Synthesis of cyclic peptides through ring-closing metathesis of photolabile protecting groups

Article abstract of DOI:10.1055/s-0034-1378908

The new photolabile protecting groups 4-allyl-5-methoxy-3-nitrobenzyl (AMNB) and 4-allyl-5-methoxy-3-nitrobenzyloxycarbonyl (AMNBOC) with an allylic side chain can easily be obtained from vanillin. These protecting groups can be introduced on the C- as well as on the N-terminus of peptides, and subsequent ring-closing metatheses provides a straightforward protocol for the synthesis of cyclic peptides. The protecting groups can be removed under UVA irradiation (365 nm).

Full text of DOI:10.1055/s-0034-1378908

SYNTHESIS
0
0
3
9
-
7
8
8
1
1
4
3
7
-
2
1
0
X
© Georg Thieme Verlag Stuttgart · New York
2015, 47, 411–420
paper
411
J. Hoffmann, U. Kazmaier
Paper
Synthesis
Synthesis of Cyclic Peptides through Ring-Closing Metathesis of
Photolabile Protecting Groups
Judith Hoffmann
Uli Kazmaier*
i-Bu
O
i-Bu
O
O
O
N
N
H
H
i-Pr
HN
N
O
HN
i-Pr
N
O
Universitaet des Saarlandes, Institut für Organische Chemie,
66123 Saarbruecken, Germany
u.kazmaier@mx.uni-saarland.de
O
O
+
4
9
6
(
8
1
3
)
0
2
2
4
0
9
Grubbs II
O
O
O2N
NO2
O
O
CH2Cl2
40 °C, 17 h
65%
MeO
NO2
O
MeO
NO2
O
O
O
OMe
OMe
Received: 20.08.2014
Accepted after revision: 07.10.2014
Published online: 12.11.2014
tion of kinases11 and ribonucleases.12 In addition to the bio-
logical activity of proteins, their conformation can be
influenced. Therefore, the caging technique can be used to
investigate conformational changes in peptides and pro-
teins.13
A wide range of photocleavable protecting groups can
be used for this purpose,1a,2 and (substituted) o-nitrobenzyl
groups (NB) (Figure 1) play a dominant role, although the
nitrosobenzaldehyde formed in the cleaving step can be
harmful in a biological context.14 The introduction of two
methoxy groups (DMNB) results in lower quantum yield,
but increases the absorbance at wavelengths longer than
320 nm, allowing cleavage at approximately 365 nm, which
is ideal for biological application.15
DOI: 10.1055/s-0034-1378908; Art ID: ss-2014-t0519-op
Abstract The new photolabile protecting groups 4-allyl-5-methoxy-3-
nitrobenzyl (AMNB) and 4-allyl-5-methoxy-3-nitrobenzyloxycarbonyl
(AMNBOC) with an allylic side chain can easily be obtained from vanillin.
These protecting groups can be introduced on the C- as well as on the
N-terminus of peptides, and subsequent ring-closing metatheses pro-
vides a straightforward protocol for the synthesis of cyclic peptides. The
protecting groups can be removed under UVA irradiation (365 nm).
Key words amino acids, metathesis, peptides, photochemistry, pho-
tocleavage, protecting groups
Photoactive molecules find widespread application in
molecular imaging and cell biology.1 In the life sciences,
‘caged compounds’ have become very popular in the last
O
R
R
AllO
AllO
O
years. According to the caging concept, bioactive com-
pounds are deactivated by introduction of a photolabile
protecting group.2 Upon irradiation, the protecting group is
cleaved off, releasing the bioactive molecule. By using flash
photolysis, the cleavage can be very fast, allowing spatially
well-defined and rapid changes in the concentration of the
bioactive compounds.3 This approach has been applied to a
range of neurotransmitters, including glutamate,4 dopa-
mine,5 serotonine,6 as well as to hormones and analoga.7
One of the first and most popular compounds was caged
ATP, e.g., for the generation of ATP in situ through flash
photolysis.8 Photoinduced Ca2+ release is widely used in
physiology to study Ca-dependent processes.9 In these ex-
amples, the photoactive group is coupled to a rather small
molecule, changing its properties significantly. But this
technique is also suitable for the modification of large mol-
ecules such as proteins and antibodies.1a,10 The incorpora-
tion of a photoactive group at functionalized side chains is
rather simple and has been used, for example, for modifica-
NO2
MeO
NO2
MeO
NO2
NB R = H
DMNB R = OMe
AMNB
AMNBOC
Figure 1 Photolabile protecting groups
Herein, we describe the synthesis and application of a
new derivative of DMNB,16 with one of the methoxy groups
being replaced by an allyl ether (AMNB, Figure 1). The dou-
ble bond would allow further modification such as ring-
closing metathesis (RCM)17 with other unsaturated posi-
tions of the photolabile protected target molecule.18 RCM
has found widespread application in peptide cyclizations
during the last years.19 The required double bonds can easi-
ly be introduced not only by esterification using unsaturat-
ed alcohols, but also through Claisen rearrangement,20 and
nitrogen21 and peptide backbone allylations.22 Based on this
concept, we envisioned that drug molecules could be con-
verted into inactive forms through RCM by changing their
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2015, 47, 411–420

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J. Hoffmann, U. Kazmaier
Paper
Synthesis
conformation. Upon irradiation, the protecting group
should be cleaved, opening the ring and releasing the bio-
logically active compound.
To this end, we focused on the 4-allyl-5-methoxy-3-ni-
trobenzyl protecting group (AMNB), which is easily accessi-
ble from vanillin (Scheme 1).16
4-(N,N-dimethylamino)pyridine (DMAP) and diisopropyl-
ethylamine (DIPEA; Table 1). Although generally high yields
were obtained and the protected amino acids and dipep-
tides gave the expected AMNB ester in enantiomerically
pure form, complete epimerization of the C-terminal amino
acid was observed in case of some tripeptides (entries 7–
10).
CHO
CHO
Table 1 C-Terminal Protection of Amino Acids (AA) and Peptides with
AMNB-OH (2)
Br
, K2CO3
EtOH, 78 °C, 16 h
OMe
OMe
100%
TBTU (1.7 equiv)
DMAP (0.5 equiv)
O
OH
Boc AA/Peptide
OH + 2
Boc AA/Peptide OAMNB
i-Pr2NEt (3 equiv)
CH2Cl2, r.t., 16 h
CHO
5
4
CF3COOH
KNO3 (1.0 equiv)
O2N
NaBH4
Entry
Substrate
AA/Peptide
Product
Yield (%)
r.t., 16 h
77%
EtOH, r.t., 4 h
92%
OMe
1
2
4a
4b
4c
4d
4e
4f
(S)-Phe
5a
5b
5c
5d
5e
5f
89
94
91
90
90
85
76a
75
93a
86a
O
(S)-Leu
1
OH
O
O
Cl
3
(R)-Leu
O2N
O2N
4
(S)-Val
COCl2 (10 equiv)
5
(R)-Val
THF, r.t., 2 h
100%
OMe
OMe
6
(S)-Pro-(S)-Leu
(S)-Pro-(S)-Leu-(S)-Val
(S)-Val-(S)-Pro-(S)-Leu
(S)-Ile-(S)-Leu-(S)-Pro
(S)-Ala-(S)-Ala-(S)-Ala
O
O
7
4g
4h
4i
5g
5h
5i
2
3
8
AMNB-OH
AMNBOC-Cl
9
Scheme 1 Synthesis of the AMNB and AMNBOC protecting group
10
4k
5k
Starting from vanillin, the O-allylated product23 was ob-
tained in quantitative yield. The next step, the introduction
of the nitro group, was found to be the critical step. Best re-
sults are obtained with KNO3 in trifluoroacetic acid. When
exactly one equivalent of KNO3 was used, the expected
product 1 was formed in good yield and purity. Reduction
of the aldehyde functionality gave rise to the required sub-
stituted o-nitrobenzylalcohol 2 (AMNB-OH) in excellent
yield. When a slight excess of KNO3 was used in the nitra-
tion step, the yield of 1 was comparable, but the 4,5-dini-
trocatechol derivative was formed as side product in 15–
20% yield, probably through ipso-substitution. This side
product could not be separated from 1, but could easily be
removed after the reduction step.
AMNB-OH can be used directly as carboxyl protecting
group. The analogous carbonate protecting group (AMN-
BOC) was available from 2 by stirring for two hours with an
excess of phosgene. After evaporation of the excess of phos-
gene, chloroformate 3 (AMNBOC-Cl) was obtained in pure
form in quantitative yield. Although 3 is highly sensitive to-
wards moisture, it can be stored under refrigeration with-
out notable decomposition.
a Complete epimerization of the C-terminal amino acid.
The introduction of AMNBOC at the N-terminus was not
found to be problematic. Cleavage of the Boc protecting
group with trifluoroacetic acid gave the deprotected pep-
tides in almost quantitative yield and subsequent treatment
with AMNBOC-Cl (3) provided the double photolabile pro-
tected peptides 6 in acceptable to good yields (Table 2).
