Synthesis and Characterization of a Photoactivatable

Bioconjugate Chem. 1999, 10, 169−175

169

Synthesis and Characterization of a Photoactivatable Glycoaryldiazirine for Surface Glycoengineering Yann Chevolot,*,† Odile Bucher,‡ Didier Le´onard,† Hans Jo¨rg Mathieu,† and Hans Sigrist‡ De´partement des Mate´riaux, LMCH, Ecole Polytechnique Fe´de´rale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland, and CSEM (Centre Suisse d’Electronique et de Microtechnique SA), Jaquet-Droz 1, CH-2007 Neuchaˆtel, Switzerland . Received May 12, 1998; Revised Manuscript Received October 15, 1998

Biological systems make considerable use of specific molecular interactions. Many biomolecules involved in biorecognition are glycosylated, the carbohydrate moiety playing an essential role. Controlled surface glycoengineering is thus of crucial importance in biosensing, cell guidance, and biomedical applications. This study describes the synthesis of an aryldiazirine-derivatized galactose and the functionalization of surfaces by carbohydrates using photochemical immobilization techniques. A photoactivatable glycosylated reagent was synthesized by addition of thiogalactopyranose to the maleimide group of N-[m-[3-(trifluoromethyl)diazirin-3-yl]phenyl]-4-maleimidobutyramide (MAD) to give N-[m-[3-(trifluoromethyl)diazirin-3-yl]phenyl]-4-[3-thio (1-D-galactopyranosyl)succinimidyl]butyramide (MAD-Gal). The structure of the newly synthesized molecule was confirmed by UV spectroscopy, photoactivation, 1H NMR, and 13C NMR. MAD-Gal was immobilized on thin diamond films by photoactivation of the diazirine function (350 nm). Surface modification was investigated by XPS (X-ray photoelectron spectroscopy) and ToF-SIMS (time-of-flight secondary ion mass spectrometry). Imaging ToF-SIMS was applied to detect glycopatterns generated by mask-assisted lithography.

INTRODUCTION

Biological systems make considerable use of surface glycosylation. Indeed glycosylated molecules are involved in, for example, cell recognition (including species discrimination), blood group typing, blood coagulation cascade, and asialoglycoprotein recognition (1-3). Tissue engineering requires control of specific cell receptor interactions at material surfaces through oriented immobilization of biologically active molecules such as carbohydrates. Carbohydrate immobilization is reported in the literature for galactose (4), melibiose (5), mannose (5, 6), lactose (7), starch (8), lactosaminide (9), and heparin (5, 10-15). This has been achieved mainly by thin-film adsorption (weak interactions) or by covalent binding. Adsorption of molecules through weak interactions (ionic in the case of heparin (13)) may reverse with time. Covalent immobilization was reported for polymer substrates by divinyl sulfone activation for mannose (5), reductive amination for melibiose on polyacrylamide (5), the bisorane method for lactose and N-acetylglucosamine (5), CNBr activation for heparin (5), reductive amination of nitrous acid treated heparin (13), and copolymerization for galactose (4) and lactosaminide (9). Galactose-derivatized polymers were used for the study of hepatocyte adhesion (e.g. PVLA,1 poly(vinylbenzyl-β-D-lactonamide) (7, 16)). However, this kind of immobilization is not surface specific, unless coated. Furthermore, it is performed on polymeric materials and is not addressable. Photochemical procedures were applied for heparin (1012, 14, 15) and dermatan (14) immobilization. Photoac* To whom correspondence should be addressed. Phone: ++ 41216934124.Fax: ++41216933946.E-mail: [email protected]. epfl.ch. † Ecole Polytechnique Fe ´ de´rale de Lausanne. ‡ Centre Suisse d’Electronique et de Microtechnique SA.

tivatable reagents generate very reactive intermediates that allow immobilization of the molecules onto various substrates, as well as addressable immobilization. Erdtmann et al. (14) used a nitrene generating reagent to achieve immobilization of heparin and dermatan on cellulose membranes. The membranes were first modified with 4-azido-1-fluoro-2-nitrobenzene. Guire (10, 11) immobilized heparin on polyurethane: albumin was first photoimmobilized on polyurethane, and heparin was then linked to albumin using 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide chemistry. These strategies combine thermochemistry and photochemistry for surface immobilization. The development of a single step photochemical process would provide a more convenient tool for light addressable surface patterning. Utilizing diazirine as a photoactivatable function, a reactive carbene is generated by thermochemical or light activation. The carbene may insert into C-H, C-C, Cd C, N-H, O-H, and S-H bonds (17). Carbene-mediated target molecule binding has been reported for TiO2/SiO2 (18), silicon (19, 20), silicon nitride (19-21), diamond (19, 20, 22) and FEP-OH (hydroxylated fluorinated ethylenepropylene) (23). Shergold et al. (22) used diazomethane, and Gao et al. (18) used an aryldiazirine-derivatized bovine serum albumin (T-BSA), while other investigations were carried out with the photoreagent MAD (1921, 23, 24) (Figure 1). The energy used for photolabel 1 Abbreviations: MAD, N-[m-[3-(trifluoromethyl)diazirin-3yl]phenyl]-4-maleimidobutyramide; MAD-Gal, N-[m-[3-(trifluoromethyl)diazirin-3-yl]phenyl]-4-[3-thio(1-D-galactopyranosyl)succinimidyl]butyramide; BSA, bovine serum albumin; FEP-OH (hydroxylated fluorinated ethylenepropylene); EDTA, ethylenediaminetetraacetic acid; TFA, trifluoroacetic acid; PVLA, poly(vinylbenzyl-β-D-lactonamide); XPS, X-ray photoelectron spectroscopy;ToF-SIMS,time-of-flightsecondaryionmassspectrometry; UHV, ultrahigh vacuum; CVD, chemical vapor deposition; fwhm, full width half maximum; ROI, region of interest.

