Microwave Assisted Synthesis Of Protected Homoserine Lactones Biology Essay

Published:

This essay has been submitted by a student. This is not an example of the work written by our professional essay writers.

In response to fluctuations in cell-population density, many bacteria regulate a diverse array of physiological activities by a process called quorum sensing. Quorum sensing bacteria release autoinducers to modify gene expression that leads to alteration in processes such as competence, conjugation, antibiotic production and biofilm formation. In general, Gram-negative bacteria (i.e., Pseudomonas aeruginosa) use N-acylated homoserine-γ-lactones (1, PG=acylated chain) as autoinducers [1]. Cystic Fibrosis (CF) is the most common autosomal recessive disorder of caucasian populations which refers to the characteristic fibrosis and cyst formation within the pancreas. Difficulty in breathing is the most common symptom which results from lung infections that are treated with, though not cured by, antibiotics and other medications. Although, most CF patients survive into adulthood through the extensive use of antibiotics, majority of them succumb to respiratory failure brought on by chronic bacterial infection from Pseudomonas aeruginosa and Burkholderia cepacia. Because of the rise of antibiotic resistant strains of bacterium commonly found in CF patients, alternative methods are now sought for the treatment of CF and other diseases which needs the use of antibiotics for the treatment [2b]. One such alternative therapy which uses chemicals that interrupt or destroy quorum sensing signals is called signal interference or quorum quenching. Recent research has shown that bacteria's ability to communicate can be disrupted and thereby disable or diminish the bacteria's ability to become pathogenic [2c]. The body is therefore not compromised by cell damage, inflammation, toxicity or other detrimental effects of the bacteria. This gives the body time to eradicate the bacteria naturally through normal immune system functions [2d]. It seems plausible that such a novel approach to the treatment of bacterial infections would compliments or replaces the use of antibiotics.

N-Protected homoserine-γ-lactones (1) are also known for their biological activities against serine proteinases as well as thiol-containing enzymes [3]. Further, N-protected homoserine-γ-lactones (1) have been used in numerous syntheses of organic natural products and drugs [4, 5]. They also serve as a precursor for a number of synthetically important compounds such as homoserine derivatives (2) [6], 2-amino-4-halo-butanoic acid derivatives (3) [7] and vinyl glycine derivatives (4) [8]. (Scheme 1)

Scheme 1 Common organic reactions of N-protected-L-homoserine-γ-lactones

Despite their proven value, N-protected homoserine-γ-lactones (1) are very expensive or not even available in their optically pure form. For example, the cost of N-benzyloxycarbonyl-L-homoserine-γ-lactone (1b) is about $ 150/g while the cost of optically impure N-tert-butylcarbonyl-homoserine-γ-lactone (1a) is about $ 102/g [9]. Other N-protected homoserine-γ-lactones (1) are not commercially available from any major chemical supplier. Several procedures exist for the synthesis of N-protected homoserine-γ-lactones (1) but their commercial scarcity may be due to the lack of a common or inexpensive synthetic protocol. N-Protected homoserine-γ-lactones (1) can be obtained from the N-protection of homoserine-γ-lactones (5) under basic conditions [10], or from homoserine (6) via N-protection followed by the cyclization to the lactone [11] (Scheme 2). The drawback in these synthetic schemes is the high cost of L-homoserine-γ-lactone (5) and L-homoserine [9 sigma]. Alternative methods use comparatively cheaper starting materials such as L-methionine (7) and L-aspartic acid (8) {9 Sigma }. For example, N-protected-L-homoserine-γ-lactones (1) can be synthesized by N-protection of L-methionine (7) followed by refluxing with an alkyl iodide under acidic conditions [12a,d] or by converting it to L-homoserine followed by N-protection and cyclization [12b]. While the synthesis from L-methionine can be done in high yield and in few steps, the amino protecting group choices in these methods are not diverse; for example, difficulties were observed in the synthesis of N-tert-butoxycarbonyl-L-homoserine-γ-lactone [14a] and there are concerns about possible racemization at the reflux temperature [14b]. Alternatively, N-protected-L-homoserine-γ-lactones (1) can also be synthesized in a multistep procedure starting with selective esterification of L-aspartic acid (8) followed by N-protection, precipitation of the dicyclohexylammonium salts, selective reduction with LiBH4, and cyclization to the corresponding-γ-lactones during isolation [13]. Isolated examples of syntheses of N-protected-L-homoserine-γ-lactones (1) from L-aspartic acid based derivatives (9-12) also exist in the literature; however, these reports did not provide a collective study and appear to be protecting group specific methods [method A/B/C- ref15, 16, 17]. For example, the synthetic protocol from 12 to 1 proceeded only if PG=trifluoroacetyl, otherwise reduction always led to undesired compound 13 [19]. Most of these methods also require additional steps and use of undesirable reagents [method b- ref18] (Scheme 3). For instance, synthetic protocol from 11 to 1 [18], requires the use of thionyl chloride which is not only environmentally damaging but also highly regulated and not easily available. Also, use of metal catalysis in this protocol is undesirable in late stages of a drug synthesis [ref, find which says so].

