Synthesis of 2-(4,6-Dimethoxy-1,3,5-triazin-2-yloxyimino) Derivatives: Application in Solution Peptide Synthesis

A new class of 1,3,5-triazinyloxyimino derivatives were prepared, characterized and tested for reactivity in solution peptide synthesis. The new triazinyloxyimino derivatives failed to activate the carboxyl group during formation of peptide bonds, but gave the corresponding N-triazinyl amino acid derivatives as a major product. The oxyma (ethyl 2-cyano-2-(hydroxyimino)acetate) uronium salt was superior to other uronium salts in terms of racemization, while 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT, 9) gave the best results.

Despite their widespread use, these reagents are not without ptoblems. The explosive properties of 1-hydroxybenzotriazoles are often not referenced in literature. Sometime, Material Safety Data Sheets warn that 1-hydroxybenzotriazoles may be unstable, with a relatively high sensitivity to friction and sparks, but in most cases, there is no mention of how sensitive such substances are to heating under confinement and no warning is given with respect to their ability to propagate a deflagration or a detonation.
More recently a new class of coupling reagents such as 7 and 8 ( Figure 2) based on changes to the carbon skeleton structure [34][35][36] gave more marked increases in coupling efficiency, as well as reduced racemization levels during the coupling step. Particularly noteworthy are the morpholino derivatives 8, because the oxygen in the carbon skeleton increases the solubility as well as the reactivity, which reduces the racemization during the coupling step [34][35][36]. 8a, X = CH 8b, X = N O 6a, X = CH 6b, X = N Although 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT, 9) has been successfully applied for the preparation of peptides, the mechanism of its participation in coupling reactions remains unknown [37,38]. Furthermore, in order to be successfully stored the reagent itself must be of high purity, the formation of gaseous products may generate a rapid pressure increase in the container, and accordingly, precautions should be taken to avoid the serious risk of blowout of toxic gases.
Successful activation of carboxylic acids by means of CDMT (9) confirmed the feasibility of a multistep process proceeding via triazinylammonium salts such as 10 and (DMTMM, 11) ( Figure 3) formed in situ in the presence of the appropriate amine [37,38].

Scheme 1. Synthesis of triazinyloxyimino derivatives.
The uronium salt coupling reagents were prepared by reaction of the chloro salts DCMH 14a or TCFH 14b with the oxime derivatives in the presence of triethylamine in DCM or with the potassium salt of the oxime derivatives in acetonitrile to give the oxyimino uronium base coupling reagents 15a-f (Scheme 2). To investigate the retention of configuration induced for the new coupling reagents, several previously studied model peptide system 16a,b and 17a-c ( Figure 4) were examined [36]. These models involve stepwise coupling and (2+1) segment coupling as well. Results comparing the coupling of Z-Phe-OH to H-Ala-OMe.HCl (18a) with different coupling agents to afford the Z-Phe-Ala-OMe (16a) are indicated in Table 1. All the uronium type coupling reagents (HOTU, 15a), (HTOPC, 15e), and (HTOPT, 15f), used gave less than 1% racemization according to the NMR data but CDMT (9) gave about 15% of the racemized product (Table 1). The other triazinyloxyimino derivative (DMTOC, 12a) failed to give the expected product Z-Phe-Ala-OMe (16a), due to the low activation for the carboxylic group and fast attack by the N-terminal of the amino acid which led to formation of the N-triazinyl amino acid derivative methyl 2-(4,6dimethoxy-1,3,5-triazin-2-ylamino)propanoate (19a) in 77-85% yield, as confirmed by NMR data (Scheme 3).
In the more sensitive cases of the formation of Z-Gly-Phe-Val-OMe (17b) and Z-Gly-Val-Val-OMe (17c) the same results were obtained. The best results obtained were when the Oxyma derivative (HOTU, 15a) was using as a coupling reagent, while CDMT (9) gave the highest racemization level and the triazine coupling reagents failed in the formation of the target product, giving the same side product 19b, which was obtained from the coupling of Z-Phe-OH with H-Val-OMe.HCl (18b) as observed from the HPLC and NMR data (Table 4 and 5). Table 4. Yield (%) and Racemization of (%) Z-Gly-Phe-Val-OMe (17b) using the Uronium Salts 15a,e,f and CDMT (9)

General
All chemicals were used without further purification. Solvents are redistilled before use. Nprotected amino acids and esters were purchased from Novabiochem. Melting points are uncorrected and were determined on a Gallenkamp hot stage. A Perkin Elmer Spectrum 1000 FT-IR Spectrometer was used for recording infrared (IR) spectra of the prepared compounds as KBr pellets or in spectroscopic grade dichloromethane. 1 H-and 13 C-NMR spectra of compounds were run on JEOL 400 MHz NMR spectrometer, in CD 3 COCD 3 , CDCl 3 or DMSO-d 6 at room temperature using TMS as internal standard. The instruments are located at King Saud University, College of Science, Chemistry Department. For analytical separations, characterization and determination of racemization, a reverse-phase Waters 2695 HPLC separation module was used (Barcelona Science Park, University of Barcelona, Spain) equipped with a SunFire C 18 column (Waters, 5 m, 4.6 × 150 mm), 10-90% linear gradient of 0.036% TFA in CH 3 CN/0.045% TFA in H 2 O and coupled to a Waters 2998 PDA-UV detector. Chromatograms were processed with Empower software. In the detection of the percent racemization as indicated by authentic samples. Peptide mass was detected by means of an HPLC-PDA system as described above, coupled to a Water Micromass ZQ mass detector, using the MassLynx 4.1 software. Elemental analyses were performed on a Perkin-Elmer 2400 elemental analyzer, and the values found were within ±0.3% of the theoretical values. Oxime derivatives 13a-c were prepared following the literature procedures [47,48].

