# Solid phase chemical ligation

S. Abboud, A.F. Delmas, V. Aucagne

► To cite this version:

S. Abboud, A.F. Delmas, V. Aucagne. Solid phase chemical ligation. Brik Ashraf, Dawson Philip, Liu Lei. Total Chemical Protein Synthesis, Wiley, 2021, 978-3-527-34660-8. hal-02974365

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Distributed under a Creative Commons Attribution 4.0 International License
SOLID PHASE CHEMICAL LIGATION

Skander Abboud, Agnès F. Delmas and Vincent Aucagne*

Centre de Biophysique Moléculaire, CNRS UPR 4301, Rue Charles Sadron 45071
Orléans, France.

*: corresponding author

Authors emails: skander.abboud@cnrs-orleans.fr; delmas@cnrs-orleans.fr;
aucagne@cnrs-orleans.fr

**Abstract**

This is an exhaustive review of the methods developed in the last 25 years for chemical
proteins synthesis through solid-supported ligation-based assembly of peptide
segments. A particular emphasis was put on the nature of solid supports, the structure
of the linkers used to link the first segment to the solid support, the different
chemoselective ligation strategies investigated, and on post-ligation solid-phase
transformations.

**Keywords**

SPCL; Solid phase; Solid support; Linker; Native Chemical ligation; Peptidomimetic
triazole ligation; Oxime ligation

**Acknowledgments:**

We thank the French Agence Nationale de la Recherche for funding (ANR-15-CE07-022 EasyMiniProt grant).
Table of contents

I- Introduction

A- The promises of solid phase peptide chemical ligation (SPCL)

B- Chemical ligation reactions used for SPCL

C- Key requirements for a SPCL strategy

II- SPCL in the C-to-N direction

A- Temporary masking groups to enable iterative ligations

B- Linkers for C-to-N SPCL

1- Use of same linker and solid support for SPPS and SPCL

2- Re-immobilization of the C-terminal fragment

III- SPCL in the N-to-C direction

A- Temporary masking groups to enable iterative ligations

B- Linkers for N-to-C SPCL

C- Case study

D- SPCL with concomitant purifications

IV- Post-ligations solid-supported transformations

A- Chemical transformations

B- Biochemical transformations

V- Solid supports
# I- Introduction

## A- The promises of solid phase peptide chemical ligation (SPCL)

The concept of solid-supported synthesis introduced by Merrifield half a century ago [1] has been a paradigm shift in the field of peptide research. The development of ligation reactions [2] was another major breakthrough: building blocks are no longer protected amino acids but unprotected peptide segments equipped with functional groups able to react regio- and chemoselectively in an aqueous environment.

As largely discussed throughout this book, the intensive optimization of the chemical toolbox of the peptide/protein chemist made considerably easier the chemical synthesis of proteins, and has led to tremendous achievements in terms of target size (several hundreds amino acid residues). Notable achievements in the field include the synthesis of the enzyme DapA (31 kDa, 312 residues) and its D-version through the convergent ligation-based assembly of six segments [3] or, more recently, the synthesis of tetra-ubiquitinated α-globulin (53 kDa, 472 residues) from six segments [4,5]. A key point for the ligation of such impressive syntheses is the requirement for one of the chemoselectively-reactive moieties of the internal segments to be temporarily masked. This calls for a careful optimization of each unmasking reactions in addition to the strictly speaking chemical ligations steps, as well as for the use of peptide segments as long as possible to minimize the overall number of steps.

As a general statement, multiple ligation-based protein synthesis is often associated with very low overall yields, essentially due to the laborious intermediate chromatographic purifications. Having in mind the idea to minimize such purifications, native chemical ligation (NCL) [6] was extended soon after its discovery to a solid-phase version by its inventors [7-9], aiming to simplify the multiple successive ligation process. Later on, despite its appealing advantages, solid phase peptide ligation (SPCL) was barely considered [10] until we and others [11-15] started to reinvestigate the field. In this review, we exhaustively describe the efforts done in the SPCL field in the past twenty years. The low-hanging fruits have already been harvested and published as proofs of concept, but many further efforts and methodology developments will probably be needed to transform SPCL into a mature technology
and fulfill its promises. In the following sections, we will try to stress the main
challenges associated with SPCL and to identify the remaining bottlenecks.

Note that the term “solid phase chemical ligation” was introduced by Muir in 1998 [16]. However it referred in this case to the use of a solid supported thioester that is released into solution during the ligation, rather than to a ligation-based assembly on a solid support. Later on, SPCL referred only to ligation-based assembly on a solid support, on which this review is concentrated.

## B- Chemical ligation reactions used for SPCL

The great potential of SPCL was initially applied to NCL [6], and not to other amide bond-forming ligation reactions so far. It was later extended to peptidomimetic triazole ligation (PTL) [17], which exploits the amide mimicking features of the 1,4-disubstituted 1,2,3-triazole products generated by of the copper-catalyzed alkyne/azide cycloaddition (CuAAC) [18,19]. Moreover, besides application to chemical protein synthesis, SPCL has also been applied to oxime ligation for the modular synthesis of complex peptide-based architectures [13] (figure 1).

These three types of chemical ligation reactions typically take place in aqueous media at room temperature or slightly above (37°C), and their kinetics rates in solution [20,21] allows to reach completion within a few hours at millimolar concentrations using a slight excess of one of the peptide segments. Note however that the solid support can have a dramatic influence on kinetics, as discussed later in part V.

