Syntheses of model oligosaccharides of biological ...

$yntheses of model oligosaccharides sf biological significance. '7. Synthesis of a fucosylated N,N'-diacetylchitcPebiosidelinked to bovi~aeserum albumin and immunochemical characterization of rabbit antisera to this structureP3' Ho-HUATLEE A N D DAVIDA. SCHWARTZ Ludwig Institute,f;?rCancer Re.rearch, Toronto Brtrnch, 9 Earl Street, Toronto, Otzt., Canada M4Y 1M4

JOHN F. HARRIS Lorzdon Reg~o~zcil Cancer Centre, 391 South Streer, Lorzdon, Ont , Canada N6A 4G5

JEREMY P. CARVER Dep~zrtmentsofMediccrl

genetic,^

~zndMediccil Biophysics, U n i v e r ~ i hqf Toronto, Toronto, Ont., Canada M5S 1AS

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AND

JIRI J . K R E P I N S K Y ~ Ludwig Institute for Cancer Research, Tororzto Branch, 9 Earl Street, Toronto, Onr., Car-rndaM4Y I M 4 , and Departments of Medical Biophysics and Mecliccil Generics, U17il,er-.sitxqf Tororzto, Toronto, Otzt.. Curzada M5S IAK Received February 3 . 1986

Ho-HUATLEE, DAVID A. S C H ~ A R T JOHN Z . F. HARRIS. JEREMYP. CAR\.ER, and JIKI J . KREPINSKY. Can. J . Chem. 64. 1912 (1986). Synthesis of methoxycarbonyloctyl 2-acetan1ido-4-O-[(2'-acetamido-2'-deoxy-P-~-plucopyranosyl-6-O-cu-1.-fucopyranosyll-2-deoxy-P-D-glucopyranoside as shown in Scheme 1 is described together with coupling it with bovine serum albumin and poly-L-1)-sine. Mouse and rabbit antisera to the BSA conjugate were prepared and partially characterized. Ho-HUATLEE,DAVIDA . SCHWAKTZ, JOHNF. HARRIS, JEREMY P. CARVER et JIKI J. KREPTNSKY. Can. J . Chern. 64. 1912 (1986). On decrit la synthese l'acetamido-2 0-4-[(acktamido-2' dioxy-2' P-D-glucopyranilosyl) 0-6-a-L-fucopyrannosyl] deoxy-2 P-D-glucopyrannoside de rnCthoxycarbonyloctyle. realisCe suivant le schkma 1, ainsi que son couplape avec l'albumine du serum de boeuf et la poly-I.-serine. On a aussi prepark et partiellement caractCrisC des antiserum de souris et de lapin du conjug6 du ASB. (Traduit par la revue] Any success of contemporary cancer therapy depends heavily on early diagnosis of the disease, i.e. before a widespread invasion and dissemination occurs ( 1 ). Since clinical symptoms appear usually when the disease is in a relatively advanced stage, it is s f importance to characterize reliable and easily detectable chemical markers (bio-, immuno-) of the onset of neoplasia and malignancy. It has been shown in numerous studies that unusual carbohydrate structures appear both in neoplasia and cancer (2-6). The carbohydrate structures in an appropriate form are usually immunogenic and consequently can be used for the preparation of antibodies which in turn can recognize the original carbohydrate structure. Appropriately modified or as such, the antibodies can serve as diagnostic reagents. The high specificity required can be best achieved by using in the immunization protocol a well-characteri~ed. chemically pure, oligosaccharide antigen. In theory, such an antigen could be available from natural sources. These oligosaccharides are, however, obtainable from natural sources in minute quantities only, and their purity can never be assured entirely even after tedious and time-consuming purification procedures. Chemical synthesis eliminates these problems, and moreover allows to vary structural parameters as necessary for different uses. Since we have centered our attention on large bowel cancer, we have initiated a program of syntheses of

or

part 6, see D. M . Whitfield, J. P. Carver, and J . J. Krepinsky, J. Carbohydr. Chem. 4, 369 (1985). 'presented at 8th International Glycoconjugate Symposium. Houston. TX, September 1985. 3Author to whom correspondence should be addrcssed.

oligosaccharide cancer markers associated with this particular site, and of studies of antibodies to these oligosaccharides. One of the structures of interest is a class of oligosaccharide structures represented certain saccharide moieties of IV-!inked glycoproteins. A characteristic invariant basic structure of this class i s portrayed in formula 1. This structural element has been shown to exist in y-glutamyltranspeptidase (in liver) and carcinoembryonic antigen ( 7 , 8 )in cancer but not in the absence of the disease. The ' h n m ~ a l " olieosaccharides lack e~ther N-acetylglucosamine or fucose or both (cf. underlined fragments in 1).We have decided to synthesize I in two parts (cf a and b in 1). and Investigate the inlrnunochemistry of both fragments separately. This approach will he!p in the recognition and elimination of antibodies cross-reacting w ~ t hthe small fragments of 1when the antibodies to the complete I are made. In order to synthesize fragment a and link it to bovine serum albumin (and poly-L-lysine as an immunoadsorbent for immunoassays) we have modified a procedure employed previously for the synthesis of a methyl glycoside of the trisaccharide u (9). We have chosen to connect the trisaccharide with the carrier. i.e. bovine serum albumine, with a linker proposed by Lemicux et al. (10) and successfully used by others as well (1 1). Ethyl 9-hydroxynonanoate has been used in the first step (ref. 10; cf. Scheme I ) since it was easier to prepare it than a corresponding methyl ester. Its preparation involves diborane reduction of the free carboxyl group in a monoester of azelaic acid, and the methyl carboxylate is reduced to a significant extent whereas ethyl carboxylate is not. In the first ZemplCn deacetylation. quantitative exchange to give the methyl ester occurs. Since the linker carries an alkali-labile methoxycarbonyl group, care had

-

Galp

P1,4

GlcpNAc

Fucp

PI ,2

-

Manp

I

I

I II

GlcpNAc

P1,4 -----d

Manp

p1.4

'

