Inorganic Chemistry in Biology and Medicine - American Chemical

14 The Iron Bleomycins JAMES C. DABROWIAK, FREDERICK T. GREENAWAY, and FRANK S. SANTILLO Department of Chemistry, Syracuse University, Syracuse, N Y 13210 STANLEY T. CROOKE and JOHN M. ESSERY

Downloaded by PURDUE UNIV on September 26, 2017 | http://pubs.acs.org Publication Date: December 22, 1980 | doi: 10.1021/bk-1980-0140.ch014

Bristol Laboratories, Thompson Road, Syracuse, N Y 13206

The bleomycins (BLM) are a group of Streptomyces-produced glycopeptides which show antitumor activity (1). Clinically employed bleomycin, marketed under the trade name Blenoxane, is a mixture of eleven antitumor-active bleomycins. The drug is widely used for the treatment of squamous cell carcinomas, lymphomas and testicular carcinomas (2). Current evidence suggests that the antibiotic functions by a unique mechanism of action. It has long been known that bleomycin is capable of causing single and double strand breaks in DNA (1), and it is now believed that DNA breakage by bleomycin occurs via a metal-mediated redox mechanism involving iron. Horwitz and co-workers (3,4,5) were the first to show that Fe(II)BLM is an air sensitive compound and that i t readily oxidizes in the atmosphere to Fe(III)BLM. They postulated that this oxidation process occurs in the tumor cell while the iron-bleomycin is bound to DNA, and that DNA damage is the result of radical production in the vicinity of the biopolymer by iron-bleomycin. Recent r e s u l t s (6-9) have shown bleomycin to be a f u n c t i o n a l . One part o f the drug i s capable o f binding to DNA (the b i t h i a z o l e moiety) while a second region ( i n v o l v i n g the amino-pyrimidineimidazole region and p o s s i b l y the carbamoyl group) i s capable o f binding metal i o n s . Although the l i g a t i n g p r o p e r t i e s o f a number of t r a n s i t i o n metal i o n s , e s p e c i a l l y Cu(II) and Z n ( I I ) , towards bleomycin have been studied ( 9 ) , the p h y s i c a l and chemical p r o p e r t i e s o f the b i o l o g i c a l l y important i r o n bleomycins are only now being i n v e s t i g a t e d . This r e p o r t d i s c l o s e s our l a t e s t f i n d i n g s on the i r o n bleomycins. Experimental Bleomycin A H C £ 1, the most abundant component o f Blenoxane, was used as obtained from B r i s t o l L a b o r a t o r i e s . The synthesis and c h a r a c t e r i z a t i o n o f the new bleomycin analogues 2-4 w i l l be r e ported elsewhere (10.). The s t r u c t u r e s o f 1-4 are shown i n F i g ure 1. 2

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1980 A m e r i c a n Chemical Society

Martell; Inorganic Chemistry in Biology and Medicine ACS Symposium Series; American Chemical Society: Washington, DC, 1980.


Figure 1. 2

The structures oj BLM-A ,

1, and the new bleomycin analogues, 2-4


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The preparation and handling of Fe(II)BLM was done i n an i n e r t atmosphere box. Unless otherwise noted, complexes were p r e pared by the a d d i t i o n of s t o i c h i o m e t r i c amounts of e i t h e r Fe(II)(C£0u) *6H 0 or F e C l I I ) ( C £ 0 } 3 * 6 H 0 to the drug, followed by the adjustment of the pH to a s p e c i f i c value. The pH (the pH meter reading without c o r r e c t i o n for the deuterium isotope e f f e c t ) of the F e ( I I ) - d r u q complex i n D 0 was adjusted to 6.5 p r i o r to o b t a i n i n g the C nmr spectrum. For nmr purposes, the Fe(III)BLM complex was prepared by o x i d i z i n g the d i v a l e n t compound with molecular oxygen. The pH of the oxygenated s o l u t i o n was adjusted to 6.5 p r i o r to o b t a i n i n g the spectrum. The v a r i a t i o n i n the amounts of the high and low s p i n forms of Fe(III)BLM as a f u n c t i o n of pH was determined using e s r . Since the linewidth of the low spin esr s i g n a l was independent of pH, the peak height of the s i g n a l was used as a measure of the amount of low spin Fe(III)BLM present. Although the linewidth of the high spin species v a r i e d as a f u n c t i o n of the pH, e s p e c i a l l y i n the presence of b u f f e r s , the peak height of the s i g n a l was taken as rough measure of the amount of high s p i n Fe(III)BLM p r e sent i n the mixture. The e s r as well as the nmr and absorption data were c o l l e c t e d i n the e a r l i e r described manner (11,12,13). The Fe(II) complexes of the new bleomycin d e r i v a t i v e s , 2 - 4 , were prepared i n the presence of the atmosphere by the a d d i t i o n of Fe(II)(CilOu) 2 to a 1 0 ' M s o l u t i o n of the bleomycin. The pH was adjusted by a d d i t i o n of small amounts of 1M HC& or 1M NaOH to the s o l u t i o n c o n t a i n i n g the complex. No o x i d a t i o n to FeUHI} o c c u r red. The dc polarographic studies were c a r r i e d out i n buffered and i n unbuffered aqueous s o l u t i o n s . P o t e n t i a l s were referenced against a Ag/AgC£ saturated NaC£ e l e c t r o d e . The electrochemical assignments and the apparatus used to c o l l e c t the polarographic data have been p r e v i o u s l y described (14,15). 2

