This document summarizes research on synthesizing anionic foldamers for macromolecule delivery across cellular membranes. Key points: - Anionic foldamers like ADM-116 have an intrinsic ability to fold into a helical structure and passively penetrate cell membranes despite being negatively charged. - ADM-158, a derivative of ADM-116, was developed with an azide group to attach fluorescent molecules via click chemistry for studying its cell-penetrating abilities. - Initial studies show ADM-158 conjugates aggregate on giant plasma membrane vesicle surfaces, indicating interaction with cell surfaces. Further studies will explore attaching other cargos like oligonucleotides, peptides, and nanoparticles using

1. Brad DeMarco, Sunil Kumar, Andrew Miranker
Synthesis of Anionic Foldamers for Macromolecule Delivery
Introduction
Cell 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foldamer, has an intrinsic capacity to fold into a helical structure (Unpublished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
O
OH
O
OMe
O
N
NH
EtO
O
N
NH
O
O
HO O
N
NO2
EtO
O
ADM-116
N
NH
O
OH
O
O
O
N3
N
NH
EtO
O
N
NH
O
O
HO O
N
NO2
EtO
O
ADM-158
Materials and Methods
NH2
NO2
O
CO2CH3N
H
NO2
CHCO2CH3
MeOH
CCO2CH3HCCO2C
N
H
O
NO2
COOMe
N
H
O
NO2
COOMe
N
NO2
O
OtBu
O
COOMe N
NO2
O
OtBu
O
COOH
N
NO2
O
OtBu
O
COOH
HO Br
N
NO2
O
OtBu
O
O
O
Br N
NH2
O
OtBu
O
O
O
Br
N
H
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
O
OtBu
O
O
O
Br
N
NO2
EtO
COOH
N
NH
O
OtBu
O
O
O
Br
N
NO2
EtO
O
N
NH
O
OtBu
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
H
N N3
OH
O
R1
O
SO3
SO3
NH2
H2N
HO
O
O
H
N
O
O
O
O
H
N
O
O
OH
NH2
NH2
SO3
SO3
O
N
N
R1
O
OH
NH
O
O
N
H
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
O
OtBu
O
O
O
Br
N
NH2
EtO
O
N
NH
O
OtBu
O
O
O
Br
N
NH
EtO
O
N
NH
O
O
tBuO O
N
NO2
EtO
O
N
NH
O
OtBu
O
O
O
Br
N
NH
EtO
O
N
NH
O
O
tBuO O
N
NO2
EtO
O
N
NH
O
OH
O
O
O
N3
N
NH
EtO
O
N
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
Acknowledgments
I 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