Development of a reactive peptide sequence for site-selective bioconjugation

Development of a Reactive Peptide Sequence for Site-selective Bioconjugation

by

Suan Lian Tuang B.S. Chemistry

Massachusetts Institute of Technology, 2014

SUBMITTED TO THE DEPARTMENT OF CHEMISTRY IN PARTIAL FULFILLMENT OF

THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN CHEMISTRY

AT THE

MASSACHUSETTS INSTITUTE OF TECHNOLOGY

February 2020

© 2019 Massachusetts Institute of Technology. All rights reserved.

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Department of Chemistry September 20 19

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r Bradley L.Pentelute' Associate Professor of Chemistry

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OF TECHNOLOGY.. Haslam and Dewey Professor of Chemistry

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Development of a Reactive Peptide Sequence for Site-selective Bioconjugation

By

Suan Lian Tuang

Submitted to the Department of Chemistry on September 20, 2019 in Partial Fulfillment of the Requirements for the

Degree of Doctor of Philosophy in Chemistry

ABSTRACT

Achieving catalyst-free, site-selective modification of proteins in water is a significant challenge in chemical biology. Issues of residue specificity, site-selectivity, reagent stability, and reaction rate are pervasive in this field, and despite advances over the past few decades, achieving fast, pinpoint modifications of complex molecules remains a tremendous obstacle. Herein, we describe the development of a nine-amino acid motif (Met-Cys-Pro-Phe-Leu-Pro-Val-Val-Tyr) termed engineered reaction (EnAct) tag. The EnAct interface, discovered by iterative screening of peptide libraries, consists of a reactive peptide (EnAct tag) that undergoes rapid (second-order rate

constant, k - 150 M-1 s-') nucleophilic aromatic substitution with a perfluoroarene-containing

peptide (EnAct probe). Bioconjugation reactions centered on peptide interfaces are emerging as promising strategies to prepare homogeneous biological conjugates, and our results with the EnAct interface represent a 210-fold increase in reaction rate over the previous standard for this class of cysteine arylation.

Furthermore, the EnAct sequence consists of all-natural amino acids and thus enables the facile genetic engineering of the sequence onto proteins of interest. We disclose here the incorporation of the EnAct sequence at the C-termini of the IgG antibody trastuzumab heavy chains, which were subsequently conjugated to the EnAct probe with excellent site-selectivity, despite the 32 other Cys residues on this protein. Remarkably, this system's rapid kinetics enabled quantitative conversion in 1.5 hours and at lower substrate concentrations. Finally, this bioconjugation reaction is still selective even in the complex environments of cell lysate mixtures, illustrating the enhanced selectivity and rapid reactivity of the EnAct interface.

The appreciable increase in cysteine arylation rate and selectivity achieved with the EnAct sequenced represents a new standard for site-selective bioconjugations using peptide interfaces. To explore the versatility of the reactive peptide sequence, we found that this reactive peptide enabled aqueous arylations of Cys with small molecule electrophiles in mostly water, which was not previously accessed with this class of electrophiles. Furthermore, the perfluoroarene on the probe was found not only to function as an electrophile for thiol arylation, but also to offer a handle for easy elimination to form dehydroalanine. Thus, the EnAct system represents a powerful, versatile, and selective bioconjugation method.

Thesis Supervisor: Bradley L. Pentelute Associate Professor of Chemistry

Acknowledgments

I owe a debt to my thesis advisor, mentors, colleagues, friends, and family. This thesis

would not have been possible without their support. Graduate school is challenging. Through these individuals, my graduate school experience became a source of tremendous personal growth. Words are not sufficient to perfectly capture my immense gratitude, but I will attempt here.

I am most grateful to my thesis advisor Brad Pentelute. I attribute my growth during

graduate school to his mentorship and guidance. I am always energized and determined after every conversation with him. His enthusiasm is infectious. I will always be thankful for his care of my personal well-being and growth.

Thank you to Alex Shalek and Matt Shoulders for serving on my thesis committee. I really appreciate their advice and support.

The highlight of my Pentelute lab experience has been working with my partner-in-crime Diomedes D.. He pushes me to achieve my potential, both in the lab and outside the lab. My time in the Pentelute lab would not be the same without him. Thank you so much. I thank Chris S. for his friendship and generosity. I could not imagine the past five months without him sitting next to me providing the calm presence and guidance. I thank Chi Z. and Peng D. for the mentorship they provided early in this project. This work would not have been possible without their help. I thank Andrei for providing scholarly advice and writing help. I thank Amy R. for being my mentor during my rotation and the subsequent life catchups in the following years.

I thank Anthony Q. for being the best roommate anyone could ask for the past three years (going onto four). I could not imagine going through graduate school without having him to lean on both in lab and in the apartment. I thank Alex L. for his friendship since day one through the ups and downs of graduate school. I thank Carly S. for always making lab fun and for entertaining all questions about American culture. I thank Rebecca H. for always being there to chat about anything from oral exams to grad school struggles. I thank Azin S. for the laughter we shared over many things, most of which revolved around being immigrants. I thank Zak G. for being the realest

OG. Thank you to all the other members in the Pentelute lab who have taught me and shared with

me many memories: Rachael F., Emily W., John A., Sarah A., Joe, Alex C., Amanda C., Kyan D., Charlotte F., Patrick H., Stephanie H., Heemal D., Nina H., Muhammad J., Yen-chun L., Changxi L., Eva V., Aaron M., Mackenzie P., Sebastian P., Jacob R., Adeline S., Xuyu T., Jason T., Nick

T., Xiyun Y., Genwei Z., Alex M., Ethan E., Justin W., Anthony R., Colin F., Mike L., Faycal T., Mark S., Surin M., Michael S., Mikael M., Katie H., Guillaume L., Alex V., Mette I., Vanessa Z.,

Ivan B., Caterina C., Anupam B., Arisa S., Puguang C., Shunying L., Bente S., Binyou W., and Cameron H.

