α,α-Disubstituted Glycines Bearing a Large Hydrocarbon Ring: Peptide Self-Assembly through Hydrophobic Recognition

Article abstract of DOI:10.1002/chem.200305492

A method was developed for synthesizing α,α-disubstituted glycine residues bearing a large (more than 15-membered) hydrophobic ring. The ring-closing metathesis reactions of the dialkenylated malonate precursors proceed efficiently, particularly when long methylene chains tether both terminal olefin groups. Surprisingly, the amino groups of these α,α-disubstituted glycines are inert to conventional protective reactions (e.g., N-tert-butoxycarbonyl (Boc) protection: Boc ₂O/4-dimethylaminopyridine (DMAP)/CH₂Cl₂; N-benzyloxycarbonyl (Z) protection: Z-Cl/DMAP/CH₂Cl₂). Curtius rearrangement of the carboxylic acid functionality of the malonate derivative after ring-closing metathesis leads to formation of an amine functionality and can be catalyzed by diphenylphosphoryl azide. However, only the intermediate isocyanates can be isolated, even in the presence of alcohols such as benzyl alcohol. The isocyanates obtained by Curtius rearrangement in an aprotic solvent (benzene) were isolated in high yields and treated with 9-fluorenylmethanol in a high-boiling-point solvent (toluene) under reflux to give the N-9-fluorenylmethoxycarbonyl (Fmoc)-protected aminomalonate derivatives in high yield. These hydrophobic amino acids can be incorporated into a peptide by Fmoc solid-phase peptide synthesis and the acid fluoride activation method. The stability of the monomeric α-helical structure of a 17-amino-acid peptide was enhanced by replacement of two alanine residues with two hydrophobic amino acid residues bearing a cyclic 18-membered ring. The results of sedimentation equilibrium studies suggested that the peptide assembles into hexamers in the presence of 100 mM NaCl.

Full text of DOI:10.1002/chem.200305492

FULL PAPER
a,a-Disubstituted Glycines Bearing a Large Hydrocarbon Ring: Peptide Self-
Assembly through Hydrophobic Recognition
Tomohiko Ohwada,*[a] Daisuke Kojima,[a] Tatsuto Kiwada,[b] Shiroh Futaki,*[b]
Yukio Sugiura,[b] Kentaro Yamaguchi,[c] Yoshinori Nishi,[d] and Yuji Kobayashi[d]
Abstract: A method was developed for
synthesizing a,a-disubstituted glycine
residues bearing a large (more than 15-
membered) hydrophobic ring. The
ring-closing metathesis reactions of the
dialkenylated malonate precursors pro-
ceed efficiently, particularly when long
methylene chains tether both terminal
olefin groups. Surprisingly, the amino
groups of these a,a-disubstituted gly-
cines are inert to conventional protec-
tive reactions (e.g., N-tert-butoxycar-
bonyl (Boc) protection: Boc2O/4-dime-
tionality of the malonate derivative
after ring-closing metathesis leads to
formation of an amine functionality
and can be catalyzed by diphenylphos-
phoryl azide. However, only the inter-
mediate isocyanates can be isolated,
even in the presence of alcohols such
as benzyl alcohol. The isocyanates ob-
tained by Curtius rearrangement in an
aprotic solvent (benzene) were isolated
in high yields and treated with 9-fluore-
nylmethanol in a high-boiling-point sol-
vent (toluene) under reflux to give the
N-9-fluorenylmethoxycarbonyl (Fmoc)-
protected aminomalonate derivatives
in high yield. These hydrophobic
amino acids can be incorporated into a
peptide by Fmoc solid-phase peptide
synthesis and the acid fluoride activa-
tion method. The stability of the mono-
meric a-helical structure of
a 17-
amino-acid peptide was enhanced by
replacement of two alanine residues
with two hydrophobic amino acid resi-
dues bearing
a cyclic 18-membered
thylaminopyridine
(DMAP)/CH2Cl2;
ring. The results of sedimentation equi-
librium studies suggested that the pep-
tide assembles into hexamers in the
presence of 100 mm NaCl.
Keywords: amino acids ¥ peptides ¥
N-benzyloxycarbonyl (Z) protection:
Z-Cl/DMAP/CH2Cl2). Curtius rear-
rangement of the carboxylic acid func-
protein
design
¥
ring-closing
metathesis ¥ self-assembly
Introduction
Hydrophobic amino acids play an essential role in the mo-
lecular architectures of proteins and peptides. These amino
acids are commonly found in the transmembrane regions of
membrane proteins and ion channels, embedded in lipid bi-
layers.[1] Farnesylation (15 carbon atoms) and geranylgerany-
lation (20 carbon atoms) of cysteine side chains are known
to have dramatic effects on the hydrophobicity of proteins
and have been proposed to be important for signal transduc-
tion.[2] Hydrophobic interaction also often plays a crucial
role in protein folding and assembly in water. Thus, the con-
trol of molecular recognition of hydrophobic surfaces is re-
garded as one of the key elements in the rational design of
artificial proteins and protein-interacting molecules with
pharmaceutical potential.[3] Various unnatural amino acids
with hydrophobic side chains have been explored as building
blocks for peptides that provide novel hydrophobic cores.[4]
Amino acids with Ca,a-disubstituted cycloaliphatic groups
are intriguing because a,a-disubstitution constrains the con-
formation of a peptide chain and also causes changes in hy-
drophobicity.[5,6] A representative Ca,a-disubstituted gly-
cine, a-aminoisobutyric acid (Aib), is known to induce a 310/
[a] Prof. Dr. T. Ohwada, D. Kojima
Graduate School of Pharmaceutical Sciences
The University of Tokyo, Hongo, Bunkyo-ku
Tokyo 113-0033 (Japan)
Fax : (+81)3-58414735
E-mail: ohwada@mol.f.u-tokyo.ac.jp
[b] T. Kiwada, Prof. Dr. S. Futaki, Prof. Dr. Y. Sugiura
Institute for Chemical Research
Kyoto University, Uji
Kyoto 611-0011 (Japan)
Fax : (+81)774-323038
E-mail: futaki@scl.kyoto-u.ac.jp
[c] Prof. Dr. K. Yamaguchi
Chemical Analysis Center
Chiba University, Yayoi-cho, Inage-ku
Chiba 263-8522 (Japan)
[d] Y. Nishi, Prof. Dr. Y. Kobayashi
Graduate School of Pharmaceutical Sciences
Osaka University, Suita
Osaka 565-0871 (Japan)
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
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Chem. Eur. J. 2004, 10, 617 626
DOI: 10.1002/chem.200305492
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

FULL PAPER
a-helical protein structure.[5] A series of systematic studies
by Toniolo and co-workers has established that oligopepti-
des containing Ca,a-disubstituted cycloaliphatic groups
show a preference for 310-helical and b-bend structures.[6]
Their work has provided considerable information about the
effects of amino acids with these side chains upon local sec-
ondary structures of the peptide molecule. However, noth-
ing is yet known about amino acids bearing a larger hydro-
carbon moiety (a 15-membered or larger ring) at the a,a-po-
sitions and the effects of such amino acids in longer peptides
of 15 20 amino acids.
oalkenylated product 5 was moderate to high, though the re-
action is accompanied by formation of the corresponding di-
alkenylated compound as a minor product (6 (except 6b),
see Table 1). The yields of the second alkenylation step are
generally high and are insensitive to the chain length of the
second alkenyl bromide (Table 2).
The ring-closing metathesis reactions of the dialkenyl pre-
cursors 6 were carried out by treatment with Grubbs× ruthe-
nium catalyst in dichloromethane (Table 3).[7] The yields of
the desired cyclized products 7 were moderate to good,
except for substrates containing a cyano group. The olefin
products were obtained as mixtures of the E and Z forms;
the ratio of these isomers was evaluated on the basis of the
13C signals of the olefin moiety. The olefins were hydrogen-
ated over Pd/C.
Herein, we describe a general approach to the synthesis
of amino acids fused with a large saturated hydrocarbon
ring at the a,a-positions (1, 2, and 3, Scheme 1).[6] Incorpo-
9-Fluorenylmethoxycarbonyl (Fmoc) solid-phase synthesis
has become the most widely used method for peptide syn-
thesis. Alternatively, the amino acid fluoride method can be
used as a coupling system for the introduction of Aib into a
peptide chain.[9] We used a combination of acid fluoride and
N-Fmoc chemistry for the synthesis of peptides containing
Ca,a-disubstituted cycloaliphatic amino acids. To prepare
the Fmoc amino acid fluorides 12A C, we started from
benzyl tert-butyl malonate 4c (Scheme 3). The cyclic olefin 7
was prepared by means of ring-closing metathesis and hy-
drogenated, then reductive debenzylation of the ester gave
the saturated half acids 8A C. Curtius rearrangement of the
carboxylic acid functionalities of 8A C to amine functionali-
ties was catalyzed by DPPA[10] but, unexpectedly, only the
intermediate isocyanates 9A C could be isolated, even in
the presence of alcohols such as benzyl alcohol.[11] We car-
ried out Curtius rearrangement of 8A C in an aprotic sol-
vent (benzene) and isolated the isocyanates 9A C in high
yields. These isocyanates were treated with 9-fluorenylme-
thanol in the high-boiling-point solvent toluene under reflux
to give the N-Fmoc-protected esters 10A C in high yield.
