Indian Journal of Chemistry

Vol. 52A, Aug-Sept 2013, pp. 973-991

Advances in Contemporary Research

Site-specific chemical modifications of proteins

Divya Agrawala,

* & Christian P R Hackenbergera, b,

*

aLeibniz-Institut für Molekulare Pharmakologie (FMP), Robert-Rössle-Str- 10, 13125, Berlin, Germany bHumboldt Universität zu Berlin, Institut für Organische und Bioorganische Chemie,

Institut für Chemie, Brook-Taylor-Str. 2, 12489 Berlin, Germany

Email: agrawal@fmp-berlin.de (DA); hackenbe@fmp-berlin.de (CPRH)

Received 8 April 2013; accepted 10 May 2013

Since long, peptides and proteins have been recognized as targets for the development of synthetic methodologies.

There have been major advances in the development of methods for the site-specific modifications. The naturally occurring

functionalities in peptides and proteins are used to modify the less common amino acid targets. The most important benefit

of chemical bioconjugation techniques using naturally occurring amino acids is that they do not require additional

biochemical techniques to install unnatural functionalities for chemoselective reactions. However, unnatural functionalities

can well be incorporated into peptides and proteins followed by the establishment of bioorthogonal coupling methodologies.

The unnatural amino acids can be incorporated into proteins either in a site-directed or residue-specific fashion.

Bioorthogonal chemistry is an important tool for the development of synthetic methodologies and for further advances in

biological research. A variety of methods exist that tag cellular components with reporters not only for visualization but

also for isolation from biological samples. In this review, we present an overview of the efforts of both chemical and

biochemical approaches to functionalize peptides and proteins with the desired molecular components. We focus on the site-

directed methods to incorporate unnatural groups into biomolecules and the development of bioorthogonal transformations

involving unnatural functional groups.

Keywords: Protein modifications, Site-selective modifications, Protein functionalities, Bioorthogonal transformations,

Chemoselective reactions, Bioconjugation, Chemical bioconjugation, Copper catalysed reactions, Biomolecules,

Amino acids, Unnatural amino acids, Canonical amino acids, Azides, Alkynes, Ketones, Aldehydes

How can one probe diverse biological functions

of proteins as well as the impact of side-chain

functionalities in complex systems? Although

innumerable successful research projects have been

carried out, this is still a major challenge for chemical

biologists and chemists which they tend to address

by the introduction of various chemical modifications

in proteins1.

The site-selective introduction of chemical

functionalities into proteins has a particular focus.

A set of enzymes present in nature leads to

the introduction of bioorthogonal functional groups

and modifications of proteins in living cells2.

The enzymes such as biotin ligase (BirA), trans-

glutaminase (TGase) and lipoic acid ligase (LplA)

have been used to site-specifically modify proteins

with biophysical probes, short chain azido fatty acids

and fluorophores3. The formylglycine-generating

enzyme (FGE) consensus is exploited as a genetically

encoded aldehyde tag for site-specific protein

modification (Scheme 1)4. The fusion proteins of

O6-alkylguanine-DNA alkyltransferase (AGT) in

mammalian cells are specifically labeled with

fluorophores using guanine derivatives in a method

termed as ‘SNAP tag’5. The other tags used

for covalent labeling of proteins inside living cells

include SNAP/CLIP tag and Halotag6.

The techniques addressing naturally occurring

amino acids do not require additional biochemical

techniques to install unnatural functionalities

for chemoselective reactions7 and are important

in constructing the majority of man-made protein

INDIAN J CHEM, SEC A, AUG-SEPT 2013

974

conjugates. However, the presence of the large

number of electrophilic and nucleophilic sites

makes the selective functionalization using naturally

occurring functional groups complicated. In addition

to the functional groups present in the 22 canonical

amino acids, posttranslational modifications provide

proteins with additional chemical moieties to

carry out their natural functions. However, there still

remains the need to develop methodologies in order

to functionalize peptides and proteins both, in vitro

and in vivo. The attachment of non-natural

functionalities to specific locations on proteins

requires a set of chemoselective reactions. Metabolic

and genetic engineering methods have provided

an alternative strategy and allowed the incorporation

of abiotic functional groups like azides and alkynes

into proteins, nucleic acids, glycans and lipids8.

The functional group acting as a chemical reporter

is first installed on a target molecule to which

various probes can be delivered exogenously9.

Metabolic oligosaccharide engineering (MOE) is

another rapidly growing technique that helps to

deliver bioorthogonal chemistry for cell surface

modification10

.

Bioorthogonal chemistry addresses the challenge

of selectively combining two functional groups in

a chemically complex environment ranging from

aqueous solutions to living animals. The components

of bioorthogonal chemical reactions react rapidly

and selectively with each other under physiological

conditions in the presence of multitude

of functionality. Bioorthogonal protein modification

reactions target chemical reporters while

ignoring native protein functionalities (Fig. 1)11

.

The challenges include that the reactions must

proceed in water at physiological pH and temperature

providing good yield under reasonably fast reaction

rates at low reagent concentrations while remaining

inert to surrounding biological electrophiles and

nucleophiles. The components must be non-toxic

to cells and organisms. It is of importance if one

of the reactive groups is small, to minimally perturb

a biomolecule into which it has been introduced.