10.1021/bc980050h CCC: $18.00 © 1999 American Chemical Society Published on Web 02/04/1999

170 Bioconjugate Chem., Vol. 10, No. 2, 1999

Figure 1. Reaction of 1-thio-β-D-galactopyranose with MAD, yielding N-[m-[3-(trifluoromethyl)diazirin-3-yl]phenyl]-4-(3-thio(1-D-galactopyranosyl)succinimidyl)butyramide (MAD-Gal).

activation does not impair biological functions ((24) and unpublished results). This work presents the derivatization of MAD with 1-thio-β-D-galactopyranose to give N-[m-[3-(trifluoromethyl)diazirin-3-yl]phenyl]-4-[3-thio(1-D-galactopyranosyl)succinimidyl]butyramide (Figure 1). The parent molecule MAD provides a maleimide function, allowing the reaction with thiogalactopyranose. Both MAD and its derivative MAD-Gal contain the diazirine function that yields carbenes upon light activation at 350 nm. The synthesis, purification, and bulk analysis of MAD-Gal are described. MAD-Gal was then immobilized on diamond. Due to its physical properties and biocompatibility (25-27), diamond and diamond-like carbon are widely used in biomedical applications. For example, thin film coatings are applied to stents and carbon is identified as a good substrate for biosensor applications (28). Therefore, surface modification of diamond functionalized by biological molecules to obtain specific interaction with analytes or cells is of great interest. Structured surfaces (photopatterning) can be used for probing or controlling interactions of cells or macromolecules at the surfaces of materials (i.e. implants and biosensing). The immobilization of MAD-Gal on diamond is confirmed by surface analysis techniques such as XPS (X-ray photoelectron spectroscopy) and ToF-SIMS (time-of-flight secondary ion mass spectrometry). Imaging ToF-SIMS is applied to detect glycopatterns generated by mask-assisted lithography. EXPERIMENTAL SECTION

Materials. Synthesis and Characterization. MAD was synthesized according to Collioud et al. (24). 1-Thio-β-Dgalactopyranose was purchased from Aldrich. Uvasol quality solvents (Merck) were used for chemical reactions. The HPLC grade solvents were purchased from Romil (Switzerland). Trifluoroacetic acid (TFA) was purchased from Sigma. MAD-Gal was purified by HPLC using a wide-pore butyl (C4) Bakerbond standard column (dimensions 4.6 × 250 mm; particle size 5 mm; Baker Inc., Phillipsburg, NJ) on a Bio-Cad system. The 1D 1H NMR, 2D 1H/1H COSY NMR, 13C NMR, and DEPT 13C NMR spectra were collected at the University of Bern on a Bruker 500 MHz NMR using D2O as solvent. Surface Analysis. XPS analysis was performed under UHV conditions (∼10-9 Torr) using a PHI 5500 (Perkin-

Chevolot et al.

