Chemistry and Physics of Lipids, 54 (1990) 99--113 Elsevier Scientific Publishers Ireland Ltd.

99

13C- and 31p-NMR studies of dioctanoylphosphatidylcholine and dioctanoylthiophosphatidylcholine

1H-,

Mufeed M. Basti and Laurine A. LaPlanche Department of Chemistry, Northern Illinois University, DeKalb, IL 60155 (U.S.A.)

(Received September 18th, 1989; revision received and accepted December 27th, 1989) Coupling constants and chemical shifts were measured for dioctanoylphosphatidylcholineand its thio analogue in a CDCI/ CD3OD solvent mixture. Replacing the bridging oxygen atom of the CH--CH2--O--P portion of the phosphatidylcholine molecule with a sulfur atom affects chemical shifts and coupling constants in the glycerolbackbone portion of the molecule as well as in the choline head group region. Preferred conformations about selected bonds in the phospholipids were determined from the vicinal ~H-~H,3~P-IHand 3~P-~3Ccoupling constants. A reduction of the 3~p T2. (effective spin-spin relaxation time) for the thio analogue, as well as changes in the relative chemical shifts of t3C nuclei in the acyl chains, suggest a somewhat greater degree of aggregation for the thio analogue. The quadrupolar coupling constant tJ(~4N-~3C) for the choline methyls of either analogue, however, indicates that aggregation of these phospholipids in the CDCI3/CD3ODsolvent mixture is not significant. Differences in conformation between dioctanoylphosphatidylcholineand its thio analogue may be responsible for their differences in chemical and physical properties. Keywords: IH-NMR; 13C-NMR;3sP-NMR;phosphatidylcholines; thiophosphatidylcholines; phospholipids.

Introduction Phospholipids are an important constituent of biological membranes. N M R has played a key role in elucidating fine details o f the structure o f several o f these phospholipids in solution, including the determination o f rotamer populations about many o f the dihedral angles in the glycerol and choline regions of the molecule. These angles determine the three-dimensional conformations allowed to phospholipids; a knowledge o f the 3D structure is believed to be important in understanding the biological function o f phospholipids in cell membranes. Knowing that dioctanoylphosphatidylcholine (dioctanoyl PC) and its thio analogue have different chemical and physical properties [1--3],

Correspondence to: Dr. Laurine A. LaPlanche.

we set out to determine the conformational differences, if any, between the two molecules. Using 'H, ~3C and 3~p one and two-dimensional NMR experiments, chemical shift assignments were made for each molecule and 'H-~H, 3tpJH, and 3~P-~3C coupling constants were determined. Karplus-like relationships were used to derive populations about bonds from theoretical and observed coupling constants. The 3j(PXCH) and 3J(PXCC) coupling constants (X = O or S) were found to be quite different for the oxygen and sulfur analogues, possibly due to a difference in populations o f the antiperiplanar vs. gauche rotamers about the C H - - C H 2 - - X - - P dihedral angle. The critical micelle concentration (CMC) in aqueous solution is an example of a difference in physical properties between dioctanoyl P C (CMC = 250 /aM [1]) and thiodioctanoyl PC (CMC = 56 /aM [2]); the latter has a greater

0009-3084/90/$03.50 © 1990Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland

100

[51.

NMR tubes were rinsed with EDTA to eliminate paramagnetic metal ions. Chloroform/methanol solutions were prepared in a glove bag and transferred to NMR tubes under a dry nitrogen atmosphere to reduce exchange of the methanold 4 with atmospheric moisture. The NMR tube was thoroughly degassed and sealed under a vacuum. ~H-NMR spectra were recorded at 20°C at either 200.132 MHz using the IBM Instruments NR/200 spectrometer at Northern Illinois University or at 500 MHz using a Bruker Instruments, Inc. AM-500 spectrometer at the NMRFAM facility at the University of Wisconsin at Madison. z3C- and 3~P-NMR spectra were recorded at 50.32 MHz or at 81.01 MHz, respectively, on the IBM Instruments spectrometer. Using the IBM Instruments spectrometer, 16K data points were collected in the quadrature mode, with typical sweep widths of 3300 Hz for 1H, 12,500 Hz for ~3C and 5000 Hz for 3~p. Tetramethylsilane was the internal reference for IH while CDCI 3 (at 77.38 ppm) was the reference for ~3C studies in the CDC1/CD3OD solvent mixture. For 3~p-NMR spectra, a melting point capillary filled with an 85°70 phosphoric acid solution served as the external reference. Bruker microprograms were used for the COSY [6,7] and heteronuclear (IH-~3C) shift correlation experiments [8]. For the latter experiments, 0.600-M solutions of the dioctanoylphosphatidylcholine in CDCI/CD3OD were used. All other solutions were 0.100 M in phospholipid.

