Improved Fmoc Solid-Phase Peptide Synthesis of Oxytocin with High Bioactivity

Article abstract of DOI:10.1055/s-0036-1589037

We described here the synthesis of oxytocin by an improved Fmoc solid-phase peptide synthesis (SPPS) method with a Rink-Amide resin as the solid support, HBTU as the coupling reagent, Fmoc-protected amino acids as the building blocks, and piperazine for Fmoc removal as a substitute for the standard reagent piperidine. Unlike previously reported syntheses, the removal of the S -Acm protecting group of Cys and cyclization forming the disulfide bond were carried out by using iodine on the resin with the fully protected peptide chains. Finally, a crude oxytocin with a purity of 92% was obtained by simultaneous cleavage of the peptide chains from the resin and removal of all side-chain protecting groups with trifluoroacetic acid containing the scavengers (yield 85%). The crude peptide was purified by using preparative RP-HPLC to obtain oxytocin (high purity 99.3%) with a bioactivity of 588 IU/mg, the highest reported so far in the literature. This investigation provides a contribution in efforts for the large-scale synthesis of oxytocin in high purity under mild conditions with iodine for on-resin disulfide bond formation and a substitute for the standard Fmoc-deprotecting reagent piperidine, a controlled substance.

Full text of DOI:10.1055/s-0036-1589037

SYNLETT
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©
Georg Thieme Verlag Stuttgart · New York
2017, 28, A–E
letter
A
en
Synlett
P. Sun et al.
Letter
Improved Fmoc Solid-Phase Peptide Synthesis of Oxytocin with
High Bioactivity
t-Bu
Pengcheng Sun
Wenli Tang
t-Bu
O
O
O
O
H
N
Yu Huang
H
N
Boc
O
Boc
O
N
N
H
H
Bi-Huang Hu*
H
H
HN
HN
Acm
HN
S
Acm
I2/DMF
a) 5% piperazine/DMF/20 min
b) Fmoc-Aa-OH, HBTU/DIEA/DMF
S
S
O
S
O
HN
2.5 h, 25 °C
O
O
H
O
O
College of Oceanography, Hainan University, Haikou,
Hainan Province, 570228, P. R. of China
bhhu@hainu.edu.cn
N
Fmoc
N
O
N
H
NH
Standard Fmoc
SPPS
N
O
NH
O
H
N
H
O
H
O
H
H
O
N
N
N
N
N
N
O
O
H
(
= Rink Amide AM Resin)
H
O
O
O
O
NH HN
Trt
NH HN
Trt
Trt
Trt
Received: 08.04.2017
Accepted after revision: 04.05.2017
Published online: 31.05.2017
benzyl-N-carbobenzoxy-L-cysteine was also used to obtain
5
0.1 g of oxytocin. Subsequently, rather than condensing the
DOI: 10.1055/s-0036-1589037; Art ID: st-2017-w0240-l
short peptide fragments, started with 50 g of S-p-methoxy-
benzly-L-cysteine protected amino acids (nitrophenyl es-
ters) were coupled one by one to the prepared peptide
Abstract We described here the synthesis of oxytocin by an improved
Fmoc solid-phase peptide synthesis (SPPS) method with a Rink-Amide
resin as the solid support, HBTU as the coupling reagent, Fmoc-protect-
ed amino acids as the building blocks, and piperazine for Fmoc removal
as a substitute for the standard reagent piperidine. Unlike previously re-
ported syntheses, the removal of the S-Acm protecting group of Cys
and cyclization forming the disulfide bond were carried out by using io-
dine on the resin with the fully protected peptide chains. Finally, a
crude oxytocin with a purity of 92% was obtained by simultaneous
cleavage of the peptide chains from the resin and removal of all side-
chain protecting groups with trifluoroacetic acid containing the scaven-
gers (yield 85%). The crude peptide was purified by using preparative
RP-HPLC to obtain oxytocin (high purity 99.3%) with a bioactivity of 588
IU/mg, the highest reported so far in the literature. This investigation
provides a contribution in efforts for the large-scale synthesis of oxyto-
cin in high purity under mild conditions with iodine for on-resin disul-
fide bond formation and a substitute for the standard Fmoc-deprotect-
ing reagent piperidine, a controlled substance.