---+

GlcpNAc

I

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Galp

P 1,4

GlcpNAc

p1,2

p1.4

1 ~ 1 ~ 6

GlcpNAc

p 1+

Asn

/

Manp

to be taken to avoid any condition causing hydrolysis of this ester group. In practice it meant maintaining anhydrous conditions even more carefully than usual in most steps. Particularly difficult was the hydrazinolysis of the phthalimido protective group, since a number of conditions led to the hydrolysis of the ester group. Eventually. hydrazine acetate was found to be compatible with the presence of the ester group. Phthalirnido protection of the amino function was used to avoid a potential problem of imidate formation (12) by an acetamido group in giycosylation reactions using strong promoters. as required for the formation of the 1.4 linkage in chitobiose. It has also been found that the simplest approach is to form a chitobiose derivative first. and to connect it with a fucosyl derivative (13) in the second stage. The reaction pathway is depicted in Schemes 1 and 2 which are seif-explanatory. Acetylatiori after complete deprotection was found to be necessary for the purification of the trisaccharide since it allowed for easy chromatographic separation on silica gel. Not only did this chromatography remove unwanted by-products but also inorganic ions introduced during the hydrogenolysis of the benzyl groups. The inorganic ions caused unacceptable broadening of 'Hnms signais (14). We have found FAB mass spectrometry extremely useful for both identification and purity determination of synthetic intervalues for M-derived mediates. We report in this article n ? / ~ ions; the complete analysis, however, of the FAD mass spectra of compounds reported here. together with other carbohydrate intermediates, will be published elsewhere. Finally, this trisaccharide ester was coupled to two different canier molecules, bovine serum albumin (BSA) or poly-Llysine, using a recent modification of the standard procedure (1 1). The trisaccharide ester 150 was first converted into the acyl hydrazide and subsequently. by treatment with dinitrogen tetroxide into the acyl azide which was then attached to BSA or poiy-L-lysine. Quantitative determination (16) of incorporation by the colorimetric pbenolsulphuric acid method showed incorporation of 10.5 molecules of carbohydrate per nlolecule of BSA and 1.9 carbohydrate molecules per poiy-L-lysine molecule (mw,,, 16 000). The synthetic antigen 15b was used to immunize (17) both rabbits and mice. The specificity of binding of rabbit antisera to the trisaccharide determinant P5a was determined in a preliminary fashion by competition analysis with various oligosaccharides using a solid-phase immunoassay (1 8) on the solid state substrate 15c. For detection of solid phase binding of the antibodies. '25~-proteinA was used. The binding specificity was expressed in terns of the molar concentrations of various oligosaccharides and their derivatives required to inhibit 50% of

the binding of a non-saturating concentration of rabbit sera (2 x lop3) and the values found are summarized in Table 1 . As can be seen from the Table 1, the only effective inhibitors were the trisaccharide derivatives 15. Other segments of the structure P or its variations (cf. Table 1) did not exhibit any binding to the rabbit antibodies. Similar results were obtained with mice. The analysis of rabbit antisera and the biochemical characterization of the purified antibodies is in progress and will be published elsewhere.

Experimental General methods Melting points were determined on a Reichert Thcrmolar ~neltrng point apparatus and are uncorrected. Optical rotations uere measured with a Perkin-Elmer polarirneter (Model 140) at 26 t 1°C. Microanalyses were performed by the Microanalytical Laboratory Ltd.. Markham. Ont. ' H nmr spectra were recorded at 360 MHz ~vitha Nicolet spectrometer at the Toronto Biomedical NMK Centre. Univcrsity of Toronto (Director: Dr. A. A. Grey). They were obtained at 23 + 2°C either in CDCI, containing 1 % TMS as the internal standard or in D 2 0 (99.996%. Merck, Sharp. and Dohnre) with acetone (0.1%. 2.225 ppm relative to internal DSS) as the internal standard. Infrared spectra were recorded with a Perkin-Elmer (Model 1430) Infrared ' polystyrene !dm. FAB Spectrometer. and calibrated at 1601 c n ~ C of mass spectrometry was performed using thioglycerol matrix and ?NaOAc with a VG Analytical ZAB HF r e ~ e r s e dgeometry instrument (for general conditions. cf. ref. 19, and references therein) at the Institute of Physiological Chemistrl-. University of Bonn through the courtesy of Dr. H . Egge. Thin layer chromatography (tlc) was performed on silica gel 60 Fz;j (Merck) plastic plates and visualized by quenching of ultraviolet fluorescence and (or) spraying with 50% aqueous sulfuric acid and heating at 100°C. Silica gel 60 (230-400 mesh: Merck) Mas used for flash column chromatography. Al! starting materials were dried overnight over KOH or P20s ill I'LICLLO prior to use. All solvents were distilled from appropriate drying agents. Unless stated otheru,ise. all reactions were carried out under anhydrous conditions and in an argon atmosphere. Solvents were removed at reduced pressures using a rotar) evaporator at temperatures not exceeding 40°C. -

-

8 - E t h o , ~ y c a r b o n ~ l o3c,~4 l, 6 - t r i - 0 - a c e ~ 1 - 2 - c i e ~ 1 . ~ ~ - 2 - ~ ~ h t h c i l i 1 1 1 i ~ i o - ~ D-glucopyrcrno.vide ($a; 15) To a mixture of the bromide 2 (1.5 g. 3.01 mmol) and silver zeolite (1.5 g) in dry dichloromethane (9 mL) was added a solution of 8-ethoxycarbonyloctylalcohol ( 3 a : 0.73 g , 3.61 mmo!) prepared according to ref. 10 in dichloromethane (9 mL) under stirring and protection from light at room temperature.%After 18 h. an additional amount of 30 (0.12 g. 0.60 mmol) in dichloromethane (2 1nL) waa

his compound and some other intermediates were purchased from TRC (Toronto Research Chemicals), 4483 Chesswood Drive. Downsview. Ont.. Canada M5J 2C3.

