Solution synthesis and characterization of aza-β3-peptides (Nα-substituted hydrazino acetic acid oligomers)

Full text of DOI:10.1021/jo001500d

J . Org. Chem. 2001, 66, 4923-4929
4923
Sch em e 1
Solu tion Syn th esis a n d Ch a r a cter iza tion of
Aza -â3-p ep tid es (Nr-Su bstitu ted Hyd r a zin o
Acetic Acid Oligom er s)
Arnaud Cheguillaume,† Arnaud Salau¨n,†
Sourisak Sinbandhit,‡ Michel Potel,§ Philippe Gall,|
Miche`le Baudy-Floc′h,† and Philippe Le Grel*,†
Ta ble 1. Yield s of Mon om er s 1 (%)
1x
1a NRhAla 1b NRhVal 1c NRhLeu 1d NRhPhe
SESO, UMR CNRS 6510, Universite´ de Rennes I,
Laboratoire du Solide et Inorganique Mole´culaire, UMR
CNRS 6511, Laboratoire de Chimie Inorganique, INSA, 20
Av. des Buttes de Coesmes, CRMPO, Campus de Beaulieu,
F-35042 Cedex, France
Boc-1x-OMe
Boc-1x-OBn
Boc-1x-OH
H-1x-OMe
H-1x-OBn
94
92
66
79
94
96
76
65
68
84
78
Phillipe.Legrel@univ-rennes1.fr
Received October 18, 2000
Following our recent work on NR-substituted hydrazino
acetic building blocks,10 we have now showed that they
can be used for the synthesis and characterization of new
pseudopeptides, which share the same characteristics
(Scheme 1). The first considerations on the ability of these
oligomers to adopt specific secondary structure in solution
are presented.
In tr od u ction
Pseudopeptidic oligomers, which can be conveniently
considered as “biopolymer mimetics”,1e are the object of
great synthetic efforts. Several recent reviews reflect the
increasing interest for such molecules.1 Among other
applications, they could be used as biomaterial sur-
rogates,2 peptidomimetics,3 immunomodulators,4 or genic
therapy agents.5 These potential properties mainly rely
on both enhanced resistance to proteolysis and specific
intramolecular or intermolecular three-dimensional ar-
rangement such as turns, â-sheets, or helices. Peptoids,6
azatides,7 ureapeptoids,8 and â-peptoids,9 all described
within the past decade, can be regarded as belonging to
a particular family of pseudopeptides. For all of them,
the side chains are connected to nitrogen atoms of the
backbone.
Resu lts a n d Discu ssion
Monomers 1 were synthesized as Nâ-Boc-protected
benzyl or methyl esters, except for 1a , which was directly
prepared as free NR-(Me)hydrazino acetic acid benzyl
ester (Table 1). Boc deprotection was obtained by TFA/
DCM (1/1) treatment for 15 min. Hydrogenolysis was
performed using 5% Pd/C in 2-propanol under 1 atm
(using methanol yields to partial transesterification).
Saponifications were run in acetonitrile (Scheme 2). Free
NR-substituted hydrazino acetic esters have to be freshly
prepared (they can be stored several days in solution in
DCM while they are unstable in the neat form).
These monomers can be very efficiently coupled under
simple DCC/DMAP activation in dry DCM. Oligomers
can be deprotected and elongated in the same way
without any particular problems. A step by step approach
and convergent procedure were combined to assemble a
variety of oligomers from dimer to dodecamer as sum-
marized in Scheme 3.
* To whom correspondence should be addressed. Phone: (33) 02 99
28 69 66. Fax: (33) 02 99 28 67 38.
† SESO, UMR 6510, Universite´ de Rennes I.
‡ CRMPO, Universite´ de Rennes I.
§ Laboratoire du Solide et Inorganique Mole´culaire, UMR CNRS
6511, Universite´ de Rennes I.
| Laboratoire de Chimie Inorganique, INSA, 20 Av. des Buttes de
Coesmes.
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10.1021/jo001500d CCC: $20.00 © 2001 American Chemical Society
Published on Web 06/19/2001

4924 J . Org. Chem., Vol. 66, No. 14, 2001
Notes
Sch em e 2a
F igu r e 1. Schematic 4J long-range COSY interactions involv-
ing NH protons.
experiments.11 It appears that each NH assumes 4J
couplings with the corresponding methylenes as il-
lustrated in Figure 1.
By comparing this information, it becomes possible to
assign all signals and to perform the full sequencing of
the oligomers. A part of such spectra is shown in Figure
2. It was obtained for the tetramer Boc-4-OMe, and some
chemical shifts are represented as illustrated below.
The title compounds are structurally related to hy-
drazinopeptides12 and â3-peptides.13 These relationships
are exemplified by comparing the structure of the corre-
sponding building blocks with NR-substituted hydrazino
acetic acid monomer 1, which clearly shows the relation-
ships (Figure 3).
a
Key: (a) NaOH, acetonitrile; (b) H2 (1 atm), 5% Pd/C, 2-pro-
panol; (c) TFA/DCM (1/1).
All the oligomers are oily products except the dodeca-
mer 7, which is an amorphous white powder. They show
good solubility in most common organic solvents but are
sparingly soluble in water. All the products were char-
acterized by H NMR; some 13C NMR spectra were also
1
recorded for the shortest members, but for the highest
oligomers, in particular dodecamer 7, the overlap of
signals becomes very important. After column chroma-
tography on silica gel, most of the compounds have
reached an appreciable level of purity despite their
polarity. Only compounds Boc-2b-OMe, H-2b-OMe, Boc-
3-OH, Boc-4-OMe, and Boc-5-OMe, could not be purified
over 85-90%. Some compounds were also characterized
using electrospray mass spectrometry.
Due to the absence of a proton in the R and R′ positions
relative to the NHs, no other 3J coupling, except those
present in the side chains, could be observed in the one-
dimensional 1H NMR spectra. On the other hand, fruitful
information can be gained by using long-range COSY
Hydrazino acids A have the same backbone as mono-
mers 1. The difference relies on the position of the side
chain, which has been formally shifted to the adjacent
nitrogen. NR-Substituted hydrazino acetic acids can also
be regarded as aza analogues of â3-amino acids B (â3-
Haa). Therefore, a full satisfying nomenclature is not
easy to adopt. The name NR-substituted hydrazinopep-
tides is ambiguous, as it does not clearly appear that the
backbone is exclusively built up from hydrazino acetic
acid units. NR-Substituted hydrazino acetic acid oligo-
mers, although explicit, is quite a long name. Thus, even
if it eclipses the relationship with hydrazino acids, aza-
â3-peptides seems the most attractive as a simple termi-
nology relative to â3-peptides.