Experimental

Results and discussion

L-a-(dioctanoyl PC) was purchased from Avanti Polar Lipids, Inc. and used without further purification. Dr. William Snyder synthesized the racemic thio analogue using published methods [3]. Thin layer chromatography was used to assure the purity of the phospholipids. Both were stored at - 2 0 ° C until used. The deuterated chloroform used as a solvent was purchased from MSD Isotopes Inc. and kept dry over Linde type 4A molecular sieves. Deuterated methanol was purchased in one gram ampoules from Wilmad Glass Inc. Before filling,

Chemical shift assignments

tendency to self-associate in aqueous solution. A difference in chemical properties between the two phospholipids is found when comparing their rate of hydrolysis by the enzyme phospholipase C. The absolute rate of hydrolysis of dioctanoyl PC is at least 100 times greater than that of its thio analogue [3]. The deuterated solvent system chosen for this study was a 2:1 by volume solvent mixture composed of chloroform and methanol. Chloroform is an excellent solvent for phospholipids. The solvent mixture will be more polar than chloroform due to the higher dielectric constant and hydrogen bonding ability of methanol relative to chloroform and will therefore reduce the degree of aggregation due to self-association of the phosphatidylcholine molecules; this in turn results in narrower NMR resonance peaks. Phospholipids are monomeric in pure methanol [4]; the form of the aggregates present in the solvent mixture is unknown, although it is believed to be "small micelles" consisting of 60--70 molecules [5]. In aqueous solution the dioctanoyl PC micelle contains i>470 monomers [1]. Analysis of the vicinal coupling constants of dihexanoylphosphatidylcholine in D20 above and below the CMC, as well as in pure CD3OD, support the hypothesis that the conformation of the lipid is quite similar under different experimental conditions of concentration and solvent; the conclusion was that intramolecular forces are primarily responsible for determination of conformation

~H-NMR spectra The chemical shift assignments for dioctanoyl PC in CDC13/CDaOD (2:1 v/v) solution, which were confirmed by proton spin decoupling and COSY experiments, are quite similar to those reported by Hauser et al. [5], for dipalmitoylphosphatidylcholine (DPPC) in the same solvent. The assignments are given in Table I; the NMR spectra at 200 MHz are shown in Fig. 2. The Gt and G 2 protons in the glycerol portion

I01 (ELK2)

(C)

(DI)

o

CH2

0

CH2

CH2

--

- -

(B)

(A)

(CH2) 4

CH3

(C~12) 4 - -

CH3

(D 2)

(F)

HC

- -

I

CH2

S

P--

(glU2)

CH2 - -

O

4-

CH2 ~

ell 2

(HH')

(II')

+ N(CH3) 3 (J)

(a)

hl O

t

II

C3--O31-- C h2 ~0

04 ~ J

C2

O21-- ~

gl

fl

e

d

C

C '

C

" C ~

g2

f2 C

C

C

c

b

a

C ~

C--

C

C _ _ C ~ C _ _ C +

k

~ al

S11~ =2

~ O" a3

012~ C l l ~ 1 a4

C 1 2 - - N(CH3) 3 • n

a5

(b) Fig. 1. Molecular structure of dioctanoyl PC (sulfur analogue shown). (a) Capital letters refer to tH nuclei. The sets E/E~ and G / G 2 refer to methylene protons which are magnetically non-equivalent, while H / H ' and I / I ' are chemical shift equivalent. (b) Small letters refer to ~3C nuclei. Greek letters refer to dihedral angles which are defined by nuclei having subscripted numbers (see Table III).

are magnetically equivalent for both dioctanoyl PC (this work) and DPPC [5] in this solvent mixture. (They are, however, non-equivalent by 0.02 ppm for dioctanoyl PC in pure CDCI3. ) Most of the protons for dioctanoyl PC and its thio analogue resonate within 0.02 ppm of each other facilitating initial ~H assignments for the thio analogue. These assignments were confirmed using the COSY experiment. The major differences between dioctanoyl PC and its thio analogue are protons GI and G: which, due to their proximity to the sulfur atom, are shifted upfield by approximately 1 ppm in the thio analogue. In addition, G t and G 2 are magnetically non-equivalent in the thio analogue with a chemical shift difference of 0.09 ppm (Table I). For the magnetically non-equivalent E I and E 2 protons, which are four bonds removed from the

sulfur atom, the chemical shift difference is 0.27 ppm for the oxygen analogue and 0.39 ppm for the sulfur analogue. The chemical shift difference (AdH) between the D methylene protons of the acyl chains increases from 0.01 ppm in dioctanoyl PC to 0.03 ppm in the thio analogue. This may simply result from the difference in chemical shielding for the protons of the two analogues.

