Identification of ortho-Substituted Benzoic Acid/Ester Derivatives via the Gas-Phase Neighboring Group Participation Effect in (+)-ESI High Resolution Mass Spectrometry

Article abstract of DOI:10.1007/s13361-017-1884-8

Benzoic acid/ester/amide derivatives are common moieties in pharmaceutical compounds and present a challenge in positional isomer identification by traditional tandem mass spectrometric analysis. A method is presented for exploiting the gas-phase neighbor

Full text of DOI:10.1007/s13361-017-1884-8

B American Society for Mass Spectrometry, 2018
J. Am. Soc. Mass Spectrom. (2018)
DOI: 10.1007/s13361-017-1884-8
RESEARCH ARTICLE
Identification of ortho-Substituted Benzoic Acid/Ester
Derivatives via the Gas-Phase Neighboring Group
Participation Effect in (+)-ESI High Resolution Mass
Spectrometry
William D. Blincoe,1 Agustina Rodriguez-Granillo,2 Josep Saurí,1 Nicholas A. Pierson,1
Leo A. Joyce,1 Ian Mangion,1 Huaming Sheng1
1Analytical Research and Development, Merck and Co., Inc., Rahway, NJ 07065, USA
2Chemistry Modeling and Informatics, Merck and Co., Inc., Rahway, NJ 07065, USA
Abstract. Benzoic acid/ester/amide derivatives are common moieties in pharmaceu-
tical compounds and present a challenge in positional isomer identification by tradi-
tional tandem mass spectrometric analysis. A method is presented for exploiting the
gas-phase neighboring group participation (NGP) effect to differentiate ortho-
substituted benzoic acid/ester derivatives with high resolution mass spectrometry
(HRMS1). Significant water/alcohol loss (>30% abundance in MS1 spectra) was
observed for ortho-substituted nucleophilic groups; these fragment peaks are not
observable for the corresponding para and meta-substituted analogs. Experiments
were also extended to the analysis of two intermediates in the synthesis of
suvorexant (Belsomra) with additional analysis conducted with nuclear magnetic
resonance (NMR), density functional theory (DFT), and ion mobility spectrometry-mass spectrometry (IMS-
MS) studies. Significant water/alcohol loss was also observed for 1-substituted 1, 2, 3-triazoles but not for the
isomeric 2-substituted 1, 2, 3-triazole analogs. IMS-MS, NMR, and DFT studies were conducted to show that the
preferred orientation of the 2-substituted triazole rotamer was away from the electrophilic center of the reaction,
whereas the 1-subtituted triazole was oriented in close proximity to the center. Abundance of NGP product was
determined to be a product of three factors: (1) proton affinity of the nucleophilic group; (2) steric impact of the
nucleophile; and (3) proximity of the nucleophile to carboxylic acid/ester functional groups.
Keywords: Benzoic acid/ester derivatives, Gas phase neighboring group participation effect, HRMS, (+)-ESI,
DFT, IMS, NMR, Structural elucidation
Received: 26 October 2017/Revised: 21 December 2017/Accepted: 23 December 2017
have been fundamental to the pharmaceutical industry in prod-
ucts that cover a bevy of therapeutic benefits, including inflam-
Introduction
ubstituted benzoic acid/ester derivatives are important
building blocks used in many pharmaceutical compounds
and in organic synthesis. They have notably been used as
preservatives for food, cosmetics, and pharmaceutical products
because of their antimicrobial and antifungal properties [1–3].
Compounds that contain the benzoic acid/benzamide moiety
mation (aspirin, salicylic acid), schizophrenia (amisulpride),
insomnia (suvorexant), and cancer therapy (pemetrexed,
imatinib).
S
Different positional isomers substituted at ortho, meta, and
para positions as well as conformational isomers such as E/Z
isomers can be generated during the synthesis of the aromatic
substituted benzoic acid/ester derivatives. Traditionally, ana-
lytical methods such as nuclear magnetic resonance (NMR)
and high performance liquid chromatography-high resolution
tandem mass spectrometry (HPLC-HRMS/MS) are able to
differentiate these sets of isomers. However, each method has
limitations. NMR studies can often be time-consuming, and
Electronic supplementary material The online version of this article (https://
doi.org/10.1007/s13361-017-1884-8) contains supplementary material, which
is available to authorized users.
Correspondence to: Huaming Sheng; e-mail: huaming.sheng@merck.com

W. D. Blincoe et al.: Identfying ortho-Substituted Benzoic Acid via NGP Effect
depend on the purity of samples making complex mixture
NGP effect in two triazole benzoic acid isomers
(23–24) as shown in Figure 1 generated during the
synthesis of suvorexant (trade name Belsomra) and ana-
lyzed here by HRMS, MS/MS, Ion mobility
spectrometry-mass spectrometry (IMS-MS), NMR spec-
troscopy, and density functional theory (DFT)
calculations.
