Synthesis of a 24-membered cyclic peptide-biphenyl hybrid

Article abstract of DOI:10.1002/ejoc.200600833

The synthesis of a 24-membered macrocyclic peptide-biphenyl hybrid with four amino acid residues and two biphenyl fragments was performed by a combination of solid-phase and solution methodologies. The acyclic precursor was prepared by a solid phase metho

Full text of DOI:10.1002/ejoc.200600833

FULL PAPER
DOI: 10.1002/ejoc.200600833
Synthesis of a 24-Membered Cyclic Peptide-Biphenyl Hybrid
Ana Montero,[a,b][‡] Fernando Albericio,*[c,d] Miriam Royo,*[b] and Bernardo Herradón*[a]
Keywords: Biaryls / Calpain inhibitors / Macrocycles / Peptide-biphenyl hybrids / Solid-phase synthesis
The synthesis of a 24-membered macrocyclic peptide-bi-
phenyl hybrid with four amino acid residues and two bi-
phenyl fragments was performed by a combination of solid-
phase and solution methodologies. The acyclic precursor was
prepared by a solid phase methodology whereas the final
macrocyclization was carried out by diisopropylcarbodiimide
(DIPCDI) and N-hydroxybenzotriazole (HOBt) in solution.
The target molecule was a moderate inhibitor of µ-calpain.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2007)
Introduction
Peptides and related compounds are the focus of intense lack of transportation across cell membranes, in vivo insta-
research activity. These compounds have been used as tools bility, and incapacity to achieve a bioactive conformation.
to study biochemical and biophysical processes,[1] as chiral As a result of these drawbacks, many peptide analogues
catalysts,[2] as building blocks for the preparation of techno- have been prepared. Two general strategies for the design
logical materials,[3] and, mainly, as pharmaceuticals.[4] Al- of peptide analogues are the insertion of non-peptidic frag-
though many peptides show biological activity, they are fre- ments into a peptide chain (either main or side)[5] and the
quently questioned as drugs because of poor absorption, generation of cyclic structures.[6]
Figure 1. Generic structures of peptide-biphenyl hybrids of types A–C.
Recently, we have focused on the synthesis, structure, and
properties of peptide-biphenyl hybrids (A–C, Figure 1),
which are derivatives of 1,1Ј-biphenyl with amino acid or
peptide chains at the positions C(2) and C(2Ј).[7–10] Since
biphenyl derivatives[11–13] and peptides possess useful char-
acteristics, the combination of the two functionalities has
resulted in the generation of compounds with structural[7]
and biological properties of great interest. Of these, the
most interesting is their capacity to inhibit calpain.[8,9] This
enzyme is a cysteine protease with an active physiological
role,[14] which, when over-activated, causes a variety of
pathological conditions.[15–16]
[a] Instituto de Química Orgánica General, CSIC,
Juan de la Cierva 3, 28006 Madrid, Spain
E-mail: herradon@iqog.csic.es
[b] Combinatorial Chemistry Unit, Barcelona Science Park, Uni-
versity of Barcelona,
Josep Samitier 1, 08028 Barcelona, Spain
E-mail: mroyo@pcb.ub.es
[c] Institute for Research in Biomedicine, Barcelona Science Park,
University of Barcelona,
Josep Samitier 1, 08028 Barcelona, Spain
E-mail: albericio@pcb.ub.es
[d] Department of Organic Chemistry, University of Barcelona,
Martí I Franqués 1, 08028 Barcelona, Spain
[‡] Present address: The Scripps Research Institute,
10550 North Torrey Pines Road (BCC-104), La Jolla, CA
92037, USA
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A. Montero, F. Albericio, M. Royo, B. Herradón
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Scheme 1. Retrosynthetic analysis of macrocycle 1.
