Sulfanylmethyldimethylaminopyridine as a Useful Thiol Additive for Ligation Chemistry in Peptide/Protein Synthesis

Article abstract of DOI:10.1021/acs.orglett.0c01383

Sulfanylmethyl-installed dimethylaminopyridine, 2-sulfanylmethyl-4-dimethylaminopyridine (2), has an acidic thiol group comparable to that in aryl thiols due to the formation of a zwitterion consisting of a thiolate anion and a pyridinium cation. It can be used as an additive for native chemical ligation. The alkyl thiol in 2 allows it to be used for the one-pot native chemical ligation-desulfurization protocol in peptide synthesis. The utility of 2 in the synthesis of cyclic peptides is demonstrated.

Full text of DOI:10.1021/acs.orglett.0c01383

pubs.acs.org/OrgLett
Letter
Sulfanylmethyldimethylaminopyridine as a Useful Thiol Additive for
Ligation Chemistry in Peptide/Protein Synthesis
Kento Ohkawachi, Daishiro Kobayashi, Kyohei Morimoto, Akira Shigenaga, Masaya Denda,
Kenzo Yamatsugu, Motomu Kanai, and Akira Otaka*
Cite This: https://dx.doi.org/10.1021/acs.orglett.0c01383
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: Sulfanylmethyl-installed dimethylaminopyridine, 2-sulfanylmethyl-4-
dimethylaminopyridine (2), has an acidic thiol group comparable to that in aryl thiols
due to the formation of a zwitterion consisting of a thiolate anion and a pyridinium
cation. It can be used as an additive for native chemical ligation. The alkyl thiol in 2
allows it to be used for the one-pot native chemical ligation−desulfurization protocol
in peptide synthesis. The utility of 2 in the synthesis of cyclic peptides is
demonstrated.
Recently, Kanai et al. developed a catalyst 1 that activates
acetyl-CoA as a relatively stable thioester with an ε-amino
group of lysine in histone proteins being regioselectively
acetylated (Figure 1).9 This catalyst 1 includes a thioester
variety of thiol additives have been utilized to promote
Anative chemical ligation (NCL) involving the chemo-
selective condensation between thioesters and peptides with
N-terminal cysteine.1 The moderate reactivity of alkyl thioester
peptides generally employed for NCL supports their shelf
stability, but completion of NCL with the alkyl thioesters
requires the use of thiols such as aryl thiols, including 4-
mercaptophenylacetic acid (MPAA).2 Thioester exchange
generating the aryl thioester enables NCL to proceed to
completion in a few hours. To date, MPAA as a water-soluble
aryl thiol has been the standard additive for NCL.
Advances in NCL protocols for protein synthesis include a
one-pot, sequential use of NCL-related technologies including
the ligation and desulfurization3 of cysteine to form an alanine
residue. Such a one-pot protocol avoids the laborious
purification steps necessary for ligation products and improves
the overall yield of the final product. Desulfurization of NCL-
derived β- or γ-sulfanyl amino acid residues has further
enhanced the utility of NCL for the synthesis of non-cysteine-
containing proteins,4 and as a result, integration of NCLs and
subsequent desulfurization in a one-pot operation is desirable.
MPAA has served as the most efficient thiol additive in NCL
protocols. A limitation of its use, however, is that MPAA
inhibits the most frequently used tris(2-carboxyethyl)-
phosphine (TCEP)-mediated radical desulfurization5 due to
the much lower bond dissociation energy of aryl thiols when
compared to that of alkyl thiols.6 Therefore, desulfurization-
applicable NCL additives have been sought.7 In this context,
sodium mercaptoethanesulfonate (MESNa), trifluoroethane-
thiol (TFET), and methyl thioglycolate categorized into alkyl
thiols have served as NCL additives compatible with the one-
pot, desulfurization operation, but there is much room for
improvement in terms of the NCL efficiency and the
malodorous character of alkyl thiols. Alternatively, an elegant
protocol using a thiol-capturing resin has been introduced.8
Figure 1. Structure of thiol-containing dimethylaminopyridines as
thioester additives.
