Synthesis and Antibacterial Activities of N-Glycosylated Derivatives of

Mar 12, 2009 - E-mail: [email protected] (Z.G.); [email protected] (Q.W.)., †. Department of Chemistry, Wayne State University. , ‡. School of ...
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2052

J. Med. Chem. 2009, 52, 2052–2059

Synthesis and Antibacterial Activities of N-Glycosylated Derivatives of Tyrocidine A, a Macrocyclic Peptide Antibiotic Honggang Hu,†,‡ Jie Xue,† Benjamin M. Swarts,† Qianli Wang,† Qiuye Wu,*,‡ and Zhongwu Guo*,† Department of Chemistry, Wayne State UniVersity, Detroit, Michigan 48202, School of Pharmacy, Second Military Medical UniVersity, Shanghai 200433, China ReceiVed December 14, 2008

An efficient and practical method for macrocyclic glycopeptide synthesis was developed and utilized to synthesize tyrocidine A and its glycosylated derivatives. The method is based on solid-phase peptide synthesis using 2-chlorotrityl resin as the solid-phase support and glycosyl amino acids as building blocks. After glycopeptides with fully protected glycans and side chains were released from the acid-labile resin, their Cand N-termini were intramolecularly coupled in solution to afford cyclic glycopeptides in quantitative yields. This synthetic method should be generally applicable to various macrocyclic glycopeptides. Biological studies of the synthetic tyrocidine A derivatives showed that linking glycans directly to the Asn residue of tyrocidine A diminished its antibacterial activity, but linking glycans to Asn via a simple spacer did not. These results revealed the important impact of glycans on the activities, and probably the structures, of glycopeptide antibiotics. Introduction The fast growing prevalence of resistance to antibiotics administered in clinics necessitates the discovery, design, and development of new antibacterial agents.1-3 In this regard, macrocyclic peptide/glycopeptide antibiotics have gained renewed attention.4-8 An example of such types of antibiotics is tyrocidine A (1, Chart 1), a cyclic decapeptide isolated from Bacillus bacteria9 that exhibits strong bactericidal activities.10 Tyrocidine A has been suggested to primarily target the bacterial membrane; it is therefore difficult for bacteria to develop resistance to this antibiotic because significant alterations of the bacterial cell wall lipid composition would be required.10 As a result, tyrocidine A represents an attractive lead compound for the development of new antibacterial drugs11-16 despite its severe side effects. Meanwhile, many natural macrocyclic peptide antibiotics are glycosylated, and the glycans are usually pivotal to the antibacterial activity.4-6 Glycosylation can have a multifaceted impact on the properties and functions of peptides, such as increasing peptide hydrophilicity and oral bioavailability, forcing peptides to adopt certain conformations and/or stabilizing the peptide conformations, conferring peptide resistance to proteolytic cleavage, and so on. The glycans of glycopeptide antibiotics may also play a critical role in interacting with or binding to the target molecules. Thus, glycosylation can be a useful tool for improving the physical and biological properties of peptide antibiotics in the development of new antibacterial agents. In view of the widespread occurrence of macrocyclic glycopeptide antibiotics in nature and the important roles of carbohydrate in these structures, we are interested in developing a general and practical strategy for the preparation of macrocyclic glycopeptides. The strategy will be used to prepare not only * To whom correspondence should be addressed. Phone: ++1 (313) 5772557 (Z.G.); ++86 (21) 2507-0381 (Q.W.). Fax: 1 (313) 577 8822 (Z.G.); 86 (21) 2507 0381 (Q.W.). E-mail: [email protected] (Z.G.); [email protected] (Q.W.). † Department of Chemistry, Wayne State University. ‡ School of Pharmacy, Second Military Medical University.

the natural products but also their derivatives bearing different carbohydrates, as well as various glycosylated forms of the naturally nonglycosylated macrocyclic peptides, for structureactivity relationship studies and for the development of novel antibiotics. To explore a new synthetic strategy, tyrocidine A was utilized as the first example in this study. Consequently, we designed and synthesized several N-glycosylated tyrocidine A derivatives 2a-c and evaluated their bactericidal activities to reveal the influence of glycosylation on tyrocidine A. Tyrocidine A has been synthesized by a number of groups employing different strategies.17-22 Recently, Walsh and co-workers13,14 have prepared and studied several glycosylated derivatives of tyrocidine A. Their synthetic strategy was based on enzyme-catalyzed cyclization of peptides14 or glycopeptides,13 which required the conversion of peptides/glycopeptides into corresponding thioesters. Moreover, for the former, glycans were attached to the resultant cylclopeptides via click chemistry.14 The present report describes a new synthetic strategy that is based on direct chemical cyclization of glycopeptides, and the glycans can be attached to the peptide backbone either through a native glycosidic linkage or through a simple linker. This synthetic strategy should be broadly applicable to the synthesis of natural and unnatural cyclic glycopeptides. Results and Discussion Synthetic Design. Our plan was to first construct linear glycopeptides by solid-phase peptide synthesis (SPPSa) and then unite their C-terminus and N-terminus via chemical condensation to obtain cyclic glycopeptides. For solid-phase glycopeptide assembly, literature results showed that glycosyl amino acids a Abbreviations: Ac2O,acetic anhydride; Bn, benzyl; Boc, t-butylcarboxyl; tBu, t-butyl; DCC, dicyclohexylcarbodiimide; DCM, dichloromethane; DIPEA, diisopropylethylamine; DMF, dimethylformamide; EtOAc, ethyl acetate; EtOH, ethanol; Fmoc, 9-fluorenylmethyloxycarbonyl; HBTU, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HOAc, acetic acid; HOBt, N-hydroxybenzotriazole; MeOH, methanol; MIC, minimum inhibition concentration; NIS, N-iodosuccinimide; NMP, N-methyl-2-pyrrolidone; OD, optical density; PyBOP, benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate; SPPS, solid-phase peptide synthesis; TES, triethylsilane; TFE, trifluoroethanol, TfOH, triflic acid; TLC, thin layer chromatography; TMS, tetramethylsilane; Trt: trityl.

