A Synthetic Isoprenoid Lipoquinone, Menaquinone-2, Adopts a Folded Conformation in Solution and at a Model Membrane Interface

Article abstract of DOI:10.1021/acs.joc.7b02649

Menaquinones (naphthoquinones, MK) are isoprenoids that play key roles in the respiratory electron transport system of some prokaryotes by shuttling electrons between membrane-bound protein complexes acting as electron acceptors and donors. Menaquinone-2 (MK-2), a truncated MK, was synthesized, and the studies presented herein characterize the conformational and chemical properties of the hydrophobic MK-2 molecule. Using 2D NMR spectroscopy, we established for the first time that MK-2 has a folded conformation defined by the isoprenyl side-chain folding back over the napthoquinone in a U-shape, which depends on the specific environmental conditions found in different solvents. We used molecular mechanics to illustrate conformations found by the NMR experiments. The measured redox potentials of MK-2 differed in three organic solvents, where MK-2 was most easily reduced in DMSO, which may suggest a combination of solvent effect (presumably in part because of differences in dielectric constants) and/or conformational differences of MK-2 in different organic solvents. Furthermore, MK-2 was found to associate with the interface of model membranes represented by Langmuir phospholipid monolayers and Aerosol-OT (AOT) reverse micelles. MK-2 adopts a slightly different U-shaped conformation within reverse micelles compared to within solution, which is in sharp contrast to the extended conformations illustrated in literature for MKs.

Full text of DOI:10.1021/acs.joc.7b02649

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Article
A synthetic isoprenoid lipoquinone, menaquinone-2, adopts a
folded conformation in solution and at a model membrane interface
Jordan T. Koehn, Estela S. Magallanes, Benjamin J. Peters, Cheryle N. Beuning, Allison
A. Haase, Michelle J. Zhu, Christopher D. Rithner, Dean C. Crick, and Debbie C. Crans
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.7b02649 • Publication Date (Web): 23 Nov 2017
Downloaded from http://pubs.acs.org on November 24, 2017
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A synthetic isoprenoid lipoquinone, menaquinone-2, adopts a folded conformation in
solution and at a model membrane interface
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Jordan T. Koehn , Estela S. Magallanes , Benjamin J. Peters , Cheryle N. Beuning , Allison A.
†
†
†
‡,§
†,‡*
Haase , Michelle J. Zhu , Christopher D. Rithner , Dean C. Crick and Debbie C. Crans
†
Chemistry Department, Colorado State University, Fort Collins, Colorado 80523, United States.
Email: Debbie.Crans@colostate.edu
‡
Cell and Molecular Biology Department, Colorado State University, Fort Collins, Colorado
80523, United States.
§
Department of Microbiology, Immunology and Pathology, Colorado State University, Fort
Collins, Colorado 80523, United States.
ABSTRACT: Menaquinones (naphthoquinones, MK) are isoprenoids that play key roles in the
respiratory electron transport system of some prokaryotes by shuttling electrons between
membrane-bound protein complexes acting as electron acceptors and donors. Menaquinone-2
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(
MK-2), a truncated MK, was synthesized and the studies presented herein characterize the
conformational and chemical properties of the hydrophobic MK-2 molecule. Using 2D NMR
spectroscopy, we established for the first time that MK-2 has a folded conformation defined by
the isoprenyl side-chain folding back over the napthoquinone in a U-shape, which depends on
the specific environmental conditions found in different solvents. We used Molecular Mechanics
to illustrate conformations found by the NMR experiments. The measured redox potentials of
MK-2 differed in three organic solvents, where MK-2 was most easily reduced in DMSO, which
may suggest a combination of solvent effect (presumably in part because of differences in
dielectric constants) and/or conformational differences of MK-2 in different organic solvents.
Furthermore, MK-2 was found to associate with the interface of model membranes represented
by Langmuir phospholipid monolayers and reverse micelles. MK-2 adopts a slightly different U-
shaped conformation within reverse micelles compared to within solution, which is in sharp
contrast to the extended conformations illustrated in literature for MKs.
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INTRODUCTION
Isoprenoids are among the most numerous
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and diverse compounds found in nature.
It was reported that 55,000 of these
naturally occurring compounds had been
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identified by 2007 ; an estimate that
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increased to 70,000 in 2015. Classes of
isoprenoids include lipoquinones, sterols,
carotenoids, prenylated proteins, dolichols,
monoterpenes, and sesquiterpenes. These
Figure 1. Structure of menaquinone-9 (MK-9), a
representative MK found in Mycobacterium
tuberculosis (top) and MK-2, a simplified MK analog
bottom), is also shown. Protons are labelled on MK-
for spectral interpretation.
(
2
compounds are involved in diverse functions such as electron transport, hormone
function, membrane structure and fluidity, vision, photoprotection, insect reproduction,
fragrance and defense. The solution structures of many of these compounds, such as the
sterols, are well studied. However, the conformations of long linear polymers of isoprene
units such as those found in the side-chains of lipoquinones involved in bacterial and
4-7
eukaryotic electron transport systems have not been characterized. Lipoquinones are
small hydrophobic molecules that shuttle electrons between the membrane-bound
protein complexes acting as electron acceptors and donors in the respiratory electron
transport systems (ETS). Two major structural groups of lipoquinones are recognized;
ubiquinones (or benzoquinones, UBQ) typically found in eukaryotes and Gram-negative
prokaryotes and menaquinones (or naphthoquinones, MK) typically found in Gram-
positive prokaryotes, including many pathogens such as Mycobacterium tuberculosis.
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The roles and applications of lipoquinone derivatives are currently being explored where
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a range of different approaches are used for combatting human diseases.
This
manuscript aims to characterize the conformation of a synthetic truncated MK analog in
organic solution and within a model membrane interface as well as document the redox
potential of these systems accompanying the conformational studies.
