Solid-Phase Synthesis of Deglycobleomycins: A C-Terminal

Tetraamine Linker That Permits Direct Evaluation of Resin-Bound. Bleomycins. Zhi-Fu Tao, Christopher J. Leitheiser, Kenneth L. Smith, Shigeki Hashimot...
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Bioconjugate Chem. 2002, 13, 426−434

Solid-Phase Synthesis of Deglycobleomycins: A C-Terminal Tetraamine Linker That Permits Direct Evaluation of Resin-Bound Bleomycins Zhi-Fu Tao, Christopher J. Leitheiser, Kenneth L. Smith, Shigeki Hashimoto, and Sidney M. Hecht* Departments of Chemistry and Biology, University of Virginia, Charlottesville, Virginia 22901. Received September 8, 2001

Deglycobleomycin analogues having different length polyamine side chains at the C-terminus were synthesized using a novel solid-phase synthesis strategy that produces fully deprotected deglycobleomycin congeners attached to the resin. Detailed studies of DNA cleavage by these compounds and their resin-bound counterparts using supercoiled plasmid DNAs and DNA restriction fragments as substrates revealed that (i) the length of the polyamine side chain of free deglycoBLM had limited effect on its DNA cleavage potency or sequence selectivity, and (ii) the nature of the linker moiety between the resin and attached deglycobleomycin had a more substantial effect on the potency of DNA cleavage, but no effect on sequence selectivity of resin-bound deglycoBLMs. Resin-bound 4 exhibited efficient DNA cleavage, indicating that its tetraamine linker moiety could be used for the elaboration and direct evaluation of bleomycin congeners attached to resins.

The bleomycins (BLMs),1 exemplified by bleomycin A5, are naturally occurring, peptide-derived antitumor agents (1, 2) that are used clinically for the treatment of several cancers, notably squamous cell carcinomas and malignant lymphomas (3). The antitumor activity of these BLMs is believed to depend on their ability to effect the selective cleavage of DNA (1, 2, 4-9) and possibly also RNA (10-12). Deglycobleomycin lacks the carbohydrate moiety but nonetheless exhibits DNA cleavage properties quite similar to those of bleomycin itself as regards potency, sequence selectivity, and actual chemistry of DNA degradation (13-15). Numerous analogues of bleomycin and deglycobleomycin have been synthesized and characterized for their properties in DNA degradation (16-35). However, with the exception of our discovery that the carbohydrate moiety of bleomycin is not essential for DNA cleavage (13-15), none of the analogues reported to date can be argued to have improved properties, simplified structures, or to be more readily accessible by synthesis such that improved antitumor agents might be realized. We have developed two solid-phase strategies for synthesis of BLM analogues (36, 37), one goal of which is the elaboration of BLM-like species having specific improved properties. It should be possible to realize this goal through the synthesis and survey of large numbers of BLM analogues. * Address correspondence to Dr. Sidney M. Hecht, Department of Chemistry, University of Virginia, Charlottesville, VA 22901. Fax: (434) 924-7856. E-mail: [email protected]. 1 Abbreviations: BLM, bleomycin; Boc, tert-butoxycarbonyl; BOP, benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate; Dde, 2-acetyldimedone; DIPEA, N,N′-diisopropylethylamine, Hu¨nig’s base; HATU, O-(7-azabenzotriazol1-yl)- N,N,N′,N′-tetramethyluronium hexafluorophosphate; Fmoc, 9-fluorenylmethoxycarbonyl; HBTU, O-benzotriazolyl-N,N,N′,N′tetramethyluronium hexafluorophosphate; HOBt, N-hydroxybenzotriazole; HOAt, 1-hydroxy-7-azabenzotriazole; TFA, trifluoroacetic acid; Tr, triphenylmethyl.

Combinatorial chemistry has become a powerful tool for the organic and medicinal chemist. There are numerous ways in which libraries can be prepared and tested; solid-phase synthesis has been utilized widely for library construction (38-43). The assay of libraries on solid supports has been used successfully for the screening of medicinally important molecules and synthetic receptors (44-50). A key parameter that defines the potential utility of resin-bound ligands for assaying ligand-receptor inter-

10.1021/bc010083o CCC: $22.00 © 2002 American Chemical Society Published on Web 03/09/2002

Resin-Bound Bleomycin Analogues

action is the accessibility of resin-bound library members to the receptors. The recent finding (51, 52) that conjugation of BLM A5 to a solid support via its C-terminus had little effect on its ability to mediate DNA cleavage encouraged us to identify a C-terminal linker applicable to direct evaluation of resin-bound DNA-cleaving bleomycin analogues. Reported herein are (i) the solid-phase syntheses of deglycoBLM analogues in which the Cterminal polyamine substituent is varied in a systematic fashion, and (ii) a characterization of DNA cleavage by these resin-free and resin-bound analogues, resulting in the identification of a C-substituent that facilitates the direct evaluation of resin-bound bleomycin analogues.

