Revealing the Mechanistic Pathway of Acid Activation of Proton Pump

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Revealing the Mechanistic Pathway of Acid Activation of Proton Pump Inhibitors to Inhibit the Gastric Proton Pump: A DFT Study Kalyanashis Jana, Tusar Bandyopadhyay, and Bishwajit Ganguly J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b09334 • Publication Date (Web): 05 Dec 2016 Downloaded from http://pubs.acs.org on December 12, 2016

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The Journal of Physical Chemistry

Revealing the Mechanistic Pathway of Acid Activation of Proton Pump Inhibitors to Inhibit the Gastric Proton Pump: A DFT Study Kalyanashis Jana1, Tusar Bandyopadhyay2 and Bishwajit Ganguly1* 1

Computation and Simulation Unit (Analytical Discipline and Centralized Instrument Facility), CSIR–Central

Salt and Marine Chemicals Research Institute, Bhavnagar–364002, Gujarat, India and Academy of Scientific and Innovative Research, CSIR–CSMCRI, Bhavnagar–364002, Gujarat, India. 2

Theorectical Chemistry Section, Chemistry Group MOD LAB, Bhabha Atomic Research Centre, Trombay,

Mumbai 400 085, INDIA * To whom correspondence should be addressed. E-mail: [email protected]

Abstract: Acid-related gastric diseases are associated with disorder of digestive tract acidification due to the acid secretion by gastric proton pump, H+,K+-ATPase. Omeprazole is one of the persuasive irreversible inhibitor of the proton pump H+,K+-ATPase. However, the reports on the mechanistic pathway of irreversible proton pump inhibitors (PPIs) on the acid activation and formation of disulfide complex are scarce in the literature. We have examined the acid activation PPIs i.e. timoprazole, S-omeprazole and R-omeprazole using M062X/631++G(d,p) in aqueous phase with SMD solvation model. The proton pump inhibitor is a prodrug and activated in the acidic canaliculi of the gastric pump H+,K+-ATPase to sulfenic acid which can either form another acid activate intermediate sulfenamide or a disulfide complex with cysteine amino acid of H+,K+-ATPase. The quantum chemical calculations suggest that the transition state (TS5) for the disulfide complex formation is the rate determining step of the multistep acid inhibition process by PPIs. The free energy barrier of TS5 is 5.5 kcal/mol higher for timoprazole compared to the S-omeprazole. The stability of the transition state for the formation of disulphide bond between S-omeprazole and cysteine

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amino acid of H+,K+-ATPase is governed by inter- and intramolecular hydrogen bonding. The disulphide complex for S-omeprazole is thermodynamically more stable by 4.5 kcal/mol in aqueous phase compared to disulfide complex of timoprazole, which corroborates the less efficacy of timoprazole as irreversible PPI for acid inhibition process. It has been speculated that sulfenic acid can either form sulfenamide or a stable disulfide complex with cysteine amino acid residue of H+,K+-ATPase. The M062X/6-31++G(d,p) level of theory calculated results reveal that the formation of tetra cyclic sulfenamide is unfavoured by ~ 17 kcal/mol for S-omeprazole and 11.5 kcal/mol for timoprazole compared to the disulfide complex formation in each case. The DFT calculations have further shed light on the acid activation process of R- and S-isomers of omeprazole. The calculated results suggest that the efficacy of these isomers lie on their metabolic pathway and excretion from human body.

Introduction: In 1905, John Edkins discovered gastrin that has subsequently stimulated the scientific exploration of the secretion of gastric acid.1 Gastric acid plays a key role in normal upper gastrointestinal functions, including protein digestion, calcium and iron absorption, as well as providing some protection against bacterial infections.2 However, inappropriate levels of gastric acid in stomach and intestine cause several pathological conditions, e.g. Gastroesophageal Reflux Disease (GERD) and peptic ulcer (PU).2-6 Heartburn is the one of the most common symptom for GERD and peptic ulcer causes pain in the stomach and small intestine.4-6 Discovery of gastrin and elucidation of the role of histamine led to the understanding of the pathogenic basis of PU, GERD and gastric acid related diseases and their subsequent treatments. The secretion gastric acid in parietal cells is due to the presence of food in stomach or intestine and also due to the taste, smell, sight or thought of food.2,7-8 Gastric acid is secreted followed by the activation of histamine, acetylcholine or gastrin receptors (the H2, M3 and 2 ACS Paragon Plus Environment

