 7.91-7.78 (m, 8 H, arom.), 5.82 (dd, 1 H, H-3[prime][prime][prime]), 5.50 (d, J = 3.7 Hz, 1 H, H-1[prime][prime][prime][prime]), 5.40 (d, J = 3.3 Hz, 1H, H-4[prime][prime][prime]), 5.38 (d, J = 2.9 Hz, 1 H, H-4[prime][prime][prime][prime]), 5.38 (d, J = 9.0 Hz, 1 H, H-1[prime][prime]), 5.25 (d, J = 3.5 Hz, 1 H, H-4[prime]), 5.23 (d, J = 3.4 Hz, 1 H, H-4), 5.19 (d, 2.9 Hz, 1 H, H-1), 2.81 (s, 3 H, OMe), 2.20-1.58, (cluster of s, 33 H, 10 × OAc and NHAc), 1.35 (d, J = 6.2 Hz, 3 H, CMe).
Anal Calc. for C69H83N3O37: C, 53.59; H, 5.41; N, 2.72. Found: C, 53.39; H, 5.63; N, 2.59.
Methyl O-(2-acetamido-2-deoxy-[beta]-d-galactopyranosyl-(1->4)- O-[[alpha]-l-fucopyranosyl-(1->3)-O]-(2-acetamido-2-deoxy-[beta]-d- glucopyranosyl)-(1->6)-O-[(3-O-sulfo-[beta]-d-galactopyranosyl sodium salt)-(1->3)-O]-2-acetamido-2-deoxy-[alpha]-d-galactopyranoside (31)
Compound 30 (0.45 g, 0.29 mmol) in N,N-dimethylformamide (20 ml) was treated with sulfur trioxide-pyridine complex (0.25 g, 1.6 mmol) at 0°C for 5 h. Excess reagent was destroyed by the addition of methanol (~5 ml), followed by pyridine (~5 ml). The mixture was then concentrated under diminished pressure and the residue was passed through a small column of silica gel by using 15-20% methanol in dichloromethane as the eluent. The fractions corresponding to product were pooled and concentrated and the residue taken in methanol-hydrazine hydrate (4:1, 50 ml) and heated at ~90°C for 5 h. The mixture was then concentrated and the crude product mixture was taken in methanol-triethylamine (2:1, 24 ml), cooled (0°C) and treated with acetic anhydride (5 ml). It was allowed to gradually attain room temperature and kept for an additional 1 h at same temperature. The mixture was concentrated, and the residue applied to a column of silica gel and eluted with chloroform/methanol/water 13:6:1 and 4:5:1 (v/v/v). Fractions corresponding to product were pooled and concentrated and the residue redissolved in water and passed through a small column of Amberlite IR-120 (Na+) cation exchange resin. Lyophilization of the eluate then furnished 31 (0.11 g, 37%), [[alpha]]D (c 1.0, H2O); 1H NMR (D2O): [delta] 5.12 (d, J = 3.9 Hz, 1 H, H-1[prime][prime][prime][prime]), 4.77 (d, J = 3.7 Hz; 1 H, H-1), 4.57 (d, J = 7.9 Hz, 1 H, H-1[prime][prime]), 4.54 (d, J = 8.3 Hz, 1H, H-1[prime][prime][prime]), 4.48 (d, J = 8.2 Hz, 1 H, H-1[prime]), 3.37 (s, 3 H, OMe), 2.07, 2.03 and 2.02 (each s, 9 H, 3 × NAc), and 1.27 (d, J = 6.6 Hz, 3 H, CMe); 13C NMR (D2O); GalNAc-[beta]-(1->4) residue: 100.37 (C-1), 51.44 (C-2), 69.79 (C-3), 66.77 (C-4), 73.57 (C-5), 59.90 (C-6), 21.14 (NAc); Fuc-[alpha]-(1->3) residue: 97.50 (C-1), 67.76 (C-2), 68.24 (C-3), 69.11 (C-4), 65.85 (C-5), 14.41 (C-6); GlcNAc-[beta]-(1->6) residue: 99.76 (C-1), 53.99 (C-2), 74.45 (C-3), 73.88 (C-4), 72.44 (C-5), 59.06 (C-6), 21.31 (NAc); 3-O-SO3Na Gal-[beta]-(1->3) residue: 103.37 (C-1), 68.21 (C-2), 79.29 (C-3), 66.42 (C-4), 73.73 (C-5), 60.48 (C-6); GalNAc-[alpha]-OMe residue: 97.25 (C-1), 47.48 (C-2), 76.55 (C-3), 65.40 (C-4), 71.07 (C-5), 67.87 (C-6), 54.62 (OMe), 21.06 (NAc). ES-MS: m/z = 1028.38 [M-Na]- .
Anal Calc. for C37H62N3O28.SNa. 2 H2O: C, 40.84; H, 6.11; N, 3.86. Found: C, 40.73; H, 6.15; N, 3.73.
Acknowledgments
This research was supported by NCI Grants CA38701 (to A.V.), CA35329 and CA63218 (to K.M.), and CA16056 (RPCI). A.K. was also supported by Training Grant (T32 DK07202; P.I. M. Kagnoff).
References
2Present address: TransCell Technologies, Inc., 8 Cedar Brook Drive, Cranbury, NJ 08512
3To whom correspondence should be addressed at: Department of Gynecologic Oncology, Roswell Park Cancer Institute, Elm & Carlton Streets, Buffalo, NY 14263
This page is run by Oxford University Press, Great Clarendon Street, Oxford OX2 6DP, as part of the OUP Journals
Comments and feedback: www-admin{at}oup.co.uk
Last modification: 10 Jun 1998
Copyright©Oxford University Press, 1998.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  E. V. Chandrasekaran, S. S. Lakhaman, R. Chawda, C. F. Piskorz, S. Neelamegham, and K. L. Matta Identification of Physiologically Relevant Substrates for Cloned Gal: 3-O-Sulfotransferases (Gal3STs): DISTINCT HIGH AFFINITY OF Gal3ST-2 and LS180 SULFOTRANSFERASE FOR THE GLOBO H BACKBONE, Gal3ST-3 FOR N-GLYCAN MULTITERMINAL Gal{beta}1,4GlcNAc{beta} UNITS AND 6-SULFOGal{beta}1,4GlcNAc{beta}, AND Gal3ST-4 FOR THE MUCIN CORE-2 TRISACCHARIDE J. Biol. Chem., March 12, 2004; 279(11): 10032 - 10041. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Dang, S. Wiehler, and K. D. Patel Increased PSGL-1 expression on granulocytes from allergic-asthmatic subjects results in enhanced leukocyte recruitment under flow conditions J. Leukoc. Biol., October 1, 2002; 72(4): 702 - 710. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Leppanen, S. P. White, J. Helin, R. P. McEver, and R. D. Cummings Binding of Glycosulfopeptides to P-selectin Requires Stereospecific Contributions of Individual Tyrosine Sulfate and Sugar Residues J. Biol. Chem., December 8, 2000; 275(50): 39569 - 39578. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||