pubs.acs.

org/JPCL Letter

Unravelling Melatonin’s Varied Antioxidizing Protection of

Membrane Lipids Determined by its Spatial Distribution
Dongmei Zhang, Chu Gong, Jie Wang, Dong Xing, Lingling Zhao, Danyang Li, and Xinxing Zhang*
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ABSTRACT: The antioxidizing capability of membrane antioxidants is strongly affected by

the submolecular regions of the membrane that they locate. However, the concurrent
determination of their location in the membranes and the consequent antioxidizing effect
remains difficult. Using our field-induced droplet ionization mass spectrometry method-
Downloaded via NANKAI UNIV on September 16, 2022 at 12:42:54 (UTC).

ology, here we show the rapid determination of the antioxidation effect and the spatial
distribution of melatonin in POPC membranes. Melatonin effectively protects the
membrane lipids against hydroxyl radicals originating from the Fenton reactions in the
water phase but cannot protect the lipids against singlet oxygen generated by a lipophilic
photosensitizer in the lipid tail region (oil phase). These varied antioxidizing behaviors
indicate that melatonin dwells at the headgroup subregion of the membranes. We anticipate
that the methodology in this study can be widely utilized in the screening of antioxidants’
spatial distribution and antioxidizing efficiency, and eventually in designing novel
antioxidants that could deliver specific functions.

O xidation and antioxidation is an ongoing and never-

ending tug of war in aerobic organisms.1−6 By the intake
or biosynthesis of a large plethora of antioxidants, reactive
oxygen species (ROS) generated in vivo can be scavenged in
order to avoid or cure oxidative stress, and the latter has been
known to directly link to many adverse health effects such as
apoptosis,7 cancer,8 neurodegenerative diseases,9 aging,10
inflammation,11 and atherosclerosis.12 The antioxidizing
efficiency of these highly reducing antioxidants depends not
only on their intrinsic properties but also on their in vivo spatial
distributions, which are generally determined by the water or
lipid solubility. For example, water-soluble vitamin C
distributes in the water phase, but lipid-soluble vitamin E
and β-carotene tend to dwell in lipophilic environments such
as in the lipid membranes, and such antioxidants are
collectively known as the membrane antioxidants.13
Nevertheless, the spatial distributions of antioxidants often
do not conform to the above-described simple black-or-white
(water-or-oil) scenarios, and exceptions may occur due to the
varied electrostatic interactions with the substrate, complex
structures and conformations of the antioxidants per se, and the
fluctuating local environments such as the changing osmotic
pressures.14−19 Taking the 1-palmitoyl-2-oleoyl-snglycero-3-
phosphocholine (POPC) membrane as an example (Figure
1a), it has three subregions: the oil phase mainly occupied by
the lipid tails, the water phase where the headgroups locate,
and the interface region between them. An antioxidant
molecule could selectively bind to any of these three
submolecular regions, resulting in variable antioxidizing effects.
A good example showing the subtle effect of the antioxidants’
selective spatial distributions in these subregions of membranes
is the well-known polyunsaturated fatty acids (PUFA). Both of
eicosapentaenoic acid (EPA) and docosahexaenoic acid
(DHA) are typical PUFAs and promise to be good membrane
antioxidants. Counterintuitively, DHA cannot deliver effective
antioxidation compared to EPA in the lipid layer, because X-
ray diffraction experiments have revealed that the polyunsatu-
rated hydrocarbon chain of DHA prefers to locate in close
proximity to the headgroup region of the membrane due to its
“curly” conformation introduced by the multiple double bonds,
but EPA exhibits a relatively straight chain that inserts deeper
into the membrane and consequently delivers a better
antioxidation effect.14,15 Molecular dynamics (MD) simula-
tions have also revealed that the penetration depths of a series
of quercetin derivatives into lipid membranes strongly depend
on the molecular charge and substitutional variations, which
causes variations of their biological activities, including
oxidative stress defense.16 Depending on the osmotic
pressures, X-ray diffraction experiments indicate that curcumin
could either cover the headgroups of membranes like a layer of
carpet or insert into the membranes, which could ultimately
deliver distinct antioxidizing capabilities.17 Being amphiphilic
in nature, the location of melatonin (M), a neurotransmitter
and an antioxidant,18−20 has also been disclosed by 2D X-ray
Received: June 19, 2021
Accepted: July 27, 2021
Published: July 30, 2021

