The Regioselective Synthesis of o -Nitrobenzyl DOPA Derivatives

Article abstract of DOI:10.1055/s-0036-1588766

Photocaged DOPA derivatives may serve for non-invasive unmasking of the catechol fragment in biological systems. This would enable efficient control of the redox and metal-coordinating properties associated with the free catechol moiety, in particular, in biosynthetically produced adhesive proteins and synthetic peptides. Synthetic routes towards photocaged DOPA derivatives are reported herein. A new method for preparing para -alkylated DOPA starting from 3,4-dihydroxybenzaldehyde is described for the first time.

Full text of DOI:10.1055/s-0036-1588766

SYNTHESIS
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© Georg Thieme Verlag Stuttgart · New York
2017, 49, A–I
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T. Schneider et al.
Paper
Synthesis
The Regioselective Synthesis of o-Nitrobenzyl DOPA Derivatives
Tobias Schneider
Joshua Martin
Patrick M. Durkin
Vladimir Kubyshkin
Nediljko Budisa*
Institute of Chemistry, Technical University of Berlin,
Müller-Breslau-Str. 10, 10623 Berlin, Germany
nediljko.budisa@tu-berlin.de
Received: 03.03.2017
Accepted after revision: 06.03.2017
Published online: 13.04.2017
candidate for this task, allowing for the protection of the
catechol moiety as well as the selective and non-invasive
deprotection through irradiation with UV light.
DOI: 10.1055/s-0036-1588766; Art ID: ss-2017-z1859-op
A suitable possibility for this, is the use of the o-nitro-
benzyl (oNB) protecting group, which can be selectively
cleaved via UV irradiation (~365 nm).11 Our aim is the syn-
thesis of photoactivatable DOPA derivatives, and their sub-
sequent use in biological settings, particularly in in vivo
protein synthesis.12 Importantly, the bio-incorporation of
O-oNB tyrosine along with the subsequent deprotection of
the hydroxyl group upon UV irradiation has been demon-
strated previously.13 The amino acid bio-incorporation
technology used in these reports is based on stop-codon
suppression with tRNA/aminoacyl-tRNA synthetase or-
thogonal pair derived from Methanocaldococcus janna-
schii.14
Since DOPA possesses two chemically similar phenolic
hydroxy groups, we initially considered three options for
protection: two single protected variants 2 and 3 and a
double protected one 4 (Figure 1).
The double-masked species 4 was easily produced by
double-alkylation of protected DOPA 5 with 2 equivalents
of oNB-bromide (Scheme 1). However, this type of modifi-
cation might significantly extend the size and hydrophobic-
ity of the amino acid side chain, which in turn makes it is
the least promising for incorporation into proteins in vivo
(see Figure 1). Therefore, we focused on a synthetic route
towards 3 as an arbitrary mono-oNB-masked DOPA deriva-
tive.
Abstract Photocaged DOPA derivatives may serve for non-invasive un-
masking of the catechol fragment in biological systems. This would en-
able efficient control of the redox and metal-coordinating properties
associated with the free catechol moiety, in particular, in biosyntheti-
cally produced adhesive proteins and synthetic peptides. Synthetic
routes towards photocaged DOPA derivatives are reported herein. A
new method for preparing para-alkylated DOPA starting from 3,4-dihy-
droxybenzaldehyde is described for the first time.
Key words amino acids, DOPA, photocaged, o-nitrobenzyl group,
protection group
The chemical synthesis of water-soluble cell-permeable
3,4-dihydroxyphenylalanine (DOPA) derivatives with a pro-
tected catechol functionality is a desirable task. This is due
to the fact, that many classes of biologically and medicinally
important molecules contain catechols as the key function-
al group. Those are, for example, siderophores1 which are
mainly non-ribosomally produced peptides. They are
known to be the strongest known binders of Fe(III) ions.
Another example is catecholamines (as derivatives of ca-
nonical amino acid tyrosine), physiologically significant
neurohormones.2 It has also been demonstrated that the
catechol function of DOPA is responsible for the adhesive
properties of mussel foot proteins.3–5 This class of natural
products is interesting for the development of biocompati-
ble adhesives for medicinal applications.6–10
A method by which one could mask the functional ef-
fects of the catechol group of DOPA, and on-demand reveal
this functionality would allow direct control of the redox,
metal-coordinating and adhesive properties of the catechol
moiety. A photo-cleavable group presents itself as a good
Several methods towards selective production of p-O-al-
kylated DOPA derivatives have been described in the litera-
ture. The action of Pd catalysts on m-iodotyrosine has been
applied for borylation or acylation in the m-position. Subse-
quently O-alkyl-DOPA derivatives were produced through
alkylation of the p-hydroxyl group followed by oxidation of
the m-substituent.15 Another common approach employs
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T. Schneider et al.
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Synthesis
NO2
NO2
O2N
OH
OH
OH
O
O
O
OH
O
NO2
CO2H
CO2H
NH2
CO2H
CO2H
NH2
NH2
NH2
1
2
3
4
logD7.40
volume [Å3]
–1.80
226
+0.01
373
+0.01
370
+1.83
520
Figure 1 DOPA (1) and its oNB derivatives: mono- (2 and 3) and dialkylated (4). The predicted values of distribution coefficients and molecular vol-
umes highlight changes introduced to the amino acid side chain by oNB functionalization
NO2
NO2
OH
OH
O
O
OH
OH
(a)
(b)
(d)
(c)
O
NO2
O
NO2
CO2H
CO2Me
CO2H
CO2Me
NHBoc
NH2
NHBoc
NH2
1
5
6
4
Scheme 1 Reagents and conditions: (a) SOCl2, MeOH, quant.; (b) Boc2O, Et3N, MeCN, 72%; (c) oNB-Br (2 equiv), K2CO3, NaI, acetone, 87%; (d) aq HCl,
1,4-dioxane, 58%.
tyrosine as a starting material. The amino acid side chain
OH
O
OH
OH
O
should be either acylated (Friedel–Crafts reaction) or
formylated (Reimer–Tiemann reaction) in the m-position.
Subsequent p-O-alkylation and oxidation of the acyl moiety
(Dakin oxidation or Baeyer–Villiger oxidation) leads to the
desired alkylation pattern of the catechol fragment.16,17 We
first applied this strategy for the synthesis of 3 (Scheme 2).
The Friedel–Crafts acylation of tyrosine 7, followed by pro-
tection of N- and C-terminal functions and alkylation of the
phenolic hydroxy group with o-nitrobenzyl bromide, pro-
duced 10 in good yields. The oxidative rearrangement with
MCPBA was successful, but was a notably slow reaction
(taking up to 5 d). With 11 in hand, further conversion into
3 was achieved using standard procedures.
