Pure&AppI. Chem., Vol. 57, No. 5, pp. 801—821, 1985.

Printed in Great Britain.

© 1985 IUPAC

NMR of carotenoids—new experimental techniques

Gerhard Englert
Central Research Units, F. Hoffmann—La Roche & Co. , Ltd. , CH—4002 Basle
(Switzerland)

Abstract - In continuation of my previous work further 1H—NMR data of

numerous end groups of retinoids and carotenoids are presented. In ad—
dition, the 'C—NflR shifts and assignments of more than 60 end groups
and derivatives thereof are compiled. Several new experimental NMR
techniques which are extremely useful for the elucidation of molecular
structures have been applied to carotenoids of known structure in order
to demonstrate their utility and great power. Included are Double Indor
Difference (DID) for the assignment of signals in complicated 1H—NMR
spectra, the pulse sequence DEPT for the investigation of the multipli—
city of carbon signals, and some recent two—dimensional (2D) NMR tech-
niques. Examples are given for the heteronuclear J—resolved 2D, the
homonuclear (COSY) and heteronuclear chemical shift correlated 2D, and
the 2D-INADEQUATE experiments. The last provides information on the
carbon framework of the molecule. The advantages and disadvantages of
these experimental techniques are discussed with special emphasis on
the spectra of carotenoids.

INTRODUCTION

During the last three years again a number of new or improved NMR techniques has been made
available to the practicing spectroscopist which will no doubt further enhance the import-
ance and efficiency of NMR in solving complicated structural problems. It is the main ob-
jective of this paper to present some selected examples of the application of such new
techniques in order to demonstrate and discuss their relative advantages and drawbacks with
special emphasis on the spectra of carotenoids. In this context the following methods will
be discussed:
— A simple modification of the long—known INDOR experiment called Double Indor Difference
(DID) which significantly simplifies the assignment of proton signals in strongly over-
lapping spectra typical for carotenoids.
— A pulse sequence called DEPT, which is frequently applied now in 13C—NMR spectroscopy
to determine the multiplicity of the signals, i.e. the number of attached protons.
— Several two—dimensional (2D) NMR methods which were found extremely useful in the NMR of
carotenoids. Although these methods are in part highly sophisticated from an experimen-
tal as well as a theoretical point of view, there is no doubt that they will be
increasingly applied in this field in the near future. Due to lack of space and time I
shall be unable, unfortunately, to discuss the physical background of these new methods
and will merely reference the pertinent literature.

NMR DATA OF END GROUPS

Spectroscopic methods such as IN, MS and NMR are well—known, effective tools for the eluci-
dation of the structure of carotenoids. An important starting point in this task, namely
the determination or identification of the structure of the specific end groups, can be
very efficiently achieved by 1H— or 13C—NMR. I previously presented 1H—NMR data on 48
end groups with 84 derivatives including some cis isomers (Ref. 1). In Table 1 are shown
further 1H—NMR shifts for 26 additional end groups and 52 derivatives; thus data are now
compiled for more than 70 different end groups and 136 derivatives.
Since 13C—NMR has also become increasingly applied in the field of retinoids and carote—
noids a similar compilation of '3C chemical shifts is given in Table 2, containing 92 de-
rivatives of 62 end groups. The data of Tables 1 and 2 were extracted from numerous spectra
of retinoids and carotenoids which were all run and assigned in our laboratory. However, I
should like to mention here that part of the data resulted from a very fruitful cooperation

801
802 G. ENGLERT

1
Table 1. Characteristic H-NMR data of some typical end groups of natural and synthetic ca—
rotenoids (solvent CDC13).*

(ic) (5e) (5f) (14a) 2.01

1 04 1.09br.578 1.09 1.14 1.20 1.15

16 22 21

2
1.96 1.51 —i.37 1.56 1.55
1.41 H 2.075
4.02 a 1.995 5.09 2.075 399 2.43c
2.35ct 2.4lcz

(14b) (14c) (14d) (43b)

2.00 2.01 2.00

HO 1.97 AcO A 1.91 AcO 1.96 1.42

2.04 2.05 6.91 6.49
1.41 2.135
''ax 2 09 ''ax ax 2.165 217
4.01 5.04 2.49a 5.06 2.52c
2.4ócx

49a 49b 50 51
2.02 2.01
0.95 2.20 1.92 1.97

1.9
::
1.98
:::21?.;;
5.60 1.73 -2.67 1.78
2.05 2.08

52 53 54a 54b

-1 44 1.04 1.09 1.97 2.04

1 31

HQo1
Hax (JJ6.12 6.16 6.29
1.755 -2.15 1.71 6.98 2.08
1 .85cx

55 56a ss a)
2.04 1.08 CH2OH

:::
0 -1.30 Hax 2.085
4.03 2.39ct

57b b) 57c 58a b) 58b

1 17 0 99 1.93 1 17 1 01 1.93 1.93 1.93
1 01 1 16 0 99 1 21

2.02
1.19 %i93 1.21 —1.65
HO
=1.19 2.02 =1.17
1.785
2.4lcx
3.92 1.635 3.88 1.895 4.92 1.875
2.4Ocx 2.2Ocx 2.30 cx

59a 1.74 59b 60a 60b

1 21 1 14 1 21 1 19 1.74 1.79 1.79
1 23 1 19

HOA AO#H5.19 AcOA £0 H5.19 HOA AO H AcO 1.52

AO H
—1.37 H 1.45 2.04
H '1.49 jj
=1.48 5.11 2.03 5.11
4.02
2.32cx 7,8
j 0 Hz 5.10 1.695
2.3Oa 7,8
0H 4.02 1.515 J
2.28a 7,8
18H 5.11 1.585 J
2.24a 7,8
1.8 Hz
 NMR of carotenoids—new experimental techniques 803

Table 1. Continued

61a b) 61b 62a b) 62b

1.34 1.20 1 81 1.28 1.20 1.81 1.33 1 17 1.72 1.26 1.18 1.72
6.50 \ 5.291 6.49
148c 1.49cx
1.8O8?16.19 1.885 ' 18 1.768 I /6.19
1.885 1
HO'tAO H
1.68 5.07 AcO9A>cT''
2.07
1.51>i%!._::.
HO 0
5.17
AcOT'AO H5.18
Het\1•62 5.07 2.07 1.58
4.24 HeqN1162
5.19 l.87cz 4.24 1.99 5.21 1.95cc
2.118 7,8
j 1.9 Hz 1.9 Hz
2.128 J78 —
0.7 Hz 1 Hz
2.198 J7,8 2.248 7,8

63 64 65 66a
2.06 1.86
1.19
1.18 6.57 2.03 1.18 1.93 --1.98
1.32 0.85 1.35 1.99 1 21 6 42
HO 571 I 659