6
chain, yielding 0.918 million units of oxytocin. In a later re-
port, du Vigneaud reported the synthesis of a tritiated oxy-
tocin using tritium-labeled L-leucine as the starting materi-
7
al. Beyerman reported with the starting material 30.2
mmol of N-benzyloxycarbonyl-S-benzyl-L-cysteine, the for-
mation of protected linear nonapeptide precursors to oxy-
tocin with a variety of protecting groups by using N,N′-di-
8
cyclohexylcarbodiimide (DCC) as a coupling agent. All the
approaches mentioned above for the synthesis of oxytocin
had the same strategy as removal of the protecting groups
with sodium/liquid ammonia, followed by formation of the
disulfide bond via air oxidation in solutions to obtain oxy-
9
tocin product. However, these procedures for formation of
the disulfide bond suffered from long reaction times, more
impurities, and low yields. Using NPS-glycine resin (0.71
mmol), 20 mg of oxytocin was obtained with the highest
bioactivity for the synthesized oxytocin reported so far
Key words oxytocin, Fmoc solid-phase peptide synthesis, on-resin cy-
clization, disulfide bond formation by iodine, piperazine
(
508 IU/mg), 10 although higher values could be reached
Oxytocin is a cyclic nonapeptide hormone containing a
disulfide bond, and released by the posterior lobe of the pi-
tuitary gland in humans and mammals to regulate the pro-
cesses such as childbirth and milk production. Oxytocin
was more recently also linked with more complex process-
with improved syntheses yielding the hormone in higher
purity. Other approaches for cyclization in the synthesis of
the cyclic nonapeptide included N-carbobenzoxy-S-benzo-
yl-L-cysteinyl-L-prolyl-L-leucyl-glycin-amide (11 mmol) as
the starting material and selective removal of the S-protect-
ing groups, followed oxidation by 1,2-diiodoethane to ob-
1
es, such as social adjustment, maternity, sexual behavior,
2
11
and regulation of algesia. It is in the essential medicines list
tain 65 mg of oxytocin with a bioactivity of 400 IU/mg, re-
of World Health Organization.
moval of the S-protecting groups and cyclization using
In 1953, du Vigneaud determined the chemical struc-
ture of oxytocin and reported the first total synthesis of
thallium(III) trifluoroacetate (Tl(tfa) ) in trifluoroacetic ac-
id, forming oxytocin 15.1 mg in 45% yield, and cleavage of
3
3
12
oxytocin by conventional solution synthesis and demon-
strated its biological activity in the same year. Fragment
condensation strategy with 7 g of the starting material S-
various S-protecting groups of cysteine (Acm, Tacm, and
But) with methyltrichlorosilane or tetrachlorosilane in tri-
fluoroacetic acid in the presence of diphenylsulfoxide,
4
©
Georg Thieme Verlag Stuttgart · New York — Synlett 2017, 28, A–E

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Synlett
P. Sun et al.
Letter
forming the cyclized peptide in 56%, 69%, and 64% yields,
respectively.
Based upon the above data, there is a need to optimize
the synthesis with mild reagents and reaction conditions
and improve the cyclization yield for large-scale synthesis
of oxytocin.
Here, we reported our improved Fmoc SPPS of oxytocin.
We used a polystyrene-based Rink Amide AM resin as a sol-
id support to build the protected linear peptide chain by
standard Fmoc/t-Bu strategy20 with HBTU as the coupling
reagent and Fmoc-protected amino acids as the building
blocks except for the N-terminal amino acid using Boc-
Cys(Acm)-OH (Scheme 1).
