Epimerization-Free Preparation of C-Terminal Cys Peptide Acid by Fmoc SPPS Using Pseudoproline-Type Protecting Group

Article abstract of DOI:10.1021/acs.joc.9b02344

Preparation of C-terminal Cys-containing peptide acid by Fmoc solid-phase peptide synthesis (SPPS) is difficult due to base-mediated epimerization at Cys. In this paper, use of a C-terminal pseudoproline structure and Trt(2-Cl) resin achieved epimerization-free direct preparation of the C-terminal Cys-containing peptide acid by Fmoc SPPS. Additionally, the C-terminal Cys(ψ^Dmp,Hpro)-containing protected peptide segment was applied to an epimerization-free segment condensation reaction.

Full text of DOI:10.1021/acs.joc.9b02344

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Note
Epimerization-free preparation of C-terminal Cys peptide acid
by Fmoc SPPS using pseudoproline-type protecting group
Shugo Tsuda, Shun Masuda, and Taku Yoshiya
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.9b02344 • Publication Date (Web): 21 Oct 2019
Downloaded from pubs.acs.org on October 21, 2019
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Epimerization-free preparation of C-terminal Cys peptide acid by Fmoc
SPPS using pseudoproline-type protecting group
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Shugo Tsuda, Shun Masuda, and Taku Yoshiya*
Peptide Institute, Inc.
7-2-9 Saito-asagi, Ibaraki-shi, Osaka 567-0085, Japan
https://www.peptide.co.jp/en
t.yoshiya@peptide.co.jp
Abstract
Preparation of C-terminal-Cys-containing peptide acid by Fmoc solid-phase peptide synthesis (SPPS)
is difficult due to base-mediated epimerization at Cys. In this paper, use of C-terminal pseudoproline
structure and Trt(2-Cl) resin proved to achieve epimerization-free direct preparation of the C-terminal-
Cys-containing peptide acid by Fmoc SPPS. Additionally, C-terminal-Cys(ΨDmp,Hpro)-containing
protected peptide segment was confirmed to be applied to an epimerization-free segment condensation
reaction.
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Solid-phase peptide synthesis (SPPS) introduced in 19631 made peptide preparation easier. Peptide
preparation was further facilitated by the emergence of 9-fluorenylmethyloxycarbonyl (Fmoc) SPPS.2
However, the indispensable repetitive base treatment, which removes the temporal Fmoc protective
group, sometimes causes undesired side reactions like aspartimide formation at the Asp-Xaa
sequence.3 Additionally, in case of the preparation of C-terminal Cys peptide acid (not peptide amide),
two side reactions at the C-terminal Cys caused by the base treatment are recognized: i) the conversion
of Cys to D/L-β-piperidinoalanine via dehydroalanine (Dha) and ii) the epimerization to D-Cys. Thus,
even when the esterification reaction attaching the first Cys on the resin was optimized to cleanly
afford Fmoc-Cys(PG)-O-resin (PG: any protecting group), these side reactions degrade the peptide
quality during peptide elongation. Especially, the undesired C-terminal-D-Cys-containing peptide thus
generated is often undetectable using high-performance liquid chromatography (HPLC), because it
tends to co-elute with the desired target peptide. Thus, precautions are required to prepare C-terminal-
Cys-containing peptide acids. Many studies have been conducted to solve these problems. It was first
reported in 1987 that the C-terminal Cys(StBu)-O-Wang type resin is hypersensitive to base
treatment;4,5 undesired C-terminal-β-piperidinoalanine-containing peptide6 was obtained nearly
quantitatively. While a bulky protecting group for Cys side chain like Trt confers resistance to side
reactions,7 the use of Wang resin is eventually recognized unsuitable for the synthesis of C-terminal-
Cys-containing peptide acids. Contextually, a research breakthrough was provided by Kiso et al.8 in
1993. They found that bulky trityl-type linker structure between peptide and the resin efficiently
prevents side reactions and enables practical preparation of C-terminal-Cys-containing peptides by the
standard Fmoc SPPS. Although many C-terminal-Cys-containing peptide acids were prepared using
this method thereafter, some amounts of the undesirable side reactions are still observed in crude
deprotected peptide mixtures. Thus, many tune-up methods are reported including side chain
anchoring methods,7c,9 use of safety-catch linker,10 development of novel side chain protecting
groups,11 and use of precursor hydrazide peptide12 (Figure 1A). These methods mitigate side reactions
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but are inconvenient and the epimerization cannot be suppressed completely. Therefore, to prevent
epimerization of C-terminal Cys, research using native chemical ligation to ligate C-terminal Cys with
a corresponding peptide thioester at the final reaction was recently reported.13 (Figure 1B). Herein, we
report that practically complete suppression of the side reactions was achieved using C-terminal
pseudoproline structures and Trt-type resin (Figure 1C). Comparison of the side reaction rate between
the C-terminal Cys(Trt)-O-Trt(2-Cl)-resin and the C-terminal pseudoproline-O-Trt(2-Cl)-resin under
standard/heated basic conditions revealed that the latter is prominent for suppressing the side reactions.
