Catalysis of Thiol-Thioester Exchange by Water-Soluble Alkyldiselenols Applied to the Synthesis of Peptide Thioesters and SEA-Mediated Ligation

Article abstract of DOI:10.1021/acs.joc.8b01903

N-Alkyl bis(2-selanylethyl)amines catalyze the synthesis of peptide thioesters or peptide ligation from bis(2-sulfanylethyl)amido (SEA) peptides. These catalysts are generated in situ by reduction of the corresponding cyclic diselenides by tris(2-carboxye

Full text of DOI:10.1021/acs.joc.8b01903

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Article
Catalysis of thiol-thioester exchange by water soluble alkyldiselenols
applied to the synthesis of peptide thioesters and SEA-mediated ligation
Marine Cargoët, Vincent Diemer, Benoit Snella, Rémi Desmet, Annick
Blanpain, Hervé Drobecq, Vangelis Agouridas, and Oleg Melnyk
J. Org. Chem., Just Accepted Manuscript • Publication Date (Web): 19 Sep 2018
Downloaded from http://pubs.acs.org on September 19, 2018
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Catalysis of thiol-thioester exchange by water soluble alkyldiselenols
applied to the synthesis of peptide thioesters and SEA-mediated liga-
tion
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Marine Cargoët, Vincent Diemer, Benoît Snella, Rémi Desmet, Annick Blanpain, Hervé Drobecq,
Vangelis Agouridas,* Oleg Melnyk*
Université de Lille, Institut Pasteur de Lille, UMR CNRS 8204, 1 rue du Pr Calmette, 59021 Lille,
France.
Corresponding authors:
oleg.melnyk@ibl.cnrs.fr
vangelis.agouridas@ibl.cnrs.fr
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Abstract
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N-Alkyl-bis(2-selenoethyl)amines catalyze the synthesis of peptide thioesters or peptide ligation
from bis(2-sulfanylethyl)amido (SEA) peptides. These catalysts are generated in situ by reduction
of the corresponding cyclic diselenides by tris(2-carboxyethyl)phosphine (TCEP). They are par-
ticularly efficient at pH 4.0 by accelerating the thiol-thioester exchange processes, which are oth-
erwise rate-limiting at this pH. By promoting SEA-mediated reactions at mildly acidic pH, they
facilitate the synthesis of complex peptides such as cyclic O-acyl isopeptides that are otherwise
hardly accessible.
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Introduction
The native chemical ligation (NCL1ꢀ2) is a powerful chemoselective peptide bond forming reacꢀ
tion that is intensively used for protein chemical synthesis (Scheme 1A).3 NCL consists in reꢀ
acting a peptide thioester with a cysteinyl peptide to produce a native peptide bond to cysteine
in water at neutral pH. Several thiol catalysts of the NCL reaction with peptide alkylthioesters
were developed among which 4ꢀmercaptophenylacetic acid (MPAA) is the most popular.4ꢀ8
This thiol additive acts through a thiolꢀthioester exchange process that is the rateꢀlimiting step
of NCL. Since the arylthiol MPAA is more acidic and therefore a better leaving group than the
departing alkylthiol, the newly formed peptide arylthioester is a better acyl donor than the startꢀ
ing peptide alkylthioester.9 Preformed peptide thio10ꢀ12 or seleno13ꢀ15 aryl esters can be used as
well in NCL and are particularly powerful acyl donors.
The use of N,Sꢀacyl shift systems based on the 2ꢀmercaptoethylamide scaffold are also popular
in the field (Scheme 1B).16ꢀ26 They can be used either as precursors of peptide thioesters by exꢀ
change with thiol additives or as thioester surrogates in NCL due to their capacity to rearrange
in situ into transient thioesters. Considering the increased use of N,Sꢀacyl shift systems alone or
in combination with NCL for protein chemical synthesis, the search for novel catalysts or conꢀ
ditions adapted to these chemical systems is of great importance. However, novel developments
in this area are complicated by the lack of mechanistic insight for these reactions.
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We are particularly interested in developing new conditions and catalysts for the bis(2ꢀ
sulfanylethyl)amido (SEA)19 thioester surrogate whose utility for protein total synthesis has
been demonstrated.27ꢀ29 A motivation for this work is to accelerate SEAꢀbased reactions but also
to exploit SEA thioester surrogates under experimental conditions that cannot be attained by
NCL for extending the range of polypeptides amenable to chemical synthesis.
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Toward this end, we performed detailed kinetic investigations of the SEA N,Sꢀacyl shift process
and of the SEA amideꢀthiol exchange reaction under mildly acidic conditions. The data enabled
to extract the apparent kinetic constants for the individual steps involved in these chemical proꢀ
cesses and reveal that thiolꢀthioester exchanges are rate limiting in the working conditions.
With this knowledge, we further designed
Nꢀalkyl diselenols which are able to catalyze SEAꢀ
mediated reactions by accelerating thiolꢀthioester exchanges (Scheme 1B). The catalysts proved
particularly useful for promoting SEAꢀmediated reactions at difficult amino acids such as Val,
Ile or Thr and also for preparing peptides that are otherwise hardly accessible such as cyclic
Oꢀ
acyl isopeptides. Therefore, the novel SEA peptide/5ꢀalkylꢀ1,2,5ꢀdiselenazepane catalyst comꢀ
bination represents a useful addition to existing ligation techniques.
Scheme 1. A) Principle of NCL. B) N,S-acyl shift systems based on the 2-
sulfanylethylamide scaffold enable the synthesis of peptide thioesters or can act as thioe-
ster surrogates in NCL. Structure of SEA peptides and of the
developed in this work.
N-alkyl diselenol catalysts
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Results and discussion
Kinetic study and mechanistic model
We first investigated the kinetics of the uncatalyzed SEA group – alkylthiol exchange reaction
in the presence of 3ꢀmercaptopropionic acid (MPA) in large excess (Scheme 2A), a useful reacꢀ
tion that enables the preparation of peptidyl MPAꢀthioesters. As this reaction was shown to
proceed optimally at pH 4.0 in previous studies,30 we carried out a detailed kinetic study at this
pH to determine the rate constants for the individual steps involved in this transformation and
identify the rateꢀlimiting step of the process.
SEA peptides are produced by Fmoc SPPS in the form of a cyclic disulfide SEAoff. Therefore,
the conversion of SEA peptides into peptide MPA thioesters in the presence of TCEP and MPA
involves three different chemical steps (Scheme 2A): i) the reduction of the SEAoff disulfide
into an active SEA dithiol (SEAon), i.e.
1 → 2,
ii) the rearrangement of the SEAon amide into
the SEA thioester by intramolecular N,Sꢀacyl migration, i.e.
2 → 3, iii) and finally the thiolꢀ
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thioester exchange enabling the exchange of the SEA thioester by MPA, i.e.
3 → 4. Throughout
this study, a model SEA peptide 1a featuring a Cꢀterminal alanine residue was used as the acyl
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donor component (Scheme 2B,C).
