Anal. Chem. 1992, 64, 1259-1263
1259
Electrochemical Characteristics of Amino Acids and Peptides Derivatized with Naphthalene-2,3-dicarboxaldehyde: pH Effects and Differences in Oxidation Potentials Mark A. Nussbaum,*JJill E. Przedwiecki,l and Daniel U. Staerks Department of Chemistry, Ripon College, Ripon, Wisconsin 54971
Susan M. Lunte and Christopher M. Riley Center for Bioanalytical Research and the Department of Pharmaceutical Chemistry, University of Kansas, 2095 Constant Avenue, Lawrence, Kansas 66047
The ekctrochemicai oxidations of the cyanobenz[/llsoindole (CBI) derlvatlvesof 18 amino acids and 15 peptides,including enkephalinsand several enkephalin fragments,were studied. Cyclic voltammetry indlcated that the oxidation potentlais of derlvatlzedamlno acids were vlrtualiy Independent of pH. The utiilty of thlr pH-independence was demonstrated by contrdilng, through varying the pH, the selectivity wlth which CBI dervatives could be detected in the presence of phenolic compounds. I n addltion, hydrodynamlc voltammograms of derivatized amino aclds and peptides were constructedfrom chromatographic data and compared. The EllZvalues among the derlvatized amlno acids covered a range of 215 mV, with the derlvatlves of the badc amlno acids belng the eadest to oxidize and those of the acidic amino acids belng the most dltflcultto oxidize. The values of the derivatizedpeptldes examlnedvarled by 270 mV, with minor variationsin structure capable of producing marked changes In oxidation potential. These result8 indlcatethat voltammetry can aid in ldentlflcation or selective detection of CBI derivatives of amlno acids or peptides.
INTRODUCTION Sensitive and selective methods for the detection of amino acids and peptides are of increasing importance in bioanalytical research. One common derivatizing reagent used to enhance detectability is o-phthalaldehyde (OPA),which reacts with primary amines in the presence of a thiol to produce N-substituted isoindoleswhich are generally both fluorescent and electrochemically However, many OPA derivatives degrade rapidly, necessitating carefully timed precolumn derivatization.3.4 The stable derivatives, for example those which employ tert-butyl mercaptan as the requisite thiol, are electroactive but exhibit negligible fluorescence.3 In all cases, peptides derivatized with OPA have been reported to show significantly less fluorescence than amino acids so derivatized.196P6
* Author to whom correspondence should be addressed.
+ Present address: Lilly Research Laboratories, Eli Lilly & Company, Pharmaceutical Sciences Division, Bldg. 82, Lilly Corporate Center, Indianapolis, IN 46285. Present address: Henri's Food Products, Milwaukee, WI 53204. I Present address: Texas A&M University, College Station,TX 77843. (1) Joseph, M. H.; Daviee, P. J. Chromatogr. 1983,277, 125-136. (2) Allison, L. A.; Mayer, G. S.; Shoup, R. E. Anal. Chem. 1984, 56, 1089-1096. (3) Lindroth, P.; Mopper, K. Anal. Chem. 1979,51, 1667-1674. (4) de Montigny, P.; Stobaugh, J. F.; Givens, R. S.; Carlson, R. G.; Srinivasachar, K.; Sternson, L. A,; Higuchi, T. Anal. Chem. 1987, 59, 1096-1101. (5) Joys, T. M.; Kim, H. Anal. Biochem. 1979,94, 371.