© 2021 American Chemical Society https://doi.org/10.1021/acs.jpclett.1c01965

7387 J. Phys. Chem. Lett. 2021, 12, 7387−7393
The Journal of Physical Chemistry Letters
Figure 1. (a) Lipid membrane arrangement using POPC as an example. (b) A schematic drawing of the spatial distribution of melatonin relative to
the lipid layer and how the distribution affects its antioxidizing effect against ROS generated from either the water phase or the oil phase. (c) The
FIDI-MS setup and the experimental procedure.
diffraction, nuclear magnetic resonance (NMR), MD simu-
lations, as well as neutron diffraction studies to be in the
headgroup subregion of lipid layers.21−23 MD simulations
show that the positioning of M, especially the indole moiety
that is vulnerable to oxidation, locates at the headgroup region
and shows a sharp boundary with the lipid tail region.21
The above discussions have pointed out that the spatial
distributions of antioxidants throughout the subregions of
membranes play an important role in determining the
effectiveness of the oxidative stress defense. On the technical
front, X-ray diffraction,17,21 NMR,22 neutron diffraction,23 and
dialysis equilibrium24 techniques have been utilized to reveal
the locations of the antioxidants relative to the lipid
membranes. MD simulation provides another powerful in-
silico tool to understand and predict the molecule−membrane
interaction at the molecular level.16,25 However, the character-
ization of the consequent antioxidizing effects resulting from
the varied spatial distributions requires a different set of
analytical techniques such as chromatographic methods.26 In
view of the above discussions, a facile, in situ technique that
can evaluate the antioxidizing effect by directly sampling the
oxidation products of membranes initiated by various ROS and
can detect the spatial distribution of the antioxidants in the
subregions of membranes seems to be in need for the
mechanistic studies of antioxidation processes and the future
development of novel antioxidants that has high specificity
targeting a certain subregion.
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In this study, we adopt our unique home-built field-induced
droplet ionization mass spectrometry (FIDI-MS) methodology
(Figure 1c), which is capable of selective “online” in situ
sampling of lipid monolayer membranes and their products
that reside at the air−water interface right after the reactions
without any sample handling or transfer and suffers minimal
influence from the bulk of the solution.27−32 We present the
antioxidizing protection of POPC membranes by M against the
hydroxyl radicals (•OH) generated by the Fenton reaction in
the water phase and singlet oxygen (1O2, SO) generated by a
lipophilic photosensitizer, 8-(4-chloromethylphenyl)-2,6-diio-
do-1,3,5,7-tetramethyl dipyrrometheneboron difluoride (DI-
BODIPY), in the lipid tail region (oil phase). Detailed
experimental procedures are provided in the Supporting
Information (SI). The structures of all the key molecules
mentioned are provided in Figure S1. Previous studies21−23
have evidenced that M prefers to bind to the headgroup region
of the lipid layer (Figure 1b). One could postulate that this
special spatial arrangement should have profound influence on
the antioxidizing effect of M: ROS generated in the water
phase has to oxidize the M protection layer first before it can
reach the double bonds in the lipid hydrocarbon chains,
exhibiting a good antioxidizing effect against oxidation initiated
in the water phase (Figure 1b). But both of the double bonds
in the lipid hydrocarbon chains and melatonin are available to
be oxidized by the ROS generated in the lipid tail region,
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The Journal of Physical Chemistry Letters
exhibiting a poor antioxidizing effect against oil-phase-initiated