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Here, we use an abbreviation system where MK with two isoprene units is MK-2 and
with nine isoprene units is MK-9, Figure 1. Naturally occurring lipoquinones are
characterized by the presence of an isoprenyl side-chain of varying length from 1-14
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1,22
isoprene units
depending on the species of origin; a characteristic long used to assist
taxonomic efforts. Textbooks often represent UBQ and MK as “Q” or “MQ” in illustrations
of the ETS and in primary literature they are generally shown in an extended
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,23,24
conformation, Figure 1.
The extended conformation of these molecules seems
highly unlikely in light of the one-step cyclization of squalene epoxide, an isoprenoid
compound containing six isoprene units, to form the protosterol cation, which contains
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the basic steroidal ring system with eight chiral centers.
Woodward reported the
cyclization of this polyisoprenoid squalene epoxide forming only one stereoisomer out of
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the possible 256 (2 ) in 1953.
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The stereospecificity of this reaction requires
preorganization of the epoxide before protonation, and such a conformer is not the
extended conformation commonly depicted in the literature.
Small angle X-ray diffraction experiments with the isoprenoid dolichol, an alcohol
with 18-20 isoprene units, indicated that the molecule adopted a helical structure in
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organic solvent. The conformation of the isoprenoid moiety in MK derivatives is likely
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important to its function as an electron transfer agent and other biological roles. Although
nothing is known about the conformation of MK derivatives, limited conformational
analysis is available on different classes of compounds including alkanes, alkenes, and
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8-42
fatty acids with much simpler structures.
The all-trans conformations of alkanes are
generally considered the most stable and therefore, it is expected that the most prevalent
28
conformation of an alkane is the fully extended alkyl chain. A few studies have been
reported with simple alkanes and a range of other, more complex compounds with large
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alkane-components such as in polyenes and fatty acids.
The U-shape in fatty acids
were described when bound to a fatty acid binding protein (FABP), where the alkyl chain
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region of two fatty acids, palmitic and oleic acids, folds over in the shape of a U.
008, X-ray crystal structure analysis, as well as NMR spectroscopy, also supported the
U-shaped conformation of amphiphilic alkyl chains bound to a synthetic receptor where
In
2
the folded C
8
, C10, and C12 conformations have anticlinal as well as gauche
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arrangements.
Based on these few examples, we hypothesized that MKs will adopt
folded conformations depending on specific environmental conditions.
In this manuscript, we describe the synthesis and characterization of a truncated MK
derivative with two isoprene units. MK-2 (Figure 1) can be characterized in detail and
serves as a representative MK reference compound as it is the simplest MK containing a
napthoquinone and repeating isoprenyl side-chain allowing comparison to more complex
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MK-derivatives found in various microorganisms.
First, the conformation of MK-2
was investigated in organic solvents (polar and hydrophobic). Next, because we are
ultimately interested in the function of these derivatives, we measured the
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electrochemical potential of MK-2 in the organic solvents where the conformations were
determined. Finally, the location and conformation of MK-2 in simple membrane model
systems was examined. Combined, our results show that MK-2 folds into a U-shaped
conformation in solution contrary to common perception, and that a folded, slightly
different U-shaped conformation also exists in the presence of a simple model lipid
membrane interface.
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RESULTS AND DISCUSSION
Synthesis of MK-2. MK-2 was synthesized using a procedure in literature (Scheme
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1).
First, menadione 1 was reduced to the corresponding menadiol 2 using aqueous
sodium dithionite. Geraniol 3 was coupled to menadiol 2 using the Lewis acid catalyst,
boron trifluoride. This preparation produced a yield of 20% overall, which is lower than
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the reported MK-2 preparation with the deuterated analog in literature. The lower yield
appears to be due to extensive alkylation at the C2 position instead of the desired C3
position; however, this side product is readily separated using column chromatography.
Highly pure material yields a yellow solid upon crystallization at -20 °C and retains this
Scheme 14.5-46Synthetic route to prepare MK-2 4, from menadiol 2 and geraniol 3 using Lewis acid catalyst
conditions.
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state when warmed to room temperature, while material with impurities does not
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crystallize and remains a red oil.
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D H NMR Spectroscopic Studies of MK-2 in Different Solvents. MK-2 was first
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characterized using 1D H NMR spectroscopy. Figure 2 shows H NMR spectra of MK-2
in isooctane (2,2,4-trimethylpentane), d -pyridine, d -benzene, d -acetonitrile, d -DMSO,
and D O. The observed chemical shifts of MK-2 vary dramatically in the different solvents
shown. Hydrophobic solvent environments including aliphatic (isooctane) and aromatic
-pyridine and d -benzene) as well as hydrophilic (d -acetonitrile, d -DMSO, and
O/D O) solvent environments generate very different spectroscopic signatures. For
example, the pairs of aromatic protons, H /H and H /H , are significantly different in the
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(d
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H
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a
b
c
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two different classes of solvents. This observed variation in chemical shift could be
attributed to conformational changes of MK-2 in the various solvent environments
investigated, alterations of the electronic state due to interaction with the solvent, or most
1
likely, a combination of both. Focusing on the H NMR spectra of MK-2 in the three
hydrophilic solvents, d
similarities are observed (Figure 2). The MK-2 spectrum in D
more scans to produce a reasonable spectrum because of the poor solubility in D
proton chemical shifts generally are observed further up field in D O, presumably
indicative of more aggregation in this solvent. The alkene H and H proton signals are
not observed in the D O spectrum because of overlap with the HOD peak. However, the
HOD signal in the d -DMSO spectrum is observed at 3.3 ppm, which allows for
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-DMSO, and D
2
O, some differences and
O required significantly
O. The
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h
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h i 6
observation of the alkene protons, H and H, at 4.9 ppm. The spectrum of MK-2 in d -
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MK-2 spectrum in D O. The aromatic protons, H
/H signals in both solvents. This suggests that these protons remain in similar
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chemical environments in these two solvent environments. Combined, these three
spectra provide a representation of the properties of the MK-2 in a hydrophilic solvent
environment.