These compounds was treated with TFA to give the acids 11
A C, and then with DAST to give the corresponding acid
fluorides 12A C.[9] Surprisingly, the amino groups of the re-
lated amine compounds 10 (R2 =H, Et ester, derived from 7
b, 7j, and 7g) were inert to protective reactions (N-tert-bu-
toxycarbonyl (Boc) protection: Boc2O/4-dimethylaminopyri-
dine (DMAP)/CH2Cl2; N-benzyloxycarbonyl (Z) protection:
Z-Cl/DMAP/CH2Cl2).
Scheme 1. Macrocyclic a,a-disubstituted amino acids.
ration of two amino acid residues 2 bearing a cyclic 18-mem-
bered ring (referred to as C18) into an a-helical peptide re-
sulted in the formation of a quaternary assembly through in-
teractions of the hydrophobic core.[4]
Results and Discussion
Synthesis of a,a-disubstituted glycines bearing a large hy-
drocarbon ring: a,a-Amino acids bearing 21-membered (1),
18-membered (2), 15-membered (3), and 14-membered
rings, respectively, were synthesized efficiently through ring-
closing metathesis reactions of the appropriate dialkenyl
precursors (6), which were derived from malonate deriva-
tives (Scheme 2).[7] Stepwise alkenylation of malonate deriv-
Single-crystal X-ray diffraction structure analysis of the
N-benzyl ethyl ester derivative of the 18-membered com-
pound 2 (Figure 1) showed a flat and unfolded 18-mem-
bered ring.[12] The crystal structure of 2 shows a left-handed
helical conformation (i.e. f (+45.48) and pseudo y(N1-C8-
C26-O3) (+41.88) are both positive). These backbone tor-
sion angles f and pseudo y are closer to the values expected
for an a helix (f=ꢀ558, y=ꢀ458) than to those expected
for a 310 helix (f=ꢀ578, y=ꢀ308).[5b,6b,6p,6q] In addition, the
bond angles indicate an asymmetric geometry for the Ca
atom in the crystal structure: the bond angles t(N1-C8-C9)
(111.48) and t(N9-C8-C25) (112.38) are greater than the
ideal tetrahedral angle (109.458), while the bond angles
t(C26-C8-C25) (107.98) and t(N1-C8-C26) (108.08) are
smaller than the tetrahedral value.
Scheme 2. General synthetic strategy for macrocyclization of malonate
derivatives. a) CH2=CH(CH2)mBr, NaH, DMSO, room temperature;
b) CH2=CH(CH2)nBr, NaH, DMSO, room temperature; c) [(PCy3)2Cl2-
Ru=CHPh], CH2Cl2, reflux. DMSO=dimethyl sulfoxide.
atives 4 in the presence of dimsyl sodium (NaH/DMSO) to
form the monoalkenylated intermediates 5 and then the dia-
lkenylated precursors 6 was more efficient than direct
double alkenylation, even when the same alkenyl bromide
was used in both steps.[8] In most cases, the yield of the mon-
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Chem. Eur. J. 2004, 10, 617 626

a,a-Disubstituted Glycines
617 626
Table 1. Alkenylation of malonate derivatives with CH2=CH(CH2)mBr.
To examine the feasibility of
solid-phase synthesis of amino
acids bearing bulky hydropho-
bic side chains, which might
cause considerable steric hin-
drance at the Ca-position, 17-
amino-acid model peptides II
IV were designed (Figure 2).
The design of these peptides
was based on the sequence of
the helical peptide I, which is
composed of alanine, lysine,
and glutamic acid residues and
has been described by Marqu-
see and Baldwin.[14] Peptide I
was shown to adopt a mono-
meric, a-helical structure by
forming Gluꢁ Lys+ salt bridges
between the side chains of
these residues. We replaced the
two alanine residues in peptide
I (positions 3 and 10) with a
C18 amino acid (peptide II) so
that the C18 rings were both
placed on the same side of the
helix. We hypothesized that this
arrangement might allow the
peptide to form an assembly
through the interaction of alter-
nate large hydrophobic side
chains with each other, as in a
zipper motif (see Figure 3).
Starting
Alkenylation products
Yield [%]
compound
m
n
X
Y
Mono-
Di-[a]
Yield [%]
4a
4b
4c
4d
4e
4a
4b
4c
4d
9
9
9
9
9
6
6
6
6
-
-
-
-
-
-
-
-
-
CO2Me
CO2Et
CO2Bn
CN
CO2Me
CO2tBu
CO2tBu
CO2Et
CN
CO2Me
CO2tBu
CO2tBu
CO2Et
5a
5b
5c
5d
5e
5f
5 g
5h
5i
64
82
68
33
42
65
79
67
51
6a
6b
6c
6d
6e
6f
6 g
6h
6i
7
0
13
23
14
11
5
CN
CO2Me
CO2Et
CO2Bn
CN
9
21
[a] Dialkenylation with CH2=CH(CH2)mBr took place in a single step.
Table 2. Second alkenylation of monoalkenylated malonate derivatives with CH2=CH(CH2)nBr.
Starting
compound
Dialkenylation
Product
m[a]
n
X
Y
Yield [%]
5a
5b
5c
5b
5d
5e
5f
5g
5h
5h
5a
5b
9
9
9
9
9
9
6
6
6
6
9
9
9
9
9
6
2CN
2CN
6
6
6
9
2
2
CO2Me
CO2Et
CO2Bn
CO2Et
CO
CN
CO2Me
CO2Et
CO2Bn
CO2Bn
CO2Me
CO2tBu
CO2tBu
CO2tBu
2Et
6a
6b
6c
6j
6k
6l
82
95
84
81
63
86
86
87
64
86
79
77
CO2Me
CO2tBu
CO2tBu
CO2tBu
CO2Me
CO2tBu
6f
6g
6h
6m
6n
6o
COMe
2
COEt
2
[a] The first alkenylation was carried out by treatment with CH2=CH(CH2)mBr (see Table 1).
Table 3. Olefin metathesis of dialkenyl glycine derivatives.
Conditions
Ring size Compound
m
n
X
Y
T[a] Time [h] Product Yield [%] E/Z ratio[b]
The energy-minimized struc-
tures of peptides I and II were
21
21
21
21
21
18
18
15
15
15
15
14
14
14
14
6a
6b
6c
6d
6e
6j
6m
6f
6g
6h
6i
9
9
9
9
9
9
6
6
6
6
6
9
9
9
9
9
9
9
9
9
6
9
6
6
6
6
2
2CN
2
2
CO2Me CO2Me
A
B
B
A
A
B
B
A
B
B
B
B
78
26
12
163
118[c]
12
20
24
16
14
7a
7b
7c
7d
7e
7j
64
8283:17
84
5
23
93
85:15
CO2Et
CO2Bn
CN
CO2tBu
CO2tBu
CO2Et
CN
obtained by using the OPLS-
64:36
77:2
79:21
50:50
[15]
3
AA force field. Two arrange-
CN
ments of the two 18-membered
rings that produce stable con-
CO2Et
CO2Bn
CO2tBu
CO2tBu
7m
7f
9245:55
43
formations
(Figure 3). Conformer
were
found
is
CO2Me CO2Me
66:34
A
CO2Et
CO2Bn
CN
CO2tBu
CO2tBu
CO2Et
7g
7h
7i
7o
7l
6249:51
7250:50
8
5
0
more stable than conformer B
by 11.2kcalmol ꢁ1 (OPLS-AA
force field and water as a sol-
vent).
We examined the effect of
the resulting hydrophobic cores
of the peptide structures on hel-
icity. For comparison, peptides
with one C18 amino acid resi-
due near the center (at posi-
67
20
nd
nd
-
6k
6l
6n
6o
CN
CO2Et
[d]
CN
COMe CO2Me
A
50
A
B
18[e]
21
7n
7k
36
32nd
52:48
2
COEt
CO2tBu
2
[a] A: room temperature; B: reflux. In both cases, the reactions were carried out in CH2Cl2. [b] Determined
from the relative intensities of the peaks of the olefinic carbon atoms in the 13C NMR spectra. [c] Additional
heating at reflux for 9 h. [d] Additional heating at reflux for 7 h. [e] Additional heating at reflux for 118 h.
Helical peptides containing the amino acid bearing an 18-
membered ring: The Fmoc solid-phase method has become
the most widely used technique of peptide synthesis because
of its easy handling.[13] Solution synthesis of oligopeptides
containing a,a-disubstituted cycloaliphatic amino acids with
medium-sized rings has been reported.[5,6] However, the ap-
plicability of the Fmoc solid-phase method to a,a-disubsti-
tuted glycines bearing a large cycloaliphatic ring has been
little studied.
tion 10, peptide III) or near the N terminus (at position 3,
peptide IV) were synthesized (Figure 2).
The peptide chains were constructed by Fmoc solid-phase
peptide synthesis on Rink amide resin.[16] Although the
steric hindrance at the Ca atom caused by the C18 ring
might lead to inhibition of peptide chain elongation, a com-
bination of Fmoc protection and acid fluoride activation al-
lowed successful addition of the C18 amino acid and the
next amino acids to the peptide chain.[9] A coupling system
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T. Ohwada, S. Futaki et al.
FULL PAPER
Figure 2. Amino acid sequences of peptides used in this study.
Scheme 3. Synthesis of the Fmoc amino acid fluorides of macrocyclic
a,a-disubstituted amino acids. a c) As in Scheme 2; d)H2, Pd/C, AcOEt;
e) DPPA, Et3N, benzene, reflux; f) 9-fluorenylmethanol, toluene, reflux;
g) TFA, CH2Cl2, room temperature; h) DAST, CH2Cl2, room tempera-
ture. DAST=diethylaminosulfur trifluoride, DPPA=diphenylphosphoryl
azide, TFA= trifluoroacetic acid.