Scheme 2 Comparison of different possible route to synthesize 1. Scheme 3. Synthetic routes to 1 from L-aspartic acid based compounds

In light of the above mentioned difficulties, we report an economical and facile way to synthesize the N-protected-L-homoserine-γ-lactones (1) in a three step process from L-aspartic acid (8). Our method involves N-protection of L‐aspartic acid (8) under basic conditions [20], followed by the acid catalyzed condensation with paraformaldehyde to yield N-protected-5-oxazolidinone-L-aspartic acid (11) (Scheme 4) [21]. While some of the N-protected-5-oxazolidinone-L-aspartic acids (11a-b,d) could be synthesized in moderate to good yields by refluxing a mixture of 14, paraformaldehyde and p-toluenesulfonic acid (p-TsOH) in toluene or benzene, similar conditions resulted in low yield of 11c or did not produce expected N-acyl or N-nosyl (N-2-nitrobenzenesulfonyl) oxazolidinone compounds (11d, f). Several attempts to obtain 11f by varying equivalents of p-TsOH (0.06-1.1 eq.) and paraformaldehyde (1-4 eq.), reaction time (1 hr- overnight), solvents (benzene, toluene, THF, DMF, acetic anhydride/acetic acid) or temperature (60-100 °C or refluxing) using conventional heating were unsuccessful. The 2-nitrobenzenesulfonyl (nosyl) group, introduced by Fukuyama [31], is important because it can be removed under very mild conditions with thiolates. N-Fmoc protection is also important from a synthetic perspective whereas N-acyl protected compounds are significant for biological studies as mentioned earlier. Following a lead by Tantry et.al. [21g] where an unmodified domestic microwave was used for the synthesis of related oxazolidinone derivatives, we attempted to use a 2.45 GHz CW (continuous wave) microwave reactor for the synthesis of the desired oxazolidinones. Microwave heating (300 W, 80-105 °C, benzene or toluene) considerably shortened the reaction time to 5-15 minutes and resulted in consistently good yields of 11a-f. The best reaction conditions standardized for the synthesis of 11a-f are described in the experimental section. Oxazolidinones 11d and 11f were new compounds while full characterization data was not reported for 11c and 11e. 1H and 13C NMR data for 11a also did not completely match with the reported data. A considerable broadening was observed for the -CH2COOH protons in the 1H NMR spectra of 11a and 11c, probably due to the presence of rotamers.26a Structures of 11a and 11c were confirmed by collecting 1H NMR spectra at higher temperatures (See Supporting Information no). N-Acyl-5-oxazolidinone-L-aspartic acid (11d) was obtained as a mixture of two rotamers at room temperature as evidenced by 1H and 13C NMR spectrum which corresponds to two sets of peaks at 295 K. These two set of peaks (ratio 1/2.5) starting to merge as 1H NMR spectra were collected at higher temperature and finally, merged completely when the temperature reached 67 °C in d5-pyridine (See Supporting Information no). This phenomenon is not surprising, since the presence of rotamers in similar N-acyl-5-oxazolidinone compounds have been reported previously.26b

Scheme 4. Synthesis of N-Protected-5-oxazolidinone-L-aspartic acid (11). (i) Solvent, base, PG-Cl or PG-anhydride (ii) Solvent, PTSA, paraformaldehyde. (a 14a,d were commercially available)

It is known that the N-protected-L‐aspartic acid derivatives (14) can be selectively reduced [22], therefore, it was anticipated that selective reduction of the COOH side chain of 11 can be similarly achieved. The N-Cbz-5-oxazolidinone-L-aspartic acid (11b) was chosen for initial experiments due to its higher yields and stability. Treatment of 11b with N-methylmorpholine and methyl chloroformate in dry THF at -15 °C led to the in situ formation of a mixed anhydride (15b, PG=Cbz) and was followed by the addition of NaBH4 to reduce the side chain and form N-Cbz-5-oxazolidinone-L-homoserine (16b, PG=Cbz). The reaction was quenched with MeOH and an acid (dil. HCl or CH3COOH) to obtain 16b in 61% yield. In an attempt to improve this yield, isobutyl chloroformate was used as an alternative to methyl chloroformate [23], which led to the synthesis of 16b in an improved yield of 71% (Scheme 5). N-Cbz-5-oxazolidinone-L-homoserine (16b) was further treated with 1.0 M citric acid and was refluxed for 1 hr, which led to the synthesis of 1b in 55% yield (Scheme 5). The yield of 1b was increased to 70% by subjecting 16b to the next step without any workup and by increasing the reaction time to 2-4 h. It was observed that increasing reaction time (20 h) or using HCl (1 M) instead of citric acid (1M) or the use of LiBH4 instead of NaBH4 as the reducing agent, all resulted in lower yields. N-Tosyl- and N-nosyl-L-homoserine-γ-lactone (1e, f) were also obtained in 68% and 70% yield respectively by using a similar protocol. However, this method failed to produce any significant quantities of 1a, c-d, presumably because of the acid sensitivity of the starting substrates (11a,c-d) under refluxing temperatures [29].

To determine at what stage the problem is occurring, the reaction of 11c was stopped after reduction step and 16c was obtained in 82% yield. However, 16c further decomposes during the cyclization step under conventional heating conditions. When microwave irradiation (300 W, 80 °C) was applied to 16c in 1M citric acid solution, it readily converted to 1c within 10 min. Lactone 1c could be obtained in 80% yield when 16c was subjected to MW conditions without workup or purification at that stage and 1d was similarly obtained in 84% yield using this protocol, while 1b and 1e-f were obtained in moderate yields.

Scheme 5. Synthesis of N-protected-L-homoserine-γ-lactones. (i) NMM (1.2 eq.) / IBCF (1.2 eq.) in THF, NaBH4 followed by MeOH (ii) 1M Citric acid, Method A: refluxing; Method B: MW, 300 W, 5-15 min., 80-100 °C.