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To simplify the determination of the kinetic constants, we first built a kinetic model for the
conversion of SEAoff disulfide into SEA thioester (
1 → 3 in Scheme 2A) which could be studꢀ
ied separately by replacing the thiol MPA by propionic acid (Scheme 2B). This reaction was
studied in the pH range 3.0ꢀ4.4 with a step of 0.2 pH units. The proportion of SEAoff peptide 1a
SEAon peptide 2a and SEA thioester 3a were quantified by LCꢀMS. The data presented in Fig.
1A correspond to the experiment carried out at pH 4.0. This figure clearly shows the reduction
of SEAoff peptide 1a into SEAon peptide 2a in the initial phase of the reaction, and then the conꢀ
version of SEAon peptide 2a into SEA thioester 3a until the establishment of an equilibrium.
The apparent kinetic constants k1, k2 and kꢀ2 were extracted by fitting the model to the data usꢀ
ing KinTek® chemical kinetics software.31 The relationship between k2 or kꢀ2 and pH can be
seen in Fig. 1B. The logarithm of the equilibrium constant for the SEAon peptide 2a/SEA thioeꢀ
ster 3a equilibrium determined experimentally and calculated from rate constants is shown in
Fig. 1C. The good correspondence between the two series of values for K shows the quality of
the fit and the pertinence of the kinetic model.
,
Scheme 2. Kinetic model for the SEA-MPA exchange reaction and experimental condi-
tions.
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SESAEoAffoff peptide 1a
SEAon peptide 2a
SEAam
A)
SESAEthA thioester peptide 3a
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Time (s)
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k2
k-2
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3,3
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3,7
pH
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4,3
4,5
C)
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0
-0,2
-0,4
-0,6
-0,8
-1
log(K)
-1,2
2,9
3,1
3,3
3,5
3,7
pH
3,9
4,1
4,3
4,5
Figure 1. Kinetic study of the reduction of SEAoff peptide 1a into SEAon peptide 2a and of the equiliꢀ
bration of SEAon peptide 2a into SEA thioester 3a. A) The proportion of each species in the mixture at
pH 4.0 was determined by HPLC using UV detection at 215 nm. k1 = 0.049 ± 1.2 10ꢀ3 Mꢀ1.sꢀ1, k2 = 8.38
10ꢀ5 ± 1.5 10ꢀ6 sꢀ1, kꢀ2 = 4.44 10ꢀ4 ± 5.9 10ꢀ5 sꢀ1. B) Rate constants k2 (red circles) and kꢀ2 (blue circles) as
a function of pH. The error bars correspond to the standard deviation. C) Logarithm of the equilibrium
[ꢀꢁ]
constant as a function of pH. Green circles: experimental K values calculated as log( ꢂꢃ) were [2a]eq
[ꢄꢁ]
ꢂꢃ
and [3a]eq are the concentrations of 2a and 3a at equilibrium; magenta circles: values of K calculated
using rate constants k2 and kꢀ2 determined by dataꢀfitting, log(ꢅꢅꢆꢇ).
ꢈꢇ
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With these data in hand we next performed additional experiments by replacing propionic acid
by MPA with the aim of building a kinetic model for the uncatalyzed conversion of SEAoff pepꢀ
tide 1a into MPA peptide thioester 4a (Scheme 2A,C; Fig. 2). This novel dataset was combined
with the rate constants k2 and kꢀ2 determined above for extracting the apparent rate constants k3
and kꢀ3 for the exchange of the SEA peptide thioester 3a by MPA (k3 = 5.21 10ꢀ7± nd Mꢀ1.sꢀ1, kꢀ3
= 6.33 10ꢀ4 ± 1.0 10ꢀ4 Mꢀ1.sꢀ1).
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The kinetic model tells us that the conversion of SEA peptide thioester 3a into peptide thioester
4a is rate limiting at the working pH of 4.0. The fact that the thiolꢀthioester exchange step of the
process shown in Scheme 1A is rate limiting is consistent with the accumulation of the SEA
peptide thioester 3a at the beginning of the reaction (Fig. 2A). Interestingly, the kinetic model
enables to predict the impact of adding a catalyst of the thiolꢀthioester process on the timeꢀ
course of the reaction (Fig. 2B). The catalyst will increase the apparent rate constants k+3 and kꢀ
without changing the equilibrium constant k+3/kꢀ3. An acceleration factor of 2 relative to the
3
uncatalyzed reaction is predicted to have already a real impact on the global rate, while the fastꢀ
est rates can be achieved for acceleration factors greater than 15. This prompted us to search for
additives that could restore a fast thiolꢀthioester regime, thereby allowing the SEAꢀbased reacꢀ
tions to fully benefit from the fast N,Sꢀacyl shift that is observed at mildly acidic pH. Such cataꢀ
lysts would be especially useful in the case of reactions at difficult amino acids such as Thr or
Val.
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SEAth
SEA thioester peptide 3a
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MPA peptide thioester 4a
MPAth
A)
on
SESAEaAm peptide 2a
SEAoff peptide 1a
SEAoff
0
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100000
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Time (s)
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Acceleration
factor
=
kCat+3 (M-1.s-1) kCat-3 (M-1.s-1)
1.66 10-5
8.32 10-6
4.16 10-6
2.08 10-6
2.02 10-3
1.01 10-3
5.06 10-4
2.53 10-4
x 32
x 16
x 8
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10
x 4
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1.27 10-4
x 2
k+3 (M-1.s-1)
k-3 (M-1.s-1)
B)
5.2 10-7
6.33 10-4
10000 15000 20000 25000 30000
Time (s)
0
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100000
150000
200000
250000
Time (s)
Figure 2. Kinetic study of the conversion of SEAoff peptide 1a into MPA peptide thioester 4a at pH 4.0.
A) Timeꢀcourse of the reaction. The proportion of each species in the mixture was determined by
HPLC using UV detection at 215 nm. B) The kinetic model enables to predict the favorable impact of
adding a catalyst of the thiolꢀthioester exchange process on the rate. Blue circles: experimental points
for the uncatalyzed reaction. Blue crosses: kinetic model fitted to experimental data. The other traces
were calculated using the kinetic model and the kCat and kCat values corresponding to the different
+3
ꢀ3
acceleration factors indicated on the right.
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Catalysis by classical arylthiol or selenol additives
We first examined the capacity of classical thiol or selenol additives, i.e. MPAA or phenylseleꢀ
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nide PhSeH, to promote the formation of peptide thioester 4a. For this purpose, SEA peptide 1a
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was treated with the thiol MPA and TCEP in 6 M Gn⋅HCl at pH 4.0 in presence or absence of
the additive (Fig. 3).30 The kinetic profiles were determined by HPLC and fitted to a pseudoꢀ
first order kinetic law from which halfꢀreaction times (t½) were extracted (Table 1).
In the absence of catalyst, formation of peptide thioester 4a proceeded with a t½ of 7.28 h (Fig.