The bioorthogonal reactions of azides mark the first

successful story, azide being a small and biostable

functional group that can act either as a soft

electrophile or a 1,3-dipole. Alkynes are considered

bioorthogonal at physiological conditions due to their

high chemical inertness. Recently, Mootz et al.12

discovered an unexpected reaction between cysteine

proteases of the SUMO and ubiquitin family and

their propargylated substrate proteins forming a vinyl

sulfide linkage inhibiting the proteases. The reaction

was driven by the close proximity and alignment

of the two functional groups.

Incorporation of Unnatural Amino Acids

The replacement of a particular canonical amino

acid by genetic-code engineering (residue-specific incorporation) involves the use of an auxotrophic bacterial strain starved for the natural amino acid and supplemented with its analogue leading to the efficient incorporation of its analogues into bacterial proteins (Fig. 2)

13. The aminoacyl tRNA synthetases

(aaRSs) correctly charges tRNA with unnatural amino acids which are structurally and electronically similar to natural analogs

14. There have been various attempts

to enrich the variety of unnatural amino acids that can

Fig. 1—Bioorthogonal chemical reactions for the modification of peptides and proteins.

Fig. 2—Incorporation of unnatural amino acids using auxotrophic cell lines.

AGRAWAL & HACKENBERGER: SITE-SPECIFIC CHEMICAL MODIFICATIONS OF PROTEINS

975

be incorporated15

. Using this methodology, amino acid analogues have been incorporated not only for phase determination in X-ray crystallography and for spectroscopic studies, but also to study protein activity, folding

16, and stability. Non-proteinogenic

functional groups are introduced into proteins for further protein modification

15(d, f),17.

Genetic-code engineering providing one type of non-canonical amino acid (NCAA) per target

protein has also been used to change the biophysical properties of a certain protein, for example, fluorescence

18. The functional groups like alkynes,

alkenes and azides have been introduced into proteins using methionine (Met) surrogates homopropargylamine (Hpg), homoallylglycine (Hag),

allylcysteine and azidohomoalanine (Aha) (Fig. 3). The incorporated alkynes and azides have been further reacted with other molecules via Cu-catalyzed azide-alkyne cycloaddition (CuAAC) and Staudinger ligation

17f,19. Virus-like capsids have also been

functionalized with azides and alkynes by this

method20

. The selective identification of newly synthesized proteins in mammalian cells has been accomplished using this method in a process termed as bioorthogonal non-canonical amino acid tagging (BONCAT)

21.

The major disadvantage of genetic-code engineering is that it is restricted to the naturally occurring amino acids. The technique also leads to chemical modification at multiple sites. Although this is an important technology, there are other

circumstances where it is required to replace only a single residue while retaining access to all natural

amino acids. Schultz and coworkers22

developed the amber suppression method for introducing non-natural amino acids into specific positions of proteins. Significant advances have been made in the development of methods for the site-specific

modifications23

. Herein we focus on the methods to introduce unnatural functional groups by site-specific incorporation of NCAAs as well as a few other promising methods followed by the development of a series of bioorthogonal reactions involving these unnatural functional moieties.

Site-Directed Incorporation of Unnatural Amino

Acids Unnatural amino acids act as handles for

subsequent modification and help to tune protein

function. The use of the existing protein biosynthetic

machinery of the cell has allowed the development

of a number of in vitro methods to incorporate

NCAAs into proteins24

. Bertozzi and coworkers4

described a method for the site-specific introduction

of aldehyde groups into recombinant proteins using

the 6-amino-acid consensus sequence recognized by

the FGE. FGE oxidizes cysteine to formylglycine

(fGly). Coexpression of the tagged protein alongside

FGE directly produces the aldehyde-functionalized

protein. The aldehyde can then be modified by

using a variety of methods, such as condensations

with aminooxy or hydrazide probes4. However,

these methods suffer from major drawbacks and

thus, broader methods for the selective amino-

acylation of tRNAs were required. Nevertheless,

misaminoacylation of the tRNA with unnatural amino

acids using aaRSs as well as direct chemical acylation

of the tRNA are not practical25

. Consequently,

truncated tRNAs were enzymatically ligated to

chemically aminoacylated mono- and dinucleotides

with the development of semisynthetic methods26

.

The three degenerate stop codons, UAA, UAG, and

UGA, termed as nonsense codons do not encode

amino acids, but signal termination of polypeptide

synthesis by binding release factors27

. However, only

one stop codon is required for the termination of

protein synthesis thereby leaving two ‘blank’ codons

in the genetic code which can be used to uniquely

specify an unnatural amino acid. This approach

was used to report a general in vitro method for

the site-specific incorporation of a large number

of unnatural amino acids into proteins with

excellent translational conformity. The generation of

mutant β-lactamases containing p-nitrophenylalanine,

Fig. 3—Non-canonical amino acids as analogues of methionine

(Met).

