Chapter 4 : Kinetic and mechanistic study

of the interaction between

diglycine and cis-diaqua (cis-

1 ,2-diaminocyclohexane)

. platinum (II) perchlorate

'71/l/l/l/l/171/l/l/l/.l/l/l/.l/17.1/l/l/l/l/17.1/171:11/l/.l/l/l./l/l/l/.l/l/l/171/l'/l/l/l/l/l/l/l/171/lliiT/I/I/I/I/I/I/1/I/I/I/I/I/.1/1/I/I/I/I/I/.I/I'/171/I/I/Ik

Chapter 4 : Kinetic and mechanistic study of the interaction between diglycine

and cis-diaqua ( cis-1 ,2-diaminocyclohexane) platinum (ll)

perchlorate

4.1 Introduction

From detailed in vitro and in vivo investigations of platinum antitumour compounds it

has been observed that any cis-diamine (chelated or non-chelated) complex of platinum

(ll) with good monodentate leaving groups may serve as the model antitumour agent.

Moreover, such compounds are aquated inside the cell before reaching the target DNA.

In vitro investigations have established that diaqua derivative is more reactive towards

DNA than the corresponding dichloro compound1. The 1,2-diamino cyclohexane

platinum (ll) complexes have been found to possess better antitumour activity but with

reduced toxicity than cisplatin2.

In order to study the nature of interactions of amino acids and peptides with anticancer

drugs, the cis-diaqua cis-1 ,2-diamino cyclohexane platinum (ll) perchlorate has been

chosen as the model compound.

During protein digestion by enzymes, polypeptides and dipeptides are obtained as

intermediate compounds, which are composed of different amino acids. The dipeptide,

diglycine made of the single amino acid, glycine is seldom obtained. So the ligand

diglycine is not a bioactive ligand in the true sense but it has been used as a model

compound to study metal-peptide interactions.

4.2 Materials and methods

The substrate cis-[Pt(lR,2S-dach)(H20)2](Cl04)2 was prepared in two steps. The pale

yellow compound cis-[Pt(cis-dach)Ch] was prepared first3. A solution of potassium

tetrachloro platinate (II) in water was prepared. An equimolar amount of cis-1 ,2-diamino

cyclohexane was added to it slowly with constant stirring. The orange coloured solution

turned hazy after 10-15 minutes. The mixture was kept for 6-8 hours on magnetic stirrer

at room temperature. The water insoluble pale yellow compound was filtered after

54

standing for sometime. It was washed successively with water, ethanol and acetone. The

solid was dried in vacuo and theIR data was recorded.

In the next step the yellow solid was sprinkled over an aqueous solution of silver

perchlorate, containing two-mol equivalent. The suspension was kept in dark for more

than 24 hours.

The water soluble cis-diaqua(1R,2S-diamino cyclohexane) platinum (ll) perchlorate was

then separated from precipitated AgCl by filtration. The precipitate was washed with

water and the volume of the solution was made up to the mark. The pH of the stock

solution was brought down to 2.5 to prevent oligomerization. The solution was

characterized by UV spectra.

The product of the reaction, cis-[Pt(1R,2S-dach)(OOCCH2NCOCH2NH3)]CI04 was

prepared by mixing the diaqua substrate and the aqueous solution of diglycine each at pH

4.0 in the molar ratios viz. 1:1, 1:2, 1:3 and 1:4 and thermostating the mixtures at 60°C

for 48 hours. The absorption spectra of all the solutions were recorded using aqueous

ligand solution of appropriate molarity in the reference cell. It was observed that all

solutions exhibit almost identical absorbance indicating the formation of one product.

The composition of the product was ascertained by Job's method of continuous variation.

The combining ratio was found to be 1:1. The Doo value was obtained by keeping the 1 :30

molar ratio of the reactant complex and diglycine at 60°C for 48 hours.

The pH of the solutions was adjusted by adding NaOHIHC104 solution and the

measurements were carried out with the help of a Systronics digital pH meter (model

355) with an accuracy of ±0.01 unit. Doubly distilled water was used to prepare all the

solutions. NaOH was AR Grade product ofBDH, HCl04 was AR Grade product of E.

