Solid-phase synthesis of "mixed" peptidomimetics using Fmoc-protected aza-β3-amino acids and α-amino acids

Article abstract of DOI:10.1021/jo051585o

A solid-phase fluorenylmethyloxycarbonyl (Fmoc)-based synthesis strategy is described for "mixed" aza-β³-peptides as well as a convenient general approach for their required building blocks, the aza-β³-amino acid residues (aza-β³-aa). These monomers allow the synthesis of relatively large quantities of pure mixed aza-β³-peptides. The required Fmoc-substituted aza-β³-amino acids are accessible by convenient synthesis, and a number of monomers including those containing side chains with functional groups have been synthesized. The method was applied toward the solid-phase ynthesis of aza-β³-peptide mimetics of a biologically active histone H4 sequence.

Full text of DOI:10.1021/jo051585o

Solid-Phase Synthesis of “Mixed” Peptidomimetics Using
Fmoc-Protected Aza-â3-amino Acids and r-Amino Acids
Olivier Busnel,† Lanrong Bi,† Hayet Dali,§ Arnaud Cheguillaume,† Soizic Chevance,‡
Arnaud Bondon,‡ Sylviane Muller,§ and Miche`le Baudy-Floc’h*,†
Groupe “Ciblage et Auto-Assemblages Fonctionnels”, UMR CNRS 6510, Institut de Chimie, Universite´ de
Rennes I, Av. du Ge´ne´ral Leclerc, F-35042 Rennes Ce´dex, France, PRISM, Equipe RMN-ILP, UMR CNRS
6026, CS 34317, Campus de Villejean, Universite´ de Rennes 1, F-35043 Rennes Cedex, France, and UPR
9021 CNRS, Institut de Biologie Mole´culaire et Cellulaire, 15 rue Rene´ Descartes, 67000 Strasbourg
F-67000 Strasbourg, France
Michele.Baudy-Floch@univ-rennes1.fr
Received July 29, 2005
A solid-phase fluorenylmethyloxycarbonyl (Fmoc)-based synthesis strategy is described for “mixed”
aza-â3-peptides as well as a convenient general approach for their required building blocks, the
aza-â3-amino acid residues (aza-â3-aa). These monomers allow the synthesis of relatively large
quantities of pure mixed aza-â3-peptides. The required Fmoc-substituted aza-â3-amino acids are
accessible by convenient synthesis, and a number of monomers including those containing side
chains with functional groups have been synthesized. The method was applied toward the solid-
phase synthesis of aza-â3-peptide mimetics of a biologically active histone H4 sequence.
Introduction
ureapeptoids,15,16 amino-oxypeptoids,17 â-peptoids,18 and
hydrazinoazapeptoids.19 The particularity of these ana-
logues is the absence of chirality in the N-substituted
residues. Peptoid analogues are peptide mimics in which
Peptides and proteins play a crucial role in virtually
all biological processes. Peptides are usually smaller than
proteins and are often important starting molecules for
the development of potential therapeutic agents.1 Nev-
ertheless, peptides have severe limitations for usage as
therapeutic agents due to degradation by proteases and
low membrane permeability. To overcome these limita-
tions and to improve metabolic stability, bioavailability,
and biological absorption, a number of backbone-modified
peptides and peptide mimics have been the focus of
research over the past several years.2,3
(6) Kessler, H. Angew. Chem., Int. Ed. Engl. 1993, 32, 543-544.
(7) Kruijtzer, J. A. W.; Liskamp, R. M. J. Tetrahedron Lett. 1995,
36, 6969-6972.
(8) Dutta, A. S.; Morley, J. S. J. Chem. Soc., Perkin Trans. 1 1975,
1712-1720.
(9) Gray, C. J.; Ireson, J. C.; Parker, R. C. Tetrahedron 1977, 33,
739-743.
(10) Gante, J. Synthesis 1989, 6, 405-413.
(11) Quibell, M.; Turnell, W. G.; Johnson, T. J. J. Chem. Soc., Perkin
Trans. 1 1993, 2843-2849.
Potential classes of peptidomimetics for broad applica-
(12) Greenlee, W. J.; Thorsett, E. D.; Springer, J. P.; Patchett, A.
A.; Ulm, E. H.; Vassil, T. C. Biochem. Biophys. Res. Commun. 1984,
122, 791-797.
tion are peptoids4-7 and analogues such as azapeptoids,8-14
(13) Han, H.; Janda, K. D. J. Am. Chem. Soc. 1996, 118, 2539-
2544.
* To whom correspondence should be addressed. Phone: 33 (0)-
223236933. Fax: 33(0)23236738.
(14) Gibson, C.; Goodman, S. L.; Hahn, D.; Ho¨lzemann, G.; Kessler,
H. J. Org. Chem. 1999, 64, 7388-7394.
† Institut de Chimie, Universite´ de Rennes I.
‡ Campus de Villejean, Universite´ de Rennes 1.
(15) Kruijtzer, J. A. W.; Lefeber, D. J.; Liskamp, R. M. J. Tetrahe-
dron Lett. 1997, 38, 5335-5338.
§ Institut de Biologie Mole´culaire et Cellulaire.
(1) Giannis, A. Angew. Chem., Int. Ed. Engl. 1993, 32, 1244-1267.
(2) Gante, J. Angew. Chem., Int. Ed. Engl. 1994, 33, 1699-1720.
(3) Gellman, S. H. Acc. Chem. Res. 1998, 31, 173-180.
(4) Kirshenbaum, K.; Zukermann, R. N.; Dill, K. A. Curr. Opin.
Struct. Biol. 1999, 9, 530-535.
(16) Wilson, M. E.; Nowick, J. S. Tetrahedron Lett. 1998, 39, 6613-
6616.
(17) Shin, I.; Park, K. Org. Lett. 2002, 4, 869-872.
(18) Hamper, B. C.; Kolodziej, S. A.; Scates, A. M.; Smith, R. G.;
Cortez, E. J. Org. Chem. 1998, 63, 708-718.
(19) Cheguillaume, A.; Lehardy, F.; Bouget, K.; Baudy-Floc’h, M.;
Le Grel, P. J. Org. Chem. 1999, 64, 2924-2927.
(5) Zukermann, R. N.; Kerr, J. M.; Kent, S. B. H.; Moos, W. H. J. J.
Am. Chem. Soc. 1992, 114, 10646-10647.
10.1021/jo051585o CCC: $30.25 © 2005 American Chemical Society
Published on Web 11/08/2005
J. Org. Chem. 2005, 70, 10701-10708
10701

Busnel et al.
SCHEME 1a
the side chain has been shifted from the R-carbon atom
in a peptide to the achiral nitrogen. Undoubtedly, this
will have consequences not only on the structure but also
on biological activity of these analogues as compared to
the parent peptide.20
As part of our effort to explore new peptoid analogues
with potentially useful biological properties, we have
developed a synthetic strategy for hydrazinopeptoids or
aza-â3-peptides.21 As is the case with the synthesis of
other peptidomimetics, the synthesis of the required
building blocks is just as important as the construction
of the oligomeric structure. Recently, we developed a
procedure for solution synthesis of aza-â3-peptides using
tert-butyloxycarbonyl2 (Boc)-protected monomers.22 Be-
cause of the usefulness of these peptide derivatives, we
have been interested in the synthesis of “mixed” peptides
using solid-phase synthesis based upon the fluorenylm-
ethyloxycarbonyl (Fmoc)/tert-butyl strategy, because the
Fmoc protection groups can easily be removed using mild
basic conditions. Moreover, when using the Fmoc protec-
tion strategy, acid-labile groups can be used for protection
of the side chains. Consequently, final deprotection of side
chains and cleavage from the resin can be carried out
under less vigorous conditions compared to using the Boc
deprotection strategy. Employment of the Fmoc groups
also makes these monomers fully compatible with usage
of other standard Fmoc-protected amino acids or other
Fmoc-protected building blocks. Thus, both aza-â3-pep-
tides and “mixed” peptidomimetics would easily be
synthesized.
