Oxime-Based Carbonates as Useful Reagents for Both N-Protection and Peptide Coupling

We have demonstrated that oxime-based mixed carbonates are very effective reagents for both N-protection and peptide coupling.


Introduction
Peptides are increasingly gaining recognition as potential bioactive ingredients in the pharmaceutical industry [1][2][3]. Peptide synthesis depends on the strategies used for protecting the α-amino group and for activating the carboxylic acid group prior to peptide coupling. The two main classes [4][5][6] of carboxylic acid group activation methods are: (i) those that require in situ activation of the carboxylic OPEN ACCESS acid and (ii) those that require an activated species that has previously been prepared (usually from an in situ activation step), isolated, purified, and characterized.
The amino group is most commonly protected by preparing the corresponding carbamate derivative. Despite the vast number of reagents reported to date for introducing the protecting group into the N-terminal amino group, there is still no universally active species capable of providing optimal protecting group introduction.
Later, we reported a series of Fmoc/Alloc-oxime carbonate reagents which are easy to prepare, stable, and highly reactive crystalline materials that afford nearly pure Fmoc/Alloc-amino acids in high yields. Among the Fmoc-oxime carbonates that we evaluated for the preparation of Fmoc/Alloc-Gly-OH, the N-hydroxypicolinimidoyl cyanide derivative 9 ( Figure 1) gave the best results [35]. More recently, our research group reported the cyanoacetamide-based oximes 10 (Figure 1), which show unusual ability to afford Fmoc-protected amino acids in high yield, high purity and at lower cost relative to compound 9 [36]. Herein, we extended our studies for the synthesis of a new family of carbonate derivatives based on OxymaPure ® , which are easy to prepare, stable, and have shown high efficiency in N-protection as well as peptide coupling.

Preparation of 4-(Ethoxycarbonylamino)benzoic Acid
To study the reactivity of prepared carbonate derivatives 13a-f and their utility for the preparation of the N-protected amino acids, we initiated our studies with 4-aminobenzoic acid (20), which on treatment with the previously synthesized carbonate derivatives 13a-f in a homogenous acetone/aqueous solvent mixture in the presence of sodium carbonate with stirring overnight at room temperature, provides the product 21. Samples of 4-(ethoxycarbonylamino)benzoic acid (21) were obtained from the different carbonate derivatives after removing the unreacted starting carbonate by extracting with ether and acidifying the aqueous layer with 1N HCl (Scheme 4). The purity of the product 21 was determined after injection onto reverse-phase HPLC are shown in Table 3.     [4,5-b]pyridin-3-yl ethyl carbonate (13e) provided high purity (95.3% and 97.0%, respectively) and moderate yield (43% and 31%), while, ethyl 2-oxopyridin-1(2H)-yl carbonate (13c) provided the highest yield (59%) with excellent purity (100%) as indicated from the HPLC traces.

HPLC Study of the Rate of Formation of the Active Ester
Before attempting simultaneous protection and activation of amino acids, we tried to prepare active esters of N-protected amino acids using the carbonate derivatives 13a, 17, 18, and 19 to ensure that these compounds can activate carboxylic acids by forming the corresponding active ester for different Fmoc-amino acids. The reaction of Fmoc-amino acids with oxime carbonate derivatives was monitored by HPLC to study the rate of formation of the active ester. Aliquots (5 µL) of the reaction mixture were taken, diluted with acetonitrile, and then analyzed by HPLC. Follow-up samples were studied at intervals of time 30 min and 1, 2, 4 and 24 h pre-activation. This enabled us to determine the optimum pre-activation time for each carbonate reagent, as excessively long times could lead to greater formation of alkyl or aryl esters.     Due to the best results obtained from the previous example with the oxime carbonate derivatives 17 and 18, Fmoc-Phe-OH 23 was tested with i BuocOXY 17 and AllocOXY 18 under the same conditions used in the previous example. From the results obtained from HPLC monitoring, the maximum levels of the active ester 27 are formed from the oxime carbonate derivatives 17 and 18 at 1 and 2 h, respectively; while the alkyl esters 26 appeared after 30 min and 1 hour, respectively. Thus, the pre-activation time in both systems should not exceed more than 30 min (Tables 8 and 9).  To ensure that we will get the same results with the oxime carbonate derivatives 17 and 18, further study was performed with the more sterically hindered amino acid Fmoc-Pro-OH 24 using the two carbonate derivatives i BuocOXY 17 and AllocOXY 18. From the results obtained by HPLC monitoring, we observed the maximum levels of the active ester 27 after 30 min and 2 h, respectively; while the alkyl esters 26 appeared after 30 min and 1 hour, respectively. Thus, the pre-activation time in both systems should not exceed more than 30 min, which in agreement with the previous results (Tables 10 and 11).  The results obtained from Tables 4-11 may indicate that, the activation of carboxylic acid group of the amino acid using oximinocarbonate derivatives may proceed through a mixed anhydride-type intermediate 25, which may react with the oxyma anion to afford the active ester 27 (Scheme 5). Its decarboxylation will afford alkyl or aryl esters 26, which is a rather slow step. All intermediate stages of the reaction are run at low temperature to prevent side reactions.