Elmer, USA) system equipped with a hemispherical analyzer and a nonmonochromatized Mg KR radiation source. The analyzed area was 0.12 mm2, and spectra were recorded with a takeoff angle of 45° (with respect to the surface). Pass energies used for survey scans and high-resolution elemental scans were 93.5 and 23.5 eV, respectively. No sample charge-up was observed. For each sample, three areas were analyzed. For evaluation of the XPS spectra, the absolute peak areas were calculated using a parabolic interpolation routine, and background subtraction was applied. Resulting values were normalized to the step size to give the intensity in electronvolts per second. Sensitivity factors (Si) were 0.296, 0.711, 0.477, 1.000, and 0.570 (29) for C 1s, O 1s, N 1s, F 1s, and S 2p, respectively. Atomic percentages of selected elements (x) were calculated using the above sensitivity factors and the equation: X% ) (Ix/Sx)/(∑Ii/ Si). Diamond chemical shift was chosen as the reference at 284.2 eV (30). It was confirmed with gold as internal standard. ToF-SIMS was performed on commercial equipment from PHI-EVANS & Associates (described in (31, 32)) equipped with a pulsed FEI Ga+ ion gun operated at 15 kV for high mass resolution. The sample surface was biased (3 kV with respect to the grounded extraction electrode for positive/negative mode SIMS, respectively. The 800-850 pA dc ion beam current was pulsed at a 5 kHz repetition rate (pulse width of about 8 ns, as measured with the unbunched beam). The analyzed area was estimated to be a square of 84 µm × 84 µm. Spectra were acquired in the high mass resolution mode (bunched beam). The total ion dose for a typical spectrum was on the order of 9 × 1011 ions/cm2, which is within the socalled “static” conditions (33). The mass resolution obtained for Si+/- on a Si wafer was m/∆m > 3600 in the positive mode and >3000 in the negative mode. Normalized values were calculated by dividing the absolute intensity of secondary ions by the corrected total intensity ()total intensity from which intensities of H+- of all inorganic peaks and of the main peaks of ubiquitous contaminants were subtracted). Images were also collected in the high mass resolution mode. The high mass resolution conditions were similar to those described above except for analyzed area (180 µm × 180 µm) and analysis time (20 min), but the ion dose was similar (8 × 1011 ions/cm2). Methods. Synthesis of N-[m-[3-(Trifluoromethyl)diazirin-3-yl]phenyl]-4-[3-thio(1-D-galactopyranosyl)succinimidyl]butyramide. 1-Thio-β-D-galactopyranose (1.5 mg) was dissolved in 150 µL of buffer A (20 mM citric acid, 35 mM Na2HPO4, 108 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA)) and 150 µL of ethanol. MAD (3.3 mg) was dissolved in 150 µL of ethanol and an additional 150 µL of buffer A. Both solutions were mixed. The mixture was stirred and allowed to react at room temperature for 90 min, to give MAD-Gal. The product was purified by HPLC. The HPLC flow rate was 2 mL/min. For gradient elution (0 min, 100% A; 5 min, 100% A; 20 min, 70% A; 30 min, 0% A; 40 min, 0% A), solvent B was added to solvent A (solvent A, 0.1% TFA in water; solvent B, 0.1% TFA in acetonitrile/water (4:1 by volume)). Detection was performed at 254 nm and fractions of the eluant were collected. 13C NMR (D2O chemical shift (δ) in parts per million): 179.14, 178.40, 177.99, 177.86, 173.30, 137.79, 129.53, 129.38, 122.46, 121.84, 118.32, 84.42, 84.27, 78.99, 78.91, 73.84, 73.66, 69.73, 69.68, 68.58, 68.49, 60.80, 60.69, 39.61, 38.66, 38.46, 37.71, 35.83, 33.57, 22.68, 22.57.

A Photoactivatable Glycoaryldiazirine

Bioconjugate Chem., Vol. 10, No. 2, 1999 171

Figure 3. Figure 2. UV absorption spectra of MAD-Gal before and after irradiation for differing lengths of irradiation time.

Immobilization of MAD-Gal on Diamond. Hot filament CVD deposited thin-film diamond (34) was used as the substrate to exploit MAD-Gal immobilization. Pristine samples (5 × 5 mm2) were washed (5 min ultrasonic bath in hexane and in ethanol) and dried for 2 h at room temperature under vacuum (∼6 mbar). Then, MAD-Gal (10 µL, 0.25 mM in ethanol) was deposited as a droplet released from a syringe. The samples were dried for 2 h at room temperature under vacuum (30-40 mbar). They were irradiated for 20 min with the Stratalinker light source (0.95 mW/cm2) and washed by ultrasound treatment twice in ethanol (5 min) and twice in hexane (5 min). The investigated samples are referenced as follows: A (washed surface), B (MAD-Gal deposited, no illumination performed before final washing), and C (MAD-Gal deposited, illumination and final washing). Patterning was performed with a mask consisting of 40 µm × 40 µm square windows separated by 40 µm bridges. MAD-Gal was deposited on the diamond surface before mask-assisted illumination and final washing. RESULTS AND DISCUSSION

Chemical Characterization of MAD-Gal. The reaction between 1-thio-β-D-galactopyranose and MAD, yielding MAD-Gal, is displayed in Figure 1. HPLC analysis of the educts revealed a retention time of 37.41 min for MAD and approximately 15 min for 1-thio-β-D-galactopyranose, which was detected with 5,5′-dithiobis(2-nitrobenzoı¨c acid) (35). HPLC separation of the reaction mixture revealed a major peak with a retention time of 33.68 ( 0.07 min (88.2% of the total area). Minor byproducts with retention times of 33.06 ( 0.02 and 35.61 ( 0.02 min (0.6% and 11.2% of the total area, respectively), were observed. We suggest that they are products of the reaction of molecular oxygen with the thiols. During concentration of the eluant, thermal degradation may occur. Indeed, a peak with retention time of 35.99 min (10% area) was observed upon rechromatography of concentrated MAD-Gal. To avoid any thermal or light degradation of MAD-Gal, it was stored at -20 °C in the dark. MAD has shown to be stable over a period of at least 8 months under such conditions (24). In the immobilization experiments, MAD-Gal was dissolved in ethanol. Exposure to artificial light, unfiltered sunlight, or heat (over 30 °C) was avoided. These solutions were also stored at -20 °C. The UV absorption spectrum of the product corresponding to the peak with the retention time of 33.68 min (major peak) shows an absorption band at 357 nm, characteristic of the diazirine function (23). Figure 2 indicates that the absorbance diminished with increasing light exposure at 357 nm, demonstrating exposure time dependent photoactivation and, thus, conversion of the diazirines into carbenes.

