Cyclopeptides containing the DEKS motif as conformationally restricted collagen telopeptide analogues: Synthesis and conformational analysis

Article abstract of DOI:10.1039/c4ob02348j

The collagen telopeptides play an important role for lysyl oxidase-mediated crosslinking, a process which is deregulated during tumour progression. The DEKS motif which is located within the N-terminal telopeptide of the α1 chain of type I collagen has been suggested to adopt a βI-turn conformation upon docking to its triple-helical receptor domain, which seems to be critical for lysyl oxidase-catalysed deamination and subsequent crosslinking by Schiff-base formation. Herein, the design and synthesis of cyclic peptides which constrain the DEKS sequence in a β-turn conformation will be described. Lysine-side chain attachment to 2-chlorotrityl chloride-modified polystyrene resin followed by microwave-assisted solid-phase peptide synthesis and on-resin cyclisation allowed for an efficient access to head-to-tail cyclised DEKS-derived cyclic penta- and hexapeptides. An N^ε-(4-fluorobenzoyl)lysine residue was included in the cyclopeptides to allow their potential radiolabelling with fluorine-18 for PET imaging of lysyl oxidase. Conformational analysis by ¹H NMR and chiroptical (electronic and vibrational CD) spectroscopy together with MD simulations demonstrated that the concomitant incorporation of a d-proline and an additional lysine for potential radiolabel attachment accounts for a reliable induction of the desired βI-turn structure in the DEKS motif in both DMSO and water as solvents. The stabilised conformation of the cyclohexapeptide is further reflected by its resistance to trypsin-mediated degradation. In addition, the deaminated analogue containing allysine in place of lysine has been synthesised via the corresponding ε-hydroxynorleucine containing cyclohexapeptide. Both ε-hydroxynorleucine and allysine containing cyclic hexapeptides have been subjected to conformational analysis in the same manner as the lysine-based parent structure. Thus, both a conformationally restricted lysyl oxidase substrate and product have been synthetically accessed, which will enable their potential use for molecular imaging of these important enzymes.

Full text of DOI:10.1039/c4ob02348j

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Cyclopeptides containing the DEKS motif as
conformationally restricted collagen telopeptide
Cite this: DOI: 10.1039/c4ob02348j
analogues: synthesis and conformational analysis†
Robert Wodtke,a,d Gloria Ruiz-Gómez,b Manuela Kuchar,a,d M. Teresa Pisabarro,b
Pavlina Novotná,c Marie Urbanová,c Jörg Steinbach,a,d Jens Pietzscha,d and
Reik Löser*a,d
The collagen telopeptides play an important role for lysyl oxidase-mediated crosslinking, a process which
is deregulated during tumour progression. The DEKS motif which is located within the N-terminal telo-
peptide of the α1 chain of type I collagen has been suggested to adopt a βI-turn conformation upon
docking to its triple-helical receptor domain, which seems to be critical for lysyl oxidase-catalysed deami-
nation and subsequent crosslinking by Schiff-base formation. Herein, the design and synthesis of cyclic
peptides which constrain the DEKS sequence in a β-turn conformation will be described. Lysine-side
chain attachment to 2-chlorotrityl chloride-modified polystyrene resin followed by microwave-assisted
solid-phase peptide synthesis and on-resin cyclisation allowed for an efficient access to head-to-tail
cyclised DEKS-derived cyclic penta- and hexapeptides. An Nε-(4-fluorobenzoyl)lysine residue was
included in the cyclopeptides to allow their potential radiolabelling with fluorine-18 for PET imaging of
lysyl oxidase. Conformational analysis by 1H NMR and chiroptical (electronic and vibrational CD) spec-
troscopy together with MD simulations demonstrated that the concomitant incorporation of a D-proline
and an additional lysine for potential radiolabel attachment accounts for a reliable induction of the
desired βI-turn structure in the DEKS motif in both DMSO and water as solvents. The stabilised confor-
mation of the cyclohexapeptide is further reflected by its resistance to trypsin-mediated degradation. In
addition, the deaminated analogue containing allysine in place of lysine has been synthesised via the
corresponding ε-hydroxynorleucine containing cyclohexapeptide. Both ε-hydroxynorleucine and allysine
containing cyclic hexapeptides have been subjected to conformational analysis in the same manner as
the lysine-based parent structure. Thus, both a conformationally restricted lysyl oxidase substrate and
product have been synthetically accessed, which will enable their potential use for molecular imaging of
these important enzymes.
Received 5th November 2014,
Accepted 4th December 2014
DOI: 10.1039/c4ob02348j
www.rsc.org/obc
enzymes that participate in modifications of amino acid side
chains the situation is less clear. Lysyl oxidase, an enzyme
Introduction
Enzymes that act on peptides and proteins very often recognise which catalyses the oxidative deamination of protein-bound
their substrates in certain favoured conformations. This is lysine residues to the corresponding aldehydes called allysines
especially well understood for proteases,1 while for other (Scheme 1), is involved in the crosslinking of extracellular
matrix proteins such as collagen and elastin.2,3 Lysyl oxidases
aInstitute of Radiopharmaceutical Cancer Research, Helmholtz-Zentrum
Dresden-Rossendorf, Bautzner Landstraße 400, 01328 Dresden, Germany.
E-mail: r.loeser@hzdr.de; Fax: +49 351 260-2915; Tel: +49 351 250-3658
bStructural Bioinformatics, BIOTEC, Technische Universität Dresden, Tatzberg 47/49,
01307 Dresden, Germany
cDepartment of Physics and Measurements, Institute of Chemical Technology,
166 28 Prague, Czech Republic
dDepartment of Chemistry and Food Chemistry, Technische Universität Dresden,
Bergstraße 66, 01069 Dresden, Germany
†Electronic supplementary information (ESI) available: NMR spectra and
Scheme 1 Lysyl oxidase-catalysed oxidative deamination of protein-
additional Figures. See DOI: 10.1039/c4ob02348j
bound lysine residues to allysine.
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have been identified as key players in the processes of hypoxia- shaped N-telopeptide of the α1 chain (Fig. 1).5 Detailed investi-
induced tumour invasion and metastasis by remodelling the gations to unravel the structural determinants contributing to
extracellular matrix mainly through crosslinking of type I the substrate properties of telopeptides concerning their con-
collagen.6–8 Therefore, these enzymes represent a highly attrac- version by lysyl oxidase have been performed by Nagan and
tive target for functional molecular imaging of tumours. Kagan. This study indicated the requirement of the interaction
Among the different imaging modalities, positron emission with the triple-helical region around Lys 930, as the negative
tomography (PET) is unparalleled in terms of sensitivity charge of the Glu preceding the lysine residue at the telopep-
and quantification and requires the development of target- tide undergoing oxidation has to be compensated for efficient
tailored molecular probes labelled with positron-emitting enzymatic conversion. Furthermore, the conformation of the
radionuclides.9
peptide chain containing the Lys residue to be deaminated
For type I collagen, one major crosslink pathway involves seems to be of importance for its recognition by the enzyme
the condensation of an allysine carbonyl with the amino group itself, as a telopeptide derivative containing Pro N-terminal
of an unmodified lysine or hydroxylysine residue to a Schiff before Lys exhibited the most favourable kinetic parameters.15
base-type linkage.10 Notably, this process seems to occur spon-
To image lysyl oxidase in vivo, the design of radiolabelled
taneously without enzymatic assistance. Especially well under- probes based on the α1(I)-N-telopeptide seems to be promising
stood is the crosslinking process where the lysine residue as their conversion will lead to aldehydes that can react with
located in the N-terminal telopeptide of the α1(I) chain under- amino groups on collagen strands which may result in a signal
goes oxidation to act as aldehyde donor towards a (hydroxy)- enrichment on sites of enzymatic activity. As this will require
lysine (930) residue located at the α1 chain in the C-terminal the interaction of the probe molecule with collagen we envi-
region of the triple-helical portion of another type I collagen saged stabilising a potential lysyl oxidase substrate derived
molecule.5,11 On the basis of the Chou–Fasman algorithm and from the α1(I)-N-telopeptide in a β-turn-like conformation.
molecular modelling studies, it has been proposed that the
The most promising approach to stabilise the DEKS
N-terminal α1(I) telopeptide adopts a hairpin-like conformation sequence in the crucial turn seems to be its incorporation in
with the central sequence Asp-Glu-Lys-Ser (DEKS) forming a head-to-tail cyclised peptides. This approach should be most
β-turn.11 This model has been supported experimentally on feasible for cyclic penta- and hexapeptides, as their con-
the basis of 1H NMR and ECD12,13 as well as IR14 spectroscopic formational properties are well understood.16–20 Nevertheless,
analysis. In detail, these investigations revealed a confor- the use of these macrocycles in biomedical applications is
mation for the DEKS motif that is in agreement with a type I rather scarce as their synthesis can be challenging and
β-turn. Extended molecular modelling studies made evident cumbersome.21–24 Besides potentially enhanced target inter-
that the triple-helical region around lysine 930 constitutes a action, the cyclisation of peptides very often leads to improved
recognition site that is complementary to the hairpin-like pharmacokinetic properties over their linear counterparts,20,25
Fig. 1 Model of the α1(I)-N-telopeptide docked to the triple-helical receptor region of bovine type I collagen. Left and Middle: The α1(I) chains are
shown in green, the α2(I) chain in yellow and the α1(I)-N-telopeptide in purple each in ribbon plots. For the zoomed area, the βI-turn forming DEKS
sequence in the α1(I)-N-telopeptide and the Lys930 in the collagen triple helix are represented in the stick model, the proposed hydrogen bond
between Asp10N and Ser7N is depicted by the dotted line. Right: the α1(I) and α2(I) chains are shown in the surface model (grey = carbon, red =
oxygen, blue = nitrogen) and the α1(I)-N-telopeptide is shown in stick representation (purple = carbon, red = oxygen, blue = nitrogen). All figures
were prepared with PyMOL4 using the model developed by Malone et al.5 based on the file “Malone_etalFig1d3ce.pdb” included in the Supporting
Information of that article.
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which was a further motivation of this work. In order to allow end, Fmoc-Lys-OAll (1) was prepared according to a published
the attachment of the radiolabel, an additional amino acid procedure29 and attached to the 2-chlorotrityl chloride resin (2-
had to be introduced. For the development of PET tracers ClTrtCl resin) resulting in a loading degree of 0.26 mmol g−1
.
based on small molecules, the positron emitter fluorine-18 The corresponding resin-bound Fmoc-protected pentapeptide
represents the most advantageous radionuclide.26,27 Therefore, ester was sequentially deprotected at its C- and N-termini. This
a 4-fluorobenzoyl group has been chosen to be attached to the procedure ensures that the macrocycle-forming amide bond
side chain of a second lysine residue to allow the perspective coupling will occur at a hydrogen-bonded peptide bond of the
labelling with fluorine-18 of the designed cyclopeptides.28
intended β-turn.30 Conditions for allyl ester cleavage were
Herein we report on the synthesis of cyclic peptides contain- selected according to Horne et al. using Pd(PPh3)4 as catalyst
ing the telopeptide-derived DEKS motif together with its ally- and a solution of N-methylmorpholine and acetic acid in
sine-containing analogue and their conformational analysis in dichloromethane as allyl scavenger.31 To obtain the desired
both DMSO and water on the basis of NMR and chiroptical spec- cyclopentapeptide, the resin-bound terminally deprotected
troscopy supported by molecular dynamics (MD) simulations. pentapeptide was treated with HATU and DIPEA. Minicleavage
To assess the intrinsic turn-forming propensities of the DEKS followed by ESI-MS analysis indicated that the cyclisation pro-
motif, a linear analogue was synthesised and its enzymatic stabi- cedure had to be repeated twice until no linear precursor was
lity was investigated in comparison to the cyclohexapeptides.
detectable. The amino group of the Dde-protected lysine was
released by repetitive treatment with 2% hydrazine/DMF moni-
tored spectrophotometrically followed by 4-fluorobenzoylation
using 4-fluorobenzoyl chloride and triethylamine in dichloro-
methane. Cleavage from the resin and concomitant side chain
deprotection yielded the desired DEKS and 4-fluorobenzoyl
Results and discussion
Synthesis
Initially, the conformational restriction of the DEKS motif was lysine containing cyclopentapeptide 2. HPLC analysis of the
attempted within the cyclic pentapeptide 2 (Scheme 2). To this crude product indicated the presence of substantial amounts
Scheme 2 Synthesis of cyclo(Ser-Lys(FBz)-Asp-Glu-Lys) (2) and cyclo(Ser-D-Pro-Lys(FBz)-Asp-Glu-Xaa) (3, 8, 9). Reagents and conditions: (a)
1. Fmoc-Lys-OAll × TFA, DIPEA, THF, 2 h, 2. CH2Cl2–CH3OH–DIPEA (17/1/2), 3 × 2 min; (b) Fmoc-Hnl-OAll, pyridine, CH2Cl2–DMF (1/1), 24 h or 65 h,
2. CH2Cl2–CH3OH–DIPEA (17/1/2), 3 × 2 min; (c) SPPS; (d) Pd(PPh3)4, CH2Cl2–NMM–CH3COOH (8 : 2 : 1), Ar, 4 h; (e) 20% piperidine/DMF, 2 × 8 min;
(f) HATU, DIPEA, DMF, 1–3 × 4 h; (g) 2% N2H4–DMF, 5–12 × 5 min; (h) 4-fluorobenzoyl chloride, TEA, CH2Cl2, 2 h; (i) TFA–TES–H2O (95 : 2.5/2.5),
3 h; ( j) TFA–TES–CH2Cl2 (1/5/94), 30 min; (k) Dess-Martin periodinane, CH2Cl2, 3 h; (l) TFA–CH2Cl2 (9/1), 1 h.
