A rapid and clean synthetic approach to cyclic peptides: Via micro-flow peptide chain elongation and photochemical cyclization: Synthesis of a cyclic RGD peptide

Article abstract of DOI:10.1039/c6ob02391f

A cyclic RGD peptide was efficiently synthesized based on micro-flow, triphosgene-mediated peptide chain elongation and micro-flow photochemical macrolactamization. Our approach enabled a rapid (amidation for peptide chain elongation <5 s, macrolactamization <5 min) and clean (only one column chromatographic separation) synthesis of a cyclic peptide.

Full text of DOI:10.1039/c6ob02391f

Organic &
Biomolecular Chemistry
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PAPER
A rapid and clean synthetic approach to cyclic
peptides via micro-flow peptide chain elongation
and photochemical cyclization: synthesis
of a cyclic RGD peptide†
Cite this: DOI: 10.1039/c6ob02391f
Yuto Mifune, Hiroyuki Nakamura and Shinichiro Fuse*
Received 3rd November 2016,
Accepted 9th November 2016
A cyclic RGD peptide was efficiently synthesized based on micro-flow, triphosgene-mediated peptide
chain elongation and micro-flow photochemical macrolactamization. Our approach enabled a rapid
DOI: 10.1039/c6ob02391f
(amidation for peptide chain elongation <5 s, macrolactamization <5 min) and clean (only one column
www.rsc.org/obc
chromatographic separation) synthesis of a cyclic peptide.
amide bond formations (1 to 2 and 3 to 4). The use of these
low-atom-economy reagents generate much waste, and tedious
Introduction
Cyclic peptides are important as drugs and tools in chemical purification steps to remove them are usually required after
1
,2
6
biology,
and their decreased level of conformational each amide bond formation. Therefore, the development of a
freedom enhances their metabolic stability and binding rapid, clean and practical synthetic approach for cyclic pep-
affinity compared with linear peptides. In general, cyclic pep- tides remains highly important.
4
,7
tides 4 are chemically synthesized via a two-stage approach:
In terms of the key macrolactamization, a native chemical
peptide chain elongation and cyclization (Fig. 1). The linear ligation (NCL) and its modified approach have been developed
8
–13
peptide 2 is prepared from the protected C-terminal amino into a clean and mild cyclization method.
acid via repetitive amide bond formation using an requires neither the addition of coupling reagents nor an iso-
N-protected amino acid and deprotection. After the de- lation of the poorly soluble precursor 3. However, the pre-
This approach
1
8
,9
10
protection at the C-terminal carboxyl group of the prepared cursors must have either a cysteine or a serine/threonine
linear peptide 2, macrolactamization of 3 affords the desired residue at their N-terminus. Several cyclization-cleavage
cyclic peptide 4. This conventional approach has several draw- approaches have been reported using oxime-, thioester-, sulfo-
backs. (1) The remaining/generated compounds in the de- namide-, hydrazine- or catechol-linkers in solid-phase peptide
14
protection step (2 to 3) often cannot be removed from the cycli- synthesis. These approaches do not require the isolation of 3.
zation precursor 3. Free amino and carboxyl groups make pre- However, the oxime- and thioester-linkers are applicable
cursor 3 poorly soluble against a variety of solvents, and this only to Boc chemistry due to their high reactivity against
complicates the removal of the impurities. These can inhibit nucleophiles. The use of sulfonamide-, hydrazine- and cate-
3
the subsequent key step of macrolactamization. (2) chol-linkers requires an excess amount of activating reagents
Macrolactamization (3 to 4) requires an excess amount of and an extended reaction time (≥1 day) for cyclization, and
coupling reagents to accelerate the activation of carboxylic these linkers also have a limited scope of amino acids and pro-
acids because high dilution conditions are usually employed tecting groups.
in order to avoid the undesired intermolecular reactions. A
Nicolaou and coworkers reported 5-acyl-7-nitroindoline as a
great deal of waste originates from excess amounts of coupling photoactivatable linker, and they demonstrated an intra-
reagents, which complicates the purification of the cyclization molecular cyclization-cleavage approach for the synthesis of
4
15,16
product 4. (3) Coupling reagents, such as carbodiimides, 7-membered heterocycles.
This approach is attractive
5
uronium salts or phosphonium salts, are usually used for because the cyclization precursor does not contain the free car-
boxyl group, which simplifies the removal of impurities from
the cyclization precursor. Moreover, the addition of an excess
Laboratory for Chemistry and Life Science, Institute of Innovative Research,
amount of activating reagent is unnecessary for photochemical
Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8503,
macrolactamization. However, this approach usually requires
Japan. E-mail: sfuse@res.titech.ac.jp
an extended reaction time (6–12 h), and it has yet to be used
†
Electronic supplementary information (ESI) available: A micro-flow reactor
setup and NMR spectra of products. See DOI: 10.1039/c6ob02391f
for the macrolactamization of peptides.
