Q00
AMI.
Ct”. 1902, 64. 960-964
barrier to decreasing sample sizes is the intensity of the precursor ion relative to the chemical background. Therefore, any technique that increases this ratio is expected to provided a similar gain in overall sensitivity, and several different possibilities can be suggested. Increasing the resolution of MS 1 may allow separation of the sample from the background, but with the reduced precursor ion intensity MS/MS analysis may not be possible. Alternatively, choice of the proper gas may allow reaction-induced dissociations’* (RID) to fragment the analyte ion selectively while neutralizing the interfering FAB background. Another approach would be the use of an ionization technique that provides a higher signal-to-background ratio than FAB, as demonstrated in this study. Regardless of the methodology, reducing the background interference yields a significant decrease in sample requirements,and permits more extensive use of the increased sensitivity provided by array detection.
ACKNOWLEDGMENT We thank J. A. Hill, J. E. Biller, and K. Biemann for providing information from their manuscript before publication and R. K. Boyd for helpful comments with this work.
(4) Hill, J. A,; Biller, J. E.; Martin, S. A.; Blemann, K.; Yoshldome, K.; Sato, K. Int. J . Mass Spectrom. Ion Processes 1888, 92. 211. (5) Cottrell, J. S.; Evans, S. Anal. Chem. 1987, 59, 1990. (6) Evans, S. In Methods in E~Z~mObgyY; McCloskey, J. A., Ed.; Academ ic Press: San Dbgo, CA 1990; Vol. 193. (7) &oss. M. L. In M e W s in €nzyfno&gy; McCloskey, J. A,, Ed.; Academic Press: Sen Dlego. CA, 1990 Vol. 193. (8) HIII, J. A.; Biller, J. E.; Biemann, K. Inf. J . Mass Spectrom. Ion Processes, in press. (9) Falick, A. M.; Medzihradszky, K. F.; Walls, F. C. f?apMC”un. Mass Specfrom. 1990, 4 . 318. (10) Bryant, D. K.; Orlando, R. RapidCommun. Mass. Spectrom. 1881, 5 . 124. (11) Wails, F. C.; Baldwin, M. A.; Fallck, A. M.; Glbson, B. W.; Kaur, S.; Maltby. D. A.; GllleceCastro, 8. L.; Medzihradszky, K. F.; Evans, S.; Burlingame, A. L. I n Biological Mass spectrometry; Burlingame, A. L., McCloskey, J. A., Eds.; Elsevler: Amsterdam, 1990. (12) Caprioli, R. M. Bhxhemlstry 1988, 27, 513. (13) Bean, M. F.; Car, S. A.; Thorne, G. C.; Reilly, M. H.; Qaskell. S. J. Anal. Chem. 1881. 63, 1473. (14) Martin, S. A.; Johnson, R. S.; Costello, C. E.; Blemann, K. In Analysk Of fepfMes and Proteins; McNeal, C. J., Ed.; Wiley: Chichester, England, 1988. (15) Johnson, R. S.; Martin, S. A.; Blemann, K. Int. J . Mass Spectrom. Ion Processes 1888, 86. 137. (16) Poulter, L.; Taylor, L. C. E. Int. J . Mass Spectrom. Ion ProCeJses 1888. 91, 183. (17) Scoble, H. A.; Martin, S. A.; Biemann, K. Biochem. J . 1987, 245, 821. (18) Orlando, R.; Fenselau, C.; Cotter, R. J. J . Am. Soc. Mass Spectrom. 1881, 2 , 189.
REFERENCES (1) Busch, K. L.; Cooks. R. 0. Anal. Chem. 1883, 55, 38A. (2) Yost, R. A,; Enke. C. 0. J . Am. Chem. Soc.1978. 100, 2274. (3) Blemann. K. Anal. Chem. 1886, 58, 1289A.
RECEIVED for review September 30,1991. Accepted February 13, 1992.
