Solid-phase synthesis of cyclic lipopeptidotriazoles

Article abstract of DOI:10.1002/ejoc.201402111

The solid-phase synthesis of cyclic lipopeptidotriazoles derived from the cyclic decapeptide c(Lys-Lys²-Leu-Lys-Lys⁵-Phe-Lys-Lys- Leu-Gln) (BPC194), incorporating a triazolyl ring at Lys² and a hexanoyl group at Lys⁵, was studied. Four different strategies that required the use of five orthogonal protecting groups (Fmoc, tBu, All, pNZ, ivDde) were explored. The influence of the side-chain protection of Lys ² and Lys⁵ with the ivDde and pNZ groups was evaluated by incorporating Lys²(ivDde)/Lys⁵(pNZ) or Lys ²(pNZ)/Lys⁵(ivDde). The order of removal of these protecting groups and of the introduction of the hexanoyl and triazolyl moieties was also studied. The best strategy included: (i) synthesis of a cyclic peptidyl resin bearing Lys²(ivDde) and Lys⁵(pNZ); (ii) pNZ group removal; (iii) acylation with hexanoic acid; (iv) ivDde group removal; and (v) acylation with propiolic acid followed by an azide-alkyne 1,3-dipolar cycloaddition. By using this protocol, a set of cyclic lipopeptidotriazoles was prepared in high purities.

Full text of DOI:10.1002/ejoc.201402111

FULL PAPER
DOI: 10.1002/ejoc.201402111
Solid-Phase Synthesis of Cyclic Lipopeptidotriazoles
Sílvia Vilà,[a] Cristina Camó,[a] Eduard Figueras,[a] Esther Badosa,[b] Emilio Montesinos,[b]
Marta Planas,*[a] and Lidia Feliu*[a]
Keywords: Synthetic methods / Solid-phase synthesis / Peptides / Lipopeptides / Protecting groups / Nitrogen heterocycles
The solid-phase synthesis of cyclic lipopeptidotriazoles de-
rived from the cyclic decapeptide c(Lys-Lys2-Leu-Lys-Lys5-
Phe-Lys-Lys-Leu-Gln) (BPC194), incorporating a triazolyl
ring at Lys2 and a hexanoyl group at Lys5, was studied. Four
different strategies that required the use of five orthogonal
protecting groups (Fmoc, tBu, All, pNZ, ivDde) were ex-
plored. The influence of the side-chain protection of Lys2 and
Lys5 with the ivDde and pNZ groups was evaluated by incor-
porating Lys2(ivDde)/Lys5(pNZ) or Lys2(pNZ)/Lys5(ivDde).
The order of removal of these protecting groups and of the
introduction of the hexanoyl and triazolyl moieties was also
studied. The best strategy included: (i) synthesis of a cyclic
peptidyl resin bearing Lys2(ivDde) and Lys5(pNZ); (ii) pNZ
group removal; (iii) acylation with hexanoic acid; (iv) ivDde
group removal; and (v) acylation with propiolic acid followed
by an azide–alkyne 1,3-dipolar cycloaddition. By using this
protocol, a set of cyclic lipopeptidotriazoles was prepared in
high purities.
hemolytic activities. In general, these sequences displayed
high activity against phytopathogenic bacteria but were
hemolytic.
Introduction
Lipopeptides have received considerable attention be-
cause of their antimicrobial and anticancer properties.[1]
Native lipopeptides are small linear or cyclic sequences (six
to seven d- and l-amino acids), with a net positive or nega-
tive charge, and to which a fatty acid moiety is covalently
attached. Within this group, cyclic lipopeptides of the itu-
rin, fengycin, surfactin, and polymyxin families exhibit sig-
nificant antimicrobial activity against phytopathogenic mi-
croorganisms.[2] Moreover, synthetic lipopeptides have been
obtained from the acylation of AMPs with fatty acids,
which has proven to be a useful approach to improve the
antibacterial activity of the parent peptides as well as to
endow them with antifungal properties.[3] In this context,
with the aim of identifying antimicrobial agents to fight
plant diseases of economic importance, we have recently re-
ported the synthesis of cyclic lipopeptides derived from the
antimicrobial cyclic decapeptide c(Lys-Lys2-Leu-Lys-Lys5-
Phe-Lys-Lys-Leu-Gln) (BPC194).[4] These cyclic lipopept-
ides were prepared by acylation of the Nε-amino group of
Lys5 with a range of fatty acids.[5] This study revealed that
the fatty acid length influences both the antimicrobial and
Our research group has also prepared analogues of
BPC194 bearing a 1,2,3-triazole ring at the side chain of an
amino acid.[6] 1,2,3-Triazoles have been reported to play an
important role in biological systems due to their favorable
physicochemical properties.[7] Moreover, their straightfor-
ward synthesis through copper-catalyzed azide alkyne cy-
cloaddition (CuAAC) has allowed the preparation of a wide
variety of biologically active peptidotriazoles.[7a,8] In par-
ticular, cyclic peptidotriazoles derived from BPC194
[a] Laboratori d’Innovació en Processos i Productes de Síntesi
Orgànica (LIPPSO), Departament de Química, Universitat de
Girona
Campus Montilivi, 17071 Girona, Spain
E-mail: marta.planas@udg.edu;
lidia.feliu@udg.edu
http://www.lippso.com
[b] Laboratory of Plant Pathology, Institute of Food and
Agricultural Technology-CIDSAV-CeRTA, Universitat de
Girona,
Campus Montilivi, 17071 Girona, Spain
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201402111.
Figure 1. General structure of cyclic lipopeptidotriazoles.
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M. Planas, L. Feliu et al.
FULL PAPER
showed significant antimicrobial activity. Interestingly, the of the peptidyl sequence, removal of the C- and N-terminal
presence of the 1,2,3-triazole ring led to a decrease in the protecting groups (All and Fmoc, respectively) and cycliza-
hemolytic activity.[6]
tion. By following this protocol, the selective derivatization
Based on these considerations, the combination of an of Lys2 and Lys5 could be achieved by using two further
acyl substituent and a 1,2,3-triazole ring in the sequence orthogonal protecting groups. For this purpose, the p-nitro-
of BPC194 would represent an interesting approach to the benzyloxycarbonyl (pNZ)[11] and the 1-(4,4-dimethyl-2,6-
identification of antimicrobial agents with low hemolysis. dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde)[12] groups
Thus, we decided to study the solid-phase synthesis of cyclic were chosen. The pNZ group can be removed by treatment
lipopeptidotriazoles derived from BPC194 incorporating a with SnCl2/HCl, whereas ivDde removal can be ac-
1,2,3-triazole ring at the side chain of Lys2 and a hexanoyl complished with NH2NH2. To evaluate the influence of the
group at the Nε-amino group of Lys5 (Figure 1). To the best derivatization of Lys2 and Lys5 with these groups, cyclic
of our knowledge, until now, the solid-phase synthesis of peptidyl resins 1 and 2 were prepared (Schemes 1 and 2).
