Chiral O-(Z-α-Aminoacyl) Sugars: Convenient Building Blocks for

Bioconjugate Chem. 2007, 18, 994−998

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Chiral O-(Z-r-Aminoacyl) Sugars: Convenient Building Blocks for Glycopeptide Libraries Alan R. Katritzky,* Parul Angrish, and Tamari Narindoshvili Center for Heterocyclic Compounds, Department of Chemistry, University of Florida, Gainesville, Florida 32611-7200. Received November 27, 2006; Revised Manuscript Received January 25, 2007

1,2:3,4-Di-O-isopropylidene-R-D-galactopyranose (2), 1,2:5,6-di-O-isopropylidene-D-glucose (5), and 2,3:5,6-diO-isopropylidene-R-D-mannofuranose (7) are efficiently O-acylated in 78-96% yields with readily available N-(ZR-aminoacyl)benzotriazoles 1a-e, 1d+1d′ under microwave irradiation to give chiral 3a-d, 4, 6a-d, 8a,b and diastereomeric mixtures (3d+3d′), (6a+6a′), and (6d+6d′). The original chirality was retained as evidenced by HPLC. The diisopropylidene protecting groups were removed from compounds 3a,d, 6d to give the free O-(ZR-aminoacyl) sugars 9a,b, 10.

INTRODUCTION Various potential biologically active molecules cannot be used in the clinic due to undesirable physical properties such as low water solubility, low permeability in biological membranes, and rapid loss by metabolic degradation (1-3). For example, the intrinsic flexibility of short peptides can lead to multiple conformations which can consequently reduce the binding efficiency to a critical receptor and/or induce undesirable effects due to binding with other receptors present (4). Many of these drawbacks can be significantly mitigated by derivatization of the biomolecules. Thus, the incorporation of saccharide units into the peptide moiety can improve the physiochemical properties of various bioactive peptides and inter alia increase their immunizing performance (5), intestinal permeability (6), resistance to enzymatic degradation (7), molecular recognition, (8, 9), and rigidity of the peptide backbone (10-12). Advantageously, sugar-amino acid conjugates can offer both improved properties and access to significant diversification. Such efficacy arises from the fact that these hybrids are made up of two biocompatible components with almost unlimited potential structural variations (13, 14). Such findings have established a pivotal role for a carbohydrate moiety in numerous biological processes ranging from cell-cell communication, cell adhesion, protein folding, and their implication in various diseases such as chronic inflammation, microbial infections, and cancers (10, 15-35). In the past several years, considerable efforts have been devoted to the synthesis of sugar-amino acid conjugates utilizing solution and solid-phase methodologies and enzymatic synthesis. In solution-phase methodology, the desired conjugates are obtained using coupling reagents like DCC/DMAP, (36-40) succinic anhydride/DMAP (40), or COCl2-pyridine in dichloromethane (41). Examples prepared using these reagents utilize hexose moieties with all hydroxyl groups protected except for 6-OH, which enters into the coupling reaction. Coupling of an unprotected sugar with an N-protected amino acid can also be achieved via an active ester such as a pentachlorophenyl ester, in the presence of imidazole as catalyst (3, 12, 42-46) which requires reaction times of 20-24 h and provides a yield of 30-50% (3, 45, 46). Recently, solid-phase methodology has also been utilized for the preparation of such * Corresponding author. E-mail: [email protected].

