Native Proteins

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Perspective

Chemistry for Covalent Modification of Endogenous/Native Proteins: From Test Tubes to Complex Biological Systems Tomonori Tamura, and Itaru Hamachi J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b11747 • Publication Date (Web): 28 Dec 2018 Downloaded from http://pubs.acs.org on January 1, 2019

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Journal of the American Chemical Society

JACS Perspective Chemistry for Covalent Modification of Endogenous/Native Proteins: From Test Tubes to Complex Biological Systems Tomonori Tamura† and Itaru Hamachi†,§* †

Graduate School of Engineering, Department of Synthetic Chemistry and Biological Chemistry,

Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan

ERATO, Japan Science and Technology Agency (JST), 5 Sanbancho, Chiyoda-ku, Tokyo

§

102-0075, Japan

1 ACS Paragon Plus Environment

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ABSTRACT Chemical modification of proteins provides powerful tools to realize a broad range of exciting biological applications, including the development of new classes of biopharmaceuticals and

functional studies of individual proteins in complex biological systems. Numerous strategies for

linking desired chemical probes with target proteins have been developed in the last two decades,

with most exploiting genetic protein engineering and/or bio-orthogonal chemistry that utilizes unnatural amino acids incorporated into proteins. Modification of native proteins in test tubes and biological contexts by site-specific and target-selective approaches remains challenging because

appropriate organic chemistry to carry out such modifications is currently limited. Nonetheless, a

variety of promising strategies have appeared recently that address this grand challenge in

chemical biology. These new chemistries yield native protein-based well-defined bioconjugations,

specific labeling of endogenous proteins in various biological crude milieus, and the establishment of chemical proteomics as a new research area in protein science. In this

perspective, we focus on recent remarkable progress in chemistry for native protein modification.

We survey chemical characteristics of the methods and describe briefly these advanced

applications to address unsolved biological issues. Current limitations and future directions of this research field are also discussed.


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Introduction Proteins are a major class of biopolymers that play pivotal roles in numerous biological events in all living organisms. Thus, structural and functional studies of proteins are a central research subject in life sciences. Chemical modification of proteins with designer synthetic probes can

create protein-synthetic molecule hybrids, which offer many exciting opportunities for protein research, biotechnology and drug development.1–5 For example, fluorescent labeling of a protein

of interest (POI) enables imaging analysis of the structure, function, dynamics and localization of the POI in biological environments.6,7 The introduction of a particular post-translational

modification (PTMs) into POIs in a site-specific fashion offers deep insights into our understanding of their fundamental roles in biological systems.8,9 Polyethylene glycol

modification of pharmaceutical proteins (PEGylation) has been used clinically to prolong the

circulatory time of these proteins and reduce immunogenicity.10 Antibody–drug conjugates (ADCs) generated by covalent modification of antibodies with cytotoxic compounds have

recently shown considerable promise in the treatment of various cancers.11 Furthermore, chemical incorporation of artificial functionalities into a POI has proven to be valuable for a wide variety

of other biological applications, such as construction of protein-based biosensors,12–14 artificial control of protein activity,15 detection of protein–protein interactions16 and specific enrichment of proteins in chemical biology.17

Covalent modification of proteins without impairing their inherent function and

structure requires careful consideration of several issues (Figure 1a and b). (1) The chemical reaction for protein modification must tolerate biological ambient conditions (pH 6–8, ≤ 37 °C, aqueous solvent). (2) Unlike standard reaction conditions of organic synthesis (typically, tens or

hundreds mM concentrations of substrates are used), proteins are generally handled as substrates

at low concentrations (on the order of μM or less) for both in vitro and in vivo applications,18

which can be a kinetic obstacle. (3) Although there are many reactive amino acids in proteins,

protecting groups, powerful tools to achieve chemoselective synthesis in conventional organic chemistry, cannot be generally used. (4) Nevertheless, in many cases, regio- and/or chemoselective incorporation of synthetic probes into a certain amino acid(s) in protein scaffolds is required to afford well structurally defined bioconjugates. (5) Moreover, modification of a POI

in complex biological environments such as in live cells gives rise to additional issues that must be overcome. There are numerous biological molecules other than the POI in highly condensed,

crowded environments where their localization and distribution are spatially and temporally 3 ACS Paragon Plus Environment


heterogeneous. Target protein-selectivity must be realized under such multi-molecular crowding conditions.

Bioorthogonal chemistry coupled with genetic protein engineering offers powerful

means to tackle these formidable requirements.3,19 This approach typically employs a two-step

protocol (Figure1c): (i) the site-specific incorporation of unnatural amino acids (UAAs) bearing

bioorthogonal reactive handles (e.g., azide, alkyne, alkene, tetrazine) through genetic code expansion strategies, or insertion of exogenous peptide/protein tag sequences into a POI,

followed by (ii) a corresponding bioorthogonal chemical reaction to selectively attach a desired

functionality to the genetically modified region of the POI. Because of the tremendous efforts to

improve and develop bioorthogonal chemistries, this strategy is now widely used for site-specific

and target-selective protein modification with outstanding rapid reaction kinetics in a variety of biological contexts, e.g., test tube, cells, tissues and animals.20 Although this approach is robust

and versatile, the major drawback is that it is applicable only to genetically engineered proteins and not to naturally occurring proteins.