Outside the Pentelute lab, I would like to thank the MIT Chemistry department for all their support. Thank you Jennifer Weisman, Rebecca Teixeira, Mitch Moise, and Jay Matthews. I'm grateful to everyone in the MDPhD office for the guidance throughout the training thus far: Loren Walensky, Amy Cohen, Robin Lichtenstein, Yi Shen, Jennifer DeAngelo, Temperance Rowell,

and Steve Obuchowski. Thanks to all from the HST MD Office: Rick Mitchell, Matt Frosch, David

part of the strong support through the Paul and Daisy Soros Fellowship and would like to thank my cohorts as well as the program staff: Craig Harwood, Yulian Ramos, and Nikka Landau.

My faith community has also been a tremendous source of spiritual and emotional support for me. Thank you Ashton G. & family, Clark W. and family, David A. & family, Dan T. & family, Finny K. & family, Jeremy H. & family, Joe W. & family, Malcolm W. & family, Marlin W. & family, Matthew M. & family, Stephen T. & family, Eric Z. & family, Timothy M. & family, Christian B., David A., Earl W., Hans H., Zack J., and Jeff T.

Thanks to all my friends (old and new) who I have leaned on throughout graduate school. Thank you Elizabeth for being my best friend. Thank you Kwadwo and Nathan for all our conversations about faith, graduate school, and life. Thank you Chidi for all the timely conversations that always put life in perspective. Thank you Anu for your friendship through college and graduate school.

Finally, I thank my family. They are the bedrock of my existence. Their love know no bound. Thanks to my parents and siblings for their nurturing care and example they have set for me.

Table of Contents Abstract 5 Acknowledgments 7 Table of Contents 10 List of Figures 12 List of Tables 14

Chapter 1: Background and Overview

1.1 Protein bioconjugation 16

1.2 Chemoselective bioconjugation 20

1.3 Site-selective protein modification 24

1.4 Cysteine arylation as an enzyme-free site-selective bioconjugation method

42

1.5 Overview of thesis 48

1.6 References 50

Chapter 2: An engineered reaction match pair for fast and selective cysteine bioconjugation

2.1 Introduction 71

2.2 Results and Discussion 72

2.3 Conclusion 99

2.4 Experimental 100

2.5 Acknowledgment 109

2.6 Appendix of tables 110

2.7 Appendix of reaction analyses 114

2.8 Appendix of LC-MS characterization 151

2.9 References 187

Chapter 3: Exploring the versatility of reactive peptide sequences toward diverse bioconjugation reactions

3.1 Introduction 188

3.2 Results and Discussion 190

3.3 Experimental 207

3.5 Appendix of reaction analyses 221

3.6 Appendix of LC-MS characterization 241

3.7 Appendix of reaction optimization 253

List of Figures

Figure 1.1. Protein surfaces have a variety of side chain functionalities, often including multiple copies of the same residues, as shown with lysozyme (PDB: 2LYZ).

Figure 1.2. Various electrophiles enable chemoselective bioconjugation of desired cargoes to lysine (Lys) and cysteine (Cys) side chains.

Figure 1.3. Chemoselective bioconjugations often yield heterogeneous product mixtures when multiple copies of the same functional group are present on the protein surface.

Figure 1.4. Methods to achieve site-selective functionalization of proteins (green, PDB: 2LYZ) with cargo of interest (red).

Figure 1.5. Genetically encoded unnatural amino acids (UAA) contain bioorthogonal reactive handles that enable site-selective protein modification.

Figure 1.6. Ligand-directed reactions enable site-selective protein modification.

Figure 1.7. Fusion of engineered self-modifying enzymes to proteins of interest enable site-selective protein modification.

Figure 1.8. Peptide tags appended to proteins of interest are recognized by enzymes and enable site-selective protein modification.

Figure 1.9. Peptide tags appended to proteins of interest enable site-selective attachment of fluorescent labels.

Figure 1.10. Electrostatic interactions between coiled-coil peptides can be leveraged to achieve site-selective protein modification.

Figure 1.11. Cysteine arylation via nucleophilic aromatic substitution (SNAr). Figure 1.12. 7-Clamp-mediated site-selective cysteine modification.

Figure 2.1. Glycine scan of a perfluoroaryl peptide ("n-probe") reveals sequence-dependent reactivity.

Figure 2.2. Combinatorial library selection was used to discover a more reactive arylation system.

Figure 2.3. Library design of each selection process.

Figure 2.4. Kyte and Doolittle hydrophobicity plot of the sequences from the 1st library selection reveals hydrophobic residues in position #1, #5, and #7 and hydrophilic residues in position #6. Figure 2.5. Selective homodimerization of EnAct tag was observed when EnAct tag and Gen 4 tag were reacted with decafluorobiphenyl.

Figure 2.6. Second order rate constants determined for EnAct tag, EnAct probe, Gly tag, and Gly probe.

Figure 2.7. The EnAct tag exhibits sequence-dependent reactivity when treated with the EnAct probe.

Figure 2.8. Gly point mutations to the EnAct probe have little effect on its reactivity with the EnAct tag.

Figure 2.9. The reactivity of the EnAct tag is dependent on both its position and its orientation in a peptide.

Figure 2.10. The reactivities of the EnAct tag and probe are not dependent on their stereochemistries.

Figure 2.11. Chromatograms of crude reaction mixtures at 0 min (black) and 60 min (red) show reactions of the EnAct tag and EnAct probe gave rapid, full conversion to arylated product. Figure 2.12. Chromatograms of crude reaction mixtures at 0 min (black) and 60 min (red) show reactions between the n-clamp and n-probe gave minimal conversion to arylated it-product under similar conditions to the EnAct interface.

Figure 2.13. EnAct tag-mediated cysteine arylation enables site-selective labeling of the C-terminal heavy chains of trastuzumab at low reagent concentrations.

Figure 2.14. EnAct-mediated cysteine arylation is selective in a complex cell lysate environment. Figure 3.1 Reaction between EnAct tag and various small molecule electrophiles.

Figure 3.2. Conversion of the reactions between the EnAct tag and various small molecule electrophiles.

Figure 3.3. Reaction between EnAct-Z33 and pentafluorophenyl sulfide.

Figure 3.4. Observed transformation and proposed mechanism of the base-mediated dehydroalanine intermediate formation.

