<figure><img src="image_126.png" /></figure>

<figure><img src="image_127.png" /><figcaption>**Fig. 89** Ligation–desulfurisation at tryptophan, reported by Payne and collaborators.⁷⁸⁶ (a) Electrophilic sulphenylation of Trp in acidic conditions with DNPS-Cl and the obtention of the corresponding 2SH-Trp derivative. (b) Auxiliary-assisted native ligation of N-terminal 2SH-Trp peptide (2SH-WSPGYS-NH₂) with a thioester of the peptide Ac-LYRANGW-SPH resulting in 12-mer peptide Ac-LYRANGWSPGYS after the desulfurisation step.</figcaption></figure>

of the native ligation with peptide thioesters, and could thus serve as Nα acyl transfer auxiliaries (see Section 2.3.1).⁷⁸⁷

The proposed mechanism for this approach is mechanistically similar to NCL. It was hypothesised that the reaction would proceed *via* an initial step of the peptide thioester transthioesterification with an indole 2-thiol functionality followed by an *S*- to *N*-acyl shift through a 7-membered ring transition state to generate a native amide bond. The last step of 2-thiol Trp desulfurisation results in obtention of a ligated product with only naturally occurring amino acid residues (Fig. 89b). Although this methodology represents a clever chemoselective approach for the ligation of completely unprotected peptide fragments through Trp moiety, the harshness of the reaction conditions of sulfenylation and desulfurisation limit it only to peptide substrated and don't allow its application on complex biomolecules.

### 2.4.2 Pictet-Spengler reaction.
Another approach for the ligation of unprotected peptides was proposed by Li *et al*.⁷⁸⁸ and exploited the Pictet-Spengler reaction: an acid-catalysed intramolecular condensation between an iminium ion and an aromatic *C*-nucleophile described in 1911.⁷⁸⁹ This non-natural ligation (peptides are linked by non-natural bonds) involves reaction of two peptide partners in acetic acid: one containing a Trp residue at its N-term and another with a C-terminal aldehyde function. The latter should generally be introduced by means of solid-phase peptide synthesis on acetal resin (described by the same authors).

### 2.4.3 N-Acyl tryptophan isopeptides.
Lastly, an interesting example of native N-terminal Trp ligation, mechanistically very similar to NCL (see Section 2.3.1), was recently reported by Popov *et al*.⁷⁹⁰ The key intermediates of this methodology – *N*-acyl tryptophan isopeptides – undergo selective acyl transfer to yield natural peptides. These are, however, not accessible

<figure><img src="image_128.png" /></figure>

<figure><img src="image_129.png" /><figcaption>**Fig. 90** N-terminal histidine labelling described by Zhang and Tam.⁷⁹¹ (a) Mechanism of thiocarboxylic acid activation by Ellman's reagent. (b) Labelling of bovine parathyroid hormone fragment (14–34, pdb: 1ZWC) with activated tetrapeptide thioacid.</figcaption></figure>

directly by methods reported so far, which substantially restricts the applicability of the methodology.

## 2.5 Histidine

Despite being known for its particular importance for the acyl transfer in many enzymatic processes,⁶⁶⁵ histidine has been rarely used for bioconjugation. The only study describing the ligation of N-terminal His peptides with activated thioester was reported by Zhang and Tam.⁷⁹¹

Ellman's regent³⁴⁸ was used to activate the C-terminal peptide thiocarboxylic acid by forming acyldisulfide derivative, which is then nucleophilically attacked by N-terminal histidine. Captured by the imidazole of the N-terminal histidine, the obtained $N_{im}$-acyl intermediate is hypothesised to undergo $N_{im} \rightarrow N_{\alpha}$ shift to form histidine at the ligation site (Fig. 90). However, the $N_{im}$-acyl intermediate has not been isolated and it is quite possible that regioselectivity is obtained simply because of anchimeric assistance of the proximal imidazole moiety at the ligation site.

