Cui catalyzed azide-alkyne intramolecular i-to-(i+4) side-chain-to-SideChain cyclization promotes the formation of Helix-Like secondary structures

Article abstract of DOI:10.1002/ejoc.200901157

A solid-phase assembly of model peptides derived from human parathyroid, hormone-related protein (11-19) containing ω-azido- and ω-yl-α-amino acid residues in positions i and i+4 was cyclised in solution by an intramolecular Cu^I-catalyzed azide-alkyne 1,3-dipolar Huisgen cycloaddition. These series of heterodetic cyclo-nonapeptides varied in the size of the disubstituted 1,2,3-triazolyl-containing bridge, the location and the orientation of the 1,2,3-triazolyl moiety within the bridge. The 1,2,3-triazolyl moiety, presented at either 1,4or 4,1-orientation, is flanked, by side chains containing 1-4 CH₂ groups that result in bridges comprised, from 4-7 CH₂ groups connecting residues 13 and 17. Comprehensive conformational analysis employing CD, NMR and molecular dynamics reveals the conformational propensities of these heterodetic cyclo-nonapeptides. Cyclo-nonapeptides containing either the 7 methylene bridge (VII and. VIII) or the 4 methylene bridge (II) are unstructured in structure-promoting solvent. Cyclo-nonapeptide I in which the 1,4-disubstituted 1,2,3-triazolyl is flanked by 3 and 1 CH₂ groups in proximity to the respective residues 13 and 17, is stabilized in a noncanonical structure. All the other heterodetic cyclo-nonapeptides (III-VI) in which the 1,2,3-triazolyl is flanked by a total of 5 or 6 CH₂ groups nicely accommodate a-helical structures and reproduce very closely the helical structure stabilized by the analogous cyclo-nonapeptide in which Lys¹³ and Asp¹⁷ are bridged by the isosteric lactam. These studies suggest that the bioorthogonal i-to-(i+4) side-chain-to-side-chain cyclization via the prototypic click reaction offers a new and powerful approach for generating stable helix mimetic struc-tures

Full text of DOI:10.1002/ejoc.200901157

FULL PAPER
DOI: 10.1002/ejoc.200901157
CuI-Catalyzed Azide–Alkyne Intramolecular i-to-(i+4) Side-Chain-to-Side-
Chain Cyclization Promotes the Formation of Helix-Like Secondary Structures
Mario Scrima,[a] Alexandra Le Chevalier-Isaad,[b,c] Paolo Rovero,[b,d] Anna Maria Papini,[b,c]
Michael Chorev,[e,f] and Anna Maria D’Ursi*[a]
Keywords: Cyclopeptides / Helical structures / Conformation analysis / Click chemistry / Human parathyroid hormone-
related protein
A solid-phase assembly of model peptides derived from hu-
man parathyroid hormone-related protein (11–19) containing
ω-azido- and ω-yl-α-amino acid residues in positions i and i+4
was cyclised in solution by an intramolecular CuI-catalyzed
azide–alkyne 1,3-dipolar Huisgen cycloaddition. These
series of heterodetic cyclo-nonapeptides varied in the size of
either the 7 methylene bridge (VII and VIII) or the 4 methyl-
ene bridge (II) are unstructured in structure-promoting sol-
vent. Cyclo-nonapeptide I in which the 1,4-disubstituted
1,2,3-triazolyl is flanked by 3 and 1 CH2 groups in proximity
to the respective residues 13 and 17, is stabilized in a non-
canonical structure. All the other heterodetic cyclo-nonapep-
the disubstituted 1,2,3-triazolyl-containing bridge, the loca- tides (III–VI) in which the 1,2,3-triazolyl is flanked by a total
tion and the orientation of the 1,2,3-triazolyl moiety within
the bridge. The 1,2,3-triazolyl moiety, presented at either 1,4-
or 4,1-orientation, is flanked by side chains containing 1–4
CH2 groups that result in bridges comprised from 4–7 CH2
of 5 or 6 CH2 groups nicely accommodate α-helical structures
and reproduce very closely the helical structure stabilized by
the analogous cyclo-nonapeptide in which Lys13 and Asp17
are bridged by the isosteric lactam. These studies suggest
groups connecting residues 13 and 17. Comprehensive con- that the bioorthogonal i-to-(i+4) side-chain-to-side-chain cy-
formational analysis employing CD, NMR and molecular dy-
namics reveals the conformational propensities of these het-
erodetic cyclo-nonapeptides. Cyclo-nonapeptides containing
clization via the prototypic “click reaction” offers a new and
powerful approach for generating stable helix mimetic struc-
tures.
Introduction
At the macromolecular interacting sites found in recep- side-chain cyclizations through intramolecular covalent
tors, enzymes, channels, and protein-protein interfaces pep- bond formation represent a frequently practiced strategy to
tide ligands assume well-defined conformations that recog- stabilize the so called bioactive conformations, which may
nize, bind and trigger specific biological responses. How- contribute to enhanced potency, improved target selectivity,
ever, in solution, linear peptides exist as ensembles of con- favourable pharmacokinetic properties, and increased meta-
formers in dynamic steady-state equilibrium. Side-chain-to- bolic stability.
[a] Dipartimento di Scienze Farmaceutiche,
via Ponte Don Melillo 11C, 84084 Salerno, Italy
Fax: +39-089-969602
Abbreviations: Boc: tert-butyloxycarbonyl, CD: circular dichroism,
DCM: dichloromethane, DMF: N,N-dimethylformamide, DQF-
COSY: double quantum-filtered correlation spectroscopy, ESCI-
MS: electrospray/chemical ionization mass spectra, Fmoc: 9-fluor-
enylmethoxycarbonyl, HFA: hexafluoroacetone, HFIP: hexa-
fluoro-2-propanol, HOBt: N-hydroxybenzotriazole, HPLC: high-
performance liquid chromatography, MD: molecular dynamics,
NMM: N-methylmorpholine, NOE: nuclear Overhauser effect,
NOESY: NOE spectroscopy, Pbf: (2,2,4,6,7-pentamethyldihydro-
benzofuran-5-yl)sulfonyl, PTHR1: parathyroid hormone recep-
tor 1, PTHrP: parathyroid hormone-related peptide, RMSD: root
mean square deviation, RP-HPLC: reversed-phase HPLC, tR: re-
tention time, SPPS: solid-phase peptide synthesis, TBTU: 2-(1H-
benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate,
TFA: trifluoroacetic acid, TFE: trifluoroethanol, TOCSY: Total
Correlated Spectroscopy, Trt: trityl, UPLC: ultra-performance li-
quid chromatography.
E-mail: dursi@unisa.it
[b] Laboratory of Peptide & Protein Chemistry & Biology, Polo
Scientifico e Tecnologico, University of Firenze,
50019 Sesto Fiorentino, Italy
[c] Dipartimento di Chimica Organica, Polo Scientifico e Tecnolo-
gico, University of Firenze,
via della Lastruccia 13, 50019 Sesto Fiorentino, Italy
[d] Dipartimento di Scienze Farmaceutiche, University of Firenze,
via Ugo Schiff 3, Polo Scientifico e Tecnologico, 50019 Sesto
Fiorentino, Italy
[e] Laboratory for Translational Research, Harvard Medical
School,
One Kendal Square, Building 600, Cambridge, Massachusetts
02139, USA
[f] Department of Medicine, Brigham and Women’s Hospital,
75 Francis Street, Boston, MA 02115, USA
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.200901157.
