Internal Activation of Peptidyl Prolyl Thioesters in Native Chemical Ligation

Article abstract of DOI:10.1021/jacs.6b01202

Prolyl thioesters have shown significantly lower reactivities in native chemical ligation (NCL) in comparison to that of the alanyl thioester. This report describes a mild and efficient internal activation protocol of peptidyl prolyl thioesters in NCL without using any thiol-based additives, where the introduction of a 4-mercaptan substituent on the C-terminal proline significantly improves the reactivity of prolyl thioesters via the formation of a bicyclic thiolactone intermediate. The kinetic data indicate that the reaction rate is comparable to that of the reported data of alanyl thioesters, and the mechanistic studies suggest that the ligation of two peptide segments proceeds through an NCL-like pathway instead of a direct aminolysis, which ensures the chemoselectivity and compatibility of various amino acid side chains. This 4-mercaptoprolyl thioester-based protocol also allows an efficient one-pot ligation-desulfurization procedure. The utility of this method has been further demonstrated in the synthesis of a proline-rich region of Wilms tumor protein 1.

Full text of DOI:10.1021/jacs.6b01202

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Article
An Internal Activation of Peptidyl Prolyl Thioesters in Native Chemical Ligation
Yue Gui, Lingqi Qiu, Yaohao Li, Hongxing Li, and Suwei Dong
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b01202 • Publication Date (Web): 16 Mar 2016
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An Internal Activation of Peptidyl Prolyl Thioesters in Native
Chemical Ligation
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Yue Gui,† Lingqi Qiu,† Yaohao Li,† Hongxing Li,† and Suwei Dong†,*
†State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking Uni-
versity, 38 Xueyuan Road, Haidian District, Beijing 100191, China
Supporting Information Placeholder
ABSTRACT: Prolyl thioesters have shown significantly lower reactivities in native chemical ligation (NCL) in comparison
to the alanyl thioester. This report describes a mild and efficient internal activation protocol of peptidyl prolyl thioesters
in NCL without using any thiol-based additives, where the introduction of a 4-mercaptan substituent on the C-terminal
proline significantly improves the reactivity of prolyl thioesters via the formation of a bicyclic thiolactone intermediate.
The kinetic data indicate that the reaction rate is comparable to the reported data of alanyl thioesters, and the mechanis-
tic studies suggest that the ligation of two peptide segments proceeds through an NCL-like pathway instead of a direct
aminolysis, which ensures the chemoselectivity and compatibility of various amino acid side chains. This 4-
mercaptoprolyl thioester-based protocol also allows an efficient one-pot ligation-desulfurization procedure. The utility of
this method has been further demonstrated in the synthesis of a proline-rich region of Wilms tumor protein 1.
Introduction
possible ligation sites have been largely expanded, and a
number of thio-amino acids have been utilized as the N-
termini in NCL,14 even the secondary amine proline.15
Protein-based biomacromolecules have been broadly
studied for their essential roles in organisms. While bio-
chemical methods are still predominantly used for pro-
tein production,1 chemical peptide synthesis has shown
its irreplaceability among several research areas ，
including D-protein-based mirror image phage display,2
racemic protein crystallography,3 and syntheses of ho-
mogeneously modified proteins, especially glycopro-
teins.4 Over the past century, the requirements of di-
verse natural and unnatural peptides and derivatives in
biochemical research and drug design have prompted
the development of a series of new synthetic methods
for polypeptides, including solid-phase peptide synthe-
sis (SPPS) pioneered by Merrifield in the 1960s,5 as well
as native chemical ligation (NCL) reported by Kent and
associates in 1994 (Figure 1).6 These advancements in
turn accelerated the development of related research
fields. As a milestone advance, in conjunction with the
broad scoped metal-free desulfurization (MFD),7 NCL is
not only one of the most powerful methods in the field
of protein chemical synthesis, but it has inspired chem-
ists on seeking new chemical methods and tools to
study biological problems. A number of ligation meth-
ods were thus invented, including auxiliary-based liga-
Figure 1. Native chemical ligation (NCL).
For the requisite N-terminal peptide segment in NCL,
numerous endeavors have been made to obtain stable
and easy to handle peptidyl thioesters or surrogates
without racemizing the corresponding C-terminal ami-
no acid residues, particularly the fragments derived
from the routinely applied Fmoc-SPPS. The develop-
ments of several epimerization-free protocols were thus
investigated, including the usage of a stable alkyl thioe-
ster,6,16 Dawson’s resin,17 as well as Liu’s acyl hydrazide-
based protocol.18 Notably, these procedures all require
exogenous
thiol
additives,
such
as
4-
mercaptophenylacetic acid (MPAA)19 to accelerate the
reaction rate and ensure satisfactory ligation efficiency.
