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Assignment of primary and secondary amide carbonyl resonances in carbon-13 NMR
JOURNAL OF MAONETK RESONANCE21,1-7
(1976)
Assignment of Primary and Secondary Amide Carbonyl Resonancesin Carbon-13 NMR RICHARD A. NEWMARK AND JAMESR. HILL Central Research Laboratories,
3M Company, St. Paul, Minnesota 55133
Received April 25,1975 Carbons a to the NH in amides show a 0.1 ppm isotope shift of the CNH compared to the CND. This results in doublets (secondary) or triplets (primary) when amides are dissolved in dimethylsulfoxide, dimethylformamide, or hexamethylphosphoramide containing equimolar exchangeable protons or deuterons. This condition is readily obtained by adding 10% Hz0 and 10 % D20 to the amide solution. Aromatic carbons a or /l to an NH-CO-R substituent are split by 0.1 ppm, whereas those a to CO-NH-R are split by 0.05 ppm under similar conditions. Examplesdescribed include acetamide. benzamide, acetyl glycine, hippnric acid, acetanilide, and the three isomers of amidophenol and acetotoludide. Deuterium substitution has been used to assign carbon-13 spectra. The a, /J’, and y carbons can be assigned by observation of a C-D multiplet, /?-isotope shift, and
long-range coupling to the D, respectively (I-4). Feeney et al. (5) showed that isotope shifts could be observed for peptide carbonyl resonances in aqueous solution. Thus, in 50: 50 H,O : D,O, a secondary amide carbonyl is a doublet due to a 0.1 ppm isotope shift of the CONH compared to the COND. We have extended this technique to amides dissolved in dimethylsulfoxide (DMSO), dimethylformamide (DMF), and hexamethylphosphoramide (HMPA). In all three cases the amide carbonyl splits into a doublet (secondary) or triplet (primary) if H,O and D,O are added at a level to obtain equal amounts of exchangeable protons and deuterium atoms. Furthermore, aliphatic carbons c(to the NH are normally split by 0.1 ppm and aromatic carbons both a and /I to the NH are also split by 0.1 ppm. EXPERIMENTAL
All samples were commercially available materials and used as received. Spectra were run in the Fourier transform mode on a Varian XL-100 NMR spectrometer. Spectra were obtained on approximately 500 mg samples in 2.0 ml DMSO to which 0.5 ml DMSO-d, was added for a lock. For convenience, about 0.25 ml H,O and 0.25 ml D,O was added routinely to most solutions to observe the carbon isotope shifts. The added D20 served as the lock in the DMF or HMPA solutions. Several samples precipitated out of solution when water was added, but redissolved upon heating. Peaks were referenced to internal DMSO (40.48 ppm (6) relative to TMS) because of the low solubility of TMS in DMSO, but chemical shifts are reported relative to TMS. Addition of 20 % water caused a downfield shift between 0. I and 1.5 ppm of most of the peaks, butabout a 2.5 ppm downfield shift of the amide carbonyl. This shift Copyright CC 1976 by Academic Press, Inc. All rights of reproduction in any form reserved Printed in Great Britain
1
2
N E W M A R K AND HILL
was proportional to the amount of water added. Consequently, less water was added to samples with peaks separated by less than 1 ppm in order to follow the differential shifts. All chemical shifts reported in this paper are for DMSO solutions in the absence of added H,O or DzO. Doublets due to possible rotational isomers were not observed in any of the secondary amide spectra. Spectra were obtained on 8K data points at sweep widths of 5000 Hz (1.25 Hz/point) for chemical shifts, at 1000 Hz sweep widths (0.25 Hz/point) for most isotopic splittings, but at 500 Hz (0.125 Hz/point) for primary amide carbonyls and the carbonyls in VI to X. Although peak broadening was usually observable for resonances in secondary amides at 1.25 Hz/point, increased resolution was necessary to measure the splittings or to observe any effect in the primary amides. RESULTS AND DISCUSSION
The acetamide (I) carbonyl is a 1:2: 1 triplet in 50: 50 H,O:D,O at pH 6, split by 0.06 ppm, but the resolution is barely sufficient to resolve the peaks (Fig. 1). The
HpO/ D20
DMF
HMPA
DMSO
FIG. 1. Acetamide (CH3-CO-NH2) carbonyl in water (pH 6), DMF, HMPA, and DMSO. Each organic solvent contained approximately lOoA HZ0 and 12% DzO.
