Miniaturization of the Structure Elucidation of Novel Natural Products - Two Trace Antibacterial Acylated Caprylic Alcohol Glycosides from Arctostaphylos pumila

• Abstract
• Introduction
• Materials and Methods
• General
• Plant material, extraction and isolation
• Antibacterial activity
• Results and Discussion
• Acknowledgements
• References

Abstract

High-throughput isolation, purification and analysis methods applied to natural products libraries from plants gave rise to the discovery of two novel acylated caprylic alcohol glycosides (1, 2) produced by Arctostaphylos pumila. The NMR spectra were acquired using the CapNMR™ probe and performed on mass-limited samples, which enabled us to elucidate the structures of 2,6-diacetyl-3,4-diisobutyl-1-O-octylglucopyranoside (1, 200 μg) and 2,6-diacetyl-3,4-dimethylbutyl-1-O-octylglucopyranosid (2, 70 μg). Compounds 1 and 2 exhibited antibacterial activity against Gram-positive methicillin-resistant Staphylococcus aureus with an MIC of 128 μg/mL and 64 μg/mL, respectively.

Introduction

Our high-throughput natural products chemistry methods [1], [2] applied to the production, purification and analysis of natural products libraries have increased the rate of discovery of novel bioactive compounds in various drug discovery collaborations. Nuclear magnetic resonance spectroscopy (NMR) is the most versatile analytical platform for the structure elucidation of those active compounds. This analytical technique conventionally requires greater than one milligram of a compound to obtain the prerequisite set of NMR spectra to fully elucidate a structure. During the bioassay-guided fractionation of biologically active fractions, micrograms are used to locate activity, but one or more milligrams of a compound are needed for structure elucidation. Obtaining these quantities often requires the recollection of plants or re-fermentation of microorganisms, large-scale extraction, and more than one purification technique performed repeatedly. This can be very time-consuming and requires considerable effort.

In recent years the development and commercialization of new probe technologies have increased NMR sensitivity [3]. We are currently using the CapNMR™ probe for all of our NMR data acquisitions. It has enabled us to acquire 1D proton and 2D gradient COSY NMR spectra on quantities of less than 10 micrograms with few non-deuterated artifacts in approximately 5 minutes and 1.5 hours, respectively. HSQC or HMQC experiments are routinely acquired on samples of 30 micrograms in approximately 5 hours. HMBC experiments are performed with approximately 70 micrograms of material in 10 to 15 hours, which typically enable internal acceptance of the structure per commercial standards. ¹³C-NMR data are acquired on amounts greater than 200 micrograms when required for specific internal and/or information releasing decisions. Based on our experiences working with the MicroCryoProbe^TM and other conventional NMR probes, the CapNMR™ probe has a higher filling factor resulting in fewer non-deuterated artifacts allowing us to obtain superior ¹H- and COSY NMR spectra on as little as 5 to 10 micrograms of material. The gain in sensitivity results from a reduced flow cell (5 μL total volume with 1.5 μL in active volume) as well as an RF coil wound into a solenoid configuration [1], [3]. This probe is easier to shim, much less expensive to purchase and operate, and consumes approximately fifty microliters of deuterated solvents for each sample without NMR tubes, so operating costs are reduced.

We describe here the high-throughput isolation of two acylated caprylic alcohol glycosides (1, 2) produced by Arctostaphylos pumila Nutt. (Ericaceae); the first application of the CapNMR™ probe for the miniaturization of the structure elucidation of the two novel mass-limited natural products 1 (200 μg) and 2 (70 μg) as well as their antibacterial assays.[]