Figure 3.5. Elimination of perfluoroarene-installed cysteine to form dehydroalanine. Figure 3.6. Conversion of the perfluoroarene-installed peptides to dehydroalanine.

List of Tables

Table 2.1. Peptides identified from the 1st round of library screening and de novo sequencing. Table 2.2. Peptides identified from the 2nd round of library screening and de novo

sequencing.

Table 2.3. Peptides identified from the 3rd of library screening and de novo sequencing. Table 2.4. Peptides identified from 4th round of library screening and de novo sequencing.

1.1 Protein bioconjugation

Proteins are the biological and chemical foundation of life, playing critical roles in cellular

processes such as signaling, growth and development, and metabolism.1 Within natural biological

systems, proteins are synthesized by the ribosome. It is quite remarkable that Nature's nearly

limitless array of proteins that performs widespread and diverse functions is constructed from the

same, simple alphabet of 20 canonical amino acid monomers. The function of these proteins is

precisely controlled by the precise folding of their primary linear sequences (typically >100

residues) into specific three-dimensional structures. Many enzymes have folded structures in

which particular amino acids are placed in a specialized active-site environment essential for the

protein's function, and even a single amino acid difference can result in a significant change to its

structure, and hence its activity.2

In addition to the amino acid sequence, further structural and functional variations is

achieved in nature through chemical modifications to specific residues in proteins post-synthesis

(i.e., post-translational modification, PTM).3 For instance, acetylation of histone proteins by

histone acetyltransferases neutralizes the positive charge on the modified lysine, resulting in

decreased binding to the negatively charged phosphate groups of DNA, and enabling the activation

of gene transcription.4 _{Life employs a wide-ranging arsenal of PTMs, including phosphorylation,5}

glycosylation,6 nitrosylation,7 lipidation, _{and ubiquitination.}9 _{These PTMs play crucial roles in}

cellular processes such as protein degradation,10 immune response,1 and signal transduction.12

Since structural alterations to a single residue on proteins can have a tremendous effect on

their functions, ample research efforts within this field of bioconjugation have been carried out to

selectively modify specific amino acid side chains in order to modulate protein function.13

molecules, a biomolecule and the modifying cargo.14 Over the past two decades, various methods

for designer bioconjugation chemistry have emerged as invaluable tools for the development of functionalized proteins. The bioconjugation of various cargoes, such as fluorophores, affinity

handles, and therapeutic drugs, have enabled the development of new therapeutic modalities,15-17

the investigation of proteins in their biological context,1 8-2 4 and the tracking of cellular events.2 5

Synthetic modifications of proteins are chemically challenging, given that methods must preserve protein structural and functional integrity, thus requiring more stringent and milder conditions than in traditional organic synthesis. Reactions must usually be performed in a buffered aqueous environment close to neutral pH, at ambient or physiological temperatures (< 37 °C), and with reaction times of a few hours. Due to the limited solubility of proteins, it is impractical to employ them as substrates at concentrations exceeding the micromolar range. Therefore, bioconjugation reactions need to be robust enough to achieve high conversions at low micromolar or nanomolar protein substrate concentrations. A desirable reaction rate for bioconjugation reactions is considered to be in the range of 1-1000 M-1 s-1.26,27

An additional consideration for bioconjugations is the challenge of controlling the site on the protein where the cargo is attached, as the location of modification can have a pronounced effect on the activity of the modified biomolecule. Given that endogenous protein surfaces are filled with numerous nucleophilic residues, many bioconjugations techniques rely on the reactivity of exogenous electrophiles. However, the competition between these various nucleophiles, such as thiols (in cysteine), hydroxyls (in serine/threonine), phenols (in tyrosine), guanidines (in arginine), imidazoles (in histidine), amines (at the N-terminus and in lysine), and carboxylates (at the C-terminus and in aspartate/glutamate), makes achieving functional group selectivity challenging (Fig. 1.1). Thus, in order to obtain homogeneous, well-defined products, the reagents

are often designed to exhibit high chemoselectivity, in which only one residue type is preferentially modified over the others (e.g., lysine side chains over other amino acids). However, even if chemoselectivity is achieved, bioconjugations could provide a heterogeneous product mixture if several copies of the same nucleophilic functional group are present, leading to product mixtures that are difficult to control, purify, and characterize. It is hence important to achieve high site-selectivity by preferentially modifying only one residue in the presence of others of the same type within the protein (e.g., a specific lysine residue over others). Developing robust and selective bioconjugation reactions is a persistent challenge in chemical biology, and the various approaches to overcome this challenge will be discussed.

NH Ny N H HO-NH H2+ -NH2 NH3 0-/OH

Figure 1.1. Protein surfaces have a variety of side chain functionalities, often including multiple copies of the same residues, as shown with lysozyme (PDB: 2LYZ).

1.2 Chemoselective protein modification

Beginning with the challenge of chemoselectivity, electrophilic reagents must exhibit specific reactivity only with matched nucleophilic side chain functional groups. Achieving such selectivity requires the rational design of these electrophiles, in addition to careful optimization of reaction conditions, such as pH, stoichiometry, and reaction time. Chemoselective protein modifications have been broadly implemented for lysine and cysteine (Fig. 1.2), while applications

with other residues such as tyrosine,28_-31 _tryptophan,3 2_-3 4 _methionine,35_,36 _{aspartic/glutamic}

acids,37

,38 _{or histidine}39_-1 _{have received less attention.}

While the lysine s-amine group is a good nucleophile, the primary competitor for its selective modification is the more nucleophilic cysteine thiol. In order to overcome this issue, Lys-selective derivatizations often involve using (1) 'harder' electrophiles, (2) reagents that form amides, leveraging the relative instability of thioesters by comparison, or (3) reagents that operate via imine formation, inaccessible for thiols (Fig. 1.2A). The most common electrophiles among this class are the N-hydroxysuccinimide (NHS) esters, which are commercially available and widely applied to modify proteins with different cargoes such as fluorescent probes and affinity reagents.4 2

-s0 Other routes to achieve lysine-selective modification involve the use of sulfonyl chlorides,5 isothiocyanates,5_2,53 _{and aldehyde derivatives.54-56 Given that lysine residues are} highly abundant in human proteins (5.9% in amino acid content), issues related to (1) polyfunctionalization and (2) disparate selectivity can be challenging to overcome and often result

* = Cargo H H H H H N N N (B) H \NSH 0 SO N R.S' S* H X' _N* X = 1, CI, Br H 0S'

Figure 1.2. Various electrophiles enable chemoselective bioconjugation of desired cargoes to lysine (Lys) and cysteine (Cys) side chains. (A) Lys-selective electrophiles: (i) N-hydroxysuccinimide esters, (ii) isocyanates and isothiocyanates, and (iii) aldehydes for reductive amination. (B) Cys-selective electrophiles: (i) maleimide Michael acceptors, (ii) disulfide exchange reagents, and (iii) halo-acetamides.