13
The early syntheses of oxytocin 32.5 mg and 70 mg by
SPPS with a Merrifield resin were reported by three
10,14
groups.
After obtaining the S-Bn-protected linear pep-
tide by SPPS, the cleavage of the protected peptide from the
Merrifield resin was achieved in a methanol solution satu-
rated with ammonia at 10 °C overnight, followed by remov-
al of all protecting groups with sodium/liquid ammonia and
15
cyclization using potassium ferricyanide to obtain a prod-
uct at 27% yield and with a bioactivity of 430 IU/mg. The
SPPS procedure of Ives obtained oxytocin with a bioactivity
After removal of the Fmoc group on the resin with 5% of
piperazine in DMF, the first amino acid was attached to the
resin using Fmoc-Gly/HBTU/DIEA (2:1.9:2.4) in DMF for
two hours, monitored by the ninhydrin test. 21 Treatment
with 5% piperazine in DMF for 20 minutes was used to re-
move the Fmoc group for the first time in Fmoc SPPS of
10
of 508 IU/mg. An S-Mebzl-protected linear peptide pre-
cursor to oxytocin by the Boc/Bn strategy on a benzhydryl-
amine (BHA) resin was synthesized and cleaved from the
resin with HF, and the disulfide bond was formed in 0.01 M
potassium ferricyanide in water and generated 72 mg of
16
22
oxytocin. N-Iodosuccinimide (NIS) was used to form the
oxytocin wherein a crystalline piperazine , easy to handle
disulfide bond, but the total yield of oxytocin was only 1%
with 4 mg of oxytocin at a scale of 0.5 mmol resin.
Syntheses of oxytocin with Boc/MBHA (4-methylben-
zhydrylamine) and Fmoc/PAL (5-(4-aminomethyl-3,5-di-
methoxyphenoxy)-valeryl) and the oxidation of the unpro-
tected thiols were investigated by several approaches in-
and readily available, successfully substituted the standard
Fmoc-deprotecting reagent piperidine, a controlled sub-
stance. For the coupling of the other amino acids, the same
protocol as in the first amino acid was followed but a short-
er reaction time (1.5 h). After completion of the synthesis of
the fully protected linear oxytocin on the resin, the cycliza-
tion reaction using iodine on the resin for removal of S-Acm
protecting group20 and formation of the disulfide bond
with fully protected peptide chains was optimized with
equivalents of iodine, reaction volume, reaction time, reac-
tion temperature, the resin loading, and the status of the α-
amino group of the N-terminal amino acid.
17
cluding oxidation with air, iodine, and Tl(tfa) , indicating
3
that the most successful oxidant for the disulfide bond for-
mation was Tl(tfa) , forming the cyclic nonapeptide in 79%
3
yield; in contrast, with iodine in 28% yield at the scales of
5
0–60 mg of the resins and about 20μmol of oxytocin.18 Re-
cently, a synthesized oxytocin (0.346 g) with a yield of 34%
was reported with no bioactivity data where the linear
peptide without protecting groups in solution was treated
with iodine through Fmoc SPPS on a Rink-Amide resin and
cleavage of the peptide chains and removal of protecting
The crude oxytocin was obtained by the removal of pro-
tecting groups and cleavage of the peptide chains from the
resin using TFA–triisopropylsilane–H O (95:2.5:2.5) for 30
2
minutes.
19
groups starting with 2 g of the resin, in which 20% piperi-
dine/DMF was used to remove the Fmoc protecting group.
