Molecular dynamics simulations of water molecule-bridges in polar domains of humic acids.

Abstract

The stabilizing effect of water molecule bridges on polar regions in humic substances (HSs) has been investigated by means of molecular dynamics (MD) simulations. The purpose of these investigations was to show the effect of water molecular bridges (WAMB) for cross-linking distant locations of hydrophilic groups. For this purpose, a tetramer of undecanoid fatty acids connected to a network of water molecules has been constructed, which serve as a model for spatially fixed aliphatic chains in HSs terminated by a polar (carboxyl) group. The effect of environmental polarity has been investigated by using solvents of low and medium polarity in force-field MD. A nonpolar environment simulated by n-hexane was chosen to mimic the stability of WAMB in a hydrophilic hotspot surrounded by a nonpolar environment, while the more polar acetonitrile environment was chosen to simulate a more even distribution of polarity around the carboxylic groups and the water molecules. The dynamics simulations show that the rigidity of the oligomer chains is significantly enhanced as soon as the water cluster is large enough to comprise all four carboxyl groups. Increasing the temperature leads to evaporization processes which destabilize the rigidity of the tetramer-water cluster. Embedding it into the nonpolar environment introduces a pronounced cage effect which significantly impedes removal of water molecules from the cluster region. On the other hand, a polar environment facilitates their diffusion from the polar region. One important consequence of these simulations is that although the local water network is the stabilizing factor for the organic matter matrix, the degree of stabilization is additionally affected by the presence of nonpolar surroundings.

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Published: August 24, 2011
r2011 American Chemical Society
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pubs.acs.org/est
Molecular Dynamics Simulations of Water Molecule-Bridges in Polar
Domains of Humic Acids
Adelia J. A. Aquino,*,†,‡Daniel Tunega,†Hasan Pa? sali? c,‡Gabriele E. Schaumann,§Georg Haberhauer,†
Martin H. Gerzabek,†and Hans Lischka‡
†Institute of Soil Research, University of Natural Resources and Applied Life Sciences Vienna, Peter-Jordan-Strasse 82, A-1190 Vienna,
Austria
‡Institute for Theoretical Chemistry, University of Vienna, W€ ahringer Strasse 17, A-1090 Vienna, Austria
§Institute for Environmental Sciences, Department of Environmental and Soil Chemistry, Universit€ at Koblenz-Landau, Fortstrasse 7,
76829 Landau, Germany
b
S Supporting Information
’INTRODUCTION
Humic substances (HSs) constitute natural components of
soils and sediments, being largely heterogeneous, control the
fate of environmental pollutants, and the biogeochemistry of
organic carbon in the global ecosystem.1Partitioning models2,3
as well as polymer and polymer distribution models4?10have
been developed to characterize the molecular structure of HSs.
Increasing evidence emphasizes the relevance of noncovalent
intermolecular interactions (e.g., π?π, CH-π, van der Waals,
charge-transfer, and hydrogen-bonding) between relatively small
organic moieties11and indicates a micellar structure of HSs
protecting hydrophobic interiors from contact with neighboring
water molecules.12?14A recent review on natural nanomaterials
insoils15reportedthatHSsareabletorearrangeandrestructurein
responsetoenvironmentalchangessuchaspH,ionicstrength,and
moisture. Within the supramolecular picture of HSs many studies
have shown that individual compounds in soil organic material
(SOM)canbebridgedviawatermoleculebridges(WAMB)16?18
and/or cation bridges (CAB).8,19?21In previous theoretical
investigations we have studied supramolecular interactions in
HSs taking into account pH effects on the carboxylic groups and
the effect of a water network interacting through hydrogen
bonding with carboxylic groups has been demonstrated.16,19,22
Schulten et al.23showed that in humic acids most hydrophilic
functional groups are too far from each other to form direct
intermolecular cross-links. They therefore require WAMB for
intermolecular cross-linking. Swelling and wetting processes can
drasticallyaffectthesorptionpropertiesofSOMandtheintrusion
of contaminants and nutrients into soils.18Differential scanning
calorimetry (DSC) experiments24on swelling peat samples
showed an antiplasticizing effect of water on the organic matrix
which increased with equilibration time and which decreased
significantly after sudden changes of moisture conditions but
Received:
Accepted:
Revised:
June 6, 2011
August 24, 2011
August 18, 2011