Table 2 Introduction of the N-Terminal AMNBOC Group
1) CF3COOH
CH2Cl2, r.t., 2 h
2) NaHCO3
Boc Peptide
AMNBOC Peptide
OAMNB
OAMNB
3) 3, DMAP, py
THF, r.t., t
6
5
Entry
5
Peptide
Time
(h)
6
Yield (%)
1
2
3
4
5
5g
5h
5i
(S)-Pro-(S)-Leu-(R/S)-Val
(S)-Val-(S)-Pro-(S)-Leu
(S)-Ile-(S)-Leu-(R/S)-Pro
72
72
90
72
72
6g
6h
6i
68
59
80
5g-1 (S)-Pro-(S)-Leu-(S)-Val
5g-2 (S)-Pro-(S)-Leu-(R)-Val
6g-1 63
6g-2 68
Introduction of AMNB at the C-terminus of protected
amino acids and peptides can be realized with a wide range
of esterification protocols. The best results so far were ob-
tained by using N,N,N′,N′-tetramethyl-O-(benzotriazol-1-
yl)uronium tetrafluoroborate (TBTU) in combination with
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2015, 47, 411–420

413
J. Hoffmann, U. Kazmaier
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Synthesis
With peptides 6 in hand, we next investigated the ring-
closing metathesis, for example, with peptide 6g. Although
the C-terminal amino acid was epimerized during synthe-
sis, we subjected 6g to cyclization using Grubbs II catalyst.24
We wanted to establish whether the cyclization proceeds at
all and whether one of the two diastereomers undergoes
cyclization preferentially. One might expect that the diaste-
reomeric peptides have different conformations in solution,
with one being more suitable for cyclization than the other.
To favor the RCM over the intermolecular process, a solu-
tion of 6g was added slowly to a highly diluted solution of
Grubbs II catalyst in dichloromethane, and the reaction
mixture was heated at reflux overnight to ensure complete
reaction. The expected cyclized product 7g was obtained in
good yield as an almost 1:1 diastereomeric mixture (Table
3, entry 1).
termine which spectrum belonged to which stereoisomer.
Therefore, we synthesized the pure diastereomers 6g-1and
6g-2 (Table 2, entries 4 and 5) from enantiopure valine de-
rivatives 5d and 5e via Boc-protected intermediates 5g-1/2
(Scheme 2).
1) CF3COOH
CH2Cl2, r.t., 30 min
2) NaHCO3
Boc-Val-OAMNB
3) Boc (S)-Pro-(S)-Leu-OH
TBTU, DMAP, i-Pr2NEt
5d (S)-Val
5e (R)-Val
CH2Cl2, r.t., 1.5 h
Boc-(S)-Pro-(S)-Leu-Val-OAMNB
5g-1 (S)-Val 45%
5g-2 (R)-Val 72%
Scheme 2 Synthesis of protected tripeptides 5g-1/2
Table 3 Cyclization of AMNBOC-Protected Tripeptides
Interestingly, the diastereomeric products behave dif-
ferently in RCM. The cyclization of the all-S peptide 6g-1
was rather slow, giving rise to cyclopeptide 7g-1, which
gave rise to the broad signal set in the 1HNMR spectrum. In
contrast, cyclization of diastereomer 6g-2 was very fast.
Complete consumption of starting material was observed
after 40 minutes, and the product 7g-2 was isolated in com-
parable yield (Table 3, entries 2 and 3). This cyclopeptide
turned out to be the stereoisomer producing the very dis-
tinct NMR spectrum, clearly indicating that the conforma-
tion of the linear peptides plays a pivotal role in the
cyclization step.
To investigate and verify the photolytic properties of
our compounds, we first recorded the UV absorption spec-
trum of 5a in acetonitrile. Subsequently, the solution was
irradiated with a UV-LED lamp (MR365PL) at 365 nm and
the UV/Vis spectra were recorded after 0.5, 1, 2, 4, and 8
hours (Figure 2). The gradual decrease of the intense band
(λmax = 350 nm) indicates the typical cleavage reaction of
DMNB derivatives.
N
N
Ar
Ar
O
Cl
Cl
Ru
i-Bu
N
Ph
HN
PCy3
(10 mol%)
CH2Cl2, 40 °C
MeO
O
O
O
O
HN
NO2
NO2
i-Pr
O
O
O
O
HN
MeO
i-Bu
N
6
MeO
O
O
O
HN
i-Pr
NO2
NO2
O
O
O
O
MeO
7
Entry
Substrate
Val
R/S
Time (h)
Product
Yield (%)
1
2
3
6g
17
7g
65
49
54
6g-1
6g-2
S
15
7g-1
7g-2
R
40 min
Whereas the 1H NMR spectra of linear peptides are gen-
erally difficult to evaluate because of the diastereomeric
mixture and the formation of rotamers, the cyclized prod-
uct 7g gave two different sets of signals. One diastereomer
gave very sharp signals, and the second was characterized
by very broad peaks. Clearly, one diastereomer exists in a
preferred conformation (resulting in sharp signals), where-
as the second appears to be a mixture of several conformers
(rotamers). Unfortunately, as a result of the epimerization
in the protection of the C-terminus, we were not able to de-
Figure 2 UV spectra of 5a in MeCN upon irradiation (10.2 mM) after
0.5, 1, 2, 4, and 8 h and before irradiation (black)
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2015, 47, 411–420

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J. Hoffmann, U. Kazmaier
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A further method to prove the formation of photolysis
product was the isolation of the generated N-Boc-protected
amino acid 4 as the corresponding methyl ester by treat-
ment of the reaction mixture with diazomethane. Purifica-
tion showed that this approach was successful, but we also
wanted to follow the formation of product during irradia-
tion. For this purpose, we monitored the progress of reac-
tion quantitatively by HPLC/MS analysis. The approach was
accomplished by the use of an external calibration of start-
ing material (5a, 5f and 5i) and the corresponding free acids
(4a, 4f and 4i) (for details see the Supporting Information).
The decreasing amount of starting materials 5, in addition
to the formation of products 4 (40–56%), demonstrated the
photolytic properties of our new photolabile protecting
groups, and showed the promise of the concept (Figure 3).
The best results were obtained with a UV-LED lamp
(365 nm); the use of a mercury-vapor lamp (even with dif-
ferent filters) caused several photochemical side reac-
tions,2b resulting in only moderate yields (22–46%).
Photolysis experiments of the cyclic compound 7g-2
showed deprotection of both C-terminal and N-terminal
protecting groups, which was indicated by the observation
of a mass peak with m/z 327.30 (calcd 327.22). Unfortu-
nately, the high polarity of the released tripeptide (formed
as a salt) prevented an exact quantification of the product
under the same conditions used for the photolysis of the C-
terminal protected peptides.
In conclusion, we have shown that new photolabile pro-
tecting groups (AMNB and AMNBOC) can easily be obtained
from vanillin. These protecting groups can be cleaved under
mild conditions using UVA light (365 nm), which are condi-
tions that are compatible with biological systems. The allyl-
ic side chain allows ring-closing metathesis of double-
protected peptides, giving rise to photocleavable cyclic pep-
tides.
Figure 3 Photolysis of AMNB-protected amino acid and peptides:
(a) cleavage of the AMNB-protecting group; (b) formation of N-Boc-
protected amino acid and peptides.
at the Saarland University. Photolysis experiments were performed
with a UV-LED lamp (MR365PL, MR Chemie Unna), 9 × 365 nm, ca.
50 W/m² in MeCN. UV-spectra were recorded with a V-650 spectrom-
eter (Jasco; d = 1.0 cm, solvent: MeCN). HPLC analysis was performed
with a Merck-Hitachi LaChrom system (7000-series, column: Chiral-
cel OD-H), HPLC/MS analysis with a Shimadzu system (LC: 10A-series
+ Autosampler, MS: LCMS-2020).
All air- or moisture-sensitive reactions were carried out in dried
glassware (>100 °C) under an atmosphere of nitrogen or argon. Dried
solvents were distilled before use: THF was distilled from LiAlH4, di-
chloromethane (CH2Cl2) was dried with CaH2 before distillation, and
DMF was purchased from Sigma–Aldrich. The products were purified
by flash chromatography on silica gel columns (Macherey-Nagel 60,
0.063–0.2 mm) and with a flash chromatography system [Reveleris
(Grace), RediSep-columns 4, 12, 24, and 40 g from Axel Semrau]. Mix-
tures of EtOAc and petroleum ether (PE) or CH2Cl2 and MeOH were
generally used as eluents. Analytical TLC was performed on pre-coat-
ed silica gel plates (Macherey-Nagel, Polygram SIL G/UV254). Visual-
ization was accomplished with UV-irradiation, or by staining with
KMnO4 solution or ninhydrin solution. Melting points were deter-
mined with a Dr. Tottoli (Büchi) melting point apparatus and are un-
corrected. 1H and 13C NMR spectra were recorded with a Bruker AV-
400 [400 MHz (1H) and 100 MHz (13C)] spectrometer in CDCl3. Chem-
ical shifts are reported in ppm relative to TMS; CHCl3 was used as the
internal standard. Diastereomeric ratios were determined by NMR
analysis. Mass spectra were recorded with a Finnigan MAT 95 spec-
trometer using the CI technique. Elemental analyses were performed
4-Allyloxy-5-methoxy-2-nitrobenzaldehyde (1)
To a solution of KNO3 (202 mg, 2.00 mmol) in trifluoroacetic acid
(TFA; 3.10 mL, 40.0 mmol), 4-allyloxy-3-methoxybenzaldehyde
(384 mg, 2.00 mmol) in TFA (3.1 mL, 40.0 mmol) was added dropwise
at 0 °C and the reaction mixture was stirred at r.t. for 20 h. The sol-
vent was evaporated in vacuo and the residue was dissolved in CH2Cl2
(10 mL) and washed with sat. aq NaCl (3 × 5 mL). Drying over Na2SO4
afforded the product as an orange solid. The crude product was puri-
fied by column chromatography (PE–EtOAc, gradient) to afford 1.
