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C 2004) Journal of Solution Chemistry, Vol. 33, No. 10, October 2004 (

Complexation of a Macrocycle Containing Thiopyrimidine and Uracil Moieties with Dicarboxylic and Amino Acids in Various Organic and Aqueous Organic Solutions A. R. Mustafina,1,∗ L. S. Kuznetsova,1 A. S. Michailov,1 V. E. Kataev,1 A. I. Konovalov,1 and W. D. Habicher1 Received March 2, 2004; received August 10, 2004 The complexation of a macrocycle containing thiopyrimidine and uracil moieties (M) with amino acids and some dicarboxylic acids was studied by pH-metric, UV-VIS, 1 H NMR spectroscopy methods in chloroform, methanol, aqueous 1,4-dioxane, and biphasic water–chloroform media. The complexation of M with acids is too weak to solubilize them from the solid state into chloroform solutions containing M. The 1 H NMR spectra and pH-metric data of aqueous 1,4-dioxane (80 vol.%) reveal the pHdependent 1:1 binding between M and the acids studied. The protonation of M is not a prerequisite for binding of fumaric, succinic, o-phtalic acids and the series of amino acids, whereas binding of maleic acid requires the protonation of both thiopyrimidine moieties of M. Therefore, M·(H+ )2 exhibits strong selectivity towards maleic acid in aqueous 1,4-dioxane and in biphasic water–chloroform media. KEY WORDS: Amino acids; nitrogen-containg macrocycles; pyrimidines; complexation; pH-potentiometry; 1 H NMR spectroscopy.

1. INTRODUCTION In the past decade, a variety of cleft and macrocyclic structures, aligned with convergent functional groups, have been prepared for selective substrate recognition. Clefts and macrocycles with nitrogen-containing building blocks are interesting for their complexing ability towards carboxylic acids and anions.(1−12) The preliminary organization of the nitrogen binding centers plays a key role in the selectivity of substrate recognition.(5) Most of the receptors, made up of amide 1 Kazan

Scientific Centre of the Russian Academy of Sciences, A.E. Arbuzov Institute of Organic and Physical Chemistry, Arbuzov Str. 8, Kazan, 420088, Russia; e-mail: [email protected]. 1257 C 2004 Springer Science+Business Media, Inc. 0095-9782/04/1000-1257/0

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Mustafina, Kuznetsova, Michailov, Kataev, Konovalov, and Habicher

groups are able to bind both anions and acids.(1−6) Macrocyclic polyamines as well as porphyrin- or supphyrin-based macrocycles can act as receptors of anions in aqueous media.(7−12) The main driving force for binding of carboxylic acids or their anions in nonpolar media is intermolecular hydrogen bonding,(1−3) just as in aqueous or aqueous organic media intermolecular hydrogen bonding is considerably hindered by hydration and both electrostatic and stacking interactions become predominant.(11) It is also known that the investigation of the interactions between polyamines and both inorganic and organic anions is a contribution to speciation studies of biofluids.(13) The synthesis and acid–base properties of the novel macrocycle M have been recently studied in our group.(14) Macrocycle M has two aminothiopyrimidine fragments, each of them can be proposed to fit to a carboxylic group. Therefore, M can be predicted to bind polyfunctional carboxylic acids, such as amino acids, oxyacids, and polycarboxylic acids via intermolecular hydrogen bonding. It has also been shown that M undergoes stepwise protonation at pH < 6 in homogeneous and biphasic aqueous organic media.(14) Thus, both M itself and its protonated forms can bind polyfunctional carboxylic acids and their anions in polar aqueous organic media. The main goal of the work presented here is to study the pH-dependent complexation of M and dicarboxylic (fumaric, maleic, o-phtalic, and succinic acids) and the series of amino acids (L-Ala, L-Val, L-His, L-Cys, L-Tyr, L-Ser, L-Orn, L-Asp, L-Glu, L-Asn) in aqueous 1,4-dioxane, methanol, and in biphasic water–chloroform media by 1 H NMR spectroscopy and pH-potentiometry methods. 2. EXPERIMENTAL The macrocycle discussed here, namely 32-trimethy1-23,36-dioxo-15,19dithio-2,9,13,20,24,31,34,35-octaaza-tetracyclo[28,3,110,14 ,120,24 ]hexatriacontahep-taen-1(34), 10(35),11,13,21,30,32 (M) was synthesized according to a procedure published earlier.(14) Methanol, 1,4-dioxane, and CHCI3 (all solvents of commercial grade) were purified by usual procedures.(15) The water was bidistilled. Dicarboxylic and amino acids were of “chemically pure” grade (Reakhim, Russia). 1 H NMR spectra were recorded on Bruker WM-250 and Bruker DRX-500 spectrometers at 250.13 and 500 MHz, respectively, in 1,4-dioxane-d 8 , D2 O with 1,4-dioxane-d 8 mixtures, CDCI3 , and CD3 OD at 25◦ C. The 1 H NMR-titration in CDCI3 was carried out by the addition of a fixed aliquot of M solution to a maleic acid solution in CDCI3 . The 1 H NMR-titrations in CD3 OD and D2 O-1,4-dioxaned 8 were carried out by the addition of a fixed aliquot of DCI, maleic, or fumaric acid solutions to M solutions in the respective solvent. The chemical shift values are reported relative to TMS.