We describe in this article a convenient and general
method for the synthesis of Fmoc-protected aza-â3-amino
acid monomers, including those with functional side
chains. Validation of the solid-phase synthesis was
demonstrated by the synthesis of mixed aza-â3-peptides
of a model peptide encompassing residues 88-99 of
histone H4 (H2N-88Tyr-Ala-Leu-Lys-Arg-Gln-Gly-Arg-
Thr-Leu-Tyr-99Gly-OH). As part of our program to study
the structural characteristics as well as the biological
activities, we are especially interested in the synthesis
of hybrid peptides using solid-phase synthesis, with the
long-term challenge of creating libraries for structure-
activity relationship and potentially drug discovery.
a Reagents and conditions: (i) RCHO; (ii) NaBH3CN; (iii)
BrCH2CO2Bn, K2CO3, toluene, ∆, 24 h; H2 (1 atm), 5% Pd/C or
BrCH2CO2t-Bu, K2CO3, toluene, ∆, 24 h; CF3CO2H; NaHCO3; (iv)
OHCCO2H, methanol, NaBH3CN, HCl (2 N).
containing nonfunctional side chains is straightforward
and is outline in Scheme 1.
For the synthesis of functionalized monomers, the most
commonly used acid-labile protections reported for solid-
phase synthesis, using the Fmoc strategy, are Boc, t-Bu,
or Trt group, depending on the functionality. Due to the
acid-labile group, protecting group requiring acid condi-
tions cannot be applied to protect the carboxy group. A
benzyl group could be used, but a partial cleavage of the
Fmoc protecting group can occur by catalytic hydrogena-
tion during the benzyl deprotection. However, through
carefully controlled hydrogenation condition we have
shown that the benzyl group can be cleaved in the
presence of Fmoc in 60% yield. Nevertheless, the nucleo-
philic substitution of benzyl bromoacetate with Fmoc
carbazate proceeds with low yield (20%) and with Fmoc
hydrazine in 40-60% yield, so to avoid the steps of
substitution and benzyl deprotection, a second approach
that relies on reductive amination of glyoxilic acid and
Nâ-Fmoc-protected NR-substituted hydrazine 2 has been
studied (Scheme 1, iv).
As an example to get the Fmoc-aza-â3-tyrosine, the
4-hydroxybenzaldehyde was first protected either as a
tert-butyl ether using tert-butyl trichloroacetimidate23 or
as a methoxy ethyl ether using chloromethyl ethyl
ether,24 after which condensation with Fmoc carbazate
1 and reduction with NaBH3CN afford the hydrazine 2
(R ) EtOCH2O-C6H4-CH2). Reductive amination of
glyoxilic acid and hydrazine 2 (R ) EtOCH2O-C6H4-
CH2) leads to Fmoc-aza-â3-Tyr(OCH2OEt)-OH 3 (R )
EtOCH2O-C6H4-CH2) in 60% yield.
For the synthesis of the aza-â3-alanine, the formalde-
hyde is not stable and must be used either in water,
which is not favorable for a condensation with hydrazine,
or as diethyl acetal. Thus, the corresponding Fmoc
hydrazine 2 (R ) CH3) was prepared in using the
enhanced nucleophilicity of the methylated N-atom of
methylhydrazine. Methylhydrazine was treated first with
an excess of di-tert-butyl dicarbonate (Boc2O), the monoa-
cylation occurred on the substituted nitrogen in 92%
yield, no acylation was observed on the unsubstituted
nitrogen, followed by addition of FmocCl and NEt3.25 The
Boc-protecting group was then cleaved by acidification
with trifluoroacetic acid (TFA), and then reductive ami-
nation of glyoxylic acid led to the Fmoc-aza-â3-Ala-OH 3
(R ) CH3) (Scheme 2).
Results and Discussion
Preparation of Monomers. Two strategies have been
employed for the synthesis of the required Fmoc-
protected monomers 3. Fmoc-carbazate 1 was converted
to the corresponding Nâ-Fmoc-protected NR-substituted
hydrazine 2 by condensation with an aldehyde and then
reduction of the hydrazone. The first approach involves
the use of nucleophilic substitution of tert-butyl or benzyl
bromoacetates by the hydrazine 2, which has been
successfully applied for the synthesis of various aza-â3-
amino esters.22 The required monomers 3 were then
obtained in satisfactory to good yields by deprotection of
the carboxy-protecting group. The synthesis of monomers
Because side reactions occurred during the coupling
of the analogue Fmoc-aza-â3-Gly-OH 3 (R ) H), due to
the formation of polymeric products during activation of
(20) Kruijtzer, J. A.; Hofmeyer, L. J. F.; Herma, W.; Verluis, C.;
Liskamp, R. M. J. Chem.-Eur. J. 1998, 4, 1570-1580.
(21) Cheguillaume, A.; Salaun, A.; Sinbandhit, S.; Potel, M.; Gall,
P.; Baudy-Floc’h, M.; Le Grel, P. J Org. Chem. 2001, 66, 4923-4929.
(22) Cheguillaume, A.; Doubli-Bounoua, I.; Baudy-Floc’h, M.; Le
Grel, P. Synlett 2000, 331-334.
(23) Amstrong, A.; Brackenridge, R. F. W.; Kirk, J. M. Tetrahedron
Lett. 1988, 29, 2483-2486.
(24) Sebhat, I. K.; Tan, Y. L.; Widdowson, D. A.; Wilhelm, R.; White,
A. J. P.; Williams, D. J. Tetrahedron Lett. 2000, 56, 6121-6134.
(25) Metz, H. J.; Neunhoeffer, H. Chem. Ber. 1982, 115, 2807-2818.
10702 J. Org. Chem., Vol. 70, No. 26, 2005

Solid-Phase Synthesis of “Mixed” Peptidomimetics
SCHEME 2a
SCHEME 4a
a Reagents and conditions: (i) Boc2O, CHCl3; (ii) FmocCl, NEt3;
(iii) CF3CO2H, NaHCO3; (iv) OHCCO2H, methanol, NaBH3CN,
HCl (2 N).
SCHEME 3a
a Reagents and conditions: (i) (Boc)2O/NEt3, 90%; (ii) AcCO2H,
98%; (iii) FmocNHNH2, 89%; (iv) NaBH3CN, 82%; (v) BrCH2CO2Bn,
K2CO3, toluene, ∆, 48 h, 60%; (vi) CF3CO2H, NEt3, 95%; (vii)
(BocNH)2NTf, NEt3, 84%; (viii) H2 (1 atm) Pd/C, 96%.
a Reagents and conditions: (i) BrCH2CO2Bz, DIEA, toluene, ∆,
48 h, 61%; (ii) CF3CO2H or HClg NEt3, 85%; (iii) (Boc)2O/NEt3,
CH2Cl2, 60%; (iv) FmocCl, 71%; (v) H2 (1 atm), 10% Pd/C, 96%.
SCHEME 5
NR-unprotected hydrazino acids,26,27 this monomer needs
to be NR-protected. Due to the insolubility of the Fmoc-
aza-â3-Gly-OH 3 (R ) H) in organic solvent, it was not
possible to protect its NR-postion. Therefore, we focused
our attention on the synthesis of Boc-aza-â3-Gly-OBn 4
by nucleophilic substitution of benzyl bromoacetate with
Boc-carbazate, followed by deprotection of the Nâ-Boc of
4, affording 5, which was acylated by using Boc2O/NEt3
in CH2Cl2. Bonnet et al.27 have shown that the acylation
of ethyl hydrazinoacetate hydrochloride using Boc2O/N-
methylmorpholine in a H2O/EtOH mixture led only to a
monoacylation in the Nâ-position. In our conditions, it is
interesting to note that the acylation occurred in the NR-
position in 60% yield together with monoacylation in Nâ-
position in 40% yield. Then, protection of 6 at the Nâ-
position with FmocCl in the presence of triethylamine
and hydrogenolysis of 7 over 10% Pd/C gave the Fmoc-
aza-â3-Gly(Boc)-OH 3 (R ) Boc) (Scheme 3).
The Fmoc-aza-â3-Lys(Boc)-OH was prepared starting
from the commercially available 4-aminobutylaldehyde
diethyl acetal (Scheme 4, n ) 4).11 The amino group was
initially protected with the t-butoxycarbonyl group by
reaction with Boc2O in chloroform. The Boc-protected
diethyl acetal was then treated with aqueous HCl in
tetrahydrofuran (THF) to give crude NR-Boc-pyrrolidin-
2-ol in good yield. The amide carbinol exists in equilib-
rium with the required aldehyde. Reaction between Fmoc
hydrazine and NR-Boc-pyrrolidin-2-ol in ether gave the
hydrazone as a mixture of geometrical isomers. Reduction
of this crude mixture with sodium cyanoborohydride gave
the required N,N′-disubstituted hydrazine 2 (R ) (CH2)4-
NHBoc), and then reductive amination of glyoxilic acid
led to the final product Fmoc-aza-â3-Lys(Boc)-OH 3 (R )
(CH2)4NHBoc).