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Fig. 1 Conventional synthetic approach and our approach for a cyclic peptide. PG = protecting group; aa = amino acid.
We demonstrated an efficient micro-flow photochemical of the acetyl group and the following simple precipitation
synthesis of vitamin D and its analogues, along with α-aryl afforded the pure Bni 9 in 4 steps with 80% yield. An acylation
1
7
esters. The narrow reaction space in a micro-flow reactor with N-Fmoc-glycine and thionyl chloride, followed by re-
enhanced the penetration efficiency of light and reduced the crystallization, provided Bni-protected N-Fmoc-glycine 10 in
1
8
reaction time. We envisaged that the use of micro-flow 73% yield.
technology would enable rapid photochemical cyclization Peptide chain elongation was performed using our devel-
Fig. 1). In addition, we planned to use our originally devel- oped micro-flow amide bond formation
oped triphosgene-mediated micro-flow amide bond formation protection (Scheme 2). The Fmoc group of the glycine deriva-
1
9
(
and batch de-
1
9
for peptide chain elongation. Herein, we report a rapid and tive 10 was removed under basic conditions. After removing
clean synthesis of a cyclic RGD peptide, which is a highly the non-polar compounds that originated from the Fmoc
2
0
potent and selective antagonist for the α
v
β
3
integrin receptor.
group via short-path chromatography, micro-flow amide bond
formation with N -Boc-N -nitro-L-arginine (12) was carried
α
ω
out. We connected two T-shaped micro-mixers with Teflon®
tubing. A solution of carboxylic acid 12 and N,N-diisopropyl-
ethylamine (DIEA) in DMF was introduced into the first micro-
Results and discussion
1
6a, mixer with a syringe pump. A solution of triphosgene in MeCN
was also introduced into the first micro-mixer with a syringe
pump. After the generation of symmetric anhydride in situ
We selected readily available 5-bromo-7-nitroindoline (Bni)
b,f,21
as a photoactivatable group. Bni-protected Fmoc-glycine
0, a precursor of peptide chain elongation, was efficiently pre-
pared from indoline (8) as shown in Scheme 1. The sequence
of acetylation/regioselective bromination/nitrization/removal
1
(
<0.5 s), a solution of amine 11 was introduced into the second
micro-mixer with a syringe pump to accomplish amidation
<4.3 s). The reaction temperature was maintained at 20 °C by
(
immersing the micro-mixers into a water bath. The reaction
was quenched by pouring the mixture into a 0.5 M solution of
HCl in CH Cl . The HCl salt of DIEA and unreacted carboxylic
2
2
acid 12 was separated by simple aqueous workup to afford a
fairly pure protected dipeptide 13. The removal of the Boc
group of the protected dipeptide 13 was performed under
acidic conditions. In the subsequent micro-flow amidation
α ε
with N -Boc-N -2-chlorocarbobenzoxy (2-Cl-Cbz)-L-lysine (15),
the TFA salt of amine 14 was basified with DIEA before intro-
duction into the micro-mixer. After aqueous workup, the crude
protected tripeptide 16 was treated with 4 M HCl in 1,4-
dioxane to provide the HCl salt of amine 17. Next, micro-flow
amidation with N-Boc-D-phenylalanine (18) was performed
under the same conditions as described above. The final coup-
ling of tetrapeptide 20 with N-Boc-L-aspartic acid 4-benzyl ester
(
21) afforded a crude mixture of the protected pentapeptide 22.
After silica-gel column chromatography and recrystallization,
Scheme 1 Synthesis of Bni-protected N-Fmoc-glycine 10.
the desired pure pentapeptide 22, which did not contain
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Scheme 3 Intramolecular cyclization of Bni-protected pentapeptide
and global deprotection.
around a 9 W portable UV lamp (wavelength: 365 nm) and con-
1
7c,d
nected with PEEK tubing.
After removal of the Boc group
of the protected pentapeptide 22, a syringe pump was used to
introduce a 1 mM solution of amine 23 in MeCN into the FEP
tube, which was then irradiated for 5 min. The desired pro-
tected cyclic pentapeptide 24 was readily obtained after recrys-
tallization in 36% yield (2 steps). The subsequent hydrogen-
ation of the protected pentapeptide 24 afforded a cyclic RGD
peptide (25) in a quantitative yield. The observed spectra of the
cyclic RGD peptide (25) were in good agreement with the pre-
2
0c,h
viously reported data.