3, entry 1, Table 1). MPAA accelerated the SEAꢀthiol exchange reaction (Fig. 3), albeit with a
limited effect (4.86 h, entry 3, Table 1). The fitting of the kinetic data to the model depicted in
Scheme 2A shows that MPAA accelerated the thiolꢀthioester exchange step by a factor of about
2.5
.
Replacing MPAA by PhSeH did not improve the reaction rate (Fig. 3, entry 4, Table 1) altꢀ
hough the concentration of nucleophilic chalcogenate species at pH 4.0 is significantly larger
for PhSeH vs MPAA due to the lower pKa of the former.5,32 Since selenophenyl esters are more
powerful acylating species than thioesters derived from MPAA,33 the poor catalytic properties
of selenophenol is suggestive of an unfavourable selenophenolꢀalkylthioester exchange process.
This lack of reactivity arises probably from the larger thermodynamic stability of alkylthioeꢀ
sters relative to selenophenylesters which disfavour the formation of the later in the reaction
mixture.13
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Time (h)
Figure 3. Exchange of the SEA group by MPA catalyzed by MPAA or selenophenol (generated by in
situ reduction of diphenyldiselenide with TCEP). The continuous curves correspond to the fitting to a
pseudo first order kinetic law from which t½ were extracted (see Table 1).
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Table 1. Catalysis of SEA-thiol exchange.a
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8
en- SEA pep- catalyst
catalyst
conc.
(mM)
ꢀ
t1/2 (h)
b
try
tide
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1
2
ø
MPAA
MPAA
PhSeH c
5a
7.28
4.08
1a
1a
1a
1a
1a
1a
1a
1a
1a
1a
1b
1b
1b
1b
1b
1c
1c
1d
1d
200
100
100
100
50
3
4.86
4
5.25
5
2.04
6
2.03
5a
7
25
1.95
5c
8
12.5
6.25
3.13
ꢀ
2.35
5c
9
3.00
5c
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13
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17
18
19
3.35
5c
ø
67.96
51.73
20.03
24.32
19.36
8.86
MPAA
5a
100
100
100
100
ꢀ
5b
5c
ø
100
ꢀ
3.12
50.0 d
5b
ø
100
31.6
5b
a Conditions: [1a] = [1b] = [1c] = 1 mM, 37 °C under inert atmosphere, 6 M Gn.HCl, MPA 5% v/v,
b
100 mM TCEP.HCl, pH 4.0. Determined by HPLC. The value corresponds to the time needed to
reach 50% conversion. c Selenophenol was formed in situ from PhSeSePh and was soluble in our conꢀ
ditions. d The data could not be fitted to a pseudoꢀfirst order kinetic law.
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Novel catalyst design and synthesis
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In search for alternative catalysts that could perform better than MPAA, we were interested by
the findings of Makriyannis et al. who showed that an alkylthioester can be exchanged by an
alkyselenol at mildly acidic pH.34 Inspired by this work, which shows the formation of an alꢀ
kylselenoester from an alkylthioester in the typical pH range for the SEAꢀmediated reactions,
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we undertook the synthesis of
Nꢀalkyldiselenides 5a-c (Scheme 3A). These compounds are
highly waterꢀsoluble due to their protonation at acidic pH. Moreover, the presence of an ammoꢀ
nium group closeby the selenol contributes to lower its pKa (estimated pKa 4.5) and thus the
formation of selenoate species at the working pH of 4.0, i.e. the active species involved in the
catalysis of the thiolꢀthioester exchange process. Moreover and as will be shown later, the
Nꢀ
alkyl group has no effect on catalysis. Therefore, it can be varied depending on the final appliꢀ
cation. For example, the carboxylic acid group of catalyst 5c enables potentially its attachment
to a solid support.
Nꢀalkylated diselenides 5a-c were initially prepared starting from 1,2,5ꢀdiselenazepane by alꢀ
kylation with 3ꢀbromoꢀ1ꢀpropanol (for 5a) or by silica gelꢀcatalyzed azaꢀMichael addition35 to
acrylamide or acrylic acid (for 5b or 5c respectively, see Experimental Section). We also develꢀ
oped the third method described in Scheme 3 that facilitates the production of catalyst 5c on a
larger scale. In this case, the tertiary amine scaffold was produced by double reductive alkylaꢀ
tion of
aldehyde
tageously, cyclic diselenide formation and
β
ꢀalanine tertꢀbutyl ester with aldehyde
, i.e. acetal
, was easily produced by adapting the method of Abbas et al.36 Advanꢀ
Bu protecting group removal were performed simulꢀ
7 (PMB: 4ꢀmethoxybenzyl). The precursor for
7
6
t
taneously in TFA in the presence of 2,2′ꢀdithiobis(5ꢀnitropyridine) (DTNP).37 This practical
route provided diselenide 5c with an overall yield of 51% and can potentially be applied to the
synthesis of other
Nꢀalkylated perhydroꢀ1,2,5ꢀdiselenazepane derivatives.
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Scheme 3. Structure of catalysts 5a-c (A) and synthesis of diselenide 5c (B).
5
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Catalysis of SEA thioester-thiol exchange reaction by N-alkyl-diselenides
Diselenide 5a which is in situ reduced into the active alkyldiselenol significantly accelerated
the formation of peptide thioester 4a (Fig. 4B) and consequently reduced the t½ of the reaction
by a factor 3.6 compared to the uncatalyzed reaction (entries 1 and 5, Table 1). The fitting of
the kinetic data to the model described in Scheme 2A indicates that the exchange of the SEA
thioester by MPA is accelerated more than 15 times compared to the uncatalyzed reaction.
Catalyst 5a was then compared to its analogs 5b and 5c which differ by the functionality preꢀ
sent on the exocyclic alkyl side chain. For this purpose, peptide 1b which features a sterically
demanding Cꢀterminal Val residue was used as starting material (Fig. 4C). The three catalysts
converted 1b into the thioester peptide 4b with almost equal efficiency (entries 13ꢀ15, Table 1),
while MPAA had a minor effect compared to the control experiment without catalyst (compare
entries 11 & 12, Table 1). Note also that catalyst 5b promoted the SEAꢀthiol exchange reaction
in the cases of SEA peptides equipped with a Cꢀterminal Thr (1c, entries 16 and 17, Table 1) or
Ile (1d, entries 18 and 19, Table 1) residues.
As mentioned above, diselenide 5a was significantly more efficient than MPAA in catalyzing
the SEAꢀthiol exchange reaction (compare entries 2 & 5, Table 1). Since the bivalency of cataꢀ
lyst 5a might introduce a bias in the comparisons, we compared the catalytic potency of
diselenide 5a at 100 or 50 mM (entries 5,6 in Table 1) with those of MPAA used at 200 or 100
mM respectively (entries 2,3 in Table 1). In each case the rate of SEAꢀthiol exchange was sigꢀ
nificantly faster with diselenide 5a than with MPAA, excluding the possibility that the differꢀ
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ence observed at 100 mM for both catalysts might be due to a doubling of selenol concentration
for 5a. We also decreased the diselenide catalyst 5c concentration to see if the SEAꢀMPA exꢀ
change is still accelerated at low catalyst concentration (see entries 7ꢀ10 in Table 1). Interestꢀ
ingly, diselenide catalyst 5c had a significant effect on the rate even at 3 mM, and was more
powerful at this concentration than MPAA at 200 mM.
