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Bioconjugate Chem. 2002, 13, 1286−1291
Synthesis and Photochemical Properties of Nitro-Naphthyl Chromophore and the Corresponding Immunoglobulin Bioconjugate Anil K. Singh* and Prashant K. Khade Department of Chemistry, Indian Institute of Technology, Bombay, Powai, Mumbai - 400 076, India. Received March 28, 2002
Synthesis, photochemistry, and biomolecular caging properties of a new chromophore namely 3-nitro2-naphthalenemethanol are described. This chromophore is photoexcitable with photons in 350-400 nm range and in several solvents including aqueous medium. On irradiation, it gives the expected nitroso-aldehyde photoproduct with high quantum yield (0.6-0.8). Further, it can be conveniently coupled to the amino residues of immunoglobulin (IgG) using diphosgene. Irradiation of the resulting IgG-nitronaphthyl chromophore bioconjugate at 380 nm causes photorelease of IgG as evidenced by Protein-A affinity binding studies. The bioconjugate showed low level of binding to Protein-A. However, the binding increases after irradiation and, thus, modifies the Fc site of the IgG. Electrophoresis studies of the irradiated bioconjugate show that IgG does not undergo fragmentation or molecular weight change under the irradiation conditions. Thus, 3-nitro-2-naphthalenemethanol can be used as a photocaging agent under physiological conditions at wavelengths, which does not cause significant damage to the biomolecule. The work provides new directions for the development of organic chromophores for biomolecular caging applications.
INTRODUCTION
Photochemical release of biologically active component from “caged biomolecule” is an important strategy in the study of numerous aspects of structure and dynamics of biochemical and medical significance (1-5). This is because of the ability of light to control the site, time, and concentration of the released molecule. A caged biomolecule is defined as a biomolecule whose activity or function is lost or altered upon its chemical modification with a photolabile group (i.e., masking of the functional group(s) of the biomolecule by a photocleavable protecting reagent), and irradiation of the caged biomolecule with light results in removal of the protecting group with simultaneous rapid generation of the active or functional biomolecule. Such photochemical deprotection of masked functional groups of biomolecules presents a simple, noninvasive technology to investigate a wide range of cellular activities and gives rise to many practical applications including site directed drug delivery. Several photocleavable nitroaryl groups have been employed as protecting groups in organic synthesis (6). Further, nitroaryl chromophore-based photoactivable caged nucleotides (7-12) and proteins and peptides (1323) have been developed to study intracellular dynamics, metabolism, and enzymatic activities. More recently, applications of photocleavable chromophores have been made in site-directed photocleavage for mapping protein architecture (24), synthesis of difficult cyclic peptides by inclusion of photolabile nitrobenzyl auxiliary in a ring contraction strategy (25), and selective inhibition, separation, and purification of serine proteases (26). In most cases, caging of the biomolecules has been done with ortho-nitrobenzyl chromophoric group, which ab* To whom correspondence should be addressed. Phone: (091-22) 576 7167. Fax: (091-22) 576 7152. E-mail: Retinal@ chem.iitb.ac.in.
