Chemo-and regioselective modification of D , L -phenylalanine with α-cyanoacetylenic alcohols in water

D , L -Phenylalanine reacts with  -cyanoacetylenic alcohols under biomimetic conditions (room temperature, water) to give a novel family of unnatural amino acids, containing 2,5-dihydro-5-iminofuranyl substituents in the amino group, in almost quantitative yields (92-95%). This “green” one-pot version of the tandem assembly of nontraditional amino acids opens an unexplored avenue to the new types of drugs and their precursors


Introduction
Among the amino acids, a special place belongs to their aromatic congeners, in particular, phenylalanine. 1 The phenylalanine derivatives are used as antidiabetic agents (nateglinide). 2 Among them there are ACE-NEP, 3 urokinase 4 and pyruvate kinase inhibitors. 5Modifications of phenylalanine are active against the infections caused by Gram-positive bacteria (e.g.vancomycin 6 ).Some of them are applicable for the treatment of rheumatoid arthritis, inflammatory bowel diseases, systemic lupus erythematosus, multiple sclerosis, Sjögren's syndrome, asthma, psoriasis, allergy, cardiovascular diseases, arterial sclerosis, restenosis, tumor proliferation and transplantation rejection. 7herefore, the development of new synthetic strategies for phenylalanine modification is of essential importance.Meanwhile, the chemo-and regioselective transformation of the phenylalanine structural unit still represents a certain challenge and remains a subject of steadily growing investigations.
In this line, regioselective postsynthetic modification of phenylalanine residue with IPy2BF4 was reported. 8An efficient arylation of phenylalanine side chains (in unprotected iodopeptides) was rendered by Suzuki-Miyaura cross-coupling. 9Recently, a microwave-assisted esterification of N-acetyl-L-phenylalanine with modified Mukaiyama's reagents was also published. 10A series of fullerene substituted phenylalanine derivatives was prepared from 1,2-(4′oxocyclohexano)fullerene and protected (4-amino)phenylalanine. 11 The RAFT-polymerization of N-acryloyl-L-phenylalanine was accomplished. 12his concise but not exhaustive overview of the latest results concerning the phenylalanine modification shows that the efforts in this area keep extending.The objective of this paper is two-fold.The first is to check whether the strategy for the modification of amino acids with αcyanoacetylenic alcohols (α,β-acetylenic γ-hydroxy nitriles) recently pioneered by us 13 is general and also valid for phenylalanine.The second one is, using this strategy, to elaborate the efficient chemo-and regioselective synthesis of novel family of unnatural amino acids of unusual structure.

Results and Discussion
As applied to D,L-phenylalanine 1, the above strategy implies the reacting of these amino acids with available 14 α-cyanoacetylenic alcohols 2a-d to afford the initial adducts 3a-d and 4a-d which might be capable of further cyclizing to iminodihydrofuran substituted amino acids 5a-d (Scheme 1).

Scheme 1. Synthesis of unnatural amino acids 5a-d.
We have started with the monitoring of the reaction conditions which has shown that the above Scheme 1 is actually realizable when the reactants 1 and 2 are allowed to contact in aqueous medium in the presence of 50 mol% of NaOH (pH ~8-9) at room temperature, i.e. under biomimetic conditions.The modified phenylalanines 5a-d are formed chemo-and regioselectively in almost quantitative yields (92-95%), Scheme 1.
As depicted in Scheme 1, in the presence of 50 mol% of NaOH, zwitterionic form of phenylalanine 1a is partially neutralized to deliver carboxylate 1b having free amino group.The latter nucleophilically attacks α-cyanoacetylenic alcohols 2a-d to give two primary adducts, correspondently zwitterions 3a-d and carboxylates 4a-d, both being in equilibrium.Then (after adopting E-configuration) follows their cyclization to iminodihydrofurans 5a-d and 6a-d which are also in equilibrium.Finally, the equilibrium mixtures of iminodihydrofurans 5a-d and 6a-d were passed through alumina, Al2O3(H2O)n, to afford pure amino acids 5a-d.Alumina is known 15 to possess Lewis and Bronsted acid centers, owing to which the salts 6a-d exchange their sodium cation for proton.
The iminodihydrofuranyl moieties are assembled involving the cyano and hydroxyl functions of the primary adducts Z-3a-d and Z-4a-d.The Z-configuration of the adducts is predicted by the known trans-mode of nucleophilic addition to mono-substituted acetylenes. 16Therefore, before cyclization, the adducts Z-3a-d and Z-4a-d should undergo the isomerization to E-isomers since Z-configuration is incapable of the ring closing.
As it is clear from the above consideration, the role of NaOH as a catalyst is to deprotonate the ammonium site of the amino acid 1a thus converting it into a true nucleophile, salt 1b, Scheme 2.

Scheme 2
The attack of the amino group of salt 1b at acetylenes 2a-d is accompanied by the concerted proton transfer (either from water molecules or intramolecularly from the hydroxyl group) to give the ammonium hydroxides 7 which after release of water molecule leads to adducts 3a-d.
Duration of the synthesis depends on the structure of starting acetylenes 2a-d: for acetylenes 2a,b the reaction takes 3 hours, while acetylenes with cycloalkyl substituents 2c,d react completely for 72 hours (control TLC).This is likely resulted from decreased solubility of these acetylenes in water and the steric hindrance to the reactants contact imposed on the reaction by the cyclic substituents.Amino acids 5a-d are light-yellow powders, soluble in water, methyl and ethyl alcohols; insoluble in most organic solvents.
Multinuclear 1 H, 13 C and 2D (HMBC) NMR spectroscopy data as well as IR and UV investigation results of the adducts 5a-d are in agreement with their structures.In the 1 H NMR spectra of the adducts 5a-d, there is an olefinic proton (H-3) signal at 4.74-4.81ppm.The protons of the СН-and СН2-groups form three double doublets of three-spin system ABX in the region 3.92-3.99,3.11-3.33and 2.86-2.96ppm, respectively, protons of the phenyl ring are shown as a multiplet at 7.08-7.27ppm.In the case of 5b, the doubling of alkyl and olefinic hydrogen signals, corresponding to diastereomeric mixture R,R(S,S):R,S(S,R) = 1:1, is observed.The 13 C NMR spectra confirm also the structure of amino acids 5a-d.
The unusual zwitterionic structure of amino acids 5a-d follows from our recent results on the modification of glycine, 13a 2-aminobenzoic acid, 13c D,L-tryptophan 13d using the same strategy, generality and feasibility of which are additionally confirmed by this work.

Conclusions
In summary, "green" chemo-and regioselective modification of D,L-phenylalanine with αcyanoacetylenic alcohols in aqueous medium has been effected to furnish a new family of unnatural amino acids, containing 2,5-dihydro-5-iminofuranyl substituents, in almost quantitative yields.The results obtained confirm the generality and feasibility of the environmentally benign strategy for unusual modification of the amino acids which allows to combine in a one molecule both amino acid and iminodihydrofuran pharmacophoric moieties.Such a strategy as well as the modified phenylalanine derivatives may find applications as prospective pharmaceuticals and precursors for drug design.