Bisphosphine-functionalized cyclic decapeptides based on the natural product gramicidin s: A potential scaffold for transition-metal coordination

Article abstract of DOI:10.1002/chem.200901127

The natural product Gramicidin S is a promising scaffold for novel oligopeptide-based bisphosphine ligands, combining the advantageous rigid chiral backbone with the close proximity of phosphine substituents. The required unnatural, phosphine-containing,

Full text of DOI:10.1002/chem.200901127

DOI: 10.1002/chem.200901127
Bisphosphine-Functionalized Cyclic Decapeptides Based on the
Natural Product Gramicidin S: A Potential Scaffold for
Transition-Metal Coordination
AHCTUNGTRENNUNG
Sebastian Burck,[a, f] Sander G. A. van Assema,[a, f] Bas Lastdrager,[b, f] J. Chris Slootweg,[a]
Andreas W. Ehlers,[a] Josꢀ M. Otero,[c] Bruno Dacunha-Marinho,[d]
Antonio L. Llamas-Saiz,[d] Mark Overhand,*[b] Mark J. van Raaij,*[c, e] and
Koop Lammertsma*[a]
8134
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Chem. Eur. J. 2009, 15, 8134 – 8145

FULL PAPER
Abstract: The natural product Gramici-
din S is a promising scaffold for novel
oligopeptide-based bisphosphine li-
gands, combining the advantageous
rigid chiral backbone with the close
proximity of phosphine substituents.
The required unnatural, phosphine-
containing, amino acid building blocks
were synthesized by means of a novel
protocol that involves the enantioselec-
tive alkylation of a chiral nickel Schiff
base template. Three Ni complexes
were prepared with different alkyl
chains between the phosphine group
and the a-carbon atom of the incorpo-
rated glycine; the absolute stereochem-
istry of two of them was determined by
single-crystal X-ray structure analysis.
By detaching the template, enantiopure
l-phosphine amino acids resulted ena-
bling the solid-phase, stepwise con-
struction of a linear sequence of the
phosphine-modified oligopeptides. On
cyclization three bisphosphine-substi-
tuted Gramicidin S analogues resulted,
differing only in the size and shape of
the linkage between the phosphine
groups and the oligopeptides backbone.
Their crystal structures suggest these
species to have potential as chelating
ligands.
Keywords: amino acids · peptides ·
phosphanes · solid-phase synthesis ·
X-ray diffraction
Introduction
functional groups in the secondary structure of the peptide
backbone. Rhodium complexes of such a-helices and palla-
dium complexes of b-turn motifs (Figure 1, left), even em-
Designer-modified oligopeptides (peptide engineering) pro-
vide access to new molecules with unique functional proper-
ties, while retaining the structural characteristics of the
parent system.[1] Exemplary is their use in asymmetric catal-
ysis both as organocatalysts[2] and as ligands for transition-
metal complexes.[3,4] In their pioneering work, Gilbertson
and co-workers showed phosphine-derivatized oligopeptides
to function as transition-metal ligands in asymmetric catalyt-
ic reactions, such as hydrogenation and allylic substitution.[4]
Crucial to the design of oligopeptide catalysts for bidentate
metal chelation is the spatial orientation of the two phos-
phine moieties, necessitating proper placements of these
Figure 1. Gilbertsonꢁs peptide-based ligand with b-turn motif (left) and
the bisphosphine-functionalized Gramicidin S analogue (right).
[a] Dr. S. Burck, Dr. S. G. A. v. Assema, Dr. J. C. Slootweg,
Dr. A. W. Ehlers, Prof. Dr. K. Lammertsma
Department of Chemistry and Pharmaceutical Sciences
Vrije Universiteit Amsterdam, De Boelelaan 1083
1081 HV Amsterdam (The Netherlands)
Fax : (+31)20-5987488
bedded on solid support, reportedly display varying degrees
of catalytic activity for different turn motifs and sequences.[4]
Intrigued by the demonstrated catalytic prospects we recog-
nized the void in structural information of the polypeptides
and the tunability of the oligopeptide ligands.
E-mail: K.Lammertsma@few.vu.nl
[b] Dr. B. Lastdrager, Dr. M. Overhand
Leiden Institute of Chemistry, Gorlaeus Laboratories
Einsteinweg 55, 2300 RA Leiden (The Netherlands)
Fax : (+31)71-5274307
In search of a system that would enforce close proximity
with some flexibility of the ligating phosphine groups, we
decided to construct a rigid cyclic oligopeptide. As a rigid
peptide scaffold, we selected the structurally well-studied
antimicrobial peptide Gramicidin S (GS, cyclo(Val-Orn-Leu-
d-Phe-Pro)2), which is a C2-symmetric cyclic decapeptide
that adopts a rigid cyclic b-hairpin structure.[5,6] The GS
structure is stabilized by four intramolecular hydrogen-
bonding interactions at the opposing Val and Leu residues
and the two 2-residue turns of the d-Phe-Pro sequences. The
two Orn residues, as part of the short b-strand sequences
(Val-Orn-Leu), have their side chains in close spatial prox-
imity and are therefore ideally suited to be replaced by
E-mail: overhand@chem.leidenuniv.nl
[c] Dr. J. M. Otero, Dr. M. J. v. Raaij
Department of Biochemistry and Molecular Biology
Faculty of Pharmacy, University of Santiago de Compostela
15782 Santiago de Compostela (Spain)
Fax : (+34)981-599157
E-mail: mark.vanraaij@usc.es
[d] Dr. B. Dacunha-Marinho, Dr. A. L. Llamas-Saiz
X-ray Diffraction Unit, RIAIDT, CACTUS building
University of Santiago de Compostela
15782 Santiago de Compostela (Spain)
[e] Dr. M. J. v. Raaij
Department of Structural Biology
Institute of Molecular Biology of Barcelona—CSIC
PCB, C/Baldiri Reixac 10-12, 08028 Barcelona (Spain)
phosphine amino acids. Such
a designer oligopeptide
(Figure 1, right) necessitates the phosphine substituents to
have minimal effect on the steric properties of the amino
acids to avoid disturbing the peptide backbone through
[f] Dr. S. Burck, Dr. S. G. A. v. Assema, Dr. B. Lastdrager
Contributed equally to the work.
Chem. Eur. J. 2009, 15, 8134 – 8145
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K. Lammertsma, M. J. van Raaij, M. Overhand et al.
ACHTUNGTRENNUNGaltered dihedral and torsional angles and hydrogen-bonding
interactions.
The synthesis of the designer cyclic decapeptides requires
the preparation of amino acid building blocks with different
lengths and rigidities of the phosphine-containing side chain.
Whereas there is a great variety in tailor-made amino acids
with which to study and modify proteins and polypeptides,
those with non-natural substituents remain rare. This is par-
ticularly true for phosphine-containing amino acids, in part
due to the sensitivity of the phosphine groups toward oxida-
tion, which would hamper its chelation to transition metals.
In this paper we report on the synthesis of three phosphine
amino acids with different substituents using a template ap-
proach for two of them. The appropriateness of a chiral NiII
Schiff base template is examined as they are used increas-
ingly in the synthetic design of tailor-made amino acids. We
further address the incorporation of the three derivatized
chiral amino acids into the backbone of the cyclic decapep-
tide GS and examine the geometrical features of the
Scheme 1. Enantioselective synthesis of 1 based on the Evansꢁ (top) and
Knochelꢁs (bottom) protocol.
volves chromatographic separation of diastereomeric azides,
while the less elaborate route based on Knochelꢁs protocol
for the preparation of zinc cuprates[9] starts with a commer-
cially available, chiral iodo amino acid. Neither route is
readily extended to phosphine-derivatized amino acids with
linkages different from methylene.
A
For 2 only the synthesis of a racemic mixture of the phos-
phine oxide functionalized free amino acid has been report-
ed.[10] This route involves the reaction of bromoethylphos-
phine oxide with diethylacetamidomalonate and subsequent
acid hydrolysis of the resulting alkylation product
(Scheme 2).
Synthesis of amino acids: To evaluate phosphine-functional-
ized amino acids for incorporation into Gramicidin S we
considered 1, 2, and 3 with their respective methylene, ethyl-
ene, and benzylene linkages, and Belekonꢁs template 4 (see
below). The three derivatized amino acids must be obtained
Scheme 2. Synthesis of racemic phosphine oxide analogue of 2.
The synthesis of a tyrosine derivative related to 3, con-
taining Ac and CO2Me instead of Fmoc and CO2H groups,
respectively, and its implementation into a linear pentapep-
tide has been reported earlier, but no structural details were
provided other than spectroscopic data (Scheme 3).[4] We
enantiomerically pure in the depicted l-configuration with
an Fmoc-protected amino function (Fmoc=9-fluorenylme-
thoxycarbonyl) and an unprotected carboxylic acid to
enable their use in standard peptide methods to synthesize
the Gramicidin S derivatives. To avoid oxidation of the
amino acids during the peptide synthesis, the phosphorus
atom is protected with a sulfur atom, which can be removed
at a later stage to obtain the desired bisphosphine-substitut-
ed cyclic decapeptides.[4,7]
Scheme 3. Synthesis of tyrosine derivative 3.
adapted this procedure and provide full details in the Exper-
imental Section to render the Fmoc-protected 3 from com-
pounds 5–7, for use in the standard peptide synthetic
Two different syntheses of enantiomerically pure 1 have
been described earlier by Gilbertson (Scheme 1).[4] The first
route utilizes Evansꢁ[8] chiral oxazolidone chemistry and in-
ACHTUNGTRENNUNG
ꢀ
ꢀ
oped that allows for varying the length of the ACHTNUGTRNEG(UN CH2)n link-
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Chem. Eur. J. 2009, 15, 8134 – 8145

Bisphosphine-Functionalized Cyclic Decapeptides
FULL PAPER
age between the a-carbon atom of a chiral amino acid and
its phosphine moiety. Belekonꢁs template, the NiII Schiff
base of (S)-2-[N-(benzylpropyl)-amino]benzophenone (4), is
an ideal candidate for enantioselective alkylation to render
enantiomerically pure phosphine-derivatized amino acids. It
has been demonstrated already that glycine incorporated in
4 can be functionalized[11] and that the resulting unnatural
amino acid can be removed readily from the template in
high yields and large quantities.[12] However, we are aware
of only one example introducing a phosphorus-containing
substituent, namely the synthesis of phosphinato- and phos-
phonato-substituted amino acids.[13] Here, we describe our
efforts to use template 4 for introducing phosphine substitu-
ents with carbon linkages ranging from n=0–2 (compounds
8a–c).