analysis difficult [4–9]. HPLC-HRMS/MS has the advantages
of chromatographic separation of mixtures, but identification of
each chromatographically resolved peak often requires the
retention time match with an authentic sample because most
positional isomers of aromatic substituted benzoic acid/ester
derivatives have indistinguishable fragmentation patterns un-
der tandem mass spectrometric analysis [10–16]. The neigh-
boring group participation (NGP) effect in organic chemistry
has been defined by IUPAC as the intramolecular interaction of
a reaction center with a lone pair of electrons in an atom or the
electrons present in a σ-bond or π-bond [17]. While NGP
influences many reactions in solution, the gas-phase NGP
effect has also been extensively studied to explain many frag-
mentation patterns during collision induced dissociation (CID)
[18–24]. Cooks et al. showed δ-cleavage from gas-phase aryl
participation in several azulene compounds after electron ion-
ization [23]. Bigler and Hesse identified cinnamic acid deriva-
tives of diamines based on electrospray ionization (ESI)-MS
fragmentation and noted the difference in reactivity correlated
with ring strain of cyclized products [19]. While these previous
studies evaluated the NGP effect under CID conditions, this
study mainly focuses on the NGP effect that can take place
during MS1 analysis of protonated ortho-substituted benzoic
acid/ester derivatives with nucleophilic functional groups con-
taining lone pair or π-bond electrons. The high energy barrier
for intramolecular cyclization as seen in CID could be over-
come if the nucleophile and electrophile is in small spatial
proximity as is the case for ortho-substituted benzoic acid/
ester derivatives. These nucleophilic groups can attack the
protonated carboxylic acid/ester moiety to facilitate the water/
alcohol neutral molecule loss. On the other hand, no significant
water/alcohol neutral loss would be expected for the meta- and
para-substituted benzoic acid/ester derivatives due to the larger
spatial distance and steric penalty for nucleophilic attack. This
observation can be used to differentiate some ortho-substituted
benzoic acid/ester derivatives from their meta- and para-
substituted isomers.
In the present study, 22 ortho-, meta-, and para-
substituted benzoic acid scaffolds (1-22) (Figure 1) were
chosen to study this effect. These compounds were incu-
bated in 0.05% H2SO4 in methanol (MeOH), ethanol
(EtOH), and isopropanol (i-PrOH) solutions in order to
form the corresponding ester products. The resulting 65
acid and ester products were separated by LC followed
by (+)-ESI/MS analysis. Three factors were determined
to impact the differentiation of isomers based on MS1
analysis: (1) the effects of the competing protonation of
the carboxyl group versus the other nucleophilic group
during ESI; (2) the steric effects of cyclization; and (3)
the nucleophilic group proximity to the electrophilic cen-
ter. Each compound was tested to see if neutral loss of
either H2O for the acids or MeOH, EtOH or i-PrOH for
the esters occurred during MS1 analysis. These losses
will be referred to as neutral group losses. Furthermore,
efforts were made toward understanding the gas-phase
Experimental
Chemicals and Reagents
Methanol, ethanol, isopropanol, acetonitrile, water with
0.1% formic acid solvents, and sulfuric acid were obtained
from Fischer Scientific (Optima LC-MS grade, Waltham,
MA, USA). Compounds were sourced as follows: 1a, 4,
7a, 12a, 13a, 15a, 17a, and 21a were obtained from
Sigma-Aldrich (St. Louis, MO, USA); 2a, 6a, 8a, 19a,
and 20a were obtained from Enamine LLC (Monmouth,
NJ, USA); 3a, 11a, and 14a were obtained from
Maybridge (Loughborough, UK); 5a was obtained from
Rieke Metals (Lincoln, NE, USA); 9 was obtained from
ChemBridge (San Diego, CA, USA); 10a was obtained
from Matrix Scientific (Elgrin, SC, USA); 16a was obtain-
ed from Chem-Impex International (Wood Dale, IL, USA);
18a was obtained from Apollo Scientific (Manchester,
UK); 22a was obtained from ArkPharm (Libertyville, IL,
USA); 23a and 24a were synthesized according to litera-
ture procedure [25–27].
Sample Preparation
Samples were prepared in 2 mL HPLC vials using an Andrew
Alliance pipetting robot. The 24 analyte compounds were
prepared in 0.05% H2SO4 in three different solvents (meth-
anol, ethanol, and isopropanol) for esterification to the b, c,
and d compounds by adding a few crystals of each analyte
compound to the vials and mixing. Dissolved analytes were
stored at room temperature for 2 d to allow for esterification
of the carboxylic acid to occur.
Instrumentation
HPLC/UV LC separation was performed on a Waters
Acquity UPLC system, consisting of a binary pump system, a
sample manager, and a PDA detector (Waters Corporation,
Milford, MA, USA). The output signal was monitored and
processed with MassLynx software designed by Waters Cor-
poration (Milford, MA, USA).
Separation was carried out on a Waters BEH C18 column
(2.1 × 50 mm, 1.7 μm particle sizes). The mobile phase
consisted of water with 0.1% formic acid (mobile phase A)
and acetonitrile (mobile phase B). The injection volume was 3
μL. Analytes were eluted using a gradient method consisting of
an initial hold at 1% mobile phase B for 0.5 min, followed by a
linear gradient to 90% B over 3.5 min, then a linear gradient to
0% B over 0.1 min, a hold at 0% B for 0.4 min, and finally a

W. D. Blincoe et al.: Identfying ortho-Substituted Benzoic Acid via NGP Effect
Figure 1. Model compounds grouped by the extent of water/alcohol loss. Nucleophilic groups are marked in red and the R group is
marked in blue. Two triazole compounds were also tested with 23 having large loss (>30%) and 24 having no loss (0%)
hold at 1% B for 0.5 min affording a total run time of 5
min. The flow rate was 0.5 mL/min. The column tem-
perature was maintained at 40 C. The PDA detector was
Mass Spectrometry The eluent was introduced directly into
the mass spectrometer via an electrospray ionization source.
