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Differentiation of Deprotonated Acyl, N- and O-Glucuronide Drug Metabolites by Using Tandem Mass Spectrometry Based on Gas-Phase Ion-Molecule Reactions Followed by Collision-Activated Dissociation Edouard Niyonsaba, Mckay Whetton Easton, Erlu Feng, Zaikuan Yu, Zhoupeng Zhang, Huaming Sheng, John Kong, Leah F. Easterling, Jacob Milton, Harry R Chobanian, Nicholas R. Deprez, Mark Cancilla, Gozdem Kilaz, and Hilkka I. Kenttämaa Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b02717 • Publication Date (Web): 05 Aug 2019 Downloaded from pubs.acs.org on August 6, 2019

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Analytical Chemistry

Differentiation of Deprotonated Acyl, N- and O-Glucuronide Drug Metabolites by Using Tandem Mass Spectrometry Based on GasPhase Ion-Molecule Reactions Followed by Collision-Activated Dissociation Edouard Niyonsaba,1 McKay W. Easton,1 Erlu Feng,1 Zaikuan Yu,1 Zhoupeng Zhang,2 Huaming Sheng,3 John Kong,3 Leah F. Easterling,1 Jacob Milton,1 Harry R. Chobanian,2 Nicholas R. Deprez,4 Mark T. Cancilla,2 Gozdem Kilaz,5 and Hilkka I. Kenttämaa1* 1Department

of Chemistry, Purdue University, West Lafayette, Indiana 47907, United States of Pharmacokinetics, Pharmacodynamics, & Drug Metabolism, Merck & Co., Inc., West Point, Pennsylvania 19486, United States 3Analytical Research & Development, Merck & Co., Inc., Rahway, New Jersey, 07065, United States 4Process Chemistry, Merck & Co., Inc., Rahway, New Jersey, 07065, United States 5Purdue University, School of Engineering Technology, West Lafayette, Indiana 47907, United States 2Department

Submitted to Analytical Chemistry

1 ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT. Glucuronidation, a common phase II biotransformation reaction, is one of the major in vitro and in vivo metabolism pathways of xenobiotics. In this process, glucuronic acid is conjugated to a drug or a drug metabolite via a carboxylic acid, a hydroxy, or an amino group to form acyl, O-, and/or Nglucuronide metabolites, respectively. This process is traditionally thought to be a detoxification pathway. However, some acyl glucuronides react with biomolecules in vivo, which may result in immune-mediated idiosyncratic drug toxicity (IDT). In order to avoid this, one may attempt in early drug discovery to modify the lead compounds in such a manner that they then have a lower probability of forming reactive acyl glucuronide metabolites. Because most drugs or drug candidates bear multiple functionalities, e.g., hydroxy, amino, and carboxylic acid groups, glucuronidation can occur at any of those. However, differentiation of isomeric acyl, N- and O-glucuronide derivatives of drugs is challenging. In this study, gas-phase ion-molecule reactions between deprotonated glucuronide metabolites and BF3 followed by collision-activated dissociation (CAD) in a linear quadrupole ion trap mass spectrometer were demonstrated to enable the differentiation of acyl, N-, and O-glucuronides. Only deprotonated Nglucuronides and deprotonated, migrated acyl glucuronides form the two diagnostic product ions: a BF3 adduct that has lost two HF molecules, [M – H + BF3 – 2 HF]–, and an adduct formed with two BF3 molecules that has lost three HF molecules, [M – H + 2 BF3 – 3 HF]–. These product ions were not observed for deprotonated O-glucuronides and unmigrated, deprotonated acyl glucuronides. Upon CAD of the [M – H + 2 BF3 – 3 HF]– product ion, a diagnostic fragment ion is formed via the loss of 2-fluoro1,3,2-dioxaborale (MW of 88 Da) only in the case of deprotonated, migrated acyl glucuronides. Therefore, this method can be used to unambiguously differentiate acyl, N- and O-glucuronides. Further, coupling this methodology with HPLC enables the differentiation of unmigrated 1-β-acyl glucuronides from the isomeric acyl glucuronides formed upon acyl migration. Quantum chemical calculations at the M06-2X/6-311++G(d,p) level of theory were employed to probe the mechanisms of the reactions of interest.