oxidation (Figure 1b).
To test these thoughts, we first investigate the antioxidizing
protection of POPC membrane by M against •OH generated
by the Fenton reaction, Fe2+ + H2O2 → Fe3+(OH−) + •OH,
from the water phase. The Fenton reaction is triggered and
enhanced by a 365 nm UV light (1.43 × 10−4 W).33 If the
relative locations of M and POPC indeed conform to the
scenario described in Figure 1b, M should be extensively
oxidized and exhibit effective antioxidation protection for
POPC. Figure 2a presents the FIDI mass spectra showing the
Figure 2. (a) FIDI mass spectra of the 1 min oxidation products of
POPC mediated by •OH from the Fenton reactions in the water
phase with different concentrations of M. Products are shaded in red.
(b) Oxidation product percentage of POPC in (a) as a function of M
concentrations. (c) A typical mass spectrum showing the 1 min
oxidation products of M by Fenton reactions.
oxidation products of POPC (red shaded) after 1 min
exposure to UV light. The addition of M significantly decreases
the oxidation of POPC, and no obvious products are observed
when the concentration of M reaches 40 μM, thus presenting a
good antioxidizing effect. The oxidation products of POPC at
the higher m/z side compared to the parent cation correspond
to the CC double bond in the oleyl chain being oxidized
into a carbonyl group (+16 Da) or other saturated C−H bonds
being oxidized by adding carbonyl (+14 Da) or hydroxyl
groups (+16 Da). The products at the lower m/z side
correspond to the CC double bond in the oleyl chain being
cleaved into aldehyde groups. The mechanisms in forming
these oxidation products of POPC by •OH have been
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investigated in a previous study33 and exhibited in Figure
S2a. Figure 2b shows the clear decrease of POPC oxidation
products as a function of the concentrations of M. Figure 2c
presents the FIDI mass spectrum of the oxidation products of
M after 1 min exposure to UV light, which are 14, 16, or 32 Da
more than the parent ion (vide infra). The mass spectra of
POPC and M are shown separately in Figure 2b and 2c in
order to clearly display the oxidation products. The total ion
chromatography (TIC) spectrum with M, POPC, and their
•
OH oxidation products is presented in Figure S3a.
We next discuss the oxidation initiated by SO generated by
the photosensitizer DI-BODIPY in the lipid tail region (oil
phase). DI-BODIPY is a lipophilic photosensitizer that
efficiently generates SO due to the enhanced intersystem
crossing (ISC) rate as a result of the high spin−orbit coupling
constant introduced by the two heavy iodine atoms.34 The
synthetic route, structure, UV−vis absorption spectrum of DI-
BODIPY, and the output (1.68 × 10−5 W) of the green laser
pointer (532 nm) used to excite it are provided in the
Supporting Information. Figure 3a shows the oxidation
Figure 3. (a) FIDI mass spectra of the 2 min oxidation products of
POPC mediated by SO generated in the oil phase with different
concentrations of M. Products are 32 Da more than the parent ions.
(b) Oxidation product percentage of POPC in (a) as a function of M
concentrations. (c) A typical mass spectrum showing the 2 min
oxidation products of M by SO.
products of POPC by SO with the presence of different
concentrations of M. Only the products 32 Da more than the
parent protonated or sodiated POPC are observed at m/z 793
and 815, corresponding to the well-known peroxidation
product POPC-OOH from the reaction between SO and
olefins (Figure. S2b).31 Surprisingly, the extent of POPC
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Figure 4. Proposed mechanisms for the oxidation of M by •OH (a) and SO (b). The products in red boxes are those observed in the mass spectra.
The collision-induced dissociation spectra of these products are provided in Figure S7.

