Contact time (ms)
III. Self Evaluation
In the past two years (Nov 2003 to Sept 2005), we had managed to prepare and characterize the target sol-gel glass systems, which were then soaked in simulated body fluid to induce the formation of HAP. The molecular mechanism of apatite formation on bioactive glass surface is studied using the techniques of XRD, EDX, SEM, FT-IR, and solid-state 31P NMR. Using the sol-gel method a bioactive glass system containing glass beads of 2 to 3 microns in size is prepared with the composition containing 30% CaO – 70% SiO2. Our experimental data support the apatite formation mechanism proposed by Hench concerning the precipitation and crystallization of calcium phosphate. The phosphate ions initially deposited on the glass surface are largely in amorphous phase and have substantial amount of water molecules in the surrounding. As the soaking time in simulated body fluid increases, some of the water molecules diffuse out of the phosphate lattice, leading to the formation of a crystalline phase. Our data show that the structure of the crystalline phase is different from type B carbonate apatite but similar to hydroxyapatite.
In addition, considerable efforts were made to develop solid-state NMR strategy to study our model compounds, viz. octacalcium phosphate (OCP) and HAp. This part of the works, which is in collaboration with Professor C. Y. Mou, has proven to be very fruitful. The molecular mechanism of OCP to HAp transformation in vitro has been studied by several physical techniques, with particular emphasis on solid-state 31P homonuclear double-quantum (DQ) NMR spectroscopy. Together with computer assisted lattice matching, our NMR data reveal that OCP crystals transform to HAp topotaxially along their c axes with anti-parallel relationship, instead of the parallel relationship as presumed in the literature. Furthermore, the data of the 31P{1H}
cross-polarization NMR suggest that water molecules enter the hydration layers of OCP crystals via the hydrolysis reaction HPO42- + OH- = PO43- + H2O, which also accounts for the deprotonation of the HPO42- ions during the transformation.
We have also made a considerable progress in the development of new solid-state nuclear magnetic resonance (SSNMR) technique for the determination of backbone torsion angle ψ of polypeptides. In particular, we have resolved two important issues. Firstly, we have achieved a good 13C-13C polarization transfer efficiency in a uniformly labeled non-crystalline polypeptide, demonstrating the applicability of our approach to the studies of real biological systems. Secondly, we show that a simultaneous high power 1H decoupling is NOT necessary for J-coupling mediated 13C-13C polarization transfer. We believe that this no-decoupling approach is quite general and can be immediately incorporated into most existing sequences. Altogether, we
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anticipate that J-coupling mediated polarization transfer can find fruitful applications in many structural problems involving non-crystalline solids and systems with significant dynamics.
Concerning the synthesis of our target peptide, we have initiated a collaboration with Dr.
Steve S. F. Yu at the Chemistry Institute of Academia Sinica. The laboratory of Dr. Yu is well-equipped for peptide synthesis. As expected, it is relatively straightforward to prepare and purify our target peptide DpSpSEEKFLRRIGRFG. However, it proves to be very difficult to attach enough amount of the target peptide to HAp crystal surface for solid-state NMR study. The main difficulty is that the zeta-potential of HAp crystal surface is of negatively charged and therefore the conventional protocol of mixing the peptide solution and HAp crystal in buffer solution is not effective. Nevertheless, we have developed a new strategy to enhance the peptide attachment. This work will provide a roadmap for the future development of the idea described in this project.
Overall, this pilot project has proven the applicability of solid-state NMR spectroscopy to the investigation of the molecular mechanism of biomineralization. Although the major objective, characterization of the conformation of peptides attached on bioactive glass surface, has not yet accomplished, we have completed most of our specific aims. Upon the completion of this project, which is the very first NSC funded project of the principal investigator, the results have been resulted in six full papers (five published and one submitted). Additional experiments are still underway to study the glass-peptide interaction. Furthermore, this project has seeded many collaboration works and the NMR methodology developed in this project has provided a very solid foundation to further our study on teeth and bones
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Appendix
Thanks to the traveling allowance provided in this project, I have attended the following international conference in solid-state NMR spectroscopy:
The 4th Alpine conference on Solid-State NMR, Charmonix, France.
September 11-14, 2005
The aim of the conference is to provide an international forum for high-level discussions to physicists, chemists, biologists and the other scientists with both an academic or industrial background, interested in the latest developments in solid-state NMR. The meeting focuses on the state of the art and theoretical and methodological developments, as well as on recent applications of solid-state NMR in fields as diverse as: organic and inorganic chemistry, catalysis, structural biology, materials science and polymer science.
Although I was not an invited speaker of the conference but I was encouraged by the organizer to give an oral presentation of my work. The “invitation” email is reproduced below:
Dear Jerry,
I am sending you this on behalf of the scientific committee for the 2005 Alpine solid-state NMR conference (http://www.alpine-conference.org/).
We hope very much that you can attend the conference and will shortly register and submit an abstract before the deadline (May 31). On the basis of the work we know about we expect to offer you a contributed talk at the conference.
The organizing committee will send out invitations after all the abstracts have come in.
Please be aware, however, that the conference does not usually pay expenses for contributing speakers. If you are eligible, you may be able to apply for a student stipend (details on the web site).
best wishes, mobile: +44 77 6652 2964
email: Malcolm.Levitt@soton.ac.uk website: http://www.mhl.soton.ac.uk
*************************************************************
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The title of my talk is
“Efficient Spin-Spin Scalar Coupling Mediated 13C-13C Polarization Transfer in Solid-State NMR Spectroscopy.”
Although the conference does not publish any proceedings, the materials of the presentation I gave there have been published:
1. Y Mou, JCH Chao, JCC Chan, 2006, “Efficient Spin-Spin Scalar Coupling Mediated C-13 Homonuclear Polarization Transfer in Solid-State NMR Spectroscopy,” Solid State Nucl.
Magn. Reson., in press.
2. Y Mou and JCC Chan, 2006, “Frequency Selective Polarization Transfer Based on Multiple Chemical Shift Precession,” Chem. Phys. Lett., in press.
My presentation is a great success and I believe the NMR pulse sequence we have developed during the execution of this project has obtained adequate exposure. Indeed, we notice that the MCSP approach described in our project has a considerably impact on the community. In the same conference, another group led by Dr. Robert Tycko (Laboratory of Chemical Physics, NIH, Bethesda) also presented a technique based on a very similar principle. In the preprint of his submitted manuscript, it has been stated that:
“Independently, Chan and coworkers have shown that the introduction of chemical shift precession periods into the R-TOBSY recoupling sequence leads to frequency-selective polarization transfers. R-TOBSY produces a scalar, zero-quantum effective coupling Hamiltonian.
Chemical shift precession periods produce a phase modulation that truncates the effective Hamiltonian to the form in Eq. (17) unless n