Sequential native chemical ligation utilizing peptide thioacids derived from newly developed Fmoc-based synthetic method

Article abstract of DOI:10.1016/j.tet.2010.03.016

The first facile Fmoc-based synthetic procedure for peptide thioacids was developed. Successful application of the resulting thioacids to sequential native chemical ligation (NCL) in the N to C direction was achieved. Conversion of the peptide thioacids to the corresponding thioesters with Ellman's reagent followed by NCL in the presence of tris(2-carboxyethyl)phosphine (TCEP) and thiophenol was accomplished in a one-pot manner.

Full text of DOI:10.1016/j.tet.2010.03.016

Tetrahedron 66 (2010) 3290–3296
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Sequential native chemical ligation utilizing peptide thioacids derived from
newly developed Fmoc-based synthetic method
*
Akira Shigenaga, Yoshitake Sumikawa, Shugo Tsuda, Kohei Sato, Akira Otaka
Institute of Health Biosciences and Graduate School of Pharmaceutical Sciences, The University of Tokushima, Shomachi, Tokushima 770-8505, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The first facile Fmoc-based synthetic procedure for peptide thioacids was developed. Successful appli-
cation of the resulting thioacids to sequential native chemical ligation (NCL) in the N to C direction was
achieved. Conversion of the peptide thioacids to the corresponding thioesters with Ellman’s reagent
followed by NCL in the presence of tris(2-carboxyethyl)phosphine (TCEP) and thiophenol was accom-
plished in a one-pot manner.
Received 9 February 2010
Received in revised form 3 March 2010
Accepted 3 March 2010
Available online 9 March 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Peptide thioacids
Peptide thioesters
Native chemical ligation
N-S Acyl transfer
Fmoc solid-phase peptide synthesis
1. Introduction
Native chemical ligation (NCL) has shown great utility in the
preparation of proteins in a synthetic or semi-synthetic manner.1
Intermolecular chemoselective reaction of the thiol group on
N-terminal cysteine (Cys) residue with thioesters followed by S-N
acyl shift allows the two peptide fragments without protecting
groups to be condensed.
That is not the case for repetitive NCL with more than one thio-
ester fragment, whereby the N and/or S functions on the N-terminal
Cys residue in the middle thioester fragment require protection to
avoid intramolecular NCL, thus leading to the formation of cyclic
peptides (Scheme 1, Method A).2 To this end, various types of pro-
tecting groups are introduced onto the N-terminal cysteine to mask
intramolecular NCL-reactivity of thioesters. An elegant alternative
developed by Kent et al. is the use of intermolecular reaction of the
free N-terminal Cys residue in peptide alkylthioesters with peptide
arylthioesters, where the sulfhydryl group reacts intermolecularly to
the arylthioester in a kinetically controlled manner (Scheme 1,
Method B).3 Masking the reactivity of thioesters is another potential
option to establish facile sequential NCL protocols, but masked
thioesters requires easy conversion to active thioesters through
simple manipulations. With this in mind, we re-evaluated the syn-
thetic applicability of peptidyl thioacids as thioester precursors to
sequential NCL in solution phase synthesis.
Scheme 1. Sequential native chemical ligation for preparation of peptides/proteins.
Method A: PN and PS¼protections for amino and sulfanyl groups, respectively; (a) first
native chemical ligation (first NCL); (b) removal of PN and/or PS; (c) second NCL (second
NCL). Method B: (d) first NCL (kinetically controlled conditions); (e) second NCL.
* Corresponding author. E-mail address: aotaka@ph.tokushima-u.ac.jp.
0040-4020/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2010.03.016

A. Shigenaga et al. / Tetrahedron 66 (2010) 3290–3296
3291
Peptidyl thioacids were first introduced into peptide chemistry
by Li et al.4 They applied the conversion of thioacids to the corre-
sponding active acyl components by treatment with silver nitrate
ligation of peptide thioacid (1(SH) or 4(SH)) via conversion to the
corresponding peptide thioester (1(SR) or 4(SR), respectively) with
N-terminal Cys fragments, such as 2 or 3 can be conducted in a one-
pot manner in the presence of TCEP without requiring purification
of the thioesters. Notably, the use of N-terminal Cys thioacid frag-
ments, such as 2, in the NCL step allows the coupling to be per-
formed in a repetitive manner in the N to C direction.8
Nevertheless, widespread use of peptide thioacids in peptide
chemistry, including ligation chemistry, has not been seen. This
seems to be partly dependent on the fact that while Fmoc SPPS
continues to increase in utility, it could not be used for preparation
of peptide thioacids. Thus, development of Fmoc-based protocols
for synthesizing peptide thioacids has been required in reinforcing
the general applicability of sequential NCL protocols using peptide
thioacids. Here, we present sequential NCL using peptide thio-
acids, including a new preparative protocol for peptide thioacids
by Fmoc SPPS. We selected one-disulfide bond-containing
32-residue human natriuretic peptide (hBNP) as the model target
peptide.
or diaryl disulfides in the synthesis of a-inhibin-92. The formation
of peptidyl thioesters from the reaction of thioacids with Ellman’s
reagent with applications to inter-5a–c and intramolecular5d NCL
was later reported. Among reports concerning peptide thioacids, it
is worth noting that reversed-type N to C elongation was achieved
using peptide thioacids.6,7 In this research, condensation of N-ter-
minal Cys thioacid fragments on resin-bound thioester followed by
activation of the thioacids to the corresponding thioesters with
alkylating agents, such as bromoacetic acid, is involved. The
resulting thioester could be subjected to further NCL reaction after
washing to remove bromoacetic acid.