conjugates, (47) utilizing coupling reagents like DCC and DIC in the presence of a catalytic amount of DMAP in DMF as solvent on a solid support bound to the protected sugar template with a free 6-OH. Finally, sugar-amino acid conjugates are also prepared by enzymatic synthesis in pyridine using commercial protease, Optimase M-440 (48). We have already used N-acylbenzotriazoles extensively for [N-acylation] (49-53), [C-acylation] (54-58), and [O-acylation] (58). Previously, 1-(R-Boc-, -Z-, and -Fmoc-aminoacyl)benzotriazoles enabled the preparation of chiral di-, tri-, and tetrapeptides in average yields of 88% from natural unprotected amino acids, both without (L-Ala, L-Phe, L-Val, L-Leu) and with unprotected side chain functionality (L-Trp, L-Tyr, L-Gln, L-Ser, L-Cys, L-Asn), in aqueous acetonitrile (59-63), as well as amino-amides from chiral amines (50). The original chirality was preserved in all cases (>95% as evidenced by NMR and >99% by HPLC) (59-63). Herein, we present a convenient and efficient formation of R-amino acid-sugar conjugates derived from N-(Z-R-aminoacyl)benzotriazoles (59-61) and sugar -OH groups under microwave irradiation.

EXPERIMENTAL PROCEDURES General Methods. Melting points were determined on a capillary point apparatus equipped with a digital thermometer. NMR spectra were recorded in CDCl3 or DMSO-d6 with TMS for 1H (300 MHz) and 13C (75 MHz) as an internal reference. N-Z-Amino acids and amino acids were purchased from Fluka and Acros and were used without further purification. All the reactions were carried out under microwave irradiation with a single mode cavity Discover Microwave Synthesizer (CEM Corporation, NC) producing a continuous irradiation at 2450 MHz. Elemental analyses was performed on a Carlo Erba-1106 instrument. Optical rotation values were measured with the use of sodium D line. Column chromatography was performed on silica gel (200-425 mesh). HPLC analyses were performed on Beckman system gold programmable solvent module 126 using Chirobiotic T column (4.6 × 250 mm), detection at 254 nm, flow rate 1.0 mL/min, and methanol as solvent. General Procedure for the Preparation of O-(Z-r-Aminoacyl)diisopropylidene Sugars 3, 4, 6, 8 under Microwave Irradiation. A dried heavy walled Pyrex tube containing a small stir bar was charged with N-(Z-R-aminoacyl)benzotriazole 1 (1.0 mmol), sugars 2, 5, 7 (1.5 mmol), DMAP (0.2 mmol), and THF (0.5 mL). For the preparation of 4, 1.0 equiv of

10.1021/bc0603687 CCC: $37.00 © 2007 American Chemical Society Published on Web 04/19/2007

Technical Notes

Bioconjugate Chem., Vol. 18, No. 3, 2007 995

Scheme 1

Scheme 2

Scheme 3

Scheme 4

1e, 2.5 equiv of 2, and 0.4 equiv of DMAP in 0.5 mL of dry THF were utilized. The reaction mixture was exposed to microwave irradiation (100 W) for 15 min (to obtain 3, 4, 6, 8) and 1 h [to obtain the diastereomeric mixtures1 (3d+3d′), (6a+6a′) and (6d+6d′)] at a temperature of 65 °C. After the irradiation, the reaction mixture was allowed to cool through an inbuilt system in the instrument until the temperature had fallen below 30 °C (ca. 10 min). The reaction mixture was subjected to silica-gel column chromatography using ethyl acetate/hexanes (2:1) as eluent to afford the desired glycosyl amino acids 3, 4, 6, 8. Compounds 3b-d, 4, 6a,d, (6a+6d′), 8a were recrystallized from CH2Cl2/hexane unless specified, otherwise Compounds 3a, 6b,c, 8b were obtained as colorless oils. 1 Compound numbers in brackets represent the diastereomeric mixtures of two components.

