37 CHAPTER - V MECHANISM OF OXIDATION OF AMINO ACIDS BY NBN Before proposing a probable mechanism for the oxidation of amino acids by NBN, the inetic results of the present investigation are summed up as follows:. The reaction is first order with respect to the oxidant (NBN), but there is a decrease in the values of the rate constants with increase in the initial concentration of the oxidant. Further a plot of the observed rate constants ( obs ) Vs the reciprocal of NBN concentration gives a straight line with a finite intercept. 2. Increase in [amino acid has a slight positive effect on the rate of oxidation. Plots of log obs Vs log [amino acids are linear with slope less than unity indicating fractional order dependence on the amino acids. 3. The rate of oxidation decreases with increase in hydrochloric acid concentration. 4. The reaction is not affected by added Cl ion. It is evident from the rates that do not change with increase in sodium chloride concentration. 5. The rate of reaction is quite sensitive to change in solvent polarity. Increasing concentration of acetic acid in the binary solvent mixtures of acetic acid and water increases the rate. 6. Initial addition of one of the products, nicotinamide, to the reaction mixture retards the reaction rate. 7. The change of concentration of Hg(OAc) 2 has no significant effect. In the present study, oxidation by bromine was completely suppressed as
38 the oxidative studies were carried out in presence of Hg(OAc) 2. 30,09 The function of added mercuric acetate 00 is therefore only to fix up 2- bromide formed in the course of the reaction as HgBr 2 or HgBr 4.The liberation of yellow colour followed by faster reaction due to bromine oxidation were suppressed by the addition of 0.005M mercuric acetate 45. 8. The change of concentration of sodium perchlorate does not change the rate appreciably. Negligible influence of ionic strength of the medium on rate indicates presence of a neutral molecule in the rate determining step 87. Added salts lie BaCl 2, KCl, Na 2 SO 4 and K 2 SO 4 do not have any effect on the rate. 9. No polymerization is observed when acrylonitrile is added to the reaction mixture. 0. Increase in temperature increases rate of reaction and plots of log obs Vs reciprocal of temperature are linear for all the amino acids. The entropy of activation is negative in all cases. The free energy of activation of all the amino acids is nearly the same.. The order of reactivity with different amino acids by NBN is as follows: Gly < Ala < Val < Leu < Pro < Ile < Phe < Tyr. The order of relative reactivities of the amino acids is in accord with the views of Porovsaya 46 who observed that amino acids with an even number of atoms in the carbon chain are more easily oxidized in acidic medium than those with an odd number of atoms. 47 It is also interesting to observe that the order of the magnitude of the activation parameters is also consistent with Porovsaya s odd-even effect. This effect arises due
39 to the structure of the given zwitter-ions and the effect of the side chain in the molecule. The presence of alyl groups at the α-carbon has a very minor effect on the amino and carboxyl group as evidenced by their p a values; for most amino acids p a () = 2. ± 0.3 and p a (2) =9.6 ± 0.7. The slight electron-releasing effect of the alyl groups should favour the stabilization of partial positive charge that develops on the α-carbon in the transition state due to the possible non-concertedness of bond-maing and bond-breaing. The non-concerted nature of the reaction may be attributed to the positive charge on the nitrogen which allows carboncarbon bond cleavage to precede carbon-nitrogen bond formation to some extent. This suggests that the oxidizing species attacs the carboxylate group rather than the amino group in the amino acid. Alanine and valine differ from glycine only in containing a methyl or isopropyl group, respectively, at the α-carbon. The presence of these groups has very little effect on the p a of the carboxyl or amino groups. 2. The stoichiometric study shows that one mole of amino acid consumes one mole of NBN. RCH(NH )COOH NBN HCl/H 2O 2 NA RCHO NH3 Cl 3. The Exner plot is linear with correlation coefficient r = 0.9999. The mechanism proposed for the oxidation of amino acids by NBN should therefore account for the observations discussed above. It is necessary to identify the active oxidizing species before any mechanistic interpretation could be advanced.