Interestingly, no sign of coupling reaction has occurred when a corresponding non-activated C-terminal thiocarboxylic acid is participating in the reaction instead of the acyldisulfide. The reaction pH plays an important role on the effectiveness of the reaction. Only when maintained at slightly acidic values (pH 5–6) and in the absence of the thiol nucleophiles, would the imidazolyl moiety of histidine be the sole nucleophile present in the polypeptide. This methodology has been applied to generate histidine-containing peptides with yields up to 75%.

## 2.6 Selenocysteine

Selenium and sulphur belong to the same main group of elements; therefore, 21st proteinogenic amino selenocysteine and Cys residue exhibit rather similar properties in terms of reactivity for bioconjugation.⁷⁹²⁻⁷⁹⁴ For instance, both Hilvert's⁷³⁸ and Raines's group⁷³⁶ demonstrated that C-terminal peptide thioesters react smoothly with peptide fragments containing a N-terminal selenocysteine in exactly the same manner as with corresponding cysteine analogues. Presumably proceeding through the same mechanism as NCL (Section 2.3.1), the first
<figure><img src="image_130.png" /></figure>

step of the ligation process consists in the nucleophilic attack on the thioester by selenolate to give a selenoester intermediate that subsequently rearranges to give a native chemical bond.

Sec ligation can be chemoselective when conducted at slightly acidic pH. Low intrinsic pKₐ values of selenocysteine (5.2)⁷⁹⁵ and consequently its higher dissociation level at low pH, endows this amino acid with unique biochemical properties, allowing regiospecific covalent conjugation with electrophilic compounds in the presence of the side chains of all other natural amino acids including the thiol group of Cys (pKₐ 8.3). For instance, the reaction rate with selenocysteine was found to be 1000 fold faster than with cysteine at pH 5.0.⁷³⁶ Moreover, the lower pH generally suppresses β-elimination of the selenol group from selenocysteine resulting in the obtaining of unreactive dehydroalanine.³⁹²

Initially, considerable efforts were made to show the applicability of selenocysteine NCL for the preparation of selenium-containing derivatives of enzymes and benchmarking activities thereof. Hilvert and associates synthesised a C38U analogue of bovine pancreatic trypsin inhibitor (BPTI). Amusingly, the wild-type BPTI and its artificial analogue folded into alike conformations and demonstrated similar inhibiting affinity of trypsin and chymotrypsin.⁷³⁸ Raines *et al*. selected 124-residue ribonuclease A (RNase A) as a model protein for the investigations.⁷³⁶ DNA recombinant technology was utilised to prepare a C-terminal thioester fragment corresponding to residues 1–109, while standard solid phase peptide synthesis methodology was used to obtain a N-terminal Cys and Sec peptides corresponding to residues 110–124 (Fig. 91). Just as in the case of BPTI, the semisynthetic wild-type RNase A and C110U RNase A presented equivalent ribonucleolytic activities. Further advances in the field of Sec-NCL have resulted in synthesis and investigations of other different proteins such as seleno-glutaredoxin,⁷⁹⁶ azurin,⁷⁹⁷,⁷⁹⁸ and thioredoxin reductase.

Ease of the post-ligation transformation of selenocysteine to alanine (by deselenisation), dehydroalanine (by β-elimination) or non-natural amino acids (by addition reaction to dehydroalanine, see Section 1.3.8) became a spur to a further propagation of Sec-mediated methodologies as very effective tools in rational design of peptides and proteins. Quaderer and Hilvert⁷⁹⁹ exploited such transmutations of selenocysteine to access a series 16-residue cyclic peptides (Fig. 92).

