Bioconjugate Chem. 1997, 8, 249−252
249
Synthesis of an Amino Acid Analogue To Incorporate p-Aminobenzyl-EDTA in Peptides Anne In. Song† and Tariq M. Rana* Department of Pharmacology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, 675 Hoes Lane, Piscataway, New Jersey 08854. Received July 24, 1996X
A convenient and straightforward synthesis of an amino acid analog, [p-(N-R-Fmoc-L-aspartic acidβ-amido)benzyl]-EDTA tetra-tert-butyl ester, compatible with Fmoc solid phase peptide synthesis strategy is described. This reagent was used to incorporate p-aminobenzyl-EDTA at an internal sequence position in an HIV-1 Tat protein fragment. After cleavage from the resin and standard deprotection, the peptide was purified by high-performance liquid chromatography and characterized by mass spectrometry. Through this methodology, flexible linkers of different lengths and containing various structures can be placed between the R-carbon backbone of peptides and metal chelates. These peptides will provide a new class of affinity cleaving reagents that can be directed against protein and nucleic acid targets.
Interaction of transition metal complexes with nucleic acids and proteins makes them a useful tool in molecular biology. Metal complexes can be synthesized for specific recognition and cleavage of nucleic acids, or they can be used as nonspecific nucleases (for an excellent review, see ref 1). Shape-selective metal complexes have been designed to target specific DNA and RNA structures (24). An iron-EDTA complex cleaves DNA nonspecifically and can be applied to determine the helical periodicity of DNA and structural details of bent DNA (5, 6). Cleavage of DNA and RNA by metal chelates is an important new approach to characterize specific structural features of nucleic acids and their complexes in solutions (7-11). The metal chelate attachment converts sequence-specific DNA-binding protein or oligonucleotide to sequence-specific DNA-cleaving molecules that function under physiological pH, temperature, and salt conditions (9, 10, 12-16). As with nucleic acids, methods for specific and nonspecific cleavage of proteins have been developed. Sitespecific cleavage of proteins is achieved by introducing a metal-binding site at one position in a polypeptide chain or attaching it to protein binding ligands (17-26). Recently, an Fe-EDTA-catalyzed protein cleavage method has been applied to complex biological systems such as membrane proteins (27). Nonspecific protein cleavage by an untethered Fe-EDTA has been recently applied to map protein domains involved in macromolecular interactions such as the surface of a DNA-binding protein (28) and interactions between subunits of the multisubunit RNA polymerase (29). Artificial proteolytic reagents would be extremely useful to characterize important structural features of proteins and their complexes under physiological conditions. To accurately probe structure-function relationships in nucleic acids, proteins, and nucleic acid-protein complexes by affinity cleaving methods, the development of both general and specific methods for single-site * Author to whom correspondence should be addressed [telephone (908) 235-4082; fax (908) 235-4073; e-mail rana@umdnj. edu]. † Present address: Department of Chemistry, Hartwick College, Oneonta, NY 13820. X Abstract published in Advance ACS Abstracts, February 1, 1997.
S1043-1802(96)00084-5 CCC: $14.00