cyclic peptides bearing both an acyl chain and a triazolyl The former incorporated the ivDde group at Lys2 and the
moiety has not been reported. The preparation of these cy- pNZ at Lys5 (Strategies 1a and 1b), whereas the latter in-
clic derivatives requires significant synthetic effort with the cluded the pNZ group at Lys2 and ivDde at Lys5 (Strategies
concurrence of five orthogonal protecting groups. The cy- 2a and 2b). For each of these peptidyl resins we analyzed
clic structure could be prepared by following a standard the order of the incorporation of the hexanoyl and triazolyl
9-fluorenylmethoxycarbonyl (Fmoc)/tert-butyl (tBu)/allyl moieties and, therefore, the order of removal of the protect-
(All) strategy that involves the synthesis of a linear peptidyl ing groups.
resin followed by its cyclization.[9] The incorporation of the
1,2,3-triazole and the acyl group could be afforded by
masking the side chain of Lys2 and Lys5 with two ad-
ditional orthogonal protecting groups. Such an approach
would easily allow the selective introduction of the 1,2,3-
Synthesis of Cyclic Lipopeptidotriazoles from Peptidyl
Resin 1 (Strategies 1a and 1b)
triazole and the acyl chain at these positions. Herein, we
The preparation of cyclic lipopeptidotriazoles starting
describe the evaluation of four different strategies for the from resin 1 was studied for BPC730 and is depicted in
solid-phase synthesis of cyclic lipopeptidotriazoles.
Scheme 1. Cyclic peptidyl resin 1 was synthesized from an
Fmoc-Rink-MBHA resin. After Fmoc removal with piperi-
dine/N,N-dimethylformamide (DMF) (3:7), Fmoc-Glu-
OAll was coupled by using N,NЈ-diisopropylcarbodiimide
(DIPCDI) and ethyl 2-cyano-2-(hydroxyimino)acetate
(Oxyma) in DMF for 1 h. This trifunctional amino acid
allows peptide anchoring onto the support and results in a
Gln residue after peptide cleavage. Elongation of the pept-
ide sequence was performed by sequential Fmoc removal
and coupling steps. Lys residues were incorporated with the
Nε-amino group protected as tert-butyl carbamate (Boc),
except for Lys2 and Lys5, which were coupled as Fmoc-
Lys(ivDde)-OH and Fmoc-Lys(pNZ)-OH, respectively. The
latter amino acid is not commercially available and was pre-
pared from Fmoc-Lys(Boc)-OH by acidolytic removal of
the Boc group followed by treatment with pNZ-N3.[11] Aci-
dolytic cleavage of an aliquot of the resulting linear peptidyl
resin 3 with trifluoroacetic acid (TFA)/H2O/triisopropyl-
silane (TIS) (95:2.5:2.5) for 2 h afforded the linear pept-
ide Fmoc-Lys-Lys(ivDde)-Leu-Lys-Lys(pNZ)-Phe-Lys-Lys-
Leu-Gln-OAll in 96% HPLC purity. Once the linear se-
quence was completed, the C-terminal allyl ester was
cleaved by treatment with Pd(PPh3)4 in CHCl3/AcOH/N-
methylmorpholine (NMM) (3:2:1) for 3 h. After Fmoc re-
moval, cyclization was carried out by using [ethyl cyano(hy-
Results and Discussion
Design of Cyclic Lipopeptidotriazoles
Cyclic lipopeptidotriazoles I were designed based on the
structure of the antimicrobial cyclic peptide c(Lys-Lys2-
Leu-Lys-Lys5-Phe-Lys-Lys-Leu-Gln) (BPC194)[4] by incor-
porating a 1,2,3-triazol-4-ylcarbonyl substituent at the side
chain of Lys2 and an hexanoyl group at the Nε-amino of
Lys5 (Figure 1). We chose to acylate Lys5 because this resi-
due is involved in the interaction of BPC194 with negatively
charged model membranes.[10] The acylation could increase
the antimicrobial activity by favoring peptide insertion into
the hydrophobic core of the membrane.[3e,3g] Among the
other Lys residues present in the sequence, Lys2 was se-
lected to be derivatized with the 1,2,3-triazol-4-ylcarbonyl
group because, on the one hand, it has been shown that this
residue is not essential for antibacterial activity[4] and, on
the other hand, it is located at a convenient position be-
tween Lys5 and the residue that will be anchored to the
solid support during the synthesis.
droxyimino)acetato-O2]
tri-1-pyrrolidinylphosphonium
hexafluorophosphate (PyOxim), Oxyma and N,N-diisopro-
pylethylamine (DIEA) in N-methyl-2-pyrrolidinone (NMP)
Synthetic Approach to Cyclic Lipopeptidotriazoles
We planned to study the synthesis of cyclic lipopeptido- for 24 h. An aliquot of the cyclic peptidyl resin 1 was sub-
triazoles I on solid-phase by following a Fmoc/tBu/All pro- jected to acydolytic cleavage, affording the cyclic pept-
tocol (Schemes 1 and 2).[5,9] This methodology involves an- ide c[Lys-Lys(ivDde)-Leu-Lys-Lys(pNZ)-Phe-Lys-Lys-Leu-
choring of Fmoc-Glu-OAll to the solid support, elongation Gln] in 84% HPLC purity.
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Solid-Phase Synthesis of Cyclic Lipopeptidotriazoles
Scheme 1. Synthesis of cyclic lipopeptidotriazole BPC730 by following strategies 1a and 1b.
With the cyclic peptidyl resin 1 in hand, we set out to ivDde) must be removed. After each key step, an aliquot of
examine the order of incorporation of the triazolyl and the resulting resin was treated with TFA/H2O/TIS
hexanoyl moieties at Lys2 and Lys5, respectively (Strategies (95:2.5:2.5) for 2 h and the crude mixture was analyzed by
1a and 1b, Scheme 1). Before the derivatization of each of HPLC and characterized by mass spectrometry. In strategy
these residues, the corresponding protecting group (pNZ or 1a we first evaluated the selective removal of the pNZ group
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Scheme 2. Synthesis of cyclic lipopeptidotriazole BPC730 by following strategies 2a and 2b.
