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Nuclear magnetic resonance spectra of dl -threonine and dl -valine
Spectrochimlca Acta,1967,Vol. !23A,pp. 1346to 1350.PergamonPressLtd. Printedin NorthernIreland
Nuclear magnetic resonance spectra of mlthreonine and DL-valhe Kerala University
G. &XJLDHAS Physics Department, (Received
20 December
Alwaye-2,
Kerala, India
1966)
Al&&-The 100 MC/S nuclear magnetic resonance spectra of nL-threonine and nL-valine in deuterium oxide at room temperature have been studied. In both the cases, the observed spectra have been analysed by assuming that each spectrum is a superposition of two spectra arising from the different conformations. 1. INTRODUCTION
nuclear magnetic resonance spectra of several amino acids have been studied [l-6] and attempts have been made to explain the spectra using the idea of rotational isomerism [6]. Some of the authors [l-3]studied mainly the chemical shifts and BOVEY and TIERS [4] estimated the coupling constants from spectra which were not well resolved. Amino acids in solution exist as a mixture of three interconverting conformations because of the internal rotation about the ChCB bond. The relative energies of the different conformations, the barriers to internal rotation and the chemical shifts and spin coupling constants of each conformation are the factors that govern the NMR spectra of such substances [7].At low temperatures each conformation is long lived and the observed spectrum is a superposition of the spectra due to the different conformations [S-lo]. At room temperature the internal rotation occurs at a rapid rate and the chemical shifts and spin coupling constants are averaged [6, 7, 111. In the present investigation, the analysis of the NMR spectra of DL-threonine and DL-valine at room temperature is reported. The observed spectra could be explained only by assuming that each spectrum is a superposition of two spectra These two cases, where complete rotational arising from the different conformations. averaging is not taking place at room temperature, are important and hence taken up for study. THE
[l] M. TAKEDA and 0. JARDET~KY, J. Chem. Phys. 26, 1346 (1967). [2] 0. JARDETZKY and C. D. JARDETZKY, J. Biol. Chem. 233, 383 (1958). [3] S. FUNJIWARA, Y. ARATA, N. HAYAKAWA and H. MONOI, Bull. Chem. Sot. Japan 36, 1658 (1962). F. A. BOVEY and G. V. D. TIERS, J. Am. Chem. Sot. 81, 1870 (1959). K. A. MCLAUCELAM, Mol. Phy.9. 5, 195 (1962). K. G. R. PACHLER, S~ectrochim. Acta 19,2086 (1963); 20, 581 (1964). J. A. POPLE, W. G. SCHNEIDER and H. J. BERNSTEIN, R@ Reeoltiion Nuclear Magnetic Reeonance, McGraw-Hill (1959). [8] D. G. THOMPSON, R. A. NEWMARK and C. H. SEDERHOLM, J. C&m. Phya. 37, 411 (1962). [9] R. A. NEK and C. H. SEDERHOLM, J. Chem. Phye. 43, 602 (1965). [lo] W. S. BREY, JR. and K. C. RAMEY, J. Chem. Phye. 39, 3646 (1963). [ll] H. S. GUTOWSKY, G. G. BELFORD and P. E. MC-ON, J. Chem. P&ye. 86,3354 (1962) and references cited therein. [4] [6] [6] [7]
12
1345
1346
G. ARULDRAS
2. EXPERIMENTAL The samples were obtained from E. Merk and 3.1 per cent solutions of these were prepared in deuterium oxide from the Atomic Energy Establishment, Bombay. The spectra were recorded on a Varian HR-100 high resolution NMR spectrometer operating at 100 MC/S. The temperature of the sample was about 28°C throughout. The separations of all peaks were measured by the usual side band technique. 3. RESULTS The amino and hydroxyl protons exchange rapidly with the deuterium of the solvent, so that the corresponding peaks coalesce with the residual water peak giving an intense line at the lowest field. 