'Bis-ornithine' (2,2-bis(aminopropyl)glycine): a new tetravalent template for assembling different functional peptides

Article abstract of DOI:10.1016/j.tetlet.2006.03.136

Synthesis of bis-ornithine, a new C^α,α-disubstituted α-amino acid bearing orthogonally protected α and δ amine groups is reported. Bis-ornithine (bisOrn) and dipeptides containing bis-ornithine have been synthesized in solution up to multigram

Full text of DOI:10.1016/j.tetlet.2006.03.136

Tetrahedron Letters 47 (2006) 3723–3726
‘Bis-ornithine’ (2,2-bis(aminopropyl)glycine): a new
tetravalent template for assembling different functional peptides
*
´
Baptiste Aussedat, Gerard Chassaing, Solange Lavielle and Fabienne Burlina
Universite´ Pierre et Marie Curie-Paris 6, Case 45, 4, Place Jussieu, 75252 Paris Cedex 05, France
`
´
UMR CNRS 7613, Synthese, Structure et Fonction de Molecules Bioactives, FR 2769, France
Received 3 February 2006; accepted 21 March 2006
Available online 12 April 2006
Abstract—Synthesis of bis-ornithine, a new Ca,a-disubstituted a-amino acid bearing orthogonally protected a and d amine groups is
reported. Bis-ornithine (bisOrn) and dipeptides containing bis-ornithine have been synthesized in solution up to multigram scale. As
a first example, one of these compounds Boc-bAla-bisOrn(Alloc)2-OH has been attached to a solid support and used as template for
the synthesis of a symmetrical assemblage of peptides.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
the parent protein is not a prerequisite and (ii) some do-
mains are not pre-folded prior to their association with
their ligand. An assemblage of peptides (AP) into a min-
imal structure that displays—like proteins—multiple
functions represents a challenging target for chemists.
Proteins are composed of sub-domains that may be in-
volved in distinct functions such as catalytic transforma-
tion, cell regulation and/or intracellular trafficking.
Some may also serve as three-dimensional scaffold to
support other domains. The identification of the do-
mains and their associated functions has been mainly
based on molecular dissections and mutagenesis experi-
ments. The activities detected with some protein frag-
ments have demonstrated that (i) the full sequence of
The initial step of our approach to build peptide assem-
blages rests on the design of a small highly functional-
ized template where the spatial distribution does not
prevent the freedom of each domain. The tetravalent
template ‘bis-ornithine’ or 2,2-bis(aminopropyl)-glycine
was selected on the basis of the conformational proper-
ties of 2,2-bis(n-propyl)glycine,1 where the alkyl side
chains and the backbone adopt a fully extended confor-
mation. The introduction of two orthogonal protections
on the a and d amino groups of bis-ornithine leads to
the construction of APs, with a C2 symmetry (Scheme
1). Indeed, domains 3 and 4, which are linked to the d
amino groups will contain the same peptidic sequence
Keywords: Peptide template; a,a-Disubstituted a-amino acid.
Abbreviations: Ac, acetyl; Alloc, allyloxycarbonyl; bisOrn, bis-orni-
thine; Boc, tert-butyloxycarbonyl; Cbz, benzyloxycarbonyl; 2-BrCbz,
2-bromobenzyloxycarbonyl; 2-ClCbz, 2-chloro benzyloxycarbonyl;
DIEA, diisopropylethylamine; DMAP, N,N-dimethylaminopyridine;
DMF, N,N-dimethylformamide; Fmoc, fluorenylmethyloxycarbonyl;
HBTU, N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-meth-
ylmethanaminium hexafluorophosphate-N-oxide; HOAt, 1-hydroxy-7-
azabenzotriazole; HPLC, high performance liquid chromatography;
IBCF, isobutylchloroformate; MALDI-TOF, matrix-assisted laser
desorption/ionization time-of-flight; MBHA-PS, 4-methylbenzhydryl-
amine polystyrene; NMM, N-methylmorpholine; NMR, nuclear
magnetic resonance; PG, protecting group; PyAOP, 7-aza-
O
PG1
Domain 2
AcKLTKPKPQAESKKKGG
NH
O
NH
H
N
YAc
PG2
NH
O
NH
Domain 3 = Domain 4
H
N
CO2H
benzotriazol-1-yloxytris(pyrrolidino)phosphonium
hexafluorophos-
CONH2
O
phate; PyBOP, benzotriazol-1-yloxytris(pyrrolidino)phosphonium
hexafluorophosphate; Tfa, trifluoroacetamide; TFFH, tetramethylflu-
oroformamidinium hexafluorophosphate; THF, tetrahydrofuran;
TLC, thin layer chromatography.