δ-lactam derivatives can occur. Herein we used Goodman
reagents N,N′-di-Boc-N′′-trifluoromethane sulfonylguani-
dine as versatile guanidinating reagents.28 Therefore, to
get Fmoc-aza-â3-Arg(Boc)-OH, we have to prepare first
the Fmoc-aza-â3-Orn(Boc)-OH, which is the homologue
of the Fmoc-aza-â3-Lys(Boc)-OH. Similarly, Fmoc hydra-
zine 1 and the commercially available diethylacetal of
3-aminopropanal were used as initial starting material
(Scheme 4, n ) 3). The latter was N-t-butoxy carbonyl-
protected with Boc2O, and the diacetal was hydrolyzed
with diluted acetic acid.29 Condensation of 3-t-butoxy
For the synthesis of the analogue of arginine, we need
to conveniently protect the trifunctional guanidine group,
which displays a strong nucleophilic character, otherwise
side reactions such as intramolecular cyclization to
(26) Guy, L.; Vidal, J.; Collet, A. J. Med. Chem. 1998, 41, 4833-
4843.
(27) Bonnet, D.; Grandjean, C.; Rosselot-Pailley, P.; Joly, P.; Bourel-
Bonnet, L.; Santraine, V.; Gras-Masse, H.; Melnyk, O. J. Org. Chem.
2003, 68, 7033-7040.
(28) Feichtinger, K.; Zapf, C.; Sings, H. L.; Goodman, M. J. Org.
Chem. 1998, 63, 3804-3805.
(29) Leuchin, I.; Rajan, P.; Borloo, M.; Bollaet, W.; Haemers, A.
Synthesis 1994, 37-39.
J. Org. Chem, Vol. 70, No. 26, 2005 10703

Busnel et al.
TABLE 1. Amino Acid Sequence of Peptide Analogues 88-99 of H4
name sequence
yield %
aza-â3-Ala 89
Y azaâ3A L K R Q G R T L Y G
Y A azaâ3L K R Q G R T L Y G
YA L azaâ3K R Q G R T L Y G
YA L K azaâ3R Q G R T L Y G
YA L K R Q azaâ3G R T L Y G
YA L K R Q G azaâ3R T L Y G
YA L K R Q G R T azaâ3L Y G
YA L K R Q G R T L azaâ3Y G
YA L K R Q G R T L Y azaâ3G
Y azaâ3A azaâ3L K R Q G R T L Y G
Y azaâ3A azaâ3L azaâ3K R Q G R T L Y G
YA L K R Q G R T azaâ3L azaâ3Y G
36
34
31
26
30
25
42
17
24
32
20
15
aza-â3-Leu 90
aza-â3-Lys 91
aza-â3-Arg 92
aza-â3-Gly 94
aza-â3-Arg 95
aza-â3-Leu 97
aza-â3-Tyr 98
aza-â3-Gly 99
aza-â3-Ala 89-Leu 90
aza-â3-Ala 89-Leu 90-Lys 91
aza-â3-Leu97-Tyr 98
TABLE 2. NMR Data (ppm) of Aza-â3-L97 in DMSO (313 K)
H2N-88Tyr-Ala-Leu-Lys-Arg-Gln-Gly-Arg-Thr-Aza-â3-Leu-Tyr-99Gly-OH
residue
15N
NH
CRH
CâH
others
Tyr 88
33.25
123.48
118.22
117.00
118.48
118.33
106.36
118.22
112.36
156.12
118.27
106.36
3.97
2.78, 3.00
1.25
1.48, 1.49
1.68
1.51, 1.53
1.78, 1.91
NH2 7.70, CHoo′ 7.05, CHmm′ 6.70, OH 9.29
Ala 89
8.59
8.06
7.88
7.96
7.95
8.12
8.06
7.84
8.92
8.26
8.24
4.43
Leu 90
Lys 91
Arg 92
Gln 93
Gly 94
4.35
CγH 1.72, CδH 0.88 0.91
4.28
CγH 1.33, CδH 1.55, CꢀH 2.77, NH 7.99, N 38.50
CγH 1.71, CδH 3.10, NH 7.52, N 85.13
CγH 2.14, NH 6.73 7.21
4.30
4.28
3.88, 3.69
4.42
Arg 95
Thr 96
aza-â3-Leu 97
Tyr 98
1.50, 1.54
3.96
2.42, 2.51
2.69, 2.95
CγH 1.72, CδH 3.11, NH 7.47, N 84.85
CγH 1.03, OH 4.88
CγH 1.63, CδH 0.83 0.85
CHoo′ 7.08, CHmm′ 6.64, OH 9.10
4.06
4.43
3.77, 3.78
Gly 99
carbonyl aminopropanal with the Fmoc hydrazine 1 and
then reduction of the hydrazone with sodium cyanoboro-
hydride afforded N,N′-disubstituted hydrazine 2 (R )
(CH2)3NHBoc). As described previously, Fmoc-aza-â3-
Orn(Boc)-OBn 8 (R ) (CH2)3NHBoc) was obtained by
nucleophilic substitution of benzyl bromoacetate with
hydrazine 2 (R ) (CH2)3NHBoc). Then, selective depro-
tection of 8 (R ) (CH2)3NHBoc) with trifluoroacetic acid
(TFA) gave 8 (R ) (CH2)3NH2) in 90% yield. The crude
amine 8 (R ) (CH2)3NH2) was guanidinylated with the
Goodman reagents (BocNH)2NTf,28 and the protected
analogue of arginine 8 (R ) (CH2)3NHC(NHBoc)NBoc)
was obtained in 85% yield. Finally, deprotection of the
benzyl-protecting group with Pd/C (10%) in ethanol was
effected successfully to give the expected Fmoc-aza-â3-
Arg(Boc)2-OH 3 (R ) (CH2)3NHC(NHBoc)NBoc).
Automated Solid-Phase Synthesis of Mixed Aza-
â3-peptides. The synthesis of hybrid peptides of the
dodecapeptide (residues 88-99 of histone H4) was next
examined using a commercial automatic peptide synthe-
sizer by coupling Fmoc-R-amino acids or Fmoc-aza-â3-
amino acid on preloaded PEG-PS or Wang resin or on
resin where we first attached a Fmoc-protected aza-â3-
amino acid. The general procedure for the solid-phase
synthesis of hybrid peptides using Fmoc/tert-butyl strat-
egy is depicted in Scheme 5. The Fmoc-Gly-OH on the
preloaded resin was first liberated upon treatment with
20% piperidine in dimethylformamide (DMF). For elon-
gating the peptide chain, the resin was treated with a
solution of 4 equiv of Fmoc-R-amino acid or Fmoc-aza-
â3-amino acid activated by diisopropylcarbodiimide (DIC)/
1-hydroxybenzotriazole (HOBt) at room temperature for
30-60 min (depending on the amino acid) to 80 min for
the analogues. No problem was observed for the coupling
of most analogues except for the time of coupling cycles,
which must be longer. In comparison to the synthesis of
â3-amino acid-containing peptides in which coupling
reactions for R/â + R peptides proceed for 3-24 h while
those for R-peptides proceed for 1-24 h,30 coupling cycles
for Fmoc-aza-â3-amino acid as well as coupling cycles for
R-amino acid following the analogue proceed for 80 min
to 2 h while those for Fmoc-R-amino acids proceed for
30-60 min. Coupling Fmoc-aza-â3-Gly-OH affords poly-
meric products during activation of NR-unprotected mono-
mer; this is in accordance with the results described
before during the coupling of hydrazino acids.26 Guy et
al. solved this problem by using Nâ-Boc-NR-Bn-hydrazino
acids, and Bonnet et al. coupled a triprotected Boc
monomer in the N-terminal position.27 In our case, the
coupling of Fmoc-aza-â3-Gly(Boc)-OH leads to the ex-
pected peptide. Deprotection cycles were realized with
20% piperidine in DMF. The hybrid peptides were then
deprotected and cleaved from the resin with TFA/phenol/
EDT/H2O (88/5/2/5), and after preparative HPLC, the
hybrid peptides (25-45% yield) were isolated in >98%
purity. During the cleavage of peptide analogue aza-â3-
Y98 either with a monomer Fmoc-aza-â3-Tyr(Ot-Bu)-OH
or with a monomer Fmoc-aza-â3-Tyr(OCH2OEt)-OH, we
observed not only the deprotection of the protecting group
but also a partial cleavage of the tyrosine chain, and thus
cleavage was realized with CH2Cl2/TFA/phenol/TIS/H2O
(40/48/5/2/5) (where TIS is triisopropylsilane) and moni-
tored by HPLC.