Sch em e 3a
a
Key: (a) NaOH, acetonitrile; (b) H2 (1 atm), 5% Pd/C, 2-propanol; (c) TFA/DCM (1/1); (d) DCC/DMAP, DCM.

Notes
J . Org. Chem., Vol. 66, No. 14, 2001 4925
Sch em e 4
peptides and demonstrated that they could adopt specific
helical secondary structure in organic solvents.
As illustrated on Scheme 4, aminoxy acids are isosteric
with â2-amino acids. Recent studies on oxapeptides17
revealed that they could also adopt helical secondary
structure. Nevertheless, the characteristics of the latter
differ from those of â2-peptides (helix 1.88 versus helix
314) due to a preference for nearest neighbor interaction.
Some questions were then raised: as NR-substituted
amino acetic acids are isosteric with â3-amino acids, do
aza-â3-peptides behave as â3-peptides or oxapeptides?
Can they adopt secondary structure despite their lack of
defined chirality?
An interesting starting point is the comparison of some
NMR characteristics (CDCl3) between monomers and
oligomers. For this purpose, we prepared two new hy-
drazinoacetic esters Ac-1b-OMe and Ac-1d -OMe (Boc
group is replaced by an acetyl) in order to have a
reference with respect to hydrazidic bonds in the oligo-
mers. In contrast to Boc-protected monomers, the corre-
sponding acetyl compounds show two clearly distinct sets
of resonances (relative intensities 60/40) in NMR spectra.
This can be reasonably interpreted as reflecting the
coexistence of both cis and trans conformations for the
acethydrazide bond. This conformational equilibrium
does not appear in the case of oligomers (except for the
ester form of the dimer), although they contain several
of these. Compounds seem constrained in a single state
(Scheme 5).
F igu r e 2. 1H long-range COSY NMR spectrum of Boc-4-OMe.
The chemical shift of a carbazic NH in a monomer is
around 5.6 ppm, whereas the hydrazidic one is around
7.3 ppm. The corresponding values in an oligomer show
that the former is almost unaffected (average variation
of 0.2 ppm) while the latter undergoes a drastic shifting
to lower fields ranging from 2 to 2.7 ppm. This is a strong
indication that they are engaged in hydrogen bond
interaction. No significant differences were observed
F igu r e 3. Structural comparisons between NR-substituted
hydrazino acetic acid monomer 1 and related compounds.
Due to the rapid improvement of the synthesis of
oligomeric pseudopeptides, it has become possible to
investigate their ability to adopt defined secondary
structure (a characteristic property of biopolymers). Some
fascinating findings and perspectives have followed.14
Seebach15 and Gellman have published important work
on â-peptides.16 Seebach worked on â2-peptides and â3-
(15) (a) Hintermann, T.; Seebach, D. Synlett 1997, 437-438. (b)
Seebach, D.; Ciceri, P. E.; Overhand, M.; J aun, B.; Rigo, D.; Oberer,
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Hommel, U.; Widmer, H. Helv. Chim. Acta 1998, 81, 932-982.
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R.; Gellman, S. H. J . Am. Chem. Soc 1996, 118, 13071-13072. (b)
Appella, D. H.; Christianson, L. A.; Klein, D. A.; Powell, D. R.; Huang,
X.; Barchi, J . J ., J r.; Gellman, S. H. Nature 1997, 387, 381-384. (c)
Barchi, J . J ., J r.; Huang, X.; Appella, D. H.; Christianson, L. A.; Durell,
S. R.; Gellman, S. H. Am. Chem. Soc 2000, 122, 2711-2718. (d)
Appella, D. H.; Christianson, L. A.; Karle, I. L.; Powell, D. R.; Gellman,
S. H. J . Am. Chem. Soc 1999, 121, 6206-6212. (e) Appella, D. H.;
Christianson, L. A.; Klein, I. L.; Richards, M. R.; Powell, D. R.;
Gellman, S. H. J . Am. Chem. Soc 1999, 121, 7574-7581.
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J . Am. Chem. Soc 1996, 118, 9794-9795. (b) Yang, D.; Qu, J .; Li, B.;
Ng, F.-F.; Wang, X.-C.; Cheung, K.-K.; Wang, D.-P.; Wu, Y. D. J . Am.
Chem. Soc 1999, 121, 589-590. (c) Wu, Y.-D.; Wang, D.-P.; Chan, K.
W. K.; Yang, D. J . Am. Chem. Soc 1999, 121, 11189-11196. (d)
Thevenet, L.; Vanderesse, R.; Marraud, M.; Didierjean, C.; Aubry, A.
Tetrahedron Lett. 2000, 41, 2361-2364.
(11) Bax, A.; Freeman, R. J . Magn. Reson. 1981, 44, 542.
(12) (a) Lecoq, A.; Marraud, M.; Aubry, A. Tetrahedron Lett. 1991,
24, 2765-2768. (b) Aubry, A.; Bayeul, D.; Mangeot, J .-P.; Vidal, J .;
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793-801. (c) Aubry, A.; Mangeot, J .-P.; Vidal, J .; Collet, A.; Zerkout,
S.; Marraud, M. Int. J . Peptide Protein Res. 1994, 43, 305-311. (d)
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687.

4926 J . Org. Chem., Vol. 66, No. 14, 2001
Notes
Sch em e 5
between the spectra that were obtained for different
concentrations (from 10-1 to 10-3 M) of oligomers, which
indicate that the molecules exhibit no tendency toward
aggregation.
Nearest neighbor hydrogen bond interactions can only
explain the “high” chemical shift of all the hydrazidic NH
of an oligomer. Two cases have to be considered; one
relying on repetitive six-membered hydrogen-bonded
pseudocycles, the other one on eight-membered rings in
an opposite direction (Figure 4a,b).
