Insights into the mechanism and catalysis of peptide thioester synthesis by alkylselenols provide a new tool for chemical protein synthesis

Article abstract of DOI:10.3390/molecules26051386

While thiol-based catalysts are widely employed for chemical protein synthesis relying on peptide thioester chemistry, this is less true for selenol-based catalysts whose development is in its infancy. In this study, we compared different selenols derived from the selenocysteamine scaffold for their capacity to promote thiol-thioester exchanges in water at mildly acidic pH and the production of peptide thioesters from bis(2-sulfanylethyl)amido (SEA) peptides. The usefulness of a selected selenol compound is illustrated by the total synthesis of a biologically active human chemotactic protein, which plays an important role in innate and adaptive immunity.

Full text of DOI:10.3390/molecules26051386

molecules
Article
Insights into the Mechanism and Catalysis of Peptide Thioester
Synthesis by Alkylselenols Provide a New Tool for Chemical
Protein Synthesis
Florent Kerdraon 1 , Gemma Bogard 1, Benoît Snella 1, Hervé Drobecq 1, Muriel Pichavant 1,
Vangelis Agouridas 1,2,
*
and Oleg Melnyk 1,
*
1
U1019-UMR 9017—CIIL—Center for Infection and Immunity of Lille, Institut Pasteur de Lille,
University of Lille, CNRS, Inserm, CHU Lille, F-59000 Lille, France; florent.kerdraon@ibl.cnrs.fr (F.K.);
gemma.bogard@inserm.fr (G.B.); benoit.snella@ibl.cnrs.fr (B.S.); herve.drobecq@ibl.cnrs.fr (H.D.);
muriel.pichavant@pasteur-lille.fr (M.P.)
2
Centrale Lille, F-59000 Lille, France
*
Correspondence: vangelis.agouridas@ibl.cnrs.fr (V.A.); oleg.melnyk@ibl.cnrs.fr (O.M.)
Abstract: While thiol-based catalysts are widely employed for chemical protein synthesis relying on
peptide thioester chemistry, this is less true for selenol-based catalysts whose development is in its
infancy. In this study, we compared different selenols derived from the selenocysteamine scaffold for
their capacity to promote thiol–thioester exchanges in water at mildly acidic pH and the production
of peptide thioesters from bis(2-sulfanylethyl)amido (SEA) peptides. The usefulness of a selected
selenol compound is illustrated by the total synthesis of a biologically active human chemotactic
protein, which plays an important role in innate and adaptive immunity.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Kerdraon, F.; Bogard, G.;
Snella, B.; Drobecq, H.; Pichavant, M.;
Agouridas, V.; Melnyk, O. Insights
into the Mechanism and Catalysis of
Peptide Thioester Synthesis by
Keywords: peptide thioester; alkylselenol; catalysis; chemical protein synthesis
Alkylselenols Provide a New Tool for
Chemical Protein Synthesis. Molecules
2021, 26, 1386. https://doi.org/
10.3390/molecules26051386
1. Introduction
Chemical protein synthesis is now established as an alternative mode of production
of tailored proteins for use in diverse fields of research, primarily biological and medicinal
research [
frequent application of the native chemical ligation (NCL [
ization methods [ 10] to the chemoselective formation of peptide junctions to cysteine or
1]. Thioester chemistry is central to the field of chemical protein synthesis by the
2–6
], Figure 1A) and desulfur-
Academic Editor: Luca D. D’Andrea
7
–
alanine in water using unprotected peptide segments as reactants [11].
Besides the assembly of the protein itself and the acceleration of peptide bond forma-
tion [12–16], one limitation of chemical protein synthesis using NCL is the production of the
Received: 11 February 2021
Accepted: 27 February 2021
Published: 4 March 2021
peptide thioester segments [17]. Therefore, facilitating the access to the peptide thioesters
using Fmoc-SPPS has been intensively pursued, especially during the last decade, and the
progress made in this area explains in part the rapid increase in the number of protein
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published maps and institutional affil-
iations.
targets assembled since 2010 [
progress of chemical protein synthesis exploits the capacity of N-(2-mercaptoethyl)amides,
i.e., N,S-acyl shift systems [18 36], to rearrange into thioesters in aqueous media. Several
1,11]. One corpus of methods that has contributed to the
–
of these systems have been validated by the synthesis of challenging proteins [37–44].
One popular mode of use of N,S-acyl shift systems is the production of alkylthioesters
by exchanging the N-(2-mercaptoethyl)amine unit by a thiol used in excess, i.e., R0SH
in Figure 1B. Such exchange reactions are equilibrated, the equilibrium being displaced
toward the thioester form at acidic pH probably due to the masking of the departing
N-(2-mercaptoethyl)amine by protonation [45]. Detailed mechanistic studies performed on
the bis(2-sulfanylethyl)amido (SEA) system established that in such conditions the thiol–
thioester exchanges are rate limiting. This is because thiol–thioester exchanges proceed
preferably through thiolate species [46], while the attacking thiol R0SH in the process
shown in Figure 1B is mostly in its neutral form at the acidic pH used for the exchange
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Licensee MDPI, Basel, Switzerland.
This article is an open access article
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Molecules 2021, 26, 1386
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reaction, typically pH 4 [47]. These mechanistic findings led to the conclusion that the
production of peptide thioesters from SEA peptides might be facilitated by catalyzing
the thiol/thioester exchange step shown in Figure 1B. Indeed, efficient catalysis could
be achieved by using diselenol catalyst 8a obtained by in situ reduction of diselenide 7a
(R00 = alkyl group, Figure 1D), which in practice was found to be superior to other classical
thiol or selenol additives for promoting the production of peptide thioesters from SEA
peptides at pH 4. The design of catalyst 8a has been inspired by the observation that the
simpler diselenol 8b (R00 = H, Figure 1D) was able to catalyze the SEA/thiol exchange
reaction, albeit with byproduct formation due to the presence in 8b of a nucleophilic
secondary amine, hence the introduction of a blocking alkyl group on the nitrogen.
Figure 1.
(A) Principle of the native chemical ligation (NCL). (B) N,S-acyl shift amide systems
rearrange into thioesters in aqueous solution and can be used for producing stable thioesters by
exchange with a thiol. (C) The bis(2-sulfanylethyl)amide (SEA) system. (D) Selenol catalysts reported
by Cargoët et al. [47].
The diverse kinetic data collected so far support that the catalysis of the SEA/thiol
exchange reaction by selenols proceeds as shown in Figure 2 [45 47]. According to this
mechanism, the SEA peptide undergoes a spontaneous N,S-acyl shift to produce the
,
9
transient SEA thioester 10. The latter reacts with the selenol catalyst probably through
selenoate species to produce selenoester 12, which then undergoes an exchange reaction
with the thiol additive used in excess. The catalytic potency of selenols stems from their
low pKa compared to thiols, which ensures that a significant fraction of the selenol is in the
form of a nucleophilic selenoate at the working acidic pH for the reaction, and also from
the strong acylating properties of selenoesters of type 12 [13,16].