Next, the aromatic and alkene protons were examined in the hydrophobic solvents,
isooctane, d
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-pyridine, and d
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-benzene (Figure 2). Here, protons H
a
, H
b
, Hc,
H
d,
H
h
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H
i
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h
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Figure 2. 1D H NMR (400 MHz) spectra of MK-2 in hydrophilic (d
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3 2
-acetonitrile, and D O) and
hydrophobic [isooctane (2,2,4-trimethtylpentane), d
corresponds to MK-2 structure in Figure 1.
-pyridine, and d -benzene] solvents. Proton peak text labelling
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both further downfield compared to the signals in hydrophilic solvents and most
interestingly, the two alkene protons become non-chemical shift equivalent in d -pyridine.
This contrasts with the aliphatic protons in the spectrum in d -benzene, which are like
those in d -DMSO except for H , H . The changes in the chemical shifts are
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q
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most distinctive in the aromatic and alkene protons, suggesting that there are some
major differences in their respective environments. Together, these differences strongly
indicate that MK-2 is sensitive to the solvent environment. However, information from 2D
NMR techniques is needed to elucidate if this change in chemical shift is due to
conformational variations or solvent effect.
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H- H 2D NOESY and H- H 2D ROESY NMR Spectroscopic Studies of MK-2 in
-DMSO and d -pyridine. Differences between the conformations observed in d
DMSO vs. d -pyridine are likely manifested by the conformation minimizing the
d
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unfavorable interactions with the solvent environment and maximizing favorable
intramolecular interactions with the isoprenyl side-chain. To investigate the conformation
of MK-2 in organic solvents, we utilized two different but complementary 2D NMR
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methods, H- H 2D NOESY and H- H 2D ROESY. Figure 3 shows the H- H 2D NOESY
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Figure 3. H- H 2D NOESY and H- H 2D ROESY NMR (400 MHz) spectra of 20 mM MK-2 in d
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pyridine at 26 °C. (A) Full H- H 2D NOESY NMR spectrum of MK-2 in d
6
-DMSO, (B) Partial H- H 2D NOESY NMR
1
1
1
1
spectrum of MK-2 in d
6
-DMSO, (C) Partial H- H 2D ROESY NMR spectrum of MK-2 in d
6
-DMSO, (D) Partial H- H
-pyrdine, (E) Partial H- H 2D ROESY NMR spectrum of MK-2 in d -pyrdine
F) Partial H- H 2D ROESY NMR spectrum of MK-2 in d -pyridine illustrating stacking interactions. Blue intensity
1
1
2
D NOESY NMR spectrum of MK-2 in d
5
5
1
1
(
5
contours represent negative NOE’s or ROE’s and red intensity contours represent positive NOE’s or ROE’s. A
standard NOESY pulse sequence was used consisting of 200 or 256 transients with 16 scans in the f1 domain using
a 500 ms mixing time and 1.5 sec. relaxation delay. A standard ROESY pulse sequence was used consisting of 200
or 256 transients with 16 scans in the f1 domain using a 400 ms mixing time and 2.0 sec. relaxation delay. The
structure of MK-2 is shown with a proton labelling scheme key. Green arrows indicate proton H
cross peaks differed the most between the two solvents studied.
w
, where the observed
6 5
and ROESY NMR spectra of MK-2 in d -DMSO and portions of the spectrum in d -
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pyridine. Figure 3A shows the complete H- H 2D NOESY spectrum of MK-2 in d
and Figure 3B-C shows zoomed in regions of interest in the NOESY and ROESY spectra
collected in d -DMSO, respectively (See Figure S3-4 for full spectra). Figure 3D-F shows
zoomed in regions of interest in the NOESY and ROESY spectra collected in d -pyridine,
respectively (see Figure S6-7 for full spectra). Figure 3B and Figure 3D illustrates that
alkene protons, H and H, are in similar environments in d -pyridine and d -DMSO due to
the observance of similar NOE cross peaks. In both solvents, Figure 3B and Figure 3D
show NOE cross peaks illustrating that H and H interact with H /H , H , H and H
Figure 3B and Figure 3D show NOE cross peaks demonstrating proton H interacts with
protons H /H and H /H in both d -DMSO and d -pyridine. Figure 3C and Figure 3E
shows partial H- H 2D ROESY NMR spectra where the focus is on proton H in d
DMSO and d -pyridine (see Figure S4 & S7 for full spectra and Figure S9 for NOE/ROE
correlation traces (slices)). Figure 3B-C for MK-2 in d -DMSO, NOE and ROE cross
peaks indicate the H proton interacts with Hq, , and H . However, the lack of ROE
cross peaks in Figure 3E shows H does not interact with H or H in d -pyridine
observed cross peaks between H and H or H in d -pyridine are likely due to TOCSY
6
-DMSO
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h
i
n
m
q
r
z
.
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h
i
m
n
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1
1
w
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-
5
6
w
H
r,
H
y
z
w
y
z
5
(
w
y
z
5
exchange). The observation of cross peaks show that there are similar interactions
between the aromatic and alkene protons in the two solvents; however, there is a twist
around the C-C bond leading to closer proximity of the H
protons in d -DMSO. The NOE and ROE cross peaks in the d
clearly supports a folded, U-shaped conformation for MK-2 in d
w
with the Hq,
-DMSO spectrum in Figure
-DMSO. However,
r, x y
H H , and H
6
6
3
6
evidence of a folded conformation for MK-2 is not as definitive in d
5
-pyridine due to the
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Figure 4. MK-2 conformations generated using MMFF94 calculations to illustrate the conformations elucidated by
1
1
the NMR studies. (A) Illustrates the MK-2 conformation in d
NMR spectral data cross peak interactions (66.8 kcal/mol, and internuclear distance H
6
-DMSO determined from H- H 2D NOESY and ROESY
-H : 2.6 Å), (B) Illustrates a
-pyridine consistent with the H- H 2D NOESY and ROESY NMR
spectral data cross peak interactions (66.7 kcal/mol, and internuclear distance H -H : 6.1 Å). See Supporting
Information for a table of selected proton to proton distances for conformations A and B.