MS analysis was performed on a Waters Premier quadrupole
time-of-flight (Q-ToF) mass spectrometer operating in positive
o
scanned from 200 to 400 nm.

W. D. Blincoe et al.: Identfying ortho-Substituted Benzoic Acid via NGP Effect
ion mode. Source temperature and desolvation temperature
including compounds that exhibited a neutral group loss greater
than 30%, Group 2 including compounds with a neutral loss
between 0% and 30%, and Group 3 with no neutral group loss
observed. Examples of the MS1 spectra observed for 6a-b and
20a-b are shown in Figure 2. All other MS1 spectra can be
found in the Supplementary material.
o
o
were set to 120 C and 400 C, respectively. Nitrogen was
used as both the cone gas (50 L/h) and desolvation gas (800
liters/h). The capillary voltage was set to 3 kV. The cone
voltage applied was 10 V. Leucine enkephalin was used as
the lock mass (m/z of 556.2772) for accurate mass calibration
and was introduced using the lock spray interface at 20 μL/min
at a concentration of 0.5 mg/mL in 50% aqueous acetonitrile
containing 0.1% formic acid. During MS scanning, data were
acquired in centroid mode from m/z 50 to 1000.
The compounds in Group 1 (1-6) showed significant neutral
group loss greater than 30%. These compounds all included
nucleophilic groups ortho to the acid/ester functional group.
These nucleophiles ranged from amine groups in 1, to oxygen
nucleophiles in 3 and 5, to π-bonds in 2. Group 2 compounds
(7-14) showed moderate neutral group loss with ortho nucleo-
philic groups and two para nucleophilic groups in 8 and 10.
Interestingly, two para compounds include longer chain nucle-
ophiles that might allow nucleophilic attack to bridge the
aromatic ring. Another explanation for the neutral group loss
observed in MS1 analysis of 8 and 10 is that the carboxylic
acid/ester is the main protonation site during ionization due to
the relative low proton affinity of the para substitution group.
Group 3 compounds (15-22) included only compounds that
had nucleophilic groups meta and para to the acid/ester, which
did not show neutral loss during ionization, as expected.
A proposed mechanism to explain the neutral group loss
from the model compounds can be found in Scheme 1. The
reaction proceeds via two pathways, a 5- or 6-member ring
formation depending on the placement of the nucleophilic
group. In the case of carboxylic acid, the reaction is initiated
by protonation on the carbonyl group of the carboxylic acid
during ionization. The NGP effect then occurs as the nucleo-
philic substituent attacks the electrophilic carbonyl with subse-
quent elimination of the neutral leaving group to form a cyclic
product ion. The three controlling factors governing this cycli-
zation are proposed as follows: (1) the proton affinity of the
carboxylic acid/ester competing with other basic sites on the
compound; (2) the steric penalty of forming the 5- or 6-member
ring; and (3) the proximity of the nucleophilic group to the
carbonyl.
Ion Mobility Spectrometry (IMS) Instrumentation IMS-MS
experiments were conducted on Agilent 6560 uniform-field ion
mobility-Q-ToF instrument (Agilent Technologies, Santa
Clara, CA, USA). Samples were infused by syringe pump (5
μL/min) and ionized by ESI (3.5 kV capillary voltage). The
instrument was tuned for fragile ions, an automated procedure
that lowers ion optics voltages to preserve fragile ion structures
as they are transported through the instrument from the source
to detector. Sheath and desolvation gas temperatures were set
to 150 °C and 100 °C, respectively. Ion mobility measurements
were performed in both helium and nitrogen drift gases (4.0
Torr) with a drift tube entrance voltage of 1200 V and a drift
tube exit voltage of 250 V. Experimental nitrogen and helium
collision cross-sections (CCS) were measured by single-field
calibration with the Agilent Tune Mix ions m/z 118 and 322.
Theoretical CCS values were calculated by the trajectory meth-
od in MOBCAL [28] from energy-minimized structures ob-
tained from the DFT calculations described below.
NMR NMR data were acquired in DMSO-d6 at room tem-
perature using a 3 mm tube at 500 MHz from a Bruker instru-
ment equipped with a triple-resonance (TCI) Prodigy Cryo-
Probe. For the 1D selROE experiment acquired for isomer 24a,
a 20 ms Gaussian selective pulse was used to excite proton Hb.
A mixing time of 300 ms was used for the ROESY step.
Proton Affinity (PA) of Nucleophilic Group
and Carboxylic Acid/Ester
Density Functional Theory (DFT) Calculations An ensemble
of molecular mechanics (MM) conformations for each inter-
mediate were generated with Openeye’s Omega [29, 30],
followed by DFT optimization using Jaguar 9.1 [31, 32]. All
calculations were performed at the B3LYP-D3/6-31++G**lev-
el in the gas phase [33–37].