[(5R,5aS,8aS,8bR)-2,2,7,7-Tetramethyltetrahydro-3aH-di(1,3)dioxolo[4,5-b:4′,5′-d]pyran-5-yl]methyl (2S)-2-[(benzyloxy)carbonyl]amino-3-phenylpropanoate, Z-L-Phe-O-galactopyranose, 3a: Colorless oil (93%), [R]23D ) -20.4 (c 2.25, CH2Cl2); 1H NMR (CDCl3) δ 1.33 (s, 6H), 1.45 (s, 3H), 1.52 (s, 3H), 3.13 (quintet, J ) 6.0 Hz, 2H), 3.97-4.01 (m, 1H), 4.07 (d, J ) 8.0 Hz, 1H), 4.26 (dd, J ) 11.5, 4.9 Hz, 2H), 4.31-4.37 (m, 1H), 4.60 (dd, J ) 7.6, 2.2 Hz, 1H), 4.67-4.73 (m, 1H), 5.06 (d, J ) 12.3 Hz, 1H, B part of AB system), 5.11 (d, J ) 12.4 Hz, 1H, A part of AB system), 5.22 (d, J ) 7.9 Hz, 1H), 5.53 (d, J ) 5.0 Hz, 1H), 7.12-7.14 (m, 2H), 7.22-7.27 (m, 3H), 7.30-7.38 (m, 5H). 13C NMR (CDCl3) δ 24.4, 24.9, 25.9, 26.0, 38.2, 54.8, 64.1, 65.8, 66.9, 70.4, 70.6, 70.8, 96.2, 108.8, 109.6, 127.0, 128.0, 128.1, 128.5, 128.6, 129.4, 135.7, 136.2, 155.6, 171.4. Anal. Calcd for C25H35NO9: C, 64.31; H, 6.51; N, 2.59. Found: C, 64.04; H, 6.62; N, 2.55.

996 Bioconjugate Chem., Vol. 18, No. 3, 2007

Katritzky et al.

Table 1. Preparation of O-(Z-r-Aminoacyl)diisopropylidene Sugars 3a-d and the Diastereomeric Mixture (3d+3d′) from 2

a

R

product

yielda (%)

tR (min)b

CH2C6H5 1a CH2(3-indolyl) 1b CH2CH2SMe 1c CH3 1d CH3 1d+d

Z-L-Phe-O-galactopyranose 3a Z-L-Trp-O-galactopyranose 3b Z-L-Met-O-galactopyranose 3c Z-L-Ala-O-galactopyranose 3d Z-DL-Ala-O-galactopyranose (3d+3d)

93 88 90 87 91

3.01 3.08 2.99 3.00 3.01, 3.35

Isolated yield. b For conditions, refer to Experimental Section.

Table 2. Preparation of O-(Z-r-Aminoacyl)diisopropylidene Sugars 6a-d and Diastereomeric Mixtures (6a+6a′), (6d+6d′) from 5

a

R

product

yielda (%)

tR (min)b

CH2C6H5 1a CH2C6H5 1a+1a CH2(3-indolyl) 1b CH2CH2SMe 1c CH3 1d CH3 1d+1d

Z-L-Phe-O-diacetone glucose 6a Z-DL-Phe-O-diacetone glucose (6a+6a) Z-L-Trp-O-diacetone glucose 6b Z-L-Met-O-diacetone glucose 6c Z-L-Ala-O-diacetone glucose 6d Z-DL-Ala-O-diacetone glucose (6d+6d)

83 82 94 93 83 91

2.99 2.99, 3.23 3.01 2.99 2.99 2.99, 3.36

Isolated yield. b For conditions, refer to Experimental Section.

