‘SHE ~ O U R N A I . OF BIOI.OGICAI. CHEMISTRY V o l 465, Nu. 7. Isue of April I O , pp. 2816-2X21. 19x0 Prmtrd ln I ’ S A

Modification of Hemoglobin with Analogs of Aspirin*

(Received for publication, July 30, 1979)

Robert H. Zaugg,S Joseph A. Walder,§ Roxanne Y, Walder,§ Jeffrey M. Steele, and Irving M. Klotz From the Department of Chemistry and Department of Biochemistry and Molecular Biology, Northwestern University, Euanston, Illinois 60201

A variety of acyl esters of salicyclic acid and 3,5- dibromosalicylic acid have been prepared and exam- ined for their ability to place the acyl group on hemo- globin. In general, short chain acyl groups (C2 and C,) are more reactive than longer chains (C4 to G o ) , but longer chains may be more effective with intact red cells because of their enhanced ability to permeate the erythrocyte membrane. The brominated salicyl esters also exhibit enhanced permeation of the membrane, as well as increased activity due to activation at the acyl site. Bis(salicy1) esters, nonbrominated and bromi- nated, are more reactive than corresponding monoes- ters, and those from C4 dicarboxylic acids connect p subunits by covalent bridges. These double-headed as- pirins have the attractive features of being bound se- lectively by hemoglobin and of forming a covalent cross-link that may influence the conformation of the tetramer.

Sickle cell disease is a hematological disorder in which erythrocytes from affected individuals undergo dramatic transformations in shape upon deoxygenation. Such abnor- mally shaped erythrocytes are readily trapped in the micro- vasculature and this blockage leads to tissue damage and to severe hemolytic anemia.

These cellular and clinical manifestations arise from the aggregation of sickle hemoglobin (Hb S) in the deoxygenated state. Prevention or reversal of this aggregation might be achieved by modification of the hemoglobin. If covalent chem- ical changes are to be made, one is largely limited, by phar- macological constraints, to reactions with amino groups of this protein. One class of such reactions utilizes aromatic alde- hydes which form Schiff base adducts with amino groups of intracellular hemoglobin (1 ,2) . A second class of amine group- specific reagents includes cyanate (3) and acetylsalicylate (4), which carbamylate and acylate hemoglobin, respectively.

Acetylation of amine groups of hemoglobin (Equation 1) does occur with moderate concentrations (0.01 M) of acetyl- salicylic acid, aspirin (4):

coo- coo-

However, the extent of acetylation resulting from even high

* This investigation was supported in part by Grant HL22719 from the National Heart, Lung, and Blood Institute, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

+Present address, Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Mass. 02139.

5 Present address, Department of Biochemistry, University of Iowa, Iowa City, Iowa 52242.

concentrations of the drug is insufficient to inhibit signif- cantly erythrocyte sickling (5,6). Aspirin, however, is only one of a broad class of acylsalicylates. Enhancements of acylating activity, with concomitant improvements in antisickling effect, might be achieved by altering either the leaving group (the salicylate) or the acyl group. We recently reported that a compound altered in the leaving moiety, acetyl-3,5-dibromo- salicylic acid, is a potent acetylating agent of intracellular sickle hemoglobin (7). Exposure of sickle er-ythrocytes in vitro to low levels of this alternative aspirin produces a sizeable increase in the oxygen affinity of sickle hemoglobin and, furthermore, significantly inhibits erythrocyte sickling a t fixed percentages of saturation with oxygen.

It is likely that other members of the broad class of acyl- salicylates will provide enhanced. acylation of intracellular hemoglobin. We have undertaken, therefore, a systematic investigation of the acylating and antisickling activities of several series of aspirin-like compounds. This report describes the effect on these activities of alterations in the acyl portion of the drug molecule.