The gas-phase PA of each functional group within a molecule
is closely related to the probability of protonation during (+)-
ESI ionization [38]. As shown in Table 1, it was observed that
the proton affinity (PA) of the neighboring nucleophilic groups
and the carboxylic acid/ester closely correlate with the extent of
neutral loss observed experimentally. On one hand, the higher
PA of the leaving group (carboxylic acid/ester) led to more
neutral loss. For example, methyl ester group (203.3 kcal/mol)
in 1b had higher PA than carboxylic acid group (196.2
kcal/mol) in 1a [38]. Hence, more protonation was expected
on the methyl ester, resulting in a larger extent of neutral loss
(100% for 1b vs 22% for 1a). On the other hand, the higher PA
of the neighboring substituent led to less neutral loss. For
example, a larger extent of neutral loss was observed for 3a
(100%) compared with 1a (22%) because the benzyl amino
Results and Discussion
MS1 Spectra of Benzoic Acids and Esters
Table 1 summarizes the MS1 data obtained for all 65 model
compounds. The stability of the ester compounds was not
uniform and some compounds were unable to form all three
ester products, as shown in Figure 1. The compounds were
divided into four groups also shown in Figure 1, with Group 1

W. D. Blincoe et al.: Identfying ortho-Substituted Benzoic Acid via NGP Effect
Table 1. MS1 Data for benzoic acid and ester model compounds. Nominal m/z provided with relative abundance in parenthesis. Compounds in blue are >30%
abundance. Compounds in green are between 0% and 30% abundance relative to the base peak. Compounds in red are 0% abundance (not detected)
#
1
R
[M+H]+
(m/z)
[M+H-ROH]+
(m/z)
[M+H-NH3/OR]+
(m/z)
a (H-)
152 (100%)
166 (0.6%)
180 (0.2%)
194 (0.4%)
179 (100%)
193 (85.5%)
189 (62.0%)
203 (15.0%)
210 (100%)
179 (27.3%)
161 (100%)
207 (27.5%)
189 (100%)
203 (100%)
217 (100%)
231 (6.4%)
243 (100%)
257 (100%)
271 (100%)
285 (100%)
179 (100%)
193 (100%)
208 (55.3%)
179 (100%)
193 (100%)
272 (100%)
217 (100%)
231 (100%)
245 (100%)
259 (100%)
189 (100%)
217 (100%)
217 (100%)
231 (100%)
245 (100%)
243 (100%)
257 (100%)
271 (100%)
152 (53.5%)
166 (52.6%)
180 (76.5%)
194 (39.3%)
152 (54.5%)
166 (39.6%)
180 (55.9%)
189 (100%)
203 (100%)
272 (100%)
286 (100%)
300 (100%)
189 (100%)
203 (100%)
217 (100%)
217 (100%)
231 (100%)
245 (100%)
259 (100%)
189 (100%)
203 (100%)
217 (100%)
231 (33.5%)
204 (3.6%)
218 (1.5%)
232 (29.4%)
204 (100%)
218 (100%)
232 (100%)
134 (22.1%)
134 (100%)
134 (100%)
134 (100%)
161 (57.7%)
161 (100%)
171 (100%)
171 (100%)
192 (59.0%)
193 (32.9%)
161 (100%)
161 (100%)
171 (60.5%)
171 (69.5%)
171 (51.2%)
171 (100%)
225 (15.9%)
(225) 15.2%
225 (7.3%)
225 (4.5%)
161 (10.7%)
161 (9.4%)
190 (4.0%)
161 (0%)
161 (1.4%)
254 (29.1%)
199 (13.3%)
199 (21.2%)
199 (3.2%)
199 (9.3%)
171 (11.9%)
171 (0%)
199 (2.6%)
199 (5.2%)
199 (0%)
225 (0%)
225 (0%)
225 (0%)
134 (0%)
134 (0%)
134 (0%)
134 (0%)
134 (0%)
134 (0%)
134 (0%)
171 (0%)
171 (0%)
254 (0%)
254 (0%)
254 (0%)
171 (0%)
171 (0%)
171 (0%)
135 (34.0%)
135 (7.0%)
135 (8.6%)
135 (6.6%)
162 (4.9%)
162 (11.2%)
172 (11.0%)
---
193 (20.2%)
162 (5.0%)
162 (10.7%)
162 (11.4%)
172 (7.0%)
172 (8.5%)
172 (6.7%)
172 (12.1%)
b (Me-)
c (Et-)
d (i-Pr-)
a (H-)
b (Me-)
a (H-)
b (Me-)
(H-)
a (H-)
b (Me-)
c (Et-)
a (H-)
b (Me-)
c (Et-)
d (i-Pr-)
a (H-)
b (Me-)
c (Et-)
d (i-Pr-)
a (H-)
b (Me-)
(H-)
2
3
4
5
6
7
8
---
---
---
---
---
---
9
10
191 (0.8%)
---
---
255 (4.1%)
200 (1.9%)
200 (2.6%)
200 (0.5%)
200 (9.6%)
172 (1.5%)
---
---
---
---
---
---
a (H-)
b (Me-)
(H-)
11
12
a (H-)
b (Me-)
c (Et-)
d (i-Pr-)
a (H-)
c (Et-)
a (H-)
b (Me-)
c (Et-)
a (H-)
b (Me-)
c (Et-)
a (H-)
b (Me-)
c (Et-)
d (i-Pr-)
a (H-)
b (Me-)
c (Et-)
a (H-)
b (Me-)
a (H-)
b (Me-)
c (Et-)
a (H-)
b (Me-)
c (Et-)
a (H-)
b (Me-)
c (Et-)
d (i-Pr-)
a (H-)
b (Me-)
c (Et-)
d (i-Pr-)
a (H-)
b (Me-)
c (Et-)
a (H-)
13
14
15
16
---
135 (100%)
149 (100%)
163 (100%)
177 (100%)
135 (100%)
149 (100%)
163 (100%)
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
17
18
19
20
21
199 (0%)
199 (0%)
199 (0%)
199 (0%)
171 (0%)
171 (0%)
171 (0%)
171 (0%)
186 (100%)
186 (100%)
186 (100%)
186 (2.5%)
186 (4.4%)
186 (0%)
22
23
24
187 (12.8%)
187 (12.1%)
187 (12.7%)
–
–
–
b (Me-)
c (Et-)

W. D. Blincoe et al.: Identfying ortho-Substituted Benzoic Acid via NGP Effect
Figure 2. The MS1 spectra of 6a and 20b under (+)-ESI. The pyrazole substitution group is highlighted in red. The acid/ester
functional group is highlighted in blue
group (218.3 kcal/mol) has higher PA than furan (192.0
kcal/mol) [38]. Interestingly, compounds 1a, 16a-d, and 17a-
c with ortho, meta, and para-amino substitutions showed more
NH3 loss rather than water/alcohol loss, which is another
indication that high PA of the neighboring substituent is not
favored in cases where the NGP effect occurs.