activation and a nucleosome recognition unit. Subsequent
inter- and intramolecular S−S and S−N acetyl transfers are
thought to be responsible for the activation of acetyl-CoA
through the formation of an N-acylpyridinium species. The
thioester activation unit in 1 forms a zwitterion consisting of
the thiolate anion and a pyridinium cation, and functioning as
a relatively acidic thiol.10 Computational prediction using ACD
Laboratories software11 indicates that the pKa of the thiol in 2-
sulfanylmethyl-4-dimethylaminopyridine (SMDMAP) 2 is 6.15
0.6, lower than that of MPAA [6.97 0.4 (predicted value)
and 6.6 (measured value2)]. Taking into consideration the fact
Received: April 21, 2020
https://dx.doi.org/10.1021/acs.orglett.0c01383
© XXXX American Chemical Society
Org. Lett. XXXX, XXX, XXX−XXX
A

Organic Letters
pubs.acs.org/OrgLett
Letter
that SMDMAP probably functions as a better leaving group
and more preferentially exists as the corresponding highly
nucleophilic thiolate anion than MPAA does, we envisioned
that at the appropriate pH, SMDMAP should be superior to
MPAA as an effective NCL additive. More importantly, due to
its alkyl thiol structure, SMDMAP was expected not to
interfere with the subsequent desulfurization.
products without significant side products, but NCL of 6c
required an increased temperature (50 °C) (Figure S3A−C).
Although an acceptable amount of epimerization product13
(<6%) was detected in the NCL of Ser thioester 6d with the
hydrolyzed material being generated, >90% conversion was
achieved (Figures S3 and S4). Reaction of 6e also gave the
desired material along with formation of ∼8% lysine-related
seven-membered ring lactam peptide (Figure S3D).
Synthesis of SMDMAP 2 is shown in Scheme 1 and begins
with 4-chloro-2-pyridinemethanol (3). Reaction of 3 with
We next compared the catalytic activity of 2 with that of
other additives (Figure 2). In the presence of 50 mM TCEP
Scheme 1. Synthesis of the Disulfide Dimer of 2-
Sulfanylmethyl-4-dimethylaminopyridine (di-SMDMAP 5)
a
a
Reagents and conditions: (i) Me2NH·HCl, NaOH, H2O, 155 °C;
(ii) SOCl2, CH2Cl2, rt; (iii) TrSH, DBU, CH2Cl2, rt; (iv) TFA,
triethylsilane, CH2Cl2, rt; (v) DMSO/H2O (9:1), rt.
dimethylamine followed by chlorination with thionyl chloride
and subsequent nucleophilic substitution with triphenylmetha-
nethiol (TrSH) afforded a sulfanyl moiety incorporating
DMAP (4) in 56% isolated yield over three steps. Treatment
of 4 in TFA/CH2Cl2 with triethylsilane gave a mixture of 2 and
the corresponding disulfide (di-SMDMAP 5). Because these
components were difficult to separate, the disulfide (5) was
isolated as its hydrochloride salt12 in 28% yield (after
recrystallization) through the HCl treatment followed by
DMSO-mediated oxidation of the mixture. The resulting
disulfide additive 5, following in situ reduction with TCEP, can
be used for NCL.
Figure 2. Comparison of SMDMAP (2) with other NCL additives.
Percentages of ligation were determined by HPLC analyses with UV
detection at 220 nm and calculated using the equation percent ligated
= 100[integration (integ.) 8b]/(integ. 7 + integ. 8b).