*
0003-2700/92/0364-1259$03.00/0
To circumvent the problems associated with OPA, naphthalene-2,3-dicarboxaldehyde(NDA) was designed and developed by de Montigny et ala4 This reagent reacts with primary amines in the presence of cyanide to produce l-cyanobenz[flisoindole (CBI) derivatives (Figure 1). Generally, these derivatives are more stable than their OPA counterparts and exhibit higher quantum efficiencies, particularly with pe~tides.49~As with OPA, the CBI derivatives are also electrochemically a ~ t i v e . ~ ~ ~ Most work utilizing OPA or NDA has made use of fluorescence detection. However, electrochemical detection has also been used and offers some advantages.19297f~ For example, compounds with multiple derivatized amine sites (e.g., lysine) show severe fluorescence quenching, but their electrochemical response is actually higher, allowing lower detection limita.7 Also, since electrochemicaldetection occurs at an electrode surface, the extremely small cell volumes necessary for microbore LC can be used without sacrifice in signal.298 Furthermore, voltammetric data can provide qualitative information that can be used to confirm peak identities.7 Finally, selectivity can be controlled by varying the electrode potential. Although a thorough oxidation mechanism has not been described, the electrochemical activity of OPA or CBI derivatives apparently arises from the isoindole ring formed in the derivatization reaction. Cyclic voltammetry of derivatized amines indicates that the oxidation of the isoindole is diffusion-controlled and chemically irreversible.2~7 The oxidation appears to be a one-electron process in nonaqueous solution,2J although a two-electron oxidation was proposed for OPA derivatives in aqueous/acetonitrile mixtures.2 Little has been reported regarding pH dependence of the oxidation, although the oxidation potential of OPA-derivatized glutathione was found to remain constant between pH 8 and 11.9 Because the isoindole structure is common to all OPA and CBI derivatives, one might expect the oxidation potentials of all the derivatives to be quite similar. However, there have been indications of differences in such oxidation potentials. For example, OPA derivatives of basic amino acids (arginine, histidine, lysine, tryptophan, and ornithine) were reported to be more readily oxidized than other OPA-amino acids.' In another report,2 the OPA derivative of tryptophan was found to be easier to oxidize than several other OPA derivatives, (6) Matuszewski, B. K.; Givens, R. S.; Srinivasachar, K.; Carlson, R. G.; Higuchi, T. Anal. Chem. 1987,59, 1102-1105. (7) Lunte, S. M.; Mohabbat, T.; Wong, 0. S.; Kuwana, T. Anal. Biochem. 1989, 178, 202-207. (8)Oates, M. D.; Jorgenson, J. W. Anal. Chem. 1989, 61, 432-435. (9)Buchberger, W.; Winsauer, K. Anal. Chim. Acta 1987,196, 251254. 0 1992 American Chemlcal Society
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ANALYTICAL CHEMISTRY, VOL. 84, NO. 11, JUNE 1, 1992 CN
CHO
CHO
RMIz
pH -pK,
CN N- R
Naphthalen.-Z,J-dlcarboxald.hyde (NW
Flgure 1. NDA derlvatiration reaction. Condltlons used are glven In the text.
and this difference was attributed to the inherent electrochemical activity of underivatized tryptophan. Finally, amino acids multiply derivatized by NDA (e.g., lysine, desmosine) were found to be more readily oxidized than those only singly derivatized.' However, there has been little work regarding the magnitudes of these differences in oxidation potentials among derivatized amino acids and no reported comparison of oxidation potentials of derivatized peptides. Such information is important in determining which derivatized amino acids and peptides are particularly easily oxidized and thus well-suited for selective electrochemical detection. In this paper, we report on electrochemical characteristics of CBI derivatives of amino acids and peptides. In particular, the effecta of pH on oxidation potentials are compared for CBI derivatives and phenolic compounds, and the ability to control detection selectivity by changing pH is demonstrated. Alao, differences in oxidation potentials (Ell2 values) of several derivatized amino acids and peptides are reported.