A. pumila is a low shrub and a rare plant species, which is closely related to bearberry (A. uva-ursi) [4]. Flavonoid, gallic acid and polyphenols were obtained previously from A. uva-ursi [5], [6], [7], and the polyphenol-type compound corilagin was found to possess antibacterial activity [7]. There are no references to compounds isolated from A. pumila. The 1D and 2D NMR data were acquired using the CapNMR™ probe on a Bruker Avance 600 MHz NMR spectrometer, allowing us to deduce the core structure of compounds 1 and 2 to be an acylated 1-O-octylglucopyranoside. To estimate the quantities of active compounds isolated by the RP semi-preparative HPLC, we have developed procedures to generate ELSD (evaporative light scattering detection) calibration curves, which were based on the separation of a mixture of seven standards (caffeine, rutin, muristerone A, hydrocortisone, NDGA, p-hydroxybenzoic acid n-butyl ester, and mevastatin) through a single channel Beckman HPLC system [3].

The resulting Arctostaphylos library was analyzed by parallel eight-channel HPLC-ELSD-MS after preparation as previously described [1], [2]. The acylated 1-O-octylglucopyranosides will frequently elute in the same chromatographic fractions of our library production process and their retention times during HPLC-ELSD-MS analysis will match when normalized using the retention times of standard compounds. Based on the full NMR data (¹H, ¹³C, DEPT, COSY, HSQC and HMBC) and the LR-/HR-ESI-MS, the structure of 1 was confirmed to be 2,6-diacetyl-3,4-diisobutyl-1-O-octylglucopyranoside. It was difficult to obtain the complete ¹³C and DEPT NMR spectra for 2 on the obtained mass, however, their 2D NMR (HSQC and HMBC) spectra were completely acquired and the chemical shifts of all the protons and carbons including the quaternary carbons were observed, and unambiguously assigned due to the fact that 2 is an analogue of 1. Two other trace components (≤ 5 μg) were obtained from the same preparative HPLC fraction containing 1 and 2. Their 1H and gCOSY NMR spectra acquired using the probe showed general features to those of 1 and 2, indicating that they are also analogues of the acylated 1-O-octylglucopyranoside. Unfortunately, the structures are still open. However, it was sufficient for our research committee to properly allocate resources based upon information available on its chemical class, biological activity, synthetic accessibility, and therapeutic index. Naturally occurring caprylic alcohol glycosides had already been found from several medicinal plants with antiallergic activity [8], [9]. The regioselective acylation of 1-O-octylglucopyranosides has been achieved through a DMAP-catalyzed acetylation in the laboratory [10], [11]. Our naturally occurring compounds 1 and 2 exhibited antibacterial activity against Gram-positive methicillin-resistant Staphylococcus aureus with MIC values of 128 μg/mL and 64 μg/mL, respectively.

Materials and Methods

General

Automated flash chromatography separations were performed on 50 gram Si flash columns (International Sorbent Technology Ltd., Mid Glamorgan, UK) using a Flash Master II automated chromatographic system (Jones Chromatography Inc., Lakewood, CO, USA) using a step gradient of (1) 75 % hexanes, 25 % EtOAc (Flash Fraction 1), (2) 50 % hexanes, 50 % EtOAc (Flash Fraction 2), (3) EtOAc neat (Flash Fraction 3), (4) 75 % EtOAc, 25 % MeOH (Flash Fraction 4), (5) 50 % EtOAc, 50 % MeOH (Flash Fraction 5). Flash Fraction 1 was extremely lipophilic (log P > 5), and was discarded. One gram of organic extracts was loaded for each Si flash column. The flow rate was set up as 30 mL/min, and the eluting time for step 1 was 13 minutes (totaled 390 mL solvent collected), and step 2 through step 5 each was 10 minutes (each 300 mL solvent collected). Preparative HPLC separations were performed on Betasil C18 columns (20 × 100 mm, 5 mm, Keystone Scientific Inc., Bellefonte, PA, USA). A parallel four-channel preparative HPLC system was assembled and consisted of 4 Beckman System Gold 126 gradient HPLC pumps (Beckman Coulter Inc., Fullerton, CA, USA) with system controllers and four-way solvent delivery modules, 4 Beckman System Gold 166 single wavelength UV detectors with preparative flow cells, a Gilson 215/849 multiple probe autosampler (Gilson Inc., Middleton, WI, USA), and 4 Gilson 204 fraction collectors. Each flash fraction was separated into 40 fractions (20 mL/min, 1 min per collection per tube) using the parallel four-channel preparative HPLC system. A different 40 min gradient was applied to each flash fraction for adequate separation: 60 - 85 % acetonitrile in water for Flash Fraction 2, 30 - 70 % acetonitrile in water for Flash Fraction 3, and 5 - 40 % acetonitrile in water for Flash Fractions 4 and 5. The system was controlled by Beckman 32 Karat chromatography software.