Domain 1
NH
O
NH
PG1
AcKLTKPKPQAESKKKGG
Bis-ornithine (bisOrn)
Target assemblage of peptides (AP)
*
Corresponding author. Tel.: +33 1 44 27 32 61; fax: +33 1 44 27 38
43; e-mail: chassain@ccr.jussieu.fr
Scheme 1.
0040-4039/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.03.136

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B. Aussedat et al. / Tetrahedron Letters 47 (2006) 3723–3726
and a total of three different sequences can be incorpo-
rated in the AP. This template will find interesting appli-
cations, for example, for ligand dimerization, with
domains 1 and 2 remaining available for the introduc-
tion of a reporter group (fluorophore or radioisotope)
and/or an affinity tag (biotin or poly-histidine).
gonally protected bis-ornithine derivative A, or into
dipeptides B and C containing bis(cyanoethyl)glycine
or bis-ornithine, respectively.
2. Synthesis of 2,2-bis(aminopropyl)glycine,
bis-ornithine A
In the present work, we report an efficient synthesis of
the achiral quaternary amino acid, bis-ornithine, which
can be achieved on a multigram scale. The preparation
in solution of various dipeptides containing bis-orni-
thine or precursors with protecting groups that are
compatible with solid-phase peptide synthesis is also
presented. As a proof of concept, one of these dipeptides
has been used in the elaboration of an AP by stepwise
solid-phase synthesis. The target AP contains a peptidic
ligand in domains 3 and 4, a tyrosine for radioactive
labeling in domain 2 and a b-alanine in domain 1
(Scheme 1).
The Boc protecting group was introduced on compound
2 by reacting (Boc)2O in THF (Scheme 3).7
The methyl ester of 3 was saponified and the nitrile
groups cleanly reduced in a one-pot procedure.8 Finally,
the d-amino groups were protected by a Fmoc or 2-chlo-
robenzyloxycarbonyl (2-ClCbz) group, leading to the
protected bis-ornithine derivatives 4 and 5, respectively.
3. Synthesis of Na-protected-bis(cyanoethyl)glycine and
dipeptidic derivative B
The choice of the strategy for the synthesis of bis-orni-
thine was dictated by the reactivity of the different steri-
cally hindered intermediates. The two-step Bucherer–
Bergs route2 leading to the bulky hydantoin was there-
fore a priori discarded. As Fu et al. have reported the
reduction of various Ca,a-disubstituted a-nitro esters,3
reduction of tert-butyl 2,2-bis(cyanoethyl)-2-nitroace-
tate to give bis-ornithine was first attempted. But, both
nitrile groups were efficiently reduced, whereas the nitro
group was nearly unaffected, whatever the conditions
used. We next planed to use a masked amino group
and start the synthesis with an aldimine glycine ester,
as described by O’Donnell for alkylations or Michael
condensations under solid–liquid phase transfer
catalysis.4
The methyl bis(cyanoethyl)glycinate 2 was submitted to
ester hydrolysis by LiOH (Scheme 4). In the same step,
the amine was protected by benzyl- or fluorenylchloro-
formate to give products 6 (Na-Cbz) or 7 (Na-Fmoc)9
or by (Boc)2O in the presence of hydroxylamine10 to give
compound 8 (Na-Boc). Noteworthy, compounds 7 and 8
may be directly used as building blocks in solid-phase
NC
CN
NC
CN
(Boc)2O / THF
1) Ni Raney / H2
EtOH / THF
NaOH
H2N CO2Me
BocHN CO2Me
3
2) Protection step
2
PG
PG
HN
NH
The synthesis of the starting compound 1 (Scheme 2)
was easily achieved by a double Michael addition of
acrylonitrile on methyl N-parachlorobenzylidene glycin-
ate.5 The methyl bis-(cyanoethyl)glycinate 2 was readily
obtained on 0.5 mol scale after aldimine hydrolysis.6
Then, this precursor was transformed into the ortho-
PG = Fmoc (4), 2Cl-Cbz (5).