13C

NMR overview spectrum of MAD-Gal.

In Figure 1, carbon and hydrogen atoms are numbered to facilitate the discussion of the NMR spectra. The 13C NMR spectrum showed four separated areas corresponding to the 13C nuclei of the amide function, the aromatic ring, the carbohydrate moiety, and the alkyl chain, respectively (Figure 3). The split signals of carbons 1, 2, 3, 4, 6, and 7 and of the carbohydrate moiety indicate the occurrence of two stereoisomers detected in similar proportions. Indeed, the sulfhydryl groups can attack the maleimide group above or below the plane of the ring, leading to two configurations of carbon 2. Consequently, the nuclei of both compounds in the vicinity of carbon 2 can appear at different chemical shifts. The DEPT (distortionless enhancement through polarization transfer) sequence analysis demonstrated that the peaks of the alkyl chain (22.57, 22.68, and 33.57 ppm) and C6′ (60.69 and 60.37 ppm, corresponding to the two isomers) correspond to secondary carbons. Other peaks appearing at 28.32, 27.99, 60.37, 71.52, 120.68, and 122.86 ppm were attributed to undefined contaminations. The 1H NMR spectrum of the purified product confirmed the presence of aromatic (6.5-7.5 ppm), alkyl (3.2-1.6 ppm), and carbohydrate protons (4.8-3.4 ppm) (Figure 4a). The 1H/1H COSY-NMR spectrum of MADGal is displayed in Figure 4b. The two-proton signal at 1.8 ppm was connected with the peaks at 2.3 and 3.4 ppm. It was attributed to proton 7. The peak at 2.3 ppm was also a two-proton signal. Peaks at 2.3 and 3.4 ppm were attributed to proton 8 and proton 6, respectively. The two peaks at 4.1 and 4.2 ppm corresponded to 1/2 of a proton signal each. The former was connected to the signal at 2.7 and 3.14 ppm, whereas the latter was connected to the peaks appearing at 2.8 and 3.14 ppm. According to the integration, peaks at 2.7 and 2.8 ppm represented the signal of a 1/2 proton each. The wide peak at 3.14 ppm was a one-proton signal. Thus, the peaks at 4.1 and 4.2 ppm corresponded to H-2 in two configurations. The peaks at 2.7 and 2.8 ppm corresponded to H-3a (one for each stereoisomer), and the peak at 3.14 ppm consisted of two peaks (shown by COSY) and corresponded to H-3b. According to the integration, the peak at 4.4 ppm was also 1/2 of a proton signal. The chemical shift corresponded to H-1′ of β-thiogalactopyranose. H-1′ for the second stereoisomer (also β) was at 4.7 ppm, within the water signal. Peaks between 3.9 and 3.4 ppm corresponded to protons 2′, 3′, 4′, and 5′. In summary, the results confirmed the structure of MAD-Gal. NMR analyses sustain that the final product is a mixture of two stereoisomers detected approximately in the same quantities. Surface Photobonding and Surface Analysis. MAD-Gal was deposited on washed diamond surfaces. The solvent was removed under vacuum to avoid the reaction of carbenes with solvent molecules. Next, the diazirines were activated by light (350 nm) and modified surfaces were extensively washed. It was expected that

172 Bioconjugate Chem., Vol. 10, No. 2, 1999

Figure 4. (a, top) 1H NMR spectrum of MAD-Gal. (b, bottom) 1H/1H NMR COSY NMR spectrum of MAD-Gal.

this final washing step removed most of the physically adsorbed but not the covalently bound MAD-Gal molecules. XPS wide scans of samples A and B exhibited intense photoelectron peaks at about 285 and 532 eV corresponding to the C 1s and O 1s lines. For sample B, on one area only, very low intensity peaks were observed around 400 and 688 eV corresponding to the N 1s and F 1s lines, respectively. According to the structure of the molecule (Figure 1), oxygen, fluorine, nitrogen, and sulfur were expected to be detected on diamond surfaces with grafted MAD-Gal. For sample C, XPS wide scans exhibited intense peaks corresponding to C 1s (285 eV), O 1s (532 eV), and F 1s (688 eV). Lower intensity peaks were observed at 165 and 400 eV corresponding to S 2p and N 1s, respectively. The binding energy of the S 2p line confirmed a C-S bond (Figure 5a) (36, 37), and that of the N1s line corresponded to the chemical shift of an amide function (Figure 5b) (38). The F 1s chemical shift confirmed the presence of C-F (Figure 5c) (36, 38). Results shown in parts a and b of Figure 5 indicated that XPS analysis of N and S on sample C was close to the instrumental detection limit (1012-1013 molecules/cm2).

Chevolot et al.

