Absolute Structure Determination of Chiral Molecules: StateoftheArt Xray Diffraction based Tools

1
Absolute Structure Determination of Chiral
Molecules State-of-the-Art X-ray Diffraction
based Tools

• A.L.Spek
• Bijvoet Centre of Biomolecular Research
• Utrecht University
• The Netherlands
• Organon, Oss, 25-January 2007

2
Outline of this Talk

• Introduction to who we are.
• Intro to Single Crystal X-ray Structure
  Determination.
• Concept of Absolute Structure (Absolute
  Configuration).
• Resonant Scattering (Anomalous Dispersion).
• Early Applications (Bijvoet, Peerdeman van
  Bommel).
• The Flack Parameter The Current De-facto
  Absolute Structure Analysis Tool (IUCr
  Approved).
• Absolute Structure Determination of Light Atom.
  Structures Problems and Tentative New Tools.
• Concluding Remarks.

3
Who are we ?

• The National facility for small molecule single
  crystal structure determination since 1971 in the
  Netherlands.
• Embedded within the Crystal and Structural
• Chemistry group in Utrecht.
• Most of the Crystal and Structural Chemistry
  research in Utrecht has moved from small molecule
  to protein crystallography (Structural Biology
  Piet Gros).

4
Small molecule and Protein XtallographyUtrecht
5
Some Statistics

• Collaboration of the National facility with most
  synthetic groups in the Netherlands (mostly
  academic and a few commercial) who send their
  samples for analysis to Utrecht.
• We handled over 3800 requests over the past 35
  years. Mainly organometallic and coordination
  chemistry, but also from organic, pharmaceutical
  and mineralogical background.
• Up to now, the results have been reported in over
  1200 (joint) papers.

6
People Involved

• The last years 3 to 4, mostly PHDs, of which one
  on a postdoc position.
• Currently a permanent staff of 2 1 postdoc.
• Dr. Martin Lutz (since 1997)
• Dr. Lars von Chrzanowski
• (postdoc since Oct 15, 2006)
• Successor of Dr. Huub Kooijman (now SHELL)
• In the past a few trained chemists in the
  context of their synthetic work.

7
Associated Functions

• Development of crystallographic software based on
  local needs collected in the PLATON package.
• Crystal Structure Validation (IUCr)
• Co-Editor of Acta Cryst. C (involved in the
  handling of more than 1000 CIF-formatted papers).

8
Single Crystal Structure Determination Routine

• Select and Mount a Suitable Single Crystal,
  preferably taken from the mother liquor (typical
  size 0.3 mm in all directions)
• Determine Lattice Parameters, Space Group
  Symmetry
• Collect (Redundant) Set of Reflection Intensity
  Data, preferably on a CCD based diffractometer
  system at 150 K (in N2 stream) (MoKa X-rays)
• Solve the Phase Problem (I.e. Recover Phases
  Associated with the Measured Intensities)
• Least Squares Refinement of a Parameter Model
  (Coordinates, Displacement parameters, etc.)
• Analysis, Reporting Archiving of the Data
  Results

9
X-Ray source, Goniometer Serial Detector
10
X-ray source, goniometer crystal, N2-cooling
and CCD Detector
11
One of the several hundreds of CCD images with
diffraction spots
12
Data Collection

• Diffraction Condition (determines the position of
  the diffracted beams on the detector)
• 2 dhkl sin(Q) n l (Bragg Equation)
• Result
• - Cell Dimensions, a,b,c, a, b, g
• - Reflection intensities by planes (hkl) in
  the crystal I(hkl) (many thousands)

13
Structure Determination

• Experiment ? Ihkl ? Fhkl Sqrt(Ihkl)
• Needed for 3D structure (approximate) Phases
  fhkl
• Current Tools for Phase Recovery from Ihkl
  with
• - Patterson Techniques (heavy atom
  structures) (DIRDIF)
• - Direct Methods (SHELXS, SIR)
• - New Charge Flipping
• Brute Force, Random Start, Ab-Initio (FFT,
  FFT-1)
• - Fhkl fhkl Fhkl? 3D-Fourier Synthesis
• r(x,y,z) Shkl Fhkl exp-2p(hx ky
  lz) / V

