Oriented and covalent immobilization of target molecules to solid

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Bioconjugate Chem. 1993, 4, 528-536

Oriented and Covalent Immobilization of Target Molecules to Solid Supports: Synthesis and Application of a Light-Activatable and Thiol-Reactive Cross-Linking Reagent? Andr6 Collioud, Jean-FranCois Clkmence, Michael Sanger, and Hans Sigrist’ Institute of Biochemistry, University of Berne, Freiestrasse 3, CH-3012 Berne, Switzerland. Received June 27, 1993’

Light-dependent oriented and covalent immobilizationof target moleculeshas been achieved by combining two modification procedures: light-dependent coupling of target molecules to inert surfaces and thiolselective reactions occurring at macromolecule or substrate surfaces. For immobilization purposes the heterobifunctional reagent N- [m- 13-(trifluoromethyl)diazirin-3-yll phenyl] -4-maleimidobutyramide was synthesized and chemically characterized. The photosensitivity of the carbene-generating reagent and its reactivity toward thiols were ascertained. Light-induced cross-linking properties of the reagent were documented (i) by reacting first the maleimide function with a thiolated surface, followed by carbene insertion into applied target molecules, (ii) by photochemical coupling of the reagent to an inert support followed by thermochemical reactions with thiol functions, and (iii) by thermochemical modification of target molecules prior to carbene-mediated insertion into surface materials. Procedures mentioned led to light-dependent covalent immobilization of target molecules including amino acids, a synthetic peptide, and antibody-derived F(ab’) fragments. Topically selective, light-dependent immobilization was attained with the bifunctional reagent by irradiation of coated surfaces through patterned masks. Glass and polystyrene served as substrates. Molecular orientation is asserted by inherently available or selectively introduced terminal thiol functions in F(ab’) fragments and synthetic polypeptides, respectively.

INTRODUCTION Covalent immobilization of biomolecules to various substrate surfaces has yielded biosurfaces applicable for molecular analysis (Bhatia et al., 1992),separation (Brocklehurst et al.; 1984;Berry et al., 1991;O’Shannessy, 19901, targeting (Martinetal., 1982),synthesis (Fodor etal., 19921, and diagnosis (Ljungquist et al., 1989). Most procedures used to date for target molecule immobilization are based on thermochemical reactions: covalent bonds are formed between surface-exposed amino, carboxyl, or thiol functions or generated aldehydes and suitably modified solid supports, membrane surfaces, or macromolecular aggregates. For optimal performance of biosurfaces, full retention of biological activities (Fassina, 1992) and molecular orientation is attempted (Wimalasena and Wilson, 1991; Matson and Little, 1988). Approaches mentioned require functionalization of either the target molecules, the substrate surface, or both (Larsson et al., 1987). In addition, thermochemical reaction conditions may affect biological activities and multiple-site attachment of individual molecules may occur. To overcome drawbacks of thermochemical reactions, noninvasive coupling procedures and mild reaction conditions are necessary to retain native biomolecule structures. Brunner

* Abbreviations used: TRIMID, 3-(trifluoromethyl)-3-(misothiocyanopheny1)diazirine; FEP-OH, Hydroxylated fluori-

nated ethylenepropylene;PPO, 2,5-diphenyloxazole; POPOP, 2,2’-p-phenylenebis(5-phenyloxazole);HEPES, 442-hydroxyethy1)piperazine-l-ethanesulfonicacid EDTA, ethylenediaminetetraacetic acid; PSA, prostate-specific antigen; DTNB, 53’dithiobis(2-nitrobenzoicacid);TEA, triethylamine; TLC, thinlayer chromatography; DMF, dimethylformamide; BSA bovine serum albumin;MAD, N- [m-[3-(trifluoromethyl)diazirin-3-yl] phenyl]-4-maleimidobutyramide;CDPGYIGSR, Cys-Asp-ProGly-Tyr-Ile-Gly-Ser-Arg-NH2. Abstract published in Adoance ACS Abstracts, November 1, 1993. 1043-1 802/93/2904-0528$04.00/0

and Richards (1980) and others (Bayley, 1983; Brunner, 1989) have introduced (trifluoromethy1)aryl diazirines as hydrophobic (membrane) protein labels. On the basis of these investigations, new routes of biomolecule immobilization have been initiated (Sigrist et al., 1992; Slinger et al., 1992). Since photoactivatable reagents are topically addressable, photoimmobilization of biomolecules became a subject of intense research toward the development of resistant materials, biosensors, and biomaterials (Fodor et al., 1992; Rosznyai et al., 1992; Yan et al., 1993). For topically addressable and oriented biomolecule immobilization, we designed a linking agent which fulfills the requirements for molecular orientation and noninvasive (light-dependent) activation. The reagent, N - [m[3-(trifluoromethyl)diazirin-3-yllphenyll-4-maleimidobutyramide (Figure lA), carries a maleimide function for thermochemical modification of cysteine thiols (Bhatia et al., 1989)and an aryldiazirine function for light-dependent, carbene-mediated binding to solid supports, biomimetic devices, or biological surfaces. Activation of a carbenegenerating aryldiazirine with a 350-nm light source has been shown to lead to covalent coupling of proteins, enzymes, immunoreagents, carbohydrates, and nucleic acids (Sigrist et al., 1992; Klingler et al., 1991; Clbmence, unpublished) under conditions such that biological activity is not impaired (Gao et al., unpublished; Kung et al., unpublished). In this study the synthesis, chemical characterization, and reactivity of the new heterobifunctional cross-linking reagent are described. Selected applications of this crosslinker are depicted in Figure 1B-D, all of which lead to thermochemical/photochemical immobilization of target molecules. Basic immobilization procedures are elaborated with amino acids and a terminally thiolated peptide. With regard to future applications in diagnostic procedures, the versatility and applicability of the new bifunctional cross-linking agent are investigated with immu0 1993 American Chemical Society

