140 Bloconjugate Chem. lQS1, 2, 148-153

Conjugation of p-Aminophenyl Glycosides with Squaric Acid Diester to a Carrier Protein and the Use of Neoglycoprotein in the Histochemical Detection of Lectinsl

Lutz F. Tietze,'J Claudia Schroter,? Sigrun Gabius,g Ulrich Brinck," Ada Goerlach-Graw,+ and Hans-Joachim Gabiust

Institut fur Organische Chemie, Tammannstrasse 2, D-3400 Gottingen, FRG, Max-Planck-Institut fur Experimentelle Medizin, Abteilung Chemie, Hermann-Rein-Strasse 3, D-3400 G6ttingen, FRG, Medizinische Universitatsklinik, Abteilung Hlimatologie-Onkologie, Robert-Koch-Strasse 40, D-3400-G6ttingen, FRG, and Zentrum Pathologie der Universitat, Robert-Koch-Strasse 40, D-3400 Gottingen, FRG. Received November 26, 1990

The coupling of p-aminophenyl2-acetamido-Z-deoxy-3- O-@-D-ga~actop~anosyl-@-~-g~actop~anoS~de (gal-@ 1,3-galNAc) to bovine serum albumin (BSA) was achieved by using 1,2-diethoxycyclobutene- 3,4-dione (squaric acid diester) as a new coupling reagent. Two selective consequential steps afforded the desired neoglycoprotein: reaction of the p-aminophenyl group of gal-@ 1,3-galNAc with squaric acid diester gave the corresponding squaric acid amide ester, which was transformed into the BSA conjugate by coupling with the lysyl c-amino groups of BSA through formation of a squaric acid 1,Bbisamide. The experimental conditions for the reactions and the optimization of average were performed by using p-anisidine as model substance, the methyl group substituting for the carbohydrate part of a p-ami- nophenyl glycoside. Neoglycoproteins have proven to be valuable tools for lectin detection. To evaluate the properties of this type of probe, the obtained neoglycoprotein with the histochemically crucial T-antigen structure was used for glycocytological and glycohistochemical studies. Three cultured human tumor cell lines and tissue sections from human breast carcinomas were chosen. Its efficiency was similar in comparison to measurements with a probe, derived by diazotization with the p-aminophenyl glycosides of gal-@ 1,3-galNAc and already shown to be a reliable marker for lectin localization in tissue sections and cultured cells.

INTRODUCTION The coupling of carbohydrates onto proteins is of great

interest, because the obtained synthetic sugar-protein conjugates, termed neoglycoproteins, can be used as tools with target specificity to cells. Especially the histochem- ical application of neoglycoproteins has underlined the value of this class of synthetic probes to localize endog- enous sugar-binding proteins (1,2). In general, the specific interaction of the carbohydrate part of glycoconjugates with receptors like lectins is supposed to be important for cell-cell interactions, growth regulation, and cell differ- entiation (3-5). Since endogenous lectins, too, have been detected in tumors, their histochemical mapping is of potential value for histopathology (6). Notably, the detection of endogenous lectins in tumors may reveal functional aspects of the alterations in abundance and structure of cellular glycoconjugates.

I t has already been emphasized that the structure of the linker between protein and carbohydrate can markedly influence the potency of the neoglycoprotein to serve as a detection device for tissue lectins (7). Thus, any expansion of the array of methods for the attachment of carbohydrates to proteins should concomitantly be tested for the efficiency of the formed neoglycoprotein in protein- carbohydrate interaction.

1 Anticancer Drugs, Part 16. This work was supported by the Bundesminister fur Forschung und Technologie (FBrderkennze- ichen 03189-52A9) and the Fonds der Chemischen Industrie. C.S. thanks the Fonds der Chemischen Industrie for a scholarship. Part 15, ref 13.

* To whom correspondence should be addressed. + Institut fur Organische Chemie. t Max-Planck-Institut fur Experimentelle Medizin. I Medizinische Universitiitaklinik. 11 Zentrum Pathologie der Universiut.

