Synthesis of Histidine-Containing Oligopeptides via Histidine-Promoted Peptide Ligation

Article abstract of DOI:10.1002/asia.201701802

Histidine-containing peptides are valuable therapeutic agents for a treatment of neurodegenerative diseases. However, the synthesis of histidine-containing peptides is not trivial due to the potential of imidazole sidechain of histidine to act as a nucleophile if unprotected. A peptide ligation method utilizing the imidazole sidechain of histidine has been developed. The key imidazolate intermediate that acts as an internal acyl transfer catalyst during ligation is generated by deprotonation. Transesterification with amino acids or peptides tethered with C-terminal thioester followed by N→N acyl shifts led to the final ligated products. A range of histidine-containing dipeptides could be synthesized in moderate to good yields via this method without protecting the imidazole sidechain. The protocol was further extended to tripeptide synthesis via a long-range N→N acyl transfer, and tetrapeptide synthesis.

Full text of DOI:10.1002/asia.201701802

CHEMISTRY
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Accepted Article
Title: Synthesis of Histidine-Containing Oligopeptides via Histidine-
Promoted Peptide Ligation
Authors: Kai-Jin Huang, Yi-Chen Huang, and Yuya Angel Lin
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10.1002/asia.201701802
Chemistry - An Asian Journal
COMMUNICATION
Synthesis of Histidine-Containing Oligopeptides via Histidine-
Promoted Peptide Ligation
Kai-Jin Huang, Yi-Chen Huang and Yuya A. Lin*[a]
Dedication ((optional))
Abstract: Histidine-containing peptides are valuable therapeutic
agents for treatment of neurodegenerative diseases. However, the
synthesis of histidine-containing peptides is not trivial due to the
potential of imidazole sidechain of histidine to act as a nucleophile if
results in mixtures of enantiomeric products. As a result, the
imidazole moiety on the sidechain of histidine is usually
protected during peptide synthesis. Common N-terminal
protecting groups like Boc or Fmoc, however, cannot be used
since protecting group orthogonality with the N-terminal amine
need to be achieved for effective coupling. Orthogonally
protected histidine derivatives are often difficult to prepare and
costly to purchase.
unprotected.
A peptide ligation method utilizing the imidazole
sidechain of histidine has been developed. The key imidazolate
intermediate that acts as an internal acyl transfer catalyst during
ligation is generated by deprotonation. Transesterification with amino
acids or peptides tethered with C-terminal thioester followed by
NN acyl shifts led to the final ligated products. A range of histidine-
containing dipeptides could be synthesized in moderate to good
yields via this method without protecting the imidazole sidechain.
The protocol was further extended to tripeptide synthesis via a long-
range NN acyl transfer, and tetrapeptide synthesis.
Imidazole has been reported as an acyl transfer catalyst in
amide bond formation of simple amines under basic
conditions.[11] Moreover, Katritzky and co-workers have shown
that the indole moiety of tryptophan sidechain can be utilized in
native peptide ligation.[12] This methodology involves the
acylation of the indole moiety of a tryptophan derivative by a
benzotriazolide. Following deprotection of the N-terminus, the
intermediate undergoes a long-range NN acyl migration under
microwave irradiation to result ligated peptide products with
native linkage. Inspired by these work, our laboratory explored
the potential of utilizing the imidazole moiety of histidine in
peptide ligation and is reported herein (Scheme 1), where the
histidine imidazolate B, generated by deprotonation of A, first
undergoes a transesterification with an activated ester (XR3) to
form intermediate C followed by NN acyl shift to result the
ligated product D.
