NMR Spectra of Glycine Isotopomers in Anisotropic Media: Subtle

Article pubs.acs.org/ac

NMR Spectra of Glycine Isotopomers in Anisotropic Media: Subtle Chiral Interactions Christoph Naumann and Philip W. Kuchel* School of Molecular Bioscience University of Sydney, Sydney, New South Wales 2006, Australia S Supporting Information *

ABSTRACT: NMR spectra of deuterated glycine-2- 13C revealed interactions between chiral anisotropic gelatin and κcarrageenan gels and the prochiral and chiral isotopomers. The 1 H, 2H and 13C NMR spectra of mixtures of racemic monoand prochiral bis-deuterated glycine-2-13C were resolved and well simulated using distinct dipolar coupling constants DCαH and DCαD for the enantiomers and also for the -13CαD2- group (DC,DA, and DC,DB). The orientation of the proton or deuteron on the 13Cα-atom of glycine was assigned by analogy with alanine and lactate assuming that the molecular orientation of glycine isotopomers is the same. The assignment of the prochiral sites was derived from chiral analogues.

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We previously24 described the interaction of the prochiral-13CαH2-group of glycine-2-13C with gelatin gel and accounted for four additional resonances in 1H NMR spectra and two in 13 C NMR spectra. Both these spectra were successfully simulated with the following three propositions: (1) the methylene protons in glycine-2-13C retain their chemical-shift degeneracy under anisotropic conditions; (2) a hidden 2JHH scalar coupling constant of −16.5 Hz applies; and (3) the RDC of the carbon−hydrogen bond (2DCH) is, under (favorable) chiral anisotropic conditions, no longer equi-partitioned, but it has two distinct RDCs (DC,HA, and DC,HB) with a characteristic ratio between them of ∼3:2. The key to these spectral analyses was the rapid and reversible adjustability of the interaction between glycine and the anisotropic electric field-gradient tensor in stretched and compressed states of the gels. Changing from stretching to compression inverts the signs of the RDC, D, (for an explanation see ref 25) but not that of scalar coupling constants J. We also showed that for monodeuterated glycine the sign of 2 JH,D can be directly inferred (being negative, −2.3 Hz at 15 °C in D2O) from a plot of 1(J + 2D)C,H vs 2(J + 2D)H,D of monodeuterated glycine-2-13C in gelatin-gel, which had been systematically stretched and compressed (with the isotropic state in between). For a similar case of a methylene group in monodeuterated ethanol in PBLG the negative sign of 2JH,D was determined by simulating the experimental spectra.14 Here we describe the spectral characteristics of isotopomers of glycine: specifically, (1) H3N+-(13)CH2-CO2−; (2) the racemic mixture H3N+-(13)CHD-CO2− or [D]-glycine; and

ver the past decade, a new NMR method has evolved, using stretched hydrogels to identify compounds in chiral mixtures.1,2 Specifically, it is possible to quantify the amounts of each of the R- and S-isomers of (bio)chemical compounds by using 1H, 2H, and 13C NMR spectroscopy. A popular test sample is a racemic mixture of alanine.3−5 Enantiomers are differentiated in 2H NMR spectra by the different magnitudes of the residual quadrupolar coupling constants (RQCs) of each deuterium atom within a chiral medium. Polymeric gels such as collagen,3 gelatin,4,5 and carbohydrates such as ι- and κcarrageenan,6,7 and cross-linked gelatin gels8 have all been used. Applying a simple experimental apparatus,9−11 each sample can be readily extended or compressed sequentially many times, as has been shown for achiral gels such as poly(ethylene oxide), using a wide range of solvent conditions.12 Our approach leads to a series of spectra in which values of peak splittings are linearly related to the extent of stretching as the guest molecules are exposed to the varied extents of molecular anisotropy. This ease of adjusting the extent of anisotropy and the absence of an anisotropic minimum are important advantages over the alternative alignment media such as achiral liquid crystals,13 chiral liquid crystals such as poly-γ-(benzyl-Lglutamate)/deuterated chloroform (PBLG/CDCl3),14,15 bicelles,16 filamentous bacteriophage,17 DNA,18 and stressed achiral polyacrylamide gel.19−21 Generally not all degrees of alignment can resolve all mixtures in these media. The chirality of gelatin means that not only alanine enantiomers3−5 but other compounds with prochiral sites, such as those that occur in deuterated dimethyl sulfoxide (DMSO-d6),10 can be differentiated once the gelatin gel is stretched or compressed. Similarly, in 1H and 13C NMR spectra, RDCs have been used to distinguish between enantiomers and prochiral molecular sites.5,10,11,14,22,23 Published XXXX by the American Chemical Society