There is a growing need for precise modification of natural proteins.2,21–23 Direct

chemical modification of natural amino acids in a target native protein is the most straightforward approach, and unlike the genetic method, should save time for the preparation of protein

conjugates and be more economical. In addition, the specific labeling of an endogenous protein

or protein group (sub-proteome exhibiting a particular common characters (e.g. localization, activity)) that is expressed in genetically intact living systems is ultimately the best scenario for

studying their true function in basic science and for therapeutic/diagnostic applications (Figure

1b). However, site-specific or target-selective chemical modification of such non-engineered

native proteins represents one of the most challenging tasks in chemical biology because it needs

to fulfill the abovementioned five requirements without any genetic engineering. Nevertheless, recent progress in this field continues to produce many promising strategies. For example, several

chemical reactions for modifying naturally occurring amino acids with elegantly controlling the position and number of probes on a protein have been developed in in vitro bioconjugation

studies (Figure 1a).1,2,4,5 These new technologies allow the generation of well-defined

biotherapeutics and PTM mimics. Recognition-driven chemical reactions enable selective

labeling of endogenous POIs even in the crowded multi-molecular environment of live cells,

which have been successfully applied for protein imaging, biosensor construction and irreversible

inhibition of protein activity (Figure 1b, top).2,12–14,23 Additionally, promiscuous covalent 4 ACS Paragon Plus Environment

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modification of a particular protein group in live cells has recently been proven to be useful for

proteome-wide analysis, being actively applied for the comprehensive identification of enzyme

families, PTMs, protein activity profiling and function/localization annotation of unknown proteins (Figure 1b, bottom).17,24

In this Perspective, we describe the current status and future directions in the field of

natural protein modification from three distinct points of view: (1) chemical modification of

natural amino acids in a single purified protein in a test tube; (2) a single target (endogenous) protein in crude live cell conditions; and (3) a class of natural proteins (proteome) as a target in

crude biological systems. We focus on recent strategies and discuss their advantages and

shortcomings. Although there are many chemical methods for covalent modification of proteins,

we have selected only the latest studies targeting natural (endogenously expressed) proteins.

More comprehensive discussions about chemical protein modification are available in excellent reviews.1–5,19–25



Figure 1. Chemical modification of natural/endogenous proteins with synthetic small molecules. (a) In vitro covalent modification of an isolated native protein in a site-specific manner. (b) Endogenous protein (proteome) labeling in biological systems containing a variety of biomolecules other than the target. (c) A complementary protein labeling strategy with genetic engineering and bioorthogonal chemistry. The first step involves the genetic incorporation of a bioorthogonal reactive handle into a protein of interest (POI) in cells. In the second step, the reactive handle selectively reacts with a designed synthetic probe through a bioorthogonal reaction. UAA, unnatural amino acid; aaRS, aminoacyl-tRNA synthetase.


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Chemo- and Regio-Selective Modification In Vitro

Chemical modification of native amino acids in proteins in test tubes is of significant value for

creating protein-based biopharmaceuticals, PTM mimics and hybrid biomaterials, as well as

structural and functional analysis of proteins. In most cases, chemo- and regio-selective reactions

are desirable because the site and the number of attached moieties critically affect the chemical

and physical properties of the conjugates, such as structural integrity, stability, activity, pharmacodynamics and pharmacokinetics. Thus, site-specific incorporation of desired

functionalities into proteins by strict control of the modification number is a primary interest in current protein bioconjugation studies. In this section, we highlight new strategies that have

emerged recently for modifying naturally occurring amino acids in proteins in a site-selective fashion with retaining their structures and activities.

Cysteine Modification. Cysteine residues naturally occurring and/or newly

incorporated by site-directed mutagenesis have long been used for chemo- and site-selective

protein modification because of their highest nucleophilicity among the canonical amino acids

and relatively low abundance (only 2.3% genome-wide).2 With appropriate electrophilic reagents,

such as maleimides and α-halocarbonyls, cysteines can be specifically modified via thiol alkylation (Michael addition and SN2 reaction, respectively)21,22,26. However, many studies have

indicated that the thioether bond generated by these reactions may decompose by undergoing thiol exchange reactions in the presence of high concentrations of thiol species (e.g., free cysteine

and glutathione).27,28 This issue is problematic, especially in applications involving ADCs, which

require stringent, high in vivo stability to avoid unexpected off-target toxicity. Therefore, recent efforts have explored advanced cysteine bioconjugation approaches that yield a very stable

covalent link. Several promising reactions have been identified, including the SNAr reaction and thiol-ene/yne coupling.22,29,30 Among them, a noteworthy example is the modification of cysteine

using an organometallic palladium reagent. Buchwald et al. developed the aryl palladium (II) complex using 2-2-dicyclohexylphosphino-2',6'-diisopropoxybiphenyl (RuPhos) as a ligand and

demonstrated that the reagent reacts very quickly and chemo-selectively with a cysteine thiol to

generate high yields of conjugates (Figure 2a).31 In general, palladium complexes are prepared in

situ from the precursor palladium source and ligands because of their instability. However, the authors found that judicious choice of an organic ligand affords stability even in water but is still

a suitable reactive palladium complex for transition-metal-based bioconjugation. The reaction 7 ACS Paragon Plus Environment


kinetics are fast and comparable to the secondary reaction rate of maleimide-thiol couplings (k2 = 103–104 M 1s 1).32 Owing to these superior kinetics, the reaction proceeds within 30 min using −

−

micromolar reagent concentrations or less with almost 100% yield. The S-arylated product obtained was more stable than the maleimide-thiol conjugate product. The authors succeeded in attaching the anticancer drug vandetanib to cysteine residues of the Trastuzumab antibody with

maintaining its binding ability. Although transition metal-based protein modifications generally suffer from the instability and insolubility of reagents in aqueous conditions, and severe

contamination of toxic metals in obtained conjugates, this work clearly demonstrated that appropriate molecular design of reagents and purification (94% of palladium can be removed by size-exclusion chromatography) can overcome such limitations.

