MAGNETIC RESONANCE IN CHEMISTRY, VOL. 24, 167-170 (1986)

Conformational Analysis of DL-, L- and D-Methionhe by Solid-State "C NMR Spectroscopy

Luis E. Diaz* Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Junin 956, Beunos Aires, Argentina

Frederick Morin, Charles L. Mayne and David M. Grant Department of Chemistry, University of Utah, Salt Lake City, Utah 84112, USA

Ching-jer Chang Department of Medicinal Chemistry and Pharmacognosy, School of Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, Indiana 47907, USA

The conformational analysis of DL-, L- and D-methionine was studied by solid-state =C NMR with the help of cross-polarization and magic-angle spinning techniques. The spectrum of commercial crystalline DL-

methionine showed two chemical shifts (18.7 and 15.8ppm) for the methyl carbon. Crystallization from water or water-ethanol (95 : 5) allowed the separation of two crystalline forms (a and fi), of which one had 6 (CH,) 18.7 ppm and the other 6 (CH,) 15.8 ppm. Therefore, each crystalline form of DL-methionine consists of a single conformer, a result in agreement with x-ray ditfraction data. This conformational difference of the two crystalline forms was also reflected in the carbon resonances of C-fi and C-y. When L- and D-methionine were crystallized from water-ethanol (3 : 2), the solid-state 13C NMR spectra showed that each isomer displayed two methyl carbon peaks at 18.1 and 16.2 ppm of equal intensities, while the a-carbon resonances showed a broad peak due to two unresolved doublets. These results suggest that the L- or D-methionine crystal contains two merent conformers in one unit cell, a conclusion that is also supported by the x-ray data.

INTRODUCTION

The analysis of the molecular architecture of a biological molecule is critical to the understanding of molecular mechanisms involved in biochemical reac- tions. It is well recognized that reaction specificity is often a consequence of unique molecular conforma- tions resulting from intra- and inter-molecular interactions in biochemical systems. NMR spectros- copy has proved to be one of the most powerful tools in the conformational analysis of biomolecules.l The most frequently used NMR parameter in conforma- tional analysis is the homonuclear or heteronuclear vicinal coupling constant (3J) of two nuclei (X-Y). These couplings are related to the dihedral angles (8) by the general equation derived from the valence bond calculation of Karplus:2

3J = A cos2 8 + B cos 8 + C

In small biomolecular systems (e.g. an amino acid), a molecule can freely undergo intramolecular rotation in solution. Theoretical calculations on vicinally substituted compounds have shown that the energeti- cally favoured forms are the three staggered state^.^ The conformation is then specified by the relative population of the different staggered conformers, and can be determined from the values of the vicinal

* Author to whom correspondence should be addressed.

0749-1581/86/020167-04$05 .OO @ 1986 by John Wiley & Sons, Ltd.

coupling constant given by 3Jg+auche and 3J;auche (Fig. 1). However, the complication implicit in this calculation may be considerable. The initial assump- tion of one 'JgaUche (3Jlauche= 3J;auche) value may be questionable, since 3J can be significantly influenced by the electronegativity of the neighbouring groups.66 Therefore , up to six different coupling parameters may have to be employed for the most commonly used three-spin system in the conformational analysis of amino acids.7 The values of 34,.a,, and 3Jgauche also vary for different amino acids, since vicinal coupling constants can be significantly influenced by the polarizability and electronegativity of attached groups and by the hybridization of bonding orbitals.66 Hence idealized values for 34ra,, and 'Jgauche are not invariant for all amino acids, and different values have been used by various g r o ~ p s . ~ , ~ , ~ - l l

In crystalline solids, rapid conformational equilibra- tion is often inhibited, revealing specific chemical environments with different chemical shifts and coupling constants. Therefore, considerable benefit may be derived from an NMR study of molecular conformation in the solid state. In the last few years, the combined techniques of high-power proton decoupling , cross-polarization (CP) and magic-angle spinning (MAS) have been routinely used in obtaining high-resolution 13C NMR spectra of solid^.'^-^^ This paper reports the use of high-resolution solid-state 13C NMR for the conformational analysis of methionine.

