Hydrating the Bispropionate Notch in Malaria Pigment: A New Structural Motif in the Iron(III)(deuteroporphyrin) Dimer

Article abstract of DOI:10.1002/chem.201805116

Treating deuterohemin, chloro(deuteroporphyrinato)iron(III), with a non-coordinating base in DMSO/methanol allows for the isolation of [(deuteroporphyrinato)iron(III)]₂, deuterohematin anhydride (DHA), an analogue of malaria pigment, the natural product of heme detoxification by malaria. The structure of DHA obtained from this solvent system has been solved by X-ray powder diffraction analysis and displays many similarities, yet important structural differences, to malaria pigment. Most notably, a water molecule of solvation occupies a notch created by the propionate side chains and stabilizes a markedly bent propionate ligand coordinated with a long Fe?O bond, and a carboxylate cluster associated with water molecules is generated. Together, these features account for its increased solubility and more open structure, with an increased porphyrin–porphyrin separation. The IR spectroscopic signature associated with this structure also accounts for the strong IR band at 1587 cm^?1 seen for many amorphous preparations of synthetic malaria pigment, and it is proposed that stabilizing these structures may be a new objective for antimalarial drugs. The important role of the vinyl substituents in this biochemistry is further demonstrated by the structure of deuterohemin obtained by single-crystal X-ray diffraction analysis.

Full text of DOI:10.1002/chem.201805116

A Journal of
Accepted Article
Title: Hydrating the Bispropionate Notch in Malaria Pigment: A New
Structural Motif in the Iron(III)(deuteroporphyrin) Dimer.
Authors: david kuter, liliana suarez, bruce c noll, erin l dodd, peter w
stephens, and Scott Bohle
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Hydrating the Bispropionate Notch in Malaria Pigment: A New
Structural Motif in the Iron(III)(deuteroporphyrin) Dimer.
David Kutera,d, Liliana Suáreza, Erin L. Dodda, Bruce C. Nollc, Peter W. Stephens*b, and
D. Scott Bohle*a
a
Department of Chemistry, McGill University, 801 Sherbrooke St. W., Montreal H3A 0B8
b
CANADA; Department of Physics and Astronomy, State University of New York, Stony
c
Brook, Stony Brook New York, 11794-3800 USA; Bruker-AXS, 5465 E Cheryl Pkwy,
d
Fitchburg, WI 53711, USA; Department of Chemistry and Polymer Science, Stellenbosch
University, Private Bag X1, Matieland, 7602, Stellenbosch, South Africa.
Abstract: Treating deuterohemin, chloro(deuteroprophyrinato)iron(III), with a non-coordinating
base in DMSO and methanol allows for the isolation of [(deuteroporphyrinato)iron(III)]2,
deuterohematin anhydride (DHA), an analog of malaria pigment, the natural product of heme
detoxification by malaria. The structure of DHA from this solvent system has been solved by X-
ray powder diffraction and displays many similarities and important structural differences with
malaria pigment. Most notably a water of solvation occupies a notch created by the propionate
sidechains and stabilizes a markedly bent propionate coordinated with a long Fe-O bond and the
formation of carboxylate cluster associated with the waters. Together these account for its
increased solubility and more open structure with an increased porphyrin-porphyrin separation.
The IR spectroscopic signature associated with this structure also accounts for the strong IR
bands at 1587 cm-1 which are present in many amorphous preparations of synthetic malaria
pigment and it is proposed that stabilizing these structures may be new motif to target
antimalarial drugs. The important role for the vinyl substituents in this biochemistry is further
demonstrated by the structure of deuterohemin from single crystal X-ray diffraction.
1
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Introduction In a brief synchronized timeline, the malaria parasite repeatedly invades human
red blood cells, imports hemoglobin into a digestive vacuole, degrades the hemoglobin peptides,
oxidizes the heme to iron(III), and crystallizes the resulting Fe(III)(protoporphyrinato) molecule
into the excretion product known as malaria pigment.1 This unique biochemistry presents many
of the known2,3 drug targets for past and future generations of antimalarials and all are the
subject of sustained intense investigation.4,5 While some aspects of this time line are now well
understood, particularly the molecular and structural biology of the peptidases which degrade
hemoglobin,6-8 other processes9,10 such as heme oxidation, heme transport, and heme
crystallization remain poorly understood.11 Even the ultimate product of heme crystallization,
malaria pigment, also termed hemozoin (HZ), has aspects of its structural and surface chemistry
still to be resolved.12
Figure 1 Propionic acid structural roles in HZ and HA, [Fe(protoporphyrinato)]2. Each heme has
one Fe-O bond to one propionate and the second propionic acid forms a H-bonded dimer. H-
bonds are shown as blue or red dashed lines with a conventional CPK atomic coloring scheme
employed. Structure shown taken from the CDC entry XETXUP and shows only one set of
positions for the disordered vinyl groups.13
2
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A particularly challenging problem in the biosynthesis of HZ is its remarkable crystallinity and
the rate at which a single phase crystallizes from the heterogeneous milieu of the digestive
vacuole.14 Following its original structural elucidation by X-ray powder diffraction in 2000,13
there have been four separate powder diffraction structure determinations15-17 as well as single
crystal structures of a DMSO solvated phase.18 In each case, either synthetic (hematin anhydride,
HA§)13,15,16,18,19 or natural (HZ),17 the structure consists of iron(III)(protoporphyrinato) dimers.