INDIAN J CHEM, SEC A, AUG-SEPT 2013

976

p-fluorophenylalanine or homophenylalanine

substitutions at Phe 66 is the first demonstration of

this general approach for the site-specific

incorporation of unnatural amino acids into a protein

in vitro22

. Many unnatural amino acids have been

incorporated into proteins using this technique despite

low yields and considerable effort involved in

synthesizing the amino-acylated tRNA28

. Varieties of

proteins have been studied by this in vitro technique29

.

The microinjection of the engineered mRNA and a

chemically aminoacylated yeast tRNAPhe

-derived

amber suppressor has led to the extension of unnatural

amino acid mutagenesis to Xenopus oocytes30

.

A completely autonomous bacterium with a 21 amino

acid genetic code was generated. This bacterium

can biosynthesize a nonstandard amino acid from

basic carbon sources and incorporate this amino

acid into proteins in response to the amber nonsense

codon31

.

In vivo systems for site-specific mutagenesis

of unnatural amino acids involve the selection of

orthogonal tRNA and aaRSs recognizing the amber

stop codon and unnatural amino acid, respectively

(Fig. 4)32

. Subsequently, many other orthogonal

aaRS:tRNA pairs were reported33

. The adaptation of

this technique to yeast34

and mammalian cell culture35

has expanded the scope to in vivo functional

interrogation studies at multiple levels. By increasing

the production of the engineered tRNA and reducing

the rate of non-sense mediated mRNA decay,

Wang et al.36

have increased the efficiency of yeast

expression to E. coli-like levels (10–20 mg L−1

).

In vivo methods have been used for the

incorporation of a variety of unnatural amino acids

into proteins. Functional groups like azides11b,37

,

alkynes38

, alkenes39

, ketones40

, aniline41

, halides42

and

boronic acids43

have been successfully installed and

are further reacted. In an interesting example, Francis

et al.44

used bacteriophage MS2 to create a targeted,

multivalent photodynamic therapy vehicle for

Jurkat leukemia T cells. The unnatural amino acid,

p-aminophenylalanine (pAF) was introduced on the

external surface and the aniline groups of the side

chains were modified with N,N-dialkylphenylene

diamine derivatives through a highly chemoselective

oxidative coupling strategy (Scheme 2)45

. This

method was also used to attach porphyrins to pAF

side chains introduced using amber stop codon

suppression46

. The post-translational modifications

are efficiently incorporated by the in vivo system47

.

An orthogonal MjtRNATyr

CUA/MjTyrRS pair

was evolved for the selective incorporation of

p-carboxymethyl-L-phenylalanine (pCMF), a mimetic

of phosphotyrosine (pTyr) into proteins in response to

an amber codon in E. coli with high conformity and

efficiency. As pCMF is resistant to hydrolysis by

protein tyrosine phosphatases (PTPs), it provides a

useful tool for the generation of stable analogues of

selectively phosphorylated proteins and peptides48

.

The cross-linking groups49

and fluorophores50

are successfully installed using this approach. The

photocaged versions of cysteine, serine and tyrosine

residues have been genetically encoded to allow the

masking of important residues51

.

In a dual-labeling approach developed by Schultz

and coworkers52

, a cysteine residue and a NCAA,

p-acetyl-L-phenylalanine coded by an amber codon

Fig. 4—In vivo site-specific incorporation of unnatural amino

acids into proteins.

AGRAWAL & HACKENBERGER: SITE-SPECIFIC CHEMICAL MODIFICATIONS OF PROTEINS

977

were used for the site-selective dual labeling of

a protein. Chin and coworkers53

generated two

mutually orthogonal M. jannaschii TyrRS:tRNACUATyr

pairs capable of suppressing UAG and AGGA, thus

making possible multiple NCAA incorporation.

Liu and coworkers54

incorporated two chemically

distinct NCAAs into green fluorescent protein

(GFPuv) by combining two orthogonal pairs in a

single expression experiment. A UAG (amber) codon

was used in combination with a UAA (ochre) codon.

Later, they applied this double NCAA incorporation

method to genetically install two different bioorthogonal

functional groups into a protein allowing catalyst-free

and site-specific one-pot labeling of the protein with

a FRET (Förster resonance energy transfer) pair55

.

An overview of the attempts at the multiple

site-specific NCAA incorporation of two different

NCAAs by the reassignment of termination and

quadruplet codons has been presented by Hoesl and

Budisa56

. Furthermore, it is demonstrated that the

suppression-based incorporation of single NCAAs

can further be improved by the use of mutated

ribosomal proteins57

, elongation factors58

, and even

mutated ribosomal RNAs59

.

Bioorthogonal Reactions

The development of synthetic methodologies

and biological research rely largely on the expansion

of the field of bioorthogonal chemistry60

. A variety

of methods exist that tag biomolecules with reporters

enabling visualization and even isolation from

biological samples61

. However, as discussed

previously, bioorthogonal reactions easily allow

selective functionalization of peptides and

proteins in the presence of a pool of functionalities.

A variety of chemical reporters possessing the

requisite qualities include azides62

, terminal

alkynes63

and peptide sequences64

. The

bioorthogonal reactions help in the chemical

modification of biomolecules in vitro and enable

real time imaging of processes in cells and live

organisms. The noteworthy examples include

Staudinger ligation62a

and click chemistry (both

Cu-catalyzed65

and strain-promoted versions66

).