In our concept (8) for the construction of selective an- ticancer agents we use acetal glycosides which consist of carbohydrates, aldehydes, or ketones and alcohols and are in fact nontoxic prodrugs of the cytotoxic aldehydes or ketones. These compounds are acid labile and they are cleaved preferentially in an acidic environment with liberation of the cytotoxic principle. Since it has been proven that the tumor tissue is selectively acidified under hyperglycemic conditions due to an increase of "aerobic" glycolysis (9), these compounds may be advantageous in the treatment of cancer. Thus, recently we have shown that by applying acetal glycosides of aldophosphamide and ketophosphamide at pH 6.2 (pH of malignant tissue under hyperglycemia) the cloning efficiency of cells is decreased by the factor of >lo4 compared to that at pH 7.4 (pH of normal tissue) (10, 11). On the other hand, the carbohydrates employed in these compounds may enhance their concentration in the tumor tissue by binding to tumor-associated lectins on the cell surface. This would increase the pH-dependent selectivity and thus addition- ally diminish toxic effects of the used chemotherapeutica on the normal cell population. Trisaccharides or disac- charides such as gal-@ l,&galNAc (2-acetamido-2-deoxy- 3 - O - @ - ~ g a l a d o p y r a n o s y l - @ - ~ g ~ ~ p ~ ~ o s i d e ) may there- fore be used as the carbohydrate part in the acetal glycosides. When immobilized onto a carrier to give a neoglycoprotein, the carbohydrate moieties of the carrier will participate in directing these compounds to certain types of organ and also to the tumor, as indicated in a model for tumor-bearing mice with an array of neogly- coprotein (12). To underline the usefulness of this concept we synthesized a BSA conjugate with p-aminophenyl gal-@ 1,3-galNAc using a newly developed technique.

Our current study was prompted by the observation that l,Z-diethoxycyclobutene-3,4-dione (2; squaric acid di-

1043-1802/9 112902-0148$02.50/0 0 1991 American Chemical Society

Conjugatlon of olycosldes wlth Squarlc Acid Diester

ester) can be used as an coupling reagent for low molec- ular weight amines (13). First, a monoamide is formed between the ligand and the squaric diester. This derivative is then attached to an amino group of another low mo- lecular weight amine or a polymer like poly(L4ysine) via controlled formation of an 1,2-bisamide of the squaric acid. Because of the different high-wavelength absorptions (A,) of mono- and bisamides of squaric acid, the progress of the coupling reaction to poly(L-lysine) could be mon- itored by UV spectroscopy. By employing this scheme, amenable preparation of neoglycoproteins can thus be performed in a highly efficient and simple way.

In this paper we describe the use of this new method for the formation of neoglycoproteins with an 1,2-bisamide moiety as linker. For immobilization we have chosen the oncologically interesting disaccharide gal-@ 1,3-galNAc to address the issue whether binding sites can effectively be localized. The concentration of protein and carbohydrate was determined by UV spectroscopy. 1,QBisamide 10 could be formed in two selective steps: by the amidation of squaric diester 2 with the primaryp-aminophenyl group of glycoside 8 and then by amidation of the resulting monoamide 9 with the lysyl e-amino groups and perhaps the amino terminal group of bovine serum albumin (BSA) 6 to form 1,a-bisamide 10. In order to obtain information about the suitability of this linkage group within protein- carbohydrate recognition, we carried out glycocytological and glycohistochemical studies in comparison to the probe 11, derived by diazotization of the p-aminophenyl glyco- side of gal-@ l,&galNAc. The probe has already proven its capacity to serve as a potent lectin-localizing marker (14).

B(oC0nlugete Chem., Vol. 2, No. 3, 1991 140

from the American Type Culture Collection (Rockville, MD). Solvents and reagents were purified by standard methods.

Synthesis of 1-[ (pMethoxyphenyl)amino]-2-ethox- ycyclobutene-3,4-dione (3). To a stirred solution of p-anisidine (0.23 g, 1.87 "01) in dry ethanol (2 mL) was added 1,2-diethoxycyclobutene-3,4-dione (0.55 mL, 3.73 mmol) at room temperature. Stirring was continued for 2 h (TLC, silica, ethyl acetate/light petroleum, 1/21 and the mixture was afterward concentrated under reduced pressure. Crystallization of the residue from hot dry methanol produced light yellow needles (0.34 g, 74%): mp 159-160 "C; thin-layer chromatography (silica, ethyl acetate/lightpetroleum, 1/21. Rf = 0.1; lH NMR (DMSO-