10.1021/jm801577r CCC: $40.75  2009 American Chemical Society Published on Web 03/12/2009

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Chart 1

Scheme 1

Chart 2

could be directly used as building blocks, just like other simple amino acids, for coupling to the peptide backbone efficiently.23,24 On the other hand, as the synthetic targets may contain free amino and/or carboxylic groups within their amino acid side chains, to secure regioselective coupling reactions between the peptide C- and N-termini, it is necessary to have the glycopeptide side chains fully protected during the cyclization reaction. In this regard, the extremely acid-sensitive 2-chlorotrityl resin (3) can be a useful SPPS support,25 as peptides can be released from this resin using 10% acetic acid, which does not affect the protecting groups of amino acid side chains.26 Another advantage of using fully protected glycopeptides for cyclic glycopeptide synthesis is that these substrates would be easily soluble in organic solvents, which should be particularly helpful for the cyclization reaction. On the basis of the above considerations, we envisioned a synthetic strategy as outlined in Scheme 1. Synthesis of Glycosylated Asparagine Derivatives. Synthetic targets 2a-c have a mono- or disaccharide attached to their asparagine side chain. The monosaccharide and disaccharide in 2a and 2b represent the inner core of N-glycans of glycoproteins and are coupled to tyrocidine A through a native N-linkage. Lactose in 2c is a binding ligand of asialoglycoprotien receptors27 and may help 2c target the liver, while the short spacer between the sugar and peptide chains can reduce the steric interactions between the ligand and the receptor during their

recognition and binding process. Glycosyl asparagine derivatives 4a-c were key intermediates for the synthesis of 2a-c, and our first undertaking was to prepare 4a-c (Chart 2). The synthesis of 4a and 4b, as shown in Scheme 2, started from 2-amino-2-deoxy-D-glucose hydrochloride 5. First, 5 was transformed into 6, 7, and 8 by well established procedures.28-30 In 6, 7, and 8, the amino group was protected with a phthalyl group, and the anomeric center was protected as either an azidoglycoside or a p-toluenethioglycoside. Glycosyl azides can be readily and selectively reduced to form glycosyl amines to facilitate coupling with amino acids,28 while thioglycosides can be directly activated and used as glycosyl donors.31 Benzylation of 6, which gave only a moderate yield of 932 due to side reactions involving the phthalyl group under basic conditions, was followed by substitution of the phthalyl group in 9 with an acetyl group through a two-step-one-pot procedure28 to afford 10 in an excellent yield (94%). Next, 10 was reduced by Lindlar catalyst-catalyzed hydrogenation, and the resultant glycosyl amine was directly used without purification for coupling with 11 to give 12 in a very good yield (85%). The β-glycosidic linkage of 12 was verified by the observed large coupling constant between H-1 and H-2 (J1,2 ) 8.0 Hz), and no R-glycoside or amino acid racemization was noticed. Finally, the t-butyl group used to protect the carboxyl group of asparagine was removed with 25% trifluoroacetic acid in dichloromethane (DCM) to produce 4a.

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Scheme 2a

a Reagents and conditions: (a) BnBr, NaH, DMF, 66%; (b) 1,2-ethylenediamine, nBuOH, refl, then Ac2O, pyr., DCM, 94% for 10 and 91% for 15; (c) Lindlar catalyst, H2, then DCC, HOBt, DCM, 85% for 12 and 70% for 16; (d) 25% TFA, DCM, 98% for 4a and 92% for 4b; (e) NaOMe, MeOH, then BnBr, NaH, DMF, 59%; (f) NIS, TfOH, DCM, 74%.

Scheme 3a

a

Reagents and conditions: (a) 11, Lindlar catalyst, H2, then DCC, HOBt, DCM, 92%; (b) 25% TFA, DCM, 91%.

On the other hand, 8 was deacetylated and then benzylated to form glycosyl donor 13. The glycosylation of 7 by 13 was promoted by N-iodosuccinimide (NIS, 2 equiv) in the presence of a catalytic amount of triflic acid (TfOH),31 and the reaction was carried out at -30 °C to room temperature to produce disaccharide 14 stereoselectively in a good yield (74%). Again, the β-glycosidic linkage of 14 was verified by a large coupling constant between H-1 and H-2 (J1,2 ) 7.5 Hz), and its spectroscopic data were consistent with those reported in the literature.32,33 Finally, the replacement of the phthalyl group in 14 with an acetyl group, the successive coupling reaction with 11, and deprotection of the amino acid carboxyl group were carried out according to the procedures described for 4a to eventually afford 4b33 in an excellent overall yield. The synthesis of 4c (Scheme 3) was straightforward. After lactose (17) was converted into 1834,35 via several conventional transformations, the azido group was reduced and the resultant amine was then coupled with 11 to afford 19. Finally, the carboxyl group of 19 was deprotected with 25% TFA to give 4c, which was ready for the assembly of glycopeptides. Synthesis of the Title Compounds. From a retrosynthetic point of view, any peptide bond of the cyclic peptide can be cleaved to offer a linear structure to work with, so theoretically SPPS can start from any amino acid in the ring. Practically, however, various synthetic plans can give different results.36 For example, if there is a Gly in the ring, it is probably more convenient to break its peptide bond and leave Gly at the C-terminus. SPPS starting with Gly can make use of the relatively inexpensive Gly-loaded resin. More importantly, if Gly is the C-terminal amino acid, the subsequent cyclization reaction would probably be easier and more effective because of relatively small steric hindrance, and the potential of C-terminal amino acid racemization during the condensation reaction can be avoided as well. In the case of glycopeptide