Conclusions
In summary, we demonstrated a rapid and clean synthesis of a
cyclic RGD peptide. Our originally developed triphosgene-
mediated micro-flow amide bond formation enabled a rapid
Scheme 2 Peptide chain elongation based on micro-flow amide bond (amidation <5 s) and clean (only one column chromatographic
formation and batch deprotection.
separation) synthesis of the linear protected pentapeptide 22.
A rapid intramolecular cyclization (<5 min) was achieved by
irradiation of the Bni-protected pentapeptide 23 in a micro-
flow reactor. Our developed process will allow the rapid and
epimers, was obtained in 8 steps with 7% yield from protected practical preparation of a variety of biologically active cyclic
glycine 10. Although the observed yield was moderate, this peptides.
result was obtained in only one trial with no optimization of
the reaction conditions. In addition, it should be noted that
the desired pentapeptide 22 was obtained using only one-
column chromatographic purification.
Experimental
General techniques
Next, we performed the intramolecular cyclization of a Bni-
protected linear peptide by irradiation using a micro-flow NMR spectra were recorded on Bruker Biospin AVANCE II 400
1
13
reactor (Scheme 3). A fluorinated ethylene propylene copoly- (400 MHz for H, 100 MHz for C)and Bruker Biospin AVANCE
1
13
mer (FEP) tube (inner diameter: 1.0 mm) was tightly wrapped III HD 500 (500 MHz for H, 125 MHz for C) instruments in
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the indicated solvent. Chemical shifts are reported in units of Synthesis of Bni-protected N-Fmoc-glycine 10
parts per million (ppm) relative to the signal (0.00 ppm) for
To a solution of N-Fmoc-glycine (5.23 g, 17.6 mmol, 1.10
equiv.) in toluene (80.0 mL), thionyl chloride (2.30 mL,
internal tetramethylsilane for solutions in CDCl
3
(7.26 ppm for
1
13
1
H, 77.0 ppm for C) or DMSO (2.50 ppm for H, 39.5 ppm for
3
2.0 mmol, 2.00 equiv.) and two drops of DMF were added
dropwise at room temperature. After being stirred at 70 °C for
.5 h, 5-bromo-7-nitroindoline (9) (3.89 g, 16.0 mmol, 1.00
equiv.) was added at room temperature. After being stirred at
0 °C for 17 h, the reaction mixture was cooled and concen-
1
3
1
13
C) or D O (4.79 ppm for H, 21.0 ppm for C using acetic
2
acid as an internal standard). Multiplicities are reported by
using the following abbreviations: s; singlet, d; doublet, t;
triplet, q; quartet, m; multiplet, br; broad, J; coupling con-
stants in Hertz (Hz). IR spectra were recorded on a JASCO FT/
IR-4100. Only the strongest and/or structurally important
1
7
trated in vacuo. The residue was recrystallized from EtOAc/
hexane to give Bni-protected N-Fmoc-glycine 10 (6.09 g,
−
1
peaks are reported as the IR data given in cm . Optical
rotations were measured with a Rudolph Research Analytical
AUTOPOL® IV. HRMS (ESI-TOF) were recorded on a Bruker
micrOTOF II.
1
1.7 mmol, 73%) as a light yellow solid.
1
H NMR (400 MHz, CDCl ): δ 7.70 (d, J = 8.0 Hz, 2H), 7.68
3
(
s, 1H), 7.55 (d, J = 7.2 Hz, 2H), 7.43 (s, 1H), 7.32 (t, J = 7.4 Hz,
2
2
H), 7.25 (t, J = 7.6 Hz, 2H), 5.96 (br, 1H), 4.31 (d, J = 6.8 Hz,
H), 4.17–4.09 (m, 5H), 3.10 (t, J = 8.0 Hz, 2H); C NMR
All reactions were monitored by thin-layer chromatography
carried out on 0.25 mm E. Merck silica gel plates (60F-254)
with UV light, and visualized by 10% ethanolic phosphomolyb-
dic acid or 0.5% ninhydrin n-butanol solution. Flash column
chromatography was performed on silica gel PSQ 60B pur-
chased from Fuji Silysia Chemical Ltd. DIEA was distilled from
ninhydrin and KOH.
13
(
100 MHz, CDCl ): δ 167.1, 156.3, 143.6, 141.0, 140.5, 138.4,
3
1
4
2
33.1, 131.6, 127.6, 127.0, 125.1, 125.0, 119.8, 116.7, 67.1, 48.6,
6.9, 44.0, 28.7; IR (KBr): 3422, 3328, 3068, 3016, 2948, 2894,
−1
360, 1716, 1685, 1539, 1459, 1218, 742 cm ; mp 120–122 °C;
+
HRMS (ESI-TOF): calcd for [C25
found 544.0487.