sorbs mainly in the ultraviolet region, a wavelength that may not be very suitable for biomolecular applications. For practical purposes, it is important to have photocleavable systems, which are useful under physiological conditions and can be photoexcited at a number of wavelength and more importantly at wavelengths which are less damaging to the biological systems. In this paper are described synthesis, photochemistry, and biomolecular caging properties of a new chromophore, namely, 3-nitro-2-naphthalenemethanol (7). It is photoexcitable in 350-400 nm range, and it shows expected photochemical reaction of ortho-nitroaryl group giving the nitroso-aldehyde photoproduct. Further, 7 can be conveniently coupled to the amino residues of immunoglobulin (IgG) using diphosgene method via chloroformate 8. Irradiation of the resulting IgG-7 biconjugate 9 at 380 nm causes photorelease of IgG, as evidenced by Protein-A affinity binding studies. It has been demonstrated that chromophore 7 can be employed to cage proteins and the conjugated proteins can be photochemically released at wavelengths which are less damaging to the biological matrix in aqueous medium. EXPERIMENTAL PROCEDURES
Chemicals, Apparatus, and General Methods. Starting materials for synthesis were obtained from M/s. Lancaster and M/s. SRL, India Ltd. Mumbai. Human immunoglobulin (IgG), gel electrophoresis chemicals, and buffers were purchased from M/s. Bangalore Genie, India Ltd. Bangalore. Diphosgene was obtained from the Indian Institute of Chemical Technology, Hyderabad. Protein-A sepharose CL-4B was purchased from Pharmacia Biotech. Synthetic compounds were purified by column chromatography using 60-120 mesh silica gel. Deionized and double distilled water was used for buffer preparation, electrophoresis, and affinity experiments. Melting points were recorded on Veego melting point apparatus and are uncorrected. UV-vis spectra were
10.1021/bc020021d CCC: $22.00 © 2002 American Chemical Society Published on Web 11/05/2002
Properties of Nitro-Naphthyl Chromophore
recorded on Shimadzu Uv-260 spectrophotometer. FTIR spectra were measured on Impact Nicolet-400 spectrophotometer. 1H NMR spectra were recorded on Varian 300 MHz NMR spectrometer using TMS as the internal standard. Mass spectra were recorded on GCD 1800A Hewlett-Packard GC-MS spectrometer. Elemental analysis was performed on Theroquest CE instrument-1112 series CHN auto-analyzer. Protein samples were centrifuged at 4 °C in L8M55 Beckman ultracentrifuge equipped with Ti-45 rotor. Electrophoresis experiments were performed on a vertical gel electrophoresis instrument (mini model) from M/s. Bangalore Genie. Irradiations were done using a 400 W medium pressure mercury lamp (Applied Photophysics Ltd., London, Model R-607), fitted either with a f/3.4 monochromator or with a glass filter to isolate the desired wavelength for irradiation. The organic solvents used for various electronic measurements or photochemical purposes were freshly distilled and thoroughly deoxygenated by bubbling dry nitrogen. The irradiated solutions were analyzed by UV-vis, FTIR, and 1H NMR methods. The relative quantum yield (ΦPR) was determined against potassium ferrioxalate actinometer (27). Synthesis of 3-Nitro-2-naphthalenemethanol (7). Compound 7 was synthesized by reacting phthalaldehyde (5) with 3-nitropropionic ethyl ester (4) in the presence of sodium ethoxide in absolute ethanol as previously described (28). Reduction of the resulting 3-nitro-2naphthoic acid (6) with NaBH4-BF3-Et2O in 1,2dimethoxyethane followed by usual workup and column chromatographic purification yielded the desired alcohol 7: yield, 40%; mp, 118-19 °C [lit. (28) mp 118-19 °C]. UV-vis: λmax, nm (, mol-1 cm-1 L) in CH3CN, 350 (2285). FTIR (KBr): vmax (cm-1) 3458-3372 (OH), 1513, 1331 (NO2). 