O
'%.650 1 2.41'—. .''L."
2.37
/ 2:09
7.24
2.43' 6.21
1 24 /1 6.38 6.20 6.39
OH
1.97
1.89 HO
1.49 H 1.55
\oHo 5.92 —2.05

2.88

66b 67a 67b 68 c)

—1.72 1.23 1.91 —1.98 —1.44 1 24 1 93 —1 98 —1.46 1.26 1.94 1.73

>JJ
1.82 1.96
HO HO I 6461
1.21
6 41 6.21 6.39 i24>1"L''L: 2.31 6.14
HO.J
1.26
5 81

—2.38 6.76 I
6.03
4.90
1-177
/1
5.19''OAc
2.11 6.21 6.36 6.74 4.96 /OAc\ \5.94 6.24

5.16 2.07 —2.06

1.98

69a 69b 70 71
2.09
1.43 1.98 1.98 2.08 1.53 1.94 1.98 1.18 1.92 —1.98 1 89 1.99 —2.00
HOJ 6.60 I 669 I 667 I
AcO>J,JJ (5 50 I 1

1 .83
'__(_—_._._\
'J\\
6.46 A45
o \
—6.62 —6.58 6.37
6.17
II 6.29 6.36
1.67 1.691.75

72a 72b 72c 73a

2.231) 2.06 2.24 2.07 2.24 2.07 2.08
2.17 I 666 I
2.15 206 6.53 I 661 I

6.24 6.20
...
-L.•
J,,,J,6. 23 6.19 ._.JL6.26 6.21
-4.66HO 6 53 2.24 H3CO 6.60
3.82 2.32
Ac
6.73 2.25 HO" L2 221
2.191)

73b 73c
2.08 2.08

'%%
6.18 6.19
%%•
6.18 6.20

3.67H3CO'' 2.191)
2.221) 2.36
AcO
2.06
2.21

*Numbering continued from Table 1 of Ref. 1. Numbers in parantheses are supplements to the
previous Table;1)corresponding assignments may be interchanged; br. broad signal, tempera-
ture dependent; n.a. not assigned. Note a): see Ref. 2. Note b): see Ref. 3. Note C): see
Ref. 4.
 NMR of carotenoids—new experimental techniques 805

Table 2. Continued.

16 17a 17b 17c

156 161.0 161.5 161 7 124.4
12 6 I 12.6 / 20.4
27.7 '124 3 124.8 35.8 35.9 27.6
39.5 P.A23.3I 124 6 u—. 27.7/i 25 71
> 3 4
II i33.3t ,
37:61
34 3 P141.21134.4 34:31
134.7 134.8
125.9
186.7 130.8 198.9 130.0 199.5° 130.0
123.2
198.8 130.9
133.1

18a 18b 19a 19b

162.1 160.4 161.4
12.6 12.6 158.0 12.6
36.8 30.7 26.2' 30.5 26 3/ 12.6
. 31 124 4
/\\/123
2 124 4 39 2 28.1 23 0 124.7
141.6
21 124 6

I Ii 142.4t135 4 20.4 143

H3CCO'('140 134.5 HO")ff37 .134.6 6 134.5
H0!114O 134.3
200.4
20.8
0 o\ 128.4
194.0
182.40128.3 168.4 0/
H3CCO'fl13
179.7
0 130.6

20 21 22 23
67.22) 55.2 55.1
28.1 11 8 29.5' 13.1 156.0 12.5
27.1 29 3 / 13.1
/
24.4
34.8 ./$\/129.8I 124.8 79 ./22 ol
1) 34.1
44 7
¼/$\/128.81 124.9
41.1''%.'.'
66.71 ,136.71 130.8 /1 138.8132 3
HOO1'T
65 9 S
HO 134.5 135 35.2 31.5
i24.61 22.8 HO12471 22.7 137CCH3135:0
41 •71 )/21 .4 197.8 137.9 138.0 1;8.8 o 30.2
66.32)

24 25 26 27a
107.9
108.3 23.0 169.6 17.9
159.3 21.0 26.0 / 12 4 143.8
134.8 171 P312.4
\j J119 9 124 9
41 .2 2?? _L,,393 20.7
21 124 8
156.1
/ 38.0
39.8 27.\3
52 2
40.6'/ 28.3 135.4
50 .0
207.4
.0 . 126 S8.lt/ll84
9.9
126:7
201.30 / 9.6
135.1
'fl'14.3
185.30 138.3
118.0 132.3

27b 28 29 30a 111 0

103.0
23.3 168.2 147.7 \ 17.6

39.8
143.5
27?'
\—L's
140 7

187.9
J
¼%. '119
12.5
0 124.4
141.7
34.0 29.1
134.7
91.3 121.0
124 0
31.0 26.)\,J9
44.3 1140.5
88 1117 6
156.Oq 1193.5 1 I 146.51137.0
126
203..2 135.0
I
U 14.3 126.1 32 i1 22 .8 -1041(4144
69.3 Q 133.5
185.30 119.4 123.5 199.5

30b 105.9
30c 110.8 30d 31a
106.0 108.0
147.0 23.0 146.2 17.6 145.8 22.9
30.8 26.\ I 123.7 30.9 26')\
\\ 147.4\\j,,,
39.7 28.3
31.2 26.3"\,L139.3
44
69 31 11934
126.8
36.8
41 5
20 9 707.8 117.3 1140.5 20.8
36.9
41.6 +1139 2
70 91 11115
125.8
145
.2 1
A. iJ.
38.4
119.8
HO14.4 H3CCO 14.5
170.2Q 135.1
H3CCO(146 126.7 H01\14Q
181.5 0 135.8
199.30 134.0 118.3 0I
170.0 0192.60 135.3
193.0
806 GENGLERT

Table 2. Continued.

31b 32 33a 33b

103.6
23.2
144 2
25.3 24.9 19 9 12.8 25:9 21.4 12.8 24.8 25.7 20.8 12 8
29 25
138 0 41.2
'%
1 24 6 44.0 7 43 . 7 \ '120.8l 124.6

i 93.6'tL6:5
123 51.0 ••fl 47.6

H3CO 39.
149:5 I

55.0 180.2 0 138.0

119. 5'
14.5
216. 48.3\ 201.133.9
55.3 148.1
141 5
70 4
HO159.0 202.91468
40.
34.1
170.6
H3CCO 4258.5202.2\
140.6

147.0
133.7

21.3 73.6

34a 34b 35a 35b

25.7 26.2
12.8 12.8 18.9
25.0 19.5 24.7 21.1 18.1 23.8 21.3 12.8 24.3 21.2 12 8
45.8 124.6 21.1 46.7
51. 4b4L::. 47. HO
81.8 H3CCO
70.2. \41.1 43.0I120.8
21.3 73 41.0
HO4 46.7\ 206.1\134.1
58.8 147.2 H3CC9
41.9\ °\1472
\ 134.0
58.6
30.8
32.O334.1 170.3 83
28.4 58.3
2.3133.9
147.0
170.9 58.1 146.7
o 201.7

36 117.5 103.3 37a 37b 38a

146.1
I
/14.0
125.7 21 7 I 12 1
147.4 12.1
22 2142.7
/ 12 4
134.1 154.6 1 A28 21
45 5 1128.5?
._I• /120.3 I 36.3 . . 126.0
126:9
37.2
21.1 682r'2.3 132.6 I I 124.3 124.1 199 0 8.31 138.0
23.0
29 0135.5 23.9 .
141.3
H3CC2921)
0 132.5?
25.5 o.ç7[io.o
38.7
I 72.2
45.