TECHNICAL NOTES Liquid Membrane Electrode for Guanosine Nucleotides Using a Cytosine-Pendant Triamine Host as the Sensory Element Koji Tohda, Masahiro Tange, Kazunori Odashima, and Yoshio Umezawa* Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo 060, Japan Hiroyuki Furuta and Jonathan L. Sessler Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, Texas 78712 INTRODUCTION The recognition and complexation of target chemical substances by synthetic host molecules and consequent signal transduction involving changes in membrane potential constitute an important approach to chemical sensing. A number of ion-selective electrodes (ISEs) based on polymer matrix liquid membranes have been investigated. Many of these display high selectivity for particular target substances and are now commercially available.’* However, most of the polymer matrix liquid membrane ISEs developed so far have focused on the recognition of alkali and alkaline earth metal cations by the use of natural and synthetic cyclic and acyclic neutral ionophores as sensory elements.’~~ Recently, a number of anion-selective electrodes have been developed by the use of alkyltin compounds? vitamin Blz and metalloporphyrin derivative^,'^'^ diphosphonium16J7and diammonium cations,ls and macrocyclic polyamines.’*21 Whereas the former two types of electrodes are based on reversible coordination of the anionic guests to the vacant coordination site(s) of the central metal ions, the latter two are based on electrostatic interaction between the anionic guests and the cationic hosts. Of these, the macrocyclic polyamine electrodes are characteristic in that the hosts
function as anion receptors by protonation at the membrane surface.1sa One of the remarkable features of the macrocyclic polyamine electrodes is their ability to discriminate among the adenosine nucleotides as a function of the number of negative charges. As a result, by far the strongest potentiometric response is observed for ATP“ as compared to ADP3and AMP2-.19 However, since these protonated macrocyclic polyamines appear to bind mainly to the phosphate group of nucleotides, it would be difficult to effect discrimination among similarly charged nucleotides bearing different kind of bases. Recently, a cytosine-pendant triamine host (la) was developed as a new type of receptor for the recognition and binding of guanosine 5’-monopho~phate.~~ This host has ditopic recognition sites for guanosine nucleotides, i.e., the cytosine moiety for complementary base pairing with the guanine base and the (protonated) triamine moiety for electrostatic binding with the phosphate group. The formation of a stable complex with the above nucleotide was observed in dimethyl sulfoxide (K,= 2.6 X lo4M-l at 23 “C for the 1:l complex25). In this paper, we report potentiometric response properties for organic and inorganic anions of a polymer matrix liquid membrane electrode using as a sensory element host
0003-2700/92/0364-0960$03.00/00 1992 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 64, NO. 8, APRIL 15, 1992
861
100 -
HvH I
la: Ib:
N\R
R =c2H5 R = (CH&.CH3
(CH2)15CH3
2
N5
OAN 0.1-N N
3
sE
Y
O-loo: -200-
-300-
2
4
6
8
1 0 1 2
PH
Figure 2. Effect of pH on the membrane potential. (1) Electrode 1 based on lipophilic cytoskrependent trlamine lb. (2) Electrode 2 based on lipophilic macrocyclic polyamine 2. (3) Electrode 3 based on Ilpophilic cytosine derivative 3. (4) Electrode 4 based on lipophlllc cytldlne derhrathre 4. The pH of the sample solution containing M H,SO, was adjusted by addltlon of NaOH.