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CCK2 receptors, respectively) located in the basolateral membrane of the parietal cells. The H2, M3 and CCK2 receptors initiate signal transduction pathways that triggers the activation of the H+,K+-ATPase which causes acid secretion.8 Acid-related gastric diseases are associated with disorder of digestive tract acidification. The gastric proton pump, H+,K+ATPase, exports H+ in the luminal cavity in exchange of K+, which generates a highly acidic environment in the gastrointestinal track, and therefore, is a target for acid suppressant drug molecules. This perhaps the commonly used pharmacological approach to regulate the acid secretion as it can inhibit acid secretion independently. Omeprazole is known to be the first proton-pump inhibitors used in clinical practice.2,9 The development of omeprazole was a new approach for the effective inhibition of acid secretion and the treatment of acid-related diseases, and was quite quickly shown to be clinically superior to the H2-receptor antagonists. Omeprazole consists of two optical isomers (enantiomers), one being the mirror image of the other, and exists as racemic mixture.11-12 The S-isomer of omeprazole subsequently proved to be a better drug that is significantly superior compared to the racemic omeprazole. Pharmacokinetics and pharmacodynamics studies have revealed that the Sisomer of the proton pump inhibitors are more superior compared to the R-isomers.12-13 Like omeprazole drug, other irreversible proton pump inhibitors e.g. timoprazole, tenatoprazole, rabeprazole, pantoprazole, lansoprazole are racemates in nature.14 It is important to note that omeprazole and other proton-pump inhibitors are prodrugs which are converted to their active form in acidic environments of parietal cell.15-21 Proton pump inhibitors are weak bases and generally concentrate in the acidic secretory canaliculi of the parietal cell, where they are activated to an acid activated complex by a proton-catalysed process (Scheme 115).22 The acid activated complex makes covalent bond with the sulphydryl groups of cysteine-813 or 822 residues in the extracellular domain of the H+,K+-ATPase to generate stable disulfide complex and thereby inhibiting the enzyme activity.



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Scheme1: Mechanism of the acid activation and finally stable disulfide complex formation of proton pump inhibitor

Scheme 1 shows the plausible mechanism to inhibit the acid secretion by PPI.15 The Scheme 1 suggests that pyridine (Py) nitrogen of neutral drug molecule gets protonated in the acidic environment in the parietal cell to form (PyH+) intermediate. Further, the N-3 nitrogen of benzimidazole (Bz) moiety is protonated via the transfer of proton from the pyridinium nitrogen (Scheme 1 and 2). The nitrogen lone-pair of pyridine unit intramolecularly attacks the C-2 carbon of the Bz moiety to form the N-C bond and a spiro intermediate i.e., dihydrobenzimidazole is formed. The unstable spiro intermediate opens up and undergoes aromatization to form sulfenic acid which further dehydrates to form tetracyclic sulfenamide (Scheme 1). Finally, the sulfenic acid or the tetracyclic sulfenamide reacts with the cysteine