In the first hypothesis, it must be assumed that the
carbazic NH does not participate to the structuration as
its chemical shift is the same in the monomer as well as
in the oligomer as previously mentioned (this was
confirmed by the addition of small amounts of DMSO,
which displaces the carbazic NH to around 8.2 ppm while
the hydrazidic NHs remain unaffected). In the second
hypothesis, this is more easily explained as the carbazic
NH has no bonding partner. Moreover, since the depro-
tection of the C-terminus induces only small variations
of NH chemical shifts (around 0.2 ppm increment), the
N-terminus deprotection leads to an upfield shielding of
one hydrazidic NH to 8 ppm (Figure 4c). This can be best
rationalized once more with the second model, as the
N-terminal hydrazidic NH has simply no more carbonyl
partner.
F igu r e 4. Nearest-neighbor hydrogen-bonding models for
hexamers 6.
Recently, we grew racemic crystals from small model
compounds that incorporate one hydrazino acetic unit.
We observed that they are structured by the formation
of a bifidic intramolecular eight-membered hydrogen-
bonded interaction very similar to “hydrazinoturn” and
“N-O” turn already described. The Nâ-Boc-NR-benzylhy-
drazinoacetic monomer Boc-1d -OH also shows an inter-
action involving the acidic proton and the carbonyl of the
carbazate as illustrated in Figure 5. The O4-H1 distance
(2.15 Å) is typical of a strong hydrogen bond, and the
N1-H1 distance (2.17 Å) unambiguously indicates the
participation of this atom to further stabilization.
All the available data demonstrate the ability of aza-
â3-peptides to fold in CDCl3 solution. Our favorite hy-
pothesis for the moment is the existence of a hydrogen
bond network relying on the formation of successive
eight-membered pseudocycles. In this model, the driving
force seems to be the reinforcement of the structure by
participation of the lone pair of electron of each NR atom.
In the proposed model, all the hydrazidic bonds have the
trans conformation as illustrated in Figure 6.
F igu r e 5. X-ray crystal structure of Boc-1d -OH. Distances
are given in angstroms.
F igu r e 6. Schematic two-dimensional representation of pos-
sible H-bond network for hexamer 6.
be assembled into a new class of pseudopeptidic com-
pounds. The procedure should be reasonably imple-
mented on solid support. The resulting aza-â3-peptides
attract attention by the presence of pyramidal nitrogen
atoms, which are as much chiral centers with free
configuration. Despite the resulting degenerated chiral-
ity, aza-â3-peptides show NMR characteristics that
Con clu sion
Using standard protective groups and a very simple
coupling procedure, NR-hydrazino acetic derivative can

Notes
J . Org. Chem., Vol. 66, No. 14, 2001 4927
Boc-Nr-Leu -OMe (Boc-1c-OMe). The reaction was carried
out according to the general procedure using NR-isobutyl-Nâ-Boc-
hydrazine (7 g, 37.23 mmol), methyl 2-bromoacetate (15 g, 98
mmol), and K2CO3 (5.4 g, 39 mmol). The residue was purified
by chromatography on silica gel (ethyl acetate/hexanes) (1/9) to
give (Boc-1c-OMe) (9.58 g, 99%) as an oil: 1H NMR (CDCl3) δ
0.87 (d, J ) 6.6 Hz, 6H), 1.37 (s, 9H), 1.63 (m, 1H), 2.57 (d, J )
7.3 Hz, 2H), 3.62 (s, 2H), 3.65 (s, 3H), 6.48 (br s, 1H).
strongly suggest their ability to self-organize through
intramolecular hydrogen bond network in solution. Con-
firmation of these preliminary results is in progress.
These easily and inexpensively obtainable oligomers
could be useful as “foldamer” models. Questions in
connection with the relative configuration of nitrogen
centers are particularly intriguing. It is interesting to
point out that the formation of a hydrogen bond network
is likely to reduce the distance between successive side
chains. Such kinds of molecular contraction could balance
the elongated backbone of aza-â3-peptides, making them
attractive surrogates with respect to their parent pep-
tides. From this point of view, some interesting questions
still need to be answered concerning their biochemical
stability in comparison with the excellent resistance of
the closely related â3-peptides to peptidolysis.
Gen er a l P r oced u r e for Sa p on ifica tion . Boc-Nr-Va l-OH
(Boc-1b-OH). To a solution of NR-isopropyl-Nâ-Boc-hydrazi-
noglycine methyl ester Boc-1b-OMe (2.93 g, 11.91 mmol) in
acetonitrile (30 mL) was added NaOH 2 N (6 mL). The mixture
was stirred for 1 h 30 min at room temperature. After evapora-
tion of acetonitrile, the residue was diluted with 30 mL of water,
washed twice with ether (2 × 30 mL), acidified by addition of
HCl 2 N, and extracted with CH2Cl2 (2 × 50 mL), and the
combined extracts were dried over Na2SO4. Filtration and
evaporation of the solvent afforded 1.80 g of Boc-1b-OH (65%)
as a viscous oil that crystallized by addition of ether: mp 73-
75 °C; IR (KBr) 3500-2500, 1700 cm-1; 1H NMR (CDCl3) δ 1.03
(d, J ) 6.5 Hz, 6H), 1.39 (s, 9H), 3.09 (m, 1H), 3.47 (s, 2H), 6.29
(br s, 1H), 8.97 (br s, 1H); 13C NMR (CDCl3) δ 18.8 (q, J ) 126
Hz), 28.6 (q, J ) 127 Hz), 56.9 (d, J ) 138 Hz), 57.7 (t, J ) 137
Hz), 65.2 (t, J ) 132 Hz), 82.1 (s), 157.8 (s), 172.6 (s); HR-MS
m/z for C11H22N2O4 calcd 232.1423 (M+), obsd 232.1434.
Exp er im en ta l Section
NMR spectra were run at 200, 300 (1H), or 75.5 MHz (13C).
HR-MS were obtained from the Centre Re´gional de Mesures
Physiques de l’Ouest, using MS/MS mass spectrometer ZAB Spec
TOF. Infrared spectra were recorded on an FT-IR spectrometer
as suspensions in KBr. Elemental analyses were performed by
the analytical laboratory, CNRS (Lyon). X-ray structure was
performed from the Centre de Cristallographie de Rennes.
