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The National Stable Isotope Resource at Los Alamos

Contents

• Contact Information
• Grant Number
• Research Emphasis
• Current Research
• Resource Capabilities
• References

Contact Information

National Stable Isotope Resource at Los Alamos
Los Alamos National Laboratory
B-3, M.S. E529
Los Alamos, NM 87544
http://sir.lanl.gov/

Principal Investigator/Contact
Clifford J. Unkefer
Phone: 505-665-2560
Fax: 505-665-5052
cju@lanl.gov

Contact
Louis A. (Pete) Silks
Phone: 505-667-0151
Fax: 505-665-5052
pete-silks@lanl.gov

Grant Number

Grant No. EB002166

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Research Emphasis

The primary function of the SIR is to develop new methods to label biomolecules. The Technology Research and Development component of the SIR develops new, efficient methods for the synthesis of labeled compounds required for modern spectroscopic methods. Specifically the SIR focuses on developing new-labeled precursors, stereospecific labeling of L-α-amino acids, and synthesis of labeled nucleotides. In addition, the SIR is developing stereospecific methods for introducing multiple stable isotope labels into carbohydrates. The Service component of the SIR supports biomedical researchers applying novel stable isotope labels to important biomedical problems by providing labeled compounds that are not readily available. We develop Collaborative Research arrangement that emphasize the development of new synthetic methods and highlight applications of isotopically labeled compounds to important biomedical problems. Finally, the SIR performs a significant technology transfer function, which has resulted in a viable stable isotope industry that serves some of the needs of the biomedical research community. 

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Current Research

Labeled Precursors

Because carbon-13 and nitrogen-15 are separated from their lighter isotopes by cryogenic distillation of CO and NO, all labeled carbons and nitrogens must be derived ultimately from these two compounds. The highly efficient conversion of CO and NO to useful chemical precursors is perhaps the most unique aspect of stable isotope labeling chemistry. Historically, the SIR has devoted considerable effort to the development of methods for the preparation of useful synthetic synthons including methane, methanol, sodium formate, potassium cyanide, and carbon dioxide. At present, we are developing a potentially powerful new set of labeled synthons based on methyl-labeled thioanisole. We have developed the capability to produce mole quantities of thioanisole in excellent yield and labeled in the methyl group with ¹³C and any combination of hydrogen isotopomer (H₃, H₂²H, H²H₂, ²H₃). We have also developed a variety of ¹³C- and/or ²H-labeled synthons from thioanisole, and are exploring the application of these synthons in the synthesis of amino acids, nucleotides, and carbohydrates.

Stereoselective Synthesis of Labeled L-α-amino Acids

We are exploring the application of Oppolzer's camphor-based chiral glycinate to the stereoselective synthesis of stable isotope labeled α-amino acids. N-protected glycine (N-[bis(methylthio)methylene]-glycine) is linked as an amide to the nitrogen in the sultam ring of Oppolzer's chiral auxiliary. This chiral glycine equivalent is metallated by treatment with n-butyl lithium. Decomposition of the enolate with electrophiles is carried out in the presence of hexamethylphosphoramide to yield L-α-amino acids. This process occurs with remarkable stereoselectivity. Starting with the (2S)-camphorsultam and using a series of alkyl iodides as electrophiles, we have produced a variety of amino acids including L alanine, L-valine, L-leucine, L-phenylalanine, and L-aspartic acid, L-proline, L-methionine, L-ornithine, (L-arginine), L-lysine, in enantiomeric excess of greater than 99.5%. The alkylation reaction was uniformly efficient and had remarkable enantioselectivity. We have developed efficient procedures for deblocking the product amino acid that minimized racemization of the product.

Synthesis of Labeled Carbohydrates

During the past decade our laboratories have been developing both the chemistry of the selenocarbonyl and its use as a ⁷⁷Se NMR probe to interrogate remotely disposed chiral centers. During the course of these investigations we uncovered a new type of aldol reaction using chiral selone reagents in which the selenocarbonyl plays a pivotal role in determining the stereoselectivity of these reactions. Initially we observed that the reaction of benzaldehyde with the titanium-based enolate of an N-acyl selone gave one predominant product. Not only did the product appear to be stable and formed in good yield, but the reaction also gave the opposite syn isomer to the one observed for an Evans-type process. The relationship between the two new chiral centers that are generated in this carbon-carbon bond forming reaction is anti. We are developing routes to labeled carbohydrates that take advantage of these aldol reactions to construct labeled carbohydrates.