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INTRODUCTION Glucuronidation is a major component of phase II drug metabolism.1 In this process, glucuronic acid is conjugated with a carboxylic acid, a hydroxyl, or an amino group of a drug or drug metabolite to form an acyl, O-, or N-glucuronide metabolite, respectively.2 This reaction is catalyzed by a superfamily of uridine 5'-diphospho-glucuronosyltransferase enzymes and it expedites the elimination of drugs via urine or bile by increasing their solubility in water.3–5 Generally, glucuronidation detoxifies drugs; however, in some cases, glucuronidation leads to the formation of reactive toxic metabolites. For instance, drugs containing a carboxylic acid often form reactive acyl glucuronides at physiological conditions.2,6–9 The toxicological effects of acyl glucuronides are associated with their ability to undergo acyl migration via intramolecular rearrangement (Scheme S1).10,11 In this process, the acyl group that is initially at the 1-hydroxyl position of the glucuronic acid moiety (1-β-acyl glucuronide) migrates to the 2-hydroxyl group, and then possibly to the 3- and 4-hydroxyl groups, of the glucuronic acid to form 2-, 3-, and/or 4β- and α-acyl glucuronides, respectively. After migration, the glucuronic acid moiety can undergo ringopening at the anomeric position to generate aldehydes.12 The β-aldehyde isomers can covalently glycate to nucleophilic macromolecules, potentially causing idiosyncratic drug toxicity (IDT).12–17 Although the toxicological effects of acyl glucuronides are well documented, the current analytical techniques cannot be used to unambiguously differentiate N-, O- and acyl glucuronides. However, knowing the site of glucuronidation is vital for the drug development process because it would allow early assessment of the safety of new drug candidates. Liquid chromatography coupled with tandem mass spectrometry based on collision-activated dissociation (CAD) has been the standard method for detecting glucuronides.18 The presence of a glucuronide can be verified by the detection of ions that are formed through the elimination of an anhydroglucuronic acid molecule (MW 176 Da) upon CAD for both positively and negatively charged glucuronide ions.18 This method, however, only detects the presence of a glucuronide and cannot be used to reliably differentiate O-, N-, and acyl glucuronides. Although chemical derivatization prior to CAD has been used to differentiate some glucuronide isomers, including estriol glucuronides,19 carvedilol glucuronides,20 morphine glucuronides,21 and some acyl glucuronides,22 this approach is limited in scope and practicality because it often is time-consuming and difficult to implement in high-throughput analysis. Tandem mass spectrometry based on gas-phase ion-molecule reactions with trichlorosilane as the reagent has recently been introduced for the differentiation of deprotonated N-glucuronides from Oglucuronides;23 however, this method cannot be used to differentiate N-glucuronides from acyl glucuronides. Additionally, some deprotonated O-glucuronides containing multiple phenol or carboxylic acid functionalities react with trichlorosilane similarly to N-glucuronides, making it difficult to differentiate these O-glucuronides from N-glucuronides (Figure S1). In this study, ion-molecule reactions with boron trifluoride followed by CAD of a diagnostic product ion are demonstrated to enable the differentiation of N-, O- and migrated acyl glucuronides. Furthermore, when this methodology is coupled with HPLC, it can be used to differentiate unmigrated 1-β-acyl glucuronides from the isomeric acyl glucuronides formed upon acyl migration. EXPERIMENTAL SECTION Chemicals. Boron trifluoride diethyl etherate (synthesis grade) was used as the source of BF3 and was purchased from Sigma-Aldrich. All acyl glucuronide (Table S1), N-glucuronide (Table S2), and Oglucuronide (Table S3) drug metabolites were purchased from Toronto Research Chemicals (TRCCanada). 18O-Probenecid acyl β-ᴅ-glucuronide was prepared by dissolving probenecid acyl β-ᴅ3 ACS Paragon Plus Environment


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glucuronide in 18O-water containing 5 % (v/v) formic acid for a week, as previously described.24 13CIbuprofen acyl β-ᴅ-glucuronide was synthesized; the details are given in the supporting information and in Scheme S1. Acetonitrile (LC/MS optima grade), water (LC/MS optima grade), and methanol (LC/MS optima grade) were purchased from Fisher Scientific. All chemicals were used as received. 100 mM Potassium phosphate buffer (pH 7.4) was prepared by mixing 80.2 mL 1.0 M potassium diphosphate (K2HPO4) and 19.8 mL 1.0 M potassium monophosphate (KH2PO4). The pH of the mixture was adjusted to 7.4 by adding either HCl or NaOH and it was diluted to 1 L with distilled water. Sample Preparation. 0.1 mM Solutions of acyl, N-, and O-glucuronide drug metabolites were prepared in 50:50 (v/v) methanol:water and analyzed promptly. Stable acyl glucuronides (telmisartan, repaglinide, and montelukast acyl glucuronides) were dissolved in above phosphate buffer at the same concentration to induce acyl migration. The stability of the acyl glucuronides was based on their previously reported half-life (t1/2; a measure of the degradation rate (hydrolysis and acyl migration) of the 1-β-acyl glucuronide isomer .25 A mixture containing acyl, N- and O-glucuronides was prepared by incubating telmisartan acyl-β-ᴅ-glucuronide, 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (abbreviated as PhIP below for simplicity), N-β-ᴅ-glucuronide, and ezetimibe O-β-ᴅ-glucuronide in the phosphate buffer at room temperature for 4 hours. Mass Spectrometry. All experiments were performed with a Thermo Scientific linear quadrupole ion trap (LQIT) mass spectrometer equipped with electrospray ionization (ESI) operated in negative ion mode. The analytes dissolved in 50:50 (v/v) methanol:water were infused into the ESI source at a rate of 10 µL/min with a 500 μL Hamilton syringe. Typical ESI conditions were as follow: The sheath and auxiliary gas (N2) were set at 30 and 10 arbitrary units, respectively. The spray voltage was set at 3.5 kV and the capillary temperature at 275 °C. The external reagent mixing manifold used to introduce the neutral reagents into the helium buffer gas line has been described previously.26–28 Boron trifluoride diethyl etherate was introduced into the manifold via a syringe pump at a flow rate of 3 µL/h. The syringe port and the surrounding area were heated to reach approximately 110 °C to ensure complete evaporation of the boron trifluoride diethyl etherate. The reagent was then diluted with a controlled amount of helium before entering the ion trap through a leak valve, which directed part of the mixture into the exhaust. Because of the high temperature, boron trifluoride diethyl etherate dissociates to form BF3 and ethoxyethane. When the experiments had been completed, the manifold was connected to a rough pump in order to facilitate removal of the remaining reagent from the manifold. No fouling nor contamination was observed either in the instrument or in the manifold. To verify that BF3 was present in the ion trap, formic acid was introduced into the ion source and deprotonated, and the deprotonated formic acid (m/z 45) was isolated (isolation width 12 m/z units) and allowed to react with BF3 for 100 ms. Trifluorohydroborate ions (HBF3¯, m/z 69) were observed with an abundance of 30 % relative to that of the deprotonated formic acid (m/z 45). After this, the analytes were introduced into LQIT, deprotonated, isolated (isolation width two m/z units), and allowed to react with BF3. For CAD experiments, the product ions of interest were isolated and subjected to CAD in the ion trap. Typical reaction, isolation and CAD conditions were as follows: reaction time 100 ms, isolation width two m/z-units, activation q 0.25, and collision energy 15 (arbitrary units). High Performance Liquid Chromatography (HPLC)/LQIT Mass Spectrometry. The analytes were dissolved in phosphate buffer, in pure water, or water containing 0.1 % (v/v) formic acid prior to HPLC analysis. The solutions were analyzed immediately as well as at different time points for up to four hours after preparing them. All HPLC experiments were performed on a Thermo Surveyor Plus HPLC. Water (A) and acetonitrile (B), each containing 0.1 % (v/v) formic acid, were used as the mobile phases 4 ACS Paragon Plus Environment

