Introduction

Carbapenems are important β-lactam antibiotics which, owing to high antibacterial activity and resistance to β-lactamase, continually attract the interest of industrial and academic laboratories.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 Recently we have shown that the Kinugasa reaction between D-lactic acid-derived acetylene 2 and five-membered cyclic nitrone 1 obtained from 2-deoxy-d-ribose14, 15, 16, 17 offers an attractive entry into carbapenems.18 The main drawback of this strategy has been related to the undesired cis configuration of H-5 and H-6 protons in the major adducts 4 or 6 (Scheme 1). We have demonstrated that the stereogenic center at C-6 of the alcohol 4 can be epimerized in the presence of 2 equiv. KHMDS to provide the trans isomer 5 in 55% yield (Scheme 1). Epimerization performed on compound with protected side-chain hydroxyl 6 caused β-elimination to the exo double bond.

As effective cis/trans epimerization requires a free hydroxyl group in the side chain and seeing that Kinugasa reaction involving commercially available alcohol 2 and its t-butyldiphenysilyl-protected derivative 319 was low yielding, we decided to change the side-chain hydroxyl group protection and reaction conditions. After conducting several experiments we found that the protection of alcohol 2 as t-butyldimethylsilyl derivative 820 and the use of 1,1,3,3-tetramethylguanidine as the base offered better yield and higher content of the trans isomer 10 which allowed for its easy separation (Scheme 1). This observation was important for the further synthesis of thienamycin because one of the structural features present in carbapenem antibiotics is the trans configuration in the four-membered β-lactam ring.

Results and discusion

Selective debenzylation of the primary hydroxyl group21 in 10 by hydrogenolysis under the pressure of 6 bar H2 provided a mixture of two regioisomers 11 and 12 (6:1), however, in a low yield (35%) only. Higher pressure led to debenzylation of both groups to afford 13 in low yield. The same deprotection of both benzyl groups can be achieved by a reduction with sodium in liquid ammonia (Figure 1).22

Figure 1
figure 1

Selective debenzylation of the primary hydroxyl group.

Full size image

This results prompted us to use partly deprotected nitrone 14 in the Kinugasa reaction with 8. Nitrone 14 was obtained from 1 by a modification of a known method,23, 24 using the complex of BCl3 with dimethyl sulfide. Under such milder conditions, only the primary hydroxyl group was deprotected in 50% yield (Scheme 2).

The reaction of 14 with 8 gave a mixture of cis and trans carbapenams 15 and 11 in a ratio of about 1:3 and with 54% overall yield. The trans adduct 11 was separated by chromatography. The primary hydroxyl group was oxidized to the aldehyde using Dess–Martin periodinane25 in 91% yield and subsequently the crude product was oxidized with sodium chlorite under Pinnick–Lindgren26, 27 conditions to afford the carboxylic acid function, which was in turn methylated with diazomethane28 to afford ester 18 (Scheme 3).

The benzyl protecting group was then removed using palladium hydroxide on carbon to afford the alcohol 19 in 97% yield. The structure and configuration of alcohol 19 was confirmed by X-ray crystallography. Compound 19 was then oxidized to ketone 20 using Dess–Martin reagent (Scheme 4). It’s important to know that the similar keto-p-nitrobenzyl ester 20a has already been transformed into thienamycin by the Hannesian group.29

The transformation of the ketone 20 into thienamycin derivative 21 according to a known procedure required the enolization of the carbonyl group followed by phosphorylation of the enol hydroxyl and subsequent addition of N-acetylcysteamine.29 The best result was obtained for diethyl chlorophosphate as a phosphorylation agent. We also found that the enolization of ketone 20 required quite a long time. Otherwise phosphorylation did not occur and compound 21 was not formed. Particularly when treating 20 with diphenyl chlorophosphate, we observed the opening of the pyrrolidine ring via retro-Dieckmann reaction and formation of thioester 22 (Scheme 5).

