Indian Journal of Chemistry Vol. 40A, May 2001, pp.509-5 13

Kinetic and mechanistic studies on the inter­action of L- glutamine with cis -diaqua

(ethylenediamine )platinum (II) perchlorate in aqueous medium

Partha Sarathi Sengupta, Ramanath Sinha & G S De*

Chemistry Department. The University of Burdwan, Burdwan 713104, India

Received 5 Decetnber 2000; revised /6 February 2001

The kinetics of the interaction of L-glutamine with cis­[Pt(en)(H20hf+ have been studied spectrophotometrically as a function of [Pt(en)(H20)/+], [L-glutamine] and temperature at a particular pH (4.0) where the substrate complex exists predomi­nantly as the diaqua species and L-glutamine as the zwitterion. The substitution reaction shows two consecutive processes; the first step is the ligand assisted anation and the second one is the chelation step. Activation parameters for both steps have been calculated. The low tJ.H 1# (43.6 1± 1.44 kJ mor 1

) and large nega­tive values of tJ.S1# (-122.9 ± 4.5 J K" 1mor 1

) as well as tJ.H/ (39.61 ± 0.8 kJ mol -1

) and tJ.S2# (- 203.8 ± 2.2 J K" 1 mor 1) indicate

associati ve mode of activation for both the ligand substitution processes in the two consecutive steps.

Substitution reactions on square planar platinum (II) complexes attracted continued attention due to their intrinsic chemical 1-4 and bio-medical applications5-7. In particular cis-platin8-13, (cis -dichlorodiamineplati­num(II)) and its structural analogues 14 have been used in cancer chemotherapy. At present it is believed that DNA is the main target to these drugs in tumour cells15-18. Binding to proteins and peptides also occurs as has been shown by many investigators 19-24 . It is widely admitted that the two non-leaving spectator cis- nitrogen ligands are still attached to platinum when it reaches its target in cellular DNA 25·26. When a chloro drug is administered, the chloride ions have been displaced by a hydrolytic process and the hy­drolytic product undergoes favourable and active nu­cleophilic substitution by the DNA base sites27·28 . But the major drawback of these chloro derivatives of N­N chelated platinum (II) complexes were their neph­rotoxicity14. Thus the search for other drugs has been continued, however, the aqua variety was found to be superior5·29 to others.

Kinetic and mechanistic description of the reac­tions between cis -(N-N)- chelated diaquaplatinum (II) ions with proteins and their amino acid fragments

are now the subject of interst30-32. In order to examine the reactivity of aqua amine complexes of platinum (II) towards amino acids and substituted amino acids , we have undertaken the present studies. In continua­tion of our study of using aqua amine complexes of platinum (II) as a binder of amino acids, this note deals with the interaction of L-glutamine, an essential amino acid, with cis-diaqua (ethylenedi­amine)platinum (II) perchlorate in aqueous medium.

Experimental

The reactant complex, cis-[Pt(en)(H20h]2+ (com­

plex I) was prepared according to the literature method33 and characterised spectroscopically3

.J

O"max=256 nm,£ =200 em·' M 1). The pH of the solu­

tion was so maintained (pH =4.0) that perchlorate salt exists as diaqua species. The product (substituted complex) of the solution was prepared in different molar ratios viz. I: I, 1:2, 1 :3, 1 :4 and I :5 and ther­mally equilibrated at 60°C for 48 h. All the five solu­tions so prepared exhibited almost identical absorban­ces at 224 nm. The composition of the product in the reaction mixture was determined by Job's method of continuous variation. The metal : ligand ratio was found to be 1: I. The spectral difference between the product complex has been shown in Fig I.

The pH of the solution was adjusted by adding NaOHI HCI04 and the measurements were carried out with the help of a Systronics digital pH meter (model 335) with an accuracy of± 0.01 unit. Doubly distilled water was used to prepare all the solutions. All other chemicals used were of either AR grade or purified before use. The reactions were carried out at constant ionic strength (0. 1 M NaCI04).

