Remarkable functions of sn-3 hydroxy and phosphocholine groups in 1,2-diacyl-sn-glycerolipids to induce clockwise (+)-helicity around the 1,2-diacyl moiety: Evidence from conformation analysis by 1H NMR spectroscopy

Article abstract of DOI:10.3762/bjoc.13.196

Cell-membrane glycerolipids exhibit a common structural backbone of asymmetric 1,2-diacyl-sn-glycerol bearing polar head groups in the sn-3 position. In this study, the possible effects of sn-3 head groups on the helical conformational property around the 1,2-diacyl moiety in the solution state were examined. ¹H NMR Karplus relation studies were carried out using a series of 1,2-dipalmitoyl-sn-glycerols bearing different sn-3 substituents (namely palmitoyl, benzyl, hydrogen, and phosphates). The ¹H NMR analysis indicated that the helical property around the 1,2-diacyl moiety is considerably affected by these sn-3 substituents. The sn-3 hydroxy group induced a unique helical property, which was considerably dependent on the solvents used. In CDCl₃ solution, three staggered conformers, namely gt(+), gg(?) and tg, were randomized, while in more polar solvents, the gt(+) conformer with (+)-helicity was amplified at the expense of gg(?) and tg conformers. The sn-3 phosphocholine in phosphatidylcholine exhibited a greater effect on the gt(+) conformer, which was independent of the solvents used. From the ¹H NMR analysis, the helical conformational properties around the 1,2-diacyl moiety conformed to a simple empirical rule, which permitted the proposal of a conformational diagram for 1,2-dipalmitoyl-sn-glycerols in the solution states.

Full text of DOI:10.3762/bjoc.13.196

Remarkable functions of sn-3 hydroxy and phosphocholine
groups in 1,2-diacyl-sn-glycerolipids to induce clockwise
(+)-helicity around the 1,2-diacyl moiety: Evidence from
conformation analysis by 1H NMR spectroscopy
Yoshihiro Nishida*1, Mengfei Yuan1, Kazuo Fukuda1, Kaito Fujisawa1, Hirofumi Dohi1
and Hirotaka Uzawa2
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2017, 13, 1999–2009.
1Nanobiology Course in Graduate School of Advanced Integration
Science & Molecular Chirality Research Center, Chiba University,
Matsudo 271-8510, Chiba, Japan and 2Nanomaterials Research
Institute, National Institute of Advanced Industrial Science and
Technology (AIST), 1-1-1 Higashi, Tsukuba 305-8565, Japan
doi:10.3762/bjoc.13.196
Received: 13 February 2017
Accepted: 01 September 2017
Published: 25 September 2017
Email:
This article is part of the Thematic Series "Chemical biology".
Guest Editor: H. B. Bode
Yoshihiro Nishida* - YNishida@faculty.chiba-u.jp
* Corresponding author
© 2017 Nishida et al.; licensee Beilstein-Institut.
License and terms: see end of document.
Keywords:
cell membrane; chirality; conformation; deuterium labeling;
sn-glycerol; glycerolipids; glycerophospholipids; helicity; Karplus
equation; proton NMR spectroscopy; staggered conformers
Abstract
Cell-membrane glycerolipids exhibit a common structural backbone of asymmetric 1,2-diacyl-sn-glycerol bearing polar head
groups in the sn-3 position. In this study, the possible effects of sn-3 head groups on the helical conformational property around the
1,2-diacyl moiety in the solution state were examined. 1H NMR Karplus relation studies were carried out using a series of 1,2-
dipalmitoyl-sn-glycerols bearing different sn-3 substituents (namely palmitoyl, benzyl, hydrogen, and phosphates). The 1H NMR
analysis indicated that the helical property around the 1,2-diacyl moiety is considerably affected by these sn-3 substituents. The
sn-3 hydroxy group induced a unique helical property, which was considerably dependent on the solvents used. In CDCl3 solution,
three staggered conformers, namely gt(+), gg(−) and tg, were randomized, while in more polar solvents, the gt(+) conformer with
(+)-helicity was amplified at the expense of gg(−) and tg conformers. The sn-3 phosphocholine in phosphatidylcholine exhibited a
greater effect on the gt(+) conformer, which was independent of the solvents used. From the 1H NMR analysis, the helical confor-
mational properties around the 1,2-diacyl moiety conformed to a simple empirical rule, which permitted the proposal of a confor-
mational diagram for 1,2-dipalmitoyl-sn-glycerols in the solution states.
