Inverse γ-Turn-Inspired Peptide: Synthesis and Analysis of Segetalin A Indole Hemiaminal

Article abstract of DOI:10.1002/ejoc.201501179

Substitution of a peptide bond for an imine transforms the irreversible macrocyclization of peptides into a reversible process. The inherent cyclization tendency of a linear peptide is then analyzable through the equilibrium between the aldehyde and the imine by virtue of the higher reactivity of the corresponding linear peptide aldehyde. The tryptophan side chain of segetalin A aldehyde forms a 12-membered cyclic indole hemiaminal instead of the 18-membered macrocyclic imine expected. Herein, we analyzed this uncommon hemiaminal that shows that the biosynthesis of cyclic peptides is not necessarily based on linear precursor peptides with a high inherent macrolactamization tendency. By substituting a peptide bond for an imine, the cyclization tendency of a linear peptide can be analyzed through equilibration of the aldehyde and imine forms. The tryptophan side chain of segetalin A aldehyde forms a 12-membered cyclic indole hemiaminal instead of the expected 18-membered macrocyclic imine. Herein, we analyze this uncommon hemiaminal.

Full text of DOI:10.1002/ejoc.201501179

FULL PAPER
DOI: 10.1002/ejoc.201501179
Inverse γ-Turn-Inspired Peptide: Synthesis and Analysis of Segetalin A Indole
Hemiaminal
Matthias Lamping,[a] Sebastian Enck,[a] and Armin Geyer*[a]
Keywords: Peptides / Aldehydes / Cyclization / Macrocycles / Nitrogen heterocycles
Substitution of a peptide bond for an imine transforms the
irreversible macrocyclization of peptides into a reversible
process. The inherent cyclization tendency of a linear pept-
ide is then analyzable through the equilibrium between the
aldehyde and the imine by virtue of the higher reactivity of
the corresponding linear peptide aldehyde. The tryptophan
side chain of segetalin A aldehyde forms a 12-membered
cyclic indole hemiaminal instead of the 18-membered macro-
cyclic imine expected. Herein, we analyzed this uncommon
hemiaminal that shows that the biosynthesis of cyclic pept-
ides is not necessarily based on linear precursor peptides
with a high inherent macrolactamization tendency.
cases, the expected macrocyclic ring with the correct ring
size was formed, although many side-chain functionalities
caused side reactions. Therefore, in the case of segetalin A
we also expected a clean reaction to the macrocyclic pept-
ide. Substitution of the amide bond can take place at any
amino acid and leads to cyclic imines; in the case of proline,
the functionality formed is an enamine. Owing to fast race-
mization of aldehydes, nonracemizable glycine is often cho-
sen as an aldehyde moiety. Another advantage of this reac-
tion is the use of water or phosphate buffer as the solvent.
This enables NMR-spectroscopy-based analysis of the reac-
tion, even at very low concentrations. Furthermore, the
aqueous solution promotes intramolecular interactions,
such as hydrogen bonding and the hydrophobic effect. On
the basis of this concept, we analyzed several cyclic peptides
with regard to their inherent cyclization tendency and
studied linear peptide aldehyde 1 of the corresponding nat-
ural product segetalin A (Figure 1). Despite the low reactiv-
ity of the indole NH group, as described already, the revers-
ible cyclization of 1 should lead to 18-membered macro-
cyclic imine 2. However, against all expectations, we iden-
tified 12-membered indole hemiaminal 3, which was exclu-
sively formed. Thus, the lower reactivity of the indole NH
Introduction
Indole derivatives have received much attention in medi-
cal chemistry and natural products synthesis.[1–3] A host of
natural products contain the indole core in the form of
alkaloids and tryptophan derivatives, which creates new
challenges for synthetic chemistry and offers new features
and surprising reactivity.[4] Indole hemiaminals, which have
already proved their antitumor properties in the inhibition
of tubuline polymerization, are also interesting as structural
motifs.[5,6] In peptide drugs, the indole skeleton is repre-
sented by the side chain of tryptophan and is based on the
slight reactivity of the indole NH group; this functional
group will not react during conventional peptide synthesis.