Yield: 363 mg (1.53 mmol, 77%); yellow powder; mp 133–134 °C; Rf =
0.45 (SiO2; PE–EtOAc, 1:1).
1H NMR (400 MHz, CDCl3): δ = 4.01 (s, 3 H), 4.75 (ddd, J = 5.5, 1.4,
1.4 Hz, 2 H), 5.43 (ddt, J = 10.5, 1.2, 1.2 Hz, 1 H), 5.54 (ddt, J = 17.3, 1.5,
1.4 Hz, 1 H), 6.06 (ddt, J = 17.3, 10.7, 5.5 Hz, 1 H), 7.41 (s, 1 H), 7.61 (s,
1 H), 10.43 (s, 1 H).
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J. Hoffmann, U. Kazmaier
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Synthesis
13C NMR (100 MHz, CDCl3): δ = 56.7, 70.4, 108.6, 109.9, 119.7, 125.6,
131.2, 143.7, 151.3, 153.5, 187.7.
Yield: 432 mg (0.89 mmol, 89%); pale-yellow solid; mp 104–105 °C;
[α]D20 +17.7 (c= 1.0, CHCl3); Rf = 0.58 (SiO2; PE–EtOAc, 1:1).
HRMS (CI): m/z [M]+ calcd for C11H11NO5: 237.0637; found: 237.0667.
1H NMR (400 MHz, CDCl3): δ = 1.39 (s, 9 H), 3.12 (m, 2 H), 3.97 (s,
3 H), 4.62–4.68 (m, 3 H), 4.95 (d, J = 8.1 Hz, 1 H), 5.36 (ddt, J = 10.5,
1.2, 1.2 Hz, 1 H), 5.44–5.63 (m, 3 H), 6.07 (ddt, J = 17.2, 10.7, 5.5 Hz,
1 H), 6.98 (s, 1 H), 7.13–7.15 (m, 2 H), 7.21–7.30 (m, 3 H), 7.73 (s, 1 H).
13C NMR (100 MHz, CDCl3): δ = 28.2, 38.2, 54.7, 56.6, 63.9, 70.1, 80.2,
109.8, 110.6, 119.2, 127.0, 127.2, 128.7, 129.2, 131.8, 135.7, 147.0,
154.6, 171.8; signals for AllylOCAr and t-BuOCO were too weak to be
detected.
MS (ESI/APCI): m/z [M + Na]+ calcd for C25H30N2O8Na: 509.19; found:
509.20.
HRMS (CI): m/z [M]+ calcd for C25H30N2O8: 486.2002; found:
486.1988.
4-Allyloxy-5-methoxy-2-nitrobenzyl Alcohol (AMNB-OH) (2)
To a suspension of 1 (5.21 g, 18.7 mmol) in EtOH (50 mL), NaBH4 (1.06
g, 28.0 mmol) was added portionwise at 0 °C, and the reaction mix-
ture was stirred at r.t. for 4 h. The reaction mixture was diluted with
Et2O (50 mL), hydrolyzed with sat. aq NH4Cl (30 mL) and extracted
with Et2O (30 mL). The combined organic layers were washed with
brine (20 mL) and dried over Na2SO4. The solvent was evaporated in
vacuo and the residue was purified by column chromatography (PE–
EtOAc, gradient) to afford 2.
Yield: 4.09 g (17.1 mmol, 92%); yellowish powder; mp 112–113 °C;
Rf = 0.25 (SiO2; PE–EtOAc, 1:1).
1H NMR (400 MHz, CDCl3): δ = 4.00 (s, 3 H), 4.67 (ddd, J = 5.5, 1.4,
1.4 Hz, 2 H), 4.95 (s, 2 H), 5.36 (ddt, J = 10.5, 1.3, 1.3 Hz, 1 H), 5.46
(ddt, J = 17.3, 1.4, 1.4 Hz, 1 H), 6.07 (ddt, J = 17.2, 10.5, 5.5 Hz, 1 H),
7.17 (s, 1 H), 7.71 (s, 1 H).
13C NMR (100 MHz, CDCl3): δ = 56.4, 62.9, 70.2, 110.0, 111.3, 119.1,
132.0, 132.3, 139.7, 146.8, 154.3.
HRMS (CI): m/z [M]+ calcd for C11H13NO5: 239.0794; found: 239.0780.
Anal. Calcd for C25H30N2O8 (486.52): C, 61.72; H, 6.22; N, 5.76. Found:
C, 61.37; H, 6.09; N, 5.96.
4-Allyloxy-5-methoxy-2-nitrobenzyl N-tert-Butoxycarbonyl-(S)-
leucinate (5b)
To a solution of Boc-(S)-leucine (249 mg, 1.00 mmol), 2 (239 mg, 1.00
mmol), DMAP (61.1 mg, 0.50 mmol), and DIPEA (349 μL, 2.00 mmol)
in anhydrous CH2Cl2 (2.0 mL), TBTU (482 mg, 1.50 mmol) was added
at 0 °C and the mixture was stirred at r.t. for 3 h. Washing with 1 M
HCl (2 mL), sat. aq NaHCO3 (2 mL), and brine (2 mL), drying over
Na2SO4, and evaporation of the solvent in vacuo afforded the crude
product as an orange oil. Purification by column chromatography
(PE–EtOAc, gradient) afforded 5b.
Anal. Calcd for C11H13NO5 (239.23): C, 55.23; H, 5.48; N, 5.86. Found:
C, 55.07; H, 5.19; N, 5.72.
4-Allyloxy-5-methoxy-2-nitrobenzyloxy Chloroformate (AMN-
BOC-Cl; 3)
Yield: 432 mg (0.94 mmol, 94%); pale-yellow oil; Rf = 0.62 (SiO2; PE–
EtOAc 1:1).
To a solution of phosgene (20% in toluene, 15.9 mL, 30.1 mmol), 2
(722 mg, 3.02 mmol) in THF (6.0 mL) was added dropwise and the re-
action mixture was stirred at r.t. overnight. The excess of phosgene
was purged out of the reaction mixture with a stream of nitrogen,
then the solvents were evaporated to give the product, which was
used without further purification.
1H NMR (400 MHz, CDCl3): δ = 0.96 (d, J = 6.4 Hz, 6 H), 1.41 (s, 9 H),
1.56–1.76 (m, 3 H), 4.02 (s, 3 H), 4.40 (dt, J = 8.8, 5.2 Hz, 1 H), 4.68
(ddd, J = 5.5, 1.4, 1.4 Hz, 2 H), 4.88 (d, J = 8.4 Hz, 1 H), 5.36 (ddt, J =
10.5, 1.3, 1.3 Hz, 1 H), 5.43–5.68 (m, 3 H), 6.06 (ddt, J = 17.2, 10.5,
5.5 Hz, 1 H), 7.07 (s, 1 H), 7.73 (s, 1 H).
13C NMR (100 MHz, CDCl3): δ = 21.8, 22.8, 24.8, 28.3, 41.4, 52.3, 56.6,
63.8, 70.1, 80.0, 109.8, 109.9, 119.2, 127.4, 131.9, 139.3, 146.9, 154.2,
155.5, 173.2.
Yield: 908 mg (3.01 mmol, 99%); pale-yellow solid; Rf = 0.55 (SiO2;
PE–EtOAc, 1:1).
1H NMR (400 MHz, CDCl3): δ = 4.00 (s, 3 H), 4.69 (ddd, J = 5.5, 1.4,
1.4 Hz, 2 H), 5.37 (ddt, J = 10.5, 1.4, 1.4 Hz, 1 H), 5.47 (ddt, J = 17.3, 1.4,
1.4 Hz, 1 H), 5.72 (s, 2 H), 6.06 (ddt, J = 17.3, 10.5, 5.5 Hz, 1 H), 7.01 (s,
1 H), 7.76 (s, 1 H).
MS (ESI/APCI): m/z [M + Na]+ calcd for C22H32N2O8Na: 475.21; found:
475.25.
HRMS (CI): m/z [M]+ calcd for C22H32N2O8: 452.2159; found:
13C NMR (100 MHz, CDCl3): δ = 56.5, 69.8, 70.2, 110.1, 110.7, 119.3,
452.2160.
124.3, 131.7, 139.7, 147.8, 150.3, 154.1.
HRMS (CI): m/z [M]+ calcd for C12H12ClNO6: 301.0353; found:
301.0332.