EXPERIMENTAL PROCEDURES

Materials Salicylic acid, 3,5-dibromosalicylic acid, and 4-hydroxybenzoic acid

were purchased from Aldrich. Aspirin was obtained from Merck. Acetic anhydride and thionyl chloride were obtained from Mallinck- rodt; propionic anhydride was from Eastman; suberic acid, dodeca- nedioic acid, and all acid chlorides were from Aldrich. (Solvents used in the synthesis and recrystallization of carboxyphenyl esters were obtained from commercial sources and were purified by fractional distillation when necessary.) Propylamine was purchased from East- man. Tris buffer was obtained from Schwarz/Mann and bis-Tris’ was from Sigma.

Carrier ampholytes (pH 6 to 8) for isoelectric focusing were ob- tained from LKB; sodium dodecyl sulfate was from Matheson, Cole- man and BeU 2-mercaptoethanol was from Eastman; acrylamide and bisacrylamide were from Aldrich; and ammonium persulfate and N,N,N’,N’-tetramethylethylenediamine was from Bio-Rad.

Synthetic Procedures Suberoyl dichloride and dodecanedioyl dichloride were prepared

from the corresponding dicarboxylic acid by refluxing in excess thionyl chloride for several hours according to established procedures (8). 2- Carboxyphenyl esters (acylsalicylates) and 4-carboxyphenyl esters were synthesized according to one of three general methods.

Method A-One equivalent of the appropriate carboxyphenol was dissolved in 2 to 3 eq of acid anhydride to which was added 5 to 10 drops of sulfuric acid. The mixture was stirred at room temperature for 15 min, then at 40-50°C for 5 min. The reaction mixture was poured into ice-cold distilled water and the product was precipitated as a white solid, collected, dried in uacuo, and recrystallized.

Method B-To 1 eq of carboxyphenol in pyridine at 0°C was added dropwise 1.1 eq of acid chloride. The mixture was stirred at room temperature for several hours, after which it was poured into ice-cold acid water. The precipitate formed was collected, dried, and recrys- tallized.

I The abbreviation used is: bis-Tris, bis(2-hydroxyethy1)iminotris- (hydr0xymethyl)methane.

2816

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Aspirin Analog Modification of Hemoglobin 2817

Method C-Two equivalents of carboxyphenol were dissolved in 40 to 50 rnl of benzene to which was added 4 eq of dimethylaniline. T o this stirred mixture at 0°C were added dropwise 1.1 eq of the appropriate dicarboxylic acid dichloride and the mixture was stirred overnight at room temperature. The mixture poured into ice-cold acid water produced a precipitate which was collected, dried, and recrystallized.

In each case, completeness of acylation of carboxyphenol was assured by analysis of the product for traces of free carboxyphenol using thin layer chromatography in benzene/dioxane/acetic acid (18 2:l). For 2-carboxyphenyl esters, a negative ferric chloride test en- sured the absence of free salicylic acid. Subsequent base hydrolysis of the salicyl ester generated the characteristic purple color in the presence of ferric chloride that provides a positive test for salicylic acid. Product purity was assessed and identification confmed by melting range and elemental analyses.

Preparation of Erythrocyte and Hemoglobin Solutions Whole blood was drawn by venipuncture from healthy adults.

EDTA was used as anticoagulant. Erythrocytes were washed twice in isotonic phosphate buffer (0.123 M sodium phosphate), pH 7.2. Packed erythrocytes were suspended to a final hematocrit of 20% (v/v) in isotonic phosphate buffer for tests requiring intracellular hemoglobin. In the preparation of extracellular hemoglobin solutions, 1 volume 01 saline-washed packed erythrocytes was hemolyzed by addition of 02 volume of 0.05 M sodium phosphate, pH 7.2, containing 0.01 M sodium cyanide to yield a stock hemolysate containing 18 g/dl of hemoglobin. For tests involving cell-free hemoglobin, this stock solution was reduced to a final hemoglobin concentration of 6 g/dl in 0.05 M sodium phosphate, 0.01 M sodium cyanide, pH 7.2. Thus, final hemoglobin concentrations were roughly 1 KIM for both intra- and extracellular tests.