modification of the peptide sidechain is required. Reagents potentially useful for such experiments are the “bifunctional chelating agents,” so called because they incorporate a strong metal-chelating group and a chemically reactive functional group. Bifunctional chelating agents have been synthesized and used to provide biological molecules with the nuclear, physical, and chemical properties of chelated metal ions (30-32). To prepare peptides labeled with metal chelates, protected derivatives of EDTA analogs compatible with Merrifield solid phase protein synthesis using N-tertbutyloxycarbonyl (Boc)-protected amino acids were developed (13, 33-35). There are two major concerns about this synthetic strategy: (a) repetitive TFA acidolysis in Boc-group deprotection could lead to acid-catalyzed side reactions and (b) cleavage and deprotection of peptides requires HF and specific laboratory setup which is not available to many researchers. Due to these concerns 9-fluorenylmethyl carbamate (Fmoc) solid phase peptide synthesis was developed which employs N-R-Fmoc amino acids (36). In this strategy, the Fmoc group is deprotected with piperidine and TFA is required only for the final cleavage and deprotection step. In this paper, we have synthesized an amino acid analog, [p-(N-R-FmocL-aspartic acid-β-amido)benzyl]-EDTA tetra-tert-butyl ester (9). We report here a new selective protection scheme for the preparation of amino acid analog 9 which is designed to be compatible with the Fmoc solid phase peptide synthesis strategy. The synthetic route for this synthesis is outlined in Scheme 1. EDTA analog 3 was synthesized with modifications of the method published by Studer and Meares (37). To test the use of reagent 9 to incorporate p-aminobenzyl-EDTA in internal sequence of a peptide, we synthesized a fragment of HIV-1 Tat protein (amino acids 56-72) on an automated peptide synthesizer. We used standard HOBt/DCC peptide syntheis protocols to incorporate reagent 9 in the peptide sequence. The amino acid sequence of the Tat peptide contained Arg-X-Pro-Pro-GlnGly-Ser-Gln-Thr-His-Gln-Val-Ser-Leu-Ser-Lys-Gln, where X was the modified amino acid 9. After standard TFA deprotection, the peptide was purified and characterized by mass spectrometry, which confirmed the incorporation of EDTA in the peptide sequence. Tat protein is a potent transactivator of transcription from the viral long terminal repeat and acts by binding to a stem-loop RNA © 1997 American Chemical Society
250 Bioconjugate Chem., Vol. 8, No. 2, 1997 Scheme 1
structure called TAR RNA (38). Tat peptides modified with metal chelates at various sequence positions would provide a class of affinity cleaving reagents that can be directed against TAR RNA, and these studies are in progress. EXPERIMENTAL SECTION
Anhydrous DMF, N,N-diisopropylethylamine, fluorenemethanol, TFA, piperidine, DMAP, and 10% Pd on charcoal were purchased from Aldrich. DCC and HOBt were obtained from Sigma. N-R-Fmoc-β-tert-butyl ester L-aspartic acid was purchased from Bachem. All chemicals were of reagent grade unless otherwise specified. NMR spectra were recorded at 200 MHz on a Gemini 200 spectrometer (Varian). The FAB MS spectrum was recorded using m-nitrobenzoic acid/NaI as matrix. IR spectra were obtained on a Perkin-Elmer FT 1600 as thin film. TLC was performed with precoated 0.2 mm silica gel 60 F-254 TLC plates (EM Reagents, Darmstadt, Germany). (p-Nitrobenzyl)-EDTA Tetra-tert-butyl Ester (2). To a 100 mL round-bottom flask were added 0.92 g (3.4 mmol) of (p-nitrobenzyl)ethylenediamine dihydrochloride and 50 mL of dry acetonitrile. The flask was flushed with nitrogen for 10 min, and to the stirring mixture were added 2.46 g (17.7 mmol) of K2CO3 and 0.64 g (3.8 mmol) of KI. Finally, tert-butyl bromoacetate (2.9 mL, 17.9 mmol) was added to the mixture slowly. The mixture was refluxed at 105 °C for 120 h in the dark and then cooled to room temperature. Evaporation of solvent gave brown oil containing powdery deposit. The crude mixture was dissolved in methylene chloride and filtered through a glass frit, and the solid was washed with more methylene chloride until the powder on top of the frit turned white. The filtrate was collected and evaporated to give a yellow oil, which was silica gel column purified with 10% ethyl acetate in methylene chloride. The 1H NMR showed several kinds of para-disubstituted phenyl groups, seemingly a mixture of mono-, di-, tri-, and tetraprotected EDTA moieties. Another silica gel column with 5% ethyl acetate in CH2Cl2 gave the final product (1.53 g, 2.4 mmol, 70.6% yield): IR 1731 cm-1 (CdO, br s); 1H NMR (CDCl3) δ 8.03 (2H, d, 8.76 Hz), 7.42 (2H, d, 8.42 Hz),
