ODN conjugate showed very little reactivity within the duplex. ... platinum complexes (10,11). ... binding to the DNA duplex, we have conjugated racemic CPI to.
1996 Oxford University Press
Nucleic Acids Research, 1996, Vol. 24, No. 4
683–687
Rapid and efficient hybridization-triggered crosslinking within a DNA duplex by an oligodeoxyribonucleotide bearing a conjugated cyclopropapyrroloindole Eugeny A. Lukhtanov, Mikhail A. Podyminogin, Igor V. Kutyavin*, Rich B. Meyer, Jr and Howard B. Gamper Epoch Pharmaceuticals, Inc., 1725 220th Street S.E., #104, Bothell, WA 98021, USA Received October 17, 1995; Revised and Accepted January 4, 1996
ABSTRACT The antitumor antibiotic CC-1065 binds in the minor groove of double-stranded DNA, and the cyclopropapyrroloindole (CPI) subunit of the drug alkylates adjacent adenines at their N-3 position. We have attached racemic CPI to oligodeoxyribonucleotides (ODNs) via a terminal phosphorothioate at either the 3′or 5′-end of the ODNs. These conjugates were remarkably stable in aqueous solution at neutral pH even in the presence of strong nucleophiles. When a 3′-CPI–ODN conjugate was hybridized to a complementary DNA strand at 37C, the CPI moiety alkylated nearby adenine bases of the complement efficiently and rapidly, with a half-life of a few minutes. The 5′-CPI– ODN conjugate showed very little reactivity within the duplex. CPI–ODN conjugates should be highly effective sequence-specific inhibitors of single-stranded viral DNA replication or gene selective inhibitors of transcription initiation. INTRODUCTION Modified oligodeoxyribonucleotides (ODNs) capable of hybridization-triggered crosslinkage in a physiologic buffer could be used as sequence specific affinity labeling reagents and might have potential as gene selective drugs. Ideally, these conjugates would be chemically inert until hybridization, triplexation or synapsis, whereupon they would rapidly and efficiently crosslink to the complexed DNA or RNA. Examples of attempts at the design of such agents are rare. Matteucci and coworkers showed that N4-ethanocytosine-containing ODNs meet some of these criteria (1–3), but are slow in the crosslinking reaction, with a half-life of ∼30 h. The alkylated base was a mismatched cytosine. Rokita et al. used a stable silylated phenolic quinone methide precursor (4), which, upon hybridization, lost the silyl group to generate the reactive species. Alkylation of the complementary strand occurred with moderate efficiency over several hours.
* To
whom correspondence should be addressed
Other crosslinkable ODNs fail to meet these specifications because the appended crosslinking moiety has inherent reactivity leading to self-alkylation or to the modification of cellular nucleophiles prior to nucleic acid binding. Such agents include the p-(N-2-chloroethyl-N-methylamino)-benzyl group (5), chlorambucil (6,7), haloacetamidoalkyl groups (8,9) and binuclear platinum complexes (10,11). Other appended crosslinking groups act under non-physiologic conditions or require unusual cofactors, such as ketal (12) (requiring low pH), thioether (13) (requiring CNBr) and silyl phenol (14) (requiring KF) groups. A variety of photo-crosslinkable ligands have been conjugated to ODNs, most notably psoralen (15,16). Since these agents covalently react upon exposure to near ultraviolet light, their use in vivo is problematic. We describe the synthesis and properties of a new class of hybridization-triggered crosslinkable ODNs which are conjugated to the reactive cyclopropapyrroloindole (CPI) subunit of the potent antitumor antibiotic CC-1065 (17,18). This DNA alkylating agent is composed of three repeating 1,2-dihydro-3Hpyrrolo[3,2-e]indole subunits (Fig. 1). The two non-reactive N-terminal subunits (B and C) confer a high binding affinity for the deep, narrow minor groove of A–T rich DNA. The C-terminal A subunit (CPI), which contains an electrophilic cyclopropyl moiety, contributes additional binding affinity and is responsible for the N-3 alkylation of adenine (19). CC-1065 is very stable in neutral aqueous solution (20), but becomes activated when bound to the minor groove of double-stranded DNA. Alkylation of adenine occurs as a result of DNA mediated general acid catalysis (21,22) within low affinity 5′-(A/T)(A/T)A* (* denotes preferred alkylation site) and high affinity 5′-PuNTTA* and 5′-AAAAA* consensus sequences (23,24). The C subunit of the natural (+)-enantiomer of CC-1065 binds to the 5′-end of these sequences, thus directing the reactive CPI subunit to the vicinity of the 3′-adenine. Alkylation of adenines in dsDNA by (+)-CPI alone is substantially less efficient and less selective than by (+)-CC-1065 (24). This is a consequence of the low binding affinity of (+)-CPI for A-T rich dsDNA. While (–)-CC-1065 binds to DNA with the opposite polarity as the (+)-enantiomer, it
684
Nucleic Acids Research, 1996, Vol. 24, No. 4 aminocaproic acid (0.31 g, 1.3 mmol) and EDC (N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride) (0.77 g, 4 mmol) were added. After stirring for 3 h, the reaction mixture was concentrated in vacuo to an oil and triturated with water (25 ml). The resulting solid was centrifuged, washed with water, centrifuged again, and dried in vacuo. The crude material was purified by flash chromatography in dichloromethane–methanol (9:1) to give 1c as an off-white solid (0.195 g, 67%): 1H NMR (CDCl3) δ 10.27 (s, 1H), 9.40 (s, 1H), 8.20 (s, 1H), 7.00 (s, 1H), 4.30 (br s, 1H), 4.4–3.8 (m, 4H), 3.4–3.1 (m, 3H), 2.7–2.45 (m, 2H), 2.42 (s, 3H), 2.0–1.7 (m, 2H),1.7–1.3 (m, 13H, partially overlapping with H2O and tBOC singlet). 2-(N-bromoacetyl-6-aminohexanoyl)-1,2,8,8a-tetrahydro-7methylcyclopropa[c]pyrrolo[3,2-e]indol-4(5H)-one (2b)
Figure 1. Structure of (+)-CC-1065 and its N-3 adenine adduct.
alkylates just as well. The (–)-enantiomer of CPI, on the other hand, reacts with a reversed binding orientation 10–100-fold more slowly than (+)-CPI (24,25). To take advantage of the catalysis of the reactivity of CPI by its binding to the DNA duplex, we have conjugated racemic CPI to the 3′- or 5′-end of ODNs. This replaces the minor groove binding B and C subunits of CC-1065 with a much more sequence specific binding agent (an ODN). The sequences of the ODNs were designed to orient the CPI moiety to a preferred crosslinking site upon hybridization to a complementary strand. The 3′-CPI–ODN conjugate rapidly alkylated the complementary strand of the hybrid under physiologically relevant conditions with ∼50% efficiency in a few minutes. MATERIALS AND METHODS All air and water sensitive reactions were carried out under argon. Anhydrous solvents were obtained from Aldrich (Milwaukee, WI). Flash chromatography was performed on 230–400 mesh silica gel. UV-visible absorption spectra were recorded on a Lambda 2 (Perkin Elmer) spectrophotometer with a PTP-6 temperature controller. 1H NMR spectra were run at 20C on a Varian 300 spectrometer, and chemical shifts are reported in p.p.m. downfield from Me4Si. Racemic compound 1a was prepared as previously described (20,24,26). 3-(N-tert-Butyloxycarbonyl-6-aminohexanoyl)-1-chloromethyl1,2-dihydro-3H-8-methylpyrrolo[3,2-e]indol-5-ol (1c) Racemic 3-(tert-Butyloxycarbonyl)-1-chloromethyl-1,2-dihydro3H-8-methylpyrrolo[3,2-e]indol-5-ol (1a) (0.22 g, 0.65 mmol) was converted into 1b by treatment with HCl in ethyl acetate according to the literature procedure (24). The hydrochloride 1b was dissolved in 10 ml of anhydrous DMF to which tBOC-N-