splitting is due to different chemical shifts for the CONH,, CONHD + CONDH, and CONDz species. The line broadening is probably due to proton exchange, which must be slow compared to the peak separation in order to resolve peaks from the different deuterated species. If a&amide is dissolved in DMSO, DMF, or HMPA, a single sharp line is observed for the carbon resonances. However, addition of approximately equal amounts of DzO and H,O results in a triplet for the carbonyl, similar to that observed in water, but with much greater resolution in DMSO and HMPA (Fig. 1). In a typical experiment, about 500 mg acetamide, 0.35 ml D20, and 0.25 ml H,O are added to 2.5 ml solvent. Excess D,O is needed to compensate for the exchangeable protons in the solute. It is only necessary to add DzO equivalent to the number of exchangeable protons in the amide. However, excessH,O plus D20 does not affect the results and vitiates the requirement of weighing the samples and knowing beforehand the structure and number of exchangeable protons. Other primary amides investigated included butyramide (II), benzamide (III), and nicotinamide (1V). In all cases triplets are observed for the amide carbonyl in DMSO plus H,O and D,O, split by 0.06 ppm. There was no evidence for splitting of any other
ISOTOPE SPLITTJNGS IN AMIDES
3
carbon atoms, but broadening of the aromatic Cl carbon in III was apparent when the peak width (1.8 Hz at half-height) was compared to a sample containing no water in which the Cl peak width was only 0.5 Hz. Similar broadening was observed in II, in which the CImethylene carbon linewidth increased from 0.85 to 3.0 Hz. Secondary amides proved more interesting because the splittings are larger and readily observed at additional carbon atoms in the molecule. In acetyl glycine (V) the methylene is split by 0.09 ppm, slightly more than the amide carbonyl splitting of 0.08 ppm (Fig. 2) when H,O and D,O are added to the DMSO solution. This result is reasonable since both carbons are equidistant from the NH (or ND). In addition, the carboxyl, fi to the NH, shows a 0.02 ppm splitting. Similar results are observed in N-acetyl valine methyl ester (VI) and N-acetyl phenylalanine ethyl ester (VII), but no splilting of the /j’carbonyl was observed in hippuric acid (VIII) or N-benzoyl alanine (IX). However, the aromatic Cl fl to the NH split by 0.03 to 0.04 ppm in VIII and IX.
FIG. 2. 13C spectrumof carbonylsand methyleneof acetylglycine(CH,-CO-NH-CH,-CO,H) in DMSO containingequimolarexchangeable protons and deuterons.
Note that the small splittings in N-aliphatic amides are only observable for p quaternary carbons because the linewidth of a CH carbon usually exceeds I .O Hz (0.04 ppm at 25 MHz). The quatemary carbon linewidths in V-IX were under 0.5 Hz. The chemical shifts and isotopic splittings are given in Table 1. In aromatic secondary amides, such as acetanilide (Xl), the aromatic carbon /I to the NH (or ND) is split by 0.1 ppm (Fig. 3). This splitting, as large as the CIsplitting, is twice the ,!3effect observed in the aliphatic systems discussed above. The remaining aromatic protons were readily assigned in acetanilide from their 2: 1 relative intensities and the aromatic substituent chemical shifts (SCS) of the amide then determined by comparing the acetanilide chemical shifts to that of benzene (128.16 ppm) in DMSO. The SCS for NHCOCH3 is: Cl, 11.2; ortho, -9.1; meta, fo.5; para, -5.2 ppm. Similar results are observed in substituted acetanilides, such as the three acetotoluidide (XII-XIV) and amidophenol (XV-XVII) isomers. The chemical shifts and isotopic splittings are given in Table 2. The assignments in these molecules is based on (1) relative peak intensities due to nuclear Overhauser enhancements (NOE) to
VIII’
A B C F GH D CH,-CO-NH-CH(CHMeJ-CO-NH,
X
1
169.3 (d, 0.08)
166.3 (0.08)
171.6 (0.07)
169.3 (0.08)
170.0 (0.08)
170.4 (0.08)
B
57.5
48.3 (0.10)
41.5 (0.10)
53.7 (0.08)
57.6 (0.10)
41.1 (0.09)
C
173.2 (t. 0.06)
174.3
166.8
171.7 (0.02.)