synthesis, it is readily imaginable that the glycosylated amino acids should be installed at a later stage so as to reduce any potential interferences caused by the glycans and, moreover, to facilitate manual installation of the glycosyl amino acids, if necessary. In our synthesis of the title compounds, we chose to construct the linear peptide/glycopeptides with 2-chlorotrityl resin as the solid-phase support and Phe as the first amino acid to install. In this case, asparagine would be the second-to-last amino acid to be introduced (Scheme 4). Thus, Phe was first anchored to the solid-phase support via reacting trityl resin 20 with FmocPhe-OH sodium salt. Next, the peptide and glycopeptides were assembled via sequential introduction of Fmoc-Pro-OH, FmocD-Phe-OH, Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Val-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Asn(Trt)-OH or glycosyl Asn derivatives 4a-c, and Fmoc-D-Phe-OH. Nonglycosylated tyrocidine A peptide (22a/23a) was completely constructed by automatic SPPS using 20% piperidine for the deprotection of Fmoc and HBTU/DIPEA for the coupling reactions. For the glycopeptide (22b-d/23b-d) assembly, amino acids 2-8 were introduced by an automatic peptide synthesizer, but glycosyl amino acids 4a-c and the last FmocD-Phe-OH were installed manually with DCC and HOBt as the condensation reagents. This design aimed to minimize the use of 4a-c; therefore, only 2 equiv of 4a-c were employed. The coupling processes were monitored by detecting Fmoc released in each deprotection step, which suggested that all coupling reactions were very effective. Subsequently, the loaded resins were treated with 10% HOAc in trifluoroethanol (TFE) and DCM (1:8) to give peptide and glycopeptides 23a-d. Their MS data verified that the protecting groups of amino acid side chains, and the carbohydrates were not affected under these conditions. The released peptide and glycopeptides 23a-d were directly used in the next step without further purification.

N-Glycosylated DeriVatiVes of Tyrocidine A

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Scheme 4a

a Reagents and conditions: (a) HOAc, TFE, DCM (1:1:8), rt; (b) PyBOP, HOBt, DIPEA, DCM; (c) TFA, Et3SiH, DCM (2:1:8) for 24a, 61% (overall after HPLC); (d) Pd/C, H2; then TFA, Et3SiH, DCM (2:1:8) for 24c-d, 59-64% (overall after HPLC).

The cyclization of 23a-d was realized with benzotriazol-1yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), HOBt, and DIPEA as the condensation reagents.36,37 To promote intramolecular reaction, the substrate peptide/glycopeptides 23a-d were added very slowly to the DCM solution of condensation reagents, which would guarantee that the substrate concentration was kept low during the reaction process. Both TLC and MS results indicated that the cyclization reactions were clean and produced only cyclic products 24a-d. Then the side chain protecting groups of 24a were removed with 20% TFA in DCM containing 10% triethylsilane (TES) to afford tyrocidine A (1). Global deprotection of 24b-d was realized in two steps, namely catalytic hydrogenation to remove the benzyl groups used to protect sugar chains and then TFA treatment to remove the amino acid side chain protecting groups to afford cyclic glycopeptides 2a-c. The final products were thoroughly purified by reverse phase HPLC to afford pure 1 and 2a-c in excellent overall yields (59-64%, starting from the step to load Phe onto the resin). All final products were fully characterized by NMR and HR MS, and the product purity was evaluated with HPLC and was more than 95%. Antibacterial Activities. The bactericidal activities of the synthetic tyrocidine A (1) and its glycosylated derivatives 2a-c were evaluated using Gram-positive bacterium Bacillus subtilis (strain CMCC-B 63501), and the minimum inhibition concentrations (MIC) were determined according to a published method.18,38 The MIC of 1 was 31 µg/mL, consistent with the reported value for natural tyrocidine A (20 µg/mL). Of the three glycosylated derivatives 2a-c, the antibacterial activity of 2c (MIC, 62 µg/mL) was comparable to that of 1, but 2a and 2b did not show obvious antibacterial activity at concentrations up to 500 µg/mL. Evidently, direct linkage of glycans to the cyclic peptide of tyrocidine A had a major influence on its antibacterial activity, while linkage of glycans to the cyclic peptide through

a simple spacer had no or very little influence on the antibacterial activity. These results suggested that the N-linked glycans in 2a and 2b might have resulted in significant structural and conformational changes of the cyclic peptide, while the glycan linked to the cyclic peptide through a short spacer did not. How N-glycosylation affected the peptide structure or the antibacterial activity of tyrocidine A is an interesting question worthy of further investigation. Conclusion In summary, a highly efficient and versatile synthetic method was developed for macrocyclic glycopeptides. The method is based on SPPS using a hyper acid-labile resin, i.e., 2-chlorotrityl resin, as the solid-phase support and glycosyl amino acids as building blocks, which allowed access to glycopeptides having fully protected glycans and peptides. The cyclization reactions of these glycopeptides gave the desired cyclic glycopeptides in practically quantitative yields. This method was employed to synthesize three glycosylated derivatives of tyrocidine A, and it should be applicable to other macrocyclic glycopeptides as well. Biological studies of the synthetic compounds suggest that N-glycosylation of tyrocidine A had a significant impact on its antibacterial activity. This discovery, as well as further studies on the structures and activities of cyclic glycopeptides that can be synthesized by the present method, can be generally useful for understanding the functions of carbohydrates in macrocyclic glycopeptide antibiotics and for the development of new antibacterial agents. Experimental Section General Procedures. 1H NMR spectra were recorded at 400 and 500 MHz with the chemical shifts reported in ppm (δ) downfield from tetramethylsilane (TMS), and 13C NMR spectra were recorded at 100 and 120 MHz with the 13C signal of CDCl3