Introduction
Glycerophospholipids, constituting the basic elements of cyto- sn-glycerol backbone. Although sn-glycerol is symmetric, an
plasm bilayer membranes, are responsible for several cell func- sn-3 phosphate group makes it chiral with an (R)-configuration
tions [1-3]. These chiral biomolecules have an asymmetric at the sn-2 position [4]. Such molecular chirality is crucial to
1999

Beilstein J. Org. Chem. 2017, 13, 1999–2009.
not only their biological activities but also for their metaphys- groups were examined. As shown in Figure 1, gt(+) is one of
ical properties, as glycerophospholipids comprise elements of the gauche conformers with a right-handed (+)-helicity around
fluid membrane [5] and nanoscale vesicles called liposomes [6]. 1,2-diol, while gg(−) is another gauche conformer with an
antipodal left-handed (−)-helicity. Harada and Nakanishi [19]
In addition, the chiral sn-glycerol backbone is composed of reported the dibenzoate chirality CD methodology, which helps
acyclic polyols that produce several conformers through the free in the analysis of the chirality originating from the disparity be-
rotation about each of the C–C single bonds. For example, the tween these two helical conformers. We have found thereby that
free rotation about the sn-1,2 and sn-2,3 C–C bonds furnishes the 1,2-dibenzoyl moiety favors the right-hand screwed gt(+)
nine conformers by the combination of three staggered conformer over the left-handed one [17]. The gt(+)-preference
rotamers, namely gt (gauche–trans), gg (gauche–gauche) and tg was kept irrespective of the sn-3 substituting groups and the sol-
(trans–gauche, Figure 1). Conformational flexibility often leads vents used. Moreover, a relation in the order as gt(+) > gg(−) >
to the ambiguous characterization of acyclic molecules, thereby tg was maintained. On the other hand, the intensity of exciton
making it difficult to precisely examine their biological activi- couplet CD bands changed remarkably among the 1,2-diben-
ties. This observation is applicable for cell-membrane glyc- zoyl-sn-glycerols [18], indicating that the disparity between
erophospholipids that have been targets in numerous conforma- gt(+) and gg(−) conformers varies widely by influences from
tional studies [7-15].
sn-3 groups.
Cell-membrane glycerophospholipids are known to adopt the Helical properties constitute one of the major factors in deter-
gt(+) and gg(−) conformations around the 1,2-diacyl moiety mining the molecular chirality [20] of not only proteins and
(Figure 1). From X-ray crystallography data, a common struc- nucleic acids but also simpler biomolecules [17-19] such as
ture in which the 1,2-diacyl chains are aligned in parallel is ob- acyclic sn-glycerols and glycerophospholipids [8,21]. In this
served, which adopts either the gt(+) or gg(−) conformer study, the helical properties of four 1,2-dipalmitoyl-sn-glyc-
[7,10,12]. An analogous conformation has been reportedly ob- erols 1−4 (Scheme 1) are examined; these 1,2-dipalmitoyl-sn-
served among α-glycosyl 1,2-diacyl-sn-glycerols in the solu- glycerols are composed of different substituents (X) at the sn-3
tion state [16]. Probably, the two gauche conformers, namely position, and each of them serves as a representative model for
gt(+) and gg(−), are stabilized in a manner so as to permit the 1,2-diacyl-sn-glycerols, as categorized in Scheme 1. Al-
stacking interactions between the 1,2-diacyl chains.
though the exciton chirality CD methodology is not applicable
for these 1,2-diacyl-sn-glycerolipids without an appropriate
In our previously reported circular dichroism (CD) studies UV/CD chromophore, 1H NMR spectroscopy will permit
[17,18], helical conformational properties of a series of 1,2- the precise determination of their helical conformational proper-
dibenzoyl-sn-glycerols bearing different sn-3 substituting ties.
Figure 1: (a) Structures of cell-membrane glycerophospholpids with a common asymmetric 1,2-diacyl-sn-glycerol-3-phosphate structure and
(b) the conformational equilibrium among three staggered conformers, namely gt(+), gg(−) and tg around the 1,2-diacyl moiety.
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Beilstein J. Org. Chem. 2017, 13, 1999–2009.
Scheme 1: The four 1,2-dipalmitoyl-sn-glycerols 1–4 examined in this study.