172.4 (0.02)
171.8 (0.02)
D
17.0
60.4
51.7
E
Chemical shifts (isotope splittings),
19.4
36.9
19.1
G
18.0
18.4
H
Cl = 134.1 (0.03), C2,6 = 127.5,
30.2
14.0
30.0
F
in ppm
’ Phenyl ring chemical shifts and Cl isotope splittings: VII, Cl = 137.2, C2, 6 = 129.0, C3, 5 = 128.2, C4 = 126.5; VIII, C3,5=128.5,C4=131.6;IX,Cl = 134.0(0.04), C2, 6= 127.5, C3,5=128,3,C4= 131.4.
B CE C.&-CO-NHCHMe-C02H
IX”
22.6
B C D C&-CO-NH-CH2-C02H D
22.3
D A B C G CH,-CO-NH-CH(CH,C,H,)-COz-CH,
VII’
F
22.3
A B C F GH D CH,-CO-NH-CH(CHMe&C01-CH3
VI E
22.6
A
A B C D CH,-CO-NH-CH,-CO,H E
TABLE ISOTOPESPLITTINGS IN N-ALIPHATIC SECONDARYAMIDES (Me Is METHYL)
V
CHEMICAL SHWTSAND NH/ND
$ r
%
E
P
5
ISOTOPESPLITTINGS IN AMIDES
distinguish the quaternary carbons, (2) calculated chemical shifts based on the SCS of the amide group given above and the SCS of OCH,, OH, and CHB given by Levy and Nelson (7), and (3) the 0.1 ppm doublets induced by the addition of H,O + D,O. The observed shifts differed by less than 3.5 ppm from those calculated for all carbons except C6 of XII in which the difference was 6.0 ppm. p-Acetotoluidide (XIV) was also studied in DMF and HMPA. The resolution of the amide carbonyl was the same in all three solvents, but that of the aromatic CH ortho to the amido was greatest in DMSO and DMSO was used for all further work. Data on o-acetoacetanisidide (XVIII), o-acetophenetidide (XIX) and benzoyl acetanilide (XX) are also reported in Table 2. Although XX was a 2 : 1 mixture of the keto and enol tautomers, addition of water greatly enhanced the keto form. The addition of equimolar amounts of H,O and D,O for the assignment of carbonyl and a and p carbons can be extended to hetero and polynuclear aromatics. In 2acetamidonaphthalene (XXI) the carbonyl (168.6 ppm) and C2 (137.0) quaternary
50 Hz
JLl..I1.J_.i co
Cl
c 3,5
c4
c 2,6
FIG. 3. W spectrum of carbonyl and aromatic carbons of acetanilide (CH3-CO-NH-C6HS) DMSO containing 10% H,O and 1076 D20.
in
carbons and Cl (120.0) and C3 (115.0) methines all split by 0.09 ppm. The remaining methines, not assigned, are at 124.4, 126.3, 127.3, 127.4, and 128.3; C9 is at 133.5 and Cl0 at 129.7 ppm. Id 3-acetamidoquinoline (Xx11) the carbonyl(l69.4) and C4 (121.9) split by 0.09 and 0.08 ppm, respectively. The quaternary C3 (133.1) and C2 (144.6) did not split into doublets, but their linewidths doubled and permitted their unambiguous assignment. The remaining methines, not assigned, are at 127.1, 128.6, and an unresolved 2-carbon peak at 127.7; C9 is at 144.2 and Cl0 is at 128.0 ppm. Assignments in XXI and XXII are based on the chemical shifts of the parent compounds reported by Pugmire et ~1. (8). Doublets were not observed for the carbonyl in cyclic amides or imides, such as pyridone, succinimide, or phthalimide. The carbonyl linewidth was 2.5,0.8, and 1.3 Hz, respectively, in the absence of added water and did not change significantly when equimolar exchangeable protons and deuterons were added. In conclusion, addition of approximately equal amounts of Hz0 and D20 to solutions of amides in DMSO provides a rapid means of assigning the carbons a to the NH and, in many cases, also carbons ,B to the NH.