Anal. Calcd for C22H32N2O8 (452.52): C, 58.39; H, 7.13; N, 6.19. Found:
C, 58.45; H, 7.08; N, 6.19.
Anal. Calcd for C12H12ClNO6 (301.68): C, 47.78; H, 4.01; N, 4.64; found:
C, 48.28; H, 3.75; N, 4.67.
4-Allyloxy-5-methoxy-2-nitrobenzyl N-tert-Butoxycarbonyl-(S)-
valinate (5d)
To a solution of Boc-(S)-valine (326 mg, 1.50 mmol), 2 (359 mg, 1.50
mmol), DMAP (92.0 mg, 0.50 mmol) and DIPEA (786 μL, 4.50 mmol)
in anhydrous CH2Cl2 (3.0 mL), TBTU (819 mg, 2.55 mmol) was added
at 0 °C and the mixture was stirred at r.t. overnight. Washing with
1 M HCl (3 mL), sat. aq NaHCO3 (3 mL), and brine (3 mL), drying over
Na2SO4, and evaporation of the solvent in vacuo afforded the product
as an orange oil. Purification by column chromatography (PE–EtOAc,
gradient) afforded 5d.
4-Allyloxy-5-methoxy-2-nitrobenzyl N-tert-Butoxycarbonyl-(S)-
phenylalaninate (5a)
To a solution of 2 (239 mg, 1.00 mmol), Boc-(S)-phenylalanine (265
mg, 1.00 mmol), DMAP (61.1 mg, 0.50 mmol), and DIPEA (349 μL,
2.00 mmol) in anhydrous CH2Cl2 (2.0 mL), TBTU (482 mg, 1.50 mmol)
was added at 0 °C and the reaction mixture was stirred at r.t. over-
night. The reaction mixture was washed with 1 M HCl (2 mL), sat. aq
NaHCO3 (2 mL) and brine (2 mL). Drying over Na2SO4 and evaporation
of the solvent in vacuo afforded the crude product as an orange oil.
The residue was purified by column chromatography (PE–EtOAc, gra-
dient) to afford 5a.
Yield: 593 mg (1.35 mmol, 90%); yellow oil; Rf = 0.57 (SiO2; PE–EtOAc,
1:1); HPLC (OD-H; hexane–i-PrOH, 90:10; 1 mL/min): tR = 7.55 min.
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J. Hoffmann, U. Kazmaier
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1H NMR (400 MHz, CDCl3): δ = 0.94 (d, J = 6.8 Hz, 3 H), 1.00 (d, J =
6.8 Hz, 3 H), 1.42 (s, 9 H), 2.17 (m, 1 H), 4.00 (s, 3 H), 4.30 (dd, J = 8.8,
5.1 Hz, 1 H), 4.67 (ddd, J = 5.5, 1.4, 1.4 Hz, 2 H), 5.00 (d, J = 8.8 Hz,
1 H), 5.36 (ddt, J = 10.5, 1.3, 1.3 Hz, 1 H), 5.46 (ddt, J = 17.3, 1.4, 1.4 Hz,
1 H), 5.58 (m, 2 H), 6.07 (ddt, J = 17.2, 10.5, 5.5 Hz, 1 H), 7.05 (s, 1 H),
7.73 (s, 1 H).
13C NMR (100 MHz, CDCl3): δ = 17.7, 19.1, 28.3, 31.0, 56.6, 58.9, 63.7,
70.1, 80.0, 109.9, 110.4, 119.2, 127.1, 131.9, 139.5, 147.1, 154.1, 155.8,
172.1.
HRMS (CI): m/z [M + H]+ calcd for C27H40N3O9: 550.2764; found:
550.2781.
4-Allyloxy-5-methoxy-2-nitrobenzyl N-tert-Butoxycarbonyl-(S)-
prolyl-(S)-leucyl-(R/S)-valinate (5g)
To a solution of Boc-(S)-prolyl-(S)-leucyl-(S)-valine (400 mg, 0.84
mmol), 2 (201 mg, 0.84 mmol), DMAP (51.4 mg, 0.42 mmol), and
DIPEA (441 μL, 2.53 mmol) in anhydrous CH2Cl2 (2.0 mL), TBTU (460
mg, 1.43 mmol) was added at 0 °C and the mixture was stirred at r.t.
overnight. Washing with 1 M HCl (2 mL), sat. aq NaHCO3 (2 mL), and
brine (2 mL), drying over Na2SO4, and evaporation of the solvent in
vacuo afforded the crude product as an orange oil. Purification by col-
umn chromatography (PE–EtOAc, gradient) afforded 5g.
HRMS (CI): m/z [M]+ calcd for C21H30N2O8: 438.2002; found:
438.2003.
4-Allyloxy-5-methoxy-2-nitrobenzyl N-tert-Butoxycarbonyl-(R)-
Yield: 415 mg (0.64 mmol, 76%); yellow resins (diastereomeric mix-
valinate (5e)
ture, 1:1); Rf = 0.55/0.60 (SiO2; EtOAc).
HRMS (CI): m/z [M]+ calcd for C32H48N4O10
648.3365.
To a solution of Boc-(R)-valine (326 mg, 1.50 mmol), 2 (359 mg, 1.50
mmol), DMAP (92.0 mg, 0.50 mmol), and DIPEA (786 μL, 4.50 mmol)
in anhydrous CH2Cl2 (3.0 mL), TBTU (819 mg, 2.55 mmol) was added
at 0 °C and the mixture was stirred at r.t. overnight. Washing with
1 M HCl (3 mL), sat. aq NaHCO3 (3 mL), and brine (3 mL), drying over
Na2SO4, and evaporation of the solvent in vacuo afforded the crude
product as an orange oil. Purification by column chromatography
(PE–EtOAc, gradient) afforded 5e.
: 648.3370; found:
Diastereoisomer 1
1H NMR (400 MHz, CDCl3): δ = 0.87–1.00 (m, 12 H), 1.45 (s, 9 H),
1.52–1.75 (m, 3 H), 1.88–2.05 (m, 3 H), 2.18–2.24 (m, 2 H), 3.38 (m,
2 H), 4.02 (s, 3 H), 4.25 (m, 1 H), 4.37–4.48 (m, 2 H), 4.66 (m, 2 H),
5.34 (m, 1 H), 5.42–5.63 (m, 3 H), 6.06 (ddt, J = 17.2, 10.7, 5.5 Hz, 1 H),
6.88 (m, 1 H), 7.04–7.08 (m, 2 H), 7.71 (s, 1 H).
Yield: 595 mg (1.36 mmol, 90%); yellow oil; Rf = 0.57 (SiO2; PE–EtOAc,
1:1); HPLC (OD-H; hexane–i-PrOH, 90:10; 1 mL/min): tR = 6.95 min.
1H NMR (400 MHz, CDCl3): δ = 0.94 (d, J = 6.8 Hz, 3 H), 1.01 (d, J =
6.8 Hz, 3 H), 1.42 (s, 9 H), 2.18 (m, 1 H), 4.00 (s, 3 H), 4.30 (dd, J = 8.8,
5.1 Hz, 1 H), 4.68 (ddd, J = 5.5, 1.4, 1.4 Hz, 2 H), 4.99 (d, J = 8.8 Hz,
1 H), 5.36 (ddt, J = 10.5, 1.3, 1.3 Hz, 1 H), 5.46 (ddt, J = 17.3, 1.4, 1.4 Hz,
1 H), 5.58 (m, 2 H), 6.07 (ddt, J = 17.2, 10.5, 5.5 Hz, 1 H), 7.05 (s, 1 H),
7.74 (s, 1 H).
13C NMR (100 MHz, CDCl3): δ = 17.8, 19.1, 28.3, 31.0, 56.6, 58.9, 63.7,
70.2, 80.0, 109.9, 110.4, 119.2, 127.1, 131.9, 139.6, 147.1, 154.1, 155.8,
172.1.
13C NMR (100 MHz, CDCl3): δ = 18.2, 19.2, 21.5, 23.0, 24.7, 24.8, 28.3,
28.5, 30.5, 39.9, 47.2, 51.8, 56.7, 58.0, 60.4, 63.6, 70.1, 80.9, 109.9,
110.3, 119.1, 131.9, 139.3, 147.0, 154.3, 155.9, 171.5, 172.2, 172.3;
signal for OCH2CAr was too weak to be detected.
Diastereomer 2
1H NMR (400 MHz, CDCl3): δ (selected signals) = 4.39 (m, 1 H), 4.52
(dd, J = 8.2, 5.4 Hz, 1 H).
13C NMR (100 MHz, CDCl3): δ (selected signals) = 57.5, 60.3.
HRMS (CI): m/z [M]+ calcd for C21H30N2O8: 438.2002; found:
438.2002.
4-Allyloxy-5-methoxy-2-nitrobenzyl N-tert-Butoxycarbonyl-(S)-
prolyl-(S)-leucyl-(S)-valinate (5g-1)
Anal. Calcd for C21H30N2O8 (438.48): C, 57.52; H, 6.90; N, 6.39. Found:
C, 57.21; H, 6.79; N, 6.86.