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at a flow rate of 500 μL/min on an Agilent ZORBAX SB-C18 5 µm, 4.6 × 250 mm column or on a Nacalai COSMOSIL PBr 5 µm, 4.6 × 100 mm column. A nonlinear gradient was used as follows: 0.0 min, 20 % B, 80 % A; 2.0 min, 20 % B, 80 % A; 20.0 min, 80 % B, 20 % A; 23.0 min, 80 % B, 20 % A; 25.0 min, 20 % B, 80% A; 30.0 min, 20 % B, 80 % A. Following HPLC separation, the analytes were ionized via (–) ESI and the ionized analytes were isolated in the linear quadrupole ion trap and allowed to react with BF3 for 30 ms. Calculations. All density functional theory calculations were performed at the M06-2X/6311++G(d,p) level of theory by using the Gaussian16 program.29,30 All transition state structures were determined to possess a vibration with one negative frequency while energy minima possessed no vibrations with negative frequencies. Zero-point energy corrections were carried out. Intrinsic reaction coordinate (IRC) calculations were performed to confirm that the transition state structures connected the correct reactant and product structures. The free energies used to construct the potential energy surfaces were calculated using ideal gas statistical mechanics. All calculations were performed at 273.15 K. RESULTS AND DISCUSSION Gas-phase reactivity of several deprotonated acyl, N- and O-glucuronides towards BF3 and CAD of selected product ions were studied in a linear quadrupole ion trap (LQIT) mass spectrometer to explore the utility of this approach for differentiating acyl, N- and O-glucuronide drug metabolites. A total of 19 acyl glucuronides, 8 N-glucuronides and 20 O-glucuronides were tested (see Supporting Information for their structures). The reactivity of different deprotonated glucuronides toward BF3 is discussed first, followed by the chromatographic separation of isomeric acyl glucuronides formed via acyl migration and their reactivity towards BF3. Finally, quantum chemical calculations employed to explore relevant reaction mechanisms are discussed. Gas-phase ion-molecule reactions of deprotonated acyl, N- and O-glucuronides with BF3 followed by CAD. Several acyl, N-, and O-glucuronides were ionized via ESI in the negative ion mode. The deprotonated analytes were allowed to react with BF3 for up to 100 ms. All of the studied deprotonated acyl, N- and O-glucuronides reacted with BF3 to form two primary products, an adduct ion, [M – H + BF3]–, and an adduct ion that had lost one HF molecule, [M – H + BF3 – HF]– (Tables S1, S2 and S3). These product ions can react with water in the ion trap to generate product ions referred here to as hydrolysis product ions. Additionally, deprotonated acyl and N-glucuronides formed a diagnostic primary product corresponding to an adduct that had lost two HF molecules, [M – H + BF3 – 2 HF]–, and a diagnostic secondary product, an adduct containing two BF3 molecules that had lost three HF molecules, [M – H + 2 BF3 – 3 HF]–. These product ions were not observed for deprotonated O-glucuronides. Therefore, the formation of these ions can be used to differentiate O-glucuronides from N-glucuronides and migrated acyl glucuronides (see discussion on their mechanisms later). Further, several small molecules containing carboxylic acid functionalities that were not glucuronides were examined, including benzoic acid, hexanoic acid, glycine, serine, isoserine, 3hydroxylbutyric acid, and glyceric acid. These ionized compounds did not form the reported diagnostic product ions ([M – H + BF3 – 2 HF]– and [M – H + 2 BF3 – 3 HF]–) when isolated and allowed to react with BF3. As it is still possible that some ionized compounds that are not glucuronides form the [M – H + BF3 – 2 HF]– and [M – H + 2 BF3 – 3 HF]– product ions, it is necessary first to verify that the analyte is a glucuronide based on CAD, as described in the literature.18



In order to explore how to differentiate deprotonated N-glucuronides from acyl glucuronides, the diagnostic secondary [M – H + 2 BF3 – 3 HF]– product ions were isolated and subjected to CAD. Indeed, only acyl glucuronides generated a fragment ion via the loss of a molecule with MW 88 Da. Among the 19 acyl (Table S1), 8 N- (Table S1), and 20 O-glucuronides (Table S3) studied here, no false positives nor negatives were observed. As an example of above analytical method, a set of sample mass spectra measured for gas-phase ion molecule and CAD reactions for deprotonated carvedilol-O-β-ᴅ-glucuronide (m/z 581), carvedilol-Nβ-ᴅ-glucuronide (m/z 581), and clofibric acyl-β-ᴅ-glucuronide (m/z 389) is shown in Figure 1. Only the deprotonated acyl and N-glucuronides formed the diagnostic primary and secondary [M – H + BF3 – 2 HF]– and [M – H + 2 BF3 – 3 HF]– product ions, while the deprotonated O-glucuronides did not. Upon CAD of the diagnostic secondary [M – H + 2 BF3 – 3 HF]– product ions, only the [M – H + 2 BF3 – 3 O O HN O

O O

HO HO

Relative Abundance

O-glucuronide

O

O

OH

NH

Carvedilol O-ß-D-Glucuronide m/z 581

A

[M–H+BF3–HF]– 629 [M–H+BF3]– 649

100 [M–H]– 581

50 0

*

675

*

695

550

650 m/z

600

700

750

O

HO HO

O O

O

N-glucuronide

N

OH

NH

O

#

50 654

656

CAD m/z 657

655

0 655

637

100

657

50 0

m/z

500

600

m/z

0 600

550 O O HO HO

O OH

O

Me Me

650

700

m/z

Cl

acyl glucuronide

O O

#

50

[M–H+BF3–2HF]– 417

[M–H]– 389

435 437

#

463

50

[M–H+BF3–HF]–

464

462 0

[M–H+BF3]–

460

– 457 [M–H+2BF3–3HF]

m/z

465

*

420

440

460

m/z

480

CAD m/z 465

377

100

421 445

-88 Da

50 353

465

0 400 m/z

465

0 400

C

465

100

Clofibric acyl-ß-D-Glucuronide (m/z 389)