EXPERIMENTAL SECTION Apparatus. Chromatography was performed using either a Perkin-Elmer (Norwalk, CT) Series 10or Shimadzu (Baltimore, MD) LC-6A HPLC pump, Rheodyne (Cotati, CA) Model 7125 injection valve with either a 20- or 6-pL loop (see specific chromatographicconditionsbelow),and ODs-Hypersil(Keystone Scientific, State College, PA) column ( 5 pm, 150 X 4.6 mm). A Rainin (Woburn,MA) Rabbit peristaltic pump was used for postcolumn addition of buffers. Electrochemical detection was accomplished using either a Shimadzu L-ECD-6A amperometric detector, a Bioanalytical Systems (W. Lafayette, IN) CV-27 voltammograph (in conjunction with a current amplifier (PacificInstruments, Concord,CA)), or a Bioanalytical Systems LC-4A amperometric detector. A glassy carbon working electrode, stainless steel counter electrode, and Ag/AgCl reference electrode were used (Bioanalytical Systems). Cyclic voltammetry was performed using a Bioanalytical SystemsCV-27voltammograph,glassycarbon workingelectrode, platinum counter electrode, and Ag/AgCl reference electrode. Chemicals. All chemicals were used as received. All peptides were obtained from Sigma (St. Louis, MO) except for LArg-Gly-Gly which was obtained from Research Plus, Inc. (Bayonne,NJ). Naphthalene-2,3-dicarboxaldehyde(NDA)was generously provided by Oread Laboratories (Lawrence, KS). Water was purified by either a Milli-Q (Millipore,Bedford, MA) or Nanopure (Sybron-Barnstead, Boston, MA) system. Acetonitrile was HPLC grade. All other chemicals are commonly available and were obtained from a variety of sources. Stock Solutions. Borate buffers (0.025-0.10 M; pH 9.5-9.9) were prepared by dissolvingboric acid in dilute aqueous sodium hydroxide and adding sodium hydroxide until the desired pH was reached. Phosphate buffer (0.050 M) of pH 2.0 for postcolumn pH adjustment was made by diluting 85% phosphoric acid and adjusting the pH with sodium hydroxide. Other phosphate buffers (0.050 or 0.010 M; pH 7.0 or 7.5-7.6) were made by dissolving potassium dihydrogen phosphate in water and adding sodium hydroxide until the desired pH was obtained. NDA solutions (5-6 mM) were prepared in acetonitrile, protected from light, and stored at 5 "C. Amino acid and peptide solutions (0.3-1.0 mM) were prepared in water and stored at 5 "C. Potassium cyanide (10 mM), phenol (0.45 mM), and resorcinol (0.55 mM) were prepared in water and stored at ambient temperature. Solutions could generally be used for several weeks and were replaced when evidence of degradation became apparent (either visually or chromatographically).
Mobile Phases. All mobile phases, except as noted below, consisted of a mixture of 0.050M phosphate buffer (pH 7.5-7.6) and acetonitrile. The percentage (by volume) of acetonitrile ranged from 20% to 45% (specific conditions given below), depending on the retention of the analytes. The conductivity of the mobile phase containing 45% acetonitrile was measured and found to be as high as that of 0.025 M NaCl; therefore, it was deemed unnecessary to add supporting electrolyte to the acetonitrile. In those cases involving a postcolumn pH adjustment, the HPLC mobile phase was more dilute: 0.010 M phosphate (pH 7.5), 24% acetonitrile. Just prior to reaching the electrochemical detector, the column effluent was mixed with a stream of 0.050 M phosphate buffer of pH 2.0 or 7.5 (both containing 20% acetonitrile),pumpedat l.OmL/min bythe peristaltic pump. Mobile phases were filtered and degassed prior to use. A flow rate of 1.0 mL/min was used throughout this work. Derivatization and Sample Treatment. For cyclic voltammetry experiments and chromatograms involving postcolumn pH adjustment (done concurrently), the following derivatization conditions were used: to 500 pL of 0.10 M borate (pH 9.5) were added 500 pL of 0.5 mM amino acid (glycine,glutamic acid, or arginine), 20 pL of 10 mM KCN, and finally 20 pL of 