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residue of H+,K+-ATPase enzyme to form the stable disulfide bond that suppress the acid secretion process. The plausible mechanism for the inhibition of acid secretion with PPI has not been examined experimentally or computationally. In this article, we have explored the mechanism of disulfide bond formation between the cysteine amino acid residue of the gastric proton pump H+,K+-ATPase and PPIs. Timoprazole one of the PPIs is the basic scaffold of many irreversible PPIs.23,15 The derivatization of timoprazole shows better efficacy compared to parent temiprazole.2,24-25 Therefore, we have examined the acid activation mechanism with timoprazole and one of the superior PPI, i.e. omeprazole.2 5-methoxy-2-(((4-methoxy-3,5dimethylpyridin-2-yl)methyl)sulfinyl)-1H-benzo[d]imidazole (omeprazole) is a substituted 2-((pyridin-2-ylmethyl)sulfinyl)-1H-benzo[d]imidazole (timoprazole) (Scheme 2). It would be interesting to see that such structural difference in PPIs can induce the activity toward the suppression of acid secretion in the acidic canaliculi. Further, the experimental observations fail to shed light on the controversy of disulfide bond formation with sulfenic acid or with sulfenamide of the drug molecules. The computational study showed a clear preference for the formation of disulphide bond between the enzyme and sulfenic acid. This is the first report to decipher this ambiguous observation in the experimental studies of PPI.

Scheme 2: a) S-isomer of the timoprazole drug molecule b) S-isomer of the omeprazole drug i.e. Someprazole and c) R-isomer of omeprazole.

Computational Methods: 5 ACS Paragon Plus Environment


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All the acid activated geometries of timoprazole, R- and S-omeprazole have been optimised in gas phase at M062X26 DFT functional with 6-31++G(d,p)27-28 Pople basis set. We have performed harmonic frequency calculations at the same level of theory to confirm minima of optimised geometries with no imaginary frequencies. The M062X is one of the accurate DFT functional for organic and ionic systems.29-30 We have located transition states on the potential energy surface with M062X/6-31++G(d,p) in gas phase and a transition state is confirmed with one imaginary frequency. Further, we have performed IRC calculation to connect the TS geometries with the initial and final complex.31 The electronic energy differences were calculated with respect to the initial neutral drug molecule as:

ΔE =EX– EN ........ (1) where EX is electronic energy of intermediates, transition states or final complex and EN is the electronic energy of neutral drug molecule molecules and ΔE is the difference in the electronic energies. The M062X/6-31++G(d,p) level of theory optimized geometries were taken for single point energy calculations in aqueous phase with Self Consistent Reaction Field (SCRF) method.32 We have performed the aqueous phase (ε=78.8) calculation using SMD solvation model with M062X/6-31++G(d,p) level of theory.33 It has been reported that SMD solvation model is a universal solvation model, where “universal” denotes its applicability to any charged or uncharged solute in any solvent or liquid medium for which a few key descriptors are known. We have calculated the free energy differences using similar equation for acid activation process of the PPIs. The Gibbs free energy in the solvent phase has been calculated as: ‫ܩ‬௔௤ = ‫ܩ‬௚௔௦ – (‫ܧ‬௔௤ – ‫ܧ‬௚௔௦ ) + ߂‫ ܩ‬ଵ௔௧௠→ଵெ …….2 where Gaq and Eaq are Gibbs free energy and electronic energy in the aqueous phase, Ggas and Egas are Gibbs free energy and electronic energy in the gas phase, ߂‫ ܩ‬ଵ௔௧௠→ଵெ is the the


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correction associated with the change in the standard state from the gas phase (1 atm) to solution (1 mol L−1) and its value at 298.15 K is 0.003012 Hatree.34-36 Free energy differences have been calculated as: ߂‫ܩ‬௔௤ = ‫ܩ‬௔௤,௑ − ‫ܩ‬௔௤,ே − ‫ܩ‬௔௤,ு శ ...............3 where Gaq,X is the free energy of the intermediates, transition states, Gaq,N is the free energy of the neutral drug molecules and ‫ܩ‬௔௤,ு శ is the free energy of the proton. The ‫ܩ‬௔௤,ு శ has been calculated as:37 ଴ ଵ௔௧௠→ଵெ ‫ܩ‬௔௤,ு శ = ‫ܩ‬௚௔௦,ு ...............4 శ + ߂‫ܩ‬௔௤,௦௢௟,ு శ + ߂‫ܩ‬