H
20BrN
3
O
5
+ Na] 544.0479,
Synthesis of 5-bromo-7-nitroindoline (Bni) (9)
Removal of the Fmoc group
To a solution of indoline (8) (5.00 mL, 44.5 mmol) in acetic
acid (66.8 mL), acetyl chloride (18.7 mL) was added at room
temperature. After being stirred at 90 °C for 1.5 h, the reaction
mixture was cooled and concentrated in vacuo. The residue was
used for the next reaction without further purification.
To a solution of Bni-protected N-Fmoc-glycine 10 (731 mg,
.40 mmol) in CH Cl (2.8 mL), diethylamine (2.8 mL) was
added at room temperature. After being stirred at the same
temperature for 1 h, the reaction mixture was concentrated
in vacuo. The residue was purified by short path column
1
2
2
To a solution of crude N-acetyl indoline (3.22 g, 20.0 mmol,
2 2
chromatography on silica gel (10% MeOH in CH Cl ) and used
for the next reaction without further purification.
1
.00 equiv.) in CH
2 2
Cl (40.0 mL), N-bromosuccinimide (3.92 g,
2
2.0 mmol, 1.10 equiv.) was added at room temperature. After
being stirred at the same temperature for 30 min, the reaction
mixture was diluted with CH Cl , washed with water, saturated
aqueous NaHCO and brine, dried over MgSO , filtered, and
concentrated in vacuo. The residue was used for the next reac-
tion without further purification.
General procedure for removal of the Boc group
2
2
2 2
To a crude peptide, TFA/CH Cl (1/3, 6.0 mL) or 4 M HCl 1,4-
3
4
dioxane (2.0–2.5 mL) was added at room temperature. After
being stirred at the same temperature for 40–60 min, the reac-
tion mixture was concentrated in vacuo. The residue was used
for the next reaction without further purification.
To a solution of crude aryl bromide (4.64 g, 19.3 mmol,
1
2
.00 equiv.) in TFA (39.0 mL), sodium nitrate (1.80 g,
1.2 mmol, 1.10 equiv.) was added at 0 °C. After being stirred
General procedure for micro-flow amide bond formation
at room temperature for 30 min, the reaction mixture was
poured into ice cold water. The precipitate was filtered, A solution of carboxylic acid (0.35 M) and DIEA (0.42 M) in
−
1
washed with water, dried under vacuum, and used for the next DMF (flow rate: 2.0 mL min ) and a solution of triphosgene
(0.093 M) in MeCN (flow rate: 1.2 mL min− ) were introduced
1
reaction without further purification.
To a solution of crude amide (5.50 g, 19.3 mmol, 1.00 into a T-shape mixer 1 (inner diameter: 0.25 mm) at 20 °C
equiv.) in MeOH (19.0 mL) and THF (97.0 mL), sodium hydro- with the syringe pumps. The resulting mixture was passed
xide (1.30 g, 32.5 mmol, 1.68 equiv.) was added at 0 °C. After through a reaction tube 1 (inner diameter: 0.8 mm, length:
being stirred at room temperature for 30 min, one third of 54 mm, volume: 27 μL, reaction time: 0.5 s) at the same temp-
THF was removed in vacuo. The resultant mixture was poured erature. Then, the resulting mixture and a solution of amine
into ice cold water. The precipitate was filtered, washed with (<0.14 M) and DIEA that was added to adjust the pH to 8–9 in
water, and dried under vacuum to give 5-bromo-7-nitroindo- DMF (flow rate: 2.0 mL min− ) were introduced to a T-shape
1
line (9) (3.89 g, 16.0 mmol, 4 steps 80%) as an orange solid.
mixer 2 (inner diameter: 0.25 mm) at 20 °C. The resulting
1
H NMR (400 MHz, CDCl
brs, 1H), 3.90 (t, J = 8.4 Hz, 2H), 3.18 (t, J = 8.4 Hz, 2H);
3
): δ 7.89 (s, 1H), 7.24 (s, 1 H), 6.79 mixture was passed through a reaction tube 2 (inner diameter:
1
3
(
C
0.8 mm, length: 742 mm, volume: 373 μL, reaction time: 4.3 s)
): δ 148.0, 136.0, 132.5, 128.9, 124.2, at the same temperature. The resulting mixture was poured
Cl suspension at room temperature.
