Design and synthesis of multivalent α-1,2-trimannose-linked bioerodible microparticles for applications in immune response studies of Leishmania major infection

Article abstract of DOI:10.3762/bjoc.15.58

Leishmaniasis, a neglected tropical disease, currently infects approximately 12 million people worldwide with 1 to 2 million new cases each year in predominately underdeveloped countries. The treatment of the disease is severely underdeveloped due to the

Full text of DOI:10.3762/bjoc.15.58

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Design and synthesis of multivalent α-1,2-trimannose-linked
bioerodible microparticles for applications in immune
response studies of Leishmania major infection
Chelsea L. Rintelmann1, Tara Grinnage-Pulley2,3,4, Kathleen Ross4,5,
Daniel E. K. Kabotso1, Angela Toepp2,3, Anne Cowell1, Christine Petersen*2,3,4,
Balaji Narasimhan*4,5 and Nicola Pohl*1,4
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2019, 15, 623–632.
1Department of Chemistry, Indiana University Bloomington, 800 E.
Kirkwood Ave., Bloomington, Indiana 47405-7102, USA, 2Department
of Epidemiology, College of Public Health, University of Iowa, 105
River Street, S444 CPHB, Iowa City, Iowa 52242, USA, 3Center for
Emerging Infectious Diseases, University of Iowa Research Park,
2500 Crosspark Road, MTF B166 Coralville, Iowa 52241, USA,
4Nanovaccine Institute, Iowa State University, 2114 Sweeney Hall,
Ames, Iowa 50011-2230, USA and 5Department of Chemical and
Biological Engineering, Iowa State University, 618 Bissell Road,
Ames, Iowa 50011-2230, USA
doi:10.3762/bjoc.15.58
Received: 26 November 2018
Accepted: 20 February 2019
Published: 11 March 2019
Associate Editor: D. Spring
© 2019 Rintelmann et al.; licensee Beilstein-Institut.
License and terms: see end of document.
Email:
Christine Petersen* - christine-petersen@uiowa.edu;
Balaji Narasimhan* - nbalaji@iastate.edu; Nicola Pohl* -
npohl@indiana.edu
* Corresponding author
Keywords:
adjuvant; carbohydrates; L. major; microparticle; PAMP
Abstract
Leishmaniasis, a neglected tropical disease, currently infects approximately 12 million people worldwide with 1 to 2 million new
cases each year in predominately underdeveloped countries. The treatment of the disease is severely underdeveloped due to the
ability of the Leishmania pathogen to evade and abate immune responses. In an effort to develop anti-leishmaniasis vaccines and
adjuvants, novel carbohydrate-based probes were made to study the mechanisms of immune modulation. In this study, a new
bioerodible polyanhydride microparticle was designed and conjugated with a glycodendrimer molecular probe. This molecular
probe incorporates a pathogen-like multivalent display of α-1,2-trimannose, for which a more efficient synthesis was designed, with
a tethered fluorophore. Further attachment of the glycodendrimer to a biocompatible, surface eroding microparticle allows for
targeted uptake and internalization of the pathogen-associated oligosaccharide by phagocytic immune cells. The α-1,2-trimannose-
linked bioerodible microparticles were found to be safe after administration into the footpad of mice and demonstrated a similar
response to α-1,2-trimannose-coated latex beads during L. major footpad infection. Furthermore, the bioerodible microparticles
allowed for investigation of the role of pathogen-associated oligosaccharides for recognition by pathogen-recognition receptors
during L. major-induced leishmaniasis.
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Beilstein J. Org. Chem. 2019, 15, 623–632.
Introduction
Recognition of parasite cell surface molecules by host immune responsible for host protection. Through a range of mecha-
cells initiates the first step in the immune response [1,2]. The nisms not entirely understood, Leishmania subverts activation
host’s immune system recognizes parasite surface glycoconju- of antimicrobial (nitric oxide) and cytokine inducible functions
gates, or pathogen-associated molecular patterns (PAMPs), to that initiate effective immunity [5]. There are no approved anti-
build an immune response against the parasite and impede leishmanial vaccines and the current therapeutics are toxic,
disease progression. Antigen presenting cells (APCs) recognize expensive and prone to induce resistance, such as, ampho-
these PAMPs as pathogenic as compared to host glycans tericin B (AmB), paromomycin and antimony-based chemother-
making these moieties feasible targets for both parasite detec- apeutics [7-9]. With growing resistance to antileishmanial drugs
tion and vaccine development [3].
