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Anal. Chem. 1987, 59, 1534-1538
(5) Stetter, J. R.; Jurs, P. C.; Rose, S. L. Anal. Chem. 1988. 5 8 , 860-866. (6) Bott, B.; Jones, T. A. Sens. Actuators 1088, 9 , 19-25. (7) Muller, R.; Lange. E. Sens. Actuators 1988, 9 , 39-48. (8) Draper. N. R.; Smith, H. Applled Regression Analysis, 2nd ed.;Wiiey: New York, 1981; Chapter 4. (9) Geladi, P.; Kowalski. B. R. Anal. Chim. Acta 1988, 185, 1-17. (IO) Mandel, J. J . Res. Net/. Bur. Stand. ( U S . ) 1985, 9 0 , 465-476. (11) Naes. T.: Martens, H. Comun . Statist .-Sirnula. Compura . 1985, 1 4 , 545-576. (12) Naes. T.; Irgen, C.; Martens, H. Appl. Statist. 1988, 35, 195-206. (13) Simpson, R. L. Ph.D. Dissertation, University of Washington, Seattie, WA, 1985.
(14) Carey. W. P.; Beebe, K. R.; Kowaiski, B. R.; Iiiman, D. L.; Hirschfeid, T. Anal. Chem. 1088, 58. 149-153. (15) Carey, W. P.; Kowaiski, B. R. Anal. Chem. 1988, 58, 3077-3084. (16) Lorber, A. Anal. Chem. 1988, 58, 1167-1172. (17) Wold, S.; Ruhe, A.; Wold, H.; Dunn, W. J. SIAM J . Stat. Comput. 1984, 5 , 735-743.
RECEIVED for review October 30, 1986. Accepted February l 7 , 1987. This work was Supported in Part by the Office of Naval Research.
Effect of Fluorine Substitution on the Anodic Oxidation of Catecholamines and Amino Acids Margaret E.Rice and Bita Moghaddam
Department of Chemistry, University of Kansas, Lawrence, Kansas 66015 Cyrus R. Creveling
Laboratory of Bioorganic Chemistry, NIDDK, NIH, Bethesda, Maryland 20892 Kenneth L. Kirk*
Laboratory of Chemistry, NIDDK, N I H , Bethesda, Maryland 20892
The electrochemlcal behavior of the 2-, 5-, and Muoro analogues of dopamine (DA), noreplnephrlne(NE), and (3,4-dlhydroxypheny1)alanlne (DOPA) have been determined by cyclic voltammetry and by measuring fluorkle release during bulk oxldations. At pH 7.4, the order of lncreaslng oxldatlon potentials (€,,2) for the DA serles Is 6-FDA < DA < 5-FDA < 2-FDA; for the NE serks, &FNE < 5-FNE < 2-FNE < NE; and for the DOPA serles, 6-FWPA < 5-FDOPA = 2-FDOPA < DOPA. The 6-fkKm, analogue in each series of conpmds Is the most easlly oxldized and appears to result from a 2electron process rather than the four-electron process (the ECE pathway) for the parent catecholamines or catecholamino acld. Potentlometric measurement wlth a fluorldeton-selective electrode contkms that oxidation of the Wiuoro analogue in each series results In the release of fluorlde Ion. Molecular schemes for the rationalltation of the unlque behavior of the 6-fluor0 analogues are presented.
2-, 5-, and 6-fluoronorepinephrine (2-FNE, 5-FNE, 6-FNE) have been performed. We report here the effects of fluorine substitution on the electrochemical behavior of DA, NE, and 3,4-dihydroxyphenylalanine (DOPA). We were prompted to initiate these studies by the subjective observation that our fluorinated analogues, in particular, compounds having fluorine in the 6-position, seemed more prone to oxidative decomposition than the unsubstituted parent. Thus, initial attempts to determine the phenolic pK, of 6-FNE by measuring ultraviolet absorption spectra as a function of pH were complicated by extremely rapid oxidation at pH values greater than 9. During storage at -20 "C,sample vials containing 6-FDA were found ruptured from internal pressure. The decision to obtain a quantitative assessment of this behavior was reinforced by the belief that knowledge of the electrochemical behavior of fluorinated catecholamines and amino acids might help in understanding aspects of their biological properties.