For the synthesis of the peptide analogue aza-â3-G99,
anchoring the first monomer Fmoc-aza-â3-Gly(Boc)-OH
was achieved by esterification of the Fmoc-aza-â3-Gly-
(Boc)-OH (5 equiv with respect to the resin loading) to
(30) Sadowsky, J. D.; Schmitt, M. A.; Lee, H.-S.; Umezawa, N.;
Wang, S.; Tomita, Y.; Gellman, S. H. J. Am. Chem. Soc. 2005, 127,
11966-11968.
10704 J. Org. Chem., Vol. 70, No. 26, 2005

Solid-Phase Synthesis of “Mixed” Peptidomimetics
dried over Na2SO4 and concentrated, giving a white solid that
was triturated with petroleum ether to afford a white powder
(13.28 g, 90%): mp 160 °C; 1H NMR (CDCl3) δ 2.68 (s, 3H,
CH3), 4.28 (t, J ) 6.6 Hz, 1H, CH), 4.50 (d, J ) 6.6 Hz, 2H,
CH2), 6.33 (brs, 1H, NH), 7.30-7.83 (m, 8H, CHar). 13C NMR
(CDCl3) δ 157.2 (s), 143.7 (s), 141.4 (s), 127.8 (d), 127.1 (d),
125.0 (d), 120.0(d), 67.0 (t), 47.2 (d), 39.2 (q). HRMS (ESI) m/z
calcd for C16H16N2O2Na [M + Na]+: 291.11095, found: 291.1104
(2 ppm).
the hydroxyl group of the resin with activation by DIC
at room temperature in DMF for 2 h.31 Completion of the
coupling was confirmed by a 2,4,6-trinitrobenzenesulfonic
acid test, and the loading was determined by Fmoc
cleavage from a resin simple by measuring the UV
absorption of the dibenzofulvene-piperidine adduct that
is formed after cleavage.32
All the peptides (Table 1) were characterized by
electrospray mass spectrometry (HR-MS, ESI) as well as
NMR spectroscopy. The NH hydrazino protons are not
detectable in D2O/H2O because of fast exchange with the
water protons; however, they are readily observed in
DMSO. We give as an example in Table 2 the chemical
shifts in DMSO for the analogue aza-â3-Leu 97.
Fmoc-Aza-â3-Ala-OH 3 (R ) Me). To a stirred solution of
2 (R ) Me) (1.50 g, 5.6 mmol) in DCM/MeOH (10/20 mL) was
added glyoxylic acid monohydrate (0.62 g, 1.2 equiv). NaBH3-
CN (0.42 g, 1.2 equiv) was added to the above mixture, and
pH was brought to 3 by slowly adding a solution of 2 N HCl.
After being stirred for 0.5 h, the pH was adjusted to 1 during
10 min and increased to 4 with solid NaHCO3. The mixture
was filtrated, concentrated and taken up with EtOAc (50 mL),
and washed with water and brine. The organic layer was dried
over Na2SO4 and concentrated to afford the final product as a
moss that was purified by chromatography on silica gel (ether/
Conclusion
We have developed an efficient synthetic strategy for
the synthesis of Fmoc-aza-â3-amino acids. When suitable
protecting groups were used, identical chains can be
incorporated into amino acid analogues. Furthermore, an
efficient solid-phase methodology for the synthesis of
hybrid peptides using a fully automated peptide synthe-
sizer has been developed. Fmoc-protected monomers
enable the preparation of hybrid peptides on automated
peptide synthesizers without any major adjustments in
the synthesis protocols, since the Fmoc-protecting group
is standard in automated synthesis. Thus, different
“mixed” peptide-aza-â3-peptides are easily accessible and
other aza â3-peptidomimetics could be envisaged. These
peptide-aza-â3-peptidomimetic hybrids have been used to
evaluate their biological activity compared with the
histone H4 dodecapeptide (residues 88-99). Evaluation
of the biological properties of these analogues of histone
H4 is currently being undertaken and will be published
elsewhere.33
1
MeOH/AcOH 95/5/0.25) to give 3 R ) Me: (1.68 g, 92%). H
NMR (CDCl3) δ 2.75 (s, 3H, CH3), 3.62 (s, 2H, NCH2), 4.21 (t,
J ) 6.6 Hz, 1H, CH), 4.49 (d, J ) 6.6, 2H, CH2), 6.57 (brs, 1H,
NH), 7.26-7.81 (m, 8H, CHar). 13C NMR (CDCl3) δ 173.1 (s),
156.6 (s), 143.5 (s), 141.3 (s), 127.9 (d), 127.2 (d), 125.1 (d),
120.1 (d), 67.4 (t), 59.0 (t), 47.0 (d), 45.1 (q). HRMS (ESI): m/z
calcd for C18H17N2O4Na2 [M - H + 2Na]+: 371.09837, found:
371.0981 (1 ppm).
Fmoc-Aza-â3-Leu-OH 3 (R ) CH2CH(CH3)2). Hydrazine
(2, R ) CH2CH(CH3)2)22 (2.00 g, 6.46 mmol), glyoxylic acid
monohydrate (0.71 g, 1.2 equiv), and NaBH3CN (0.48 g, 1.2
equiv) were reacted according to the procedure of 3 R ) CH3.
1
Yield: 64%; (1.52 g); mp 131 °C; H NMR (CDCl3) δ 0.93 (d,
6H, J ) 6.4 Hz, CH3), 1.59 (m, 1H, CH), 2.70 (d, 2H, J ) 7.0
Hz, CH2), 3.55 (s, 2H, CH2), 4.23 (t, 1H, J ) 6.1 Hz, CH), 4.58
(d, 2H, J ) 6.1 Hz, CH2), 6.02 (s, 1H, NH), 7.28-7.79 (m, 8H,
CHar). 13C NMR (CDCl3) δ 173.5 (s), 157.1 (s), 143.6 (s), 141.4
(s), 127.8 (d), 127.2 (d), 125.1 (d), 120.1 (d), 67.1 (t), 65.8 (t),
59.4 (t), 47.2 (d), 26.5 (d), 20.6 (q). HRMS (ESI) m/z calcd for
C19H22N2O2Na [M + Na]+: 333.15790, found: 333.1580 (0
ppm).
Fmoc-Protected Tyrosine Hydrazine 2 (R ) EtOCH2O-
C6H4-CH2). Fmoc carbazate (7.64 g, 30 mmol) was added to
a stirred solution of 4-ethyloxymethylbenzaldehyde (5.41 g, 1
equiv) in DCM (100 mL) at rt. The reaction mixture was stirred
for 24 h and concentrated under vacuum to give crude solid
that was purified by crystallization from ethanol to afford (1)
as a white powder (10.00 g, 80%): mp 159 °C; 1H NMR (CDCl3)
δ 1.24 (t, 3H, J ) 7.0 Hz, CH3), 3.76 (q, 2H, J ) 7.0 Hz, CH2),
4.36 (t, 1H, J ) 7.1 Hz, CH), 4.58 (d, 2H, J ) 7.1 Hz, CH2),
5.28 (s, 2H, CH2), 7.08 (d, J ) 8.2 Hz, 1H, CH), 7.32-8.1 (m,
12H, CHar). 13C NMR (CDCl3) δ 158.9 (s), 144.8 (s), 143.7 (s),
141.3 (s), 128.7 (d), 127.4 (d), 116.3 (d), 128.8 (d), 127.8 (d),
125.2 (d), 120.0 (d), 92.9 (t), 67.8 (t), 64.5 (t), 47.0 (d), 15.1 (q).