these structures are the dimethyl substitution of the pyrazole
rings in 12a-d where as 6a-d do not have these substituents.
These methyl groups also create steric interference that reduces
the stability of the ring formation.
Proximity of the Nucleophilic Group
to the Carbonyl
Steric Effect of Ring Formation
The ortho, meta, and para relationships of the nucleophilic
groups were also investigated in this study. As shown for 1a-d,
16a-d, and 17a-c, the ortho-substituted primary amine exhib-
ited significant neutral group loss that was not seen in the meta-
and para-amines. Cyclization resulting from nucleophilic at-
tack appears to be constrained to ortho-positioned nucleophilic
groups. This effect was visible in numerous other positional
isomeric compound pairs in this study, including 2a-b and 8a-
c, 3a-b and 18a-b, 5a-d and 10a-c, 7a-d and 15a-d, and 12a-d
and 21a-d. This differentiation based on nucleophilic group
The formation of the 5- or 6-member rings following nucleo-
philic attack of the carbonyl was also seen to be impacted by
the bulkiness of the groups attached to the nucleophile. As seen
in 4 and 9, the water loss was much greater in 4 (59.0%) than in
9 (4.0%). This difference in reactivity can be attributed to the
steric penalty of the bulky isopropyl group attached to 9 com-
pared with the smaller methoxyl group attached to 4. It was also
observed that 12a-d had reduced neutral group losses (3.2%–
21.2%) compared with 6a-d (51.2%–100%). The differences in
5-Member Ring Formation
H
N
H
N
H
H
H
O
H
H2N
O
-H2O
ESI
O
1a
O
H
Ionization
O
H
6-Member Ring Formation
O
O
O
H
O
H
O
-H2O
HN
NH
O
HN
O
ESI
O
9
Ionization
OH
O
O
H
Scheme 1. Proposed mechanism for neutral group loss based on the NGP effect. The protonation is on the carbonyl group to
facilitate the subsequent intramolecular nucleophilic addition

W. D. Blincoe et al.: Identfying ortho-Substituted Benzoic Acid via NGP Effect
Figure 3. Intermediate step in synthesis of suvorexant [25]
placement proves to be an effective means for differentiating
ortho-placed nucleophilic groups from meta and para groups
by neutral group loss.
reactivity of 24a-c: (1) The main protonation site is on position
3 nitrogen instead of position 2 due to the PA difference, which
would inhibit the following proton transfer from triazole to
acid/ester group; (2) the triazole and phenyl ring were not
favored to be synperiplanar due to the steric hindrance of the
two hydrogens (Figure 5). A rotational barrier must be over-
come to form cyclized neutral loss product for 24a but not for
23a. This hypothesis was further investigated using NMR
spectroscopy, IMS-MS, and DFT analysis.
Studies of Two Triazole Acid 23a and 24a (Synthetic
Intermediates for Suvorexant)
This research was also extended to an analysis of a benzoic acid
building block of suvorexant (Belsomra), a commercially
available drug produced by MSD approved by the FDA in
2014 for the treatment of insomnia. The synthesis of
suvorexant includes two benzoic acid isomer intermediates as
shown in Figure 3 after the triazole acid addition step [25–27].
These intermediates (shown in Figure 1) were also converted to
esters and tested by UPLC-HRMS in order to investigate if MS
spectrum differences can be found based on NGP effects. The
MS1 spectra and IMS drift time distributions for 23a and 24a
can be found in Figure 4. The products were also studied with
IMS, NMR, and density functional theory (DFT) calculations.
NMR Data
To investigate the orientation of 24a towards the acid group,
we acquired a 1D selective ROE applying selective inversion at
the aromatic proton Hb. As shown in Figure 5 and Figure S49
(in Supporting Information), a strong ROE correlation was
observed between protons Hb and Ha, indicating that they are
close in the space (~2–3 Å distance), which would be consis-
tent with the nitrogen at the 2-position oriented toward the
carboxylic acid moiety. However, it must be noted that NMR
could not determine the exact rotamer of 24a in solution
because the C-N bond was expected to rotate in solution.