The conditions required to acylate the aromatic nitrogen
atom are quite harsh and are not achieved during peptide
macrocyclization.[7]
In many cases, macrocyclization in peptide natural prod-
uct synthesis is the limiting step, and in biosynthetic routes,
this cyclization is often supported by complex enzyme ma-
chineries.[8] The substitution of a nonhydrolyzable amide
bond [CONH] through a pH-dependent hydrolyzable iso-
steric imine Ψ[CH=N] or iminium Ψ[CH=NH+] bond in a
macrocyclic peptide has already been established by our
group for the cyclopeptide tyrocidine A and the nostocyclo-
peptides.[9,10] Through this substitution, the irreversibility
of the macrocyclization will be converted into a reversible
process, which can answer the question whether the linear
or cyclic peptide is preferred. Additionally, in the cases of
tyrocidine A and the nostocyclopeptides, thermodynamic
analysis of the cyclization process was possible.[9,10] In both
[a] Institute of Chemistry, Philipps-University Marburg
Hans-Meerwein-Strasse, 35032 Marburg, Germany
E-mail: geyer@staff.uni-marburg.de
Figure 1. The linear peptide aldehyde (yellow, 1) can react with the
N-terminal amine (red, 2) or with the indole NH (green, 3). The
competition reaction between both functionalities is monitored by
1H NMR spectroscopy. In this case, we could only detect a new
signal for the addition of the indole NH group.
https://www.uni-marburg.de/fb15/ag-geyer
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201501179.
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© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
7443

M. Lamping, S. Enck, A. Geyer
FULL PAPER
group is compensated by the high reactivity of the aldehyde.
In this work, we analyzed the reversible reaction and the
1
resulting structural motif by H NMR spectroscopy.
Results and Discussion
To perform the NMR experiments, we synthesized pept-
ide aldehyde 1, a linearized derivative of segetalin A cyclo-
[–Val–Pro–Val–Trp–Ala–Gly]. The synthesis route started
with 9-fluorenylmethoxycarbonyl (Fmoc) protected glycine
4, which was transformed into the corresponding Weinreb
amide 5 (Scheme 1). After selective lithium aluminum
hydride (LAH) reduction, aldehyde 6 formed an oxazolid-
ine, which was directly tert-butoxycarbonyl (Boc) protected.
The corresponding amino alcohol is unprotected threonine
7, and it was covalently linked through glycine as a spacer
to a NovaSyn TG resin. The loaded resin 8 was then used
in a conventional Fmoc-based solid-phase peptide synthesis
Scheme 1. Synthesis of Fmoc-glycine aldehyde loaded TG resin 8.
The black dot symbolizes the TG resin. (A) Reagents and condi-
tions: (a) N,O-dimethylhydroxylamine (2.6 equiv.), 2-(1H-benzotri-
azol-1-yl)-1,1,3,3-tetramethyluronium hexauorophosphate (HBTU,
1.1 equiv.), hydroxybenzotriazole (HOBt, 1.1 equiv.), N,N-diiso-
propylethylamine (DIPEA, 2.6 equiv.), DMF, r.t., 4 h. (b) LAH
(2.5 equiv.), THF, 0 °C, 20 min. (B) Reagents and conditions: THF,
4 h, 50 °C.
Under standard cyclization conditions, it cannot be dis-
(SPPS). After cleavage with AcOH/H2O/CH2Cl2/MeOH tinguished whether a macrocyclic ring has a constrained
(10:5:63:22), the peptide aldehyde was purified by semipre- conformation or a low-energy conformation stabilized by
parative HPLC. The cyclic hexapeptide segetalin A was iso- intramolecular hydrogen bonds. So, if aldehyde 1 is sub-
lated from the seeds of Vaccaria segetalis (Caryophyllaceae), jected to reversible imine cyclization, neither the structure
and it shows potent estrogen-like activity.[11] The structure of 12-membered ring 3 nor the type of covalent bond –
of native segetalin A has been well studied by crystal-struc- an indole hemiaminal – is comparable to known peptides
ture analysis and in solution by NMR spectroscopy.[12,13] In (Figure 1 and Scheme 2). Surprisingly, peptide aldehyde 1
the solid state, segetalin A is characterized by two β turns, does not show any tendency to form the anticipated 18-
one type I (Trp4 and Ala5) and one type VI (Val1 and Pro2) membered ring 2a, although segetalin A is known as a cy-
with a cis-Pro bond.[12] The structure is fixed by two trans- clic natural product. All molecular species involved can be
annular hydrogen bonds formed between Gly6 and Val3, differentiated by NMR spectroscopy and can, thus, be clas-
which results in an overall antiparallel β-sheet structure. In sified by their pH and temperature dependency. Achiral gly-