It is well established that the intramolecular tethering of was efficient, releasing the free carboxylic acid by basic hy-
the N- and C-terminals of peptide chains, generating a mac- drolysis. However, this strategy was not viable in this case
romolecular structure, provides compounds with lowered since the link to the resin would also be hydrolyzed. There-
conformational flexibility as well as increased stability vs. fore, we used the allyl ester as carboxylic protection.
peptidases. As a step forward in our research on peptide-
biaryl hybrids, here we have prepared a cyclic analogue of
a peptide-biphenyl hybrid of type C, with the objective to
assess the effect of this structural restriction on its proper-
ties as well as to prove the feasibility of the synthetic strat-
egy. The target model molecule was 1, which was intended
to be prepared by cyclization, in solution, of the lineal pre-
cursor 2 (Scheme 1), which in turn would be synthesized in
the solid-phase mode following an adapted methodology
from that recently developed by our group.[9]
Thus, the acid 6 was coupled to H-Phe-OAll (PyBOP/
HOAt/DIEA) to provide 7, which, by treatment with a cata-
lytic amount of Pd(PPh3)4 in the presence of PhSiH3,[19]
gave the hybrid 8. This acid was activated with CDI and
treated with excess of 2,2Ј-biphenyl-1,1Ј-diamine (13) to
furnish the peptide-biphenyl hybrid 9 with two biphenyl
fragments. The amine 9 was reacted with the anhydride 14
(Et3N) to give an N-protected peptide-biphenyl hybrid 10,
which was deprotected (piperidine) to the amine 11. The
structure and purities of all the reaction products were as-
sessed by a combination of HPLC and MS of the corre-
sponding acid obtained after treatment with TFA used to
remove the solid support. HPLCs of all intermediates 4–11
showed high purities (Ͼ 90%). Once the synthetic sequence
in the solid phase was completed, the peptide was removed
from the resin (TFA) to give, after solvent treatment
(CH3CN-H2O) and lyophilization, the target acyclic com-
pound 2 as its trifluoroacetate salt (2·TFA).
With the acyclic precursor 2 in hand, we performed the
key macrocyclization step in solution.[20] Preliminary ex-
periments were performed to determine the most suitable
conditions.[21] We found that the treatment of a 2 mM solu-
tion of 2 in DCM with DIPCDI/HOBt/DIEA gave the
macrocycle 1 in 70% yield, after purification by preparative
HPLC. Overall, the complete synthetic sequence (12 steps)
was done starting from 500 mg of MBHA-PS-resin, with
an initial loading of 0.7 mmol/g, affording 81.8 mg of the
macrocycle 1 in 27% overall yield. The macrocyclic peptide-
biphenyl hybrid 1 was tested as an inhibitor of µ-calpain.
Our results show that this hybrid showed modest inhibitory
capacity, with an IC50 value of 29 µM.[22]
Results and Discussion
Our synthetic plan for the solid-phase synthesis of the
target peptide-biphenyl hybrid is based on a standard strat-
egy for the solid-phase synthesis of peptides,[17] with the
initial amino acid linked to the resin by the C-terminal,
although taking into account that when the biphenyl unit,
which contains two carboxylic moieties, is incorporated, the
chain changes sense.[5c] In our previous synthesis of pep-
tide-biphenyl hybrids, we used methyl ester as temporary
protection of carboxylic acid and a Rink amide linker
bonded to a MBHA-PS-resin, which resulted in primary
amides after resin cleavage. Although the solid-phase syn-
thesis of the target acyclic peptide-biphenyl 2 could be per-
formed in a similar way, two significant modifications were
made. On the one hand, the Wang acid linker (AB) was
used, thereby allowing us to obtain a carboxylic acid after
removal from the resin; and, on the other hand, an orthogo-
nal protection for the carboxylic group was required.