The configuration of the stereocenter of 1a-e was assigned to be S by using a variety of methods. Optical rotation values for 1b, 1d and 1e correlated well with literature values [24]. To confirm the optical purity of 1c, racemic 1c was obtained (Scheme 5). HLPC analysis of racemic 1c on a CHIRACEL-OJ chiral HPLC column revealed two peaks while only one peak was observed for 1c, confirming the optical purity of 1c.

Scheme 6. Synthesis of racemic N-protected-L-homoserine-γ-lactones.

The absolute configuration of previously unknown 1f was determined to be S with the help of single crystal X-ray analysis (Figure 1) [30].

Figure 1. X-ray structure of compound 1f. (ORTEP diagram with 50% probability, H atoms have been deleted except at C2 position)

In summary, we have developed a new and facile method for the synthesis of N-protected-L-homoserine-γ-lactones. This method is an advancement of the previous methods and uses cheap starting material to produce optically pure N-protected-L-homoserine-γ-lactones in moderate to high yields. Use of MW conditions also provides an innovative synthesis with less reaction time and less solvent with an eye on green synthesis.

Experimental:-

N-Protected-L-aspartic acid derivatives (14a,d) were purchased from Sigma Aldrich. Compounds 14b and 14c were synthesized using reported protocols [20]. M.P., IR, 1H NMR, 13C NMR and optical rotation for 14b and 14c matched literature reports [20].

Synthesis of (2S)-N-Tosylaspartic acid (14e) synthesized following the procedure from ref [20h]. yield= 92%, Mp. 112-114°C (lit. 114-116°C,20h 112-114°C20i); : +12.3° (c = 4.0, MeOH) (lit. +12.1° (c = 4.0, MeOH)20i); 1H NMR (MeOH-d4) δ: 8.13-8.10 (m, 1H), 7.91-7.88 (m, 1H), 7.80-7.77 (m, 2H), 4.42 (t, J = 5.4 Hz, 1H), 2.85 (m, 2H); 13C NMR (MeOH-d4) δ: ; IR (neat, ν max): 3500-2500, 3302, 1716, 1595, 1405, 1341, 1305, 1242, 1212, 1186, 1163, 1126, 1089, 1041, 1018, 950, 911 (cm-1).

Synthesis of (2S)-N-Nosylaspartic acid (14f) To a solution of L-aspartic acid (2.0 g, 15.0 mmol) in aq. NaOH solution (16.0 mL, 1.20 g, 30.0 mmol) at 0°C was added Ns-Cl (3.66 g, 16.5 mmol), (i-Pr)2EtN (2.87 mL, 16.5 mmol) and acetone (16.0 mL). After stirring for 10 min, the mixture changed from cloudy to clear yellow and was left to stir at RT for 18 hrs. The mixture was then diluted with water and the aqueous layer was washed with diethyl ether (3 x 10 mL). The aqueous layer was then acidified to pH 1 with 1 M HCl and was extracted with EtOAc (3 x 10 mL). The organic layers were then combined, dried over Na2SO4, filtered and concentrated under reduced pressure. The Ns protected aspartic acid, 14f, (3.18 g, 83%) was obtained as yellow oil after purification by flash chromatography on silica gel (70% EtOAc/n-hexane). Recrystallization from EtOAc gave 14f as pale yellow crystals. Mp. 182-184°C; : -92.1° (c = 1, MeOH); 1H NMR (MeOH-d4) δ: 8.13-8.10 (m, 1H), 7.91-7.88 (m, 1H), 7.80-7.77 (m, 2H), 4.42 (t, J = 5.4 Hz, 1H), 2.85 (m, 2H); 13C NMR (MeOH-d4) δ: 173.8, 173.2, 149.2, 135.4, 135.1, 133.9, 131.7, 126.2, 54.3, 38.5; IR (neat, ν max): 3293, 2990, 2933, 2879, 1706, 1541, 1415, 1404, 1358, 1341, 1298, 1228, 1195, 1160, 1124, 1068, 919 (cm-1). Elemental

General procedure for N-Protected-5-oxazolidinone-L-aspartic acid (11)

Method A:- To a solution of 14 (1.0 eq.) in benzene (10 mL/mmol for 14a) or toluene (10 mL/mmol for 14b-e) was added paraformaldehyde (2.0 eq.) and p-TsOH.H2O (0.06 eq.). The mixture was heated to 60 °C (for 14a) or refluxed (for 14b-e) with removal of water using a Dean-Stark trap filled with MgSO4, The reaction was stopped after disappearance of starting material as judged by TLC analysis (approximately 1-2 hr, 4hr for 14c). The reaction mixture of 14a was cooled to RT and EtOAc was added. The organic layer was washed with aq. K2CO3 (0.3 M, 2 mL) and brine and dried over MgSO4. The solution was filtered and concentrated under reduced pressure. Recrystallization from diethyl ether provided 11a as a light yellow solid. For 14b-e, after consumption of starting material the solvent was removed under reduced pressure and the corresponding oxazolidinones (14b-e) were immediately purified by flash chromatography on silica gel using EtOAc/hexanes as the eluant [21].

Method B (Microwave Assisted Method):- To a solution of 14 (1.0 eq.) in benzene (5 mL/g for 14a-b, d-f) or toluene (5 mL/mmol for 14c) was added paraformaldehyde (7.0 eq.) and p-TsOH.H2O (0.06 eq.). The mixture was subjected to microwave irradiation (300 W) at 80°C (for 14a-b, d-f) or 105°C (for 14c) for 5-15 min. The reaction mixture was concentrated in vacuo and the corresponding oxazolidinones (11) were immediately purified by flash chromatography on silica gel using EtOAc/hexanes as the eluant.

