New building blocks or dendritic pseudopeptides for metal chelating

Dendritic oligopeptides have been reported as useful building blocks for many interactions. Starting from hydrazine, we described an approach to create new dendritic pseudopeptides linked with biological systems, such as cell membrane, as chelate metal, Ni2+-nitrilotriacetic acid moieties which could target histidine rich peptides or proteins. Depending on the nature of these new chemical recognition units, they could be integrated into a peptide by coupling in C or N-termini.Graphical abstract: Dendrimer formation


Background
Unnatural amino acids constitute attractive targets for drug design. Disposing of a wide variety of unnatural amino acids allows the modulation of physical and chemical properties of the resulting peptide depending on the selected side chains (Gentilucci et al. 2010). The aza-β 3amino acids represent an exciting type of analogs of β 3amino acids in which the CH β is replaced by a nitrogen stereocenter conferring a better flexibility to the pseudopeptide due to the side chain borne on a chiral nitrogen atom with non-fixed configuration (Busnel et al. 2005). Moreover, the backbone modification makes these molecules more stable towards proteolytic degradation (Dali et al. 2007;Laurencin et al. 2012).
Herein we aimed to design new amino acid analogues or building blocks that can be incorporated into any polypeptide by solid-phase peptide synthesis. Potential applications of these metal-chelating units will be as metal sensors for synthetic receptors that interact specifically with histidine-tagged peptides.

Results and discussion
As part of our research program we develop new peptide analogues with potentially useful biological properties. For this purpose, we have developed synthetic strategy for aza-β 3 -aspartic acid Abbour and Baudy-Floc'h 2013). We observed that during this process a double substitution of benzyl carbazate 1 occurred to afford Z-aza-β 3 -Asp(Ot-Bu)-Ot-Bu 4 in 19 % yield. By using tert-butyl bromoacetate (3 eq) 2 and N,N-Diisopropyl ethylamine (DIPEA) (2 eq) 3 was obtained in 80 % yield (Scheme 1). The hydrogenolysis of 3 over 10 % Pd/C gave our precursor 4. A nucleophilic substitution of 4 by tert-butyl bromoacetate (1 eq) in the presence of N,N-Diisopropyl ethylamine (DIPEA) (1 eq) afforded the expected building block 5 with one azanitrilotriacetic acid which could be coupled in C-termini (Scheme 1) with 20 % yield, we observed the formation of a secondary product 5′. To increase the yield of compound 5, we tried different solvents and different bases. The yield of 5 with acetonitrile/DIPEA or NEt 3 was 18 %, with Toluene/potassium carbonate K 2 CO 3 in suspension 20 %, and with μWaves (150 W, 90 °C, 45 min) 5 %.
Reductive amination of trisubstituted hydrazine 5 with glyoxylic acid in the presence of NaBH 3 CN led to the tetrasubstituted hydrazine 6 as new building block with one aza-NTA, which could be coupled in N-termini.
To create more flexibility to the aza-NTA, we first prepared the substituted aza-β 3 -glutamic ester 9. Compound 8 was obtain by nucleophilic substitution of methyl 3-bromopropanoate 7 and benzyl carbazate 1 in the presence of DIPEA with only 17 % yield. The same reaction without solvent realized under microwaves activation provided 8 with 35 % yield. Then a second nucleophilic substitution of tert-butyl bromoacetate 2 with compound 8 and DIPEA led to Z-aza-β 3 Glu(OMe)-Ot-Bu 9 with 96 % yield after stirring at 80 °C for 5 days. Then hydrogenolysis of 9 over 10 % Pd/C gave the monomer H-azaβ 3 Glu(OMe)-Ot-Bu 10. Nucleophilic substitution with two equivalents of tert-butyl bromoacetate 2, H-azaβ 3 Glu(OMe)-Ot-Bu 10 and DIPEA gave 11 (94 % yield). Methyl ester of 11 could be saponified (Pascal and Sol 1998) by sodium hydroxide in MeOH in the presence of CaCl 2 affording the expected aza-NTA 12, which could be coupled in N-termini of a peptide (Scheme 2).
To obtain a new ligand with an amine function, which could be coupled on C-termini peptide we choose to work on ornithine analogue. The 1-amino-3,3-diethoxypropane precursor 13 was first N-protected with a benzyl group by reaction with benzylchloroformate under the presence of sodium hydroxide to afford benzyl 3,3-diethoxypropylcarbamate 14 with excellent yield (99 %). The acetal 14 was then treated with acetic acid and water (2/1) to give benzyl 2-formylethylcarbamate 15. The condensation of 15 with our precursor 4 led to the hydrazone 16. Reduction with sodium cyanoborohydride (NaBH 3 CN) gave the hydrazine 17. Nucleophilic substitution of tert-butyl bromoacetate by hydrazine 17 afforded substituted aza-NTA 18. Hydrogenolysis of 18 under 10 % Pd/C, gave a new ligand aza-NTA 19, bearing a long amino chain with more flexibility (Scheme 3).
Our goal was to get multimeric aza-NTA in order to increase the affinity to histidine tag proteins. Thus we built the dendritic pseudopeptides starting from our two building blocks 18 and 19. Deprotection of acid functions of 18 with TFA afforded 20. Then dendritic pseudopeptides or Z-aza-tris-NTA-tBu 21 were synthesized via standard EDCI coupling of one equivalent of the C-deprotected intermediate 18 with three equivalent of the N-deprotected one 19. We showed that it is possible to deprotect 21 either on C-ter to give Z-aza-tris-NTA-OH 22, or on N-ter to lead to H-aza tris-NTA-tBu 23. NMR and HMRS mass spectrometry were used to verify the structure and purity of the amphiphilic dendritic peptides (Scheme 4).