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Figure 1. Chemical structure and reaction routes of MAD-mediated target-molecule immobilization: (A) Structure of MAD (Nphenyl amide); (B) reaction route 1,thermochemical coupling of MAD (4-maleimidobutyryl)-N-[m-[ 3-(trifluoromethyl)diazirin-3-y1] to thiolated surfaces is followed by photochemical target molecule coupling; (C) reaction route 2, photochemical coupling of the reagent to an inert surface is followed by thermochemical reactions with thiol functions; (D) reaction route 3, thermochemical modification of target molecules prior to carbene-mediated insertion to surface materials. T: target molecule.

noreagents. F(ab') fragmentsof monoclonalanti-prostatespecific-antigen antibodies (anti-PSA antibodies) are covalently attached through photoimmoblization on polystyrene. EXPERIMENTAL PROCEDURES

Materials. 'H-NMR spectra were recorded on a Varian EM 360L and absorption measurements were carried out on an Uvicon 810 or a Beckmann DU 64 spectrophotometer. IR spectra were recorded on a Perkin-Elmer 782 infrared spectrophotometer. Melting points were determined with a Buchi 510 instrument and a CH 7A Varian MAT was used for mass spectroscopy. Radioactive disintegrations were measured in 5 mL of scintillation fluid (1080 mL of toluene, 5.4 g of PPO, 0.2 g of POPOP, 920 mL of Triton X-100, and 40 mL of acetic acid) on a Betamatic V liquid scintillation counter. Thin-layer chromatography (TLC) was performed with silica gel 60 F ~ plates M (Merck). Silica gel 60 (particle size 0.040-0.063 mm, 230-400 mesh ASTM) for flash chromatography was from Merck. Light sources for irradiation were either a HBO 350 mercury lamp from Osram (filtered with a Schott WG 320 and a 1-cm layer of saturated CuSO4 in H20; transmission bandwidth 320-550 nm) or the Stratalinker UV 2400 (Stratagene) with five 365-nm bulbs. The SVX 1530 mercury lamp power supply (constant power output of 200 W) and the MU4 timer were from Mueller Electronics (Moosinning, Germany). Light intensities were measured with a Suss UV intensity meter Model 1000 (Hilpert AG). HPLC was performed with a widepore butyl (C4)Bakerbond standard column (particle size, 5 pm; dimensions 4.6 X 250 mm; Baker Inc., Phillipsburg, NJ) on a Hewlett-Packard 1090 liquid chromatograph equipped with a Perkin-Elmer LC 75 detector (280 nm) and a Hewlett-Packard 3396A integrator. The UVmax ELISA microplate reader was from Molecular Device. FEP-OH membranes (3 cm in diameter) were a gift of Prof. Aebischer. The photomask with 300-pm slits sep-

arated by 500-pm spacings was purchased from Towne Laboratories Inc. Somerville, NJ. Bovine serum albumin (BSA)was purchased from Fluka. F(ab')2 fragments of monoclonalanti-PSA antibodieswere provided by Hoffmann-La Roche, Basel, Switzerland. 3-(Trifluoromethy1)-3-(m-aminophenyl)diazirine was synthesized according to Dolder et al. (1990). The peptide Cys-Asp-Pro-Gly-Tyr-Ile-Gly-Ser-Arg-NH, (CDPGYIGSR) was from Sigma. 4-Maleimidobutyric acid, ethylchloroformate (purum), TEA (puriss), DMF, (3-mercaptopropy1)trimethoxysilane(purum),and DTNB were from Fluka. Sodium cyanoborohydride and dithiothreithol (DTT) were from Merck. [14C]Tyrosine(468 mCi/mmol), [14C]formaldehyde(46.7 mCi/mmol), and [35S]cysteine (20-150 mCi/mmol) were from Amersham. All other chemicals were reagent grade. PD-10 (Sephadex G-25, medium) columns were obtained from Pharmacia. For sandwich type ELISA, reagents provided with the Cobas Core PSA assay kit were used (Hoffmann-La Roche, Basel, Switzerland). Polystyrene Microwell modules polysorb F8 and Immunowash 8 were from NUNC (Intermed). Microplate wells were coated with BSA prior to antibody photoimmobilization. Methods. Synthesis and Chemical Characterization of N-[m-[3-(Trifluoromethyl)diazirin-3-yl]phenyll-4-maleimidobutyramide.4-Maleimidobutyricacid (40 mg, 0.22 mmol) was dissolved in 4 mL of DMF and cooled to -20 "C under nitrogen. Triethylamine (56 pL, 0.4 mmol) was added to the stirred solution followed by dropwise addition of ethyl chloroformate (37.8 pL, 0.4 "01) in 500 pL of DMF. The reaction mixture was stirred for 15 min and freshly prepared 3-(trifluoromethyl)-3(m-aminopheny1)diazirine(45.9 mg, 0.22 mmol) in 500 pL of DMF was slowly added. The solution was allowed to warm to ambient temperature and was stirred for 1.5 h. The reaction mixture was poured into 20 mL of 1M NaCl on ice under vigorous stirring and extracted four times with 5-mL aliquots of ether. Ether extracts were washed twice with 1M HCl(5 mL each) and four times with water