yield after purification. White powder: mp 218-219 O C (chloroform); 'H NMR (400 MHz, CDC13-CD30D(lOl), ppm downFigure 1. Structures of the host compounds used in the present study. field of tetramethylsilane) 6 0.88 (t, J = 6.8 Hz, 3 H, NCH2CH2(CH2)&H3), 1.20-1.38 (m, 30 H, NCH2CH2(CH2)&H3), 1.69 (m, 2 H, NCH2CH2),3.76 (t, J = 6.4 Hz, 2 H, lb, a lipophilic derivative of la. The most interesting feature NCHZCHZ), 5.79 (d, J = 7.3 Hz, 1 H, C-5 CH), 7.32 (d, J = 7.3 of this electrode is the potentiometric discrimination between Hz, 1H, C-6 CH); IR (KBr) 3348,1665,and 1620cm-'; MS m / z guanosine and adenosine nucleotides. To the best of our 364 (M+ + 1). Anal. Calcd for CZ2H4'N30:C, 72.68; HI 11.37; knowledge, this is the first example of such potentiometric N, 11.56. Found: C, 72.81; H, 11.45; N, 11.49. discrimination based on the use of complementary base Electrode Preparation and EMF Measurement. PVC pairing, an interaction that, of course, plays a critical role in matrix liquid membrane electrodes containing hosts lb, 2-4 the duplication and transcription of DNA and RNA in living (electrodes 1-4, respectively) were prepared according to the systems. previously reported pro~edure.'~ The membrane composition was 2 wt % host compound, 70 wt % DBS as a membrane solvent, EXPERIMENTAL SECTION and 28 wt % PVC as a polymer matrix. From the membrane thus prepared (ca. 0.1-mm thickness), a circle of ca. 7-mm diameter Reagents. All reagents were of the highest grade commercidy was cut out and mounted on an electrode body (Model IS-561, available and used without further purification. Guanosine 5'Philips Electronic Instruments Co., Mahwah, NJ). A lo-' M triphosphate (5'-GTP; catalog no. G-5881), guanosine 5'-monopotassium chloride solution containing M tetra-n-pentylphosphate (5'-GMP; G-8377),guanosine 2'-monophosphate (2'ammonium chloride (TPA+Cl-)(1.5 mL) was used as an internal GMP; G-7752),adenosine 5'4riphosphate (5'-ATP; A-5394), and solution to obtain a stable membrane potential. The addition adenosine 5'-monophosphate @'-AMP; A-1752) were purchased of TPA+ ion in this case is justified for the reason described from Sigma Chemical Co. (St. Louis, MO). Dibutyl sebacate earlier.1g The reference electrodewas a double-junctiontype based (DBS 10107-00)was purchased from Kanto Chemical Co. (Tokyo, on an Ag/AgCl electrode (Model HS-305DS, TOA Electronics Japan). N-(2-Hydroxyethyl)piperazine-N'-2-ethanesulfonicacid Ltd., Tokyo, Japan). Thus, the electrode cells for the emf (HEPES; 342-01375) was purchased from Dojindo Laboratories measurements were as follows: Ag/AgC1(3 M KClIO.1 M (Kumamoto, Japan). Poly(viny1 chloride) (PVC; nav= 1100; K2S041samplesolutionlmembrane(0.1M KC1 M TPA+223-00255),sodium salicylate (195-03145),and sodium phosphate Cl-(AgCl/Ag. (dibasic;Na2HP0,; 197-02865)were purchased from Wako Pure All of the potentiometric measurements were carried out at Chemical Industries (Osaka, Japan). Other inorganicanions were room temperature (ca. 20 O C ) . The potentiometric selectivity also purchased as sodium salts. All solutionswere prepared with coefficients (qt) were calculated for the series of mono- and milli-Q water with a resistance greater than 17.5 Mil. dianions which gave appreciable potentiometric responses. The Host Compounds. The structures of the host compounds used calculation was made at a concentration of 1.0 X M with in the present study are shown in Figure 1. Lipophilic macrocyclic polyamine 2 (15-hexadecyl-1,4,7,10,13-pentaazacyclohexade~ane~~salicylate-or 5'-GMP2- as the primary ions for the series of monoand dianions, respectively. The separate solution methodn" was was kindly provided by Eiichi Kimura, Hiroshima University used with the Nicolsky-Eisenman equation, although the primary School of Medicine. The synthesis of lipophilic cytidine derivative and interfering anions tasted did not give calibration curves with 4 (2',3',5'-tri4-O-(triisopropylsilyl)cytidine)is described elsewhere." ideal linearity and Nernstian slope (see ref 28 for the relevant The lipophilic cytosine-pendant triamine host l b (4-aminodiscussion). The response time t(At, aE)estimated in the present 1-[4-[Nfl-bis[2-(Nfl-dihexylamino)ethyl]amino]butyl] -2(1H)study is, as defined in previous articles,2g31the time at which the pyrimidinone) was synthesized in accord with the general prodifferential quotient (aE/At)of the potential-time curve becomes cedures given previously for host la.% Colorless caramel: 'H smaller than a prechosen value ( A E / A t < 1.0 or aE < 1.0 mV NMR (400 MHz, CDC13-CD30D(2:1), ppm downfield of tetramethylsiie) S 0.91 (br t, J = 6.4 H z , 12 H, NCH2CH2(CH2)3CH3), within At = 1.0 min in the present study). 