2. Results and discussion
As shown in Scheme 2, we speculated that thioesterification of
peptide thioacid 1(SH) by aryl disulfides, such as Ellman’s reagent,
followed by the first NCL with middle thioacid fragment 2 in the
presence of tris(2-carboxyethyl)phosphine (TCEP) for reduction of
excess Ellman’s reagent should result in formation of ligated pep-
tide thioacid 4(SH) in a one-pot manner. After purification of 4(SH),
the resulting thioacids could be brought to the second NCL with
C-terminal fragment 3 according to the procedure described for the
first NCL to give sequential NCL product. In this envisioned scheme,
First, Fmoc-based synthesis of peptide thioacids was established
for preparation of N-terminal hBNP (1–9)- and middle hBNP
(10–25)-fragments (10b and 11) as shown in Scheme 3.9 Successive
Scheme 3. Fmoc-based preparation of peptide thioacids using (N-Fmoc-glycyl-N-sul-
fanylethyl)aminobenzoic acid linker 5.10a
condensation of Fmoc-Ala-OH and 4-[Fmoc-glycyl-(2-tritylsulfa-
nylethyl)amino]benzoic acid 510a on aminomethyl ChemMatrix
resin afforded the linker-incorporated resin 6. Standard Fmoc SPPS
on the resulting resin 6 followed by deprotection with TFA-based
reagent cocktail gave deprotected peptide amide resins 8, which
was then treated with 4 M HCl/DMF in the presence of 1% (w/v)
Scheme 2. Envisioned sequential native chemical ligation utilizing peptide thioacids
as N-terminal and middle fragments. TCEP¼tris(2-carboxyethyl)phosphine.

3292
A. Shigenaga et al. / Tetrahedron 66 (2010) 3290–3296
TCEP to give peptide thioester resins 9 via N-S acyl transfer on
resin.10 Treatment of the resulting thioesters 9 with NaSH in 1 M
phosphate buffer (pH 9.2)/MeCN (85:15) yielded Na-Fmoc pro-
tected peptide thioacid 10a and peptide thioacid 10b within
15 min.11 To avoid the intramolecular NCL-mediated release of
middle peptide fragment from the resin, protection on the a-amino
group was required. To this end, we selected the Fmoc group
among the potential candidates that are stable under TFA-
mediated deprotection conditions. Here, we were pleased to
find that the continuous NaSH-treatment in the presence of
peptide-detached resin (linker) resulted in the formation of
Fmoc-deprotected peptide thioacid 11, whereas reaction without
the resin gave little Na-amine peptide (Fig. 1).12 The Na-Fmoc
protection exerted its utility in the Fmoc-based preparation of
peptide thioacids, such as 11, because conversion of the thioester
to thioacid proceeded more rapidly than deprotection of the
Figure 1. HPLC monitoring of release of peptide thioacids from resin by treatment
with NaSH in 1 M phosphate buffer (pH 9.2)/MeCN (85:15). (A) After 1 h incubation of
resin 9a, the filtrate was analyzed. (B) After 8 h incubation of the resin. HPLC condi-
tions: Cosmosil 5C18-AR-II column (4.6ꢀ250 mm) with a linear gradient of 0.1% TFA/
MeCN/0.1% TFA aq (5:95–65:35 over 30 min) at a flow rate of 1.0 mL minꢁ1, detection
at 220 nm.
Fmoc group. The C-terminal fragment (H-CKVLRRH-OH 12: hBNP
(26–32)) was prepared by standard Fmoc SPPS. Generality of
the treatment with NaSH for preparation of N-terminal Cys
peptide thioacids was confirmed by successful application to the
synthesis of H-CYAVTGRGDSPAASSG-SH. Alternatively, addition
of piperidine (to 5% (v/v)) into the reaction mixture containing
NaSH also afforded the desired peptide thioacid (Fig. 2).
Having three requisite fragments for construction of the hBNP
backbone, we attempted repetitive NCL in the N to C directive
manner as shown in Scheme 4.
Figure 2. HPLC monitoring of release of peptide thioacid (Fmoc-CYAVTGRGD-
SPAASSG-SH) and deprotection of the Fmoc group by additional NaSH treatment or
piperidine treatment. (A) After 15 min treatment of resin (Fmoc-CYAVTGRGD-
SPAASSG-S-Ar-resin) with 120 mM NaSH in 6 M guanidine-0.1 M sodium phosphate
buffer (pH 9.2) at 37 ꢂC. (B) After additional treatment with NaSH aq for 24 h in the
presence of the peptide-detached resin. (C) Fifteen min hydrothiolitic release of the
peptide followed by addition of piperidine (5%, (v/v)) with additional 15 min treat-
ment. HPLC conditions identical to that of Figure 1 were used.