The result in entry 1 (Table 1) indicates that tripalmitin 1 favors
gt(+) with right-handed (+)-helicity compared to gg(−) with
left-handed helicity (helical disparity = +6%−7%). According to
our previously reported study [18], the disparity, as estimated
from Equation 2, is linear with respect to the magnitude and in-
Results and Discussion
1. Helical conformational properties of
tripalmitin 1 and 3-O-benzyl 1,2-dipalmitoyl-
sn-glycerol (2) in CDCl3 solutions
First, the helical property of tripalmitin 1 (entry 1, Table 1) is tensity of exciton coupling CD bands, indicating that the 1,2-
examined according to a previously reported method [18]. diacyl moiety in 1 exhibits (+)-chirality corresponding to the
Briefly, fractional populations (%) of the three staggered equilibrium imbalance between gt(+) and gg(−) conformers as
conformers [gt(+), gg(−) and tg] are calculated using two indicated by the helicity index (entry 1 in Table 1). The helical
Karplus equations, Equation 1 [22] and Equation 2 [18]. From volume of 1 (76% by Equation 2 and 81% by Equation 1) indi-
the conformer populations (%), the “helicity index” is deter- cates that this glycerolipid favors the two helical conformers in
mined according to the method previously reported by our addition to the antiperiplanar tg conformer (ca. 25% by Equa-
group [18].
tion 2) at equilibrium.
Next, the helical property of chiral 3-O-benzyl derivative 2 is
examined. In our previously reported CD study [17], the inten-
sity of the exciton couplet CD bands for 3-O-benzyl-1,2-diben-
zoyl-sn-glycerol is greater than those of 3-palmitoyl-1,2-diben-
zoyl-sn-glycerol. From the preceding result, the replacement of
the sn-3 palmitoyl group in 1 with a benzyl ether is expected to
enhance the helical property. As can be seen from the result of 2
(1)
(2)
Table 1: 1H NMR data and helical conformational properties of tripalmitin 1 and 3-O-benzyl derivative 2 in the solution state.
Entry Compound Solventa
(head X = )
1H NMR data
δ (ppm)
Populations (%) of staggered conformers
Helicity index in sn-1,2
in sn-1,2 position
position
3J (Hz)
Equation 1
H1proR H1proS gt(+) gg(−)
Equation 2
tg gt(+) gg(−)
Equation 2 (Equation 1)
tg Sign Disparity
(+/−) [gt−gg]%
Volume
[gt+gg]%
1
2
3
1b (palmitoyl) CDCl3
4.15
6.0
4.29
4.4
44
52
53
37
37
36
19
11
11
41
49
50
35
34
33
24
17
17
+
+
+
6 (7)
76 (81)
83 (89)
83 (89)
2 (-CH2Ph)
CDCl3
4.19
6.4
4.34
3.8
15 (15)
17(17)
C/M
(10:1)
4.19
6.5
4.34
3.8
aC/M (v/v) represents the ratios of the mixed solvents CDCl3 (C) and methanol-d4 (M). bDiscrimination between HproR and HproS as well as the acqui-
sition of their 1H NMR data are carried out according to our previously reported studies [23,24] and in the Materials and methods section of this paper.
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Beilstein J. Org. Chem. 2017, 13, 1999–2009.
(Table 1, entries 2 and 3), the helical disparity (+15%, These observations are in good agreement with the 1H NMR
Equation 1 and Equation 2) increases with the introduction of a data of 3 reported by Vilceze and Bittman [29].
benzyl group. This result is in good agreement with our expec-
tation. In addition, the helical volume (%) was increased by
7–8% as compared with that of 1. The 3-O-benzyl group appar-
ently enhances the (+)-chirality around the 1,2-diacyl moiety.
To examine the possible effects of solvents, the helical prop-
erty of 2 is also examined in a mixed solvent containing
ca. 10% methanol-d4 in CDCl3 (C/M 10:1, v/v). The result in
entry 3 (Table 1) indicates that the helical property of 2 is
marginally affected by protic solvents.
2. Helical conformational property of chiral
1,2-dipalmitoyl-sn-glycerol (3) using different
solvents
Next, the helical property of 1,2-dipalmitin 3 with a hydroxy
(OH) group in the sn-3 position is examined. This compound is
selected as a representative model of 1,2-diacyl-sn-glycerols,
which play essential roles in the metabolism and anabolism of
glycerolipids [25-28]. Compound 3 is prepared by the catalytic
hydrogenolysis of benzyl ether 2 (for the synthetic details, see
Supporting Information File 1).
Figure 2: 1H NMR spectra of 1,2-dipalmitin (3) in CDCl3 after partial
isomerization into the 1,3-isomer. (a) The expanded spectrum of 3 in
CDCl3, (b) 3 in a mixed solvent with ca. 10% methanol-d4 in CDCl3
(C/M ca 10:1, v/v). The signal marked with an asterisk * corressponds
to a 1,3-diacyl isomer, which is derived from 3 during storage in a
CDCl3 solution.
