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Bioconjugate Chem. 1996, 7, 536−540
6-Fluoropyridoxal Polymer Conjugates: Novel Magnetic Resonance Spectroscopy
19F
pH Indicators for
Vimal D. Mehta,† Aravind Sivasubramanian, Padmakar V. Kulkarni,* Ralph P. Mason, and Peter P. Antich Department of Radiology, University of Texas Southwestern Medical Center, Dallas, Texas 75235. Received March 8, 1996 X
Fluorinated macromolecular probes (6-fluoropyridoxal-polymer conjugates) have been synthesized and characterized as potential pH indicators for magnetic resonance spectroscopy and imaging applications. The 19F pH sensor 2-fluoro-5-hydroxy-3-(hydroxymethyl)-6-methyl-4-pyridinecarboxaldehyde (6-fluoropyridoxal; 2) has been conjugated to carrier molecules (polyamino dextran, polylysine, and albumin) by reductive alkylation for enhanced vascular retention and tissue targeting. The pH indicator polymer conjugates were purified by exhaustive dialysis and isolated in good yields (6684%). The 6-fluoropyridoxal-polymer conjugates exhibit excellent 19F pH sensitivity and pKa suitable for in vivo studies. The potential application of these polymeric indicators has been demonstrated in whole blood. These novel macromolecular pH probes offer a new approach for studying tissue physiology.
The important role of pH in developmental processes has stimulated the need for better methods of measuring pH in cells, organelles, and microenvironments (1, 2). Deviations of tissue pH and the pH gradient are found to accompany many disease states and may cause dysfunction. In addition, the efficacy of various therapeutic modalities in cancer therapy, notably chemotherapy and hyperthermia, is strongly influenced by pH. Thus, methods of measuring pH could have far-reaching applications in biomedicine, both for physiology research and in the clinic. Consequently, a variety of new methods have been introduced for measurement of cellular pH employing exogenous pH reporter molecules (indicators). Several specific attributes apply to an ideal pH indicator. It should be nontoxic and nonmetabolizable, and the pKa should be of the same order of magnitude as the pH to be determined. For NMR indicators the chemical shift difference between protonated and deprotonated species should be large to resolve small pH differences (ideally >1 ppm/pH unit) and the pH-dependent chemical shift should be independent of other physiological parameters (e.g., ionic strength, temperature, and plasma proteins). Preferably, the ionized forms are in fast exchange yielding a single narrow resonance. Traditional techniques of pH measurement are invasive (microelectrodes) or are limited to surface tissue (fluorescence). Methods based on positron emission tomography (PET) or nuclear magnetic resonance (NMR) are much less invasive and more suitable for human studies. PET of radiolabeled compounds (e.g., [11C]CO2 and [11C]dimethyloxazolidinedione) provides an estimate of aggregate pH (intra- and extracellular compartments), and the measurements may be difficult to interpret (3). In contrast, NMR may provide simultaneous intra- and extracellular pH determinations and can be entirely * Address correspondence to this author at the Department of Radiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, Texas 75235-9058 [telephone (214) 648-2957; fax (214) 648-2991]. † Present address: CuraGen Corp., 322 E. Main St., Branford, CT 06405. X Abstract published in Advance ACS Abstracts, August 15, 1996.