A. Shigenaga et al. / Tetrahedron 66 (2010) 3290–3296
3293
adjustment of the reaction pH to around 7.4 at 4 ꢂC.15 The first NCL
completed in 2 h to yield ligated peptide thioacid 13 corresponding
to hBNP (1–25) (Fig. 3, C and D). After HPLC purification of the crude
material, purified thioacid 13 was subjected to the second NCL step
in the presence of thiophenol after conversion to the corresponding
Ar thioester 130 by treatment with Ellman’s reagent followed by
TCEP reduction16 as mentioned in the transformation of 10b (Fig. 3,
E and F). Attempted second NCL with C-terminal fragment 12 also
efficiently proceeded to yield 2-Cys-SH hBNP (Fig. 3, G and H). Then,
HPLC-purified Cys-SH peptide was oxidized to hBNP by the action
of DMSO.
Scheme 4. Sequential NCL for the preparation of hBNP. (a) Ellman’s reagent, KHCO3,
H2O/DMF (8:2); (b) TCEP, thiophenol; (c) NCL.
In order to evaluate epimerization of chiral C-terminal amino
acids, model thioacid fragments were synthesized for application to
NCL. Using 4-[Fmoc-
benzoic acid, protected peptide resins corresponding to H-HRFA(
or -)A-SH were constructed on aminomethyl ChemMatrix resin by
standard Fmoc SPPS. After on-resin deprotection of the completed
L- (or D-)alanyl-(2-tritylsulfanylethyl)-amino]-
First, N-terminal thioacid fragment 10b (1 mM) was converted
to the corresponding Ar thioester 10b0 by reaction with Ellman’s
reagent (3 mM) in the presence of KHCO3 (3 mM) in H2O/DMF
(80:20) at room temperature (Fig. 3, A and B).13,14 After reduction of
L-
D
Figure 3. HPLC monitoring of sequential NCL. (A) HPLC analysis of N-terminal thioacid fragment 10b. (B) Conversion of 10b to the corresponding Ar thioester 10b0 by the reaction of
10b with Ellman’s reagent in the presence of KHCO3 in H2O/DMF (2:8) at room temperature for 1.5 h. Ar¼–C6H4(NO2)(CO2H). (C) The first NCL (before adjustment of pH): To the
crude reaction mixture of 10b and Ellman’s reagent were successively added TCEP, thioacid fragment 11 and thiophenol followed by the adjustment of pH to 7.5. (D) The first NCL
x
(t¼2 h) at 37 ꢂC, H-SPKMVQGSGCFGRKMDRISSSSGLG-SH (13). Hydrolyzed product of 10b0. (E) HPLC-purified 13. (F) Conversion of 13 to the corresponding Ar thioester 130
(H-SPKMVQGSGCFGRKMDRISSSSGLG-SAr) by the reaction of 13 with Ellman’s reagent in the presence of KHCO3 in H2O/DMF (2:8) at room temperature for 1.5 h followed by
addition of TCEP. (G) The second NCL (before adjustment of pH): To the crude reaction mixture containing 130 were added C-terminal fragment 12 and thiophenol followed by the
adjustment of pH to 7.5. (H) The second NCL (t¼2 h) at 37 ꢂC. HPLC conditions: Cosmosil 5C18-AR-II column (4.6ꢀ250 mm) with a linear gradient of 0.1% TFA/MeCN/0.1% TFA aq
(5:95–55:45 over 30 min) at a flow rate of 1.0 mL minꢁ1, detection at 220 nm. *5-mercapto-2-nitrobenzoic acid, **Non-peptidic impurities.
excess Ellman’s reagent with TCEP, the first NCL was initiated by
successive addition of equimolar amount of middle thioacid frag-
ment 11 and thiophenol5d to the reaction mixture followed by
resins, N-S acyl transfer on the resin was achieved by treatment of
the amide-type resins with TFA at ambient temperature for 20 h in
the presence of TCEP (1% (w/v)).17 Treatment of the resulting

3294
A. Shigenaga et al. / Tetrahedron 66 (2010) 3290–3296
thioester resin with NaSH in 1 M phosphate buffer (pH 9.2)/MeCN
(85:15) afforded H-HRFA( - or -)A-SH peptide. Each peptide thi-
oacid resulting from - or -Ala-linked resin was converted to the
corresponding thioester by reaction with Ellman’s reagent followed
by NCL with peptide 12 in the presence of TCEP and thiophenol to
give the desired ligated peptide while accompanied by no epi-
merized material (Fig. 4).
column (Nacalai Tesque, 4.6ꢀ250 mm, flow rate 1 mL/min) or
a 5C18-AR-II semi-preparative column (Nacalai Tesque,
L
D
L
D
10ꢀ250 mm, flow rate 3.0 mL/min) was employed, and eluting
products were detected by UV at 220 nm. A solvent system con-
sisting of 0.1% TFA aq solution (v/v, solvent A) and 0.1% TFA in MeCN
(v/v, solvent B) was used for HPLC elution. All Chemicals were
purchased from either Kanto Chemicals, Sigma Aldrich Japan, Tokyo
Chemical Industry, Wako Pure Chemical Industries or Watanabe
Chemical Industry.