Figure 5. (a, top) S 2p XPS spectra of samples B and C (B, MAD-Gal on diamond, not irradiated, washed; C, MAD-Gal on diamond, irradiated and washed). The chemical shift corresponds to a C-S bond. S is only detected on sample C. (b, middle) N 1s XPS spectra of samples B and C. Nitrogen is only present on sample C (B, MAD-Gal on diamond, not irradiated, washed; C, MAD-Gal on diamond, irradiated and washed). The chemical shift corresponds to an amide. (c, bottom) F 1s XPS spectra of samples B and C (B, MAD-Gal on diamond, not irradiated, washed; C, MAD-Gal on diamond, irradiated and washed). Fluorine is only present on sample C. The chemical shift corresponds to a C-F bond. F is only detected on sample C.

The elemental compositions of samples A, B, and C are displayed in Table 1. For sample A, oxygen has an atomic percentage of 6%. Its presence at diamond surfaces can be explained by the fact that, during the hot filament CVD process, oxygen was incorporated to give a very complex surface with various functionalities (39, 40): lactone, ether, carboxylic acid, and alcohol. The presence of oxygen on diamond surfaces was consistent with the various contributions in the C 1s envelope (data not shown) and the full width half maximum (fwhm) of diamond contribution to C 1s (39). Except on one area, the sample B spectrum did not exhibit fluorine or

A Photoactivatable Glycoaryldiazirine

Bioconjugate Chem., Vol. 10, No. 2, 1999 173

Table 1. XPS C, O, N, F, and S Atomic Percentages for Samples A, B, and Ca C O N F S

A 93.9 ( 0.6 6.0 ( 0.6 0(0 0(0 0(0

B 92.3 ( 1.39 7.5 ( 1.05 0.42* 0.16* 0(0

C 88.7 ( 1.7 9.8 ( 1.61 0.6 ( 0.07 0.8 ( 0.19 0.08 ( 0.07

a Mean values for three areas except for values marked with an asterisk, for which only one area is displayed (see text). The investigated samples are denoted as follows: A, washed surface; B, MAD-Gal deposited, no illumination performed before final washing; C, MAD-Gal deposited, illumination and final washing.

Table 2. Negative Mode ToF-SIMS Corrected Total Intensity and Normalized ToF-SIMS Intensities (‰) for Samples A, B and C (Average of Three Spectra)a cor tot.b COFCF3SSO4C4H4NO2C5H5O2S-

A 313 ( 12.9 74 ( 2.5 200 ( 7.2 29 ( 2.53 7 ( 0.75 1.87 ( 0.18 0.78 ( 0.20 0.07 ( 0.01 0.11 ( 0.01

B 409 ( 11.9 108 ( 1.6 162 ( 2.1 6 ( 0.33 0.7 ( 0.02 0.9 ( 0.05 1.34 ( 0.07 0.08 ( 0.01 0.03 ( 0.00

C 408 ( 20.24 62.9 ( 1.2 184.9 ( 0.7 197 ( 3.1 8.59 ( 0.29 10.3 ( 0.05 1.47 ( 0.02 1.05 ( 0.06 0.15 ( 0.02

a

The investigated samples are denoted as follows: A, washed surface; B, MAD-Gal deposited, no illumination performed before final washing; C, MAD-Gal deposited, Illumination and final washing. b Negative mode ToF-SIMS corrected total intensity (×104).

nitrogen, illustrating the efficiency of the washing step to remove physisorbed molecules. O and C atomic percentages were intermediate between those of samples C and A. The elemental composition of sample C was characterized by significant amounts of fluorine and nitrogen at the surface. Sulfur was also detected, but at a very low intensity, and the oxygen content was higher for sample C than for sample B. XPS data revealed an F/N ratio of 1.3, which was close to the theoretical value (1.5). However, the F/S ratio was higher than 3, indicating a possible partial loss of sulfur during XPS analysis. Table 2 displays the corrected total intensity (×104, denoted “cor tot.”) of negative mode ToF-SIMS spectra and normalized ToF-SIMS intensities (‰) of selected peaks for samples A, B, and C. F-, CNO- (an amide related ion), and CF3- fragments were related to the MAD moiety of the molecule (20). C4H4O2N- (20) was identified as part of the succinimidyl group. S- (not correlated to SO4-) and C5H5O2S- were specifically related to the carbohydrate moiety (41, 42). It should be noted that the mass resolution allowed the separation between S- and O2- and between the peak at 129.01 amu (C5H5O2S-) and those appearing at 129.06 and 129.13 amu (not illustrated). Due to its high sensitivity, ToFSIMS analysis revealed F where XPS analysis failed (samples A and B). Moreover, S was easily detected compared to XPS for sample C. All the characteristic ions of MAD-Gal showed lower intensities on sample B than on sample C. The results further confirmed that the washing procedure removed most of the physisorbed molecules. The oxygen XPS signal and oxygen-containing ToFSIMS ions were expected characteristic fragments of MAD-Gal. The data presented here (Tables 1 and 2) are of low information value, due to signals contributed by the substrate. MAD-Gal surface patterning was performed using contact mode, mask-assisted lithography. Figure 6 pre-

