WO1993023031A9 - Multivalent mimetics and peptide mimetics for blocking carbohydrate-dependent cellular interaction and for eliciting anticarbohydrate t-cell response - Google Patents

Abstract

A stabilized carbohydrate epitope having more resistance to metabolic degradation than a corresponding naturally occurring carbohydrate epitope, or those assembled in multivalent structures. A mimetic of a carbohydrate epitope, wherein said mimetic has a structure such that the mimetic has about the same or higher antibody-binding or selectin-binding activities, immunogenicity and antigenicity as that of a corresponding naturally occurring carbohydrate epitope. A process for preparing the above-described mimetic of a carbohydrate epitope is described as well as medicaments and methods for inhibiting carbohydrate-mediated cell adhesion and a vaccine and method of vaccinating to induce an anti-carbohydrate T cell response.

Description

Multivalent mimetics and peptide mimetics for blocking carbohydrate-dependent cellul interaction and for eliciting anticarbohydrate T-cell response

FIELD OF THE INVENTION

The instant invention relates to stable multivalent carbohydrate epitopeε and mimetics of carbohydrate epitopes and their uses. The stable carbohydrate epitopeε are prepared by chemically modifying the structure of known carbohydrate epitopes using, for example, 6-trifluoromethylfucose, carbocyclic fucose, N-trifluoroacetyl or N-carbamylneuraminic acid, or S-glycosideε of sialic acid and fucose. The peptide mimetics complementary to carbohydrates can be based on amino acid sequences of complementarity-determining regions (CDR) 1, 2 or 3 of the variable heavy or variable light regions of anti-carbohydrate idiotype antibodies which mimic carbohydrate structure. The chemically modified carbohydrate epitopes and the carbohydrate mimetics are useful for inhibiting carbohydrate-mediated cell adhesion. The peptide mimetics complementary to carbohydrates also are useful to induce a T cell response directed to carbohydrates.

BACKGROUND OF THE INVENTION

Specific complex carbohydrates such as sialosyl-Le* (SLe^x) (1) . sialosyl-Le^a (SLe^a) (2), Le^x (3) , Le^a (4) , Le^y (5) , Le^b (7) , GM3 (8) , GD3 (9) , GD2 (10) , Gg3Cer (11) , Tn (13) , sialosyl-Tn (14) , T (15) and sialosyl-T (16) (see Table I) are important epitopes recognized as tumor-associated carbohydrate antigens (TACA'ε) . SLe and SLe^a have been identified as the epitopeε recognized by selectinε. Expression of the antigens listed above may be instrumental in the ability of tumor cells to invade surrounding tissues, and metastasize in vivo, based on the following types of observations: i. Strong correlation between expression of TACA in primary tumors and grade of subsequent tumor progression. ii. Identification of some of those antigens as adhesion molecules recognized by glycosphingolipids or other glycoconjugates expressed on a particular type of cell (e.g., microvascular endothelial cell). There is strong evidence for specific adhesion of tumor cells expressing GM3 (Table I, structure 8) to non-activated endothelial cells expressing LacCer (structure 12) and for adhesion of tumor cells expressing H/Lev/Leb to endothelial cells expressing H (structure 6) iii. Recognition of tumor cells expressing SLe X,

SLea or analogue thereof by selectins expressed on activated platelets or endothelial cells.

Selectin-dependent adhesion of leukocytes to microvascular endothelial cells and platelets also is regarded as an important initial event during inflammatory processes. (Hakomori, Curr. Opin. Immunol.. 3:646-653, 1991; Cancer Cells. 3:461-470, 1991)

For prevention of various pathobiologic processes involved in cancer progression and inflammation, general approaches have been proposed, such as follows, (i) Application of carbohydrates as listed in Table I, or their conjugates, to block pathologic cell adhesion, (ii) Application of monoclonal antibodies (mAb's) directed to those carbohydrates and having appropriate affinity to block pathologic cell adhesion. Approaches (i) and (ii) could be applied for blocking adhesion of tumor cells to endothelial cells or to neighboring cells, or excessive accumulation of recruited neutrophils or monocytes at sites of inflammation, which is caused by adhesion of leukocytes or endothelial cells followed by transendothelial migration of leukocytes. Those anti-adhesion approaches could prevent or reduce metastasis and invasivenesε of tumor cellε on one hand and prevent excessive inflammatory processes following infection, myocardial infarction (heart attack) , traumatic tissue injury etc. on the other.

Active immunization with carbohydrates or derivatives thereof is designed primarily to elicit humoral immune responses (mainly IgM or IgG₃) (which may not be sufficient to eliminate tumor cells) or to block activity of leukocytes and monocytes recruited at sites of inflammation. Active immunization with Le^x glycolipid may reduce inflammatory myelocytic response at the inflammatory lesion of rheumatoid arthritis (Ochi et al., J. Rheumatol. , 15:1609-1615, 1988). So far, the active immunization approach for suppression of tumor growth has been hampered by the facts that (a) the immune response against most carbohydrate antigens is a T cell-independent B cell response; (b) almost all antibodies produced are IgM or IgG₃ isotype, with relatively low affinity; and (c) it is extremely rare to obtain an IgG₁ response or T cell response. One exceptional case involved injection of mice with desialosylated ovine submaxillary mucin. The mice exhibited a weak T cell response, apparently against Tn, the simplest carbohydrate antigen (Singhal et al., Cancer Res.. 51:1406-1411, 1991). Despite extensive searching, no other cases of T cell response to carbohydrate antigens have been found.

Carbohydrate epitopes designed to block carbohydrate-carbohydrate interaction or selectin-dependent adhesion should be stable and not destroyed in vivo. It should also be designed to gain high affinity to carbohydrates or to lectin domains of selectin. Tritiated galactosylβl->4 glucose (lactose) has a half-life of only 3-5 min (degraded and recovered as ³H-labeled Gal) when injected into mice. Similarly, if sialosyl or fucosyl carbohydrate derivatives (such as SLe^a or SLe^x, the epitopes recognized by selectin) are injected, they are degraded rapidly. Other εtudieε by the instant inventors also have shown that bivalent sialosyl-Le^x or bivalent Le^x had higher binding affinity to selectin, which indicates that stable carbohydrate epitopes should be designed in a multivalent structure.

SUMMARY OF THE INVENTION

Accordingly, important objects of the instant invention are: (i) preparation of stable, conformationally-restrictedcarbohydrateoligosaccharide epitopes which has high affinity to carbohydrateε or to selectin and can efficiently block carbohydrate-dependent cell adhesion (i.e., based on carbohydrate-carbohydrate or carbohydrate-selectin interaction) ; (ii) preparation of oligosaccharide analogues which negatively effect the normal expression of carbohydrates that mediate intercellular adhesion; (iii) bivalent or multivalent structures of carbohydrate mimetics as described hereinabove; and (iv) preparation of peptide mimetics having a peptide conformational surface structure the same as specific carbohydrate antigens. Such peptide/non-carbohydrate mimetics are useful not only for blocking carbohydrate-dependent cell adhesion but also for inducing a T cell response against carbohydrate antigens since many non-peptide/ non-carbohydrate epitopes are known to elicit a T cell response quite well (Kochibe et al., Proc. Natl. Acad. Sci. USA. 72:4582-4587, 1975; Handa et al. , J. Immunol.. 135:1564, 1985). Accordingly, the present invention provides a stabilized carbohydrate epitope having more resistance to metabolic degradation than a corresponding naturally occurring carbohydrate epitope and having high affinity to block cell adhesion based on carbohydrate-carbohydrate interaction and carbohydrate-selectin interaction. Such a high affinity structure could be based on multimeric mimetics.

Furthermore, the instant invention provides a preparation of oligosaccharide analogues which negatively effectε the normal expression of carbohydrates that mediate intercellular adhesion.

The present invention also provides a mimetic of a peptide epitope, wherein the mimetic has a structure such that the mimetic has about the same antibody-binding or selectin-binding activities, immunogenicity and antigenicity as that of a corresponding naturally occurring carbohydrate epitope.

The present invention further provides a process for preparing the above-described mimetic of a peptide epitope having the same surface structure as carbohydrate, the process comprising:

(A) making a monoclonal antibody (Abl) directed against the naturally occurring carbohydrate epitope, (B) making an anti-idiotype monoclonal antibody

(Ab2) directed against the internal image structure of said Abl,

(C) determining the amino acid sequence of the variable heavy (V_H) and/or the variable light (^vι) regions corresponding to complementarity-determining region (CDR) l, 2 and 3 of said Ab2,

(D) determining the conformational structures of peptides in said CDR 1, 2 and 3, (E) identifying regions of said CDR 1, 2 and 3 that are complementary to the conformational structure of the naturally occurring carbohydrate epitope and (F) synthesizing a peptide analogue of the naturally occurring carbohydrate epitope.

The present invention further provides a medicament for inhibiting metastasis of tumor cells, inhibiting inflammatory processes and inhibiting microbial infection caused by carbohydrate-mediated cell adhesion, the medicament comprising:

(A) a stabilized carbohydrate epitope having more resistance to metabolic degradation than a corresponding naturally occurring carbohydrate epitope or a mimetic of a carbohydrate epitope, wherein said mimetic has a structure such that the mimetic has about the same or higher antibody-binding or selectin-binding activities, i unogenicity and antigenicity as that of a corresponding naturally occurring carbohydrate epitope,

(B) bivalent or multivalent assembly of stabilized carbohydrate epitope and

(C) a pharmaceutically acceptable carrier, diluent or excipient.

The present invention further provides a method for inhibiting carbohydrate-mediated cell adhesion including metastasis of tumor cells, inflammatory processes and microbial infection, the method comprising contacting cells with an inhibitory amount of a stabilized carbohydrate epitope having more resistance to metabolic degradation than a corresponding naturally occurring carbohydrate epitope or with a mimetic of a carbohydrate epitope wherein the mimetic has a structure such that the mimetic has about the same antibody-binding or selectin-binding activities, immunogenicity and antigenicity as that of a naturally occurring carbohydrate epitope.

The present invention additionally provides a vaccine for induction of an anti-carbohydrate T cell immune response, the vaccine comprising:

(A) a mimetic of a carbohydrate epitope, wherein the mimetic has a structure such that the mimetic has about the same or higher antibody-binding or selectin-binding activities, immunogenicity and antigenicity as that of a corresponding naturally occurring carbohydrate epitope,

(B) Bivalent or multivalent stable carbohydrate epitope as above and

(C) a pharmaceutically acceptable carrier, diluent or excipient.

The present invention also provides a method of vaccinating to induce an anti-carbohydrate T cell immune response, the method comprising vaccinating a host with an anti-carbohydrate T cell inducing amount of a vaccine comprising a mimetic of a carbohydrate antigen, wherein the mimetic has a structure such that the mimetic has about the same antibody-binding or selectin-binding activities, immunogenicity and antigenicity as that of. a corresponding naturally occurring carbohydrate epitope.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures IA and IB graphically depict inhibition of B16 melanoma cell adhesion of HUVEC's (Fig. IA) and LacCer (Fig. IB) by methyl-β-lactoside (Me-β-Lactoside) , mimetic 6-deoxy-6-fluoro-galactopyranosyl-βl->4- glucopyranosyl-βl-methylglycoside (compound (1')) and galactopyranosyl-βl->4-6-deoxy-6-deoxy-6-fluoro- glucopyranosyl-βl-methylglycoside (compound (2')). The abscissa represents concentration of added compound and the ordinate represents cell binding expressed as radioactivity per well.

Figures 2A, 2B and 2C show various synthesis schemes involved in preparing compound (1') and compound (2¹) shown in Fig. IA. In Figs. 2A through 2C, the following abbreviations have the following meanings: Me = methyl; Bn = benzyl; PrCO = butyryl; Ac = acetyl;

MEM = (2-methoxyethoxyl)methyl; Bz = benzoyl; MeBz = 4-methylbenzoyl; Me₂Bz = 2,4-dimethylbenzoyl;

Me₃Bz = 2,4,6-trimethylbenzoyl; and Tr = trityl. Figure 3 shows eleven examples of primary mimetics of sialosyl-Le^x. Figures 4A, 4B, 5, 6A, 6B, and 7 are synthetic schemes for the eleven mimetics shown in Fig. 2. The references referred to in Figs. 4A through 7 are listed in Example II.

Figures 4A and 4B are the synthetic schemes for compounds 1-6 shown in Fig. 3.

Figure 5 is the synthetic scheme for compounds 7 and 8 shown in Fig. 3.

Figures 6A and 6B show the synthetic scheme for compound 9 shown in Fig. 3. Figure 7 is the synthetic scheme for compounds 10 and 11 shown in Fig. 3.

Figure 8 shows a structural analogue common to both the sialosyl-Le^x and sialosyl-Le^a structureε.

Figures 9A and 9B show the synthetic scheme for synthesizing the cyclohexanediol analogue (87) shown in Fig. 8.

Figure 10 depicts a synthetic scheme, beginning with fucose (3) , to make a carbocyclic derivative thereof. In the Figure, (a) is DMSO, Ac₂0, rt, overnight; (b) is LiCH₂P(0) (OMe)₂, THF, N₂, -77°C, 30 min; (c) is NaBH₄, THF, rt, overnight; (d) is DMSO, TFAA, Et₃N, CH₂C1₂, -77°C, 1.5 h; (e) is NaH, diglyme, N₂, 65°C, 1 h; (f) is (Ph₃PCuH)₆, H₂0, THF, N₂, rt, 48 h; (g) is NaBH₄, CeCl₃, MeOH, rt, 5 min; (h) is NaBH₄, EtOH, rt, 4 h; and (i) is 10% Pd/C, H₂ (1 atm) , EtOH, rt, overnight. Percent values indicate yield. Numbers identify compounds. Figure 11 depicts a continuation of the scheme set forth in Figure 10. In Figure 11, (a) is NaBH₄, CeCl₃, MeOH, rt, 5 min; (b) is 9-BBN, THF, N₂, 0°C for 2 h and rt for 1 h; (c) is NaBH₄, MeOH/THF, -20°C, 1 h; and (d) is Li, liq. NH₃, THF, 2 h. Percent values indicate yield. Numbers identify compounds.

Figure 12 depicts a continuation of the schemeε set forth in Figures 10 and ll. In Figure 12, (a) is (BnO)₂PN(i-Pr)₂, lH-tetrazole, CH₂C1₂, rt, 2 h; (b) is Li, liq. NH₃, THF, 2h; (c) is Dowex 50X8-400 (Et₃HN⁺) ; (d) is m-CPBA, -40°C-+0°C, 45 min; and (e) is GMP-morpholidate, pyridine, rt, 5 d. The HPLC separation comprised RP-18; 24:10.05M aq Et₃HNHC0₃-MeCN, isocratic and the remaining chromatographic treatment (the triethyl ammonium salt to the sodium salt) comprised Bio-Rad AG 50 -X2 (Na⁺) . Percent values indicate yield. Numbers identify compounds.

Figure 13 depicts a scheme for obtaining an intermediate of a carbocyclic derivative of a selectin epitope.

Figure 14 (Scheme IV, Route 1) depicts a continued synthesis to yield carbocyclic derivative intermediates, Compounds (23) and (24) . Figure 14 also shows continuous synthesis from Compounds (24) to (26) by extension of the supporting arm of Le^x having carbocyclic fucose.

Figure 15 (Scheme V, Route 2) depicts another route for synthesis of a carbocyclic compound of Le^x, having carbocyclic fucose (Compound (26) and Compounds (28) or (29)).

Figure 16 (Scheme VI, Plan 1) depicts synthesis of a carboxyl group linked at the 3 position of the terminal galactose of Le^x having carbocyclic fucose with appropriate arms (Compound (32)). Figure 17 (Scheme VII, Plan 2) shows another scheme for synthesis of Le^x having carbocyclic fucose that has a carboxyl group at the 3 position (Compound (34)). Figure 18 (Scheme VIII, Plan 3) shows another plan for synthesis of a sulfonated group at the terminal Gal of Le^x having carbocyclic fucose (Compound (39)).

Figure 19 (Scheme IX, Plan 4) shows another plan for synthesis of Le^x having carbocyclic fucose that has a sulfonyl group linked through an intermediate carbon (Compound (41) ) .

Figure 20 (Scheme X, Plan 5) depicts a phosphono group at the galactose residue of Le^x having carbocyclic fucose (Compound (43)).

Figure 21 (Scheme XI, Plan 6) depicts synthesis of any alkyl group with acidic functionalities at the 3 position of the galactose of Le^x having carbocyclic fucose using an alkyl halide. Figure 22 depicts Scheme XII for synthesis of a ^• trifunctional stabilized carbohydrate epitopes in which M represents any carbohydrate mimetic structure of SLe^x, SLe^a, HLe^y, Le etc. The structures have arms with an amino group to make trivalent structures as depicted. Figure 23 depicts possible synthesis of lipids which carry various stabilized carbohydrate mimetics (Compound (46) ) which can be incorporated readily into liposomes.

Figure 24 (Scheme XIV) depicts a synthetic scheme for multimerization of carbohydrate mimetics (Compound (48) ) .

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention, the phrase "having more resistance to metabolic degradation than a corresponding naturally occurring carbohydrate epitope (or carbohydrate antigen) " means that the half-life of the structure, when tested by any art-recognized test for measuring a half-life of a metabolite, is more than the half-life of the corresponding naturally occurring carbohydrate epitope or carbohydrate antigen, the difference being statistically significant.

In the context of the instant invention, multivalent and multimeric are considered equivalents.

