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Solid state structures of phenylpyruvates as studied by high resolution 13C NMR spectroscopy
0584-@39/93 $5.00 + 0.00 @ 1992 Pergamon Press Ltd
Sjmfm&imica Acta, Vol. 49A, No. 1, pp. 125-133, 1993 Printed in Great Britain
Solid state structures of phenylpyruvates as studied by high resolution 13c NMR spectroscopy AKIO KUWAE* College of General Education, Nagoya City University, Miiho-ku,
Nagoya 467, Japan
and KAZUHIKO Gifu Pharmaceutical
HANAI
University, Mitahora-higashi,
Gifu 502, Japan
and KAORU OYAMA,
MASAZUMI
UCHINO
Yokohama Research Laboratory, Chisso Corporation,
and HO-HI
LEE
Kamariya, Kanaxawa-ku, Yokohama 236, Japan
(Receiued 26 March 1992; accepted 26 April 1992) ,kb&raet--High resolution solid state 13C NMR measurements were made on phenylpyruvic acid and its radium, lithium and calcium salts, using the total suppression of spinning side bands and the dipolar diphasing technique. The spectra of their 2-“C enriched analogs were also recorded. The NMR data were discussed by reference to the solution spectra, and the following definitive evidence was obtained: the acid, the hydrated sodium and lithium salts and the dehydrated sodium salt take the enol, the diol and the keto form, respectively, but the hydrated calcium salt exists in the keto form. The i3C NMR signal of the gem-diol carbon was found to appear at 98 ppm. The calcium salt has two doublets at 166 and 134 ppm which originate from the carbons C(1) and C(4); this splitting suggests that the carboxylate group and/or the phenyl ring in the phenylpyruvate anion are oriented in two different ways.
attention has been focused on a-keto acids such as phenylpyruvic acid (PPA) because of their biological roles. PPA occupies a central position in several metabolic sequences and is a substrate for microbial production of phenylalanine [l-3]. From a structural point of view, the tautomeric behavior of a-keto acids has sustained a long-standing interest both in solution and in the solid state. Spectroscopic studies have been extensively carried out to investigate the structures and the tautomeric equilibrium of PPA [4-81. Owing to the chemical nature of the a-keto carbonyl group, in addition to the keto and enol tautomers (I and II), the existence of the hydrated keto form (diol form III) is also perceived under certain conditions, as shown in Scheme 1. UV, NMR and vibrational studies have revealed that PPA and its salts exist in a complex equilibrium of these three forms in solution. As for the solid state structure we have established, on the basis of IR and Raman spectra, that PPA takes the enol form I, while CONSIDERABLE
I
II Scheme 1. Structures of PPA and its salts.
* Author to whom correspondence
should be addressed. 12.5
III
126
AKIO KUWAE etal. Table 1. 13CNMR chemical shifts (ppm) of phenylpyruvates and their r3Ccompounds in solution PPA
PPA-2-“C*
in CDrOD
in DMSO-d,
188.44
193.58
168.38
166.24
142.32 136.44 130.78 129.28 128.38 131.62 128.95 127.74
141.75 134.86 129.15 128.20 127.05 129.81
111.53
109.42
C(3), en01
44.70
C(3), keto
PPA-2-“C-Na.H*O* in D,O
134.06 130.68 129.71 128.17 131.13 129.12 127.66 115.79
193.59 166.64 165.89> + 141.79 (134.89)
C(2), keto C(l), en01 C(2), en01 C(4), en01 C(5), C(6), C(7), enol
C(5), C(6), C(7), keto
in DrO
170.93
Assignment
126.70
PPA-Na.H,O
205.16
in DMSO-d,
205.15 171.26 170.64 I + 145.56 (134.07) (130.66) (129.71) (128.17) I (131.12) (129.11) (127.65) 96.72
C(2), keto C(l), keto C(2), en01 C(4), keto C(5), C(6), C(7), keto
C(5), C(6), C(7), diol C(3), en01 C(2), diol
46.68
C(3), keto
45.20
C(3), diol
*Weak signals denoted in parentheses arise from the natural abundance carbons. + Weak doublets due to the one bond coupling, Jcc
its sodium salt assumes either the keto form II or the diol form III depending on the hydration state [7]. Although IR and Raman studies provide some information on molecular species in the solid, discussion on the molecular detail is limited by difficulty in analyzing rather unresolved features of vibrational spectra. X-Ray crystallography, though conclusive in solving the crystal structure, is a time-consuming process and not always feasible. No reports have appeared on the X-ray structures of PPA and its salts. High resolution solid state i3C NMR, under conditions of cross polarization (CP), magic angle spinning (MAS) and high power ‘H decoupling, has emerged as a powerful tool for investigating the structure in the solid state [9]. This approach allows discussion of the structure of the solid samples that might be altered substantially by dissolution