Complexation of a Macrocycle Containing Thiopyrimidine and Uracil Moieties

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The pH-metric measurements were carried out in a thermostatically controlled cell at (25.0 ± 0.1)◦ C using an “I-130 Ionomer” meter with a precision less than 0.05 pH-units. HCI (1 × 10−2 mol-L−1 ) solution was used as the titrant. The pH-meter was calibrated with a series of buffer solutions. The measurement of the pH of mixed aqueous-organic solutions was based on the standardization of a glass electrode with aqueous buffer solutions followed by an evaluation of the liquid-junction potential according to an established procedure.(16) To evaluate the binding constants and the stoichiometry of complexation, the pH-titration of M, guest, and the mixture of M and guest in the concentration ratio 1:1 and 1:2 were recorded in the range of pH 2.5–9.0 with the M concentration being 2 × 10−3 mol-L−1 . No extra salts were added in the pH-measurements to maintain a definite ionic strength value in order to avoid undesirable association equilibria.(17) Consequently, the value of ionic strength results from the ions involved in the titrations and is in the range 2 × 10−3 to 4 ×10−3 mol-L−1 . The titrations were carried out at a complexation degree between 20 and 85%. The mathematical treatment of the pH-metric data (30–40 experimental points) to calculate the K (i) -values (K (i) is the constant of ith equilibrium) was performed iteratively using the CPESSP computer program.(18) The mathematical treatment by CPESSP is based on a search for the best fit between the experimentally observed Bierrum function (n˜ exp ), Eq. (1), and their theoretical analogs calculated from the law of mass action (n˜ calc ) by finding the minimum F function, Eq. (2), by iteration. C0M ·V0 VHCl ·CHCl −pH n˜ = 2 − (1) − 10 / V0 + VHCl V0 + VHCl F= [(n˜ exp − n˜ calc )wm ]2 (2)

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where m varies from 1 to N (N—number of experimental points), wm —a meansquare error. The validity of the chosen model was evaluated using transformed Fisher’s criteria, Fmin : Fmin ≤ F·σ 2 (N − 2k)

(3)

where σ represents the variance and k is the number of complex species. All pH-metric measurements have been performed twice. The pH measurements of M aqueous dioxane solutions with various quantities of maleic and fumaric acids added were performed at the same concentration conditions as for the NMR titrations. The pK and log K(i) values obtained from parallel measurements are within the combined standard deviations (Table I). The extraction procedure is as follows: 5 mL of chloroform solution of M 2 × 10−3 mol-L−1 was mixed with an equal volume of aqueous solution, which contains the guest acid (2 × 10−3 mol-L−1 ) and a definite concentration of hydrochloric acid (4 × 10−3 mol-L−1 and 1 × 10−2 mol-L−1 for the data presented on the Figs. 12 and 13, respectively). The mixture was stirred for 60 min at room temperature (25.0 ± 0.2)◦ C with a further pH-measurement of the aqueous phase after phase separation. The equilibrium concentration of M in the organic phase was determined spectrophotometrically at 260 nm. The UV-spectra were recorded on a “Specord UV-VIS" spectrophotometer. 2