served in the crude reaction mixtures. Single-crystal X-ray
structure determinations established the stereochemistry of
two of the products. Suitable red needles of solvate 8b were
obtained from a solution of the compound in acetonitrile at
48C. Compound 8c crystallized as orange needles with two
independent molecules per unit cell on vapor diffusion of
n-hexane into a solution of 8c in ethyl acetate. Their molec-
ular structures (Figure 2, Table 1) show pseudo square-
Deprotonation of 4 with nBuLi, NaH or NaOH rendered
the corresponding anion. In situ phosphanation with 9 and
alkylation with the phosphine-containing reagents 10 and
11, followed by sulfurization in the case of 9 and 10, afford-
ed after column chromatography the functionalized tem-
plates 8a–c as red powders in yields of about 90%
(Scheme 4). The need for using slightly different reaction
Scheme 4. Enantioselective alkylation of template 4. i) nBuLi, THF,
ꢀ788C. ii) NaH, acetonitrile. iii) NaOH, acetonitrile. iv) addition of 9,
ꢀ788C; addition of S8 at RT, 16 h. v) addition of 10, after 8 h addition of
S8, 16 h. vi) addition of 11, 16 h.
Figure 2. Molecular structure of 8b (top) and 8c (bottom). H atoms have
been omitted for clarity. Selected bond lengths (ꢂ) and angles (8), 8b:
ꢀ
ꢀ
ꢀ
ꢀ
Ni(1) N(1) 1.946(7), Ni(1) N(8) 1.847(7), Ni(1) N(22) 1.859(7), Ni(1)
O(26) 1.865(6); N(8)-Ni(1)-N(22) 94.5(3), N(8)-Ni(1)-O(26) 175.0(3),
N(22)-Ni(1)-O(26) 87.1(3), N(8)-Ni(1)-N(1) 88.4(3), N(22)-Ni(1)-N(1)
173.4(3), O(26)-Ni(1)-N(1) 90.5(3). 8c: Ni(1)-N(5) 1.845(7), Ni(1)-N(9)
1.822(7), Ni(1)-N(12) 1.940(7), Ni(1)-O(1) 1.848(6), N(9)-Ni(1)-N(5)
93.7(3), N(9)-Ni(1)-O(1) 176.2(3), N(5)-Ni(1)-O(1) 87.0(3), N(9)-Ni(1)-
N(12) 84.1(3), N(5)-Ni(1)-N(12) 174.4(3), O(1)-Ni(1)-N(12) 94.9(3).
conditions (see Experimental Section) is ascribed to the dif-
ference in reactivity of 9, 10, and 11. Whereas chlorophos-
phine 9 reacts readily with Li+4ꢀ, the more reactive Na+4ꢀ
ion pair is needed for chloromethylphosphine 10. Interest-
ingly, the corresponding phosphine sulfide (10·S) displayed
no reactivity, which we attribute to deactivation of the leav-
ing group by the electron-withdrawing nature of the phos-
phine sulfide. Attempts to extend the alkylation with the
next higher homologue (n=3), the propyl-containing 12a or
its bromo derivative 12b, was unsuccessful, probably due to
self alkylation of the substrate under the reaction condi-
tions.[14]
planar coordination of nickel by three nitrogen atoms and
one oxygen atom, like that for the non-functionalized tem-
plate 4.[14] The bond lengths and angles around nickel are
very similar for 8b and the two independent molecules for
8c. A notable difference is the manner in which the N-
benzyl and alkylphosphinyl groups are rotated, which may
be due to packing effects in the solid state. Importantly, the
desired and expected S configuration at the a-carbon atom
of the embedded glycine fragment is confirmed for both 8b
and 8c.
Stereochemistry of amino acids: Products 8a–c were each
obtained as single diastereomers, whereas the phosphana-
tion and alkylation of the anion of chiral 4 might have given
two. The high selectivity is evident from the single, sharp
31P NMR resonance (8a 49.3; 8b 36.7; 8c 42.3 ppm) ob-
Chem. Eur. J. 2009, 15, 8134 – 8145
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8137

K. Lammertsma, M. J. van Raaij, M. Overhand et al.
HCl for the red color of the sol-
Table 1. Selected crystallographic data for compounds 8b, 8c, 19b and 19c.
8b 8c 19b
C40H36N3NiO3PS 2(C41H38N3NiO3PS) 2(C82H104N10O10P2S2) C92H108N10O10P2S2
utions to fade to pale green or
yellow (Scheme 5). After
19c
formula
4
(C2H3N) H2O
7
E
H2O
simple work-up, including the
recovery of the free ligand of
the template for reuse towards
4, formation of the phosphine
substituted amino acid inter-
mediate 13, and removal of the
nickel salts, methyl esters 14b
and 14c were isolated in respec-
tively 93 and 82% yield, respec-
tively, and were fully character-
ized by NMR spectroscopy.
However, in case of 8a the de-
sired product 14a was not ob-
tained. The 1H NMR spectrum
did not show the presence of an
ester or carboxylic acid group
and the 13C-DEPT NMR spec-
Mr [gmolꢀ1
l [ꢂ]
crystal system
space group
a [ꢀ]
b [ꢀ]
c [ꢀ]
a [8]
b [8]
]
910.69
0.71073
orthorhombic
P212121
11.8645(7)
18.2225(8)
21.1364(13)
90
742.47
1.5418
triclinic
P1
9.2751(5)
13.7542(6)
14.2109(6)
79.029(2)
88.427(3)
87.292(5)
1777.5(1)
1.387
3157.78
1.5418
monoclinic
P21
19.936(4)
19.008(4)
23.812(5)
90
100.03(3)
90
8886(3)
1.175
2
1.39
3344
0.01ꢃ0.02ꢃ0.09
100.0(1)
1.90–50.40
64214
17558
0.0620
17558
1012
1006
2.43
0.1753
0.4868
0.1792
0.4990
0.30(5)
1.77/ꢀ0.72
1657.99
1.5418
monoclinic
P21
9.3998(6)
27.6013(19)
17.1335(12)
90
95.552(4)
90
4424.4(5)
1.245
90
90
g [8]
V [ꢂ3]
4569.7(4)
1.324
4
0.557
1912
1calcd [gcmꢀ3
]
Z
1
2
m [mmꢀ1
]
2.110
776
1.407
1764
0.02ꢃ0.02ꢃ0.11
100.0(1)
2.59–47.48
24272
7899
0.0777
7899
1407
1069
F
N
crystal size [mm]
T [K]
q range [8]
reflns collected
reflns unique
R(int)
0.08ꢃ0.10ꢃ0.70 0.03ꢃ0.08ꢃ0.18
100.0(1)
1.48–20.81
32704
4801
0.0892
4775
4
318
1.341
0.0671
0.1491
0.0912
0.1599
0.01(3)
110.0(1)
3.17–54.24
71873
8607
0.1214
8607
32
902
1.124
0.0805
0.1839
0.0956
0.2033
0.00(3)
1.46/ꢀ0.56
trum revealed
a
methylene
data
restraints
group. We identify the product
as amino-phosphine 15, formed
by decarboxylation of the inter-
mediate phosphine-substituted
amino acid 13a. Decarboxyla-
tion was reported earlier in at-
tempts to synthesize a phos-
phinyl glycine[16] and represents
a common degradation of phos-
phine acetic acids.[17]
parameters
GOF on F2
1.327
R1 indices [I>2s(I)]
wR2 indices [I>2s(I)]
R1 indices (all data)
wR2 indices (all data)
absolute structure
0.1098
0.2904
0.1279
0.3123
0.08(5)
0.53/ꢀ0.50
largest difference peak/hole 0.59/ꢀ0.44
[eꢂꢀ3
]
Base-catalyzed ester cleavage
of 14b and c, followed by the
Enantiomerically pure amino acids are generally obtained
from Schiff base NiII complexes by simple hydrolysis, requir-
ing little purification, but there is diversity in reaction condi-
tions, yield, and stereoselectivity.[12] We wondered how the
phosphine-containing compounds 8a–c would sustain the
hydrolysis. Liberation of the derivatized amino acids re-
quired prolonged heating in a mixture of MeOH and 2m
standard protocol for introducing the Fmoc group in pro-
tected amino acids,[4] gave after purification by column chro-
matography analytically pure 1 and 2 in 72 and 62% yield,
respectively (Scheme 5, step iii). Their enantiomeric purity
was established by the Mosherꢁs ester procedure for the
methyl esters of the amine-deprotected 16b and
c
(Scheme 6). Based on the integration of the 19F NMR reso-
nances of the two possible diastereomeric products 17b and
c resulting from the reaction of the free amines with (R)-a-
methoxy-a-(trifluoromethyl)phenylacetic
acid
chloride
(MTPACl) and one equivalent of 4-dimethylaminopyridine
(DMAP) in chloroform (Scheme 6), verified by the reaction
with racemic MTPACl, the enantiomeric ratios of 1 and 2
were determined to be 94:6 and >200:1, respectively.
Scheme 5. Liberation of PAAꢁs
1 and 2. i) MeOH, HCl (2n), 2 h.
ii) MeOH, Et2O/HCl, 16 h. iii) LiOHaq, THF, 2 h; FmocOSu, NaHCO3,
dioxane, H2O, 0 8C, 4 h.
Scheme 6. Determination of the enantiomeric purity of the PAAs by deri-
vatization with Mosherꢁs acid chloride.
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Bisphosphine-Functionalized Cyclic Decapeptides
FULL PAPER
Synthesis of cyclic decapeptides: The phosphine-functional-
ized amino acids (PAAs) 1–3 are now available for the con-
struction of the desired bisphosphine-substituted oligopepti-
des. Previous studies have shown that cyclic decapeptides
such as Gramicidin S and derivatives can be readily synthe-
sized by cyclization of their corresponding linear precursors.