in 1 by employing 6 m SnCl2 and 1.6 mm HCl/dioxane (4ϫ was then removed with NH2NH2·H2O/NMP (2:98, 6ϫ
30 min) followed by coupling of hexanoic acid (10 equiv.) 20 min) and the free amino group was acylated with propi-
mediated by DIPCDI (10 equiv.) and Oxyma (10 equiv.) in olic acid (5 equiv.) in the presence of DIPCDI (5 equiv.) and
NMP for 1 h (Scheme 1). Cleavage of the resulting resin Oxyma (5 equiv.) in NMP for 1 h. An aliquot of the re-
4 gave c[Lys-Lys(ivDde)-Leu-Lys-Lys(CO-C5H11)-Phe-Lys- sulting resin 5 was cleaved and the expected alkynyl lipo-
Lys-Leu-Gln] in 84% purity. The ivDde group of resin 4 peptide c[Lys-Lys(CO-CϵCH)-Leu-Lys-Lys(CO-C5H11)-
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Solid-Phase Synthesis of Cyclic Lipopeptidotriazoles
Phe-Lys-Lys-Leu-Gln] was obtained in 88% purity. The an aliquot of 5 afforded c[Lys-Lys(CO-CϵCH)-Leu-Lys-
alkynyl lipopeptidyl resin 5 was subjected to a cycloaddition Lys(CO-C5H11)-Phe-Lys-Lys-Leu-Gln] in 87% purity. Final
reaction with BnN3 (5 equiv.), CuI (5 equiv.), and ascorbic cycloaddition reaction of 5 with BnN3 and cleavage with
acid (5 equiv.) in piperidine/DMF (2:8) for 5 h. After acido- TFA rendered BPC730 in 80% purity. ESI-MS analysis
lytic cleavage, the cyclic lipopeptidotriazole c[Lys-Lys(CO- showed the presence of the expected peptide together with
Tr-Bn)-Leu-Lys-Lys(CO-C5H11)-Phe-Lys-Lys-Leu-Gln] traces of the product without the triazolyl ring.
(BPC730) was obtained in 86% purity (Tr stands for a tri-
azolyl moiety). ESI-MS analysis revealed only the presence olyl moiety by pNZ group removal, acylation with propiolic
of the peak corresponding to the expected peptide. acid, and cycloaddition with BnN3. The first two steps led
Strategy 2b involved derivatization of Lys2 with a triaz-
In strategy 1b, we evaluated the synthesis of BPC730 by to alkynyl peptidyl resin 10, which was cleaved to yield
first removing the ivDde group of Lys2 from resin 1 and c[Lys-Lys(CO-CϵCH)-Leu-Lys-Lys(ivDde)-Phe-Lys-Lys-
subsequently introducing the triazole ring at this position Leu-Gln] as shown by mass spectrometry. The CuI-cata-
(Scheme 1). With this aim, resin 1 was treated with lyzed cycloaddition with BnN3 afforded triazolyl resin 11,
NH2NH2·H2O/NMP (2:98) and then acylated with propi- which, after acidolytic cleavage, rendered c[Lys-Lys(CO-Tr-
olic acid, DIPCDI and Oxyma to afford peptidyl resin 6. Bn)-Leu-Lys-Lys(ivDde)-Phe-Lys-Lys-Leu-Gln] in 74% pu-
Cleavage of 6 gave c[Lys-Lys(CO-CϵCH)-Leu-Lys-Lys- rity. BPC730 was obtained in 73% purity from 11 through
(pNZ)-Phe-Lys-Lys-Leu-Gln] as characterized by ESI-MS. ivDde group removal, acylation with hexanoic acid, and ac-
The alkynyl resin 6 was treated with BnN3 under the condi- idolytic cleavage. ESI-MS analysis revealed the presence of
tions described above for resin 5. Analysis of the crude mix- BPC730 together with the product without the triazolyl
ture from cleavage of the resulting triazolyl resin 7 showed ring.
the presence of c[Lys-Lys(CO-Tr-Bn)-Leu-Lys-Lys(pNZ)-
Analysis of these results established that all strategies
Phe-Lys-Lys-Leu-Gln] in 84% purity. The next step was the were efficient for the solid-phase synthesis of cyclic lipopep-
removal of the pNZ group from resin 7 as described above tidotriazoles. However, strategy 1a turned out to be the
for 1, followed by acylation with hexanoic acid and acido- most satisfactory, leading to BPC730 with the highest pu-
lytic cleavage. BPC730 was obtained in 77% purity. ESI- rity. This strategy starts from cyclic peptidyl resin 1 incor-
MS analysis showed the presence of a byproduct that does porating the ivDde group at Lys2 and pNZ at Lys5. Regard-
not incorporate the hexanoyl chain, together with an un- ing the order of introduction of the hexanoyl and triazolyl
identified compound.
moieties, this strategy involves first the acylation of Lys5
with the fatty acid, followed by incorporation of propiolic
acid at Lys2 and subsequent 1,3-dipolar cycloaddition.
BPC730 was purified by reverse-phase column chromatog-
raphy (100% purity) and was characterized by H NMR
spectroscopy.
Synthesis of Cyclic Lipopeptidotriazoles from Peptidyl
Resin 2 (Strategies 2a and 2b)
1
Alternatively, the synthesis of the cyclic peptidotriazole
BPC730 was studied starting from cyclic peptidyl resin 2
(Strategies 2a and 2b, Scheme 2). This resin was prepared
by following the same protocol described for resin 1 but
incorporating Fmoc-Lys(pNZ)-OH and Fmoc-Lys(ivDde)-
OH at Lys2 and Lys5, respectively. The synthesis of 2 in-
cluded the preparation of linear peptidyl resin 8. Cleavage
of an aliquot of 8 afforded Fmoc-Lys-Lys(pNZ)-Leu-Lys-
Extension of the Methodology to the Synthesis of a Set of
Cyclic Lipopeptidotriazoles
The conditions developed in strategy 1a were applied to
Lys(ivDde)-Phe-Lys-Lys-Leu-Gln-OAll in 87% purity. Allyl the preparation of the cyclic lipopeptidotriazoles depicted
and Fmoc group removal of 8 followed by cyclization in Scheme 3. Thus, resin 1 was subjected to pNZ group re-
yielded peptidyl resin 2. An aliquot of this resin was cleaved moval, coupling of hexanoic acid, ivDde group removal,
and
c[Lys-Lys(pNZ)-Leu-Lys-Lys(ivDde)-Phe-Lys-Lys- and coupling of propiolic acid to afford alkynyl cyclic pept-
Leu-Gln] was obtained in 76% purity.
idyl resin 5. This alkynyl resin was treated with the corre-
As for resin 1, the order of incorporation of the triazolyl sponding azide p-Me-C6H4-N3, p-NH2-C6H4-N3, NaN3,
and hexanoyl moieties in 2 was also evaluated (Strategies PhN3, or p-MeO-C6H4-N3 (5 equiv.) in the presence of
2a and 2b, Scheme 2). Removal of protecting groups, acyla- ascorbic acid (5 equiv.) and CuI (5 equiv.) in piperidine/
tions and the cycloaddition step were performed under the DMF (2:8) while stirring overnight at room temperature.
same reaction conditions described for strategy 1. Thus, we Finally, cyclic lipopeptidotriazoles were individually cleaved
assayed strategy 2a, which first involved acylation of Lys5 from the resin with TFA/H2O/TIS (95:2.5:2.5), analyzed by
with a hexanoyl group through ivDde removal and hexa- HPLC, and characterized by ESI-MS and HRMS. BPC740,
noic acid coupling. When the resulting lipopeptidyl resin 9 BPC742, BPC744, BPC746, and BPC748 were obtained in
was cleaved, c[Lys-Lys(pNZ)-Leu-Lys-Lys(CO-C5H11)-Phe- excellent purities (80–99%). These cyclic lipopeptidotria-
1
Lys-Lys-Leu-Gln] was obtained in 88% purity. Alkynyl zoles were purified and characterized by H NMR spec-
peptidyl resin 5 was prepared from 9 by pNZ removal and troscopy. These results confirmed the suitability of strategy
subsequent propiolic acid coupling. Acidolytic cleavage of 1a for the synthesis of cyclic lipopeptidotriazoles.
