Analysis of Natural Products

Bruker BioSpin

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High Performance NMR Solutions

NMR

NMR Spectroscopy

Recent advances in Bruker’s NMR technology now deliver the high performance

solutions necessary to address the complex structural questions confronting natural

products researchers. Improvements in probe design and receivers provide the

highest NMR sensitivity available on the market. These hardware developments,

in combination with our new software packages, provide the perfect complement

of tools for optimizing your productivity and making new discoveries.

Sensitivity

Bruker’s most sensitive NMR spectrometer is able to detect nanograms of isolated natural products in just a few minutes. This remarkably high sensitivity is achieved through the use of small volume probe technology and CryoProbe™ technology. Probes of greatest interest to natural products researchers include: the 1.7 mm TCI MicroCryoProbe for ultimate mass sensitivity and versatility combining MicroProbe and CryoProbe technology and performance; the 1 mm TXI MicroProbe as the probe with the highest proton mass sensitivity achieved with conventional room temperature technology for very small sample amounts; the 1.7 mm MicroProbe as the all-purpose small volume room temperature probe with excellent 1H and 13C mass sensitivity; the 5 mm DCH CryoProbe, a dual configuration CryoProbe for the highest carbon sensitivity available.

Advances in receiver technology add to the benefits provided by probe developments give exceptional dynamic range enabling the detection of low level components in mixtures. Let our innovative NMR products assist you in studying the molecules that were previously unapproachable.

25 µg of Jaspamide isolated from sponge acquired in CDCl3 on a 1 mm MicroProbe at 700 MHz. 1H TOCSY spectrum acquired using 32 scans. Total acquisition time 2 hours 15 min. Sample was kindly provided by Tadeusz Molinski, UCSD.

700 MHz MicroProbe™ Results

1.7 mm MicroCryoProbe™ Results

(A) 5 µg Quinine in 30 ul DMSO-d6: edited HSQC acquired on a 1.7mm MicroCryoProbe at 600 MHz with 8 scans per increment in 15 minutes. (B) 90 µg of Muironolide A* isolated from a marine sponge acquired in CDCl3 in a 1.7 mm MicroCryoProbe at 600 MHz. 1H NMR spectrum kindly provided by Tadeusz Molinski, UCSD.*Dalisay et al., J. Am. Chem. Soc., May 15, 2009.

B

App m

8 .5 8.0 7 .5 7.0 6 .5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2 .0 1.5 1 .0 0.5 p pm

20

40

60

80

100

120

140

N

OCH3

OHN

CH2

NMR results from quantification of specific components in crude extracts

Weighing a very small amount of a precious natural product is difficult but without weight information the actual yield or biological activity is difficult to ascertain. Determining the amount of sample of a pure material, or as part of a mixture, is both easy and accurate using the quantitative NMR tools provided by Bruker. By using calibrated integral values, a simple integration yields a concentration based on a specific resonance. Use this in manual mode or in automation for high throughput screening.

Quantification

Spectra showing quantification

**

**

32.8 mM

**

**

*Scutellarin

S. Incana Extract

*

Quantitative Techniques

The integral values are scaled to a reference sample of known concentration or a synthesized NMR signal that is calibrated using a sample of known concentration. Once the integral values are compared to the calibrated sample, individual components may be quantified as desired. A pure sample of the target material is not needed.

S. laterafloria nd

42.1

4.6

nd

nd

98.1 10.0

29.6

4.5

107.5*

nd

32.8

9.8

10.3*

6.2*

Extract Origin Baicalein (mM) Baicalin (mM) Scutellarin (mM)

S. incana

S. alpina

87.5 7.9 7.9S. baicalensis

T. canadense

T. chamaedrys

nd = not detected, *Baicalin derivative or Scutellarin derivative observed

AVANCE™ III Spectrometer Architecture

Precise quantification measurements are also enhanced with a high performance spectrometer architecture. The AVANCE III is the latest generation in the very suc-cessful AVANCE series product line. The AVANCE III spectrometer is designed around an advanced digital concept that provides an optimized pathway for highest speed RF generation and data acquisition with a highly modular and scalable trans-mitter and receiver channel architecture.

Quantification of Scutellarin in crude Skull Cap extract from Scutellaria incana was determined to be 32.8 mM based on 1H NMR spectra, using quantitative NMR. The presence of Scutellarin was determined through a match against a spectral base of potential botanical extract components using AMIX.

Screening

Dereplication is an integral part of a natural products discovery program. Quality control promotes the safety and efficacy of the botanical supplements. Bruker software and hardware assists in both of these processes by providing the automation, the instrument stability and the analysis capabilities necessary to complete these tasks on both pure materials and on complex mixtures. Spectral bases (SBASE) may be generated from any spectrum, such as 1D proton, 1D carbon, COSY, HSQC, HMBC, for rapid comparisons

with new samples to determine sample similarities and differences. Sample information, such as the site or date of collection or the originating organism, may be imported and linked to the SBASE entries to aid the rapid interpretation of search results. Analysis capabili-ties allow one to find known compounds or fragments, categorize spectra, and find unique NMR spectra. Statistical analysis delivers rapid and easy-to-visualize comparisons between large series of samples.