Bioconjugate Chem., Vol. 13, No. 3, 2002 427

All synthetic reactions were carried out under nitrogen or argon. Anhydrous grade DMF, anhydrous grade piperidine, HATU, HOAt, trifluoroacetic acid, hydrazine monohydrate, and anhydrous grade Hunig’s base were purchased from Aldrich chemicals. TentaGel resin, HBTU, HOBt, BOP, 9-fluorenylmethoxycarbonyl-β-alanine, and 9-fluorenylmethoxycarbamoylthreonine were purchased from NovaBiochem. Methylene chloride was distilled from calcium hydride. Fmoc cleavage was determined by UV measurement of the dibenzylfulvene-piperidine adduct formed upon treatment of the resin with piperidine (54). The optical density of 5540 M-1 at 290 nm and 7300 M-1 at 300 nm was used to calculate the loading from a known weight of dry resin. Molecular beacon DNA oligodeoxynucleotide, labeled with 5′ FAM and 3′ BHQ dyes, was custom-synthesized by Integrated DNA Technology, Inc. BHQ is a trademark of Biosearch Technologies, Inc.; the chemical structure has not been disclosed. Fluorescence image analysis was performed at the W. M. Keck Center for Cellular Imaging, University of Virginia, using an Olympus Infinity corrected inverted microscope.

EXPERIMENTAL PROCEDURES

Materials and General Techniques. Calf intestinal phosphatase (one unit is the amount of enzyme that hydrolyzes 1 µmol of p-nitrophenyl phosphate to pnitrophenol in 1 min at 37 °C in a total reaction volume of 1 mL), EcoRV, HindIII (one unit is the amount of enzyme required to digest 1 µg of λ DNA in 1 h at 37 °C in a total reaction volume of 50 µL), T4 polynucleotide kinase (one unit is the amount of enzyme catalyzing the production of 1 nmol of acid-insoluble 32P in 30 min at 37 °C under the following conditions: 70 mM Tris-HCl, pH 7.6, 10 mM MgCl2, 5 mM DTT, 66 µM [γ-32P]ATP (5 × 106 cpm/mmol), 0.26 mM 5′-OH terminating salmon sperm DNA). Plasmid pSP64 was cultured in E. coli and isolated using a Qiagen resin. Plasmid pBR322 DNA was purchased from New England Biolabs. [γ-32P]ATP (7000 Ci/mmol) was obtained from ICN Radiochemicals, Inc. Fe(NH4)2(SO4)2‚6H2O (Aldrich) was used to prepare aqueous solutions for admixture with bleomycin; these were made immediately prior to use. Agarose gel electrophoresis was carried out in 90 mM Tris-borate buffer, pH 8.3, containing 5 mM EDTA. The agarose gel loading solution contained 30% glycerol and 0.05% (w/v) bromophenol blue. Polyacrylamide gel electrophoresis was carried out in 90 mM Tris-borate buffer, pH 8.3, containing 5 mM EDTA. The loading solution contained 80% formamide, 2 mM EDTA, 0.05% (w/v) xylene cyanol, and 0.05% (w/v) bromophenol blue. Chemical sequencing was carried out according to the method of Maxam and Gilbert (53). Distilled and deionized water from a Milli-Q system was autoclaved prior to use in all aqueous solutions in the DNA cleavage experiments. The 5′-32P end labeled 158-bp DNA restriction fragment was prepared from pBR322 plasmid DNA according to reported procedures (51, 52). Ultraviolet spectra were recorded on a Perkin-Elmer Lambda 20 UV-Vis spectrophotometer. 1H NMR spectra were recorded on a General Electric QE-300 MHz NMR spectrophotometer using the residual HOD resonance at 4.80 ppm as an internal standard. HPLC purification was performed on a Varian 9012 HPLC gradient programmer system with a Perkin-Elmer LC-235 diode array detector.

Synthesis of Resin-Bound DeglycoBLM 4. DeglycoBLM analogues 1-4 were prepared using closely related methods; these are exemplified by the synthesis of deglycoBLM 4. To a suspension containing 200 mg (0.45 mmol/g) of TentaGel amino functionalized resin (5; Scheme 1) (55) was added a DMF solution containing 362 mg (0.90 mmol) of protected spermine and 313 µL (232 mg, 1.8 mmol) of Hunig’s base. The reaction mixture was shaken for 24 h, and then the resin was filtered and washed successively for 30 s each with three 5-mL portions of DMF, three 5-mL portions of CH2Cl2, three 5-mL portions of methanol, and again with three 5-mL portions of DMF. A DMF solution containing 128 mg (0.27 mmol) of Fmoc-bithiazole, 102 mg (0.27 mmol) of HBTU, and 94 µL (70 mg, 0.54 mmol) of Hunig’s base was added. After 30 min, the resin was filtered and washed successively for 30 s each with three 5-mL portions of DMF, three 5-mL portions of CH2Cl2, and again with three 5-mL portions of DMF. Quantitation of Fmoc cleavage indicated a loading of 0.17 mmol/g (57% yield for three steps). The resulting resin was treated for 5 min each with three 2-mL DMF solutions containing 20% piperidine in DMF. The resulting resin was washed for 30 s each with seven 5-mL portions of DMF, five 5-mL portions of CH2Cl2, and three 5-mL portions of DMF. A solution containing 35 mg (0.10 mmol) of Fmoc-threonine, 38 mg (0.10 mmol) of HBTU, 15 mg (0.10 mmol) of HOBt, and 36 µL (26 mg, 0.20 mmol) of Hunig’s base in 0.5 mL of DMF was added. After 30 min the resin was filtered and washed with three 5-mL portions of DMF, three 5-mL portions of CH2Cl2, and again with three 5-mL