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We further examined the stability of our catalysts in the presence TCEP. While the metalꢀfree
desulfurization of Cys thiols requires a phosphine such as TCEP and a radical initiator to proꢀ
ceed at an appreciable rate,38 the radical initiator is not necessary for the deselenization of alꢀ
kylselenols by TCEP.39 The deselenization of the catalysts could dramatically alter their catalytꢀ
ic potency over long reaction times. We therefore determined the extent of catalyst deselenizaꢀ
tion by quantifying the amount of TCEPꢀderived selenophosphine produced in the reaction mixꢀ
ture. This study indicated that less than 5% of the catalyst was deselenized after 24 h even for
the lowest catalyst concentrations tested (see Supporting Information).
The usefulness of catalyst 5c was then confirmed on preparative scale by the synthesis of pepꢀ
tide 4b with 63% yield (Scheme 4). Chiral GC analysis of peptide 4b after acid hydrolysis indiꢀ
cated a DꢀVal content below detectable levels (< 0.1%), and thus that no epimerization occurred
during the exchange process. In another example, the 46 amino acids SEA peptide 1e which is
equipped with a Cꢀterminal Thr residue was successfully converted into thioester 4e using the
same protocol (36%, Scheme 4).
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Figure 4. Exchange of the SEA group by MPA catalyzed by MPAA, selenophenol or diselenides 5a-c.
A) Model reactions. B) Conversion of peptide 1a into 4a catalyzed by MPAA or diselenide 5a. C)
Conversion of peptide 1b into 4b uncatalyzed, catalyzed by MPAA or diselenides 5a-c. The continuous
curves correspond to the fitting to a pseudo first order kinetic law from which t½ were extracted (see
Table 1).
Scheme 4. Preparative synthesis of peptide thioesters using diselenide 5c.
MPA 5% vol
100 mM TCEP
6 M Gn.HCl
O
peptide
S
peptide
N
CO2H
S
5c
100 mM
O
S
pH 4.0
4b (63%,
4e
D-Val < 0.1%)
1b,e
(36%)
b: 50 h, 37 °C. peptide = ILKEPVHGV-
e: 19 h, 30 °C. peptide = RLSPEYYDLARAHLRDEEKSC(StBu)PC(StBu)LAQEGPQGDLLTKT
4e
QELGRDYRT- (StBu groups are absent in
)
Catalysis of SEA-mediated ligation by N-alkyl-diselenides
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Having demonstrated the usefulness of diselenides
5 for catalyzing the SEAꢀthiol exchange reꢀ
action, we next examined if they could accelerate the mechanistically related SEAꢀmediated
ligation as well. The optimal pH for such ligation catalyzed by MPAA is 5.5 and its rate deꢀ
creases as the pH is lowered from 5.5 to 4.0 (Fig. 5). As for the SEAꢀMPA exchange reaction,
diselenide 5b was more powerful in promoting the SEAꢀbased ligation of peptide 1b at pH 4.0
than MPAA at pH 4.0 or 5.5. To illustrate the synthetic usefulness of diselenide 5c, peptide 10
was synthesized on preparative scale (59%). Here again, no significant epimerization (0.19%)
was observed for the valine involved in the formed junction. In another example, the 38 amino
acids SEA peptide 1f which is equipped with a Cꢀterminal Val residue was successfully cyꢀ
10
11
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clized into cyclic peptide 11 in the presence of diselenide 5c (Scheme 5). In this case
glucoside was used as a nonꢀionic detergent to avoid the gelification of the ligation mixture that
had been observed when ligation was attempted in 6 M Gn HCl. Note also that the high hydroꢀ
Nꢀoctyl
⋅
philicity of catalyst 5c enabled the direct purification of the crude ligation mixtures by HPLC.
This is a great advantage over MPAA which must be removed from the ligation mixture prior to
the isolation step.7,29,40
100
80
60
40
pH 4
pH 5.5
pH 4.0
200 mM MPAA
20
0
100 mM 5b
0
20
40
60
80
100
120
140
160
Time (hour)
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Figure 5. Catalysis of the SEAꢀmediated ligation by diselenide 5b or MPAA. Peptide concentration
was 7 mM.
5
6
7
8
9
The possibility to promote SEAꢀmediated ligations at pH 4.0 broadens the scope of the peptides ameꢀ
nable to chemical synthesis. We undertook the synthesis of cyclic peptide 12 (Scheme 5) whose linear
precursor 1g features a Cꢀterminal Thr residue and an internal Oꢀacyl isopeptide unit.41ꢀ43 The later reꢀ
arranges spontaneously by O,Nꢀacyl shift above pH 5 to produce a native peptide bond.41ꢀ43 Therefore,
the classical conditions for NCL (MPAA, pH 7), SEA ligation (MPAA, pH 5.5) or for any other N,Sꢀ
acyl shift mediated ligation cannot cyclize peptide 1g without inducing the rearrangement of the Oꢀacyl
isopeptide unit. In contrast, the combination of a SEA peptide precursor and diselenide 5c successfully
yielded cyclic peptide 12 in about 10 h. Here again as for the other examples discussed above, the
crude product could be directly purified by HPLC. The presence of the Oꢀacyl isopeptide unit in the
final product and the cyclic structure of peptide 12 was demonstrated by rearranging peptide 12 at neuꢀ
tral pH and by analyzing the resulting cyclic peptide c[CTGSKDCFEA TTCTGSTNCYKATT] 13 after
alkylation and trypsin digestion (see Supporting Information).
10
11
12
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Scheme 5. Cyclative SEA-mediated ligation catalyzed by diselenide 5c.
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Conclusion
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5
6
7
8
In conclusion, we describe a novel family of waterꢀsoluble alkyldiselenol catalysts for the bis(2ꢀ
sulfanylethyl)amido (SEA)ꢀthiol exchange reaction or the SEAꢀmediated ligation which are
particularly useful for promoting the reactions at difficult amino acids. The performance of the
novel catalysts at pH 4.0 is superior to those of MPAA at any pH. They act by accelerating thiꢀ
olꢀthioester exchanges which are rateꢀlimiting at pH 4.0. The catalysts have an excellent waterꢀ
solubility due to the presence of a protonated tertiary amine within their structure and enable
the direct purification of the reaction mixtures by HPLC. Besides accelerating the SEAꢀ
mediated reactions, working at pH 4.0 is potentially an advantage when the peptide segments
are not soluble at higher pH. Ligation at pH 4.0 also extends the scope of peptides amenable to
9
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chemical synthesis such as cyclic
Oꢀacyl isopeptides which cannot be produced at neutral or
slightly acidic pH. The novel catalysts should find various applications in the synthesis of comꢀ
plex peptides or challenging proteins.