5 mM NDA. The mixture was allowed to react at room temperature for at least 1 h. The reacted mixture was placed on a Sep-Pak (Waters; Millford, MA) C-18 cartridge and rinsed with approximately 2 mL of 5 % acetonitrile-95% water. (Note: the entire 2 mL of the 5% acetonitrile rinse could not be used for the derivatized glutamic acid because of its weak retention on the cartridge; rinsing with pure water would have been preferable). This removed the excess borate, amino acid, and KCN, thereby allowing for lower background currents and simpler pH adjustment. For cyclic voltammetryexperiments,the derivatizedamino acid was eluted with 1.4 mL of 90% acetonitrilelo% water into the electrochemical cell and diluted with 4.0 mL of 0.050 M phosphate (pH 3.1, 7.5, or 12.9). For HPLC experiments, the derivatized amino acid was eluted form the cartridge with 1mL of 90% acetonitrile-10% water and mixed with 1 mL of 0.010 M phosphate (pH 7.5) so that it would more nearly resemble the composition of the HPLC mobile phase. The chromatographic sample consisted of a mixture of 500 pL of each of the three derivatized amino acids, treated as above, 200 pL of 0.45 mM phenol, and 60 p L of 0.55 mM resorcinol. For all other work, conditions involving excess NDA and cyanide were used. These conditions varied somewhat, but the followingwas typical for amino acids: to 1.0 mL of borate buffer (pH 9.5-9.9) were added 40-pL aliquots of 0.3 mM amino acid(@, 200 pL of 10 mM potassium cyanide, and 200 pL of 5 mM NDA. For peptides or mixtures of amino acids and peptides, the derivatization conditions were the same except that phosphate buffer (pH 7.0 for all except di-, tri-, and tetraglycine and ArgGly-Gly, for which the pH was 7.5-7.6) was used in place of the borate and twice as much potassium cyanide was used. The lower pH and increased amount of potassium cyanide were used to minimize a cyclization side product which can form when derivatizing peptides.1° For each sample, NDA and cyanide were in excess by at least a factor of 10 (generally about 40) relative to the total amino acid and/or peptide concentration. The solutions were mixed and stored in the dark at ambient temperature for several minutes and then refrigerated. Although the derivatization reaction is generally complete in 5 min under optimum conditions, the samples were not used until after at least 60 min to ensure complete reaction. This was especially important for samples containing both amino acids and peptides, since the rate of derivatization for amino acids is decreased at the lower pH used for peptides.'JO All samples were used on the same day as derivatized. A blank (i.e., containing all derivatizing reagents with no amino acid or peptide) was prepared and chromatographed with each sample. Hydrodynamic Voltammopams. Hydrodynamic voltammograms were constructed by obtaining multiple chromatograms of a given sample at 50-mV increments in electrode potential. Signals were normalized to the maximum attained for each analyte and plotted vs applied potential. Half-wavepotentials (El,, values) were measured from these plots. (10) de Montigny, P.; Riley, C. M.; Sternson, L. A.; Stobaugh, J. F.J . Pharm. Biomed. Anal. 1990,8,419-430.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 11, JUNE 1, 1992
1261
3
I M
F-
E (mV vs. Ag/AgCi)
I Flguro 2. Cyclic voltammograms of CBIglycine: 25% acetonltrlle; 75% 0.050 M phosphate, pH 7.5.
To compensate for variations in the reference electrode potential, solution conductivity, and other contributors to apparent potential shifts, half-wave potentials were measured of CBI-glycine. CBI-glycineor another with reference to the El/* amine whose half-wavepotentialvs that of CBI-glycinewas known was included in each analyte mixture as a voltammetric internal standard. Half-wave potentials are thus reported with respect to CBI-glycine and not as independent values.