Received: July 7, 2015 Accepted: September 23, 2015

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DOI: 10.1021/acs.analchem.5b02542 Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry

Article

(3) prochiral H3N+-(13)CD2-CO2− or [D2]-glycine (Scheme 1), each with varying extents of deuteration of their methylene

and using different delays for inversion recovery (d7, 2.8 s for mono- and nondeuterated glycine; 0.65 s for the bisdeuterated isotopomer). The gelatin concentration was 65% (w/w), and T1 relaxation times in this matrix were estimated to be 1.6 and 4.5 s for mono- and bisdeuterated isotopomers, respectively.

Scheme 1. Alanine, Lactate, and Glycine Isotopomers: Racemic Mixtures and Prochiral Glycine Molecules That Differ in the Configuration of Atoms Bonded to the CαAtom Were Resolved in Various NMR Experiments with the Solutes as Guests in Anisotropic Gelatin Gels

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RESULTS AND DISCUSSION H NMR Spectra of Deuterated Glycine in Stretched and Compressed Gelatin Gels. Unstretched gelatin gel, made with [D2]-glycine (see Scheme 1) dissolved in D2O, showed two single 2H NMR resonances that corresponded to those from glycine and D2O in free solution. When the sample was stretched, up to six signals (three quadrupolar-split doublets) became apparent, with the degree of spectral resolution depending on the extent of stretching. The prochiral deuterons interacted differently with the chiral anisotropic gel: a diastereomeric complexation was inferred to exist, so a quadrupolar doublet appeared for each glycine deuteron. In fact, the low resolution resulting from the small chemical shift difference between the 2H spins (e.g., D2O/HDO and [D2]glycine) could be overcome by changing the RQCs for each deuteron by adjusting the anisotropy of the medium. There was no additional splitting of the peaks arising from possible dipolar interactions brought about by the anisotropy (e.g., between the two D atoms) but a dipolar interaction probably contributed to line broadening. Plotting the distinct RQC values of the two deuterons of [D2]-glycine recorded for a range of anisotropies (see Figure S1 in the Supporting Information for a definition) from stretched to compressed vs each other gave a straight line (black squares and fitted line in Figure 1) with an “anisotropy

groups. Dipolar interactions of the spin-1/2 nuclei 1H and 13C, as well as quadrupolar interactions of the spin-1 nuclei 2H, have to be considered. Monodeuterated glycine is especially interesting because it is chiral, and its two enantiomers are expected to be spectroscopically resolved, as with the enantiomers of alanine and lactate. Thus, we studied the glycine isotopomers in chiral gelatin and carrageenan gels as well as in achiral cromolyn liquid crystals26,27 as a contrasting medium.