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Resource Capabilities

High Resolution NMR Spectrometers

• Bruker Avance-500 NMR Spectrometer (500 MHz)
• Bruker DRX-500 NMR Spectrometer (500 MHz)
  Both 500 instruments have pulsed field gradients, 2H decoupling, and four channel capabilities with a triple axis gradient triple resonance probe suitable for the 13C and 15N measurements. The instruments are equipped with seven probes including a broadband inverse probe and an inverse triple resonance probe (1H, 13C, and 15N).
• Bruker Avance-300 NMR spectrometer (300 MHz 1H)
• Bruker DPX-300 NMR Spectrometer (300 MHz 1H)
• Bruker AMX-400 wide-bore NMR spectrometer (400 MHz 1H)

Mass Spectrometers

• Thermo Finnigan FT Mass Spectrometer
  • Thermo Finnigan LTQ
  • Thermo Finnigan Surveyor quaternary liquid chromatograph
  • Thermo Finnigan Surveyor diode array UV/VIS detector
  • Thermo Finnigan Surveyor autosampler
• Thermo Finnigan LCQ DECA XP Plus Ion trap mass spectrometer
  • Thermo Finnigan Surveyor quaternary liquid chromatograph
  • Thermo Finnigan Surveyor diode array UV/VIS detector
  • Thermo Finnigan Surveyor autosampler
• Thermo Finnigan Polaris Q Ion trap mass spectrometer
  • Thermo Finnigan Trace gas chromatograph
  • Combi-PAL autosampler for head-space and liquid sampling

Chemical Synthesis Equipment

• High pressure and vacuum lines required for transfer of ¹³C-labeled CO, CO₂, CH₄, and ¹⁵N-labeled NO, NH₃, N₂, etc.
• Two Labeled Methanol Synthesis plants: High pressure reactors used to make ¹H, ²H, ¹²C and ¹³C isotopomers of methanol.
• 1-liter High Pressure Hastelloy C reactors (Autoclave Engineers): for  the synthesis of ¹³C-labeled formate, acetate, propionate, ethanol, etc.
• Applied Biosystems Model 433a Peptide Synthesizer
• Advanced ChemTec  APEX Model 396 peptide synthesizer
• Applied Biosystems Model 394 DNA/RNA oligonucleotides synthesizer
• B/R Instruments Spinning Band Distillation apparatus.

Analytical and Chromatography Equipment

• Thermo Flash 112 Series EA (Elemental analysis)
• Nicolet 550 FT IR spectrometer
• Automated Amino Acid Analyzer, Beckman model 7300
• High pressure Liquid Chromatographs
  • Varian ProStar HPLC
  • Waters model 600 preparative HPLC 
  • Model 2996 diode array detector
  • Model 2767 Sample Manager
  • Parmacia FPLC

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References

1. Martinez RA, Alvarez MA, Velarde SP, Silks LA, Stotter PL, Schmidt JG, Unkefer CJ. Large-scale preparation of [¹³C]methyl phenyl sulfide from [¹³C]methanol by a one-step process. Organic Process Research and Development 2002;6:851-854.
2. Kim SH, Aznar C, Brynda M, Silks LA, Michalczyk R, Unkefer CJ, Woodruff WH, Britt RD. An EPR, ESEEM, structural NMR, and DFT study of a synthetic model for the covalently ring-linked tyrosine-histidine structure in the heme-copper oxidases. Journal of the American Chemical Society 2004;126:2328-2338.
3. Tomson F, Bailey JA, Gennis RB, Unkefer CJ, Li ZH, Silks LA, Martinez RA, Donohoe RJ, Dyer RB, Woodruff WH. Direct infrared detection of the covalently ring linked His-Tyr structure in the active site of the heme-copper oxidases. Biochemistry 2002;48:14383-14390.
4. Ollivault-Shiflett M, Kimball DB, Silks LA. Synthesis of chiral ¹³C, ⁷⁷Se-labeled selones. Journal of Organic Chemistry 2002;69:5150-5152.
5. Kimball DB, Michalczyk R, Moody E, Ollivault-Shiflett M, De Jesus K, Silks, LA. Determining the solution state orientation of a Ti enolate via stable isotope labeling, NMR spectroscopy, and modeling studies. Journal of American Chemical Society 2003;48:14666-14667.