oxidation stays almost unchanged with increasing M
concentrations (less than 5% fluctuation in Figure 3b),
suggesting that M does not deliver antioxidation in this case,
which agrees well with the situation depicted in Figure 1b.
Figure 3c presents the FIDI mass spectrum of the oxidation
products of M after 2 min oxidation by SO, which also displays
extensive oxidation (vide infra). The TIC spectrum with M,
POPC and their SO oxidation products is presented as Figure
S3b.
Even though it is not the major topic of this study, here we
briefly discuss the oxidation mechanisms by •OH and SO. The
mechanisms for POPC oxidation have been previously
reported31,33 and provided in Figure S2. The proposed
mechanisms for the oxidation of M by •OH and SO are
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exhibited in Figure 4a and 4b, respectively. It is been reported
that •OH preferentially attacks the C2, C3, and C7 positions of
the indole moiety of M (Step 1 in Figure 4a),20,35,36 resulting
in 2-hydoxy-, 3-hydroxy-, and 7-hydroxymelatonin, 16 Da
more than the parent ion (m/z 249). 3-Hydroxymelatonin is
further followed by Step 2, where the N atom on the amide
group adds to C2 through a nucleophilic addition, yielding a
cyclic 3-hydroxymelatonin molecule. The cyclic 3-hydroxyme-
latonin molecule can further be oxidized to form the well-
known N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK)
product (Step 3), which is 32 Da more than the parent
ion.37−40 Steps 1−3 have been very-well documented in
previous studies,35−40 however, to the best of our knowledge,
this study is the first that observes the +14 Da product at m/z
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J. Phys. Chem. Lett. 2021, 12, 7387−7393
The Journal of Physical Chemistry Letters
247. We postulate that the m/z 247 product is attributable to
an H+ catalyzed Aldol reaction that yields an α,β-unsaturated
ketone (since both the Fenton system and air−water interface
are acidic), resulting in the loss of H2O compared to AFMK
(Step 4).
We next discuss the oxidation mechanisms of M by SO. Step
5 depicts the addition of SO to C2C3 of M to form a
dioxetane intermediate, which readily cleaves into AMFK41−43
(Step 6, +32 Da), a same product as that in Step 3. AMFK
further undergoes the Aldol reaction to give the m/z 247
product again (+14 Da, Step 7). SO could also add to C3 of M
(Step 8), which shifts the position of the double bond and
yields a 3-hydroperoxymelatonin molecule, and the latter
undergoes a similar nucleophilic addition as in Step 2 to yield a
cyclic 3-hydroperoxymelatonin molecule (Step 9). The cyclic
3-hydroperoxymelatonin molecule could then rearrange itself
in Step 10 and yield two products, one again the AFMK
molecule, the other an N-acetyl-N-formyl-5-methoxykynur-
amine (AFMK-2) molecule.43 The fact that the collision-
induced dissociation (CID) mass spectrum of the m/z 265
product from the SO oxidation is more complicated than that
from the •OH confirms the coexistence of AFMK and AFMK-
2. The CID spectra of all the oxidation products of M are
provided in Figure S7. These mechanistic steps have also been
very-well documented in previous studies41−43 except the ones
forming the m/z 247 product.
It is worth mentioning that Bolmatov et al.44 reported that
melatonin was able to stabilize the phase separation and lipid
rafts in lipid membranes even at 45 °C. In a previous study of
ours that also used FIDI-MS as the main probe,29 we
discovered that the formation of lipid rafts stiffened the lipid
membranes, lowered the permeability of potent oxidizers into
the membranes, and delivered a physical antioxidizing effect.
Taken together, the addition of melatonin might be able to
bring dual protection of lipid membranes: the stabilization of
lipid rafts provides a physical protection and the high reducing
nature of melatonin provides a chemical protection, manifest-
ing the important biophysical and biochemical roles of
melatonin in vivo. As a result, the interaction of melatonin
and lipid rafts and the consequent protection effect should be a
great FIDI-MS study in the near future.