13C-NMR spectra Preliminary 1ac chemical shift assignments for dioctanoyl PC in CDC13/CD3OD (2:1 v/v) were made by comparison with those of Burns and Roberts who assigned ~3C chemical shifts for micellar dioctanoyl PC in aqueous solution [9]. All eight carbons of the acyl chains are resolved at 50.32 MHz in dioctanoyl PC as well as in the

102

thio analogue (Fig. 3). '3C assignments for each analogue (Table I) were verified using 2D ~3C-tH heteronuclear shift correlation experiments. The only uncertainty in assignments is for peaks d and e which are separated by only 0.1 ppm, since they are correlated to protons which overlap at 1.28 ppm in the heteronuclear correlation diagram. The carbonyl h~ and h 2 resonances of dioctanoyl PC are expected to have the same relative assignment as for the analogous t3C nuclei in dihexanoylphosphatidylcholine (DHPC) in CDC13 or CD3OD; in both solvents, h~ (on the acyl chain attached to C3) resonates downfield of h 2 [10]. Interestingly, the two t3C= O resonances cross over as a function of DHPC concentration in D20. At low DHPC concentration (monomeric), the assignment is the same as in the organic solvents. At higher DHPC concentration (micellar) the assignment reverses. Due to the variability of the a3C=O chemical shifts (a range of over 2 ppm for DHPC) we regard our assignment for hj and h 2 in the thio analogue as tentative. A long-range heteronuclear correlation experiment may allow h~ and h 2 t o be assigned with certainty. The largest differences in assignments between the two analogues were found in the glycerol backbone region, with t3C peak k, being directly bound to the sulfur atom, shifted upfield by 32.3 ppm (Table I) in the thio analogue. The other two z3C resonances most affected by the sulfur substitution were i and j, both experiencing downfield shifts of 1.75 ppm and 1.04 ppm, respectively. These shifts are consistent with heteroatom 13C substitutions [11]. Just as the sulfur substitution caused a greater chemical shift difference in the D methylene protons of the acyl chains, the aiiphatic laC nuclei of the chains also become more non-equivalent. If Ad c is the chemical shift difference between the same t3C nucleus in the C 3 vs. the C2 acyl chain, one may see that for the ~3C(f) nuclei, A6 c = 0.03 ppm in dioctanoyl PC and 0.12 ppm in the thio analogue (Table I). Similarly, for the ~3C(g) nuclei, A6 c = 0.15 ppm in dioctanoyl PC and 0.25 ppm in the thio analogue. The quantity A6 c is related to the state of aggregation in short-chain phospholipids with A6 c generally increasing for

TABLE I Muitinuclear chemical shifts for the oxygen and thio analogues of dioctanoylphosphatidylcholine. Nucleus

Label

~H"

A B C DI D2 E] E2 F G~ Gz H I J a b c d d fl f2 g~ g2 hi h2 i j k 1 m n

~;Cc

3~pd

Oxygen analogue

Thio analogue

0.83 1.28 1.60 (2.32) (2.33) 4.15 4.42 5.22 4.00b 4.0(P 4.24 3.61 3.22 13.68 22.44 31.56 28.93 28.81 24.75 24.78 33.96 34. l 1 173.87 173.50 62.60 70.40 63.54 58.96 66.52 54.00 - 0.33

0.85 1.28 1.59 (2.30) (2.33) 4.04 4.43 5.25 2.94 3.03 4.26 3.65 3.24 14.00 22.67 31.75 29.16 29.01 24.96 25.08 34.22 34.47 074.07) (173.87) 64.35 71.44 31.24 58.94 66.73 54.46 + 18.9

.Concentration of the oxygen analogue is 0.100 M; the sulfur analogue is 0.100 M; both in CDCI3/CD3OD solution (2:1 v/v). Proton sets labeled (E I, E 2) and ((3: Gz) refer to magneticaBy non-equivalent protons on the same carbon atom. Protons labeled Da, D 2 refer to protons on the acyl chain attached to glycerol carbon C 3 or C 2, respectively (see Fig. 1). Assignment of Dt and D, may be reversed. ~H chemical shifts are referenced to TMS. ¢I'he Or, G, protons for the oxygen analogue are magneticaily equivalent in this solution, but non-equivaient by 0.02 ppm in CDCI 3. ~Concentration is 0.100 M phosphatidylcholine in C D C I / CD3OD solution (2:1 v/v). ~3C chemical shifts are referenced to CDCI 3 at 77.38 ppm. Carbons labeled f : g~, and hi refer to nuclei on the acyl chain attached to C r while those labeled f2, g2, and h~ refer to the acyl chain attached to C 2 (see Fig. 1). Assignment of h~ and h 2 may be reversed in the thio analogue. dConcentration is 0.044 M pbosphatidylcholine in CDCI 3 solution. Chemical shift is relative to 85% HjPO, external reference. (3tp in dioctanoyl PC resonates upfieid of ~P in the thio analogue.)

103

(a)

G

I

A

CD3OH

A

i

D2'DI

F

4

5

I .........

I,

3

2

•

I

. . . .

i

D2,D 1 (b)

CD3OH G2,G 1

H

:2'D1 F

. . . . .

E2 H

C

'

t . . . . . . . . .

I

5

4

. . . . . . . . .

L

I . . . . . . . . .

I . . . . . .

3

2

,,I . . . . . . . . . 1

I 0

PPM Fig. 2. 200 Mi-iz ~H spectra of an 0.100-M solution of (a) dioctanoyl PC and (b) its sulfur analogue in CDCI/CD3OD (2:1 v/v).

104

CD3OD

(a)

n

CDCI 3

j

k

d,e b

g2gl f2fl

i

m

,,,,I

....

80

l,,,d,,,J

70

....

....

I ....

I ....