In this initial report, the deselenisation step was conducted rather harshly (RANEY® Ni, H₂), implying that all Cys residues (if there were any) would have been reduced as well. Recently, however, Dawson and collaborators⁷³⁵ have demonstrated that

<figure><img src="image_131.png" /><figcaption>Fig. 91 Selenocysteine native chemical ligation applied for the preparation of C110U mutant of RNase A (wild-type – pdb: 7RSA).⁷³⁶</figcaption></figure>

<figure><img src="image_132.png" /><figcaption>Fig. 92 Synthesis of cyclic 16-residues selenopeptides by native chemical ligation and following transformation thereof to (a) alanine (RANEY® Ni, H₂, TCEP, 20% AcOH); (b) dehydroalanines (H₂O₂, 10% ACN).</figcaption></figure>

selenocysteine can be chemoselectively deselenated with TCEP in the presence of cysteines. This allowed overpassing the main limitation of the NCL-desulfurisation strategy (see Section 2.3.1.4), namely the inability to control regioselectivity of desulfurisations if several cysteines are present in the peptide or protein, and yielded in a pool of NCL-deselenisation strategies for mild incorporation of alanine, phenylalanine and proline into the ligation site by classic NCL approaches (see Table 2). Finally, selenocysteine peptides were found to efficiently undergo reverse NCL at acidic pH and thus to be of particular interest for the generation of thioesters by sequential N → Se acyl-transfer and substitution of the obtained selenoester by exogenous thiol (see Section 2.3.1.3).⁷⁴⁵

Because the incorporations of selenocysteine by the cell translational machinery are generally very laborious,⁸⁰⁰,⁸⁰¹ selenopeptides are mainly obtained by SPPS.⁷³⁶,⁷³⁷,⁷⁹⁴

## 2.7 Proline

The oxidative coupling of *o*-aminophenols and *o*-catechols recently reported by Francis and collaborators⁸⁰² represents an interesting approach for selective N-terminal conjugation on proteins. The strategy consists in prior *in situ* oxidation of *o*-aminophenols and *o*-catechols to active coupling species using potassium ferricyanide (Fig. 93a) followed by their reaction with a protein (Fig. 93a). This approach was shown to work particularly well with proline (due to increased nucleophilicity thereof) and can therefore be considered as N-terminal Pro selective methodology.⁸⁰² The key advantage of the approach compared to the majority of N-terminal selective methodologies is its fast second-order

<figure><img src="image_133.png" /><figcaption>Fig. 93 Selective modification of 1P-GFP (Pro residue is introduced on N-terminus) with *o*-aminophenol-PEG reagent (wild-type – pdb: 1GFL).⁸⁰² (a) *In situ* oxidation of *o*-aminophenol derivatite. (b) Reaction of the active coupling species with N-terminus of 1P-GFP.</figcaption></figure>
kinetics in a single step transformation, which does not require prior oxidation of the N-terminus. Free cysteine residues, however, are also reactive during the oxidative coupling and must thus be protected, which represents the main drawback of the approach.

### 3. C-terminal conjugation

Chemical methods for C-termini conjugation are rather scarce. Definitely, previously mentioned approaches for the conjugation of side-chain carboxylates (see Section 1.5) can also be applied effectively to modify the terminal carboxylates. The selectivity, however, would be the main issue, for up to date there is no described method for somewhat selective activation of N-terminal carboxylates by any activating reagent.

Because of the impetuous development of NCL (see Section 2.3.1), requiring C-terminal thiolates as reacting partners for N-terminal Cys proteins, those became widely accessible, namely by means of SPPS. A perspective approach, exploiting these advances was proposed by Goody⁸⁰³ and collaborators, who developed a protocol for selective transformation of C-terminal thioesters to corresponding hydroxylamines, enabling thus the application of aldehyde- and ketone-selective methodologies on the C-terminus.

On the other hand, the unique position of protein C-termini has stimulated numerous efforts to target this location, which resulted in numerous enzymatic and intein-based approaches for C-terminal-selective protein modification.⁸⁰⁴⁻⁸⁰⁹ These methodologies are, however, not covered by this review devoted to chemical methods of bioconjugation.

