526
Anal. Chem. 1982, 5 4 , 526-529
Conformational and Acid-Base Equilibria of Captopril in Aqueous Solution Dallas L. Rabensteln* and Anvarhuseln A. I s a b Department of Chemistry, Unlverslty of Alberta, Edmonton, Alberta, Canada T6G 202
'H NMR spectra have been measured at 400 MHr and ''C spectra at 50.3 MHr for captoprll, 1-[2(5)-3-mercapt0-2methyl-1-oxopropyi]-~-prollne,in aqueous solution as a function of pH. The NMR spectra indlcate that captoprll exlsts as two isomers wlth respect to the conformation across the amide bond. The conformatlon having the C, carbon of the proline resldue and the C2 carbon of the 2(5)-3-rnercapto-2methyl-I-oxopropyl part trans across the amide bond is the most abundant, wlth the actual mole fractlon In this form dependent on the protonation states of the carboxylic acld and sulfhydryl groups. The carboxyllc acld group of the trans Isomer Is less acidic than that of the CISIsomer by 0.66 pK, units, and the proportlon of captopril in the trans form decreases from 0.86 to 0.58 upon titratlon of Its carboxylic acid group. These resutls are Interpreted to lndlcate Intramolecular hydrogen bonding between the carbonyl oxygen of the amide group and the carboxylic acld proton In the trans Isomer.
obtain the acid dissociation constants for the carboxyl and sulfhydryl groups of both conformations, while the conformational equilibrium (I + 11) was characterized over this pH range from the 'H NMR measurements.
EXPERIMENTAL SECTION Chemicals. The captopril was a gift from The Squibb Institute for Medical Research, Princeton, NJ. The 99.7% DzO,40% NaOD in D20,and 35% DC1 in D20 were obtained from Merck Sharp and Dohme, Ltd. Hydrochloric acid and sodium hydroxide were analytical grade reagents. NMR Measurements. 'H NMR spectra were measured at 400 MHz on a Bruker WH-400 spectrometer operating in the pulsed Fourier transform mode. The probe temperature was 25 "C. 'H chemical shifts were measured relative to internal tertbutyl alcohol and are reported relative to the methyl proton (DSS). resonance of sodium 2,2-dimethyl-2-silapentane-5-sulfonate The methyl resonance of tert-butyl alcohol is at +1.23 ppm (pH 7.0) relative to DSS. All lH measurements were made on DzO solutions of captopril. 13C NMR spectra were measured at 50.3 MHz on a Bruker WH-200 spectrometer operating in the pulsed Fourier transform Captopril, l-[2(S)-3-mercapto-2-methyl-l-oxopropyl]-~- mode. The 13C measurements were made with coherent offresonance lH decoupling or with broad band 'H decoupling. To proline, is a recently developed drug for the treatment of high minimize temperature gradients in the sample and to maintain blood pressure (1-3). High blood pressure can result from the temperature near 25", 5-mm NMR tubes were used with a the overproduction of angiotensin 11from inactive angiotension stream of nitrogen passing through the probe (14). All 13C I, the conversion being catalyzed by angiotensin-converting measurements were made on captopril in HzO solution. Since enzyme ( 4 , 5 ) . The mechanism by which captopril is thought the HzO solutions do not contain sufficient deuterium for a to be active involves inhibition of angiotensin-converting deuterium lock signal, the spectrometer was run in the unlocked mode with no detectable drift over a period up to 12 h. 13C enzyme, presumably through interaction with the enzyme at chem2al shifts were measured relative to internal dioxane but its active site (1-3), are reported relative to (CH3)@i(the dioxane resonance is 67.4 I t is well-known that proline-containing peptides normally ppm to higher frequency from (CH3)4Si).Positive shifts correexist as an equilibrium mixture of cis and trans isomers with spond to less shielding than in (CH3)4Si. respect to the peptide