3 a , Ag zeolite Br

CH2C12

+

Ac AcO Ac

0(CH2)8COOEt

NPhth

NPhth

2

4a

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NaOMe MeOH

I

Benzyl bromide, THF, NaH

H

pOMePh

'7'

pOMeC6H4CH20 Sod~urncyanoborohydnde

0 Bn

0(CH2)8COOMe NPhth

CF3COOH. DMF

+ 0(CH2)8COOMe NPhth

7

8

1

2 ,CH3N02,collidine, Ag triflate, -25°C to +23"C

0(CH2)xCOOMe NHPhth

Ammonium Ce(IV) nitrate, aq. CHiCN Ac AcO Ac \

0(CH2)xCOOMe

NPhth

H-6),4.18(dd. l H , J = 1 2 . 3 a n d 2 . 3 H z . H - 6 ' ) , 3 . 1 3 ( q . J = 7.1 Hz. added and stirring in the dark was continued for 22 h. The mixture was 0CH2CH3),3.81-3.90and3.40-3.47 (2n1,3H, H-5. andOCHz(CH2),), then filtered through Celite, the Celite was washed with dichloro2.21 (t, 2H, J = 7.4 Hz, CH,COOEt). 2.12. 2.04. 1.87 (3s. 9H, methane, the combined filtrates and washings (100 mL) were washed CH3COO), 1.26 (t, 3H. J = 7.1 Hz. CH2CHI), 1.35- 1.58 and 0.90) water (2 X with cold aqueous sodium bicarbonate (100 I ~ L and 1.16 (2m, 12H. [CH2I6). Anul. calcd. for C3,H4,NOI2(619.263): 50 mL), dried over Mg2S04. and concentrated to give a light yellow C 60.09, H 6.67, N 2.26; found: C 59.83, H 6.42. N 2.18. Molecular syrup (2.13 g). The latter was subjected to chromatography on a silica ions observed: [MH]' 620. [ M N ~ ] '642. gel column using hexanelethyl acetate (3:l) to give slightly yellow syrupy 4 a (1.0 g, 54%), R f0.42 (sixfold development in the above 8-Methox?tarbonqlochl3,4,6-trl-0-aten /-2-cleo\~-2-phthal~mlnosolvent mixture); +21.2" (c. 1.3, CHCI?): ir (neat) 1780, 1750, P-D-gl~ctop?ranoszde( 4 b ) 1720 cm-' (CO); ' H nmr 6: 7.75-7.92 (m. 3H. N[C012ChH4).5.80 To a mixture of the bromide 2 (0 5 g. 1 00 mmol) and silver reolite (dd, l H , J = 1 0 . 7 a n d 9 . 1 H z , H - 3 ) . 5 . 3 6 ( d , I H . J = 8 . 5 H z . H - l j , (0 5 g) In dry dlchloromethane (3 mL) was added a solution of 5.19(dd. I H , J = lO.Oand9.3Hz.H-4).4.29-4.37(m,?H.H-2and

OBn 10

OBn

+

OBn Br

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\

OBn AcO.

J

1. NH2NH2.CH3COOH, MeOH 2 . AczO, pyridine

NHAc

1 . Hz, Pd/C 2 . Ac20, pyridine 3. NaOMe, MeOH

TABLE1. Competition analyses of two rabbit sera raised against 1 5 b a

Competitor a

Rabbit 1

Rabbit 2

HO

,'

concentrated to give a light brown syrup (0.63 g) which was subjected to chromatography on a silica gel column using hexanelethyl acetate (2:l) giving slightly yellow syrupy 4b (0.33 g, 54%), R f 0.45 (fourfold development in the above solvent mixture); [ a ] + 18.5" (c. 1.5, CHC13); 'Hnrnr 6: 7.75-7.92 (m, 4H, N[C0]2C6H4],5.79 (dd, lH, J = 10.7and9.1Hz,H-3),5.36(d, l H , J = 8 . 5 H z , H - 1 ) , 5 . 1 8 ( b r t , lH, J = 9.5 Hz, H-4), 4.27-4.37 (m, 2H, H-2 andH-6), 4.18 (dd, lH, J = 12.2 and 2.3 Hz, H-6'), 3.80-3.90 and 3.39-3.47 (2m, 3H, H-5, and 0CH2(CH2)7), 3.68 (s, 3H, 0CH3), 2.23 (t, 2H, J = 7.5 Hz, CH2COOMe), 2.12, 2.03, 1.87 (3s, 9H, CH3COO), and 0.92-1.57 (m, 12H, [CH2I6).Anal. calcd. for C30H39N012 (605.247): C 59.50, H 6.49, N 2.31; found: C 59.71, H 6.61, N 2.28. Molecular ions observed: [MH]' 606, [MNa]' 628, [MNH4]+ 623.

"C,, is the molar concentration of carbohydrates and glycoconjugates required to inhibit 50% of the binding of a non-saturating concentration of rabbit using a solid phase immunoassay. The solid phase substrate sera (2 x was 15c and the detection of solid phase binding of antibodies was with 8-Methoxycarbonylociyl2-deoxy-2-phthalimidow-glucopy'Z51-proteinA. Compounds used in competition experiments were preincubated ranoside ( 5 ) with the dilution of rabbit sera for 16 h in the presence of 1% BSA prior Treatment of 4a (6.15 g, 9.94 mmol) in dry methanol (100 mL) with to the assay at 4°C. 16 is GlcNAcpl,4(Fucal,6)GlcNaCpl,OMe;17 is 1% sodium methoxide in methanol (100 mL) for 30 min. deionization GlcNAcPl ,BSA. Other compounds tested were not effective competitors with with Dowex-1 (H', 37 mL, prewashed with methanol) at O°C, addition C50 > G ~ c N A c P ~ , ~ G I c N1,OBn A c ~ ~(18); Fucal,6GlcNAcp 1,OMe; of ethyl acetate (200 mL), filtration, and evaporation of the filtrate and ~ucal,4(Fucal,6)GlcNAc~l,OMe; Fucal,3Galpl,OMe; Fuca1,OMe: of added toluene (150 mL) gave a thick syrup of 5 (4.77 g, 99%); R f GlcNAcP1,4GlcNAc~l,OMe (9); Mana1,3Manc*l ,OMe: Mana1,6Manal ,OMe; Manal,3(Mancul,6)Manal,OMe (21); GlcNAcpl,2Manal,3(GlcNAc~1,2- 0.43 (ethyl acetate); ir (neat): 3430 (OH). 1770, 1735, 1714 cm-' Manal,6)Manp1,4GlcNAcpl,4GlcNAc; as well as purified oligosaccharides (CO); 'H nrnr6: 7.78 (m, 4H, N[C0]2C6H4),5.20 (d, lH, J = 8.4 Hz, from fetuin, fibrinogen A l , and ovalbumin (these compounds were kindly H-1), 3.66 (s, 3H, 0CH3), 2.22 (t, 2H, J = 7.5 Hz, CH2COOCH3). provided by Dr. D. A. Cumming). Note that 3 X lo-' of 18 for rabbit 2 was This product was used without further purification. the next most effective concentration.