100

750

Relative Abundance

Relative Abundance

Carvedilol N'-ß-D-Glucuronide [M–H+BF3–HF]– m/z 581 [M–H+BF3]– [M–H+BF3–2HF]– 649 # 100 [M–H+2BF3–3HF]– 627 * 629 50 [M–H]– 657 581 609

B

657

100

OH

Relative Abundance

O

Relative Abundance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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500

520

540

Figure 1. (A) Mass spectra measured after 100 ms reaction of a deprotonated O-, N-, and acyl glucuronide with BF3. Only the deprotonated N- and acyl glucuronides formed the diagnostic product ions [M – H + BF3 – 2 HF]– and [M – H + 2 BF3 – 3 HF]– (highlighted in red). (B) For the deprotonated N-glucuronide, CAD (collision energy 15 arbitrary units) of the diagnostic secondary [M – H + 2 BF3 – 3 HF]– product ion yielded a major fragment ion (m/z 637) corresponding to elimination of HF with MW 20 Da. (C) For the deprotonated acyl glucuronide, a diagnostic fragment ion (m/z 377) due to the loss of C2H2O2BF (MW 88 Da) was observed. In addition, elimination of HF (m/z 445) and CO2 (m/z 421) were observed. # Hydrolysis product ions of [M – H + BF3 – HF]– and [M – H + 2 BF3 – 3 HF]–: [M – H + BF3 – 2 HF + H2O]– and [M – H + 2 BF3 – 3 HF + H2O]– , respectively. * Secondary product ions. 6 ACS Paragon Plus Environment

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HF]– product ions formed from deprotonated acyl glucuronides but not those formed from deprotonated N-glucuronides formed a fragment ion via the loss of a C2H2O2BF molecule (MW 88 Da; the identification of this molecule is addressed later). This general procedure for distinguishing acyl-, N- and Oglucuronides is illustrated in Scheme 1. O O HO HO

O

F B

OH

O

O

O O

O

O

O HO HO

O

H O

F

R

O O

CAD

B

O O

F B

O

O

O

F F

+

O

O 2-fluoro-1,3,2-dioxaborole MW 88 Da

O

R

R O

F B

O

O

N

OH

BF3 R

F B

O O

O O

F B

F

OH N R

O O HO HO

O OH

O R

Scheme 1. A scheme illustrating how deprotonated migrated acyl, N- and O-glucuronides can be differentiated by using ion-molecule reactions and CAD methodology. HPLC/MS3 of acyl glucuronides. Due to acyl migration, the acyl glucuronides studied above may have been mixtures of unmigrated and migrated isomers. In an attempt of determining whether this was the case, it was important to figure out whether unmigrated acyl glucuronides (1-β-acyl glucuronides) and their isomers formed via acyl migration react similarly with BF3. This was explored by dissolving lumiracoxib acyl-β-ᴅ-glucuronide (t1/2 = 6.5 hours in plasma31; the structure is shown in Table S1) in water containing 0.1 % (v/v) formic acid (to inhibit migration) and in 100 mM phosphate buffer pH 7.4 (to facilitate migration). The two solutions were kept at room temperature for a variable length of time. HPLC was then used to separate possible isomeric acyl glucuronides before (–) ESI/MS3 analysis. The HPLC chromatograms measured for the analyte in acidified water showed only one peak, suggesting that acyl migration did not occur within the four-hour time span studied (Figure 2A). However, multiple peaks were observed when the analyte was dissolved in the phosphate buffer even for one hour, suggesting that the lumiracoxib acyl-β-ᴅ-glucuronide rapidly underwent acyl migration to generate several isomers (Figure 2B). The unmigrated acyl glucuronide eluted at the same time independent of the solvent used. In contrast, the isomeric migrated acyl glucuronides eluted at different times While the isomeric glucuronides eluted from the HPLC column, they were ionized and isolated, and the deprotonated, isomeric glucuronides (m/z 468) were allowed to react with BF3 in the ion trap for up to 30 ms. Only the migrated, deprotonated acyl glucuronides were found to form the diagnostic product ions [M – H + BF3 – 2 HF]– (m/z 496) and [M – H + 2 BF3 – 3 HF]– (m/z 544) while the unmigrated, deprotonated acyl glucuronide did not (Figures 2C and 2D). CAD of the [M – H + 2 BF3 – 3 HF]– product ions formed for the migrated acyl glucuronides yielded the diagnostic fragment ion via the loss a molecule with MW 88 Da (Figure 2D). Similar results were obtained for probenecid acyl-β-ᴅ-glucuronide (t1/2 = 0.3 hours in phosphate This compound was dissolved either in water or water containing 0.1 % (v) formic acid and stored for one hour. When pure water was used, the deprotonated compound formed the diagnostic product ions upon reactions with BF3. However, when acidified water was employed, the diagnostic product ions were not observed. These findings suggest that probenecid acyl β-ᴅ-glucuronide underwent

buffer25).



acyl migration only when it was dissolved in water but not when it was dissolved in acidified water (Figure S2). O O HO HO

O

O

OH

O HN Cl

F

Lumiracoxib acyl-ß-D-Glucuronide m/z 468

B

Water pH 2.7

16.30

100

0 Hour

0 100

Unmigrated acyl

16.28 1 Hour

0 100

16.27 2 Hours

0 100

16.25 3 Hours

0 100

4 Hours

16.27

0

100 0 100

Relative Abundance

A

Relative Abundance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0 100 0 100 0 100

C

Phosphate buffer pH 7.4

16.29

Unmigrated acyl

0 Hour

15 20 Time (min)

50

536

[M-H]468

516

0

1 Hour

D

2 Hours

16.28

Migrated acyl

50

16.29

536 [M–H+BF3–2HF]– [M-H]468 450

Migrated acyl

550

Migrated acyl

100

0

3 Hours

500 m/z

450

16.30

516

[M–H+2BF3–3HF]– 544

496 500 m/z

550 m/z 544

E 16.66

4 Hours

100

Migrated acyl

15

Time (min)

