Binding ability of thiocarbonyl-containing dipeptides. Potentiometric and spectroscopic studies of copper(II) and nickel(II) coordination

Binding Ability of Thiocarbonyl-containing Dipeptides. Potentiometric and Spectroscopic Studies of Copper(I1) and Nickel(I1) Coordination Katalin Vamagy, Henryk Kozlowski, Imre Sovago, Teresa Kowalik-Jankowska, Marian Kruszynski, and Jolanta Zboinska KV, IS. Institute of Inorganic and Analytical Chemistry, Kossuth University, Debrecen, Hungary.-HK, TKI. Institute of Chemistry, University of Wrocfaw, Wroclaw,

Poland.--MK,

JZ. Institute of Chemistry, University of Gdansk, Gdansk, Poland.

ABSTRACT Complex formation reactions of thioamide-containing dipeptides (Alat-Ala-OMe and Asp-Phet-NHr) with copper(H) and nickel(R) were studied by the pH-metric and spectroscopic methods. Sulfur donation was established in acidic and neutral media, and it resulted in the enhanced stability of the respective complex species. Already, at pH < 3, the S to metal charge transfer band indicated the { NHr,S} coordination in cupric complexes. The high stability constants of CuA and CuA2 complexes in comparison to respective complexes with glycine-amide (two and five orders, respectively) also indicate the involvement of sulfur in the metal ion coordination. The thiocarbonyl donor is involved in coordination of nickel@) ions as well. The stability constants of respective complexes are enhanced, and, according to the spectroscopic data, the NiAr species is octahedral. It was also found that the coordination of thiocarbonyl donor although it is very effective, cannot prevent an amide nitrogen deprotonation and its coordination to metal ion in basic solutions. According to absorption and c.d. spectra in basic solutions, metal ions are bound to { NH2,N - } donor sets, and resulting complexes MArH _ r are tetragonal for copper and square-planar for nickel ions.

INTRODUCTION Recent studies on the thioamide analogues of amino acids and peptides have shown that presence of the -CS-NHmoieties has a considerable effect on the biological

Address reprint requests to Prof. H. Kozlowski, Joliot-Curie 14, 50383 Wroclaw, Poland.

Instytut Chemi,

Uniwersytetu

Wroclawskiego,

Journal of Inorganic Biochemistry 34, 83-93 (1988) 0 1988 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, NY, NY 10010

ul. F.

83 0162-0132/88/$3.50

84 K. Varnagy et al.

activity and on the coordination ability of various molecules [l-3]. Equilibrium and spectroscopic results obtained for the copper(B) and nickel(B) complexes revealed that the thiocarbonyl group is a much more effective donor than carbonyl oxygen, and its introduction into a peptide linkage can significantly change the binding ability of a peptide ligand [3]. Although the { NHz,CS} coordination is very favorable when compared to the corresponding { NH*,CO } chelation, the sulfur donation cannot prevent an amide deprotonation, and the coordination of an amide nitrogen to a metal ion in the basic solutions is observed. The sulfur donation is largely dependent on the neighboring amino acid residues [46]. Also, the peptide linkage binding ability may depend considerably on the peptide sequence [4, 7, 81; therefore, we have undertaken further study on the metal ion interaction with dipeptide thiocarbonyl analogues: L-alanyl-alanine-thiopeptidemethylester (Alat-Ala-OMe), L-aspartyl-L-phenylalanine-thioamide (Asp-Phet-NHa), and its nonthio analogue; the results are presented in this paper. For the details of the thioamide nomenclature, see Ref. 191.