Over the past few years, we have reported results of several studies regarding the biological properties of ring-fluorinated biogenic amines ( I ) . In addition, we and others have been pursuing actively the use of fluorinated phenolic amines and amino acids as biological tracers, in recognition of the potential of I9F NMR techniques in biological problems (2) and the importance of 18Flabeled compounds as scanning agents for positron emission transaxial tomography (3). Despite the remarkable biological effecta of fluorine substitution on certain of these biogenic amines-the adrenergic agonist properties of ring-fluorinated norepinephrines (NE) is a notable example-relatively little is known about the effect of fluorine substitution on the chemical behavior of these analogues. Fluorine substitution has the expected acid strengthening effect on phenol acidities, as we reported for fluorinated serotonins (4), dopamines (DA) (51, and NE'S (6). Calculations of the effect of fluorine substitution on the electronic charge distribution (7) and molecular electrostatic potential (8)of
EXPERIMENTAL SECTION Fluorinated analogues of DA, NE, and DOPA were prepared as previously described (5,6,9). Stock solutions of 2-FNE oxalate, 5-FNE hydrochloride, and 6-FNE oxalate were made 10 mM in 0.1 N HC104 Stock solutions of NE, DA, 6-FDA hydrobromide, and 5-FDA hydrochloride were made 20 mM in 0.1 N HC104 All electrochemical measurements were made in 0.1 M phosphate buffer, pH 7.4, with 0.9% NaCl (phosphate buffered saline, PBS) unless otherwise stated. Aliquots of stock solutions were added to a final concentration of 100 pM,unless otherwise stated. An IBM EC 225 voltammetric analyzer with a Houston Instruments Omnigraphic 200 X-Y recorder was used for the cyclic voltammetry with a large carbon-paste electrode (GP-38graphite:hexadecane pasting liquid, 2:l ratio) (IO) having a surface area of 0.21 Cm2. Hexadecane-based carbon paste can be used to make a stable electrode surface that exhibits faster electrontransfer rates than conventional Nujol paste surfaces. A platinum-gauze electrode was used for bulk oxidations. A saturated calomel electrode (SCE) was used as the reference, and the auxiliary electrode was a platinum wire. Oxidation Ellz values were determined according to the Nicholson and Shane theory (11). A fluoride-selectiveelectrode (Orion) was used to measure
This article not subject to U.S. Copyright. Published 1987 by the American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 59, NO. 11, JUNE 1, 1987
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Table I. Relative Oxidation Potentials of Fluorocatechols"
fluorine substitution
DOPA
norepinephrine pH 7.4 pH 3.2 E l l z ,V vs. SCE AEp, V Ellz, V vs. SCE
dopamine pH 7.4 Ellz, V vs. SCE AE,,V
pH 7.4 Ell29
V vs. SCE
none
0.32
0.22
0.21
0.12
0.08
0.19
2 5 6
0.30 0.30 0.30
0.12
0.15
0.14
0.20 0.16
0.27
0.12
0.10
0.06 0.05 0.15
0.16 0.16 0.12
0.14
"All E , and AEn were determined bv using a 5OmV/s scan rate. Scheme I
A
PH
OH
HO
\
CHEMICAL REACTION
" W o H b - + 2 H +
-
3.01L 2.0
ELECTRON
TRANSFER HO
0 H
t
H
fluoride release following bulk oxidations. Potentiostatic bulk oxidations were carried out in a 10-mL beaker by using a Pbgauze electrode. Catechol solutions were typically 300 rM in PBS. A Pt-wire auxiliary and SCE reference electrode completed the circuit. A potential of +0.5 V vs. SCE was applied for 5 min. After longer times the solutions darkened from subsequent polymerization reactions. Continuous stirring accompanied the oxidation. Tyrosinase (polyphenol oxidase [EC 1.4.18.11) was purchased from Sigma Chemical Co. (St. Louis, MO). Stock solutions of substrates (20 mM) were prepared in 0.025 N HC1. The rate of oxidation catalyzed by tyrosinase was followed spectrophotometrically at 25 "C by using standard assay conditions provided by Sigma.