8-methoxycarbonyloctylalcohol (3b; 0.24 g, 1.28 mmol) prepared according to ref. 10 in dichloromethane (3 rnL) under stirring and protection from light at room temperature. After continued stirring for 19 h in dark, the mixture was filtered through Celite, the Celite was washed with dichloromethane, the combined filtrates and washings were washed with water (2 x 50 mL), dried over MgS04, and

8-Methoxycarbonyloctyl2-deoxy-4,6-0-(4-methoq1benzylidene)-2 phthalimido- P - D-glucopyranoside ( 6 ) To a mixture of 5 (4.46 g, 9.94 mmol) and 4-methoxyberizaldehyde dimethyl acetal (3.3 rnL, 19.9 mmol) in acetonitrile (150 mL) was added p-toluenesulfonic acid monohydrate (120 mg) (20). The reaction mixture was stirred at room temperature and with light exclusion for 50 min, then it was poured into water (300 mL) and the product was

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1916

CAN. J. CHEM.

extracted into diethyl ether (3 x 250 mL). The ethereal solution was 8-Methoxycarbonyloctyl3-O-beizzyl-2-deox~-6-0-(4-rnethoxybeniyl)washed with cold aqueous sodium bicarbonate (200 mL) and water 2-phthalimido-4-0-(3',4',6'-tri-0-ace'-phthali(200 mL), dried over MgS04, and evaporated to a light brown syrup mido-/3-D-glucopyranosyl)-0-glucopyraoide( 9 ) which was subjected to chromatography on a silica gel column in A mixture of 8 (323 mg, 0.469 mmol), collidine (0.26 mL. hexanelethyl acetate (2:l) yielding 6 (3.81 g, 64%) as a pale yellow 1.92 mmol), dry nitromethane (1 mL), and silver triflate (482 mg. syrup, [ a I D -36.2" (c. 0.87, CHC13), R f 0.22 (solvent as above); 1.87 mmol) was stirred at -25OC, and a solution of 2 (934 mg, ir (neat): 3487 (OH), 1778,1735,1715 c m ' (CO); ' H nmr 6: 7.89 and 1.87 mmol) in dry nitromethane (1 mL) was added. After 30 min the 7.75 (m, 4H, N[C0]2c6H4)r7.44 and 6.93 (2m, 4H, C6H40CH3), mixture was warmed up slowly to room temperature, and stirring was 5.53 (s, IH, C6H5CH),5.26 (d, lH, J = 8.5Hz, H-I), 4.63 (ddd, 1H, continued for 4.5 h. Then the mixture was diluted with ethyl acetate J = 10.4, 8.6, and3.2Hz,H-3),4.38(dd, lH, J = 10.4and4.2Hz, (50 mL) and filtered through Celite, which was washed with ethyl H-6), 4.24 (dd, 1H, J = 10.5 and 8.5, H-2), 3.81 (s, 3H, C6H40CH3), acetate (3 X 20 mL), and the combined filtrates and washings were 3.66 (s, 3H, COOCH3), 3.37-3.87 (m, 5H, H-4, H-5, H-6', and washed with cold water (100 mL), cold 1 N HCl (100 mL), cold OCH2[CH2I7),2.44 (d, lH, J = 3.3 Hz, exchangeable wiih D20, aqueous sodium bicarbonate (100 mL), and again cold water (100 ml), OH), 2.23 (t, 2H, J = 7.4 Hz, CH2COOMe), 0.90-1.54 (m, 12H, and dried over MgSO4. The residue obtained after solvent evaporation (GH2)6). Anal. calcd. for C32H39N010 (597.369): C 64.31, H 6.58, was subjected to chromatography on a silica gel column with N 2.34; found: C 64.42, H 6.61, N 2.39. Molecular ions observed: hexanelethyl acetate (3:2) and gave 9 (345 mg, 66%) as an amorphous [MH]' 598, [MNa]' 620. solid, [ a I D +3.3" (c. 0.95, CHCI3); Rf0.30 (two-fold development in 8-Methoxycarbonyloctyl3-0-benzyl-2-deoxy-4,6-0-(4-methoxyben- hexanelethyl acetate 2:l); ir (neat) 1780, 1750, 1717 cm-' (CO); 'H nmr 6: 6.85-7.90 (m, 17H, N[C0I2C6H4, C6H4OcH3, and zy1idene)-2-phthalimido-/3-~-glucopyranoside (7) C6H5CH2),5.78 (dd, IH, J = 10.7 and 9.1 Hz, H-3'), 5.49 (d, J = A mixture of 6 (3.63 g, 6.07 mmol) and benzyl bromide (14.3 mL, 8.4Hz,H-lr),5.11 (dd, lH, J = 10.0and9.2Hz,H-4'),4.92(d, lH, 121 mmol) in dry THF (20 mL) was added to solid sodium hydride J = 8.2 HZ, H-I), 4.82 (d, IH, J = 12.6 HZ, C6H5CH2),4.46-4.49 (390 mg, 9.71 rnmol, prewashed with dry hexane), and the resulting (m, 3H, C6H5CH), 4.32 (dd, l H , J = 10.7 and 8.4, H-2'). 3.83 reaction mixture was stirred in the dark at room temperature for 4 h. (s, 3H, C6H40&(H3),3.65 (s, 3H, COOCH3), 3.19-4.23 (m, I l H , Additional sodium hydride (180 mg, 4.48 mmol) was added and the H-2, H-3, H-4, H-5, 2H-6, H-5', 2H-6', and 0CH2[GH2],), 2.19 (t, reaction mixture stirred for another 17 h. Then it was diluted with ethyl 2H, J = 7.5 Hz, CH2COOMe), 2.01, 1.97, 1.85 (3s, 9H, CH3COO), acetate (50 mL), filtered through Celite, washed withethyl acetate (3 x and 0.80-1.50 (m, 12H, (CH2)6). Anal, calcd. for C59H66N2019 50 mL), and the combined filtrate and washings were washed with (1 106.426):C 64.01, H 6.01. N 2.53; found: C 63.98, H 5.88, N 2.49. water (300 mL). The organic layer was separated, the aqueous layer Molecular ions observed: [MH]+ 1107, [MNa]' 1129, [MNH41T extracted with ethyl acetate, all organic extracts were combined, 1124. washed once again with water (200 mL), dried over MgSQ4, and evaporated to dryness. The residue was subjected to chromatography 8-Methoxycarbonyloctyl3 - 0 -benzyl-2-deoxy-2-phthalirnido4- 0 on a silica gel column with hexanelethyl acetate (3: 1) to give 7 (3.76 g, (3 ',4 ', 6'-tri-0-acetyl-2 '-deoxy-2 '-phthalimido-/3-~-glucop?.90%) as colorless plates, mp 88-89°C (hexanelethyl acetate), [ a ] " ranosy1)-P-D-glucopyranoside1 8 (20) +27.4" (c. 1.0, CHC13); R f 0.37 (two-fold development, the same To a stirred solution of 9 (419 mg, 0.379 mmol) in acetonitrile/water solvent as above); ir (neat): 1778, 1736, and 1716 cm-' (CO); w nnx (9:1, 1.5 1nL) at O"C was added ammonium cerium(1V) nitrate 6: 5.58 (s, 1H, C6H5CH), 5.18 (d, IH, J = 8.5 HZ, H-I), 4.78 (415 mg, 0.758 mrnol) and stirring was continued at O"C for 10 min and (2d, 2H, J = 12.3 HZ, C6H5Cff2), 4.49 (2d, 2H, J = 12.3 Hz, then at room temperature for 45 min. The mixture was diluted with C&sCH2), 3.82 (s, 3H, C6H40CH3),3.66 (s, 3H, COOCH3), 2.22 ethyl acetate (100 mL), washed with water (2 X 50 mL), dried with (t, 2H, J = 7.5 Hz, CH2COOMe), 0.90-1.54 (m, 12M, (CH&). MgS04, and the solvent evaporated to a syrup which was subjected to Anal. calcd. for C39H45N010(687.445): C 68.11, H 6.60, N 2.04; chromatography on a silica gel column. Elution with hexanelethyl found: C 68.33, H 6.65, N 2.18. Molecular ions observed: [MH]" 688, acetate (3: 1) gave unreacted 9 (24 mg, 6%), followed by 80 [MNa]' 7 10. (319 mg, 86%) which was a colorless solid, mp 142.5-144.6"C 8-Methoxycarbonyloctyl3-0 -benzyl-2-deoxy-6-0-(4-methoqbenZy0(ethyl etherlhexane); [ a I D +10.3" (c. 1 . I , CHCI3); Rf 0.17 (ethyl 2-phthalimido-/3-D-glucopyranoside ( 8 ) (20) etherlhexane, 1:1); ir (neat) 3485, 3525 (OH), 1777, 1750, and A mixture of 7 (3.62 g, 5.27 mmol), sodium cyanoborohydride 1718 cm-' (CO); '1-1 nmr 6: 6.80-7.95 (m, 13H, N[C0I2C6H4and (1.66 g, 26.4 mmol), and powdered molecular sieve 3A (5 g) in C6ff5CH2),5.80 (dd, lH, J = 10.6 and 9.1 Hz, H-3'), 5.65 (d, 1H, N,N-dimethylformawide (DMF, 40 mL) was stirred for 15 niin at room J = 8.3 Hz, H-l'), 5.16 (dd, IH, J = 10.0 a n d 9 . 2 Hz, H-4'), temperature, and a solution of trifluoroacetic acid (4 mL, 52.7 mmol) in 12.7 HZ, C6H5CffZ), 4.99 (d, IH, J = 8.6 Hz, H-I), 4.86 (d, IH, J DMF (35.7 mL) was then added; stirring was continued for 19 h and, 4.46 (d, 1H, C6H5CH), 4.35 (dd, IW, J = 10.6 and 8.3, H-2'), after addition of further trifluoroacetic acid (3.8 mL, 50 mmol), for an 3.65 (s, 3H, COOCH3), 3.22-4.32 (m, 11H, H-2, H-3, H-4, H-5, additional 24 h. The mixture was then cooled to O°C, diluted with ethyl 2H-6, H-5', 2H-6', and 0CH2[CH2I7), 2.19 (t, 2H, J = 7.5 Hz, acetate (50 mL) and methanol (5 mL), stirred for 5 min and filtered, and CH2COOMe), 2.00, 1.99, 1.84 (3s, 9H, CH,COO), 1.73 (dd, 1H. the residue was washed wiih ethyl acetate (3 X 50 mL). The combined J = 8.8 Hz and 4.3 HZ, exchangeable with D20, OH), and 0.80-1.50 filtrates were washed with cold water (200 mL). cold aqueous sodium (m, 12H, (CH2)6). Anal. calcd. for C51H58N2018 (986.368): C 62.06, bicarbonate (200 mL), and water (2 X 100 mL), dried over MgS04, H 5.92, N 2.84; found: C 62.21, H 6.03, N 2.73. Molecular ions and evaporated to dryness. Chromatography of the syrupy residue on a observed: [MH]+ 987, [MNa]' 1009, [MNH,]' 1004. silica gel column with hexanelethyl acetate (2: 1) gave 8 (3.01 g, 83%); [ a I D + 16.5" (c. I . 1, CHC13), R f 0.36 (same solvent as above); 8-Methoxycarbonyloctyl4-0-(3',4',6'-tri-0-acetyl-2 '-deoq-2'ir (neat): 3487 (OH), 1778, 1737, and 1713 cm-' (CO); 'H nmr 6: phthalimido-/3-D-glucopyranosyl)-6-0-(2'' 3",4"-tri-0 -benzyl6.90-7.90 (m, 13H, N[C0]2C6H4,C6H40CH3,and C6H5CH2),5.11 a -L-fucopyranosy1)-3-0-benzyl-2-deoxy-2-phthalimido- P-D(d, 1H, J = 8.3 HZ, H-I), 4.75 (d, J = 12.2 HZ, C6H5Cff), glucopyranoside ( d l ) A mixture of 10 (312 mg, 0.315 mmol), tetraethylammonium 4.50-4.60 (m, 3H, C6H5CH), 4.21 (dd, IH, J = 10.7 and 8.3 Hz, H-3), 4.12(dd, l H , 3 = 10.7 and8.3, H-21, 3.82(s, 3H, C6H40Cff3)s bromide (1.32 g, 6.30 mmol), and powdered molecular sieve 4 8 3.66 (s, 3H, COOCN3), 3.26-3.85 (m, 6H, H-4, H-5, H-6, H-6'. and (2.33 g) in dry dichiorornethane (6 mL) was stirred in the dark at room OCH2[CH2I7), 3.02 (d, 1H, J = 2.4Hz, exchangeable with D 2 0 , temperature for 1 h. A solution of freshly prepared tri-0-benzyl-a-LOH), 2.21 (t, 2H, 3 = 7.6 Hz, CH2COOMe), 0.85-1.53 (m, 12W, fucopyranosyl bromide (ref. 13a; 626 mg, 1.26 mmol) in dry dichloro(CH&). Anal. calcd. for C39H47N010(689.320): C 67.91, H 6.87, methane (ref. 13b; 5 mL) was then added, and the reaction mixture was N 2.03; found: C 67.83, H 6.64, N 1.97. Molecular ions observed: slowly stirred for 44 h. It was diluted with dichloromethane (60 mL) [MHIf 690, [MNa]' 712, [MNH4]' 707. and filtered through Celite, which was washed with dichloromethane

