Highly efficient synthetic method on pyroacm resin using the boc SPPS protocol for C-terminal cysteine peptide synthesis

Article abstract of DOI:10.1002/bkcs.11045

A very effective process on Pyroacm resin was developed for solid-phase peptide synthesis (SPPS) of C-terminal cysteine and cysteine ester peptides. The process uses cysteine side chain anchoring to the Pyroacm resin and the Boc protocol for SPPS. The Pyroacm resin showed remarkable stability under standard trifluoromethanesulfonic acid (TFMSA) cleavage condition. TFMSA cleavage of protecting groups generates a peptide-linked resin, which can be subjected to peptide modification reactions. Finally, the peptide can be cleaved from the resin using methoxycarbonylsulfenyl chloride. The utility of this protocol was demonstrated by its applications to the synthesis of model peptides, key intermediates in the preparation of natural products riparin 1.2 and a-factor.

Full text of DOI:10.1002/bkcs.11045

Article
DOI: 10.1002/bkcs.11045
BULLETIN OF THE
V. Juvekar et al.
KOREAN CHEMICAL SOCIETY
Highly Efficient Synthetic Method on Pyroacm Resin Using the Boc
SPPS Protocol for C-terminal Cysteine Peptide Synthesis
*
Vinayak Juvekar, Kang-Tae Kim, and Young-Dae Gong
Innovative Drug Library Research Center, Department of Chemistry, College of Science, Dongguk
University, Seoul 100-715, Korea. *E-mail: ydgong@dongguk.edu
Received October 10, 2016, Accepted November 19, 2016, Published online January 5, 2017
A very effective process on Pyroacm resin was developed for solid-phase peptide synthesis (SPPS) of
C-terminal cysteine and cysteine ester peptides. The process uses cysteine side chain anchoring to the Pyr-
oacm resin and the Boc protocol for SPPS. The Pyroacm resin showed remarkable stability under standard
trifluoromethanesulfonic acid (TFMSA) cleavage condition. TFMSA cleavage of protecting groups gener-
ates a peptide-linked resin, which can be subjected to peptide modification reactions. Finally, the peptide
can be cleaved from the resin using methoxycarbonylsulfenyl chloride. The utility of this protocol was
demonstrated by its applications to the synthesis of model peptides, key intermediates in the preparation
of natural products riparin 1.2 and a-factor.
Keywords: Side chain anchoring, Solid-phase peptide synthesis, C-terminal cysteine, Thiol-sulfenyl pro-
tection, β-Elimination
Introduction
synthesis and the side chain anchoring method displays
decreased yields owing to piperidine-promoted
C-terminal cysteine and cysteine ester peptides hold a prominent
position in peptide chemistry because of their wide occurrence
in nature and applications as important bioactive agents. Con-
β-elimination and racemization. Finally, a technique, which
involves initial preparation of the peptide sequence attached
to the C-terminal cysteine followed by introduction of the
cysteine using native chemical ligation methods, has been
developed. However, this protocol requires additional
chemical steps.
1
sequently, the development of methods to form solid-phase syn-
thesis of these peptides is an important component of studies in
this area. Boc-based solid-phase peptide synthesis (SPPS) found
instant acceptance in the early years of peptide chemistry owing
7
In the previous efforts, we designed and synthesized an
acetamidomethyl (Acm)-derived resin named Pyroacm
resin (2d). In addition, we demonstrated that the new resin
can be used for anchoring cysteine through its side chain
thiol as part of an Fmoc protocol for synthesis of C-
2,2a–c
to its simplicity and adaptability to automation.
However,
the need for strong acids (e.g., Hydrofluoric acid (HF)) for the
final cleavage step requires the use of special expensive appa-
2d,e
ratus in order to account for safety issues. Use of HF in the
final cleavage process also affects the ability to tag peptides with
certain types of fluorophores or saccharides (oligo). As a result
of these drawbacks, the Fmoc SPPS procedure, in which the
Fmoc group is readily cleaved using mild base (piperidine)
6d
terminal cysteine peptides. Furthermore, we found that
the inexpensive and mild reagent methoxycarbonylsulfenyl
chloride (ScmCl) can be used for resin cleavage to produce
8
the crude peptide with a high degree of purity. However,
treatment followed by treatment with a Trifluoro acetic acid
prolong treatment with piperidine in the Fmoc removal step
was found to result in decreased yields as a result of
β-elimination. And also, during Fmoc deprotection of sec-
ond residue leads to diketopiperazine (DKP) side reaction.
To avoid the use of extended piperidine treatment required
in the Fmoc protocol and to lower the atom economy of
this approach, we continued in detail study of the Boc pro-
tocol in conjunction with the Pyroacm resin for the synthe-
sis of peptides containing C-terminal cysteine and cysteine
esters. In addition, to make the process have wide-ranging
applications, we searched for an alternate to HF for promot-
3
(TFA) cocktail, has become prominent.
Nevertheless, the synthesis of peptides containing C-terminal
cysteine and cysteine esters employing the Fmoc SPPS method
remains problematic. For example, in most cases, these peptides
undergo ready β-elimination and racemization (ca. 0.5%) dur-
4
ing the piperidine treatment step. In recent years, a few proto-
cols to avoid the formation of C-terminal dehydroalanine
β-elimination induced elimination have been developed includ5-
ing masking the C-terminal carboxylic group (Figure 1, 1),
6
,6a,b
and anchoring the cysteine side chain through a trityl (2a),
6b
6c
9
chlorotrityl (2b), Xanthenyl amide linker (XAL) (2c), or
ing the Boc-based SPPS. This effort led to the discovery
6
d
Pyroacm-based resins (Figure 1, 2d).
that trifluoromethanesulfonic acid (TFMSA) is viable alter-
10
Unfortunately, the masked carboxylic acid group
approach is not compatible with cysteine ester peptide
native to HF for this purpose. As TFMSA does not
require the use of a specialized apparatus, it compatible
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KOREAN CHEMICAL SOCIETY
Figure 1. Strategies for C-terminal cysteine peptide synthesis.
with both small- and large-scale SPPS. Below, we describe
the results of this investigation, which have led to the
development of Boc and Pyroacm-based SPPS using both a
one and two step cleavage protocol for the preparation of
C-terminal cysteine peptides.
Scheme 2. Model peptides on resin using Pyroacm resin.
Results and Discussion
In order to evaluate the proposals presented above, we first
immobilized HCl.H-Cys-OMe 4 (4 equiv) on Pyroacm
resin 3 by using acidic conditions involving 20 vol % of
TFA in CH Cl for 2 h (Scheme 1). The extent of Cys-
(
76–84%) with high degrees of purity by cleavage from the
resin using ScmCl in AcOH. When a 18:1:1 mixture of TFA:
PhOH:H O containing 2 equiv of ScmCl was used for the
cleavage process, the unprotected peptides were produced in
yield in the 87–91% range.