However, in addition to the long reaction time of the
Baeyer–Villiger oxidation, this synthesis produced rather
low yields of our desired compound 3. Therefore we further
aimed to develop a novel synthetic strategy, starting from
3,4-dihydroxybenzaldehyde (12) (Scheme 3).
The incorporation of the oNB group at the start of the
synthesis was proposed to be selective for the para-pheno-
lic position due to the higher acidity of this position arising
from mesomeric stabilization by the aldehyde functional-
ity. Indeed the alkylation of 3,4-dihydroxybenzaldehyde
(12) formed selectively 13, which was purified by crystalli-
zation. Subsequent reduction of the aldehyde group to 14
and exchange of the aliphatic hydroxyl group with bromine
(a)
(b)
(c)
HCl
CO2H
CO2H
NH2
CO2Me
(d)
NH2
NHBoc
8
7
9
NO2
NO2
NO2
O
O
O
O
OH
HCl
OH
(e)
(f)
(g)
CO2H
NH2
3·HCl
CO2H
CO2Me
NHBoc
10
NHBoc
11
Scheme 2 Reagents and conditions: (a) AcCl, AlCl3, nitrobenzene, 59%;
(b) SOCl2, MeOH, 97%; (c) Boc2O, Et3N, H2O/1,4-dioxane (1:1), 67%; (d)
oNB-Br, K2CO3, TBAB, MeCN, 57%; (e) MCPBA, CH2Cl2, 67%; (f) LiOH,
acetone/H2O, 22%; (g) aq HCl, 1,4-dioxane, quant.
was achieved expediently to form 15. Subsequently, we at-
tempted to introduce an amino acid moiety by alkylation of
diethyl acetamidomalonate into 15. However, this transfor-
mation was found to be problematic, affording a complex
mixture of products. Analysis of the NMR spectra of the
crude reaction mixture revealed that under the conditions
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T. Schneider et al.
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Synthesis
NO2
NO2
NO2
NO2
OH
O
O
O
O
OH
O
(d)
OH
(b)
(c)
(a)
OH
OH
Boc
O
H
Br
Br
OH
O
H
12
13
14
15
16
(e)
(h)
NO2
NO2
NO2
NO2
O
O
O
O
OH
OH
O
OH
Boc
(f)
(i)
(g)
CO2H
NHAc
CO2Et
NHAc
CO2Et
CO2Et
NHAc
CO2Et
CO2H
NH2
rac-19
rac-3
18
17
(j)
(k)
NO2
NO2
O
O
OH
OH
+
CO2H
NH2
CO2H
NHAc
(R)-19
3
Scheme 3 Reagents and conditions: (a) oNB-Br, K2HPO4, NaI, DMF, 42%; (b) NaBH4, EtOH, 99%; (c) PBr3, CH2Cl2, 95%; (d) Boc2O, Et3N, DMAP (cat.), Et2O,
84%; (e) diethyl acetamidomalonate, NaH, DMF, 93%; (f) EtOH, H2O, quant.; (g) aq HCl, 1,4-dioxane, 56%; (h) KOH, EtOH/H2O; then aq HCl, THF, reflux
(i) Ac2O, Et3N, MeCN, 97%; (j) Acylase I Asp. melleus, 0.1 M pH 8, 33%; (k) Ac2O, NaOAc, AcOH, then NaHCO3, H2O, 98%.
employed (NaH, DMF, 0 °C) the phenolic hydroxyl group at-
tacks the benzylic bromide preferentially. To circumvent
this problem, the free hydroxyl group in 15 was temporarily
masked with a Boc protecting group yielding 16. Alkylation
of diethyl acetamidomalonate with 16 then proceeded
smoothly, producing 17 in a good yield (Scheme 3, step e).
The regiochemistry of the O-alkylation was initially
confirmed by analysis of the 1H NOESY NMR spectra (NOE:
O-CH2 ↔ catechol CH). We were then able to isolate crystals
of compounds 14, 16, and 17 suitable for X-ray diffraction.
Analysis of the crystal structures unequivocally confirmed
that the alkylation had indeed occurred on the para-posi-
tion (Figure 2).
It was anticipated, that compound 18 could be convert-
ed into N-acetylated amino acid rac-19 using aqueous base
and subsequent decarboxylation under acidic conditions.
However, the action of aqueous alkali metal hydroxides
(LiOH, NaOH, and KOH were tested) at elevated tempera-
tures on 18 lead to the cleavage of the oNB group; these ba-
sic conditions were therefore not suitable for the substrate.
Nevertheless, 18 was converted into the free amino acid
rac-3 by refluxing in a mixture of hydrochloric acid and 1,4-
dioxane. It is noteworthy that oNB group cleavage was also
observed under these conditions (20% after 4 h), although it
was distinctly slower than under basic conditions. The
product of this reaction, rac-3 (acidic hydrolysis also result-
ed in the loss of the N-acetyl group), was isolated simply by
crystallization. For resolution of the racemic mixture, the
amino group was re-acetylated with acetic anhydride. Sub-
sequently, kinetic resolution with acylase I from Aspergillus
melleus afforded the desired (S)-amino acid 3.
Phenolic O-Boc groups, similar to that of 17, have been
previously reported to be rather unstable, as they can be
easily deprotected under mild conditions.18 In our case,
heating to 80 °C in a water/ethanol mixture led to complete
deprotection within 20 hours yielding 18.
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2017, 49, A–I

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T. Schneider et al.
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Synthesis
m-OH side chain group; the latter remained unprotected in
course of the synthesis.
In summary, we reported the regioselective synthesis
and isolation of O-oNB-alkylated DOPA derivatives 3 and 4.
The classical approach of tyrosine modification, aromatic
acylation–O-alkylation–Baeyer–Villiger oxidation of the
acetyl group worked, although with sub-optimal yields. For
this reason we developed an alternative strategy. The latter
was based upon selective alkylation of 3,4-dihydroxybenz-
aldehyde, and subsequent construction of the amino acid
moiety through manipulation of the aldehyde group. Amino
acid (S)-3 was prepared on gram scale using this method.
Microbiological studies with this compound are ongoing,
and will be reported elsewhere.
All organic solvents were distilled before use. Dry solvents were pur-
chased from Acros Organics. TLC was carried out on aluminum sheets
coated with silica gel 60 F254 (Merck, Darmstadt, Germany). TLC
plates were inspected under 254 nm irradiation. 1H and 13C NMR
spectra were recorded on Bruker Avance DRX 400 and Bruker Avance
III 500 spectrometers calibrated by the residual solvent signal.19 ESI
and APCI mass spectra were recorded in the positive mode (unless
stated otherwise) using a LTQ Orbitrap XL spectrometer. Optical rota-
tions were measured using a Jasco P-2000 Polarimeter. Distribution
coefficient values were calculated in Marvin Sketch 15.4.27 (ChemAx-
on Ltd). Molecular volumes were calculated for aqueous medium in
Scigress Modeling suite (Fujitsu).