1H NMR (DMSO): δ 4.91 (2H, d, J ) 5.12 Hz, -CH2), 5.90 (1H, t, -OH), 7.75-7.59 (2H, m, H-C6 and H-C7), 8.06 (1H, d, J ) 8.24, H-C8), 8.17 (1H, d, J ) 8.05 Hz, H-C5), 8.24 (1H, s, H-C1), 8.75 (1H, s, H-C4). MS m/z (%): 205 (M+ + 2), 171 (74), 143 (100), 126 (55), 115 (58). Anal. Calcd for C11H9NO3: C, 65.05; H, 4.47; N, 6.90. Found: C, 65.49; H, 4.33; N, 6.72. Preparation of 3-Nitro-2-naphthyloxycarbonyl chloride (8). In a typical procedure, 3-nitro-2-naphthalenemethanol (7, 0.005 g, 0.25 mmol) was treated with diphosgene (20 µL) and dry pyridine (8 µL) as a catalyst in dried and freshly distilled 1,4-dioxane (150 µL). A yellow precipitate was immediately formed. The reaction mixture was magnetically stirred at ambient temperature for 24 h. The reaction was monitored by TLC using 20% ethyl acetate in petroleum ether (60-80 °C fraction). 1,4-Dioxane, pyridine, and excess of diphosgene were evaporated by using a stream of dry nitrogen. The residue was taken-up in dry 1,4-dioxane (125 µL), and the resulting solution was used for protein conjugation. The chloroformate 8 showed expected IR peaks at 1738 (-OCO) and 1535 and 1377 (-NO2) cm-1. Coupling of Chloroformate 8 with IgGsPreparation of Bioconjugate 9. In a typical experiment, human immunoglobulin (IgG, 0.004 g) was taken in sodium bicarbonate (4 mL, 0.1 M; pH, 8.3) in a 10 mL roundbottom flask. To this was added 80 µL of chloroformate 8. The reaction mixture was left overnight for coupling at 4 °C. Subsequently, the mixture was dialyzed twice against 200 mL each of 0.153 M sodium chloride (pH, 6.7). The white insoluble complexes were removed by centrifugation at 15 000g for 15 min at 4 °C to obtain bioconjugate 9 as clear solution. To this was added a solution of 9 4 mL of 0.153 M aq. sodium chloride (pH 6.7), and the resulting solution was used for further
Bioconjugate Chem., Vol. 13, No. 6, 2002 1287
study. The bioconjugate thus prepared showed UV-vis band extending up to 410 nm. To determine the maximum number of chromophores that bind to IgG, the coupling of IgG to chloroformate 8 was done by using different amounts of 8 (0 µL as the control, and 5,10, and 20 µL aliquots of chloroformates) in 0.001 g of IgG solution in 1 mL of sodium bicarbonate buffer (0.1 M, pH, 8.3). It has been found that 20 µL of 1,4-dioxane solution of chloroformate 8 is sufficient to maximally modify the IgG. The number of chromophores coupled to the protein was determined as follows. The concentration of chromopohre in bioconjugate was obtained from the absorbance value at the absorption maximum at 350 nm due to the chromophore. The concentration of protein in the bioconjugate was determined by considering its absorbance at 280 nm. However, in bioconjugate there is present absorption contribution by the chromophore also. Therefore, first the absorption contribution of the chromophore at 280 nm was determined from its absorbance at 280 nm in the bioconjugate, followed by subtracting it from the total absorbance of the bioconjugate at 280 nm. In calculating the concentration of chromophore from the absorbances obtained from bioconjugates, the extinction coefficients of chromophore at 280 and 350 nm in ethanol were used. From the concentrations, the number of molecules of IgG and the chromophore were calculated. Thus, the relative number of chromophores attached to the protein was obtained. Irradiation of Compound 7 and Bioconjugate 9. A solution of 7 in acetonitrile (3 mL of 1.0 × 10-4 M) taken in a quartz cuvette and degassed by passing dry nitrogen was irradiated at 380 nm at ambient temperature. The distance between the sample and the lamp was kept 35 cm. The progress of the photochemical reaction was monitored spectrophotometrically by observing the changes at 350 nm. The photomixture was subsequently analyzed by FTIR and 1H NMR analysis. The dialyzed solution of bioconjugate 9 (3 mL, 0.153 M NaCl, pH, 6.7) was irradiated at 380 nm under the same conditions as described above. The bioconjugate was also