Having the hydrochloride salt of the requisite thiol additive
5·2HCl as a crystalline and shelf-stable compound, we
examined its applicability to NCL using model thioesters [H-
LYRANX-S-CH2CH2CO-L-NH2 (6)] and an N-terminal
cysteine peptide [H-CSPGYS-NH2 (7)]. Initially, the NCL
of an Ala-containing thioester [X = Ala (6a), 1 mM] with 7 (1
mM) in 6 M guanidine·HCl (Gn·HCl)/0.2 M phosphate
buffer in the presence of 50 mM TCEP and 10 mM 5 at pH
6.9 and 37 °C for 3 h was found to go almost to completion,
affording the ligation product [H-LYRANA-CSPGYS-NH2
(8a)] in 94% conversion (isolated yield of 85%) (see Figure
S1A). At pH 4, no NCL product was observed and the starting
materials remained (Figure S1B). The reaction at pH 6
required >3 h to reach completion (Figure S1C), and the
reactions at pH 8 or 10 were accompanied by the formation of
hydrolyzed side products 9 (Figure S1D,E). No acylation of
the ε-amino group of lysine was observed at pH 6.9 in an
attempt using a Lys-containing amine component (Figure S2).
Accordingly, we employed the NCL at pH 6.9 as optimized
conditions for further examination of the substrate scope.
Several thioesters 6 with different C-terminal amino acids [X
= Ala (6a), Leu (6b), Val (6c), Ser (6d), and Lys (6e)] were
subjected to the reaction with 7 (Figure S3). Ligations at
aliphatic amino acid sites (6a−6c) afforded the desired
and each of active additives at 20 mM, the NCL of 6b (2 mM)
with 7 (1 mM) at pH 6.9 was performed. SMDMAP showed
NCL-promoting activity comparable or slightly superior to that
of MPAA, as was expected from the pKa values. In the case of
low concentrations (20 mM) of desulfurization-compatible
additives, SMDMAP facilitated the NCL more efficiently than
MESNa, TFET, or 1,2,4-triazole.14 Although the reaction using
a high concentration (250 mM) of the volatile TFET (bp 35−
37 °C) reached the level comparable to that of the MPAA or
SMDMAP-mediated reaction, careful handling of TFET in a
chemical fume hood is necessary. In contrast to TFET, use of
the nonmalodorous SMDMAP allows for the ligations without
ventilating facilities.
We next evaluated the applicability of 2 to desulfurization
reactions (Figure S5). NCL of 6a with 7 in the presence of 50
mM TCEP and 10 mM 5 at pH 6.9 and 37 °C for 3 h,
followed by addition of desulfurization buffer containing 200
mM TCEP, 80 mM VA-044, and 80 mM glutathione and
subsequent reaction for 3 h at 37 °C, yielded the
desulfurization peptide [H-LYRANA-ASPGYS-NH2 (10)] in
77% isolated yield. Because additive 2 proved to be compatible
with the one-pot NCL−desulfurization protocol, we next
evaluated the applicability of 2 to one-pot, sequential NCL−
B
https://dx.doi.org/10.1021/acs.orglett.0c01383
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
pubs.acs.org/OrgLett
Letter
desulfurization protocols that are indispensable for protein
chemical synthesis using a model peptide.
As an N-to-C protocol, we employed the phosphate-
controllable N-sulfanylethylanilide (SEAlide)-mediated se-
quential NCLs.16 Three fragments [thioester 6a, N-terminal
cysteinyl SEAlide peptide H-CLYRANA-SEAlide-G-NH2 (12),
and 7] were sequentially ligated as shown in Scheme 2. The
first NCL of 6a with 12 in 6 M Gn·HCl/0.1 M N-(2-
hydroxyethyl)piperazine-N′-propanesulfonic acid (HEPPS) in
the presence of 50 mM TCEP and 10 mM 5 at pH 6.9 and 37
°C afforded the ligated SEAlide peptide 13 in 4 h. Addition of
a phosphate solution of 7 to the reaction mixture allowed the
SEAlide unit to work as a thioester, resulting in the formation
of fully ligated peptide 14 in 7 h. Then, desulfurization buffer
was added to the mixture to initiate the TCEP-mediated
radical desulfurization. After 6 h at 37 °C, the desired 11 was
obtained in 44% isolated overall yield (Figure 3A−C).