38b 39 40 41a
143 8 168.3 25.1 70.3 25.1
/ 12 4 14.6 1 12 4 29 6 / 12 9 70.412 9
148.41 /120 7 126.9 35.4\/1252( 125.2
479 472
2110' I 126.61137.7 630
199 52./,I1139.6 5 jl7/.o 140 8 64.O\t132.2
HO
00 128.81
155.5
25.4 46.3
41.8A 67.0 \134.5
137.2
•/2o.1 \134.3
68.8 137.5
20.3

41b 42 153.3 43 154.1 44

26.8 71.1
24.8 / 12 8 31.3111118.8
I / 29 11)! 120.0 1526 120.5
35.2 138.7 /12.6
33.7 29:01)! /12.6 30.7 26
$/1J//,,24.7 34 2 30.61)//13.4
47.s2\.k::..
21.3
H3CCO)
66./K21.4 \134.1
170.7
35.6 64.5
138.4
47:,/>
HO
67.9/
/
4/ 87.2
88.4
6771

.
468
HO' 'A\O
2)
I /\127.2

31.41)\86.9
87.8
21.3
H39COV'.?O
68.61

170.3 o 46.11)127.1
I
\ 137.4
88.3
47.5 28.2 87.3

45 46 47 48a
151 8 119.5 119 7 103.1 117 9 103.4
124.3 98.6 1
29.7 28.61;//12.6
31.5' 14 2
28J/
30.0\
2)
6
21.0 68 9
1124.2

126.4
34. 7$-
46.7
#- 5.4) %
28.0
0
36.6
30.5

[\135.1

H39CdvQol37.6?
67 4
ZZ.34.0
HO'4281"/99 200.8
21.2
H3CCO" 7OO'\133.0 HO
64.91 1189.1
22.5
119.1

170.3 o 46.61'T7.4 88.8

200.7
I 1) 25.9 170.4 40.91 19.7 41.51
26.4 0 29.3 137.3
 NMR of carotenoids—new experimental techniques 807

Table 2. Continued.

48b 48c 48d 49

17 0 12.7
20 41 1265 1248

20.9 135.5?
127.5
126.5

18.6 13.0 12.9

136
26.2 131.2
131.21
134.9 137.5
137.1

55 56 57 58
203.7 119.9 131.5 198.0
18 8 17 2 12.9 12.7 12.7 12.8
0 19.3
/ 12.9 1270 11284 125 ol 124 0 I
24.5"\ . H
193.1
69.3/ 1126.4/ 2084 0 156.11141.1 1 147.61140.4 1 135.7
36.8 136.01 29.71I147.8r:
46.5
134.0
39.4 133.5 135.3 12.9 133.2 142.2
137.9 136.6

59 60 61 62
9.6 131.9 141.2 9.7 12.8 168.3 12.8 12.8 27.4 12.8
128 0 122.4
.. 60 3 123.1 HO 29.8 125 0
H HCHOC..'L.'L 72.7
194.1 u t148.4 27 0
/ 14. 4 1138.9 1
01 01261 144.0
138.01131
137.3 137.413.0 94..8t1137.8
136.6 146.0
134.9
1;3.0 I 28.8 138.5
25.8
132.0

1Corresponding assignments may be reversed.

808 GENGLERT

with the groups of Dr. Britton (Liverpool), Prof. Eugster (Zurich), Prof. Jensen (Trond—
heim), Prof. Kleinig (Freiburg i.Br.) and Dr. Pfander (Berne), who kindly provided quite a
number of interesting samples.
The assignments given in Table 1 were supported by the comparison of a great number of
reference spectra, by decoupling and DID experiments (see example below) and by numerous
homonuclear Overhauser difference experiments, a few examples of which were discussed
previously (Ref. 1). Similarly, the assignments in Table 2 were based on the application of
shift reagents, measurement of CW—offset and 1H—coupled spectra (see Refs. 5, 6), and in
several cases on the application of 13C—1H chemical shift correlated 2D experiments
(see example below). In both Tables the chemical shifts of the first protons or carbons of
the side—chains were included because they give additional information on the specific end
group. Since these data were partly extracted from the spectra of retinoids and related
compounds with only 4 or 5 conjugated double bonds the shifts of protons H(lO) and H(ll) as
well as of carbons C(9) to C(ll) had to be corrected in some of these cases by simple addi—
tivity rules in order to take into account the influence of strongly anisotropic terminal
groups such as carbonyls or carboxyls.
The assignments were given as complete as possible and were carefully checked for
consistency within the entire set of spectra available in our laboratory.
I hope that this extended chemical shift compilation will be of practical use in the iden—
tification of retinoids and carotenoids.