14
Contoured 2D-Section Through the 3D Structure
15
Abstracted and Interpreted Structure
16
Refinement of a 3D Model

• Extract the 3D Coordinates (x, y, z) of the
  atoms.
• Assign Atom Types (Scattering type C, O etc.)
• Assign Additional Parameters to Model the Thermal
  Motion (T) of the Atoms.
• Other Parameters Extinction, Twinning, Flack x
• Model Fhkl Sj1,n fj T exp2pi(hx ky lz)
• Non-linear Least-squares Parameter Refinement
  until Convergence.
• Minimize Shkl w (Fhklobs)2 (Fhklcalc)22
• Agreement Factor R S Fobs Fcalc / SFobs

17
ORTEP Presentation of Model Parameters
18
A-Priori Info Needed

• In principle nothing needs to be known about the
  composition.
• Any available (correct) info may speed up the
  analysis and interpretation.
• Often a service analysis turns up the structure
  of a different compound than intended, either
  boring or an interesting surprise. The only good
  crystal in a batch may be a contamination.

19
Newly Obtained Info

• Confirmation of proposed structure
• Unexpected new structure or chemistry
• Detailed info on the geometry (bonds, angles,
  torsion, ring puckering)
• Polymorphism
• Molecular interactions
• Absolute Configuration

20
X-ray Analysis Routine ?

• Yes under optimal circumstances in the hands of a
  professional.
• No in many cases due to
• Poor Crystal Quality (fine needle bundles etc.)
• Complicated Twinning
• Disorder in part of the molecule
• Disordered (unknown) included lattice solvent
• Pseudo Symmetry, Incommensurate Structures

21
Absolute Structure of Chiral Compounds

• Question before 1951 how to correlate
  microscopic absolute configurations to
  macroscopic properties such as the sign of the
  optical rotation of polarised light.
• Emil Fischer relative system assign D
  configuration to () Glyceraldehyde.
• His lucky choice was later confirmed by
  calculations and physical methods.

22
Example of a Macroscopic Property
23
Absolute Structure
D-()-Glyceraldehyde
Emil Fisher Arbitrary D assignments (50 chance
to be correct)
24
Natural Isomer L-(R,R-() Tartaric Acid)
CIP-Nomenclature for Chiral Atoms R,S
25
Bijvoet used Anomalous Dispersion (Resonant
Scattering) to Solve the Absolute Structure
Problem around 1950
Prof. Dr. J.M. Bijvoet (1892-1980)
26
Resonant Scattering

• X-rays interacting with the electrons in an atom
  scatter in all directions or in crystals in only
  certain directions due to interference.
• The phase of the waves scattered by the outer
  electrons of an atom is shifted by 180 degrees .
• This is no longer true for the inner electrons in
  heavier atoms resulting in a phase shift less
  than 180 degrees
• Therefore scattering factors are real numbers
  only in a first order approximation (I.e. with
  phase 0 or 180o)
• Second order effects become prominent when the
  frequency of the X-rays is close to the resonance
  frequency of the inner electrons of a heavy atom
• (e.g. K shell).

27
Complex Scattering Factors

• Scattering factor f f0 f if
• Where
• f0 a function of diffraction angle Q and
  equal to the number of electrons in the atom at Q
  0.
• f and f atom type and l dependent
• i sqrt(-1)
• Note A phase shift is often represented
  mathematically as a complex number.

28
Selected f - values
29
Friedel Pairs

• It can be derived from the expression for the
  calculated structure factor that for
  non-centrosymmetric crystal structures
• Fhkl not necessarily equal to F-h-k-l
• for f gt 0, thus breaking the earlier
  assumed Friedel Law Fhkl F-h-k-l
• (The Friedel Law still holds for
  centro-symmetric structures containing racemic
  mixtures of chiral compounds).

30
H,K,L
-H,-K,-L
Friedel Pair of Reflections
31
Early Applications

• Around 1930 Coster, Knol Prins determined that
  the shiny side of ZnS corresponds to the Sulfur
  layer and the dull side to the Zink layer.
• Around 1950 Bijvoet et al. generalized this
  method and showed that the arbitrarily assigned
  absolute L configuration of () tartaric acid was
  the correct one. Later this result was also
  confirmed with other techniques.

32
The First page of the famous 1951 Article in
Nature - E. Fischer turned out to have made the
correct choice by luck - Nobel Price ? Dorothy
Hodgkin
33
Experiments of Bijvoet et al.