S1043-1802(96)00052-3 CCC: $12.00
noninvasive. 31P NMR is relatively appealing since pH is determined on the basis of endogenous metabolites (relative chemical shift of inorganic phosphate and phosphocreatine) (4). However, measurements of pH using the chemical shift of inorganic phosphate (Pi) require caution because the pKa is affected by ionic strength, protein solvation, and temperature (5). In addition, 31P NMR suffers from low metabolite concentrations and the crowded overlapping resonances (5). Intracellular pH has also been reported on the basis of the chemical shift of histidine observed by 1H NMR, but this suffers severely from the intense water signal and multiple overlapping metabolite resonances (6). Thus, a variety of NMR pH reporter molecules have been synthesized as exogenous indicators possessing enhanced spectral characteristics. Although 1H (7), 13C (8), and 31P (9) nuclei have been exploited, the NMR characteristics of the 19F nucleus are particularly attractive. 19F is 100% naturally abundant and has NMR sensitivity approaching that of protons. There is a large chemical shift range with minimal background signal in vivo. Various fluorinated NMR indicators have been reported; however, early molecules were poorly sensitive to changes in pH (10) and others lacked the appropriate pKa (11). Recently several new classes of 19F indicators have been reported with significantly improved properties: these include derivatives of fluoroaniline (12), fluoroaminophenol (13), difluoroisobutyric acid (14), and 6-fluoropyridoxol [6-FPOL (1); a vitamin B6 analog] (15). The measurements of pH using NMR indicators generally require 19F concentrations in the range of a millimole (∼1 mM). We are actively pursuing the development of 19F MR agents (15-19) for mapping dynamic functional parameters such as pH, vascular properties, and perfusion with NMR. We have previously demonstrated the use and reliability of 6-FPOL (1) as a 19F pH indicator in whole blood (15) and the perfused heart (20). However, for in vivo applications it is desirable that the pH indicator undergo tissue-specific or, at least, compartment-specific distribution to reduce the effective dose of the indicator and achieve high temporal and spatial resolution. Enhanced vascular retention and tissue targeting have been © 1996 American Chemical Society
Bioconjugate Chem., Vol. 7, No. 5, 1996 537
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proposed on the basis of conjugating small molecules such as drugs (21, 22) and imaging agents (16, 18, 23-25) to polymers. Indeed, this concept was demonstrated for fluorescent pH indicators (e.g., SNARF-1) coupled to dextran (26). We have now adopted this approach to couple fluorinated NMR pH indicators to carrier molecules. We have previously conjugated a prototype indicator 3-fluorosalicylaldehyde (3-FSA) to polymers and observed an unusual reversal in the titration curve of the 19F signal for 3-FSA conjugates (17). Here, we have investigated the feasibility of conjugating the highly sensitive 19F pH sensing agents “fluorinated vitamin B6 analogs” to carrier polymers without compromising the pH sensing characteristics. The macromolecular pH indicators could be useful in assessment of disease states such as tumors, ischemia, stroke, and regions of inflammation. The fluorinated vitamin B6 analogs are ideally suited for attachment to carrier macromolecules due to their high 19F chemical shift sensitivity to pH (∼10 ppm), useful pKa, and multiple choices of functional groups in the pyridoxine ring for conjugation. The change in 19F chemical shift in vitamin B6 analog (1) with pH is mainly due to the protonation/deprotonation of the 3-position hydroxy group, which influences the electronic environment around fluorine at the 6-position (the effect being more pronounced due to the para location). Assuming that this structure sensitivity relationship holds, the remaining three positions (2, 4, and 5) in the pyridoxine ring could be exploited for conjugation to polymers without sacrificing the pH sensing capability of the pH indicator. As our first design option, we have conjugated 2-fluoro5-hydroxy-3-(hydroxymethyl)-6-methyl-4-pyridinecarboxaldehyde (6-fluoropyridoxal; 2) to carrier polymers through the aldehyde group at the 4-position in the pyridoxine ring by reductive alkylation. 6-Fluoropyridoxal (2) was obtained by selective oxidation (27) of 1 with MnO2. We
have used three different classes of polymers, namely polyaminodextran (40K) (16), polylysine (98K), and naturally occurring bovine serum albumin. Reaction of 6-fluoropyridoxal (6-FPAL; 2) with polymer1,2 produced a Schiff base, which on subsequent sodium borohydride reduction yielded 6-FPAL-polymer conjugates (3-5) (Scheme 1). Attempts to maximize the degree of labeling of polylysine with 6-FPAL (2-3 M excess) resulted in a polymer conjugate that was insoluble in water. By controlling the amounts of reagent (6-FPAL; 0.5 mol) in a reaction mixture, water soluble 6-FPAL-polylysine conjugate (4) was obtained. We attribute this to more 1 Reagents and Materials. Polyamino dextran (40K) was synthesized as previously reported (16). Polylysine (98K) and bovine serum albumin (BSA) were purchased from Sigma Chemical Co. S-Ethyl trifluorothioacetate, sodium trifluoroacetate (CF3COONa), phenitidine, and sodium bicarbonate (NaHCO3) were purchased from Aldrich Chemical Co. Spectra Por dialysis tubings (Fisher Scientific) with molecular weight cutoffs (MWCO) of 6-8K and 12-14K were used. All other reagents and solvents were of reagent grade unless otherwise specified.