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(5 mL each) and dried over anhydrous NazS04. Solvents were removed under reduced pressure, and the crude product was purified by flash chromatography on silica gel 60 with ethyl acetate/hexane 1/1 (v/v) as eluent. Product-containing fractions were pooled and organic solvents evaporated. The procedure described yielded 65.2 mg of MAD (0.17 mmol, 77%), which recrystallized as white needles. The cross-linking reagent was stored at -20 “C. Chemical homogeneity and the molecular structure of the bifunctional reagent were confirmed as follows. TLC: Rf0.34 in ethyl acetate/hexane, 1/1 (v/v).Mp: 96.5-98 “C. UV absorption spectrum (in ethanol): MAD shows a characteristic absorption shoulder at 360 nm constituted by both the diazirine (e347 = 446 M-’ cm-l; Dolder et al. 1990) and the maleimide absorption band (e300 = 620 M-l cm-1; Roberts and Rouser, 1958). IR (CHC13): 3040 (C=CH), 1720 (C=O), 1605 cm-1 (N=N). MS (FAB): mle (re1 intensity) 366 ( M + H, 3.191, 338 (26.281, 228 (29.58),173 (loo), 172 (58.95), 166 (49.03),138 (45.97), 110 (61,14), 98 (15.32), 28 (16.49), 18 (28.49). HRMS (FAB): mle calcd for (C16F3H13N403 + H) 366.0939, found 366.0936. ‘H NMR (CDCl3, chemical shifts (6) in parts per million): 8.2 (s, lH), 6.9-7.8 (m, 4H), 6.7 (s, 2H), 3.6 (t, 2H), 1.9-2.5 (m, 4H). Preparation of Anti-PSA F(ab’)Fragments. F(ab’)z fragments of anti-PBA monoclonal antibodies (270pg, 2.05 mg/mL) in 0.1 M sodium phosphate buffer, pH 6.8, were combined with DTT (final concentration 1 mM) and incubated for 50 min at 37 “C. F(ab’) fragments were separated from excess reducing agent by chromatography on PD-10 in 0.1 M sodium acetate, 0.5 M NaC1, 1 mM EDTA, pH 5.0. Fractions (600 pL) were collected and protein contents were analyzed by measuring the 280-nm absorption (Azml% = 14.0). Generated fragments were used immediately after separation to avoid thiol oxidation. Preparation of [ 14C]-MethylatedF(ab’)zand F(ab’) Fragments. F(ab’)z fragments of F5 anti-PSA antibody (540 pg, 2.05 mg/mL) in 0.1 M sodium phosphate buffer pH 6.8 were transferred to 0.1 M HEPES buffer, pH 7.5, by chromatography on PD-10. [WIFormaldehyde (2 pmol, 100 pCi) and sodium cyanoborohydride (final concentration 24 mM ) were added to the pooled protein fractions, and the reaction mixture was stirred for 4 h at ambient temperature. [14C]-Methylated protein was purified on PD-10 (equilibrated with 0.1 M sodium phosphate buffer, pH 6.8) or by HPLC on Ultrapac TSK 3000 SW (7.6 x 300 mm; elution conditions, 1mM EDTA, 0.1 mM sodium phosphate buffer, pH 6.8; flow, 0.75 mL/ min; detection, 280-nm absorbance). [W]-Methylated F(ab’) was prepared by reduction of [I%]-methylated F(ab’)Z with 1mM DTT for 1h a t 37 “C before HPLC separation. Conditions for the thermochemical reaction of the [l4C1-methylated F(ab’) with MAD or maleimide supports were identical to those described below for nonradiolabeled F(ab’) fragments. PSA Microplate ELISA Assay. The COBAS Core PSA solid-phase enzyme immunoassay was modified to the extent that F(ab’) fragments of anti-PSA antibodies were photoimmobilized on the bottom of microplate wells. Samples were dried, placed 4 cm distant from the light source, and illuminated with an irradiance of 0.7 mW/ cm2. Titer plates were mounted on a reflecting mirror and agitated perpendicular to the light bulbs with a deflection of 4 cm and a frequency of 26 cycledmin. In all experimental protocols, wells were saturated with 1% (w/v) BSA in PBS, pH 7.4, for 2 h at 37 “C and washed three times with doubly distilled water before ELISA

Collioud et al.

procedures were carried out. Aliquot samples of the PSA standard (10 pL, 0.6 ng/well) and 40 pL of anti-PSA-POD conjugate were added to coated wells. Following incubation for 15 min at 37 “C with permanent shaking, wells were washed with doubly distilled water (3 X 300 pL/ well). Enzyme-catalyzed reactions were initiated with the addition of 50 p L of substrate solution (3,3’,5,5’-tetramethylbenzidine) and terminated after 15min at 37 OC with stop solution (4.9% sulfuric acid, 200 pL/well). Color development was monitored at 450 nm with the UVmax microplate reader. Functionalization of Solid Supports with the Maleimide-Diazirine Bifunctional Reagent. ( a ) Glass Supports with Terminal Diazirines (Preparation of MAD-Derivatized Glass-Fiber Disks). GF-C Whatman glass-fiber disks (9 cm in diameter) were thiopropylated according to Aebersold and collaborators (1986) using (3mercaptopropy1)trimethoxysilane instead of the corresponding (3-aminopropy1)trimethoxysilane.“Curing” of the support was omitted to avoid thiol oxidation. Surface thiolation yielded 12 f 2 nmol of thiol groups per mg of glass fiber quantitated by the Ellman reagent (Riddles et al., 1983). Glassware was treated with chromic acid and DTNB-containing solutions were degassed and stored under nitrogen. Dried thiopropylated glass-fiber sheets were cut into disks (12 mm diameter) and incubated in acetone containing 0.1% TEA (v/v) for 1 h at room temperature with a 2-fold molar excess MAD (assuming a fiber volume of 25 pL). After removal of excess reagent with 20 mL of acetone, disks were dried under reduced pressure at ambient temperature. As quantitated by Ellman procedures, 95% of the SH groups were modified by MAD. MAD-functionalized glass fibers were stored at -20 OC in the dark. ( b ) Polystyrene with Terminal Maleimide Functions (Preparation of MAD-DerivatizedPolystyrene). MAD (30 pL, 79 pg, 216 nmol) dissolved in MeOH was placed in modular microplate wells. MeOH was removed by evaporation at 37 “C during 1h followed by additional drying at room temperature for (1 h, 0.02 mbar). Wells were then irradiated in the Stratalinker for 20 min. Control samples were treated identically but omitting the irradiation step. Wells were then washed four times with 250 pL of methanol. ( c ) Preparation of BSA-Precoated Polystyrene with Terminal Maleimide Functions. To form an adsorptive protein adlayer on polystyrene (Andrade and Hlady, 1986), modular microplate wells were precoated with BSA (2 73 solution (w/v) in PBS, pH 7.4). Aliquots (300 pL) were distributed to each well and incubated for 1.5 h at 37 OC. Excess solution was removed, and modules were dried at room temperature (1.5 h, 0.02 mbar). MAD was dissolved in 50 pl of 2 % EtOH in 10 mM sodium phosphate buffer, pH 5.0 (v/v), and applied in indicated concentrations (Figure 6) to BSA-precoated wells. Liquids were removed under reduced pressure (0.02 mbar) at ambient temperature and wells were irradiated with the Stratalinker for various lengths of time. Noncovalently bound reagent was removed by washing the wells with 2 % EtOH in 10 mM sodium phosphate buffer, pH 5.0 (v/v) (four times), and doubly distilled water (twice). Thermochemical Functionalization of Cysteine, a Terminally ThiolatedPeptide, and F(ab’)Fragments with MAD. ( a ) M A D Modification of [=S]Cysteine. MAD was reacted with [35SlCysin EtOH/2O mM citric acid, 35 mM Na~HP04,108mM NaC1,l mM EDTA, pH 6.5 ( l / l , v/v). [35SlCys(8 pL, 0.64 pmol) and Cys (6 pL, 0.35 pmol) were added to 500 pL of a stirred MAD solution