These bioorthogonal reactions help to probe a

biomolecule labeled with an azide using

complementary reagents in vitro, in cells and in live

organisms (Fig. 5)67

.

Recently, chemical transformations like 1,3-dipolar

cycloadditions (Scheme 3A)68

, Diels-Alder reactions69

,

metal-catalyzed couplings70

and nucleophilic

additions71

have also been exploited towards

bioorthogonal chemistry. For selective protein

labeling, Sletten and Bertozzi72

have demonstrated

quadricyclane ligation as a promising bioorthogonal

reaction (Scheme 3B). In the subsequent section we

present an overview of the existing chemical reporters

and bioorthogonal reactions.

Fig. 5—A phosphine-biotin (Phos-biotin) probe, FRET-based fluorogenic phosphine, phosphine-luciferin probe and cyclooctyne-biotin

(OCT-biotin) probe for detection of azides reported by Bertozzi and coworkers.

INDIAN J CHEM, SEC A, AUG-SEPT 2013

978

Ketones and aldehydes

Ketones and aldehydes are placed into proteins

by a variety of methods including enzymatic

modification of peptide motifs, incorporation of

unnatural amino acids and sugars and chemical

oxidation of terminal residues73

. Ketones and

aldehydes were installed on the tyrosine residues of

the protein capsid of the tobacco mosaic virus

(TMV)74

and on the lysine residues on the exterior

of the MS2 capsid75

,

respectively. As mentioned

previously, Schultz et al.40a

created a cell line that

introduced ketones site-specifically into proteins by

using an orthogonal tRNA and tRNA synthetase

couple incorporating p-acetyl-phenylalanine as a

21st amino acid. Hutchins et al.

76 expressed a Fab

fragment bearing an unnatural keto amino acid

to which a maleimide-functionalized linker was

conjugated by oxime formation. This was further

conjugated to a single cysteine residue engineered

into a protein toxin leading to the synthesis of a

site-specific heterodimeric antibody-toxin conjugate76

.

The transamination reaction of N-terminal amino

acids upon exposure to pyridoxal 5’-phosphate (PLP)

at 37 °C in an aqueous solution (pH 6.5) reported by

Francis et al.77

results in the corresponding pyruvamide

derivative introducing ketone at a single location.

Ketones and aldehydes are the bioorthogonal

chemical reporters that are readily introduced into

a variety of scaffolds and can tag proteins, glycans

as well as other secondary metabolites. They are

mild electrophiles and are inert to the reactive

functionalities normally found in proteins. Unlike the

formation of reversible Schiff base with primary

amines such as with lysine side chains that is not

favored in water with the equilibrium favoring

the free carbonyl, the stabilized Schiff bases with

hydrazide, thiosemicarbazide and aminooxy groups

are quite stable under physiological conditions78

.

Hydrazide79

or hydroxylamine80

groups react with

ketones and aldehydes forming a hydrazone81

or

oxime82

bond, respectively. Hence, these groups

have been in widespread use in the field of

protein modification4a,83

. Aniline-catalyzed hydrazone

ligation between surface-immobilized hydrazines

and aldehyde-modified antibodies was reported by

Bailey et al.84

as an efficient method for attaching

protein capture agents to model oxide-coated

biosensor substrates. Ketones and aldehydes have

been used to modify mammalian cell surfaces3a,85

.

Sadamoto and coworkers86

introduced ketones into

bacterial cell walls and labeled them with a

hydrazide-based fluorophore. Several early attempts

to create fully synthetic proteins relied upon this

strategy, since for some time this represented the

best method for peptide ligation without the need for

side chain protection87

. Kent and coworkers88

created

AGRAWAL & HACKENBERGER: SITE-SPECIFIC CHEMICAL MODIFICATIONS OF PROTEINS

979

a fully synthetic mimic of human erythropoietin,

a glycosylated protein hormone that stimulates the

production of erythroid cells. Native chemical ligation

(NCL) was used to create the full-length polypeptide

product with specifically placed ketones that were

used as attachment points for aminooxy terminated

polymers designed to mimic natural glycans.

Ketones and aldehydes (best considered as

‘biorestricted’ chemical reporters), although suitable

in the presence of cultured cells, have limitations in

living organisms. The optimum pH for condensation

reactions is 5−6 that cannot be achieved in most

tissues in vivo and are not truly bioorthogonal in more

complex physiological settings. However, ketones and

aldehydes serve as unique chemical reporters89

.

Paulson and coworkers90

introduced aldehydes

into cell-surface sialic acid residues by mild

periodate oxidation and then captured the modified

glycoproteins by reaction with aminooxybiotin

followed by streptavidin chromatography.

Azides

The aldehydes and ketones have limited

intracellular applicability. Azides, however, are

more versatile chemical reporters91

. They are totally

absent from biological systems except one naturally

occurring azido metabolite isolated from unialgal

cultures92

. The azide group being small, minimally

perturbs the modified substrate93

. Azides are

easily approachable synthetically. They are mild

electrophiles and do not react with hard nucleophiles

abundant in biological systems. Azides do not react

with water, are resistant to oxidation and stable at

physiological temperatures. Covalently bound azides

decompose at elevated temperatures and are

considered explosive classes of compounds. For

organic azides to be non-explosive, the rule is that

the number of nitrogen atoms must not exceed that of

carbon and that (NC + NO)/NN ≥ 3 (N = number of

atoms)94

. The azide anion is a widely used cytotoxin,

however, organic azides have no intrinsic toxicity.

