Hanna S- Gracz PhD.NMR Facility

North Carolina State University

Using ACD/2D NMR Processor Under the Virtual Computing Lab (VCL )

Outline

1. LITRE funding for ACD/2D Processor

2. ACD/2D processing software under VCL

3. NMR data distribution and accessibility -application tovariety of problems at

4. 2D NMR more powerful than 1D NMR for fast testing2D demo course

5. Collaborators- Lipidomics-core Facility at MUSC

Ad 1. LITRE funding for ACD/2D Processor

Motivations

1. Provide common platform –NMR Fourier transformation processing software for users in thirteen (13) different Departments over two Campuses

2. Facilitate communication, improve access and increase the use of available NMR resources within the NMR Facility and within NC State University

3. Bring closer Faculty with the NMR expertise and needs, to facilitate interdepartmental collaboration and project sharing

4. Improve general student learning skills in a fields of physical and analytical chemistry

5. Help Faculty to accommodate students individual learning style by enhancing available teaching and learning NMR tools

6. Provide students with modern electronic tools for the NMR data analysis, spectra prediction and report writing

NMR Spectrometers and Software

Bruker AVANCE 500 MHzRed Hat LinuxTopspinBruker DMX 300 MHzWindows NT WorkstationXWINNMR 3.6Varian INOVA 600MHzSun workstationVnmrsysVarian Gemini 300 MHzVarian 300 MHz wide boreVarian Mercury 400MHzVarian Mercury 300MHzVarian Gemini 200 MHz

NMR Equipment and SoftwareNMR Equipment and Software

College of Engineering

Working in cooperation with NC State colleges and departments, Information Technology Division (ITD) staff take pride in providing IT infrastructure, services and expertise that enable students, faculty and staff to achieve -

Innovation in Action

Ad 2. ACD/2D processing software under VCL

Virtual Computing Laboratory

What is it?An environment delivery service

Remote access to high-end softwareMotivation

Student owned computingDistance Education

Traditional lab modelShared compute resources

Custom compute environments

WebInterface Scheduler Schedule

DB

ManagementNode

Servers

ApplicationImageLibrary

Internet

NCSU Virtual Computing Lab Model

User gets accessthrough web interfaceand requeststhe application

Scheduler findsa server with therequested applicationor has management node load requested application on a server

Server with requestedapplication is made available to user

VCL Manager Software

WebInterface Scheduler Schedule

DB

ManagementNode

Institution AServersManagement

NodeServers

Server aggregation in sharedVCL Data Center B

Internet

ManagementNode

Servers

Fully distributed VCL Data Center A

ApplicationImage Library

Institution BServers

WebInterfaceSchedulerSchedule

DB

Remotely scheduled

The Virtual Computing Initiative: Anytime, Anywhere On Demand Computing

VCL step by step introductionfor Microsoft Windows and Linux

A snapshot of the VCL reservation page

This is a snapshot of the VCL reservation status page

A snapshot of the VCL current reservation

After each reservation the machine is sanitized and dynamically reloaded for the next user reservation

A snapshot of the VCL connect protocol using Remote Desktop and ssh

A snapshot of the VCL remote computer info

Program>Communication>Remote Desktop>Connection>Options

A snapshot of the VCL reservation Remote Desktop

ACD/2D NMRVersion 9.0 for Microsoft Windows

Tutorial1. Basic Processing of the FID Spectrum

• Load raw data into ACD/2D NMR Processor;FID.• Process the free induction decay file (FID) into a FT spectrum;• View a spectrum in two-dimensional coordinate system.

2. Analyzing the FT spectrum using phase correction tools;• Correct the baseline;• Symmetrize the FT spectrum;• Merge defined multiplets in accordance with the previously chosenoptions;

• Edit an experimental spectrum.3. Analyzing the FT Spectrum

• Define a peak-picking area;• Pick peaks, both manually and automatically;• Measure the distance between peaks;• Adjust the spectrum to a reference point;• Integrate peaks, either manually or automatically;

This system has recently been implemented . We are upgrading to 10.0

ACD/labs 2D NMR processor

2D processor

Phasing 2D spectra manually

Horizontal phase correction

Vertical phase correction

3.2.2. Manual Peak Picking

3.3 Adjusting the Spectrum to a Reference Point

3.4 Measuring Distance

Ad 3. 2D demo course

2D NMR more powerful than 1D NMR for fast testing

NMRNMR

chemistrychemistry physicsphysics

biologybiology medicinemedicine

Jean Jeener, Lecture at Ampère Summer School, BaskoPolje, Yugoslavia – 1971

Proposed to introduce a true second frequency dimensionProposed a two-pulse experiment in the time-domain for which Fourier transformation of the response would yield a 2D spectrum (unpublished)

Richard Ernst, published the first 2D experiments– W. P. Aue, E. Bartholdi and R. R. Ernst (1976)

J. Chem. Phys. 64, 2229.

Who Invented 2D NMR?Who Invented 2D NMR?

