Bioconjugate Chem. 1995, 6, 319-322
319
Strategies for the Synthesis and Screening of Glycoconjugates. 2. Covalent Immobilization for Flow Cytometry Dirk Vetter, Emily M. Tate, and Mark A. Gallop* Affjmax Research Institute, 4001 Miranda Avenue, Palo Alto, California 94304. Received September 28, 1994@
Glycosylamines are readily available carbohydrate derivatives that undergo acylation reactions with homobifunctional N-hydroxysuccinimidyl esters. The product glycosylamides carry a spacer group equipped with one active ester functionality. This route provides well-defined glycoconjugates, which may be cross-linked to various amino-functionalized resins. Carbohydrate recognition of the resulting sugar-bead conjugates is probed by lectin immunostaining or flow cytometry using a fluorescently labeled lectin.
INTRODUCTION
Carbohydrates play various physiological roles and have drawn attention as potential constituents of pharmacophores (11. The systematic screening of the carbohydrate pool would greatly facilitate the drug discovery process in this area. In one development, the use of flow cytometry has been introduced for the assessment of carbohydrate-protein specificities and affinities (2-4). Instead of cells, carbohydrate-functionalized latex or polystyrene beads are subjected to fluorescence-activated particle scanning and sorting techniques. We are currently interested in generating combinatorial libraries of sugar-bead conjugates that may be screened for receptor binding using flow cytometry methods. Our group has been using a similar approach to screen large libraries of peptides synthesized on spherical microbeads, wherein the structure of the ligand on any bead is encoded by the parallel synthesis of a single-stranded DNA molecule (5). Individual beads with appropriate receptor binding characteristics can be sorted from the population, and the DNA tag can be amplified and sequenced to reveal the structure of the cognate ligand. As a prerequisite to the synthesis of encoded carbohydrate libraries, this paper describes a simple two-step method for the immobilization of a variety of glycosylamines on amino-functionalized resin, using mild amide bond-forming chemistry. Sugar-bead conjugates are subjected to lectin binding assays, and flow cytometry is employed to select binding populations from pools of immobilized carbohydrates. EXPERIMENTAL SECTION
Mono(succinimidy1)Mono(sialyllactosy1amido)suberate. A 304 mg portion of disuccinimidyl suberate (0.83 mmol, Pierce) and 107 mg of 1-hydroxybenzotriazole were dissolved in DMSO (1.25 mL, heat). After the mixture was cooled to room temperature, 50 mg of sialyllactosylamine (79 pmol) was added and the resulting mixture stirred for 2 h. The reaction was followed by TLC (l-propanol/ethyl acetatdwater, 6:1:3, Rfeduct 0.40, Rf product 0.67). After complete conversion, product was precipitated with acetone/ether (1:2, 10 mL). Following centrifugation, the residue was washed with acetone/
* To whom correspondence should be addressed. Phone:
(415)
812-8706. Fax: (415) 424-9860. @
Abstract published in Advance ACS Abstracts, April 15,
1995.
1043-1802/95/2906-0319$09.00/0
ether ( l : l , 10 mL) and dried in vacuo. Yield: 60 mg (86%). For analytical data see Table 2. Other active ester-sugar conjugates were prepared accordingly. A similar strategy was applied to the biotinylation of a library of 50 glycosylamines (unpublished results), and some influence of carbohydrate structure on the yields of conjugate obtained was observed. Most critical was the conjugate precipitation step: with monosaccharides of decreased hydrophilicity (for instance, Xyl or GlcSNAc), addition of acetonelether either did not precipitate the glycoconjugate or only very low yields were obtained. However, all oligosaccharide-conjugates prepared here were efficiently precipated, due to their high polarity. Lactose-TentaGel. A 102 mg portion of mono(succinimidyl) mono(lactosy1amido)suberate (171 pmol) was dissolved in 1 mL of DMF and mixed with 156 mg of TentaGel S NHz (Rapp Polymere, 0.25 mmol/g, 39 pmol of amino groups). After sonication for 1h and vortexing