From the analysis of the 1H NMR data using Equations 1 and 2,
1,2-dipalmitin 3 in CDCl3 exhibits a very unique helical confor-
The 1H NMR spectrum of 3 in a CDCl3 solution (Figure 2a) mational property. That is, the populations of the gt(+) and
shows a pair of double doublet signals of H1proS (δ 4.32 ppm) gg(−) conformers are almost equal to give a helical disparity of
and H1proR (δ 4.23 ppm), which exhibit a spectral feature simi- around 0% (Table 2, entries 1 and 2). A helical volume of
lar to that of 1 [23]. On the other hand, the signals of H3proR around 75% (Equation 2) is analogous to that observed in 1. In
and H3proS in 3 collapse in a narrow region around δ 3.73 ppm. contrast to the 1H NMR data of 2, those of 3 showed remark-
Table 2: 1H NMR data and helical conformational properties of 1,2-dipalmitin 3 using different solvents.
Entry Compound Solventa
(head X = )
1H NMR data
δ (ppm)
Populations (%) of staggered conformers Helicity index in sn-1,2 position
in sn-1,2 position
3J (Hz)
Equation 1
H1proR H1proS gt(+) gg(−)
Equation 2
tg gt(+) gg(−)
Equation 2 (Equation 1)
tg Sign Disparity
(+/−) [gt−gg]%
Volume
[gt+gg]%
1
2
3
4
5
6
3 (-H)
CDCl3
CDCl3
4.23b
5.6
4.33b
4.5
40
41
48
52
53
55
40
40
38
38
37
38
20
19
13
9
35
37
45
49
50
53
39
39
35
35
34
34
26
24
20
16
16
13
–
–/+
+
−4 (0)
−2 (1)
74 (80)
76 (81)
80 (86)
84 (90)
84 (90)
87 (93)
4.23
5.7
4.32
4.4
C/M (10:1)
C/M (5:1)
C/M (2:1)
4.20
6.2
4.33
4.0
10 (10)
14 (14)
16 (16)
19 (17)
4.19
6.4
4.34
3.7
+
4.19
6.5
4.37
3.7
10
7
+
C/M (2:1) +
D2O
4.18
6.6
4.37
3.5
+
aC/M (v/v) represents the ratios of the mixed solvents CDCl3 (C) and methanol-d4 (M). b1H NMR data from the study reported by Vilceze and Bittman
[29].
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Beilstein J. Org. Chem. 2017, 13, 1999–2009.
able changes in the “mixed solvents” containing methanol-d4 in 33% methanol-d4 (C/M 2:1, v/v), the addition of one aliquot of
CDCl3. With the addition of methanol-d4, the H1proR and D2O to this solution further changes the gt(+) and tg popula-
H1proS signals shift to high and low fields, respectively tions by a few percent (Table 2, entry 6 and Figure 3). More-
(Figure 2b). Simultaneously, the H3 signals shift upfield by over, the H2 signal of 3 shifts downfield by 0.03 ppm in the
0.04 ppm. The shift of these H1 signals increases with an presence of D2O, although this signal marginally changes in the
increase in the content of methanol-d4 in the mixed solvents, mixed solvents without D2O.
while the H3 signals are marginally changed; thereafter, their
positions are maintained at δ 3.69 ppm (Figure 2b). As shown in From the 1H NMR spectra in Figure 2, a part of 3 is isomerized
Table 2, entries 1–6, the change in the chemical shifts is related to 1,3-isomer during storage in solutions. To examine the
to that in the vicinal coupling constants, indicative of a change possible effects from this isomer, the isomerization is promoted
in the dynamic conformations occurring around the 1,2-diacyl up to 50%, and the 1H NMR spectrum of the isomeric mixture
moiety in 3.
is analyzed. This experiment indicates that the presence of the
1,3-isomer marginally affects the 1H NMR signals of 3.