Kinetics Kinetic measurements were cruTied out on a Shi­

madzu spectrophotometer (UV -2101 PC) equipped with a Shimadzu TB thermobath (accuracy ± 0.1 °C).The absorption due to L-glutamine was sub­tracted by using 1:1 (molar ratio) ligand: water mi x­ture in the reference cell. The progress of the reaction was monitored by following the increase in absorb­ance at 224 nm where the spectral difference between the reactant complex (complex 1) and the product complex was maximum (Fig. I) . Conventional mixing

510 INDIAN J. CHEM., SEC A, MAY 2001

technique was followed and pseudo- first order con­ditions were maintained throughout the course of the

reaction . The plot of In (D~ -D,) where D, and D~ are absorbances at time t and after the completion of the reaction against time (t) is found to be non-linear; it is curved at the initial stage and subsequently of con­stant slope indicating that the reaction proceeds through two consecutive steps . The method of Weyh and Hamm35 was adopted to calculate the rate con­stants for two consecutive steps. From the limiting

linear portion of In (D~ -D,) versus time (t) curve k2

values were obtained . The k1(obs) values were obtained from the plot of In fl versus time (t) where time (t) is small. Rate data, represented as an average of dupli­

cate runs, are reproducible within± 4 %. Typical plots of In (D~ -D,) versus time (t) and In fl versus time (t) are shown in Figs 2 and 3.

Results and discussion The pK1 and pK2 of L-glutamine36 are 2 .17 and 9.01

at 25°C respectively , which refer to the following dis­sociation processes

+ H2NCOCH2CH2CH( N H3) (COOH)¢::>

+ H2NCOCH2CH2CH( N H3) Coo· + H+: pK1= 2.17

+ H2NCOCH2CH2CH( N H3)COO ¢::>

H2NCOCH2CH2CH(NH2) coo·+ H+; pK2 =9.01

so that at pH 4.0, the major species involved in the kinetic process is the zwitterionic form of L-

..

2·6-

N

2-2

1·6

g l ·lt 0 ~ 1·2 0

~ 1·0

0 ·8

0·6

0•4

M

0·0 1--~---'-~---:-'":---'--:-:::--'---::-:--'--:-:::-:--'--:::-:----' 200 220 240 260 280 300 320

Wovclcngth (nm)

Fig. !-Spectral differences between complex (I) and L­glutamine substituted product; [complex (1)] 2.96 xlO -4 mol dm -3 (2) complex (I)= 2.96 xI 0-4 mol dm -3

, [L-glutamine] = 0.0148 mol dm - ; cell used = lcm quartz.

glutamine. Since the pK/ and pK/ of cis-diaqua (eth­ylenediamine)platinum (JI)34 are 5.8 and 7.6 respec­tively, we can assume that at pH 4.0 the reactant ex­ists as the diaqua ion. At constant temperature, con­stant pH (4.0) and fixed concentration of complex ( 1),

the In (D~ -D,) versus time (t) plot for different ligand concentrations is curved at the initial stage and subse­quently of constant slope. It indicates that the reaction involves a two step consecutive process. the first step is dependent on ligand concentration while the second step is independent of ligand concentration . In the first step one aqua ligand was replaced from cis -[Pt(en)(H20hf+ (complex l) by L-glutamine. The second step is the slower step, where another aqua ligand is substituted. This is the ring closure step.

The rate constants for such process can be evalu­ated by assuming the following scheme :

A~ B ~ C where A is the diaqua species,

L0.15

-0.20

;;-·0.25

~'d :S -o.3o

-0.35

-0.40

0 10 20 30 40 50 Time(min)

Fig.2-A typical kinetic plot of In (D~ -D,) versus time (t) , [com­plex (I)]= 2.96 xlO -4 mol dm -3

, [L-glutamine ]= 0.011 84 mol dm -3

, T= 400C

-2.3

-2.4

-2.5

-2.6

~ -2.7

-2.8

-2.9

-3.0

·3.1

6 10 Time (min)

Fig.3-A typical kinetic plot of In D. versus time, [complex (1)]= 2.96 xlO -4 mol dm -3

, [L-glutamine ]= 0.01184 mol dm -3, T=

400C

NOTES 511

B is the single substituted species and C is the final product (complex 2); [Pt(en) (L-L)t. Formation of C from B is predominant after some time has elapsed.