428 Bioconjugate Chem., Vol. 13, No. 3, 2002 Scheme 1. Solid-Phase Synthesis of Resin-Bound and Free DeglycoBLM 4

portions of DMF. Quantitation of Fmoc cleavage indicated a loading of 0.17 mmol/g (>95%). The resin was treated for 5 min each with three 2-mL DMF solutions containing 20% piperidine in DMF. The resin was washed for 30 s each with seven 5-mL portions of DMF, five 5-mL portions of CH2Cl2, and three 5-mL portions of DMF. The resin was treated with a solution containing 37 mg (0.10 mmol) of Fmoc-methylvalerate, 38 mg (0.10 mmol) of HBTU, 15 mg (0.10 mmol) of HOBt, and 36 µL (26 mg, 0.20 mmol) of Hunig’s base. After 30 min, the resin was filtered and washed for 30 s each with three 5-mL portions of DMF, three 5-mL portions of CH2Cl2, and again with three 5-mL portions of DMF. Quantitation of Fmoc cleavage indicated a loading of 0.15 mmol/g (94%). The resulting resin was treated for 5 min each with three 2-mL DMF solutions containing 20% piperidine in DMF and then washed for 30 s each with seven 5-mL portions of DMF, five 5-mL portions of CH2Cl2, and three 5-mL portions of DMF. The resin was treated with a solution containing 63 mg (0.10 mmol) of tritylated Fmoc histidine, 38 mg (0.10 mmol) of HATU, 16 mg (0.10 mmol) of HOAt, and 36 µL (26 mg, 0.20 mmol) of Hunig’s base. After 30 min, the resin was filtered and washed for 30 s each with three 5-mL portions of DMF, three 5-mL portions of CH2Cl2, and with three 5-mL portions of methanol. The resulting resin was dried under diminished pressure over KOH pellets. A small sample (∼20 mg) was treated for 5 min each with three 1-mL portions of 2% hydrazine in DMF (v/v). The resulting solution was concentrated under diminished pressure. The resulting oil was dissolved in 1 mL of an aq 0.1% TFA solution, frozen, and lyophilized to give an off-white solid: mass spectrum (electrospray) m/z 1249.4 (M + H)+ and 1273.6 (M + Na)+. A suspension containing 40 mg of the above resin swollen in 0.5 mL of DMF was treated for 5 min each with three 2-mL DMF solutions containing 20% piperidine in DMF. The resulting resin was washed for 30 s each with seven 5-mL portions of DMF, five 5-mL portions of CH2Cl2, and three 5-mL portions of DMF. The

Tao et al.

resin was treated with a cooled (0 °C) solution containing 5.0 mg (12 µmol) of Boc-pyrimidoblamic acid, 15 mg (35 µmol) of BOP, and 12 µL (9.0 mg, 70 µmol) of Hunig’s base in 0.5 mL of DMF. After 16 h, the resin was filtered and washed for 30 s each with three 5-mL portions of DMF, three 5-mL portions of CH2Cl2, and three 5-mL portions of methanol. The resin was treated with a solution consisting of 100 µL of triisopropylsilane and 100 µL of dimethyl sulfide. The resin was shaken for 5 min and then 4 mL of trifluoroacetic acid was added to the suspension. After 4 h, the resin was filtered and washed with five 3-mL portions of CH2Cl2 and three 3-mL portions of methanol. The resulting resin was dried under diminished pressure over KOH pellets to give resinbound deglycoBLM 4. Synthesis of DeglycoBLM 4. To a suspension containing 30 mg of resin-bound deglycoBLM 4 was added a 0.5 mL of DMF containing 2% hydrazine. The resin was filtered and then treated with three additional 0.5-mL portions of DMF containing 2% hydrazine. Each treatment was carried out for 5 min. The combined filtrate was concentrated under diminished pressure. The resulting oil was dissolved in 2 mL of CF3COOH and added dropwise to cold (-20 °C) ether. The suspension was centrifuged and the ether decanted. The resulting solid was dissolved in water, frozen and lyophilized. HPLC of the crude material on an Alltech Alltima semipreparative C18 reversed-phase HPLC column (250 × 10 mm, 5 µm) was carried out using 0.1% aqueous CF3COOH and CH3CN as mobile phases. A linear gradient was employed starting from 88:12 0.1% aqueous CF3COOH-CH3CN to 83:17 0.1% aqueous CF3COOH-CH3CN over a period of 30 min at a flow rate of 4 mL/min. Fractions containing the desired product eluted after 12 min; these were collected and lyophilized. The yield was determined by 1 H NMR spectroscopy, by comparing the integration of the exocyclic methyl group on pyrimidoblamic acid vs the methyl signal resulting from a known concentration of tert-butyl alcohol, and then relating the calculated concentration to the amount of the di-tert-butoxycarbonyl spermine determined to have been attached to the resin initially. Yield 3.1 mg (66%); 1H NMR (D2O) δ 0.94 (d, 3H, J ) 7.0 Hz), 0.99 (d, 3H, J ) 7.0 Hz), 1.07 (d, 3 H, J ) 7.0 Hz), 1.69 (m, 4H), 1.93 (s, 3H), 1.98 (m, 4H), 2.53 (t, 1H, J ) 7.5 Hz), 2.63 (m, 2H), 3.03 (m, 12H), 3.21 (m, 4H), 3.44 (t, 2H, J ) 7.0 Hz), 3.58 (t, 1H, J ) 5.5 Hz), 3.64 (t, 1H, J ) 5.0 Hz), 3.77 (t, 1H, J ) 6.5 Hz), 3.97 (t, 2H, J ) 6.0 Hz), 4.04 (t, 1H, J ) 5.5 Hz), 4.12 (d, 1H, J ) 5.5 Hz), 7.27 (s, 1H), 7.98 (s, 1H), 8.13 (s, 1H) and 8.59 (s, 1H); mass spectrum (electrospray), m/z 1114.6 (M + H)+ (calculated 1114.4). General Procedure for Relaxation of Plasmid DNA by DeglycoBLM Analogues. The reactions were performed in a 25 µL (total volume) reaction mixture containing 300 ng of pSP64 DNA, 10 mM sodium cacodylate, pH 7.0, and various concentrations of BLM (analogues) or Fe2+. The concentrations of resin-bound deglycoBLM analogues are expressed as the concentration that would have been present in solution had the analogues not been attached to the resin. Each reaction mixture was incubated at 37 °C for 30 min, terminated by the addition of 5 µL of loading dye (30% glycerol containing 0.125% bromophenol blue) and applied to 1% agarose gel containing 0.5 µg/mL ethidium bromide. The gel was run in 90 mM Tris-borate buffer, pH 8.3, containing 5 mM EDTA at 138 V for 3-3.5 h; the DNA bands were visualized under UV light. General Procedure for Degradation of 5′-32P End Labeled DNA Duplexes by DeglycoBLM Analogues.