Additionally, the protected peptide segment with the C-terminal pseudoproline structure obtained by
our method was applied successfully to epimerization-free segment condensation reactions.
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Figure 1. Preparation of C-terminal-Cys-containing peptide acid.
Pseudoprolines were first developed by Mutter et al. for effective preparation of difficult sequence-
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containing peptides.14a,b In this method, Ser/Thr/Cys in pseudoprolines are cyclized with a single C
atom between Nα atom and the side chain functionality (O or S atom) as a (thio)acetal, and the final
TFA-mediated deprotection reaction simultaneously releases intact Ser/Thr/Cys through acid-
mediated hydrolysis of (thio)acetal. A feature of this method is the “kink” in peptide on the resin
induced by the insertion of a Pro-like structure at Ser/Thr/Cys; an amino acid residue like Pro disrupts
undesired secondary structure formation on the resin. Simply put, such pseudoprolines are structurally
unique compared with other protecting groups introduced only on the side chain. Therefore, we
envisioned that pseudoproline affords a new paradigm of the effective protection group for Cys related
to the suppression of side reactions during the Fmoc SPPS of the C-terminal Cys peptide acid.
Pseudoproline-containing protected peptides are generally produced on the resin using pseudoproline
dipeptides unit,14c,d because direct preparation of such peptides via on-resin acylation of the
pseudoproline is often difficult. Thus, we also decided to adopt commercially available Cys-
pseudoproline dipeptides for this study. (Please note that, according to the general nomenclature of
products on the market, the Fmoc-protected Cys-pseudoproline dipeptide unit is denoted as Fmoc-
Xaa-Cys(ΨDmp,Hpro)-OH in this study.)
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Scheme 1. Comparison study of epimerization rate at C-terminal Cys of protected peptide acid on the
resin.
Scheme 2. Synthetic scheme of hCNP22 using Cys-pseudoproline dipeptide unit.
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To compare the epimerization rate of C-terminal Cys of the protected peptide acid on the resin, we
constructed Boc-Phe-Ala-Cys(ΨDmp,Hpro)-O-Trt(2-Cl)-resin (1) and Boc-Phe-Ala-Cys(Trt)-O-Trt(2-
Cl)-resin (2), respectively. The Cys-pseudoproline dipeptide was loaded to the Cl-Trt(2-Cl)-resin in
the presence of DIEA without any problem, and then Boc-Phe-OH was introduced to afford resin 1.
