Bioconjugate Chem. 2002, 13, 893−897
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Improving the Labeling of S-Acetyl NHS-MAG3-Conjugated Morpholino Oligomers Guozheng Liu,* Surong Zhang, Jiang He, Zhihong Zhu, Mary Rusckowski, and Donald J. Hnatowich* Division of Nuclear Medicine, Department of Radiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655. Received April 15, 2002
S-Acetyl MAG3 (S-acetylmercaptoacetyltriglycine) has been used as a chelator for the 99mTc labeling of a variety of biomolecules. The objective of this study was to improve upon the labeling of morpholino (MORF), a DNA analogue, as a model biomolecule. A 15mer MORF with a primary amine was conjugated with NHS-MAG3 in the usual manner, and the MORF-MAG3 was purified over a P4 column as before. The conjugate was radiolabeled using stannous ion as usual, and the impurities were identified using size exclusion high-performance liquid chromatography (SE HPLC). Various methods were then investigated to remove the impurities. With tartrate as the transchelator, two impurities were identified as labeled MAG3 and labeled tartrate. The labeled MAG3 could not be removed by simply repurifying the conjugate using the usual pH 5.2 NH4OAc buffer before labeling. However, this impurity could be completely removed if the conjugate was adjusted to pH 7.6 and heated before repurification. The labeled tartrate impurity was removed by heating during labeling. On the basis of these observations, the following procedure for purification of the conjugation mixture and subsequent labeling was adopted. After MORF was conjugated with NHS-MAG3 and purified over P4 with pH 5.2 NH4OAc eluant, the oligomer fractions were combined, adjusted to pH 7.6, and heated in a boiling water bath for 20 min. The conjugated oligomer was then repurified over P4 for storage at refrigerator temperatures. Labeling is achieved simply by adding fresh stannous ion to a solution of the MORF-MAG3 in pH 7.6 containing tartrate followed by 99mTc-pertechnetate. After the mixture is heated for 20 min in boiling water, the labeling efficiency is always over 90% as determined by size exclusion HPLC and paper chromatography and the specific activities can exceed 7 mCi/µg. By making several relatively simple changes to the routine procedure used to conjugate and radiolabel biomolecules with 99mTc via MAG3, a modified procedure was developed that results in labeling efficiency high enough to avoid postlabeling purification.
INTRODUCTION
Among other bifunctional chelators, this laboratory has used an activated NHS ester of S-acetyl MAG3 (Sacetylmercaptoacetyltriglycine) for the radiolabeling of a variety of biomolecules (1-15). An acetyl group protecting the sulfur on MAG3 was substituted for the benzoyl group to avoid the harsh conditions of alkaline pH and elevated temperatures required for the removal of this group (14). Labeling is successful at neutral pH (normal labeling efficiency 50-80%), but separation after labeling is always required to achieve acceptable radiochemical purities (1-15). Furthermore, because tartrate is used as a transchelator, the labeled biomolecule is often contaminated with labeled tartrate, which may then coelute with the biomolecule during postlabeling purification and subsequently become oxidized to 99mTc-pertechnetate. Heating can encourage transchelation of the labeled tartrate to the desired product. Heating can also accelerate the oxidation of stannous ion and, in the presence of low concentrations of this reducing agent, can result in oxidation of the labeled tartrate to 99mTcpertechnetate. The pertechnetate will then be retained on the size exclusion column during postlabeling purification (1, 2). * To whom correspondence should be addressed. D.J.H.: Tel: (508)856-4256. Fax: (508)856-4572. E-mail: Donald.
[email protected].