Peptides are important biomolecules in nature and many
have interesting therapeutic properties which have been
exploited in medicinal chemistry.[1] Peptide drugs are known to
be highly selective, lower in toxicity for the body, and have
reduced propensity for developing resistance. Histidine-
containing peptide sequences have excellent affinity for metals
and are found in many metalloproteins such as superoxide
dismutase,[2] dopamine β-monooxygenase[3] and amyloid-like
protein 2.[4] Recently, peptides that bind copper(II) ions are
particularly of high interest because research has shown that the
aggregation and precipitation of amyloid-β, a major cause of
Alzheimer’s disease (AD), could be induced by binding of Cu2+ in
the brain under certain conditions.[5] Therefore, histidine-
containing peptides can be considered as valuable therapeutic
agents for AD, owing to their ability to bind copper efficiently,
biocompatibility and modifiable metal-binding properties.[6]
Conventional synthetic methods for peptides are wasteful as
the reactions require the activation of carboxylic acid using
coupling reagents such as carbodiimides,[7] phosphoniums[8] and
uranium salts,[9] followed by nucleophilic attack of a free amine.
While many novel amidation reactions have been discovered,[10]
developing efficient and atom-economical methods for synthesis
of peptides remain an ongoing challenge in chemistry. Synthesis
of histidine-containing peptide usually require more care as the
imidazole sidechain of histidine, if unprotected, can react with
activated carboxylic acid during coupling thus reducing the
amount of activated acid for reaction. Moreover, the imidazole
moiety of histidine promotes epimerization during coupling and
Scheme 1. Concept of Peptide Ligation Mediated by Histidine Imidazolate.
Investigation began with the screening of coupling conditions
using histidine derivative 1 and alanine methyl ester 2 as there
were already reports for imidazolate catalyzing amidation
reaction of unactivated esters.[11] As shown by Table 1, no
reaction occurred in the presence of
a weak base like
triethylamine (entry 1). This outcome was as expected since
triethylamine would not be strong enough to generate
imidazolate, the proposed active catalyst. However, product
formation still could not be observed even when 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU) was used as the base nor
when the temperature was raised to 90 C (entry 2-4). Finally, by
performing the reaction in a more concentrated solution at 90 C
[a]
K.-J. Huang, Y.-C. Huang, Prof. Y. A. Lin*
Department of Chemistry
National Sun Yat-sen University
70 Lienhai Rd., Kaohsiung 80424, Taiwan
E-mail: yylin@mail.nsysu.edu.tw
Supporting information for this article is given via a link at the end of
the document.
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the ligated product was formed albeit only 28% yield (entry 5).
Low yields and the harsh reaction conditions suggested poor
reactivity of the unactivated ester in ligation.
9
SEt
SEt
SEt
SEt
SEt
SEt
SEt
SEt
SEt
DBU (1.5)
DBU (1.5)
DBU (1.5)
DBU (1.5)
DBU (1.5)
Imidazole (1.5)
TMG (1.5)
TBD (1.5)
MeCN
MeCN
DMF
25
60
60
60
60
60
60
60
60
68
84
83
47
33
11
29
23
72
10
11
12
13
14
15
16
17
Therefore, the more reactive thioester was examined next to
see if more practical conditions could be developed. When
thioester 3a was used to screen reaction conditions, no reaction
occurred in the absence of base and only 4% of product was
formed under elevated temperature (entry 6-7). However, in the
presence of DBU at room temperature the product yield was
increased to 48-68% (entry 8-9). These results indicated that the
more reactive thioester alone was not enough and that the
formation of imidazolate was vital for the ligation to occur.
Optimal yields of 83-84% could be obtained using 1.5
equivalents of DBU in acetonitrile or N,N-dimethylformamide
(DMF) at 60 C (entry 10-11). Product yield diminished when
tetrahydrofuran (THF) or chloroform was used as the solvent
(entry 12-13). Moreover, imidazole was used instead of DBU as
the base to see if the enhanced ligation was simply due to the
action of imidazole moiety (entry 14). Only 11% of the ligation
product was obtained. This result showed that there was indeed
some degree of direct N-acylation by the N-terminus of histidine
with thioester, and that the generation of imidazolate anion on
histidine was essential to achieve good yields, consistent with
proposed mechanism. Attempts to improve the yield by using
other organic bases of similar pKas to DBU such as 1,1,3,3-
tetramethylguanidine (TMG) and 1,5,7- triazabicyclo[4.4.0]dec-5-
ene (TBD) were unsuccessful (entry 15-16). A reasonably good
THF
CHCl3
MeCN
MeCN
MeCN
MeCN
MTBD (1.5)
[a] Isolated yield. [b] Reaction carried out at 3 M; n.r. = no reaction.