2
2
2
OMe loading in 5 was determined to be approximately
.83 mmol/g using quantitative ninhydrin analysis. The
ATR-FTIR spectrum of 5 contains a sharp peak at
0
Post-modification of peptides, such as the introduction of
functional groups, taking place while they are attached to
resins is typically efficient because it avoids difficulties associ-
ated with purification, characterization of intermediates, and
extensive synthetic operations. In order to determine the effec-
tiveness of this approach, model Pyroacm-linked peptide con-
taining a Lys(2-Cl-Z) residue 9 (in Scheme 3) was prepared
and subjected to the standard TFMSA deprotection conditions
to obtain resin intermediate 10 (see Figure S1, Supporting
Information). Introduction of a biotin or acetylene group was
then carried out through use of lysine amide bond forming
reactions. Finally, cleavage of both resins promoted using
ScmCl in general TFA condition generated the respective bio-
tin and acetylene modified peptides 10a and 10b in respective
yields 67 and 52% after preparative High performance liquid
chromatography (HPLC) purification.
−
1
1
748 cm (see Supporting Information) corresponding to
the ester group in the amino acid linked to the resin.
To examine its uses, the Cys-OMe-linked resin 5 was
employed to prepare resins containing the model pentapep-
tides 6, 7, and 8 utilizing the Boc SPPS protocol (Scheme
2). Method A, in which cleavage from the corresponding
resins 6, 7, and 8 utilizing 2 equiv of ScmCl in AcOH gave
the fully protected pentapeptides 6a, 7a, and 8a in high
yields and purities. In addition, Method B (in Scheme 2),
we examined the procedure in which deprotection peptide
side chain was carried out prior to cleavage from the resin.
This process was performed on the Pyroacm-linked, protected
pentapeptides 6, 7, and 8 using the standard TFMSA depro-
tection conditions with 1:10:1:0.5 TFMSA:TFA:thioanisole:
ꢀ
Triisopropyl silane (TIPS) ratio for 2.5 h at 0 C. Under these
In an earlier effort, we demonstrated that an Fmoc-based
SPPS can be used to prepare riparin 1.2 in high purity but
in low yield caused by competing β-elimination in the
conditions, the Pyroacm resin showed excellent stability.
Finally, 6b, 7b, and 8b (Scheme 2) are generated efficiently
6d,11
piperidine induced Fmoc removal step.
In the current
study, we probed the comparative efficiencies of Pyroacm
resin approaches to intermediate of riparin 1.2 peptide 13
that utilize Boc and Fmoc-based SPPS methods. For this
purpose, the Cys-allyl-linked resin 11 (Scheme 4) was pre-
pared and subjected to the Boc and Fmoc protection and
peptide assembly sequences. The allyl groups in both pep-
tides prepared in this manner were cleaved using palladium
to form intermediates 12a and 12b. Upon being subjected
Scheme 1. Anchoring cysteine onto the resin.
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C-Terminal Cysteine Peptide Synthesis Using Pyroacm Resin
KOREAN CHEMICAL SOCIETY
Scheme 3. Pre-cleavage modification on the resin.
to the two step protocol using first TFMSA and then ScmCl
in TFA:PhOH:H O, the linked Boc-protected peptide 12b
2
produced polypeptide 13 in a 73% yield after preparative
HPLC (Scheme 4). In contrast, the yield of 13 generated
using the Fmoc protection and removal was 46%.
The two step Boc-based protocol described above was
employed to synthesize the key intermediate 16 (Scheme 5)
in the pathway employed for the preparation of the a-factor,
1
2
which contains a tryptophan residue. Typically, unpro-
tected tryptophan residues in peptides undergo sulfenyla-
1
3
tion under ScmCl cleavage conditions. However, we
observed incorporation of a formyl protecting group on the
tryptophan residue in the Pyroacm-linked peptide 14 pre-
vents this process. Consequently, Method B in which treat-
ment of 14 with first TFMSA and then ScmCl in TFA:
Scheme 5. Synthesis of a key intermediate in the preparation of a-
factor.
PhOH:H O efficiently (83%) generated the highly pure S-
2
presence of 10 vol % TIPS does not lead to an improve-
ment of the efficiency of the process (entry 6). Finally, we
found that increasing the ratio of TFMSA in the cleavage
cocktail led to generation of 15 in a highly pure state
sulfenyl formyl-protected peptide 15. Peptide 16 then pro-
duced with high crude purity using a one-pot reaction
involving treatment of 15 with 5 vol % DTT in piperidine:
DMF (1:5) (Scheme 5). The crude purified via HPLC to
obtain pure 16 (Figure 2).
(Table 1, entries 7 and 8). The main drawback of one-pot
cleavage process utilizes 70 vol % TFMSA (entry 8) due to
Lys(2-Cl-Z) can be avoided, if orthogonal Lys(Z) or
Lys(Alloc) side chain protection could be exploited instead
of Lys(2-Cl-Z) in which Lysine side chain protective group
can easily cleaved prior to one-pot cleavage process
The one-pot cleavage procedure can be employed to
form the side chain deprotected target 15 directly from 14
(Scheme 5). However, we observed that treatment of 14
with a 9:1 mixture of TFA:TFMSA containing 2 equiv of
ScmCl generated 15 in only a 9% crude purity along with
lysin (2-Cl-Z) containing peptide 15a (Scheme 5) in a 91%
crude purity (Table 1, entry 1). Addition of 10 vol % of
scavengers such as thioanisole or dimethylsulfide (DMS)
resulted in deactivation of ScmCl (entries 2 and 3).
For
W D P A
DTT:Piperidine:DMF
1:4:16), 15 min, rt
(
Y
I
I
K
G
V
F
Y
G
I I K V F W D P A
O
C
S
S
O
C
SH
H
3
O
O
CH
3
H
3
O
O
Although addition of 10 vol % anisole or 10 vol % p-
cresol as scavengers did not result in deactivation of
ScmCl, 15a was still the major product (71 and 75%,
respectively, entries 4 and 5) in crude. Likewise, the
Figure 2. Liquid chromatography mass spectrometer (LCMS)
chromatogram of purified 16 (Retention time (RT) = 8.4 min)
after the addition of 5 vol % of DTT to 15 (RT = 8.9 min) in
piperidine:DMF (1:4).
Scheme 4. Comparison study for synthesis of riparin 1.2 interme-
diate on Pyroacm resin with Boc vs. Fmoc protocols.