Scheme 5.
Mechanism of the formation of the oxime active ester using oxyme carbonates.

Synthesis of Dipeptide Fmoc-Val-Ala-OMe
As an initial model to examine the reactivity of the oxime carbonate 13a, 17, 18 and 19 as coupling reagents, these reagents were examined in the stepwise coupling of a previously studied model system [29] Fmoc-Val-Ala-OMe 28. The pre-activation time was determined for each of these coupling reagents according to the previous studies obtained from HPLC for the rate of formation of active ester. All coupling reactions were performed in the presence of 2 equiv. pyridine as a base and in DMF as solvent. The results for each coupling reagent are given in Table 12. The purity of the dipeptide 28 was determined by HPLC, and was found to be 96.1 to 100% at t R LL [Fmoc-Val-Ala-OMe] = 8.32 min. From Table 12, the highest yield and purity was obtained with EtocOXY 13a, while the lowest yield and purity was obtained with i BuocOXY 17 and ZOXY 19. While, the AllocOXY 18 had moderate yield and purity. None of the D,L-isomer was detected by HPLC or NMR spectra in all the cases, but the impurities were related to the alkyl ester and oxime.

Materials
The solvents used were of HPLC reagent grade. Melting points were determined with a Mel-Temp apparatus and are uncorrected. Infrared (IR) spectra were recorded on a Perkin-Elmer 1600 series Fourier transform instrument as KBr pellets. Nuclear Magnetic resonance spectra ( 1 H-NMR and 13 C-NMR spectra) were recorded on a JOEL 500 MHz and on a Mercury 400 MHz spectrometer with chemical shift values reported in δ units (ppm) relative to an internal standard. Elemental analyses were performed on Perkin-Elmer 2400 elemental analyzer, and the values found were within ±0.3% of the theoretical values. Follow-up of the reactions and checks of the purity of the compounds was done by TLC on silica gel-protected aluminum sheets (Type 60 GF254, Merck, Barcelona, Spain) and the spots were detected by exposure to UV-lamp at λ 254 nm for a few seconds. The compounds were named using ChemDraw Ultra version 11, CambridgeSoft Corporation (Cambridge, MA, USA).

General Method for Preparation of Ethyloxycarbonyl Derivatives 13(a-d, f)
A solution of ethyloxycarbonyl chloride (11, 0.95 mL, 10 mmol) in CH 2 Cl 2 (30 mL) was added slowly to a solution of sodium carbonate (2.12 g, 20 mmol) and 10 mmol of oximes (12a, 12b), 1-hydroxypyridin-2(1H)-one (12c), or benzotriazole derivatives (12d or 12f) in H 2 O (20 mL) with stirring at 0 °C. The resulting clear mixture was stirred at 0°C for 30 min and then at room temperature for 2 h. After dilution with CH 2 Cl 2 (50 mL), the organic phase was collected and washed with water and saturated aqueous NaCl (30 mL), dried over anhydrous Na 2 SO 4 and then filtered, and the solvent was then removed on a rotary evaporator. The residue was recrystallized from CH 2 Cl 2 /hexane to give the ethyloxycarbonyl derivatives 13(a-d, f) [1,2,3]

