Design of PolyMOCs and the synthesis of crosslinkers for the BASP platform

Design of PolyMOCs and the Synthesis of Crosslinkers for the BASP Platform by

Jiwon Victoria Park Submitted to the Department of Chemistry

in Partial Fulfillment of the Requirements for the Degree of Bachelor of Science

at the

ts the MASSACHUSETTS INSTITUTE

OF TECHNOLOGY Massachusetts Institute of Technology FTCNLG

June 2017

_JUL

_{0 6 2017}

C 2017 Massachusetts Institute of Technology

LIBRARIES

The author hereby grants to MIT permission to reproduce and to distribute publicly paper and electronic copies of this thesis document in whole or in part in any medium now

known or hereafter created.

Signature redacted

Signature redacted

Signature redacted

Accepted by

Troy Van Voorhis Haslam and Dewey Professor of Chemistry Undergraduate Officer, Department of Chemistry

Design of PolyMOCs and the Synthesis of Crosslinkers for the BASP Platform

by Jiwon V. Park

Submitted to the Department of Chemistry

on May 26, 2017 in Partial Fulfillment of the Requirements for the Degree of Bachelor of Science in Chemistry

ABSTRACT

In Chapter 1, two stepwise assembly strategies for the integration of metal-organic cages (MOCs) into polymers were explored. The first strategy creates Block Co-PolyMOCs (BCPMOCs), which feature the integration of MOCs into block copolymers (BCPs). In the first assembly step, BCPs functionalized with a bispyridyl ligand on the chain end undergo Pd induced MOC assembly. In the second step, microphase separation of BCPs is induced, introducing a physical cross-link between the star polymers and producing the desired BCPMOC networks in the bulk or gel state. In the second strategy, another orthogonal interaction is explored to create a different type of polyMOC. In this case, poly(methyl acrylate) (PMA) homopolymers are synthesized from initiators featuring a diene or dienophile on one end and functionalized with a bispyridyl ligand on the other end. Diels-Alder (DA) cycloaddition is used in the second step to create a polyMOC network. Given the functional diversity of MOCs, both strategies should enable access to materials with a wide range of properties and applications.

Chapter 2 outlines the synthesis of norbornene macromonomers (MMs) with varying anchor groups and crosslinkers that are stimuli-sensitive for the brush-arm star polymer (BASP) drug delivery platform. Variation of MM anchor groups modifies the rate of propagation of ring-opening metastasis polymerization (ROMP), while the design of crosslinkers that are acid- and photo-labile contributes to the expansion of a wide-ranging library of crosslinkers for drug loading and release. The brush-first ROMP

polymerization strategy allows for the synthesis of BASPs for single drug or multiple drug combinations.

Thesis Supervisor: Jeremiah A. Johnson

Acknowledgements

I would like to thank Professor Johnson, Yufeng Wang, Hung Van Thanh Nguyen, and Junpeng Wang for their continual support and mentorship for my research endeavors. This research was done in the Johnson Research Group as part of the MIT Undergraduate Research Opportunities Program (UROP).

TABLE OF CONTENTS

Chapter I. PolyMOCs by Orthogonal Crosslinking Strategies Introduction

Results and Discussion Conclusion

Experimental Methods Spectral Data

Chapter II. Extending the Brush Arm Star Polymer (BASP) Platform for Drug Delivery

Introduction

Results and Discussion Conclusion

Experimental Methods Spectral Data

Chapter III. References References

Chapter I. PolyMOCs by Orthogonal Crosslinking Strategies Introduction

Coordination-directed self-assembly of metal ions and organic ligands is a process in which multiple subunits spontaneously join together to form three-dimensional molecular architectures, such as metal-organic cages (MOCs) and metal-organic frameworks (MOFs).' MOCs are assembled from various stoichiometries of metal ions and organic ligands. By manipulating the design of the ligands and selecting proper metal ions, MOCs can be self-assembled through metal coordination and incorporate well-defined geometries, sizes, and cavities potentially useful in encapsulation, catalysis, drug delivery, and sensing.2 _{These cage-like structures are comprised of nanocavities with}

multiple open windows allowing for passage of small molecules. The cavities can also be leveraged to encapsulate molecules or bind to other guest species.