Next, the substrate scope of histidine-promoted ligation was
investigated. Various amino acid thioesters were synthesized
following previously reported procedures (see SI).[13] These
compounds were selected as substrate for screening to see how
steric hindrance of the sidechain affect the reaction outcome.
The results are summarized in Scheme 2. Apart from alanine,
the substrate used in model reaction, amino acids such as
glycine, β-alanine and leucine resulted in good yields ranging
from 70 to 79% (4a-4d). Reaction with glycine as substrate was
found to produce better result at room temperature likely due to
increased level of undesired deprotonation of the more acidic -
hydrogen by DBU at higher temperature. The -hydrogen of
amino acids that contain sidechains are less acidic thus not
affected by this side reaction. Valine, phenylalanine, proline and
methionine all contain bulkier sidechains which are expected to
influence the efficiency of the reaction. Indeed, the yields of the
ligated product for these amino acids declined to between 59
and 64% (4e-4h). It should be noted that less efficient ligations
could lead to some degree of racemization on the two α-centers
of the dipeptide product. For instance, 1:1 inseparable
diastereomeric mixtures were obtained in reactions with leucine,
methionine and phenylalanine while ligation with valine resulted
in diastereomers with a ratio of 1:5. To examine the efficiency of
this ligation method in tripeptide synthesis, a peptide sequence
(GGH) that has been shown to bind copper(II) ions effectively
was selected as an example.[14] Dipeptide thioester 2i worked
well in the ligation under optimized reaction conditions and 74%
of the desired tripeptide 4i was obtained. Amino acids such as
serine, cysteine, lysine and glutamic acid, which contain either
nucleophilic, basic or acidic sidechains, were not tested in
histidine-promoted ligation as the unprotected sidechains are
highly likely to interfere with the action of imidazolate leading to
poor ligation results. Even with protected sidechains, side
reaction involving the transfer of protecting groups to the
imidazole moiety of histidine is expected to occur.
yield of 72%
was obtained when 7-Methyl-1,5,7-
triazabicyclo[4.4.0]dec-5-ene (MTBD) was used as the base
(entry 17) though still not as good as when DBU was used. Thus
the remainder of the investigation were carried out using the
optimized ligation conditions: 1.5 equivalents of DBU in
acetonitrile or DMF at 60 C. It should be noted that all the
experiments were carried out under anhydrous conditions and
nitrogen atmosphere since the presence of water would quench
the imidazolate species formed during the reaction.
Table 1. Optimization of Reaction Conditions
Entry
XR
Base (equiv.)
NEt3 (1.5)
DBU (1.5)
DBU (1.5)
DBU (1.5)
DBU (1.5)
None
Solvent
MeCN
MeCN
MeCN
DMF
T (C)
25
% Yield[a]
n.r.
n.r.
n.r.
n.r.
28
1
OMe
OMe
OMe
OMe
OMe
SEt
2
25
3
60
4
90
5[b]
6
DMF
90
To further validate the importance of imidazolate moiety in
the ligation, control experiments using N-methyl histidine
derivative 1-Me and phenylalanine derivative 5 was conducted
(Scheme 3). The control ligation with N-methyl histidine and
thioester 3a led to a low yield (14%) comparable to the case
MeCN
MeCN
MeCN
25
n.r.