The reported variations in structural models are solely related to the presence/absence of solvent
interrupting the H-bonded propionic acid chain, and differing models for the vinyl disorder.
Thus, regardless of source, natural or synthetic, HA and HZ are isostructural and have the base
structure shown in Figure 1, a chain of propionate linked dimers. Inhibiting the formation of HZ
is the consensus drug target for the quinoline family of antimalarials.5,20 There has been a
sustained effort to synthetically mimic and inhibit this crystallization step and one resulting
general observation is that unless crystallization is slowed with a competing solvolysis, by
performing the reaction in DMSO for example,21 substantial amounts of amorphous products are
obtained. Thus, synthetic HA is usually contaminated to varying degrees by other amorphous
phases characterized by strong IR bands at 1585 cm-1 in addition to those at 1712 and 1664 cm-1
in HZ and purified HA.22 The malaria parasite avoids the formation of these amorphous phases
by an as yet unknown mechanism. In synthetic materials, these amorphous phases are usually
treated as a nuisance and their higher solubility/reactivity is exploited in subsequent vigorous
purification/washing steps with bicarbonate buffer, pyridine, water and methanol. In many
conditions, these amorphous phases are the main product of the reaction and once posed severe
limitations on early efforts to structurally characterize HA with X-ray diffraction.23 To date,
little is known of these materials apart from their unique IR signature, their reactivity/solubility
3
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and their lack of crystallinity. In this contribution we present a structure of a related phase of
HA, with deuteroporphyrin instead of protoporphyrin, which has the IR signature and
reactivity/solubility of these amorphous phases. We propose that this new variant of the HZ
structure/motif adds to our understanding of heme crystallization in HZ and may ultimately aid
in the development of new antimalarials.
Results and Discussion
Deuterohematin anhydride, DHA, the deuteroporphyrin devinylated analog of HA, is prepared
by the dehydrohalogenation of deuterohemin, DH, with a non-coordinating base, 2,6-lutidine
(equation 1).15 Solvent effects are important in these transformations with other solvents and
bases leading to different semi-crystalline materials, Figure S6.24 The structure of the
DMSO/MeOH product was solved as a hydrate from synchrotron powder diffraction data,
Figures 2, 3, S3, with Figure S2 showing the diffraction pattern and refined final Rietveld fit. As
shown in Figure 2A, the deuteroporphyrin forms an inversion related dimer pair. Thus, as with
HZ, Figure 1, DHA shares the common 1 iron bound propionate bound dimer with the iron 0.5
Å out of the plane of the porphyrin and displaced towards the propionate ligand. It does not,
however, form a chain of propionic H-bonded dimers. Further refinement details are given in the
4
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Table S1 and S6 with a detailed contrast of HA and DHA is given in Table S5. Also shown in
Figure S1 and Table S5 are related bispropionate linked dimers.
(A)
(B)
Figure 2. Structure of DHA. (A) Tilting of the intradimer pair in DHA, hydrates not shown.
(B) Packing of propionate linked dimers showing H-bonds (black dashed lines) and close
contacts (blue dashed lines) between free propionic acid and non-iron bound propionate oxygen.
Note the water occupying the gap or notch between the two propionic acid side chains.
5
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While the structures of HA and DHA share similarity in being comprised of inversion dimers
for the iron and porphyrin core, there are notable differences in their hydration, intra-dimer
geometry, the inter-dimer interactions, as well as crystallographic symmetry. Within the dimer
of DHA, the iron carboxylates are tilted or canted by 61.0 away from the orthogonal orientation
found in HA and in many metallocarboxylates.25 This tilt is accompanied by an increase in the
iron-oxygen bond length to 1.94(7) Å, an increase in the separation of the porphyrin planes to
4.82 Å, and a lateral offset of the porphyrins by 4.47 Å. Additionally, there is almost no overlap
of the two intra-dimer porphyrin planes. One result of this “opening up” of the structure is that it
allows for the most unusual feature of the DHA structural motif – the formation of a hydrated
carboxylic acid cluster with weak H-bonds between free propionic acid groups on neighboring
dimers as well as the hydrate to the non-iron bound oxygen of the coordinated propionate. The
later O---[H]---O separation of 2.614 Å, suggests that the iron bound propionate ligand could be
described as an oxygen coordinated propionic acid derivative. The collected structure of the two
propionates, two propionic acids, and two waters is shown schematically in Figure 3. This
unusual H-bonded cluster accounts for many of the structural changes found in DHA.