for 20 more hours, beads were recovered by centrifugation, washed with DMF and water, and lyophilized. Conjugates of maltose, sialyllactose, chitobiose, LacNAc, GlcNAcGSOy, and Gl~NAc6P03~with TentaGel were prepared analogously. Lactose-Microresin. A 1.5 mg portion of mono(succinimidyl) mono(lactosy1amido)suberate (2.5 pmol) was dissolved in 50 pL of DMF with 0.3 mg of HOBt and 5 pL of DIEA and vortexed with 1mg (0.1 pmol of amino groups) of 10 pm dodecylamine-grafted polystyrene resin (5)for one hour. Beads were washed thoroughly with DMF. Conjugates of maltose, sialyllactose, chitobiose, LacNAc, GlcNAc6S03-, and GlcNA~6P03~with this resin were prepared analogously. Solid Phase Assays. Carbohydrates were detected on beads using adapted digoxigenin immunoassays (Glycan Detection Kit, Glycan Differentiation Kit, Boehringer Mannheim, see product sheets for detailed information) or direct fluorescent lectin staining. (A) Ca. 5 mg of TentaGel beads were treated as follows: 1mL of periodate solution (14 mmoVL) for 1h, 10 pL of digoxigenin hydrazide (5 mmoVL in DMF) plus 0.2 mL of acetate buffer for 1 h, 1 mL of casein solution (2% w/v in Tris-buffered saline (TBS)) for 1 h, 1 mL of antidigoxigenin-alkaline peroxidase conjugate (1ng/mL) for 1h, 1mL of nitro-blue tetrazolium staining solution, overnight. (B) Ca. 2.5 mg of TentaGel-beads was incubated in the following order: 1mL of casein (2% w/v) in TBS for 1h, 0.1 mL of digoxigenin-lectin conjugate (RCA 120 a t 1 0 1995 American Chemical Society
Vetter et al.
320 Bioconjugate Chem., Vol. 6,No. 3, 1995 Table 1. Literature Studies Describing the Solution-PhaseAcylation of Unprotected 1-Amino, 1-deoxy Sugars acylating agent acryloyl chloride Fmoc-Asp(0Pfp)-OtBu Asp-containing peptide Fmoc chloride Asp-containing peptide dansyl chloride fluorescein-OSu chloroacetic anhydride
condns
ref Kallin et al., 1989 (8) Otvos et al., 1989 (9) Anisfeld and Lansbury, 1990 (10) Kallin et al., 1991 (11) Cohen-Anisfeld and Lansbury, 1993 (14) Manger et al., 1992 (15)
THFMeOHNa2C03 DMFIwater BOP or HBTU in DMSO or DMF in presence of DIEA aqueous NazC03/dioxane HBTU, HOBt or HBTU, DIEA aqueous NazC03/acetone aqueous Na2COdDMF aqueous NazC03
Table 2. Diagnostic Analytical FAB-MSand 300 MHz (DzO) 'H-NMR Data for Glycosylamide-ActiveEster Conjugates m l z (calcdfound for FAELMS),
product
6 and J for the N-linked anomeric proton 682/683.2 (M
ethylene glycol mono(succinimidy1 succinate) mono(maltosy1amidosuccinate) ethylene glycol mono(succinimidyl succinate) mono(lactosy1amidosuccinate) mono(succinimidy1)mono(lactosy1amido)suberate mono(succinimidy1)mono(sialyllactosy1amido)suberate mono(succinimidy1)mono(lactosy1-N-acetylamidolsuberate mono(succinimidy1)mono(chitobiosy1amido)suberate mono(succinimidy1)mono(GlcNA~6P03~amidohberate mono(succinimidyl) mono(GlcNAc6SO~--amido)suberate
+ H+), 4.894 ppm (d, 8.3 Hz)
nd,n 4.916 ppm (d, 8.6 Hz)
+
594/595.3 (M H+), 4.906 ppm (d, 8.6 Hz) nd, 4.988 ppm (d, 8.7 Hz) 6351561.2 (M - Su Na+),b5.062 ppm (d, 9.1 Hz) 67U602.3 (M - Su + Na'),b 5.091 ppm (d,8.9 Hz) 597/501.1 (Mdlsodlum salt - Su + H+),b5.091 ppm (d, 8.9 Hz) 575/501.1 (Msodlum salt - Su + Na+),b 5.091 ppm (d, 8.9 Hz)
+
M - Su is product without the succinimidyl (Su) group. The corresponding free acid appears to be a FABa nd: not determined. MS-induced artifact as the intact succinimidyl ester is detected in the 'H-NMR: 2.878 (4 H, s, N-succinimidylate); free N-hydroxysuccinimide resonates a t higher field (2.173 ppm, 4 H, s). Also indicative is the shift of the methylene protons of suberic acid moiety: the methylene groups neighboring the glycosylamide linkage resonate at 2.663 ppm (2 H, t, a-CH2) and 1.665 ppm (2 H, m, P-CHB), the methylene groups next to the succinimidyl ester resonate at 2.264 ppm (2 H, t, a'-CH2) and 1.569 ppm (2 H, m, P-CH2). OH
H o m o OH
-0H O -
OH
Ho
HO
OH
OH
OH
NHz
\
aq. ammonium carbonate, 5 days
1 \
+ DSSI
Figure 1. Structures of lactose, lactosylamine, mono(lactosy1amido) mono(succinimidyl)suberate, and lactosyl resin, illustrating the reaction sequence for the covalent immobilization of carbohydrates on polystyrene beads.