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Figure 2. Mechanism of the SEA/thiol exchange catalysis by selenols.
The ease of synthesis, the catalytic potency and the water solubility are certainly
key points to consider in the design of catalysts acting on unprotected peptide segments.
Diselenol catalysts of type 8a are efficient and water soluble but their synthesis is complex
and low yielding. This latter limitation prompted us to search for novel selenol catalysts
whose production would be easier. Evolving catalyst 8a structure into easier to make selenol
catalysts while maintaining similar catalytic potencies is not obvious. This is because the
importance of the bis selenol and tertiary amine functionalities in the catalysis by 8a
remains unknown. In addition, the consequences of changing catalyst structure on selenol
nucleophilicity and pKa are hard to predict.
In this work, we evaluated the interest of selenol compounds 13 and 14 for cat-
alyzing the SEA/thiol exchange process (Figure 3). These compounds, which share the
2-(selanylethyl)amino motif with original catalyst 8a, have been studied in the past as
analogs of choline [48–50]. They have the advantage of being easily produced in multigram
quantities from cheap starting materials. Detailed kinetic studies showed the capacity
of selenol 13 to act as a substitute of diselenol 8a for the synthesis of peptide thioesters.
Its usefulness is illustrated by the first chemical synthesis of a biologically active 9 kDa
granulysin protein, an antibacterial human protein secreted by cytotoxic cells in response
to various infectious agents [51–53].
Figure 3. Selenol catalysts studied in this work.
2. Results and Discussion
2.1. Catalyst Synthesis
Catalysts 13 and 14 were produced as their corresponding diselenides, i.e., diselenides
17 and 18 respectively, starting from 2-chloro-N,N-dimethylethylamine hydrochloride
according to known procedures [50,54]. Briefly, 2-chloro-N,N-dimethylethylamine was
neutralized with potassium carbonate and then reacted with potassium selenocyanate
to produce N,N-dimethyl-2-selenocyanatoethan-1-amine 16, which upon treatment with
sodium hydroxide afforded diselenide 17 (Scheme 1). Diselenide 17 was easily alkylated
with methyl iodide to produce diselenide 18.

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Scheme 1. Synthesis of diselenide precatalysts 17 and 18.
2.2. Kinetic Studies
The SEA/thiol exchange reaction used for studying the potency of the different
catalysts is shown in Scheme 2. The water soluble tris(2-carboxyethyl)phosphine (TCEP)
enables an in situ reduction of the cyclic SEAoff disulfide 19 and of the diselenide precatalyst
17 or 18. The thiol additive used in this reaction is 3-mercaptopropionic acid (MPA), which
is a classical thiol used for preparing peptide thioesters.
Scheme 2. The bis(2-sulfanylethyl)amido (SEA)/thiol exchange reaction investigated by kinetic studies.
The exchange reactions were monitored by HPLC (Figure 4A). The kinetic data were
fitted to extract the apparent second order rate constants (Figure 4B). In this figure and in
the following paragraphs, the data are expressed as a function of total selenol concentration.
As suggested by the experimental data shown in Figure 4B, the rate constants obtained for
13 and 8a, which are formed by in situ reduction of diselenides 17 and 7a, plateau at the
same value above 50 mM. However, examination of Figure 4B or of Table 1, which gives
the half-reaction times for the different experiments, shows that the potencies of catalysts
13 and 8a are not the same for concentrations below 50 mM. In such a case, the SEA/thiol
exchange reaction proceeds faster with catalyst 8a. The difference is particularly striking
by looking at the data obtained at the lowest catalyst concentration tested (compare in the
insert of Figure 4A blue circles and triangles for 13 and 8a respectively used at 6.25 mM
concentration). We can conclude at this point that catalyst 8a can be substituted by selenol
13 with the condition of being used at a minimal concentration of 50 mM, under which
conditions the highest rate for the SEA/thiol exchange reaction can be achieved.