Chemical Modifications All compounds were tested a t a concentration of 5 mM in 20%

erythrocyte suspensions in isotonic phosphate buffer, pH 7.2, and in 6% solutions of cell-free hemoglobin in 0.05 M sodium phosphate, 0.01 M sodium cyanide, pH 7.2. The reagent was dissolved in buffer, to a concentration 5 times that desired in the final mixture with erythro- cytes, and titrated with base to restore the pH to the original value in the buffer and to ensure complete solubilization. One volume of this solution of reagent was mixed with 4 volumes of red cell suspension. Single-dose experiments were supplemented by multiple-dose tests. After each of four exposures of 20% erythrocyte suspensions to 5 mM doses of compound, the preceding dose was removed after separation of the erythrocytes by centrifugation. Several of the more reactive compounds were tested a t lower concentrations. All incubations took place for 2 h a t 37°C in a water-bath shaker. Reactions were termi- nated by rapid freezing of solutions in a dry ice/methanol bath.

Extents of acylation of hemoglobin were assessed by isoelectric focusing in polyacrylamide gels as described previously (2). Acylated species appeared as focused bands. The extents of acylation were assessed quantitatively by integration of peaks in densitometric scans of the gels.

Each of the bis(2-carboxyphenyl) esters was mixed with 6% Hb A to investigate the possible formation of chemical cross-links. Incuba- tions involved 20 mM compound in 0.05 M phosphate with 0.01 M cyanide, or in 0.05 M bis-Tris.HCI, pH 7.2, for 2 h a t 37°C. The occurrence of cross-links between subunits of the hemoglobin tetra- mer was detected by polyacrylamide gel electrophoresis in the pres- ence of sodium dodecyl sulfate as described previously (2). The extents of cross-linking were determined quantitatively by integration of peaks in densitometric scans of the stained gels.

Aminolysis by Propylamine The rates of aminolysis by propylamine of several 2-carboxyphen-

ylesters were determined in 0.02 M Tris-HCI, pH 8.5. At a fixed concentration of propylamine, greatly in excess of carboxyphenyles- ter, a pseudo-fmt order rate constant was calculated from early observations of the extent of aminolysis. Corresponding experiments were carried out at other fixed, excess concentrations of amine (loo-, 200-, and 300-fold molar excess over ester). The apparent second order rate constant for aminolysis of each ester was obtained from the slope of the line relating the pseudo-first order rate constant to the concentration of propylamine.

C 1 2 3 4

FIG. 1. Polyacrylamide gels demonstrating the cross-linking of hemoglobin. Solutions of cell-free Hb A were treated with 20 m M bis(salicy1)fumarate (Compound 15) for 2 h at 37°C in 0.05 M phos- phate, 0.01 M cyanide, pH 7.2. Gel electrophoresis in the presence of sodium dodecyl sulfate was performed as described under “Experi- mental Procedures.” The anode is at the hoffom. C , untreated Hb; I to 4. treated Hb samples containing 0.6, 1.2.3.0, and 6.0 g/dl of Hb A, respectively.

RESULTS

The twenty-three carboxyphenylesters examined in the present study are listed in Table I. Compounds 1 through 6 are simple chain-length homologs of aspirin (1) with acyl groups having 2, 3, 4, 6, 8, and 10 carbon atoms (4), respec- tively. Compound 7 has a branched, five-carbon acyl group. We also tested several dibrominated derivatives of acylsali- cylates since the dibromosalicylate leaving group has been shown to greatly facilitate the transfer of the acetyl moiety to hemoglobin (7). Compounds 8 through 11 (Table I) contain dibrominated phenyl rings but are identical to Compounds 1 to 4, respectively, with regard to the acyl group. Compounds 12 to 14 were examined to see if placement of the acyl group para to the carboxyl functionality enhances reactivity. The remaining compounds in Table I (Compounds 15 to 23) are diesters of a series of dicarboxylic acids that contain 4, 6, 8, 10, or 12 carbon atoms. These “double-headed” reagents have the potential to react with pairs of adjacent amino groups of hemoglobin to form covalent molecular cross-links (15).