Calculation of k 1 for A--; B step The rate constant k l(obsl for the A-; B step can be

evaluated by the method of Weyh and Hamm35 using the usual consecutive rate law,

(Doc-D,) = a,exp(-k, (obsJ t) + a2 exp(-k2(obsJt) ... (1)

whence (Doc -D,)- a2 exp(-k2(obsl) = a,exp(-ki (obsl 1) ... (2)

where a1 and a2 are constants dependent upon the rate constants and extinction coefficients . Values of (Doc - D,) - a2 exp(- k2(obs)O are obtained from X-Y at di fferent time (t ), so that

!J. =a, exp(-kl (obs) l)

or, In !J. =constant- ki (obs)l ... (3)

k1 (obsl is derived from the slope of In !J. versus time (t ), when t is small (Fig. 3).

A simi lar procedure is applied for each ligand in the concentration range 0 .00296 to 0 .0148 mol dm -3

at constant complex ( I ) concentration of 0 .000296 mol dm-3 at pH 4.0 and at 40, 45 ,50,55 and 60°C re­spectively. The k1(obsl values thus obtained are linearly dependent on the studied concentration range.The k1 (obs) values for different ligand concentrat ions at dif­ferent temperatures are g iven in Table 1. The following scheme can be proposed :

[Pt(en)(H20 h f+ + L-L H k, , ,tow

LH f+ + H20

[Pt (en) (H20) L- LH )2+ k 2 .. 110

"'

H3o +

[Pt(en) (H20) L­... (4)

[Pt (en) L-Lt + ... (5)

where L-LH is the zwitterionic form of L- glutamine +

(H2NCO CH2 CH2CH( N H3) COO -) Based on the above scheme a rate expression (6)

can be derived for the A~B step :

dB ldt = k 1 [Pt(en) (H20) 2 2+]total [L-glu] ... (6)

where [Pt(en) (H 20) 2 2+] 10131 is the concentration of the

unreacted complex and [L-glu ] is the concentration of L-glutamine.

Hence we can write

kl (obs) = k1 [L- glu] ... (7)

where k1 is the second order rate constant for the first

aqua ligand substitution. A plot 0f k I(obsl (s-1) versus [L- glu] should be lin­

ear passing through the origin. This was found to be so at all temperatures studied. The second order rate constants (k 1) calculated from the slope of k I(obsl (s-1) versus [L- glu] (mol dm-3) plot at different tempera­tures are collected in Table 2 .

Calculation of kdor B-.; C step values obtained The B~ C step is the ring closure step in which the

amino group of the amino acid , L-glutamine binds the metal centre. This step is slow and independent of ligand concentration variation . At a particular tem­perature the k2 values were calculated from the limit­ing linear portion (when t is large) of the In (Doc -D,) versus time (t ) curve.For different temperatures k1

values obtained directly from the limiting slope of the In (Doc -D,) versus time (t) are given in Table 2.

Effect of temperature on reaction rate The reaction was studied at five different tempera­

tures for different ligand concentrations and the ana­tion rate constants for both A~ B (k 1) and B~C (k2) steps are collected in Table 2. The activation parame­ters for both steps calculated from Eyring plots are given in Table 3 and compared with those for analo­gous systems (Table 3).

Mechanism and conclusion The ligand L-glutamine exists as a zwitterion at the

experimental pH (4.0). The present investigation of

Table I - 10 3 kiCot>s> (s - I) values for different [L-glutamine 1 at different temperatures

[Pt(en) (H20h 2+1 = 2.96 x I 0 -4 mol dm -3, pH =4.0,!-1 = 0.1 mol

dm -3 NaCI0 4

10 3[L-glutamine1 Temp (0 C)

(mol dm -3) 40 45 50 55 60

2.96 0.35 0.51 0.65 0.82 1.12

5.92 0.69 1.04 1.31 1.63 2.25

8.88 1.05 1.53 1.97 2.47 3.35

11.84 1.41 2.12 2.69 3.29 4.49

14.80 1.75 2.59 3.25 4.21 5.61

Table 2-Yalues of k1 and k2 for the substitution reaction

Temp 102k, 105k, (OC) (dm3mol- ' s- 1

) (s· ' )-

40 12.61±0.03 3.64±0.03 45 17.72±0.04 4.58±0.04 50 22.52±0.01 5.96±0.04 55 28.5 1±0.02 7.47±0.05 60 37.9±0.02 9.59±0.04