Due to the dynamic nature and versatility of MOCs, there has been an ongoing effort to introduce them into polymer networks to provide polyMOCs. These new hybrid materials can have tunable viscoelastic properties and are capable of having functions originally only possible for MOFs and MOCs. 5 For example, there have been reports of the synthesis of polyethylene glycol (PEG) hydrogels cross-linked via assembly of M.LY clusters (x metal ions and y ligands for each junction).' Despite advances in the field, an understanding of how the polyMOC microstructure translates to bulk material properties, such as modulus and relaxation dynamics, is necessary. To gain further understanding, detailed and modular polyMOC synthesis strategies that enable access to various structures and properties are essential.

Previously, the Johnson Group described a polyMOC design, in which a linear polymer chain was functionalized with an identical bispyridine-based ligand on both chain ends. With the introduction of Pd and thermal annealing, the metal ions and PEG terminated with para-bispyridyl ligands formed ML24 cages with varying defects, such

as primary loops.7 _{While they were able to design highly branched and loop-rich gels, the}

process still had a few limitations. Since the polymers had metal-coordinating ligands on both chain ends, the resulting network was assembled in one-step, resulting in polyMOCs with a high percentage of topological defects.7

To address this issue, a two-step assembly strategy was designed to introduce orthogonal interactions after MOC formation (Figure 1).' In this approach, the ligands attached on the end of the polymer chain first undergo metal-coordination, driving the formation of MOCs. Next, the network structure is formed through interactions that are orthogonal to metal coordination. In this chapter, two new classes of polyMOCs based on this strategy are described.

First, we developed block co-polyMOCs (BCPMOCs), which are synthesized via stepwise self-assembly first of MOCs and then of BCPs. More specifically, we demonstrated the synthesis of BCPMOCs featuring Fujita-sphere ML24 MOCs and

paddlewheel M2L4 MOCs (Figure 2). These materials were able to undergo phase

separation and physical cross-linking to form non-covalent orthogonal crosslinks at polymer junctions. The BCPMOCs that we synthesized featured one glassy block, one rubbery block, and a pyridyl ligand on the end of the rubbery block. ATRP was used for the preparation of the BCPs. These BCPs underwent metal-coordination-driven assembly in solution, resulting in star polymers with well-defined MOC cores and precise number

of polymer arms. After removing the solvent or adding a solvent that is selective for one block of the BCP, the polymers underwent phase-separation and physically cross-linked to yield BCPMOCs as thermoplastic elastomers (TPE) or thermoresponsive organogels.'

Motivated by this work, we decided to alter the assembly strategy from physical BCP crosslinking to covalent orthogonal crosslinks via Diels-Alder (DA) reactions. The ends of poly(methyl-acrylate) PMA polymers were functionalized with a pyridyl ligand on one end and either a diene or dienophile on the other end. The Diels-Alder reaction (which involves a [4+2] cycloaddition between a diene and dienophile) was explored because of its versatile substrate scope, high efficiency, and compatibility with MOCs.9- 0

and _2nd assembly

Metal-organic cages

Orthogonal crosslinks

Figure 1. Orthogonal interactions and step-wise self-assembly of polyMOCs.' N

Pd2*

.-4-- R 120* VS. R 7 o

"Fujita sphere" L-para L-meta

Figure 2. Structure of Fujita sphere and paddlewheel MOCs. Pd2. M2L4 paddlewheel LI refers to L-meta

1

Results and Discussion

1.1 PolyMOCs by Phase Separation 1.1.1 Design of Ligands

For the synthesis of BCPMOCs, poly(methyl methyacrylate)-block-poly(n-butyl acrylate) (PMMA-PBA) with a pyridyl ligand at the chain end was used. PMMA-PBA is well known to phase separate in the bulk state and in appropriate solvents."-" We

incorporated the meta and para pyridyl ligands as shown in Figure 2. In the presence of Pd 2 _{, these ligands formed PdL, complexes with either paddlewheel or Fujita-sphere}

geometries.'

1.1.2 BCP Synthesis

Atom transfer radical polymerization (ATRP) followed by post-polymerization

functionalization was used to synthesize the BCPs.' The PMMA block was synthesized using ethyl a-bromophenylacetate (EBPA) as the initiator, yielding polymers with low dispersities (D = 1.05-1.10) and controlled molecular weights." The obtained PMMA then served as a macroinitiator for the synthesis of PMMA-PBA by consecutive chain extension. To functionalize PMMA-PBA with the desired bispyridyl ligand, a

nucleophilic substitution reaction was used, displacing the polymer chain-end bromide with the appropriate pyridyl phenol ligand. The functionalized polymers were purified by

silica gel chromatography. 'H nuclear magnetic resonance (NMR) spectroscopy verified the presence of protons from the initiator and bispyridine chain end.