20

CAD 15

456 -88 Da

50

0 10

Unmigrated acyl

100

500

544

0 450

500 m/z

550

Figure 2. HPLC chromatograms (detection using the signal measured for ions of m/z 468) and MS2 and MS3 mass spectra measured at different times for lumiracoxib acyl-β-ᴅ-glucuronide incubated in (A) water spiked with 0.1 % formic acid (v/v) at pH 2.7 and (B) in phosphate buffer at pH 7.4. (C) Based on the measured MS2 mass spectra, the deprotonated unmigrated isomer (m/z 468) did not form the diagnostic product ions after 30 ms reaction with BF3. Ions of m/z 536 and m/z 516 correspond to [M – H + BF3]– and [M – H + BF3 – 2 HF]– product ions, respectively. (D) Based on the measured MS2 mass spectra, all deprotonated migrated isomers (m/z 468) formed the diagnostic product ions (highlighted in red) after 30 ms reaction with BF3. (E) The CAD (MS3) mass spectrum (collision energy 15 arbitrary units) measured for the [M – H + 2 BF3 – 3 HF]– product ion (m/z 544) yielded a fragment ion (m/z 456) corresponding to the loss of a molecule with MW 88 Da. Above two studies indicate that only migrated, deprotonated acyl glucuronides react with BF3 to form the diagnostic products ions. Therefore, this HPLC/MS3 methodology can be used to quickly differentiate migrated acyl glucuronides from their unmigrated 1-β-acyl glucuronide isomers. Furthermore, since all acyl glucuronides studied (Table S1) showed the diagnostic products, all of them had undergone some acyl migration prior to analysis. Mechanisms for the formation of the diagnostic [M – H + BF3 – 2 HF]– and [M – H + 2 BF3 – 3 HF]– product ions. Based on quantum chemical calculations (M06-2X/6-311++G(d,p) level of 8 ACS Paragon Plus Environment

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theory) for reactions of deprotonated glucuronic acid with BF3 (Figure S3), the formation of the primary [M – H + BF3 – 2 HF]– product ions is proposed to involve two steps: ring-opening at the anomeric position of the glucuronic acid and elimination of a HF molecule followed by bicyclization and elimination of the second HF molecule (Figure 3A). The reaction is initiated by covalent bond formation between BF3 and the deprotonated carboxylic acid moiety of the glucuronic acid to generate adduct ions, [M – H + BF3]–. The adduct ions undergo ring-opening at the anomeric position of the glucuronic acid, followed by the elimination of one HF molecule to form the [M – H + BF3 – HF]– product ions. The opening of the glucuronic acid ring generates [M – H + BF3 – HF]– ions that are more flexible, enabling a hydroxyl group of the glucuronic acid moiety to add to the boron atom, which leads to elimination of another HF molecule and generation of bicyclic product ions [M – H + BF3 – 2 HF]–. Deprotonated glucuronic acid was used as the model compound in order to obtain the desired computational results in a reasonable time because it is smaller than the glucuronides studied but still forms the diagnostic product ions upon reactions with BF3 (Figure S4A). Further, based on quantum chemical calculations (M06-2X/6-311++G(d,p) level of theory) carried out for reactions of deprotonated erythronic acid and BF3 (Figure S5), the primary [M – H + BF3 – HF]– product ions generate the secondary [M – H + 2 BF3 – 3 HF]– product ions upon reactions with BF3. Before the bicyclization step discussed above, the [M – H + BF3 – HF]– ions may interact with another BF3 molecule via formation of a covalent bond at the 2-, 3- or 4-hydroxyl group of the glucuronic acid moiety, generating [M – H + 2 BF3 – HF]– adduct ions. These product ions can undergo rearrangements, eventually leading to the elimination of a HF molecule to form the secondary [M – H + 2 BF3 – 2 HF]product ions. These secondary product ions can undergo another intramolecular reaction involving another hydroxyl group of the glucuronic acid moiety to form the diagnostic secondary [M – H + 2 BF3 – 3 HF]– product ions (Figure 3B). Deprotonated erythronic acid was used as the model compound for the calculations because it forms the same product ions with BF3 (Figure S6) as deprotonated glucuronic acid, deprotonated N-glucuronides and deprotonated migrated acyl glucuronides but it is much smaller. A similar mechanism for the formation of the diagnostic [M – H + BF3 – 2 HF]– and [M – H + 2 BF3 – 3 HF]– product ions can be depicted for deprotonated N-glucuronides because they contain a free amino group at the anomeric position (Figure S7). The amino group is involved in the ring-opening step for the formation of the [M – H + BF3 – 2 HF]– and [M – H + 2 BF3 – 3 HF]– product ions in the same

Ring-opening

A

O HO

O

+BF3

O

O R

O HO

OH O

O

[M-H]

-HF

F

O

R

F

O

O

OH

O

F

O O

OH

H

R

[M-H+BF3]

O

O

F B

O

HO

O

F

O H

O

O

F B

HO F

O

O

B

HO

O H

OH R

O

B F

O

O

Bicylization

F

O

O

+HF

O O

R

O

[M-H+BF3-2HF]

[M-H+BF3-HF]

3--acyl glucuronide

B

O

O

F B

HO

O

F

OH

O R

O

O

O

[M-H+BF3-HF] 3--acyl glucuronide

+BF3

H O

F F B O F R

O

B O

O O

F

F F

-HF

H O

O

B

F B O

OH

F

O

R

F

O

F F

B F

OH

O

R

O

O

O

O

F B

OH

O O

O

O

O

[M-H+2BF3-2HF]

[M-H+2BF3-HF]


-HF

F

F

B

F B

O

O O

F

+3HF

O

+HF O

O

O

R

[M-H+2BF3-3HF]


