Peptide o-aminoanilides as crypto-thioesters for protein chemical synthesis

Article abstract of DOI:10.1002/anie.201408078

Fully unprotected peptide o-aminoanilides can be efficiently activated by NaNO₂ in aqueous solution to furnish peptide thioesters for use in native chemical ligation. This finding enables the convergent synthesis of proteins from readily synthesizable peptide o-aminoanilides as a new type of crypto-thioesters. The practicality of this approach is shown by the synthesis of histone H2B from five peptide segments. Purification or solubilization tags, which are sometimes needed to improve the efficiency of protein chemical synthesis, can be incorporated into the o-aminoanilide moiety, as demonstrated in the preparation of the cyclic protein lactocyclicin Q.

Full text of DOI:10.1002/anie.201408078

Angewandte
Chemie
DOI: 10.1002/anie.201408078
Native Chemical Ligation
Peptide o-Aminoanilides as Crypto-Thioesters for Protein Chemical
Synthesis**
Jia-Xing Wang, Ge-Min Fang, Yao He, Da-Liang Qu, Min Yu, Zhang-Yong Hong, and Lei Liu*
Abstract: Fully unprotected peptide o-aminoanilides can be
efficiently activated by NaNO2 in aqueous solution to furnish
peptide thioesters for use in native chemical ligation. This
finding enables the convergent synthesis of proteins from
readily synthesizable peptide o-aminoanilides as a new type of
crypto-thioesters. The practicality of this approach is shown by
the synthesis of histone H2B from five peptide segments.
Purification or solubilization tags, which are sometimes needed
to improve the efficiency of protein chemical synthesis, can be
incorporated into the o-aminoanilide moiety, as demonstrated
in the preparation of the cyclic protein lactocyclicin Q.
P
eptide thioesters are key intermediates in protein chemical
synthesis.[1] Although the direct solid-phase peptide synthesis
(SPPS) of peptide thioesters is possible by the use of tert-
butoxycarbonyl (Boc) chemistry,[2] the preparation of peptide
thioesters bearing acid-sensitive groups requires the 9-fluor-
enylmethyloxycarbonyl (Fmoc) method.[3] Among the Fmoc
methods for thioester preparation, an efficient approach
developed by Blanco-Canosa and Dawson involves the use of
a 3,4-diaminobenzoic acid (Dbz) linker (Scheme 1A).[4] Key
to this approach is the on-resin transformation of an o-
aminoanilide into an N-acylurea derivative upon treatment
with p-nitrophenylchloroformate and a base. Subsequently,
the resin-bound peptide N-acylurea can be deprotected and
cleaved from the resin with trifluoroacetic acid (TFA). The
resulting unprotected peptide N-acyl benzimidazolinone
(peptide-Nbz) can undergo thiolysis in a neutral aqueous
buffer to generate a peptide thioester for use in native
Scheme 1. Two different methods for the activation of peptide
o-aminoanilides: A) the method described by Blanco-Canosa and
Dawson;[4] B) our method. PG=protecting group.
chemical ligation (NCL).[5] As the Dbz-functionalized resins
are commercially available,[6] this approach has been applied
to the chemical synthesis of many proteins, such as ubiquiti-
nated histone H2B,[7] exon 1 of the Huntingtin protein,[8] and
tetraubiquitin.[9] However, as p-nitrophenylchloroformate is
incompatible with many nucleophilic groups, the activation
step must be conducted with full side-chain protection. It
remains a challenge to activate peptide o-aminoanilides
under protecting-group-free conditions so that peptide o-
aminoanilides can be used with more flexibility for the
chemical synthesis of proteins from multiple peptide seg-
ments.
Herein, we describe a new strategy based on the NaNO2-
promoted activation of fully unprotected peptide o-amino-
anilides to thioesters in an aqueous buffer (Scheme 1B). This
approach enables peptide o-aminoanilides to serve as a new
class of peptide crypto-thioesters (i.e. masked peptide thio-
esters which, unless activated, are inert in NCL) that are
useful in the condensation of multiple peptide segments.[10] In
a few cases, the Fmoc-SPPS of peptide hydrazides was
[*] J.-X. Wang,[+] G.-M. Fang,[+] Prof. Dr. L. Liu
Tsinghua-Peking Center for Life Sciences, Key Laboratory of
Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry
of Education), Department of Chemistry, Tsinghua University
Beijing 100084 (China)
E-mail: lliu@mail.tsinghua.edu.cn
Y. He, D.-L. Qu
School of Life Sciences
University of Science and Technology of China
Hefei 230026 (China)
M. Yu, Prof. Z.-Y. Hong
State Key Laboratory of Medicinal Chemical Biology
College of Life Sciences, Nankai University
Tianjin 300071 (China)
[+] These authors contributed equally.