NEW EXPERIMENTAL TECHNIQUES

Double Indor Difference — Recently, the utility of the interproton Overhauser difference
technique for the assignment of proton signals of carotenoids and the solution of stereo—
chemical problems was strongly emphasized (Ref. 1). In this technique a sufficiently isola—
ted signal (singlet or multiplet) is strongly irradiated for a few seconds by a second
radiofrequency. After switching off the irradiation, the observation pulse is applied and
the free induction decay (FID) is accumulated as usual. If a second FID is subtracted with
the pre—irradiation set outside the absorption range the difference spectrum contains only
those signals which have altered intensities in the two FID's. Signals with increased (po—
sitive) intensities are caused by the Overhauser effect and reflect a close spatial proxi—
mity of the two protons.
The long—known Fourier INDOR difference experiment (Ref. 7) uses a similar experimental
set—up, however, only a short ( 0.5 sec) and very weak (j H2 1 Hz) pre—irradia—
tion is applied to one selected component of a multiplet. This causes a change of the popu—
lation of these two energy levels which is reflected by a change in intensity of all other
transitions which start or end on one of the two previous levels. In the INDOR difference
spectrum only these so—called connected transitions within a coupled spin system appear as
positive or negative signals reflecting an increase or decrease of the excess population of
the corresponding transitions. The observation of these difference signals allows one to
locate approximately the signals of coupled protons. The main advantage of INDOR is the
fact that in contrast to decoupling or Overhauser experiments the perturbation is
selectively applied only in a very narrow frequency range of the order of 1 Hz. This
strongly extends the applicability of the method in crowded spectra.
Kessler and co—workers recently proposed a simple modification of the INDOR difference ex-
periment called Double Indor ifference (DID; Ref. 8). A first modification is that the
difference FID is obtained from two on—resonance experiments, namely by irradiating two
suitably chosen components of a given multiplet, e.g. the first and second component of a
doublet, or the first and fourth component of a doublet of doublets. The second modifica-
tion is that instead of positive and negative signals, only positive ones are produced by
applying a magnitude calculation to the final difference spectrum. The main advantage of
DID is the fact that in simple, coupled spin systems as frequently observed in carotenoids
the true multiplet structure of the coupled protons appears in the difference spectrum. As
will be shown, this method greatly simplifies the assignment of protons in crowded spectra
of carotenoids.
As an example shown in Fig. 1, DID is used to perform or confirm some assignments in the
olefinic part of the 400 MHz spectrum of (5'R)—cryptocapsone (Ref. 6). As usual the assign-
ments of the protons near the end groups are easily and unambiguously performed by compari-
son with the data of the two symmetrical compounds 13—carotene and (5R,5'R)—capsorubone (see
Tables 1 here and in Ref. 1). These comprise H(7), H(8), H(lO), H(l4), H(l4') and H(8).
Additional assignments can be obtained by a few selected DID experiments. The first experi-
ment, namely pre—irradiation of H(8') near 7.4 ppm as indicated by the two arrows, results
in the appearance only of the doublet for H(7') near 6.5 ppm. Irradiation of the first and
fourth component of the doublet of doublets near 6.7 ppm gives rise to a further doublet of
doublets and the broadened doublet of H(l4), thus the two former multiplets must be
assigned to H(l5) and H(l5') as it is confirmed by the fourth and sixth experiment. In the
third experiment the signals of H(ll), H(l2) and H(lO) are linked. From these and the fol—
 NMR of carotenoids—new experimental techniques 809

19
%1O
18'
4'ci

LL 20
18

2.0 1.5
10 ' 11' 12

48
1L_ 7'
_________

-----
A
141
151

—--------------

11

14 1.99 ppm
H20
14' 10.-
H1:+H20,

7:0 6.5 6.lppm

Fig. 1. Top: High and low—field sections of the 400 MHz 1H—NMR of
(5'R)—cryptocapsone in CDC13. Below: Sequence of DID experiments with
pre—irradiation of 0.5 sec duration at positions marked by arrows;
*
impurity.
810 G.ENGLERT

lowing experiments It Is been that by a series of DID experiments all but one sub—group of
three coupled protons can be easily identified. These are the three very strongly coupled
protons H(lO'), H(ll) and H(12') all absorbing near 6.6 ppm as revealed by the integral.
It is worthwile mentioning here that DID can also be successfully applied to detect con—
nectivities between the protons of the in—chain methyl groups and their olefinic coupling
partners although the corresponding 4—bond coupling is only ca. 1.3 Hz. This is demonstra—
ted in the last two experiments of Fig. 1. A difference spectrum obtained by pre—irradia—
tion of the methyl signal at 1.992 ppm (H(20)) just before and after the peak maximum
(difference ca. 1 Hz) gives rise to the appearance of the signal of H(l4). Similarly, pre—
irradiation of the 6—proton singlet near 1.98 ppm reveals connectivities of the correspond—
ing methyl protons H(l9) and H(20') with H(lO) and H(l4') leaving the assignment of the
signal of the remaining in—chain methyl protons H(l9') at slightly higher field.
In conclusion it is seen that DID is a very selective method for locating the signals of
coupled spin systems in strongly overlapping spectra as those of carotenoids. Assuming nor—
mal sample quantities of a few mg a fully automated sequence of experiments can be per—
formed in less than one hour (see example of Fig. 1). If longer accumulation times are
acceptable, experiments in the pg—range are also possible. We have found that a few
selected DID experiments which can be performed even on older instruments can replace, in
certain cases, a much more time—consuming and complicated 2D experiment.

g! — The assignment of 13C—NMR signals to the corresponding carbons in complicated

molecules relies heavily on the correct determination of their multiplicities reflecting
the number of directly attached protons. This information was previously derived from
proton coupled spectra or from CW—offset decoupled spectra. The former method gives addi—
tional information on the proton—carbon couplings J, the latter on relative chemical
shifts of the corresponding protons if the magnitude of the couplings are roughly pre—
dictable. However, it is frequently difficult, even at high magnetic fields, to identify
the different multiplets in crowded spectra and hence reliably to derive the individual
multiplicities. In addition, a considerable loss of intensity is implied due to the split—
ting of the carbon signals into several components; thus much longer acquisition times are
needed.
In the last few years a number of multi—pulse sequences has been proposed for the rapid de—
termination of the multiplicities of 13C signals (Refs. 9, 10). For almost two years we
have routinely applied the pulse sequence called DEPT (Distortionless Enhancement by
Polarization Transfer; Ref. 10) which has several advantages compared to previous methods
(Ref. 11). In this technique which uses two carbon—13 and three proton pulses, polarization
is transferred from protons to carbons, i.e. the excess population between the C levels
and hence the intensity of these transitions is enhanced considerably, thus reducing the
acquisition time. The separating delay between the pulses is set equal to l/(2J). This
one—bond 13C—1H coupling may vary in carotenoids between ca. 130 and 170 Hz. A further
advantage of DEPT is that the results do not critically depend on the value used for J
and therefore a typical average value of ca. 140 Hz is suitable. If the usual broad—band
proton decoupling is switched on during the accumulation of the FID, the intensity of the
decoupled 13C singlets depends on the magnitude of the last proton pulse in the pulse
sequence, called theta pulse P0, and on the number of protons attached to the carbons.
This dependence is caused by the different rotation frequencies and phases of the trans-
verse magnetizations of the different types of carbon—l3 due to the couplings to the
attached protons (Ref. 10).
A typical example is shown in Fig. 2 wherein trace A represents the normal l3C NMR spec-
trum of astaxanthin diacetate, the assignments of which are known (Ref. 1). Also presented
is a set of typical DEPT spectra obtained for 90° (trace B), 45° (trace C) and 135° theta
pulses (trace D). The first observation is that in all these DEPT spectra the quaternary
carbon signals (designated by s in trace A) including the solvent signals are eliminated.
In addition, in trace B only GI signals are retained, in trace C all prtonated carbons are
seen with positive intensities, in trace D CH and CH3 are positive, CH2 negative. Thus,
the clear—cut differentiation of all the signals according to their multiplicities is
possible even in very crowded spectra. The normal l3C—NMR was obtained from 128 scans,
the DEPT spectra from 256 scans with a relaxation delay of 4 sec. It is clearly seen that
only a small fraction of this number of scans is actually needed for a signal—to—noise
ratio comparable to the normal spectrum.
Useful for daily routine applications is the fact that, in general,a single experiment,
namely the one in trace D (P0 = 135°), is sufficient to give all needed information, if
the CH and CH3 signals can be differentiated by consideration of their chemical shifts at
low and high field, respectively.
However, if all three DEPT spectra are available one can calculate linear combinations of
the three spectra yielding so—called edited DEPT spectra presenting separated sub—spectra
for all three types of carbon as shown in Fig. 3. Thus, trace C of Fig. 3 with the CH2
sub—spectrum was obtained by substracting trace D from C of Fig. 2 with a suitable scaling
factor. Similarly, the CH3 sub—spectrum was calculated from all three spectra of Fig. 2.
 NMR of carotenoids—new experimental techniques 811