Figure 2 X-ray crystal structures: carbon, grey; hydrogen, light grey;
oxygen, red; nitrogen, blue; bromine, green. CCDC deposition num-
bers: 14: 1521796; 16: 1521798; 17:1521797.
The undesired enantiomer of N-acetyl amino acid (R)-
19 was then subjected to racemization to afford rac-19 with
excellent yields (Scheme 3, step k). We were then able to
perform a second acylase kinetic resolution and so increase
the total yield of this reaction sequence.
Overall, the presented method thus offered an alterna-
tive to the synthesis starting from tyrosine and afforded an
improved overall yield (10% vs. 3%).
Methyl (S)-2-Amino-3-(3,4-dihydroxyphenyl)propanoate Hydro-
chloride
This compound was synthesized according to the literature.20
1H NMR (500 MHz, DMSO-d6): δ = 8.96 (s, 1 H), 8.93 (s, 1 H), 8.55 (br,
3 H), 6.67 (d, J = 8.0 Hz, 1 H), 6.59 (d, J = 2.1 Hz, 1 H), 6.45 (dd, J = 8.0,
2.1 Hz, 1 H), 4.10 (t, J = 6.5 Hz, 1 H), 3.68 (s, 3 H), 2.99 (dd, J = 14.1, 5.8
Hz, 1 H), 2.91 (dd, J = 14.1, 7.0 Hz, 1 H).
Further functionalization with suitable N-protecting
group enables the use of the obtained photocaged DOPA de-
rivatives in peptide synthesis. To demonstrate this possibili-
ty, we prepared N-Fmoc-protected amino acid 20, and syn-
thesized a short peptide H-Leu-3-Val-OH using convention-
al Fmoc-solid phase peptide synthesis approach (Scheme
4). HPLC and NMR analysis of the crude product did not re-
veal any side reactions, which might have occurred on the
The NMR data is consistent with the literature.20
Methyl (S)-2-[(tert-Butoxycarbonyl)amino]-3-(3,4-dihydroxyphe-
nyl)propanoate (5)
The compound was synthesized according to the literature.16 In addi-
tion the crude product was purified by column chromatography (sili-
ca gel, n-hexane/EtOAc 1:1). Evaporation of the solvents gave the
product as a white solid; yield: 5.70 g (72%).
NO2
NO2
NO2
O
O
O
OH
OH
OH
(b)
(a)
O
N
CO2H
CO2H
CO2H
H
NH
NH2
NH
H3N
H-Leu-3-Val-OH
Fmoc
O
3
20
Scheme 4 Reagents and conditions: (a) TMSCl, DIPEA, CH2Cl2; then Fmoc-Cl, 53%; (b) solid-phase peptide synthesis conditions.
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2017, 49, A–I

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1H NMR (500 MHz, DMSO-d6): δ = 8.71 (s, 1 H), 8.66 (s, 1 H), 7.11 (d,
J = 7.9 Hz, 1 H), 6.63–6.60 (m, 2 H), 6.44 (d, J = 7.9, 1.8 Hz, 1 H), 4.08–
4.02 (m, 1 H), 3.59 (s, 3 H), 2.78 (dd, J = 13.7, 5.3 Hz, 1 H), 2.67 (dd, J =
13.7, 9.5 Hz, 1 H), 1.34 (s, 9 H).
1H NMR (400 MHz, D2O): δ = 7.71 (d, J = 2.3 Hz, 1 H), 7.36 (dd, J = 8.5,
2.2 Hz, 1 H), 6.87 (d, J = 8.6 Hz, 1 H), 4.15–4.05 (t, J = 7.0 Hz, 1 H),
3.22–2.97 (m, 2 H), 2.55 (s, 3 H).
HRMS (ESI): m/z [M + H]+ calcd for C11H14NO4: 224.0917; found:
224.0916.
The NMR data is consistent with the literature.20
The found data are in agreement with literature.16
Methyl (S)-3-[3,4-Bis(2-nitrobenzyloxy)phenyl]-2-[(tert-butoxy-
carbonyl)amino]propanoate (6)
Methyl (S)-3-(3-Acetyl-4-hydroxyphenyl)-2-aminopropanoate Hy-
Compound 5 (311 mg, 1 mmol), 2-nitrobenzyl bromide (432 mg, 2
mmol), K2CO3 (415 mg, 3 mmol), and NaI (45 mg, 0.3 mmol) were
heated to 50 °C for 48 h in acetone (30 mL). The solvent was evaporat-
ed under reduced pressure. To the residue, 1 M aq KOH (10 mL) was
added and the mixture was extracted with CH2Cl2 (20 mL). The organ-
ic phase was dried (Na2SO4). After evaporation of the solvent under
reduced pressure, the product was obtained as a pale yellow solid;
yield: 505 mg (87%).
1H NMR (500 MHz, CDCl3): δ = 8.17–8.13 (m, 2 H), 8.00–7.95 (m, 2 H),
7.70–7.63 (m, 2 H), 7.51–7.45 (m, 2 H), 6.95 (d, J = 8.3 Hz, 1 H), 6.76
(d, J = 2.0 Hz, 1 H), 6.72 (dd, J = 8.3, 2.0 Hz, 1 H), 5.56–5.47 (m, 4 H),
4.98 (d, J = 7.5 Hz, 1 H, NH), 4.59–4.50 (m, 1 H), 3.71 (s, 3 H), 3.10–
2.92 (m, 2 H), 1.41 (s, 9 H).
drochloride
A solution of 8 (6.32 g, 24.3 mmol) in MeOH (120 mL) was cooled to 0
°C, and SOCl2 (2.00 mL, 27.6 mmol) was added dropwise over 15 min.
After 30 min at 0 °C, the mixture was allowed to warm up to r.t. and
stir for 18 h. The solvent was removed under reduced pressure to
yield the product as a light yellow solid which was used without any
further purification; yield: 6.46 g (97%).
1H NMR (400 MHz, D2O): δ = 7.80 (d, J = 2.3 Hz, 1 H), 7.43 (dd, J = 8.6,
2.3 Hz, 1 H), 7.00 (d, J = 8.6 Hz, 1 H), 4.42 (dd, J = 7.4, 6.0 Hz, 1 H), 3.83
(s, 3 H), 3.32–3.19 (m, 2 H), 2.66 (s, 3 H).