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Figure 4. Study of the SEA/thiol exchange reaction catalyzed by selenols 8a or 13. Conditions:
◦
SEA peptide = 1 mM, 37 C under inert atmosphere, 6 M Gn
·
HCl, MPA 5% v/v, 100 mM tris(2-
) Experimental data as obtained by
) Apparent second-order rate constants obtained by fitting experimental data.
carboxyethyl)phosphine hydrochloride (TCEP
HPLC monitoring. (
·
HCl), pH 4.0. (A
B
The error bars correspond to the standard error of the fit. (C) Kinetic profile in the presence of catalyst
14 (200 mM).
The ability to accelerate the SEA/MPA exchange reaction was also assessed according
to the same conditions for selenol 14 obtained from in situ reduction of 18. Whether at a

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concentration of 200 mM, 100 mM or 50 mM, selenol 14 was found to be significantly less
potent than catalysts 8a and 13 (Table 1). We noticed the formation of a red solid in the
reaction mixture after a few hours at the highest concentration tested (200 mM), probably
elemental selenium (see Supplementary Information, Figure S17). This observation strongly
suggests that catalyst 14 decomposes throughout the course of the reaction. We also noticed
an increase of the pH of the reaction mixture, a phenomenon that was not observed with
the other selenols. Since previous studies established that the SEA/thioester ratio at
equilibrium increases with pH [45,47], the pH drift observed in these experiments might
explain in part the diminished conversion observed with catalyst 14. Taken together, the
lower conversion and rates observed with catalyst 14 preclude its use as an interesting
alternative to 8a for the production of peptide thioesters from SEA peptides.
Table 1. Catalysis of bis(2-sulfanylethyl)amido (SEA)–thiol exchange using selenols 8a, 13 or 14 a,b
.
Catalyst t1/2 (h)
13
Total Selenol Concentration
8a, 13, 14 (mM)
Entry
8a c
14 d
1
2
3
4
5
6
200
100
50
25
12.5
6.25
1.97
2.03
1.95
2.35
3.00
3.35
1.68
2.08
2.22
3.10
4.60
5.87
2.51
2.87
3.60
nd
nd
nd
Conditions: SEA peptide = 1 mM, 37 ◦C under inert atmosphere, 6 M Gn
·
HCl, MPA 5% v/v, 100 mM tris(2-
a
carboxyethyl)phosphine hydrochloride (TCEP
·
HCl), pH 4.0. Selenol catalysts were added in the form of the
corresponding diselenides. The half-reaction time (t 12 ) for the uncatalyzed reaction is 7.28 h [47]. Data from
b
c
d
ref [47]. Measured pH at equilibrium: pH 4.45 at 200 mM 14, pH 4.19 at 100 mM 14, pH 4.14 at 50 mM 14.
Another striking observation made during the HPLC-MS monitoring of the exchange
process catalyzed by selenol 14 was the formation of the peptide selenoester derived from
selenocholine in substantial amounts (Figure 4C). In contrast, the amount of selenoester
formed during the SEA/thiol exchange catalyzed by selenols 8a or 13 was below HPLC-MS
detection limits. The persistence of the selenoester derived from 14 in the reaction mixture
suggests that the quaternarization of the basic nitrogen significantly alters the reactivity
of the carbonyl group nearby. This observation is reminiscent of the work of Mautner
and coworkers, who noticed that the hydrolysis of 2-dimethylaminoethyl selenobenzoate
proceeded significantly faster at pH 7 than hydrolysis of benzoylselenocholine. The authors
proposed that the formation of an intramolecular hydrogen bond between the protonated
secondary amine and the selenoester carbonyl oxygen might promote the hydrolysis of the
selenoester carbonyl [49]. Such an intramolecular activation of the selenoester group by
the protonated amine as illustrated in Figure 5 might contribute to the catalytic potency of
selenols 8a and 13.
Figure 5. The presence of a protonated amine nearby the peptide carbonyl might promote the attack
of thiol nucleophiles.