Design of stable carbohydrate epitopes. particularly sialosyl-Le^x and sialosyl-Le^a

The epitopes recognized by E-selectin have been identified as SLe (Phillips et al., Science. 250:1130-1132, 1990) and SLe^a (Takeda et al., Biochem. Biophys. Res. Commun. , 179:713-719, 1991; Berg, E.L. et al., J. Biol. Chem.. 266:14869-14872, 1991). Those recognized by P-selectin also were shown to be SLe^x (Polley et al., Proc. Natl. Acad. Sci. USA. 88:6224-6228, 1991) and SLe^a (Handa et al., Biochem. Biophvs. Res. Commun.. 181:1223-1230, 1991). Thus, those two carbohydrate structures are crucial for inhibition of E-selectin-dependent and P-selectin-dependent cell adhesion. However, the carbohydrates are unstable and easily destroyed in vivo by, for example, sialidase and fucosidase. The half-life of SLe^x C-labeled at sialic acid was 1.5 min when injected intravenously.

According to the present invention, the following structural modifications are suggested to increase stability of SLe^x and SLe^a and thereby ensure more effective inhibition of cell adhesion. (i) Replace fucose by 6-trifluoromethylfucose. Synthesis of the 6-trifluoromethyl analogue of L-fucose was previously reported (Bansal et al., J. Chem. Soc. Chem. Commun.. 12:796-798, 1991 and copending patent application No. 07/693292, filed 30 April 1991, expressly incorporated herein by reference) . (ii) Replace N-acetyl sialic acid with an N-trifluoroacetyl or N-carbamyl group as described in copending patent application No. 467458, filed 19 January 1990, expressly incorporated herein by reference) . N-modified carbohydrates can be obtained readily by known methods (Hakomori et al. (1980) "Cell Biological and Immunological Significance of Ganglioside Changes Associated With Transformation" in Structure-Function of Ganαliosideε. (Svennerhol et al., Eds.) Plenum Publishing Corp., N.Y., pp. 247-261). (iii) Instead of O-glycosylation of sialic acid and fucose, replace with S-glycoside, which is resistant to sialidase or fucosidase. (iv) Use carbasugar derivatives, such as carbocyclic fucose. (v) Uεe εialic acid having a lactone or a lactam ring structure which is resistant to hydrolysis by sialidase. The lactone of SLe^x does not bind E-selectin; however, the lactone of SLe^a has not been tested for E-selectin binding, and neither SLe^x nor SLe^a lactone has been tested for P-selectin binding.

Those modifications can be applied to various carbohydrate epitopes as listed in Table I. For example, stable Le^y or Le^x can be constructed using 6-trifluorofucose; stable STn can be constructed using lactones or lactams, or with N-substituted sialic acid. Carbohydrates constructed in those ways are more stable and therefore better able to inhibit carbohydrate-dependent cell adhesion. Six-trifluorofucose is capable of inhibiting H-hemagglutination induced by anti-H lectin. Therefore, the H structure or Lewis structure in which fucose is replaced by 6-trifluorofucose also is capable of binding to antibodies or lectins which bind to fucose.

Design of peptide mimetics that have the same or similar surface structures as carbohydrate epitopes

The following procedure allows the artisan to estimate and predict mimetic structures presenting a conformational surface structure such that the mimetic has about the same, i.e., within experimental error, antibody-binding or lectin-binding activities, immunogenicity and antigenicity as that of the native carbohydrate epitopes.

1. A monoclonal antibody (mAb) directed against the carbohydrate epitope of interest (Abl) is made by known methods. In fact, that step already has been accomplished for all the epitopes shown in Table I.

2. An anti-idiotype mAb (Ab2) directed against the internal image structure of Abl is made by known methods. To confirm that the desired Ab2 has been made, it is essential to demonstrate inhibition by Ab2 of Abl binding to the carbohydrate epitope of interest, or inhibition by the carbohydrate of interest of Ab2 binding to Abl. 3. A specific peptide region

(complementarity-determining region; CDR) of Ab2 should have the same surface profile as the original carbohydrate epitope used to establish Abl. The amino acid sequence can be determined directly or deduced from the nucleotide sequence of the variable heavy (V_H) or of the variable light (V_L) region corresponding to CDR 1, 2 or 3 of Ab2. Further, the amino acid sequence of the CDR's should have the same surface structure found in the original carbohydrate epitope, which is naturally rich in hydroxyl groups. Therefore, clusters of hydroxylated amino acids will be found.

4. Based on the amino acid sequence focused on hydroxylated amino acid clusters found in either CDR 1, 2 or 3, the conformational structure thereof can be determined by a minimum-energy modeling program (e.g., Sybyl 5.5, Tripos Associates) . The conformational structure is compared to that of the original carbohydrate epitope. In general, more than two sequences at different CDR's of the V_H or of the V_L region will cooperate for complete satisfaction of complementarity. 5. Based on a defined sequence in CDR 1, 2 or 3 of the V_H or of the V_L region showing conformational surface structure similar to that of the original carbohydr ate ep itope , a r ig id , conformationally-restricted peptide mimetic is synthesized by cross-linking or by substitution of appropriate amino acids as described. In particular, a large β-loop structure can be maintained by appropriate cross-linking. The same side chains of the essential peptide structure as in the original should be maintained. Thus, a peptide analogue which mimics the surface structure of the original carbohydrate epitope can be synthesized. Such carbohydrate mimetics made from anti-carbohydrate idiotype CDR sequences should demonstrate the same antibody-binding or lectin-binding activities, as well as the same immunogenicity, as the original carbohydrate epitope. However, the mimetics are more stable than the original carbohydrate epitope, or the original CDR peptide of Ab2, in terms of hydrolyzability with glycosidases and peptidases.

TABLE I. Specific carbohydrate structures recognized as tumor-associated antigens or functioning as adhesion molecules.

trivial name structure

sialosyl-Le NeuAcα2→3Galβl-+4GlcNAcβl-R*

3 t

Fucαl

2 . sialosyl-Le^a NeuAcα2→3Galβl-3GlcNAcβl→R

4

Fucαl

Le' Galβl→4GlcNAcβl→3Galβl-+4GlcNAcβl→R 3 3 t T

Fucαl ± ( Fucαl )

Le° Galβl→3GlcNAcβl→3Galβl→R 4 t Fucαl

Le' Galβl→4GlcNAcβl→3Galβl→R 2 3 t t

Fucαl Fucαl

6. H Fucαl→2Galβl→4GlcNAcβl→3Galβl→R

7. Le Galβl- 3GlcNAcβl→3Galβl→R 2 4 t t

Fucαl Fucαl

8. GM3 NeuAcα2→3Galβl-*4Glcβl->lCer

9. GD3 NeuAcα2-»8NeuAcα2→3Galβl-+4Glcβl→lCer 10. GD2 NeuAcα2→8NeuAcα2→3Galβl→4Glcβl→lCer

4

T

GalNAcβl

11. Gg3Cer GalNAcβl-*4Galβl→4GLcβl→lCer

12. LacCer Galβl→4Glcβl→lCer

13. Tn GalNAcαl→O-Ser/Thr-peptide

14. sialosyl-Tn NeuAcα2→6GalNAcαl→0-Ser/Thr-peptide 15. T Galβl- GalNAcαl→O-Ser/Thr-peptide

16. sialosyl-T Galβl→3GalNAcαl→0-Ser/Thr-peptide

6 t NeuAcα2

17. Fucαl

^■l 3 Galβl→4GlcNAcβl •.

6

Galβl→4GlcNAcβl→3Galβl→R 3 Galβl→4GlcNAcβl

3

T

Fucαl

18. NeuAcα2 Fucαl I I

3 3 Galβl→4GlcNAcβl

6 Galβl→4GlcNAcβl→3Galβl→R

3 Galβl→4GlcNAcβl 3 3 t T

NeuAcα2 Fucαl

19. Fucαl i 3 Galβl→4GlcNAcβl

6

Galβl→4GlcNAcβl→3Galβl-R 3 --

Galβl→4GlcNAcβl 3 3 t t NeuAcα2 Fucαl

*/ R represents a carrier molecule Example of procedure for design of carbohydrate (CHO) mimetics

Step 1: Obtain hybridoma 1 producing anti-CHO mAb

splenocytes x HAT-sensitive myeloma

h y b r i d o m a 1 producing Abl which binds to CHO Ag

Step 2: Obtain hybridoma 2 producing Ab2 (anti-anti-CHO) immunization with Abl or hybridoma 1

plenocytes x

HAT-sens it ive myeloma

for Ab2 inhibits of Abl to

Step 3: Sequence information for Ab2 sequence the V_H and v_L regions, focus on CDR 1, 2 and 3

Example: A cluster of hydroxylated amino acids is found in CDR 1 and 2 of the V_μ region, as shown below, and no such cluster is found in CDR 3. Conformational analysis of the CDR 1 and CDR 2 peptides is performed. CDR 1 Ala-Gly-Leu-Ser-Ser-Tyr-Tyr-Leu-Thr-Thr-Tyr- Arg-Pro (SEQ. ID NO. 1)

CDR 2 Leu-Trp-Ser-Thr-Tyr-Tyr-Glv-Ser-Tyr-Arg-Arq- Ala-Gln (SEQ ID NO. 2) CDR 3 no obvious cluster of hydroxylated amino acids was found

Step 4: Comparison of conformational structures of peptides in CDR 1, 2 and 3, focusing on regions where hydroxylated amino acids (Ser, Thr and Tyr) are clustered, which may mimic the CHO Ag

(d) none CDR 3

The conformational structure of the original CHO epitope based on hard sphere exano eric calculation (a) is compared with the conformational structures of the hydroxy-amino acid cluster sequences of regions CDR 1 and 2 (b and c) . The majority of the surface structure of (a) is shared with b (top to right-side portion as shown) . A part of the surface structure of (a) is shared with (c) (lower part) . Since CDR 3 in the above case does not show a cluster of hydroxylated amino acids, no structure can be assigned. Similar conformational analysis can be applied to the V_L region.

Step 5: Chemical synthesis of peptide mimetics based on a defined peptide sequence found in CDR 1 and 2 above, whose conformation mimics that of the original CHO epitope

Based on careful examination of the conformation of CDR 1 and 2, appropriate cross-linking within the region is performed, or modification of peptide linkages is performed, to achieve a conformationally more stable form. Cross-linking or a change of peptide linkage should not change the arrangement of side chains or general conformation of CDR 1 and 2. Thus, the mimetics will retain the essential structure of the original peptide (i.e., orientation and conformation of side chains) but will present a stable and rigid structure which is restricted highly in terms of conformational change. The essential amino acid sequence of the peptide essentially should be the same as that found in the original CDR 1 and 2 (in the above case) .

Step 6: Determination of biological activity of peptide mimetics created in step 5 Peptide mimetics obtained as in step 5 should have the same biologic properties as the original CHO antigen. That is, the mimetics should: (i) bind the appropriate lectin or inhibit binding of lectin to the original antigen; (ii) (if the original epitope is SLe^x or SLe^a) bind to E-selectin or P-selectin, or inhibit binding of E-selectin or P-selectin to SLe or SLe^a; (iii) bind to appropriate anti-CHO mAb's or inhibit binding of those mAb's to the original CHO epitope; (iv) induce antibody response when conjugated with macromolecular carriers and injected into the body. In some cases, T cell response should be observed, or even predominate over humoral immune response. Humoral antibody response, or T cell response after immunization, should be stimulated equally by the mimetic as by the original CHO epitope.

Testing for biological properties is conducted according to well known protocols.

Example of Procedure for Design of Peptide Mimetics

No studies on carbohydrate mimetics have been published, but there have been a few reports on synthesis of peptide mimetics. One recent example (Saragovei et al.. Science. 253:792-795, 1991) involving peptide mimetics of the reovirus 3 receptor peptide sequence, is described below. Reovirus infection of various host cells is based on initial interaction between a reovirus agglutinin which recognizes host cell receptors. In a series of experiments, mAb 9BG5 directed to the agglutinin was established. mAb 9BG5 binds to reovirus 3 hemagglutinin, that represents a cell-binding site, and therefore inhibits binding of reovirus to the host cell. Anti-idiotype mAb 87.92.6, which binds to mAb 9BG5 as well as to the cell-binding site of reovirus, subsequently was established and thus mimics the cell-surface receptor function. mAb 87.92.6 also down-regulates receptor function and inhibits DNA synthesis in cells. The essential peptide sequence of the CDR region of mAb 87.92.6 was found in the V_L region and has the sequence shown below (Williams et al., Proc. Natl. Acad. Sci. USA. 86:5537-5541, 1989). Lys-Pro-Gly-Lyε-Thr-Aεn-Lyε-Leu-Leu-Ile-

Tyr-Ser-Gly-Ser-Thr-Leu-Gln (SEQ. ID NO. 3) The pentapeptide Tyr-Ser-Gly-Ser-Thr (SEQ. ID NO. 4) shown in bold hereinabove is the essential site for binding to mAb 9BG5, and also binds to the cell receptor, down-regulates receptor function and inhibits DNA synthesis in cells and inhibits binding of reovirus to cells. Those conclusionε were baεed on a number of inhibition studies using various peptides with altered sequences, substitutions and other modifications. To fix the pentapeptide conformation, a mimetic was synthesized by using a cross-linking molecule. The mimetic inhibited binding of the long peptide representing the V_L region to mAb 9BG5, strongly down-regulated expression of reovirus receptor at the surface and inhibited reovirus-induced cellular DNA synthesis. The peptide mimetic was resistant completely to proteolysis (Saragovi et al., Science. 253:792-795, 1991) .

Medicaments and methods for inhibiting carbohydrate-mediated cell adhesion; vaccine for induction of anti-carbohydrate T cell immune response and method of vaccinating

As discussed above, there has been increasing evidence that specific carbohydrates (oligosaccharides) and conjugates thereof, which block carbohydrate-dependent cell adhesion, are useful for preventing tumor cell invasion and metastasis, inflammatory processes and microbial infection. The major obstacle to that approach has been the fact that those carbohydrates are unεtable, with unexpectedly short half-lives in vivo.

Accordingly, the present invention also provides a medicament for inhibiting metastasis of tumor cells, inflammatory processes and microbial infection caused by carbohydrate-mediated cell adhesion, the medicament comprising:

(A) a stabilized carbohydrate epitope having more resistance to metabolic degradation than a corresponding naturally occurring carbohydrate epitope or a mimetic of a carbohydrate epitope, wherein the mimetic has a structure such that the mimetic has about the same antibody-binding or selectin-binding activities, immunogenicity and antigenicity as that of a correspondingly naturally occurring carbohydrate epitope, and

(B) a pharmaceutically acceptable carrier, diluent, or excipient. The present invention also provides methods for inhibiting carbohydrate-mediated cell adhesion, which include inhibiting metastasis of tumor cells, inhibiting inflammatory processes and inhibiting microbial infection caused by carbohydrate-mediated cell adhesion. The method comprises contacting the cells with or administering to a host in need of treatment an inhibitory amount of a stabilized carbohydrate epitope having more resistance to metabolic degradation than a corresponding naturally occurring carbohydrate epitope or a mimetic of a carbohydrate epitope, wherein the mimetic has a structure such that the mimetic has about the same antibody-binding or selectin-binding activities, immunogenicity and antigenicity as that of a corresponding naturally carbohydrate. A majority of relevant carbohydrates comprise one or more fucose residues. Generally, fucosyltransferases catalyze transfer of L-fucopyranose from GDP-fucose at appropriate sites on putative glycoconjugates. Generally fucoεe occurε at nonreducing termini of glycoconjugateε. Accordingly, inhibitors of fucosylation, by affecting the proper expression of cell adhesion molecules, can affect profoundly intercellular interactions, such as cell adhesion.

One such inhibitor is based on the replacement of the glycosyl moiety of a sugar nucleotide by a more stable carba-sugar. For example, carbocyclic analogues of GDP-fucose serve as suitable inhibitors of fucosyltransferases. The conversion of L-fucose to the carbocyclic analogues thereof can be achieved by intramolecular olefination. For example, a suitable reaction scheme is intramolecular Emmons-Horner-Wadsworth olefination (Paulsen et al., Liebigs Ann. Chem. 1987:125; Marquez et al., J. Or . Chem. 53: 5709, 1988; Fukase & Horii, J. Org. Chem. 57: 3651, 1992; Becker, Tetrahedron 36: 1717, 1980) of the 2,6-dioxophosphonate intermediate. The reaction proceeds with retention of the stereogenic carbons.

Thus, carbohydrates comprising a fucose, such as Le^x, Le⁸, Le^y, SLe^x, SLe^a and the like, can be manipulated using a carbocyclic fucose derivative. Further, the carbocyclic fucose can be attached to the carbohydrate chain through a variety of linkages but preferably is linked in the α configuration at any of the available sites on the sugars comprising the backbone.

It has been observed that multivalent carbohydrate structures bind to selectin more strongly than do the monovalent structures under high shear stress conditions. Thus, bivalent Le^x (structure 17 in Table 1) , bivalent SLe^x (structure 18 in Table 1) and Le^x linked to SLe^x (structure 19 of Table 1) bind more strongly to selecting than do Le^x or SLe^x alone. Accordingly, it is desirable to produce bivalent or multivalent derivatives of carbohydrate mimics as a enhanced means to influence cell-cell interactions dependent on carbohydrate structures for recognition and adhesion.

Multivalent structures can be prepared conveniently by either use of a multifunctional molecule, liposomeε or polymerization. Since the Le mimic (26) and the SLe^x mimics (32), (34), (39), (41), (43) and (44) all possess a spacer arm with an amino group, derivatizations of those mimics are performed readily by reaction with a carboxyl group of another compound. For (43) , for convenience, the mimics are depicted by an "M" bonded to an NHBoc group or to a NH₂ group, thus, M-NHBoc or M-NH₂.

