Stereoselectively synthesis and structural confirmation of dehydrodipeptides with dehydrobutyrine

Most of polypeptides containing α,β-dehydroamino acids have important biological activity, so exploration of synthetic method has practical significance. In this paper, dipeptides were prepared from l-threonine by protecting of c-terminal allyl acetate, and condensing reaction with a series of N-Boc amino acid. Then, treatment of dipeptides obtained with DMAP, (Boc)2O and tetramethylguanidine in the acetonitrile occured β-elimination reaction to yield stereoselectively dehydrodipeptides. Structures of dehydrodipeptides were confirmed by 1H NMR, 13C NMR and MS. Analysis of 1H NMR, 2D NMR and crystal structure showed that the dehydrodipeptides were Z-configuration.Graphical abstract Dehydrodipeptides were prepared from l-threonine. Their structures were confirmed by 1H NMR, 13C NMR and MS Electronic supplementary material The online version of this article (doi:10.1186/s40064-016-2005-z) contains supplementary material, which is available to authorized users.

Background α,β-Dehydroamino acids, as unnatural amino acids, are found in many natural products including the fungal metabolites, β-lactam antibiotic, sulfide antibiotics, anticarcinoma antibiotics, phytotoxin, the antrimycins, tentoxin, and the phosphatase inhibitors microcystin, nodularin and the synthetic drugs (Gross and Meienhofer 1983;Valentekovich and Schreiber 1995;Botes et al. 1984). In the active peptide, α,β-dehydroamino acids are usually used to fixed peptide main chain and side chain, and make its conformation keeping relatively stable, also can inhibit its biological degradation process. So α,β-dehydroamino acids play an important role in the design and synthesis of biological peptide and the study of its structure-activity relationship (Chang et al. 1998;Rappoport 1994;Harburn et al. 1998;Kohno et al. 1996;Bierbaum 1999).
Many methods available for synthesis of α,βdehydroamino acids have been reported (Bonauer et al. 2006;Poisel and Schmidt 1976;Kolasa 1983;Maekawa et al. 2004;Schmidt et al. 1992;Trost and Dake 1997;Nagano and Kinoshita 2000;Chen et al. 2005;Goodall and Parsons 1995;Li et al. 1996;Somekh and Shanzer 1983;Miller 1980;Stohimeyer et al. 1999). However, these methods often are low yielding, multistep, require tedious purification steps to remove reagent side products, or incorporate unusual, difficultly obtained amino acid intermediates. Elimination of water from β-hydroxyα-amino acids is a well-established route to obtain β-dehydroamino acids. This method has been used for the preparation of dehydroalanine and dehydroaminobutanoate from serine and threonine. Shioiri demonstrated Martin's sulfurane is a mild, neutral and stereospecific dehydrative agent for the dehydrative elimination, which give stereospecific polypeptide with Z-configurational unsaturated amino acid, through removing hydroxyl group of β-hydroxy-α-amino acids in dipeptide or tripeptide (Yokokawa and Shioiri 2002). However, the disadvantage is that the Martin's sulfurane is expensive. Ferreira reported one important and well-used approach involves the β-elimination reactions of serine and threonine derivatives with  pyridine (DMAP) (Ferreira et al. 1999;Ferreira et al. 1998). Furthermore, they use the base N,N,N,Ntetra-methylguanidine (TMG)  the tert-butyl carbonate group from the O-(tert butyloxycarbonyl)-β-hydroxyamino acid derivatives, give the corresponding dehydroamino acid derivative. This twostep method can be carried out as a one-pot procedure and is stereoselective, giving only the Z isomer (Ferreira et al. 2007). In this study, We now wish to synthesize a variety of dehydroamino acid derivatives by Ferreira's synthetic approach and the double bond formed by dehydration reaction is determined Z isomer by NOESY (Shimohigashi et al. 1982;Duhamel et al. 1972) and X-ray crystal diffraction.