We first studied the influence of the equivalents of io-
dine: 2.5 equivalents (Table 1) and 5 equivalents (Table 2)
O
O
Fmoc-Gly-OH
HBTU/DIEA
5% piperazine/DMF
5% piperazine/DMF
H
H
N
H
H
Fmoc
NH2
N
N
Fmoc
NH2
N
Standard Fmoc
SPPS
(
= Rink Amide AM Resin)
t-Bu
a) 5% piperazine/DMF/20 min
b) Fmoc-Aa-OH, HBTU/DIEA/DMF/1.5 h
t-Bu
O
O
OH
O
O
O
H
N
O
H2N
Boc
O
H
O
H
N
Boc
N
O
N
H
H
H
N
TFA/TIS/H2O = 95:2.5:2.5
HN
HN
I2/DMF
H
H
S
HN
HN
S
Acm
HN
HN
S
Acm
S
O
S
2.5 h, 25 °C
S
O
O
O
O
O
O
H
N
O
N
O
N
N
H
NH
N
O
O
N
H
NH
N
H
O
H
H
O
O
H
N
N
H
H
O
O
O
NH2
N
O
N
O
N
N
N
O
H
O
N
O
H2N
H
O
H
O
NH HN
Trt
O
NH2
O
HN
NH
Trt
Trt
Trt
Scheme 1 Synthesis of oxytocin by Fmoc SPPS via on-resin cyclization
©
Georg Thieme Verlag Stuttgart · New York — Synlett 2017, 28, A–E

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Synlett
P. Sun et al.
Letter
with different reaction time, two different solvent volumes
DMF) of the on-resin cyclization reaction on the product
purity (Scheme 1) at 25 °C, and a resin loading of 0.55
mmol/g. The results indicated that the oxytocin purity with
iodine 5 equivalents was significantly higher than those
with 2.5 equivalents while a higher concentration in DMF
Table 3 The Purity of Oxytocin with Different Reaction Temperatures
(
Reaction time (h)a
–25 °Cb
87.28%c
0 °C
87.16%
25 °C
2.5
92.01%
a
b
c
Time of cyclization reaction.
Temperature of cyclization reaction.
Purity of crude oxytocin.
(0.28 mM) afforded a slightly purer product with 2.5 equiv
of iodine (Table 1). In contrast, the highest purities were
achieved at lower concentration (0.20 mM) with 5 equiva-
lents of iodine. So far, the optimized conditions iodine 5
equivalents, reaction time 2.5 hours, and the iodine con-
centration of 0.2 mM generated the product with the high-
est purity (92.01%; Table 2).
Table 4 The Purity of Oxytocin with Different Resin Loadings
Reaction time (h)a
0.37 mmol/gb
0.55 mmol/g
92.01%
0.87 mmol/g
83.38%
87.71%c
2.5
a
b
c
Time of cyclization reaction.
The resin loading.
Purity of crude oxytocin.
We next investigated the effect of reaction temperature
with the above optimized conditions (5 equiv of iodine, re-
action time 2.5 hours, 0.20 mM) on the purity, indicating
that the purity was not significantly changed over a tem-
perature range of –25 °C to 25 °C (Table 3).
The resin loading was also studied with the above de-
termined conditions. It showed that the resin loading (0.55
mmol/g) among the studied was spatially favored for cy-
clization on the resin (Table 4).
Finally, we investigated the effect of the status of the
amino group of the N-terminal amino acid (Boc protected
or unprotected) on the purity and yield of oxytocin. Boc
protection of the α-amino group of the N-terminal amino
acid led to the formation of the product in considerably
higher purity (92% vs. 68% for the unprotected amine; Fig-
ure 1 (a, b), respectively), and in slightly higher yield (Table
5). In addition, Boc-Cys(Acm)-OH for the N-terminal amino
acid as a building block avoided an extra step to remove the
Fmoc group after the cyclization instead of Fmoc-
Cys(Acm)-OH.