HRMS (ESI): m/z [M + H]+ calcd for C12H16NO4: 238.1047; found:
238.1073.
The found data are in agreement with literature.21
13C NMR (125 MHz, CDCl3): δ = 172.2, 155.1, 148.2, 147.4, 146.9 (2 C),
134.0, 134.0, 133.9, 130.1, 129.0, 128.7, 128.6, 128.4, 128.3, 125.0,
124.9, 123.0, 115.7, 114.7, 78.0, 68.1, 54.4, 52.2, 37.8, 28.3.
Methyl (S)-3-(3-Acetyl-4-hydroxyphenyl)-2-[(tert-butoxycarbon-
yl)amino]propanoate (9)
HRMS (ESI): m/z [M – CO2C4H8]+ calcd for C24H24N3O8: 482.1552;
Methyl (S)-3-(3-acetyl-4-hydroxyphenyl)-2-aminopropanoate hydro-
chloride (6.46 g, 23.6 mmol) was dissolved in H2O/1,4-dioxane (1:1,
100 mL) and cooled to 0 °C. Et3N (8.3 mL, 62. 3 mmol) was added, fol-
lowed by di-tert-butyl dicarbonate (5.68 g, 26.0 mmol). The mixture
was stirred for 18 h, concentrated under reduced pressure to 50 mL,
then acidified to pH 4 with 1 M HCl and extracted with EtOAc (3 × 50
mL). The combined organic phases were washed with sat. NaHCO3
solution (2 × 25 mL), H2O (2 × 25 mL), and brine (25 mL), dried
(Na2SO4), and evaporated under reduced pressure. Purification by
flash chromatography (silica gel, hexanes/EtOAc 2:1) delivered the
desired product as an light brown oil; yield: 5.36 g (67%).
1H NMR (400 MHz, CDCl3): δ = 7.49 (d, J = 2.1 Hz, 1 H), 7.24 (dd, J = 8.5,
1.7 Hz, 1 H), 6.92 (d, J = 8.5 Hz, 1 H), 5.04 (d, J = 7.7 Hz, 1 H), 4.58 (d, J =
7.3 Hz, 1 H), 3.74 (s, 1 H), 3.15–2.95 (m, 2 H), 2.62 (s, 3 H), 1.42 (s, 9
H).
HRMS (ESI): m/z [M + H]+ calcd for C17H24NO6: 338.1598; found:
338.1599.
found: 482.1563.
(S)-2-Amino-3-[3,4-bis(2-nitrobenzyloxy)phenyl]propanoic Acid
Hydrochloride (4·HCl)
Compound 6 (480 mg, 0.82 mmol) was dissolved in 1,4-dioxane (10
mL) and 32% aq HCl (5 mL). The mixture was heated to 60 °C for 16 h.
Subsequently, 32% aq HCl (20 mL) was added. The precipitate was fil-
tered and washed with MeCN (10 mL). The solid was then collected
and dried under vacuum to give the product as a white powder; yield:
240 mg (58%); [α]D20 +2.3 (c 1.00, MeOH/MeCN, 2:1).
1H NMR (500 MHz, DMSO-d6): δ = 13.80 (br, 1 H), 8.44 (br, 3 H), 8.14
(t, J = 7.3 Hz, 2 H), 7.90–7.81 (m, 2 H), 7.81–7.75 (m, 2 H), 7.65–7.59
(m, 2 H), 7.12 (s, 1 H), 7.06 (d, J = 8.3 Hz, 1 H), 6.86 (d, J = 8.3 Hz, 1 H),
5.48 (s, 2 H), 5.46 (s, 2 H), 4.19–4.07 (m, 1 H), 3.17–3.00 (m, 2 H).
13C NMR (125 MHz, DMSO-d6): δ = 170.8, 148.1, 147.7, 147.6, 147.5,
134.5, 134.4, 133.3, 133.2, 129.6, 129.5 (2 C), 129.4, 128.9, 125.3 (2 C),
123.3, 116.4, 115.0, 67.8, 67.7, 53.7, 35.6.
The found data are in agreement with literature.16
HRMS (APCI): m/z [M + H]+ calcd for C23H22N3O8: 468.1407; found:
468.1395.
Methyl (S)-3-[3-Acetyl-4-(2-nitrobenzyloxy)phenyl]-2-[(tert-bu-
toxycarbonyl)amino]propanoate (10)
(S)-3-(3-Acetyl-4-hydroxyphenyl)-2-aminopropanoic Acid Hydro-
Compound 9 (5.36 g, 15.8 mmol) was dissolved in MeCN (100 mL), 2-
nitrobenzyl bromide (3.77 g, 17.5 mmol), K2CO3 (4.60 g, 33.4 mmol),
and TBAB (0.51 g, 1.6 mmol) were added to the mixture and the reac-
tion mixture was stirred at r.t. for 72 h. The mixture was filtered, and
the filtrate was subsequently washed with MeCN (2 × 50 mL) and the
combined organic washings were concentrated under reduced pres-
sure. Purification by flash chromatography (silica gel, hexanes/MTBE
2:1) delivered the product as a dark amber-colored oil; yield: 4.28 g
(57%).
chloride (8)
AlCl3 (22.07 g, 165.5 mmol) was added to a suspension of L-tyrosine
(7.5 g, 41.4 mmol) in nitrobenzene (150 mL) at 0 °C. After the addi-
tion, the mixture was stirred for 15 min and then allowed to warm up
to r.t. Then AcCl (3.53 mL, 3.9 g, 49.6 mmol) was added dropwise and
the mixture was heated to 100 °C for 6 h. The resulting brown slurry
was added to ice (200 g) and 32% aq HCl (40 mL). CH2Cl2 (50 mL) was
added to help solvation of the organic phase, which was then extract-
ed with 6 M HCl (2 × 100 mL). The combined aqueous phases were
concentrated under reduced pressure to 250 mL and allowed to stand
for 18 h at 0 °C. The resulting precipitate was recrystallized (6 M HCl)
to afford the product as an off-white powder; yield: 6.32 g (59%).
1H NMR (400 MHz, CDCl3): δ = 8.18 (dd, J = 8.2, 1.2 Hz, 1 H), 7.85 (d, J =
7.8 Hz, 1 H), 7.70 (td, J = 7.6, 1.2 Hz, 1 H), 7.52 (ddd, J = 8.4, 7.3, 1.1 Hz,
1 H), 7.49 (d, J = 2.0 Hz, 1 H), 7.21 (dd, J = 8.5, 2.4 Hz, 1 H), 6.91 (d, J =
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8.5 Hz, 1 H), 5.57 (s, 2 H), 5.04–4.94 (m, 1 H), 4.60–4.51 (m, 1 H), 3.73
(s, 3 H), 3.11 (d, J = 13.9, 5.2 Hz, 1 H), 3.00 (d, J = 13.9, 6.3 Hz, 1 H), 2.61
(s, 3 H), 1.41 (s, 9 H).