6 Hz, CH20), 7.10 (2 d, AA'BB' system, 6~ = 6.90, 6~ = 7.30, JAB = 8.0 Hz, Hmmatic), 10.60 (s, br, NH); l3C NMR

de) 6 1.42 (t, J = 6 Hz, CH3), 3.75 (8, OCH3), 4.75 (9, J =

(DMs0-d~) 6 15.6 (CH3), 55.2 (OCHs), 69.3 (CHzO), 114.2 (c-3aromatio c-5aromatic), 121.3 (C-2aromatict C&omatic), 131.0 (C-laromatic), 156.2 (c-4,o,tic), 169.2, 177.3, 177.5, 177.6, 177.7, 183.2, 188.0 (cyclobutene-C); IR (KBr) 3270 (OH, NH), 3086 (aromatic-H), 2996, 2938 (CH2, CH3), 1798, 1696, 1616, 1582 ((2-0, C-C), 1068, 1026 (C-O), 828 (aromatic-H) cm-l; UV A, (methanol) 296 (log E 4.413) and 221 nm (log e 4.114); MS m/z 247 (89) [MI+, 191 (11)

(291, 64 (18). Anal. Calcd for C13H13N04: C, 63.15; H, 5.30. Found C, 63.07; H, 5.38.

Synthesis of 1-[ (pMethoxypheny1)aminol-2-[ [5-ac- etamido-5-(methoxycarbonyl)pentyl]amino]cy- clobutene-3,4-dione (5). To a stirred solution of 3 (0.15 g, 0.61 mmol) in dry ethanol (6 mL) a t room temperature was added triethylamine (1.5 mL) andN-l-acetyldysine methyl ester (0.19 g, 0.92 mmol). Stirring was continued for 90 min and the solvent removed under reduced pressure. Chromatographic purification of the residue (50 g of silica, tert-butyl methyl ether/light petroleum, 2/ 1) afforded 0.22 g (89% ) of bisamide 5 as a light yellow solid; mp 286290 "C dec; thin-layer chromatography (silica, chloroform/methanol, 10/1) Rf = 0.3; 'H NMR (DMSO-

3.68 (m, l'-H2), 3.62 (s, C02CH3), 3.75 (s, phenyl-OCH3), 4.18-4.32 (m, 5'-H), 7.15 (2 d, AA'BB'-system, SA = 6.94,

[M - 2 CO]+, 163 (17) [M - 2 CO - CzH4]+, 134 (100) [M - 2 CO - C2H4 - OCH3]+, 122 (13), 107 (17), 92 (19), 77

de) S 1.22-1.80 (m, 2'-H2,3'-Hzt4'-H2), 1.84 (8, NAc), 3.51-

6~ 7.36, JAB = 8.0 Hz, Hmomatic), 7.55 (s, br, N'H), 8.22 (d, J = 8.0 Hz, NHAc), 9.51 (5, NIH); 13C NMR (DMSO- de) 6 22.0 (CH3), 22.2 (c-3'), 30.1, 30.5 (c-2', c-4'), 43.3

(C-3aromatic, c-5aromatiA9 119.6 (C-2aromatio c&romatic), 132.1 (C-l'), 51.5 (CHsO), 51.7 (C-5'),55.2 (phenyl-OCH3), 114.4

180.3,183.4 (cyclobutene-C, C=O); IR (KBr) 3280 (NH), 3056 (aromatic-H), 2952 (CH2, CHs), 1794, 1740, 1660, 1612 (C=O), C=C), 1586,1544,1516 (aromatic-H, NH), 1374 (CH&O), 828 (aromatic-H) cm-'; UV Am, (methanol) 229 (log c 4.384) and 303 nm (log E 4.470); MS m/z 403 (6) [MI+, 175 (32), 173 (26), 138 (15),84 (62), 73 (29), 44 (63). Anal. Calcd for C~H2,&06: C, 59.54; H, 6.25. Found C, 59.68; H, 6.23.