Song and Rana
3.37 (4H, s), 3.34 (4H, s), 3.30 (2H, m), 2.95 (2H, m), 2.34 (1H, m), 1.36 (36H, s); 13 C NMR δ 171.42, 171.09, 149.41, 146.66, 130.69, 123.64, 63.52, 56.89, 56.08, 53.81, 37.54, 28.54, 28.49. (p-Aminobenzyl)-EDTA Tetra-tert-butyl Ester (3). To dried compound 2 (1.5 g, 2.3 mmol) in 100 mL of THF were added 415 mg (3.0 mmol) of K2CO3 and 400 mg of 10% Pd/C under nitrogen flow at room temperature. After addition, hydrogen inlet was used instead of nitrogen, and with vigorous stirring and strong hydrogen flow, the mixture was allowed to react overnight at room temperature under H2 atmosphere. The reaction mixture was filtered through a glass frit, and the solid was washed with more THF. The collected solution was evaporated to give a yellow oil. Silica gel column separation with ethyl acetate and another column with methylene chloride gave yellow oil, which was identified as pure product by 1H and 13C NMR spectra (0.78 mg, 1.3 mmol, 56.5% yield): IR 1731 cm-1 (CdO, br s); 1H NMR in CDCl3 δ 6.98 (2H, d, 8.18 Hz), 6.57 (2H, d, 8.22 Hz), 5.29 (2H, s), 3.48 (4H, s), 3.43 (4H, s), 3.03 (1H, m), 2.81 (2H, m), 2.55 (2H, m), 1.43 (18H, s), 1.40 (18H, s); 13C NMR δ 172.05, 171.58, 145.11, 130.23, 115.62, 63.54, 56.69, 55.35, 53.92, 35.69, 20.50. N-r-Fmoc-β-tert-butyl Ester L-Aspartic Acid 9-Fluorenemethyl Ester (5). To a 25 mL round-bottom flask was added compound 4 (0.411 g, 1 mmol) dissolved in 5 mL of methylene chloride. At 0 °C, 206 mg (1 mmol) of DCC and 2 mg of DMAP were added to the mixture and stirred for 1 h at the same temperature. Prepared solution of 9-fluorenemethanol (216 mg, 1.1 mmol) in 2 mL of CH2Cl2 was added, and the mixture was stirred overnight at room temperature. Filtration to remove white salt (DCU) and evaporation of the filtrate gave a light yellowish oil. Silica gel column with 30% ethyl acetate in petroleum ether gave 0.5 g of colorless oil and 0.1 g of white solid. NMR confirmed the oily compound as the product (0.5 g, 0.85 mmol, 85% yield): 1H NMR (CDCl3) δ 7.50 (16H, m), 6.00 (1H, d, 8.5 Hz, NH), 4.76 (1H, dt), 4.48 (4H, 2d), 4.29 (2H, 2t), 2.94 (2H, dd, 4.48 Hz, 15.10 Hz), 1.48 (9H, s); 13C NMR δ 171.49, 170.54, 156.55, 144.41, 144.27, 144.03, 141.81, 128.42, 128.27, 127.74, 127.62, 125.59, 125.56, 120.60, 120.54, 68.33, 67.84, 51.24, 47.66, 47.23, 38.27, 28.57. N-r-Fmoc-r-9-fluorenemethyl Ester L-Aspartic Acid (6). At 0 °C, 1.5 mL of trifluoroacetic acid was added to 0.309 g (0.52 mmol) of 5 in 4 mL of methylene chloride. The mixture was stirred for 2 h at 0 °C, and the solvent was evaporated under vacuum. Complete dryness gave 0.25 g (0.47 mmol, 90.4% yield) of white powder, which was confirmed to be 6 by mass spectrum: 1H NMR (CD OD) δ 7.80-7.27 (16H, m), 4.70-4.20 (7H, 3 m), 2.80 (2H, m). [p-(N-r-Fmoc L-aspartic acid 9-fluorenemethyl ester β-amido)benzyl]-EDTA Tetra-tert-butyl Ester (7). To a 25 mL round-bottom flask containing 126 mg of 6 (0.24 mmol) was added 50 mg (0.37 mmol) of HOBt in 5 mL of 1:1 mixture of THF and DMF. DCC (100 mg, 0.48 mmol) in 3 mL of methylene chloride and 0.5 mL of IPEA were added to keep the pH about 8. The reaction mixture was stirred for 30 min at 0 °C and for 2 h at room temperature. Without filtration, compound 3 (0.14 g, 0.225 mmol) in 3 mL of THF was added to the mixture and left for stirring overnight. TLC in 15% petroleum ether/ethyl acetate showed two spots, one corresponding to the mixture of Fmoc derivatives and another to the coupled product. Spin chromatotron separation with 80% petroleum ether in ethyl acetate gave 150 mg of pure product (0.13 mmol) as yellow oil, which was identified by NMR and high-resolution mass spectra to be com-
