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Amino acids are protein building components that include both acid and base groups. β-Alanine was used in alkaline medium for the current investigation. β-Alanine is a naturally occurring β-amino acid, meaning that the amino group is connected to the β-carbon rather of the more common α-carbon for alanine. 3-aminopropanoic acid is the IUPAC designation for β-alanine. Unlike its homologue, α-alanine, it lacks a stereocenter. The kinetics of β-Alanine oxidation by alkaline Os (VIII), which was continuously regenerated by hexacyanoferrate (III) ion, were investigated in the 0.01- 0.07 M and 298 K - 318 K temperature ranges.The rate of reaction was determined to be zero with regard to [Fe(CN6)]^3- and one with respect to Os (VIII).

To investigate the kinetics of β-alanine oxidation by osmium (Os (VIII)) in an alkaline medium, with Os (VIII) being regenerated by hexacyanoferrate (III), focusing on determining the reaction order and the influence of temperature and reactant concentrations.

Amino acids can have carboxylic, sulphonic, or other acid groups. The -NH₂ group can be found in any location in amino acids.In general, amino acids are commonly used in reference to α-amino acids derived from natural sources. Amino acids exist as zwitterions in solution^{11,12,13,14,15}.

In general, all amino acids are water soluble but insoluble in non-polar solvents such as ethers, benzene, and so on^14,26.The hexacyanoferrate (III) ion is distinct from other oxidants that have more than one electron equivalent^17,18. In acidic and alkaline environments, it is an inert substitute^1,2,,6,8,16.As a result, the readily accessible and water-soluble hexacyanoferrate (III) ion is a very effective oxidant for a wide range of inorganic and organic molecules^{21,25,28,30,39,40}.

Standardisation of osmium tetroxide

One g of OsO₄ glass tube was collected. It was broken up and placed in a beaker with 0.5 N of sodium hydroxide solution. Glass tubing fragments have been removed from the solution. These pieces were washed with distilled water many times in the same beaker until the solid was totally dissolved. The solution was then transferred to a 1 L measuring flask. Conductivity water was utilised to bring the volume of the solution up to the desired level after dissolving the solid OsO₄ solution. The strength of sodium hydroxide was determined by titrating the solution against a reference solution of potassium hydrogen phthalate with phenolphthalein as an indicator. The OsO₄ solution was also iodometrically standardised^19,20.

 λ_max of potassium hexacyanoferrate (III):

The absorbance of unreacted (0.001M) potassium hexacyanoferrate (III), which was to be used as the oxidant, was measured at different wavelengths using a double beam Biosync Teknology Spectrophotometer, model number BST 2800, and an Elico-Digital Spectrophotometer, model number CL-27. The absorbance vs wavelength graph was constructed. The value of max for potassium hexacyanoferrate (III) was 420 nm^24,31.

Molar absorption coefficient of potassium hexacyanoferrate (III):

The absorbance values of different [potassium hexacyanoferrate (III)] were measured at 420 nm

The molar absorbance coefficient for potassium hexacyanoferrate (III) at 420 nm was measured to be 990 dm³ mol^-1 cm^-1, which corresponds to the quoted value (1030 dm³ mol^-1 cm^-1)^24,31,32,33.

Measurement of the rate constants:

The reaction mixtures were created using standard techniques. A combination of the predicted substrate and NaOH contents was added to the reaction vessels. Standard NaCl solutions kept their ionic strength. A solution of potassium hexacyanoferrate (III) in a volumetric flask of 250 mL was prepared. These were kept at the desired temperature in a thermostatic water bath with a 0.1⁰C precision. The reaction was started by adding the required amount of hexacyanoferrate (III) to the reaction mixture once it had attained temperature equilibrium, which took at least 20 to 30 minutes in all cases. The reaction mixture was promptly extracted and transferred to the spectrophotometer's sample tube using a 5 mL pipette. The absorbance of the reaction mixture at 420 nm was measured using a double beam Biosync Teknology Spectrophotometer, model number BST 2800and an Elico-Digital Spectrophotometer, model number CL-27^22,23,37.The absorbance was measured quickly, with no interval between the removal of the reaction mixture and the recording of the results. To trace the course of the reaction until it was completed, the absorbance of the reaction mixture was measured over time. The reactions were found to be too sluggish to study in alkaline media in the absence of a catalyst. Without the use of a catalyst in alkaline media, the oxidation of β-Alanine took nearly a month to complete. Hence Os (VIII) catalyst was added to the reaction mixtures to speed up the reactions. It was observed that in the presence of an Os (VIII) catalyst, alkaline medium reactions may be completed in less than 4 to 5 hours. The absorbance versus time charts for more than two half-lives of the reactions were almost linear, indicating a zero-order reaction with potassium hexacyanoferrate (III). The slope of the linear plot between absorbance and time was calculated in each case. The following formula was used to compute the rate constant k_o:

                                                            

                                                   Fig. 1: Zero order plot for the oxidation of B-Alanine

The same method was used in the kinetic experiment with Os (VIII) as a catalyst.

Stoichiometery of the reactions:The stoichiometry of the reactions was calculated under the following two conditions.

(i)            [Substrate]            >>        [K₃[Fe(CN)₆]

(ii)          [Substrate]            <<       [K₃[Fe(CN)₆]

The experimental circumstance Condition (i) specifies the initial oxidation product of the substrate, whereas Condition (ii) specifies the total amount of oxidation.The stoichiometry was obtained under experimental condition (ii) as detailed in Table 1 while the reaction mixtures were kept at the desired temperature in a thermostat. Before beginning the process, the [hexacyanoferrate (III)] concentration was determined, and the absorbance at 420 nm was calculated until it reached a constant value. Equation (I) was used to compute the quantity of unreacted [hexacyanoferrate (III)]. The stoichiometry was then determined as shown in the Table1.

Table 1:  Stoichiometry of the reaction between potassium hexacyanoferrate (III)[P] and β-Alanine[β].

      

β-Alanine, osmium tetroxide, sodium hydroxide, sodium chloride, acetone, potassium hydrogen phthalates, oxalic acid, potassium hydroxide, and phenolphthalein were all manufactured by E. Merck (India) Ltd. Fluka Puriss was the source of potassium hexacyanoferrate (III). All of the solutions were made in Pyrex glass receptacles with triple distilled conductivity water.

Product Analysis:

The product was tested in two different ways.

(a)               [substrate]    >>    [hexacyanoferrate]

(b)               [substrate]    <<    [hexacyanoferrate]

When substrate]    <<    [hexacyanoferrate]

Under this experimental condition, the final products of β-Alanine oxidation were keto acid and ammonia. Nesseler's reagent verified the presence of ammonia³⁸. Spot testing using phenylhydrazine and an oxidising agent (potassium ferricyanide) revealed keto acid⁵. The presence of carboxylic and keto groups is shown by the ir peaks at 1760 cm^-1 and 1591 cm^-1, respectively.

Order with Respect to Hexacyanoferrate (III):

The reaction order with respect to hexacyanoferrate (III) was determined at 298K with constant [substrate], [Os (VIII)], [OH^-], and ionic strength.The [hexacyanoferrate (III)] ranged from 8.0x10^-4 to 16x10^-4. The higher concentrations in the initial [hexacyanoferrate (III)] were not applicable because Beer's law does not apply at higher concentrations. In the current study, the absorbance vs time plot for the oxidation of β-Alanine indicated that order with respect to hexacyanoferrate (III) was zero. Fig.1 depicts a representative plot. The observation that the k_o values remained unchanged with the change in the initial [hexacyanoferrate (III)] while the t_1/2 values increased with the change in the initial [hexacyanoferrate (III)] provided additional support to the zero order reaction. Table 2 shows the results.