w
y
1
1
potential U-shaped MK-2 conformation in d
5
w
y
lack of NOE and ROE cross peaks between H
shaped conformation in d -pyridine, it would have a conformation as shown in Figure 4B,
where the distance between H
and ROESY NMR experiment. This conformation for MK-2 in d
other NOE/ROE cross peaks observed (i.e. H to H , H to H or H
illustrates π-π stacking between pyridine and part of the napthoquinone ring, which could
possibly explain the lack of interaction between H and H or H
w y z
and H or H . If MK-2 did adopt a U-
5
1
1
w
and H
y
/H
z
is beyond observation in the H- H 2D NOESY
-pyridine is based on
, H to H ) Figure 3F
5
w
h
w
q
r
w
x
w
y
z
.
Illustrating MK-2 Conformations using Molecular Mechanics. MK-2 has a short
repeating isoprenyl chain (C10) but enough carbons with numerous degrees of rotational
freedom; therefore, even the truncated version of MK, MK-2 can assume many different
conformations. Using Molecular Mechanics calculations, we explored the energy surface
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and visualized specific conformations defined by the H- H 2D NOESY and ROESY NMR
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experiments of MK-2 in d -DMSO and d -pyridine. Specific conformations were
5
6
generated based on distances imposed by the NMR data and then energy optimized to
produce realistic bond lengths and angles for these conformations. Finally, an energy
was calculated to assure that the conformation was at a reasonable energy. Longer
calculations generally lead to rotations around bonds and conformations not exactly as
those suggested from the NMR data (see below and Supporting Information). The intent
with the Molecular Mechanics calculations was to illustrate conformations corresponding
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to the H- H 2D NOESY and ROESY NMR spectral data in d
1
6 5
-DMSO and d -pyridine
constrained by all cross peak interactions observed as well as to provide visual aids
(
Figure 4). One of the low energy conformations generated for MK-2 has a conformation
Figure 4A, 66.8 kcal/mol, H -H : 2.6 Å) consistent with the NOESY and ROESY spectral
-DMSO. Figure 4B illustrates a potential conformation of MK-2
that is consistent with NOESY and ROESY spectral parameters observed in d -pyridine
(66.7 kcal/mol, H -H : 6.1 Å). See Table S1 for selected proton to proton distances for
conformations seen in Figure 4A-B consistent with 2D NOESY and ROESY NMR
(
w
y
parameters observed in d
6
5
w
y
6
spectral parameters. The MK-2 conformation determined in d -DMSO adopts a folded, U-
shaped conformation.
Fewer NOE and ROE cross peaks were observed in d
folded conformation and although this does not preclude a U-shaped conformation, it
does suggest that the H -H internuclear distance is longer and outside the observable
range using the H- H 2D NOESY and ROESY NMR experiment. We based this folded
5
-pyridine that would indicate a
w
y
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conformation off other NOE and ROE cross peaks observed, which were suggestive of
folding (see Figure 3 and related discussion). However, a U-shaped conformation has
6
been observed by NMR analysis in another aromatic solvent, d -benzene, for n-
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dodecane. The U-shaped conformations shown in Figure 4 are representative of many
possible stable conformations MK-2 could adopt. For comparison, Figure S12C shows
another favorable conformation for MK-2 that has a characteristic U-shape. The energy
of the conformation shown in Figure S12C was the lowest energy we found. This
conformation was obtained by subjecting an extended MK-2 conformation to 10,000
iterations followed by an energy minimization, illustrating that non-extended
conformations are readily reached and energetically favorable.
Combined, the stability of the U-shaped conformations shown by these simple
1
calculations are in line with work reported that showed H NMR spectra of n-pentane and
n-hexane measured in polar and spherical solvents such as DMSO favor gauche
conformations as well as the reported U-shaped conformations of alkyl chains bound to
3
9,40,47
synthetic receptors.
The folded conformations demonstrated herein from
experiment for MK-2 align with reported computationally determined conformations for
4
MK analogs. However, it is contrary to the general expectation that the all-trans
8
7
,23,24,28
conformations of alkanes or alkyl components are most stable.
In addition to the U-shaped conformations seen in conformations A and B in Figure 4,
a series of alternative conformations of MK-2 (Figure S12D-E) were also generated from
the structures most often depicted by representations in the literature and then were
energy optimized. Conformations D-H in Figure S12 were generated to compare these
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conformations energetically to the two U-shaped conformations shown in Figure 4A-B.
The energies of conformations D-H in Figure S12 were all higher (~11 to ~23 kcal/mol)
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than the conformations found in d -DMSO, d -pyridine, and conformation C. Additional
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analysis of conformations and a table of selected internuclear distances of MK-2
conformations from Figure 4 and Figure S12 are given in Table S1.
Selected simple alkanes and a few other, more complex compounds with long alkyl-
segments such as in polyenes and fatty acids report that the extended and folded forms
2
9,30
are comparable in energy to the all-trans form.
The “pentane effect” for longer chains
+
destabilizes the trans conformations because of unfavorable g g arrangements, which
-
2
9,30
increases the energy by ~0.55 kcal/mol with each kink in the chain.
In contrast,
gauche arrangements are often energetically favored because of entropic
3
1,32
contributions.
and can be justified using limited sphere-considerations. Gauche arrangements tend to
favor folded conformations where the degree of folding of n-alkanes (C -C32) in solution
This is in part due to the spatial requirements that all molecules exert
3
3
5
3
depends on the strength of the dispersion force of the solvent and the degree of folding
5
34,36
increases with increasing chain length of the solute.