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(77.23 ppm) as reference. Coupling constants (J) are reported in hertz (Hz). Thin layer chromatography (TLC) was performed on silica gel plates detected by charring with phosphomolybdic acid in EtOH or 5% H2SO4 in EtOH. Commercial anhydrous solvents and reagents were used without further purification. 3,4,6-tri-O-Benzyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl Azide (9). After 6 (1.0 g, 3.0 mmol), Bu4NI (100 mg, 0.25 mmol) and BnBr (3 mL, 24.1 mmol) were dissolved in DMF (30 mL), and NaH (500 mg, 20.8 mmol) was added slowly at 0 °C. The reaction mixture was stirred at 0 °C for 1 h and then at rt overnight. The reaction was quenched with saturated NH4Cl solution and extracted with DCM. The organic phase was washed with NH4Cl solution, brine and H2O, dried over Na2SO4, and then condensed in vacuum to give crude product, which was purified by flash column chromatography to give 1.2 g of pure 9 (66%) as a white solid. 1H NMR (400 MHz, CDCl3): δ 7.92-7.73 (m, 4 H), 7.20-7.41 (m, 10 H), 6.80-7.00 (m, 5 H), 5.36-5.40 (d, J ) 10.0 Hz 1 H), 4.78-4.85 (m, 2 H), 4.58-4.70 (m, 3 H), 4.11 (t, J ) 10.0 Hz, 1 H), 3.76-3.85 (m, 3 H), 3.72-3.75 (m, 1 H). 13C NMR (100 MHz, CDCl3) δ 138.16, 138.04, 138.02, 128.77, 128.68, 128.33, 128.22, 128.17, 128.06, 127.97, 127.65, 123.67, 85.92, 79.35, 79.25, 77.55, 75.33, 75.16, 73.82, 68.45, 55.58. The NMR data were consistent with those reported.32 ESI MS: calcd for C35H32N4NaO6 [M + Na]+ m/z, 627.2; found, 627.3. 2-Acetamido-3,4,6-tri-O-benzyl-2-deoxy-β-D-glucopyranosyl Azide (10). After a solution of 9 (500 mg, 0.83 mmol), NH2CH2CH2NH2 (5 mL), and n-butanol (25 mL) was stirred at 90 °C overnight, it was concentrated to dryness under vacuum. The residue was dissolved in Ac2O and pyridine (12 mL, 1:2) and stirred at rt overnight. The reaction mixture was diluted with EtOAc (50 mL), and the solution was washed with saturated NaHCO3 and brine, dried over Na2SO4, and concentrated in vacuum. The residue was purified on a silica gel column to give 10 as a white solid (403 mg, 94%). 1H NMR (500 MHz, CDCl3): δ 7.35-7.19 (m, 15 H), 5. 42 (d, J ) 9.6 Hz, 1 H), 4.93 (d, J ) 6.8 Hz, 1 H), 4.85-4.78 (m, 2 H), 4.64-4.54 (m, 4 H), 3.97 (t, J ) 7.6 Hz, 1 H), 3.80-3.71 (m, 2 H), 3.70 (t, J ) 7.6 Hz, 1 H), 3.63-3.55 (m, 1 H), 3.45 (dd, J ) 7.2 Hz, 1 H), 1.85 (s, 3 H). 13C NMR (100 MHz, CDCl3): δ 170.85, 1 38.42, 138.15, 138.04, 128.81, 128.70, 128.66, 128.31, 128.18, 128.15, 128.07, 127.95, 80.71, 78.43, 77.33, 75.07, 75.00, 73.79, 68.63, 56.42, 23.68. The NMR data were consistent with those reported.32 ESI MS: calcd for C35H32N4NaO6 [M + Na]+ m/z, 539.2; found, 539.1. p-Methylphenyl 3,4,6-tri-O-Benzyl-2-deoxy-2-phthalimido-1thio-β-D-glucopyranoside (13). After the solution of 8 (5 g, 9.23 mmol) in 40 mL of 0.05 M NaOMe in methanol was stirred at rt for 2 h, it was neutralized to pH 6-7 using Amberlyst H+ resin. The solution was filtered off and concentrated to afford a white powder, which was directly used for next step. The crude product (3.00 g, 7.22 mmol) was dissolved with TBAI (300 mg, 0.76 mmol) and BnBr (5.71 mL, 45.88 mmol) in DMF (60 mL) at 0 °C, and then NaH (1.6 g, 40.00 mmol) was added slowly. The reaction mixture was stirred at 0 °C for 1 h and then at rt overnight. Workup of the reaction was completed as described for 9, and the crude product was purified by column chromatography to afford pure 13 (2.9 g, 59%) as a white solid. 1H NMR (400 MHz, CDCl3): δ 7.907.60 (m, 4 H), 7.42-7.26 (m, 10 H), 7.04-6.82 (m, 5 H), 5.51 (d, J ) 8.0 Hz, 1 H), 4.76-4.90 (m, 2 H), 4.74-4.56 (m, 3 H), 4.50-4.36 (m, 2 H), 4.26 (t, J ) 7.6 Hz, 1 H), 3.90-3.66 (m, 4 H), 2.29 (s, 3 H). 13C NMR (125 MHz, CDCl3): δ 138.56, 138.30, 138.22, 138.05, 134.06, 133.91, 133.65, 129.76, 128.57, 128.29, 128.18, 128.06, 127.92, 127.77, 127,58, 123.66, 123.50, 83.56, 80.54, 79.65, 79.63, 75.23, 75.19, 73.69, 69.11, 55.24, 21.34. ESI MS: calcd. for C42H39NNaO6S [M + Na]+ m/z, 708.3; found, 708.4. O-(-3,4,6-tri-O-Benzyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-(1f4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl Azide (14). To a mixture of 13 (1.0 g, 1.46 mmol), 7 (514 mg, 1.0 mmol), and MS 4 Å (1.0 g) in dry CH2Cl2 (50 mL) were added NIS (520 mg, 2.31 mmol) and then slowly TfOH (41 µL, 0.46 mmol) under an Ar atmosphere at -30 °C. After the mixture was stirred for 20 min, it was filtered off through celite, and the

Hu et al.