From a more practical point of view, reaction conditions for solid-supported ligations do not really differ from solution phase ones. Typical conditions for NCL includes a pH 6.5-7.5 phosphate buffer and 6 M guanidinium chloride (to increase solubility of segments), in conjunction with TCEP to keep a reducing environment, and an arylthiol catalyst such as 4-mercaptophenylacetic acid (MPAA). CuAAC proceeds well at neutral pH and various co-solvents, such as NMP, HFIP or methanol can be used in case of low solubility of segments. It requires a copper(I) catalyst which can lead to Fenton-type oxidation in the presence of oxygen, which is particularly detrimental for methionine and histidine side chains. Therefore, the reaction mixtures should be
carefully deoxygenated, and use of copper(I) ligands such as tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) in conjunction with radical scavengers [22] is sometimes necessary. Acidic sodium acetate buffer (pH 4.5) is well suited for oxime ligations, which can proceed without any additives. However, aniline has been shown to considerably accelerate oxime formation kinetics in solution [20].

<figure><img src="image_2.png" /><figcaption>Figure 1 – Chemoselective ligation reactions that have been used for SPCL</figcaption></figure>

## C- Key requirements for a SPCL strategy

If SPPS can be conducted only in one way, from the C-terminus to the N-terminus, for reasons of epimerization during amide formation, solid-supported ligations of unprotected peptide segments can be conducted either in the N-to-C or C-to-N directions (figure 2) as illustrated in the seminal publication of Kent and coworkers introducing the SPCL concept in 1999 [8].
In this review, we will illustrate pros and cons of both methods, which share a number
of common requirements:

- the use of masking groups for one of the chemoselective reactive moieties to enable successive ligation, by preventing segment cyclization and oligomerization, such as for solution phase multiple segments assembly;

- a strategy to immobilize the starting material, a pure and unprotected peptide segment;

- a SPCL linker cleavable under conditions compatible with a large spectrum of proteins, while being stable to ligation and unmasking conditions;

- a SPCL solid support compatible with the aqueous environment used for ligations, with the unmasking reactions and the SPCL linker cleavage.

In addition, we will highlight post-ligation solid phase synthetic transformations, taking
further advantage of the solid support. A table recapitulating all the publications related
to SPCL can be found at the end of this manuscript.
<figure><img src="image_3.png" /><figcaption>Figure 2 – General principles of solid phase chemical ligation</figcaption></figure>

## II- SPCL in the C-to-N direction

### A-Temporary masking groups to enable iterative ligations

As discussed earlier, the key tactic for multiple successive ligations is the temporary masking of one of the reactive ends of internal peptide segments. Importantly for the
SPCL strategies, the unmasking conditions have to be compatible with the stability of
the SPCL linker.

The masking groups that have been used for solid phase ligations in the C-to-N
direction are listed in Figure 3. Most reports of C-to-N SPCL are based on NCL
reactions between N-terminal cysteine and C-terminal thioesters segments: as a
consequence, a Cys-masking strategy is necessary. Three different Cys protecting
groups have been exploited for this purpose, all three being commonly used in solution-
phase multi-segment ligation strategies. The methylsulfonylethyloxycarbonyl (Msc) [23]
amine protecting group can be removed under aqueous basic conditions [9]. The
nowadays most used strategy is N,S bis-protection as a thiazolidine ring (Thz) that can
be opened using aqueous methoxyamine [10, 15, 24]. The acetamidomethyl group
(Acm) [25] thiol protecting group used by Kent in the first SPCL report can be removed
on resin by treatment with mercury(II) acetate [4]. Despite its wide applicability, one of
the drawbacks of the Acm protecting group is its removal requiring extended reaction
times and harsh conditions. Recently, Brik reported the use of Pd(II) salts to remove
this protecting group within minutes in aqueous media [26]. This method is very
efficient for Acm deprotection in solution and looks promising for SPCL, although this
has not been demonstrated yet.

As an alternative to protecting groups, Muir employed an N-terminal appendage of a
17-mer propeptide sequence (MASSRVDGGRSDLIEGR) after the cysteine residue,
this sequence being selectively removed by the protease factor Xa [7].

Besides NCL, Aucagne and Delmas reported multiple solid supported oxime ligation
[13] using segments bearing an N-terminal aminoxy function protected by an N-
allyloxycarbonyl (Aloc) group [27]. Aloc can be removed by a Tsuji-Trost reaction using a Pd(0) complex [28] in combination with N,N-dimethyl barbituric acid (NDMBA). Note that NDMBA was preferred to classical hydride donors as they caused partial cleavage of the N-O bond.

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /><figcaption>Figure 3 – Temporary masking groups used for SPCL in the C-to-N direction</figcaption></figure>
## B- Linkers for C-to-N SPCL

<figure><img src="image_6.png" /><figcaption>Figure 4 – Two possible routes for SPCL in the C-to-N direction</figcaption></figure>

Two different strategies have been explored for SPCL in the C-to-N direction, calling for different SPCL linkers design. The same linker and solid support employed for the SPPS elongation can be also used for SPCL after selective side chains deprotection (Figure 4, route A), or the C-terminal segment can be re-immobilized on a second solid
support through a chemoselective reaction, after its cleavage from the SPPS support (route B).