N-Boc-5-oxazolidinone-L-aspartic acid (11a) (Method A/B)

To a solution of 14a (2.00 g for method A or 200 mg for method B) in benzene/EtOAc (65.0/5.00 mL for Method A) or benzene (2 mL for method B) was added paraformaldehyde (2.0 eq., 515 mg for method A or 7.0 eq., 231 mg for method B) and p-TsOH·H2O (0.06 eq., 89 mg for method A or 0.06 eq., 12 mg for method B). The mixture was either refluxed for 2 hr (method A) or subjected to microwave irradiation (300 W) at 65°C for 10 min. 11a was obtained as white solid after workup as reported earlier.21b recrystallization from diethylether. yield= 1.47 g, 70% (Method A), 130 mg, 62% (Method B), Mp. 128-129°C (lit.21b 132-134°C); : +151.3° (c = 1, CHCl3) (lit.21b: +153.1° (c = 1, CHCl3); 1H NMR(400 MHz, CDCl3) δ: (at 328 K) 10.50 (s, 1 H), 5.43 (d, J = 2.0 Hz, 1H), 5.23 (d, J = 3.6 Hz, 1H), 4.32 (s, 1H), 3.25-3.15 (br, 1H), 3.05-3.02 (two d, J = 3.0 Hz, 1H), 1.49 (s, 9H); (at 293 K) 10.00-8.50 (br s, 1 H), 5.50-5.42 (br, 1H), 5.24 (d, J = 3.1 Hz, 1H), 4.31 (s, 1H), 3.40-3.15 (br, 1H), 3.09-3.03 (two d, J = 2.9 Hz, 1H), 1.49 (s, 9H). 13C NMR (100 MHz, CDCl3) δ: (at 328 K) 174.2, 172.0, 152.2, 82.6, 78.8, 78.5,78.2, 51.6, 51.4, 34.5, 28.2, 28.1; (at 293 K) 175.3, 172.0, 152.2, 82.6, 78.8, 51.7, 34.7, 28.2, IR (neat, ν max): 3500-2800, 3064, 2980, 2933, 1804, 1712, 1499, 1478, 1398, 1370, 1313, 1290, 1265, 1169, 1141, 1100, 1052, 988, 926, 861, 846, 819, 770, 736, 702, 684 (cm-1).

N-Cbz-5-oxazolidinone-L-aspartic acid (11b) (Method A/B)

To a solution of 14b (2.00 g for method A or 200 mg for method B) in benzene (56.0 mL or 1 mL for method B) was added paraformaldehyde (449 mg, 2 eq. for method A or 157 mg, 7 eq. for method B) and p-TsOH·H2O (77.0 mg, 0.06 eq. or 8 mg, 0.06 eq. for method B). The mixture was either refluxed for 2 hr (method A) or subjected to microwave irradiation (300 W) at 80 °C for 6 min (method B). 11b was obtained as clear oil after purification with flash chromatography on silica gel using EtOAc/hexanes (3/7) as the eluant. Recrystallization from EtOAc/n-hexane gave 11b as a clear crystal. yield= 1.90 g, 91% (Method A), yield= 182 mg, 87% (Method B), Mp. 82-84°C (lit.21e 85-87°C); : +124° (c = 3.53, MeOH) (lit.21e : +125.7° (c = 3.53, MeOH)); 1H NMR (CDCl3) δ: 9.78 (br s, 1H), 7.34 (s, 5H), 5.48 (br s, 1H), 5.28 (d, J = 3.5 Hz, 1H), 5.21-5.11 (m, 2H), 4.34 (s, 1H), 3.30-3.02 (m, 2H); 13C NMR (CDCl3) δ: 175.0, 171.7, 152.9, 135.2, 128.8, 128.4, 78.5, 68.3, 51.4, 34.0; IR (neat, ν max): 3500-2600, 3030, 1802, 1721, 1422, 1360 (cm-1).

N-Fmoc-5-oxazolidinone-L-aspartic acid (11c) (Method B)

A mixture of 11c (300 mg), paraformaldehyde (7.0 eq., 177 mg ) and p-TsOH.H2O (0.06 eq., 9 mg) in toluene (1 mL) was subjected to microwave irradiation (300 W) at 105°C for 6 min. The reaction mixture was cooled to rt and EtOAc was added. The organic layer was washed with water and brine and dried over MgSO4. The solution was filtered and concentrated under reduced pressure. Recrystallization from acetone/CH2Cl2/hexanes (2/1/3) or EtOAc/n-hexane gave 11c as a white solid. Alternatively, 11c can also be purified by flash chromatography on silica gel using EtOAc/hexanes (5/5) as the eluant. yield= 263 mg, 85 % (Method B), Mp. 185-187°C (lit.21g 175-177°C); : +103.4° (c = 1, MeOH); 1H NMR (Acetone-d6, 318 K) δ: 7.85 (d, J = 7.6 Hz, 2H), 7.66 (d, J = 7.3 Hz, 2H), 7.41 (t, J = 7.4 Hz, 2H), 7.33 (t, J = 7.5 Hz, 2H), 5.41 (d, J = 3.5 Hz, 1H), 5.19 (s, 1H), 4.62-4.53 (m, 2H), 4.33 (t, J = 5.7 Hz, 1H), 4.26 (m, 1H), 2.84 (br s, 2H); 13C NMR (DMSO-d6, 318 K) δ: 172.1, 171.3, 152.4, 143.6, 140.8, 127.7, 127.2, 127.1, 125.0, 120.1, 77.9, 67.1, 51.4, 46.6, 34.2; IR (neat, ν max): 3300, 3047, 2955, 2923, 1772, 1727, 1692, 1449, 1431, 1411, 1360, 1299, 1260, 1173, 1131, 1053, 1131, 1053, 992 (cm-1).