RESULTS AND DISCUSSION Cyclic Voltammetry. Cyclic voltammograms for CBIglycine a t four different scan rates are shown in Figure 2. Each scan was initiated a t 0 mV and reversed at +900 mV. An oxidation wave is clearly seen at approximately +570 mV. No distinct reduction wave is seen, although there is a faint indication of one a t approximately +510 mV (this feature was not seen in all voltammograms of CBI-glycine, however, and did not become prominent at higher scan rates). Other scans (not shown) were extended as far negative as -900 mV and showed no distinct reduction wave for the oxidized product. In addition, the oxidation potential was largely independent of scan rate. These results indicate that the oxidationof CBI-glycine is chemically irreversible and involves facile electron transfer. Finally, cyclic voltammograms of CBI-glycine obtained a t pH 3.1 and 12.9 were virtually identical to those shown in Figure 2 (obtained a t pH 7.9, indicating that the oxidation mechanism involves no gain or loss of H+. It seems likely, therefore, that the oxidation involvesthe loss of an electron from the heterocyclic nitrogen forming a resonance-stabilized radical cation, which goes on to react further and is thus unavailable for reduction. Cyclic voltammograms of CBI-glutamic acid and CBI-arginine were also obtained at pH 3.1 and 12.9 and gave similar results. The peak potential of CBI-glutamic acid remained virtually constant at these two pH values, while that of CBI-arginine shifted negatively by 80mV in going to the more alkaline pH. This small shift was presumably due to the acid-base characteristics of the arginine side chain (deprotonation possibly leading to greater stabilization of the radical cation formed in the oxidation)and is far less than would be expected for an oxidation mechanism directly involving H+. p H Control of Selectivity. Since many organic compounds (e.g., phenols, aromatic amines, thiols) which undergo electrochemical oxidation do so with the loss of H+, their oxidation potentials are highly dependent on pH (59 mV per pH unit if the number of hydrogen ions lost equals the number of electrons lost). The fact that the oxidation of the isoindole ring in CBI derivatives is virtually pH-independent suggested that pH could be used to increase the selectivity of their detection. By operating at a low pH, the oxidation potentials of many possible interferences could be shifted to more positive values, while the oxidation potentials of the CBI derivatives would remain unchanged. Thus, it should be possible to detect the CBI derivatives at a potential where
0
4
8
12
Time (min.)
Figure 3. Chromatograms of CBIglutamlc acM (I), resorcinol (2). phenol (3), CBIglyclne (4), and CBI-arglnlne (5) detected at pH 7.5. Mobile phase: 0.010 M phosphate, pH 7.5; 24% acetonltrlk postcolumn addltion of 0.050 M phosphate, pH 7.5. Electrode potentlals: (A) +750 mV; (B) +550 mV. 2
A
3
1
I
I
0
4
I
a
I
12
Time (mln.)
Figure 4. Chromatograms of CBIglutamic acM (l), resorcinol (2), phenol (3),CBI-glyclne (4), and CBI-arglnlne (5) detected at pH 2.0. Mobile phase: 0.010 M phosphate, pH 7.5; 24% acetonltrlk postcolumn addltlon of 0.050 M phosphate, pH 2.0. Electrode potentlals: (A) +950 mV; (B) +750 mV.
compounds which interfere at higher pH values would no longer be detected. To demonstrate this phenomenon without confusing the results by changes in chromatographic retention, a postcolumn pH adjustment was used. A sample containing phenol, resorcinol, and CBI derivatives of glutamic acid, glycine, and arginine was separated using a dilute (0.010 M) pH 7.5 phosphate buffer. A postcolumn pH adjustment was then made by mixing the eluent with 0.050 M phosphate of pH 2.0 or pH 7.5 prior to reaching the electrochemical cell. Multiple injections of the same sample were made a t various electrode potentials. Resulting chromatograms are shown in Figures 3 and 4. Figures 3A and B show the results obtained at pH 7.5 a t +750 and +550 mV, respectively. All five compounds were readily detected a t +750 mV or higher potentials a t this pH. At +550 mV, the peaks for CBI-glutamic acid and phenol were
1262
ANALYTICAL CHEMISTRY, VOL. 64, NO. 11, JUNE 1, 1992
-
Table I. Half-Wave Potentials for Derivatized Amino Acids vs CBI-Glycine (&2 +630 m V vs Ag/AgCl)
E1 zvs
amino acid
CBI-dly, m V
Lysa Arg TrP Met Leu
-105 -65 -65
Ile Thr
His Ala a
-150
-50 -40 -40
-35 -35
amino
E1 2
~ s
acid
CBI-dly, mV
Ser Val TYr
-30 -30 -10 -5 -5
Gln
ASil GlY Phe ASP
Glu
T
8.4
nA
1
0
0 +15 +65
Lysyl side chain amine group also derivatized.