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of side products that eluted at significantly greater retention
times represented by a broad peak. These major impurities
were assigned as oligomerisation/polymerisation products
resulting from processes competing with cyclisation. The
occurrence of these side products, despite the low initial
loading of 0.26 mmol g−1, may reflect the poor tendency for
cyclisation of the linear pentapeptide precursor. Isolation of
the product from the crude mixture furnished the 4-fluoroben-
zoylated cyclopentapeptide 2 in 22% yield. 1H NMR analysis of
2 in DMSO at varying temperatures revealed chemical shift-
temperature gradients for the amide protons <−2.5 ppb K−1
,
which indicates the absence of a defined secondary structure
in this cyclic peptide (data not shown).
The synthesis of 2 resulted in low yields and no evidence
for a stabilised structure was discernible. For this reason, it
was envisaged to include a further amino acid acting as turn-
inducing element. As both D-amino acids and proline are to be
found frequently in position i + 1 of β-turns, the incorporation
of one of these amino acids into the peptide chain can strongly
support or induce turn formation.32,33 D-Proline combines
Scheme 3 Synthesis of Fmoc-Hnl-OAll (7). Reagents and conditions:
(a) sodium nitroprusside, 4 M NaOH, pH = 9–10, 6 h at 60 °C; (b) allyl
bromide, DIPEA, DMF, 16 h at rt; (c) CH2Cl2–TFA (1 : 1, v/v), 2 h at rt; (d)
Fmoc-OSu, 0.62 M Na2CO3, DME, 2 h at 4 °C to rt.
both structural features and has therefore the potential to removal of the Boc group of 5 by treatment with trifluoroacetic
acid was accompanied by trifluoroacetylation at the ε-hydroxyl
induce turn-like conformations in cyclic hexapeptides as
demonstrated by Matter and Kessler.34 Thus, it was decided to group. As compound 6 was Fmoc-protected at its α-amino
insert a D-proline between the N-terminal serine and the lysine group under slightly basic aqueous conditions, the trifluoro-
acetic acid ester was quantitatively hydrolysed to the corres-
to be fluorobenzoylated (Scheme 2). Assuming the added
residue will adopt the i + 1 position of a β-turn, Ser, Lys(FBz), ponding alcohol 7, which was the required building block for
and Asp would be found in the positions i, i + 2, and i + 3, resin attachment.
Loading of 7 to the 2-ClTrtCl resin was tried under the
respectively. Potentially, this arrangement could lead to hydro-
gen bonding between the carbonyl oxygen of Ser and the same conditions as for the amino analogue Fmoc-Lys-OAll (1),
amide NH of Asp, which would preorganise these residues for but it resulted in no detectable resin attachment due to the
low nucleophilicity of the hydroxyl group. Applying conditions
the formation of a second β-turn where they adopt the posi-
tions of i + 3 and i, respectively. Cyclisation of the linear hexa- developed for the functionalisation of the 2-ClTrtCl resin with
peptidic precursor was done as in the synthesis of 2. Notably, Fmoc-protected amino alcohols, which employ pyridine in
excess as base and prolonged reaction times,36 led to a loading
no repetition of the cyclisation procedure was necessary, which
might reflect its stronger turn-forming propensity compared to degree of 0.86 mmol g−1 when one equivalent of 7 and a reac-
the linear pentapeptide precursor. According to HPLC analysis, tion time of 65 h was used. Construction of the hexapeptide
sequence, cyclisation, and fluorobenzoylation was done
according to the synthesis of the amino analogue. The
the content of 3 in the crude product was 80%, whereas that of
2 was only 22%.
As the oxidation reaction catalysed by lysyl oxidase leads to increased resin loading of 0.86 mmol g−1 compared to
0.26 mmol g−1 in the synthesis of the lysine-containing cyclo-
the transformation of a primary amine to its corresponding
aldehyde, the synthesis of the allysine derivative 9 of cyclo- hexapeptide 3 did not have detrimental effects on the cyclisa-
hexapeptide 3 via its hydroxy analogue 8 was envisaged tion, as confirmed by ESI-MS analysis of the product obtained
by minicleavage of the Dde-protected cyclohexapeptide. Analy-
(Scheme 2). To conserve the preparative strategy, Fmoc-Hnl-
OAll (7) (Hnl: ε-hydroxynorleucine) was intended to be sis of the fluorobenzoylated L-ε-hydroxynorleucine-containing
attached to the 2-ClTrtCl resin. Subsequent construction of the cyclo-hexapeptide 8 released from the resin indicated the pres-
ence of an undesired component exhibiting a molecular mass
hexapeptide, cyclisation, and cleavage under conservation of
the tBu side chain protection groups should be followed by oxi- that is 96 units higher than the product, which has been
dation of the primary alcohol to the allysine-containing cyclo- assigned to result from trifluoroacetylation at a free hydroxyl
group. This esterification must occur at the hydroxyl group of
hexapeptide and final deprotection. The synthesis of amino
acid 7 was achieved in four steps (Scheme 3). Nα-Boc-protected L-ε-hydroxynorleucine rather than that of serine, as trifluoro-
L-ε-hydroxynorleucine (4) was accessible in satisfying yields by acetylation has not been observed for cyclohexapeptide 3. Gen-
a chiral pool synthesis based on the diazotation of Nα-Boc-
erally, trifluoroacetylation in solid-phase peptide synthesis
lysine with sodium nitroprusside and hydrolysis of the inter- during TFA-mediated cleavage has been reported only for
mediate diazonium salt in a one-pot sequence published by N-terminal serine and threonine residues but not for internal
ones.37 As trifluoroacetylation was also encountered along the
Glenn et al.35 Treatment of 4 with allyl bromide furnished the
Boc-protected L-ε-hydroxynorleucine allyl ester (5). Surprisingly, route to building block 7 during TFA-mediated Boc deprotection
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of 5, it can be concluded that the nucleophilicity of the
hydroxyl group of the L-ε-hydroxynorleucine must be increased
due to sterical and/or electronical reasons. To hydrolyse the
TFA ester, the crude material was dissolved in a 1 : 1 mixture of
acetonitrile and water and kept at room temperature overnight.
ESI-MS analysis clearly confirmed the complete hydrolysis of
the TFA ester under these conditions. Purification by prepara-
tive HPLC revealed the existence of side products that eluted at
greater retention times than the product 8, which was already
observed for cyclopentapeptide 2 but not for cyclohexapeptide
3. Since the initial loading degree for the synthesis of 8
(0.86 mmol g−1) was significantly higher compared to 3
(0.23 mmol g−1), the generation of oligomerisation/polymeris-
ation products was probably favoured and competed against
the intramolecular cyclisation, even in the presence of
D-proline. After purification by preparative HPLC and lyophilisa-
tion of the product-containing fractions, the desired cyclohexa-
peptide 8 was obtained in 30% yield.
Fig. 2 Structure of the linear hexapeptide 10.
formally resulting from a cut between the Lys(FBz) and D-Pro
moieties of the corresponding cyclic peptide, the central resi-
dues of the intended β-turn, was synthesised. To mask the
terminal charges, the required linear hexapeptide 10 (Fig. 2)
was N- and C-terminally acetylated and amidated, respectively,
and assembled by standard Fmoc solid-phase synthesis on
Rink amide resin. The 4-fluorobenzoyl residue was introduced
in analogy to the synthesis of the cyclic peptides.
For the synthesis of the allysine-containing cyclohexapep-
tide 9, the loading procedure of Fmoc-Hnl-OAll (7) to the
2-ClTrtCl resin was repeated with a reduced amount of the
amino acid (1 eq. → 0.6 eq.) and shortened reaction time (65 h
Conformational analyses
→ 24 h). This variation resulted in a loading degree of The successful synthesis of the three cyclohexapeptides 3, 8
0.46 mmol g−1, which is still higher than that obtained with and 9 allowed the investigation of their solution confor-
Fmoc-Lys-OAll (1; 0.26 mmol g−1) but should be sufficient to mations by 1D and 2D 1H NMR experiments at 298 K in
attenuate intermolecular reactions. To synthetically access the DMSO-d6. On the basis of TOCSY experiments, it was possible
allysine-containing cyclohexapeptide 9, the selective oxidation to assign all amide and CαH resonances in the 1H NMR
of 8 was necessary. Oxidation of primary alcohols that stop at spectra of these cyclic peptides unambiguously. Notably, no
the aldehyde level is reported to work very well by employing cis-amide bonds were detected. The larger dispersion in the
the Dess-Martin periodinane as oxidising agent.38 As the chemical shifts of the NH and CαH protons of the constrained
deprotected cyclohexapeptide 8 also contains another hydroxyl peptide 3 compared to the low chemical shift dispersion
group at its serine residue, this side chain should be protected observed on its linear version 10 indicates a more defined
to prevent oxidation there. Release of peptides linked to structure for the cyclopeptide. This is particularly obvious for
2-ClTrtCl resin via their C-terminal carboxy group without the region of the CαH chemical shifts, which in the case of
affecting the acid-labile side-chain protecting groups can be cyclopeptide 3 shows distinctive signals for every proton
conveniently accomplished by incubation with a 1 : 4 mixture whereas the signals of the Asp and Ser as well as the Glu, Lys
of hexafluoroisopropanol and dichloromethane.39 However, and D-Pro Cα protons in the linear analogue 10 are overlapping
when the resin containing fully protected 8 was subjected to with each other, respectively. Values greater than 8 Hz for the
these conditions, the yield of the released peptide was very 3JNHCHα coupling constants of the residues Lys(FBz), Asp, and
low, as the 2-chlorotrityl ethers are obviously more acid-stable Lys, Aly or Hnl are consistent with a structure of restricted
than the corresponding esters. A good yield for cleavage of the flexibility.43 This assumption is supported by the presence of
fully protected cyclohexapeptide (tri-tBu-8) was achieved with medium to strong sequential dNN (i,i + 1) ROEs for the residues
TFA–TES–CH2Cl2 (1 : 5 : 94). Oxidation of tri-tBu-8 with Dess- Glu, Lys/Aly/Hnl and Lys(FBz) each in position i, which lose
Martin periodinane and subsequent deprotection with TFA led the connectivity on the Asp residue. In addition, strong-
to the desired allysine-containing cyclohexapeptide 9. Analysis medium and weak dαN (i,i + 1) ROEs were detected for D-Pro
1
by ESI-MS and H NMR clearly confirmed its identity as alde- and Asp, and Lys/Aly/Hnl, Lys(FBz), Glu, respectively (Fig. 3).
hyde. This is worth to note, as allysine-containing peptides Values for the chemical shift temperature coefficients more
have been reported to be unstable due to dehydrative cyclisa- positive than −4 ppb K−1 were observed for the amide protons
tion to tetrahydropyridine derivatives.40,41 Even over several of Asp, Glu, Lys/Hnl/Aly and Ser residues in all constrained
months at room temperature in DMSO-d6, no changes were hexapeptides (Fig. 4). Generally, temperature coefficients for
discernible for the aldehyde group from the 1H NMR spec- amide protons in DMSO more positive than −2 ppb K−1 are
trum. This result is in agreement with a more recent study in considered indicative of intramolecular hydrogen bonds,
which no intrinsic instability of peptide-incorporated allysine whereas values more negative than −4 ppb K−1 allow to con-
residues was observed either.42
clude solvent-exposed NH-groups.17,44 The most positive tem-
To gain insight into the intrinsic conformational properties perature coefficients are exhibited by the amide protons of the
of the DEKS motif, the linear version of cyclohexapeptide 3 Asp and Ser residues, which indicates their involvement in
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3
Fig. 3 Left: summary of amide NH temperature coefficients (Δδ/T > −2 ppb K−1 shown by ), JNHCHα coupling constants (>8 Hz represented by ↑
and <6 Hz shown by ↓) and ROEs correlations summary for cyclohexapeptide 3 in DMSO at 298 K. Right: 20-lowest energy structures of 3. For
clarity only the side chain of pro is shown. For summary and structures of cyclohexapeptides 8 and 9 see ESI (Fig. S2†). For distance restraints of
compounds 3, 8 and 9 see Tables S1–S3 in ESI.†
●
Applied to the cyclohexapeptides 3, 8 and 9, this allows to
infer that also the telopeptide-derived DEXS (X = Lys, Hnl, Aly)
motif must form a β-turn-like structure, as Ser and Asp are
the terminal residues of both the tetrapeptide sequences
SpK(FBz)D and DEXS. This conclusion is in accordance with the
observed temperature coefficient for the serine amide protons.