3.1 nn-threonine The different conformations are schematically shown in Fig. 1 and the observed spectrum is shown in Fig. 2. The protons A and B together with the methyl protons constitute an ABX, system. As the proton A has appreciable interaction with B and X, protons, lines l-9 are assigned to A proton. Lines lo-13 are assigned to proton B and 14-17 to X, protons. General expressions have been reported for the transitions and relative intensities in an ABX, system [12, 131. In the X, group of the ABX, case there are 12 transitions excluding the combination lines. An inspection of the frequencies shows that six of these give rise to one field independent doublet whose splitting value gives lJax + Jsxl. The two lines of this doublet are of the same intensity. When (va - vs) > JAB, the other six transitions give rise to another doublet symmetric with the previous one, with splitting equal to lJax - J,,I. The lines of this doublet are also of equal intensity. In the present case (va - vs) > JAB and J,, < 0.05 c/s [14, 151 and therefore the two doublets may coalesce and give a single one. In the experimental X spectrum (Fig. 2b), there are two doublets (lines 14, 15; 16, 17) and they are not symmetric. Hence we can assume that J,, is very nearly zero giving a single doublet for the X spectrum, of the ABX, and the experimental spectrum is a superposition of two ABX, spectra. We can arrive at this conclusion from the B transitions also. The intensity distribution suggests that lines l-7,10,11, 16 and 17 form one set, ABX, and the lines 4-9, 12-15 form the other set, A’B’X,‘; the ratio of the relative proportion of the two forms being 1.4: 1. The analysis gives the following parameters in c/s. ABX,
system J,, = 3.9 J AX = 6.5 J BX = 0.0
A’B’X,’
J;,;, R. V. C. J.
vB -
vx =
vB =
263 & 3.0 270 4 3.0
system J A'B' = 4.9 J Idt = 6.5
[12] [13] [14] [15]
va - vB = 52.5 * 0.5
=
0-o
vA, vs. -
vB, = 66.2 & 0.5 vx, = 227 f 3.0 VB’ =
245 f
3.0
W. FESSENDEN and J. S. WAUGH, J. Chem. Phys. 30, 944 (1959). J. KOWALEWSKI and D. G. DEKOWALEWSKI, J. Chem. Phys. 33, 1794 (1960). A. REILLY and J. D. SWALEN, J. Chem. Phys. 35, 1522 (1961). I. MUSHER and R. G. GORDON, J. Chem. Phys. 36, 3097 (1962).
1347
Spectra of DL-threonine and DL-valine
Fig. 1. Conformations c-x
3
of DL-threonine.
IO
6
75
50
7.5
0
C/S
Fig. 2(a).
Observed spectra of A (lines l-9) and B (lines 19-13) threonine. Field increasing from right to left.
protons of
DL-
16
Fig. 2(b). Methyl group of DL-threonine.
Field increasing from left to right.
1348
G. ARULDHAS
The accuracy in the spin coupling constants is ho.1 c/s. The shifts vs and vnS are from cyclohexane which appears at a higher field than B and B’, used as an internal standard. As only two lines for the B protons are obtained and the spectra due to A and A’ overlapping, it is almost impossible to determine the relative sign of JAB and J,,. 3.2 nr,-valine The three conformations are drawn in Fig. 3 and the experimental spectrum is shown in Fig. 4. The coupling between the two methyl groups is negligible. The experimental spectrum shows that (vs - va) and (va - vX) are large compared to the coupling constants, suggesting a first order case. If the two methyl groups are distinct, one has to get two doublets with all the lines equal in intensity. The spectrum (Fig. 4b) is showing two doublets of unequal intensities indicating that both
C&3H
COOH
C@bH
Fig. 3. Conformations
of DL-valine.
the methyl groups are almost equivalent and the observed spectrum is a superposition of two spectra as in DL-threonine, AB(X,), and A’B’(X,‘),; the ratio of the relative population of the two forms being 1.2: 1. The large width of about 35 c/s of group 2 suggests that the spectra of the protons A and A’ overlap to give group 2. The two lines of proton B coincide with the two lines of proton B’ and give group 1. In the X spectrum of rn,-valine the more intense doublet occurs at the low field side. The analysis gives the following parameters in c/s. AB(X,), system
J AR = 4.6
vB -
vx
= 255 & 3
vB = 247 & 3
J *x = 5.0 J BX = 0.0 A’B’(X,‘),
system
J A’B’
=
4.6 5.1
J B’X’
=
vB, -
vx’ = 262 f
3
247 f
3
vB’ =
0.0
The shifts vB and vBSare from cyclohexane and the accuracy in the spin coupling constants is &lo-l c/s. The group 2 in Fig. 4 is broad and the centre is 115 c/s from c yclohexane . 4. DISCUSSION We shall first consider the DL-threonine results. In conformation I the two protons are trans and in II and III they are gauck. In substituted ethanes, J,, the coupling constant between two trans hydrogens and J, that between two gauche hydrogens are respectively in the range of lo-17 and l-3 C/S [S, 11, 161. As the values [16] N. SHEPHERD and J. J. TURNER,
Proc. Roy. Sot. (London) A252, 506 (1959).