446
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Helix-Like Secondary Structures
Intramolecular side-chain-to-side-chain cyclizations chain cyclization. The inherent low reactivity of the azide
through bridges include those formed by the redox suscepti- and alkynyl functions toward an expansive range of func-
ble and hydrophobic disulfide bonds between pairs of cyste- tional groups and reagents eliminates the need for elaborate
ines,[2] the polar lactams between pairs of ω-amino- and ω- protection schemes. In addition, the 1,4-disubstituted 1,2,3-
carboxyl-α-amino acids,[3] and the all hydrophobic “sta- triazolyl bridging moiety which is isosteric to the lactam
pled” rings between pairs of non-coded ω-olefin-derivatized bridge is also inert toward a vast variety of synthetic condi-
α-amino acids.[4] To enable unambiguously targeted intra- tions and its proteolytic stability is of great importance for
molecular side-chain-to-side-chain disulfide- and lactam- applications in biological and material sciences.[6,8–13]
bridged cyclizations, orthogonal protection strategies are
Recently, we have reported on different approaches to
employed. Moreover, insertion of cysteines or non-biogenic synthesize a model i-to-(i+4) side-chain-to-side-chain 1,4-
amino acids such as ω-alkenyl-α-amino acid residues leads disubstituted 1,2,3-triazolyl-bridged cyclo-nonapeptide [Nα-
to highly hydrophobic bridges (e.g. disulfide and olefin Ac-Lys-Gly-Xaa(&1)-Ser-Ile-Gln-Yaa(&2)-Leu-Arg-NH2]-
bridges).[5] Evidently, side-chain-to-side-chain cyclization [{&1(CH2)4-1,4-(1,2,3)triazolyl-CH2&2}] derived from para-
via amide bond isosteres that can be carried out without thyroid hormone-related peptide (PTHrP)[1] (Scheme 1, III)
elaborated orthogonal protection schemes is of interest and and compared its conformational preferences with the cor-
will fulfil important unmet needs. To this end, the recently responding i-to-(i+4) side-chain-to-side-chain lactam-
introduced CuI-catalyzed azide–alkyne 1,3-dipolar cycload- bridged cyclo-nonapeptide, [Nα-Ac-Lys-Gly-Lys(&1)-Ser-
dition, a prototypic “click chemistry” reaction,[6–8] presents Ile-Gln-Asp(&2)-Leu-Arg-NH2]. Both the heterodetic cy-
an interesting opportunity to develop a new paradigm for clopeptides III and the corresponding lactam, which is a
generating heterodetic cyclopeptides through pseudopep- truncated version of the α-helical and potent parathyroid
tidic and bioorthogonal intramolecular side-chain-to-side- hormone receptor 1 (PTHR1) agonist [Lys13(&1),Asp17-
Scheme 1. Linear precursors IЈ–VIIIЈ (excluding IIIЈ that was reported previously[1]): linear peptides in which Lys13 and Asp17 were
replaced with ω-azido- and ω-yl-α-amino acid residues. i-to-(i+4) side-chain-to-side-chain 1,4-disubstituted 1,2,3-triazolyl-bridged cyclo-
nonapeptides (I–VIII, excluding III that was reported previously[1]) are derived from the model peptide Nα-Ac-PTHrP(11–19)NH2.
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(&2)]PTHrP(1–34)NH2,[14,15] share the same spatial orienta- CuSO4·5H2O and ascorbic acid in tBuOH/H2O (1:2, v/v).[1]
tion of the side-chains of Ser, Gln, and Ile located at the Complete and clean conversion of all linear precursors into
i+1 to i+3 positions of the ring-forming sequences.[13]
the desired heterodetic 1,2,3-triazolyl-containing cyclo-nona-
Herein we report on the synthesis and a comprehensive peptides I–VIII was observed. Under these condition there
conformational analysis of a series of i-to-(i+4) side-chain- is no formation of oligomeric products resulting from inter-
to-side-chain 1,4-disubstituted 1,2,3-triazolyl-bridged cyclo- molecular click reactions, thus suggesting formation of a
nonapeptides (Scheme 1, I–VIII, except III that was re- CuI/acetylide/azide complex with high preference for intra-
ported previously)[1] derived from the model peptide Nα- molecular cyclizations.[17] In an attempt to identify putative
Ac-PTHrP(11–19)NH2. Solid phase peptide synthesis gen- non-catalyzed spontaneous click reaction, leading to intra-
erated a series of linear peptides in which Lys13 and Asp17 molecular cyclization or oligomerization, via intermo-
were replaced with ω-azido- and ω-yl-α-amino acid residues lecular interaction we have incubated the pure linear pep-
(Scheme 1, structures IЈ–VIIIЈ, except IIIЈ that was reported tides IЈ–VIIIЈ in water pH = 6 at 1 mg/mL for 12 days at
previously).[1,16] Solution phase intramolecular CuI-cata- room temperature and monitored changes daily, by RP-
lyzed azide–alkyne 1,3-dipolar Huisgen cycloaddition[6,8] HPLC and when warranted, also by electrospray ionization
resulted in cyclo-nonapeptides comprised from side-chains (ESI)-MS analyses. At the end of the incubation we were
bridged by either 1,4- or 4,1-disubstituted 1,2,3-triazolyl able to detect only traces of the heterodetic 1,2,3-triazolyl-
moieties (I, III, V, and VII, and II, IV, VI, and VIII, respec- containing cyclo-nonapeptide (0.5%–2.8% relative to the
tively). The bridging 1,2,3-triazolyl rings are flanked by linear precursor) (Figure 1). Only in the case of the linear
alkyl chains (CH2)m and (CH2)n (where m ϶ n, n and m = peptide IIIЈ we were able to detected the characteristic m/z
1–4 and m+n = 4, 5, 6, and 7) located at the Cα of residues = [M + 3H]+ attributed to the formation of the 1,2,3-triaz-
13 and 17, respectively. Conformational analysis that in- olyl-containing dimer (not shown). Therefore, in the rela-
cluded circular dichroism (CD) and 1H-nuclear magnetic tively short catalyzed cyclization we can rule out significant
resonance (NMR) in water and water/hexafluoroacetone contributions from non-catalyzed spontaneous intramolec-
(HFA) mixture, and molecular dynamics simulations (MD) ular cyclizations to the formation of the heterodetic 1,2,3-
revealed the impact of the size of the 1,2,3-triazolyl-con- triazolyl-containing cyclo-nonapetides I–VIII.
taining bridge, the orientation and the position of the 1,2,3-
triazolyl moiety within the connecting bridge on the confor-
mational propensities of these heterodetic cyclo-nonapep-
tides. These results offer an initial set of guidelines for the
use of this new class of side-chain-to-side-chain intramolec-
ular cyclization in the rational design of conformationally
stabilized peptides important in the development of novel
biomaterial and peptide-based drugs.