irradiated with light filtered with a glass filter having 47% and 96% transmittance at 370 and 420 nm, respectively. Under this irradiation condition, the distance between the sample and the 400 W mediumpressure mercury lamp was kept 2 cm. The progress of the photolysis was monitored spectrophotometrically. One aliquot of bioconjugate sample being irradiated was removed time to time and eluted from Protein-A affinity column. In a control experiment, the native IgG solution (3 mL, 0.153 M NaCl, pH 6.7) was subjected to irradiation under conditions identical to the irradiation conditions of conjugate 9, and its binding affinity to protein-A was also studied. Affinity Chromatography Studies of Bioconjugate Samples. A 3 cm long and 0.5 cm wide column was packed with Protein-A and equilibrated with 0.05 M Trisbuffer (pH, 7.4). One aliquot (0.5 mL) of irradiated bioconjugate sample was removed time to time and loaded on the affinity column, which was washed with the binding buffer until the absorbance of the eluted solution at 280 nm approached background level. The column was then eluted with 0.05 M glycine buffer (pH, 3.0) to remove the bound IgG protein and washed until the absorbance of elute at 280 nm approached the background level. The fractions collected were neutralized by addition of 100 µL of 1.0 M glycine buffer (pH, 9) in each test tube. Subsequently, the column was washed with 25 mL of binding buffer (0.05 M Tris-buffer, pH 7.4)
1288 Bioconjugate Chem., Vol. 13, No. 6, 2002 Scheme 1
Singh and Khade Table 1. UV-Vis Absorption Spectrala and Quantum Yield (ΦPR) Data of 7 solvent
λ/nm
/M L-1 cm-1
1,4-dioxane
217 258 265 302 345 215 256 263 301 350 222 256 263 303 346 217 259 306 350
70 000 28 000 26 667 8 466 3 333 32 000 24 000 24 000 5 547 2 285 25 000 30 000 26 667 6 655 3 000 12 323 7 298 2 435 1 289
acetonitrile
ethanol
0.153 NaCl
Scheme 2
and equilibrated with it. From the absorbance at 280 nm, the concentration of IgG protein was calculated. Protein-A binding affinity studies showed that unirradiated protein conjugate 9 binds to Protein-A to the extent of 18%. Electrophoresis Studies of IgG, Bioconjugate, and Photoreleased IgG. The sample buffer (50 µL, 1.0 × 10-7 M) containing 10% glycerol and SDS, 0.5% bromophenol blue, and 0.5 M Tris-HCl (pH, 6.8) was added separately to irradiated bioconjugate, unirradiated bioconjugate, and unconjugated, native IgG. The mixture was heated to 85 °C for 10 min. The various protein samples, including 50 µL each of irradiated bioconjugate, unirradiated bioconjugate, unconjugated, native IgG, and the marker proteins in 14-200 kD MW range, which contain 5 µL of denaturing buffer were loaded on 10% SDS-polyacrylamide discontinuous gel. The electrophoresis was done in nonreducing conditions by applying 90120 V and 10 A current. The electrophoresis was stopped when bromophenol blue reached to the bottom of the gel and the gels were stained (brilliant blue G stain, 0.1% w/v brilliant blue G, 25% methanol, 7% acetic acid). The destaining was done by 40% methanol, 10% acetic acid and 50% deionized water. RESULTS AND DISCUSSION
Synthesis of 3-Nitro-2-naphthalenemethanol (7) and IgG-7 Bioconjugate (9). Synthesis of compound 7 and its IgG-bioconjugate 9 was done by following the steps outlined in Schemes 1 and 2. Esterification of 3-bromopropanoic acid (1) with ethanol gave ester 3, which on reaction with silver nitrate yielded the nitroester 4. The reaction of nitroester 4 with phthaldehyde 5 in the presence of sodium ethoxide, followed by acidic hydrolysis (of the ester group), resulted in the formation of nitro-naphthoic acid 6, which on subsequent reduction
ΦPR at 380 nm
0.82
0.796
0.75
0.63
a Absorption coefficient at 280 nm: 6 826 (1,4-dioxane), 5 124 (acetonitrile), 6 200 (ethanol) ,and 2 124 (0.153 M NaCl). At 380 nm: 1 415 (1,4-dioxane), 1 264 (acetonitrile), 1 350 (ethanol), and 809 (0.153 M NaCl).