From the analysis of the 1H NMR data using the Karplus equa-
tions (Equation 1 and 2), an equilibrium shift mainly occurs be- As shown in Table 1, entries 2 and 3, the solvents marginally
tween the gt(+) and tg conformers. In the mixed solvents with affect the 1H NMR signals of 2. Clearly, sn-3 OH plays an
high methanol-d4 contents, the population of the gt(+) essential role in the conformational dynamics, as shown above.
conformer seemingly increases at the expense of the tg The dynamic change is probably caused by solvation by metha-
conformer. The population of the gg(−) conformer decreases by nol-d4 and/or D2O around the 3-OH group as well as the in-
several percent after the addition of ca. 10% of methanol-d4 creasing polarity of the mixed solvent. As judged from the
(Table 2, entry 3). Thereafter, the gg(−) population remains chemical shift change in the H3 signals, the solvation is
constant at around 35% irrespective of the solvents.
possibly saturated in the mixed solvent with 10% methanol-d4
(C/M = 10:1). In the solvent containing more than 33% metha-
Because of the shift in the equilibrium from tg to gt(+) in the nol-d4 (C/M = 2:1), the solvation by methanol-d4 might be
mixed solvents with high methanol contents, the helical partly replaced with D2O.
disparity (%) and helical volume (%) increase. With an increase
in the methanol-d4 content to 17% (C/M 5:1), the helical prop- Hamilton et al. [30] employed 13C NMR spectroscopy to exam-
erty of 3 becomes similar to that of 2 (Figure 3). Although this ine the dynamic molecular behavior of 1,2-dilauroyl-sn-glycer-
change seems to be saturated in the mixed solvent containing ol located in liposomes mixed with glycerophospholipids. Their
Figure 3: Fractional populations (%) of the three staggered conformers around the sn-1,2 C–C single bond in 1,2-dipalmitoyl-sn-glycerols 1–3 bear-
ing different substituents (X) at the sn-3 position. Populations (%) are calculated from Equation 2 and each of the populations possibly includes devia-
tions within ±3% by digital resolution (<0.12 Hz) of 1H NMR spectroscopy (500 MHz).
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Beilstein J. Org. Chem. 2017, 13, 1999–2009.
13C NMR analysis revealed that the hydration occurring around
the carbonyl groups in the 1,2-diacyl moiety triggers the dy-
namics of the molecular alignments in liposomes. Probably, an
analogous phenomenon related to the solvation around sn-3 OH
was observed. Thus, solvation is thought to play a key role in
the dynamic conformation change around the 1,2-diacyl moiety.
3. Helical conformational properties of 1,2-
dipalmitoyl-sn-glycero-3-phosphocholine
(4, DPPC) and other glycerophospholipids in
the solution state
Figure 4: Partial 1H NMR spectrum of 4 in a mixture of CDCl3 and
methanol-d4 (C/M = 10:1, v/v).
The current 1H NMR analysis is extended to four 1,2-dipalmi-
toyl-sn-glycerophospholipids (Scheme 2) bearing different ter-
minal groups (Y). Large portions of their 1H NMR data were
collated by Hauser et al. [10]. In our experiment, the 1H NMR In fact, the 1H NMR Karplus analysis indicates that the helical
data of phosphatidylcholine 4 are obtained using the mixed sol- disparity of 4 increases above 30% (Table 3, entries 1 and 2);
vent C/M = 10:1.
the disparity is greater than that observed thus far in previously
reported studies [16-18]. When previously reported 1H NMR
data for 4 are examined [8,10,31], the strong (+)-chirality is in-
dependent of the solvents used (Table 3, entries 1–4). More-
over, the data in entries 5−7 (Table 3) indicate that this prop-
erty is commonly observed in the glycerophospholipids listed in
Scheme 2, indicating that an sn-3 phosphate group plays a key
role. From Table 3, the sn-3 phosphate group can also simulta-
neously increase the helical volume (%). The helical volumes
(%) of 4 using Equation 1 nearly reach the theoretical limit
(100%). This result is in good agreement with the conforma-
tional properties of cell-membrane glycerophospholipids re-
ported previously [10-15]. On the other hand, in our calcula-
tions using Equation 2 as the advanced Karplus equation [18],
the helical volumes of these glycerophospholipids are around
90%, which permits the presence of the tg conformer by
ca. 10%. Note, that the tg conformer is crucial [32,33] because
the antiperiplanar relation is thought to deform lamellar phases
and trigger membrane fusion.
Scheme 2: Structures of glycerophospholipids with a common struc-
tural skeleton of 1,2-dipalmitoyl-sn-glycerol 3-phosphate. Abbrevia-
tions: DPPC =1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPE =
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, DPPS = 1,2-
dipalmitoyl-sn-glycero-3-phospho-L-serine, DPPA = 1,2-dipalmitoyl-sn-
glycerol 3-phosphate.
With respect to the antiperiplanar tg conformer, Hauser et al.
