Feasibility study on the reaction of 1 , 4-diazabicyclo [ 2 . 2 . 2 ] octane ( DABCO ) with ( L-Serine-L-Serine ) and ( L-Phenylalanine-L-Serine ) diketopiperazines

Article history: Received January 21, 2016 Received in revised form July 10, 2016 Accepted 13 July 2016 Available online 13 July 2016 This paper summarizes the reaction of DABCO with the enol tosylate derivatives made from (L-Ser-L-Ser) and (L-Phe-L-Ser) diketopiperazines (DKP’s). The reaction between DABCO and EE-di-tosylate (L-Ser-L-Ser) DKP (2), results in the isomerization of the serine di-tosylate from EE-2 to ZZ-2. This is the first direct example of the utility of DABCO as a reagent demonstrating the successful isomerization in a DKP derivative. The E-enol tosylate of (LPhe-L-Ser) DKP (4) upon reaction with DABCO provided a unique bis-ylidiene product (5). © 2016 Growing Science Ltd. All rights reserved.


Introduction
2][3][4][5][6][7][8][9] The presence of these 2,5-DKP's in bioactive natural products, their synthetic utility and applications in medicinal chemistry is excellently reviewed by Borthwick. 10A detailed description of the application of these 2,5-DKP's in peptide and combinatorial chemistry is thoroughly reviewed by P.M. Fischer. 112,5-DKP's have continued to establish impact on the frontiers of organic, structural, and medicinal chemistry. 12DKP derivatives, such as enol di-tosylate (2) and enol tosylate (4), can serve as building blocks for a class of important compounds called endiamino peptides [compounds bearing a 1,3-diaminoethenyl functional group]. 13Endiamino bonds can induce a conformational rigidity in peptides and hydrogen bonding assisted enzyme sulfatases catalysis.It is worth to mention, that Callynormine A, is a recently isolated marine metabolite from Kenyan sponges Callyspongia abnormis, appears as a rare N-atom containing heterodetic peptide that has the Z-endiamino group as the key structural element that is proposed to induce conformational rigidity. 14The role of 1,3-diaminoethenyl structural unit in enzyme sulfatases activity via a serine modification (prokaryotes) or a cysteine modification (eukaryotes and prokaryotes) is studied also. 15n example of a serine type sulfate is the well characterized bacterial arylsulfatase of Klebsiella pneumoniae. 16,17is paper details the preparation of the (L-Ser-L-Ser) DKP (Scheme 1), followed by oxidation to a stable EE-(L-Ser-L-Ser) DKP enol di-tosylate also referred here as EE-serine di-tosylate, using the dimethyl sulfoxide solution in dimethylformamide as oxidants in the presence of activator paratoluenesulfonyl chloride, i.e. the modified Moffat conditions (Scheme 2).Furthermore, the successful conversion of EE serine di-tosylate by reaction with DABCO to its ZZ isomer (Schemes 3 & 4) is probed by NMR studies and we now present the details of this isomerization reaction.The presented work serves as the first direct example where DABCO facilitates isomerization of the (L-Ser-L-Ser) DKP enol di-tosylate.
We also studied the reaction of DABCO with 4, the enol tosylate prepared from (L-Phe-L-Ser) DKP.Scheme 5, shows the preparation of compound 3, (L-Phe-L-Ser) DKP.Modified Moffat oxidation of 3 produced the E-enol tosylate 4 as shown in Scheme 6.We have previously reported, that compound 4 undergo reaction with 1˚and 2˚ amines to yield products bearing 1,3-diaminoethenyl moiety. 13,18However, when 3 equivalents of DABCO were used as a tertiary amine to react with compound 4 (Scheme 7), it resulted in a unique elimination bis-ylidiene product 5.

Results and Discussion
Scheme 1, shows the preparation of compound 1, (L-Ser-L-Ser) DKP, obtained by passing of a solution of L-Serine methyl ester hydrochloride in methanol through an ion exchange resin column, that was pre-treated with 5% sodium bicarbonate.The desired dimer was obtained in 66% yield.

Scheme 1. Synthesis of (L-Ser-L-Ser) diketopiperazine
The oxidation of the L-Serine dimer (1) was carried out by employing modified Moffat oxidation conditions (Scheme 2) to yield 2 in 31% yield.The dimethyl sulfoxide solution in dimethylformamide was applied as oxidants in the presence of the para-toluenesulfonyl chloride and triethylamine at -5 ºC.