4.2. Preparation of resin bound peptide thioesters 9a and 9b
On aminomethyl ChemMatrixÒ resin (1.0 mmol amine/g, 40 mg,
0.040 mmol) was coupled Fmoc-Ala-OH (40 mg, 0.12 mmol) as an
internal standard amino acid in the presence of DIPCDI (19 mL,
0.12 mmol) and HOBt$H2O (21 mg, 0.13 mmol) in DMF at room
temperature for 2 h followed by Fmoc removal by 20% piperidine/
DMF to afford internal standard-incorporated resin. The resulting
resin was treated with a preactivated acid 5 (59 mg, 0.080 mmol)
with HATU (30 mg, 0.075 mmol) and DIPEA (14 mL, 0.075 mmol) to
yield aniline-linked resin 6. On the resulting resin 6, standard Fmoc
SPPS (Fmoc amino acid (3.0 equiv), DIPCDI (3.0 equiv) and
HOBt$H2O (3.3 equiv) in DMF for acylation and 20% piperidine/DMF
(10 min) for Fmoc removal) was performed for the chain elongation
to give protected peptide resin 7a or 7b. The resulting completed
resin 7a or 7b was treated with TFA-thioanisole-m-cresol-H2O-
EDT-Et3SiH (80:5:5:5:2.5:2.5, (v/v)) at room temperature for 1.5 h
to give deprotected peptide resin 8a or 8b, respectively. After being
washed with CH2Cl2, each amide-type resin 8a or 8b was subjected
to N-S acyl transfer reaction in 4 M HCl/DMF in the presence of
TCEP (1% (w/v)) at room temperature for 20 h followed by washing
with CH2Cl2 to yield the corresponding on-resin thioester peptide
9a or 9b, respectively.
4.3. Hydrothiolytic release of peptide N-Cys thioacid 11
On-resin peptide thioester 9a was incubated with 120 mM NaSH
in 1 M sodium phosphate buffer (pH 9.2)/MeCN (85:15) at 37 ꢂC.
Release of Na-Fmoc protected peptide thioacid 10a was confirmed
by HPLC analysis within 15 min of incubation. After additional
incubation for 8 h, the Fmoc group on peptide 10a was completely
removed to give crude materials, which were then subjected to
semi-preparative HPLC purification to afford the desired Fmoc-
deprotected peptide thioacid 11. The residual resin was hydro-
lyzed with 6 M HCl aq in the presence of phenol (0.2%, (v/v)). The
resulting hydrolysate was subjected to amino acid analysis (AAA)
and the release yield was estimated to be 62% based on the
internal Ala.
Figure 4. HPLC examination of epimerization of C-terminal chiral amino acid. (A)
HPLC analysis of crude material after NCL completion (H-HRFA-L-Ala-SHþH-
CKVLRRH-OH). (B) Co-injection of the crude L-Ala peptide and purified D-Ala peptide.
HPLC conditions: Cosmosil 5C18AR-II column (4.6ꢀ250 mm) with a linear gradient of
0.1% TFA/MeCN/0.1% TFA aq (5:95–30:70 over 30 min) at a flow rate of 1.0 mL/min,
detection at 220 nm. Only a critical retention time region of the HPLC charts was
enlarged.
3. Conclusion
We established the first facile Fmoc-based synthetic protocols
for peptide thioacids using the N-S acyl transfer methodology.
Resulting peptide thioacids have been successfully applied to
sequential NCL procedures. The thioacid moiety serves as a masked
thioester with its ready conversion to the corresponding active
thioester via reaction with aryl disulfide and participates in the NCL
step in the presence of TCEP in a one-pot manner. Recent growing
interest of thioacids in peptide/protein chemistry18 allows our
presented protocols to gain unequivocal utility in protein synthesis.
Extension of our methods for preparation of proteins, including
modification of proteins, is currently under investigation.
Compound 10a: Analytical HPLC condition: linear gradient of
solvent
B in solvent A, 5 to 65% over 30 min, retention
time¼21.7 min. MS (ESI-TOF) m/z calcd for ([Mþ2H]2þ) 969.9,
found 970.0.
Compound 11: Analytical HPLC condition: linear gradient of
solvent
B in solvent A, 5 to 65% over 30 min, retention
time¼13.7 min. Semi-preparative HPLC condition: linear gradient
of solvent B in solvent A, 5 to 15% over 30 min. MS (ESI-TOF) m/z
calcd for ([Mþ2H]2þ) 858.8, found 858.8.
