Replacement of Ala by Aib improves structuration and biological stability in thymine-based α-nucleopeptides

Article abstract of DOI:10.1039/b920211k

Three thymine-based nucleo-heptapeptides, each containing two nucleo-amino acids and zero, one or four Aib residues, respectively, have been synthesized. A single Aib residue is enough to promote the adoption of a helical structure in our nucleopeptides a

Full text of DOI:10.1039/b920211k

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Replacement of Ala by Aib improves structuration and biological stability in
thymine-based a-nucleopeptides
Piero Geotti-Bianchini,a,b Alessandro Moretto,a Cristina Peggion,a Julien Beyrath,b Alberto Bianco*b and
Fernando Formaggio*a
Received 28th September 2009, Accepted 24th November 2009
First published as an Advance Article on the web 21st January 2010
DOI: 10.1039/b920211k
Three thymine-based nucleo-heptapeptides, each containing two nucleo-amino acids and zero, one or
four Aib residues, respectively, have been synthesized. A single Aib residue is enough to promote the
adoption of a helical structure in our nucleopeptides and to increase significantly their resistance
towards enzymatic degradation. The insertion of four Aib residues, out of seven residues in the
sequence, affords a rigid, 310-helical nucleopeptide that is substantially unaffected by serum enzymes
and is not cytotoxic.
of these conformationally controlled nucleopeptides11 had to be
Introduction
performed by time-consuming solution methodologies, since the
Naturally occurring amino acids carrying nucleobases at their side
faster solid-phase synthesis approach gives unsatisfactory results
chains were discovered several decades ago,1 giving rise to the
when a large number of sterically hindered Aib residues is present.
first interest in the biological properties of peptides containing
Therefore, in the present work we combine the faster solid-phase
such amino acids, which were named nucleopeptides.2 However,
only in the last two decades the research on nucleopeptides has
synthesis technique with the advantages offered by the insertion
of Aib residues into our (i, i+3)-functionalized nucleopeptides. We
significantly developed, mainly in view of the possibility to specifi-
aim at verifying if as few as one Aib residue is sufficient to impart
cally design synthetic peptide/nucleotide hybrids for gene therapy
applications.3–7 The most known and investigated oligonucleotide
conformational and biological stabilities to a nucleo-heptapeptide
containing two b-thyminyl-alanine (AlaT, Fig. 1) residues. In
particular, we have synthesized and studied H-(Ala-AlaT-Ala)2-
Lys-NH2 (T2), H-Ala-AlaT-Ala-Aib-AlaT-Ala-Lys-NH2 (T2U),
with one Aib in the middle of the sequence, and H-(Aib-AlaT-
Aib)2-Lys-NH2 (T2U4), with all Ala replaced by Aib (Fig. 1).
analogues with a polyamide backbone are the peptide nucleic acids
(PNA),8 in which the nucleobases are anchored to the a-nitrogen
through a linker. Nucleopeptides consisting of a-amino acids are
often called aPNA, as they share with the PNA the polyamide
backbone.4d,7
Peptide-based constructs have remarkable advantages with
respect to the phosphate–sugar backbone of the natural polynu-
cleotides in view of in vivo applications. Indeed, peptide backbones
are not degraded by nucleases (active on phosphodiester bonds
and ubiquitous). In addition, they can often more easily cross
the cell membranes, as they lack the large negative charge of
polynucleotides. Finally, hydrophobicity, chemical stability, 3D-
structure and additional features can be tailored to specific needs
by an appropriate choice of the amino acid building blocks.
We have recently reported that sequential nucleopeptides,
characterized by the insertion of a-alanyl nucleoamino acids
at (i, i+3) positions, are able to penetrate the cells and reach
the nucleus without cytotoxic effects.9 As conformation plays a
major role in determining the functionality of oligonucleotides10
and peptide/nucleobase hybrids,4d we have also explored11 the
possibility to build nucleopeptides with rigid and predictable
3D-structures by exploiting the helix induction properties of a-
aminoisobutyric acid (Aib, Fig. 1).12 This latter achievement is
quite remarkable as it offers a tool to spatially arrange the
nucleobases at well defined positions. However, the syntheses
aDepartment of Chemistry, University of Padova, 35131 Padova, Italy.
E-mail: fernando.formaggio@unipd.it
bCNRS, Institut de Biologique Mole´culaire et Cellulaire, Laboratoire
d’Immunologie et Chimie The´rapeutiques, 67000 Strasbourg, France.
E-mail: A.Bianco@imbc-cnrs.unistra.fr
Fig. 1 Chemical structures of Aib, AlaT and the three nucleo-heptapep-
tides synthesized.
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Results and discussion
Design
According to the encouraging results of our previous work on
nucleopeptides,9,11 we decided to maintain the two AlaT residues
at the (i,i+3) relative positions. This arrangement would allow for
the alignment of the two nucleobases on the same side of the helix,
provided the peptide adopts a 310-helical conformation. We added
one Lys residue to impart water solubility, as it has been shown
to be sufficient for peptides of this length.4d Finally, we chose to
have a primary amide at the C-terminus since the negative charge
of the carboxylate could hamper the membrane penetration and
decrease the affinity towards the polyanionic nucleotides.