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C-Terminal Cysteine Peptide Synthesis Using Pyroacm Resin
KOREAN CHEMICAL SOCIETY
a
Table 1. One-pot cleavage cocktail to obtain fully deprotected Scm-peptide.
b
c
Entry
Cocktail composition
TFA:TFMSA (9:1)
Ratio (%) 15:15a
Yield (%)
1
2
3
4
5
6
7
8
a
9:91
—
49
TFA:TFMSA:thioanisole (8:1:1)
TFA:TFMSA:DMS (8:1:1)
TFA:TFMSA:anisole (8:1:1)
TFA:TFMSA:p-cresol (8:1:1)
TFA:TFMSA:TIPS (8:1:1)
TFA:TFMSA:anisole (5:4:1)
TFA:TFMSA:anisole (2:7:1)
Traces
Traces
57
56
19
—
29:71
25:75
38:62
89:11
99.7:0.3
52
46
Reactions carried out on 0.01 mmol scale.
b
c
Percent product and byproducts in the crude mixtures (small peaks in HPLC have been ignored).
Yields after preparative HPLC.
(
Scheme 5). Resulting resin intermediate can be subjected
30 to 40 min, and the gradient raised from 45% to 90% of
ACN (0.1% TFA). An Agilent 6460 triple Quad LCMS
coupled to an Agilent 1290 infinity LC instrument equipped
with a Poroshell 120 EC-C18 analytical reversed-phase column
(3.0 mm × 50 mm, 2.7 μm) was used for analysis. Linear gra-
dients of ACN into water (5 mM NH HCO ) were used at a
to condition (Table 1, entry 4), which involves 10 vol % of
TFMSA, to obtain target peptide with high crude purity.
Conclusion
4
3
In the study described above, we developed a mild protocol
for SPPS of terminal cysteine and cysteine ester containing
peptides in high purities. Moreover, we observed the Pyr-
oacm resin showed excellent stability under standard
TFMSA cleavage condition, which is useful for on resin
peptide modification. The developed method eliminates
unwanted β-elimination and racemization reactions that
occur when using the Fmoc protocol. In addition, we
observed that formyl groups on formyl-protected trypto-
phan residues in peptides are cleaved during the disulfide
bond reduction process. Surprisingly, during the one-pot
cleavage process, the stability of formyl group on trypto-
phan and very high cleavage potential of ScmCl observed
even in the presence of the high concentrations of TFMSA.
Finally, the one-pot cleavage process can be utilized in con-
junction with Boc-based SPPS protocol employing the
Acm protecting group, thus avoiding the need for tedious
flow rate of 0.3 mL/min in the ESI mode. An Agilent 6550
iFunnel Q-TOF coupled to an Agilent 1290 infinity II LC spec-
trometer, equipped with a Poroshell 120 EC-C18 analytical
reversed-phase column (3.0 mm × 50 mm, 2.7 μm), was used
for High resolution mass spectrometer (HRMS) measurements.
Linear gradients of ACN into water (5 mM NH HCO ) were
4 3
used at a flow rate of 0.4 mL/min and a run time of 7 min in
the ESI mode. All LC eluents were monitored at 220 nm. An
Ilshin Biobase Co. Ltd, freeze dryer (Gyeonggi-do, Republic
1
of Korea) was used for lyophilization. H NMR spectra were
recorded on a Bruker Ascend™500 instrument (Bruker, MA,
USA) at 500 MHz. Residual chloroform (7.26 ppm) was used
13
as an internal standard. C NMR spectra were measured at
126 MHz with residual chloroform (77.16 ppm) as an internal
standard. A Waters PrepHPLC containing Waters 2525 Binary
Gradient System with a Waters 2487 dual wavelength UV–vis
detector and reverse-phase column (19 mm × 250 mm,
5 μM) was used to purify peptides. Analytical thin layer chro-
matography (TLC) was performed using plates coated with a
2
+
3+
+ 14
processes utilizing metals such as Hg , Tl , and Ag .
0
.25 mm thickness of silica gel, and compounds were visua-
Experimental
lized using UV light, iodine, or KMnO staining. Normal
4
phase flash chromatography was performed on an automated
MPLC of Biotage IsoleraOne Instrument (Biotage, Uppsala,
Sweden) (silica gel 230–400 mesh size).
General Information. All reactions were conducted in
flame- or oven-dried glassware under an atmosphere of argon.
SPPS was carried out manually in a syringe containing a poly-
ethylene frit. Solvents and soluble reagents were removed by
using suction. Infrared (IR) spectra were recorded on a Smiths
IdentityIR ATR-FTIR spectrometer. A Scinco S4100 PDA
UV–vis spectrophotometer was used to record UV spectra.
An Agilent 1260 infinity instrument, equipped with an Eclipse
plus-C18 reversed-phase analytical column (4.6 mm × 250
mm, 5 μM), was used for HPLC analysis. HPLC separation
used a flow rate of 1 mL/min and 40 min method, which
involves an isocratic mixture of water: Acetonitrile (ACN):
SPPS Using the Boc Protocol. Resin was swollen in DMF
for 10 min. Pre-activation of the amino acid involves
HBTU/HOBt/DIPEA (4/4/8) treatment of 0.4 M Boc-
protected amino acids (4.0 equiv) in DMF for 2 min. In
each case, the mixture was taken into the resin and the
ꢀ
syringe was tumbled on a shaker at 25 C and 160 rpm for
1 h. The resin was washed with DMF (5 × 1 min), CH Cl2
2
(5 × 1 min), DMF (5 × 1 min), and CH Cl (5 × 1 min).
2
2
A small portion of the resin was assayed using the Kaiser
test. In the case of a positive test, the process was repeated.
Removal of the Boc group was achieved by treatment of
(95:5) containing 0.1% TFA for 5 min, gradient raised from
5% to 45% of ACN (0.1% TFA) from 5 to 30 min and from
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C-Terminal Cysteine Peptide Synthesis Using Pyroacm Resin
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the resin with 50 vol % TFA in CH Cl containing 0.5%
(3 mL/g) followed by TFA (30 mL/g). The mixture was
stirred for 5 min and 2 equiv of ScmCl (per Acm mimic) in
TFA was added very slowly. The mixture was stirred for
4 h at RT and filtered. The resin was washed with TFA,
concentrated under reduced pressure, and precipitated using
cold diethyl ether. The precipitate was centrifuged and lyo-
philized to obtain the crude peptide, which subjected to
HPLC and LCMS analysis.