solution, the CO–Val3 group of the cyclic hexapeptide cine could be used as the perfect ring-closing position for
forms two hydrogen bonds to NH–Gly6 and NH–Val1. That the cyclization of segetalin A aldehyde 1. If 1 is dissolved
changes the type II turn into a type I turn.[12] It is known in aqueous buffer at pH = 3 (Figure 2), the conformation
1
that it adopts a rigid main conformation without the forma- adopted is almost exclusively open chain. In the H NMR
tion of a second set of NMR signals for the trans-Pro rot- spectrum, the resonance for the NH protons is found at δ
1
amer.[13] The H NMR spectrum of the linear peptide alde- = 0.78 ppm; this is quite low relative to the same signal for
hyde 1 shows instead only the trans conformer. The config- the cyclic peptide (δ = 1.61 ppm).[12,13] Increasing the pH
uration of the Val–trans-Pro rotamer was assigned on the value stepwise to 6.5 should lead to head-to-tail cyclization
basis of the chemical shift of the β- and γ-CH2 groups (δ = of the macrocyclic 18-membered imine. Indeed, the ring
29.0 and 24.5 ppm, respectively) of the proline by HSQC.[14] that is formed could not be assigned to the expected macro-
In contrast to most peptides containing Pro, no other signal cycle 2a. Instead, the addition of the indole NH group to
1
set for the cis-Pro rotamer was observed in the H NMR the
glycine
aldehyde,
which
forms
indole
spectrum of 1, 1a, or 3. Additionally, no exchange signals hemiaminal 3, is identified in the HMBC spectrum through
were visible for a secondary set of NMR signals in the a long-range correlation (Figure 3a). The observation of
homonuclear 2D NMR spectra (TOCSY and ROESY, Fig- this cross peak is due to the antiperiplanar arrangement of
ure 3). The stretched conformation was, furthermore, con- the bonds, as shown in the energy-minimized 3D structure
firmed through the relatively weak NHi–Hαi ROEs and in- (Figure 4). The side chain of Trp is flexible enough to ap-
tense contacts of Hαi–NHi+1 across the whole peptide chain proach the C-terminus and to cyclize the peptide through
of 1. In this context, we analyzed the influence of the i–1 the indole NH group. In the HMBC spectrum, the cross
and i+1 amino acids to proline by synthesizing tripeptides. peak caused by JC,H coupling between HaminalR–Gly and
3
Determination of the cis/trans ratio by 1H NMR spec- C7a–Trp can be recognized. The corresponding HMBC and
troscopy showed that β-branched amino acids lead to a ROESY spectra are displayed in Figure 3. The covalent
higher amount of trans-Pro. The valine residues in both po- structure of the postulated hemiaminal 3 is proven by this
sitions in segetalin A could explain the trans configuration coupling and the subsequent ROE signals. The low cycliza-
(for more details, see the Supporting Information). With tion tendency of segetalin A aldehyde is, therefore, ex-
this information in hand, it was exciting to analyze the cy- plained by the preferred stretched conformation of the N-
clization tendency of linear peptide aldehyde 1.
terminal amino acids. The maximum amount of the formed
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Eur. J. Org. Chem. 2015, 7443–7448

Synthesis and Analysis of Segetalin A Indole Hemiaminal
Scheme 2. Reversible cyclization of segetalin A aldehyde in aqueous phosphate buffer. Peptide 1 cyclizes reversibly at pH = 6.5 to 12-
membered hemiaminal 3 with a diastereomeric ratio of 60:40 at the stereocenter of the hemiaminal instead of forming 18-membered cis-
Pro cyclic imine 2a. Oxidation of hemiaminal 3 with tetrapropylammonium perruthenate (TPAP) and N-methylmorpholine N-oxide
(NMO) in acetonitrile yields cyclic amide 9, which hydrolyzes quickly to linear segetalin A (10). Owing to methylation, peptide 1a cannot
form the indole hemiaminal and also shows no head-to-tail cyclization to 2b.
cyclic peptide is 30% at pH = 6.5 and a temperature of cyclization of the side chain, which, therefore, should lead
320 K in a R/S stereoisomer ratio of 60:40 (Scheme 2). The to the formation of the macrocyclic ring. The correspond-
selective N-methylation of the indole NH group suppresses ing amino acid was synthesized in two steps, and the syn-
thesis started with Boc-protected tryptophan 11. The indole
NH group was deprotonated by using tBuOK in THF and
was methylated by using MeI. After aqueous workup, the
methylated amino acid was directly deprotected with tri-
fluoroacetic acid (TFA)/CH2Cl2 (3:1), and the correspond-
ing TFA salt 12 was obtained in 95% yield.
