Indian Journal of Chemistry Vol. 42A, January 2003, pp. 116-120

Improved selectivity to orthophosphate, pyrophosphate and triphosphate by adding

metal ions to a tripodal ligand

Van-He Guo", Qing-Chun Ge", Hai Linb, Hua-Kuan Lin".c,\

Shou-Rong ZhLl, Chang-Yue Zhou"

"Department or Chemistry of Nankai University, Tianjin , 300071 , P. R. China

"State Key Laboratory or Functional Polymer Materials ror Adsorption and Separation. Nankai University, Tianjin, 300071,

P. R. China

cThe National Key Laboratory of Coordinati on Chemistry, Nanjing University, 210093. p. R. China

Receil 'eil 26 Decelllber 2001 .. revised 31 JlIly 2002

The , [ru ng and selec ti ve binding of phosphate (PO), pyrophosphate (PP) and tri phosphate (tP) by tripodal li gand N, N, N-tri«2-aminoethyl )thioethyl)-amine (L) has been stud ied in aq ueous so lution at 1 = 0.1 mol dm·J KNO.1 and 25 °C. The interacti ons between L ;lIld the inorganic substrates are desc ri bed and eq uilibrium constants ror each spec ies formed have been determined. The stabi lity constants of I: I: I M (M = Cu~+ or Zn2+):L:S (S = PO. PP, tP) are determined by potentiometric pH titrati ons. The interactions of Zn(II)/L/phosphate systems and Cu(II)/L/phosphate systems are inves ti gated by J lp NMR and EPR spectra respectively. The results show that metal ion acts as "messenger" or bridge between the receptor and the substrate. and the exi stcnce of metal ion in the ternary systems leads to competition between the mixed li ga nds. The metal ion improves the selecti vity and recog niti on of the substrates by protonated tripodal li gand .

Phosphate-type of anions are ubiquitous in biological structure, function , and regulation; thus, their interactions with the corresponding receptors are of special interest I. Studies have explored the design and synthesis of polyammonium li gands both as receptors for phos phate-containing substrates and as catalysts for phosphoryl transfer reactions2

. 3.

Since catalytic efficiency depends largely on the recognition of the receptor to the substrate, the field o f anion-coordination chemistry has gained much attention". It has been reported that coulombic interactions, hydrogen bonding, geometrical fit of the substrate wi th respect to the receptor and/or 7t-7t

stacking forces are the main elements of the binding between receptors and substrates5

.

The tripodal ligands can form octahedral

E- mail: hklill @nankai.edu.cn Fax: 086-022-23502458

complexes by wrapping around the metal ion and leaving an open face with three groups positioned, which can be capped by suitable reagents . However, varying the length of the arms or the nature of the donor atoms produces ligands whose complexes have di fferent cavity sizes or chemical properties. In this paper, determination of the s;:ability of the supramolecules formed by a tripodal ligand L with the substrate S (S = PO/-, p20 t and p}oll) in the presence or absence of metal ions is ~x pected to show some new coordination characteristics of receptor/substrate interactions.

Experimental The starting materials were obtained commercially

and were purified prior to use. The tripodal li gand L was prepared according to the literature6

.

Triphosphate was synthesized according to the published work and was purifi ed by repeated crystallization from water/ methanol so lution 7. The aqueous stock solutions of the phosphates were fresh ly prepared daily and the exact concentrations were measured each ti me by ti tration wi th K 0 H. A II other soluti ons were prepared with redi stilled water and stocked in jacked flask.

C, H, N elemental analyses were made on a Perkin­Elmer 240C analyzer (American). tH and 31p NMR spectra were taken on a Varian Unity-p lus 400MHz spec trometer. IR spectra were recorded by a Euinox 55 FT spectrometer (Bruker). Titrations were carried

out using a Beckman pH meter (model <P71) equipped wi th a 39481 type combination g lass electrode. The EPR spectra were reco rded on an EMX-6/ I (B rukcr) spectrometer.

Potentiometric determination was carried out at 25.0 ± 0.1 °C according to the method reported 8. The ionic strength of the so lutions used was adj usted to 0.1 mol.dm-3 KN03• The values of kcv =1.08x I0· 1

", Yi/+

= 0.851 were used for calculation. For each system at least three titrations were performed and the calculations were carried out through the curve-fitting computer program (TITFITt

The 31p NMR chemical sh ifts of polyphosphare in LIS (S = PO, orthophosphate; PP, pyrophosphate; tP, triphosphate) and Zn(ll)/LIS systems were measured

at 25±0.5°C according to literature". In a typical experiment, a 0.5ml solution containing 0 .0 I mol.dm-.1

NOTES 11 7

tP and 0.0 I mol.dm·3 of tripod in the presence or absence of Zn(ll) (0.01 mol.dm·3) were placed in the NMR probe in 5-mm tubes at the temperature indicated.