Boc-Nr-Leu -OH (Boc-1c-OH). The reaction was carried out
according to the general procedure using NR-isobutyl-Nâ-Boc-
hydrazinoglycine methyl ester Boc-1c-OMe (4 g, 15.38 mmol)
to afford Boc-1c-OH (2.97 g, 79%) as a viscous oil: IR (KBr)
3457-3189, 1738, 1705 cm-1 1H NMR (CDCl3) δ 0.97 (d, J )
;
The following were prepared according to literature proce-
6.8 Hz, 6H), 1.46 (s, 9H), 1.70 (m, 1H), 2.64 (d, J ) 7.1 Hz, 2H),
3.57 (s, 2H), 5.97 (br s, 1H), 9.35 (br s, 1H).
dures: substituted hydrazines,18 H-NRhAla-OBn, H-1a -OBn.19
Gen er a l P r oced u r e for th e P r ep a r a tion of Nr-Su bsti-
tu ted Nâ-Boc-h yd r a zin oglycin e Ester 1. Boc-Nr-Va l-OMe
(Boc-1b-OMe). To a solution of NR-isopropyl-Nâ-Boc-hydrazine
(5.76 g, 33.10 mmol) and methyl 2-bromoacetate (13 g, 85 mmol)
in toluene (30 mL) was added dry K2CO3 (4.56 g, 33.10 mmol).
The mixture was refluxed under stirring for 12 h. After filtration,
the organic layer was concentrated in vacuo. The excess of
methyl 2-bromoacetate was conveniently eliminated by coevapo-
ration with fresh toluene. The residue was purified by chroma-
tography on silica gel (ether/petroleum ether) (3/1) to give (4.97
g, 61%) of Boc-1b-OMe as an oil: IR (KBr) 3695-3140, 1740,
1725 cm-1; 1H NMR (CDCl3) δ 1.27 (d, J ) 6.3 Hz, 6H), 1.63 (s,
Boc-Nr-P h e-OH (Boc-1d -OH). The reaction was carried out
according to the general procedure using NR-benzyl-Nâ-Boc-
hydrazinoglycine methylester Boc-1d -OMe (6 g, 20.41 mmol) to
afford Boc-1d -OH (5.48 g, 96%) as white crystals: mp 125 °C;
1H NMR (CDCl3) δ 1.41 (s, 9H), 3.64 (s, 2H), 4.08 (s, 2H), 5.98
(br s, 1H), 7.39 (s, 5H), 7.65 (br s, 1H). X-ray crystal data of
Boc-1d -OH: C14H20N2O4, M ) 280.32 g‚mol-1crystallizes in
space group P21/c with lattice parameters a ) 16.5786(3) Å, b
) 9.3781(2) Å, c ) 20.6580(4) Å, â ) 112.4555(8)°, V ) 2968.29
Å3 , Z ) 8, Dc ) 1.255 g‚cm-3. From a crystal with dimensions
0.47 × 0.19 × 0.055 mm3 7482 unique reflections were collected
at T ) 95(1) K over a 2θ range (2-55.8°) with a Nonius
KappaCCD diffractometer equipped with a graphite-monochro-
matized Mo KR (λ ) 0.710 t73 Å) radiation. The crystal structure
was solved by direct method using SIR97 program. All hydrogen
atoms were included at calculated positions excepted for H2N
located by difference Fourier syntheses and full-matrix least-
squares refinement using 4804 reflections with I > 2σ(I) were
performed with SHELXL97 and converged to final agreement
factors R ) 0.0489 (R ) 0.0908 for all reflections), GOF ) 1.038.
Boc-Nr-Va l-Nr-Ala -Nr-Leu -Nr-Va l-OH (Boc-4-OH). The
reaction was carried out according to the general procedure using
Boc-4-OMe (0.5 g, 0.87 mmol) to give Boc-4-OH (0.31 g, 64%) as
9H), 3.42 (m, 1H), 3.91 (s, 3H), 3.94 (s, 2H), 6.72 (s br, 1H); 13
C
NMR (CDCl3) δ 20.4 (q, J ) 126.3 Hz), 28.3 (q, J ) 126.7 Hz),
51.6 (q, J ) 147.5 Hz), 54.6 (t, J ) 136.7 Hz), 54.7 (d, J ) 133.7
Hz), 79.7 (s), 155.3 (s), 171.7 (s); HR-MS m/z for C11H22N2O4 calcd
246.15796 (M), obsd 246.15838.
Boc-Nr-P h e-OMe (Boc-1d -OMe). The reaction was carried
out according to the general procedure using NR-benzyl-Nâ-Boc-
hydrazine (1.73 g, 7.8 mmol), methyl 2-bromoacetate (3 g, 19.5
mmol), and K2CO3 (1.1 g, 7.8 mmol). The residue was purified
by chromatography on silica gel (ethyl acetate/hexanes) (1/9) to
give Boc-1d -OMe (2.15 g, 94%) as an oil: IR (KBr) 3370, 2946,
1
1752, 1719 cm-1; H NMR (CDCl3) δ 1.32 (s, 9H), 3.60 (s, 2H),
1
a viscous oil: IR (KBr) 3457-3189, 1738, 1705 cm-1; H NMR
3.66 (s, 3H), 4.04 (s, 2H), 6.57 (br s, 1H), 7.16-7.37 (m, 5H).
(CDCl3) δ 0.88 (d, J ) 6.5 Hz, 6H), 0.98 (d, J ) 6.4 Hz, 6H), 1.05
(d, J ) 6.5 Hz, 6H), 1.38 (s, 9H), 1.39 (m, 1H), 2.50 (d, J ) 7.1
Hz, 2H), 2.58 (s, 3H), 3.07 (m, 2H), 3.23 (s, 2H), 3.34 (s, 2H),
3.41 (s, 2H), 3.44 (s, 2H), 5.90 (br s, 1H), 6.20 (br s, 1H), 9.29
(br s, 1H), 9.48 (br s, 1H), 9.90 (br s, 1H).
