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