2002 Nobel Prize in Chemistry2002 Nobel Prize in Chemistry

Showed that NMR was possible for proteinsFrom Nobel Web page http://www.nobel.se/chemistry/laureates/2002/public.html

Kurt WüthrichSwiss Federal Institute of Technology (ETH), Zürich, Switzerland and The Scripps Research Institute, La Jolla, USA

”for his development of nuclear magnetic resonancespectroscopy for determining the three-dimensionalstructure of biological macromolecules in solution”.

TwoTwo--dimensional NMR spectroscopydimensional NMR spectroscopyuutilizetilize nuclear couplingsnuclear couplings

2D NMR pulse sequence

t2t1t1 t2

Types of internuclear couplings

Through-bond (J,scalar) Through-space (dipolar)

General 2D General 2D eexperimentxperiment

The nuclear spins are preparedThe nuclear spins are prepared for the experiment by establishing for the experiment by establishing some wellsome well--defined statedefined state..

o Steady-state condition from thermal equilibrium or from rapid RF pulsing.

o final part of preparation period: one or more pulses that place magnetization at right angles to the direction of the magnetic field axis (this allows to discriminate between chemically different nuclei on the basis of their respective precession rates during the evolution period

o Requirement: spin system brought to well-defined state that is the same for all separate values of t1.

Preparation time

Do not confuse t1 (evolution) and t2 (detection) with T1 (spin-lattice relaxation time: longitudal)and T2 (spin-spin relaxation time: transverse).

Magnetization precesses in environment that might have:

o refocusing pulses to decouple J-couplings and/or refocus chemical shifts.

o during all or part of this time homonuclear or heteronucleardecoupling may be applied.

o pulsed field gradients may be applied.

Interactions examined in the 2D experiment are active during this time.

General 2D General 2D eexperimentxperiment

Evolution time (t1)

General 2D General 2D eexperimentxperiment

Mixing time

Redistribution of nuclear magnetization among the spinso distribution may involve the use of pulses and/or time periods

The idea is to allow spin communication for a fixed periodo two examples of mechanisms of spin communication :

o J-couplings o dipolar relaxation

General 2D General 2D eexperimentxperiment

Detection

The detection part of 2D experiment :o recording of normal FID producing a chemical shift spectrum.

The appearance of the spectrum:o usually differ in phase and intensity from the ordinary spectrum

The phase and/or intensity variations can be investigated in a complete manner by systematically and regularly varying the evolution time (t1) from zero to some upper limit

TwoTwo--ddimensionalimensional NMR NMR sspectroscopypectroscopy..Experimental Experimental sschemecheme

Evolution (t1): arrayed

Array of evolution time (t1); acquire spectra for each t1 value

2D NMR 2D NMR mmechanicsechanics

2D spectra are collected “point by point” in t1, the evolution time. Series of free induction delays (FIDs) are collected, one for each value of t1, which is incremented systematically by fixed value, starting from t1=0.A data matrix is produced, where the rows represent actually acquired FIDs.

Row represents a different value of t1 - columns give the intensity of the signal as a function of t1. After fourier transforming (FT) the rows, each column shows the intensity of a particular signal in F2 as a function of t1. A second FT is performed to the columns - convert the columns into the second frequency dimension, which is called F1.

Acquire a series of FIDs:starting with t1=0, the evolution time t1 is incremented byΔt1 from one FID to the next one.

A series of spectra in Ω2,an interferogram ("FID") in the orthogonal direction (t1).