EXPERIMENTAL Thiocarbonyl analogues were prepared with Lawesson’s reagent [ 1, 10, 1 l] using the classical procedure in solution. All were in hydrochloric form. The purity of the ligands has been checked by pH-metric titration. In the pH-metric studies, the ligand concentration varied between 1 and 4 x 10m3 mol dme3, while the metal ion to ligand ratios ranged from 1: 1 to 1:4. The 5 cm3 samples were titrated with carbonate free potassium hydroxide, and the “reversibility” of the complex formation was checked by the “back titration” with the hydrochloric acid. Argon was bubbled through the samples to ensure the absence of molecular oxygen and carbon dioxide and for the stirring of the solutions. All pHmetric studies were carried out at 298 K, at a constant ionic strength of 0.2 mol drnm3 KCl. The measured pH was inverted to H + -concentrations by the method of Irving et al. [ 121. Measurements were made with a Radiometer pHM 84 pH-meter with GK2421C combined electrode and ABU 80 autoburette. Stability constants were calculated with a general computational program (PSEQUAD) that was published recently [13]. Absorption spectra were recorded on a Beckman UV5240 spectrophotometer. C.d. spectra were recorded on an automatic JASCO-J-20 spectropolarimeter in the 200-800 nm region. Results are expressed in terms of Ae = eL - CR.The ligand and metal ion concentrations were similar to those used in the potentiometric measurements.

RESULTS

AND DISCUSSION

The stability constants of proton, copper( and nickel(I1) complexes of Alat-AlaOMe are presented in Table 1. Since there is presence of the methylester residue on the C-terminal, this dipeptide may act as the bidentate ligand with formation of one 5membered chelate ring via { NH2,CS} donor set. Therefore, the equilibrium data of glycine-amide (GA) [ 141 are also included for comparison. It can be seen from Table 1 that copper(B) forms similar complexes with both ligands, i.e., GA and Alat-AlaOMe. Their stability constants, however, are considerably different. The high stability constants of CuA and CuAz complexes (charges are omitted for simplicity) of

THIOCAKBONYL-CONTAINING

DIPEPTIDES

85

TABLE 1. Stability Constants of the Copper(B) and Nickel(B) Complexes with Alat-Ala-OMeand Glycine amide (Gly-NH*) Alat-Ala-OMe PK

Gly-NH2”

7.88 + 0.02 Cu(Q

MA MA2 MA2H_, MAzH_2

7.19 14.21 7.12 -0.82

log(K&)

0.17

PK-I* PK-2’ PK-I/

* f f f

0.02 0.02 0.03 0.03

8.01

Ni(II)

WB)

Ni(II)

7.41 * 0.03 -7.69 f 0.03

5.30 9.56 2.49 -5.84

3.80 6.88 - 12.13

1.04

7.09 7.94

7.07 8.33 7.55

T= 298K,

9.46

1 = 0.2 mol dm-‘.

lM,&B,I PP9’=[M]p[A]q[H]’ 0 Data taken from Ref. 14. 6 pK _ , represents reaction MA2 * MAzH _, + H + . c pK_* represents reaction MA*H_, 4 MA2H-2 + H + d pK_,* represents reaction MA2 + MA2H_Z + 2H+ (average

value).

thioamide-containing dipeptide suggest { NH2,CS} coordination of this ligand, while { NH2,CO} coordination is established for the GA ligand. The species distribution for the Cu(II)-Alat-Ala-OMe system is given in Figure 1. The spectroscopic data summarized in Table 2 clearly support the thioamide coordination and the concentration distribution presented in Figure 1. Already, at pH < 3, the charge-transfer band characteristic of thioamide sulfur coordination [3] was observed suggesting {NH&S} chelation. This coordination was seen for both the CuA and CuA2 complexes (Table 2, Fig. 1). The low value of logKI/Kz shows the enhanced stability of the bis complex, similar to the complexes of amino acid thioamides [3]. On the other hand, it is also evident from the equilibrium and spectroscopic data that the sulfur donation cannot prevent the amide nitrogen deprotonation and coordination to metal ion. (Tables 1 and 2). Therefore, the two complex species present in the basic solutions, i.e., CuAzH- 1 and CUA*H_~, have one or two ligands bound via { NH2,w donor set. The thiocarbonyl donor is also involved in the coordination of nickel(E) ions. The NiA2 species, which, according to the spectroscopic data, is an octahedral complex, seems to have { NH,,S ) donor set bound to metal ion (Table 6). Such coordination is supported by the enhanced stability to this complex when compared to the NiGA (Table 1, Fig. 2). This binding mode is not able to prevent an amide nitrogen deprotonation and its coordination to nickel ion, as has been discussed above for the cupric complexes. The NiA2H_2 complex, with (NH,,N- } coordination, is, according to the spectroscopic data, the square planar complex (Table 6), and it is formed at lower pH than the species with GA [14].