We then decided to insert one Aib in the middle of the sequence
of the reference nucleopeptide T2, in the hope that this residue
could exert its conformational bias on the entire nucleopeptide.
Given the lower reactivity of Aib at its amino function, as
compared to its carboxylic moiety, the Ala3–Aib4 coupling was
expected to be easier than the AlaT2–Aib3, so that position 4 was
chosen for the insertion.
Scheme 1 Synthetic strategy adopted for the solution synthesis of T2U4.
(i) EDC/HOBt, NH3(g) in DCM; (ii) H2, Pd/C, DCM; (iii) EDC/HOBt,
NMM, pH 8, DCM; (iv) EDC/HOAt, NMM, pH 8, DMF; (v) H2, Pd/C,
MeOH; (vi) EDC, DCM; (vii) NMM, pH 8, MeCN; (viii) 50% TFA in
DCM; (ix) EDC in DMF; (x) NMM, pH 8 in MeCN, reflux; (xi) 20% TFA
in DCM.
Synthesis
Z- and Boc-L-AlaT-OH were prepared according to literature
procedures,11,13 starting from the appropriate Na-protected L-Ser
b-lactone.
Conformationally informative data were obtained from the
ROESY spectra. As summarized in Table 1, for all nucleo-
peptides we observed most of the possible sequential cross-
peaks of the types NH(i)→NH(i+1) and CaH(i)→NH(i+1)
[CbH(i)→NH(i+1) in the case of the Aib residues], indicative
of folded conformations.14 In particular, none of such cross-
peaks is missing in the ROESY spectrum of T2U4. Moreover,
in this latter case two medium range, CbH(i)→NH(i+2) spatial
connectivities were observed (Fig. 2), which strongly support14
the conclusion that T2U4 adopts a 310-helical15 conformation in
solution. Medium range connectivities are not observed for T2
and only two quite weak (and between nuclei three residues apart)
are found for T2U. Overall, our NMR data indicate that T2U4
The synthesis of T2 and T2U was accomplished on solid phase
by applying the Boc/Bzl strategy on a MBHA resin. Yields in
AlaT introduction were much lower as compared to an Ala residue,
probably because of the relatively large side-chain and to a possible
H-bonding interference by the thymine moiety. No significant
improvement was observed when the BOP/HOBt activation
protocol was replaced by the often more efficient HATU/HOAt
mixture. As Aib reacts sluggishly in amide bond formation, triple
coupling, longer reaction times and the HATU/HOAt activation
were used. Longer reaction times were applied also for the
introduction of Ala3. These procedures allowed us to obtain T2
and T2U in comparable yields (29% T2, 26% T2U).
Because of the four Aib residues, two of them consecutive,
T2U4 was prepared in solution by a segment condensation
approach (Scheme 1), previously successfully applied to a sim-
ilar nucleopeptide.11 Firstly, the C-terminal tetrapeptide amide
Z-Aib-AlaT-Aib-Lys(Boc)-NH2 and the N-terminal tripeptide
Z-Aib-AlaT-Aib-OtBu were synthesized step-by-step. Then the
N-terminal segment was C-deprotected by acidolysis, transformed
into its reactive 4H-oxazolidin-5-one by treatment with a carbo-
diimide, and coupled with the N-deprotected tetrapeptide amide
to yield the protected T2U4. The pure final nucleopeptide was
obtained after removal of the Z and Boc protecting groups
(by catalytic hydrogenation and TFA acidolysis, respectively),
followed by a HPLC purification step.
Table 1 Sequential cross-peaks detected in the 2D-NMR ROESY spectra
of T2, T2U and T2U4
Cross-peak
i
T2
T2U
T2U4
b
b
b
NH(i)-NH(i+1)
1
2
3
4
5
6
7
+
+
+
+
+
+
+
+
+
+
+
+
+
+
n. d.c
+
+
+
CaH(i)-NH(i+1)a
1
2
3
4
5
6
7
n. d.
+
+
n. d.
+
+
+
n. d.
+
+
+
+
+
+
+
+
+
+
Conformational analysis
++d
++d
++d
The three nucleo-heptapeptides were studied by 2D-NMR tech-
niques in d6-DMSO solution, at 5 mM concentration. All proton
resonances could be assigned with the only exception of the
heterocyclic N(3)H proton of every couple of thymines, due to
a In the case of Aib, lacking the Ca proton, the CbH(i)→NH(i+1) cross
peaks are considered. b The rapid exchange of the ammonium protons with
the solvent moisture suppresses most of their cross-peaks. c n. d.: cross-peak
not detected. d Cross-peaks with both non-equivalent CONH2 protons are
observed
+
overlapping, and of the two NH3 in T2U4.