Technical Notes
pound 7 (MS ) 1137.581, theor ) 1137.580; 58% yield): IR 1731 cm-1 (CdO, br s); 1H NMR (CDCl3) δ 7.50 (20H, m), 6.25 (1H, d), 4.50 (7H, m), 3.49 (4H, s), 3.44 (4H, s), 2.85 (8H, m), 1.44 (18H, s), 1.41 (18H, s); 13C NMR δ 171.83, 171.41, 168.40, 156.80, 144.30, 144.16, 144.09, 144.00, 141.74, 137.31, 135.78, 130.29, 128.28, 128.20, 127.69, 127.60, 125.65, 125.51, 120.47, 120.28, 68.18, 67.88, 63.81, 60.92, 56.81, 55.94, 54.06, 51.31, 47.55, 47.15, 39.03, 36.83, 28.63, 21.58, 14.70. [p-(L-Aspartic acid β-amido)benzyl]-EDTA Tetratert-butyl Ester (8). To 150 mg of 7 (0.13 mmol) was added 5 mL of 20% piperidine in methylene chloride, and the mixture was stirred for 2.5 h. The solvent was evaporated completely. The obtained yellow solid was washed with a minimal amount of ether and filtered through a micropipet with cotten. White solid, which was not ether soluble, was washed again with 2 mL of ether. The filtrate, which contains the product and Fmoc derivatives, was concentrated. The resulting yellow oil was fractionally crystallized in petroleum ether (72.4% yield; M + Na ) 759.3065, theor ) 759.3061): IR 1731 cm-1 (CdO, br s); 1H NMR (CDCl3) δ 7.55 (2H, d), 7.10 (2H, d), 3.42 (8H, 2br s), 3.20-250 (8H, m), 1.42 (18H, s), 1.40 (18H, s). [p-(N-r-Fmoc-L-aspartic acid β-amido)benzyl]EDTA Tetra-tert-butyl Ester (9). To a round-bottom flask was transferred compound 8 (405 mg, 0.55 mmol), and 10 mL of 10% Na2CO3 was added. A solution of 270 mg of Fmoc-OSu (0.8 mmol) in 20 mL of DME was also added to the mixture. The reaction mixture was stirred for 5 h at room temperature. After the solvent was evaporated, 10 mL of H2O was added to the crude mixture and the pH was adjusted to 7 with 1 N HCl. The mixture was extracted with ethyl acetate and washed with H2O and brine. Spin chromatotron purification with 50% ethyl acetate in methanol gave 200 mg (0.21 mmol) of yellow oil as the product (38% yield; M + Na ) 959.503, theor ) 959.502): IR 1731 cm-1 (CdO, br s); 1H NMR (CDCl3) δ 7.7-7.0 (12H, m), 3.32 (8H, m), 3.20-2.20 (14H, m), 1.53 (18H, s), 1.47 (18H, s). PEPTIDE SYNTHESIS
All Fmoc-amino acids, piperidine, 4-(dimethylamino)pyridine, dichloromethane, N,N-dimethylforamide, 1-hydroxybenzotriazole (HOBT), 2-(1H-benzotriazo-1-yl)1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), diisopropylethylamine, and HMP-linked polystyrene resin were obtained from Applied Biosystems Division, PerkinElmer. Trifluoroacetic acid, 1,2-ethanedithiol, phenol, and thioanisole were from Sigma. Tat-derived peptide (from amino acids 56-72) was synthesized on an Applied Biosystems 431A peptide synthesizer using standard FastMoc protocols. Reagent 9 was incorporated in the peptide sequence by HOBt/DCC peptide coupling procedures. Cleavage and deprotection of the peptide was carried out in 2 mL of reagent K for 6 h at room temperature. Reagent K contained 1.75 mL of TFA, 100 µL of thioanisole, 100 µL of water, and 50 µL of ethanedithiol (39). After cleavage from the resin, peptide was purified by HPLC on a Zorbax 300 SB-C8 column. The mass of fully deprotected and purified peptides was confirmed by FAB mass spectrometry: calculated mass for Tat (56-72 containing Asp-aminobenzyl-EDTA at position 57) ) 2494.6, found 2495.6 (M + H). ACKNOWLEDGMENT
We thank Xilu Wang for critical reading of the manuscript. This research was supported by Research Grant AI 34785-01 from the National Institutes of Health.