3. RESULTS AND DISCUSSION 3.1. 1 H NMR Spectroscopic Data The NMR titration is the most useful method to study host–guest complexation. According to literature data chloroform is a suitable medium for studying the interaction between M and carboxylic acids, driven by intermolecular hydrogen bonding.(1−3) The series of amino acids and some dicarboxylic acids were treated as guests. However, only maleic acid (H2 Mal) is soluble enough in chloroform to carry out the 1 H NMR-titration in CDCl3 . The titration of M by H2 Mal results in the downfield shift of the protons of aminothiopyrimidine fragments (Fig. 1). The pyr pyr signals of Har and CH3 protons undergo a detectable down-field shift, but the most shifted is the signal of NH-protons. The dependence obtained reveals that saturation is not achieved at nearly a twofold excess of H2 Mal, and a larger excess cannot be obtained because of the restricted solubility of H2 Mal in CDCl3 . None of the amino and dicarboxylic acids studied were efficient in the solubilization and binding with M in CHCl3 -d. Therefore, a more polar medium should be used to enhance the solubility of carboxylic acids. As shown recently,(14) the NMR spectra of M changes on going from CHCl3 -d to methanol-d 4 , the signal (broad singlet) of NH-protons collapses and two triplets of α-CH2 protons overlap into one broad singlet. The spectrum of m in 1,4-dioxane-d 8 is similar to those in

“guests”-amino acids (HA± )

4.0 ± 0.1 3.4 ± 0.1 2.7 ± 0.1

6.53 ± 0.04

orto-phtalic 5.19 ± 0.02

7.60 ± 0.02

fumaric

4.0 ± 0.1 3.8 ± 0.1 3.0 ± 0.1 3.7 ± 0.1 2.7 ± 0.4 3.7 ± 0.1 3.90 ± 0.04 5.90 ± 0.05 4.4 ± 0.1 5.6 ± 0.1

4.37 ± 0.04

4.36 ± 0.03

2.71 ± 0.07

3.93 ± 0.03

4.50 ± 0.02

4.24 ± 0.02

3.64 ± 0.02

4.74 ± 0.02

4.04 ± 0.03

4.3 ± 0.1

Ala

Val

His

Cys

Tyr

Ser

Orn

Asp

Glu

Asn

log K(4) [M + HA± (M·HA± )]

—

3.73 ± 0.03

maleic

succinic

log K(4) [M + H2 A M·H2 A]

pK1

(H2 A)

4.31 ± 0.09

4.4 ± 0.2

4.03 ± 0.03

4.94 ± 0.03

—

—

4.8 ± 0.1

6.18 ± 0.07

—

4.2

4.62

—

4.35

—

—

4.9

—

—

—

log K(10) [M·H+ )· (HA± ) + H+ M·(H+ )·(H2 A+ )]

log K(9) [(M·HA± ) + H+ M·(H+ )·(HA± )] —

—

—

—

3.24 ± 0.05

5.1 ± 0.1

4.9 ± 0.1

4.8 ± 0.1

—

log K(5) log K(8) [M·H2 A + H+ M·(H+ )·H2 A] [M·(H)2 + H2 A M·(H+ )2 ·H2 A]

8.5 ± 0.1

9.0 ± 0.1

—

9.29 ± 0.06

9.38 ± 0.07

10.3 ± 0.1

9.7 ± 0.1

—

9.5 ± 0.1

9.5 ± 0.1

log K(11) [(M·HA± ) + 2H+ M·(H+ )2 ·(HA± )]

—

—

—

—

pK1 and log K(i) Values, where K1 is the Dissociation Constant of Dicarboxylic and Amino Acids, K(i) is the Constant of Equilibria Presented

“guests”-dicarboxylic acids

Table I.

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Fig. 1. Plot of chemical shift changes (δobs ) of M protons (Har , NH, CH3 ) versus Cmaleic acid /CM concentration ratio in CDCl3 and D2 O/1,4-dioxane-d8 (80 vol.%).