Recently, morpholino oligomers (MORF) and their complementary forms (cMORFs) have been under investigation as potentially useful agents for pretargeting applications after radiolabeling via MAG3 (1, 2). These oligomers were therefore selected as model biomolecules to search for methods leading to efficient labeling with no need for postlabeling purification. MATERIAL AND METHODS
The 15mer MORF and its complement used herein were identical to those used by us earlier (1-3). The P4 separation gel (Bio-Gel Medium, Bio-Rad Laboratories, Hercules, CA) was purchased and used as received; S-acetyl NHS-MAG3 was synthesized in house (14), and the structure was confirmed by elemental analysis, proton nuclear magnetic resonance (NMR), and mass spectroscopy. All other chemicals were reagent grade and were used without purification. The 99mTc-pertechnetate was eluted from a 99Mo-99mTc generator (Dupont, Billerica, MA). Size Exclusion High-Performance Liquid Chromatography (HPLC) and Paper Chromatography. Size exclusion HPLC analysis was performed on a Superdex Peptide column (optimal separation range: 1 × 102 to 7 × 103 Da; Amersham Pharmacia Biotech, Piscataway, NJ) with 0.10 M pH 7.2 phosphate buffer as eluant at a flow rate of 0.60 mL/min. 99mTc-pertechnetate is retained on this column. The HPLC was equipped with both an inline radioactive detector and an
10.1021/bc0255384 CCC: $22.00 © 2002 American Chemical Society Published on Web 06/27/2002
894 Bioconjugate Chem., Vol. 13, No. 4, 2002
inline Waters 2487 dual wavelength absorbance detector. Recovery was routinely measured. The presence of 99m Tc-pertechnetate was confirmed by paper strip chromatography on Whatman No. 1 with acetone as the mobile phase. Only 99mTc-pertechnetate migrates with the solvent while labeled MORF and other labeled impurities remain at the origin. Conjugating MORF with MAG3 and Radiolabeling. MORF derivitized on the 3′ equivalent end with a primary amine was conjugated with S-acetyl NHSMAG3 as previously described (1-3). A solution of 1000 µg of MORF in 200 µL of 0.2 M, pH 8.0, N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid (HEPES) buffer was added to a vial containing 1700 µg of S-acetyl NHSMAG3. The vial was immediately vortexed thoroughly, incubated for 1 h at room temperature, and purified on a 0.7 cm × 20 cm P4 column with 0.25 M, pH 5.2, NH4OAc buffer as eluant. MORF-MAG3 was originally labeled by adding 5 µL (∼300 µCi) of 99mTc-pertechnetate generator eluate to a solution of 30 µL of MORF-MAG3 in the pH 5.2 ammonium acetate buffer, 10 µL of 50 µg/µL Na2‚tartrate‚ 2H2O in a pH 9.2 buffer (0.5 M Na2HCO3, 0.25 M NH4OAc, and 0.175 M NH3), and 4 µL of 1 µg/µL SnCl2‚ 2H2O in ascorbate-HCl solution (1 µg/µL sodium ascorbate in 10 mM HCl). The final pH was 7.6. To better understand the original labeling method, several studies were performed as follows: (i) The pH 7.6 solution of labeled MORF was divided, one-half was stored at room temperature for 10 h while the other was heated in a boiling water bath for 20 min before storage at room temperature for 10 h. (ii) In addition, some of the heated half was purified again over P4. The three samples were then analyzed by HPLC. As will be shown below, the radioimpurities after labeling by the original method are labeled tartrate and labeled MAG3. If a 20 min heating step is introduced at the end of the labeling method, the only radioimpurity remaining is labeled MAG3. Thus, if labeled MAG3 can be prevented, postlabeling purification will be unnecessary. The labeled MAG3 may arise from inefficient purification of the free MAG3 remaining after the conjugation or it may be generated by cleavage during labeling of some relatively unstable MAG3 conjugate. To better understand the origin of this impurity, (i) the purified MORF-MAG3 in pH 5.2 was purified again on P4 before labeling and (ii) the purified MORF-MAG3 in pH 5.2 was adjusted to pH 7.6, heated for 20 min, and then purified over P4 again using the same pH 5.2 NH4OAc buffer as eluant. MAG3 Groups per MORF. After conjugation, the acetyl group on S-acetyl MAG3 may no longer be necessary to protect the thiol group; nevertheless, its continued presence has been demonstrated following conjugation to one peptide (10). By introducing a 20 min heating step at boiling water temperature, especially at pH 7.6, it is possible that most of the acetyl groups are hydrolyzed (16, 17). Because rapid kinetics of labeling may depend on the presence of the unprotected free sulfhydryl on MORF-MAG3 (18, 19), the number of unprotected MAG3 groups on MORF-MAG3 was determined. The assay employed 4,4′-dithiodipyridine (4-DTDP) according to the following reaction (5, 20, 21):
Liu, et al.