13C NMR (126 MHz, CDCl3): δ = 199.2, 172.1, 156.3, 155.0, 147.0,
134.4, 134.2, 132.9, 131.5, 129.2, 128.8, 128.6, 125.2, 113.3, 67.8, 54.4,
52.4, 49.5, 37.3, 31.6, 28.3.
added. The organic phase was separated and washed with H2O (3 ×
200 mL), it was then filtered, and the filtrate was dried (Na2SO4), and
the solvent was removed under reduced pressure. The crude yellow
product (35 g) was refluxed in CH2Cl2 (1 L) for 1 h, and the hot mix-
ture was filtered. The precipitate contained pure starting material
(2.95 g), which was later recycled. The filtrate volume was reduced to
600 mL under reduced pressure, and the solution was kept at +3 °C for
2 d. The precipitate formed was filtered and washed with cold CH2Cl2
(20 mL). After drying under vacuum the product was obtained as a
light yellow solid; yield: 14.72 g (42% brsm).
1H NMR (500 MHz, DMSO-d6): δ = 9.79 (s, 1 H), 9.76 (br, 1 H), 8.17 (dd,
J = 8.2, 1.0 Hz, 1 H), 7.86 (dd, J = 8.0, 1.1 Hz, 1 H), 7.82 (dt, J = 7.5, 1.0
Hz, 1 H), 7.64 (ddd, J = 8.5, 7.0, 1.0 Hz, 1 H), 7.38 (dd, J = 8.3, 2.0 Hz, 1
H), 7.32 (d, J = 2.0 Hz, 1 H), 7.20 (d, J = 8.3 Hz, 1 H), 5.59 (s, 2 H),
HRMS (ESI): m/z [M + H – Boc]+ calcd for C19H21N2O6: 373.1394;
found: 373.1389.
(S)-2-[(tert-Butoxycarbonyl)amino]-3-[3-hydroxy-4-(2-nitroben-
zyloxy)phenyl]propanoic Acid (11)
To a solution of 10 (4.28 g, 9.1 mmol) in CH2Cl2 and MCPBA (3.91 g,
22.6 mmol) was added at r.t. The mixture was stirred for 7 d, and then
the mixture was washed with 50% NaHCO3 solution (3 × 50 mL), H2O
(50 mL), and brine (25 mL). The organic phase was concentrated un-
der reduced pressure to yield a yellow oil (3.05 g). This was then dis-
solved in H2O/acetone (20 mL, 1:1) and LiOH (0.45 g, 18.8 mmol) was
added. The mixture was stirred at r.t. for 4 h. The mixture was neu-
tralized to pH 7 with 1 M HCl and subsequently lyophilized, to afford
the crude product. This was dissolved in MeCN and purified using Re-
diSep® Combiflash setup. A C-18 reverse phase column (130 g, linear
gradient 20–100% H2O/MeCN, 10 min, 85 mL/min). The solvents were
removed under reduced pressure to afford the product as a light
brown powder; yield: 0.47 g (22%).
1H NMR (500 MHz, CDCl3): δ = 8.15 (d, J = 8.2 Hz, 1 H), 7.77 (d, J = 7.6
Hz, 1 H), 7.67 (t, J = 7.5 Hz, 1 H), 7.51 (t, J = 7.7 Hz, 1 H), 6.76–6.82 (m,
2 H), 6.63 (dd, J = 8.2, 1.5 Hz, 1 H), 5.52 (s, 2 H), 5.03 (d, J = 7.9 Hz, 1 H),
4.55 (d, J = 6.7 Hz, 1 H), 2.98–3.11 (m, 2 H), 1.42 (s, 9 H).
13C NMR (126 MHz, CDCl3): δ = 175.7, 155.5, 147.1, 145.8, 144.5,
134.1, 133.1, 130.1, 128.7, 128.6, 125.1, 121.3, 116.3, 113.0, 80.4, 68.1,
54.2, 37.1, 28.3.
13C NMR (126 MHz, DMSO-d6): δ = 192.0, 152.3, 147.8, 147.7, 134.6,
132.8, 130.9, 129.6 (2 C), 125.5, 124.5, 114.6, 113.9, 67.5.
HRMS (APCI): m/z [M + H]+ calcd for C14H12NO5: 274.0715; found:
274.0708.
5-(Hydroxymethyl)-2-(2-nitrobenzyloxy)phenol (14)
Compound 13 (14.72 g, 53.9 mmol) was dispersed in EtOH (150 mL)
and NaBH4 (2.04 g, 53.9 mmol) was added in small portions. The mix-
ture was stirred for 1 h at r.t. Then H2O (200 mL) and sat. NH4Cl (200
mL) were added. The mixture was extracted with EtOAc (3 × 150 mL).
The combined organic phases were dried (Na2SO4) and the solvent
was evaporated under reduced pressure. The product was obtained as
a light yellow solid; yield: 14.68 g (99%). Crystals for X-ray diffraction
were prepared by gas-phase diffusion of n-pentane into a solution of
16 in pyridine.
1H NMR (400 MHz, DMSO-d6): δ = 9.13 (s, 1 H), 8.14 (dd, J = 8.2, 1.2
Hz, 1 H), 7.90 (dd, J = 7.9, 0.9 Hz, 1 H), 7.80 (dt, J = 7.5, 1.2 Hz, 1 H),
7.64–7.58 (m, 1 H), 6.87 (d, J = 8.2 Hz, 1 H), 6.83 (d, J = 2.0 Hz, 1 H),
6.64 (dd, J = 8.2, 2.0 Hz, 1 H), 5.43 (s, 2 H), 5.02 (t, J = 5.7 Hz, 1 H), 4.34
(d, J = 5.7 Hz, 2 H).
HRMS (ESI): m/z [M + H]+ calcd for C21H24N2O8·NH4: 450.1871; found:
450.1869.
13C NMR (100 MHz, DMSO-d6): δ = 147.2, 146.9, 144.9, 136.4, 134.0,
133.3, 129.1, 128.9, 124.8, 117.2, 114.7, 114.5, 67.3, 62.6.
HRMS (ESI, neg. mode): m/z [M + Cl]– calcd for C14H13ClNO5:
310.0488; found: 310.0482.