General Procedure for the Cleavage of Diisopropylidene Group Present in O-(Z-r-Aminoacyl)diisopropylidene Sugars 3a,b, 6d. TFA-H2O mixture was added to 3a,b, 6d (0.200 g) at room temperature and were then stirred until 3a,b, 6d were completely consumed as monitored by TLC. The resulting mixture was concentrated under vaccum followed by washings with diethyl ether (5-6 times) and drying under reduced pressure to afford the desired compound 9a,b, 10 with free OH groups. (3,4,5,6-Tetrahydroxytetrahydro-2H-pyran-2-yl)methyl (2S)2-[(benzyloxy)carbonyl]amino-3-phenylpropanoate, Z-L-PheO-D-glucose, 9a: White solid (92%), mp 110 °C, decomposed and turned black brown: [R]23D ) +3.4 (c 2.50, DMF); 1H NMR (DMSO-d6) δ R/β: 2.81-2.92 (m, 1H), 3.00-3.14 (m, 1H), 3.44-3.62 (m, 2H), 3.68-3.95 (m, 2H), 4.05-4.18 (m, 2H), 4.23-4.38 (m, 2H), 4.40-4.80 (m, 3H), 4.92-4.94 (m, 1H), 4.98 (s, 2H), 7.19-7.37 (m, 10H), 7.78-7.85 (m, 1H). 13C NMR (DMSO-d ) δ R/β: 36.5, 55.5, 64.1, 65.4, 67.4, 67.5, 6 71.7, 71.8, 73.0, 75.9, 81.2, 82.6, 92.7, 97.4, 101.8, 126.5, 127.6, 127.6, 127.8, 128.3, 128.3, 129.1, 136.9, 137.4, 137.6, 155.9, 171.8. Anal. Calcd for C23H27NO9: C, 59.86; H, 5.90; N, 3.04. Found: C, 59.58; H, 5.92; N, 2.94.

RESULTS AND DISCUSSION Preparation of O-(Z-r-aminoacyl)diisopropylidene sugars 3a-d, the diastereomeric mixture (3d+3d′) and Bis-O-(Z-raminoacyl)diisopropylidene Sugar 4 from 1,2:3,4-Di-Oisopropylidene-r-D-galactopyranose 2. O-(Z-R-Aminoacyl)diisopropylidene sugars 3a-d, (3d+3d′), and 4 were prepared by coupling N-(Z-R- aminoacyl)benzotriazoles 1a-d, 1d+1d′, and 1e, respectively, with 6-OH present in 2. In the presence of a catalytic amount of DMAP (0.2 equiv), under 100 W microwave irradiation at 65 °C for 15 min; the acylating reagent 1a-d was completely consumed (Scheme 1). However, for the preparation of (3d+3d′) under similar conditions, the reaction takes 1 h to complete. This could be attributed to the slow reactivity of the D-component present in the DL-amino acid moiety. For the preparation of 4, 0.4 equiv of DMAP were utilized (Scheme 2). The crude products 3a-d, (3d+3d′), 4 were subjected to silica-gel column chromatography using ethyl acetate/hexane (2:1) as an eluent. The desired products 3a-d, 4, and the diastereomeric mixture (3d+3d′) were obtained in yields of 88-96%. NMR and HPLC analysis of 3a-d and 4 revealed no detectable racemization. 1H NMR of 3a-d, 4 showed a prominent doublet for the R-NH proton of the amino acid fragment in the range of 5.30-5.45 ppm in CDCl3. All the methyl protons from the diisopropylidene group in sugar

Table 3. Preparation of O-(Z-r-Aminoacyl)diisopropylidene Sugars 8a,b from 7 R

product

yielda (%)

tR (min)b

CH2C6H5 1a CH2CH2SMe 1c

Z-L-Phe-O-mannofuranose 8a Z-L-Met-O-mannofuranose 8b

78 93

2.99 3.00

a

Isolated yield. b For conditions, refer to Experimental Section.

Table 4. Cleavage of Diisopropylidene Groups Present in 3a,d Using TFA-H2O (9:1; 5 mL) entry

O-(Z-R-aminoacyl) diisopropylidene sugars

O-(Z-R-aminoacyl) sugars

yielda

1 2

3a 3d

9a 9b

92 94

a

Isolated yield.