Dependence on [Os (VIII)]:

The oxidation of β-Alanine by alkaline hexacyanoferrate (III) was studied at different [Os (VIII)] at constant concentrations of all other reagents. It was discovered that the plots between k_o and [Os (VIII)] for a ten-fold variation in the initial [Os (VIII)] were linear and almost passed through the origin, as shown in Fig. 3 for the Os (VIII) catalysed hexacyanoferrate (III) oxidation of β-Alanine in alkaline medium. Table 4 summarises the findings.                           

                        

Dependence on [amino acids]:

The order of the reaction with respect to β-Alanine was determined by studying the effect of different [β-Alanine] on the values of k_o at constant concentrations of all other reagents. Only a seven-fold change in the [substrate] was carried out in β-Alanine. Because of the large amounts of β -Alanine, complicated kinetics was observed, and the rate constants fell. The plot of k_o^-1 versus [β-Alanine]^-1 was found to be linear in each case, with an intercept on the k_o^-1 axis^22,23,37. Fig.4 demonstrate these graphs. The results are shown in Table 5.

                                     

Effect of the ionic strength:

The effect of ionic strength on β-Alanine was examined by varying the ionic strength in a standardized sodium chloride solution. The k_o values improved as the ionic strength increased. The plot of log k_o was found to be linear with a positive slope, Fig. 5, indicating that the reaction was observed between similarly charged ions. Table 6 summarises the results.

                                      

Mechanism of oxidation of β-Alanine:

The following process is suggested for the oxidation of β-Alanine in alkaline medium^23,37 by hexacyanoferrate (III) employing Os (VIII) as a catalyst.

The aforementioned process clearly shows that product (1) is generated as indicated in Eq. (5) and is then oxidised to the appropriate keto acid (the end product) in a fast step under the experimental circumstances, which is confirmed by stoichiometry and product analysis.

Therefore, the rate of disappearance of Os (VIII) be derived as:

parameters for the β-Alanine amino acid. The activation energy and entropy values were determined using  the slope and intercept values of the Arrhenius plot (Fig.5), which was practically linear for β- Alanine⁷.

In view of Eq. (6) and valid assumptions

K₂ K₄>>  K₁K₃  and

1 + K₂K₄[OH^-][NH₂-CH₂-CH₂-COOH]>> K₂[OH^-] +  K₁K₃ [NH₂-CH₂-CH₂-COOH]

Eq. (11) reduces to Eq. (12)

The following are the important features of the Os (VIII) catalysed hexacyanoferrate (III) oxidation of β -Alanine:

1. The reaction has a zero order in regard to hexacyanoferrate (III).
2. The reaction rate with reference to catalyst is one because the k_o vs [Os (VIII)] plot is linear and almost reaches the origin.
3. As [OH]^- levels rise, consequently does the rate of oxidation.
4. The linearity of the k_o^-1 vs [substrate]^-1 plot results in a positive k_o^-1 axis intercept.
5. The rate of oxidation increases with increasing ionic strength, and the log k_o vs√ µ plot suggested that the reaction occurred between ions with similar charges.