(S)-2-Amino-3-[3-hydroxy-4-(2-nitrobenzyloxy)phenyl]propano-
ic Acid Hydrochloride (3·HCl)
Compound 11 (0.47 g, 1.21 mmol) was dissolved in 1,4-dioxane (3
mL) with 32% aq HCl (1 mL). The mixture was stirred for 3 h and the
progress was monitored via TLC (BuOH/EtOAc/AcOH/H2O 2:1:1:1). Af-
ter completion, the mixture was diluted with H2O (10 mL) and lyo-
philized to afford the product as a brown powder; yield: 0.44 g
(quant.); [α]D20 –5.8 (c 1.00, 1 M HCl).
1H NMR (500 MHz, CD3OD): δ = 8.14 (dd, J = 8.1, 1.1 Hz, 1 H), 7.89 (dd,
J = 7.8, 0.8 Hz, 1 H), 7.73 (td, J = 7.5, 1.2 Hz, 1 H), 7.56 (td, J = 7.6, 1.4
Hz, 1 H), 6.89 (d, J = 8.2 Hz, 1 H), 6.83 (d, J = 2.2 Hz, 1 H), 6.70 (dd, J =
8.2, 2.2 Hz, 1 H), 5.52 (s, 1 H), 4.18 (dd, J = 7.9, 5.2 Hz, 1 H), 3.21 (dd,
J = 14.7, 5.2 Hz, 1 H), 3.03 (dd, J = 14.7, 7.9 Hz, 1 H).
5-(Bromomethyl)-2-(2-nitrobenzyloxy)phenol (15)
Compound 14 (14.68 g, 53.3 mmol) was dispersed in anhyd CH2Cl2
(180 mL). The mixture was cooled to 0 °C and PBr3 (5.13 mL, 14.61 g,
53.3 mmol) in CH2Cl2 (20 mL) was added dropwise. After complete
addition, the solution was stirred at 0 °C for 15 min. Then the mixture
was washed with H2O (100 mL), the organic phase was separated, and
the aqueous phase was extracted with CH2Cl2 (2 × 25 mL). The com-
bined organic phases were dried (Na2SO4). The solvent was evaporat-
ed under reduced pressure. The product was obtained as a white
powder; yield: 17.72 g (95%).
13C NMR (126 MHz, CD3OD): δ = 171.2, 148.9, 148.7, 147.5, 134.9,
134.6, 130.2, 129.9, 129.3, 125.9, 121.8, 118.1, 115.6, 69.1, 55.2, 36.7.
1H NMR (500 MHz, CDCl3): δ = 8.18 (d, J = 8.3 Hz, 1 H), 7.73–7.66 (m, 2
H), 7.54 (dt, J = 7.8 Hz, 2.2 Hz, 1 H), 7.03 (d, J = 2.0 Hz, 1 H), 6.85 (dd,
J = 8.3, 2.0 Hz, 1 H), 6.77 (d, J = 8.3 Hz, 1 H), 5.68 (br, 1 H), 5.56 (s, 2 H),
4.44 (s, 2 H).
13C NMR (126 MHz, CDCl3): δ = 147.2, 146.0, 145.3, 134.2, 132.7,
132.1, 129.0, 128.6, 125.3, 121.1, 161.1, 112.6, 68.2, 33.6.
HRMS (ESI, neg. mode): m/z [2 M – H]– calcd for C32H31N4O12
663.1944; found: 663.1943.
:
3-Hydroxy-4-(2-nitrobenzyloxy)benzaldehyde (13)
3,4-Dihydroxybenzaldehyde (12, 20 g, 144.8 mmol) and 2-nitroben-
zyl bromide (31.28 g, 144.8 mmol) were dissolved in DMF (70 mL)
and anhyd K2HPO4 (30.26 g, 173.7 mmol, 1.2 equiv) was added fol-
lowed by addition of NaI (2.16 g, 14.5 mmol, 0.1 equiv). The mixture
was stirred for 20 h at r.t. under a N2 atmosphere. EtOAc (500 mL) was
HRMS (ESI, neg. mode): m/z [M – H]– calcd for C14H11BrNO4:
335.9877; found: 335.9866.
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2017, 49, A–I

G
T. Schneider et al.
Paper
Synthesis
5-(Bromomethyl)-2-(2-nitrobenzyloxy)phenyl tert-Butyl Carbon-
Diethyl 2-Acetamido-2-[3-hydroxy-4-(2-nitrobenzyloxy)ben-
ate (16)
zyl]malonate (18)
Compound 15 (17.27 g, 51.1 mmol) was dispersed in Et2O (750 mL)
and the mixture was cooled to 0 °C. Di-tert-butyl dicarbonate (16.72
g, 76.8 mmol, 1.5 equiv) and DMAP (312 mg, 2.56 mmol, 0.05 equiv)
were added. After this, Et3N (7.08 mL, 5.17 g, 51.1 mmol) in Et2O
(100 mL) was added dropwise to the solution, the mixture was al-
lowed to warm to r.t. and was stirred for 18 h. The mixture was
washed with H2O (200 mL). The aqueous phase was then extracted
with Et2O (2 × 250 mL). The combined organic phases were dried
(Na2SO4) and the solvent volume was reduced to 50 mL under reduced
pressure. The product precipitated from the solvent, and was filtered.
n-Hexane (200 mL) was added to the filtrate and the volume was re-
duced to 150 mL under reduced pressure; the resulting mixture was
kept at 0 °C for 2 h. The resulting precipitate was combined with the
previous. The combined solids were washed with n-hexane (2 ×
20 mL) and dried under vacuum. The product was obtained as a white
powder; yield: 18.91 g (84%). Crystals for X-ray diffraction were pre-
pared gas-phase diffusion of n-pentane into a solution of 16 in CHCl3.
1H NMR (500 MHz, CDCl3): δ = 8.19 (dd, J = 8.2, 1.2 Hz, 1 H), 7.90 (d, J =
7.8 Hz, 1 H), 7.68 (dt, J = 7.7, 1.1 Hz, 1 H), 7.49 (t, J = 7.7 Hz, 1 H), 7.24–
7.19 (m, 2 H), 6.93 (d, J = 8.3 Hz, 1 H), 5.55 (s, 2 H), 4.49 (s, 2 H), 1.53
(s, 9 H).
13C NMR (126 MHz, CDCl3): δ = 151.5, 150.0, 146.8, 140.5, 134.3,
133.5, 131.4, 128.6, 128.5, 127.8, 125.2, 123.9, 114.0, 83.9, 67.6, 32.8,
27.8.
Compound 17 (22.95 g, 40 mmol) was mixed with EtOH (500 mL) and
H2O (250 mL). The mixture was heated to 80 °C and stirred for 20 h.
The solvents were removed under reduced pressure. The product was
obtained as a white powder; yield: 19.0 g (quant.).