148.7 (0.07) 149.2 (0.08)
139.3 (0.09) 136.9 (0.09) 126.5 (0.09) 140.4 (0.10) 130.8 (0.09) 127.7 (0.09) 127.3 (0.10) 139.0 (0.09)
168.2 (0.09) 168.1 (0.09) 169.3 (0.08) 168.3 (0.10) 167.8 (0.10) 168.4 (0.09) 165.2 (0.08) 165.4 (0.09)
24.0 24.0 23.1 24.1 23.8 23.9 51.7 48.1
3-CHJ
4-CHJ
2-OH
3-OH
4-OH
2-OCHzCHs
2-OCH3
H
H
H
H
H
H
H
COCHS
COC.~HS
XIII
XIV
xv
XVI
XVII
XVIII
XIX
xx
128.8
111.1
112.1
115.2
157.7
116.2
129.0
137.8
130.3
123.4
124.4
124.2
153.3
110.3
124.9
131.9
123.7
125.9’
123.0
c4
in ppm C5
128.8
120.3
120.1
115.2
129.4
119.2
129.0
128.5
125.0’
128.7
n R chemical shifts: XIX, 203.6 (CO) and 30.2 (CH3); XX, 194.6 (CO), 136.3 (Cl), 128.4 (C2, 6), (128.8 (C3,5), and 133.6 (C4). b X chemical shifts: XII, 17.9; XIII, 21.2; XIV, 20.4; XVIII, 63.8 (CH,) and 14.6 (CH3); XIX, 203.6 (CO) and 55.7 (CH,). c May be interchanged. 1 Not measurable due to overlap with C5.
119.1 (0.10)
121.1 (0.10)
106.3 (0.08)
148.1 (0.08)
119.1 (0.09)
119.6 (0.09)
131.6 (0.09)
136.6 (0.11)
168.2 (0.08)
23.3
128.7
119.2 (0.09)
139.4 (0.10)
168.4 (0.09)
24.0
2-CHa
H
H
c3
c2
Cl
co
Chemical shifts (isotope splittings),
H
CHzR
2
XI
Xb
TABLE ISOTOPESPLITTINGS IN AROMATIC SECONDARYAMIDES, X-A-NHCOCH2R
XII
R”
CHEMICAL SHIFTSAND NH/ND
119.1 (0.10)
121.4 (0.12)
122.0 (0.13)
121.1 (0.10)
109.9 (0.09)
122.5 (0.10)
119.1 (0.09)
116.3 (0.09)
125.06
119.2 (0.09)
C6
E
zi 0 x
z
z
ISOTOPE SPLITTINGS IN AMIDES
7
REFERENCES I. D. DODDRELL AXD I. BURFITT, Aust. /. Chem. 25,2239 (1972). 2. J. B. STOTHERS,C. T. TAN, A. NICKON, F. HUANG, R. SRIDHAR, AND R. WEGLEIN, .I. Amer. Chem. Sot. !M,8581 (1972). 3. A. P. TULL~CH AND M. MAZUREK, J. Chew. Sot., Chem. Commun. 692 (1973). 4. R. H. MARTIN, J. MORIAU, AND N. DEFAY, Tetrahedron 30,179 (1974). 5. J. FEENEY, P. PARTINGTON, AND G. C. K. ROBERTS,J. Magn. Resonance 13,268 (1974). 6. G. C. LEVY AND .I. D. CARUIOLI, .I. Magn. Resonmce 6,143 (1972). 7. G. C. LEVY AND G. L. NELSON, “Carbon-13 Nuclear Magnetic Resonance for Organic Chemists,” Wiley-Interscience, New York, 1972. 8. R. J. PUGMIRE, D. M. GRAhT, M. J. RORTNS,ANU R. K. ROBINS, J. Amer. Chem. Ser. 91,6381(1969).