When dissolved in acetonitrile with palladium nitrate, PMMA-PBA-L coordinated with Pd to assemble into clusters upon thermal annealing. When Pd is added, most of the peaks corresponding to the ligand shifted downfield. After 4 h of annealing at 80 'C, a

sharper set of peaks formed, indicating the formation of well-defined cage structures (Figure 6). Since the EBPA initiator is far from the MOC junction, the peaks

corresponding to the initiator did not change before and after the assembly.

Moreover, ligand exchange during the MOC assembly process was investigated by 'H NMR. M2L4 and M2L2 4 MOCs were mixed at room temperature and then heated at 50 'C

overnight. The cages were stable at 50 'C. A small amount of free ligand was added to the M2L4 MOC solution to enhance ligand exchange at 80 'C overnight. When heated at

80 *C overnight, new peaks developed, indicating ligand exchange or MOC degradation (Figure 9).

1.2 Diels-Alder Reaction 1.2.1 Design of Ligands

For the synthesis of PolyMOCs (PMOCs), poly(methyl acrylate) (PMA) with the L2 bispyridine ligand at the chain end was used. L2 was synthesized according to reported a literature procedure.' PMA is known to have interesting thermoresponsive properties." In the presence of Pd , these ligands formed M12L24, Fujita spheres, complexes.

Several routes were explored to design a PMOC system utilizing DA chemistry. The less successful attempts are summarized below.

I. A furfuryl alcohol and protected maleimide DA linkage was explored initially. The polymers were synthesized using a furfuryl alcohol and protected maleimide initiator using ATRP at 70 *C. The synthesis of the protected maleimide-PMA polymer at 70 'C proved to be problematic due to biradical termination. GPC analysis showed a slight shoulder next to the major peak. In addition, a gel formed when Pd(II) was added to deprotected-maleimide-PMA-L2.

II. Different dienophile initiators were explored, including an electrophilic alkyne. I explored the synthesis of the electrophilic alkyne by coupling NHS by EDC and DCC. The coupling conditions I used either led to no coupling or a very low yield.

As a result, we are now attempting to change the conditions of ATRP for the synthesis of the protected maleimide-PMA polymer. UV-induced polymerization of maleimide (Mal)-PMA allows the polymerization to be performed at room temperature so that the

protected maleimide does not degrade.'7 _{We explored the synthesis of Mal-PMA-L2 and}

anthracene (Anth)-PMA-L2 homopolymers using photoinduced ATRP modified from the procedure reported by Hao et. al in 2017 (Figure 3)." Following their procedure, a

protected maleimide initiator was added to methyl acrylate monomer in the presence of Cu(II)Br and Me6Tren and exposed to UV light at room temperature. The synthesis of

various time points and scaled up to undergo Fujita sphere assembly and to test mechanical properties.

Based on initial model DA reactions of Furan-MalOH and Anthracene-OH in toluene at 70 *C for 24 h, it appears that the conversion is incomplete, resulting in the formation of two other byproducts (Figure 4). The synthesis of the appropriate polymers and optimization of the conditions for DA chemistry are ongoing.

a.N

0

S

I

Figure 3. (a) Synthesis of Mal-PMA-L2 (b) and Anth-PMA-L2 (c) Design of PMOCs by DA reaction.

0 RPVP

OH OH 0 700C N

Toluene

Figure 4. Model DA reaction of furan-protected maleimide and Anthracene-OH

Conclusion

This chapter describes two different strategies for the design of PMOC networks: one makes use of microphase separation and the other DA reactions. We developed a new class of block co-polyMOCs (BCPMOCs) based on stepwise assembly of MOCs followed by BCP microphase separation. The BCPMOC design elucidated here can be utilized for various other functional materials. In addition, a DA linkage was tested as another option for orthogonal crosslinking of PMOCs. Although not yet successful, this strategy may lead to the formation of materials with interesting dynamic properties by making use of the reversible nature of MOC formation and DA reactions.