38 ଴ where ‫ܩ‬௚௔௦,ு kcal/mol and ߂‫ ܩ‬ଵ௔௧௠→ଵெ =1.89 శ = − 6.28 kcal/mol, ߂‫ܩ‬௔௤,௦௢௟,ு శ = − 261.85

kcal/mol are obtained from literature. The ߂‫ܩ‬௔௤,௦௢௟,ு శ is debated in the literature and difficult to either measure or predict accurately. In

this article, we have considered

߂‫ܩ‬௔௤,௦௢௟,ு శ =−261.85 kcal/mol and the ‫ܩ‬௔௤,ு శ =−266.24 kcal/mol value has been used in (3).38 Further, we have additionally performed aqueous phase calculations of all the acid activated intermediates and transition states of timoprazole drug molecule using the same level of theory. The free energy differences calculated using zero-point energy as well as the entropy term for timoprazole using Equation-3 (Table S1). We have observed that ΔGaq obtained using Gaq calculated by Equation-2 and the optimization of timoprazole in aqueous phase showed similar results. All DFT calculations were performed using the G09 package. 39

Results and Discussion: The mechanism of acid activation and disulphide bond formation with cysteine of H+K+ ATPASE has been examined with M062X/6-31++G(d,p) level of theory in aqueous 7 ACS Paragon Plus Environment


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phase. To investigate the effect of substituents on the overall acid activation process timoprazole and S-omeprazole molecules have been considered. The basic scaffold of timoprazole has been substituted with the methoxy groups at the 4-position in pyridine (Py) and 5-position benzimidazole (Bz) rings and with methyl group at 3, 5 position of the Py in Someprazole molecule (Scheme 2). The M062X/6-31++G(d,p) calculated results show that the pyridine Py and Bz rings are co-planner for timoprazole molecule (Figure 2). Such arrangement of rings facilitates the proton transfer process in the inhibitor molecule to form the acid activated intermediate (Figure 2). The oxygen of the sulfinyl group of timoprazole forms a weak intramolecular hydrogen bond (2.37 Ǻ) with N-H proton of the Bz unit and the bond angle between the -O--H-N is ~103º. The protonation free energy of pyridine nitrogen is -3.8 kcal/mol compared to the neutral timoprazole drug molecule (Figure 1 and Table 1). The protonated intermediate (Timo-PyH+) also experiences the intramolecular hydrogen bonding interaction between the oxygen of sulfinyl group and N-H proton of the benzimidazole moiety. The transition state free energy barrier (Timo-TS1) for the protonation of the benzimidazole nitrogen has been found to be 4.2 kcal/mol compared to preceding TimoPyH+ and the intermediate Timo-BzH+ is less stable by 0.4 kcal/mol compared to the neutral drug molecule. The free energy profile suggests that the formation of Timo-BzH+ could be barrierless, where Timo-BzH+ and Timo-TS1 are energetically higher by 0.4 kcal/mol than timoprazole, however, electronic energy (∆Eaq) results show that Timo-BzH+ is stable by 1.4 kcal/mol compared to Timo-TS1 (Table S2). On the other hand, there are

reports

available in the literature, which suggests that the protonation of the Bz moiety might be favorable compared to the Py unit.16,40 Therefore, we have examined the pKa of both Bz and Py rings using the M062X/6-31++G(d,p) level of theory. The calculated pKa values suggest that the N1 nitrogen of the Py moiety is more basic than the N3 nitrogen of the Bz unit which is in agreement with the experimental report of Shin et al. (Table S3).15


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Figure 1: Free energy profile of the acid activation process further disulfide complex formation of the timoprazole drug molecule.