Scheme 2. Synthesis of EE-Serine di-tosylate
The 1 H and 13 C NMR spectra for compound 2 are consistent with the structure of di-tosylate.The 1 H-NMR spectrum showed the characteristic vinyl proton at δ 6.80 ppm, and the amide proton located downfield at δ 10.87 ppm.The stereochemistry at the double bonds was determined through 1D differential NOE experiments.Upon irradiating the amide proton at 10.87 ppm, we observed an NOE signal build up at the vinyl positioned at δ 6.80 ppm.The reciprocal experiment of irradiating the vinyl proton and the corresponding NOE signal build up at amide proton site at δ 10.87 ppm, confirmed and attested to the observation that only the E,E geometry assignment for the compound di-tosylate could give rise to such NOE signal effects.Based on these two correlating NOE signal results, the geometry around the double bond center is confirmed to be E,E.The ESI/MS mass spectrum of 2 contained a molecular ion [M+NH4] + peak at 496 Da.
The isomerization reaction of serine di-tosylate (Scheme 3) was carried out in an NMR tube on a micro scale level.Experimentally, the E,E-2 serine di-tosylate was dissolved in 0.6 mL of DMSO-d6, and 3 equivalents of DABCO was added.The reaction, monitored by means of 1 H-NMR spectroscopy, was complete in less than 2 hours furnishing the isomeric product Z,Z-2.
The 1 H-NMR spectra recorded at times ranging from 5 minutes to 2 hours of reaction run, led to a series of events that helps us to understand the molecular process behind the mechanism of this reaction.At first, upon addition of DABCO, we noticed that within 5 minutes the amide proton at 10.87 ppm for the EE isomer disappeared completely from the 1 H-NMR spectra either by chemical exchange or by deprotonation.Also, we noticed no change in chemical shift of vinyl group observed at δ 6.80 ppm.After the 10 minutes of reaction run, two new signals evolved at 6.59 ppm and 5.58 ppm.The intensity of the two signals appeared similar.Because of the proximity of the 6.59 ppm signal to EE isomer at 6.80 ppm, we assigned this to the E of EZ isomer in the test sample.The other signal which corresponds to the Z of EZ in the test sample was observed at 5.58 ppm.During this period, we observed the evolution of a less intense neighbor signal at 5.85 ppm, which we assign as the ZZ isomer.
With the progress of time (47 min), the intensity of the Z,Z signal at 5.85 ppm continued to grow and the intensity of the signal for the Z of EZ part at 5.58 ppm diminished.Finally, after 110 minutes, we observed complete loss of the EZ component in the test sample.The reaction was complete with ZZ isomer as the sole product with a chemical shift of 5.85 ppm.Thus, we were able to monitor the molecular evolution for this isomeric conversion of EE di-tosylate to ZZ product including the formation of intermediate components and identify the signals for the E and Z features of EZ isomer, using simple 1 H NMR techniques.
Based on the above details of the sequence of 1 H NMR events, the following mechanism of conversion of EE di-tosylate to its ZZ isomer is depicted on Scheme 4. The further NOE experiments concentrated on observation of interaction on NH and CH protons in the ZZ form, as expected, have not shown any NOE correlations.3).The free amino acid methyl ester of L-Phenylalanine was extracted by diethyl ether from a solution of L-phenylalanine methyl ester hydrochloride in 50% potassium carbonate.This freshly prepared L-Phenylalanine methyl ester was immediately dissolved in chloroform, and N-Boc-Serine as well as dicyclohexyl carbodiimide were added.The mixture was stirred at room temperature for 24 hours.Formed dicyclohexyl urea was filtered off, and after the solvent removal protected dipeptide was obtained.

N-Boc-L-Serine
Ion Exchange L-Phenylalanine methyl ester 1 eq.DCC, CHCl 3 24 hr,RT.,stir TFA salt ( 95%) 1. Treatment of the dipeptide with an excess of trifluoroacetic acid in dichloromethane resulted in the deprotection of Boc-group from the dipeptide.The triflouroacetate salt, obtained as a white powder, was next refluxed in methanol, and chloroform; however, these attempts to obtain the diketopiperazine product were unsuccessful.Removal of the triflouroacetate counter ion seems to be critical for the cyclization step.The triflouroacetate salt was dissolved in methanol and passed through an ionexchange column, that was pre-treated with 5% NaHCO3.Subsequent solvent evaporation resulted in a white precipitate.The precipitate was refluxed in chloroform for 24 hours to yield the DKP product -S,S-3-benzyl-6-hydroxymethyl-piperazine-2,5-dione, in 80% yield.This methodology offers simple synthetic procedure with improved yield in comparison to existing DKP literature methods. 19,20e synthesis of the enol tosylate 4 was completed in 80% yield (Scheme 6) using the modified Moffatt conditions as it was mentioned above. 18According to the NOE correlations we have state that enol tosylate 4 adopts an E-stereochemistry of the double bond. 13heme 6. Synthesis of E-enol tosylate of (L-Phe-L-Ser) DKP