When using a multifunctional molecule, such as a trifunctional molecule as depicted in Scheme XII

(Figure 22) , oxidation of a commercially available tris(3-hydroxypropyl)aminomethane (A) (Aldrich Chemical, Milwaukee, WI) to (B) , followed by esterification with NHS, yields the tris(active ester) (C) . A typical coupling reaction between M-NH₂ and (C) gives the trivalent derivative (45) . The Boc group can be cleaved readily by TFA for further derivatization.

When using liposomes (Scheme XIII) (Figure 23) , a suitable lipid, such as the commercially available 2-tetradecylhexadecanoic acid (D) (Wako Chemical Co., Japan) , is converted to the active ester (E) and coupled to M-NH₂. The resulting neoglycolipid (46) can be used to prepare a liposome.

If polymerization is desired to produce a multimeric or multivalent structure, a number of possible known schemes and materials can be used. For example, as depicted in Scheme XIV (Figure 24) , free radical polymerization of the acrylamide derivative of (47) , prepared from M-NH₂ and commercially available (F) (Eastman Kodak Co., Rochester, NY) with acrylamide and its derivatives yields the copolymer (48) in which the composition and structure can be varied by the artisan by mere design choice. The carba-sugar analogue can be exposed to or administered to cells and hosts using known methods. As is known for pharmaceuticals, the amounts and routes of administration of the active agent can be determined by the artisan using methods known in the art. Thus, the presentation form of the active agent, for example, in a pill, liquid and the like, is at the discretion of the artisan practicing known methods and using known non-critical pharmaceutically accepted compounds to enhance the efficacy and delivery of the active agent.

Alternatively, carbohydrates can be made comprising such carbocyclic analogues. Replacement of the ring oxygen in a sugar pyranoside by a methylene group transforms a glycosidic linkage into an ether linkage, which is resistant to the glycohydrolase reaction. • Hence, incorporation of such an analogue will increase the metabolic stability of the parent carbohydrates without altering the stereochemistry. The stereochemistry of the carbohydrate is one of the most important factors for molecular recognition.

Since the negative charge of sialic acid plays an important role in cell adhesion processes (Tyrrell et al., Proc. Natl. Acad. Sci. USA 88:10372, 1991; Kelm et al., Eur. J. Biochem. 205:147, 1992; Yuen et al., Biochem. 31:9126, 1992), the carbocyclic analogue of Le^x can be modified further to mimic SLe^x by an incorporation of simple anionic functionalities. Thus, the terminal sugar of the backbone of carbocyclic analogues may be derivatized to contain a negatively charged substituent, such as a carboxyl group, sulfono group, phosphono group and the like.

A specific use of the method of inhibiting tumor cell metastasis includes treatment of malignancies. The method of inhibiting inflammation is applicable to any inflammation which is due to neutrophil motility and invasion into blood vessel walls. Another important application of peptide mimetics of carbohydrate epitope baεed on anti-idiotype monoclonal antibodies against anti-carbohydrate monoclonal antibodies is for induction of an anti-carbohydrate T-cell immune reεponse for suppression of tumor growth. Most carbohydrate antigens induce humoral antibody response, i.e., a T cell-independent B cell response. Apparently carbohydrates are not processed by the host immune machinery by regular antigen pathways. Therefore, peptide mimetics based on the CDR region of anti-idiotype monoclonal antibodies against anti-carbohydrate monoclonal antibodies can provide useful antigens for induction of a T cell response against carbohydrate antigens. Accordingly, the present invention also provides a- vaccine for inducing of an anti-carbohydrate T cell immune response, the vaccine comprising:

(A) a peptide mimetic of a carbohydrate epitope, wherein the mimetic has a structure such that the mimetic has about the same antibody-binding or selectin-binding activities, immunogenicity and antigenicity as that of a corresponding naturally occurring carbohydrate antigen, and (B) a pharmaceutically acceptable carrier, diluent or excipient. The present invention also provides a method of vaccinating to induce an anti-carbohydrate T cell immune response, the method comprising vaccinating a host with an anti-carbohydrate T cell inducing amount of a vaccine comprising the above-described mimetic of a carbohydrate antigen.

The inhibitory effective amount and the anti-carbohydrate T cell inducing amount of stabilized carbohydrate epitope or of the carbohydrate mimetic according to the present invention can be determined using art-recognized methods, such as by establishing dose response curves in suitable animal models and extrapolating to humans; extrapolating from in vitro data; or by determining effectiveness in clinical trials. Suitable doses of the stabilized carbohydrate epitope or of the carbohydrate mimetic according to the instant invention depend on the particular medical application, i.e., inhibiting carbohydrate-mediated cell adhesion or inducing anti-carbohydrate T cells, the severity of the disease, the weight of the individual, the age of the individual, the half-life in circulation etc. , and can be determined readily by the skilled artisan.

The number of doses, daily dosage and course of treatment may vary from individual to individual. Depending on the particular medical application, the stabilized carbohydrate epitope or the carbohydrate mimetic can be administered in a variety of ways, such as orally, parenterally and topically. Suitable pharmaceutically acceptable carriers, diluents or excipients which can be combined with the stabilized carbohydrate epitopes and the carbohydrate mimetics for administration depend on the particular medical use and can be determined readily by the skilled artisan. The stabilized carbohydrate epitopes and the carbohydrate mimetics with or without carrier can take a variety of forms, such as tablets, capsules, bulk or unit dose powders or granules; may be contained with liposomes; or may be formulated into solutions, emulsions, suspensions, ointments, pastes, creams, jells, foams or jellies.

Parenteral dosage forms include solutions, suspensions and the like.

Additionally, a variety of art-recognized carriers, excipients, diluents, fillers etc., are likely to be included in the dosage forms. Such subsidiary ingredients include disintegrants, binders, lubricants, surfactants, e ulsifiers, buffers, moisturizerε, solubilizers and preservatives.

The artisan can configure the appropriate formulation comprising stabilized carbohydrate epitopes or carbohydrate mimetics seeking guidance from numerouε authorities and references, such as "Goodman & Gilman'ε, The Pharmaceutical Basis of Therapeutics" (6th Ed. , Goodman et al., MacMillan Publ. Co., NY 1980).

In body sites that are characterized by continual cell growth or that require cell growth inhibition because of dysfunction and that are relatively inaccessible, stabilized carbohydrate epitopes or carbohydrate mimetics can be administered in a suitable fashion to ensure effective local concentrations. For example, the stabilized carbohydrate epitopes or the carbohydrate mimetics may be injected in a depot or adjuvant, carried in a surgically situated implant or reservoir that slowly releases a fixed amount of the substance over a period of time or may be complexed to recognition molecules with the ability of binding to a site presenting with abnormal cell growth.

An example of such a contemplated scenario is a recognition molecule that is an antibody with binding specificity for a bone marrow specific antigen, wherein the bone marrow-specific antibody is complexed to the stabilized carbohydrate epitope or the carbohydrate mimetic, the complex being administered to a patient with leukemia.

The invention now will be described by reference to specific examples, which are not to be considered to limit the invention. EXAMPLES

EXAMPLE I

Inhibitory Activity of Fluorinated Mimetic

Inhibition of B16 Melanoma Cell Binding

Human umbilical vein endothelial cells (HUVEC'ε) were grown in 20-well plates. Radiolabeled B16 melanoma cells (2 x 10 /well) were added in the presence of various concentrations of methyl-β-lactoside, compound (1') or compound (2') (Fig. IA) and the cells bound by the compound were measured, in terms of radioactivity.

A similar experiment was performed using lactosylceramide (LacCer)-coated plates (Fig. IB).

Figs. IA and IB graphically depict inhibition of B16 melanoma cell adhesion to HUVEC's (Fig. IA) and LacCer (Fig. IB) by methyl-β-lactoside (Me-β-Lactoside) , mimetic 6-deoxy-6-fluoro-galactopyranosyl-βl-+4- glucopyranosyl-βl-methylglycoside (compound (1')) and galactopyranosyl-βl->4-6-deoxy-6-deoxy-6-fluoro- glucopyranosyl-βl-methylglycoside (compound (2¹)). The abscissa represents concentration of added compound and the ordinate represents cell binding expressed as radioactivity per well.

In both experiments, compound (1') showed much stronger inhibition of B16 cell adhesion than did methyl-β-lactoside. Further compound (2') had no inhibitory effect.

Compound (l¹) therefore is considered to be a useful anti-adhesion reagent for suppression of tumor cell metastasis.

Synthesis of compound (!') and compound (2') Compound (1') (Fig. IA) , a lactose mimetic, was synthesized by subtiliεin-catalyzed preferential acylation of the 6-primary, OH group of the galactopyranosyl residue of lactose in dimethylformamide (DMF) . The 6-butyryl-galactopyranosyl-βl-*4-glucose derivative was formed (structure 6 in Scheme I, Fig. 2A) .

The compound then was treated with various reagents to compare the best yield of various derivatives which have a free OH group at the 6-position of the galactopyranosyl residue of lactose, but with all other OH groups substituted with various residueε (structures (11)-(18) in Scheme II, Fig. 2B) . Among those, structure (18) (i.e., trimethylbenzyl-substituted) showed the best yield.

The compound then was treated with diethylamino sulfur trifluoride (DAST) in dry CH₂C1₂ to achieve fluorination of the 6-primary OH group of the galactopyranosyl residue, followed by deprotection, thus yielding compound (1') in Fig. IA (structure (22) in Scheme II, Fig. 2B) .

Structure (6) in Scheme I (Fig. 2A) was tritylated at the primary OH group of the glucopyranosyl residue, was acylated at all unsubstituted OH groups and then treated with DAST, to give structure (29) in Scheme III

(compound (2') shown in Fig. IA) .

The details of the syntheses are as follows. Reference should be made to Figs. 2A to 2C for structures of compounds.

General

Melting points were measured with a Fisher-Johns melting point apparatus and uncorrected.

¹H NMR spectra were measured at 24°C, unless otherwise noted, on a Bruker WM-500 spectrometer with TMS (CDC1₃) or DSS (D₂0) as an internal standard. 13C and 19F NMR spectra were recorded on a Varian VXR-300 spectrometer. References used were internal TMS for ¹³C and internal CFC1₃ (CDC1₃) or external CF₃COOH (D₂0) for ¹ F. FABMS, including HRMS, were obtained using a JEOL

JMS-HX 110 mass spectrometer.

Specific rotations were determined at the 589 n (Na line) at rt with a Perkin-Elmer 241MC polarimeter. TLC was performed on Merck silica gel 60F₂₅₄ plates (0.25 mm thickness).

Flash column chro atography (Still et al., J _ Org. Chem. 1978, 43, 2923) was performed over Merck silica gel 60 (230-400 mesh ASTM) .

Solutions were concentrated below 40° under reduced pressure.

Subtilisin (protease N) was purchased from Amano

International Enzyme Co. (Troy, VA) and activated by lyophilization from a 0.1M phosphate solution (pH 7.8) prior to use (Riva et al., J. Am. Chem. Soc. 1988, 110. 584).

2,4-Dimethylbenzoyl chloride (Ador & Meier, Ber. 1879, .12., 1970) was prepared by reaction of 2,4-dimethylbenzoic acid with thionylchloride by a standard methodology (Vogel et al., "Vogel's Textbook of Practical Organic Chemistry"; Longman: New York, 1978; p 498) .

Synthesis of Compound fi¹)

Subtilisin-Catalyzed Esterification of β-Lactosides

β-Lactoside (8 mmol) , prepared by known methods, (Oguchi et al.. Cancer Commun. 1990, 2., 311; Koike et al., Carbohydr. Res. 1987 163. 189) and 2,2,2-trichloroethyl alkanoate (24 mmol) were dissolved in dry DMF (45 mL) . Subtilisin (protease N, 2 g) then was added and the suspension was shake incubated at 37°C for 5 d. After removal of the enzyme by filtration, the filtrate was concentrated to dryneεs. The crude product was purified by flash column chro atography (34:9:3 EtOAc/MeOH/H₂0) . One of the products was compound (6) (Fig. 2A) , which was characterized as follows.

Methyl 6 '-O-butyryl-β-lactoside (6): a colorless solid (73%); [α]_D +4.78° (c 2.0, MeOH) ; ¹H NMR (D₂0) δ 0.88 (t, 3, butyryl Me, J = 7.4 Hz), 1.59 (sixtet, 2, butyryl β-CH₂, J = 7.4 Hz), 2.36 (t, 2, butyryl α-CH₂, J = 7.4 Hz), 3.26 (dd, 1, H-2, J = 8.2, 8.2 Hz) , 3.51 (dd, 2, H-2', J = 7.9, 10.0 Hz), 3.53 (s, 3, OMe), 3.63 (dd, 1, H-3', J = 3.3, 10.0 Hz), 3.75 (dd, 1, J = 4.1, 11.5 Hz) and 3.93 (br d, 1, J = 11.5 Hz) (2 x H-6) , 3.90 (dd, 1, H-5' , J = 4.3, 8.0 Hz), 3.92 (br d, 1, H-4 ' , J = 4.0 ^■ HZ), 4.24 (dd, 1, J = 8.0, 11.7 Hz) and 4.29 (dd, 1, J = 4.3, 11.5 Hz) (2 X H-6'), 4.35 (d, 1, H-l, J = 8.2 Hz), and 4.41 (d, 1, H-l', J = 7.9 Hz); HRMS calc for C₁₇H₃₀O_l2-H 425.1659, found 425.1661.

Compound (6) then was used to prepare methyl 2,2',3,3',4', 6-hexa-O-acy1-6 ' -O-butyryl-β-lactosides (compounds (13), (14) , and (15)) as follows. A mixture of (6) (90 mg, 0.21 mmol) and the corresponding acyl chloride (3.7 mmol) in dry pyridine (3 mL) was refluxed for 3 h. Evaporation and co-evaporation with toluene yielded a crude product, which was purified by flash column chromatography (10:1 toluene/EtOAc) .

Compound (13): 217 mg (98%); [α]_D - 0.14 (c 1.0, CHC1₃) ; ¹H NMR (CDC1₃) <S 0.92 (t, 3, butyryl Me, J = 7.4 Hz), 1.59 (sixtet, 2, butyryl β-CH₂, J = 7.4 Hz), 2.21 (t, 2, butyryl α-CH₂, J = 7.4 Hz), 3.45 (s, 3, OMe), 3.50 (dd, 1, J = 7.4, 11.4 Hz) and 3.55 (dd, 1, J = 6.3, 11.4 Hz) (2 X H-6'), 3.79 (dd, 1, H-5 ' , J = 6.3, 7.4 Hz) , 3.82 (ddd, 1, H-5, J = 2.5, 4.5, 9.5 Hz), 4.21 (dd, 1, H-4, J = 9.5, 9.5 Hz), 4.49 (dd, 1, J = 4.5, 12.2 Hz) and 4.60 (dd, 1, J = 2.5, 12.2 Hz) (2 x H-6) , 4.61 (d, 1, H-l, J = 7.9 Hz) , 4.86 (d, 1, H-l¹, J = 8.0 Hz) , 5.34 (dd, 1, H-3', J = 3.5, 10.4 Hz) , 5.40 (dd, 1, H-2 , J = 7.9, 9.8 HZ) , 5.63 (d, 1, H-4 ' , J = 3.5 Hz) , 5.67 (dd, 1, H-2', J = 8.0, 10.4 Hz) , and 5.76 (dd, 1, H-3 , J =

9.5, 9.8 Hz) ; HRMS calc for C₅₉H₅₄0₁₈ +Na 1073.3208, found 1073.3210.

Compound (14): 237 mg (99%); [α]_D +111.5° (c 1.5, CHC1₃) ; ¹H NMR (CDC1₃) δ 0.92 (t, 3, butyryl Me, J = 7.3 Hz) , 1.60 (sixtet, 2, butyryl β-CH₂, J = 7.3 Hz) , 2.21

(s, 3) , 2.22 (s, 3) , 2.27 (ε, 3) , 2.36 (ε, 3) , 2.45 (ε, 3) , and 2.46 (ε, 3) (6 x PhMe) , 3.44 (ε, 3, OMe) , 3.53 (d, 2, 2 X H-6', J = 6.6 Hz) , 3.75 (t, 1, H-5 • , J = 6.6 Hz) , 3.79 (br dd, 1, H-5, J = ca. 4, 9.4 Hz) , 4.18 (dd, 1, H-4 J = 9.4 Hz), 4.45 (dd 1, J = 4.4 12.1 Hz) and

4.57 (m, 1) (2 x H-6) , 4.68 (d, 1, H-l, J = 8.6 Hz), 4.83 (d, 1, H-l', J = 7.8 Hz), 5.29 (dd, 1, H-3 ' J = 3.3, 10.3 Hz), 5.36 (dd, 1, H-2 , J = 8.6, 9.4 Hz) , 5.60 (d, 1, H-4' J = 3.3 Hz) , 5.63 (dd, 1, H-2 ' , J = 7.8, 10.3 HZ), and 5.73 (dd, 1, H-3 , J = 9.4, 9.4 Hz) ; HRMS calc for C^H^O._g+Na 1157.4150, found 1157.4123.