## 1-Use of same linker and solid support for SPPS and SPCL

In this case, the C-terminal segment is synthesized on a solid support which is compatible to both SPPS and SPCL conditions: organic solvents and aqueous buffers. The other crucial point is the use of side-chain protecting groups that can be cleaved without affecting the SPPS linker, in order to obtain a fully deprotected peptide still attached to the solid support. To date, all C-to-N SPCL strategies exploiting the same support as SPPS have been developed on Fmoc SPPS. They differ in the nature of the linker, side-chain protecting groups and ligation type.

<figure><img src="image_7.png" /><figcaption>Figure 5 – Linkers exploited for SPCL in the C-to-N direction using the same support for both SPPS and SPCL</figcaption></figure>

This strategy was introduced by Kent [10] for the synthesis of the 28 amino acid knottin EETI-II [29] on the superpermeable organic combinatorial chemistry (SPOCC) resin [30,31] equipped with a TFA-labile Wang-type linker. The first segment was here limited to a Cys-Gly dipeptide, the cysteine being protected as a Thz. Two successive
native chemical ligations gave the expected cysteine-rich peptide with a 80 to 90 %
yield based on the initial loading of the resin.

Brik [15] extended the approach by the synthesis of a 135 amino acid HA-tagged human histone 2B (H2B) derivative, using a Rink amide-PEGA solid support, through four successive NCL reactions using Thz as the N-terminal Cys masking group. In this case, the first segment was a single cysteine residue, protected as a Thz. On-resin desulfurization of Cys residues into Ala, followed by Rink linker cleavage, gave the target polypeptide (10% isolated yield), which slightly differs from the native sequence by the appendage of a C-terminal alaninamide residue.

Besides applications to chemical proteins synthesis, it is worth mentioning that Cys-
Rink amide PEGA resin was recently exploited by Olsen [32] for the chemoselective
trapping and enrichment of cyclic peptide thioesters from a complex mixture of natural
products secreted by bacteria. Release through TFA treatment allowed the
identification of previously unknown auto-inducing peptides that regulate population-
wide behavior by acting on quorum sensing.

With the objective to increase the amino acid diversity in the C-terminal segments, Aucagne and Delmas [13] explored the use of highly acid-labile trityl-type side chain protecting groups. The key point was to use acidic deprotection conditions that would not cleave the TFA-labile linker. The nonapeptide PPAHGVSTA derived from the human mucin protein MUC1 was synthesized on a PEGA resin equipped with a Wang-type linker, with the appendage of a N-terminal aminoxyacetic (Aoa) residue. Trityl (Trt) groups were used to protect Aoa, Ser and Thr, and methoxytrityl (Mmt) for His. Mild treatment using dilute TFA in dichloromethane in the presence of *iPr*3SiH was used to
cleave the protecting groups without affecting the linker. Optimization of the linker
structure revealed that the 4-(hydroxymethyl)phenoxypropionic linker (HMPP) was
cleaved 3.5 time slower than 4-(hydroxymethyl)phenoxyacetic (HMPA) under these
conditions. Notably, Mmt was preferred over Trt for His protection, as the later required
prolonged 2% TFA treatment that is detrimental to the linker integrity. Two successive
oxime ligations using Aloc-protected Aoa as a masked aminooxy group gave a 61
amino acid polypeptide construct in 52% yield without the need of any further
purification after TFA cleavage of the SPPS/SPCL resin. Note that, this strategy could
not be generalized to peptide fragments containing Arg residues, due to the lack of
compatible highly-acid labile protecting groups for the side chain guanidine.

## 2- *Re-immobilization of the C-terminal fragment*

The single support C-to-N SPCL strategy is inherently limited by the necessity to obtain
a C-terminal segment devoid of any co-products arising from side-reactions or
incomplete couplings (truncations or deletions). A more versatile, though more
sophisticated, strategy implies the synthesis of the C-terminal fragment on a SPPS
resin, followed by re-immobilization on a second, water-compatible SPCL resin. In this
case, a SPCL linker is introduced during SPPS and is functionalized by a group
allowing its chemoselective grafting on the SPCL support (red moiety, Figure 6). The
SPCL linker obviously has to be stable to SPPS elongation and cleavage conditions,
in addition to the ligation and unmasking steps used for SPCL.

This double support strategy is compatible with the twenty proteinogenic amino acids,
and allows the intermediate purification of the C-terminal segment prior to re-
immobilization.
<figure><img src="image_8.png" /><figcaption>Figure 6 – Different cleavable linkers used to immobilize the C-terminal fragment on a SPCL solid support</figcaption></figure>

In the first-ever SPCL report, Kent [8] proposed the use of a base-labile linker derived from the carboxamidomethyl ester (CAM) [-OCH₂CONH₂] protecting group for carboxylic acids (Figure 6A) [33]. Esterification was carried out in solution to provide building blocks for Boc-SPPS. After elongation of the peptide segment, a ketone functional group was introduced through solid-supported coupling of levulinic acid on the side chain of a Fmoc-protected Lys. After cleavage from the SPPS resin, the segment was immobilized by oxime ligation onto an aminooxy-functionalized cellulose solid support. The 118 amino acids human group V secretory phospholipase A2 (GV-PLA2) was synthesized through three successive NCL, using Acm as Cys-masking
groups. Cleavage from the SPCL resin was achieved through a short treatment with
aqueous sodium hydroxide and the crude product was then folded to give a six disulfide
bonds-containing protein with full enzymatic activity.