1.35 (24 H, br, NCH2CH2(CH2)3CH3), 1.46 (m, 2 H, cytosy1-NRESULTS AND DISCUSSION (CH2)2CH2),1.69 (br m, 10 H, cytosyl-NCH2CH2 and NCHZCH2(CH2)&HJ, 2.57 (t, J = 7.3 Hz, 2 H, cyt0syl-NpH Profile of the Electrodes. The pH profiles of elec(CH2),CHZN),2.90 (t, J = 6.8 Hz,4 H, NCH2CH2N),3.09 (m, 8 trodes 1-4 based on hosta lb, 2-4, respectively, are shown in H, NCHZCH&CHZ)&H3), 3.20 (t,J = 6.8 Hz, 4 H, NCHZCHZN), Figure 2. The membrane potential of electrode 2 increased 3.78 (t, J 6.8 Hz, 2 H, cytosyl-NCHZ),5.88 (d, J 7.2 Hz, 1 with a decrease in pH from 11to 6 (curve 2). This is due to H, C-5 CH), 7.66 (d, J = 7.2 Hz, 1H, C-6 CH); IR (CHC13) 3346, successive protonation of the macrocyclic polyamine a t the 3212,1674, and 1605cm-'; HRMS m / z for C36H73N60 (M+ + 1) membrane surface, increasing its capability to form host-guest calcd 605.5845, obsd 605.5837. complexes with anionic guests.'"21 Electrode 3 showed a The lipophilic alkyl derivative of cytosine, 3 (4-amino-1-octapH-dependent potential increase below pH 7 (curve 3), redecyl-2(lH)-pyrimidinone)was prepared by alkylation of cytosine with 1-iodooctadecaneand NaH in DMF and obtained in 18% flecting protonation of the cytosine amino group. Electrode
3
4
+
B62
ANALYTICAL CHEMISTRY, VOL. 64, NO. 8, APRIL 15, 1992
-20 A-A----_
A-A
Flgwe 3. Potentiometricresponse curves for 5'oMP obtained at three different pH condkbns (pH 4.0, 6.6 and 8.0) for (a) electrode 1 based on cytosine-pendant triamine l b and (b) electrode 2 based on macrocyclic polyamine 2. The buffer solutions used were 0.1 M AcONaAcOH for pH 4.0, 0.1 M HEPES-NaOH for pH 6.6, and 0.1 M TrisH~SO,for p~ 8.0.
=
I -90
-110
-6
I
-5
,
I
I
I
-3
-4
Y ,
I
-2
c'oi 5.IICylali I
1
log c Figure 5. Potentiometric response cuves for 5 ' W and other organic and inorganic anions measured at pH 6.6 (0.1 M HEPES-NaOH) for electrode 1 based on cytosine-pendant triamine lb. The guest anbns tested were salicylate- (o),C04- ( O ) ,5'OEvlP- (e),Hpo," SCN(A),SO4'- (U),Br- (O),NO,- (A),and Ci- (0).
m,
Flgure 4. Potentlal vs time curves for the potentlometric responses by electrode 1 based on cytosine-pendant triamine 1b. (a) 5'oMP and (b) HP042-as the guests (0 or 10 mM). 0.1 M HEPES-NaOH buffer (pH 6.8).
4 also showed similar potential increase but starting from the alkaline region (pH = 10). With electrode 1 based on the cytosine-pendant triamine lb, a characteristic pH profile was observed. In the alkaline region, the membrane potential increased with a decrease in pH from 11 to 8 in a manner similar to that of electrode 2. However, in the region below pH 8, the potential decreased with an increase in pH and then became constant in the acidic region below pH 6. The reason for such a pH profile is not clear, but a possible explanation might be the formation of a highly lipophilic dimeric complex by hydrogen bonding between the partially protonated ammonium moiety and the cytosine residue, resulting in an inhibition of charge separation with the hydrophilic counter anion. Potentiometric Response to 5'-GMP.Upon addition of 5'-GMP as a guest, a decrease in the membrane potential (anionic potentiometric response) was observed for electrodes 1and 2 at pH 6.6. Such a potentiometric response was not observed for electrodes 3 and 4. Figures 3a and 3b show the potentiometric responses of electrodes 1and 2, respectively, for 5'-GMP at three different pH conditions examined, Le., pH 4.0,6.6, and 8.0. The potentiometricresponses of electrode 2 to 5'-GMP at pH 4.0 and 6.6 were greater than that at pH 8.0 (Figure 3b). These results suggest that protonation of the macrocyclic polyamine host plays an indispensable role in the potentiometricresponse to the nucleotide. On the other hand, electrode 1 showed a moderate potentiometric response to 5'-GMP at pH 6.6, but not at either pH 8.0 or pH 4.0. Whereas the lack of response at pH 8.0 can be explained by the loss of anion receptor function of the triamine host due to insufficient protonation, the reason for the lack of response at pH 4.0 remains recondite. A possible explanation might be the loss of charge separation due to intermolecular dimerization as mentioned above. Figure 4a shows a typical example of the response behavior of electrode 1 to 5'-GMP examined as a function of time at pH 6.6. Upon the addition of 5'-GMP to a 10 mM concentration, the membrane potential of electrode 1 decreased rapidly and then increased gradually and equilibrated after several minutes. Similar behavior was observed upon the
