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Brad DeMarco, Sunil Kumar, Andrew MirankerSynthesis of Anionic Foldamers for Macromolecule Delivery
IntroductionCell penetrating peptides (CPP’s) have the capacity to permeate through cellular membranes through an active form of transport. They have been used as cargo to deliver macromolecules and drugs across cellular membranes. Constructs such as HIV1-TAT protein have shown that small polypeptides (7-9 residues) can effectively transport an array of macromolecules and have eliminated many obstacles in drug design and delivery (Brooks, 2004). Typically, CPP’s possess two important characteristics: cationic character and helicity. However, CPP’s are limited by their biological half-life in which their degradation can lead to loss of cell penetrating function. Recently, our lab discovered a potent antagonist (ADM-116) of islet amyloid polypeptide (IAPP) fibrillation. ADM-116, an anionic oligoquionline-based foldamer, has an intrinsic capacity to fold into a helical structure (Unpublished results, Kumar, 2015). The folding behavior gives ADM-116 the unique ability to cross cellular membranes passively despite its anionic character. To explain the cell-penetrating dynamics of ADM-116, a derivative (ADM-158) was developed to investigate the viability of ADM-116 as a transport moiety. Azide functionality was introduced at the C-terminus of ADM-158 to covalently append alkyne-based molecules of interest via click chemistry. These oligoquinoline-based conjugates were then introduced to giant plasma membrane vesicles (GPMV’s) and were characterized using confocal scanning laser microscopy (CSLM). Initial studies have shown aggregation of ADM-158 fluorescent conjugates on GPMV surfaces, suggesting targeting and interaction with cell surfaces.
N
NH
OOH
O
OMe
ON
NH
EtO
ON
NH
O
O
HO O
N
NO2
EtO
O
ADM-116
N
NH
OOH
O
O
O
N3
N
NH
EtO
ON
NH
O
O
HO O
N
NO2
EtO
O
ADM-158
Materials and Methods
NH2
NO2
O
CO2CH3NH
NO2
CHCO2CH3
MeOH
CCO2CH3HCCO2C
NH
O
NO2
COOMe
NH
O
NO2
COOMe N
NO2
OOtBu
O
COOMe N
NO2
OOtBu
O
COOH
N
NO2
OOtBu
O
COOH
HO Br
N
NO2
OOtBu
O
O
O
Br N
NH2
OOtBu
O
O
O
Br
NH
O
NO2
COOMe N
NO2
O CH3
COOMe N
NO2
O CH3
COOH
?
1.) LiOH, THF 2.) Acetic Acid
DIAD, THF Triphenyl Phosphine
Pd/C, H2 Atm
DIAD, THF, EtOH Triphenyl Phosphine
1.) LiOH, THF 2.) Acetic Acid
BrCH2CO2tBu, Na2CO3 NaI Acetone/DMF, 70°
Tetrameric Oligoquinoline Synthesis
Results
ConclusionN
NH2
OOtBu
O
O
O
Br
N
NO2
EtO
COOHN
NH
OOtBu
O
O
O
Br
N
NO2
EtO
O
N
NH
OOtBu
O
O
O
Br
N
NH2
EtO
O
2-Chloro-1-Methylpyrridinum iodide Triethylamine Dichloromethane
+
Pd/C, H2 Atm
Fluorescent ADM-158 Conjugate Synthesis
NH
O
O
HN N3
OH
O
R1
O
SO3
SO3
NH2
H2N
HOO
O
HN
O
O
OO
HN
OO
OH
NH2
NH2
SO3
SO3
O
N
N
R1
O
OH
NH
O
O
NH
N
+
Alexa-488 Alkyne
Department of Molecular Biophysics and Biochemistry, Yale University, 266 Whitney Avenue, New Haven CT 06520
References
Synthesis of Oligoquinoline Precursors
Click Chemistry
Fig 1. X-ray Crystallography Structure of ADM-116 (Kumar, 2015)
• A successful click-chemistry active ADM-116 derivative has been synthesized
• ADM-158 is extremely active in click reactions with a high yield
• Appending Alexa-488 alkyne has proven successful
• Aggregation of ADM-158 fluorescent conjugate on cellular membranes has been
observed in initial GMPV exposure
N
NH
OOtBu
O
O
O
Br
N
NH2
EtO
O
N
NH
OOtBu
O
O
O
Br
N
NH
EtO
ON
NH
O
O
tBuO O
N
NO2
EtO
O
N
NH
OOtBu
O
O
O
Br
N
NH
EtO
ON
NH
O
O
tBuO O
N
NO2
EtO
O
N
NH
OOH
O
O
O
N3
N
NH
EtO
ON
NH
O
O
HO O
N
NO2
EtO
O
1.) NaN3, DMF, 70°
2.) DCM, TFA, TES
ADM-158
Future Applications• Fluorescently labelled alkyne oligonucleotides• Fluorescently labelled peptides• Large organic molecules with alkyne modification• Gold nanoparticles• Lipid nanoparticles
Fig 2. GPMV’s introduced to Alexa 488-ADM-158 conjugate (10mM). Pictures taken via CSLM. (Melissa Birol). (A) GPMV only; (B) Multiple GPMV’s with ADM-158 Alexa 488 conjugate exposure and aggregation; (C) Single GPMV with ADM-158 Alexa 488 conjugate exposure and aggregation.
- Jafari, Samira, Solmaz Maleki Dizaj, and Khosro Adibkia. “Cell-Penetrating Peptides and Their Analogues as Novel Nanocarriers for
Drug Delivery.” BioImpacts : BI 5.2 (2015): 103–111. PMC. Web. 6 Aug. 2015.
- Hilary Brooks, Bernard Lebleu, Eric Vivès, Tat peptide-mediated cellular delivery: back to basics, Advanced Drug Delivery Reviews,
Volume 57, Issue 4, 28 February 2005, Pages 559-577, ISSN 0169-409X, http://dx.doi.org/10.1016/j.addr.2004.12.001.
- Kumar, et al. 2015, Unpublished results.
Future Work• Finding solvent system to prevent premature ADM-158 folding and aggregation
in GPMV exposure
• Addition of azide functionality to N-terminus region to maintain methoxy functionality of the C-terminus region
• Attachment of macromolecules as outlined in future application section
A B
C
AcknowledgmentsI would like the thank Dr. Sunil Kumar and Professor Andrew Miranker for their guidance throughout this project. Without his
investment of time this project would not have been possible. I want to thank the National Science Foundation for providing funding
for this project as well as Yale for providing funding and facilities. Moreover, I would like to thank the rest of the Miranker lab for
assisting in imaging and preparation of GMPV samples. Finally, thank you to Doro Noble for organizing the REU program and
providing addition guidance in future endeavors