Application of MPAA expanded the scope of the C-
termini of peptidyl thioesters to almost all native amino
acids, including a number of previously problematic
residues,20 such as glutamate,21 valine,14g and isoleu-
cine.3c However, the inherent radical quenching proper-
ty of aryl thiols was incompatible with the radical-based
desulfurization method, which led to a required opera-
8
9
tion methods,
Staudinger ligation,
α-ketoacid-
hydroxylamine (KAHA) ligation,10 seleno-amino acids
based ligation,11 serine-threonine ligation (STL),12 and
peptide hydrazides-based ligation,13 etc. However, na-
ture always poses new challenges to chemists. For in-
stance, the synthesis of highly diverse peptide sequences
requires various possible ligation sites in the events
where two peptide segments joint together selectively.
Under the conditions of metal-free desulfurization, the
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a) Origins of the low reactivity of prolyl thioesters
tion of removing such species between the ligation and
dethiylation steps, compromising the overall efficiency
of the powerful ligation-desulfurization strategy. Chem-
ical synthesis of large polypeptides and complex pro-
teins in a more practical and efficient manner calls for
solutions to address this issue.
Peptide 1
Peptide 1
N
O
O
SR
O
H2N
HS
Peptide 2
ref. 24
+
extremely slow reaction
= 0.00038 M-1s-1
N
H
O
O
An alternative strategy to circumvent epimerization
at the ligation sites when preparing the peptidyl thioe-
sters, is activating peptide fragments at their C-terminal
proline or glycine sites, taking notes from the direct
condensation method for peptidyl fragments coupling,
where the peptidyl prolyl acids were epimerization-free
under typical coupling conditions.22 It would thus be
ideal to conduct NCL at the proline sites, as the corre-
sponding thioesters (or other derivatives) could be pre-
pared using common coupling reagents without the
concern of epimerization, and not require strictly con-
trolled reaction conditions.23 However, prolyl thioesters
have been suggested to be extremely unreactive under
typical NCL conditions (Figure 2a),24 which significantly
limited the use of peptidyl prolyl thioesters in the chem-
ical synthesis of polypeptides and proteins. Aiming to
exploit the reactivity of C-terminal prolyl esters under
NCL conditions, a number of research groups attempted
to tackle this problem. For instance, Danishefsky et al.
noticed that the p-nitrophenolester of proline was able
to react with cysteinyl peptides in weak acidic buffers, in
spite of the competing hydrolysis.25 In 2011, Durek et al.
reported that the use of prolyl selenoesters enabled rap-
id ligation with cysteine-containing peptides in the
presence of a selenol catalyst under mild buffered condi-
tions, accompanied by a competing side reaction that
forms unreactive thioesters on unprotected nonligation
site cysteines (Figure 2b).26 More recently, the Otaka
group developed a protocol where the prolyl thioesters
were activated in suitable ligation conditions, which
required the addition of MPAA (200 mM) and elevated
temperature (50 °C) (Figure 2b).27 Despite these ad-
vancements, a more effective and mild, thiol-additive-
free proline-ligation method would be desirable in the
preparation and studies of various peptide sequences
and proteins, including the ones containing proline-rich
regions (PRRs).28 Herein, we report the development of
such an optimized method based on a design of proline-
derived active intermediate.
SR
9
b) Previous work
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O
SeR
H2N
N
Peptide 2
DPDS buffer
Durek, et. al
O
+
O
Peptide 1
H
HS
SH
N
(5 equiv)
N
O
Peptide 2
O
Peptide 1
O
O
SR
H2N
HS
N
O
Peptide 2
200 mM MPAA
50
Otaka, et. al
+
C
O
Peptide 1
c) Our design
Peptide 2
HS
1) intramolecular
transthio-
esterification
HS
O
S
SR
NH2
Peptide 1
N
N
O
O
O
O
Peptide 1
thiolactone intermediate 2
1
2) intermolecular
transthio-
esterification
HS
HS
3) S
N
HS
H
acyl transfer
S
N
N
Peptide 2
N
O
O
Peptide 2
O
NH2
Peptide 1
O
O
O
Peptide 1
MFD
H
N
Pro-Ala
Peptide 1
Peptide 2
N
O
Peptide 2
O
Peptide 1
O
Figure 2. Prolyl thioesters vs. thioprolyl thioesters in native
chemical ligation.
interaction, and form a strained ring system, both of
which may promote the intermolecular transthioesteri-
fication, and the subsequent irreversible S→N acyl trans-
fer would afford a ligated peptide containing the thio-
Pro-Cys segment. The resulting peptides may be con-
verted to the native sequences with Pro-Ala residues
using the desulfurization protocol. Considering the po-
tential lability of 2 during the preparation of the corre-
sponding peptides, thio-prolyl thioester 1 was designed
as the precursor to 2.