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Fig. 2 Section of the ROESY spectrum of T2U4 (5 mM in d6-DMSO). The two medium range CbH(i)→NH(i+2) connectivities, diagnostic of a 310-helix,
are highlighted.
is folded into a helical conformation, T2U folds to a significant
extent, whereas T2 has the lowest degree of helical content.
A 310-helical disposition would locate the two thymine moieties
on the same side of the helix, after one complete helix turn.
This 3D arrangement is indeed confirmed for T2U4, in the
ROESY spectrum of which 4 spatial connectivities between the
two thymines are observed (Fig. 3). Only one of these is seen in
the spectrum of T2U and none in that of T2.
Fig. 4 CD spectra of T2, T2U and T2U4 in phosphate buffer, pH 7.4
(room temperature, 40 mM nucleopeptide concentration).
unaffected by the temperature variation, whereas the spectra of
T2 and T2U undergo a significant change, particularly in the low-
wavelength spectral region (l < 230 nm). Fig. 5 illustrates the effect
of the temperature on the total molar ellipticity value at 208 nm,
the typical absorption wavelength for the amide p→p* transition
(parallel component) of a helical peptide.16,17 It is clear that T2U4
is endowed with a superior structural stability as compared to T2
and T2U.
Concerning the nature of the secondary structures adopted
by the three nucleopeptides, we must rely mainly on the NMR
data. Indeed, it is difficult to estimate the contribution of the
nucleobase chromophore to the amide absorption region (190–
250 nm), the most conformationally informative for a peptide.
However, keeping this advice in mind we can tentatively draw
some conclusions, as previously done by other authors on different
nucleopeptides.5c,6 We observe (Fig. 4) a negative maximum for
T2U4, at about 215 nm, and for T2U, at about 203 nm, but
not for T2 in the same spectral region. The presence of such a
Fig. 3 The four connectivities (indicated by the arrows) observed between
the two thymines in the ROESY spectrum of T2U4 (5 mM in d6-DMSO).
Circular dichroism spectra of the three nucleo-heptapeptides
were recorded in phosphate buffer (10 mM NaH2PO4/Na2HPO4,
pH 7.4), at 40 mM peptide concentration (Fig. 4). All spectra dis-
play a negative band, mostly due to the nucleobase chromophore,
in the range 250–300 nm, and a more intense negative band in
the low-wavelength region (l < 230 nm). The nucleobase band
is slightly more intense for T2U than for T2, but definitely much
larger and 10 nm red-shifted in the case of T2U4. The increased
intensity of the dichroic signal is likely due to a lower degree of
conformational freedom experienced by the two nucleobases in the
side chain of T2U4,5c as a consequence of the rigidified backbone
structure.
Informative hints come also from the CD spectra recorded in
the same environment, but at different temperatures (0–60 ◦C
range). Shape and intensity of the spectrum of T2U4 are almost
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Human lymphoid tumor Raji cells were incubated for 24 h with
different concentrations of T2U4 and their viability was evaluated
by the spectrophotometric MTS assay.20 As shown in Fig. 7, T2U4
has no cytotoxic effects up to 100 mM concentration.
Fig. 5 [q]T values at 208 nm for T2, T2U and T2U4 at different
temperatures.
Fig. 7 Raji cells viability, evaluated by the MTS assay, after 24 h
incubation with different amounts of T2U4 or doxorubicin (positive
control). The average of three experiments and their standard deviation
are plotted.
negative maximum is often indicative of a helical (a or 310) peptide
structure, whereas extended (“unordered”, or poly-Pro-II type)
conformations are characterized by a negative maximum below
200 nm (usually around 195 nm).17 Therefore, T2U and T2U4 are
likewise folded into a helical structure, as indicated also by our
NMR investigation. Altogether, our CD analysis suggests that
nucleobase rigidity and helical content markedly increase when
Ala residues are replaced by Aib.
Conclusions
Three thymine-based nucleo-heptapeptides (T2, T2U, T2U4)
containing two AlaT residues at (i,i+3) relative positions and
zero, one or four Aib residues, respectively, have been synthesized
by solid-phase (T2, T2U) or solution (T2U4) procedures. The
conformational analysis indicates that T2U4 adopts a very stable
helical structure, most likely of the 310 type. Also T2U, containing
only one Aib residue, is folded to a significant extent, thus
confirming the known ability of Aib to promote helical structures.
The nucleopeptide T2 appears to be the less folded among the
three. Tests in mouse serum have shown that a single Aib residue is
sufficient to impart a tenfold resistance increase towards enzymatic
degradation. The insertion of four Aib, out of seven residues,
affords a nucleopeptide that is stable even after 48 h incubation
without detected cytotoxicity.
At this stage of our research it would be premature to conclude
that these biological results are directly due to the presence of
Aib residues. Indeed, the observed behavior could arise from the
(helical) secondary structure formed as a consequence of the Aib
introduction. In any case, the encouraging results of this work
stimulate us to develop longer Aib-containing nucleopeptides,
amenable to be synthesized by means of the faster solid-phase
technique. We believe that a small percentage of Aib residues could
be sufficient to impart resistance to enzyme degradation and a well
defined 3D-structure. This latter feature, in turn, might allow us
to design nucleopeptides with a nucleobase spatial arrangement
specifically tailored for interactions with a given target (e.g., a
polynucleotide sequence).