In conclusion, we have demonstrated that the cyclic five-membered nitrone 1, easily available from 2-deoxy-d-ribose represents an attractive substrate for the stereocontrolled synthesis of thienamycin—an important natural carbapenem antibiotic. The configuration of the stereogenic center next to the nitrogen atom has a decisive role in the asymmetric induction at the C-5 carbon atom of the carbapenam scaffold. The predominance of the isomer with anti configuration of H-5 and H-6 protons was achieved by the use of tetramethylguanidine as a base in the Kinugasa reaction.

Experimental Procedure

Melting points were determined using Köfler hot-stage apparatus with microscope and were uncorrected. Proton and carbon NMR spectra were recorded on a Varian VN MRS Spectrometer (Varian Inc., St Clara, CA, USA) at 600 and 150 MHz, respectively, in CDCl3 or C6D6. IR spectra were obtained on an FT-IR-1600 Perkin-Elmer spectrophotometer (Perkin Elmer, Waltham, MA, USA). The optical rotations were measured with a JASCO J-2000 digital polarimeter (Jasco Inc., Easton, MD, USA). High-resolution mass spectra were recorded on ESI-TOF Mariner spectrometer (Perspective Biosystem, Framingham, MA, USA). X-ray analysis were performed on Nonius MACH3 diffractometer (Bruker, Madison, WI, USA). The HPLC analysis were carried out on Hitachi chromatograph (Hitaschi, Tokyo, Japan) with L-2130 pump and L-2450 DAD detector (Brucker, Karlsruhe, Germany) equipped with LiChrospher Si60 analytical column (Merck, Darmstadt, Germany).

Thin-layer chromatography (TLC) was performed on aluminum sheets silica gel 60 F254 from Merck. Column chromatography (CC) was carried out using Merck silica gel (230–400 mesh) or Florisil (100–200 mesh). The TLC spots were visualized in UV (254 nm) and by treatment with alcoholic solution of ninhydrine, aqueous solution of KMnO4 or with ceric sulphate/phosphomolybdic acid solution.

All solvents were dried and purified applying standard techniques.

scheme 1

Synthesis of carbapenams via Kinugasa reaction with Et3N and 1,1,3,3-tetramethylguanidine as a base. Attempted epimerization at C-6.

scheme 2

Deprotection of nitrone 1 and Kinugasa reaction with alkyne 8.

scheme 3

Preparation of carbapenam 18.

scheme 4

Synthesis of ketone 20. A full color version of this scheme is available at The Journal of Antibiotics journal online.

scheme 5

Synthesis of protected thienamycin 21 and side reactions in the phosphorylation-addition sequence.

(2S,3S)-3-(benzyloxy)-2-((benzyloxy)methyl)-3,4-dihydro-2H-pyrrole-1-oxide (1) Compound 1 was prepared according to literature methods

Step 1: To a solution of 3,5-di-O-benzyl-2-deoxy-D-ribofuranoside15 (8.91 g, 28.3 mmol)16 in dry toluene (70 ml) was added MgSO4 (10.3 g) under argon. The suspension was stirred at reflux temperature for 5 min, and then O-tert-butyldiphenylsilylhydroxylamine (8.6 g, 32 mmol) and pyridinium p-toluenesulfonate (98 mg) were added. The reaction mixture was heated at the same temperature for 30 min, and then it was filtered. The filtrate was washed with a saturated aqueous solution of NaHCO3 and with brine and dried over MgSO4 to give a residue, which was use in the next step without further purification.

Step 2: To a solution of oxime with previous step (12.0 g, 21.2 mmol) in CH2Cl2 (70 ml) placed at 0 °C under argon atmosphere were added successively triethylamine (4.5 ml, 32.8 mmol) and mesyl chloride (1.9 ml, 24.6 mmol). The solution was stirred for 30 min, and then water (15 ml) was added. The aqueous layer was extracted three times with CH2Cl2. The organic layers were washed with brine, dried over MgSO4, and concentrated under vacuum to give a residue, which upon short column chromatography over silica gel (pentane/AcOEt 9/1, 4/1, then 1/1) yielded the desired unstable mesylate (13.4 g, 97.6%: 3/1 mixture of E/Z isomers) as an yellow oil. To a solution of mesylate (13.4 g, 20.7 mmol) in THF (400 ml) placed at 0 °C under argon was added tetrabutylammonium fluoride (24.2 ml, 1 M solution in THF, 24.2 mmol). The reaction mixture was stirred for 5 min at 0 °C, and then the solvent was removed under vacuum. The residue was dissolved in ethyl acetate, water was added, and the aqueous layer was extracted three times with ethyl acetate. The organic layers were washed with brine, dried over MgSO4, and concentrated under vacuum to give a residue, which was use in the next step without further purification.