Evaluation of potential hydrogen bonding was carried out by
acquiring 1D proton NMR for both 23a and 24a. Assuming
that there is no hydrogen bonding in 23a and due to the fact that
proton spectra revealed that the OH proton had the same
chemical shift in both isomers (Figures S49-S51), it was con-
cluded that there was no hydrogen bonding in isomer 24a
MS1 Data
Table 1 summarizes the MS1 data obtained for 23a-c and 24a-
c. Among all acids and esters, it can be seen that significant
neutral group loss of 100% occurs for 23a-c while a minimal
loss of 0%–4.4% occurs for 24a-c. Model compound analysis
suggests that significant neutral group loss would occur in both
compounds due to the ortho-triazole ring to the carboxylic acid
in both cases. Two factors were proposed to explain the low
Figure 4. (Left) MS1 spectra of 23a and 24a under (+)-ESI. (Right) IMS difference of 23a and 24a in drift time

W. D. Blincoe et al.: Identfying ortho-Substituted Benzoic Acid via NGP Effect
with the carbonyl reactive site. Thus, no nucleophilic attack can
occur without a conformational rearrangement. Additionally,
the 3-position is the most basic site in 24a. As shown in
Figure 6, in order for the proton transfer to occur, the nitrogen
at the 2-position needs to be protonated, which is 6.5 kcal/mol
higher in energy than the protonated isomer at position 3. This
pathway was compared with the corresponding one for 23a in
which significant water loss is observed experimentally. DFT
calculations show that the neutral species of 23a has the triazole
pointing towards the carbonyl group. Initial protonation of the
triazole is likely leading to a rapid proton transfer to the
hydroxyl portion of the carboxylic acid with subsequent addi-
tion and elimination of the water.
Ion Mobility Spectrometry-Mass Spectrometry
Analysis
Figure 5. DFT optimization (B3LYP-D3-631++G**, gas phase)
Ion mobility spectrometry (IMS) provides an additional dimen-
sion of analysis through gas-phase shape and charge separation
of ionized analytes prior to MS detection [39]. In this study,
IMS-MS was utilized to compliment NMR and computational
efforts to distinguish isomer structures 23a and 24a. Ion mo-
bility drift time distributions extracted from precursor m/z 204
are shown in Figure 4 for both triazole benzoic acid isomers
analyzed in nitrogen drift gas. The faster drift time for isomer
23a indicates a more compact structure compared to 24a. The
proposed mechanism for intramolecular cyclization and subse-
quent neutral loss prevalent in MS1 of compound 23a suggests
the amine-containing neighboring group is positioned in close
proximity to the acidic functional group, which is supported by
the observed relative ordering of a higher mobility isomer 23a.
Despite the close structural similarity in these isomers, IMS
allows for differentiation between the two protonated precursor
ions. When combined with computational modeling, experi-
mentally and theoretically derived collision cross-section
(CCS) values can be compared to proposed all-atom structures
of relevant species for 23a
either. Note that in the case of hydrogen bonding, the OH
proton would have resonated at a different, more downfield
chemical shift. In summary, the 1D selROE data were consis-
tent with the nitrogen at the 2-position in 24a pointing toward
the carboxylic acid group. However, this could not be further
confirmed by hydrogen bonding formation as it was not ob-
served in proton NMR spectra.
DFT Calculations
The protonation and cyclization processes of 23a and 24a were
also investigated using DFT calculations with results shown in
Figures 5 and 6. The most stable species of 24a in gas phase
corresponds to an anti-planar conformation, where the triazole
group is orthogonal to the phenyl ring. Therefore, the lack of
neutral group loss in 24a can be partially attributed to the
position of the nucleophilic triazole group, which is not aligned
Figure 6. DFT optimization (B3LYP-D3-631++G**, gas phase) of relevant species for 24a

W. D. Blincoe et al.: Identfying ortho-Substituted Benzoic Acid via NGP Effect
[40]. Energy minimization of the DFT-generated protonated
23a and 24a structures, followed by calculation of CCS
values by the trajectory method in MOBCAL yields
theoretical CCS values in agreement with the experimen-
tally measured CCS values; 23a (77.7 Å2) has a smaller
calculated trajectory method CCS than 24a (80.1 Å2).
MOBCAL calculates CCS with helium as the neutral
collision gas, and thus experimentally derived CCS
values in helium are reported for direct comparison with
the calculations with values of 78.3 Å2 for 23a and 79.7
Å2 for 24a. Experimental and theoretical CCS (trajectory
method) values are typically considered to be in agree-
ment when values are within 2% relative difference [41];
this is the case for these two isomers, with 23a (0.8%
difference) and 24a (0.5% difference). Characterization
of subtle structural differences between these two triazole
benzoic acid isomers through a combination of NMR,
IMS-MS, and computational modeling helps reinforce
the conclusions drawn from each of these complementary
structure elucidation approaches, and supports the hy-
pothesis of water loss due to a gas-phase NGP effect
for the scaffolds investigated in this study.
Thomas Williamson and Dr. Caroline McGregor for their
managerial support.
References
1. Chipley, J.R.: 2 Sodium benzoate and benzoic acid. In: Davidson, P.M.,
Sofos, J.N., Branen, A.L. (eds.) Antimicrobials in food, 3rd ed., p. 11.