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Scheme 3. Synthesis of cyclic lipopeptidotriazoles.
were purified and dried by passing them through an activated alu-
mina purification system (MBraun SPS-800) or by conventional
distillation techniques.
Conclusions
Herein, we have explored four strategies for the solid-
phase synthesis of cyclic decapeptides incorporating a tria-
zolyl moiety and an acyl group at the side chain of Lys2
and Lys5, respectively. These strategies involved the use of
five orthogonal protecting groups: Fmoc, tBu, All, pNZ,
and ivDde. The last two groups masked the Nε-amino
group of Lys2 and Lys5. Best results were obtained by pro-
tecting Lys2 with ivDde and Lys5 with pNZ, and by incor-
porating first the acyl group at Lys5 and then the triazolyl
ring at Lys2. This strategy was applied to the preparation
of a set of cyclic lipopeptidotriazoles that were obtained in
excellent purities. These results demonstrate that this strat-
All compounds were analyzed under standard analytical high-per-
formance liquid chromatography (HPLC) conditions with a Di-
onex liquid chromatography instrument. Analysis was carried out
with a Kromasil 100 C18 (40 mmϫ4.6 mm, 3.5 μm) column with a
2–100% B linear gradient over 7 min at a flow rate of 1 mL/min.
Solvent A was 0.1% aqueous trifluoroacetic acid (TFA), and sol-
vent B was 0.1% TFA in CH3CN. Detection was performed at
220 nm.
Reverse-phase column chromatography purification was performed
with a CombiFlash Rf200.
ESI-MS analyses were performed with an Esquire 6000 ESI ion
egy constitutes a general methodology that could be easily Trap LC/MS (Bruker Daltonics) instrument equipped with an elec-
trospray ion source (University of Girona). The instrument was
operated in the positive ESI(+) ion mode. Samples (5 μL) were in-
troduced into the mass spectrometer ion source directly through an
HPLC autosampler. The mobile phase (80:20 CH3CN/H2O at a
flow rate of 100 μL/min) was delivered by a 1100 Series HPLC
pump (Agilent). Nitrogen was employed as both the drying and
nebulizing gas. HRMS were recorded under conditions of ESI with
a Bruker MicroTof-Q IITM instrument using a hybrid quadrupole
time-of-flight mass spectrometer (University of Girona). Samples
were introduced into the mass spectrometer ion source by direct
extended to the preparation of libraries of such peptide de-
rivatives.
Experimental Section
General Methods: Manual peptide synthesis was performed in poly-
propylene syringes fitted with a polyethylene porous disk. Solvents
and soluble reagents were removed in vacuo. Commercially avail-
able reagents were used throughout without purification. Solvents
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Solid-Phase Synthesis of Cyclic Lipopeptidotriazoles
infusion through a syringe pump and were externally calibrated
using sodium formate. The instrument was operated in the positive
ESI(+) ion mode.
1H and 13C NMR spectra were measured with a Bruker 300 or a
Bruker 400 MHz NMR spectrometer. Chemical shifts were re-
ported as δ values (ppm) directly referenced to the solvent signal.
using standard Fmoc chemistry. Fmoc-Rink-MBHA resin
(0.4 mmol/g) was used as solid support. Fmoc-Leu-OH, Fmoc-Lys-
(Boc)-OH, Fmoc-Lys(ivDde)-OH, Fmoc-Lys(pNZ)-OH, Fmoc-
Phe-OH, and Fmoc-Glu-OAll were used as amino acid derivatives.
Couplings of the Fmoc-amino acids (4 equiv.) were mediated by
ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma) (4 equiv.), and
N,NЈ-diisopropylcarbodiimide (DIPCDI) (4 equiv.) in DMF at
room temperature for 1 h. The completion of the reactions was
checked by the Kaiser test.[13] Fmoc group removal was achieved
with piperidine/DMF (3:7, 2 + 10 min). After each coupling and
deprotection step, the resin was washed with DMF (6ϫ 1 min) and
CH2Cl2 (3ϫ 1 min), and air-dried. An aliquot of each resulting
peptidyl resin was treated with TFA/H2O/triisopropylsilane (TIS)
(95:2.5:2.5) for 2 h. Following TFA evaporation and diethyl ether
extraction, the crude peptides were dissolved in H2O, lyophilized,
analyzed by HPLC, and characterized by mass spectrometry.
Synthesis of Fmoc-Lys-OH: Fmoc-Lys(Boc)-OH (2.0 g, 4.40 mmol)
was dissolved in a solution of TFA/CH2Cl2 (1:1, 32 mL) and stirred
for 2 h at room temperature. Following TFA evaporation and di-
ethyl ether extraction, Fmoc-Lys-OH (1.54 g, 98% yield) was ob-
tained as a white powder. Rf = 0.5 (CHCl3/MeOH/AcOH, 5:3:1);
tR = 7.16 min. IR (neat): ν = 3066.47 (ϵCH st), 1670.64 (C=O st),
˜
1519.64 (C=C), 1449.63 (δ CH2), 1181.57 (δ C–H ip), 789.19 (γ
1
CH2), 738.60 (δ NH opp) cm–1. H NMR (400 MHz, [D6]DMSO):
δ = 1.34–1.43 [m, 2 H, CH2(γ)], 1.49–1.74 [m, 4 H, CH2(β),
CH2(δ)], 2.73–2.78 [m, 2 H, CH2(ε)], 3.90–3.96 [m, 1 H, CH(α)],
4.21–4.26 [m, 1 H, CH (Fmoc)], 4.28–4.34 [m, 2 H, CH2 (Fmoc)],
7.33 (td, J = 0.7 and 7.2 Hz, 2 H, 2CHAr), 7.42 (t, J = 7.2 Hz, 2
H, 2CHAr), 7.63 (d, J = 7.2 Hz, 1 H, CHAr), 7.71–7.74 (m, 1 H,
CHAr), 7.79 (br., 1 H, CONH), 7.89 (d, J = 7.2 Hz, 2 H,
2CHAr) ppm. 13C NMR (100 MHz, [D6]DMSO): δ = 22.57
[CH2(γ)], 26.51 [CH2(β)], 30.17 [CH2(δ)], 38.59 [CH2(ε)], 46.66 [CH
(Fmoc)], 53.61 [CH(α)], 65.58 [CH2 (Fmoc)], 120.13 (CHAr), 120.14
(CHAr), 125.26 (2CHAr), 127.07 (CHAr), 127.08 (CHAr), 127.65
(2CHAr), 140.73 (CAr), 140.76 (CAr), 143.79 (CAr), 143.81 (CAr),
156.20 (CONH), 173.85 (COOH) ppm. MS (ESI): m/z = 369.1 [M
+ H]+.