A 2D NMR spectrum:diagonal peaks (Ω1=Ω2)and cross peaks (Ω1≠ Ω2)

TwoTwo--ddimensionalimensional NMR NMR sspectrumpectrum

Graphical representation of twoGraphical representation of two--dimensional spectradimensional spectra

Stacked plotSeries of F2 spectra for different F1-

values plotted one above another

Top view of the stacked plot Contour plotA section in a plane at a fixed height

through the peaks of the stacked plot

Graphical representation of twoGraphical representation of two--dimensional spectradimensional spectra

TwoTwo--ddimensionalimensional NMR NMR sspectrumpectrum

Spectrum

Coupled spinscross-peaks

t2t1

diagonalpeaks

2D NMR: resolving overlapping signals2D NMR: resolving overlapping signals

1D

2D

2 signalsoverlapped

2 cross peaksresolved

W. Chazin (Vanderbilt University). NMR notes: http://structbio.vanderbilt.edu/chazin/classnotes/

COSY = Correlated spectroscopyDQ-COSY = Double quantum filtered COSYECOSY = Exclusive correlation spectroscopyNOESY = Nuclear Overhauser and exchange spectroscopyROESY = Rotating-frame Overhauser spectroscopyTOCSY (HOHAHA)= Total correlation spectroscopy (Homonuclear Hartmann-Hahn)EXSY = Exchange spectroscopySECSY = Spin-echo correlated spectroscopyHETCOR = Heteronuclear CorrelationCOLOCS = Correlation spectroscopy for long-rang couplingsHOESY = Heteronuclear Overhauser enhancement spectroscopyHET2DJ = Heteronuclear two-dimensional J-correlated spectroscopyHMQC = Heteronuclear Multiple Quantum CoherenceHSQC = Heteronuclear Single Quantum CoherenceHMBC = Heteronuclear Multiple Bond CorrelationINADEQUATE = Incredible natural abundance double quantum transfer

experimentAnd more.........

2D NMR Spectroscopy2D NMR SpectroscopyAcronyms for basic experimentsAcronyms for basic experiments

2D NMR spectroscopy2D NMR spectroscopyAcronyms for basic experimentsAcronyms for basic experiments

Homonuclear(1H-1H)

COSYScalar Coupling(directly bonded atoms)

Dipolar Coupling(spatially close atoms)

Heteronuclear(1H-13C, 1H-15N)

TOCSY

NOESY

HSQCHMQC

NOESY-HSQC

NOESY-HMQC

Differs only by nature of mixing

HETCOR

COLOC

HMBC

An important effect which depends on how close in space nuclei are•Usually only up to about 5 Å

The 2D COSY experiment is the most simple and widely used 2D experiment. It is an homonuclear chemical shift correlation experiment based on the transfer polarization by a mixing pulse between directly J-coupled spins. Thus, homonuclear through-bond interactions can be trace out by simple analysis of the 2D map

The 2D COSY experiment is a simple two-pulse sequence in which a variable evolution period is inserted between these two pulses. During this period, both J-coupling and chemical shift evolutions take place.

2D 2D 11HH--11H H COSY (COSY (COrrelationCOrrelation SpectroscopYSpectroscopY))

N

OO

O

N

NN

N

HH

HH

H

H

H

H

H

H

H

HH

1'2'3'

4'

5'

4

5

2

6

8

11HH--11H COSY spectrum H COSY spectrum ofof deoxyadenosinedeoxyadenosineH8 H2 NH2

H1’H3’-OH

H5’-OH H4’H2”H2’H5’,H5”H3’

OO

OH

H

HH

H

H

H

H

H

Base

1'

2'3'

4'

5'

2''

3'-OH

5'-OH

5"

ExpandedExpanded region region ofof 11HH--11H COSY H COSY spectrum spectrum ofof deoxyadenosinedeoxyadenosine H1’

H3’-OHH5’-OH H4’

H2”H2’H5’,H5”H3’

2D 2D 11HH--X HSQC (X HSQC (HeteronuclearHeteronuclear SingleSingle--Quantum Correlation)Quantum Correlation)

The 2D HSQC experiment permits to obtain a 2D heteronuclear chemical shift correlation map between directly-bonded 1H and X-heteronuclei (commonly, 13C and 15N). It is widely used because it is based on proton-detection, offering high sensitivity when compared with the conventional carbon-detected 2D HETCOR experiment. Similar results are obtained using the 2D HMQC experiment.

The HSQC spectrum correlates chemical shifts of heteronucleus X (F1 dimension) and protons (F2 dimension) via the direct heteronuclear coupling 1J(XH). Carbon decoupling is usually performed during proton acquisition and the corresponding satellites collapse to a single resonance showing all proton-proton couplings.

