An N-protection free ligation of the peptide thioester and the peptide with an N-alkoxy- or N-aryloxyamino group at its N-terminus

Article abstract of DOI:10.1016/j.tetlet.2017.10.074

Peptides with an N-alkoxy or N-aryloxy amino acid at their N-terminus were synthesized and successfully ligated with a peptide thioester by silver ion activation under a slightly acidic condition without requiring protection of the side chain amino groups

Full text of DOI:10.1016/j.tetlet.2017.10.074

Accepted Manuscript
An N-protection free ligation of the peptide thioester and the peptide with N-
alkoxy- or N-aryloxyamino group at its N-terminus
Hironobu Hojo, Toru Kawakami, Yuta Hiroyama, Saburo Aimoto
PII:
S0040-4039(17)31378-3
https://doi.org/10.1016/j.tetlet.2017.10.074
TETL 49436
DOI:
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
8 September 2017
23 October 2017
27 October 2017
Please cite this article as: Hojo, H., Kawakami, T., Hiroyama, Y., Aimoto, S., An N-protection free ligation of the
peptide thioester and the peptide with N-alkoxy- or N-aryloxyamino group at its N-terminus, Tetrahedron Letters
(2017), doi: https://doi.org/10.1016/j.tetlet.2017.10.074
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An N-protection free ligation of the peptide
thioester and the peptide with N-alkoxy- or
N-aryloxyamino group at its N-terminus
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Hironobu Hojo*, Toru Kawakami, Yuta Hiroyama, Saburo Aimoto

1
Tetrahedron Letters
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An N-protection free ligation of the peptide thioester and the peptide with N-alkoxy-
or N-aryloxyamino group at its N-terminus
Hironobu Hojoa,* Toru Kawakamia, Yuta Hiroyamab, Saburo Aimotoa
aInstitute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan
bHamari Chemicals, Ltd., 1-4-29, Kunijima, Higashi-Yodogawa-ku, Osaka, 533-0024, Japan
ARTICLE INFO
ABSTRACT
Article history:
Received
Peptides with an N-alkoxy or N-aryloxy amino acid at their N-terminus were synthesized and
successfully ligated with a peptide thioester by silver ion activation under a slightly acidic
condition without requiring protection of the side chain amino groups. The N-methoxy group
was easily cleaved by the SmI2 reduction in CH3OH aq. to obtain the desired peptide with a
native peptide bond. This method was successfully applied to the synthesis of the human atrial
natriuretic peptide showing the efficiency of the novel ligation.
Received in revised form
Accepted
Available online
Keyword:
2009 Elsevier Ltd. All rights reserved.
Peptide thioester
Ligation method
N-methoxy group
Samarium(II) iodide reduction
Atrial natriuretic peptide
1. Introduction
Ligation chemistries for protein synthesis have been
continuously improved and used for the synthesis of proteins as
well as post-translationally modified ones, such as glycoproteins.
The present synthetic results mainly originate from the
application of the native chemical ligation method (NCL)
developed by Kent in 1994.1 In this method, a peptide thioester is
condensed with the C-terminal peptide having a cysteine residue
at its N-terminus via the intermolecular thioester exchange
reaction followed by the intramolecular S-to-N acyl shift to form
the native peptide bond (Figure 1). This method has the
advantage that the unprotected peptide segments obtained by the
solid-phase method can be directly used for the ligation reaction.
Due to this simplicity and the efficiency of the reaction, the NCL
method has gained wide acceptance and its advantage and
limitation have been extensively studied. The major problem
with the NCL method is that the cysteine residue is required at
the ligation site. As the natural abundance of the cysteine residue
is very low (1.5%), there are cases when the suitable ligation
sites cannot be found in the target proteins. Significant efforts
have been made and this problem has been considerably
overcome by using various amino acids carrying thiol auxiliary
groups, which can be converted to natural amino acids.2
Figure 1. Example of the ligation methods; a) Native chemical
ligation and b) Thioester method.
proteins. However, the complexity of this method is that
protection of the side chain amino and thiol groups is required. In
the original method, the Boc group was used for the amino
protecting group. As the Boc group is cleaved during the final
deprotection of the solid-phase synthesis, it has to be re-
introduced prior to the ligation. Recently, we found that the 4-
pyridylmethoxycarbonyl (iNoc) group,5 which is acid-stable, but
cleavable by zinc treatment in acetic acid, is suitable for the
amino protecting group. By using the iNoc group, peptide
segments prepared by the solid-phase method can be directly
used for the ligation like the NCL method. This improved
thioester method was successfully used for the synthesis of
glycoproteins, such as human interleukin-2.4a-c However, if the
On the other hand, the thioester method, developed by our
group in 1991,3 has also been used for (glyco)protein synthesis
mainly by our group.4 In this method, the ligation is achieved by
the aminolysis of a peptide thioester via an active ester by the
side chain N- and S-protected peptide segment (Figure 1 b). In
principle, this method can realize the condensation at any site,
which enables the flexible design of the synthetic route of