Conclusion
In summary, depending on the nature of our new chemical recognition units, these could be introduced by coupling in a peptide in C or N-termini as well as on peptidic chain. These new Ψ-NTA could open new ways to control protein-protein interactions, to design peptidebased interaction pairs or to generate switchable protein functions. Moreover it would be interesting to look at the self-assembly of our new dendric pseudopeptides.
Yield: 0.5 g (17 % Compound aza-NTA 6. To a solution of substituted hydrazine 5 (1.9 g, 5 mmol) in DCM/MeOH (10/25 mL), glyoxylic acid monohydrate (0.44 g, 1.2 equiv) was added. Then NaBH 3 CN (0.46 g, 1.5 eq) was added fractionally into the above mixture, which was maintained under stirring for 1 h, and the pH was maintain at 3 by addition of 2 N HCl. Then HCl was added until pH 1 over 10 min and finally increased to 4-5 with a saturated NaHCO 3 solution. The mixture was filtered, concentrated, taken up with EtOAc (10 mL) and washed with 2 N HCl solution and brine. The organic Compound Aza NTA 12. 11 (1.2 g, 6 mmol) was dissolved in MeOH (14 mL) and CaCl 2 (2.6 g, 0.4 M), NaOH (0.125 g, 3.1 mmol) was dissolved in H 2 O (6 mL). These two solutions were mixed and stirred at room temperature for 6 h. Then, 2 N HCl solution was added to get a neutral pH. Evaporation of methanol under vacuum and extraction with EtOAc (20 mL × 2) led to an organic phase, which was washed with 2 N HCl solution (20 mL) and brine (20 mL). The solvent was evaporated under vacuum and the residue was purified by column chromatography on silica gel with DCM/EtOAc (8/1) to afford the triester 12.

Hydrogenolysis procedure
Hydrazine (18 mmol) was dissolved in MeOH (50 mL) and 10 % Pd/C (0.7 g) was added. The mixture was stirred under hydrogen atmosphere at room temperature for 6 h. The catalyst was eliminated by filtration through a Celite ® pad and the solvent removed under vacuum to obtain colorless product 4, 10, 19 and 23 enough pure.
Compound 4 A solution of 1-Amino-3,3-diethoxypropane 13 (2 g, 13.6 mmol) was added into a solution of NaOH (0.55 g, 13.6 mmol) in water (20 mL) and cooled at 0 °C. The solution of benzylchloride (2.32 g, 13.6 mmol) in DCM (20 mL) was slowly added into the cooled solution. The mixture was stirred at room temperature for 12 h. After washing with H 2 O, the organic phase was dried and concentrated under vacuum to give benzyl 3,3-diethoxy propyl carbamate 14.
Benzyl (3-oxopropyl) carbamate 15 (2.6 g, 12.6 mmol) and 5 (3.25 g, 12.6 mmol) were dissolved into DCM (30 mL), Na 2 SO 4 was added to absorb the water and accelerated the reaction. The solution was stirred overnight at room temperature and filtrated to remove Na 2 SO 4 . The filtrate was concentrated and purified by chromatography over silica gel with PE/EtOAc (7/3) first and then (6/4) to give pure hydrazone 16.
The hydrazone 16 (2.1 g, 4.68 mmol) was dissolved in MeOH (30 mL), NaBH 3 CN (0.35 g, 1.2 eq) was added by portions. 2 N HCl solution was used to maintain a pH 3 and then the mixture was stirred for 2 h. HCl 2 N was added until pH 1, and after 10 min, the pH was increased to 7-8 by adding NaHCO 3 . The solid was filtrated after 2 min, and the solvent was removed under vacuum and the crude product was dissolved into EtOAc (30 mL) and washed by H 2 O (2 × 20 mL). The organic phase was dried under Na 2 SO 4 and the solvent was removed under vacuum to afford hydrazine 17.

Authors' contributions
MR carried out all the synthesis and performed the analysis. IN have made substantial contributions to conception and performed some analysis. MBF conceived of the study, and participated in its design and coordination and have been involved in drafting the manuscript. All authors read and approved the final manuscript.

Competing interests
The authors declare that they have no competing interests.