Other molecules that contain
alkyl chains such as fatty acids and polyethylene derivatives have also been reported as
either extended chains or in folded conformations depending on physical and
3
1,32,37,38
environmental conditions.
Conformational analysis of long chain allylic
polyunsaturated fatty acid chains using Hartree-Fock calculations showed that
conformational differences between the all-trans and helical folded structures are only a
3
few kcal/mol. Ultimately, there are a few but strongly convincing examples
7
30,39-42
that
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folded and/or U-shaped conformations are likely to become much more important than
have been recognized previously. The studies presented herein represent a new class of
compounds that adopt a U-shaped conformation, illustrating that these folded
conformations are likely to be important and may impact the biological function of these
lipoquinone systems. It is very important to recognize the molecular plasticity, which
allows the alkyl chain to assume shapes depending on the complementary structures or
influences of the immediately surrounding environment.
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Electrochemistry of MK-2 in Different Solvents. Our interest in these systems
relates to the fact that the redox potential of MK is important in shuttling electrons
between protein complexes. Although the focus of this manuscript has been on the
conformations of these compounds, we are particularly interested in redox properties
because we are aiming to investigate the function of these systems as well. For MKs to
carry out this process, MKs must associate with the transmembrane proteins involved in
4
the electron transfer processes. The electron transfer processes all take place within or
9
near the membrane interface with two one electron reduction processes sequentially
50-52
forming the radical anion (semiquinone) and then the dianion.
Even though there has
been many reports in literature on the redox potentials of MK derivatives, the origin of
5
0-52
these changes are not well understood.
These processes are particularly poorly
understood when the quinones are present in a hydrophobic molecule that is entirely or
partly located in the membrane of a biological system. Although, it is premature to
correlate conformation with redox potential, we are seeking systematic information where
redox potentials and conformations are known (i.e. within different organic solvents) and
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the electrochemical data on this system becomes an important frame of reference
because the conformational analysis has been carried out. We hypothesized that the
redox potential of MK-2 will vary based on organic solvent environment.
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Although this hypothesis would be anticipated to be confirmed, it is important to
measure how and if the magnitude of the redox potential changes as the solvent
environment changes because the function of MK-2 is tied to the redox potentials.
Initially, redox studies were planned to be carried out in DMSO, acetonitrile, and
benzene; however, because of low solubility of all of the electrolytes examined in
5
3
benzene, benzene was replaced with pyridine as a representative aromatic solvent.
1
The electrolyte tetrabutylammonium perchlorate (TBAP) was chosen because 1D H
NMR studies showed that the addition of TBAP to MK-2 solutions (solvents: d -DMSO,
-pyridine, d -acetonitrile) did not affect the observed chemical shifts of the MK-2
6
d
5
3
protons and thus no evidence for artifacts due to ion pairing would be observed (see
Supporting Information for further description of the electrochemical analysis, and see
Figure S13 for MK-2/TBAP NMR studies).
MK-2’s first electrochemical potential is the one electron reduction of the quinone to
•
semiquinone (Q/Q ) and the second is the one electron reduction of the semiquinone to
-
•
the dianion (Q /Q ), Scheme S1.
-
2-
50-52
The cyclic voltammogram’s (CVs) of MK-2 and an
+
internal standard of ferrocene (Fc /Fc) in the three solvents are shown in Figure 5. The
•
-
+
Q/Q E1/2 vs Fc /Fc for MK-2 in DMSO, CH
3
CN, and pyridine were measured to be -1.155
(±0.001) V, -1.230 (±0.003) V, and -1.331 (±0.001) V, respectively. As shown in Figure 5,
+
we included the Fc /Fc reference in the data presented. These results are consistent with
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+
•-
Figure 6. Measured E1/2 (vs Fc /Fc in V) of MK-2 Q/Q
Figure 5. Three representative cyclic voltammogram’s
(CVs) of 2 mM MK-2 in CH CN, DMSO, and pyridine.
•
-
2-
and Q /Q redox processes versus solvent. Added
lines show the distinction between each solvent for
each redox process. Each solvent was run in triplicate
with error bars shown. Student’s t test indicated the half
wave potentials of each redox process are significantly
different in each solvent (p<0.01 for Q /Q CH CN-
3
DMSO and p<0.0001 for all other comparisons). See
Supporting Information for details.
3
+
The potentials are referenced to the Fc /Fc couple (2
mM) determined in each solvent. From left to right,
•-
2-
•-
+
redox processes are Q /Q , Q/Q , and Fc /Fc. Each
sample has 0.1 M TBAP and was degassed with argon
gas for 10 minutes at ambient room temperature before
spectra were recorded. Current sweeps are in the
anodic direction from -2V to 1V and back to -2V. A 100
mV scan rate was used.
•
-
2-
•
-
+
50,54
Q/Q E1/2 vs Fc /Fc values given for 1,4-naphthoquinone.
The values determined
CN, and pyridine were
measured to be -1.863 (±0.008) V, -1.902 (±0.012) V, and -2.075 (±0.003) V, respectively
Table S2). In Figure 6, the half-wave potentials are plotted showing the trend that both
redox processes are more readily reduced in DMSO than CH CN or pyridine similar to
•
-
2-
+
experimentally for Q /Q E1/2 vs. Fc /Fc for MK-2 in DMSO, CH
3
(
3
5
4
the trend observed in literature for 1,4-napthoquinone potentials.
In summary, during the first electrochemical process producing the semiquinone, MK-2
has the most positive potential in DMSO and the most negative potential in pyridine,
3
showing MK-2 is slightly more reducible in DMSO than pyridine or CH CN. The
observation of differing redox potentials between organic solvents supports the second
hypothesis that the redox potential of MK-2 is influenced by the specific organic solvent,
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where a contributing factor to the observed
changes is due to the differences between
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the dielectric constant of each solvent. The
different organic solvents influence the
observed redox potential of MK-2 and from
the 2D NMR studies on the conformation of
MK-2, slight differences in conformation
were observed, which may suggest that a
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combination
of
solvent
effect
and
conformational differences of MK-2 may
affect redox potential.