The nucleophilic thiol of cysteine can be post-translationally converted to

dehydroalanine (Dha) as an electrophilic reactive handle by oxidative or bis-alkylation-induced

β-elimination of the thiolate from the cysteine.1,8,33,34 This “umpolung” strategy provides a powerful way for site-specific protein modification with nucleophile-tethered reagents (Figure 2a). For example, the Davis group reported a number of methods to create various PTM mimics

(e.g., phosphorylation, acetylation, methylation and glycosylation) and attach functional molecules to proteins through a Michael-type addition between Dha and thiol nucleophiles.8 A

potential drawback of this method is the use of high concentrations of thiol-containing reagents that may perturb disulfide bonds present in the protein scaffold by forming mixed disulfides. To

circumvent this issue, aza-Michael ligation to Dha with amine nucleophiles was reported recently.35 While the reaction rate is slow (k2 < 6.1×10 4 M 1s 1) owing to the relatively low −

−

−

nucleophilicity of the amine group under physiological pH, and thus a high concentration of

reagent is required (more than mM), this new technique has been applied to modify proteins containing many disulfide bonds.

The products generated by Dha-based conjugations using thiol- or amine nucleophiles

contain an unnatural thioether or sec/tert-amine linkages in the side chain, respectively, which are

not natural PTM structures. Recently, impressive studies to address this shortcoming were

independently reported by the Davis and Park groups.36,37 These new strategies exploit the

chemoselective reaction of Dha toward a variety of alkyl carbon free radicals prepared from corresponding alkyl halides with sodium borohydride or Zn(0)/Cu(II). They allowed C(sp3)-C(sp3) bond formation on proteins under aqueous buffer conditions, which realizes the

“posttranslational chemo-mutagenesis” application, whereas the conjugate will be obtained as 1:1 8 ACS Paragon Plus Environment

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D/L-diastereomers.8,36,37 The power of these approaches was demonstrated by the site-specific incorporation of PTMs previously inaccessible by genetic methods (e.g., ornithine, di- and trimethylated Lys).

Lysine Modification. The ε-amino group of lysine side chains is also commonly used

as a reactive handle for protein bioconjugation because of the rich chemistry available for

chemoselective reactions with primary amines. For example, Lys can be modified efficiently with

activated esters, sulfonyl chlorides, isocyanates and isothiocyanates, as well as reductive aminations and 6π-aza electron cyclic reactions. Basic pH conditions (pH > 8) are usually required to obtain the best yields because of the high pKa of the amino group (pKa ~10).21,22 A

major challenge in Lys-targeted conjugation is the control of the modification number because of

its high natural abundance on protein surfaces (5.9% of all sites in human proteins).38 Only

heterogeneous products with multiple modifications are usually obtained under standard conjugation conditions that use a large excess amount of reagents. While the degree of modifications can be controlled to some extent by limiting the reagent dose, such equivalent- or

sub-stoichiometric reaction conditions lead to a drastic decrease in modification yields. In general, some proteins have highly reactive Lys residues (i.e., exhibiting much lower pKa value of the

amino group than others in protein) on their surfaces, not active pockets, which may give site-specific homogeneous conjugates without impairing protein activities.39 A more robust

strategy was reported recently by Jiménez-Osés and Bernardes, in which computer-designed sulfonyl acrylate reagents enabled single lysine modifications on native proteins under

biocompatible conditions (37 °C, pH 8.0, 1 h) (Figure 2b).38 The reagent prefers lysine to other

nucleophilic amino acids mainly because of the stabilization of the intermediate caused by transient hydrogen bonding between the sulfonyl group and the ε-amino group of lysine side chains. Such an exquisite reaction mechanism also enhanced the reaction efficiency with a low

concentration of reagent. This allowed the selective modification of the most reactive lysine, which is predictable in silico by calculating solvent accessibilities and pKa values, in proteins

when using a single molar equivalent of the reagent. This successful work clearly shows that

computer-assisted reaction development is an important future direction in chemistry for covalent

protein modification. In particular, the precise prediction of amino acid reactivity and the pH level of the microenvironment on the surface of a protein, and/or molecular dynamics simulations

of the protein structure during the reaction could facilitate rational design of the reagent and 9 ACS Paragon Plus Environment


control of its chemo- and regioselectivity to generate homogenous conjugates with high

efficiency.

Aromatic Amino Acids. Modification of aromatic amino acids is neither common nor

straightforward when compared with that of cysteine and lysine modifications because of their

weaker reactivity. Nevertheless, it is important to expand the diversity of the conjugation

structures and functions. A wide variety of chemical reactions for selective Tyr modification have

been developed, including the azo coupling reaction with diazonium salts,40 the Mannich reaction with tyrosine/aldehyde/aniline,41 π-allyl palladium complex-mediated alkylation of the phenolic

hydroxyl group of tyrosine42 and the ene-like reaction with cyclic diazodicarboxamides.43

Conversely, only a limited number of studies on Trp-selective modification exist with a few

outstanding examples using transition metal reagents reported.44,45

In addition to these approaches, modern organic chemistry offers new opportunities to

efficiently modify aromatic residues of proteins. Ball and coworkers recently reported Tyr side

chain metallation through a three-component coupling of tyrosine, probe-appended boronic acid

and rhodium salt (Figure 2c).46 This reaction is selective toward Tyr even in the presence of basic side chains (Lys, His, Met) or other aryl side chains (Phe, Trp), and applicable to several protein substrates including antibodies. More interestingly, the resultant organometallic linkage (η6-arene Rh complex) is cleaved by treatment with dithiothreitol (DTT) or H2O2. While this

susceptibility toward nucleophilic reductants may cause unfavorable problems in some situations

that require stable conjugates (e.g., ADC applications), it may provide a unique strategy for antibody-mediated drug delivery or traceless release of native antibodies in reducing environments in vivo.

For chemo-selective Trp bioconjugation, Kanai and coworkers exploited a transition

metal-free

method

using

an

N-oxyl

radical,

9-azabicyclo[3.3.1]nonane-3-one-N-oxyl

(keto-ABNO), as a Trp-reactive electrophile precursor (Figure 2d).47 Although the reactions require acidic aqueous solutions (0.1–0.5% AcOH) and the less bioorthogonal NaNO2, the

absence of toxic transition metals is invaluable, especially for avoiding metal contamination of

semi-synthesized biopharmaceuticals. More recently, another radical-based Trp modification

method was reported by the Davis group.48 Here, sodium trifluoromethanesulfinate (NaTFMS, also known as Langlois’ reagent) and tert-Butyl hydroperoxide (TBHP) as a CF3 source and a

radical initiator, respectively, were used for direct trifluoromethylation of proteinogenic amino 10 ACS Paragon Plus Environment

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acids. The new radical-based protein fluorination approach was shown to prefer Trp to other canonical amino acids and is useful for 19F-NMR-based protein conformational analysis.

