Synthesis and conformational analysis of an expanded cyclic ketoxime-hexapeptide

Article abstract of DOI:10.1002/psc.2873

In this work the synthesis of a linear hexapeptide with a hydroxylamine functionality at the N-terminus and a ketone instead of the carboxylic acid at the C-terminus is described. Cyclization by ketoxime formation yields the 19-membered ring-expanded cyclic hexapeptide cyclo[Goly-Val-Ala-Pro-Leu-Kly] which adopts a main conformer with two intramolecular hydrogen bonds. The hydrolytic stability of a ketoxime lies between the inert amide and the labile imine. The substitution of an amide bond for an iminium bond transforms the irreversible macrocyclization into a reversible process, but macrocyclic imines are difficult to isolate because they are prone to hydrolysis. The enhanced chemical stability of the ketoxime justifies its application in ligation protocols. The detailed NMR analysis of a ketoxime linkage presented here identifies its local conformational preferences in a constrained peptide environment.

Full text of DOI:10.1002/psc.2873

Research Article
Received: 14 January 2016
Revised: 16 February 2016
Accepted: 16 February 2016
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/psc.2873
Synthesis and conformational analysis
of an expanded cyclic ketoxime-
hexapeptide
Matthias Lamping, Yvonne Grell and Armin Geyer*
In this work the synthesis of a linear hexapeptide with a hydroxylamine functionality at the N-terminus and a ketone instead of the
carboxylic acid at the C-terminus is described. Cyclization by ketoxime formation yields the 19-membered ring-expanded cyclic
hexapeptide cyclo[Goly-Val-Ala-Pro-Leu-Kly] which adopts a main conformer with two intramolecular hydrogen bonds. The hy-
drolytic stability of a ketoxime lies between the inert amide and the labile imine. The substitution of an amide bond for an iminium
bond transforms the irreversible macrocyclization into a reversible process, but macrocyclic imines are difficult to isolate because
they are prone to hydrolysis. The enhanced chemical stability of the ketoxime justifies its application in ligation protocols. The
detailed NMR analysis of a ketoxime linkage presented here identifies its local conformational preferences in a constrained pep-
tide environment. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
Keywords: peptide; cyclization; NMR; oxime; conformation
The oxime, as a functional group in cyclic peptides, has the charac-
teristic that the peptide backbone is expanded by one oxygen
Introduction
Several synthetic methods have been developed for the chemo-
and regioselective ligation of unprotected peptides in high yields
over the years. Common examples are disulfide formations, Michael
additions with maleimides, alkylations with iodoacetamides and
condensation reactions which form hydrazones, imines or oximes
[1–7]. The oxime formation between aldehyde and hydroxylamine
is the most widely used method in all these ligations because of
the high stability at a physiological pH and the selectivity of the re-
action [8,9]. Another advantage of this condensation reaction is the
simple handling and the fast reaction rate at pH5–6 [8,9]. The syn-
thesis of oxime/ketoxime-ligated peptides is based on unnatural C-
terminal α-oxo aldehyde peptides and pyruvic acid peptides, which
can be synthesized via solid-phase peptide synthesis [6,10,11]. The
advantages of the chemoselective ligation of unprotected peptides
lead to a variety of improved methods, such as the α-ketoacid-
hydroxylamine amide ligation, which was introduced in 2006 and
further developed over the next few years [12–17].
atom, which leads to uncommon 19-membered ring structures in-
stead of cyclic 18-membered hexapeptides. We obtained the cyclic
ketoxime peptide 2 cyclo[Goly-Val-Ala-Pro-Leu-Kly] from the linear
precursor 1 (Figure 1) and analyzed the oxime E/Z and the proline
cis/trans isomerism in solution by nuclear magnetic resonance
(NMR) spectroscopy. The standard three-letter code Gly is supple-
mented to the four letter code Goly, which represents the ex-
panded backbone. The six-letter code for unnatural dipeptides is
adapted here for the four atom building block Goly [28]. Kly stands
for keto glycine and is a blend of the ketone and the code Gly. The
peptide 2 shown in Figure 1 can form four different structures, be-
cause of the E/Z isomerism of the ketoxime and the cis/trans isom-
erism of the proline.
Results and Discussion
The macrocyclization of peptides is an irreversible process which
can be transformed into a reversible process by the substitution of
an amide bond for an iminium bond. Through this substitution, the
inherent cyclization tendency has already been analyzed for the
peptides tyrocidine A, nostocyclopeptide and segetalin A by our
group [18–20]. However, cyclic imino peptides are prone to hydro-
lysis and are, therefore, hard to isolate in pure form. By substituting
the amide bond by an oxime linkage, the cyclic peptides become
more stable and isolable, especially in the case of ketoximes [21].
Several approaches have been made to synthesize amino acid side
chain aldehydes/ketones for the ligation or cyclization with hydrox-
ylamines [22–27].