Results and Discussion
Synthetic Design. In 2011, on the basis of a system-
atic study, Kent et al. proposed that the n→π* orbital
Synthesis of Unnatural Amino Acids and Peptide
Segments. We initiated our investigation by synthesiz-
ing the thio-proline derivatives 11 (Scheme 1). Starting
from the commercially available N-Boc-4-hydroxyl pro-
line (3), selective allylation of the carboxylate afforded
compound 4,30 followed by the activation of hydroxyl
with 4-(trifluoromethyl)benzene-1-sulfonyl chloride (5)31
to produce sulfonate 6. The thio substituent was intro-
duced in cis-configuration to the carboxylate using po-
interaction29 and steric hindrance of the N-carbonyl of
proline may reduce the electrophilicity of the prolyl thi-
oester carbonyl, thus resulting in an extremely low reac-
tivity (Figure 2a).24 We envisioned that a hetero-
bicyclo[2.2.1]septane structure 2 (Figure 2c) may pre-
clude the N-carbonyl oxygen/thioester carbonyl n→π*
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tassium thioacetate32 to form compound 7, ensuring the
DIEA (Scheme 2). The resulting preloaded resins 12 were
further employed in SPPS under Fmoc-based condi-
tions,34 followed by cleavage from the resin using a cock-
tail containing DCM/TFE/AcOH (3:1:1), which afforded
peptide 14 as a peptidyl acid with N-terminal and side-
chain protecting groups untouched. The C-terminal
carboxylic acid was then coupled with ethyl 3-
mercaptopropionate (15) to generate prolyl thioesters 16.
formation of the requisite cyclic bridge in the ligation
reactions. Hydrolysis of the thioacetate and carboxylate
was accomplished simultaneously using aqueous lithi-
um hydroxide, followed by protection of the free thiol
with methylthio group in the same reaction flask, af-
fording carboxylic acid 10a. The corresponding Fmoc
derivative 11a was generated from 10a in two steps, and
was ready for use in Fmoc-based SPPS.
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Scheme 2. Preparation of peptidyl thioprolyl thioesters. a)
2-Chlorotritylchloride resin, DIEA, DCM; b) Fmoc-based
SPPS; c) DCM/TFE/AcOH (3:1:1); d) 3-mercaptopropionate
(15), HATU, DIEA, DCM; e) TFA/TIS/H2O (95:2.5:2.5). TFE
= trifluoroethane, HATU = 1-[Bis(dimethylamino)methyl-
ene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluoro-
phosphate ， TFA = trifluoroacetic acid, TIS = triiso-
propylsilane, PG = protecting groups.
Scheme 1. Synthesis of thioproline derivatives 11a and 11b.
a) 3-Bromo-1-propene, DIEA, DMF, rt, 14 h, 93%; b) 5, TEA,
DMAP, DCM, rt, 2 h, 95%; c) KSAc, DMF, 40 °C, 3 h, 96%;
d) LiOH•H2O, THF/H2O (1:1), rt, 6 h; then 8, 2 h, 71%; e)
DCM/TFA/TES (8:2:1), rt, 3 h; f) FmocOSu, TEA, MeCN, rt,
6 h, 87% over two steps; g) LiOH•H2O, THF/H2O (1:1), rt, 7
h, 99%; h) TrtCl, DCM, rt, 19 h, 91%; i) 4 M HCl in 1,4-
dioxane, rt, 1 h; j) FmocCl, TEA, DCM, rt, 15 h, 97% over
two steps. DIEA = N,N-diisopropylethylamine, DMF = N,N-
dimethylformamide, TEA = triethylamine, DMAP = 4-
dimethylaminopyridine, DCM= dichloromethane, THF =
tetrahydrofuran, TFA = trifluoroacetic acid, TES = tri-
ethylsilane. Boc = tert-butyloxycarbonyl, All = propenyl, Trt
= triphenylmethyl, Fmoc = 9-fluorenylmethoxycarbonyl.
Table 1. Optimization of thioester formation.
a
Estimated conversions based on calculations of the ana-
lytical HPLC integrations integrations of free peptides ob-
tained from the global deprotection of 14b and 16b.
In a similar manner, a trityl-protected thio-proline
derivative 11b was synthesized, where carboxylic acid 9
with a free thiol group was isolated after hydrolysis of 7,
and was further protected with a trityl group to provide
10b. The final N-protecting-group manipulation afford-
ed Fmoc amino acid 11b in decent yield, thus providing
an alternative 4-mercaptan-proline derivative for further
evaluation of peptide synthesis and ligation conditions.
In these coupling reactions, several activating rea-
gents, including EDC (Table 1, Entry 1), PyBOP (Entry 2)
and HATU (Entry 3), were evaluated, and the HATU-
mediated condition was found to be optimal.35 After
global deprotection using TFA/TIS/H2O (95:2.5:2.5),
purification by preparative reverse-phase high perfor-
mance liquid chromatography (RP-HPLC) provided the
desired peptidyl thioesters 17 with either a methylthio-
protected thiol on the C-terminal proline (17a), or a free
thiol in the case of peptide 17b. On the other hand, the
east-side peptides containing Cys or thio-amino acid
derivatives at the N-termini were synthesized following
the Fmoc-based SPPS protocol and deprotection proce-
dures.36
While it was plausible to incorporate the thio-
proline moiety via direct condensation with side-chain
protected peptide fragment,33 we decided to preload the
proline derivatives on resin to take full advantage of the
convenience of SPPS, and to circumvent any possible
racemization in the preparation of peptidyl thioesters.