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The Iron Binding S i t e of Bleomycin The l o c a t i o n of the i r o n binding s i t e on bleomycin i s c r i t i cal to understanding the r o l e that i r o n plays i n the DNA-degrading process. The C nmr spectra shown i n Figure 2 and the s t r u c t u r e shown i n Figure 3 c l e a r l y show that the p y r i m i d i n e imidazole-sugar regions of both Fe(II)BLM and Fe(III)BLM are i n volved i n metal b i n d i n g . C nmr resonances a t t r i b u t e d to carbon atoms i n that area of the drug are e i t h e r missing from or are broadened i n the spectra of the i r o n complexes. Only the narrow unshifted resonances were assigned. The f o l l o w i n g carbon atoms of Fe(II)BLM were observed as narrow resonance l i n e s (Figure 2b): C, 41-49 and C, 51-55. Broadened and unassigned resonances f o r Fe(II)BLM occurred at 23.2, 5 7 . 5 , 9 3 . 0 , 168.5 and 192.5 ppm. A number of broad, overlapping resonances a l s o occurred i n the sugar region of the nmr spectrum (60-75 ppm). Fe(III)BLM e x h i b i t e d narrow resonance l i n e s f o r carbon atoms 1 3

1 3



Figure 3. The structure of BLM-A missing from the spectra (A) 2

showing which C NMR resonance lines are of Fe(II)BLM-A and Fe(III)BLM-A 13

2

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C, 41-49 and C, 51-55 (Figure 2 c ) . Broad unassignable r e s o n ances were observed a t 64 and 71 ppm but the sugar region was simpler than was observed f o r Fe(II)BLM. In a d d i t i o n , broad and apparently s h i f t e d resonances were found a t 14, 34, and 101 ppm. The C nmr spectrum o f Fe(III)BLM a l s o contained a number o f weak but narrow resonances a t 1 4 . 2 , 1 5 . 2 , 3 3 . 9 , 34.2 and 188.5 ppm. These resonances have not been p r e v i o u s l y observed i n the spectrum o f the drug or any o f i t s m e t a l ! o d e r i v a t i v e s and they may be due to bleomycin fragments which occur upon o x i d a t i o n o f Fe(II)BLM (16). Thus, the C nmr r e s u l t s o f Fe(II)BLM and Fe(.III)BLM are s i m i l a r to those e a r l i e r obtained f o r Cu(II) and Zn(.II)BLM. A l though the b i t h i a z o l e i s an important group f o r DNA binding (171 9 ) , the C nmr data f o r the i r o n complexes g r a p h i c a l l y show that the n i t r o g e n - s u l f u r heterocycle o f BLM i s not involved i n iron binding. Its resonance l i n e s are narrow and unshifted and are not a f f e c t e d by the presence of the paramagnetic c a t i o n s . S p e c i f i c bleomycin donor atoms o f Fe(.II) and Fe(III) have been i d e n t i f i e d . Potentiometric t i t r a t i o n studies by us (6J and by Sugiura e t al_. (20) have shown that the primary amine f u n c t i o n and the imidazole moiety are bound to Fe(_II) i n Fe(II)BLM. Sugiura e t a K (20) have a l s o pointed out that the secondary amine function i s bound to F e ( I I ) . The assignment o f the fourth and f i n a l proton l o s s accompanying the b i n d i n g Fe(II) to the a n t i b i o t i c has been a t t r i b u t e d to the removal o f an amide proton. UV v i s i b l e absorption and electrochemical studies have i d e n t i f i e d an a d d i t i o n a l l i g a t i n g group o f both Fe(II)BLM and Fe(III)BLM. Polarographic measurements show that the reduction a t - 1 . 