After obtaining the protected peptide resins, we applied them to a basic condition (20% piperidine in
NMP) over 24 h at room temperature (Scheme 1). Then, the production of impurities was monitored
by HPLC after TFA-mediated deprotection (See SI). Consequently, no detectable side product arising
from C-terminal Cys residue exists in the crude peptide mixture, when Cys(ΨDmp,Hpro) was adopted
as Cys protection. By contrast, D-Cys-containing peptide (3b, 4.2%) along with D/L-β-
piperidinoalanine-containing peptides (3c, 2.0% and 2.9%, respectively) was observed when Cys(Trt)
was adopted as Cys protection. In both cases, no byproduct containing Dha was detected. The D/L-β-
piperidinoalanine-containing peptides were apparently produced by addition of piperidine to Dha-
containing peptides on the resin. On the basis of these positive results, we further applied two resins
to harsh condition of 60 °C for 4 h (Figure 2). Under this accelerated test condition, D-Cys-containing
peptide (3b, 17.7%) along with D/L-β-piperidinoalanine-containing peptides (3c, 11.0% and 13.6%,
respectively) was observed, when Cys(Trt) was adopted as Cys protection. By contrast, when
Cys(ΨDmp,Hpro) was adopted as Cys protection, D-Cys-containing peptide remained negligible (0.1%),
whereas D/L-β-piperidinoalanine-containing peptides were slightly observed (0.6% and 0.3%,
respectively). The Dha-containing peptide was not detected this time too. To validate the applicability
of our method, we then synthesized human C-type natriuretic peptide 22 (hCNP22, 4) containing a
single disulfide bond between Cys6 and Cys22 by the modified Fmoc SPPS15 using Fmoc-Gly-
Cys(ΨDmp,Hpro)-OH for C-terminal 2 residues (Scheme 2 and SI). Since the D-Cys epimer peptide
often co-elutes with the target peptide, the authentic D-Cys-containing peptide was synthesized
separately and analyzed with the target peptide by HPLC. Consequently, although separation of the
two peptides before oxidation by HPLC could not be achieved, the two peptides after oxidation were
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successfully analyzed by HPLC as reported previously.8b Thus, we evaluated epimerization of C-
terminal Cys using an oxidized peptide without purification. As shown in Figure S4, D-Cys-containing
peptide was not confirmed in the oxidized crude mixture synthesized by our method. Additionally, no
significant side products were observed arising from the use of our method. These results indicated the
applicability of our method to the Fmoc SPPS.
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Scheme 3. (A) Possible mechanism of epimerization at C-terminal Cys from 5a to 5b via 6 and
conversion to D/L-β-piperidinoalanine 8 via Dha 7. (B) In our method using pseudoprolines,
abstraction of Hα to afford D-Cys epimer would be mainly restricted by steric hindrance in the structure
of enol intermediate corresponding to 6.
Next, we investigated the chemistry behind this phenomenon, but, surprisingly, the mechanism of the
side reactions of Cys(Trt)-Trt(2-Cl)-resin has remained poorly discussed. Thus, we initially focused
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on the side reaction that occurred against the Boc-Phe-Ala-Cys(Trt)-O-Trt(2-Cl)-resin (2) under basic
conditions. In such a structure containing Cys-ester, Hα acidity of Cys is probably relatively high in
comparison with a general C-terminal protected amino acid residue, because interaction between Sβ
and the carbonyl group of Cys itself activates the ester linkage.16 Under such conditions, a possible
reaction diagram underlying side reactions is shown in Scheme 3A, and it includes two main pathways:
i) epimerization of Cys from 5a to 5b via transition state enol 6 and ii) irreversible amination to afford
D/L-β-piperidinoalanines 8 via Dha-containing compound 7. To evaluate whether β-elimination
producing 5a and 5b is reversible or not, we planned a simple experiment, i.e., basic treatment of the
Boc-Phe-Ala-Cys(Trt)-O-Trt(2-Cl)-resin in the presence of an excess amount of Trt-SH (See SI). In
this experiment, if β-elimination is reversible and Trt-SH can re-enter the Dha, epimerization should
be accelerated while β-piperidinoalanine is decreased. As a result, the production rate of the D-Cys-
containing compound and D/L-β-piperidinoalanine-containing compound was not clearly affected by
the presence of the excess amount of Trt-SH, suggesting that β-elimination is almost irreversible.17
These results indicate epimerization occurred mainly via an enol-type compound 6 accompanied by
abstraction of the Hα. On the basis of this observation, the absence of epimerization in Cys(ΨDmp,Hpro)
in our method is fairly reasonable, because an enol-type compound 6 containing two successive atoms
(Nα and Cα) with double-bond character would be difficult to be formed with a cyclized imine structure
(Scheme 3B).18 Additionally, the interaction between Sβ and the carbonyl group of Cys itself activating
the ester linkage is cancelled due to a structurally constraint, and the C-terminal ester group of the
Xaa-Pro peptide-ester is de-activated by the interaction between the carbonyl moiety of Xaa and that
of Pro.19 Eventually, it is conceivable that multiple factors affected our Cys(ΨDmp,Hpro) system to
achieve epimerization-free preparation of C-terminal-Cys-containing peptides. Additionally, β-
elimination reaction to afford Dha is suppressed in the structure of Cys(ΨDmp,Hpro), because
antiperiplanar conformer that should be involved during β-elimination reaction from Cys(Trt) to Dha
is prevented by the ring constraint in Cys(ΨDmp,Hpro).