The synthesis is shown in Scheme 2.[18] The MBHA-PS
resin was modified with the AB linker (DIPCDI/HOBt) and
The macrocyclic peptide-biphenyl hybrid compound 1 is
reacted with the first protected amino acid, Fmoc-Val-OH a mixture of atropisomers. Thus, it was observed that, at
(DIPCDI/DMAP), to give 3, which was sequentially treated room temperature, the HPLC of 1 presents two close broad
with piperidine and coupled with Fmoc-Phe-OH (DIPCDI/ peaks center at 10.44 and 10.61 min with identical UV spec-
HOBt) to afford the dipeptide 4. This dipeptide, in turn, tra. These two peaks tend to coalesce (10.35 min) on in-
was N-deprotected (piperidine) to 5 and treated with di- creasing the temperature to 60 °C (Figure 2), which indi-
phenic anhydride (12) (Et3N) to give the peptide-biphenyl cates that these peaks correspond to equilibrating conform-
hybrid 6. The next amino acid had to be coupled by the ers. The presence of several conformers was confirmed by
amino group with a suitable protection at the carboxyl 1H NMR (Figure 3b); thus, 1 is a mixture of 4 conformers
group. Previously, we found that protection as methyl ester in DMSO at 60 °C, probably because of the restricted rota-
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© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Cyclic Peptide-Biphenyl Hybrid
FULL PAPER
Scheme 2. Synthesis of macrocyclic peptide-biphenyl hybrid 1. (i) (a) DIPCDI (2.0 equiv.), HOBt (2.0 equiv.), DMF, (b) Fmoc-Val-OH
(10.0 equiv.), DIPCDI (10.0equiv.), DMAP (0.1 equiv.), DCM/DMF (9:1); (ii) (a) piperidine/DMF (1:4), (b) Fmoc-Phe-OH (3.0 equiv.),
DIPCDI (3.0 equiv.), HOBt (3.0 equiv.), DMF; (iii) piperidine/DMF (1:4); (iv) 12 (5.0 equiv.), Et3N (5.0 equiv.), DMF; (v) HCl·H-Phe-
OAll (3.0 equiv.), PyBOP (3.0 equiv.), HOAt (3.0 equiv.), DIEA (6.0 equiv.), DMF; (vi) Pd(PPh3)4 (0.1 equiv.), PhSiH3 (10.0 equiv.), DCM;
(vii) (a) CDI (10.0 equiv.), DMF, (b) 13 (5.0 equiv.), DIPCDI (5.0 equiv.), HOBt (5.0 equiv.), DMF; (viii) (a) 14 (3.0 equiv.), Et3N
(3.0 equiv.), DCM, (b) piperidine/DMF (1:4); (ix) TFA/DCM (95:5); (x) DIPCIDI (3.0 equiv.), HOBt (3.0 equiv.), DIEA, (3.0 equiv.),
DCM (2 mM).
tion around the aryl-aryl bond of the biphenyl moieties, as the preparation of other macrocyclic peptide-biphenyl hy-
found in other acyclic peptide-biphenyl hybrids.[7a] How- brids as well as the determination of their conformational
ever, the macrocycle 1 showed lower conformational mo- preferences and properties.
bility than its acyclic analogue, as inferred from the com-
1
parison of their H-NMR spectra (Figure 3).
Experimental Section
Conclusions
General Methods: 4-Methylbenzhydrylamine resin VHL (0.7 mmol/
g, 100–200 mesh), AB handle and Fmoc-amino acids were pur-
chased from NovaBiochem (Läufelfingen, Switzerland). Dimethyl-
The macrocyclic peptide-biphenyl hybrid 1, which con-
tains two fragments of biphenyl and four amino acid resi-
formamide (DMF), dichloromethane (DCM) and tetrahydrofuran
dues, was synthesized in high overall yield by a combination
(THF) were purchased from SDS (Peypin, France) and used as
of solid-phase and solution methodologies, including an ef-
received. Analytical HPLC was carried out on a Waters® 2695 Sep-
ficient macrocyclization to a 24-membered ring. The pres-
ent strategy provides access to macrocyclic compounds with
amino acid and biaryl fragments which, like the acyclic
counterpart, may show structural, biological, and techno-
arations Module instrument, including a Waters® 996 Photodiode
Array Detector with UV detection from 210 to 500 nm. Linear gra-
dients of CH3CN and H2O were run at 1 mL/min flow rate, from
100:0 (0.1% of TFA in H2O: 0.1% of TFA in CH3CN) to 0:100
logical properties of interest. We are currently working on (0.1% of TFA in H2O: 0.1% of TFA in CH3CN) over 10 or 15 min,
Eur. J. Org. Chem. 2007, 1301–1308
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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A. Montero, F. Albericio, M. Royo, B. Herradón
FULL PAPER
Figure 2. Crude product, HPLC analysis (a) at room temperature and (b) at 60 °C; purified macrocycle 1, (c) HPLC analysis at room
temperature; (d) mass spectrum.