EXPERIMENTAL SECTION
General Methods
Reagents and solvents
2ꢀ(1HꢀBenzotriazolꢀ1ꢀyl)ꢀ1,1,3,3ꢀtetramethyluronium fluorophosphate (HBTU) and NꢀFmoc protected
amino acids were obtained from Iris Biotech GmbH. Sideꢀchain protecting groups used for the amino
acids were FmocꢀAlaꢀOH, FmocꢀArg(Pbf)ꢀOH, FmocꢀAsn(Trt)ꢀOH, FmocꢀAsp(OtBu)ꢀOH, Fmocꢀ
Glu(OtBu)ꢀOH, FmocꢀGlyꢀOH, FmocꢀHis(Trt)ꢀOH, FmocꢀIleꢀOH, FmocꢀLeuꢀOH, FmocꢀLys(Boc)ꢀ
OH, FmocꢀProꢀOH, FmocꢀPheꢀOH, FmocꢀSer(tBu)ꢀOH, FmocꢀThr(tBu)ꢀOH, FmocꢀTyr(tBu)ꢀOH,
FmocꢀValꢀOH,
FmocꢀCys(StBu)ꢀOH
or
FmocꢀCys(Trt)ꢀOH.
Synthesis
of
bis(2ꢀ
sulfanylethyl)aminotrityl polystyrene (SEA PS) resin was carried out as described elsewhere.19
4ꢀmercaptophenylacetic
acid
(MPAA),
3ꢀmercaptopropionic
acid
(MPA),
tris(2ꢀ
carboxyethyl)phosphine hydrochloride (TCEP), guanidine hydrochloride (Gn·HCl) were purchased
from SigmaꢀAldrich. All other reagents were purchased from Acros Organics or Merck and were of the
purest grade available.
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Peptide synthesis grade N,Nꢀdimethylformamide (DMF), dichloromethane (CH2Cl2), diethylether
(Et2O), acetonitrile (CH3CN), heptane, LC–MSꢀgrade acetonitrile (CH3CN, 0.1% TFA), LC–MSꢀgrade
water (H2O, 0.1% TFA), N,Nꢀdiisopropylethylamine (DIEA), acetic anhydride (Ac2O) were purchased
from Biosolve and FisherꢀChemical. Trifluoroacetic acid (TFA) was obtained from Biosolve. Water
was purified with a MilliꢀQ UltraꢀPure Water Purification System.
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Analyses
The reactions were monitored by analytical LC–MS (Waters 2695 LC/ZQ 2000 quadripole) on a reꢀ
verse phase column XBridge BEH300 C18 (3.5 µm, 300 Å, 4.6 × 150 mm) unless otherwise stated.
The elutions were carried out at 30 °C using a linear gradient of 0ꢀ100% of eluent B in eluent A over
30 min at a flow rate of 1 mL/min (eluent A = 0.1% TFA in H2O; eluent B = 0.1% TFA in
CH3CN/H2O: 4/1 v/v). The column eluate was monitored by UV at 215 nm and by evaporative light
scattering (ELS, Waters 2424 detector). The peptide masses were measured by onꢀline LC–MS: Ionizaꢀ
tion mode: ES+, m/z range 350–2040, capillary voltage 3 kV, cone voltage 30 V, extractor voltage 3 V,
RF lens 0.2 V, source temperature 120 °C, dessolvation temperature 350 °C.
MALDIꢀTOF mass spectra were recorded with a BrukerAutoflex Speed using alphaꢀcyanoꢀ4ꢀ
hydroxycinnaminic acid, sinapinic acid or 2,5ꢀdihydroxybenzoic acid (DHB) as matrix. The observed
m/z corresponded to the monoisotopic ions, unless otherwise stated.
High resolution mass spectra (HRMS) were recorded with an Orbitrap mass spectrometer (Therꢀ
moFischer). ESI spectra (positive mode, 100ꢀ600 uma) were obtained by direct injection of the comꢀ
pounds.
1H and 13C NMR spectra were recorded on a Bruker Advanceꢀ300 spectrometer operating at 300 MHz
and 75 MHz respectively. The spectra are reported as parts per million (ppm) down field shift using
tetramethylsilane or dimethylselenide as internal references. The data are reported as chemical shift (δ),
multiplicity, relative integral, coupling constant (J Hz) and assignment where possible.
The determination of optical purity for the Cꢀterminal amino acid was done by chiral GCꢀMS following
total acid hydrolysis in deuterated aqueous acid (C.A.T. GmbH & Co. Chromatographie und Analysenꢀ
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technik KG, Heerweg 10, Dꢀ72070 Tübingen, Germany).44
4
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HPLC purification
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Preparative reverse phase HPLC of crude peptides were performed with a preparative HPLC Waters
system using a reverse phase column XBridge BEH300 Prep C18 (5 µm, 300 Å, 10 × 250 mm) and
appropriate linear gradient of increasing concentration of eluent B in eluent A (flow rate of 6 mL/min).
Selected fractions were then combined and lyophilized.
Preparative reverse phase HPLCs of crude selenium compounds were performed with an Autopurificaꢀ
tion prep HPLC–MS Waters system using a reverse phase column XBridge ODB prep Cꢀ18 (5 µm, 300
Å, 19 × 100 mm) and appropriate gradient of increasing concentration of eluent B in eluent A (flow
rate of 25 mL/min). The fractions containing the purified target compound were identified onꢀline usꢀ
ing MS (ZQ 2000 quadripole). Selected fractions were combined and lyophilized.
Kinetic studies
Kinetic model (see Scheme 2)
For the kinetic study of the MPA – SEA thioester exchange process (see Scheme 2), all reactions were
set up from a single batch of lyophilized SEAoff peptide 1a. Also, freshly prepared buffers and newly
opened reagents were used for the whole series of experiments.
In order to determine the rate constants involved in the different steps of the SEA thioester – MPA exꢀ
change process, the system was decomposed to simpler reactions and the protocol was gradually comꢀ
plexified. Data were fitted and rate constants were determined using KinteK Explorer SoftwareTM (Verꢀ
sion 7.2.180216., Kintek Corporation ꢀ https://kintekcorp.com/software/)
Step 1. Study of the SEA amide / SEA thioester equilibrium (see Figure 1)
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The reduction and the rearrangement of the SEA peptide were investigated in the absence of MPA. In
order to maintain the concentration of carboxylic acids to the same level in all reactions, MPA was reꢀ
placed by propionic acid (574 mM).
7
8
9
The reaction mixture was prepared as follows: TCEP (28.65 mg, 100 mM) was dissolved in a 6 M
Gn.HCl solution in phosphate buffer 0.1 M (1 mL). 958 ꢁL of this solution was added to propionic acid
(42 ꢁL, 574 mM) and the pH was adjusted to the desired value by adding a 6 M NaOH solution. Lyꢀ
ophilized SEAoff peptide 1a (~ 850 ꢁg, ~0.6 ꢁmol) was weighed directly in an HPLC vial and dissolved
in the above described buffer (~200 ꢁL, 3 mM).