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Figure 3. Proposed mechanisms for the reaction between migrated, deprotonated 3-β-acyl glucuronides and BF3 to yield (A) the diagnostic primary product ions ([M – H + BF3 – 2 HF]–) and (B) the diagnostic secondary product ions [M – H + 2 BF3 – 3 HF]–. way a free hydroxyl group is involved in the mechanisms discussed above. On the other hand, deprotonated unmigrated acyl glucuronides and O-glucuronides do not form the diagnostic product ions as they do not contain a hydroxyl group at the anomeric carbon of the glucuronic acid moiety. Mechanism for the loss of a molecule with MW 88 Da upon CAD of the diagnostic secondary [M – H + 2 BF3 – 3 HF]– product ions. To determine the structure of the molecule with MW 88 Da that is eliminated upon CAD of the diagnostic secondary [M – H + 2 BF3 – 3 HF]– product ions, quantum chemical calculations (M06-2X/6-311++G(d,p) level of theory) were performed on possible unimolecular dissociation reactions of the [M – H + 2 BF3 – 3 HF]– product ions. Based on the calculations (Figure S8), the molecule with MW 88 Da is 2-fluoro-1,3,2-dioxaborale and it is eliminated via a mechanism involving a four-membered transition state (Figure 4). This mechanism is likely to be somewhat different from those for deprotonated migrated 2-β- and 3-β-acyl glucuronides (Figure S9). O R

C O

C

O

B O

O B

O

O

F F

CAD

O

R

C O

O

F

4--Acyl glucuronide (0.0 kcal/mol)

O B O

O

F F

R

C O

O

O

[M-H+2BF3-3HF] -

C

O

B

O

C

O

O B O

F F

+

O B

O

F

O

Diagnostic ions

C2H2O2BF (MW 88 Da)

F

83.8 kcal/mol

9.8 kcal/mol

Figure 4. Proposed mechanism for the loss of 2-fluoro-1,3,2-dioxaborale (C2H2O2BF; MW 88 Da) from the diagnostic secondary [M – H + 2 BF3 – 3 HF]– product ions generated upon reactions between deprotonated 4-β-acyl glucuronide and BF3. Values shown are ΔG in kcal/mol (M06-2X/6-311++G(d,p) level of theory). Experimental support for the identity of the 2-fluoro-1,3,2-dioxabolane as the eliminated neutral molecule was obtained by considering isotopes of B, C and O. For example, the relative abundances of the boron isotopes in the CAD product ions are in agreement with their proposed elemental composition as the abundance of 10B relative to that of 11B decreased from 50 % in the [M – H + 2 BF3 – 3 HF]– product ions to 25 % in the fragment ions, suggesting that the [M – H + 2 BF3 – 3 HF]– product ions contained two boron atoms while their fragment ions contained only one (Figure S10). Further, examination of 18Oprobenecid acyl-β-ᴅ-glucuronide (the 18O label was placed on one of the two oxygens of the carboxylic acid group in the glucuronic acid moiety) and 13C-ibuprofen acyl-β-ᴅ-glucuronide glucuronide (the 13C label was at the carbon of the carboxylic acid group in the ibuprofen moiety; for structures, see Figure S11) indicated that the ions formed upon CAD of their the [M – H + 2 BF3 – 3 HF]– product ions retained the 18O- and 13C-labels (Figure S11), thus providing further support for the assignment of 2-fluoro-1,3,2dioxaborale as the structure of the eliminated neutral molecule. Although the reactions between deprotonated glucuronic acid and BF3 yield the diagnostic secondary [M – H + 2 BF3 – 3 HF]– product ions with the aldehyde group at the anomeric position (MS2; Figure S4A), CAD of these [M – H + 2 BF3 – 3 HF]– product ions (MS3) resulted in the elimination of HF to yield [M – H + 2 BF3 – 4 HF]– fragment ions instead of the expected elimination of 2-fluoro-1,3,2dioxaborale. This is likely due to the extra hydroxyl group present in above [M – H + 2 BF3 – 3 HF]– 10 ACS Paragon Plus Environment

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product ions, which facilitates elimination of HF, possibly as shown in Figure S4B. The same applies for CAD of the [M – H + 2 BF3 – 3 HF]– product ions formed upon reactions of deprotonated N-glucuronides and BF3 (Figure 1B and 5F). For the same reasons as for deprotonated glucuronic acid, this is likely due to the extra hydroxyl group present in above [M – H + 2 BF3 – 3 HF]– product ions, which facilitates elimination of HF (Figure S12).

7.13

0

50

O[M-H]

-

10 15 Time (min)