By measuring on a UV spectrophotometer (U-2000, Hitachi Instruments Inc., Danbury, CT), it was determined that MORF and cysteine show low to nonexistent absorption at the 323 nm absorption maximum of the indicator 4-thiopyridone (4-TP). Only 4-DTDP shows a small but measurable absorbance at this frequency. However, 4-DTDP was present at large excess such that its concentration was effectively unchanged by the reaction. Accordingly, free sulfhydryl groups could be quantitated in the presence of 4-DTDP. A standard curve of absorbance vs free sulfhydryl concentration was obtained by adding 40 µL of 4-DTDP (80 pmol) in 0.1 M, pH 7.2, phosphate buffer to each of a series of cysteine solutions in the range of 5-45 µM in 100 µL of pH 5.2 acetate buffer and measuring the absorbance against a blank 4-DTDP solution. The concentration of free sulfhydryls in the MORF-MAG3 sample was then determined by applying its absorbance to the standard curve. In this measurement, the concentration of MORF was obtained by its absorbance at 265 nm using the molar absorbance (1.583 × 105 M-1 cm-1) provided by the manufacturer. Optimization. Optimization of labeling conditions was accomplished by varying the amount of MORF-MAG3, stannous ion, temperature, and time using a MORF solution with 0.13 unprotected MAG3 per molecule, determined as described above. Thus, 5 µL (∼300 µCi) of 99m Tc generator eluant was added to a mixture of 35 µL of 0.25 M, pH 5.2, NH4OAc buffer containing various amounts of MORF-MAG3, 10 µL of 50 µg/µL tartrate solution in a pH 9.2 buffer (0.5 M NaHCO3, 0.25 M NH4OAc, and 0.175 M NH3 buffer), and 5 µL (4 µg/µL) of SnCl2‚2H2O solution in 10 mM HCl containing 1 µg/µL sodium ascorbate followed by heating in boiling water for 20 min. Thereafter, by holding the MORF-MAG3 concentration constant, the influence of the tin, temperature, and time was evaluated. Finally, the influence of radioactivity levels was established by adding varying volumes of the pertechnetate generator eluant to the labeling solution. All samples were measured by HPLC, and in all cases, the recovery of radioactivity off the column was greater than 90% unless otherwise stated. RESULTS
Removal of Labeled Tartrate. Figure 1A presents a HPLC radiochromatogram of labeled MORF-MAG3 prepared from MORF-MAG3 purified on P4 only once and incubated at room temperature for 1 h before analysis. The peak at 20.8 min is due to labeled MORF, that at 26 min is due to labeled tartrate, and that at 40.6 min is due to labeled MAG3. Identification was made by comparison to radiochromatograms of labeled tartrate and commercial labeled MAG3. The high molecular weight shoulder on the labeled MORF peak may be due to the 2:1 complex duplex 99mTcO(MORF-MAG3)2 (22, 23). Despite a reasonable separation by HPLC, we were unable to remove labeled tartrate from the radiolabeled MORF using P4. However, increasing the room temperature incubation time from 1 to 5 h resulted in a decrease in labeled tartrate as shown in Figure 1A,B and an increase in HPLC recovery from 85 to 92%. Heating in boiling water for 20 min in place of longer room temperature incubations was more successful in decreasing this radiocontaminant as shown in Figure 1C. Because 99m Tc-pertechnetate is retained on the HPLC column and because the recovery was quantitative, the decrease
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Figure 2. UV spectra of cysteine, MORF-MAG3, 4,4′-dithiodipyridine, and the reaction mixture (i.e., 4-TP) in the determination of MAG3 groups per MORF.
Figure 1. Size exclusion HPLC radiochromatograms of radiolabeled MORF-MAG3 incubated at room temperature for 1 h (panel A) and 5 h (panel B) and after heating the radiolabeled MORF-MAG3 for 20 min (panel C) followed by P4 purification (panel D).
in labeled tartrate must have been to the benefit of labeled MORF, presumably by increasing transchelation. Heating also removed the high molecular weight shoulder probably by its transformation from the 2:1 complex to a 1:1 complex (22, 23). Figure 1D was obtained following postlabeling purification on P4 of the labeled MORF after heating for 20 min and shows that labeled MAG3 can be removed by P4 purification of the heated labeled mixture. Removal of Labeled MAG3. Although heating during labeling of MORF-MAG3 could remove the labeled tartrate radioimpurity, radiolabeled MAG3 remained. Repurification on P4 before labeling lowered but did not eliminate this radiocontaminant. This indicated that the labeled MAG3 was not solely due to insufficient purification of the conjugate. However, by adjusting the purified MORF conjugate to pH 7.6 from pH 5.2 and heating for 20 min followed by purification again on P4, the labeled MAG3 radioimpurity was completely absent after labeling. This suggests that the labeled MAG3 radioimpurity may be the result of some weak conjugation of MAG3 to MORF that is susceptible to dissociation during heating in ammonium buffer at pH 7.6. Therefore, the subsequent investigations were conducted using MORF-MAG3 conjugates, which after conjugation and purification on P4 were adjusted to pH 7.6 and heated for 20 min before repurification on P4. Determination of Deprotected MAG3 Groups per MORF. Figure 2 presents the absorption spectra for cysteine, MORF, 4-DTDP, and the reaction mixture (i.e., 4-TP) in the range of 280-400 nm. Both the free cysteine and the MORF-MAG3 therefore have no absorption at the 323 nm peak absorbance of the indicator product 4-TP. Only 4-DTDP has measurable absorbance at this
Figure 3. UV HPLC chromatograms of native MORF (panel A) and two MORF-MAG3 preparations with an average of 0.13 (panel B) and 0.41 (panel C) groups per molecule.