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2.3. Total Chemical Synthesis of 9 kDa Granulysin
To illustrate the usefulness of the new catalyst 13, we undertook the total chemical
synthesis of 9 kDa granulysin (9-GN, Figure 6A). 9-GN is a human cytotoxic and proin-
flammatory protein that belongs to the NK-lysin family of proteins [53,55,56]. The protein
is stabilized by two disulfide bonds. 9-GN protein is secreted by specialized cells from the
immune system in response to infection by various agents such as bacteria, parasites, fungi
and viruses. Designing an efficient and modular synthetic approach toward 9-GN can
facilitate the development of novel antibacterial proteins of potential therapeutic interest.
Figure 6.
(A) Primary structure of 9 kDa granulysin (9-GN) and pattern of disulfide bonds. (B) General strategy used for
assembling the linear 9-GN polypeptide (9-GN-l).
The synthetic strategy that was followed for accessing the linear 9-GN polypeptide
(
9-GN-l) is described in Figure 6B. 9-GN-l polypeptide was assembled from three peptide
segments using NCL, which are highlighted by orange (N-terminal), blue (middle) and
green (C-terminal) colors in Figure 6. The preceding residue of all the cysteines present
in 9-GN sequence are β-branched amino acid residues, meaning that regardless of the
AA-Cys junctions chosen for 9-GN assembly, the formation of difficult peptidic junctions
will have to be dealt with. The assembly was done in the C-to-N direction and therefore
required the protection of the N-terminal cysteine residue of the middle segment to avoid
its cyclization or oligomerization during the first ligation step. The acetoacetyl (AcA) group
was chosen for this purpose due to its ease of installation and removal [37–39,57].

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The preparation of the middle thioester segment 21 in blue in Figure 6B proved to
be challenging. First, the classical Fmoc SPPS of the SEA peptide precursor resulted in
low yields due to difficult couplings after Arg94. This problem was solved by introducing
Ala92 and Thr93 as a pseudoproline dipeptide unit, i.e., Fmoc-Ala-Thr(Ψ(Me,Me)Pro-OH
(Figure 7) [58]. The second issue was the sluggish SEA/MPA exchange process enabling
the installation of the thioester group, due to the presence of a sterically demanding
valine residue at the C-terminus. Catalyst 13 proved to be particularly useful in this case.
Interestingly, the protection of the N-terminal cysteine residue using methyl-S-(acetoacetyl)-
thioglycolate (AcA-MTG) in the presence of 4-mercaptophenylacetic acid (MPAA) and the
diselenide-catalyzed SEA/MPA exchange reaction could be performed in one-pot with a
pH change in between, thereby saving one intermediate purification step.
Figure 7. Synthesis of the middle thioester segment 21 by alkyl selenol-catalyzed SEA/MPA exchange.
The successful synthesis of thioester segment 21 set the stage for the assembly of the
linear 9-GN-l polypeptide (Figure 8). The ligation of middle 21 and right 22 segments
was followed by the removal of the AcA group in one pot by adding hydroxylamine
hydrochloride to the reaction mixture and adjusting the pH to mildly acidic values. The
second ligation step with peptide thioester 24 provided the full length 9-GN-l polypeptide
which was purified by HPLC.

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Figure 8. Total synthesis of 9-GN (the structure of 9-GN shown in this figure was prepared using
Pymol and pdb entry 1L9L [59]).
◦
The folding step was performed at 20 C in a phosphate buffered saline/glycerol
9/1 mixture in the presence of glutathione (1 mM)/glutathione disulfide (0.2 mM) redox
system and guanidine hydrochloride as a solubilizing additive. The use of guanidine
hydrochloride during this step was mandatory to enable the solubilisation of the linear
9-GN-l polypeptide. The formation of the native pattern of disulfide bonds as shown
in Figure 6A has been demonstrated by identifying the peptides produced upon trypsin
digestion under non-reducing conditions by mass spectrometry (see Supplementary Infor-
mation). The UPLC-MS analysis of 9-GN shown in Figure 9 highlights the quality of the
protein obtained by the designed synthetic route.

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Figure 9. UPLC-MS analysis of synthetic 9-GN. UV detection at 215 nm. M (average mass) calcd.
8431.79, obs. 8431.9. 9-GN is a cationic protein that shows several TFA adducts in its deconvoluted
MS spectrum. The structure of 9-GN shown in this figure was prepared using PyMol and pdb
entry 1L9L [59].
2.4. Biological Activity of Synthetic 9-GN
The successful synthesis and folding of 9-GN set the stage for examining its biological
activity. One hallmark of 9-GN is its capacity to induce the migration of immune cells to the
site of infection. Typically, 9-GN is a chemoattractant for monocytes in the low nanomolar
range in a transwell migration assay performed in Boyden micro chambers.
In such an assay, monocytes are placed on the upper layer of a cell permeable mem-
brane inserted in a multi-well plate, which separates the cells from the solution containing
the chemoattractant. After incubation (1 h), the cells that have migrated through the mem-
brane are stained and counted. In such an assay, the synthetic 9-GN protein displayed a
strong migration index in the 1–100 nM range as expected from literature data (Figure 10),
showing the biological functionality of the protein obtained by chemical synthesis [56].
Figure 10. Monocyte migration assay in Boyden micro chambers. The data are expressed relative
to the cell migration observed in the Roswell Park Memorial Institute medium (RPMI 1640) sup-
plemented with 0.1% fetal calf serum. Monocyte chemotactic protein-1 (MCP-1, also called CCL2,
23 nM) was used as a positive control. The data for 0 nM of 9-GN were obtained using the protein
dialysis buffer (PBS containing 10% by vol of glycerol). The bars correspond to the mean for six
donors, from whom monocytes were isolated. The error bar correspond to the standard error of the
mean. The data for each donor can be found in the Supplementary Information.