mg/mL, MAA a t 0.2 mg/mL) for 1h, 1 mL of antidigoxigenin-alkaline peroxidase conjugate (1 ng/mL) for 30 min, 1mL of nitro-blue staining solution for 5 min (RCA 120), or overnight (MAA). (C) Ca. 0.2 mg of 10 pm diameter polystyrene resin was incubated in the following order: 0.35 mL of 2% BSM TBS for 30 min, 0.3 mL of WGA-fluorescein (Molecular Probes) or RCA 120-fluorescein (Sigma) in TBS (each a t 0.5 mg/mL) for 30 min. Bead suspensions were then subjected to fluorescence activated particle analysis on a FACScan instrument (Becton-Dickinson). For each histogram, 10 000 events were acquired. RESULTS AND DISCUSSION
In the preceding paper we described a glycosylamine library consisting of 50 mono- and oligosaccharides (6).
Forty-five 1-amino-1-deoxy sugars were generated from the unprotected parent saccharides in a simple one-step conversion (7). Five glycosylamines from commercial sources were added to this group. Glycosylamines are very attractive precursors for glycoconjugate chemistry. A primary amino function is introduced into the reducing terminus of a carbohydrate without affecting ring structure and conformation, allowing for the selective derivatization with a variety of reagents. When we started this study, glycosylamines had been used as substrates for various conjugation reactions (814). See Table 1 for a survey of preceding work on the acylation of 1-amino-1-deoxysugars. Recently, however, concerns were raised in the literature that the acylation of glycosylamines might be an unreliable reaction. Ex-
Technical Notes
Bioconjugate Chem., Vol. 6,No. 3, 1995 321
1!L 4.5 4.0 3.5 3.0 G/PPm Figure 2. Typical shift displacement of the anomeric proton of the reducing terminus of an oligosaccharide (lactose) upon amination and acylation. Top: lactose, the free reducing terminus displays alp stereoisomerism. Center: lactosylamine, amination shifts H-1 ca. -0.6 ppm upfield, yielding the /3 anomer exclusively. Bottom: lactosylamide, the anomeric proton is shifted ca. 0.9 ppm downfield, fully retaining its p configuration upon acylation.
5.0
plicitly, Manger et al. stated "that direct modification of the 1-amino function of a glycosylamine is both inefficient and difficult to control'' (15). This led Manger et al. to suggest 1-N-glycyloligosaccharides as superior intermediates for the formation of glycoconjugate probes. Using 1-amino-1-deoxy-@-lactose,we set out to investigate the glycosylamine reactivity toward common hetero- or homobifunctional conjugation reagents, such as N-succinimidyl S-acetylthioacetate, 2-iminothiolane, Nsuccinimidyl bromoacetate, disuccinimidyl suberate (DSS), and ethylene glycol bis(succinimidyl succinate) (EGS). Although unprotected glycosylamines contain both primary and secondary hydroxyl groups, we anticipated that the primary amino group of these sugars should undergo
mild, chemoselective acylation by the N-hydroxysuccinimidyl esters of these conjugating reagents. Conversions are followed by lH-NMR as exemplified in Figure 2. Water was found to be detrimental to the amidation reactions of glycosylamines. As mentioned in our previous communication (6), glycosylamines exhibit various stabilities toward water, with half-lives of minutes in aqueous media being quite common. This may explain the problems encountered by Manger et al. in attempting glycosyl amidations in aqueous carbonate buffer. Of the reagents employed in our study, DSS clearly gave the best reaction profile. With conjugation reagents other than DSS or EGS, typical side products included glycosylamine dimers and epimeric glycosylamides. The condensation reactions with DSS were carried out in the presence of a large excess of the homobifunctional reagent and yielded the unsymmetrical amido ester exclusively (Figure 1). The resulting products are versatile glycosylamide conjugation reagents because one N-hydroxysuccinimidyl ester is left intact, allowing for further derivatization. To demonstrate t h e , scope of this approach, seven