Biological tests
The stability of the three nucleopeptides towards enzymatic hy-
drolysis was tested in murine serum. HPLC analysis at increasing
incubation times showed that T2 is rapidly degraded, while T2U
is significantly more resistant and T2U4 is completely stable even
after 48 h incubation (Fig. 6). In particular, after 1 h incubation
only 5% of the initial amount of T2 is left, whereas the quantity of
non-degraded T2U is 10 times higher and T2U4 is still intact.
The observed behavior is probably due to the higher efficacy
of hydrolytic enzymes in cleaving amide bonds among coded
residues, rather than peptide bonds involving non-coded amino
acids.18 Indeed, similar results have been recently reported for Aib-
containing peptides.19
Experimental
Fig. 6 Fraction of intact nucleopeptide after different incubation times
in 50% murine serum, evaluated by analytical HPLC.
Materials and methods
Solvents. Reagent grade N,N-dimethylformamide (DMF)
˚
We have previously shown that Ala-rich nucleopeptides bearing
AlaT residues at (i,i+3) positions are not cytotoxic.9 Therefore, a
cytotoxicity test was performed only on T2U4, the nucleopeptide
with the highest content of Aib residues.
from Baker (Phillipsburg, USA) was stored over 3 A molecular
sieves under N2. Reagent grade dichloromethane (DCM) from
Carlo Erba (Milan, Italy), freshly distilled over phosphoric
anhydride, was used for the couplings.
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Acetic acid (AcOH), 1-butanol, chloroform, dichloromethane
(DCM), diethyl ether (Et2O), ethanol, ethyl acetate (AcOEt),
methanol (MeOH), petroleum ether, boiling range 40–60 ◦C (PE)
and toluene were at least reagent grade and used without further
purification.
instrument, provided with a SDS C18 Macherey-Nagel column
-1
˚
(10 ¥ 250 mm, 100 A, flow 6.0 mL min ) and a Beckmann System
Gold UV detector operating at 230 nm, or on a Shimadzu SCL-
6B instrument, provided with Shimadzu LC-8A pumps, a Delta
-1
˚
Pack C18 column (19 ¥ 300 mm, 100 A, flow 17 mL min ) and a
Acetonitrile (MeCN) was HPLC grade and used without further
purification.
Shimadzu SPD-64 detector operating at 265 nm. As eluents, the
following solvent mixtures were used: (1) A: H2O + 0.1% TFA,
B: MeCN + 0.08% TFA (HPLC Varian and Beckmann); (2) A¢:
H2O–MeCN 9 : 1 + 0.05% TFA; B¢: MeCN–H2O 9 : 1 + 0.05%
TFA (HPLC Agilent and Shimadzu). MilliQ H2O was used for
eluent preparation.
Reagents. Para-cresol and trifluoroacetic acid (TFA) were
purchased from Acros-Janssen (Geel, Belgium). Potassium hy-
drogensulfate, sodium hydrogencarbonate, sodium hypochlorite
and sodium sulfate anhydrous were purchased from Carlo
Erba (Milan, Italy). N,N-diisopropylethylamine (DIEA), N-
methylmorpholine (NMM), ninhydrin and palladium catalyst
(10% on activated charcoal) were purchased from Fluka (Buchs,
Switzerland). 7-Aza-1-hydroxy-1H-benzotriazole (HOAt) and O-
(7-aza-benzotriazol-1-yl)-N,N,N¢,N¢-tetramethyluronium hexa-
fluorophosphate (HATU) were purchased from GL Biochem
Lt (Shangai, China). N-Ethyl-N¢-(3-dimethylamino)propyl-
carbodiimide chlorohydrate (EDC·HCl), O-(benzotriazol-1-
yl)- N,N,N¢,N¢-tetramethyluronium hexafluorophosphate (BOP)
were purchased from Iris Biotech (Marktredwitz, Germany).
Disodium hydrogenphosphate dihydrate and sodium dihydrogen-
phosphate monohydrate were purchased from Merck (Darm-
stadt, Germany). Formic acid, iodine, N,N,N¢,N¢-tetramethyl-
4,4¢-diamino-diphenylmethane (TDM) and trimethylsilyl triflu-
oromethansulfonate (TMSOTf) were purchased from Sigma-
Aldrich (Milwaukee, USA).
1D 1H-NMR spectra were recorded on Bruker AC 200, Bruker
AC 250 or DRX 400 spectrometers. 2D NMR spectra were
recorded on AMX 500 Bruker or DMX 600 spectrometers.
Chemical shifts (d) are expressed in parts per million (ppm)
with regard to the tetramethylsilane signal. Solvent residual
peaks (CHCl3, d 7.26 ppm, or d5-DMSO, d 2.50 ppm) were
used for calibration. Peak multiplicity is described as follows: s
(singlet), br s (broad singlet), d (doublet), dd (doublet of doublets),
m (multiplet), coupling constant (J) values are given in Hz.