4.4. Hydrothiolytic release of peptide thioacid 10b
4. Experimental section
4.1. General methods
On-resin peptide thioester 9b was incubated with 120 mM
NaSH in 1 M sodium phosphate buffer (pH 9.2)/MeCN (85:15) at
37 ꢂC for 15 min to yield hydrothiolytically released peptide thio-
acid 10b. The crude material was purified by semi-preparative
HPLC. The residual resin was hydrolyzed with 6 M HCl aq in the
presence of phenol (0.2%, (v/v)). The resulting hydrolysate was
Exact mass spectra were recorded on Waters MICROMASS LCT
PREMIER. For HPLC separations, a Cosmosil 5C18-AR-II analytical

A. Shigenaga et al. / Tetrahedron 66 (2010) 3290–3296
3295
subjected to AAA and the release yield was estimated to be 69%
based on internal Ala.
around 7.5 by addition of 10% K2CO3 solution (31
m
L) at 4 ꢂC. After
incubation of the mixture at 37 ꢂC for 2 h, the starting materials
were disappeared and the crude material was purified by semi-
preparative HPLC to give ligated peptide 2-Cys-SH hBNP (1.0 mg,
63%). Oxidation of the purified 2-Cys-SH hBNP (0.3 mM) was per-
formed in 0.1 M sodium phosphate buffer containing 6 M guani-
dine hydrochloride (pH 7.3)/DMSO (9:1). After incubation of the
mixture at 37 ꢂC for 12 h, hBNP was isolated by semi-preparative
HPLC.
Compound 10b: Analytical HPLC condition: linear gradient of
solvent B in solvent A, 5 to 45% over 30 min, retention time-
¼10.3 min. Semi-preparative HPLC condition: linear gradient of
solvent B in solvent A, 5 to 10% over 30 min. MS (ESI-TOF) m/z calcd
for ([MþH]þ) 906.4, found 906.5.
4.5. Confirmation of generality of the preparative method for
N-terminal Cys peptide thioacid
Compound 10b0: Analytical HPLC condition: linear gradient of
solvent B in solvent A, 5 to 45% over 30 min, retention time-
¼16.3 min. MS (ESI-TOF) m/z calcd for ([MþH]þ) 1071.4, found
1071.5.
Compound 12: Analytical HPLC condition: linear gradient of
solvent B in solvent A, 5 to 45% over 30 min, retention time-
¼10.3 min. MS (ESI-TOF) m/z calcd for ([Mþ2H]2þ) 456.3, found
456.2.
Compound 13: Analytical HPLC condition: linear gradient of
solvent B in solvent A, 5 to 45% over 30 min at a flow rate of
1.0 mL minꢁ1, retention time¼17.8 min. Semi-preparative HPLC
condition: linear gradient of solvent B in solvent A, 15 to 35% over
30 min. MS (ESI-TOF) m/z calcd for ([Mþ3H]3þ) 863.4, found 863.4.
Compound 130: Analytical HPLC condition: linear gradient of
On-resin peptide Fmoc-Cys(Trt)-Tyr(tBu)-Ala-Val-Thr(tBu)-Gly-
Arg(Pbf)-Gly-Asp(tBu)-Ser(tBu)-Pro-Ala-Ala-Ser(tBu)-Ser(tBu)-Gly-
N-Ar-resin was prepared on Leu-aminomethyl ChemMatrixÒ resin
as mentioned above using Fmoc protocol. Deprotection of the
completed resin with TFA-thioanisole-m-cresol-H2O-EDT-Et3SiH
(80:5:5:5:2.5:2.5, (v/v)) at room temperature for 1.5 h followed by
the treatment with 4 M HCl/DMF in the presence of TCEP (1% (w/v))
at room temperature for 20 h gave deprotected on-resin peptide
thioester Fmoc-Cys-Tyr-Ala-Val-Thr-Gly-Arg-Gly-Asp-Ser-Pro-Ala-
Ala-Ser-Ser-Gly-S-Ar-resin. The on-resin peptide thioester was
incubated with 120 mM NaSH in 6 M guanidine-0.1 M sodium
phosphate buffer (pH 9.2) at 37 ꢂC for 15 min to yield the hydro-
thiolytically released peptide thioacid Fmoc-Cys-Tyr-Ala-Val-Thr-
Gly-Arg-Gly-Asp-Ser-Pro-Ala-Ala-Ser-Ser-Gly-SH (Fig. 2-A). After
additional incubation for 24 h, complete removal of the Fmoc group
was confirmed by HPLC analysis (Fig. 2-B). When piperidine was
added to the crude mixture of the Na-Fmoc protected peptide
thioacid obtained after hydrothiolysis, removal of the Fmoc group
was completed within 15 min of incubation (Fig. 2-C).
solvent
B in solvent A, 5 to 45% over 30 min, retention
time¼19.8 min. MS (ESI-TOF) m/z calcd ([Mþ3H]3þ) 918.4, found
918.5.
hBNP (2-Cys-SH form): Analytical HPLC condition: linear gra-
dient of solvent B in solvent A, 5 to 45% over 30 min, retention
time¼16.5 min. Semi-preparative HPLC condition: linear gradient
of solvent B in solvent A, 15 to 35% over 30 min. MS (ESI-TOF) m/z
calcd for ([Mþ4H]4þ) 866.9, found 866.9.
Fmoc-Cys-Tyr-Ala-Val-Thr-Gly-Arg-Gly-Asp-Ser-Pro-Ala-Ala-
Ser-Ser-Gly-SH: Analytical HPLC condition: linear gradient of sol-
vent B in solvent A, 5 to 65% over 30 min, retention time¼21.4 min.