and poor patient compliance, the development of safe,
efficacious and inexpensive alternatives to current therapeutics
Leishmania, an obligate intracellular parasite with varying sur- is needed. Understanding the mechanisms by which
face glycoconjugates, infects an estimated 12 million people Leishmania regulates host immune responses are crucial for the
worldwide and has 350 million people at risk in endemic areas development of vaccines, which may induce adaptive
(predominately in underdeveloped countries). The World immunity that may prove beneficial for combating other intra-
Health Organization estimates that this neglected tropical cellular pathogens, such as Mycobacterium tuberculosis
disease causes between 1–2 million new cases annually [4]. [10,11].
Leishmaniasis manifests into three different forms, cutaneous,
mucosal, or visceral, depending on parasite species, host Carbohydrate-based probes provide one method to investigate
immune system and location of infection [5]. Cutaneous, the parasitic mechanisms of immune suppression and evasion. The
most common form, causes severe nodular and ulcerative skin cell surface glycoconjugates on Leishmania have been implicat-
lesions, while mucocutaneous destroys mucus membranes, and ed in the ability of the parasite to infect host cells, then evade
the most deadly form, visceral leishmaniasis, often results in and suppress host immune responses [5,12-15]. The most
organ failure [6]. L. major, the parasite used in our in vivo prominent of these cell surface glycoconjugates, lipophospho-
model, causes the cutaneous disease. In each pathogenesis, the glycan (LPG), consists of a glycosylphosphatidylinositol (GPI)
parasite infects and propagates in immune cells, the very cells anchor and variable sugar capping structures (Figure 1) [15-20].
Figure 1: Leishmania lipophosphoglycan (LPG), with Gal-β-(1→4)-[Man-α-(1→2)-Man-α-(1→2)-Man] variant capping structure highlighted in blue and
R = H or species dependent oligosaccharide [25].
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Beilstein J. Org. Chem. 2019, 15, 623–632.
The capping sugar tropisms vary with Leishmania species through toll-like receptor 2 (TLR-2) and mannose receptor
(>20 species) and life stage of the pathogen, making it chal- (MR)-dependent pathways by increasing the production of
lenging to define how the capping sugars interact and modulate IL-12, a key cytokine involved in recruiting a T cell-mediated
host immune responses [21-24]. Since these cell-surface adaptive immune response [11,40,42]. Furthermore, our acid-
glycans are structurally diverse and difficult to isolate in appre- labile probe was found to increase T cell production, suggesting
ciable quantities, synthetic pathogen-associated carbohydrate α-1,2-trimannose antigen presentation at the major histocompat-
probes are necessary to understand the LPG structure-to-func- ibility complex II (MHC II) [41].
tion relationship. These synthetic oligosaccharides also provide
a structurally homogeneous standard to study how these glycans Based on our previous studies [11,40-42], we sought to design
interact with the host immune system in an effort to develop and synthesize a new Leishmania-associated molecular probe
effective anti-Leishmania vaccines.
that could allow intracellular monitoring and validation of T
cell antigen presentation. This new probe would also be linked
Previously, discrete structural components of LPG have been to the surface of a polyanhydride copolymer microparticle
synthesized [26-38] and their involvement in modulating an based on 1,6-bis(p-carboxyphenoxy)hexane (CPH) and sebacic
immune response of these reduced motifs has been investigated anhydride (SA) to allow for enhanced internalization and uptake
[34-36,39]. The Pohl and Petersen groups have previously in- of the immunogenic glycoconjugate through recognition by
vestigated how truncated oligosaccharide capping structures of phagocytic cells [43-45]. Here we discuss the design and syn-
LPG interact with macrophage pattern recognition receptors thesis of this new bioerodible microparticle 2 compared to our
(PRRs) to modulate an innate immune response to L. major-in- previously designed [11] trimannose-coated latex bead
duced leishmaniasis in vitro and in vivo [11,40-42]. These construct 1 (Figure 2) and evaluate the biocompatibility of the
studies showed that a neutral α-1,2-trimannose capping struc- microparticle in spared dose administration to L. major infected
ture alone is sufficient to modulate an innate immune response mice (Figure 4).