filtrate was washed with aqueous NaHCO3 and Na2S2O3 solutions and brine sequentially. The organic layer was dried over Na2SO4, filtered, and concentrated to give the crude product, which was purified by silica gel chromatography to afford pure 14 (800 mg, 74%) as colorless oil. 1H NMR (400 MHz, CDCl3): δ 7.91-7.53 (m, 8 H), 7.40-7.20 (m, 15 H), 7.01-6.83 (m, 10 H), 5.34 (d, J ) 8.8 Hz, 1 H), 5.17 (d, J ) 10.0 Hz, 1 H), 4.90 (d, J ) 12.8 Hz, 1 H), 4.87-4.76 (m, 2 H), 4.70 (d, J ) 10.8 Hz, 1 H), 4.62-4.35 (m, 7 H), 4.30-4.13 (m, 3 H), 4.06 (t, (t, J ) 9.6 Hz, 1 H), 3.87 (t, J ) 9.6 Hz, 1 H), 3.80-3.64 (m, 2 H), 3.57 (d, J ) 10.0 Hz, 1 H), 3.50-3.35 (m, 3 H). 13C NMR (125 MHz, CDCl3): δ 138.75, 138.53, 138.30, 133.99, 131.78, 128.68, 128.61, 128.54, 128.29, 128.15, 128.07, 127.97, 127.77, 127.67, 127.57, 127.17, 123.58, 97.18, 85.78, 79.86, 79.26, 77.04, 76.79, 75.37, 75.12, 75.06, 74.79, 73.48, 72.98, 68.23, 67.95, 56.93, 55.43. NMR data were consistent with those reported.33 MALDI-TOF MS: calcd for C63H57N5O12Na [M + Na]+ m/z, 1098.2; found, 1098.4. O-(2-Acetamido-3,4,6-tri-O-benzyl-2-deoxy-β-D-glucopyranosyl)(1f4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-β-D-glucopyranosyl Azide (15). After the mixture of 14 (200 mg, 0.19 mmol), NH2CH2CH2NH2 (1 mL), and n-butanol (5 mL) was stirred at 90 °C overnight, it was concentrated to dryness in vacuum. The residue was dissolved in Ac2O and pyridine (3 mL, 1:2) and stirred at rt overnight. The reaction mixture was diluted with EtOAc (50 mL), and the solution was washed with saturated NaHCO3 and brine, dried over Na2SO4, and concentrated in vacuum. The residue was purified on a silica gel column to give 15 as a white solid (156 mg, 91.2%). 1H NMR (400 MHz, CDCl3): δ 7.40-7.16 (m, 25 H), 6.33 (d, J ) 7.2 Hz, 1 H), 5.01 (d, J ) 7.8 Hz, 1 H), 4.85-4.72 (m, 3 H), 4.71-4.62 (m, 2 H), 4.63-4.54 (m, 3 H), 4.53-4.43 (m, 3 H), 4.35 (d, J ) 7.2 Hz, 1 H) 4.01 (t, J ) 6.4 Hz, 1 H), 3.92 (m, 1 H), 3.83-3.57 (m, 8 H), 3.55-3.45 (m, 1 H), 3.38-3.31 (m, 1 H). 2.01 (s, 3 H) 1.73 (s, 3 H). 13C NMR (100 MHz, CDCl3): δ 170.88, 170.83, 138.65, 138.40, 138.16, 138.12, 138.08, 128.88, 128.80, 128.72, 128.64, 128,56, 128.50, 128.30, 128.22, 128.15, 128.03, 127.97, 127.92, 127.76, 100.10, 88.58, 81.15, 78.71, 78.06, 76.61, 75.12, 75.06, 74.66, 73.94, 73.86, 73.71, 72.99, 69.29, 68.77, 55.70, 52.15, 23.73, 23.35. NMR data were consistent with those reported.33 MALDI-TOF MS: calcd for C51H57N5O10Na [M + Na]+ m/z, 922.5; found, 922.8. Preparation of Glycosyl Amino Acids 4a-c (Common Procedures). To a solution of 11 in DCM (10 mL) and NMP (1 mL) was subsequently added HOBt and DCC, and the mixture was stirred at rt for 1 h to form the active ester of 11. Meanwhile, a mixture of glycosyl azide 10, 15, or 18 and Lindlar catalyst in MeOH was stirred at rt for 2.5 h under a H2 atmosphere. After filtration of the reaction mixture through celite to remove the catalyst and then concentration of the solution in vacuum, to the resulting residue were added DCM (2 mL) and the freshly prepared solution of the active ester. The mixture was stirred at rt overnight, concentrated in vacuum, and directly applied to silica gel column chromatography to give 12, 16, and 19, respectively, as white solids. 12 (85%): 1H NMR (400 MHz, CDCl3) δ 7.76 (d, J ) 7.2 Hz, 2 H), 7.61 (d, J ) 7.2 Hz, 2 H), 7.42-7.14 (m, 19 H), 5.97 (d, J ) 8.8 Hz, 1 H), 4.96-4.73 (m, 4 H), 4.59 (d, J ) 11.5 Hz, 1 H), 4.56 (d, J ) 12.5 Hz, 1 H), 4.48-4.34 (m, 4 H), 4.70-4.54 (m, 3 H), 4.53-4.32 (m, 3 H), 4.29 (t, J ) 8.0 Hz, 1 H), 4.22 (t, J ) 7.2 Hz, 1 H), 3.93-3.66 (m, 4 H), 3.54-3.40 (m, 2 H), 2.82 (ddd, J ) 16 and 4 Hz, 2 H), 1.71 (s, 3 H), 1.42 (s, 9 H). 13C NMR (100 MHz, CDCl3) δ 172.54, 171.06, 170.26, 156.36, 144.20, 144.12, 141.48, 138.20, 137.98, 129.18, 129.07, 128.73, 128.63, 128.32, 128.29, 128.21, 128.00, 127.89, 127.31, 125.49, 120.16, 82.22, 80.73, 78.56, 76.71, 75.26, 74.49, 73.86, 68.29, 67.37, 53.86, 51.25, 47.38, 38.05, 28.13, 23.33. NMR data were consistent with those reported.39 MALDI-TOF MS: calcd for C52H57N3O10Na [M + Na]+ m/z, 906.4; found, 906.9. 16 (70%): 1H NMR (500 MHz, CD3OD/ CDCl3 1:5) δ 7.71 (d, J ) 8.0 Hz, 2 H), 7.56 (d, J ) 7.5 Hz, 2 H), 7.40-7.06 (m, 29 H), 4.79-4.67 (m, 4 H), 4.61-4.53 (m, 3 H), 4.51 (d, J ) 10.5 Hz, 1H), 4.42 (t, J ) 5 Hz, 1 H), 4.40-4.31 (m, 5 H), 4.23 (t, J ) 7.5 Hz, 1 H), 4.16 (t, J ) 7.0 Hz, 1 H), 4.04 (t, J ) 7.0 Hz, 1 H), 3.96 (t, J ) 8.5 Hz, 1 H), 3.84 (t, J ) 10.0 Hz,