N-Acyl-5-oxazolidinone-L-aspartic acid (11d) (Method B)

A mixture of 14d (200 mg), paraformaldehyde (7.0 eq., 231 mg ) and p-TsOH.H2O (0.06 eq., 12 mg) in benzene (1 mL) was subjected to microwave irradiation (300 W) at 80 °C for 15 min. The reaction mixture was concentrated in vacuo and 11d was purified by flash chromatography on silica gel using EtOAc/hexanes (1/1) as the eluant. 11d was dissolved in MeOH and added hexanes/diethyl ether. This solution was triturated to obtain white solid after filteration, yield= 160 mg, 75 % for two rotamers (1/2.5) (Method B), Mp. 132-133°C; : +210.2° (c = 1.0, MeOH); 1H NMR (300 MHz, 295 K, MeOH-d4) δ: 5.68 (d, J = 4.6 Hz, 1H*), 5.64 (d, J = 3.2 Hz, 1H), 5.50 (d, J = 3.1 Hz, 1H), 5.25 (d, J = 4.5 Hz, 1H*), 4.66 (d, J = 3.4 Hz, 1H*), 4.50 (t, J = 3.3 Hz, 1H), 3.14 (ABX, J = 18.2, 4.1, 2.3 Hz, 2H), 3.12 (d, J = 3.1 Hz, 2H*), 2.15 (s, 3H*), 2.07 (s, 3H) *Minor rotamer; 13C NMR (100 MHz, MeOH-d4) δ: 174.3, 174.0, 173.5, 172.9, 171.7, 170.5, 80.1, 80.1, 53.7, 52.6, 36.6, 34.7, 21.6, 21.3; IR (neat, νmax): 3500-2600, 2989, 2955, 2926, 2854, 1791, 1716, 1605, 1462, 1455, 1427, 1413, 1350, 1316, 1288, 1253, 1206, 1184, 1107, 1093, 1058, 1031, 994, 940, 921, 830, 807, 765, 688, 654, 617, 603 (cm-1). Elemental

N-Tosyl-5-oxazolidinone-L-aspartic acid (11e) (Method A/B)

A mixture of 14e (200 mg), paraformaldehyde (2.0 eq., 42 mg for Method A; 7.0 eq., 146 mg for Method B) and p-TsOH.H2O (0.06 eq., 7 mg) in toluene (6 mL for method A and 1 mL for method B) was refluxed for 3 hr or subjected to microwave irradiation (300 W) at 100°C for 5 min. The reaction mixture was concentrated invacuo and 11e was purified by flash chromatography on silica gel using EtOAc/hexanes (7/3 to 3/7) as the eluant. sticky solid, yield= 137 mg, 66 % (Method A), 150 mg, 72 % (Method B), Mp. 117-118°C(lit.21i 130-131°C); : +252° (c = 1.0, Acetone);21i 1H NMR (300 MHz, CDCl3) δ: 8.41 (br s, 1H), 7.93 (m, J = 8.2 Hz, 2H), 7.40 (m, J = 8.0 Hz, 2H), 5.38 (dd, J = 5.1 Hz, 2H), 4.06 (t, J = 3.6 Hz, 1H), 3.16 (d, J = 3.7 Hz, 2H), 2.46 (s, 3H); 13C NMR (100 MHz, CDCl3) δ: 175.0, 170.9, 145.8, 132.0, 130.58, 127.7, 79.6, 52.9, 36.2, 21.6; IR (neat, νmax): 3500-2500, 3241, 3092, 3062, 2986, 2930, 1803, 1718, 1597, 1493, 1401, 1357, 1307, 1293, 1268, 1215, 1165, 1105, 1045, 966, 922, 817, 761(cm-1).

N-Nosyl-5-oxazolidinone-L-aspartic acid (11f) (Method B)

A mixture of 14f (200 mg), paraformaldehyde (7.0 eq., 132 mg ) and p-TsOH.H2O (0.06 eq., 3.2 mg) in benzene (1 mL) was subjected to microwave irradiation (300 W) at 80°C for 10 min. The reaction mixture was concentrated in vacuo and 11f was purified by flash chromatography on silica gel using EtOAc/hexanes (1/1) and EtOAc as the eluant. The solid was recrystallised with ethyl acetate /diethyl ether to yield awhite solid, yield= 178 mg, 86% (Method B), Mp. 180-181°C; : +232.4° (c = 1.0, MeOH); 1H NMR (400 MHz, MeOH-d4) δ: 8.21-8.19 (m, 1H), 7.93-7.86 (m, 3H), 5.73 (d, J = 4.5 Hz, 1H), 5.43 (dd, J = 4.5 Hz, J = 0.7 Hz, 1H), 4.61 (t, J = 3.5 Hz, 1H), 3.04 (ABX, J = 18.1 Hz, J = 4.2 Hz, J = 3.4 Hz, 2H); 13C NMR (400 MHz, MeOH-d4) δ: 173.0, 149.8, 136.7, 133.8, 132.2, 131.4, 126.0, 80.7, 54.6, 36.6; IR (neat, νmax): 3500-2500, 3101, 3024, 2955, 2930, 2854, 1793, 1763, 1726, 1540, 1406, 1372, 1330, 1301, 1223, 1201, 1170, 1127, 1101, 1050, 1041, 971, 852, (cm-1).