Deuterated solvents (CDCl3, d6-DMSO) were purchased from
Cambridge Isotope Laboratories (Cambridge, England).
ESI-TOF mass spectra were collected on a Mariner ESI-TOF
mass spectrometer (Perseptive Biosystems). Prior to injection,
samples were dissolved in a 1 : 1 mixture of H2O–MeOH con-
taining 0.1% formic acid. The positive ions were accelerated at 10,
15, 20 or 30 keV.
Circular dichroic spectra were recorded on a Jasco (Tokyo,
Japan) model J-715 dichrograph provided with a Haake model
F3 thermostat. Fused quartz cells (Hellma, Mu¨llheim, Germany)
of 0.5 cm path length were employed. Data are expressed in terms
of total molar ellipticity [qT] (deg cm2 dmol-1).
Silica gel 60 (40-63 mm diameter, mesh 230-400) from Merck
was used for flash-chromatographic purifications.
Characterization. Melting points were measured on a Leitz
apparatus model Laborlux 12 and are uncorrected.
Silica gel 60 F254 (Merck) on glass was used for TLC character-
ization. Retention factors (Rf) were measured using the following
solvent systems as eluents: CHCl3–ethanol 9 : 1 (Rf1), 1-butanol–
AcOH–H2O 3 : 1 : 1 (Rf2), toluene–ethanol 7 : 1 (Rf3). Products
were detected either by UV lamp irradiation or by exposition
to I2 vapors or by warming with a heat gun and spraying
firstly with a 1.5% NaClO solution, then with a ninhydrin–
TDM solution.21 All compounds were chromatographically
pure.
Optical rotations were measured on a Perkin-Elmer model 241
polarimeter at the sodium D line wavelength, using a cell with
an optical pathlength of 10 cm. Concentrations are expressed in
g/100 mL. [a]2D5 are calculated using the formula [a] = a/(c·l),
where c is the concentration (in g mL-1) and l is the optical path
(in dm). Spectrophotometric grade MeOH (Fluka) was used as a
solvent.
Solid-phase peptide synthesis
Solid-phase synthesis of the nucleopeptide sequences was ac-
complished using a PSP 4000 semiautomatic peptide synthesizer
for Boc/Bzl strategy.22 4-Methylbenzhydrylamine hydrochloride
salt resin (loading 0.62 mmol g-1), purchased from Applied
Biosystems (Carlsbad, USA) was used as solid support. Boc-
Ala-OH, Boc-Lys(2Cl-Z)-OH and Boc-Aib-OH were purchased
from NeoMPS (Strasbourg, France). Boc-AlaT-OH was prepared
according to literature procedures.13 Couplings were allowed
to proceed for 20 min for activated protein amino acids on
N-terminal protein amino acids, 40 min for activated protein
amino acids on N-terminal AlaT, 60 min for activated protein
amino acids on N-terminal Aib, for activated Boc-AlaT-OH and
for activated Boc-Aib-OH. Each coupling was repeated twice
(three times for activated Boc-Aib-OH) and subsequently the
qualitative Kaiser test23 was used to verify the completion of
the reaction. Boc deprotection was accomplished by treating
with neat TFA twice (firstly for 1 min then for 3 min). After
completing the peptide sequences, the resin beads were transferred
to syringes provided with sintered glass filters, a TFA–TMSOTf–
para-cresol (80 : 25 : 10, v/v/w) cleavage mixture24 was added and
cleavage was allowed to proceed overnight at room temperature.
Crude nucleopeptides were recovered by precipitation with cold
Et2O, dissolved in milliQ H2O and lyophilized prior to HPLC
purification (Beckmann HPLC, 1-21% B in 30 min).
IR absorption measurements on KBr pellets were performed on
a Perkin-Elmer model 580 B spectrophotometer, equipped with a
Perkin-Elmer 3600 IR data station.
Analytical chromatograms were recorded either on an Agilent
Technologies 1200 Series instrument, provided with a Kromasil
-1
˚
Phenomenex C18 column (4.6 ¥ 250 mm, 100 A, flow 1.0 mL min )
or on a Varian ProStar 410 instrument, provided with a Nucleosil
-1
˚
100-5 C18 column (4.6 ¥ 150 mm, 100 A, flow 1.2 mL min ) and
a Varian ProStar 330.71 PDA detector.