50

60

I ....

l,,,,l~,,,Iu

40

,~I

30

....

I~

20

CD~ OD (b)

d,e

CDCI3

•

i

3

, = ~ I

a

n

....

g2gl

i

[ ....

70

I ....

I ....

60

k

I ....

I ....

50

I ....

l~,,

40

I ....

I ....

30

[,,,LI

....

I,,

20

PPM

Fig. 3. 50.32 MHz '3C spectra of an 0.100-M solution of (a) dioctanoyl PC and Co) its sulfur analogue in CDCI/CD3OD (2:1

v/v).

the acyl chain ~3C nuclei in the micellar state as compared with the monomer [9]. Thus the greater A6 c for 13C(f) and ~3C(g) in the thio analogue as compared with dioctanoyl PC may indicate a greater degree of aggregation. Ad c will,

however, also be affected by the difference in magnetic shielding of the phosphate vs. the thiophosphate moiety in the two analogues. The present work provides evidence that the conformations of the two molecules differ, spe-

105

cifically with regard to the rotarner populations about the dihedral angles a~ and 02 (Fig. 1). Such a change in conformation would change the average distance between the acyl chains and the phosphate or thiophosphate groups, resulting in a change in chemical shielding for the acyl m3C nuclei. This could be the reason for the greater downfield shift of ~3C(f2), ~3C(g2) and ~3C(h2) (relative to ~C(f~), ~3C(g~) and l~C(hm)) in the thio analogue resulting in a larger A6 c for nuclei f and g of this molecule.

TABLE II ~H-~H, 3~P-IH, 31P-t3C and t4N-~3C coupling constants in 0.100-M solutions of the oxygen and sulfur analogues of dioctanoyl PC in CDCI~/CD~OD (2:1 v/v)'.

1H.mH 2J(E;E 2)

3J(EI*F) 3J(E2-F) 3J(Gt'F) 3J(G2-F) 2J(GI_G2 ) 3J(H-I) = 3J(I"I'-I') ~J(H-I') = 3J(H'-I) 2J(H-H') 2J(I-I')

Oxygen analogue

Sulfur analogue

12.14 6.81 3.12 4.23 4.23 b 2.38 6.76 16.40 16.47

12.13 6.99 2.94 6.25 6.25 13.72

31p_aH 3J(31P-Gi) 3J(3JP-G2) 3J(31P-H) 3J(3~P-H') 31p_t3C 3J(~P-j) 2J(31p-k) 2J(31P-i) Jjpip_m)

c c c

6.99 6.99 5.15 5.15

14.72 14.72 4.78 4.78

7.89 5.26 4.74

2.21 3.67 5.15

c

c

~(N.JH

3J()'N-H') J,N.13C tJ(l'N-n)

3.24

3.68

3.68

•See Fig. 1 for letter code for IH and 13C resonances. bGl, G 2 are chemical-shift equivalent in the oxygen analogue of dioctanoyl PC in CDCI3/CD3OD (2:1 v/v). ~Not resolved.

31P-NMR spectra

The 3~p nucleus, being directly bound to the sulfur or oxygen atom in these phosphatidylcholines, resonates d o w n f i e l d by 19.2 ppm in the thio analogue relative to dioctanoyl PC (Table I). The 3~p chemical shift is affected by a combination of factors including substituent electronegativity, bond angles, dihedral angles, and phosphorus p and d orbital occupation [12,13]. All of these factors may contribute to the observed 19.2 ppm 31p chemical shift difference between the two analogues. The appearance of the 3~p resonance is also quite different in the two molecules. For the oxygen analogue, the resonance is a well-resolved quintet due to threebond coupling with protons G and H (Table II). For the sulfur analogue, however, the 3JpH couplings are not resolved in the 31p spectrum and only a very broad peak is observed. 31p spin-lattice relaxation times (T l) were measured by the inversion-recovery method; the values are 3.14 s for dioctanoyl PC and 2.17 s for the thio analogue in CDC13/CD3OD. The T~ values for egg phosphatidylcholine vesicles and liposomes, measured at different magnetic fields are constant at TI = 1.3 s [14]; thus chemical shift anisotropy (CSA) is not an important source of T~ relaxation for these 3~p nuclei (at least up to a phosphorous resonance frequency of 120 MHz). The mechanism which gives rise to Tx of phosphorus nuclei is predominantly a dipole-dipole interaction between 3~p and mH nuclei which is affected by the correlation time (T) for molecular reorientation and the throughspace distance between the 3~p nucleus and a given proton. The motion giving rise to T~ relaxation for the 3~p nucleus in phospholipids is in the fast correlation time region [14] with COoTc < 1 (w o = Larmor precessional frequency). The phosphorus Tm (measured at 109.5 MHz) for dioctanoyl PC micelles in aqueous solution is 1.7 s; for dibutyrylphosphatidyl monomers it is 6.5 s [15]. Thus, our values of 3.14 s (dioctanoyl PC) and 2.17 s (thio analogue) in CDC13/CD3OD fall between the values for micelles in aqueous solution and those for monomers. The 3~p linewidths at half-maximum intensity for dioctanoyl PC and its thio analogue, which differ by ten-fold, give an estimate of T2*, the