Step 3: To a solution of crude oxime with previous step (6.2 g, 18.8 mmol) in a 4:1 mixture of methanol and water (200 ml) were added NaHCO3 (8.5 g, 102.0 mmol) and hydroxylamine hydrochloride (6.7 g, 96.5 mmol). The reaction mixture was heated overnight at reflux temperature. After concentration under vacuum, the residue was dissolved in CH2Cl2, and water was added. The aqueous layer was extracted three times with CH2Cl2, and the organic layers were washed with brine, dried over MgSO4 and concentrated under vacuum to give a residue, which upon column chromatography over silica gel (acetone/DCM 1/9 then methanol/DCM 5/95) yielded the nitrone 1 (5.1 g, 87%) as an olive syrup.

[α]D+23,7 (c 1, CHCl3), IR (film) ν: 737, 1113, 2866, 3062, 3239 cm−1

1H NMR (600 MHz, CDCl3) δ: 2.74–2.78 (m, 1H, H-3), 2.79-2.85 (ddd J=2.6 Hz, 7,1 Hz, 17,9 Hz, 1H, H-3), 4.00–4.05 (m, 1H, H-5), 4.07–4.12 (m, 2H, CH2OBn), 4.45–4.50 (m, 1H, H-4), 4.54–4.62 (m, 2H, 2 × CH2Ph), 6.85–6.88 (m, 1H, H-2). 13C NMR (150 MHz, CDCl3) δ: 35.03, 64.86, 72.20, 73.63, 73.66, 74.32, 127.56, 127.60, 127.66, 127.94, 128.31, 128.50, 133.23, 137.41, 137.99.

HR MS (ESI): m/z [M+Na]+calculated. for C19H21NO3Na: 334,1419; found: 334,1427

(2S,3S)-3-Benzyloxy-2-hydroxymethyl-3,4-dihydro-2H-pyrrole-N-oxide (14)

To a solution of nitrone 1 (0.46 g, 1.50 mmol) in CH2Cl2 (30 ml), cooled to 0 °C, BCl3•Me2S complex (1 ml, 7.50 mmol) was added. The reaction mixture was stirred at room temperature overnight. Subsequently the mixture was evaporated, treated with methanol and evaporated again (4 × 10 ml). The crude mixture was purified by silica gel chromatography to afford the nitrone 14 (0.17 g, 0.75 mmol) in 50% yield.

Colorless syrup; [α]D+23,7 (c 1, CHCl3), IR (film) ν: 1453, 2925, 3030, 3334 cm−1

1H NMR (600 MHz, CDCl3) δ: 2.70-2.77 (m, 1H, H-4), 2.85–2.94 (m, 1H, H-4), 4.01–4.07 (m, 1H, CHHOH), 4.09–4.15 (m, 2H, H-2, OH), 4.20-4.45 (m, 1H, CHHOH), 4.43-4.62 (m, 3H, CH2Ph, H-3), 6.89 (s, 1H, H-5), 7.24–7.40 (m, 5H, Ar). 13C NMR (150 MHz, CDCl3) δ: 34.93, 60.35, 71.78, 73.53, 76.79, 127.60, 128.26, 128.52, 128.68, 136.72.