CHCl3 -d with the exception of the NH-protons, whose resonances appear as two triplets in 1,4-dioxane and a broad singlet in CHCl3 -d. The addition of water on going to aqueous 1,4-dioxane provides similar changes to that observed in the case of methanol (Fig. 2). So, both methanol and aqueous 1,4-dioxane (80 vol.%) are the most convenient media for carrying out the 1 H NMR titration. However, intermolecular hydrogen bonding becomes less efficient on going from CDCl3 to more polar media,(11) though the protonation of M may enhance its complexation with carboxylic anions. The protonation of M in aqueous methanol and 1,4-dioxane was found(14) to proceed stepwise. An analysis of the distribution of the various species as a function of pH in aqueous 1,4-dioxane (Fig. 3) reveals that the accumulation of M·H+ is no more than 50% and overlaps with M and M·(H+ )2 in neutral media, where dicarboxylic acids occur in both acidic and monoanionic forms. Thus, to evaluate the interaction of M, M·H+ , and M·(H+ )2 with dicarboxylic acids the 1 H NMR data should be analyzed at various pH values and protonation– deprotonation equilibria occurring in host–guest mixture should be taken into account. The protonation in aqueous 1,4-dioxane results in a more enhanced downfield shift (0.48 ppm) (Fig. 4) than in methanol (0.35 ppm).(14) The proton signals of two pyrimidine residues are very close to each other in aqueous 1,4-dioxane pyr pyr (δ = 0.01 for Har and δ = 0 for CH3 ), being differentially displaced during


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Fig. 2. 1 H NMR spectra of M in 1,4-dioxane-d8 (a) and in aqueous (D2 O) 1,4-dioxane-d 8 with 80 vol.% (b), 70 vol.% (c), and 60 vol.% (d) of organic component.

the titration by DCl. As is evident from Fig. 5, the difference between the δ values of pyrimidine residues (δ1 –δ2 ) correlates with the formation degree of M·H+ (α1 = [M·H+ /CM , where [M·H+ ] is the equilibrium concentration of M·H+ and C M is the total concentration of M), being the greatest during the addition of one equivalent of HCl, when α1 is at a maximum. According to the data presented in Fig. 6, the signals of M protons experience a pronounced down-field shift with increasing maleic (H2 Mal) and fumaric

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Fig. 2. Continued

(H2 Fum) acid concentrations in aqueous 1,4-dioxane-d 8 (80 vol.%) and methanold 4 . The down-field shifts experienced by protons of the pyrimidine fragments pyr pyr (Har , CH3 ), δ- and α -CH2 protons next to the pyrimidine fragments are much more significant than the downfield shifts of Har and CH3 protons of the uracil moiety. H2 Fum was found to provide the least enhanced downfield shift of the M proton signals compared to those provided by H2 Mal (Fig. 6). The saturation conditions for H2 Fum in aqueous 1,4-dioxane-d 8 (80 vol.%) do not occur even at a 16-fold excess of titrant, whereas in the case of H2 Mal saturation is achieved at


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Fig. 3. The distribution of various species versus pH in aqueous 1,4-dioxane (80 vol.%) solution of M.

Fig. 4. The plot of δobs (Har , CH3 , CH2 -δ protons) versus CDCl /CM in aqueous 1,4-dioxaned8 (80 vol.%), δobs = δobs − δfree , where δobs is the averaged δ-values of thiopyrimidine moieties (δ1 and δ2 ).

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Fig. 5. The dependence of both (δ1 − δ2 ) values (the difference between proton signals from two pyrimidine fragments) and formation degrees of M, M·(H+ ), and M·(H+ )2 (αM , α1 , and, α2 ) on CDCl /CM in aqueous 1,4-dioxane (80 vol.%).


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Fig. 6. The plot of chemical shift changes (δobs ) of M protons (Har , CH3 , CH2 -α, CH2 − δ) versus Cacid /CM (fumaric and maleic acids) in (a) CH3 OH-d4 and (b) D2 O-1,4-dioxane-d8 (80 vol.%), pyr pyr δobs = δobs − δfree , where δobs for Har and CH3 protons is the averaged δ-values of thiopyrimidine moieties (δ1 and δ2 ).