2
2
TIPS for (1 × 5 min) and (1 × 15 min). Washings between
deprotection and coupling steps were performed with
CH Cl (5 × 1 min) and DMF (5 × 1 min). In situ neutral-
2
2
ization Boc-chemistry for SPPS protocol was followed.
After completion of the final coupling or deprotection step,
the resin was washed with CH Cl (5 × 1 min), DMF
2
2
(
5 × 1 min), CH Cl (5 × 1 min), and dried under high
2 2
vacuum.
One-Pot Cleavage Protocol. To the peptide resin in a glass
vial was added anisole (3 mL/g). The mixture was placed
in an ice bath and TFA (30 mL/g) was added. The mixture
was stirred for 10 min and TFMSA (3 mL/g) was added
very slowly, followed by 2 equiv of ScmCl (per Acm
SPPS Using the Fmoc Protocol. Resin was swollen in
DMF for 10 min. Amino acids were pre-activated for
2
min by HBTU/HOBt/DIPEA (4/4/8) treatment of 0.4 M
Fmoc-protected amino acids (4.0 equiv) in DMF. In each
case, the mixture was added to the resin and the syringe
tumbled on a shaker for 1 h at 25 C and 160 rpm. The
ꢀ
mimic) in TFA. The mixture was stirred for 5 h at 0 C and
ꢀ
filtered into cold diethyl ether. Centrifugation followed by
lyophilization gave crude peptide, which subjected to
HPLC and LCMS analysis prior to preparative HPLC
purification.
resin was washed with DMF (5 × 1 min), CH Cl2
2
(5 × 1 min), and DMF (5 × 1 min). A small portion of the
resin was assayed using the Kaiser test. In the case of a
positive test, the process was repeated. Removal of Fmoc
groups was achieved by treatment of the resin with 20 vol
Experimental Details
1
-(Methoxymethyl)pyrrolidin-2-one (2e). To pyrrolidon-2-
%
piperidine in DMF (2 × 10 min). Washings between
deprotection and coupling steps were performed with DMF
5 × 1 min), CH Cl (5 × 1 min), and DMF (5 × 1 min).
one (25 g, 0.294 mol) in 1 M KOH (0.01 equiv) at RT was
slowly added 36% aqueous formaline (24.5 mL,
(
2
2
0
.294 mol). The mixture was stirred at RT for 2 h, and con-
Following the final coupling or deprotection step, the resin
was washed with DMF (5 × 1 min), CH Cl (5 × 1 min),
and dried under high vacuum.
Removal of the Allyl Group. To the resin swollen in 50×
centrated under reduced pressure. The crude solid contain-
ing 1-(hydroxymethyl)pyrrolidin-2-one (11.5 g, 0.1 mol)
2
2
was dissolved in THF (575 mL), and then NaH (4.4 g,
ꢀ
0
.11 mol) was added in portions at 0 C. When H evolu-
2
dry CH Cl for 15 min under argon atmosphere was slowly
2
2
tion ceased, MeI (6.85 mL, 0.11 mol) was added dropwise.
added via syringe Pd(PPh3)4 (0.75 equiv) and Ph SiH
3
ꢀ
The mixture was stirred at 0 C overnight. After the con-
(30 equiv) in 20× CH Cl . The resin was tumbled in a
2 2
ꢀ
sumption of starting material, MeOH (5 mL) was added
dropwise to quench unreacted NaH. The mixture was con-
centrated under reduced pressure giving a residue that was
mixed with CH Cl (200 mL). The organic layer was fil-
shaker for 1.5 h at 25 C, 160 rpm and washed with CH Cl
2
2
(5 × 1 min), DMF (5 × 1 min), CH Cl (5 × 1 min), and
2 2
dried under high vacuum. The process was repeated twice.
Two Step Cleavage Process
2
2
tered, dried over MgSO and concentrated under reduced
4
Standard TFMSA Deprotection Conditions. The peptide
resin was placed in a glass vial with a stirring bar. Thioani-
sole/TIPS (2:1) was added (3 mL/g) and following place-
ment of the glass vial in an ice bath, TFA (20 mL/g) was
added. The mixture was stirred for 10 min and 2 mL of
TFMSA (2 mL/g) was added very slowly. The mixture stir-
red for 2.5 h at 0 C and filtered to give the resin, which
washed with TFA and CH Cl (10 × 1 min) and dried
under high vacuum.
pressure. The residue was subjected to flash silica chroma-
tography eluting with a gradient of CH Cl :methanol
2
2
(
100:0 ! 90:10) to yield the product 2e as a pale yellow
liquid (10.96 g, 84%). R = 0.25 (CH Cl :methanol,
9
(t, J = 7.1 Hz, 2H), 3.29 (s, 2H), 2.45 (t, J = 8.1 Hz, 2H),
2
7
f
2
2
1
.5:0.5) H NMR (500 MHz, CDCl ) δ 4.69 (s, 2H), 3.49
3
ꢀ
1
3
.13–2.03 (m, 2H). C NMR (126 MHz, CDCl ) δ 176.3,
3
2
2
4.0, 56.1, 46.0, 31.2, 18.1.
General Cleavage Procedure Comprised of ScmCl in
AcOH. To the resin (0.01 mmol) in 0.75 mL of AcOH (let
resin swell for 2 min) in a syringe with a polyethylene frit
was added 2 equiv of ScmCl (per Acm mimic) in 0.05 mL
Pyroacm Resin (3). To a solution of 1-(methoxymethyl)
pyrrolidin-2-one (4.8 g, 37.07 mmol) in dry THF (90 mL)
under an argon atmosphere was added 2 M LDA (18.5 mL,
ꢀ
37.07 mmol) at −78 C. The mixture was raised above the
ꢀ
ꢀ
of AcOH. The syringe was tumbled at 25 C and 160 rpm
cooling bath for 10 min and then cooled to −78 C. After
on a shaker for 5 h. The resin was separated by using filtra-
10 min, 2.2 mmol/g of Merrifield resin (3.37 g, 7.41 mmol,
100–200 mesh size) was added in three portions with
5 min interval between additions. The mixture is stirred at
tion and washed with AcOH (1 × 1 mL) and H O
2
(1 × 1 mL). The filtrate was diluted with water (made 20%
ꢀ
aqueous) and lyophilized to obtain the crude product,
which was analyzed using HPLC and LCMS.
General Cleavage Procedure Using the General ScmCl in
TFA Condition. To 0.01 mmol resin in a glass vial with a
stirring bar was added a 1:1 mixture of water and phenol
−78 C and 200 rpm for 1 h and then the temperature was
ꢀ
increased slowly to 25 C. The mixture was stirred for 4 h
ꢀ
before the temperature was reduced to −20 C and 10%
aqueous NH Cl was slowly added. The mixture was filtered
4
and the resin was washed with THF (5 × 50 mL), DMF
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C-Terminal Cysteine Peptide Synthesis Using Pyroacm Resin
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(
5 × 50 mL), and CH Cl (5 × 50 mL) and then dried
yielded 6b as a white solid (5.9 mg, 88%). ESI-MS: for
2
2
+
+
under high vacuum for overnight.