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The reaction mixture was left at 37°C in a temperatureꢀcontrolled well plate autosampler (Dionex Ulꢀ
timate 3000 RS) and 1 ꢁL was injected for analysis at regular time intervals (eluent A 0.1% TFA in
water, eluent B 0.1% TFA in CH3CN, Column Acquity UPLC® Peptide BEH C18 300 Å, 1.7 µm, 2.1
mm x 100 mm, gradient 10ꢀ35% B in 3 min, 0.7 mL/min, 37° C). The chromatograms were processed
on the basis of the absorbance signal at 215 nm to deduce the conversion ratio of the different species
(no absorbance correction was applied as SEAoff, SEAon amide and SEA thioester were considered to
have the same molecular extinction coefficient).
All datasets were fitted with a fourꢀphase exponential analytical function to obtain an estimate of the
standard deviation. A subsequent global fit allowed determining rate constants k1, k+2, kꢀ2 as well
standard deviations at each studied pH (see Supporting Information).
Step 2. Kinetic study of the MPA ꢀ SEA thioester exchange (see Figure 2)
TCEP (28.65 mg, 100 mM) was dissolved in a 6 M Gdn•HCl solution in phosphate buffer 0.1 M (1
mL). 950 ꢁL of this solution was added to mercaptopropionic acid (50 ꢁL, 574 mM) and the pH was
adjusted to 4.0 by adding a 6 M NaOH aqueous solution.
SEAoff peptide 1a (~ 850 ꢁg, ~0.6 ꢁmol) was weighed in an eppendorf tube and dissolved in the above
described buffer (~200 ꢁL, 3 mM). The reaction mixture was left at 37°C under nitrogen atmosphere. 3
µL of the reaction mixture were collected every ~20 minutes and added to 100 µL of a 20% acetic acid
aqueous solution. The sample was extracted four times by Et2O and the aqueous layer was analyzed by
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HPLC (eluent A 0.1% TFA in water, eluent B 0.1% TFA in CH3CN/water: 4/1 by vol. C18 Xbridge
BEH 300 Å 5 ꢁm (4.6 × 250 mm) column, gradient 0ꢀ50% B in 15 min, 1 mL/min, 30° C, detection at
215 nm)
8
9
The chromatograms were processed on the basis of the absorbance signal at 215 nm to deduce the conꢀ
version ratio of the different species (no absorbance correction was applied as SEAoff, SEAon amide and
SEA thioester were considered to have the same molecular extinction coefficient). The data were proꢀ
cessed as described above for Step 1.
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SEA thioesterꢀMPA exchange (see Figure 3 and 4)
General procedure illustrated with peptide 1a and MPAA as catalyst
To a solution of TCEP·HCl (28.67 mg, 100 µmol, 100 mM final concentration) in 6 M Gn·HCl, 0.2 M,
pH 7.2 sodium phosphate buffer (1 mL) was added MPAA (16.82 mg, 0.1 mmol, 100 mM final conꢀ
centration) dissolved in 20 µL 5 M NaOH. Note that the presolubilization step in NaOH was not reꢀ
quired for diselenide catalysts.
The SEA peptide 1a (0.994 mg, 0.6 µmol) was dissolved in the previous solution (final peptide concenꢀ
tration 1 mM). To the resulting mixture was added MPA (5% v/v). The pH was adjusted to 4.0 by addiꢀ
tion of 5 M NaOH and the reaction mixture was then stirred at 37 °C under nitrogen atmosphere.
The progress of the reaction was monitored by HPLC. For each point, a 2 ꢁL aliquot was taken from
the reaction mixture and quenched by adding 50 ꢁL of 0.1% aqueous TFA. The sample was then exꢀ
tracted with Et2O to remove MPAA prior to HPLC analysis. Note that this extraction step was not reꢀ
quired for diselenide catalysts.
SEAꢀmediated ligation (see Figure 5)
General procedure illustrated with peptide 1b, Cys peptide 8 and MPAA as catalyst
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To a solution of TCEP·HCl (11.47 mg, 40 µmol, 200 mM final concentration) in 6 M Gn·HCl, pH 7.2,
0.1 M sodium phosphate buffer (200 µL) was added MPAA (6.72 mg, 40 µmol, 200 mM final concenꢀ
tration) dissolved in 20 µL 5 M NaOH. The pH was then adjusted to 4ꢀ5.5 by addition of 5 M NaOH.
Note that the presolubilization step in NaOH was not required for diselenide catalysts.
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The SEA peptide 1b (1.498 mg, 1 µmol, 7 mM final concentration) and Cys peptide 8 (2.22 mg, 1.5
µmol, 10.5 mM, 1.5 equiv) were dissolved in the previous solution. The reaction mixture was stirred at
37 °C under nitrogen atmosphere.
The progress of the reaction was monitored by HPLC. For each point, a 2 ꢁL aliquot was taken from
the reaction mixture and quenched by adding 50 ꢁL of 0.1% aqueous TFA. The sample was then exꢀ
tracted with Et2O to remove MPAA prior to HPLC analysis. Note that this extraction step was not reꢀ
quired for diselenide catalysts.
Peptide synthesis
General protocol for automated peptide synthesis
Peptide elongation was performed using standard Fmoc/tertꢀbutyl chemistry on an automated peptide
synthesizer (0.2 mmol scale). Couplings were performed using 5ꢀfold molar excess of each FmocꢀLꢀ
amino acid, 4.5ꢀfold molar excess of HBTU, and 10ꢀfold molar excess of DIEA. A capping step was
performed after each coupling with Ac2O/DIEA in DMF. At the end of the synthesis, the resin was
washed with CH2Cl2, diethylether (3 × 2 min) and dried in vacuo.
Synthesis of SEAoff peptide segments
Peptide elongation was performed on SEA PS resin19 (0.2 mmol, 0.16 mmol/g). Typical procedures for
the synthesis of SEAoff peptide segments were described in previous papers.19,29,45
For the synthesis of SEAoff peptide 1g, BocꢀThr(FmocꢀAla)ꢀOH and the following amino acid were
coupled manually using HBTU/DIEA activation in DMF.
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Peptides 1a-d were already described elsewhere.19
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RLSPEYYDLARAHLRDEEKSC(StBu)PC(StBu)LAQEGPQGDLLTKTQELGRDYRTꢀSEAoff (pepꢀ
tide 1e): 26 mg (8% yield, 0.05 mmol scale). MALDIꢀTOF m/z calcd for [M+H]+ (average): 5618.48,
found: 5617.2.
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C(StBu)LTIVQKLKKMVDKPTQRSVSNAATRVC(StBu)RTGRSRWRDVꢀSEAoff (peptide 1f): 11.5
mg (4% yield, 0.05 mmol scale). MALDIꢀTOF m/z calcd for [M+H]+ (average): 4683.8, found: 4683.3.