The efficiency of the cyclization reaction of an activated
linear decameric precursor depends strongly on the ability
to preorganize into a b-hairpin conformation.[19] These pre-
viously cyclized peptide precursors generally have two bulky
protected ornithine side chains presumable comparable in
size with bulky phosphine-modified side chains and an oth-
erwise identical sequence as our target peptide derivatives.
Thus, a standard solid-phase peptide synthesis protocol,[19]
starting with leucine as the first immobilized amino acid and
the tailor-made Fmoc- and sulfur-protected PAAs 1–3 was
followed toward the synthesis of the immobilized dimeric
sequence 18a–c (Scheme 7). The phosphorus-containing
amino acids could be readily incorporated and are stable
under Fmoc deprotection conditions, as anticipated. After
acid treatment, cyclization, and purification the cyclic deca-
mers 19a–c were obtained in yields ranging from 45–65%.
The three cyclic decamers 19a–c were fully characterized
and their structures analyzed with high-field 1D and 2D
NMR spectroscopy. Of the three cyclic peptides, 19a proved
to be less soluble, requiring more methanol and an elevated
temperature to effectively record the NMR spectrum. Be-
cause of this, possibly in combination with the close proximi-
ty of the bulky CH2P(S)Ph2 groups, the NH signals of the
amides appear broader and shifted as relative to those of
peptides 19b and c. The overall NMR data of compounds
19a–c, however, are in full accord with the anticipated cyclic
b-hairpin secondary structure,[20] which was established un-
equivocally for compounds 19b and 19c by single-crystal X-
ray analysis. The structures (Figure 3) show that the mole-
Figure 3. Molecular structure of functionalized GS compounds 19b (top)
and 19c (bottom). Hydrogen atoms were omitted for clarity. Dashed
lines display the “inner” (green) and “outer” (black) hydrogen bonds.
The distance between phosphorus atoms is indicated by orange dashed
lines.
cules maintain the general b-strand arrangement of the
parent GS molecule with twist angles of 158 and 378 for 19b
(both independent molecules) and 19c, respectively
(Figure 4). The twist angle is defined as the angle between
straight lines drawn through opposing C-a atoms of the
valine and leucine residues of the GS backbone. For native
GS these angles are 18 and 478 for the structures deter-
mined by Llamas-Saiz et al. and Tishchenko et al., respec-
tively,[5,6] suggesting that compound 19b has a conformation
less twisted than any of the
native GS structures. The more
twisted backbone conformation
of 19c is possibly induced by
the more bulky phenyl group in
the spacer. The side chains of
the modified amino acids are
more flexible in compound 19b
with respect to compound 19c,
as evidenced by disordered
multiple conformations ob-
served in the crystal structure.
The distance between the phos-
phine atoms in 19b is 6.2–6.5 ꢂ,
depending on the conformation,
while for compound 19c the
distance is about 6.1 ꢂ. Upon
deprotection, the flexible phos-
phine-containing side chains
Scheme 7. Synthesis of bisphosphine-functionalized Gramicidin S analogues 19a–c. i) Fmoc-Leu-OH, DIC,
DMAP. ii) Repetitive deprotection: 20% piperidine in NMP, peptide bond formation with 1–3 (denoted as
PAA1–3), HATU or the corresponding Fmoc-AA-OH, HCTU, DiPEA. iii) a) 20% piperidine in NMP, b) TFA/
TIS/H2O (95:2.5:2.5, v/v/v). iv) PyBOP, HOBt, DiPEA.
should be able to act as chelat-
ing ligands. Preliminary model-
ing studies show indeed that
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8139

K. Lammertsma, M. J. van Raaij, M. Overhand et al.
logues was proven by NMR spectroscopic studies and in two
cases X-ray structures provided unique insight in the struc-
tural conformations of the phosphine-modified oligopepti-
des. The rigid peptide backbone enforces a close proximity
of the two phosphorus atoms, which are available for transi-
tion-metal coordination after removal of the protecting
sulfur atoms. The synthesis of the free ligands and their
metal complexes as well as their potential in enantioselec-
tive catalysis is currently under active investigation.
Experimental Section
General: 1H, 13C, 19F and 31P NMR spectra were recorded on Bruker
Avance 250, Bruker AV 400, and MSL 400 spectrometers at 298 K,
unless stated otherwise. NMR chemical shifts are internally referenced to
tetramethylsilane (1H and 13C NMR) and externally for 19F to CFCl3 and
for 31P to 85% H3PO4. Coupling constants (J) are given in Hz. Where in-
dicated, NMR peak assignments were made using COSY and TOCSY ex-
periments. Infrared spectra were recorded on a Paragon-PE 1000 and
Shimadzu FTIR-84005 spectrophotometer and data are reported in cmꢀ1
.
High-resolution mass spectra were recorded on a Finnigan LTQ Orbitrap
system, a JEOL JMS SX/SX 102A four-sector mass spectrometer, cou-
pled to a JEOL MS-MP9021D/UPD system program and electrospray
ionization (ESI) mass spectrometry was carried out with a micrOTOF-Q
instrument in positive ion mode. Melting points were measured on sam-
ples in sealed capillaries and are uncorrected. Analytical LC-MS analysis
was conducted on a Jasco system (detection simultaneously at 214 nm
and 254 nm) equipped with an Alltima C-18 analytical column (Alltech,
4.6ꢃ150 mm, 5 mm particle size). Solvent system: A: 100% water, B:
100% acetonitrile, C: 1% aq. trifluoroacetic acid (TFA). Purification of
the cyclic decapeptides was conducted on a BioCAD “Vision” automated
HPLC system (PerSeptive Biosystems, Inc.), supplied with a semiprepar-
ative Alltima CN column (Alltech, 10.0ꢃ250 mm, 5 mm particle size).
Solvent system: A: 100% water, B: 100% acetonitrile, C: 1% aq. TFA
(linear gradient of 50%!80% B). Compounds 4,[22, 11] and 12b[14] were
synthesized according to literature procedures.
Figure 4. Side view of the molecular structure of functionalized GS com-
pounds 19b (top) and 19c (bottom) indicating the twist angles.
these bisphosphine-functionalized Gramicidin S analogues
are ideally suited for transition-metal complexation favoring
the elusive trans-bisphosphine coordination over the typical
cis configuration.[21] The synthesis of these metal complexes
and their application in transition-metal catalysis is under
active investigation.
Synthesis of chloromethyldiphenylphosphine (10):[23a] nBuLi (0.63 mL of
a 1.6m solution in n-hexane; 1.0 mmol) was added slowly to a solution of
Ph2PH (0.19 g, 1.0 mmol) in dry THF (15 mL) at ꢀ788C, after which the
solution turned orange. Subsequently, the mixture was allowed to warm
up to room temperature and the in situ generated Ph2PLi was added
slowly to CH2Cl2 (50 mL) at ꢀ788C and stirred until the orange color dis-
appeared. The resulting colorless solution was allowed to warm up to
room temperature and evaporated to dryness. The product was extracted
into toluene (25 mL) and filtered. Evaporation of all volatiles in vacuo
yielded 10 as a colorless oil (1.83 g, 78%), which was used without fur-
ther purification. 31P{1H} NMR (CDCl3): d=ꢀ8.9 ppm (s); 1H NMR
(CDCl3): d=7.57–7.30 (m, 10H; PhH), 4.10 ppm (d, 2JPH =5.0 Hz, 2H;
CH2).
Conclusions
The synthesis and structural characterization of three novel
bisphosphine-substituted oligopeptides based on the natural
product Gramicidin S were achieved by cyclization of their
linear precursors obtained by a solid-phase peptide-coupling
protocol. The notable difference between the GS analogues
is found in the size and shape of the linkage between the
phosphine group and the oligopeptide backbone. As a start-
ing point, two of the three synthesized phosphine-containing
amino acid building blocks were obtained by the enantiose-
lective alkylation of a chiral nickel Schiff base complex with
phosphine-containing reagents, which allows the introduc-
tion of varying alkyl chains between the phosphine group
and the amino acid part. The selective formation of one dia-
stereomer was observed by 31P NMR spectroscopy and the
absolute stereochemistry of the a-carbon atom of the gener-
ated phosphine amino acid was established by X-ray struc-
ture analysis. Acid-catalyzed liberation gave access to enan-
tiopure free phosphine amino acids, which were purified and
properly protected to enable their use in standard peptide-
coupling protocols. The cyclic b-hairpin structure of GS ana-
Synthesis of (2-chloroethyl)diphenylphosphine sulfide (11): nBuLi
(3.38 mL of a 1.6m solution in n-hexane; 5.4 mmol) was added slowly to
a solution of Ph2PH (1.00 g, 5.4 mmol) in dry THF (10 mL) at ꢀ788C,
after which the solution turned orange. Subsequently, the mixture was al-
lowed to warm up to room temperature, diluted with more THF
(50 mL), and the in situ generated Ph2PLi was added slowly to 1,2-di-
chloroethane (50 mL) at ꢀ208C. After complete addition, the orange
color disappeared and the reaction mixture was allowed to warm up to
room temperature. Elemental sulfur (0.43 g, 13.5 mmol) was added and
the resulting mixture was stirred for 16 h. Evaporation to dryness and
chromatography of the remaining white solid over silica gel eluting with
n-hexane/dichloromethane (2:1) and subsequent recrystallization from di-
ethyl ether/n-hexane gave 11 as colorless needles (0.94 g, 62%). M.p.