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Scheme 4. Epimerization-free segment condensation reaction using C-terminal Cys-pseudoproline
structure.
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Finally, we evaluated whether the protected segment containing C-terminal Cys-pseudoproline enables
epimerization-free segment condensation reactions. This was already prospected in the early stages of
the development of pseudoproline technology, but experimental evidence is nonexistent to our best
knowledge.14b Similar to C-terminal-Pro- and Ser/Thr-pseudoproline-containing peptide,15,20 C-
terminal Cys-pseudoproline peptides would be condensed with the amino group without epimerization.
We decided to confirm whether such an epimerization-free segment condensation is achievable in the
C-terminal Cys. As shown in Scheme 4, a protected segment containing C-terminal Cys(ΨDmp,Hpro) 9
was prepared using a method like that earlier described and applied to the standard segment
condensation reaction with 10 using PyBOP-HOBt. Therefore, it was confirmed that the protected
peptide segment containing C-terminal Cys(ΨDmp,Hpro) is synthesized without epimerization and
applied to the segment condensation without epimerization (Figure S8).
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In summary, we discovered that C-terminal-Cys-containing peptide acid can be synthesized without
epimerization by Cys(ΨDmp,Hpro) and Trt(2-Cl) resin. Epimerization during basic treatment was not
detected even under accelerated test conditions. To our knowledge, this is the first report of an
epimerization-free direct preparation of C-terminal-Cys-containing peptide acid by Fmoc SPPS.
Additionally, C-terminal-Cys(ΨDmp,Hpro)-containing protected peptide segment was confirmed to be
applied to an epimerization-free segment condensation reaction without problem.
Experimental Section
General Information
All reagents and solvents were obtained from Peptide Institute, Inc. (Osaka, Japan), FUJIFILM Wako
Pure Chemical Corporation (Osaka, Japan), Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan),
Nacalai Tesque, Inc. (Kyoto, Japan), Watanabe Chemical Industries, Ltd. (Hiroshima, Japan), Merck
KGaA (Darmstadt, Germany) and Sigma-Aldrich Co. LLC. (St. Louis, MO).
Preparative HPLC was carried out on a Shimadzu liquid chromatograph Model LC-8A (Kyoto, Japan)
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with a YMC-Pack ODS-A (30 × 250 mm) and the following solvent systems: 0.1% TFA in H2O and
0.1% TFA in CH3CN at a flow rate of 20 mL min-1 with detection at 220 nm. Analytical HPLC was
performed on a Shimadzu liquid chromatograph Model LC-10A (Kyoto, Japan) with a DAISO-PAK
SP-120-5-ODS-BIO (4.6 × 150 mm) or a YMC-Pack ODS-A(4.6 × 150 mm) and the following solvent
systems: 0.1% TFAin H2O and 0.1% TFAin CH3CN at a flow rate of 1 mLmin-1 (40 °C) with detection
at 220 nm. Low resolution mass spectra (LRMS) were observed with an Agilent G1956B LC/MSD
detector using an Agilent 1100 series HPLC system; for deconvolution, observed masses were
calculated using the experimental m/z values (most abundant masses) for each protonation states of a
target peptide. Heating reaction was performed using an EYELA Dry Block Bath MG-2200. Amino
acid analyses were carried out on a Hitachi amino acid analyzer L-8900 after hydrolysis of the peptides
with 6 M HCl at 110 °C for 22 h in an evacuated sealed tube.