Azides possess orthogonal reactivity to the

majority of biological functional groups. The large

energy content of azides is used for the development

of bioorthogonal reactions for selective labeling of

azide-functionalized biomolecules. Azides are largely

introduced by metabolic incorporation of unnatural

amino acids and monosaccharides17e,32a

. The diazo

transfer agents can also be used to introduce azide

group into both peptides and proteins. Triflyl azide

in the presence of divalent Cu ions can easily

convert solid-phase bound peptide amines into

azides95

. The water-soluble diazo transfer agents are

used to substitute the amines of both lysine residues

and the N-terminus by azide groups on proteins96

.

Pedersen and Johansson97

have nicely compiled

available methods for the synthesis of optically active

azide- and alkyne-functionalized α-amino acids.

Staudinger ligation

Staudinger98

reported the reaction of azides and

triphenylphosphines under mild conditions to produce

aza-ylide intermediates that can subsequently be

hydrolyzed in water or trapped by electrophiles to

produce an amine and the corresponding phosphine

oxide. Later, Bertozzi and Saxon62a

modified the

classic Staudinger reaction by introduction of an

intramolecular trap, a proximal ester into the phosphine,

now known as Staudinger ligation. The Staudinger

ligation can be used to covalently attach probes to

azide-bearing biomolecules99

. However, oxidation

of the phosphine may reduce the amount of probe

available in biological systems. The major limitation of

Staudinger ligation is relatively slow reaction kinetics

necessitating high concentrations of triarylphosphine100

.

All efforts to improve the reaction kinetics by

increasing the nucleophilicity of the phosphine reagents

lead to increased phosphine oxidation in air.

The Staudinger ligation has been used for

site-selective immobilization of azide-labeled proteins

on surfaces101

and to impart new functionality to

recombinant proteins102

. The selective in vivo covalent

modification of cell-surface glycans with chemical

probes was achieved in live mice by Staudinger

ligation103

. Lemieux et al.104

reported a fluorogenic

phosphine by replacing one of the aryl rings

with a coumarin dye104

. Phosphine oxidation during

Staudinger ligation relieves the quenching effect

caused by the lone pair of electrons on the phosphorus

atom producing a highly fluorescent biomolecule-

bound product (Scheme 4). However, the oxidation

of phosphine in air caused background fluorescence.

To overcome this problem, Bertozzi et al.105

incorporated a FRET quenching group at the ester

position. Recently, they reported bioluminogenic

phosphine reagent that releases luciferin during

Staudinger ligation. Luciferin readily enters cells

wherein heterologously expressed luciferase

catalyses its oxidation and the concomitant emission

of light106

. The Staudinger ligation was also used for

the site-specific PEGylation of azido-homoalanine

containing trombomodulin107

.

INDIAN J CHEM, SEC A, AUG-SEPT 2013

980

Hackenberger et al.108

demonstrated the Staudinger

reaction of unprotected azido-peptides with silylated

phosphinic acids and esters in solution as well as

on the solid support (Scheme 5A). The protocol

offers a straightforward acid-free entry to different

phosphonamidate peptide esters or acids under

mild conditions in high yield and purity108

.

They identified another Staudinger-type reaction

for the chemoselective transformations of azides

in peptides and proteins (Scheme 5B)109

.

This Staudinger-phosphite reaction (SPR) utilizes

phosphites and hence is very easy to perform.

The phosphites can be prepared by standard organic

synthesis protocols and are stable against oxidation

upon exposure to air. SPRs can be performed in

various solvents and buffers at room temperature,

conditions suitable for quantitative modification

reactions in proteins. They also showed that not

AGRAWAL & HACKENBERGER: SITE-SPECIFIC CHEMICAL MODIFICATIONS OF PROTEINS

981

only symmetrical but also unsymmetrical phosphites

can be used for the modification of proteins via

aqueous SPR (Scheme 5B)110

. Additionally, the first

example of Staudinger reaction of aryl-phosphonites

(Staudinger-phosphonite reaction) for the chemoselective

transformation of azido-containing peptides and

proteins in aqueous systems at room temperature in

high conversions was developed (Scheme 5A)111a

.

Since alkyl-phosphonites appeared to oxidize rapidly,

aryl-substituted analogues were used in which the

sp2-hybridized carbon at phosphorus accounts for

a higher stability upon air exposure. Also, the

phosphonites used were obtained as borane adducts

in a one-pot process to allow prolonged storage.

The phosphonite hydrolysis to a phosphinic acid ester

as observed by 31

P NMR was a limiting factor in the

Staudinger-phosphonite reaction. This limitation was

addressed by introducing OEG-(oligo ethylene glycol)

groups at phosphorus to further enhance the water

solubility and reduce the probability of nucleophilic

attack at the phosphorus. The Staudinger-phosphonite

reaction was then successfully applied to the

site-specific modification of the protein, calmodulin.