virtually non-existent, whereas resorcinol, CBI-glycine, and CBI-arginine were still detected. Note that it is impossible to obtain a chromatogram of the CBI derivatives alone a t this pH by adjusting the electrode potential. Resorcinol and phenol have oxidation potentials similar to those of the CBI derivatives; in fact, resorcinol is actually easier to oxidize than CBI-glutamic acid at this pH. Figures 4A and B show the results for the same sample at pH 2.0. Note that all five compounds were detected at +950 mV (Figure 4A). However, a t this lower pH, the oxidation potentials of the phenolic compounds were shifted far positive of the CBI derivatives (phenol was not all oxidized even at +950 mV, as noted by its smaller peak compared to Figure 3A). At +750 mV, neither phenol nor resorcinolwas detected, and a clean chromatogram of the CBI derivatives was readily obtained (Figure 4B). Thus, the selectivity of electrochemical detection of CBI derivatives is enhanced by operating at a lower pH. Of course, in most cases a postcolumn addition of buffer would not be used; rather, this utility of a lower pH should be considered along with retention factors in the selection of a chromatographic mobile phase. In cases where satisfactory separations can be achieved using an acidicmobile phase, the enhanced selectivity of detection of CBI derivatives over compounds with pH-dependent oxidation potentials should prove beneficial. Presumably, oxidations of OPA derivatives and other similar isoindoles are also largely independent of pH, and thus they could also be detected most selectively in complex matrices by using a low pH. OxidationPotentials. Amino Acids. Hydrodynamic voltammograms were constructed for 18 CBI-amino acids to compare their oxidationpotentials. Half-wavepotentials were measured relative to CBI-glycine to account for any potential shifts due to instrumental factors. The resulting E1p values are listed in Table I in order of increasing difficulty of oxidation. The uncertainty of each Ell2 value is approximately f15 mV because of the graphical method of measurement. Many of the CBI-amino acids had similar halfwave potentials; however, some significant differences were apparent (Table I). The derivatives of lysine and arginine were unusually easy to oxidize, while those of aspartic and glutamic acids were the most difficult. Thus it would be simple, for example, to distinguish between these CBI-amino acids based on their oxidation potentials or to selectively detect the derivatives of lysine or arginine in the presence of other CBI-amino acids by adjusting the detector potential. Although it is difficult to discern definite trends among all the amino acids, those with basic side chains appear easier to oxidize than those with acidic side chains. This coincides with earlier observations regarding OPA.1 Lysine is unique in that it is doubly derivatized, and its low half-wave potential is, in part at least, due to this double derivatizationy rather than the presence of a basic side chain as may be the case for arginine. Also of note is the differenceof about 50 mV between the half-wave potentials of CBI-aspartic and CBI-glutamic
0
5
10
15
20
25
Time (mln.)