A Mega 1200 evaporator (Genevac Technologies, Suffolk, UK) was used to remove solvents from the preparative HPLC fractions. The preparative HPLC fractions were transferred from tubes to 96-deep-well plates by a Packard MultiProbe II liquid handling system (Packard BioScience Company, Meriden, CT, USA). A Genevac HT-12 evaporator was used to remove solvents from the 96-well plates. A parallel eight-channel HPLC-ELSD-MS system was assembled and consisted of an LCT time-of-flight mass spectrometer with an eight-way MUX electrospray interface (Micromass Ltd, Manchester, UK), a Waters 600E Multisolvent Delivery System (Waters Corporation, Milford, MA, USA) to pump solvents through an eight-way manifold which splits the flow to 8 HPLC columns (4.6 × 50 mm, 3 mm, Keystone Betasil C-18), a Gilson 215/889 multiple probe autosampler, and 8 Alltech 500 ELSD detectors (Alltech Associates Inc., Deerfield, IL, USA). HPLC chromatographic conditions of 5 % acetonitrile in water for the first 1.0 minute, a linear gradient of acetonitrile from 5 % to 95 % in 8.0 minutes, followed by 95 % acetonitrile in water for 1.0 minute. After each analysis the column was equilibrated at 5 % acetonitrile in water for 2.5 minutes. The system was controlled by Waters MassLynx software version 4.0. The instrument was operated in both positive and negative electrospray modes with the capillary voltage set to 3.1 kV in positive electrospray mode and 2.7 kV in negative electrospray mode. Desolvation temperatures of 200 °C with a source temperature of 120 °C were used for all experiments. The nitrogen desolvation and nebulizer gas flow rates were set to 1200 and 6 L/h, respectively. The sample cone voltage was set to 25 V with extraction cone voltages set at 1.0 V for positive mode and 2.0 V for negative mode. The RF lens voltage was set to 300 V. Data analysis was performed using the Waters OpenLynx software followed by Extractor, a customized software package developed for Sequoia Sciences by Koch Associates, La Jolla, CA, USA.

Semi-preparative HPLC isolation for individual compounds was performed on a single channel Beckman HPLC system consisting of a Keystone BetaMax Neutral C18 column (8 × 250 mm I.D., 5 mm), a Beckman Coulter System Gold^® 508 autosampler, a Beckman 168 diode array UV detector and a Sedex 75 ELSD detector (Sedere, France), and a Foxy Jr. fraction collector (Isco, Inc., Lincoln, NE, USA) equipped with minitubes loaded in 96-well plate (flow rate: 3 mL/min, 0.33 min per collection time per minitube). A splitter was used to split the flow in 10 : 90 to ELSD and fraction collector, respectively. For ELSD Sedex 75 conditions: the gas flow was set to achieve a pressure of 3.5 psi ± 2 psi, gain 9, and temperature 40 °C ± 1 °C.

NMR data for the structure elucidation of compounds were acquired utilizing a Bruker Avance 600 MHz NMR system (Bruker, Rheinstetten, Germany) and a CapNMR^TM probe with 5 μL volume flow cell (Magnetic Resonance Microsensors, Savoy, IL, USA). The probe was operated at a temperature of 298 K. Pulse widths were 5.5 μs at a power of 24 dB for the ¹H spectra. Pure compound was dissolved into 6.5 μL deuterated solvent and loaded manually into the CapNMR^TM flow probe.

