New building blocks for peptide analogue synthesis: Reduced diaza-β3-dipeptides

Article abstract of DOI:10.1055/s-2005-917100

Reduction of N-protected aza-β³-amino esters and oxidation of the alcohols afford aza-β³-amino aldehydes. Condensation of unprotected aza-β³-amino esters to the resultant aldehydes leads to hydrazones, which have been conv

Full text of DOI:10.1055/s-2005-917100

LETTER
2591
New Building Blocks for Peptide Analogue Synthesis: Reduced
Diaza-b3-Dipeptides
Reduced
D
iaza-
b
-
3l
D
ipept
i
ides vier Busnel, Michèle Baudy-Floc’h*
Groupe ‘Ciblage et Auto-Assemblages Fonctionnels’, UMR 6510 CNRS, Institut de Chimie, Université de Rennes I, 263 Av. du Général
Leclerc, 35042 Rennes Cedex, France
Fax +33(2)3236738; E-mail: michele.baudy-Floch@univ-rennes1.fr
Received 4 August 2005
Several attempts were done to overcome these problems
Abstract: Reduction of N-protected aza-b3-amino esters and oxida-
such as introduction of chirality to PNA by linking chiral
tion of the alcohols afford aza-b3-amino aldehydes. Condensation
and functionalized amino acids,5 peptides6 and oligo-
nucleotides7 to the PNA or by using chiral amino acids in
of unprotected aza-b3-amino esters to the resultant aldehydes leads
to hydrazones, which have been converted by reduction to useful
synthetic building blocks for solid-phase synthesis of new peptido- the backbone itself.8 To our knowledge, analogues of
mimetics.
PNAs have not been described yet except constrained
PNAs.9
Key words: aza-b3 amino aldehydes, pseudopeptides, reductive
amination, reduced aza-b3-peptides, PNA aza analogues
The aim of the work described here was to synthesize new
building blocks that we could call N-hydrazinoethyl
hydrazinoglycine units or reduced diaza-b3-dipeptide.
These new analogs could be integrated in a peptide in con-
struction or coupled on themselves to give aza analogues
of PNA (Figure 2). This nitrogen-enriched backbone
could permit to introduce various side chains allowing cell
membrane penetration.
Unnatural oligomers are synthetic compounds designed to
mimic peptides and others biopolymers and increased
their bioavailability.1 Within the past decade, these com-
pounds have emerged as important research targets in
drug discovery as well as conformational analysis. Re-
search on solid-phase syntheses of oligomers with a
defined sequence is proliferating as a consequence. Well-
designed oligomers can be formed on a support by repeat-
ing the same types of coupling reactions. They can have
chemically diverse side chains, but they need to have un-
proteolytically labile amide bonds. One class of non-hy-
drolysable peptides are reduced peptides.2 Peptide nucleic
acid (PNA), which is a DNA analogue (the phosphodi-
ester backbone has been replaced by a pseudopeptide
backbone), is composed of a backbone built up from N-
aminoethyl glycine units or reduced dipeptide backbone
(Figure 1), that makes them very stable in biological
fluids.3
R1
N
R2
N
O
O
N
N
R3
N
H
H
n
Figure 2 Aza analogues of PNA or reduced aza-b3-peptides
In a previous paper10 we have shown that Nb-Boc-protect-
ed aza-b3-amino esters could be reduced into the corre-
sponding alcohols by NaBH4/LiCl in THF–EtOH which
then have been oxidized by the SO3–pyridine complex in
a CH2Cl2–DMSO mixture to give Nb-Boc-aza-b3-amino
aldehydes (Scheme 1). With the aim to enable the synthe-
sis of hybrids peptides as well as analogues of PNA using
solid-phase synthesis, we undertook to prepare Nb-Fmoc-
aza-b3-amino aldehydes.