Results
Synthesis of 1,2,3-Triazolyl-Containing Cyclo-Nonapeptides
Synthesis of Linear Precursors
Figure 1. RP-HPLC chromatograms of the linear peptide precursor
The assembly of the resin-bound linear peptide precur-
sors IЈ to VIIIЈ by TBTU/HOBt/NMM-mediated couplings
was carried out in a straightforward manner, by the Fmoc/
tBu solid phase peptide synthesis (SPPS) methodology. As
previously reported, the incorporation of the building
blocks Nα-Fmoc-Xaa(ω-N3)-OH and Nα-Fmoc-Yaa(ω-yl)-
OH during the SPPS was found to be the most convenient
approach to introduce the functions essential for the subse-
quent solution phase, CuI-catalyzed intramolecular azide–
alkyne cycloaddition.[1]
incubated in tBuOH/H2O (1:2, v/v) in the absence of catalyst. Evol-
ution of the chromatograms during 12 days incubation of linear
peptide precursors IЈ in water pH = 6 at 1 mg/mL. The chromato-
grams were obtained employing a linear gradient of 10% to 60%
of B in A for 20 min, A = 0.1% TFA in H2O, B = 0.1% TFA in
CH3CN.
CD Analysis
A preliminary screening of the conformational prefer-
ences of the 1,2,3-triazolyl-containing cyclo-nonapeptides
I–II and IV–VIII as a function of the solvent system was
performed by means of CD spectroscopy.
Cyclization of the Linear Precursors via Intramolecular
Click Reaction
HPLC-purified linear peptide precursors IЈ to VIIIЈ were
CD spectra were recorded in water solution and in a
subjected to solution-phase intrachain CuI-catalyzed side- water/HFA mixture (1:1, v/v). Figure 2 shows the CD spec-
chain-to-side-chain azide–alkyne 1,3-dipolar cycloaddition. tra of 1,2,3-triazolyl-containing cyclo-nonapeptides (I–II
The click reaction conditions were identical to those re- and IV–VIII) recorded in water (pH 6.6) (Figure 2, upper
ported by us previously and used 10-fold molar excess of panel) and in water/HFA (1:1, v/v) (Figure 2, lower panel).
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Helix-Like Secondary Structures
Table 1. Quantitative evaluation of the CD spectra using DICHROWEB website (ContinLL). Relative abundance of conformational
populations are reported for cyclopeptides I–II and IV–VIII recorded in water (pH 6.6) and in HFA/water (1:1, v/v).
Cyclopeptide
Secondary structure
Helix
Strand
Turn
Disordered
HFA/water water
HFA/water
water
HFA/water
water
HFA/water water
I
0.900
0.800
1
1
1
1
1
0.339
0.340
0.524
0.843
0.553
1
0
0
0
0
0
0
0
0.235
0.100
0.05
0.009
0.040
0
0.100
0.200
0
0
0
0
0
0.298
0.330
0.196
0.148
0.279
0
0
0
0
0
0
0
0
0.128
0.230
0.230
0
0.128
0
II
IV
V
VI
VII
VIII
0.793
0.010
0.197
0
All CD spectra present the typical shapes of turn-helical To exclude potential aggregation, we recorded the 1D pro-
structures. The quantitative analysis of CD curves using ton spectra of the 1,2,3-triazolyl-containing cyclo-nonapep-
ContinLL algorithm on DICHROWEB website,[18] tides I–II and IV–VIII at a concentration range spanning
(Table 1) shows that cyclo-nonapeptides I–II and IV–VIII 1.0–0.1 m. At a peptide concentration of 1.0 m there
in water are present in similar proportions of α-helical, turn were not any noticeable effects of aggregation. Therefore,
and random coil conformations, suggesting that in water our NMR analyses were carried at sample concentrations
the cyclo-nonapeptides are characterized by flexible confor- of 0.1 m. Chemical shift assignments of the proton spectra
mations in steady-state equilibrium.
of 1,2,3-triazolyl-containing cyclo-nonapeptides I–II and
IV–VIII in water and in water/HFA mixture were achieved
via the standard systematic application of DQF-COSY,
TOCSY and NOESY experiments,[19–21] using the
SPARKY software package[22] according to the procedure
of Wüthrich.[23]
The values of the proton chemical shifts for each 1,2,3-
triazolyl-containing cyclo-nonapeptide in water and in
water/HFA (1:1, v/v), are reported in the Supporting Infor-
mation Section (Tables S5–S7). These tables also include
the chemical shifts differences between Hα values of I, II
and IV–VIII cyclo-nonapeptides analyzed by us and those
reported for random coil conformation.[24] The analysis of
chemical shift differences of cyclopeptides in water and
water/HFA mixture displays a remarkable up-field Hα
chemical shifts as compared to the standard values reported
for random coil conformations. This up-field shift, known
as indicative of turn-helical structures,[24] supports the con-
clusion drawn from the CD data on the presence of turn-
helical structures for all cyclo-nonapeptides I, II and IV–
VIII in water as well as in water/HFA solution.
Amide and fingerprint regions of NOESY spectra of I,
II and IV–VIII cyclo-nonapeptides collected in water, and
in water/HFA (1:1, v/v) are reported in Supporting Infor-
mation Section (Figures S15–S18). Figure 3 and Figure 4
summarize NOE data for 1,2,3-triazolyl-containing cyclo-
nonapeptides I, II and IV–VIII in water and water/HFA
solution, respectively. NOESY spectra of cyclo-nonapep-
tides in water include few significant NOE connectivities
suggesting a low contribution of regular secondary struc-
tures. On the contrary, NOESY spectra of cyclo-nonapep-
tides I, II and IV–VIII in water/HFA mixture display a high
number of sequential and medium range NOEs. In particu-
lar sequential α-N(i,i+1) and NH-NH(i,i+1) NOE connec-
tivities, as well as medium range α-N(i,i+2), NH-NH(i,i+2)
and NH-NH(i,i+3) NOE connectivities, define patterns
Figure 2. CD spectra of i-to-(i+4) side-chain-to-side-chain 1,2,3-
triazolyl-bridged cyclo-nonapeptides I–II and IV–VIII recorded in
water (upper-panel) and water/HFA (1:1, v/v) (lower-panel).
In water/HFA mixtures, a prevalent contribution of heli-
cal conformations is detectable, indicating the presence of
ordered structures.
NMR Analysis
NMR spectra were acquired in water and water/HFA which are diagnostic of specific canonical secondary struc-
(1:1, v/v), the same solvents used for the CD measurement. tures.[23]
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Figure 3. NOE connectivities diagrams of i-to-(i+4) side-chain-to-side-chain 1,2,3-triazolyl-bridged cyclo-nonapeptide I–II (panels a and
b) and IV–VIII (panels c–g) in water solution.
Figure 4. NOE connectivities diagrams of i-to-(i+4) side-chain-to-side-chain 1,2,3-triazolyl-bridged cyclo-nonapeptide I–II (panels a and
b) and IV–VIII (panels c–g) in water/HFA (1:1 v/v).