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3. Materials and Methods
3.1. Synthesis of Diselenide Precatalyst 17
To a solution of potassium carbonate (11.5 g, 0.083 mol) in water (30 mL), was added
2-chloroethyl-N,N-dimethylamine hydrochloride (6.0 g, 0.042 mol). The resulting mixture
was stirred at room temperature for 10 min. The resulting crude mixture was extracted
3 times with 30 mL of Et2O, dried over MgSO4 and concentrated under reduced pressure.
1.0 g of the resulting pale yellow oil (9.3 mmol) was diluted in 90 mL of acetonitrile and
potassium selenocyanate (1.47 g, 10.2 mmol) was added portionwise. The resulting solution
was stirred at room temperature for 16 h. Et2O (100 mL) was added to the reaction mixture
which was then filtered through a pad of celite. The filtrate was evaporated in vaccuo
and then redissolved in 120 mL of absolute ethanol. Aqueous sodium hydroxide (60 mL,
1 M) was slowly added under stirring and the reaction mixture was stirred 3 h at room
temperature. The resulting mixture was concentrated in vacuo to remove the maximum
of ethanol and extracted Et2O (3
MgSO4 and concentrated under reduced pressure to afford diselenide 17 as a yellow oil
×
30 mL). The organic layers were combined, dried over
(1.24 g, 4.09 mmol, 88%).
1H NMR (300 MHz, CDCl3)
δ (ppm) 3.10–2.96 (m, 4H), 2.68–2.55 (m, 4H), 2.25 (s, 12H)
(see Figure S12).
13C NMR (75 MHz, CDCl3) δ (ppm) 60.32, 45.20, 28.20 (see Figure S13).
MALDI-TOF Matrix 2,5-dihydrobenzoic acid, positive detection mode, calcd. for
[M+H]+ (exact mass): 305.00, found: 304.92.
3.2. Synthesis of 9-GN
Step 1. Synthesis of peptide thioester 21
To a solution of Gn
MPAA (17 mg, 50 mM). SEAoff peptide 26 (15 mg, 2.6
(77 L of a 0.10 M solution in ACN, 7.7 mol, 3.0 equiv) were then successively dissolved
·
HCl (1.1 g) in 0.1 M pH 7.4 phosphate buffer (1.2 mL) was added
µmol, 1.0 equiv) and AcA-MTG
µ
µ
in the MPAA solution (1.3 mL) and the pH of th◦e mixture was adjusted to 7.2 by addition
of 6 M NaOH. The mixture was stirred at 37 C for 8 h. 3.0 additional equivalents of
◦
AcA-MTG were added and the mixture was stirred an additional 16 h at 37 C under
nitrogen atmosphere. Completion of the reaction was ascertained by UPLC-MS analysis.
After protection of the N-terminal Cys residue (24 h), the subsequent SEA/MPA
exchange reaction was carried out in one pot. TCEP
·
HCl (34 mg, 0.10 M), diselenide
catalyst 17 (20 mg, 50 mM) and MPA (65 L, 5% by vol) were added to the previous
µ
mixture. The pH was adjusted to 4.0 by addition of 6 M NaOH and the reaction mixture
was stirred at 37 ◦C for 72 h. After completion of the reaction, the mixture was diluted in
10 mL of water containing 0.1% TFA. The crude was purified by HPLC (eluent A = water
containing 0.1% TFA, eluent B = acetonitrile in water 4/1 v/v containing 0.1% TFA, 50 ◦C,
detection at 215 nm, 6 mL min−1, 0–37% eluent B in 15 min, then 37–57% eluent B in 60 min,
C18XBridge column) to give peptide thioester 21 as a white solid after lyophilization
(5.09 mg, 34%).
Peptide 21 was analyzed by UPLC-MS (see Figure S18) and MALDI-TOF mass spec-
trometry (see Figure S19).
ESI (positive detection mode, see Figure S18): calcd. for [M] (average): 4561.40,
found: 4561.79.
MALDI-TOF (positive detection mode, matrix 2,5-dihydrobenzoic acid, see Figure S19):
calcd. for [M + H]+ (average): 4562.40, found: 4562.87.
Step 2. Synthesis of peptide amide 23
To a solution of Gn
added MPAA (34 mg, 0.20 mmol) and the pH of the mixture was adjusted to 7.2 by addition
of 6 M NaOH. Peptide 21 (3.5 mg, 0.61 mol, 1.0 equiv) and peptide 22 (2.8 mg, 0.67 mol,
·
HCl (0.57 g) in 0.1 M, pH 7.4 phosphate buffer (0.60 mL) was
µ
µ
1.1 equiv) were then successively dissolved in the MPAA solution (0.12 mL) and the pH
of the mixture was adjusted to 7.2 by addition of 6 M NaOH. The mixture was stirred at