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Automated Fmoc SPPS was performed on an ABI 433A peptide synthesizer (Applied Biosystems,
MA). The deprotection of Fmoc group was performed by treatments of piperidine/DMF (1:4) (2.5 min
× 5). The peptide chain was elongated using the coupling protocol of Fmoc-amino
acid/DIC/OxymaPure.21 The following side-chain-protected amino acids were employed: Arg(Pbf),
Asp(OMpe), Cys(Trt), Lys(Boc), Ser(tBu).
Boc-Phe-Ala-Cys(ΨDmp,Hpro)-O-Trt(2-Cl)-resin (1)
Cl-Trt(2-Cl)-Resin (133 mg, 0.2 mmol) was placed in a reaction vessel. Fmoc-Ala-Cys(ΨDmp,Hpro)-
OH (56 mg, 0.1 mmol, 1 equiv, Peptide Institute, Inc. (Osaka, Japan)) and DIEA (68 µL, 0.4 mmol, 4
equiv) dissolved in dry CH2Cl2 (5 mL) were added. The slurry was shaken for 1 h. The resin was
washed with a mixture of CH2Cl2/MeOH/DIEA (v/v/v, 17/2/1, 1 min × 3). After washing with CH2Cl2,
the peptide was assembled using automated Fmoc SPPS procedure as described in general information
(Boc-Phe: 5 equiv).
A small aliquot of the resin was treated with TFA cocktail and the obtained peptide was analyzed by
mass spectrometry. LRMS (M+H) calcd for C15H22N3O4S 340.1, found 340.1.
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Boc-Phe-Ala-Cys(Trt)-O-Trt(2-Cl)-resin (2)
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The peptide was assembled on an Fmoc-Cys(Trt)-O-Trt(2-Cl)-Resin (0.2 mmol) using automated
Fmoc SPPS procedure as described in general information (Fmoc-Xaa: 5 equiv, Boc-Phe is used as N-
terminal Xaa).
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A small aliquot of the resin was treated with TFA cocktail and the obtained peptide was analyzed by
mass spectrometry. LRMS (M+H) calcd for C15H22N3O4S 340.1, found 340.1.
hCNP22 = Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Met-Ser-Gly-Leu-
Gly-Cys (Disulfide bond between Cys6-Cys22) (4) 22
Cl-Trt(2-Cl)-Resin (133 mg, 0.2 mmol) was placed in a reaction vessel. Fmoc-Gly-Cys(ΨDmp,Hpro)-
OH (55 mg, 0.1 mmol, 1 equiv) and DIEA (68 µL, 0.4 mmol, 4 equiv) dissolved in dry CH2Cl2 (6 mL)
were added. The slurry was shaken for 3 h. The resin was washed with a mixture of
CH2Cl2/MeOH/DIEA (v/v/v, 17/2/1, 5 min × 2). After washing with CHCl3, the resin was dried over
vacuum pump to yield Fmoc-Gly-Cys(ΨDmp,Hpro)-O-Trt(2-Cl)-resin (192 mg, 0.1 mmol). The
obtained resin (96 mg, 0.05 mmol) was placed in a reaction vessel and treated with 10 mL of a mixture
of 140 mM DBU and 129.5 mM HOBt in DMF (DBU/HOBt/DMF = 2.1/1.7/96.2, v/w/v) for 30 min.15
The peptide was assembled on the resin using automated Fmoc SPPS procedure as described in general
information (Fmoc-Xaa: 20 equiv). The subsequent deprotection of the resin was carried out by
TFA/TIS/H2O (v/v, 95/2.5/2.5) for 1.5 h to give a crude reduced form product. The obtained crude
peptide was dissolved in 50 mL of DMSO/buffer solution (6 M Gn·HCl, 0.1 M Na2HPO4, pH 7.0)
(1/1). After standing for 20 h, the reaction mixture was subjected to preparative HPLC to yield the title
peptide as a lyophilized white powder (31 mg, 28%). Analytical HPLC: tR = 14.0 min (20–40%
CH3CN/0.1% TFA for 25 min); LRMS (ESI) calcd for C93H157N27O28S3 2197.1, found 2197.2; Amino
acid analysis: Asp 1.01 (1), Ser 2.71 (3), Gly 5.92 (6), Met 1.03 (1), Ile 1.00 (1), Leu 3.93 (4), Phe
0.97 (1), Lys 1.93 (2), Arg 1.00 (1), 1/2(Cys)2 1.81 (2).