106 effective spin-spin relaxation time. Measured at 81.01 MHz, with broadband proton decoupling, the linewidth is 2.4 Hz (T2* = 0.13 s) for dioctanoyl PC and 27.9 Hz (T2* = 0.011 s) for the thio analogue. This ten-fold difference in the phosphorous T2* values cannot be totally attributed to motional differences between the analogues. The large linewidth for the thio analogue is larger than the linewidths of much more aggregated systems. For example, for micellar dioctanoyl PC in aqueous solution the linewidth is 16.3 Hz (at 36.4 MHz for 3~p, [16]). For 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine vesicles, the linewidth is 5.8 Hz (at 32.2 MHz for 3~p, [17]). While a portion of the very small T2* value of the thio analogue may be attributed to increased aggregation, another significant contribution may come from chemical shift anisotropy of the thiophosphate group vs. the phosphate group [18]. The substitution of sulfur for oxygen in a phosphate can cause a large change in the chemical shift tensor [19]. Such a change leads to a difference in the CSA between the two analogues. Since CSA contributes to the T2* relaxation of phospholipids [19], (causing the linewidth to increase as the square of the magnetic field), relaxation via CSA is a likely cause of the large difference in 3~p linewidths between the oxygen and sulfur analogues. Thus, the decrease in T2* for the 3~p nucleus of the thio analogue as compared with dioctanoyl PC is indicative of a decreased mobility for the headgroup region of the thio analogue, perhaps due to both a greater degree of aggregation in the thio analogue, and a greater contribution from CSA to the 3~p relaxation.

Coupling constants Table II contains the ~H-~H, 3~p-~H, 3~P-~3C, ~4N-~H and '4N-~3C coupling constants for dioctanoyl PC and its thio analogue. Coupling constants among the glycerol protons (E through G) were determined by analyzing the CH2CHCH2OP spin system using the Bruker program PANIC. The 3Jan couplings from 3~p to the G protons, which, for the thio analog, were not measurable from the phosphorus resonance, were determined in this manner. The methylene

protons in the choline head group region of dioctanoyl PC were analyzed as part of an XAA'BB'Y spin system (X = 31p, y = 14N)" It was not possible to treat the methylene protons in the choline head group of the thio analogue in a similar manner due to line broadening. Figure 2(a) reveals complex multiplet structure for protons labeled H and I in dioctanoyl PC, while Fig. 2(b), for the thio analogue, shows a very broad line with no multiplet structure for the H protons, and a broad triplet for the I protons. This lack of structure, which was present at 200 MHz as well as at 500 MHz in both solvents (pure CDCI 3 as well as the CDCI/CD3OD mixture) prevented determination of the coupling constants between the methylene protons for the choline group of the thio analogue. Line broadening of the 3~p resonance in the thio analogue also precluded an independent measurement of the two 3~P-~H three-bond coupling constants. The 31p resonance of dioctanoyl PC at 81.01 MHz is a well-resolved quintet due to coupling with protons G and H. The observation of t4N-x3C quadrupolar coupling in the trimethyl group of the choline region of phospholipids is an indication that the lipid is not aggregated [20]. The 13C methyl triplets collapse for egg yolk phosphatidylcholine when aggregated as reverse micelles (in pure CDCI3) or liposomes (in D20) due to the reduction in 14N T~ and T2 relaxation times. For nonaggregated phosphatidylcholines, the average ~J(~4N-t3C) is equal to 3.7 Hz and the ~4N relaxation times are much longer than for the aggregates. In both dioctanoyl PC and the thio analogue, ~J(14N-~3C) = 3.68 Hz, indicating that significant aggregation (large micelles or vesicles) does not occur in these molecules in this solvent system. Thus, 3~p T2. relaxation times (already cited) indicate that the thio analogue may be somewhat more aggregated than dioctanoyl PC. One-bond ~4N-~3C coupling constants, however, show that neither is significantly aggregated since the experimental ~J(~4NJ3C) value is the same as that for non-aggregated phosphatidylcholines in CD3OD or CDCI/CD3OD/D20 (50:50:15, v / v /

v) [20].

107

04 o o

CH2SP

CHaSP

CH2SP

DOPC and A B C thio-DOPC Calculated Coupling Constants (Hz) J (E1F) 5.01 10.68 0.90 J (E2F) 10.68 3.07 2.84 Fig. 4. Calculated coupling constants (3JHH) for the three rotamers about 04. DOPC is dioctanoyl PC and thio-DOPC is its sulfur analogue.