1349
Spectra of DL-threonineand DL-valine 1
4
-x 3
2
L
i
I
I
I
150
I
100
50
0
C/S
Fig. 4(a). Observed spectra of A and B protons of DL-valine. Field increasing from right to left.
Fig. 4(b). Methyl group of
DL-Vdh3.
Field increasing from left to right.
1350
G.
ARULDHAS
obtained here for JAB and J,*,, are 3.9 ad 4.9, we can conclude that the conformation I in which the two hydrogens are trans is not giving a separate spectrum but it exchanges rapidly with II and III. The fact that we are observing two separate spectra implies that the exchange between II and III is not rapid. The above conclusion can also be arrived at in a qualitative way. The results on halogen substituted ethanes [9] indicate that if two large halogens are eclipsed, then the barriers are much larger. In Fig. 1 when HCH,OH in conformation I rotates 120” to form conformation II, it is necessary that the H moves past the COOH, the CH, past H and the OH past NH,. However, when this group in II rotates to form III two large groups are eclipsing two other large groups. Hence, the highest barrier is between II and III. It is therefore reasonable to assume that interconversion of conformations II and III is slower than the exchange between I and II or I andII1. These conclusions are also true for DL-valine. To substantiate the conclusions arrived in this investigation and for the determination of the barriers to internal rotation, it is desirable to study the spectra at various temperatures and different spectrometer frequencies and this is being investigated. Acknowledgements-The spectra were recorded with the the Indian Institute of Technology, Kanpur. The VENKATESWARLU for his generous help in recording the K. VE~TESWABLU for his interest in the progress of the
high resolution NMR spectrometer at author is grateful to Prof. PUTCHA spectra. Thanks are also due to Prof. work.
Nuclear magnetic resonance spectra of mlthreonine and DL-valhe Kerala University
G. &XJLDHAS Physics Department, (Received
20 December
Alwaye-2,
Kerala, India
1966)
Al&&-The 100 MC/S nuclear magnetic resonance spectra of nL-threonine and nL-valine in deuterium oxide at room temperature have been studied. In both the cases, the observed spectra have been analysed by assuming that each spectrum is a superposition of two spectra arising from the different conformations. 1. INTRODUCTION
nuclear magnetic resonance spectra of several amino acids have been studied [l-6] and attempts have been made to explain the spectra using the idea of rotational isomerism [6]. Some of the authors [l-3]studied mainly the chemical shifts and BOVEY and TIERS [4] estimated the coupling constants from spectra which were not well resolved. Amino acids in solution exist as a mixture of three interconverting conformations because of the internal rotation about the ChCB bond. The relative energies of the different conformations, the barriers to internal rotation and the chemical shifts and spin coupling constants of each conformation are the factors that govern the NMR spectra of such substances [7].At low temperatures each conformation is long lived and the observed spectrum is a superposition of the spectra due to the different conformations [S-lo]. At room temperature the internal rotation occurs at a rapid rate and the chemical shifts and spin coupling constants are averaged [6, 7, 111. In the present investigation, the analysis of the NMR spectra of DL-threonine and DL-valine at room temperature is reported. The observed spectra could be explained only by assuming that each spectrum is a superposition of two spectra These two cases, where complete rotational arising from the different conformations. averaging is not taking place at room temperature, are important and hence taken up for study. THE