Scheme 1a
a0.1
M NaHCO3, 6-fluoropyridoxal (2); NaBH4.
Table 1. Characteristics of pH Indicators (1, 2) and Conjugates (3-7) pH indicator 1 2 3 4 5 6 7
19F
pH 2.00 11.35 2.00 11.40 2.00 11.60 2.00 12.00 2.00 11.00 2.00 11.50 2.00 11.50
NMRa (δ) (ppm) -9.85 -19.61 -8.65 -19.30 -8.92 -19.44 -8.87 -19.75 -8.79 -19.14 -9.08 -19.38 -8.76 -19.48
pH sensitivity (∆δ) (ppm)
pKa
yield (%)
9.76
7.9
40
10.65
8.2
26
10.52
6.6
72b
10.88
5.9
62b
10.35
6.8
84b
10.30
9.3c
65
10.72
NDd
80b
a Chemical shift w.r.t. NaTFA (0 ppm). bBased on amount (mg) of derivatized product recovered in coupling procedure. c pKa of compound 6 was determined in 50% water and dimethyl sulfoxide solution. d Precise pKa of compound 7 could not be determined because this material came out of solution at neutral pH. ND, not determined.
extensive labeling which reduced the availability of free amino groups and hence water solubility. We did not encounter this problem with polyamino dextran and albumin. The pH indicator conjugates (3-5) were purified by exhaustive dialysis and isolated in good yields (62-84%, Table 1). A control experiment showed that dialysis successfully removed the reaction byproducts. 6-FPAL (1) was dissolved in 0.1 M NaHCO3 and stirred overnight followed by addition of NaBH4. The mixture was stirred for another 4 h and subjected to exhaustive dialysis. Lyophilization of the 6-FPAL dialyzate yielded no visible product. Purity and integrity of the polymers were assessed using gel permeation chromatography (GPC). GPC analyses showed a smooth profile similar to the starting macromolecules without formation of additional low or high molecular weight products. The IR spectra of conjugates (3-5) differed from those of starting materials, though there were no distinct wellresolved bands. The pH indicators (1, 2) and conjugates (3-5) exhibit a single sharp 19F NMR signal in acid and base as shown in Figure 1. Typical line widths for 6-FPAL (2) and the conjugate 3 are 23 and 55 Hz, respectively. We have determined the relative 19F NMR sensitivity of conjugate 4 compared to 6-FPOL (1). Relative sensitivity (16) is 2 Conjugation Procedure. Reaction of 6-FPAL (2) with polymers: the appropriate polymer (polyamino dextran, polylysine, or albumin) (100-200 mg) was dissolved in 0.1 M NaHCO3 (4050 mL). 6-FPAL (2) was added (0.5-3 M excess) to the polymer solution at room temperature with stirring and allowed to proceed overnight. NaBH4 (2 M excess) was added to the reaction mixture, and stirring was continued for another 4 h. The conjugate was extensively dialyzed using MWCO 6-8K and 12-14K dialysis tubings (sequentially) against water to remove low molecular weight materials (excess reagents). The solution was lyophilized to yield 6-FPAL-polymer conjugates (3-5).
538 Bioconjugate Chem., Vol. 7, No. 5, 1996
Figure 1. 282.3-MHz 19F NMR spectra of 6-FPAL (2) and conjugates 3 and 6 in acid (A, C, and & E) and base (B, D, and F), respectively, with CF3COONa as an external reference. Conjugates 3 and 6 show a single sharp 19F signal and similar pH sensitivities to the native indicator 6-FPAL (2).
