This document summarizes research on synthesizing anionic foldamers for macromolecule delivery. It discusses how charge and secondary structure influence cell penetration, and how the research group synthesized quinoline oligomers and conjugated them to Alexa fluorophores via click chemistry. The conjugates could deliver cargo like oligonucleotides, peptides, and be used to assay for successful cell penetration. The overall goal is to develop non-toxic anionic molecules that can transport therapeutic macromolecules into cells.

1. Synthesis and Identification
of Anionic Foldamers for
Macromolecule Delivery
Brad DeMarco Sackler/REU
Miranker Research Group

2. Membrane Active Peptides
IAPP
• Involved in Type II Diabetes
• Passes through cell membranes
• Toxic To Cells
Cell Penetrating
Peptides
• Non-toxic
• Studied as cargo transporters

3. Membrane Active Peptides
IAPP
• Involved in Type II Diabetes
• Passes through cell membranes
• Toxic To Cells
Cell Penetrating
Peptides
• Non-toxic
• Studied as cargo transporters
Our Project

4. Charge and Secondary Structure are Important in Cell Penetrating
Behavior
David B. Thompson, James J. Cronican, David R. Liu, Chapter twelve - Engineering and Identifying Supercharged Proteins for Macromolecule Delivery into Mammalian Cells
• Positive character is very important in cell penetrating peptides
• Charge alone can help transport molecules across the membrane
• Electrostatic interactions with cell surfaces are a predominate factor in cell penetration
- +

5. Folding is Equally Important in Cell Penetration
Martín, I., Teixidó, M. & Giralt , E. Design, Synthesis and Characterization of a New Anionic Cell‐Penetrating Peptide: SAP(E). ChemBioChem 12, 896–903 (2011).
• Secondary structure is also directly responsible for the penetrating ability of a
molecule
• High helical propensities relate to its ability to cross membranes

6. ADM-116 Rescues IAPP Toxicity Intracellularly
N
O
NH
N
O
NH O
O
N
O
NH O
N
O
O
O OH
O OH
CH3
CH3
NO2
OMe
• Inhibitor of IAPP
• Cell penetrating property is attributed to its folding behavior
• Takes a passive mode of diffusion
• Anionic
Dr. Melissa Birol
ADM-116

7. Building Monomeric Quinoline Units
NH2
NO2
O
CO2CH3N
H
NO2
CHCO2CH3
MeOH
CCO2CH3
HCCO2C
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°
Dr. Sunil Kumar
∆
95%
~90% 20%
60%~90%
~90%
95%

8. ADM-116 Azide Functionalized Analog
Quinoline Tetrameric Synthesis Scheme
Dr. Sunil Kumar
N
NH
O
OH
O
O
O
N3
N
NH
EtO
O
N
NH
O
O
HO O
N
NO2
EtO
O
ADM-158
N
NH2
O
OtBu
O
O
O
Br
N
NO2
EtO
COOH
N
NH
O
OtBu
O
O
O
Br
N
NO2
EtO
O
2-Chloro-1-Methylpyrridinum iodide
Triethylamine
Dichloromethane
+
Pd/C, H2 Atm
N
NH
O
OtBu
O
O
O
Br
N
NH2
EtO
O
1.) Dimer Formation and Reduction

9. ADM-116 Azide Functionalized Analog
Dr. Sunil Kumar
N
NH
O
OH
O
O
O
N3
N
NH
EtO
O
N
NH
O
O
HO O
N
NO2
EtO
O
ADM-158
N
NH
O
OtBu
O
O
O
Br
N
NH2
EtO
O
N
NO2
O
COOH
tBuO O
+
N
NH
O
OtBu
O
O
O
Br
N
NH
EtO
O
N
NO2
O
O
tBuO O
2-Chloro-1-Methylpyrridinum iodide
Triethylamine
Dichloromethane
Pd/C, H2 Atm
N
NH
O
OtBu
O
O
O
Br
N
NH
EtO
O
N
NH2
O
O
tBuO O
Quinoline Tetrameric Synthesis Scheme
2.) Trimer Formation and Reduction

10. ADM-116 Azide Functionalized Analog
Dr. Sunil Kumar
N
NH
O
OH
O
O
O
N3
N
NH
EtO
O
N
NH
O
O
HO O
N
NO2
EtO
O
ADM-158
N
NH
O
OtBu
O
O
O
Br
N
NH
EtO
O
N
NH2
O
O
tBuO O
N
NO2
EtO
COOH
+
2-Chloro-1-Methylpyrridinum iodide
Triethylamine
Dichloromethane
N
NH
O
OtBu
O
O
O
Br
N
NH
EtO
O
N
NH
O
O
tBuO O
N
NO2
EtO
O
Quinoline Tetrameric Synthesis Scheme
3.) Tetramer Synthesis and Reduction

11. ADM-116 Azide Functionalized Analog
Dr. Sunil Kumar
N
NH
O
OH
O
O
O
N3
N
NH
EtO
O
N
NH
O
O
HO O
N
NO2
EtO
O
ADM-158
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
DCM, TFA, TESNaN3, DMF,
Quinoline Tetrameric Synthesis Scheme
4.) Substitution and Deprotection

12. Synthesis of Tetramer Alexa® Conjugates via Click Chemistry
R2
N3
HC
R1
N
N N
R2
R1
+
Cu(I) (cat)
H2O
• High yield and easy to carry out
• Creates byproducts that can be separated without column chromatography
• Uses inert solvents
• Allows addition of vast library of substituents
NH
O
O
H
N N3
OtBu
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
tBuO
NH
O
O
N
H
N
+
Cu (I) Cat.
Alexa-488 Alkyne

13. Future Applications
R Groups
Alkyne Alexa 488 Fluor®
FAM-Oligonucleotides
FAM-Labelled Neutral Peptides (Gly-Ser)
FAM-Labelled Postive Peptides (His-Lys)
N
NH
O
OH
O
O
O
N
N
NH
EtO
O
N
NH
O
O
HO O
N
NO2
EtO
O
N
N
R

14. Future Applications
N
NH
O
OH
O
O
O
N
N
NH
EtO
O
N
NH
O
O
HO O
N
NO2
EtO
O
N
N
R
GPMV
• Allows for easy assay of successful cell penetration
• Possible fluors can be Alexa™ and Fluorescein alkyne nucleotide/peptide conjugates
(Large Vesicle From Cell Membranes)

15. Acknowledgements
• Sackler REU Program
• National Science Foundation
Dr. Sunil Kumar Professor Andrew Miranker
Miranker LabMentor