The extents of chemical modification of hemoglobin pro- vided by 5 mM doses of each carboxyphenylester are shown in Table 11. Hemoglobin was present either within erythrocytes (intracellular), so that the modifying reagent had to permeate the cell membrane prior to reaction, or free in solution (extra- cellular). In the absence of a membrane barrier in the extra- cellular incubations one can measure the intrinsic reactivity of each compound towards hemoglobin. In the intracellular experiments, erythrocyte suspensions were exposed to both a single 5 mM dose of compound and to four successive 5 mM doses.’

‘Long chain acylsalicylates (e.g. Compound 6) appear to have detergent-like properties and cause considerable hemolysis. The CI? homolog (not shown) was synthesized and tested but was found to be both hemolytic and water-insoluble.

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2818 Aspirin Analog Modification of Hemoglobin

TABLE I Acylsalicylates: chemical structures, synthetic procedures, melting points

Class Compound" Melting point

R= Synthetic procedure" Found Literature (Ref.) value

Salicyl esters

coo-

3.5-Dibromosalicyl esters

coo -

Br

4-Carboxyphenyl esters /-z3

0 Bis(salicy1) diesters

Bis(3,5-dibromosalicyl) diesters

1 Acetate (Cz) 2 Propionate (C:]) 3 Butyrate (C4) 4 Caproate (CS)

5 Caprylate (Ca) 6 Caprate (CIO) 7 Isovalerate (branched C,)

8 Acetate (Cz) 9 Propionate (C3)

10 Butyrate (C4) 11 Caproate (C,)

12 Acetate (Cz) 13 Caproate (C6) 14 t-Butylacetate (branched CS)

15 Fumarate (unsaturated C,) 16 Succinate (C4) 17 Adipate (C,) 18 Suberate (C,) 19 Sebacate ( G O )

20 Dodecanedioate ( C I ~ )

21 Fumarate (unsaturated C,) 22 Succinate (C4) 23 Sebacate ( G O )

" Length of acyl group is shown in parentheses. 'See "Experimental Procedures" for details.

The bis(dibromosalicy1) diesters (Compounds 21 to 23) are the most reactive compounds included in the present study. This potency of these agents necessitated the use of lower test concentrations.

To examine the formation of chemical cross-links between amino groups on different subunits of hemoglobin, the protein was treated with high concentrations (20 mM) of each of the diesters (Compounds 15 to 23) listed in Table 11. The extents of cross-linking, as assessed by polyacrylamide gel electropho- resis in the presence of sodium dodecyl sulfate, are shown in Table 111. Among bis(salicy1) compounds, only those diesters having four-carbon acyl chains (Compounds 15 and 16) pro- duced cross-links in detectable amounts. A similar, albeit less strict, dependence of cross-linking on short connecting groups was also observed in the b1~(3,5-dibromosalicyl) series of dies- ters.

As shown in Fig. 1, only p chains of the intact tetramer are linked to form /3/3 dimers to the exclusion of aa and Cyp pairs. Furthermore, the extent of cross-linking is independent of hemoglobin concentration over the range of 0.6 to 6 g/d. Essentially all the p chains are cross-linked; free /3 subunits are not seen. Such behavior is consistent with formation of

A B B

B B B

A A A A

A B B

C C c C C C

C C C

commercially obtained 96-98 95 (9) 79-80 82 (9) 69-73 75 (9)

70 (IO) 79-81 81 (10) 69-70 88-89 95 (11)

155-157 156 (12) 114-116 118-120 88-90

186-189

195-198 152-153

178-180 183-184 171-174 150-152

134-136 139-142

215 (dec.) 195-196 138-142

190 (13)

180 (14)

covalent connections between subunits of the same tetramer rather than between distinct tetramer^.^

Intact erythrocytes were exposed to four successive 1 mM doses of bis(3,5-dibromosalicyl) succinate (Compound 22), after which hemoglobin was extracted from the cells and subjected to gel electrophoresis in the presence of sodium dodecyl sulfate. Approximately 15% of the monomers of intra- cellular hemoglobin were crosslinked by this procedure. This finding indicates that substantial amounts of the bis(dibromosalicy1) diesters penetrate the erythrocyte mem- brane with both ester functions intact.