2 2 V , assigned to the two e l e c t r o n i r r e v e r s i b l e reduction o f the 4-amino pyrimidine moiety o f the a n t i b i o t i c (J_5) i s s e n s i t i v e to iron binding. Figure 4 shows that t h i s wave i s absent from the dc polarogram of the F e ( I I , I I I ) - d r u g complexes. The binding o f the 4-amino pyrimidine moiety to Fe(11) and Fe(III) d r a m a t i c a l l y s h i f t s the reduction p o t e n t i a l o f the h e t e r o c y c l e out o f the p o l a r o g r a p h i c window o f o b s e r v a t i o n . S i m i l a r e f f e c t s have observed f o r other metal 1 obieomycins and m e t a l l o t a l l y s o m y c i n s (15). Further evidence that the pyrimidine i s bound i n the i r o n de r i v a t i v e s can be seen i n the d i f f e r e n c e s p e c t r a shown i n Figure 5. Both o f the complexes e x h i b i t e d a pyrimidine π-π* e l e c t r o n i c t r a n s i t i o n which was s h i f t e d from i t s o r i g i n a l p o s i t i o n i n bleomy cin. This band occurs a t -230 nm i n the free drugs and s h i f t s to -250 nm i n t h e i r metal 1 o d e r i v a t i v e s . In a d d i t i o n , the i r o n com plexes e x h i b i t a second strong envelope a t -300 nm. Since t r a n s i t i o n s due to two chromophores present i n the a n t i b i o t i c s , the η->π* o f the pyrimidine and the π-π* o f the b i t h i a z o l e moiety, overlap a t 290 nm, the o r i g i n o f the band a t -300 nm i n the d i f ference spectra o f the metal 1oderivatives i s d i f f i c u l t to d e t e r mi ne. Fe(II)BLM e x h i b i t s two weak absorptions at 475 nm (εΜ 360) and 375 nm (εΜ -600). The p o s i t i o n s and i n t e n s i t i e s o f these two 1 3

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Figure 5.

Difference spectra: (

; Fe(II)BLM vs. BLM

vs. BLM; (

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Fe(III)BLM


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absorptions are i n good agreement with those e a r l i e r reported by S a u s v i l l e e t al_. Q ) . Although the bands appear to be o f d-d o r i g i n the assignment o f the t r a n s i t i o n s must await the d e t e r mination o f the ground s t a t e . Fe(III)BLM has two strong (εΜ 1,000-2,000) but poorly defined absorptions a t 341 and 365 nm. The o r i g i n o f these bands i s unknown. The d i f f e r e n c e spectra o f Fe(III)-BLM produced by a i r o x i d a t i o n o f Fe(II)BLM and the com plex formed by combining F e ( I I I ) ( C & 0 ) 3 and the a n t i b i o t i c a t pH, 7.0 were i d e n t i c a l . The s p e c t r o s c o p i c evidence taken together with the t i t r a t i o n r e s u l t s f o r Fe(II)BLM d e f i n i t i v e l y e s t a b l i s h the amino-pyrimidineimidazole-sugar region o f bleomycin as important f o r b i n d i n g Fe(II) and F e ( I I I ) . The d e t a i l e d geometry and stereochemistry of the i r o n b i n d i n g s i t e remains to be e l u c i d a t e d .