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Fig. 7. The dependence of the (δ1 –δ2 ) values of M proton signals during the addition of (a) maleic and (b) fumaric acids on Cacid /CM concentration ratio.

a 5–6-fold excess (Fig. 6b). The downfield shift of the M proton signals provided by H2 Mal and H2 Fum can be induced by the protonation of M, which is greater in the case of H2 Mal than in the case of H2 Fum, because the K1 value of H2 Fum is smaller (Table I). However, a comparison of the down-field shift of M caused by both acids in methanol and in aqueous 1,4-dioxane reveals that they are more enhanced in methanol than in aqueous 1,4-dioxane (Fig. 6). This is not in accordance with the acidity of H2 Fum and H2 Mal in aqueous 1,4-dioxane ( pK 1 = 6.53 for H2 Fum and 3.73 for H2 Mal; Table I) and in methanol(19) ( pK 1 = 8.01 for H2 Fum and 6.35 for H2 Mal). The dependence of the difference (δ1 − δ2 ) between the chemical shifts of the protons of two pyrimidine moieties of M on the H2 Fum and H2 Mal concentrations (Fig. 7) indicates that H2 Fum protonates M mainly in one step, whereas both pyrimidine moieties are protonated in an excess of H2 Mal. If no interaction occurs between M and dicarboxylic acids the dependence of the down-field shifts of M on pH, caused by the increase of H2 Fum and H2 Mal concentrations should fit to those provided by HCl. Figure 8 illustrates the chemical shifts (δobs ) and pH values during the addition of various amounts of H2 Fum and H2 Mal as well as the chemical shifts (δ p ) and saturation extents (α) of M, M·H+ , and M·(H+ )2 , produced by HCl at the same pH values. The deviation between δobs and δ p indicates that the protonation is not the only phenomenon providing the down-field shift of M in the presence of maleic and fumaric acids. 3.2. pH-Metric Data 3.2.1. Dicarboxylic Acids The acid–base properties of both M and the guests studied provide the applicability of pH-potentiometry for evaluation of binding constants. When mixing in a 1:1 molar ratio in aqueous organic solutions, the guest-acid dissociates and M


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pyr

Fig. 8. The experimentally observed chemical shifts of Har protons (δobs ) of M and those calculated for (αM , α1 , and α2 ) as a function of various Cacid /CM and associated pH values, where the acid is maleic (a) and fumaric (b).

is protonated with the deprotonation and protonation degrees depending on both pH and the K (i) values of M·(H+ )2 and the guest-acid. As mentioned above, the protonation degree depends on the pK1 value of the “guest." Thus, H2 Fum being in a 1:1 molar ratio does not lead to the essential protonation of M, whereas H2 Mal does in accordance with its K n values (Table I). The titration of a host–guest mixture by HCl results in further protonation of both participants. In this case, the experimentally observed Bjerrum function values (n˜ exp ) should be fitted to those calculated for the dissociation of the guest acid and protonation of M, which are independent from each other (n˜ calc ). However, the detectable difference in n˜ exp and n˜ calc versus pH occurs in aqueous 1,4-dioxane (80 vol.%) at a 1:1 concentration ratio of M and H2 A (A = Fum or Mal) (Fig. 9). The difference in pH values at the same values of n˜ exp and n˜ calc in the case of H2 Fum is not within the combined experimental error (up to 0.45), indicating an interaction in a host–guest system. Thus, the n˜ exp versus pH curve lying above the n˜ calc versus pH (Fig. 9) curve indicates that mixing of M with H2 Fum, as well as with succinic and o-phtalic acids in a 1:1 concentration ratio, results in acidifying the medium, in turn showing

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Fig. 9. The plot of n˜ obs and n˜ calc versus pH for the pH-metric titration of M with maleic, fumaric, o-phtalic, and succinic acids in 1:1 concentration ratio (CM = 2 × 10−3 mol-l−1 , CHCl = 1 × 10−2 mol-l−1 ) in aqueous 1,4-dioxane (80 vol.%).

that some equilibria prevent the protonation of M in these systems. To accomplish the fitting between n˜ exp and n˜ calc , the equilibria (4) and (5) should be taken into account. M + H2 A M·H2 A

(4)

M·H2 A + H+ M·(H+ )·H2 A

(5)