NMR Structure Calculations: Three-dimensional struc- mixing times. Interprotonic distances were derived from
tures of cyclo-nonapeptides I, II and IV–VIII were calcu- 200 ms mixing time NOESY spectrum. The best 20 struc-
lated by simulated annealing procedures based on sequen- tures out of 50 calculated with the DYANA software pack-
tial and medium-range NOE-derived restraints extracted age were chosen according to the lowest values of the pen-
from NOESY spectra. To avoid overestimation of NOE ef- alty (f) for the target function. These structures were energy
fects, NOESY spectra were collected using 150, 200, 250 ms minimized using the distance restraints with a progressively
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Helix-Like Secondary Structures
Figure 5. NOESY spectra were collected using 150, 200, 250 ms mixing times. Interprotonic distances were derived from 200 ms mixing
time NOESY spectrum. The best 20 structures out of 50 calculated with the DYANA software package were chosen according to the
lowest values of the penalty (f) for the target function. These structures were energy minimized using the distance restraints with a
progressively smaller force constant. This minimization procedure yielded an improved helical geometry and a lower total energy of the
structure.
smaller force constant. This minimization procedure yielded tion of the bundles of the 20 best DYANA calculated con-
an improved helical geometry and a lower total energy of formers corresponding to cyclo-nonapeptides I, II and IV–
the structures. Figure 5 shows an all-heavy atom presenta- VIII in water/HFA. The 20 low-energy conformers of each
Figure 6. Ramachandran plots of NMR structures of i-to-(i+4) side-chain-to-side-chain 1,2,3-triazolyl-bridged cyclo-nonapeptides I–II
(panel a and b) and IV–VIII (panels c–g) in water/HFA (1:1, v/v).
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cyclo-nonapeptide were overlapped at level of the residues analysis of cyclo-nonapeptide I confirms these data, by
in positions 13 and 17 that carry the side chains involved identifying the presence of non-canonical secondary struc-
in the 1,2,3-triazolyl-containing bridge. NMR structure tures. Same analysis of cyclo-nonapeptide II confirms a
bundles in water/HFA are well-defined, with local displace- major contribution of disordered structures with a minor
ment of Ͻ0.6 Å as calculated for the heavy atoms. Figure 6 presence of γ-turn conformations comprising different tri-
shows the Ramachandran plots of NMR structure bundles ads along the Ser14-Ile-Gln-Nva(δ-1-1,2,3-triazolyl)-Leu-
of the low-energy conformers in water/HFA. A pronounced Arg19-NH2 sequence.
clustering of dihedral angles is evident, with very few of
them located in disallowed regions of the maps.
The bundle formed by superimposition of the 20 low-
energy NMR structures of cyclo-nonapeptide IV by over-
Conversely, the NMR structure bundles of the cyclo- lapping the Ser14-Ile-Gln-Nle17(ε-1-1,2,3-triazolyl) segment
nonapeptide I, II and IV–VIII in water display local dis- (Figure 5, panel c) reveals a high structural similarity (back-
placement of Ͼ1.0 Å as calculated for the heavy atoms, con- bone RMSD Յ 0.54 Å). The dihedral angles are clustered
firming the CD data on the presence of disordered struc- within allowed regions of the Ramachandran map (Fig-
tures that are much more flexible than the structures of the ure 6, panel c), indicating the recurrence of regular second-
same cyclo-nonapeptide in water/HFA. The Ramachandran ary structures; these, according to PROMOTIF analysis,[25]
plots of NMR structure bundles in water display wide scat- are identified as regular type II β-turns located at the Gln16-
tering of dihedral angles with some of them in disallowed Nle(ε-1-1,2,3-triazolyl)-Leu-Arg19 segment. NMR structure
regions (not shown).
bundles of cyclo-nonapeptide V and VI (Figure 5, panels d
and e, respectively) display a good overlap of the low-energy
conformers at the sequence spanning residues 13 to 17
NMR Analysis in Water
Analysis of NMR structures in water shows that cyclo- (RMSD Յ 0.5). In agreement with these data, a large ma-
nonapeptides I, V and VIII are characterized by non-regu- jority of backbone torsion angles are located within the al-
lar, disordered conformations. Accordingly, Ramachandran lowed regions of the Ramachandran plots (Figure 6, panels
plots show significant scattering of backbone dihedral d and e), thus confirming the propensity of these cyclo-
angles, some of them falling within disallowed regions of nonapeptides to assume regular secondary structures. In-
the maps. According to PROMOTIF procedure, a weak deed, PROMOTIF analysis of backbone dihedral angles
presence of γ-turns in the Gly12-Nva(δ-1-1,2,3-triazolyl)- points to the prevalence of ordered α-helical structures
Ser-Ile15 segment of cyclo-nonapeptide I, and type I β-turn spanning almost the entire sequence Ac-Lys11-Gly-hAla(γ-
in the Ser14-Ile-Gln-Nle17(δ-4-1,2,3-triazolyl) segment of cy- 1-1,2,3-triazolyl)-Ser-Ile-Gln-Nle(δ-4-1,2,3-triazolyl)-Leu18
clo-nonapeptide V are observed. For cyclo-nonapeptide of cyclo-nonapeptide V. Same analysis identifies type I β-
VIII no regular conformation was identified. Ramachand- turn structures at the Nle13(δ-4-1,2,3-triazolyl)-Ser-Ile-
ran plots of cyclo-nonapeptides II, IV, VI and VII reveals Gln16 segment for cyclo-nonapeptide VI.
an appreciable presence of γ-turn and β-turn conforma-
The bundles formed by superimposition of the 20 low-
tions. In particular, according to PROMOTIF analysis, γ- energy NMR structure of cyclo-nonapeptides VII and VIII
and type I β-turns are found in the C-terminal segment of by overlapping the Nva13(δ-1-1,2,3-triazolyl)-Ser-Ile-Gln-
cyclo-nonapeptide II and IV, as well as in the Gly12-Nle(δ- Nle17(δ-4-1,2,3-triazolyl) sequence (Figure 5, panels f and
4-1,2,3-triazolyl)-Ser-Ile15 and Gly12-Nva(δ-1-1,2,3-triaz- g) indicate conformational disparity among the calculated
olyl)-Ser-Ile15 segments of cyclo-nonapeptide VI and VII, structures. Accordingly, in the corresponding Ramachand-
respectively.