General Method for Preparation of Oxime Carbonate Derivatives 17-19
A solution of chloroformate (10 mmol) [isobutyloxycarbonyl chloride (14), allyloxycarbonyl chloride (15) or benzyloxycarbonyl chloride (16)] in CH 2 Cl 2 (30 mL) was added slowly to a solution (10 mmol) of oxima 12a and sodium carbonate (2.12 g, 20 mmol) in H 2 O (20 mL) with stirring at 0 °C. The resulting clear mixture was stirred at 0 °C for 30 min and then at room temperature for 2 h. After dilution with CH 2 Cl 2 (50 mL), the organic phase was collected, washed with water and saturated aqueous NaCl (30 mL), and then dried over anhydrous MgSO 4 . It was then filtered, and the solvent was removed with a rotary evaporator. The residue was recrystallized from CH 2 Cl 2 /hexane to give oxime carbonate derivatives 17-19. (17). The product was obtained as a white solid  (19). The product was obtained as white crystals (2.76 (21) A solution of ethyloxycarbonyl derivative 13(a-f) (1 mmol) in acetone (10 mL) was added dropwise to a stirring solution of 4-aminobenzoic acid 20 (0.14 g, 1 mmol) and sodium carbonate (0.32 g, 3 mmol) in acetone (20 mL) and H 2 O (10 mL). After stirring overnight, the reaction mixture was concentrated under reduced pressure, and then extracted with CH 2 Cl 2 (20 mL) to remove the unreacted ethyloxycarbonyl derivatives. The reaction mixture was acidified with 1 N HCl (detected with Congo red litmus paper) to give a white solid, which was filtered, washed with water several times, dried and then recrystallized (ethyl acetate/n-hexane) to give a white solid. The purity of 21 was determined by reverse-phase HPLC. Conditions: detection at 220 nm (Waters 996 PDA detector); Sunfire C 18 column (3.5 µm 4.6 × 100 mm); linear gradient over 14 min (10 to 100% CH 3

General Method for the Synthesis of Dipeptide Fmoc-Val-Ala-OMe 28
A solution of Fmoc-Val-OH 22 (0.339 g, 1 mmol) and the appropriate coupling reagent (1 mmol) in DMF (2 mL) was cooled to 0 °C and treated dropwise with pyridine (0.088 mL, 1.1 mmol). The reaction mixture was stirred for pre-activation at different times, depending on the conditions of the entry studied, and then treated with a solution of H-Ala-OMe.HCl (0.139 g, 1 mmol) and pyridine (0.088 mL, 1.1 mmol) in DMF (1 mL). The reaction mixture was stirred overnight. After dilution with 25 mL of ethyl acetate, the organic phase was washed with 5% citric acid (3 × 15 mL), saturated aq. NaHCO 3 (3 × 15 mL) and saturated aq. NaCl (3 × 15 mL), and then dried over anhydrous Na 2 SO 4 and filtered. The solvent was removed with a rotary evaporator, and the residue was recrystallized from CH 2 Cl 2 /hexane to give the dipeptide Fmoc-Val-Ala-OMe 28. The purity of 28 was by reverse-phase HPLC. Conditions: detection at 220 nm (Agilent 1200 PDA detector); Eclipse plus C 18 column (3.5 µm 4.6 × 100 mm); linear gradient over 14 min (10 to 100% CH 3

Conclusions
Protection of the amino group and activation of the carboxylic acid groups are the most important steps associated with peptide synthesis. A possible strategy is to use oxime carbonate derivatives to simultaneously protect the amino group as a carbamate derivative and activate the carboxylic acid group as an active oxime ester was performed. A detailed study is carried out to understand the scope and limitations of this method using different oxime carbonate derivatives. The efficiency of these derivatives depends on the nature of oxime carbonates and also on the nature of the amino acids. From our studies we determined that the new family of oximes are useful reagents for both N-protection and activation of the protected amino acid. As a final conclusion from our studies, the i BuocOXY compound 17 and AllocOXY compound 18 both give the best results for formation of the active ester with less alkyl ester formation, while the EtocOXY compund 13a gave the best results for the coupling step. The ZOXY reagent 19 might be not useful in either the activation or coupling steps.