BocHN CO2H
4 / 5
Scheme 3.
PG
PG2
2 NH HN
PG1 orthogonal to PG2
PG
1 N CO2H
H
(A)
NC
CN
PG
PG2
2 NH HN
NC
CN
N
CO2Me
H2N CO2Me
PG1AACO N CO2H
2
1
H
Cl
(C)
NC
PG
CN
H
N
CO2Me
1 N
H
O
(B)
Scheme 2.

B. Aussedat et al. / Tetrahedron Letters 47 (2006) 3723–3726
3725
tion of the dipeptides containing bis-ornithine was
found here to be quite efficient. However complete cou-
pling reactions are required for solid-phase synthesis.
For the synthesis of the APs on solid support, we thus
decided to directly incorporate a dipeptidic unit (Boc-
AA-bisOrn(PG)2-OH) into the elongating peptide to
avoid aborted sequences. First, the coupling conditions
were optimized by synthesizing on solid-phase model
polymers containing a central dipeptidic unit 10a–12b.
We compared the activation of the dipeptides by
HBTU, TFFH, PyBOP, HATU and PyAOP reagents.
In each case, the subsequent activated intermediate
was reacted for 12 h at 50 ꢁC with the elongating poly-
mer and double or triple couplings were performed.
The best result was obtained using an equimolar mixture
of PyAOP and HOAt for activation. In this case no
aborted sequence was detected after cleavage from the
solid support.
NC
CN
NC
CN
LiOH / CH3CN / H2O
Cbz-Cl
HBTU
HCl.GlyOMe
DIEA / DMF
H2N CO2Me
CbzHN CO2H
6
2
NC
CbzHN
CN
H
N
CO2Me
O
9
Scheme 4.
peptide synthesis (Fmoc or Boc strategy) with the nitrile
functions as precursors of amino groups.
The amino and carboxylic groups of Ca,a-disubstituted
a-amino acids are known to be poorly reactive for
amino acids coupling.11 To check the reactivity of the
carboxylic group of bis(cyanoethyl)glycine derivatives,
compound 6 was activated by HBTU/DIEA and cou-
pled in solution to glycine methyl ester (Scheme 4). This
procedure led to dipeptide 9 in good yields (80%).12
The synthesis of the target assemblage of peptides was
started by manual coupling of Boc-b-alanine on a
MBHA-PS resin (domain 1, Scheme 6). Dipeptide Boc-
bAla-bisOrn(Alloc)2-OH 10a activated by PyAOP/
HOAt was then coupled14 for 12 h at 50 ꢁC (double cou-
pling). After Boc elimination, tyrosine was classically
incorporated
(Boc-Tyr(2-BrCbz)-OH/HBTU/DIEA)
4. Synthesis of dipeptides containing bis-ornithine C
followed by acetylation to give domain 2. The Alloc pro-
tecting groups of bis-ornithine side chains were
removed.15 Two glycines serving as spacer and the C-
terminal lysine of the selected peptidic sequence consti-
tuting domains 3 and 4 (AcKLTKPKPQAESKKK)
were incorporated manually to allow a monitoring of
the reactions (Kaiser test). Efficient couplings for these
residues were obtained using a standard protocol
(HBTU/DIEA amino-acid activation). Finally, the rest
of the peptide sequence was assembled by stepwise
solid-phase synthesis on an ABI 433A peptide synthesizer.