Interaction
of
Langmuir
Phospholipid Monolayers with MK-2. The
physiologically relevant environment for
MKs is in association with membranes;
therefore, we also studied the interaction
and conformation of MK-2 with a more
Figure 7. Schematic diagram of (A) a Langmuir
phospholipid monolayer and (B) a RM present in a
microemulsion. Labelling for the RM is as follows: the
water pool (A), the Stern layer (B), the surfactant tails
direct model membrane interface,
a
(
C), and the organic solvent, isooctane (D).
phospholipid monolayer. Several membrane
models exist including Langmuir monolayers, liposomes, micelles, and reverse micelles
55-61
(RM) (Figure 7).
Each model system has advantages and disadvantages, and we
have chosen to investigate the phospholipid monolayer because of their direct
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association with a membrane and the RM system because RMs allow for molecular
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placement of MK-2 with regard to the interface.
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First, we describe our studies with Langmuir monolayers
consisting of
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phospholipids to gain insight into the interactions between two classes of phospholipids
(dipalmitoylphosphatidylcholine (DPPC) or dipalmitoylphosphatidylethanolamine (DPPE))
and MK derivatives. These studies will demonstrate how the phospholipid packing is
affected by the presence of MK derivatives as it interacts with the phospholipid interface
6
0,62-65
(Figure 7A).
Due to the low solubility of MK-2 in aqueous environments, the
Langmuir monolayer studies involve experimental protocols used for other materials with
6
6,67
low solubility in water, such as UBQ.
Figure 8 shows the change in surface pressure
as a function of area per molecule of DPPC or DPPE in the presence of various amounts
of MK-2. The resulting compression moduli as functions of surface pressure are shown in
Figure S14.
Figure 8. Compression isotherms of MK-2 films (dotted lines), DPPC or DPPE phospholipid films (solid lines), or
a 50:50 mixture of MK-2 and phospholipid (dashed line). On the left (A) are the resulting compression isotherms
of MK-2 and DPPC, while on the right (B) are the resulting compression isotherms of MK-2 and DPPE.
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The compression isotherm of DPPC shows a transition at ~8 mN/m and both DPPC
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and DPPE collapse at ~55 mN/m similar to findings reported in literature. The
compression isotherm of the 1:1 mixture of DPPC and MK-2 shows a disappearance of
the gas to liquid phase transition at 8 mN/m, suggesting there is a condensing effect of
MK-2 with DPPC at a high area per molecule/low surface pressure. Moving to DPPE, the
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:1 mixture of MK-2 with DPPE reduces the collapse pressure of the film to 50 mN/m
showing destabilization of the DPPE film. The pure MK-2 films had limited stability shown
by a maximum surface pressure of ~20 mN/m. This result suggests MK-2 forms a less
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stable film than the phospholipids.
To understand the interactions of MK-2 with DPPE
and DPPC further, the compression moduli were calculated using eq. (4) (see Supporting
Information) where the compression modulus is the measure of the compressibility of the
film.
Our results are interpreted as MK-2 having a condensing effect on DPPC phospholipid
films at high area per molecule/low pressure and destabilization of DPPE phospholipid
films. The resulting mixed films (DPPC/MK-2 or DPPE/MK-2) were also consistent with
reported UBQ suggesting similar trends where UBQ was shown to be “squeezed” out of
6
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the phospholipid interface.
In summary, MK-2 has a condensing effect on DPPC
phospholipid films, a destabilization of DPPE phospholipid films, and is most likely
6
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compressed out of the phospholipid film similar to UBQ.
Unfortunately, these
studies do not directly show whether a folded or extended conformation of MK-2 exists. A
folded conformation occupies a larger volume than the extended conformation, which
would affect packing within the phospholipid interface. Our studies do demonstrate that
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MK-2 interacts with the phospholipid model
membrane interfaces of DPPC and DPPE
even above physiologically relevant
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pressures. It is possible that the observation
of the destabilization of the DPPE
phospholipid films (not observed for DPPC)
1
Figure 9. 1D H NMR (400 MHz) spectra of MK-2’s
by the presence of the MK may suggest a aromatic protons, H , H , H , and H in D O, isooctane
a
b
c
d
2
and different sized RMs. Proton peak text labelling
corresponds to MK-2 proton labelling scheme key
found in Figure 3. H
a chemical shift upon inclusion inside RMs.
folded MK-2 conformation that does not fit in
the organization of the DPPE film (possibly
a b c d
, H , H , and H protons undergo
related to packing of the phospholipid).
1
D H NMR Spectroscopic Studies of MK-2 in RMs. Our ultimate objective was to
1
characterize the molecular details of MK-2’s association with membrane-like interfaces.
This characterization includes determining the location and conformation of MK-2;
6
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therefore, the RM model system was investigated (Figure 7B).
In the RM, a
ternary microemulsion system forms when a surfactant is dissolved in an organic solvent
and the addition of water creates nanosized water droplets encased in
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surfactant.
This dynamic, yet simple, model can provide molecular detail on
location and conformation of MK at an interface using NMR spectroscopy, whereas the
use of phospholipids such as DPPC have overlapping proton signals with key MK proton
7
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signals making interpreting conformational information very complex.
RM systems
provide information regarding the location, conformation, and physical interactions of MK
with lipid films and potentially membranes, which allowed us to examine our third
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hypothesis that the membrane interface influences the conformation of the MK
derivative.