Bioconjugate Chem., Vol. 8, No. 2, 1997 251 LITERATURE CITED (1) Tullius, T. D. (1989) In Metal-DNA Chemistry (T. D. Tullius, Ed.) pp 1-23, American Chemical Society, Washington, DC. (2) Pyle, A. M., Long, E. C., and Barton, J. K. (1989) Shapeselective targeting of DNA by (phenanthrequinone diimine) rhodium (III) photocleaving agents. J. Am. Chem. Soc. 111, 4520-4522. (3) Chow, C. S., and Barton, J. K. (1990) Shape-selective cleavage of tRNAphe by transition-metal complexes. J. Am. Chem. Soc. 112, 2839-2841. (4) Chow, C. S., Behlen, L. S., Uhlenbeck, O. C., and Barton, J. K. (1992) Recognition of tertiary structure in tRNAs by Rh(phen)2phi3+, a new reagent for RNA structure-function mapping. Biochemistry 31, 972-982. (5) Tullius, T. D., and Dombroski, B. A. (1985) Iron(II) EDTA used to measure the helical twist along any DNA molecule. Science 230, 679-681. (6) Price, M. A., and Tullius, T. D. (1992) Using hydroxyl radical to probe DNA structure. Methods Enzymol. 212, 194-219. (7) Latham, J. A., and Cech, T. R. (1989) Defining the Inside and Outside of a Catalytic RNA Molecule. Science 245, 276282. (8) Celander, D. W., and Cech, T. R. (1991) Visualizing the higher order folding of a catalytic RNA molecule. Science 251, 401-407. (9) Dreyer, G. B., and Dervan, P. B. (1985) Sequence-specific Cleavage of Single-stranded DNA: OligodeoxynucleotideEDTA. Fe(II). Proc. Natl. Acad. Sci. U.S.A. 82, 968-972. (10) Moser, H. E., and Dervan, P. B. (1987) Sequence-Specific Cleavage of Double Helical DNA by Triple Helix Formation. Science 238, 645-650. (11) Han, H., Schepartz, A., Pellegrini, M., and Dervan, P. B. (1994) Mapping RNA regions in eukaryotic ribosomes that are accessible to methidiumpropyl-EDTA-Fe(II) and EDTAFE(II). Biochemistry 33, 9831-9844. (12) Chen, C.-H. B., and Sigman, D. S. (1987) Chemical conversion of a DNA-binding protein into a site-specific nuclease. Science 237, 1197-1201. (13) Sluka, J. P., Horvath, S. J., Bruist, M. F., Simon, M. I., and Dervan, P. B. (1987) Synthesis of a sequence-specific DNA-cleaving Peptide. Science 238, 1129-1132. (14) Franc¸ ois, J.-C., et al. (1989) Sequence-Specific recognition and Cleavage of Duplex DNA via Triple-Helix Formation by Oligonucleotides Covalently Linked to a Phenanthrolinecopper Chelate. Proc. Natl. Acad. Sci. U.S.A. 86, 9702-9706. (15) Ebright, R. H., Ebright, Y. W., Pendergrast, P. S., and Gunasekera, A. (1990) Conversion of a helix-turn-helix motif sequence-specific DNA binding protein into a site-specific DNA cleavage agent. Proc. Natl. Acad. Sci. U.S.A. 87, 28822886. (16) Ebright, Y. W., Chen, Y., Ludescher, R. D., and Ebright, R. H. (1993) N-(iodoacetyl)-p-phenylenediamine-EDTA: a reagent for high-efficiency incorporation of an EDTA-metal complex at a rationally selected site within a protein. Bioconjugate Chem. 4, 219-225. (17) Rana, T. M., and Meares, C. F. (1990) Specific Cleavage of a Protein by an Attached Iron Chelate. J. Am. Chem. Soc. 112, 2457-2458. (18) Schepartz, A., and Cuenoud, B. (1990) Site-specific cleavage of the protein calmodulin using a trifluoperazine-based affinity reagent. J. Am. Chem. Soc. 112, 3247-3249. (19) Hoyer, D., Cho, H., and Schultz, P. G. (1990) A new strategy for selective protein cleavage. J. Am. Chem. Soc. 112, 3249-3250. (20) Rana, T. M., and Meares, C. F. (1991) Iron ChelateMediated Proteolysis: Protein Structure Dependence. J. Am. Chem. Soc. 113, 1859-1861. (21) Rana, T. M., and Meares, C. F. (1991) Transfer of Oxygen from an Artificial Protease to Peptide Carbon During Proteolysis. Proc. Natl. Acad. Sci. U.S.A. 88, 10578-10582. (22) Cuenound, B., Tarasow, T. M., and Schepartz, A. (1992) A new strategy for directed protein cleavage. Tetrahedron Lett. 33, 895-898. (23) Ermacora, M. R., Delfino, J. M., Cuenoud, B., Schepartz, A., and Fox, R. O. (1992) Conformation-dependent cleavage of staphylococcal nuclease with a disulfide-linked iron chelate. Proc. Natl. Acad. Sci. U.S.A. 89, 6383-6387.