(A) H ₂ N 3H2 0 o X=C=N* X = S0 a H 11 (then NaBH3CN) (Ili)

In contrast, cysteine residues occur less often on protein surfaces (1.9% in amino acid content),58 59 and this lower abundance coupled with its increased nucleophilicity make cysteine an attractive option for residue-specific protein modification.6 _-2 _{Furthermore, under} physiological conditions, cysteine thiols are more acidic (pKa ~8) than other common protein nucleophiles, such as hydroxyls (pKa -13), phenols (pKa -10), or amines (pKa -10), and thus predominantly exist as more nucleophilic thiolates.63 The sulfur atom is also more polarizable (soft) than oxygen or nitrogen, enabling chemoselectivity in reactions with soft electrophiles. Examples of such reagents include various classes of Michael acceptors (maleimides,64 65 _vinyl sulfones,66 _{and acrylonitriles) and ax-halocarbonyls} 67

,68 (iodoacetamides and chloroacetamides) (Fig. 1.2B). Many of these cysteine-reactive electrophiles are commercially available and

routinely used in different applications to conjugate drugs, affinity labels, and fluorescent dyes to peptides and proteins.69-73

Given the lower natural abundance of cysteine compared to other amino acids in proteins, these methods are particularly amenable to site-selective modification by site-directed mutagenesis of cysteine at desired locations.7₄76 _{If a protein is expressed with a single Cys residue mutated at} a desired position, chemoselective Cys-selective reagents can easily add unnatural cargo to this position. These methodologies, however, are not suitable for proteins containing more than one cysteine, as they often afford heterogeneous reaction mixtures (Fig. 1.3). The current inability to selectively target one cysteine residue among many in the same protein demands the development of new chemistries to perform site-selective protein modification on native proteins.

o0 Cysteine-0~ selective bioconjugation NHOH bis-functionalized product

Figure 1.3. Chemoselective bioconjugations often yield heterogeneous product mixtures when multiple copies of the same functional group are present on the protein surface. For example, a protein surface that contains three cysteine thiols can react with a cysteine-selective reagent to give numerous mono-, bis-, and tris-functionalized conjugates.

triS-functionaIized product

1.3 Site-selective protein modification

To overcome the challenge of modifying one residue among multiple copies in the same molecule, recent efforts to achieve site-selective modifications of proteins have built on the chemoselective methods described above, and include: (1) unnatural amino acid (UAA) incorporation, (2) ligand-directed protein modification, (3) fusion of engineered, self-modifying enzymes, and (4) peptide tag-based modification (Fig. 1.4). These methods generally rely on

(A) 7

Unnatural amino acid incorporation (B) Protein of interest (D) Ligand-directed reaction Fusion of engineered, self-modifying enzyme Peptide tag-based modification

Figure 1.4. Methods to achieve site-selective functionalization of proteins (green, PDB: 2LYZ) with cargo of interest (red). (A) Unnatural amino acids that display biorthogonal reactivity can be inserted into proteins. (B) Electrophiles appended to protein-binding ligands can direct reactivity to particular sites. (C) Self-modifying proteins attached to POIs will rapidly react with particular reagents. (D) Short peptide sequences appended to POIs can exhibit strong reactivity with matched electrophiles.

-0

1.3.1 Unnatural amino acid incorporation

Since differentiating between multiple nucleophilic canonical amino acid side chains is a persistent challenging, extensive research has focused on the incorporation of unnatural amino acids (UAA) into proteins through genetic code expansion (Fig. 1.5).26,77-80 The inserted UAAs generally possess abiotic side-chain functionalities that exhibit bioorthogonal reactivity compared to natural residues, and react rapidly, selectively, and quantitatively with matched reagents in transformations that have been termed "click chemistry." For example, in a broadly utilized strategy, trans-cyclooctynes-containing amino acids embedded in proteins react rapidly with tetrazines in inverse electron demand Diels-Alder reactions, exhibiting exquisite selectivity both

in vitro and in vivo (Fig. 1.5A).s 83 _{Other unnatural amino acids have been incorporated by genetic}

code expansion to perform bioconjugation reactions that utilize various bioorthogonal transformations: (1) Staudinger ligation between azides and triarylphosphines,8₄88 ₍₂₎ copper(I)-catalyzed azide-alkyne cycloaddition,89 9 3 _{and (3) strain-promoted azide-alkyne 1,3-dipolar} cycloadditions9_4-10 _{(Fig. 1.5B). A wide variety of probes, including fluorescent dyes, biotin,} lipids, sugars, spin labels, and crosslinkers, can be attached using this strategy even in a complex biological milieu and in living cells.78

Incorporation of UAAs featuring unique functional groups offers a tremendous opportunity for site-selective protein modification. The success of this method relies on both the efficiency of

UAA incorporation into the protein of interest and the robustness of the reaction between the

incorporated reactive handle with its reaction partner. Leveraging bioorthogonal chemistry between the reactive handles has been successful in addressing the latter requirement.78 _The incorporation of UAAs, however, can be a cumbersome procedure that often affords moderate expression yields depending on the protein or the UAA.10 2_{Furthermore, the engineered expression}

system remains challenging to implement for many laboratories.1

°

interest in broadening the applicability of this method,' 04 _{alternative techniques have also been}

H N

(A)

<IN-N

Genetically encoded

0

trans-cyclooctene

(B) (i)

+ro

Figure 1.5. Genetically encoded unnatural amino acids (UAA) contain bioorthogonal reactive handles that enable site-selective protein modification. (A) An inverse electron-demand Diels-Alder reaction between trans-cyclooctene and tetrazine. (B) Other examples of bioorthogonal reactions include: (i) Staudinger ligation between azide and triarylphosphine, (ii) copper(I)-catalyzed azide-alkyne cycloaddition, and (iii) strain-promoted azide-alkyne 1,3-dipolar cycloadditions.