N-Glycosylated DeriVatiVes of Tyrocidine A

1 H), 3.64-3.49 (m, 7 H), 3.49-3.41 (m, 2 H), 3.28-3.23 (m, 1 H), 2.77 (ddd, J ) 16 and 3.5 Hz, 2 H), 1.78 (s, 3 H), 1.73 (s, 3 H), 1.37 (s, 9 H). NMR data were consistent with those reported.33 MALDI-TOF MS: calcd for C74H82N4O15Na [M + Na]+ m/z, 1289.6; found, 1289.8. 19 (92%): 1H NMR (400 MHz, CDCl3) δ 7.73 (d, J ) 7.6 Hz, 1 H), 7.60 (t, J ) 7.2 Hz, 1 H), 7.42-7.09 (m, 39 H), 6.41 (m, 1 H), 6.03 (d, J ) 8.8 Hz, 1 H), 5.03 (d, J ) 10.4 Hz, 1 H), 4.98 (d, J ) 11.2 Hz, 1 H), 4.83-4.64 (m, 7 H), 4.56 (d, J ) 11.6 Hz, 1 H), 4.49-4.16 (m, 10 H), 3.93-3.66 (m, 7 H), 3.60-3.26 (m, 10 H), 2.58 (ddd, J ) 15.2 and 4 Hz, 2 H), 1.44 (s, 9 H). 13C NMR (100 MHz, CDCl3) δ 170.33, 170.26, 156.38, 144.15, 141.48, 139.24, 139.00, 138.75, 138.71, 138.31, 138.06, 128.61, 128.50, 128.41, 128.24, 128.04, 127.98, 127.80, 127.75, 127.67, 127.35, 127.08, 125.55, 120.17, 104.19, 103.23, 83.23, 82.76, 82.28, 81.99, 80.13, 75.64, 75.51, 75.37, 75.03, 74.97, 73.74, 73.63, 73.37, 73.29, 72.78, 70.29, 68.76, 68.29, 67.38, 51.56, 47.36, 40.21, 37.53, 28.18. MALDI-TOF MS: calcd for C86H92N2O16Na [M + Na]+ m/z, 1431.6; found, 1431.7. The respective solutions of 12, 16, and 19 in TFA (1 mL) in anhydrous DCM (4 mL) were stirred in the dark at rt for 2 h, and the mixtures were concentrated in vacuum. The residues were subjected to flash column chromatography to give 4a-c as white solids. 4a (98%): 1H NMR (400 MHz, CD3OD) δ 8.41 (d, J ) 9.6 Hz, 1 H), 8.04 (d, J ) 9.2 Hz, 1 H), 7.84 (d, J ) 7.2 Hz, 2 H), 7.72 (d, J ) 7.2 Hz, 2 H), 7.56-7.08 (m, 20 H), 5.02 (t, J ) 7.6 Hz, 1 H), 4.76-4.58 (m, 4 H), 4.58-4.11 (m, 9 H), 3.80 (m, 1 H), 4.48-4.34 (m, 4 H), 2.82 (dd, J ) 4.0, 16.0 Hz, 2 H), 1.76 (s, 3 H). NMR data were consistent with those reported.39 ESI MS: calcd for C48H49N3O10 [M - H]- m/z, 827.3; found, 826.6. 4b (96%): 1 H NMR (400 MHz, CD3OD-CDCl3 1:5) δ 7.76 (d, J ) 7.2 Hz, 2 H), 7.63 (d, J ) 7.2 Hz, 2 H), 7.37-7.16 (m, 32 H), 5.71 (d, J ) 2.4 Hz, 1 H), 4.78-4.52 (m, 9 H), 4.49-4.44 (m, 4 H), 4.39-4.31 (m, 2 H), 4.26-4.16 (m, 3 H), 3.93-3.73 (m, 6 H), 3.66-3.53 (m, 5 H), 3.37 (s, br, 1 H), 2.86 (dd, J ) 5.6, 16.4 Hz, 1 H), 2.76 (dd, J ) 7.2, 15.2 Hz, 2 H), 2.04 (s, 3 H), 1.86 (s, 3 H). NMR data were consistent with those reported.33 MALDI-TOF MS: calcd for C70H74N4O15Na [M + Na]+ m/z, 1233.5; found, 1233.7. 4c (99%): 1 H NMR (400 MHz, CD3OD-CDCl3 1:5) δ 7.70 (d, J ) 7.6 Hz, 1 H), 7.58 (t, J ) 7.2 Hz, 1 H), 7.42-7.09 (m, 41 H), 4.95 (d, J ) 10.4 Hz, 1 H), 4.90 (d, J ) 11.2 Hz, 1 H), 4.80-4.62 (m, 7 H), 4.58-4.10 (m, 11 H), 3.88-3.56 (m, 7 H), 3.50-3.26 (m, 10 H), 2.69 (dd, J ) 4.0, 15.2 Hz, 2 H). ESI MS: calcd for C82H84N2O16 [M - H]- m/z, 1352.6; found, 1351.9. Synthesis of the Title Compounds 1 and 2a-c (Common Procedures). After a mixture of trityl resin 20 (200 mg, 0.24 mmol) and Fmoc-Phe-ONa (98 mg, 0.24 mmol) was shaken on a vortex mixer at rt for 2 h, the resin was filtered off and washed several times with MeOH and DCM. The Phe-linked resin was then loaded onto the automatic peptide synthesizer for the construction of full length peptide/glycopeptides. The protocols employed were: deprotection of Fmoc with 20% piperidine in NMP and peptide coupling using 5 equiv of amino acid and 5 equiv of HBTU/DIPEA. All deprotection and coupling reactions were set to perform 2 h at rt. Amino acids Fmoc-Pro-OH, Fmoc-D-Phe-OH, Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Val-OH, Fmoc-Tyr(tBu)-OH, and FmocGln(Trt)-OH were sequentially installed to construct an octapeptide on the resin. In the synthesis of 22a, the peptide chain was further elongated by automatic peptide synthesis with Fmoc-Asn(Trt)-OH and Fmoc-D-Phe-OH as building blocks. In the synthesis of glycopeptides 22b-d, after the octapeptide was constructed, the loaded resin was removed from the peptide synthesizer and the remaining amino acids were installed manually. Glycosyl amino acids 4a-c (2 equiv) were respectively mixed with DCC and HOBt in DCM, and the mixture was transferred to a vessel containing the resin-bound octapeptide, which was shaken with a vortex mixer at rt overnight. After the resin was washed sequentially with NMP, MeOH, and DCM, it was treated with 20% piperidine in NMP at rt for 2 h. The resin was washed again with NMP, MeOH, and DCM and was finally subjected to coupling with Fmoc-D-Phe-OH (5 equiv) and de-Fmoc condition by the same methods to form the desired glycopeptide on resin (22b-d).