The building block for the SPPS was Fmoc-protected,
and 13 was afforded in an overall yield of 67% (Scheme 3).
The peptide synthesis of the methylated derivative was ac-
complished in analogy to the synthesis of peptide aldehyde
1. The blocked indole nitrogen atom cannot react, and the
formation of the 12-membered hemiaminal is, therefore,
suppressed. Instead, we expected the formation of the 18-
membered cyclic imine, which is the only possible product.
Upon performing the NMR experiment as described pre-
viously, we could not detect any new NMR signals; conse-
quently, the linear peptide aldehyde also does not form any
cyclic product or oligomers, only the hydrate. This also con-
firmed the already described stretched conformation of the
Val–trans-Pro rotamer of 1.
Owing to their importance as model systems with a mini-
mum antiparallel β-sheet structure, cyclic hexapeptides are
among the most intensively studied macrocyclic rings.[15,16]
An explanation for the formation of hemiaminal 3 could be
the structure motif itself, which shows surprising analogy
1
Figure 2. Excerpts from the WATERGATE H NMR spectra of 1
to an inverse γ turn. The definition of a γ turn includes a
and 3 at different temperatures and pH values (600 MHz; H2O/
D2O, 5:1; H3PO4/KH2PO4). (A) The indole NH group and the
aldehyde signal between δ = 9 and 10.5 ppm (highlighted in yellow)
hydrogen bond between the CO group of the ith amino acid
and the (i+2)th amino acid.[17] Most of the time, classic γ
at 300 K and pH = 3 are characteristic for open-chain peptide 1.
(B) At pH = 6.5 and 300 K, a new signal set for cyclic hemiaminal
3 is detectable, and the corresponding signals are highlighted in
green. The ratio between linear and cyclic under these conditions
is about 85:15. (C) We raised the temperature to 320 K at pH = 6.5
to increase the ratio up to 70:30.
turns can be found at the ends of β hairpins, whereas the
inverse γ turn can be involved in the folding of β strands as
intermediates.[18,19]
The proposed structure shown in Figure 4 is based on
ROE signals that are assigned in the ROESY spectra start-
Eur. J. Org. Chem. 2015, 7443–7448
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M. Lamping, S. Enck, A. Geyer
FULL PAPER
Scheme 3. Synthesis of Fmoc-protected, methylated tryptophan
building block 13. Reagents and conditions: (a) (1) tBuOK
(2.0 equiv.), MeI (1.05 equiv.), DMF/THF (1:1), 0 °C, 2 h; (2) TFA/
CH2Cl2 (3:1), 0 °C, 3 h. (b) Fmoc-succinimide (1.50 equiv.), 10%
aq. Na2CO3, acetone, r.t., 16 h.
bars. The distinction between the two possible planar chiral
stereoisomers is made through the local network of ROEs,
which is formed by the 2H of the indole side chain (arrows
in Figure 4). The ROE signals of Hα–Trp could not be iden-
tified, because the chemical shift is located next to water
suppression. The ROE signals of indole-2H to HβProS–Trp,
Hα–Gly, Hα–Ala, HaminalR–Gly, and NH–Gly are assigned.
Detailed information of the ROE signals is given in the
Supporting Information. Accordingly, hemiaminal 3 dis-
played in Figure 4 shows close similarity to a γi loop struc-
tural motif, in which Ala takes the central i+1 position.
Trp–NH possesses a large temperature dependency
(–10 ppbK–1) of the chemical shift, which is typical for sol-
vent-oriented NH groups. The temperature gradient of
Ala–NH (–6 ppbK–1) is significantly lower, which can be
explained by a lower solvent accessibility. The value for
Gly–NH is –3 ppbK–1, which is indicative of coordination
with Trp–CO, and this is typical for a hydrogen bond. With
a value of φ = –102°, the φ torsion angle (COi–Ni+1–Cαi+1
–
COi+1) of Ala differs by about –20° from the idealized value
of a γi loop. However, this is largely compensated by the
opposite deviation of the ψ torsion angle (Ni+1–Cαi+1
–
Figure 3. Excerpts from HMBC and ROESY spectra of 1/3
(600 MHz; 310 K; H2O/D2O, 5:1; H3PO4/KH2PO4; pH = 6.5).