1c (150 mg, 0.33 mmol) was treated with 5 ml of 3 M HCl in ethyl acetate. The reaction mixture was stirred for 15 min and then evaporated in vacuo to dryness. To a solution of the resulting amine hydrochloride (1d) in 3 ml of anhydrous DMF was added NaH (41 mg, 1.7 mmol) suspended in 1 ml of DMF. After stirring for 30 min, a solution of N,N-diisopropylethylamine hydrochloride (150 mg, 1.1 mmol) in 0.5 ml of DMF was added to quench excess NaH. The reaction mixture containing 2a was immediately used for the next step. Bromoacetic acid N-hydroxysuccinimide ester (180 mg, 0.7 mmol) was added and the mixture was stirred for 3 h. The solvent was removed in vacuo and the resulting mixture was separated by reverse phase HPLC (PRP-1, Hamilton Co, 7 × 300 mm) in a gradient of acetonitrile in water (30–100%). The desired compound 2b was obtained as a colorless solid after removal of the solvent in 15% yield (from 1c): 1H NMR (CDCl3) δ 9.84 (s, 1H), 6.85 (s, 1H), 6.69 (br s, 1H), 4.15 (m, 1H), 4.06 (s, 2H), 4.00 (m, 1H), 3.34 (m, 2H), 2.89 (m, 1H), 2.52 (m, 2H), 2.02 (s, 3H), 1.99 (m, 1H, overlapping with CH3 signal), 1.74 (m, 2H), 1.57 (m, 2H), 1.40 (m, 2H), 1.21 (m, 1H). ODN synthesis The unmodified target ODN was prepared from 10 µmol of polymeric support (Pharmacia) on an OligoPilot DNA synthesizer (Pharmacia) using the protocol supplied by the manufacturer. Standard reagents for the β-cyanoethyl phosphoramidite coupling chemistry were purchased from Glen Research. 5′- and 3′-thiophosphate modifications were introduced using a phosphorylating phosphoramidite in combination with a sulfurizing reagent (Glen Research). Synthesis of CPI–ODN conjugates To a solution of an ODN with a terminal phosphorothioate (50 U A260, ∼0.25 µmol) in 20 µl of water were added triethylamine (0.5 µl) and 2b (20 µl of a 33 mM solution in DMF, 0.66 µmol). After 2 h, the solution was diluted with 0.8 ml of water and loaded onto a reverse phase HPLC column (PRP-1, 7 × 300 mm). The conjugates were resolved in 50–60% yields as cleanly separated peaks using an acetonitrile gradient (0–30%, 50 mM triethylammonium acetate pH 8). DNA crosslinking reaction The 30mer target ODN was 5′-end-labeled by using T4 polynucleotide kinase and [γ-32P]ATP and purified by 8% denaturing PAGE. CPI–ODNs (10 µM) were incubated with the
685 Nucleic Acids Acids Research, Research,1994, 1996,Vol. Vol.22, 24,No. No.14 Nucleic
685
desired 3′- or 5′-(+/–)-CPI–ODN conjugates in good yield. The integrity of the cyclopropapyrroloindole system was clearly indicated by lack of change in the characteristic UV absorption at 350 nm (29). The CPI–ODNs were purified by reverse phase HPLC, where they were identified by a characteristic long wavelength absorption (300–400 nm) and a significant increase in retention time. A minor but closely eluting side product was resolved by HPLC. This peak, which exhibited minimal absorption in the near ultraviolet, probably resulted from reaction of the cyclopropyl function of 2b with a competing nucleophile, possibly the phosphorothioate. Stability and solvolysis
Figure 2. Synthesis of the CPI reagent (2b) and CPI–ODN conjugates. Reagents: (a) saturated HCl in ethyl acetate; (b) BOC-N-aminocaproic acid, EDC, DMF; (c) NaH, DMF; (d) bromoacetic acid N-hydroxysuccinimide ester.