• Accurate measurement of the Intensities of
  Friedel Pairs
• On Mixed salts of () Tartaric Acids
• NaRb Tartrate
• NaH Tartrate
• Using X-ray Film techniques

34
Qualitative Long Exposures Unstable X-Ray
Sources
35
Quantitative Methods

• Hamilton Test Refine both enantiomorph models
  and statistically test whether the difference in
• R-value is significant.
• Refine a multiplicative h parameter with value in
  the range 1 to 1 to f .
• Beurskens B parameter (from DIRDIF)
• Scatter Plot of Bijvoet Pair Differences
• Refine Inversion Twin Parameter x (Flack,1983)

36
Scatter Plot of Bijvoet Differences

• Plot of the Observed Bijvoet Differences against
  the Calculated Differences.
• A Least-Squares line and Correlation Coefficient
  are calculated
• The Least-squares line should run from the lower
  left to to upper right corner for the correct
  enantiomorph and the Correlation close to 1.0

37
Excellent Correlation
38
Flack Parameter

• The current official method to establish the
  absolute configuration of a chiral molecule
  calls for the determination of the Flack x
  parameter.
• Flack, H.D. (1983). Acta Cryst. A39, 876-881.
• Twinning Model (mixture model and image)
• Ihklcalc (1 x) Fhkl2 x F-h-k-l2
• Result of the least-squares refinement x(u)
• Where x has physically a value between 0 and
  1
• and u the standard uncertainty (esd)

39
Interpretation of the Flack x

• H.D.Flack G. Bernardinelli (2000)
• J. Appl. Cryst. 33, 1143-1148.
• For a statistically valid determination of the
  absolute structure
• u should be lt 0.04 and x lt 2u
• For a compound with known enantiopurity
• u should be lt 0.1 and x lt 2u

40
Practical Aspects of Flack x

• The structure should contain atoms with
  sufficiently strong anomalous dispersion
  contributions for the radiation used (generally
  MoKa) in the experiment (e.g. Br).
• Preferably, but not nesessarily, a full set of
  Friedel pairs is needed.
• Unfortunately, many relevant pharmaceuticals
  contain in their native form only light atoms
  that at best have only weak anomalous scattering
  power and thus fail the strict Flack conditions.

41
Light Atom Targets

• Options for the Absolute Structure
  Determination of Light Atom Compounds
• Add HBr in case of tertiary N.
• Co-crystallize with e.g. CBr4.
• Co-crystallize with compound with known. absolute
  configuration.
• Alternative Statistical analysis of Bijvoet pair
  differences.

42
My First Abs. Struct. Determination Nature 1971
Dextro-Benzetimide HBr
43
Statistical Analysis of Bijvoet Pairs

• Many experimentalists have the experience that
  the official Flack x method is too conservative,
  based on multiple carefully executed experiments
  with compounds with known absolute structure.
• The feeling is that also in light atom structures
  the average of thousands of small Bijvoet
  differences will point in the direction of the
  correct enantiomorph.

44
Example Ammonium Bitartrate Test
45
Ammonium BiTartrate (MoKa)
46
Bayesian Approach

• Rob Hooft (Former PhD student in Utrecht now with
  Bruker-AXS) came up with a new statistical method
  based on Bayesian statistics.
• E.g. Assuming that the material is enantiopure,
  the probability that the assumed absolute
  structure is correct, given the set of observed
  Friedel Pair Differences, is calculated.
• This probability P2 is by a Delft colleague
  dubbed to be called the swallow parameter.
• An extension of the method offers the Hooft y
  (or Fleq) parameter, comparable with the Flack x.

47
Natural Vitamin C, L-()Ascorbic Acid
48
L-() Ascorbic Acid
49
Current Status of the Bayesian Method

• Supporting Paper
• Determination of Absolute Structure using
  Bayesian Statistics on Bijvoet Differences
• R.W.W. Hooft, L.H. Straver A.L.Spek
• Rejected by J. Appl. Cryst., mainly on the basis
  of the verdict of one well-known crystallographer
  as a referee.

50
Concluding Remarks

• The Rob Hooft approach works well for the
  multiple examples we tested but is not officially
  accepted (yet).
• WarningThe absolute structure determination on a
  single crystal is not necessarily representative
  for the absolute structure of all crystals in the
  batch. In principle, multiple crystals should be
  investigated by testing a number of
  representative Friedel Pair differences.
• An absolute structure determination is
  meaningless if not related to a macroscopic
  property such as the sign of the optical rotation
  or special crystal faces etc..

51
END

• THANK YOU
• http//www.cryst.chem.uu.nl

52
(No Transcript)