[**] This study was supported by the 863 Program of the Ministry of
Science and Technology (2012AA02A700), the National Basic
Research Program of China (973 Program, No. 2013CB932800), and
the NSFC (No. 91313301).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201408078.
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Table 1: Scope of the ligation of peptide o-aminoanilides.[a]
reported to be difficult, and peptide o-aminoanilides had to
be made.[11] Moreover, the carboxy group of the o-amino-
anilide moiety provides an ideal site for the attachment of
[12]
auxiliary groups, such as an Arg6 or His6[13] tag.
Our study started with the ligation of H-Leu-Tyr-Arg-Ala-
Asn-Dbz-Ala-OH (2a) with H-Cys-Lys-Tyr-Ala-His-NH2 (3;
Figure 1). Peptide 2a was synthesized by the procedure
described by Blanco-Canosa and Dawson.[4] The two peptides
(1.0 :1.4) were dissolved in an aqueous buffer containing 6m
Entry
Xaa (2)
Ligation time [h]
Conversion [%][b]
1
2
3
4
5
6
7
8
9
Asn (2a)
Ala (2b)
Arg (2c)
Gln (2d)
Lys (2e)
Met (2 f)
Ser (2g)
Tyr (2h)
Val (2i)
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
7.0
1.5
80
78
90
84
89
88
89
85
88
0[c]
10
Asp (2j)
[a] The reactions were performed in an aqueous buffer containing 6m
Gn·HCl and 0.2m Na2HPO4. The activation step was conducted at pH 3
for 20 min in an ice–salt bath. The ligation step was performed at pH 6.9
at room temperature with MPAA. [b] Conversion was determined by
HPLC with benzamide as the internal standard. [c] Only the hydrolysis
product H-Leu-Tyr-Arg-Ala-Asp-OH was observed.
To examine the extent of racemization in the process, we
compared 4b produced from 2b and 3 with authentic H-Leu-
Tyr-Arg-Ala-(l-)Ala-Cys-Lys-Tyr-Ala-His-NH2 and H-Leu-
Tyr-Arg-Ala-(d-)Ala-Cys-Lys-Tyr-Ala-His-NH2. HPLC anal-
ysis showed that the extent of racemization is less than 1%.
This observation is similar to the situation with peptide
hydrazides,[10b] thus confirming that NaNO2 activation does
not cause racemization at the C-terminal amino acid.
Furthermore, in a ligation reaction of 2a with 3, we added
free alanine (20 equiv). We did not observe any reaction
between 2a and alanine, thus confirming the chemoselectivity
of the ligation.
LC–MS analysis of the reaction mixture after the activa-
tion of 2b by NaNO2 revealed the formation of a peptide
benzotriazole 1. Thus, we propose that activation/ligation of
the o-aminoanilide proceeds through the mechanism depicted
in Scheme 1. The NaNO2-promoted conversion of an acyl o-
aminoanilide into an acyl benzotriazole was observed pre-
viously.[16] Gilley and Kobayashi also showed that an acyl
benzotriazole could be converted into a thioester by thiol-
ysis.[17] Despite these previous observations, the present study
established for the first time the practicality of using fully
unprotected peptide o-aminoanilides for protein chemical
synthesis.
As well as intermolecular ligation, we also tested the
intramolecular ligation/cyclization of peptide o-aminoanilides
in the synthesis of the cyclic peptide trypsin inhibitor SFTI-
1[18] (Figure 2). The linear precursor 5 was prepared by Fmoc-
SPPS. After the activation of 5 by NaNO2 and the addition of
MPAA, the cyclization reaction proceeded smoothly with
concomitant formation of the disulfide bond to afford SFTI-1,
which was isolated in 40% yield (Figure 2A). NMR spectro-
scopic structural analysis of the synthetic SFTI-1 (Figure 2B)
showed an ordered b-hairpin structure consistent with
previous reports.[18]
Figure 1. Reaction of 2a with 3. Analytical HPLC chromatograms
(l=214 nm) are shown: A) before the addition of NaNO2; B) after
ligation for 1.5 h. Benzamide was used as an internal standard to
measure conversion. The peak marked with an asterisk corresponds to
the benzotriazole released in the reaction.
guanidine hydrochloride (Gn·HCl) and 0.2m Na2HPO4 at
pH 3. In an ice–salt bath at À108C, a solution of NaNO2
(6 equiv) was added to activate 2a. After 20 min, an aqueous
solution of 4-mercaptophenylacetic acid (MPAA, 60 equiv)
was added to the reaction mixture.[14] The pH value was then
adjusted to 6.9 to initiate NCL at room temperature. The
ligation proceeded smoothly to give 4a with 80% conversion
after 1.5 h. A similar conversion was observed when activa-
tion with NaNO2 was conducted at pH 4. At pH 5, the
activation was inefficient and led to less than 10% conversion.