Experimental Methods

All monomers used, methyl methacrylate (MMA), methyl acrylate (MA), and n-butyl acrylate (BA) were purchased from Sigma-Aldrich and passed through a basic alumina column to remove inhibitor before use. Tris(2-(dimethylamino)ethyl)amine (Me6Tren),

copper(II) bromide (CuBr2), and ethyl u-bromophenylacetate (EBPA) were purchased

from Sigma-Aldrich used as received. 3,5-Dibromoanisole, 3-pyridylboronic acid pinacol ester, 4-pyridylboronic acid pinacol ester, and tetrakis(triphenylphosphine) palladium(O) (Pd(PPh3)4) were purchased from Ark Pharm, Inc. and used as received. All other

Ligand synthesis:

L2 was synthesized following literature procedures. LI was synthesized using a similar protocol as described below.19-20

Synthesis of 3,5-di(pyridin-3-yl)phenol (LI)

1,3-Dibromo-5-methoxybenzene (2.66 g, 10.0 mmol), 4-pyridineboronic acid pinacol ester (5.0 g, 24.4 mmol), tetrakis(triphenylphosphine) palladium(0) (1.16 g, 1 mmol), and potassium carbonate (13.8 g, 0.1 mol) were added into a 100 mL single-neck round-bottom flask equipped with a magnetic stir bar and capped with a septum. Under nitrogen atmosphere, 60 mL of DMF along with 2 mL of H20 were added and the

resulting mixture was stirred for 60 h at 100 'C. After cooling to room temperature, the reaction residue was diluted with chloroform/methanol (10/1, v/v) and filtered through Celite. The filtrate was extracted with brine, dried over anhydrous Na2SO4, and

concentrated under vacuum. Using silica gel column chromatography, the crude product was purified to yield a light yellow solid. The solid was further dried under high vacuum, and was dissolved in 100 mL anhydrous dichloromethane (DCM) in a 250 mL round bottom flask equipped with a stir bar. The solution was cooled to -78 'C, and BBr₃ (50.0 mL, 1.0 M in CH2Cl2, 50 mmol) was added slowly to the flask over the course of 30 min.

The reaction was stirred for another 10 min before it was gradually warmed to room temperature and stirred for 8 h. The reaction residue was poured into 400 mL of iced sodium bicarbonate saturated aqueous solution to quench. A light-yellow precipitate formed and was collected by filtration. The solid was further purified by washing with

deionized water, ethyl ether, and a large amount of CH2Cl2 to yield 1.1 g of L2 as a white

solid (65 %). 'H NMR is shown below.

Synthesis of PMMA-PBA block copolymer'

PMMA-PBA was synthesized via activator regenerated by electron transfer (ARGET) ATRP. A representative procedure is as follows. A long-neck Schlenk flask equipped with a magnetic stir bar was charged with 1.0 g of PMMA, 10 mL of n-butyl acrylate, 2 mL of 0.047 M Me6Tren in anisole, and 300 RL of 0.076 M CuBr2 in DMF.

The resulting solution was degassed by bubbling nitrogen for 45 min. After that, 1.0 mL of tin(II) 2-ethylhexanoate in anisole (0.1 M, degassed) was added. The reaction mixture was then submerged in an oil bath that was maintained at 70 *C. Reaction aliquots were withdrawn at timed intervals to calculate the conversion based on 'H NMR analysis. The reaction was quenched after 4 h by opening the flask to air and cooling to room

temperature. Immediately after that, the reaction mixture was diluted in THF and passed through a column of neutral alumina to remove the copper catalyst. The resulting solution was then concentrated and dried under high vacuum at 50 *C.

Block copolymer end-functionalization (synthesis of PMMA-PBA-L)

A long-neck Schlenk flask equipped with a magnetic stir bar was charged with 1.0

g of PMMA-PBA block copolymer, 200 mg of Ligand LI or L2, 300 mg Cs2CO3,

and a catalytic amount of KI. The Schlenk flask was then evacuated and back-filled with nitrogen for three cycles. Under nitrogen, 3.0 mL of DMF was added and the reaction mixture was heated to and maintained at 60 *C in an oil bath. After 8 h, the DMF was

removed under high vacuum. The functionalized block copolymer was isolated via silica gel column chromatography (3 % methanol in DCM). Due to the pyridine group,

the functionalized block copolymers have a higher polarity, making it straightforward to purify the polymer using silica gel chromatography. The 'H NMR spectrum of

PMMA4kPBA19k-L3 is shown in Figure 7.