Figure 1. Single-crystal X-ray diffraction structure of the N-benzyl ethyl
ester derivative of compound 2, which contains an 18-membered ring.
involving benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphoni-
um hexafluorophosphate (PyBOP),[17] 1-hydroxybenzotria-
zole (HOBt), and 4-methylmorpholine (NMM) was used for
the introduction of other amino acids to the peptide chain.
The construction of the peptide was straightforward. No
double coupling experiments were carried out for the intro-
duction of amino acids in this experiment. The N terminus
of the peptide chain was acetylated by treatment with acetic
anhydride in the presence of NMM. The peptide resin was
treated with trifluoroacetic acid/ethanedithiol (95:5) at 258C
Figure 3. Energy-minimized a-helical structures of a) peptide I (original
Baldwin peptide), b) conformer A of peptide II, c) confomer B of pep-
tide II. Conformers A and B differ in the orientations of the two 18-
membered rings. Conformer A is more stable than conformer B. Water
was used as a solvent in the force field calculations.
for 2h and the sample was then purified by HPLC separa-
tion to give a pure peptide. The proposed structure of the
product was supported by MALDI-TOF MS data. Total
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Chem. Eur. J. 2004, 10, 617 626

a,a-Disubstituted Glycines
617 626
yields of the peptides calculated on the basis of the amount
of starting resin were 48% (peptide I), 10% (peptide II), 16
% (peptide III), and 32% (peptide IV).[18] The retention
times of the peptides in reversed-phase HPLC analyses in
the same eluent provide information about the hydrophobic-
ity of the peptides: those containing the C18 amino acid
have a longer retention time than those without such a resi-
due. Peptide II has the longest retention time and is there-
fore likely to be the most hydrophobic: 12.0 min (peptide I),
29.0 min (peptide II), 20.0 min (peptide III), 22.3 min (pep-
tide IV).[19]
while the helicities of the other peptides (I, III, and IV) at
608C were approximately 30% of those at 58C.
The helicity of peptide II is highly dependent on salt con-
centration (Figure 5a). The [q]222/[q]208 ratio calculated from
the CD spectrum of peptide II in the presence of 100 mm
NaCl was approximately 1.7 ([q]222
:
ꢁ2.73î104 degcm2
dmolꢁ1; [q]208: ꢁ1.64î104 degcm2 dmolꢁ1), while the corre-
sponding value in the presence of 10 mm NaCl was 0.98
([q]222: ꢁ2.37î104 degcm2 dmolꢁ1; [q]208: ꢁ2.42î104 degcm2
dmolꢁ1). This considerable change in [q]222/[q]208 ratio sug-
gests that the peptide may have an aggregated structure in
100 mm NaCl.[20] In contrast, the [q]222/[q]208 ratio of peptide
I in the presence of 100 mm NaCl was not significantly dif-
The CD spectra of peptides I IV in the presence of
10 mm NaCl were all consistent with a helical structure
(Figure 4a). The helical content of these peptides can be
ferent from that with 10 mm NaCl (100 mm NaCl: [q]222
=
ꢁ1.85î104 degcm2 dmolꢁ1; [q]208 =ꢁ1.65î104 degcm2 dmolꢁ;
[q]222/[q]208 =1.1. 10 mm NaCl: [q]222 =ꢁ2.03î104 degcm2
dmolꢁ; [q]208 =ꢁ2.31î104 degcm2 dmolꢁ; [q]222/[q]208 =0.90;
Figure 5b).
Figure 4. a) CD spectra of the peptides containing a,a-disubstituted gly-
cine (2, C18) residue(s); b) the thermal stabilities of these peptides. Red
circles, peptide I (20 mm); green squares, peptide II (16 mm); blue dia-
monds, peptide III (12 mm); black triangles, peptide IV (13 mm). All pepti-
des were in a solution of 1 mm sodium citrate, 1 mm sodium borate, 1 mm
sodium phosphate, 10 mm sodium chloride (pH 7.3). Spectra were taken
at 208C (a), and with temperature elevation at 18Cminꢁ1 (b). The helical
Figure 5. The effect of salt concentration on the CD spectra of a) peptide
II, which contains a,a-disubstituted amino acid 2 (C18) and b) peptide I
(does not contain the C18 residue). Spectra were measured with the pep-
tides in solutions containing 100 mm sodium chloride (black) or 10 mm
sodium chloride (colored) and in both cases 1 mm sodium citrate, 1 mm
sodium borate, and 1 mm sodium phosphate (pH 7.3). The peptide con-
centration was 20 mm.
content of the peptides is compared in (b) in the form of the ratio [q]222
[qini]222, where [qini]222 denotes molecular ellipticity at 222 nm and 58C.
/
evaluated from the intensity of the absorption at 222 nm.
The [q]222 values in order of magnitude are: peptide II
(ꢁ2.37î104 degcm2 dmolꢁ1)>peptide I (ꢁ2.03î104 degcm2
dmolꢁ1)>peptide III (ꢁ1.65î104 degcm2 dmolꢁ1)ꢂpeptide
IV (ꢁ1.57î104 degcm2 dmolꢁ1). Peptide II showed signifi-
cantly higher thermal stability than the other peptides (in-
cluding the native peptide I; Figure 4b); although no signifi-
cant cooperative transition between the folded and unfolded
states was observed between 5 and 608C, the helical content
of peptide II at 608C was 62% of that observed at 5 8C,
The assembly of peptide II was assessed by sedimentation
equilibrium experiments at 40000 rpm (Figure 6a).[21,22]
Nonlinear least-squares fitting to Equation (1) (see the Ex-
perimental Section) gave an apparent molecular weight
(Mapp
)
of (1.13ꢀ0.056)î104, (1.19ꢀ0.060)î104, (1.27ꢀ
0.064)î104, or (1.24ꢀ0.062)î104 at peptide concentrations
of 195, 220, 400, and 900 mm, respectively. The results ob-
tained with a rotor speed of 30000 rpm gave similar values
621
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T. Ohwada, S. Futaki et al.
FULL PAPER
er. Although further study is necessary to elucidate the pre-
cise conformation of peptide II, it is clear that these a,a-di-
substituted glycines with large hydrophobic rings will be
useful as building blocks of peptide hyperstructure.
Experimental Section
General methods: All melting points were measured with a Yanaco
Micro melting point apparatus and the uncorrected values are reported.
Proton NMR spectra were measured on a JEOL Caliber-GX400 NMR
spectrometer at 400 MHz with tetramethylsilane as an internal reference
and CDCl3 as the solvent, unless otherwise specified. High-resolution
mass spectra (HRMS, EI+) and FAB mass spectra (FAB+) were record-
ed on a JEOL JMS-SX 102A spectrometer. Flash column chromatogra-
phy was carried out on silica gel (silica gel 60, 40 63 mm, Merck). The
combustion analyses were carried out in the microanalytical laboratory
of the Graduate School of Pharmaceutical Sciences, The University of
Tokyo. Matrix-assisted laser desorption ionization time-of-flight mass
spectra (MALDI-TOF MS) were recorded on an Applied Biosystems
Voyager-DE STR instrument. CD spectra were recorded on a Jasco J-600
spectrometer in a 2-mm cuvette. The full data for the synthetic materials
are given in the Supporting Information.
Monoalkenylation of malonate derivatives
Dimethyl monoundecenylmalonate (5a): A solution of dimethyl malonate
(4a; 141.1 mg, 1.07 mmol) in DMSO (5 mL) was added dropwise to a so-
lution of NaH (33.6 mg, 1.40 mmol, 1.3 equiv) in DMSO (4 mL) at room
temperature. A solution of 11-bromo-1-undecene (315.3 mg, 1.35 mmol,
1.3 equiv) in DMSO (5 mL) was added and the mixture was stirred at
room temperature for 4 h then added to ice-water and extracted with di-
ethyl ether (4î30 mL). The organic phase was washed with brine and
dried over Na2SO4. The residue (326.7 mg) obtained after evaporation of
the solvent was flash chromatographed (silica gel; EtOAc/nhexane
(1:18)) to give 6a (dialkenyl product; 30.9 mg, yield: 7%; colorless oil)
and 5a (monoalkenyl product; 194.2mg, yield: 64%; yellow oil).
5a: 1H NMR: d=5.813 (d, d, t, J=17.05, 10.27, 6.78 Hz, 1H; CH2=CH),
4.992(d, d, d, J=17.05, 3.67, 1.47 Hz, 1H; CH2=CH-cis), 4.929 (m, 1H;
CH2=CH-trans), 3.738 (s, 6H; 2îCOOCH3), 3.359 (t, J=7.52Hz, 1H;
CH), 2.063 2.009 (m, 2H; CH2=CHꢁCH2), 1.903 1.885 (m, 2H), 1.368
1.262 ppm (m, 14H).
6a: 1H NMR: d=5.881 (d, d, t, J=16.86, 10.08, 6.60 Hz, 2H; CH=CH2),
4.990 (d, d, d, J=17.05, 3.67, 1.47 Hz, 2H; CH=CH2-cis), 4.928 (m, 2H;
CH=CH2-trans), 3.704 (s, 6H; CO2CH3), 2.062 2.009 (m, 4H; CH2=CHꢁ
CH2), 1.877 1.834 (m, 4H), 1.367 1.117 ppm (m, 28H). HRMS: calcd for
C27H48O4: 436.3554; found: 436.3551.