We synthesized the linear peptide Boc-Goly-Val-Ala-Pro-Leu-Kly-
Smc 1a (Smc: semicarbazone) to perform the macrocyclization
reaction. The N-terminal Boc-protected building block Goly repre-
sents the natural amino acid glycine, which is expanded through
the formal insertion of an oxygen atom in the N―C bond. The syn-
thesis was carried out in two steps starting with N-Boc-
hydroxylamine (3), which forms the corresponding methyl ester 4
*
Correspondence to: Armin Geyer, Institute of Chemistry, Philipps-University
Marburg, Hans-Meerwein-Straße, 35032 Marburg, Germany. E-mail: geyer@staff.
uni-marburg.de
Here, we integrated the oxime bond into a common cyclic
hexapeptide in order to study in detail whether it fits into a β-turn.
Institute of Chemistry, Philipps-University Marburg, Hans-Meerwein-Straße, 35032,
Marburg, Germany
J. Pept. Sci. 2016; 22: 228–235
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CONFORMATIONAL ANALYSIS OF AN EXPANDED CYCLIC KETOXIME-HEXAPEPTIDE
Figure 1. Condensation of the linear hexapeptide 1 to the cyclic ketoxime 2. Based on the E/Z conformation (red) formed by the ketoxime and the cis/trans-
isomerism (blue) of Pro4, the formation of four different conformers is possible. The conformers formed are identified and analyzed via NMR spectroscopy. The
peptide 2 is shown here as the E-trans-conformer.
in a nucleophilic substitution. After hydrolysis of the ester, the Boc-
protected hydroxylamino acid 5 is obtained in good yield. The C-
terminal ketone is synthesized in two steps from the natural amino
acid glycine (6). In the first step, the corresponding glycine is trans-
formed into the ketone 7 through a Dakin-West reaction, and the
Scheme 2. Solution-phase synthesis of the protected peptide 1a. The acid,
following cleavage of the acetyl groups yields the amino acetone
in the first step Goly, was activated by IBCF under basic conditions at ꢀ20 °C.
hydrochloride. The ketone is protected as the semicarbazone 8
with semicarbazide (Scheme 1) for peptide synthesis. The synthesis
of 8 has already been described in the literature [29].
After the addition of the amino acid methyl ester, the amide formed was
isolated and the ester cleaved for the next coupling. These steps were
repeated until the protected peptide 1a was obtained.
The peptide 1a was assembled stepwise from the N- to the C-
terminus with the relatively reactive semicarbazone as a temporary
protecting group. Because of this labile protecting group, the un-
conventional N- to C-terminus synthesis strategy was used, and
the small amount of stereoisomers (less than 10%, estimated from
NMR) during the synthesis were removed via HPLC. The synthesis
started with Boc-Goly-OH, which was activated by isobutyl
chloroformate (IBCF) as a mixed anhydride under basic conditions.
The next amino acid valine was introduced as a methyl ester and,
after isolation of the corresponding amide, the ester was saponified
with LiOH in THF/H2O (1:1). This procedure was very efficient, the
side-products were removed easily, and it was repeated until the
complete hexapeptide was obtained (Scheme 2). The protected lin-
ear peptides which contain proline show the expected two signal
sets in the 1H NMR with a cis/trans ratio at the proline amide bond
of approximately 2:3.
Before the final macrocyclization, the terminal protecting groups
had to be cleaved and the peptide was purified by high perfor-
mance liquid chromatography (HPLC). The acid labile protecting
groups were cleaved with aqueous TFA (95%) for 1 h at 45 °C. The
crude product was then dissolved in water and purified by prepar-
ative HPLC, and the peptide was cyclized directly. The synthesis was
not optimized for maximum yield, and the 3% yield refers to the
purest HPLC fraction which was used for NMR analysis to detect
the E/Z and cis/trans isomers. For higher yields, the protecting
group strategy of the ketone should be improved to avoid building
block 8 which has a low solubility. The isolated macrocyclic
ketoxime peptide 2 was analyzed by 1D and 2D NMR spectroscopy.
The 1H NMR shows a dominating signal set instead of the possible
four which would be expected for an equilibrium of several
conformers.
The cross signals in red show the NH/NH ROE contacts of NH-
Leu/Kly and NH-Val/Ala of the major conformer. The trans con-
figuration in the case of the cis/trans isomerism of the Pro amide
bond was assigned by the chemical shift of the β-CH2 and γ-CH2
methylene groups of the proline (δ = 28.8 and 25.5 ppm, respec-
tively) for the major conformer. This method could not be used
in this case based on the low intensity of the minor conformer.