86 K. Varnagy et al.

30

5.0

6P

7P

BP

9.0

pH

FIGURE 1. Concentration distribution of the complexes formed in the Cu(II)-Alat-Ala-OMe system. [Cu] = 0.001 and [A] = 0.002 mol dm-3.

TABLE 2. Spectroscopic Data for the Copper@) Complexes with Alat-Ala-OMe Absorption Spectra Species

X[nm] e[dm3 mol-’ cm-‘]

C.d. Spectra X[nm]

Ae[dm3 mol-’ cm-‘]

CUA

672 360

174” 22006

Cm%

656 362

260” 5020b

690 560 342 298

+0.05 Be -0.08 EC - 3.036 +0.55d

CuA,H _ ,

620 361

205” 38OOb

605 510 358 305

- 0.29 B +0.03 E -2.55b + 0.91”e

CuArH_r

526

200”

603 490 325

-0.64 B -0.1 E +2.1ge

e, Ae values are calculated for the total concentration of metal ion for the pH at which the highest concentration of the respective species is found from the species distributions. 0 d-d transition. b S to Cu(II) charge transfer transition. c B and E are the splitted d-d transitions. d NH2 to Cu(B) charge transfer transition. c N- to Cu(II) charge transfer transitions.

THIOCARBONYL-CONTAINING

O/O

DIPEPTIDES

87

Ni(ll1

FIGURE! 2. Concentration distribution of the complexes formed in the Ni(lI) Alat-Ala-OMe system. mi] = 0.001 and [A] = 0.002 mol dmT3.

The coordination equilibria in the metal Asp-Phe-NH2 system and especially in the solutions containing thioderivative Asp-Phet-NH2 are much more. complicated than those mentioned above. The presence of aspartic acid residue may change considerably the binding ability of the peptide ligand [ 15 181due to possible coordination of the side-chain carboxyl group. It may remind one of the &&tnine-like binding, while the deprotonation and binding of amide nitrogens should follow the glycilglycine-amide coordination mode. Hence, the stability constants of the copper complexes presented in Table 3 were collected for all mentioned above ligands. These data clearly indicate that complex formation processes in the metal Asp-Phe-NH* and metal Asp-Phet-NH* solutions are distinctly different. It is much more obvious from the species distribution of the respective system depicted in Figures 3 and 4. The stability constants of the CuA and CuAz complexes of Asp-Phe-NH2 indicate that in slightly acidic medium, ligand behaves like /3-alanine, i.e., cupric ion is chelated by (lQ-&,COO} donor set of Asp residue (see also [16]). The deprotonation and coordination of amide nitrogens begins at pH > 5, and it results in the formation of CuAH _ , and CWU-I_~ complexes with characteristic GGA-like coordination. The CuA complex of Asp-Phet-NH2 is formed at considerably lower PH. It suggests the involvement of thiocarbonyl sulfur in the metal ion binding. The sulfur coordination at pH < 3 is seen in C.d. spectra in which the S + Cu charge transfer band at 348 nm appears already at pH below 3. This transition is observed in the pH range below 9, reaching maximum intensity at pH about 5, and then decreasing distinctly at pH > 7. This intensity variation corresponds well to the species distribution presented in Figure 4. There are two possible binding modes that are in agreement with the CuA stoichiometric composition (this species has one positive charge), and they are represented by structures I and Il. Taking into