T[C(StBu)TGSKDC(StBu)FEA]TC(StBu)TGSTNC(StBu)YKATTꢀSEAoff (peptide 1g): 0.1 mmol
scale, 36.1 mg (11%). MALDIꢀTOF m/z calcd for [M+H]+ (monoisotopic): 2962.1, found: 2962.2
Synthesis of Cys peptides
The synthesis of peptide 9 has been described elsewhere.19
Synthesis of diselenide catalysts 5a-c
Synthesis of diselenide 5a (alkylation of 1,2,5ꢀdiselenazepane with 3ꢀbromopropanol/CsOH)
Cesium hydroxide monohydrate (509 mg, 3.00 mmol, 2 equiv) was added to activated powdered 4 Å
molecular sieve (400 mg) in anhydrous N,Nꢀdimethylformamide (1 mL). The white suspension was
vigorously stirred for 10 min at rt. Then, the 1,2,5ꢀdiselenazepane trifluoroacetate salt46ꢀ47 (350 mg, 1.5
mmol) was dissolved in DMF (2.5 mL) and added to the suspension of molecular sieve. The mixture
was stirred for 30 min before adding 3ꢀbromopropanol (402 µL, 4.55 mmol, 3 equiv). The reaction
mixture was stirred for 20 h at rt, diluted with water containing 0.1% TFA (20 mL) and centrifuged.
The supernatant was collected, filtered and purified by RPꢀHPLC (XBridge C18 OBD column, eluent
A: water containing 0.1% TFA by vol, eluent B: water/acetonitrile: 1/4 by vol containing 0.1% TFA by
vol, detection at 215 nm, 25 mL/min, 0ꢀ15% eluent B in 10 min). The purified fractions were collected,
frozen and lyophilized to give 19.53 mg (4.5%) of 5a as the trifluoroacetate salt.
The synthesis of 5a was unsatisfatory and was discontinued.
Catalyst 5a: HRMS calcd. for [M+H]+ C7H16ONSe2 289.9557, found 289.9552 (ꢀ1.67 ppm).
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1H NMR (300 MHz) spectrum for compound 5a (DMFꢀd7, 300 K). δ 3.97 (m, 4H), 3.69 – 3.65 (m,
3H), 3.46 – 3.40 (m, 6H), 2.00 (m, 2H) ppm.
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13C JMOD NMR (75 MHz) spectrum for compound 5a (DMFꢀd7, 300 K). δ 59.2 (CH2), 54.6 (4 ×
CH2), 22.30 (CH2) ppm. One 13C signal is not seen due to overlap with the residual signal from the solꢀ
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vent.
Synthesis of diselenide catalyst 5b (reaction of 1,2,5ꢀdiselenazepane with acrylamide)
The 1,2,5ꢀdiselenazepane trifluoroacetate salt47 (43.2 mg, 0.125 mmol, 1 equiv) and acrylamide (44.4
mg, 0.625 mmol, 5 equiv) were dissolved separately in acetonitrile (1 mL for each reactant). The two
solutions were combined and silica gel (50 % w/w relative to the diselenide) and triethylamine (17 µL,
0.125 mmol, 1 equiv) were then successively added to the mixture. Note that the 1,2,5ꢀdiselenazepane
trifluoroacetate salt precipitates partially upon Et3N addition but after a few time the precipitate disapꢀ
pears probably due to its adsorption on silica gel.
The suspension was stirred at 60 °C and the progress of the reaction was monitored by LCꢀMS. After
24 h, more acrylamide (5 equiv) was added to the reaction mixture and the resulting suspension was
stirred at 60 °C for two further days. When the reaction was completed, the supernatant was collected
and 2 mL of waterꢀTFA 0.1% were added to the residue of silica gel. The mixture was stirred for 30
min and the supernatant was collected. This washing procedure was repeated twice. All supernatants
were combined and concentrated in vacuo to remove the acetonitrile. This operation must be carefully
controlled to avoid evaporation to dryness.
The solution was filtered and purified by RPꢀHPLC (XBridge C18 OBD column, eluent A : water conꢀ
taining 0.1% TFA by vol, eluent B: water/acetonitrile : 1/4 by vol containing 0.1% TFA by vol, detecꢀ
tion at 215 nm, 25 mL/min, 0ꢀ15% eluent B in 20 min). The purified fractions were collected, frozen
and lyophilized to give 30.9 mg (40%) of 5b as the trifluoroacetate salt.
Catalyst 5b: HRMS calcd. for [M+H]+ C7H15ON2Se2 302.9509, found 302.9503 (ꢀ1.98 ppm).
1H NMR (300 MHz) spectrum for compound 5b (DMFꢀd7, 333 K). δ 7.59 (s, 1H), 6.84 (s, 1H), 3.77 –
3.74 (m, 4H), 3.42 – 3.36 (t, J = 6.9 Hz, 2H), 3.33 – 3.29 (m, 4H), 2.73 – 2.64 (t, J = 6.9 Hz, 2H) ppm.
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13C JMOD NMR (75 MHz) spectrum for compound 5b (DMFꢀd7, 291 K). δ 172.49 (C), 55.93 (2 ×
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CH2), 53.90 (CH2), 23.33 (2 × CH2) ppm. One C signal is not seen due to overlap with the residual
signal from the solvent.
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Synthesis of catalyst 5c (Scheme 3)
Synthesis of (2,2ꢀdiethoxyethyl)(4ꢀmethoxybenzyl)selane 6
To a solution of 1,2ꢀbis(2,2ꢀdiethoxyethyl)diselane36 (1.94 g, 4.94 mmol) in ethanol (34 mL) was added
portionwise (30 min) at 0 °C NaBH4 (500 mg, 13.2 mmol, 2.7 equiv). Once the emission of H2 stopped,
the reaction mixture was warmed to 25 °C for 30 min. Then the reaction mixture was cooled to 0 °C
and 4ꢀmethoxybenzyl chloride (1.24 mL, 9.19 mmol, 1.9 equiv) was added. The reaction mixture was
stirred at room temperature for 2 h. Water (150 mL) was added afterwards and the obtained aqueous
layer was extracted with Et2O (2 × 100 mL). The combined organic layers were dried over MgSO4 and
the solvent was evaporated under reduced pressure. Purification of the crude by column chromatogꢀ
raphy (cyclohexane/ethyl acetate 95:5) provided the expected selenide 6 (2.75 g, 94%) as an oil.
Compound 6: ESIꢀMS [M+Na]+ calcd. 341.06, found 341.09.
1H NMR (300 MHz) spectrum for compound 6 (CDCl3, 293 K). δ 7.23 (d, J = 8.6 Hz, 2H), 6.82 (d, J =
8.6 Hz, 2H), 4.59 (t, J = 5.6 Hz, 1H), 3.82 (s, 2H), 3.78 (s, 3H), 3.59ꢀ3.71 (m, 2H), 3.45ꢀ3.57 (m, 2H),
2.63 (d, J = 5.6 Hz, 2H), 1.21 (d, J = 7.0 Hz, 6H) ppm.