Figure 5. Chromatogram of derivatized enkephalin fragments (20 pL injected: 100 pmol of each). Electrode potential: +700 mV. Mobile phase: 0.050 M phosphate, pH 7.5; 29% acetonitrile.
acids, despite the similarity of their structures. Finally, it should be noted that the voltammograms for CBI-tryptophan and (CBI)2-lysinewere were more drawn out than those of the other derivatized amino acids, making their half-wave values less certain. This was not simply an artifact of uncompensated solution resistance because the voltammograms of other derivatized amino acids obtained under the same chromatographic conditions did not exhibit the same effect. It is likely that these extended voltammetric waves resulted from the presence of two electroactive moieties in CBI-tryptophan and (CB1)z-lysinewhich differed slightly in oxidation potential, although not enough to yield two distinct waves. CBI-tyrosine also contains two such sites, but did not show this effect, presumably because the oxidation potentials of the two sites were too similar. The response factors of CBItryptophan and CBI-tyrosine were larger than those of other derivatized amino acids, indicating oxidation at both electroactive sites in each case. For example, the response for CBI-tyrosine was about 37% higher than that for CBI-glycine. Peptides. Hydrodynamic voltammograms of several CBIpeptides were also constructed, and half-wave potentials were measured relative to CBI-glycine. The effect of simply increasing peptide chain length was briefly examined by comparing derivatives of di-, tri-, and tetraglycine to CBIGly. Only a slight increase in half-wave potential was seen as the chain length was increased. (E112 values compared to CBI-glycinewere +15, +20, and +35 mV, respectively.) The present study included enkephalins and enkephalin fragments because of this research group’s experience and interest in these biologically significant compounds.lOJ1 Methionineand leucine-enkephalin both contain tyrosine and so can be detected electrochemically even without derivatization;12-14 however, derivatization allows simultaneous detection of fragments not containing tyrosine. Figure 5 is a chromatogram of four derivatized enkephalin fragments, including one lacking tyrosine. Using the conditions indicated, derivatives (11) Mifune,M.;Krehbiel,D.K.;Stobaugh, J. F.;Riley,C.M. J.Chromatogr. 1989, 496, 55-70. (12) Sauter, A.; Frick, W. J. Chromatogr. 1984,297, 215-223. (13) Spatola, A. F.;Benovitz, D. E. J.Chromatogr. 1985,327,165-171. (14) Fleming, L. H.; Reynolds, N. C., Jr. J. Chromatogr. 1988, 431, 65-76.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 11, JUNE 1, 1992 1263 Table 11. Half-Wave Potentials of Derivatized Enkephalin Frawents peptide E112 vs CBI-Gly, mV Tyr-Gly-Gly-Phe-Met(ME)" -+15* Tyr-Gly-Gly-Phe-Leu(LE)" -+15*
Table 111. Half-Wave Potentials of Other Derivatized PeDtides peptide Ell2 vs CBI-Gly, mV ~
Tyr-Gly-Gly
+15
Tyr-Gly Gly-Gly-Phe Gly-Gly-Phe-Leu
+20
Arg-Gly-Gly Gly-Gly-Arg Gly-Gly-Tyr-A r g b Leu-Arg
+15
Leu-Ala
-70
a ME = Met-enkephalin; LE = Leu-enkephalin. Estimated because voltammogram did not reach a level plateau.
Gly-His-LysC
~~
+30 --loo0 -160, +45 +305
--+w -180
Approximate because of less direct comparison with CBI-Gly. bTwo voltammetric waves. c Lysyl side chain amine group also derivatized.
1*2
I
Y
J
L
POTENTIAL (HV)
Flgurr 6. Hydrodynamic voltammogramsof CBI-Oly-His-LysCBI (m), CBIQiyQly-Tyr-Arg (O), and CBIOly-Oly-Phe-Leu(A). Mobile phases: 0.050 M phosphate, pH 7.5; 32-35% acetonitrile.