Boc-Phe-Lys(Boc)-Val-Ala-Cys(ΨDmp,Hpro)-OH (9)
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Cl-Trt(2-Cl)-Resin (67 mg, 0.1 mmol) was placed in a reaction vessel. Fmoc-Ala-Cys(ΨDmp,Hpro)-OH
(56 mg, 0.1 mmol, 1 equiv) and DIEA (68 µL, 0.4 mmol, 4 equiv) dissolved in dry CH2Cl2 (6 mL)
were added. The slurry was shaken for 1 h. The resin was washed with a mixture of
CH2Cl2/MeOH/DIEA (v/v/v, 17/2/1, 2 min × 3). The peptide assembly was conducted in a similar
manner described above. The subsequent deprotection of the obtained resin was carried out by
HFIP/CHCl3 (v/v, 20/80) for 1.5 h to give a crude title peptide (27.3 mg, 30%). The protected peptide
was used to the next reaction without further purification. Analytical HPLC: tR = 24.3 min and 24.6
min (10–60% CH3CN/0.1% TFA for 25 min); LRMS (M+H) calcd for C45H67N6O12S 915.5, found
915.4.
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Leu-Pro-Val-Gly-Phe-NH2 (10)
The peptide was assembled on a Rink Amide Resin (0.25 mmol) using automated Fmoc SPPS
procedure as described in general information (Fmoc-Xaa: 4 equiv). The subsequent deprotection of
the resin was carried out by TFA/TIS/H2O (v/v, 95/2.5/2.5) for 1.5 h and purified by HPLC to give the
title peptide as a lyophilized white powder (73 mg, 55%). Analytical HPLC: tR = 10.0 min (10–60%
CH3CN/0.1% TFA for 25 min); LRMS (M+H) calcd for C27H43N6O5 531.3, found 531.3; Amino acid
analysis: Gly 1.00 (1), Val 1.00 (1), Leu 1.00 (1), Phe 1.00 (1), Pro 1.00 (1).
Phe-Lys-Val-Ala-Cys-Leu-Pro-Val-Gly-Phe-NH2 (12)
Boc-Phe-Lys(Boc)-Val-Ala-Cys(ΨDmp,Hpro)-OH (1.0 mg, 1.09 µmol), Leu-Pro-Val-Gly-Phe-NH2
(0.58 mg, 1.09 µmol) and HOBt (0.15 mg, 1.09 µmol) were dissolved in 30 µL of DMF. PyBOP (0.68
mg, 1.31 µmol) dissolved in 10 µL of DMF was added into the mixture and the pH was adjusted with
DIEA to 7. After stirring for 1 h, the reaction mixture was monitored by analytical HPLC. Then, the
reaction mixture was concentrated and then deprotected by TFA/TIS/H2O (v/v, 95/2.5/2.5). After 30
min, the solution was concentrated. Then, the residue was dissolved in 50% AcOH in H2O and washed
with hexane. Aqueous layer was monitored by analytical HPLC. Analytical HPLC: tR = 14.7 min (10–
60% CH3CN/0.1% TFA for 25 min); LRMS (M+H) calcd for C53H83N12O10S 1079.6, found 1079.5.