Dawson [9] later extended the double support C-to-N SPCL approach to C-terminally-
amidated peptides, exploiting the safety catch acid-labile linker (SCAL) [34] . This linker
is stable to Boc-SPPS elongation and cleavage conditions in the form of *bis*-sulfoxide,
and TFA-labile after reduction into sulfides. A thioester moiety located on the SCAL
linker (figure 6B) mediated NCL immobilization of the segment onto a Cys-
functionalized sepharose solid support. The 71 amino acids vMIP I chemokine was
assembled through two consecutive NCL, the use of either S-Acm or N-Msc as Cys-
masking group demonstrating comparable efficiencies. Quantitative cleavage of the
SCAL linker was achieved through a treatment with 1 M SiCl₄ in TFA in the presence
of scavengers. The crude polypeptide product was purified in a 21% recovered yield.

More recently, Ottesen [25] extended the double support C-to-N SPCL approach to
Fmoc-SPPS segment synthesis, making use of the base-labile 4-
hydroxymethylbenzoic acid (HMBA) linker [35], functionalized with a thioester moiety
(figure 6C). In order to allow simple monitoring of the different ligation and Cys
unmasking steps, a Rink amide linker was incorporated between the Cys and the
PEGA support. Two histone proteins, H4 (102 aa) and CENP-A (139 aa), N-acetylated
on some of their Lys residues, were synthesized through NCL-based assembly on a
Cys-PEGA resin, using Thz-protected segments. In a first approach, both proteins
were synthesized through solid-supported NCL using four and five segments,
respectively, followed by on resin desulfurization and HMBA cleavage by a short
aqueous sodium hydroxide treatment. Even though both products were obtained in a
good purity, recovery yields were extremely low. Based on the observation that the
recovery severely dropped down after the ligation of the fourth segment, a hybrid phase
approach was designed, in which the first three segments of each protein were
assembled on resin, and the last ones in solution.

Muir applied C-to-N SPCL to expressed protein ligation (EPL) [36–38], to assemble
synthetic C- and N-terminal segments with recombinant ones [7]. C-terminal segment
consisted in a 19-mer cysteinyl peptide incorporating the LEVLFQGP sequence,
referred to as “PreSciss linker”, and a biotinylated Lys residue (figure 6D). This
segment was immobilized on avidin-functionalized sepharose beads, followed by EPL
with a recombinant thioester with an N-terminal MASSRVDGGRSDLIEGRC
appendage. Selective cleavage of the R/C peptide bond by factor Xa generated an N-
terminal Cys, which was further ligated with a synthetic thioester. Release of the
semisynthetic protein in solution was finally achieved through treatment with
PreScission protease that cleaves the Q/G peptide bond of the PreSciss linker. This
process was used to dual-label the amino and carboxyl termini of the 304 amino acids
Crk-II adapter protein with a FRET pair for application as a protein–protein sensor.

The double support N-to-C SPCL approach was also exploited by Seitz for the NCL-
based assembly of proteins on microtiter plates without using any cleavable linker, with
the aim to generate small libraries of proteins that can be subsequently screened on-
surface [39,40].
### III- SPCL in the N-to-C direction

SPCL can also be accomplished in a N-to-C direction. In this approach, the SPCL linker is introduced at the N-terminus of the N-terminal fragment after SPPS elongation, followed by linker cleavage and re-immobilization on a SPCL solid support (figure 7) [41].

<figure><img src="image_9.png" /><figcaption>Figure 7 – SPCL in the N-to-C direction</figcaption></figure>

### A- *Temporary masking groups to enable iterative ligations*

A challenge in N-to-C NCL assembly of proteins is the design of masked thioesters, and many groups investigated such thioesters precursors that could be activated on demand. The first report of masked thioesters for N-to-C SPCL was the use of peptide thiocarboxylate segments by Kent [8] : the absence of a proton can be considered as
a protecting group for the thioacid, because the ionized thiocarboxylate is almost unreactive under NCL conditions. The thiocarboxylate can be easily converted into a reactive thioester, through chemoselective S-alkylation with bromoacetic acid at pH 4-5 (figure 8A). At this low pH, other potentially nucleophilic functional groups in the unprotected peptide segments are unreactive.

In the last decade, a new strategy for the Fmoc-SPPS-based generation of thioesters has been developed that exploits the spontaneous conversion of β-mercato amides into β-amino thioester through a *N*→*S* acyl shift. The equilibrium between the two species can be shifted towards a thioester by trans-thioesterification with an exogenous thiol used in large excess. Melnyk and Liu exploited these properties for *N*-to-C SPCL, through a latent thioester surrogate the *bis*--(2-sulfanylethyl)amido group (SEA) [42,43]. The disulfide form of SEA (SEA<sup>off</sup>) is unreactive under NCL conditions [44], providing that no strong reducing agent is used, but can be converted on demand into an active thioester by treatment with a combination of TCEP and mercaptoacetic acid as the exogenous thiol, through an intermediate SEA<sup>on</sup> species (figure 8B).