[10] examined the effect of self-assembly using 1,2-dihexanoyl
(C6) homologs of glycerophospholipids. They added these acyl
homologs into D2O at concentrations less than or greater than
As shown in Figure 4, the 1H NMR spectrum of 4 shows a pair the critical micellar concentration. In their 1H NMR spectrosco-
of well-separated double doublet signals of H1proR py analysis, the tg conformer is almost absent under the self-
(δ 4.14 ppm) and H1proS (δ 4.40 ppm). Compared to the other assembled conditions [10]. In addition, in our calculation by
1,2-diacyl-sn-glycerols 1–3, this phospholipid exhibits a higher Equation 2, the helical volume (%) reaches the theoretical limit
vicinal coupling constant to H1proR (3JH1R,H2 = 7.2 Hz) and a (100%), and the helical disparity (%) is greater 40% [18]. Prob-
lower one to H1proS (3JH1S,H2 = 3.5 Hz). In addition, the ably, cell-membrane glycerophospholipid 4 can adopt the
difference in the chemical shift (Δδ = 0.26 ppm) between the unusual rotational mode, where the 1,2-diacyl chains swing be-
H1proR and H1proS signals increases in 4. These observations tween gt(+) and gg(−) conformers. However, such extraordi-
predict that the 1,2-diacyl moiety in 4 exhibits an extremely nary rotation would be possible only when molecules are locat-
unique conformational property.
ed under self-assembled conditions.
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Beilstein J. Org. Chem. 2017, 13, 1999–2009.
Table 3: 1H NMR data of 1,2-dipalmitoyl-sn-glycero-3-phospholipids and their helical conformational properties in solution states.
Entry Compound Solventa
1H NMR
δ (ppm)
3J (Hz)
Populations (%) of staggered conformers
Helicity index in sn-1,2
around sn-1,2
position
Equation 1
H1proR H1proS gt(+) gg(−)
Equtation 2
tg gt(+) gg(−)
Equation 2 (Equation 1)
tg Sign Disparity
(+/−) [gt−gg]%
Volume
[gt+gg]%
1
2
3
4
5
6
7
4 (DPPC)
CDCl3
4.13b
7.3
4.40b
2.9
66
62
61
61
59
64
61
35
32
38
36
36
36
33
−1
6
64
59
59
59
57
63
59
30
27
33
31
31
30
28
6
13
8
+
+
+
+
+
+
+
34 (31)
32 (30)
26 (23)
28 (25)
26 (23)
33 (28)
31 (28)
94 (101)
86 (94)
92 (99)
91 (97)
88 (95)
93 (100)
87 (94)
C/M
(10:1)
4.14
7.2
4.40
3.5
C/M
(2:1)
4.16c
6.9
4.42c
3.1
1
CD3OD
4.18d
7.0
4.42d
3.2
3
10
12
7
DPPEc
DPPSc
DPPAc
C/M
(2:1)
4.18
6.9
4.40
3.4
5
C/M
(4:3)
4.19
7.2
4.43
3.0
0
C/M
(2:1)
4.21
7.1
4.40
3.5
6
13
aC/M (v/v) represents the ratios of the mixed solvents CDCl3 (C) and methanol-d4 (M). b1H NMR data obtained from a database of Spectral Database
for Organic Chemistry (SDBS), No. 16108HSP-45-792 in http://sdbs.db.aist.gojp/sdbs/vgi-bin/direct_frame_top.cgi [31]. c1H NMR data from a paper of
Hauser et al. [10]. d1H NMR data from a paper of Bruzik et al. [8].
4. General trend in the helical conformational
When the gt(+) population is arbitrarily changed between 30%
properties of 1,2-dipalmitoyl-sn-glycerols 1–4
in the solution state
By plotting the helical disparity (%) obtained by Equation 2
against the population (%) of the gt(+) conformers
for glycero1ipids 1–4 examined herein, a linear relation
(y = 1.34x − 50.8, R2 = 0.976) is obtained (Figure 5).
(B1 section) and 75% (C2 section) in these empirical formulae,
a diagram shown in Figure 6 is obtained. The derived diagram
is apparently useful for summarizing the overall helical confor-
mational properties of the four 1,2-dipalmitoyl-sn-glycerols
1–4.