Figure 2. 19F chemical shift of 6-FPAL (2, 4) and 6-FPALpolymer conjugate (4, 9) vs pH in water. The pKa for conjugate is shifted toward the physiological range.
defined here as the number of milligrams of conjugate 4 needed to give the same intensity of 19F NMR signal as that obtained with 1 mg of 6-FPOL (1). The NMR sensitivity provides an estimate of the average degree of molar substitution and in vivo NMR detectability of derivatized polymer. The average molar substitution ratio was found to be ∼25 6-FPAL per carrier molecule in the case of polylysine (4). We have assessed the chemical shift sensitivity and pKa using 19F NMR spectroscopy for each of the fluorinated vitamin B6 analogs (1, 2) and conjugates (3-5) over the pH range 2.0-12.0 (Figure 2 and Table 1) using sodium trifluoroacetate (CF3COONa) as an external reference.3,4 The 6-FPAL-
Mehta et al.
polymer conjugates (3-5) have chemical shift sensitivity in the range 10.35-10.88 ppm, which is similar to that of the starting molecule 6-FPAL (2) (Table 1). This indicates that conjugation at the 4-position of the pyridoxine ring to different classes of polymers with molecular weights in the range 40-98K does not perturb the chemical shift range of the 19F signal in response to pH. The pKa was found to move toward the physiological range (5.9-6.8) for conjugates, from a more basic starting value (pKa ∼ 8.0) for 6-FPAL (2) (Figure 2 and Table 1). The ability to conjugate the pH sensor to carrier molecules is an attractive approach in terms of targeting specific tissues, enhancing vascular retention, and differentiating intra- and extracellular pH. However, it is important to investigate the possible effect on pH sensing characteristics of the indicator molecule (6-FPAL, 2) upon coupling to the polymer, i.e., the effect of large molecular size, electric charge, shape, and lypophilicity/hydrophilicity. Thus, 6-FPAL (2) was coupled to a small molecule (p-phenetidine)5 using linkages similar to those used for polymers, and the pH properties of the 6-FPAL-phenitidine conjugate (6) were investigated. The pH response obtained for 6 was very similar to that of 3-5 (Figure 1; Table 1). These results suggest that polymers do not exert any unusual effect on the pH sensing capability of the indicator (6-FPAL, 1) as compared with monomer (Figure 1; Table I). However, 19F signal line broadening was observed at certain pH values for the polymer conjugates. The 19F chemical shift sensitivities to pH for 6-FPAL (2) and all of the conjugates (both polymeric and monomeric; 3-6) were found to be very similar, contrasting an unusual reversal observed for conjugates of a prototype indicator 3-fluorosalicylaldehyde (17). To demonstrate the potential use of these polymeric indicators, we have evaluated the new conjugate (4) in 3 19F NMR Spectroscopy (15). 19F chemical shift titration curves were established for the samples in distilled water. Sodium trifluoroacetate (CF3COONa) was used as an external chemical shift reference standard (0 ppm). Typically, 6-FPOL (1), 6-FPAL (2), and 6-FPAL conjugates (2-20 mg) were dissolved in water (500 µL). The pH of the solution in water was altered with aliquots of diluted HCl or NaOH solutions. The pH of the sample was measured in the NMR tube using a combination pH electrode (Wilmad, Buena, NJ) attached to a pH meter (Corning 220, Sudbury, U.K.). 19F NMR detection sensitivity was estimated using 6-FPOL (1) solution as an internal standard with relative sensitivity per milligram of material. Shimming was performed on the water signal, and 19F NMR spectra were typically obtained with 4K data points across (10 000 Hz. Data were apodized with a 10-20 Hz exponential line broadening prior to Fourier transformation. 4 Equipment and Physical Measurements. 19F NMR spectroscopy used a 7 T (282.3 MHz, 19F) Oxford vertical bore magnet under control of a Tecmag console. Infrared spectra (IR) were recorded in KBr on a Mattson Galaxy (2000) FT-IR spectrometer. Gel permeation chromatography was carried out using two Waters ultrahydrogel columns (500 and 250 Å) at 25 °C. Phosphate buffer (2 M) was used for elution at a flow rate of 0.8 mL/min with a Waters 484 (UV-vis) and 410 differential refractometer. 5 Reaction of 6-FPAL (2) with Phenitidine. p-Phenetidine (55.5 mg, 0.4 M) dissolved in 1 N HCl (1 mL) was added to an aqueous solution (50 mL) of 6-FPAL (2, 75 mg, 0.4 M). The reaction mixture was stirred and brought to pH 8.5 by adding 1 N NaOH. The solution was left in an ice bath for 2 h, and the solid separated out (27) was filtered (40 mg): MS (m/z, FAB) 305 (M + 1)+. The Schiff base (40 mg, 0.13 M) was dissolved in 20 mL of MeOH followed by sodium borohydride (5 mg, 0.13 M) addition. The resulting mixture was stirred for 4 h. The methanol was evaporated and the residue chromatographed on silica gel to obtain 6 by elution with ethyl acetate and chloroform (26 mg): MS (m/z, FAB) 307 (M + 1)+.