with a Symmetry® C-18 column (5.0 µm, 4.6 mmϫ150 mm). All til a negative test was obtained. IR spectra were measured in a
HPLC-MS spectra were obtained on a Micromass ZQ mass spec- Perkin–Elmer Spectrum One FT-IR spectrometer; the frequencies
1
trometer in electrospray positive ionization (ES+) mode in-line with
in the IR spectra are shown in cm–1. H NMR spectra were mea-
sured in Bruker DRX-600, Varian UNITY 500 and Varian INOVA
300 spectrometers. Chemical shifts (δ) are reported in parts per
a Waters® 2795 HPLC system (Separations module, AllianceTM
)
and Waters® 2487 Dual absorbance Detector (PDA). Linear gradi-
ents of CH3CN and H2O were run at a flow rate of 0.5 mL/min,
from 100:0 (0.1% of HCOOH in H2O:CH3CN) to 0:100 (0.1% of
HCOOH in H2O:CH3CN) over 10 or 15 min, with a X-Terra® C-
18 column (5.0 µm, 4.0ϫ50 mm). All reactions were performed in
polypropylene syringes (10 mL), each fitted with a polyethylene po-
rous disc. Ninhydrine tests were performed in the reactions involv-
ing resin-bonded amines[23] and the reactions involving resin-
bonded carboxylic acids were monitored by the malachite green
test,[24] and when necessary, the coupling reaction was repeated un-
1
million and the coupling constants are indicated in Hz. H-NMR
spectra were referenced to the chemical shift of either TMS (δ =
0.00 ppm) or the residual proton in the deuterated solvent. MS
analyses were recorded in Hewlett–Packard 1100 MSD (ESI) spec-
trometers. Combustion analyses were performed in a Carlo–Erba
EA 1180-Elemental Analyzer by E. Barbero (CSIC). Optical rota-
tions were determined in a Perkin–Elmer 241 MC polarimeter at
room temperature (ca. 295 K). Melting points were measured on a
Kofler hot-stage apparatus.
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© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Cyclic Peptide-Biphenyl Hybrid
FULL PAPER
1
Figure 3. H-NMR spectra (600 MHz, [D6]DMSO) of acyclic peptide-biphenyl hybrid 2·TFA at 60 °C (a) and of macrocycle 1 at 50 °C
(b).
Incorporation of the AB Acid Linker into the PS-MBHA Resin
(preparation of MBHA-AM): After washing the PS-MBHA resin
next Fmoc-aa-OH (Fmoc-Phe-OH) was introduced by reaction
with a solution of Fmoc-Phe-OH (3.0 equiv.), DIPCDI (3.0 equiv.)
(500 mg, 0.35 mmol) with DCM (5ϫ 1 min), TFA 40% / DCM (1ϫ and HOBt (3.0 equiv.) for 2.5 h. The Fmoc-protecting group was
1 min, 1ϫ 30 min), DCM (5ϫ 1 min), 10% DIEA / DCM (3ϫ
3 min) and DCM (5ϫ 1 min), a solution of the AB handle
(137.2 mg, 0.7 mmol) and HOBt (94.6 mg, 0.7 mmol) in DMF was
added, followed by DIPCDI (108.4 µL). The mixture was stirred
overnight.
removed with piperidine/DMF (1:4) (2ϫ 15 min) to give amine 5,
which was used in the next step.
Incorporation of the First Biphenyl Fragment (preparation of 6): The
peptidyl resin 5 was swollen in DMF. Diphenic anhydride (12,
5.0 equiv.) dissolved in the minimum amount of DMF was then
added followed by Et3N (5.0 equiv.). The mixture was shaken at
room temperature for over 2.5 h and the solvent was removed by
filtration. The resin was washed with DMF (5ϫ 1 min), DCM (5ϫ
1 min) and dried in vacuo.