Previous investigations have shown that CPI derivatives are very stable in neutral aqueous solution. Solvolysis of these compounds is very slow above pH 3 (20). Realizing that covalent attachment of the CPI residue to an ODN could alter its stability, we monitored the long wavelength band in the UV spectrum of these conjugates over time to determine the solvolytic stability of the cyclopropane ring. Loss of this absorbance indicates reaction of the CPI. No detectable reaction was observed at pH 7.2 for 3 days for any of the conjugates. In the presence of 10 mM glutathione, the conjugated CPI moiety reacted slowly at 37C (t = 36 h) but not at room temperature (data not shown). Although 2b was found to be stable for days in aqueous solution at pH 4.5, the CPI residue in the conjugates underwent slow acid-catalyzed hydrolysis, with a t = 170 min at 21C in a pH 4.5 solution. DNA crosslinking activity
complementary or non-complementary 30mer target ODNs (2 µM) in 10 mM HEPES, pH 7.4 and 100 mM NaCl either at 25C for 90 min or at 37C for 30 min. Crosslinked products were detected by direct analysis of reaction aliquots in an 8% denaturing polyacrylamide gel. Alkylation sites were further converted into nicks with 5′- and 3′-phosphate termini by incubation at 95C for 30 min in the reaction buffer, followed by treatment with 10% piperidine (19,27). Sites of cleavage in the target 30mer were mapped relative to G and G+A sequencing ladders (28) in an 8% denaturing polyacrylamide gel. The percent of alkylation was determined by quantitative phosphorimage analysis (Bio-Rad) of the cleavage products. RESULTS Preparation of CPI–ODN conjugates The conjugation chemistry is based on alkylation of a terminal thiophosphate residue on the ODN by a CPI derivative (2b) with a bromoacetamide group attached. We presumed that, at neutral pH, this latter electrophilic group would be more reactive in solution than CPI to sulfur nucleophiles. The preparation of 2b is shown in Figure 2. Racemic chloromethyl derivative 1a (24) was converted into amine hydrochloride 1b, which in turn was reacted with N-tBOC-aminocaproic acid to give 1c. Deprotection afforded 1d and cyclization in the presence of NaH gave 2a. The reaction mixture containing 2a was condensed with bromoacetic acid N-hydroxysuccinimide ester to provide the desired bromoacetamide derivative 2b in 15% yield (unoptimized) from 1c. Reaction of bromoacetamide 2b with the sulfur anion of thiophosphate-tailed ODNs in aqueous DMF provided the
Alkylation of a complementary ssDNA by the (+/–)-CPI–ODN conjugates was initially evaluated spectrophotometrically using 4 × 10–6 M target ODN and 8 × 10–6 M CPI–ODN conjugate in 100 mM NaCl and 10 mM HEPES, pH 7.4. The structures of the target ODN and CPI–ODN conjugates used in this study are shown in Figure 3. Two 18mer CPI–ODNs were designed to target the same oligoadenylate sequence embedded in the middle of the complementary target ODN. One of the reactive ODNs had the CPI residue attached at the 3′-end while the other had it attached at the 5′-end. A rapid reaction (t = 2 min at 37C) was observed when the 3′-CPI–ODN conjugate was incubated with the target strand. Upon completion of the reaction the long wavelength absorption had been reduced by half (Fig. 4). When assayed by UV monitoring, no reaction was observed for the 5′-CPI–ODN. In a similar experiment conducted with a 30+18mer duplex, unconjugated compound 2b did not show any detectable changes in long wavelength absorption over 2 h. This underscores the importance of affinity targeting by conjugation. We were concerned that the conjugated CPI moiety would exhibit an ‘anchor’ effect by binding to the minor groove of dsDNA, independently of the tethered ODN, and alkylating the DNA non-specifically. To test this, we incubated the 3′-CPI– ODN used in this work with poly(dT)–poly(dA). No changes in the long wavelength region of the UV were observed after 8 h (data not shown). This would provide an optimal opportunity for the non-specific reaction to occur, and the result indicates that this is not a major issue in the use of these agents in more complex systems. Electrophoretic analysis of the reaction products (Fig. 5) confirmed the preliminary spectrophotometric results. The
686
Nucleic Acids Research, 1996, Vol. 24, No. 4
Figure 3. Sequence of the target ODN and of the CPI–ODN conjugates. The shaded oval represents the CPI moiety, showing its expected location in the duplex formed between the conjugate and the target. The arrows show the nucleobases alkylated by the 3′-CPI–ODN, with the length of the arrows representing the relative amount of alkylation. The conditions for the crosslinking reaction are given in Materials and Methods.