In this diagram, an intersection, denoted by B2, is observed, in-
dicating that the helical disparity becomes 0% when both
gt(+) and gg(−) populations are 38%. At this point, the helical
volume is 76%, and the tg population is 24%. 1,2-Dipalmitin 3
exhibits a similar behavior when dissolved in CDCl3 (Table 2,
entry 2). When methanol-d4 is added to the CDCl3 solution of
3, the gt(+) population increases from 37% up to 50% at the
expense of the gg(−) and tg conformers. The observed change is
From the linearity, we obtain Equation 3 and Equation 4:
(3)
(4)
Equation 3 indicates that the helical disparity (%) increases as a well reproduced in this diagram. Glycerophospholipid 4 shows
function of gt(+) population (%). Equation 4 indicates that the the largest gt(+) population (64%) in the CDCl3 solution
population (%) of the gt(+) conformer increases at the expense (Table 3, entry 1). A similar situation is denoted by a section
of the gg(−) conformer. When the rule of 100 > gt(+) > 0 (%) is C1, where the populations of gt(+), gg(−) and tg are 64%, 29%
applied to Equation 4, the gg(−) population can assume values and 7%, respectively. These values are in good agreement with
in a narrow range between 25% and 51%. At a gg(−) popula- the experimental results (Table 3, entry 1).
tion of 25%, the gt(+) population and helical volume (%) reach
their theoretical limits (75% and 100%, respectively). At a In Table 4, the applicability of Equation 3 and Equation 4 is
gg(−) population of 51%, the gt(+) population reaches 0% evaluated using α-D- and α-L-glucopyranosyl 1,2-dipalmitoyl-
(tg = 49%).
sn-glycerols (Table 4, entries 1–4). The helical conformational
2005

Beilstein J. Org. Chem. 2017, 13, 1999–2009.
Figure 5: Linear relation between the helical disparity (%) and gt(+) population (%) as observed for the helical conformational properties of 1,2-
dipalmitoyl-sn-glycerols 1–4 in the solution state.
Conclusion
In this study, a 1H NMR spectroscopy analysis of 1,2-dipalmi-
toyl-sn-glycerols 1–4 in the solution state was carried out to elu-
cidate their helical conformational properties around the 1,2-
diacyl moiety. In addition, the possible effects from the substit-
uents at the sn-3 position were evaluated. In the current analy-
sis, the chiral 2H-labeled triacylglycerols [23,24] provided a key
basis to discriminate between the H1proR and H1proS signals
(Materials and methods). Throughout this study, each of the
1,2-diplamitoyl-sn-glycerols 1–4 exhibited a unique helical
property, indicating that not only sn-configurations but also
sn-3 substituents govern the helical conformational property
around the 1,2-diacyl moiety. The biological systems in nature
effectively utilize the sn-3 substituents. For example, the sn-3
OH group in 1,2-diacyl-sn-glycerols is essential for the dynamic
conformational behavior, which possibly plays major roles in
their biological functions as transmembrane second messengers
[25-30,34]. The sn-3 phosphocholine in phosphatidylcholine in-
duced strong (+)-chirality regardless of the solvents used, which
should considerably contribute to their functions as activators of
Figure 6: An empirical diagram showing helical conformational proper-
ties around 1,2-diacyl moiety in asymmetric 1,2-dipalmitoyl-sn-glyc-
erols in solution states.
properties of these α-glycolipids are determined by Equation 2 membrane-bound glycoproteins [35-37].
applying the 1H NMR data reported in a preceding paper [16].
The results of the 1H NMR analyses are compared with those The helical conformational properties observed in the four 1,2-
calculated by Equation 4. Entries 1–4 (Table 4) indicate that dipalmitoyl-sn-glycerols (Scheme 1) conformed to an empirical
Equation 4 can reproduce also the helical conformational prop- rule, as shown in Equation 3 and in the diagram shown in
erties of these α-glycolipids.
Figure 6. This rule revealed that the helical disparity (%)
2006

Beilstein J. Org. Chem. 2017, 13, 1999–2009.
Table 4: Helical conformational properties of α-D- and α-L-glucopyranosyl 1,2-dipalmitoyl-sn-glycerols in the solvent mixture of CDCl3 and
methanol-d4 (C/M = 10:1).
Entry
Compounda
(head groups at sn-3)
Resultsb (%) from 1H NMR spectroscopic
analyses by Equation 2
Calculated valuesc (%) with Equation 4
gt
gg
tg
dispariity
volume
gt
gg
tg
disparity
volume
1
2
α-D-Glc
53
53
36
36
11
11
17
17
89
89
53
53
33
33
14
14
20
20
86
86
6-phosphocholine
α-D-Glc
3
4
6-palmitoyl
α-D-Glc
49
55
37
33
14
12
12
22
86
88
49
55
34
32
17
13
15
23
83
87
6-phosphocholine
α-L-Glc
aAbbreviations: α-D- or α-L-Glc = α-D- or α-L-glucopyranoside, b1H NMR data in our preceding study [16] are analyzed with Equation 2; ccalculated
values (%) from Equation 4 by adapting the gt population (%) in the 1H NMR spectroscopy analysis.