Molecules 2021, 26, 1386
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◦
37 C for 24 h under nitrogen atmosphere. Completion of the reaction was insured by
UPLC-MS analysis.
After completion of the NCL ligation (24 h), the AcA protecting group was removed
by reaction with NH2OH. The reaction mixture was first acidified to pH 3.0 with 6 M
HCl and extracted 3 times with 1.0 mL of Et2O to remove MPAA. A 0.5 M solution of
NH2OH
·
HCl in H2O (26
µ
L,◦13
µmol, 20 equiv) was then added and the pH was adjusted
to 5.5. After 4 h stirring at 37 C, pH was adjusted to 3.0 with 0.25 g mL−1 aqueous solution
of TCEP HCl. The crude was further diluted in 4.0 mL of water containing 0.25% TFA and
·
purified by HPLC (eluent A = water containing 0.25% TFA, eluent B = acetonitrile in water
4/1 v/v containing 0.25% TFA, 50◦C, detection at 215 nm, 6 mL min−1, 0–40% eluent B
in 10 min, then 40–50% eluent B in 45 min, C18XBridge column) to afford peptide 23 as a
white solid after lyophilization (3.49 mg, 56%).
Peptide 23 was analyzed by UPLC-MS (see Figure S20) and MALDI-TOF mass spec-
trometry (see Figure S21).
ESI (positive detection mode, see Figure S20): calcd. for [M] (average): 7899.22,
found: 7899.94.
MALDI-TOF (positive detection mode, matrix 2,5-dihydrobenzoic acid, see Figure S21)
calcd. for [M+H]+ (average): 7900.22, found: 7901.49.
Step 3. Synthesis of 9-GN-l
:
To a solution of Gn
MPAA (34 mg, 0.20 mmol) and the pH of the mixture was adjusted to 7.7 by addition of
6 M NaOH. Peptide 23 (3.5 mg, 0.36 mol, 1.0 equiv) and peptide thioester 24 (0.38 mg,
0.36 mol, 1.0 equiv) were then successively dissolved in the MPAA solution (91 L) The
·
HCl (0.57 g) in 0.1 M pH 7.4 phosphate buffer (0.60 mL) was added
µ
µ
µ
◦
mixture was stirred at 37 C for 24 h under nitrogen atmosphere. Completion of the
reaction was insured by UPLC-MS analysis.
After completion of the NCL ligation (24 h), TCEP HCl (5.2 mg, 18 µmol) was added to
·
the reaction medium to reduce any mixed disulfide formed during ligation. The resulting
mixture was diluted in 3.0 mL of water containing 0.1% TFA. The crude was purified
by HPLC (eluent A = water containing 0.1% TFA, eluent B=acetonitrile in water 4/1 v/v
containing 0.1% TFA, 50 ◦C, detection at 215 nm, 6 mL min−1, 0–38% eluent B in 10 min,
then 38–58% eluent B in 60 min, C18XBridge column) to give 9-GN-l as a white solid after
lyophilization (2.92 mg, 78%).
Peptide 9-GN-l was analyzed by UPLC-MS (see Figure S22) and MALDI-TOF mass
spectrometry (see Figure S23).
ESI (positive detection mode, see Figure S22): calcd. for [M] (average): 8435.79,
found: 8435.45.
MALDI-TOF (positive detection mode, matrix 2,5-dihydrobenzoic acid, see Figure S23)
:
calcd. for [M + H]+ (average): 8436.79, found: 8436.65.
3.3. Folding
The linear polypeptide 9-GN-l (0.558 mg, 0.054
µmol) was dissolved in PBS containing
10% by vol of glycerol, 2 M of Gn HCl, 1 mM reduced glutathione, 0.2 mM oxidized
·
glutathione (1 mL/mg final peptide concentration). The reaction mixture was let 1 h at
0 ◦C, then 8 days at 20 ◦C.
The protein was dialyzed twice (respectively 2 h and 24 h) at 20 ◦C against 200 mL
PBS containing 10% in vol of glycerol. 0.71 mL of the dialyzed solution were recovered and
the concentration of the folded protein was determined using UV quantification at 280 nm
(60.7 µM, 0.043 µmol, 80%)
Folded protein 9-GN was analyzed by UPLC-MS (see Figure S24).
Experimental determination of the disulfide bridge pattern was achieved by trypsin
digestion of 9-GN and identification of the resulting fragments by mass spectrometry using
non-reducing conditions. Fragments obtained by digestion with trypsin permitted the
direct assignment of Cys69-Cys132 and Cys96-Cys107 disulfide bonds (see Figure S26 in
the Supplementary Information).