The chemical shift of the methyl group is characteristic of the as-
signment of the E/Z conformation of the ketoxime. We synthe-
sized the model compound acetophenone O-benzyloxime as a
mixture of the E and Z conformers to compare the chemical
shifts of the E and Z isomer. The chemical shift of the methyl
group was 12.6 and 21.3ppm for the E conformer and the Z
conformer, respectively, a difference of 8.7ppm. We could com-
pare the chemical shifts in both conformers because of the high
intensity of the ketoxime methylgroups in peptide 2. The methyl
group has a chemical shift of 11.7 and 12.7 ppm for the major
conformer and minor conformer, respectively, a difference of just
1.0ppm. Based on these chemical shifts, the major conformer
must be the E-ketoxime and the trans-proline, and the minor
Scheme 1. Synthesis of the N-terminal Boc-Goly-OH (5) and the C-terminal H2N-Kly-Smc (8). A) Reagents and conditions: 1) Methylbromoacetate (2.0 eq)
and DIPEA (2.0 eq), DCM, RT, 14 h. 2) LiOH (2.5 eq), THF/H2O (1:1), 45 °C, 2 h. B) Reagents and conditions: 1) Ac2O/pyridine (3:1), reflux, 4 h. 2) aq. HCl (6 M),
reflux, 4 h. 3) Semicarbazide hydrochloride (1.31 eq), ethanol/H2O (1:1), RT, 2 h.
J. Pept. Sci. 2016; 22: 228–235
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LAMPING, GRELL AND GEYER
conformer also contains the E-ketoxime, but the cis-proline
(Figure 2).
Table 1. 3J-NH/α couplings and temperature gradients of the amide
protons of both conformers (major and minor) determined by 1H NMR
spectra at different temperatures (300, 310 and 320 K)
The complete assignment of the major conformer was accom-
plished by 2D NMR spectroscopy (TOCSY, HSQC and ROESY). Only
the NH signals could be assigned from the exchange peaks in the
ROESY spectrum for the minor isomer. The temperature gradients
of the amide protons of the major isomer identify two intramolec-
ular hydrogen bonds (NH-Kly and NH-Ala) and one hydrogen bond
for NH-Val for the minor isomer (Table 1).
Amide
proton
3J-NH/α
(Hz)
Major
[ppb/K]
3J-NH/α
(Hz)
Minor
[ppb/K]
NH-Leu
NH-Kly
NH-Val
NH-Ala
7.86
5.37
2.54
7.64
1.67
7.42
4.45
6.55
0.79
3.92
8.70/2.88
8.28
7.56/4.11
8.97
7.44
7.41
The complete NOE list of the major isomer is contained in the SI.
Two pairs of NH/NH contacts NH-Val/NH-Ala and NH-Leu/NH-Kly
are characteristic for two β-turns. Further NOEs from the NH-αH se-
quential walk identify the βII turns. Even the transannular NOE be-
tween βCH3-Ala and αH-Kly(proS) is visible. The strong sequential
NOE contact αH-Pro/NH-Leu and a medium NH-Leu/αH-Leu iden-
tify the βII turn, although the contribution from fast rotation about
the central peptide bond (i + 1)–(i + 2) about 180° – the so-called
βI/II flip – cannot be excluded [30,31]. The structure in Figure 3A dis-
plays the β-turn type II with alanine, proline and leucine in the po-
sitions i, i + 1 and i + 2 of the turn. Pro is expected to occupy the i + 1
position comparable to one of the first cyclic hexapeptides eluci-
dated by NMR and other additional cyclic β,β-hexapeptides [32].
The structure calculations of 2 were carried out as described pre-
viously using the program package HyperChem with MM+ force
field and without explicit water included [18,33,34]. Ten snapshots
from a 100-ps molecular dynamics simulation (step size 1 fs,
300 K) were averaged, and the resulting structure was allowed to re-
lax without restraints to verify its plausibility [35]. The NOE contacts
of the minor conformer between NH-Ala/NH-Val and NH-Leu/αH-
Ala indicate the formation of β-turn type VIa structure. In contrast
to the major conformer, the sequence APL in this turn structure ro-
tated clockwise by one position. Proline takes now the i + 2 position
in the turn and the central peptide bond (i + 1)–(i + 2) changes from
trans to cis. This confirms the assumption that the minor conformer
gets structured by an E-ketoxime and cis-proline (Figures 3B and 4).
β-turns, as secondary structures, are stabilized by intramolecular
hydrogen bonds, which are formed between the i + 4 position and
Figure 2. A) (600 MHz, 300 K, DMSOd6) NH region of the 1H NMR spectrum of peptide 2. The equilibrium between both conformers – the major conformer
(86%, green) and the minor conformer (14%, orange) – is visible from the exchange signals. B) The difference between both conformations is the Pro amide
bond, which is trans for the major conformer and cis for the minor conformer. Only one conformer is populated by the E/Z isomerism of the ketoxime. The E-
configured ketoxime was identified in both conformers. The ROE contacts in the expansion of the ROESY spectrum (mixing time 300 ms) are marked in red,
and the black cross signals in black display the chemical exchange between both conformations.
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CONFORMATIONAL ANALYSIS OF AN EXPANDED CYCLIC KETOXIME-HEXAPEPTIDE
Figure 3. Illustration of the major conformer (A) and the minor conformer (B) as sphere models. Based on the cis/trans isomerism at the proline (grey) of both
conformers, the APL sequence is located at different positions in the template. The hydrogen bonds are indicated by arrows.