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 2057

To cleave the linear peptides 23a-d from the resin, the peptideloaded resin was treated with a mixture of HOAc and TFE in DCM (1:2:16, 15 mL) for 1 h at rt. The resin was filtered off and was washed with DCM and DCM/MeOH mixture. The washings were combined and condensed in vacuum to give 23a-d. 23a, MALDITOF MS: calcd. for C113H133N13O16Na [M + Na]+ m/z, 1950.9; found, 1950.8. 23b, MALDI-TOF MS: calcd for C123H150N14O21Na [M + Na]+ m/z, 2182.1; found, 2183.2. 23c, MALDI-TOF MS: calcd for C145H175N15O26Na [M + Na]+ m/z, 2565.2; found, 2564.8. 23d, MALDI-TOF MS: calcd for C157H185N13O27Na [M + Na]+ m/z, 2707.3; found, 2706.7. These crude products were applied to cyclization reactions without purification. For peptide/glycopeptide cyclization, after PyBOP (3 equiv), HOBt (3 equiv) and DIPEA (6 equiv) were dissolved in DCM (200 mL) in a round-bottom flask, 23a-d dissolved in DCM (0.5 mg/ml) was added to the solution over a period of 2 h. After the addition was finished, the mixture was stirred for another 10 h. Then, DCM was removed and the resulting residue was briefly purified by Sephadex LH-20 to afford 24a-d. 24a, MALDI-TOF MS: calcd for C113H131N13O15Na [M + Na]+ m/z, 1932.9; found, 1932.9. 24b, MALDI-TOF MS: calcd for C123H148N14O20Na [M + Na]+ m/z, 2164.1; found, 2164.5. 24c, MALDI-TOF MS: calcd for C145H173N15O25Na [M + Na]+ m/z, 2547.2; found, 2547.2. 24d, MALDI-TOF MS: calcd for C157H183N13O26Na [M + Na]+ m/z, 2689.3; found, 2688.8. For the deprotection of 24a, it was dissolved in a mixture of TFA, DCM, and Et3SiH (2:8:1, 5 mL), and the mixture was stirred at rt for 2 h. The solvent was removed in vacuum. After the residue was coevaporated with toluene for three times, it was dissolved in water (5 mL) and extracted with ether (3 × 3 mL) and finally purified by HPLC (Supelco Discovery C18 250 mm × 10 mm column, acetonitrile in water containing 0.1% TFA, gradient from 20% to 80% in 20 min, then 100% in 10 min, 2 mL/min) to give 1 (6.5 mg, 61%, Rt ) 26.5 min) as a white solid. 1H NMR: (400 MHz, CD3OD) δ 9.26-7.49 (signal of amide protons), 7.32-7.10 (m, 15 H), 6.94 (s, 2 H), 6.61 (m, 2 H), 5.78 (m, 1 H), 5.42(m, 1 H), 4.86-4.40 (m, 6 H), 4.09 (m, 1 H), 4.03 (m, 1 H), 3.42-2.76 (m, 9 H), 2.45-2.35 (m, 3 H), 2.15-1.94 (m, 5 H), 1.92-1.50 (m, 6 H), 1.49-0.62 (m, 16 H), 0.43 (s, 1 H). The results were fully consistent with those of natural tyrocidine A. MALDI-TOF MS: calcd for C66H88N13O13 [M + H]+ m/z, 1270.6, found 1270.6; calcd for C66H87N13O13Na [M + Na]+ m/z, 1292.6, found 1292.7; calcd for C66H87N13O13K [M + K]+ m/z, 1308.6, found 1308.7. HR-ESI MS: calcd for C66H88N13O13 [M + H]+ m/z, 1270.6625, found 1270.6674. For the deprotection of cyclic glycopeptides 24b-d, first, they were mixed with 10% Pd-C (10 mg) in MeOH (10 mL) and then the mixture was stirred under a H2 atmosphere at rt for 24 h. After the catalyst was filtered off, the solution was condensed in vacuum. The residue was then dissolved in a mixture of TFA, DCM, and Et3SiH (2:8:1, 5 mL). After the mixture was stirred at rt for 2 h, the solvent was removed in vacuum and the residue was coevaporated with toluene for three times. The crude product was dissolved in water (5 mL) and extracted with ether (3 × 3 mL), and was then purified by HPLC to give 2a-c. 2a (6.5 mg, 59.1%, white solid, Rt ) 15.0 min): 1H NMR (400 MHz, D2O): δ 8.15-7.40 (signal of amide protons), 7.39-7.05 (m, 15 H), 7.04 (d, J ) 8.0 Hz, 2 H), 6.82 (d, J ) 8.0 Hz, 2 H), 5.67 (d, J ) 10.8 Hz, 1 H), 5.28 (m, 1 H), 5.15 (d, J ) 9.6 Hz, 1H), 4.86-4.35 (m, 6 H), 4.18 (m, 1 H), 4.00 (m, 1 H), 3.69 (t, J ) 9.6 Hz, 1 H), 3.60 (t, J ) 9.6 Hz, 1 H), 3.51(t, J ) 8.4 Hz, 1 H), 3.41 (d, J ) 12.4 Hz, 1 H), 3.35-3.22 (m, 3 H), 3.20-2.72 (m, 8 H), 2.60 (m, 1 H), 2.28 (m, 1 H), 2.14-1.84 (m, 10 H), 1.82-1.18 (m, 10 H), 1.12-0.94 (m, 12 H), 0.32 (m, 1 H). MALDI-TOF MS: calcd for C74H101N14O18 [M + H]+ m/z, 1473.7, found 1473.9; calcd for C74H100N14O18Na [M + Na]+ m/z, 1495.7, found 1495.9. HR-ESI MS: calcd for C74H101N14O18 [M + H]+ m/z, 1473.7418, found 1473.7423. 2b (8.7 mg, 63.5%, white solid, Rt ) 29.0 min): 1H NMR (400 MHz, CD3OD): δ 9.30-7.40 (signal of amide protons), 7.39-7.08 (m, 15 H), 6.99 (s, 2 H), 6.59 (s, 2 H), 5.90-5.74 (m, 2 H), 5. 47 (s, 1 H), 4.86-4.35 (m, 6 H), 4.12-3.98 (m, 3 H), 3.96 (d, J ) 9.6 Hz, 1 H), 3.82-3.57 (m, 7 H), 3.54-2.78 (m, 10 H), 2.40-2.24