Resin-Bound Bleomycin Analogues

Reactions were carried out in 25 µL (total volume) of 10 mM sodium cacodylate, pH 7.0, containing 32P-labeled DNA duplex (104 cpm) and the concentrations of reagents indicated in the figure legends. Each reaction mixture was incubated at 37 °C for 30 min, and then the reaction mixture was frozen in dry ice, lyophilized, and dissolved in gel loading solution (80% formamide, 2 mM EDTA, 0.05% (w/v) xylene cyanol, and 0.05% (w/v) bromophenol blue). The resulting solution was heated at 90 °C for 3 min, chilled on ice, and then applied to a 10% denaturing polyacrylamide gel. The gel was visualized by autoradiography or by use of a phosphorimager. The sites of cleavage mediated by (resin-bound) 1-4 were determined by comparison with Maxam-Gilbert DNA sequencing lanes (53). Fluorescence Microscope Analysis of Beacon DNA Cleavage. A suspension of TentaGel-conjugated deglycoBLM 4 and agarose gel solution were prepared in 50 mM sodium cacodylate, pH 7.0, for the fluorescence microscopy studies. To a single well on a 96-well plate was added 35 µL of 2.4% agarose gel solution in 50 mM sodium cacodylate, pH 7.0; after solidification, this was followed by 20 µL of 500 µM TentaGel-conjugated deglycoBLM A6 suspension in the same buffer, and an additional 35 µL of buffered 2.4% agarose gel solution. The resulting 55-µL suspension above the solidified gel was immediately mixed with a pipet tip. The plate was placed on ice for 20 min to facilitate solidification of the agarose mixture. A 30-µL solution of 1.5 µM beacon DNA solution was added to the agarose mixture containing the TentaGel beads. The plate was maintained on ice in the absence of light for 12 h to allow the beacon DNA to diffuse through the agarose gel. The final concentrations of deglycoBLM A6 and beacon DNA were 0.11 mM and 0.37 µM, respectively. The supernatant on the agarose mixture was removed, and a gel piece containing the bleomycin bound resin was excised from the gel. The gel was sandwiched between two cover glass plates (22 × 22 mm), and the bleomycin was activated with 10 µL of 5 mM Fe(NH4)2(SO4)2 solution. Fluorescence images were taken every 5 min through an FITC filter (excitation at 480 ( 20 nm; fluorescence emission at 535 ( 25 nm). RESULTS AND DISCUSSION

Solid-Phase Synthesis of DeglycoBLM Analogues. We have described a solid-phase synthesis of deglycoBLM A5 (36), in which the final deprotection of acid-labile groups and detachment of the deglycoBLM from the solid support were accomplished simultaneously by treatment with CF3COOH; therefore, fully deprotected resin-bound bleomycin analogues could not be obtained by this approach. Access to resin-bound, fully deprotected BLM analogues has been accomplished by the use of a solidphase methodology (37) that employs a Dde-based linker (54). This linker has been shown to be stable to conditions used to remove both base and acid-labile protecting groups. As outlined in Scheme 1 starting from TentaGelderived resin 5, which was prepared according to reported procedures (55), Boc-protected spermine (56) was attached to the resin via a transamination procedure. The resulting primary amine was then condensed with the bithiazole moiety of bleomycin. For this purpose, Fmocprotected bithiazole was activated with HBTU and Hunig’s base in DMF; this solution was added to the resin containing a free primary amino group, and the mixture was shaken under N2 at 25 °C for 30 min. The resin was then washed with DMF and CH2Cl2, and a small sample was utilized for qualitative verification of coupling by colorimetric assays using the Kaiser (57) and bromophe-