0
0

C P0=45 CH, CH2, CH3I

1LIi -

D P0=135°

CH2j

150 100 50 0 ppm

Fig. 2. A: 100 MHz 13C—NMR of astaxanthin diacetate (60 mg in 0.4 ml

CDC13, 128 scans, 45° flip angle); S quaternary carbons. B,C,D: DEPT
spectra obtained for proton pulses P0 = 90°, 45° and 135°; 256
scans, 4 sec relaxation delay, J 130 Hz.

13
A

LJ ii
1fliJt IH
10 10 50 0 ppm

C
CH2

D
Cm3

10 13 50 0 ppm

Fig. 3. 100 MHz 13C—NMR and edited DEPT spectra of astaxanthin diacetate.
812 G. ENGLERT

It should be mentioned that the DEPT sequence can be used also for the measurement of
proton—coupled 13C—spectra if the decoupler remains switched off during the accumulation
of the FID. Several other pulse sequences have been published including those for the
generation of l3C sub—spectra of quaternary carbons only (Ref. 12). This might be useful
in cases where quaternary and other carbons have exactly identical chemical shifts.

Two—dimensional NMR (2D) — The basic idea of 2D NMR was first proposed in 1971 by J. Jeener
(Ref. 13). In more recent years this promising technique was mainly developed and extended
by the research groups of R. Ernst and K. Wüthrich in Zurich and R. Freeman in Oxford (for
an introduction see Ref. 14).
In a normal lD Fourier experiment a short rf—pulse is applied to the spin system. The
resulting x,y—magnetization rotating around the magnetic field axis z is detected as a
function of time, the acquisition time t2, and stored in the computer memory. By a
Fourier transform with respect to t2 the intensities and frequencies of the normal 1D
spectrum are calculated by the computer within a few seconds. The greater the number N of
data points used in the experiment. the better is the digital resolution of the spectrum
measured in Hz per data point.
In a 2D experiment two or more pulses are applied for the preparation of the spin system
during a first time domain, called evolution time t1, before the free induction decay FID
is acquired during t2. In addition, the timing of the pulses and hence the evolution
period t1 is systematically incremented, thus N subsequent FID's are now acquired instead
of only one. In practice N is chosen mostly between ca. 50 and 1000 depending on the type
of experiment and the desired resolution. It is clearly evident that if the number of ex—
perirnents and the digital resolution is not kept reasonably small the storage requirement
N * N of the data matrix as well as the acquisition and processing times may be excessive.
The processing consists of two consecutive steps. First, N subsequent Fourier transforms of
all the rows of the data matrix are performed with respect to t2 yielding a set of N
spectra. Since t was systematically incremented the amplitudes and phases of a specific
signal in subsequent rows can also vary as a function of t1, i.e. the signal along a
specific column may be modulated e.g. by spin couplings, either directly (J—resolved 2D) or
indirectly due to transfer of polarization between coupled spins (çrrelated 2D
pectroscop, COSY). The frequencies f1 involved in the amplitude variation of the dif—
ferent columns are calculated by subsequent Fourier transforms with respect to ti. In
general, a magnitude calculation of the data is performed to obtain only positive intensi—
ties. The result, namely the intensities as a function of the two frequencies f1 and
f2, is called a 2D spectrum. It can be presented as an impressive stacked plot as shown
in Fig. 4. Here, a section of a proton—proton chemical shift correlated 2D spectrum is
shown. The two axes cover the range between S = 6.1 and 6.8 ppm and thus contain the che-
mical shift range of all olefinic protons except one (H(lO')). The normal spectrum is con-
centrated along the diagonal running from top—right to bottom—left. In this homonuclear
COSY spectrum, which is symmetrical with respect to the diagonal, cross—peaks at coordi-
nates 6A in f1 and 6B in f2 (as well as 6B In f1 and A in 2)
reflect the fact that the two protons at chemical shifts 6A and 6B are spin—coupled
to each other. Stronger couplings generally give more intense cross—peaks. This spectrum
will be discussed later in more detail.
A stacked plot is not normally suited for extracting the information contained in a 2D
spectrum, but it gives a good picture of the different intensities and the achieved signal—
to—noise ratio. The information is much better revealed by so—called contour plots where
the intensities are shown as closed contour levels of equal intensity, starting from a
suitably chosen minimum level above the noise and incrementing normally by a factor of 2
from level to level.

J—resolved 2D — In this relatively simple 2D technique only the chemical shift information
is retained in the horizontal f2—axis, the coupling information solely along the perpen-
dicular f1—axis. It appears as if the spin multiplets were simply rotated by 900 into a
second frequency axis. The technique can be applied to homonuclear (mostly proton—proton)
and heteronuclear (mostly carbon—proton) systems. Two examples of the former were already
discussed previously (Ref. 1). It is felt that the homonuclear variant will not find many
useful applications in the field of carotenoids, because severe difficulties are en-
countered in strongly coupled spin systems.
More useful in specific cases is the heteronuclear J—resolved 2D experiment, an example of
which is shown in Fig. 5. Here, the contour plot of the 13C—1H J—resolved 2D of ethyl
8'—apo--13—caroten—8'—oate is presented on bottom. Since the width of the horizontal
f2—axis is more than 18 kHz according to the wide range of 13C chemical shifts, the
single contour lines representing the different intensities levels are, unfortunately, not
resolved in this picture. The perpendicular f1—axis containing the coupling information
covers a range of 290 Hz. Although 128 experiments from 128 different t1 increments were
performed the digital resolution in f1 (after zero—filling to 256 points) was only 1.1 Hz
per data point. This is more than sufficient clearly to distinguish the different singlets,
doublets, triplets and quartets in the contour plot but is insufficient for the detection
of very small long—range couplings.
 NMR ofcarotenoids—new experimental techniques 813

20

18

6.1

6.7 ppm

I
6.7 6.5 6.3 6.1 ppm

Fig. 4. Stacked plot (section between 6.1 and 6.8 ppm) of the sym—
metrized proton—proton chemical shift correlated 2D (400 MHz, COSY—90,
N—type) of ethyl 8—apo—13—caroten—8'—oate (22 mg in 0.4 ml CDC13).
Experimental data: Spectral width W 601.7 x 601.7 Hz, 2K x 470, zero—
filled to 1K, 470 t1 increments, 0.6 and 0.6 Hz per data point, po-
larization transfer delay 0.1 sec. relaxation delay 2.3 sec, 16 scans
and 1 dummy scan per increment, sine—bell windows, magnitude spectrum.
9.5 h acquisition time, 1024 K file size. A corresponding contour plot
is shown in Fig. 7.