To a solution of 5d (200 mg, 0.46 mmol) in CH2Cl2 (1.0 mL), TFA (430
μL, 5.58 mmol) was added and the mixture was stirred at r.t. for 30
min. The solvents were evaporated in vacuo to afford the TFA salt of
N-deprotected 5d. The free amine was obtained by addition of sat. aq
NaHCO3, extraction with CH2Cl2, and evaporation of the solvent in
vacuo.The residue was dissolved in anhydrous CH2Cl2 (2.0 mL), and
Boc-L-prolyl-L-leucine (149 mg, 0.46 mmol), DMAP (27.8 mg, 0.23
mmol), and DIPEA (238 μL, 1.37 mmol) were added, followed by the
addition of TBTU (248 mg, 0.77 mmol) at 0 °C. The mixture was
stirred at r.t. for 1.5 h then washed with 1 M HCl (2 mL), sat. aq
NaHCO3 (2 mL), and brine (2 mL). Drying over Na2SO4 and evaporation
of the solvent in vacuo gave the desired product as a yellow oil. Purifi-
cation by column chromatography (PE–EtOAc, gradient) afforded 5g-1.
4-Allyloxy-5-methoxy-2-nitrobenzyl N-tert-Butoxycarbonyl-(S)-
prolyl-(S)-leucinate (5f)
To a solution of Boc-(S)-prolyl-(S)-leucine (328 mg, 1.00 mmol), 2
(239 mg, 1.00 mmol), DMAP (61.1 mg, 0.50 mmol), and DIPEA (524
μL, 3.00 mmol) in anhydrous CH2Cl2 (3.0 mL), TBTU (546 mg, 1.70
mmol) was added at 0 °C and the mixture was stirred at r.t. overnight.
After washing with 1 M HCl (2 mL), sat. aq NaHCO3 (2 mL), and brine
(2 mL), drying over Na2SO4, and evaporation of the solvent in vacuo,
the crude product was obtained as an orange oil. Purification by col-
umn chromatography (PE–EtOAc, gradient) afforded 5f.
Yield: 466 mg (0.85 mmol, 85%); yellow solid; mp 108–109 °C; Rf =
0.27 (SiO2; PE–EtOAc, 1:1); [α]D20 –48.1 (c= 1.0, CHCl3).
Yield: 134 mg (0.21 mmol, 45%); yellow foamy resin; Rf = 0.15 (SiO2;
PE–EtOAc, 1:1).
1H NMR (400 MHz, CDCl3): δ = 0.93 (m, 6 H), 1.44 (s, 9 H), 1.59–1.77
(m, 3 H), 1.81–1.93 (m, 3 H), 2.22 (m, 1 H), 3.40 (m, 2 H), 4.02 (s, 3 H),
4.26 (m, 1 H), 4.58–4.67 (m, 3 H), 5.34 (m, 1 H), 5.44 (m, 1 H), 5.57 (m,
2 H), 6.06 (ddt, J = 17.2, 10.7, 5.5 Hz, 1 H), 6.38 (m, 1 H) 7.08 (s, 1 H),
7.72 (s, 1 H).
13C NMR (100 MHz, CDCl3): δ = 21.7, 22.8, 24.8, 24.9, 27.4, 28.4, 41.0,
47.0, 51.1, 56.7, 59.7, 63.8, 70.1, 80.6, 109.9, 110.1, 119.1, 127.5,
131.9, 139.3, 146.9, 154.3, 156.0, 172.3, 172.4.
1H NMR (400 MHz, CDCl3): δ = 0.85–0.91 (m, 6 H), 0.94–0.97 (m, 6 H),
1.45 (s, 9 H), 1.53–1.72 (m, 3 H), 1.77–1.98 (m, 3 H), 2.16–2.33 (m,
2 H), 3.40 (m, 2 H), 4.00 (s, 3 H), 4.24 (m, 1 H), 4.41 (m, 1 H), 4.52 (dd,
J = 8.3, 5.3 Hz, 1 H), 4.66 (ddd, J = 5.5, 1.3, 1.3 Hz, 2 H), 5.34 (ddt, J =
10.5, 1.2, 1.2 Hz, 1 H), 5.44 (ddt, J = 17.3, 1.4, 1.4 Hz, 1 H), 5.56 (m,
2 H), 6.06 (ddt, J = 17.2, 10.5, 5.5 Hz, 1 H), 6.91 (m, 1 H), 7.04 (s, 1 H),
7.71 (s, 1 H).
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13C NMR (100 MHz, CDCl3): δ = 17.9, 19.1, 21.0, 23.0, 24.7, 24.8, 28.3,
28.4, 30.8, 39.9, 47.1, 51.9, 56.6, 57.5, 60.3, 63.8, 70.1, 80.7, 109.9,
110.5, 119.1, 131.9, 139.5, 147.0, 154.2, 171.1, 171.2, 172.3; signals
for OCH2CAr and t-BuOCO were too weak to be detected.
13C NMR (100 MHz, CDCl3): δ = 17.4, 19.4, 21.9, 22.8, 24.8, 25.2, 26.8,
28.3, 31.5, 41.1, 47.1, 51.3, 56.7, 56.8, 59.6, 63.9, 70.2, 79.7, 109.9,
110.2, 119.1, 127.3, 131.9, 139.4, 147.0, 154.2, 155.8, 171.1, 172.4,
172.8.
MS (ESI/APCI): m/z [M + H]+ calcd for C32H49N4O10: 649.35; found:
HRMS (CI): m/z [M + H]+ calcd for C32H49N4O10: 649.3449; found:
649.30.
649.3449.
Anal. Calcd for C32H48N4O10 (648.75): C, 59.24; H, 7.46; N, 8.64. Found:
C, 58.96; H, 7.25; N, 8.11.
4-Allyloxy-5-methoxy-2-nitrobenzyl N-tert-Butoxycarbonyl-(S)-
isoleucyl-(S)-leucyl-(R/S)-prolinate (5i)
To a solution of Boc-(S)-isoleucyl-(S)-leucyl-(S)-proline (1.31 g, 2.96
mmol), 2 (707 mg, 2.95 mmol), DMAP (181 mg, 1.48 mmol), and
DIPEA (1.55 mL, 8.87 mmol) in anhydrous CH2Cl2 (10.0 mL), TBTU
(1.61 g, 5.02 mmol) was added at 0 °C and the mixture was stirred at
r.t. overnight. Washing with 1 M HCl (5 mL), sat. aq NaHCO3 (5 mL),
and brine (5 mL), drying over Na2SO4, evaporation of the solvent in
vacuo, and purification by column chromatography (PE–EtOAc, gradi-
ent) afforded 5i.
4-Allyloxy-5-methoxy-2-nitrobenzyl N-tert-Butoxycarbonyl-(S)-
prolyl-(S)-leucyl-(R)-valinate (5g-2)
To a solution of 5e (200 mg, 0.46 mmol) in CH2Cl2 (1.0 mL), TFA (422
μL, 5.47 mmol) was added and the mixture was stirred at r.t. for 20
min. The solvents were evaporated to afford the TFA salt of N-depro-
tected 5e. The free amine was obtained by addition of sat. aq NaHCO3,
extraction with CH2Cl2 and evaporation of the solvent in vacuo.The
residue was dissolved in anhydrous CH2Cl2 (2.0 mL), and Boc-L-prolyl-
L-leucine (149 mg, 0.46 mmol), DMAP (27.8 mg, 0.23 mmol) and
DIPEA (238 μL, 1.37 mmol) were added, followed by TBTU (248 mg,
0.77 mmol) at 0 °C. The mixture was stirred at r.t. for 1.5 h, then
washed with 1 M HCl (2 mL), sat. aq NaHCO3 (2 mL) and with brine
(2 mL). Drying over Na2SO4 and evaporation of the solvent in vacuo
gave the desired product as a yellow oil. Purification by column chro-
matography (PE–EtOAc, gradient) afforded 5g-2.
Yield: 1.82 g (2.75 mmol, 93%); yellow oil; Rf = 0.25 (SiO2; PE–EtOAc,
1:1).
1H NMR (400 MHz, CDCl3): δ = 0.88–0.94 (m, 12 H), 1.12 (m, 1 H), 1.43
(s, 9 H), 1.44–1.49 (m, 3 H), 1.65 (m, 1 H), 1.84 (m, 1 H), 2.03–2.14 (m,
3 H), 2.27 (m, 1 H), 3.63 (m, 1 H), 3.81 (m, 1 H), 3.93 (m, 1 H), 4.05 (s,
3 H), 4.61 (m, 1 H), 4.67 (ddd, J = 5.5, 1.4, 1.4 Hz, 2 H), 4.79 (td, J = 8.6,
5.2 Hz, 1 H), 4.99 (d, J = 8.9 Hz, 1 H), 5.35 (ddt, J = 10.5, 1.3, 1.3 Hz,
1 H), 5.43–5.64 (m, 3 H), 6.07 (ddt, J = 17.3, 10.5, 5.3 Hz, 1 H), 6.40 (d,
J = 8.4 Hz, 1 H), 7.08 (s, 1 H), 7.72 (s, 1 H).
Yield: 213 mg (0.33 mmol, 72%); yellow foamy resin; Rf = 0.25 (SiO2;
PE–EtOAc, 1:1).
