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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-3O-@-D-ga~actop~anosyl-@-~-g~actop~anoS~de (gal-@1,3-galNAc) to bovine serum albumin (BSA) was achieved by using 1,2-diethoxycyclobutene3,4-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-galNAcwith 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-aminophenyl 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 histochemical application of neoglycoproteins has underlined the value of this class of synthetic probes to localize endogenous 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 differentiation (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. It 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 efficiencyof the formed neoglycoprotein in proteincarbohydrate interaction. 1 Anticancer Drugs, Part 16. This work was supported by the Bundesminister fur Forschung und Technologie (FBrderkennzeichen 03189-52A9)and the Fonds der ChemischenIndustrie. 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.
1043-1802/9 112902-0148$02.50/0
In our concept (8) for the construction of selective anticancer 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 additionally diminish toxic effects of the used chemotherapeutica on the normal cell population. Trisaccharides or disaccharides such as gal-@l,&galNAc (2-acetamido-2-deoxy3 - O - @ - ~ g a l a d o p y r a n o s y l - @ - ~ g ~ ~ pmay ~ ~ othereside) 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 neoglycoprotein (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 di0 1991 American Chemical Society
Conjugatlon of olycosldes wlth Squarlc Acid Diester
ester) can be used as an coupling reagent for low molecular 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 molecular weight amine or a polymer like poly(L4ysine) via controlled formation of an 1,2-bisamideof 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 monitored 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-galNActo 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 proteincarbohydrate recognition, we carried out glycocytological and glycohistochemicalstudies in comparisonto the probe 11, derived by diazotization of the p-aminophenyl glycoside of gal-@l,&galNAc. The probe has already proven its capacity to serve as a potent lectin-localizing marker (14).
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 spectrophotometer Model Lambda 2. Mass spectra of monoamide 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 MikroanalytischesLaboratorium, GGttingen, FRG. Chromatographic 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 monitored by thin-layer chromatography using Machery, Nasilica gel TLC plates SILG/UV~M, and gel & Co. 0.25" spots were determined under ultraviolet light (A = 254 nm). The neoglycoproteins were purified with Pharmacia Sephadex G-25M columns (PD-10 columns) (diameter 1cm, 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 commerciallyavailable purity from Biomol (Ilvesheim, FRG). ABC kit was from Camon (Wiesbaden, FRG). The cell lines were obtained
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. Synthesisof 1-[(pMethoxyphenyl)amino]-2-ethoxycyclobutene-3,4-dione (3). To a stirred solution of p-anisidine (0.23 g, 1.87 "01) in dry ethanol (2 mL) was (0.55 mL, 3.73 added 1,2-diethoxycyclobutene-3,4-dione 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 (DMSOde) 6 1.42 (t, J = 6 Hz, CH3), 3.75 (8, OCH3), 4.75 (9, J = 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 (DMs0-d~) 6 15.6 (CH3),55.2 (OCHs),69.3 (CHzO), 114.2 (c-3aromatioc-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) [M - 2 CO]+, 163 (17) [M - 2 CO - CzH4]+, 134 (100) [M - 2 CO - C2H4 - OCH3]+, 122 (13), 107 (17), 92 (19), 77 (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-acetamido-5-(methoxycarbonyl)pentyl]amino]cyclobutene-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. Chromatographicpurification 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 (DMSOde) S 1.22-1.80 (m, 2'-H2,3'-Hzt4'-H2), 1.84 (8, NAc), 3.513.68 (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, 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); 13CNMR (DMSOde) 6 22.0 (CH3), 22.2 (c-3'), 30.1, 30.5 (c-2', c-4'), 43.3 (C-l'), 51.5 (CHsO),51.7 (C-5'),55.2 (phenyl-OCH3),114.4 (C-3aromatic,c-5aromatiA9 119.6 (C-2aromatio c&romatic), 132.1 (C'lmmatic), 155.2 (C-4mmatjC)p 163.6, 168.7, 169.2, 172.5, 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 dimethyl sulfoxide (DMSO)was added dropwise with vigorous stirring a 10-fold to 100-fold molar excess of 3. According to Table I model substance 3was 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 1mg/83 pL) control,
150 Bloconlcngate Chem., Voi. 2, No. 3, 1991
Tietze et ai.