Taking into account the threshold value for temperature coeffi-
cients in DMSO outlined above, the value of −2.2 ppb K−1
observed for the amide proton of the unmodified lysine
residue is more ambiguous in regard to intramolecular
H-bonding, but it is unlikely that this amide proton is exposed
to the solvent. The chemical shift temperature coefficients exhibi-
ted by the amide protons of the linear DEKS-containing hexa-
Fig. 4 Amide temperature coefficients of the cyclohexapeptides 3, 3a,
peptide 10 were all more negative than −4 ppb K−1 except for
8–9 and the linear hexapeptide 10 in DMSO or water (marked with *).
Temperature coefficients more positive than −2.0 ppb K−1 (dashed red
the Glu NH (see MD section below), which allows to conclude
their solvent exposure and thus the absence of turn-like struc-
tures in this compound (Fig. 4).
line) or −4.5 ppb K−1 (dashed blue line) indicate that the amide proton is
part of an intramolecular hydrogen bond in DMSO and water, respect-
ively. For the values of the amide temperature coefficients and plots of
the temperature dependence of chemical shifts for the amide protons
Solution structures were calculated from a total of 37 (15
inter-residue), 34 (11 inter-residue), and 29 (11 inter-residue)
of 3, 3a, 8, 9 and 10 see ESI (Fig. S1†).
ROEs of 3, 8 and 9 in DMSO, respectively, four backbone ϕ-di-
hedral angle restraints derived from the amide coupling con-
3
intramolecular hydrogen bonds. This data combined with the stants (−120
30° for JNHCHα > 8 Hz and −65
30° for
information obtained from the ROESY spectra allows to con- 3JNHCHα < 6 Hz), and two H-bond restraints i,i + 3 involving the
clude the presence of a β-turn formed by the residues Ser, Asp and Ser residues (Fig. 3, S2 and Tables S1–S3 in ESI†).
D-Pro, Lys(FBz) and Asp stabilised by a i,i + 3 hydrogen bond Structures were calculated in XPLOR-NIH47 using a dynamic
between Ser CO (i) and Asp NH (i + 3). Generally, the criterion simulated annealing (SA) protocol,48 followed by energy mini-
for the presence of a β-turn in a peptide sequence requires the misation in the CHARMm force field.49 The 20 lowest-energy
Cα atoms of the first (i) and fourth (i + 3) residues to be less structures of 3, 8 and 9 did not have any distance (≥0.2 Å) or
than 7 Å apart from each other.18,45,46 This feature implies for dihedral angle (≥2.4°) violations, converging with a backbone
a cyclic hexapeptide that the presence of one β-turn accounts RMSD of 0.25, 0.21, and 0.24 Å, respectively. The presence of
for the existence of a second β-turn for topological reasons.
two β-turns flanked by Ser and Asp residues involving the
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Table 1 Phi (ϕ) and Psi (ψ) dihedral angles (°) defining two β-turns in cyclo(Ser-D-Pro-Lys(FBz)-Asp-Glu-Xaa) with Xaa = Lys (3), Hnl (8), Aly (9)
Compound
ϕ(D-Pro)
ψ(D-Pro)
ϕ(Lys(FBz))
ψ(Lys(FBz))
3
8
9
63.6 3.0
65.1 2.3
64.9 2.5
−108.0 10.6
−113.7 8.1
−110.4 8.6
−91.5 6.2a
−92.5 6.9
−90.4 4.5
−5.9 15.5
−3.3 13.7
−10.7 13.1
Compound
ϕ(Glu)
ψ(Glu)
ϕ(Xaa)
ψ(Xaa)
3
8
9
−57.4 4.3
−58.1 2.7
−53.5 4.9
−12.4 15.3
−19.7 3.9
−20.1 9.2
−90.5 2.7b
−89.9 1.3c
−88.7 1.3d
−23.2 21.6
−26.7 28.2
−18.4 28.5
a 90% of the 20 ensemble structures, 10% ϕ = −131.6 9.1. b 80% of the 20 ensemble structures, 20% ϕ = −150.2 0.1. c 95% of the 20 ensemble
structures, 5% ϕ = −144.9. d 90% of the 20 ensemble structures, 10% ϕ = −139.9 8.2.
sequences SpK(FBz)D and DEXS (X = Lys, Hnl, Aly) was conso-
lidated in all three constrained peptides. The Cα distances
between these flanking residues were 5.2 Å, which confirms
the compliance with the criterion for β-turns as noted
above.18,45,46 As expected, the turn involving D-Pro (i + 1) and
Lys(FBz) (i + 2) residues exhibited ϕ and ψ angle values consist-
ent with a type II reverse (II′) β-turn (Table 1). As previously
observed,50 the geometrical characteristics of this turn impose
short distances between the Cα atoms of the Ser and Asp flank-
Fig. 5 Secondary chemical shifts of Cα protons in cyclohexapeptide 3
ing residues in the cyclic structures, which allows to nucleate a
in aqueous solution referenced to tabulated random coil values (Δδ =
second β-turn around Glu (i + 1) and Xaa (Xaa = Lys, Hnl, Aly)
(i + 2) (Table 1). Their ϕ and ψ angles showed values which are
in accordance with a type I β-turn46 in 80–95% of the ensemble
structures.
δ
CHα(3) − δCHα(random coil)).52
deshielding of these protons at higher temperatures (Fig. 4).
DMSO is known to favour the formation of secondary struc- This result suggests that the changes in the chemical shifts of
tures in peptides.51 Given that the interaction of peptides with these protons over the observed temperature range is not
other biomolecules in vivo occurs in aqueous solution, the con- exclusively determined by their hydrogen bond behaviour, as
formational behaviour of 3 was also exemplarily studied in both intramolecular and intermolecular hydrogen bonds are
water. NMR spectra of 3 at varying temperatures were acquired weakened with increasing temperature.
in water, and the signals were assigned based on the 2D
However, the NH protons of the residues that stabilise the
TOCSY spectrum. As for 3 in DMSO, the dNN(i,i + 1) corre- two β-turns by acting presumably as H bond donors, Asp and
lations in the 2D ROESY spectrum in water between Asp-Lys Ser, exhibit the most positive values for Δδ/T. This finding is in
(FBz) and Lys-Ser clearly indicate the presence of the two accordance with the results obtained for DMSO as solvent,
β-turns (Fig. S3 in ESI†). Due to spectra acquisition with which indicates the similarity of the backbone conformations
solvent suppression by water presaturation, it was not possible of 3 in DMSO and water. Generally, the chemical shift tem-
to obtain quantitative ROE signals for the Cα protons. There- perature coefficients should be interpreted cautiously to gain
fore, no solution structure could be calculated. Further evi- structural information, especially when the amide protons are
dence for structural order in the peptidic backbone of 3 in in the vicinity to an aromatic moiety, as their chemical shift
water is provided by considering the differences in the Cα changes can be influenced by ring current effects.53 To investi-
proton chemical shifts between the values observed and the gate the influence of the 4-fluorobenzoyl moiety on the amide
ones tabulated for random-coil conformations in water.52 As chemical shift temperature coefficients, the analogue of 3
Fig. 5 shows, the secondary chemical shifts of the “inner resi- lacking this group (compound 3a) was prepared. The NMR
dues” in the proposed turn (Glu/Lys and D-Pro/Lys(FBz)) are analysis of 3a in water revealed that its amide protons are
negative, whereas those of Asp and Ser, the residues bridging slightly more deshielded than in 3 due to the lack of the aro-
both turns, are positive. To evaluate whether amide protons in matic ring current. Interestingly, the temperature coefficients
peptides and proteins participate in intramolecular hydrogen for the chemical shifts of the amide protons in 3a did not
bonds a temperature chemical shift coefficient of −4.5 ppb K−1 differ significantly from those in 3, and the sequence of the
is considered as threshold value in aqueous solution.53 Tem- values was the same in both derivatives, which suggests that
perature coefficients more negative than this value indicate the 4-fluorobenzoyl group has minor influence on the chemi-
solvent-exposed amide protons. Interestingly, all amide cal shift temperature coefficients of the amide protons (Fig. 4).
protons of 3 in water display positive values, i.e. their chemical
The presence of a stabilised secondary structure within
shifts increase with temperature, which is equivalent to greater the cyclohexapeptides 3, 8 and 9 was further supported by
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Fig. 6 ECD spectra of the cyclohexapeptides 3, 8, 9 and the linear ana-
logue 10 in aqueous solution in the absence and presence of trifluoro-
ethanol. The concentration of each peptide was 0.125 mg mL−1 The
spectra for 0.5 mg mL−1 can be found in Fig. S4 in ESI.†
electronic circular dichroism (ECD) spectra recorded in
aqueous solution at different concentrations and in the
absence and presence of 50% trifluoroethanol (TFE; Fig. 6).
The spectra of 3 and 8 show a strong negative maximum at
201 nm and a weaker positive maximum at 185 nm. This ECD
signature is in accordance with a type II reverse β-turn.54
Worth of note, no changes in the shape and intensity were
observed upon addition of trifluoroethanol. The spectra of the
allysine-containing analogue 9 are of similar shape even
though lower absolute values for the molar ellipticities are
exhibited. In contrast, the shape of the ECD spectrum of the
linear hexapeptide 10 is of distinct character and resembles
that of a type II poly(L-Pro) (PII) helix with a strong negative
maximum at 197 nm and a weak positive maximum at 231 nm
(Fig. 6).54 This motif of secondary structure has been very
Fig. 7 IR (top) and VCD (bottom) spectra of the cyclohexapeptides 3, 8,
9 and the linear analogue 10, and listing of characteristic signals in the
amide I region (max = maximum, pos = positive, neg = negative).
often observed in short peptides.55 Similar as for 3, 8 and 9, calculations clearly indicated a high propensity for compound
no major changes in the shape and intensity within the ECD 3 to adopt a turn-extended-turn structure. Therefore, the
spectrum of 10 were observable upon addition of TFE.
absorption bands at 1660 cm−1 and the VCD band at
Further evidence for the existence of a β-turn conformation 1658 cm−1 may result from the weak β-sheet character of this
in 3, 8 and 9 can be derived from their IR absorption and kind of structure. In this context, it should be noted that the
vibrational circular dichroism (VCD) spectra recorded in IR and VCD bands of 3, which are attributed to the β-sheet
DMSO (Fig. 7). Compared to ECD spectra, which reflect the and βII′-turn proportions, respectively, are of approximately
conformation of an oligopeptide as a whole, VCD spectra are equal intensities, in contrast to the β-hairpin structures investi-
more sensitive to local differences in conformation as they are gated by Zhao et al. where the β-sheet proportion is dominat-
derived from vibrational states and resemble an average con- ing.57 The VCD spectrum of the allysine-containing
formation in the oligopeptide molecule.56 The absorption cyclohexapeptide 9 is of similar signature in the amide
bands in the IR spectrum of 3 are 1691, 1660, 1635, 1540, and I-region as the one of 3. Obviously, the β-sheet-related IR and
1504 cm−1. The corresponding VCD spectrum exhibits a VCD bands show stronger intensities in the case of the
slightly positively biased negative couplet in the amide I ε-hydroxynorleucine analogue 8, which otherwise exhibits the
region with the positive component at 1689 cm−1 and the same band positions in the IR and VCD spectra as cyclohexa-
negative component at 1658 cm−1. This signal is followed by a peptide 3. It is striking that the VCD spectrum of the linear
negative couplet in the amide II region consisting of a positive analogue 10 is of similar shape as those of 3, 8 and 9.
band at 1554 cm−1 and a negative band at 1523 cm−1. On the However, as deduced from the NMR results and the ECD
basis of VCD investigations on small peptides with a β-hairpin spectra, the structure of 10 must be distinct from that of the
structure containing D-Pro in the position i + 1 of the central cyclohexapeptides. The negatively biased negative amide I
β-turn,57 the absorption bands at 1691 cm−1 and 1635 cm−1 couplet consisting of a positive component at 1683 cm−1 and a
in the IR spectrum and the VCD band at 1689 cm−1 can be negative component at 1660 cm−1 may indicate the presence
attributed to the β-turn structure. The NMR-based structure of a PII helix,58 which is in agreement with the ECD results.