11HH--1313C HSQC spectrum C HSQC spectrum ofof deoxyadenosinedeoxyadenosine

N

CH

CH2

O

CH

CH

CH2OH

OH

C

C

C

N

N

CHCH

N

NH2

C5’

C2’

C3’

C4’C1’

C8

C2

H8H2

NH2H1’ H3’-OH H5’-OH H4’ H2”H2’

H5’,H5”H3’

The 2D HMBC experiment permits to obtain a 2D heteronuclear Chemical Shift correlation map between long-range coupled 1H and heteronuclei (commonly, 13C).

It is widely used because it is based on proton-detection, offering high sensitivity when compared with the carbon-detected COLOC experiment.

In addition, long-range proton-carbon coupling constants can be measured from the resulting spectra.

11HH-- X HMBX HMBCC ((HeteronuclearHeteronuclear Multiple Bond CorrelationMultiple Bond Correlation))

11HH--1313C HMBC spectrum C HMBC spectrum ofof deoxyadenosinedeoxyadenosine

N

CH

CH2

O

CH

CH

CH2OH

OH

C

C

C

N

N

CHCH

N

NH2

H8 H2 NH2

H1’

H3’-OH

H5’-OH H3’ H2”H2’H5’,H5”H4’

11HH--1515N HSQC spectrum N HSQC spectrum ofof adenosineadenosine

NO

OH

OHOH

N

NN

NH2

1

3

9

7

N9

N7N1N3

H8 H2

NH2H1’ H2’

NO

OH

OHOH

N

NN

NH2

1

3

9

7

11HH--1515N HMBC spectrum N HMBC spectrum ofof adenosineadenosine

College of Textile

We are using 2D NMR spectroscopy to characterize the complexes of inclusion compound cloprestenol with beta-cyclodextrins

T.Uyar, M.A.Hunt, H.S. Gracz, A.E.Tonelli (2006)Crystal Growth&Design vol 6,No 5, 1113-1119

Department of Chemistry at Centennial Campus-liganddevelopment project

Targeting RNA with cyclic peptides

•Non coding sequences regulate the function of mRNA and DNA. In animal mRNA, IRON REGULATORY ELEMENTS (IRE’s) regulate the synthesis of proteins for iron storage, uptake and red cell heme formation. The ferritin iron responsive element (IRE), a conserved sequence of 30 nucleotides is a hairpin loop. It is a conserved mRNA specific translational regulatory sequence which forms 9-17 base pairs close to 5’ cap. IRE’s bind specific regulatory proteins IRP.

Background

Z.Gdaniec, H.S-Gracz, E.C. Theil (1998) Biochemistry 37, 1505-1512

2D NMR 1H-15N HSQC / IRE Imino Region

Ad 4. Lipidomics-core Facility at MUSC

The Lipidomics Core at MUSC provides conceptual and practical training in various aspects of lipidology, qualitative and quantitative analysis of lipid components from different biological materials (cells, tissue, biological fluids), synthetic molecular tools to study lipid metabolism (functionalized and fluorescent ceramides, site-specific and radioactive sphingolipids), diversified synthetic lipids and analogs for cellular, in vitro, and in vivo studies (organelle-targeting sphingolipids and organelle-targeting inhibitors of sphingolipid metabolizing enzymes). They also assist investigators in experimental design, selection of lipid of interest and interpretation of the analytical results.

• Ceramides are non-charged, densely functionalized sphingosine-derived lipids.

•They are involved in the regulation of several cellular processes: differentiation, growth suppression, cell senescence and apoptosis.

•Ceramide, by itself, or as the lipid backbone of sphingomyelin and glycosphingolipid, is a structural and functional component of cell membranes.• We have noticed a strong correlation between the electronic structure of ceramides and their in vivo and in vitro activity..

INTRODUCTION

Zdzislaw M. Szulc Jacek Bielawski, Hanna Gracz, Yusuf A. Hannun and Lina Obeid , Alicja Bielawska *Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 294251

HO

N

OH

H

O

N Br

HO

N

OH

H

O D-erythro-C16-Ceramide

D-erythro-C16 - Pyridinium-Ceramide Bromide (LCL - 30)

+

CERAMIDES

Determination of the molecular geometry of ceramides is an important step in understanding their cellular function.

However, due to the very poor solubility in water, their aqueous solution structure was never investigated under physiological conditions.