To probe the scope of the method, we tested a number of
model peptides with various C-terminal amino acid residues
(Ala, Arg, Gln, Lys, Met, Ser, Tyr, Val). All of these ligations
afforded the desired products with high conversion (78–
90%), although the ligation at a sterically hindered site (i.e.
Val) required a longer reaction time (Table 1). Previously, we
could not produce C-terminal hydrazides of Asn, Asp, and
Gln owing to the intramolecular cyclization that occurred
when the peptide was released from the resin.[10b] In this study,
we found that peptide o-aminoanilides with C-terminal Asn
and Gln residues can be readily prepared by Fmoc-SPPS and
can be smoothly ligated with Cys peptides. Nonetheless, Asp
still cannot be used in the o-aminoanilide/NaNO2 approach,
because only hydrolysis occurred with a C-terminal Asp
substrate (compound 2j).[15]
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histone proteins constituting the histone octamer in nucleo-
somes.[19,20] In our synthesis, we divided H2B into five
segments with Ala21, Ala58, Ala81, and Ala107 temporarily
mutated to Cys (Scheme 2A).
The right half of H2B was made through the ligation of
Tbeoc-Thz81-Leu106-Dbz-Ala-OH (6) with Cys107-Lys125-OH
(7). Tbeoc-Thz was used to avoid the self-cyclization and
oligomerization of 6. HPLC monitoring showed that the
ligation of 6 (activated in situ) with 7 was complete in 4 h.
Treatment with TCEP (tris(2-carboxyethyl)phosphine) fol-
lowed by methoxyamine hydrochloride[21] gave Cys81-Cys107
-
Lys125-OH (9), which was isolated in 53% yield. The left half
of H2B was made through sequential ligation of Pro1-Lys20-
Dbz-Ala-OH (10), Cys21-Lys57-Dbz-Ala-OH (11), and Cys58-
Leu80-Dbz-Ala-OH (12) in the N-to-C direction. In both
ligations we observed some lactamization of the C-terminal
Lys residue.[22] Nonetheless, Pro1-Cys21-Lys57-Dbz-Ala-OH
(13) was obtained from 10 and 11 in 52% yield, and Pro1-
Cys21,58-Leu80-Dbz-Ala-OH (14) was obtained from 13 and 12
in 33% yield. Remarkably, in both of the above ligation steps,
the Dbz moiety in the Cys-peptide segment survived the
reaction well and therefore could be used as a crypto-
thioester for the next step.
Figure 2. Synthesis of SFTI-1. A) Synthetic route. B) Structure deter-
mined by NMR spectroscopy.
In comparison to the on-resin p-nitrophenylchlorofor-
mate activation method described by Dawson, NaNO2
activation under protecting-group-free conditions enables
the use of peptide o-aminoanilides as peptide crypto-thio-
esters. This feature extends the utility of peptide o-amino-
anilides in the convergent synthesis of proteins from multiple
peptide segments. As a demonstration, we examined a con-
vergent synthesis of histone H2B, which is one of the core
To test the compatibility of the Dbz moiety with the free-
radical-based desulfurization reaction,[23] we treated 14 with
Scheme 2. Convergent synthesis of histone H2B. A) Synthetic route. Tbeoc=2-(tert-butyldisulfanyl)ethyloxycarbonyl, MESNa=sodium 2-mercap-
toethanesulfonate, Thz=l-thiazolidine-4-carboxylic acid. B) The amino acid sequence of histone H2B. C) HPLC chromatogram showing the
complete consumption of 15 in the ligation between 15 and 9 at pH 6.8 (t=5 h). Benzamide was added as an internal standard. D) Analytical
HPLC chromatogram (l=214 nm) of purified histone H2B. E) Mass spectrum of histone H2B. The deconvoluted spectrum gave an observed
mass of 13856.0 Da (calcd: 13857.9 Da, average isotopes).
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TCEP, tBuSH, and 2,2’-azobis[2-(2-imidazolin-2-yl)propane]
dihydrochloride (VA-044). The desulfurized product was
isolated in 79% yield with the Dbz moiety intact. We also
tested the possibility of generating an isolated peptide
thioester from the peptide o-aminoanilide by treating the
product of the desulfurization of 14 with MESNa after NaNO2
activation. The desired thioalkyl ester Pro1-Ala21,58-Leu80-
MESNa (15) was isolated in 75% yield. The final ligation
between 15 and 9 was conducted at pH 6.8. After 5 h
(Scheme 2C), the desired product 16 was obtained in 72%
yield. The Cys81 and Cys107 residues of 16 were then converted
back into Ala residues by free-radical-based desulfurization
to give histone H2B in 79% yield (or an overall yield of the
isolated product of 6%).