Solutions containing Ix lO·3 mol.dm·3 tP and Ix lO·3

mol.dm·3 tripod in water in the presence or absence of

Zn(I1) (lxlO·3mol.dm·3) were put into tubes with a much smaller diameter than standard EPR tubes to reduce the dielectric losses caused by water. The calibrated microwave frequency is 9.854 GHz and the

records were carried out at 25°C.

Results and discussions The tripodal ligand was synthesized according to

the literature 6 with a modification. The synthesis process is shown in Fig. I . Reaction of I with 2-aminoethylenethiol in a ratio of 1:3 gave a mixture of several materi als which were indicated by lR tind MS analysis. On increasing the ratio of IINH2C H2C H2S' to I :6, the concentration of the more nucleophilic HS' was found to be comparatively higher. The 1 H NMR and elemental analysis data are li sted as below: 1 H

NMR (400MH z, CDCb) 82.58-2.89 (24H, m, all CH2); 3.75 (6H, brand s, NH2). M.S. , M+ 326.41 (326. 16, theoretical) . Elemental analysis: obtai ned (calcd.) C, 44.13 %(43 .86). H, 9.07%(9.26) N,17.31%(17.17).

The protonation constants of L 'have been determined by potentiometric pH titration in a concentration of I x l 0.3 mol.dm·3. The stepwi se protonation constants are found to be I 0.05, 9.34, 8.56 and 5.76 respectively.

For the tripodal ligand, the tertiary "cap" amino nitrogen atom is considered the most ac idic group due to the linkage to three S atoms. The existence of sulphur atom significantly decreases the basicity of the ami no nitrogen atoms, which results from the protonation constants of its structural building block, di(2-aminoethyl)su lphur (DA DS). Stepwise protonation constants of DADS are 9.64 and 8.84 respectively 10, and the first va lue is lower than that of ethylamine, 10.63 .

The stabi lity constants of complexes of ligand L

with metal ions Cu(lI) or Zn(il) in 1: 1 ratio were studied. It is observed that there are five metal complexes, CuLH24+, CuHL3+, CUL2+, CuLR 1+ and CuLH.2 in the Cu(II)/L system, while ZnH2L 4+ is not stable in solution due to the repulsion between Zn2+ and protons. The stability constants for the species ZnHL3+. ZnL2+, ZnLR t and ZnLR2 are 15.01 ,8.1 9, - 1.22 and - 10.68 respectively. For the corresponding

,OH HCIN~ ./'-. ~OH

OH

SOCI). ~

~ NaO"Bu I + 3 H2N SH ~

II

CI

HeINL ~CI

CI

Fig. 1- Synthesis process of the tripodal ligand

Cu(lI) complex, the stability constants are 2 1.10, 14.43, 4.16 and -5 .59. The stability content for CUL2 4+ is found to be 27.72.

The single crystal structure of NiL has been reported 6. All the three pairs of sulphur and nitrogen atoms of the tripod are coordinated to the nickel in a slightly distorted octahedral fas hion (Fig. 2), while the "cap" nitrogen lies 3 .5A from the Ni(ll) and is uncoordinated. The possible coordination structure of Cu(II) complexes are shown in Fig. 2 . Since the thioether sites act as weak donors , it probably binds to metal ion at lower p H values because the more basic and stronger nucleophi le, nitrogen atoms, are restrained by H+ there.

Binding ojphosphales by prolonaled Iripodalligand Poly ammonium li gands can form strong

I . h' I' . h h 1110 I comp exes Wit simp e 1I10rgalllc p osp ate . -, SUC 1

as orthophosphate (PO) , pyrophosphate (PP) and triphosphate (tP) as well as nucleotides such as AMP. ADP and ATP, and so on2.3. 13. It is observed that L can be protonated in the pH range 3- 10 . So, it is reasonable that the polyprotic species of L have the tendency to bind phosphate substrates when some proximity has been suitable for the counter-parts . The binding constants of the protonated li gand with polyphosphates are li sted in Table I . The protonation constants of phosphates and the stability constants of their metal complexes involved in this study are the reported values l4

.