Figure 6. a) Sedimentation equilibrium analysis of peptide II. Peptide II
(195 mm) was run at 40000 rpm at 208C in 1 mm sodium citrate, 1 mm
sodium borate, 1 mm sodium phosphate, and 10 mm sodium chloride
(pH 7.3). b) Relationship between the peptide concentration and ob-
served molecular weight (Mapp). The mean molecular weight (Mw), which
correponds to the reciprocal of the intercept on the ordinate at zero con-
centration, was calculated as (1.30ꢀ0.065)î104, which suggests that pep-
tide II exists as a hexamer in the given solution.
Benzyl tert-butyl undecenylmalonate (5c): Colorless oil (68% yield).
1H NMR: d=7.355 7.310 (m, 5H; Ph), 5.808 (d, d, t, J=17.04, 10.08,
6.60 Hz, 1H; CH2=CH), 5.163 (d, d, J=12.27, 12.27 Hz, 2H; PhCH2),
4.990 (d, d, d, J=17.04, 3.11, 1.47 Hz, 1H; CH2=CH-cis), 4.939 4.908 (m,
1H; CH2=CH-trans), 3.267 (t, J=7.51 Hz, 1H; CH), 2.034 (m, 2H; CH2=
CHꢁCH2), 1.855 (m, 2H), 1.381 (s, 9H; tert-butyl), 1.348 1.241 ppm (m,
total 14H).
6c (diundecanyl derivative): Colorless oil (13% yield). 1H NMR: d=
7.355 7.298 (m, 5H; Ph), 5.807 (d, d, t, J=16.85, 10.08, 6.60 Hz, 2H;
CH2=CH), 5.137 (s, 2H; PhCH2), 4.988 (d, d, d, J=17.04, 3.66, 1.65 Hz, 2
H; CH2=CH-trans), 4.925 (m, 2H; CH2=CH-cis), 2.034 (m, 4H; CH2=
CHꢁCH2), 1.822 (m, 4H), 1.325 (s, 9H; tert-butyl), 1.381 1.241 ppm (m,
total 28H).
of Mapp within the range of experimental error (5%). The
weight-average molecular weight for the peptide at infinite
dilution (Mw) was calculated as (1.30ꢀ0.065)î104 (Figure 6
b), which is very close to the molecular weight of the hex-
amer (12348). The symmetrical residuals and the small 95%
confidence intervals indicate the absence of the monomer
and aggregates other than the hexamer in solution.
In conclusion, we have developed a novel approach for
the preparation of a,a-disubstituted glycines bearing a large
hydrophobic ring. These amino acids could be readily incor-
porated into a peptide chain by Fmoc solid-phase peptide
synthesis and the acid fluoride activation method. A 17-
amino-acid peptide containing two residues substituted with
a C18 ring formed a stable helical structure, which suggests
a potential helicogenic effect of these new amino acids. This
result is consistent with the increased magnitude and ther-
mal stability of helicity indicated by CD spectroscopy for
this peptide. The 17-amino-acid peptide was also shown by
sedimentation equilibrium analysis to aggregate to a hexam-
Benzyl tert-butyl octenylmalonate (5h): Colorless oil (67% yield).
1H NMR: d=7.355 7.309 (m, 5H; Ph), 5.792(d, d, t, J=16.86, 10.08,
6.59 Hz, 1H; CH2=CH), 5.163 (d, d, J=12.27, 12.27 Hz, 2H; PhCH2),
4.983 (d, d, d, J=17.04, 3.48, 1.47 Hz, 1H; CH2=CH-trans), 4.23 (m, 1H;
CH2=CH-cis) 3.267 (t, J=7.51 Hz, 1H; CH), 2.023 (m, 2H; CH2=CH-
CH2), 1.857 (m, 2H), 1.382 (s, 9H; tert-butyl), 1.348 1.241 ppm (m, total
8H).
6h (dioctenyl derivative): Colorless oil (9% yield). 1H NMR: d=7.348
7.300 (m, 5H; Ph), 5.789 (d, d, t, J=17.04, 10.26, 6.78 Hz, 2H; CH2=CH),
5.137 (s, 2H; PhCH2), 4.926 (d, d, d, J=17.04, 3.66, 1.47 Hz, 2H; CH2=
622
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2004, 10, 617 626

a,a-Disubstituted Glycines
617 626
CH-trans), 4.922 (m, 2H; CH2=CH-cis), 2.016 (m, 4H; CH2=CHCH2),
1.824 (brt, J=8.43 Hz, 4H), 1.325 (s, 9H; tert-butyl), 1.354 1.115 ppm
(m, total 16H).
1.882 1.755 (m, 4H), 1.304 1.297 (s, 9H; tert-butyl), 1.422 1.052 ppm (m,
total 16H). E/Z=50/50 (determined from the 13C NMR signals).
Dimeric product: 1H NMR: d=7.343 7.289 (m, 10H; Ph), 5.352 5.320
(m, 4H; CH=CH), 5.136 (s, 4H; PhCH2), 2.044 1.953 (m, 8H; CH2ꢁCH=
CHꢁCH2), 1.835 (m, 8H), 1.318 (s, 18H; tert-butyl), 1.495 1.084 ppm (m,
total 32H).
Second alkenylation of malonate derivatives
Dimethyl diundecenylmalonate (6a): A solution of dimethyl undecenyl-
malonate (5a; 187.3 mg, 0.66 mmol) in DMSO (2mL) was added drop-
wise to a solution of NaH (29.6 mg, 1.2 mmol, 1.9 equiv) in DMSO
tert-Butyl 1-(benzoyloxycarbonyl)cyclooctadec-8-ene carboxylate (7m): A
solution of diene 6m (2.2836 g, 4.46 mmol) in dichloromethane (250 mL)
was added over a period of 30 min to a solution of Grubbs× catalyst
(196.8 mg, 0.24 mmol, 5 mol%) in dichloromethane (750 mL) bubbled
through with argon, at room temperature. The mixture was heated at
reflux with stirring under an Ar atmosphere for 20 h. After evaporation
of the solvent, the obtained residue was flash chromatographed (silica
gel; EtOAc/nhexane (1:30!1:20)) to give 7m (1.9908 g, 92%, colorless
oil), together with an undefined dimeric product (152.2 mg, 8% yield,
brown oil).
7m: 1H NMR: d=7.346 7.259 (m, 5H; Ph), 5.283 (m, 2H; CH=CH),
5.140 (s, 2H; PhCH2), 2.033 1.981 (m, 4H; CH2-CH=CHꢁCH2), 1.883
1.822 (m, 4H), 1.310 (s, 9H; tert-butyl), 1.423 1.080 ppm (m, total 22H).
E/Z=45/55 (determined from the 13C NMR signals).
(3 mL) at room temperature.
A solution of 11-bromo-1-undecene
(211.0 mg, 0.91 mmol, 1.4 equiv) in DMSO (1 mL) was added dropwise
and the mixture was stirred for 5.5 h at room temperature. The whole
mixture was poured into ice-water and extracted with diethyl ether. The
organic layer was washed with brine, dried over Na2SO4, and the solvent
was evaporated to give a residue (332.7 mg), which was flash chromato-
graphed (silica gel; EtOAc/nhexane (1:18)) to give 6a (237.3 mg, 82%
yield, colorless oil).
Benzyl tert-butyl diundecenylmalonate (6c): Colorless oil (84% yield).
Benzyl tert-butyl dioctenylmalonate (6h): Colorless oil (64% yield).
Benzyl tert-butyl octenylundecenylmalonate (6 m): Colorless oil (86%
yield). 1H NMR: d=7.355 7.298 (m, 5H; Ph), 5.798 (m, 2H; CH2=CH),
5.136 (s, 2H; PhCH2), 4.982(m, 2H; C H2=CH-trans), 4.923 (m, 2H;
CH2=CH-cis), 2.035 (m, 4H; CH2=CHꢁCH2), 1.823 (m, 4H), 1.325 (s, 9
H; tert-butyl), 1.398 1.092ppm (m, total 22H).
Dimeric product: 1H NMR: d=7.345 7.257 (m, 10H; Ph), 5.360 5.298
(m, 4H; CH=CH), 5.137 (s, 4H; PhCH2-), 2.006 1.961 (m, 8H; CH2-
CH=CHꢁCH2), 1.832(m, 8H), 1.302 (s, 18H;
tert-butyl), 1.495
Olefin ring-closing metathesis of dialkenyl glycine derivatives
1.084 ppm (m, total 44H).