1.3.2 Ligand-directed protein modification

An alternative approach to the genetic mutations of expressed proteins is the direct,

site-selective modification of native proteins. Ligand-directed protein modifications fall within this paradigm, which leverages the affinity of specific protein-binding moieties (ligands) for proteins of interest (Fig. 1.6). The reagents employed for this chemistry are composed of the ligand (small molecule or peptide), the reactive group matched with the functionality present on the protein surface, and the cargo to be conjugated. The binding of the ligand to the target protein brings the reactive group in close proximity to the protein, enabling reactivity with nearby amino acid residues. A prominent example of this approach is ligand-directed tosyl (LDT) chemistry (Fig.

1.6A).105_-"0 _{The tosyl groups are normally unreactive under optimized conditions, except through} proximity-induced reactivity promoted by ligand binding, enabling site-selectivity modification. An added feature of this method is that the ligand dissociates from the protein with the tosylate leaving group, thus making this a "traceless" modification. Site-selective protein modification using LDT chemistry has been applied in photo-crosslinking of protein complexes0 5_{, small} molecule screening against a therapeutic target0 8_{, and live cell imaging"}0_{. In addition to tosyl} electrophiles, ligand-directed methods have also leveraged employing chemistries with 5-sulfonyl tetrazoles''", acyl-phenols'12, 0-nitrobenzoxadiazoles"', acyl imidazoles,1 1

4-116 and various catalysts2 8

,11 7,"'s (Fig. 1.6C). Beyond small molecule ligands, peptides have also emerged as

valuable affinity handles (Fig. 1.6B).119

-12 4 _{For instance, human immunoglobulin G fragment} (IgG-Fc)-binding peptides were derivatized with various reactive groups designed to modify a specific residue near the binding region, resulting in homogeneous antibody-drug conjugates.' 25 ,126

The success in site-selectively modifying proteins in their native state has made ligand-directed bioconjugation a compelling approach. A central requirement in this method is the unique

recognition between a binding pocket in the native protein and a robust affinity molecule. In addition, it is also crucial to have an appropriately positioned nucleophilic residue on the protein surface that would react with the introduced moiety upon binding. For proteins of interest that do not have a native binding pocket or for which a specific ligand has yet to be discovered, it is desirable to develop a more general bioconjugation approach to complement this method.

(A)Nu

P o4

Protein of interest

~O0

reactive tosyl linker 0 = Affinity ligand

NU u

_

(B) V

0

Small molecule ligand of FKBP12 protein

ArN

Minimized Z domain of protein A (affinity peptide)

(C) j? 0 04 0--f N J0 V& _N '-N Pq N.-Ii

5-sulfonyl tetrazole Acyl imidazole

Figure 1.6. directed reactions enable site-selective protein modification. (A) Ligand-directed tosylation of proteins. (B) Examples of small molecule ligands and affinity peptides. (C) Other electrophiles include: 5-sulfonyl tetrazole, acyl imidazole, and acyl phenol linkers.

Acyl phenol

---1.3.3 Fusion of engineered, self-modifying enzymes

While ligand-directed techniques are able to achieve exquisite site-selectivity, it may be desirable to develop a complementary approach that is more generalized and does not require unique binders for each protein of interest. One such avenue involves the fusion of these proteins with engineered enzymes specific toward known substrates (Fig. 1.7). For instance,

06-alkylguanine-DNA-alkyltransferase (hAGT) is a 20 kDa human DNA repair protein whose active site cysteine undergoes an irreversible alkyl group transfer from its substrate (06-alkyguanine) to the cysteine thiol.12 7

,128 Taking advantage of its alkylguanine specificity, hAGT was engineered through multiple rounds of directed evolution to expand its substrate scope and impart reactivity with 06_{-benzylguanine (BG) derivatives with varying cargo, resulting in a system known as the} SNAP-tag.129 _{Upon appending the SNAP-tag onto protein of interests, a wide variety of} BG-functionalized cargoes can be efficiently transferred to the SNAP-tag fused proteins through the self-labeling reaction (Fig. 1.7A). Other routinely used self-modifying enzymes and their substrates include the CLIP-tag (02_{-benzylcytosine} derivatives),"' _{the TMP-tag (trimethoprim} derivatives),3 1_"1 32 _{and the Halo-tag (haloalkane derivatives)} 3 _{(Fig. 1.7B). These types of} site-selective protein modifications have been widely used in live cell imaging as well as the development of antibody-based diagnostics and therapeutics.

The use of engineered, self-modifying enzymes takes advantage of the high specificity and robust reactivity inherent to the native enzyme-substrate pair. Given that the fusion of these enzymes brings along a massive increase in size (18-33 kDa) to the protein of interest, the attachment may alter the structure, function, or localization of the proteins studied.34 _{For proteins}

(A)

06-benzylguanine derivative

POI (green) fused to SNAP-tag (blue)

(B) (i) NH2(u

N 4OCH3

TMP-tag derivatie Halo-tag Haloalkane derivative

Figure 1.7. Fusion of engineered self-modifying enzymes to proteins of interest enable site-selective protein modification. (A) The reactive cysteine of SNAP-tag (blue, PDB:3KZY) fused to the protein of interest (green, PDB: 2LYZ) reacts with an 06-benzylguanine modified cargo. (B) Examples of other self-modifying enzyme tags and their respective cargo-derivatized substrates: (i) TMP-tag (PDB: ILUD) and trimethoprim derivatives, and (ii) Halo-tag (PDB:

1.3.4 Peptide tag-based site-selective bioconjugation

One such approach to minimize the fingerprint of the reactive protein species involves the

development of smaller peptide-based tags. These short sequences (0.6 - 6 kDa) confer a specific

mode of reactivity that can in turn be leveraged to conjugate virtually any desired cargo to proteins. Furthermore, since these tags consist of canonical amino acids, they do not require genetic code expansion, and can easily be appended to the termini of numerous proteins of interest by recombinant expression. Furthermore, the small size of these tags only minimally perturbs the protein structure, a desirable feature that reduces the likelihood of significantly affecting its native function. Peptide tag-based bioconjugation could be divided into four classes: (1) enzymatic labeling, (2) recognition of metal ions and small molecules, (3) peptide and protein-based recognition, and (4) enzyme-free reactive peptide tags.