HRMS (ESI) m/z calcd for C25H24N2O4Na [M + Na]+: 439.16338,
found: 439.1630 (1 ppm).
Experimental Section
N-Boc-N-methylhydrazine.25 To a solution of methylhy-
drazine (3.07 g, 66.7 mmol) in CHCl3 60 mL cooled in ice bath,
Boc2O (13.11 g, 60 mmol) in CHCl3 was slowly added dropwise.
The mixture was then stirred at room temperature (rt)
overnight and washed respectively with aqueous NaHCO3 1
M, water, and brine and dried over Na2SO4. Evaporation of
solvent gave crude Boc-N-methylhydrazine (8.10 g, 92%)
suitable for the next step without purification. 1H NMR
(CDCl3) δ 1.5 (s, 9H, CH3), 3.09 (s, 3H, CH3), 4.08 (brs, 2H,
NH2).
Fmoc-Protected Alanine Hydrazine 2 (R ) Me). To a
stirred solution of Boc-N-methylhydrazine (8.10 g, 55.4 mmol)
in THF/water (70/70 mL) was first added solid NaHCO3 (9.3
g, 2 equiv) and then, dropwise, a mixture of FmocCl (15.72 g,
1.1 equiv) in THF (70 mL). The mixture was stirred overnight
at rt, and ether (100 mL) was added. The organic layer was
washed with brine, dried over Na2SO4, and concentrated. The
residual oil was taken up with dichloromethane (DCM) (200
mL) and HClg overnight to remove Boc protection. The mixture
was concentrated and taken up with ether, giving by filtration
the hydrochloride of 2 (R ) Me) as a white powder. We
generated hydrazine in solution of water-ethyl acetate (EtOAc)
by adding saturated aqueous NaHCO3. The organic layer was
Then Fmoc-protected tyrosine hydrazone (8.24 g, 19.8 mmol)
was dissolved in a solution of DCM/MeOH 40/60 mL. Sodium
cyanoborohydride (1.5 g, 1,2 equiv) was added, and we brought
the pH to 3 or 4 by slowly adding a solution of 2 N HCl. The
mixture was stirred for 0.5 h, and then the pH was adjusted
to 1. After 10 min of being stirred, the solution was neutralized
with solid NaHCO3, the mixture was concentrated under
vacuum, and the residue was taken up with EtOAc (50 mL)
and washed with water and brine. The organic layer was dried
over Na2SO4, and the solvent was removed to give crude oil
that was purified by chromatography on silica gel (PE/EtOAc
1/1) to afford (2 R ) EtOCH2O-C6H4-CH2) as a white solide
(31) Sieber, P. Tetrahedron Lett. 1987, 28, 6147-6150.
(32) Hancock, W. S.; Battersby, J. E. Anal. Biochem. 1976, 71, 260-
264.
(33) Dali, H.; Busnel, O.; Bi, L.; Decker P.; Briand, J. P.; Baudy-
Floc’h, M.; Muller, S. Submitted for publication.
1
(6.12 g 69%): mp 109 °C; H NMR (CDCl3) δ 1.25 (t, 3H, J )
7.1 Hz, CH3), 3.76 (q, 2H, J ) 7.1 Hz, CH2), 3.96 (brs, 2H,
CH2), 4.25 (t, 1H, J ) 6.6 Hz, CH), 4.39 (d, 2H, J ) 6.6 Hz,
CH2), 5.24 (s, 2H, CH2), 6.28 (brs, 1H, NH), 7.01-7.82 (m, 12H,
J. Org. Chem, Vol. 70, No. 26, 2005 10705

Busnel et al.
CHar). 13C NMR (CDCl3) δ 157.8 (s), 144.4 (s), 142.0 (s), 131.2
(s), 130.9 (s), 128.4 (d), 127.7 (d), 125.6 (d), 120.7 (d), 116.9
(d), 93.8 (t), 67.5 (t), 64.9 (t), 55.6 (t), 47.8 (t), 15.8 (q). HRMS
(ESI) m/z calcd for C25H26N2O4Na [M + Na]+: 441.17903,
found: 441.1788 (0 ppm).
(ESI) m/z calcd for C14H20N2O4Na [M + Na]+: 303.13208,
found: 303.1327 (2 ppm).
Aza-â3-Gly(Boc)-OBn (6). A solution of 4 (2.23 g, 7.95
mmol) in DCM 30 mL was saturated by HClg and stirred at
rt overnight. The mixture was concentrated and taken up with
ether, giving by filtration the hydrochloride of 5 as a white
powder (1.47 g), which was added into a stirred solution of
Et3N (0.76 g, 1.1 equiv) in 20 mL of DCM. Boc2O (1.47 g, 1
equiv) was added, and the mixture was stirred overnight,
concentrated, and taken up with ether. After cooling for 4 h,
the hydrochloride of Et3N was removed by filtration, and the
filtrate was concentrated. The crude oil was purified by
chromatography on silica gel (EtOAc/EP 3/7) to give 6 (1.15 g,
Fmoc-Aza-â3-Tyr(OCH2OEt)-OH 3 (R ) EtOCH2O-
C6H4-CH2). Hydrazine 2 (R ) EtOCH2O-C6H4-CH2) (1.50
g, 3.60 mmol), glyoxylic acid monohydrate (0.40 g, 1.2 equiv),
and NaBH3CN (0.34 g, 1.5 equiv) were reacted according to
the procedure of 3 R ) CH3. Yield: 60% (1.03 g); mp 114 °C;
1H NMR (CDCl3) δ 1.21 (t, 3H, J ) 7.0 Hz, CH3), 3.63 (s, 2H,
CH2), 3.70 (q, 2H, J ) 7.0 Hz, CH2), 4.00 (s, 2H, CH2), 4.17 (br
t, 1H, CH), 4.48 (br d, 2H, CH2), 5.18 (s, 2H, CH2), 6.39 (br s,
1H, NH), 7.00-7.80 (m, 12H, CHar). 13C NMR (CDCl3) δ 173.3
(s), 157.2 (s), 143.7 (s), 141.4 (s), 130.8 (s), 128.9 (d), 116.3 (d),
127.9 (d), 127.2 (d), 125.2 (d), 120.1 (d), 93.1 (t), 67.3 (t), 64.3
(t), 60.4 (t), 56.6 (t), 47.1 (d), 15.2 (q). HRMS (ESI) m/z calcd
for C27H28N2O6Na [M + Na]+: 499.18451, found: 499.1843 (0
ppm).
1
52%) as a colorless oil. H NMR (CDCl3) δ 1.40 (s, 9H, CH3),
4.17 (s, 2H, CH2), 5.17 (s, 2H, CH2), 7.34-7.41 (m, 5H, CHar).
13C NMR (CDCl3) δ 170.4 (s), 158.0 (s), 136.1 (s), 129.2 (d),
129.0 (d), 128.9 (d), 81.9 (s), 67.4 (t), 54.1 (t), 28.8 (q). HRMS
(ESI) m/z calcd for C14H20N2O4Na [M + Na]+: 303.13208,
found: 303.1309 (4 ppm).
Fmoc-Aza-â3-Gly(Boc)-OBn (7). To a stirred solution of
6 (1.83 g, 6.52 mmol) in THF/water (15/15 mL) was first added
solid NaHCO3 (1.09 g, 2 equiv) and then, dropwise, a mixture
of FmocCl (2.02 g, 1.2 equiv) in THF (15 mL). The mixture
was stirred overnight at rt, and then ether (50 mL) was added.
The organic layer was washed with brine, dried over Na2SO4,
and concentrated to afford an oil that was purified by chro-
matography on silica gel (EP/EtOAc 9/1 and 7/3) to give 2.31
g (71%) of 7 as a white powder: mp 140 °C; 1H NMR (CDCl3)
δ 1.44 (s, 9H, CH3), 4.26 (brs, 2H, CH2), 4.35 (brs, 1H, CH),
4.47 (br, 2H, CH2), 5.21 (s, 2H, CH2), 6.96 (br s, 1H, NH), 7.30-
7.79 (m, 13H, CHar). 13C NMR (CDCl3) δ 170.0 (s), 156.5 (s),
155.4 (s), 144.1 (s), 141.9 (s), 135.8 (s), 129.3 (d), 129.2 (d),
129.0 (d), 128.9 (d), 128.5 (d), 125.6 (d), 120.7 (d), 83.1 (s), 68.6
(t), 67.8 (t), 53.5 (t), 47.7 (d), 28.7 (q). HRMS (ESI) m/z calcd
for C29H30N2O6Na [M + Na]+: 525.20016, found: 525.2001 (0
ppm).