1. Bellaki M.B., Mahesh R.T. and Nandibeoor S.T., Kinetics of oxidative decarboxylation and deamination of l- glutamate by diperiodatonickelate (IV) in aqueous alkaline medium, Asian J. Chem., 13A, 1639-1644 (2004)
2. Chimatadar S.S., Nandibewoor S.T. and Salunke M.S., A kinetic and mechanistic study of oxidation of arsenic (III) by hexacyanoferrate (III) in aqueous ethanoic acid, Transition Metal Chemistry,29, 743–750 (2004)
3. Culbertson J.Y., Kreider R.B., Greenwood M., and Cooke M., Effects of beta-alanine on muscle carnosine and exercise performance: a review of the current literature, Nutrients. 2(1), 75–98 (2010)
4. Dunnett M., Harris R.C., Influence of oral beta-alanine and L-histidine supplementation on the carnosine content of the gluteus medius, Equine Vet. J. Suppl., 30, 499–504 (1999)
5. Feigl F., Spot Test in Organic Analysis, New York, Elsevier, 6^th ed. (1975)
6. Garg D. and Kothari S., Kinetics and mechanism of the oxidation of some-amino acids by benzyltrimethylammonium tribromide, Indian J. Chem., 44(B), 1909-1914 (2005)
7. Goel A., Shakunj and Shivani, Kinetics and mechanism of iridium (iii) catalyzed oxidation of some amino acids by hexacyanoferrate (iii) ions in aqueous alkaline medium, International journal of chemical science, 6(4), 1891-1899 (2008)
8. Gowda J. I. et al., Oxidation of glycine by diperiodatocuprate (III) in aqueous alkaline medium, Indian J. Chem., 52(A), 200-206 (2013)
9. Harris R.C, Tallon M.J., Dunnett M., Boobis L., Coakley J., Kim H. J., et al., The absorption of orally supplied beta-alanine and its effect on muscle carnosine synthesis in human vastus lateralis. Amino Acids, 30(3), 279–289  (2006)
10. Harris R.C., Jones G., Hill C. H., Kendrick I. P., Boobis L., Kim C. K., et al., The carnosine content of vastus lateralis in vegetarians and omnivores, FASEB J., 21, 76 (2007)
11. Heinzpenzlin, The riddle of “life,” a biologist’s critical view, Naturewissenschaften, 96, 1- 23 (2009)
12. Hiremath D.C., Sirsalmath K.T., Nandibewoor S.T., Osmium(VIII)/Ruthenium(III) catalysed oxidation of L-lysine by diperiodatocuprate(III) in aqueous alkaline medium: A comparative mechanistic approach by stopped flow technique, Cata. Lett., 122, 144-154 (2008)
13. Hosmani R.R. and Nandibewoor S.T., Mechanistic study of ruthenium (III) catalysed oxidation of Lysine by diperiodatoargentate (III) in aqueous alkaline medium, J. Chem. Sci., 121 275- 281 (2009)
14. Insausti M.J. and Mata-perez F., Alvarez-Macho P. Permanganate oxidation of glycine: Influence of amino acid on colloidal manganese dioxide,  International J. Chem. Kinet., 24(5), 411-419 (1992)
15. Jacimirskij K.B., Jovan V, Ankica J., et al., Uvod Bioneorgansku Hemiju. Serbia: Beograd Privredni pregled, 1980.
16.  Lancaster J.M. and Murray R.S., The ferricyanide- sulphite reaction, J. Chem. Soc. A, 2755-2758 (1971)
17.  Mahanti M.K. and Laloo D.J., Kinetics of oxidation of amino acids by alkaline hexacyanoferrate (III), Chem. Soc., Dalton Trans., 1, 311-313 (1990)
18.  Mehrotra U.S., Agrawal M.C. and Mushram S.P., Kinetics of the reduction of hexacyanoferrate (III) by ascorbic acid, J. Phys. Chem., 73(6), 1996–1999 (1969)
19.  Mehta S.P.S., Ph.D. Thesis, Kumaun University, Nainital (1986)
20. Mohan D. and Gupta Y.K., Kinetics and mechanism of the osmium (VIII)-catalyzed oxidation of phosphite by hexacyanoferrate (III) ion in aqueous alkaline media, Journal of the Chemical Society, Dalton Transactions, 11, 1085-1089 (1977)
21. Nowduri A., Adari K.K., Gollapalli N.R. and Parvataneni V., Kinetics and mechanism of oxidation of l- cysteine by hexacyanoferrate (III) in alkaline medium, E. J. Chem., 6(1), 93- 98 (2009)