Table I. Ligand Attachment of BSA and Yields of BSA Conjugates, Determined by UV Spectroscopy and in Different Experiments average no. average % no. molar yield of of ligands no. of exp excess of 3 attached to BSA BSA conjugates
1 2 3 4 5 6 7
4 4 4 4 3 3 3
0
10 20 40 60 80 100
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
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
Table 11. Binding of Neoglycoproteins 10. and l l bto 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 2 2' 3 4 4e 5 6 6e 7 7e 8 9 10 11 12 13 13O 14 14e
-10 +++I2 +++I1 +++I1 +++I1 +/2 +++I1
-10 +++/1-2 -10 -10 +++/2
-10
+++/2-3 +++I2 +++I1
+/ 1
+/1-2 +++I2 -10 +++I2 -10
0/20 p L of pure DMSO; 10/2.57; 20/5.15; 40/10.29; 60/ -10 15.36; 80/20.50; 100/25.65. The reaction mixtures were +++/2-3 -10 frequently shaken 3-4 h at room temperature. The -/Q +++I1 +++/1 mixtures were individually processed by chromatography +++/1-2 +++/1-2 on PD-10 columns. The samples were given onto the +++I1 +++I1 columns and these were eluated with buffer (pH 7.3) (5 -10 -10 X 0.5 mL each). The eluate was thrown away. Eluation -10 -10 was continued using 3-4 X 0.5 mL of buffer for each column +++I1 +++/1 +++/2-3 +++/2 to give an eluate containing the coupling product 7. For UV measurement the eluate of 7 was diluted with buffer Coupled by squaric acid. * Coupled by diazotization; synthesis [ l mL of eluate/buffer (pH 7.3),1/2] and the absorbance and results described elsewhere (14). e Cytoplasmic staining is was determined for each sample a t X = 270 and 310 nm. evaluated by the percentage of positive cella, grouped into the categories -, 0%; +, 0-5%; +, 5-20%; ++, 20-5055, and +++, 50Standard Curves and Calculation of the Molar 100% ,and by theintensityoftheindividualstainingreaction,grouped Extinction Coefficients. For BSA 6. The standard into the categories 0, no staining; 1,weak but significant staining; 2, curve was established at a wavelength of X = 270 nm with medium staining; 3, strong staining;and 4, very strongstaining.d The BSA concentrations of 1-10 X lo* M (in buffer pH 7.3). tumor cases were classified as invasiveductal carcinoma (1-111, nonThe calculated extinction coefficient was t270nm = 31 100 invasive intraductal carcinoma (12), tubular carcinoma (13), and invasive lobular carcinoma (14). a Denotes lymph node metastasis of M-1. the same primary tumor were the metastasis in the same patient For 1,2-Bisamide 5. The standard curves were meaoriginated from an apocrine carcinoma. sured at wavelengths of X = 270 and 310 nm in the initially aliquota onto 11separate PD-10 columns and product 10 chosen concentration range of 1-7 X M, because the was obtained in eluate portion of 2 mL per column. The concentration of the bisamide is 10-fold higher in the total volume of product solution was 22 mL. For the UV coupling product. The following extinction coefficients determination 1.2 mL of the product solution was diluted were calculated: f 2 7 h m = 10 500 M-' and e31hm = 25 300 with pH 7.3 buffer (product solution/buffer, 1/21 and the M-1. Because of 1,Qbisamide 5 was not easily soluble in absorbance was measured by X = 270 and 310 nm. buffer, it was first dissolved in methanol (1mL) and the The number of residues of squaricacid per BSA molecule solution was then diluted with buffer (pH 7.3). This was 15 f 2 and the yield of the BSA conjugate was 93%. standard solution was used. Synthesis of p[(4-Ethoxy-2,3-dioxocyclobut-l-en- Analysis of Neoglycoprotein-Binding Sites in Tuy1)aminolphenyl 2-Acetamido-2-deoxy-3-0-8-~-gal-mor Cells and Tissue Sections. The glycocytological and glycohistochemical procedure for visualizing binding actopyranosyl-8-D-galactopyranoside (9). To a stirred sites for the carbohydrate moiety of the biotinylated neosolution of p-aminophenyl2-acetamido-2-deoxy-3-0-/3-~glycoprotein and the control reactions for specificity were galactopyranosyl-/3-D-galactopyranoside (14) (27.4 mg, 58 described in detail elsewhere (14). Briefly, the cytospin mol) in dry ethanol (10 mL) a t room temperature was preparations of the human colon adenocarcinoma cell line (17rL, added dropwise 1,2-diethoxycyclobutene-3,4-dione COL0205, the human breast carcinoma cell line DU4475, 115 pmol). Stirring was continued for 10 h (TLC, established from a metastatic cutaneous nodule, and the chloroform/methanol/water, 13/8/2) and the mixture human erythroleukemia cell line HEL 92.1.7 as well as