50 0

Unmigrated acyl 757 [M–H]–

689 700

737 m/z

750

Relative Abundance

100

632

584

0 600

E

F 100 50 0

C

652

m/z

Migrated acyl

689 700

717

737

m/z

750

50

[M–H]–

0

m/z 765 CAD 15

399

447 427

400

450

m/z

100 50 0

D

467 [M–H+2BF3–3HF]–

475

G

757 765

N-

650

[M–H+2BF3–3HF]–

[M–H]–

100

628 600

-88 Da 721 677

m/z 475 CAD 15

455

100

Relative Abundance

B 100

Relative Abundance

50

Acyl 13.06 O14.83

N-

Relative Abundance

A 100

Relative Abundance

Relative Abundance

Application of the HPLC/MS3 methodology for differentiation of N-, O- and acyl glucuronides in a mixture. To further demonstrate the utility of the above method for differentiating deprotonated O-, N- and migrated acyl glucuronides, a mixture containing approximately 0.2 mM of telmisartan acyl-β-ᴅ-glucuronide, PhIP N-β-ᴅ-glucuronide, and ezetimibe O-β-ᴅ-glucuronide (for structures, see Figure S13) was stored in the phosphate buffer for 4 hours at room temperature prior to HPLC/MS3 analysis in order to induce acyl migration for telmisartan acyl-β-ᴅ-glucuronide. PhIP N-β-ᴅglucuronide (denoted by N-) and telmisartan acyl-β-ᴅ-glucuronide (denoted by acyl) showed a single HPLC peak each while ezetimibe O-β-ᴅ-glucuronide (denoted by O-) showed two peaks (Figure 5A). It is not surprising that only a single peak was observed in the HPLC chromatogram of telmisartan acyl-βᴅ-glucuronide (see further discussion below) because this compound is among the most stable acyl glucuronides.25,32,33 Furthermore, degradation of telmisartan acyl-β-ᴅ-glucuronide has been reported to occur primarily through hydrolysis and not through acyl migration.34 The two peaks observed for ezetimibe O-β-ᴅ-glucuronide are proposed to correspond to two diastereomers. As expected, following HPLC separation, ions corresponding to the two peaks of ezetimibe O-β-ᴅ-glucuronide did not form the [M – H + BF3 – 2 HF]– nor [M – H + 2 BF3 – 3 HF]– product ions upon reactions with BF3 (Figure 5E). However, deprotonated PhIP N-β-ᴅ-glucuronide produced both the [M – H + BF3 – 2 HF]– (m/z 427) and the [M – H + 2 BF3 – 3 HF]– (m/z 475) diagnostic product ions (Figure 5B), as expected. Surprisingly, the same was observed for some of the telmisartan acyl-β-ᴅ-glucuronide molecules in spite of the observation of only a single HPLC peak (Figures 5A, S14, and S15). No diagnostic product ions were observed upon reactions with BF3 for ions derived from the molecules eluting during the first half of the peak. However, ions derived from the molecules eluting during the latter half of the peak showed the diagnostic product ions ([M – H + BF3 – 2 HF]– (m/z 717) and [M – H + 2 BF3 – 3 HF]– (m/z 765)) upon reactions with BF3 (Figures 5C and 5D). Because only migrated deprotonated acyl glucuronides form the diagnostic product ions with BF3 (Figure

Relative Abundance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


50 0

411 400

431

m/z

475

450

765

m/z 700

Figure 5. (A) HPLC chromatogram measured for a mixture of an N-, O-, and acyl glucuronides after 4 hours in phosphate buffer (pH 7.4). Mass spectra measured after 30 ms reaction of BF3 with (B) deprotonated ezetimibe O-β-ᴅ-glucuronide, (C) PhIP-N-β-ᴅ-glucuronide, (E) unmigrated deprotonated telmisartan acyl-β-ᴅ-glucuronide, and (F) migrated deprotonated telmisartan acyl-β-ᴅ-glucuronide. Both deprotonated PhIP-N-β-ᴅ-glucuronide and migrated deprotonated telmisartan acyl-β-ᴅ-glucuronide formed the diagnostic [M – H + 2 BF3 – 3 HF]– product ions while deprotonated ezetimibe O-β-ᴅglucuronide and unmigrated deprotonated telmisartan acyl-β-ᴅ-glucuronide did not. (D) The CAD 11 ACS Paragon Plus Environment


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(collision energy 15 arbitrary units) mass spectrum measured for the [M – H + 2 BF3 – 3 HF]– product ions of deprotonated PhIP-N-β-ᴅ-glucuronide does not show a fragment ion that is formed via the loss of a molecule with MW 88 Da. (G) The CAD mass spectrum measured for the [M – H + 2 BF3 – 3 HF]– product ions of migrated deprotonated telmisartan acyl-β-ᴅ-glucuronide show the diagnostic fragment ion. 2), this observation demonstrates that telmisartan acyl-β-ᴅ-glucuronide had undergone partial acyl migration prior to the analysis in spite of showing only one HPLC peak. It should be noted here that the use of Ultra-Performance Liquid Chromatography (UPLC; details are given in Figure S15) did not result in a better resolution of the single peak observed. As expected, CAD of the [M – H + 2 BF3 – 3 HF]– product ions yielded fragment ions via the elimination of 2-fluoro-1,3,2-dioxaborale. These results demonstrate that in spite of the fact that telmisartan acyl-β-ᴅ-glucuronide is among the most stable acyl glucuronides, it nevertheless underwent partial acyl migration within 4 hours. Furthermore, the results obtained by coupling an HPLC with ion-molecule reactions suggest that the use of HPLC alone or HPLC coupled with MS may not enable unambiguous determination of whether an acyl glucuronide has undergone acyl migration. CONCLUSIONS N- and O- as well as migrated acyl glucuronide metabolites can be differentiated by allowing their deprotonated forms react with BF3 followed by CAD of the diagnostic secondary product ions (MS3 experiment; Scheme 1). Deprotonated N- and migrated acyl glucuronides can be distinguished from deprotonated O-glucuronides and unmigrated acyl glucuronides by the formation of diagnostic primary [M – H + BF3 – 2 HF]– and secondary [M – H + 2 BF3 – 3 HF]– product ions while deprotonated, migrated acyl glucuronides can be differentiated from deprotonated N-glucuronides by isolating the diagnostic secondary [M – H + 2 BF3 – 3 HF]– product ions and subjecting them to CAD. A fragment ion due to the loss of 2-fluoro-1,3,2-dioxaborale (MW 88 Da) is formed for deprotonated, migrated acyl glucuronides but not for N-glucuronides. Therefore, the fact that all acyl glucuronides studied here yielded the diagnostic product ions demonstrates that they all had at least partially undergone acyl migration. The reactivity differences observed between deprotonated acyl, N- and O-glucuronides were rationalized via quantum chemical calculations. The above MS3 method was successfully coupled with HPLC for structural characterization of mixtures of acyl glucuronide isomers. This methodology enables the differentiation of unmigrated 1-β-acyl glucuronides from the isomeric acyl glucuronides formed upon acyl migration. Further, the results obtained by coupling an HPLC with ion-molecule reactions suggest that the use of HPLC alone or HPLC coupled with MS may not enable unambiguous determination of whether an acyl glucuronide has undergone acyl migration. However, this is critically important because the extent of acyl migration for some acyl glucuronides may be underestimated; hence, their IDT may also be underestimated. The approach presented here enables the fast identification of unstable and therefore undesired acyl glucuronide metabolites, which provides valuable insights into potential safety concerns for compounds with carboxylic acid functionalities during drug discovery and development. ASSOCIATED CONTENT Supporting Information The supporting information associated with this manuscript is attached. Tables illustrating product ions formed between different deprotonated glucuronides and BF3; ion/molecule reaction MS/MS spectra; HPLC/MS/; computational data. AUTHOR INFORMATION Corresponding Author 12 ACS Paragon Plus Environment