ran maps (Figure 6, panels f and g), an appreciable scat-
tering of the backbone dihedral angles is evident. PROMO-
TIF evaluation of the NMR structures of cyclo-nonapep-
NMR Analysis in Water/HFA
Bundles of 20 low-energy conformers representing the tide VII, suggests the presence of α-helical structures span-
backbones of cyclo-nonapetides I and II are superimposed ning the Ac-Lys11-Gly-Nva(δ-1-1,2,3-triazolyl)-Ser-Ile-Gln-
at the level of Nva13(δ-1-1,2,3-triazolyl)-Ser-Ile-Gln-Pra17 Nle(δ-4-1,2,3-triazolyl)-Leu18 segment, with a noticeable
and Pra13-Ser-Ile-Gln-Nva17(δ-1-1,2,3-triazolyl) segments, presence of type I β-turn in the segment Gln16-Nle(δ-4-
respectively (Scheme 1 and Figure 5, panels a and b, respec- 1,2,3-triazolyl)-Leu-Arg19-NH2. For cyclo-nonapeptide
tively). The Ramachandran plots show a tight fit of the VIII a significant presence of γ-turn conformations en-
bundle for cyclo-nonapeptide I but not for II (Figure 6, compassing the Gly12-Nle(δ-4-1,2,3-triazolyl)-Ser-Ile15 seg-
panels a and b, respectively), suggesting that the structure ment is observed. To complete the characterization of the
of the former is defined with high precision in the overlap- conformational preferences of the cyclo-nonapeptides we
ping region, while the structure of the latter is more disor- analysed the H-bond patterns of the most representative
dered. Accordingly, the NMR structure-derived bundles NMR-derived structure for cyclo-nonapeptide I, II and IV–
show clustering of backbone dihedral angles for cyclo- VIII. A list of H-bonds involving amino acid backbone,
nonapeptide I (Figure 6, panel a) and scattering of back- side chains and the 1,2,3-triazolyl ring is reported in Sup-
bone dihedral angles for cyclo-nonapeptide II (Figure 6, porting Information (Table S9). H-bonds correlate with the
panel b). For cyclo-nonapeptide I several backbone dihe- structural regularity of the cyclo-nonapeptides and they in-
dral angles are in disallowed regions suggesting the occur- volve backbone atoms as well as atoms belonging to the
rence of non-regular secondary structures. PROMOTIF side-chain-to-side-chain bridge that includes the 1,2,3-tri-
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Helix-Like Secondary Structures
azolyl ring. In 1,2,3-triazolyl ring C5-H and N2 play as H location and orientation of the 1,2,3-triazolyl moiety within
bond donor and acceptor respectively.[9,26,27] In cyclo-nona- the bridge, and the conformational propensities of the het-
peptides I and II C5-H proton of the 1,2,3-triazolyl is H- erodetic cyclo-nonapeptides I–II and IV–VIII (Scheme 1).
bond donor to the CO of the backbone of residues 13.
Combination of stepwise solid-phase assembly of the lin-
However, this pattern does not correspond to any regular ear peptide, Fmoc/tBu strategy employing preformed ω-azi-
secondary structure. In cyclo-nonapeptides VII and VIII H- do- and ω-yl-modified Nα-Fmoc-protected amino acids,
bonds involve the backbone atoms with a negligible partici- and solution phase CuI-catalyzed click reactions of the
pation of the 1,2,3-triazolyl-containing bridge. In cyclo- purified linear peptide resulted in good yields of the hetero-
nonapeptides IV, V and VI, regular H-bond patterns in- detic 1,2,3-triazolyl-bridged cyclo-nonapeptides. As antici-
volve backbone as well as 1,2,3-triazolyl atoms. C5-H pro- pated, in the absence of catalyst intramolecular cyclization
ton is H-bond donor for the CO of the backbone of resi- and oligomerization are negligible. The CD and NMR mea-
dues 13 and/or 16, confirming the participation of both surements of the cyclo-nonapeptides I–VIII were carried
non-cyclised and cyclised portions of cyclo-nonapeptides in out in water and water/HFA mixture. Water is considered
intramolecular H-bond formation. Interestingly, similar H- the most bio-compatible medium to perform solution-phase
bond patterns were reported previously for the cyclo- conformational studies since it is the predominant compo-
nonapeptide III. Analysis of NOEs involving C5-H proton nent of most biological compartments. Nevertheless, water
of the 1,2,3-triazolyl ring, the only 1,2,3-triazolyl atom ob- solutions enhance the conformational flexibility of short
served in the proton NMR spectra (see Supporting Infor- peptides compromising the collection of a sufficient number
mation, Table S8), shows that the triazolyl ring is close to of NOE-based interprotonic distances needed for 3D-model
the backbone of amino acid residues 13 and 17 participat- building. Therefore, in order to enhance the prevalence of
ing in the side-chain-to-side-chain cyclization. Moreover, in ordered, compact and relevant conformers over extended
cyclo-nonapeptides I, II, V and VII, C5-H protons are also and/or disordered and biologically irrelevant ones, water-
proximal to protons that do not belong to the bridge, HN cosolvent mixtures with a viscosity higher than that of pure
and H2β on Ser14 or H2γ on Gln16, depending on the orien- water are frequently used.[28] Mixtures of water with fluori-
tation of the 1,2,3-triazolyl ring in the bridge, whether it is nated cosolvents such as water/trifluoroethanol (TFE),
incorporated in the 1,4- or 4,1-orientation, respectively. In water/HFA and water/hexafluoroisopropyl alcohol (HFIP),
cyclo-nonapeptides V C5-H proton of the 1,2,3-triazolyl are frequently used as media to induce “environmental con-
ring is also in contact with protons that are not included in straints” in peptides.[29] Fluorinated cosolvents exert a helix
the cyclic portion, such as Leu18 and Arg19. This pattern of inducing/stabilizing effect, which does not override the in-
interatomic distances, which is dependent on the conforma- trinsic conformational tendency dictated by the specific se-
tional feature of the cyclopeptide V, is similar to that found quence.[28,30,31] In addition, these mixtures are compatible
in the closely related 1,2,3-triazolyl-containing cyclo- with NMR experiments, allowing measurements of the
nonapeptide III.[1] Both cyclo-nonapeptides III and V share same samples in both CD and NMR and subsequently ob-
the 1,4-orientation of the 1,2,3-triazolyl ring, which is re- taining compatible data.
moved from the Cα of residue 13 by more than one methyl-
ene.
Analysis of CD spectra suggests that in water, heterodetic
cyclo-nonapeptides I–II and IV–VIII present different en-
sembles of turn-helical conformers presented in different
proportions. This finding is diagnostic of the flexibility of
the cyclo-nonapeptides in water. Confirming the CD data,
NOESY spectra in water contain NOEs suggesting the for-
Discussion
The recently introduced CuI-catalyzed azide–alkyne 1,3- mation of only incipient turn-helical conformations, which
dipolar Huisgen cycloaddition as a prototypic “click chem- are insufficient to calculate high resolution 3D models of
istry reaction”[6,8,10] offers the opportunity for introducing cyclo-nonapeptides I–II and IV–VIII. CD data in water/
the 1,4-disubstituted 1,2,3-triazolyl moiety as a new isoster HFA mixture show a dramatic decrease of turn and random
for peptide bonds.[6,8–13] We postulated that intramolecular coil conformations and point to a high prevalence of helical
side-chain-to-side-chain click reaction between two side structures. Accordingly, high quality NMR structures were
chains, one substituted by an ω-azido and the other by an calculated on the basis of NOE data in HFA/water. In any
ω-yl function, will generate an heterodetic cyclopeptide that event, by comparing the incipient conformational tendency
will mimic an analogous lactam-bridged cyclopeptide. Sub- of cyclo-nonapetides in water and in HFA/water a general
sequently, we reported the synthesis and conformational agreement seems to exist between the two sets of structural
analysis of a model i-to-(i+4) side-chain-to-side-chain 1,4- data, thus supporting the contention that water/HFA is a
disubstituted 1,2,3-triazolyl-bridged cyclo-nonapeptide III suitable solvent system to enhance and stabilize, but not to
(Scheme 1) structurally related to the helical i-to-(i+4) lac- override the intrinsic conformational propensities of pep-
tam-bridged cyclopeptide [Lys13(&1),Asp17(&2)]PTHrP(1– tides. The comparison of the conformational features of the
34)NH2.[1] Our current study aims to systematically explore cyclo-nonapeptides in water/HFA mixture indicates that:
the relationship between the features of the i-to-(i+4) intra- 1) cyclo-nonaopeptide I, in which the 1,2,3-triazolyl moiety
molecular bridge containing the 1,4-disubstituted 1,2,3-tri- is flanked by chains comprised of a total of 4 methylene
azolyl moiety, as it relates to the size of the bridge and the groups (CH2)(m+n = 4) (Scheme 1), forms a well defined con-
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A. M. D’Ursi et al.