22. Parmar R. and Bhatt S., kinetics and mechanism of oxidation of some α-amino acids by chromic acid in presence of chloro substitutedacetic acids,Journal of Emerging Technologies and Innovative Research,4(9), 305-316  (2017)
23. R. Sayee Kannan, D. Easwarmoorthhi, K. Vijaya and M.S. Ramachandran, Autocatalytic oxidation of β‐alanine by peroxomonosulfate in the presence of copper (II) ion, International journal of chemical kinetics, 40(1), 44-49 (2008)
24.  Rajchel-Mieldzioc P., Tymkiewicz R., Sołek J., Secomski W., Litniewski J. and Fita P., Reaction kinetics of sonochemical oxidation of potassium hexacyanoferrate (II) in aqueous solutions, Ultarsonics Sonochemistry, 63, 10912 (2020)
25.  Rao K.V.C., Neema S.K., Krishnamurthy V.N., Swaminathan V. and Gowariker V.R., Kinetics of oxidation of hydroxyl ion by potassium hexacyanoferrate (III), J. Indian Chem. Soc., 61(9), 751 (1984)
26. Rosenberg L.E., Berman M. and Segal S., Studies of the kinetics of amino acid transport, incorporation into protein and oxidation in kidney-cortex slices, Biochimica et Biophysica Acta., 71, 664-675 (1963)
27. Sengupta K.K. and Basu B., Kinetics of Os (VIII)- catalysed oxidation of As (III) by alkaline hexacyanoferrate (III), Indian J. Chem., 16(A), 808-809 (1978)
28. Sharanabasamma K., Mahantesh A., Angadi and Tuwar S.M., Kinetics and mechanism of Ruthenium (III) catalyzed oxidation of l-proline by hexacyanoferrate (III) in aqueous alkali, The Open Catal. J., 4, 1-8 (2011)
29. Shimpi R., A review of kinetics of oxidation of organic compounds by hexacyanoferrate (iii), Research journal of life science. bioinformatics, pharmaceutical and chemical science, 5(2), 164(2019)
30. Singh A.K., Singh R.K., Srivastava J., Rahmani S. and Yadav S., Kinetic and mechanistic studies of oxidation of glycine and valine by N-bromosuccinimide using chloro complex of Rh (III) in its nano-concentration range as homogenous catalyst, Indian J. Chem., 51(A), 681-689 (2012)
31. Singh T, Kinetics of oxidation of l-proline by hexacyanoferrate (III) ion in presence of Os (VIII), International journal of chemical engineering and processing, 7(1), 12-24 (2021)
32. Singh T., Kinetic study of l-lysine and l-arginine by hexacyanoferrate (III) ion in presence of Os (VIII), Research journal of chemistry and environment, 27 (2) ,62-72 (2023)
33.  Singh T., Ph.D. Thesis, Kumaun University, Nainital (2014)
34. Stegen S., Bex T., Vervaet C., Vanhee L., Achten E. and Derave W., Beta-Alanine dose for maintaining moderately elevated muscle carnosine levels, Med. Sci .Sports Exerc., 46(7), 1426–1432 (2014)
35. Stout J. R., Cramer J. T., Zoeller R. F., Torok D., Costa P., Hoffman J. R., et al., Effects of beta-alanine supplementation on the onset of neuromuscular fatigue and ventilatory threshold in women, Amino Acids, 32(3), 381–386 (2007)
36. Stout J.R., Graves B.S., Smith A.E., Hartman M.J., Cramer J.T., Beck T. W., et al., The effect of beta-alanine supplementation on neuromuscular fatigue in elderly (55–92 Years): a double-blind randomized study, J. Int. Soc. Sports Nutr., 5, 21 (2008)
37. Verma A., Pandey J. Srivastava S., Kinetic and Mechanistic Study of Oxidation of L-Alanine by Acidic Solution of Chloramine-T in Presence of Chloro- Complex of Ir (III) as Homogeneous Catalyst, Canadian chemical transactions, 4(1), 1-16 (2016)
38. Vogel A.I., A textbook of practical organic chemistry including qualitative organic analysis, Longmans,3, 332 (1973)
39. Waldemar I., Alicja W., Zdzislawa N. and Grzegorz S., Mass spectrometric study of amino acid–triethylborane chelates, Monatsh Chem., 140, 359-364 (2009)
40. Williard H.H. and Manalo G.D., Some formal oxidationreduction potentials, Anal. Chem., 19, 462-463 (1947)