Accordingly, Fmoc-amino acids 11 were loaded on 2-
chlorotritylchloride resin in DCM in the presence of
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Experimental Evaluation and Optimization. We
be either isolated separately using prep-HPLC, or as a
mixture subjected to the next step.
chose two peptide segments 18a and 20a (Scheme 3) to
investigate suitable reaction conditions, where the ligat-
ed peptide product would resemble the sequence of a
proline-rich region in cornifin-B protein.37 With the
requisite peptide segments in hand, we next investigat-
ed the ligation reaction under a typical NCL condition
(6 M Gn∙HCl, 200 mM NaH2PO4, 20 mM TCEP∙HCl, pH
7.0, room temperature),24 and the reaction progress was
monitored using HPLC-MS. When equal amounts of
peptides 18a and 20a were mixed in buffer, it was ob-
served that a new peak formed almost immediately after
the addition of buffer (Figure 3), and analysis of the
mass spectra suggested a thiolactone-containing inter-
mediate 19a as we originally designed, this observation
indicated a rapid intramolecular trans-thioesterification
favored the formation of such a bicyclic structure in the
reaction buffer.36
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a
SSMe
StBu
PKSKEP SR
H
H
CHPKV OH
+
18a: R = CH2CH2CO2Et
20a
S
H
CHPKV OH
H
AHPKV
OH
N
19a
O
H
PKSKE
20b
20c
Figure 3. HPLC-MS traces of ligation between 18a and 20a.
Reaction conditions: 18a (3 mM), 1.0 equiv 20a, 400 μL of
NCL buffer (6 M Gn⋅HCl, 200 mM NaH2PO4, 20 mM
TCEP⋅HCl, pH 7.0), room temperature. a UV trace from LC-
MS analysis of the reaction quenched right after the
addition of buffer.
Conversion: 70%b
Isolated yield: 62%c
Conditionsa
SH
SH
+
+
H
H
PKSKEPCHPKV OH
PKSKEPAHPKV
H
OH
PKSKEPAHPKV OH
21a
22a
23a
Scheme 3. Ligation between 18a and 20a. a Reaction condi-
tions: 18a (3 mM), 1.0 equiv 20a, 400 μL of NCL buffer (6 M
Gn⋅HCl, 200 mM NaH2PO4, 20 mM TCEP⋅HCl), room tem-
perature, 8 h. b Estimated conversion based on calculations
of the analytical HPLC integrations of peptides 20b and 20c,
and products 21a, 22a, and 23a. c Isolated yield after HPLC
purification. TCEP = Tris(2-carboxyethyl)phosphine.
In order to further probe the desulfurization process,
hoping to obtain the product with native Pro-Cys seg-
ment, we elucidated the structure of the mono-
desulfurized product by comparing its HPLC retention
time
PKSKEP(SH)AHPKV-OH (s22a) and H-PKSKEPCHPKV-
OH The co-injection experiments
(s22a’).36
against
two
authentic
samples,
H-
As the reaction proceeded, the desired ligation
product 21a was observed, along with the formation of
products 22a and 23a, which were desulfurized peptides
as indicated by the mass spectra. Based on the integra-
tions of UV signals of reactive starting peptide segment
H-CHPKV-OH (20b), desulfurized east-side peptide H-
AHPKV-OH (20c), and all ligation products (21a-23a),
the conversion of this reaction after 8 h was calculated
as ca. 70%. The desulfurized products may be resulted
unambiguously assigned that under the reaction
conditions, the –SH group of Cys was reduced prior to
the –SH on Pro, which suggested that it would be
difficult to selectively remove the thiol on Pro and leave
Cys untouched.
To improve the ligation efficiency, an alternative
peptide 18b (Scheme 4) and several reaction conditions
were evaluated.36 The ligation between protection-free
thioester 18b and 20a was found to be cleaner than that
between 18a and 20a, leading to higher isolated yield of
desired products (70%). We found that using a slight
excess (1.2 equiv) of either starting material improved
the conversion under neutral pH conditions.
from
a
TCEP-promoted reaction involving
a
phosphoranyl radical-based mechanism,7, 38, 39 which was
supported by the fact that formation of side products
was suppressed when using a phosphine-free DTT-
buffer.36,40 Our observation was also in accord with a
previous report where prolonged ligation time led to
desulfurized product under NCL conditions.26 In our
case, the extraneous thiol groups needed to be
eventually removed to reveal the native amino acid
residues, thus desulfurization occuring in the ligation
step was inconsequential. Regardless, all products could
StBu
SH
Conditionsa
+
PKSKEP SR
H
CHPKV OH
21a
22a 23a
+ +
H
20a
18b
R = CH2CH2CO2Et
Scheme 4. Ligation between 18b and 20a. a Reaction condi-
tions refer to the Supporting Information.