Characterization Methods

Nuclear magnetic resonance spectroscopy (NMR). I H NMR was recorded using a Bruker AVANCE-400 NMR spectrometer. Chemical shifts are reported in parts per million (ppm), and are referenced to residual solvent peaks. Scalar coupling

Spectra Data d -M n _b e -. a e a b, c d o 46 -4 I I I I 10 9 f I I I I 8 7 6 5 4 3 2 1 0 IH Chemical Shift (ppm)

Figure 5.'H NMR spectrum (400 MHz) of PMMA4k-PBA9K-L2. Solvent:

acetonitrile-d3.

b

JLAdd

Pe

IH chemical shift (ppm) 'H chemical shift (ppm)

Figure 6. (a) 1H NMR characterization of the self-assembly of PMMA-PBA-L1 to form

a

I

x

self-assembly of PMMA-PBA-L2 into 24-arm star polymers containing a Fujita-sphere core. a b d

...

9

8

7

6

Figure 7. 1H NMR spectra comparing the formation of paddlewheel M2L4 MOC based

on small molecules in DMSO-d6 (blue) and block copolymers in acetonitrile-d3 (red).

Spectra before (a and b) and after (c and d) assembly are shown. Block copolymer

a OH b

I

S01%A

Figure 8. 1H NMR spectra comparing the formation of Fujita sphere-like M12L24 MOC

based on small molecules in DMSO-d6 (blue) and block copolymers acetonitrile-d3 (red).

Spectra before (a and b) and after (c and d) assembly are shown. Block copolymer PMMA4k-PBA19k-L2 was used to obtain the spectra. The molecular weight of the

assembled star polymer (shown in d) is estimated to be 552,000 (23000x24), thus explaining the broadening of the MOC-associated resonances.

-a Paddle Wheel+ Extra free L2 b Spherical cage C Mixture of a and b +L+ r. t _{+ +} d duMixture of a and b heat at 50 C an+ ₊ overnight eh Mixture of a and b heat at 80 *C overnight 1 I .O 9'5 9.0 8' 0 7. 5 7'0

Figure 9. 'H NMR spectra showing the M2L4 and M,2L' MOCs separately, as a mixture at room temperature, and after heating at 50 *C and 80 *C overnight. (a) Spectrum of paddlewheel MOC; small amount of extra free ligand is added to enhance ligand exchange (if any) purposely. (b) Spectrum of Fujita sphere MOC. (c) Spectrum of the mixture of a and b. (d) Spectrum shows no changes after the mixture was heated at 50 *C indicating that the cages are stable under these conditions. (e) New peaks developed after the mixture was heated at 80 *C overnight, suggesting ligand exchange or MOC

Chapter II. Extending the Brush Arm Star Polymer (BASP) Platform for Drug Delivery

Introduction

Single-nanoparticle (NP) combination chemotherapeutics are an attractive alternative to traditional cancer chemotherapy and a promising class of drug delivery vehicles .2' Nanoparticle designs offer many advantages for multiple drug delivery

compared with combinations of free drugs, including improved drug solubility, controlled release of drugs, reduced off-target toxicity, enhanced blood circulation lifetime, and increased amount of drug delivered to specific cells and tissues. Traditional cancer treatments can lead to resistance or significant toxicity, limiting the amount of chemotherapy that a patient can receive and leading to negative health effects even decades after exposure.2_{' Although some existing clinical protocols are being used to}

decrease side effects from specific drug regimens,24 _{NP-based drug carriers can increase}

the quantity of drug delivered to a patient while minimizing side effects. In addition, NP platforms for cancer therapy have been expanded from single-drug loading to multiple-drug loading to allow for improved control of multiple-drug-multiple-drug interactions and time-dependent drug synergy.24 _{Long-circulating formulations can continuously release drugs at}

controlled ratios or allow independent regulation of release rates of particular drugs, unlike rapidly clearing free drugs. Recent successes of NP therapies, such as the CPX-351 liposomal formulation of cytarabine/daunorubicin targeting leukemia cells,25 _show

the potential of NP platforms for cancer treatment and motivate further exploration of the design of multidrug-containing NPs.