1H NMR (400 MHz, CDCl3): δ = 0.88–0.91 (m, 6 H), 0.96 (m, 6 H), 1.45
(s, 9 H), 1.52–1.71 (m, 3 H), 1.88 (m, 2 H), 2.02 (m, 1 H), 2.19–2.24 (m,
2 H), 3.41 (m, 2 H), 4.02 (s, 3 H), 4.26 (m, 1 H), 4.40–4.48 (m, 2 H),
4.66 (ddd, J = 5.5, 1.4, 1.4 Hz, 2 H), 5.34 (ddt, J = 10.5, 1.3, 1.3 Hz, 1 H),
5.42–5.63 (m, 3 H), 6.05 (ddt, J = 17.2, 10.5, 5.5 Hz, 1 H), 6.92 (br. s,
1 H), 7.08–7.11 (m, 2 H), 7.71 (s, 1 H).
13C NMR (100 MHz, CDCl3): δ = 11.4, 15.5, 21.7, 23.3, 24.5, 24.8, 25.0,
28.3, 29.1, 37.2, 41.6, 46.9, 48.9, 56.8, 58.9, 59.3, 64.0, 70.2, 79.9,
109.9, 110.6, 119.1, 127.2, 131.9, 139.4, 147.1, 154.3, 155.7, 170.9,
171.4, 171.7.
HRMS (CI): m/z [M + H]+ calcd for C33H51N4O10: 663.3605; found:
663.3615.
13C NMR (100 MHz, CDCl3): δ = 18.2, 19.2, 21.5, 23.0, 24.7, 24.8, 28.3,
28.5, 30.4, 39.9, 47.3, 51.8, 56.7, 58.0, 60.4, 63.6, 70.1, 80.9, 109.8,
110.2, 119.1, 127.5, 131.9, 139.9, 146.9, 154.2, 156.0, 171.5, 172.2,
172.4.
Anal. Calcd for C33H50N4O10 (662.78): C, 59.80; H, 7.60; N, 8.45. Found:
C, 59.82; H, 7.43; N, 8.70.
4-Allyloxy-5-methoxy-2-nitrobenzyl N-tert-Butoxycarbonyl-(S)-
alanyl-(S)-alanyl-(R/S)-alaninate (5k)
Anal. Calcd for C32H48N4O10 (648.75): C, 59.24; H, 7.46; N, 8.64. Found:
C, 59.48; H, 7.24; N, 8.39.
To a solution of Boc-(S)-alanyl-(S)-alanyl-(S)-alanine (735 mg, 2.22
mmol), 2 (531 mg, 2.22 mmol), DMAP (135 mg, 1.11 mmol), and
DIPEA (1.16 mL, 6.65 mmol) in anhydrous CH2Cl2 (5.0 mL), TBTU (1.21
g, 3.77 mmol) was added at 0 °C and the mixture was stirred at r.t.
overnight. Washing with 1 M HCl (5 mL), sat. aq NaHCO3 (5 mL), and
brine (5 mL), drying over Na2SO4, evaporation of the solvent in vacuo
and purification by column chromatography (PE–EtOAc, gradient) af-
forded 5k.
4-Allyloxy-5-methoxy-2-nitrobenzyl N-tert-Butoxycarbonyl-(S)-
valyl-(S)-prolyl-(S)-leucinate (5h)
To a solution of Boc-(S)-valyl-(S)-prolyl-(S)-leucine (1.00 g, 2.34
mmol), 2 (559 mg, 2.34 mmol), DMAP (143 mg, 1.17 mmol), and
DIPEA (1.23 mL, 7.02 mmol) in anhydrous CH2Cl2 (5.0 mL), TBTU (1.28
g, 3.98 mmol) was added at 0 °C and the mixture was stirred at r.t.
overnight. Washing with 1 M HCl (5 mL), sat. aq NaHCO3 (5 mL), and
brine (5 mL), drying over Na2SO4 and evaporation of the solvent in
vacuo afforded the product as an orange oil. Purification by column
chromatography (PE–EtOAc, gradient) afforded 5h.
Yield: 1.05 g (1.91 mmol, 86%); yellow foamy resin; Rf = 0.05 (SiO2;
PE–EtOAc, 1:1).
1H NMR (400 MHz, CDCl3): δ = 1.34–1.37 (m, 6 H), 1.40 (s, 9 H), 1.50
(m, 3 H), 4.01 (s, 3 H), 4.10 (m, 1 H), 4.45–4.62 (m, 2 H), 4.67 (m, 2 H),
4.06 (m, 1 H), 5.35 (m, 1 H), 5.45 (m, 1 H), 5.55 (m, 2 H), 6.06 (ddt, J =
17.3, 10.5, 5.3 Hz, 1 H), 6.71 (m, 1 H), 7.00–7.17 (m, 2 H), 7.72 (s, 1 H).
Yield: 1.13 g (1.75 mmol, 75%); yellow oil; Rf = 0.20 (SiO2; PE–EtOAc,
1:1).
1H NMR (400 MHz, CDCl3): δ = 0.90–0.99 (m, 12 H), 1.43 (s, 9 H), 1.58
(m, 1 H), 1.64–1.73 (m, 2 H), 1.88 (m, 1 H), 1.93–2.01 (m, 2 H), 2.07
(m, 1 H), 2.35 (m, 1 H), 3.57 (m, 1 H), 3.71 (m, 1 H), 4.02 (s, 3 H), 4.28
(dd, J = 9.0, 6.7 Hz, 1 H), 4.55 (m, 1 H), 4.60 (m, 1 H), 4.67 (ddd, J = 5.5,
1.4, 1.4 Hz, 2 H), 5.19 (d, J = 9.2 Hz, 1 H), 5.35 (ddt, J = 10.5, 1.3, 1.3 Hz,
1 H), 5.43–5.65 (m, 3 H), 6.07 (ddt, J = 17.3, 10.5, 5.3 Hz, 1 H), 7.08 (s,
1 H), 7.24 (d, J = 7.2 Hz, 1 H), 7.73 (s, 1 H).
13C NMR (400 MHz, CDCl3): δ = 17.7, 17.7, 17.8, 48.3, 48.4, 48.4, 56.6,
64.1, 70.2, 80.1, 109.9, 110.4, 119.1, 126.6, 131.9, 139.5, 147.1, 154.2,
155.6, 172.1, 172.7, 172.7.
HRMS (CI): m/z [M]+ calcd for C25H36N4O10
: 552.2431; found:
552.2434.
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J. Hoffmann, U. Kazmaier
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Synthesis
4-Allyloxy-5-methoxy-2-nitrobenzyl N-(4-Allyloxy-5-methoxy-2-
nitrobenzyloxycarbonyl)-(S)-prolyl-(S)-leucyl-(R/S)-valinate (6g)
4-Allyloxy-5-methoxy-2-nitrobenzyl N-(4-Allyloxy-5-methoxy-2-
nitrobenzyloxycarbonyl)-(S)-prolyl-(S)-leucyl-(R)-valinate (6g-2)
To a solution of Boc-(S)-Pro-(S)-Leu-(R/S)-Val-OAMNB (5g; 399 mg,
0.62 mmol) in CH2Cl2 (1.0 mL), TFA (600 μL, 7.44 mmol) was added
and the mixture was stirred at r.t. for 2 h. The solvents were evaporat-
ed to afford the TFA salt of N-deprotected 5g. The free amine was ob-
tained by addition of sat. aq NaHCO3 (2 mL), extraction with CH2Cl2
(5 mL) and evaporation of the solvent in vacuo. The residue was dis-
solved in anhydrous THF (1.5 mL) under a nitrogen atmosphere, anhy-
drous pyridine (199 μL, 2.46 mmol), DMAP (37.6 mg, 0.31 mmol), and
4-allyloxy-5-methoxy-2-nitrobenzyl chloroformate (3; 196 mg, 0.62
mmol) were added and the mixture was stirred at r.t. for 72 h. Wash-
ing with 1 M HCl (2 mL), sat. aq NaHCO3 (2 mL), and brine (2 mL), dry-
ing over Na2SO4, evaporation of the solvent in vacuo, and purification
by column chromatography (PE–EtOAc, gradient) afforded 6g.
To a solution of Boc-L-Pro-L-Leu-D-Val-OAMNB (5g-2; 200 mg, 0.31
mmol) in CH2Cl2 (1.0 mL) TFA (300 μL, 3.72 mmol) was added and the
mixture was stirred at r.t. for 2 h. The solvents were evaporated to af-
ford the TFA salt of N-deprotected 5g-2. The free amine was obtained
by washing with sat. aq NaHCO3 (2 mL), extraction with CH2Cl2 (5 mL)
and evaporation of the solvent in vacuo. The residue was dissolved in
anhydrous THF (1.5 mL) under a nitrogen atmosphere, anhydrous
pyridine (100 μL, 1.24 mmol), DMAP (19.4 mg, 0.16 mmol) and 3
(92.9 mg, 0.31 mmol) were added, and the mixture was stirred at r.t.
for 72 h. After washing with 1 M HCl (2 mL), sat. aq NaHCO3 (2 mL)
and brine (2 mL), drying over Na2SO4, evaporation of the solvent in
vacuo, and purification by column chromatography (PE–EtOAc, gradi-
ent), 6g-2 was obtained.