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EXPERIMENTAL SECTION Gelatin gel (Gelita Asia Pacific, West Krugersdorp, South Africa) and carrageenan gels (Sigma-Aldrich, St Louis, MO) were prepared as previously described.6,7,10 Cromolyn sodium salt (Aldrich) was dissolved in deionized water at neutral pH containing isotopomer mixtures of glycine-2- 13 C at a concentration of 16% (w/w). Such a mixture was gently warmed to ∼50 °C, transferred to a 5 mm NMR tube, warmed again, and held at room temperature until NMR measurements were carried out. Anisotropic gel samples were made by drawing gelatin, or carrageenan solutions, containing polar guest solutes into an elastic silicone rubber tube that allows (after the setting of the gel) the rapid and reversible adjustment of the length of the tube.10 Stretched samples were rapidly and reversibly converted into compressed ones by melting the stretched gelatin gel at 37 °C, allowing the gel to reset by cooling, and then adjustably releasing the tense silicone tubing containing the now isotropic sample. This compresses the gel and reverses the sign of D. Mono- and bisdeuterated glycine2-13C samples were prepared using acetic acid-salicylic aldehyde28 or by leaving a sealed D2O solution of glycine2-13C at pH 10 at 100 °C for 2 months. All NMR spectra were recorded at 15 °C on a Bruker (Karlsruhe, Germany) 400 MHz wide-bore (Oxford Instruments, Oxford, U.K.) spectrometer. The spectra of mono- and bisdeuterated glycine-2-13C isotopomers were separated by applying one-dimensional T1 inversion recovery experiments (Bruker pulse sequence, t1ir1d)

Figure 1. Anisotropy line (AL) graph of CαD RQCs of (R)- vs (S)enantiomers of solutes in variably stretched gelatin gels (30−150% w/ w): [D2]glycine, black squares; racemic alanine [D]- and/or [D4]alanine, red discs; and or [D4]-lactate, green triangles. Similar anisotropy slope (AS) values were estimated indirectly by plotting the RQCs of HDO vs CαD RQCs for each enantiomer and calculating the ratio of the obtained AS values.

slope” (AS) of 1.87 ± 0.03 (large RQC values vs smaller ones). This “anisotropy line” (AL) for [D2]-glycine was compared to that obtained with a mixture of enantiomers, viz., racemic deuterated-alanine that had one deuteron attached to the Cαatom in each of its two enantiomers. The AL constructed for deuterated D-(R)-alanine vs L-(S)-alanine (red circles and fitted B



Article 1

H NMR Spectra of (Partially) Deuterated Glycine2- C under Isotropic and Anisotropic Conditions. The 1 H NMR spectrum of monodeuterated glycine-2-13C, [D]glycine dissolved in cromolyn liquid crystal had two triplets of equal intensities from the -13CαD- group with a scalar-coupling enhanced by an RDC for the 13Cα-D bond (Figure 2A). The

line in Figure 1), from comparable gelatin concentrations, yielded an AS of 1.77 ± 0.04. This value was virtually indistinguishable from the previous one for [D2]-glycine. The same was true for the AL of deuterated D-(R)-lactate and L-(S)lactate (green triangles, AS = 1.80 ± 0.01). The agreement between these AS values suggests similarity of the binding environments around the Cα-atom in the three compounds. The idea of comparing the anisotropy patterns of compounds with similar structure has been shown previously for a series of epoxides in the homopolypeptide liquid crystals PBLG−CHCl3 and poly-γ-carbobenzoxy-L-lysine (PCBLL) in various organic solvents29 and more recently for two cholesterol derivatives in stretched polydimethylsiloxane/CDCl3 gels thus distinguishing diastereomers.30 We also estimated AS values indirectly for both enantiomers without both being present in the same mixture. This was done by relating each AL to the range of anisotropies obtained for a shared resonance such as that of D2O/HDO. This was successful because the relationship between the extent of stretching and the RQC of guests like D2O/HDO in gelatin-gel is linear (Table 1). For alanine, the indirectly estimated AS

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Table 1. Quadrupolar AS Values for CαD and CβD3 Groups in Deuterated Alanine, Lactate, and Glycinea anisotropy slopes (ASs) of RQCs of HDO vs methine D (S)-enantiomer or pro-R stereoisomer