3.0
6
t th
RESULTS AND DISCUSSION The relative oxidation potentials (indicated by Eijz) for the three series of compounds studied are given in Table I. At pH 7.4, the order of increasing values of Ellzwere 6-FNE < 5-FNE < 2-FNE < NE in the N E series, 6-FDA < DA < 5-FDA < 2-FDA in the DA series, and 6-FDOPA < 5-FDOPA = 2-FDOPA < DOPA in the DOPA series. Thus, in agreement with ow subjective observations, the 6-fluor0 analogue in each series was the most readily oxidized, although air oxidation and electrochemical oxidations are not necessarily comparable processes. In the NE and DOPA series, fluorine substitution in any position lowered the oxidation potential, presumably as a result of increased electron density of the aromatic ring resulting from back donation of a electrons of fluorine. In contrast, fluorine substitution on DA did not seem to have as marked an effect on E l I 2 .This difference may reflect the greater inherent ease of oxidation of DA, so that the increased electron density of the aromatic ring had a lesser net effect. To explain the particular sensitivity of the 6-fluor0 analogues to oxidation, it is instructive to consider the mechanism of electrochemical oxidation of catecholamines and amino acids and the effect of fluorine substitution on that mechanism. Thus, the oxidation pathway of a catecholamine such as NE follows what is known as an ECE reaction path, consisting of an electron transfer (oxidation), followed by a chemical reaction (cyclization), followed by another electron transfer (12) (Scheme I). In the electrochemical oxidation of NE, if the potential is scanned slowly, allowing for complete conversion of the original quinone to the cyclized intermediate, a maximum of four electrons per molecule can be transferred at the electrode surface. If, however, the potential is scanned quickly, or the cyclization reaction is inhibited by amine protonation a t low
5
10
15
20
25
VX
Figure 1. Dependence of anodic peak current upon scan rate. Profiles of iplV,,, vs. V,,,: (A) at pH 3.2 for NE (0),pH 7.4 for NE (O),P-FNE 5-FNE (0),and 6-FNE (A);(6)for DA (O),2-FDA ( O ) ,5-FDA (0), and 6-FDA (A). See text for conditions.
(o),
pH, a maximum of two electron transfers is observed. Substitution of fluorine in the 6-position in all three series of compounds fundamentally altered this behavior. Thus, for 6-FNE, the anodic peak current (ips) at a given scan rate, when normalized for concentration, was only about half that of NE and the other fluoro analogues. This, in itself, suggests that less than the typical four-electron process occurred. 6-FDA and 6-FDOPA showed similar behavior. In cyclic voltammetry, ipa is proportional to the square root of the scan rate ( v ' / ~(13). ) Hence, in the absence of chemical follow-up reactions after oxidation, a plot of ipalv1/2vs. u1J2 should produce a straight line. This result was obtained for the oxidation of NE at pH 3.2 (Figure 1A) at which pH no cyclization of the intermediate quinone was expected to occur. At pH 7.4 (Figure 1A) where cyclization occurs, an increase in iPlu1l2with decreasing scan rate was seen, reflecting the greater amount of cyclization with subsequent oxidation of the cyclized intermediate. In contrast, 6-FNE a t pH 7.4 shows a relationship between the scan rate and anodic current similar to NE a t pH 3.2 (Figure 1A). This strongly suggested that the oxidation of 6-FNE (as well as 6-FDA and 6-FDOPA) was also a two-electron process (Figure 1B). At either pH, the 2-F and 5-F analogues behaved similarly to NE, suggesting the
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 11, JUNE 1, 1987
i
B
C
+05
W L
10.7
+0.3
I
10.1
+02
00
-02
E(V) vs ECV
I
10.5
to4
-0.1
-0.3
E(V) vs SCE Figure 2. Consecutive cyclic voltammograms of (A) P-FNE, (B) 5-FNE, and (C) 6-FNE. The concentrations of 2- and 5-FNE were 200 pM in PBS, pH 7.4; the Concentration of 6-FNE was 100 pM in PBS, pH 7.4; the scan rate was 50 mV/s. The origin of the numbered anodic and cathodic peaks is discussed in the text.