MS (ESI-TOF) m/z calcd for ([MþH]þ) 1735.7, found 1735.8.
hBNP: Analytical HPLC condition: linear gradient of solvent B in
solvent A,
5
to 45% over 30 min, retention time¼15.3 min.
Semi-preparative HPLC condition: linear gradient of solvent B in
solvent A, 15 to 35% over 30 min. MS (ESI-TOF) m/z calcd for
([Mþ4H]4þ) 866.5, found 866.5.
H-Cys-Tyr-Ala-Val-Thr-Gly-Arg-Gly-Asp-Ser-Pro-Ala-Ala-Ser-
Ser-Gly-SH: Analytical HPLC condition: linear gradient of solvent B
in solvent A, 5 to 65% over 30 min, retention time¼10.0 min. MS
(ESI-TOF) m/z calcd for ([MþH]þ) 1513.6, found 1513.6.
4.7. Examination of epimerization during hydrothiolytic
release of peptide thioacid followed by thioesterification
and NCL
4.6. Synthesis of hBNP-32 by sequential native chemical
ligation utilizing peptide thioacids
On-resin peptide H-His(Trt)-Arg(Pbf)-Phe-Ala-
L-Ala-N-Ar-resin
First cycle. Peptide thioacid 10b (630
solved at a final concentration of 1.0 mM in DMF/H2O (690
(v/v)) containing 3.0 mM Ellman’s reagent and 3.0 mM KHCO3. The
resulting mixture was shaken at room temperature for 1.5 h and
conversion of thioacid 10b to thioester 10b0 was confirmed by HPLC
analysis. After reduction of an excess Ellman’s reagent with 1% (w/v)
m
g, 0.69
m
mol) was dis-
or H-His(Trt)-Arg(Pbf)-Phe-Ala- -Ala-N-Ar-resin was prepared on
D
mL, 2:8
Leu-aminomethyl ChemMatrixÒ resin as mentioned above using
Fmoc protocol. Deprotection of the completed resin with TFA-
thioanisole-m-cresol-H2O-EDT-Et3SiH (80:5:5:5:2.5:2.5, (v/v)) at
room temperature for 1.5 h followed by the treatment with TFA in
the presence of TCEP (1% (w/v)) at room temperature for 20 h gave
TCEP (6.9 mg), N-Cys thioacid fragment 11 (1.2 mg, 0.69
thiophenol (6.9 L) were added to the mixture. Then 10% K2CO3 aq
solution (50
L) was added to the reaction mixture at 4 ꢂC to adjust
the pH around 7.3. After incubation of the mixture at 37 ꢂC for 2 h,
the starting materials were disappeared and the crude material was
purified by semi-preparative HPLC to give ligated peptide thioacid
13 (1.2 mg, 67% yield).
m
mol) and
deprotected on-resin peptide thioester H-His-Arg-Phe-Ala-
L-Ala-S-
m
Ar-resin or H-His-Arg-Phe-Ala- -Ala-S-Ar-resin, respectively. The
D
m
on-resin peptide thioester was incubated with 120 mM NaSH in 1 M
sodium phosphate buffer (pH 9.2)/MeCN (85:15) at 37 ꢂC for 15 min
to yield the hydrothiolytically released peptide thioacid H-His-Arg-
Phe-Ala-L-Ala-SH or H-His-Arg-Phe-Ala-D-Ala-SH, respectively.
Each peptide thioacid was transformed into a corresponding Ar
thioester by treatment with Ellman’s reagent, and it was ligated
with N-Cys peptide 12 using sequential NCL protocol as mentioned
above. After completion of NCL, the reaction mixtures were
analyzed by HPLC and no epimerization was observed after these
reactions (Fig. 4).
Second cycle. The purified peptide thioacid 13 (1.2 mg,
0.46
mmol) was dissolved at a final concentration of 1.0 mM in DMF/
H2O (460
mL, 2:8(v/v)) containing 3.0 mM Ellman’s reagent and
3.0 mM KHCO3. The resulting mixture was shaken at room tem-
perature for 1.5 h. Then 1% (w/v) TCEP (4.6 mg) was added to the
reaction mixture to reduce an excess Ellman’s reagent and the
hetero disulfide generated from the cysteinyl sulfhydryl group and
5-mercapto-2-nitrobenzoic acid. The conversion of thioacid 13 to
thioester 130 was confirmed by HPLC analysis. N-Cys fragment 12
H-His-Arg-Phe-Ala-L-Ala-SH and H-His-Arg-Phe-Ala-D-Ala-SH:
Analytical HPLC condition: linear gradient of solvent B in solvent A,
5 to 25% over 30 min, retention time¼17.6 min. MS (ESI-TOF) m/z
calcd for ([MþH]þ) 617.3, found 617.3.