L-Phe
The E-enol tosylate 4, dissolved it in a minimum amount of dimethyl sulfoxide and in the presence of 3 equivalents of DABCO at room temperature during 5 days, undergo the transformation which after the quenching with cold water furnish bis-ylidiene 5 as an yellow tinted solid precepitate in 84% yield (Scheme 7).

Scheme 7. Formation of bis-ylidiene product 5
The 1 H, 13 C, and COSY NMR spectra of 5 acquired in DMSO-d6 were consistent with the structure of ylidiene product.In the 1 H NMR spectrum, the signals at 4.95 and 5.30, and 6.75 ppm are assigned to the methylene and the vinyl groups, respectively.The signals of two amide protons are observed at 10.11 and 10.95 ppm.In the 13 C NMR spectrum the characteristic vinyl group carbon appears at 100.97 ppm.The ( 1 H-1 H) COSY showed correlation between proton signals at 4.95 and 5.30 ppm, which confirmed the coupling between the two methylene protons, associated with this ylidiene product.The structure was further confirmed by mass spectrum, which contained a molecular ion peak of 214 Da.

Conclusions
2,5-diketopiperazines (DKP's) such as compounds 1 and 3 as well as corresponding enol tosylates 2 and 4 continue to generate interest in the chemical community through their presence in diverse chemical compounds having potential applications in organic and medicinal chemistry.We have demonstrated herein that, in the presence of DABCO, symmetric EE enol di-tosylate, derived from DKP of (L-Ser-L-Ser), undergo the isomerization reaction to form the ZZ enol di-tosylate.In the same time, the enol tosylate, derived from DKP of (L-Phe-L-Ser), in the presence of DABCO and DMSO undergo an unique transformation to yield bis-ylidiene product 5.Such DKP modifications have potential utility as synthons and can induce a conformational rigidity in peptides.

Materials and Methods
1 H and 13 C NMR spectra were recorded on a JEOL 400 NMR instrument operating at 400 and 100 MHz, respectively.Decoupling 1 H experiments were acquired on BRUKER AM 300 MHz.All spectra were recorded using DMSO-d6 as the NMR solvent.Proton and carbon spectra were referenced to δ 2.50 and 39.50 ppm.Nuclear Overhauser (NOE) experiments were performed after measuring the necessary T1 relaxation for protons under study.Finnigan TSQ instrument with a direct probe insertion module, Mariner ESI/MS, and GC/MS from HP, were used to determine mass measurements.Optical rotations were measured by using either Rudolph Autopol III or Polyscience SR-6 polarimeter.Melting points are obtained using MEL-TEMP capillary melting point apparatus and are uncorrected.All solvents and amines were distilled prior to use and stored under nitrogen.

Preparation of (1), (L-Ser-L-Ser) DKP
A 32 g ion-exchange column bed was made in a regular column chromatography set up.This was pretreated with 200 mL of 5% aqueous sodium bicarbonate, 240 mL of distilled water and 150 mL of methanol.5 g of L-Serine methyl ester hydrochloride was dissolved in 50 mL methanol.This methanolic solution was passed through the treated ion exchange column and additional 200 mL of methanol was used to run the column to collect the free ester.The solvent was evaporated and the resulting oily substance was stored at room temperature for 7 days.A faint yellow colored solid was obtained and this was co washed with 2.5 mL each of ether and methanol and filtered, air dried to get (3.68 g) 66% yield of product cyclo (L-Ser-L-Ser) DKP. 1 H NMR (400 MHz, DMSO-d6): δ = 3.6 (s, 6H), 5.0 (s, 2H), 8.0 (s, 2 NH's) ppm. 13C NMR (100 MHz, DMSO-d6): δ = 56.8,63.4, 166.0 ppm.