Figure 4. Illustration of the major conformer as a β-turn type II and the minor conformer as a β-turn type VIa. The NOE contacts served as distance restraints
in a molecular dynamics simulation. In the major conformer the oxime nitrogen forms the electron donor for the intramolecular hydrogen bond with NH-Leu.
amino acid in the i position. In the case of the major conformer,
these amino acids are Kly and Ala. The temperature gradients of
the four amide protons support the structure proposed. NH-Ala
possesses the lowest temperature dependency of the chemical
shift, followed by the gradient of the NH-Kly. Both values are indic-
ative of the hydrogen bond between each of them. The tempera-
ture gradients of NH-Leu indicate solvent-oriented NH-groups
(Table 1). The determined torsion angels φ and ψ are listed in
Table 2.
The black cross signals of the ROESY spectrum shown in Figure 2
can be further analyzed to determine the chemical exchange rate
between both conformers. Based on the ratio determined, the acti-
vation energy which is necessary for the conformational change
can be calculated. In our special case, the populations are unequal,
and we have to consider the mole fractions XA = 0.86 and XB = 0.14
for the calculation of r [36,37]. Therefore, the intensities of the cross
signals of both NH-Ala signals are determined and, with the mole
fractions, a ratio of 13.33 was calculated. This ratio in combination
J. Pept. Sci. 2016; 22: 228–235
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LAMPING, GRELL AND GEYER
Table 2. Based on the illustration of the major conformer in Figure 4,
we determined the ϕ and ψ torsion angels the β-turn type II (left) and
compared them with the ideal values for a β-turn type II (right)
gel 60 F254 (Merck KGaA), and detection was carried out by fluores-
cence quenching under UV light (λ = 254 nm) or by staining with
ninhydrin solution followed by heating to 500 °C. Flash chromatog-
raphy was performed on silica gel 60 (0.040–0.063mm) from Merck
KGaA. Semi-preparative HPLC was performed on a Dionex HPLC sys-
tem with a diode array detector using an ACE HPLC C18 RP,
7.75× 150mm, and analytical HPLC was performed on the same
system using an Intersil ODS-4, C18 RP, 3.0 × 150 mm. The NMR
spectra were recorded on Bruker AV 600 spectrometers. Chemical
shifts (δ) are given in ppm referring to the solvent signal. The cou-
Amino acid
φ angle
ψ angle
φ angle
ψ angle
(ideal)
(ideal)
Goly
Val
ꢀ171.5, -8.5
73.9
111.9
-3.5
-60
80
120
0
Ala
Pro
Leu
Kly
-125.2
-46.8
160.1
100.9
15.0
—
—
120
0
-60
80
79.4
3
pling constants are J couplings, unless otherwise indicated. All
mass spectra were recorded on a Finnigan LTQ-FT spectrometer.
-116.9
-97.0
—
—
Boc-2-(Aminooxy)methylacetate (4)
DIPEA (9.70 ml, 56.8mmol, 2.00 eq) and methylbromoacetate
(2.70 ml, 28.4mmol, 1.00 eq) were added to a solution of N-
Boc-hydroxylamine (3, 3.78 g, 28.4mmol, 1.00 eq) in DCM (40 ml)
at RT. After stirring for 14h, the solvent was removed under re-
duced pressure, and a colorless solid was obtained. The residue
was solved in EtOAc (100 ml), washed three times with 5% aq.
NaHCO3, twice with 10% aq. citric acid and three times with brine.
The organic phase was dried over MgSO4, filtered and concentrated
in vacuo to afford 4 in 58% yield (3.37 g, 16.4mmol) as a colorless
Figure 5. Calculation of the activation energy of the conformational
exchange [36,37]. The ratio r determined combined with the mixing time
tm of 300 ms lead to the constant k = 0.5012 sꢀ1. An energy of 75.1 kJ/mol
(ca. 18 kcal/mol) is required for the change between both conformers.
oil. Rf = 0.86 (EtOAc/pentane 3:1); 1H-NMR (300 MHz, DMSO-d6
,
300 K): δ = 10.17 (s, 1H, NH), 4.36 (s, 2H, CH2), 3.67 (s, 3H, OCH3),
1.40 (s, 9H, Boc) ppm; 13C-NMR (75 MHz, DMSO-d6, 300 K): δ =
169.1 (COester), 156.3 (COcarbamate), 80.1 (Bocquat.), 72.0 (CH2), 51.5
(OCH3), 27.9 (Boc-CH3) ppm; HRMS (ESI): calcd. for [C8H15NO5Na+]:
228.0842, found: 228.0843.
with the mixing time of the ROESY experiment of 300 ms leads to
an equilibrium constant k =0.5012 sꢀ1. Based on this value, the ac-
tivation energy of this process of 75.1kJ/mol was determined
(Figure 5). This value is comparable with the activation barrier of
the cis/trans isomerism (75–85 kJ/mol) [38–40].