Synthesis of N-Cbz-5-oxazolidinone-L-homoserine (16b)28

This protocol was adapted from a previous method.27 To a solution containing 11b (500 mg 1.78 mmol, 1.0 eq.) and distilled THF (20 mL) at -15°C was added NMM (235 µL, 2.14 mmol, 1.2 eq.) and IBCF (282 µL, 2.14 mmol, 1.2 eq.). The mixture was left to stir under argon for 30 min at -15°C. NaBH4 (0.203 g, 5.35 mmol, 3.0 eq.) was then added in one portion followed by MeOH (20.0 mL), which was added dropwise to the mixture over a period of 10 min at 0°C. The solution was stirred for additional 10 min, and then neutralized with 1 M HCl (1 mL). The organic solvents were evaporated under reduced pressure while making sure the water bath did not exceed 35-40°C. EtOAc (25 mL) was added to the mixture and the organic layer was washed consecutively with 0.1 M HCl (2 mL), H2O (5 mL), 5% aq NaHCO3 (5 mL), H2O (2 x 5 mL), dried over MgSO4 and concentrated under reduced pressure. The product was further purified directly by flash chromatography on silica gel (50% EtOAc/hexane) leaving the product as a clear oil (335 mg, 72% yield). 1H NMR (CDCl3) δ: 7.32 (s, 5H), 5.15 (s, 2H), 4.88 (br s, 1H), 4.80 (d, J = 11.2 Hz, 1H), 4.57-4.47 (m, 3H) 4.24-4.21 (m, 2H), 3.61 (br s, 1H), 2.49 (br s, 2H); 13C NMR (CDCl3) δ: 175.17, 174.71, 154.80, 135.55, 135.18, 128.41, 128.17, 128.06, 127.85, 72.29, 71.78, 68.07, 67.81, 65.88, 65.41, 56.04, 55.33, 27.43, 26.85; IR (neat, ν max): 3446 (br), 2956, 1779, 1705, 1482, 1436, 1355, 1258, 1184, 1024, 737, 700 (cm-1) MS, ESI, m/z (%): 288 ([M+Na]+, 100); HRMS, CI (+ve), m/z: calcd for C12H13NO4 [M-H2CO]+: 235.0845; found: 235.0836.

Synthesis of N-Fmoc-5-oxazolidinone-L-homoserine (16c)

This protocol was adapted from the previous method.27 To a solution containing 11c (380 mg, 1.03 mmol, 1.0 eq.) and distilled THF (10 mL) at -15°C was added NMM (125 µL, 1.14 mmol, 1.1 eq.) and IBCF (150 µL, 1.14 mmol, 1.1 eq.). The mixture was left to stir under argon for 30 min at -15°C. Afterwards, the mixture was then cooled to -78°C where upon NaBH4 (117 mg, 3.10 mmol, 3.0 eq.) was added in one portion, followed by the dropwise addition of MeOH (10.0 mL). The mixture was left to stir at -78°C for 1.5 hrs and then quenched with the addition of 1-2 mL of AcOH. The mixture was then stirred for an additional 30 min at -78°C before warming to RT. The organic solvents were evaporated under reduced pressure while making sure the water bath did not exceed 35-40°C. To the mixture was added EtOAc (25 mL) and the organic layer was washed 0.1 M HCl (2 mL), H2O (5 mL), 5% aq NaHCO3 (5 mL), H2O (2 x 5 mL), dried over Na2SO4 and concentrated under reduced pressure. The product was further purified directly by flash chromatography on silica gel (50% EtOAc/hexane) leaving the product as a clear oil (230 mg, 63% yield). 1H NMR (400 MHz, CDCl3) 13C NMR (CDCl3, 313K) δ: 174.81, 154.85, 143.5, 143.3, 141.2, 127.8, 127.7, 127.1, 124.8, 124.5, 119.9, 119.8, 71.9, 67.7, 65.8, 56.2, 47.1, 27.1; IR (neat, ν max): 3445, 3065, 2962, 2917, 1780, 1704, 1479, 1451, 1391, 1370, 1354, 1254, 1180, 1102, 1023, 952, 810, 761, 740 (cm-1); MS, ESI, m/z (%): 376 ([M+Na]+, 100); HRMS, ESI (+ve), m/z calcd for C20H19NO5Na [M+Na]+: 376.1161; found: 376.1148.

General procedure for the preparation of N-protected-L-homoserine-γ-lactones (1)

Method A: To a dry THF (40.0 mL/g) solution of 11 (1.0 eq.) at -10 to -15°C was added N‐methylmorpholine (1.2 eq.) and isobutyl chloroformate (1.2 eq.). The mixture was left to stir under nitrogen for 30 min at -15°C. NaBH4 (3.0 eq.) was then added in one portion and the reaction was stirred for 15-20 min followed by the dropwise addition of methanol (40.0 mL/g), and the stirred for additional 10 min at 0°C, and then neutralized with 1 M acetic acid or citric acid until solution becomes clear (pH=5-7). The organic solvents were evaporated under reduced pressure while making sure the water bath did not exceed 35-40°C. To the concentrate was added 1.0 M citric acid (~10 mL/g) and the mixture was refluxed for 2-4 hr and then cooled to rt. It was quickly extracted with ethyl acetate (4 x). The organic layers were combined and washed with water, brine, dried over MgSO4, filtered and concentrated under reduced pressure. The corresponding lactones, 1 were obtained after purification by flash chromatography on silica gel (hexanes/ethyl acetate).