[l] M. TAKEDA and 0. JARDET~KY, J. Chem. Phys. 26, 1346 (1967). [2] 0. JARDETZKY and C. D. JARDETZKY, J. Biol. Chem. 233, 383 (1958). [3] S. FUNJIWARA, Y. ARATA, N. HAYAKAWA and H. MONOI, Bull. Chem. Sot. Japan 36, 1658 (1962). F. A. BOVEY and G. V. D. TIERS, J. Am. Chem. Sot. 81, 1870 (1959). K. A. MCLAUCELAM, Mol. Phy.9. 5, 195 (1962). K. G. R. PACHLER, S~ectrochim. Acta 19,2086 (1963); 20, 581 (1964). J. A. POPLE, W. G. SCHNEIDER and H. J. BERNSTEIN, R@ Reeoltiion Nuclear Magnetic Reeonance, McGraw-Hill (1959). [8] D. G. THOMPSON, R. A. NEWMARK and C. H. SEDERHOLM, J. C&m. Phya. 37, 411 (1962). [9] R. A. NEK and C. H. SEDERHOLM, J. Chem. Phye. 43, 602 (1965). [lo] W. S. BREY, JR. and K. C. RAMEY, J. Chem. Phye. 39, 3646 (1963). [ll] H. S. GUTOWSKY, G. G. BELFORD and P. E. MC-ON, J. Chem. P&ye. 86,3354 (1962) and references cited therein. [4] [6] [6] [7]
12
1345
1346
G. ARULDRAS
2. EXPERIMENTAL The samples were obtained from E. Merk and 3.1 per cent solutions of these were prepared in deuterium oxide from the Atomic Energy Establishment, Bombay. The spectra were recorded on a Varian HR-100 high resolution NMR spectrometer operating at 100 MC/S. The temperature of the sample was about 28°C throughout. The separations of all peaks were measured by the usual side band technique. 3. RESULTS The amino and hydroxyl protons exchange rapidly with the deuterium of the solvent, so that the corresponding peaks coalesce with the residual water peak giving an intense line at the lowest field. 3.1 nn-threonine The different conformations are schematically shown in Fig. 1 and the observed spectrum is shown in Fig. 2. The protons A and B together with the methyl protons constitute an ABX, system. As the proton A has appreciable interaction with B and X, protons, lines l-9 are assigned to A proton. Lines lo-13 are assigned to proton B and 14-17 to X, protons. General expressions have been reported for the transitions and relative intensities in an ABX, system [12, 131. In the X, group of the ABX, case there are 12 transitions excluding the combination lines. An inspection of the frequencies shows that six of these give rise to one field independent doublet whose splitting value gives lJax + Jsxl. The two lines of this doublet are of the same intensity. When (va - vs) > JAB, the other six transitions give rise to another doublet symmetric with the previous one, with splitting equal to lJax - J,,I. The lines of this doublet are also of equal intensity. In the present case (va - vs) > JAB and J,, < 0.05 c/s [14, 151 and therefore the two doublets may coalesce and give a single one. In the experimental X spectrum (Fig. 2b), there are two doublets (lines 14, 15; 16, 17) and they are not symmetric. Hence we can assume that J,, is very nearly zero giving a single doublet for the X spectrum, of the ABX, and the experimental spectrum is a superposition of two ABX, spectra. We can arrive at this conclusion from the B transitions also. The intensity distribution suggests that lines l-7,10,11, 16 and 17 form one set, ABX, and the lines 4-9, 12-15 form the other set, A’B’X,‘; the ratio of the relative proportion of the two forms being 1.4: 1. The analysis gives the following parameters in c/s. ABX,
system J,, = 3.9 J AX = 6.5 J BX = 0.0
A’B’X,’
J;,;, R. V. C. J.
vB -
vx =
vB =
263 & 3.0 270 4 3.0
system J A'B' = 4.9 J Idt = 6.5
[12] [13] [14] [15]
va - vB = 52.5 * 0.5
=
0-o
vA, vs. -
vB, = 66.2 & 0.5 vx, = 227 f 3.0 VB’ =
245 f
3.0
W. FESSENDEN and J. S. WAUGH, J. Chem. Phys. 30, 944 (1959). J. KOWALEWSKI and D. G. DEKOWALEWSKI, J. Chem. Phys. 33, 1794 (1960). A. REILLY and J. D. SWALEN, J. Chem. Phys. 35, 1522 (1961). I. MUSHER and R. G. GORDON, J. Chem. Phys. 36, 3097 (1962).
1347
Spectra of DL-threonine and DL-valine
Fig. 1. Conformations c-x
3
of DL-threonine.
IO
6
75
50
7.5
0
C/S
Fig. 2(a).