Further, pyridine nitrogen attacks the C-2 carbon atom of the Bz moiety (Scheme 2), which leads to the formation of the transition state (Timo-TS2) with free energy barrier of 13.9 kcal/mol compared to the preceding intermediate Timo-BzH+ and consequently yields spiro intermediate dihydrobenzimidazole (Timo-spiro, Figure 2). The DFT calculated distance between C-2 carbon of Bz moiety and nitrogen of Py unit in the transition state (Timo-TS2, Figure 2) geometry is 1.90 Ǻ. The spiro complex is also an unstable intermediate compared to the timoprazole drug molecule (5.7 kcal/mol). Table 1: The M062X/6-31++G(d,p) level of theory calculated free energy difference calculated using Equation-3 given in kcal/mol. PyH+ Timoprazole S-Omeprazole R-Omeprazole

∆Gaq ∆Gaq ∆Gaq

-3.8 -5.4 -3.6

TS1 0.4 -0.5 -0.2

BzH+ 0.4 -1.9 -1.0

TS2 14.3 11.1 10.0

Spiro 5.7 1.7 2.6

TS3 11.4 7.1 7.6

Sulfenic Acid -9.9 -12.6 -10.1


TS4 51.6 48.8 50.2

Sulfenamide -14.9 -17.3 -17.9

TS5 40.1 31.9 32.4

Disulfide -29.4 -33.9 -30.4


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The N-H proton of Bz ring transferred to the sulfinyl oxygen to form sulfenic acid and the computed free energy barrier is 5.7 kcal/mol compared to the previous spiro intermediate (Figure 2). The corresponding intermediate sulfenic acid is 9.9 kcal/mol lower compared the neutral drug molecule. Intramolecular hydrogen bonding interaction N-H---O (1.93 Ǻ) was observed, which presumably stabilizes the sulfenic acid intermediate. Dehydration of sulfenic acid can lead to the formation of tetracyclic sulfenamide, however, activation barrier for such dehydration process is 61.5 kcal/mol compared to the sulfenic acid intermediate. The DFT calculations suggest that the tetra cyclic sulfenamide is stable by only 5.0 kcal/mol compared to the sulfenic acid. The water elimination and S-N bond formation occurs in a concerted way through a four membered ring formation in the transition state geometry (Timo-TS4, Figure 2). On the other hand, the acid activated complex sulfenic acid can form stable disulfide bond with cysteine amino acid of H+,K+-ATPase followed by the elimination of water molecule, which is the desired complex for the acid inhibition process (Figure 2). We have considered neutral cysteine amino acid to mimic the cysteine amino residue of the gastric proton pump H+,K+ATPase, whereas zwitterionic form of cysteine might exist, however, as discrete amino acid.41 The free energy barrier for the disulphide complex formation (Timo-TS5) is 50.0 kcal/mol compared to the sulfenic acid and disulfide complex is remarkably stable (-29.4 kcal/mol) compared to the tetra cyclic sulfenamide (Figure 1). Dehydration and S-S bond formation takes place through the formation of four membered transition state (Timo-TS5) and intramolecular hydrogen bond N-H---O (2.00 Ǻ) becomes weaker compared to timoprazole sulfenic acid. The DFT calculated results reveal that the formation of disulfide complex with cysteine amino acid of H+,K+-ATPase is kinetically and thermodynamically more stable than sulfenamide intermediate.


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Figure 2: Mechanism of the acid activation and finally stable disulfide complex formation of timoprazole proton pump inhibitor.

We have extended our quantum chemical calculations with the potent proton pump inhibitor S-omeprazole drug molecule. We have performed the calculation in aqueous phase with M062X/6-31++G(d,p) level of theory. Protonation of pyridine nitrogen of S-omeprazole forms S-Ome-PyH+ and the protonation free energy of S-omeprazole is -5.4 kcal/mol (Figure 3 and 4). The PES of S-omeprazole showed the similar pathway to form sulfenic acid as observed with timoprazole (Figure 2 and 4).



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Figure 3: Free energy profile of the acid activation process and further disulfide complex formation of the S-omeprazole drug molecule.

S-Ome-sulfenic acid is stable by -12.6 kcal/mol compared to the S-omeprazole in the free energy surface in the acid activation process. Further, dehydration can either form a tetracyclic sulfenamide intermediate or a stable disulfide complex with cysteine amino acid of H+,K+-ATPase through a four membered ring transition state (S-Ome-TS4/5). The free energy barrier (S-Ome-TS4) for the formation of sulfenamide is 61.4 kcal/mol compared to the preceding intermediate sulfenic acid and dehydrated sulfenamide is stable by only -4.7 kcal/mol compared to the sulfenic acid.