Along those lines, Hackenberger and coworkers

have also used alkyne-phosphonites in a modular

azide-azide coupling strategy by sequential CuAAC

and Staudinger reactions110b

.

Copper-catalyzed [3 + 2] azide-alkyne cycloaddition

Azides also act as 1,3-dipoles and undergo

electrocyclization reactions with dipolarophiles such

as activated alkynes112

to provide stable triazole

adducts, first described by Huisgen113

. Both the

reactants are stable under physiological conditions

and the reaction proceeds readily in water and

tolerates a wide range of functionalities. Despite the

exothermic nature of these reactions, without alkyne

activation the process requires elevated temperatures

or pressure incompatible with living systems114

and

produces a mixture of regioisomers. Sharpless and

coworkers65,115b

and Meldal and others115a

however,

demonstrated that the rate of cycloaddition can be

accelerated using catalytic amounts of Cu(I). The

cycloaddition proceeds with almost complete

regioselectivity (1,4-regioisomer) at physiological

temperatures in the presence of biological materials

providing 1,4-disubstituted triazoles62b

. Initially,

active Cu(I) was generated in situ from Cu(II)

salts, (Cu wire) and a reductant, ascorbic acid

or tris(2-carboxyethylphosphine) (TCEP). However,

Cu(I) salts in the presence of a ligand (commonly,

tris-triazole) have shown to increase the rate

of reaction quite significantly116

. The use of an

electrochemical cell to generate and protect

catalytically active-Cu-ligand species for CuAAC

bioconjugation and synthetic coupling reactions

has been reported by Finn and coworkers117

. The

Cu-accelerated azide-alkyne cycloaddition (CuAAC)

more appropriately termed as ‘click’ reaction118

has

found broad application as ligation tool in polymer

science119

and in combinatorial organic synthesis120

.

The azides incorporated within proteins121

,

fucosylated glycoproteins (Scheme 6)122

, nucleic

acids123

and virus particles124

are tagged using this

approach. The first examples of directly radiolabeled

([18

F]-glyco)proteins were reported by Davis and

coworkers125

. They used this ‘tag and modify’

approach to synthesize homogenous fluorinated

glyco-amino acids, peptides and proteins carrying

a fluorine label in the sugar.

Metalloproteases are a diverse class of enzymes

involved in many physiological and disease processes

and are regulated by post-translational mechanisms

that reduce the effectiveness of conventional

genomic and proteomic methods for their functional

characterization. Click chemistry is used for

the activity-based protein profiling (ABPP) of

metalloproteases126

. CuAAC finds excellent use

where high reaction yields are required at low

substrate concentrations. The major advantage of

click reaction is its faster rate compared to Staudinger

ligation, whereas the cellular toxicity of the

metal catalyst and copper-induced denaturation of

proteins are the biggest disadvantages17f,127

. Finn and

coworkers128

described two immobilized forms of

a Cu-binding ligand shown to accelerate triazole

formation under many different conditions using

different resin supports that are appropriate for

aqueous or organic solvents. CuAAC was used for

the site-specific PEGylation of human superoxide

dismutase-1 (SOD), a key enzyme in preventing

INDIAN J CHEM, SEC A, AUG-SEPT 2013

982

the formation of reactive oxygen species in cells129

.

There are also examples of CuAAC available

where the azide and alkyne functionalities switched

places130

. Bertozzi and coworkers130d

identified

potential fluorogenic azidofluoresceins. Later,

in order to evaluate them as biological imaging

reagents, Chinese hamster ovary (CHO) cells

were metabolically labeled with peracetylated

N-(4-pentynoyl)-mannosamine (Ac4ManNAl). These

cells convert Ac4ManNAl to the corresponding

alkynylsialic acid, which is then incorporated

into cell-surface glycoproteins. Control cells were

incubated with peracetylated N-acetylmannosamine

(Ac4ManNAc). The cells were then fixed

with paraformaldehyde and incubated with

azidofluorescein under CuAAC conditions.

After washing, the cells showed robust alkyne-

dependent labeling with azidofluorescein.

The low concentrations at which biomolecules

are manipulated bring challenges for any

ligation methodology. A generally applicable

CuAAC procedure solving many click bioconjugation

problems was described by Finn and coworkers131

.

They used the new CuAAC conditions to couple

a protein to the outer surface of the Qβ virus-like

particle. The major improvements involve

the addition of a water-soluble member of

the tris(triazolylmethyl)amine family, tris(3-hydroxy-

propyltriazolylmethyl) amine (THPTA) and

aminoguanidine. This allows ascorbate to be

used as reducing agent while eliminating problems

caused by copper ascorbate side-reactions.

The addition of at least five equivalents of THPTA

relative to Cu helps to quickly reduce reactive

oxygen species generated by the ascorbate-driven

reduction of dissolved O2 without affecting

the CuAAC reaction rate to a large extent.

Aminoguanidine intercepts byproducts of ascorbate

oxidation that can covalently modify or crosslink

proteins.