Figure 2: Pathogen-associated bioerodible microparticles 2 and trimannose-coated latex beads 1.
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Beilstein J. Org. Chem. 2019, 15, 623–632.
Results and Discussion
the dendrimer would allow for standard immunofluorescence
To further probe the biological mechanisms responsible for assays, to track the microparticle following cellular uptake and
L. major immune regulation, a new adjuvant-like pathogen-as- intracellular processing. TAMRA’s high photostability, low pH
sociated molecular probe was designed to promote pathogen sensitivity and ease of incorporation through amide bond cou-
recognition receptor (PRR)-mediated internalization and slow pling, facilitates synthetic manipulation and biocompatibility
erosion in the acidic phagolysosome of macrophages. This [48,49].
bioerodible microparticle 2 design evolved from earlier synthe-
tic probes with improved translatability for therapeutic develop- In an effort to make a direct comparison to our previous biolog-
ment [11,41,42]. To mimic the Leishmania pathogen, the size of ical studies, trimannose-coated latex beads also were synthe-
our probe needed to be approximately the same size as the sized using 1 μm FluoSpheres© carboxylate-modified latex
pathogen (1 μm in diameter) and provide a multivalent display beads labeled with a red fluorophore (emission maximum
of the synthetic α-1,2-trimannose similar to the Leishmania 580 nm). The latex-bead synthesis was modified slightly from
promastigote cell-wall glycans. Furthermore, a tethered fluoro- previous synthesis for improved ease of synthesis of triman-
phore would allow for immunofluorescence assays, intracel- nose and comparability to this study’s bioerodible microparti-
lular trafficking, and validation of T cell-mediated antigen cles by coupling the reducing end of trimannose with the afore-
recognition. To allow for both surface functionalization of this mentioned amino alcohol spacer [11,41]. Biological analysis of
glycan to the bioerodible microparticles and conjugation of the the trimannose-coated latex beads was described previously
fluorophore, two amine handles would be necessary (Figure 3). [42] and used to compare the new construct.
The design would also benefit from the development of a more
efficient synthesis of the mannose oligomer sidechain.
Synthesis of α-1,2-trimannose
The synthesis of α-1,2-trimannose, required for both the glyco-
From the general design elements in Figure 3, we proposed the dendrimer 2 and latex beads 1, was achieved through iterative
following synthetic motifs. For PRR binding, a dendrimeric glycosylation with known trichloroacetimidate mannosyl
scaffold provides a multivalent display of the α-1,2-trimannose donors 3 and 4 [47,50] and the use of a fluorous tag containing
glycoconjugate, similar to Leishmania LPG and additionally an amino alcohol spacer rather than an alkene that would
may promote glycodendrimer antigen presentation when hydro- require late-stage modifications. The aminopentanol spacer 6
lyzed in the acidic phagolysosome [39,41,46]. To improve the used at the reducing end to conjugate to the dendrimer was first
overall synthetic sequence from our previous syntheses protected with fluorous CbzF-NHS 5 to allow for purification
[11,41,42], an amino alcohol spacer was utilized to connect the by FSPE of the mannosyl intermediates and to facilitate future
trimannose reducing end to the dendrimer by means of amide automated syntheses to produce α-1,2-trimannose in appre-
bond coupling. Furthermore, the amino alcohol spacer provides ciable quantities similar to previous efforts (Scheme 1)
a handle for the incorporation of a fluorous-tag for ease of [11,47,51].
purification by fluorous solid phase-extraction (FSPE). The use
of the fluorous tag enables solution-phase automated synthesis To synthesize the oligosaccharide, activation of TCA donor 3
of α-1,2-trimannose in the future as shown previously with a with TMSOTf and glycosylation of the CbzF-protected
related spacer and fluorous tag [47]. Incorporation of the com- aminopentanol 7 afforded the monosaccharide 8 (Scheme 2).
mercially available rhodamine dye, TAMRA (555ex/580em), to The 2-O-position was then deacylated, followed by iterative
Figure 3: General design of the bioerodible microparticles.
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