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Figure 4: Acid activation and further disulfide bond formation mechanistic pathway of the Someprazole drug molecule.

On the other hand, the disulfide bond formation with cysteine amino acid of H+,K+ATPase by dehydration is kinetically and thermodynamically more favorable compared to the sulfenamide formation (Figure 3). The free energy barrier calculated for the disulfide bond formation is 44.5 kcal/mol compared to the previous intermediate S-Ome-sulfenic acid for Someprazole molecule.

The transition state (TS5) is the rate determining step for the

formation of disulphide complex between the PPI and the cysteine residue of H+,K+-ATPase in both timoprazole and S-omeprazole systems. It is to note that the activation barrier for the formation of (TS5) is lower (5.5 kcal/mol) in the case of S-omeprazole compared to 13 ACS Paragon Plus Environment


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timoprazole (Figure 1, 2, 3 and 4). The stability of TS5 S-omeprazole is governed by the hydrogen bonding interactions between the PPI and cysteine molecule. The intermolecular hydrogen bonding interaction (N-H---O, 1.8 Ǻ) between carboxylic oxygen of the cysteine amino acid residue and N-H proton of Bz moiety was observed and the intramolecular Hbonding interaction (C-H---N, 2.29 Ǻ) was also observed between the methylene proton of sulfenic acid and N-1 nitrogen of Bz unit. The torsional angle calculated (N3-C2-N1’-C2’) for S-omeprazole is ~48º, which is much smaller compared to the corresponding torsional angle of ~60º (Figure S2). Analysing the geometry of sulfenic acid and the TS5 for timoprazole and omeprazole suggests that the substituents at remote position of the Bz and Py moiety helps to align the two aromatic rings (Py and Bz) in S-omeprazole. Such a difference in the rate determining activation step can augment the efficacy of S-omeprazole compared to timoprazole molecule in acid inhibition process. The disulfide complex formed in the acidic environment of the parietal cell is energetically preferred compared to the corresponding tetra cyclic sulfenamide intermediate. The disulfide complex formed from the S-omeprazole is stable by -33.9 kcal/mol compared to the S-omeprazole drug molecule. The formation of the tetracyclic sulfenamide is less likely in this case presumably due to the formation of puckered six membered ring through a four membered transition state geometry (S-Ome-TS4). It has been reported that S-isomer i.e. S-omeprazole is more efficient compared to the R-isomer of omeprazole.42,12-14 The pharmacokinetic and pharmacodynamics suggests that the activity of R- and S-isomer of omeprazole might differ in the metabolic pathway.43-44 However, the mechanistic pathway for the formation of disulfide complex between cysteine and R- and S-isomer of omeprazole remain unexplored. At the cellular level, both R- and Sisomers are protonated and converted in the acidic compartment of the parietal cell in exactly the same way to form acid activated achiral sulfenic acid or tretra cyclic sulphonamide which 14 ACS Paragon Plus Environment

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makes disulfide bond with thiol group of the Cys-813 to inhibit H+,K+-ATPase.42 We have computed the acid activation and the disulfide bond formation process of omeprazole at molecular level using M062X/6-31++G(d,p) level of theory in aqueous phase. We have not also observed any significant difference in the acid activation and disulfide complex formation (Table 1, Scheme S1, Figure S1 and Figure S3). The free energy differences with respect to neutral omeprazole show that the free energy difference for the acid activation process and final disulfide complex formation is similar for these two isomers i.e. the acid activation of the R-omeprazole and S-omeprazole is equally favorable in the acidic canaliculi of the parietal cell (Table 1). Therefore, it appears that the acid activation process of R- and S- isomers of omeprazole starts to differ in their metabolic pathway and excretion from human body.