Boc-Nr-Leu -OBn (Boc-1c-OBn ). The reaction was carried
out according to the general procedure using NR-isobutyl-Nâ-Boc-
hydrazine (4.50 g, 23.94 mmol), benzyl 2-bromoacetate (14.14
g, 61.48 mmol), and K2CO3 (3.29 g, 23.94 mmol). The residue
was purified by chromatography on silica gel (ethyl acetate/
hexanes) (1/9) to give Boc-1c-OBn (5.25 g, 66%) as an oil: IR
Gen er a l P r oced u r e for Boc Dep r otection . H-Nr-Leu -
OBn (H-1c-OBn ). A solution of Boc-1c-OBn (2.08 g, 6.19 mmol)
in CH2Cl2 (10 mL) and TFA (10 mL) was stirred for 2 h at room
temperature. After evaporation of the solvent, the residue was
diluted with water (20 mL) and washed with ether (2 × 25 mL).
The aqueous phase was treated with NaHCO3 1 N until pH 7-8
and extracted by CH2Cl2 (3 × 30 mL). The organic layers were
dried (Na2SO4), and the solvent was removed in vacuo to afford
H-1c-OBn (1.14 g, 78%) as an oil: IR (KBr) 3340, 3080-3020,
1750-1700, 1580 cm-1; 1H NMR (CDCl3) δ 0.90 (d, J ) 6.6 Hz,
6H), 1.78 (m, 1H), 2.49 (d, J ) 7.4 Hz, 2H), 3.29 (br s, 2H), 3.52
(s, 2H), 5.14 (s, 2H), 7.34 (m, 5H); 13C NMR (CDCl3) δ 20.6 (q, J
) 125.1 Hz), 26.4 (d, J ) 125.7 Hz), 61.1 (t, J ) 136.4 Hz), 66.2
(t, J ) 149.5 Hz), 68.3 (t, J ) 131.2 Hz), 128.3 (d, J ) 158.7 Hz),
128.4 (d, J ) 163 Hz), 128.6 (d, J ) 161.1 Hz), 135.7 (s), 170.7
(s).
(KBr) 3440-3220, 3080-3020, 1725, 1700 cm-1 1H NMR
;
(CDCl3) δ 0.95 (d, J ) 6.25 Hz, 6H), 1.46 (s, 9H), 1.73 (m, 1H),
2.67 (d, J ) 7.25 Hz, 2H), 3.75 (s, 2H), 5.17 (s, 2H), 6.56 (br s,
1H), 7.37 (s, 5H); 13C NMR (CDCl3) δ 20.6 (q, J ) 125.1 Hz),
26.6 (d, J ) 122.1 Hz), 28.3 (q, J ) 126.5 Hz), 56.0 (t, J ) 137.9
Hz), 64.9 (t, J ) 132.5 Hz), 66.4 (t, J ) 147.8 Hz), 79.8 (s), 128.4
(d, J ) 157.5 Hz), 128.5 (d, J ) 155.7 Hz), 128.7 (d, J ) 159.8
Hz), 135.4 (s), 154.9 (s), 170.9 (s); HR-MS m/z for C11H22N2O4
calcd 336.2049 (M+), obsd 336.2047.
(18) Cheguillaume, A.; Lehardy, F.; Bouget, K.; Baudy-Floc’h, M.;
Le Grel, P. J . Org. Chem 1999, 64, 2924-2927.
(19) Masters, J . J .; Hegedus, L. S. J . Org. Chem. 1993, 58, 4547-
4554.

4928 J . Org. Chem., Vol. 66, No. 14, 2001
Notes
H-Nr-Va l-OMe (H-1b-OMe). The reaction was carried out
according to the general procedure using Boc-1b-OMe (5 g, 20.33
mmol) to give H-1b-OMe (2.01 g, 68%) as an oil: 1H NMR
(CDCl3) δ 1.07 (d, J ) 6.5 Hz, 6H), 2.97 (m, 1H), 3.25 (br s, 2H),
3.71 (s, 2H), 3.72 (s, 3H).
OH (150 mg, 89%) as a viscous oil: 1H NMR (CDCl3) δ 0.93 (m,
12H), 1.07 (m, 12H), 1.45 (s, 9H), 1.46 (m, 2H), 2.52 (d, J ) 6.2
Hz, 2H), 2.62 (s, 3H), 2.67 (m, 5H), 3.05 (m, 2H), 3.22 (s, 2H),
3.33 (s, 2H), 3.42 (s, 2H), 3.44 (s, 2H), 3.47 (s, 2H), 3.52 (s, 2H),
9.32 (br s, 1H), 9.53 (s, 2H), 9.60 (s, 1H), 9.79 (br s, 1H).
H-Nr-P h e-OMe (H-1d -OMe). The reaction was carried out
according to the general procedure using Boc-NR-Phe-OMe Boc-
1d -OMe (5 g, 17.01 mmol) to give H-1d -OMe (2.5 g, 76%) as an
oil: 1H NMR (CDCl3) δ 3.39 (br s, 2H), 3.56 (s, 2H), 3.74 (s, 3H),
3.97 (s, 2H), 7.35 (m, 5H).
Gen er a l P r oced u r e for Cou p lin g. Boc-Nr-Va l-Nr-Ala -
OBn (Boc-2a -OBn ). To a solution of Boc-NR-Val-OH (Boc-1b-
OH) (0.7 g, 3.02 mmol), H-NR-Ala-OBn (H-1a -OBn) (0.59 g, 3.02
mmol), and DMAP (14 mg, 0.11 mmol) in CH2Cl2 (50 mL) cooled
at 0 °C was added DCC (0.93 g, 4.53 mmol). After 5 min, the
mixture was stirred at room temperature for 12 h. The DCU
was filtered on Celite, and the solvent was evaporated. The
residue was purified by column chromatography on silica gel
using ether and then methanol. The crude product eluted by
MeOH was washed with HCl 2 N, dried over Na2SO4, and
evaporated to afford Boc-2a -OBn (0.82 g, 67%) as a viscous oil:
H-Nr-Leu -Nr-Va l-OMe (H-2b-OMe). The reaction was car-
ried out according to the general procedure using Boc-2b-OMe
(2.5 g, 6.68 mmol) to give H-2b-OMe (1.51 g, 83%) as an oil: 1H
NMR (CDCl3) δ 0.85 (d, J ) 6.6 Hz, 6H), 1.02 (d, J ) 6.4 Hz,
6H), 1.72 (m, 1H), 2.25 (d, J ) 7.4 Hz, 2H), 2.30 (br s, 2H), 3.10
(s, 2H), 3.21 (m, 1H), 3.62 (s, 3H), 3.69 (s, 2H), 8.57 (br s, 1H).