Coupling of 3 to BSA. The reactions were performed in Eppendorf tubes. To a solution of BSA (0.25 mL of 3.3 mg/mL = 5 X 10-5 M in buffer pH 9.0) dissolved in dim- ethyl sulfoxide (DMSO) was added dropwise with vigorous stirring a 10-fold to 100-fold molar excess of 3. According to Table I model substance 3 was diluted in a concentration of 1 mg/83 pL in DMSO, and to achieve the respective molar excess the following microliters of this solution were added to 0.25 mL of BSA solution: (fold molar excess/ microliters of DMSO solution with 1 mg/83 pL) control,

(C'lmmatic), 155.2 (C-4mmatjC)p 163.6, 168.7, 169.2, 172.5,

EXPERIMENTAL PROCEDURES

General Procedures. lH and 13C NMR spectra of chemical compounds were recorded on a Varian FT-BOA for monoamide 3 and Varian XL 200 for 1,2-bisamide 5 and carbohydrate 9 in either D M s 0 - d ~ or acetone-de/ D2O as solvents with TMS as internal standard. Melting points were measured on a Reichert Heiztischmikroskop Model Kofler, and the data are corrected. Infrared spectra were recorded on a Bruker infrared spectrometer Model IFS 25. UV spectra were recorded on a Perkin-Elmer spec- trophotometer Model Lambda 2. Mass spectra of monoa- mide 3 and 1,a-bisamide 5 were obtained on Varian MAT 311 mass spectrometer by using an electronic ionization energy of 70 eV. Microanalyses were performed by the Mikroanalytisches Laboratorium, GGttingen, FRG. Chro- matographic separation of product 5 and 9 was carried out by "flash chromatography" with Machery, Nagel & Co. 0.032-0.063 silica gel. Chemical reactions were mon- itored by thin-layer chromatography using Machery, Na- gel & Co. 0.25" silica gel TLC plates SILG/UV~M, and spots were determined under ultraviolet light (A = 254 nm). The neoglycoproteins were purified with Pharma- cia Sephadex G-25M columns (PD-10 columns) (diameter 1 cm, high 5 cm, bed volume 9.1 mL). Before using a new PD-10 column, it was washed with a 0.5% BSA solution (1-2 mL), and after use it was regenerated by washing with phosphate buffer, pH 7.3 (50 mL). Bidistilled water was used to prepare the phosphate buffer with pH 7.3. The buffer with pH 9.0, which was used for the coupling reaction, was bought from Merck, Puffer-Titrisol, Art. 9889 (Darmstadt, FRG). The carrier protein bovine serum albumin (BSA) was of the highest commercially available purity from Biomol (Ilvesheim, FRG). ABC kit was from Camon (Wiesbaden, FRG). The cell lines were obtained

150 Bloconlcngate Chem., Voi. 2, No. 3, 1991

Table I. Ligand Attachment of BSA and Yields of BSA Conjugates, Determined by UV Spectroscopy and in Different Experiments

Tietze et ai.

no. 1 2 3 4 5 6 7

- no.

of exp 4 4 4 4 3 3 3

molar excess of 3

0 10 20 40 60 80

100

average no. of ligands

attached to BSA 0.2 f 0.1 7.3 f 1.3

12.8 f 1.5 20.0 f 2.2 20.7 f 2.5 22.0 f 2.5 24.7 f 2.6

average % yield of

BSA conjugates 90.5 f 7.4 77.5 f 6.9 63.8 f 9.9 57.3 f 9.0 60.3 f 2.9 56.0 f 9.3 52.0 f 5.7

0/20 pL of pure DMSO; 10/2.57; 20/5.15; 40/10.29; 60/ 15.36; 80/20.50; 100/25.65. The reaction mixtures were frequently shaken 3-4 h at room temperature. The mixtures were individually processed by chromatography on PD-10 columns. The samples were given onto the columns and these were eluated with buffer (pH 7.3) (5 X 0.5 mL each). The eluate was thrown away. Eluation was continued using 3-4 X 0.5 mL of buffer for each column to give an eluate containing the coupling product 7. For UV measurement the eluate of 7 was diluted with buffer [ l mL of eluate/buffer (pH 7.3),1/2] and the absorbance was determined for each sample a t X = 270 and 310 nm.

Standard Curves and Calculation of the Molar Extinction Coefficients. For BSA 6. The standard curve was established at a wavelength of X = 270 nm with BSA concentrations of 1-10 X lo* M (in buffer pH 7.3). The calculated extinction coefficient was t270nm = 31 100 M-1.