778C; 31P{1H} NMR (CDCl3): d=37.8 ppm (s); 13C{1H} NMR (CDCl3):
d=132.1 (d, 1JCP =81.3 Hz; ipso-Ph), 132.1 (d, 4JCP =3.0 Hz; p-Ph), 131.1
2
(d, 2JCP =10.4 Hz; o-Ph), 129.0 (d, 3JCP =12.6 Hz; m-Ph), 38.4 (d, JCP
=
8140
www.chemeurj.org
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2009, 15, 8134 – 8145

Bisphosphine-Functionalized Cyclic Decapeptides
FULL PAPER
4.0 Hz; CH2Cl), 36.5 ppm (d, 1JCP =52.1 Hz; PCH2); 1H NMR (CDCl3)
d=7.88–7.82 (m, 4H; o-PhH), 7.51–7.47 (m, 6H; m/p-PhH), 3.83–3.74
(m, 2H; CH2Cl), 3.02–2.91 ppm (m, 2H; PCH2); HR FAB-MS: m/z calcd
for C14H15PSCl: 281.0321; found: 281.0320; MS: m/z (%): 281.0 (100)
[M]+, 218.0 (39) [MꢀC2H3Cl]+; IR: n˜ =3051 (w), 1677 (w), 1557 (w),
1477 (m), 1437 (s), 1404 (w), 1313 (m), 1296 (m), 1199 (w), 1116 (m),
1099 (s), 1068 (m), 1026 (s), 997 (m), 923 (m), 833 cmꢀ1 (m).
tonitrile (10 mL) was added and the reaction mixture was stirred for 8 h.
Elemental sulfur (0.32 g, 10.0 mmol) was added and the resulting mixture
was stirred for an additional 16 h. The reaction mixture was added to
H2O (100 mL) and the product was extracted into CH2Cl2 (3ꢃ100 mL).
The combined organic layers were dried over MgSO4 and all volatiles
were removed in vacuo. Chromatography of the residue over silica gel
eluting with CHCl3/acetone (4:1) yielded 8a as a red solid (3.23 g, 89%).
M.p. 155–1568C; 31P{1H} NMR (C6D6): d=36.7 ppm (s); 13C{1H} NMR
Synthesis of (3-chloropropyl)diphenylphosphine sulfide (12a): nBuLi
(15.6 mL of a 1.6m solution in n-hexane; 1.0 mmol) was added slowly to
a solution of Ph2PH (4.65 g, 25.0 mmol) in dry THF (50 mL) at ꢀ788C,
after which the solution turned orange. Subsequently, the mixture was al-
lowed to warm up to room temperature and the in situ generated Ph2PLi
was slowly added over a period of 2 h to a solution of 1,3-dichloropro-
pane (4.00 g, 20.0 mmol) in toluene (10 mL) at 08C. Elemental sulfur
(1.12 g, 35.0 mmol) was added and the resulting mixture was evaporated
to dryness. Chromatography of the remaining white solid over silica gel
eluting with n-hexane/dichloromethane (2:1) and subsequent recrystalli-
zation from ethanol/n-hexane gave 12a as colorless needles (1.87 g,
(C6D6): d=180.5 (s; CO), 176.1 (d, JCP =1.8 Hz; CO2), 171.2 (s; C=N),
1
144.6 (s; C), 135.2 (s; C), 134.6 (s; C), 134.1 (s; C), 133.6 (d, JCP
=
1
80.9 Hz; ipso-C), 133.5 (s; CH), 133.1 (d, JCP =88.0 Hz; ipso-C), 132.0 (d,
2JCP =10.6 Hz; CH), 131.9 (s; CH), 131.5 (d, 2JCP =10.6 Hz; CH), 131.2
(d, 3JCP =3.1 Hz; CH), 131.0 (d, 3JCP =3.0 Hz; CH), 129.5 (s; CH), 129.3
(s; CH), 129.1 (s; CH), 128.9 (s; CH), 128.8 (s; CH), 128.7 (s; CH), 128.6
(s; CH), 127.2 (s; CH), 126.4 (s; CH), 125.6 (s; C), 124.3 (s; CH), 120.1
(s; CH), 70.6 (s; aCPro) 66.9 (d, 2JCP =1.7 Hz; CH), 63.1 (s; CH2Ph), 57.4
(s; dCPro), 38.0 (d, 1JCP =50.6 Hz,;PCH2), 31.1 (s; bCPro), 24.1 ppm (s;
gCPro); 1H NMR (C6D6): d=9.03 (d, 3JHH =8.7 Hz, 1H; CH), 8.04–7.90
(m, 4H; CH), 7.80 (d, 3JHH =7.2 Hz, 2H; CH), 7.19–6.78 (m, 13H; CH),
6.53 (dd, 3JHH =8.2 Hz, 4JHH =1.5 Hz, 1H; CH), 6.46 (dd, 3JHH =4.6 Hz,
4JHH =1.5 Hz, 2H; CH), 6.29 (t, 3JHH =8.0 Hz, 1H; CH), 4.64–4.48 (m,
1H; CH), 4.14 (d, 2JHH =12.5 Hz, 1H; CH2Ph), 3.58–3.25 (m, 4H; PCH2,
aHPro, dHPro, gHPro), 3.23–3.05 (m, 1H; bHPro), 3.14 (d, 2JHH =12.5 Hz,
1H; CH2Ph), 3.03–2.87 (m, 1H; PCH2), 2.22–2.06 (m, 1H; bHPro), 1.63–
32%). M.p. 81–828C; 31P{1H} NMR (CDCl3): d=42.1 ppm (s); 13C{1H}
4
NMR (CDCl3): d=132.4 (d, 1JCP =80.5 Hz; ipso-Ph), 131.5 (d, JCP
=
3.1 Hz; p-Ph), 130.9 (d, 2JCP =10.7 Hz; o-Ph), 128.5 (d, 3JCP =12.6 Hz; m-
1
Ph), 45.2 (d, 2JCP =5.3 Hz; CH2), 30.0 (d, JCP =58.5 Hz; PCH2), 25.6 ppm
(s; CH2Cl); 1H NMR (CDCl3): d=7.89–7.83 (m, 4H; o-PhH), 7.49–7.43
3
(m, 6H; m/p-PhH), 3.60 (t, JHH =6.0 Hz, 2H; CH2Cl), 2.67–2.56 (m, 2H;
1.43 ppm (m, 2H; dHPro
, gHPro); HR FAB-MS: m/z calcd for
PCH2), 2.17–2.07 ppm (m, 2H; CH2); HR ESI-MS: m/z calcd for
C15H17PSCl [M+H]+: 295.0472; found: 295.0460; IR: n˜ =1703 (m), 1653
(m), 1437 (m), 1105 (m), 740 (m), 666 (m), 513 (m), 477 cmꢀ1 (m).
C40H37N3O3PSNi [M+H]+: 728.1647; found: 728.1639; m/z (%): 750 (11)
[M+Na]+, 728 (63) [M+H]+, 510 (21) [MꢀC12H10PS]+, 466 (13) [Mꢀ
C13H10O2PS]+,
91
(28)
[MꢀC33H29N3O3PSNi]+,
77.0
(15)
[MꢀC34H31N3O3PSNi]+; IR: n˜ =1686 (w, br), 1633 (s, br), 1585 (m), 1541
(m), 1467 (w), 1435 (s), 1357 (m), 1332 (m, br), 1253 (s), 1163 (m), 1128
(w), 1099 (m), 1060 (w), 1028 (w), 1016 (w), 999 (w), 962 (w), 842 (m),
748 (m), 734 (m), 692 cmꢀ1 (s, br).
Synthesis of 8a: nBuLi (0.55 mL of a 1.6m solution in n-hexane) was
added slowly to a solution of 4 (0.40 g, 0.8 mmol) in dry THF (40 mL) at
ꢀ788C, after which the deep red solution turned green. Subsequently, the
reaction mixture was stirred for 1 h at the same temperature followed by
the slow addition of Ph2PCl (0.20 g, 0.88 mmol) that resulted in a color
change to red. After complete addition, the reaction mixture was allowed
to warm up to room temperature. Elemental sulfur (0.06 g, 2.0 mmol)
was added and the resulting mixture was stirred for 16 h. The reaction
mixture was added to H2O (200 mL) and the product was extracted into
CH2Cl2 (3ꢃ75 mL). The combined organic layers were dried over MgSO4
and all volatiles were removed in vacuo. Chromatography of the remain-
ing red solid over silica gel eluting with CHCl3/acetone (5:1) and subse-
quent recrystallization by layering a saturated ethyl acetate solution with
n-hexane gave 8a as red crystals (0.43 g, 77%). M.p. 1988C (decomp.);
31P{1H} NMR (CDCl3): d=49.3 ppm (s); 13C{1H} NMR (CDCl3): d=180.9
(s; CO), 173.3 (d, JCP =1.7 Hz; C=N), 172.5 (d, JCP =6.3 Hz; CO2), 143.6
(s; C), 134.2 (s; CH), 133.7 (d, JCP =1.1 Hz; C), 133.3 (s; CH), 133.2 (s;
CH), 132.7 (s; CH), 132.6 (s; C), 132.2 (d, JCP =3.1 Hz; CH), 131.8 (s;
CH), 131.7 (s; CH), 131.5 (s; CH), 131.3 (s; C), 130.1 (s; CH), 129.9 (s;
C), 129.6 (s; CH), 128.9 (s; CH), 126.7 (s; CH), 128.5 (s; CH), 128.5 (s;
CH), 128.3 (s; CH), 128.2 (s; CH), 128.0 (s; CH), 126.7 (s; CH), 126.0 (d,
Synthesis of 8c: Carefully ground NaOH (0.33 g, 8.3 mmol) was added to
a solution of 4 (1.65 g, 3.3 mmol) in dry acetonitrile (50 mL) at room
temperature and the resulting mixture was stirred for 1 h, during which
the red solution turned deep red. Subsequently, 11 (1.39 g, 5.0 mmol) was
added and the reaction mixture was stirred for 16 h. The reaction mixture
was quenched with 0.1m HCl (50 mL) and the product was extracted into