C H N O S [M + H ] ; cald 667.2426, found 667.3
25 42 6 11 2
+
+
MeO-Cys-NH ꢁHCl (4). Acetyl chloride (16 mL,
[M + Na ] ; cald 689.2245, found 689.3.
2
2
20 mmol) was added to MeOH (100 mL) and the solution
Ac-Gly-Thr(Bn)-Glu(Bn)-Ala-Cys(Scm)-OCH3
(7a).
ꢀ
was cooled to 0 C. The solution stirred for 5 min. Cysteine
Model pentapeptide 7a synthesis started with 5. Subsequent
coupling with Boc-Ala-OH, Boc-Glu(Bn)-OH, Boc-Thr
(Bn)-OH, and Ac-Gly-OH via standard Boc protocol.
(2.42 g, 20 mmol) was added to reaction mass and stirred
at RT for 36 h under argon atmosphere. The reaction was
evaporated under reduced pressure, added ether, and the
solid thus obtained was filtered and washed with ether.
Then dried under high vacuum to yield the product 4 as a
50 vol % TFA in CH Cl2 containing 0.5 vol % TIPS
2
(1 × 5 min) and (1 × 15 min) treated with the resin to
remove Boc group. Cleavage via ScmCl in AcOH: Resin
7 (0.012 mmol) was subjected to the general cleavage con-
dition using ScmCl in AcOH to obtain crude 7a. The crude
product analyzed using HPLC and LCMS, and purified by
using prepHPLC. The process yielded 7a as a white solid
1
pale green amorphous solid (3.28 g, 94%). H NMR
(
2
500 MHz, MeOD) δ 4.34 (m, 1H), 3.83 (s, 3H), 3.09 (m,
13
H).
C NMR (126 MHz, MeOD) δ 169.1, 55.7,
5
3.9, 25.1.
+
+
Anchoring Cysteine onto the Resin (5). To Pyroacm resin
(8.9 mg, 93%). ESI-MS: for C H N O S [M + H ] ;
36 47 5 12 2
+
+
3
4
8
(1 g, 1.82 mmol) under argon atmosphere was added
(1.25 g, 7.28 mmol) in CH Cl (30 mL). After 15 min,
cald 806.2736, found 806.2736 [M + Na ] ; cald
828.2555, found 828.2547.
2
2
mL of TFA was slowly added. The mixture was shaken
Ac-Gly-Thr-Glu-Ala-Cys(Scm)-OCH3 (7b). Model penta-
peptide 7b synthesis started with 5. Subsequent coupling
with Boc-Ala-OH, Boc-Glu(Bn)-OH, Boc-Thr(Bn)-OH,
and Ac-Gly-OH was carried out via standard Boc protocol.
for 2 h before being filtered. The resin was washed with
CH Cl2 (5 × 20 mL), DMF (5 × 20 mL), and CH Cl2
2
2
(5 × 20 mL). Loading of 5, calculated using Ninhydrin-
quantitative analysis, was found to be 0.83 mmol/g.
50 vol % TFA in CH Cl2 containing 0.5 vol % TIPS
2
Ac-Gly-Asp(Bn)-Val-Lys(2-Cl-Z)-Cys(Scm)-OCH3
Model pentapeptide 6a synthesis started with 5. Subsequent
coupling with Boc-Lys(2-Cl-Z)-OH, Boc-Val-OH, Boc-Asp
(6a).
(1 × 5 min) and (1 × 15 min) treated with the resin to
remove Boc group. 0.025 mmol resin 7 subjected to stand-
ard TFMSA deprotection condition to remove side chain
protective groups. Cleavage via ScmCl in AcOH: The resin
(0.01 mmol) was subjected to the general cleavage condi-
tions using ScmCl in AcOH to obtain crude 7b. The crude
product analyzed using HPLC and LCMS, and purified by
using prepHPLC. The process yielded 7b as a white solid
(Bn)-OH, and Ac-Gly-OH was carried out via the standard
Boc protocol. 50 vol % TFA in CH Cl containing 0.5 vol
2
2
%
TIPS (1 × 5 min) and (1 × 15 min) treated with the
resin to remove Boc group. Cleavage via ScmCl in AcOH:
Resin 6 (0.011 mmol) was subjected to the general cleav-
age conditions using ScmCl in AcOH to obtain 6a. The
crude product was analyzed using HPLC and LCMS, and
purified by using prepHPLC. This process yielded 6a as a
white solid (9.2 mg, 90%). ESI-MS: for C H ClN O S
2
+
+
(5.3, 84%). ESI-MS: for C H N O S [M + H ] ; cald
2
2 35 5 12 2
+
+
626.1797, found 626.1790 [M + Na ] ; cald 648.1616,
found 648.1604. Cleavage via standard ScmCl in TFA:
The resin (0.0135 mmol) was subjected to the general
cleavage conditions using ScmCl in TFA to obtain crude
7b. The crude product analyzed using HPLC and LCMS,
and purified by using prepHPLC. The process yielded 7b
as a white solid (8 mg, 91%). ESI-MS: for C H N O S
5 12 2
4
0
53
6
13
+ +
+
+
[
M + H ] ; cald 925.2874, found 925.2893 [M + Na ] ;
cald 947.2693, found 947.2684.
Ac-Gly-Asp-Val-Lys-Cys(Scm)-OCH3 (6b). Model penta-
peptide 6b synthesis started with 5. Subsequent coupling
with Boc-Lys(2-Cl-Z)-OH, Boc-Val-OH, Boc-Asp(Bn)-
OH, and Ac-Gly-OH was carried out via the standard Boc
protocol. 50 vol % TFA in CH Cl containing 0.5 vol %
2
2
35
+ +
+
+
[M + H ] ; cald 626.1797, found 626.2 [M + Na ] ; cald
648.1616, found 648.2.
Ac-Gly-Tyr(2-Br-Z)-Leu-Ser(Bn)-Cys(Scm)-OCH3
(8a).