It is observed that there are four supramolecul ar species in the LltP system. Although potentiometric pH titration cannot be an unambiguous method to assign the species configuration, however, based on electrostati c predictions, the more stab le complexes are expected to form between the most charged species. A comparison among the data present in Table 1 shows that the binding strength is in the order

118 INDIAN J CHEM, SEC. A, JANUARY 2003

CuLH, CuLH CuL

CuLH., CuUl,

Fig. 2- Possible structures of metal complexes in Cu(lI)1L system.

of H2LS<H)LS<l-LtLS (S = PO, PP and tP) . The protons may be located at the tripod, leading to the maximum charge interactions and the biggest number of H-bonds between positively charged ammonium and negatively charged sites of the polyphosphate chain . On the other hand, neutralization of anions will decrease the negati ve charge densi ty as well as the hydrogen binding strength, which follows the order H4LS>HsLS>H6LS (S=PP and tP) except for the substrate orthophosphate (PO) due to its high protonation constant of almost 1013

.5

• So in basi c and slight ac idic solution the guest is HPO/-, while H2P04- can only exist stably in highly acidic solution. The results above indicate that both electrostatic affects ancl hydrogen-bond network play an important role in the supramolecular in teractions.

The chemical actions of the UPO ancl UPP systems are approximately analogous to those of UtP apart from the different amount of complex which contains the same protons in different US system. Despite the difference, the fact that the interactions occur only when the ligand has been bi-protonated, and the small amount of adduct at pH> lOis due to the formation of the mono-protonated or free ligand cannot be ignored .. Table 1 shows that the binding affinity of anions to the protonated ligand is in the order PO<PP<tP. On the other hand , for example, it is seen that the substrates prefer H4L 4+ to a greater extent than it does to H)L3

+, especially for the largest difference of 4.42 pH unit in the stabi lity constant for

Table I--Binding strength for the complexes present in US systems (S=PO, pol; PP, P20 7

4- and fP, P3o IOS

)

Equilibria LogK

PP tP

[H2LS]/[H2L][S] 4. 15 [H3LS]/[H3L][S] 5.28 5.87 [H4LS]/[H3L ][HS] 8.70 9.70 [HsLS1/[~L][HS] 6.64 6.71 [H6LS]/~L ][H2S] [H2LPO]l[HL][HP04

2-] 3.33

[H3LPO]/[H2L][HPO/ -] 3.53 [~LPO]/[H3L][HPO/-] 4. 18 [HsLPO]/[H4L][HP04

2-] 4.84

[H6LPO]/[~L][H2P04- 1 3.37

L-P30 105-. Thus, the substrates can be recognized and

selected by the protonated ligand via multiply interactions. The strongest interactions between triphosphate and L accounts for the longest phosphate chain of ATP which has the largest negative density.

Interactions between metaL ion/tripod/phospha te On comparing the distribution diagrams of the

ternary systems (Fig. 3) with the relative binary systems, it is apparent that considerable interactions takes place when the metal ion is introduced to the I: I US systems. It is interesting to note that the presence of the substrate stabilizes the system so that species involving up to four protons can form, and the binding constants (- logK of equilibrium I) are larger than those of Uphosphate. On the other hand, the comparison between the stability constants for MLSHn and LSHn shows the extent of stability of the ternary complex compared to amine-phosphate interaction in the absence of the metal ion. For example, the stability constants f r CuLSH2 and ZnLSH2 are 35.19 and 28.7 respecti vely, while that for LSH2 is only 23.54.

(I)

The tripodal ligand can coordinate with a metal ion bound to the phosphate substrate, and this in turn may form up to two hydrogen bonds between the substrate and the tripod. Such binding, involves a coordination bond and hydrogen bonds that can bind the phosphate anion quite strongly. For the mononuclear complexes it is seen that the binding constants of CuLS (Table 2) steadi ly increase with protonation till the formation of CuLSH2. The distribution diagrams (Fig. 3) of the I: I: 1 systems reveal that the ternary species CuLSH3

(S=tP) is domjnant at pH about 7.0. However, further protonation occurring at the substrate may cause a

NOTES 11 9

Table 2-Binding strength for the complexes present in I: I: I CU2+ (Znh):L:S systems (S=PO, P04); pp, P20 7

4- and tP, P)OI05)

Equilibria

PP

Cu Zn IMLS]lIMLJ[S] 4.70 4.23

IMLSH]/IMHL][S] 6.95 5.52

IMLSH2l1IMH2L][S) 7.47 5. 13

IMLSHJI/IMH2LJ[HSI 6.73 3.35

IMLSI141/IMH2L][H2S] 4.56

Cu I MLPO III M(OH)Lll HP04)·] 3.23 I MLPOH]/IML][HP04)·] 7.44 [MLPOH2J1[MHL][HP043

-] 7.56 I MLPOH.1J1IMH 2L]1 HP04)-] 4.06

KNOJ=O.I mol.dm·J; Temp. 25°C; IM2+]=ISI=[L]=lx I 0·) mol.dm·)

LogK tP

Cu Zn

5.34 4.48

8.16 6.49 8.92 6.01 7.58 3.8 1 5.63

Zn

2.87 5.49 3.64

--------------------------

decrease in the number of hydrogen bonds as well as the stability of the complex. The same is true for the binding of pyrophosphate and triphosphate with mononuclear Cu(ll)-L complexes except for the fact that the species CuLPOH4 does not appcar in Cu(II)IUPO system. It is observed that the maximum stability exists in the species whose poly phosphate group is not protonated. In addition, the behaviours of ZnlLlS systems are similar to those of Cu/US systems apart from the weaker stability of Zn(H) complexes than those relative species of Cu(II).