Plant material, extraction and isolation

The stem of Arctostaphylos pumila was collected from Monterey County, California in April 2000. Plant samples were shipped frozen to Sequoia Sciences and lyophilized upon arrival. They were identified by John Stone (Missouri Botanical Garden Herbarium, St. Louis, MO, USA). A voucher specimen (No. 2991) is deposited at the Herbarium of the Missouri Botanical Garden. Dried stem (100 g) was extracted with EtOH:EtOAc (50 : 50) followed by H₂O:MeOH (30 : 70), to obtain 5 g and 9 g dry organic and aqueous extracts, respectively. The organic extracts were loaded on the Flash Master II automated chromatographic system using our standard elution gradient described above. The ethyl acetate fraction (Flash Fraction 3) totaled 60 mg; 50 mg were fractionated by preparative C18 HPLC from 30 % to 70 % acetonitrile in water collecting 40 one minute fractions. Compounds 1 and 2 resided in preparative HPLC fraction 37, which exhibited antibacterial against Gram-positive methicillin-resistant Staphylococcus aureus. Review of the HPLC-ELSD-MS data acquired on all of the preparative fractions from the Flash Fraction 3 suggested preparative HPLC fraction 37 contained compounds with molecular weights less than 600 daltons which could readily be isolated using reverse phase chromatography. The initial mobile phase gradient applied to isolating compounds 1 and 2 from fraction 37 was based on the elution profile observed during the preparative HPLC separation that created the fraction. A semi-preparative HPLC method was developed which resulted in an isocratic gradient of 75 % acetonitrile in water acidified with 0.01 % formic acid for 32 minutes to obtain pure compounds 1 (totaled 200 μg; yield: 0.0002 %) and 2 (totaled 70 μg; yield: 0.00006 %) with retention times at 11.53 min and 13.99 min, respectively.

2,6-Diacetyl-3,4-diisobutyl-1-O-octylglucopyranoside (1): ESI-MS: m/z = 515 [M - H]^-, 517 [M + H]⁺, 539 [M + Na]⁺; HR-ESI-MS: m/z = 539.2847 (C₂₆H₄₄O₁₀Na requires 539.2832); ¹H- and ¹³C-NMR data for 1 see Tables [1] and [2].

2,6-Diacetyl-3,4-dimethylbutyl-1-O-octylglucopyranoside (2): ESI-MS: m/z = 567 [M + Na]⁺. ¹H- and ¹³C-NMR data for 2 see Tables [1] and [2].

Antibacterial activity

The in vitro antibacterial activities of the compounds 1 and 2 were determined against Gram-positive methicillin-resistant Staphylococcus aureus (ATCC 33 591). The assay was performed at MDS Pharma Services (Taipei, Taiwan). Methods employed in this study have been adapted from the scientific literature [12], [13] to maximize reliability and reproducibility. Reference standards were run as integral part of each assay to ensure the validity of the results obtained. For primary assays, only the lowest concentration with a significant response was judged by the assays’ criteria. Culture medium: Mueller Hinton Broth. Vehicle: 1 % DMSO. Incubation time/temp: 1 day @ 37 °C. Incubation/administration volume: 1 mL/10 μL. Time of assessment: 1 day. Quantitation method: Turbidity measurement. Evaluated in in vitro antibacterial assays at concentrations ranging from 1 to 128 μg/mL. Compounds 1 and 2 exhibited significant antibacterial activity, in accordance with in-house criteria, against Gram-positive methicillin-resistant Staphylococcus aureus (ATCC 33 591) with MIC values of 128 μg/mL and 64 μg/mL, respectively. Concurrently, gentamicin was used as a positive control and had an MIC 2 μg/mL.