PO1* In this work we measured the 13CCPMAS NMR spectra of PPA, its two sodium salts (the hydrate and anhydrous forms, PPA-Na.H*O and PPA-Na) and two other hydrated salts (PPA-Li-HZ0 and PPA-Ca-H,O). Chemical shifts of all the carbon resonances were discussed by reference to the solution data. 13C CPMAS spectra of the 2-13C enriched samples were also measured to assist the assignments. Each phenylpyruvate exhibits a
CPMAS NMR spectra of phcnylpyruvatcs
127
Table 2. UC NMR chemical smith (ppm) of phenylpyruvatesand their “C compounds in the solid state PPA
PPA-2-“C
TOSS
TOSWDD
CPMAS
Assignment
170.0 138.0 133.8 131.9 129.0 127.2 115.9
170.3 138.6 134.5
138.0
C(l), enol C(2), enol c(4), ewl C(5). c(6). c(7), end C(3), en01
PPA-Na-Hz0
PPA-2-=C-Na-H20
175.4 136.1
175.5 136.2 131.2 128.0 98.6 46.2
C(l), diol c(4), diof C(5), C(6), Co,
98.5
PPA-Na 205.7 170.6 131.7 128.8 126.0 > 47.0
205.7 170.5 132.4
C(2). diol C(3), diol
PPA-2-‘%Na* 205.5
C(2), keto C(l), keto C(4). keto c(5), C(6), c(7), keto c(3), keto
PPA-Li-Hz0 178.5 135.8 132.0 128.5 98.1 47.9
98.8
dial
PPA-2-‘%Xi*Hz0
178.5 135.9
C(l), diol C(4), dial C(5), C(6). C(7). dial
98.2
97.9
C(2). diol C(3). diol
PPA-Ca.H*O C(2), keto C(l), keto C(4). keto C(5). C(6). c(7). keto C(3). keto l Measured in a TOSS sequence. ’ Doublets, see text.
unique “C CPMAS spectral pattern with decent resolution, which can he used for characterization of the three forms. EXPERIMENTAL Materials PPA-Na-Hz0 was purchased from Tokyo Kasei Kogyo and recrystallizedfrom water. PPA was preparedfrom PPA-Na-Hz0 and purifiedby dissolution of the crude product in acetone and then
AKIO KUWAEet al.
128
(a)
lb)
Fig. 1. Solid state ‘%ZNMR spectra of PPA (a and b) and PPA-2-‘v TOSSIDD.
(c). (a): TOSS; (b):
by precipitation with carbon tetrachloride. Its purity was checked by elemental analysis. PPA-Na was obtained from the hydrated salt by dehydration in uucuo at 100°C for 15 h. PPA-Li.H,O was prepared from the hydrated sodium salt by the ion exchange method using Dowex SOW(Li+ ) and recrystallized from water. PPA-Ca=H20 was obtained commercially (Tokyo Kasei Kogyo) and recrystallized from water. In the IR spectrum of this salt two bands are observed at 3456 and 3256 cm-‘, indicating that the salt has at least two kinds of water of crystallization. In the thermal analysis one mole of water was detected, but further dehydration was not confirmed owing to thermal degradation. PPA-2-13C, PPA-2-13C-Na*H20, PPA-2-13C-Na and The four 13C-labelled compounds, PPA-2-“C-Li.H20, were prepared to establish the NMR assignment; first the acid, PPA-Z”C, was synthesized from glycine-2-13C (99.2 atom% 13C, ISOTEC Inc.) by the azlactone method [ll], and then its salts were obtained by neutralization or ion exchange and purified by the above method.
Spectra ‘H and 13CNMR spectra of solutions were recorded on JEOL JNM-GSX 400 and JNM-EX 400 NMR spectrometers by using standard pulse sequences; tetramethylsilane was used as an internal reference for organic solutions and dioxane for DzO solutions. *“CNMR spectral measurements of the solid samples were carried out on a JEOL JNM-GSX 400 NMR spectrometer operating at 100.4 MHz for ‘v in the CPMAS mode. The samples (ca 200 mg) contained in a cylinder-type rotor of zirconium ceramic were spun at a spinning rate of about 6.0 kH.z. The spectral conditions were as follows: spectral width, 30 kHz; proton 90” pulse, 5.6,~s; contact time, 5 ms; repetition time, 7 s; 8 K data points; and the number of acquisitions, 2ooO12,ooO depending on the sample. A TOSS (total suppression of spinning side bands) sequence was applied to eliminate spinning side bands [12]. In separate experiments, spectra were also recorded in a combined sequence of TOSS and the dipolar dephasing (TOSS/DD) [ 131. Chemical shifts were calibrated using adamantane (29.5 ppm) as an external standard.