Boc-2-(Aminooxy)acetic Acid (5)
LiOH (0.96 g, 40.0 mmol, 2.47 eq) was added to a solution of Boc-
2-(Aminooxy)methylacetate (4, 3.33 g, 16.2 mmol, 1.00 eq) in
THF/H2O (1:1, 100 ml) at RT. After stirring for 4 h at 45 °C, the solvent
was removed under reduced pressure and the residue was diluted
with aq. HCl (2M) until the pH reached ~2. After extraction with
EtOAc (200 ml), the organic phase was dried over MgSO4, filtered
and concentrated in vacuo to afford the acid 5 in 79% yield
Conclusions
A linear peptide ketone with an N-terminal hydroxylamine was
cyclized to a 19-membered macrocyclic peptide. Two-dimensional
NMR spectroscopy identifies a minor conformer from the rotation
about the Pro tertiary amide bond, but not around the oxime link-
age. The facile macrocyclization process leads to a stable and isola-
ble cyclic peptide, and this method could be used in other
macrocyclizations where the oxime can be part of a β-turn struc-
ture. Broader application will be possible after integration of the ox-
ime cyclization into solid-phase peptide synthesis, for which the
C-terminal ketone must be covalently linked to a resin by a suitable
linker. Instead of the flexible linkage expected, we identify a well-
defined main conformer very similar to the canonical double β-turn
cyclic hexapeptides. The oxime nitrogen serves as the electron do-
nor in a hydrogen bond to the i + 2 amide hydrogen atom in a
βII-type reverse turn. Although the ketoxime linker expands the pep-
tide backbone by the four atom building block Goly, the ketoxime is
compatible with a hairpin conformation in the hexapeptide exam-
ple presented here. This conformational preference here can serve
as a template for the future design of fortunate ligation sites.
1
(2.43 g, 12.7 mmol) as a colorless solid. H-NMR (300 MHz, DMSO-
d6, 300 K): δ = 12.80 (s, 1H, OH), 10.10 (s, 1H, NH), 4.26 (s, 2H, CH2),
1.40 (s, 9H, Boc) ppm; 13C-NMR (75 MHz, DMSO-d6, 300 K): δ =
170.0 (COacid), 156.4 (COcarbamate), 80.0 (Bocquat.), 72.0 (CH2), 28.0
(Boc-CH3) ppm; HRMS (ESI): calcd. for [C7H13NO5Na+]: 214.0686,
found: 214.0688.
Diacetyl Aminoacetone (7)
Based on a synthesis of Hepworth et al., glycine (6, 10.0 g, 0.13 mol,
1.00eq) was solved in acetic anhydride (150ml, 1.55 mol, 11.7 eq)
and pyridine (65.0ml, 0.80 mol, 6.00 eq), and this mixture was
refluxed for 6 h. After stirring for 14 h at RT, the solvent was re-
moved under reduced pressure and the residue was purified by
vacuum distillation. The product 7 was obtained in 48% yield as a
yellow oil (9.79g, 62.3 mmol). Boiling point: 125 °C (1mbar); 1H-
NMR (300 MHz, DMSO-d6, 300 K): δ = 4.60 (s, 2H, CH2), 2.26 (s, 6H, 2
x Ac), 2.16 (s, 3H, CH3) ppm; 13C-NMR (75 MHz, DMSO-d6, 300 K):
δ = 202.9 (COketone), 172.6 (COAc), 53.8 (CH2), 26.9 (Ac), 25.8 (CH3)
ppm; HRMS (ESI): calcd. for [C7H11NO3Na+]: 180.0631, found:
180.0636 [29].
Experimental Section
General Information
The reactions were executed under a nitrogen atmosphere at ambi-
ent pressure. Thin-layer chromatography was performed on silica
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CONFORMATIONAL ANALYSIS OF AN EXPANDED CYCLIC KETOXIME-HEXAPEPTIDE
Aminoacetone-Semicarbazone Hydrochloride (8)
Boc-Goly-Val-Ala-OMe (10)
Based on a synthesis of Hepworth et al., the acetamide (7, 9.59g,
61.0mmol, 1.00 eq) was solved in aqueous HCl (6M, 70 ml) and
refluxed for 6 h. After stirring for 14 h at RT, the solvent was re-
moved under reduced pressure and the dark red oil (6.95g) ob-
tained was used directly in the next step. The residue was
solved in ethanol (45 ml) and a solution of semicarbazide hydro-
chloride (8.93 g, 80.0mmol, 1.31 eq) in H2O (20 ml) was added.