HR MS (ESI): m/z [M+Na]+calculated for C12H15NO3Na: 244,1086; found: 244,1088

(2S,3S,5R,6S,1’R)-3-Benzyloxy-6-[1’-(tert-butyldimethylosiloxy)ethyl]-2-(hydroxymethyl)-1-azabicyclo[3.2.0]heptan-7-one (15) and (2S,3S,5R,6R,1’R)-3-benzyloxy-6-[1’-(tert-butyldimethylosiloxy)ethyl]-2-(hydroxymethyl)-1-azabicyclo [3.2.0]heptan-7-one (11)

To a suspension of CuI (0.5 mmol, 95 mg) in dry, degassed MeCN (3 ml), 253 μl (2.0 mmol) of 1,1,3,3-tetramethylguanidine was added. After cooling to 0 °C a solution of acetylene 820 (92 mg, 0.5 mmol) in 1 ml of MeCN was added and thus obtained mixture was stirred for 15 min. Then a solution of nitrone 14 (221 mg, 1.0 mmol) in MeCN (2 ml) was added slowly and the mixture was kept at 0 °C for additional 15 min. After removal of the cooling bath, the mixture was stirred at ambient temperature under an inert atmosphere for 24 h. The progress of the reaction was monitored by TLC. Removal of the solvent gave a residue, which was chromatographed on silica gel (hexane/AcOEt 4:6 v/v) to afford a mixture of 15 and 11 in ratio 1:3 (110 mg, 54%, according to 1H NMR of crude reaction mixture).

Compound 11; colorless syrup; [α]D +64,3 (c 1, CHCl3); IR (film) v: 1099, 1757, 2856, 3437 cm−1; 1H NMR (600 MHz, CDCl3) δ: 0.06 (s, 6H, Si(CH3)2), 0,87 (s, 9H, t-Bu), 1.22 (d, J=6.1 Hz, 3H, CH3), 1.68-1.73 (m, 1H, H-4), 2.31-2.36 (m, 1H, H-4), 2.80 (dd, J= 2.0 Hz, 6,0 Hz, 1H, H-6), 3.73 (d, J= 5.6 Hz, 2H,CH2OH), 3,86-3.89 (m, 1H, H-5), 4.00 (q,1H, J=5.8 Hz, H-2), 4,15-4.20 (m, 1H, CHOSi), 4.44-4.46 (m, 1H, H-3,), 4.45–4.64(m, 2H,CH2Ph), 7.28–7.32 (m, 3H, Ar), 7.34-7.37 (m, 2H, Ar); 13C NMR (150 MHz, CDCl3) δ: −4.94, −4.22, 17,95, 22,61, 25,68, 53.81, 61.28, 62.65, 65.23, 66.18, 72.33, 84.95, 127.57, 128.64, 137.33, 177.16; HR MS(ESI) m/z [M+Na]+ calculated for C22H35NO4SiNa: 428.2233; found: 428.2234.

Compound 15: colorless syrup; [α]D +39,1 (c 1, CHCl3); IR (film) v: 1092, 1744, 2926, 3400 cm−1; 1H NMR (600 MHz, CDCl3) δ: 0.06 (s, 6H, Si(CH3)2), 0,88 (s, 9H, t-Bu), 1.20 (d, J=6.2 Hz, 3H, CH3), 1.82-1.87 (m, 1H, H-4), 2.15–2.20 (m, 1H, H-4), 3.39(dd, J=5.7 Hz, 7,7 Hz, 1H, H-6), 3.71–3.3.78(m, 2H, CH2OH), 3.90(q, J=5.7 Hz, 1H, H-2), 3.91-3.95 (m, 1H, H-5), 4.00–4.05 (m, 1H, CHSi), 4.42–4.45(m, 1H, H-3), 4.46–4.63(m, 2H, CH2Ph), 7.35–7.37(m, 5H, Ar); 13C NMR (150 MHz, CDCl3) δ: −4.55, −4.33, 22.84, 25.81, 29.68, 32.08, 54.20, 58.12, 61.52, 61.94, 65.36, 72.27, 84.66, 126.97, 127.64, 128.55, 140.87, 178.37; HR MS(ESI) m/z [M+Na]+calculated. for C22H35NO4SiNa: 428.2233; found: 428.2227.