512 INDIAN J. CHEM. , SEC A, MAY 2001

Table 3-Activation parameters for analogous systems

Systems !1H1# !1S1# CisPt(NH3)z(H20 h 2+ (kJmol-1) (JKmor1)

s'· dG MPH2 31.2±4.3 - 117± 15

51. GMPH2 40.64±4.4 -106±16

Cis-[Pt(en) (H20)f+

DL- Methionine 15 .58±0.9 -230±3.0

Thioglycolic acid 29.5 1±4.8 -1 88±15.0

Adenosine 63 .7±1.6 -58±5.0

Thiourea 61.95±1.7 -71±5.8

Thiosemicarbazide 35 .69±0.8 -166±2.54

Pyridine -2-thiol 36.0±3.0 -166.0±8.0

DL- Penicillamine 46.5±4.6 - 143± 15

L-Giutamine 43.61±1.44 -122.9±4.5

aqua ligand substitution by L-glutamine shows that L­glutamine interacts in an associative mode of activa­tion in the transition state. The second step is the ring closure step which is slower than the first step and is independent of ligand concentration.

The two cis - positions of platinum (II) ion are blocked by nitrogen ligands and in view of the prefer­ence of platinum for square planar configuration in its complexes, it is unlikely that L-glutamine behaves like a tridentate ligand in this complex formation. As­suming the bidentate behaviour of L -glutamine, one can envisage two different ways in which the amino acid can bind platinum.The ligand may coordinate through the carboxyl and the terminal amino groups with the amide group in the side chain free, thus be­having like a simple amino acid i.e. glycine 2

, alanine. Alternatively, the ligand can also coordinate through the terminal amino group and the carbonyl oxygen of the amide group. The second mode of coordination would mean that a seven membered ring would be formed between platinum and L-glutamine. The latter would probably be more strained and less favoured . The affinity of platinum for nitrogen donor centres provides the driving force for the deprotonation in the second step.

The activation parameters (D..H1# = 43.61±1.44 kJ mor 1

, f.. S1# =-122.9 ± 4.5 J K 1mor 1) for the first step

and second step (f.. H2# = 39.61 ± 0.8 kJ mol - 1, f.. S/ =

- 203.8 ± 2.2 J K 1 mor 1) suggest an associative mode

of activation for the substitution processes. The low f.. H1# and f.. H/ values implies a good degree of ligand participation in the transition state. The large negative values of f.. sl# and f.. s 2# suggests a more compact tran­sition state than that present in the starting reactants.

!1H/ 11S/ Ref (kJm~r 1 ) (Jkm~r 1 )

60.8±5.3 - 55±17 .9 37

62.8±1.5 -46.3±5.2 38

19.43± 1.2 -225.5±4.0 3 1

48.99±1.0 - 130.0±3.0 39

41.0±0.5 - 197.0±1.5 27

26.7±0.8 - 186.8±2.7 40

44.54±1 .32 - 182±4. 18 41

26.0±1.0 -218±4.0 42

44.3±1.3 - 189.0±4.20 43

39.61±0.8 -203.8±2.2 This work

Acknowledgement

The authors thank the UGC, New Delhi for pro­viding financial assistance under DSA project in Chemistry. One of the authors (PSS) is grateful to the UGC for the award of teacher fellowship.

References Basolo F & Pearson RG , Mechanism of inorganic reactions. A study of metal complexes in soli/lions (Wil ey, New York) 1967, 351.

2 Chaloner P A, Coord chem Rev, 72 ( 1986) I. 3 Chaloner P A, Coord chem Rev, 89 (1988) I. 4 Basolo F, Coord chem Rev 154 ( 1996) 151. 5 Johnson N P, Hoeschele J P & Rahn R 0. Chem Bioi

Interactions, 30 ( 1980) 151 . 6 Keppler B K, Metal complexes in cancer chemotherapy

edited by B K Keppler, (VCH, Weinheim) 1993, I. 7 Arpalathi J, Metal ions in biological systems, edi ted by A

Sigel and H Sigel, Vol.32, (Marcel -Dekker, New York). 1996, 379.