For the purification of the crude oxytocin, we explored
five solvent systems: 1) 0.1%TFA in water–MeCN, 2) pH =
Table 1 The Purity of Oxytocin under Different Reaction Conditions
with 2.5 Equivalents I2
Reaction time (h)a
0.28 mMb
0.20 mMc
1
2
3
6
.5
19.39%d
26.23%
27.92%
31.82%
0
2.25 Teap buffer–MeCN, 3) pH = 4.25 Teap buffer–MeCN, 4)
.5
20.03%
21.87%
23.99%
pH = 6.25 Teap buffer–MeCN, and 5) 0.1 M NaH PO in wa-
2
4
.5
ter–MeCN) with identical HPLC column and stationary
phase and selected 0.1 M NaH PO in water–MeCN system
.0
2
4
a
with the highest theoretical plate number (53270/m). After
purification with a RP-HPLC DAC column, oxytocin was ob-
tained with 99.3% purity and 51% yield (1.2 g). The bioactiv-
ity of this oxytocin was measured according to the Europe-
Time of cyclization reaction.
The molar concentration of iodine in DMF (1 mmol of peptidyl resin in 9
b
mL DMF).
c
The molar concentration of iodine in DMF (1 mmol of peptidyl resin in
2.7 mL DMF).
Purity of crude oxytocin, analyzed by RP-HPLC (Agilent 1260 Infinity, UV
1
d
23
an pharmacopoeia 9.0 with the authentic standard and
detection at 220 nm, column (kromasil C18, 5 μm, 100 Å, 4.6 × 250 mm).
Column temperature at 25 °C and a flow rate of 1.0 mL/min: eluent A (0.1
M NaH
A–40% A, analytical HPLC methods for purity of oxytocin in this paper are
identical).
was found to be 588 IU/mg, the highest bioactivity reported
2
PO
4
/H
2
O), eluent B (MeCN–H
2
O = 1:1); gradient (0–30 min): 70%
thus far in the literature.
10
In addition, six oxytocin-related impurities (Figure 2, a–
f) were synthesized using the same protocol as in the above
synthesis of oxytocin to confirm the purity of the final puri-
fied oxytocin, and analyzed with oxytocin by RP-HPLC. The
Table 2 The Purity of Oxytocin under Different Reaction Conditions
with 5 Equivalents I2
Reaction time (h)a
0.28 mMb
0.20 mM
Table 5 The Purity and Yield of Crude Oxytocin with an Unprotected
1
2
3
.5
84.97%c
81.33%
86.46%
87.10%
92.01%
87.04%
(Method 1) or Boc-Protected N-Terminal Amino Acid (Method 2)
.5
Method 1
Method 2
.5
a
b
c
Time of cyclization reaction.
The molar concentration of iodine in DMF.
Purity of crude oxytocin.
Purity (%)
Yield (%)
68.15
79.36
92.25
85.22
©
Georg Thieme Verlag Stuttgart · New York — Synlett 2017, 28, A–E

D
Synlett
P. Sun et al.
Letter
possible oxytocin-related impurities in this report were
listed as the followings: a) unsuccessful removal of S-Acm;
b) truncated N-terminal amino acid; c) removed S-Acm
without disulfide formation; d) truncated N-terminal ami-
no acid without removal of the S-Acm; e) single removal of
S-Acm in the N-terminal Cys; f) the N-terminal Cys with S-
Acm (see Table 6).
Table 6 Oxytocin-Related Impurities a–f (Figure 2)
The sequences of the impurities
a
b
c
d
e
f
H-Cys(Acm)-Tyr-Ile-Gln-Asn-Cys(Acm)-Pro-Leu-Gly-NH
H-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH
H-Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH
H-Tyr-Ile-Gln-Asn-Cys(Acm)-Pro-Leu-Gly-NH
2
2
2
2
H-Cys-Tyr-Ile-Gln-Asn-Cys(Acm)-Pro-Leu-Gly-NH
2
2
H-Cys(Acm)-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH
As shown in Figure 2, oxytocin and related imputities a–
f were completely resolved. It is obvious that the purified
oxytocin excluded those impurities.
In summary, despite the advances in Fmoc SPPS, the
large-scale synthesis of oxytocin remained a challenge on
account of the low yield and reaction conditions involved in
dangerous sodium/liquid ammonia or highly toxic thalli-
um(III) trifluoroacetate.