Sequential Coupling of MBHA-AM with Fmoc-Amino Acids and
Removal of Fmoc Group (preparation of 5): The resin was washed
with DCM (5ϫ 1 min) and DMF (5ϫ 1 min) and sequentially
treated with solutions of DIPCDI (2.0 equiv.) and HOBt
(2.0 equiv.), and with a solution of Fmoc-Val-OH (10.0 equiv.), and
left to react for 2.5 hours. The Fmoc group was then removed from
the resin by treatment with piperidine/DMF (1:4) (2ϫ 15 min). The
Coupling with HCl·H-Phe-OAll (preparation of 7): The resin 6 with
the free carboxylic functionality was swollen in DMF and then
Eur. J. Org. Chem. 2007, 1301–1308
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A. Montero, F. Albericio, M. Royo, B. Herradón
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DIEA (3.0 equiv.), PyBOP (3.0 equiv.) and HOAt (3.0 equiv.), dis-
solved in the minimum amount of DMF, were added. The mixture
was shaken for 5 min and then a solution of HCl·H-Phe-OAll
(3.0 equiv.) and DIEA (3.0 equiv.) in DMF was added. The reac-
tion mixture was stirred at room temperature for 2.5 h and the
resin was then filtered and washed with DMF (5ϫ 1 min).
Synthesis of Macrocycle 1: A solution of 2·TFA (134.9 mg,
0.14 mmol) in dry DCM (70 mL, 2 mM) was sequentially treated
with DIPCDI (64 µL, 3.0 equiv.), HOBT (56 mg, 3.0 equiv.), and
DIEA (70 µL, 3.0 equiv.). The mixture was stirred at room tem-
perature overnight. The solvent was then removed to give a crude
product, which was purified by HPLC to give 81.8 mg (70% yield,
27% overall yield for the whole 12-steps sequence) of macrocycle
1. White solid; m.p. 165–168 °C. [α]D = +40 (c = 0.02, MeOH). IR
Removal of the Allyl Group (preparation of 8): The peptide resin 7
was washed with DCM (5ϫ 1 min), Ar was passed through the
resin, a solution of PhSiH3 (10.0 equiv.) in DCM was added and
the resin was manually stirred. Then, a solution of Pd(PPh3)4
(0.10 equiv.) in DCM was added and the reaction was shaken for
10 min. The peptide resin was washed with DMC (8ϫ 30 s) and the
process was repeated once to assure completeness of the reaction to
obtain the acid 8.
(KBr): ν = 3416, 3057, 3028, 2963, 2927, 1646, 1518, 1439, 1201,
˜
1174, 753, 699. 1H NMR (500 MHz, 27 °C, CDCl3, mixture of
atropisomers): δ = 9.24 (br. d, J = 8.4), 8.80 (br. s), 8.69 (br. s),
8.10–7.85 (m), 7.65–6.90 (m), 6.85 (m), 6.75 (m), 6.58 (br. d, J =
6.9), 6.40 (br. s), 6.16 (br. s), 6.09 (br. s), 6.02 (br. d, J = 6.8), 5.96
(br. d, J = 7.8), 5.79 (br. d, J = 6.3), 5.55 (m), 4.94 (m), 4.79 (m),
4.65 (m), 4.52 (m), 4.45–4.25 (m), 4.09 (m), 3.90 (m), 3.82 (br. t, J
= 6.3), 3.70 (br. t, J = 5.2), 3.63 (br. d, J = 8.3), 3.50 (br. d, J =
6.8), 3.47 (br. d, J = 5.4), 3.40 (br. t, J = 5.1), 3.16 (m), 3.01 (m),