B
O
O
O
N
N
N
H
H
n
R
R
N
O
B = nucleobase
NaBH4/LiCl
PG
N
PG
N
OH
N
OMe
THF/EtOH
70–90%
Figure 1 Structure of PNA
H
H
1
2
PG-aza-β3aa-OMe
In spite of its resistance to cellular enzymes such as nu-
cleases and proteases, the major limitations confounding
its application are poor solubility in aqueous media, in-
efficient cellular uptake, due to the uncharged backbone
and ambiguity in orientational selectivity of binding.4
R
N
O
SO3/pyridine
CH2Cl2/DMSO
40–50%
PG
N
H
H
3
Scheme 1 Synthesis of aza-b3-amino alcohols 2 and aldehydes 3
SYNLETT 2005, No. 17, pp 2591–2594
1
7
.1
0
.2
0
0
5
Advanced online publication: 05.10.2005
DOI: 10.1055/s-2005-917100; Art ID: G25005ST
© Georg Thieme Verlag Stuttgart · New York

2592
O. Busnel, M. Baudy-Floc’h
LETTER
R1
N
In our initial investigations, we applied the conditions em-
ployed in Scheme 1, so the synthesis of Nb-Fmoc-aza-b3-
amino alcohols 2 by reduction of the corresponding
Fmoc-aza-b3-amino ester 1 using NaBH4/LiCl proceeds
with 90% yield, but no reaction occurred during the
oxidation of Nb-Fmoc-aza-b3-amino alcohol 2 with the
SO3–pyridine complex, the starting alcohol was recov-
ered. We examined alternative methods to generate the al-
dehyde, Swern oxidation was the best way to get the Nb-
Fmoc-aza-b3-amino aldehydes 3.11 Fmoc-aza-b3-amino
alcohol 2 was added to oxalyl chloride in DMSO–CH2Cl2
under nitrogen atmosphere at –78 °C for 15 minutes, ad-
dition of Et3N and stirring at room temperature for 45
R1
N
(COCl)2
O
Fmoc
Fmoc
N
H
OH
CH2Cl2/DMSO
Et3N
N
H
H
2
3
R2
R2
N
O
O
CF3CO2H
Boc
N
NaHCO3
H2N
N
H
OPG
OPG
1
4
R1
N
R2
N
O
Fmoc
N
H
N
OPG
5
minutes afforded Fmoc-aza-b3-amino aldehyde
3
Fmoc-aza-β3aaΨ(CH=N)-aza-β3aa-OPG
(PG = Fmoc). While 3 could be isolated pure by chroma-
tography in 40% yield, due to its instability, it was used
directly in the next step following the removal of solvent.
NaBH3CN
2 N HCl
R1
N
R2
N
With the aldehydes in hand, we next turned our attention
to their reductive amination to get Fmoc-aza-b3-aa-
y(CH2NH)-aza-b3-aa-OPG 6. Crude Fmoc-aza-b3-amino
aldehyde 3 was mixed with H-aza-b3-amino ester 4 in
CH2Cl2 to give, after 12 hours, hydrazone 5 in an overall
yield from the alcohol of 50%.12 Reduction of the hydra-
zone 5 with NaBH3CN and 2 N HCl led to the expected
Fmoc-aza-b3-aa-y(CH2NH)-aza-b3-aa-OPG 6 (Scheme 2
and Table 1).13
O
Fmoc
N
N
OPG
H
H
6
Fmoc-aza-β3aaΨ(CH=N)-aza-β3aa-OPG
R3X
DIEA
R1
N
R2
N
O
Fmoc
N
N
R3
OPG
The R1, R2 groups, mimicking the peptidic chain, the
nucleobase or favorizing the solubilization in biological
medium, the cell membrane crossing, could be introduced
on the starting compound following our previous
method.14 The R3 group could be incorporated by nucleo-
philic substitution considering the good nucleophilicity of
the unsubstituted nitrogen atom of reduced analogue 6.