The actual measurement of the resolved couplings and the visual presentation of the dif-
ferent multiplets can be achieved in the following way. First, a horizontal projection of
the 2D spectrum is calculated (see top of Fig. 5). It looks very similar to the usual
broad—band decoupled 13C spectrum but its digital resolution is. in general. necessarily
smaller (here 9 instead of ca. 1 Hz per point). Since the maxima of the horizontal projec-
tion represent the chemical shift of the different carbons, the corresponding multiplets
can be plotted as so—called J—cross—sections as normal intensity plots along the f1—axis.
All J—cross—sections (except COO) which were resolved are shown in Fig. 6 together with the
assignments, the intensity scaling factor, and the chemical shifts. In carotenoids the
1J—values of olefinic carbons are in a rather narrow range between ca. 149 and 155 Hz
and, therefore, they are not very useful for the purpose of assignment. However, the number
of long—range couplings or the width of the unresolved components of a multiplet can be
rather characteristic. Thus C(7). c( 11) and the corresponding c( 11 ) usually appear as
sharp doublets with high intensities. They can already be distinguished from other carbons
by inspection of the horizontal projection as seen in Fig. 5.
A further point should be noted here. If a singlet and a doublet appear at (almost) the
same chemical shift they can be completely separated in a J—resolved spectrum as is seen
from two examples in Fig. 6. The intensities and the magnitude of the measured J—coup—
ling allows a safe differentiation from a single triplet signal.
It is evident from this example that any meaningful application of this special 2D method
will be essentially limited to those cases where the number and magnitude of the carbon—
proton couplings are of importance for the solution of a structural problem and the normal
1H—coupled 13C—spectrum is too crowded for a clear—cut differentiation.
814 G. ENGLERT

150 100 50 0 ppm

f.i

f
—100 $

I liii III III I t ii

0 IIIt I III I

I 1111 111111
I i ii
•
I
100 I
Hz

150 100 50 0 ppm

613c

Fig. 5. Ethyl 8'—apo—13—caroten—8'—oate (100 mg in 0.5 ml CDC13):

Contour plot and horizontal projection of the 13C-1H J-resolved 2D
spectrum. Experimental conditions: V 18518 x 290 Hz, 4K x 128 data
points, zero—filled to 256 points, 128 t1—increments, digital
resolution 9 and 1.1 Hz, 3.5 sec. relaxation delay, sine—bell windows,
magnitude spectrum, file size 512 K, ca. 14 h acquisition time.

C ,C 12.78 ppm 14.35 19.31 ppm

130.76
C7 C10
CH3
C2Q,C, A 150.4H5 129.33

___ ___
A

J/2
100
C18

C1
0 -100 Hz
21.71 C16

34.28 C2
28.96 C4

39.73 OCH2
33.13

60.34
C14

C9
.
x2
•_•__•_-.

131.92 C3

—,---
136.35 C12
.

135.26 C14,

136.98 C8
_ .,
135.70

137.68

...T

Fig. 6. J—cross—sections of the J—resolved 2D spectrum of Fig. 5. The

assignments are given together with the chemical shifts and the inten-
:9JJL _
sity scaling factors. The COO—signal is not shown.
 NMR of carotenoids—new experimental techniques 815

Homonuclear chemical shift correlated 2D spectroscopy (COSY) — In this experiment a spin

coupling between two protons is revealed by the existence of cross—peaks in the 2D plot
(Ref. 14). Since it is actually the homonuclear spin coupling which causes the cross—peaks
the method is also called homoscalar correlated 2D. It is often possible to derive the full
sequence of coupled protons even in a complicated compound, thus providing important
information on its molecular structure. As will be shown the coupling pathway or connec—
tivity of protons can even in such cases be detected where the couplings are very small.
The previous 400 MHz COSY spectrum of Fig. 4 from ethyl 8'—apo—13—caroten—8'—oate is n
presented in Fig. 7 as a contour plot which is much more appropriate for the deduction of
the connectivities. Since the olefinic part is highly crowded a very good digital resolu—
tion of only 0.6 Hz per point was used. The normal 1D spectrum and the assignments are
given in the upper 1D spectrum. The 2D contour plot is symmetrical with respect to the dia—
gonal on which the normal spectrum is seen. Those peaks which are very close to the dia—
gonal are caused by transfer of magnetization between the transitions of the same proton.
Thus, the doublets appear as 2x2, all doublets of doublets as 4x4 signals. More distant
from the diagonal are found the cross—peaks indicating that the two corresponding protons
are coupled to each other. Thus, the two off—diagonal peaks at coordinates 6A'6B
and (6B'6A) prove the presence of a J—coupling between proton A at (6A'6A)
and B at (6B'6B)• One of the advantages of a COSY in the case of highly crowded
signals near the diagonal is the fact that the multiplet structures can be derived by
inspection of the cross—peaks which are often sufficiently separated.
If a good signal—to—noise ratio has been achieved in a COSY spectrum by application of a
longer accumulation time it is often possible to detect very weak cross—peaks due to long—
range couplings. As will be seen later in this COSY spectrum a long—range coupling of the
order of 0.1 Hz or less extending over 7 intervening bonds can be detected although such
small couplings can not normally be seen or resolved in the lD spectrum. In Fig. 7 all
long—range connectivities were designated in the upper—left triangle, the 3—bond connecti—
vities in the lower—right triangle of the symmetrical 2D spectrum.
The procedure for revealing the sequences of coupled protons can start e.g. from the isola—
ted signal of H(lO') at lowest field. Horizontal and vertical lines lead to the strong
cross—peaks with H(ll'), the diagonal signal of which is now found at the intersections of
the horizontal and vertical lines with the diagonal. A further much weaker cross—peak due
to 4J10. ,12' identifies the signal of H(l2'). It can also be located from the much
stronger, direct cross—peaks between H(ll') and H(l2') via 3J11.12.. Further connecti—
vities are visible between H(l5), H(l5') and H(l4), between H(ll), H(12) and H(lO), between
H(15) and H(14') etc. Some further long—range connectivities are marked in the upper—left
triangle of Fig. 7. Interestingly, a cross—peak H(15)/H(ll') is clearly visible,
corresponding to a 7—bond coupling between these two protons (see also Fig. 8). From the 2D
spectrum of Fig. 7 the whole sequence of protons between H(lO) and H(lO') is deduced. The
connectivity between H(7) and H(8) can also be found in an expanded plot of the right upper
corner (not shown) although their chemical shifts are very close to each other.
A very convenient way of visualizing the different connectivities is to plot selected rows
(or columns) of the 2D data. Three selected rows chosen at 6.59 ppm through H(l2'), at 6.74
ppm through H(l5') and 7.30 ppm through H(lO') (see arrows in Fig. 7) are presented as
examples in Fig. 8. The form and intensities of the diagonal peak and its cross—peaks are
clearly seen as well as the above—mentioned long—range connectivity.
In a completely analogous way the connectivities between the protons of in—chain methyl
groups and olefinic protons are detectable if the experiment is run with a spectral width
covering the whole spectrum. However, in this case the digital resolution has to be reduced
in order to avoid an excessive size of the data matrix. Despite several drawbacks of this
technique, namely long accumulation and processing times and large data storage require-
ments, it is strongly recommended for the solution of complex structural problems if
simpler methods such as DID cannot provide all necessary information.