Results and Discussion

The molecular weight of compound 1 and its chemical formula of C₂₆H₄₄O₁₀ were deduced from the positive mode high-resolution electrospray ionization mass spectrum (HR-ESI-MS), which showed the [M + Na]⁺ ion peak (m/z = 539.2847; C₂₇H₄₄O₁₀Na requires 539.2832). Supporting evidence was obtained from both negative and positive low-resolution ESI mass spectra which displayed peaks at m/z = 515 [M - H]^- and 517 [M + H]⁺, 539 [M + Na]⁺. The ¹³C-NMR and DEPT spectral data (Table [2]) showed twenty-six carbon signals, indicating the presence of four acylic carbonyl groups at δ = 176.11, 175.23, 171.12, and 169.36, seven carbon bearing oxygens at δ = 98.89 (CH), 70.25 (CH), 69.92 (CH₂), 69.43 (CH), 68.14 (CH), 66.24 (CH) and 62.37 (CH₂), and seven methyl groups at δ = 20.66, 20.53, 18.94, 18.89, 18.53, 18.51, and 13.94. One bond proton-carbon connectivities were determined by a heteronuclear single quantum coherence (HSQC) NMR experiment, which showed an anomeric proton at δ = 4.80 (1H, d, J = 7.9 Hz, H-1′; δ_C = 8.89, CH). Analysis of the ¹H-¹H COSY spectrum (600 MHz, CDCl₃) showed the existence of a seven-proton spin system for the sugar part in the low field with coupling between the anomeric H-1′ and H-2′ at δ = 4.89 (1H, dd, J = 7.9, 3.0 Hz), between H-2′ and H-3′ at δ = 5.70 (1H, dd, J = 3.0, 2.9 Hz), between H-3′ and H-4′ at δ = 5.03 (1H, dd, J = 10.4, 2.9 Hz), between H-4′ and H-5′ at δ = 4.13 (1H, m), between H-5 and H_{2 -} 6′ at δ = 4.39 (1H, dd, J = 11.6, 2.2 Hz) and 4.31 (1H, dd, J = 11.6, 5.2 Hz). The observed chemical shifts and the coupling constants showed that the core structure of 1 is a full substituted β-glucopyranoside with a twisted chair conformation. The β configuration in the sugar was deduced from the coupling constant (7.9 Hz) of the anomeric proton in glucose. The abnormal coupling constants between H-2′ and H-3′ (J = 3.0 Hz) as well as between H-3′ and H-4′ (J = 2.9 Hz) in the glucose moiety are due to the space strength attributed by highly substituted acylic moieties. Two separated proton spin systems of the two isobutyl moieties were clearly observed in the gCOSY spectrum. The two acetyl methyl groups were found at δ = 2.11 (3H, s) and 2.03 (3H, s). Similar to the reported caprylic alcohol glycoside [8], the terminal methyl group at δ = 0.90 (3H, t, J = 6.4, H-8), the signals at δ = 1.27 - 1.33 (10H, overlapped) and 1.58 (2H, m, H₂ - 2) indicated the presence of a saturated long-chain hydrocarbon. The couplings between the oxygenated methylene protons at δ = 3.89 and 3.52 (each 1H, dt, J = 9.4, 6.5 Hz, H₂ - 1) and the methylene protons at δ = 1.58 were also observed in the gCOSY spectrum, which together with the molecular formula suggested the presence of 1-octanol as the aglycone. A heteronuclear multiple bond correlation (HMBC) NMR experiment indicated long-range proton-carbon couplings (Fig. [1]), which enabled us to confirm all the linkage positions of the octylation, acetylation and the isobutylation. Therefore, compound 1 was elucidated to be 2,6-diacetyl-3,4-diisobutyl-1-O-octylglucopyranoside.