88

K. Varnagy et al.

TABLE

3. Stability Constants of Cu(lI) Complexes Asp-Phet-NH1, &Ala, and Gly-Gly-NH2 Asp-Phet-NH*

PKI PKZ MA

MAH MAz MAH_, MAH_* MA2H_ , MA2H_2 WWKz) pK-lb pK-2’

Asp-Phe-NH1

3.01 + 0.02

3.46 + 0.02

7.64 It 0.03

1.45 f 0.03

7.81 * 0.02

5.67 f 0.04

with Asp-Phe-NH*,

P-Ala Gly-Gly-NH*” 3.42 10.11 6.93

7.84 4.88

9.54 f 0.06

3.66 f 0.03 -4.44 + 0.04

4.21 8.10

T = 298 K, Z = 0.2 mol dm-‘. 0 Data taken from Ref. 14. b pK_, represents reaction MA + c PK-~ represents reaction MAIL,

10.51 f 0.05 -0.46 i 0.04 -8.37 f 0.05 3.04 f 0.06 -5.44 f 0.06 0.83 6.13 7.91

12.21 -0.61

-0.19 - 8.20

0.65 5.07 8.01

MAH_I + H+ --t MAH_2 + H+.

9.0 PH FIGURE 3. Concentration distribution of. the complexes formed in the Cu(II)-AspPhe-NH2 system. [Cu] = 0.00069 and [A] = 0.001482 mol dme3.

THIOCARBONYL-CONTAINING

DIPEPTIDES

89

9.0 PH

6.0

FIGURE 4. Concentration distribution of the complexes formed in the Cu@)-Asp-Phet-NH* system. [Cu] = 0.000666 and [A] = 0.001618 mol dmm3. account the fact that in the absorption and C.d. spectra tbe distinct band around 306-

310 nm, which corresponds to the N(amide) + Cu charge, tranfer transition is observed already at pH 2 S, and it seems that structnre I is more likely. Figure 4 indicates that the CuAH _ I species predominates at pH region 4.5-8. This complex can be unambigously assigned, as the species in which ligand binds cupric ion via

l-e

CH

I

\NH;

NH/ C /

R’/

CH

I O\

\ NH2YCU

structure +

II

90 K. Varnagy et al.

TABLE 4. Spectroscopic Data for the Copper@) Complexes with Asp-Phet-NH2 and Asp-Phe-NH* Absorption Spectra Species Asp-Phet-NH* c&AH_,

C&AH_*

X[nm] e[dm3 mol-’ cm-‘]

C.d. Spectra X[nm]

Ae[dm3 mol-’ cm-‘]

589 307

180” 46OOb*d*’

665 490 351 310

-0.30 B’ -0.07 EC + 2.OOb -2.12e

563

172’

550

+0.15

B

440

-0.08

Ef

87”

627

-0.22

B + E

106”

323 585 315

+0.12’ -0.51 B + E +0.27’

Asp-Phe-NHr CuAH _ , CuAH_r

640 567

0--pAs in Table 2. 1 Higher energy component

of the splitted E transition,

(see, e.g., Ref. 19).

{ NH2,N,S} donor set as it is seen both from stability constants and the spectroscopic data (Tables 3 and 4). The distinct decrease of the broad band at 306 nm in the absorption spectra (it contains both N and S to metal charge transfer transitions) and complete disappearing of the 350 run C.d. band [S to Cu@) charge transfer band] indicates, however, that thiocarbonyl coordination does not prevent the deprotonation and coordination of the terminal amide nitrogen. This results in the formation of the CuAH_r species similarly to the system with Asp-Phe-NH2 with three nitrogen coordination. The data for the corresponding nickel(II) complexes are collected in Tables 5 and 6. The equilibrium data (Table 5) for NiA and NiA2 complexes of Asp-Phe-NH2 and