Method B (Microwave Assisted Method):- To a dry THF (40.0 mL/g) solution of 11 (1.0 eq.) at -10 to -15°C was added N‐methylmorpholine (1.2 eq.) and isobutyl chloroformate (1.2 eq.). The mixture was left to stir under nitrogen for 30-45 min at -10°C. NaBH4 (3.0 eq.) was added in one portion and the reaction was allowed to slowly warm while it stirred for 15-20 min followed by the dropwise addition of methanol (50.0 mL/g), and the stirred for additional 10 min at 0°C, and then neutralized with 1 M acetic acid (~ 8 mL/g) until solution becomes clear (pH ~ 7). The reaction mixture was then concentrated in vacuo, transferred to a microwave vial with the help of additional methanol and the solvent quickly evaporated in vacuo without exposing it to the heat (< 40 °C). The reaction mixture was then added 0.5-1M citric acid (~ 4-6 mL/g) solution and the mixture was exposed to the microwave irradiation (300W) for 5-10 min at 80°C. The mixture was extracted with ethyl acetate (4 x). The organic layers were combined and washed with water, brine, dried over MgSO4, filtered and concentrated under reduced pressure. The corresponding lactones, 1 were obtained after purification by flash chromatography on silica gel (hexanes/ethyl acetate).

N-Cbz-L-homoserine-γ-lactone (1b) A solution of 11b (200 mg) in dry THF (8.0 mL) was treated with NMM (1.2 eq., 94 µL) and IBCF (1.2 eq., 113 µL), followed by the addition of NaBH4 (3.0 eq., 80 mg) and then, quenched with MeOH (10.0 mL) and 1 M acetic acid (1.5 mL). The solvent was evaporated in vacuo and added 1M citric acid (4 mL for Method A or 1 mL for Method B). The reaction mixture was either refluxed for 2 hr (Method A) or exposed to the microwave irradiation (300W) for 15 min at 80°C. After the general workup procedure, 1b was purified by flash chromatography on silica gel using hexanes/ethyl acetate (7/3 to 1/1) as eluent. Recrystallization from EtOAc/n-hexane gave 11b as white needles. yield= 118 mg, 70% (Method A), 109 mg, 65% (Method B),, Mp. 122-124°C (lit.16 126-127°C); : -32.1° (c = 1.0, MeOH) (lit.16 : -30.5° (c = 1.0, MeOH)); 1H NMR (400 MHz, CDCl3) δ: 7.33 (s, 5H), 5.39 (br s, 1H), 5.11 (s, 2H), 4.44-4.35 (m, 2H), 4.24-4.17 (m, 1H), 2.78-2.70 (m, 1H), 2.26-2.11 (m, 1H); 13C NMR (100 MHz, CDCl3) δ: 175.1, 156.0, 135.8, 128.4, 128.1, 128.0, 67.1, 65.6, 50.2, 29.6; IR (neat, νmax): 3328, 3058, 3029, 2944, 1778, 1695, 1542, 1385, 1298, 1265, 1225, 1180, 1073, 1013, 1007, 946 (cm-1).

N-Fmoc-L-homoserine-γ-lactone (1c) A solution of 11f (200 mg) in dry THF (8.0 mL) was treated with NMM (1.2 eq., 73 µL) and IBCF (1.2 eq., 105 µL), followed by the addition of NaBH4 (3.0 eq., 62 mg) and then, quenched with MeOH (10.0 mL). The solvent was quickly evaporated in vacuo and added 0.5 M citric acid (2 mL). The reaction mixture was exposed to the microwave irradiation (300W) for 10 min at 80°C. After the general workup procedure, 1f was purified by flash chromatography on silica gel using hexanes/ethyl acetate (1/1) as eluent followed by recrystallization from 95% EtOH, white solid, yield= 141 mg, 80% (Method A), Mp. 208-209°C (Lit.7b 208-209°C);: +12.0° (c = 0.25, CHCl3); 1H NMR (400 MHz, CDCl3) δ: 7.76 (d, J = 7.5 Hz, 2H), 7.58 (d, J = 7.4 Hz, 2H), 7.40 (t, J = 7.4 Hz, 2H), 7.31 (t, J = 7.4 Hz, 2H), 5.39 (br s, 1H), 4.47-4.41 (m, 4H), 4.26-4.20 (m, 3H), 2.78 (m, 1H), 2.20 (m, 1H); 13C NMR (100 MHz, CDCl3) δ: 174.9, 156.1, 143.7, 143.6, 141.3, 127.8, 127.1, 125.0, 120.0, 67.3, 65.8, 50.5, 47.1, 30.5; IR (neat, νmax): 3328, 3061, 3017, 2948, 2918, 2851, 1789, 1686, 1534, 1461, 1384, 1243, 1285, 1263, 1222, 1187, 1161, 1103, 1081, 1017, 995, 971, 947, 909, 756, 738, 692, 644, 621(cm-1). Elemental