Figure 6. Negative mode ToF-SIMS image of F- after maskassisted patterning of MAD-Gal on thin film diamond. The pattern was generated with a mask consisting of 40 µm × 40 µm square windows separated by 40 µm bridges. The mask was placed on a MAD-Gal-coated diamond surface before irradiation. Negative ToF-SIMS images were recorded after irradiation and final washing of the surfaces. Table 3. Negative Mode Normalized ToF-SIMS Intensities (‰) for the Spectra Corresponding to the Three Regions of Interest (ROIs) Displayed in Figure 6a FCF3SSO4CNOC4H4O2NC5H5O2S-

ROI 1 141.3 6.6 7.4 1.0 12.7 0.3 0.1

ROI 2 97.7 4.3 3.6 1.1 8.8 0.2 0.01

ROI 3 145.3 5.7 6.2 0.8 12.0 0.3 0.1

a ROIs 1 and 3 were light-exposed, whereas ROI 2 was a masked region.

sents the image of F- ions and documents addressable immobilization of the photoreagent MAD-Gal. The figure shows three regions-of-interest (ROI) areas from which spectra were collected. Table 3 displays the normalized ToF-SIMS intensities (‰) of MAD-Gal characteristic ions for the three ROIs. ROIs 1 and 3 were light-exposed, whereas ROI 2 was a masked region. Areas where characteristic signatures of MAD-Gal were observed corresponded to the light-exposed domains. However, it was noted that the nonexposed ROI 2 exhibited detectable amounts of MAD-Gal fragments (higher than those observed on sample B), indicating the limitations of contact mode, mask-assisted patterning. DISCUSSION

Great efforts are made in biomaterial science to generate bioactive surfaces with grafted oriented biomolecules. Molecular orientation is needed for biological interactions with modified surfaces. Carbohydrates are known to be involved in many biological phenomena. Linear and complex carbohydrate structures are present at biosurfaces in specific orientations and in defined structural (3-D) arrangements. In this study, oriented surface immobilization is implicated in the asymmetric molecular structure of MAD-Gal. Maleimido groups are known to react readily with accessible sulfhydryl groups (24). Chemical and structural analyses have demonstrated that the addition of 1-thio-β-D-galactopyranose to MAD occurred at the maleimido group. The reaction produced two stereoisomeric glycosylated photoreagents. No attempts have been made to separate the isomers. Diamond surfaces possess various types of surface functions (39) which figure as candidates for carbene

174 Bioconjugate Chem., Vol. 10, No. 2, 1999

insertion. Surface analysis techniques provided strong evidence that MAD-Gal was successfully grafted to the diamond surface. The surface analyses and related phenomena are described in detail in (42). They also indicated that the applied wash procedure removed most but not all of the physically adsorbed molecules. A total of 10-8 mol/cm2 (6.02 × 1015 molecules/cm2) of MAD-Gal was deposited at the sample surfaces. The XPS signal of the molecule after photoimmobilization and washing was close to the detection limit of this technique. Therefore, the surface density of the molecule was estimated to be 1012-1013 molecules/cm2 (20). The efficiency of the grafting is estimated to be between 1% and 1‰. Optimization of the grafting will be considered. However, for future purposes (i.e. biological applications), such densities should not be a problem. Biomolecules can exhibit biological activity at very low concentration, as demonstrated by Massia et al. (43) in the case of surface-grafted peptides. ToF-SIMS imaging allowed visualization of topically addressed MAD-Gal. However, the patterns obtained were not as distinct as expected. A possible explanation is the position of the mask during the illumination, which was positioned above but not close enough to the surface, and the diffraction of light under the mask. In summary, the new photoreagent MAD-Gal was successfully synthesized as demonstrated by 13C NMR, 1H NMR, and UV absorption spectroscopy. Photoimmobilization of MAD-Gal on diamond surfaces via diazirine insertion was illustrated by XPS and ToF-SIMS surface analyses. The feasibility of surface patterning was demonstrated, although the lateral resolution was limited. Biological tests are in progress to identify the remaining biological activity of photoimmobilized MAD-Gal molecules. Primary results with rat hepatocyte cultures are encouraging. However, the implicated surface orientation of MAD-Gal awaits further experimental documentation with biological systems. ACKNOWLEDGMENT

Financial support from the Swiss Priority Program on Material Research is gratefully acknowledged. LITERATURE CITED (1) Lee, Y. C., and Lee, R. T. (1995) Carbohydrate-Protein Interaction: Basis of Glycobiology. Acc. Chem. Res. 28, 321327. (2) Monsigny, M. (1995) Glycotechnologies: les Biotechnologies Douces. Biofutur 142, 27-32. (3) Petrak, K. (1994) Introduction: Physical Basis of Specific Interactions Involving Carbohydrates. Adv. Drug Deliv. Rev. 13, 211-213. (4) Hatanaka, K., Takeshige, H., and Akaike, T. (1994) Synthesis of New Polymer Containing Uridine and Galactose as Pendent Groups. Carbohydr. Chem. 13, 603-610. (5) Hermanson, G. T., Mallia, A. K., and Smith, P. K. (1992) Immobilized Affinity Ligand Techniques (G. T. Hermanson, A. K. Mallia, and P. K. Smith, Eds.), pp 158-167, Academic Press, London. (6) Largent, B. L., Walton, K. M., Hoppe, C. A., Lee, Y. C., and Schnaar, R. L. (1984) Carbohydrate-Specific Adhesion of Alveolar Macrophages to Mannose-Derivatized Surfaces. Biol. Chem. 259, 1764-1769. (7) Kobayashi, K., Kobayashi, A., and Akaike, T. (1994) Culturing Hepatocytes on Lactose Carrying Polystyrene Layer via Asialoglycoprotein Receptor-Mediated Interactions. Methods Enzymol. 247, 409-419. (8) Onyiriuka, E. C. (1996) Modification of Polystyrene Cell Culture Surfaces by Grafting a Thin Film of Starch. J. Adhes. Sci. Technol. 10, 617-633.