In another series of experiments, we checked the reactiv-
ity of the amine function of bis(cyanoethyl)glycine and
synthesized in solution various dipeptides. Compound
2 was reacted with the mixed anhydride of Boc- or
Cbz-protected amino acids obtained by treatment at
0 ꢁC with isobutyl chloroformate (Scheme 5). Three
dipeptides containing either b-alanine (10), glycine (11)
or alanine (12) were prepared in good yields using this
procedure (60–65%).13 The nitrile functions were then
reduced by H2/Raney-Ni under basic conditions to give
the products with the free amine and carboxylic groups.8
The d-amino groups were subsequently protected either
by Alloc, 2-ClCbz, Fmoc, Tfa or Boc group, suitable for
solid-phase peptide synthesis.
introduction of
domains 3 and 4.
Alloc
Alloc
NH
O
HN
5. Synthesis of an assemblage of peptides AP by
solid-phase strategy
H
N
H
N
introduction of
domain 2.
Boc
N
N
MBHA
H
H
O
O
As mentioned before, acylation onto the N-terminus of
C
a,a-disubstituted a-amino acids during solid-phase
compound 10a Domain 1
peptide synthesis is known to be very difficult, because
of the sterical hindrance of carbon a.11 Synthesis in solu-
Scheme 6.
NC
CN
NC
CN
CO Me
IBCF / BocAAOH
NMM / CH2Cl2
1) Ni Raney / H2
EtOH / THF
NaOH
H2N CO2Me
BocAACO-NH
2
2) Protection step
2
10 / 11 / 12
2-ClCbz
10b
Alloc
Fmoc Tfa Boc
PG
NH
PG
Boc-bAla
-
10a
-
10c 10d
HN
Boc-Gly
11a
11b 11c 11d
-
-
-
Boc-Ala 12a
12b
BocAACO-NH CO2H
Scheme 5.

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B. Aussedat et al. / Tetrahedron Letters 47 (2006) 3723–3726
13.9 Hz, 2H); 1.91–1.83 (ddd, J = 6.3, 8.8, 14.7 Hz, 2H).
13C NMR (100 MHz, MeOD): d 173.9, 119.1, 59.5, 53.1,
35.5, 12.2. Anal. Calcd for C9H13N3O2: C, 55.37; H, 6.71;
N, 21.52. Found: C, 55.35; H, 6.85; N, 21.5.
The designed AP (Scheme 1) was cleaved from the resin
by treatment with anhydrous HF, purified by HPLC and
characterized by MALDI-TOF mass spectrometry.
7. To a solution of 2 (3 g, 15.3 mmol) in THF (20 mL) was
added (Boc)2O (3.7 g, 17 mmol), the mixture was stirred
for 8 days at room temperature. Then, water (5 mL) and a
catalytic amount of DMAP were introduced, the solution
was stirred for 20 min. THF was evaporated, CH2Cl2
(50 mL) was added. The organic layer was washed with a
saturated solution of NH4Cl in water (30 mL), dried over
MgSO4 and evaporated. The compound 3 was purified
on silica gel (CH2Cl2/MeOH) (80% yield). 1H NMR
(400 MHz, MeOD): d 3.79 (s, 3H); 2.46–2.33 (m, 6H);
2.27–2.19 (m, 2H); 1.44 (s, 9H). 13C NMR (100 MHz,
MeOD): d 173.4, 156.2, 120.4, 81.2, 62.2, 53.5, 31.5, 28.6,
12.5.
8. In a typical procedure, the dinitrile-ester 3, 10, 11, or 12
(8 mmol) was dissolved in a mixture of absolute ethanol
(146 mL) and THF (34 mL). Raney-Nickel (4 g, 34 mmol)
as a 50% slurry in water was added together with 2 N
NaOH (18 mL). The mixture was stirred under 50 psi
hydrogen pressure at room temperature for 8 h. The
catalyst was filtered off, pH was increased to 7 (with 1 N
HCl) and the solvents were removed in vacuo. The crude
product was used without further purification.
In conclusion, we have developed a straightforward
strategy to prepare the acyclic a-quaternary a-amino
acid bis-ornithine. We have demonstrated that dipep-
tides containing bis(cyanoethyl)glycine can be synthe-
sized in solution by coupling an amino acid on its
a-carboxylic or amino group. From these compounds,
bis-ornithine containing dipeptides can be obtained as
crystalline products in good yields up to the multigram
scale. The incorporation of these dipeptides is achiev-
able by solid-phase strategy, leading to new tetravalent
templates for the synthesis of peptide assemblages.