2
2
TIPS (1 × 5 min) and (1 × 15 min) treated with the resin
to remove Boc group. 0.025 mmol resin 6 subjected to the
standard TFMSA deprotection condition to remove side
chain protective groups. Cleavage via ScmCl in AcOH:
Resin (0.0114 mmol) was subjected to the general cleavage
conditions using ScmCl in AcOH to obtain crude 6b. The
crude product was analyzed using HPLC, LCMS and puri-
fied by using prepHPLC. This process yielded 6b as a
white solid (6.4 mg, 84%). ESI-MS: for C H N O S
2
Model pentapeptide 8a synthesis started with 5. Subsequent
coupling with Boc-Ser(Bn)-OH, Boc-Leu-OH, Boc-Tyr(2-
Br-Z)-OH, and Ac-Gly-OH was carried out via the standard
Boc protocol. 50 vol % TFA in CH Cl containing 0.5 vol
% TIPS (1 × 5 min) and (1 × 15 min) treated with the
resin to remove Boc group. Cleavage via ScmCl in AcOH:
Resin 8 (0.0098 mmol) was subjected to the general cleav-
age conditions using ScmCl in AcOH to obtain crude 8a.
The crude product was analyzed using HPLC and LCMS,
and purified by using prepHPLC. The process yielded 8a
2
2
2
5
42
6
11
+ +
+
+
[M + H ] ; cald 667.2426, found 667.2453 [M + Na ] ;
cald 689.2245, found 689.2235 Cleavage via standard
ScmCl in TFA: The resin (0.01 mmol) was subjected to the
general cleavage conditions using ScmCl in TFA to obtain
crude 6b. The crude product was analyzed using HPLC
and LCMS, and purified by using prepHPLC. The process
as
a
white solid (9 mg, 92%). ESI-MS: for
+
+
C H BrN O S [M + H ] ; cald 990.2259, found
4
3
52
5 13 2
+
+
990.2252 [M + Na ] ; cald 1012.2078, found 1012.2076.
Ac-Gly-Tyr-Leu-Ser-Cys(Scm)-OCH3 (8b). Model penta-
peptide 8b synthesis started with 5. Subsequent coupling
Bull. Korean Chem. Soc. 2017, Vol. 38, 54–62
© 2017 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de
59

BULLETIN OF THE
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C-Terminal Cysteine Peptide Synthesis Using Pyroacm Resin
KOREAN CHEMICAL SOCIETY
with Boc-Ser(Bn)-OH, Boc-Leu-OH, Boc-Tyr(2-Br-Z)-OH,
and Ac-Gly-OH was carried out via the standard Boc proto-
col. 50 vol % TFA in CH Cl containing 0.5 vol % TIPS
general cleavage conditions using ScmCl in TFA to obtain
crude 10b. The crude product analyzed using HPLC and
LCMS, and purified by using prepHPLC. The process
yielded 10b as a sticky solid (5.1 mg, 52%). ESI-MS: for
2
2
(1 × 5 min) and (1 × 15 min) treated with the resin to
+
+
remove Boc group. 0.025 mmol resin 8 subjected to stand-
ard TFMSA deprotection condition to remove side chain
protective groups. Cleavage via ScmCl in AcOH: Resin
C H N O S
[M + H ] ; cald 795.3164, found
34 50 8 10 2
+
+
795.3152 [M + Na ] ; cald 817.2983, found 817.2963.
Ac-Gly-Lys-His-Pro-Cys-OMe (10c). The resin
(
0.0095 mmol) was subjected to the general cleavage con-
(0.02 mmol) was subjected to the standard TFMSA depro-
tection condition to remove side chain protective groups.
Finally, the resin (0.0114 mmol) was subjected to the gen-
eral cleavage condition using ScmCl in TFA to obtain
crude 10c. The crude product analyzed using HPLC and
LCMS, and purified by using prepHPLC. The process
yielded 10c as a sticky solid (6.5, 84%). ESI-MS: for
ditions using ScmCl in AcOH to obtain crude 8b. The
crude product analyzed using HPLC and LCMS, and puri-
fied by using prepHPLC. The process yielded 8b as a
sticky solid (5 mg, 76%). ESI-MS: for C H N O S
2
2
8
41
5
11
+ +
+
+
[
M + H ] ; cald 688.2317, found 688.2307 [M + Na ] ;
cald 710.2136, found 710.2126. Cleavage via standard
ScmCl in TFA: The resin (0.0089 mmol) was subjected to
the general cleavage conditions using ScmCl in TFA to
obtain crude 8b. The crude product analyzed using HPLC
and LCMS, and purified by using prepHPLC. The process
yielded 8b as a sticky solid (5.3 mg, 87%). ESI-MS: for
+
+
C H N O S [M + H ] ; cald 687.2589, found 687.2574
2
7 42 8 9 2
+
+
[M + Na ] ; cald 709.2408, found 709.2389.
Synthesis of Intermediate Riparin 1.2 Resin (12a) via
Fmoc SPPS. For synthesis of intermediate 12a, the synthe-
sis started with Fmoc cleavage of 11 using 20 vol % of
piperidine in DMF wherein Fmoc SPPS protocol used to
couple Fmoc-Thr(t-Bu)-OH, Fmoc-Gly-OH, Fmoc-Lys
(Boc)-OH, Fmoc-Tyr(t-Bu)-OH, Fmoc-Ala-OH, Fmoc-Cys
(Acm)-OH, twice Fmoc-Pro-OH, Fmoc-Leu-OH, and
Fmoc-Phe-OH, respectively. 20% piperidine in DMF
(2 × 10 min) treated with resin to remove Fmoc group.
Allyl group is removed by given method followed by Fmoc
removal with standard protocol.
+
+
C H N O S [M + H ] ; cald 688.2317, found 688.3
[
2
8 41 5 11 2
+
+
M + Na ] ; cald 710.2136, found 710.3.
Ac-Gly-Lys(2-Cl-Z)-His(Tos)-Pro-Cys-OMe (9). Model
pentapeptide 9 synthesis started with 5. Subsequent cou-
pling with Boc-Pro-OH, Boc-His(Tos)-OH, Boc-Lys(2-Cl-
Z)-OH, and Ac-Gly-OH was carried out via the standard
Boc protocol. 50 vol % TFA in CH Cl containing 0.5 vol
2
2
%
TIPS (1 × 5 min) and (1 × 15 min) treated with the
resin to remove Boc group.