Compound (15): 253 mg (98%); [α]_D +83.1° (c 1.0, CHC1₃) ; ¹H NMR (CDC1₃) δ 0.92 (t, 3, butyryl Me, J = 7.4 Hz), 1.59 (sixtet, 2, butyryl β-CH₂, J = 7.4 Hz), 2.15 (s, 3), 2.20 (s, 3), 2.21 (s, 3), 2.29 (s, 3), 2.32 (s, 3), 2.35 (br s, 6), 2.36 (s, 3), 2.38 (s, 3), 2.43 (s, 3), 2.44 (S, 3), and 2.55 (S, 3) (6 X PhMe₂) , 3.44 (S, 3, OMe), 3.48 (dd, 1, J = 6.1, 11.1 Hz) and 3.71 (dd, 1, J = 7.3, 11.1 Hz) (2 X H-6'), 3.78 (ddd, 1, H-5, J = 1.8, 5.1, 9.4 HZ), 3.81 (dd, 1, H-5' , J = 6.1, 7.3 Hz) , 4.12 (t, 1, H-4, J = 9.4, 9.4 HZ), 4.39 (dd, 1, J = 5.1, 12.1 HZ) and 4.58 (dd, 1, J = 1.8, 12.1 Hz) (2 X H=6) , 4.57 (d, 1, H-l, J = 7.9 Hz) , 4.81 (d, 1, H-l', J = 7.9 Hz) , 5.31 (dd, 1, H-2, J = 7.9, 9.6 Hz), 5.34 (dd, 1, H-3 ' ,J = 3.5, 10.4 Hz), 5.63 (dd, 1, H-2 ' , J = 7.9, 10.4 Hz) , 5.68 (d, 1, H-4', J = 3.5 Hz), and 5.69 (dd, 1, H-3 , J = 9.4, 9.6 Hz); HRMS calc for C₇₁H₇₈0₁₈+Na 1241.5090, found 1241.5129.

Methyl 6'-O-butyryl-2,2 '-3,3 ' ,4 ' ,6-hexa-O-mesitoyl- β-lactoεide (16) . A mixture of (6) (0.5 g, 1.17 mmol), 2,4, 6-trimethylbenzoic acid (mesitoic acid) (2.35 g, 14.3 mmol) and trifluoroacetic anhydride (2.3 mL, 16.3 mmol) in dry benzene (90 mL) was stirred at rt under dry N₂ for 2 h and then poured into a pre-cooled solution of sat. aq NaHC0₃. The organic layer was separated, washed with H₂0 and dried over anhyd Na₂S0₄.

After filtration, the filtrate was concentrated and the residue was purified by flash column chromatography (10:1 toluene/EtOAc) to yield compound (16) (1.5 g, 100%) as a colorlesε εolid: [α]₀ -16.9° (c 1.0, CHC1₃) ; ¹H NMR (CDC1₃) δ 0.93 (t, 3, butyryl Me, J = 7.4 Hz), 1.59 (sixtet, 2, butyryl β-CH₂, J = 7.4 Hz), 2.05-2.35 (m, 6 x PhMe₃ and butyryl α-CH₂) , 3.26 (m, 2, 2 x H-6'), 3.43 (s, 3, OMe), 3.53 (br d, 1, H-5, J = ca. 9.3 Hz), 3.76 (dd, 1, H-5^«, J = 6.6, 6.6 Hz) , 3.98 (dd, 1, H-4, J = 9.4, 9.4 Hz), 4.16 and 4.43 (AB-q, 2, 2 X H-6, J = 12.3 Hz), 4.51 (d, 1, H-l, J = 7.1 Hz), 4.68 (d, 1, H- 1', J = 7.6 HZ), 5.30 (dd, 1, H-l, J = 7.6, 8.1 Hz), 5.52 (m, 2, H-2' and H-3) , 5.58 (dd, 1, H-3 ' , J = 3.5, 10.5 Hz), and 5.71 (br s, 1, H-4 * ) ; HRMS calc for C^H^O-a-CH-_jO 1271.5948, found 1271.5919.

Selective De-O-butyrylation of (13) . (14) . (15) or (16) to yield compound (18)

The reaction conditions and yield are shown in Table II. Table II. Selective de-O-butyrylation of (13) -(16)

substrate reaction condition product (yield )

(13) Et₃N-MeOH-H₂0 (1:5:1) no selectivity 30 min, 0°C

(14) Et₃N-MeOH-H₂0 (1:5:1) no selectivity 30 min, 0°C

(15) 0.01M NaOMe in MeOH 17 (84%) overnight, 0°

(16) 1M NaOH-MeOH (1:10) 18 (87%) 1 h, rt

Isolated yield.

TLC indicated that, in addition to the starting material (> 60%) , a mixture of several compounds was formed due to partial de-O-acylation.

The reaction mixture was neutralized with Amberlite IR-120 (H⁺) resin and purified by flash column chromatography (10:1 toluene/EtOAc) . The following compounds were obtained.

Methyl 2,2^»,3,3',4',6-hexa-O-(2,4-dimethylbenzoyl)- β-lactoside (17): [α]_D +83.1° (c 1.5, CHC1₃) ; ¹H NMR (CDC1₃) δ 2.16 (s, 3), 2.21 (s, 3), 2.22 (s, 6), 2.29 (s, 3), 2.36 (s, 3), 2.39 (s, 6), 2.43 (ε, 3), 2.47 (s, 3) , 2.48 (s, 3), and 2.55 (s, 3) (6 x PhMe₃) , 2.93 and 3.00 (ddd, 1, J = 6.6, 6.6, 13.8 Hz) (2 X H-6^• ) , 3.45 (s, 3, OMe), 3.65 (t, 1, H-5 ' , J = 6.6 Hz), 3.78 (m, 1, H-5) , 4.14 (dd, 1, H-4, J = 9.5, 9.5 Hz) , 4.35 (dd, 1, J = 5.2, 11.9 Hz) and 4,58 (m, 1,) (2 x H-6) , 4.57 (d, 1, J = 7.9 Hz) , 4.78 (d, 1, H-l¹, J = 8.0 Hz) , 5.36 (dd, 1, H-3', J = 3.4, 10.3 Hz) , 5.38 (dd, 1, H-2, J = 7.9, 9.5 HZ) , 5.58 (d, 1, H-4',J = 3.4 Hz) , 5.62 (dd, 1, H-3 , J = 9.5, 9.5 Hz) , and 5.71 (dd, 1, H-2 ' , J = 8.0, 10.3

Hz) ; HRMS calc for C₆₇H₇₂0₁₇+Na 1171.4670, found 1171.4612.

Methyl 2, 2', 3, 3' ,4', 6-hexa-O-meεitoyl-β-lactoεide (18) : [α]_D +1.7° (c 1.5, CHC1₃) ; ¹H NMR (CDC1₃) <S 2.11 (ε, 9), 2.15 (s, 6) , 2.19 (s, 6) , 2.21 (ε, 6) , 2.22 (ε, 3) , 2.24 (ε, 3) , 2.28 (s, 6) , 2.30 (s, 3) , 2.32 (S, 6) , 2.33

(s, 3), and 2.35 (ε, 3) (6 x PhMe₃) , 2.85 (dd, 1, J = 8.2, 11.7 Hz) and 3.14 (br m, 1) (2 x H-6') , 3.44 (ε, 3, OMe) . 3.50 (ddd, 1, H-5, J = 2.1, 4.6, 9.5 Hz) , 3.66 (dd, 1, H-5', J = 3.7, 8.2 Hz) , 4.20 (dd, 1, J = 4.6,. 12.2 Hz) and 4.44 (dd, 1, J = 2.1, 12.2 Hz) (2 X H-6) ,

4.48 (d, 1, H-l, J = 7.5 Hz) , 4.75 (d, 1, H-l', J = 6.9 Hz) , and 5.69 (d, 1, H-4 ' , J = 3.3 Hz) . HRMS calc for ^C7₃ ^H ₈₄°₁7^+Na 1255.5610, found 1255.5642.

Fluorination of (18) with DAST.

A solution of (18) (1.54 g, 1.24 mmol) in dry CH₂C1₂

(35 mL) was treated with a εolution of DAST (0.45 mL, 3.4 mmol) in dry CH₂C1₂ (35 mL) at 0°C under dry N₂. After stirring at rt for 3 d, DAST (0.4 mL, 3.0 mmol) was added further and stirring continued another 2 d. The mixture was poured into a pre-cooled solution of sat. aq NaHC0₃. The organic layer was separated, washed with H₂0 and dried over anhyd Na₂S0₄.

After filtration, the filtrate was concentrated and the residue was purified by flash column chromatography (10:1 toluene/EtOAc) which yielded methyl 6'-deoxy-6'- fluoro-2 ,2*,3,3^,,4', 6-hexa-O-mesitoyl-β-lactoside (compound 21) (1.45 g, 94%) as colorless crystals (from EtOH): mp 93-95°C; [α]_D -8.6° (c 2.0, CHC1₃) ; ¹H NMR (CDC1₃) δ 2.06 (s, 6), 2.15 (br s, 6), 2.20 (br ε, 12), 2.23 (S, 3), 2.24 (Ξ, 9) , 2.28 (s, 6) , 2.30 (s, 3), 2.31 (s, 3) , and 2.33 (br s, 6) (6 x PhMe,) , 3.44 (s, 3, OMe) , 3.45 (ddd, 1, J = 7.3, 9.3, 46.5 Hz) and 3.57 (ddd, 1, J = 6.1, 9.3, 46.5 Hz) (2 X H-6 ' ) , 3.52 (m, 1, H-5) , 3.78 (br ddd, 1, H-5 ' , J = 6.1,7.3, 7.6 Hz) , 4.00 (dd, 1, H-4, J = 9.5, 9.5 HZ), 4.11 (dd, 1, J = 4.0, 12.2 Hz) and 4.43 (dd, 1, J = 2.1, 12.2 Hz) (2 x H-6) , 4.50 (d, 1, H-l, J = 7.4 Hz), 4.69 (d, 1, H-l', J = 7.7 Hz) , 5.30 (dd, 1, H-2, J = 7.7, 9.4 Hz), 5.49 (dd, 1, H-3, J = 9.4, 9.5 Hz), 5.51 (dd, 1, H-2 ' , J = 7.7, 10.5 Hz), 5.60 (dd, 1, H-3', J = 3.6, 10.5 Hz), and 5.75 (d, 1, H-4 ^• , J = 3.6 Hz); HRMS calc for C-^H^FO^+Na 1257.5560, found 1257.5591.

Reductive De-O-mesitoylation of (21) The reaction condition, workup and yield are described in Table III.

Table III. Reductive De-O-mesitoylation of (21)

reagent (equiv ) solvent time iεolated yield,

Reactions were performed using a solution of (21) (0.02 mmol) in an appropriate solvent (2 mL) under dry N₂ at rt and terminated by the addition of pre-cooled H₂0. An equiv amount of one acyl group. ^c The reaction mixture was concentrated and purified by LH-20 column chromatography (MeOH) to give (23) and then (22) . ^d Not detected in the reaction mixture. ^e The same result was obtained when ten mmol of (21) waε used. Prepared in situ from LiAlH₄ and H₂S0₄ (see Brown & Yoon, Jj_ Am. Chem. SOC 1966, 88., 1464).

The physical data of one of the products, compound (22), was as follows.

Methyl 6'-deoxy-6'-fluoro-β-lactoside (22) (Compound (1') in Fig. IA) : an amorphous solid; [α]_D - 12.5° (c 1.0, MeOH); ¹H NMR (D₂0, 47°C) δ 3.40 (dd, 1, H- 2, J = 7.9, 8.3 Hz), 3.65 (dd, 1, H-2 - , J = 7.8, 9.9 Hz), 3.66 (s, 3, OMe), 3.77 (dd, 1, H-3' , J = 3.2, 9.9 Hz) , 3.89 (dd, 1, J = 4.3, 12.3 Hz) and 4.07 (br d, 1, J = 12.3 Hz) (2 X H-6) , 4.08 (d, 1, H-4 ' , J = 3.2 Hz) , 4.10 (ddd, 1, H-5', J = 4.0, 7.4, 15.1 Hz) , 4.48 (d, 1, H-l, J = 7.9 Hz) , 4.57 (d, 1, H-l', J = 7.8 Hz) , and 4.74 (ddd, 1, J = 7.4, 10.3, 46.6 Hz) and 4.78 (ddd, 1,

J = 4.0, 10.3 45.3 HZ) (2 X H-6 ' ) ; ¹ F NMR (D₂0) -5 - 153.3 (ddd, F-6', J = 15.1, 45.3, 46.6 Hz); HRMS calc for C₁₃H₂₃F0_1fJ-H 357.1197, found 357.1194.

Synthesis of Compound 2'

Methyl 2,2'3,3' ,4'-penta-O-acetyl-6'-O-butyryl-6-O- trityl-β-lactoside (26) .

A mixture of (6) (273 mg, 0.64 mmol) and triphenylmethyl chloride (trityl chloride) (197 mg, • 0.7 mmol) in dry pyridine (10 mL) was heated to 100°C. After 1 h, trityl chloride (100 mg, 0.35 mmol) was added further and heating continued for another 35 min. Then Ac₂0 (8 mL) waε added and heating continued for an additional 1 h. The reaction was terminated by the addition of EtOH (8 mL) . Concentration of the mixture, followed by co-evaporation with toluene, left a crude product which was purified by flash chromatography (1:1 hexane/EtOAc) to give compound (26) (450 mg, 80%) as a colorless glass: [α]_D -26.8° (c 2.0, CHC1₃) ; ¹H NMR (CDC1₃) «S 1.01 (t, 3, butyryl Me, J = 7.4 Hz), 1.7 sixtet, 2, butyryl β-CH₂, J = 7.4 HZ) , 1.93 (s, 3) , 2.04 (s?, 3) , 2.07 (s,3) , 2.10 (s,3), and 2.35 (s,3) (5 x OAc, 2.36 (t, 2, butyryl α-CH₂, J = 7.4 Hz), 3.07 (dd, 1, J = 2.1, 10.4 Hz) and 3.71 (br d, 1, J = 10.4 HZ) (2 X H-6) , 3.39 (br d, 1, H- 5, J = ca. 10 Hz), 3.56 (s, 3, OMe), 3.61 (br t, 1, H- 5', J = ca. 6.8 Hz), 4.04 (dd, 1, J = 6.6, 11.3 Hz) and 4.22 (dd, 1, J = 6.8, 11.3 Hz) (2 X H-6^•) , 4.32 (dd, 1, H-4, J = 9.5, 9.5 HZ), 4.40 (d, 1, H-l, J = 7.8 Hz), 4.45 (d, 1, H-l', J = 8.1 HZ), 4.66 (dd, 1, H-3 ' , J = 3.5, 10.3 HZ), 4.84 (dd, 1, H-2' , J = 8.1, 10.3 Hz), 5.06 (dd, 1, H-2, J = 7.8, 9.5 Hz) , 5.13 (dd, 1, H-3 = 9.5, 9.5 Hz), and 5.22 (d, 1, H-4 ' J = 3.5 Hz); HRMS calc for C₄₆H₅₄0₁₇+Na 901.3259, found 901.3242.

Methyl 2,2',3,3',4'-penta-O-acetyl-6'-O-butyryl-β- lactoside (27) .

A mixture of (26) (280 mg, 0.32 mmol) was treated with 80% aq AcOH (10 mL) at 80 °C for 5 h. After concentration, the reεidue was purified by flash column chromatography (1:2 hexane/EtOAc) to give (27) (158 mg, 78%) aε a colorless syrup: [α]_D -13.1° (c 1.0, CHC1₃) ; ¹H NMR (CDC1₃) δ 0.95 (t, 3, butyryl Me, 1 = 7.4 Hz), 1.64 (sixtet, 2, butyryl β-CH₂, J = 7.4 Hz), 1.84 (dd, 1, OH, J = 3.9, 9.6 Hz), 1.96 (s, 3), 2.04 (s, 6), 2.05 (s, 3), and 2.15 (s, 3) (5 x OAc) , 2.28 (t, 2, butyryl α-CH₂, J = 7.4 HZ), 3.41 (br d, 1, H-5, J = 9.6 Hz), 3.49 (s, 3, OMe) 3.76 (br dd 1, H-6a, J - ca. 9.6, ca. 9.6 Hz), 3.90 (br dd, 1, H-5', J = ca. 6.1, ca. 6.1 Hz), 3.93 (dd, 1, H-4, J = 9.6, 9.6 Hz), 4.09 (dd, 1, J = 6.4, 10.9) and 4.13 (dd, 1, J = 6.4, 10.9 Hz) (2 x H-6' ) , 4.42 (d, 1, H-l, J = 8.2 Hz) , 4.61 (d, 1, H-l', J = 7.9

HZ) , 4.85 (dd, 1, H-2, J = 8.2, 9.5 Hz) , 4.99 (dd, 1, H- 3', J = 2.8, 9.9 HZ) , 5.11 (dd, 1, H-2 ' , J = 7.9, 9.9 Hz) , 5.19 (dd, 1, H-3, J = 9.5, 9.6 Hz) , and 5.34 (d, 1, H-4', J = 2.8 HZ) ; HRMS calc for C₂₇H₄₀O₁₇+Na 659.2163, found 659.2151.

Fluor ination of (27) with DAST

In_CH₂Cl₂

A solution of (27) (50 mg, 0.078 mmol) in dry CH₂C1₂ (2 mL) was treated with a solution of DAST (0.1 mL, 0.75 mmol) in dry CH₂C1₂ (2 mL) at 0 °C under dry N₂. After stirring at rt for 30 min, the mixture was poured into a pre-cooled solution of sat. aq NaHC0₃. The organic layer was separated, washed with H₂0 and dried over anhyd Na₂S0₄. After filtration, the filtrate was concentrated and the residue was purified by flash column chromatography.