(2R,3S,5R,6S,1’R)-3-Benzyloxy-6-[1’-(tert-butyldimethylsiloxy)ethyl]-2-methoxycarbonyl-1-azabicyclo[3.2.0]heptan-7-one (18)

To a solution of 11 (166 mg, 0.4 mmol) in CH2Cl2 (15 ml) sodium bicarbonate (134 mg, 1.6 mmol) and Dess–Martin periodinane (340 mg, 0.8 mmol) were added. The reaction mixture was stirred at room temperature for 1 h. Subsequently, a saturated solution of Na2S2O3 was added. The water phase was extracted with CH2Cl2 (3 ×). The combined extracts were washed with brine and dried over MgSO4. The solution was evaporated and the crude aldehyde 16 was used in the next step without purification.

The crude 16 was dissolved in t-BuOH (6 ml) and treated with 2-methyl-2-butene (6 ml). Subsequently NaClO2 (0.21 g, 2.3 mmol) and a solution of NaH2PO4 (0.26 g, 1.90 mmol) in water (11 ml) were added. The reaction mixture was stirred overnight at room temperature. NH4Cl was added and the aqueous solution was extracted with ethyl acetate (3X). The combined extracts were dried over MgSO4, filtered and evaporated. The crude acid 17 was dissolved in Et2O (20 ml), cooled to 0 °C and treated with a solution of diazomethane. After 5 min the mixture was treated with drop of acetic acid and evaporated. The product was purified on a silica gel column using hexane-EtOAc, 6:4 v/v as an eluant to give 18 in 58% yield.

Colorless syrup; [α]D +121,1(c 1, CHCl3); IR (film) v: 1099, 1746, 1792, 2929, cm−1; 1H NMR (500 MHz, CDCl3) δ: 0.08 (s, 6H, Si(CH3)2), 0,89 (s, 9H, t-Bu), 1.24 (d, J=6.0, 3H, CH3), 1.62-1.69 (m, 1H, H-4),2.32-2.38 (m, 1H, H-4), 2.82 (dd, J=2.1 Hz, 6,4 Hz, 1H, H-6), 3.70 (s, 3H, CO2CH3), 4.03-4.08 (m, 1H, H-5), 4.19-4.25 (m, 1H, CHOSi), 4.55 (s, 2H, OCH2Ph), 4.57-4.61 (m, 1H, H-3), 4.61-4.64 (m, 1H, H-2), 7.25-7.36 (m, 5H, Ar); 13C NMR (150 MHz, CDCl3) δ: -4.21, 17.93, 22.62, 25.69, 36.02, 52.03, 55.28, 63.70, 65.34, 66.25, 72.61, 85.47, 127.54, 127.88, 128.42, 137.33, 168.59, 175.88.

HR MS(ESI) m/z [M+Na]+calculated. for C23H35NO5SiNa: 456.2182; found: 456.2185.

(2R,3S,5R,6S,1’R)-6-[1’-(tert-Butyldimethylsiloxy)ethyl]-3-hydroxy-2-methoxycarbonyl-1-azabicyclo[3.2.0]heptan-7-one (19)

Compound 18 (0.10 g, 0,30 mmol) in ethyl acetate (10 ml) was treated with Pd(OH)2 (0.15 g) and hydrogenated under 1 atm H2 at room temp. for 3 h. Subsequently the catalyst was filtered off through a short path of celite. The solution was evaporated and purified on a silica gel column using hexane/EtOAc 4:6 v/v as an eluent to give 19 (0.10 g, 0.29 mmol) in 97% yield.

Colorless crystals; [α]D +103, 8 (c 1, CHCl3); m.p.=151–153 °C;

IR (film) v: 1718, 1735, 2927, 3337 cm−1;

1H NMR (500 MHz, CDCl3) δ: 0.09 (s, 6H, Si(CH3)2), 0,89 (s, 9H, t-Bu), 1.25 (d, J=6.2 Hz, 3H, CH3), 1.69-1.70 (m, 1H, H-4), 2.34–2.36 (m, 1H, H-4), 2.69 (bs,1H, OH), 2.85 (dd, J=1.8 Hz, 5,8 Hz, 1H, H-6), 3.77(s, 3H, CO2CH3), 4.09–4.10 (m, 1H, H-5), 4.24–4.26 (m, 1H, CHOSi), 4.5 (d, J=4.8 Hz, 1H, H-2), 4.94-4.95 (m, 1H, H-3);

13C NMR (150 MHz, CDCl3) δ: −4.99, −4.22, 17.94, 22.64, 25.67, 39.41, 52.45, 55.33, 64.56, 64.84, 66.00, 79.06, 169.91, 176.13.