Sequential NCLs are classified as N-to-C and C-to-N
directed protocols.15 Compatibility with a one-pot, N-to-C
directed NCL−desulfurization protocol was initially evaluated
through the one-pot synthesis of model peptide H-LYRGA-
ALYRANA-ASPGYS-NH2 (11) (Scheme 2, Figure 3A−C, and
Figures S6 and S7).
Scheme 2. One-Pot N-to-C Directed NCL−Desulfurization
a
Protocol
The C-to-N operation using SMDMAP thioester 15 of a
thiazolidine (Thz) peptide17,18 is summarized in Scheme 3.
Scheme 3. One-Pot C-to-N Directed NCL−Desulfurization
a
Protocol
a
Conditions: (i) Peptides (6a and 12, 1 mM each), 6 M Gn·HCl/0.1
M HEPPS, 50 mM TCEP, 10 mM 5, pH 6.9, 37 °C, 4 h; (ii) peptide
7, 0.5 M phosphate, pH 6.9, 50 °C, 7 h; (iii) desulfurization buffer
(200 mM TCEP, 80 mM VA-044, and 80 mM glutathione), 37 °C, 6
h.
a
Conditions: (i) peptides (7 and 15, 1 mM each), 6 M Gn·HCl/0.1
M HEPPS, 5 mM TCEP, pH 6.9, 37 °C, 3 h; (ii) (a) CuSO4 (5 mM);
(b) methoxyamine (200 mM), pH 4.2, 37 °C, 4 h; (iii) peptide 6a
(0.75 mM, 1.5 equiv), 6 M Gn·HCl/0.2 M phosphate, 50 mM TCEP,
10 mM 5, pH 6.9, 37 °C, 2 h; (iv) desulfurization buffer (200 mM
TCEP, 80 mM VA-044, and 80 mM glutathione), 37 °C, 2 h.
The first NCL of 15 with 7 in 6 M Gn·HCl/0.1 M HEPPS in
the presence of 5 mM TCEP under well-degassed conditions
gave a ligated product that was converted to the ring-opening
product 16 by the action of CuSO4.19 Subsequent NCL with a
thioester 6a added to the mixture proceeded successfully to
give a ligated peptide 14, but the attempted one-pot
desulfurization failed to proceed to completion (Figures S8
and S9).20 An alternative protocol using 200 mM methoxy-
amine as a ring-opening reagent at pH 4.2,7b,16 followed by the
second NCL with 6a and subsequent desulfurization in a one-
pot reaction, yielded the desired product 11 in 41% isolated
yield after HPLC purification. The progress of the sequence of
reactions including the methoxyamine-mediated ring opening
is shown in Figure 3D−F and Figure S7.
Figure 3. (A−C) HPLC monitoring of the one-pot N-to-C directed
NCL−desulfurization protocol for the synthesis of 11: (A) first NCL
(t = 4 h), (B) second NCL (t = 7 h), and (C) one-pot desulfurization
(t = 6 h). Asterisks denote nonpeptide impurities. (D and E) HPLC
monitoring of the one-pot C-to-N directed NCL−desulfurization
protocol using the ring opening by methoxyamine. (D) Ring opening
by methoxyamine (t = 4 h). (E) Second NCL (t = 2 h). (F) One-pot
desulfurization (t = 2 h). Asterisks denote the remaining SMDMAP
thioester of 6a.