Strain-promoted [3 + 2] azide-alkyne cycloaddition

The use of ring strain to activate dipolarophile62c

for electrocyclization involves constraining the alkyne

within an eight-membered ring creating strain

that is released in the transition state upon [3+2]

cycloadditoon with an azide132

. The strain-promoted

cycloaddition of cyclooctynes with azides, commonly

referred to as Cu-free click chemistry, has been

used to label biomolecules both in vitro and on cell

surfaces without the need for a catalyst (Fig. 6)62c,133.

The major drawback, however, is the slow rate

of reaction. In order to improve reaction

kinetics cyclooctyne derivatives with added fluorines

were designed134

. Later, a series of cyclooctynes

were investigated in living systems through the

strain-promoted azide-alkyne cycloaddition (SPAAC)

(Fig. 6)135

. Three coumarin-cyclooctyne conjugates

were used to label proteins tagged with Aha in Rat-1

fibroblasts leading to the visualization of biomolecule

dynamics in living cells136

. BODIPY-cyclooctyne

(BDPY), a membrane-permeant fluorophore was used

to label intracellular proteins in live mammalian

cells137

.

Lemke and coworkers138

genetically encoded

strained alkynes into E. coli by use of an engineered

pyrrolysine amber suppressor tRNA/synthetase

pair from Methanosarcina mazei. As a result

cyclooctynyl lysine derivatives were incorporated

site-specifically in proteins allowing efficient labeling

with fluorogenic azide-bearing dyes by means of

a SPAAC under physiological conditions in vitro and

in vivo (Scheme 7A)138

. The difluorinated cyclooctyne

(DIFO) showed kinetics comparable to CuAAC

in biomolecule labeling experiments and has been

used for imaging azide-labeled biomolecules

within complex biological systems134,139

. Boons and

coworkers140

fused two aryl rings to the cyclooctyne

core to form dibenzocyclooctyne (DIBO) for

rate enhancement, and later Bertozzi and

coworkers141

added an amide bond to it forming

biarylazacyclooctynone (BARAC). Later, many

versions of DIBO and BARAC were reported142

.

Hinderlich and Hackenberger et al.143

tested the

potential of Ac3-4-azido-ManNAc as a tool for

glycan labeling in living animals. They injected

Ac3-4-azido-ManNAc into the hindbrain vesicle of

zebrafish larvae at 24 hpf (hours post fertilization).

AlexaFluor 488-conjugated DIBO which reacts

specifically with the azido group of modified sialic

acids was injected at 48 hpf and live embryos

were analyzed at 72 hpf. In embryos injected with

Ac3-4-azido-ManNAc, a distinct labeling of the

mid-brain and the hindbrain was detected. In addition,

a faint staining of the dorsal myosepta was observed,

which was absent in the control embryos.

The fluorescence staining of regions of the central

nervous system and the myosepta reflected high

incorporation of Ac3-4-azido-ManNAc into sialic

acids of heavily O-glycosylated proteins. The use of

dibenzocyclooctyn-ol in strain-promoted cyclization

AGRAWAL & HACKENBERGER: SITE-SPECIFIC CHEMICAL MODIFICATIONS OF PROTEINS

983

Fig. 6—Strain-promoted azide-alkyne cycloaddition; a series of cyclooctynes for copper-free click chemistry.

INDIAN J CHEM, SEC A, AUG-SEPT 2013

984

has been reported by Boons and coworkers140

(Scheme 7B). Ting et al.144

optimized two-step

enzymatic/chemical labeling scheme to tag and image

a variety of cellular proteins fused to a 13-amino acid

recognition sequence (LAP) in multiple mammalian

cell lines with diverse fluorophore conjugates to

aza-dibenzocyclooctyne (ADIBO). The theoretical

studies by Houk and coworkers145

enable to

predict and optimize cyclooctyne reactivity.

Studies also demonstrated that scaffolds like

monobenzocyclooctyne (MOBO) and bicyclononyne

(BCN) find an optimal balance between

strain enhancement and minimization of steric

hinderance146

. Difluorobenzocyclooctyne (DIFBO)

that was found to be almost 20 times more

reactive than MOBO, was unstable and prone

to oligomerization in concentrated solution147

.

Bertozzi and coworkers148

synthesized thiaOCT and

thiaDIFBO (the sulfur-containing analogues) in order

to assess the effects of an endocyclic sulfur atom

on cyclooctyne reactivity. Thiacycloheptynes also

form a promising class of reagents for bioorthogonal

Cu-free click chemistry. Banert and Plefka149

reported

that 3,3,6,6-tetramethylthiacycloheptyne (TMTH)

is more reactive than a DIBO-like cyclooctyne

in a 1,3-dipolar cycloaddition with nitrous oxide

(Scheme 7C). Bertozzi et al.148

tested the reactivity

of TMTH with azide-functionalized biomolecules.

Alkenes and iodides

Alkene acts a versatile chemical reporter/probe

capable of multiplexed applications due to

various possible organic transformations including

1,3-dipolar cycloaddition, Diels-Alder reaction

and olefin metathesis. Alkene-containing amino acids

incorporated site-specifically into proteins include

Hag19e

, O-allyl-tyrosine39

and dehydroalanine150

.

Several groups have investigated the potential

of strained bicyclic alkenes for bioconjugation151

.