We overcame this predicament by:

1. mixing ceramide with a selected

cationic surfactant,

2. modifying its hydrophobic interface .

Ceramides were synthesized and used in the following two model systems: D-erythro-C16-ceramide, L-threo-C16-ceramide and their mixtures with a fully deuterated cationic surfactant: cetyl pyridinium bromide (CPB-d38). For the comparison studies we used D-erythro-C6-ceramide, D-erythro- 4,5-dihydro-C16-ceramide and a cationic analogs of D-erythro- C16- ceramide and L- threo-C16-ceramide with identical cationic moiety incorporated as tethers into N-fatty acid parts.For NMR studies, lipid samples were dissolved in CDCl3, MeOD or D2O.

EXPERIMENTAL

Zdzislaw M. Szulc Jacek Bielawski, Hanna Gracz, Yusuf A. Hannun and Lina Obeid , AlicjaBielawska *(2006) Bioorganic&medicinal Chemistry ,14 (18) 7083-7104

O

N

O

O

H

H

H

D- erythro -C16-Ceramide

Interfaces of Ceramide and Their Four Distinct Points

12 3

4

518

1'2'

16'

D-erythro C16 ceramides in MeOD and CDCL3

D-erythro and L-threo ceramides in water

2D HMQC D-Erythro L-Threo C16 ceramides

ConclusionConclusion

• Confomational preferences in the polar interface of ceramides are controlled by their key structural factors in the following order: stereochemistry > saturation at the C4-C5 positions > molecular geometry of the hydrophobic interface. • Mixing ceramides with CPB-d38 or modifying their structure by the incorporation of the cationic moiety as a tether in the hydrophobic part, does not interfere with the conformation of the ceramide. • Profound differences were observed for the methyleneprotons at the C(1)H2 position located in the polar interface of ceramides and for the protons vicinal to the amide group (NHCOCH2, intermediate polarity interface

Summary

1. Using ACD/2D processor under Virtual environment gives researchers access to tools they normally wouldn’t have /use

2. This allows breaking prejudice against use of 2D experiments routinely

3. This gives the researchers access to tools which they might not have in another software

4. This type of virtual system brings hope for attracting more users and generating enough revenue to be able to afford next year total ACD package including famous ACD/Structure Elucidator

AcknowledgementsNCSU Team

Dr. Ken Hanck

Barry Eriksen

Aaron Peeler

Zachary Cook

Dariusz Schmidt

ACD/Labs

Dr. Anthony Williams

Kelly Turnbull

Tricia Corrin

Ryan Sasaki

Financial SupportNCSU-LITRE to Hanna GraczNCSU NMR Facility

Collaborators

Dr. hab.Zofia Gdaniec

IBCH-Poznan, PolandDr. Jim Beery BASF

http://vcl.ncsu.edu

Users

Database Management node NManagement node 1

Management node 2

University Labs

Bladecenter Bladecenter

Image Library Image Library

VCL Infrastructure

Hardware Blades, servers, desktops, storage

OS:

Apps

Win Linux Other …

VirtualLayer

OS: Win Linux …

Apps

e.g.,Web

Sphere

e.g., Web

Sphere

SOARDP,VNC,

…

e.g., VMWare,

XEN, MSVS2500,..

X-WinClient

Apps.WorkFlow

Services

End-UserAccess

VisServices Other …

Middlewaree.g. LSF

VCLManager

“Applic

atio

n”

Imag

e Sta

ck

xCAT VCL code IBM TM

WebServer DataBase Etc.

Users“Images”

H/W ResourcesLocal or distributed

Differentiator: User to Image to Resource Mapping, Management & Provenance

Simplicity, Flexibility, ReliabilityScalability, Economy

EvolutionEvolution ofof 1313C C magnetizationmagnetization vectorsvectors for for twotwo spin AX systemspin AX system(A=(A=11H H andand X=X=1313C) C) atat differentdifferent timestimes tt11

a: t1=0 b: t1=[4J(C,H)]-1

c: t1=[2J(C,H)]-1

d: t1=3[4J(C,H)]-1

e: t1=[J(C,H)]-1

FT with respect to t2 (with 1H decoupling) gives single 13C peak whose amplitude depends on t1.

Amplitude is modulated at a frequency related to J(C,H)

FT with respect to t1 yields two peaks with a separation J(C,H)