N-to-C SPCL also applicable to the peptidomimetic triazole ligation strategy developed by Aucagne and Delmas, based on the protection of the alkyne moiety by a tri-isopropylsilyl group (TIPS) [45]. This masking group can be cleanly removed under aqueous conditions through protiodesilylation upon treatment with silver nitrate, to generate a terminal alkyne suitable for CuAAC (figure 8C) [14].
<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /><figcaption>Figure 8 – Temporary masking strategies used for solid-supported iterative ligations in the N-to-C direction</figcaption></figure>

## B- Linkers for N-to-C SPCL

The N-to-C SPCL strategy requires the introduction of the SPCL linker at the N-terminus, which is more straightforward for peptide chemists than the functionalization at the C-terminus. Kent on his first report [8] on SPCL derivatized the N-terminal peptide segment at its N-terminus by a base-labile SPCL linker derived from the Msc amine protecting group. The linker featured a ketone functionality for oxime ligation-mediated immobilization on a cellulose-based solid support containing an aminoxy group (figure 9A). The linker was incorporated in three steps after Boc-SPPS elongation, making use of a *N*-Boc protected amine-containing sulfonylethylcarbonate building block [46] followed by coupling of levulinic acid. This linker was applied to the synthesis of several polypeptides (68 to 115 amino acids) through the NCL-based solid phase assembly of up to three peptide segments, exploiting thiocarboxylates as thioester precursors. The linker is cleaved by aqueous base (NaOH, pH 12-14 for 2 min) to release the polypeptide into solution.
More than ten years later, Aucagne and Delmas introduced the (2-[2-(2-azido-ethoxy)-ethyl-sulfonyl]-2-ethoxycarbonyl linker (N₃-Esoc) derived from the same Msc protecting group, and functionalized with an azide moiety (figure 9B) [11]. N₃-Esoc can be incorporated on the N-terminal amine of peptide segments using an activated carbonate derivative. Peptide equipped with this linker can be immobilized on an alkyne-functionalized solid support by either CuAAC or strain-promoted azide/alkyne cycloaddtion (SPAAC) [47]. This linker was first applied to the synthesis of a triazole-containing analogue of the extracellular domain of the human mucin 1 (MUC1), composed of a densely O-glycosylated twenty amino acids tandem repeat. Three successive peptidomimetic triazole ligations using segments equipped with TIPS-masked alkynes, followed by linker cleavage at pH 11.5 afforded an unglycosylated 160 residue polypeptide in a 60 % overall yield. This constitutes the longest sequence that has been assembled through SPCL to date.

The same linker has been exploited for NCL-based SPCL in collaboration with Melnyk [12], using segment incorporating a SEA<sup>off</sup> moiety as masked thioester. Assembly of five peptide segments gave a 136 amino acids polypeptide derived from hepatocyte growth factor (HGF) in 6.5 % yield.

The basic conditions required to cleave the sulfonylethyl carbamate are relatively harsh, and were shown to be incompatible with *O*-glycosylated serine residues, known to be sensitive to base-mediated beta-elimination. With the objective to extend N-to-C SPCL to the synthesis of *O*-glycoproteins, Aucagne and Delmas developed another azide-functionalized linker, 1-azido-5-[1,3-dimethyl-2,4,6(1H,3H,5H)-trioxopyrimidine-5-ylidene]pentyl (N₃-Dtpp), [14] (figure 9C) derived from the enamine-based amine protecting group DTPM [48,49] which can be cleaved by hydrazinolysis under mild conditions. N₃-Dtpp was introduced on a peptide segment through the coupling of a derivative of the last amino acid N-protected with this linker. Assembly of a 160 residues triazole-containing MUC1 polypeptide on a controlled pore glass (CPG) solid support was realized through four successive PTL. Linker cleavage can be achieved by aqueous hydrazine at neutral pH, but these conditions were shown to partially reduce TIPS-protected C-terminal alkyne groups, which complicated the monitoring of each ligation and unmasking step through analytical scale cleavages. Optimized
conditions using aqueous hydroxylamine allowed clean and efficient cleavage of the linker.

More recently, Melnyk investigated the use of an acetoacetyloxime (AcA) linker (figure 9D) [50] which was introduced by solution phase NCL of a N-terminal cysteinyl segment with an AcA thioester. The segment can then be grafted onto an aminooxy-functionalized SPCL solid support by oxime ligation. This strategy was used to synthesize polypeptides derived from the K1 domain of HGF on a PEGA solid support. Treatment with a mixture of hydroxylamine and aniline at a mildly acidic pH liberates an acetoacetyloxime in solution, which undergoes spontaneous cyclizative amide bond cleavage affording the native protein. Hydroxyamate formation resulting from the reaction with Asn/Gln was observed during the linker cleavage, however decreasing the pH and hydroxylamine concentration was found to nearly abolish this side reaction.

<figure><img src="image_12.png" /><figcaption>Figure 9 – Different cleavable linkers used to immobilize the N-terminal segment on a SPCL solid support</figcaption></figure>
C- Case study

In order to illuminate SPCL from a more practical point of view, we have chosen one
example of N-to-C assembly of a protein to illustrate the performance of the process
by showing the HPLC analysis of the intermediates and final products (Figure 10). This
case study is based on the 2013 report by Melnyk and our group [12] exploiting the N₃-
Esoc linker along with the blocked thioester properties of the SEAoff group for the
assembly of five segments to give a 15 kDa polypeptide derived from human
hepatocyte growth factor 1 (HGF1). To date, this constitutes the only example of more
than three successive ligations conduced on a solid support.