CRC Press Taylor and Francis Group, Boca Raton (2005)
2. Shrewsbury, R.P.: University of North Carolina Eshelman School of
Pharmacy. The Pharmaceutics and Compounding Laboratory. Available:
http://pharmlabs.unc.edu/appendix_resources.htm. Accessed 11
Oct 2017
3. United States Food and Drug Administration (FDA). Code of Federal
Regulations Title 21. Volume3. Available: http://www.accessdata.fda.
gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?fr=184.1021. Accessed 11
Oct 2017
4. Malz, F., Jancke, H.: Purity assessment problem in quantitative NMR-
impurity resonance overlaps with monitor signal multiplets from stereo-
isomers. Anal. Bioanal. Chem. 385, 760–765 (2006)
5. Szafran, Z.: Structural Isomer Identification via NMR. J. Chem. Educ. 62,
260–261 (1985)
6. Queiroz, S.L., de Araujo, M.P., Batista, A.A., Macfarlane, K.S., James,
B.R.: Synthesis of [RuCl2(dppb)(PPh3)] and identification of the cis- and
trans-[RuCl2(dppb)(phen)] geometrical isomers via 31P{1H} NMR spec-
troscopy. J. Chem. Educ. 78, 87–88 (2001)
7. Kikuchi, K., Nakahara, N., Wakabayashi, T., Suzuki, S., Shiromaru, H.,
Miyake, Y., Saito, K., Ikemoto, I., Kainosho, M., Achiba, Y.: NMR
characterization of isomers of C78, C82, and C84 fullerenes. Nature.
357, 142–145 (1992)
8. Forseth, R.R., Schroeder, F.C.: NMR-spectroscopic analysis of mixtures:
from structure to function. Curr. Opin. Chem. Biol. 15, 38–47 (2011)
9. Caldearelli, S.: Chromatograhpic NMR: a tool for the analysis of small
molecules. Magn. Reson. Chem. 45, 548–555 (2007)
Conclusion
Ortho-substituted benzoic acid/ester derivatives were iden-
tified among other positional isomers using HRMS1 by
the neighboring group participation (NGP) effect. A total
of 65 model compounds were tested, and it was deter-
mined that the NGP effect depends on three factors: the
proton affinity of the nucleophile, the bulkiness of the
nucleophile, and the positional isomer (ortho- compared
to meta- and para-nucleophile). The trends found for the
model compounds were then extended to investigate two
triazole isomers that form during the synthesis of the
active pharmaceutical ingredient (API) suvorexant
(Belsomra®). MS1 spectra differentiated the isomers
based on the NGP effect, as 23 showed large neutral
group loss whereas 24 did not. Further IMS-MS, NMR
and DFT studies were also conducted to show the non-
reactivity of 24 (in terms of neutral loss during MS1) can
be attributed to two main factors: 1) higher PA of the
nitrogen at the 3-position of the triazole; and 2) the
protonated triazole in 24 is likely oriented 90 degrees,
orthogonal to the carboxylic acid in the gas phase,
disfavoring nucleophilic attack by the nitrogen at the 2-
position. The results from this work aid structural eluci-
dation of substituted benzoic acid/ester isomers through
facile interrogation of the gas-phase NGP effect in MS1
analysis.
10. Slabizki, P., Legrum, C., Meusinger, R., Schmarr, H.G.: Characterization
and analysis of structural isomers of dimethyl methoxypyrazines in cork
stoppers and ladybugs (Harmonia axyridis and Coccinella
septempunctata). Anal. Bioanal. Chem. 406, 6429–6439 (2014)
11. Babushok, V.I.: Chromatographic retention indices in identification of
chemical compounds. Trends Anal. Chem. 69, 98–104 (2015)
12. Pacakova, V., Feltl, V.: Chromatographic retention indices, an aid to
identification of organic compounds. E. Horwood, New York (1992)
13. Wenig, P., Odermatt, J.: Efficient analysis of Py-GC/MS data by a large
scale automatic database approach: an illustration of white pitch identifi-
cation in pulp and paper industry. J. Anal. Appl. Pyrolysis. 87, 85–92
(2010)
14. Zhang, L., Tang, C., Cao, D., Zeng, Y., Tan, B., Zeng, M., Fen, W., Xiao,
H., Liang, Y.: Strategies of structure elucidation of small molecules using
gas chromatography-mass spectrometric data. Trends Anal. Chem. 47,
37–46 (2013)
15. Alam, M.S., Stark, C., Harrison, R.M.: Using variable ionization energy
time-of-flight mass spectrometry with comprehensive GCxGC to identify
isomeric species. Anal. Chem. 88, 4211–4220 (2016)
16. Zhang, L., Cheng, X.L., Liu, Y., Liang, M., Dong, H., Lv, B., Yang, W.,
Luo, Z., Tang, M.: Identification of the related substances in ampicillin
capsule by rapid resolution liquid chromatography coupled with
electrospray ionization tandem mass spectrometry. J. Anal. Methods
Chem. 2014, 1–15 (2014)
17. International Union of Pure and Applied Chemistry (IUPAC). Compen-
dium of Chemical Terminology, 2nd ed. (the BGold Book^). Complied by
A. D. McNaught and A. Wilkinson. Blackwell Scientific Publications:
Oxford (1997) XML on-line corrected version: http://goldbook.iupac.org
(2006-) created by M. Nic, J. Jirat, B. Kosata; updates complied by A.
Jenkins. Accessed 11 July 2016
18. Capon, B.: Neighboring group participation. Q. Rev. Chem. Soc. 18, 45–
111 (1964)
19. Bigler, L., Hesse, M.: Neighboring group participation in the electrospray
ionization tandem mass spectra of polyamine toxins of spiders. Part 1:
α,ω-Diaminoalkane compounds. J. Am. Soc. Mass Spectrom. 6, 634–637
(1995)
20. Darshan, D.V., Chandar, B.G.N., Srujan, M., Chaudhuri, A., Prabhakar,
S.: Electrospray ionization tandem mass spectrometry study of six
Acknowledgments
The authors thank the MSD Summer Internship Program for
funding, Dr. Gary Martin for helpful discussions, and Dr. R.