Communications
Figure 3. 282.3-MHz 19F NMR spectrum of 6-FPAL-polylysine (4) together with 6-FPOL (1) in whole blood (pH 7.36) with CF3COONa as an external reference. Conjugate 4 shows a single resonance (δ -17.89) corresponding to extracellular pH (7.4), while 6-FPOL (1) shows two signals (δ -10.7 and -11.4) representing the pH from intracellular (pH 7.18) and extracellular (pH 7.48) compartments. This spectrum was acquired in 20 min from 500 µL of whole pig blood. Scheme 2b
b0.1
M NaHCO3, EtSCOCF3.
whole blood and compared its behavior with that of 6-FPOL (1). Conjugate 4 together with 1 were added to whole pig blood, and a capillary of CF3COONa solution was included as an external chemical shift reference. 4 showed a single signal, whereas 6-FPOL (1) showed two signals attributed to intra- and extracellular compartments. When the external pH was altered by addition of acid or base, 19F signals from the indicators moved as expected. Figure 3 shows a representative spectrum of conjugate 4 and 1 using whole blood with extracellular pH 7.36 (electrode). The signals from 6-FPOL (1) occurred at δ -10.7 (pHi 7.18) and δ -11.4 (pHe 7.48), while conjugate 4 resonated at δ -17.89 (pH 7.4). Thus, the chemical shift indicated essentially the same pH as observed with the electrode and the extracellular resonance of 6-FPOL (1). Indeed, centrifugation of the blood sample showed 4 in the supernatant only, verifying restricted distribution (extracellular) of this polymeric indicator (4). Hitherto, we have used an external chemical shift reference (CF3COONa) to determine relative chemical shift of the pH indicators and hence pH. As a further advance we have now incorporated an intramolecular reference signal (-COCF3) on the 6-FPAL-albumin conjugate (5) to provide direct chemical shift quantitation. This was achieved by selective trifluoroacetylation6 of remaining amino groups on 5 with S-ethyl trifluorothioacetate (Scheme 2) (16). This indicates that there is still scope to enhance the labeling efficiency of 6-FPAL conjugation reaction (i.e., residual free NH2 groups). The fluorine signal from the intramolecular reference (-COCF3) was found to be insensitive to changes in pH 6 Reaction of Conjugate (5) with S-ethyl trifluorothioacetate. 5 was dissolved in 0.1 M NaHCO3 followed by S-ethyl trifluorothioacetate addition (5 M excess), while maintaining the pH at ∼8.0. The solution was stirred overnight. The reaction mixture was extensively dialyzed using MWCO 6-8K and 1214K dialysis tubings (sequentially) against water to remove low molecular weight materials. The solution was lyophilized to yield TFA-[6-FPAL-albumin] conjugate (7).