N- or C-Terminal Modification. Since single-chain proteins have only one N- and

C-termini, targeting such unique sites serves as a powerful solution to obtain homogeneous

conjugates. There are several approaches for N-terminal modification, such as pH-controlled

acylation and reductive alkylation, native chemical ligation and cyclic condensation at N-terminal cysteines, and transamination using pyridoxal-5'-phosphate (PLP).49 As a more general and

simple approach, the Francis group recently reported a one-step modification of a native protein

N-terminus with 2-pyridinecarboxaldehyde (2PCA) derivatives (Figure 3a).50 The reaction

proceeds via imine formation between 2PCA and the N-terminus followed by the attack of the

neighboring amide nitrogen in the protein backbone to form a stable imidazolidinone product. This method allows direct incorporation of desired functionalities to native protein N-termini with the exception of proteins with proline at position 2 or N-terminal acylated proteins.

In contrast to many successful examples of N-terminal modification, chemical

strategies for selective transformation of protein C-terminal carboxylates remain sparse. In 2018, however, a significant breakthrough was achieved by the MacMillan group (Figure 3b).51 They

exploited the subtle difference in oxidation potentials between side chain alkyl carboxylates (i.e., Asp and Glu) [E1/2Red = ~1.25 V (vs SCE)] and C-terminal α-amino carboxylates [E1/2Red = ~0.95 V (vs SCE)] to attain C-terminal selective modification via oxidative decarboxylation. Among

several candidates, flavins were selected as a water-soluble and efficient photocatalyst for single-electron transfer. The authors demonstrated the C-terminal-selective photoredox

decarboxylative conjugation with the flavin catalyst and the probe-linked α,β-unsaturated

carbonyl compounds (Michael acceptors). Although further investigations are required to clarify

the protein scope of this method and identify side reactions observed in some cases, this novel strategy sheds light on the development of photoredox catalyzed protein modifications.

Other Compelling Methods. Methionine is the second least abundant amino acid in

mammals, and solvent-exposed methionines are rarely found on the surface of proteins because

of their hydrophobicity. Thus, methionine-selective modification should be a promising strategy

to obtain well-defined bioconjugates. A robust Met-selective bioconjugation method has been recently reported by Toste, Chang and coworkers, called redox-activated chemical tagging 11 ACS Paragon Plus Environment


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(ReACT) (Figure 2e).52 The authors focused on the oxidative sulfur imidation reaction with

oxaziridine derivatives for direct conversion of methionines to the corresponding sulfimide conjugation products. The reaction proceeded rapidly when BSA was used as a model protein, and the second order rate constant was measured to be 18.0 ± 0.6 M 1s 1. They also demonstrated −

−

the ReACT technology is applicable for the synthesis of ADCs and identification of

hyperreactive Met residues in whole proteomes (as discussed below). Most recently, Gaunt and

coworkers reported another methionine-selective bioconjugation strategy with hypervalent iodine

reagents.53

In addition, direct arylation/alkenylation of amide N−H bonds of a protein main chain

has emerged recently as a new class of protein modifications. Ball and coworkers discovered that a specific N−H bond of the peptide backbone immediately before a histidine residue undergoes the Chan-Lam coupling reaction with copper (II) acetate and boronic acid derivatives (Figure

3c).54 The remarkable reactivity and selectivity likely relies on the amino-terminal Cu and Ni

(ATCUN) motif-like intermediate. The reaction proceeds under benign conditions (pH 7.4,

aqueous buffer, room temperature) with millimolar reagent concentrations. Using this method, despite the low yield (~25%), the authors demonstrated a single modification of a backbone N−H

bond in lysozyme. This approach has also been used for selective modification of N-terminal

pyroglutamate (a naturally occurring PTM)-His tagged proteins in E. coli cell lysates55 and photocaging protein backbones56.

Recognition-driven chemical reactions also provide a powerful way to site-specifically

modify proteins (discussed in more detail in the following section).57 Notably, this approach affords the efficient modification of less reactive amino acid side chains that are inert with

normal intermolecular reactions, because the inherent reaction rate is dramatically accelerated by the proximity effect. For example, ligand-directed tosyl (LDT) chemistry57–59 developed by

Hamachi, Tsukiji and coworkers can modify protein surface Glu, Asp, His, Tyr and Cys residues

only near the ligand-binding site of a protein via a proximity-enhanced SN2 reaction, and its variant acyl imidazole (LDAI) chemistry60 primarily targets Lys and Ser side chains to form

carbamate or carbonate esters in water. The acyl transfer catalyst-mediated Tyr specific modification has been demonstrated by using affinity-guided DMAP (AGD) chemistry.61 A wider scope of the side chains (Trp, Tyr, Phe, Gln, Asn, Glu, Asp, Arg, Ser, and His, as well as Cys and

Lys) can be modified using affinity-guided dirhodium catalysis reported by the Ball group.62 It is

noteworthy that this work represents the first example of Gln and Asn modification, highlighting 12 ACS Paragon Plus Environment

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the power of the proximity effect. Taking advantage of the prominent reactivity and a specific

interaction between the Fc regions of antibodies and the Z domain of protein A, the same group

elegantly succeeded in site-specific antibody functionalization (at an Asn in the Fc region) and in

generating a single-modified homogeneous ADC (Herceptin-doxorubicin).63 More recently,

another type of proximity-driven protein modification method, called linchpin-directed modification (LDM), has been reported.64 In this work, reagents containing a carbonyl group and

an epoxide initially react with amino group of Lys residues in protein through Schiff base formation, which facilitate the second reaction between the epoxide and a His residue only close

to the Lys via a proximity effect. This method allows chemo- and site-selective labeling of His

residues of several native proteins, while conversions are modest in many cases.