(1976)
Assignment of Primary and Secondary Amide Carbonyl Resonancesin Carbon-13 NMR RICHARD A. NEWMARK AND JAMESR. HILL Central Research Laboratories,
3M Company, St. Paul, Minnesota 55133
Received April 25,1975 Carbons a to the NH in amides show a 0.1 ppm isotope shift of the CNH compared to the CND. This results in doublets (secondary) or triplets (primary) when amides are dissolved in dimethylsulfoxide, dimethylformamide, or hexamethylphosphoramide containing equimolar exchangeable protons or deuterons. This condition is readily obtained by adding 10% Hz0 and 10 % D20 to the amide solution. Aromatic carbons a or /l to an NH-CO-R substituent are split by 0.1 ppm, whereas those a to CO-NH-R are split by 0.05 ppm under similar conditions. Examplesdescribed include acetamide. benzamide, acetyl glycine, hippnric acid, acetanilide, and the three isomers of amidophenol and acetotoludide. Deuterium substitution has been used to assign carbon-13 spectra. The a, /J’, and y carbons can be assigned by observation of a C-D multiplet, /?-isotope shift, and
long-range coupling to the D, respectively (I-4). Feeney et al. (5) showed that isotope shifts could be observed for peptide carbonyl resonances in aqueous solution. Thus, in 50: 50 H,O : D,O, a secondary amide carbonyl is a doublet due to a 0.1 ppm isotope shift of the CONH compared to the COND. We have extended this technique to amides dissolved in dimethylsulfoxide (DMSO), dimethylformamide (DMF), and hexamethylphosphoramide (HMPA). In all three cases the amide carbonyl splits into a doublet (secondary) or triplet (primary) if H,O and D,O are added at a level to obtain equal amounts of exchangeable protons and deuterium atoms. Furthermore, aliphatic carbons c(to the NH are normally split by 0.1 ppm and aromatic carbons both a and /I to the NH are also split by 0.1 ppm. EXPERIMENTAL
All samples were commercially available materials and used as received. Spectra were run in the Fourier transform mode on a Varian XL-100 NMR spectrometer. Spectra were obtained on approximately 500 mg samples in 2.0 ml DMSO to which 0.5 ml DMSO-d, was added for a lock. For convenience, about 0.25 ml H,O and 0.25 ml D,O was added routinely to most solutions to observe the carbon isotope shifts. The added D20 served as the lock in the DMF or HMPA solutions. Several samples precipitated out of solution when water was added, but redissolved upon heating. Peaks were referenced to internal DMSO (40.48 ppm (6) relative to TMS) because of the low solubility of TMS in DMSO, but chemical shifts are reported relative to TMS. Addition of 20 % water caused a downfield shift between 0. I and 1.5 ppm of most of the peaks, butabout a 2.5 ppm downfield shift of the amide carbonyl. This shift Copyright CC 1976 by Academic Press, Inc. All rights of reproduction in any form reserved Printed in Great Britain
1
2
N E W M A R K AND HILL
was proportional to the amount of water added. Consequently, less water was added to samples with peaks separated by less than 1 ppm in order to follow the differential shifts. All chemical shifts reported in this paper are for DMSO solutions in the absence of added H,O or DzO. Doublets due to possible rotational isomers were not observed in any of the secondary amide spectra. Spectra were obtained on 8K data points at sweep widths of 5000 Hz (1.25 Hz/point) for chemical shifts, at 1000 Hz sweep widths (0.25 Hz/point) for most isotopic splittings, but at 500 Hz (0.125 Hz/point) for primary amide carbonyls and the carbonyls in VI to X. Although peak broadening was usually observable for resonances in secondary amides at 1.25 Hz/point, increased resolution was necessary to measure the splittings or to observe any effect in the primary amides. RESULTS AND DISCUSSION
The acetamide (I) carbonyl is a 1:2: 1 triplet in 50: 50 H,O:D,O at pH 6, split by 0.06 ppm, but the resolution is barely sufficient to resolve the peaks (Fig. 1). The
HpO/ D20
DMF
HMPA
DMSO
FIG. 1. Acetamide (CH3-CO-NH2) carbonyl in water (pH 6), DMF, HMPA, and DMSO. Each organic solvent contained approximately lOoA HZ0 and 12% DzO.