Flash column chromatography (3:2 hexane/EtOAc) yielded two fractions. The first fraction gave penta-O- acetyl-6*-butyryl-6-θ-methyl-β-lactosyl fluoride (30) (18 mg, 36%) as a colorless syrup: ¹H NMR (CDC1₃) δ 0.95 (t, 3, butyryl £fe, J = 7.5 Hz), 1.64 (sixtet, 2, butyryl, β-Cfi₂, J = 7.5 Hz), 1.97 (s, 3), 2.05 (s, 3), 2.06 (s,3), 2.08 (s,3) , and 2.15 (ε, 3) 5 x OAc), 2.29 (t, 2, butyryl α-CH₂, J = 7.5 Hz), 3.44 (s, 3, OMe), 3.63 (br d, 1, J = 10.6 Hz) and 3.69 (br dd, 1, J = ca. 1.0, 10.6 Hz) (2 X H-6) , 3.65 (m, 1, H-5) , 3.89 (5, 1, H-5 ' , J = 6.6, 7.1 Hz), 4.06 (dd, 1, H-4 , J = 9.4, 9.4 Hz) , 4.10 (dd, 1, J = 7.1, 11.1 Hz) and 4.13 (dd, 1, J = 6.6, 11.1 Hz) (2 X H-6') , 4.56 (d, 1, H-l', J = 8.0 Hz) , 4.98 (dd, 1, H-3', J = 3.5, 10,3 HZ), 5.00 (ddd, 1, H-2, £ = 6.0, 8.0, 10.1 HZ), 5.10 (dd, 1, H-2 ' , J - 8.0, 10.3 HZ), 5.16 (dd, 1, H-3, J = 8.0, 9.4 Hz) , 5.31 (dd, 1, H- 1, J = 6.0, 52.9 Hz) , and 5.34 (d, 1, H-4 ' , J = 3.5 Hz) ; ¹³C NMR (CDC1₃) δ 60.7 (C-6) , 66.7 (C-6'), 71.3 (d, C-2 ,

J = 29.1 HZ), 72.1 (d, C-3, J = 7.6 Hz) , 100.80 (C-l') , and 106.2 (d, C-l, J = 217 Hz) ; ¹⁹F NMR (CDC1₃) δ -135.5 (dd, F-l, J = 10.1, 52.9 Hz) ; HRMS calc for C₂₇H₃₉FO_H+Na 661.2120, found 661.2099. The second fraction gave methyl 2,2' ,3,3' ,4' -penta-

O-acetyl-6 ' -0-butyryl-6-deoxy-6-f luoro-β-lactoside (28 ) (16 mg, 32%) as a colorless syrup: [α]_D —24.0° (c l.o, CHCl_j) ; ¹H NMR (CDC1₃) δ 0.94 (t, 3, butyryl Me, J = 7.4 Hz), 1.63 (sixtet, 2, butyryl β-CH₂, J = 7.4 Hz), 1.96 (s, 3), 2.04 (S, 6) , 2.06 (s, 3) , and 2.14 (s, 3) (5 X

OAc) , 2.28 (t, 2, butyryl α-CH₂, J = 7.4 Hz), 3.49 (s, 3, OMe), 3.52 (br dd, 1, H-5, J = ca. 9.6, 24.8 HZ), 3.89 (t, 1, H-5', J = 6.6, 7.5 Hz) , 3.92 (dd, 1, H-4 , J « 9.6, 9.6 HZ) , 4.09 (dd, 1, J = 7.5, 11.0 Hz) and 4.13 (dd, 1, J = 6.6, 11.0 Hz) (2 x H-6 • ) , 4.42 (d, 1, H-l,

J = 7.8 HZ) , 4.58 (d, 1, H-l', J = 7.8 Hz) , 4.59 (dd, 1, J = 10.5, 48.6 Hz) and 4.69 (dd, 1, J = 10.5, 48.2 Hz) (2 X H-6) , 4.87 (dd, 1, H-2, J = 7.8, 9.4 Hz) , 4.99 (dd, 1, H-3', J = 3.1, 10.4 HZ), 5.12 (dd, 1, H-2 ' , J = 7.8, 10.4 Hz), 5.22 (dd, 1, H-3 , J = 9.4, 9.6 Hz) , and 5.33 (d, 1, H-4', J = 3.1 Hz) ; HRMS calc for C₂₇H₃₉F0₁₆+Na 661.2120, found 661.2108.

In diglyme

Fluorination was carried out, similarly to that described above, by mixing a solution of (27) (110 mg, 0.17 mmol) in dry diglyme (5 mL) and a solution of DAST (0.23 mL, 1.7 mmol) in dry diglyme (5 mL) . After stirring at rt overnight, the same workup as above gave (28) (75 mg, 68%) as the sole product.

Methyl 6-Deoxy-6-fluoro-β-lactoside (29) (Compound (2') in Fig. IA) A mixture of 28 (118 mg, 0.19 mmol) in 0.01M methanolic NaOMe (6 mL) was left at 0°C overnight. The mixture was neutralized with Amberlite IR-120 (H^*) resin and passed through a column of Bio-Gel P-2 with H₂0. The eluate was lyophilized to give (29) (54 mg, 82%) as an amorphous solid: [α]₀ -0.07° (c 1.0, H₂0) ; ¹H NMR (D₂0, 47 °C) δ 3.43 (dd, 1, H-2, J = 8.1, 8.1 Hz) , 3.66 (br dd, 1, H-2' , J = ca. 8.0, ca. 8.0 Hz), 3.68 (s, 3, OMe) , 3.77 (br dd, 1, H-3¹, J = 3.0, 10.0 Hz) , 4.04 (d, 1, H- 4' , J = 3.0 HZ) , 4.54 (d, 2, H-l and H-l¹, J = 8.1 Hz), and 4.89 (br dd, 1, J = 10.7, 48.1 Hz) and 4.94 (ddd, l, J = 2.5, 10.7, 46.6 Hz) (2 X H-6) ; ¹⁹F NMR (D₂0) δ -156.9 (ddd, F-6, J = 30.0, 46.6, 48.1 Hz) ; HRMS calc for C₁₃H₂₃FO₁₀-H 357.1197, found 357.1198.

EXAMPLE II

Examples of Stabilized Carbohydrate Epitopes of

Sialosyl-Le^x

To reveal the minimum structure recognized by ELAM-1, some analogues of SLe^x were synthesized. The analogues syntheεized fall into four groupε: (1) deoxy analogueε (3) and (5) ; (2) deoxyfluoro analogueε (2) , (4) and (6); (3) metabolically εtable analogueε (7), (8) and (9) ; and (4) some analogues of the NeuAc residue (10) and (11) . On the basiε of a recent finding that the internal GlcNAc residue can be replaced by a Glc residue without losing any binding activity, methyl β-D-lactoside waε selected as a core structure.

Various substitutions of SLe" are shown as structures (1) through (11) in Fig. 3. The synthetic schemes are presented in Figs. 4A and 4B (for structures (l)-(6)), Fig. 5 (structureε (7)-(8)), Figε. 6A and 6B (structure (9)) and Fig. 7 (structureε (lθ)-(ll)). The compoundε all are εtable and are more effective aε inhibitors than are native SLe^x and SLe^a in view of the finding that the 6-fluoro-6-deoxy-galactopyranosyl substituted form of methyl-β-lactoside is much more effective than native methyl-β-lactoside for inhibition of cell adhesion.

I. Synthesis of (l)-(6) (Figs. 4A and 4B) ♦

6'-deoxy, 6'-deoxy-6'-fluoro, 6-deoxy and 6-deoxy- 6-fluoro analogues of methyl β-D-lactoside, that is, compounds (13), (14), (15) and (16), respectively, were synthesized from (12) by applying protease-catalyzed regioselective esterification. Acetonation, followed by selective benzoylation which relies on the lowest reactivity of the 3-OH group, yields the corresponding 3-O-unprotected lactosideε ((12)-(16) → (17)-(21) → (22)-(26)). For introducing the Fuc residue, thioglycoside-mediated glycosidation between (22)-(26) was employed along with the properly protected methyl 1-thio-β-L-fucopyranoside (31) , which is readily available from L-Fuc (27) ((27) → (29) → (31)). The glycosidation was initiated by the addition of dimethyl(methylthio)sulfoniu triflate⁷ • (DMTST) to construct the trisaccharides, (33) and (35)-(38).

A similar glycosidation also was practiced using the trifluoromethyl Fuc analogue ((22) + (32) → (34)). The glycosyl donor (32) waε prepared from (28)⁵ via (30) . Acidic treatment of (33) -(38) gave the dihydroxyglycosides (39)-(44) , which then were subjected to sialosylation by using methylthiosialoside (48) , prepared from (45)⁸ ((45) → (46)⁹ → (47)⁹ → (48)) and DMTST. The glycosidation condition is reported¹⁰ to give an α-isomer in a high yield.

The regioselectivity of the reaction dependε on the selective reaction of the equatorial hydroxyl group. Subsequent deprotection of the tetrasaccharides (49)-(54) yields the deoxy analogues (3) and (5) and the deoxyfluoro analogues (2), (4) and (6).

For further chemical and biological explorations, the methyl glycosides (49)-(54) can be converted to the glycosyl chlorides (55)-(60) by treatment¹¹ with dichloromethyl methyl ether and ZnCl.

II. Synthesis of (7) and (8) (Fig. 5)

Metabolically stable analogues of SLe^x containing a thiofucopyranoside linkage or a carba-Fuc residue also were synthesized. Both analogues are expected to inhibit α-fucosidase activities.

The synthetic plan for the incorporation of a thioglycoside linkage involves inversion of the configuration at the glycosyl position of the acceptor by nucleophilic attack of a thiolate ion. Therefore it is necessary to epimerize the 3-OH group of lactoside. The dibutylstannylene-mediated oxidation of 3' ,4•-isopropylidene lactoside (17) results in the selective oxidation of the 3-OH group ((17) -> (65)¹³). Benzoylation of the residual hydroxyls ((65) → (66) followed by hydride reduction, epimerizes the 3-OH group yielding β-D-galactopyranosyl-β-D-glucopyranoside (68) . The 3-OH group of (68) then is converted to a better leaving group (triflate, Tf) ((68) - (70)). Condensation of (64) and (70) leads to the thioglycoside (72) , which, after a series of transformations including sialosylation and deprotection, gives the SLe^x analogue (7) containing the α-thiofucopyranoεyl residue.

Carba-α-L-fucopyranose from L-fucopyranoεe from L-Fuc (27) was synthesized. One of the intermediates of the synthesis, (73) , is considered a properly protected glycosyl donor. The construction of the pseudotrisaccharide (75) followed steps similar to those for the preparation of (72) , except that the lithio derivative (74) is coupled with the benzyl-protected disaccharide (71) which is prepared from (65) ((65) → (67) → (69) → (71)). Subsequent sialosylation, followed by deprotection, gives SLe^x analogue (8) possessing a carba-α-Jj-fucopyranosyl residue.

III. Synthesis of (9) (Figs. 6A and 6B)

The thiosialoside analogue of SLe which is expected to resist sialidases also is syntheεized. The principle of introducing the thioglycoεide linkage iε baεically the same as that described above. Thus, the triflate derivative (84) is required as a thiosialosylation acceptor. The 3*-0-allyl derivative (76) is prepared from (12) through stannylation. After acetonation ((76) → (77)¹⁵) and selective benzoylation ((77) → (78)), the α-L-Fuc residue is introduced to the 3-position of (78) by use of benzyl-protected fucopyranosyl fluoride (79) as a glycosyl donor ((78) + (79) → (80)). The fluoride (79) is prepared readily from (31) by treatment with NBS and HF-pyridine.¹⁶ Isomerization of the allyl group and subsequent hydrolysis yield (81) . The hydroxyl group then is epimerized by an oxidation-reduction procedure ((81) -> (82) → (83)). Condensation of the thiolate (47) and the trif late ( 84 ) proceeds in the S_N2 fashion to produce the tetrasaccharide ( 85 ) . De-O-benzylation with N a i n l i q . NH₃ , ε a p o n i f i c a t i o n a n d de-O-isopropylidenation give the thiosialoside analogue of SLe^x ( 9) .

IV. Synthesis of (10) and (11) (Fig. 7)

The N-acetyl group on the NeuAc residue appears to be important for binding through hydrophobic interactions of the methyl group. Therefore, the acetyl group of the NHAc group is replaced by trifluoroacetyl and benzoyl groups, expecting that those hydrophobic groups increase binding activity. De-N-acetylation is carried out by refluxing (1) under strong basic ccoonnddiittiioonnss ((((11)) →→ ((8866)))) .. SSeelleeccttiivvee trifluoroacetylation ((86) → (10)) and benzoyllaat- ion ((((8866)) →→ ((1111)))) aarree possible by employing CF₃C0SEt and Bz₂0, respectively.

EXAMPLE III

Structural Analogue Common to Both the

SLe^x and SLe^a Structures

On the basis of the recent finding that ELAM-1 contains a binding site which recognizes a carbohydrate domain common to both SLe^x and SLe^a antigens, it is believed that the internal Glc residue may be necessary only for presenting the Fuc and NeuAcα2-3Gal residues in proper orientation. If the Glc residue is substituted for by (1R, 2R)-1,2-cyclohexanediol, the SLe-like structure and the SLe^a-like structure become identical (see (87)) (Fig. 8).

The cyclohexanediol analogue (87) is synthesized as shown in Figs. 9A and 9B. The coupling of (31) with (1R*, 2R*)-l,2-cyclohexanediol, followed by column chromatography, gives a mixture of two isomerε (88) and

(89) ((88) being the deεired one) . The εyntheεiε of the

NeuAcα2-+3Gal portion followε an established procedure¹⁰

((48) + (91) → (92)), except that the primary hydroxyl group is protected selectively by protease-catalyzed esterification² ((90)²¹ → (91)). After acetylation ((92)

→ (93)), the 2-(trimethylsilyl) ethyl group is transformed sequentially to the acetate and methylthio ² groups ((93) -+ (94) → (95)). The coupling reaction of (88) and (95) yields, after deprotection, pseudo-tetrasaccharide (87) ((88) + (95) → (96) → (87)).

The references referred to in the various synthetic schemes hereinabove are listed below.

References

1. Oguchi et al., Cancer Commun. 1990, 2., 311.

2. Cai et al., j Org. Chem. 1992, 57, in press.

3. For example: Yoshino et al., Glvcoconiugate i. 1988, 5, 377.

4. For example: Bhatt et al., J_j_ Chem. Soc.. Perkin I 1977, 2001.

5. Bansal et al., J^. Chem. Soc.. Chem. Commun. 1991, 796.

6. Kameyama et al. , J_j_ Carbohydr. Chem. 1991, 10. 549. 7. Fvigedi & Garegg, Carbohvdr. Res. 1986, 149.

C9.

8. Kuhn et al., Chem. Ber. 1966, 9_£, 611.

9. Hasegawa et al., __Ϊ_ Carbohvdr. Chem. 1986, 5_, 11. 10. For example: Murase et al., Carbohvdr. Res.

1989, 188, 71.

11. For example: Kovac et al., J^ Org. Chem. 1985, 50, 5323. 12. Schmidt & Stu pp, Liebigs Ann. Chem. 1983, 1249.

13. Fernandez-Mayoralas et al., Tetrahedron 1988, 44. 4877. 14. Cai et al., 204th ACS National Meeting,

Washington, D.C., Aug. 23-28, 1992.

15. Jung et al., Liebigs Ann. Chem. 1989, 1099.

16. Nicolaou et al., .J_^. Am. Chem. Soc. 1990, 112. 3693. 17. For example: Kanie et al. , Jj_ Carbohydr. Chem.

1987, 6, 117.

18. For example: Jennings et al., ^. Immunol. 1986, 132, 1708.

19. Schallenberg & Calvin, J Am. Chem. Soc. 1955, 77, 2779.

20. Berg et al., J___ Bio. Chem. 1991, 266. 14869.

21. Alais et al.. Tetrahedron Lett. 1983, 24. 2383.

22. Jansson et al., J^. Org. Chem. 1988, 5_3_, 5629.

EXAMPLE IV

The conversion of L-fucose to its carbocyclic analogues was achieved by intramolecular Emmons-Horner-Wadsworth olefination (Paulsen & Von Deyn, Liebigs Ann. Chem. 1987, 125; Marquez et al., J^. Org. Chem. 1988, 53, 5709; Fukase & Horii, J^. Org. Chem. 1992, 57, 3651; Becker, Tetrahedron 1980, 36, 1717) of the 2,6-dioxophoεphonate (7) (Figure 10), which proceeded with retention of the stereogenic centers at C-2, C-3 and C-4 in L-fucopyranose. The synthesis of (7) started from the known hemiacetal (Dejter-Juszynski & Flowers, Carbohydr. Res. 1971, 18, 219) (3), readily accessible from L-fucose in three steps (Zehavi & Sharon, J^. Org. Chem. 1972, 37, 2141) (61% yield) . Albright-Goldman oxidation (Albright & Goldman, J^. Am. Chem. Soc. 1965, 87, 4214; 1967, 89, 2416) of (3) to the 1,5-lactone (4) (All new compounds, except the unstable dioxophosphonate (7) were characterized fully on the basis of the H NMR spectra and MS analysis thereof.) followed by a nucleophilic substitution reaction with the carbanion derived from dimethyl methylphosphonate, afforded the heptulopyranose (5) as a single anomeric isomer. (The H NMR spectrum indicated (5) to exist in a single anomer. The nucleophile likely approached from the lesε hindered side of the carbonyl group to form an axially disposed hydroxyl group, i.e., an α-anomer.)

Reductive ring opening of (5) with NaBH₄ to the heptitol (6) and subsequent Swern oxidation (Huang et al., Synth. 1978, 297) yielded the unstable dioxophosphonate (7) . The ensuing intramolecular olefination of (7) occurred smoothly by treatment with NaH in diglyme (Grieco & Pogonowski, Synth. 1973, 425) to give the unsaturated inosose (8) ([α]_D-105°, c. 1.0, CHC1₃) in 94% yield. The use of K₂C0₃ as base in the presence of 18-crown-6 ^,c afforded the product in a lower yield («60 %) .

