Journal of MOLECULAR STRUCTURE

Journal of Molecular Structure 377 (1996) 105-l 12

The zwitterion structure of imidazol- I-ylacetic acids in the solid state and in solution

Pilar L6peza, Paula Zaderenko”, Jose Luis Balcazarb, Isabel Fonsecac, FClix Hernindez Cane’, Paloma Ballesteros”‘*

‘Departamento de Quhica Orgcinica y Biologia, Facultad de Ciencias, UNED, Senda del Rey s/n, 28040-Madrid, Spain ‘Departamento de Ciencias Analiticas, Facultad de Ciencias. UNED. Senda de1 Rey s/n, 28040-Madrid, Spain

‘Departamento de Cristalograjia, Institute de Quitnica Fisica ‘Rocasolano*, CSIC, Serrano 119. 28OO&Madrid, Spain

Received 7 June 1995; accepted in final form 20 September 1995

Abstract

The crystal zwitterion structures of imidazol-1-ylacetic acid 1 and (f)-3-ethoxycarbonyl-2-imidazol-1-ylpropionic acid 2 have been determined by X-ray diffraction analysis. Spectroscopic 13C NMR studies in the solid state, by the CP/MAS technique, and in solution have revealed the presence of the zwitterion structure in Hz0 and DzO solutions. Results have been complemented with I70 NMR and IR data.

Keywords: X-ray crystallography; Solid state NMR spectroscopy; Zwitterion structure; Imidazol-I-ylacetic acid>

1. Introduction

The analgesic and hypnotic activity of imidazol- 1-ylacetic acid 1 was formerly related to the pre- sence of an intramolecular hydrogen bond between the carboxylic group and the nitrogen atom at position 3 of the imidazole ring [I]. Later and considering the ratio of ionic molecules to neutral ones obtained from Ebert’s equation, a zwitterion structure was proposed [2]. Also pioneer ‘H NMR work suggested a similar zwitterion structure in D20 solution [3].

We have described recently a series of imidazol- l-ylalkanoic acids as new probes to determine the intracellular and extracellular .pH and cell volume

* Corresponding author.

by ‘H NMR [4]. Among them, compounds l-5 and the related 6 were prepared.

C02H

1

CO#Bu

4

CO*Et HO&

HO&

COpMe

3

COgH

6

0022-2860/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved SSDI 0022-2860(95)09118-l

106 P. Ldpez et al.lJournal of Molecular Slrmlure 377 (1996) 105-112

Table 1 Crystal data, data collectior? and structure refinementb of compounds 1 and 2

Parameter Compound 1 Compound 2

Formula WdW2 CgHuN204 Crystal size (mm) 0.38 x 0.32 x 0.21 0.38 x 0.32 x 0.21 Symmetry Monoclinic, P2,/n Monoclinic, C2/c Unit ceil determination Least-squares fit from 58 reflexions Least-squares fit from 64 reflexions

(10 < 28 < 90”) (12 < 28 < 90”) Unit cell dimensions 14.328(l), S.SSO(l), 4.618(l) A 26.927(2), 5.426(l), 14.555(l) A

90.0”, 98.777(5)“, 90.0” 90.0”, 104.043(l)“, 90.0 Packing: V (A’), 2 561.1(l), 4 2063.0(4), 8

D, &cm-‘), M, F (000) 1.4930, 126.115, 264.0 1.3664, 212.205, 896 /I (cm-‘) 9.575 8.801

Number of reflexions: Measured 958 1738 Observed 862 (I > 20 (I) criterion) 1699 (I > 2o (I) criterion) Range of hkl -16/16 O/l1 O/6 -31/31 o/7 o/17

H atoms Fourier syntheses, Coord. and thermal Fourier synthesis, Coord. and thermal parameters refined parameters refined

Parameters 151 256 (A/o) max 0.002 0.07 Final R and R,,. 0.041, 0.049 0.047, 0.058 Ap max 0.18 e Am3 0.24 e A-3

’ Technique: Four circle diffractometer, Philips PW 1100, Bisecting geometry, Graphite oriented monochromator, Cu Ko 1.5418 A, w/28 scan. Scanning range for 6’: 2 < 0 < 65. Absorption correction, None. Two standard reflexions, frequency, 90 min.

b Solution: Direct methods. Refinements: L.S. on F&s. w-Scheme: Empirical as to give no trends in < wA2F > vs. < IF01 > and < sine/X >. Computer and programs: Vax 1 l/750, MULTANBO [8], XRAYBO [9], PESOS [IO], PARST [l 11. Scattering factors and anomalous dispersion: Int. Tables for X-Ray Crystallog [12].