2058 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7

(m, 2 H), 2.22-1.88 (m, 9 H), 1.86-1.24 (m, 12 H), 1.16-0.82 (m, 12 H), 0.32 (s, 1 H). MALDI-TOF MS: calcd for C82H114N15O23 [M + H]+ m/z, 1676.8, found, 1676.9; calcd. for C82H113N15O23Na [M + Na]+ m/z, 1698.8, found 1698.9. HR-ESI MS: calcd for C82H114N15O23 [M + H]+ m/z, 1676.8212, found 1676.8215. 2c (10.3 mg, 58.5%, white solid, Rt ) 28.4 min): 1H NMR (400 MHz, CD3OD): δ 9.24-7.42 (signal of amide protons), 7.39-7.03 (m, 15 H), 6.88-6.80 (m, 2 H), 6.58 (s, 2 H), 5.75 (s, 1 H), 5. 42 (s, 1 H), 4.96-4.39 (m, 6 H), 4.28 (d, J ) 8.4 Hz, 1 H), 4.22 (d, J ) 8.0 Hz, 1 H,), 4.09 (m, 1 H), 4.04 (s, 1 H), 3.96 (s, 1 H), 3.85-2.75 (m, 29 H), 2.50-1.88 (m, 8 H), 1.86-1.54 (m, 6 H), 1.52-1.20 (m, 4 H), 1.19-0.80 (m, 12 H), 0.42 (s, 1 H). 13C NMR (DEPT) (CD3OD, 100 MHz): CH carbons 129.80, 129.46, 129.42, 129.96, 128.48, 128.30, 128.17, 127.35, 126.73, 126.57, 115.17 (CH of Tyr), 103.90 (1-CH of lactose), 102.82 (1′-CH of lactose), 79.35, 75.94, 75.17, 73.69, 73.54, 71.32, 69.08, 60.36, 58.75, 57.26, 55.64, 54.89, 54.25, 53.71, 51.11, 50.01, 32.38 (β-C of Val), 25.20 (R-C of Val). MALDI-TOF MS: calcd for C80H112N13O24 [M + H]+ m/z, 1638.8, found, 1638.8; calcd for C80H111N13O24Na [M + Na]+ m/z, 1660.8, found 1660.9. HR-ESI MS: calcd for C80H112N13O24 [M + H]+ m/z, 1638.7943, found 1638.7969. Evaluation of the Antibacterial Activities of 1 and 2a-c. The culture derived from a single colony of B. subtilis (CMCC-B 63501) was prepared by overnight incubation in LB medium on an orbital shaker under the optimal conditions (30 °C, 200 rpm). After the culture was diluted to a density of 5 × 105 colony-forming units/ mL, it was transferred onto a 96-well plate, and 200 µL of the culture was added to each well. Then, 2-fold serially diluted solutions of 1 and 2a-c, with the concentrations ranging from 1 to 500 µg/mL, were added to the wells. The negative control was prepared and treated under the same conditions without 1 and 2a-c. The plate was incubated at 30 °C on an orbital shaker (200 rpm) until the optical density (OD) at 600 nm of the negative control reached ca. 1.0. Finally, the OD600 values of all wells were measured with a microplate reader, and the MIC was determined from a plot of OD600 values versus the concentrations of 1 and 2a-c, as described in the literature.18,38

Acknowledgment. This work was supported by NSF (CHE0407144 and 0715275). We thank Drs. B. Shay and L. Hryhorczuk (WSU) for some MS measurements and Dr. B. Ksebati (WSU) for some NMR measurements. We thank Dr. Jiyang Li, School of Pharmacy, Fudan University, China, for evaluating the antibacterial activities of the title compounds. Supporting Information Available: NMR and MS spectra of all compounds and HPLC chromatograms of the title compounds 1 and 2a-c. This material is available free of charge via the Internet at http://pubs.acs.org.

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