addition of 5'-GMP to other concentrations (1.0 and 5.0 mM). The response time t(l.0 min, 1.0 mV) was calculated from Figure 4a to be 4.6 min. In contrast, the response time for inorganic phosphate HPO2-was much shorter (t(l.O min, 1.0 mV) = 0.8 min; Figure 4b), although the potentiometric response was weaker than that induced by 5'-GMF' (vide infra; Figure 5). This fact suggests that the factor dominating the establishment of equilibrium a t the surface of the liquid membrane might be different between 5'-GMP and HPOa2-. The reversibility of the potentiometric response of electrode 1was checked with 5'-GMP as a guest. A set of measurement with a conditioning solution (0.1 M HEPES-NaOH buffer, pH 6.6) and a sample solution containing 5'-GMP (10 mM) in the same buffer was successivelyrepeated four times. Stable potentials with good reversibility and repeatability were obtained without interference by the memory effect (hysteresis); the average potentials with mean deviations were -47.4 f 0.6 mV and -72.4 f 0.6 mV, respectively, for the conditioning and sample solutions. Selectivities of Potentiometric Response t o S'-GMP and Other Organic and Inorganic Anions. Potentiometric responses of electrode 1based on cytosine-pendant triamine host l b to organic and inorganic anions are shown in Figure 5. This electrode displayed a potentiometric response order of salicylate- >> C104- > 5'-GMP > HP04*-> SCN- > Sod21 Br- 1 NO3- 1 C1-. The potentiometric selectivity coefficients for the series of mono- and dianions which gave appreciable responses were as follows. Monoanions: log Pdw~ats,~l~, = -0.70, log PAwhk,scN = -1.55. Dianion: log IQ?&P,HPO = -0.73. The selectivity pattem is different from the electrdes based on either conventional ion exchangers or other types of sensory elements in the following sense. (i) For monovalent inorganic anions, the magnitude of the potentiometric response of electrode 1was C104- >> SCN- > Br-, NO3-,C1-, which is in accord with the Hofmeister series generally observed with the ion-exchanger type electrodes based on lipophilic quaternary ammonium salts (for representative results, see refs 9, 10, 11,and 13). (ii) The response of electrode 1to divalent inorganic anions (HP042-,S042-)seems to be somewhat stronger than that to some monovalent anions with weak response (Br-, NO,, C1-). This is in contrast to the selectivity of the conventional quaternary ammonium electrodes, which generally give much stronger responses to the above monovalent anions as compared to the divalent anions (general response order: NO, > Br- > C1- >> S042-2 HPo42-).9-10311313 Such a reversal of selectivity has not been observed with other nonconventional anion-selective electrodes such as those based on alkyltin compoundsg or vitamin B12 and metalloporphyrin derivat i ~ e s . ' ~ JA~ tendency J~ of the selectivity reversal between the
063
ANALYTICAL CHEMISTRY, VOL. 64, NO. 8, APRIL 15, 1992 4I
wLz2d
-70-6
-5
-4
-3
log c
.2
-1
0 " .6
"
"
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-4
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1
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-8
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(a) (b) Figure 6. Potentiometric response curves for 5'-GMP, 2'-GMP, and 5'-AMP obtained at pH 6.6 (0.1 M HEPES-NaOH) for (a) electrode 1 based on cytosine-pendant triamine l b and (b) electrode 2 based on macrocyclic polyamine 2.
(a) (b) Figure 7. Potentiometric response curves for 5'-GTP and 5'-ATP obtained at pH 6.6 (0.1 M HEPES-NaOH) for (a) electrode 1 based on cytosine pendant triamine l b and (b) electrode 3 based on cytosine derivative 3.