The copper(I) hydride hexamer (Ph₃PCuH)₆ (Brestensky & Stryker, Tetrahedron Lett. 1989, 30, 5677; Koenig et al., Tetrahedron Lett. 1990, 31, 3237) allowed the stereoselective conjugate reduction of (8) yielding the desired inosose (9) (J_H-_5,H-_6ax' ^{= 13}-8 Hz) as the only detectable diastereoisomer in 93% yield. Apparently, the hydride was delivered to the less-hindered side of (8) . The NaBH₄-CeCl₃ reduction in MeOH (Gemal & Luche, J__j. Am. Chem. Soc. 1981, 103, 5454) furnished an almost quantitative conversion of (9) to the equatorial alcohol (10) (J,,., _H_₂ = 9.3 Hz), which was the suitably protected carba-β-L-fucopyranose required for the subsequent phosphorylation. In the absence of CeCl₃ the same reduction resulted in a poor stereoselectivity giving a mixture of (10) and its epimeric alcohol (11) (J_H-ι_,H-₂ ⁼ 2.4 Hz) in a ratio of 1.3:1. Hydrogenolysis of (11) yielded 5a-carba-α-L-fucopyranose (12) (mp 142-143°C; [α]_D -81°, c 1.0, H₂0) .

Stereoselective 1,2-reduction of the carbonyl moiety in (8) was successful by treatment with NaBH₄-CeCl₃ in MeOH, (Gemal & Luche, J_^. Am. Chem. Soc. 1981, 103, 5454) which afforded exclusively the desired pseudoequatorial alcohol (13) («5_H.₆5.47, J_H.. _H.₆ = 1.2 Hz) (On the basis of the H NMR εtudies on the antibiotic valienamine, ID-(1,3,6/2)-6-amino-4-hydroxymethyl-4- cyclohexene-1,2,3-triol, and its derivatives, it is reported that a vicinal pseudoequatorial substituent (i.e., 1-OH in (13)) causeε the reεonance of an olefinic proton (H-6) to move upfield relative to the pseudoaxial epimer (i.e., (14)): Toyokuni et al., Bull. Chem. Soc. Jpn. 1983, 56, 1161) in 91% yield (Figure 11).

However, the reduction with NaBH₄ alone produced a mixture of diastereoisomerε (13) and (14) (<5_H.₆5.53,J_H_. _H.₆ = 4.3 Hz) together with the saturated alcohol (10) in a ratio of 3:1:1.5. The 1,2-reduction was effected also with 9-BBN in THF, (Krishnamurthy & Brown, J^ Org. Chem. 1975, 40, 1864) but with less satisfactory results, producing a 5:1 mixture of (13) and (14). The Birch reduction (McCloskey, Adv. Carbohydr. Chem. 1957, 12, 137) of (14) afforded the unεaturated analogue of carba-α-L-fucopyranose (15) ([α]_D-272°, c 1.0, H₂0) .

Phosphorylation of (10) and (13) proceeded smoothly in high yields by phosphitylation using N,N-diisopropyl dibenzyl phosphoramidite and IH-tetrazole, followed by oxidation with m-CPBA (Yum & Fraser-Reid, Tetrahedron Lett. 1988, 29, 979) (Figure 12). Subsequent Birch reduction (McCloskey, Adv. Carbohvdr. Chem. 1957, 12, 137) of the resulting perbenzylated phosphate (16) and (18) yielded carba-β-L-fucopyranosyl phosphate (17) ([α]_D -1.6°, c 1.0, H₂0) and its unsaturated analogue (19) ([α]_D -55°, c 0.7, H₂0) , respectively in excellent yields. The phosphates (17) and (19) then were coupled to GMP-morpholidate according to a standard procedure (Nunez et al., Can. J. Chem. 1981, 59, 2086; Gokhale et al., Can. J. Chem. 1990, 68, 1063; Schmidt et al., Liebigs Ann. Chem. 1991, 121) to give target compounds (1) ([<*]„ -14.0°, c 1.0, H₂0) and (2) ([α]_D - 19.9°, c 0.7, H₂0) , respectively.

An inhibition assay waε carried out againεt α(l-+3/4) fucosyltransferaεe obtained and solubilized from human colonic adenocarcinoma Colo205 cells maintained in culture uεing lacto-N-fucopentaoεe 1 (LNF 1: Fucαl→2Galβl→3GlcNAcβl-3Galβl→4Glc) as a substrate (The fucosyltranεferaεe catalyzeε the tranεfer of L-fucoεe from GDP-Fuc to the 4-OH of the GlcNAc residue in LNF 1 yielding Le -hexasaccharide [Fucαl→2Galβl-3(Fucαl→4)GlcNacβl→3Galβl->4Glc]) . Both carbocyclic analogues (1) and (2) exhibited a potent inhibitory activity. The half-chair confirmation of the cyclohexane ring in (2) probably could mimic the fucoεyltranεferaεe tranεition εtate by adopting, albeit not perfectly, the flattened anomeric conformation of the fucosyl intermediate.

Melting points were measured with a Fisher-Johnε melting point apparatuε and were uncorrected. H NMR spectra were measured at 36°C on a Bruker WM-500 spectrometer with TMS (CDC1₃) or DSS (D₂0) as an internal standard. FABMS including HRMS was obtained using a JEOL JMS-HX 110 mass spectrometer. For compoundε (1) and (2) , electroεpray (ES) MS waε recorded on a Sciex Biomolecular Mass Analyzer API III mass spectrometer in the Mass Spectrometer Analysis Laboratory, Department of Biochemistry, University of Washington. Specific rotations were determined at 589 nm (Na line) at 23°C with a Perkin-Elmer 24IMC polarimeter. TLC was performed on Merck silica gel 60F₂₅₄ plates (0.25 mm thickness) and flash column chromatography on Merck silica gel 60 (230-400-mesh ASTM) . Solutionε were concentrated uεing a rotary evaporator below 40°C. GDP-Fuc waε εyntheεized according to a known procedure. GDP-[U- 14C]Fuc was purchased from Amersham (Arlington Heights, IL) and LNF 1 from Oxford GlycoSystems, Inc. (Roεedale, New York) . Colo205 cellε were obtained from the American Type Culture Collection (Rockville, MD) . Radioactivity waε meaεured on a Beckman LS 3801 Liquid Scintillation Counter.

Tri-0-Benzyl-L-fucono-l,5-lactone (4) . A mixture of tri-O-benzyl-L-fucopyranose (Dejter-Juszynski & Flowers, Carbohydr. Res. 1971, 18, 219) (3) (3.85 g, 8.86 mmol), acetic anhydride (27 mL) and DMSO (40 mL) was stirred at rt overnight. Lyophilization, followed by flaεh column chromatography (5:2 hexane/toluene) , gave (4) (3.22 g, 84%) as a colorless syrup: [α]_D -89.5° (c 1.0, CHC1₃) ; H NMR (CDC1₃) δ 1.34 (d, 3, Me, J = 6.5 Hz) , 3.79 (dd, 1, H-4, J = 1.4, 2.2 Hz), 3.89 (dd, 1, H-3, J = 2.2, 9.5 Hz), 4.33 (dq, 1, H-5, J = 1.4, 6.5 Hz) , 4.46 (d, 1, H-2, J = 9.5 Hz); HRMS calc for C₂₇H₂₈Na0₅ (M + Na) + 455.1834, found 455.1852.

3,4,5-Tri-0-benzyl-l-deoxy-l-(dimethylphosphoryl)- α-L-fuco-2-heptulopyranose (5) .

A 2.5M solution of n-BuLi in hexane (21.2 mL) was added dropwise to a solution of dimethyl methylphosphonate (8.11 mL, 74.8 mmol) in THF (150 mL) with stirring at -77°C (dry ice-acetone) under N₂. After 30 min at -77°C, a solution of (4) (7.80 g, 18 mmol) in THF (80 L) was added dropwise. The mixture was stirred at -77°C for 30 min, warmed to 0°C and poured into chilled 10% aq NH₄C1 (200 mL) and dried over anhyd Na₂S0₄. After evaporation, the residue was purified by flash column chromatography (4:1 toluene/EtOAc) to give (5) (9.12 g, 91%) as a colorless syrup: [α]_D + 16.8° (c 0.9, CHC1₃) ; ¹H NMR (CDC1₃) δ 1.12 (d, 3, Me, J = 6.5 Hz), 1.77 (dd, 1, J = 15.3, 18.6 Hz) and 2.38 (dd, 1, j = 15.3, 17.6 Hz) (CH₂P) , 3.62 (d, 3, J = 11.0 Hz) and 3.74 (d, 3, J = 11.0 Hz) (P(OMe)₂) , 3.68 (br s, 1, H-5) , 3.74 (d, 1, H-3, J = 9.7 HZ), 4.09 (dd, 1, H-4, J = 2.8, 9.7 Hz), 4.15 (br q, 1, H-6, J = 6.5 Hz) ; HRMS calc for C₃₀H₃₆O₇P (MH - H₂0) = 539.2199, found 539.2221.

A mixture of 3,4,5-tri-0-benzyl-l,7-dideoxy-7- (dimethoxyphosphoryl)-D-glycero-d-galacto-hepitol and 3,4,5-tri-O-benzyl,1,7-dideoxy-7-(dimethoxyphosphoryl)- L-glycero-D-galacto-heptitol (6) .

A mixture of (5) (9.12 g, 16.4 mmol) and NaBH₄ (0.8 g, 21.1 mmol) in THF (100 mL) was stirred at rt overnight. After evaporation, the residue was dissolved in EtOAc (200 mL) , washed with H₂0 (3 x 150 mL) , dried over anhyd Na₂S0₄ and concentrated to dryness. The reεidue waε purified by flaεh column chromatography (2:1' toluene/EtOAc) to give a homogeneous mixture of C-6 epimers (6) (8.50 g, 93%) as a colorlesε εyrup: H NMR (CDC1₃) for (6a): δ 1.21 (d, 3, Me, J = 6.3 Hz) , 1.84 (dd, 1, J = 3.9, 15.5, 19.1 Hz) and 2.08 (ddd, 1, J = 8.3, 15.5, 17.6 Hz) (CH₂P) , 3.67 (d, 3, J =11.0 Hz) and 3.69 (d, 3, J = 11.0 Hz) (P(OMe)₂). For (6b): <5 1.24 (d, 3, Me, J = 6.5 Hz), 1.92 (ddd, 1, J = 10.3, 15.5, 15.5 Hz) and 2.24 (ddd, 1, J = 3.0, 15.5, 18.9 Hz) (CH₂P) , 3.67 (d, 3, J = 11.0 Hz) and 3.69 (d, 3, J = 11.0 Hz) (P(OMe)₂); HRMS calc for C₃₀H₄₀O₈P (MH) = 559.2461, found 559.2476.

4L-(4,5/6)-4,5, 6-Triε (benzyloxy) -3-methyl-2- cyclohexenone (8) .

A solution of TFAA (8.8 mL, 62.3 mmol) in CH₂C1 (360 mL) was added dropwise to a solution of DMSO (70 mL, 98.6 mmol) in CH₂C1₂ (75 mL) with stirring at -77°C (dry ice-acetone) . After 30 min at -77°C, a solution of (6) (8.5 g, 15.2 mmol) in CH₂CL₂ (70 mL) was added and the mixture was stirred at -77°C for lh. Et₃N (20.5 mL, 146 mmol) then was added and stirring continued at -77°C for another 30 min. The reaction mixture was warmed to 0°C, and poured into a chilled mixture of CH₂C1₂ (260 mL) and 2M aq HC1 (220 mL) . The organic layer was separated, washed with sat. aq NaHC0₃ (2 x 400 mL) and H₂0 (2 x 400 mL) and dried over anhyd Na₂S0₄. After filtration, the filtrate was concentrated to give unstable 3,4, 5-tri-0-benzyl-l,7-dideoxy-l- (dimethoxyphosphory1)-D-arabino-2,6-heptodiulose (7) («90% purity on TLC, R_f0.3d with 2:1 toluene EtOAc) : LRMS 577 (MNa) + 555 (MH)+. A mixture of (7) , prepared above, and NaH (637 mg of 60% oil dispersion, 15.9 mmol) in diglyme (90 mL) was stirred at rt for 30 min and warmed to 65°C for 1 h under N₂. The mixture was diluted with brine (200 mL) and extracted with Et₂0 (3 x 100 mL) . The combined organic layers were washed with H₂0 (100 mL) , dried over . anhyd Na₂S0₄ and concentrated to dryness. Flash column chromatography (100:7 toluene/EtOAc) of the residue afforded (8) (5.02 g, 77% from (6) as a colorlesε εyrup; [α]_D-105° (c 1.0, CHC1₃) ; ¹H NMR (CDC1₃) <S 1.94 (ε, 3, Me), 3.95 (dd, 1, H-5, J = 3.2, 8.3 Hz) , 4.24 (d, 1, H-4, J - 3.2 HZ), 4.33 (d, 1, H-6, J = 8.3 Hz) , 5.83 (br s, 1, H-2); HRMS calc for C₂₈H₂₈Na0₄ (MNa)+451.1886, found 451.1881.

2L-(2/3,4,5) -2 , 3 , 4-Tri-0-benzyl-5-methyl-2 ,3,4- trihydroxycyclohexanone (9) .

A mixture of (8) (210 mg, 0.49 mmol) and (Ph₃PCuH)₆ (230 mg) in THF (8 mL) containing H₂0 (250 μL) was stirred at rt under N₂. After 12 h, Ph₃PCuH)₆ (460 mg) was added further and stirring continued another 48 h. The reaction mixture was exposed to air for 1 h and the resulting precipitates were removed by filtration with the aid of a celite bed. After evaporation, the residue was purified by flash column chromatography (10:1 toluene/EtOAc) to yield (9) (196 mg, 93%) as a colorless syrup: [α]_D - 75° (c 1.0 CHCI^) ; ¹H NMR (CDC1₃) δ 1.05 (d, 3, Me, J - 6.7 Hz) , 1.77-1.85 (m, 1, H-5) , 2.16 (dd, 1, H-6eq', J = 4.1, 13.8 Hz) , 2.60 (dd, 1, H-6ax', J - 13.8, 13.8 Hz) , 3.64 (dd, 1, H-3 , J = 2.2, 10.2 Hz) , 3.83 (br s 1, H-4) , 4.55 (d, 1, H-2 , J = 10.2 Hz) ; HRMS calc for C₂₈H₃₀NaO₄ (MNa) + 453.2042, found 453.2047.

ID-(1,3,4,5/2)-2,3,4-Tri-0-benzyl-5-methyl-l,2,3,4- cyclohexanetetrol (10) .

NaBH₄ (10.5 mg, 0.28 mmol) was added portionwise during 2 min to a solution of (9) (105 mg, 0.24 mmol) in MeOH (2 mL) containing CeCl₃ (197 mg, 0.80 mmol) with stirring at rt. After an additional 3 min, the mixture was poured into H₂0 (10 mL) and extracted with Et₂0 (3 x 5 mL) . The organic layer waε dried over anhyd Na₂S0₄ and concentrated to dryness. Flash column chromatography (20:3 toluene/EtOAc) of the residue yielded 10 (104 mg, 99%) as a colorless syrup: [α]_D-4.9° (c 1.0, CHC1₃; ¹H NMR (CDC1₃) δ 1.01 (d, 3, Me, J = 6.5 Hz), 1.54-1.60 (m, 1, H-5) , 1.60-1.68 (m, 2, 2x H-6) , 3.42 (dd, 1, H-3, J = 2.2, 9.6 Hz) , 3.70 (br q 1, H-l, J = «9 HZ), 3.72 (dd, 1, H-4, J = 1.7, 2.2 Hz) , 3.78 (dd, 1, H-2, J = 9.3, 9.6 Hz) ; HRMS calc for C₂₈H₃₂Na0₄ (MNa) + 455.2198, found 455.2168.

A mixture of 10 and 1L-(1,2/3,4,5)-2,3,4-tri-O- benzyl-5-methyl-l,2,3,4-cyclohexanetetrol (11) .

A mixture of (9) (79 mg, 0.18 mmol) and NaBH₄ (3.4 mg, 0.09 mmol) in EtOH (1 mL) was stirred at rt for 4 h. Work-up and purification as described above yielded (11) (34 mg, 43%) as a colorless syrup and then (10) (44 mg, 55%). (11): [α]_D-42° (c 1.0, CHC1₃) ; Η NMR (CDC1₃) δ 0.97 (d, 3, Me, J = 6.9 Hz) , 1.59-1.70 (m, 2, 2X H-6) , 1.98-2.06 (m, 1, H-5) , 2.48 (br S 1, OH), 3.75 ((br s, 1, H-4), 3.77 (dd, 1, H-2, J = 2.4, 9.6 Hz) ; 3.90 (dd, 1, H-3, J = 3.4, 9.6 Hz) , 4.12 (br d, 1 H-l, J = «3 Hz); HRMS calc for C₂₈H₃₂Na0₄ (MNa) + 455.2198, found 455.2195. 1L-(1,2/3,4,5)-5-Methy1=1,2,3,4-cyclohexanetetrol (5a-carba-α-L-fucopyranose) (12) .

A mixture of (11) (12 mg, 0.03 mmol) and 10% Pd/C (3 mg) in EtOH (1 mL) was stirred under H₂ (1 atm) at rt overnight. After removal of the catalyst by filtration, the filtrate waε concentrated to dryness. Purification of the reεidue by Bio-Gel P2 column chromatography (H₂0) gave (12) (3 mg, 67%) aε white crystals: mp 142-143°C; [α]_D-81° (c 1.0, CHCL_j) (mp 115°C; [α]_D-58° (c 1, CHCI^) ) .

1D-(1, 3 , 4/2) -2 , 3 , 4-Tri-O-benzy1-5-methy1-5- cyclohexene,1,2,3,4-tetrol (13).