4

The Spin State o f the Complexes The spin s t a t e o f the d i v a l e n t complex, Fe(II)BLM, has not y e t been determined. However, the f a c t that the C nmr spectrum of the Fe(II) a n t i b i o t i c complex has many carbon resonance l i n e s missing and others that are broadened (Figure 2 b ) , shows t h a t Fe(II)BLM i s paramagnetic. Diamagnetic complexes such as Z n ( I I ) BLM y i e l d s h i f t e d but narrow C nmr l i n e s (Jl_). Thus, the only s p i n s t a t e s p o s s i b l e f o r Fe(II)BLM are S = 1 and S = 2. Mag n e t i c , and Mossbauer measurements are under way to r e s o l v e t h i s issue. The s p i n s t a t e o f the Fe(III) complex was e a s i l y determined by e s r . Preparing Fe(III)BLM by combining molar equivalents o f F e ( I I I ) ( C £ 0 ) 3 and the a n t i b i o t i c i n water i n the absence o f a buffer y i e l d e d a t l e a s t two e s r - a c t i v e forms o f F e ( I I I ) . One form, with a g value o f 4.3 has been assigned to a high s p i n (S = 5/2) Fe(III) ion i n a completely rhombic c r y s t a l f i e l d (6,7,20). Since the peak a t g , 4.3 o c c a s i o n a l l y shows some s p l i t t i n g , p a r t i c u l a r l y i n the presence o f b u f f e r s , the s i g n a l may be due to the presence o f more than one form o f high s p i n F e ( I I I ) . The f a c t t h a t F e ( I I I ) ( C i l 0 ) d i s s o l v e d i n water a t pH, 7.0 gives only a very weak s i g n a l a t g , 4.3 while the s i g n a l observed i n F e ( I I I ) - d r u g s o l u t i o n s i s strong suggests t h a t the high spin s i g n a l i s due to Fe(III) which i s i n some manner bound to the drug. The e s r s i g n a l s having g values o f 2.412, 2.182 and 1.898 emanate from a low spin (S = 1/2) Fe(III) i o n i n a rhombic c r y s t a l f i e l d (21). No other e s r a c t i v e species were observed b e tween pH 3.0 and 1 0 . 5 . As has been p r e v i o u s l y reported ( 6 , 7 ) , producing Fe(III)BLM by a i r o x i d a t i o n o f Fe(II)BLM y i e l d s a t r a n s i e n t low s p i n Fe(III) species having g values o f 2.254, 2.171 and 1.939. The e s r signatures o f that species and those o f the p r e v i o u s l y mentioned compounds are shown i n Figure 6. 1 3

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Esr studies have shown that the r e l a t i v e amounts o f low and high s p i n Fe(III) are dependent on pH, and on the presence o f buffer ions. The pH dependency o f the s p i n states o f Fe(III)BLM i s shown i n Figure 7. Within the pH range 7-9 the low s p i n form o f Fe(III) dominates and the amounts o f the two s p i n forms o f Fe(III) are r e l a t i v e l y i n s e n s i t i v e to pH. At pH values outside t h i s range, i n c r e a s i n g amounts o f high s p i n Fe(III) form, a t the expense o f the low s p i n s p e c i e s , u n t i l a t pH - 3 and -11 high s p i n Fe(II) p r e dominates. These processes are r e v e r s i b l e but a t low pH the rate of conversion o f the low s p i n species to the high s p i n species upon lowering the pH i s much greater than the reverse process. These r e s u l t s suggest t h a t Fe(III)BLM i s a low s p i n complex and that i t e x i s t s i n e q u i l i b r i u m with high s p i n forms which predomi n a t e a t high and low pH. E l e c t r o c h e m i s t r y shows that a t l e a s t one metal l i g a t i n g group, the pyrimidine moiety, i s metal bound i n the pH range 4 - 9 . The low s p i n - h i g h s p i n e q u i l i b r i u m i s a l s o a f f e c t e d by the presence o f b u f f e r s . At pH, 7.0 and 8.0 i n e i t h e r a phosphate or a borate buffer the high s p i n e s r s i g n a l o f the metal complexes dominates. A pH study using phosphate buffers has shown that the l o s s o f the low spin signal and the increase i n the high s p i n s i g n a l f o r Fe(III)BLM can be c o r r e l a t e d with the presence o f the buffer species H P 0 ~ . I t i s p o s s i b l e that t h i s buffer species i s binding to the metal i o n thereby reducing the c r y s t a l f i e l d and y i e l d i n g a high s p i n F e ( I I I ) complex. At pH values >10, where H P 0 " i s i n low c o n c e n t r a t i o n and HP0Û2 predominates, the e s r spectra of Fe(III)BLM are nearly i d e n t i c a l to those obtained i n water a t pH, 1 0 . 0 . S i m i l a r counter i o n e f f e c t s were observed f o r borate b u f f e r s . The v i s i b l e absorption spectra o f both Fe(II)BLM and Fe(III)BLM were found to be i n s e n s i t i v e to pH w i t h i n the pH range pH 4 - 9 . However, outside o f t h i s range the drug appeared to d e metal l a t e as i s evidenced by the loss o f the absorption spectrum of the metal 1 o d e r i v a t i v e . The absorption s p e c t r a o f both Fe(II) and Fe(III) complexes were a f f e c t e d by the presence o f buffer ions. In g e n e r a l , the spectra taken i n t r i s buffer s o l u t i o n s , as opposed to those obtained i n phosphate and borate media, most c l o s e l y approximate spectra obtained i n unbuffered s o l u t i o n s . Since DNA degrading by BLM has been shown to be s e n s i t i v e to the nature and concentration o f the buffer ions which are present i n s o l u t i o n (5J, we examined the e f f e c t s o f various buffers o f the e s r spectra and the electrochemical p r o p e r t i e s o f the Fe(III) complexes. E l e c t r o c h e m i s t r y allows a c l e a r view o f how one o f the metal l i g a t i n g s i t e s o f BLM, the 4-amino pyrimidine moiety, b e haves i n the presence o f buffer i o n s . I f Fe(III)BLM i s s y n t h e s i z ed from the free drug and F e ( I I I ) ( C £ 0 ) i n the absence o f buffers, e l e c t r o c h e m i s t r y shows that the 4-amino pyrimidine remains bound to the metal i o n w i t h i n the pH range 4-9 where e s r shows that low 2