Chevolot et al. (9) Kobayashi, K., Akaike, T., and Usui, T. (1994) Synthesis of Poly(N-Acetyl-β-Lactosaminide-Carrying Acrylamide): Chemical-Enzymatic Process. Methods Enzymol. 242, 226-235. (10) Guire, P. E. (1990) Method of Improving the Biocompatibility of Solid Surfaces (Bio-Metric Systems, Inc.). U.S. Patent 4,973,493. (11) Guire, P. E. (1990) Biocompatible Coating for Solid Surfaces (Bio-Metric Systems, Inc.). U.S. Patent 4,979,959. (12) Anderson, A. B., Enrico, L. S., Melchior, M. J., Pietig, J. A., Tran, L. V., Tran, T. H., and Duquette, P. H. (1994) Photochemical Immobilization of Heparin to Reduce Thrombogenesis. Proceedings of 20th Annual Meeting in conjunction with the 26th International Biomaterials Symposium, Boston (Society of Biomaterials, Ed.), p 75. (13) Courtney, J. M., Yu, J., and Sundaram, S. (1993) Immobilization of Macromolecules for Obtaining Biocompatible Surfaces. Immobilised Macromolecules: Application Potentials (U. B. Sleytr, Ed.), pp 175-193, Springer, London. (14) Erdtmann, M., Keller, R., and Baumann, H. (1994) Photochemical Immobilization of Heparin, Dermatan Sulphate, Dextran Sulphate and Endothelial Cell Surface Heparan Sulphate onto Cellulose Membrane for the Preparation of Athrombogenic and Antithrombogenic Polymers. Biomaterials 15, 1043-1049. (15) Anderson, A. B., Tran, T. H., Hamilton, M. J., Chudzik, S. J., Hasting, B. P., Melchior, M. J., and Hergenrother, R. W. (1996) Platelet Deposition and Fibrinogen Binding on Surfaces Coated with Heparin or Friction-Reducing Polymers. Am. J. Neuroradiol. 17, 859-863. (16) Adachi, N., Maruyama, A., Ishihara, T., and Akaike, T. (1994) Cellular Distribution of Polymer Particles Bearing Various Densities of Carbohydrate Ligands. J. Biomater. Sci. Polymer Edn. 6, 463-479. (17) Sigrist, H., Collioud, A., Cle´mence, J. F., Gao, H., Luginbuhl, R., Sanger, M., and Sundarababu, G. (1995) Surface Immobilization of Biomolecules by Light. Opt. Eng. 34, 23392348. (18) Gao, H., Sanger, M., Luginbuhl, R., and Sigrist, H. (1995) Immunosensing with Photoimmobilized Immunoreagents on Planar Optical Wave Guides. Biosensors and Bioelectronics 10, 317-328. (19) Le´onard, D., Chevolot, Y., Bucher, O., Tang, X. M., Mathieu, H. J., and Sigrist, H. (1997) Aryldiazirine photoimmobilisation on silicon, silicon nitride and diamond substrates studied by XPS and ToF-SIMS. Proceedings of ECASIA, Go¨teborg (I. Olefjord, L. Nyborg, and D. Briggs, Eds.), pp 135-138, Wiley, Chichester, U.K. (20) Le´onard, D., Chevolot, Y., Bucher, O., Sigrist, H., and Mathieu, H. J. (1998) ToF-SIMS and XPS Study of Photoactivatable Reagents designed for Surface Glycoengineering: Part 1: N-(m-(3-(trifluoromethyl) diazirine-3-yl) phenyl)-4maleimido-butyramide (MAD) on silicon, silicon nitride and diamond. Surf. Interface Anal. 26, 783-792. (21) Gao, H., Luginbuhl, R., and Sigrist, H. (1997) Bioengineering of Silicon Nitride. Sens. Actuators, B 38-39, 38-41. (22) Shergold, H. L., and Hartley, C. J. (1982) The Surface Chemistry of Diamond. Int. J. Miner. Process. 9, 219-233. (23) Cle´mence, J. F., Ranieri, J. P., Aebischer, P., and Sigrist, H. (1995) Photoimmobilization of a Bioactive Laminin Fragment and Pattern-Guided Selective Neuronal Cell Attachment. Bioconjugate Chem. 6, 411-417. (24) Collioud, A., Cle´mence, J. F., Sanger, M., and Sigrist, H. (1993) Oriented and Covalent Immobilization of Target Molecules to Solid Supports: Synthesis and Application of a Light-Activable and Thiols Cross-Linking Reagent. Bioconjugate Chem. 4, 528-536. (25) Dion, I., Baquey, C., and Monties, J. R. (1993) Diamond: the Biomaterial of the 21st Century? Int J. Artif. Organs 16, 623-627. (26) Butter, R. S., and Lettington, A. H. (1995) Diamond-Like Carbon for Biomedical Applications (review). J. Chem. Vapor Deposition 3, 182-192. (27) Tang, L., Tsai, C., Gerberich, W. W., Kruckeberg, L., and Kania, D. R. (1995) Biocompatibility of Chemical-VaporDeposited Diamond. Biomaterials 16, 483-488.