Methyl 1-(methoxycarbonyl)cyclohenicos-11-enecarboxylate (7a): A solu-
tion of diene 6a (234.3 mg, 0.54 mmol) in dichloromethane (10 mL) was
added over a period of 1 min to a solution of Grubbs× catalyst (benzyli-
Synthesis of acid fluorides 13A C
1-(tert-butoxycarbonyl)cyclohenicosane carboxylic acid (8A): A solution
of tbutyl 1-(benzyloxycarbonyl)cyclohenicos-11-enecarboxylate (7c;
2.0146 g, 3.83 mmol) in ethyl acetate (450 mL) was hydrogenated over 10
% Pd/C (265.0 mg) at room temperature for 24 h. Filtration and evapora-
tion of the solvent gave a residue (1.8042g), which was flash chromato-
graphed (CHCl3/MeOH (40:1)) to give the saturated product 8A
(1.3996 g, 83% yield) as a colorless solid. M.p.: 89.2 91.38C. (colorless
plates, recrystallized from nhexane). 1H NMR: d=1.870 (m, 4H), 1.474
(s, 9H; tert-butyl), 1.290 1.126 ppm (m, total 36H). Elemental analysis
calcd (%) for C27H50O4: C 73.92, H 11.49; found: C 73.77, H 11.54.
dene-bis(tricyclohexylphosphine)dichlororuthenium;
30.9 mg,
0.038 mmol, 7.0 mol%) in dichloromethane (50 mL) bubbled through
with argon and the mixture was stirred under an Ar atmosphere at room
temperature for 16.5 h. Since the reaction was incomplete, Grubbs× cata-
lyst (21.6 mg, 0.025 mmol, 4.6 mol%) was added again and the whole
mixture was stirred for 24 h. After evaporation of the solvent, the ob-
tained residue (290.0 mg) was flash chromatographed (silica gel; EtOAc/
n-hexane (1:22)) to give 7a (140.4 mg, 64%). M.p.: 71.3 72.38C (color-
less plates, recrystallized from methanol). 1H NMR: d=5.330 (m, 2H;
CH=CH), 3.700 (s, 6H; CO2CH3), 2.010 (m, 4H), 1.882 (m, 4H), 1.326
1.090 ppm (m, 28H). E/Z=83/17 (determined from the 13C NMR sig-
nals). Elemental analysis calcd (%) for C25H44O4: C 73.48, H 10.85;
found: C 73.25, H 10.87. MS (EI): 408 [M]+.
1-(tert-butoxycarbonyl)cyclooctadecane carboxylic acid (8B): A solution
of tert-butyl 1-(benzyloxycarbonyl)cyclooctadec-8-enecarboxylate (7m;
1.9908 g, 4.11 mmol) in a mixture of ethyl acetate (250 mL) and methanol
(50 mL) was hydrogenated over 10% Pd/C (268.8 mg) at room tempera-
ture for 24 h. Filtration and evaporation of the solvent gave a residue
(1.8042g), which was flash chromatographed (CHCl 3/MeOH (40:1)) to
give the saturated product 8B (1.3295 g, 82% yield) as a colorless solid.
M.p.: 110.5 112.08C (colorless plates, recrystallized from nhexane).
1H NMR: d=1.890 (m, 4H), 1.460 (s, 9H; tert-butyl), 1.401 1.192ppm
(m, total 30H). Elemental analysis calcd (%) for C24H44O4: C 72.68, H
11.18; found: C 72.56, H 11.27.
tert-Butyl 1-(benzyloxycarbonyl)cyclohenicos-11-enecarboxylate (7c): A
solution of diene 6c (2.5219 g, 4.55 mmol) in dichloromethane (250 mL)
was added over a period of 30 min to a solution of Grubbs× catalyst
(194.2mg, 0.24 mmol, 6 mol%) in dichloromethane (750 mL) bubbled
through with argon at room temperature. The mixture was heated at
reflux with stirring under an Ar atmosphere for 12h. After evaporation
of the solvent, the obtained residue (290.0 mg) was flash chromatograph-
ed (silica gel; EtOAc/nhexane (1:30)) to give 7c (2.0146 g, 84%, colorless
oil), together with an unidentified dimeric product (228.1 mg, yield: 10
%, pale yellow solid).
7c: 1H NMR: d=7.342 7.289 (m, 5H; Ph), 5.326 (m, 2H; CH=CH),
5.137 (s, 2H; PhCH2), 2.019 (m, 4H; CH2ꢁCH=CHꢁCH2), 1.844 (m, 4
H), 1.309 (s, 9H; tbutyl) 1.339 1.078 ppm (m, total 32H). E/Z=36/64
(determined from the 13C NMR signals).
1-(tert-butoxycarbonyl)cyclopentadecane carboxylic acid (8C): A solution
of tert-butyl 1-(benzyloxycarbonyl)cyclopentadec-8-enecarboxylate (7h;
1.0065 g, 2.28 mmol) in a mixture of ethyl acetate (300 mL) was hydro-
genated over 10% Pd/C (130.8 mg) at room temperature for 28 h. Filtra-
tion and evaporation of the solvent gave a residue (812.0 mg), which was
flash chromatographed (CHCl3/MeOH (30:1)) to give the saturated prod-
uct 8C (712.7 mg, 88% yield) as a colorless solid. M.p.: 137.2 138.68C
(colorless plates, recrystallized from nhexane). 1H NMR: d=1.698 (m, 4
H), 1.498 (s, 9H; tbutyl), 1.477 1.242 ppm (m, total 24H). Elemental
analysis calcd (%) for C21H38O4: C 71.15, H 10.80; found: C 71.03, H
10.93.
Dimeric product: 1H NMR: d=7.343 7.297 (m, 10H; Ph), 5.382 5.328
(m, 4H; CH=CH), 5.136 (s, 4H; PhCH2), 2.015 1.956 (m, 8H; CH2ꢁCH=
CHꢁCH2), 1.831 (m, 8H), 1.320 (s, 18H; tert-butyl), 1.436 1.082ppm (m,
total 64H).
tert-Butyl 1-isocyanatocyclohenicosanecarboxylate (9A): A solution of 1-
(tert-butoxycarbonyl)cyclohenicosane carboxylic acid (8A; 1.2402 g,
2.83 mmol), DPPA (1.0423 g, 3.79 mmol, 1.3 equiv), and triethylamine
(293.1 mg, 2.90 mmol, 1.0 equiv) in benzene (15 mL) was heated at reflux
under an argon atmosphere for 2h. After evaporation of the solvent, the
residue was flash chromatographed (silica gel; EtOAc/nhexane (1:25)) to
give the product 9A (1.1827 g, 96% yield) as a colorless oil. IR (neat):
tert-Butyl 1-(benzoyloxycarbonyl)cyclopentadec-8-ene carboxylate (7h):
A
solution of diene 6h (1.5216 g, 3.22 mmol) in dichloromethane
(200 mL) was added over 20 min to a solution of Grubbs× catalyst
(136.7 mg, 0.17 mmol, 5 mol%) in dichloromethane (500 mL) bubbled
through with argon, at room temperature. The mixture was heated at
reflux with stirring under an Ar atmosphere for 14 h. After evaporation
of the solvent, the obtained residue was flash chromatographed (silica
gel; EtOAc/nhexane (1:25!1:10) to give 7h (1.0259 g, 72%, colorless
oil), together with an undefined dimeric product (299.8 mg, 22% yield,
brown oil).
1
n˜ =2250 cmꢁ1 (N=C=O). H NMR: d=1.761 1.608 (m, 4H), 1.497 (s, 9H;
tert-butyl), 1.469 1.205 ppm (m, total 36H).
tert-Butyl 1-isocyanatocyclooctadecanecarboxylate (9B): A solution of 1-
(tert-butoxycarbonyl)cyclooctadecane carboxylic acid (8B; 1.2158 g,
3.07 mmol), DPPA (1.1067 g, 4.02mmol, 1.3 equiv), and triethylamine
7h: 1H NMR: d=7.347 7.257 (m, 5H; Ph), 5.398 5.321 (m, 2H; CH=
CH), 5.135 5.123 (s, 2H; PhCH2), 2.027 1.974 (m, 4H; CH=CHꢁCH2),
623
Chem. Eur. J. 2004, 10, 617 626
www.chemeurj.org
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

T. Ohwada, S. Futaki et al.
FULL PAPER
(319.8 mg, 3.17 mmol, 1.0 equiv) in benzene (15 mL) was heated at reflux
under an argon atmosphere for 3 h. After evaporation of the solvent, the
residue was flash chromatographed (silica gel; EtOAc/nhexane (1:25)) to
give the product 9B (1.1813 g, 98% yield) as a colorless oil. IR (neat):
n˜ =2250 cmꢁ1 (N=C=O). 1H NMR: d=1.698 (m, 4H), 1.498 (s, 9H; tert-
butyl), 1.477 1.242 ppm (m, total 30H).
and the organic layer was washed with brine and dried over Na2SO4. The
residue obtained by evaporation of the solvent was flash chromatograph-
ed (CHCl3/MeOH (30:1)) to give the acid 11B (361.8 mg, 98% yield) as
a colorless solid. M.p.: 184.0 185.18C (colorless needles, recrystallized
from nhexane/CH2Cl2). IR (KBr suspension) n˜ =1723 cmꢁ1 (C=O).
1H NMR: d=7.762(d, J=7.51 Hz, 2H; H4, H5), 7.577 (d, J=7.33 Hz, 2
H; H1, H8), 7.398 (d, d, J=7.33, 7.33 Hz, 2H; H3, H6), 7.310 (d, d, d, J=
7.51, 7.51, 1.10 Hz, 2H; H2, H7), 5.006 (brs, 1H; NH), 4.442(brm, 2H;
H10), 4.212 (t, J=6.96 Hz, 1H; H9), 1.876 (m, 4H), 1.295 1.209 ppm (m,
total 30H). Elemental analysis calcd (%) for C34H47NO4: C 76.51, H 8.88,
N 2.62; found: C 76.33, H 9.09, N 2.62.
tert-Butyl 1-isocyanatocyclopentadecanecarboxylate (9C): A solution of
1-(tert-butoxycarbonyl)cyclooctadecane carboxylic acid (8C; 641.0 mg,
1.81 mmol), DPPA (645.5 mg, 2.35 mmol, 1.3 equiv), and triethylamine
(188.1 mg, 1.86 mmol, 1.0 equiv) in benzene (10 mL) was heated at reflux
under an argon atmosphere for 2h. After evaporation of the solvent, the
residue was flash chromatographed (silica gel; EtOAc/nhexane (1:20)) to
give the product 9C (570.5 mg, 90% yield) as a colorless oil. IR (neat):
1-(Fmoc-amino)cyclopentadecanecarboxylic acid (11C): TFA (1 mL) was
added to a solution of tert-butyl 1-(Fmoc-amino)cyclopentadecanecarbox-
ylate (10C; 256.3 mg, 0.469 mmol) in dichloromethane (2 mL) at 08C.