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spectrum of Fe(IU)BLM taken immediately after oxidation of Fe(II)BLM with molecular oxygen

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The relative amounts of (A) high and (%) low spin Fe(III)BLM function of pH as determined by ESR


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s p i n Fe(III) predominates. In a pH 7 o r 8 phosphate buffer (0.1Ml e l e c t r o c h e m i s t r y shows that the pyrimidine moiety i s not bound to the metal i o n . E s r confirms that these s o l u t i o n s contain only high s p i n (S = 5/2) F e ( I I I ) . Synthesizing Fe(III)BLM by anaerob i c a l l y combining Fe(II)(C£0 )2 and the drug i n a pH 7 or 8 phos phate b u f f e r , followed by a i r o x i d a t i o n o f the r e s u l t i n g F e ( I I ) drug complex y i e l d s a low s p i n Fe(III)BLM complex which contains a bound pyrimidine f u n c t i o n . I f the o x i d i z e d s o l u t i o n s are a l lowed t o stand a t room temperature f o r a few hours, e s r and v i s i b l e absorption spectra show that a r e d i s t r i b u t i o n o f the s p i n states o c c u r s ; high s p i n Fe(III) again forms a t the expense o f low s p i n F e ( I I I ) . E l e c t r o c h e m i s t r y , however, shows that the pyrimidine i s s t i l l bound to the metal i o n . Thus, our r e s u l t s show t h a t Fe(III)BLM i s high s p i n when the pyrimidine moiety i s not bound to the m e t a l , but that some high s p i n forms a l s o e x i s t where the pyrimidine i s bound to the c a t i o n . These o b s e r v a t i o n s , when viewed i n the l i g h t that almost a l l o f the DNA degrading ex periments with the a n t i b i o t i c have been c a r r i e d out i n buffered media, underscore the importance o f c a r e f u l l y d e f i n i n g the metal l i g a t i n g properties o f the drug. I t i s i n t e r e s t i n g to note t h a t the highest DNA degrading a c t i v i t y achieved by i r o n bleomycin i s i n a phosphate b u f f e r - a medium which apparently s i g n i f i c a n t l y a l t e r s the primary c o o r d i n a t i o n sphere o f the metal i o n (5J.