The reaction was stirred for 2 h, and the colorless precipitate
was filtered and washed with ethanol. The solid was purified by
recrystallization (H2O/ethanol 1:1), and the hydrochloride 8
(6.94 g, 41.7mmol) was obtained in 68% yield as a colorless crys-
The peptide was synthesized as described above in two steps and
was obtained in 48% yield as a colorless solid. Rf = 0.33 (EtOAc/pen-
tane 3:1); 1H-NMR (300 MHz, DMSO-d6, 300K): δ = 10.32 (s, 1H, Goly-
NH), 8.49 (d, 1H, 3J = 6.60Hz, Ala-NH), 7.97 (d, 1H, 3J = 8.10 Hz, Val-
3
3
NH), 4.28 (dd, 1H, J = 7.11 Hz, J = 9.20 Hz, Val-αH), 4.22–4.26 (m,
1H, Ala-αH), 4.19 (d, 2H, 2J = 15.67 Hz, Goly-αH), 3.61 (s, 3H, OCH3),
1.94–2.00 (m, 1H, Val-βH), 1.41 (s, 9H, Boc), 1.28 (d, 3H, 3J = 6.60 Hz,
3
3
Ala-βH), 0.89 (d, 3H, J = 6.72 Hz, Val-γH), 0.91 (d, 3H, J = 6.78 Hz,
Val-γH) ppm; 13C-NMR (75 MHz, DMSO-d6, 300 K): δ = 172.8 (Ala-
CO), 170.4 (Val-CO), 167.8 (Goly-CO), 156.9 (Boc-CO), 80.6 (Bocquat.),
74.5 (Goly-αH), 56.7 (Val-αC), 51.7 (O-CH3), 47.5 (Ala-αC), 30.8 (Val-β
C), 27.9 (Boc-CH3), 18.9 (Val-γC), 18.0 (Val-γC), 16.7 (Ala-βC) ppm;
HRMS (ESI): calcd. for [C16H29N3O7H+]: 376.3078, found: 376.3075.
talline solid. Melting point: 193 °C; 1H-NMR (300 MHz, DMSO-d6
,
300 K): δ = 9.35 (s, 1H, NH), 8.13 (s, 3H, NH3+), 6.78 (s, 1H, NH2),
6.45 (s, 2H, NH2), 3.32 (s, 2H, CH2), 1.82 (s, 3H, CH3) ppm; 13C-
NMR (75MHz, DMSO-d6, 300 K): δ = 157.3 (CO), 141.2 (C¼N), 42.6
(CH2), 14.6 (CH3) ppm; HRMS (ESI): calcd. for [C4H10N4OH+]:
131.0927, found: 131.0929 [29].
Boc-Goly-Val-Ala-Pro-OMe (11)
The peptide was synthesized as described above in two steps and
was obtained in 26% yield as a colorless oily solid. Rf = 0.14 (EtOAc);
1H-NMR (300 MHz, DMSO-d6, 300 K): δ = 10.33 (s, 1H, Goly-NH), 8.25
3
3
(d, 1H, J = 6.69 Hz, Ala-NH), 7.97 (d, 1H, J = 9.03Hz, Val-NH), 4.47
Peptide Synthesis
3
(t, 1H, J = 6.89 Hz, Pro-αC), 4.25–4.34 (m, 2H, Val-aH, Ala-aH), 4.19
2
The synthesis of peptide 2 was accomplished linearly in solution
from the N-terminus to the C-terminus via IBCF coupling of the
Boc-Goly-OH and the corresponding amino acid methyl ester. In
general, the carboxylic acid (1.00 eq) was solved in THF (conc. =
0.2M) and NMM (1.00 eq) was added for the peptide coupling.
The solution was cooled to ꢀ20°C, IBCF (1.00 eq) was added slowly,
and the suspension was stirred for 10 min. The corresponding
amine hydrochloride was solved in THF (10–20 ml), neutralized with
NMM (1.00 eq) and added to the suspension. The reaction was
stirred for 14 h at RT, and the solvent was removed under reduced
pressure. The residue was solved in EtOAc (100 ml), washed three
times with 5% aq. NaHCO3, twice with 10% aq. citric acid and three
times with brine. The organic phase was dried over MgSO4, filtered
and concentrated in vacuo to afford the corresponding peptide.
(d, 2H, J = 15.67 Hz, Goly-αH), 3.63–3.70 (m, 2H, Pro-dH), 3.60 (s,
3H, OCH3), 2.12–2.23 (m, 1H, Val-βH), 1.78–1.95 (m, 4H, Pro-βH,
3
Pro-γH), 1.41 (s, 9H, Boc), 1.20 (d, 3H, J = 7.00 Hz, Ala-βH), 0.86 (d,
3H, 3J = 6.78 Hz, Val-γH), 0.81 (d, 3H, 3J = 6.75 Hz, Val-γH) ppm; 13C-
NMR (75 MHz, DMSO-d6, 300 K): δ = 172.2 (CO), 170.5 (CO), 170.0
(CO), 167.7 (CO), 156.9 (Boc-CO), 80.6 (Bocquat.), 74.5 (Goly-αH),
58.4 (Pro-αC), 56.8 (Val-αC), 51.7 (O-CH3), 46.3 (Pro-δC), 46.1 (Ala-α
C), 30.6 (Val-βC), 28.4 (Pro-βC), 27.9 (Boc-CH3), 24.6 (Pro-γC), 19.0
(Val-γC), 18.0 (Val-γC), 16.4 (Ala-βC) ppm; HRMS (ESI): calcd. for
[C21H36N4O8Na+]: 492.2425, found: 492.2424.