Oriented Coupling of Target Molecules

(256 pg , 0.7 pmol) and incubated for 30 min. Aliquot samples (50 pL) were withdrawn from the mixture and analyzed by HPLC on butyl (C4) Bakerbond. Linear gradient elution protocols were followed replacing solvent A (0.1 % v/v TFA in HzO) with solvent B (0.1% v/v TFA in CH&N/H20,3/1 by vol) from 0 to 100% in 30 min with a flow rate of 1.3 mL/min. After completion of the reaction, the product was purified by HPLC as detailed above, and solvents were removed under reduced pressure. The product (MAD-P5S1Cys) was quantified by its diazirine UV adsorption (€357 = 446 M-' cm-') and stored at -20 OC in MeOH. The yield was 47.5% (0.33 pmol). (b) Modification of CDPGYIGSR with MAD. The peptide (CDPGYIGSR, 1mg, lyophilized with buffer salts and 2-mercaptoethanol) was dissolved in 1 mL of H20. The solution was stored at -20 "C until use. Aliquot samples (155 nmol peptide 200 pL) were brought to pH 6.5, and 187 pg (500 nmol) of MAD in 200 pL of EtOH was added. The mixture was incubated for 1 h at room temperature. Product formation was surveyed by HPLC under the same conditions as described for MAD-Cys. For quantitation purposes the synthetic peptide was radiolabeled by reductive methylation accordingto Jentoft and Dearborn (1979). To attain this, the followingreagents were added to the above reaction mixture upon completion of the reaction with MAD: NaCNBH3 (100 pL, 240 mM), 100 pL of 1M HEPES buffer, pH 7.5, and 20 pL of [l4C1formaldehyde in water (4.6 pCi, 153 nmol). After incubation for 3 h at room temperature the product was isolated by HPLC as above. The radiolabeled product eluted with the same retention time as MAD-CDPGYIGSR. Solvents were evaporated under reduced pressure, and the product was dissolved in 1 mL of EtOH/H20 1/1 (v/v). Radiolabeled MAD-[l4C1 CDPGYIGSR was quantitated by diazirine absorption (e347 = 446 M-' cm-l) at 347 nm. These procedures yielded 69 nmol (45% ) MAD-[l4C1-methylated-CDPGYIGSR with a specific radioactivity of 14.05 nCi/nmol and 48 % methylated amino functions. ( c ) Thermochemical Coupling of MAD to F(ab') Fragments. Freshly prepared F(ab') fragments (130 pg, 2.6 nmol) were dissolved in 950 pL of 0.1 M sodium acetate, 0.5 M NaC1,l mM EDTA, pH 5.0, and combined with the cross-linking reagent (19 pg, 52 nmol dissolved in 9.6 pL of EtOH). The reaction mixture was incubatedat ambient temperature for 16 h in the dark. Excess MAD was removed by chromatography on PD 10 in 15 mM sodium phosphate buffer, pH 6.5. Fractions containing protein ( A ~ N were ) pooled and used immediately (MAD-F(ab') fragments). Photochemical Coupling of Target Molecules to MAD-Derivatized Supports. (Reaction Route 1, Figure 1B). Photocoupling of [I4ClTyrosine to M A D Derived Glass Supports. MAD-derived glass-fiber disks (see previous text for preparation) were soaked with 10pL of EtOH before [l4C1Tyr(0.66 nmol, 305 nCi) was applied. Disks were then dried under vacuum (50 mbar) for 1h at ambient temperature in the dark. Photolabeled glass disks were placed between two quartz slides under argon in a custom-made holder and mounted at a distance of 45 cm from the light source. Samples were photoactivated for 12 min (6 min each side) with the filtered light of the high pressure mercury lamp (150 mW/cm2). Control samples were treated identically, but light exposure was omitted. Photoactivated and control disks were then washed extensively (Sigrist et al., 1990) and analyzed for radioactivity by liquid scintillation counting. Net lightdependent amino acid binding was determined by sub-