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Hilkka I. Kenttämaa Tel.: +1 (765) 494 0882; fax: +1 (765) 494 9421 E-mail: [email protected] ORCID Edouard Niyonsaba: 0000-0002-3847-0935 Hilkka I. Kenttämaa: 0000-0001-8988-6984 Notes The authors declare no competing financial interests. ACKNOWLEDGMENT We thank Merck & Co., Inc., Kenilworth, NJ, USA, for financial support. REFERENCES (1) King, C.; Rios, G.; Green, M.; Tephly, T. UDP-Glucuronosyltransferases. Curr. Drug Metab. 2000, 1, 143–161. (2) Lemke, T. L.; Williams, D. A.; Roche, V. F.; Zito, W. S. Foye’s Principles of Medicinal Chemistry, 7th ed.; Lippincott Williams & Wilkins: Baltimore, MD, 2013. (3) Kato, Y.; Igarashi, T.; Sugiyama, Y.; Nishino, A. Both CMOAT/MRP2 and Another Unknown Transporter (s) Are Responsible for the Biliary Excretion of Glucuronide Conjugate of the Nonpeptide Angiotensin II Antagonist, Telmisaltan. Drug Metab. Dispos. 2000, 28, 1146–1148. (4) Mackenzie, P. I.; Somogyi, A. A.; Miners, J. O. Advances in Drug Metabolism and Pharmacogenetics Research in Australia. Pharmacol. Res. 2017, 116, 7–19. (5) Sabordo, L.; Sallustio, B. C.; Evans, A. M.; Nation, R. L. Hepatic Disposition of the Acyl Glucuronide 1-O-Gemfibrozil-β-D-Glucuronide: Effects of Clofibric Acid, Acetaminophen, and Acetaminophen Glucuronide. J. Pharmacol. Exp. Ther. 2000, 295, 44–50. (6) Mulder, G. J. Pharmacological Effects of Drug Conjugates: Is Morphine 6-Glucuronide an Exception? Trends Pharmacol. Sci. 1992, 13, 302–304. (7) Yi, L.; Dratter, J.; Wang, C.; Tunge, J. A.; Desaire, H. Identification of Sulfation Sites of Metabolites and Prediction of the Compounds’ Biological Effects. Anal. Bioanal. Chem. 2006, 386, 666–674. (8) Langguth, H. S.; Benet, L. Z. Acyl Glucuronides Revisited: Is the Glucuronidation Proces a Toxification as Well as a Detoxification Mechanism? Drug Metab. Rev. 1992, 24, 5–47. (9) Bailey, M. J.; Dickinson, R. G. Acyl Glucuronide Reactivity in Perspective: Biological Consequences. Chem. Biol. Interact. 2003, 145, 117–137. (10) Regan, S. L.; Maggs, J. L.; Hammond, T. G.; Lambert, C.; Williams, D. P.; Park, B. K. Acyl Glucuronides: The Good, the Bad and the Ugly. Biopharm. Drug Dispos. 2010, 31 (7), 367–395. (11) Horng, H.; Spahn-Langguth, H.; Benet, L. Z. Mechanistic Role of Acyl Glucuronides. In DrugInduced Liver Disease (Third Edition); Elsevier, 2013; pp 35–70. (12) Smith, P. C.; Benet, L. Z.; McDonagh, A. F. Covalent Binding of Zomepirac Glucuronide to Proteins: Evidence for a Schiff Base Mechanism. Drug Metab. Dispos. 1990, 18, 639–644. (13) Van Breemen, R.; Fenselau, C.; Mogilevsky, W.; Odell, G. Reaction of Bilirubin Glucuronides with Serum Albumin. J. Chromatogr. B. Biomed. Sci. App. 1986, 383, 387–392.



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(30)

(31) (32) (33) (34)


Liang, W.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Throssell, K.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M. J.; Heyd, J. J.; Brothers, E. N.; Kudin, K. N.; Staroverov, V. N.; Keith, T. A.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A. P.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Millam, J. M.; Klene, M.; Adamo, C.; Cammi, R.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Farkas, O.; Foresman, J. B.; Fox, D. J. Gaussian 16, Revision B.01. Gaussian, Inc., Wallingford CT, 2016. Zhao, Y.; Truhlar, D. G. The M06 Suite of Density Functionals for Main Group Thermochemistry, Thermochemical Kinetics, Noncovalent Interactions, Excited States, and Transition Elements: Two New Functionals and Systematic Testing of Four M06-Class Functionals and 12 Other Functionals. Theor. Chem. Acc. 2008, 120 (1–3), 215–241. Kang, P.; Dalvie, D.; Smith, E.; Renner, M. Bioactivation of Lumiracoxib by Peroxidases and Human Liver Microsomes: Identification of Multiple Quinone Imine Intermediates and GSH Adducts. Chem. Res. Toxicol. 2008, 22, 106–117. Ebner, T.; Heinzel, G.; Prox, A.; Beschke, K.; Wachsmuth, H. Disposition and Chemical Stability of Telmisartan 1-O-Acylglucuronide. Drug Metab. Dispos. 1999, 27 (10), 1143–1149. Jinno, N.; Ohashi, S.; Tagashira, M.; Kohira, T.; Yamada, S. A Simple Method to Evaluate Reactivity of Acylglucuronides Optimized for Early Stage Drug Discovery. Biol. Pharm. Bull. 2013, 36, 1509–1513. Lassila, T.; Hokkanen, J.; Aatsinki, S.-M.; Mattila, S.; Turpeinen, M.; Tolonen, A. Toxicity of Carboxylic Acid-Containing Drugs: The Role of Acyl Migration and CoA Conjugation Investigated. Chem. Res. Toxicol. 2015, 28, 2292–2303. Table of Contents Artwork Deprotonated glucuronides O

O HO O O

O OH

O

R O

O HO HO

O BF F O

O O F B O

OH

BF3

H O

N

OH

R

O O F B O

88 Da loss

O O R

CAD

O BF F O

OH N R

O O HO HO

O OH

O R


Differentiation of Deprotonated Acyl, N- and O ... - ACS Publications

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