HR MS(ESI) m/z [M+Na]+calculated for C16H29NO5SiNa: 366.1713; found: 366.1707.

Crystallographic data: C16H29NO5Si, MW=343.49 Da, 0.40 × 0.40 × 0.05, monoclinic, space group. P=21, Z=2, T=100(2) K, a= 7.3250(2), b= 7.5160(2), c= 17.4905(5) Å, V=962.89(5) Å3, λ(Cu, Kα)= 1.54184 Å, μ=1.271 mm−1.Intensity data were collected on SuperNova area detector system. The structure was solved by direct mehods and refined by the full-matrix least-squares on F2 (SHELXL-97). A total of 2389 reflections were measured and 2345 were independent. Final R1=0.0479, wR2=0.1325 (2389 references; I>2σ(I), and GOF=1.080 (for all data, R1 =0.0483, wR2=0.1333).

(2R,5R,6S,4’R)-6-[1’-(tert-Butyldimethylosiloxy)ethyl]-2-methoxycarbonyl-1-azabicyclo[3.2.0]heptan-3,7-di-one (20)

To a solution of compound 19 (84 mg, 0,24 mmol) in CH2Cl2 (10 ml), NaHCO3 (81 mg, 0.96 mmol) and Dess–Martin periodinane (0.20 g, 0.48 mmol) were added. The reaction mixture was stirred at room temperature for 40 min. Subsequently, saturated aqueous solution of Na2S2O3 was added. The aqueous phase was extracted with CH2Cl2 (3 ×). The combined organic extracts were washed with brine, dried over MgSO4 and evaporated. The product was purified on a silica gel column with hexane–EtOAc, 4:6 (v/v) as the eluent to afford 20 (40 mg, 0.11 mmol) in 48% yield.

Colorless crystals m.p. 151–154 °C; [α]D +125,5 (c 1, CHCl3);

IR (film) v: 1256, 1770, 2856, 2929 cm−1;

1H NMR (600 MHz, CDCl3) δ: 0.08 (s, 6H, Si(CH3)2), 0,90 (s, 9H, t-Bu), 1.26 (d, J=6.2 Hz, 3H, CH3), 2.40 (dd, J=7.7 Hz, 18,8 Hz, 1H, H-4), 2.86 (dd, J=6.9 Hz, 18.8 Hz, 1H, H-4), 3.10 (dd, J=2.0 Hz, 5,1 Hz, 1H, H-6), 3.75 (s, 3H, CO2CH3), 4.11-4.15 (m, 1H, H-5), 4.30 (dq, J=6.15 Hz, 1H, CHOSi), 4.64 (s, 1H, H-2);

13C NMR (150 MHz, CDCl3) δ: -5.09, -4.25, 17.88, 22.59, 25.58, 27.14, 41.17, 46,87, 50.98, 53.02, 63.93, 65.35, 68.76, 165.46, 172.47, 207.32.

HR MS(ESI) m/z [M+Na]+calculated. for C16H27NO5SiNa: 364.1556; found: 364.1557

(5R,6S,1’R)-Methyl 3-[(2-acetamidoetyl)thio]-6-[1’-(tert-butyldimethylsiloxy)ethyl)-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate (21)

Compound 20 (14 mg, 0.04 mmol) in acetonitrile (2 ml) was cooled to 0 °C and treated with N,N-diisopropylethylamine (8 μl, 0.05 mmol), diethyl chlorophosphate (7 μl, 0.05 mmol) and catalytic amount of DMAP. The mixture was stirred for 1 h at room temperature. Subsequently it was cooled to 0 °C and treated with a another portion of N,N-diisopropylethylamine (11 μl, 0.06 mmol) and N-acetylcysteamine (6,5 μl, 0.06 mmol). The mixture was stirred overnight at room temperature. Subsequently it was evaporated under reduced pressure and purified on a silica gel column using toluene-i-propanol, 4:1(v/v) as an eluent to afford 21 (8 mg, 0.018 mmol) in 44% yield.