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tracting nonspecifically adsorbed reagent (control) from the radioactivity recovered on irradiated disks. Thermochemical Immobilization of Target Molecules to Surfaces with Terminal Maleimides. (Reaction Route 2, Figure 1C). (a) [%SICysteine Coupling to Polystyrene. MAD-derivatized polystyrene microplate wells (see previous text for preparation) were incubated with 5 nmol(112.5 nCi) of [35SlCysdissolved in MeOH/20 mM citric acid, 35 mM Na2HP04, 108 mM NaC1,l mM EDTA buffer, pH 6.5 ( l / l , v/v), for 2 h at ambient temperature. To remove free reagent, wells were washed four times with 250 pL of H20, four times with 250 pL of MeOH/H20 ( l / l , v/v), and four times with 250 pL of MeOH. Washed wells were analyzed for radioactivity by liquid scintillation counting, and coupling efficiencieswere determined. ( b ) Thermochemical Coupling of F(ab') Fragments to BSA-Precoated MAD-Deriuatized Microplates. Freshly prepared F(ab') fragments were dissolved in 0.1 M sodium acetate, 0.5 M NaC1, 1 mM EDTA, pH 5.0, to a final concentration of 30 pg of F(ab')/mL. Aliquot samples (50 pL, 1.5 pg F(ab')) were applied to BSA-precoated, MADderivatized wells and microplates were incubated in the dark for 24 h under argon at 4 "C. After this thermochemical reaction, wells were washed twice with the coupling buffer, once with PBS pH 7.4, and once with doubly distilled water to remove free F(ab') fragments. Immunological activity of the immobilized F(ab') fragments was analyzed by sandwich type ELISA procedures, and binding efficiencywas quantified by liquid scintillation counting. Photochemical Coupling of MAD-DerivatizedTarget Molecules to Solid Supports. (Reaction route 3, Figure 1D). (a) Photocoupling of MAD-[s5SlCys to Polystyrene. MAD-[35SlCys in MeOH (27.5 pL, 10nmol, 19 140 dpm) was added to microplate wells and dried for 1 h at 50 mbar and ambient temperature. Coated wells were irradiated for 15 min in the Stratalinker (1.3 mW/ cm2,in this experiment the distance of the support surface to the light bulbs was 1cm). Control samples were treated identically, but irradiation was omitted. Wells were washed four times with 250 pL of MeOH. Retained radioactivity was measured by liquid scintillation counting. ( b )Photocoupling of ML~.D-[~~CICDPGYIGSR to Polystyrene. MAD-[l4C1CDPGYIGSR (30 pL, 1.8 nmol, 64820 dpm) in EtOH/H20 (l/l,v/v) was placed into microplate wells and dried for 1 h at 37 OC and 1 h at ambient temperature at 0.02 mbar. Wells were irradiated for 20 min in the Stratalinker (0.7 mW/cm2). Wash procedures and determination of coupling yields were carried out as described for MAD-[3%lCys. ( c ) Photocoupling of MAD-F(ab') Fragments onto BSA-Precoated Microplate Wells. Various concentrations (0-1.0 pg/well) of MAD-F(ab') fragments in 15 mM sodium phosphate buffer, pH 6.5, were applied to BSAprecoated microplate wells and adjusted to a final volume of 70 pL with the same buffer. Wells were dried at ambient temperature and 0.02 mbar. Photoactivation was carried out with the Stratalinker (0.7 mW/cm2). Wells were then washed four times with doubly distilled water to remove buffer salts and noncovalently coupled protein. Control samples were treated identically except that irradiation was omitted. Photocouplingof MAD-[W]Cys to Hydroxylated FEP Membranes through a Patterned Mask. MAD[35SlCys(0.13 pmol, 3.4 pCi) was dissolved in 200 pL of EtOH and spread on a FEP-OH membrane. The solvent was evaporated (1h at 40 "C followed by drying for 2 h

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at 0.02 mbar and ambient temperature). Dried membranes were covered with the patterned mask and placed between two quartz glasses. Photoactivation for 10 min with the mercury lamp (lamp to sample distance of 47 cm, 11.6 mW/cm2). Adsorbed MAD-P5S1 Cys was removed by sonication for 1 min in 60 mL of EtOH (three times), MeOH/HZO ( U l , v/v), MeOH, HzO, and MeOH (once each). Membranes were exposed for 2 days to an X-ray film (Hyperfilm MP, Amersham). RESULTS

Synthesis and Characterization of MAD. Condensation of the amino function of 3-(trifluoromethyl)-3-(maminopheny1)diazirine with 4-maleimidobutyric acid required activation. Carboxyl group activation with dicyclohexylcarbodiimideand 4-(dimethy1amino)pyridinewas not successful. Synthesis of MAD was achieved via a mixedanhydride intermediate. Chemical reactions were monitored by TLC. All methods including 'H NMR used for chemical characterization of MAD confirmed the structure depicted in Figure 1A. Thiol groups reacted within minutes (Figure 2A) with the maleimide moiety of the heterobifunctional photo-cross-linker in aqueous media, leaving the diazirine function intact for light-dependent reactions. When irradiated with UV light (2320 nm), diazirines decayed to highly reactive carbene intermediates and nitrogen (Figure 2B). Crystalline MAD was stored at -20 "C in the dark over a period of more than 8 months without appreciable deterioration. MAD is soluble in most organic solvents. In the experiments described, MAD was dissolved in ethanol before addition to aqueous solutions. Stock solutions in ethanol were stored at -20 "C and used within1 week. Exposure to intense sunlight and artificial light was avoided. Thermochemical Functionalization of Cysteine and a Terminally Thiolated Peptide. Modification of [%']Cysteine with MAD. The reaction of substoichiometric amounts of MAD with cysteine was complete within minutes. Product formation was analyzed by HPLC. Under the conditions used for product separation, the retention times were 2.5 min for Cys, 17.1 min for MADCys, and 20.5 min for MAD. Eluted products were identified by reference analysis (Cys, MAD) and on the basis of the radioactivity elution pattern ([35SlCy~, MAD[35SlCys). Pool fractions of MAD-Cys were further characterized by UV spectroscopy. MAD-Cys showed a characteristic diazirine absorption band at 357 nm, which disappeared after irradiation. Since MAD-Cys mimics the thermochemical reaction product of MAD with any thiolated target molecule, the derivative was used to investigate the kinetics of the photoreaction. For kinetic studies, the concentration Ct of the diazirine at a defined irradiation time ( t ) was quantitated by the absorption difference At - A, at 357 nm, where A, is the absorption under saturating light conditions (300-9 irradiation). log ([At - A,I/[Ao - A,]}, expressed as a function of the irradiation time, yielded a straight line (Figure 3), consistent with the anticipated first-order kinetic process (Nassal, 1983). Modification of CDPGYIGSR with MAD. Commercially available CDPGYIGSR was stabilized with salts and 2-mercaptoethanol. Initially, attempts were made to remove the stabilizing agents by HPLC procedures prior to the thermochemical reaction with MAD. It was noted that reactive thiol functions were modified-most probably oxidized-during this HPLC separation, and the HPLCpurified material failed to react with MAD. Therefore, educt purification was not carried out, and CDPGYIGSR