bond involving the proline amino group pH Measurements. All pH measurements were made at 25 (6). Thus, captopril is expected t o be present in aqueous "C with a Fisher Model 520 Accumet pH meter equipped with solution as the trans and cis forms, I and 11,respectively, with a Fisher microprobe combination pH electrode. Fisher certified the relative populations of the two forms dependent on the buffers of nominal pH values 4.00,7.00, and 10.00were used for protonation state of the molecule (6-8). calibration of the pH meter. The exact pH of the buffers was determined by comparison with freshly prepared N.B.S. pH H02C, ,CH standard solutions (15). pH measurements made on DzOsolutions CH 'CH2 I I have been corrected for deuterium isotope effects with the relation HSCH2FH,r,N-CH 2 pD = pH meter reading + 0.40 (16). Sample Preparation. Solutions for 'H NMR measurements were prepared in 99.7% DzO containing 0.16 M NaN03 and I II approximately 0.0001 M tert-butyl alcohol. Typically, the captopril concentration was 0.005 M. Samples for the measurement It is not known if both I and I1 are active as inhibitors of of lH NMR spectra as a function of pD were prepared by adding angiotension-converting enzyme. The characterization of the sufficient concentrated DC1 to give an initial pD of approximately aqueous solution chemistry of captopril is a necessary first 1. The first sample was withdrawn, and then subsequent samples step toward answering this question. Since the rate of inwere withdrawn as the pD was increased with NaOD. terchange between the cis and trans conformations by rotation Solutions for 13Cmeasurements were prepared in distilled HzO around the C-N bond of the peptide linkage is slow on the containing 0.16 M NaN03 and approximately 0.1 M dioxane. The nuclear magnetic resonance (NMR) time scale (7-13), NMR concentration of captopril was around 0.5 M. Samples were provides a convenient method for detecting the presence of prepared as a function of pH as described above for the 'H NMR cis-trans isomers and for studying simultaneously the solution measurements. chemistry of the two isomers (8). In the present paper, we RESULTS AND DISCUSSION describe the results of an IH and 13CNMR study of captopril The 400-MHz lH NMR spectra and the 10-70 ppm region in aqueous solution. The two isomers of captopril can be of the 50.3-MHz 13Cspectra for the H,A, HA-, and A2- forms observed from pH 1to p H 13 in both the IH and the 13CNMR of captopril are shown in Figures 1 and 2, respectively. spectra. The 13C NMR titration curves were analyzed t o 0003-2700/82/0354-0526$0 1.25/0
0 1982 American Chemical Soclety
ANALYTICAL CHEMISTRY, VOL. 54, NO. 3, MARCH 1982
Table I.
13C
527
Chemcal Shifts for Captoprila
H2A
HA-
A=-
c,
trans 27.630 27.730 29.689 cis b 29.101 27.560 44.832 c* trans 42.553 42.333 cis 43.141 4 2.850 b CH, trans 16.898 16.901 16.751 cis b 16.750 16.089 CON trans 177.153 176.505 17 8.476 cis b 179.726 177.373 C, trans 60.049 62.550 62.475 cis b 63.210 63.211 trans 29.836 30.433 30.497 CP cis 31.673 32.190 32.261 C, trans 25.204 25.130 25.131 cis b 23.290 23.293 C6 trans 48.655 48.800 48.802 cis 47.722 47.921 47.772 CO, trans 177.006 180.534 178.402 cis b b 179.729 a From 13C spectra measured at pH values of 0.59, 7.43, and 12.30. Resonance either too small to detect or not resolved from resonance for trans isomer. I
*
I 4.00
3.00
I
,
,
,
2.00
1
1.00
porn
Figure 1. The 400-MHz 'H NMR spectra of 0.005 M captoprii in D20 solution containing 0.16 M NaCi as a function of pD. Resonance assignments are given in the text.
1.45
h'
Figure 2. The 10-70 ppm region of the 50.3-MHz I3C spectrum of 0.55 M captopril as a function of pH in H,O solution containing 0.16 M NaCI. Resonance assignments are given in the text.