Several salicyl esters were treated with propylamine to assess the inherent susceptibility of these acyl groups towards aminolysis (see Table IV). The concentration of ester in all cases was 0.1 mM. Additional aminolyses of sdicylcaprylate (5 ) at 0.01 and 5.0 m~ c o n f i e d that rate constants were independent of ester concentration within this range. Rate constants tend to decrease with increasing chain length within

Cross-linking between amino groups within the same subunit (a or f3 chain) may also occur but these cannot be detected by this electrophoretic procedure.

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Aspirin Analog Modification of Hemoglobin 2819

TABLE I1 Percentages of modification of intracellular and extracellular hemoglobins by acylsalicylates

Modification of hemoglobin

Compound Intracellular"." Extracellular (1 dose)" ' 1 dose 4 doses

%

Salicyl esters 1 Acetate (C2) 2 Propionate (Cd 3 Butyrate (C4) 4 Caproate (CS) 5 Caprylate (Cd 6 Caprate (CIO) 7 Isovalerate (branched Cg)

3,5-Dibromosalicyl esters 8 Acetate (CZ) 9 Propionate (C3) 10 Butyrate (C4) 11 Caproate (CS)

<5 <5

0 7

14 Lysis

0

77 35 8 9

4-Carboxyphenyl esters 12 Acetate (CZ) <5 13 Caproate (CS) 11 14 t-Butylacetate (branched CS) 5

Bis(salicy1) diesters 15 Fumarate (unsaturated C4) 0 16 Succinate (C4) 0 17 Adipate (C,) e 5 18 Suberate (C8) 10 19 Sebacate ( G O ) 24 20 Dodecanedioate ( C I ~ ) 35

16 20 <5 29 36

<5 Lysis

100 €44 26

Lysis

22 33 22

0 0

15 35 42 65

7 5

<5 5 9

13 t 5

77 31 6

16

10 14 5

70 14 16 14 14 17

Bis(3,5-dibromosalicyl) diesters 21 Fumarate (unsaturated C.,) 15 (1 mM) 93 (1 mhi) 22 Succinate (C4) 18 (1 mM) 64 (1 mM) 23 Sebacate ( G O ) 29 (0.5 mM) 63 (0.5 m h i )

"Each dose involved exposure of 20% (v/v) erythrocyte suspensions in isotonic phosphate buffer, pH 7.2, to 5 mM compound (unless

88 (1 mM) 70 (1 mM) 30 (0.5 mM)

otherwise noted) for 2 h a t 37°C. Reproducibility of extent of modification was +5%.

'Dose involved treatment of 6 g/dl of hemoglobin solutions in 0.05 M sodium phosphate, 0.01 M sodium cyanide, pH 7.2, with 5 mM compound (unless otherwise noted) for 2 h at 37°C.

TABLE I11 Percentages of hemoglobin fl chains that are cross-linked by

various bis(sa1icyl) diesters Compound % Cross-linked"

Bis(salicy1) diesters 15 Fumarate (unsaturated C4) 85 16 Succinate (C4) 10 17 Adipate (C,) 0 18 Suberate (Ca) 0 19 Sebacate (Clo) 0 20 Dodecanedioate ((212) 0

Bis(3,5-dibromosalicyl) diesters 21 Fumarate (unsaturated C4) 22 Succinate (C4) 23 Sebacate ( G o )

70 40 0

a Reactions carried out for 2 h a t 37°C in solutions containing 20

and 6 g/dl of extracellular hemoglobin in 0.05 M sodium phosphate mM ester for nonbrominated compounds, 1 mM for brominated ones,

buffer, 0.01 M sodium cyanide, pH 7.2.

each group (Table IV). The dibrominated salicyl esters are 2- to &fold more reactive towards propylamine than are the corresponding salicyl esters. One bis ester examined previ- ously for reactivity with propylamine (16) showed a fist rate constant comparable to that of the corresponding monoester.