Finally, another potential utility of SMDMAP in ligation
chemistry was explored. Expecting that additive 2 should be
applicable to the synthesis of lactam or lactone peptides
because the parent catalyst 1 promotes the acylation of histone
C
https://dx.doi.org/10.1021/acs.orglett.0c01383
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
pubs.acs.org/OrgLett
Letter
Authors
proteins, we attempted to use SMDMAP for the synthesis of
cyclic peptides. Suitable conditions for lactam formation were
explored by using the substrate peptide H-GALYRGFA-
SCH2CH2-CO-G-NH2 (17). Cyclization of 17 (0.5 mM)
proceeded quantitatively in the presence of 20 mM 5, 30 mM
TCEP, and 170 mM diisopropylethylamine (DIPEA) in N-
methyl-2-pyrolidone (NMP)/H2O (4:1)21 to afford the cyclic
peptide cyclo(-GALYRGFA-) (18) in 98% isolated yield
without epimerization (Figure 4A,B and Figure S10; for
Kento Ohkawachi − Institute of Biomedical Sciences and
Graduate School of Pharmaceutical Sciences, Tokushima
University, Tokushima 770-8505, Japan
Daishiro Kobayashi − Institute of Biomedical Sciences and
Graduate School of Pharmaceutical Sciences, Tokushima
University, Tokushima 770-8505, Japan
Kyohei Morimoto − Institute of Biomedical Sciences and
Graduate School of Pharmaceutical Sciences, Tokushima
University, Tokushima 770-8505, Japan
Akira Shigenaga − Institute of Biomedical Sciences and
Graduate School of Pharmaceutical Sciences, Tokushima
University, Tokushima 770-8505, Japan
Masaya Denda − Institute of Biomedical Sciences and Graduate
School of Pharmaceutical Sciences, Tokushima University,
Tokushima 770-8505, Japan; orcid.org/0000-0001-6820-
2328
Kenzo Yamatsugu − Graduate School of Pharmaceutical
Sciences, The University of Tokyo, Tokyo 113-0033, Japan;
orcid.org/0000-0002-4260-414X
Motomu Kanai − Graduate School of Pharmaceutical Sciences,
The University of Tokyo, Tokyo 113-0033, Japan;
orcid.org/0000-0003-1977-7648
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.orglett.0c01383
Figure 4. HPLC monitoring of the cyclization reaction for (A and B)
lactam and (C and D) the lactone peptide: (A) t < 5 min, (B) t = 3 h,
(C) t < 5 min, and (D) t = 12 h. Asterisks denote nonpeptide
impurities.
Notes
The authors declare no competing financial interest.
reaction optimization, see Table S1). Optimized conditions
could be applied to the cyclization of other linear peptides with
chains of different lengths and ligation junctions (Table S2).
The presence of thiol and pyridine units in the molecule was
proven to be critical through a comparative study using other
additives (Figure S11). In addition, lactone formation of the
serine-containing thioester [Ac-SAFYG-S-CH2CH2-CO-L-
NH2 (19)] almost reached completion in NMP with 40 mM
5, 40 mM TCEP, and 80 mM DIPEA at 50 °C to give the
corresponding lactone peptide 20 in 95% conversion and 86%
isolated yield (Figure 4C,D and Table S3).
In conclusion, in addition to NCL-accelerating activity
comparable to that of MPAA, SMDMAP is also compatible
with radical desulfurization and thus can be applied to the one-
pot sequential NCL−desulfurization protocol that is central to
modern protein chemical synthesis. In addition, syntheses of
lactam or lactone peptides from peptide thioesters using
SMDMAP were achieved.
ACKNOWLEDGMENTS
■
This research was supported in part by a Grant-in-Aid for
Scientific Research (KAKENHI) and The Canon Foundation.
A.S. is grateful for support from the Research Foundation for
Pharmaceutical Sciences.
REFERENCES
■
(1) (a) Dawson, P.; Muir, T.; Clark-Lewis, I.; Kent, S. Synthesis of
proteins by native chemical ligation. Science 1994, 266, 776−779.
(b) Kent, S. B. H. Total chemical synthesis of proteins. Chem. Soc.
Rev. 2009, 38, 338−351.
(2) Johnson, E. C. B.; Kent, S. B. H. Insights into the Mechanism
and Catalysis of the Native Chemical Ligation Reaction. J. Am. Chem.
Soc. 2006, 128, 6640−6646.
̈
(3) Agouridas, V.; El Mahdi, O.; Diemer, V.; Cargoet, M.; Monbaliu,
J.-C. M.; Melnyk, O. Native Chemical Ligation and Extended
Methods: Mechanisms, Catalysis, Scope, and Limitations. Chem.
Rev. 2019, 119, 7328−7443.