The 'H spectra consist of two highly coupled spin systems: the -CH2CH2CH2CH- spin system from the proline residue and the -CHzC€ICH3 spin system from the 3-mercapto-2methyl-1-oxopropyl group. The spectra are further complicated by the presence of two sets of resonances for each type of hydrogen, which indicates the existence of both the cis and trans isomers of captopril. This doubling of resonance patterns is seen clearly, for example, in the 3.4-3.8 ppm region of the pD 7.28 spectrum. Comparison of this region of the three spectra in Figure 1 reveals that the fractional concentrations of the two forms are pH dependent. Resonances in the lH spectrum were assigned on the basis of results from spin decoupling experiments and from the
effect of titration of acidic protons on chemical shifts. For the pD 1.37 spectrum, the assignments for the most abundant form are as follows: CH3, the doublet at 1.143 ppm; the two hydrogens on C, of the proline residue and one of the hydrogens on C, of the proline residue, 2.0-2.1 ppm; the other hydrogen on C, of the proline residue, 2.2-2.4 ppm; the two hydrogens on C3 and the one hydrogen on C2 of the 3mercapto-2-methyl-1-oxopropyl part, 2.55-2.7 ppm and 2.9-3.1 ppm, respectively; and the two hydrogens on C6and the single hydrogen on C, of the proline residue, 3.67-3.84 and 4.44-4.47, respectively. No attempt has been made to extract the exact chemical shifts for the various resonances of the two formls due to the complexity of the multiplet patterns and the extensive overlap of resonances, even with the high dispersion a t 400 MHz. Resonances in the 13C spectra were assigned to specific carbons by selective 'H decoupling experiments, by knowing the lH assignments discussed above, and by the effect of titration on 13C chemical shifts. Separate resonances are clearly visible for some of the carbons of the two isomers for the HA- and A2- forms (Figure 2); however, they are more difficult to detect for the minor isomer for the H2A form due to its low fractional concentration. Resonance assignments and chemical shifts are given in Table I. Also listed in Table I are the chemical shifts for the amide and carboxylate carbons, which are found in the 176-181 ppm region. The redionance at 67.4 ppm i s due to dioxane. The fractional concentrations of the two isomers were determined as a function of pD by using the relative intensities of the two multiplet patterns in the 3.4-3.9 ppm region. The fractional concentrations are shown as a function of pD in Figure 3. The equilibrium constant for the I ~ rI1! equilibrium, KBq= [II]/[I],is 0.17, 0.69, and 0.30 for the H2A, HA-, anld A2- forms, respectively. The more abundant isomer has been assigned the trans conformation on the basis of the following: (i) The trans isomer is the most abundant isomer for the related molecules glycyl-I,-proline, glycyl-L-hydroxyproline, L-alanyl-L-proline, N-acetylsarcosine, and glycylsarcosine and the fractional concentration of the trans isomer for these molecules decreases upon titration of the carboxylic acid group (8),as is also found for captopril. For example, the fractional concentration of the trans isomer of glycyl-L-proline decreases from 0.84 to 0.65 (8) as compared ta the decrease from 0.86 to 0.58 for the major isomer of captopril. (ii) The chemical shift of the amide carbon of the trans isomer undergoes a characteristic deshielding (8)
528
ANALYTICAL CHEMISTRY, VOL. 54, NO. 3, MARCH 1982
t " " " " " " ' I
1
t
0.01
30
-
28
-
J
C '
-e
"
1
3
'
"
5
"
7
9
"
1
'
1
1
c3 ( C I S )
' ' 1
1
''ro
3
PD
Flgue 3. Fractional concentrationsof the trans (I) and cis (11) isomers of captopril as a function of pD.
1
3
5
7
9
11
13
PH
upon deprotonation of the carboxylic acid group. For example, the amide carbons of the trans isomers of glycyl-L-proline, glycyl-L-hydroxyproline,and L-alanyl-L-proline are deshielded by 0.70, 0.63, and 0.75 ppm, respectively (8). The amide resonance for the isomer of captopril assigned the trans conformation is deshielded by 0.65 ppm. The chemical shifts for the carbon resonances of captopril are dependent on its protonation state (Table I). The 13C chemical shift titration curves for C, of the proline residue and C3 of the 3-mercapto-2-methyl-1-oxopropyl group are plotted in Figure 4. The curves for the C, carbons reflect the titration of the directly bonded carboxylic acid group, whereas the curves for the C3 carbons reflect titration of the sulfhydryl group and, to a much smaller extent, titration of the carboxylic acid group. Since exchange-averaged resonances are observed for all carbons of each isomer from pH 1to 13, the observed chemical shifts at a given pH are the weighted averages of the chemical shifts of the various protonated forms between which a particular isomer is exchanging
where f and 6 represent, respectively, the fractional concentration and the chemical shift of the species indicated. Acid dissociation constants for the carboxylic acid and sulfhydryl groups of the cis and trans isomers were calculated from the data in Figure 4. Since titration of the carboxylic acid and sulfhydryl groups occurs in widely separated pH regions, eq 1 can be simplified to include only the H2A and HA- species for the calculation of K1, the acid dissociation constant for the carboxylic acid group, and only the HA- and A2- species for the calculation of K , (8). Thus, for the pH region 1-5, eq 1 becomes 6o = fH2A6H2A + fm6m which, when combined with the relationship 1 = fH2A + fHA leads to
~ H was ~ A
calculated at each pH value where 6o was between and then, with eq 3, a value was calculated for the acid dissociation constant from each value of fH2A (8). ~ H and ~ A 6HA,
(3) The average values obtained for pKl for the trans and cis
Figure 4. 13C chemical shift titration curves for the C, carbon of the proline residue and the C, carbon of the 3-mercapt~2-rnethyl-l-oxopropyl group of the cis and trans conformations of captopril.