For 1,2-Bisamide 5. The standard curves were mea- sured at wavelengths of X = 270 and 310 nm in the initially chosen concentration range of 1-7 X M, because the concentration of the bisamide is 10-fold higher in the coupling product. The following extinction coefficients were calculated: f 2 7 h m = 10 500 M-' and e31hm = 25 300 M-1. Because of 1,Qbisamide 5 was not easily soluble in buffer, it was first dissolved in methanol (1 mL) and the solution was then diluted with buffer (pH 7.3). This standard solution was used.

Synthesis of p[ (4-Ethoxy-2,3-dioxocyclobut-l-en- y1)aminolphenyl 2-Acetamido-2-deoxy-3-0-8-~-gal- actopyranosyl-8-D-galactopyranoside (9). To a stirred solution of p-aminophenyl2-acetamido-2-deoxy-3-0-/3-~- galactopyranosyl-/3-D-galactopyranoside (14) (27.4 mg, 58 mol) in dry ethanol (10 mL) a t room temperature was added dropwise 1,2-diethoxycyclobutene-3,4-dione (17 rL, 115 pmol). Stirring was continued for 10 h (TLC, chloroform/methanol/water, 13/8/2) and the mixture then concentrated under reduced pressure. Purification by chromatography (20 g of silica, chloroform/methanol/ water, 13/8/2) afforded20a6mg(60%) of9asalightyeUow solid: 187-188 "C dec; thin-layer chromatography (silica, chloroform/methanol/water, 13/8/2) Rf = 0.5; lH NMR (acetone-de/DzO) 6 1.33 (t, J = 7.0 Hz, OCH2CH3), 1.90 (s, NAc), 3.20-4.02, 4.12-4.78 (2 m, 2-H, 3-H, 4-H, 5-H, 6-H2, 1'-H, 2'-H, 3'-H, 4'-H, 5'-H, 6'-H2), 4.68 (9, J = 7.0 Hz, OCH&Ha), 5.04 (d, J = 8.5 Hz, 1-H), 1.70 (AB system, b~ = 6.98, 6~ = 7.22, JAB 8.0 Hz, Hwomatie); IR (KBr) 3422 (OH, NH), 2930 (CH2, CH3), 1806,1714,1618,1588 (C-0, C=C), 1512 (NH), 1382 (CHsCO), 832 (aromatic-H) cm-l; UV A,, (methanol) 295 (log t 4.338) and 219 nm (log 6

4.042). Coupling of 9 to BSA. To a stirred solution of BSA

(18.2 mg, 2.75 X mmol) in buffer solution (pH 9.0,5.5 mL) was added dropwise at room temperature a solution of 9 (14 mg, 2.34 X mmol) in DMSO (1.12 mL). After stirring for 3.5 h the mixture was given in 11 X 0.6 mL

Table 11. Binding of Neoglycoproteins 10. and llb to Tumor &lle in Sections of Malignant Breast Lesions

cytoplasmic staining cased gal-j3 Ib-galNAc-BSA 10 gal-j3 1,3-galNAc-BSA 11

1 -10 -10

2' +++I1 +++I2 3 +++I1 +++I1 4 +++I1 +/ 1

+/1-2 +++I2

4e +/2 5 +++I1

-10 +++I2

6 -10 6e +++/1-2 7 -10 -10 7e -10 -10

9 -10 -/Q 10 +++I1 +++/1 11 +++/1-2 +++/1-2 12 +++I1 +++I1 13 -10 -10

-10 +++/1

13O -10 14 +++I1 1 4e +++/2-3 +++/2

2 +++I2 +++/2-3

8 +++/2 +++/2-3

0 Coupled by squaric acid. * Coupled by diazotization; synthesis and results described elsewhere (14). e Cytoplasmic staining is evaluated by the percentage of positive cella, grouped into the categories -, 0%; +, 0-5%; +, 5-20%; ++, 20-5055, and +++, 50- 100% ,and by theintensityoftheindividualstainingreaction,grouped into the categories 0, no staining; 1, weak but significant staining; 2, medium staining; 3, strong staining; and 4, very strongstaining.d The tumor cases were classified as invasive ductal carcinoma (1-111, non- invasive intraductal carcinoma (12), tubular carcinoma (13), and in- vasive lobular carcinoma (14). a Denotes lymph node metastasis of the same primary tumor were the metastasis in the same patient originated from an apocrine carcinoma. aliquota onto 11 separate PD-10 columns and product 10 was obtained in eluate portion of 2 mL per column. The total volume of product solution was 22 mL. For the UV determination 1.2 mL of the product solution was diluted with pH 7.3 buffer (product solution/buffer, 1/21 and the absorbance was measured by X = 270 and 310 nm.