Bioconjugate Chem., Vol. 7, No. 5, 1996 539
and, thus, the assessment of chemical shifts for the resulting conjugate (7) no longer required an external reference. The 19F chemical shift sensitivity to pH for 7 was similar to that of the starting conjugate (5), suggesting that incorporation of an additional reference fluorine on the indicator may not have any effect on its pH sensing properties. In addition, the stronger CF3 signal may facilitate 19F MR imaging to reveal the distribution of the molecules and hence “localize” the pH values. In summary, we have introduced a new class of fluorinated macromolecules for assessment of pH in vivo using 19F MRS/MRI. Fluorinated polymeric pH probes were synthesized by conjugation of a 19F pH sensor to carrier polymers. 6-FPAL-polymer conjugates exhibit excellent 19F pH sensitivity and pKa suitable for assessment of pH in vivo with 19F MRI/MRS. The novel macromolecular pH probes open a new line of approach for studying tissue physiology. We believe this approach could serve the dual purpose of restricting the pH sensing molecules to specific biological compartments (e.g., extracellular) and targeting specific tissue (e.g., tumors). Further ongoing investigations are aimed at (i) application of different chemical linkages or conjugation of the pH sensing moiety at different positions (in the pyridoxine) to polymers for enhancing efficiency of labeling and reduction in 19F signal line width, (ii) exploring active targeting of lesions (labeling antibodies and peptides), as opposed to nonspecific targeting provided by leakage of polymers, and (iii) demonstrating the in vivo utility of these fluorinated probes. Besides pH mapping, an immediate extension of the methodology will be to develop other 19F physiological markers, e.g., pO2, temperature, redox state, cation concentration, and perfusion using 19F MRI/MRS. ACKNOWLEDGMENT
This work was supported by a grant from the Texas Higher Education Coordinating Board (ATP 003660-032) (V.D.M.) and The Whitaker Foundation (R.P.M.). NMR experiments were performed at the Mary Nell and Ralph B. Rogers MR Center, an NIH Biotechnology Research Facility 5-P41-RR02584. LITERATURE CITED (1) Nuccitelli, R. (1982) Intracellular pH: Its measurement, regulation and utilization in cellular function (R. Nuccitelli and D. W. Deamer, Eds.) p 161, Liss, New York. (2) Roos, A., and Boron, W. P. (1981) Intracellular pH. Physiol. Rev. 61, 296. (3) Kearfott, K. J., and Junck, L. (1983) C-11 Dimethyloxazolidinedione (DMO): biodistribution, radiation absorbed dose, and potential for PET measurement of regional brain pH: concise communication. J. Nucl. Med. 24, 805. (4) Moon, R. B., and Richards, J. H. (1973) Determination of intracellular pH by 31P magnetic resonance. J. Biol. Chem. 248, 7276. (5) Robitaille, P. M. L., Robitaille, P. A., Brown, G. G. J., and Brown, G. G. (1991) An analysis of the pH-dependent chemical-shift behavior of phosphorous-containing metabolites. J. Magn. Reson. 92, 73. (6) Brown, F. F., Campbell, I. D., Kuchel, P. W., and Rabenstein, D. C. (1977) Human erythrocyte metabolism studies by 1H spin echo NMR. FEBS Lett. 82, 12. (7) Gil, M. S., Cruz, F., Cerdan, S., and Ballesteros, P. (1992) Imidazol-1-ylalkanoate esters and their corresponding acids. A novel series of extrinsic 1H NMR probes for intracellular pH. Biomed. Chem. Lett. 2, 1717. (8) Chacko, V. P., and Weiss, R. G. (1993) Intracellular pH determination by 13C-NMR spectroscopy. Am. J. Physiol. 264, C755.