8 Rosenberg B, Van Camp L, Trosko J E & Mansour V H, Nature, 222, (1969) 385.

9 Hindmarsh K, House D A &Turnbull M M, lnorg chim Acta. 257 ( 1997) I I.

I 0 Baird C L, Griffitus A E, Baffic S, Bryant P, Wolf B, Lutton J, Beradini M & Arvantis G M, lnorg chim Acta. 256 (1997)253.

II Gisseifa lt C L, Akrman B & Jonson M. Electrophoresis, 18 (1997) 663.

12 Yoshi T, Kojima M& Yoshikawa Y, J coord Chem, 37 (1996) 305.

13 Zaludova R, Zakovska A, Kasparkova J, Balcarova Z, Kleinwachter V, Vrana 0 , Farrel N & Brabec V, European J Biochem, 246 ( 1997) 508.

14 Umapathy P, Coord chem Rev, 95 (1989) 129. I 5 Reedijk J, J. Pure appl Chem, 59 (1987} 18 1. 16 Taka hara PM, Rosenzweig A C, Frederick C A& Lippard S

J, Nature, 377( 1995) 649. 17 Takahara PM, Frederick C A & Lippard S J, J Am chem Soc.

118(1996) 12309.

NOTES 513

18 Talman E G, Bruning W, Reedijk J, Spek AL &Veldman N, European Journal, 6 (2000) 2002. lnorg Chem, 36 ( 1997)854. 30 Ghosh S, De G S & Ghosh A K, Indian J Chem, 36 (1997)

19 Roberts J J& Thomson A J, Prog Nuc/ Acid Res Mol Bioi, 22 836. ( 1979) 71. 3 1 Ghosh S, Sengupta P S &De G S, Trans metal Chem, 24

20 Cis platin, wrrelll status and new developments edited by A (1999) 59. W Prestayko, S T Crooke & S K Karter, (Academic Press, 32 Appleton T G & Ross F B, lnorg chim Acta 256 (1996) 79. New York) l980. 33 Lim M C & Martin R B, J inorg nuc/ Chem, 38 ( 1976) 1911 .

21 Platinum coordination compounds in cancer chemotherapy 34 Coley R F & Martin Jr, D S, lnorg chim Acta, 7 ( 1973) 573. ed ited by M P Hacker, E B Douple & I H Krakoff (Martinus 35 Wyeh J A & Hamm R E, lnorg Chem, 8 (1969) 2298 Nijhoff, Boston) 1984. 36 Marte ll A E & Smith R M, Critical stability constants, Vol I,

22 Guo Z J, Hambley T W, Murdoch P D, Sadler P J &Frey U, J Amino acids, (Plenum Press, New York) ( 1974) 41 . chem Soc Dal Trans , 4 ( 1997) 469. 37 Evans D J, Green M& Eldik R V, lnorg chim Acta, 27 (1987)

23 Ji ang J L, Chen Y, Tang G Z & Tang W X. J inorg Bio chem, 128. 65 ( 1997) 73 . 38 Eapen S S, Green M & Esmail I M, J inorg Biochem, 23

24 Katsarou E, Trogani s A & Hadjili adis N, lnorg chim Acta, (1985) 233. 256 (1997) 2 1. 39 Ghosh S, De G S & Ghosh A K, J Indian chem Soc, 76

25 Burton J K & Lippard S J, Nucleic acid - metal ion (1999) 41. interaction, edited by T.G.Spiro (Wiley, New York) Chp 2 40 Sengupta P S, Ghosh S & De G S, Trans metal Chem, 25 (1980). (2000) 279.

26 Cleare M J & Hoeschele J P, Bioinorg chem, 2 (1973) 187. 41 Ghosh S, Sengupta P S & De G S, Indian J Chem, 38 A 27 Ghosh S, De G S & Ghosh A K, lnorg reaction Mech, (1999)453.

(1999) 189. 42 Sengupts P S & De G S, Indian J Chem, 39 A (2000) 928 28 Cleare M J, Coord chem Rev, 12 (1974) 349. 43 Sengupta P S, Sinha R & De G S, (In press. Trans metal 29 Legendre F, Bas V, Kozelka J & Chotterd J C, Chemistry- A Chem)