Statistical Analysis using AMIX

PCA of 1H NMR spectra from pine bark extract (blue) and grape seed extract (black) rapidly, and clearly, showed that one sample was adulterated.

adulterated sample

AMIX™

AMIX MATCH successfully classified the unknown natural product extract sample to the proper taxonomy using a user generated spectral base linked with known taxonomy data. Spectra and taxonomy data kindly supplied by Cherie Motti, AIMS, Australia

Combined viewing of molecule, LC-MS and NMR data in AMIX. Cursors are linked where appropriate.

AMIX

AMIX provides a collection of powerful tools that enable you to get the most out of your data. AMIX includes many integrated routines for spectroscopic and statistical analyses, significantly enhancing productivity across a wide variety of applications, such as small molecule research, metabolomics and mixture analysis. A unique feature is the combined analysis of NMR and MS data. In many applications, such as structure verification, NMR and MS confirm each other, while in other appli-cations, such as structure elucidation, they are also complementary.

Automation is available for even the smallest of sample sizes and smallest

NMR tubes. With Bruker, the automation workflow begins with the submission

of a sample, followed by sample preparation, transfer to the spectrometer,

automatic probe tuning, data acquisition and processing, and finally with

data distribution and archiving. All of the automation features are designed to

increase your efficiency and seamlessly integrate with your standard workflow.

ICON-NMR is the graphical user interface for fully automated acquisition and processing. ICON-NMR supports sample changers, sample preparation robots and manual operation. The user can set up or supervise measurements remotely via WEB browser from desktop or pocket PC.

ICON-NMR™

The SampleJet is Bruker’s most versatile sample changer. It is designed for open access operation as well as for high throughput batch-automation. It supports a variety of NMR tube diameters and lengths. This sample changer accepts five well plates with 96 NMR tubes each and up to 96 single NMR tubes without the need for spinners for open access laboratories. The SampleJet is also available in a version where the samples may be cooled for longer time storage in the sample changer.

Automation

ICON-NMR’s screening setup window provides easy acquisition and analysis of samples, ranging from crude extracts to pure material, using a user defined SBASE and screening profile.

Hyphenation

Major tools for natural products research include HPLC, SPE, NMR and MS. Bruker

offers combined systems to meet various research needs. Our Metabolic Profiler

is a dedicated, integrated system featuring an AVANCE NMR spectrometer and a

micrOTOF-Q II™ from Bruker Daltonics. By combining the structural resolving power

of NMR with the mass accuracy of the micrOTOF, we can offer the most complete

system for structural analysis and integrated NMR/MS statistics available today.

Optional equipment can combine a third technique resulting in an LC-NMR-MS

system. The use of solid phase extraction provides an efficient interface between

chromatography and NMR and is easily integrated into this system to enable

concentration of low-level metabolites.

LC-SPE-NMR-MS Results

LC-(SPE)-NMR-MS of Apple Juice (A) high resolution mass spectra from m/z 355.1034 (upper part) of chlorogenic acid and the comparison with ion trap library (lower part) (B) 1H NMR spectrum of chlorogenic acid and the comparison with reference compound commercially available.

LC-(SPE)-NMRTwo different main workflows are possible by either (1) coupling the chromatography system directly to the NMR spectrometer, or (2) by collecting the samples into loops that are transferred to the NMR after the chromatographic separation. Direct coupling allows stop-flow or on-flow analysis. Loop transfer minimizes sample handling and limits exposure to air or light. Collection on SPE cartridges is also possible.

A

B

NMR -- Liquid Handler -- SPE -- micrOTOF-Q II

TopSpin™

TopSpin is our software for acquiring, processing and analyzing NMR data in one convenient, streamlined package. TopSpin is designed for Windows® and Linux® users, and features a highly intuitive interface utilizing the most widespread standards from word processing, graphics, or presentation programs, providing the same look-and-feel for your NMR applications.

TopSpin

The molecular structure verification tool uses a powerful combination of experimental NMR data and computational tools. The measured 1H, 13C-HSQC shift correlation spectrum is compared to predicted values. The structure/data consistency rating is reported back to the user in an easy-to-read visual format. Spectral simulation using PERCH™ assists 1D proton and carbon analysis.

TopSpin’s cursor linked multiple-spectrum display allows for easy data evaluation. Its window consists of a data area, a data browser, toolbars and a menu bar for a simple interface to the data and spectrometer. The data area provides easy access to all aspects of the dataset, including the spectrum, parameters, pulse program, title, peak lists, integral list, structure, and FID.

600 MHz AVANCE III CryoProbe System with console running TopSpin software

Bruker’s solutions for natural product research incorporates various analytical methods, each with unique features to benefit your specific area of study. These include: accurate complex structure information through NMR, high throughput analysis with X-ray crystallography and molecular identification by MS/MS.

Bruker is your ideal partner in finding the most suitable solution for your natural products research.

Some of our products that are particularly useful for natural products research include:

NMR Spectroscopy

CryoProbe Technology

Small Volume Probes

AVANCE III

LC-(SPE)-NMR-MS

SampleJet

Mass Spectrometry

micrOTOF-Q II

Atmospheric Pressure Chemical Ionization (APCI) accessory

SmartFormula 3D

X-ray

Smart X2S

Solutions at a Glance

Bruker BioSpin

info@bruker-biospin.comwww.bruker-biospin.com

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