Nanoparticle designs leverage polymers, liposomes, micelles, and proteins. In the design, drugs are either encapsulated or conjugated to domains on the carrier. For

co-delivery of drugs, chemical and physical properties need to be considered in order to tune the loading and release profiles. For clinical applications, FDA-approved materials, such as polyethylene glycol (PEG) and polyvinyl alcohol (PVA), are often used for

nanoparticle design.2 _{The Johnson Group has developed a brush-first ring-opening}

metathesis polymerization (ROMP) strategy to enable efficient synthesis of brush-arm star polymer (BASP) NPs of well-defined size that can carry precise ratios of two, three, or potentially more drugs with differing mechanisms of release and action .229 BASPs synthesized using PEG are particularly versatile for clinical drug delivery applications due to the formation of a unimolecular micelle-like structure with easily tunable core and

shell functionality 28 In this polymerization strategy, surface- or core-functionalized star

polymers and miktoarm star polymers are prepared from brush-first macromonomer (MM) cross-linking via ROMP .2129 For example, ROMP of a norbornene-terminated MM is followed by in situ cross-linking with a bis-norbornene derivative crosslinker (XL). These polymer architectures can be easily synthesized using the appropriate MM and

cross-linker precursors. Nanoscopic PEGylated BASPs have been developed to degrade in response to various stimuli, such as mildly acidic tumor microenvironments, UV light, and hydrolytic enzymes." The Johnson Group also developed a strategy to design drug conjugates as building blocks for parallel construction of a series of multi-drug-loaded NPs, allowing for precise tuning and rapid variation of drug loading ratios and release kinetics. In this chapter, we describe the development of a library of macromonomers and crosslinkers compatible with multiple drugs to expand the BASP drug delivery platform.

Results and Discussion

Reaction rate modification via tuning of macromonomer anchor group

BASP synthesis is achieved through the design of MMs and XLs, which are functionalized with norbornene units to enable brush-first ROMP. The desired MMs are polymerized by exposure to bispyridyl-modified third generation Grubbs initiator, generating bottlebrush polymers comprised of defined ratios of the branched MM drug conjugates and PEG-MM .2627 Next, the living bottlebrush polymers are cross-linked by

addition of an XL, creating BASP NPs. The brush-first ROMP polymerization strategy allows for the synthesis of BASPs from any single drug or multiple drug combination that is suitable for a particular treatment regimen with excellent control over relative drug loading ratios and high reproducibility and yield.

The rate of propagation of ROMP by MMs depends on the chemical structure of the anchor group-the atoms connecting the polymer side chain to the polymerizable group. This difference is likely due to varying steric demands and electronic structure among the anchor groups." By rational selection of the anchor group, a high MM

conversion can be achieved to prepare pure, high molecular weight bottlebrush polymers by ROMP.

In order to modify the rate of propagation for BASP synthesis, PEG MMs with different moieties anchoring the polymer unit to the polymerizable norbornene chain end were synthesized (Figure 10). These anchor groups were selected due to their extensive use in literature and ability to provide varying degrees of proximity of the ester or imide carbonyl to Ru. The norbornene imide and norbornene carboxylic acid derived groups position the carbonyl to chelate the metal center through a six-membered ring.3'

HN t* u F- HN 1Hee *-0 0 a DCM EDC-HCI 0 DMAP 0 0 0 MA 0 NHS N: toeH +O O Dt OK oH --- - ' O O O O O HCI O 0 H2N4< ,.OlH 0 0 0 NDH 0 OH rO)<Y N"_ _N KO H 0 DMQK 0) N n ON IH ) D OK N 0 n --->roIZ 0 02 HN tBu TFA:DCM (1 1) OH -- -- -- - HN . . . 0 0 DCM 0 _-__N OH PPTS DCM _EDC-HCI 1OH + C0 0 C NHS, DMAP, DCM 0 0 0 0 _N Nt OOH H n O OO' H n -0

Figure 10. Synthesis of MMs with varying anchor groups.