Various applications in peptide/protein interactions are
now under investigation. In addition, linear polymers
containing bis-ornithine derivatives have already been
employed for drug delivery in culture cells.14
Acknowledgements
`
This work was supported by the Ministere de l’Enseign-
9. To a solution of 2 (1.95 g, 10 mmol) in water/CH3CN (1:1,
50 mL) was added LiOH (420 mg, 10 mmol). After 25 min
no remaining ester was detected on TLC. Alkyl chloro-
formate (30 mmol) was added and pH kept at 9 (with
LiOH) during 3 h. Then pH was decreased to 3 (with HCl
1 N) and the organic layer was extracted with ether
(30 mL). Compounds 7 and 8 were purified on silica gel
(CH2Cl2/MeOH/AcOH) (60–65% yield over two steps).
10. Harris, R. B.; Wilson, I. B. Tetrahedron Lett. 1983, 24, 231.
11. Humphrey, J. M.; Chamberlain, A. R. Chem. Rev. 1997,
97, 2243.
12. To a solution of 6 (1.26 g, 4 mmol) in DMF (10 mL) were
added HBTU (1.52 g, 4 mmol) and DIEA (1.72 mL,
10 mmol), the mixture was stirred for 30 min at room
temperature. Then glycine methyl ester chlorhydrate
(625 mg, 5 mmol) was added in DMF (10 mL) and the
solution was stirred overnight. A saturated solution of
NH4Cl in water was added and the aqueous layer was
extracted with ether (6·). The organic layer was dried over
MgSO4 and evaporated. Compound 9 was purified on
silica gel (AcOEt/cyclohexane) (80% yield).
13. Boc-amino acid or Cbz-amino acid (61.5 mmol) and
NMM (7.8 mL, 71 mmol) in CH2Cl2 (250 mL) was cooled
to 0 ꢁC and stirred during the dropwise addition of
isobutyl chloroformate (9.25 mL, 71 mmol) in CH2Cl2
(100 mL) over 10 min. The solution was allowed to warm
up to room temperature, then 2 (8 g, 41 mmol) in CH2Cl2
(50 mL) was added. After 4 h, the solution was washed
with a saturated solution of NH4Cl in water, dried over
MgSO4 and evaporated. Compounds 10, 11 and 12 were
purified on silica gel (AcOEt/cyclohexane). Compound 11:
(65% yield) 1H NMR (400 MHz, CDCl3): d 7.36 (br s,
1H); 5.33 (br s, 1H); 3.91 (s, 3H); 3.75–3.74 (d, J = 5.8 Hz,
2H); 2.99–2.92 (dt, J = 6.6 Hz, 13.6 Hz, 2H); 2.36–2.24
(m, 4H); 2.2–2.13 (dt, J = 7.3, 14.2 Hz, 2H); 1.47 (s, 9H).
13C NMR (100 MHz, CDCl3): d 171.8, 169.6, 156.2, 118.4,
80.8, 62.5, 53.9, 45.1, 30.2, 28.2, 12.1. Anal. Calcd for
C16H24N4O5: C, 54.54; H, 6.86; N, 15.9. Found: C, 54.45;
H, 6.83; N, 15.83.
´
ement superieur et de la Recherche and CNRS.
References and notes
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(5.2 g, 38 mmol) was first added, then acrylonitrile (50 mL,
380 mmol) was added dropwise at 0 ꢁC. The solution was
stirred overnight at room temperature. MeOH was evap-
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washed with brine (3 · 50 mL), dried over MgSO4, filtered
and the solvent removed in vacuo. The resulting imine was
stirred with 1 N HCl (190 mL) in THF (190 mL) at 0 ꢁC
for 1 h. The THF was removed, the aqueous layer was
washed with ether (2 · 50 mL) the pH was increased to 9
with Na2CO3 (saturated solution in water) and extracted
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´
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