### 4. Sequence-selective approaches

Several especially useful methodologies in bioconjugation exploit not a specific property of the residue, or its peculiar position in protein, but rather the synergetic effect of a batch of neighbouring amino acid residues. For instance, an example of such selectivity is the aforementioned selective modification of His-AA-Ser and His-AA-Thr peptide motifs (see Section 1.2) by usually promiscuous activated esters. In this case, the His imidazolyl side chain located in close proximity to Ser/Thr side chains increased the reactivity thereof towards electrophiles.

Metal-chelation methodologies are perhaps the most elaborated among sequence-selective approaches (Fig. 94).⁸¹⁰ The oligo-histidine sequence (usually $H_6$), called His-tag, is known to interact robustly with transition-metal complexes, including a nitrilotriacetic acid (NTA) complex of $Ni^{2+}$, therefore the sequence is widely used for protein purification by affinity chromatography. Similarly, oligo-aspartates (most often ($D_4$)$_n$, $n = 1–3$) were developed for selective labelling with multinuclear $Zn^{2+}$ complexes.⁸¹¹,⁸¹² The tetracysteine motif (CCPGCC) was reported to especially effectively chelate biarsenical dyes such as 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein (FlAsH),⁵,⁸¹³ while a similar structural tetraserine sequence (SSPGSS) was found to be selective for diborate-scaffolds.

<figure><img src="image_134.png" /><figcaption>Fig. 94 Strategies for the selective conjugation of proteins based on metal-chelation: tetracysteine/biarsenical system, oligohistidine/nickel-complex system, tetraserine-borate system, oligo-aspartate/zinc-complex system.²⁷</figcaption></figure>

### 5. Proximity-driven modifications

All the above-mentioned methodologies are mainly residue-specific. That is to say, they exploit specific reactivity of the functional group of interest or of an assembly of residues. As a result, bioconjugations of highly nucleophilic Cys, Lys, and Tyr residues with electrophilic reagents are definitely prevalent among described methodologies.

The inherent reactivity is rare to be overcome, which largely limits of the scope of known methods for bioconjugation of the amino acids possessing low nucleophilicity. However, bringing the reaction partners into close proximity can accelerate a reaction thereof, which would not otherwise be possible because of the presence of other more reactive species. Routinely exploited by enzymes, this approach enables selectivity on the basis of the molecular shape rather than reactivity or the local environment (Fig. 95).

<figure><img src="image_135.png" /><figcaption>Fig. 95 Schematic representation of the proximity-driven approach for selective modification of macromolecules. Combining of promiscuous reactivity of the reactive group (RG, shown in red) with specific molecular recognition results in specific modification of the functional group of interest (FG, shown in blue).</figcaption></figure>

<figure><img src="image_136.png" /></figure>
Review Article

Developed by Hamachi and collaborators in 2006,⁴⁶² the post-affinity-labelling approach can be used for the selective tethering a functional molecule at the proximity of the active site of enzymes. This method was then used on numerous substrates and enlarged the scope of selective conjugation especially on histidine and tyrosine (see Section 1.5.1).⁴⁶²,⁴⁶³,⁴⁸⁶,⁸¹⁴ Despite these successes, a prevalent limitation of the applicability of the method due to the possible modification of the residues situated only in the vicinity of ligand-binding pocket of the target protein prevents this methodology from becoming a general approach for protein labelling.