a

PCy2 OiPr Pd X

iPrO

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S

10 µM for 1 µM protein pH 7.5, Tris buffer, 5% DMF 30 min, ~100% yield (k2 = ca. 103 – 104 M-1 s-1) S

HS ~mM SH

pH 8.0 0.5–2 h >95% yield

CONH2 Br Br

H 2N

pH 8.0 1–3 h

pH 8–9, NaPi buffer 1–24 h, >95% yield (k2 = 6.1 x 10-4 M-1 s-1)

X

b

O

4

NH2

NH

~mM

CONH2

NaBH4, pH 4–8 or Cu(OAc)2, Zn0, pH 4.5

Single Lys modification

MeO

SO2Me

10 µM for 10 µM protein (1 equiv.)

4

H N

OMe O

~mM

H2NOC OH

(1 mM)

(HO)2B

4

NH OMe O

pH 8.0, 1–2 h

pH 8.0, 37 °C 1–2 h ~100% yield

c

H 2N

H N

OH H N Rh

RhCl3 (1 mM) Na2CO3 (pH 9.4) 37 °C, overnight

O

X

DTT or H2O2

d

N

O

NH

e

N

O

(5 equiv.)

N

O

NaNO2 (3 equiv.) HO

H2O : AcOH = 200 : 1 RT, 30 min 64% (for Lysozyme)

N O

N

NH

O N S

O

S

O

N H

N H

(100 µM) PBS buffer, RT 1–2 min, >95% yield (k2 = 18 M-1 s-1)

Figure 2. Modern protein bioconjugation reactions through side chain modification of naturally occurring amino acids. (a) Representative cysteine-specific modifications. (b) Single lysine modification by sulfonyl acrylate reagents followed by aza-Michael addition. (c) Reversible tyrosine modification through three-component organometallic complexation. (d) Metal-free tryptophan modification with N-oxyl radical. (e) Chemo-selective methionine bioconjugation using oxaziridine-based reagents. 14 ACS Paragon Plus Environment

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a R H 2N

H

N

O

5–10 mM

H N

N

pH 7.5 RT–37 °C, 8–16 h 43% – 96%

O

-terminus

b

Me O

O

R

N H

R

OH

Me N

Me Me

10 equiv.

O

H N

Me O

N

HN

N

N

O

O NH

O

3 equiv.

34 W blue light

Me H N

c

Me O N H

R

O

pH 3.5, RT, 8 h Caesium formate buffer : glycerol = 95 : 5 31% – 66%

C-terminus

O

BF3K R

O N H

H N

1 mM

O

1 mM Cu(OAc)2

N H

O N

NH

pH 7.4 RT, overnight ~ 25%

R

O N

H N

O N H

O N

NH

Figure 3. Recent strategies for modifying the main chain of native proteins. (a) One-step N-terminal modification with 2-pyridinecarboxyaldehydes. (b) C-terminal-selective photoredox decarboxylative conjugate addition with a flavin photo-catalyst and radicalphile (3-methylene-2-norbornanone derivatives). (c) Copper (II)-mediated direct arylation of backbone N−H bonds in proteins.



Selective Chemical Modification of Endogenous Proteins in Live Cells by a Ligand-Directed Approach

Most of the above-mentioned bioconjugation methods exhibit amino acid selectivity but not

protein selectivity, which therefore limits their use to in vitro applications for a single isolated

protein. Affinity-based labeling is particularly useful for implementing target-selective and

site-specific labeling of endogenous proteins in their natural environments.65,66 However, there remain limited chemistry approaches and these generally suffer from low reaction yields. In

addition, the labeled protein obtained by traditional affinity labeling often loses its native activity

because the affinity ligand permanently occupies the ligand-binding pocket, which hampers suitable applications. This situation is changing with the advent of a series of traceless affinity labeling and affinity-guided catalysts (Figure 4a and Figure 5a), both of which our group

pioneered.2,23,57,58,67 These new technologies enable efficient incorporation of various

functionalities into endogenous proteins while retaining their original functions in intact cells.

As the reaction conditions of native protein modifications shift from test tubes to living

cells, tissues, and even in vivo, several new challenges not faced in in vitro conjugation methods

must be addressed. (1) Since the expression levels of intracellular endogenous proteins are

extremely low (generally, 8) and low bioorthogonality of the thioester acyl donors), our group recently developed a

newly designed affinity-guided labeling system, in which a pyridinium oxime and a NASA

reactive group were used as a highly nucleophilic acyl transfer catalyst and a bioorthogonal acyl donor, respectively.89 This method, termed affinity-guided oxime (AGOX) chemistry, allowed

more efficient and low-background labeling of native-form proteins in test tubes and cell lysates 20 ACS Paragon Plus Environment

Page 21 of 56 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


than that of AGD chemistry (Figure 5c). Furthermore, the improved biocompatibility and bioorthogonality enabled target-selective and site-specific labeling of an endogenous

neurotransmitter receptor (AMPAR) in mouse hippocampal and cerebellar slices (Figure 5c and d). Nonetheless, selective modification of native proteins has not been achieved by catalyst-based traceless protein labeling. This might be because of insufficient bioorthogonality of acyl donors

and the kinetic disadvantage of this approach relying on a multi-component reaction, that is, the

rate-determining activation step of the acyl donor is a conventional intermolecular reaction. Thus

continuous efforts are required to explore better chemistry or a strategy that can enhance the

activation step. While a ligand-tethered transition metal catalyst offers a promising alternative for the selective modification of native proteins in crude biological samples, there are only a few reports that have demonstrated the feasibility of transition metal-mediated affinity labeling in

live-cell environments.90



Figure 5. Endogenous protein labeling through affinity-guided (AG) catalysts in biological environments. (a) General reaction scheme of the AG catalyst strategy. (b) Selective labeling of cell surface receptors using the DMAP-tethered antibody and thioester-type acyl donor. (c) Selective labeling of neurotransmitter receptors in brain slices by AG oxime (AGOX) chemistry. AMPAR, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor (d) Location of the labeling sites on the crystal structure of AMPAR (PDB ID: 3KG2).