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(2 X 30 mL), and the combined filtrate and washings were washed with water (2 x 100 mL), dried over MgS04, and evaporated to give a syrupy residue that was subjected to chromatography on a silica gel column with hexanelethyl acetate (2: 1). Together with unreacted 10 (19 mg, 2%), 11 (347 mg, 79%) was obtained as an amorphous solid, [ a I D -51.9" (c. 1.0, CHC13); R f 0.30 (two-fold development in the above solvent); ir (neat) 1779, 1750, and 1718 cm-' (CO); 'H nmr 6: 6.80-7.95 (m, 28H, N(C0)2ChH4 and C6H5CH2),5.72 (dd, lH, J = 10.7 and 9.2 Hz, H-3'), 5.63 (d, lH, J = 8.4 Hz, H-1'), 5.09 (d, l H , J = 3.3 HZ, H-1"), 4.91 (d, lH, J = 8.3 Hz, H-1), 3.64 (s, 3H, COOCH3), 2.19 (t, 2H, J = 7.5 Hz, CH2COOMe), 1.91, 1.87, and 1.83 (3s, 9H, CH3COO), and 1.04 (d, 3H, J = 6.5 Hz, H-6"). Anal. calcd. for C78H86N2022 (1402.567): C 66.75, H 6.18, N 2.00; found: C 66.61, H 6.11, N 1.94. Molecular ions observed: [MH]+ 1403, [MNal+ 1425, [MNH4]+ 1420.

1917

5.68 (d, l H , J = 7.9 Hz, exchangeable with D20, NH), 5.60 (d, lH, J = 8.6 Hz, exchangeable with D 2 0 , NH), 5.07 (d, 1H, J = 3.6 Hz, H-1"), 4.96 (d, lH, J = 8.4 Hz, H-1'), 4.51 (d, lH, J = 7.8 Hz, H-1), 4.30 (br, q, lH, J = 6.2 Hz, H-5"), 3.66 (s, 3H, COOCH3), 2.30 (t, 2H, J = 7.5 HZ, CH2COOCH3), 2.18, 2.15, 2.07, 2.01, 2.00, 1.96, and 1.91 (7s, 27H, CH,COO), 1.28-1.61 (m, 12H, CH2[CH2I6CH2COOCH3),and 1.13 (d, 3H, J = 6.4Hz, H - 6 ) . Anal. calcd. for C46H70N2024 (1034.432): C 53.38, H 6.82, N 2.71; found: C 53.46, H 6.91, N 2.83. Molecular ions observed: [MH]' 1035, [MNa]' 1057.