Received 25 May 1985 Accepted 23 August 1985

168

Y Y Y

L. E. DiAZ ET AL.

X A

TRANS GAUCHE+ GAUCHE-

Figure 1. The three staggered states in an amino acid.

EXPERIMANTTAL

DL-Methionine was purchased from Matheson, Cole- man and Bell (Cincinnati? OH) and D- and L-methionine from Sigma Chemical Co. (St. Louis, MO). The solid-state NMR spectral data were obtained on a Bruker CXP-100 spectrometer using the combined techniques of CP-MAS with a 3 ms mixing time, a 200ms acquisition time and a recycle delay of 3 s. Andrew-type rotors were used at a spinning speed of 3.6 kHz, packed with approximately 220 mg of sample.

All chemical shifts are expressed externally referenced to the 13C resonance of SiMe,. The actual reference compound was hexamethylbenzene, either periodically mixed with the sample or run separately in the rotor prior to running the sample alone. The high-field peak of solid hexamethylbenzene is assumed to be 17.6 ppm downfield from SiMe,.

RESULTS AND DISCUSSION

The solid-state CP-MAS 13C NMR spectrum of commercial DL-methionine is shown in Fig. 2A. The chemical shift assignments can be made on the basis of the previous assignments for methionhe in solution. l5 The a-carbon is split into a broad doublet because both the 13C-14N dipolar and 14N quadrupolar interactions cannot be eliminated simultaneously by magic-angle spinning.16J7 The most prominent change in the spectral pattern from the liquid (Fig. 2D) is the splitting of the methyl carbon in the solid (Fig. 2A). This splitting, arising from a chemical shift difference, reflects molecular differences in the crystalline state, and can be explained by either the existence of asymmetric unit cells or the coexistence of two different crystalline forms. Recrystallization of DL- methionhe from water or water-ethanol (95 : 5 ) solution provides one crystalline form, as indicated by the solid-state 13C spectrum [Fig. 2B; 6 (CH,) = 18.7ppml. Further cooling of the mother liquor in liquid nitrogen yields predominantly another crystal- line form [Fig. 2C; 6 (CH,) = 15.8ppml. X-ray diffraction data on DL-methionine corroborates the suggestion that two different crystalline forms ( a and p) are indeed p r e ~ e n t . ’ ~ . ~ ~ The difference between the dimorphs is the conformation around the terminal C-y-S bond (Fig. 3 ) . The low-frequency shift of the methyl carbon resonance in the a-form compared with the p form is probably attributable to the steric interaction.

A 18.7 I

H, csc r o e H, c H c H coy C”3

I + N H ~ c=o *

I

C

Figure 2. Proton-decoupled 25 MHz solid-state CP-MAS %NMR spectra (ppm) of DL-methionine: (A) before re- crystallization; (B) after recrystallization from water-ethanol (95 : 5) solution; (C) after precipitation of the mother liquor in liquid nitrogen; (D) 75MHz solution spectrum in D,O. The solid-state spectra were recorded on a Bruker CXP-100 spectrometer by cross-polarization with a 3 ms mixing time, 200 ms acquisition time and a recycle delay of 3s. Approximately 220 mg of material, packed in an Andrew-type rotor, were rotated at 3.6 kHz at the magic angle. *Dioxane reference.

This conformational difference is also revealed by the carbon resonances of C-p and C-y (Fig. 2B and C). The chemical shift difference between these two carbons is too small to make unambiguous assign- ments, even though their chemical shifts in aqueous solution have been determined.” Nevertheless, the chemical shift difference between C-/3 and C-y for the a form (A6 = 2.3 ppm) is greater than that for the /3 form (A 6 = 0.7 ppm) . The dihedral angles around

SOLID STATE 13CNMR OF METHIONINE 169

H H

c H 3 6 90.4' h8:350

H CHZ$HCOF H CH2FC02-

P I NH3 +AH3

ar Figure 3. The conformation at the C-y-S bond in the two crystalline forms of DL-methionine.