The comparison of the interactions strength of ternary complexes indicates that the M-L complexes favour the phosphate anions in the order PO<PP<tP. The largest interaction for Cu-L-tP is due to the longest backbone and the highest charge density of tP.

3/ P NMR spectra .l Ip NMR I5 chemical shifts have been used to

demonstrate the binding site of metal ion with phosphate group. The single .l Ip NMR signal observed for P207v- in the PP/H+ or UPP system is divided into two signals in the Zn(JI)IUPP system. Splitting of the .l Ip signals in P20 7

4- ternary system may be because of formation of H bonds with different strength with [he 0- of the phosphorous centers, and the difference in the H bonding may be because of the orientation of the Sand L on binding with the metal ion . This non­equivalence for pyrophosphate has been observed previously in the 31 p NMR spectrum of the [Co(lIl)(tren)P2074-] 16. For P30 10

5-, two phosphate

groups mainly participate in the binding of Zn(U) while the last one is dangling.

100

80

60

% 40

20

0 CutP 6 8 10 4

plJ

Fig. 3--Distribution diagrams for the species present in I x 1 0·' mol.dm·) Cu2+, Ix10·3 mol.dm·) L, Ix 10·3 mol .dm·3 orthophosphate

(tP) solution at 25°C and 1= 0.1 mol.dm·3 KNOJ.

The chemical shifts of polyphosphate in the presence of L are smaller than those caused by Zn(ll ). The fact that the signals of phosphate in ZnlS system significantly shift down field when compared with ZnlUS system reveals the strong competition between L and phosphates in binding Zn(II). Since Zn(lI ) prefers amino nitrogen atom rather than phosphate oxygen atoms, the positive e lectron cloud on Zn(lI ) shifts to the stronger L and then decreases its ability to attract phosphate oxygen atoms. Thus, the role of adding metal ion to the US system probably not only leads to the change in conformati0n of complex, but also takes competition between the mixed ligands, i. c. phosphates and L.

EPR oftP containing systems The EPR spectra of the binary Cu2+/tripod and the

ternary Cu2+/tripod/tP system were investigated, and the values of a (Iinewidth) and g were calcu lated

It was observed that, both the g values of the CulL and CulUtP systems beeol1le smaller, indicating the increase in covalent properties of the electron on Cu(II) as expected. The assisting coordination of the tP molecule to Cu(II)/tripod leads to smaller covalent character betwcen Cu(ll) and nitrogen atoms of tripoJ due to the stronger ionic component formed between negatively charged oxygen and the Cu(II). While the linewidth value a of the Cu(I1)/L or Cu(II)/UtP system is increased, indicating that the Fermi hyperfine interactions become stronger. So there must be some factors that make contribution to the extra stabi lity of the ternary systems. When regard to the equi librium (2),

(2)

120 INDIAN J CHEM, SEC. A, JANUARY 2003

LllogK=logK=u Cusw 10gK=uLHx+z CuLSHx+z

= 10gBCuLHx -logBCuLSHx+z+ 10gBcuHy (3) for example, the LllogK values for species CuLPP, CuLPPH, CuLPPH2 are 2.90, 0.65, 0.13 respectively (MSHz, Z=O), and for species CuLPPSH3 and CuLPPSH4 is -0.96 and -1.78 respectively (MSHz,

Z= I or 2). However, due to Jahn-Teller effect, the expected val ue for LllogK is in the range -0.9--0.3 (Refs 17& 18. LllogK > 0 indicates that there exists extra stabilization due to the intermolecular H bonding or electrostatic interaction In species CuL-CuLH2.

Conclusion The degree for tripodal receptor binding with

similar inorganic anions, orthophosphate, pyrophosphate and triphosphate is directly related to their basicity as well as the size compatibility between the "semi-cavity", tripod, and the substrates. The addition of metal ion to Llsubstrate increases the stabi lity of the ternary complexes and improves the selectivity for substrate. Thus, metal ion can act as "messenger" between the receptor and the substrate. The results received in this study make it possible to obtain a quantitativ€ assessment of the different factors governing the receptor-substrate binding interactions, and this study extends the coordination chemistry of ions to a class of anionic substrates which play a fundamental role in the bioenergetics of all living organi sms .

Acknowledgements This project 299710 18 \Vas supported by the

National Science Foundation of P. R. China and the National Science Foundation of Tianjin .

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