then concentrated under reduced pressure. Purification paraffin-embedded and formaldehyde-fixed sections of by chromatography (20 g of silica, chloroform/methanol/ malignant breast lesions were processed by a series of steps water, 13/8/2) afforded20a6mg(60%)of9asalightyeUow including blocking endogenous peroxidase activity and solid: 187-188 "C dec; thin-layer chromatography (silica, nonspecific protein-binding sites, subsequent incubations chloroform/methanol/water, 13/8/2) Rf = 0.5; lH NMR with the labeled probe at a concentration of 100 pg/mL (acetone-de/DzO) 6 1.33 (t, J = 7.0 Hz, OCH2CH3), 1.90 and with ABC reagents as well as enzyme substrates for (s, NAc), 3.20-4.02, 4.12-4.78 (2 m, 2-H, 3-H, 4-H, 5-H, development of the colored product to allow visualization 6-H2, 1'-H, 2'-H, 3'-H, 4'-H, 5'-H, 6'-H2), 4.68 (9, J = 7.0 of specific binding sites and with hemalum for counterHz, OCH&Ha), 5.04 (d, J = 8.5 Hz, 1-H),1.70 (AB system, staining. Binding capacity for the carbohydrate part in b~ = 6.98, 6~ = 7.22, JAB 8.0 Hz, Hwomatie); IR (KBr) 3422 the tumor cells in sections was semiquantified with respect (OH, NH), 2930 (CH2, CH3), 1806,1714,1618,1588 (C-0, to the cell percentage within the total cell compartment C=C), 1512 (NH), 1382 (CHsCO),832 (aromatic-H) cm-l; as well as with respect to the intensity, and these (methanol) 295 (log t 4.338) and 219 nm (log 6 UV ,A, parameters were grouped into categories, as detailed in 4.042). the footnote to Table 11. 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 RESULTS AND DISCUSSION mL) was added dropwise at room temperature a solution of 9 (14 mg, 2.34 X mmol) in DMSO (1.12 mL). After Neoglycoprotein Synthesis. To develop a generally stirring for 3.5 h the mixture was given in 11 X 0.6 mL usable method for the coupling of p-aminophenyl glyco0
Bloconlugate Chem., Vol. 2,
Conjugation of Glycosides with Squaric Acid Dlester
No. 3, 1991 151
Scheme I. Synthesis of the Model Substances OEt
MeO-GHb-NH
-
H$4 Lys- BSA
8 j4Me 1
Figure 1. UV spectrum of 1,2-bisamide5. 0
OL
tc
6
r
i
I I
Scheme 11. Coupling of the Disaccharide Gal-@ lP3-GalNActo BSA
OH
@
I
c
NHAc 0
,DOH
0
\
Lm
3w
nm
m
l
Figure 2. UV spectrum of BSA 6. Scheme 111. Neoglycoprotein Diazotization (14)
HO
11
Derived
by
0- p-CeHq-N=N*BSA OH
NHAc
0 0h
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-bisamide 5 that was needed as reference to determine the UV absorbance of one 18-bisamide was obtained via amidation of monoamide 3 withN-1-acetyl-L-lysinemethyl ester 4. Each product was checked by 'H NMR, 13CNMR, MS, IR, UV, and microanalysis. Coupling monoamide 3 to BSA 6 afforded product 7. Investigations to infer a possible structural rearrangement 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-10columns) and examination by UV spectroscopy. Scheme I1 summarizes 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. Neoglycoprotein 10 was obtained via amidation of monoamide 9 with the lysyl r-aminogroupsor 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 chromatographyon silica plates.
UV Spectroscopic Determination. In order to measure 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-bisamide 5 and of BSA. 1,2-Bisamidesof squaric acid usually have a broad absorption that clearly overlaps with the UV spectra of BSA. However, comparison of the two spectra (Figures 1and 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. Consequently, 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 bisamide 5 has a relative minimum. On the basis of the standard curves and calculation of the extinction coefficients 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-folddid 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 concomitantlyreduced. 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-specificbinding sites, (b) after coincubationwith 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 enzymesubstrates 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 evidencefor precipitation caused by the presence of dimethyl sulfoxide. Investigationof the stability of coupled product 7 showed that it is stable for more than 2 days in buffer solution (pH 7.3) at room temperature. Time-course experiments revealed that the optimal time in terms of coupling and yield is about 3-4 h. Determinationof the Number of Gal-@1,3-GalNAc Residues per BSA in 10. For the coupling of disaccharide 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