CPMAS NMR spectra of phenylpyruvates
129
RESULTS ANDDISCUSSION
Solution spectra It is important to understand the solution ‘e NMR spectra because they provide a basis for understanding the solid spectra. Table 1 summarizes the 13Cchemical shifts and assignments of PPA in organic solution and PPA-Na*H*O in DzO solution, together with those of PPA-2-13C analogs. PPA dissolved in organic solvents has been shown to exist in an equilibrium involving the enol and keto forms (955 in DMSO-d,) [5,7]. In accordance with the ‘H NMR result, PPA in DMSO-d6 shows “C NMR signals assignable to the enol and keto forms. The spectrum of PPA-2-13C exhibits two 13C-enriched carbon peaks at 141.79 and 193.59 ppm, the former and the latter being assigned to C(2) of the enol and the keto form, respectively. It is noteworthy that the C(1) and C(3) signals of the enol form show splittings of 75 and 83 Hz, respectively. These splittings originate from the homonuclear carbon-carbon coupling as a result of the 2-13C labelling. The magnitude of the observed Jcc s falls within the typical range for the C(sp’)-C(sp*) coupling [14]. Thus the enol structure is further confirmed from these Jcc values. The question on the configuration around the C(3)=C(2) bond is still to be answered. SCIACOVELLI ef al. reported on the configuration of the enol PPA[S]; they concluded that PPA in DMSO-11, solution takes the Z configuration on the basis of the value of the vicinal 1H-C=C-‘3COOH coupling constant [15]. We also obtained a similar value (3.8 Hz) for this coupling. PPA-Na.H20 in D20 solution shows mixed features of the keto, diol and enol forms as shown in Table 1. All the hydrated and dehydrated salts of PPA give the same spectrum as that of PPA-Na-N20 in D20 solution. In contrast to the PPA case, the keto form is predominant in solution. The molar ratio of the three forms was estimated to be
(a)
,....,,
(b)
k
Fig. 2. Solid state “C NMR spectra of PPA-N&H20 (a and b) and PPA-2-“C-Na.H20 (a): TOSS; (b): TOSSIDD.
(c).
AKIO KUWAEet al.
130
(a)
lb)
Fig. 3. Solid state “C NMR spectra of PPA-Na (a and b) and PPA-2-“C-Na (c). (a) and (c): TOSS; (b): TOSSIDD.
9O:lO:trace by ‘H NMR spectra [8]. PPA-2-‘3C-Na-H20 in DzO exhibits three signals arising from “C enriched carbons at 205.16, 145.56 and 96.72 ppm which are assigned to C(2) of the keto, the enol and the diol form, respectively. The CH2 carbon signal of the coupling, again keto form is split with a Jcc of 37 Hz typical for the C(sp’)-C($) confirming the existence of the a-keto moiety. Solid spectra As shown in the preceding section, 13CNMR are well understood in terms of an equilibrium however, no a priori reason to expect molecular as those in solution. Table 2 lists the 13CNMR
spectra of PPA and PPA-Na in solution of the three molecular species. There is, species in the solid state to be the same chemical shifts of the phenylpyruvates.
(1) PPA. The solid state structure of PPA proposed by the IR and Raman spectroscopies is the enol form. Fig. l(a) shows the *%JCPMAS spectrum of PPA measured under TOSS condition. As expected, no 13Cresonances are observed in the CH, carbon region and the carboxylic acid carbon resonates at 170.0 ppm. The spectrum shows signal-tosignal correspondence with that observed in CD30D solution, though the signals for C(1) and C(2) in the solid state tend to shift to the lower- and higher-field by 2 and 4 ppm, respectively. These shifts could be explained by the effects of hydrogen bondings involving the carboxylic acid and the hydroxyl groups [7]. All these observations are consistent with the IR and Raman results that solid PPA exists in the enol form. There is considerable complexity in the 130-134ppm region, where both phenyl and olefinic carbon resonances are expected. The dipolar dephased (DD) spectrum was measured to assist the assignment [Fig. l(b)]. In this case, the use of no ‘H decoupling during a short period prior to an acquisition allows the appearance of only quaternary carbon and
131
CPMAS NMR spectra of phenylpyruvates
methyl group peaks. The signals observed at 170.3, 138.6 and 134.5 ppm are enhanced under this pulse condition, indicating that these resonances come from non-hydrogen attached carbons. There is, however, still ambiguity in the assignment of the two resonances at 138.6 and 134.5 ppm. The CPMAS spectrum of 2-“C enriched PPA was measured to clarify this point. As shown in Fig. l(c), the 138.0 ppm resonance is the only signal observed in this sample, confirming the assignment of this resonance to the enolic carbon C(2). (2) PPA-Na*H20. Figure 2 shows the solid 13CNMR spectra of PPA-Na.H,O. IR and Raman studies from our laboratories suggested that solid PPA-Na.H,O exists in the diol form. In accordance with the vibrational findings, the results from the solid 13CNMR support the diol form. The methylene carbon resonates at 46.2 ppm. The resonances at 175.5, 136.2 and 98.6ppm are assigned to the carbons without hydrogen atom(s) attached because of the observation of these peaks in the TOSWDD spectrum [Fig. 2(b)]. The first and the second peak can be assigned to C(1) and C(4), respectively. The signal at 98.6 ppm is ascribed to C(2). There has been little information about “C NMR chemical shifts of the diol type, -C(OH)*- carbon. Thus we measured the CPMAS spectrum of ninhydrin, which has a -C(OH)*- moiety, as a reference compound. In the TOSWDD spectrum, ninhydrin shows a resonance assignable to the -C(OH)T carbon at 89.2 ppm, in addition to two carbonyl carbon signals at 199.3 and 194.6 ppm and the phenyl quaternary carbon at 139.7 ppm. The present finding together with the results of TOSWDD and that of CPMAS of the 13C-enriched sample leads to the conclusion that PPA-Na.H20 takes the diol form. (3) PPA-Na. The solid 13CNMR spectra of PPA-Na are shown in Fig. 3. In contrast to the PPA spectrum, the olefinic C(3) resonance is not detected and, instead, a peak I
I
(a)
4 I
.