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Table 2 Phi (ϕ) and Psi (ψ) dihedral angles (°) obtained from MD simulations of 3 and 9 in DMSO and water
ϕ(D-Pro)
ψ(D-Pro)
ϕ(Lys(FBz))
ψ(Lys(FBz))
Turnc (%)
3a
3b
9a
9b
66.3 9.7
63.5 10.0
66.1 9.9
63.7 9.8
−122.1 11.9
−124.0 13.4
−119.9 12.3
−123.7 13.5
−83.3 12.8
−82.3 14.1
−84.4 13.2
−81.8 13.8
3.1 17.2
0.1 19.3
0.8 16.3
−0.6 19.2
97
98
96
98
ϕ(Glu)
ψ(Glu)
ϕ(Xaa)
ψ(Xaa)
Turnc (%)
3a
3b
9a
9b
−59.9 10.4
−60.9 11.5
−65.9 9.1
−62.5 11.3
−17.6 11.4
−24.8 16.8
−14.7 10.6
−22.9 15.4
−92.0 14.0
−103.2 22.0
−93.6 15.2
−103.7 22.3
−33.8 12.3
−28.8 18.8
−30.4 13.5
−28.6 19.0
82
87
86
86
a DMSO. b Water. c Calculated according to the angle deviations allowed around the ideal dihedral angles that define the classical type II reverse
β-turn and type I β-turn, respectively.46
The most significant hint for the absence of a β-turn-like struc-
ture in 10 is included in its IR spectrum, in which the band
around 1635 cm−1 is missing, while the cyclic counterparts
feature this vibrational transition (Fig. 7). Taken together, it
can be concluded that in this case ECD provides more infor-
mation regarding the secondary structure compared to VCD,
as the VCD spectra of the cyclopeptides and the linear hexa-
peptide 10 differ only subtly from each other.
To gain further insight into the conformational behaviour
of the constrained peptides 3 and 9, MD simulations were per-
formed. Their conformational space was explored in DMSO
and water as solvents. One of the best NMR-calculated struc-
tures was selected in each case and simulated without
restraints for 100 ns using the ff99SB force field59 and stan-
dard protocols as implemented in the AMBER12 package.60
The trajectories were analysed in terms of backbone RMSD,
backbone dihedral angles and H-bonding. The turn-extended-
turn conformations observed from NMR-calculations were also
dominant during the simulations in both DMSO and water
(Table 2, Fig. 8 and 9).
Comparison of the backbone dihedral angles involving
D-Pro, Lys(FBz), Glu, and Xaa (Xaa = Lys and Aly) in DMSO and
water showed consistency with the two β-turn types previously
identified (Table 2, ESI Fig. S5†). The type II reverse β-turn
with D-Pro at position i + 1 was well maintained during at least
96% of the simulation time in each solvent. The type I β-turn
involving Glu and Xaa (Xaa = Lys and Aly) residues was also
consistent for 82–87% of the sampled conformations. Hydro-
gen bond analysis shed further light about the conformational
space visited by the constrained cyclohexapeptides 3 and 9 in
the different solvent environments (Fig. 9). The geometric cri-
Fig. 8 Backbone RMSD of 3 (black) and 9 (grey) in DMSO (top) and
water (bottom). Backbone RMSD referred to the mass-weighted least-
squares fitting of the average structures after removal of overall transla-
tional and rotational motions of 100 ns MD simulations.
terion used to calculate the presence of intramolecular hydro- Ser and the CO group of Asp was also observed, although with
gen bonds in 3 and 9 involved distance and angle cutoffs of lower occupancy (68% and 67% for 3 and 9 in DMSO, respect-
3.5 Å and 120°, respectively. The hydrogen bond involving the ively). The presence of this hydrogen bond dropped consider-
amide NH of Asp and the CO group of Ser showed similar ably in water (38% and 39% for 3 and 9, respectively).
occupancy (88–92%) in both compounds. These data are in Consistent with the experimental data, the MD studies illus-
good agreement with the experimentally determined amide trate the presence of a highly populated H-bond in DMSO
temperature coefficients and provide evidence for the high (>90%) between the side chain of Asp and the amide NH of
stabilisation of the type II reverse β-turn in these cyclic hexa- Lys/Aly in 3 and 9, respectively (see Fig. S6 in ESI†). This result
peptides. In addition, the H-bond involving the amide NH of is in accordance with previous studies on β-turns in peptides
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have similar propensities as in the lysine analogue 3. The
strong tendency for the formation of the Asp-Lys side chain
contacts in DMSO can be explained by the highly populated
H-bond between the Asp side chain and the amide NH of Lys,
which may preorganise the Asp residue for the formation of
that salt bridge while the side chain of Glu prefers to form
H-bond contacts with its amide NH (see ESI Fig. S6†).65 In con-
trast, the occupancy of both H-bonds is significantly decreased
in water. The intraresidual (backbone-side chain) H-bond in
the Glu residue has been also observed for the linear hexapep-
tide 10, as indicated by the low temperature coefficient for the
amide NH of Glu in DMSO (Fig. 4).
The increased structural flexibility of the cyclohexapeptides
3 and 9 in aqueous solution compared to DMSO as solvent is
also reflected by the B factors for the backbone atoms of the
individual residues (see Fig. S7 in ESI†).
Enzymatic stability
To obtain information on the biological stability of cyclohexa-
peptide 3, its susceptibility against enzymatic cleavage was
investigated by HPLC in comparison to its linear counterpart
10. The presence of a lysine residue in these peptides renders
the peptide bond between this residue and the following
serine potentially prone towards proteolysis by the serine pro-
tease trypsin (EC 3.4.21.4). Compound 3 resisted degradation
in the presence of bovine trypsin over a time of 30 min, even
when stoichiometric amounts of the enzyme were employed
(Fig. 10).
In contrast, the linear analogue 10 was completely con-
verted within the same time already at a trypsin concentration
of 1 µM (see Fig. S8 in ESI†). These results provide further evi-
dence for a stabilised structure of 3, as most proteases are
unable to cleave peptide bonds within turn structures1 and,
furthermore, demonstrate the advantages of small cyclic pep-
tides with regard to metabolic stability.
Fig. 9 H-bond analysis derived from the 100 ns trajectories in DMSO
(red) and water (blue). Top: cyclo(Ser-D-Pro-Lys(FBz)-Asp-Glu-Lys) (3),
Bottom: cyclo(Ser-D-Pro-Lys(FBz)-Asp-Glu-Aly) (9).
and proteins, which pointed out that a hydrogen bond
between an acceptor atom in the functional side chain of the
residue at position i and the amide NH of residue at i + 2 is a
common feature of type I β-turns.34,61–63 Moreover, the posi-
tion i of type I β-turns in proteins is occupied by Asp in high
statistical probability.63,64 Upon changing the solvent from
DMSO to water the presence of the hydrogen bond between
the Asp side chain and the amide NH of Lys/Aly was reduced
by ca. 40%. To a similar extent (ca. 30%) dropped the occu-
pancy of the SerNH–AspCO H-bond, which indicates the con-
tribution of the i,i + 2 side chain-main chain interaction to the
Concluding remarks
stabilisation of the βI-turn. The loss of this H-bond was com- The results of this study demonstrate that the collagen α1(I)-N-
pensated by an alternative contact between the Asp side chain telopeptide-derived DEKS motif can be successfully stabilised
and the amide NH of Glu. As opposed to the results obtained in a type I β-turn as a part of a turn-extended-turn confor-
from NMR studies on the linear DEKS sequence, in which it mation in cyclic hexapeptides and thus preorganised to inter-
has been concluded that a salt-bridge between the side chains act with its receptor region at type I collagen. D-Pro together
of Glu and Lys could stabilise a type I β-turn (in CD3OH–H2O with Lys(FBz) were introduced strategically in the cyclohexa-
(60/40) as solvent),13 low H-bond occupancy was observed peptide to favour a type II′ β-turn and to nucleate a type I
between these side chains in the constrained analogue 3 in β-turn in the DEKS collagen recognition sequence. Further-
DMSO. In fact, the Asp and Lys residues showed a tendency to more, the introduction of a second Lys residue allows the
form H-bond contacts between their side chains, which was attachment of a reporter group to enable the convenient func-
twice as high compared to the side chains of Glu and Lys. The tionalisation with the radionuclide fluorine-18 for PET
fact that the Glu-Lys side chain contact seems to be of negli- imaging studies. This together with the evaluation of the sub-
gible importance for the stabilisation of the βI-turn is further- strate properties towards lysyl oxidase-catalysed deamination is
more supported by the results of H-bond analysis from the MD subject of ongoing investigations. The replacement of the
simulation for the allysine-derived cyclohexapeptide 9 (Fig. 9). DEKS-derived Lys residue by ε-hydroxynorleucine and allysine
These indicate that the H-bond contacts between Asp(CO) and did not compromise the structural stabilisation of the βI-turn
Ser(NH), as well as between the Asp carboxylate and Aly(NH), within this motif. The building block Fmoc-Hnl-OAll (7)
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and water, the occupancy of the H-bond involving the amide
NH Ser residue and the CO group of Asp decreased consider-
ably in the aqueous environment, which may have impli-
cations for the understanding of the collagen cross-linking
process. Spectroscopic investigations of the linear analogue 10
did not reveal an inherent turn-forming propensity for the
DEKS motif. Furthermore, the cyclic counterpart 3 was resist-
ant towards tryptic cleavage, as opposed to 10. These results
demonstrate the power of cyclic hexapeptides to constrain
tetrapeptide sequences in β-turn-like conformations with con-
comitantly achieving stability against proteolytic degradation.
Moreover, the combination of D-Pro as turn inducer together
with an adjacent lysine for functionalisation with reporter
groups might be useful as a general approach for the design of
other imaging probes. Potentially, this approach may stimulate
the design of other turn mimetic-based radiotracers, especially
those targeting G-protein coupled receptors, as many ligands
of peptide-activated GPCRs bind to their receptors in β-turn-
like conformations.66,67
Experimental section
General
All commercial reagents and solvents were used without
further purification unless otherwise specified. Melting points
were determined on a Galen III Boetius apparatus from Cam-
bridge Instruments. Nuclear magnetic resonance spectra were
recorded on a Varian Unity 400 MHz or an Agilent Techno-
logies 400 MR spectrometer. Spectra were processed by using
MestreNova (version 6.1.1-6384).68 NMR chemical shifts were
referenced to the residual solvent resonances relative to tetra-
methylsilane (TMS). Mass spectra (ESI) were obtained on a
Micromass Quattro LC or a Waters Xevo TQ-S mass spectro-
meter each driven by the Mass Lynx software. Elemental analy-
sis was performed on
a LECO CHNS-932 apparatus.