Preparative scale chromatographic purifications were per-
formed either on a on a Beckmann System Gold HPLC 166
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2*TFA·H-(Ala-AlaT-Ala)2-Lys(H)-NH2 [T2]. Synthesis scale:
25 mmol. Activation protocol: 5 eq each of Boc-protected protein
amino acid, BOP, HOBt and 15 eq DIEA or 3 eq each of Boc-AlaT-
OH, HATU, HOAt, 10 eq DIEA. Yield: 5.9 mg (29%). HPLC
(Varian, 0–50% B in 20 min): tr 8.41 min. NMR dH (500 MHz;
d6-DMSO) 11.22-11.20 (2H, 2 br s, N(3)H thymine AlaT2 and
AlaT5), 8.80-8.79 (1H, d, J 7.0, aNH AlaT2), 8.30-8.28 (1H, d, J
6.5, NH Ala3), 8.25-8.24 (1H, d, J 6.5, NH Ala4), 8.19 (3H, br s,
derivative) was added and the pH was adjusted to 8 with NMM.
The mixture was stirred at room temperature for 6 days. The
solvent was removed at reduced pressure, the resulting oil was
taken up with AcOEt, washed with KHSO4 5%, H2O, NaHCO3
2% and H2O, then desiccated on Na2SO4 and evaporated to an oil.
The crude product was purified by flash-chromatography (eluent:
DCM–MeOH, gradient from 18 : 1 to 9 : 1) to yield a colorless
solid (0.46 g, 58%). Mp: 142-145 ◦C (from PE). [a]D25 = -35.9 (c =
0.34, MeOH). TLC: Rf1 0.30, Rf2 0.85, Rf3 0.20. IR (cm-1): 3410,
1675, 1521. NMR dH (200 MHz, d6-DMSO) 8.32 (1H, br s, N(3)H
thymine), 7.64-7.60, 7.47-7.43 (2H, 2 d, J 7.6 and 7.8, aNH AlaT
and aNH Lys), 7.35-7.30 (7H, m, C6H5-Z, C(6)H thymine and NH
Aib), 7.11 (1H, br s, 1 CONH), 7.00 (1H, br s, 1 CONH), 6.76-6.72
(1H, t, J 4.2, eNH Lys), 5.06-4.89 (2H, m, CH2-Z), 4.41-4.33 (1H,
m, 1 aCH), 4.23-4.19 (2H, m, 1 aCH and 1 bCH AlaT), 3.64-3.55
(1H, m, 1 bCH AlaT), 2.88-2.80 (2H, m, eCH2 Lys), 1.69-1.65
(4H, m, 4H, C5H3 thymine and 1 bCH Lys), 1.59-1.49 (1H, m, 1
bCH Lys), 1.38-1.33 (16H, m, 1 CH3 Aib, 3 CH3 Boc, gCH2 and
dCH2 Lys), 1.30 (3H, s, 1 CH3 Aib).
NH3 Ala1), 8.14-8.13 (1H, d, J 5.5, NH Ala6), 8.06-8.04 (1H,
+
d, J 8.0, aNH AlaT5), 7.95-7.93 (1H, d, J 4.5, aNH Lys7), 7.87
(3H, br s, NH3 Lys7), 7.44 (1H, s, C(6)H thymine AlaT2), 7.40
+
(1H, s, C(6)H thymine AlaT5), 7.31 (1H, br s, 1 CONH), 7.01 (1H,
br s, 1 CONH), 4.66-4.63 (1H, m, aCH AlaT2), 4.61-4.57 (1H,
m, aCH AlaT5), 4.27-4.24 (1H, m, aCH Ala3), 4.24-4.21 (1H, m,
aCH Ala6), 4.17-4.14 (1H, m, aCH Ala4), 4.13-4.11 (1H, m, aCH
Lys7), 4.09-4.06 (1H, m, 1 bCH AlaT5), 4.04-4.03 (1H, m, 1 bCH
AlaT2), 3.84-3.79 (2H, m, aCH Ala1 and 1 bCH AlaT2), 3.74-3.69
(1H, m, 1 bCH AlaT5), 2.77-2.74 (2H, m, eCH2 Lys7), 1.72 (3H, s,
C5H3 thymine AlaT2), 1.71 (3H, s, C5H3 thymine AlaT5), 1.68-1.64
(2H, m, bCH2 Lys7), 1.55-1.52 (2H, m, dCH2 Lys7), 1.34-1.33 (3H,
d, J 6.5, CH3 Ala1), 1.31-1.25 (2H, m, gCH2 Lys7), 1.23-1.21 (6H,
2 d, CH3 Ala3 and Ala6), 1.19-1.18 (3H, d, J 7.0, CH3 Ala4). m/z
(ESI) 820.50 (M + H+; C34H54N13O11 requires 820.41).
Z-Aib-AlaT-Aib-Lys(Boc)-NH2. (Z-Aib)2O
(0.58
g,
1.26 mmol) dissolved in anhydrous MeCN and cooled in
a water/ice bath was added to H-AlaT-Aib-Lys(Boc)-NH2
(obtained by catalytic hydrogenolysis of 0.46 g, 0.65 mmol, of
the corresponding Z-protected peptide). The pH was adjusted to
8 with NMM and the mixture was stirred at room temperature
for 7 days. The solvent was evaporated at reduced pressure, the
residue was taken up with AcOEt, washed with KHSO4 5%, H2O,
NaHCO3 2%, and H2O, desiccated on Na2SO4 and evaporated
to a solid foam, which was purified by flash-chromatography
(eluent: DCM–MeOH, gradient from 15 : 1 to 13 : 1) to afford a
colorless solid (0.36 g, 75%). Mp: 124–126 ◦C (from Et2O–PE).