FULL PAPER
formational ensemble that does not correspond to any ca- structure. In the absence of this structural constraint, cyclo-
nonical secondary structure (Figure 6, panel a); 2) cyclo- nonapeptides V and VI, in which the total number of CH2
nonapeptides IV/VI, which contain bridges comprised of a groups in the 1,2,3-triazolyl-containing bridge is greater
total of 5 and 6 methylene groups (CH2)(m+n = 5 and 6) than in cyclo-nonapeptide IV (6 vs. 5) and the number of
(Scheme 1), are characterized by ordered and regular sec- the CH2 groups in the chains flanking the 1,2,3-triazolyl
ondary structures (Figure 5, panels c–e); and 3) cyclo- ring is always greater than 1, assume regular conformations
nonapeptides II, VII and VIII, in which the 1,2,3-triazolyl that span the full sequence. These canonical conformations
moiety is flanked by chains comprised of a total of 4 and 7 form regardless the 1,4- or 4,1-orientation of the 1,2,3-tri-
methylene groups (CH2)(m+n = 4 and 7) (Scheme 1), form a azolyl moiety in respective cyclo-nonapeptides V and VI.
wide spread of low-energy conformations (Figure 5, panels This optimized structural regularity of cyclopeptides V and
b, f and g). These results suggest that i-to-(i+4) side-chain- VI is also reflected in their extensive H-bonds patterns
to-side-chain 1,2,3-triazolyl-bridged cyclo-nonapeptides where the participation of backbone as well as 1,2,3-triaz-
comprised of bridges containing a total of 4 and 7 methyl- olyl ring atoms is evident. Comparison of the low-energy
ene groups tend to form either non-canonical (cyclo-nona- conformations of heterodetic 1,2,3-triazolyl-containing cy-
peptide I) or disordered (cyclo-nonapeptides II, VII and clo-nonapeptides IV–VI with either the previously reported
VIII) secondary structures. Apparently, cyclo-nonapeptides low-energy conformation of the parent α-helical i-to-(i+4)
constrained by a smaller bridge containing a total of only lactam-bridged cyclo-nonapeptide 1 (Figure 7) or its iso-
4 methylens will be forced into either a stable non-regular steric 1,2,3-triazolyl-containing cyclo-nonapeptide III (Fig-
structure or fail to form a tight ensemble of closely related ure 8) reveals remarkable structural similarities.[1] Pairwise
conformations (cyclo-nonapeptides I and II, respectively). superposition of the backbone heavy atoms of Xaa13-Ser-
At the same time, cyclo-nonapeptides linked by a larger Ile-Gln-Yaa17 sequences of the low-energy NMR conform-
bridge that contains a total of 7 CH2 groups will entertain ers of cyclo-nonapeptides IV–VI with either the lactam 1
larger molecular flexibility and will form a divergent ensem- or the homologous cyclo-nonapeptide III reveals
ble of non-regular conformations (cyclo-nonapeptides VII RMSDϽ0.8 Å. In particular, the best structural overlap is
and VIII). Evidently, cyclo-nonapeptides IV/VI, which are observed for cyclo-nonapetides V and VI. For these con-
cyclized through bridges containing a total of 5 and 6 CH2 formers the overlapping with the corresponding segments
groups, represent the optimal size to assume regular sec- of lactam 1 (Figure 7, panels b and c) and cyclopeptide III
ondary structures. Similar to cyclo-nonapeptides IV/VI, the (Figure 8, panels b and c) shows RMSDϽ0.1, which is ex-
conformational analysis of the previously reported cyclo- tended to include the methylene chain of the bridging por-
nonapeptide III, containing a total of 5 CH2 groups in the tion. In Figures 7 and 8 the orientation of the side chains
1,2,3-triazolyl containing bridge, revealed formation of a of the residues included in the cyclic portions of cyclo-
well defined canonical structure.[1] The comparison of low- nonapetides IV, V and VI are compared to the correspond-
energy 3D models of cyclo-nonapeptides IV/VI clarify the ing side chains of lactam 1 and the homologous cyclo-nona-
role of the location of the 1,2,3-triazolyl moiety within the peptide III, respectively. We observe good overlap of Ser14,
bridge on the stabilization of the conformational propen- Gln15 and Ile16 side chains located within the cyclic portion
sities of the cyclopeptides. The close proximity of the 4,1- for each pair of conformers analysed. We anticipate that in
disubstituted 1,2,3-triazolyl ring to the backbone in cyclo- addition to the requirements for specific side chains disposi-
nonapeptide IV explains the constraints imposed on the tion different macromolecular targets will differentially ac-
proximal sequence Gly12-Ala(β-4-1,2,3-triazolyl)-Ser14 lead- commodate the various orientations of the 1,2,3-triazolyl
ing to a slight distortion from a canonical conformation. In rings present in the i-to-(i+4) bridge. Taken all together, our
this compound, there is a particular structural effect related study suggest that heterodetic 1,4- or 4,1-disubstituted
to the presence of Ser14 γ-OH. The separation between the 1,2,3-triazolyl-bridged cyclo-nonapeptides obtained via i-
Cα of residue 13 and the 1,2,3-triazolyl ring by only one to-(i+4) side-chain-to-side-chain cyclization can be success-
methylene induces the distortion of α-helical torsion angles fully designed to reproduce and stabilize α-helical confor-
due to a favourable dipolar interaction between Ser14 γ-OH mations. Bridges containing 1,2,3-triazolyl moieties flanked
and triazolyl ring N2. Conversely, in the same cyclo-nona- by stretches of (CH2)(m = 1,2, and 4) and (CH2)(n = 1,2, and 4)
peptide the longer chain of 4 CH2 groups separating the where m+n = 5 or 6 will nicely accommodate α-helical
1,2,3-triazolyl ring from the Cα of residue 17 allows the structures.