linearly changes by the function of gt(+) populations, albeit in Acquisition of the 1H NMR spectral data of H1proR
an allowed range. Probably, the range between B2 and C1 and H1proS signals
sections in the diagram covers the conformational properties of Each of the four glycerolipids 1–4 is dissolved in either CDCl3
most 1,2-diacyl-sn-glyceols in the solution state. The conforma- or the mixed solvents containing methanol-d4 in CDCl3
tional properties in this region can be characterized by the rela- (deuterium content > 99.5%) at ca. 10 mM concentrations.
tion of gt(+) > gg(−) > tg (%), which has been commonly ob- 1H NMR spectroscopy is measured on a JEOL 400 MHz or
served in our preceding studies [16-18].
500 MHz instruments at temperatures between 22–25 °C.
Chemical shifts (δ, ppm) and coupling constants (3J, Hz) of
The 1H NMR spectroscopy analysis was carried out in organic H1proR and H1proS signals are obtained manually with
solvents. It is possible that the conclusions obtained herein 1H NMR spectra expanded in the region between δ 4.0 ppm and
deviate from those examined under physiological conditions. δ 4.5 ppm. The manual process is of high significance for the
For example, glycerophospholipids are located in self-assem- current 1H NMR analysis since a peak top by computer system
bled lamellar structures that show liquid crystalline properties. does not always point at a weighted center correctly.
Plasma membranes comprise glycerophospholipids which
interact with other membrane components such as glyco- The discrimination between H1proR and H1proS signals is
proteins and sterols [38,39]. Moreover, natural glycerolipids are another crucial process. In our former studies [22,23], chiral
composed of heterogeneous acyl chains with different 2H-labelled triacylglycerols were prepared (Scheme 3) and
alkyl lengths and alkenyl –C=C– bonds. Thus, it will be applied for the assignment of these diastereomeric protons,
of high significance in extensional studies to evaluate the namely H1proR and H1proS. The results have shown an empir-
helical conformational properties of 1,2-diacyl-sn-glycerols ical relation between the two H1 signals; the H1proS signals
assuming these heterogeneous situations which may occur in appear downfield from the H1proR signals (δ H1proS >
nature.
δ H1proR ppm) and have lower smaller coupling constants
(3JH1proR,H2 > 3JH1proS,H2 Hz). This rule is maintained among
1,2-diacetyl-, 1,2-dipalmitoyl-, and 1,2-dibenzoyl-sn-glycerols
and substituents at the sn-3 position. The validity of this rule is
Materials and Methods
Model compounds
Tripalmitin 1 was prepared together with chirally deuterated confirmed in a comparative analysis using circular dichroism
sn-glycerols and identified in our former studies [22,23]. 1,2- (CD) spectroscopy [17,18]. The current study applies these rela-
Dipalmitoyl-sn-glycerol (3) and its 3-O-benzyl derivative 2 tions established in our preceding 1H NMR and CD studies.
were prepared in a reported manner [8,29] (for details, see Sup-
porting Information File 1). 1,2-Dipalmitoyl-sn-glycero-3-phos- Calculation of fractional populations (%) of three
phocholine (4 DPPC) was purchased from Tokyo Kasei Co. staggered conformers around the 1,2-diacyl group
Ltd. and used without purification. All the compounds studied with a Karplus relation
here have chemical purities over 95% (1H NMR) except for 3 A general Karplus equation of Haasnoot et al. [40] is extended
which isomerizes into the 1,3-diacyl isomer during storage in into the simultaneous linear equations Equation 1 [22] and
CDCl3 solution.
Equation 2 [18].
2007

Beilstein J. Org. Chem. 2017, 13, 1999–2009.
Supporting Information
Supporting Information File 1
Experimental and copies of spectra.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-13-196-S1.pdf]
Acknowledgements
This work was supported by Grant in Aid from the Japan
Society of the Promotion of Science (KAKENHI 25450146,
16K07711). We thank all staffs at Center for Analytical Instru-
mentation of Chiba University for their technical supports for
NMR and other spectroscopic measurements. The authors
would like to thank Enago (http://www.enago.jp) for the
English language review.
Scheme 3: Chirally 2H-labelled tripalmitins (1S)- and (1R)-1-[2H]-1
[23].
From the vicinal coupling constants (3J Hz) of H1proR and
H1proS signals, the fractional populations (%) of the three stag-
gered conformers are calculated. Equation 1 is a standard equa-
tion, in which the three staggered conformers have the dihedral
angles of ± 60° or 180° around 1,2-diols.
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