Yield: 340 mg (0.42 mmol, 68%); yellow foamy resin (1:1 diastereo-
Yield: 171 mg (0.21 mmol, 68%); yellow foamy resin; Rf = 0.11 (SiO2;
meric mixture); Rf = 0.09 (SiO2; PE–EtOAc, 1:1).
PE–EtOAc, 1:1).
1H NMR (400 MHz, CDCl3): δ = 0.86–1.01 (m, 12 H), 1.48–1.67 (m,
2 H), 1.77 (m, 1 H), 1.93 (m, 2 H), 2.07–2.27 (m, 3 H), 3.54 (m, 2 H),
3.98–4.00 (m, 6 H), 4.31 (m, 1 H), 4.39–4.55 (m, 2 H), 4.65–4.67 (m,
4 H), 5.22–5.75 (m, 8 H), 6.01–6.10 (m, 2 H), 6.98–7.06 (m, 2 H), 7.65–
7.72 (m, 2 H).
1H NMR (400 MHz, CDCl3): δ = 0.76–0.99 (m, 12 H), 1.57–1.77 (m,
3 H), 1.92–1.98 (m, 2 H), 2.08–2.25 (m, 3 H), 3.49 (m, 2 H), 3.95, 3.98
(2 × s, 6 H), 4.31 (m, 1 H), 4.47 (m, 1 H), 4.51–4.63 (m, 4 H), 5.20–5.72
(m, 8 H), 6.02 (ddt, J = 17.3, 10.7, 5.5 Hz, 2 H), 6.61 (d, J = 6.9 Hz, 1 H),
6.97–7.18 (m, 2 H), 7.18 (d, J = 7.9 Hz, 1 H), 7.62 (s, 1 H), 7.68 (s, 1 H).
13C NMR (100 MHz, CDCl3): δ = 19.0, 19.2, 21.6, 23.0, 24.6, 24.7, 29.6,
30.8, 39.8, 47.1, 51.6, 56.4, 56.7, 57.6, 61.3, 63.7, 64.7, 70.1, 70.1,
109.9, 109.9, 110.5, 110.8, 119.1, 119.1, 127.1, 127.3, 131.9, 131.9,
139.3, 139.5, 146.9, 146.9, 154.1, 154.2, 155.0, 171.2, 171.7, 172.2.
13C NMR (100 MHz, CDCl3): δ = 17.6, 18.3, 19.1, 21.3, 23.0, 24.6, 25.0,
29.6, 30.4, 39.7, 47.0, 51.5, 56.3, 56.6, 58.0, 61.2, 63.5, 64.5, 65.2, 70.0,
109.6, 109.8, 110.2, 111.5, 119.0, 119.1, 126.8, 127.3, 131.8, 131.98
139.1, 140.0, 146.8, 147.2, 153.6, 154.1, 155.5, 171.5, 171.7, 172.1.
HRMS (CI): m/z [M + 2H]+ calcd for C39H53N5O14: 815.3589; found:
HRMS (CI): m/z [M + 2H]+ calcd for C39H53N5O14: 815.3589; found:
815.3587.
815.3561.
4-Allyloxy-5-methoxy-2-nitrobenzyl N-(4-Allyloxy-5-methoxy-2-
nitrobenzyloxycarbonyl)-(S)-prolyl-(S)-leucyl-(S)-valinate (6g-1)
4-Allyloxy-5-methoxy-2-nitrobenzyl N-(4-Allyloxy-5-methoxy-2-
nitrobenzyloxycarbonyl)-(S)-valyl-(S)-prolyl-(S)-leucinate (6h)
To a solution of Boc-(S)-Pro-(S)-Leu-(S)-Val-OAMNB (5g-1; 175 mg,
0.27 mmol) in CH2Cl2 (1.0 mL), TFA (262 μL, 3.26 mmol) was added
and the mixture was stirred at r.t. for 2 h. The solvents were evaporat-
ed to afford the TFA salt of N-deprotected 5g-1. The free amine was
obtained by washing with sat. aq NaHCO3 (2 mL), extraction with
CH2Cl2 (5 mL) and evaporation of the solvent in vacuo. The residue
was dissolved in anhydrous THF (1.5 mL) under a nitrogen atmo-
sphere, anhydrous pyridine (87.5 μL, 1.09 mmol), DMAP (17.0 mg,
0.14 mmol) and 3 (85.8 mg, 0.27 mmol) were added, and the mixture
was stirred at r.t. for 72 h. Washing with 1 M HCl (2 mL), sat. aq
NaHCO3 (2 mL), and brine (2 mL), drying over Na2SO4, evaporation of
the solvent in vacuo, and purification by column chromatography
(PE–EtOAc, gradient) afforded 6g-1.
To a solution of Boc-(S)-Pro-(S)-Leu-(S)-Val-OAMNB (5h; 701 mg,
1.09 mmol) in CH2Cl2 (3.0 mL), TFA (1.06 mL, 13.1 mmol) was added
and the mixture was stirred at r.t. for 2 h. The solvents were evaporat-
ed to afford the TFA salt of N-deprotected 5h. The free amine was ob-
tained by washing with sat. aq NaHCO3 (3 mL), extraction with CH2Cl2
(7 mL) and evaporation of the solvent in vacuo. The residue was dis-
solved in anhydrous THF (1.5 mL) under a nitrogen atmosphere, anhy-
drous pyridine (354 μL, 4.37 mmol), DMAP (66.8 mg, 0.55 mmol) and
3 (329 mg, 1.09 mmol) were added and the mixture was stirred at r.t.
for 72 h. After washing with 1 M HCl (3 mL), sat. aq NaHCO3 (3 mL)
and brine (3 mL), drying over Na2SO4, evaporation of the solvent in
vacuo and purification by column chromatography (PE–EtOAc, gradi-
ent), 6h was obtained.
Yield: 138 mg (0.18 mmol, 63%); yellow foamy resin; Rf = 0.11 (SiO2;
Yield: 525 mg (0.65 mmol, 59%); yellow resin; Rf = 0.15 (SiO2; PE–
PE–EtOAc, 1:1).
EtOAc, 1:1).
1H NMR (400 MHz, CDCl3): δ = 0.86–0.96 (m, 12 H), 1.58–1.94 (m,
5 H), 2.11–2.23 (m, 3 H), 3.45 (m, 2 H), 3.99, 4.01 (2 × s, 6 H), 4.31 (m,
1 H), 4.39 (m, 1 H), 4.54 (m, 1 H), 4.67 (ddd, J = 5.4, 1.3, 1.3 Hz, 4 H),
5.34–5.68 (m, 8 H), 6.07 (ddt, J = 17.3, 10.7, 5.5 Hz, 2 H), 6.77 (m,
0.5 H), 6.91 (m, 0.5 H), 6.99–7.05 (m, 3 H), 7.69 (s, 1 H), 7.72 (s, 1 H).
13C NMR (100 MHz, CDCl3): δ = 18.3, 19.0, 21.6, 23.0, 24.7, 24.9, 29.6,
30.4, 39.8, 47.0, 51.6, 56.4, 56.7, 58.1, 61.3, 63.5, 64.7, 70.1, 70.1,
109.8, 109.9, 110.5, 111.6, 119.1, 119.1, 126.8, 127.1, 131.9, 131.9,
139.2, 139.3, 146.9, 147.0, 154.1, 154.2, 155.6, 171.2, 171.7, 172.1.
1H NMR (400 MHz, CDCl3): δ = 0.91–1.02 (m, 12 H), 1.59 (m, 1 H),
1.67–1.76 (m, 2 H), 1.86–2.01 (m, 3 H), 2.09 (m, 1 H), 2.34 (m, 1 H),
3.59 (m, 1 H), 3.72 (m, 1 H), 3.98–4.02 (m, 6 H), 4.35 (dd, J = 9.1,
6.3 Hz, 1 H), 4.54–4.59 (m, 2 H), 4.67 (m, 4 H), 5.34–5.36 (m, 2 H),
5.43–5.64 (m, 7 H), 6.01–6.10 (m, 2 H), 7.01 (s, 1 H), 7.06 (s, 1 H), 7.10
(d, J = 7.3 Hz, 1 H), 7.70–7.72 (m, 2 H).
13C NMR (100 MHz, CDCl3): δ = 17.5, 19.4, 21.9, 22.8, 24.8, 25.1, 27.0,
31.4, 41.2, 47.8, 51.3, 56.5, 56.6, 57.6, 59.8, 63.8, 64.0, 70.2, 70.2,
109.7, 109.9, 110.2, 110.3, 119.1, 119.1, 127.1, 128.3, 131.9, 131.9,
139.4, 139.5, 146.9, 147.0, 154.1, 154.2, 155.9, 170.9, 172.1, 172.3.
HRMS (CI): m/z [M + H]+ calcd for C39H52N5O14: 814.3511; found:
814.3505.
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2015, 47, 411–420

419
J. Hoffmann, U. Kazmaier
Paper
Synthesis
Anal. Calcd for C39H51N5O14 (813.86): C, 57.56; H, 6.32; N, 8.61. Found:
C, 57.47; H, 6.34; N, 8.09.
ed to reflux overnight. The solvent was removed in vacuo and the
crude product was purified by column chromatography (CH2Cl2–
MeOH, gradient, then PE–EtOAc, gradient) to afford cyclopeptide 7g-1.