(a) The 3JC,H correlation between C7a–Trp and HaminalR–Gly in the
HMBC spectrum is clearly separated from the cross peaks within
the indole ring. The arrangement of the two antiperiplanar atoms
leads to a large coupling, which causes an intensive cross peak. No
cross peak is observed for the gauche arrangement in the dia-
stereomeric ring with HaminalS–Gly. (b) In the ROESY spectrum
shown, the cross peaks of HaminalR–Gly and the indole-2H are high-
lighted by bars.
COi+1–Ni+2) by about +20° (ψ = +93°). The two larger nu-
meric values, relative to the idealized value of a γi loop, lead
to an overall straighter conformation of the γi loop, without
detracting from the characteristic intramolecular hydrogen
bond Gly–NH–Trp–CO. The 12-membered ring was oxid-
ized to promote and analyze hemiaminal formation more
precisely. The oxidation of indole hemiaminal 3 to amide 9
was realized by the method of Scheidt and Maki involving
catalytic dehydrogenative coupling of indoles and primary
alcohols.[7] There, the resulting hemiaminal, which was also
generated in organic solvents, was oxidized to the corre-
sponding amide 9 by using TPAP and NMO as co-oxidants
(Scheme 2). Here, the reaction was performed in acetonitrile
at room temperature to yield crude product 9 of the oxid-
ized hemiaminal, which could only be detected by ESI-MS:
m/z = 674.3276 [M + Na+] (calcd. m/z = 674.3273).
Figure 4. Energy-minimized conformation of the 12-membered
ring of hemiaminal 3. The NOE contacts from the rotating frame
NOESY spectrum are displayed by arrows (ROESY: Figure 3b).
The oxidation was performed with N-acylated segeta-
lin A aldehyde. The reason for the low yield and difficult
isolation of the corresponding amide is the high reactivity
of 12-membered cyclic acylindoles. Such motifs were al-
ing from the indole-2H of the R stereoisomer. The corre- ready described by Katrizky et al. as active acylating esters
R
sponding signals are blue, and the ROE signals of Haminal
–
in a similar system.[20] It was found that these 12-membered
Gly and diastereomeric HaminalS–Gly are highlighted by rings occur as intermediates in inter- and intramolecular
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Synthesis and Analysis of Segetalin A Indole Hemiaminal
(s, 3 H, NCH3) ppm. 13C NMR (75 MHz, [D6]DMSO, 300 K): δ
= 156.6 (Fmoc-CO), 140.7, 143.8 (each Fmoc-Carom.,quart.), 120.1,
125.2, 127.0, 127.6 (each Fmoc-CHarom.), 65.7 (Fmoc-CH2), 61.1
(OCH3), 46.6 (Fmoc-CH), 41.1 (αCH), 32.1 (NCH3) ppm. HRMS
(ESI): calcd. for C19H20N2O4Na+ 363.1315; found 363.1313.
acylation reactions and are, therefore, prone to hydrolysis.
This could also be observed by ESI-MS: m/z = 692.3377
[M + Na+] (calcd. m/z = 692.3378).
Nα-Fmoc-Gly-H (6): A solution of Nα-Fmoc-Gly N,O-dimethyl-
hydroxamic acid (5; 1.10 g, 3.23 mmol, 1.0 equiv.) in THFabs
(10 mL) was cooled to 0 °C under nitrogen. LiAlH4 (306 mg,
8.08 mmol, 2.5 equiv.) was added in small portions under vigorous
stirring over 15 min. After complete addition, the mixture was
stirred at 0 °C for 5 min. It was then carefully diluted with EtOAc
(10 mL) and 10% aq. citric acid (10 mL) and stirred at room tem-
perature for another 15 min. The phases were separated, and the
aqueous phase was extracted with EtOAc (3ϫ 10 mL). The com-
bined organic phases were washed with 5% aq. NaHCO3, H2O,
and 2 n aq. HCl. After washing with brine (2ϫ) and drying with
MgSO4, the solution was filtered and concentrated in vacuo. The
crude product was purified by flash chromatography (petroleum
ether/EtOAc, 2:1) to yield aldehyde 6 (435 mg, 1.55 mmol, 48%) as
a crystalline colorless solid. Rf = 0.32 (petroleum ether/EtOAc, 2:1).