ABSTRACT: The stabilizing effect of water molecule bridges on polar regions in humic
substances (HSs) has been investigated by means of molecular dynamics (MD)
simulations.Thepurposeoftheseinvestigationswastoshowtheeffectofwatermolecular
bridges (WAMB) for cross-linking distant locations of hydrophilic groups. For this
purpose, a tetramer of undecanoid fatty acids connectedto a network of water molecules
has been constructed, which serve as a model for spatially fixed aliphatic chains in HSs
terminated by a polar (carboxyl) group. The effect of environmental polarity has been
investigated by using solvents of low and medium polarity in force-field MD. A nonpolar
environment simulated by n-hexane was chosen to mimic the stability of WAMB in a
hydrophilic hotspot surrounded by a nonpolar environment, while the more polar
acetonitrile environment was chosen to simulate a more even distribution of polarity
around the carboxylic groups and the water molecules. The dynamics simulations show
that the rigidity of the oligomer chains is significantly enhanced as soon as the water
cluster is large enough to comprise all four carboxyl groups. Increasing the temperature leads to evaporization processes which
destabilize the rigidity of the tetramer-water cluster. Embedding it into the nonpolar environment introduces a pronounced cage
effect which significantly impedes removal of water molecules from the cluster region. On the other hand, a polar environment
facilitates their diffusion from the polar region. One important consequence of these simulations is that although the local water
network is the stabilizing factor for the organic matter matrix, the degree of stabilization is additionally affected by the presence of
nonpolar surroundings.

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reincreased after some time of storage.18,25These phenomena
were rationalized by formation and disruption of WAMBs17and
may further be responsible for the reduced dissolution rate of
dissolvedorganicmatter(DOM)frompremoistenedsoilmaterial.26
In the same way, it could prevent organic molecules from
entering or leaving certain microregions in SOM17unless those
molecules disrupt the WAMB via competition for hydrogen
bonds. It is, therefore, important to obtain advanced under-
standing of interactions between water, small organic molecules,
and organic material forming intermolecular cross-links to sup-
port the above-mentioned models. While the former studies
demonstrated therelevanceofWAMBsforintermolecularcross-
linking under various spatial andpHconditions,itisunknown to
which extent the nanospatial heterogeneity in the organic matter
affects the relevance and stability of the WAMB. Furthermore,
the effect of the polarity of the nanoenvironment on the stability
of the WAMB still remains unresolved, and the direct link to
experimental data, e.g., the DSC step transition temperature
T*,17,24,27is still open.
Modeling of water-OM interactions in humic substances
requires simulation of situations of limited mobility and large
distances between functional groups, for example by using a
reduced molecular structure and at the same time fixing certain
points in space as done in the two preceding studies.19,28This
concept reduces the complexity of the molecule to the char-
acteristics of interest without having to model complete poly-
meric structures. As prototype for modeling polar interactions
the carboxyl group represents a good choice since it is found in
humic substances in a high content. For example, it has been found
thattheSwanneeRiverhumicsubstancecontains9.6molkg?1of
carboxyl carbon.29In this context classical molecular dynamics
simulations have been performed, and interactions with water
havebeenstudied forfulvicacid model.30Italso should benoted
that carboxyl groups are responsible for forming negatively charged
sitesinHSsalreadyatrelativelylowpHvalues(dissociationstarts
at pH > 4.4).29
The concept of limited mobility and focus on the carboxyl
group is also used in this work, where a region of a polar center
interacting with a water cluster has been constructed by using a
set of four medium-sized fatty acid segments which serve as
models for spatially fixed aliphatic chains in HSs terminated by a
polar (carboxyl) group. The arrangement of these four chains is
shown in Figure 1. Each chain has a great torsional flexibility due
toalongaliphatictail.Thenonpolarendsofthechainswerekept
fixed in order to simulate the situation of a stiff backbone such
that the carboxyl groups cannot approach each other to undergo
direct interactions but have some spatial mobility. Only this
fixationofthenonpolarendsofthefattyacidsegmentsallowsfor
mimicking the crucial situation in humic substances this study
aims for.