[a]2D5 = -55.6 (c = 0.25, MeOH). TLC: Rf 1 0.25, Rf 2 0.80, Rf 3
0.15. IR (cm-1): 3329, 1675, 1525. NMR dH (250 MHz, CDCl3)
9.22 (1H, br s, N(3)H thymine), 8.63-8.62 (1H, d, J 3.5, aNH
AlaT), 7.92 (1H, s, C(6)H thymine), 7.34-7.33 (5H, m, C6H5-Z),
7.17-7.13 (1H, d, J 8.0, aNH Lys), 7.03 (2H, br s, CONH2),
6.59 (1H, br s, NH Aib3), 5.78 (1H, br s, NH Aib1), 5.05 (2H, s,
CH2-Z), 4.62 (1H, br s, eNH Lys), 4.28-4.15 (3H, m, aCH Lys,
aCH and 1 bCH AlaT), 4.06-3.98 (1H, m, bCH AlaT), 3.14-3.09
(1H, m, 1 eCH Lys), 3.03-2.95 (1H, m, 1 eCH Lys), 2.05-1.95
(1H, m, 1 bCH Lys), 1.88 (3H, s, C5H3 thymine), 1.87-1.80 (1H,
m, 1 bCH Lys), 1.52-1.43 (13H, m, 3 CH3 Aib, gCH2 and dCH2
Lys), 1.42-1.41 (12H, 2 s, 1 CH3 1 Aib and 3 CH3 Boc). m/z (ESI)
745.38 (M+H+; C35H53N8O10 requires 745.39).
2*TFA·H-Ala-AlaT-Ala-Aib-AlaT-Ala-Lys(H)-NH2
[T2U].
Synthesis scale: 15 mmol. Activation protocol: 5 eq each of
Boc-protected protein amino acid, BOP, HOBt and 15 eq DIEA,
5 eq each of Boc-Aib-OH, HATU, HOAt and 15 eq DIEA or 3
eq each of Boc-AlaT-OH, BOP, HOBt and 10 eq DIEA. Yield:
3.4 mg (26%). HPLC (Varian, 0-50% B in 20 min): tr 9.10 min.
NMR dH (500 MHz, d6-DMSO) 11.24-11.23 (2H, 2 br s, N(3)H
thymine AlaT2 and AlaT5), 8.86-8.84 (1H, d, J 8.0, aNH AlaT2),
8.43-8.42 (1H, d, J 6.0, NH Ala3), 8.36 (1H, br s, NH Aib4), 8.21
(3H, br s, NH3 Ala1), 7.92-7.91 (1H, d, J 8.0, aNH AlaT5),
+
7.86-7.84 (1H, d, J 8.5, aNH Lys7), 7.84-7.82 (3H, br s, NH3
+
Lys7), 7.74-7.72 (1H, d, J 7.0, NH Ala6), 7.50 (1H, d, J 1.5,
C(6)H thymine AlaT2), 7.36 (1H, s, C(6)H thymine AlaT5), 7.22
(1H, br s, 1 CONH), 7.02 (1H, br s, 1 CONH), 4.66-4.62 (1H, m,
aCH AlaT2), 4.49-4.44 (1H, m, aCH AlaT5), 4.28-4.24 (1H, m,
1 bCH AlaT5), 4.22-4.21 (1H, m, aCH Ala3), 4.21-4.19 (1H, m,
aCH Ala6), 4.15-4.11 (1H, m, aCH Lys7), 4.02-3.98 (1H, m, 1
bCH AlaT2), 3.92-3.87 (1H, m, 1 bCH AlaT2), 3.86-3.80 (2H, m,
aCH Ala1 and 1 bCH AlaT5), 2.78-2.72 (2H, m, eCH2 Lys7), 1.73
(3H, d, J 1.5, C5H3 thymine AlaT2), 1.71 (3H, s, C5H3 thymine
AlaT5), 1.68-1.65 (2H, m, bCH2 Lys7), 1.58-1.52 (2H, m, dCH2
Lys7), 1.36-1.34 (2H, m, gCH2 Lys7), 1.33-1.32 (3H, d, J 7.0, CH3
Ala1), 1.29 (6H, s, 2 CH3 Aib4), 1.27-1.25 (3H, d, J 7.0, CH3
Ala6), 1.25-1.24 (3H, d, J 7.5, CH3 Ala3). m/z (ESI) 834.49 (M +
H+; C35H56N13O11 requires 834.42).