Fmoc-Lys(Boc)-Lys(ivDde)-Leu-Lys(Boc)-Lys(pNZ)-Phe-Lys(Boc)-
Lys(Boc)-Leu-Glu(Rink-MBHA)-OAll (3): This peptidyl resin was
prepared from Fmoc-Rink-MBHA resin (100 mg) by following the
procedure described above. Acidolytic cleavage of an aliquot of this
peptidyl resin afforded Fmoc-Lys-Lys(ivDde)-Leu-Lys-Lys(pNZ)-
Phe-Lys-Lys-Leu-Gln-OAll in 96 % purity. tR = 7.64 min. MS
(ESI): m/z = 969.1 [M + 2H]2+, 1937.2 [M + H]+, 1959.2 [M +
Na]+. HRMS (ESI): m/z calcd. for C101H154N18O20 [M + 4H]4+
484.7891; found 484.7878; calcd. for C101H153N18O20 [M + 3H]3+
646.0497; found 646.0488; calcd. for C101H152N18O20 [M + 2H]2+
968.5710; found 968.5712.
Synthesis of Fmoc-Lys(pNZ)-OH:[11] p-Nitrobenzyl chloroformate
(301 mg, 1.35 mmol) was dissolved in 1,4-dioxane (590 μL) and a
solution of NaN3 (106 mg, 1.62 mmol) in H2O (420 μL) was added.
The resulting emulsion was stirred for 2 h and the formation of p-
nitrobenzyl azidoformate was monitored by thin-layer chromatog-
raphy (TLC) (CH2Cl2). A solution of Fmoc-Lys-OH (500 mg,
1.40 mmol) in 1,4-dioxane/2% aqueous Na2CO3 (1:1, 1.69 mL) was
then added dropwise and the resulting mixture was stirred for 24 h.
The pH was maintained at 8–9 by adding 10% aqueous Na2CO3.
The reaction was monitored by TLC (EtOAc/MeOH/NH3, 5:2:1).
Once the reaction was finished, H2O (40 mL) was added and the
resulting suspension was washed with tert-butyl methyl ether (3ϫ
20 mL). The aqueous phase was acidified to pH 2–3 with 3 n HCl
and was extracted with EtOAc (3ϫ 20 mL). The organic fractions
were combined, washed with an aqueous HCl solution (pH 2) (2ϫ
40 mL) and dried with anhydrous MgSO4. Evaporation of the or-
ganic solvent under reduced pressure gave Fmoc-Lys(pNZ)-OH
(309 mg, 42%) as a yellow oil, which solidified to a white solid
upon addition of diethyl ether. Rf = 0.5 (AcOH/MeOH/NH3,
Fmoc-Lys(Boc)-Lys(pNZ)-Leu-Lys(Boc)-Lys(ivDde)-Phe-Lys(Boc)-
Lys(Boc)-Leu-Glu(Rink-MBHA)-OAll (8): This peptidyl resin was
prepared from Fmoc-Rink-MBHA resin (100 mg) by following the
procedure described above. Acidolytic cleavage of an aliquot of this
peptidyl resin afforded Fmoc-Lys-Lys(pNZ)-Leu-Lys-Lys(ivDde)-
Phe-Lys-Lys-Leu-Gln-OAll in 87 % purity. tR = 7.56 min. MS
(ESI): m/z = 969.1 [M + 2H]2+, 1937.2 [M + H]+, 1959.2 [M +
Na]+. HRMS (ESI): m/z calcd. for C101H154N18O20 [M + 4H]4+
484.7891; found 484.7870; calcd. for C101H153N18O20 [M + 3H]3+
646.0497; found 646.0475; calcd. for C101H152N18O20 [M + 2H]2+
968.5710; found 968.5691.
Synthesis of the Cyclic Peptidyl Resins 1 and 2: Prepared from the
linear peptidyl resins 3 and 8, respectively. The C-terminal allyl
ester of 3 or 8 was cleaved by treatment with Pd(PPh3)4 (5 equiv.) in
CHCl3/AcOH/N-methylmorpholine (NMM) (3:2:1) under nitrogen
for 3 h, and the resins were washed with tetrahydrofuran (THF)
(3ϫ 2 min), N-methyl-2-pyrrolidinone (NMP) (3ϫ 2 min), N,N-
diisopropylethylamine (DIEA)/CH2Cl2 (1:19, 3ϫ 2 min), sodium
N,N-diethyldithiocarbamate (0.03 m in NMP, 3ϫ 15 min), NMP
(10ϫ 1 min) and CH2Cl2 (3ϫ 2 min). Fmoc was removed with
piperidine/DMF (3:7, 2 + 10 min) followed by washes with DMF
(6ϫ 1 min) and CH2Cl2 (3ϫ 1 min). Cyclization was carried out by
treating the resulting resin with [ethyl cyano(hydroxyimino)acetato-
O2] tri-1-pyrrolidinylphosphonium hexafluorophosphate (PyOxim)
(5 equiv.), Oxyma (5 equiv.), and DIEA (10 equiv.) in NMP whilst
stirring for 24 h. Following washes with NMP (6ϫ 1 min) and
CH2Cl2 (3ϫ 1 min), an aliquot of each cyclic peptidyl resin was
cleaved by treatment with TFA/H2O/TIS (95:2.5:2.5) for 2 h. Fol-
lowing TFA evaporation and diethyl ether extraction, the crude
peptides were dissolved in H2O, lyophilized, analyzed by HPLC
and characterized by mass spectrometry.