TABLE IV Apparent second order rate constants for the aminolysis of various

acylsalicylates by propylamine in 0.02 M Tris-HCI, pH 8.5 Compound

- kr.

M-' min-' Salicyl esters 1 Acetate (C,) 2 Propionate (Ca) 3 Butyrate (C4) 4 Caproate (C,) 5 Caprylate (C")

0.098 0.051 0.027 0.030 0.031

3,5-Dibromosalicyl esters 8 Acetate (C,) 0.770 9 Propionate (Ca) 0.157 10 Butyrate (C4) 0.096 11 Caproate (C,) 0.075

DISCUSSION

In the present study, the effects on acylation of hemoglobin of variations in structure of the acyl portion of aspirin analogs have been examined. Fourteen different acyl groups were surveyed. Six of these derived from straight chain monocar- boxylic acids, form adducts containing 2 t o 10 carbon atoms upon transfer of the acyl group to amines of hemoglobin (Equation 1). Two additional derivatives (Compounds 7 and

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2820 Aspirin Analog Modification of Hemoglobin

14) place branched acyl groups at NHz sites of the protein. Among salicyl esters (Compounds 1 to 7), increases in the

chain length from Cz to Clo led to slight improvements in acylation of extracellular hemoglobin (Table 11) despite low- ered susceptibilities of these esters towards aminolysis by propylamine (Table IV). The butyrate ester, however, be- haved contrarily. The presence of a branched acyl chain (Compound 7) also diminished reactivity. The dominant trend with chain length may reflect increased binding of the longer chain derivatives near reactive sites on the hemoglobin mol- ecule. Furthermore, additional enhancements in modification with increasing chain length were observed with regard to the acylation of intracellular protein. This finding suggests that longer chain salicyl esters permeate the erythrocyte mem- brane more readily. (Indeed, the extensive hemolysis produced by esters having acyl chains of 10 carbons or more attests to the lipophilicity of the longer chain derivatives.) Thus, salicyl caprylate (Compound 5) acylated 2- to %fold more intracel- lular hemoglobin than did aspirin (Compound 1) despite the 3-fold lower intrinsic susceptibility of the Cs ester towards aminolysis.

A different trend emerges upon examination of the behavior of the 3,5-dibromosalicyl esters (Compounds 8 to 11). The reactivity of these compounds towards intra- and extracellular hemoglobin and towards propylamine was found to decrease with increasing chain length (Tables I1 and IV). That the electron-withdrawing bromine substituents activate the car- bonyl group towards nucleophilic attack by amines is clearly evident in the 8-fold greater aminolysis rate for dibromoaspi- rin (Compound 8 ) as compared to aspirin (Table IV). Fur- thermore, bromination leads to enhanced membrane perme- ability (Table 11). Dibromoaspirin, at 5 mM concentration, modified intracellular and extracellular hemoglobin to equiv- alent extents. Obviously the membrane presents little barrier to the free diffusion of this Cz analog. Longer chain dibromo- salicyl esters likewise possess high lipid solubility as evidenced by the first appearance of hemolysis with the (26 dibromo derivative (Compound 11) whereas, among the nonbromi- nated salicyl esters, hemolysis first appears with the Clo com- pound (Compound 6).