The number of residues of squaric acid per BSA molecule was 15 f 2 and the yield of the BSA conjugate was 93%.

Analysis of Neoglycoprotein-Binding Sites in Tu- mor Cells and Tissue Sections. The glycocytological and glycohistochemical procedure for visualizing binding sites for the carbohydrate moiety of the biotinylated neo- glycoprotein and the control reactions for specificity were described in detail elsewhere (14). Briefly, the cytospin preparations of the human colon adenocarcinoma cell line COL0205, the human breast carcinoma cell line DU4475, established from a metastatic cutaneous nodule, and the human erythroleukemia cell line HEL 92.1.7 as well as paraffin-embedded and formaldehyde-fixed sections of malignant breast lesions were processed by a series of steps including blocking endogenous peroxidase activity and nonspecific protein-binding sites, subsequent incubations with the labeled probe at a concentration of 100 pg/mL and with ABC reagents as well as enzyme substrates for development of the colored product to allow visualization of specific binding sites and with hemalum for counter- staining. Binding capacity for the carbohydrate part in the tumor cells in sections was semiquantified with respect to the cell percentage within the total cell compartment as well as with respect to the intensity, and these parameters were grouped into categories, as detailed in the footnote to Table 11.

RESULTS AND DISCUSSION Neoglycoprotein Synthesis. To develop a generally

usable method for the coupling of p-aminophenyl glyco-

Conjugation of Glycosides with Squaric Acid Dlester

Scheme I. Synthesis of the Model Substances OEt MeO-GHb-NH

H$4 - Lys- BSA

8 j 4 M e

1

Scheme 11. Coupling of the Disaccharide Gal-@ lP3-GalNAc to BSA

OH NHAc

Bloconlugate Chem., Vol. 2, No. 3, 1991 151

@ I ,DOH

0

0 h 0

sides to BSA we performed initial studies with model substance 1 (see Scheme I) in which the methyl group substitutes the carbohydrate moieties.

Formation of monoamide 3 was achieved by reaction of p-anisidine 1 with squaric acid diester 2. The 1,a-bisa- mide 5 that was needed as reference to determine the UV absorbance of one 18-bisamide was obtained via amida- tion of monoamide 3 withN-1-acetyl-L-lysine methyl ester 4. Each product was checked by 'H NMR, 13C NMR, MS, IR, UV, and microanalysis. Coupling monoamide 3 to BSA 6 afforded product 7.

Investigations to infer a possible structural rearrange- ment of 1,Zbisamide 5 to a corresponding 1,3-bisamide raised evidence that 5 is stable. In solution [methanol/ buffer (pH 7.3)) 1/91 no reaction was seen during 10 days despite heating to 50 "C twice for 4 h during this time, followed by routinely applied purification (PD-10 columns) and examination by UV spectroscopy. Scheme I1 sum- marizes the analogous synthetic route that has been used to prepare the T-antigen-bearing neoglycoprotein 10. Reaction of the p-aminophenyl group of disaccharide 8 (14) with squaric diester 2 afforded monoamide 9, which was checked by lH NMR, IR, and UV spectroscopy. Neo- glycoprotein 10 was obtained via amidation of monoam- ide 9 with the lysyl r-aminogroups or terminal amino group of BSA. All reactions, except the coupling with BSA were followed, and the purity of the various compounds was determined by thin-layer chromatography on silica plates.

Figure 1. UV spectrum of 1,2-bisamide 5.

0 6 r i t I O L c I

c \ 0 L l m 3w m nm

Figure 2. UV spectrum of BSA 6. Scheme 111. Neoglycoprotein 11 Derived by Diazotization (14)

0- p-CeHq-N=N*BSA HO OH NHAc

0 UV Spectroscopic Determination. In order to mea-

sure the number of squaric acid residues per BSA molecule in BSA conjugates 7 and 10 we used UV-difference spectroscopy.

