Synthesis of the Fmoc-aza-Arg ( Boc ) 2 precursor via hydrazine alkylation

The aza-arginine precursor Fmoc-aza-Arg(Boc)2 was synthesized starting from mono-protected hydrazines via an azaornithine precursor. This reaction path is shorter and more efficient than the reductive alkylation reaction.


INTRODUCTION
* Substitution of DNA encoded amino acids (1) by their aza analogues (2) (Fig. 1) is a feasible structural modification of biologically active peptides for obtaining peptidomimetics with an increased effective lifetime in biological systems [1,2].
Incorporation of aza-amino acids into peptides can be done by using protected precursors, which can be presented as hydrazine derivatives (see 2 in Fig. 1).In * Corresponding author, anton.mastitski@ut.eemost cases these precursors have been prepared from carbonyl compounds via reductive alkylation of protected hydrazines [3][4][5] (Scheme 1, A).However, there are other synthetic routes, mainly based on alkylation of N′-mono-or di-substituted hydrazine derivatives with alkyl halides [4,[6][7][8] (Scheme 1, B), or introducing a protecting group into commercially available alkyl hydrazines [4,[7][8][9][10] (Scheme 1, C).However, in the case of some aza-amino acid precursors these straightforward synthetic routes cannot be used.For example, synthesis of an aza-arginine precursor is complicated by guanidyl moiety introduction and protection.Therefore guanylation of the side-chain amino group of aza-ornithine, already included into peptide sequence, was used in the first attempt to synthesize the aza-arginine containing peptidomimetics [5].According to this methodology, ω-N-Aloc-protected aza-ornithine was incorporated into peptide and thereafter the Fmoc-protected resin-bound peptide was treated to remove Aloc protection followed by guanylation with N,N ′-bis-Boc-1-guanylpyrazole [11].Later, pre-paration of the Fmoc-protected aza-arginine precursor was accomplished according to Scheme 1A, using reductive amination of 3-(N ω ,N ω -di-Boc-guanidyl)propanal with Fmoc hydrazine, while the required carbonyl compound was prepared by guanylation of 3-aminopropanol with N,N ′-bis-Boc-S-methylisothiourea, followed by oxidation with the Dess-Martin reagent [12].
In this report we suggest an alternative synthesis of the aza-arginine precursor, using alkylation of N-protected hydrazine with N-protected 3-bromopropyl amine (Scheme 2), and thereafter converting the protected aza-ornithine precursor into a precursor of azaarginine (Scheme 3).Compared with the previously published method of reductive alkylation, this synthetic pathway is shorter, allows avoiding condensation and reduction steps and does not require relatively expensive oxidizing reagents.

Chemicals and analytical equipment
All solvents and reagents were purchased from Merck or Sigma-Aldrich and were at least ACS grade.The NMR spectra were measured on a 200 MHz spectrometer and on a 700 MHz spectrometer (Bruker, Germany) in DMSO-d6 or CDCl 3 as the solvent and using the internal reference.The HRMS of the products were measured on a LTQ Orbitrap (Thermo Electron) spectrometer (positive ionization, static nanospray, boron silicate emitters Proxeon, resolution 100 000 at m/z 400, external calibration), using acetonitrile as the solvent.
N-protected 3-aminopropyl bromides (1-4) were obtained by the modified procedure described in [15][16][17][18].Shortly: 3-bromopropylammonium bromide (1 equiv.) was dissolved in chloroform (to give ca 0.2-0.5 M soln.),N,N-diisopropylethylamine (1.5-2.2 equiv., 1.05 equiv. in case of acylation with Boc anhydride) was added, the solution was cooled to 0 °C, and alkoxycarbonyl chloroformate (1 equiv.)or Boc anhydride (1.05 equiv.) was added slowly.The mixture was allowed to stir overnight.Then the reaction mixture was diluted with EA, washed with water and brine, dried over anhydrous magnesium sulphate, and concentrated in vacuo to obtain a crude product in nearly quantitative yield, which could be used without purification.

RESULTS AND DISCUSSION
As the first step of aza-arginine precursor synthesis, the double-protected aza-ornithine precursor was prepared via alkylation of protected hydrazine as shown in Scheme 2.
The reaction was performed by analogy with Bochydrazine alkylation in DMF, as was described by Hansen [19]; however, two modifications were introduced.Firstly, DMF was replaced by NMP to avoid formation of the formulated side product PG-NHNHCHO.Secondly, excess of the hydrazine derivative was used to minimize the formation of the dialkylated compound PG-NHNAlk 2 .If equivalent amounts of the alkylating reagent and Fmoc-hydrazine were used, the yield of the mono-alkylated product was around 35%.In the case of 3-fold carbazate excess, the yield was up to 70% as listed in Table 1.
Further, different combinations of protecting groups were tested in both reagents, in N-protected 3-bromopropyl amines and protected hydrazines, keeping in mind that these combinations may be needed for selective removal of one protecting group in the presence of another protecting group (the orthogonality requirement) in the following steps of aza-peptide synthesis.These experiments did not reveal any significant influence of the protecting group structure on the formation of the mono-alkylated product, except the reaction of Z-protected hydrazine.Therefore both ornithine precursors FmocNHNH(CH 2 ) 3 NHBoc and BocNHNH(CH 2 ) 3 NHFmoc can be used for the synthesis of aza-amino acid containing peptidomimetics.However, as the Fmoc strategy was selected for peptide synthesis in this project, the Fmoc-aza-Arg(Boc) 2 precursor was prepared, as summarized in Scheme 3.

CONCLUSIONS
Synthesis of FmocNHNH(CH 2 ) 3 NHBoc, which is the precursor for aza-Arg incorporation into a peptidomimetic structure, was made by alkylating hydrazine derivatives.The alkylation reaction was performed on substrates with various combinations of protecting groups to meet different requirements of the orthogonality condition, which depends upon the applied strategy of peptide synthesis.

Scheme 1 .Scheme 2 .Scheme 3 .
Scheme 1. Possible synthetic routes for the preparation of N ′-protected N-alkyl hydrazine derivatives PG stands for protecting group.