1
5:2:1); tR = 8.48 min. H NMR (300 MHz, [D6]DMSO): δ = 1.29–
1.40 [m, 4 H, CH2(δ), CH2(γ)], 1.56–1.71 [m, 2 H, CH2(β)], 2.99
[q, J = 6.3 Hz, 2 H, CH2(ε)], 3.87–3.94 [m, 1 H, CH(α)], 4.19–4.29
[m, 3 H, CH2 (Fmoc), CH (Fmoc)], 5.15 (s, 2 H, OCH2), 7.31 (t,
J = 7.5 Hz, 2 H, 2CHAr), 7.39–7.44 (m, 3 H, NH, 2CHAr), 7.59 (d,
J = 9.0 Hz, 2 H, 2CHAr), 7.63 (d, J = 7.8 Hz, 1 H, NH), 7.72 (d,
J = 7.5 Hz, 2 H, 2CHAr), 7.89 (d, J = 7.2 Hz, 2 H, 2CHAr), 8.23
(d, J = 9.0 Hz, 2 H, 2CHAr), 12.55 (br. s, 1 H, COOH) ppm. 13C
NMR (100 MHz, [D6]DMSO): δ = 23.90 [CH2(γ)], 29.87, 31.34
[CH2(β), CH2(δ)], 41.90 [CH2(ε)], 47.60 (CH-Fmoc), 54.73
[CH(α)], 64.90 (OCH2), 66.55 (OCH2), 121.10 (2CHAr), 124.10
(2CHAr), 126.25 (2CHAr), 128.04 (2CHAr), 128.61 (2CHAr), 129.04
(2CHAr), 141.67 (CAr), 141.69 (CAr), 144.77 (CAr), 144.80 (CAr),
146.31 (CAr), 147.84 (CAr), 156.73 (NHCOO), 157.15 (NHCOO),
174.99 (COOH) ppm. MS (ESI): m/z = 548.1 [M + H]+.
c[Lys(Boc)-Lys(ivDde)-Leu-Lys(Boc)-Lys(pNZ)-Phe-Lys(Boc)-Lys-
(Boc)-Leu-Glu(Rink-MBHA)] (1): Prepared from the linear pept-
idyl resin 3 (300 mg) by following the procedure described above.
Acidolytic cleavage of an aliquot of the resulting cyclic peptidyl
Synthesis of the Linear Peptidyl Resins 3 and 8: The linear peptidyl
resins were synthesized manually by the solid-phase method by
Eur. J. Org. Chem. 2014, 4785–4794
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
4791

M. Planas, L. Feliu et al.
FULL PAPER
resin 1 afforded c[Lys-Lys(ivDde)-Leu-Lys-Lys(pNZ)-Phe-Lys-
Lys-Leu-Gln] in 84% purity. tR = 7.65 min. MS (ESI): m/z = 828.5
piperidine/DMF (2:8). The reaction mixture was stirred overnight
at room temperature. The resin was washed with sodium N,N-di-
[M + 2H]2+, 1657.1 [M + H]+, 1678.1 [M + Na]+. HRMS (ESI): ethyldithiocarbamate (0.03 m in NMP, 3 ϫ 3 min), DMF (6 ϫ
m/z calcd. for C83H138N18O17 [M + 4H]4+ 414.7616; found
1 min) and CH2Cl2 (20 min). The resulting cyclic lipopeptidotri-
414.7587; calcd. for C83H137N18O17 [M + 3H]3+ 552.6798; found azole was cleaved from the resin with TFA/H2O/TIS (95:2.5:2.5)
552.6779; calcd. for C83H136N18O17 [M + 2H]2+ 828.5160; found for 2 h, and analyzed by HPLC and mass spectrometry. The crude
828.5137.
product was then purified by reverse-phase liquid chromatography
and the cyclic lipopeptidotriazole was analyzed by HPLC and char-
acterized by MS (ESI), HRMS (ESI) and H NMR spectroscopy.
c[Lys(Boc)-Lys(pNZ)-Leu-Lys(Boc)-Lys(ivDde)-Phe-Lys(Boc)-Lys-
(Boc)-Leu-Glu(Rink-MBHA)] (2): Prepared from the linear pept-
idyl resin 8 (300 mg) by following the procedure described above.
Acidolytic cleavage of an aliquot of the resulting cyclic peptidyl
resin 2 afforded c[Lys-Lys(pNZ)-Leu-Lys-Lys(ivDde)-Phe-Lys-
Lys-Leu-Gln] in 76% purity. tR = 7.58 min. MS (ESI): m/z = 829.0
[M + 2H]2+, 1656.9 [M + H]+, 1677.9 [M + Na]+. HRMS (ESI):
m/z calcd. for C83H138N18O17 [M + 4H]4+ 414.7616; found
414.7627; calcd. for C83H137N18O17 [M + 3H]3+ 552.6798; found
552.6811; calcd. for C83H136N18O17 [M + 2H]2+ 828.5160; found
828.5191.
1
Cyclic Lipopeptidotriazole c[Lys-Lys(CO-Tr-Bn)-Leu-Lys-Lys(CO-
C5H11)-Phe-Lys-Lys-Leu-Gln] (BPC730): Alkynyl resin 5 was
treated with BnN3 by following the general procedure described
above. c[Lys-Lys(CO-Tr-Bn)-Leu-Lys-Lys(CO-C5H11)-Phe-Lys-
Lys-Leu-Gln] (BPC730) was obtained in 86% purity before purifi-
cation. Reverse-phase column chromatography provided BPC730
in 100% purity (36% yield). tR = 7.81 min. 1H NMR (400 MHz,
CD3OD): δ = 0.86–0.93 [m, 15 H, 4 CH3(δ)-Leu, CH3-Hex], 1.57–
2.03 [m, 54 H, 6 CH2(β)-Lys, 6 CH2(γ)-Lys, 6 CH2(δ)-Lys, CH2(β)-
Gln, CH2(γ)-Gln, 2 CH2(β)-Leu, 2 CH(γ)-Leu, 4 CH2-Hex], 2.93
[m, 8 H, 4 CH2(ε)-Lys], 3.07–3.13 [m, 4 H, 2 CH2(ε)-Lys], 3.30–
3.50 [m, 2 H, CH2(β)-Phe], 4.03–4.24 [m, 10 H, 10 CH(α)], 5.61–
5.65 (m, 2 H, CH2-Bn), 7.22–7.25 (m, 5 H, Ph), 7.34–7.37 (m, 5 H,
Ph) ppm. MS (ESI): m/z = 777.5 [M + 2H]2+, 1553.9 [M + H]+,
1575.9 [M + Na]+. HRMS (ESI): m/z calcd. for C78H131N20O13 [M
+ 3H]3+ 518.6729; found 518.6724; calcd. for C78H130N20O13 [M +
2H]2+ 777.5058; found 777.5068.
Synthesis of c[Lys(Boc)-Lys(ivDde)-Leu-Lys(Boc)-Lys(CO-C5H11)-
Phe-Lys(Boc)-Lys(Boc)-Leu-Glu(Rink-MBHA)] (4): Cyclic peptidyl
resin 1 (250 mg) was swollen with CH2Cl2 (20 min), and DMF
(20 min), and the pNZ group was removed with 6 m SnCl2 in DMF
containing 1.6 mm HCl/dioxane (4ϫ 30 min). The resin was sub-
sequently washed with DMF (3ϫ 30 seg), H2O/DMF (1:1, 3ϫ 30
seg), H2O/THF (1:1, 3ϫ 30 seg), DMF (3ϫ 30 seg), and CH2Cl2
(3 ϫ 30 seg), and then treated with hexanoic acid (10 equiv.),
DIPCDI (10 equiv.) and Oxyma (10 equiv.) in NMP for 1 h. Com-
pletion of the reaction was checked with the Kaiser test.[13] Follow-
ing washes with NMP (6ϫ 1 min) and CH2Cl2 (6ϫ 1 min), an
aliquot of the resulting resin 4 was cleaved by treatment with TFA/
H2O/TIS (95:2.5:2.5) for 2 h. After TFA evaporation and diethyl
ether extraction, the crude peptide was dissolved in H2O and lyo-
philized. c[Lys-Lys(ivDde)-Leu-Lys-Lys(CO-C5H11)-Phe-Lys-Lys-
Leu-Gln] was obtained in 84% purity. tR = 7.46 min. MS (ESI):
m/z = 788.0 [M + 2H]2+, 1575.1 [M + H]+, 1597.1 [M + Na]+.