Delft et al.152

described the reaction of

oxanorbornadienes with azides via a tandem

cycloaddition-retro-Diels-Alder (crDA) reaction and

used the technology for protein modification. Lin and

coworkers68a

developed photoinducible bioorthogonal

reactions known as ‘photoclick’ chemistry to modify

tetrazole-containing proteins in biological media by

their 1,3-dipolar cycloaddition with simple alkenes to

form pyrazolines. The reaction required light with a

wavelength of 302 nm to produce the nitrile-imine

dipole. In order to avoid photodamage caused

to exposed cells, they designed diaryltetrazoles that

could undergo ring-opening at 365 nm to generate

reactive nitrile imine dipoles153

. The utility of this

approach is demonstrated by the selective labeling

of an alkene-containing Z-domain protein in

E. coli with diaryl tetrazoles68e

. In addition to the mild

reaction conditions, the approach offers convenient

fluorescent monitoring due to the formation of

the fluorescent cycloadducts. An unusually

fast bioorthogonal reaction based on the inverse-

electron-demand hetero-Diels-Alder reaction between

dipyridyltetrazine and trans-cyclooctene (TCO)

was reported by Fox and coworkers69a

and higher

rate constant was reported in phosphate buffered

saline (PBS) at 37 oC (Scheme 8A)

154. Tetrazine-TCO

ligation was also utilized in a fluorogenic reaction

and 18

F-scintigraphic imaging155

. Hilderbrand and

coworkers69b

developed the reaction of norbornene-

conjugated antibody, SKBR3 and fluorescent

VT680-tethered tetrazine, both in serum and live

cells. Later, they reported the use of this reaction in

selectively imaging TCO-labeled cancer cells156

.

Prescher and coworkers157

used a new chemical

reporter–cyclopropene–to target biomolecules in vitro

and in live cells. The small size and selectivity make

cyclopropenes a versatile class of chemical reporters

to label biomolecules in living systems. A variety of

substituted cyclopropene scaffolds were synthesized

and were found to be stable in aqueous solution and

in the presence of biological nucleophiles. In order

to investigate whether cyclopropenes would be

useful for cellular labeling studies, they constructed

a methylcyclopropene–sialic acid conjugate

(9-Cp-NeuAc). These modified sialic acids were

metabolically introduced into cell surface glycans.

The presence of these cell surface cyclopropenes was

subsequently probed by reaction with a tetrazine–

biotin conjugate (Tz-Biotin) (Scheme 8B).

With the development of water-soluble olefin

metathesis catalysts, chemoselective modification

of alkenes by cross-metathesis is emerging as

an important bioorthogonal reaction158

. Davis and

coworkers19g,159

modified proteins containing allyl

sulfide groups through cross-metathesis (Scheme 9A).

Later,

allyl selenides were found to be the

most reactive substrates for olefin metathesis

(Scheme 9B)160

.

With the ability to genetically incorporate

cross-coupling partners on proteins, palladium

catalyzed cross-coupling has found applications

on biomolecules. Schultz and coworkers161

developed

AGRAWAL & HACKENBERGER: SITE-SPECIFIC CHEMICAL MODIFICATIONS OF PROTEINS

985

INDIAN J CHEM, SEC A, AUG-SEPT 2013

986

a method to site-specifically incorporate 4-iodo-

L-phenylalanine (iodoPhe) into proteins in response

to an amber TAG codon. Yokoyama and coworkers162

genetically engineered a Ras protein carrying an

iodophenylalanine that serves as an orthogonal

functionality for selective conjugation with a biotin-

tethered alkene via the Mizoruki-Heck reaction.

Later, a related site-specific biotinylation via the

Sonogashira reaction was reported70

. Davis et al.163

disclosed a convenient catalyst for Suzuki-Miyaura

cross-coupling on proteins under aqueous conditions

(Scheme 9C) and later demonstrated the use of small

molecule palladium scavenger, 3-mercaptopropionic

acid (3-MPrAc)164

.

Conclusions Till date a number of bioorthogonal strategies have

been reported, each with a particular strength and

utility, changing our ways of studying biomolecules

in vitro and in vivo. Among these, in vivo site-specific

incorporation of NCAAs proves to the most potential

method for ongoing development and widespread

application. However, the competition with naturally

occurring functional moieties leads to incomplete

substitution with the reporter-modified building

block. Also, some of the existing bioorthogonal

reactions proceed either at slow rates or require

high concentrations of reactants. There exists a need

for additional reactions that have faster reaction

rates and high overall yields. There is also a need

to develop additional orthogonal functional groups

as well as new chemical reporters to produce

multidimensional experiments. The methodology for

robust and efficient incorporation of unnatural amino

acids into proteins is evolving very rapidly and the

newer methods that take advantage of our present

knowledge are constantly underway.

Acknowledgement

The authors acknowledge financial support

from the German Science Foundation within the

Emmy-Noether program (HA4468/2–1), the SPP

1623, the Einstein Foundation Berlin (Leibniz-

Humboldt Professorship), the SFB 765, the

Boehringer-Ingelheim Foundation (Plus 3 award)

and the Fonds der chemischen Industrie (FCI).

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