Figure 5. Crosslinking of CPI–ODN conjugates to complementary ssDNA. 5′or 3′-CPI–ODN conjugates were incubated at 25C for 90 min with the target ODN or control non-complementary 30mer and analyzed by 8% denaturing PAGE for crosslinkage products (see Methods). Lane 1, target ODN alone. Lane 2, 5′-CPI–ODN conjugate + target ODN. Lane 3: 5′-CPI–ODN conjugate + non-complementary 30mer. Lane 4: 3′-CPI–ODN conjugate + target ODN. Lane 5: 3′-CPI–ODN conjugate + non-complementary 30mer. Lanes 6–10: the same reaction mixtures as in lanes 1–5, but treated with hot piperidine to cleave target DNA at sites of alkylation as described in Materials and Methods. The Maxam and Gilbert G and A+G sequencing reactions of the target ODN were added to lanes G and A. The sequence of the non-complementary 30mer was 5′-TCGTTGTCAGAAGTAAGTTGGCCGCAGTGT. Figure 4. Time course for the crosslinking reaction of the 3′-CPI–ODN conjugate with the target ODN. The spectra were taken in a buffer containing 10 mM HEPES, pH 7.4 and 100 mM NaCl at 37C in 1 min intervals. The lowest spectrum was recorded after addition of 30 µl 2 M Na-Tosylate buffer pH 3.0 to the reaction mixture to consume unreacted enantiomer.
DISCUSSION 3′-CPI–ODN conjugate showed remarkable crosslinking activity when incubated with the complementary 5′-end-labeled target ODN, and was totally inactive when incubated with a non-complementary control 30mer. Under the same conditions the 5′-CPI–ODN conjugate showed only traces of activity. Quantification of the products was conducted after treatment with heat/piperidine, since direct analysis of the crosslinked products was complicated by their unusual behavior on a denaturing gel. The heat/piperidine cleavage fragments revealed one major and three minor crosslink sites. The total crosslinking efficiency was close to 50%. The adenine residue complementary to the fifth thymidine from the 3′-end of the reactive ODN was the major alkylation site, constituting 60% of the reaction. Alkylation of adjacent adenines accounted for the remaining cleavage fragments. Although the reaction envelope obtained using the 3′-CPI–ODN conjugate was broader than would be expected using (+)-CC-1065 and (+)-CPI (24), all reagents predominately alkylate the fifth adenine from the 5′-end of an oligoadenylate run on the target strand.