The improved acylation of intracellular hemoglobin by the dibromosalicyl derivatives of acetate ( 8 ) and propionate (9), compared to the nonbrominated derivatives (Compounds 1 and 2), reflects, therefore, both an enhanced interaction of the brominated acylating agent with -NHz sites of hemoglobin and an augmented intrinsic reactivity towards aminolysis. As pointed out previously (7), dibromoaspirin (Compounds 8 ) acetylates hemoglobin at a rate 50-fold greater than does aspirin (Compound 1). Only part of this large differential can be accounted for by the &fold greater rate of aminolysis of dibromoaspirin as compared to aspirin (Table IV). The addi- tional rate enhancement may be a manifestation of increased binding of the dibromo derivatives by hemoglobin, probably by virtue of the high polarizability of the bromine substituents.

Dibromosalicyl esters having acyl chains longer than three carbon atoms (Compounds 10 and 11) exhibit markedly de- creased levels of acylation (Table 11). Reference to Table IV indicates, however, that the greatest decline in aminolysis rate occurs between Cz (Compound 8 ) and Ca (Compound 9). Taken together, these findings suggest that the protein local environments encompassing the susceptible -NHz sites of hemoglobin are sterically confined. The combination of the long chain with the adjacent bromine and COO- substituents hinders access to the amine nucleophile. A similar steric hindrance seems to be operative with bulky, branched chain acyl groups (Compounds 7 and 14) which are poorly trans- ferred to hemoglobin.

The 4"rboxyphenylesters (Compounds 12 to 14) are somewhat more reactive than the corresponding 2-carboxy- phenylesters. Thus, Compounds 12 and 13 produce slightly more modification of hemoglobin than do Compounds 1 and 4, respectively. Again a branched acyl group (Compound 14) diminishes reactivity.

Nine additional reactants contain acyl groups of dicarbox- ylic acids, 4 to 12 carbon atoms in length. The bis(salicy1) esters of these diacids (Compounds 15 to 23) could react with hemoglobin according to Equation 2:

coo- 0 9 coo O-C-(CHAn"C"O

Hb

(111)

The intermediate (I) will not likely persist since it is suscep- tible either to hydrolysis, forming (10, or to aminolysis by a second amine to form a cross-linked species (114. If a cross- link arises between amino groups on different subunits of the hemoglobin tetramer, stable dimers will persist under condi- tions that normally dissociate native hemoglobin into constit- uent a and /3 monomers. The extent of such intersubunit cross-linking, therefore, can be assessed by techniques that distinguish proteins on the basis of sue, such as polyacryl- amide gel electrophoresis in the presence of sodium dodecyl sulfate (see Table I11 and Fig. 1).

Among bis(salicy1) diesters (Compounds 15 to 20), the fumarate derivative (Compound 15) produced vastly superior acylation of extracellular hemoglobin. Its effectiveness prob- ably arises from the facilitation of electron withdrawal from the reacting acyl C=O and from resonance stabilization of the anion intermediate of aminolysis provided by the conjugated double bond. Such assistance is not furnished by the diesters derived from saturated dicarboxylic acids (Compounds 16 to 20), all of which exhibited lower, roughly equivalent levels of modification.

Upon exposure of intact erythrocyte suspensions to these latter reagents (Compounds 16 to 20), however, clear differ- ences in acylation emerged. The extent of modification of intracellular hemoglobin rose with increasing length of the acyl chain. This trend is most readily explained on the basis of differences in membrane permeability. The erythrocyte membrane is virtually impermeable to large dianions and cations (17). The four-carbon bis(salicy1) diesters (Compounds 15 and 16) are large dianions and are thus unable to penetrate the membrane. On the other hand, permeability is achieved with the c6 derivative and it becomes greater with increasing chain length thereafter. This altered behavior may result from the greater distance between negative charges for the longer chain diesters (Compounds 17 to 20), this separation allowing each diester to act as two distinct monoanions in penetrating the membrane.

The longest chain diesters (Compounds 19 and 20), at 5 mM concentration, provided greater acylation of intracellular than of extracellular hemoglobin (see Table 11). This finding

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Aspirin Analog Modification of Hemoglobin 2821

suggests that these compounds were accumulated within the erythrocyte, where the hemoglobin concentration is about 5 mM. Accumulation of acylation agent by erythrocytes would diminish the amount of free agent that would otherwise be available to cause unwanted side-effects elsewhere.