(500
mg, 0.55
mmol) and thiophenol (4.6
mL) were added to the
H-His-Arg-Phe-Ala-L-Ala-Cys-Lys-Val-Leu-Arg-Arg-His-OH: An-
reaction mixture and then the pH of the reaction was adjusted
alytical HPLC condition: linear gradient of solvent B in solvent A,

3296
A. Shigenaga et al. / Tetrahedron 66 (2010) 3290–3296
thioacid fragment 10b to evaluate the feasibility of repetition of the thioacid-
employing NCL protocol.
5 to 30% over 30 min, retention time¼17.7 min. MS (ESI-TOF) m/z
calcd for ([Mþ2H]2þ) 747.4, found 747.2.
10. (a) Tsuda, S.; Shigenaga, A.; Bando, K.; Otaka, A. Org. Lett. 2009, 11, 823–826;
Other Fmoc-based synthetic methodologies for peptide thioester should be
applicable to the preparation of peptide thioacid, see: (b) Alsina, J.; Yokum, T. S.;
Albericio, F.; Barany, G. J. Org. Chem. 1999, 64, 8761–8769; (c) Ollivier, N.; Behr,
J.-B.; El-Mahdi, O.; Blanpain, A.; Melnyk, O. Org. Lett. 2005, 7, 2647–2650; (d)
Hojo, H.; Onuma, Y.; Akimoto, Y.; Nakahara, Y.; Nakahara, Y. Tetrahedron Lett.
2007, 48, 25–28; (e) Li, L.; Wang, P. Tetrahedron Lett. 2007, 48, 29–32; (f) Mende,
F.; Seitz, O. Angew. Chem., Int. Ed. 2007, 46, 4577–4580; (g) Ficht, S.; Payne, R. J.;
Guy, R. T.; Wong, C.-H. Chem.dEur. J. 2008, 14, 3520–3629; (h) Hojo, H.;
Murasawa, Y.; Katayama, H.; Ohira, T.; Nakahara, Y.; Nakahara, Y. Org. Biomol.
Chem. 2008, 6, 1808–1813.
H-His-Arg-Phe-Ala-D-Ala-Cys-Lys-Val-Leu-Arg-Arg-His-OH: An-
alytical HPLC condition: linear gradient of solvent B in solvent A,
5 to 30% over 30 min, retention time¼18.2 min. MS (ESI-TOF) m/z
calcd for ([Mþ2H]2þ) 747.4, found 747.2.
Acknowledgements
This research was supported in part by a Grand-in-Aid for Sci-
entific Research (KAKENHI). Authors are grateful for research grants
from Nagase Science and Technology Foundation (A.O.), Takeda
Science Foundation (A.S.) and The Science and Technology Foun-
dation of Japan (A.S.).
11. (a) Camarero, J. A.; Adeva, A.; Muir, T. W. Lett. Pept. Sci. 2000, 7, 17–21; (b) Zhang,
X.; Lu, X.-W.; Liu, C.-F. Tetrahedron Lett. 2008, 49, 6122–6125.
12. Various attempts at evaluating the effect of peptide-detached resin for re-
moval of the Fmoc group were conducted; however, the reason for the facile
cleavage of the Fmoc group has yet to be disclosed. For example, treatment
of 10a in the presence of (N-sulfanylethyl)aminobenzoic acid linked resin
with NaSH in 1 M phosphate buffer (pH 9.2)/MeCN (85:15) have no effect in
Fmoc removal.
References and notes
13. Ellman’s reagent showed the most suitable nature (reactivity to thioacids and
stability of the resulting thioester) in the examined aryl disulfides including
phenyl disulfide and pyridyl disulfide.
14. Treatment of peptide thioacids with Ellman’s reagent under acidic conditions
(around pH 4) for more than 15 min resulted in the formation of peptide
thioesters via more active acyl intermediates such as acyl disulfides.
15. Since the formed peptide arylthioester is susceptible to hydrolysis under
neutral conditions, addition of thiophenol and the ligation partner followed by
adjustment of pH is critical.
16. Conversion of peptide thioacid 13 by reaction with Ellman’s reagent (3 mM
Ellman’s reagent in 3 mM KHCO3 in H2O/DMF) accompanied the formation of
a non-negligible amount of disulfide peptide resulting from the reaction of
cysteine sulfhydryl group with the aryl disulfide. Furthermore, the remaining
excess Ellman’s reagent should induce hindrance of the N-terminal Cys residue
in the following NCL step under neutral conditions. To prevent these undesir-
able reactions, the addition of TCEP as a reducing agent for disulfides is crucial
for achieving the sequential NCL in a one-pot manner.
17. For the N-S acyl transfer on peptidyl aminobenzoic acid linker, treatment with
4 M HCl/DMF most efficiently induced the acyl transfer thus far in the exami-
nation; however, application of this system to chiral amino acid attached resin
accompanied the partial epimerization of the C-terminal amino acid. On the
other hand, the reaction with TFA encountered no epimerization although the
progress of the acyl transfer is slower than that observed in the reaction with
4 M HCl/DMF. See Ref. 10a.
1. (a) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H. Science 1994, 266,
776–779; (b) Muir, T. W.; Dawson, P. E.; Kent, S. B. H. Methods Enzymol. 1997,
289, 266–298; (c) Dawson, P. E.; Kent, S. B. H. Annu. Rev. Biochem. 2000, 69, 923–
960; (d) Tam, J. P.; Xu, J.; Eom, K. D. Biopolymers 2001, 60, 194–205; (e) Kent, S.