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Since desulfurization of starting peptide 20a was also
thiolproline-containing peptide 20g, hoping to conquer
the challenging Pro-Pro ligation, neither the ligated
thioester intermediate from intermolecular trans-
thioesterification, nor the final amide bond forming
product, was detected (Entry 5). Further attempts under
elevated temperature (60 °C) did not generate any
desired product, but promoted an intramolecular
condensation. In this case, the free amino group at
either the N-terminus, or the side chain of the west-side
peptide, directly reacted with the thiolactone moiety,
producing a cyclic peptide as indicated by mass
observed in the reaction, where the N-terminal Cys was
converted to Ala to afford an unreactive material, we
wondered whether this process was competing with the
desired ligation, and possibly diminishing the reaction
conversion. To test this hypothesis, tert-butylthiol was
added as a scavenger of the free radicals in the reaction
to eliminate the dethiylations.36 As a result, no desulfu-
rized product was detected (Figure 4), and the conver-
sion was approximately the same as the one without the
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t
addition of BuSH. This finding suggests that formation
of 2oc may have no significant impact on the reaction
conversion. Based on these results, and in consideration
of reaction scales in most cases of protein chemical syn-
thesis, we conducted further experiments at the concen-
tration of 3 mM for west-side peptidyl thioesters, and
the reactions were stirred in pH 7.0 buffer at room tem-
perature for 8 h.
spectrometry.36
It
was
noticeable
that
this
intramolecular aminolysis process was not observed at
room temperature, which underscores the distinct
reactivity of the bicyclic thiolactone structure in NCL
conditions, in contrast to the observed aminolysis in
THF reported by Brands, et al.41 The unsuccessful Pro-
Pro ligation suggested that two prolines might be too
strained to adopt a suitable conformation for the S→N
acyl transfer, and whether the transthioesterification
would be affected was unclear thus far.
SH
Further explorations of a number of sequences
consisting of commonly used natural amino acids
demonstrated the compatibility of diversed functional
groups in the thiolactone-mediated ligation (Entries 6-
10). In particular, Lys, Ser and Thr were found to be
tolerated and no direct aminolysis or esterification was
H
PKSKEPCHPKV OH
21a
observed,
which
further
underscored
the
chemoselectivity of this internal activation of prolyl
thioesters, and suggested an NCL-type process. Peptides
containing O-mannosylated Ser (Entry 7), or Acm-
protected Cys (Entry 10), were also proven to be
compatible under the reaction conditions, suggesting
potential applications of this strategy in the synthesis of
cysteine-containing proteins, or glycopeptides and
glycoproteins.
S
N
O
H
PKSKE
19a
Figure 4. HPLC-MS traces of ligation between 18b and 20a.
18b (3 mM), 1.2 equiv 20a, 200 μL of NCL buffer (6 M
Gn⋅HCl, 200 mM NaH2PO4, 20 mM TCEP⋅HCl, pH 7.0), 20
μL of tBuSH, room temperature.
In order to further evaluate this thioprolyl thioester-
based method in different sequences, in particular the
problematic ones in Otaka’s studies,27 two peptides 18g
(Entry 11) and 18h (Entry 12) with Gly and Ser adjacent to
prolyl thioester, respectively, were prepared. While 18g
was found to be prone to forming an amino-acid-
deleted byproduct in the reported procedure, under our
standard conditions the ligation reaction with peptide
20i generated the desired product in decent yield
without any observed amino acid deletion. However,
ligation of peptide 18h afforded the product in only 37%
isolated yield, similar to the previously reported result,27
indicating that such poor yield was probably due to an
unstable sequence under the buffered conditions.
Scope and limitations. To evaluate the applicability of
peptidyl thioproline thioester in reactions with peptides
containing non-cysteine N-termini, several sequences
with previously reported N-terminal β- or γ-thio-
containing amino acid derivatives were tested (Table 2).
Peptide 20d containing β-methyldisulfide-Val,14q
a
representative sterically hindered ligation site, reacted
with 18b to afford the ligation products in a combined
conversion of 92% within 8 h (Entry 2). Considering that
the ligation of penicillamine usually requires more than
12 h,14q this result demonstrated the high reactivity of
the thiolactone intermediate.