.
.
.
-IL
(b)
Fig. 4. Solid state ‘%I NMR spectra of PPA-Li-H20 (a and b) and PPA-2-“C-Li.H20 (a): TOSS; (b): TOSSIDD.
(c).
AKIO KUWAE et al.
132
(b)
am
Fig. 5. Solid state “C NMR spectra of PPA-Ca-H20.
(a): TOSS; (b): TOSSIDD.
assignable to the methylene carbon appears at 47.0 ppm. A low-field resonance is observed at 205.7 ppm in Fig. 3(a-c). This signal is straightforwardly assigned to the a-keto carbonyl carbon. All the resonances for PPA-Na are unambiguously understood on the basis of the keto form. Chemical shifts observed for the PPA-Na solid are close to those of PPA-Na-Hz0 in DzO solution, suggesting a similarity of conformation between the solution and the solid state PPA-Na. (4) Other salts of PPA. The lithium and calcium salts contain water molecules in the crystals [7]. Figure 4 shows the solid NMR spectra of PPA-LieH,O. The spectral pattern is quite similar to that of PPA-Na-H20, indicating that the solid lithium salt also exists in the diol form. The spectra of PPA-Ca+H,O (Fig. 5) show a close resemblance to those of PPA-Na. The a-keto carbonyl resonance is observed at 204.2 ppm. The splitting features at 166.9 and 165.5 ppm are assigned to the carboxylate ion carbon. The phenyl group carbon C(4) is also split (135.3 and 133.3 ppm). These splittings suggest that the carboxylate group and/or the phenyl ring in the phenylpyruvate anion are oriented in two different ways. The 13Csolid NMR technique has been shown to be useful for discussing the structural characteristics of the tautomeric forms.
REFERENCES [l] A. Meister, Biochembrry of the Amino Acids, Vol. II. Academic Press, New York (1%5). [2] S. Sakurai, J. Biochem. 43, 851 (1956). [3] T. Asai, K. Aida and K. Oishi, Amino Acids 2, 114 (1960). [4] M.-L. Josien, M. Joussot-Dubien and J. Vizet, Bull. Sot. Chim. Fr. 1148 (1957). [5] 0. Sciacovelli, A. Dell’Atti, A. De Giglio and L. Cassidei, Z. Naturforsch. 31c, 5 (1976). [6] L. Cassidei, A. Dell’Atti and 0. Sciacovelli, Z. Naturforsch. 31c, 641 (1976); 35c, 1 (1988). [7] K. Hanai, A. Kuwae, S. Kawai and Y. Ono, J. Whys. Chem. 93,6013 (1989). [8] K. Hanai, S. Kawai and A. Kuwae, J. Molec. Struct. 245, 21 (1991). [P] J. R. Lyerla, C. S. Yannoni and C. A. Fyfe, Act. Chem. Res. 15,208 (1982). [lo] J. A. Ripmeester, Chem. Whys. Letr. 74, 536 (1988). [ll] R. M. Herbst and D. Shemin, in Organic Syntheses, Collect., Vol. 2 (Edited by A. H. Blatt), pp. 1,519. Wiley, New York (1966). [12] W. T. Dixon, J. Schaefer, M. D. Sefcik, E. 0. Stejskal and R. A. McKay, J. Magn. Reson. 49,341(1982).
CPMAS NMR spectra of phenylpyruvates
133
[13] S. J. OpeIIa and M. H. Frey, J. Am. Chem. Sot. 101,5854 (1979). [14] G. E. Maciel, J. W. McIver, Jr, N. S. Ostlund and J. A. Pople, L Am. Chem. Sot. !U, 1.11 (1970); F. J. Weigert and J. D. Roberts, J. Am. Chem. Sot. 94,6021(1972). [15] K. M. Crecely, R. W. Crecely and J. H. Goldstein, J. Molec. Specfmsc. 37,252 (1971); J. L. Marshall and R. Seiwell, .I. Mugn. Reson. 15,150 (1974); A. Stubbe and G. L. Kenyon, Biochetiby IO,2669 (1971); R. M. Letcher and R. M. Acheson, Org. Mugn. Reson. 16,316 (1981).