(R)-enantiomer or pro-S stereoisomer

alanineb

1.90

1.15

lactate

2.18

1.22

glycine

2.23

1.21

anisotropy slope (AS)c (R) vs (S): 1.77 (1.65) (R) vs (S): 1.80 (1.79) pro-S vs pro-R: 1.87 (1.84)

anisotropy slope (AS) of RQC of HDO vs CD3 group alanineb lactate

(S)-enantiomer

(R)-enantiomer

anisotropy slope (AS)c

7.64 5.06

25.5 9.78

(S) vs (R): 3.42 (3.34) (S) vs (R): 1.75 (1.93)

Figure 2. Sections of 1H NMR spectra showing peaks from the protons of the methylene group in mixtures of glycine-2-13C isotopomers, under anisotropic conditions embedded in achiral matrix cromolyn liquid crystal (part A), and chiral stretched gelatin (part B) (D2O residual quadrupolar coupling constant of 1350 Hz). The superimposed spectra in part C were calculated for the individual components of the glycine mixture: black peaks correspond to glycine2-13C (-13CαH2-); blue and red lines denote the peaks from the monodeuterated enantiomers -13CαHD-; blue denotes the peaks of (R)- and red those of the (S)-enantiomer. A line-broadening factor of 7.5 Hz was used for the calculated NMR spectra. The color of each chemical structure is the same as for its corresponding spectrum. (For parts A and B, the relative amounts of glycine isotopomers in the samples differed.)

a

The AS values were estimated directly by plotting the larger vs the smaller RQCs for a range of chiral anisotropic conditions in stretched to melted-set-and-compressed gelatin gels (Figure 1) or indirectly by estimating AS values from the RQC values of HDO vs individual CαD or CβD3 RQCs, and in a second step deriving the enantiomer-vsenantiomer AS values. bNote: D-alanine = (R)-alanine, L-alanine = (S)alanine. cIndirect calculation via the HDO RQC in parentheses.

value for the two enatiomers was 1.65 (Table 1); and there was no isomer-effect evident in the value of the RQC in 2H NMR spectra for monodeuterated and bisdeuterated glycine. Specifically, the geometry/stereoisomerism was seemingly much more important in defining the spectral features. The pro-S deuteron of [D2]-glycine and the deuteron in (S)-[D]glycine is expected to elicit similar anisotropic behavior to (R)alanine and (R)-lactate. (See the illustration in ref 22 for a hypothetical molecule.) The AS values for CD3 groups were not always similar to those recorded for the Cα-atom. The AS values for the two CD3 groups in alanine were at least twice those for the CD group (in favor of the (S)-enantiomer); whereas in lactate it was comparable to that of its methine deuterons. This indicates that the alignment tensors for the methyl groups in lactate and alanine differed.

separation of the two triplets corresponded to the 13Cα-H splitting (JCH + 2DCH). Using chiral anisotropic media (stretched gelatin and κ-carrageenan gels) introduced more peaks due to the additional interactions of the enantiomers with the chiral matrix (Figure 2B, stretched gelatin gel). Instead of one set of a doublet of triplets, there were now two; one for each enantiomer (Figure 2C for calculated spectra) with distinct values for -13Cα-H- and -13Cα-D- splittings. Similarly, when the gelatin was compressed, two sets of doublets of equalintensity triplets emerged (Figure S2 in the Supporting Information). 13 C NMR Spectra of Partially Deuterated Glycine-2-13C in Chiral and Achiral Anisotropic Media. The 13C NMR spectrum of monodeuterated glycine-2-13C in solution showed C