2
Tetrahedron
target protein has a significant number of lysine residues, there
were not natural amino acids.9
would be the potential danger of their incomplete deprotection
during the final stage of the synthesis. In this regard, extension of
the thioester method to an N-protection-free method would be
desirable.
We thus examined the ligation of peptide 1 with peptide
thioesters using silver activation in the absence of an active ester
component as shown in Table 1. For this purpose, the peptide
aryl (3) or alkyl (4) thioester was synthesized by the N-
alkylcysteine (NAC)-assisted thioesterification method.10 We
then examined the acylation of 1 by the reactive aryl thioester 3
in acetic acid using silver triflate (AgOTf) as an activator. To our
delight, the aryl thioester was activated within 30 min and the
selective acylation indeed proceeded to give the desired peptide,
pESEEGG(MeO)GGLEFKAGR-NH2 5, as shown in Table 1
entry 1. The acylation at the N-terminal N-methoxyglycine, not at
the side chain amino group, was confirmed by the Glu-C
digestion followed by mass analysis. However, CH3CO-
(MeO)GGLEFKAGR-NH2 was also obtained in about a 40%
yield, which suggests that the reaction proceeded via the
asymmetric anhydride between the N-terminal segment and
acetic acid. The change in the solvent from acetic acid to the
more acidic trifluoroacetic acid in DMSO or formic acid did not
give the product (Entry 2, 3). Thus, it was speculated that the
appropriate acidity is required to realize the N-terminal specific
ligation. We then found that the reaction efficiently proceeded in
the slightly acidic hexafluoroisopropyl alcohol (HFIP) (Entry 4),
We previously demonstrated that if we introduce the N-
methoxyglycine at the N-terminus or the side chain amino group
of a peptide, we can selectively label the N-methoxyamino group
by the fluorescent dye having an isothiocyanate structure under
acidic conditions.6 The reason for this selectivity seems to be
derived from the significantly lower pKa (4-5) of the N-
methoxyamino group7a compared to that of the α-amino group
(about 9) and the ε-amino group of the Lys residue (about 10.5).7b
Based on this fact, we speculated that a peptide having N-
methoxyglycine at its N-terminus can be selectively acylated by
the other peptide segment in the presence of free side chain
amino groups as long as they are protonated under slightly acidic
conditions (Figure 2). In this paper, we proved the basic concept
of this method using the human atrial natriuretic factor (hANP)8
as a model.
1
4
T 0
2 h
Figure 2. The novel ligation concept based on the thioester method.
2. Results and Discussion
We first examined the reactivity of the N-methoxyamino
group toward the acylation by N-succinimidyl acetate (Ac-OSu)
using (MeO)GGLEFKAGR-NH2 1 as shown in Scheme S1.
Peptide 1 was previously used as a model peptide for the
selective reaction with phenylisothiocyanate (PITC). Under the
basic and slightly acidic conditions (pH 8.6 – pH 4.7, Buffer A to
C in Scheme S1), acetylation at the side chain amino group
24 h
30
5
48 h*
20
10
selectively occurred to give (MeO)GGLEFK(Ac)AGR-NH2
2
(See Figure S1). On the other hand, in 0.5 M acetic acid in 80%
CH3CN aq. (pH 2.9), in which the selective reaction between the
N-methoxyamino group with PITC occurred,6 the acetylation to
the side chain amino group was completely suppressed.
However, the N-methoxyamino group did not react either. Based
on these results, it was suggested that the selective acylation at
the N-methoxyamino group requires reagents with a higher
reactivity than the active esters as well as under certain acidic
conditions. In fact, the acylation of the N-alkoxy or aryloxy
group has been performed by acid chloride or acid chloride with
silver ions in previous syntheses, though the acyl components
0
10 20
Retention time (min)
Figure 3. HPLC profile of the reaction between peptides 1
and 4 in HFIP. *After 24 h, one eq. of AgOTf was added and
kept for another day. Elution conditions: column, Cosmosil
5C18 ARII (4.6 × 150 mm, Nacalai Tesque, Japan) at the flow
rate of 1 mL /min; eluent, A, 0.1% TFA, B, acetonitrile
containing 0.1% TFA.
Table 1. Results of ligation
Entry
Thioester
pESEEGG-SC6H5 3
pESEEGG-SC6H5 3
pESEEGG-SC6H5 3
C-Terminal peptide
(MeO)GGLEFKAGR-NH2 1
(MeO)GGLEFKAGR-NH2 1
(MeO)GGLEFKAGR-NH2 1
Solvent
CH3COOH
Product
Yield*
35%
0%
1
2
3
5
5
1% TFA/DMSO
HCOOH
HFIP
0%
5
4
5
6
7
8
9
pESEEGG-SC6H5 3
(MeO)GGLEFKAGR-NH2 1
56%
80%
29%
87%
58%
16%
5
pESEEGG-SCH2CH2COOH 4
pESEEGG-SCH2CH2COOH 4
pESEEGG-SCH2CH2COOH 4
pESEEGL-SCH2CH2COOH 10
pESEEGG-SCH2CH2COOH 4
(MeO)GGLEFKAGR-NH2 1
HFIP
5
(Me2NCOCH2O)GGLEFKAGR-NH2
6
HFIP
8
(pMeC6H4O)GGLEFKAGR-NH2
(MeO)GGLEFKAGR-NH2 1
GGLEFKAGR-NH2 12
7
HFIP
9
HFIP
11
13
5% CH3COOH/DMF
*The yield was calculated based on the HPLC area of the product over the sum of the areas of the peaks derived from the C-terminal
peptide.