The constants for equilibria (4) and (5) for fumaric, succinic, and o-phtalic acids are presented in Table I. The distribution of the various species on pH in aqueous 1,4-dioxane solutions of M, and M with H2 Fum in the 1:1 concetration ratio is shown in Figs. 3 and 10a, respectively. The accumulation of M·H2 A reaches a maximum at pH = 6, turning into M·(H+ )·H2 A with the acidification of the medium. The accumulation of M·(H+ )·H2 A reaches its maximum at pH = 4.5, decreasing and turning into M·(H+ )2 with further decrease of pH. According to the data presented, the protonation constant of M·H2 A is smaller than the constant of the first-step protonation of M, Eq. (6), log K (6) = 5.40,(14) but similar to the


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Fig. 10. The distribution of various species versus pH in aqueous 1,4-dioxane (80 vol.%) solution of M with H2 Fum in a 1:1 concentration ratio (a) and M with H2 Mal in a 1:1 concentration ratio (b).

constant for the second-step protonation of M, Eq. (7), log K (7) = 4.58.(14) M + H+ M·(H+ ) +

+

(6) +

M·H + H M·(H )2

(7)

So, it seems that only one thiopyrimidine moiety of M participates in the binding of H2 A, because the one-step protonation of M·H2 A occurs with a constant very close to log K(7) and the second-step protonation destroys the complex. In accordance with its pK1 value (Table I), H2 Mal when mixed with M results in more enhanced protonation of M. The curve n˜ exp versus pH lies below that for n˜ calc versus pH (Fig. 9), indicating that maleic acid promotes the protonation of M. The complex M·(H+ )2 ·H2 Mal formed according to Eq. (8) with log K(8) presented in Table I provides the best fit between n˜ exp and n˜ calc . M·(H+ )2 + H2 Mal M·(H+ )2 ·H2 Mal

(8)

As is evident from Fig. 10b, the accumulation of M·(H+ )2 ·H2 Mal increases with the decrease of pH. The down-field shifts induced by complex formation of M with H2 Fum and H2 Mal (δcomplex ) were evaluated from the 1 H NMR data using binding constants extracted from the pH-metric data and are presented in the Table II. The data obtained indicate that binding with H2 Fum practically does pyr not change the chemical shifts of the Har resonances of both M (compare 5.88 and 5.86 ppm) and M·H+ (compare 6.21 and 6.31 ppm), whereas complexation of pyr M·(H+ )2 with H2 Mal decreases the chemical shift of Har from 6.36 to 6.16 ppm. So, the data obtained reveals the peculiar features of the binding between M and H2 Mal. It seems very probable that both protonated thiopyrimidine fragments of M·(H+ )2 participate in binding with H2 Mal.

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Mustafina, Kuznetsova, Michailov, Kataev, Konovalov, and Habicher pyr

Table II. δcomplex Values of Har Resonances Calculated from the 1 H NMR Titration Data for Complexes with Maleic and Fumaric Acids Complex

δcomplex

M·H2 Fum M·(H+ )·H2 Fum M·(H+ )2 ·H2 Mal

5.86 ± 0.02 6.31 ± 0.05 6.16 ± 0.06

3.2.2. Amino Acids According to the pH-metric data amino acids also exhibit the deviations between n˜ exp and n˜ calc , which are similar to those produced by fumaric acid. The deviation between n˜ exp and n˜ calc is different for various amino acids, as it is shown for L-Asp and L-Glu (Fig. 11). According to the data presented in Table I the tightness of binding is greatest for Asn and Asp and decreases in the following order: Asp > Asn > Glu > Ala = Orn = Ser = Cys = Val > His = Tyr. The log K(4) value for Asp is much larger than for succinic acid, indicating the importance of the NH+ 3 moiety in complexation with M. The log K(4) value is much greater for Asp than that for Glu, indicating that the fitting of the two binding centers of the guest to those of the host is also very important. So the data obtained indicate that the availability of the two carboxylic groups of the guest results in a more pronounced effect on the log K (4) value than the presence of aliphatic or aromatic moieties of the guest. Unlike the dicarboxylic acid complex, M·(HA± ) (HA± is an amino acid) undergoes, both one-step and two-step protonation, Eqs. (9)

Fig. 11. The plot of n˜ obs and n˜ calc versus pH for the pH-metric titration of M with aspartic (a) and glutaric (b) acids in a 1:1 concentration ratio (CM = 2 × 10−3 mol-l−1 , CHCl = 1 × 10−2 mol-l−1 ) in aqueous 1,4-dioxane (80 vol.%).