Remarkably this crystallographic structure is closely related to the optimized DFT geometry for
a hydrated dimer, Table 1 and Figures S8. The structural contrast of HA and DHA structural
motifs illustrates some of key interactions within the propionate bonding of the dimer. As
shown in Figures 3 and S4 the two parallel propionates in HA adopt a more open and kinked
arrangement characterized by the torsion angles in Table S5. The contrast of the free and the
metal bound propionates in HA and DHA is
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Figure 3 Water and carboxylate cluster in DHA. Oxygens from four different propionates are
shown as red spheres O32, O33, O37 and O38 and two water as Ow. In grey are the
carboxylate carbons and in blue the Fe‐N bonds to the two deuterohemes. Iron atoms are
depicted in brown/rust‐orange.
seen in the similarity of the conformation of the metal bound group while in free propionate
large differences of 90, 100, and 40 are seen in the respective torsion angles of the two
structures. The gap created by the propionates in HA provides a notch or a groove for a
neighboring iron bound propionate to pack closely in the lattice with key metric parameters
collected in Table 1. In DHA this site is blocked by the water of hydration and the resulting
carboxylates form the cluster shown in Scheme S1 and Figure S3. This key site is also occupied
by the side chain of chloroquine, CQ, in the crystal structure of the model gallium analog,
.
[Ga(protoporphyrinato)(OMe)]2 CQ2,26 and this site also exhibits significant porphyrin/drug
interactions by NOESY/NMR studies in solution.27 Perhaps most importantly of all in the
structure of HA/HZ this site is filled by the propionate of an adjacent heme dimer and in the
single crystal structure of the DMSO solvate of HA the one of the DMSO methyl groups is at the
7
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entrance to this notch.18 The bispropionate notch is an important drug target of the
heme/antimalarial drug binding interaction.
Table 1 Metrical Parameters of the Propionate Notch
Structure
HA
[Ga(PP)]2.2CQ
DHA.2H2O
Experimental
This work
DHA.H2O
Mono-Theory
B3LYP/cc-pvdz
DHA.2H2O
Di-Theory
B3LYP/cc-pvdz
CCDC Code
XETXUP
162267
DONRAB
904065
Metric
O-------O (Å)a
C-------C (Å)b
Xc
6.94
6.65
(C)==O
3.65,
4.14
6.44
6.46
CH2
7.38
8.05
H2O
5.00
5.85
H2O
5.00, 5.00
5.95, 5.95
H2O
2.82, 3.05
2.82, 3.04
2.36, 2.36
116.95,116.95
120.80,120.79
1.880, 1.880
134.65, 134.65
X-----O (Å)d
2.50, 2.93
2.62, 4.91
2.83; 3.03
X-----HCm (Å)e
O--X--O ()f
C--X--C ()g
M-O (Å)h
4.85
4.02
130.9
103.5
2.007
168.1
3.16
2.34
117.4
120.7
153.0
173.8
1.893
166.5
159.3
143.2
1.945
143.8
1.881; 1.852
140.7;162.5
M--O--O ()i
Stationary points for hydrates with two high spin heme for the gas phase B3LYP/cc-pvdz.
a
b
c
Closest propionate oxygen atom separation; Separation of propionate carbons; Functional
group held in notch. –C(O)-OFe oxygen of HA, a methylene of CQ in its gallium adduct, and
the water of the DHA solvate. d X-oxygen separations for propionates that define the notch. For
each pair listed the first and the shorter distance corresponds to the distance to the iron bound
e
oxygen, and the second to FeOC=O; The X ---H separation for the proton on the Cm between
f
g
pyrroles C and D; O-X-O angle for the propionate oxygens, and The C-X-C angle for the
propionate carbons. h,i Fe-O given for the water bound propionate pair and the water free pair of
propionates.