Determination of resin loading was performed on a Thermo
Scientific Helios α UV/Vis spectrophotometer.
Chromatography
Thin-layer chromatography (TLC) was performed on Merck
silica gel F-254 aluminium plates with visualisation under UV
(254 nm) and/or staining with a 0.1% (m V−1) ninhydrin solu-
tion in ethanol. Preparative column chromatography was
carried out using Merck silica gel (mesh size 230–400 ASTM)
with solvent mixtures as specified for the particular com-
pounds. Preparative HPLC for the purification of peptides 3,
3a, 8–10 was performed on a Varian Prepstar system equipped
with UV detector (Prostar, Varian) and automatic fraction col-
lector Foxy 200. A Microsorb C18 60-8 column (Varian
Dynamax 250 × 21.4 mm) was used as the stationary phase
and a binary gradient system of 0.1% CF3COOH–water (solvent
A) and 0.1% CF3COOH–CH3CN (solvent B) at a flow rate of
10 mL min−1 served as the eluent. The conditions for the gra-
dient elution are as follows: 0–3 min 90% A, 3–35 min gradient
Fig. 10 Stability of cyclohexapeptide 3 against cleavage by trypsin as
determined by RP-HPLC. Compound 3 was incubated with bovine
trypsin at varying concentrations over a time of 30 min. To distinguish
the signals that originate from the enzyme, trypsin alone was analysed
under the same conditions.
required for the construction of these analogues was obtained
by an efficient synthetic procedure which will account for the
synthesis of further Hnl/Aly-containing cyclopeptides.
Although the geometrical characteristics that define the type I
β-turn around DEKS sequence is maintained in both DMSO
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to 35% B, 35–40 min gradient to 100% B, 40–45 min 100% B, modifications. To
a
suspension of Boc-Lys-OH (5.00 g,
45–50 min gradient back to 90% A.
20.3 mmol) in water (78.5 ml) was added 4 M NaOH (7.85 mL)
Analytical HPLC for both analyses of peptidic products as at 60 °C followed by small portions of sodium nitroprusside
well as stability assays outlined below was performed on an (9.23 g, 31.0 mmol) as solid. After complete addition of
Agilent Technologies 1200 LC system consisting of gradient sodium nitroprusside, more of 4 M NaOH (7.85 mL) was
pump G1311A combined with degasser G1322A, autosampler added resulting in a pH of 9 to 10. The resulting red-brown
G1329A, column heater G1316A and diode array detector suspension was stirred for 6 h at 60 °C, then cooled in an ice
G1315D. The system was operated by the Agilent Chemstation bath and treated with 1 M HCl until pH ≈ 1 was reached
2008 software.
A Nucleosil Standard C18 100-7 column (78.5 mL). The resulting dark brown suspension was extracted
(Macherey-Nagel EC 250 × 4.6) was used as stationary phase with ethyl acetate (3 × 80 mL), the combined organic layers
and a binary gradient system of 0.1% CF3COOH–water (solvent were dried over Na2SO4 and the solvent was removed in vacuo.
A) and 0.1% CF3COOH–CH3CN (solvent B) at a flow rate of The crude product was obtained as yellow oil, which spon-
1 mL min−1 served as the eluent. The stationary phase was taneously crystallised upon trying to dissolve in ethyl acetate
kept at 30 °C and the programme for elution was as follows: again. The precipitate was filtered off to obtain 2 (1.93 g, 38%)
0–3 min 90% A, 3–35 min gradient to 35% B, 35–40 min gradi- as a fluffy white powder. Rf 0.23 (ethyl acetate–CH3COOH
ent to 100% B, 40–45 min 100% B, 45–50 min gradient back to 99 : 1); m.p. 110–113 °C (Lit.72 112–113 °C); 1H-NMR (DMSO-
3
90% A.
d6) δ: 12.37 (s, 1H, COOH), 7.02 (d, J = 8.0 Hz, 1H, NH), 4.35
(broad s, 1H, OH), 3.87–3.78 (m, 1H, CαH), 3.39–3.33 (m, 2H,
CεH2), 1.69–1.47 (m, 2H, CH2), 1.45–1.22 (s, 13H, 3 × CH3 of
Synthetic procedures
Fmoc-Lys-OAll × TFA (1). The synthesis was accomplished in Boc, 2 × CH2); 13C-NMR (DMSO-d6) δ: 174.25, 155.57, 77.89
orientation to procedures published in ref. 29, 69 and 70 with (quart. C of Boc), 60.48 (Cε), 53.48 (Cα), 32.04, 30.66, 28.21 (3 ×
slight modifications. To a solution of Fmoc-Lys(Boc)-OH CH3 of Boc), 22.25; ESI-MS (ESI+) m/z: calc. for C11H22NO5
(2.00 g, 4.3 mmol, 1 equiv.) in CH3CN (10 mL) was added allyl [M + H]+, 248.15, found 248.4; Elemental analysis: calc. for
bromide (9.3 mL, 108 mmol, 25 equiv.) and DIPEA (1.5 mL, C11H21NO5, C, 53.43; H, 8.56; N, 5.66; found C, 53.65; H, 8.61;
8.6 mmol, 2 equiv.). After stirring for 5 h at 40 °C the reaction N, 5.61.
mixture was diluted with ethyl acetate (100 mL) and succes-
Boc-Hnl-OAll (5). The synthesis was accomplished according
sively washed with 0.1 M HCl (3 × 50 mL), saturated NaHCO3 to the C-terminal allyl-protection of Fmoc-Ser-OH described in
(3 × 50 mL) and saturated NaCl (3 × 50 mL). The organic phase ref. 73. To a solution of 4 (1.60 g, 6.5 mmol, 1 equiv.) in DMF
was dried over Na2SO4 and evaporated to yield intermediary (40 mL) DIPEA (2.2 mL, 13 mmol, 2 equiv.) and allyl bromide
Fmoc-Lys(Boc)-OAll as a yellow oil. The crude material was dis- were added dropwise at 0 °C. The resulting light yellow solu-
solved in CH2Cl2–TFA (1 : 1 v/v, 50 mL) and the resulting tion was stirred over night at room temperature. The reaction
orange solution was stirred for 2 h at room temperature. The mixture was then diluted with ethyl acetate (110 mL), washed
volatile components were removed in the N2 stream followed with water (4 × 100 mL) and saturated NaCl (1 × 100 ml). The
by repeated evaporations from diethyl ether (3 × 30 mL). The organic phase was dried over Na2SO4 and evaporated to obtain
obtained brownish oily residue was purified via column a yellow oil. The crude product was purified via column chrom-
chromatography (gradient from CH2Cl2–CH3OH 95 : 5 to atography (gradient from petroleum ether–ethyl acetate 3 : 1
CH2Cl2–CH3OH 80 : 20) to afford 1 (2.03 g, 91%) as an off- over 3 : 2 to 1 : 1). The product-containing fractions were com-
white, adhesive solid. Rf 0.55 (CHCl3–CH3OH–CH3COOH bined and evaporated to afford 5 (1.22 g, 65%) as a yellow oil.
1
3
75 : 25 : 2); H-NMR (DMSO-d6) δ: 7.89 (d, J = 7.5 Hz, 2H, 2 × Rf 0.16 (petroleum ether–ethyl acetate 3 : 2); 1H-NMR (CDCl3)
CH of Fmoc), 7.83 (broad s, 3H, NH3 ), 7.79 (d, 3J = 7.9 Hz, 1H, δ: 5.97–5.84 (m, 1H, CHvCH2), 5.33 (d, 3J = 17.2 Hz, 1H,
+
NH), 7.71 (dd, 3J = 7.4 Hz, 4J = 3.5 Hz, 2H, H-1,8 of Fmoc), 7.42 CHvCHH), 5.25 (d, 3J = 10.4 Hz, 1H, CHvCHH), 5.08–5.00
(t, 3J = 7.4 Hz, 2H, 2 × CH of Fmoc), 7.33 (t, 3J = 7.4 Hz, 2H, 2 × (m, 1H, NH), 4.68–4.57 (m, 2H, CH2O of allyl), 4.33–4.28 (m,
CH of Fmoc), 5.95–5.82 (m, 1H, CHvCH2), 5.30 (dd, J = 17.2 1H, CαH), 3.64 (t, 3J = 6.3 Hz, 2H, CεH2), 1.90–1.39 (m, 15H, 3 ×
3
Hz, J = 1.5 Hz, 1H, CHvCHH), 5.20 (dd, J = 10.5 Hz, J = 1.4 CH3 of Boc, 3 × CH2); 13C-NMR (CDCl3) δ: 172.67 (CO), 155.59
2
3
2
3
Hz, 1H, CHvCHH), 4.58 (d, J = 5.3 Hz, 2H, CH2O of Fmoc), (CONH), 131.78 (CH of allyl), 118.94 (CH2 of allyl), 80.08
4.38–4.26 (m, 2H, CH2O of allyl), 4.23 (t, 3J = 6.8 Hz, 1H, H-9 of (quart. C of Boc), 65.98, 62.63, 53.48 (Cα), 32.76, 32.24, 28.47
Fmoc), 4.08–4.00 (m, 1H, CαH), 2.84–2.68 (m, 2H, CεH2), (3 CH3 of Boc), 21.70; ESI-MS (ESI+) m/z: calc. for C14H26NO5
1.78–1.46 (m, 4H, 2 × CH2), 1.45–1.26 (m, 2H, CH2); 13C-NMR [M + H]+, 288.18, found 288.3; Elemental analysis: calc. for
2
(DMSO-d6) δ: 172.00, 157.98 (q, JC,F = 31.2 Hz, CO of TFA), C14H25NO5, C, 58.52; H, 8.77; N, 4.87; found C, 58.01; H, 8.64;
156.19, 143.75, 140.75, 132.39, 127.65, 127.07, 125.22, 120.14, N, 4.78.
117.72, 65.63, 64.76, 53.76 (Cα), 46.65 (C-9 of Fmoc), 38.52,
H-Hnl(Tfa)-OAll × TFA (6). The synthesis was accomplished
30.04, 26.44, 22.44; 19F-NMR (DMSO-d6) δ: −74.13 (s, CF3); according to the Boc-deprotection of Fmoc-Lys(Boc)-OH in ref.
ESI-MS (ESI+) m/z: calc. for C24H29N2O4 [M + H]+, 409.21, 29, 69 and 70. Compound 5 (1.17 g, 4.1 mmol) was dissolved
found 409.6.
in CH2Cl2–TFA mixture (1 : 1 v/v, 50 mL) and the resulting solu-
Boc-Hnl-OH (4). The synthesis was accomplished in orien- tion was stirred for 2 h at room temperature. The volatile com-
tation to procedures published in ref. 35 and 71 with slight ponents were removed in the N2 stream followed by repeated
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evaporations from diethyl ether (3 × 20 mL). The obtained (17/1/2, 4 mL, 3 × 2 min) and finally with CH2Cl2 (4 mL, 4 ×
residue was exposed to oil pump vacuum to remove remaining 1 min) again. The resin was dried in vacuo over night.
TFA. 1.84 g (quantitative yield containing 12% of residual TFA)
Loading of Fmoc-Hnl-OAll (7) onto 2-ClTrtCl resin
1
High loading. The synthesis was accomplished according to
ref. 36. A solution of 7 (634.7 mg, 1.55 mmol, 1 equiv.) in
CH2Cl2–DMF (1 : 1, 6 mL) was added to the preswollen
(CH2Cl2, 6 mL, 30 min) 2-ClTrtCl resin (1.00 g, 1.55 mmol,
1 equiv.) in a PP filter vessel. Pyridine (0.5 mL, 6.2 mmol,
4 equiv.) was added subsequently and the mixture was sealed
and agitated for 65 h at room temperature. The resin was suc-
cessively washed with DMF (4 mL, 4 × 1 min), CH2Cl2 (4 mL,
4 × 1 min), CH2Cl2–CH3OH–DIPEA (17 : 1 : 2, 5 mL, 3 × 2 min)
and finally with CH2Cl2 (4 mL, 4 × 1 min). The resin was dried
in vacuo over night.