1H MR (300 MHz, [D6]DMSO, 300 K): δ = 9.46 (s, 1 H, CHO),
Conclusions
We synthesized a linear peptide aldehyde precursor of the
cyclic peptide segetalin A and analyzed its cyclization as a
reversible process in water. In previous examples, the revers-
ible cyclization of a peptide aldehyde led to the expected
macrocyclic ring despite the presence of reactive side chains.
In this work, the peptide aldehyde surprisingly showed no
tendency for macrocyclic ring closure in the equilibrium;
instead, we observed the formation of a 12-membered cyclic
hemiaminal. The indole-based structure of this unexpected,
inverse γ-turn-like structure motif was elucidated by 2D
NMR spectroscopy. The high reactivity of the aldehyde
shows the great potential of this type of reaction and pro-
vides new perspectives for studies regarding the inherent
cyclization tendency of natural products with a peptide
backbone. Furthermore, this approach enables the analysis
of topologically challenging knotted peptides, such as
microcin J25 and capistruin, which can also be synthesized
as peptide aldehydes.
7.90 (d, JHH = 7.4 Hz, 2 H, Fmoc-CHarom.), 7.72 (d, JαH/NH
=
7.2 Hz, 2 H, Fmoc-CHarom.), 7.31–7.44 (m, 4 H, Fmoc-CHarom.),
4.22–4.35 (m, 3 H, Fmoc-CH2, Fmoc-CH), 3.84 (m, 2 H, αH) ppm.
13C NMR (75 MHz, [D6]DMSO, 300 K): δ = 200.2 (CHO), 156.6
(Fmoc-CO), 143.8–140.7, (each Fmoc-Carom.,quart.), 120.1, 125.1,
127.0, 127.6 (each Fmoc-CHarom.), 65.7 (Fmoc-CH2), 59.7 (αCH),
46.6 (Fmoc-CH) ppm. HRMS (ESI): calcd. for C17H15NO4Na+
304.0944; found 304.0944.
Experimental Section
H-L-Trp-(NMe)-OH (12): A solution of Boc-l-Trp-OH (11;
300 mg, 986 μmol, 1.0 equiv.) in DMF/THF (1:1, 10 mL) was
cooled to 0 °C under nitrogen. 1 n tBuOK in THF (2.00 mL,
2.00 mmol, 1.05 equiv.) was added at 0 °C. The mixture was stirred
for 10 min, and MeI (64.5 μL, 1.03 mmol, 1.03 equiv.) was added
in one portion. After stirring at room temperature for 3 h, the reac-
tion was quenched with 2 n aq. HCl, the mixture extracted with
EtOAc (3ϫ 10 mL) and washed with 10% aq. citric acid and H2O.
After washing with brine (2ϫ) and drying with MgSO4, the solu-
tion was filtered and concentrated in vacuo. The crude product [Rf
= 0.31 (EtOAc)] was dissolved in TFA/CH2Cl2 (3:1; 4 mL) at 0 °C
and stirred for 4 h. The mixture was coevaporated with toluene and
General Information: The reactions were performed under nitrogen
at ambient pressure. Thin-layer chromatography was performed on
silica gel 60 F254 (Merck KGaA), and detection was performed by
fluorescence quenching under UV light (λ = 254 nm) or by staining
with ninhydrin solution followed by heating to 500 °C. Flash
chromatography was performed on silica gel 60 (0.040–0.063 mm)
from Merck KGaA. Semipreparative HPLC was performed with a
Dionex HPLC system with a diode array detector by using an ACE
HPLC C18 RP, 7.75ϫ150 mm, and analytical HPLC was per-
formed with the same system by using an Intersil ODS-4, C18 RP,
3.0ϫ150 mm. The NMR spectra were recorded with Bruker AV
600 spectrometers. Chemical shifts (δ) are given in ppm and are
referenced to the solvent signal. The coupling constants are 3J cou-
plings unless otherwise indicated. All mass spectra were recorded
with a Finnigan LTQ-FT spectrometer. The SPPS was performed
on an H–Thr–Gly–NovaSyn TG resin from Merck KGaA.
chloroform to afford the TFA salt of S5 (310 mg, 933 μmol, 95%).