Article

the same peak-pattern as its 1H NMR spectrum, viz., two sets of 1:1:1 triplets with a splitting that corresponded to 1JCD, separated by 1JCH. [D2]-glycine-2-13C in solution showed five peaks with an integral ratio of 1:2:3:2:1 split by 1JCD; this was in contrast to the three peaks of glycine-2-13C split by 1JCH. Increasing deuteration shifted the peaks from the isotopomers to lower frequency by ∼24 Hz per D atom. This made the 13C spectrum of the mixture readily interpretable (Figure S3 in the Supporting Information). Dissolution in aqueous cromolyn liquid crystal did not change the basic appearance or pattern of the resulting 13C NMR spectrum, apart from increased splitting due to the dipolar interactions (Figure S3C). With a mixture of isotopomers (two enantiomers and two prochiral compounds, -13CαD2- and -13CαH2-) the resulting 13C NMR spectra from the solutes in stretched gelatin gel were very complex. However, we succeeded in making peak assignments by using the large (2.8-fold) differences in longitudinal relaxation time, T1, of the 13C nuclei in -13CαD2- vs -13CαHDand -13CαH2-groups. Figure 3A shows the 13C NMR resonance complexes for the mixture of isomers, while Figure 3B shows the spectrum of [D2]-glycine-2-13C. Figure 3C,D shows the spectra of the complete mixture superimposed upon those calculated for each of the four isotopomers. It is evident that monodeuterated glycine showed two pairs of 1:1:1 triplets for each enantiomer, with distinct -13Cα-H- and -13Cα-D-splittings, whereas the 13C NMR spectrum of bisdeuterated glycine yielded four additional peaks compared with the achiral situation. The dipolar contributions to the C−D splitting of both deuterons became different as the gel was stretched (see Figure 3B inset). The ratio of DCD,A/DCD,B for the two methylene deuterons was 1.72 in stretched gelatin; in compressed gelatin only five major peaks were resolved, but the additional eight small peaks identified in SpinWorks spectral calculations31 were not resolved, especially considering our use of 7.5 Hz linebroadening (Figure S4 in the Supporting Information). As described above for 2H NMR spectroscopy of deuterated enantiomers, the orientation of the deuteron determined the magnitude of the RDCs: (R)-alanine showed larger -13Cα-HRDCs than (S)-alanine. The AS (see 2H NMR Spectra of Deuterated Glycine in Stretched and Compressed Gelatin Gels section) was 1.80 and 1.77 when determined indirectly using the RQC of HDO (Table 2). For monodeuterated glycine2-13C there were different 13Cα-H and 13Cα-D RDCs for each stereoisomer. The (R) enantiomer (by analogy with D-alanine) had a larger -13Cα-H-splitting than the (S) enantiomer. When plotted over a range of anisotropies from the stretched to compressed gel, a linear relationship was apparent with a slope (AS) of 1.74, or indirectly via HDO it was 1.73 (Table 2). And consistent with this finding, monodeuterated glycine showed a peculiar AS behavior: the enantiomer with the larger -13Cα-HRDC, postulated to be (R), had a smaller -13Cα-D-RDC (because its deuteron is situated in an (S) stereoscopic position, which has smaller RQC and RDC values). The RDC of the carbon-deuteron duplex was not influenced by the number of deuterons: a deuteron in the pro-S configuration yielded AS values that were similar to that of a deuteron in (S)monodeuterated glycine. Stretched κ-carrageenan gels resolved the 13C NMR peaks of two [D]-glycine enantiomers, whereas stretched and compressed ι-carrageenan gels (although chiral) did not.

Figure 3. Section of the 13C NMR spectra of a mixture of glycine2-13C isotopomers in stretched gelatin gel (D2O residual quadrupolar coupling constant, 1350 Hz) showing the peaks from the methylene group, for (A) the monodeuterated and nondeuterated glycine; (B) the deuterated isomer; and (C) the spectrum from a complete mixture of the isotopomers. Parts A and B were able to be discriminated from part C on the basis of their different T1 relaxation times (see text). The superimposed spectra in part D were calculated for the individual components of the glycine mixture: glycine-2-13C (-13CαH2-), black; deuterated glycine-2-13C (-13CαD2-), green; peaks from monodeuterated enantiomers -13CαHD-, denoted by blue and red; and (R) blue and (S) red. The color of each chemical structure is the same as for the corresponding spectrum. A line-broadening factor of 7.5 Hz was used in the calculated NMR spectra.