Bioconjugate Chem., Vol. 13, No. 3, 2002 429

Figure 1. Relaxation of supercoiled pSP64 plasmid DNA by deglycoBLM analogues 1-4. Lane 1: DNA alone; lane 2: 1.5 µM Fe2+; lane 3: 3 µM 1; lane 4: 1 µM 1 + 1.5 µM Fe2+; lane 5: 3 µM 1 + 1.5 µM Fe2+; lane 6: 3 µM 2; lane 7: 1 µM 2 + 1.5 µM Fe2+; lane 8: 3 µM 2 + 1.5 µM Fe2+; lane 9: 3 µM 3; lane 10: 1 µM 3 + 1.5 µM Fe2+; lane 11: 3 µM 3 + 1.5 µM Fe2+; lane 12: 3 µM 4; lane 13: 1 µM 4 + 1.5 µM Fe2+; lane 14: 3 µM 4 + 1.5 µM Fe2+; lane 15: 3 µM deglycoBLM A5; lane 16: 1 µM deglycoBLM A5 + 1.5 µM Fe2+; lane 17: 3 µM deglycoBLM A5 + 1.5 µM Fe2+.

nol blue (58) tests for amines. The extent of coupling was quantified by UV measurement following Fmoc cleavage with piperidine (54); this procedure indicated a loading of 0.17 mmol/g of resin, which represented a coupling efficiency of 57% for three steps. Successive treatments of the resin with 20% piperidine in DMF, followed by Fmoc threonine, resulted in successful synthesis of the tripeptide. The same procedure was used for attachment of Fmoc-methylvaleric acid, resulting in successive attachments of these two amino acids in yields of >95% (0.17 mmol/g) and 94% (0.15 mmol/g), respectively. The resin-bound oligopeptide was deblocked with piperidine in DMF and coupled with Fmoc histidine in the presence of the highly reactive coupling reagent HATU; the coupling efficiency was calculated as 95%, based on spectrophotometric assay of the dibenzylfulvene-piperidine adduct formed following treatment of a portion of the resin with piperidine (54). Boc pyrimidoblamic acid (59-61) was then coupled to the resin using BOP reagent and Hunig’s base at 0 °C in the absence of light for 16 h. The resin was washed with DMF and methylene chloride, and the acid-sensitive trityl protecting group on histidine and Boc protecting group on pyrimidoblamic acid were removed simultaneously by treatment with 90:5:5 CF3COOH-triisopropylsilane-Me2S. This afforded fully deprotected resin-bound deglycoBLM 4. A portion of resin-bound 4 was treated with 2% hydrazine in DMF to give deglycoBLM 4; the structure was confirmed by 1H NMR spectroscopy and electrospray ionization mass spectrometry. The yield of deglycoBLM 4 prepared by solid-phase synthesis was 66%. DeglycoBLMs 1, 2, and 3 were synthesized and characterized by following procedures analogous to those employed for analogue 4. DNA Cleavage Mediated by DeglycoBLM Analogues. The abilities of deglycoBLM analogues 1-4 to cleave DNA were investigated initially using supercoiled plasmid DNA as a substrate. As shown in Figure 1, deglycoBLM analogues 2-4 mediated DNA relaxation to roughly the same extent in the presence of Fe2+; the cleavage mediated by Fe(II)‚deglycoBLM A5 was also determined for comparative purposes and shown to be quite similar. Thus the efficiency of DNA cleavage mediated by deglycoBLM analogue 4, having three basic amino groups within the C-terminal substituent, was not significantly different in this assay than that of 2 or 3, each of which contain two amino groups within the C-substituent. In comparison, analogue 1, having a single amine within the C-substituent, was less efficient as a DNA cleaving agent. This finding was anticipated based on earlier studies of the effects of the number and spacing of positively charged groups in the C-substituent on the efficiency of DNA binding (62) and degradation (63).

430 Bioconjugate Chem., Vol. 13, No. 3, 2002

Figure 2. Cleavage of a 5′-32P end-labeled 158-bp DNA restriction fragment by 1 and resin-bound 1. Lane 1: DNA alone; lane 2: 15 µM Fe2+; lane 3: 15 µM 1; lane 4: 1.5 µM 1 + 15 µM Fe2+; lane 5: 15 µM 1 + 15 µM Fe2+; lane 6: 15 µM deglycoBLM A2; lane 7: 1.5 µM deglycoBLM A2 + 15 µM Fe2+; lane 8: 15 µM deglycoBLM A2 + 15 µM Fe2+; lane 9: 200 µM resin-bound 1; lane 10: 50 µM resin-bound 1 + 30 µM Fe2+; lane 11: 100 µM resin-bound 1 + 30 µM Fe2+; lane 12: 200 µM 1 + 30 µM Fe2+.