2.99–2.83 (m), 2.77–2.51 (m), 2.29 (m), 1.93 (m), 1.32 (d, J = 7.2),
1.27 (d, J = 6.9), 1.22 (d, J = 5.4), 1.17 (d, J = 7.2), 0.97 (d, J =
7.3), 0.92–0.77 (m), 0.73 (d, J = 6.5), 0.71 (d, J = 5.7), 0.66 (d, J
= 6.9), 0.60 (d, J = 7.5), 0.49 (dd, J = 6.8, 2.3). 1H NMR (300 MHz,
40 °C, CD3CN, mixture of atropisomers): δ = 8.77 (s), 8.68 (br. d,
J = 9.3), 8.27 (m), 8.16 (m), 8.05 (m), 7.92 (d, J = 8.1), 7.84 (dd, J
= 7.8, 1.2), 7.70 (m), 7.62 (td, J = 7.7, 1.4), 7.55–6.93 (m), 6.89
(m), 6.87–6.76 (m), 6.68 (m), 6.60 (br. d, J = 7.8), 6.53 (m), 6.39
(m), 4.67 (td, J = 9.3, 4.4), 4.58–4.42 (m), 4.34 (m), 4.11 (m), 3.78
(m), 3.67 (t, J = 5.4), 3.35 (m), 3.12 (t, J = 6.1), 2.76 (d, J = 3.8),
2.71 (d, J = 4.4), 2.57 (dd, J = 14.2, 9.7), 2.30 (d, J = 9.3), 2.25
(dd, J = 9.3, 3.3), 1.32 (m), 1.23 (d, J = 7.3), 1.13 (d, J = 4.9), 1.10
(d, J = 8.8), 0.95–0.85 (m), 0.83–0.74 (m), 0.71 (d, J = 5.8), 0.64
(d, J = 6.9). 1H NMR (600 MHz, [D6]DMSO, 50 °C, mixture of
atropisomers): δ = 9.48 (d, J = 7.9, 1 H, NH-Phe2), 9.42 (d, J =
8.7, 1 H, NH-Phe1), 8.98 (s, 1 H, NH-biph.), 8.42 (s, 1 H, NH-
biph.), 8.25 (broad d, 1 H, NH-Ala), 7.76 (d, J = 7.8, 1 H, Harom),
7.71 (d, J = 7.8, 1 H, Harom), 7.61 (t, J = 7.5, 1 H, Harom), 7.65 (d,
J = 8.3, 1 H, NH-Val), 7.45–7.35 (m, 5 H, Harom.), 7.29–7.18 (m,
6 H, Harom.), 7.15–7.06 (m, 7 H, Harom.), 6.96 (d, J = 7.4, 1 H,
Harom), 6.89 (d, J = 7.4, 1 H, Harom), 6.54 (m, 2 H, Harom), 6.19 (d,
J = 7.5, 1 H, Harom), 4.61 (m, 1 H, CHα-Phe1), 4.33 (m, 1 H, CHα-
Val), 4.17 (m, 1 H, CHα-Phe2), 3.78 (m, 1 H, CHα-Ala), 2.60 [m,
1 H, C(HαCHβ)-Phe1; 1 H, C(HαCHβ)-Phe2], 2.13 [m, 1 H,
C(HαCHβ)-Phe1], 1.95 [m, 1 H, C(HαCHβ)-Phe2; 1 H, CH(CH3)2-Val],
1.19 (d, J = 7.0, 3 H, CH3-Ala), 0.58 [d, J = 6.8, 3 H, C(CH3)2-Val],
0.52 [d, J = 6.8, 3 H, C(CH3)2-Val]. ESI-MS: m/z = 855.3 ([MH]+,
100%). C52H50N6O6 (854.99) calcd. C 73.05, H 5.89, N 9.83; found:
C 73.28, H 5.91, N 9.63.
Incorporation of the Second Biphenyl Fragment (preparation of 9).
Activation of the Carboxylic Acid: The resin-linked acid 8 was
swollen in DMF and a solution of CDI (10.0 equiv.) in DMF was
added. The mixture was shaken at room temperature over 2.5 h to
assure complete activation of the acid. The resin was then filtered,
washed with DMF (5ϫ 1 min) and DCM (5ϫ 1 min) and dried in
vacuo.
Coupling with Biphenyl-2,2Ј-diamine (13): The activated resin was
swollen in DMF and then a solution of 13 (5.0 equiv.) in DMF
was added followed by DIPCDI (5.0 equiv.) and HOBt (5.0 equiv.),
dissolved in the minimum amount of DMF. The mixture was
shaken for over 2 h at room temperature. The resin was finally
filtered, washed with DMF (5ϫ 1 min), DCM (5ϫ 1 min) and
dried in vacuo to give 9, which was used in the next step.