Low loading. A solution of 7 (270.3 mg, 0.66 mmol, 0.6
equiv.) in CH2Cl2–DMF (1 : 1, 5 mL) was added to the pre-
swollen (CH2Cl2, 6 mL, 30 min) 2-ClTrtCl resin (712 mg,
1.10 mmol, 1 equiv.) in a PP filter vessel. Pyridine (213 µL,
2.64 mmol, 2.4 equiv.) was added subsequently and the
mixture was sealed and agitated for 24 h at room temperature.
The resin was successively washed with DMF (4 mL, 4 ×
1 min), CH2Cl2 (4 mL, 4 × 1 min), CH2Cl2–CH3OH–DIPEA
(17 : 1 : 2, 5 mL, 3 × 2 min) and finally with CH2Cl2 (4 mL, 4 ×
1 min). The resin was dried in vacuo over night.
of a brown oil were yielded. H-NMR (DMSO-d6) δ: 8.42 (s, 3H,
+
3
NH3 ), 5.99–5.87 (m, 1H, CHvCH2), 5.38 (dd, J = 17.3 Hz, 2J =
3
2
1.5 Hz, 1H, CHvCHH), 5.28 (dd, J = 10.5 Hz, J = 1.3 Hz, 1H,
CHvCHH), 4.70 (d, 3J = 5.0 Hz, 2H, CH2O of allyl), 4.38 (t, 3J =
6.3 Hz, 2H, CεH2), 4.16–4.07 (m, 1H, CαH), 1.91–1.64 (m, 4H, 3
× CH2), 1.56–1.31 (m, 2H, CH2); 19F-NMR (DMSO-d6) δ: −74.75
(s, CF3 of TFA), −74.79 (s, CF3 of Tfa); ESI-MS (ESI+) m/z: calc.
for C11H17F3NO4 [M + H]+, 284.11, found 284.3.
Fmoc-Hnl-OAll (7). The synthesis was accomplished accord-
ing to the Fmoc-protection of Lys(Dnp) described in ref. 74.
Fmoc-OSu (1.72g, 5.1 mmol) was dissolved in DME (30 mL)
and a suspension of 6 (1.84g containing residual TFA,
4.1 mmol) in 0.62 M Na2CO3 (10 mL; 1.5 equiv. with respect to
6) was added slowly under ice cooling. The reaction mixture
was stirred for 2 h at 4 °C and afterwards over night at room
temperature. The suspension was filtered and the filtrate was
adjusted to pH ≈ 3 with 1 M HCl. DME was evaporated and
the residual aqueous solution was extracted with ethyl acetate
(3 × 20 mL). The combined organic phases were washed with
water (1 × 10 mL), saturated NaHCO3 (1 × 10 mL) and saturated
NaCl (1 × 10 mL), dried over Na2SO4 and evaporated in vacuo.
The obtained oily material was purified via column chromato-
graphy (gradient from petroleum ether–ethyl acetate 3 : 1 to
1 : 1). The product-containing fractions were combined to
yield 7 (0.97 g, 58%) as colourless oil that solidified in the
refrigerator. Rf 0.22 (petroleum ether–ethyl acetate 1 : 1);
Determination of the loading yield
The determination was accomplished according to ref. 75 with
two aliquots of the resin. About 5 mg of the resin (exact value
has to be noted) containing the Fmoc-protected amino acid
allyl esters were placed into a PP reaction vessel and swollen in
DMF (2 mL) for 30 min. The suspension was filtered and the
resin was treated with 2% DBU in DMF (2 mL) and stirred for
another 30 min. The suspension was filtered into a graduated
10 mL flask and the resin was washed with CH3CN (3 × 1 mL).
Each filtrate was added to the flask and the solution was
diluted to 10 mL with CH3CN. 2 mL of this solution were
transferred to a graduated 25 ml flask and diluted with CH3CN
to 25 mL. A reference solution was prepared the same way but
without addition of the resin. The sample solutions were
measured against the reference at 294 nm in the UV/Vis
spectrometer. The loading yield was calculated by using the
following equation.
1
3
m.p. 100–103 °C; H-NMR (CDCl3) δ: 7.77 (d, J = 7.6 Hz, 2H,
3
H-4,5 of Fmoc), 7.60 (d, J = 7.0 Hz, 2H, 2 × CH of Fmoc), 7.40
(t, J = 7.4 Hz, 2H, 2 × CH of Fmoc), 7.35–7.29 (m, 2H, 2 × CH
3
of Fmoc), 5.98–5.84 (m, 1H, CHvCH2), 5.40–5.23 (m, 2H,
CHvCHH, NH), 5.27 (dd, 3J = 10.4 Hz, 2J = 1.0 Hz, 1H,
3
CHvCHH), 4.65 (d, J = 5.5 Hz, 2H, CH2O of allyl) 4.45–4.35
3
(m, 3H, CH2O of Fmoc and CαH), 4.23 (t, J = 7.0 Hz, 1H, H-9
3
of Fmoc), 3.65 (t, J = 6.3 Hz, 2H, CεH2), 1.95–1.40 (m, 6H, 3 ×
CH2); 13C-NMR (CDCl3) δ: 172.34, 156.10, 143.96, 141.46,
131.64, 127.85, 127.20, 125.22, 120.13, 119.14, 67.16 (CH2O of
Fmoc), 66.16, 62.58, 53.95 (Cα), 47.32 (C-9 of Fmoc), 32.63,
32.19, 21.64; ESI-MS (ESI+) m/z: calc. for C24H28NO5 [M + H]+,
410.20, found 410.2; Elemental analysis: calc. for C24H27NO5,
C, 70.49; H, 6.65; N, 3.42; found C, 69.89; H, 6.67; N, 3.43.
Loading ðmmol gꢀ1Þ294 nm ¼ ðE ꢁ 14 214 μmolÞ=mresin
Finally the average value was calculated.
Procedures for loading of allyl esters 1 and 7 onto 2-ClTrtCl
resin
Procedures for the syntheses of the peptides 2, 3, 3a, 8–10 on
the 2-ClTrtCl resin
Loading of Fmoc-Lys-OAll × TFA (1) onto 2-ClTrtCl resin.
The synthesis was accomplished according to ref. 29. A solu-
Synthesis of the linear peptide precursors. The linear penta-
tion of 1 (226.8 mg, 0.43 mmol, 0.5 equiv.) and DIPEA and hexapeptides were assembled on the 2-ClTrtCl resin
(0.15 ml, 0.87 mmol, 1 equiv.) in THF (5 mL) was added to the loaded with the side-chain attached Fmoc/allyl-protected
preswollen (5 mL THF, 1 h) 2-ClTrtCl resin (560.0 mg, amino acid derivatives by microwave-assisted solid phase
0.87 mmol, 1 equiv., 1.55 mmol g−1) in a PP filter vessel. The peptide synthesis using the CEM Liberty 12-channel peptide
PP filter vessel was sealed and agitated for 2 h at room tem- synthesizer with integrated microwave reactor following a stan-
perature. The resin was successively washed with DMF (4 mL, dard Fmoc protocol. The resin (0.1 mmol amino acid, 1 equiv.)
4 × 1 min), CH2Cl2 (4 mL, 4 × 1 min), CH2Cl2–CH3OH–DIPEA was swollen in DMF (5 mL) for 30 min outside the peptide
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synthesizer. After transfer to the reaction vessel, the resin was (29.5 µL, 0.25 mmol, 2.5 equiv.) were added to the suspension.
swollen in CH2Cl2–DMF (1 : 1, 10 mL) for another 15 min. The mixture was agitated for 2 h at room temperature. The
Removal of the Fmoc groups (except for N-terminal serine) was peptidyl resin was washed with CH2Cl2 (5 mL, 4 × 1 min) and
performed by using a solution of 20% piperidine in DMF with dried in vacuo over night.
0.1 M HOBt (1 × 7 mL at 35 W for 30 s and 1 × 7 mL at 44 W
for 180 s, both at 75 °C). After each treatment, the resin was
washed with DMF (5 + 28 mL). Coupling of each amino acid
Procedures for cleavage of the peptides from the 2-ClTrtCl
resin and/or the protecting groups
was performed with two solutions: 0.45 M HBTU in DMF
Simultaneous cleavage of resin bond and t-butyl protecting
(1 mL, 4.5 equiv.) and 2 M DIPEA in NMP (0.5 mL). The amino groups. The cleavage was accomplished according to ref. 29.
acids were applied as 0.2 M solutions in DMF. 2.5 mL The synthesis steps after assembly of the linear protected pre-
(5 equiv.) of these solutions were taken for the coupling steps cursors were monitored by subjecting small portions to clea-
(300 s at 21 W and 75 °C). The peptidyl resin containing the vage from resin after each step and analysis by ESI-MS.
protected linear precursor peptide was then washed with DMF Therefore, the volumes of the mixtures used for cleavage and
(21 mL) and outside the peptide synthesizer with ethanol washing are different compared to the complete release of the
(4 × 5 mL) and CH2Cl2 (4 × 5 mL).
peptide from the resin.
Removal of Allyl and Fmoc protecting groups. The deprotec-
The dry peptidyl resin was suspended in 1 or 3 mL of a
tions were accomplished according to ref. 31. The peptidyl solution of TFA–TES–H2O (95 : 2.5 : 2.5) for 1 or 3 h for small-
resin (0.1 mmol peptide, 1 equiv.) was swollen in CH2Cl2 scale analytical or complete cleavage, respectively. After filter-
(6 mL) for 30 min and then suspended in CH2Cl2–NMM– ing, the resin was washed with TFA (2 × 1 or 5 mL for analytical
CH3COOH (8 : 2 : 1, 6 mL). After 2 min of degassing with Ar, and complete cleavage, respectively) and the combined fil-
Pd(PPh3)4 (57.8 mg, 0.05 mmol, kept under Ar atmosphere) was trates were evaporated in a N2 stream. Ice-cold diethyl ether
added to the mixture and the newly formed yellow suspension (10 mL) was added to the oily residue and the mixture was
was degassed for another 2 min with Ar. The PP filter vessel cooled on ice for 30 min. After this, the newly formed white,
was sealed and agitated for 4 h. The suspension was filtered voluminous precipitate was filtered off and dried in vacuo.
and the peptidyl resin was successively washed with CH2Cl2
Release of side-chain protected cyclohexapeptide 8 from
(5 mL, 4 × 1 min), DMF (5 mL, 4 × 1 min), 0.5% w/v sodium resin. The dry peptidyl resin (0.11 mmol peptide) was sus-
diethyldithiocarbamate in DMF (5 mL, 6 × 1 min) and finally pended in 3 mL of a solution of TFA–TES–CH2Cl2 (1 : 5 : 94) for
with DMF (5 mL, 5 × 1 min) again. The removal of the Fmoc 30 min. After filtering and washing of the resin with CH2Cl2
group was performed by treatment of the peptidyl resin with (1 × 4 mL), NaHCO3 (52 mg) was added to the combined filtrates
20% piperidine in DMF (6 mL, 2 × 8 min). Then the peptidyl and the suspension was stirred for 10 min. After renewed fil-
resin was washed with DMF (6 mL, 3 × 1 min), 5% DIPEA in tration, the filtrate was evaporated in a N2 stream. The oily
DMF (6 mL, 3 × 1 min), DMF (6 mL, 1 × 1 min) and CH2Cl2 residue was dissolved in CH3CN–H2O (1 mL, 1 : 1) and stirred
(6 mL, 5 × 1 min). The peptidyl resin was dried in vacuo over over night. Finally, the solution was evaporated in vacuo and
night.
Cyclisation. The cyclisation was accomplished according to
lyophilised.
Cleavage of t-butyl protecting groups from tri-tBu-9. The dry
ref. 29. The peptidyl resin (0.1 mmol peptide, 1 equiv.) was fully protected peptidyl aldehyde 9 (43 µmol) was dissolved in
swollen in DMF (4 mL) for 30 min. DIPEA (34 µL, 0.05 mmol, 2 TFA–CH2Cl2 (5 mL, 9 : 1) and stirred for 1 h. The solution was
equiv.) and HATU (57.0 mg, 0.15 mmol, 1.5 equiv.) were added evaporated in a N2 stream. Ice-cold diethyl ether (10 mL) was
and the newly formed orange suspension was agitated for 4 h added to the oily residue and the mixture was kept on ice for
at room temperature. The peptidyl resin was washed with DMF 30 min. After this, the newly formed white, voluminous pre-
(5 mL, 5 × 1 min) and CH2Cl2 (5 mL, 4 × 1 min) and dried in cipitate was filtered off and dried in vacuo.
vacuo over night. The cyclisation was repeated until three
times with respect to the cyclisation yield.