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Covalent Modification of Endogenous “Proteomes” for Chemical Proteomics In addition to the specific chemical modification of a single targeted protein, “broad” or “promiscuous” labeling of a protein group (proteome) bearing a particular common character is

also important in current chemical biology (Figure 1b, bottom).17,24 This approach allows a wide

range of chemical proteomic applications, such as the investigation of chemical and biological properties of a proteome of interest, characterization of less-known protein families, global profiling of enzymatic activity, and identification of a drug target (and off-targets). Further, the

labeling site analysis, if possible, also provides proteome-wide information on the chemoselectivity of the labeling reaction in biological contexts. Clearly, these studies rely mostly on targeting natural amino acids in endogenous proteins, while UAAs-based methods also serve

as powerful strategies for chemical proteomics.91–93 Many interesting chemical proteomics studies

have been reported because of the recent growth of both biocompatible protein chemistry and mass-based proteomic technologies. In this section, we discuss several recent research efforts on

proteome-targeted chemical proteomics with reflection on endogenous protein labeling.

Activity-Based Protein Modification. Traditional proteomics have focused on

comprehensive identification of proteins and quantification of changes in their abundances using

gel- and/or mass-based analysis. However, since the function of a protein is exquisitely regulated by posttranslational modification/truncation and interaction with other biomolecules in live cells, a method for direct evaluation of “protein activity” is also required to accurately reveal the

biological roles of each individual protein. Active site-directed protein labeling, represented by activity-based protein profiling (ABPP), provides a powerful approach for the proteome-wide

monitoring of enzyme functional states in complex biological systems (Figure 6a).17 The Cravatt

group, pioneers of this approach, devised activity-based probes (ABPs) that generally have three

elements: (i) a reactive group to form a covalent linkage with natural amino acids located at the protein active site; (ii) a spacer to avoid steric hindrance or a binding group that directs the

reactive group toward certain classes of proteomes; and (iii) a reporter tag (e.g., fluorescent dye,

biotin, bioorthogonal reactive handles) for detection and purification of binding proteins. ABPs

exclusively react with active enzymes and not with inactivated or inhibited states. This

exclusivity allows quantitative and comprehensive readout of a class of enzyme activity without

relying on the expression levels through gel- or contemporary LC-MSMS-based proteomics

technologies. A vast array of ABPs has been developed for many enzyme classes, including 23 ACS Paragon Plus Environment


hydrolases, kinases, phosphatases, glycosidases, histone deacetylases and oxidoreductases.17

These ABPs typically employ an electrophile or photoreactive group as the warhead, such as

fluorophosphonates, Michael acceptors, epoxides, phenyl sulfonates, vinyl sulfonates, sulfonyl fluorides, α-halo carbonyls, acyloxymethyl ketones, benzophenones and diazirines (Figure 6b).

Most of these warheads have been developed originally as irreversible inhibitors or for bioconjugation of natural proteins in vitro, which explicitly highlights that future expansion of

the chemistry for natural protein covalent modification will involve further enrichment of the

ABPP strategy.

In addition to enzyme activity profiling, the ABPP approach extends to global analysis

of amino acid reactivity in proteomes. Weerapana, Cravatt and coworkers established a new

method for quantitative profiling of cysteine reactivity at the proteome level (Figure 7a).94 In this method, cysteines in proteins are initially alkylated with an alkyne-tethered iodoacetamide probe

(IA). Subsequently, using click chemistry, a cleavable purification tag (TEV-tag) containing a

biotin and an isotopically labeled valine (heavy or light) is ligated to the alkylated protein. This TEV-tag thus functions both as a cleavable linker after affinity purification with avidin beads and

as a probe for quantitative mass spectrometry of the labeled peptides (isotopic tandem orthogonal

proteolysis-activity-based protein profiling, isoTOP-ABPP). Millimolar concentrations of IA are

typically required to modify all cysteine thiols in the proteome. In contrast, if a lower concentration (~μM) of IA is used, only high-nucleophilic cysteines preferentially react.

Therefore, the reactivity of cysteines in a proteome can be quantified by comparing the extent of alkylation in the two proteomes treated with high and low concentrations of IA. Using this elaborate method, a large number of hyper-reactive cysteines were identified in several proteins

including functionally unknown proteins, some of which were ascertained to be involved in

enzyme biosynthesis, activity and redox reactions. This strategy has been used for characterizing

the unique roles of reactive cysteines in lipid modification95 and strong coordination of zinc96.

The Weerapana group also reported a more biocompatible photo-caged probe for cysteine

alkylation, enabling global labeling and profiling of cysteine reactivity in live cell environments (Figure 7b).97 More recently, reactivity profiling focusing on lysine98 and methionine52 has also

been conducted with sulfotetrafluorophenyl ester and oxaziridine compounds as the reactive group of ABPs, respectively (Figure 7c and d). Furthermore, reverse-polarity ABPP, where nucleophilic hydrazine probes were used, enabled the discovery and characterization of novel

electrophilic PTMs that are naturally generated on canonical amino acids in proteins under live 24 ACS Paragon Plus Environment

Page 24 of 56

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cell contexts (Figure 6c).99 Such a series of exhaustive inquiries into amino acid reactivity in

native proteomes further enhances our understanding of the molecular mechanisms of protein

activities and also provides helpful guidelines for the rational design and future development of new synthetic reagents for chemical proteomics and target- and site-selective protein

modification.