Merck, K~Cl4 and AgCl04 were purchased from Sigma Chemical Company and 1R,2S­

dach from Aldrich Chemical Company. Diglycine was purchased from SRL, India.

4.3 Experimental Procedure I Kinetic Study

The progress of the reaction was monitored by the absorbance measurement at different

interval of time with a Shirhadzu spectrophotometer (UV-21 01 PC) equipped with a

Shimadzu TB 85 thermo bath (accuracy ±0.1 °C). The absorption due to ligand was

subtracted by using a 1:1 (volume ratio) ligand:water in the reference cell. The

55

absorbance of the product complex was measured at 230 nm where the spectral

difference between the reactant complex and the product complex is large i.e. M is

1.506 (Fig. 4.1 ). Before each kinetic run the pH of each of the reactant complex and the

ligand was adjusted to 4.0 and a pseudo-first order condition was maintained

althroughout. Conventional mixing technique was followed and the absorbance was

noted after allowing a small interval of time to attain the experimental temperature. Rate

data represented as an average of duplicate runs, are reproducible within ±4%.

4.4 Results and Discussion

Diglycine is the smallest of aU the dipeptides and contains three separated functional

groups: terminal- amino group (-NH3}, carboxylate group (-COO} and amide group

(-CONH-), which is referred to as peptide linkage. The two dissociation constants are

pK14 (-COOH) 3.21 and pKl (-NH3} 8.13 at 298 K At the experimental pH (4.0),

diglycine exists as dipolar ion. The dissociation constants of cis-diaqua(cis-1,2-diamino

cyclohexane) platinum (ll) ion, evaluated by Irving-Rossotti titration technique6, are 6.25

and 7.80 respectively. So, at pH 4.0 the reactant complex exists as the diaqua species.

At constant ionic strength (0.1 M with respect to NaCl04), temperature, pH (4.0) and

fixed concentration of the substrate (3.0 x 104 mol dm-3), the In (Da)- D1) values were

plotted against time (t) measured in second. Dt is the value of the absorbance of the

product complex at time t and Doo is the value of absorbance of the product complex after

completion of the reaction. The plot is initially curved and subsequently is linear in

nature (Fig. 4.2). This clearly indicates the biphasic nature of the substitution, one aqua

ligand is replaced by the dipeptide giving the monoaqua product. In the second phase of

substitution two possibilities may arise:

(a) The aqua ligand may be replaced by the second molecule of the dipeptide giving

ML2 type product (his peptide complex) where M represents [Pt(cis-dach)f+ and

L is -OOCCH2NHCOCH2NH3 +.

(b) The aqua ligand may be replaced by chelation involving amide deprotonation

giving ML type product (mono peptide complex).

The reaction between the substrate, cis-[Pt(cis-dach)(H20hf+ and the ligand, diglycine

may be explained on the basis of the following scheme.

56

1.8

1.6 (\ 1.4

\ 1.2 I I

(I)

\) 0 1.0 c: cv

..0 '- 0.8 0

\ (/)

.0 <( 0.6

0.4 ~ ~' "'· 0.2 ' ~

0.0 200 220 240 260 280 300

Wavelength J nm

Fig. 4.1 Spectral difference between the product complex (2) and the substrate

complex (1) [Pt(dach)(H20)ll = 3.0 x 104 mol dm.J, (Diglycine] =9.0 x 10-J

mol dm.J, pH== 4.0, temp.= 60 °C.

0.50

0.48

a-'s 0.46

0 -0.44

0.42

0 1000 2000 3000 4000 5000

Time in Sec

Fig. 4.2 A typical kinetic plot ofln (Da~-:- Dt) versus time, [Pt(dach)(H20)l2+] = 3.0 x

104 mol dm-3, (Diglycine] = 3.0 x 10-3 mol dm-3, pH= 4.0 Temp. 40°C.