Synthesis
In the synthesis of dehydrodipeptides, first, we obtained dehydroamino acids using N-protected β-hydroxyamino acid esters as raw material. Then, we attempted to obtain dehydrodipeptides by condensation reaction of dehydroamino acids and amino acid. However, owing to the low reactivity of the α-amine group of dehydroamino acids and to the instability of its N-deprotected derivatives, byproducts of reaction are very much, and difficult to separate by this methods. Therefore, this led us to investigate the applicability of Ferreira's methodology to the dehydration of peptides containing β-hydroxyamino acids as precursors of dehydropeptides. First, dipeptides were prepared from two amino acid, then dehydrodipeptides were obtained by β elimination dehydration of dipeptides.
In order to prepare dipeptides containing l-threonine, the protection of functional groups were very important because of containing three activity groups in l-threonine. Generally, protected groups were selected according to the final products. In this studies, allyl group were used to protect carboxy terminus of amino acid. However, if l-threonine was treated with allyl bromide in dry DMF, besides carboxyl group, the amino group was reacted with allyl bromide. Thus, amino group of l-threonine was protected firstly by Boc group, When N-Bocprotected l-threonine 2 was reacted with allyl bromide in the presence of K 2 CO 3 in dry DMF, it was converted quantitatively into 3 within 12 h. However, in the presence of NaOH, only traces of 3 were detected. This suggests that the by-product may result from a base-induced side-reaction. Thus, the use of an alternative base could possibly reduce this side reaction, so K 2 CO 3 was substituted for NaOH in the reaction of 2 with allyl bromide. Therefore, N-Boc-protected l-threonine 2 were treated with 1.2 equiv. of allyl bromide and K 2 CO 3 in dry DMF to give the corresponding allyl esters of N-protected l-threonine 3.
Dipeptides were readily prepared from 3 by N-deprotection and coupling with the N-protected amino acid using DIPEA/HBTU in higher yield (Scheme 1). Compound 4 was treated with 1.2 molar amounts of N-protected amino acid in the presence of 1.2 molar amounts of HBTU and DIPEA in dichloromethane (DCM) and DMF at room temperature to afford the desired dipeptides 5a-5l in high yield (>90 %).
Ferreira's method, one of the noteworthy stereospecific feature of the method was reported for the elimination with Boc-anhydride and 4-(N,N-dimethylamino) pyridine (DMAP) and TMG by Ferreira et al. (1999). Therefore, We treated N-Boc protected dipeptides with 3.3 equiv. of (Boc) 2 O in the presence of DMAP, gave O-tertbutyl carbonates of dipeptides, followed by direct reaction with N,N,N,N-tetra-methylguanidin (TMG) without isolation, afforded the corresponding dehydrodipeptides in good yields. This two-step method can be carried out as a one-pot procedure. However, In order to compare with Ferreira's method, we tried to synthesize dehydrodipeptides by β-elimination of dipeptides containing β-hydroxyl group using Martin's sulfurane. Unfortunately, the treatment of dipeptides 5 with Martin's sulfurane did not led to the desired products. Therefore, Ferreira's synthetic approach could be used efficiently stereoselective synthesis of dehydrodipeptides. In comparison with previous methods, this procedure is a onestep process, since separation of the intermediates is unnecessary and theatment of intermediates with TMG gave the corresponding dehydrodipeptides in good yield. This mildness of the mothod is compatible with the presence of a variety of functional groups.

Structure characterization
To further investigate the configuration of the dehydrodipeptides, 6c was subjected to the 2D NMR measurements. Since the NOE cross-peaks between the protons that are closer than 0.4 nm in space will be observed in NOESY spectrum and the relative intensities of these cross-peaks depend on the spaces between the corresponding protons. As can be seen from Fig. 1, the NOESY spectra of 6c showed clear NOE cross-peaks A of H1 of methyl group in double bonds and H2 of amino group in amide bonds, demonstrating that the substituents of methyl goup and amino group located on the same side of the double bond. As well as no correlation between H3 of double hand and H2 of amino group, would provide us further information about the orientation of the H proton and amino group in the double bonds. These indicated distinctly that the double bond in dehydrodipeptides was Z configuration. In addition, We obtained single crystals suitable for X-ray crystallography by slowly evaporating a ethyl acetate solution of 6c. Interestingly, X-ray crystallographic analysis of 6c reveals that the double bond C7-C8 in dehydrodipeptides was Z configuration (Fig. 2). Therefore, the dehydro-dipeptides was synthesized stereoselectively by β-elimination reaction using DMAP, (Boc) 2 O and tetramethylguanidine.