TABLE 5. Stability Constants of the Nickel(B) Complexes with Asp-Phet-NHz, Asp-Phe-NH2, &Ala, and Gly-Gly-NH2a Asp-Phet-NH2 MA MA2 MA3 MAHm2 MA&r log(KJKr) PK-12’

4.91 f 0.05 8.50 f 0.10 - 10.09 f 0.03 1.37 7.50

a See Tables 1 and 2 for explanations. b data taken from Ref 20. c pK_,r represents reaction MA + MAH_2

Asp-Phe-NH2 4.61 f 8.34 + -10.9 f - 13.53 f - 10.71 + 0.88 9.07

&Ala

Gly-Gly-NHzb

4.52 7.84 9.91

3.42 6.21 8.6 - 14.44

1.20

0.63 8.93

0.04 0.06 0.1 0.05 0.07

+ 2H+ (average

value).

THIOCARBONYL-CONTAINING

DIPEPTIDES

91

TABLE 6. Spectroscopic Data for the Nickel@) Complexes with Alat-Ala-OMe, Asp-Phet-NH2 and Asp-Phe-NH*’ Absorption Spectra X[nm] e[dm3 mol-’ cm-t] Ni@boh

Alat-Ala-OMe NiA* NiA2H_2 Asp-Phet-NH2 NiA NiAH-2

AspPhe-NH2 NiAH-2

1156 720 660 392

2.0b 2.0b l.Sb 5.0b

1020

627 467

lO.Ob 8.0” 82.0’

1036 625 452

8.0b lO.Ob 382.0’

430

70.0’

C.d. Spectra X[nm]

Ae[dm3 mol-’ cm-t]

575 465

- 0.27 Ad - 0.26 Ed

535 453

- 1.52 A + 1.13 E

451

-0.345A

+ E

a e and AE values calculated as described in Table 2. b d-d transitions of an octahedral nickel(II) complexes c d-d transition of a square-planar nickel(U) complexes d A and E are the components of the d-d transitions

Asp-Phet-NH2 are very similar to each other and to those of @lanine suggesting the same type of coordination in all complexes. There is, however, a significant difference in the pK_ i2 values of the amide deprotonation of dipeptide ligands indicating easier coordination of amide nitrogen in the Ni(II)-Asp-Phet-NH2 system (formation of NiAH _ z species at lower pH) (Table 5, Figs. 5,6). The coordination in NiA and NiA2 species could be as that shown above in structure II for cupric complex. Such coordination explains the easier terminal amide coordination by nickel ions. The spectral data that clearly indicate the formation of square-planar complexes in basic solutions (Table 6) are not very informative at pH < 7 and cannot be used as univocal evidence for the Ni@) ion coordination to sulfur donor in the discussed system at lower pH. It is clear, however, that complex formed at pH < 7 is an octahedral species (Table 6), and the distinct increase of the d-d transition intensities (one order) when compared to the aquo-ion may indicate the coordination of sulfur already in the octahedral complex NiA. Similar to the other peptides, the cooperative deprotonation process takes place when square-planar complex is formed in basic media [4]. The detailed data for the planar complexes are presented in Tables 5 and 6. CONCLUSIONS The results obtained in the presented work clearly indicate the specific binding ability of the thiocarbonyl donor inserted in the oligopeptide molecule. This donor promotes

K. Varnagy et al.

'10NitI\)

5.0

3.0 FIGURE 2.

O/O

5.

Species distribution

6.0

7.0

8fl

for the Ni(II) Asp-Phe-NH2

9.0

10.0 pH

system; other data as in Figure

Ni(l I)

0

1

:

3.0 FIGURE 2.

CO 6.