8-Methoq~carbonyloctyl2-acetamido-4-O-(2'-acetamido-2'-deoq-~o-g1ucopyranosyl)-6-O- a -L-fucopyranosyl-2-deoq-P- D-glucopyranoside (15a) ZemplCn deacetylation of 14 (78 mg, 0.075 mmol) in methanol (2 mL) was performed as described for the preparation of 5. The 8-Merhoxycarbonyloctyl2-acetamido-4-0-(2'-acetarnido-3',4',6'-tritrisaccharide 15a (50 mg, 91%) was obtained as colorless solid, O-acetyl-2 '-deoxy-P-D-glucopyranosyl)-3-Obenzyl- 6 - 0 mp 283-286°C (aqueous ethanol), [ e l D -63.5" (c. 0.42, 50% (2",3",4"-tri-O-benzyl-a-~-fucopyranosyl)-2-deoqlP-D-glucoaqueous ethanol); 'H nmr 6: 4.90 (d, lH, J = 3.8 Hz, H-1"), 4.64 pyranoside ( 1 2 ) (d, lH, J = 8.4 Hz, H-1'), 4.49 (d, lH, J = 7.8 Hz, H-1), 4.13 A solution of 11 (591 mg, 0.422 mmol) and hydrazine acetate (br, t, 1H, J = 6.5 Hz, H-5"), 3.68 (s, 3H, COOCH,), 3.40-3.95 (1.17 g, 12.7 mmol) in dry methanol (20 mL) was boiled under reflux (m, 17H, H-2, H-3, H-4, H-5, 2H-6, H-2', H-3', H-4', H-5', for 3 h, then another portion of hydrazine acetate (1.17 g, 12.7 mmol) 2H-6', H-2", H-3", H-4", 0CH2[CH7]), 2.39 (t, 2H, J = 7.4 HZ, was added and the refluxing continued for 4.5 h. After cooling. the CH2COOCH3), 2.08 and 2.03 (2s, 6H, CH3COO), 1.58 and 1.30 reaction mixture was evaporated to dryness and treated with acetic (m, 12H, CH2[CH2]hCH2C(30CH3),and 1.23 (d, 3H, J = 6.6 Hz, anhydride (14 mL) in pyridine (15 mL) at room temperature for 19 h. H-6"). Anal. calcd. for C32H56N2017 (740.358): C 51.88, H 7.62, The mixture was diluted with ethyl acetate (200 mL) and washed with N 3.78; found: C 51.92, H 7.83, N 3.88. Molecular ions observed: cold water (100 mL), 1 N HCl(2 X 100 mL), and water (100 mL). The [MH]' 741, [MNa]' 763. organic layer was dried over MgS04, the solvent evaporated, and the Hydrazide pale yellow solid residue was subjected to chromatography on silica Compound 15a (35 mg, 0.047 mmol) and hydrazine hydrate gel using ethyl acetatelhexane (3: l), to give pure 12 (423 mg, 82%) (1.6 mL) were stirred in dry ethanol (4.6 mL) at room temperature for as acolorless solid, mp 186-188°C; [ a I E -42.5" (c. 0.76, CHC13);Rf 24 h. The mixture was evaporated to dryness, and the residue was 0.12 (the above solvent); ir (neat) 3305 (NH), 1748 (COOR), and 1662 evaporated from water (2 X 12 mL) to give the hydrazide as a colorless (CONH); 'H nrnr 6: 7.20-7.50 (m, 20H, C6H5CH2),6.22 (d, l H , J = solid (36 mg) that was used in the next step without purification. 9.4Hz,NH),5.56(d, l H , J = 8 . 7 H z , N H ) , 5 . 0 0 ( d , lH, J = 9 . 6 H z , H-lf),4.66(d, lH, J = 2 . 5 H z , H - l U ) , 4 . 4 3 ( d ,l H , J = 6 . 9 H z , H - 1 ) ; Coupling reaction with BSA 3.65 (s, 3H, COOCH3), 2.28 (t, 2H, J = 7.5 Hz, CH2COOMe), To a solution of the hydrazide (21 mg, 0.029 mmol) in DMF 2.03, 1.98, 1.94, 1.87, and 1.82 (5s, 15H, CH3COO), 1.20-1.65 (0.4 mL) stirred at -40 to -30°C, was added a solution of dinitrogen (m, 12H, [CH2I6),and 1.04 (d, 3H, J = 6.4 Hz, H-6"). Anal. calcd. tetroxide (1 1) in dichloromethane (0.46 M, 0.08 mL). Stirring was for C66H86N2020(1226.577): C 64.59, H 7.06, N 2.28; found: continued at -20 to - 10°C for 45 min, then this solution was added C 64.67, H 7.20, N 2.33. Molecular ions observed: [MH]' 1227, to a buffered solution (KHC03/Na2B407;pH 8.93; 9.4 mL) of bovine [MNa] 1249. serum albumin (purified, 94 mg, 0.00145 mmol) under stirring at O°C, and the stirring was continued for 25 h. The mixture was then dialyzed 8-Methoxycarbonyloctyl2-acetamido-4-0-(2'-acetamido-3',4',6'-triagainst water (5 x 100 mL) using an Amicon ultrafiltration cell O-acetyl-2'-deoq-~-~-glucopyranosyl)-6-O-(a-~-fucopyrano(Model 402) equipped with a YM-10 membrane. After lyophilization, sy1)-2-deony-P- D-glucopyanoside (13) 15b (97 mg) was obtained as a colorless solid. The carbohydrate Hydrogenolysis of 12 (179 mg, 0.146 mmol) on 10% Pd/C in ethyl content was determined according to ref. 16 as 10.5 carbohydrate acetate/methanol(1:3, 20 mL) gave, after filtration through Celite and equiv. per BSA molecule. evaporation of the solvent, colorless 13 (118 mg, 94%), which was unstable on standing. Rf0.40 (ethyl acetate/methanol, 5:l); 'H nmr 6: Coupling reaction with poly- I.-lysine 5.11 (t, IH, J = 9.8Hz, H-3'), 5.02 (t, lH, J = 9.7 Hz, H-4'), Hydrazide (17 mg, 0.023 mmol) was treated with dinitrogen 4.81 (d, lH, J = 1.8 Hz, H-1"), 4.73 (d, lH, J = 8.6 Hz, H-1'), tetroxide as in the previous reaction; however, instead of BSA, poly4.39 (d, lH, J = 7.8 HZ, H-I), 3.67 (s, 3H, COOCH3), 3.40-4.28 L-lysine (mw 16 000); 96 mg, 0.006 mmol) in buffer (9.6 mI,) at O°C (m, 16H, H-2, H-3, H-4, H-5, 2H-6, H-2', H-5', 2H-6', H-2", H-3", was used. After 44 h the reaction mixture was dialyzed against water H-4", H-5", CH2[CH2I7),2.30 (t, 2H, J = 7.5Hz, CH2COOCH3), (5 x 100 mL) using a YM-2 membrane, and lyophilized to give 15c 2.09,2.02,2.01,2.00, and 1.96 (5s, 15H, CH3COO), and 1.20-1.65 as a pale yellow solid (47 mg). The incorporation level was determined (m, 15H, H-6" and cH2[CH2]hCOOCH3). as above as 1.9 carbohydrate equiv. per poly-L-lysine molecule. +