CH2Cl2 (4ꢃ40 mL). The combined organic layers were dried over MgSO4
and all volatiles were removed in vacuo. Chromatography of the residue
over silica gel eluting with CHCl3/acetone (5:1) yielded 8a as a red solid
(2.20 g, 90%). M.p. 230–2318C; 31P{1H} NMR (CDCl3): d=42.3 ppm (s);
13C{1H} NMR (CDCl3): d=180.5 (s; CO), 178.7 (s; CO2), 171.2 (s; C=N),
142.5 (s; C), 133.5 (s; CH), 133.4 (s; C), 133.3 (s; C), 133.1 (s; C), 133.0
(s; C), 132.5 (s; CH), 131.7 (s; CH), 131.4 (d, 2JCP =10.2 Hz; CH), 131.2
2
(d, JCP =10.2 Hz; CH), 129.8 (s; CH), 129.5 (s; CH), 129.1 (s; CH), 129.1
(s; CH), 129.0 (s; CH), 128.9 (s; CH), 128.8 (s; CH), 127.7 (s; CH), 126.9
(s; CH), 126.3 (s; C), 124.0 (s; CH), 120.9 (s; CH), 70.4 (s; aCPro), 70.3
3
(d, JCP =17.4 Hz; CHGly), 63.2 (s; CH2Ph), 57.2 (s; dCPro), 30.8 (s; bCPro),
CP =2.4 Hz; C), 123.5 (s; CH), 120.6 (s; CH), 75.3 (d, 1JCP =35.7 Hz;
29.3 (s; CH2), 29.0 (d, 1JCP =57.2 Hz; PCH2), 23.8 ppm (s; gCPro);
J
3
1H NMR (CDCl3): d=8.15 (d, 3JHH =8.6 Hz, 1H; CH), 8.03 (d, JHH
=
CGly), 71.3 (s; aCPro), 63.7 (s; CH2Ph), 57.8 (s; dCPro), 31.5 (s; bCPro),
24.0 ppm (s; gCPro); 1H NMR (CDCl3): d=8.41 (d, 3JHH =8.6 Hz, 1H;
CH), 8.11–7.96 (m, 4H; CH), 7.70–7.58 (m, 2H; CH), 7.54–7.04 (m, 13H;
CH), 6.74 (t, 3JHH =7.4 Hz, 1H; CH), 6.66–6.48 (m, 3H; CH), 5.08 (d,
3JPH =7.1 Hz, 1H; HGly), 4.35 (d, 2JHH =12.6 Hz, 1H; CH2Ph), 3.58–3.34
(m, 3H; aHPro, dHPro, gHPro), 3.51 (d, 2JHH =12.6 Hz, 1H; CH2Ph), 3.15–
3.09 (m, 1H; bHPro), 2.55–2.46 (m, 1H; bHPro), 2.12–1.99 ppm (m, 2H;
dHPro, gHPro); HR FAB-MS: m/z calcd for C39H35N3O3PSNi [M+H]+:
714.1490; found: 714.1500; MS: m/z (%): 736 (2) [M+Na]+, 714 (15)
[M+H]+, 670 (75) [MꢀCO2]+, 497 (14) [MꢀC12H10PS]+, 482 (10)
[MꢀC26H21N2NiPS]+, 217.0 (13) [MꢀC27H25N3O3Ni]+, 160 (100)
[MꢀC28H21N2O3PSNi]+, 91 (62) [MꢀC32H28N3O3PSNi]+, 77 (12)
[MꢀC33H29N3O3PSNi]+; IR: n˜ =1668 (m, br), 1645 (s, br), 1585 (m, br),
1541 (m), 1437 (m), 1332 (m, br), 1255 (s), 1163 (m), 1130 (w), 1103 (m),
1062 (w, br), 1014 (w), 997 (w), 923 (w, br), 841 cmꢀ1 (w).
7.2 Hz, 2H; CH), 7.92–7.81 (m, 2H; CH), 7.79–7.68 (m, 2H; CH), 7.53–
7.29 (m, 11H; CH), 7.23–7.08 (m, 3H; CH), 6.78 (d, 3JHH =7.5 Hz, 1H;
CH), 6.69–6.56 (m, 2H; CH), 4.37 (d, 2JHH =12.6 Hz, 1H; CH2Ph), 3.80
(dd, 3JHH =9.2 Hz, 3JHH =3.9 Hz, 1H; HGly), 3.40–3.36 (m, 2H; dHPro
,
,
aHPro), 3.52 (d, 2JHH =12.6 Hz, 1H; CH2Ph), 3.26–3.18 (m, 2H; gHPro
CH2), 2.60–2.45 (m, 2H; dHPro, bHPro), 2.43–2.30 (m, 2H; bHPro, PCH2),
2.05–1.92 ppm (m, 3H; dHPro, gHPro, CH2); HR FAB-MS: m/z calcd for
C41H39N3O3PSNi [M+H]+: 742.1803; found: 742.1805; MS: m/z (%): 764
(11) [M+Na]+, 742 (100) [M+H]+, 511 (5) [MꢀC13H12PS]+, 160 (92)
[MꢀC30H25N2O3PSNi]+, 91.0 (62) [MꢀC34H32N3O3PSNi]+, 77.0 (16)
[MꢀC35H33N3O3PSNi]+; IR: n˜ =1670 (s), 1637 (s), 1589 (m), 1548 (w),
1441 (m), 1473 (m), 1363 (m), 1350 (m), 1334 (m), 1319 (w), 1292 (m),
1259 (s), 1209 (m), 1161 (m), 1130 (w), 1099 (m), 1070 (w), 1031 (w), 989
(w), 929 (w), 883 (w), 812 cmꢀ1 (w).
Synthesis of 8b: NaH (0.18 g, 7.5 mmol) was added to a solution of 4
(2.48 g, 5.0 mmol) in dry acetonitrile (100 mL) at room temperature and
the resulting mixture was stirred for 1 h, during which the red solution
turned deep red. Subsequently, a solution of 10 (1.76 g, 7.5 mmol) in ace-
General procedure for the liberation of 14a–c: Compound 8a–c
(2 mmol) was dissolved in a mixture of MeOH (30 mL) and 2m HCl
(20 mL) and heated at 658C for 3 h, which resulted in a color change of
the reaction mixture to pale green or yellow. All volatiles were removed
Chem. Eur. J. 2009, 15, 8134 – 8145
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
8141

K. Lammertsma, M. J. van Raaij, M. Overhand et al.
in vacuo to give a pale yellow residue that was redissolved in H2O
(40 mL) to remove the insoluble, white (S)-2-[N-(benzylpropyl)-amino]-
benzophenone·HCl salt (BPB·HCl) by filtratation; residual BPB·HCl can
be removed by extraction with Et2O. After removal of all volatiles in
vacuo, the residue was dissolved in a mixture of MeOH (30 mL) and
with HCl(g) saturated Et2O (15 mL) and the reaction mixture was stirred
for 16 h at room temperature. Again, all volatiles were removed in vacuo
and the residue was extracted into CH2Cl2 (20 mL) to separate it from
the yellow, insoluble NiCl2, which was filtered off. Evaporation to dryness
yielded the methyl esters 14b and c as a pale yellow solid. Compound 15
was isolated instead of 14a.
25.9 ppm (s; CH2); 1H NMR (CDCl3): d=8.60 (brs, 1H; CO2H), 7.86–
7.70 (m, 8H; CH), 7.65–7.46 (m, 2H; CH), 7.53–7.33 (m, 8H; CH), 7.32–
7.23 (m, 2H; CH), 5.60 (d, 3JHH =7.6 Hz, 1H; NH), 4.40 (brs, 3H; CH/
CH2), 4.46–4.33 (m, 3H; CH/CH2), 4.24–4.13 (m, 1H; CH), 2.71–2.45 (m,
2H; CH2), 2.29–1.95 ppm (m, 2H; CH2); HR FAB-MS: m/z calcd for
C31H29NO4PS [M+H]+: 542.1555; found: 542.1548; MS: m/z (%): 564
(39) [M+Na]+, 542 (62) [M+H]+, 496 (9) [MꢀCO2]+, 363 (14)
[MꢀC14H11]+, 77.0 (18) [MꢀC25H23NO4PS]+; IR: n˜ =1714.8 (s, br) 1647.3
(s), 1510.3 (s, br), 1437.0 (s), 1340.6 (w), 1209.4 (m, br), 1140.0 (w),
1103.2 (m), 1047.4 (m, br), 999.2 (w), 935.5 cmꢀ1 (w).
Synthesis of 5b: Compound 5b was synthesized according to literature
procedures[4d] in 94% yield. 1H NMR (CDCl3): d=7.36–7.17 (m, 4H;
CH), 5.00 (brm, 1H; NH), 4.59 (brm, 1H; CH), 3.71 (s, 3H; OCH3),
Data for 14b: Yield: 0.66 (93%); m.p. 758C; 31P{1H} NMR (CDCl3): d=
1
38.4 ppm; 13C{1H} NMR (CDCl3): d =170.3 (s; CO2), 132.5 (d, JCP
=
1
3.21–2.99 (m, 2H; CH2), 1.46 (s, 9H; C
d=171.7 (s; CO2), 154.8 (s; CO), 148.4 (s; Ph), 136.8 (s; Ph), 130.9 (s;
Ph), 121.7 (s; CF3), 121.1 (s; Ph), 80.0 (s; C(CH3)3), 54.0 (s; CH), 52.1 (s;
(CH3)3); 13C{1H} NMR (CDCl3):
ACHTUNGTRENNUNG
81.5 Hz; ipso-Ph), 131.9 (d, JCP =81.2 Hz; ipso-Ph), 131.6 (s; p-Ph), 131.6
(s; p-Ph), 131.3 (d, 2JCP =10.6 Hz; o-Ph), 131.1 (d, 2JCP =10.6 Hz; o-Ph),
128.8 (s; m-Ph), 128.7 (s; m-Ph), 53.7 (d, 2JCP =16.1 Hz; CH), 53.2 (s;
OCH3), 28.1 ppm (d, 1JCP =57.2 Hz; CH2); 1H NMR (CDCl3): d=8.03–
7.68 (m, 4H; o-PhH), 7.65–7.35 (m, 6H; m/p-PhH), 4.55–4.32 (m, 1H;
CH), 3.61–3.28 ppm (m, 5H; CH2, CH3); HR FAB-MS: m/z calcd for
C16H19NO2PS [M+H]+: 320.0874; found: 320.0871; MS: m/z (%): 320
(100) [M+H]+, 217 (52) [MꢀC4H8NO2]+, 77 (7) [MꢀC10H13NO2PS]+;
IR: n˜ =2891 (br), 1747 (s), 1481 9m0, 1437 (s), 1400 (w), 1315 (w), 1236
(m), 1187 (w), 1144 (w), 1101 (s), 1043 (m), 997 (m), 930 (w), 879 (m),
827 (s), 741 (s), 700 cmꢀ1 (s).