splitting is due to different chemical shifts for the CONH,, CONHD + CONDH, and CONDz species. The line broadening is probably due to proton exchange, which must be slow compared to the peak separation in order to resolve peaks from the different deuterated species. If a&amide is dissolved in DMSO, DMF, or HMPA, a single sharp line is observed for the carbon resonances. However, addition of approximately equal amounts of DzO and H,O results in a triplet for the carbonyl, similar to that observed in water, but with much greater resolution in DMSO and HMPA (Fig. 1). In a typical experiment, about 500 mg acetamide, 0.35 ml D20, and 0.25 ml H,O are added to 2.5 ml solvent. Excess D,O is needed to compensate for the exchangeable protons in the solute. It is only necessary to add DzO equivalent to the number of exchangeable protons in the amide. However, excessH,O plus D20 does not affect the results and vitiates the requirement of weighing the samples and knowing beforehand the structure and number of exchangeable protons. Other primary amides investigated included butyramide (II), benzamide (III), and nicotinamide (1V). In all cases triplets are observed for the amide carbonyl in DMSO plus H,O and D,O, split by 0.06 ppm. There was no evidence for splitting of any other
ISOTOPE SPLITTJNGS IN AMIDES
3
carbon atoms, but broadening of the aromatic Cl carbon in III was apparent when the peak width (1.8 Hz at half-height) was compared to a sample containing no water in which the Cl peak width was only 0.5 Hz. Similar broadening was observed in II, in which the CImethylene carbon linewidth increased from 0.85 to 3.0 Hz. Secondary amides proved more interesting because the splittings are larger and readily observed at additional carbon atoms in the molecule. In acetyl glycine (V) the methylene is split by 0.09 ppm, slightly more than the amide carbonyl splitting of 0.08 ppm (Fig. 2) when H,O and D,O are added to the DMSO solution. This result is reasonable since both carbons are equidistant from the NH (or ND). In addition, the carboxyl, fi to the NH, shows a 0.02 ppm splitting. Similar results are observed in N-acetyl valine methyl ester (VI) and N-acetyl phenylalanine ethyl ester (VII), but no splilting of the /j’carbonyl was observed in hippuric acid (VIII) or N-benzoyl alanine (IX). However, the aromatic Cl fl to the NH split by 0.03 to 0.04 ppm in VIII and IX.
FIG. 2. 13C spectrumof carbonylsand methyleneof acetylglycine(CH,-CO-NH-CH,-CO,H) in DMSO containingequimolarexchangeable protons and deuterons.
Note that the small splittings in N-aliphatic amides are only observable for p quaternary carbons because the linewidth of a CH carbon usually exceeds I .O Hz (0.04 ppm at 25 MHz). The quatemary carbon linewidths in V-IX were under 0.5 Hz. The chemical shifts and isotopic splittings are given in Table 1. In aromatic secondary amides, such as acetanilide (Xl), the aromatic carbon /I to the NH (or ND) is split by 0.1 ppm (Fig. 3). This splitting, as large as the CIsplitting, is twice the ,!3effect observed in the aliphatic systems discussed above. The remaining aromatic protons were readily assigned in acetanilide from their 2: 1 relative intensities and the aromatic substituent chemical shifts (SCS) of the amide then determined by comparing the acetanilide chemical shifts to that of benzene (128.16 ppm) in DMSO. The SCS for NHCOCH3 is: Cl, 11.2; ortho, -9.1; meta, fo.5; para, -5.2 ppm. Similar results are observed in substituted acetanilides, such as the three acetotoluidide (XII-XIV) and amidophenol (XV-XVII) isomers. The chemical shifts and isotopic splittings are given in Table 2. The assignments in these molecules is based on (1) relative peak intensities due to nuclear Overhauser enhancements (NOE) to
VIII’
A B C F GH D CH,-CO-NH-CH(CHMeJ-CO-NH,
X
1
169.3 (d, 0.08)
166.3 (0.08)
171.6 (0.07)
169.3 (0.08)
170.0 (0.08)
170.4 (0.08)
B
57.5
48.3 (0.10)
41.5 (0.10)
53.7 (0.08)
57.6 (0.10)
41.1 (0.09)
C
173.2 (t. 0.06)
174.3
166.8
171.7 (0.02.)