The aqueous layer was extracted twice with CH Cl . The com-
NMR (100 MHz, CDCl
3
1
1
07.0, 46.9, 27.9; IR (KBr): 3369, 2352, 1621, 1581, 1507, 1482, into a 0.5 M HCl and CH
392, 1317, 1284, 1230, 1157, 965, 861, 763 cm−1
2
2
.
2
2
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1
3
bined organic layer was washed with 1 M HCl, twice with satu- 1H), 1.59–1.20 (m, 7H), 1.08–0.92 (m, 2H); C NMR (125 MHz,
rated aqueous NaHCO and with brine, dried over Na SO , fil- DMSO): δ 172.0, 171.2, 170.5, 169.9, 169.7, 169.6, 159.2, 155.8,
3
2
4
tered and concentrated in vacuo. The residue was used for the 137.2, 136.1, 134.6, 132.3, 129.8, 129.7, 129.3, 129.1, 128.4,
next reaction without further purification.
128.1, 128.0, 127.8, 127.3, 126.3, 65.6, 62.6, 54.6, 54.4, 51.9,
8.8, 43.3, 40.2, 40.2, 37.4, 35.2, 30.9, 28.7, 28.7, 24.8, 22.7;
4
Pentapeptide 22
−1
IR (KBr): 3284, 3071, 2937, 2364, 2337, 1643, 1542, 1261 cm
;
2
8
Purification conditions: column chromatography on silica gel [α] = −38.1 (c 0.156, CHCl
3
/CH
3
OH = 1/1); mp 206–209 °C;
D
+
(
5% MeOH in CH Cl ), then recrystallization from EtOH/ HRMS (ESI-TOF): calcd for [C H ClN O + Na] 929.3320,
2
2
42 51
10 11
2 2 2
CH Cl /Et O/hexane, 120 mg, 0.0960 mmol, 8 steps 7% from found 929.3318.
Bni-protected N-Fmoc-glycine 10
1
Yellow solid; H NMR (500 MHz, CDCl /CD OD = 1/1): Synthesis of a cyclic RGD peptide (25)
3
3
δ 8.01 (brd, J = 5.0 Hz, 1H), 7.93 (brd, J = 5.0 Hz, 1H), 7.85 (br,
To
a
solution of the cyclic pentapeptide 24 (3.0 mg,
2
7
4
H), 7.74 (s, 1H), 7.61 (s, 1H), 7.42–7.17 (m, 14H), 6.65 (brd, J =
.0 Hz, 1H), 6.55 (br, 1H), 5.17 (s, 2H), 5.10–5.05 (m, 2H),
.55–4.46 (m, 2H), 4.41 (br, 1H), 4.34–4.08 (m, 5H), 3.30–3.16
0
(
.0033 mmol, 1.0 equiv.) in MeOH (0.4 mL) and acetic acid
0.4 mL), 10% Pd/C (8.8 mg, 0.0083 mmol, 2.5 equiv.) was
added at room temperature under argon. After being stirred at
the same temperature for 18 h under H , the reaction mixture
(
(
m, 4H), 3.14–3.04 (m, 3H), 2.99 (dd, J = 8.0, 12.5 Hz, 1H), 2.82
dd, J = 6.5, 17.5 Hz, 1H), 2.77 (dd, J = 6.5, 17.5 Hz, 1H),
2
was filtered through a pad of Celite and concentrated in vacuo
to give a cyclic RGD peptide (25) (4.2 mg, quant.) as a colorless
solid.
