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 ADM-116, an anionic oligoquionline-based foldamer, has an intrinsic capacity to fold into a helical structure (Unpublished results, Kumar, 2015).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