2
1H NMR (300 MHz, [D6]DMSO, 300 K): δ = 3.02 (dd, JHH
=
14.5 Hz, JHH = 9.4 Hz, 1 H, βHdiast.), 3.15–3.21 (m, 1 H, βHdiast.),
3.70 (s, 1 H, NCH3), 4.19–4.23 (m, 1 H, αH), 7.14 (s, 1 H, 2-Hindol),
7.24–7.73 (m, 4 H, CHindole), 7.88 (d, JNH/αH = 7.6 Hz, 3 H, NH),
12.68 (br. s, 1 H, COOH) ppm.
Nα-Fmoc-Gly-N,O-dimethylhydroxamic Acid (5): HBTU (2.62 g,
6.92 mmol, 1.1 equiv.), HOBt·H2O (1.06 g, 6.92 mmol, 1.1 equiv.),
DIPEA (2.78 mL, 16.4 mmol, 2.6 equiv.), and N,O-dimethylhy-
Fmoc-
L-Trp-(NMe)-OH (13): Fmoc-OSu (365 mg, 1.08 mmol,
1.2 equiv.) was added to a solution of H-l-Trp-(NMe)-OH as the
droxylamine hydrochloride (1.60 g, 16.4 mmol, 2.6 equiv.) were TFA salt (12; 300 mg, 903 μmol, 1.0 equiv.) in acetone/10% aq.
added at room temperature to a solution of Fmoc-Gly-OH (S1;
1.87 mg, 6.29 mmol, 1.0 equiv.) in DMF (20 mL). After stirring for
4 h, the mixture was diluted with EtOAc (200 mL) and then washed
with 5% aq. NaHCO3 (3ϫ), 10% aq. citric acid (2ϫ), and brine
(3ϫ). The organic phase was dried with MgSO4, filtered, and con-
centrated in vacuo to afford Weinreb amide 5 in quantitative yield
(2.12 g, 6.24 mmol) as a pale yellow waxy solid. Rf = 0.62 (EtOAc/
toluene, 7:1). 1H NMR (300 MHz, [D6]DMSO, 300 K): δ = 7.90
(d, JHH = 7.3 Hz, 2 H, Fmoc-CHarom.), 7.72 (d, JHH = 7.7 Hz, 2
Na2CO3 (1:1; 10 mL). After stirring for 16 h, the mixture was di-
luted with H2O (10 mL) and extracted with Et2O (3ϫ 10 mL). The
aqueous phase was diluted with 2 n aq. HCl to pH = 2 and ex-
tracted with EtOAc (3ϫ 10 mL). After washing with brine (2ϫ)
and drying with MgSO4, the solution was filtered and concentrated
in vacuo. The crude product was purified by flash chromatography
(EtOAc) to yield the methylated tryptophan S6 (280 mg, 635 μmol,
70%) as a colorless solid. Rf = 0.26 (EtOAc). 1H NMR (300 MHz,
2
[D6]DMSO, 300 K): δ = 3.02 (dd, JHH = 14.6 Hz, JHH = 9.6 Hz,
H, Fmoc-CHarom.), 7.48 (t, JNH/αH = 6.1 Hz, 1 H, NH), 7.31–7.44 1 H, βHdiast.), 3.17–3.27 (m, 1 H, βHdiast.), 3.70 (s, 1 H, NCH3),
(m, 4 H, Fmoc-CHarom.), 4.20–4.30 (m, 3 H, Fmoc-CH2, Fmoc- 4.01–4.29 (m, 4 H, αH, Fmoc-CH, Fmoc-CH2), 6.95–7.89 (m, 14
CH), 3.91 (d, JαH/NH = 5.8 Hz, 2 H, αH), 3.68 (s, 3 H, OCH3), 3.09
H, Fmoc-CHarom., indole-CHarom., NH), 12.65 (br. s, 1 H, COOH)
Eur. J. Org. Chem. 2015, 7443–7448
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
7447

M. Lamping, S. Enck, A. Geyer
FULL PAPER
ppm. 13C NMR (75 MHz, [D6]DMSO, 300 K): δ = 26.8 (βCH2),
Cyclic Amide 9: TPAP (0.5 mg, 1.50 μmol, 0.1 equiv.), NMO
32.3 (NCH3), 46.6 (Fmoc-CH), 55.0 (αCH), 65.6 (Fmoc-CH2), (5.3 mg, 46.0 μmol, 3.0 equiv.), DIPEA (2.7 μL, 15.1 μmol,
109.6, 118.5, 120.0, 121.0, 125.3, 127.0, 128.0, 140.7, 143.8 (Fmoc- 1.0 equiv.), and molecular sieves (4 Å; 5 beads) were added to a
CHarom., Fmoc-Carom.,quart., indole-CHarom, indole-Carom.), 156.0
(Trp-CO), 173.6 (Fmoc-CO) ppm. HRMS (ESI): calcd. for
C27H24N2O4H+ 439.1652; found 439.1668.