Heteronuclear chemical shift correlated 2D — This technique (Ref. 15) which is mostly
applied to carbon—l3 and protons is even more useful in practice since it provides connec—
tivities between 13C and 1H chemical shifts. The two portions of a heteronuclear 2D
from (5'R)—cryptocapsone are presented in Fig. 9 and 10, showing separately the olefinic—
olefinic and the aliphatic—aliphatic sections of the same experiment. The proton spectrum
runs along the vertical f1—axis, the carbon spectrum along the horizontal f2—axis. For
convenience, both lD spectra of the two nuclei and the horizontal projection of the 2D
spectrum are also presented. Since the delay for polarization transfer between the two
nuclei was chosen to enhance connectivities by one—bond couplings of 140 Hz, cross—peaks
are seen only between carbons and their directly attached protons. Correspondingly, in the
horizontal projections only signals of protonated carbons are detectable. Thus, starting
from a known proton signal in f1 one obtains in a simple and mostly unambiguous way the
assignment of the corresponding carbon in f2. In a strongly crowded proton spectrum it
frequently suffices to know the relative order of the chemical shifts to assign the carbon
signals. Conversely, if the carbon assignment is known the chemical shift of the coupled
proton is deduced.
816 G. ENGLERT

12' 12
11 10
1cJ111
10' 15 ' 14'
ñ
rLl
rrL1r11 14
I HJJ L
7,4 65 ppm

Fig. 7. Top: Olefinic range of the resolution enhanced 400 MHz

1H—NMR spectrum of ethyl 8'—apo—13-caroten—8'—oate. Bottom: Contour
plot of the symmetrized COSY—90 spectrum. Experimental conditions see
Fig. 4; * folded signal. Arrows indicate selected rows shown in Fig. 8.
 NMR of carotenoids—new experimental techniques 817

20

II

12' 12
iif'i
71
10

I I I I J I I

70 6.5 6.0

10' 12
14 l2 ,11'

6.59 ppm________ 11'

3J
15,15 I
15

19L 3
Jiol ,11I

7.30_..!JJL
10 ,12'

7.0 6.5 6.0 ppm

Fig. 8. Top: Olefinic section of the 400 MHz 1H—NMR spectrum of ethyl
8'—apo—B—caroten—8'—oate. Bottom: Selective horizontal rows of the COSY
spectrum of Fig. 7 plotted at 6.59, 6.74 and 7.30 ppm (see arrows in
Fig. 7) indicating all cross—peaks of H12', H15 and H10',
respectively.

In the case of ambiguities important aid is given by additional plots of horizontal rows at
selected proton chemical shifts, revealing the position of the connected carbon signal, or
conversely, the plot of selected columns yielding the multiplet structure of the signal of
the proton coupled to a specific carbon.
A very useful supplement to the one-bond coupling experiment is obtained by a subsequent
experiment tuned to connectivities mediated by two— and three—bond couplings which are
frequently between 5 and 10 Hz. This gives further relevant information since connectivi—
ties between more distant nuclei, including the quaternary carbons, are involved. As an
example, the aliphatic—aliphatic portion of the 13C—1H correlated 2D of the same com-
pound is shown in Fig. 11. The delay for polarization transfer was chosen to enhance cross—
peaks caused by long—range couplings of ca. 8 Hz. However, some of the cross—peaks caused
by the much larger one—bond couplings can be still detectable.
For purposes of interpretation it is important to note that only two— or three—bond carbon—
proton couplings are sufficiently large to cause long—range cross—peaks. Applying this rule
and the results from Fig. 10, all the cross—peaks of Fig. 11 can be readily assigned. It is
evident that not only the assignments of proton and carbon signals can be deduced or con-
firmed, but also important information on the molecular structure is revealed by the con—
nectivities caused by the two— and three—bond couplings. Thus, as an example, in this
section of the 2D plot, protons H(l6) and H(l7) of the 13—ring have cross—peaks with C(2),
C(l) and C(l7), C(l6); protons H(l8) only with C(4) and C(l8). In the 5—membered ring, on
the other hand, protons H(l6') and H(l7') have cross—peaks with C(5'), c(2') and C(l'),
protons H(l8') with C(S), C(4') and C(l').
Further relevant information of this type can be deduced from the other sections of the
heterocorrelated 2D, namely the aliphatic—olefinic and olefinic—olefinic sections which are
not shown here. This includes e.g. cross—peaks between H(l9) and C(8), C(9) and C(lO) as
well as between H(20) and C(l2), C(l3) and C(l4).
818 G. ENGLERT

19 20

t16 I
18
150 120 ppm

10 io ppm

.11

14
6.5
10' 11' 12 ii

'7

7.5

150 140 130 120 ppm

Fig. 9. A: 100 MHz 13C—NMR spectrum (olefinic section) of (5'R)—cryp—

tocapsone (45 mg in 0.5 ml CDC13); *impurity. B: Horizontal projec-
tion of the 13C—1H correlated 2D spectrum. C: 400 MHz 1H—NMR
spectrum measured with the decoupler coil. Bottom: 13C—1H correla-
ted 2D spectrum (olefinic—olefinic section); Experimental conditions:
W 21740 x 3334 Hz, 4K x 256, zero—filling to 512, 10.6 and 6.5 Hz per
data point, 2.5 sec relaxation delay, polarization transfer delay 3.6
msec (J 140 Hz), 48 scans, 1 dummy scan, sine—bell windows,
magnitude spectrum, 1024 K file size, 9 h acquisition.