appeared as singlets in the NMR analysis, supporting the absence of any undesired isomers of 3a-d, 4. In the 1H NMR of 3d, the doublet arising from the methyl protons present in the Ala fragment overlapped with one of the methyl protons of the sugar moiety. In addition, a clear doublet arising from the R-NH proton was observed. No splitting of the peaks was observed in the 13C NMR of 3d, in contrast to its corresponding diastereomeric mixture (3d+3d′), supporting the enantiopurity of 3d. HPLC analysis of 3a-d, 4 gave single retention times whereas (3d+3d′) gave two retention times, confirming the enantiopurity of 3a-d, 4 (Table 1 and Scheme 2). Preparation of O-(Z-r-Aminoacyl)diisopropylidene Sugars 6a-d, 8a,b and Diastereomeric Mixtures (6a+6a′), (6d+6d′) Using 1,2:5,6-Di-O-isopropylidene-D-glucose 5 and 2,3:5,6Di-O-isopropylidene-r-D-mannofuraanose 7. O-(Z-R-Aminoacyl)diisopropylidene sugars 6a-d were prepared by coupling N-(Z-R-aminoacyl)benzotriazoles 1a-d with 3-OH of 5, and the products 8a,b were prepared by coupling 1a and 1c with 1-OH present in 7, using similar reactions conditions as used for 3 (Scheme 3 and 4). However, for the preparation of (6a+6a′) and (6d+6d′) under similar microwave conditions, the reaction takes 1 h to complete (Scheme 3). The products 6a-d, 8a,b and the diastereomeric mixtures (6a+6a′) and (6d+6d′) were obtained in yields 78-94%. NMR analysis of 6a-d, 8a,b revealed no detectable racemization. 1H NMR demonstrated a prominent doublet for the R-NH proton of the amino acid fragment in the range of 5.445.66 ppm in CDCl3. All the methyl protons from the diisopropylidene groups in the sugar appeared as singlets in the NMR spectra indicating the absence of any undesired isomers of 6ad, 8a,b. For example, in 6d, the methyl groups gave the desired singlet at 1.39 ppm and a doublet at 1.43 ppm as compared to the corresponding mixture (6d+6d′) which gave a multiplet at

Technical Notes

Bioconjugate Chem., Vol. 18, No. 3, 2007 997

Scheme 5

Scheme 6

1.37-1.42 ppm and two doublets at 1.43 and 1.45 ppm. HPLC analysis of 6a-d, 8a,b each gave single retention times whereas (6a+6a′) and (6d+6d′) gave two retention times, confirming the enantiopurity of 6a-d, 8a,b (Tables 2 and 3). Cleavage of the Diisopropylidene Groups Present in O-(Zr-aminoacyl)diisopropylidene Sugars 3a,d, 6d. The diisopropylidene protecting groups were cleaved using TFA-H2O (9:1; 5 mL) (Scheme 5 and 6). The TFA-H2O mixture was added to O-(Z-R-aminoacyl)diisopropylidene sugars 3a,d, 6d (0.200 g) at room temperature and was stirred until 3a,d, 6d were completely consumed as monitored by TLC. Each resulting mixture was concentrated under reduced pressure followed by subsequent washings with diethyl ether (5-6 times). The free amino acid-sugar conjugates 9a,b, 10 were obtained in yields of 92-94% (Table 4 and Scheme 6) and characterized by 1H and 13C NMR spectroscopy, elemental analysis, melting point, and ORP. The other diastereomer was also observed in the NMR analysis, to the extent of 40-50%.

CONCLUSIONS In conclusion, we have demonstrated a convenient and efficient preparation of chiral O-(Z-R-aminoacyl)diisopropylidene sugars 3a-d, 4, 6a-d, 8a,b and diastereomeric mixtures (3d+3d′), (6a+6a′), (6d+6d′) in yields of 78-96%. The original chirality of the resulting products was preserved in >97% ee as evidenced by NMR and HPLC. These advantages also suggest N-protected-(R-aminoacyl)benzotriazoles as a preferred choice for esterification among conventional coupling agents. Supporting Information Available: Compound characterization data for all the compounds. This material is available free of charge via the Internet at http://pubs.acs.org

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