4
7
SEt
None
60
8
SEt
DBU (1.0)
25
48
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where only direct N-acylation by N-terminus is in action (Table 1,
entry 14). This result in turn suggested that the formation of
histidine imidazolate species was vital for the enhanced yields
observed in peptide ligation and that a potential mechanism
which involves histidine sidechain acting as a hydrogen bond
donor to bring the thioester into proximity for reaction could be
eliminated. Phenylalanine was chosen as another candidate for
control ligation experiment because it has an aromatic sidechain
of similar steric bulk to the imidazole moiety on histidine
sidechain but does not contain any acidic proton under the
conditions employed. The control ligation between phenylalanine
5 and thioester 3a under identical reaction conditions resulted in
low yield as expected and only 12% of the ligated product was
obtained.
ligation was performed using the optimized reaction conditions
as before. The corresponding dipeptide was then subjected to
standard Boc-deprotection procedures using 10 M hydrochloric
acid in methanol to free up the N-terminal amino group.
Gratifyingly, the second histidine-promoted ligation also
proceeded smoothly under previously optimized conditions with
a yield of 69%. The less efficient second ligation presumably is
due to the higher degree of freedom and in the 9-member ring
transition state. Application of histidine-promoted ligation in
tetrapeptide synthesis was also examined. Despite the absence
of bioactivity, the tetrapeptide GVHG is a truncated analogue of
alloferon,[15] an antiviral peptide isolated from the blow fly
Calliphora vicina.[16] Routine tetrapeptide syntheses in solution
phase usually consist of coupling reactions between two
dipeptides. Thus, dipeptide 8 and 9 were synthesized by
standard peptide coupling reaction using TBTU as the coupling
reagent (see SI). After deprotection of histidine derivative 8
using 10 M HCl and methanol, the resulting dipeptide and
thioester 9 successfully produced the target tetrapeptide 10
when the optimized ligation conditions were applied (Scheme 5).
Histidine-promoted ligation is sensitive to steric hindrance as
seen from the investigation of the substrate scope. Therefore, it
was not surprising that the tetrapeptide was only obtained in a
moderate yield of 41%.
N
N
NH
3a-i
NH
RCOSEt
(1.5 equiv)
O
H
H
N
N
DBU (1.5 equiv),
MeCN, 60 °C, 18 h
H2N
R
N
5
5
H
O
O
1
4a-i
(1 equiv)
N
N
N
O
NH
NH
NH
O
O
O
O
O
H
N
H
N
H
N
N
H
N
N
5
5
5
H
H
BocHN
BocHN
BocHN
O
, 84%
BocHN
O
BocHN
[a]
[a],[b]
[a]
4a
4b
4c
, 70%
, 74%
N
N
N
NH
NH
NH
S
O
O
H
H
H
N
N
N
N
N
N
H
5
5
5
H
H
O
, 74%
BocHN
O
, 64%
BocHN
O
, 62%
[a],[c]
[a],[c]
[a],[c]
4d
4e
4f
NHBoc
N
N
N
H
NH
NH
NH
N
O
H
H
H
N
N
O
O
N
N
H
N
N
H
5
5
5
H
O
, 61%
NBoc
O
O
, 74%
[a],[c]
[a]
[a]
4g
4h
, 59%
4i
Scheme 2. Scope of Peptide Synthesis via Histidine-Promoted Peptide
Ligation. [a] Isolated yield. [b] Reaction performed at room temperature. [c]
Racemization observed.
Scheme 4. Tripeptide Synthesis via Long-Range NN Acyl Transfer.
Scheme 5. Synthesis of Tetrapeptide via Histidine-Promoted Ligation.
Small to medium sized cyclic peptides exhibit wide range of
therapeutic properties including anticancer and antimicrobial
activities.[17] Biologically active cyclic tetrapeptides, especially,
mimic reverse turns that are important structural motif in protein-
protein interaction.[18] However, synthesis of cyclic tetrapeptides
is not trivial due to the preference of linear peptides for trans-
amide bonds which could impede the ring-like transition state
required for cyclization.[19] Therefore, as a final extension of
Scheme 3. Control Ligation Experiment Using: a) N-methyl histidine derivative;
b) phenylalanine derivative.