2+ Hz ex:'\. /OHz

Pt N/ 'oH Hz 2

A

0 0 II II +

+ Q-(-{HzNH-f-CHzNH3 --~

kz cy, chelation

0 2+ Hz II ex:'\. ))-C'\.

Pt CH N/ 'oHz I 2 + Hz NHCOCHzNH3

B

2+

Similar to the reactions of glycine and L-glutamic acid, which are (N, 0) donors,

diglycine also forms the stable 5-membered (N, 0) chelate complex through amide

deprotonation7 (step B ~ C). The step B ~ D shows the formation of his-peptide

complex, which is probably reversible in nature. This has been assumed to explain the

result of Job's method which shows 1:1, substrate : ligand ratio at the completion of

reaction. With the advancement of chelation the product B is removed and B ~ D step

57

shifts the equilibrium to the left, leading to the formation of more stable Pt- N bond8. In

our experiment a shoulder at ca. 240 nm has been observed in addition to the peak at ca.

227 nm at low-concentration_ The shoulder, develops to a band at higher temperature and

after the reaction has advanced to a significant extent with a maximum at 235_5 nm, is

due to the monopeptide complex C9. The peak, which also changes to a band with a

maximum at 227 nm represents the mono aqua complex.

4.4.1 Calculation o/k1 ·

The present substitution reaction is a two step (biphasic) consecutive reaction. The rate

constant for the first phase of the reaction A ~ B was calculated from absorbance data

using Weyh and Hamm10 equation.

. .. (1)

where a1 and a2 are constants for a given reaction. The equation may be rearranged to

... (2)

The values of a2 exp(-k2(obs)t) at different 't' values are obtained from the limiting linear

portion ofln (Da)- Dt) vs. t plot.

H (Da)- Dt)- a2 exp(-k2(obs)t) = atexp(-ki(obs)t) = L\

then In L\ = In a1 - k1(obs)t

Here L\ = (Da)- Dt)overaiJ- (Da)- Dt)timiting

... (3)

.... (4)

The k1(obs) values, therefore, are obtained from the slopes ofln L\ vs. t plots (Fig. 4.3).

The substitution reaction was investigated for each . of the following ligand

concentrations:

0.0030, 0.0045, 0.0060, 0.0075 and 0.0090 mol dm-3 at constant substrate concentration

of0.0003 mol dm-3, at constant pH (4.0) and at five temperatures viz. 40, 45, 50, 55 and

60°C. Every k1(obs) value showed dependence on the ligand concentration and on the

temperature. The values of kt(obs) are given in Table 4 .1_

The k1 (the second order rate constant for the first phase of substitution) value at each

temperature is obtained from the slope ofkt(obs) versus [Giycylglycine] linear plot (Fig. 4.

4).

58

-3.10

-3.15

<l -3 20 c .

-3.25

0 100 200 300 400 500 600

Time in Sec

Fig. 4.3 A typical kinetic plot ofln A versus time, [Pt(dach)(H20)/+] = 3.0 x 104 mol

dm-3, [Diglycine] = 3.0 x 10-3 mol dm-3, pH= 4.0, Temp. 40°C.

0.0040

0.0035

0.0030

0.0025 ..,. ~0.0020

.::E 0.0015

0.0010

0.0005

0.0000 -f"'l!=--.---...--..---r----..r-----r---r---r--,-----, 0.000 0.002 0.004 0.006 0.008 O.Q10

[Diglycine}

Fig. 4.4 A linear plot of kt(obs) versus ( Diglycine} at different temperatures

( 40°C - 60°C).