frequency, but its concentration was unchanged during the measurement so that determination against the blank solution of 4-DTDP could eliminate its influence. The standard curve obtained provided a straight line over nine concentrations with a coefficient of regression of 0.998 (data not presented). In this manner, the average number of deprotected MAG3 groups per MORF was determined for two preparations as 0.13 and 0.41. These values are in qualitative agreement with the results of HPLC analysis of these samples. As shown in Figure 3, the UV chromatogram of native MORF shows a singlet at the retention time 20.8 min of the oligomer in panel A while those of MORF-MAG3 conjugated at an average of 0.13 groups per molecule in panel B and MORF-MAG3 conjugated at an average of 0.41 groups per molecule in panel C show a shoulder at 17.7 min whose intensity increases with increasing conjugation. The closely spaced doublet on HPLC could not be adequately separated for quantitating the relative amount of the new peak. Optimization. The following studies were performed on the MORF-MAG3 sample conjugated at an average of 0.13 groups per molecule. The results presented in Table 1 were obtained by HPLC analysis. The weight of MORF-MAG3 with 300 µCi of 99mTc is clearly a determinent of labeling efficiency as shown in the table. The poor recovery at low weights of MORF-MAG3 was
896 Bioconjugate Chem., Vol. 13, No. 4, 2002
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Figure 5. Size exclusion HPLC radiochromatograms of labeled MORF-MAG3 vs time without heating during labeling (panel A) and with heating for 20 min (panel B).
Figure 4. Influence of stannous ion on labeling efficiency. Table 1. Influence of the Amount of MORF-MAG3 on the Labeling MORF-MAG3 (µg)
0.02
0.04
0.08
0.20
0.78
3.90
recovery (%) area of labeled MORF (%) labeling efficiency (%)
67 67 45
90 75 68
99 90 89
105 94 94
100 96 96
103 97 97
labeling efficiency of MORF will be greater than 90% after the introduction of 5 µL (300 µCi) of pertechnetate followed by heating for 20 min. With 12 µg of SnCl2‚2H2O and 0.39 µg of MORF-MAG3, the labeling efficiencies were, respectively, 96, 91, 38, and 17% for 5, 20, 50, and 100 µL of 99mTc-pertechnetate (145 µCi/µL). The specific activity of at least 7400 µCi/µg (i.e., 38 Ci/µmol) can be achieved without postlabeling purification. DISCUSSION
assumed to be due to the presence of pertechnetate from oxidation of labeled tartrate, which is retained on the column. After the addition of 300 µCi of 99mTc to the labeling solution at pH 7.6 containing 0.2 µL of MORFMAG3 (i.e., 0.08 µg, 0.28 µM MORF, 0.036 µM MAG3 as MORF-MAG3) or more and a fixed amount of tin (20 µg of SnCl2‚2H2O), the labeling efficiency was greater than 90%. Figure 4 presents the influence of stannous ion on labeling efficiency and shows that labeling efficiency drops with increasing tin. For 0.5 µL of MORF-MAG3 (i.e., 0.20 µg of MORF, 0.026 µg of pure MORF-MAG3), labeling efficiency drops below 90% when the amount of SnCl2‚2H2O exceeds 40 µg, possibly by increasing the concentration of technetium in oxidation states lower than +5 that cannot be directly transchelated to MORFMAG3. Heating is necessary for efficient labeling as shown in Figure 5. Panel A presents radiochromatograms obtained over time for the labeled MORF-MAG3 prepared without heating (0.5 µL of 0.20 µg of MORF-MAG3 and 20 µg of SnCl2‚2H2O). As shown, the radiolabeled tartrate peak at 26 min is still prominent even after 10 h postlabeling. In addition, recovery in this sample fell below 80% at 10 h presumably because of oxidation of labeled tartrate to pertechnetate. However, as shown in panel B, by introducing a 20 min heating step, the radiochemical purity is greater than 93% even after 8 h postlabeling. Thus, at pH 7.6 with tartrate as the transchelator, if the amount of MORF-MAG3 is greater than 0.08 µg and the amount of SnCl2‚2H2O is between 4 and 20 µg, the