solution of N-acylated segetalin A aldehyde as TFA salt (1; 10 mg,
15.1 μmol, 1.0 equiv.) in acetonitrile (0.15 mL). After stirring for
16 h, the mixture was purified by HPLC. The product and the hy-
drolyzed amide were verified by mass spectrometry only. MS (ESI):
m/z = 652.34 [M + H]+. HRMS: calcd. for C33H46N7O7 [M + H]+
652.3453; found 652.3452.
General Protocol for Loading of H-Thr-Gly-NovaSyn TG Resin:
(1) Swelling of the resin in 1% AcOH in CH2Cl2/MeOH (1:1) for
30 min; (2) addition of Fmoc-protected amino acid aldehyde
(5.0 equiv.) in CH2Cl2; (3) 4 h incubation at r.t.; (4) washing with
CH2Cl2, DMF, and THF (3ϫ each); (5) 3 h incubation with Boc2O
(5.0 equiv.) and N-methylmorpholine (NMM, 5.0 equiv.) in THF
at 50 °C; (6) washing with CH2Cl2, DMF, and THF (3ϫ each).
Acknowledgments
We thank the Evonik-Stiftung for a Werner–Schwarze scholarship
and financial support.
General Protocol for Manual Synthesis of Peptide Aldehydes:
(1) Swelling of the amino acid aldehyde loaded resin in DMF for
30 min; (2) Fmoc deprotection: 10 min and 20 min incubation
with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)/piperidine/DMF
(2:2:96); (3) washing with THF, iPrOH, and DMF (3ϫ each);
(4) chain elongation: 50 min incubation with the Fmoc-protected
amino acid (3.0 equiv.), HBTU (3.0 equiv.), HOBt (1.0 equiv.), and
DIPEA (6.0 equiv.) in DMF; (5) washing with THF, iPrOH, and
DMF (3ϫ each); repeat steps (2)–(5) until assembly of target se-
quence; (6) Fmoc deprotection: 10 min and 20 min incubation with
DBU/piperidine/DMF (2:2:96); (7) removal of Boc and side-chain
protecting groups: 10 min incubation with 100% TFA (2ϫ);
(8) washing with CH2Cl2 (2ϫ); (9) cleavage of the peptide alde-
hyde: 30 min incubation with AcOH/H2O/CH2Cl2/MeOH
(10:5:63:21).
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General Protocol for the Purification of the Peptide Aldehydes: The
peptide solutions, which were obtained from cleavage, were concen-
trated in vacuo. The peptides were precipitated from dry Et2O at
–30 to –40 °C and then lyophilized after centrifugation. Segetalin A
aldehyde (1) was characterized by HPLC and ESI-MS; the purity
without further purification was approximately 90%. The peptide
was directly analyzed by NMR spectroscopy.
Segetalin A1-CHO (1): Yield: 87% (20 mg, 29.8 μmol) pale yellow
solid. HPLC [r.t.; 15–65% CH3CN in 0.05% TFA/H2O, in 20 min
(gradient)]: tR = 11.54 min. MS (ESI): m/z = 612.5 [M + H]+.
HRMS: calcd. for C31H46N7O6 [M + H]+ 612.3504; found
612.3508.
Segetalin A1 Aldehyde (1a): The peptide was characterized by
HPLC and ESI-MS, and the purity without further purification
was approximately 70%. The peptide was purified by semiprepar-
ative HPLC to a purity of approximately 97%. After purification,
the peptide was analyzed by NMR spectroscopy. The ESI MS data
showed mainly methyl acetal [Seg A-CH(OMe)2]. Yield: 77%
(18 mg, 26.3 μmol), colorless solid. HPLC [r.t.; 15–65% CH3CN in
0.05% TFA/H2O, in 20 min (gradient)]: tR = 13.09 min. MS (ESI):
m/z = 658.4 [M + MeOH + H]+. HRMS: calcd. for C33H52N7O7
[M + MeOH + H]+ 658.3923; found 658.3918.
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Received: September 10, 2015
Published Online: October 28, 2015
7448
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© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2015, 7443–7448