AHCTUNGTRENNUNG
OCH3), 37.7 (s; CH2), 28.0 ppm (s; CH3); 19F{1H} NMR (CDCl3): d=
ꢀ73.3 ppm (s); HR FAB-MS: m/z calcd for C16H21F3NO7 [M+H]+
428.0991; found: 428.0995; MS: m/z (%): 428 (4) [M+H]+, 372 (80)
[MꢀC4H8]+, 328 (100) [MꢀC5H8O2]+, 268 (55) [MꢀC7H12O4]+, 239 (4)
[MꢀC9H16NO2]+, 57 (45) [MꢀC12H11NO7F3S]+.
Synthesis of 7b: Compound 7b was synthesized according to literature
procedures[4d] in 61% yield. 31P{1H} NMR (CDCl3): d=43.6 ppm (s);
1
13C{1H} NMR (CDCl3): d=171.8 (s; CO2), 154.9 (s; CO), 140.1 (d, JCP
=
Data for 14c: Yield: 0.61 (82%); m.p. 878C; 31P{1H} NMR (CDCl3): d=
42.6 ppm; 13C{1H} NMR (CDCl3): d =170.3 (s; CO2), 132.9 (s; ipso-Ph),
132.2 (d, 1JCP =24.3 Hz; ipso-Ph), 131.6 (s; p-Ph), 131.6 (s; p-Ph), 131.3
(d, 2JCP =10.6 Hz; o-Ph), 131.1 (d, 2JCP =10.6 Hz; o-Ph), 128.8 (s; m-Ph),
1
2
3.0 Hz; ipso-Ph), 133.5 (d, JCP =3.0 Hz; ipso-Ph), 132.3 (d, JCP =11.1 Hz;
o-Ph), 132.1 (d, 2JCP =10.7 Hz; o-Ph), 131.4 (d, 3JCP =12.6 Hz; m-Ph),
4
3
130.7 (s; p-Ph), 129.4 (d, JCP =12.8 Hz; p-Ph), 128.4 (d, JCP =12.6 Hz; m-
Ph), 80.0 (s; C), 54.1 (s; CH), 52.2 (s; OCH3), 38.2 (s; CH2), 28.1 ppm (s;
CH3); 1H NMR (250.1 MHz, CDCl3): d=7.72–7.64 (m, 6H; CH), 7.46–
7.40 (m, 6H; CH), 7.24–7.20 (m, 2H; CH), 5.05 (brd, 3JHH =7.6 Hz, 1H;
NH), 4.55 (brm, 1H; CH), 3.67 (s, 3H; OCH3), 3.15–3.07 (m, 1H; CH2),
3.04–2.90 (m, 1H; CH2), 1.37 ppm (s, 9H; CH3); HR FAB-MS: m/z calcd
for C27H31NO4PS [M+H]+: 496.1711; found: 496.1712; MS: m/z (%): 496
(15) [M+H]+, 440 (100) [MꢀC4H8]+, 336 (6) [MꢀC7H10O4]+, 308.1 (11)
[MꢀC8H14NO4]+, 217 (7) [MꢀC15H20NO4]+, 57.0 (9) [MꢀC23H21NO4]+.
3
1
128.7 (s; m-Ph), 53.7 (d, JCP =16.1 Hz; CH), 53.2 (s; CH3), 28.1 (d, JCP
=
57.0 Hz; CH2), 24.3 ppm (s; CH2); 1H NMR (CDCl3): d=8.02–7.86 (m,
4H; o-PhH), 7.47–7.34 (m, 6H; m/p-PhH), 4.22 (m, 1H; CH), 3.58 (s,
3H; CH3), 2.91 (m, 2H; CH2), 2.48–2.33 (m, 1H; CH2), 2.32–2.14 ppm
(m, 1H; CH2); HR FAB-MS: m/z calcd for C17H21NO2PS [M+H]+:
334.1037; found: 334.1031; MS: m/z (%): 334 (100) [M+H]+, 217 (17)
[MꢀC5H10NO2]+, 77.0 (18) [MꢀC11H15NO2PS]+; IR: n˜ =2901 (br), 1740
(m), 13736 (m), 1437 (m), 1307 (w), 1229 (s), 1180 (w), 1103 (s), 1028
(w), 977 (w), 982 (m), 734 (s), 709 cmꢀ1 (s).
Synthesis of 3: LiOH (10 mL of a 0.2m solution in H2O) was added drop-
wise to a solution of 7b (1.0 mmol, 0.50 g) in THF (3 mL) at 08C, and
the resulting mixture was stirred for 2 h. Neutralization with conc. HCl
and evaporation to dryness gave a pale yellow residue that was extracted
into CH2Cl2 (2ꢃ 10 mL). The combined organic layers were dried over
MgSO4 and the solution was concentrated to approximately 5 mL and
cooled to 08C, after which TFA (1 mL) was added and the reaction mix-
ture was stirred for 1 h. Removal of all volatiles in vacuo gave the free
phosphino amino acid as a yellow solid, which was dissolved in a mixture
of H2O (7 mL) and dioxane (15 mL) and cooled to 08C. After addition
of NaHCO3 (0.17 g, 2.0 mmol) and Fmoc-OSu (0.34 g, 1.0 mmol), the re-
sulting mixture was stirred for 18 h. Subsequently, the reaction mixture
was neutralized with conc. HCl and the product was extracted into ethyl
acetate (2ꢃ20 mL). The combined organic layers were dried over MgSO4
and all volatiles were removed in vacuo. The remaining pale yellow foam
was purified by column chromatography over silica gel eluting with
CH2Cl2/MeOH/TFA (20:5:1). The obtained salt was dissolved in ethyl
acetate (25 mL) and washed with H2O until the washing water was neu-
tral. Evaporation of all volatiles in vacuo gave 3 as a pale yellow foam
(0.50 g, 82%). M.p. 908C; 31P{1H} NMR (CDCl3): d=43.4 ppm (s);
13C{1H} NMR (CDCl3): d=174.9 (s; CO2), 156.1 (d, JCP =1.4 Hz; CO),
Data for 15: 31P{1H} NMR (CDCl3): d=42.1 ppm; 13C-DEPT
ACTHNUTRGNEU(GN 135) NMR
4
3
(CDCl3): d =132.2 (d, JCP =2.9 Hz; p-Ph), 131.7 (d, JCP =9.6 Hz; m-Ph),
129.2 (d, 2JCP =11.7 Hz; o-Ph), 46.6 ppm (d, 1JCP =51.6 Hz; CH2).
1H NMR (CDCl3): d=7.93–7.75 (m, 4H; o-PhH), 7.58–7.43 (m, 6H; m/p-
PhH), 3.61 (s, 2H; NH2), 1.46 ppm (s, 2H; CH2).
General procedure for the synthesis of 1 and 2: LiOH (10 mL of a 0.2m
solution in H2O) was added dropwise to a solution of methyl ester 14b,c
(1 mmol) in THF (5 mL) at 08C and the resulting mixture was stirred for
2 h. Neutralization with conc. HCl and evaporation to dryness gave a
pale yellow residue that was dissolved in a mixture of H2O (7 mL) and
dioxane (15 mL) and cooled to 08C. After addition of NaHCO3 (0.17 g,
2.0 mmol) and Fmoc-OSu (0.34 g, 1.0 mmol), the resulting mixture was
stirred for 4 h. Subsequently, the reaction mixture was neutralized with
conc. HCl, after which H2O (10 mL) was added and the product was ex-
tracted into CH2Cl2 (2ꢃ 20 mL). The combined organic layers were dried
over MgSO4 and all volatiles were removed in vacuo. Chromatography of
the remaining pale yellow foam over silica gel eluting with CH2Cl2/
MeOH/TFA (20:5:1) gave 1,2 as a white solid.
Data for 1:[4] Yield: 0.38 g (72%); 31P{1H} NMR (CDCl3): d=38.3 ppm;
1H NMR (CDCl3): d=7.97–7.84 (m, 4H; CH), 7.84–7.75 (m, 3H; CH),
7.69–7.64 (m, 1H; CH), 7.55–7.31 (m, 10H; CH), 5.94 (d, 3JHH =5.9 Hz,
1H; NH), 4.77 (m, 1H; CH), 4.61 (d, 3JHH =7.6 Hz, 1H; CH2), 4.39
144.0 (d, JCP =6.8 Hz; C), 141.7 (s; C), 140.1 (s; C), 133.2 (d, JCP
85.4 Hz; C), 133.1 (d, JCP =85.2 Hz; C), 132.9 (d, JCP =11.9 Hz; CH),
132.7 (d, JCP =10.8 Hz; CH), 132.6 (d, JCP =10.6 Hz; CH), 132.0 (d, JCP
=
=
3
(pseudo-t, JHH =7.1 Hz, 1H; CH2), 4.22–4.12 (m, 1H; CH), 3.47–3.35 (m,
3.0 Hz; CH), 130.1 (s; CH), 129.9 (s; CH), 129.1 (d, JCP =0.5 Hz; CH),
128.8 (d, JCP =0.5 Hz; CH), 128.2 (s; CH), 127.5 (s; CH), 125.4 (s; CH),
125.3 (s; CH), 120.4 (d, JCP =1.2 Hz; CH), 67.3 (s; CH2), 54.7 (s; CH),
47.5 (s; CH), 37.9 ppm (s; CH2); 1H NMR (CDCl3): d=9.58 (brs, 1H;
CO2H), 7.88–7.81 (d, 3JHH =7.4 Hz, 2H; CH), 7.76–7.31 (m, 10H; CH),
7.28–7.22 (d, 3JHH =7.4 Hz, 2H; CH), 5.74 (s, 1H; NH), 4.30–4.05 (brm,
4H; CH, CH2), 3.18 (dd, 2JHH =13.3 Hz, 3JHH =3.2 Hz; CH2), 2.94 ppm
(dd, 2JHH =13.3 Hz, 3JHH =10.7 Hz; CH2); HR FAB-MS: m/z calcd for
C36H31NO4PS [M+H]+: 604.1711; found: 604.1721; MS: m/z (%): 626
1H; PCH2), 3.30–3.13 ppm (m, 1H; PCH2).