Species distribution

5.0

6.0

for the Ni(I1) Asp-Phet-NH1

I, 7.0

40

PH

system; other data as in Figure

THIOCARBONYL-CONTAINING

DIPEPTIDES

93

the metal ion binding at significantly lower pH that it is found for carbonyl analogues. The thiocarbonyl sulfur may easily compete in the metal ion binding with -COO group of Asp residue. The latter donor was found to be very effective binding site especially for cupric ions [ 15- 181. Also, the position of thiocarbonyl group in the oligopeptide sequence has critical influence on the coordination equilibria as well as on the structure and binding sites in the formed complexes. Two of us (H.K. and T.K.-J.) wish to thank Polish Academy of Sciences (Project CPBP 01.12.) for financial support.

REFERENCES 1. K. Rolka, M. Kruszynski, G. Kupryszewski, U. Ragnarson, K. Kolasa, W. Turski, and Z. Klinrok, Acta Pharm. Scienc. 21, 173 (1984). 2. K. Klausen, A. F. Spatola, C. Lemieux, P. Schiller, and S. 0. Lawessen, Biochem. Biophys. Res. Commun. 120, 305 (1984). 3. T. Kowalik, H. Kozlowski, I. Sovago, K. Vamagy, G. Kupryszewski, and K. Rolka, J. Chem. Sot. Dalton Trans. 1 (1987). 4. H. Sigel and R. B. Martin, Chem. Rev. 82, 385 (1982). 5. I. Sovago and R. B. Martin, J. Znorg. Nucl. Chem. 43, 425 (1981). 6. H. Kozlowski, B. Decock-Le Reverend, D. Ficheaux, C. Loucheux, and I. Sovago, J. Znorg. Biochem. 29, 187 (1987). 7. L. D. Pettit, I. Steel, T. Kowalik, H. Kozlowski, and M. Bataille, J. Chem. Sot. Dalton Trans. 1201 (1985). 8. M. Bataille, L. D. Pettit, I. Steel, H. Kozlowski, and T. Tatarowski, J. Znorg. Biothem. 24, 211 (1985). 9. W. C. Jones, Jr., J. J. Nestor, Jr., and V. du Vigneaud, J. Am. Chem. Sot. 95, 5677 (1973). 10. S. Scheibye, B. S. Pedersen, and S. 0. Lawesson, Bul. Sot. Chim. Belg. 87, 229 (1978). 11. K. Clausen, M. Thorsen, and S. 0. Lawesson, Tetrahedron 37, 3635 (1981). 12. H. M. Irving, M. H. Miles, and L. D. Pettit, Anal. Chim. Acta 38, 479 (1967). 13. L. Zekany and I. Nagypal, in Computational Methods for the Determination of Stability Constants, D. Leggett, Ed., Plenum Press, N.Y., 1985. 14. I. Sovago, B. Harman, A. Gergely, and B. Radomska, J, Chem. Sot. Dalton Trans. 235, (1986). 15. B. Decock-Le Reverend, L. Andrianarijaona, C. Livera, L. D. Pettit, I. Steel, and H. Kozlowski, J. Chem. Sot. Dalton Trans. 2221 (1986). 16. B. Decock-Le Reverend, F. Liman, C. Livera, L. D. Pettit, S. Pybum, and H. Kozlowski, J. Chem. Sot. Dalton Trans. (in press). 17. I. Sovago, T. Kiss, and A. Gergely, Znorg. Chim. Acta 93, L53, (1984). 18. B. Decock-Le Reverend, A. Lebkiri, C. Livera, and L. D. Pettit, Znorg. Chim. Acta, 124, L19, (1986). 19. G. Formicka-Kozlowska, H. Kozlowski, I. Z. Siemion, K. Sobczyk, and E. Nawrocka, J. Znorg. Biochem. 15,201 (1981). 20. T. F. Dorigatti and E. J. Billo, J. Znorg. Nucl. Chem. 37, 1510 (1973). Received February 12, 1988; accepted April 17, 1988