Moreover, in a previous work we have shown that side
reaction occurred during the coupling of the analogue
Fmoc-aza-b3-Gly-OH, due to the formation of polymeric
products during activation of Na-unprotected hydrazino
acids,15 so it was necessary to protect the monomer in Na-
position. Coupling the protected monomer Fmoc-aza-b3-
Gly(Boc)-OH has led to the expected peptide. To avoid
side reactions during the coupling of our new building
blocks and to control the possibility of substitution on the
CH2NH of the reduced dimer 6, we realized the acylation
of 6 with (Boc)2O in the presence of DIEA. Fmoc-aza-b3-
aa-y(CH2NBoc)-aza-b3-aa-OBn 7 was obtained in 41–
66% yield.16 Finally, deprotection of the carboxylic group
by catalytic hydrogenation affords the free acid 817 which
could be then coupled on solid-phase synthesis to afford
hybrid peptides or PNA analogues.
H
7
Fmoc-aza-β3aaΨ(CH2NR3)-aza-β3aa-OPG
PG = Bn
H2/Pd
R1
N
R2
N
O
Fmoc
N
N
R3
OH
H
8
Fmoc-aza-β3aaΨ(CH2NR3)-aza-β3aa-OH
Scheme 2 Synthesis of reduced diaza-b3-dipeptides
non-standard nucleobases are under progress. These new
dimers will facilitate investigation for the preparation of
new oligomers or mixed peptides on solid-phase support.
The solid-phase synthesis of oligomers and hybrid pep-
tides are under investigations.
References
(1) (a) Liskamp, R. M. J. Angew. Chem., Int. Ed. Engl. 1994, 33,
633. (b) Zuckermann, R. N.; Kerr, J. M.; Kent, S. B. J.;
Moos, W. H. J. Am. Chem. Soc. 1992, 114, 10646.
(c) Seebach, D.; Abele, S.; Gademann, K.; Guichard, G.;
Hintermann, T.; Jaun, B.; Matthews, J. L.; Schreiber, J. V.;
Hommel, U.; Widmer, H. Helv. Chim. Acta 1998, 81, 932.
(d) Appella, D. H.; Christianson, L. A.; Karle, I. L.; Powell,
D. R.; Gellman, S. H. J. Am. Chem. Soc. 1999, 121, 6206.
(e) Burgess, K.; Linthicum, K. S.; Shin, H. Angew. Chem.,
Int. Ed. Engl. 1995, 34, 907.
In conclusion, we have shown that new building blocks
corresponding to reduced diaza-b3-dipeptides, Fmoc-aza-
b3-aa-y(CH2NH)-aza-b3-aa-OH, can be conveniently
prepared by reductive amination of the corresponding
Fmoc-aza-b3-amino aldehydes. Moreover, substitution of
the nitrogen atom (CH2NH) was possible, this result
offers the opportunity to introduce various side chains to
control either the solubility or the cell membrane per-
meation. The syntheses of analogues bearing natural or
Synlett 2005, No. 17, 2591–2594 © Thieme Stuttgart · New York

LETTER
Reduced Diaza-b3-Dipeptides
2593
Table 1 Yields of Reduced Diaza-b3-dipeptides 6–8
Reduced diaza-b3-dipeptides
R1
R2
R3
H
Yield (%)
6a
6b
Fmoc-aza-b3-Leu-y(CH2NH)-aza-b3-Ala-OBn
CH2CH(CH3)2
CH2CH(CH3)2
CH3
96
72
Fmoc-aza-b3-Leu-y(CH2NH)-aza-b3-
CH2C6H4OCH2OEt
H
Tyr(OCH2OEt)-OBn
7a
7b
Fmoc-aza-b3-Leu-y(CH2NBoc)-aza-b3-Ala-OBn
CH2CH(CH3)2
CH2CH(CH3)2
CH3
Boc
Boc
66
41
Fmoc-aza-b3-Leu-y(CH2NBoc)-aza-b3-
Tyr(OCH2OEt)-OBn
CH2C6H4OCH2OEt
8a
8b
Fmoc-aza-b3-Leu-y(CH2NBoc)-aza-b3-Ala-OH
CH2CH(CH3)2
CH2CH(CH3)2
CH3
Boc
Boc
72
75
Fmoc-aza-b3-Leu-y(CH2NBoc)-aza-b3-
Tyr(OCH2OEt)-OH
CH2C6H4OCH2OEt
(2) (a) Martinez, J.; Bali, J. P.; Rodriguez, M.; Castro, B.;
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1874. (b) Quesnel, A.; Zerbib, A.; Connan, F.; Guillet, J. G.;
Briand, J. P.; Choppin, J. J. Peptide Sci. 2001, 7, 157.