Boc-Goly-Val-Ala-Pro-Leu-OMe (12)
The peptide was synthesized as described above in two steps and
was obtained in 36% yield as a colorless oil. Rf = 0.40 (DCM/MeOH
20:1); 1H-NMR (300 MHz, DMSO-d6, 300 K): δ = 10.32 (s, 1H, Goly-
NH), 8.35 (d, 1H, 3J = 7.54Hz, Ala-NH), 8.14 (d, 1H, 3J = 7.51 Hz, Val-
Ester Hydrolysis
3
NH), 7.94 (d, 1H, J = 8.62 Hz, Leu-NH), 4.44–4.59 (m, 1H, Ala-αH),
For the hydrolysis of the peptide methyl esters the corresponding
esters were solved in THF/H2O (1:1, 20 ml) and LiOH (2.47 eq) was
added. The solution was stirred at 45°C until the reaction was com-
plete. The solvent was removed under reduced pressure, and the
residue was diluted with aq. HCl (2M) until the pH reached ~2. After
extraction with EtOAc (100 ml), the organic phase was dried over
MgSO4, filtered and concentrated in vacuo to afford the corre-
sponding acid.
4.18–4.37 (m, 3H, Val-αH, Pro-αH, Leu-αH), 4.19 (d, 2H, 2J =
15.64 Hz, Goly-αH), 3.60 (s, 3H, O-CH3), 3.54–3.58 (m, 2H, Pro-δH),
1.78–2.07 (m, 5H, Pro-βH, Pro-γH, Val-βH), 1.60–1.68 (m, 1H, Leu-γ
3
H), 1.46–1.55 (m, 2H, Leu-βH), 1.41 (s, 9H, Boc-H), 1.17 (d, 3H, J =
3
3
6.84Hz, Ala-βH), 0.89 (d, 3H, J = 6.51Hz, Val-γH), 0.83 (d, 3H, J =
6.42 Hz, Val-γH), 0.80–0.85 (m, 6H, Leu-δH) ppm; 13C-NMR (75 MHz,
DMSO-d6, 300 K): δ =173.2–167.7 (7 x CO), 156.9 (Boc-CO), 80.6
(Bocquat.), 74.5 (Goly-αH), 56.8 (Val-αC), 51.7 (O-CH3), 50.2 (Leu-αC),
46.6 (Pro-dC), 46.2 (Ala-αC), 39.5 (Leu-βC), 30.7 (Val-βC), 28.8 (Pro-β
C), 27.9 (Boc-CH3), 24.2 (Pro-γC), 24.1 (Leu-γC), 22.2 (Leu-δC), 21.3
(Leu-δC), 19.1 (Val-γC), 18.0 (Val-γC), 16.7 (Ala-βC) ppm; HRMS (ESI):
calcd. for [C27H47N5O9Na+]: 608.3266, found: 608.3272.
Boc-Goly-Val-OMe (9)
The peptide was synthesized as described above in two steps and
was obtained in 69% yield as a colorless solid. Rf = 0.59 (EtOAc/pen-
tane 3:1); 1H-NMR (300 MHz, DMSO-d6, 300K): δ = 10.41 (s, 1H, Goly-
NH), 8.36 (d, 1H, 3J = 8.16 Hz, Val-NH), 4.26 (dd, 1H, 3J=6.32Hz,
Cyclo-(Goly-Val-Ala-Pro-Leu-Kly) (2)
2
3J = 8.45 Hz, Val-αH), 4.24 (d, 2H, J=15.88Hz, Goly-αH), 3.64 (s, 3H,
The peptide was synthesized as described above in two steps and
directly treated with aqueous TFA (95%). The linear peptide was
solved in MeCN/H2O (1:1) and cyclized spontaneously. After purifi-
cation via semi preparative HPLC the cyclic ketoxime peptide was
obtained in 3% yield as a colorless solid. HPLC-Rt = 9.37 min (10%–
90% MeCN in 20 min, flow: 0.6ml/min); 1H-NMR (600 MHz, DMSO-
OCH3), 2.02–2.13 (m, 1H, Val-bH), 1.42 (s, 9H, Boc), 0.91 (d, 3H,
3
3J = 4.77 Hz, Val-γH), 0.88 (d, 3H, J=4.74Hz, Val-γH) ppm; 13C-NMR
(75 MHz, DMSO-d6, 300K): δ = 171.5 (Val-CO), 168.5 (Goly-CO), 80.8
(Bocquat.), 74.5 (Goly-αH), 57.0 (Val-αC), 51.7 (O-CH3), 29.9 (Val-βC),
27.9 (Boc-CH3), 18.8 (Val-γC), 18.0 (Val-γC) ppm; HRMS (ESI): calcd.
for [C13H24N2O6H+]: 305.1707, found: 305.1706.