TABLE IIl Fractional populations about selected dihedral angles in dioctanoyl PC and its thio analogue. Dihedral angl eb

Defining atoms b

Fractional populations" A

B

C

O2t-C2-C3-O31

Dioctanoyl PC Thio analogue

0.02 0.00

0.60 0.62

0.38 0.38

OlI-C1-C2"O21 S.-C~-C2-O21

Dioctanoyl PC Thio analogue

0.17 0.39

0.27 0.36

0.56 0.25

a/

P-O.-CI-C 2 P-S~,-C~-C2

Dioctanoyi PC Thio analogue

(A + B) 0.30 > 0.30

~4t

P-OI2-CII-CI2

Dioctanoyl PC Thio analogue

0.27 0.23

0.73 0.77

a~s

OI2-CII-CI2-N

Dioctanoyl PC Thio analogue

1.00 --

0.0 --

04 °

0~d

0.70 < 0.70

•See text for rotamer diagrams. Solutions are 0.100M phospholipid in CDCI/CD3OD (2:1 v/v). bSee Fig. l for dihedral angle definition and atom numbering. Notation follows Sundaralingam [24]. cValues calculated from 3J(EtF) and 3J(E2F). aValues calculated from 3J(FG,) and 3J(FG2). •Values calculated from 3J(POCC(j)) or 3J(PSCC(j)). tValues calculated from 3J0aOCH ) or ~J(PSCH). ,Values calculated from 3J(HCCI) for dioctanoyi PC. Line broadening precluded determination of coupling constants for the thio analogue.

108

Rotamer populations The Haasnoot equation [21,22], which takes into account substituent electronegativity and orientation of substituents relative to the coupled protons, was used to calculate coupling constants (3JHH) for each of the three possible rotamers about dihedral angles 04, 02 and %. The coupling constants to phosphorus (3Jpc and 3Jpn) were calculated using the Lankhorst equations [23] for the rotamers about dihedral angles a~ and a 4. Rotamers have been drawn for the naturally occurring L-enantiomer. The thio analogue is racemic; identical results would be obtained with either enantiomer.

Dihedral angle O~ Dihedral angle 04 (O21-C2-C3-O31)controls the relative orientation of the dioctanoyl chains (Figs. 1 and 4). Rotamer populations about 04 are essentially equal for dioctanoyl PC and its thio analogue (Table III). Similar populations were found for other phospholipids in solution [5,25--27]. A great deal of evidence from X-ray crystallography [28--30] indicates that the acyl chains in diacylphospholipids extend in a parallel orientation and that 04 is gauche as in rotamers BorC.

Dihedral angle 02 The calculated coupling constants given in Fig. 5 between protons F and G allow calculations of the rotamer populations about the dihedral angle 02 (O.-CI-C2-O21 , Fig. 1). In dioctanoyl PC, protons G 1 and G 2 are accidentally chemical shift equivalent in the CDC13/ CD3OD solvent mixture. Upon spin decoupling at the resonance frequency of the E protons, the F multiplet collapses into a triplet, due to coupling with the G protons. Only the sum (JAx + JBx) may be determined from this triplet [31]; however since J(FG~) and J(FG2) are so close for dipalmitoylphosphatidylcholine (DPPC) in the same solvent (5.44 Hz vs. 5.54 Hz), we will assume that they are equal in dioctanoyl PC (4.23 Hz, Table II). The resulting rotamer populations about 02 in dioctanoyl PC are given in Table III. Protons GI and G z are non-equivalent in the thio analogue (Table I); the PANIC program was used to find J(F(]l) and J(FG2). Within 0.01 Hz, these coupling constants were each equal to 6.25 Hz (Table II). The resulting rotamer populations about 02 together with those for dioctanoyl PC (Table III) show that the polar head groups of these phospholipids have three accessible conformations about 02.

02 s-P-O

G2

G~

O-C-R U o

O-C-R

O-C-R II

A

B

DOPC J(FG1) 5.01 J(FG2) 10.68 thio-DOPC J(FG1) 4.11 J(FG2) 11.52

~

o

C

Calculated Coupl!ng Cor)stants (Hz) 10.68 0.90 3.07 2.84 11.52 2.17

1.93 3.88

Fig. 5. Calculated coupling constants (3j..) for the three rotamers about 02. Same abbreviationsas in legend to Fig. 4.

109 Dihedral angle 02 is quite close to the site of sulfur substitution. The replacement of oxygen by sulfur causes the G~ and G 2 protons to have very different chemical shifts in the two analogues (Table I). It also alters the rotamer populations about 02 (Table III). This is a significant change, since 02 affects the orientation of the choline head group relative to the acyl chains. The shift in populations between the two analogues may be due to the difference in steric interactions of the C 2 acyl chain with an oxygen atom vs. a sulfur atom; rotamer A places the acyl oxygen atom and the sulfur atom antiperiplanar. Other NMR studies [5,25,26] of phosphatidylcholines show that rotamer C predominates, as in dioctanoyl PC. In X-ray crystal structure studies of phospholipids, all three values for the dihedral angle 02 have been found [28--301.

Table III. NMR studies of other phospholipids in both organic and aqueous solutions [5,25,26] also show that the conformation about a 5 is predominantly gauche; and X-ray crystal structure studies of phospholipids give similar results [28 --30,32]. The gauche conformation for % allows the positively charged quaternary nitrogen to fold back toward the negatively charged phosphate group. The fact that this conformation is found in the crystal as well as in organic and aqueous solution stresses the importance of intramolecular forces in the choline region of the phospholipid molecule.