The first segment was immobilized onto PEGA1900 resin through a SPAAC reaction, then the N-to-C elongation was based on a cycle of (1) conversion of the C-terminal SEA<sup>off</sup> group into a thioester and (2) NCL reactions. For the coupling of the last segments, these two steps were realized concomitantly. The target polypeptide was obtained in an overall 6.5 % yield after a final HPLC purification. The remarkable HPLC purity of all the different intermediates highlights the efficiency of the SPCL process.
<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /><figcaption>Figure 10 – Case study of the synthesis of HGF1 NK1 [1-136] through five successive solid-supported ligations (adapted from ref [12] with permission from The Royal Society of Chemistry)</figcaption></figure>
## D- SPCL with concomitant purifications

Protein elongation in the N-to-C direction provides an important advantage over the alternative C-to-N direction, which is the “self-purification” of the incoming peptide segments, provided that a capping step is used after each amino acid coupling during SPPS. The side products are normally truncated sequences rather than deleted ones. This self-purification effect is based on the fact that only the full-length peptide segments contain the N-terminal reactive group needed for immobilization or segment ligation, i.e. the linker, a cysteine for NCL or an azide for PTL. Truncated peptides, which constitutes the major co-products from Fmoc-SPPS, are unreactive and are simply washed off after the immobilization and ligation steps, allowing to work with crude, unpurified peptide segments providing that no other side reaction occurred during SPPS (e.g. aspartimide formation, oxidation etc...). This self-purification effect has been largely exploited for the non-chromatographic purification of peptides from the 70's to the 90's, [46, 51–56] and the extension of this concept for SPCL with self-purification has been pioneered by Aucagne and Delmas in 2012 [11], then exploited by other groups [50,57].

## IV- Post-ligations solid-supported transformations

Taking significant advantage of the solid support used for SPCL, different chemical or biochemical transformations can be implemented after ligation-based protein elongation, in order to simplify the work-up and to limit purification steps.

### A- Chemical transformations

To bypass the limitation of the requirement of the rare Cys residue at the ligation disconnection, desulfurization reactions after ligation has been developed, but generate extra handling and purification steps. The metal-free desulfurization approach developed by Danishefsky [58] using a radical initiator in combination with tris-(2-carboxyethyl)phosphine (TCEP) is the most popular method. It was successfully
applied to SPCL by Brik [15] and then Ottesen [25] to convert cysteine and
penicilamine residues into Cys and Val, repectively.

Probes can be chemoselectively conjugated to the synthesized supported-protein, as
demonstrated by Aucagne and Delmas with the CuAAC-based conjugation of a biotin
[14].

Photochemical processes can also be conducted on the solid support. Brik used
thioester segments incorporating a C-terminal Lys protected with 6-
nitroveratryloxycarbonyl (Nvoc) group on side-chain to prevent lactamization during
the ligation step [15]. The Nvoc group was successfully removed before SPCL linker
cleavage through exposure to UV.

B- Biochemical transformations

Formation of disulfide bonds is a post-translational modification crucial for the structure stabilization and function of many proteins. The total chemical synthesis of disulfide-containing proteins involves a final oxidative folding step, i.e. formation of the disulfide bond concomitantly with the folding of the peptide backbone. Solid-supported oxidative folding would benefit from the pseudo-dilution effect [59], which favors intramolecular reactions, thus potentially avoiding intermolecular aggregation, a common problem during protein folding. In 2006, Kent [10] explored this idea to fold the disulfide rich miniprotein EETI-II (28 amino acids, three disulfide bonds) by air oxidation after SPCL, while it was still covalently attached to the solid support. Note that in this case the oxidative folding processed slower on solid phase than in solution.

In order to provide a simple access to long glycoproteins, Aucagne and Delmas
combined SPCL of peptide fragments with enzymatic glycosylation [14]. In this work,
they performed a solid-supported enzymatic glycosylation by using recombinant
polypeptide α-N-acetyl-galactosaminyl transferase 1 (pp-GalNAc-T1). In order to
facilitate monitoring of enzymatic glycosylation and the different ligation and
unmasking steps, a TFA-labile PAL linker [71] was incorporated between the alkyne
used for the CuAAC-mediated immobilization and the solid support. Different solid
supports were screened, and the best results were obtained using controlled pore
glass (CPG) beads with pore size around 100 nm. The enzymatic reaction was much
slower than in solution, and kinetics were improved by the introduction of a PEG spacer (3000 Da) between the polypeptide chain and the solid support. The expected product incorporating 160 amino acids and 20 carbohydrate residues was purified in a rather moderate 6% yield, due to the presence in the crude product of small amounts of under-glycosylated sequences (18 or 19 GalNAc residues) difficult to separate from the target.

## V- Solid support

The ideal support for solid-phase ligation must be compatible with aqueous conditions required for the solvation of unprotected peptides, ideally including a large range of buffers and pH, and to both ligations and unmasking conditions. A solid support does not behave as an inert carrier but rather acts as a co-solvent [60, 61] and this feature will affect any parameters of a chemical reaction, yield, purity, speed and cost.

Resin bead size has an effect on the reaction rate as well. Many studies conducted on solid phase organic chemical reactions showed that the diffusion rate increased with decreasing bead size.[62–64] What was observed in organic solvents is certainly also true in aqueous media that are used for SPCL. The degree of cross-linking can also have a large impact on reaction yield, as shown by Meldal [62] and Lippens [65], who observed that increasing cross-linking slowed down reactions by lowering the diffusion rates. Diffusion of peptide segments could be a limiting process in SPCL likewise.

The nature of the SPCL resin can impair the ligation reaction as observed by Dawson [9] with the PEGA resin. He attributed poor NCL yields to peptide aggregation on the support due to the high loading of the PEGA resin used. Yields were not improved by adding a large excess of peptide segment or increasing the temperature and ligation time. Aggregation on PEGA was also hypothesized by Ottesen [25] who found that a tenfold reduction of the resin loading significantly improved kinetic reaction rates. In this special case, this is however proved to be insufficient for long polypeptide chains: a first series of three solid-supported ligations were successfully carried out whereas the last one had to be performed in solution to avoid presumable aggregation issues.