40 The possible oxidizing species in acidified aqueous acetic acid solution are NBN, NBNH, NBNBr, Br 2, HOBr, H 2 OBr 48.The relative concentration of each species depends on the concentration of N-halogeno compound, the nature (polar or not) and ph of the medium. The observed first order dependence of the reaction rate on NBN rules out NBNBr and molecular Br 2 as the reactive oxidizing species. NBNH and H 2 OBr may be discarded because of inverse dependence of reaction rate on [H. 97 The same observation has been made in the oxidation of amino acids under study. Of the remaining two, HOBr and not NBN should be the oxidizing species, since the rate is also inverse function of nicotinamide concentration. A plot of inverse of the observed rate constant against the nicotinamide concentration is linear (r = 0.9920). The retardation of the rate of oxidation with added nicotinamide suggests that the pre-equilibrium step involves a process in which nicotinamide is one of the products [Eq.2. Another possible reaction, namely, disproportionation of NBN to N, N-dibromonicotinamide, can be ruled out in view of the strict first order dependence of the reaction rate on NBN. Thus the most liely oxidizing species is HOBr. 33,34 NBN H 2 O - NA HOBr ()
4 In acid medium, amino acid exists in its protonated form (SH ) which is resistant to attac by NBN. It is observed that the rate has inverse dependence on [H. Thus the only species possibly controlling the rate of oxidation seems to be RCH(NH 2 )COOH. It is a well nown fact that in acid medium amino acids exist in a mixture of zwitter-ionic and cationic form. Cationic forms of amino acids can be considered to be the reactive species in acid medium. The attac of NBN on the α-carbon of zwitter-ionic form of amino acid is more feasible since it has relatively lower electron density than the anionic form 97. The first order dependence on [substrate and [oxidant reveals that overall rate may involve the interaction of HOBr and amino acid in the rate determining step 49. But first order in the [substrate and a definite intercept in the Vs obs [sub plot, suggest that the decomposition of the complex formed from the substrate and HOBr is the rate determining step as shown in Scheme 3. SH 2-2 S H (2) S HOBr 3-3 [RCH (NH 2 ) COO-Br H 2 O Complex (3)
42 [RCH (NH 2 ) COO-Br R-C H (NH 2 )CO 2 Br (4) R-C H (NH 2 ) RCH=NHH (5) RCH=NHH 2 O RCHONH 3 (6) Here SCHEME 3 R=-C 3 H 7 forval,-c 4 H 9 for Leu,Ile, Ph CH 2 - for Phe, H for Gly,-CH 3 for Ala, -OH Ph CH 2 - Tyr, CH 2 -( CH 2 ) 3 in cyclic structure Pro, (CH 2 ) 4 NH 2 for Lys, -CH 2 C 3 N 2 H 3 for His and -CH 2 C 3 N 3 H 8 for Arg, (CH 2 ) OH for Ser and CH 3 (CHOH) for Thr, (CH 2 ) 2 COOH for Glu and CH 2 COOH for Asp The rate law for the above mechanism may be derived as follows Rate = d[nbn dt = d [Complex (7) Rate = [NBN[SH d 2 3 [NA[H (8) [NBN T = [NBN [HOBr [complex (9) = [NBN d 2 3[NBN[SH [NBN (0) [NA [NA[H [NBN = [NBN T 2 [NA [NA 3 [SH [H ()
43 Substituting [NBN in equation (8) d[nbn T dt = d 23[NBN [NA[H T [SH [H 2 3 [SH (2) On rearranging the equation [NA[H d 2 3 [H d2 3 [SH d = obs (3) The rate-determining step proposed in the above mechanism predicts negligible salt effect which has been experimentally observed. The formation of the complex involves the charge separation which leads to a negative solvent effect. This has been confirmed by the increase in rate with decreasing dielectric constant of the medium. The involvement of amino acid molecule in the rate-determining step leads to different values of obs for different initial concentrations of amino acids under study. The proposed mechanism is well supported by the moderate values of energy of activation and thermodynamic parameters. The negative entropy of activation indicates the complex formation as suggested in the above reaction mechanism, and also indicates that the complex is more ordered than reactants. 50 High positive values of the free energy of activation and the enthalpy of activation show that the transition state is highly solvated. The postulated establishment of prior equilibrium involving NBN and NA may also explain the observation that the rate constant for the oxidation of glycine with NBN decreases linearly with increase in initial
44 concentration of oxidant (Table 4.2). It may be pointed out that the increase in the initial concentration of the oxidant leads to an increase in the concentration of nicotinamide which has been shown to have a distinct retarding effect on oxidative reaction (Table 4.8). Fairly high positive values of the free energy of activation DG # and the enthalpy of activation DH # indicate that the transition state is highly solvated while the negative entropy of activation DS # suggests the formation of an activated complex with a reduction in the degree of freedom of molecules.