Oxidation with Dess-Martin periodinane. To a solution of
the fully protected peptidyl alcohol 8 (43 µmol) in CH2Cl2
Removal of the Dde protecting groups. The deprotection (5 mL) was added DMP suspended in CH2Cl2 (1.5 mL). The
was accomplished according to the procedure published in ref. resulting turbid solution was stirred at room temperature and
76. The peptidyl resin (0.1 mmol peptide) was swollen in DMF the oxidation was followed by ESI-MS analysis of a small solu-
(4 mL) for 30 min before treated with 2% N2H4 in DMF (5 mL, tion sample. After 3 h, no remaining alcohol was detectable
5–12 × 5 min). The filtrates were collected and the extinctions and 1.3 M NaOH (20 mL) and CH2Cl2 (10 mL) were added and
measured against the N2H4 solution at λ = 300 nm. Constant the solution was stirred for 15 min. The organic layer was sepa-
extinction values close to zero indicate complete removal of rated and washed successively with 1.3 M NaOH (2 × 10 mL)
the Dde group. The peptidyl resin was washed with DMF and brine (1 × 10 mL). After that, the organic layer was dried
(4 mL, 4 × 1 min) and CH2Cl2 (4 mL, 4 × 1 min) and dried over NaSO4 and evaporated in vacuo.
in vacuo over night.
Fluorobenzoylation. The peptidyl resin (0.1 mmol peptide,
Analytical data for the peptides
1 equiv.) was swollen in CH2Cl2 (5 mL, 30 min) and TEA
Cyclo(Ser-Lys(FBz)-Asp-Glu-Lys) × TFA (2). Initial loading of
(34.8 µL, 0.25 mmol, 2.5 equiv.) and 4-fluorobenzoyl chloride 0.26 mmol g−1; 36.1 mg crude product resulted in 8.1 mg
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(22%) purified peptide; RP-HPLC analysis: tR = 15.8 min; (m, CβHH), 1.89–1.79 (m, CβHH), 1.78–1.60 (m, CδH2),
ESI-MS (ESI+) m/z: calc. for C31H45FN7O11, [M + H]+, 710.32, 1.53–1.32 (m, CγH2); ESI-MS (ESI+) m/z: calc. for
found 711.2.
C29H49FN8O11, [M + H]+, 685.35, found 685.2.
Cyclo(Ser-D-Pro-Lys(FBz)-Asp-Glu-Lys)
×
TFA (3). Initial
Cyclo(Ser-D-Pro-Lys(FBz)-Asp-Glu-Hnl) (8). Initial loading of
loading of 0.23 mmol g−1; 34.4 mg crude product resulted in 0.86 mmol g−1; 114.4 mg crude product resulted in 34.3 mg
27.5 mg (80%) purified peptide; RP-HPLC analysis: tR
=
(30%) purified peptide; RP-HPLC analysis: tR = 14.9 min;
1
3
3
14.2 min; H-NMR (DMSO-d6) δ: Ser: 6.83 (d, J = 6.0 Hz, NH), 1H-NMR (DMSO-d6) δ: Ser: 6.79 (d, J = 6,1 Hz, NH), 4.57–4.48
3
2
4.57–4.49 (m, CαH), 3.71–3.62 (m, CβHH), 3.35 (t, J = J = 9.4 (m, CαH), 3.71–3.62 (m, CβHH), 3.39–3.28 (m, CβHH) D-Pro:
Hz, CβHH) D-Pro: 4.31–4.26 (m, CαH), 3.79–3.71 (m, CδHH), 4.31–4.25 (m, CαH), 3.80–3.71 (m, CδHH), 3.62–3.53 (m, CδHH),
3.62–3.53 (m, CδHH), 2.12–1.73 (m, CβH2, CγH2) Lys (FBz): 8.51 2.12–1.71 (m, CβH2, CγH2) Lys (FBz): 8.52 (d, 3J = 8.2 Hz,
(d, 3J = 8.4 Hz, CαNH), 8.46 (t, 3J = 5.6 Hz, CεNH), 7.90 (dd, 3JH, CαNH), 8.46 (t, 3J = 5.4 Hz, CεNH), 7.90 (dd, 3JH,H = 8.7 Hz, 4JH,F
4
3
3
3
= 8.8 Hz, JH,F = 5.5 Hz, H-2,6 of FBz), 7.28 (t, JH,H = JH,F
=
= 5.6 Hz, H–2,6 FBz), 7.28 (t, JH,H = 3JH,F = 8.8 Hz, H–3,5 FBz),
H
8.9 Hz, H-3,5 of FBz), 4.08–4.00 (m, CαH), 3.29–3.16 (m, CεH2), 4.07–4.00 (m, CαH), 3.27–3.18 (m, CεH2), 2.12–1.71 (m, CβHH),
2.12–1.73 (m, CβH2), 1.67–1.41 (m, CδH2), 1.41–1.15 (m, CγH2) 1.63–1.43 (m, CδH2, CβHH), 1.43–1.11 (m, CγH2) Asp: 7.97 (d, 3J
3
2
Asp: 7.98 (d, J = 9.3 Hz, NH), 4.80–4.71 (m, CαH), 3.11 (d, J = = 9.4 Hz, NH), 4.81–4.71 (m, CαH), 3.12 (d, 2J = 17.1 Hz, 3J = 2.7
2
3
2
3
14.0 Hz, CβHH), 2.86 (dd, J = 17.2 Hz, J = 9.9 Hz, CβHH) Glu: Hz, CβHH), 2.88 (dd, J = 17.3 Hz, J = 9.8 Hz, CβHH) Glu: 8.17
8.21 (d, 3J = 3.0 Hz, NH), 3.71–3.62 (m, CαH), 2.41–2.25 (m, (d, J = 2.6 Hz, NH), 3.71–3.62 (m, CαH), 2.38–2.27 (m, CγH2),
3
CγH2), 2.12–1.73 (m, CβH2) Lys: 7.87 (d, J = 8.6 Hz, NH), 7.65 2.12–1.71 (m, CβH2) Hnl: 7.82 (d, 3J = 8.4 Hz, NH), 4.17–4.08
3
+
(broad s, NH3 ), 4.19–4.10 (m, CαH), 2.80–2.68 (m, CεH2), (m, CαH), 3.39–3.28 (m, CεH2), 2.12–1.71 (m, CβHH), 1.63–1.43
2.12–1.73 (m, CβH2), 1.67–1.41 (m, CδH2), 1.41–1.15 (m, CγH2); (m, CβHH), 1.43–1.11 (m, CγH2, CδH2); 13C-NMR (DMSO-d6) δ:
13C-NMR (DMSO-d6) δ: 172.43, 171.08, 170.60, 170.56, 170.50, 173.74, 172.49, 171.96, 171.91, 171.89, 171.77, 170.65, 168.72,
1
1
4
170.20, 169.45, 167.35, 163.78, 162.50 (d, JC,F = 247.9 Hz, C-4 165.07, 163.76 (d, JC,F = 248.2 Hz, C-4 FBz), 131.15 (d, JC,F
=
of FBz), 156.90 (d, 2JC,F = 34.6 Hz, CO of TFA), 130.22 (d, 4JC,F
=
2.9 Hz, C-1 FBz), 129.76 (d, 3JC,F = 9.0 Hz, C-2,6 FBz), 115.12 (d,
2.9 Hz, C-1 of FBz), 128.83 (d, JC,F = 8.9 Hz, C-2.6 of FBz), 2JC,F = 21.7 Hz, C-3,5 FBz), 61.96, 60.55, 60.00, 56.08, 52.95,
3
2
114.34 (d, JC,F = 21.7 Hz, C-3,5 of FBz), 73.99, 61.63, 59.75, 52.85, 48.46, 47.45, 38.74, 36.91, 31.86, 31.29, 30.19, 30.10,
55.87, 52.80, 52.71, 52.33, 48.35, 47.32, 38.71, 38.66, 36.91, 28.74, 25.80, 24.84, 23.09, 22.24. Two signals are missing (Cα
30.81, 30.23, 28.83, 28.79, 26.40, 25.86, 24.94, 23.22, 22.63; and methylene group); 19F-NMR (DMSO-d6) δ: −110.33 (tt, 3JF,H
4
19F-NMR (DMSO-d6) δ: −75.02 (s, CF3 of TFA), −110.30– = 8.9 Hz, JF,H = 5.6 Hz, FBz); ESI-MS (ESI+) m/z: calc. for
−110.40 (m, FBz); ESI-MS (ESI+) m/z: calc. for C36H52FN8O12, C36H51FN7O13, [M + H]+, 808.35, found 809.0.
[M + H]+, 807.37, found 808.1.
Cyclo(Ser-D-Pro-Lys(FBz)-Asp-Glu-Aly) (9). Initial loading of
3
1H-NMR (water) δ: Ser: 7.46 (d, J = 6.5 Hz, NH), 3.87–3.76 0.46 mmol g−1; 22.6 mg crude product resulted in 5.7 mg
(m, CβH2), CαH lies under the water signal D-Pro: 4.37–4.32 (m, (25%) purified peptide; RP-HPLC analysis: tR = 15.1 min;
3
CαH), 3.76–3.65 (m, CδH2), 2.21–1.55 (m, CβH2, CγH2) Lys 1H-NMR (DMSO-d6) δ: Ser: 6.83 (d, J = 6.2 Hz, NH), 5.09–5.02
3
3
(FBz): 8.72 (d, J = 7.4 Hz, CαNH), 8.46 (t, J = 5.8 Hz, CεNH), (m, OH), 4.57–4.48 (m, CαH), 3.71–3.62 (m, CβHH) (second
7.79 (dd, 3JH,H = 8.7 Hz, 4JH,F = 5.4 Hz, H-2,6 FBz), 7.24 (t, 3JH,H CβH of Serine lies under water signal) D-Pro: 4.32–4.25 (m,
3
= JH,F = 8.8 Hz, H-3,5 FBz), 4.32–4.21 (m, CαH), 3.45–3.34 (m, CαH), 3.80–3.71 (m, CδHH), 3.62–3.53 (m, CδHH), 2.11–1.70 (m,
3
CεH2), 2.21–1.55 (m, CβH2, CδH2), 1.54–1.31 (m, CγH2) Asp: CβH2 und CγH2) Lys (FBz): 8.51 (d, J = 8.2 Hz, CαNH), 8.46 (t,
3
3
4
8.24 (d, J = 9.1 Hz, NH), 3.07–2.89 (m, CβH2), CαH lies under 3J = 5.4 Hz, CεNH), 7.90 (dd, JH,H = 8.8 Hz, JH,F = 5.6 Hz,
the water signal Glu: 8.41 (d, 3J = 4.0 Hz, NH), 4.04–3.97 (m, H-2,6 FBz), 7.28 (t, JH,H = JH,F = 8.8 Hz, H-3,5 FBz), 4.08–3.99
CαH), 2.58–2.37 (m, CγH2), 2.21–1.55 (m, CβH2) Lys: 8.31 (d, J (m, CαH), 3.27–3.19 (m, CεH2), 2.11–1.70 (m, CβH2), 1.64–1.20
= 7.5 Hz, NH), 7.53 (broad s, NH3 ), 4.32–4.21 (m, CαH), (m, CγH2, CδH2) Asp: 7.97 (d, J = 9.3 Hz, NH), 4.80–4.71 (m,
3.07–2.89 (m, 2H, CεH2), 2.21–1.55 (m, CβH2 and CδH2), CαH), 3.10 (d, J = 17.1 Hz, J = 2.5 Hz, CβHH), 2.87 (dd, J =
1.54–1.31 (m, CγH2).