Gln16-Nle(ε-1-1,2,3-triazolyl)-Leu18 to assume a much
Moreover, the larger bridges containing a total of 6 CH2
greater structural regularity than that of the preceding se- groups formed by flanking the 1,2,3-triazolyl ring between
quence. Interestingly, the structural distortion observed in chains of 2 and 4 CH2 groups, as in cyclo-nonapeptides V
cyclopetide IV is not evident in the related cyclopeptide III and VI, will accommodate the triazolyl ring without im-
both have a total of 5 CH2 groups in the bridge formed by posing any destabilizing spatial interactions with the back-
combinations of either 1+4 or 4+1 CH2 groups flaking the bone leading to larger propensity for ordered structures.
4,1- and 1,4-(1,2,3-triazolyl) moiety, respectively. In cyclo- These conformationally favoured cyclo-nonapeptides V and
nonapeptide III the contacts between the 1,2,3-triazolyl VI show great conformational similarity with the helical
ring and the backbone in Gln16-Ala(β-4-1,2,3-triazolyl)- model lactam-bridged cyclo-nonapeptide 1 and with its iso-
Leu18 do not interfere with the regularity of the backbone steric heterodetic 1,2,3-triazolyl-containing cyclo-nonapep-
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Helix-Like Secondary Structures
Figure 7. Superposition of low-energy NMR conformers of the heterodetic 1,2,3-triazolyl containing cyclo-nonapeptides IV, V, and VI
(a, b, and c, respectively) with low-energy NMR conformer of isosteric lactam-bridged cyclo-nonapeptide 1. The backbone of cyclo-
nonapeptides 1 is colored in green and those of the clicked cyclo-nonapeptides IV, V and VI are coloured in red, blue and yellow,
respectively. In all cyclo-nonapetides the bridged side chains are colour-coded by atom types (C: green; N: blue; and O: red).
Figure 8. Superposition of low-energy NMR conformers of the heterodetic 1,2,3-triazolyl-containing cyclo-nonapeptides IV, V, and VI
(a, b and c, repectively) with low-energy NMR conformer of the homologous 1,2,3-triazolyl-containing cyclo-nonapeptide III. Side chains
of the macrocycle forming sequence Ser14-Gln-Ile16 are dispayed in stick rendering. Backbone of clicked cyclo-nonapeptides III–VI are
colour-coded in white, red, blue and yellow, respectively. In all clicked cyclo-nonapeptides the bridged side chains are colour-coded by
atom types (C: green and N: blue).
chased from Aldrich. High performance liquid chromatography
(HPLC)-grade acetonitrile (MeCN) was purchased from Carlo–
Erba (Italy).
Synthesis of Nα-Fmoc-Xaa(ω-N3)-OH and Nα-Fmoc-Yaa(ω-yl)-OH:
The detailed syntheses of these alkynyl- and azido-containing
building blocks are described elsewhere.[16]
Solid Phase Synthesis of Ac[Xaa13(ω-N3),Yaa17(ω-yl)]hPTHrP(11–
19)NH2 (IЈ, VЈ and VIIЈ) and Ac[Yaa13(ω-yl),Xaa17(ω-
N3)]hPTHrP(11–19)NH2 (IIЈ,IVЈ,VIЈ and VIIIЈ)
tide III. Importantly, our studies suggest that the i-to-(i+4)
side-chain-to-side-chain 1,4- and 4,1-disubstituted 1,2,3-tri-
azolyl-bridged cyclo-nonapeptide, in addition to success-
fully reproducing the α-helical structure, can be fine tuned
to support a wide range of distorted and non-canonical
structures. Future studies conducted in our laboratories will
provide additional insight on the relationship between con-
formation and the separation between residues participat-
ing in the side-chain-to-side-chain cyclization, size of the
connecting bridge, position of the 1,2,3-triazolyl ring in the Synthesis of Rink amide resin-bound Ac[Lys11(Nε-Boc),Xaa13(ω-
N3),Ser14(tBu),Gln16(Trt),Yaa17(ω-yl),Arg19(NG-Pbf)]hPTHrP(11–
bridge and last but not least, the orientation of the triazolyl
ring in the bridge.
19) and Ac[Lys11(Nε-Boc),Yaa13(ω-yl),Ser14(tBu),Gln16(Trt),Xaa17-
(ω-N3),Arg19(NG-Pbf)]hPTHrP (11–19).
Peptides IЈ–VIIIЈ were synthesized on a manual batch synthesizer
(PLS 4ϫ4, Advanced ChemTech) using a Teflon reactor (10 mL),
Experimental Section
applying the Fmoc/tBu solid phase peptide synthesis (SPPS) pro-
cedure. The Nα-Fmoc-Arg(NG-pbf)-Rink amide resin was swelled
Materials: 9-Fluorenylmethyloxycarbonyl (Fmoc)-protected -
with DMF (1 mL/100 mg of resin) for 20 min before use.
amino acids and Rink amide resin (0.63 mmolg–1) were obtained
from Novabiochem AG (Laufelfingen, Switzerland). 2-(1H-Benzo- Stepwise peptide assembly was performed by repeating for each
triazol-1-yl)-1,1,3,3-tetramethyluronium
tetrafluoroborate
added amino acid the following deprotection-coupling cycle:
1) Swelling: DMF (1 mL/100 mg of resin) for 5 min; 2) Fmoc-de-
protection: resin is washed twice with 20% piperidine in DMF
(1 mL/100 mg of resin, one wash for 5 min followed by another
wash for 20 min); 3) Resin washings: DMF (3ϫ5 min); 4) Cou-
pling: scale employing TBTU/HOBt/NMM (2.5:2.5:3.5 equiv.) as
(TBTU), N-Hydroxybenzotriazole (HOBt) were purchased from
Iris-Biotech. Peptide-synthesis grade N,N-dimethylformamide
(DMF) was purchased from Scharlau (Barcelona, Spain). Tri-
fluoroacetic acid (TFA), dichloromethane (DCM), piperidine, ace-
tic anhydride (Ac2O), and N-methylmorpholine (NMM) were pur-
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A. M. D’Ursi et al.
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the coupling system and 2.5 equiv. of the Fmoc protected amino
acids, except for Xaa(ω-N3) and Yaa(ω-yl) (1.5 equiv.), in DMF
(1 mL/100 mg of resin) for 40 min. Each coupling was monitored
by Kaiser test[32] and was negative, therefore recouplings were not
NMR Spectrometry: Samples were prepared by dissolving
about 1.2 mg of the heterodetic 1,2,3-triazolyl-containing cyclo-
nonapeptides in 0.5 mL of aqueous phosphate buffer (pH 6.6,
100 m). To prepare samples for measurements in water/HFA, the
needed; 5) Resin washings: DMF (3ϫ5 min) and DCM samples in aqueous phosphate buffer were lyophilized and dis-
(1ϫ5 min).
solved in a mixture of water/HFA (0.5 mL, 1:1, v/v). NMR spectra
were recorded on a Bruker DRX-600 spectrometer. One-dimen-
sional (1D) NMR spectra were recorded in the Fourier mode with
quadrature detection. The water signal was suppressed by a low-
power selective irradiation in the homogated mode. Double Quan-
tum- Filtered Correlation Spectroscopy (DQF-COSY), Total
Correlated Spectroscopy (TOCSY) and Nuclear Overhauser Effect
Spectroscopy (NOESY)[19–21] experiments were run in the phase-
sensitive mode using quadrature detection in ω1 by time-pro-
portional phase incrementation of the initial pulse.[33] Data block
sizes comprised of 2048 addresses in t2 and 512 equidistant t1 val-
ues. Before Fourier transformation, the time domain data matrices
were multiplied by shifted sin2 functions in both dimensions. A
mixing time of 70 ms was used for the TOCSY experiments.