8-Methoxycarbonyloctyl2-acetamido-4-0-(2'-acetamido-3',4',6'-triImmunization protocol (17) (3 -acetyl-2 '-deoq-/3-~-glucopyranosyl)-6-O-(2", 3", 4"-tri-0Rabbits were immunized subcutaneously with 200 yg of 156 acetyl- a - L fucopyranosyl)-3-0 -acetyl-2-deoxy-P-D-glucop?ra(0.2 mL in 0.15 M NaCl) and equal volume of Freund's complete noside (14) adjuvant at four sites at the back of the neck. Boosting immunizations Compound 1 3 (120 mg, 0.139 mmol) was treated with acetic were performed with the same amount of 15b and incomplete Freund's anhydride (1.5 mL) in dry pyridine (2 mL) for 19 h. The reaction adjuvant weekly for 3 weeks. After a resting period of 2 weeks, the mixture was diluted with ethyl acetate (100 mL), washed with cold animals were again immunized with 400 yg of 15b in PBS (0.4 mL) water (50 mL), 1 N HCl (2 x 50 mL), and water (50 mL), dried in the ear vein, and blood samples were taken after 1 week. (MgS04), and evaporated to give a colorless, syrupy residue. The latter was subjected to chromatography on a silica gel column using ethyl Solid-phase radioimmunoassay (RIA) and competition analysis acetate to give 14 (113 mg, 79%); [ a I D -69.2" (c. 1.0, CHC13); The solid phase RIA was performed using 86-well acrylic plastic Rf 0.12 (ethyl acetate), 0.64 (ethyl acetatelmethanol 9: 1); 'H nmr 6: plates (Flow laboratories) that were incubated with 15c at 20 yg/mL

1918

CAN. J . CHEM. VOL. 64. 1986

for 16 h at room temperature. Following washing in ST buffer (0.15 M NaCI, 0.01 M TRIS, pH 8 ) , the plates were blocked with 0.5% gelatin in ST. The solid-phase binding of rabbit sera was measured by incubating the plates for 4 h at room temperature with 50 mL rabbit sera diluted in 1% BSA-ST, washing in ST buffer, amplifying for 2 h with '25~-proteinA prepared according to ref. 18 ( 3 X lo4 cpm/well). A non-saturating concentration of rabbit sera was determined for use in the competition analysis. The compounds used in competition experiments prepared in our laboratories ( 9 , 14, 21) were preincubated with this dilution of rabbit sera (2 X in 1% BSA-ST buffer for 16 h at 4°C. The residual activity was determined in the solid phase RIA, and the percent inhibition was calculated for a titration of the competitor.

5.

6. 7. 8. 9. 10.

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11.

Acknowledgements This work was in part supported b y a grant from NCI Canada (to J.F.H.). Our thanks are due to D r . W. R. Bruce, Director of the Toronto Branch of the Ludwig Institute for Cancer Research for continuous encouragement and interest in this work. W e thank to Dr. H. Egge for allowing u s t o record the mass spectra at his instrument, Mr. J. McGoey and Mr. A. Lee for recording the nmr spectra, and Dr. H. Pang for recording mass spectra. We also thank Dr. D. A . Cumming for the generous gift of several competing compounds, M r . J. Baptista for measuring optical rotations, and Mr. M . Best for technical assistance.

12. 13. 14. 15. 16. 17. 18. 19.

1. J. D. HARDCASTLE and K. D. VELLACOTT. Rec. Results Cancer Res. 83, 86 (1982). Cancer Res. 45, 2405 (1985). 2. S.-I. HAKOMORI. 3 . T. FEIZI.Nature, 314, 53 (1985'). 4 . H. KOPROWSKI and M. HERLYN. In Molecular biology of tumor

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cells. Edited by B. Wahren. Raven Press, New York, NY. 1985. p. 123. S.-I. HAKOMORI. In Molecular biology of tumor cells. Edited by B. Wahren. Raven Press, New York, NY. 1985. p. 139. T. FEIZIand R. A. CHILDS.Trends Biochem. Sci. 10, 24 (1985). A. KOBATAand K. YAMASHITA. Pure Appl. Chem. 56, 821 (1984). K. YAMASHITA, I. UEDA,M. KUROKI,Y. MATSUOKA, and A. KOBATA. Proc. 8th Int. Symp. Glycoconjugates, Houston, TX. 1985. D. A. SCHWARTZ, H. H. LEE,J. P CARVER, andJ. J. KREPINSKY. Can. J. Chem. 63, 1073 (1985). R. U . LEMIEUX, D. R. BUNDLE, andD. A. BAKER. J. Am. Chem. SOC.97, 4076 (1975). B. M. PINTOand D. R . BUNDLE.Carbohydr. Res. 124, 313 (1983). J.-R. POUGNY,U . KRASKA, and P. SINAY.Carbohydr. Res. 60, 383 (1978);50, 181 (1976). and H. M. FLOWERS. Carbohydr. Res. ( a )M . DEJTER-JUSZYNSKI 18, 219 (1971); ( b ) R. U. LEMIEUX, K. B. HENDRICKS, R. V. STICK,and K. JAMES.J . Am. Chem. Soc. 97,4056 (1975). R. N . SHAH,D. A. CUMMING: A. A. GREY.J. P. CARVER, and J. J. KREPINSKY. Carbohydr. Res. In press. ( a )P. J. GAREGG and P. O s s o w s ~Acta ~ . Chem. Scand. B37,249 (1983);( b ) D . M. WHITFIELD,R. N. SHAH,J . P. CARVER, and J. J. KREPINSKY. Synth. Commun. 15, 737 (1985). S. C. KUSHAWA and M. KATES.Lipids, 10, 372 (1985). J. H . PAZUR.Carbohydr. Res. 107, 243 (1982). J. J. LANGONI, M. D. T. BOYLE,and T. B o ~ s o s J. . Immunol. Meth. 18, 281 (1977). H. EGGE,J. PETER-KATALINIC, J. PAZ-PARENTE, G. STRECKER, J. MONTREUIL, and B. FOURNET. FEBS Lett. 156, 357 (1983). R. JOHANSSON and B. SAMUELSSON. J. Chem. Soc. Chem. Commun. 201 (1984). F . M . WINNIK,J.-R. BRISSON,J. P. CARVER.and J. J. KREPINSKY. Carbohydr. Res. 103, 15 (1982).