17.3 Hz, 3J = 9.7 Hz, CβHH) Glu: 8.19 (d, 3J = 2.5 Hz, NH),
3
3
3
+
3
2
3
2
Cyclo(Ser-D-Pro-Lys-Asp-Glu-Lys) × 2TFA (3a). Initial loading 3.71–3.62 (m, CαH), 2.38–2.28 (m, CγH2), 2.11–1.70 (m, CβH2)
of 0.23 mmol g−1, 19.9 mg crude product resulted in 8.6 mg Aly: 9.64 (s, CHO), 7.86 (d, 3J = 8.8 Hz, NH), 4.16–4.09 (m,
1
3
(43%) purified peptide; H-NMR (water) δ: Ser: 7.45 (d, J = 6.7 CαH), 2.45–2.38 (m, CδH2), 2.11–1.70 (m, CβH2), 1.64–1.20 (m,
Hz, NH), 3.86–3.81 (m, CβH2), CαH lies under the water signal CγH2); 13C-NMR (DMSO-d6) δ: 203.10, 173.69, 172.41, 171.95,
D-Pro: 4.43–4.38 (m, CαH), 3.79–3.69 (m, CδH2), 2.33–2.21 (m, 171.87, 171.82, 171.41, 170.71, 168.66, 165.02, 163.73 (d, 1JC,F
CβHH), 2.21–2.07 (m, CγHH), 2.07–1.89 (m, CβHH, CγHH) Lys: 247.7 Hz, C-4 FBz), 131.14 (d, JC,F = 2.7 Hz, C-1 FBz), 129.73
=
4
3
+
3
2
8.77 (d, J = 7.5 Hz, CαNH), 7.53 (broad s, NH3 ), 4.35–4.22 (m, (d, JC,F = 9.0 Hz, C-2,6 FBz), 115.09 (d, JC,F = 21.6 Hz, C-3,5
CαH), 3.05–2.94 (m, CεH2), 2.07–1.89 (m, CβHH), 1.78–1.60 (m, FBz), 61.83, 59.96, 55.98, 52.96, 52.84, 52.74, 48.45, 47.39,
CβHH, CδH2), 1.53–1.32 (m, CγH2) Asp: 8.27 (d, 3J = 9.1 Hz, 42.33, 38.73, 36.89, 30.78, 30.17, 30.09, 28.72, 25.73, 24.81,
3
NH), 4.98–4.94 (m, CαH), 3.11–3.06 (m, CβH2) Glu: 8.44 (d, J = 23.07, 18.38 (one signal of a methylene group is missing);
4.0 Hz, NH), 4.04–3.97 (m, CαH), 2.61–2.41 (m, CγH2), 19F-NMR (DMSO-d6) δ: −109.95 (tt, JF,H = 8.8 Hz, JF,H = 5.2
3
4
2.21–2.07 (m, CβH2) Lys: 8.31 (d, J = 7.4 Hz, NH), 7.53 (broad Hz, FBz); ESI-MS (ESI+) m/z: calc. for C36H49FN7O13, [M + H]+,
3
+
s, NH3 ), 4.35–4.22 (m, CαH), 3.05–2.94 (m, CεH2), 2.07–1.89 806.34, found 806.5.
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Ac-Lys(FBz)-Asp-Glu-Lys-Ser-D-Pro-NH2
Organic & Biomolecular Chemistry
×
TFA (10). Initial tivity of 100 mdeg. The final spectrum was obtained as an
loading of 0.60 mmol g−1 (Rink-Amid resin); 140.5 mg crude average of 5 accumulations. The spectra were corrected for a
product resulted in 103.1 mg (74%) purified peptide. RP-HPLC baseline by subtracting the spectra of the corresponding poly-
analysis: tR = 12.9 min; mixture of D-Pro s-trans/s-cis isomers peptide-free solution. The ECD measurements were conducted
(ratio 7 : 3), NMR signals are only listed for the major isomer: at room temperature.
1H-NMR (DMSO-d6) δ: Lys (FBz): 8.46 (t, 3J = 5.5 Hz, CεNH),
3
3
4
IR and VCD spectroscopy
8.09 (d, J = 6.9 Hz, CαNH), 7.90 (dd, JH,H = 8.8 Hz, JH,F = 5.6
3
Hz, H-2,6 FBz), 7.28 (t, JH,H
=
3JH,F = 8.8 Hz, H-3,5 FBz), Solutions of peptides 3, 8, 9, and 10 were prepared in DMSO to
4.16–4.07 (m, CαH), 3.27–3.18 (m, CεH2), 1.94–1.80 (m, CH3), a concentration of 20 mg mL−1 in a total volume of 100 µL
1.66–1.56 (m, CβHH), 1.56–1.42 (m, CβHH, CδH2), 1.41–1.20 each. The VCD and infrared absorption spectra in the
(m, CγH2) Asp: 8.29 (d, 3J = 7.7 Hz, NH), 4.56–4.46 (m, CαH), 1800–1400 cm−1 region were measured on a FTIR IFS 66/S
2.80–2.69 (m, CβHH), 2.59–2.49 (m, CβHH) Glu: 7.65 (d, 3J = 8.1 spectrometer equipped with a PMA 37 VCD/IRRAS module
Hz, NH), 4.34–4.16 (m, CαH), 2.34–2.12 (m, CγH2), 1.94–1.80 (Bruker, Germany). The samples were placed in a demountable
3
(m, CβHH), 1.80–1.67 (m, CβHH) Lys: 7.85 (d, J = 7.8 Hz, NH), cell (A145, Bruker, Germany) composed of CaF2 windows sepa-
+
7.72–7.57 (broad s, NH3 ), 4.34–4.16 (m, CαH), 2.80–2.69 (m, rated by a 23 µm Teflon spacer. All of the VCD spectra were
CεH2), 1.66–1.56 (m, CβHH), 1.56–1.42 (m, CβHH, CδH2), recorded at a spectral resolution of 8 cm−1 and are the average
1.41–1.20 (m, CγH2) Ser: 8.20 (d, 3J = 6.8 Hz, NH), 4.56–4.46 of the 15 blocks of 3686 scans (20 minutes). The OPUS 6.5 soft-
(m, CαH), 3.67–3.55 (m, CβHH), 3.53–3.43 (m, CβHH) D-Pro: ware (Bruker, Germany) was used for the VCD spectra calcu-
7.05 (s, NHH), 6.91 (s, NHH), 4.34–4.16 (m, CαH), 3.75–3.67 (m, lations. The VCD spectra were corrected for a baseline, which
CδHH), 3.67–3.55 (m, CδHH), 2.08–1.95 (m, CβHH), 1.94–1.80 was obtained as the VCD spectrum of the polypeptide-free
(m, CβHH, CγH2); 13C-NMR (DMSO-d6) δ: 174.06, 173.59, solution measured under the same experimental conditions.
172.16, 171.97, 171.55, 170.64, 170.41, 170.02, 169.11, 165.06,
1
2
Stability studies for trypsin-mediated degradation
163.74 (d, JC,F = 248.1 Hz, C-4 FBz), 158.08 (q, JC,F = 34.7 Hz,
4
3
CO TFA), 131.10 (d, JC,F = 2.9 Hz, C-1 FBz), 129.73 (d, JC,F
=
A solution of trypsin from bovine pancreas (Serva 37290, lyo-
9.0 Hz, C-2,6 FBz), 115.11 (d, JC,F = 21.7 Hz, C-3,5 FBz), 61.06, philised powder, 4 U mg−1) was freshly prepared in 0.3 M Tris-
59.63, 53.15, 53.03, 51.85, 51.78, 49.55, 46.79, 38.75, 35.58, HCl pH 7.5 at a concentration of 1 mM and kept on ice. Stock
31.31, 29.99, 29.28, 28.84, 27.22, 26.56, 23.99, 22.86, 22.40, solutions of 3 and 10 (2 mM each) were prepared in 0.3 M Tris-
21.84, signal for one methylene group is missing; 19F-NMR HCl pH 7.5. The peptide stock solutions were diluted with the
2
3
(DMSO-d6) δ: −74.93 (s, CF3 TFA-Anion), −110.28 (tt, JFH = 8.8 trypsin solution and 0.3 M Tris-HCl to reach a final peptide
4
Hz, JFH
= 5.6 Hz, FBz); ESI-MS (ESI+) m/z: calc. for concentration of 500 µM and enzyme concentrations of 0.1,
C38H57FN9O13, [M + H]+, 866.41, found 866.1.
1.0, and 500 µM in a total volume of 100 µL. If necessary, the
enzyme stock solution was prediluted with 0.3 M Tris-HCl pH
7.5. The reaction mixtures were incubated at 37 °C for 30 min.
NMR spectroscopy
The compounds 3, 8, 9 and 10 were dissolved in 600 µL of After this, an aliquot of 50 µL was withdrawn and added to 5 µL
DMSO-d6. Additionally, compounds 3 and 3a were dissolved in of 6 M HCl, vortexed and centrifuged at 1000g for 30 s. 40 µL of
600 µL of H2O. In this case, an capillary insert containing D2O the supernatant was analysed by RP-HPLC as specified above.
was placed into the sample tube to allow shiming. The H2O
Structure calculations
peak was suppressed by water presaturation. The 1D and 2D
1H NMR experiments (COSY, TOCSY (mixing time/spin-lock Distance restraints used in calculating a solution structure for
time 120 ms), and ROESY (mixing time 300 ms)) were acquired 3, 8, and 9 in DMSO-d6 were derived from ROESY spectra. ROE
on a Varian 400 MHz or an Agilent Technologies 400 MR cross-peak volumes were classified manually as strong (upper
spectrometer at 298 K. VT-NMR experiments were performed distance constraint ≤2.7 Å), medium (≤3.5 Å), weak (≤5.0 Å)
to monitor amide exchange rates at varying temperatures up to and very weak (≤6.0 Å). Standard pseudoatom distance correc-
3
318 K in 5 K intervals. JNHCHα coupling constants were tions were applied for non-stereospecifically assigned
1
measured from H NMR spectra and ROE intensities were col- protons.77 Conformational averaging was tackled by classifying
lected manually.
the intensities conservatively. Only upper distance limits were
included in the calculations to allow the largest possible
number of conformers to fit the experimental data. JNHCHα-
ECD spectroscopy
3
Stock solutions of the peptides 3, 8, 9, and 10 were prepared in coupling constants measured from 1H NMR were used to
a concentration 0.5 mg mL−1 in 10 mM sodium phosphate pH apply backbone dihedral angle restraints as follows: ϕ was
7.4. The ECD spectra were measured in a quartz cuvette with restrained to −120 30° for JNHCHα ≥ 8 Hz and to −65 30°
3
3
an optical path length of 1 mm (Starna, USA) using a J-810 for JNHCHα ≤ 6 Hz. Starting structures with randomised ϕ and
spectropolarimeter (Jasco, Japan). The conditions of the ψ angles and extended side chains were generated using an ab
measurements were as follows: a spectral region of 200 (180)– initio simulated annealing protocol.48 The calculations were
400 nm, a scanning speed of 20 nm min−1, a response time of performed using the standard force field parameter set and
8 s, a resolution of 1 nm, a bandwidth of 1 nm and a sensi- topology file included in Xplor-NIH 2.34 package47,78 within
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house modifications to generate the backbone connection enabled by partial funding within the Helmholtz-Portfolio
between the N-terminus of residue 1 and the C-terminus of Topic “Technologie und Medizin – Multimodale Bildgebung
residue 6 as well as the unnatural amino acids Lys(FBz), Hnl, zur Aufklärung des In vivo-Verhaltens von polymeren Biomater-
and Aly. The final energy minimisation included 2000 steps of ialien” by the Helmholtz Association. Partial support by the
the conjugate gradient Powell algorithm and energy mini- Fonds der Chemischen Industrie to RL is cordially acknowl-
mised using CHARMm force field.49 Final structures were visu- edged. GRG is grateful for the award of a postdoctoral fellow-
alised with PyMOL4 and had no distance (>0.2 Å) or angle ship by the Alexander von Humboldt foundation. MTP group
(>2.4°) violations.
is funded by the German Research Council (DFG, SFB-TRR67;
project A7).
Molecular dynamics (MD) simulations
MD simulations of cyclohexapeptides 3 and 9 were carried out
using the ff99SB force field59 and standard protocols as
implemented in the AMBER12 package (see below).60 Each
system was solvated in a truncated octahedral box of DMSO or
TIP3P water molecules and neutralised with Na+ counterions.
In the case of DMSO, the solvent dielectric constant was set to
47. Molecular dynamic simulations were preceded by two
energy-minimisation steps: (i) only the solvent and ions were
relaxed and position restraints were applied to the cyclohexa-
Notes and references
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The authors wish to thank Mr Jerome Kretzschmar for advice
in solvent suppression NMR experiments. This work was
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