H-Nr-Va l-Nr-Ala -Nr-Leu -OBn (H-3-OBn ). The reaction was
carried out according to the general procedure using Boc-3-OBn
(0.94 g, 1.75 mmol) to give H-3-OBn (0.68 g, 90%) as an oil: 1H
NMR (CDCl3) δ 0.91 (d, J ) 6.1 Hz, 6H), 1.01 (d, J ) 6.4 Hz,
6H), 1.63 (m, 1H), 2.72 (m, 6H), 3.10 (s, 2H), 3.41 (s, 2H), 3.72
(s, 2H), 5.19 (s, 2H), 7.36 (s, 5H), 8.19 (br s, 1H), 8.80 (s, 1H).
H-(Nr-Va l-Nr-Ala -Nr-Leu )2-OBn (H-6-OBn ). The reaction
was carried out according to the general procedure using Boc-
6-OBn (190 mg, 0.22 mmol) to give H-6-OBn (160 mg, 95%) as
an oil: 1H NMR (CDCl3) δ 0.87 (m, 12H), 1.00 (m, 12H), 1.51
(m, 2H), 2.51 (m, 5H), 2.64 (m, 5H), 2.76 (m, 1H), 2.99 (m, 1H),
3.05 (s, 2H), 3.28 (s, 2H), 3.31 (s, 2H), 3.37 (br s, 4H), 3.65 (s,
2H), 5.12 (s, 2H), 7.31 (s, 5H), 8.15 (br s, 1H), 9.09 (s, 1H), 9.24
(s, 1H), 9.40 (s br, 1H), 9.52 (s, 1H).
1
IR (KBr) 3370-3120, 1710, 1705, 1670 cm-1; H NMR (CDCl3)
δ 1.02 (d, J ) 6.5 Hz, 6H), 1.43 (s, 9H), 2.77 (s, 3H), 3.04 (m,
1H), 3.28 (s, 2H), 3.68 (s, 2H), 5.22 (s, 2H), 5.59 (br s, 1H), 7.3-
7.5 (m, 5H), 9.52 (br s, 1H); 13C NMR (CDCl3) δ 18.2 (q, J ) 126
Hz), 28.6 (q, J ) 126.7 Hz), 44.8 (q, J ) 135.1 Hz), 57.3 (d, J )
136.4 Hz), 58.6 (t, J ) 136.1 Hz), 59.2 (t, J ) 136.4 Hz), 66.8 (t,
J ) 148.2 Hz), 80.8 (s), 128.7 (d, J ) 160.5 Hz); 128.9 (d, J )
158.1 Hz), 128.9 (d, J ) 158.9 Hz); 136.0 (s); 156.3 (s); 168.1 (s),
169.8 (s). HR-MS FAB m/z for C20H32N4O5 calcd 431.2270 [M +
Na]+, obsd 431.2270.
Boc-Nr-Leu -Nr-Va l-OMe (Boc-2b-OMe). The reaction was
carried out according to the general procedure for coupling using
Boc-NR-Leu-OH Boc-1c-OH (2.75 g, 11.18 mmol), H-NR-Val-OMe
H-1b-OMe (1.63 g, 11.18 mmol), DMAP (56 mg, 0.42 mmol), DCC
(3.46 g, 16.76 mmol), and CH2Cl2 (40 mL) to give Boc-2b-OMe
(3.56 g, 85%) as a viscous oil: 1H NMR (CDCl3) δ 0.97 (d, J )
6.6 Hz, 6H), 1.09 (d, J ) 6.4 Hz, 6H), 1.42 (s, 9H), 1.67 (m, 1H),
2.52 (d, J ) 7.2 Hz, 2H), 3.29 (m, 1H), 3.35 (s, 2H), 3.68 (s, 2H),
3.71 (s, 3H), 5.58 (s br, 1H), 9.16 (s br, 1H); 13C NMR (CDCl3) δ
20.1 (q, J ) 123.6 Hz), 20.9 (q, J ) 124.7 Hz), 26.8 (d, J ) 126.3
Hz), 28.6 (q, J ) 126.5 Hz), 52.1 (q, J ) 147.3 Hz), 55.2 (t, J )
138.6 Hz), 55.4 (d, J ) 138.3 Hz), 62.2 (t, J ) 136.2 Hz), 68.3 (t,
J ) 132.8 Hz), 80.7 (s), 155.85 (s), 168.5 (s), 171.1 (s); HR-MS
FAB m/z for C17H34N4O5 calcd 397.2427 [M + Na]+, obsd
397.2428.
Gen er a l P r oced u r e for Acetyla tion . Ac-Nr-Va l-OMe (Ac-
1b-OMe). To a solution of H-1b-OMe (1 g, 6.85 mmol) in CH2-
Cl2 (10 mL) was added NEt3 (0.7 g, 6.9 mmol). The mixture was
cooled to 0 °C, and acetyl chloride (0.55 g, 6.9 mmol) was added
dropwise. The mixture was allowed to warm to room tempera-
ture and was stirred for 4 h. The solvent was removed in vacuo.
The residue was dissolved in water and extracted with several
portions of ether. Drying of combined extracts with Na2SO4,
evaporation of the solvent in vacuo, and purification of the
residue by silica gel chromatography (ether/petroleum ether: 50/
50) gave (50%) of Ac-1b-OMe as a mixture of two isomers cis
and trans: 1H NMR (CDCl3) δ (major isomer) 1.12 (m, 6H), 2.16
(s, 3H), 3.28 (m, 1H), 3.75 (s, 3H), 3.82 (s, 2H), 6.95 (br s, 1H).
Ac-Nr-P h e-OMe (Ac-1d -OMe). The reaction was carried out
according to general procedure using H-1d -OMe (1 g, 5.23 mmol)
to give Ac-1b-OMe as a viscous oil: 1H NMR (CDCl3) δ (major
isomer) 1.95 (s, 3H), 3.78 (s, 3H), 3.80 (s, 2H), 4.09 (s, 2H), 7.36
(m, 6H).