with sodium borohydride gave the desired compound 7 in an overall yield of 40%. Compound 7 showed a mp identical to the mp reported in the literature (28). However, compound 7 was satisfactorily characterized additionally by other physicochemical methods, including electronic absorption, FTIR, 1H NMR, mass, and elemental analysis as described in the Experimental Section. Bioconjugate 9 could conveniently be prepared via chloroformate 8. As outlined in Scheme 2, reaction of compound 7 with diphosgene yielded chloroformate 8, which reacted with the amino residues of IgG to give the desired bioconjugate 9 in about 40% yield. Under the present preparative conditions, about 22 ((2) molecules of 8 coupled to a molecule of IgG. The IgG has a total of 50 lysine units and it is possible that 22 ((2) -amino groups of lysine in IgG underwent coupling with 8 in the present experimental conditions. Electronic Absorption and Photochemical Properties of 3-Nitro-2-naphthalenemethanol (7). The UV-vis absorption spectral data and the ΦPR of 7 are summarized in the Table 1. This compound exhibits major absorption bands in 220-250 and 300-350 nm range with tail end absorption extending up to 410 nm for a 1.0 × 10-4 M solution. The longer wavelength absorptions of 7 can be due to the 1La (vertically polarized) transition of the naphthyl group. The molar absorption coefficient of the band in 220-250 range is larger than the band in 300-350 nm range. The UV-vis absorption of 7 remains similar in a variety of solvents including acetonitrile, ethanol, 1,4-dioxane, and 0.153 M aq. NaCl (pH, 6.7). However, the molar absorption coefficient varies with the solvent. An ethanolic solution of 7 shows absorption at 346 nm with molar absorptivity of about 3000 mol-1 cm-1 L. However, for the same solution, the molar absorptivity is 1350 mol-1 cm-1 L for absorption at 380 nm. Compound 7 in aq. NaCl medium shows absorption at 350 nm with a molar absorptivity of about 1 289 mol-1 cm-1 L. Its molar absorptivity at 380 nm is found to be about 809 mol-1 cm-1 L. Typical UV-vis absorption changes during 380 nm irradiation of a 1.0 × 10-4 M acetonitrile solution of 7 are shown in Figure 1. The FTIR spectrum of the photomixture is characterized by the presence of bands at 1 532 and 1 341 cm-1 due to nitroso group and 1 703
Properties of Nitro-Naphthyl Chromophore
Bioconjugate Chem., Vol. 13, No. 6, 2002 1289
Figure 1. UV-vis absorption spectral changes during 380 nm photolysis of a 1.0 × 10-4 M acetonitrile solution of 7.
cm-1 due to aldehyde group. The alcohol group peak (3 372-3 458 cm-1) that was present in the starting chromophore disappeared after irradiation. The 1H-NMR spectrum of the photomixture showed peak at δ 10.50 due to an aldehyde proton. Similar photobehavior was observed when 7 was irradiated in protic ethanol, nonpolar 1,4-dioxane, and 0.153 M aq. NaCl solutions, indicating the absence of any solvent effect on the photochemical properties of 7. Irradiation of 7 at 300, 350, and 400 nm also showed photochemical product profile similar to its irradiation at 380 nm. The above photochemical and spectroscopic results suggest that compound 7, on irradiation with photons in 300 to 400 nm range, undergoes the expected lightinduced internal oxidation-reduction reaction of aromatic nitro compounds containing a carbon-hydrogen bond ortho to the nitro group (6). The primary photochemical process in this reaction is an intramolecular hydrogen abstraction by the nitro group generating free radical species; this is then followed by electron redistribution to the aci-nitro form, which subsequently rearranges to the nitroso-aldehyde photoproduct (10) (Scheme 3). Thus, it has been possible to develop a chromophore that can be photoexcited with desired photochemistry at wavelengths greater than 350 nm. Electronic Absorption and Photochemical Properties of Bioconjugate 9. The UV-vis absorption spectrum of bioconjugate 9 in 0.1 M aq. NaCl solution is characterized by absorption bands at 198 and 266 nm, with extended absorption up to 410 nm. The IgG protein peak at 278 nm is masked due to the absorption by chromophoric group of 7 having absorption in 250-300 nm range. The bioconjugate can, however, be photoexcited by taking the advantage of absorption in 350-410 nm, where unconjugated protein has insignificant absorption. Thus, a 1.0 × 10-5 M solution of bioconjugates 9 in 0.9% (0.153 M) aq. NaCl was irradiated at 380 nm. The irradiation resulted in changes in the absorption at 266 nm (Figure 2). The irradiated bioconjugate samples were analyzed by affinity chromatography on a Protein-A