mono- and divalent anions has also been observed for the electrode based on macrocyclic polyamine 2 that showed a response order of NO; > Br- >> 502-= HP02- > Cl-.19 The selectivity reversal is more distinct for electrode 1in the sense that SO4" and HPO4> give stronger responses than NO,. The clear preference for HP02- over S042- is also interesting, although the reason is not clear. (iii) Electrode 1 is characteristic in that it displays an outstanding response to salicylate-, an organic anion. The response was even stronger than that to C104-,which is one of the inorganic anions that induces the strongest response to conventional ion-exchanger type electrodes. Again, this is in contrast to the response order of C10, > SCN- > salicylate> Br-, which is generally observed for the conventional quaternary ammonium type e1ectrodes.l2J8 Some of the Mn(II1) complexes of porphyrin derivatives have also been found to show preference for salicylate- over inorganic anions.12 The preference for organic anions by the present electrode is also demonstrated by the stronger response to 5'-GMP2- as compared to HP042-. Selectivities of Potentiometric Response to Guanosine and Adenosine Nucleotides. To examine the possibility of potentiometric discrimination between similarly charged nucleotides, the responses of electrodes 1and 2 were measured at pH 6.6 for three kinds of nucleotides, namely, 5'-GMP, 2'-GMP, and 5'-AMP. The potential vs concentration curves for electrodes 1 and 2 are shown in Figure 6. Electrode 2, based on macrocyclic polyamine 2 which has a receptor site only for the nucleotide phosphate group, gave an anionic potentiometric response that was similar for all of the nucleotides tested (Figure 6b). The lack of selectivity in this potentiometric response can be reasonably explained by the absence of a base-pairing site in host 2. However, in contrast to electrode 2, electrode 1, based on host lb that contains a base-pairing cytosine residue, gave a potentiometric response that was selective for the guanosine nucleotides (5'- and 2'GMP). On the other hand, the response to V-AMP was almost negligible (Figure 6a). This characteristic response behavior for the guanosine nucleotides was observed from ca. 5 X M concentration with a slope of 10 mV/decade. Considering that theae guanosine nucleotides, as well as AMP,are expected to exist predominantly as the dianionic forms at pH 6.6, the slope of 10 mV/decade is smaller than the theoretical Nernstian value (29.1 mV/decade at 20 "C). The potentiometric selectivity coefficient of electrode 1for 5'-GMP (primary ion) vs 2'-GMP (interfering ion) was calculated to be log @?&Mp,1t-GMp = -0.10. Whereas no substantial discrimination was observed between the positional isomers of guanosine monophosphate, nearly all-or-none selectivity was attained between the guanosine and adenosine monophosphates. Figure 7 shows the potential vs concentration curves a t pH 6.6 for electrodes 1and 2 with 5'-GTP and 5'-ATP as guests. The potentiometric response of electrode 1to 5'-GTP began from ca. 1 X M concentration, and a linear response was
observed above ca. 1 X lo4 M with a slope near to the theoretical Nernstian value for a tetravalent anion (14.5 mV/ decade at 20 'C). At this experimental pH, both GTP and ATP are expected to exist predominantly as tetravalent anions. The response to 5'-ATP was almost negligible (Figure 7a). In addition, electrode 1 showed a stronger response to 5'-GTP as compared to 5'- and 2'-GMP in the sense that the response started from a lower concentration with the triphosphate guest. This again reflects a selectivity based on the magnitude of electrostatic interaction pertaining between the host and guest, as clearly seen in the previous s t ~ d y l ~ * ~ ~ with adenosine nucleotides for the electrode based on host 2. In contrast, electrode 3, based on the cytosine derivative 3 that lacks an anion receptor site, did not show any appreciable response to 5'-GTP, 5'-ATP (Figure 7b), or any of the monophosphates (figure not shown). Thus, a potentiometric discrimination between guanosine and adenosine nucleotides was attained only by electrode 1. This is the electrode that is based on the cytosine-pendant triamine host lb that has both a complementary base-pairing site and an electrostatic binding site. In addition, the selectivity based on the magnitude of electrostatic interaction is retained. Furthermore, it is interesting to note ihat, in contrast to electrode 2 based on macrocyclic polyamine 2, this electrode did not show appreciable potentiometric response to the adenosine nucleotides. These results, taken together, thus demonstrate the following points. (i) Ditopic recognition at the membrane surface based on both complementary base-pairing and electrostatic binding interactions is a prerequisite for potentiometric discrimination between guanosine and adenosine nucleotides. (ii) The anion receptor function of the triamine host lb seems to be moderately strong at pH 6.6. Thus, this system is only able to effect potentiometric response to nucleotides when a complementary base-pairing interaction is also possible. For the guests showing potentiometric response, the selectivity based on the magnitude of electrostatic interaction is observed. (iii) On the other hand, in contrast to the system based on host lb, the anion receptor function of pentaamine host 2 is stronger a t pH 6.6 so that potentiometric response to nucleotides is effected in the absence of any added complementary base-pairing interaction. In conclusion, we have developed a new type of liquid membrane electrode using cytosine-pendant triamine host lb that can discriminate guanosine and adenosine nucleotides by complementary base pairing. This demonstrates a novel possibility of potentiometric discrimination of nucleotides based on the ditopic recognition, i.e., complementary basepairing and phosphate-ammonium electrostatic binding interactions at the membrane surface of the electrode. Although the sensitivity for the guanosine nucleotides as well as the selectivity over some lipophilic anions of the present electrode is not enough for practical use, the negligible response to C1satisfies one of the important requirements for the application
Anal. Chem. 1QQZ.6 4 , 964-967
961
to physiological samples. Further design of the host compounds to enhance the base-pairing ability and increase the lipophilicity would afford more sensitive and selective potentiometric sensors for nucleotides.