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and (10). M·(HA± ) + H+ M·(HA± )·(H+ ) ±

+

+

±

(9) +

M·(HA )·H + H M·(HA )·(H )2

(10)

Assuming that amino acids are able to bind protons through protonation of their carboxylate group, the second-step protonation of the complex M·(HA± )·(H+ ), Eq. (10), may bind via the carboxylate group of HA± or via the thiopyrimidine fragment of M, resulting in M·(H+ )·(H2 A+ ) and M·(H+ )2 ·(HA± ), respectively. The constant for equilibrium (9) (Table I) is rather close to the constant for the second-step protonation of M, Eq. (7), for Cys and Orn. The two protonation steps are indistinguishable for the complexes M·(HA± ), where HA± is Ala, Val, Tyr, and Ser, and protonation occurs according to Eq. (11). The log K(11) values are also presented in Table I. (M·HA± ) + 2H+ M·(H+ )2 ·(HA± )

(11)

The log K(9) value for complex M·(His± ) is greater, whereas the corresponding values for the complexes M·(HA± ), HA± = Glu, Asp, and Asn, (Table I) are smaller than log K(7) , indicating that protonation of M·(His± ) strengthens and protonation of M·(HA± ), HA = Glu, Asp and Asn, weakens the host–guest binding. Therefrore, for the most of amino acids the protonation of thiopyrimidine fragments of M is not a prerequisite for their binding, indicating that the thiopyrimidine fragments act as hydrogen-bond acceptors in host–guest binding. 3.3. Complexation in the Biphasic System Chloroform/Water No detectable extraction occurs from aqueous solutions of all the acids studied into chloroform solutions of M. The acidification of the aqueous phase was found to result in the extraction of M·(H+ )2 into the aqueous phase.(14) The quantitative analysis of the distribution data (q = [M]aq /[M]org , where [M]aq and [M]org are equilibrium concentrations of M in aqueous and organic media) reveals that the presence of fumaric, o-phtalic, and succinic acids, and the series of amino acids (L-Ala, L-Val, L-His, L-Cys, L-Tyr, L-Ser, L-Orn, L-Asp, L-Glu, and L-Asn) does not affect the extraction of M·(H+ )2 . Figure 12 shows that the extraction of M into aqueous solutions of L-Asp, succinic, fumaric, and o-phtalic acids is determined by the pH of the aqueous phase. H2 Mal was found to be the only one among those studied that enhances the extraction of M·(H+ )2 . This fact indicates that the complex between M·(H+ )2 and maleic acid exists even in the aqueous phase. The quantitative analysis of the extent of extraction of M·(H+ )2 versus H2 Mal concentration at pH = 2 with the help of the so-called “log–log" procedure proves the 1:1 stoichiometry for binding (Fig. 13) according to Eq. (8). Based on the

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Fig. 12. The plot of log q versus pH in the presence of different acids in the aqueous phase.

data obtained it appears interesting to verify the effect of the water content in aqueous 1,4- dioxane mixtures on the interaction between M·(H+ )2 and H2 Mal. The deviation between n˜ exp and n˜ calc is still detectable for H2 Mal in aqueous 1,4dioxane solutions with 50 vol.% of organic component (Fig. 14), just as the lack of a deviation is observed for the other acids studied. 4. CONCLUSIONS The 1 H NMR spectroscopic data for aqueous 1,4-dioxane (80 vol.%) establishes that the protonation of M occurs in a stepwise manner. The quantitative analysis of 1 H NMR and pH-metric data provide evidence for the interaction between M, as well as its protonated forms, and some dicarboxylic acids and the series of amino acids in aqueous 1,4-dioxane (80 vol.%). The pH-metric data reveal that the protonation of M is not a prerequisite for binding of fumaric, succinic, and o-phtalic acids, and the series of amino acids, whereas binding of maleic acid requires the protonation of both thiopyrimidine moieties of M. Consequently, M·(H+ )2 exhibits sharp selectivity towards maleic acid in aqueous 1,4-dioxane and in biphasic water–chloroform media. The dependence of the binding constants of M with carboxylic and amino acids on the structure of the acid indicates that this binding is determined mainly by polar interactions, such as electrostatic or hydrogen bonding between the thiopyrimidine moieties of the host and the NH+ 3 moiety of the guest, whereas aromatic ring stacking interactions, which are