Design of stimuli-sensitive crosslinkers

A library of bis-norbornene crosslinkers was developed based on both existing and new strategies. The crosslinkers were responsive to two different stimuli: UV light, and acid. The use of a photocleavable crosslinker facilitates BASP degradation in

response to UV light.26 _{The nitrobenzylcarbonyl (NBOC) group is known to cleave upon}

UV irradiation. This chemistry is based on the photochemically-induced photoconversion of o-nitrobenzyl alcohol derivatives into o-nitrosobenzaldehyde.32 _{Esters and carbonates}

are converted into an acetal derivative, which spontaneously collapses into an aldehyde and a liberated fragment (Figure 11). When the NBOC core of BASPs degrade, the parent brush polymers regenerate with short blocks of NBOC cleavage products appended to the chain ends.26 _{Figure 12 lists the photo-labile crosslinkers synthesized for formation of}

BASPs. NO₂ OV X 0

1i~l

NO₂ OH + HOOC COOH O 0 H2N H NO₂ HOOC N COOH - , BH₃ THF N -jrOH + HO N 0 EDC 0 NO2 250C 24h 0 E 3N N OH CAH 0 0 NO₂ HO OH EDH 0 DMAP 'H DCM I N RT 20h ₀

Figure 12. Synthesis of photocleavable XLs.

PEGylated BASPs that degrade in mildly acidic conditions (pH ~ 4.5-6.5) were also synthesized (Figure 13) .2' Acid-cleavable functional groups in nanostructures and polymers are commonly used for tumor-targeted drug delivery." Acid degradability is achieved through the use of acetal-based and ester-based bis-norbornene cross-linkers. Crosslinker 3 was prepared in a three-step strategy from cis-5-norbornene-exo-2,3-dicarboxylic anhydride.29Acetal cleavage occurs via hydrolysis as shown in Figure 12.

0

0

0 _N

H30+ 1IIr'~ O 0 '0 H20 +0 cr OH 0 H +HO H HO +H20 0 HO

Figure 12. Mechanism for cleavage of acid-sensitive XL.

HOOC COOH

+ H

0 0

DMAP it

DCM__ _ _ _ _ _ _ _ _ _

Figure 13. Synthesis of acid-cleavable XLs.

Drug modification and conjugates

In previous studies, drugs conjugated onto BASPs were chosen due to their

non-overlapping toxicity profiles. The next steps will be to explore drug combinations that are clinically relevant for particular diseases. For example, the three-drug combination, comprising of melphalan, dexamethasone, and lenalidomide, is now being tested on the BASP platform for treatment of multiple myeloma.29

OH

OH

Y

Conclusion

This chapter outlines the synthesis of MMs with varying anchor groups to modify the rate of propagation, as well as the synthesis of XLs with acid-cleavable and photo-cleavable properties. Alternative anchor groups bound to norbornene can be used to control the rate of reaction for the BASP drug delivery platform. Additionally, the design of crosslinkers that are acid- and photo-labile contributes to the expansion of a wide-ranging library of crosslinkers for drug loading and release. Unique combinations of XLs and MMs will allow for synthetic ease and functional versatility, expanding the range of applications for brush-first BASP synthesis.

Experimental Methods

Synthesis of MMs with varying anchor groups.

PPTS

OH + DCM o

12h

1

Compound Al. Bicyclohept-5-en-2-ylmethanol (200.00 mg, 0.161 mmol) and pyridinium p-toluenesulfonate (40.15 mg, 0.159 mmol) were dissolved in anhydrous DCM (20 mL) was added to a dried, 100 mL round-bottom flask. The solution was cooled down to 00C in an ice bath. Next, 1 (0.82 mL) was added dropwise. The reaction was stirred for 12 h under nitrogen then concentrated via rotary evaporation. The crude product was washed with NaHCO3, water, and brine, then purified via silica gel

0 0

OH + 0

O ' u e y O OH _{OH +DMAP}

_I

2 toluene

D(A2)

Compound A2. A solution of bicyclohept-5-en-2-ylmethanol (300 mg, 2.42 mmol),

1,3-cycloheptanedione (1.55 g, 12.09 mmol), and 4-(dimethylamino)pyridine (DMAP, 29.54 mg, 0.24 mmol) was dissolved in toluene (40 mL) and added to a dried, 150 mL round-bottom flask. The reaction was refluxed for 24 h then concentrated via rotary

evaporation, giving a yellow oil. The crude product was purified via silica gel chromatography (4% MeOH/DCM) to give A2.