Dihedral angle % Dihedral angle a z may be defined by atoms P-O.-CI-Cz(j), (Fig. 1). The calculated ~JPa and 3Jpc coupling constants are given in Fig. 7. The experimental vicinal 3~P-~3C and 3~P-~H coupling constants may be measured for each analogue (Table II). These coupling constants are stereospecific; the most recent equations linking aJpH and aJpc to the dihedral angle are those of Lankhorst [23]. These equations, which were derived from coupling constants to the 3~p nucleus in oligoribonucleoside phosphates, are applicable in a qualitative way to the phosphate group of dioctanoyl PC because the electronegativities of the groups attached to the coupled nuclei are

Dihedral angle % Figure 6 gives the calculated coupling constants for the three rotamers about dihedral angle a 5 (O~2-C~,-Cn-N, Fig. 1). The choline region of dioetanoyl PC was analyzed as an XAA'BB'Y spin system. Values of J(HI) = J(H'I') = 2.38 Hz and J(HI') = J(H'I) = 6.76 Hz were obtained using the PANIC program. The resulting rotamer populations are given in

O~5 H

P'£/"

~

"H'

N+ DOPC and

A

th/o-DOPC J(HI) J(HI' )

H

O-P

He

N÷

1.65 10.61

H N+

B Calculated Couplin 9

3.35 2.09

O-P

H'

C Constants (Hz) 10.97 4.47

Fig. 6. Calculatedcouplingconstants (3Jl.ia) for the three rotamersabout %. Sameabbreviationsas in legendto Fig. 4.

110

C

C

H

H

H

C

H

H

H P

A

DOPC J(POCH) J(POCC)

B

C

Calculated Coupling Constants (Hz) 12.7 2.4 0.7 11.0 Fig. 7. Calculated coupling constants (3Jpc and 3JpH) for the three rotamers about a 1. Same abbreviations as in legend to Fig. 4. 12.7 0.7

similar. Using Eqn. 1 [31] and 3j(POCC(j)) 7.89 Hz for dioctanoyl PC gave 70070 of rotamer C about a r 3Jpc [c]

-

3Jgauche

3 J tran s -

(1)

3 Jgauch e

Using Eqn. 2 [31] and 3J(POCG) = 6.99 Hz, gives a population of 55070 for rotamer C.

(JG + J+) - (JA.x + JAx,) [C] =

(2)

JT -- JG

The difference of 15070 in the population of C obtained from 3 J ( P O C H ) vs. 3 J ( P O C C ) may reflect the qualitative nature of the calculation as applied to phospholipids. It may also indicate dihedral angles other than the _ 60 °, 180 ° values assumed. Nevertheless, the overall result that the predominant conformation for a~ is antiperiplanar is generally valid. Hauser et al. [5] in NMR studies of DPPC in CDCI3/CD3OD (2:1 v/v) found 68070 of rotamer C. X-ray structural studies of phospholipids [28--3~,32] yield values near 180 ° for dihedral angle a r Empirical energy calculations on diheptanoylphosphatidic acid-c, however, [33] show that all values of a~

( ± 6 0 ° and 180 ° ) can correspond to low energy conformations. Calculating the rgtarner populations about a~ from coupling constants is uncertain for the thio analogue. Although 3j(PSCH) and aJ(PSCC) are stereospecific [34], equations relating vicinal coupling constants to dihedral angle are not available. Also, the quantitative effect on the coupling constant of replacing an oxygen atom by a sulfur atom in the coupling pathway is unknown. An example in which 3J(PSCH) is considerably larger than 3J(POCH) in the analogous compound is 5'-deoxy-5'-thioadenosine 5'monophosphate, 1, in D20 [35]. In this compound, J(PSCH) = 9.8 Hz, O

II

HO'P'S'(~H2

adenine

OH O H

while in the oxygen analogue, 3j(POCH) = 4.6 Hz [36]. It therefore appears that the presence of sulfur m a y increase the coupling constant

111

3J(PSCH) over 3J(POCH). For dioctanoyl PC and its thio analogue (Table II), the difference between these coupling constants is 7.73 Hz. While it is possible that the difference is solely due to S vs. O in the coupling pathway, it may also be that the thio analogue contains a higher percentage of gauche rotamers about a r A comparison of the 3~P-~3C coupling constants 3j(POCC0)) = 7.89 Hz and J(PSCC(j)) = 2.21 Hz between the two analogues also indicates that the percentage of gauche rotamers about a t may be larger in the thio analogue. While, as for the vicinal 3tP-H coupling constant, the change in coupling constant may be due only to a change in coupling pathway, a decrease in 3J(PC) could also signal an increase in the percentage of gauche rotamers about a~. A gauche conformation about a t has the effect of bringing the head group of the phospholipid closer to the acyl chains (than does the antiperiplanar conformation). Such a result, if also true in aqueous solution, may contribute to the differences in aggregation properties [1,2] as well as to the decreased interaction with phospholipase C [3] observed with the thio analogue as compared with dioctanoyl PC. This enzyme specifically hydrolyzes the OH-C ~ bond.