Figures 1 and 2 illustrate the UV spectra of 1,a-bisa- mide 5 and of BSA. 1,2-Bisamides of squaric acid usually have a broad absorption that clearly overlaps with the UV spectra of BSA. However, comparison of the two spectra (Figures 1 and 2) reveals that BSA has nearly no absorption at X = 310 nm, whereas bisamide 5 shows a strong absorption at this wavelength, which is close to ita A, (306.8 nm). Thus, in the coupled products 7 and 10 the absorption at X = 310 nm can be attributed exclusively to the attached squaric acid bisamide moieties. Conse- quently, this wavelength was used for the assessment of the concentration of squaric acid bisamide moieties in the probe. The determination of the concentration of BSA was carried out at a wavelength of 270 nm, where BSA (A,,,= = 280 nm) has a strong absorption, whereas bisa- mide 5 has a relative minimum. On the basis of the standard curves and calculation of the extinction coeffi- cients the concentration of BSA and the molar ratios of ligand to carrier was accessible. In order to optimize the ligand attachment of BSA, the influence of the ratio protein/squaric acid of protein (BSA) and squaric acid amide ester 3 were respectively quantitated (Table I).

With a 30-40-fold molar excess of monoamide 3 a covalent attachment of almost 20 moieties was achieved. Increases of the molar excess of 3 to about 50-fold or even 100-fold did not result in a further increase of squaric acid bisamide moieties on BSA. When the number of squaric acid residues per BSA molecule increased, the yield of the BSA conjugate was concomitantly reduced. We suppose that the reason for this decrease can be the reduced

Figure 3. Light micrographs of sections of an invasive ductal carcinoma, referred to as case 2 in Table 11, after incubation with 50 pg/mL biotinylated peanut lectin to localize presence of T-antigen in the tissue: (a) after incubation with 100 pg/mL neoglycoprotein 10 to localize carbohydrate-specific binding sites, (b) after coincubation with a 50-fold excess of label-free neoglycoprotein 10 in relation to the marker to show specificity of protein-carbohydrate interaction, and (c) after incubation with ABC reagents and enzyme substrates and hemalum counterstaining (14); Original magnification X380 (figure reduced 90% for publication).

solubility of the protein conjugate due to the hydrophobic nature of the squaric acid bisamide moieties on the surface of the protein. Control experiments with pure BSA proved that the solubility of BSA was not affected by the organic solvent. Thus, there is no evidence for precipitation caused by the presence of dimethyl sulfoxide.

Investigation of the stability of coupled product 7 showed that it is stable for more than 2 days in buffer solution (pH 7.3) a t room temperature. Time-course experiments revealed that the optimal time in terms of coupling and yield is about 3-4 h.

Determination of the Number of Gal-@ 1,3-GalNAc Residues per BSA in 10. For the coupling of disaccha- ride 9 with BSA a &-fold molar excess was used. The yield of BSA conjugate 10 was 93% and the ratio of carbohydrate (squaric acid bisamide) attachment was 15.3 f <1 units per BSA molecule. Thus the ligand attachment of BSA in 10 is lower than that in 7 and the yield much better. Apparently, the free reactive amino functions in BSA will be blocked to a higher extent by the polar bulky carbohydrate moiety in 10 than by the small nonpolar

methyl group in 7. It can also be assumed that the conformation of the BSA conjugates 7 and 10 may be quite different due to hydrogen bonding of the hydroxy groups in the carbohydrate moiety to the peptide functionalities. The better yield in the formation of 10 is in accordance with our given explanation. Thus the solubility of 10 should be much better compared to that of 7 due to the hydrophilic character of the carbohydrate moieties.

Detection of T-Antigen-Specific Receptors by La- beled Neoglycoprotein 10 and Comparison to the Results with Neoglycoprotein 11 (See Scheme 111). Most of the cells of the two carcinoma cell lines and the erythroleukemia line were found to react positively. A similar result was seen after processing paraffin-embedded tissue sections from malignant breast lesions. A repre- sentative result is illustrated in Figure 3. The detailed pattern of cytoplasmic staining with respect to percentage of positive tumor cells in the total cell population as well as staining intensity for each case is given in Table 11.