The hydrophilic carboxyl heads were immersed into a cluster
of water molecules to investigate local wetting and drying effects
on the structural and energetic stability of the system (Figure 1).
Environmentaleffectswereincludedbymeansofembeddingthis
aggregate into solvents (n-hexane and acetonitrile) presenting
different polarity to test the stability of the WAMB toward an
excess of small organic molecules. The nonpolar environment
simulated by n-hexane was chosen to mimic the stability of
WAMB in a hydrophilic hotspot surrounded by a nonpolar
environment, while the more polar acetonitrile environment
was chosen to simulate a more even distribution of polarity
around the carboxylic groups and the water molecules. Several
temperatures ranging from 300 K to 420 K were used in the
dynamics simulations in order to investigate temperature stabi-
lityontherigidityoftheWAMBnetworkandtoobtainfirstlinks
with the experimental WAMB transition temperature T*.17,24,27
In addition to modeling the interactions with neutral carboxyl
groups, selected cases of deprotonation (carboxylate) were
studied as well in order to account for the importance of
negatively charged sites mentioned above.
In continuation of our previous philosophy, molecular dy-
namics (MD) simulations are performed, primarily at quantum
mechanical level using the semiempirical density functional
based tight-binding31,32(DFTB) method using the self-consis-
tentcharge(SCC)approach.33Theuseofanabinitioapproachis
too expensive for such big systems especially in view of the
dynamics simulations. However, it has been shown16,19that the
DFTB method represents a very good compromise in terms of
accuracy for describing hydrogen-bonded interactions and com-
putational efficiency for performing MD simulations. Explicit
inclusion of solvent molecules would lead to MD simulation
times, which even at the DFTB level were not practical. There-
fore, in this case the molecular mechanics (MM) method based
on empirical interatomic force field (FF) has been used.
’METHODS
The structural model used to describe the interactions of HS
segments (Figure 1) is composed of four fatty acid chains
Figure 1. Top and side view of the tetramer-water cluster.

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(tetramer Tm) using the undecanoid acid, CH3(CH2)9COOH.
The positions of the terminal methyl carbon atoms were kept
fixed during the simulations at a distance of 20 Å. The carboxylic
groups represent a hydrophilic center which is immersed into a
cluster consisting of 20 water molecules (Tm-(H2O)20model).
Wetting and drying processes were simulated by removing
twowatermoleculesfromtheinitialTm-(H2O)20modelinsteps
of two creating models Tm-(H2O)n, where n = 18...2. Two water
molecules were taken arbitrarily from ten different positions in
the model Tm-(H2O)20. In this way ten different structures
Tm-(H2O)18werecreated,andMDsimulationswere performed
bystartingfromthem.Basedontheaverageenergycomputedfor
each MD the five most stable cases were selected. Two water
molecules were randomly taken from different positions of the
final structures of each of the aforementioned five selected MD
simulations constructing new ten Tm-(H2O)16models. In this
waywecontinuedintheconstructionofTm-(H2O)ndownton=
2 always using ten independent MD simulations.
A single deprotonation was performed at each of the four
carboxylgroupsindividually.Testcalculationsshowedthatinsuch
acasetheSCCiterationsintheDFTBapproachdidnotconverge
due to the negative overall charge. To achieve charge neutrality,
the carbon atom of the terminal methyl group in the chain
containing the carboxylate group was replaced by N+in the sense
ofaninternalcounterion, leading tooverall chargeneutrality. The
SCC calculationsconverged,andthistechniquewasused success-
fullyintheDFTBMDcalculations.Inthefinalanalysisanaverage
over all four deprotonation cases was performed.
TheMDsimulationswerecarriedoutattheDFTBlevelinthe
canonical (NVT) ensemble at T = 300 K using the Andersen
thermostat.34A time step of 1 fs was used in the Velocity Verlet
integration algorithm35for a simulation time of 50 ps for each
case.TheanalysisoftheMDdatawasperformedforthelast30ps
of the simulations. Each two hundredth configuration was
selected for this purpose.