of Met- and Leu-enkephalin and Gly-Gly-Phe-Leu had retention times of 46-60 min but were readily eluted more rapidly by using higher concentrations of acetonitrile in the mobile phase. Hydrodynamic voltammograms of the derivatized enkephalins and enkephalin fragments were constructed and halfwave potentials measured. The resuling Ell2 values are listed in Table 11. The hydrodynamic voltammograms for derivatized Met- and Leu-enkephalin did not give diffusion-limited plateau regions, so their Ell2 values are more approximate. The half-wave potentials of all the fragments examined were similar except for CBI-Gly-Gly-Phe-Leu,which was strikingly easier to oxidize than the others, including the very similar peptide CBI-Gly-Gly-Phe. A few other peptides were also examined. Most of the ones chosen contained either arginine or lysine because of the significant effect these amino acids had on the oxidation potentials of their CBI derivatives (Table I). Hydrodynamic voltammograms for the two most easily oxidized-CBI-GlyHis-Lys-CBI (doubly derivatized) and CBI-Gly-Gly-TyrArg-are shown in Figure 6, along with CBI-Gly-Gly-PheLeu for comparison purposes. Both peptides were markedly easier to oxidize than CBI-Gly-Gly-Phe-Leu, which was the most easily oxidized of the enkephalin fragments examined. Interestingly, CBI-Gly-Gly-Tyr-Argyielded two waves. Presumably, one was due to the isoindole ring and one was due to tyrosine. Underivatized Gly-Gly-Tyr-Arg gave an E112 value similar to the more positive of these two waves;therefore, it seems likely that the more positive wave resulted from oxidation of tyrosine and the less positive from the isoindole ring. This assignment is not certain, however, since derivatization of the peptide may also alter the half-wave potential of the tyrosine. The other tyrosine-containing peptides examined (Le., the enkephalins and some fragments) did not show two distinct waves, probably because of similar oxidation
potentials of the two electroactive portions. The Ell2 values for the peptides examined are listed in Table 111. As noted above, CBI-Gly-His-Lys-CBI and CBIGly-Gly-m-Arg are dramatically easier to oxidize than many other derivatized amino acids or peptides. Among other observations, it seems clear that the presence of arginine in a peptide can significantly affect the oxidation potential of the CBI derivative, but the effect is dependent on the position of arginine in the sequence, as well aa other amino acids present. Compare, for example, the results obtained for CBIArg, CBI-Arg-Gly-Gly, CBI-Gly-Gly-Arg, and CBI-Gly-GlyGly (see Tables I and I11and text). CBI-Arg-Gly-Glyhad an oxidation potential similar to CBI-Gly-Gly-Gly,whereas CBIGly-Gly-Arg was much easier to oxidize. Also, CBI-Leu-Ala and CBI-Leu-Arg were both harder to oxidize than most others, but CBI-Leu-Arg waa the easier to oxidize of the two. These limited data suggest that the presence of arginine at the carboxyl terminus may lower the oxidation potential, although such a conclusion must be considered tentative and empirical. Finally, the low potential a t which CBI-Gly-HisLye-CBI was oxidized supports the finding reported earlier' that multiply derivatized compounds are more readily oxidized than those only singly derivatized. More data are required to know whether the conclusions reached here hold for large numbers of compounds. In general, the differences in oxidation potentials observed among derivatized amino acids and peptides are likely to be due to the presence of electron-donating or withdrawing groups near the heterocyclic nitrogen ,thereby stabilizing or destabilizing the radical cation formed. However, without knowing the exact conformation of the derivatives at the electrode/solution interface, it is difficult to explain the observed differences in oxidation potentials. It is nevertheless clear, based on the observations reported here, that the oxidation potentials of CBI derivatives vary significantly and thus both qualitative information and enhanced selectivity can be obtained by control of the electrode potential.
ACKNOWLEDGMENT We acknowledge with gratitude the donation of an LC-4A amperometric detector by Bioanalytical Systems, Inc. In addition, we are grateful to Oread Laboratories for donation of NDA. We thank Cheryl El-Hilali for verifying some of the voltammetric data. We acknowledge funding by an NSF Macro-ROA Grant, the Kansas Technology Enterprise Corporation, and the National Institute on Drug Abuse (DA04740). This work was presented in part a t the Fourteenth International Symposium on Column Liquid Chromatography, Boston, MA, May 20-25,1990, RECEIVED for review November 8, 1991. Accepted March 17, 1992.