HRMS (ESI): m/z calcd. for C81H143N17O14 [M + 4H]4+ 394.5245;
found 394.5243; calcd. for C81H142N17O14 [M + 3H]3+ 525.6969;
found 525.6972; calcd. for C81H141N17O14 [M + 2H]2+ 788.0416;
found 788.0419.
Cyclic Lipopeptidotriazole c[Lys-Lys(CO-Tr-C6H4-Me)-Leu-Lys-
Lys(CO-C5H11)-Phe-Lys-Lys-Leu-Gln] (BPC740): Alkynyl resin 5
was treated with p-Me-C6H4-N3 by following the general procedure
described above. c[Lys-Lys(CO-Tr-C6H4-Me)-Leu-Lys-Lys(CO-
C5H11)-Phe-Lys-Lys-Leu-Gln] (BPC740) was obtained in 99% pu-
1
rity (34% yield). tR = 7.37 min. H NMR (400 MHz, CD3OD): δ
= 0.87–0.94 [m, 15 H, 4 CH3(δ)-Leu, CH3-Hex], 1.70–2.07 [m, 54
H, 6 CH2(β)-Lys, 6 CH2(γ)-Lys, 6 CH2(δ)-Lys, CH2(β)-Gln,
CH2(γ)-Gln, 2 CH2(β)-Leu, 2 CH(γ)-Leu, 4 CH2-Hex], 2.43 (s, 3
H, Me), 2.93 [m, 8 H, 4 CH2(ε)-Lys], 3.06–3.14 [m, 4 H, 2 CH2(ε)-
Lys], 3.46–3.56 [m, 2 H, CH2(β)-Phe], 4.06–4.30 [m, 10 H, 10
CH(α)], 7.20–7.28 (m, 5 H, Ph), 7.42 (d, J = 8.2 Hz, 2 H, C6H4),
7.73 (d, J = 8.2 Hz, 2 H, C6H4) ppm. MS (ESI): m/z = 777.6 [M +
2H]2+, 788.5 [M + H + Na]2+, 1554.0 [M + H]+, 1577.0 [M +
Na]+. HRMS (ESI): m/z calcd. for C78H132N20O13 [M + 4H]4+
389.2565; found 389.2566; calcd. for C78H131N20O13 [M + 3H]3+
518.6729; found 518.6733; calcd. for C78H130N20O13 [M + 2H]2+
777.5058; found 777.5063.
Synthesis of c[Lys(Boc)-Lys(CO-CϵCH)-Leu-Lys(Boc)-Lys(CO-
C5H11)-Phe-Lys(Boc)-Lys(Boc)-Leu-Glu(Rink-MBHA)] (5): Cyclic
peptidyl resin 4 (250 mg) was swollen with CH2Cl2 (20 min) and
DMF (20 min), and the ivDde group was removed with
NH2NH2·H2O/NMP (2:98, 6ϫ 20 min) whilst stirring. The resin
was subsequently washed with NMP (2 ϫ 1 min), CH2Cl2 (2ϫ
1 min), MeOH (2ϫ 1 min), and NMP (2ϫ 1 min), and then treated
with propiolic acid (5 equiv.), DIPCDI (5 equiv.) and Oxyma
(5 equiv.) in NMP for 1 h. Completion of the reaction was checked
with the Kaiser test.[13] Following washes with NMP (6ϫ 1 min)
and CH2Cl2 (6ϫ 1 min), an aliquot of the resulting resin 5 was
cleaved by treatment with TFA/H2O/TIS (95:2.5:2.5) for 2 h. After
TFA evaporation and diethyl ether extraction, the crude peptide
was dissolved in H2O and lyophilized. c[Lys-Lys(CO-CϵCH)-Leu-
Lys-Lys(CO-C5H11)-Phe-Lys-Lys-Leu-Gln] was obtained in 88%
p u r i t y. t R = 7 . 0 6 m i n . M S ( E S I ) : m / z = 7 1 0 . 9 [ M +
2H]2+, 1420.9 [M + H]+, 1442.9 [M + Na]+. HRMS (ESI): m/z
calcd. for C71H124N17O13 [M + 3H]3+ 474.3183; found 474.3192;
calcd. for C71H123N17O13 [M + 2H]2+ 710.9738; found 710.9739.
Cyclic Lipopeptidotriazole c[Lys-Lys(CO-Tr-C6H4-NH2)-Leu-Lys-
Lys(CO-C5H11)-Phe-Lys-Lys-Leu-Gln] (BPC742): Alkynyl resin 5
was treated with p-NH2-C6H4-N3 by following the general pro-
cedure described above. c[Lys-Lys(CO-Tr-C6H4-NH2)-Leu-Lys-
Lys(CO-C5H11)-Phe-Lys-Lys-Leu-Gln] (BPC742) was obtained in
99 % purity (37 % yield). tR = 7.00 min. 1H NMR (400 MHz,
CD3OD): δ = 0.94 [m, 15 H, 4 CH3(δ)-Leu, CH3-Hex], 1.47–2.03
[m, 54 H, 6 CH2(β)-Lys, 6 CH2(γ)-Lys, 6 CH2(δ)-Lys, CH2(β)-Gln,
CH2(γ)-Gln, 2 CH2(β)-Leu, 2 CH(γ)-Leu, 4 CH2-Hex], 2.94 [m, 8
H, 4 CH2(ε)-Lys], 3.05–3.13 [m, 4 H, 2 CH2(ε)-Lys], 3.47–3.56 [m,
2 H, CH2(β)-Phe], 4.05–4.24 [m, 10 H, 10 CH(α)], 6.89–6.91 (m, 2
H, C6H4), 7.22–7.26 (m, 5 H, Ph), 7.50–7.54 (m, 2 H, C6H4) ppm.
MS (ESI): m/z = 778.1 [M + 2H]2+, 789.0 [M + H + Na]2+, 1555.2
[M + H]+, 1577.1 [M + Na]+. HRMS (ESI): m/z calcd. for
C77H131N21O13 [M + 4H]4+ 389.5053; found 389.5059; calcd. for
C77H130N21O13 [M + 3H]3+ 519.0047; found 519.0054; calcd. for
C77H129N21O13 [M + 2H]2+ 778.0034; found 778.0036.