When peptides containing γ-thio-Glu14e (Entry 3) and
β-thio-Asp14b,14c (Entry 4) as the N-termini were
subjected to the optimized conditions, the reactions
proceeded smoothly, and generated the ligated products
in good yields. However, when we tested the trans-4-
It is important to point out that the desulfurized
products were observed in most of our examples,
although the amount of dethiylation varied in different
peptide sequences. Such desulfurization process during
ligation was not substantial in previous studies,14,27
presumably because the –SH groups were isolated and
exogenous thiols (e.g. tert-butylmercaptan) were
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Table 2. Substrate scope and limitations.a
1
2
3
4
SH
R2S
H2N
R
O
N
+
peptide 2
XH
CO2Et
X = O or NH
peptide 1
H
R1
=
SR1
20
O
18
5
6
7
8
SH
SH
H
H
R
R
SH
R
N
N
N
peptide 2
XH
peptide 2
peptide 2
XH
XH
+
peptide 1
+
peptide 1
peptide 1
H
H
H
N
N
N
21
22
23
O
O
O
O
O
H
O
H
H
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
West-side
peptide
East-side
peptide
Conversionb
%
Yieldc %
Entry
1
total
85
(21, 22, 23)
HS
tBuSS
H2N
SR
SR
SR
SR
18b
20a
85
(23, 27, 35)
OH
OH
HPKV
N
O
PKSKE
H
H
H
H
O
O
HS
MeSS
H2N
HPKV
2
3
18b
18b
20d
20e
92
85
78
71
(28, 25, 35)
(14, 31, 26)
N
N
O
O
O
PKSKE
HS
COOH
SStBu
SPGYS
NH2
H2N
PKSKE
HS
O
HS
H2N
COOH
SPGYS
4
5
18b
18b
20f
80
69
(69, 0, 0)
N
N
NH2
PKSKE
HS
O
MeSS
SR
OH
HPKV
NDd
20g
NA
NA
N
O
H
PKSKE
HS
H
H
O
HO
OH
O
HO
HO
tBuSS
H2N
O
SR
SR
SR
SR
6e
66
82
(66, 0, 0)
72
84
N
N
N
N
18b
18c
20h
20a
OH
GY
GP
O
O
O
O
PKSKE
HS
N
H
O
O
tBuSS
OH
OH
HPKV
HPKV
7
(40, 23, 19)
H2N
EISKG
HS
H
O
O
tBuSS
H2N
8
18d
18e
20a
78
52
(12, 18, 22)
MGQAL
HS
H
H
tBuSS
H2N
OH
OH
HPKV
HPKV
9
20a
20a
93
77
90
75
(13, 41, 36)
(35, 25, 15)
WASKD
AcmS
O
O
O
SH
tBuSS
H2N
H
EATK
N
10
18f
N
H
SR
O
O
O
HS
tBuSS
H2N
SR
SR
YRANK NH2
N
N
11
12
18g
18h
20i
20i
97
96
78
37
(0, 0, 78)
(0, 0, 37)
LYRG
HS
H
H
tBuSS
H2N
NH2
YRANK
O
LYRS
O
a Reaction conditions: 18 (3 mM), 1.2 equiv 20, 200 μL of NCL buffer (6 M Gn.HCl, 200 mM NaH2PO4, 20 mM TCEP.HCl, pH 7.0),
room temperature, 8 h. b Estimated conversion based on calculations of the analytical HPLC integrations of peptides 20 and the
corresponding desulfurized peptide, and products 21, 22 and 23. c Isolated yield after HPLC purification. Not detected on
d
HPLC-MS. e 20 μL of tBuSH was added.
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Mechanistic Studies. To further probe the reactions
required as hydrogen donors to accelerate radical
1
2
3
4
5
6
7
8
using 4-mercapto-prolyl thioester, we carried out
mechanistic investigations through several control
experiments (Table 4). Firstly, the poor reactivity of
normal prolyl thioesters was verified by conducting the
reaction between the peptidyl prolyl thioester 24a and
the N-terminal cysteinyl peptide 20a in a ligation buffer
containing 30 mM MPAA (Entry 1). No ligation product
was observed, which was consistent with the results
obtianed by the Kent group.24 When an N-terminal
alanyl peptide 20c was subjected to the ligation with 18b,
the reaction did not afford any ligated peptide, which
further confirmed that the thioprolyl thioester react via
an NCL-like process instead of direct aminolysis (Entry
2). Moreover, the peptidyl thioester 24b containing a
trans-4-mercapto substituted C-terminal proline was
also found to be unreactive in the reaction conditions
(Entry 3). These experimental results, along with the
kinetic data we obtained (Figure 5), clearly indicate the
significantly improved reactivity of prolyl thioesters by
the introduction of a cis-4-mercapto substituent.
propagation of the desulfurization. It could be possible
that in our cases the -SH on proline may act as an
internal hydrogen donor, leading to an accelerated rate
of radical-based desulfurization on the adjacent
thioamino acids, particularly the sequences that better
suit the conformational requirements for such
intramolecular hydrogen delivery.
Desulfurization. The formation of native proline resi-
dues requires the removal of 4-mercaptan group on the
ligation site prolines. Accordingly, Danishefsky’s proto-
col was employed on the obtained ligation products
(Table 3).7 In the representative cases, the dethiylation
proceeded efficiently to reveal the native Pro-Ala (Entry
1), Pro-Val (Entry 2), Pro-Glu (Entry 3) and Pro-Asp
(Entry 4) residues in full conversion and excellent
isolated yields. Furthermore, as the external activation
using aryl thiol additives was not necessary in the
thiolactone-mediated procedure, the ligation and
desulfurization steps were able to be streamlined into a
straightforward one-pot protocol. In the case of reaction
between 18b and 20a, we found that eliminating the
purification step after ligation, and directly conducting
desulfurization in the same reaction flask afforded
product 23a in 85% isolated yield, which was more
efficient in comparison to the stepwise procedure (ca.
80% of 23a over two steps, cf. Table 3, entry 1). This
improved yield using a one-pot operation versus a
multistep reaction-purification procedure was in accord
with previous studies. 42 The nonessential of radical
quenching additives19,43 eased the operation in our case,
where after the ligation step only the addition of
reagents for dethiylation under an argon atmosphere
was required, thus improving the overall efficiency.14e,44
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 4. Mechanistic studies.a
Table 3. Metal-free desulfurization on thioproline-
containing peptides.a
a
Reaction conditions: 24 or 18b (3 mM), 1.0 equiv 20, 200
μL of NCL buffer (6 M Gn.HCl, 200 mM NaH2PO4, 20 mM
TCEP.HCl, pH 7.0), room temperature, 8 h. b The reaction
buffer contained 30 mM MPAA except for standard condi-
tions. c Not detected on HPLC-MS.