Further, the synthesis of tripeptide 7 via multiple histidine-
promoted ligations was investigated to determine whether long-
range NN acyl transfer is feasible or not (Scheme 4). The first
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histidine-promoted ligation, we were keen to see if the
methodology could be utilized in cyclic tetrapeptide synthesis
(Scheme 6). Accordingly, tetrapeptide 11 was synthesized by
standard solution phase peptide synthesis using either TBTU or
PyBOP as the coupling reagent (see SI). The peptide sequence
HPGA was designed to act as a model. Proline was specifically
incorporated in the sequence to induce cisoid conformation that
may facilitate the cyclization. The protection of the imidazole
moiety on histidine was found to be essential during the
synthesis of 11 to ensure reasonable yields. Next, tetrapeptide
11 was first subjected to deprotection using TFA in DCM
followed by dilute ligation conditions to prevent oligomerization.
DMF was used as the solvent to aid solubility and 36 hours of
reaction time were required for complete consumption of the
starting material. Interestingly, six-membered cyclic compound
13 and 14 were obtained in 78% and 80% yield, respectively,
instead of the desired cyclic tetrapeptide 12. There are several
possible mechanisms which could lead to the formation of the
2,5-diketopiperazine products. Based on our proposed
mechanism, we speculate that one possible route could be via
the formation of cyclic intermediate 15, though alternative
pathways are equally likely. Further experiments including
computational calculations would be required to determine the
exact mechanism of diketopiperazine formation.
Acknowledgements
We gratefully acknowledge the financial support from the
Ministry of Science and Technology (MOST 103-2113-M-110-
012-MY2, MOST 105-2738-M-110-001- and MOST 105-2113-M-
110-009-MY2).
Keywords: chemical ligation • acyl transfer • peptide synthesis •
histidine • copper-binding peptides
[1]
[2]
a) A. Greenberg, C. M. Breneman, J. F. Liebman, The Amide Linkage:
Structural Significance in Chemistry, Biochemistry, and Materials
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V. C. Culotta, Proc. Natl. Acad. Sci. 2014, 111, 5866-5871.
S. T. Prigge, R. E. Mains, B. A. Eipper, L. M. Amzel, Cell. Mol. Life Sci.
2000, 57, 1236-1259.
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A. I. Bush, Trends Neurosci. 2003, 26, 207-214.
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Scheme 6. Attempted Synthesis of Cyclic Tetrapeptide by histidine-promoted
ligation. a One possible reaction pathway.
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In summary, a peptide ligation method utilizing the imidazole
moiety of histidine as nucleophilic catalyst has been developed.
Under the optimized conditions, this ligation enabled the
synthesis of a range of histidine-containing dipeptide, tripeptides
and tetrapeptides in moderate to good yields without the need
for protection of histidine sidechain. The ligation method was
found to be not applicable to the synthesis of cyclic tetrapeptides.
We suspect the cause of this outcome might be sequence-
dependent and further studies are underway in our laboratory.
[15] M. Kuczer, A. Majewska, R. Zahorska, Chem. Biol. Drug Des. 2013, 81,
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Rose, L. M. Glerasch, J. A. Smith, Adv. Protein Chem. 1985, 37, 1-109.
[19] U. Schmidt, J. Langner, J. Pept. Res. 1997, 49, 67-73.
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Chemistry - An Asian Journal
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COMMUNICATION
Kai-Jin Huang, Yi-Chen Huang and
Yuya A. Lin*
Page No. – Page No.
Synthesis of Histidine-Containing
Oligopeptides via Histidine-Promoted
Peptide Ligation
A peptide ligation method utilizing the imidazole sidechain of histidine has been
developed. A range of histidine-containing dipeptides could be synthesized in
moderate to good yields without protecting the imidazole sidechain. The protocol
was further extended to tripeptide synthesis via a long-range NN acyl transfer,
and tetrapeptide synthesis.
For internal use, please do not delete. Submitted_Manuscript
This article is protected by copyright. All rights reserved.