540 Bioconjugate Chem., Vol. 7, No. 5, 1996 (9) Gillies, R. J., Liu, Z., and Bhujwalla, Z. (1994) 31P-MRS measurements of extracellular pH of tumors using 3-aminopropylphosphonate. Am. J. Physiol. 267, C195. (10) Deutsch, C. J., and Taylor, J. (1987) NMR spectroscopy of cells and organism (R. Gupta, Ed.) p 55, CRC Press, Boca Raton, FL. (11) Deutsch, C. J., and Taylor, J. S. (1989) New class of 19F pH indicators: fluoroanilines. Biophys. J. 55, 799. (12) Frenzel, T., Kobler, S., Bauer, H., Niedballa, U., and Weinmann, H. J. (1994) Noninvasive in vivo pH measurements using a fluorinated pH probe and fluorine-19 magnetic resonance spectroscopy. Invest. Radiol. 29, S220. (13) Rhee, C. K., Levy, L. A., and London, R. E. (1995) Fluorinated o-aminophenol derivatives for measurement of intracellular pH. Bioconjugate Chem. 6, 77. (14) Bental, M., and Deutsch, C. (1994) 19F-NMR study of primary human T lymphocyte activation: effects of mitogen on intracellular pH. Am. J. Physiol. 266, C541. (15) Mehta, V. D., Kulkarni, P. V., Mason, R. P., Constantinescu, A., Sivasubramanian, A., Goomer, N., and Antich, P. P. (1994) 6-Fluoropyridoxol: a novel probe of cellular pH using 19F NMR spectroscopy. FEBS Lett. 349, 234. (16) Mehta, V. D., Kulkarni, P. V., Rajur, S. B., Mason, R. P., Babcock, E. E., Constantinescu, A., and Antich, P. P. (1992) Novel molecular probes for 19F magnetic resonance imaging; synthesis and characterization of fluorinated polymers. Biomed. Chem. Lett. 2, 527. (17) Mehta, V. D., Kulkarni, P. V., Mason, R. P., and Antich, P. P. (1993) Fluorinated macromolecular probes for noninvasive assessment of pH by magnetic resonance spectroscopy. Biomed. Chem. Lett. 3, 187. (18) Mehta, V. D., Kulkarni, P. V., Mason, R. P., Constantinescu, A., and Antich, P. P. (1994) Fluorinated proteins as potential 19F magnetic resonance imaging and spectroscopy agents. Bioconjugate Chem. 5, 257. (19) Mehta, V. D., Mason, R. P., Kulkarni, P. V., Lea, P., Constantinescu, A., and Antich, P. P. (1995) 19F MR characterization of fluorinated proteins and relaxation rateenhance-
Mehta et al. ment with Gd-DTPA for faster imaging, Proceedings of the International Symposium on Chemists View of Imaging Centers, p 305, Plenum Press, New York. (20) Hunjan, S., Mason, R. P., Mehta, V. D., Kulkarni, P. V., Sivasubramanian, A., Arora, V., and Antich, P. P. (1995) Intracellular pH measurement in the perfused heart with 6-fluoropyridoxol using 19F NMR. Proceedings, Third Meeting of the Society of Magnetic Resonance, Nice, France, Vol. 3, p 1933. (21) Maeda, H., Seymour, L. W., and Miyamoto, Y. (1992) Conjugates of anticancer agents and polymers: advantages of macromolecular therapeutics in vivo. Bioconjugate Chem. 3, 351. (22) Sezaki, H., and Hashida, M. (1984) CRC Crit. Rev. Ther. Drug Carrier Syst. 1, 1. (23) Sinn, H., Schrenk, H. H., Friedrich, E. A., Schilling, U., and Maier-Borst, W. (1990) Design compounds having an enhanced tumor uptake, using serum albumin as a carrier. Part I. Nucl. Med. Biol. 17, 819. (24) Ogan, M. D., Schmiedl, U., Moseley, M. E., Grodd, W., Paajanen, H., and Brasch, R. C. (1987) Albumin labeled with Gd-DTPA an intravascular contrast-enhancing agent for magnetic resonance blood pool imaging: preparation and characterization. Invest. Radiol. 22, 665. (25) Weissleder, R., Lee, A. S., and Fischman, A. J. (1991) Polyclonal humanimmunoglobulin G labeled with polymeric iron oxide: antibody MR imaging. Radiology 181, 245. (26) Thiebaut, F., Currier, S. J., Whitaker, J., Haughland, R. P., Gottesman, M. M., Pastan, I., and Willingham, M. C. (1990) Activity of the multidrug transporter results in alkalinization of the cytosol: measurement of cytosolic pH by microinjection of a pH-sensitive dye. J. Histochem. Cytochem. 38, 685. (27) Korytnyk, W., and Srivastava, S. C. (1973) Synthesis and physicochemical and biological properties of 6-halogensubstituted vitamin B6 analogs. J. Med. Chem. 16, 638.
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