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To explore the location of MK-2 at a lipid/water interface further, studies were
conducted using a simple microemulsion interfacial system, which allows for identification
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of molecular placement. Figure 9 shows a stack plot of 1D H NMR spectra for the
aromatic protons (H
solutions of MK-2 containing RMs (D
: 4, 8, 12, 16, and 20 where w
AOT/isooctane RM. The aromatic MK-2 protons in the AOT/isooctane RMs are similar to
the signals observed in isooctane but very different from the signals observed in D O.
The distance between H /H and H /H is 0.52 ppm in isooctane, 0.47 ppm in the RM,
and 0.27 ppm in D O. This suggests that the environment of the MK-2 protons in the
AOT/isooctane RMs is more like an environment in isooctane than D O, where MK-2 is
no longer in isooctane but associating with the AOT molecules. The signals from the
/H protons in the AOT/isooctane RMs do change slightly as the RM size increase or
a
/H
b
and H
c
/H
d
) of MK-2 in solutions of D
O/AOT/isooctane microemulsions) of various sizes
=[D O]/[AOT]) and for comparison with an empty
2
O, isooctane, or isooctane
2
(w
0
0
2
2
a
b
c
d
2
2
H
c
d
decreases. This observation is also consistent with MK-2 penetrating and residing in the
interface.
To verify that the RMs formed in the samples used and that the properties of the
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1,63,70,77,78
samples are consistent with previous studies
, Dynamic Light Scattering (DLS)
experiments were carried out. These studies confirmed the formation of RMs, and that
the RM did not change significantly upon the addition of MK-2. This was carried out by
measuring the sizes in the presence and absence of MK-2 and confirming that the size
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Figure 10. Partial H- H 2D NOESY and ROESY NMR (400 MHz) spectra of MK-2 inside w
0
12 RM at 26 °C. (A)
1
1
1
1
Partial H- H 2D NOESY NMR spectrum in w
0
12 RM and (B) Partial H- H 2D ROESY NMR spectrum in w 12 RM.
0
Blue to blue proton text labelling shows MK-2 to MK-2 interactions, teal to teal proton text labelling shows AOT to
AOT interactions, and blue to teal proton text labelling shows MK-2 to AOT interactions. Blue intensity contours
represent negative NOE’s or ROE’s and red intensity contours represent positive NOE’s or ROE’s. A standard
NOESY pulse sequence was used consisting of 256 transients with 16 scans in the f1 domain using a 200 ms
mixing time and 1.5 sec. relaxation delay. A standard ROESY pulse sequence was used consisting of 256
transients with 16 scans in the f1 domain using a 200 ms mixing time and 2.0 sec. relaxation delay. See Figure 3 for
MK-2 proton labelling scheme key and Figure 12 for AOT proton labelling scheme key.
63
was as those reported previously (data not shown). Overall, these results are
consistent with MK-2 penetrating the RM model membrane interface; however, 2D
studies are described below to get more information regarding the placement and
conformation of MK-2 within the RM system.
1
1
H- H 2D NOESY and ROESY NMR Spectroscopic Studies of MK-2 in a RM Model
Membrane System. To provide further evidence regarding the location, orientation, and
1
conformation of MK-2 within RMs, we obtained the H- H 2D NOESY NMR spectrum and
1
1
1
0
the H- H 2D ROESY NMR spectrum of MK-2 in a w 12 RM, Figure 10 and Figure 11.
1
1
Figure 10 shows a partial H- H 2D NOESY and ROESY NMR spectra indicating both
AOT and MK-2 protons, whereas Figure 11 focused in on selected proton interactions
specifically in the NOESY spectrum (see Figure S10-11 for full spectra). Figure 11A
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Figure 11.
interactions of MK-2 with the AOT surfactant tails. (A) Interactions between MK-2’s aromatic protons and AOT.
B) Interactions between MK-2 alkene protons and AOT. The blue proton text labelling shows MK-2 to MK-2
0
Partial H- H 2D NOESY NMR (400 MHz) spectra of MK-2 inside w 12 RM at 26 °C illustrating
(
interactions, teal proton text labelling shows AOT to AOT interactions and blue to teal proton text labelling shows
MK-2 to AOT interactions. Blue intensity contours represent negative NOE’s and red intensity contours represent
positive NOE’s. A standard NOESY pulse sequence was used consisting of 256 transients with 16 scans in the f1
domain using a 200 ms mixing time and 1.5 sec. relaxation delay. See Figure 3 for MK-2 proton labelling scheme
key and Figure 12 for AOT proton labelling scheme key.
shows cross peaks between the aromatic H
a
/H
tail groups (i.e. H8 and H10 protons, see Figure 12 for AOT proton labeling key).
Figure 11B shows cross peaks between alkene protons, H and H , with HOD and AOT’s
b c d 2
and H /H and parts of the AOT CH and
’
’
CH
3
i
h
H1, H1’, H3, and H3’ protons and only evidence of a weak interaction between MK-2 and
isooctane. These interactions are consistent with placement of MK-2 between the AOT
tails as shown using a depth perception drawing in Figure 12.
Regarding the conformation of MK-2 associated with the RM water-interface, the NOE
and ROE cross peaks in Figure 10 show interactions between H
and the alkene protons, H /H, which is consistent with a folded, U-shaped
conformation. In the proposed conformation, the C2-C6 atom to atom distance in MK-2’s
w z y
and H /H and between
H
w
h
i
+
isoprenyl side-chain was found to be 3.6 Å, which is consistent with a U-shape (g g
-
conformation, Figure 13) for MK-2 placed at the RM interface (see Supporting
Information for further discussion and internuclear distances of different MK-2
conformations). An illustration of an MK-2 conformation consistent with these
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Figure 13.