Molecules 2021, 26, 1386
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3.4. Chimiotaxis Assay
Blood monocytes were purified from six donors by positive selection over a MACS col-
umn using anti-CD14-conjugated microbeads (Miltenyi Biotec) as previously described [60].
Monocyte migration (5
FCS) was performed in a 48-well Boyden microchamber (Neuroprobe) through a standard
5-
m pore filter (Neuroprobe), at 37 ◦C for 1 h. Migration was evaluated in response to
×
104 cells/well in 50
µL of RPMI 1640 medium with 0.1%
µ
synthetic 9-GN (1 to 100 nM in RPMI 1640 medium containing 0.1% FCS), CCL2 used as
positive control (200 ng/mL, 23.1 nM, R&D Systems), and medium alone used as negative
control. The filter was stained using Diff–Quik reagent. Migrated cells were counted on the
lower side of the filter in three randomly selected high power fields (magnification
Each assay was performed in triplicate. Results are expressed as the difference between
mean numbers of cells per high power fields minus the negative control (medium alone).
×
100).
4. Conclusions
We conducted detailed kinetic studies of the catalysis of the bis(2-sulfanylethyl)amido
(SEA)/thiol exchange process by selenols derived from selenocysteamine. These data pro-
vide important clues regarding the role played by the basic amino group of the selenocys-
teamine scaffold in catalysis and how to evolve the catalyst to achieve useful catalytic poten-
cies while minimizing synthetic cost. Catalysis by bis(N,N-dimethylaminoethyl)diselenide
proved particularly useful and involves presumably an intramolecular general acid cataly-
sis by the proton of the dimethylammonium group. Bis(N,N-dimethylaminoethyl)diselenide
can be easily produced in multigram scale from cheap starting materials. Its usefulness is
illustrated by the chemical synthesis of a biologically actice 9 kDa granulysin.
Supplementary Materials: The detailed experimental procedures and characterization data for all
compounds are provided in a separate PDF file, Figure S1: General approach for the preparation
of peptide amides, Figure S2: Synthesis of SEAoff peptides and MPA peptide thioesters, Figure S3:
UPLC-MS analysis of CRNFMRRYQSRVTQGLVAGETAQQICEDLR-NH2 peptide 22. LC trace:
eluent A 0.10% TFA in water, eluent B 0.10% TFA in CH3CN. C18 column, gradient 0–70% B in
20 min, 50 ◦C, 0.4 mL min-1, UV detection at 215 nm. MS trace: m/z 1765.33 ([M+2H]2+), 1177.33
([M+3H]3+), 883.33 ([M+4H]4+), 706.75 ([M+5H]5+); calcd. for [M] (average mass): 3529.04, found:
3529.04, Figure S4: MALDI-TOF analysis of peptide 22. Matrix 2,5-dihydrobenzoic acid, positive
detection mode, calcd. for [M+H]+ (average): 3530.04, found: 3529.8, Figure S5: UPLC-MS analysis of
C(StBu)LTIVQKLKKMVDKPTQRSVSNAATRVC(StBu)-RTGRSRWRDV-SEAoff peptide 26. LC trace:
eluent A 0.10% TFA in water, eluent B 0.10% TFA in CH3CN. C18 column, gradient 0–70% B in 20 min,
◦
50 C, 0.4 mL min-1, UV detection at 215 nm. MS trace: m/z 1636.25 ([M+2MTFA+3H]3+), 1598.92
([M+MTFA+3H]3+), 1199.42 ([M+MTFA+4H]4+), 1171.75 ([M+4H]4+), 959.92 ([M+MTFA+5H]5+),
937.58 ([M+5H]5+), 781.67 ([M+6H]6+), 670.67 ([M+7H]7+); calcd. for [M] (average mass): 4682.77,
found: 4682.95, Figure S6: MALDI-TOF analysis of peptide 26. Matrix 2,5-dihydrobenzoic acid,
positive detection mode, calcd. for [M+H]+ (average): 4683.77, found: 4684.22, Figure S7: UPLC-MS
analysis of DYRT-SEAoff peptide. LC trace: eluent A 0.10% TFA in water, eluent B 0.10% TFA in
◦
CH3CN. C18 column, gradient 0–70% B in 20 min, 50 C, 0.4 mL min-1, UV detection at 215 nm. MS
trace: m/z 671.42 ([M+H]+); calcd. for [M+H]+ (monoisotopic): 671.26, found: 671.42, Figure S8:
MALDI-TOF analysis of DYRT-SEAoff peptide. Matrix 2,5-dihydrobenzoic acid, positive detection
mode, calcd. for [M+H]+ (monoisotopic): 671.26, found: 671.15, Figure S9: UPLC-MS analysis of
DYRT-MPA peptide 24. LC trace: eluent A 0.10% TFA in water, eluent B 0.10% TFA in CH3CN.
C18 column, gradient 0–70% B in 20 min, 50 ◦C, 0.4 mL min-1, UV detection at 215 nm. MS trace:
m/z 1283.17 ([2M+H]+), 642.42 ([M+H]+); calcd. for [M+H]+ (monoisotopic): 642.25, found: 642.42,
Figure S10: MALDI-TOF analysis of peptide 24. Matrix 2,5-dihydrobenzoic acid, positive detection
mode, calcd. for [M+H]+ (monoisotopic): 642.25, found: 642.18, Figure S11: Synthesis scheme for the
preparation of diselenide precatalyst 17, Figure S12: 1H NMR (300 MHz) spectrum of compound
17 (CDCl3, 293 K), Figure S13: 13C APT NMR (75 MHz) spectrum of compound 17 (CDCl3, 293 K),
Figure S14: 1H NMR (300 MHz) spectrum of compound 18 (D2O, 293 K), Table S1: Kinetic data
extracted from curve fitting.a, Table S2: Fitting of MPA thioester formation in the presence of
catalysts.a, Figure S16: Fitting of SEA/MPA exchange in the presence of catalyst 13 (200 mM). Data
were fitted according to equation (1), Figure S17: Red selenium precipitate formed during catalysis