Fig. 13. The plot of log q versus log [H2 Mal] in the presence of various amounts of H2 Mal in the aqueous phase at pH = 2.

Fig. 14. The plot of n˜ obs and n˜ calc versus pH for the pH-metric titration of M with maleic acid in a 1:1 concentration ratio (CM = 2 × 10−3 mol-L−1 , CHCl = 1 × 10−2 mol-L−1 ) in aqueous 1,4-dioxane (50 vo.%).

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predominant for supphyrines,(12,20) play a minor role in the complexation of M with amino acids in aqueous 1,4-dioxane. ACKNOWLEDGMENT This work was supported by the Russian grant NSh-2030.2003.3. REFERENCES 1. V. Alcazar, J. R. Moran, and F. Diederich, Isr. J. Chem. 32, 69 (1992). 2. T. Hirose, K. Naito, H. Shitara, H. Nohira, and B. W. Baldwin, Tetrahedron: Assymetry 12, 375 (2001). 3. I. Stibor, D. S. M. Hafeed, P. Lhotak, J. Hodacova, J. Koca, and M. Cajan, Gazz. Chim. Ital. 127, 673 (1997). 4. U. Lucking, D. M. Rudkevich, and J. Rebek Jr., Tetrahedron Lett. 41, 9547 (2000). 5. P. Lustenberg, R. Welti, and F. Diederich, Helv. Chim. Acta 81, 2190 (1998). 6. F. Werner and H. J. Schneider, Helv. Chim. Acta 83, 465 (2000). 7. R. W. Hoffmann, F. Hettche, and K. Harms, Chem. Commun. 782 (2002). 8. A. Bencini, M. A. Bernardo, A. Bianchi, V. Fusi, C. Giorgi, F. Pina, and B. Valtancoli, Eur. J. Inorg. Chem. 1911 (1999). 9. Yo. Shi and H.-J. Schneider, J. Chem. Soc., Perkin Trans. 2, 1797 (1999). 10. C. Bazzicalupi, A. Beconcini, A. Bencini, V. Fusi, C. Giorgi, A. Masotti, and B. Valtancoli, J. Chem. Soc., Perkin Trans. 2, 1675 (1999). 11. T. Mizutani, K. Wada, and S. Kitagawa, J. Am. Chem. Soc. 121, 11425 (1999). 12. J. L. Sessler, A. Andrievsky, V. Kral, and V. Lynch, J. Am. Chem. Soc. 119, 9385 (1997). 13. A. De Robertis, C. De Stefano, C. Foti, O. Giuffre, and S. Sammartano, Talanta 54, 1135 (2001). 14. L. S. Kuznetsova, A. R. Mustafina, A. S. Michailov, V. E. Kataev, and V. S. Reznik, J. Solution Chem. 31, 895 (2002). 15. A. J. Gordon and R. A. Ford, The Chemist’s Companion. A Handbook of Practical Data, Techniques and References (Wiley-Interscience, 1972, 542 p.). 16. F. Jordan, J. Phys. Chem. 77, 2681 (1973). 17. M. Sirish and H.-J. Schneider, Chem. Commun. 23 (2000). 18. Yu. I. Sal’nicov, F. V. Devyatov, N. E. Zhuravleva, and D. V. Golodnitskaya, Zh. Neorg. Khim. 29, 2273 (1984) [J. Inorg. Chem. USSR (Trans.) 29, 1299 (1984)]. 19. F. Rived, I. Canals, E. Bosch, and M. Roses, Anal. Chim. Acta 439, 315 (2001). 20. J. L. Sessler and J. M. Davis, Acc. Chem. Res. 34, 989 (2001).