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C-terminal Cys epimerization study
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The Boc-Phe-Ala-Cys(PG)-O-Trt(2-Cl)-resins, where PG is Trt or ΨDmp,Hpro, were treated with 20%
piperidine/NMP at room temperature for 24 h or at 60 °C for 4 h. The obtained resin was treated with
TFA/TIS/H2O (v/v, 95/2.5/2.5), respectively. The obtained peptide was dissolved in 50% AcOH in
H2O and monitored by analytical HPLC.
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Mechanistic study of side reactions of Cys(Trt)-O-Trt(2-Cl)-resin
Boc-Phe-Ala-Cys(Trt)-O-Trt(2-Cl)-resin was treated with a mixture of Trt-SH (10 equiv) and 20%
piperidine/NMP at 60 °C for 4h. The obtained resin was treated with TFA/TIS/H2O (v/v, 95/2.5/2.5).
The obtained peptide was dissolved in 50% AcOH in H2O and monitored by analytical HPLC.
Analytical HPLC: 1–60% CH3CN/0.1% TFA for 25 min.
Supporting Information: MS spectra of deprotected compounds 1 and 2 and compounds 4, 9, 10 and
12. HPLC chromatograms of compounds 4, 9 and 10. HPLC chromatograms and graph of C-terminal
Cys epimerization study at room temperature for 24 h. HPLC chromatograms of mechanistic study of
side reactions of Cys(Trt)-O-Trt(2-Cl)-resin.
References
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(7) (a) Atherton, E.; Hardy, P. M.; Harris, D. E.; Matthews, B. H. Racemisation of C-terminal cysteine
during peptide assembly. Peptides 1990 1991, 21, 243−244. (b) Wade, J. D.; Bedford, J.; Sheppard, R.
C.; Tregear, G. W. DBU as an Nα-Deprotecting Reagent for the Fluorenylmethoxycarbonyl Group in
Continuous Flow Solid-Phase Peptide Synthesis. Pept. Res. 1991, 4, 194−199. (c) Barany, G.; Han, Y.;
Hargittai, B.; Liu, R. Q.; Varkey, J. T. Side-Chain Anchoring Strategy for Solid-Phase Synthesis of
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(8) (a) Fujiwara, Y.; Akaji, K.; Kiso, Y. Studies on racemization of C-terminal cysteine in Fmoc-based
solid phase peptide synthesis. Pept. Chem.1993 1994, 31, 29−32. (b) Fujiwara, Y.; Akaji, K.; Kiso, Y.
Racemization-free synthesis of C-terminal cysteine-peptide using 2-chlorotrityl resin. Chem. Pharm.
Bull. 1994, 45, 724−726.
(9) (a) Huang, Z.; Derksen, D. J.; Vederas, J. C. Preparation and Use of Cysteine Orthoesters for Solid-
Supported Synthesis of Peptides. Org. Lett. 2010, 12, 2282−2285. (b) Diaz-Rodriguez, V.; Mullen, D.
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Esters Using Trityl Side-Chain Anchoring: Application to the Synthesis of a-Factor and a-Factor
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Anchoring: Applications to the Synthesis of C-Terminal Ester Analogs of the Saccharomyces
cerevisiae Mating Pheromone a-Facto. J. Org. Chem. 2015, 80, 11266−11274. (d) Juvekar, V.; Gong,
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(10) Ni, S.; Zhang, H.; Huang, W.; Zhou, J.; Qian, H.; Chen, W. The application of an aryl hydrazine
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(11) (a) Hibino, H.; Nishiuchi, Y. 4-Methoxybenzyloxymethyl Group, a Racemization-Resistant
Protecting Group for Cysteine in Fmoc Solid Phase Peptide Synthesis. Org. Lett. 2012, 14, 1926−1929.