8
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Vibrational spectroscopy remains one of the best tools for deciphering the propionic acid
interactions in the heme family of condensed phases15,28 and the structure of DHA provides a
valuable new reference point for this technique. As shown in Figure 4, the IR spectrum of DHA
has strong propionic sidechain bands at 1729, 1654, 1587, and at 1206 cm-1. All of these bands
correspond well with bands present in the IR spectrum of HA prepared without extensive
purification/washing (crude HA), Figure 4A. However, on purification, the feature at 1584 cm-1is
lost (Figure 4B). This behavior is in contrast to DHA, where the 1587 cm-1 band persists even
after extensive bicarbonate washes during its work up. Many preparations of synthetic HA have
similar bands at ~1585 cm-1 upon their initial isolation, but these are lost (along with much of the
product!) during the subsequent workup steps. We propose that this band is diagnostic of the
type of bonding depicted in Figure 3, that is with a partial formal structure of –CH2-C(OH)=O-
Fe, with its relative durability being due to it being an integral part of the lattice. It is significant
that in biomimetic syntheses of HA, as well as in
hemozoin isolated from the parasite, that this
Figure 4. IR spectra of DHA and HA. (A) DHA (red)
versus crude HA (black); and (B) Extensively washed HA
(blue) versus crude HA (black). Spectra measured with a
diamond cell ATR and show the common strong carboxyl
band at 1587 cm-1 (black arrow) which is present in DHA
and crude HA. The band 1584 cm-1 is absent in
purified HA.
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band has little, if any, intensity. Additional support for this assignment is from the B3LYP/cc-
pvdz calculated gas phase vibrational modes for the HA dimer with pendant propionic acids and
the DHA internally bonded bent propionic acids, Figure S9 and Table S8. Here there is a net
decrease in the carboxylate stretching modes by ~ 90 cm-1 for the DHA structure. Further
theoretical studies of the hydrated model are underway. The implication is that the parasite
prevents these types of interactions from forming or, if formed, from propagating in the final
product. DHA is much more soluble than HA, and HA/HZ crystallinity and insolubility may
stem from the absence of these interactions as well as the converse that poorly
aggregated/crystallized synthetic HA has a large fraction of these phases. We note that the
structure of DHA is more consistent with spectroscopic results than prior trimeric and polymeric
models proposed for these phases.29
Clearly the two vinyl substituents in protoporphyrin are an important factor in controlling the HZ
crystallization and phase selectivity.30,31 Similar considerations are also found in the structural
biology of heme proteins where both the propionic acids on one side of the porphyrin as well as
the two vinyl group on the opposing side orient the heme with its binding pocket.30,31 To further
understand the structural role of the vinyl groups in hemin chemistry, we have also structurally
characterized deuterohemin (DH), the starting material of DHA synthesis. This readily
accessible derivative is obtained by a resorcinol thermolytic vinyl cleavage from
Fe(III)(protoporphyrinato)chloride, hemin, Figure S5.32 As shown in Figure 5, the absence of
the two vinyl groups at carbon atoms C(13) and C(16) does not appear to markedly influence the
porphyrin and iron geometry. However, when compared to its hemin precursor, the
intermolecular packing arrangement in the solid state is markedly affected. In DH, the hydrogen
bonding between the propionic acids leads to an offset chain, Figure 5A, whereas in hemin (not
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shown) the propionic acids form a simple dimer with another single hemin. This offset chain
propagates along the crystallographic 21 screw axis present in DH but is absent in the P-1 lattice
of hemin.
(A)
(B)
(C)
Figure 5. (A) Single crystal X-ray diffraction structure of the deuteroheme in DH viewed down
the Cl-Fe axis. Intermolecular interactions in DH viewed (B) perpendicular to and (C) along the
2/1 screw axis.
Conclusion
The structure of DHA in Figure 2 suggests an important new approach or motif the design of
antimalarial drugs. Numerous computational and synthetic efforts have focused on the putative
11
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drug targets on the faces of the growing hemozoin crystal.33-38 However, on the other side of the
equilibrium are heme dimers in solution, either linked by propionates, -oxo-bridges, or by
London dispersive forces14 and if these can be stabilized, then heme sequestration and
detoxification will also be disrupted. Alternatively, if heme precipitation can be directed
towards DHA-like phases and away from the HA structure then there may also be increased
solubility of the heme. Either mechanism might in principle accomplish the same net result:
parasite homeostasis and diminished proliferation leading to decreased parasitemia in the host.
As the pressing need for new antimalarials becomes ever more critical,39 the new structural motif
of DHA represents a complimentary new approach to the extensive efforts currently underway to
control malaria.
Competing Interests
The authors declare no competing interests.
Author Contributions
DSB and PWS conceived the study, PWS and BCN measured the diffraction data and solved the
powder and single crystal structures respectively. DSB and DK performed density functional
studies, ELD, LS, and DK performed the experiments, and analyzed the data. DSB, PWS, DK,
ELD, and LS wrote the manuscript with input from all authors.