(4) Malins, L. R.; Payne, R. J. Modern Extensions of Native
Chemical Ligation for Chemical Protein Synthesis. In Protein Ligation
and Total Synthesis I; Liu, L., Ed.; Topics in Current Chemistry 362;
Springer International Publishing, 2015; pp 27−87.
(5) Wan, Q.; Danishefsky, S. J. Free-Radical-Based, Specific
Desulfurization of Cysteine: A Powerful Advance in the Synthesis
of Polypeptides and Glycopolypeptides. Angew. Chem., Int. Ed. 2007,
46, 9248−9252.
(6) Rohde, H.; Schmalisch, J.; Harpaz, Z.; Diezmann, F.; Seitz, O.
Ascorbate as an Alternative to Thiol Additives in Native Chemical
Ligation. ChemBioChem 2011, 12, 1396−1400.
(7) (a) Siman, P.; Blatt, O.; Moyal, T.; Danieli, T.; Lebendiker, M.;
Lashuel, H. A.; Friedler, A.; Brik, A. Chemical Synthesis and
Expression of the HIV-1 Rev. Protein. ChemBioChem 2011, 12,
1097−1104. (b) Thompson, R. E.; Liu, X.; Alonso-García, N.; Pereira,
P. J. B.; Jolliffe, K. A.; Payne, R. J. Trifluoroethanethiol: An Additive
for Efficient One-Pot Peptide Ligation−Desulfurization Chemistry. J.
ASSOCIATED CONTENT
* Supporting Information
■
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.orglett.0c01383.
Detailed experimental procedures and charts for the
HPLC analyses of the attempted reactions (PDF)
AUTHOR INFORMATION
Corresponding Author
■
Akira Otaka − Institute of Biomedical Sciences and Graduate
School of Pharmaceutical Sciences, Tokushima University,
Tokushima 770-8505, Japan; orcid.org/0000-0002-0311-
5992; Email: aotaka@tokushima-u.ac.jp
D
https://dx.doi.org/10.1021/acs.orglett.0c01383
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
pubs.acs.org/OrgLett
Letter
Am. Chem. Soc. 2014, 136, 8161−8164. (c) Huang, Y.-C.; Chen, C.-
C.; Gao, S.; Wang, Y.-H.; Xiao, H.; Wang, F.; Tian, C.-L.; Li, Y.-M.
Synthesis of L- and D-Ubiquitin by One-Pot Ligation and Metal-Free
Desulfurization. Chem. - Eur. J. 2016, 22, 7623−7628.
(8) Moyal, T.; Hemantha, H. P.; Siman, P.; Refua, M.; Brik, A.
Highly efficient one-pot ligation and desulfurization. Chem. Sci. 2013,
4, 2496−2501.
Acetamidomethyl-cysteine under Aqueous Conditions. Angew.
Chem., Int. Ed. 2019, 58, 5729−5733.
(9) Amamoto, Y.; Aoi, Y.; Nagashima, N.; Suto, H.; Yoshidome, D.;
Arimura, Y.; Osakabe, A.; Kato, D.; Kurumizaka, H.; Kawashima, S.
A.; Yamatsugu, K.; Kanai, M. Synthetic Posttranslational Modifica-
tions: Chemical Catalyst-Driven Regioselective Histone Acylation of
Native Chromatin. J. Am. Chem. Soc. 2017, 139, 7568−7576.
(10) Yamatsugu, K.; Furuta, M.; Xi, S.; Amamoto, Y.; Liu, J.;
Kawashima, S. A.; Kanai, M. Kinetic analyses and structure-activity
relationship studies of synthetic lysine acetylation catalysts. Bioorg.
Med. Chem. 2018, 26, 5359−5367.
́
(11) Meloun, M.; Bordovska, S. Benchmarking and validating
algorithms that estimate pKa values of drugs based on their molecular
structures. Anal. Bioanal. Chem. 2007, 389, 1267−1281.