NaBH₄ (49.7 mg, 1.31 mmol) was added portionwise during 2 min to a solution of (8) (560 mg, 1.31 mmol) in MeOH (5 mL) containing CeCl₃ (444 mg, 1.8 mmol) with stirring at rt. After 3 min, the mixture was poured into H₂0 (10 mL) and extracted with Et₂0 (3 x 5 mL) . The combined organic layers were dried over anhydr Na₂S0₄, concentrated and purified by flash column chromatography (10:1 toluene/EtOAc) to give (13) (514 mg, 91% as a colorless syrup: [α]_D-55° (c 1.0, CHC1₃) ; ¹H NMR (CDC1₃) δ 1.72 (s, 3, Me), 3.95 (dd, 1, H-3, J = 3.5, 9.3 Hz) 3.99 (dd, 1, H-4, J = 3.5 Hz) ; 4.03 (dd, 1, H-2, J = 4.3, 9.3 Hz) 4.03 (dd, 1 H-2, J = *3 Hz) ; HRMS calc for C₂₈H₃₂Na0₄ (MNa) + 453.2042 found 455.2048.

A mixture of (10), (13) and (14).

A mixture of (8) (44 mg, 0.10 mmol) and NaBH₄ (5 mg, 0.13 mmol) in MeOH (or THF) (1 mL) was stirred at -20°C for 1 h. After evaporation, the residue was dissolved in EtOAc (5 mL) , washed sequentially with 2M aq HC1 (2 x 3 mL) , sat. aq NaHC0₃ (2 3 mL) and H₂0 (2 x 3 mL) and finally dried over anhyd Na₂S0₄. Purification as described above yielded an inseparable 1.8:1 mixture of (10) and (14) (12 mg) and then (13) (13 mg, 30%) . 1L-(1,2/3,4)-5-Methyl-5-cyclohexene-l,2,3,4-tetrol (15).

A solution of (14) (50 mg, 0.12 mmol) in THF (1 mL) was added to a solution of Li (5 mg) in liq. NH₃ (20 mL) NH₃ (20 mL) with stirring at -33°C. Several 5 mg portionε of Li were added further until the dark blue color perεiεted and then the mixture waε stirred at -33°C for 2 h. The NH₃ then was evaporated, the residue was dissolved in H₂0 and the solution was treated with Amberlite 1R-120 (H+) resin. After filtration, the filtrate was concentrated to dryness. The reεidue waε purified by Bio-Gel P2 column chromatography (H₂0) to give, after lyophilization, (15) (15.1 mg, 79%) as an amorphous solid: [α]_D-272° (c 1.0, H20) ; ¹H NMR (CDC1₃) δ 1.76 (s, 3, Me), 3.78 (dd, 1, H-2, J = 3.8, 10.3 Hz) 3.83 (dd, 1, H-3, J = 4.0, 10.3 Hz), 4.06 (d, 1, H-4, J = 4.0 Hz) 4.19 (br t, 1, H-l, J = «4 Hz), 5.59 (br d, 1, H-6, J = 4.3 Hz); HRMS calc for C₇H₁₂Na0₄ (MNa) + 183.0633, found 183.0645.

lD-(l,3,4,5/2) -2,3,4-Tri-0-benzyl-l-0- dibenzylphoεphoryl)-5-methy1-1,2,3,4-cyclohexanetetrol (16).

A mixture of (10) (280 mg, 0.65 mmol), IH-tetrazole (136 mg, 1.94 mmol) and N,N-diisopropyl dibenzyl phosphoramidite (Yum & Fraser-Reid, Tetrahedron Lett. 29, 979, 1988) (335 mg, 0.97 mmol) in. CH₂C1₂ (8 mL) waε stirred at rt for 2 h and then cooled to -40°C. A solution of m-CPBA (50-60% purity, 395 mg, « 1.3 mmol) in CH₂C1₂ (4 mL) was added and stirring continued for another 45 min at 0°C. The mixture was diluted with CH₂C1₂ (20 mL) , washed sequentially with 10% aq Na₂S0₃ (2 x 20 mL) , sat. aq NaHC0₃ (2 x 20 mL) and H₂0 (2 x 20 mL) and then dried over anhyd Na₂S0₄.

Concentration and purification by flash column chromatography (15:1 toluene/EtOAc) afforded (16) (435 mg, 97%) aε a colorleεε εyrup: [α]_D-2.3° (c 1.3, CHC13) ; ¹H NMR (CDC1₃) δ 0.96 (d, 3, Me, J = 6.6 Hz) , 1.47-1.55 (m, 1, H-5) , 1.78 (ddd, 1, H-6ax, J = 12.3, 12.3, 12.5 Hz), 1.87 (ddd, 1, H-6eq, J = 4.3, 4.3, 12.9 Hz) , 3.44 (dd, 1, H-3, J = 3.1, 10.1 Hz) , 3.56 (dd, 1, H-2, J = 7.8, 10.1 Hz) , 3.75 (br ε, 1, H-4) , 3.87-3.94

(m, 1, H-l) ; HRMS calc for C₇H₁₄0₇P (M - H) 241.0477, found 241.0472.

ID- ( 1 , 3 , 4/2 ) -2 , 3 , 4-Tr i-O-benzy l-l-O- (dibenzylphosphoryl) -5-methyl-5-cyclohexene-l, 2,3,4- tetrol (18) .

A mixture of (13) (170 mg, 0.39 mmol), IH-tetrazole (100 mg, 1.43 mmol) and N,N-diisopropyl dibenzyl phophoramidite (245 mg, 0.71 mmol) in CH₂C1₂ (5 mL) was stirred at rt for 2 h and then cooled to -40°C. A solution of m-CPBA (50-60% purity; 165 mg, ~ 0.5 mmol) in CH₂C1₂ (3 mL) was added and stirring continued for an additional 45 min at 0°C. Work-up and purification as described for the preparation of (16) gave (18) (258 mg, 94%) as a colorless syrup: [α]D+ 4.4° (c 1.5, CHC13) ; H NMR (CDC1₃) δ 1.68 (s, 3, Me), 3.57 (dd, 1, H-3, J = 3.5, 10.2 Hz), 3.93 (d, 1, H-4, J = 3.5 Hz), 4.17 (dd, 1, H- 2, J - 7.2, 10.2 Hz), 5.51 (br s, 1, H-6) ; HRMS calc for C₃₅H₃₆0₇P (M - Bn)- 599.2198, found 599.2266.

ID-(1,3,4/2)-5-Methy1-5-cyclohexene-l,2,3,4-tetrol 1-phosphate (19) .

Treatment of (18) (120 mg, 0.17 mmol) with Li in liq. NH₃ as described for the preparation of (15) yielded (19) (41 mg, 97%) as an amorphous solid: [α]D-55° (c 0.7, H₂0) ; ¹H NMR (D_2fJ) -5 1.78 (s, 3, Me), 3.58 (dd, 1, H-3, J = 4.0, 10.9 Hz) , 3.70 (dd, 1, H-2, J = 7.4, 10.9 HZ), 4.01 (d, 1, H-4, J = 4.0 Hz) , 4.38-4.44 (m, 1, H- 1), 5.53 (br s, 1, H-6); HRMS calc for C₇H₁₂0₇P (M - H)- 239.0321, found 239.0317. Guanosine 5 ' - (5a-carba-β-L-fucopyranosyl diphosphate) (1) .

The dihydrogen phoεphate (17) waε converted into the bis(triethylammonium) salt by pasεing itε aqueous solution over a column of Dowex 50X8-400 (Et₃HN+) . The eluate was lyophilized and the resulting amorphouε εolid was dried over P₂0₅ overnight prior to use.

A mixture of the bis(triethylammonium) salt of (17) (26 mg , 0.058 mmol) and guanosine 5'-monophosphomorpholidate (51 mg, 0.070 mmol) in pyridine (2 mL) was stirred at rt for 5 d. The mixture was concentrated to dryness and the residue was purified by preparative HPLC (24:1 0.05M aq Et₃HNHC0₃-MeCN, isocratic) . The desired fractionε were combined, lyophilized and passed over a column of Bio-Rad AG 50W-X2 (Na+) to give the disodium εalt of (1) (17.3 mg, 47% based on the amount of (17) used) aε an amorphous solid: [α]D-14.0° (c 1.0, H₂0) ; ¹H NMR (D₂₀₎ δ 0.88 (d, 3, Me, J = 6.9 Hz), 1.45 (ddd, 1, H-6ax, J = 11.6, 12.7, 12.7 Hz), 1.51-1.60 (m, 1, H-5) , 1.84 (ddd, 1, H-6eq, J = 4.0, 4.3, 12.7 Hz), 3.40 (dd, 1, H-3, J = 3.0, 9.7 Hz), 3.58 (dd, 1, H-2, J = 9.6, 9.7 Hz) 3.69 (br s, 1, H-4) 3.93-4.02 (m, 1, H-l), 4.14-4.18 (m, 2, ribose CH₂) , 4.29-4.32 (m, 1, ribose H-4) , 4.49 (dd, 1, ribose H-3, J = 3.3, 5.2 HZ), 4.76 (dd, 1, ribose H-2, J - 5.2, 5.9 Hz), 5.89 (d, 1, ribose H-l, J = 5.9 Hz), 8.06 (s, 1, guanine H-8) . ESMS 630.3 (M-H) , 608.4 (M-Na)-, 586.4 (M-2Na+H) .

Guanosine 5'-[ID-(1,3,4/2)-5-methyl-5-cyclohexene- 1,2,3,4-tetrol 1-diphosphate] (2).

The dihydrogen phosphate (19) was first converted into the bis(triethylammonium) salt as described above.

A mixture of the bis(triethylammonium salt of (19)

(53 mg, 0.12 mmol) and guanosine 5'-monophosphomorpholidate (260 mg, 0.35 mmol) in pyridine (4 mL) was stirred at rt for 5 d. Work-up and purification aε described above yielded the disodiu salt of (2) (37.7 mg, 50% based on the amount of (19) used): [α]D-19.9° (c 0.7, H₂0) ; ¹H NMR (D₂₀₎ δ 1.67 (s, 3, Me), 3.53 (dd, 1, H-3, J = 4.1, 11.0 Hz), 3.72 (dd, 1, H-2, J = 7.4, 11.0 Hz) , 3.94 (d, 1, H-4, J = 4.1 Hx) , 4.15-4.19 (m, 2, ribose CH₂) , 4.29-4.32 (m, 1, ribose H- 4), 4.48 (dd, 1, ribose H-3, J = 3.2, 5.9 Hz) , 4.78 (dd, 1, riboεe H-2, J = 5.9, 6.4 Hz), 5.52 (br, ε, 1, H-6) , 5.88 (d, l, ribose H-l, J - 6.4 Hz), 8.05 (s, 1, guanine H-8) . ESMS 628.0 (M-H)-; 606.0 (M-Na)-, 584.1 (M- 2Na+H)-.

Enzyme Preparation

Colo205 cells (ATCC) were grown to confluency in RPMI 1640 medium containing 10% fetal calf εerum, were trypsinized, centrifuged, washed twice with PBS (pH 7.4) and counted using a hemacytometer. Cells (4 x 10 ) were injected subcutaneously into athymic (nude) mice. Tumors were excised after 2 weeks and stored frozen at -80°C. The tumors then were homogenized at 4°C in two volumes of 50 mM HEPES (pH 7.2), 0.5M sucrose and ImM EDTA. The crude homogenate was centrifuged at 30,000 g for 30 min and the pellet waε rehomogenized in the presence of the above buffer containing 0.2% Triton-XlOO. The homogenate was centrifuged at 100,000 g for 1 h and the supernatant was concentrated to the original volume of the tumors by dialysis. The enzyme preparation was stored at -80°C until needed.

The inhibition assay was performed at a 25 μL scale. The mixture contained the following components: HEPES (pH 7.2; 0.625 μmol) , MnCl₂ (0.125 μmol) , GDP-[U-^UC]Fuc (20,000 cpm/nmol; 2.5 nmol) , LNF 1 (50 nmol) , enzyme preparation (10 μL) and inhibitor (5 nmol, 10 nmol, 20 nmol and 40 nmol) . The mixture was incubated at 37°C for 20 min and stopped by addition of ice-cold H₂0 (1 mL) . The entire mixture was passed over an aminopropyl column (1 mL; Analytichem International, Harbor City, CA) to remove unreacted GDP-[U-^UC]Fuc. The combined eluent and water-washings (2 mL) were counted on a liquid scintillation counter.

EXAMPLE V

The carboxylic acid derivative of β-D-lactoside

(17) is a useful precursor for the preparation of the 3-OH unprotected β-D-N-acetyllactosaminide (21) . Treatment of (17) with TFAA, which forms the -5-lactone, followed by a conventional acetylation and work-up with MeOH, yielded the 2-OH unprotected β-lactoside (18) . The 2-OH group in (18) was transformed into the 2-N₃ group (compound (20)) in good yield via the iodo derivative (19) .

Reduction of the azide group is accompanied by an P→N acetyl migration affording the N-acetyllactosaminide derivative (21) . Treatment of (21) with PPh₃ and I₂ yields the iodo compound (22) with the inversion of the stereochemistry at C-3.

The Le^x-mimic (26) which contains an α-carba-fucose residue is constructed either by condensation reactions between (9) and (22) and between (12) , the Tf derivative of (10) , and (21) (Scheme IV, Route l, Figure 14) or by coupling of (13) and (21) followed by saturation of a double bond (Scheme V, Route 2, Figure 15). The sialosyl Le^x mimics are elaborated from (25) aε depicted in Schemes VI-XI as set forth in the Figures. The reactions schemes are based on the regioselective alkylation at the 3-OH group in the galactose residue via the stannylene complex. To obtain a carboxyl derivative, compound (25) is refluxed with dibutyltin oxide in MeOH and subsequently reacts with allyl bromide will yield, after perbenzylation, the 3-O-allyl derivative (30) (Scheme VI, Plan I) (Figure 16). Oxidative cleavage of the double bond by ozonolysiε yields the carboxylic acid derivative (32) . Alternatively, the direct conversion of the double bond of the allyl group into a carboxylic acid can be obtained via an alkylboronic acid, affording (34), a homologue of (32) (Scheme VII, Plan 2, Figure 17) .

To obtain a sulfono derivative, the 3-OH group of the galactose reεidue in (35) , prepared from (30) by treatment with (PPh₃)₃RhCl, iε replaced with a εulfonic acid by oxidation of a thioacetate with Oxone (Scheme VIII, Plan 3) (Figure 18) . Treatment with an α-lithio εulfonate with (36) yields (41) , a homologue of (39) (Scheme IX, Plan 4, Figure 19).

To obtain a phosphate derivative, nucleophilic substitution reaction by dimethyl methylphosphonate yields the phosphonate derivative (43) (Scheme X, Plan 5, Figure 20) .

Since regioselective alkylation of (25) at the 3-OH group in the galactose residue occurs via the stannylene complex, any acidic group can be added directly at the 3-OH by reacting (25) with R-X (R = alkyl groups with acidic functionalities; X = halides) (Figure 21) .

While the invention has been deεcribed in detail with reference to a preferred embodiment, various modifications within the spirit of the invention will be apparent to those of working skill in this technical field. Accordingly, the invention should be considered as limited only by the scope of the appended claimε.

All references cited herein are incorporated herein by reference. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: HAKOMORI, SEN-ITIROH

KANNAGI, REIJI TOYOKUNI, TATSUSHI

(ii) TITLE OF INVENTION: STABLE CARBOHYDRATE EPITOPES AND THEIR MIMETICS USEFUL FOR BLOCKING CARBOHYDRATE-DEPENDENT CELLULAR INTERACTION AND FOR ACTIVE IMMUNIZATION ELICITING ANTI-CARBOHYDRATE T CELL RESPONSE

(iii) NUMBER OF SEQUENCES: 4

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Sughrue, Mion, Zinn, Macpeak &

Seas

(B) STREET: 2100 Pennsylvania Avenue, NW

(C) CITY: Washington

(D) STATE: D.C.

(E) COUNTRY: U.S.A.

(F) ZIP: 20037

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patent In Release #1.0,

Version #1.25

(Vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US

(B) FILING DATE: 08-MAY-1992

(C) CLASSIFICATION: (VUi) ATTORNE /AGENT INFORMATION:

(A) NAME: Mack, Susan J.

(B) REGISTRATION NUMBER: 30,951

(C) REFERENCE/DOCKET NUMBER: A6092

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (202) 293-7060

(B) TELEFAX: (202) 293-7860

(C) TELEX: 6491103

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: YES

(v) FRAGMENT TYPE: internal

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

Ala Gly Leu Ser Ser Tyr Tyr Leu Thr Thr Tyr Arg Pro

1 5 . 10

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: YES

(v) FRAGMENT TYPE: internal

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Leu Trp Ser Thr Tyr Tyr Gly Ser Tyr Arg Arg Ala Gin

1 5 10

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 amino acidε

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Lys Pro Gly Lys Thr Asn Lys Leu Leu lie Tyr Ser Gly Ser Thr 1 5 10 15

Gin

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acidε

(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Tyr Ser Gly Ser Thr 1 5

Claims

WHAT IS CLAIMED IS: 1. A stabilized carbohydrate epitope having more resistance to metabolic degradation than a corresponding naturally occurring carbohydrate epitope.

2. The εtabilized carbohydrate epitope according to claim 1, wherein fucoεe in the corresponding naturally occurring carbohydrate epitope is replaced by 6-trifluoromethylfucose, and/or N-acetylsialic acid is replaced with an N-trifluoroacetyl or an N-carbamyl group.

3. The stabilized carbohydrate epitope according to claim 2, wherein the corresponding naturally occurring carbohydrate epitope is sialosyl-Le^x, sialosyl-Le^a, Le^y, Le^x, Le^a or H.

4. The stabilized carbohydrate epitope according to claim 1, wherein O-glycosylation of sialic acid and fucose is replaced with S-glycoside.