The bis(dibromosalicy1) diesters (Compounds 21 to 23) were generally much more reactive than the corresponding nonbrominated diesters (Compounds 15 to 20), a finding that parallels that found for the monoester derivatives. Further- more, the presence of the bromine substituents increased the membrane permeability of these agents so that the four- carbon brominated derivatives (Compounds 21 and 22) pro- vided significant acylation of intracellular hemoglobin in marked contrast to the corresponding nonbrominated com- pounds. However, this improved lipid solubility was accom- panied by considerable hemolysis, even at reduced concentra- tions. As with the nonbrominated diesters, the fumarate dies- ter (Compound 21) proved most reactive in the series of brominated compounds. Also intracellular modification rose with increase in chain length of the acyl group to Clo.

Inasmuch as the diesters (Compounds 15 to 23) contain two activated acyl functionalities, each susceptible to nucleo- philic attack by amines, these derivatives have the potential to cross-link sites of hemoglobin. Several of these compounds did produce cross-links, as assessed by gel electrophoresis in the presence of sodium dodecyl sulfate. Since this procedure resolves proteins on the basis of size, only cross-links between subunits of hemoglobin can be detected; intrachain links are not revealed. Table I11 shows the extents of monomer cross- linking for each diester. With nonbrominated diesters, only four-carbon acyl groups (Compounds 15 and 16) provided cross-linking bridges. Among the brominated esters, the fu- marate (Compound 21) and succinate (Compound 22) were very effective, but a little cross-linking was also achieved with the CIO sebacate ester (Compound 23).

In all cases examined, cross-linking by four-carbon diesters occurred almost exclusively betwen subunits of the same hemoglobin tetramer (see Fig. 1). The bridges formed thus must be in some area where the two f l subunits are in close approximation. This is the region of the /3 cleft.

Within the /3 cleft of deoxyhemoglobin the distances be- tween pairs of amino groups on opposite f l chains are as follows (18, 19): Lys 82 . . . Lys 82,8.1 A; Lys 82 . . . Val 1, 11 A; Val 1 . . . Val 1, 18 A. Molecular models supply distances between nitrogen atoms attached to opposite ends of the fully extended dicarboxylic acids examined in this study:

0 II II II

0 0 0 II

"C"CH=CH"C-, 6.8 A; "C"(CH~"CH~),-C--, 6.8,

9.2, 11.6, 14.0, and 16.4 Ai for n = 1, 2, 3, 4, and 5, respectively. The Val 1 . . . Val 1 and Lys 82.. . Val 1 distances are large for the span of the C4 diesters, and consequently a Lys 82 + Lys 82 bridge should be favored. It should be noted, however, that the adipate (C,) and larger nonbrominated diesters fail to form /3"p bridges despite reasonable matches of their spans with distances between pairs of, "NH2 esters. Perhaps the p portal of hemoglobin is sharply selective in the structures permitted steric access to this cavity.

The cross-linking diesters may perturb the conformation of deoxyhemoglobin S. If this perturbed conformation pulls groups at inter-tetramer contact interfaces out of register, it is possible that interactions leading to aggregation of tetra- mers will be weakened and sickling diminished. The high selectivity of hemoglobin in the chain length of the double- headed aspirin with which it will react also offers the prospect of minimizing the potential for reaction in vivo with other proteins.

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252,8542-8548 3. Cerami, A,, and Manning, J . M. (1971) Proc. Natl. Acad. Sci. U.

S. A . 68,1180-1183 4. Klotz, I. M., and Tam, J . W. 0. (1973) Proc. Natl. Acad. Sci. U .

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R H Zaugg, J A Walder, R Y Walder, J M Steele and I M KlotzModification of hemoglobin with analogs of aspirin.

1980, 255:2816-2821.J. Biol. Chem. 

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