Synthesis of aza-phenylalanine , aza-tyrosine , and aza-tryptophan precursors via hydrazine alkylation

Aza-amino acid precursors with an aromatic side chain were synthesized using hydrazine alkylation. This synthetic pathway avoided use of hydrogen gas and expensive hydrogenation catalysts. For the optimization of this alkylation reaction various solvents and different reaction conditions were used. Aza-phenylalanine, aza-tyrosine, and aza-tryptophan precursors with different Nand side-chain protecting groups were synthesized starting from N-protected hydrazines.


INTRODUCTION
* Replacement of the α-carbon atom with nitrogen in the structure of encoded amino acids yields peptide-like compounds with interesting structural, chemical, and biological properties such as non-chiral structure and increased stability towards biodegradation [1][2][3].However, these peptidomimetics cannot be synthesized directly from aza-amino acids, as these compounds are not stable.Therefore N-protected alkylhydrazines have  2. Reduction R'-X, X = Cl, Br, I ; base Scheme 1. Two main synthetic routes for the preparation of N -protected N-alkyl hydrazine derivatives (PG -protecting group).
reductive hydrazine alkylation (Scheme 1, left side).This method includes hydrazone formation from N-protected hydrazine and appropriate aldehyde and its subsequent reduction into alkyl hydrazine.However, this approach has some drawbacks in the case of aromatic aza-amino acid precursors, because reduction of strongly electron deficient hydrazone formed in the reaction of carbazate with benzaldehyde (aza-Phe precursor), O-protected 4-hydroxybenzaldehyde (aza-Tyr precursor), and N-protected indole-3-aldehyde (aza-Trp precursor) is rather complicated.Certainly, this reduction step can be performed by using a Pd/C/H 2 or Pd(OH) 2 /C/H 2 system [3,10,13].However, this procedure has a non-selective character [14], requires expensive catalysts, and most importantly, needs a sophisticated hydrogen supply system and special equipment to work with this gas under elevated pressure.Therefore we decided to use for the preparation of aza-Phe, aza-Tyr, and aza-Trp precursors another known synthetic pathway, which includes direct N-alkylation of protected hydrazine with appropriate alkyl halides (Scheme 1, right side).As this alkylation method is not widely applied for the preparation of azaamino acid precursors it required optimization.
After a systematic study of the reaction conditions this synthetic method was optimized and applied for the preparation of the aromatic aza-amino acid precursors as shown in Schemes 2, 3A, 3B, and 4.

RESULTS AND DISCUSSION
The main goal of the present study was to optimize the hydrazine alkylation reaction.The reaction of benzylation of different carbazates was chosen as the model reaction.Different conditions, solvents, and benzyl halogenides were tested.The obtained results are described in Table 1 and Table 2.In the case of an equimolar or 2/1 ratio of protected hydrazine and benzyl bromide, relatively poor yields ranging from 13% to 30% were obtained at room temperature.At the same time formation of a significant amount of the dialkylated product was observed.This can be explained by the fact that introduction of alkyl moiety into protected hydrazine increases nucleophilicity of hydrazine, making the monoalkylated compound prone to undergo a second alkylation.In order to suppress polyalkylation, an at least 3-fold excess of hydrazine was used in further experiments.
Besides this, addition of bases such as pyridine or 2,4,6-trimethylpyridine (1-1.3 eq) was found to be useful to increase the yield of the monoalkylated product.It was important to consider that the type of the suitable base was dependent on the properties of the hydrazine
It was also found that solvent played an important role in the alkylation reaction.In early hydrazine alkylation studies it was found that the use of ethanol as a solvent for hydrazine alkylation reaction helps to increase monoalkylated hydrazine yield [15].The same conditions were used in later works involving hydrazine alkylation [16].In the current study it was found that in 96% ethanol the yields of alkylation of Boc-NHNH 2 and Fmoc-NHNH 2 were 57% and 61%, respectively.At the same time in methanol solution the yield of the monoalkylated product was significantly lower: 21% in the case of Fmoc-NHNH 2 (2 eq) and 11% in the case of Boc-NHNH 2 (3 eq excess).
We also tested benzyl chloride as an alkylating reagent for benzylation of N-Fmoc-protected hydrazine.In this case the monoalkylated product was obtained in 14% yield in methanol.However, using benzyl iodide as the alkylating agent gave the monoalkylated product with 75% yield in the case of BocNHNH 2 and 74% in the case of Fmoc-NHNH 2 , which was the highest yield of the synthesis of N-Fmoc-N -benzyl hydrazine (Scheme 2, Table 1, Table 2).
For the preparation of protected aza-Tyr and aza-Trp precursors (Schemes 3A, 3B, and 4) we proceeded from different aldehydes (compounds 9 and 10 in Scheme 3B and 18 in Scheme 4).The functional groups of the side chain of these compounds were protected with different protecting groups that allowed reduction of the aldehyde group with NaBH 4 in methanol and production of corresponding alcohols (compounds 4, 11, 12, and 20).Yields of these syntheses were very good.Syntheses of the corresponding bromides from these alcohols were performed by using PBr 3 /DCM in the presence of TEA or a combination of TEA and NaHCO 3 .Bromides 13 and 14 were unstable and decomposed producing HBr, which in turn cleaved protecting groups and resulted in very complex mixtures.Therefore the bromides were used for the following steps immediately after their preparation.
The obtained bromides were used for the alkylation of monoprotected hydrazine in refluxing ACN solution containing 1 eq of 2,4,6-trimethylpyridine as a base.The yields of monoalkylated hydrazines were in the range of 34-61% (Schemes 3A, 3B, and 4).
We also tested alternative and less harsh methods for the preparation of bromides (compounds 5, 13, 14, and 21).One of the possibilities was the free-radical bromination of alkyl groups of readily available p-cresole and 3-methylindole.Our attempts to obtain 4-(tert-butyloxycarbonyloxy)benzyl bromide and N-Boc-(3-bromomethyl)indole from Boc-protected p-cresole and N-Boc-3-methylindole via radical bromination, using NBS/AIBN in refluxing CCl 4 [17], was not successful.As a result of bromination of Bocprotected p-cresole the mixtures of different bromides were formed.Experiments with N-Boc-3-methylindole resulted in complete degradation of the reaction mixture. (3) HN NH PG O a.