Table 4.1: 104 x k1(obs) (s-1) values at different ligand concentrations and at

different temperatures. (Pt(cis-dach)(HlO)lj = 3.0x104 mol dm-3, pH=4.0

Temperature 103 x concentrations of diglycine in mol dm -3 Slope

oc 3.00 4.50 6.00 7.50 9.00 k, dm3mor's·1

40 2.17 3.25 4.33 5.41 6.50 0.072

45 3.00 4.50 6.00 7.50 9.00 0.10

50 4.00 6.00 8.00 10.00 12.00 0.13

55 5.50 8.25 11.00 13.75 16.50 0.18

60 7.31 10.95 14.62 18.28 21.95 0.24

4.4.2 Calculation of k2

The second phase of aqua ligand substitution reaction is composed of two simultaneous

reactions; one of them (B ~ C) is the chelation reaction giving mono peptide complex C

and is ligand concentration independent. The other (B ~D) shows the formation ofbis

peptide complex due to bonding of second molecule of glycylglycine to platinum through

carboxylate-0. This step is, however, ligand concentration dependent. This step has been

shown to be reversible as the monopeptide complex is the ultimate product of the reaction

when sufficient time is allowed for the reaction.

The k2(obs) values are obtained directly from the limiting slopes of In (Da) - Dt) vs. t plots

for different temperatures. When k2(obs) values at a given temperature are plotted against

molar concentration of the incoming ligand, straight lines are obtained with slopes and

intercept. The intercept of each plot is ligand concentration independent and represents

the rate constant k2cy for the chelation reaction. The slope of the linear plot gives the

value of the rate constant k2' ofbis peptide complex formation (Fig. 4.5). The values of

k2cy and k2' are included in Table 4.2.

4.5 Effect of temperature

All the three rate constants k1, k2cy and k2' are sensitive to changes of temperature. The

variation of rate constants with temperature are shown in Eyring plots (Fig. 4.6, Fig. 4. 7,

Fig. 4.8). From the slope of each Eyring plot the value of enthalpy of activation (Lllf) is

obtained, while from the intercept the value of entropy of activation (t\S~) is calculated.

The vaiues of activation parameters of the present reaction together with those of related

systems are given in Table 4.3.

4.6 Mechanism and conclusion

Diglycine is a model dipeptide. It has one carboxyl group and one amino group at the

opposite ends. Because of neutrality of the amide group the two terminal groups are the

most effective bonding sites for metal coordination, but steric requirements preclude the

simultaneous coordination of these groups to the same metal ion.

The complexes of diglycine with various metal ions have been thoroughly studied. The

metal ions, which are able to promote amide deprotonation are the most interesting in this

59

0.00014

0.00012

0.00010

!0.00008

~ 0.00006

0.00004

0.00002

0.00000 -+--.....--.....---r--..---.,.---,..---.---....----,....----. 0.000 0.002 0.004 0.006 0.008 0.010

[Diglycine]

Fig. 4.5 A linear plot of kz(obs) versus (Diglycine] at different temperatures ( 40°C - 60°C).

-30.8

-31.0

-31.2

..-. j::' -31.4 ~ -::::.. -31.6 .s::. ~ ~ -31.8 c

-32.0

-32.2

Fig. 4.6 Eyring plot of kt.

-40.0

-40.2

-40.4

~ -40.6 ID ~

- -40.8 -~-41.0 ....... ...... .E -41.2

-41.4

-41.6

0.00300

'"' ·,,~"""' '•

~~' 0.00305 0.00310 0.00315 0.00320

Fig. 4.7 Eyring plot ofkzcy.

-34.0

-34.2

-34.4

~ -34.6 ID

.;:,(.

--- -34.8 _.r::. N

.;:,(.

::::::::: -35.0 c:

-35.2

-35.4

0.00320

Fig. 4.8 Eyring plot of k2'.

Table 4.2: 105 x k2(obs) (s-1) values at different ligand concentrations and at

different temperatures. [Pt(cis-dach)(H20)l) = 3.0xlO""' mol dm-3, pH=4.0

Tempe- I 05 x concentrations of dig]ycine in mol dm-5 Slope Intercept

rature 3.00 4.50 6.00 7.50 9.00 k2'xl !f k cy 105. -1 2 x ms

oc dm3mor1s-1

40 1.46 1.88 2.29 2.74 3.13 2.78 0.625

45 2.08 2.65 3.22 3.79 4.36 3.80 0.94

50 3.02 3.86 4.69 5.53 6.36 5.57 1.35

55 4.25 5.40 6.55 7.70 8.85 7.67 1.95

60 5.94 7.53 9.13 10.72 12.32 10.63 2.75

Tab

le 4

.3:

Act

ivat

ion

para

met

ers

for

the

rela

ted

syst

ems.