3
avoiding the acetylation problem observed during the ligation in
acetic acid. Instead, the remaining N-terminal peptide thioester
was hydrolyzed or formed an ester with HFIP in a small amount.
The best yield, under the conditions we used, was obtained using
the less reactive alkyl thioester 4 in HFIP, although the reaction
proceeded more slowly than that with the aryl thioester 3 (Figure
3, Entry 5).
purification was 55%, which was calculated based on the
peptide content obtained by amino acid analysis. The methoxy
group was then removed by SmI2 reduction (Figure 5). As in the
case of the model peptide, the reaction efficiently proceeded to
give the demethoxy product 17, which was confirmed by
a)
14
H-Ser-Leu-Arg-Arg-Ser-Ser-Cys(Acm)-Phe-Gly-SC6H5
The reactivity of peptides having N-Me2NCOCH2O- (6) and
N-4-CH3C6H4O- (7) groups were also examined. When peptide 6
was used, the reactivity decreased and the yield of the product
was 30%, which might be due to the electron withdrawing nature
and the steric hindrance of the functional group. Interestingly, by
using peptide 7 also having an electron withdrawing group, the
selective reaction efficiently proceeded. The difference in the
reactivity between these functional groups is not currently
understood. Further optimization of the functional group is under
investigation. As the synthesis of the N-methoxyglycine
derivative is much simpler than that of the N-4-tolyloxyglycine
derivative, further experiments were done using the N-methoxy
group.
H-(MeO)Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-
Gly-Cys(Acm)-Asn-Ser-Phe-Arg-Tyr-OH 15 , AgOTf / NFTB
Acm
H-Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-(OMe)Gly-Arg-Met-
Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-
Tyr-OH
16
Acm
b)
15
14
T 0
0.5 h
2 h
The ligation at the Leu-(MeO)Gly site (Entry 8) using an alkyl
thioester having Leu at its C-terminus 10 also proceeded with an
acceptable efficiency, showing that the sterically moderately
demanding amino acid can also be accommodated as the ligation
site. However, the rate of racemization of the C-terminal Leu was
relatively high at 11.6% (Figure S3, S4). As the thioester is
directly activated by silver ions, a part of the reaction would
proceed via the highly reactive oxazolone formation. This might
be the major cause of the high racemization rate. Currently,
several methods for suppression of the racemization, such as the
addition of copper ions, is being examined.11
35
16
25
15
20 h
0
10
Retention time (min)
20
Figure 4. Ligation of N- and C-terminal segments to obtain the
entire sequence of hANP: a) Reaction Scheme, b) Time course of
the ligation. Elution conditions are the same as those of Figure 3.
As a control experiment, the peptide without the N-methoxy
group 12 was reacted with the alkyl thioester 4 under much
milder acidic conditions, i.e. 5% acetic acid in DMF, as shown in
Entry 9. Although the acylation to the side chain amino group
was not observed, the yield of the desired product
pESEEGGGGLEFKAGR-NH2 13 was quite low, showing the
importance of the N-methoxy group to increase the reactivity of
the α-amino group. In addition, the α-amino group of the acid
component has to be protected to realize the acylation to the N-
terminus of the amino component under this condition.
a)
Acm
H-Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-(R)Gly-Arg-Met-Asp-Arg
-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr-OH
Acm
16: R=OCH3
17: R=H
SmI2 in 80% MeOH aq.
We next examined the removal of the N-methoxy group after
the ligation. Unfortunately, the zinc treatment in acetic acid
frequently used failed to remove it.12 We also examined the
recently reported radical based reduction using 4-
mercaptophenylacetic acid,13 but it also did not function well.
Finally, the reduction using SmI214 in methanol aq. gave quite
satisfactory results. The removal of the N-methoxy group was
completed within 10 min without serious side reactions (See
Figure S2). To the best of our knowledge, this is the first report
on the use of SmI2 to the free polypeptide.
I2 in aq. MeOH cont. HCl
H-Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-
Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr-OH
18
b)
16
m/z 3255.9
SmI2 T 0
17
m/z 3225.4
SmI2 10 min
I2 ox. 10 min
35
Having obtained the successful result for the model peptide,
we next applied the method to the synthesis of hANP as shown in
Figure 4. The sequence was divided at Gly9-Gly10 and two
peptide segments were synthesized by the solid-phase method.
The N-terminal segment 14 was synthesized as an aryl thioester
by the NAC method to achieve the higher reactivity during the
ligation. The C-terminal segment 15 was prepared by the Fmoc
method. For the solvent of the ligation, the nonafluoro-t-butyl
alcohol (NTFB) was used to suppress the esterification of the N-
terminal segment with the solvent, which was observed for the
ligation in HFIP. Two segments were then ligated in NFTB using
AgOTf as an activator. The reaction efficiently proceeded to give
the ligation product 16 within 20 h without serious side reactions
as shown in Figure 4b. The yield of the product after the HPLC
25
15
18
m/z 3082.0
0
10
Retention time (min)
20
Figure 5. Removal of N-methoxy group and disulfide bond
formation to obtain hANP 18: a) Reaction Scheme, b) Time
course of the reaction. Elution conditions are the same as those
of Figure 3.