In comparison to the peptide hydrazide method, we
observed an interesting advantage of the o-aminoanilide/
NaNO2 approach in our synthesis of the cyclic bacteriocin
lactocyclicin Q:[24] Peptide o-aminoanilides can be readily
modified with auxiliary groups, such as an Arg6 or His6 tag in
the Dbz linker, whereas the corresponding hydrazides are
structurally conservative, thus prohibiting any modification.
As shown in Scheme 3, in our first synthetic route for
lactocyclicin Q we used three peptide hydrazides: Cys-
(Acm)1-Ala27-NHNH2 (17), Cys28-Trp42-NHNH2 (18), and
Cys43-Leu61-NHNH2 (20a). Ala1, Ala28, and Ala43 were
temporarily mutated to Cys to enable the NCLs, whereas
Cys1 was protected as Cys(Acm)1 to avoid the self-cyclization
or oligomerization of 17. The ligation of 17 with 18 proceeded
smoothly to afford Cys(Acm)1-Cys28-Trp42-NHNH2 (19) in
55% yield. Unfortunately, in the ligation of 19 and 20a, the
latter segment was so poorly soluble that the yield only
reached 11%.
To solve this problem, we designed Cys43-Leu61-Dbz-Arg6-
OH (20b), in which a solubilizing Arg6 tag[12] was incorpo-
rated. The Arg6 tag was assembled by SPPS prior to the
coupling of Fmoc-Dbz-OH. Gratifyingly, 20b showed
remarkably higher solubility. No obvious precipitation was
observed in the reaction with 19. Consequently, the ligation
product Cys(Acm)1-Cys28,43-Leu61-Dbz-Arg6-OH (21b) was
obtained in 47% yield. After the removal of Acm by
treatment with AgOAc (yield of the isolated product:
81%), intramolecular ligation by the NaNO2 activation
protocol afforded the cyclized product in 47% yield. Finally,
free-radical-based desulfurization[23] furnished lactocyclicin Q
in 73% yield upon isolation (or in 7% overall yield). The
circular dichroism (CD) spectrum of synthetic lactocyclicin Q
(Scheme 3D) exhibited double negative peaks in the 200–
230 nm region that are consistent with a highly helical
secondary structure,[25] as predicted on the basis of previous
studies.[24]
In summary, fully unprotected peptide o-aminoanilides
can be selectively and efficiently activated by NaNO2 in an
aqueous buffer and constitute a novel type of peptide crypto-
thioesters. The use of the o-aminoanilide/NaNO2 method was
Scheme 3. Total synthesis of lactocyclicin Q. A) Synthetic route. The use of 20b in place of 20a led to a great increase in the yield of the second
ligation step. Acm=acetamidomethyl. B) Analytical HPLC chromatogram (l=214 nm) of purified lactocyclicin Q. C) Mass spectrum of
lactocyclicin Q. The spectrum gave an observed mass of 6058.4 Da (calcd: 6060.1 Da, average isotopes). D) CD spectrum of lactocyclicin Q in
50% trifluoroethanol in water (v/v).
4
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b) G.-M. Fang, Y.-M. Li, F. Shen, Y.-C. Huang, J.-B. Li, Y. Lin,
demonstrated by the convergent synthesis of histone H2B
from five peptide segments. We also showed in the total
synthesis of the bacteriocin lactocyclicin Q that the o-amino-
anilide/NaNO2 method enables the incorporation of a solubi-
lizing tag. The o-aminoanilide/NaNO2 method overcomes the
limitation of the peptide hydrazide method regarding ligation
at Asn and Gln sites. As peptide o-aminoanilides have been
reported to be more readily synthesizable than peptide
hydrazides in some cases,[11] we envisage that the o-amino-
anilide/NaNO2 method will be very useful in chemical protein
synthesis.
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Received: August 8, 2014
Revised: November 18, 2014
Published online: && &&, &&&&
Keywords: native chemical ligation · peptide o-aminoanilides ·
.
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Communications
Native Chemical Ligation
J.-X. Wang, G.-M. Fang, Y. He, D.-L. Qu,
M. Yu, Z.-Y. Hong, L. Liu*
&&&& — &&&&
Peptide o-Aminoanilides as Crypto-
Thioesters for Protein Chemical Synthesis
Making it easier to make the link: The
activation of fully unprotected peptide o-
aminoanilides with NaNO2 in an aqueous
buffer enabled their use as peptide
tion (see scheme; MPAA=4-mercapto-
phenylacetic acid). The practicability and
unique advantages of this method were
demonstrated by the total chemical syn-
thesis of histone H2B and lactocyclicin Q.
crypto-thioesters in native chemical liga-
6
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