1.3.4.1 Enzymatic labeling of peptide tags

Many naturally occurring enzymes catalyze the transfer of functional chemical moieties

onto specific peptide substrates, and several bioconjugation methodologies have been developed

by leveraging these activities (Fig. 1.8). This bioconjugation strategy can be implemented by: (1)

identifying the minimal amino acid sequence in the enzyme's natural substrate onto which the

chemical moiety is transferred, (2) optimizing the sequence to enhance the reactivity and expand

the scope of the transferred moieties, and (3) appending the optimized peptide tag to the protein of

interest to be modified. These enzyme catalysts often belong to families of peptidases, transferases,

ligases, and oxidoreductases, and the chemical moieties are normally substrates capable of being

further derivatized with various cargoes. For instance, biotin ligases (BirA) are highly conserved

enzymes that catalyze the covalent attachment of biotin to the c-amino group of particular lysine

residues in target proteins.1 35 _{Combinatorial methods and sequence optimizations were employed}

to develop a minimal peptide substrate for BirA-catalyzed biotinylation, resulting in the discovery

of a 15-mer called the AP-tag

(Gly-Leu-Asn-Asp-Ile-Phe-Glu-Ala-Gln-Ly-Ile-Asp-Trp-His-Glu)136 (Fig. 1.8A). Upon genetic encoding of the AP-tag onto proteins of interest, followed by

attachment of biotin with BirA, the ligated biotin can be further captured with streptavidin attached

to a desired cargo.?1 3 _{The AP-tag/BirA system has been widely used to study protein-protein}

interactions, homo- and heterodimerization of proteins, and protein trafficking."7-14 3 Other

examples of enzymatic systems for protein modification include lipoic acid ligase,144- 5 1 tubulin

tyrosine ligase, phospho-pantetheinyl transferase,153-159 sortase A, 160-16 transglutaminase,1 69-1 77

and formylglycine-generating enzyme (Fig. 1.8B).178-181

Appending the minimal substrate amino acid sequence into proteins of interest affords

enzymes. The practical application of these methods is limited by the need to employ long reaction times and/or high concentration of substrates to achieve high labeling yields. In addition, these reactions are catalyzed by enzymes that require the use of co-factors (ATP, Ca", etc.) that may interfere with the biological process under investigation. Thus, in many cases, it may be desirable to develop a peptide tag that (1) has shorter reaction times, (2) enables the use of lower concentrations of substrates, and (3) facilitates the transformation without the use of enzymes.

0 H HN HNNH

(A) I H

B S S

Bibiotin H~H SA* £ A

BiAenzyme, ATP

'~

iL~

AP-tag fused to protein of interest

(B) (i) NN FGEH Aldehyde tag 01tgTGase, Ca2 W Q1-tag HO W ~ X NN HP 7 LpIA, ATP LAP-tag0 V

Figure 1.8. Peptide tags appended to proteins of interest are recognized by enzymes and enable site-selective protein modification. (A) BirA enzyme recognizes the AP-tag

(GLNDIFEAQKIEWHE) and catalyzes the covalent attachment of biotin onto the lysine side

chain. The biotin handle can then be further modified with cargo attached to streptavidin (SA). (B) Other examples of enzyme-peptide tag systems include: (i) formylglycine-generating enzyme

(FGE) and its aldehyde tag (LCXPXR), (ii) transglutaminase (TGase) and the QI-tag (PNPQLPF),

1.3.4.2 Recognition of metal ions by peptide tags

An alternative peptide tag-based approach involves the specific recognition of particular metal ions to achieve site-selective modifications. Peptide sequences designed with electron-rich residues can chelate Lewis-acidic metal ion probes that contain the desired cargo (Fig. 1.9). For instance, a tetracysteine core motif (CCXXCC, most commonly CCGPCC) was developed to form covalent bonds with a range of organic arsenic compounds that bear chromogenic moieties (Fig.

1.9A).182 _{In their initial unbound state, the fluorescein arsenical helix binder (FlAsH) and resorufin} arsenical helix binder (ReAsH) are non-fluorescent.182_,183 _{Upon binding to the thiol groups of the}

peptide tag, these arsenical probes become fluorescent (green for FlAsH, red for ReAsH). This method found broad application towards intracellular fluorescent labeling of proteins, in which the short and minimally disruptive tetracysteine sequence is typically genetically inserted. However, the intrinsic toxicity of these arsenic reagents hinders the widespread use of these compounds in

vivo. Beyond Lewis acidic arsenical reagents, boronic acid-containing dyes have recently been

developed as an attractive alternative. One such example is rhodamine-derived bisboronic acid (RhoBo) that can recognize and bind to the tetraserine motif SSPGSS for fluorescent imaging (Fig.

1.9B).'84 _{Other metal ions for which peptide sequences have been developed to enable} peptide-mediated protein modification include Ni(II),'85_{-191 Zn(II),192-1}9 7 _{and Tb(III).'}98 20

1 Given the specificity of these peptide tags for the chelation of particular Lewis acids, it would be desirable to further expand the utility of reactive peptide sequences alternatively for small molecules, which

(A)

SH HS SH

FlAsH tagged protein

4OOH HO O O As As. FlAsH-EDT2 (non-fluorescent) Fluorescent H complex As As s/\ g\ (B) COOH Fluorescent complex HN 0O 4 N HO0 OH i HO OHI o 0 C B(OH)2 (HO)2BONi RhoBo RhoBo tagged protein (non-fluorescent)

Figure 1.9. Peptide tags appended to proteins of interest enable site-selective attachment of

fluorescent labels. (A) Non-fluorescent fluorescein arsenical hairpin (FlAsH) binds to the tetracysteine motif ofthe FlAsH tag (CCPGCC) to restore green fluorescence emission. (B) RhoBo tag (tetraserine, SSPGSS) binds to rhodamine-derived bisboronic acid (RhoBo) to turn on fluorescence.