1
.99–1.90 (m, 1H), 1.83–1.72 (m, 2H), 1.71–1.52 (m, 3H),
1
3
1
.48–1.35 (m, 11H), 1.18–1.08 (m, 2H); C NMR (125 MHz,
CDCl /CD OD = 1/1): δ 172.5, 172.2, 172.1, 170.6, 167.1, 158.8,
1
3
3
H NMR (500 MHz, D O): δ 7.46–7.22 (m, 5H), 4.71 (dd, J =
2
1
1
1
5
2
1
56.6, 155.7, 155.7, 140.2, 138.8, 135.7, 134.9, 133.8, 132.8,
32.4, 131.4, 128.7, 128.6, 128.5, 128.5, 128.0, 127.8, 127.6,
27.4, 126.4, 126.3, 124.5, 116.2, 79.7, 66.1, 63.0, 54.9, 53.6,
2.3, 50.2, 48.6, 41.8, 39.9, 39.8, 36.5, 35.5, 30.0, 28.4, 28.2,
7.8, 27.3, 24.0, 22.2; IR (KBr): 3286, 3087, 2932, 2362, 2343,
7
7
.0, 7.0 Hz, 1H), 4.57 (dd, J = 5.0, 10.0 Hz, 1H), 4.40 (dd, J =
.0, 7.0 Hz, 1H), 4.20 (d, J = 15.0 Hz, 1H), 3.88 (br, 1H), 3.48 (d,
J = 14.5 Hz, 1H), 3.19 (br, 2H), 3.12 (dd, J = 5.5, 13.0 Hz, 1H),
.95 (dd, J = 12.0, 12.0 Hz, 1H), 2.87 (t, J = 8.0 Hz, 2H), 2.70
dd, J = 7.0, 15.5 Hz, 1H), 2.56 (dd, J = 5.5, 14.5 Hz, 1H),
1.92–1.82 (m, 1H), 1.74–1.60 (m, 2H), 1.58–1.39 (m, 5H),
2
(
−
1
28
699, 1636, 1538, 1459, 1257, 1165 cm ; [α]D = +6.73 (c 1.12,
); mp 154–157 °C; HRMS (ESI-TOF): calcd for
CHCl
3
13
0
1
5
2
6
2
.96–0.78 (m, 2H); C NMR (125 MHz, D O): δ 178.3, 173.2,
+
55
[C H66BrClN12O15 + Na] 1271.3535, found 1271.3548.
72.5, 171.5, 171.5, 170.3, 155.7, 135.0, 128.3, 127.9, 126.2,
4.6, 53.9, 51.4, 49.7, 42.8, 39.5, 38.1, 36.6, 35.7, 28.7, 26.5,
Intramolecular cyclization
+
5.0, 23.4, 21.2; HRMS (ESI-TOF): calcd for [C27
04.3202, found 604.3202.
41 9 7
H N O + H]
To pentapeptide 22 (30.3 mg, 0.0242 mmol), 4 M HCl
1
,4-dioxane (1.5 mL) was added at room temperature. After
being stirred at the same temperature for 40 min, the reaction
mixture was concentrated in vacuo. The residue was dissolved
in CH Cl , and the organic layer was washed with saturated
Acknowledgements
2
2
aqueous NaHCO
concentrated in vacuo. The residue was used for the next reac- Research Fellow, a Grant-in-Aid for Young Scientists (B),
tion without further purification.
Scientific Research on Innovative Areas 2707 Middle molecular
3 2 4
and brine, dried over Na SO , filtered and This work was partially supported by a Grant-in-Aid for JSPS
A solution of crude amine 23 in MeCN (24.0 mL) was intro- strategy form MEXT (no. 15H05849), and a Naito Foundation
duced into the tube reactor (FEP tube, inner diameter: Natural Science Scholarship.
1
.0 mm, length: 3821 mm, volume: 3.00 mL) at room tempera-
−
1
ture with a syringe pump (flow rate: 600 μL min ) and irra-
diated with a 9 W UV lamp (wavelength: 365 nm) for 5 min.
The resultant mixture was collected over 40 min and con-
centrated in vacuo. The residue was recrystallized from MeOH
to give the cyclic pentapeptide 24 (7.8 mg, 0.00860 mmol,
Notes and references
1 (a) V. J. Hruby, Nat. Rev. Drug Discovery, 2002, 1, 847;
(b) Y. Hamada and T. Shioiri, Chem. Rev., 2005, 105, 4441;
(c) M. Katsara, T. Tselios, S. Deraos, G. Deraos,
M.-T. Matsoukas, E. Lazoura, J. Matsoukas and
V. Apostolopoulos, Curr. Med. Chem., 2006, 13, 2221;
(d) D. J. Craik, D. P. Fairlie, S. Liras and D. Price, Chem.
Biol. Drug Des., 2013, 81, 136; (e) P. Thapa, M. J. Espiritu,
C. Cabalteja and J.-P. Bingham, Int. J. Pept. Res. Ther., 2014,
20, 545; (f) A. Tapeinou, M.-T. Matsoukas, C. Simal and
T. Tselios, Biopolymers, 2015, 104, 453.
2
steps 36%) as a colorless solid.