The knowledge of the precise ionization state of these new probes in solution is essential to the determination of their membrane permeability properties and to the interpretation of their bio- logical behaviour. In this report we have estab- lished unambiguously the zwitterion structures of 1 and 2 in the solid state by X-ray diffraction analysis and the relation with those observed in solution by combined studies of 13C NMR in solid state and in solution.

2. Experimental

Imidazol-1-ylacetic acid (1) [4], (f)-3-ethoxy- carbonyl-Zimidazol- 1 -ylpropionic acid (2) [4], methyl irnidazol-1-ylacetate (3) [4], tert-butyl imidazol-1-ylacetate (4) [5], (f)-2-imidazol-l-ylsuc- citric acid (5) [6j, pyrazol-1-ylacetic acid (6) [7], were prepared according to literature procedures.

2.1. Crystal structure determination

Crystals of compounds 1 and 2 were obtained from aqueous ethanol and absolute ethanol respec- tively. Crystal and experimental data are given in Tables 1 and 2. The data were collected on a Philips PW 1100 diffractometer, with graphite- monochromated radiation, bisecting geometry, and 1.5” scan width in the w/28 mode. Two reflec- tions were monitored every 90 min, showing no significant variation in the crystals nor in the experimental conditions. The structures were solved by direct methods and refined by least- squares based on Fobs only. All hydrogen atoms were unambiguously obtained in a difference synthesis and included isotropically in the final cycles of refinement. Weights were chosen to give no trends in < wA2/F > versus < [Fobs1 > and < sine/X >, by functions of the w = K/[f(F,&]m[g(sin e/x)] type, K being a scale factor to ensure that < wA2F > N 1. Final fractional

P. Ldpez et al.l.Iournal of Molecular Structure 377 (1996) 105-112 107

(b) Fig. 1. (a) A ORTEP [13] view of compound 1 showing the atomic numbering; displacement ellipsoids are drawn at the 50% probability level; (b) packing of the molecules of compound 1 viewed down the c axis.

atomic coordinates, the full list of bond lengths and angles, and the list of thermal parameters of compounds 1 and 2 are deposited with the B.L.L.D. as Supplementary Publication No. SUP26561 (48 pages).

2.2. Infrared measurements

IR spectra were recorded in KBr on a Bomem- DA3 FT-IR spectrophotometer.

2.3. NMR determinations

13C NMR spectra in solution were recorded

at SO.33 MHz using a Bruker AC200 spec- trometer at 25°C. Samples were dissolved in DZO, H20 or DMSO-d6 and the concentrations were 0.5 to 1 M. Chemical shifts are expressed in p.p.m. from tetramethylsilane using DMSO-d6 as external or internal reference. Typical conditions were as follows: pulse width, 3 ps (ca. 45”); relaxa- tion delay, 1 s, spectral width, 10 KHz; data points, 32768. I70 NMR spectra were taken at 42.82 MHz on a Bruker AM-360. The enriched compounds 1, 2 and 5 were obtained by partial and total hydrolysis of the corresponding esters with enriched I70 (28%) “0 (32%) water, and spectra conditions were the same as those

I’. Ldpez et al./Journal of Molecrclar Smtcture 377 (1996) 105-l 12

Fig. 2. (a) A ORTEP [13] view of compound 2 showing the atomic numbering; Displacement ellipsoids are drawn at the 50% level; (b) packing of the molecules of compound 2 viewed down the b axis.

probability

P. Lbpez et al./Journal of Molecular Structure 377 (1996) 105-112 109

Table 2 Geometric parameters (A, deg) for compounds 1 and 2

Bond Bond lengths (A) Angle @eg)

Compound 1 Nl-C2 Nl-C5 Nl-C6 C2-N3 N3-C4 c4-cs C6-C7 C7-08 c7-09

Compound 2 NlLC2 Nl-C5 Nl-C6 C2-N3 N3-C4 c4-c5 C6-C7 C6-Cl0 C7-08 c7-09 CIO-Cl I Cl l-012 Cl l-013 Ol3-Cl4 Cl4-Cl5

1.324(2) I .379(3) I .459(2) I .325(3) 1.373(3) 1.351(3) 1.529(3) I .259(2) 1.231(2)

1.335(2) 1.378(2) I .468(2) 1.314(2) I .370(2) I .350(2) 1.544(2) I .525(2) 1.229(2) 1.257(2) 1.502(2) I .202(2) 1.332(2) 1.456(3) 1.496(5)

Hydrogen bonds

Compound 1 Compound 2

D-H...A N3-H3.. .08’ N3-H3. . Ogb

Neighbouring chain interactions Compound 2

O12...H142 2.69(3) O12...Hl51 2.82(6) O13...H142 3.01(4) 013...H151 3.04(7) O13...Hl52 2.69(4)