Sys

tem

s m

t .1

St

m2°

Y"'

LlS2

oy.o

,1

,H{,

. .1

.S{,

. R

ef.

' k_

j m

or1

JK"1 m

or1

k J

mol

"1 JK

"1 mo

r1 'l

:J m

or1

JK"1 m

or1

ClS

-

[Pt(~3)2(ll2())2]2

+

5'dG

MP

H2

31.2

±4.

3 -1

17±

15

-----

-----

60.8

±5.

3 -5

5±17

.9

14

5'G

MP

H2

40.6

4±4.

4 -1

06±

16

·----

-----

62.8

±1.

5 -4

6.3±

5.2

15

cis-

[Pt(

en)(H

2C>)

z]"+

L-G

luta

min

e 43

.61±

1.44

-1

22.9

±4.

5 39

.61±

0.8

-203

.8±

2.2

-----

------

16

Gua

nosi

ne

36.2

8±0.

5 -1

25.3

±0.

5 ----

----

--36

.03±

0.6

-149

.2±

1.9

17

5'-A

MP

42

.76±

1.64

-1

12.1

±5.

1 ---

------

-58

.1±

1.4

-84.

2±4.

4 18

ClS

-

[Pt(

dach

)(H

2C> )

2]2 +

Dig

lyci

ne

49.7

1±.0

.55

-108

.3±

1.71

61

.72±

0.38

-1

47.7

±1.

2 56

.16±

0.98

-1

14.9

3±3.

02

Thi

s I

wor

k I

-----

field. A literature survey shows that Pd (IT), Cu (IT) and Ni (IT) are the most effective in

this respect. Potentiometric and spectroscopic studies, however, have definitely proved

that Pd (IT) forms diamagnetic, planar complexes with oligopeptides11. In this case a

tridentate behaviour of the ligand (N, N, 0) has been observed. The existence ofPt (ll)­

peptide N-bonding was proved in an X-ray study of platinum (II) complex of the peptide

GlyMet12.

Diglycine may coordinate to Pt (IT) ion in three ways:

(1) Monodentate coordination through carboxylate-0 to give 1:1 and his products.

(2) Bidentate coordination involving amino-N and carbonyl-0 is expected to occur

at low pH where amide deprotonation is difficult. The 5-member chelate so

obtained is moderately stable13.

(3) Bidentate coordination involving terminal carboxylate-0 and peptide-N gives a 5

-membered chelated product of high stability. This type of chelation occurs at pH 7 4.0 .

(4) Tridentate coordination involving terminal carboxylate-0, amide-Nand amino­

N groups gives rise to two fused 5-membered rings. This has been observed with

Cu (D) complexes. However, such type of coordination has not been found at pH

4.0 with [Pt(NH3)2(H20)2]2+ ion7.

In conclusion it may be said that the substrate cis-[Pt(cis-dach)(H20)2f+ is an (-N-N-)

chelated complex and the dipeptide glycylglycine binds to the platinum centre through

carboxylate-0 and peptide-N, because amide-N" is a soft base showing high affinity for

Pt (ll), a soft acid. The product C is the monopeptide complex, showing a characteristic

band with a maximum at 235.5 nm in the UV-Vis spectra. A similar situation has been

observed in the monopeptide complex oftriglycine with divalent platinum9.

From the calculated values of activation parameters it is obvious that an associative mode

of activation controls the present substitution reaction.

60

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England, (1990).

14. D. J. Evans, M. Green and R. V. Eldik. Inorg. Chim. Acta. 27, 128, 1987.

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17. P. S. Sengupta, R Sinha, S. K. Bera and G. S. De. Ind. J. Chern. 41A, 712,2002. ' 18. P. S. Sengupta, R. Sinha and G. S. De. Trans. Met. Chern. 27, 550,2002.

61