Fmoc-ProtectedLysineHydrazine2(R)(CH2)4NHBoc).
Fmoc carbazate 1 (9.50 g, 37.3 mmol) was added to a stirred
solution of 4-t-butoxycarbonylaminobutanal27 (7.00 g, 37.3
mmol) in DCM (150 mL) at rt. The reaction mixture was
stirred for 24 h and concentrated under vacuum to give crude
solid that was triturated in petroleum ether to afford as a
white solid (14.3 g, 90%): mp 165 °C; 1H NMR (DMSO) δ 1.38
(s, 9H, CH3), 1.56 (m, 2H, CH2), 2.16 (m, 2H, CH2), 2.94 (m,
2H, CH2), 4.26 (t, 1H, J ) 6.9 Hz, CH), 4.39 (d, 2H, J ) 6.9
Hz, CH2), 6.84 (brs, 1H, NH), 7.29-7.92 (m, 9H, Ar + CH),
10.72 (s, 1H, NH). 13C NMR (DMSO) δ 155.9 (s), 153.7 (s),
148.2 (s), 144.1 (s), 141.1 (s), 128.0 (d), 127.5 (d), 125.5 (d),
120.5 (d), 77.8 (s), 65.8 (t), 46.9 (d), 39.7 (t), 29.6 (t), 28.6 (q),
26.89 (CH2). HRMS (ESI) m/z calcd for C24H29N3O4Na [M +
Na]+: 446.20558, found: 446.2053 (1 ppm).
Then Fmoc-protected lysine hydrazone (1.25 g, 2.94 mmol)
was reduced as described before with sodium cyanoborohydride
(0.53 g, 1.5 equiv). The crude solid was purified by chroma-
tography on silica gel (EtOAc/DCM 4/6) to give 2 (R ) (CH2)4-
Fmoc-Aza-â3-Gly(Boc)-OH 3 (R ) Boc). Catalytic hydro-
genolyse with 10% Pd/C (80 mg) of 7 (1.40 g, 2.79 mmol)
afforded 1.10 g (96%) of 3 (R ) Boc) as a moss. 1H NMR
(CDCl3) δ 1.47 (m, 9H, CH3), 4.05-4.38 (br m, 3H, CH + CH2),
4.52 (d, 2H, J ) 6.8 Hz, CH2), 5.21 (s, 2H, CH2), 6.96 (br s,
1H, NH), 7.30-7.79 (m, 8H, CHar). 13C NMR (CDCl3) δ 169.8
(s), 156.5 (s), 155.2 (s), 144.0 (s), 141.7 (s), 135.6 (s), 129.1 (d),
128.9 (d), 128.8 (d), 128.3 (d), 127.6 (d), 125.5 (d), 120.5 (d),
82.9 (s), 68.4 (t), 67.6 (t), 53.3 (t), 47.4 (d), 28.49 (q). HRMS
(ESI) m/z calcd for C22H24N2O6Na [M + Na]+: 435.1532,
found: 435.1533 (0 ppm).
1
NHBoc) as a white solid (0.70 g 56%): mp 150 °C; H NMR
(CDCl3) δ 1.46 (s, 9H, CH3), 1.52 (m, 4H, CH2), 2.88 (m, 2H,
CH2), 3.16 (m, 2H, CH2), 4.00 (brs, 1H, NH), 4.24 (t, 1H, J )
6.6 Hz, CH), 4.47 (d, 2H, J ) 6.9 Hz, CH2), 4.62 (brs, 1H, NH),
6.37 (brs, 1H, NH), 7.29-7.80 (m, 8H, Ar). 13C NMR (CDCl3)δ
157.3 (s), 156.1 (s), 143.7 (s), 141.3 (s), 127.8 (d), 127.1 (d),
124.0 (d), 120.0 (d), 79.1 (s), 66.9 (t), 51.5 (t), 47.21 (d), 40.40
(t), 28.5 (q), 27.6 (t), 24.8 (t). HRMS (ESI) calcd for C24H31N3O4-
Na [M + Na]+: 448.22123, found: 448.2222 (2 ppm).
1-Boc-amino-3,3-diethoxypropane.29 A mixture of Boc2O
(9 g, 40 mmol) in dioxane (40 mL) was added dropwise to a
stirred solution of 1-amino-3,3-diethoxypropane (5.52 g, 37
mmol) and Et3N (4.04 g, 40 mmol) in dioxane (5 mL) at 0 °C.
After 2 h, the mixture was allowed to warm to rt. Stirring was
continued overnight, and the solvent was evaporated. The
residual oil was taken up in water (10 mL), and the mixture
was acidified with aqueous solution of 1 N HCl (pH 3-4) and
extracted with EtOAc (60 mL × 3). The organic layer was dried
(Na2SO4) and evaporated to give crude oil of 1-Boc-amino-3,3-
diethoxypropane 8.89 g (90%) suitable for further work without
purification. 1H NMR (CDCl3) δ 1.25 (t, 6H, J ) 8.8 Hz, CH3),
1.48 (s, 9H, CH3), 1.85 (q, 2H, J ) 6.3 Hz, CH2), 3.26 (q, 2H,
J ) 6.1 Hz, CH2), 3.22-3.77 (m, 6H, CH2), 4.58 (t, 1H, J ) 6.3
Hz, CH), 4.95 (br s, 1H, NH).
Fmoc-Aza-â3-Lys(Boc)-OH 3 (R ) (CH2)4NHBoc). Hy-
drazine (2, R ) (CH2)4NHBoc) (2.89 g, 6.76 mmol), glyoxylic
acid monohydrate (0.75 g, 1.2 equiv), and NaBH3CN (0.51 g,
1.2 equiv) were reacted according to the procedure of 3 R )
CH3. Yield 59% (2.00 g); 1H NMR (CDCl3) δ 1.44 (s, 9H, CH3),
1.40-1.60 (m, 4H, CH2), 2.89 (m, 2H, CH2), 3.09 (m, 2H, CH2),
3.66 (s, 2H, CH2), 4.21 (br t, 1H, CH), 4.46 (br d, 2H, CH2),
4.87 (br s, 1H, NH), 7.04 (br s, 1H, NH), 7.29-7.80 (m, 8H,
Ar), 9.72 (brs, 1H, CO2H). 13C NMR (CDCl3) δ 173.0 (s), 157.3
(s), 156.1 (s), 143.6 (s), 141.3 (s), 127.8 (d), 127.1 (d), 125.0 (d),
120.0 (d), 79.3 (s), 67.0 (t), 58.4 (t), 56.5 (t), 47.2 (d), 40,2 (d),
28.4 (q), 27.2 (t), 24.3 (t). HRMS (ESI) m/z calcd for C26H33N3O6-
Na [M + Na]+: 506.22671, found: 506.2276 (2 ppm).
Boc-Aza-â3-Gly-OBn (4). To a solution of Boc carbazate
(2.00 g, 15.13 mmol) in toluene (25 mL) were added diisopro-
pylethylamine (DIEA) (1.96 g, 1 equiv) and benzyl 2-bromoac-
etate (3.47 g, 1 equiv). The mixture was stirred at 75 °C for 4
days, filtrated, and concentrated to give a crude oil that was
purified by chromatography on silica gel (EtOAc/DCM 1/9) to
give a colorless oil (2.60 g, 61%) that slowly crystallized; mp
40 °C. 1H NMR (CDCl3) δ 1.47 (s, 9H, CH3), 3.72 (s, 2H, CH2),
5.19 (s, 2H, CH2), 6.43 (br s, 1H, NH), 7.31-7.42 (m, 5H, CHar).