4

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Derivatives

In an e f f o r t to more c l e a r l y understand the mechanism by which bleomycin degrades DNA, three new bleomycin analogues were s y n t h e s i z e d , 2-4. A l l three analogues were formed by the c o n densation o f the primary amine function o f BLM-A with e i t h e r benzoyl c h l o r i d e (2) o r a s u l f o n y l c h l o r i d e (3,4) using SchottenBaumann c o n d i t i o n s (10.). The compounds were p u r i f i e d v i a chroma tographic means were c h a r a c t e r i z e d using H and C nmr and po t e n t ! ome t r i e t i t r a t i o n methods. P a r t i c u l a r l y noteworthy i n the t i t r a t i o n curves o f the d e r i v a t i v e s i s the absence o f i n f l e c t i o n s at pH values o f 2.9 and 7.4. For BLM-A , i n f l e c t i o n s a t these pH values correspond t o the l o s s o f a proton from the protonated, but hydrogen bonded, secondary amine function ( E q . 1) and a loss of a proton from the protonated primary amine function r e s p e c t i v e l y ( E q . 2). 2

l

1 3

2

-NH

-NH+ H pK,2.9 (1) a -NH > - N H + H p K , 7.4 (2) α In c o n t r a s t to BLM-A , n e i t h e r o f the new d e r i v a t i v e s f a c i l i tated the o x i d a t i o n o f Fe(II) to Fe(III) i n the atmosphere Q 0 ) . Esr studies o f aqueous s o l u t i o n s c o n t a i n i n g equimolar amounts F e ( I I ) ( C £ 0 ) and the analogues, 2-4 a t pH, 7.0 showed the lack o f formation o f F e ( I I I ) . S i m i l a r observations have been reported f o r 2

3

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dep BLM (22). However, more i m p o r t a n t l y , incubation of closed c i r c u l a r PM2 DNA with any o f the analogues, 2-4 i n the presence of Fe(II) s a l t s and reducing agents d i d not lead to DNA breakage. I d e n t i c a l r e a c t i o n c o n d i t i o n s f o r B L M - A , on the other hand, lead to DNA breakage. These recent observations s u b s t a n t i a l l y strengthen the proposed metal-mediated mechanism of a c t i o n of the a n t i b i o t i c and underscore i n importance of the primary amine f u n c t i o n of bleomycin i n the DNA degrading process. 2


Conclusions Spectroscopic evidence shows that f o r the Fe(11,111) complexes of BLM the metal ions are bound to the p y r i m i d i n e - i m i d a z o l e sugar region o f the drug l e a v i n g the b i t h i a z o l e moiety free to bind to DNA. The paramagnetic Fe(II) complex can be o x i d i z e d to to a low spin (S = 1/2) Fe(III) complex which i s r a p i d l y and i r r e v e r s i b l y transformed to a second low-spin Fe(III) complex. The low s p i n species i s i n a pH- and buffer-dependent e q u i l i b r i u m with high s p i n (S = 5/2) F e ( I I I ) . Electrochemical studies show that the binding of the 4-amino pyrimidine moiety of bleomycin to Fe(III) i s s e n s i t i v e to both pH and the presence of c e r t a i n buffer species. UV v i s i b l e absorption spectra show s i m i l a r e q u i l i b r i a f o r the Fe(II) complexes. Because i n v i t r o DNA-cleavage occurs i n a wide range of b u f f e r s o l u t i o n s i t appears that the displacement of some of the metal l i g a t i n g atoms does not i n h i b i t the a c t i o n of the i r o n - d r u g complex. F i n a l l y , studies with three new bleomycin analogues have sharply defined the importance of the primary amine f u n c t i o n of bleomycin i n the drugs a b i l i t y to degrade DNA by a metal mediated mechanism. Abstract Using electrochemistry, uv-visible absorption, esr, and C nmr spectroscopy, and with the help of three new bleomycin analogues, the Fe(II) and Fe(III) binding sites of the a n t i tumor antibiotic bleomycin have been located. The drug appears to u t i l i z e the amine-pyrimidine-imidazole region for iron binding. The physical properties of the metal complexes and how those properties relate to the proposed mechanism of action of the anticancer agent is also discussed. 13

Acknowledgement We acknowledge the National I n s t i t u t e of Health f o r support of t h i s work (Grant, CA-25112-01). F . T . G . is also grateful for support through an N . I . H . I n s t i t u t i o n a l Award.

American Chemical Society Library 1155 16th St. N. W. Washington, D. C. 20036 Martell; Inorganic Chemistry in Biology and Medicine ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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