We reported the synthesis of oxytocin with the advan-
tages for potential large-scale production under mild condi-
tions and without use of dangerous or toxic reagents. For
the first time, the removal of S-Acm and cyclization for the
disulfide bond formation of oxytocin using iodine was per-
formed on the resin with fully protected peptide chains. For
each coupling, utilization of two equivalents of Fmoc amino
acid to the resin loading largely reduced production cost. To
remove the Fmoc group for the first time in Fmoc SPPS of
oxytocin, a crystalline piperazine, easy to handle and readi-
ly available, successfully substituted the standard Fmoc-
deprotecting reagent piperidine, a controlled substance.
With the optimized reaction conditions, high-purity oxyto-
cin with the highest bioactivity (588 IU/mg) reported so far,
was obtained. We believed that the results from this inves-
tigation represented a valuable contribution toward the de-
velopment of a practical methodology for the large-scale
synthesis of oxytocin.
Figure 1 RP-HPLC chromatograms of crude oxytocin by method 1 (a)
and method 2 (b)
Funding Information
This work was supported by the Hainan Provincial Program for High-
level Innovative Talents Grant HN2011
H
an
i
a
n
Porvniacil
P
orgra
m
o
f
r
Hgihe-lv
e
l
n
I
n
o
vaite
Taelnst
Grant
H( N
2
0
1
1)
Acknowledgment
Lanzhou Kaibo Pharmaceuticals Co., Ltd. (Gansu Province, P.R. China)
provided the authentic oxytocin European Pharmacopoeia Commis-
sion Reference Substance (EPCRS) from EDQM.
Supporting Information
Supporting information for this article is available online at
Figure 2 RP-HPLC chromatograms of oxytocin and related impurities
https://doi.org /10.1055/s-0036-1589037.
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P. Sun et al.
Letter
References
(11) Photaki, I. J. Am. Chem. Soc. 1966, 88, 2292.
(
(
(
12) Fujii, N.; Otaka, A.; Funakoshi, S.; Bessho, K.; Watanabe, T.; Akaji,
(1) Robinson, D. A.; Wei, F.; Wang, G. D.; Li, P.; Kim, S. J.; Vogt, S. K.;
K.; Yajima, H. Chem. Pharm. Bull. 1987, 35, 2339.
13) Akaji, K.; Tatsumi, T.; Yoshida, M.; Kimura, T.; Fujiwara, Y.; Kiso,
Y. J. Chem. Soc., Chem. Commun. 1991, 3, 167.
14) (a) Manning, M. J. Am. Chem. Soc. 1968, 90, 1348. (b) Spatola, A.
F.; Cornelius, D. A.; Hruby, V. J.; Blomquist, A. T. J. Org. Chem.
Muglia, L. J.; Zhuo, M. J. Physiol. 2002, 540, 593.
(
2) (a) Honnebier, M. B.; Figueroa, J. P.; Rivier, J.; Vale, W.;
Nathanielsz, P. W. J. Dev. Physiol. 1989, 12, 225. (b) Shukovski, L.
Reprod., Fertil. Dev. 1992, 4, 99. (c) Gurrieri, F.; Neri, G. BMC
Med. 2009, 7, 1. (d) Sasayama, D.; Hattori, K.; Teraishi, T.; Hori,
H.; Ota, M.; Yoshida, S.; Arima, K.; Higuchi, T.; Amano, N.;
Kunugi, H. Schizophr. Res. 2012, 139, 201. (e) McGregor, I. S.;
Bowen, M. T. Horm. Behav. 2012, 61, 331. (f) Seng, J.; Miller, J.;
Sperlich, M.; van de Ven Cosmas, J. M.; Brown, S.; Carter, C. S.;
Liberzon, I. J. Trauma Dissociation 2013, 14, 40. (g) Poulin, M. J.;
Holman, E. A. Horm. Behav. 2013, 63, 510. (h) Calcagnoli, F.; de
Boer, S. F.; Althaus, M.; den Boer, J. A.; Koolhaas, J. M. Psycho-
pharmacology (Heidelberg, Ger.) 2013, 229, 639. (i) Peltola,
Mikko. J.; Yrttiaho, S.; Puura, K.; Leppanen, Jukka. M.; Proverbio,
Alice. M.; Mononen, N.; Lehtimaki, T. Emotion 2014, 14, 469.