Sjmfm&imica Acta, Vol. 49A, No. 1, pp. 125-133, 1993 Printed in Great Britain
Solid state structures of phenylpyruvates as studied by high resolution 13c NMR spectroscopy AKIO KUWAE* College of General Education, Nagoya City University, Miiho-ku,
Nagoya 467, Japan
and KAZUHIKO Gifu Pharmaceutical
HANAI
University, Mitahora-higashi,
Gifu 502, Japan
and KAORU OYAMA,
MASAZUMI
UCHINO
Yokohama Research Laboratory, Chisso Corporation,
and HO-HI
LEE
Kamariya, Kanaxawa-ku, Yokohama 236, Japan
(Receiued 26 March 1992; accepted 26 April 1992) ,kb&raet--High resolution solid state 13C NMR measurements were made on phenylpyruvic acid and its radium, lithium and calcium salts, using the total suppression of spinning side bands and the dipolar diphasing technique. The spectra of their 2-“C enriched analogs were also recorded. The NMR data were discussed by reference to the solution spectra, and the following definitive evidence was obtained: the acid, the hydrated sodium and lithium salts and the dehydrated sodium salt take the enol, the diol and the keto form, respectively, but the hydrated calcium salt exists in the keto form. The i3C NMR signal of the gem-diol carbon was found to appear at 98 ppm. The calcium salt has two doublets at 166 and 134 ppm which originate from the carbons C(1) and C(4); this splitting suggests that the carboxylate group and/or the phenyl ring in the phenylpyruvate anion are oriented in two different ways.
attention has been focused on a-keto acids such as phenylpyruvic acid (PPA) because of their biological roles. PPA occupies a central position in several metabolic sequences and is a substrate for microbial production of phenylalanine [l-3]. From a structural point of view, the tautomeric behavior of a-keto acids has sustained a long-standing interest both in solution and in the solid state. Spectroscopic studies have been extensively carried out to investigate the structures and the tautomeric equilibrium of PPA [4-81. Owing to the chemical nature of the a-keto carbonyl group, in addition to the keto and enol tautomers (I and II), the existence of the hydrated keto form (diol form III) is also perceived under certain conditions, as shown in Scheme 1. UV, NMR and vibrational studies have revealed that PPA and its salts exist in a complex equilibrium of these three forms in solution. As for the solid state structure we have established, on the basis of IR and Raman spectra, that PPA takes the enol form I, while CONSIDERABLE
I
II Scheme 1. Structures of PPA and its salts.
* Author to whom correspondence
should be addressed. 12.5
III
126
AKIO KUWAE etal. Table 1. 13CNMR chemical shifts (ppm) of phenylpyruvates and their r3Ccompounds in solution PPA
PPA-2-“C*
in CDrOD
in DMSO-d,
188.44
193.58
168.38
166.24
142.32 136.44 130.78 129.28 128.38 131.62 128.95 127.74
141.75 134.86 129.15 128.20 127.05 129.81
111.53
109.42
C(3), en01
44.70
C(3), keto
PPA-2-“C-Na.H*O* in D,O
134.06 130.68 129.71 128.17 131.13 129.12 127.66 115.79
193.59 166.64 165.89> + 141.79 (134.89)
C(2), keto C(l), en01 C(2), en01 C(4), en01 C(5), C(6), C(7), enol
C(5), C(6), C(7), keto
in DrO
170.93
Assignment
126.70
PPA-Na.H,O
205.16
in DMSO-d,
205.15 171.26 170.64 I + 145.56 (134.07) (130.66) (129.71) (128.17) I (131.12) (129.11) (127.65) 96.72
C(2), keto C(l), keto C(2), en01 C(4), keto C(5), C(6), C(7), keto
C(5), C(6), C(7), diol C(3), en01 C(2), diol
46.68
C(3), keto
45.20
C(3), diol
*Weak signals denoted in parentheses arise from the natural abundance carbons. + Weak doublets due to the one bond coupling, Jcc
its sodium salt assumes either the keto form II or the diol form III depending on the hydration state [7]. Although IR and Raman studies provide some information on molecular species in the solid, discussion on the molecular detail is limited by difficulty in analyzing rather unresolved features of vibrational spectra. X-Ray crystallography, though conclusive in solving the crystal structure, is a time-consuming process and not always feasible. No reports have appeared on the X-ray structures of PPA and its salts. High resolution solid state i3C NMR, under conditions of cross polarization (CP), magic angle spinning (MAS) and high power ‘H decoupling, has emerged as a powerful tool for investigating the structure in the solid state [9]. This approach allows discussion of the structure of the solid samples that might be altered substantially by dissolution