NOESY experiments were run at 300 K with mixing times in the
range of 100–250 ms. The qualitative and quantitative analyses of
DQF-COSY, TOCSY and NOESY spectra were obtained using the
SPARKY[22] interactive program package. Complete proton reso-
nance assignments were achieved with the Wüthrich procedure.[23]
Amino Terminal Acetylation. General Procedure: The free N-ter-
minal α-amino of the resin-bound peptides was acetylated by two
consecutive steps. A 30 min exposure to Ac2O/NMM in DCM
(20 equiv. 1.6 mL of DCM) was followed by a additional 1.5 h
treatment with a fresh acetylation mixture. The reaction was moni-
tored by Kaiser test.[32]
Deprotection, Cleavage and Purification of Free Peptide. General
Procedure: Peptides cleavage from the resin and simultaneous de-
protection of the amino acid side chains were carried out with a
mixture of TFA/anisole/1,2-ethanedithiol/phenol/H2O (94:1:1:1:1
v/v/v/v/v, 1 mL/100 mg of resin-bound peptide). The cleavage was
carried out for 3 h with vigorously shaking at room temperature.
Resins were filtered and washed with TFA. The filtrate was concen-
trated under N2 stream, addition of cold diethyl ether resulted in
a precipitate that was separated by centrifugation, dissolved in H2O
and lyophilized on an Edwards apparatus, model Modulyo.
Peptides were purified by semipreparative RP-HPLC on a Waters
600 equipped with Jupiter C18 (10 µm, 250ϫ10 mm), at a flow
rate of 4 mL/min employing a linear gradient 10% to 60% of B in
A in 20 min were the solvent system used was A = 0.1% TFA in
H2O and B = 0.1% TFA in CH3CN. Fractions were analyzed by
ACQUITY UPLC (Waters Corporation, Milford, Massachusets)
coupled to a single quadrupole ESCI-MS (Micromass ZQ) using
an ACQUITY BEH C18 column (1.7 µm, 2.1ϫ50 mm) at 30 °C,
with a flow rate of 0.45 mL/min employing a linear gradient of
10% to 60% of B in A in 3 min (A and B are the same as above).
NMR Structure Calculation: Sequential and medium range NOE-
derived distances were used to generate 3D models of the hetero-
detic 1,2,3-triazolyl-containing cyclo-nonapeptides. These struc-
tures were subjected to simulated annealing procedure using the
DYANA software package[34] and energy minimized with the SAN-
DER module of the AMBER 5 program[35] using for first 1000
steps the steepest descent method and for the following 4000 steps
the conjugate gradient method. A non-bonded cut-off of 12 Å and
a distance-dependent dielectric term (e = 4·r) were used. The mini-
mization protocol included three steps in which NOE-derived dis-
tances were used as constraints with a force constant, of 1000, 100
and 10 kcal/molÅ, respectively. The final pdb files were analyzed
and validated using PROMOTIF software.[25]
Solution phase CuI-catalyzed intramolecular azide–alkyne 1,3-di-
polar Huisgen cycloaddition: Synthesis of Ac[Xaa13(1),Yaa17(2)]-
hPTHrP(11–19)NH2[{&1(CH2)(n = 2 and 3)-1,4-(1,2,3)triazolyl-
(CH2)(n = 1 and 4)&2}] (I, V and VII) and Ac[Yaa13(1),Xaa17(2)]-
hPTHrP(11–19)NH2[{&1(CH2)(n = 1 and 4)-4,1-(1,2,3)triazolyl-
(CH2)(n = 2–4)&2}] (II, IV, VI and VIII).
Molecular Dynamics: Molecular dynamics runs were performed
using the SANDER module of the AMBER 5 software at a con-
stant temperature of 300 K, using a non-bonded cut-off of 12 Å
and a distance-dependent dielectric term (e = 4·r). NMR mean
structures of the heterodetic 1,2,3-triazoyl-containing cyclo-non-
apeptides were chosen as starting structures. The molecular dynam-
ics simulations were performed with a rather drastic limitation of
allowed movements for backbone atoms whereas side-chain atoms
were allowed to move according to a small value of force constant
restraints. A force constant of 1000 kcal/molÅ was applied on the
NOE derived distance restraints of the backbone atoms, whereas a
force constant of 10 kcal/molÅ was used to constrain side-chain
atoms. Heating the system for 1 ps and 10 ps of initialization time
was followed by 500 ps simulation with 1 fs time steps. Structures
were saved every 5 ps. The average AMBER energy was 1180 kcal/
mol for the 1,2,3-triazolyl-containing cyclo-nonapeptides. The all
atoms root mean square deviation (RMSD) from the start of the
trajectory was 1.25 Å for the 1,2,3-triazolyl-containing cyclo-
nonapeptides. The final structures were analyzed using the Insight
98.0 program (Molecular Simulations, San Diego, CA).
Cyclization of Ac[Xaa13(ω-N3),Yaa17(ω-yl)]hPTHrP(11–19)NH2
(IЈ, VЈ and VIIЈ) and Ac[Yaa13(ω-yl),Xaa17(ω-N3)]hPTHrP(11–19)-
NH2 (IIЈ,IVЈ,VIЈ and VIIIЈ).
To a solution of the linear peptide (3.1 µmol) in H2O/tBuOH
(4 mL, 2:1 v/v) were added CuSO4·5H2O (43.4 µmol) and ascorbic
acid (40.3 µmol). The mixture was stirred overnight at room tem-
perature, concentrated and lyophilized. The crude heterodetic cy-
clo-nonapeptides were purified (Ն 97% purity) by RP-18 column
LiChroprep employing the same solvent system as mentioned
above.
Circular Dichroism Spectroscopy: All CD spectra were recorded on
a Jasco J-810 spectropolarimeter using cells of 1 mm path length.
The pH of the samples was adjusted to 6.6 with aqueous phosphate
buffer (100 m, pH 7.2). After pH adjustment, samples were lyo-
philized and dissolved in water, or in water containing 50%
(v/v) hexafluoroacetone (HFA) to obtain a peptide concentration
of 0.02 m. Spectra were the average of two scans from 190 to
260 nm, recorded with a band width of 0.5 nm at scan rate of 5 nm/
min.
Supporting Information (see also the footnote on the first page of
this article): HPLC tracing and MS of the linear precursors and
cyclo-nonapeptides I–II and IV–VIII; Proton Chemical Shifts of
clicked cyclo-nonapeptides I–II and IV–VIII in water and water/
HFA. Amide and fingerprint regions of NOESY spectra for clicked
For estimation of secondary structure content, CD spectra were
analyzed using CONTINN algorithm of DICHROWEB on-line
server.[18]
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Received: October 14, 2009
Published Online: December 16, 2009
Eur. J. Org. Chem. 2010, 446–457
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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