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Figure 2. (A, top) Thermochemical reaction of the maleimide function of MAD. Spectroscopic analysis of MAD (0.9 pmol, 0.33 mg in 1mL of EtOHI20 mM citric acid, 35 mM Na2HP04, 108 mM NaC1, 1 mM EDTA, pH 6.5 (l/l,v/v)). Absorption spectra were recorded before (1)and after the addition of (2) 0.1 pmol, (3) 0.2 pmol, (4) 0.4 pmol, (5) 0.6 rmol, and (6) 0.9 pmol of cysteine; (trace 7, baseline (solvents only). (B, bottom), Spectroscopic analysis of the photoreaction of cysteine-modified MAD. After completion of the thermochemicalreaction described in Figure 2A, sample 6 was irradiated for differing lengths of time: (1)0 s, (2) 10 s, (3) 20 s, (4) 30 s, (5) 40 8 , (6) 50 s, (7) 300 s, (8, baseline). A high-pressure mercury lamp with a constant 200-W output was used for irradiation. The irradiance was 150 mW/cm2.

was used as purchased. It was modified with a 3.2-fold molar excess of MAD. MAD-CDPGYIGSR was purified by HPLC. It eluted with a retention time of 16.6 min under the conditicns described in the Experimental Procedures. Thermochemical and Photochemical Coupling of Amino Acids and Peptides to Functionalized Supports. Before investigations of oriented F(ab') fragment immobilization were carried out, various routes of MADmediated target molecule immobilization were tested with

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Time [sec] Figure 3. Kinetics of MAD photoactivation. MAD (0.9 pmol) was dissolved in 1mL of EtOH/ 20 mM citric acid, 35 mM NazHP04, 108 mM NaC1, 1 mM EDTA, pH 6.5 (l/l,v/v), and irradiated for varous lengths of time under conditions described in Figure 2B.

low molecular weight reference substances, including radiolabeled [l4C1Tyr, [35SlCys, MAD-[35SlCys, and MAD-[WICDPGYIGSR. Results obtained in these studies are summarized in Table I. Preparation of MAD-Derived Glass Supports and Photochemical Coupling of [ W l T y r (Reaction Route 1, Figure 1B). Procedures for glass thiolation and determination of the coupling efficiency of route 1 reactions (Figure 1B) were necessary. Glass-surface thiolation was monitored analytically by measuring accessible thiol functions with the Ellman reagent. Surface thiolation of glass fibers yielded 12 f 2 nmol of SH groups/mg of glass support. To avoid thiol oxidation, thiopropylated glass fibers were processed after preparation. Thermochemical functionalization of thiopropylated glass fibers was nearly quantitative: 95 5% of available thiol functions were blocked by MAD. The integrity and the amount of introduced diazirine functions could not be quantitated directly. Therefore, the photocoupling capacity was tested by analyzing the light-dependent binding of [l4C1Tyr. Coupling efficiency was 4 % , and the extent of nonphotochemical, physically adsorbed [l4C1Tyrwas 20-fold lower. Effective removal of this physically adsorbed amino acid was attained with washing procedures described earlier (Sigrist et al., 1990). Coupling efficiencies match those for light-dependent tyrosine binding to TRIMID-glass

surfaces (Sigrist et al., 1990). Bochariov and Kogon (1992) reported 8% cross-linking of modified Phe-tRNA to ribosomeswith an amino group specific,cleavable, carbenegenerating, cross-linking reagent. In the latter system, molecular interactions among the constituents were enforced by the inherent bioaffinity. Photoimmobilization of M A D to Polystyrene (Reaction Route 2, Figure IC). Reaction route 2 procedures do not require functional groups on the support surface. Efficiency of photoimmobilization was assessed indirectly by thermochemical [35SlCys coupling via the maleimide function. The treatment yielded a target molecule density of approximately 60 pmol of [35SlCys/cm2(20 pmol/well). Functionalization of surfaces by reaction route 2 is advantageous for the immobilization of biomolecules in that the primary step is easily attained by photochemical procedures and thermochemical target molecule coupling is effected by thermochemical reactions in aqueous media. The procedure does not require target molecule dehydration. Photocoupling of MAD-[%SICys and MAD-[W]CDPGYIGSR to Polystyrene. The reaction route 3 applied for photocoupling of MAD-[3Ql Cys and MAD[l4C1CDPGYIGSR is depicted in Figure 1D. Target molecules were photolinked to microplate wells under slightly different, yet saturating, irradiation conditions: MAD-[35SlCys was irradiated for 15 min at 1.3 mW/cm2, whereas MAD-[l4C1CDPGYIGSR was photocoupled for 20 min with 0.7 mW/cm2. Coupling efficiencies were 7 5% and 295, respectively, and the extent of removal of physically adsorbed target molecules was similar for both systems (Table I). I t was noted that the efficiency of photoimmobilization depended on the inherent physicochemical properties of the target molecules and their molecular interaction with the surface material. Topically AddressedPhotoimmobilization of MAD[35S]Cys. Addressability by light is a genuine property of photoactivatable reagents. Topically selective, lightdependent activation of the radiolabeled target MAD[35SlCyshas been assessed to qualify the reagent for future multidomain-coating applications. Figure 4 depicts the 300-pm-sized slit pattern obtained after exposure of a MAD-[%] Cys-coatedFEP-OH surface to activating light. Autoradiography of the washed support shows that the irradiated and nonirradiated areas are distinctly separated. MAD-Mediated Immobilization of F(ab’) Fragments. Immunoreagent Immobilization via MAD-F(ab’).