The mixture was stirred at room temperature for 4 h then poured into
ice-water (40 mL). The solution was extracted with chloroform (3î
40 mL) and the organic layer was washed with brine and dried over
Na2SO4. The residue obtained by evaporation of the solvent was flash
chromatographed (CHCl3/MeOH (30:1)) to give the acid 11C (222.5 mg,
97% yield)) as a colorless solid. M.p.: 198.5 199.88C (colorless needles,
recrystallized from nhexane/CH2Cl2). 1H NMR: d=7.755 (d, J=7.51 Hz,
2H; H4, H5), 7.573 (d, J=7.14 Hz, 2H; H1, H8), 7.392(d, d, J=7.33,
7.33 Hz, 2H; H3, H6) 7.304 (d, d, d, J=7.51, 7.51, 1.28 Hz, 2H; H2, H7),
4.906 (brs, 1H; NH), 4.434 (brm, 2H; H10), 4.209 (m, 1H; H9), 1.820 (m,
3H), 1.344 1.295 ppm (m, total 25H). Elemental analysis calcd (%) for
C31H41NO4: C 75.73, H 8.41, N 2.85; found: C 75.54, H 8.45, N 2.82.
1
n˜ =2250 cmꢁ1 (N=C=O). H NMR: d=1.819 1.653 (m, 4H), 1.496 (s, 9H;
tert-butyl), 1.420 1.317 ppm (m, total 24H).
tert-Butyl 1-(Fmoc-amino)cyclohenicosanecarboxylate (10A): A solution
of tert-butyl 1-isocyanatocyclohenicosanecarboxylate (9A; 1.1748 g,
2.70 mmol) and 9-fluorenylmethanol (689.3 mg, 3.51 mmol, 1.3 equiv) in
dry toluene (10 mL) was heated at 1308C under an argon atmosphere for
42h. Flash chromatography (EtOAc/ nhexane (1:20)) of the mixture led
to the recovery of 9A (185.6 mg, 16%) and gave the N-Fmoc ester 10A
(1.4204 g, 73% yield) as a colorless oil. 1H NMR: d=7.757 (d, J=
7.51 Hz, 2H; H4, H5), 7.598 (d, J=7.14 Hz, 2H; H1, H8), 7.389 (d, d, J=
7.51, 7.51 Hz, 2H; H3, H6), 7.303 (d, d, d, J=7.51, 7.51, 1.10 Hz, 2H; H2,
H7), 5.568 (brs, 1H; NH), 4.344 (brd, J=6.05 Hz, 2H; H10), 4.213 (t, J=
6.59 Hz, 1H; H9), 2.076 (brm, 2H), 1.817 (brm, 2H), 1.292 (s, 9H; tBu),
1.509 1.088 ppm (m, total 36H).
1-(Fmoc-amino)cyclohenicosanecarboxylic acid fluoride (12A): A solu-
tion of DAST (133.7 mg, 0.829 mmol, 1.8 equiv) in dichloromethane
(2mL) was added to a solution of 1-(Fmoc-amino)cyclohenicosanecar-
boxylic acid (11A; 268.1 mg, 0.466 mmol) in dry dichloromethane (2 mL)
at 08C. The mixture was stirred at room temperature for 20 min then
poured into ice-water (20 mL). The solution was extracted with dichloro-
methane (2î20 mL) and the organic layer washed with brine and dried
over Na2SO4. The organic solvent was evaporated and the residue was
flash chromatographed (EtOAc/nhexane (1:12)) to give colorless solid 12
A (228.5 mg, 85% yield). IR (KBr suspension): n˜ =1838 cmꢁ1 (C=O).
1H NMR: d=7.763 (d, J=7.51 Hz, 2H; H4, H5), 7.568 (d, J=7.69 Hz, 2
H; H1, H8), 7.402(d, d, J=7.51, 7.51 Hz, 2H; H3, H6), 7.314 (d, d, d, J=
7.51, 7.51, 1.10 Hz, 2H; H2, H7), 4.961 (brs, 1H; NH), 4.477 (brm, 2H;
H10), 4.212 (t, J=6.41 Hz, 1H; H9), 1.880 (m, 4H), 1.374 1.241 ppm (m,
total 36H).
tert-Butyl 1-(Fmoc-amino)cyclooctadecanecarboxylate (10B): A solution
of tert-butyl 1-isocyanatocyclooctadecanecarboxylate (9B; 1.1703 g,
2.98 mmol) and 9-fluorenylmethanol (757.9 mg, 3.86 mmol, 1.3 equiv) in
dry toluene (3 mL) was heated at 1308C under an argon atmosphere for
12h. The mixture was flash chromatographed (EtOAc/ nhexane (1:12!
1:10)) to give N-Fmoc ester 10B (1.4454 g, 82% yield) as a colorless
amorphous solid. 1H NMR: d=7.759 (d, J=7.51 Hz, 2H; H4, H5), 7.598
(d, J=7.51 Hz, 2H; H1, H8), 7.392(d,d, J=7.33, 7.33 Hz, 2H; H3, H6),
7.303 (d, d, d J=7.51, 7.51, 1.10 Hz, 2H; H2, H7), 5.217 (brs, 1H; NH),
4.353 (brm, 2H; H10), 4.216 (t, J=6.78 Hz, 1H; H9), 1.882(m, 4H), 1.453
(s, 9H; tBu), 1.310 1.242 ppm (m, total 30H).
tert-Butyl 1-(Fmoc-amino)cyclopentadecanecarboxylate (10C): A solu-
tion of tert-butyl 1-isocyanatocyclopentadecanecarboxylate (9C;
561.2mg, 1.60 mmol) and 9-fluorenylmethanol (413.4 mg, 2.11 mmol,
1.3 equiv) in dry toluene (2mL) was heated at 130 8C under an argon at-
mosphere for 20 h. The mixture was flash chromatographed (EtOAc/
nhexane (1:8)) to give N-Fmoc ester 10C (859.1 mg, 98% yield) as a col-
orless amorphous solid. 1H NMR: d=7.759 (d, J=7.51 Hz, 2H; H4, H5),
7.598 (d, J=7.51 Hz, 2H; H1, H8), 7.393 (d, d, J=7.33, 7.33 Hz, 2H; H3,
H6), 7.304 (d, d, d, J=7.51, 7.51, 1.28 Hz, 2H; H2, H7), 4.888 (brs, 1H;
NH), 4.368 (brm, 2H; H10), 4.220 (t, J=6.76 Hz, 1H; H9), 1.889 1.739
(brm, 3H), 1.425 (s, 9H; tBu), 1.366 1.241 ppm (m, total 25H).
1-(Fmoc-amino)cyclooctadecanecarboxylic acid fluoride (12B): A solu-
tion of DAST (101.5 mg, 0.630 mmol, 1.3 equiv) in dichloromethane
(1 mL) was added to a solution of 1-(Fmoc-amino)cyclooctadecanecar-
boxylic acid (11B; 250.9 mg, 0.471 mmol) in dry dichloromethane (1 mL)
at 08C. The mixture was stirred at room temperature for 45 min then
poured into ice-water (20 mL). The solution was extracted with dichloro-
methane (2î20 mL) and the organic layer washed with brine and dried
over Na2SO4. The organic solvent was evaporated and the residue was
flash chromatographed (EtOAc/nhexane (1:12)) to give colorless solid 12
B (208.8 mg, 83% yield). IR (KBr suspension): n˜ =1838 cmꢁ1 (C=O).
1H NMR: d=7.762(d, J=7.51 Hz, 2H; H4, H5), 7.568 (d, J=7.51 Hz, 2
H; H1, H8), 7.402(d, d, J=7.51, 7.51 Hz, 2H; H3, H6), 7.314 (d, d, d, J=
7.33, 7.33, 1.28 Hz, 2H; H2, H7), 4.878 (brs, 1H; NH), 4.500 (brm, 2H;
H10), 4.210 (t, J=6.23 Hz, 1H; H9), 1.929 1.790 (m, 4H), 1.426
1.209 ppm (m, total 30H).