In conclusion it is found that the heteronuclear chemical shift correlated 2D is extremely

useful for the assignment of signals and the deduction of structural information on the
nuclear framework and hence for the elucidation of molecular structures. In practice, we
normally acquire both the one—bond and the long—range type of spectra in two subsequent ex-
periments normally during an overnight run from about 20 to 50 mg of sample.

INADEQUATE—2D — This technique, also called Carbon—Carbon Connectivity (CCC), is a 2D

variant of the former iD—INADEQUATE experiment which was used to measure 13C—13C
couplings constants in natural abundance (Ref. 16).
In normal 13C—NMR the spectrum from molecules containing 13C at ca. 1 % natural
abundance is acquired. The probability of having two adjacent 13C nuclei in a molecule is
about 100 times less. Since two neighbouring carbons are spin—coupled the resulting two
doublets have roughly 200 times smaller intensities. Despite this extreme loss of intensity
 NMR of carotenoids—new experimental techniques 819

A
I II j
Oppm
16 19
19 20

C
BILJJIILLJ
0

16 1

19 2

ppm

Fig. 10. Aliphatic—aliphatic section, continued from Fig. 9.

it is possible in favourable cases to acquire an INADEQUATE spectrum with a reasonable

signal—to—noise if very long acquisition times can be accepted and sufficiently large
sample quantities of several 100 mg are available and soluble in ca. 2 ml of solvent. In
the 1D version, all the pairs of doublets are superimposed along the only frequency axis
f2. Furthermore, the much more intense singlet signals of molecules with only one
carbon—l3 are partly superimposed because they are mostly incompletely suppressed by appli-
cation of complicated phase cycles.
In the 2D—INADEQUATE the different AB or AX spectra are separated along the second
frequency axis f1 according to their so—called double quantum frequency. This is the sum
of their frequency distances from the transmitter frequency which is normally placed in the
middle of the f2—axis.
An example, using a quite useful experimental variant called refocused 2D—INADEQUATE (Ref.
17), is shown in Fig. 12. It was obtained from 640 mg of astaxanthin diacetate with an
acquisition time of 64 hours. This long acquisition time is mainly caused by the fact that
a relaxation delay of (at least) 2.5 sec had to be interleaved after each scan because the
13c spin lattice relaxation time T1 of the unprotonated carbons of this compound can
exceed 3 sec (Ref. 1). An average value for J of 55 Hz was adopted for this measurement
although the actual coupling constants vary between roughly 35 and 65 Hz. The pairs of
doublets are symmetrically displaced with respect to the diagonal. This simplifies the
identification of the different connectivities and, in addition, artifacts having no sym-
metrical counter—parts can be removed by symmetrization. Unfortunately, the doublets are
very poorly resolved in this picture since the total range of f2 covers 20.8 kHz. On the
diagonal some stronger residual artifacts are seen which are caused by imperfectly
suppressed singlets of slowly relaxing carbon—l3 nuclei.
The connectivities of the carbon—carbon skeleton are revealed by the procedure shown in
Fig. 12. Thus, starting e.g. from the quaternary carbon C(l), its neighbours C(l7), C(l6),
C(2) and C(6) are clearly revealed by the existence of the corresponding pairs of doublets.
Similarly, C(2) is connected to C(3), C(3) to C(4), C(4) to C(S), C(S) to C(6), and so
forth. The CCC is interrupted at C(9)/C(lO), which have almost identical chemical shifts,
820 G. ENGLERT

A
i
50
[ sj ill 11

20 ppm

1920 16

16 17

19 20'

50 40 30 20 ppm

Fig. 11. (5'R)—cryptocapsone, aliphatic sections of the 100 MHz

NMR (A), the horizontal projection (B) of the 13C—1H correlated 2D
(bottom—right). C: aliphatic section of the 400 MHz 1H—NMR. Polariza-
tion transfer delay chosen to enhance long—range couplings 1J
8 Hz. Other experimental conditions as in Figs. 9 and 10.

and at the intervening oxygen to the substituent at C(3). The position of the acetyl group
is, however, evidenced by consideration of the chemical shift. This example clearly demon-
strates the great power of this 2D method to reveal in a fully unambiguous way not only all
the assignments but also the full sequence of carbons in the molecular structure including
ring size, position of double bonds and of methyl groups. It should be mentioned that expe-
riments with reduced sample quantities or reduced accumulation time of the order of 24 h
can also be quite useful. However, in this case only connectivities between protonated car-
bons with short relaxation times are likely to be detected as e.g. the olefinic carbons
with T1s of 0.3 sec or less.

CONCLUSION

From these selected examples it is clearly evident that NMR spectroscopy has again made
some steps forward increasing its power and utility for the solution of structural problems
in the field of carotenoids. Although some of the recent 2D techniques have become
increasingly complex and sophisticated, there is no doubt that they will be more and more
applied even on a routine basis. All the experiments described here were performed in a
fully automated manner under computer control on a Bruker WM 400 FT NMR spectrometer using
standard software.

Acknowledgement —I am indebted to several colleagues who provided the compounds

used in this study. I should like to specifically mention Drs. K. Bernhard, H.
Mayer, A. Rüttimann, E. Widmer, R. Zell and Mrs. K. Schiedt. Thanks are also due
to Mr. W. Grunauer for his skilful technical assistance.

REFERENCES

1. G. Englert, in Carotenoid Chemistry & Biochemistry, G. Britton and T.W. Goodwin,

Editors, Pergamon Press, 107—134 (1982).
2. E. Märki—Fischer, P.—A. Bütikofer, R. Buchecker, and C.H. Eugster, Helv. Chim. Acta
, 1175—1182 (1983).
 NMR of carotenoids—new experimental techniques 821

16 17 19 20
11 15

HCCO 18
0
13C 100MHz

190 150 100 5b Ôppm

I
10416
Hz

Fig. 12. Top: 100 MHz 13C—NMR spectrum of astaxanthin diacetate.

Bottom: Contour plot of the symmetrized, refocused 2D—INADEQUATE. The
carbon connectivities are indicated starting from C(l). Experimental
conditions: 640 mg in 1.8 ml CDC13, W 20.8 x 10.4 kHz, 4K x 256,
zero—filling to 2K, 10.2 and 5.1 Hz digital resolution, relaxation
delay 2.5 sec, echo—delay 4.5 msec (CC = 55 Hz), '3C pulses 90°
(14 psec), 180° and 120°, Lorentz—to—Gauss filtering, LB = —4,
GB = 0.1, LB1 = —4, GB1 = 0.1, 320 scans per 4—increment, 4096 K
data file, acquisition time 64 h.

References (contd.)
3. E. Märki—Fischer, R. Buchecker, C.H. Eugster, G. Englert, K. Noack, and M. Vecchi,
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