Ac-Gly-Lys(Biotin)-His-Pro-Cys-OMe (10a). The resin
Synthesis of Side Chain Deprotected Di-Sulfenyl Riparin
1.2 (13) via Two Step Cleavage Protocol. Resin 12a
(0.015 mmol) was swollen in 3 mL of TFA:TIPS:PhOH:
(0.02 mmol) was subjected to the standard TFMSA depro-
tection conditions to remove the side chain protective
groups. To the dried resin in HBTU/HOBt/DIPEA (4/4/8)
was added 0.4 M Biotin (4.0 equiv) in DMF. The mixture
H O of ratio (88:2:5:5) and stirred for 2.5 h at RT, filtered
2
and washed with TFA (1 × 1 min) and CH Cl2
2
(10 × 2 min). The resin (0.0065 mmol) was subjected to
the general cleavage condition of ScmCl in TFA to obtain
crude 13 as a sticky solid. The product analyzed using
HPLC and LCMS, and purified by using prepHPLC. The
process yielded 13 as a white solid (4.1 mg, 46%). ESI-
ꢀ
was tumbled on a shaker at 25 C and 160 rpm for 1 h. The
resin was washed with DMF (5 × 1 min), CH Cl2
2
(
5 × 1 min), DMF (5 × 1 min), and CH Cl (5 × 1 min).
2 2
A small portion of the resin was assayed using the Kaiser
test and if the test was positive, the process was repeated.
Finally, the resin (0.011 mmol) was subjected to the gen-
eral cleavage conditions using ScmCl in TFA to obtain
crude 10a. The crude product was analyzed using HPLC
and LCMS, and purified by using prepHPLC. The process
yielded 10a as a white solid (6.6 mg, 67%). ESI-MS: for
+
+
MS/MS: for C H N O S [M + H ] ; cald 1379.5139,
5
9 86 12 18 4
+
+
found 1379.5 [M + Na ] ; cald 1401.4958, found 1401.5.
Synthesis of Intermediate Riparin 1.2 Resin (12b) via Boc
SPPS. The synthesis of resin 12b started with Fmoc cleav-
age of 11 using 20 vol % of piperidine in DMF. The Boc
SPPS protocol was used to couple Boc-Thr(Bn)-OH, Boc-
Gly-OH, Boc-Lys(2-Cl-Z)-OH, Boc-Tyr(2-Br-Z)-OH, Boc-
Ala-OH, Boc-Cys(Acm)-OH, twice Boc-Pro-OH, Boc-Leu-
OH, and Boc-Phe-OH, respectively. 50 vol % TFA in
+
+
C H N O S
9
[M + H ] ; cald 913.3365, found
3
7 56 10 11 3
+
+
13.3353 [M + Na ] ; cald 935.3184, found 935.3170.
Ac-Gly-Lys(Hexyn)-His-Pro-Cys-OMe (10b). The resin
0.02 mmol) was subjected to the standard TFMSA depro-
(
CH Cl2 containing 0.5 vol % TIPS (1 × 5 min) and
2
tection condition to remove side chain protective groups.
The dried resin in HBTU/HOBt/DIPEA (4/4/8) was treated
with 0.4 M hexynoic acid (4.0 equiv) in DMF for 2 min
(1 × 15 min) treated with the resin to remove Boc group.
The allyl group was removed by utilizing the previously
reported method.
ꢀ
and tumbled on a shaker at 25 C and 160 rpm for 1 h. The
Synthesis of Side Chain Deprotected Di-Sulfenyl Riparin
1.2 (13) via Two Step Cleavage Protocol. Resin 12b
(0.015 mmol) was swollen in 3 mL of TFA:TFMSA:thioa-
resin was washed with DMF (5 × 1 min), CH Cl2
2
(
5 × 1 min), DMF (5 × 1 min), and CH Cl (5 × 1 min).
2 2
ꢀ
A small portion of the resin was assayed using the Kaiser
test and if the test was positive, the process was repeated.
Finally, the resin (0.0123 mmol) was subjected to the
nisole:TIPS of ratio (9:1:1:0.5) and stirred for 2.5 h at 0 C,
filtered and washed with TFA (1 × 1 min) and CH Cl2
2
(10 × 2 min). The resin (0.0074 mmol) was subjected to
Bull. Korean Chem. Soc. 2017, Vol. 38, 54–62
© 2017 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de
60

BULLETIN OF THE
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C-Terminal Cysteine Peptide Synthesis Using Pyroacm Resin
KOREAN CHEMICAL SOCIETY
the general cleavage condition of ScmCl in TFA to obtain
crude 13 as a sticky solid. The product analyzed using
HPLC and LCMS, and purified by using prepHPLC. The
process yielded 13 as a white solid (7.4 mg, 73%). ESI-
Supporting Information. The Supporting Information of
characterized data of all reactions via NMR, IR, HPLC,
LC/MS, HRMS data is available free of charge through
Internet at http://onlinelibrary.wiley.com/journal.
+
+
MS/MS: for C H N O S [M + H ] ; cald 1379.5139,
59 86 12 18 4
+
+
found 1379.5117 [M + Na ] ; cald 1401.4958, found
401.4944.
References
1
Intermediate a-Factor Resin (14). The synthesis of 14
started with 5. The Boc SPPS protocol was used to couple
Boc-Ala-OH, Boc-Asp(Bn)-OH, Boc-Trp(For)-OH, Boc-
Phe-OH, Fmoc-Val-OH, Boc-Gly-OH, Boc-Lys(2-Cl-Z)-
OH, twice Boc-Ile-OH, and Boc-Tyr(2-Cl-Z)-OH respec-
tively. 50 vol % TFA in CH Cl containing 0.5 vol % TIPS
1
2
. (a) X. Xu, R. Lai, Chem. Rev. 2015, 115, 1760;
b) K. B. Akondi, M. Muttenthaler, S. Dutertre, Q. Kaas,
D. J. Craik, R. J. Lewis, P. F. Alewood, Chem. Rev. 2014,
114, 5815; (c) S. Clarke, Annu. Rev. Biochem. 1992, 61, 355;
(d) F. L. Zhang, P. J. Casey, Annu. Rev. Biochem. 1996,
(
6
5, 241.
2
2
. (a) R. B. Merrifield, J. Am. Chem. Soc. 1963, 85, 2149;
(1 × 5 min) and (1 × 15 min) treated with the resin to
(b) R. B. Merrifield, Science 1986, 232, 341;
(c) R. B. Merrifield, Biochemistry 1964, 3, 1385;
(d) M. Muttenthaler, F. Albericio, P. E. Dawson, Nat. Protoc.
remove Boc group.
Synthesis of Side Chain Deprotected Trp(CHO) Sulfenyl
a-Factor (15) via Two Step Cleavage Process. Resin 14
2
1
015, 10, 1067; (e) M. W. Pennington, Method Mol. Biol.
994, 35, 41.
(0.015 mmol) was subjected to the standard TFMSA
deprotection condition to deprotect side chain protective
groups. The dried resin (0.007 mmol) was subjected to the
general cleavage condition of ScmCl in TFA to obtain
crude 15 as a white solid. The crude product was analyzed
using HPLC and LCMS, and purified by using prepHPLC.