PO1* In this work we measured the 13CCPMAS NMR spectra of PPA, its two sodium salts (the hydrate and anhydrous forms, PPA-Na.H*O and PPA-Na) and two other hydrated salts (PPA-Li-HZ0 and PPA-Ca-H,O). Chemical shifts of all the carbon resonances were discussed by reference to the solution data. 13C CPMAS spectra of the 2-13C enriched samples were also measured to assist the assignments. Each phenylpyruvate exhibits a
CPMAS NMR spectra of phcnylpyruvatcs
127
Table 2. UC NMR chemical smith (ppm) of phenylpyruvatesand their “C compounds in the solid state PPA
PPA-2-“C
TOSS
TOSWDD
CPMAS
Assignment
170.0 138.0 133.8 131.9 129.0 127.2 115.9
170.3 138.6 134.5
138.0
C(l), enol C(2), enol c(4), ewl C(5). c(6). c(7), end C(3), en01
PPA-Na-Hz0
PPA-2-=C-Na-H20
175.4 136.1
175.5 136.2 131.2 128.0 98.6 46.2
C(l), diol c(4), diof C(5), C(6), Co,
98.5
PPA-Na 205.7 170.6 131.7 128.8 126.0 > 47.0
205.7 170.5 132.4
C(2). diol C(3), diol
PPA-2-‘%Na* 205.5
C(2), keto C(l), keto C(4). keto c(5), C(6), c(7), keto c(3), keto
PPA-Li-Hz0 178.5 135.8 132.0 128.5 98.1 47.9
98.8
dial
PPA-2-‘%Xi*Hz0
178.5 135.9
C(l), diol C(4), dial C(5), C(6). C(7). dial
98.2
97.9
C(2). diol C(3). diol
PPA-Ca.H*O C(2), keto C(l), keto C(4). keto C(5). C(6). c(7). keto C(3). keto l Measured in a TOSS sequence. ’ Doublets, see text.
unique “C CPMAS spectral pattern with decent resolution, which can he used for characterization of the three forms. EXPERIMENTAL Materials PPA-Na-Hz0 was purchased from Tokyo Kasei Kogyo and recrystallizedfrom water. PPA was preparedfrom PPA-Na-Hz0 and purifiedby dissolution of the crude product in acetone and then
AKIO KUWAEet al.
128
(a)
lb)
Fig. 1. Solid state ‘%ZNMR spectra of PPA (a and b) and PPA-2-‘v TOSSIDD.
(c). (a): TOSS; (b):
by precipitation with carbon tetrachloride. Its purity was checked by elemental analysis. PPA-Na was obtained from the hydrated salt by dehydration in uucuo at 100°C for 15 h. PPA-Li.H,O was prepared from the hydrated sodium salt by the ion exchange method using Dowex SOW(Li+ ) and recrystallized from water. PPA-Ca=H20 was obtained commercially (Tokyo Kasei Kogyo) and recrystallized from water. In the IR spectrum of this salt two bands are observed at 3456 and 3256 cm-‘, indicating that the salt has at least two kinds of water of crystallization. In the thermal analysis one mole of water was detected, but further dehydration was not confirmed owing to thermal degradation. PPA-2-13C, PPA-2-13C-Na*H20, PPA-2-13C-Na and The four 13C-labelled compounds, PPA-2-“C-Li.H20, were prepared to establish the NMR assignment; first the acid, PPA-Z”C, was synthesized from glycine-2-13C (99.2 atom% 13C, ISOTEC Inc.) by the azlactone method [ll], and then its salts were obtained by neutralization or ion exchange and purified by the above method.
Spectra ‘H and 13CNMR spectra of solutions were recorded on JEOL JNM-GSX 400 and JNM-EX 400 NMR spectrometers by using standard pulse sequences; tetramethylsilane was used as an internal reference for organic solutions and dioxane for DzO solutions. *“CNMR spectral measurements of the solid samples were carried out on a JEOL JNM-GSX 400 NMR spectrometer operating at 100.4 MHz for ‘v in the CPMAS mode. The samples (ca 200 mg) contained in a cylinder-type rotor of zirconium ceramic were spun at a spinning rate of about 6.0 kH.z. The spectral conditions were as follows: spectral width, 30 kHz; proton 90” pulse, 5.6,~s; contact time, 5 ms; repetition time, 7 s; 8 K data points; and the number of acquisitions, 2ooO12,ooO depending on the sample. A TOSS (total suppression of spinning side bands) sequence was applied to eliminate spinning side bands [12]. In separate experiments, spectra were also recorded in a combined sequence of TOSS and the dipolar dephasing (TOSS/DD) [ 131. Chemical shifts were calibrated using adamantane (29.5 ppm) as an external standard.