1.3.4.3 Peptide and protein-based recognition by peptide tags

Finally, peptide sequences are also endowed with the ability to bind other peptide motifs with high affinity. This approach leverages the non-covalent interactions between two sequences to bring in close proximity two different reactive moieties incorporated on the peptide side chains (Fig. 1.10). One such example includes coiled-coil peptides, some of which were designed de

novo, to promote helical secondary structures that enable dimerization to form a superhelix.202-2 04

Relying primarily on strong electrostatic interactions between two unique coils, the dimerization domains of the matched peptides consists of three repeating EIAALEK motifs for the negatively charged E-coil and three repeating KIAALKE motifs for the positively charged K-coil (Fig.

1.10A). Using the coiled-coil scaffold, covalent bond-forming methods were developed in which

reactive groups were inserted at appropriate positions on the backbones of the two peptides. For instance, a cysteine thiol inserted on the E-coil reacts with an x-chloroacetyl moiety installed on the K-coil to enable crosslinking of the two peptide chains following the non-covalent binding

(Fig. 1.10B).2 0s _{Other chemistries employed for this strategy include: (1) dirhodium catalysts to}

transfer a diazo agent to a tryptophan residue206_,2 0 7 _{and (2) carboxy sulfosuccinimidyl esters to} modify c-amine of lysine residues (Fig. 1.10B).208 _{Collectively, these strategies were utilized to} perform cell surface labelling and live cell imaging.20 4208 2 09

(A)

K-tagged cargo E-tagged protein

= E-tag, (EIAALEK)3

V

CI SH OaS 00 (iii) Me Me. _{O Ph} O >0i'R4 0Rh 0 P 0 S 0 HN N 0 Si Ph me 01 me 11,00 4 H ₌

Figure 1.10. Electrostatic interactions between coiled-coil peptides can be leveraged to achieve site-selective protein modification. (A) The E-tag (EIALEK)3 appended to the protein of interest non-covalently interacts with the K-tag (KIAALKE)3 bearing the cargo to modify the protein. (B) Covalent linkage between the two coils can be achieved by: (i) proximity induced crosslinking between an electrophilic a-chloroacetyl group and a thiol side chain, (ii) proximity induced amine-reactive crosslinking with an activated ester, and (iii) rhodium-catalyzed alkylation of tryptophan with a cargo bearing diazo reagent.

(B) (i)

1.4 Cysteine arylation as an enzyme-free site-selective bioconjugation method

In developing complementary and more powerful methods for site-selective protein modifications with peptide tags, our group has explored nucleophilic aromatic substitution (SNAr) reactions between Cys-containing peptide sequences and perfluoroarenes (ArF).2 10 _This

transformation involves the addition of a thiol nucleophile to a perfluoroaromatic reagent, producing a highly stable sulfur-sp2 _{carbon bond (Fig. 1.11A).}2 _{The electron-withdrawing} fluorines serve to help stabilize the negative charge of the intermediate Meisenheimer complex. Fundamental studies on SNAr reactions with this class of reagents were initially performed in the 1960s,212 but few reports on the practical utility of this transformation followed in the subsequent decades.21

1,2 11 Our group set out to explore the use of perfluoroarenes as a new

enzyme/catalyst-free chemoselective strategy toward the modification of unprotected peptides and proteins. Initial efforts focused on the chemoselective SNAr reaction between an unprotected cysteine thiol within a peptide and ArF reagents (Fig. 1.11B).215 _{The perfluoroaryl-thiol SNAr}

reaction was found to be robust in organic solvents, but sluggish in water. Thus, this chemistry was further developed to operate in aqueous environments via a site-selective, enzyme-mediated approach. Specifically, we sought to leverage the catalytic activity of glutathione-S-transferase

(GST) which catalyzes the conjugation of activated aromatic electrophiles to the cysteine thiol

group of glutathione-containing peptides (GSH, y-Glu-Cys-Gly).2 16 _{Indeed, GST was found to be} tolerant to the scope of electrophile arenes, and promoted reactions between perfluoroarenes and and the Cys residue of a glutathione tag (y-Glu-Cys-Gly) appended to the protein of interest (Fig.

1.11C).216

However, it would still be desirable to develop technologies that proceed rapidly in aqueous buffer without the need for enzymes.

(A)

OH COOH FOF OH ( COOH

H2N eptide C(O)NH2 F F O H2N peptide C(0)NH2

? Tris, DMF

H2N NH NH

NH N:/ NH N: /

unprotected peptide chemoselective cysteine_arylation

(C

SH F S SH

F F F F

H₃N biomole-ule -C0o H3N biomol -Coo

Glutathione S- F

SH transferase _ F -F

(GST) s s

F F F F

protein with two site-selective cysteine

modification

Figure 1.11. Cysteine arylation via nucleophilic aromatic substitution (SNAr). (A) SNAr

reaction between a thiol and electron-deficient aromatic reagents under basic conditions. (B) Peptide-based "click" modification through a SNAr reaction between the unprotected thiol of a cysteine residue and a perfluoroarene. DMF: N,N-dimethylformamide, Tris: 2-amino-2-hydroxymethylpropane-1,3-diol. (C) Glutathione S-transferase (GST) catalyzes the site-selective modification of an unprotected cysteine residue in an N-terminal y-Glu-Cys-Gly sequence with cargoes bearing perfluorobiphenyl moieties.