A Photoactivatable Glycoaryldiazirine (28) Go¨pel, W., and Heiduschka, P. (1995) Interface Analysis in Biosensor Design. Biosens. Bioelectron. 10, 853-883. (29) Perkin-Elmer (1992) Multi-Technique ESCA Operator’s Reference Manual (625612: C), Perkin-Elmer, Eden Prairie, USA. (30) Benndorf, C., Hadenfeldt, S., Luithardt, W., and Zhukov, A. (1996) Photoelectron Spectroscopic Investigations and Exoelectron Emission of CVD Diamond Surfaces Modified with Oxygen and Potassium. Diamond Relat. Mater. 5, 784789. (31) Franzreb, K., Mathieu, H. J., and Landolt, D. (1995) Reactive Ion Sputter Depth Profiling of Tantalum Oxides: a Comparative Study Using ToF-SIMS and Laser-SNMS. Surf. Interface Anal. 23, 641-651. (32) Schueler, B. W. (1992) Microprobe Imaging by Time-ofFlight Secondary Ion Mass Spectrometry. Microsc. Microanal. Microstruct. 3, 119-139. (33) Briggs, D. (1992) Static SIMS-Surface Analysis of Organic Materials. Practical Surface Analysis (D. Briggs and M. P. Seah, Eds.) pp 367-423, Wiley, Chichester, U.K. (34) Mueller, C., Haenni, W., Binggeli, M., and Hintermann, H. E. (1993) Uniform Diamond Coatings on 4 in Si Wafers. Diamond Relat. Mater. 2, 1211. (35) Habeeb, A. F. S. A. (1972) Reaction of Protein Sulfhydryl Groups with Ellman’s Reagent. Methods Enzymol. 25, 457464. (36) Moulder, J. F., Stickle, W. F., Sobol, P. E., and Bomben, K. D. (1992) Handbook of X-ray Photoelectron Spectroscopy (J. Chastain, Ed), Perkin-Elmer, Eden Prairie, MN. (37) Davies, M. C., Lynn, R. A. P., Davis, S. S., Hearn, J., Watts, J. F., Vickerman, J. C., and Paul, A. J. (1993) Preparation of

Bioconjugate Chem., Vol. 10, No. 2, 1999 175 Polymer Latex Particles with Immobilized Sugar Residues and their Surface Characterisation by X-ray Photoelectron Spectroscopy and Time-of-Flight Secondary Ion Mass Spectroscopy. Langmuir 9, 1637-1645. (38) Beamson, G., and Briggs, D. (1992) High-Resolution XPS of Organic Polymers, Wiley, Chichester, U.K. (39) Evans, S. (1992) Surface Properties of Diamond. The Properties of Natural and Synthetic Diamond (J. E. Field, Ed.), pp 181-214, Academic Press, London. (40) MacNamara, K. M., Williams, B. E., Gleason, K. K., and Scruggs, B. E. (1994) Identification of Defects and Impurities in Chemical-Vapor-Deposited Diamond through Infrared Spectroscopy. J. Appl. Phys. 76, 2466-2472. (41) Le´onard, D., Chevolot, Y., Bucher, O., Tang, X. M., Mathieu, H. J., and Sigrist, H. (1998) ToF-SIMS Study of Photoactivatable Reagents Designed for Surface Glycoengineering. Proceedings of SIMS XI, Orlando (G. Gillen, J. B. R. Lareau, and F. Stevie, Eds.), pp 529-532, Wiley, Chichester, U.K. (42) Le´onard, D., Chevolot, Y., Bucher, O., Sigrist, H., and Mathieu, H. J. (1998) TOF-SIMS and XPS Study of Photoactivatable Reagents designed for Surface Glycoengineering: Part 2: N-(m-(3-(trifluoromethyl) diazirine-3-yl) phenyl)4-(-3-thio (1-D-galactopyranosyl)-succinimidyl) -butyramide (MAD-Gal) on diamond. Surf. Interface Anal. 26, 793-799. (43) Massia, S. P., and Hubbell, J. A. (1991) An RDG Spacing of 440 nm is Sufficient for Integrin, Rvβ3-mediated Fibroblast Spreading and 140 nm for Focal Contact and Stress Fiber Formation. J. Cell Biol. 114, 1089-1100.

BC980050H