Acknowledgments
We gratefully acknowledge support from the Burroughs Wellcome Fund, NSERC, FQRNT, and
CRC. DK acknowledges the South African NRF for funding. Use of the National Synchrotron
12
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Light Source, Brookhaven National Laboratory, was supported by the U.S. Department of
Energy, Office of Basic Energy Sciences, under Contract No. DE-AC02-98CH10886.
13
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Methods
Crystallography: Synchrotron powder X-ray diffraction (SPXD) data were collected at
ambient temperature the National Synchrotron Light Source (NSLS), Brookhaven National
Laboratory. Diffraction data analysis and Rietveld refinement were performed with TOPAS-
Academic.40 The diffraction pattern was indexed to a monoclinic cell, and the structure was
solved using simulated annealing. Details are given in Table S1. In the Rietveld refinement,
restraints based on literature values of similar iron porphyrins were placed on bond distances and
angles, and on the planarity of the porphyrin moiety. However, no restraints were placed on the
position of the Fe atom or the geometry of the axial Fe-O bond. The Rietveld refinement is
shown in Figure S2 and the structure is illustrated in Figure 2 of the main text. The data is
available from the CCDC 1494078.
Single Crystal diffraction: Dark red plate-like crystals of C30H28ClFeN4O4 were obtained from
slow evaporation from a chloroform solution. The crystal selected had the approximate
dimensions 0.040 mm x 0.090 mm x 0.210 mm, was used for the X-ray crystallographic analysis.
The X-ray intensity data were measured on a Bruker PROSPECTOR diffractometer system
equipped with a multilayer mirror monochromator and a Cu K1 microfocus sealed tube ( =
1.54178 Å). The data is available from the CCDC 1868333.
Theoretical Methods: The optimized gas phase structures of the non-hydrated dimers for HA
and DHA were calculated using density functional theory, B3LYP/cc-pvdz, with the
experimental structures as starting points. Both correspond to local minima with all positive
vibrational modes. The optimized structures are shown in Figure S9 and the key vibrational
modes collected in Table S8. All of the calculations described above were performed using
14
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Gaussian 98.41 Computations were carried out at the restricted Hartree-Fock (RHF),42 and
Density Functional Theory (DFT) levels .
Supporting Information Supporting information is available online. The structures of
deuterohemin chloride and deuterohematin anhydride have been deposited with the Cambridge
Crystallographic Data Centre, acquisition numbers 1471969 and 1494079 respectively.
§
The nomenclature of the Fe(III)(porphyrin) materials is poorly consolidated. For the
protoporphyrin family Fe(III)(PP)X are named hemin when X = chloride, and hematin with X
= OH. The phase present in malaria pigment, HZ, has X = propionate but the synthetic material
is often incorrectly referred to as β-hematin, that is the second isomeric phase of hematin. In
analogy with the anhydrides of the carboxylic and sulfonic acids we term the propionate dimer
phases as anhydrides; thus hematin anhydride is HA. By analogy the synthetic related material of
deuteroporphyrin is DHA, deuterohematin anhydride. Ferriprotoporphyrin anhydride has also
been suggested as a better name than -hematin.
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10.1002/chem.201805116
Chemistry - A European Journal
Graphical Abstract:
A new structural motif for heme dimerization relevant to malarial heme processing and aggregation has
been found for the iron(deuteroprophyrin) dimer.
Abstract:
Treating deuterohemin, chloro(deuteroprophyrinato)iron(III), with a non-coordinating base in
DMSO and methanol allows for the isolation of [(deuteroporphyrinato)iron(III)]2,
deuterohematin anhydride, DHA, an analog of malaria pigment, the natural product of heme
detoxification by malaria. The structure of DHA from this solvent system has been solved by X-
ray powder diffraction and displays many similarities and important structural differences with
malaria pigment. Most notably the two waters sit in two notches created by the propionate
sidechains and stabilize a markedly bent propionate coordinate with a long Fe-O bond and the
formation of carboxylate cluster associated with the waters. Together these account for its
increased solubility and more open structure with an increased porphyrin-porphyrin separation.
18
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10.1002/chem.201805116
Chemistry - A European Journal
This structure also accounts for the strong IR bands at 1587 cm-1 which are also found in many
amorphous preparations of synthetic malaria pigment and it is proposed that stabilizing these
structures may be new antimalarial drug targets. The important role for the vinyl substituents in
this biochemistry is further demonstrated by the structure of deuterohemin from single crystal X-
ray diffraction.
19
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