Ammann. D.: Huser. M.: Krautler. 6.;Rusterholz, 8.; Schubeas, P.; Lindemann, E.; Halder, E.; Simon, W. Helv. Chim. Acta 1988, 69, 849-854. Chaniotakis, N. A.; Chasser, A. M.; Meyerhoff, M. E.; Groves, J. T. Anal. Chem. 7988s 60. 185-188. Hodlnir, A.: Jyo, A. Chem. Len. 1888. 993-998. Hodinir, A.; JYO, A. Anal. Chem. 1889, 67, 1169-1171. Daunert, S.; Bachas, L. 0. Anal. Chem. 1989, 61, 499-503. Ohki, A.; Yamura, M.; Kumamoto, S.; Maeda, S.; Takeshlta, T.; Takagi, M. Chem. Lett. 1989, 95-98. Ohki, A.; Yamura, M.; Takagi, M.: Maeda, S. Anal. Sci. 1990, 6 , 505-588. Wotring, V. J.; Johnson, D. M.; Bachas, L. 0. Anal. Chem. 1990, 62, 1506-1510. Umezawa, Y.; Kataoka, M.; Takami, W.; Kimura, E.; Koike, T.; Nada, H. Anal. Chem. 1888, 6 0 , 2392-2396. Kataoka, M.; Naganawa, R.; Odashlma, K.; Umezawa, Y.; Klmura, E.; Koike, T. Anal. Lett. 1989. 22, 1089-1105. Naganawa, R.; Kataoka. M.; Odashima, K.; Umezawa, Y.; Klmura, E.; Koike, T. Bunsekl Kagaku 1990, 39, 871-676. Umezawa, Y.; Sugawara, M.; Kataoka, M.; Odashima, K. I n Ion-Selectlve Electrodes, 5 ; Pungor, E., Ed.; A k a h l e i Kiad6 (Pergamon Press): Budapest (Oxford), 1989; pp 21 1-234. Odashlma, K.; Umezawa, Y. I n Biosensor Technology; Buck, R. P., Hatfield, W. E.. Umak, M., Bowden, E. F., Eds.; Marcel Dekker: New York, 1990: Chapter 6. Odashima, K.; Sugawara, M.; Umezawa, Y. Trends Anal. Chem. 1990, 10, 207-215. Furuta, H.; Magda, D.; Sessler, J. L. J . Am. Chem. Soc. 1981, 773, 978-985. Furuta. H.; Furuta, K.; Sessler, J. L. J . Am. Chem. SOC. 1991, 713. 4708-4707. Recommendations for Nomenclature of Ion-Selective Electrodes. Pure Appl. Chem. 1976, 46, 129-132. Umezawa, K.; Umezawa, Y. I n CRC Handbook of Ion-Seknve Electrodes: SelecdMtyCoefticients; Umezawa, Y., Ed.; CRC Press: Boca Raton, FL, 1990; pp 3-9. Uemasu, I.; Umezawa, Y. Anal. Chem. 1982. 54, 1198-1200. Lindmr, E.; T&h, K.; Pungor, E.; Umezawa. Y. Anal. Chem. 1884, 56, 808-810. Lindner, E.: Tbth, K.; Pungor, E. Pure Appl. Chem. 1988, 56, 469-479.