The object of this study was to optimize the labeling efficiency with 99mTc of a model oligomer conjugated with MAG3. Our goal was to achieve 90% or greater radiochemical purity and thus avoid the need for postlabeling purification. Because in this investigation tartrate was selected as the transchelator, the possible radiochemical impurities are radiolabeled tartrate, radiolabeled MAG3, and pertechnetate. In fact, using our original MORFMAG3 labeling method, both radiolabeled MAG3 and tartrate were evident as shown in Figure 1A. Furthermore, because any labeled tartrate present will be susceptible to oxidation, it may be continually oxidized during HPLC analysis to explain the poor recoveries of about 85% that we have occasionally observed. If sufficient time is allowed, radiolabeled tartrate can continue its transchelation, in this case to MORF-MAG3, as shown in Figure 1B. However, the process can be greatly accelerated by heating as shown in Figure 1C. The removal of labeled MAG3 is more difficult. All attempts to purify free MAG3 from MORF-MAG3 by P4 were unsuccessful in that radiolabeling of the repurified oligomer consistently provided a product contaminated with labeled MAG3. This radiocontaminant could be removed, however, if the purified conjugated oligomer was placed in the pH 7.6 buffer and heated for 20 min or more before a second P4 purification and the subsequent labeling. We believe that the NHS-MAG3 used in the conjugation of the amine derivitized MORF is capable of conjugating to the sulfhydryl on the deprotected MAG3 group. The resulting thio ester bond is relatively unstable
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against hydrolysis and aminolysis and may dissociate in neutral pH solution containing a large amount of ammonium (16, 17). Originally (1-3), the labeled MORFMAG3 was placed in neutral pH only during labeling with the results that this dissociation introduced the observed radiolabeled MAG3 impurity. On the basis of these observations, the following procedure for purification of the conjugated biomolecule and subsequent labeling was adopted. After conjugation of MORF with NHS-MAG3 (molar ratio 1:20) in pH 8.0 HEPES buffer and purification over P4 with pH 5.2 NH4OAc eluant, the oligomer fractions were combined, adjusted to pH 7.6, and heated for 20 min. The conjugated oligomer was then purified over P4 again for storage at refrigerator temperatures. Labeling is achieved simply by adding fresh stannous ion to a solution of MORFMAG3 and tartrate in pH 7.6 buffer followed by 99mTcpertechnetate. After the solution is heated for 20 min in boiling water, the labeling efficiency is always over 90% as determined by HPLC and paper chromatography. With the proper selection of MORF-MAG3, stannous ion, and pertechnetate concentration, specific activities more than 7000 µCi/µg have been achieved. ACKNOWLEDGMENT
Financial support for this investigation was provided by the National Institutes of Health (CA79507 and CA94994). LITERATURE CITED (1) Liu, G., Zhang, S., He, J., Liu, N., Gupta, S., Rusckowski, M., and Hnatowich, D. J. (2002) The influence of chain length and base sequence on the pharmacokinetic behavior of 99mTc-Morpholinos in mice. Q. J. Nucl. Med., in press. (2) Liu, G., Mang’era, K., Liu, N., Gupta, S., Rusckowski, M., and Hnatowich, D. J. (2002) Tumor pretargeting in mice using technetium-99m labeled morpholinos, a DNA analogue. J. Nucl. Med. 43, 384-391. (3) Mang’era, O. K., Liu, G., Wang, Y., Zhang, Y., Liu, N., Gupta, S., Rusckowski, M., and Hnatowich, D. J. (2001) Initial investigation of 99mTc-labeled morpholinos for radiopharmaceutical applications. Eur. J. Nucl. Med. 28, 1682-1689. (4) Wang, Y., Chang, F., Zhang, Y., Liu, N., Liu, G., Gupta, S., Rusckowski, M., and Hnatowich, D. J. (2001) Pretargeting with amplification using polymeric peptide nucleic acid. Bioconjugate Chem. 12, 807-816. (5) Zhu, Z., Wang, Y., Zhang, Y., Liu, G., Liu, N., Rusckowski, M., and Hnatowich, D. J. (2001) A novel and simplified route to the synthesis of N3S chelators for 99mTc labeling. Nucl. Med. Biol. 28, 703-708. (6) Rusckowski, M., Qu, T., Gupta, S., Ley, A., and Hnatowich, D. J. (2001) A comparison in monkeys of 99mTc labeled to a peptide by 4 methods. J. Nucl. Med. 42, 1870-1877. (7) Zhang, Y. M., Wang, Y., Liu, N., Zhu, Z. H., Rusckowski, M., and Hnatowich, D. J. (2001) In vitro investigations of tumor targeting with 99mTc-labeled antisense DNA. J. Nucl. Med. 42, 1660-1669.
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