1H NMR (400 MHz, CDCl3): δ = 8.16 (d, J = 8.2 Hz, 1 H), 7.74–7.66 (m, 2
H), 7.52 (t, J = 7.5 Hz, 1 H), 6.71 (d, J = 8.2 Hz, 1 H), 6.63 (d, J = 1.9 Hz, 1
H), 6.58 (s, 1 H), 6.45 (dd, J = 8.2, 1.9 Hz, 1 H), 5.61 (br, 1 H), 5.52 (s, 2
H), 4.31–4.20 (m, 4 H), 3.54 (s, 2 H), 2.04 (s, 3 H), 1.29 (t, J = 7.2 Hz, 6
H).
13C NMR (100 MHz, CDCl3): δ = 169.1, 167.5, 147.2, 145.7, 144.4,
134.1, 133.0, 129.6, 128.8, 128.6, 125.2, 121.7, 116.8, 112.5, 68.2, 67.2,
62.7, 37.2, 23.1, 14.0.
HRMS (ESI): m/z [M + H]+ calcd for C23H27N2O9: 475.1711; found:
475.1698.
rac-2-Amino-3-[3-hydroxy-4-(2-nitrobenzyloxy)phenyl]propano-
ic Acid Hydrochloride (rac-3)
Compound 18 (18.7 g, 40 mmol) was mixed with 32% aq HCl (160 mL)
and 1,4-dioxane (100 mL). The mixture was refluxed for 4 h whilst
shielding the mixture from direct sunlight with aluminum foil. After
completion the mixture was concentrated to 100 mL under reduced
pressure and cooled to 0 °C. After 2 h the formed precipitate was fil-
tered, washed with 32% HCl (2 × 30 mL) and EtOAc (2 × 50 mL). After
drying under vacuum the product was obtained as a white powder;
yield: 8.25 g (56%). Spectroscopic data are identical with (S)-3.
HRMS (APCI): m/z [M – Br – Boc]+ calcd for C14H12NO4: 258.0766;
found: 258.0757.
rac-2-Acetamido-3-[3-hydroxy-4-(2-nitrobenzyloxy)phenyl]pro-
panoic Acid (rac-19)
Diethyl 2-Acetamido-2-[3-(tert-butoxycarbonyloxy)-4-(2-nitro-
benzyloxy)benzyl]malonate (17)
Compound rac-3 (4 g, 10.7 mmol) was dispersed in MeCN (70 mL).
Then Et3N (2.96 mL, 2.16 g, 21.3 mmol) and Ac2O (1.01 mL, 1.09 g,
10.7 mmol) were added. The mixture was stirred at r.t. for 20 h. The
solvent was removed under reduced pressure. H2O (80 mL) and 1 M
HCl (20 mL) were added to the residue. The mixture was extracted
with EtOAc (3 × 80 mL) and the combined organic phases were dried
(Na2SO4). The solvent was removed under reduced pressure and the
crude product was dried under vacuum to give the product as a white
solid; yield: 3.88 g (97%).
1H NMR (500 MHz, DMSO-d6): δ = 12.59 (br, 1 H), 9.07 (s, 1 H), 8.16–
8.09 (m, 2 H), 7.91 (d, J = 7.7 Hz, 1 H), 7.80 (t, J = 7.6 Hz, 1 H), 7.61 (t,
J = 7.8 Hz, 1 H), 6.85 (d, J = 8.3 Hz, 1 H), 6.72 (d, J = 2.0 Hz, 1 H), 6.58
(dd, J = 8.3, 2.0 Hz, 1 H), 5.41 (s, 2 H), 4.36–4.30 (m, 1 H), 2.89 (dd, J =
13.8, 5.0 Hz, 1 H), 2.71 (dd, J = 13.8, 9.3 Hz, 1 H), 1.80 (s, 3 H).
NaH (1.90 g, 47.5 mmol, 1.1 equiv, 60% in mineral oil) was dispersed
in anhyd DMF (50 mL) at 0 °C under a N2 atmosphere. Then diethyl
acetamidomalonate (9.37 g, 43.1 mmol) in anhyd DMF (40 mL) was
added dropwise. After complete addition the solution was stirred for
30 min at 0 °C. Then compound 16 (18.91 g, 43.1 mmol) in anhyd
DMF (60 mL) was added dropwise. The solution was allowed to warm
up to r.t., and it was stirred for 4 h. H2O (400 mL) was added. The mix-
ture was extracted with EtOAc (2 × 300 mL). The organic phase was
washed with aq NaCl solution (36 g/L, 3 × 300 mL) and dried (Na2SO4).
The solvent was removed under reduced pressure and the product
was obtained as a white powder; yield: 22.95 g (93%). Crystals for X-
ray diffraction were collected after recrystallization (EtOAc/n-hexane
1:2) of 17.
Note: 36 g/L NaCl solution is employed to ensure efficient phase sepa-
ration while performing the extraction, and minimizing the DMF con-
tent in the final product.
1H NMR (400 MHz, CDCl3): δ = 8.18 (dd, J = 8.2, 1.3 Hz ,1 H), 7.90 (dd,
J = 7.9, 1.0 Hz, 1 H), 7.68 (dt, J = 7.6, 1.3 Hz, 1 H), 7.48 (ddd, J = 8.4, 7.6,
1.3 Hz, 1 H), 6.89–6.78 (m, 3 H), 6.56 (s, 1 H), 5.50 (s, 2 H), 4.31–4.19
(m, 4 H), 3.59 (s, 2 H), 2.02 (s, 3 H), 1.51 (s, 9 H), 1.28 (t, J = 7.2 Hz, 6
H).
13C NMR (126 MHz, DMSO-d6): δ = 173.6, 169.6, 147.6, 147.2, 145.3,
134.5, 133.7, 131.8, 129.6, 129.3, 125.2, 120.2, 117.4, 115.0, 67.8, 54.1,
36.7, 22.8.
HRMS (ESI): m/z [M + H]+ calcd for C18H19N2O7: 375.1192; found:
375.1193.
(S)-2-Amino-3-[3-hydroxy-4-(2-nitrobenzyloxy)phenyl]propano-
ic Acid Hydrochloride (3·HCl) and (R)-2-Acetamido-3-[3-hydroxy-
4-(2-nitrobenzyloxy)phenyl]propanoic Acid [(R)-19]
13C NMR (100 MHz, CDCl3): δ = 169.4, 167.5, 151.6, 149.1, 146.7,
140.22, 134.3, 133.8, 128.9, 128.5, 128.5, 128.4, 128.1, 125.1, 124.8,
113.9, 83.7, 67.5, 67.3, 62.9, 37.0, 27.8, 23.2, 14.2.