Conclusion: Irreversible acid suppressants are common therapeutic option for the treatment of acid-related diseases since the discovery of omeprazole. However, the reports on the mechanism of acid activation and the inhibition of these prodrugs are scarce in literature. In this work, we have reported the mechanistic pathway for the acid activation and the disulfide bond formation with PPIs i.e. timoprazole, S-omeprazole and R-omeprazole computationally with M062X/6-31++G(d,p) level of theory. The effect of solvation has been studied with SCRF method using single point SMD solvation model in aqueous phase. The reactant, intermediates, transition states, and products on the potential energy surface of timoprazole was also optimized using the same solvation model and the results were found to be similar in both cases. The acid activation and the inhibition of omeprazole is superior over timoprazole molecule. Omeprazole is the substituted derivative of timoprazole and hence warranted a study to shed light on the efficacy of these drug molecules. The DFT calculations reveal that



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the acid activation of the PPIs and further gastric proton pump inhibition process follow a multistep process in which the drug molecules form a disulphide bond with the cysteine residue of H+,K+-ATPase. The free energy surface calculated for timoprazole suggests that the disulphide bond formation is energetically unfavored compared to omeprazole. The transition state (TS5) for the formation of disulfide complex is the rate determining step and one of the key steps of the acid inhibition process by PPIs. The TS5 free energy barrier for timoprazole is 5.5 kcal/mol higher than S-omeprazole. The stabilization of TS5 for Someprazole occurs due to strong inter- and intramolecular hydrogen bonding while forming the disulphide bond with cysteine residue. Such interactions and the remote substituents further helps to align the two aromatic rings (Py and Bz) in S-omeprazole to achieve more planarity compared to timoprazole molecule and hence to augment the stability. Further, the disulphide complex for S-omeprazole is also stable by 4.5 kcal/mol in aqueous phase compared to disulfide complex of timoprazole. These computed results corroborate the less efficacy of timoprazole compared to S-omeprazole for irreversible acid suppression process. The experimental observations indicate that during the acid activation process sulfenic acid can either produce sulfenamide or a stable disulfide complex with cysteine of H+,K+ATPase. The DFT results clearly suggest that the free energy barrier for the formation of tetra cyclic sulfenamide is energetically unfavoured by ~ 17 kcal/mol for S-omeprazole and 11.5 kcal/mol for timoprazole compared to the disulfide complex formation in each case. The reaction energies also show that the desired disulfide complex is ~15 kcal/mol more stable than the acid activated intermediate sulfenamide. The M062X/6-31++G(d,p) level of theory calculated results further suggests that the acid activation process of R- and S-isomers of omeprazole and their inhibition effect is very similar and the efficacy of S-omeprazole presumably depends on metabolic pathway and


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excretion from human body. Further work is under progress to examine the metabolic pathways of R- and S-isomers of omeprazole.

ASSOCIATED CONTENT: Supporting Information M062X/6-31++G(d,p) level of theory optimized Cartesian coordinates have been given in SI. The M062X/6-31++G(d,p) theory calculated aqueous phase and gas electronic energy and calculated pKa values have been given in SI. The acid activation mechanism and free energy profile of R-Omeprazole is also available in SI. This information is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION: Corresponding Author *Fax:

(+91)-278-2567562.

Telephone:

+91-278-2567760,

ext

6770.

E-mail:

[email protected]

ACKNOWLEDGMENTS: CSMCRI communication no: 089/ 2016. K. J. is thankful to UGC, New Delhi, India, for awarding a senior research fellowship. K. J. acknowledges to AcSIR for his PhD registration. B.G. thanks MSM, SIP (CSIR, New Delhi) and BRNS, Mumbai, for financial support. This work is supported by Department of Atomic Energy, Government of India, Board

of

Research

in

Nuclear

Sciences

(DAE-BRNS)

under

grant

no.

2013/37C/54/BRNS/2278. K. J. would like to thank Dr. Rabindranath Lo for helpful discussions and valuable suggestions to design this project. We thank the anonymous reviewer’s for their valuable comments/suggestions that have helped us to improve the paper.

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Graphical Abstract


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Revealing the Mechanistic Pathway of Acid Activation of Proton Pump

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