C5-Nl-C6 C2-Nl-C6 C2-Nl-C5 Nl-C2-N3 C2-N3-C4 N3-C4-C5 Nl-C5-C4 Nl-C6-C7 C6-C7-09 C6-C7-08 08-C7-09

C5-Nl-C6 C2-Nl-C6 C2-Nl-C5 Nl-C2-N3 C2-N3-C4 N3-C4-C5 Nl-C5-C4 NI-C6-Cl0 Nl-C6-C7 C7-C6-Cl0 C6-C7-09 C6-C7-08 08-C7-09 C6-ClO-Cl I ClO-Cl I-013 ClO-Cl l-012 Ol2-Cll-013 Cl I-013-Cl4 Ol3-Cl4-Cl5

H...A 1.66(2) 1.57(3)

c--x + 1, -y, -z + 1) t-x + 1, -y + 1, -z + 1) t-x + 1, -y + 1, -z + 1) c-x + 1, -y + I, -z + I) (-x+Ly,--z+1/2+1)

125.6(2) 125.4(2) 108.9(l) 108.4(2) 109.0(2) 107.0(2) 106.7(2) 113.4(l) 119.6(l) 113.9(l) 126.4(2)

124.2(l) 128.3(l) 107.5(l) 109.7(l) 108.2(l) 107.6( 1) 107.0(l) 111.6(l) 113.1(l) 113.3(l) 111.6(l) 120.5(l) 127.8(l) 114.0(l) 110.4(l) 125.8(2) 123.8(2) 116.4(2) 106.2(3)

D...A 2.645(2) 2.550(2)

D-H...A 172(2) 169(2)

n +x - 112, -y + 112 + I, i-z - l/2. b +X3 -Y. i-Z - t/2. Symmetry code..

110 P. Ldper et aLlJournal o/Molecular Structure 377 (1996) 105-112

Table 3 “C NMR chemical shifts (6, ppm)’ of compounds 1-6 at SO.33 MHz

Compound pD or pH ‘k NMR c2 c3 c4 C5 C02H C02R CH CH2

1 5.60 D20 134.4 121.9 118.5 171.5 50.8 4.70 H2O 135.2 122.6 119.1 172.1 51.4

“0: 271.1’ CP/MAS 135.2 123.5 119.9 171.4 51.2

2 3.57 D2O 134.3 120.6 118.6 171.5 171.0 59.5 36.4 3.33 H20 133.9 120.1 118.2 170.8 170.4 58.9 35.9

“0: 257.0’ CP/MAS 136.4 121.4 121.4 172.9 171.1 59.7 38.8

3 D20 137.8 126.7 120.3 170.0 46.8 CP/MAS 138.4 130.0 121.0 172.0 47.7

4 DMSO-d,” 138.1 120.9 120.5 167.5 47.7 CP/MAS 138.5 127.6 121.9 169.1 48.0

5 3.29 D20 134.3 121.6 118.4 171.8 173.2 59.6 36.6 “0: 267.1’

(R=H) CP/MAS 133.7 122.9 116.2 169.8 171.8 60.0 37.9

(R=H) 6 D20 139.9 105.0 131.0 174.5 53.6

CP/MAS 140.7 106.5 129.5 174.4 56.2

’ Imidazole ring assignments have been made considering the data described in the literature [21]. b Very insoluble in D20. ’ Performed in H:‘O.

previously reported [5]. 13C Cross-polarization magic angle spinning (CP/MAS) NMR spectra were obtained at 50.33 MHz on a Bruker CP-200 spectrometer. Samples were rotated at 3.5 or 2.5 kHz in a 7 mm ZrOz rotor; the initial contact time was 1 ms; 90” pulse lengths for protons were 7 ps; 4 s delay between scans and 200 transients were used. Chemical shifts were measured using glycine (176.1 ppm) as external reference.

2.4. Determination of pK H1 of compounds 1 and 2

The pK, of the carboxylic group of com- pounds 1 and 2 in Hz0 (pKH’) were determined by 13C NMR (50.33 MHz) using DMSO as external reference. pH titrations (25’C) were per- formed using 0.8 M solutions of the appropriate compound in Hz0 adjusting the pH with the addition of HCI. The dependence of chemical shifts of carbons N-CHz of compound 1 and N-CH of compound 2 vs pH were obtained to calculated the pK,.

3. Results and discussion

Structural study by X-ray diffraction analysis of compounds 1 and 2 (Table 1) revealed that both acids existed in zwitterion form in the solid state (Figs. 1 and 2).