Another important aspect of the ABPP strategy is that it serves as a versatile platform

for the discovery of enzyme inhibitors. For example, the competitive ABPP application, where

proteomes are pretreated with inhibitors, allows easy evaluation of inhibitor selectivity and potency by detecting their ability to block chemical labeling of enzymes with SDS-PAGE.100

Moreover, straightforward identification of the inhibitor targets is possible by excision of the

corresponding protein bands from the gel followed by mass-finger printing analysis. Recent studies clearly demonstrate that competitive ABPP coupled with quantitative LC-MSMS analysis

offers a simple and robust assay to profile both “on-”and “off-” targets of objective inhibitors in

live cells.80 More recently, an innovative competitive ABPP application for identifying “druggable” proteins was reported by the Cravatt group (Figure 7e).101 In this method,

competitive isoTOP-ABPP with a cysteine-reactive IA-alkyne probe was used for screening a

chemical library containing fragment electrophiles (predominantly containing cysteine reactive groups, average molecular weight of 284 Da) to identify ligandable cysteines in human cancer

proteomes. This approach identified 758 liganded cysteines in 637 distinct proteins, among which

545 proteins were newly identified as druggable (ligandable) proteins. This study broadened our

knowledge of the scope of drug discovery targets tremendously. Future efforts along this line

should accelerate further drug development on the basis of new binding mechanisms and also

enable exciting applications in current chemical biology, such as expansion of targetable proteins with LD chemistry, development of irreversible PROTAC,102,103 and artificial control of the

spatial location of endogenous proteins in living cells104.



Figure 6. Covalent labeling of a specific protein family with activity-based probes (ABPs). (a) General concept of activity-based protein profiling (ABPP) (b) Representative electrophilic warheads of ABPs. (c) Chemical probes for reverse-polarity ABPP.


Page 26 of 56

Page 27 of 56

a

Hyper-reactive Cys I

SH

O

N H

Light TEV-tag

S

IA-alkyne

Sample A

N N

Click reaction

S

N N

S

S

1. Mix 2. Avidin enrichment 3. Trypsin digestion 4. TEV digestion

N

N3

High concentration

SH

S

N

S

I

SH

O

Heavy TEV-tag

S

N H

S

N3

Low concentration

Click reaction

SH

N

N N

N N

N

N

Quantitative LC-MS/MS N

Intensity

SH

N N

N N

S

Less-reactive Cys

SH

Light Heavy

Sample B

b O2N Br

O

UV

O

Br

c

NaO3S

O

F

Caged bromomethyl ketone

F

F

F

d O

O

Light TEV-tag

Nonliganded Liganded

N3

Cells or lysates

Cys-reactive library I

O

Click reaction

N H

DMSO

N H

Methionine-reactive oxaziridine

Amine-reactive sulfotetrafluorophenyl ester

e

O N O

1. Combine 2. Enrich 3. Tryptic Digest 4. LC/LC-MS/MS

MS1 Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Heavy Light

N3

Heavy TEV-tag

Figure 7. isoTOP-ABPP applications. (a) Global profiling of cysteine reactivity in proteomes. (b) A photocaged bromomethyl ketone for cysteine reactivity analysis in live cells. (c) An amine-reactive sulfotetrafluorophenyl ester probe for proteome-wide quantification of lysine reactivity. (d) An oxaziridine probe for reactive methionine identification with TOP-ABPP. (e) Proteome-wide screening of covalent fragments on the basis of competitive isoTOP-ABPP.

Proximity-Dependent Endogenous Proteome Labeling. To understand the

pleiotropic functions of proteins in live cells, unveiling the subcellular localization and

interacting proteins of POIs, as well as their activities, is also invaluable. Traditionally,

organelle-focused proteomics relied on biochemical fractionation to dissect and extract the cellular components, which often suffers from heavy contamination of other organelles and some

subcellular components are difficult or impossible to isolate by the fractionation method. The conventional co-immunoprecipitation technique for identifying interacting proteins is not able to

trap weakly and/or transiently interacting proteins of POIs. As a powerful alternative, proximity-driven protein labeling has emerged recently for mapping endogenous proteins at a

specific subcellular location and identifying an interactome related to POIs (Figure 8).24 Typically, 27 ACS Paragon Plus Environment


this method uses engineered enzymes, by which information of a proteome on localization or

interaction can be converted to chemical tagging by covalent bond formation. The enzymes are usually linked by genetic fusion to a specific POI or targeted into subcellular organelles by a

signal sequence, which enable modification of endogenous proteins with biotin in close

proximity (within nanometers) of the enzymes. After the reaction, tagged proteomes are purified

and enriched by avidin-beads and identified by LC-MSMS analysis.

Two enzymes are popularly used for this approach: engineered ascorbate peroxidase

(APEX or APEX2)105.106 and a biotin ligase mutant (BioID)107. The APEX-based method was

developed by Ting et al. and relies on the activity of the enzyme to oxidize a biotin-phenol

substrate into a phenoxyl radical with the aid of 1 mM H2O2. The radical has a short half-life (95% yield

CONH2 Br Br

H 2N

pH 8.0 1–3 h

pH 8–9, NaPi buffer 1–24 h, >95% yield (k2 = 6.1 x 10-4 M-1 s-1)

X

b

O

4

NH2

NH

~mM

CONH2

NaBH4, pH 4–8 or Cu(OAc)2, Zn0, pH 4.5

Single Lys modification

MeO

SO2Me

10 µM for 10 µM protein (1 equiv.)

4

H N

OMe O

~mM

pH 8.0, 1–2 h

pH 8.0, 37 °C 1–2 h ~100% yield

c

H2NOC OH

H 2N

(1 mM)

(HO)2B

OH H N Rh

RhCl3 (1 mM) Na2CO3 (pH 9.4) 37 °C, overnight

O

X

DTT or H2O2

d

N

O

NH

e

N

O

(5 equiv.)