3
3
d6, 300 K): δ = 8.67 (d, 1H, J = 7.86 Hz, Leu-NH), 7.84 (dd, 1H, J =
J. Pept. Sci. 2016; 22: 228–235
Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jpepsci

LAMPING, GRELL AND GEYER
2.88 Hz, 3J = 8.70 Hz, Kly-NH), 7.65 (d, 1H, 3J = 8.28 Hz, Val-NH), 7.11
(d, 1H, 3J = 7.44 Hz, Ala-NH), 4.58–4.60 (m, 1H, Ala-αH), 4.42 (d, 2H,
2J = 14.41Hz, Goly-αH), 4.23–4.26 (m, 2H, Pro-αH, Kly-αH), 4.15 (dd,
Biomol. Chem. 2012; 10: 5837. DOI: http://dx.doi.org/10.1039/
c2ob25129a.
13 Ogunkoya AO, Pattabiraman VR, Bode JW. Sequential α-ketoacid-
hydroxylamine (KAHA) ligations: synthesis of C-terminal variants of the
modifier protein UFM1. Angew. Chemie Int. Ed. 2012; 51: 9693–9697.
DOI: http://dx.doi.org/10.1002/anie.201204144.
14 Murar CE, Thuaud F, Bode JW. KAHA ligations that form aspartyl
aldehyde residues as synthetic handles for protein modification and
purification. J. Am. Chem. Soc. 2014; 136: 18140–18148. DOI: http://dx.
doi.org/10.1021/ja511231f.
15 Bode JW, Fox RM, Baucom KD. Chemoselective amide ligations by
decarboxylative condensations of N-alkylhydroxylamines and α-
ketoacids. Angew. Chemie Int. Ed. 2006; 45: 1248–1252. DOI: http://dx.
doi.org/10.1002/anie.200503991.
16 Ju L, Bode JW. A general strategy for the preparation of C-terminal
peptide alpha-ketoacids by solid phase peptide synthesis. Org. Biomol.
Chem. 2009; 7: 2259–2264. DOI: http://dx.doi.org/10.1039/b901198f.
17 Pusterla I, Bode JW. The mechanism of the α-ketoacid–hydroxylamine
amide-forming ligation. Angew. Chemie Int. Ed. 2012; 51: 513–516. DOI:
http://dx.doi.org/10.1002/anie.201107198.
18 Enck S, Kopp F, Marahiel MA, Geyer A. The entropy balance of
nostocyclopeptide macrocyclization analysed by NMR spectroscopy.
ChemBioChem 2008; 9: 2597–2601. DOI: http://dx.doi.org/10.1002/
cbic.200800314.
3
3
1H, J = 5.37 Hz, J = 8.25Hz, Val-αH), 4.04–4.08 (m, Leu-αH), 3.71–
2
3.75 (m, 1H, Pro-δH), 3.43–3.47 (m, 1H, Pro-δH), 3.33 (dd, 1H, J =
15.36Hz, 3J = 3.16Hz, Kly-αH), 2.18–2.24 (m, 1H, Val-βH), 2.02–2.11
(m, 2H, Pro-βH, Pro-γH), 1.84–1.88 (m, 1H, Pro-γH), 1.81 (s, 3H, Kly-
CH3), 1.78–1.79 (m, 1H, Pro-βH), 1.65–1.73 (m, 1H, Leu-γH), 1.50–
3
1.73 (m, 2H, Leu-βH), 1.11 (d, 3H, J = 6.84Hz, Ala-βH), 0.89 (d, 3H,
3
3J = 6.51 Hz, Val-γH), 0.88 (d, 3H, J = 7.14Hz, Val-γH), 0.82 (d, 3H,
3J = 7.14 Hz, Val-γH), 0.81–0.90 (m, 6H, Leu-δH) ppm; 13C-NMR
(75 MHz, DMSO-d6, 300 K): δ = 72.5 (Goly-αH), 59.9 (Pro-αC), 57.7
(Val-αC), 51.5 (Leu-αC), 46.8 (Pro-δC), 45.9 (Ala-αC), 42.0 (Kly-αC),
38.7 (Leu-βC), 28.7 (Val-βC), 28.1 (Pro-βC), 24.9 (Pro-γC), 24.2 (Leu-γ
C), 22.9 (Leu-δC), 20.6 (Leu-δC), 19.0 (Val-γC), 17.4 (Ala-βC) 17.3
(Val-γC), 11.7 (Kly-CH3) ppm; HRMS (ESI): calcd. for [C24H40N6O6Na+]:
531.2902, found: 531.2910.
Acknowledgements
19 Enck S, Kopp F, Marahiel MA, Geyer A. The reversible macrocyclization of
Tyrocidine A aldehyde: a hemiaminal reminiscent of the tetrahedral
intermediate of macrolactamization. Org. Biomol. Chem. 2010; 8: 559–
563. DOI: http://dx.doi.org/10.1039/B917549K.
We thank the Evonik-Stiftung for a Werner-Schwarze scholarship
and financial support.
20 Lamping M, Enck S, Geyer A. Inverse γ-turn-inspired peptide:
synthesis and analysis of segetalin a indole hemiaminal. European J.
Org. Chem. 2015; 7443–7448. DOI: http://dx.doi.org/10.1002/
ejoc.201501179.
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Supporting Information
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