The process yielded 15 as a white solid (9.1 mg, 83%).
3
4
5
. (a) L. A. Carpino, G. Y. Han, J. Am. Chem. Soc. 1970, 92,
5748; (b) E. Atherton, H. Fox, D. Harkiss, C. J. Logan,
R. C. Sheppard, B. J. Williams, J. Chem. Soc., Chem. Com-
mun. 1978, 537; (c) G. B. Fields, R. L. Noble, J. Chem. Soc.,
Chem. Commun. 1990, 35, 161.
. (a) J. Lukszo, D. Patterson, F. Albericio, S. A. Kates, Lett.
Pept. Sci. 1996, 3, 157; (b) Y. Fujiwara, K. Akaji, Y. Kiso,
Chem. Pharm. Bull. 1994, 42, 724; (c) V. Diaz-Rodriguez,
E. Ganusova, T. M. Rappe, J. M. Becker, M. D. Distefano,
J. Org. Chem. 2015, 80, 11266.
+
+
ESI-MS/MS: for C H N O S [M + H ] ; cald
7
3 102 14 19 2
+
+
1
543.6960, found 1543.6927 [M + Na ] ; cald 1565.6779,
found 1565.6743.
Synthesis of Side Chain Deprotected Trp(CHO) Sulfenyl
a-Factor (15) via One-Pot Cleavage Process. Resin 14
. Z. Huang, D. J. Derksen, J. C. Vederas, Org. Lett. 2010,
12, 2282.
(
0.01 mmol) was subjected to the general one-pot cleavage
6. (a) B. H. Rietman, R. H. P. H. Smulders, I. F. Eggen, A. Van
Vliet, G. Van De Werken, G. I. Tesser, Int. J. Pept. Protein
Res. 1994, 44, 199; (b) V. Diaz-Rodriguez, D. G. Mullen,
E. Ganusova, J. M. Becker, M. D. Distefano, Org. Lett. 2012,
condition (i.e., 2 equiv of ScmCl in TFMSA:TFA:anisole
of ratio 7:2:1) to obtain crude 15 as a white solid. The
crude product was analyzed using HPLC and LCMS, and
purified by using prepHPLC. The process yielded 15 as a
1
4, 5648; (c) G. Barany, Y. Han, B. Hargittai, R. Q. Liu,
J. T. Varkey, Biopolymers 2003, 71, 652; (d) V. Juvekar,
Y. D. Gong, Org. Lett. 2016, 18, 836.
white
solid
(6.7 mg,
+46%).
ESI-MS/MS:
for
+
C H N O S [M + H ] ; cald 1543.6960, found
1
7
3 102 14 19 2
+
7
. D. Lelièvre, V. P. Terrier, A. F. Delmas, V. Aucagne, Org.
Lett. 2016, 18, 920.
+
543.7 [M + Na ] ; cald 1565.6779, found 1565.7.
Synthesis of Trp, S-Scm Reduced a-Factor (16). The crude
resin 15, generated from 0.0015 mmol of 14 via two step
cleavage process, was placed in 0.5 mL of argon purged
8
. (a) R. Hiskey, N. Muthukumaraswamy, R. Vunnam, J. Org.
Chem. 1975, 40, 950; (b) J. W. Wollack, N. A. Zeliadt,
D. G. Mullen, G. Amundson, S. Geier, S. Falkum,
E. V. Wattenberg, G. Barany, M. D. Distefano, J. Am. Chem.
Soc. 2009, 131, 7293; (c) D. G. Mullen, B. Weigel,
G. Barany, M. D. Distefano, J. Pept. Sci. 2010, 16, 219.
5
(
vol % DTT (ca., 100 equiv) in piperidine:DMF of ratio
1:4). After 15 min, it was diluted with ACN and analyzed
with HPLC before purification to obtain 16 of 95% purity.
+
+
9. (a) H. Yajima, N. Fujii, H. Ogawa, H. Kawatani, J. Chem.
Soc., Chem. Commun. 1974, 107; (b) N. Fujii, A. Otaka,
S. Funakoshi, K. Bessho, H. Yajima, J. Chem. Soc., Chem.
Commun. 1987, 163; (c) M. Nomizu, Y. Inagaki,
T. Yamashita, A. Ohkubo, A. Otaka, N. Fujii, P. P. Roller,
H. Yajima, Int. J. Pept. Protein Res. 1991, 37, 145.
ESI-MS/MS: for C H N O S [M + H ] ; cald
1
7
0 100 14 16
+
+
425.7235, found 1425.7208 [M + Na ] ; cald 1447.7054,
found 1447.7036.
Acknowledgments. This research was supported by the
Bio & Medical Technology Development Program of the
National Research Foundation (NRF) funded by the Minis-
try of Science, ICT & Future Planning (no. 2014M
1
1
1
0. J. P. Tam, W. F. Heath, R. B. Merrifield, J. Am. Chem. Soc.
1
986, 108, 5242.
1. V. M. Maselli, D. Bilusich, J. H. Bowie, M. J. Tyler, Rapid
Commun. Mass Spectrom. 2006, 20, 797.
2. R. Anderegg, R. Betz, S. A. Carr, J. W. Crabb, W. Duntze,
J. Biol. Chem. 1988, 263, 18236.
3A9A9073847), and also financially supported by the Min-
istry of Trade, Industry & Energy (MOTIE), and the Korea
Institute for Advancement of Technology (KIAT) through
the industrial infrastructure program for fundamental tech-
nologies (grant no. M0000338).
13. E. Scoffone, A. Fontana, R. Rocchi, Biochemistry 1968,
7, 971.
Bull. Korean Chem. Soc. 2017, Vol. 38, 54–62
© 2017 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de
61

BULLETIN OF THE
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KOREAN CHEMICAL SOCIETY
1
4. (a) N. Fujii, A. Otaka, O. Ikemura, K. Akaji, S. Funakosho,
Y. Hayashi, Y. Kuroda, H. Yajima, J. Chem. Soc., Chem.
Commun. 1987, 274; (b) H. Tamamura, A. Otaka,
J. Nakamura, K. Okubo, T. Koide, K. Ikeda, N. Fujii,
Tetrahedron Lett. 1993, 34, 4931; (c) R. Okamoto, S. Souma,
Y. Kajihara, J. Org. Chem. 2009, 74, 2494; (d) D. Veber,
J. Milkowski, S. Varga, R. Denkewalter, R. Hirschmann,
J. Am. Chem. Soc. 1972, 94, 5456.
Bull. Korean Chem. Soc. 2017, Vol. 38, 54–62
© 2017 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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62