B. H. Curr. Opin. Biotechnol. 2004, 15, 607–614; (f) Hackenberger, C. P. R.;
Schwarzer, D. Angew. Chem., Int. Ed. 2008, 47, 10030–10074; (g) Kent, S. B. H.
Chem. Soc. Rev. 2009, 38, 338–351.
2. Examples of multi-step NCL reactions using a peptidyl thioester possessing
a protected Cys residue at N-teminal, see: (a) Hackeng, T. M.; Griffin, J. H.;
Dawson, P. E. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 10068–10073; (b) Brik, A.;
Keinan, E.; Dawson, P. E. J. Org. Chem. 2000, 65, 3829–3835; (c) Bang, D.;
Chopra, N.; Kent, S. B. H. J. Am. Chem. Soc. 2004, 126, 1377–1383; (d) Bang, D.;
Kent, S. B. H. Angew. Chem., Int. Ed. 2004, 43, 2534–2538; (e) Ueda, S.; Fujita, M.;
Tamamura, H.; Fujii, N.; Otaka, A. ChemBioChem 2005, 6, 1983–1986; (f) Gang,
D.; Kent, S. B. H. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 5014–5019; (g) Johnson, E.
C. B.; Durek, T.; Kent, S. B. H. Angew. Chem., Int. Ed. 2006, 45, 3283–3287.
3. (a) Band, D.; Pentelute, B. L.; Kent, S. B. H. Angew. Chem., Int. Ed. 2006, 45, 3985–
3988; (b) Durek, T.; Torbeev, V. Y.; Kent, S. B. H. Proc. Natl. Acad. Sci. U.S.A. 2007,
104, 4846–4851; (c) Torbeev, V. Y.; Kent, S. B. H. Angew. Chem., Int. Ed. 2007, 46,
1667–1670.
4. (a) Blake, J.; Yamashiro, D.; Ramasharma, K.; Li, C. H. Int. J. Pept. Protein Res.
1986, 28, 468–476; (b) Yamashiro, D.; Li, C. H. Int. J. Pept. Protein Res. 1988, 31,
322–334.
18. Recently, many literatures about the utilization of thioacids in peptide chem-
istry have appeared, for reaction involving aromatic nucleophilic substitution,
see: (a) Crich, D.; Banerjee, A. J. Am. Chem. Soc. 2007, 129, 10064–10065; (b)
Crich, D.; Sana, K.; Guo, S. Org. Lett. 2007, 9, 4423–4444; (c) Crich, D.; Bowers,
A. A. Org. Lett. 2007, 9, 5323–5325; (d) For a reaction with azide compounds,
see: Crich, D.; Sharma, I. Angew. Chem., Int. Ed. 2009, 48, 7591–7594; (e)
Shangguan, N.; Katukojvala, S.; Greenberg, R.; Williams, L. J. J. Am. Chem. Soc.
2003, 125, 7754–7755; (f) Merkx, R.; Brouwer, A. J.; Rijkers, D. T. S.; Liskamp, R.
M. J. Org. Lett. 2005, 7, 1125–1128; (g) Barlett, K. N.; Kolakowski, R. V.; Katu-
kojvala, S.; Williams, L. J. Org. Lett. 2006, 8, 823–826; (h) Kolakowski, R. V.;
Shangguan, N.; Sauers, R. R.; Williams, L. J. J. Am. Chem. Soc. 2006, 128, 5695–
5702; (i) Rijkers, D. T. S.; Merkx, R.; Yim, C.-B.; Brouwer, A. J.; Liskamp, R. M. J.
J. Pept. Sci. 2010, 16, 1–5.
5. (a) Bruick, R. K.; Dawson, P. E.; Kent, S. B. H.; Usman, N.; Joyce, G. F. Chem.
Biol. 1996, 3, 49–56; (b) Zhang, L.; Tam, J. P. Tetrahedron Lett. 1997, 38, 3–6;
(c) Offer, J.; Boddy, C. N.; Dawson, P. E. J. Am. Chem. Soc. 2002, 124, 4642–
4646; (d) Camarero, J. A.; Muir, T. W. Chem. Commun. 1997, 1369–1370.
6. Canne, L. E.; Botti, P.; Simon, R. J.; Chen, Y.; Dennis, E. A.; Kent, S. B. H. J. Am.
Chem. Soc. 1999, 121, 8720–8727.
7. N to C directed chemoenzymatic ligation, see: Tan, X.-H.; Zhang, X.; Yang, R.;
Liu, C.-F. ChemBioChem 2008, 9, 1052–1056.
8. The N to C ligation shows utility in preparation of C-terminal part-modified
protein analogues. More importantly, this protocol allows convergent synthe-
sis of proteins3 to be achieved in conjunction with standard C to N ligation.
9. Although the use of thioesters as N-terminal fragments corresponding to hBNP
(1–9) is reasonable for the practical synthesis of hBNP, we utilized peptide