General Synthesis of Triazine Coupling Reagents 12a-d from Oxime Derivatives
2-Chloro-4,6-dimethoxy-1,3,5-triazine (9, 20 mmol) was added to a solution of oxime (20 mmol) and triethylamine (20 mmol) in dichloromethane (DCM, 200 mL) at 0 ºC. The reaction mixture was stirred at the same temperature for 1 h and then at room temperature for 5-8 h. DCM (400 mL) was added, and then the reaction mixture was washed twice with saturated aqueous NaCl (200 mL each). Finally, the organic solvent was dried with anhydrous Na 2 SO 4 , filtered, and the solvent was removed under reduced pressure. The crude product was recrystallized from DCM/hexane.   (DMTOPy, 12d). The product was obtained as a light tan powder, yield 4.

Synthesis of 16a,b (1+1)
The coupling reagents 15a,e,f (0.25 mmol) were added to a mixture of Z-Phe-OH (0.25 mmol), the appropriate amino acid 18a,b (0.25 mmol) and N-methylmorpholine (0.75 mmol), in dry acetonitrile (5 mL) at 0 ºC. The reaction mixture was stirred at the same temperature for 1 h and then at room temperature for 2 h. Ethyl acetate (EtOAc, 50 mL) was added and the mixture was subsequently washed with 10% aqueous HCl (v/v), saturated aqueous Na 2 CO 3 and saturated aqueous NaCl solution (2 × 10 mL each). Finally, the organic solvent was dried with anhydrous Na 2 SO 4 , filtered, and the solvent was removed under reduced pressure. The gummy residue obtained 16a,b (DL < 1%) were dried under vacuum. Both products were obtaind when the coupling chlorotriazine derivative 9 was used (15% racemization). Using the triazine coupling reagent 12a,c,d instead of 15a,e,f and following the different procedure whereby Z-Phe-OH was activated with 12a,c,d for 1 hour at room temperature and then added to a solution of 18a,b in the presence of NMM as a base in acetonitrile, the reaction mixture was stirred at the same temperature for 24 h gave compounds 19a,b.

General Method for Synthesis of 17a-c (2+1) Using Different Coupling Reagents
The coupling reagents 15a,e,f (0.25 mmol) were added to a mixture of Z-Gly-Phe-OH (0.25 mmol), 18a,b (0.25 mmol) and N-methylmorpholine (0.75 mmol), in dry ACN (5 mL) at 0 ºC. The reaction mixture was stirred at the same temperature for 1 h and then at room temperature for 24 h. Ethyl acetate (EtOAc, 50 mL) was added and the mixture was subsequently washed with 10% aqueous HCl (v/v), saturated aqueous Na 2 CO 3 and saturated aqueous NaCl solution (2 × 10 mL each). Finally, the organic solvent was dried with anhydrous Na 2 SO 4 , filtered, and the solvent was removed under reduced pressure. The gummy residue obtained 17a,b (DL < 1% in the case of using 15a) was dried under vacuum. Both products were obtained when the coupling chlorotriazine 9 was used. Using the triazine coupling reagent 12a,c,d instead of 15a,e,f and following the different procedure whereby Z-Gly-Phe-OH was activated with 12a,c,d for 1 h at 0 ºC and then added to a solution of 18a,b in the presence of NMM as a base in acetonitrile, the reaction mixture was stirred at the same temperature for 1 h, then at room temperature for 24 h gave compounds 19a,b. Z-Gly-Val-Val-OMe (17c). Prepared following the same procedure above for 17a,b by replacing Z-Gly-Phe-OH with Z-Gly-Val-OH, and the amino acid 18b was used. The gummy residue of 17c obtained when the coupling reagents 15a,e,f (DL < 0.1% in the case of using 15a) was dried under vacuum. The product was obtained (47.3% racemization) when the coupling chlorotriazine 9 was used. Using the triazine coupling reagent 12a,c,d instead of 15a,e,f and following the different procedure whereby Z-Gly-Val-OH was activated with 12a,c,d for 1 hour at 0 ºC and then added to a solution of 18b in the presence of NMM as a base in acetonitrile, the reaction mixture was stirred at room temperature for 24 h to give compound 19b.

Conclusions
In conclusion, herein a new family of 1,3,5-triazinyloxyimino derivatives 12a-d was compared with uronium salts. The morpholino and tetramethyluronium salts are more reactive than triazinyloxyimino derivatives for activation of the carboxylic group and formation of peptide bonds. The new triazinyloxyimino derivatives 12a-d failed to give the expected products, due to the low activation for the carboxylic group and fast attack by the N-terminal of the amino acid, which led to formation of the corresponding N-triazinyl amnio acid derivatives as confirmed by NMR and HPLC. Oxyma (ethyl 2-cyano-2-(hydroxyimino)acetate) uronium salts were confirmed to be superior to other oxime derivatives, HOAt (4) and HOBt (1), in terms of both coupling yield and retention of configuration. Finally, the new triazinyloxyimino derivatives may be useful in the preparation of biologically active N-triazinyl amino acids as well as N-triazinyl peptides which is now going to be tested in our laboratory.