The ESI mass spectrum of 2 showed a clear [M + Na]⁺ peak at m/z = 567. This together with its ¹H- and ¹³C-NMR spectral data (Tables [1] and [2]) indicated the molecular formula to be C₂₈H₄₈O₁₀. The ¹H-NMR spectral data of 2 showed general features similar to those of 1. The ¹H-¹H COSY NMR spectrum also showed a seven-proton spin system coupling between the anomeric proton H-1′ at δ = 4.80 (1H, d, J = 8.1 Hz; δ_C = 99.19) and H-2′ at δ = 4.89 (1H, dd, J = 8.1, 2.9 Hz; δ_C = 69.75), between H-2′ and H-3′ at δ = 5.71 (1H, dd, J = 2.9, 2.8 Hz; δ_C = 68.39), between H-3′ and H-4′ at δ = 5.06 (1H, dd, J = 10.1, 2.8 Hz; δ_C = 66.32), between H-4′ and H-5′ at δ = 4.13 (1H, m; δ_C = 70.51), between H-5′ and H₂ - 6′ at δ = 4.35 (2H, m; δ_C = 63.99). In the high field region of the ¹H-NMR spectrum, there are three terminal methyl groups at δ = 0.89 (3H, t, J = 7.3 Hz, H-4′d; δ_C = 12.05), 0.90 (3H, t, J = 6.5 Hz, H-8; δ_C = 14.52) and 0.99 (3H, t, J = 7.3 Hz, H-3′d; δ_C = 11.93). The two separated proton spin systems of the two methylbutyl moieties were clearly observed with coupling between H-3′b at δ = 2.51 (1H, m) and H₂ - 3′c at δ = 1.76 (1H, m) and 1.56 (1H, m), between H-3′b and H-3′e at δ = 1.26 (3H, d, J = 7.1 Hz), between H₂ - 3′c and H-3′d at δ = 0.99, as well as between H-4′b at δ = 2.31 (1H, m) and H₂ - 4′c at δ = 1.63 (1H, m) and 1.45 (1H, m), between H-4′b and H-4′e at δ = 1.12 (3H, d, J = 7.3 Hz), between H₂ - 4′c and H-4′d at δ = 0.89. An HMBC NMR experiment also allowed us to confirm the linkage positions of the octylation, acetylation and the methylbutylation (Fig. [1]). In accordance to the result of 1 and with the assumption of a common biosynthetic pathway for the acylated 1-O-octylglucopyranosides, the stereochemistry for the sugar moiety in 2 should be the same as 1. Thus, compound 2 was elucidated to be 2,6-diacetyl-3,4-dimethylbutyl-1-O-octylglucopyranoside.

In conclusion, this is the first known example to utilize a capillary-scale NMR probe for the elucidation of novel chemical structures of mass-limited materials with reasonable NMR data acquisition times. This probe will usher in a new era in the miniaturization of structure elucidation of natural products.

Acknowledgements

The authors acknowledge Dr. Jim Miller, John Stone, Adam Bradley, Gretchen Walters from Missouri Botanical Garden for the plant collections and identifications, Fong-Chi Cheng, Ching-Chui Lin, Peter Chiu and Jiann-Wu Wei from MDS Pharma Services-Taiwan Ltd. for the antibacterial in vitro assays. We would like to acknowledge Tim Peck, Dean Olson and Jim Norcross from Magnetic Resonance Microsensors (Savoy, IL) for making the first 5 μL proton indirect carbon gradient CapNMR^TM probe available to Sequoia Sciences.

References

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Dr. Jin-Feng Hu

Lead Discovery & Rapid Structure Elucidation Group

Sequoia Sciences, Inc.