isomers are 3.52 f 0.08 and 2.86 f 0.08, respectively. K2 was calculated by the same procedure using eq 4 and 5 . The (4)
average values obtained for pK2 are 9.71 f 0.05 and 9.99 f 0.06 for the trans and cis isomers, respectively. The finding that pK1 is larger for the major isomer by 0.66 pK units provides further support for the assignment of the trans conformation to this isomer, since the carboxylic acid groups of the related molecules glycyl-L-proline, glycyl-Lhydroxyproline, etc., are less acidic than those of the cis isomers (8). For example, pKl values for the trans and cis isomers of glycyl-L-proline are 3.05 and 2.58, respectively. The peptide bond normally exists in the trans conformation with respect to the disposition of the a-carbons of the amino acid residues joined by the peptide bonds (17). When the peptide bond is N-substituted, as in peptides containing proline, the cis form can be of comparable stability (6). The results in Figure 3 indicate that for captopril, the relative stability of the trans and cis conformations depends markedly on protonation state. The enhanced stability of the trans conformation of the H2Aform relative to that of the HA- form is probably due to stabilization of the trans form of H2A through intramolecular hydrogen bonding between the carboxylic acid hydrogen and the amide carbonyl oxygen (7,8, 18). Such a hydrogen bond is not possible in the cis isomer. Stabilization of the trans isomer of the carboxylic acid forms of related small peptides having proline or sarcosine as the C-terminal residue has also been accounted for in terms of the same intramolecular hydrogen bond (7, 8). Since the hydrogen involved in the proposed hydrogen bond is the one titrated when the trans isomer goes from H2A to HA-, the carboxylic acid group of the trans isomer would be expected to be somewhat less acidic than that for the cis isomer (8), as is found to be the case. The enhanced stability of the trans isomer of the A2- form relative to that of the HA- form is presumably due to the
Anal. Chem. 1982, 5 4 , 529-533
increased chargecharge repulsion between the deprotonated sulfhydryl and carboxylic acid groups in the cis conformation of the A2- form. Space-filling molecular models show the separation of these two groups to be larger in the trans conformation. ACKNOWLEDGMENT We thank The Squibb Institute for Medical Research, Princeton, NJ, for their generous gift of captopril.
LITERATURE CITED Ondetti, M. A.; Rubin, 6.; Cushman, D. W. Science 1977, 196, 441-444. Cushrnan, D. W.; Cheung, H. S.; Sabo, E. F.; Ondetti, M. A. 6iOChemisfry1977, 16, 5484-5491. Cushman, D. W.; Cheung, H. S.; Sabo, E. F.; Ondetti, M. A. frog. Cardiovasc. Dis. 1978, 21. 176-182. Skeggs, L. T.; Kahn, J. P.; Shumway, N. P. J. Exp. Med. 1958, 103, 295-307. Skeggs, L. T.; Dorer, F. E.; Kahn, J. R.; Lentz, K. E.; Levine. M. Am. J . Med. 1976, 6 0 , 737-748. Madison, V.; Scheliman, J. 6iopo/ymers 1970, 9 , 511-567. Gerig, J. T. Siopo/ymers 1971, 10, 2435-2443.
529
(8) Evans, C. A,; Rabenstein, D. L. J. Am. Chem. SOC. 1974, 96, 7312-73 17. (9) Stewart, W. E.; Siddal, T. H. 111, Chem. Rev. 1970, 7 0 , 517-5!51. (10) Bovey, F. A.; Ryan, J. J.; Hood, F. B. Macromolecules 1988, 1 , 305-307. (11) Deber, C. M.; Bovey, F. A.; Carver, J. P.; Blout, E. R. J. Am. Chem. SOC. 1970, 92, 6191-6198. (121 Thomas, W. A.; Wliiiams, M. K. J. Chem. SOC.,Chem. Commiin. 1972, 994-996. (13) Dorman, D. E.; Torohia, D. A.; Bovey, F. A. Macromolecules 1973, 6 , 80-82. (14) Bock, K.; Meyer, 6.; Vignon, M. J. Magn. Reson. 1980, 38, 545-5!51. (15) Bates, R. G. "Determination of pH: Theory and Practice", Wiley: NIBW York. 1973. (16) Giasoe, P. K.; Long, F. A. J. fhys. Chem. 1980, 64, 188-190. (17) Marsh, R. E.; Donohue, J. Adv. Profein Chem. 1987, 2 2 , 235-2!56. (18) Mizushima, S. Adv. Protein Chem. 1954, 9 , 299-324.