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CONCLUSIONS Here we provide for the first time NMR spectroscopic evidence that the configuration of atoms bonded to the Cα-atom of glycine, alanine and lactate are reflected in the extent of anisotropic splitting that can be elicited by stretching and compressing gelatin gels. We posit that this situation would pertain with many other low molecular weight solutes and even peptides when embedded under similar gel conditions (type and concentration). In principle, all that is required is the determination of the RDCs of the 13Cα-H or RQCs of CαD as a function of the anisotropy (stretching or compression); this would be done in the presence of D2O. For the solutes used here the nature of the groups attached to Cα did not significantly influence the resulting AS values. A large interaction between 1H and 2H nuclei attached to Cα and the anisotropic electric field-gradient tensor in stretched and D



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Table 2. 13C NMR AS Values for 13Cα-H and 13Cα-D Groups in Glycine and Alaninea RQC of HDO vs RDC of 13 CαH

AS of HDO vs AS of13CαD

enantiomer

enantiomer b

(S)-

(R)-

anisotropy slope (AS)b

43.5

70.7

pro-S vs pro-R: 1.62 (S) vs (R): 1.47 (1.63)

(S)-

(R)-

anisotropy slope (AS)

11.3 11.6

6.53 6.57

(R) vs (S): 1.74 (1.73) (R) vs (S): 1.80 (1.77)

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[D2]-glycine-2- C [D]-glycine-2-13C alanine

HDO vs 13CαH2 main peak

chirally induced peak

8.43 8.95

9.67c d

glycine-2-13C glycine-2-13C in current gels a

These AS values were obtained directly by plotting larger vs smaller RDCs for a range of chiral anisotropic conditions from stretched to melted gelatin gels (Figure 4) or were estimated indirectly from the RQC of HDO vs the RDC of Cα-H and Cα-D, and in a second step deriving the ratios of the former AS values. bIndirect calculation via RQC of HDO in parentheses. cAS values for stretched and melted states were very close to each other. d Superimposed by other peaks, due to a small amount of glycine-2-13C.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b02542. Additional information as well as experimental and calculated 1 H and 13 C NMR spectra of glycine isotopomers in chiral compressed gelatin and achiral cromolyn liquid crystal (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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Figure 4. Anisotropy line (AL) graph of: Cα-H splittings (J + 2D) of the (R)- vs (S)-enantiomers for variably stretched gelatin gels (30− 100% w/w) containing racemic alanine (black squares) and monodeuterated glycine (red discs). Similar anisotropic slope (AS) values were estimated indirectly by plotting the RQCs of HDO vs the 13 Cα-H splittings for each enantiomer and calculating the ratio of the obtained AS values.

ACKNOWLEDGMENTS The work was funded by an Australian Research Council Discovery Project Grant DP10877789 to P.W.K. Dr. Ann Kwan is thanked for assistance with the NMR spectrometer.

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REFERENCES

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compressed gelatin gels suggests this will be a valuable tool for quickly characterizing partially deuterated glycine amino-acid residues in peptides and proteins. The underlying assumption of similar dynamic behavior30,32 of the Cα group, however, needs to be tested in future. As communicated earlier,24 the accuracy of estimates of any NMR parameter can be rapidly confirmed by simulating the experimental spectra from stretched and compressed states of the same sample (by inverting the signs of D while leaving J unchanged). In the course of the present studies it became clear why the additional 1H and 13C NMR peaks/transitions recorded for prochiral glycine-2-13C in anisotropic gelatin led to the peculiar ratio of DC,HA:DC,HB of ∼3:2. This closely corresponded to the AS values for the two enantiomers of the Cα in [D]-glycine, (deuterated) alanine and deuterated lactate. It was also directly observed for the other prochiral compound [D2]-glycine-2-13C. E



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NMR Spectra of Glycine Isotopomers in Anisotropic Media: Subtle

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