In order to represent differences in nanospatial heterogeneity,
wedecidedtosimulateenvironmentsofdifferingpolarityaround
the WAMB. Calculations in two solvents were performed using
acetonitrile andn-hexane as representatives ofnonpolar(relative
dielectricconstantsεr=2.02)andpolarenvironment(εr=37.7).
The simulated system consisted of the Tm-(H2O)20structure
embedded in 357 acetonitrile and 143 n-hexane molecules,
respectively, arranged in a cubic box with length of 32 Å in order
to reproduce experimental densities36of the solvents at T = 300
K. Periodic boundary conditions combined with the minimum
image convention37were applied in all three dimensions. Classi-
cal force field MD (FF-MD) simulationsinthecanonical (NVT)
ensembleatT=300Kwereperformedinthiscase.Atimestepof
1 fs and the Nos? e-Hoover38,39thermostat were used. Both the
intra- and intermolecular interactions were described by the all
atom optimized parameters for liquid simulations (OPLS?AA)
force field.40The total simulation time was 1 ns.
Toinvestigatethechangesinenergeticstabilitywithrespectto
temperature as observed e.g. in differential scanning calorimetry
(DSC) experiments,18MDcalculationswereperformed bothfor
the isolated cluster (DFTB-MD) and in solvent (FF-MD) for
temperatures of 300, 340, 380, and 420 K, respectively. The
simulation time was 200 ps in the former case and 1 ns in the
latter. Since the simulation including an environment aimed at
the representation of a rigid matrix structure of varying polarity,
the density of the solvent and the size of the unit cell were kept
fixed at the values used for T = 300 K.
The structural analysis comprises normalized one-dimen-
sional atomic density profiles calculated along the z direction
as indicated in Figure 1, the distribution of interchain C?C
distances of carbon atoms belonging to neighboring carboxyl
groups, the average number of hydrogen bonds, and the incre-
mentalenergycalculations withrespect tothenumberof added/
removed water molecules. The atomic density profiles are
normalized with respect to the total number of atoms in the
cluster and the number of configurations.
The DFTB calculations were carried out using the DFTB+
code.41For the FF-MD calculations with the solvents n-hexane
and acetonitrile the program TINKER42was used.
’RESULTS AND DISCUSSION
Density Profiles. In this section the average spatial distribu-
tion of the water molecules and carboxyl groups obtained from
MD simulations is examined by means of z density profiles and
the distribution of interchain C?C distances of the carboxyl
carbon atoms. Atomic density profiles were calculated for the
oxygen atoms belonging both to the water molecules and the
carboxyl groups as well as profiles of the carbon atoms of the
carboxylicgroups.ThemajorresultsarepresentedinFigures2-8;
moredetailscan befound intheFigures 1S-6S of theSupporting
Information.
Figure 2. z-density profiles for carbon atoms belonging to the carboxyl
group and of all oxygen atoms for selected tetramer-water clusters.

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The Tm-(H2O)nComplexes. Starting the discussion with the
Tm-(H2O)4cluster (Figure 2a) one can see that the distribution
of the carbon atoms is structured into two peaks, covering a
region of about 4 Å. The distribution of oxygen atoms is
somewhat broader than that for the C atoms. The carboxyl
C?Cdistances(Figure3a)extendoverabroadrangewithmajor
contributions between 10 and 18 Å characterizing the large
flexibilityinthemotionoftheindividualchains.Additionalpeaks
at about 4 Å indicate specific, direct interactions between chain
pairs. For n = 14 the water cluster is large enough to connect the
carboxyl groups of all four chains. Both z-profiles and C?C
distance histograms (Figures 1Sb and 2Sb) exhibit stronger
localization of the carboxyl groups illustrating nicely the stabiliz-
ing capability of the water clusters on restricting the motion of
these groups. This trend is preserved in the 18-water complex
(Figures 2b and 3b). In contrast to the structural changes
observed for the carboxyl groups, the water molecules remain
spread out with increasing number but always exhibit a strong
overlap with the distribution of the carbon atoms (Figure 2 and
Figure 1S). Especially for the 14- and 18-water complexes the
oxygen distribution is significantly wider than the carbon dis-
tribution because of the high fluctuation of molecules in clusters
with larger water content.