3) du Vigneaud, V.; Ressler, C.; Trippett, S. J. Biol. Chem. 1953, 205,
1974, 39, 2207.
(
15) (a) Takashima, H.; Du Vigneaud, V.; Merrifield, R. B. J. Am. Chem.
Soc. 1968, 90, 1323. (b) Baxter, J. W. M.; Manning, M.; Sawyer,
W. H. Biochemistry 1969, 8, 3592. (c) Manning, M.; Coy, E.;
Sawyer, W. H. Biochemistry 1970, 9, 3925.
(16) Live, D. H.; Agosta, W. C.; Cowburn, D. J. Org. Chem. 1977, 42,
3556.
(17) Shih, H. J. Org. Chem. 1993, 58, 3003.
(
18) Albericio, F.; Hammer, R. P.; García-Echeverría, C.; Molins, M. A.;
Chang, J. L.; Munson, M. C.; Pons, M.; Giralt, E.; Barany, G. Int. J.
Pept. Protein Res. 1991, 37, 402.
(
(
(19) Li, S. T.; Zhao, H. L.; Gao, Y.; Wang, X. Q.; Wang, L. Y. Chinese J.
Phar. 2015, 01, 7.
949.
4) Vigneaud, V. D.; Ressler, C.; Swan, C. J. M.; Roberts, C. W.;
Katsoyannis, P. G.; Gordon, S. J. Am. Chem. Soc. 1953, 75, 4879.
5) Bodanszky, M.; du Vigneaud, V. J. Am. Chem. Soc. 1959, 81, 2504.
6) (a) Bodanszky, M.; du Vigneaud, V. J. Am. Chem. Soc. 1959, 81,
(
20) (a) Barlos, K.; Gatos, D. Pept. Sci. 1999, 51, 266. (b) Chan, W.;
White, P. D. Fmoc Solid Phase Peptide Synthesis: A Practical
Approach, 1st ed; Oxford University Press: Oxford, 2000.
(
(
(c) Hughes, A. B. Amino Acids, Peptides and Proteins in Organic
5688. (b) Sakakibara, S.; Nobuhara, Y.; Shimonishi, Y.; Kiyoi, R.
Chemistry, V3: Building Blocks, Catalysis and Coupling Chemistry;
Wiley-VCH: Weinheim, 2011.
Bull. Chem. Soc. Jpn. 1965, 38, 120.
(
(
(
7) Vigneaud, V. D.; Schneider, C. H.; Stouffer, J. E.; Murti, V. V. S.;
Aroskar, J. P.; Winestock, G. J. Am. Chem. Soc. 1962, 84, 409.
8) Beyerman, H. C.; Bontekoe, J. S.; Koch, A. C. Recl. Trav. Chim.
Pays-Bas. 1959, 78, 935.
9) du Vigneaud, V.; Winestock, G.; Murti, V. V. S.; Hope, D. B.;
Kimbrough, R. D. J. Biol. Chem. 1960, 235, 64.
(
(
(
21) Sarin, V. K.; Kent, S. B.H.; Tan, J. P.; Merrifield, R. B. Anal. Bio-
chem. 1981, 117, 147.
22) Ralhan, K.; KrishnaKumar, V. G.; Gupta, S. RSC Adv. 2015, 5,
104417.
23) EDQM, European Pharmacopoeia 9.0. 2017, 3250
(10) Ives, D. A. J. Can. J. Chem. 1968, 46, 2318.
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Georg Thieme Verlag Stuttgart · New York — Synlett 2017, 28, A–E