N-Acyl-L-homoserine-γ-lactone (1d)32 A solution of 11d (200 mg) in dry THF (8.0 mL) was treated with NMM (1.2 eq., 142 µL) and IBCF (1.2 eq., 121 µL), followed by the addition of NaBH4 (3.0 eq., 121 mg) and then, quenched with MeOH (10.0 mL) and 1 M acetic acid (1.5 mL). The solvent was evaporated in vacuo and added 1M citric acid (1 mL). The reaction mixture was exposed to the microwave irradiation (300W) for 15 min at 80°C. The reaction mixture was cooled to RT and aq. NaHCO3 was added to reach the pH~7. It was extracted with ethyl acetate (4 x). The organic layers were combined and washed with water, brine, dried over MgSO4, filtered and concentrated under reduced pressure. 1d was purified by flash chromatography on silica gel using EtOAc and EtOAc/MeOH (95/5) as eluent followed by repeated recrystallization from CH2Cl2/hexanes, spot detection in I2-Chamber, white solid, yield= 137 mg, 84% (Method B), Mp. 82-84°C (Lit.32b 82-84°C); : -69° (c = 1.6, DMF) (Lit. -69.0°, c = 1.6, DMF 32c; -54.7°, DMF32d); 1H NMR (400 MHz, CDCl3) 32e δ: 6.13 (s, 1H), 4.61-4.54 (m, 1H), 4.48 (m, 1H), 4.32-4.25 (m, 1H), 2.89-2.82 (m, 1H), 2.28-2.11 (m, 1H), 2.08 (s, 3H); 13C NMR (100 MHz, CDCl3) δ: 175.8, 170.9, 170.8, 66.0, 48.8, 48.7, 29.4, 22.6, 22.5; IR (neat, νmax): 3305, 3072, 2991, 2920, 1777, 1656, 1542, 1450, 1431, 1379, 1300, 1221, 1180, 1154, 1020, 951, 816, 791, 749, 708, 669, 651, 597 (cm-1).

N-Tosyl-L-homoserine-γ-lactone (1e) A solution of 11f (250 mg) in dry THF (10.0 mL) was treated with NMM (1.2 eq., 111 µL) and IBCF (1.2 eq., 134 µL), followed by the addition of NaBH4 (3.0 eq., 95 mg) and then, quenched with MeOH (12.5 mL) and 1 M acetic acid (2 mL). The solvent was evaporated in vacuo and added 1M citric acid (5 mL for Method A or 1.5 mL for Method B). The reaction mixture was either refluxed for 2 hr (Method A) or exposed to the microwave irradiation (300W) for 10 min at 65°C. After the general workup procedure, 1f was purified by flash chromatography on silica gel using hexanes/ethyl acetate (7/3 to 1/1) as eluent followed by recrystallization from CH2Cl2/hexanes, white crystals, yield= 145 mg, 68% (Method A), 100 mg, 46% (Method B), Mp. 129-130°C (Lit.12a 130-133°C); : +8.5° (c = 1.0, MeOH); 12a 1H NMR, 13C NMR and IR matched with the literature.25a

N-Nosyl-L-homoserine-γ-lactone (1f) A solution of 11f (200 mg) in dry THF (8.0 mL) was treated with NMM (1.2 eq., 81 µL) and IBCF (1.2 eq., 97 µL), followed by the addition of NaBH4 (3.0 eq., 69 mg) and then, quenched with MeOH (10.0 mL) and 1 M acetic acid (1.5 mL). The solvent was evaporated in vacuo and added 1M citric acid (4 mL for Method A or 1 mL for Method B). The reaction mixture was either refluxed for 2 hr (Method A) or exposed to the microwave irradiation (300W) for 5-10 min at 80°C. After the general workup procedure, 1f was purified by flash chromatography on silica gel using hexanes/ethyl acetate (1/1) as eluent followed by recrystallization from CHCl3, transparent crystals, yield= 121 mg, 70% (Method A), 73 mg, 42% (Method B), Mp. 131-132°C;: -350° (c = 0.1, CHCl3); 1H NMR (400 MHz, CDCl3) δ: 8.16 (m, 1H), 7.97 (m, 1H), 7.80-7.75 (m, 2H), 6.16 (d, J = 5.7 Hz, 1H), 4.47 (d, J = 9.0 Hz, 1H), 4.30-4.23 (m, 2H), 2.77-2.83 (m, 1H), 2.34-2.45 (m, 1H); 13C NMR (100 MHz, CDCl3) δ: 173.8, 147.8, 134.1, 133.8, 133.2, 130.8, 125.9, 66.0, 52.7, 31.0; IR (neat, νmax): 3345, 3089, 2998, 2960, 2924, 2883,1782, 1593, 1539, 1442, 1376, 1355, 1221, 1167, 1124, 1058, 1021, 999, 953, 912, 854, 798 (cm-1). Elemental

Writing Services

Essay Writing
Service

Find out how the very best essay writing service can help you accomplish more and achieve higher marks today.

Assignment Writing Service

From complicated assignments to tricky tasks, our experts can tackle virtually any question thrown at them.

Dissertation Writing Service

A dissertation (also known as a thesis or research project) is probably the most important piece of work for any student! From full dissertations to individual chapters, we’re on hand to support you.

Coursework Writing Service

Our expert qualified writers can help you get your coursework right first time, every time.

Dissertation Proposal Service

The first step to completing a dissertation is to create a proposal that talks about what you wish to do. Our experts can design suitable methodologies - perfect to help you get started with a dissertation.

Report Writing
Service

Reports for any audience. Perfectly structured, professionally written, and tailored to suit your exact requirements.

Essay Skeleton Answer Service

If you’re just looking for some help to get started on an essay, our outline service provides you with a perfect essay plan.

Marking & Proofreading Service

Not sure if your work is hitting the mark? Struggling to get feedback from your lecturer? Our premium marking service was created just for you - get the feedback you deserve now.

Exam Revision
Service

Exams can be one of the most stressful experiences you’ll ever have! Revision is key, and we’re here to help. With custom created revision notes and exam answers, you’ll never feel underprepared again.