DeglycoBLM analogues 2 and 3 have the same (spermidine) C-substituent, but differ in that analogue 2 contains a histidine moiety while 3 was synthesized using the amino acid analogue (S)-erythro-β-hydroxyhistidine; the OH group of the latter is the point of attachment of the disaccharide moiety in the BLMs (cf BLM A5 and deglycoBLM A5). The similar potencies of these two analogues in the experiment shown in Figure 1 argues that this OH group does not contribute to the efficiency of deglycoBLM-mediated DNA relaxation, a finding that is entirely consistent with a previous report from the Boger laboratory (64). Also investigated was the sequence selectivity of DNA cleavage by these four deglycoBLMs. The substrate employed for this study was a 158-base pair DNA restriction fragment that has been employed previously as a substrate for Fe(II)‚BLMs (33,51,52). As shown in Figures 2-4, and Figures 1 and 2 of the Supporting Information, each of the four analogues mediated sequence-selective cleavage of the DNA substrate in the same fashion as BLM itself. Thus the nature of the C-terminal substituent had little if any effect on sequence selectivity of DNA cleavage, as might have been anticipated based on earlier work (28, 65). The potency of DNA cleavage by 1-4 was investigated in direct comparison with a deglycoBLM congener obtained by HF-mediated cleavage of the sugar moiety (66). Thus deglycoBLM analogue 1 was compared directly with deglycoBLM A2; both of these have a single positively charged group within the C-substituent, and the distance of these charged groups from the bleomycinic acid nucleus (1) is quite similar. As shown in Figure 2 and Supporting Information Figure 1, deglycoBLM analogue 1 and deglycoBLM A2 were of comparable potency as DNA cleaving agents. An analogous result was obtained for deglycoBLMs 2 and 3, both of which contain spermidine C-substituents.

Tao et al.

Figure 3. Cleavage of a 5′-32P end-labeled 158-bp DNA fragment by 2, 3, resin-bound 2, and resin-bound 3. Lane 1: DNA alone; lane 2: 1 µM 2 + 10 µM Fe2+; lane 3: 10 µM 2 + 10 µM Fe2+; lane 4: 1 µM resin-bound 2 + 10 µM Fe2+; lane 5: 10 µM resin-bound 2 + 10 µM Fe2+; lane 6: 100 µM resin-bound 2 + 10 µM Fe2+; lane 7: 1 µM 3 + 10 µM Fe2+; lane 8: 10 µM 3 + 10 µM Fe2+; lane 9: 1 µM resin-bound 3 + 10 µM Fe2+; lane 10: 10 µM resin-bound 3 + 10 µM Fe2+.

Figure 4. Cleavage of a 5′-32P end-labeled 158-bp DNA fragment by 4 and resin-bound 4. Lane 1: DNA alone; lane 2: 10 µM 4; lane 3: 10 µM Fe2+; lane 4: 1 µM 4 + 10 µM Fe2+; lane 5: 10 µM 4 + 10 µM Fe2+; lane 6: 0.1 µM BLM A5 +10 µM Fe2+; lane 7: 1 µM BLM A5 +10 µM Fe2+; lane 8: 10 µM resinbound 4 + 10 µM Fe2+; lane 9: 100 µM resin-bound 4 + 10 µM Fe2+.

Their potencies of DNA cleavage assayed on a polyacrylamide gel (Figure 3) were the same, as had been noted in the supercoiled plasmid DNA relaxation assay (Figure 1). Further, assay of BLM 3 in direct comparison with

Resin-Bound Bleomycin Analogues

Figure 5. Relaxation of supercoiled pSP64 DNA by 1 and resinbound 1. Lane 1: DNA alone; lane 2: 1.5 µM Fe2+; lane 3: 3 µM deglycoBLM A2; lane 4: 1 µM deglycoBLM A2 + 1.5 µM Fe2+; lane 5: 3 µM deglycoBLM A2 + 1.5 µM Fe2+; lane 6: 3 µM 1; lane 7: 1 µM 1 + 1.5 µM Fe2+; lane 8: 3 µM 1 + 1.5 µM Fe2+; lane 9: 10 µM resin-bound 1; lane 10: 10 µM resin-bound 1 + 1.5 µM Fe2+.

authentic deglycoBLM A5, with which it was putatively identical, resulted in the same pattern and potency of cleavage of the 158-base pair DNA substrate when the BLMs were employed at each of two different concentrations (Supporting Information Figure 2). Finally, comparison of deglycoBLM analogue 4 with BLM A5 indicated good potency of cleavage by both (Figure 4), although the range of concentrations tested was not designed to permit a definitive assessment of the relative cleavage potencies of these two species. DNA Cleavage Mediated by Resin-Bound DeglycoBLM Analogues. The successful synthesis of fully deprotected resin-bound deglycoBLM analogues has permitted an assessment of the ability to characterize these BLM analogue conjugates as DNA cleaving agents directly (37). It was anticipated that on-resin characterization might prove difficult for any of a few different reasons. These include simple lack of accessibility of the ligand to the target, as noted previously (49). Our earlier finding that BLMs tethered to each of two solid supports by linkers of three different lengths could all mediate DNA cleavage (51, 52) argued that the accessibility problem could be overcome by an appropriate choice of linkers. Potentially more serious was the observation that the attachment of BLM A5 to a solid support resulted in a severalfold diminution in the potency of cleavage (51, 52). This effect was attributed to the attachment of BLMs to beads whose concentration in solution was (1012-1013fold) lower than that usually employed for DNA cleavage by BLM, thus possibly diminishing the efficiency of encounter of BLMs by the DNA substrate. Since BLM A5 was attached to the solid supports by utilizing a side chain amine to form an amide bond, it is also the case that the number of positively charged groups on the C-substituent would be reduced from 2 to 1; as illustrated in Figure 1, this could also potentially affect the efficiency of DNA binding and cleavage (62, 63). Accordingly, we sought to assess the DNA cleaving characteristics of deglycoBLM analogues having different numbers of positively charged functionalities within their C-substituents when the analogues were tested in resin-free or resin-bound form. As shown in Figure 5, resin-bound deglycoBLM 1 gave only minimal cleavage of supercoiled plasmid DNA even when employed at high (i.e., 10 µM) concentration. An analogous effect was observed when resin-bound 1 was used in the presence of a 32P-end labeled DNA duplex; no significant cleavage was observed in the presence of 200 µM resin-bound 1 + 30 µM Fe2+ (Figure 2). In comparison, the resin-free 1 gave detectable cleavage in the presence of 1.5 µM free 1 + 15 µM Fe2+, and strong cleavage when 15 µM concentrations of free 1 and Fe2+ were present (Figure 2). Thus, attachment of this analogue to a solid support resulted in a much greater diminution in cleavage potency than had been observed previously for BLM A5 (51, 52).