glycosylamide-activeester conjugates were prepared from the glycosylamines of maltose, chitobiose, lactose, sialyllactose, LacNAc, GlcNAcGS03-, and GlcNA~6P03~(16). Subsequently, the glycosylamides were immobilized on amino-functionalized ethyleneglycol-grafted polystyrene resin (TentaGel). The extent of conjugate immobilization was probed by periodate oxidation of the carbohydrate resins: the aldehydes generated from the oxidation were labeled with digoxigenin hydrazide and detected immunochemically by means of a n antidigoxigenin-alkaline phosphatase antibody conjugate and a precipitative stain (17)(data not shown). In order to probe these sugar-bead conjugates in lectin binding assays, populations of each of the seven sugarbeads were treated with maackia amurensis agglutinin (MAA) separately. The assay involved incubation with digoxigenin-labeled lectin and detection with antidigoxigenin-alkaline phosphatase and a precipitative stain (18). The lectin is specific for NeuEiAc(a2-3)Gal and unambiguously recognized the epitope in the ligand sialyllactose (see Figure 3). For flow cytometry studies, the seven glycosylamide N-hydroxysuccinimidyl ester derivatives were separately conjugated to a 10 pm diameter, monodisperse polystyrene resin (5). The beads were then mixed and incubated with fluorescently labeled lectin (wheat germ agglutinin (WGA)-fluorescein or ricinus communis agglutinin (RCA 120)-fluorescein). Fluorescence-activated particle scanning (Figure 4) showed that the different lectins selectively bound fractions of the pool. These subpopulations were identified as chitobiosylamide or
Figure 3. Solid phase lectin binding assay. Beads were treated with digoxigenin-labeled maackia amurensis agglutinin (sialic acid specific lectin). Bound lectin was detected by means of an antidigoxigenidperoxidase conjugate and precipitative stain formation. The lectin selectively recognized its corresponding ligand Neu5Aca(2-3)Gal from a pool of several different sugar-bead conjugates. From left to right: TentaGel, maltosyl-TentaGel, sialyllactosyl-TentaGel, lactosyl-TentaGel.
322 Bioconjugate Chem., Vol. 6, No. 3, 1995
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WGA
I
~Fluorescence
RCA
loo
10' 10' Fluorescence
101
I
10'
Fluorescence
Figure 4. Flow cytometry histograms of sugar-bead conjugates incubated with fluorescently labeled lectin: wheat germ agglutinin (WGA, top histogram) and ricinus communis agglutinin (RCA 120, bottom histogram) each one selected out of a mixture of seven different bead-bound carbohydrates. The seven saccharide structures presented were as follows: maltose, 1; lactose, 2; sialyllactose, 3; LacNAc, 4; chitobiose, 5; GlcNAc6P0z2-,6; GlcNAc6S03-, 7. WGA recognizes its ligand chitobiose (no. 5) and RCA 120 selects lactose (no. 2) as inferred from assaying each sugar-bead conjugate individually (3D histogram arrays).
lactosylamide beads, respectively, as inferred from assaying the seven sugar-bead conjugates individually. These disaccharides are known ligands for WGA or RCA. These results indicate that fluorescence-activated particle sorting can be employed for the detection and isolation of bioactive members from a library of immobilized synthetic carbohydrate epitopes. We have subsequently extended this work to the generation of encoded glycopeptide libraries in which various glycosylamines are used to amidate the y-carboxyl of a glutamate residue embedded in a randomly synthesised pentapeptide (19). The amino acid sequence of the peptide is recorded by the parallel synthesis of a PCR-amplifiable oligonucleotide tag, a s previously described (5). As our method requires the use of milligram quantities of saccharide, the constituents of such a carbohydrate library would generally be based on various mono- to pentasaccharides rather than on less abundant complex multiantennary carbohydrates. LITERATURE CITED (1) Alper, J. (1993) Carbohydrates surge through clinical trials.
BIOITECHNOLOGY 11, 1093.