(c) Guichard, G.; Calbo, S.; Muller, S.; Kourilsky, P.;
Briand, J.-P.; Abastado, J.-P. J. Biol. Chem. 1995, 270,
26057. (d) Benkirane, N.; Guichard, G.; Briand, J.-P.;
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(3) (a) Nielsen, P. E.; Haaima, G. Chem. Soc. Rev. 1997, 73.
(b) Planas, M.; Bardaji, E.; Jensen, K. J.; Barany, G. J. Org.
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(4) (a) Wittung, P.; Kajanus, J.; Edwards, K.; Nielsen, P.;
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Am. Chem. Soc. 1993, 115, 6477. (b) Bentin, T.; Nielsen, P.
E. J. Am. Chem. Soc. 2003, 125, 6378.
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Overhand, M.; van der Marel, G. A.; van Boom, J. H. Synlett
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(8) Haaima, G.; Lohse, O.; Buchardt, O.; Nielsen, P. E. Angew.
Chem., Int. Ed. Engl. 1996, 35, 1939.
(9) D’Costa, M.; Kumar, V. A.; Ganesh, K. N. Org. Lett. 1999,
1, 1513.
(10) Cheguillaume, A.; Doubli-Bounoua, I.; Baudy-Floc’h, M.;
Le Grel, P. Synlett 2000, 331.
(11) Mancuso, A. J.; Swern, D. Synthesis 1981, 165.
(12) To a solution of CH2Cl2 (20 mL, freshly distilled on CaH2),
under nitrogen atmosphere, was added oxalyl chloride
freshly distilled (1.35 mL, 1.2 equiv). The solution was
cooled at –78 °C, and DMSO (2.23 mL, 2.4 equiv, freshly
distilled on KOH) was carefully added. The solution was
stirred for 15 min and Fmoc-aza-b3-Leu alcohol (2, 4.63 g, 1
equiv) in CH2Cl2 (20 mL) was added dropwise, the mixture
was stirred for 15 min at –78 °C. After adding Et3N (5.52
mL, 3 equiv, freshly distilled on CaH2) the mixture was
given up to r.t. during 45 min. Then, CH2Cl2 was added (50
mL) with a solution of NaHCO3 (1 M, 20 mL). The organic
layer was washed with brine, dried over Na2SO4 and
concentrated to give a crude oil suitable for the next step.