172.4 (0.02)
171.8 (0.02)
D
17.0
60.4
51.7
E
Chemical shifts (isotope splittings),
19.4
36.9
19.1
G
18.0
18.4
H
Cl = 134.1 (0.03), C2,6 = 127.5,
30.2
14.0
30.0
F
in ppm
’ Phenyl ring chemical shifts and Cl isotope splittings: VII, Cl = 137.2, C2, 6 = 129.0, C3, 5 = 128.2, C4 = 126.5; VIII, C3,5=128.5,C4=131.6;IX,Cl = 134.0(0.04), C2, 6= 127.5, C3,5=128,3,C4= 131.4.
B CE C.&-CO-NHCHMe-C02H
IX”
22.6
B C D C&-CO-NH-CH2-C02H D
22.3
D A B C G CH,-CO-NH-CH(CH,C,H,)-COz-CH,
VII’
F
22.3
A B C F GH D CH,-CO-NH-CH(CHMe&C01-CH3
VI E
22.6
A
A B C D CH,-CO-NH-CH,-CO,H E
TABLE ISOTOPESPLITTINGS IN N-ALIPHATIC SECONDARYAMIDES (Me Is METHYL)
V
CHEMICAL SHWTSAND NH/ND
$ r
%
E
P
5
ISOTOPESPLITTINGS IN AMIDES
distinguish the quaternary carbons, (2) calculated chemical shifts based on the SCS of the amide group given above and the SCS of OCH,, OH, and CHB given by Levy and Nelson (7), and (3) the 0.1 ppm doublets induced by the addition of H,O + D,O. The observed shifts differed by less than 3.5 ppm from those calculated for all carbons except C6 of XII in which the difference was 6.0 ppm. p-Acetotoluidide (XIV) was also studied in DMF and HMPA. The resolution of the amide carbonyl was the same in all three solvents, but that of the aromatic CH ortho to the amido was greatest in DMSO and DMSO was used for all further work. Data on o-acetoacetanisidide (XVIII), o-acetophenetidide (XIX) and benzoyl acetanilide (XX) are also reported in Table 2. Although XX was a 2 : 1 mixture of the keto and enol tautomers, addition of water greatly enhanced the keto form. The addition of equimolar amounts of H,O and D,O for the assignment of carbonyl and a and p carbons can be extended to hetero and polynuclear aromatics. In 2acetamidonaphthalene (XXI) the carbonyl (168.6 ppm) and C2 (137.0) quaternary
50 Hz
JLl..I1.J_.i co
Cl
c 3,5
c4
c 2,6
FIG. 3. W spectrum of carbonyl and aromatic carbons of acetanilide (CH3-CO-NH-C6HS) DMSO containing 10% H,O and 1076 D20.
in
carbons and Cl (120.0) and C3 (115.0) methines all split by 0.09 ppm. The remaining methines, not assigned, are at 124.4, 126.3, 127.3, 127.4, and 128.3; C9 is at 133.5 and Cl0 at 129.7 ppm. Id 3-acetamidoquinoline (Xx11) the carbonyl(l69.4) and C4 (121.9) split by 0.09 and 0.08 ppm, respectively. The quaternary C3 (133.1) and C2 (144.6) did not split into doublets, but their linewidths doubled and permitted their unambiguous assignment. The remaining methines, not assigned, are at 127.1, 128.6, and an unresolved 2-carbon peak at 127.7; C9 is at 144.2 and Cl0 is at 128.0 ppm. Assignments in XXI and XXII are based on the chemical shifts of the parent compounds reported by Pugmire et ~1. (8). Doublets were not observed for the carbonyl in cyclic amides or imides, such as pyridone, succinimide, or phthalimide. The carbonyl linewidth was 2.5,0.8, and 1.3 Hz, respectively, in the absence of added water and did not change significantly when equimolar exchangeable protons and deuterons were added. In conclusion, addition of approximately equal amounts of Hz0 and D20 to solutions of amides in DMSO provides a rapid means of assigning the carbons a to the NH and, in many cases, also carbons ,B to the NH.