(12) Liu, Z.; Ma, Q.; Liu, Y.; Wang, Q. 4-(N,N-Dimethylamino)-
pyridine Hydrochloride as a Recyclable Catalyst for Acylation of Inert
Alcohols: Substrate Scope and Reaction Mechanism. Org. Lett. 2014,
16, 236−239.
(13) Kawakami, T.; Aimoto, S. The use of a cysteinyl prolyl ester
(CPE) autoactivating unit in peptide ligation reactions. Tetrahedron
2009, 65, 3871−3877.
(14) Sakamoto, K.; Tsuda, S.; Nishio, H.; Yoshiya, T. 1,2,4-Triazole-
aided native chemical ligation between peptide-N-acyl-N′-methyl-
benzimidazolinone and cysteinyl peptide. Chem. Commun. 2017, 53,
12236−12239.
(15) Raibaut, L.; Ollivier, N.; Melnyk, O. Sequential native peptide
ligation strategies for total chemical protein synthesis. Chem. Soc. Rev.
2012, 41, 7001−7015.
(16) (a) Sato, K.; Shigenaga, A.; Tsuji, K.; Tsuda, S.; Sumikawa, Y.;
Sakamoto, K.; Otaka, A. N-Sulfanylethylanilide Peptide as a Crypto-
Thioester Peptide. ChemBioChem 2011, 12, 1840−1844. (b) Sato, K.;
Shigenaga, A.; Kitakaze, K.; Sakamoto, K.; Tsuji, D.; Itoh, K.; Otaka,
A. Chemical Synthesis of Biologically Active Monoglycosylated GM2-
Activator Protein Analogue Using N-Sulfanylethylanilide Peptide.
Angew. Chem., Int. Ed. 2013, 52, 7855−7859.
(17) Bang, D.; Kent, S. B. H. A One-Pot Total Synthesis of Crambin.
Angew. Chem., Int. Ed. 2004, 43, 2534−2538.
(18) SMDMAP thioester 15 was readily prepared by the reaction of
the precursor peptide azide with 2 formed in situ by partial reduction
of 5 with a smaller stoichiometric amount of TCEP, and the
synthesized SMDMAP 15 remains intact for several weeks under
refrigeration at 4 °C.
(19) (a) Naruse, N.; Kobayashi, D.; Ohkawachi, K.; Shigenaga, A.;
Otaka, A. Copper-Mediated Deprotection of Thiazolidine and
Selenazolidine Derivatives Applied to Native Chemical Ligation. J.
Org. Chem. 2020, 85, 1425−1433. (b) Zhao, Z.; Metanis, N. Copper-
Mediated Selenazolidine Deprotection Enables One-Pot Chemical
Synthesis of Challenging Proteins. Angew. Chem., Int. Ed. 2019, 58,
14610−14614.
(20) The reason for the incomplete desulfurization in the one-pot
protocol including Cu-mediated ring opening of thiazolidine is
probably due to the presence of Cu ions in the reaction mixture.
However, the coexistence of reagents such as thiols, EDTA, DTT, and
diethyldithiocarbamate (DTC)22 that could trap the Cu ions did not
improve the reaction (Figure S9).
(21) (a) Payne, R. J.; Ficht, S.; Greenberg, W. A.; Wong, C.-H.
Cysteine-Free Peptide and Glycopeptide Ligation by Direct
Aminolysis. Angew. Chem., Int. Ed. 2008, 47, 4411−4415. (b) Thomas,
G. L.; Hsieh, Y. S. Y.; Chun, C. K. Y.; Cai, Z.-L.; Reimers, J. R.; Payne,
R. J. Peptide Ligations Accelerated by N-Terminal Aspartate and
Glutamate Residues. Org. Lett. 2011, 13, 4770−4773.
(22) Laps, S.; Sun, H.; Kamnesky, G.; Brik, A. Palladium-Mediated
Direct Disulfide Bond Formation in Proteins Containing S-
E
https://dx.doi.org/10.1021/acs.orglett.0c01383
Org. Lett. XXXX, XXX, XXX−XXX