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RESULTS ANDDISCUSSION
Solution spectra It is important to understand the solution ‘e NMR spectra because they provide a basis for understanding the solid spectra. Table 1 summarizes the 13Cchemical shifts and assignments of PPA in organic solution and PPA-Na*H*O in DzO solution, together with those of PPA-2-13C analogs. PPA dissolved in organic solvents has been shown to exist in an equilibrium involving the enol and keto forms (955 in DMSO-d,) [5,7]. In accordance with the ‘H NMR result, PPA in DMSO-d6 shows “C NMR signals assignable to the enol and keto forms. The spectrum of PPA-2-13C exhibits two 13C-enriched carbon peaks at 141.79 and 193.59 ppm, the former and the latter being assigned to C(2) of the enol and the keto form, respectively. It is noteworthy that the C(1) and C(3) signals of the enol form show splittings of 75 and 83 Hz, respectively. These splittings originate from the homonuclear carbon-carbon coupling as a result of the 2-13C labelling. The magnitude of the observed Jcc s falls within the typical range for the C(sp’)-C(sp*) coupling [14]. Thus the enol structure is further confirmed from these Jcc values. The question on the configuration around the C(3)=C(2) bond is still to be answered. SCIACOVELLI ef al. reported on the configuration of the enol PPA[S]; they concluded that PPA in DMSO-11, solution takes the Z configuration on the basis of the value of the vicinal 1H-C=C-‘3COOH coupling constant [15]. We also obtained a similar value (3.8 Hz) for this coupling. PPA-Na.H20 in D20 solution shows mixed features of the keto, diol and enol forms as shown in Table 1. All the hydrated and dehydrated salts of PPA give the same spectrum as that of PPA-Na-N20 in D20 solution. In contrast to the PPA case, the keto form is predominant in solution. The molar ratio of the three forms was estimated to be
(a)
,....,,
(b)
k
Fig. 2. Solid state “C NMR spectra of PPA-N&H20 (a and b) and PPA-2-“C-Na.H20 (a): TOSS; (b): TOSSIDD.
(c).
AKIO KUWAEet al.
130
(a)
lb)
Fig. 3. Solid state “C NMR spectra of PPA-Na (a and b) and PPA-2-“C-Na (c). (a) and (c): TOSS; (b): TOSSIDD.
9O:lO:trace by ‘H NMR spectra [8]. PPA-2-‘3C-Na-H20 in DzO exhibits three signals arising from “C enriched carbons at 205.16, 145.56 and 96.72 ppm which are assigned to C(2) of the keto, the enol and the diol form, respectively. The CH2 carbon signal of the coupling, again keto form is split with a Jcc of 37 Hz typical for the C(sp’)-C($) confirming the existence of the a-keto moiety. Solid spectra As shown in the preceding section, 13CNMR are well understood in terms of an equilibrium however, no a priori reason to expect molecular as those in solution. Table 2 lists the 13CNMR
spectra of PPA and PPA-Na in solution of the three molecular species. There is, species in the solid state to be the same chemical shifts of the phenylpyruvates.
(1) PPA. The solid state structure of PPA proposed by the IR and Raman spectroscopies is the enol form. Fig. l(a) shows the *%JCPMAS spectrum of PPA measured under TOSS condition. As expected, no 13Cresonances are observed in the CH, carbon region and the carboxylic acid carbon resonates at 170.0 ppm. The spectrum shows signal-tosignal correspondence with that observed in CD30D solution, though the signals for C(1) and C(2) in the solid state tend to shift to the lower- and higher-field by 2 and 4 ppm, respectively. These shifts could be explained by the effects of hydrogen bondings involving the carboxylic acid and the hydroxyl groups [7]. All these observations are consistent with the IR and Raman results that solid PPA exists in the enol form. There is considerable complexity in the 130-134ppm region, where both phenyl and olefinic carbon resonances are expected. The dipolar dephased (DD) spectrum was measured to assist the assignment [Fig. l(b)]. In this case, the use of no ‘H decoupling during a short period prior to an acquisition allows the appearance of only quaternary carbon and
131
CPMAS NMR spectra of phenylpyruvates
methyl group peaks. The signals observed at 170.3, 138.6 and 134.5 ppm are enhanced under this pulse condition, indicating that these resonances come from non-hydrogen attached carbons. There is, however, still ambiguity in the assignment of the two resonances at 138.6 and 134.5 ppm. The CPMAS spectrum of 2-“C enriched PPA was measured to clarify this point. As shown in Fig. l(c), the 138.0 ppm resonance is the only signal observed in this sample, confirming the assignment of this resonance to the enolic carbon C(2). (2) PPA-Na*H20. Figure 2 shows the solid 13CNMR spectra of PPA-Na.H,O. IR and Raman studies from our laboratories suggested that solid PPA-Na.H,O exists in the diol form. In accordance with the vibrational findings, the results from the solid 13CNMR support the diol form. The methylene carbon resonates at 46.2 ppm. The resonances at 175.5, 136.2 and 98.6ppm are assigned to the carbons without hydrogen atom(s) attached because of the observation of these peaks in the TOSWDD spectrum [Fig. 2(b)]. The first and the second peak can be assigned to C(1) and C(4), respectively. The signal at 98.6 ppm is ascribed to C(2). There has been little information about “C NMR chemical shifts of the diol type, -C(OH)*- carbon. Thus we measured the CPMAS spectrum of ninhydrin, which has a -C(OH)*- moiety, as a reference compound. In the TOSWDD spectrum, ninhydrin shows a resonance assignable to the -C(OH)T carbon at 89.2 ppm, in addition to two carbonyl carbon signals at 199.3 and 194.6 ppm and the phenyl quaternary carbon at 139.7 ppm. The present finding together with the results of TOSWDD and that of CPMAS of the 13C-enriched sample leads to the conclusion that PPA-Na.H20 takes the diol form. (3) PPA-Na. The solid 13CNMR spectra of PPA-Na are shown in Fig. 3. In contrast to the PPA spectrum, the olefinic C(3) resonance is not detected and, instead, a peak I
I
(a)
4 I
.