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Figure 6. Relaxation of supercoiled pSP64 plasmid DNA by 4 and resin-bound 4. Lane 1: DNA alone; lane 2: 1.5 µM Fe2+; lane 3: 5 µM 4; lane 4: 0.5 µM 4 + 1.5 µM Fe2+; lane 5: 1 µM 4 + 1.5 µM Fe2+; lane 6: 3 µM 4 + 1.5 µM Fe2+; lane 7: 5 µM 4 + 1.5 µM Fe2+; lane 8: 50 µM resin-bound 4; lane 9: 0.5 µM resin-bound 4 + 1.5 µM Fe2+; lane 10: 1 µM resin-bound 4 + 1.5 µM Fe2+; lane 11: 3 µM resin-bound 4 + 1.5 µM Fe2+; lane 12: 5 µM resin-bound 4 + 1.5 µM Fe2+; lane 13: 10 µM resinbound 4 + 1.5 µM Fe2+; lane 14: 50 µM resin-bound 4 + 1.5 µM Fe2+.

Figure 7. Relaxation of supercoiled pSP64 plasmid DNA cleavage by 3, resin-bound 3 and resin-bound 4. Lane 1: 100 µM resin-bound 3 + 1.5 µM Fe2+; lane 2: 50 µM resin-bound 3 + 1.5 µM Fe2+; lane 3: 10 µM resin-bound 3 + 1.5 µM Fe2+; lane 4: 100 µM resin-bound 3; lane 5: DNA alone; lane 6: 1.5 µM Fe2+; lane 7: 5 µM 3; lane 8: 1 µM 3 + 1.5 µM Fe2+; lane 9: 3 µM 3 + 1.5 µM Fe2+; lane 10: 5 µM 3 + 1.5 µM Fe2+; lane 11: 50 µM resin-bound 4; lane 12: 1 µM resin-bound 4 + 1.5 µM Fe2+; lane 13: 5 µM resin-bound 4 + 1.5 µM Fe2+; lane 14: 10 µM resin-bound 4 + 1.5 µM Fe2+; lane 15: 25 µM resin-bound 4 + 1.5 µM Fe2+; lane 16: 50 µM resin-bound 4 + 1.5 µM Fe2+.

Although deglycoBLM analogues 2 and 3 exhibited greater potency than 1 in relaxing supercoiled plasmid DNA (Figure 1), these analogues also gave disappointing results when tested in their resin-bound forms. As shown in Figure 3, in the presence of 10 µM Fe2+, analogues 2 and 3 gave readily detectable sequence selective DNA cleavage at 1 and 10 µM concentrations. In comparison, resin-bound 2 and 3 gave much weaker cleavage at all tested concentrations; the former species was utilized at concentrations up to 100 µM in the presence of 10 µM Fe2+. Although 2 and 3 are formally analogues of BLM A5, the conjugates of which do produce strong DNA cleavage in resin-bound form (51, 52), the (2-3-fold) diminution of DNA cleavage potency known to result from the absence of the BLM disaccharide moiety (1315) was sufficient to compromise the ability of resinbound 2 and 3 to produce a useful level of DNA damage. In contrast, resin-bound 4 cleaved DNA very efficiently. A plasmid DNA relaxation assay (Figures 6 and 7) revealed that, in the presence of 1.5 µM Fe2+, resin-bound 4 degraded the substrate significantly even at low micromolar concentrations (Figure 7, lanes 9-12) and completely degraded the DNA at 50 µM concentration. The potency of DNA cleavage by resin-bound 4 was further confirmed by high-resolution gel electrophoresis analysis of a 5′-32P end labeled 158 base pair DNA fragment that had been treated with resin-bound 4. As is clear from Figure 4, resin-bound 4 (lanes 8 and 9) cleaved the duplex with the same sequence selectivity and comparable potency as resin-free 4 (lanes 4 and 5) and natural BLM A5 (lanes 6 and 7). The difference in potencies of DNA cleavage by resin-free and resin-bound analogue 4 was clearly less than 10-fold, demonstrating the ability of additional positively charged groups in the C-substituent to compensate for the absence of the BLM disaccharide moiety, as well as the attachment of the analogue to the TentaGel resin. It may be noted also that