148.7 (0.07) 149.2 (0.08)
139.3 (0.09) 136.9 (0.09) 126.5 (0.09) 140.4 (0.10) 130.8 (0.09) 127.7 (0.09) 127.3 (0.10) 139.0 (0.09)
168.2 (0.09) 168.1 (0.09) 169.3 (0.08) 168.3 (0.10) 167.8 (0.10) 168.4 (0.09) 165.2 (0.08) 165.4 (0.09)
24.0 24.0 23.1 24.1 23.8 23.9 51.7 48.1
3-CHJ
4-CHJ
2-OH
3-OH
4-OH
2-OCHzCHs
2-OCH3
H
H
H
H
H
H
H
COCHS
COC.~HS
XIII
XIV
xv
XVI
XVII
XVIII
XIX
xx
128.8
111.1
112.1
115.2
157.7
116.2
129.0
137.8
130.3
123.4
124.4
124.2
153.3
110.3
124.9
131.9
123.7
125.9’
123.0
c4
in ppm C5
128.8
120.3
120.1
115.2
129.4
119.2
129.0
128.5
125.0’
128.7
n R chemical shifts: XIX, 203.6 (CO) and 30.2 (CH3); XX, 194.6 (CO), 136.3 (Cl), 128.4 (C2, 6), (128.8 (C3,5), and 133.6 (C4). b X chemical shifts: XII, 17.9; XIII, 21.2; XIV, 20.4; XVIII, 63.8 (CH,) and 14.6 (CH3); XIX, 203.6 (CO) and 55.7 (CH,). c May be interchanged. 1 Not measurable due to overlap with C5.
119.1 (0.10)
121.1 (0.10)
106.3 (0.08)
148.1 (0.08)
119.1 (0.09)
119.6 (0.09)
131.6 (0.09)
136.6 (0.11)
168.2 (0.08)
23.3
128.7
119.2 (0.09)
139.4 (0.10)
168.4 (0.09)
24.0
2-CHa
H
H
c3
c2
Cl
co
Chemical shifts (isotope splittings),
H
CHzR
2
XI
Xb
TABLE ISOTOPESPLITTINGS IN AROMATIC SECONDARYAMIDES, X-A-NHCOCH2R
XII
R”
CHEMICAL SHIFTSAND NH/ND
119.1 (0.10)
121.4 (0.12)
122.0 (0.13)
121.1 (0.10)
109.9 (0.09)
122.5 (0.10)
119.1 (0.09)
116.3 (0.09)
125.06
119.2 (0.09)
C6
E
zi 0 x
z
z
ISOTOPE SPLITTINGS IN AMIDES
7
REFERENCES I. D. DODDRELL AXD I. BURFITT, Aust. /. Chem. 25,2239 (1972). 2. J. B. STOTHERS,C. T. TAN, A. NICKON, F. HUANG, R. SRIDHAR, AND R. WEGLEIN, .I. Amer. Chem. Sot. !M,8581 (1972). 3. A. P. TULL~CH AND M. MAZUREK, J. Chew. Sot., Chem. Commun. 692 (1973). 4. R. H. MARTIN, J. MORIAU, AND N. DEFAY, Tetrahedron 30,179 (1974). 5. J. FEENEY, P. PARTINGTON, AND G. C. K. ROBERTS,J. Magn. Resonance 13,268 (1974). 6. G. C. LEVY AND .I. D. CARUIOLI, .I. Magn. Resonmce 6,143 (1972). 7. G. C. LEVY AND G. L. NELSON, “Carbon-13 Nuclear Magnetic Resonance for Organic Chemists,” Wiley-Interscience, New York, 1972. 8. R. J. PUGMIRE, D. M. GRAhT, M. J. RORTNS,ANU R. K. ROBINS, J. Amer. Chem. Ser. 91,6381(1969).