In 2010, exploiting similar idea, Popp and Ball⁴⁵⁴ envisioned the combination of two previously described techniques: the coiled-coil based molecular recognition of complementary peptides⁴⁵⁵,⁸¹⁵⁻⁸¹⁷ and high catalytic activity of dirhodium complexes on carbene C–H insertion, previously reported by Francis and collaborators⁴⁵² for the selective modification of tryptophan (see Section 1.4.2). Two complementary peptides: one, containing a dirhodium catalytic centre (precomplexed through two glutamate side chains), and another, containing a side chain to be modified are thus involved in this methodology (Fig. 96a). Because of the compelled proximity of the side chain of interest and the active catalytic centre in the obtained supramolecular assembly, the reaction of rhodium-catalysed C–H insertion is largely accelerated (more than 10³ times).⁸¹⁸

As a result, the conjugation of amino acid residues with lower reactivities becomes possible. This allowed to expand the scope of originally tryptophan-selective dirhodium carbene methodology first to the other aromatic residues, phenylalanine and tyrosine,⁴⁵⁴ and then to over half of the naturally occurring amino acid residues (Fig. 96b).⁸¹⁸ To date, dirhodium metallopeptide represent the only reported method for selective modification of Gln, Asn, and Phe side chains. The authors have also demonstrated the possibility to apply their methodology on chimeric proteins, containing fused coils,⁴⁵⁶ as well as on full-sised natural proteins possessing coiled-coils in their structures.⁸¹⁸

Despite its important potential, the metallopeptides methodology is however not devoid of drawbacks. Because binding to dirhodium is nonselective and thus cannot be performed in the presence of other carboxylate-containing peptides, rhodium-peptide complexes must be synthesised beforehand, which is often challenging mainly due to their poor solubility.⁸¹⁹ Moreover, the method is restricted to proteins containing coiled coil fragments in their structures, which in case of the vast majority of targets would mean the need for resource- and time-consuming expression of fused proteins.

Another approach, developed by Silverman and colleagues, exploits self-assembling of complementary DNA to bring into proximity two reacting fragments and allowed, although only on simple substrates, selective phosphorylation of tyrosine and serine, otherwise not feasible.⁵³⁸

Beyond coiled coils and DNA-based preorganisation, the principles of proximity-driven selectivity should be extended to other helix-binding protein domains and to biological molecular recognition generally. A significant broadening of the applicability of this elegant approach for protein modification,

<figure><img src="image_137.png" /><figcaption>Fig. 96 (a) Modification of c-Fos (shown in red) catalysed by Max(Rh₂) (shown in blue) metallopeptide described by Popp and Ball.⁸¹⁸ Two possible coiled-coil alignments result in modification of GLU-14 and GLU-21 residues, located in close vicinity to the dirhodium catalytic center. (b) Proposed product bond connectivity.</figcaption></figure>

biochemistry and biomaterials engineering is anticipated in the nearest future.
Conclusions

The field of bioconjugation has expanded in the last 100 years and passed from a blind-eye modification of proteins one could found in nature to a well-established independent domain full of approaches allowing precise and reliable introduction of various tags into proteins' structure.

Many, if not most of these methods, however, often possess drawbacks limiting their general applicability. This fact has become of special consideration with a rise of novel exigent applications for bioconjugation, namely the preparation of new therapeutic conjugates, vaccines, and biomaterials. Also tremendous progress in the sensitivity of analytical methodologies as well as the need to work with smaller and smaller amounts of sample, often-unstandardized patient samples, highlighted the need for more efficient, selective and reliable bioconjugation methods.

Moreover, some parameters of the mode of conjugation, previously completely neglected, were recently revealed to be of paramount importance. For instance, the stability of the generated linkage and the distribution of products generated upon conjugation can be determining for the overall efficiency of the conjugate.

Overall, we believe that intensive ongoing research in the field of bioconjugation will result in more efficient and selective methodologies allowing specific conjugation of native proteins in complex biological media, and ultimately in living organisms.

Acknowledgements

We thank Dr S. Kolodych (Syndivia SAS), Dr J.-Y. Bonnefoy (Syndivia SAS), Dr A. Osypenko (UDS), and Dr M. Recher (UDS) for fruitful discussions that led to the redaction of this manuscript. We also acknowledge the financial support from SATT Conectus "BioReLiable" project, CNRS, University of Strasbourg, and International Center for Frontier Research in Chemistry (icFRC).

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