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Figure 8. Fluorescence microscopy of TentaGel beads containing conjugated Fe(II)‚deglycoBLM 4. The images were recorded prior to Fe2+ addition (left) and 30 min after Fe2+ addition (right).

the sequence selectivity of DNA cleavage by resin-bound 4 was identical with that of resin-free 4 (Figure 4). To evaluate the relative importance of the number of positive charges in the C-substituent vs conjugation to a resin on the efficiency of DNA cleavage, the ability of free 3 to effect plasmid DNA relaxation was compared to that of resin-bound 3 and resin-bound 4. Resin-bound 3 has one fewer positively charged group than unbound 3 at physiological pH, while resin-bound 4 should have the same number as free 3. As shown in Figure 7, neither resin-bound BLM analogue was nearly so potent as free 3 in effecting DNA relaxation, but resin-bound 4 was significantly more efficient than resin-bound 3. Thus actual conjugation of a BLM to a solid support would seem to lower the efficiency of DNA cleavage primarily for reasons of localization of BLM to the (very small number of) resin beads, rather than by reducing the number of positively charged groups within the Csubstituent. Nevertheless, it is clear from the foregoing results that the incorporation of additional positively charged groups can be effective in compensating for the loss of overall cleavage efficiency and can do so without altering the sequence selectivity of DNA cleavage. Resinbound 4 exhibited strong DNA cleavage, indicating that the spermine-based linker in this analogue should be sufficient to permit the evaluation of many (deglyco)BLM analogues while they are still bound to the resin employed for their elaboration. Molecular Beacon Assay Utilizing Fluorescence Microscopy. DNA cleavage by TentaGel-conjugated BLM 4 was also monitored by molecular beacon assay utilizing fluorescence microscopy (37, 67). A molecular beacon designed for bleomycin cleavage was shown to fluoresce following BLM-mediated DNA cleavage (67). The sequence of the molecular beacon used for the microscopy was 5′d(CGCT3A7GCG); the beacon contained the fluorophore carboxyfluorescein (FAM) at the 5′-end and the “Black Hole Quencher” (BHQ) at the 3′-end. Though DABCYL has been used routinely as a quencher for commonly used fluorophores (68-70), the quenching efficiency is sometimes not high due to poor spectral overlap with the fluorescent dye (71). A new class of quencher, BHQ dye, has an absorption spectrum essentially superimposable with the emission maximum of FAM, and thus provided a significant increase in fluorescence resonance energy transfer (FRET). Therefore, a molecular beacon made with this quencher exhibited very low background fluorescence, enabling enhanced detection sensitivity. Beacon cleavage for visualization was carried out with the TentaGel embedded in an

agarose gel medium to retard diffusion of the formed fluorescent cleavage products from the gel. Figure 8 shows the fluorescence images of beacon DNA cleavage by deglycoBLM A6 without added Fe2+, and 30 min after the addition of Fe2+. As can be seen from the figure, fluorescence emission from each gel increased markedly after the addition of Fe2+; the increase was also time dependent (not shown). The fluorescence intensity reached a maximum after about 40 min. No significant fluorescence increase was observed for beacon DNA treated with Fe2+ + TentaGel lacking a BLM A6 moiety. These results demonstrate that beacon DNA cleavage by Fe(II)‚BLM 4 can be monitored visually on individual beads, thus enabling the monitoring of BLM libraries elaborated on beads by split and mix synthesis. ACKNOWLEDGMENT

We thank Michael Rishel for the β-hydroxyhistidine derivative employed in this study, and Dr. Ammasi Periasamy and Colten Noakes of the University of Virginia W. M. Keck Center for Cellular Imaging for technical assistance with the fluorescence microscopy. This work was supported by NIH Research Grants CA76297 and CA77284, awarded by the National Cancer Institute. Supporting Information Available: Gel electrophoreses of the cleavage of a 5′-32P end-labeled 158-bp DNA by 3 and synthetic deglycoBLM 1. This material is available free of charge via the Internet at http:// pubs.acs.org. LITERATURE CITED (1) Hecht, S. M. (1995) Bleomycin-group antitumor agents. Cancer Chemotherapeutic Agents (Foye, W. O., Ed.) pp 369388, American Chemical Society, Washington, DC. (2) Hecht, S. M. (2000) Bleomycin: new perspectives on the mechanism of action. J. Nat. Prod. 63, 158-168. (3) Sikic, B. I., Rozencweig, M., and Carter, S. K., Eds. (1985) Bleomycin Chemotherapy, Academic Press, New York. (4) Hecht, S. M. (1986) The chemistry of activated bleomycin. Acc. Chem. Res. 19, 383-391. (5) Kozarich, J. W., and Stubbe, J. (1987) Mechanism of bleomycin-induced DNA degradation. Chem. Rev. 87, 11071136. (6) Natrajan, A., and Hecht, S. M. (1993) Bleomycin: mechanism of polynucleotide recognition and oxidative degradation. Molecular Aspects of Anticancer Drug-DNA Interactions (Neidle, S., Waring, M. J., Eds.) pp 197-242, Macmillan, London.

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