H NMR (500 MHz, DMSO): δ 8.54 (brs, 1 H), 8.39 (br, 1H),
.12 (d, J = 8.0 Hz, 1H), 8.08 (d, J = 7.0 Hz, 1H), 8.02 (d, J = 7.0
1
8
Hz, 1H), 7.54 (d, J = 7.5 Hz, 1H), 7.50–7.46 (m, 2H), 7.43–7.27
(
(
m, 8H), 7.24 (t, J = 7.0 Hz, 2H), 7.17 (d, J = 7.0 Hz, 1H), 7.13
d, J = 7.0 Hz, 2H), 5.09 (s, 2H), 5.06 (s, 2H), 4.70 (ddd, J = 8.0,
8
.0, 7.0 Hz, 1H), 4.45 (ddd, J = 7.0, 7.0, 7.0 Hz, 1H), 4.17 (ddd,
J = 7.0, 7.0, 7.5 Hz, 1H), 4.03 (dd, J = 7.5, 14.5 Hz, 1H),
3
2
.94–3.87 (m, 1H), 3.23 (dd, J = 3.5, 14.5 Hz, 1H), 3.14 (br, 2H),
.97–2.75 (m, 5H), 2.54 (dd, J = 5.5, 16.0 Hz, 1H), 1.76–1.65 (m,
2 (a) E. M. Driggers, S. P. Hale, J. Lee and N. K. Terrett, Nat.
Rev. Drug Discovery, 2008, 7, 608; (b) E. Marsault and
This journal is © The Royal Society of Chemistry 2016
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Paper
Organic & Biomolecular Chemistry
M. L. Peterson, J. Med. Chem., 2011, 54, 1961;
c) J. Mallinson and I. Collins, Future Med. Chem., 2012, 4,
409.
Simultaneous removal of alloc and allyl protecting groups
from the cyclization precursor using Pd (0) and 1,3-di-
methylbarbituric acid (DMBA) was performed, however,
DMBA could not be removed by column chromatographic
separations. The remaining DMBA inhibited macrolactami-
zation. See: S. Fuse, H. Koinuma, A. Kimbara,
T. Fujimoto and N. Izumiya, Chem. Lett., 1992, 191;
(c) L. S. Richter, J. Y. K. Tom and J. P. Burnier, Tetrahedron
Lett., 1994, 35, 5547; (d) L. Yang and G. Morriello,
Tetrahedron Lett., 1999, 40, 8197; (e) C. Rosenbaum and
H. Waldmann, Tetrahedron Lett., 2001, 42, 5677;
(f) G. T. Bourne, S. W. Golding, R. P. McGeary,
W. D. F. Meutermans, A. Jones, G. R. Marshall,
P. F. Alewood and M. L. Smythe, J. Org. Chem., 2001, 66,
7706.
(
1
3
M. Izumikawa, Y. Mifune, H. He, K. Shin-ya, T. Takahashi 15 K. C. Nicolaou, B. S. Safina and N. Winssinger, Synlett,
and T. Doi, J. Am. Chem. Soc., 2014, 136, 12011. 2001, 900.
S. Jiang, Z. Li, K. Ding and P. P. Roller, Curr. Org. Chem., 16 The 7-nitroindoline derivatives were used as the photoacti-
4
5
2
008, 12, 1502.
(a) S.-Y. Han and Y.-A. Kim, Tetrahedron, 2004, 60, 2447;
b) C. A. G. N. Montalbetti and V. Falque, Tetrahedron,
005, 61, 10827; (c) E. Valeur and M. Bradley, Chem. Soc.
vatable carboxyl protecting groups in peptide synthesis.
See: (a) Sh. Pass, B. Amit and A. Patchornik, J. Am. Chem.
Soc., 1981, 103, 7674; (b) C. M. Kaneshiro and K. Michael,
Angew. Chem., Int. Ed., 2006, 45, 1077; (c) J.-L. Débieux and
C. G. Bochet, J. Phys. Org. Chem., 2010, 23, 272;
(d) J.-L. Débieux and C. G. Bochet, Chem. Sci., 2012, 3, 405;
(e) C. G. Bochet, Chimia, 2013, 67, 896; (f) B. A. F. Le Bailly,
L. Byrne and J. Clayden, Angew. Chem., Int. Ed., 2016, 55,
2132.
(
2
Rev., 2009, 38, 606; (d) A. El-Faham and F. Albericio, Chem.
Rev., 2011, 111, 6557.
6
(a) D. J. C. Constable, P. J. Dunn, J. D. Hayler,
G. R. Humphrey, J. L. Leazer, Jr., R. J. Linderman,
K. Lorenz, J. Manley, B. A. Pearlman, A. Wells, A. Zaks and
T.
b) V. R. Pattabiraman and J. W. Bode, Nature, 2011, 480,
71.
J. S. Davies, J. Pept. Sci., 2003, 9, 471.
(a) L. Zhang and J. P. Tam, J. Am. Chem. Soc., 1997, 119,
Y.