EXPERIMENTAL General
All solvents and reagents were purchased from Merck, Sigma-Aldrich, or Lach-Ner.NMR spectra were measured on 200 MHz and 700 MHz instruments (Bruker, Germany) in DMSO-d6 or CDCl 3 as solvent and using tetramethylsilane as the internal reference.High resolution and low resolution ESI-FT-ICR mass spectra were obtained on a Varian 910-FT-ICR-MS spectrometer using ACN as solvent.IR spectra were determined using the attenuated total reflectance (ATR) measuring technique on a Perkin-Elmer Spectrum BX spectrometer.All yields in multistep syntheses were calculated for each step separately proceeding from the mass of the obtained product.
General procedure for the reduction of aldehydes to corresponding alcohols: 1 eq of aldehyde was dissolved in methanol (about 1 g of aldehyde in 20 mL of CH 3 OH); the solution was isolated from the atmosphere with Ar and cooled to 0 °C on ice bath.Sodium borohydride (1 eq) was added to the solution and stirred for 1 h.The resulting mixture was quenched with 1 M HCl; methanol was evaporated under reduced pressure.The resulting mixture was diluted with EA, washed with 1 M NaHCO 3 ,  2 H 2 O, and brine.The water phase was extracted 3 times with EA; combined organic solutions were washed with brine, dried over anhydrous Na 2 SO 4 , and concentrated using a rotatory evaporator.The crude product was used for following reactions without purification.
General procedure for N-protected hydrazine alkylation: 3-4 eq of N-protected hydrazine was dissolved in ACN (0.1 M solution), heated to reflux; 1 eq of 2,4,6-trimethylpyridine was added.Solution of 1 eq of alkylbromide in ACN (approximately 0.1 g of bromide in 1 mL of ACN) was added dropwise and the reaction mixture was refluxed overnight.ACN was evaporated under reduced pressure; the residue was dissolved in EA, washed with 1 M NaHCO 3 ,  2 H 2 O, and brine.The water phase was extracted 3 times with EA; the combined organic solutions were washed with brine, dried over anhydrous Na 2 SO 4 , and concentrated using a rotatory evaporator.The crude product was purified on silica gel using EA/PE 1 : 2 mixture as eluent.
During the study of the alkylation reaction various solvents and reaction conditions were used (Table 1 and  Table 2).

CONCLUSIONS
A detailed study of mono-protected hydrazines using benzyl bromide was performed, and the most suitable conditions for these reactions were found.Aza-tyrozine, aza-phenylalanine, and aza-tryptophane precursors with different protecting groups were prepared from various aldehydes via the hydrazine alkylation reaction.
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Fig. 1 .
Fig. 1.General structure of the aza-amino acid precursor (PG stands for protecting group and R for alkyl group).