conventional ECE reaction scheme was applicable for these analogues. The NE series was studied a t pH 3.2 to compare electrochemical behavior under conditions where follow-up cyclization should not occur. From Table I it can be seen that all four compounds were more difficult to oxidize at lower pH and that the three fluorinated analogues oxidized at virtually the same potential. All three, however, were significantly more readily oxidized than NE itself. Thus, while fluorine substitution increases the ease of oxidation, the position of the fluorine became critical only when the follow-up cyclization could occur. At pH 3.2, it was also possible to determine the effect of fluorine substitution on the reversibility of the redox cycle in the NE series. The reversibility is indicated by the millivolt separation between the anodic and cathodic peaks of a cyclic voltammogram, or Mp.At pH 7.4, cyclization was too rapid to obtain comparable data by using conventional scan rates (500 mV/s). The resulta, shown in Table I, suggest that fluorine substitution increased the electrochemical reversibility of 2- and 5-FNE and decreased the reversibility of 6-FNE. The increased reversibility with 2- and 5-FNE reflects not only the increased ease of oxidation, but an increased ease of quinone reduction as well. The anomalous behavior of 6-FNE was entirely due to a negative shift of the cathodic reduction peak, compared to those of 2-FNE and 5-FNE, since the oxidation peak potentials were similar for all three species. This may indicate that the quinone of the 6-substituted analogue was more stable than that of 2- or 5-FNE of NE itself or that a different oxidation product was formed. As previously reported, the rate of internal cyclization after oxidation is much faster for NE (0.36 s-l) than for DA (0.038 s-l) (12). This difference is reflected in the cyclic voltammograms of fluorine-substituted NE and DA in the present
Flgure 3. Cyclic voltammograms of (A) 2-FDA, (B) 5-FDA, and (C) 6-FDA. The amine concentration was 100 WMin PBS at pH 7.4; the scan rate was 50 mV/s.
study as well, illustrated in Figures 2 and 3. In the NE series (Figure 2), no cathodic current a t +0.1 to 0.2 V vs. SCE (position 2) corresponding to the reduction of the noncyclized quinone was seen for either NE (not shown) or 2-FNE (Figure 2A) at 50 mV/s. The reduction and reoxidation of the cyclization product occur a t the peaks labeled 3 and 4, respectively. Fluorine in the 5-position apparently inhibited cyclization, however, as indicated by the appearance of a small peak 2 in the 5-FNE voltammogram (Figure 2B). Although a large reduction peak of about -0.02 V was seen in the 6-FNE voltammogram (Figure 2C), this peak was associated with appearance of anodic peak 5. This new redox couple was adsorbed onto the electrode surface and was still evident when the electrode was subsequently scanned in buffer alone (not illustrated). In contrast to NE and its analogues, all members of the DA series showed a significant reduction peak a t +0.1 V a t 50 mV/s, consistent with a slower rate of cyclization (Figure 3). The anodic to cathodic peak ratio for unsubstituted DA (not illustrated) a t this scan rate was 0.51. The reduced amount of the DA cyclization product formed, compared to that from NE, as well as a reduction potential too near the cathodic solvent limit, prevented the reliable detection of the DA cyclization redox couple, as was possible with NE. Fluorine in position 2 apparently enhanced the rate of cyclization because the ratio of cathodic to anodic peak height was reduced to 0.35 (Figure 3A). Substitution in the 5-position again inhibited cyclization, as indicated by an enhanced peak ratio of 0.64 (Figure 3B). An enhanced ratio was also seen for 6-FDA, but as with 6-FNE, the peak at position 2 occurred at about -0.030 V (Figure 3C) and may have been caused by the reduction of product other than the noncyclized quinone. Further, the 6-FDA peak separation of 150 mV, in comparison with the 50-80 mV peak separation typically seen for the quasi-reversible DA, 2-FDA, and 5-FDA oxidations (Table I), and the appearance of an early shoulder (labeled 3) on the 6-FDA oxidation wave offer further support for this hypothesis. It should be noted, however, that the new redox couple for 6-FDA does not adsorb onto the surface to the extent seen with 6-FNE (Figure 2C).