Materials and instrumentation
All of the org. solvents used in this study were dried over appropriate drying agents and distilled prior to use. All analytical grade chemicals were purchased commercially and used without further purification. Compound 2 (Goodall and Parsons 1995) was prepared according to the literature procedures. 1 H NMR and 13 C NMR spectra were recorded on a Bruker AVANCE II500 instrument in CDCl 3 solution, using tetramethylsilane as an internal reference. Elemental analyses were performed on a Perkin-Elmer 2400C instrument. The X-ray diffraction data were collected by using a Rigaku Mercury CCD AFC10 system with monochromated Mo Ka radiation.

Synthesis
Boc-L-Thr-OAllyl (3) 21.9 g Boc-l-threonine-OH (0.1 mol) was dissolved in 70 mL DMF, 16.6 g (0.12 mol) K 2 CO 3 was added and cooled to 0 °C in an ice bath. 14.4 g (0.12 mol) Allyl bromide was added dropwise with stirring by means of a separatory funnel. After the mixture was stirred for 1 h, the two phase solution was allowed to warm slowly to room temperature with vigorous stirring over 12 h. The solid residue was isolated by filtration. The solvent in the filtrate was removed in vacuo and the residue was taken up in 80 mL of a saturated NaCl solution, and the aqueous solution was extracted with ethyl acetate (30 mL × 5). The organics were combined and washed with 1 M KHSO 4 solution, water, saturated KHCO 3 and saturated NaCl, dried over MgSO 4 , the solvent removed under reduced pressure, evaporated in vacuo to give a viscous liquid, which was purified by means of silica-gel chromatography (Petroleum ether/Ethyl acetate = 5:1) to give Boc-L-Thr-OAllyl 3, the white solid with yield 91 %. Boc-N-AA-L-Thr-OAllyl (5) 1.29 g (5 mmol) Boc-L-Thr-OAllyl was taken up into a solution of trifluoroacetic acid (20 mL) in DCM (30 mL) and stirred for 3 h to remove the Boc group. After removing the solvent and TFA in vacuo, the crude product 4 was obtained (yield > 90 %), which was used to next step reaction without purification. The resulting compound was dissolved in 150 mL DCM and 15 mL DMF and 6 mmol BocNH-AA-OH and 2.25 g (6 mmol) HBTU were added, followed by gradual addition of 7.5 mL DIPEA, the mixture was stirred at room temperature overnight. The solution was  (1) CH 3 CN

Scheme 1
The synthetic route of dehydrodipeptides concentrated in vacuo, then 100 mL water were added, and the aqueous solution was extracted with ethyl acetate (30 mL × 5). The organics were combined and washed with water, 5 % K 2 CO 3 , 2 % HCl and water, dried over MgSO 4 , the solvent removed under reduced pressure, evaporated in vacuo, the crude product was purified by means of silica-gel chromatography (Petroleum ether/ Ethyl acetate = 5:1) to give white solids.  Figure S9).  Figure S13).  Figure S17).  Figure S21).  Figure S40).  Figure S43).

Conclusions
α,β-Dehydroamino acids play an important role in the design and synthesis of biological peptide and the study of its structure-activity relationship, while The synthesis of peptides containing dehydroamino acids were a challenge. A variety of dehydroamino acid derivatives were synthesized. The results showed that this methods could be carried out as a one-pot procedure, and had high stereoselectivity. 2D NMR NOESY and X-ray crystal diffraction determined Z configuration of double bond.

Supplementary information
All additional information pertaining to characterization of the complexes using 1 H NMR, 13 C NMR and EI-MS spectra are given in the supporting information available at XXX.