To conclude, using our unique home-built FIDI-MS
methodology, we have conducted a mechanistic survey of
melatonin’s antioxidation against •OH and SO, which is
strongly determined by the subregions of membranes where
the antioxidant prefers to locate. It is demonstrated that
melatonin prefers to dwell at the submolecular headgroup
region of the lipid membranes, consequently, it can effectively
protect the lipid membranes against the oxidizers that originate
from the water phase but cannot protect the membrane lipids
against the oxidizers generated in the oil phase (lipid tail
region). Moreover, the chemical identities and structures of the
oxidation products of both of the substrates and the
antioxidants can also be easily identified. We anticipate that
the methodology developed in this study can be widely utilized
in the screening of the spatial distribution of membrane
antioxidants, assessing the consequent antioxidizing effect,
unraveling the mechanisms of oxidation and antioxidation
reactions, and eventually designing novel antioxidants that
could specifically target a certain subregion of biological
systems.
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■
pubs.acs.org/JPCL Letter
ASSOCIATED CONTENT
*
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jpclett.1c01965.
Experimental procedures, structures of all the chemicals,
POPC oxidation mechanisms by •OH and SO, TIC
spectra of the oxidation products, synthetic route of DI-
BODIPY, UV−vis absorption spectrum of DI-BODIPY,
the output of the green laser pointer, and CID spectra
(Figures S1−S7) (PDF)
■
AUTHOR INFORMATION
Corresponding Author
Xinxing Zhang − College of Chemistry, Key Laboratory of
Advanced Energy Materials Chemistry (Ministry of
Education), Renewable Energy Conversion and Storage
Center (ReCAST), Frontiers Science Center for New Organic
Matter, Nankai University, Tianjin 300071, China;
orcid.org/0000-0001-5884-2727; Email: zhangxx@
nankai.edu.cn
Authors
Dongmei Zhang − College of Chemistry, Key Laboratory of
Advanced Energy Materials Chemistry (Ministry of
Education), Renewable Energy Conversion and Storage
Center (ReCAST), Frontiers Science Center for New Organic
Matter, Nankai University, Tianjin 300071, China
Chu Gong − College of Chemistry, Key Laboratory of
Advanced Energy Materials Chemistry (Ministry of
Education), Renewable Energy Conversion and Storage
Center (ReCAST), Frontiers Science Center for New Organic
Matter, Nankai University, Tianjin 300071, China
Jie Wang − College of Chemistry, Key Laboratory of Advanced
Energy Materials Chemistry (Ministry of Education),
Renewable Energy Conversion and Storage Center
(ReCAST), Frontiers Science Center for New Organic
Matter, Nankai University, Tianjin 300071, China
Dong Xing − College of Chemistry, Key Laboratory of
Advanced Energy Materials Chemistry (Ministry of
Education), Renewable Energy Conversion and Storage
Center (ReCAST), Frontiers Science Center for New Organic
Matter, Nankai University, Tianjin 300071, China
Lingling Zhao − College of Chemistry, Key Laboratory of
Advanced Energy Materials Chemistry (Ministry of
Education), Renewable Energy Conversion and Storage
Center (ReCAST), Frontiers Science Center for New Organic
Matter, Nankai University, Tianjin 300071, China
Danyang Li − College of Chemistry, Key Laboratory of
Advanced Energy Materials Chemistry (Ministry of
Education), Renewable Energy Conversion and Storage
Center (ReCAST), Frontiers Science Center for New Organic
Matter, Nankai University, Tianjin 300071, China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jpclett.1c01965
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
X.Z. acknowledges the National Natural Science Foundation of
China (22003027), the National Key R&D Program of China
(2018YFE0115000), the NSF of Tianjin City
https://doi.org/10.1021/acs.jpclett.1c01965
J. Phys. Chem. Lett. 2021, 12, 7387−7393
The Journal of Physical Chemistry Letters
(19JCYBJC19600), and the Frontiers Science Center for New
Organic Matter at Nankai University (Grant Number
63181206).
■
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