.
.
.
-IL
(b)
Fig. 4. Solid state ‘%I NMR spectra of PPA-Li-H20 (a and b) and PPA-2-“C-Li.H20 (a): TOSS; (b): TOSSIDD.
(c).
AKIO KUWAE et al.
132
(b)
am
Fig. 5. Solid state “C NMR spectra of PPA-Ca-H20.
(a): TOSS; (b): TOSSIDD.
assignable to the methylene carbon appears at 47.0 ppm. A low-field resonance is observed at 205.7 ppm in Fig. 3(a-c). This signal is straightforwardly assigned to the a-keto carbonyl carbon. All the resonances for PPA-Na are unambiguously understood on the basis of the keto form. Chemical shifts observed for the PPA-Na solid are close to those of PPA-Na-Hz0 in DzO solution, suggesting a similarity of conformation between the solution and the solid state PPA-Na. (4) Other salts of PPA. The lithium and calcium salts contain water molecules in the crystals [7]. Figure 4 shows the solid NMR spectra of PPA-LieH,O. The spectral pattern is quite similar to that of PPA-Na-H20, indicating that the solid lithium salt also exists in the diol form. The spectra of PPA-Ca+H,O (Fig. 5) show a close resemblance to those of PPA-Na. The a-keto carbonyl resonance is observed at 204.2 ppm. The splitting features at 166.9 and 165.5 ppm are assigned to the carboxylate ion carbon. The phenyl group carbon C(4) is also split (135.3 and 133.3 ppm). These splittings suggest that the carboxylate group and/or the phenyl ring in the phenylpyruvate anion are oriented in two different ways. The 13Csolid NMR technique has been shown to be useful for discussing the structural characteristics of the tautomeric forms.
REFERENCES [l] A. Meister, Biochembrry of the Amino Acids, Vol. II. Academic Press, New York (1%5). [2] S. Sakurai, J. Biochem. 43, 851 (1956). [3] T. Asai, K. Aida and K. Oishi, Amino Acids 2, 114 (1960). [4] M.-L. Josien, M. Joussot-Dubien and J. Vizet, Bull. Sot. Chim. Fr. 1148 (1957). [5] 0. Sciacovelli, A. Dell’Atti, A. De Giglio and L. Cassidei, Z. Naturforsch. 31c, 5 (1976). [6] L. Cassidei, A. Dell’Atti and 0. Sciacovelli, Z. Naturforsch. 31c, 641 (1976); 35c, 1 (1988). [7] K. Hanai, A. Kuwae, S. Kawai and Y. Ono, J. Whys. Chem. 93,6013 (1989). [8] K. Hanai, S. Kawai and A. Kuwae, J. Molec. Struct. 245, 21 (1991). [P] J. R. Lyerla, C. S. Yannoni and C. A. Fyfe, Act. Chem. Res. 15,208 (1982). [lo] J. A. Ripmeester, Chem. Whys. Letr. 74, 536 (1988). [ll] R. M. Herbst and D. Shemin, in Organic Syntheses, Collect., Vol. 2 (Edited by A. H. Blatt), pp. 1,519. Wiley, New York (1966). [12] W. T. Dixon, J. Schaefer, M. D. Sefcik, E. 0. Stejskal and R. A. McKay, J. Magn. Reson. 49,341(1982).
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[13] S. J. OpeIIa and M. H. Frey, J. Am. Chem. Sot. 101,5854 (1979). [14] G. E. Maciel, J. W. McIver, Jr, N. S. Ostlund and J. A. Pople, L Am. Chem. Sot. !U, 1.11 (1970); F. J. Weigert and J. D. Roberts, J. Am. Chem. Sot. 94,6021(1972). [15] K. M. Crecely, R. W. Crecely and J. H. Goldstein, J. Molec. Specfmsc. 37,252 (1971); J. L. Marshall and R. Seiwell, .I. Mugn. Reson. 15,150 (1974); A. Stubbe and G. L. Kenyon, Biochetiby IO,2669 (1971); R. M. Letcher and R. M. Acheson, Org. Mugn. Reson. 16,316 (1981).