1-(Fmoc-amino)cyclohenicosanecarboxylic acid (11A): TFA (3 mL) was
added to a solution of tert-butyl 1-(Fmoc-amino)cyclohenicosanecarboxy-
late (10A; 1.2376 g, 1.96 mmol) in dichloromethane (4 mL) at 08C. The
mixture was stirred at room temperature for 27 h then poured into ice-
water (50 mL). The solution was extracted with dichloromethane (4î
50 mL) and the organic layer was washed with brine and dried over
Na2SO4. The residue (1.0358 g) obtained by evaporation of the solvent
was flash chromatographed (CHCl3/MeOH (40:1) to give the acid 11A
as a colorless solid. M.p.: 146.2 147.58C (colorless needles, recrystallized
from methanol). IR (KBr suspension): n˜ =1709 cmꢁ1 (C=O). 1H NMR:
d=7.732(d, J=7.51 Hz, 2H; H4, H5), 7.581 (d, J=7.33 Hz, 2H; H1, H8),
7.396 (d, d, J=7.14, 7.14 Hz, 2H; H3, H6), 7.308 (d, d, d, J=7.51, 7.51,
1.10 Hz, 2H; H2, H7), 5.249 (brs, 1H; NH), 4.412 (brm, 2H; H10), 4.214
(t, J=6.41 Hz, 1H; H9), 2.007 1.888 (m, 4H), 1.371 1.285 ppm (m, total
36H). Elemental analysis calcd (%) for C37H53NO4: C 77.18, H 9.28; N
2.43; found: C 77.05, H 9.43, N 2.41.
1-(Fmoc-amino)cyclopentadecanecarboxylic acid fluoride (12C): A solu-
tion of DAST (96.1 mg, 0.596 mmol, 1.6 equiv) in dichloromethane
(1 mL) was added to a solution of 1-(Fmoc-amino)cyclopentadecanecar-
boxylic acid (11C; 188.2mg, 0.383 mmol) in dry dichloromethane (2mL)
at 08C. The mixture was stirred at room temperature for 30 min then
poured into ice-water (20 mL). The solution was extracted with dichloro-
methane (3î20 mL) and the organic layer was washed with brine and
dried over Na2SO4. The organic solvent was evaporated and the residue
was flash chromatographed (EtOAc/nhexane (1:9)) to give colorless solid
12C (138.0 mg, 73% yield). IR (KBr suspension): n˜ =1839 cmꢁ1 (C=O).
1H NMR: d=7.755 (d, J=7.51 Hz, 2H; H4, H5), 7.573 (d, J=7.14 Hz, 2
H; H1, H8), 7.392(d, d, J=7.33, 7.33 Hz, 2H; H3, H6), 7.304 (d, d, d, J=
7.51, 7.51, 1.28 Hz, 2H; H2, H7), 4.906 (brs, 1H; NH), 4.434 (brm, 2H;
H10), 4.209 (m, 1H; H9), 1.820 (m, 3H), 1.344 1.295 ppm (m, total 25H).
1-(Fmoc-amino)cyclooctadecanecarboxylic acid (11B): TFA (2mL) was
added to a solution of tert-butyl 1-(Fmoc-amino)cyclooctadecanecarboxy-
late (10B; 409.4 mg, 0.757 mmol) in dichloromethane (2mL) at 0 8C. The
mixture was stirred at room temperature for 27 h then poured into ice-
water (50 mL). The solution was extracted with chloroform (4î50 mL)
624
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2004, 10, 617 626

a,a-Disubstituted Glycines
617 626
Curtius rearrangement of a,a-disubstituted monoethyl malonates[11]
ðr0 2ꢁr0 1Þð1ꢁn1 Þlnðc2=c1Þ ¼ ðr2ꢁr1Þð1ꢁn1Þlnðc2=c1Þ
2
2
0
2
2
0
0
ꢀ
ꢀ
ð3Þ
a) Monoethyl a,a-dipropylmalonate (13a; R1 =R2 =propyl in ref. [11]): A
solution of monoethyl a,a-dipropylmalonate (13a; 216.9 mg, 1.00 mmol),
DPPA (284.4 mg, 1.03 mmol, 1.0 equiv), and triethylamine (112.5 mg,
1.11 mmol, 1.1 equiv) in benzene (5 mL) was heated under reflux in an
argon atmosphere for 1.25 h. A solution of benzylalcohol (120.6 mg,
1.12mmol, 1.1 equiv) in benzene (1 mL) was then added. The mixture
was heated under reflux for 47 h. After evaporation of the solvent, the
residue was dissolved in ethyl actetate (30 mL) and the organic layer was
washed sequentially with 5% aqueous HCl, water, 2n aqueous NaHCO3,
and brine, then dried over Na2SO4. After evaporation of the solvent, the
residue was flash chromatographed (silica gel; EtOAc/nhexane (1:12)) to
give N-Z-dipropylglycine ethyl ester (14a; 219.2 mg, 68% yield) as a col-
From the results of sedimentation equilibrium experiments in H2O (1=
1.00 gcmꢁ3) and in D2O/H2O (60:40; 1’=1.06 gcmꢁ3), we determined n¯ to
be 0.760 cm3 gꢁ1
.
Acknowledgements
This work was supported in part by a Grant-in-Aid from the Ministry of
Education, Science, Sports, Culture and Technology, Japan, and a grant
from the Cosmetology Research Foundation (to T.O.). The authors thank
Professor Takaysuki Shioiri (Nagoya City University and Meijo Universi-
ty) for valuable advice on the chemistry of their coupling reagents.
1
orless oil. H NMR: d=7.353 (m, 5H; Ph), 5.869 (s, 1H; NH), 5.071 (s, 2
H; PhCH2), 4.214 (q, J=7.15 Hz, 2H; CO2CH2CH3), 2.292 (m, 2H;
CH2CH2CH3), 1.695 (m, 2H; CH2CH2CH3), 1.293 (m, 2H; CH2CH2CH3),
1.275 (t, J=6.97 Hz, 3H; CO2CH2CH3), 1.009 (m, 2H; CH2CH2CH3),
0.865 ppm (t, J=7.33 Hz, 6H; CH2CH2CH3). HRMS: calcd for
C18H27NO4: 321.1941; found: 321.1934. A trace amount of the isocyanate
15a was detected.
[1] S. Mitaku, T. Hirokawa, T. Tsuji, Bioinformatics 2002, 18, 608 616.
[2] S. B. Long, P. J. Casey, L. S. Beese, Nature 2002, 419, 645 650.
[3] W. DeGrado, A. Wasserman, J. B. Lear, Science 1989, 243, 622 628.
[4] a) N. A. Schnarr, A. J. Kennan, J. Am. Chem. Soc. 2001, 123, 11
081 11082; b) N. A. Schnarr, A. J. Kennan, J. Am. Chem. Soc. 2003,
125, 667 671; c) J. T. Blanchfield, J. L. Dutton, R. C. Hogg, O. P.
Gallagher, D. J. Craik, A. Jones, D. J. Adams, R. J. Lewis, P. F. Ale-
wood, I. Toth, J. Med. Chem. 2003, 46, 1266 1272.
Preparation of peptides I IV: Peptides were synthesized by the Fmoc
solid-phase method[13] on Rink amide resin.[16] The Fmoc amino acid fluo-
ride method[9] was employed to introduce the C18 amino acid and the
successive amino acids to the peptide chain. The peptide resin was treat-
ed with 10 equivalents 12B (for the introduction of C18), Fmoc-Ala-F
(for the introduction of Ala in position 10 of peptides II and III), and
Fmoc-Glu(OtBu)-F (for the introduction of Glu in position 2of peptides
II and IV), respectively, in dimethylformamide (DMF) for 12h. Other
amino acids were introduced to the peptide resin by using a Shimadzu
PSSM-8 synthesizer. The standard protocol with a PyBOP-HOBt-NMM
coupling system was employed.[17] N-terminal acetylation was achieved
by treatment with acetic anhydride and NMM (10 equiv each) in DMF at
room temperature for 30 min. The obtained protected peptide resin was
treated with TFA/1,2-ethanedithiol (95:5) at 208C for 2h. HPLC purifi-
cation yielded the pure peptides. Total yields of the peptides calculated
with respect to the initial amount of resin were as follows: peptide I, 48
%; peptide II, 10%; peptide III, 16%; peptide IV, 32%. The fidelity of
the products was ascertained by MALDI-TOF MS: [M+H]+ found
(calcd): peptide I: 1614.5 (1613.8); peptide II: 2060.1 (2059.6); peptide
III: 1836.8 (1836.2); peptide IV: 1836.9 (1836.3).
[5] a) C. Toniolo, A. Polese, F. Formaggio, M. Crisma, J. Kamphuis, J.
Am. Chem. Soc. 1996, 118, 2744 2745; b) C. Toniolo, M. Crisma, F.
Formaggio, C. Peggion, Biopolymers (Pept. Sci.) 2001, 60, 396 419.
[6] a,a-Amino acids bearing a ring of less than 12members, as well as
those with other appendants such as Aib have been reported. 12-
membered ring: a) M. Saviano, R. Iacovino, V. Menchise, E. Bene-
detti, G. M. Bonora, M. Gatos, L. Graci, F. Formaggio, M. Crisma,
C. Toniolo, Biopolymers 2000, 53, 200 212; 11-membered ring:
b) M. Saviano, R. Iacovino, E. Benedetti, V. Moretto, A. Banzato, F.
Formaggio, M. Crisma, C. Toniolo, J. Pept. Sci. 2000, 6, 571 583; 10-
membered ring: c) A. Moretto, F. Formaggio, M. Crisma, C. Toniolo,
M. Saviano, R. Iacovino, R. M. Vitale, E. Benedetti, J. Pept. Sci.
2001, 7, 307 315; nine-membered ring: d) M. Gatos, F. Formaggio,
M. Crisma, G. Valle, C. Toniolo, G. M. Bonora, M. Saviano, R. Iaco-
vino, V. Menchise, S. Galdiero, C. Pedone, E. Benedetti, J. Pept. Sci.
