Andrew Durgan

Department of Chemistry & Biochemistry Gonzaga UniversitySeptember 24, 2009

• Introduction– Carbon-Deuterium Labeled Amino Acids– Methods: Vibrational Techniques– Infrared Spectroscopy (IR)

• IR versus Raman Spectroscopy• IR Sampling

• Results– IR of C-D Proline

– 13C NMR of Proline– Calculations of Proline Conformations

• Conclusion/Future Research

-C-D

O-H, C-H, N-H Amide bands & C=C, C=O, C=N

• C-D frequency shifted away from unlabeled vibrations• Site-specific isotopic labeling improves structural resolution• C-D bonds report on their local environment• C-D bonds report on ψ/Φ dihedral changes• Deuterium labeling produces essentially no perturbation

Protein IR Spectrum

• Unique ring structure imposes backbone conformational constraints

• Proline appears in many neuropeptides• cis-trans Amide isomerization is hypothesized to play a role in

neuropeptide selectivity

D

Cα

Raman

Pro/ConDoes not cause local heating or photodecomposition of the sample

Can cause local heating or photodecomposition of the sample

More accessible instrumentation Less accessible instrumentation due to laser requirement

Con/ProSpectral observations can be obscured by H2O (g) and CO2 (g) absorptions

Spectral observations are not obscured by H2O (g) and CO2 (g) absorptions.

Conclusion: Both spectroscopic techniques can be useful to C-D labeled amino acid investigations.

Infrared (IR)

•Potassium Bromide Pellets

2 mg of sample were homogenized with 80 mg of KBr

•Transmission Spectra

20 µL of aqueous samples (0.050 – 1.0 M) were dispensed between CaF2 plates with a 75 m

spacer.

•Scan Parameters

The sample chamber was purged with N2 and 8000

scans at 4 cm-1 resolution were collected using an MCT detector.

At least four absorptions related to Pro-a-d1 were

readily observed at approximately 2245, 2202,

2140, and 2060 cm-1

2000205021002150220022500

1

2

3

x 10-3

Wavenumber (cm-1)

Absorb

ance

At least 4 absorptions

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2

3

x 10-3

Wavenumber (cm-1)

Absorb

ance

At least 4 absorptions

Similar to solid sampling, at least four absorptions related to Pro--d1 were observed at approximately 2240, 2199,

2135, and 2050 cm-1

Solid: 2245 2202 2140 2060

Solution: 2240 2199 2135 2050

406080100120140160180 ppm

a

b

cde

e

d abc

406080100120140160180 ppm

a

b

cde

e

d abc

D-Cα-C′-OHDihedral

D-Cα-Cβ-Hsyn

Dihedral

Cβ Cα

Hsyn

D

C'

• B3LYP/6-31G* calculations with anharmonic corrections (Gaussian 03) were used to explore frequency changes

• Protonated proline was used as a model

• The D-C-C-Hsyn and D-C-C'-OH dihedrals capture the ring flip and CO2H rotation respectively

CO2H rotation

CO2H rotation

ring flip

ring flip

5.2 kcal/mol

2225 cm-1

0 kcal/mol

2244 cm-1

0.5 kcal/mol

2246 cm-1

5.4 kcal/mol

2239 cm-1

Relative electronic energies and C-D stretching frequencies are presented. For zwitterionic Pro, only two conformations might be expected due to

symmetry of the carboxylate group.

CH2 / NH2 twisting and H-O-C' bend

1220 cm-1

CH2 / NH2 twisting and D-C-N bend

1035 cm-1

A combination band at 2255 cm-1 was predicted to be in

resonance with the C-D stretch for all

four conformations.

These calculations predict possible resonances associated with CH2 / NH2 twisting and/or CH2 / NH2 twisting overtones.

• Pro ring flipping is faster than the NMR timescale but not the IR timescale, which is consistent with the observation of multiple IR absorptions and only one set of NMR signals.

• Calculations predict that the observed absorption bands arise from Pro ring flipping, resonances associated with CH2 / NH2 twisting, and possible CH2 / NH2 twisting overtones.

• Specific assignments are in progress.

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2

3

x 10-3

Wavenumber (cm-1)

Absorb

ance

At least 4 absorptions

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1

2

3

x 10-3

Wavenumber (cm-1)

Absorb

ance

At least 4 absorptions

O

NH3+

O-

D

D

D

D

D

O

NH3+

NH

O-

D

D

D

D

D

• Trp & Phe show absorptions at 2310 / 2280 & 2275 cm-1

• Future work includes incorporating C-D labeled amino acids (Pro, Phe, & Trp) into neuropeptides (e.g. Try-Pro-Trp-Phe).

• Dr. Matthew Cremeens, Gonzaga University• Christy Watson, Gonzaga University• Matt Deslman, Gonzaga University• Dr. Steven Corcelli, Notre Dame• Gonzaga Science Research Program• Howard Hughes Medical Institute