MK-2 conformation at an interface
visualized using Merck Molecular Force Field 94
(MMFF94) calculations. Molecular Mechanics
simulations generating the 3D conformation of MK-2 in
a RM that is consistent with H- H 2D NOESY and
ROESY NMR spectral data. (A) Side view of energy
1
1
Figure 12. Illustration of MK-2’s proposed folded, U-
shaped conformation and placement in the RM
1
1
interface. This arrangement is consistent with H- H
2D NOESY and ROESY NMR spectral data obtained
minimized
internuclear distance H
conformation
-H
(63.5
kcal/mol,
and
w
z
: 4.0 Å) showing MK-2’s
0
in w 12 RM; however, MK-2 likely tumbles freely
isoprene chain adopting a hook-like shape also known
as a folded, U-shaped conformation. (B) Top view of
energy minimized conformation showing the terminal
isoprene methyl-groups overlapping the carbonyl
groups on the napthoquinone.
within the interface but maintains interactions with the
region of AOT shown in the illustration. Colour depth
fading legend shows dark red as closer in distance
and dark blue as farther in distance from the reader.
AOT proton labelling scheme key is shown.
observations is shown in Figure 13. This U-
shaped conformation observed when MK-2 is associated with the RMs changed slightly
from that observed in d -DMSO regarding where the double bond overlaps with the
6
napthoquinone moiety. The overlap of the alkene and napthoquinone functionalities in
the conformation associated with the interface is best illustrated by the top view in Figure
1
3B. Importantly, the U-shape fold remains, but slight rotation around the C-C bond
facilitates the conformational changes in the U-shape.
Implications of Folded Conformations for MK Derivatives. Natural MKs contain a
naphthoquinone and an isoprenyl side-chain with some variable number of repeating
isoprene units, where MKs support the electron transfer reactions in a range of
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systems.
MK-2 was synthesized allowing for detailed characterization of the molecule.
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The results presented here for MK-2 suggests that other important MK derivatives in
biology will adopt folded conformations as well and demonstrates the need to prepare
and study such derivatives both experimentally and computationally.
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The extended conformations of long chain biological molecules such as fatty acids and
7,23,24
polyenes are generally shown in the literature if the structure is presented.
However,
a few studies of isoprene conformers and 1,3 acyclic dienes show the extended trans
7
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alkane conformations compare in energy with folded or helical conformations.
Although the folded conformations in those studies may be slightly higher in energy than
unfolded conformations, the difference is surprisingly small. The C-C single bond lengths
42
reported for stearic acid decreases under high pressure. These bond shrinkages cause
4
2
conformational and shape changes in stearic acid favoring folded conformations.
Previous studies using high temperature UV-absorption spectroscopy, molecular orbital
calculations, and gas-phase electron diffraction found that isoprene and chloroprene
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conformations did, in fact, favor folded conformations.
Although these conditions are
non-physiological, they do demonstrate that changes in a physical parameter can affect
the preferred conformation. Previous work has been carried out with fatty acids where the
experimental studies showed that the conformations of the alkane portion of the fatty
3
7
acids can and do change similar to conformations of MK-2 as shown in this work. Our
studies confirm the first hypothesis that MKs have different conformations depending on
specific environmental conditions, which was achieved by first synthesizing MK-2 and
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then demonstrating that a U-shaped, folded conformation was adopted in d -DMSO,
5
which was different from the conformation in d -pyridine.
Conformations can affect reactivity, and as in this case, the electron transfer
reactions facilitated by MK. MK forms complexes with several enzymes during the
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electron transfer processes, and although kinetic characterization of these systems have
83-85
been reported,
less is known structurally. The redox potentials of MK-2 depicted in
Figure 6 illustrate the trend where the most positive potential is in DMSO and the most
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negative potential is in pyridine, which agrees with literature reports on menadione.
These results have identified that the redox potential of MK-2 changes as a function of
solvent, and the origin of this change (presumably in part due to a change in dielectric
constant between solvents) and whether these observed solvent effects can be
associated with changes in conformation should be investigated in the future using both
suitable high level computational methods as well as electrochemistry. Indeed, the
change in redox potential is likely to be important, and since there is no conjugation
between the isoprenyl side-chain and napthoquinone, these effects are very interesting
and may reflect that the energy required for the reduction is determined by the energy
8
6
difference of the HOMO-LUMO gap in the two different conformations. Orbital analysis
of the HOMO-LUMO gap could potentially be informative and describe the origin of the
observed varied biological effects of a series of N-alkyl MK derivatives that have been
8
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reported.
2
For example, replacement of a CH group with a N-atom modifies the
parent system creating a class of compounds with a nitrogen bound directly to the
“naphthoquinone group.” These molecules have dramatically different biological
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properties than the parent MK derivatives
and it is possible that these activities could
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be related to the change in fundamental physical and chemical properties that occurs
with N-alkyl isoprenoid substitution of the naphthoquinone ring. Combined, these results
confirm the second hypothesis that the redox potential of MK-2 is influenced by organic
solvent environment, which is likely to be important for understanding the mechanistic
details in MK metabolism.
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The third hypothesis involved determining if the association of MK with the membrane
interface influences the conformation of the MK derivative. We investigated the
interaction of MK-2 with a phospholipid Langmuir monolayer, which is a simple 2D model
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9,89,90
of a single leaflet of a cell membrane.
The fact that MK-2 interacts with the
phospholipid monolayer is consistent with the expected MK affinity for the hydrophobic
phospholipids that make up the cell membrane. Langmuir monolayer studies support
interaction of MK-2 with the phospholipid monolayer interface; however, the lipoquinone
is likely pushed out of the interfacial portion of the phospholipid monolayer into the more
hydrophobic alkyl tail groups on the top of the monolayer as the pressure increases.
Unfortunately, Langmuir phospholipid monolayer studies could not determine the
conformation of MK-2; however, the RM surfactant interface complements studies with
the phospholipid monolayer interface. The question of folding was further investigated by
1
combining H NMR spectroscopic studies of MK-2 in organic solution and in a simple
model membrane system, RMs. The conformation of MK-2 was characterized in organic
solvents and was found to differ slightly from the conformation when it was associated
with the surfactant interface within RMs. It is to be expected that MK-2 would have an
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