Addition of an electrophilic moiety to a ligand such as an enzyme inhibitor or a receptor antagonist has often proven to dramatically enhance the potency of the ligand by formation of a covalent bond with the target macromolecule. It is important, however, to maintain specificity by minimizing non-target reactivity of the electrophile. The best way of accomplishing this is by design of groups that are activated in the course of reversible binding to the target. Elegant examples of this are found in several mechanismbased enzyme inhibitors (30). CC-1065 is a potent inhibitor of DNA function for exactly the same reason: it binds first in a reversible manner to the minor groove of dsDNA and, once bound, alkylates the nucleobases (especially adenine) of the nucleic acid much more rapidly than it reacts randomly with nucleophiles in solution. Presumably, general acid catalysis at the binding site is responsible for the enhanced reactivity (20). Unfortunately for therapeutic purposes, it lacks the sequence specificity in this action to allow targeting to genes of interest. As a result, it is a highly toxic agent (17,24). This cytotoxicity is attributed to a general interference of DNA
687 Nucleic Acids Acids Research, Research,1994, 1996,Vol. Vol.22, 24,No. No.14 Nucleic replication and transcription. In addition, the compound has a delayed hepatotoxicity associated with the B and C subunits (31). We have now, in effect, replaced the B and C subunits of CC-1065 with an ODN to create a new class of highly sequence-specific, hybridization-triggered DNA crosslinking agents. The advantageous properties of the reactive CPI moiety were unchanged at neutral pH when it was conjugated to the ODN. There was no loss of the CPI chromaphore in physiologic buffer. Most importantly, the 3′-CPI–ODN conjugate alkylated the complementary strand in a duplex very rapidly and efficiently. Within the minor groove of the hybrid, the tethered CPI group binds with the same polarity and preference for A–T rich regions as observed for CC-1065. Based on the known binding mode of the (+)-enantiomer of CC-1065, one would predict that the 3′-(+)-CPI–ODN should exhibit optimal crosslinking activity and the 5′-(+)-CPI–ODN should react with itself in the model system used here. This expectation was borne out by the experimental results. The 3′-(+)-CPI–ODN reacted quantitatively within a few minutes based on the UV assay and electrophoretic analysis. Hurley, Warpehoski and coworkers (24) have shown that unconjugated (–)-CPI is 10-fold less active than (+)-CPI. They have also shown that, since these two enantiomers of CPI alkylate the same sites, they must bind in the minor groove in opposite directions. We expected, therefore, that the (–)-CPI–ODN conjugates should be unreactive with the opposite strand when the (–)-enantiomer was linked to the 3′-end of the ODN, but modestly reactive when it was linked to the 5′-end. The small amount of alkylation detected by electrophoresis (see Fig. 5) when using the 5′-CPI–ODN could be due to this (–)-enantiomer. These properties suggest that (+)-CPI–ODN conjugates could have application as inhibitors of single-stranded viral DNA replication (e.g., hepatitis B virus) or as gene selective inhibitors of transcription initiation (e.g., by binding to an open promoter complex) (32). These same conjugates might also alkylate double-stranded DNA, either as part of a classical triple-stranded complex (33) or, as we have recently shown, in a recombinasestabilized synaptic joint (7). Preliminary experiments, however, have shown that the CPI–ODN conjugates described here do not efficiently alkylate a complementary RNA target and, therefore, may not be antisense candidates. Other approaches to improve the crosslinkage efficiency of CPI–ODN conjugates with DNA are underway and include the evaluation of conjugates which contain the optically pure (+)-enantiomer of CPI. At the same time we intend to verify the expectation that the sequence context which supports alkylation is less restrictive for CPI–ODNs than for CC-1065. In summary, CPI–ODNs would appear to be ideal sequencespecific hybridization-triggered crosslinking agents which have significant potential for use as gene modification agents. ACKNOWLEDGEMENTS We wish to thank Dr Vladimir Gorn for oligonucleotide synthesis. A portion of this work was funded by grant GM52774 from the National Institutes of Health, USPHS.