Boc-Nr-Va l-Nr-Ala -Nr-Leu -OBn (Boc-3-OBn ). The reaction
was carried out according to the general procedure for coupling
using Boc-NR-Val-NR-Ala-OH Boc-2a -OH (1.37 g, 4.31 mmol),
H-NR-Leu-OBn H-1c-OBn (1 g, 4.3 mmol), DMAP (20 mg, 0.14
mmol), DCC (1.34 g, 6.46 mmol), and CH2Cl2 (85 mL) to give
Boc-3-OBn (2.10 g, 93%) as a viscous oil: 1H NMR (CDCl3) δ
0.90 (d, J ) 6.63 Hz, 6H), 1.02 (d, J ) 6.49 Hz, 6H), 1.42 (s,
9H), 1.57 (m, 1H), 2.60 (s, 3H), 2.69 (d, J ) 7.01 Hz, 2H), 3.00
(m, 1H), 3.26 (s, 2H), 3.38 (s, 2H), 3.69 (s, 2H), 5.18 (s, 2H), 5.55
Gen er a l P r oced u r e for Hyd r ogen olysis. Boc-Nr-Va l-Nr-
Ala -OH (Boc-2a -OH). To a solution of Boc-2a -OBn (0.65 g, 1.59
mmol) in 2-propanol (15 mL) was added Pd/C 10% (60 mg per
mmol, 93 mg). After 18 h of stirring under H2 atmosphere, the
solution was filtered on Celite and the solvent was evaporated
to afford Boc-2a -OH (0.48 g, 95%) as a viscous oil: IR (KBr)
3400-3100, 1800, 1740, 1600 cm-1; 1H NMR (CDCl3) δ 0.98 (d,
J ) 4.77 Hz, 6H), 1.35 (s, 9H), 2.67 (s, 3H), 2.99 (m, 1H), 3.34
(s, 2H), 3.49 (s, 2H), 6.18 (br s, 1H), 8.57 (br s, 1H), 9.76 (br s,
1H); 13C NMR (CDCl3) δ 17.9 (q, J ) 125.5 Hz), 28.2 (q, J )
126.9 Hz), 45.7 (q, J ) 136.4 Hz), 57.2 (t, J ) 136.7 Hz), 57.8 (d,
J ) 137.9 Hz), 61.0 (t, J ) 138.8 Hz), 81.1 (s), 156.9 (s), 170.1
(s), 171.3 (s); HR-MS m/z for C13H26N4O5 calcd 318.1903 (M+),
obsd 318.1901.
(br s, 1H), 7.3-7.37 (m, 5H); 9.15 (br s, 1H); 9.34 (br s, 1H); 13
C
NMR (CDCl3) δ 17.7 (q, J ) 123.3 Hz), 20.7 (q, J ) 124.9 Hz),
26.45 (d, J ) 131.2 Hz), 28.3 (q, J ) 126.9 Hz), 46.7 (q, J )
136.5 Hz), 57.2 (d, J ) 137.9 Hz), 58.2 (t, J ) 134.9 Hz), 58.4 (t,
J ) 138.5 Hz), 62.4 (t, J ) 140.4 Hz), 64.3 (t, J ) 147.7 Hz,
CH2), 66.3 (t, J ) 148.3 Hz), 81.1 (s), 128.3 (d), 128.4 (d), 128.5
(d), 135.8 (s), 156.6 (s), 167.1 (s), 168.3 (s), 169.9 (s); HR-MS
FAB m/z for C26H44N6O6 calcd 559.3220 [M + Na]+, obsd
559.3223.
Boc-Nr-Va l-Nr-Ala -Nr-Leu -Nr-Va l-OMe (Boc-4-OMe). The
reaction was carried out according to the general procedure for
coupling using Boc-NR-Val-NR-Ala-OH Boc-2a -OH (1.07 g, 3.37
mmol), H-NR-Leu-NR-Val-OMe H-2b-OMe (0.93 g, 3.39 mmol),
DMAP (16 mg, 0.13 mmol), DCC (1.05 g, 5.09 mmol), and CH2-
Cl2 (50 mL) to give Boc-4-OMe (1.60 g, 83%) as a viscous oil: 1H
NMR (CDCl3) δ 0.96 (d, J ) 6.5 Hz, 6H), 1.03 (d, J ) 6.5 Hz,
6H), 1.07 (d, J ) 6.4 Hz, 6H), 1.44 (s, 9H), 1.49 (m, 1H), 2.53 (d,
J ) 7.1 Hz, 2H), 2.63 (s, 3H), 3.08 (m, 1H), 3.28 (m, 1H), 3.31 (s,
2H), 3.39 (s, 2H), 3.41 (s, 2H), 3.64 (s, 2H), 3.74 (s, 3H), 5.72 (s
br, 1H), 9.06 (s br, 1H), 9.29 (s br, 1H), 9.38 (s br, 1H); 13C NMR
(CDCl3) δ 17.8 (q, J ) 126.1 Hz), 19.7 (q, J ) 128.1 Hz), 20.9 (q,
J ) 134.6 Hz), 26.7 (d, J ) 121.5 Hz), 28.6 (q, J ) 126.9 Hz),
47.1 (q, J ) 136.6 Hz); 52.1 (q, J ) 147.2 Hz), 55.3 (d, J ) 137.8
Hz), 55.6 (t, J ) 137.0 Hz), 57.5 (d, J ) 124.8 Hz), 58.9 (t, J )
135.3 Hz), 62.2 (t, J ) 134.7 Hz), 62.5 (t, J ) 135.1 Hz), 67.4 (t,
J ) 133.1 Hz), 81.5 (s), 157.1 (s), 168.0 (s), 168.4 (s), 169.5 (s);
Boc-Nr-Va l-Nr-Ala -Nr-Leu -OH (Boc-3-OH). The reaction
was carried out according to the general procedure for hydro-
genolysis using Boc-3-OBn (0.71 g, 1.33 mmol) to give Boc-3-
OH (0.56 g, 95%) as a viscous oil: 1H NMR (CDCl3) δ 0.93 (d, J
) 6.61 Hz, 6H), 1.03 (d, J ) 6.51 Hz, 6H), 1.42 (s, 9H), 1.55 (m,
1H), 2.63 (d, J ) 6.96 Hz, 2H), 2.66 (s, 3H), 3.02 (m, 1H), 3.31
(s, 2H), 3.47 (s, 2H), 3.50 (s, 2H), 5.69 (br s, 1H), 9.45 (br s, 1H),
9.58 (br s, 1H); 13C NMR (CDCl3) δ 17.6 (q, J ) 124.5 Hz), 20.5
(q, J ) 126.0 Hz), 26.4 (d, J ) 131.2 Hz), 28.2 (q, J ) 126.6 Hz),
46.9 (q, J ) 136.6 Hz), 57.3 (d, J ) 138.5 Hz), 58.0 (t, J ) 134.9
Hz), 61.1 (t, J ) 137.0 Hz), 61.5 (t, J ) 138.9 Hz), 66.3 (t, J )