No pronounced difference was seen in the extent of staining between this neoglycoprotein and the neoglyco-

Conjugation of Glycosides with Squarlc Acid Dlester

protein obtained product of diazotization with the p-ami- nophenyl glycoside of gal-j3 1,3-galNAc. Semiquantitative analysis of these two markers in tumor cells of tissue sections from malignant breast lesions revealed no ap- parent correlation to metastasis formation. This appli- cation substantiated that this new type of linker will lead to an efficiently applicable probe for reliable lectin detection. Indeed, there are many advantages of this new linker compared to other methods; thus, (i) two amino groups can be coupled without interference by esters, hy- droxy groups, and nearly all other functionalities in the substrates; (ii) the use of the linker is simple, requires no harsh conditions, and gives excellent yields; moreover, (iii) the coupling can be performed in slightly basic media; and (iv) finally the quantity of the coupled groups can be determined by UV spectroscopy.

Bioconjugate Chem., Vol. 2, No. 3, 1991 153

(7) Gabius,H.-J., Gabius, S.,Brinck, U., andSchauer, A. (1990) Endogenous lectins with specificity to 8-galactosides and a- or fl-N-acetyl-galact”hides in human breast cancer. Pathol. Res. Pract. 186, 597-607.

(8 ) Tietze, L. F. (1990) New concepts for the development of selective ant icancer drugs. Molecular Aspec ts of Chemotherapy (E. Borowski, and D. Shugar, Eds.) pp 55-70, Pergamon Press, New York.

(9) Review: Tannock, I. F., and Rotin, D. (1989) Cancer Res. 49,

(10) Tietze, L. F., Neumann, M., Fischer, R., Mollers, T., Gliisen- kamp, K.-H., Rajewsky, M. F., and Jahde, E. (1989) Proton- mediated liberation of aldophosphamide from a non-toxic prodrug. A strategy for tumor-selective activation of cyto- cidal drugs. Cancer Res. 49, 4179-4184.

(11) Tietze, L. F., Beller, M., Fischer, Logers, M., Jhhde, E., Gliisenkamp, K.-H., Rajewsky, M. F. (1990) Development of tailor-made cytostatics activable by acid-catalyzed hydrolysis for selective tumor therapy. Angew. Chem. Znt. Ed. Engl. 29,

(12) Kojima, S., Gabius, H.-J. (1988) Biodistribution of neo- glycoproteins in mice bearing solid Ehrlich tumor. J. Cancer Res. Clin. Oncol. 114, 468-472.

(13) Tietze, L. F., Arlt, M., Beller, M., Gliisenkamp, K.-H., J h d e , E., Rajewsky, M. F. (1991) Squaric acid diethyl ester: a new coupling reagent for the formation of drug biopolymer con- jugates. Synthesis of squaric acid ester amides and squaric acid diamides. Chem. Ber. 124, 1215-1221.

(14) Gabius, H.-J., Schroter, C., Gabius, S., Brinck, U., andTietze, L. F. (1990) Binding of T-antigen-bearing neoglycoprotein and peanut agglutinin to cultured tumor cells and breast carci- nomas. J. Histochem. Cytochem. 38, 1625-1631.

Registry No. 1,104-94-9; 2,5231-87-8;3,132751-38-3; 4,6072- 02-2; 5,132751-39-4; 9,132751-40-7; p-aminophenyl 2-acetamido- 2-deoxy-3-0-~-~-galact~py~anosy~-~-~-galactopyranceide, 131339- 22-5; 1,2-diethoxycyclobutene-3,4-dione, 5231-87-8.

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(2) Gabius, Ha-J., and Bardosi, A. (1991) Neoglycoproteins as tools in glycohistochemistry. Prog. Histochem. Cytochem. 22,

(3) Gabius, H.-J. (1991) Detection and functions of mammalian lectin with emphasis on membrane lectins. Biochim. Bio-

(4) Sharon, N., and Lis, H. (1989) Lectins as cell recognition molecules. Science 246, 227-234.

(5) Barondes, S. H. (1981) Lectins: their multiple endogenous cellular functions. Annu. Rev. Biochem. 50, 207-231.

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