Table I. MAD-Mediated Photoimmobilization’ reagent, target MAD MAD MAD- [‘Bl -Cy8 MAD-[W]-CDPGYIGSR MAD-F(ab’) MAD-[14C] -F(ab’) SH-F(ab’)

support

analysis/detection route method 1 photocoupled [WI-Tyr 2 thermocoupled [%]-Cys 3 radioactivity 3 radioactivity 3 ELISA procedures radioactivityd 2 ELISA procedures

7J light-dependent coupling VS efficiency adsorptive bindingb 4 20.61 0.4 5.5:l 7 13.1:l 2 13.8:l 5od 61C (3:l)d 30-4od 6:lC

target molecule density/activity 20 pmol/mg 60 pmol/cm2 2.1 nmol/cm2 111pmol/cmZ 0.82 Edwellc 11.2 pmol/cm2d 1.8Edwellc

glass polystyrene polystyrene polystyrene BSA-precoated polystyrene BSA-precoated, MADmodified polystyrene SH-[14C]-F(ab’) modified polystyrene radioactivityd (3:l)d 50 pmol/cm2 a Coupling efficiency is defined as the amount of reagent which is retained due to light activation. The total amount of reagent applied is set to 100%. Coupling efficiency values do not include target-molecule immobilization due to physisorption. Ratios given describe the quality of the coupling process. Net light-dependent immobilization is compared with the extent of immobilization recovered in nonirradiated control samples (physisorption),the latter being set to 1. Immunoreagent immobilization is quantitated by the extent of immunocomplex formation. Data obtained with [l‘CI-methylated F5 anti-PSA F(ab’) fragments. Methylation of lysines may lead to surface polarity change and affect the immunological activity of antibodies. e Reaction route 1: Thermochemical coupling of MAD to a thiolated surface is followed by photochemical target-molecule coupling. Reaction route 2: Photochemical coupling of the reagent to an inert surface is followed by thermochemical coupling to target thiol functions. Reaction route 3: Thermochemical target modification occurs prior to carbene-mediated insertion into surface materials.

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”

0

0.1

1

10

100

MAD (nmol/well)

Figure 4. Topically immobilized MAD-[35S]Cys on hydroxylated FEP membranes. Spreading of MAD-[WICys on hydroxylated FEP was carried out as described in the Methods section. The coated membrane was covered with a photomask (slit width, 300 pm; spacings, 500 pm) and irradiated for 10 min with a mercury lamp (11.6 mW/cm2).Physically adsorbed MAD-[35S]Cys was removed and residual (photoimmobilized) label distribution was analyzed by autoradiography. White (radioactive) areas correspond to the slit openings (300 pm X 1.5 cm) of the mask. 1

0.8 h

o

E s

0.6

.-

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0.4

ln

0.

:I

2 0.2 a

0 0

0.1

0.2 0.3 0.4 0.5 0.6 0.7 0.8

0.9

1

MAD-F(ab’) ( g/well)

Figure 5. Immobilization of MAD-F(ab’) onto BSA precoated microwells (reaction route 3).Indicated amounts of MAD-F(ab‘) fragments were applied to BSA-precoated wells and irradiated for 20 min (*). Analogous experiments were carried out with BSA-precoated wells and MAD-F(ab’), but the samples were not irradiated (M). Antibody binding was analyzed by sandwichtype ELISA procedures.

MAD was thermochemically linked to freshly prepared F(ab’) fragments following the conditions described by Prisyazhnoy and collaborators (1988). Since MAD is not soluble in water, it was dissolved in ethanol prior to addition to the protein. MAD was added in a 20-fold molar excess over available thiol functions, and excess reagent was removed by gel chromatography (PD-10). The photoreactivity of MAD-F(ab’) fragments was investigated by analyzing the light-dependent coupling to BSAprecoated microplates. Efficiency of photoimmobilization was measured by liquid scintillation counting using [14C]methylated F(ab’). Up to 50% of radiolabeled antibody was covalently linked to BSA-precoated polystyrene. Immunoreactions carried out in BSA-precoated wells yielded activities (450-nm absorption) which were 6 times higher (3times for [14C]-methylatedF(ab’)) than nonirradiated control samples. Enzyme-linked immunoreactions saturated at 0.5-1.0 pg MAD-F(ab’)/well (Figure 5). Immunoreagent Immobilization to BSA-Precoated and Subsequently MAD-Derivatized Surfaces. F(ab’) fragments were successfully coupled to MAD-derived BSA surfaces following reaction route 2. BSA-precoated microplate wells were modified with the indicated amounts

Figure 6. Photoimmobilization of MAD onto BSA-precoated microplates (reaction route 2). MAD immobilization procedures were optimized by varying the amount of applied cross-linker and the irradiation time. Indicated quantities of MAD were dried orlto BSA-precoated microwells and irradiated for the following time lengths: 0, 5, 10, 15, 20 and 30 min (from dark to bright bars). Freshly prepared F(ab’) fragments (1.5 pg/well) were then thermochemically coupled to modified supports and antibody binding was analyzed by sandwich type ELISA procedures.