134.9 Hz), 81.4 (s), 157.0 (s), 169.4 (s), 169.4 (s), 171.7 (s).
Boc-(Nr-Va l-Nr-Ala -Nr-Leu )2-OH (Boc-6-OH). The reaction
was carried out according to the general procedure for hydro-
genolysis using Boc-6-OBn (190 mg, 0.22 mmol) to give Boc-6-

Notes
J . Org. Chem., Vol. 66, No. 14, 2001 4929
171.0 (s); HR-MS FAB m/z for C25H50N8O7 calcd 597.3700 [M +
Na]+, obsd 597.3705.
3.26 (s, 2H), 3.31 (s, 2H), 3.37 (s, 2H), 3.41(br s, 4H), 3.70 (s,
2H), 5.17 (s, 2H), 5.83 (br s, 1H), 7.36 (br s, 5H), 9.13 (br s, 1H),
9.26 (br s, 1H), 9.49 (br s, 3H); HR-MS FAB m/z for C40H72N12O9
calcd 887.5443 [M + Na]+, obsd 887.5434.
Boc-Nr-Va l-Nr-Ala -Nr-Leu -Nr-Va l-Nr-P h e-OMe (Boc-5-
OMe). The reaction was carried out according to the general
procedure for coupling using Boc-NR-Val-NR-Ala-NR-Leu-NR-Val-
OH Boc-4-OH (500 mg, 0.89 mmol), H-NR-Phe-OMe H-1d -OMe
(170 mg, 0.89 mmol), DMAP (4 mg, 0.03 mmol), DCC (280 mg,
1.33 mmol), and CH2Cl2 (10 mL) to give Boc-5-OMe (0.36 g, 55%)
as a viscous oil: 1H NMR (CDCl3) 0.93 (d, J ) 6.24 Hz, 6H),
1.06 (d, J ) 6.38 Hz, 6H), 1.08 (d, J ) 6.38 Hz, 6H), 1.43 (s,
19H), 2.41 (d, J ) 7.0 Hz, 2H), 2.63 (s, 3H), 2.95 (m, 1H), 3.36
(s, 2H), 3.38 (s, 2H), 3.48 (s, 2H), 3.64 (s, 2H), 3.74 (s, 2H), 4.13
(s, 2H), 5.88 (br s, 1H), 7.31 (m, 5H), 9.10 (br s, 1H), 9.42 (br s,
1H), 9.46 (br s, 1H), 9.66 (br s, 1H).
Boc-(Nr-Va l-Nr-Ala -Nr-Leu )4-OBn (Boc-7-OBn ). The reac-
tion was carried out according to the general procedure for
coupling using Boc-(NR-Val-NR-Ala-NR-Leu)2-OH Boc-6-OH (150
mg, 0.19 mmol), H-(NR-Val-NR-Ala-NR-Leu)2-OBn H-6-OBn (150
mg, 0.19 mmol), DMAP (1 mg, 0.01 mmol), DCC (60 mg, 0.29
mmol), and CH2Cl2 (10 mL) to give Boc-7-OBn (0.21 g, 72%) as
a
white powder: mp 118-121 °C; HR-MS FAB m/z for
C68H128N24O15 calcd 1543.9889 [M + Na]+, obsd 1543.9835.
Su p p or tin g In for m a tion Ava ila ble: Tables of X-ray
structural data for Boc-1d -OH. Copies of 1H NMR spectra for
compounds Boc-1b-OMe, Boc-1b-OH, H-1b-OMe, Boc-1c-OMe,
Boc-1c-OH, Boc-1c-OBn, H-1c-OBn, Boc-1d -OMe, H-1d -OMe,
Boc-1d -OH, Ac-1b-OMe, Ac-1d -OMe, Boc-2a -OBn, Boc-2a -OH,
Boc-2b-OMe, H-2b-OMe, Boc-3-OBn, Boc-3-OH, Boc-4-OMe,
Boc-4-OH, Boc-5-OMe, Boc-6-OBn, Boc-6-OH, H-6-OBn, Boc-
7-OBn. This material is available free of charge via the
Internet at http://pubs.acs.org.
Boc-(Nr-Va l-Nr-Ala -Nr-Leu )2-OBn (Boc-6-Bn ). The reac-
tion was carried out according to the general procedure for
coupling using Boc-NR-Val-NR-Ala-NR-Leu-OH Boc-3-OH (420
mg, 0.94 mmol), H-NR-Val-NR-Ala-NR-Leu-OBn 3c (490 mg, 0.94
mmol), DMAP (4 mg, 0.03 mmol), DCC (290 mg, 1.41 mmol),
and CH2Cl2 (25 mL) to give Boc-6-OBn (0.37 g, 46%) as a viscous
oil: 1H NMR (CDCl3) 0.90 (d, J ) 6.45 Hz, 6H), 0.92 (d, J )
6.45 Hz, 6H), 1.04 (d, J ) 6.38 Hz, 6H), 1.05 (d, J ) 6.38 Hz,
6H), 1.44 (s, 9H), 1.52 (m, 2H), 2.45 (d, J ) 7.0 Hz, 2H), 2.56 (s,
3H), 2.66 (s, 3H), 2.68 (d, 2H, J ) 7.0 Hz, CH2), 2.95 (m, 2H),
J O001500D