Synthesis of (2), di-tosylate of (L-Ser-L-Ser) DKP
In a 10 mł round bottom flask cyclo L-Ser-L-Ser 0.0872 g (1 eq.) was dissolved in 1 mL of dry and distilled dimethyl sulfoxide in hot condition and 0.5 gm of 4 Aº of molecular sieves was added and stored overnight.In a separate 35 mL oven dried round bottom flask that was maintained under nitrogen at -5 ºC, para-toluensulfonyl chloride 0.958 g (2.5 eq.) was added and to this 1:1 (1.25 mL each) of dry dimethyl formamide and dry dimethyl sulfoxide was added to dissolve the solids.To this solution was added the dried DKP alcohol and continuously stirred under nitrogen at -5 ºC.After 18 minutes triethylamine 1130 µL was added and the reaction flask was brought to room temperature and was stirred for additional 1 hour.The reaction mixture was quenched with 25 mL pre cooled ice cold water to allow for the precipitation of the product di-tosylate 0.0568 g, 31% yield. 1 H NMR (400 MHz, DMSO-d6): δ = 2.4 (s, 6H), 7.5 (4H, d, J = 8.04 Hz), 7.9 (4H, d, J = 8.04 Hz), 10.8 (s, 2 NH's) ppm. 13

Isomerization reaction of (2), E,E Serine di-tosylate by DABCO
In a typical micro-scale setup the reaction was done in a NMR tube.The EE di-tosylate (2) was dissolved in 0.6ml of DMSO-d6 in an NMR tube and to this 3 eq. of DABCO was added and monitored using 1 H NMR at room temperature.The reaction of di-tosylate of dimer of L-Serine with DABCO is relatively much faster than in comparison to the enol tosylate made from Phenylalanine.The reaction was completed in less than 2 hours to form the ZZ Serine di-tosylate product isomer as judged by 1 H, and further confirmed by 13 C NMR. 1 H NMR (400 MHz, DMSO-d6): δ = 2.3 (s, 6H), 7.5 (4H, d, J = 7.72 Hz), 7.9 (4H, d, J = 7.68 Hz) ppm. 13

Preparation of the methyl ester of L-Phenylalanine
L-Phenylalanine methyl ester hydrochloride 5.0 g. (25 mmol), was dissolved in 20 mL water.This was treated with a solution of potassium carbonate K2CO3 (5.0 g, in 10 mL water).The mixture was extracted with ether (4 x 25 ml).The ether extracts are pooled and dried over magnesium sulfate and the solvent was evaporated to obtain 3.67 g, (20.11 mmol, 80.4% yield) of methyl ester.Note: The free ester is freshly prepared and used immediately.The free ester can potentially dimerize if allowed to stand for a long time even with storing in refrigerator.

Dipeptide formation
In a 250 mL round bottom flask, 3.6 g (20.11 mmol) of freshly prepared L-phenylalanine methyl ester was added to 50 mL chloroform.This was charged with 4.127 g (20.11 mmol) of N-Boc-L-Serine and (4.12 g, 20.11 mmol) of DCC (dicyclohexyl carbodiimide) and additional 100 mL of chloroform was added.The contents of the flask were stirred for 24 hours during which pronounced formation of white solid is seen.This obtained dicyclohexyl urea (DCU) was carefully filtered and the solvent was evaporated to get the protected dipeptide 10.9 gm by weight as a semi-solid and was used as such for further deprotection.

Deprotection of the Boc-group from the dipeptide.
The above dipeptide was dissolved in 50 mL dichloromethane and to this 25 mL of Trifluoroacetic acid was added in one portion.An additional 70 mL dichloromethane was added and stirred for 24 hours.The progress of the reaction can be monitored by TLC, chloroform: methanol (9:1) for the deprotection of the Boc-group as evidenced by the single spot for the product.Subsequent removal of the solvent yielded a yellow oily substance and this upon triturating with cold ether gave 7.35 g, (94.47%) of the triflouroacetate salt as a white solid.M.pt.= 145 ºC.

4 .
Scheme 4. Mechanism of DABCO reaction with enol di-tosylate of (L-Ser-L-Ser) DKP Scheme 5, details the synthesis of (3).The free amino acid methyl ester of L-Phenylalanine was extracted by diethyl ether from a solution of L-phenylalanine methyl ester hydrochloride in 50% potassium carbonate.This freshly prepared L-Phenylalanine methyl ester was immediately dissolved in chloroform, and N-Boc-Serine as well as dicyclohexyl carbodiimide were added.The mixture was stirred at room temperature for 24 hours.Formed dicyclohexyl urea was filtered off, and after the solvent removal protected dipeptide was obtained.