Although the observed mass from HPLC-MS analysis
provided evidence for the thiolactone intermediate 19a
in the reaction, at this stage we could not rule out the
possibility that the activation was originated from the
conformational change of proline resulting from the 4-
substitution.45 In order to determine whether 19a was
the actual active intermediate, we prepared peptide 24c
containing a proline with 4-methylthio substituent,
a
Reaction conditions: 21 (3 mM), 200 μL of NCL buffer (6
M Gn.HCl, 200 mM NaH2PO4, 20 mM TCEP.HCl, pH 7.0),
200 μL of 0.5 M bond-breaker® TCEP solution (Pierce), 20.0
μL of 2-methyl-2-propanethiol, and 10.0 μL of radical initia-
b
tor (0.1 M VA-044 in water), 37 °C, 1 h. Estimated conver-
sion based on calculations of the analytical HPLC integra-
c
which would presumably introduce
a
similar
tions of peptides 21 and 23. Isolated yield after HPLC
purification.
conformational change as that in 18b, but could not
form a bridged bicyclic structure (Table 4, entry 4).
From the HPLC-MS analysis of the reaction between
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24c and 20a under our optimized conditions, we could resulting thioester could not proceed further to generate
1
2
3
4
not detect any ligation product, indicating that the key
bicyclic thiolactone 19a is most likely the reactive
intermediate, while the effect from the 4-substitution
was not obvious in this case.
the corresponding Pro-Pro segment.
HS
HS
OH
5
6
7
8
N
O
O
N
H2N
Peptide 1
H2O
O
C
B
HN
Peptide 1
c
b
O
c
S
HS
a
H2N Peptide 1
N
9
SR
O
HS
N
b
O
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
2
Peptide 2 OH
H2N
Peptide 1
O
H2N
thiolactoneintermediate
1
O
a
HS
HS
O
H
N
Peptide 2 OH
SH
S
Peptide 2 OH
N
N
O
O
H2N
H2N Peptide 1
O
A
H2N Peptide 1
O
O
A'
Scheme 5. Proposed mechanism for the thiolactone-
mediated ligation reaction. Pathway a: intermolecular
transthioesterification with with C-terminal peptide;
Pathway b: intramolecular aminolysis; Pathway c:
hydrolysis of intermediate 2.
Synthetic Application. The cis-4-thiol-prolyl thioester-
mediated ligation protocol was employed to synthesize
a proline-rich region (PRR) of Wilms tumor protein 1
(WT1), in order to evaluate its applicability in the
synthesis of proline-rich polypeptides (Scheme 6). WT1
is a zinc finger transcription factor that has an essential
role in the development of urogenital system, and
regulates several reproductive genes.48 It has also been
reported that WT1 may have a potential role in
luteinizing hormone β (LHβ) transcription in clonal
mouse gonadotrope LβT2 cells.49 However, the detailed
function of WT1, as well as the proline-rich regions
presented in this protein, has not been fully understood.
The full length of WT1 contains 449 amino acids, where
prolines make up approximately 10.5% of the protein.
We chose one of the most proline-rich segments, Ala25-
Gln87, as our synthetic target, which includes a stretch of
nine contiguous prolines and contains 19 prolines in
total.
Figure 5. a) Reaction conversion as a function of time for
the reactions between 18b and 20a, b) Reciprocal of con-
centration as a function of time for the reactions between
18b and 20a: [A], combined concentration of 20a and 20b;
[A0], initial concentration of 20a. Data are the average of
three replicates. The determined second order rate con-
stant k = 0.0961 mM-1∙h-1 = 0.027 M-1∙s-1, which is approxi-
mately in the same magnitude as the reported data of ala-
nyl thioester (0.087 M-1∙s-1).24 Reaction conditions: 18b (3
mM), 1.0 equiv 20a, 200 μL of NCL buffer (6 M Gn.HCl, 200
mM NaH2PO4, 20 mM TCEP.HCl, pH 7.0), room tempera-
ture.
On the basis of all experimental results, a reaction
mechanism was proposed as shown in Scheme 5. The
equilibrium between thioprolyl thioester 1 and the
thiolactone intermediate 2 preferred the latter bicyclic
46
structure in buffer,
which possessed
a
more
Retrosynthetically, the sequence could be disected
into a C-terminal thio-prolyl thioester Ala25-thioPro54 (25)
and an N-terminal cysteinyl peptide Cys55-Gln87 (26).