Data for 2: Yield: 0.30 g (67%); m.p. 958C; 31P{1H} NMR (CDCl3): d=
42.6 ppm (s); 13C{1H} NMR (CDCl3): d=175.9 (s; CO2H), 156.7 (s; CO),
1
144.2 (s; C), 143.9 (s; C), 141.7 (s; C), 141.7 (s; C), 132.5 (d, JCP
=
81.2 Hz; C), 132.1 (d, JCP =2.9 Hz; CH), 131.5 (d, JCP =10.1 Hz; CH),
131.5 (d, JCP =10.3 Hz; CH), 129.3 (s; CH), 129.0 (s; CH), 128.2 (s; CH),
127.6 (s; CH), 125.5 (d, JCP =6.5; CH), 120.4 (s; CH), 67.7 (s; CH2), 54.2
(d, 3JCP =14.8 Hz; CH), 47.4 (s; CH), 29.1 (d, 1JCP =57.2 Hz; CH2),
8142
www.chemeurj.org
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2009, 15, 8134 – 8145

Bisphosphine-Functionalized Cyclic Decapeptides
FULL PAPER
(21) [M+Na]+, 604 (59) [M+H]+, 425 (35) [MꢀC14H11]+, 77 (18)
[MꢀC30H25NO4PS]+; IR: n˜ =3047 (m, br), 1716 (s, br), 1683 (s), 1652 (s),
1549 (m), 1521 (m), 1506 (s), 1448 (w), 1437 (s), 1244 (w, br), 1213 (w,
br), 1180 (w, br), 1099 (s), 1045 cmꢀ1 (m, br).
cyclo-[Pro-Val-PAA1-Leu-d-Phe]2 ACHTNGUTRENNU(G 19a): The synthesis was performed as
outlined above to obtain 350 mg (106 mmol) immobilized decapeptide
18a. Cleavage from the resin, followed by cyclization and purification
yielded compound 19a (110 mg, 67%). 31P NMR (162 MHz, MeOD/
CDCl3 (1:1), T=298 K): d=38.9 ppm (s); 1H NMR (400 MHz, MeOD/
CDCl3 (1:5), T=330 K): d=8.20 (d, 1H; JNH,Ha =8.7 Hz, NH Leu), 7.91–
7.82 (m, 5H; NH d-Phe, 4ꢃHar), 7.69, (d, 1H; JNH,Ha =5.8 Hz, NH PAA),
7.47–7.37 (m, 6H; Har), 7.28 (d, 1H; 1H; JNH,Ha =6.9 Hz, NH Val), 7.26–
7.17 (m, 3H; Har), 7.13–7.11 (m, 2H; Har), 5.06 (m, 1H; Ha PAA), 4.61
(m, 1H; Ha d-Phe), 3.91, (m, 1H; Ha Val), 3.64 (m, 1H; Hd Pro), 3.44
(m, 1H; Hb PAA), 3.06–2.99 (m, 2H; Hb PAA, Hb d-Phe), 2.87 (dd, 1H;
JHb,Hb =12.8, JHb,Ha =5.5 Hz, Hb d-Phe), 2.62 (m, 1H; Hd Pro), 2.09 (m,
1H; Hb Pro), 2.00 (m, 1H; Hb Val), 1.70–1.60 (m, 3H; Hb Leu, Hb Pro,
Hg Pro), 1.56 (m, 1H; Hg Pro), 1.49 (m, 1H; Hg Leu), 1.32 (m, 1H; Hb
Leu), 0.85–0.80 ppm (m, 12H; 6ꢃHd Leu, 6ꢃHg Val); 13C NMR
(100 MHz, MeOD/CDCl3 (1:5), T=330 K): d=173.2, 173.2, 172.0, 171.6,
171.5 (5ꢃC=O, Leu, PAA, d-Phe, Pro, Val), 137.0 (Cq Ar), 132.6, 132.2,
132.1, 131.8, 131.7, 130.0, 129.5, 129.4, 129.3, 129.1, 127.8 (CHar), 61.2 (Ca
Pro), 54.7 (Ca d-Phe), 52.0 (Ca Leu), 50.8 (Ca PAA), 47.3 (Cd Pro), 40.9
(Cb Leu), 38.6 (Cb d-Phe), 33.8 (Cb PAA), 32.2 (Cb Val), 29.6 (Cb Pro),
25.5 (Cg Leu), 24.5 (Cg Pro), 22.8, 22.6, 19.5, 18.4 ppm (2ꢃCd Leu, 2ꢃCg
Val); HRMS (ESI): m/z calcd for C80H101N10O10P2S2 [M+H]+:
1487.66133; found: 1487.66205; IR (thin film): n˜ =3290, 2958, 1700, 1688,
1684, 1668, 1662, 1645, 1635, 1563, 1558, 1554, 1544, 1539, 1532, 1520,
1516, 1506, 1490, 1472, 1456, 1436, 1312, 1158, 1104, 799, 746, 694, 668, 6–
General procedure for the determination of the enantiomeric purity of
1–3: In an NMR tube, amino acid methyl esters 1, 2, or C-deprotected
7b (0.035 mmol) and DMAP (8.5 mg, 0.070 mmol) were dissolved in
CDCl3 (0.5 mL) yielding the free amino acids. Subsequent cooling to 08C
was followed by addition of Mosherꢁs acid chloride (8.0 mL, 0.043 mmol)
and the reaction mixture was allowed to warm up to room temperature
and after 1 h 19F{1H}-NMR spectra were recorded. This reaction scheme
was carried out twice: a) with enantiomerically pure (R)-Mosherꢁs acid
chloride, and b) with a 1:1 mixture of (R)- and (S)-Mosherꢁs acid. The
ratio between the diastereomeric pairs was determined by integration of
the 19F NMR signals.
For 1: a) 19F{1H} NMR (CDCl3): d=ꢀ68.8, ꢀ69.0 ppm (ratio 94:6); b)
19F{1H} NMR (CDCl3): d=ꢀ68.8, ꢀ69.0 ppm (ratio 1:1).
For 2: a) 19F{1H} NMR (CDCl3): d=ꢀ69.54, ꢀ69.86 ppm (ratio 200:1); b)
19F{1H} NMR (CDCl3): d=ꢀ69.54, ꢀ69.86 ppm (ratio 1:1).
For
3
(C-deprotected 7b): a) 19F{1H} NMR (CDCl3): d=ꢀ69.21,
ꢀ69.32 ppm (ratio 200:1); b) 19F{1H} NMR (CDCl3): d=ꢀ69.21,
ꢀ69.32 ppm (ratio: 1:1).
General procedure for the synthesis of cyclic decapeptides 19a–c
Loading of resin: Commercially available Wang (polystyrene) resin
(2.00 g, 1.1 mmolgꢀ1, 2.2 mmol) was allowed to swell in CH2Cl2 (50 mL).
A solution was prepared of N,N’-diisopropylcarbodiimide (DIC; 1.12 mL,
0.92 g, 7.26 mmol, 3.3 equiv), Fmoc-Leu-OH (2.33 g, 6.60 mmol,
3.0 equiv) and DMAP (cat.) in CH2Cl2. The mixture was left overnight
with occasional shaking. The resin was filtered, washed subsequently with
DMF and CH2Cl2 and dried (air). The loading of the resin was deter-
mined by treatment of the dried resin (2.69 mg) with a solution of piperi-
dine in N-methylpyrrolidone (NMP; 1:4, v/v, 1.0 mL). After stirring for
10 min followed by dilution to 10.00 mL with EtOH, the absorption of
the solution was measured at 300 nm. The loading was calculated to be
8, 592, 506 cmꢀ1
cyclo-[Pro-Val-PAA2-Leu-d-Phe]2 ACTHNGUTRNE(NGU 19b): The synthesis was performed
.
as outlined above to obtain 336 mg (101 mmol) immobilized decapeptide
18b. Cleavage from the resin, followed by cyclization and purification
yielded compound 19b (86 mg, 56%). 31P NMR (162 MHz, MeOD/
CDCl3 (1:5), T=302 K): d=45.5 ppm (s); 1H NMR (400 MHz, MeOD/
CDCl3 (1:5), T=302 K): d=8.73 (d, 1H; JNH,Ha =9.3 Hz, NH Leu), 8.41
(d, 1H; JNH,Ha =9.0 Hz, NH PAA), 8.33 (d, 1H; JNH,Ha =4.3 Hz, NH d-
Phe), 8.04–7.98 (m, 2H; Har), 7.82–7.76 (m, 2H; Har), 7.47–7.43 (m, 4H;
NH Val, 3ꢃHar), 7.40–7.33 (m, 3H; Har), 7.24 (m, 3H; Har), 7.04 (m, 2H;
Har), 4.88 (m, 1H; Ha PAA), 4.57 (m, 1H; Ha Leu), 4.38 (m, 1H; Ha d-
Phe), 4.08 (m, 1H; Ha Pro), 4.04 (m, 1H; Ha Val), 3.62 (M, 1H; Hd Pro),
2.96–2.86 (m, 3H; 2ꢃHb d-Phe, Hg PAA), 2.65 (m, 1H; Hg PAA), 2.37
(m, 1H; Hg Pro), 2.23 (m, 1H; Hb Val), 2.15 (m, 1H; Hb PAA), 2.01–1.91
(m, 2H; Hb PAA, Hb Pro), 1.62 (m, 1H; Hb Leu), 1.56–1.48 (m, 4H; Hb
Leu, Hb Pro, 2ꢃHg Pro), 1.40 (m, 1H; Hb Leu), 0.91–0.84 ppm (m, 12H;
6ꢃHd Leu, 6ꢃHg Val); 13C NMR (100 MHz, MeOD/CDCl3 (1:5), T=
302 K): d=173.5, 173.0, 172.5, 171.9, 171.9 (5ꢃC=O, Leu, PAA, d-Phe,
Pro, Val), 136.7, 134.7, 133.8, 133.0 (Cq Ar), 132.7, 132.6, 132.2, 132.1,
132.0, 130.2, 129.5, 129.4, 129.3, 129.2, 128.0 (CHar), 61.3 (Ca Pro), 60.1
(Ca Val), 55.4 (Ca d-Phe), 53.8 (Ca PAA), 51.1 (Ca Leu), 47.1 (Cd Pro),
41.0 (Cb Leu), 37.3 (Cb d-Phe), 31.5 (Cb Val), 29.9 (Cb Pro), 28.2 (Cg
PAA), 26.3 (Cb PAA), 25.4 (Cg Leu), 24.2 (Cg Pro), 23.1, 23.0, 19.4,
18.7 ppm (2ꢃCd Leu, 2ꢃCg Val); HRMS (ESI): m/z calcd for
C82H105N10O10P2S2 [M+H]+: 1515.69263; found: 151.69324; IR (thin film):
n˜ =3272, 2958, 2364, 2338, 1657, 1652, 1532, 1506, 1436, 1105, 737, 693,
0.59 mmolgꢀ1, using the formula: Loading (mmolgꢀ1)=[A300]ꢃ10/
(m=2.69 mg).