Scheme 3
sepharose CL4B column, and the extent of binding of photoreleased IgG was determined (Figure 3). The bioconjugate sample irradiated for 3 h showed 70% binding affinity to Protein-A. However, bioconjugate samples irradiated for 5 and 7 h showed binding affinity of only 50% and 40%, respectively, to Protein-A. Thus, the binding affinity was increased from 18% (the extent to which unirradiated IgG binds to Protein-A) to 70% after 3 h of irradiation. However, the binding affinity decreased to 40% after 7 h of irradiation. As control experiments, uncoupled and unirradiated native IgG was irradiated for 0 (control), 3, 5, and 7 h at 380 nm, and the binding affinity for such samples was found to be 100%, 85%, 83%, and 73% respectively. Further, a sample of bioconjugate 9 irradiated for 40 min using a glass filter having 47% and 96% transmittance at 370 and 420 nm, respectively, showed binding affinity to Protein-A to the extent of 74%. Continued irradiation up to 60 min caused further decrease in binding affinity to 51%. This shows that as the intensity of the photons is increased, the rate of the photoreaction was increased. Thus, photorelease of IgG from the
1290 Bioconjugate Chem., Vol. 13, No. 6, 2002
Singh and Khade
Figure 2. UV-vis absorption spectral changes during 380 nm photolysis of a 1.0 × 10-5 M aq. NaCl (0.153 M) solution of bioconjugate 9. Figure 4. Electrophoresis of various protein samples. (1) Irradiated bioconjugate. (2) Unirradiated bioconjugate. (3) Native IgG. (4) Marker proteins. Scheme 4
Figure 3. A plot of irradiation time vs percent Protein-A binding affinity. (O) Photo-released IgG (upon irradiation of bioconjugate 9), and (b) native IgG (which was subjected to irradiation under conditions identical to the irradiation of bioconjugate 9).
bioconjugate can be effected in much shorter time by use of appropriate light source (e.g. lasers). It is believed that irradiation of bioconjugate 9 results into the photoconversion of its nitronaphthyl chromophore to nitroso-aldehyde and subsequent release of IgG, as outlined in Scheme 4. The primary photochemical process of bioconjugate 9 is similar to the photochemical process of ortho-nitroaryl chromophores having abstractable hydrogen atoms in the vicinity (6). Overall, the photochemical reaction of bioconjugate 9 results into release of IgG via the formation of nitroso-aldehyde 10 and carbon dioxide. The decrease in binding can be due to the artificial irradiation in case of control protein and in case of coupled protein it may be due to the artificial irradiation as well as due to reaction or association of the nitrosoaldehyde photoproduct (10) with the photoreleased free protein. These results further show that the nitro-naphthyl chromophore modifies the FC site of IgG molecule. The modified protein showed low level of binding, and after irradiation, binding of released protein to Protein-A was increased. This can be due to the fact that irradiation of the conjugate leads to photorelease of the nitro-naphthyl chromophore from the Fc site of IgG, which then becomes available for binding to Protein-A. Electrophoresis studies of unconjugated native IgG (as control), unirradiated bioconjugate (9 as the control), and irradiated bioconjugate in discontinuous gel and nonreducing conditions showed that irradiation does not cause
any fragmentation or molecular weight changes in IgG (Figure 4). UV-vis absorption and electrophoresis studies thus indicate IgG protein molecules are not fragmented under the present irradiation conditions. In conclusion, it can be said that 3-nitro-2-naphthalenemethanol 7 can be used for caging of biomolecules. It can be photoexcited in aqueous solution and at wavelengths that do not cause significant damage to the biomolecules. Further, this chromophore is photoexcitable at many wavelengths in 300-400 nm range. This work provides new directions for the development of organic chromophores for biomolecular caging applications. ACKNOWLEDGMENT
A research grant [01/1509/98-EMR-II] for this work from the Council of Scientific and Industrial Research, New Delhi, Government of India, is gratefully acknowledged. LITERATURE CITED (1) McCray, J. A., and Trentham, D. R. (1989) Properties and uses of photoactive caged compounds. Annu. Rev. Biophys. 18, 239-270. (2) Kaplan, J. H. (1990) Photochemical manipulation of divalent cation levels. Annu. Rev. Physiol. 52, 897-914. (3) Adams, S. R., and Tsien, R. Y. (1993) Controlling cell chemistry with caged compounds. Annu. Rev. Physiol. 55, 755-784.
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BC020021D