Accordingly, segments 25 and 26 were prepared using
the optimized protocols described above. Ligation
reaction was conducted under the standard conditions
to afford the ligated peptide 27 in 82% isolated yield
after eight hours. After desulfurization, native segment
28 was obtained in 74% overall yield. Noticeably, our
attempts to directly prepare this 63 amino acids
sequence failed to afford any desired peptide under the
same SPPS conditions, which further underscores the
synthetic difficulties inherent in proline-rich sequences.
electrophilic thioester carbonyl. At room temperature,
Pathway a was favored for peptides containing N-
terminal cysteine or several other thio-amino acid
analogs, where the reversible intermolecular trans-
thioesterification followed by irreversible S→N acyl
transfer led to the elongated peptide, similar as in the
original NCL. At the same time, the intramolecular
aminolysis (Pathyway b) is suppressed under such
conditions. The hydrolysis of 2 (Pathway c) highly
depends on the acidity of the reaction buffer, where
increasing pH would promote such a process.47 In the
case of east-side peptide with an N-terminal trans-4-
thiol-proline residue, although we did not observe the
A’-type cross-linked thioester, the generation of A’ could
not be completely ruled out.15a Nevertheless, the
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 6. Synthesis of PRR Ala25-Gln87 (28) of WT1. a Reaction conditions: 25 (3 mM), 1.2 equiv 26, 200 μL of NCL buffer (6 M
Gn·HCl, 200 mM NaH2PO4, 20 mM TCEP·HCl, pH 7.0), room temperature, 8 h. b Reaction conditions: 200 μL of NCL buffer (6 M
Gn·HCl, 200 mM NaH2PO4, 20 mM TCEP·HCl, pH 7.0), 200 μL of 0.5 M bond-breaker® TCEP solution (Pierce), 20.0 μL of 2-
methyl-2-propanethiol, and 10.0 μL of radical initiator (0.1 M VA-044 in water), 37 °C, 1 h.
Conclusion. Among all natural amino acids, glycine
and proline have attracted significant attentions in pep-
tide synthesis, due to their tolerance in various activa-
tion conditions without the concern of racemization.22
In particularly before the era of native chemical ligation,
syntheses of peptides/proteins using a direct fragment
condensation strategy were mostly conducted at a Gly
or Pro site.50 As we have shown in this work, the suc-
cessful utilization of proline as the C-terminal reaction
site in NCL offered the convenience during the prepara-
tion of the corresponding thioester, and at the same
time took full advantage of the mild and highly
chemoselective ligation conditions without side-chain
protections. Ensured by the highly effective metal-free
desulfurization protocol, the activation of otherwise less
reactive prolyl thioester was accomplished using an easi-
ly removable internal mercaptan group, which allowed
for the ligation and desulfurization in the same flask
with satisfactory overall efficiency. As demonstrated by
synthesizing a proline-rich sequence, we believe this
strategy will have further utility in the preparation of
important proteins and glycoproteins, and would be
complementary to the existing toolbox of chemical liga-
tions. The once challenging and avoided proline site is
now a synthetically useful, or in some cases even more
effective choice as the ligation site under the thiol addi-
tive-free conditions. The strategy of utilizing intramo-
lecular reactions and strained structures to accelerate
desired transformations may be further applied to reac-
tions of other difficult C-terminal amino acid residues,
e.g. Val, Leu, Thr, etc., as well as possible developments
of novel bio-orthogonal transformations. This strategy
has been inspired not only by the logic of ligation-
desulfurization,7,51 more importantly, also the rational of
tuning bio-level large molecules utilizing chemical prin-
ciples and reactivities used on small molecules, which
has also been the most valuable inspiration from NCL to
the methodology developments in the field of pep-
tide/protein synthesis, and will continue generating
huge impacts on research at the chemistry-biology in-
terface.
ASSOCIATED CONTENT
General experimental procedures, including spectro-
scopic and analytical data for new compounds. This ma-
terial is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
* dongs@hsc.pku.edu.cn
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENT
1
The authors are grateful for financial support from Pe-
king University Health Science Center (BMU20130354),
State Key Laboratory of Natural and Biomimetic Drugs,
the National Recruitment Program of Global Youth Ex-
perts (1000 Plan), and the National Natural Science
Foundation of China (21502005). We thank Dr. Yuan
Wang, Weiqing Zhang, and Xulin Sun for spectroscopic
assistance, Professor Qian Wan (Huazhong University
of Science and Technology) for helpful discussions, and
Yuankun Dao and Changdong He (Peking University)
for the experimental assistance.
2
3
4
5
6
7
8
9
7 Wan, Q.; Danishefsky, S. J. Angew. Chem. Int. Ed. 2007, 46,
9248-9252.
8 (a) Canne, L. E.; Bark, S. J.; Kent, S. B. H. J. Am. Chem. Soc.
1996, 118, 5891-5896; (b) Offer, J.; Boddy, C. N.C.; Dawson, P. E.
J. Am. Chem. Soc. 2002, 124, 4642-4646; (c) Wu, B.; Chen, J.;
Warren J. D.; Chen, G. Hua, Z.; Danishefsky, S. J. Angew. Chem.
Int. Ed. 2006, 45, 4116-4125; (d) Brik, A.; Yang, Y.; Ficht, S.;
Wong, C. J. Am. Chem. Soc. 2006, 128, 5626-5627; For a recent
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York, 2015; p 36-46.
10
11
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14
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16
17
18
19
20
21
22
23
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2000, 2, 1939-1941.
10 Bode, J. W.; Fox, R. M.; Baucom, K. D. Angew. Chem. Int.
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