Purification by chromatography on silica gel (EtOAc–PE
3:7) gave 40% yield of 3a (1.84 g). 1H NMR (CDCl3): d =
0.95 (br, 6 H, CH3), 1.69 (m, 1 H, CH), 2.67 (br, 2 H, CH2),
3.72 (br, 2 H, CH2), 4.23 (br t, 2 H, J = 6.3 Hz, CH), 4.53 (br
d, 2 H, J = 6.3 Hz, CH2), 6.39 (br, 1 H, NH), 7.31–7.83 (m,
8 H, CHar), 9.12 (s, 1 H, CHO) ppm. The crude oil was
diluted in CH2Cl2 (50 mL) and aza-b3-Tyr-OBn (0.9 equiv)
was added, the mixture was stirred overnight at r.t. on
Na2SO4. After filtration, the solution was concentrated and
purified by chromatography on silica gel (EtOAc–PE 3:7) to
give 2.64 g (55%) of corresponding hydrazone (5b) as a
colorless oil. 1H NMR (CDCl3): d = 0.93 (d, 6 H, J = 6.6 Hz,
CH3), 1.23 (t, 3 H, J = 7.0 Hz, CH3), 1.72 (m, 1 H, CH), 2.47
(br, 2 H, CH2), 3.50 (s, 2 H, CH2), 3.73 (q, 2 H, J = 7.0 Hz,
CH2), 3.97 (s, 2 H, CH2), 4.24 (t, 1 H, J = 6.9 Hz, CH), 4.36
(s, 2 H, CH2), 4.42 (d, 2 H, J = 6.9 Hz, CH2), 5.11 (s, 2 H,
CH2), 5.18 (s, 2 H, CH2), 5.82 (br s, 1 H, NH), 6.66 (br, 1 H,
CH), 6.93–7.80 (m, 17 H, CHar) ppm. 13C NMR (CDCl3):
d = 170.08, 156.85, 155.81, 144.07, 141.38, 135.63, 131.66,
129.15, 128.61, 128.37, 127.73, 116.45, 127.73, 127.11,
125.28, 120.00, 93.14, 66.49, 65.20, 64.22, 60.44, 56.54,
54.61, 47.37, 26.37, 20.84, 15.21 ppm. HRMS (ESI): m/z
calcd for C40H46N4O6Na [M + Na]+: 701.33151; found:
701.3331 (2 ppm).
(13) The hydrazone 5b (2.64 g, 3.9 mmol) was dissolved in
MeOH (20 mL). Sodium cyanoborohydride (0.62 g, 2.5
equiv) was added and pH was brought to 3 by slowly adding
a solution of 2 N HCl. The mixture was stirred for 2 h, then
the pH was adjusted to 1. After 10 min of stirring, the
solution was neutralized with solid NaHCO3, the mixture
was filtrated, concentrated under vacum and the residue was
taken up with EtOAc (50 mL) and washed with H2O and
brine. The organic layer was dried over Na2SO4 and the
solvent was removed to give crude oil which was purified by
chromatography on silica gel (EtOAc–PE 3:7 and 5:5) to
give 1.91 g (72%) of corresponding hydrazine 6b as a
colorless oil. 1H NMR (CDCl3): d = 0.93 (d, 6 H, J = 6.7 Hz,
CH3), 1.24 (t, 3 H, J = 7.0 Hz, CH3), 1.66 (m, 1 H, CH), 2.43
(d, 2 H, J = 6.6 Hz, CH2), 2.86 (br, 4 H, CH2), 3.45 (s, 2 H,
CH2), 3.74 (q, 2 H, J = 7.0 Hz, CH2), 3.90 (s, 2 H, CH2), 4.20
(t, 1 H, J = 6.3 Hz, CH), 4.48 (d, 2 H, J = 6.3 Hz, CH2), 5.16
(s, 2 H, CH2), 5.21 (s, 2 H, CH2), 5.75 (br s, 1 H, NH), 6.95–
7.82 (m, 17 H, CHar) ppm. 13C NMR (CDCl3): d = 171.02,
Synlett 2005, No. 17, 2591–2594 © Thieme Stuttgart · New York

2594
O. Busnel, M. Baudy-Floc’h
LETTER
141.59, 136.25, 130.90, 128.42, 128.28, 128.05, 116.37,
127.56, 127.00, 125.14, 119.87, 95.33, 80.13, 66.19, 66.05,
64.87, 63.96, 58.62, 58.45, 54.18, 49.41, 47.79, 28.41,
26.41, 20.54, 14.88 ppm. HRMS (ESI): m/z calcd for
C45H56N4O8Na [M +Na]+: 803.39959; found: 803.3992 (0
ppm).
156.78, 155.89, 143.98, 141.40, 135.96, 130.40, 128.55,
128.33, 128.24, 116.17, 127.66, 127.11, 125.04, 120.01,
93.14, 66.20, 66.03, 65.90, 64.16, 58.93, 56.52, 55.93,
47.48, 46.10, 26.30, 20.83, 15.21 ppm. HRMS (ESI): m/z
calcd for C40H49N4O6 [M + H]+: 681.36521; found: 681.3656
(1 ppm).