Synthesis of the Cyclic Lipopeptidotriazoles: The alkynyl cyclic
peptidyl resin 5 was swollen with CH2Cl2 (20 min) and DMF
(20 min), and then treated with the corresponding azide (5 equiv.)
in the presence of ascorbic acid (5 equiv.) and CuI (5 equiv.) in
Cyclic Lipopeptidotriazole c[Lys-Lys(CO-Tr)-Leu-Lys-Lys(CO-
C5H11)-Phe-Lys-Lys-Leu-Gln] (BPC744): Alkynyl resin 5 was
4792
www.eurjoc.org
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 4785–4794

Solid-Phase Synthesis of Cyclic Lipopeptidotriazoles
treated with NaN3 by following the general procedure described
above. c[Lys-Lys(CO-Tr)-Leu-Lys-Lys(CO-C5H11)-Phe-Lys-Lys-
Leu-Gln] (BPC744) was obtained in 86% purity before purifica-
tion. Reverse-phase column chromatography provided BPC744 in
100 % purity (35 % yield). tR = 7.64 min. 1H NMR (400 MHz,
CD3OD): δ = 0.88–0.91 [m, 15 H, 4 CH3(δ)-Leu, CH3-Hex], 1.37–
2.17 [m, 54 H, 6 CH2(β)-Lys, 6 CH2(γ)-Lys, 6 CH2(δ)-Lys, CH2(β)-
Gln, CH2(γ)-Gln, 2 CH2(β)-Leu, 2 CH(γ)-Leu, 4 CH2-Hex], 2.94
[m, 8 H, 4 CH2(ε)-Lys], 3.12–3.14 [m, 4 H, 2 CH2(ε)-Lys], 3.32–
3.49 [m, 2 H, CH2(β)-Phe], 3.84–4.25 [m, 10 H, 10 CH(α)], 7.23–
7.25 (m, 5 H, Ph) ppm. MS (ESI): m/z = 732.7 [M + 2H]2+, 1464.4
[M + H]+, 1486.4 [M + Na]+. HRMS (ESI): m/z calcd. for
C71H126N20O13 [M + 4H]4+ 366.7448; found 366.7455; calcd. for
C71H125N20O13 [M + 3H]3+ 488.6573; found 488.6579; calcd. for
C71H124N20O13 [M + 2H]2+ 732.4823; found 732.4820.
Ministerio de Economía y Competitividad (MINECO) (AGL2009-
13255-C02-02/AGR and AGL2012-39880-C02-02). The authors
also acknowledge the Serveis Tècnics de Recerca of the University
of Girona for the NMR and mass spectrometry analysis.
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Cyclic Lipopeptidotriazole c[Lys-Lys(CO-Tr-Ph)-Leu-Lys-Lys(CO-
C5H11)-Phe-Lys-Lys-Leu-Gln] (BPC746): Alkynyl resin 5 was
treated with PhN3 by following the general procedure described
above. c[Lys-Lys(CO-Tr-Ph)-Leu-Lys-Lys(CO-C5H11)-Phe-Lys-
Lys-Leu-Gln] (BPC746) was obtained in 88% purity before purifi-
cation. Reverse-phase column chromatography provided BPC746
in 100% purity (39% yield). tR = 7.82 min. 1H NMR (400 MHz,
CD3OD): δ = 0.88–0.97 [m, 15 H, 4 CH3(δ)-Leu, CH3-Hex], 1.29–
1.99 [m, 54 H, 6 CH2(β)-Lys, 6 CH2(γ)-Lys, 6 CH2(δ)-Lys, CH2(β)-
Gln, CH2(γ)-Gln, 2 CH2(β)-Leu, 2 CH(γ)-Leu, 4 CH2-Hex], 2.94
[m, 8 H, 4 CH2(ε)-Lys], 3.12–3.14 [m, 4 H, 2 CH2(ε)-Lys], 3.41–
3.49 [m, 2 H, CH2(β)-Phe], 3.93–4.24 [m, 10 H, 10 CH(α)], 7.23–
7.25 (m, 5 H, Ph), 7.55–7.60 (m, 3 H, Ph), 7.84–7.90 (m, 2 H,
Ph) ppm. MS (ESI): m/z = 1541.0 [M + H]+, 1561.9 [M + Na]+.
HRMS (ESI): m/z calcd. for C77H130N20O13 [M + 4H]4+ 385.7526;
found 385.7541; calcd. for C77H129N20O13 [M + 3H]3+ 514.0011;
found 514.0036; calcd. for C77H128N20O13 [M + 2H]2+ 770.4979;
found 770.5005.
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S. Vilà, E. Badosa, E. Montesinos, L. Feliu, M. Planas, Org.
Biomol. Chem. 2013, 11, 3365–3374.
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Cyclic Lipopeptidotriazole c[Lys-Lys(CO-Tr-C6H4-OMe)-Leu-Lys-
Lys(CO-C5H11)-Phe-Lys-Lys-Leu-Gln] (BPC748): Alkynyl resin 5
was treated with p-MeO-C6H4-N3 by following the general pro-
cedure described above. c[Lys-Lys(CO-Tr-C6H4-OMe)-Leu-Lys-
Lys(CO-C5H11)-Phe-Lys-Lys-Leu-Gln] (BPC748) was obtained in
80% purity before purification. Reverse-phase column chromatog-
raphy provided BPC748 in 93% purity (42% yield). tR = 6.16 min.
1H NMR (400 MHz, CD3OD): δ = 0.89–0.90 [m, 15 H, 4 CH3(δ)-
Leu, CH3-Hex], 1.48–2.03 [m, 54 H, 6 CH2(β)-Lys, 6 CH2(γ)-Lys,
6 CH2(δ)-Lys, CH2(β)-Gln, CH2(γ)-Gln, 2 CH2(β)-Leu, 2 CH(γ)-
Leu, 4 CH2-Hex], 2.94 [m, 8 H, 4 CH2(ε)-Lys], 3.12–3.15 [m, 4 H,
2 CH2(ε)-Lys], 3.39–3.48 [m, 2 H, CH2(β)-Phe], 3.87–4.24 [m, 13
H, 10 CH(α), OCH3], 7.10–7.12 (m, 2 H, C6H4), 7.23–7.25 (m, 5
H, Ph), 7.72–7.74 (m, 2 H, C6H4) ppm. MS (ESI): m/z = 786.3 [M
+ 2H]2+, 1571.0 [M + H]+, 1592.5 [M + Na]+. HRMS (ESI): m/z
calcd. for C78H132N20O14 [M + 4H]4+ 393.2552; found 393.2560;
calcd. for C78H131N20O14 [M + 3H]3+ 524.0046; found 524.0050;
calcd. for C78H130N20O14 [M + 2H]2+ 785.5032; found 785.5030.
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1
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Acknowledgments
S. V. is a recipient of a predoctoral fellowship from the Generalitat
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Received: February 20, 2014
Published Online: June 24, 2014
4794
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© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 4785–4794