252 Bioconjugate Chem., Vol. 8, No. 2, 1997 (24) Ermacora, M. R., Ledman, D. W., Hellinga, H. W., Hsu, G. W., and Fox, R. O. (1994) Mapping staphylococcal nuclease conformation using an EDTA-Fe derivative attached to genetically engineered cysteine residue. Biochemistry 33, 13625-13641. (25) Wu, J., Perrin, D. M., Sigman, D. S., and Kaback, H. R. (1995) Helix packing of lactose permease in Escherichia coli studies by site-directed chemical cleavage. Proc. Natl. Acad. Sci. U.S.A. 92, 9186-9190. (26) Parac, T. N., and Kostic´, N. M. (1996) Effects of linkage isomerism and of acid-base equilibria on reactivity and catalytic turnover in hydrolytic cleavage of histidyl peptides coordinated to palladium(II). Identification of the active complex between Palladium(II) and histidyl residue. J. Am. Chem. Soc. 118, 5946-5951. (27) Ghaim, J. B., Greiner, D. P., Meares, C. F., and Gennis, R. B. (1995) Proximity mapping the surface of a membrane protein using an artificial protease: demonstration that the quinone-binding domain of subunit I is near the N-terminal region of subunit II of cytochrome bd. Biochemistry 34, 11311-11315. (28) Heyduk, E., and Heyduk, T. (1994) Mapping protein domains involved in macromolecular interactions: a novel protein footprinting approach. Biochemistry 33, 9643-9650. (29) Greiner, D. P., Hughes, K. A., Gunasekera, A. H., and Meares, C. F. (1996) Binding of σ70 protein to the core subunits of Escherichia coli RNA polymerase, studies by ironEDTA protein footprinting. Proc. Natl. Acad. Sci. U.S.A. 93, 71-75. (30) Sundberg, M. W., Meares, C. F., Goodwin, D. A., and Diamanti, C. I. (1974) Selective binding of metal ions to macromolecules using bifunctional analogs of EDTA. J. Med. Chem. 17, 1304-1307.
Song and Rana (31) Meares,C. F., and Wensel, T. G. (1984) Metal Chelates as Probes for Biological Systems. Acc. Chem. Res. 17, 202-209. (32) Meares, C. F., and Goodwin, D. A. (1984) Linking Radiometals to Proteins with Bifunctional Chelating Agents. J. Protein Chem. 3, 215-227. (33) Sluka, J. P., Griffin, J. H., Mack, D. P., and Dervan, P. B. (1990) Reagents and methods for the solid-phase synthesis of Protein-EDTA for use in affinity cleaving. J. Am. Chem. Soc. 112, 6369-6374. (34) Cuenoud, B., and Schepartz, A. (1991) Synthesis of N-RBoc-N--tribenzyl EDTA-L-lysine. An amino acid analogue suitable for solid phase peptide synthesis. Tetrahedron 47, 2535-2542. (35) Arya, R., and Garie´py, J. (1991) Rapid synthesis and introduction of a protected EDTA-like group during the solidphase assembly of peptides. Bioconjugate Chem. 2, 323-326. (36) Fields, G. B., and Noble, R. L. (1990) Int. J. Pept. Protein Res. 35, 161-214. (37) Studer, M., and Meares, C. F. (1992) A convenient and flexible approach for introducing linkers on bifunctional chelating agents. Bioconjugate Chem. 3, 420-423. (38) Jones, K. A., and Peterlin, B. M. (1994) Control of RNA Initiation and Elongation at The HIV-1 Promoter. Annu. Rev. Biochem. 63, 717-743. (39) King, D. S., Fields, C. G., and Fields, G. B. (1990) A cleavage method which minimizes side reactions following Fmoc solid phase peptide synthesis. Int. J. Pept. Protein Res. 36, 255-266.
BC960084U