of MAD by photoimmobilization procedures. The light dependency of the process was evident. BSA-precoated wells, without MAD treatment, showed marginal F(ab’) retention. Both, irradiation time and MAD concentration were varied to delineate optimal conditions for F(ab’) binding to MAD-BSA. Generally, with increased quantities of MAD and longer irradiation times, higher immunoresponses were attained. This applies to samples which were modified with more than 0.1 nmol of MAD/ well. With 1 nmol MAD/well, light exposure saturated after 10min. Irradiation times of 15-20 min were required for samples which were modified with 10 and 100 nmol MAD/well (Figure 6). Quantitation of [14C]-methylated F(ab’) binding by liquid scintillation counting yielded binding efficiencies of 30-40% (100 nmol MAD/well, irradiation time of 20 min). Oriented Coupling. Binding differences between [14C]methylated and native F(ab’) fragments were ascribed to changes of surface polarity and possible modification of the binding domain of the antibody. Residual immunological activity of radiolabeled F(ab’) was analyzed with photoimmobilized [14C]-methylatedF(ab’). To delineate the oriented couplingcapacity, the specific immunoactivity (absorbance 450nm/mg of immobilized [14C]-methylated F(ab’)) of immobilized samples was determined. Specific immunoactivity was 2-4 times higher with photoimmobilized [14C]-methylatedF(ab’) than with adsorbed (not photoactivated) [14C]-methylatedF(ab’). These results apply to both [14C]-methylatedF(ab’) binding to MADBSA and MAD-[ 14C]-methylated-F(ab’)photocoupling to BSA-precoated wells. DISCUSSION

Due to the specificity of biomolecules and macromolecular aggregates, there is no doubt that biocatalysts and biosensors will find increasing applications in analytical diagnostics, bioprocessing, control systems, medical and material sciences. Applications mentioned require, however, covalent attachment and molecular orientation. Orientation of biomolecules in vivo is crucial for cell survival. Living systems accomplish unique biomolecule orientation by concerted actions of topical molecular association, compartimentalization, and timed biosynthesis. These parameters are not easily reconstructed for the fabrication of biomimetic devices, and new regimes

Oriented Coupling of Target Molecules

are needed to establish molecular orientation. Lightdependent immobilization procedures were introduced to circumvent drawbacks imposed by thermochemical reactions and, at the same time, gain topical addressability (Sigrist et al., 1992;Rozsnyai et al., 1992). Photochemical reactions are advantageous in that covalent coupling is initiated by light and functionalization of the target molecules is not required if photoactivatable surfaces are provided (reaction route 1). Recent studies which compare physical adsorption and photoimmobilization of F(ab’) fragments have shown that photolinker-polymer-mediated immobilization is superior to physical adsorption (Gao et al. unpublished) and photolinker-polymer-mediated immobilization of oligonucleotides is attained by light exposure of photolinker-polymer-coated surfaces ( Kung et al., unpublished). The heterobifunctional photo-cross-linker described in this study combines photoreactivity and the selectivity of reaction with thiols. Most biomolecules carry functional groups at the surface of their native folded structures. Reactive functional groups are asymmetrically distributed at the surface and bear individual chemical reaction characteristics. Site-specific orientation and immobilization is easy to obtain if uniquely reactive groups are available at the surface of biomolecules. However, selectively reactive functional groups, preferably located at a single site, are seldom. Multi-site attachment and random coupling of biomolecules is most probable by targeting at ubiquitous surface exposed amino functions (Domen et al., 1990). Reactive thiol groups located at the surface of proteins are relatively rare. They can be generated (bio)chemically, obtained through chemical synthesis of biopolymers, or constructed by genetic engineering. Examples are as follows: (i) F(ab’)z fragments produced by pepsin treatment of antibodies, (subsequent reduction of disulfide bridges in the hinge region yields immunoreagents which provide a selectively reactive thiol function opposite to the binding domain of the antibody.), (ii) synthetic biopolymers designed for oriented coupling with reactive thiol functions at the N-terminal end of receptor binding peptides (Graf et al., 19871, and finally, (iii) unique thiol functions (T8C or T65C) introduced by site-directed mutagenesis into cytochrome b5 (Stayton et al., 1992), a similar approach has been chosen by Aggeler and Capaldi (1992)to prepare the molecular basis for sitedirected cross-linking of the ECFl y-subunit. In both biological and biomimetic systems, molecular interactions between biomolecules and material surfaces are initiated by physical contacts at chemical bond distances. These interactions ultimately lead to biomolecule immobilization which is either physical adsorption, protein (or receptor) binding mediated adsorption, or covalent immobilization. For biomolecule immobilization on material surfaces, it is of particular importance that noncovalent adsorption and random orientation are suppressed. Data presented in this study show that MADmediated immobilization of low molecular weight target molecules can be attained by various routes; all of them lead to the ultimate goal of covalent immobilization. The extent of physical adsorption is generally low, given by the target molecule’s surface characteristics, the material surface properties, and the interfacial behavior of both system constituents. Covalent, light-dependent, and oriented immobilization of F(ab’) fragments using MAD as the cross-linking agent has been achieved by both reaction routes 2 and 3. Molecular orientation was attained with the thermochemical reaction of the maleimide with F(ab’) sulfhydryl

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groups. Covalent binding to support materials was effected by carbene insertion. MAD-mediated target molecule immobilization can be controlled by selective irradiation of defined micron-sized areas. Thus, multiple-domain functionalization of surfaces with target (bio)molecules was feasible. Due to the unique molecular structure of antibodies, MAD-based immobilization procedures can be applied to any antibody derived F(ab’) fragment. Through usage of MAD, any biomolecule (proteins, peptides, nucleotides) which contains reactive and accessible cysteine residues (thiol functions) at the surface can be immobilized photochemically. With the diazirine as the photoactivatable group, there are no restrictions with respect to the nature of the solid support except the availability of covalent chemical bonds. ACKNOWLEDGMENT

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