A I CH, r P a

H, CSCH,CH,CHCO, I I

+NH,

c=o I r I 1 * 1 1

200 160 120 80 40 0

16.2

18.1 1

C

Figure 4. Proton-decoupled solid-state 13C NMR spectra (ppm) of (A) DL-methionhe, (8) L-methionine and (C) o-methionine. All compounds (1 g each) were recrystallized from 35 mi of water-ethanol (3 : 2) solution. The NMR experimental condi- tions are given in Fig. 2.

H H

+NH3 +AH3

H H

H CHCOF

H NH;

A B Figure 5. The conformations at the C-y-S and C-P-C-y bonds of the two different crystalline forms (A and B) of L-methionine.

their C-y-C-p, C-6-C-a and C-a-C(=O) bonds for the a and /3 forms are similar, so that the resonance signals for the a-carbon and carbonyl carbon are indistinguishable.

The solid-state l3CNMR spectra of L- and D-methionine were also studied in order to allow comparison with DL-methionine. The three forms of methionine were recrystallized from the same solution (water-ethanol, 3 : 2), and their I3C spectra are shown in Fig. 4. DL-Methionhe gives almost exclusively the p form. However, the spectra of both L- and D-methionine display two methyl carbon peaks at 18.1 and 16.2ppm (Fig. 4B and C). The relative areas of both peaks are similar in L- and D-methionine, although their intensities are different. This suggests that both L- and D-methionine crystals consist of two different conformers (A and B) in one unit cell, and their conformations around the C- y-S bond are similar to those of the p and a forms of DL-methionine. Once again, this conclusion is supported by the x-ray data.20 The crystal of L-methionine contains two crystallographically different molecules, A and B, having different conformations. The conformation at the C-y-S bond is trans in A and gauche in B. The conformations at the C-p-C-y bond of L-methionine are trans in A and gauche in B (Fig. 5). These conformational differences are shown by the solid-state spectral pattern of the a-carbon. The a-carbon signal shows as an identical pattern for all DL-methionine crystalline forms (Figs 2 and 4A) because of the similar conformation at the C-P-C-y bond for both a and p forms. On the other hand, the a-carbon resonance of both L- and

Table 1. Carbon-13 chemical shifts of methionine"

D L - ~ 175.3 54.7 30.4 29.6 14.6 P-DL- 178.9 55.7 33.0 32.3 18.7 ~ - D L - 178.3 55.5 34.0 31.7 15.8 A-L- 178.0 53.0' 34.1 32.0 18.3 B-L- 178.0 53.0' 33.2 32.0 16.3

a Chcmical shift expressed in ppm from SiMe,. bChemical shift assignment in D,O solution using dioxane as an internal reference (6, 67.4ppm). See Ref. 15. ' Broad peak; estimated error rt2 ppm.

Compound CO C-ru C-@ C-y CH,

170 L. E. D ~ A Z E T A L .

D-methionine shows a broad peak (Fig. 4B and C) due to two unresolved doublets , corresponding to different chemical shifts for the A and B forms of methionine. The 13C chemical shifts of the different methionine conformers are summarized in Table 1.

In conclusion, the results from our groups12 and other labora tor ie~~l -~~ strongly indicate that solid-state NMR can be a powerful and unique approach for studying molecular conformations in the solid state. Because of the ease of obtaining spectra, and since single crystals are not required, solid-state NMR

complements the more powerful x-ray crystallographic methods. Further, such data provide information which will also be very useful in specifying configurational and conformational features in liquids undergoing rapid equilibration.

Acknowledgment We were supported in part by the National Institute of Health under grant No. GM-08521.

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