687
REFERENCES 1 Webb, T.R. and Matteucci, M.D. (1986) J. Am. Chem. Soc., 108, 2764–2765. 2 Webb, T.R. and Matteucci, M.D. (1986) Nucleic Acids Res., 19, 7661–7674. 3 Shaw, J.-P., Milligan, J.F., Krawczyk, S.H., and Matteucci, M. (1991) J. Am. Chem. Soc., 113, 7765–7766. 4 Li, T., Zeng, Q., and Rokita, S.E. (1994) Bioconjugate Chem., 5, 497–500. 5 Knorre, D.G. and Vlassov, V.V. (1985) Prog. Nucleic Acids Res. Mol. Biol., 32, 291–320. 6 Kutyavin, I.V., Gamper, H.B., Gall, A.A., and Meyer, R.B. (1993) J. Am. Chem. Soc., 115, 9303–9304. 7 Podyminogin, M.A., Meyer, R.B., and Gamper, H.B. (1995) Biochemistry 34, 13098–13108. 8 Tabone, J.C., Stamm, M.R., Gamper, H.B., and Meyer, R.B. (1994) Biochemistry, 33, 375–383. 9 Povsic, T.J. and Dervan, P.B. (1990) J. Am. Chem. Soc., 112, 9428–9430. 10 Vlassov, V.V., Gorn, V.V., Ivanova, E.M., Kazakov, S.A., and Mamaev, S.V. (1983) FEBS Lett., 162, 286–289. 11 Gruff, E.S. and Orgel, L.E. (1991) Nucleic Acids Res., 19, 6849–6854. 12 Summerton, J. and Bartlett, P.A. (1978) J. Mol. Biol., 122, 145–162. 13 Iverson, B.L. and Dervan, P.B. (1987) J. Am. Chem. Soc., 109, 1241–1243. 14 Li, T. and Rokita, S.E. (1991) J. Am. Chem. Soc., 113, 7771–7773. 15 Gamper, H.B., Cimino, G.D., and Hearst, J.E. (1987) J. Mol. Biol., 197, 349–362. 16 Takasugi, M., Guendouz, A., Chassignol, M., Decout, J.L., Lhomme, J., Thuong, N.T., and Helene, C. (1991) Proc. Natl. Acad. Sci. USA, 88, 5402–5606. 17 Reynolds, V.L., McGovren, J.P., and Hurley, L.H. (1986) J. Antibiotics (Tokyo), 39, 319–334. 18 Boger, D.L. and Johnson, D.S. (1995) Proc. Natl. Acad. Sci. USA, 92, 3642–3649. 19 Hurley, L.H., Reynolds, L.L., Swenson, D.H., and Scahill, T. (1984) Science, 226, 843–844. 20 Warpehoski, M.A., Gebhard, I., Kelly, R.C., Krueger, W.C., Li, L.H., McGovren, J.P., Prairie, M.D., Wicnienski, N., Wierenga, W. (1988) J. Med. Chem., 31, 590–603. 21 Warpehoski, M.A. and Hurley, L.H. (1988) Chem. Res. Toxicol., 1, 315–333. 22 Lin, C.H., Beale, J.M., and Hurley, L.H. (1991) Biochemistry, 30, 3597–3602. 23 Reynolds, V.L., Molineux, I.J., Kaplan, D.J., Swenson, D.H., and Hurley, L.H. (1985) Biochemistry, 24, 6628–6237. 24 Hurley, L.H., Warpehoski, M.A., Lee, C.-S., McGovern, J.P., Scahill, T.A., Kelly, R.C., Mitchell, M.A., Wicnienski, N.A., Gebhard, I., Johnson, P.D., and Bradford, V.S. (1990) J. Am. Chem. Soc., 112, 4633–4649. 25 Boger, D.L., Johnson, D.S., Yun, W., and Tarby, C.M. (1994) Bioorg. Med. Chem., 2, 115–135. 26 Kelly, R.C., Gebhard, I., Wicnienski, N., Aristoff, P.A., Johnson, P.D., and Martin, D.G. (1987) J. Am. Chem. Soc., 109, 6837–6838. 27 Sugiyama, H., Fujiwara, T., Ura, A., Tashiro, T., Yamamoto, K., Kawanishi, S., and Saito, I. (1994) Chem. Res. Toxicol., 7, 673–683. 28 Maxam, A.M. and Gilbert, W. (1977) Proc. Natl. Acad. Sci. USA, 74, 560–564. 29 Boger, D.L. and Palanki, M.S.S. (1992) J. Am. Chem. Soc., 114, 9318–9327, and references cited therein. 30 Silverman, R. B. (1988) Mechanism-Based Enzyme Inactivation: Chemistry and Enzymology. CRC Press: Boca Raton, FL. 31 Lin, C.H., Sun, D., and Hurley, L.H. (1991) Chem. Res. Toxicol., 4, 21–26. 32 Perrin, D.M., Mazumder, A., Sadeghi, F., and Sigman, D.S. (1994) Biochemistry, 33, 3848–3854. 33 Helene, C. (1991) Anti-Cancer Drug Des., 6, 569–584.