Molecules 2021, 26, 1386
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of SEA/MPA exchange in the presence of catalyst 14 (200 mM), Figure S18: UPLC-MS analysis of
peptide 21. LC trace: eluent A 0.10% TFA in water, eluent B 0.10% TFA in CH3CN. C18 column,
gradient 0–70% B in 20 min, 50 ◦C, 0.4 mL min-1, UV detection at 215 nm. MS trace: m/z 1558.25
([M+MTFA+3H]3+), 1521.42 ([M+3H]3+), 1169.00 ([M+MTFA+4H]4+), 1141.58 ([M+4H]4+), 913.58
([M+5H]5+), 761.58 ([M+6H]6+), 652.83 ([M+7H]7+); calcd. for [M] (average mass): 4561.40, found:
4561.79, Figure S19: MALDI-TOF analysis of peptide 21. Matrix 2,5-dihydrobenzoic acid, positive
detection mode, calcd. for [M+H]+ (average): 4562.40, found: 4562.87, Figure S20: UPLC-MS analysis
of peptide 23. LC trace: eluent A◦0.10% TFA in water, eluent B 0.10% TFA in CH3CN. C18 column,
gradient 0–70% B in 20 min, 50 C, 0.4 mL min-1, UV detection at 215 nm. MS trace: calcd. for
[M] (average mass): 7899.22, found: 7899.94 (8012.58 [M+MTFA], 8124.97 [M+2MTFA], 8238.28
[M+3MTFA]), Figure S21: MALDI-TOF analysis of peptide 23. Matrix 2,5-dihydrobenzoic acid,
positive detection mode, calcd. for [M+H]+ (average): 7900.22, found: 7901.49, Figure S22: UPLC-MS
analysis of peptide 9-GN-l. LC trace: eluent A 0.10% TFA in water, eluent B 0.10% TFA in CH3CN. C18
◦
column, gradient 0–70% B in 20 min, 50 C, 0.4 mL min-1, UV detection at 215 nm. MS trace: calcd. for
[M] (average mass): 8435.79, found: 8435.45 (8547.50 [M+MTFA], 8659.48 [M+2MTFA]), Figure S23:
MALDI-TOF analysis of peptide 9-GN-l. Matrix 2,5-dihydrobenzoic acid, positive detection mode,
calcd. for [M+H]+ (average): 8436.79, found: 8436.65, Figure S24: UPLC-MS analysis of 9-GN. LC
trace: eluent A 0.10% TFA in water, eluent B 0.10% TFA in CH3CN. C18 column, gradient 0–70% B
in 20 min, 50 ◦C, 0.4 mL min-1, UV detection at 215 nm. MS trace: calcd. for [M] (average mass):
8431.79, found: 8431.92 (8543.73 [M+MTFA], 8654.61 [M+2MTFA], 8770.43 [M+3MTFA], 8882.98
[M+4MTFA]), Figure S25: Disulfide bridge pattern of 9-GN, Figure S26: MALDI-TOF analysis of the
trypsic digest of 9-GN. Matrix 2,5-dihydroxybenzoic acid (DHB), positive detection mode.VC96R
linked to DVC107R by disulfide bridge, calcd. (monoisotopic) [M+H+] 866.40, found 866.39; VC96R
linked to WRDVC107R by disulfide bridge, calcd. (monoisotopic) [M+H+] 1208.58, found 1208.57;
TC69LTIVQK linked to VTQGLVAGETAQQIC132EDLR by disulfide bridge, calcd. (monoisotopic)
[M+H+] 2932.52, found 2932.49; DYRTC69LTIVQK linked to VTQGLVAGETAQQIC132EDLR by
disulfide bridge, calcd. (monoisotopic) [M+H+] 3366.71, found 3366.73, Table S3: Results of the
Boyden micro chamber assay.
Author Contributions: Catalyst synthesis and kinetic measurements, 9-GN total synthesis, writing—
review, F.K.; chemotactic assays, G.B.; Catalyst synthesis and kinetic measurements, B.S.; Proteomic
experiments, H.D.; Chemotactic assays, writing—review, M.P.; Conceptualization, project administra-
tion, supervision, methodology, kinetic modelizations, writing—review & editing, V.A.; Conceptu-
alization, project administration, supervision, methodology, writing—review & editing, O.M. All
authors have read and agreed to the published version of the manuscript.
Funding: This work was supported by the Centre National de la Recherche Scientifique (CNRS), the
University of Lille, the Pasteur Institute of Lille and by Agence Nationale de la Recherche (ANR)
grant ANR-18-CE44-0010.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
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