4-Allyloxy-5-methoxy-2-nitrobenzyl N-(4-Allyloxy-5-methoxy-2-
Yield: 28.4 mg (0.036 mmol, 49%); yellow resin; Rf = 0.42 (SiO2;
nitrobenzyloxycarbonyl)-(S)-isoleucyl-(S)-leucyl-(R/S)-prolinate
EtOAc).
(6i)
1H NMR (400 MHz, CDCl3): δ = 0.63–0.99 (m, 12 H), 1.34–2.32 (m,
8 H), 3.54 (m, 2 H), 3.93–3.98 (m, 6 H), 4.26–4.92 (m, 6 H), 5.09–5.54
(m, 4 H), 5.84–6.05 (m, 3 H), 6.31–6.79 (m, 2 H), 6.87–6.91 (m, 2 H),
7.50–7.70 (m, 2 H).
To a solution of Boc-(S)-Ile-(S)-Leu-(R/S)-Pro-OAMNB (5h; 629 mg,
0.95 mmol) in CH2Cl2 (3.0 mL), TFA (924 μL, 11.4 mmol) was added
and the mixture was stirred at r.t. for 2 h. The solvents were evaporat-
ed to afford the TFA salt of N-deprotected 5h. The free amine was ob-
tained by washing with sat. aq NaHCO3 (2 mL), extraction with CH2Cl2
(5 mL) and evaporation of the solvent in vacuo. The residue was dis-
solved in anhydrous THF (2.0 mL) under a nitrogen atmosphere, anhy-
drous pyridine (308 μL, 3.81 mmol), DMAP (58.1 mg, 0.48 mmol) and
3 (287 mg, 0.95 mmol) were added and the mixture was stirred at r.t.
for 72 h. After washing with 1 M HCl (3 mL), sat. aq NaHCO3 (3 mL),
and brine (3 mL), drying over Na2SO4, evaporation of the solvent in
vacuo, and purification by column chromatography (PE–EtOAc, gradi-
ent) afforded the title compound 6i.
Cyclopeptide 7g-2
To a solution of Grubbs II catalyst (5.0 mg, 5.84 μmol) in CH2Cl2 (1.0
mL) heated to reflux, 6g-2 (50.0 mg, 0.061 mmol) in CH2Cl2 (3.0 mL)
was added dropwise under argon atmosphere and the reaction mix-
ture was heated to reflux for 40 min. The solvent was removed in vac-
uo and the crude product was purified by column chromatography
(CH2Cl2–MeOH, gradient, then PE–EtOAc, gradient) to afford cyclo-
peptide 7g-2, which could be crystallized from EtOH–CH2Cl2.
Yield: 25.0 mg (0.032 mmol, 54%); yellow solid; m.p. 165–167 °C; Rf =
0.44 (SiO2; EtOAc).
Yield: 633 mg (0.77 mmol, 80%); yellow resin (1:1 diastereomeric
mixture; Rf = 0.11 (SiO2; PE–EtOAc, 1:1).
1H NMR (400 MHz, CDCl3): δ = 0.92 (d, J = 6.6 Hz, 3 H), 0.97 (d, J =
6.6 Hz, 3 H), 1.03 (d, J = 7.2 Hz, 3 H), 1.05 (d, J = 7.2 Hz, 3 H), 1.58 (m,
1 H), 1.72–1.90 (m, 3 H), 1.98 (m, 1 H), 2.09 (m, 1 H), 2.22–2.40 (m,
2 H), 3.15 (m, 1 H), 3.30 (m, 1 H), 3.93, 4.01 (2 × s, 6 H), 4.37 (dd, J =
8.9, 4.2 Hz, 1 H), 4.68 (d, J = 12.2 Hz, 1 H), 4.64–4.75 (m, 4 H), 4.99 (m,
2 H), 5.27 (d, J = 16.2 Hz, 1 H), 5.52 (d, J = 16.3 Hz, 1 H), 5.97–6.03 (m,
2 H), 6.19 (d, J = 12.3 Hz, 1 H), 6.55 (d, J = 9.2 Hz, 1 H), 6.73 (s, 1 H),
6.78 (s, 1 H), 7.20 (s, 1 H), 7.22 (s, 1 H), 7.54 (d, J = 9.5 Hz, 1 H).
13C NMR (100 MHz, CDCl3): δ = 17.9, 19.3, 20.8, 23.4, 24.4, 25.4, 30.2,
30.5, 39.6, 46.9, 51.5, 56.8, 56.9, 57.6, 61.5, 62.6, 66.6, 67.8, 68.1,
109.0, 109.7, 110.6, 113.8, 125.8, 127.3, 131.3, 131.9, 137.5, 138.9,
144.8, 146.5, 154.1, 154.5, 155.6, 171.0, 171.4, 172.1.
1H NMR (400 MHz, CDCl3): δ = 0.86–0.93 (m, 12 H), 1.16 (m, 1 H),
1.44–1.53 (m, 3 H), 1.62 (m, 1 H), 1.84 (m, 1 H), 2.05–2.14 (m, 3 H),
2.28 (m, 1 H), 3.63 (m, 1 H), 3.78 (m, 1 H), 3.97–4.07 (m, 7 H), 4.62 (m,
1 H), 4.66–6.67 (m, 4 H), 4.75 (m, 1 H), 5.33–5.36 (m, 2 H), 5.43–5.61
(m, 7 H), 6.01–6.11 (m, 2 H), 6.31 (d, J = 7.9 Hz, 1 H), 6.99 (s, 1 H), 7.05
(s, 1 H), 7.70–7.71 (m, 2 H).
13C NMR (100 MHz, CDCl3): δ = 11.4, 15.4, 21.7, 23.2, 24.6, 24.9, 25.0,
29.1, 37.8, 41.6, 46.9, 49.1, 56.4, 56.7, 58.9, 59.6, 63.9, 64.0, 70.1, 70.1,
109.9, 109.9, 110.8, 110.8, 119.1, 119.1, 127.0, 127.0, 131.9, 131.9,
139.5, 139.5, 146.9, 147.1, 154.0, 154.2, 156.6, 170.8, 170.8, 171.5.
HRMS (CI): m/z [M + H]+ calcd for C40H54N5O14: 828.3667; found:
828.3678.
HRMS (CI): m/z [M + H]+ calcd for C37H48N5O14: 786.3198; found:
786.3214.
Cyclopeptide 7g
Anal. Calcd for C37H47N5O14 (785.81): C, 56.55; H, 6.03; N, 8.91. Found:
C, 56.81; H, 5.74; N, 8.49.
To a solution of Grubbs II catalyst (4.7 mg, 5.53 μmol) in CH2Cl2 (1.0
mL) heated to reflux, 6g (45.0 mg, 0.055 mmol) in CH2Cl2 (5.0 mL) was
added dropwise under argon and the reaction mixture was heated to
reflux overnight. The solvent was removed in vacuo and the crude
product was purified by column chromatography (CH2Cl2–MeOH, gra-
dient, then PE–EtOAc, gradient) to afford cyclopeptide 7g.
Acknowledgment
Financial support from the Deutsche Forschungsgemeinschaft (Ka
880/10-2) is gratefully acknowledged. J. Hoffmann gratefully ac-
knowledges a fellowship of the Studienstiftung des Deutschen Volkes.
Yield: 28.3 mg (0.036 mmol, 65%); yellow resin; Rf = 0.42 (SiO2;
EtOAc).
1H NMR (400 MHz, CDCl3): δ = 0.94–1.05 (m, 12 H), 1.61–2.38 (m,
8 H), 3.18–3.58 (m, 2 H), 3.94–4.03 (m, 6 H), 4.26–4.75 (m, 6 H), 4.83–
5.03 (m, 2 H), 5.27–5.55 (m, 2 H), 5.96–6.02 (m, 2 H), 6.19 (m, 1 H),
6.57–7.05 (m, 3 H), 7.22–7.70 (m, 3 H).
13C NMR (100 MHz, CDCl3): δ = 17.9, 19.3, 20.7, 23.4, 24.4, 25.3, 30.2,
30.4, 39.5, 46.9, 51.5, 56.8, 56.8, 57.6, 61.4, 62.6, 66.5, 67.7, 68.1,
109.0, 109.7, 110.6, 113.7, 125.7, 127.2, 131.3, 131.8, 137.5, 138.8,
144.7, 146.4, 154.0, 154.4, 155.6, 171.0, 171.4, 172.0.
Supporting Information
Supporting information for this article is available online at
http://dx.doi.org/10.1055/s-0034-1378908. Included are details of
the photocleavage experiments and copies of NMR spectra.
S
u
p
p
ortiInfogrmoaitn
S
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p
p
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MS (ESI/APCI): m/z [M + H]+ calcd for C37H48N5O14: 786.32; found:
786.35.
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© Georg Thieme Verlag Stuttgart · New York — Synthesis 2015, 47, 411–420

420
J. Hoffmann, U. Kazmaier
Paper
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