Synthesis of (S,S,S,S)-2-{2-[(2Ј-{1-[2Ј-(2-Aminopropionylamino)bi-
phenyl-2-ylcarbamoyl]-2-phenylethylcarbamoyl}biphenyl-2-carbonyl)-
amino]-3-phenylpropionylamino}-3-methylbutyric Acid (2). Prepara-
tion of the Symmetric Anhydride 14: DCC (3.0 equiv.) was added to
a solution of Fmoc-Ala-OH (6.0 equiv.) in DCM and the reaction
mixture was stirred at room temperature for over 30 min. The DCU
formed was filtered off and the resulting solution was used in the
next step.
Coupling with the Symmetric Anhydride, Removal of the Fmoc
Group and Cleavage from the Resin: The resin 9 with the free amino
functionality was swollen in DCM and the solution of the symmet-
ric anhydride, previously prepared, was added, followed by Et3N
(3.0 equiv.). The mixture was shaken for 4.5 h. The resin was finally
filtered, washed with DCM (5ϫ 1 min) and dried in vacuo to fur-
nish 10. The resin was then washed with DMF (5ϫ 1 min) and
treated with piperidine/DMF (1:4) (2ϫ 15 min) to give the amine
11. Cleavage was accomplished by treatment of the resin 11 with
TFA/DCM (95:5) for over 2 h. The filtrate was collected, concen-
trated to dryness and the resulting residue was dissolved in the
minimum amount of CH3CN. Water was then added and after ly-
ophilization compound 2 was obtained as the trifluoroacetate salt.
1H NMR (600 MHz, [D6]DMSO, 60 °C): δ = 9.31 (broad s, 1 H,
NH-biph.), 8.85 (s, 1 H, NH-biph.), 8.59 (broad s, 1 H, NH-Phe1;
1 H, NH-Phe2), 8.02 (broad s, 1 H, NH2-Ala), 7.96 (broad s, 1 H,
NH-Val), 7.47 (m, 1 H, Harom.), 7.39 (m, 2 H, Harom.), 7.27–7.10
(m, 21 H, Harom.), 6.89 (m, 2 H, Harom.), 4.60 (m, 1 H, CHα-Phe),
4.33 (m, 1 H, CHα-Phe), 4.14 (m, 1 H, CHα-Val), 3.83 (m, 1 H,
CHα-Ala), 2.92 [m, 1 H, C(HαHβ)-Phe1; 1 H, C(HαHβ)-Phe2], 2.69
[m, 1 H, C(HαHβ)-Phe1; 1 H, C(HαHβ)-Phe2], 2.01 [m, 1 H,
CH(CH3)2-Val], 1.09 (m, 3 H, CH3-Ala), 0.85 [d, J = 6.9, 3 H,
C(CH3)2-Val], 0.83 [d, J = 6.9, 3 H, C(CH3)2-Val]. ESI-MS: m/z =
872.93 ([MH]+, 100%). C52H52N6O7 (873.01) calcd. C 71.54, H
6.00; N 9.63; found: C 71.36, H 5.88, N 9.44.
Acknowledgments
This work was partially supported by Ministerio de Ciencia y Tec-
nología (MCyT) (BQU2003-00089 and 2002-02047), Ministerio de
Educacíon y Ciencia (MEyC) (CTQ2004-01978), and the Fundació
Parc Científic de Barcelona (Barcelona Science Park Foundation).
We thank Mercedes Alonso (CSIC) for determining the biological
activity of 1 and Mónica Luengo (Barcelona Science Park, Univer-
sity of Barcelona) for assistance in the HPLC purification.
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(the distance between the two ends varies from 3.28 to 5.47 Å)
were quite abundant.
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Received: September 23, 2006
Published Online: January 17, 2007
[22] As previously reported,[8] a spectrofluorimetric method using
labeled-casein as substrate was used to determine the biological
activity of 1.
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