N

N

NaNO2 (3 equiv.) H2O : AcOH = 200 : 1 RT, 30 min 64% (for Lysozyme) O

S

O

N O

N H

(100 µM)

NH

HO

O N S

N H

PBS buffer, RTACS Paragon Plus Environment 1–2 min, >95% yield (k2 = 18 M-1 s-1)

O

4

H N

NH OMe O

a

Page 49 of 56 1 R 2 H N 3 H 2N O 4 5 -terminus 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 O 21 22 23 24 25

Journal of the American Chemical Society H

N

O

5–10 mM

N

pH 7.5 RT–37 °C, 8–16 h 43% – 96%

b

Me O

O

R

N H

R

Me

Me N

Me

N

10 equiv.

O

H N

Me O

OH

N

HN

N

O

O NH

O

3 equiv.

34 W blue light

Me H N

c

R

O

pH 3.5, RT, 8 h Caesium formate buffer : glycerol = 95 : 5 31% – 66%

C-terminus

O

BF3K R N H

H N

1 mM

O

1 mM Cu(OAc)2

N H

O N

NH

ACS Paragon pH 7.4 Plus Environment RT, overnight ~ 25%

R

O N

H N

O N H

O N

NH

Me O N H

a 1 2 3 4 5 ePOI 6 7 8 9 10 11 LDT (2009 ) Probe 12 13 O 14 O S 15 O O 16 Reactive 17 group 18 Ligand 19 Tosylate 20 21 22 ~101 23k2 (M-1s-1)* 24 25 His, Tyr, Glu,Asp, Labeled 26 Cys 27amino acid 28 test tube, inside cells 29 Labeling 30 condition Mucus tissue, lysates 31 in vivo (mouse), etc. 32 33Application 19 F-NMR biosensor, 34 Fluorescent biosensor, 35 FRET analysis, 36 37 Photo-cross-link, 38 Ligand-binding site 39 identification 40 41 42 43 44 45 46 47 48 49 50 51 52 Staudinger 53 ligation SPAAC 54 O 55 OMe + + N3 56 PPh3 57 58 10-3 10-2 10-1 59 60

Cleavable electrophiles

Ligand


Page 50 of 56

Probe

Nu

Protein-ligand interaction

“Proximity-driven” labeling reaction

ePOI

ePOI

b

LDAI (2012

)

LDBB (2015

O N

)

LDSP (2017

Br O

O

N

O

O

O

O N

S

)

O

Dibromophenyl benzoate

N-sulfonyl pyridone

~101

~102

N.D (10 min ~ 6 h)

Lys, Ser, Thr, Tyr

Lys, His test tube, cell surface, inside cells

Fluorescent biosensor, Live cell imaging of membrane proteins, Pulse chase analysis, Photo-controlling of enzyme activity, Drug screening

Live cell imaging of intracellular proteins

O O

O O

Lys

test tube, cell surface, inside cells

test tube, cell surface, inside cells

FRET-based biosensor construction in cells

Covalent inhibition of HSP90

O

O S O

N

~104

LDNASA

LDT

)

CN

N-acyl-N-alkyl sulfonamide

Tyr, Lys

test tube, cell surface, mouse brain tissue (membrane proteins)

S

O

Acyl imidazole

c

O

O

O

Br

,

LDNASA (2018

O

LDBB

CN S

N O O

Br O

LDAI

O N

O

O

O

Enzymatic labeling (SNAP/CLIP-tag, etc.)

Br

N

Tetrazine-BCN

CuAAC

+

N3

1

N3

+ Cu(I)

O

101 Plus Environment 102 ACS Paragon

rate constant / M-1s-1

+

N N

N N R’

103

Tetrazine-TCO

R

O

104

+

R N N

N N R’

105

106

Page 51 of 56 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43


ACS Paragon Plus Environment

Journal of the American Chemical Society 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25


Page 52 of 56


Hyper-reactive Cys O

Light TEV-tag

S

S

N N

1. Mix 2. Avidin enrichment 3. Trypsin digestion 4. TEV digestion

N

N3

Click reaction

S

N N

S

S

N

S

Heavy TEV-tag

S

N3

Click reaction

SH

b

Br

S

c

O

NaO3S F

N N

F

F

N

d

O

O

N O

O

Light TEV-tag

Nonliganded

N3

I

O

Click reaction

N H

1. Combine 2. Enrich 3. Tryptic Digest 4. LC/LC-MS/MS

ACS Paragon Plus Environment N3

Heavy TEV-tag

N H

Methionine-reactive oxaziridine

Amine-reactive sulfotetrafluorophenyl ester

e

N

N N

N N

N

N

Quantitative LC-MS/MS

SH

F

N N

S

Intensity

I 1 N SH H 2 IA-alkyne 3 4 High SH 5 concentration 6 7 Sample A Less-reactive 8 Cys 9 10 11 O 12 SH I N 13 H 14 15 Low SH 16 concentration 17 18 Sample B 19 20 21 22 UV O2N 23 O O 24 Br 25 26 Caged bromomethyl ketone 27 28 29 30 31 32 33 34 Cys-reactive 35 library 36 Cells 37 or 38 lysates 39 40 DMSO 41 42 43

MS1 Intensity

a

Page 53 of 56

Liganded

Heavy Light

Light Heavy

Journal of the American Chemical Society Spatially-restricted enzymatic labeling 1 2 nzyme-coding 3 plasmid 4 5 6 7 Transfection 8 9 10 11 12 13 14 15 16 17 18 19 20

Biotin

H2O2

H 2O O

OH Biotin-phenol

1 min

Highly reactive Phenoxyl radical

APEX

Labeled proteome

O HN S

ATP

NH

PPi

NH2

O HN

OH O

Page 54 of 56

proteome near the enzyme

S

N

NH

O

O O P O O

Biotin-AMP BioID ACS Paragon Plus Environment TurboID (miniTurbo)

N

N N

O OH OH

6–24 h (BioID) 10 min (TurboID) Labeled proteome

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TOC 66x34mm (300 x 300 DPI)


Page 56 of 56

Native Proteins

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