(2) Yednock, T. A., Stoolman, L. M., and Rosen, S. D. (1987) Phosphomannosyl-derivatized beads detect a receptor involved in lymphocyte homing. J . Cell Biology 104, 713-723. (3) Handa, K., Nudelman, E. D., Stroud, M. R., Shiozawa, T., and Hakomori, S. (1991) Selectin GMP-140 (CD62; PADGEM) binds to sialosyl-Lea and sialosyl-Lex, and sulfated glycans modulate this binding. Biochem. Biophys. Res. Commun. 181, 1223-1230. (4) de Bruijne-Admiraal, L. G., Modderman, P. W., Von dem Borne, A. E. G. K., and Sonnenberg, A. (1992) P-Selectin mediates Ca2+-dependentadhesion of activated platelets to many different types of leukocytes: Detection by flow cytometry. Blood 80, 134-142. (5) Needels, M. C., Jones, D. S., Tate, E. M., Heinkel, G. L., Kochersperger, L. M., Dower, W. J., Barrett, R. W., and Gallop, M. A. (1993) Generation and screening of a n oligonucleotide-encoded synthetic peptide library. Proc. Natl. Acad. Sci. U.S.A. 90, 10700-10704. (6) Vetter, D., and Gallop, M. A. (1995) Strategies for the synthesis and screening of glycoconjugates. 1. A library of glycosylamines. Bioconjugate Chem. 6, 316-318. (7) Likhosherstov, L. M., Novikova, 0. S., Derevitskaja, V. A,, and Kochetkov, N. K. (1986) A new simple synthesis of amino sugar P-D-glycosylamines. Carbohydrate Res. 146, C1-C5. ( 8 ) Kallin, E., Lonn, H., Norberg, and T., Elofsson, M. (1989) Derivatization procedures for reducing oligosaccharides, Part 3: Preparation of oligosaccharide glycosylamines, and their conversion into oligosaccharide-acrylamide copolymers. J . Carbohydr. Chem. 8, 597-611. (9) Otvos, L., Jr., Wroblewski, K., Kollat, E., Perczel, A., Hollosi, M., Fasman, G. D., Ertl, H. C. J., and Thurin, J. (1989) Coupling strategies in solid-phase synthesis of glycopeptides. Pept. Res. 2, 362-366. (10) Anisfeld, S. T., and Lansbury, P. T., Jr. (1990) A convergent approach to the chemical synthesis of asparagine-linked glycopeptides. J. Org. Chem. 55, 5560-5562. (11) Kallin, E., Lonn, H., Norberg, T., Sund, T., and Lundqvist, M. (1991) Derivatization procedures for reducing oligosaccharides, Part 4: Use of glycosylamines in a reversible derivatization of oligosaccharides with the 9-fluorenylmethoxycarbonyl group, and HPLC separations of the derivatives. J. Carbohydr. Chem. 10, 377-386. (12) Urge, L., Kollat, E., Hollosi, M., Laczko, I., Wroblewski, K., Thurin, J.,and Otvos, L, Jr. (1991) Solid-phase synthesis of glycopeptides: synthesis of Na-fluorenylmethoxycarbonyl L-asparagine N/?-glycosides. Tetrahedron Lett. 32,3445-3448. (13) Urge, L., Otvos, L., Jr., Lang, E., Wroblewski, K., Laczko, I., and Hollosi, M. (1992) Fmoc-protected, glycosylated asparagines potentially useful as reagents in the solid-phase synthesis of N-glycopeptides. Carbohydr. Res. 235, 83-93. (14) Cohen-Anisfeld, S. T., and Lansbury, P. T., Jr. (1993) A practical, convergent method for glycopeptide synthesis. J . Am. Chem. SOC. 115,10531-10537. (15) Manger, I. D., Rademacher, T. W., and Dwek, R. A. (1992) 1-N-Glycyl ,&oligosaccharide derivatives as stable intermediates for the formation of glycoconjugate probes. Biochemistry 31, 10724-10732. (16) Trivial names or abbreviations represent Glc(a1-4)Glc (maltose), GlcNAc(,81-4)GlcNAc (chitobiose), Gal(pl-4)Glc (lactose), Neu5Ac(a2-3)Gal(/31-4)Glc (sialyllactose), and Gal(/31-4)GlcNAc (LacNAc). (17) Haselbeck, A., and Hosel, W. (1990) Description and application of an immunological detection system for analyzing glycoproteins on blots. Glycoconjugate J. 7, 63-74. (18) Haselbeck, A., Schickaneder, E., von der Eltz, H., and Hosel, W. (1990) Structural characterization of glycoprotein carbohydrate chains by using digoxigenin-labeled lectins on blots. Anal. Biochem. 191, 25-30. (19) Vetter, D., Heinkel, G., Raab, R., Cheng, G., Schunk, C., Sugarman, J., and Gallop, M. A. (manuscript in preparation). BC9500150