ANALYTICAL CHEMISTRY, VOL. 59, NO. 11, JUNE 1, 1987
I
V I
+0.6 +0.4 +2.0
I
0
I
I
I
1
-0.2 +0.6 +0.4 +2.0
I
0
1537
1
-0.2
E(V) vs SCE Flgure 4. Cyclic voltammograms of (A) DOPA, (B) 2-FDOPA, (C) 5-FDOPA, and (D) SFDOPA. The amino acM concentratlon was 100 pM in PBS at pH 7.4; the scan rate was 50 mV/s.
The slower DA cyclization rate allowed us to examine the effect of fluorine substitution on both the oxidation and reduction Ellz of the DA/DA-quinone system for each of the analogues at pH 7.4. This examination was not possible for the NE series. This further indicated the effect of fluorine on the reversibility of the electron-transfer process. Theory predicts that the anodic and cathodic peak potentials should be separated by 28 mV. The 50- and 60-mV separation seen for 2- and 5-FDA (Table I) are within experimental error of each other and suggest a more reversible system than that of unsubstituted, quasi-reversible DA (80 mV separation). Because both the anodic and cathodic Ellis of these fluorine analogues are more positive than those of DA, fluoro-substitution appears to stabilize the reduced form of the catechol as well. As noted above, the 150-mV separation seen for 6-FDA suggests that the cathodic current may not correspond to reduction of the original oxidation product (Figure 3A). Further, this peak does not appear until about -30 mV vs. SCE, compared to a reduction peak at +70 mV for the uncyclized quinone. This 100-mV difference suggests either that fluorine in the 6-position dramatically decreases the ease of quinone reduction or that, more likely, following oxidation, a product is rapidly formed which has different electrochemical properties than the normal DA oxidation product, as suggested above. Some possible candidates are discussed below. The cyclic voltammograms for the oxidation of DOPA, 2-, and 5-FDOPA appeared to reflect a four-electron process showing only the primary DOPA oxidation peak (Figure 4AC). The oxidation of 6-FDOPA gave only half of the current seen for the oxidation peaks of the other DOPA'S (Figure 4D). Thus the oxidation of 6-FDOPA appeared to be a two-electron process similar to that of 6-FDA and 6-FNE. It is interesting that no unusual reduction peaks were seen with 6-FDOPA, although a rapid cyclization obviously does occur (Figure 4D). This would be consistent with stabilization of the DOPAaminochrome by the presence of a carboxyl group in the 2-position. From these results, it is evident that fluorine substitution has a substantial effect on the electrochemical behavior of catecholamines and amino acids. Not only are oxidation potentials affected, but also events subsequent to the original oxidation process are clearly altered, particularly in the
analogues having fluorine substituted in the 6-position. The greater ease of oxidation of fluorinated analogues presumably indicates the ability of fluorine to donate electrons to the already electron-rich catechol ring through resonance (para 6' = -0.07). In each series the 6-fluor0 analogue is the most easily oxidized. This may reflect a relationship between oxidation potential and rate of cyclization in each series. Thus, through its inductive effect, fluorine makes the carbon to which it is bound relatively electron deficient. Nucleophilic attack of the amino group on this carbon would be facilitated by this effect. We suggest that through a "push-pull" of electrons, the oxidation and cyclization process may be energetically connected. Thus, even though these steps are not necessarily synchronous, follow-up cyclization could lower the oxidation potential of the 6-substituted analogues. The fact that the three fluorinated NE analogues oxidize with comparable potentials at pH 3.2 is consistent with this proposal (Table I). Since DA is more readily oxidized but cyclizes more slowly than NE, this rationale clearly is applicable only within each series. Further, a lack of correlation between cyclization rate and oxidation potential has also been reported for several of 6-amino- and 6-hydroxydopamine analogues (14). The unique behavior of the 6-fluor0 analogues can be rationalized by considering the likely fate of products formed subsequent to the initial oxidation. As shown in Scheme 11, cyclization of the original quinone oxidation product, as in the ECE mechanism, gives the inte