Synthesis of photo-labile crosslinkers

NO2 EDC 0 NO2 0

OH + HOOC COOH DMAP 0 0

250C

24h

(B1)

2 Compound B1

A solution of bicyclohept-5-en-2-ylmethanol (100.00 mg, 2.20 mmol), 2 (77.30 mg, 0.37

mmol), 4-(dimethylamino)pyridine (DMAP, 8.95 mg, 0.07 mmol), and EDC (154.48 mg,

0.81 mmol) was dissolved in DCM (25 mL) and added to a dried, 150 mL round-bottom

flask. The reaction was stirred for 30 h in room temperature then concentrated via rotary evaporation. The crude product was purified via silica gel chromatography (10 %

0 040 0 + H2N OH 0 NO₂ HOOC COOH -H -BH₃ THF 0 _NO 2 N ,,OH + HO 'N 0 0 0 0 E N OH 06H6 0 0 NO 2 HO OH EDC 0 0 DMAE 0 Or H DCM NN RT 0O 0 20h 0 0N20 (B2) (B3) (B4)

Compounds B2, B3, and B4 were synthesized following the procedure in Liu et. al.

2012.

Synthesis of acid-labile crosslinkers

HOOC COOH 0 'N H AN 0 0 EDO DMAP DCM ____________

Compound C1. A solution of bicyclohept-5-en-2-ylmethanol (1.20 g, 1.21 mmol), malonic acid (46.59 mg, 0.45 mmol), 4-(dimethylamino)pyridine (DMAP, 54.70 mg,

0.45 mmol), and EDC (257.47 mg, 1.343 mmol) was dissolved in DCM (15 mL) and

added to a dried, 100 mL round-bottom flask. The reaction was stirred for 24 h in room temperature then concentrated via rotary evaporation. The crude product was purified via silica gel chromatography (2% MeOH/DCM) to give C1.

OH

OH +

(CI)

'NO

Compound C2. A solution of bicyclohept-5-en-2-ylmethanol (100.00 mg, 0.98 mmol), benzaldehyde (28.50 mg, 0.40 mmol), and 4-(dimethylamino)pyridine (DMAP, 29.54 mg, 0.24 0 mmol), was dissolved in toluene (20 mL) and added to a dried, 100 mL round-bottom flask. The reaction was stirred for 24 h in room temperature then concentrated via rotary evaporation. The crude product was purified via silica gel chromatography (30% EtOAC/hexanes) to give C2. Spectra Data 16 15 14 13 12 11 10 9 8 7 6 5 4 16 15 14 13 12 11 10 9 8 75 4 fC (ppm) Compound ALE' NMR (400 MHz, CDC 13, r.t.) -17000 -16000 -15000 -14000 -13000 -12000 -11000 -10000 -9000 -8000 -7000 -6000 -5000 -4000 -3000 -2000 -1000 -0 --1000 3 2 1 0 -1 -2 -3 "

--.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 fl (ppm) Compound A2.'H NMR (400 MHz, CDC 13, r.t.) 8.5 .0 75 70 6. 6. 5.5 5.0 4.5 4. 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 f1 (ppm) Compound Bi. 'H NMR (400 MHz, CDC 13, r.t.) 3.0 2.5 2.0 1.5 1.0 0.5 3.5 3.0 2 -11000 -10000 -9000 -8000 -7000 -6000 -5000 -4000 -3000 -2000 -1000 -0 1-1000 -5500 -5000 4500 -4000 -3500 3000 2500 -2000 -1500 1000 500 -0 --500 - --- ---r .5 2.0 1.5 1.0 0.5

8.5~ ~~~~ 8. .. .. 6. 6. 5. 5. 4. 40 3. 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 fi (ppm) Compound Cl.'H NMR (400 MHz, CDC 13, r.t.) -24000 -22000 -20000 -18000 -16000 -14000 -12000 -10000 -8000 -6000 -4000 -2000 -0 --2000 3.0 2.5 2.0 1.5 1.0 0.5 -3200 -3000 -2800 -2600 -2400 -2200 2000 -1800 -1600 -1400 1200 -1000 -800 -600 -400 -200 0 -200 1.5 1.0 0.5 0.0 .8 8 .5 .0 . . 6 .0 I . 4 . . .I .5 2 .0 ).0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 fi (ppm) Compound C2. 'H NMR (400 MHz, CDC 13, r.t.)

-L-- -t

- dL---,,

-l-LJL

16fi

I

_j

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