The proton from carboxylic oxygen migrates to the N3 in the imidazole ring forming the zwitterion. The charge was delocalized along Nl-C2-N3, and the imidazole ring was planar with maxima deviations of this plane of -0.0008(20) (C5 compound 1) and -0.0016(19) (C4 compound 2). Bond distances and angles (Table 2) were very similar in the two compounds and were on the same range of values as those of the imidazolium cation in imidazolium sulphate dihydrate [14]. Both zwitterion structures were also stabilized by strong hydrogen bonds (dis- tances N3 . . ~~0 = 2.645(2) A for compound 1 and 2.549(2) A for compound 2) (Table 2). In this situation, both compounds formed infinite chains along the a axis (see Figs. 1 and 2) similar to those observed in some salts of hydroxybenzoic acids

P. Ldpez et al./Journal o$Molecular Structure 377 (1996) 105-112 111

[15]. Besides, in compound 2 there are some inter- actions of note between neighbouring chains (Table 2). Moreover, compound 2 was further linked into antiparallel pairs through ring inte!- actions (distance between centroids of 3.52 A) [16], and by a short contact between the centroid of the ring and Cl5 (3.686 A), and H152 (3.38 A).

Infrared data in the solid state confirmed the zwitterion structure of compounds 1 and 2. Pro- tonation of N3 was observed by the two broad stretching absorptions in the regions at ca 2500 and ca 1900 cm-‘. These bands corresponded to +NH stretching linked by strong hydrogen bond- ing as it has been found previously for similar compounds [17-191.

As it has been stated, NMR solid-state chemical shifts generally are very similar to those found in solution. Thus, the CP/MAS data can be used as a ‘bridge’ between solid-state structures deter- mined by diffraction techniques and those which exist in solution [20]. 13C NMR chemical shifts values of compounds l-6 in the solid state and in solution are depicted in Table 3. Although it has been reported previously [3], that compound 1 presented a zwitterion structure in DzO, it must be taken into account that the pH of the solution is decisive in the formation of the zwitterion structure, as is observed in amino acids. We have recently reported that compound 1 presented a pKD2 (N+) value of 7.23 [4,5]. Considering the pKD (pK, in DzO) and pKH (pK, in H20) relation- ship [22] pKD = 1.018 pKH + 0.43; pKHz of com- pound 1 is 6.68. On the other hand, the pKH’ (COOH) calculated here is 2.1. If we consider com- pound 1 as an amino acid, these values corre- sponded to an isoelectric point (Ip) of 4.4 The value of the pH (4.70) obtained when compound 1 was dissolved in H20 suggested that the zwit- terion form is a maximum in Hz0 solution. This fact is confirmed because of the values of the chemi- cal shifts of all carbons fitted well with those found in solid state (Table 3). Furthermore, the “0 chemi- cal shift value (27 1.1 ppm) of the carboxylic group, is also in agreement with those found in other amino acids in zwitterionic form [23].

The described pKD2 value of compound 2 was 6.86 [4] which corresponds with a pKHZ of 6.32. The carboxylic group has a pKH1 of 1.3.

These values give an Ip of 3.8. This value suggest that at 3.30, pH of the solution of compound 2 in Hz0 the contribution of the zwitterion form is slightly lower than that observed in compound 1. This fact is also corroborated by the data obtained in the solid state and the “0 NMR chemical shift value (257.0 ppm) which corresponds well with those values reported for other amino acids with protonated carboxyl O-atoms [23].

In the absence of X-ray data it could be inferred that IR data of diacid 5 in KBr suggested that the a-COOH (1597 cm-‘) is ionized in the solid state but not the ,&COOH, as has been described for other dicarboxylic mono-amino acids [24]. The 13C NMR data in the solid state and in solution also suggested a similar behaviour to compound 2 in solution.

Finally, we can conclude that this work unam- biguously proves that imidazol-1-ylacetic acids behave as amino acids and present zwitterion structures in the solid state and in solution within a certain pH range. In the solid state the packing of ions is governed by strong hydrogen bonds which play a significant role in the structural organization. The presence of rigid hydrogen- bonded aggregate structures in these acids makes them especially attractive from the crystal engi- neering point of view [25].

Acknowledgements

This work was supported in part by D.G.I.C.Y.T. (PM-92-01 1, PB-93-0037, and PB-93-0125) and Community of Madrid (AE- 00219/94). P.L. and P.Z. received F.P.U. fellow- ships from the Spanish Ministry of Education and Science. We are indebted to Dr C. Lopez for her valuable technical assistance in the per- formance of CP/MAS spectra.

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