13C JMOD NMR (75 MHz) spectrum for compound 6 (CDCl3, 293 K). δ 158.4 (C), 131.3 (C), 130.0 (2
× CH), 113.9 (2 × CH), 103.7 (CH), 62.0 (2 × CH2), 55.2 (CH3), 27.1 (CH2), 26.3 (CH2), 15.3 (2 ×
CH3) ppm.
Synthesis of tertꢀbutyl 3ꢀ(bis(2ꢀ((4ꢀmethoxybenzyl)selanyl)ethyl)amino)propanoate 8
The selenide 6 (1.24 g, 3.90 mmol) was suspended in formic acid (1 M, 16 mL). The reaction mixture
was stirred at 50 °C overnight. Water (50 mL) was added and the obtained aqueous layer was extracted
with Et2O (2 × 50 mL). The combined organic layers were dried over MgSO4 and the solvent evapoꢀ
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rated under reduced pressure. The aldehyde 7 contained in the residue was dissolved under argon atꢀ
mosphere in anhydrous 1,2ꢀdichloroethane (8 mL). Activated powdered 3 Å molecular sieves (1 g),
sodium triacetoxyborohydride (700 mg, 4.3 mmol, 3.3 equiv) and a solution of βꢀalanine tꢀbutyl ester
hydrochloride (236 mg, 1.3 mmol) in DCE (5 mL) were successively added to the solution of aldehyde
7. After 24 h stirring at RT, the reaction mixture was filtrated on Büchner funnel and the filtered solid
was washed with additional portion of DCM. The filtrate was evaporated under reduced pressure.
Then, 10% K2CO3 (25 mL) and brine (30 mL) were added to residue and the obtained aqueous layer
was extracted with DCM (3 × 50 mL). The combined organic layers were dried over MgSO4 and the
solvent was evaporated under reduced pressure. Purification of the crude by column chromatography
(cyclohexane/ethyl acetate 95:5) provided the amine 8 (695 mg, 89% from the βꢀamino acid).
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Compound 8: ESIꢀMS [M+H]+ calcd. 602.1, found 602.2.
1H NMR (300 MHz) spectrum for compound 8 (CDCl3, 305 K). δ 7.20 (d, J = 8.7 Hz, 4H), 6.82 (d, J =
8.7 Hz, 4H), 3.76 (s, 6H), 3.76 (s, 4H), 2.69 (t, J = 7.1 Hz, 2H), 2.56ꢀ2.62 (m, 4H), 2.46ꢀ2.51 (m, 4H),
2.28 (t, J = 7.1 Hz, 2H), 1.44 (s, 9H) ppm.
13C JMOD NMR (75 MHz) spectrum for compound 8 (CDCl3, 293 K). δ 171.6 (C), 158.2 (2 × C),
131.1 (2 × C), 129.7 (4 × CH), 113.7 (4 × CH), 80.1 (C), 55.0 (2 × CH3), 54.1 (2 × CH2), 49.0 (CH2),
33.8 (CH2), 28.0 (3 × CH3), 26.3 (2 × CH2), 21.4 (2 × CH2) ppm.
Synthesis of catalyst 5c
To the amine 8 (50 mg, 0.08 mmol) dissolved in TFA (0.5 mL) was added dropwise a solution of
DTNP (51.36 mg, 0.16 mmol, 2 equiv) in TFA (0.5 mL). The reaction mixture was stirred during 1 h at
r.t. Then, cold Et2O was added (40 mL) and the mixture was evaporated under reduced pressure. Water
(10 mL) was added and the solution was purified by RPꢀHPLC (XBridge C18 OBD column, eluent A:
water containing 0.1% TFA by vol, eluent B: water/acetonitrile: 1/4 by vol containing 0.1% TFA by
vol, detection at 215 nm, 25 mL/min, 0ꢀ15% eluent B in 15 min). The purified fractions were collected,
frozen and lyophilized to give 19.8 mg (57%) of 5c as the trifluoroacetate salt.
Catalyst 5c: HRMS calcd. for [M+H]+ C7H14O2NSe2 303.9350, found 303.9348 (ꢀ0.13 ppm).
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1H NMR (300 MHz) spectrum for compound 5c (DMFꢀd7, 310 K). δ 3.78 – 3.75 (t, J = 5.3 Hz, 4H),
3.41 – 3.36 (t, J = 7.2 Hz, 2H), 3.31 – 3.27 (t, J = 5.4 Hz, 4H), 2.81 – 2.76 (t, J = 7.2 Hz, 2H) ppm
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13C JMOD NMR (75 MHz) spectrum for compound 5c (DMFꢀd7, 310 K). δ 172.50 (C), 55.66 (2 ×
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CH2), 52.16 (CH2), 24.61 (2 × CH2) ppm. One C signal is not seen due to overlap with the residual
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signal from the solvent.
Preparative synthesis of peptide thioesters by SEA-thiol exchange
Synthesis of the MPA thioester 4b
TCEP·HCl (14.33 mg, 50 µmol, 100 mM final concentration) was dissolved in 6 M Gn·HCl, pH 7.2
0.2 M sodium phosphate buffer (0.5 mL). To the resulting solution (448 µL) were added diselenide 5c
(18.64 mg, 50 µmol, 100 mM final concentration), peptide 1b (4.55 mg, 3.1 µmol, final peptide conꢀ
centration 7 mM) and MPA (22 µL, 5% v/v). The pH was adjusted to 4.0 using 5 M NaOH and the
reaction mixture was stirred at 37 °C under nitrogen atmosphere. After completion of the reaction, the
mixture was diluted with waterꢀTFA 0.1% (5 mL) and purified by reversedꢀphase HPLC using a linear
waterꢀacetonitrile gradient containing 0.1% TFA to give the peptide 4b (2.8 mg, 63% yield).
The determination of the optical purity for the Cꢀterminal Val residue was performed by chiral GCꢀMS
analysis after acid hydrolysis in deuterated acid. The analysis was done by C.A.T. GmbH & Co. comꢀ
pany (Chromatographie und Analysen technik KG, Heerweg 10, Dꢀ72070 Tübingen, Germany). The
analysis indicated a DꢀVal content < 0.1%.
Peptide 4b: ESIꢀMS m/z = 1079.8 ([M+H]+), 540.6 ([M+2H]2+); calcd. for M (average): 1079.3, found:
1079.0. MALDIꢀTOF matrix = αꢀcyanoꢀ4ꢀhydroxycinnamic acid, positive detection mode, m/z calcd
for [M+H]+ (monoisotopic): 1079.6, found: 1079.3.
Synthesis of the MPA thioester 4e
TCEP·HCl (8.6 mg, 30 µmol, 100 mM final concentration) was dissolved in 6 M Gn·HCl, 0.2 M pH
7.2 sodium phosphate buffer (300 µL). To the solution were added diselenide 5c (12.48 mg, 30 µmol,
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