ACKNOWLEDGMENT We are grateful for the collaboration made possible by the JSPS Joint Research Program, organized by Eiichi Kimura, Department of Medicinal Chemistry, Hiroshima University School of Medicine, Hiroshima, Japan, and sponsored by the Japan Society for the Promotion of Science. The macrocyclic polyamine 2 used in the present study was kindly provided by Eiichi Kimura. We gratefully acknowledge support for the present study from the Grant-in-Aids for Scientific Research from the Ministry of Education, Science and Culture to Y.U. and from the Texas Advanced Research Program to J.L.S. J.L.S. also acknowledges the National Science Foundation (P.Y.I. Award 1986), the Sloan Foundation (Fellowship 1989), and the Camille and Henry Dreyfus Foundation (TeacherScholar Award 1988). REFERENCES Koryta, J. Anal. Chlm. Acta 1980, 223,1-30. Soisky, R. L. Anal. Chem. 1990, 62, 21R-33R. Janata, J. Anal. Chem. 1990, 62,33R-44R. Colllson, M. E.; Meyerhoff, M. E. Anal. chem. 1990, 62, 425A-437A. Pungor, E.; Lindner, E.; Tbth, K. Fresenius J . Anal. Chem. 1990, 337, 503-507. Janata, J. Chem. Rev. 1980, 90, 691-703. Ammann, D.; Mod, W. E.; Anker. P.; Meier, P. C.; Pretsch, E.; Simon, W. Ion-Selective Electrode Rev. 1983, 5 , 3-92. Shono, T. Bunsekl Kagaku 1984, 33,E449-E458. Wuthler, U.; Pham, H. V.; Zund, R.; WeRi, D.;Funck, R. J. J.; Bezegh, A.; Ammann, D.; Pretsch, E.; Simon, W. Anal. Chem. 1984. 56, 535-538. SchuRhess, P.; Ammann, D.;Krautler, B.; Caderas, C.; Stepinek, R.; Simon, W. Anal. Chem. 1985, 57, 1397-1401.
RECEIVED for review August 9, 1991. Accepted January 31, i992.
Raman Spectrometry with Metal Vapor Filters R. Indralingam,+J. B. Simeonsson,* G.A. Petrucci, B. W.Smith, and J. D. Winefordner*
Department of Chemistry, University of Florida, Gainesville, Florida 3261 1-2046 INTRODUCTION Perhaps the most important instrumental consideration in the detection of Raman scattering is the rejection of specular and Rayleigh scattering of the excitation source. In conventional scanning Raman systems, this entails a sacrifice in detection efficiency through the use of large double spectrometers with relatively low optical throughput. In Fourier transform based approaches, reduction of specular and Rayleigh scattering is critical due to the so-called multiplex disadvantage inherent in the FT technique. A highly efficient filter would make possible more compact Raman instrumentation using modem multichannel detection methods such as the CCD.’ The ideal fiter for either dispersive or FT methods would attenuate very efficiently only within the spectral profile of the laser source and be highly transparent a t wavelengths removed from the exciting wavelength. This requires a narrow bandpass filter with a very steep absorption edge or a ‘notch” absorption filter with an exceptionally narrow absorption profile. In practice, neither has yet been achieved. *Author to whom reprint requests should be sent. Present Address: Department of Chemistry, Stetson University, Deland, FL 32720. Present Address: Department of the Army, U.S. Army Laboratory Command, Ballistic Research Laboratory, Aberdeen Proving Ground, MD 21005-5066.
*
0003-2700/92/0384-0964$03,00/0
The most commonly used optical filter for Rayleigh scatter is the double or triple monochromator which is a part of routine Raman i n s t r u m e n t a t i ~ n . ~Since ~ ~ a broad, tailed Rayleigh scatter peak is observed even with these spectrometers, other filters have been developed for use in both dispersive and FT-Raman spectrometry. An iodine vapor cell has been used with an argon ion laser source and a single monochromator.*v5 Molecular iodine has an absorption band a t the argon ion laser wavelength of 514.5 nm and has been shown to remove elastically scattered light in surface-enhanced Raman spectroscopy6 as well as spontaneous Raman spectrometry. A filter spectrograph’ combines a line rejection filter with a dispersive spectrograph. The instrument is a modified double monochromator having a cylindrical mirror positioned so that the laser line will exit through an aperture in the mirror after preliminary dispersion of the scattered radiation. All other spectral lines are reflected and reformed into an output light signal containing all of the Raman spectral lines except the rejected laser line. The dispersion spectrograph is coupled to the line rejection filter and produces the Raman spectrum from the output light signal. A variation of this instrument is the variable band pass filter.* Ruby laser radiation is absorbed in an unpumped ruby crystal, and an array of ruby crystal cubes has been used as 0 1992 American Chemical Society