Zhang,
Green
Chem.,
2007,
9,
411; 17 (a) S. Fuse, N. Tanabe, M. Yoshida, H. Yoshida, T. Doi and
(
4
T. Takahashi, Chem. Commun., 2010, 46, 8722; (b) S. Fuse,
Y. Mifune, N. Tanabe and T. Takahashi, Org. Biomol.
Chem., 2012, 10, 5205; (c) Y. Mifune, S. Fuse and
H. Tanaka, J. Flow Chem., 2014, 4, 173; (d) S. Fuse, Y. Otake,
Y. Mifune and H. Tanaka, Aust. J. Chem., 2015, 68, 1657.
18 For recent reviews on micro-flow photochemical synthesis,
see: (a) M. Oelgemöller and O. Shvydkiv, Molecules, 2011,
16, 7522; (b) J. P. Knowles, L. D. Elliott and K. I. Booker-
Milburn, Beilstein J. Org. Chem., 2012, 8, 2025; (c) Y. Su,
N. J. W. Straathof, V. Hessel and T. Noël, Chem. – Eur. J.,
2014, 20, 10562.
7
8
2
1
363; (b) J. A. Camarero and T. W. Muir, Chem. Commun.,
997, 1369.
9
After NCL-based macrolactamization, the cysteine residue
was converted to an alanine residue via desulfurization.
See: L. Z. Yan and P. E. Dawson, J. Am. Chem. Soc., 2001,
1
23, 526.
1
1
0 For serine and threonine ligation, see: C. T. T. Wong,
H. Y. Lam, T. Song, G. Chen and X. Li, Angew. Chem., Int. 19 (a) S. Fuse, N. Tanabe and T. Takahashi, Chem. Commun.,
Ed., 2013, 52, 10212.
2011, 47, 12661; (b) S. Fuse, Y. Mifune and T. Takahashi,
Angew. Chem., Int. Ed., 2014, 53, 851; (c) S. Fuse, Y. Mifune,
N. Tanabe and T. Takahashi, Synlett, 2014, 2087.
1 The use of acyl transfer auxiliary in NCL-based macrolacta-
mization enabled the cyclization of peptides containing
various amino acids on their N-terminus, however pre- 20 (a) M. Aumailley, M. Gurrath, G. Müller, J. Calvete,
modification of peptides is required. See: J. Offer,
C. N. C. Boddy and P. E. Dawson, J. Am. Chem. Soc., 2002,
R. Timpl and H. Kessler, FEBS Lett., 1991, 291, 50;
(b) M. Gurrath, G. Müller, H. Kessler, M. Aumailley and
R. Timpl, Eur. J. Biochem., 1992, 210, 911; (c) R. Haubner,
R. Gratias, B. Diefenbach, S. L. Goodman, A. Jonczyk and
H. Kessler, J. Am. Chem. Soc., 1996, 118, 7461;
(d) R. Haubner, D. Finsinger and H. Kessler, Angew. Chem.,
Int. Ed. Engl., 1997, 36, 1374; (e) X. Dai, Z. Su and J. O. Liu,
Tetrahedron Lett., 2000, 41, 6295; (f) M. Schottelius,
B. Laufer, H. Kessler and H.-J. Wester, Acc. Chem. Res.,
2009, 42, 969; (g) S. Liu, Bioconjugate Chem., 2009, 20, 2199;
(h) J. Paleček, G. Dräger and A. Kirschning, Synthesis, 2011,
653; (i) C. Mas-Moruno, R. Fraioli, F. Rechenmacher,
S. Neubauer, T. G. Kapp and H. Kessler, Angew. Chem., Int.
Ed., 2016, 55, 7048.
1
24, 4642.
1
1
2 NCL-based macrolactamization of peptides containing
selenocysteine at their N-terminus was reported.
Selenocysteine could be converted into cysteine or alanine.
See: R. Quaderer and D. Hilvert, Chem. Commun., 2002,
2
620.
3 Decarboxylative macrolactamization between α-ketoacids
and hydroxylamines without external reagents was
reported. However, premodification of amino acids is
necessary. See: (a) T. Fukuzumi, L. Ju and J. W. Bode,
Org. Biomol. Chem., 2012, 10, 5837; (b) F. Rohrbacher,
G. Deniau, A. Luther and J. W. Bode, Chem. Sci., 2015, 6,
4
889.
4 (a) G. Ösapay, A. Profit and J. W. Taylor, Tetrahedron Lett.,
990, 31, 6121; (b) M. Xu, N. Nishino, H. Mihara,
21 Y. Hiroshi, O. Takashi, O. Tatsuo, Y. Kentaro, S. Naoko,
N. Takeshi, O. Yoichi, K. Nozomu and K. Kyosuke, US Pat,
US5721246(A), 1998.
1
1
Org. Biomol. Chem.
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