2*TFA·H-(Aib-AlaT-Aib)2-Lys(H)-NH2
[T2U4]. Z-Aib-
AlaT-Aib·Oxl (0.26 g, 0.51 mmol, obtained by activation of
0.30 g, 0.58 mmol, of Z-Aib-AlaT-Aib-OH with 0.13 g, 0.64 mmol
EDC·HCl) was suspended in anhydrous MeCN (5 mL) and added
to H-Aib-AlaT-Aib-Lys(Boc)-NH2 (obtained by catalytic
hydrogenolysis of 0.33 g, 0.40 mmol, of the corresponding
Z-protected peptide) and the resulting mixture was heated at
gentle reflux, with gradual dissolution, for 6 days. The solvent was
evaporated at reduced pressure to a reddish oil, which underwent
flash chromatography (eluent: CHCl3–MeOH, gradient 15 : 1 to
7 : 1), yielding crude Z-(Aib-AlaT-Aib)2-Lys(Boc)-NH2 (0.13 g,
28%) as a waxy solid. The protected nucleopeptide, m/z (ESI)
1110.55 (M + H+; C51H76N13O15 requires 1110.56), was used in the
next steps without further purification (HPLC analysis, Agilent
Solution-phase syntheses
The syntheses of Z-Aib-AlaT-Aib-OtBu and Z-Aib-AlaT-Aib-OH
have been described elsewhere.11
Z-AlaT-Aib-Lys(Boc)-NH2. Z-AlaT-OH (0.41 g, 1.2 mmol)
was dissolved in anhydrous DMF, cooled in a water/ice bath,
and HOAt (0.19 g, 1.4 mmol) and EDC·HCl (0.28 g, 1.5 mmol)
were added. H-Aib-Lys(Boc)-NH2 (obtained by catalytic hy-
drogenolysis of 0.89 g, 1.7 mmol, of the corresponding Z-protected
1320 | Org. Biomol. Chem., 2010, 8, 1315–1321
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20–50% B¢ in 30 min: 69% tr 27.07, 12% tr 26.45 min, 10% tr
27.84 min). Crude Z-(Aib-AlaT-Aib)2-Lys(Boc)-NH2 (0.10 g,
0.09 mmol) was Na-deprotected by catalytic (Pd) hydrogenolysis
in MeOH solution. After removal of the solvent, the residue was
dissolved in a DCM–TFA–H2O mixture (25 : 25 : 1, 1 mL) and
stirred for 1 hour at room temperature. The solvent was stripped
with N2, the residue was taken up with DCM and stripped
again with N2 2 times, then taken up with MeOH, yielding
crude T2U4 (0.09 g, 95%), which was purified by preparative
HPLC (Shimadzu, 1.5–4.8% B¢ in 22 min), then lyophilized,
affording 18 mg (18%) of a colorless solid. Mp: 176–178 ◦C (from
H2O–MeOH). [a]2D5 = -27 (c = 0.18, MeOH). TLC: Rf1 0.00,
Rf2 0.05, Rf3: 0.00. IR (cm-1): 3418, 1674, 1532. HPLC (Agilent,
1.5-6% B¢ in 30 min) tr 18.52 min. NMR dH (600 MHz, d6-DMSO)
11.33-11.31 (2H, 2 br s, 2 N(3)H thymine AlaT2 and AlaT5), 8.56
(1H, br s, NH Aib3), 8.38-8.37 (1H, d, J 6.0, aNH AlaT2), 8.14
measured at 490 nm wavelength using a Wallac Victor2 microplate
reader (Waltham, MC, USA). Results are the average of three
independent experiments.
Acknowledgements
We wish to thank the French-Italian University (UIF-UFI) for the
PhD scholarship of P. G.-B. and a travel grant.
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+
J 7.2, aNH AlaT5), 7.64 (4H, br s, NH Aib6 and 1 NH3 ), 7.42
+
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7.31 (1H, s, C(6)H thymine AlaT5), 7.04 (1H, br s, 1 CONH),
6.94 (1H, br s, 1 CONH), 4.68 (1H, m, aCH AlaT2), 4.36 (1H,
m, aCH AlaT5), 4.28-4.26 (1H, m, 1 bCH AlaT2), 4.11 (1H, m,
aCH Lys7), 4.01-3.96 (2H, m, bCH2 AlaT5), 3.73 (1H, m, 1 bCH
AlaT2), 2.73 (2H, m, eCH2 Lys7), 1.80 (1H, m, 1 bCH Lys7), 1.72
(3H, s, C5H3 thymine AlaT2), 1.71 (3H, s, C5H3 thymine AlaT5),
1.59 (1H, m, 1 bCH Lys7), 1.48 (2H, m, dCH2 Lys7), 1.42 (3H, s, 1
CH3 Aib1), 1.40 (6H, s, 1 CH3 Aib1 and 1 CH3 Aib6), 1.36 (3H, s,
1 CH3 Aib3), 1.34-1.33 (7H, m, 1 gCH Lys7, 1 CH3 Aib3 and 1
CH3 Aib6), 1.29-1.27 (7H, m, 1 gCH Lys7 and 2 CH3 Aib4). m/z
(ESI) 876.52 (M+H+;C38H62N13O11 requires 876.47).
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The Royal Society of Chemistry 2010
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