Easily solidifing syrup; [α]D+52,3 (c 0.69, CHCl3); IR (film) v: 1659, 1748, 1758, 2898, 2929, 2953 cm−1;

1H NMR (600 MHz, CDCl3) δ: 0.06 (s, 6H, Si(CH3)2), 0,90 (s, 9H, t-Bu), 1.24 (d, J=6.1 Hz, 3H, CH3), 1.97 (s, 3H, CH3), 2.88-2.94 (m, 1H, SCHH), 2.99-3.06 (m, 2H, SCHH, H-4), 3.10 (dd, J=2.7 Hz, 6,1 Hz,1H, H6), 3.22-3.29 (m, 1H, H-4), 3.39-3.49 (m, 2H, CHHNHAc), 3.81(s, 3H, CO2CH3), 4.14-4.21 (m, 2H, H5, CHOSi), 5.96 (bs, 1H, NHAc);

13C NMR (150 MHz, CDCl3) δ: -4.93, -4.26, 17.94, 22.51, 23.17, 25.62, 25.68, 31.87, 39.70, 40.10, 52.16, 52.64, 66.23, 67.38, 124.92, 145.93, 161.90, 170.44, 175.98.

HR MS(ESI) m/z [M+Na]+ calculated. for C20H34N2O5SNaSi: 465.1855; found: 465.1858

(3S,4R,1’R)-4-(2’’-acetamidoethyltiocarbonylmethyl)-3-[1’-(tert-butylodimethylsiloxy) ethyl]-azetidin-2-one (22)

Compound 20 (45 mg, 0,13 mmol) in acetonitrile (2 ml) was cooled to 0 °C and treated with N,N-diisopropylethylamine (25 μl, 0.14 mmol), diphenyl chlorophosphate (30 μl, 0.14 mmol) and a catalytic amount of DMAP. After 1 h an another portion of N,N-diisopropylethyloamine (25 μl, 0.14 mmol) and N-acetylcysteamine (15 μl, 0.14 mmol) were added. The mixture was stirred overnight. Subsequently it was evaporated under reduced pressure and the residue was purified by chromatography using hexane-acetone, 4:6 (v/v) as an eluent to give 22 (8 mg, 0.017 mmol) in 13% yield.

Syrup; [α]D +12,3 (c 0,6, CH2Cl2); IR (film) v: 1660, 1682, 1748 2930, 2954, 3202 cm−1; 1H NMR (600 MHz, C6D6) δ: −0.01 (s, 3H, Si(CH3)2), 0.08 (s, 3H, Si(CH3)2), 0,88 (s, 9H, t-Bu), 1.07 (d, J=6.1 Hz, 3H, CH3), 1.47(s, 3H, Ac), 2.56 (dd, J=2.4 Hz, 6,1 Hz, 1H, H-3), 2.58–2.73 (m, 4H, SCH2, CH2COS), 3.03-3.12 (m, 2H, CH2NH), 3.19 (s, 3H, CO2CH3), 3.89 (m, 2H, NCH2CO2), 4.04–4.10 (m, 1H, CHOSi), 4.10-4.14 (m, 1H, H-4), 4.72 (bs, 1H, NH); 13C NMR (150 MHz, CDCl3) δ: −5.22, −4.63, 22.28, 22.30, 25.58, 28.60, 38.70, 42.20, 47.61, 51.32, 52.13, 63.71, 66.03, 128.05, 166.53, 168.53, 168.88, 197.08.

HR MS(ESI) m/z [M+Na]+ calculated for C20H36N2O6SiNaS: 483.1961; found: 483.1958.