An analysis of the analogous dynamics of deprotonated
carboxyl groups in contact with a cluster of 20 water molecules
is presented in Figure 4. Comparison of the z-profiles shown in
Figure 4a with those of Figure 2b indicates similar localization of
thecarboxyl/carboxylategroupsintheneutralanddeprotonated
cases.The distributionobtainedinthe latter case is slightly more
extended. More significant differences are found for the water
distributions. For the carboxylate case the water distribution
appears smoother but also shows a significant overlap with the
distribution of the carboxyl/carboxylate groups. Finally, the
C?C distance histograms (Figure 4b) display the same com-
pactness of the carboxyl/carboxylate groups due to the water
networkasinthecaseoftheneutralcarboxylgroups(Figure3b).
Thus, the simulations show that also in the single deprotonation
case the surrounding water network is capable of keeping the
polar groups together.
Figure 3. Histograms of interchain CC distances of carbon atoms
belonging to neighboring carboxyl groups for selected tetramer-water
clusters.
Figure 4. DFTB simulations on singly deprotonated carboxyl groups
for a 20 water cluster: (a) z-density profiles for carbon atoms belonging
to the carboxyl/carboxylate groups and of all oxygen atoms and (b)
histograms of interchain CC distances of carbon atoms belonging to
neighboring carboxyl/carboxylate groups.

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Temperature Effects. Figures 5 and 3S display the oxygen
and carbon z-profiles derived from the dynamics of the
Tm-(H2O)20cluster computed at different temperatures. These
calculations should set a first simple step toward the interpreta-
tionoftheDSCexperimentson samplesofsoil organic matter.18
The z-profiles for the oxygen atoms display an increasing
fluctuation and evaporation of the water molecules with increas-
ingtemperaturewithintheMDsimulationperiod.Withdecreas-
ing number of water molecules available for linking the carboxyl
groups, the still unvaporized water molecules remain located in
the bridging region, covering, however, only two to three
carboxyl groups.
Effect of Temperature and Environment. The density
profiles for Tm-(H2O)20in n-hexane (Figures 6, 4S, 7 and 5S)
and acetonitrile (Figure 8) were computed at the FF level for
different temperatures. The C?C distance profiles for the
isolated T-(H2O)18complex (Figure 3b) and that for n-hexane
solution (Figure 7a (T = 298 K)) display a similar spatial
compactness of the carboxyl groups. This range of 4?12 Å
remainsalmost unchangedupto380 K (Figures 7and5S). Only
at 420 K a distinct additional peak appears at around 20 Å
because of the starting separation of one chain from the others.
Visual inspection of the evolution of the cluster geometries and
thefactthatthemaximumofthez-profileforthecarboxylcarbon
atoms is located at about zero in the FF dynamics (Figure 6)
demonstrate that the whole carboxyl-water cluster undergoes an
inversionmotionalongthezaxis,aprocess whichisnotfoundin
the DFTB dynamics (c.f. e.g. Figure 2b). However, as the above
discussion shows, the major effect of the spatial confinement of
thecarboxylgroupsbythewatercluster,whichisthefocusofthis
work, is very similar in both methods.
Comparison of the z-profiles for the oxygen atoms computed
for the isolated cluster (Figure 2b) and for n-hexane solution
(Figures 6 and 4S) manifests a remarkable cage effect of the
hydrophobicenvironmentofn-hexaneonthewatercluster.Even
though the cases of the free cluster (DFTB) and the n-hexane
solution(FF)arenotexactlycomparablesincedifferentmethods
are used, one can see for the free cluster that the distribution of
the water molecules is significantly wider than that of the
carboxyl groups (much broader oxygen distribution in compar-
ison to the carbon distribution), whereas in n-hexane the water
distribution is very similar to the one of the carboxyl groups
(similarextensionofthedistributions).Thisfactindicatesthatin
the latter case the water molecules stay always closer to the
Figure 5. Temperature dependence of z-density profiles for carbon
atoms belonging to the carboxyl group and of all oxygen atoms for the
Tm-(H2O)20cluster.
Figure 6. Temperature dependence of z-density profiles for carbon
atoms belonging to the carboxyl group and of all oxygen atoms for the
Tm-(H2O)20cluster in n-hexane.