(b) Hibino, H.; Miki, Y.; Nishiuchi, Y. Evaluation of acid-labile S-protecting groups to prevent Cys
racemization in Fmoc solid-phase peptide synthesis. J. Pept. Sci. 2014, 20, 30−35. (c) Ramos-
Tomillero, I.; Rodriguez, H.; Albericio, F. Tetrahydropyranyl, a Nonaromatic Acid-Labile Cys
Protecting Group for Fmoc Peptide Chemistry. Org. Lett. 2015, 17, 1680−1683.
(12) Zuo, C.; Yan, B. J.; Zhu, H. Y.; Shi, W. W.; Xi, T. K.; Shi, J.; Fang, G. M. Robust synthesis of C-
terminal cysteine-containing peptide acids through a peptide hydrazide-based strategy. Org. Biomol.
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Overcome Side Reactions during Fmoc-Based Synthesis of C-Terminal Cysteine-Containing Peptides.
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Stockdill, J. L. Epimerization-free access to C-terminal cysteine peptide acids, carboxamides,
secondary amides, and esters via complimentary strategies. Chem. Sci. 2018, 9, 350−355.
(14) (a) Haack, T.; Mutter, M. Serine derived oxazolidines as secondary structure disrupting,
solubilizing building blocks in peptide synthesis. Tetrahedron Lett. 1992, 33, 1589−1592. (b) Woehr,
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T.; Wahl, F.; Nefzi, A.; Rohwedder, B.; Sato, T.; Sun, X. C.; Mutter, M. Pseudo-Prolines as a
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(15) We adopted DBU/HOBt method for Fmoc deprotection of pseudoproline dipeptides to prevent
diketopiperazine formation according to this paper: Heinlein, C.; Varon Silva, D.; Troster, A.; Schmidt,
J.; Gross, A.; Unverzagt, C. Fragment Condensation of C-Terminal Pseudoproline Peptides without
Racemization on the Solid Phase. Angew. Chem. Int. Ed. 2011, 50, 6406−6410.
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J. Org. Chem. 1982, 47, 4996−5002.
(17) A similar theory was reported in an epimerization study during the coupling reaction of Cys:
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Derivatives. Rate of Racemization and Peptide Bond Formation of Cysteine Active Esters. J. Org.
Chem. 1970, 35, 1810−1815.
(18) Barber, M.; Jones, J. H.; Witty, M. J. Structural Factors Affecting the Direct Exchange
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Compounds by Triethylamine in Chloroform. J. Chem. Soc., Perkin Trans. 1 1979, 10, 2425−2428.
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reactivity of peptide-prolyl-thioesters in native chemical ligation. Chem. Commun. 2011, 47,
2342−2344. (b) Newberry, R. W.; Raines, R. T. The n→π* Interaction. Acc. Chem. Res. 2017, 50,
1838−1846.
(20) Coin, I.; Schmieder, P.; Bienert, M.; Beyermann, M. The depsipeptide technique applied to peptide
segment condensation: Scope and limitations. J. Pept. Sci. 2008, 14, 299−306.
(21) Subirós-Funosas, R.; Prohens, R.; Barbas, R.; El-Faham, A.; Albericio, F. Oxyma: An Efficient
Additive for Peptide Synthesis to Replace the Benzotriazole-Based HOBt and HOAt with a Lower
Risk of Explosion. Chem. Eur. J., 2009, 15, 9394–9403.
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(22) (a) Sudoh, T.; Minamino, N.; Kangawa, K.; Matsuo, H. C-Type natriuretic peptide (CNP): A new
member of natriuretic peptide family identified in porcine brain. Biochem. Biophys. Res. Commun.,
1990, 168, 863−870. (b) Fujiwara, Y.; Yokotani, J.; Akaji, K.; Kiso, Y. Synthesis of Human C-Type
Natriuretic Peptide 22 Using Chlorotrityl Resin and Tetrafluoroboric Acid Deprotection. Chem. Pharm.
Bull., 1996, 44, 1326−1331.
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