(14) Cheguillaume, A.; Lehardy, F.; Bouget, K.; Baudy-Floc’h,
M.; Le Grel, P. J. Org. Chem. 1999, 64, 2924.
(15) Busnel, O.; Bi, L.; Dali, H.; Cheguillaume, A.; Chevance, S.;
Bondon, A.; Muller, S.; Baudy-Floc’h, M. manuscript
submitted for publication.
(17) To a solution of 7b (0.30 g, 0.39 mmol) in MeOH (10 mL)
was added 10% Pd/C (20 mg). After purging 3 times with
H2, the resulting suspension was stirred for 30 min (end of
reaction controlled by TLC). The catalyst was removed by
filtration through Celite. The filtrate was evaporated and
purified by chromatography on silica gel (EtOAc–CH2Cl2,
1:1 and Et2O–MeOH, 40%) to afford 0.2 g (75%) of 8b as a
yellow oil. 1H NMR (CDCl3, 353 K): d = 1.01 (d, 6 H,
J = 5.4 Hz, CH3), 1.13 (t, 3 H, J = 6.8 Hz, CH3), 1.50 (s, 9 H,
CH3), 1.81 (m, 1 H, CH), 2.56 (br, 2 H, CH2), 2.98 (br, 2 H,
CH2), 3.26 (br, 2 H, CH2), 3.60 (q, 2 H, J = 6.8 Hz, CH2),
4.02 (br, 2 H, CH2), 4.21 (s, 2 H, CH2), 4.31 (br, 1 H, CH),
4.60 (br, 2 H, CH2), 5.08 (s, 2 H, CH2), 6.50 (br s, 1 H, NH),
7.12–7.75 (m, 12 H, CHar) ppm. 13C NMR (CDCl3, 348 K):
d = 174.78, 157.54, 156.27, 144.39, 141.61, 131.28, 127.99,
127.70, 127.45, 116.38, 127.36, 127.07, 125.19, 119.86,
93.32, 80.84, 66.43, 63.94, 59.15, 58.36, 55.86, 47.83,
47.73, 28.33, 26.52, 20.62, 14.86 ppm. HRMS (ESI):
m/z calcd for C38H50N4O8Na [M + Na]+: 713.35263; found:
713.3523 (0 ppm).
(16) To a solution of 6b (1.69 g, 2.5 mmol) in dioxane (15 mL)
was added successively DIPEA (33 mg, 0.1 equiv) and
Boc2O (1.25 g, 2.3 equiv). The mixture was stirred during 24
h at 50 °C then added to a solution 1 M of NaHSO4 (40 mL)
and extracted twice with Et2O. The organic layer was
washed with brine, dried over Na2SO4 and concentrated. The
crude oil was purified by chromatography on silica gel
(EtOAc–PE 1:9 and 2.5:7.5) to afford 0.79 g (41%) of 7b as
a colorless oil. 1H NMR (CDCl3, 348 K): d = 0.96 (d, 6 H,
J = 5.8 Hz, CH3), 1.11 (t, 3 H, J = 7.0 Hz, CH3), 1.54 (s, 9 H,
CH3), 1.79 (m, 1 H, CH), 2.65 (br, 2 H, CH2), 2.93 (br, 2 H,
CH2), 3.13 (br, 2 H, CH2), 3.59 (q, 2 H, J = 7.0 Hz, CH2),
4.18 (s, 2 H, CH2), 4.25 (s, 2 H, CH2), 4.38 (br, 1 H, CH),
4.53 (br, 2 H, CH2), 5.06 (s, 2 H, CH2), 5.10 (br s, 2 H, CH2),
6.41 (br s, 1 H, NH), 7.12–7.70 (m, 17 H, CHar) ppm. 13
C
NMR (CDCl3, 348 K): d = 170.56, 157.50, 155.3, 144.41,
Synlett 2005, No. 17, 2591–2594 © Thieme Stuttgart · New York