The nature of the solid support can also affect the SPCL linker cleavage kinetics as demonstrated by Aucagne and Delmas in their work on N₃-Esoc linker [11]. Base-mediated cleavage of this linker was found to be considerably slower when the peptide
was attached to polyethylene glycol-based water-compatible resins than in solution phase. TRIS buffer at pH = 8.5 was sufficient to cleave the linker in solution ($t_{1/2}$ = 7 h), whereas pH has to be raised up to 10.7 (50 mM arginine) on solid support, with polymer-dependant rates: $t_{1/2}$ = 13.7 h, 5.4 h and 2.1 h for ChemMatrix [66], PEGA800 and PEGA1900, respectively.

Besides non-covalent effects, the chemical structure of the polymer has obviously to be compatible with the chemical reaction used. For example Dawson [9] observed that the CLEAR solid support (Coss-Linked Ethoxylate-Acrylate Resin) developed by Barany [67] completely dissolved upon treatment with mercury(II) acetate in acetic acid used for Acm deprotection, probably resulting from ester hydrolysis. Furthermore, this resin is known to be unstable under the basic conditions used to remove the cysteine-masking N-Msc group [9].

Up to now, three families of solid support have been used for SPCL (Figure 11): i) biosourced oligosaccharide-based polymers, ii) PEG-based synthetic polymers, iii) inorganic matrixes (i.e. control pore glass, CPG).
<figure><img src="image_15.png" /><figcaption>ChemMatrix</figcaption></figure>

<figure><img src="image_16.png" /><figcaption>SPOCC</figcaption></figure>

<figure><img src="image_17.png" /><figcaption>PEGA</figcaption></figure>

<figure><img src="image_18.png" /><figcaption>Agarose</figcaption></figure>

<figure><img src="image_19.png" /><figcaption>Chitin</figcaption></figure>

<figure><img src="image_20.png" /><figcaption>Cellulose</figcaption></figure>

<figure><img src="image_21.png" /><figcaption>aminopropyl CPG</figcaption></figure>

<caption>Figure 11 – Different supports that have been employed for SPCL</caption>

Kent employed a spherilose resin [8], which is a cellulose-based solid support originally developed for affinity chromatography of proteins. Similarly, Dawson reported the use of a sepharose resin consisting of cross-linked agarose [9]. Muir [7] used a chitin-based
solid support, which has physicochemical properties similar to cellulose. These three
matrices classically used in biochemistry, have been used in the first reports on SPCL
[7,8,9,] and more recently by Seitz [57]. These polymers have been shown to be very
suitable for the aqueous conditions used in SPCL. However, their main drawback
concerns their limited chemical stability.

Polar PEG-based polymers emerged in the late 90s with the search for resins compatible with both solid phase organic chemistry and enzymatic reactions. Kent [10] reported the use of a PEG-poly(oxetane)copolymer, SPOCC [30,31], which was commercially-available at that time. In more recent works, PEG-polyacrylamide PEGA resins [68] have widely been used. PEGA are characterized by high swelling properties in both non polar and hydrophilic solvents and by high functional group reactivity facilitating the gel phase reaction to go to completion. Several PEGA resins have been developed by Meldal [69], differing in the length of the PEG chain. Two different ones have been previously used for SPCL: PEGA800 (800 Da PEG) [11,13,15, 25, 32, 50] and the no longer commercially available but very SPCL-suitable PEGA1900 (1900 Da) [12]. Note that the size of the PEG chain was omitted as in many reports on works done with a PEGA resin, and we suppose that PEGA800 was used because of its commercial availability. Important to note, the industrial production processes of PEGA polymers may have been changed as a result of successive licence transfers to different companies, as we have observed with the evolution of physical behavior of PEGA800 in the past decade.

An alternative to organic polymers is the use of controlled pore glass CPG 1000 Å [70]
as reported by Aucagne and Delmas [14]. CPG is a non-swelling inorganic matrix, a
silica glass forming porous beads with pores of 75 to 3000 angstroms Å in diameter.
This solid support has been originally developed for solid-phase oligonucleotide
synthesis. CPG is chemically stable and is compatible with both aqueous and organic
conditions, but not well suited for SPPS.
## VI- Conclusion and perspectives

About 20 years after the first report on the solid-supported version of chemical ligation *aka* SPCL, the method is still a compelling and highly challenging field of research. The adaptation of ligation chemistry to the solid phase may have the potential to considerably extend the limits of protein chemical synthesis. However, despite the significant gains made in advancing the technology, there are many remaining limitations. SPCL is still typically limited to less than three successive solid-supported ligation reactions, this being probably –at least in part– due to aggregation-prone segments or intermediates. From a technical perspective, monitoring SPCL reactions using on-resin-analysis would facilitate the optimization of the ligation and unmasking reactions, but that remains a very tricky task and physical analytic tools are not available in routine. For example FT-IR is not resolutive enough to give information for SPCL, and instrumental developments would be needed to use MAS-NMR [65] as a routine tool to analyse SPCL reactions. Alternatives are based on destructive approaches such as analytical scale “micro-cleavage” followed by LC-MS analysis of the released peptide. For SPCL reaction optimization purposes, working on sub-micromole analytical scale is still facing technical limitations related to resin handling, available glassware or maintaining oxygen-free conditions necessary for NCL and CuAAC.