13C NMR (CDCl3) δ 171.5 (s), 156.7 (s), 135.8 (s), 129.0 (d),
128.8 (d), 128.7 (d), 80.9 (s), 67.0 (t), 53.2 (t), 28.7(q). HRMS
3-Boc-aminopropanal.29 A solution of 1-Boc-amino-3,3-
diethoxypropane (8.89 g, 36 mmol) in AcOH (15 mL) and H2O
(4 mL) was stirred at rt for 10 h, neutralized with Na2CO3,
taken up in ether, and washed with water and brine. The
organic phase was evaporated under vacuum to give a yellow
oil used as such in the next step (6.31 g, 98%). 1H NMR (CDCl3)
δ l.47 (s, 9H, CH3), 2.75 (t, 2H, CH2), 3.46 (m, 2H, CH2), 4.95
(br s, 1H, NH), 9.85 (s, 1H, CHO).
Fmoc-Protected Ornitine Hydrazine 2 (R ) (CH2)3-
NHBoc). Fmoc carbazate 1 (9.24 g, 36.4 mmol) was added to
10706 J. Org. Chem., Vol. 70, No. 26, 2005

Solid-Phase Synthesis of “Mixed” Peptidomimetics
a stirred solution of 3-Boc-aminopropanal (6.31 g, 36.4 mmol)
in DCM (150 mL) at rt. The reaction mixture was stirred for
12 h and concentrated under vacuum to give crude solid that
was triturated with petroleum ether to afford the hydrazone
as a white solid (13.2 g, 89%). 1H NMR (DMSO) δ 1.38 (s, 9H,
CH3), 2.31 (m, 2H, CH2), 3.11 (m, 2H, CH2), 4.28 (t, 1H, J )
6.8 Hz, CH), 4.41 (d, 2H, J ) 6.8 Hz, CH2), 6.91 (br, 1H, NH),
7.28-7.93 (m, 9H, Ar + CH), 10.84 (s, 1H, NH). 13C NMR
(DMSO) δ 155.5 (s), 153.2 (s), 146.2 (s), 143.7 (s), 140.72 (s),
127.6 (d), 127.0 (d), 125.1 (d), 120.1 (d), 77.5 (s), 65.4 (t), 46.6
(d), 37.3 (t), 28.8 (t), 28.2 (q).
Then, Fmoc-protected hydrazone (3 g, 7.31 mmol) was
reduced with sodium cyanoborohydride (0.55 g, 1.2 equiv) as
described previously. The crude solid was triturated with
petroleum ether (PE) to afford a white solid of 2 (R ) (CH2)3-
NHBoc) (2.48 g 82%): mp 101 °C; 1H NMR (DMSO) δ 1.40 (s,
9H, CH3), 1.50 (m, 2H, CH2), 2.68 (m, 2H, CH2), 2.98 (m, 2H,
CH2), 4.24 (t, 1H, J ) 6.9 Hz, CH), 4.32 (d, 2H, J ) 6.9 Hz,
CH2), 4.70-4.85 (br s, 1H, NH), 6.78 (br s, 1H, NH), 6.80 (br
s, 1H, NH), 7.25-7.90 (m, 8H, Ar). 13C NMR (DMSO) δ 157.2
(s), 155.9 (s), 144.2 (s), 142.9 (s), 128.0 (d), 127.6 (d), 125.6 (d),
120.5 (d), 77.7 (s), 65.8 (t), 47.1 (d), 38.4 (t), 28.6 (q), 28.2 (t).
Anal. Calcd for C23H29N304 (411.22): C, 67.12; H, 7.11; N,
10.22. Found: C, 67.22; H, 7.19; N, 10.24.
Fmoc-Aza-â3-Orn(NBoc)-OBn (8, R ) (CH2)3NHBoc)).
A mixture of Fmoc-protected ornitine hydrazine 2 (R ) (CH2)3-
NHBoc) (3.7 g, 9 mmol), benzyl 2-bromoacetate (2.66 g, 11.6
mmol), toluene (20 mL), and dry K2CO3 (870 mg, 0.7 equiv)
was refluxed under stirring for 28 h. Usual workup afforded
after chromatography on silica gel (EtOAc/EP 1/3) 3.02 g (60%)
of 8 (R ) (CH2)3NHBoc) as a colorless oil that slowly crystal-
lized in ether: mp 107 °C; 1H NMR (CDCl3) δ 1.50 (s, 9H, CH3),
1.62 (m, 2H, CH2), 2.97 (m, 2H, CH2), 3.24 (m, 2H, CH2), 3.75
(m, 2H, CH2), 4.20 (t, 1H, J ) 6.9 Hz, CH), 4.50 (d, 2H, J )
6.9 Hz, CH2), 5.19 (s, 2H, CH2), 6.88 (brs, 1H, NH), 7.25-7.90
(m, 13H, Ar). 13C NMR (CDCl3) δ 171.23 (s), 156.55 (s), 155.50
(s), 144.13, 141.77 (s), 135.54 C (s), 129.13, 129.04, 128.84 (d),
128.14, 127.47, 125.44, 120.40 (d), 79.38 (s), 67.09 (t), 64.47
(t), 57.63 (t), 54.56 (t), 47.67 (d), 38.83 (t), 28.85 (q), 27.83 (t).
Anal. Calcd for C32H37N306 (559.27): C, 68.66; H, 6.67; N, 7.51.
Found: C, 68.59; H, 6.86; N, 7.28.
Fmoc-Aza-â3-Arg(Boc)2OBn (8, R ) (CH2)3NHC(NHBoc)-
NBoc). To a solution of 8 (R ) (CH2)3NHBoc) (1 g, 1.79 mmol)
in DCM (5 mL), 5 mL of TFA was added. After stirring at rt
overnight, 25 mL of DCM and 10 mL of water were added.
The mixture was neutralized with solid Na2CO3 (pH 8-9), and
the organic layer was washed with brine, dried over Na2SO4,
and partly concentrated, giving 10-15 mL of DCM solution.
Then NEt3 (1.1 equiv, 251 µL) and (BocNH)2CdNTf (0.9 equiv,
0.64 g) were added. After being stirred at rt for 15 h, the
solution was washed, respectively, with a 2 N aqueous solution
of NaHSO4 (10 mL), Na2CO3 (10 mL), and brine (10 mL). The
organic layer was dried over Na2SO4 and concentrated, giving
a crude oil that was purified by chromatography (EP/EtOAc
7/3) to afford the guanidine 8 (R ) (CH2)3NHC(NHBoc)NBoc)
(1.00 g, 80%). 1H NMR (CDCl3) δ 1.39 (s, 18H, CH3), 1.60 (m,
2H, CH2), 2.80 (m, 2H, CH2), 3.32 (m, 2H, CH2), 3.66 (m, 2H,
CH2), 4.24 (t, 1H, J ) 7.1 Hz, CH), 4.45 (d, 2H, J ) 7.1 Hz,
CH2), 5.05 (s, 2H, CH2), 6.95 (brs, 1H, NH), 7.27-7.82 (m, 13H,
Ar), 8.28 (br s, 1H, NH), 11.45 (br s, 1H, NH). 13C NMR CDCl3
δ 171.45 (s), 170.80 (s), 163.97 (s), 156.57 (s), 153.59 (s), 144.17
(s), 141.73 (s), 135.67 (d), 129.06, 128.91, 128.78 (d), 128.09,
127.45, 125.47, 120.34 (d), 83.35 (s), 79.48 (s), 66.93 (t), 60.74
(t), 58.04 (t), 53.89 (t), 47.64 (d), 38.83 (t), 28.67 (q), 28.44 (q),
27.45 (t). Anal. Calcd for C38H47N5O8 (701.34): C, 65.02; H,
6.75; N, 9.98. Found: C, 65.10; H, 6.83; N, 9.98.
7.1 Hz, CH), 4.40 (d, 2H, J ) 7.1 Hz, CH2), 7.27-7.82 (m, 8H,
Ar), 7.95 (brs, 1H, NH), 8.50 (brs,1H, NH), 11.50 (brs, 1H, NH).
13C NMR (CDCl3) δ 171.35 (s), 170.70 (s), 163.90 (s), 157.45
(s), 153.57 (s), 143.96 (s), 141.75 (s), 128.18 (d), 127.52 (d),
125.44 (d), 120.38 (d), 83.35 (s), 79.60 (s), 67.42 (t), 58.04 (t),
53.89 (t), 47.61 (d), 38.83 (t), 28.59 (q), 28.46 (q), 27.47 (t).
HRMSm/zcalcdforC31H41N508 (M+): 611.2955,found: 611.2958.