ACHTUNGTRNE[NUNG 7.8ꢃm]
Stepwise elongation on a 0.2 mmol scale: 1) The Fmoc-Leu loaded resin
was swollen with NMP followed by treatment with piperidine in NMP
(1:4, v/v, 4 mL 2ꢃ10 min) to effect Fmoc cleavage. The resin was filtered,
washed with NMP and CH2Cl2, filtered, and dried (air); 2a) coupling of
1–3 (1.5 equiv) in the presence of O-(7-azabenzotriazol-1-yl)tetramethyl-
uronium hexafluorophosphate (HATU; 1.45 equiv) and N,N-diisopropyl-
ethylamine (DiPEA; 3.0 equiv) which was pre-activated for 1 min in
NMP (4 mL) and subsequently shaken for 16 h; or 2b) coupling of the
appropriate commercially available amino acid (Fmoc-Val-OH, Fmoc-
Pro-OH, Fmoc-d-Phe-OH, Fmoc-Leu-OH) (4.0 equiv) in the presence of
O-(6-chlorobenzotriazole-1-yl)tetramethyluronium hexafluorophosphate
(HCTU; 4.0 equiv) and DiPEA (8.0 equiv), which was pre-activated for
1 min in NMP (4 mL) and subsequently shaken for 90 min; 3) washing
with NMP and CH2Cl2. Couplings were monitored for completion by the
Kaiser test[23b] or by a chloranil test after couplings with Pro-residues.[23c]
By means of this procedure the immobilized linear decapeptides (18a–c)
were obtained.
668, 610, 515 cmꢀ1
.
cyclo-[Pro-Val-PAA3-Leu-d-Phe]2 ACTHNGURTNE(NUG 19c): The synthesis was performed as
outlined above to obtain 384 mg (119 mmol) immobilized decapeptide
18c. Cleavage from the resin, followed by cyclization and purification
yielded compound 19c (91 mg, 46%). 31P NMR (162 MHz, MeOD/
CDCl3 (1:1), T=298 K): d=44.1 ppm (s); 1H NMR (400 MHz, MeOD/
CDCl3 (1:1), T=298 K): d=8.82 (d, 1H; JNH,Ha =9.1 Hz, NH Leu,), 8.39
(d, 1H; JNH,Ha =4.2 Hz, NH d-Phe), 7.96 (d, 1H; JNH,Ha =9.1 Hz, NH
PAA), 7.65–7.57 (m, 7H; Har), 7.50–7.36 (m, 7H; NH Val, 6ꢃHar), 7.29–
7.19 (m, 6H; Har), 5.32 (m, 1H; Ha PAA), 4.51 (m, 1H; Ha Leu), 4.43
Cleavage from the resin: Resin 18a–c was swollen with NMP followed by
treatment with piperidine in NMP (1:4, v/v, 4 mL 2ꢃ10 min) to effect
Fmoc cleavage. The resin was filtered, washed with NMP and CH2Cl2, fil-
tered, and dried (air). The immobilized peptide was treated with mixture
of TFA/TIS/H2O (95:2.5:2.5, 8.0 mL) and shaken for 45 min and filtered.
The resin was rinsed with
a mixture of TFA/TIS/H2O (95:2.5:2.5,
2.0 mL). All fractions were collected and co-evaporated with toluene.
(m, 1H; Ha d-Phe), 4.37 (m, 1H; Ha Pro), 4.00 (dd, 1H; JHa,NH =JHa,Hb
=
Cyclization: The appropriate crude linear decapeptide was taken up in
DMF (5.0 mL) and added dropwise over the course of 1 h to a solution
of benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate
(PyBOP; 5.0 equiv), 1-hydroxybenzotriazole (HOBt; 5.0 equiv) and
DiPEA (15.0 equiv) in DMF (150 mL) and allowed to stir for 16 h. The
mixture was concentrated, directly applied to a LH20 Sephadex size-ex-
clusion column eluted with MeOH and purified by RP-HPLC.
9.2 Hz, Ha Val), 3.66 (m, 1H; Hd Pro), 3.03 (m, 3H; 2ꢃHb d-Phe, Hb
PAA), 2.95 (m, 1H; Hb PAA), 2.33 (m, 1H; Hd Pro), 2.25 (m, 1H; Hb
Val), 1.66–1.52 (m, 6H; 2ꢃHb Leu, Hg Leu, Hb Pro, 2ꢃHg Pro), 0.93–
0.88 ppm (m, 12H; 6ꢃHd Leu, 6ꢃHg Val); 13C NMR (100 MHz, MeOD/
CDCl3 (1:1), T=298 K): d=173.1, 172.5, 171. 9, 171.8, 171.6 (5ꢃC=O,
Leu, PAA, d-Phe, Pro, Val), 136.2, 134.5, 133.9 (Cq Ar), 132.7, 132.6,
132.6, 132.5, 131.8, 130.1, 130.0, 129.8, 129.0, 129.0, 128.9, 128.8, 127.4
Chem. Eur. J. 2009, 15, 8134 – 8145
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
8143

K. Lammertsma, M. J. van Raaij, M. Overhand et al.
(CHar), 60.9 (Ca Pro), 59.7 (Ca Val), 55.0 (Ca d-Phe), 52.7 (Ca PAA), 51.0
(Ca Leu), 46.8 (Cd Pro), 40.4 (Cb Leu), 37.6, 37.2 (Cb d-Phe, Cb PAA),
30.5 (Cb Val), 29.8 (Cb Pro), 25.0 (Cg Leu), 24.0 (Cg Pro), 22.9, 22.7, 19.2,
19.1 ppm (2ꢃCd Leu, 2ꢃCg Val); HRMS (ESI): m/z calcd for
C92H109N10O10P2S2 [M+H]+: 1639.72393; found: 1639.72534; IR (thin
film): n˜ =3272, 2958, 2363, 1718, 1700, 1684, 1657, 1652, 1563, 1558, 1540,
eyda, J. Am. Chem. Soc. 2001, 123, 755–756; d) C. A. Luchaco-
Cullis, H. Mizutani, K. E. Murphy, A. H. Hoveyda, Angew. Chem.
2001, 113, 1504–1508; Angew. Chem. Int. Ed. 2001, 40, 1456–1460;
e) H. Mizutani, S. J. Degrado, A. H. Hoveyda, J. Am. Chem. Soc.
2002, 124, 779–780; f) A. W. Hird, A. H. Hoveyda, Angew. Chem.
2003, 115, 1314–1317; Angew. Chem. Int. Ed. 2003, 42, 1276–1279;
g) A. H. Hoveyda, A. W. Hird, M. A. Kacprzynski, Chem. Commun.
2004, 1779–1785; h) M. K. Brown, S. J. Degrado, A. H. Hoveyda,
Angew. Chem. 2005, 117, 5440–5444; Angew. Chem. Int. Ed. 2005,
44, 5306–5310; i) C. Gennari, U. Pinarulli, Chem. Rev. 2003, 103,
3071–3100.
1520, 1506, 1472, 1436, 1190, 1101, 747, 715, 692, 668, 612, 510 cmꢀ1
.
Crystallization: 8b: Suitable red needles were obtained from a solution
of 8b in acetonitrile at 48C. 8c: Orange needle-shaped crystals were ob-
tained after vapor diffusion of the compound dissolved in ethyl acetate at
15 mgmLꢀ1 against hexane. 19b: Colorless plate-shaped crystals were ob-
tained after slow cooling (from 658C to 48C) of a solution of 19b in
methanol at 9.7 mgmLꢀ1. 19c: Colorless prism-shaped crystals were ob-
tained after slow evaporation of 2 mL droplets of 29 mgmLꢀ1 peptide in
dimethyl formamide (DMF) plus 1 mL of tert-butanol under paraffin oil
in Terasaki plates.
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Crystal structure determination of 8b, 8c, 19b, and 19c: A crystal was
mounted in air and then rapidly transferred to a low-temperature nitro-
gen-gas stream. Data were collected on a Bruker KappaAPEXII diffrac-
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and refined using a riding atom model except those corresponding to the
water molecules in 8b and 19c that were located in difference-Fourier
maps. There were two crystallographically independent molecules per
asymmetric unit in the crystal structures of 8c and 19b. Selected crystal-
lographic data are reported in Table 1. There were several disordered
parts in the 19b structure: both leucine side chains in molecule 1, one
complete alkylphosphinyl group in molecule 2 and all water solvent mol-
ecules. The extensive degree of disorder together with the pseudo-trans-
lation symmetry present in the 19b structure hinder the possibility of ani-
sotropic refinement; therefore all atoms were refined using isotropic dis-
placement parameters and with restraints in the geometry.
CCDC-718718 (8b), 718719 (8c), 718720 (19b) and 718721 (19c) contain
the supplementary crystallographic data for this paper. These data can be
obtained free of charge from The Cambridge Crystallographic Data
Centre via www.ccdc.cam.ac.uk/data_request/cif.
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Received: April 28, 2009
Published online: July 14, 2009
Chem. Eur. J. 2009, 15, 8134 – 8145
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