1

Our group hypothesized that given the compatibility of perfluoroarenes with glutathione tag arylation techniques, it may be possible to develop a new cysteine-containing peptide sequence capable of directly reacting with the ArF reagents. Through peptide library screening, a small four amino acid reactive sequence, known as the "n-clamp" (Phe-Cys-Pro-Phe), was found to undergo cysteine arylation with ArF reagents at a rate >1000-fold faster than control peptides and without any additives (Fig. 1.12A).2 17 _{Computational analysis revealed that the a-clamp promoted the} reaction both kinetically (lowering the activation energy) and thermodynamically (generating a more stable product) compared to control peptides.2 17 _{Systematic mutation studies and solid-state}

NMR characterization suggest that (1) the hydrophobic side chains of the a-clamp interact with the perfluoroarylated reagents, and (2) the prolyl amide bond favors a trans conformation.218 _{It is} also possible that this peptide sequence facilitates rapid reactivity due to a hydrophobic effect, in which the lipophilic perfluoroarenes are drawn toward the hydrophobic pocket of the R-clamp. This phenomenon was further studied through the addition of a variety of simple inorganic salts, which were observed to change the rate constant of a-clamp arylation by over four orders of magnitude.219 _{The observed trend of this "salt effect" on the rate constant followed the Hofmeister}

series, a phenomenon known for over a century but not used for bioconjugation prior to this investigation.220221 _{The addition of salts likely tunes the reaction rates by modulating hydrophobic}

interactions between the it-clamp and the perfluoroaryl probe, as supported by computational studies. The advantage afforded by the salt effect is practical for biomolecule modification and, importantly, can be applied to reactions beyond arylation. For example, ammonium sulfate was also utilized to enhance the rate of an alkylation reaction of the n-clamp with an aryl chloride, thereby providing additional electrophilic substrates compatible with this peptide.219

(A)F F SH F _ SH __ __ _ __ __ __CF F F

F

site-selective antibody modification

Figure 1.12. n-Clamp-mediated site-selective cysteine modification. (A) x-clamp (Phe-Cys-Pro-Phe) enables selective arylation at its cysteine thiol in the presence of other competing thiols on the same biomolecule. (B) n-Clamp appended at the C-terminus of immunoglobulin heavy chains mediates site-selective antibody modification.

Given the impressive rate accelerations afforded by the a-clamp for arylations, compared with control peptides, our group next applied this system toward the site-selective modification of more complex substrates, such as antibodies. Given that the a-clamp is composed entirely of genetically encodable amino acids, it could be conveniently inserted into an antibody heavy chain C-terminus and readily conjugated to perfluoroaryl-linked probes in a single step under reducing conditions. Despite there being 32 free Cys residues, only n-clamp cysteine residues were labeled in this manner, whereas other thiols from the antibody interchain disulfides remained unmodified (Fig. 1.12B).m' The resulting site-selective antibody conjugates retained their target-binding affinity, indicating that the insertion of the n-clamp and the reaction conditions did not significantly affect the structural and functional integrity of the antibody. Compared to other site-selective antibody modification methods that require unnatural amino acids, enzymes, or multiple chemical steps, the n-clamp provides an enzyme/catalyst-free method for chemo- and regioselective modification of a natural amino-acid sequence, thus generating a fundamentally new route toward next-generation homogeneous antibody-drug conjugates.

However, a-clamp-mediated cysteine arylation is relatively slow (k= 0.73 + 0.01 M` _s-'), and thus exhibits poor selectivity in biological milieu, such as in HeLa cell lysates. This shortcoming has limited the application of this chemistry in complex biological systems. Ideally, a rate constant that is at least two orders of magnitude higher than that of n-clamp is desired. This enhanced reaction rate would enable the use of lower concentrations of reagents while still achieving quantitative conversion, and also confer greater selectivity of the conjugation reaction in complex protein mixtures. Most known reactive peptides have second-order rate constants below 1 M-1 s-1, which requires the use of high micromolar concentrations of reagents for reactions

to proceed in a practical length of time. Thus, there is a need for a faster peptide tag if this type of technology is to be universally implemented for various bioconjugation applications.

The potential of peptide libraries for discovering short-to-medium sized peptide sequences with carefully tuned chemical environments is exemplified by other studies in our group which led to the discovery of 29-residue abiotic peptide capable of site-selective Cys arylation with a ArF reagent2 2

2-2 24 and a 7-residue tag for site-selective Cys modification via thiol-yne reaction with

1.5 Overview of thesis

Building on recent progress in developing cysteine arylation as an efficient method for enzyme-free site-selective bioconjugations, this work aims to design and develop the next generation of short, reactive cysteine-containing peptide sequences. Chapter 2 focuses on the discovery of a nine-amino acid motif (Met-Cys-Pro-Phe-Leu-Pro-Val-Val-Tyr) termed the engineered reaction (EnAct) interface through a one-bead-one-compound combinatorial library selection approach. The SNAr reaction between EnAct tag and EnAct probe is 210 times faster than the first generation 7r-clamp under similar conditions (k = 152 ± 3 M- s-I vs k = 0.73 ± 0.01

M` s-'). Furthermore, due to the increased kinetics of the bioconjugation reaction, appending the EnAct sequence at the C-termini of the IgG antibody trastuzumab heavy chains enables site-selective conjugation to other biomolecules, such as biotin. Finally, Western blot analysis of bioconjugation reaction mixtures performed in cell lysates illustrates the enhanced selectivity of EnAct relative to 7t-clamp. The appreciable increase in cysteine arylation rate and selectivity achieved with the EnAct system introduces a new standard for regioselective bioconjugation under mild conditions.

Since both the nucleophilic tag and the electrophilic probe contains the same peptide sequence, we sought to explore the versatility of the peptide sequence to different chemistry. In

Chapter 3, we investigated the general reactivity of the EnAct tag for other substrates and

transformations. We found that this reactive peptide enabled aqueous arylations of Cys with minimal amounts of organic cosolvent, more aqueous than previously accessed with this class of electrophiles. We then turn to the probe, where the perfluoroarene was found not only to function as an electrophile for thiol arylation, but also to offer a handle for easy elimination to form dehydroalanine. From these advances, the EnAct interface appears poised to function as a highly

versatile and privileged peptide scaffold capable of facilitating a variety of different bioconjugation reactions.

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