Anal. Calcd for C31H41N508 (611.29): C, 60.85; H, 6.76; N,
11.45. Found: C, 60.95; H, 6.78; N, 11.47.
H2N-Tyr-Aza-â3-Ala-Leu-Lys-Arg-Gln-Gly-Arg-Thr-Leu-
Tyr-Gly-OH (Aza-â3-A89). Aza-â3-A89 peptide was synthe-
sized on a resin loaded with the first amino acid: Fmoc-Gly-
PEG-PS (1 g, 0.2 mmol; loading 0.2 mmol/g) on the Millipore
9050 Plus PepSynthesizer. The resin was swelled with DMF
for 3 min with 1.5 mL/min flow rate. After cleavage of the Fmoc
group using 20% piperidine in DMF (1 and 5 min, 5 mL/min),
the resin was washed with DMF during 7 min (5 mL/min).
Subsequently, 4 equiv of the appropriate amino acid were
dissolved in HOBt/DMF (1.4 mL, 0.6 M) and DIC/DMF (1.4
mL, 0.6 M), and the activated amino acid was then transferred
to the reaction vessel. After 30-80 min for the analogue the
reaction vessel was washed by DMF during 4 min (5 mL/min).
The deprotection and the coupling were followed by monitoring
the dibenzofulvene-piperidine adduct at 301 nm. The peptide
backbone was elongated step by step as described in Scheme
5. Finally, the Fmoc protective group was removed by treat-
ment with 20% piperidine in DMF (1 and 5 min, 5 mL/min),
and the resin was washed with DMF (7 min, 5 mL/min),
removed from the reaction vessel, washed with DCM and
ether, and dried in vacuo. The anchored hybrid peptide thus
obtained was cleaved from the solid support by treatment with
TFA/phenol/TIS/water (88/5/2/5) for 2 h. The mixture was then
filtered, and the resin was washed thoroughly with TFA and
then with DCM. The total filtrate was concentrated in vacuo
to a volume of approximately 1 to 2 mL, and then cold ether
(10 mL) was added to precipitate the peptide. The precipitate
peptide was collected by filtration through a fritted glass
funnel and dried in vacuo. The crude product was purified by
reversed-phased preparative HPLC (XTerra RP18 19 × 300
10 µm) (5-65% B in 30 min). HPLC fraction was freeze-dried
to give the target hybrid peptide as a bis-trifluoroacetate salt
(118 mg, 36%). Analytical RP-HPLC (5-65% B in 30 min) tr
) 17.45 min, purity > 99%. HRMS (ESI) m/z calcd for
C22H24N2O6Na [M + H]+: 1440.8076, found: 1440.8074.
H2N-Tyr-Ala-Leu-Lys-Aza-â3-Arg-Gln-Gly-Arg-Thr-Leu-
Tyr-Gly-OH (aza-â3-R92). Aza-â3-R92 was synthesized on a
resin loaded with the first amino acid (Fmoc-Gly-Wang) (200
mg, 0.60 mmol) using the Advanced Chem Tech 440 Mos
synthesizer. A quantity of 200 mg of resin was swelled with
DMF (2.5 mL) during 15 min. After cleavage of the Fmoc group
by 20% piperidine solution in DMF (2 × 2.5 mL; 5 and 10 min),
the resin was washed with DMF (5 × 2.5 mL). Subsequently,
4 equiv of the appropriate amino acid were dissolved in DMF/
HOBt (960 µL, 0.5 M), and the activated amino acid was then
transferred to the reaction vessel as well as DIC in DMF (960
µL, 0.5 M). After 30-80 min for the analogue, the reaction
vessel was drained and the resin was washed by DMF (5 ×
2.5 mL). The peptide backbone was elongated step by step as
described in Scheme 5. Finally, the Fmoc protective group was
removed by the treatment with 20% piperidine in DMF (2 ×
2.5 mL; 5 and 10 min), and the resin was washed with DMF
(5 × 2.5 mL) and DCM (6 × 2.5 mL), removed from the reaction
vessel, washed with ether, and dried in vacuo. The anchored
hybrid peptide thus obtained was cleaved from the solid
support and purified as described before to afford the peptide
showing a purity > 99% (84 mg, 26%). Analytical RP-HPLC
(5-65% B in 30 min) tr ) 18.01 min, purity > 99%. HRMS
(ESI) m/z calcd for C64H106N21O17 [M + H]+: 1440.8076,
found: 1440.8053 (2 ppm).
Fmoc-Aza-â3-Arg(Boc)2-OH 3 (R ) (CH2)3NHC(NHBoc)-
NBoc). Catalytic hydrogenolysis with 10% Pd/C (50 mg) of 8
(R ) (CH2)3NHC(NHBoc)NBoc) (0.86 g, 0.5 mmol) afforded
0.72 g (96%) of 7 as a colorless oil. 1H NMR (CDCl3) δ 1.38 (s,
9H, CH3), 1.39 (s, 9H, CH3), 1.62 (m, 2H, CH2), 2.89 (m, 2H,
CH2), 3.38 (m, 2H, CH2), 3.60 (m, 2H, CH2), 4.14 (t, 1H, J )
H2N-Tyr-Ala-Leu-Lys-Arg-Gln-Gly-Arg-Thr-Leu-Tyr-
Aza-â3-Gly-OH (Aza-â3-G99). Fmoc-aza-â3-Gly(Boc)-OH was
first loaded on resin. All manipulations were carried out under
J. Org. Chem, Vol. 70, No. 26, 2005 10707

Busnel et al.
N2. Wang resin (1 g, 200-400 mesh, 0.68 mmol/g) was swelled
in DMF (10 mL) for 30 min. Fmoc-aza-â3-Gly(Boc)-OH (1.12
g, 4 equiv relative to resin loading) was dissolved in dry DCM
(10 mL) (one or two drops of DMF may be needed to aid
complete dissolution). DIC (5 equiv) was added to the solution,
which was stirred for 20 min at 0 °C under N2. The DCM was
removed by evaporation, and the residue was dissolved in the
minimum of DMF (3 mL) and added to the resin with 0.1 equiv
of DMAP (8 mg). The mixture was mixed for 2 h by N2
bubbling. A resin sample was removed to determine the level
of Fmoc-aza-â3-Gly(Boc)-OH attachment by Fmoc cleavage.
The dry Fmoc-aza-â3-Gly(Boc) resin was weighed (3-5 mg) into
each of two UV cells, 20% of piperidine in DMF (3 mL) was
added, and the solution was agitated for 3 min. The absorbance
of the UV absorption of the dibenzofulvene-piperidine adduct
gave the estimate loading of the Fmoc-aza-â3-Gly-OH mono-
mer, which is 0.61 mmol/g. Aza-â3-G99 was then synthesized
in a procedure analogous to that of aza-â3-R92 (80 mg, 24%).
Analytical RP-HPLC (5-65% B in 30 min) tr ) 18.08 min,
purity > 99%. HRMS (ESI) m/z calcd for C64H106N21O17 [M +
H]+: 1440.8076, found: 1440.8057 (2 ppm).
Acknowledgment. This research was supported by
postdoctoral financing from the Ministe`re de la Recher-
che (No. 2001-1520, Ref SAR No. 28).
Note Added after ASAP Publication. There were
errors in Scheme 3, the footnotes of Schemes 3 and 4,
the discussion of compounds 6 and 7 below Scheme 3,
and the compound numbering for 8 in the Experimental
Section in the version published ASAP November 8, 2005;
the corrected version was published ASAP November 17,
2005.
Supporting Information Available: General consider-
ations, procedure used for “mixed” peptide synthesis, and
analytical data for aza-â3-L90, aza-â3-K91, aza-â3-G94, aza-
â3-R95, aza-â3-L97, aza-â3-Y98, aza-â3-A89-L90, aza-â3-A89-
1
L90-K91, and aza-â3-L97-Y98. H NMR spectra of N-Boc-N-
methylhydrazine, 1-Boc-amino-3,3-diethoxypropane, and 3-Boc-
aminopropanal and 1H, 13C NMR of Fmoc-protected ornithine
hydrazone (R ) (CH2)3NHBoc), and 13C NMR spectra of Aza-
â3Leu97. This material is available free of charge via the
Internet at http://pubs.acs.org.
JO051585O
10708 J. Org. Chem., Vol. 70, No. 26, 2005