5. The stabilized carbohydrate epitope according to claim 4, wherein the corresponding naturally occurring carbohydrate epitope is sialosyl-Le^x or sialosyl-Le^a.

6. The stabilized carbohydrate epitope according to claim 1, wherein the corresponding naturally occurring carbohydrate has εialic acid and said sialic acid is modified to have a lactone or lactam ring structure.

7. The stabilized carbohydrate epitope according to claim 6, wherein the corresponding naturally occurring carbohydrate is sialosyl-Le^x, sialosyl-Le^a or sialosyl-Tn. 8. The stabilized carbohydrate epitope according to claim 1, which is selected from the groups consisting of compound 1, 2, 3, 4, 5, 6, 7,,

8, 9, 10 and 11 shown in Figure 3.

9. The stabilized carbohydrate epitope as defined in claim 8 compriεing a bivalent or multivalent structure.

10. A peptide mimetic of a carbohydrate epitope, wherein said mimetic has a structure such that the mimetic has about the same or more antibody-binding or selectin-binding activities, immunogenicity and antigenicity aε that of a correεponding naturally occurring carbohydrate epitope.

11. The mimetic of claim 10, which has more resistance to metabolic degradation than the corresponding naturally occurring carbohydrate epitope.

12. A process for preparing a peptide mimetic of a carbohydrate epitope according to claim 10, said process comprising: (A) making a monoclonal antibody (Abl) directed against the naturally occurring carbohydrate epitope, (B) making an anti-idiotype monoclonal antibody (Ab2) directed against the internal image structure of said Abl, (C) determining the amino acid sequence of the variable heavy (V_H) and/or the variable light (^V _L) regions corresponding to complementarity-determining region (CDR) 1, 2 and 3 of said Ab2, (D) determining the conformational structures of peptideε in said CDR 1, 2 and 3, (E) identifying regions of said CDR 1, 2, and 3 that are complementary to the conformational structure of the naturally occurring carbohydrate epitope and (F) synthesizing a peptide analogue of the naturally occurring carbohydrate epitope.

13. A medicament for inhibiting metastaεiε of tumor cells caused by carbohydrate-mediated cell adhesion, said medicament comprising: (A) A stabilized carbohydrate epitope having more resistance to metabolic degradation than a corresponding naturally occurring carbohydrate epitope, or a mimetic of a carbohydrate epitope, wherein said mimetic has a structure such that the mimetic has about the same antibody-binding or selectin-binding activities, immunogenicity and antigenicity as that of a corresponding naturally occurring carbohydrate epitope; (B) bivalent or multimeric structure of the stabilized carbohydrate epitope as in A; the εtructureε are deεigned aε deεcribed in Schemeε XII, XIII and XIV; and (C) a pharmaceutically acceptable carrier, diluent, or excipient.

14. The medicament of claim 13, wherein fucose in the corresponding naturally occurring carbohydrate epitope is replaced by 6-trifluoromethylfucose, and/or N-acetylsialic acid is replaced with an N-trifluoroacetyl or an N-carbamyl group.

15. The medicament of claim 14, wherein the corresponding naturally occurring carbohydrate epitope is sialosyl-Le^x, sialoεyl-Le^a, Le^y, Le^a, H or Le^x.

16. The medicament of claim 13, wherein O-glycosylation of εialic acid and fucose is replaced with S-glycoside.

17. The medicament of claim 16, wherein the corresponding naturally occurring carbohydrate epitope is sialosyl-Le^x or sialosyl-Le^a.

18. The medicament of claim 13, wherein the corresponding naturally occurring carbohydrate has sialic acid and said sialic acid is modified to have a lactone or lactam ring εtructure.

19. The medicament of claim 18, wherein the corresponding naturally occurring carbohydrate is sialosyl-Le , sialosyl-Le^a or sialosyl-Tn.

20. The medicament of claim 13, wherein said stabilized carbohydrate epitope is selected from the group consisting of compound 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 shown in Fig. 3.

21. The medicament of claim 13, wherein said mimetic has more resistance to metabolic degradation than the corresponding naturally occurring carbohydrate antigen.

22. The medicament of claim 13, comprising a bivalent or multimeric structure.

23. A medicament for inhibiting inflammatory processes caused by carbohydrate-mediated cell adhesion, said medicament comprising: (A) A stabilized carbohydrate epitope having more resistance to metabolic degradation than a corresponding naturally occurring carbohydrate epitope, or a mimetic of a carbohydrate epitope, wherein said mimetic has a structure such that the mimetic has about the same antibody-binding or selectin-binding activities, immunogenicity and antigenicity as that of a corresponding naturally occurring carbohydrate epitope; (B) bivalent or multimeric structure of the stabilized carbohydrate epitope aε are deεigned in Schemes XII, XIII and XIV; and (C) a pharmaceutically acceptable carrier, diluent, or excipient.

24. The medicament of claim 23, wherein fucose in the corresponding naturally occurring carbohydrate epitope is replaced by 6-trifluoromethylfucose, and/or N-acetylsialic acid is replaced with an N-trifluoroacetyl or an N-carbamyl group.

25. The medicament of claim 24, wherein the corresponding naturally occurring carbohydrate epitope is sialosyl-Le^x, sialosyl-Le^a, Le^y, Le^a, H or Le^x.

26. The medicament of claim 23, wherein O-glycosylation of sialic acid and fucoεe iε replaced with S-glycoside.

27. The medicament of claim 26, wherein the corresponding naturally occurring carbohydrate epitope is sialosyl-Le^x or sialosyl-Le^a.

28. The medicament of claim 23, wherein the corresponding naturally occurring carbohydrate has sialic acid and said sialic acid is modified to have a lactone or lactam ring structure.

29. The medicament of claim 28, wherein the corresponding naturally occurring carbohydrate is sialosyl-Le^x, sialosyl-Le^a or sialosyl-Tn.

30. The medicament of claim 23, wherein said stabilized carbohydrate epitope is selected from the group consisting of compound 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 shown in Fig. 3.

31. The medicament of claim 23, wherein said mimetic has more reεistance to metabolic degradation than the corresponding naturally occurring carbohydrate antigen.

32. A medicament for inhibiting microbial infection caused by carbohydrate-mediated cell adhesion, said medicament comprising: (A) A stabilized carbohydrate epitope having more resistance to metabolic degradation than a corresponding naturally occurring carbohydrate epitope, or a mimetic of a carbohydrate epitope, wherein said mimetic has a structure such that the mimetic has about the same antibody-binding or selectin-binding activities, immunogenicity and antigenicity as that of a corresponding naturally occurring carbohydrate epitope; (B) bivalent or multimeric structures of the stabilized carbohydrate epitope as in A; the structures are designed as described in Scheme XII, XIII XIV; and (C) a pharmaceutically acceptable carrier, diluent, or excipient.

33. The medicament of claim 32, wherein fucose in the corresponding naturally occurring carbohydrate epitope is replaced by 6-trifluoromethylfucose, and/or N-acetylsialic acid is replaced with an N-trifluoroacetyl or an N-carbamyl group.

34. The medicament of claim 33, wherein the corresponding naturally occurring carbohydrate epitope is sialosyl-Le^x, sialosyl-Le^a, Le^a, H, Le^y or Le^x.

35. The medicament of claim 32, wherein O-glycosylation of sialic acid and fucose is replaced with S-glycoside.

36. The medicament of claim 35, wherein the corresponding naturally occurring carbohydrate epitope is sialosyl-Le^x or sialoεyl-Le^a.

37. The medicament of claim 32, wherein the corresponding naturally occurring carbohydrate has sialic acid and said sialic acid is modified to have a lactone or lactam ring structure.

38. The medicament of claim 37, wherein the corresponding naturally occurring carbohydrate is sialosyl-Le^x, sialosyl-Le^a or sialosyl-Tn.

39. The medicament of claim 32, wherein said stabilized carbohydrate epitope is selected from the group consisting of compound 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 shown in Fig. 3.

40. The medicament of claim 32, wherein said mimetic has more reεistance to metabolic degradation than the correεponding naturally occurring carbohydrate antigen.

41. A method for inhibiting carbohydrate-mediated cell adhesion, said method comprising contacting εaid cells with an inhibitory amount of a stabilized carbohydrate epitope having more reεiεtance to metabolic degradation than a correεponding naturally occurring carbohydrate epitope or with a mimetic of a carbohydrate epitope, wherein said mimetic has a structure such that the mimetic has about the same or more antibody-binding or selectin-binding activities, immunogenicity and antigenicity as that of a corresponding naturally occurring carbohydrate epitope.

42. The method of claim 41, wherein fucoεe in the corresponding naturally occurring carbohydrate epitope is replaced by 6-trifluoromethylfucose, and/or N-acetylsialic acid is replaced with an N-trifluoroacetyl or an N-carbamyl group.

43. The method of claim 42, wherein the corresponding naturally occurring carbohydrate epitope is sialosyl-Le^x, sialosyl-Le^a, Le^y, Le^a, H or Le^x.

44. The method of claim 41, wherein O-glycosylation of sialic acid and fucose is replaced with S-glycoside.

45. The method of claim 44, wherein the corresponding naturally occurring carbohydrate epitope is sialosyl-Le^x or sialosyl-Le^a.

46. The method of claim 41, wherein the corresponding naturally occurring carbohydrate haε sialic acid and said sialic acid is modified to have a lactone or lactam ring structure.

47. The method of claim 46, wherein the corresponding naturally occurring carbohydrate is sialosyl-Le^x, sialosyl-Le^a or sialosyl-Tn.

48. The method of claim 41, wherein said stabilized carbohydrate epitope is selected from the group consisting of compound 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 shown in Fig. 3.

49. The method of claim 48, wherein said epitope comprises a bivalent or multimeric structure.

50. The method of claim 41, wherein said mimetic has more resistance to metabolic degradation than the corresponding naturally occurring carbohydrate epitope.

51. A method for inhibiting metaεtasis of tumor cells caused by carbohydrate-mediated cell adhesion, said method comprising administering to a host in need of treatment an inhibitory amount of a stabilized carbohydrate epitope having more resistance to metabolic degradation than a corresponding naturally occurring carbohydrate epitope, or a mimetic of a carbohydrate epitope, wherein said mimetic haε a structure such that the mimetic has about the same or more antibody-binding or selectin-binding activities, immunogenicity and antigenicity as that of a corresponding naturally occurring carbohydrate epitope.

52. The method of claim 51, wherein fucose in the corresponding naturally occurring carbohydrate epitope is replaced by 6-trifluoromethylfucose, and/or N-acetylsialic acid is replaced with an N-trifluoroacetyl or an N-carbamyl group.

53. The method of claim 52, wherein the corresponding naturally occurring carbohydrate epitope is sialosyl-Le^x, sialosyl-Le^a, Le^y, Le^a, H or Le^x.

54. The method of claim 51, wherein O-glycosylation of sialic acid and fucose is replaced with S-glycoside.

55. The method of claim 54, wherein the corresponding naturally occurring carbohydrate epitope is sialosyl-Le^x or sialosyl-Le^a.

56. The method of claim 51, wherein the corresponding naturally occurring carbohydrate has sialic acid and said sialic acid iε modified to have a lactone or lactam ring structure.

57. The method of claim 56, wherein the corresponding naturally occurring carbohydrate is sialosyl-Le^x, sialosyl-Le^a or sialosyl-Tn.

58. The method of claim 51, wherein said stabilized carbohydrate epitope is selected from the group consisting of compound 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 shown in Fig. 3.

59. The method of claim 51, wherein said mimetics comprise a bivalent or multimeric structure.

60. The method of claim 51, wherein said mimetic has more resistance to metabolic degradation than the corresponding naturally occurring carbohydrate epitope.

61. A method for inhibiting inflammatory processes caused by carbohydrate-mediated cell adhesion, said method comprising administering to a host in need of treatment an inhibitory amount of a stabilized carbohydrate epitope having more resistance to metabolic degradation than a corresponding naturally occurring carbohydrate epitope or of a mimetic of a carbohydrate epitope, wherein said mimetic has a structure such that the mimetic has about the same antibody-binding or selectin-binding activities, immunogenicity and antigenicity aε that of a corresponding naturally occurring carbohydrate epitope.

62. The method of claim 61, wherein fucose in the corresponding naturally occurring carbohydrate epitope is replaced by 6-trifluoromethylfucose, and/or N-acetylsialic acid is replaced with an N-trifluoroacetyl or an N-carbamyl group.

63. The method of claim 62, wherein the corresponding naturally occurring carbohydrate epitope is sialosyl-Le^x, sialosyl-Le^a, Le^y, Le^a, H or Le^x.

64. The method of claim 61, wherein O-glycosylation of sialic acid and fucose is replaced with S-glycoside.

65. The method of claim 64, wherein the corresponding naturally occurring carbohydrate epitope is sialosyl-Le^x or sialosyl-Le^a.

66. The method of claim 61, wherein the corresponding naturally occurring carbohydrate has sialic acid and said sialic acid is modified to have a lactone or lactam ring structure.

67. The method of claim 66, wherein the corresponding naturally occurring carbohydrate iε sialosyl-Le^x, sialosyl-Le^a or sialosyl-Tn.

68. The method of claim 61, wherein said stabilized carbohydrate epitope is selected from the group consisting of compound 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 shown in Fig. 3.

69. The method of claim 61, wherein said stabilized carbohydrates comprise a bivalent or multimeric structure.

70. The method of claim 61, wherein said mimetic has more resistance to metabolic degradation than the corresponding naturally occurring carbohydrate epitope.

71. A method for inhibiting microbial infection caused by carbohydrate-mediated cell adhesion, said method comprising adminiεtering to a host in meed of treatment an inhibitory amount of a stabilized carbohydrate epitope having more resistance to metabolic degradation than a corresponding naturally occurring carbohydrate epitope, or of a mimetic of a carbohydrate epitope, wherein said mimetic has a structure such that the mimetic has about the same antibody-binding or selectin-binding activities, immunogenicity and antigenicity as that of a corresponding naturally occurring carbohydrate epitope.

72. The method of claim 71, wherein fucose in the corresponding naturally occurring carbohydrate epitope is replaced by 6-trifluoromethylfucose, and/or N-acetylsialic acid is replaced with an N-trifluoroacetyl or an N-carbamyl group.

73. The method of claim 72, wherein the corresponding naturally occurring carbohydrate epitope is sialosyl-Le , sialosyl-Le^a, Le^y, Le^a, H or Le^x.

74. The method of claim 71, wherein O-glycosylation of sialic acid and fucose is replaced with S-glycoside.

75. The method of claim 74, wherein the corresponding naturally occurring carbohydrate epitope is sialosyl-Le^x or sialosyl-Le^a.

76. The method of claim 71, wherein the corresponding naturally occurring carbohydrate has sialic acid and said sialic acid iε modified to have a lactone or lactam ring structure.

77. The method of claim 76, wherein the corresponding naturally occurring carbohydrate iε sialosyl-Le^x, sialosyl-Le^a or εialoεyl-Tn.

78. The method of claim 71, wherein εaid stabilized carbohydrate epitope iε selected from the group consisting of compound 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 shown in Fig. 3.

79. The method of claim 71, wherein said mimetic has more resistance to metabolic degradation than the corresponding naturally occurring carbohydrate epitope.

80. The method of claim 71, wherein said stabilized carbohydrate epitopes comprise a bivalent or multivalent structure.

81. A vaccine for induction of an anti-carbohydrate T cell immune response, said vaccine comprising: (A) a mimetic of a carbohydrate antigen, wherein said mimetic has a structure such that the mimetic has about the same antibody- binding or selectin-binding activities, immunogenicity and antigenicity as that of a corresponding naturally occurring carbohydrate epitope, and (B) a pharmaceutically acceptable carrier, diluent or excipient.

82. The vaccine of claim 81, wherein said mimetic has more reεistance to metabolic degradation than the corresponding naturally occurring carbohydrate epitope.

83. A method of vaccinating to induce an anti-carbohydrate T cell immune response, said method comprising vaccinating a host with an anti-carbohydrate T cell inducing amount of a vaccine comprising a mimetic of a carbohydrate antigen, wherein said mimetic has a structure such that the mimetic has about the same antibody-binding or selectin-binding activities, immunogenicity and antigenicity as that of a corresponding naturally occurring carbohydrate epitope.

84. The method of claim 83, wherein said mimetic has more resistance to metabolic degradation than the corresponding naturally occurring carbohydrate epitope.

85. An analogue of a cell surface carbohydrate comprising a carbocyclic compound, wherein said carbohydrate mediates intercellular adhesion or is expressed on tumor cells.

86. The analogue of claim 85, wherein said carbocyclic compound is a derivative of fucose.

87. The analogue of claim 85, wherein said carbohydrate is H, Le , Le^y, Le^a, Le^b, sialosyl Le^a or sialosyl Le^x.

88. The analogue of claim 86, wherein said fucose derivative has an acidic substituent at the three position of a terminal galactose.

89. The analogue of claim 88, wherein said acidic substituent is selected from the group consisting of a carboxyl group, a sulfono group and a phosphoro group.

90. The analogue of claim 85, wherein said carbohydrate is recognized by a selectin.

91. The analogue of claim 85 comprising a bivalent or multivalent structure.

92. A carbocyclic derivative of fucose.

93. A method of preventing intercellular adhesion comprising exposing cells to a physiologically effective amount of a carbocyclic derivative of fucose.

94. A conjugate comprising guanosine diphosphate and a carbocyclic derivative of fucose.

95. A method of preventing expression of a cell surface carbohydrate containing fucose, comprising exposing a cell to a physiologically effective amount of a carbocyclic derivative of fucose.

96. A method of inhibiting intercellular adhesion comprising exposing cells to a physiologically effective amount of a carbohydrate comprising a carbocyclic derivative of fucose.