Dihedral angle a~ The vicinal coupling constants U(POCC(m)) and U(POCH), where all atoms are part of the choline group, are each related to the rotamer populations about a 4 (P-Ot2-Clt-C12, Fig. 1). Line broadening, however, precluded measurement of U(POCC(m)) in the CDC13/CD3OD solvent mixture, therefore populations were calculated from ~J(POCH) using the Lankhorst equation [23]. The coupling constants of 5.15 Hz and 4.78 Hz (Table II), yield 73°70 of the antiperiplanar rotamer C for dioctanoyl PC and 77070 C for the thio analogue (See Fig. 7 for %). The populations may be compared with the 77o7o C obtained for dihexanoylphosphatidylcholine in CD3OD, and 68070 C for DPPC in CDC13/ CD3OD [5]. The sulfur atom, three bonds away, apparently has little affect upon the rotamer populations about t~4. X-ray crystal studies yield

values of 130°--150 ° for this dihedral angle in similar phospholipids [28--30,32].

Dihedral angles % and % Two of the more important dihedral angles for determining the head group conformations in phospholipids, a 2 (Ct-Slt-P-O~2) and a 3 (SH-PO~2-CH), cannot be determined from vicinal coupling constants. Rotamer populations about a 2 and % in the two analogues are expected to be somewhat different due to a change in the degree of hybridization of the phosphorous atom, a change in electronegativity between sulfur and oxygen, and different bond lengths and angles in the two analogues. Phosphate ester bond angles are known to be coupled to dihedral angles a 2 and a 3 [13]. In a series of phospholipids similar in structure to dioctanoyl PC, a 2 and a 3 were found to be gauche via X-ray crystallography [28--30,32].

A comparison of :J(POC) and 3J(POCC) A final point of interest relating to the orientation about a t is the comparison of 2j(POC) to 3J(POCC). For a series of 26 phospholipids [37], and for 10 mono- and dialkylphosphate ions [38], 3J(POCC) > 2j(POC). This is also true for dioctanoyl PC (Table II, 7.89 Hz > 5.26 Hz); however, it is not the case for the thio analogue, where 3J(PSCC) = 2.21 Hz and 2j(PSC) = 3.67 Hz. The general rule of thumb that 3j > 2j [39] will not be true when the dihedral angle takes on values near _+60° since, at these values, for most Karplus-like relationships, aj is at or near its minimum value, which may be l w3 Hz. The fact that 2j(PSC) > 3j(PSCC) for the thio analogue may be further evidence for a higher percentage of gauche conformation for a~ as compared with dioctanoyl PC. Conclusions There is evidence (from 3~p T2. values, Adc values and tH choline methylene broadening in the thio analogue) that the replacement of oxygen by sulfur in the C H q C H 2 - - S - - P thiophosphate group of dioctanoyl PC in CDCI3/CD3OD solution causes an increase in the

112 degree of intermolecular aggregation for the thio analogue. Since 1j(14N-13C) for the 13C choline methyls is a triplet for both compounds, the aggregation is not into typical micelles (as in aqueous solution), reverse micelles (as in CDCI3) or larger aggregates. Aggregation into small micelles o f perhaps 60--70 molecules, as suggested by Hauser [5] is supported, with the thio analogue being somewhat m o r e aggregated than dioctanoyl PC, as it is in aqueous solution [1,2]. The extremely short 3tp T2, for the thio analogue m a y be due to both a decrease in mobility due to aggregation and an increase in CSA relaxation due to the substitution o f a sulfur a t o m for a bridging phosphate oxygen atom. Two significant changes were found for the rotamer populations o f dioctanoyl PC as compared with its thio analogue: for dihedral angle 02, the antiperiplanar rotamer A (Fig. 5) is more highly populated in the thio analogue; and, for dihedral angle a 1, the gauche rotamers A and B (Fig. 7) are more highly populated in the thio analogue. Both angles are important for positioning the choline head group relative to the rest o f the molecule. It is conceivable that the change in preferred c o n f o r m a t i o n about these angles is related to the decreased ability o f phospholipase C [3] to hydrolyze the phospholipid at the OH-C ~ site. The very small (2.21 Hz) coupling constant 3J(3tP-S-C-C(j)) for the thio analogue indicates conformational change about dihedral angle a~, hut not enough vicinal coupling constants of this type are known for rigid phosphate groups to be absolutely certain that this small coupling results f r o m a population change about a t . Given the importance o f the phosphorus-sulfur bond in organic, inorganic and biochemical applications, these molecules deserve continued evaluation via quasielastic light scattering (to determine particle size in solution), variable temperature N M R studies (to shift rotamer populations), and X-ray crystal structure determination.

is supported in part by N I H grant RR02301 f r o m the Biomedical Research Technology Program, Division o f Research Resources. We especially wish to thank Dr. Brian J. Stockman for his help in obtaining the 500 M H z spectra, and Dr. William Snyder for synthesizing the sulfur analogue o f dioctanoyl PC.

Acknowledgements

21

This study m a d e use of the National Magnetic Resonance Facility at Madison, Wisconsin which

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