Thus, there is still room for major technical breakthroughs such as development of new polymers, new linkers, new ligations reactions with improved kinetics, new masking/unmasking strategies to broaden the scope of SPCL applications to more ambitious targets. New instrumentation would probably be also needed in order to allow the routine use of SPCL by non-specialists in a comparable way as SPPS. Why not one day even come to an automated version and bring to the market proteins synthesizers to complement peptide synthesizers?
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<table><thead><tr><td>Ref</td><td>year</td><td>Direction</td><td>Immobilization</td><td>Solid support</td><td>Cleavable linker(s)</td><td>Linker cleavage</td><td>Ligation type</td><td>Nb of solid phase ligations</td><td>Overall yield (%)</td><td>Prominent targets</td></tr></thead><tbody><tr><td>[8]</td><td>1999</td><td>C-to-N</td><td>Oxime</td><td>Cellulose</td><td>ester (CAM)</td><td>pH 12-14</td><td>NCL</td><td>3</td><td>nd</td><td>GV-PLA2 (118 aa)</td></tr><tr><td>[8]</td><td>1999</td><td>N-to-C</td><td>Oxime</td><td>Cellulose</td><td>Msc</td><td>pH 12-14</td><td>NCL</td><td>2</td><td>nd</td><td>MIF (115 aa)</td></tr><tr><td>[9]</td><td>2000</td><td>C-to-N</td><td>NCL</td><td>Sepharose</td><td>SCAL</td><td>SiCl₄ in TFA</td><td>NCL</td><td>2</td><td>20</td><td>vMIP I (71 aa)</td></tr><tr><td>[7]</td><td>2000</td><td>C-to-N</td><td>Biotin/avidin</td><td>Agarose</td><td>PreScission protease substrate</td><td>PreScission protease</td><td>NCL (EPL)</td><td>1</td><td>55</td><td>Crk-II (304 aa)</td></tr><tr><td>[10]</td><td>2006</td><td>C-to-N</td><td>SPCL on SPPS resin</td><td>SPOOC</td><td>PHB</td><td>TFA</td><td>NCL</td><td>2</td><td>80-90*</td><td>EETI-II (28 aa)</td></tr><tr><td>[11]</td><td>2012</td><td>N-to-C</td><td>CuAAC</td><td>PEGA800</td><td>Esoc / PAL</td><td>pH 11.5 / TFA</td><td>PTL</td><td>3</td><td>60</td><td>MUC1 (160 aa)</td></tr><tr><td>[12]</td><td>2013</td><td>N-to-C</td><td>CuAAC or SPAAC</td><td>PEGA1900</td><td>Esoc</td><td>pH 11.5</td><td>NCL</td><td>4</td><td>6.5</td><td>HGF fragment (136 aa)</td></tr><tr><td>[15]</td><td>2014</td><td>C-to-N</td><td>SPCL on SPPS resin</td><td>PEGA</td><td>Rink</td><td>TFA</td><td>NCL</td><td>3</td><td>10</td><td>HA-tagged histone H2B (135 aa)</td></tr><tr><td>[13]</td><td>2014</td><td>C-to-N</td><td>SPCL on SPPS resin</td><td>PEGA800</td><td>HMPA</td><td>TFA</td><td>Oxime</td><td>2</td><td>33</td><td>Padre-MUC1 (61 aa)</td></tr><tr><td>[14]</td><td>2015</td><td>N-to-C</td><td>CuAAC</td><td>CPG100</td><td>Dtpp</td><td>hydroxylamine</td><td>PTL</td><td>3</td><td>6</td><td>Glycosylated MUC1 (160 aa)</td></tr><tr><td>[25]</td><td>2016</td><td>C-to-N</td><td>Oxime</td><td>PEGA</td><td>MBHA / Rink</td><td>pH 13 / TFA</td><td>NCL</td><td>3</td><td>>90% and 16%</td><td>CpA (70 aa) and histone H4 (65 aa) fragments</td></tr><tr><td>[39]</td><td>2016</td><td>C-to-N</td><td>Hydrazone</td><td>Polypropylene</td><td>No cleavable linker</td><td>-</td><td>NCL</td><td>1</td><td>-</td><td>SH3 domain (61 aa)</td></tr><tr><td>[57]</td><td>2016</td><td>C-to-N</td><td>His tag / Ni-NTA</td><td>Agarose</td><td>No cleavable linker</td><td>-</td><td>NCL</td><td>1</td><td>-</td><td>MUC1 (126 aa)</td></tr><tr><td>[50]</td><td>2017</td><td>N-to-C</td><td>Oxime</td><td>PEGA</td><td>acetoacetyloxime</td><td>hydroxylamine</td><td>NCL</td><td>3</td><td>23</td><td>HGF fragment (87 aa)</td></tr><tr><td>[40]</td><td>2017</td><td>C-to-N</td><td>His tag / Ni-NTA or CuAAC or hydrazone</td><td>Polypropylene</td><td>No cleavable linker</td><td>-</td><td>NCL</td><td>1</td><td>-</td><td>SH3 domain (61 aa)</td></tr><tr><td>[41]</td><td>2018</td><td>N-to-C</td><td>SPCL on SPPS resin</td><td>Polystyrene</td><td>Rink linker on Asn side chain</td><td>TFA</td><td>NCL</td><td>1</td><td>>90**</td><td>Protected model peptides (max 15 aa)</td></tr></tbody></table>

**Appendix Table** – Recapitulation of the SPCL strategies developed to date (nd: not determined *recovered yield **based on HPLC peak integration)