RECEIVED for review August 10, 1981. Accepted December 3, 1981. The authors gratefully acknowledge the financial support provided by the Natural Sciences and Engineeriing Research Council of Canada and by an Alberta Heritage Foundation for Medical Research Fellowship (A.A.I.).
Acid-Catalyzed Reactions of 2,2,2-Trifluorodiazoethane for Analysis of Functional Groups by I9F Nuclear Magnetic Resonance Spectrometry K. L. Koller and H. C,, Dorn" Department of Chemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virghia 2406 I
The acld-catalyzed reactlons of trlfluorodlazoethanewith alcohols, phenols, thlols, and carboxylic acids are reported. The yleld data for these trlfluoroethyl derlvatlves suggest a simple, and In many cases, quantitative method for Introduction of a fluorine tagglng group. The ''F chemlcal shlfts lndlcate that most functional groups (e.g., phenols, alcohols, etc.) have falrly well resolved chemical shlft regions. I n addltlon, paramagnetlc shlft reagents have been utlllzed to selectlvely dlfferentlate carboxyllc acids from other active hydrogen functional groups.
Presently there are several methods available utilizing nuclear magnetic resonance for identification and quantitation of various functional groups. Most NMR analytical techniques involve characterization of functional groups using either 'H (1-8) or lBF (9-22) NMR. Unfortunately the l9F NMR tagging reagents presently available have several limitations which have restricted their widespread applicability. For example, one basic limitation of the trifluoroacetate (9-14, 22) and hexafluoroacetone (15-20) derivatives is their chemical lability. A second disadvantage of these reagents is the poor yields obtained in many cases. With this in mind, an oxytrifluoroethylation method using fluorinated diazoalkanes has been investigated. The general reaction for this reagent is analogous to the well-known acid-catalyzed reactions of diazoalkanes, for example, diazomethane (23). The general reaction for the 2,2,2-trifluorodiazoethane reagent is illustrated in eq 1.
CF,C(H):=N=N
HBF,
CF3CH2XR+ N,t (I) where X = 0 and S
+ HXR
The 2,2,2-trifluorodiazoethaneprovides modest to high yields with carboxylic acids, alcohols, phenols and thiols to provide the corresponding trifluoroethyl esters or trifluoroethyl ethers. One of the major advantages of this reagent is tlhe inherent chemical stability of the ether and ester derivative in comparison with other fluorine tagging reagents. For example, trifluoroacetate derivatives are very susceptible to hydrolysis (24). A second advantage of this reagent is the ease of derivative preparation and absence of major byproducts except for innocuous nitrogen and reaction with water (see Results and Discussion section). A possible disadvantage is the necessity of an acid catalyst (fluoroboric acid) which normally excludes derivative preparation of amines by formation of an acid-base salt between the fluoroboric acid and any amines present in the sample. Solvents which can be utilized with this reagent include diethyl ether, carbon tetrachloride, and chloroform. However, certain solvents (e.{;., tetrahydrofuran) react with the reagent in the presence of the acid catalyst. In this paper we report conditions for derivative preparations, yield, and 19F NMR chemical shifts for these ti-ifluoroethyl derivatives. In addition, the potential utility of enhanced l9FNMR spectral resolution of certain derivatives (e.g., trifluoroethyl esters of carboxylic acids) is explored via the use of paramagnetic shift reagents.
EXPERIMENTAL SECTION Varian EM-390 and Jeolco PS-100 nuclear magnetic resonance spectrometers were used to obtain 19Fspectra at 84.7 MHz and 94.1 MHz, respectively. The 19FNMR spectra were taken using 1,2-difluorotetrachloroethaneas the reference and integration standard. A stock solution containing a known weight of 1,2difluorotetrachloroethane was made in CDC13 and l/z mL of this
0003-2700/82/0354-0529$01.25/00 1982 Amerlcan Chemical Society