Observed spectra of A (lines l-9) and B (lines 19-13) threonine. Field increasing from right to left.
protons of
DL-
16
Fig. 2(b). Methyl group of DL-threonine.
Field increasing from left to right.
1348
G. ARULDHAS
The accuracy in the spin coupling constants is ho.1 c/s. The shifts vs and vnS are from cyclohexane which appears at a higher field than B and B’, used as an internal standard. As only two lines for the B protons are obtained and the spectra due to A and A’ overlapping, it is almost impossible to determine the relative sign of JAB and J,,. 3.2 nr,-valine The three conformations are drawn in Fig. 3 and the experimental spectrum is shown in Fig. 4. The coupling between the two methyl groups is negligible. The experimental spectrum shows that (vs - va) and (va - vX) are large compared to the coupling constants, suggesting a first order case. If the two methyl groups are distinct, one has to get two doublets with all the lines equal in intensity. The spectrum (Fig. 4b) is showing two doublets of unequal intensities indicating that both
C&3H
COOH
C@bH
Fig. 3. Conformations
of DL-valine.
the methyl groups are almost equivalent and the observed spectrum is a superposition of two spectra as in DL-threonine, AB(X,), and A’B’(X,‘),; the ratio of the relative population of the two forms being 1.2: 1. The large width of about 35 c/s of group 2 suggests that the spectra of the protons A and A’ overlap to give group 2. The two lines of proton B coincide with the two lines of proton B’ and give group 1. In the X spectrum of rn,-valine the more intense doublet occurs at the low field side. The analysis gives the following parameters in c/s. AB(X,), system
J AR = 4.6
vB -
vx
= 255 & 3
vB = 247 & 3
J *x = 5.0 J BX = 0.0 A’B’(X,‘),
system
J A’B’
=
4.6 5.1
J B’X’
=
vB, -
vx’ = 262 f
3
247 f
3
vB’ =
0.0
The shifts vB and vBSare from cyclohexane and the accuracy in the spin coupling constants is &lo-l c/s. The group 2 in Fig. 4 is broad and the centre is 115 c/s from c yclohexane . 4. DISCUSSION We shall first consider the DL-threonine results. In conformation I the two protons are trans and in II and III they are gauck. In substituted ethanes, J,, the coupling constant between two trans hydrogens and J, that between two gauche hydrogens are respectively in the range of lo-17 and l-3 C/S [S, 11, 161. As the values [16] N. SHEPHERD and J. J. TURNER,
Proc. Roy. Sot. (London) A252, 506 (1959).
1349
Spectra of DL-threonineand DL-valine 1
4
-x 3
2
L
i
I
I
I
150
I
100
50
0
C/S
Fig. 4(a). Observed spectra of A and B protons of DL-valine. Field increasing from right to left.
Fig. 4(b). Methyl group of
DL-Vdh3.
Field increasing from left to right.
1350
G.
ARULDHAS
obtained here for JAB and J,*,, are 3.9 ad 4.9, we can conclude that the conformation I in which the two hydrogens are trans is not giving a separate spectrum but it exchanges rapidly with II and III. The fact that we are observing two separate spectra implies that the exchange between II and III is not rapid. The above conclusion can also be arrived at in a qualitative way. The results on halogen substituted ethanes [9] indicate that if two large halogens are eclipsed, then the barriers are much larger. In Fig. 1 when HCH,OH in conformation I rotates 120” to form conformation II, it is necessary that the H moves past the COOH, the CH, past H and the OH past NH,. However, when this group in II rotates to form III two large groups are eclipsing two other large groups. Hence, the highest barrier is between II and III. It is therefore reasonable to assume that interconversion of conformations II and III is slower than the exchange between I and II or I andII1. These conclusions are also true for DL-valine. To substantiate the conclusions arrived in this investigation and for the determination of the barriers to internal rotation, it is desirable to study the spectra at various temperatures and different spectrometer frequencies and this is being investigated. Acknowledgements-The spectra were recorded with the the Indian Institute of Technology, Kanpur. The VENKATESWARLU for his generous help in recording the K. VE~TESWABLU for his interest in the progress of the
high resolution NMR spectrometer at author is grateful to Prof. PUTCHA spectra. Thanks are also due to Prof. work.