4
Tetrahedron
Kanda, M.; Suzuki, A.; Katayama, H.; Nakahara, Y.; Hojo, H.
Org. Biomol. Chem. 2013, 11, 7199–7207; c) Asahina, Y.;
Komiya, S.; Ohagi, A.; Fujimoto, R.; Tamagaki, H.; Nakagawa,
K.; Sato, T.; Akira, S.; Takao, T.; Ishii, A.; Nakahara, Y.; Hojo, H.
Ang. Chem. Int. Ed. 2015, 54, 8226-8230; d) Hojo, H. Org.
Biomol. Chem. 2016, 14, 6368–6374.
MALDI-TOF MS analysis. Finally, the disulfide bond was
formed by the iodine oxidation to give the final product, hANP
18. The product was completely co-eluted with the commercially
available standard showing that the hANP with the correct
structure was obtained (Figure S5). The ligation of Leu-Gly site
was also performed in the case of hANP using Leu9-hANP(1-9)-
SPh 19 as the thioester. The ligation of 19 and the C-terminal
segment 15 was also tested and the desired product
[Cys(Acm)7,23, Leu9, (MeO)Gly10]-hANP(1-28) 20 was obtained
in 26% isolated yield.
5. Veber, D.F.; Paleveda, Jr., W. J.; Lee, Y. C.; Hirschmann, R. J.
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In conclusion, we demonstrated that by modification of the
terminal amino group, selective ligation of two peptide segments
is realized under slightly acidic conditions. The N-methoxy group
was efficiently removed by SmI2 reduction in methanol aq. After
I2 oxidation, hANP was successfully obtained. The racemization
during the ligation is to be suppressed, which will be realized by
several methods. The ligation can be carried out in acidic
fluorous solvents, which will be advantageous for the synthesis
of hydrophobic polypeptides, such as membrane proteins. Along
this line, further studies are underway.
8. Kangawa, K., Matsuo, H. Biochem. Biophys. Res. Commun., 1984,
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Acknowledgments
This research was partly supported by JSPS KAKENHI Grant
Number JP16H04180 (to H.H.) and JP15K05565 (T.K.). We
thank Mr. Takashi Ohtani of Hamari Chemicals, Ltd. for helpful
discussions.
13. Weller, C.E.; Dhall, A.; Ding, F.; Linares, E.; Whedon, S.D.;
Senger, N.A.; Tyson, E.L.; Bagert, J. D.; Li, X.; Augusto, O.;
Chatterjee, C. Nat. Commun., 2016, 7, 12979.
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55, 11755-11772; b) Nakano, J.; Ichiyanagi, T.; Ohta, H.; Ito, Y.
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Supplementary Material
Supplementary material (Experimental details, HPLC)
associated with this article can be found in the online version.
References and notes
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5
Highlights
An N-alkoxy or N-aryloxyamino group at the N-
terminus of a peptide is selectively acylated under
acidic condition.
As an acylating agent, peptide thioester with silver
ion activation can be used.
N-methoxy group can be cleaved by samarium(II)
iodide treatment.
Using this method, human atrial natriuretic peptide
was successfully synthesized.