W. D. Blincoe et al.: Identfying ortho-Substituted Benzoic Acid via NGP Effect
isomeric cationic amphiphiles with ester/amide linker. Rapid Commun.
Mass Spectrom. 28, 1209–1214 (2014)
using high quality structures from the Protein Databank and Cambridge
Structural Database. J. Chem. Inf. Model. 50, 572–584 (2010)
31. Jaguar, version 9.1, Schrödinger, Inc.: New York, NY (2016)
32. Bochevarov, A.D., Harder, E., Hughes, T.F., Greenwood, J.R., Braden,
D.A., Philipp, D.M., Rinaldo, D., Halls, M.D., Zhang, J., Friesner, R.A.:
Jaguar: a high-performance quantum chemistry software program with
strengths in life and materials sciences. Int. J. Quantum Chem. 113, 2110–
2142 (2013)
21. Tu, Y.-P., Harrision, A.G.: Fragmentation of protonated amides through
intermediate ion-neutral complexes: neighboring group participation. J.
Am. Soc. Mass Spectrom. 9, 454–x (1998)
22. Hu, W., Reder, E., Hesse, M.: Neighboring-group participation in the
mass-spectral decomposition of 4-hydroxycinnamoyl-spermidines. Helv.
Chir. Acta. 79, 2137–2151 (1996)
23. Cooks, R.G., Wolfe, N.L., Curtis, J.R., Petty, H.E., McDonald, R.N.:
Neighboring-group participation reactions in the mass spectral fragmen-
tations of some azulenes. Comparisons with solvolytic processes. J.
Organomet. Chem. 35, 4048–4054 (1970)
24. Reid, G.E., Simpson, R.J., O’Hair, R.A.J.: Leaving group and gas phase
neighboring group effects in the side chain losses from protonated serine
and its derivatives. J. Am. Soc. Mass Spectrom. 11, 1047–1060 (2000)
25. Mangion, I.K., Sherry, B.D., Yin, J., Fleitz, F.J.: Enantioselective syn-
thesis of a dual orexin receptor antagonist. Org. Lett. 14, 3458–3461
(2012)
26. Strotman, N.A., Baxter, C.A., Brands, K.M.J., Cleator, E., Krska, S.W.,
Reamer, R.A., Wallace, D.J., Wright, T.J.: Reaction development and
mechanistic study of a ruthenium catalyzed intramolecular asymmetric
reductive amination en route to the dual orexin inhibitor Suvorexant (MK-
4305). J. Am. Chem. Soc. 133, 8362–8371 (2011)
27. Baxter, C.A., Cleator, E., Brands, K.M.J., Edwards, J.S., Reamer, R.A.,
Sheen, F.J., Stewart, G.W., Strotman, N.A., Wallace, D.J.: The first large-
scale synthesis of MK-4305: a dual orexin receptor antagonist for the
treatment of sleep disorder. Org. Process. Res. Dev. 15, 367–375 (2011)
28. Mesleh, M.F., Hunter, J.M., Shvartsburg, A.A., Schatz, G.C., Jarrold,
M.F.: Structural information from ion mobility measurements: effects of
the long-range potential. J. Phys. Chem. 100, 16082–16086 (1996)
29. OMEGA 2.5.1.4: OpenEye Scientific software, Santa Fe, NM. Available:
http://www.eyesopen.com. Accessed 3 Mar 2017
33. Lee, C.T., Yang, W.T., Parr, R.G.: Development of the Colle-Salvetti
correlation-energy formula into a functional of the electron-density. Phys.
Rev. B. 37, 785–789 (1988)
34. Becke, A.D.: A new mixing of Hartree-Fock and local density-functional
theories. J. Chem. Phys. 98, 1372–1377 (1993)
35. Johnson, B.G., Gill, P.M.W., Pople, J.A.: The performance of a family of
density functional methods. J. Chem. Phys. 98, 5612–5626 (1993)
36. Grimme, S., Antony, J., Ehrlich, S., Krieg, H.: A consistent and accurate
ab initio parametrization of density functional dispersion correction
(DFT-D) for the 94 elements H-Pu. J. Chem. Phys. 132, 154104 (2010)
37. Grimme, S., Ehrlich, S., Goerigk, L.: Effect of the damping function in
dispersion corrected density functional theory. J. Comput. Chem. 32,
1456–1465 (2011)
38. National Institute of Standards and Technology (NIST). Proton affinity
search. Available: http://webbook.nist.gov/chemistry/pa-ser.html.
Accessed 27 July 2016
39. Wyttenbach, T., Pierson, N.A., Clemmer, D.E., Bowers, M.T.: Ion mo-
bility analysis of molecular dynamics. Annu. Rev. Phys. Chem. 65, 175–
196 (2014)
40. Kim, H., Kim, H.I., Johnson, P.V., Beegle, L.W., Beauchamp, J.L.,
Goddard, W.A., Kanik, I.: Experimental and theoretical investigation into
the correlation between mass and ion mobility for choline and other
ammonium cations in N2. Anal. Chem. 80, 1928–1936 (2008)
41. Ho, K.-M., Shvartsburg, A.A., Pan, B., Lu, Z.-Y., Wang, C.-Z., Wacker,
J.G., Fye, J.L., Jarrold, M.F.: Structures of medium-sized silicon clusters.
Nature. 392, 582–585 (1998)
30. Hawkins, P.C.D., Skillman, A.G., Warren, G.L., Ellingson, B.A., Stahl,
M.T.: Conformer generation with OMEGA: algorithm and validation