1997, 3, 367 382; eight-membered ring: e) V. Moretto, F. Formag-
gio, M. Crisma, G. M. Bonora, C. Toniolo, E. Benedetti, A. Santini,
M. Saviano, B. Di Blasio, C. Pedone, J. Pept. Sci. 1996, 2, 14 2 7;
seven-membered ring: f) E. Benedetti, B. Di Blasio, R. Iacovino, V.
Menchise, M. Saviano, C. Pedone, G. M. Bonora, A. Ettorre, L.
Graci, F. Formaggio, M. Crisma, G. Valle, C. Toniolo, J. Chem. Soc.
Perkin Trans. 2 1997, 2023 2032; six-membered ring: g) R. Bardi,
A. M. Piazzesi, C. Toniolo, M. Sukumar, P. A. Raj, P. Balaram, Int.
J. Pept. Protein Res. 1985, 25, 628 639; h) M. Crisma, G. M. Bonora,
C. Toniolo, A. Bavoso, E. Benedetti, B. Di Blasio, V. Pavone, C.
Pedone, Macromolecules 1988, 21, 2071 2074; five-membered ring:
i) M. Crisma, G. M. Bonora, C. Toniolo, E. Benedetti, A. Bavoso, B.
Di Blasio, V. Pavone, C. Pedone, Int. J. Biol. Macromol. 1988, 10,
300 304; four-membered ring: j) M. Gatos, F. Formaggio, M.
Crisma, C. Toniolo, G. M. Bonora, E. Benedetti, B. Di Blasio, R. Ia-
covino, A. Santini, M. Saviano, J. Kamphuis, J. Pept. Sci. 1997, 3,
110 122; general interest: k) T. S. Yokum, T. J. Gauthier, R. P.
Hammer, M. L. McLaughlin, J. Am. Chem. Soc. 1997, 119, 1167
1168; l) E. C. Schafmester, J. Po, G. L. Verdine, J. Am. Chem. Soc.
2000, 122, 5891 5892; m) C. Garcia-Echeverria, B. Gay, J. Rahuel,
P. Furet, Bioorg. Med. Chem. Lett. 1999, 9, 2915 2920; o) M. Savia-
no, E. Benedetti, R. M. Vitale, B. Kaptein, Q. B. Broxterman, M.
Crisma, F. Formaggio, C. Toniolo, Macromolecules 2002, 35, 4204
4209; p) C. Toniolo, E. Benedetti, Macromolecules 1991, 24, 4004
4009; q) Y. Paterson, S. M. Rumsey, E. Benedetti, G. Nÿmethy,
H. A. Scheraga, J. Am. Chem. Soc. 1981, 103, 2947 2955.
Single-crystal X-ray diffraction structural analysis: A Bruker Smart 1000
CCD diffractometer employing graphite-monochromated MoKa radiation
was used. The structure was solved by direct methods with the program
SIR97 and was refined by full-matrix least-squares techniques. All calcu-
lations were performed with the teXsan crystal structure solution soft-
ware package.
Analytical ultracentrifugation: Sedimentation equilibrium studies were
performed in a Beckman Coulter Optima XL-I analytical ultracentrifuge
with a double-sector centerpiece and sapphire windows, at rotor speeds
of 30000 and 40000 rpm and at 208C. Absorbance scans were carried out
at 230 nm in the radial step mode at 0.001-cm intervals and the data were
collected as the average of 16 measurements at each radial distance.
Equilibrium was considered to have been reached when replicate scans
separated by 6 h were indistinguishable. The weight-average molecular
weight (Mapp) was estimated from Equation (1),[21] where r is the radius, c
2
2
2
ꢀ
ð1Þ
Mapp ¼ 2RT=½ðr2ꢁr1Þð1ꢁn1Þw Šlnðc2=c1Þ
is the concentration of the peptide, n¯ is the partial specific volume of the
peptide, 1 is the density of the solvent, w is the angular velocity of the
rotor (in radianssꢁ1), R is the universal gas constant, T is the absolute
temperature, and Mapp is the apparent molecular weight.
The value of n¯ was estimated from two sets of data obtained from sedi-
mentation equilibrium experiments. We assumed that Mapp does not
depend on the density of the solution, in which case, Equation (2) ap-
plies.
[7] a) S. J. Miller, H. E. Blackwell, R. H. Grubbs, J. Am. Chem. Soc.
1996, 118, 9606 9614; b) review: R. Grubbs, S. Chang, Tetrahedron
1998, 54, 4413 4450c) review: A. F¸rstner, Angew. Chem. 2000, 112,
3140 3172; Angew. Chem. Int. Ed. 2000, 39, 3012 3043; d) K.
Hammer, K. Undheim, Tetrahedron 1997, 53, 2309 2322; e) S.
2
2
0
2
0
0
ꢀ
Mapp ¼ 2RT=½ðr0 2ꢁr01Þð1ꢁn1 Þw Šlnðc2=c1Þ
ð2Þ
By combining Equations (1) and (2), Equation (3) is produced.
625
Chem. Eur. J. 2004, 10, 617 626
www.chemeurj.org
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

T. Ohwada, S. Futaki et al.
FULL PAPER
Kotha, N. Sreenivasachary, Bioorg. Med. Chem. Lett. 1998, 8, 257
260.
[12] Crystallographic data: C28H45NO3, Mr =443.67, 0.48î0.42î0.15 mm,
monoclinic, space group P21/n, a=8.290(2), b=34.275(9), c=
10.128(3) ä, b=113.102(4)8, V=2647(1) ä3, Z=4, 1calcd =1.113 g
cmꢁ3, 2qmax =56.98, T=173 K, m(MoKa)=0.71 cmꢁ1, F(000)=976.00,
Bruker Smart 1000 CCD diffractometer, w 2q scans, 2270 reflections
measured, 5941 unique, 3747 with I>3.0s(I), 290 variables, R=
[8] Direct dialkenylation of dimethylmalonate (4a) in the presence of
an excess amount of a base (5 equiv Na in methanol) and allyl bro-
mide (5 equiv) resulted in the formation of the monoallyl malonate
(55% yield), together with some diallyl malonate (12% yield).
[9] a) C. Kaduk, H. Wenschuh, M. Beyermann, K. Froner, L. A. Carpi-
no, M. Bienert, Lett. Pept. Sci. 1995, 2, 285 288; b) L. A. Carpino,
D. Ionescu, A. El-Faham, P. Henklein, H. Wenschuh, M. Bienert, M.
Beyermann, Tetrahedron Lett. 1998, 39, 241 244; c) L. A. Carpino,
M. Beyermann, H. Wenschuh, M. Bienert, Acc. Chem. Res. 1996, 29,
268 274; d) S. Futaki, M. Fukuda, M. Omote, K. Yamauchi, T.
Yagami, M. Niwa, Y. Sugiura, J. Am. Chem. Soc. 2001, 123, 12 12 7
12134.
[10] a) T. Shioiri, K. Ninomiya, S. Yamada, J. Am. Chem. Soc. 1972, 94,
6203 6205; b) K. Ninomiya, T. Shioiri, S. Yamada, Tetrahedron
1974, 30, 2151 2157.
[11] This phenomenon is also seen with acyclic long aliphatic substituents
at the a position. In the case of n-propyl disubstitution (13a), the in-
termediate isocyanate (15a) can react with benzyl alcohol to give
the N-Z derivative (14a).
0.045, Rw =0.057, S=1.98, (D/s)max =0.291, 1Dmax =0.24 eäꢁ3
,
D1min =ꢁ0.19 eäꢁ3
.
[13] a) E. Atherton, R. C. Sheppard, Solid Phase Peptide Synthesis, A
Practical Approach, IRL, Oxford, 1989; b) S. Futaki, T. Ishikawa,
M. Niwa, K. Kitagawa, T. Yagami, Bioorg. Med. Chem. 1997, 5,
1883 1891.
[14] S. Marqusee, R. L. Baldwin, Proc. Natl. Acad. Sci. USA 1987, 84,
8898 8902.
[15] Structure minimizations were performed with a suite of programs:
MacroModel version 8.1., Schrˆdinger, L. L. C. OPLS-AA force
field: W. L. Jorgensen, D. S. Maxwell, J. Tirado-Rives, J. Am. Chem.
Soc. 1996, 118, 11225 11235.
[16] H. Rink, Tetrahedron Lett. 1987, 28, 3787 3790.
[17] J. Coste, D. Le-Nguyen, B. Castro, Tetrahedron Lett. 1990, 31, 205
208.
[18] Increased hydrophobicity of the peptides caused greater nonspecific
adsorption onto the glass and plastic surfaces of the equipment used
for their purification, which lowered the yield.
[19] Analysis conditions: column: Waters Symmetry 300 C18, 5 mm
(4.6î150 mm); gradient: 10 90% B in A (A: H2O containing 0.1%
CF3CO2H; B: CH3CN containing 0.1% CF3CO2H) over 40 min;
flow: 1 mLminꢁ1; detection: 215 nm.
[20] N. E. Zhou, C. M. Kay, R. S. Hodges, J. Biol. Chem. 1992, 267,
2664 2670.
[21] For details, see the Experimental Section and H. K. Schachman, Ul-
tracentrifugation in Biochemistry, Academic Press, New York, 1959.
[22] M. Doi, Y. Nishi, S. Uchiyama, Y. Nishiguchi, T. Nakazawa, T.
Ohkubo, Y. Kobayashi, J. Am. Chem. Soc. 2003, 125, 9922 9923.
Received: September 1, 2003 [F5492]
626
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2004, 10, 617 626