11199 Sorrento Valley Road, Suite H

San Diego

CA 92121

USA

Fax: +1-858-623-0805

Email: jhu@sequoiasciences.com

References

• 1 Eldridge G R, Vervoort H C, Lee C M, Cremin P A, Williams C T, Hart S M, Goering M G, O’Neil-Johnson M, Zeng L. High-throughput method for the production and analysis of large natural product libraries for drug discovery.  Anal Chem. 2002;  74 3963-71
  CrossrefPubMedSearch in Google Scholar
• 2 Cremin P A, Zeng L. High-throughput analysis of natural product compound libraries by parallel LC-MS evaporative light scattering detection.  Anal Chem. 2002;  74 5492-500
  CrossrefPubMedSearch in Google Scholar
• 3 Hu J F, Garo E, Yoo H D, Cremin P A, Zeng L, Goering M G, O’Neil-Johnson M, Eldridge G R. Application of capillary scale NMR for the structure determination of Phytochemicals. Phytochem. Anal. in press
  Search in Google Scholar
• 4 Wells P V. In: The Jepson Manual: Higher Plants of California. Hickman JC editor University of California Press Berkeley; 1993: pp 544-59
  Search in Google Scholar
• 5 Geiger H, Schucker U, Waldrum H, Vandervelde G, Mabry T J. Quercetine-3-beta-D-(6-O-galloyl-galactoside), a constituent of Arctostaphylos uva-ursi (L) Spreng (Ericaceae).  Z Naturforsch C. 1975;  30 296
  PubMedSearch in Google Scholar
• 6 Slaveska-Raicki R, Rafajlovska V, Rizova V, Spirevska I. HPTLC determination of gallic acid and tannin in the extracts of bearberry leaves.  J Planar Chrom. 2003;  16 396-401
  PubMedSearch in Google Scholar
• 7 Shiota S, Shimizu M, Sugiyama J, Morita Y, Mizushima T, Tsuchiya T. Mechanisms of action of corilagin and tellimagrandin I that remarkably potentiate the activity of beta-lactams against methicillin-resistant Staphylococcus aureus .  Microbiology and Immunology. 2004;  48 67-73
  PubMedSearch in Google Scholar
• 8 Kim Y C, Kingston D GI. A new caprylic alcohol glycoside from Circaea lutetiana ssp. canadensis.  J Nat Prod. 1996;  59 1096-8
  CrossrefPubMedSearch in Google Scholar
• 9 Yoshikawa M, Shimada H, Shimoda H, Matsuda H, Yamahara J, Murakami N. Rhodiocyanoside-A and rhodiocyanoside-B, new antiallergic cyanoglycosides from Chinese natural medicine Si-Li-Hong-Jing-Tian, the underground part of Rhodiola quadrifida (Pall.) Fisch. et Mey.  Chem Pharm Bull. 1995;  43 1245-7
  PubMedSearch in Google Scholar
• 10 Kurahashi T, Mizutani T, Yoshida J. Effect of intramolecular hydrogen-bonding network on the relative reactivities of carbohydrate OH groups. J Chem Soc Perkin Trans 1 1999: 465-73
  Search in Google Scholar
• 11 Kurahashi T, Mizutani T, Yoshida J. Functionalized DMAP catalysts for regioselective acetylation of carbohydrates.  Tetrahedron. 2002;  58 8669-77
  CrossrefPubMedSearch in Google Scholar
• 12 DiModugno E, Erbetti I, Ferrari L, Galassi G, Hammond S M, Xerri L. In-vitro activity of the Tribactam GV 104 326 against Gram-positive, Gram-negative, and anaerobic bacteria.  Antimicrobial Agents and Chemotherapy. 1994;  38 2362-8
  PubMedSearch in Google Scholar
• 13 Misiek M, Pursiano T A, Leitner F, Price K E. Micobiological properties of a new cephalosporin, BL-S 339 : 7-(phenylacetyimidoyl-aminoacetamido)-3-(2-methyl-1,3,4-thiadiazol-5-ylthiomethyl)ceph-3-em-4-carboxylic acid.  Antimicrobial Agents and Chemotherapy. 1973;  3 40-8
  PubMedSearch in Google Scholar

Dr. Jin-Feng Hu

Lead Discovery & Rapid Structure Elucidation Group

Sequoia Sciences, Inc.

11199 Sorrento Valley Road, Suite H

San Diego

CA 92121

USA

Fax: +1-858-623-0805

Email: jhu@sequoiasciences.com