Compound rac-19 (3.88 g, 10.4 mmol) was dissolved in 0.2 M
Na2HPO4 solution (200 mL). Acylase I from Aspergillus melleus (80 mg,
>0.5 U/mg) was added. The mixture was stirred for 18 h at 40 °C. After
this EtOAc (200 mL) and 1 M aq HCl (280 mL) were added. The organic
layer was separated and the aqueous phase was extracted with EtOAc
t
HRMS (APCI): m/z [M + H – Bu]+ calcd for C24H27N2O11: 519.1609;
found: 519.1595.
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2017, 49, A–I

H
T. Schneider et al.
Paper
Synthesis
(2 × 200 mL). The organic phase was dried (Na2SO4) and the solvent
was removed under reduced pressure to give (R)-19 (2.1 g) as a light
brown powder.
resin by mixing with HFIP/CH2Cl2 (1:3) for 20 min. The NMR analysis
was performed directly on the sample of the crude peptide in CD3OD
in order to reveal possible side products of the synthesis.
The aqueous layer was adjusted to pH 6 with NaHCO3 (Note: strong
foam formation) and then filtered. The residue was washed with H2O
(2 × 30 mL). For the formation of the hydrochloride the product was
dissolved in 1 M HCl (20 mL) and MeCN (10 mL) and the solvent was
evaporated. After drying under vacuum 3·HCl was obtained as a light
yellow powder; yield: 1.25 g (33%). Spectroscopic data: see above.
HRMS (ESI): m/z [M + H]+ calcd for C27H38N4O8: 545.2611; found:
545.2589.
Acknowledgment
We gratefully acknowledge the UniCat Cluster for supporting this
project, and T.S. acknowledges the SFB-765 for financial support.
Racemization of (R)-2-Acetamido-3-[3-hydroxy-4-(2-nitrobenzyl-
oxy)phenyl]propanoic Acid
Compound (R)-19 (2.1 g, 4.8 mmol) was dissolved in AcOH (80 mL).
NaOAc (2.3 g, 28.1 mmol) and Ac2O (2.65 mL, 2.87 g, 28.1 mmol) were
added. The mixture was heated to 50 °C and stirred for 18 h. The sol-
vent was removed under reduced pressure and the residue was dried
under vacuum. Sat. aq NaHCO3 solution was added to adjust the final
solution to pH 8; this was then heated to 50 °C and stirred for 18 h.
The mixture was acidified to pH 1 using 32% aq HCl and extracted
with EtOAc (4 × 30 mL). The combined organic phases were dried
(Na2SO4) and the solvent was removed under reduced pressure; the
residue was additionally dried under vacuum. The product was ob-
tained as a light brown powder; yield: 2.05 g (98%).
Supporting Information
Supporting information for this article is available online at
http://dx.doi.org/10.1055/s-0036-1588766.
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References
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Compound (S)-3 (200 mg, 0.54 mmol) was dispersed in anhyd CH2Cl2
(10 mL). The mixture was cooled to 0 °C and DIPEA (0.566 mL, 420
mg, 3.25 mmol) and TMSCl (0.275 mL, 236 mg, 2.17 mmol) were add-
ed and the mixture was stirred for 30 min. Next, Fmoc-Cl (154 mg,
0.54 mmol) was added, and the solution was allowed to warm up to
r.t., and stirring was continued for 3 h. The solvent was removed un-
der vacuum. The resulting oil was taken up in sat. NaHCO3 solution
(20 mL), H2O (20 mL) and acetone (10 mL) were added. This mixture
was washed with Et2O (2 × 20 mL), and the aqueous phase was acidi-
fied to pH 1 with 1 M aq HCl. The aqueous phase was extracted with
EtOAc (3 × 20 mL). The combined organic phases were dried (Na2SO4)
and the solvent was removed under reduced pressure to yield the
product as a white powder; yield: 160 mg (53%).
1H NMR (400 MHz, CDCl3): δ = 8.14 (d, J = 8.1 Hz, 1 H), 7.73–7.61 (m, 3
H), 7.62 (t, , J = 7.2 Hz, 1 H), 7.54 (t, J = 7.5 Hz, 2 H), 7.48 (t, J = 7.8 Hz, 1
H), 7.40–7.34 (m, 2 H), 7.31–7.26 (m, 2 H), 6.79 (s, 1 H), 6.73 (d, J = 8.0
Hz, 1 H), 6.57 (d, J = 8.0 Hz, 1 H), 5.49 (s, 2 H), 5.26 (d, J = 8.0 Hz, 1 H),
4.66 (s, 1 H), 4.43 (dd, J = 10.3, 7.3 Hz, 1 H), 4.43 (t, J = 8.2 Hz, 1 H),
4.18 (t, J = 6.9 Hz, 1 H), 3.15–2.80 (m, 2 H).
(8) (a) Hwang, D. S.; Yoo, H. J.; Jun, J. H.; Moon, W. K.; Cha, H. J. Appl.
Environ. Microbiol. 2004, 70, 3352. (b) Hwang, D. S.; Gim, Y.;
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J.; Lu, Q.; Zeng, H.; Cha, H. J. Angew. Chem. Int. Ed. 2014, 53,
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13C NMR (100 MHz, CDCl3): δ = 175.7, 156.0, 147.3, 146.0, 144.7,
143.9, 143.8, 141.4, 134.2, 133.2, 129.8, 128.9, 128.7, 127.9, 127.2,
125.3, 121.5, 120.1, 116.4, 113.1, 68.2, 67.3, 54.6, 47.3, 37.3.
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HRMS (ESI): m/z [M + H]+ calcd for C31H27N2O8: 555.1767; found:
555.1752.
(11) (a) Klán, P.; Šolomek, T.; Bochet, C. G.; Blanc, A.; Givens, R.;
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Peptide Synthesis
The synthesis was performed starting from a Fmoc-Val-preloaded 2-
chlorotrityl chloride resin (70 mg) using a previously described proto-
col.22 Fmoc-Leu (4 equiv) and 20 (1.3 equiv) were used for coupling,
while TBTU/HOBt/DIPEA (0.98:1:2 equiv to the Fmoc-amino acid)
were used for activation, which was performed by mixing in DMF pri-
or to adding to the resin. The coupling time was 45 min for Fmoc-Leu
and 2 h for 20. For Fmoc-deprotection the resin was mixed with 22
wt% piperidine in DMF for 20 min. The peptide was cleaved from the
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2017, 49, A–I

I
T. Schneider et al.
Paper
Synthesis
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© Georg Thieme Verlag Stuttgart · New York — Synthesis 2017, 49, A–I