Synthesis and NMR identification of isobutyl analogs of phospholipids designed for the modeling of biomembrane fragments

Colloids and Surfaces A: Physicochemical and Engineering Aspects I I 5 (1996) 83 87

ELSEVIER

COLLOIDS AND X SURFACES

Synthesis and NMR identification of isobutyl analogs of phospholipids designed for the modeling of biomembrane fragments V.A. Karasev,

N.A. Korovnikova,

V.A. Gindin, V.E. Stefanov *

Department of Biochemistry. St. Petersburg State Unicersity, St. PetersbutN I99034. Russi~ Received 11 September 1995; accepted 11 Septimber 1995

Abstract

Three isobutyl analogs of phospholipids - - isobutyl phosphoric acid t IPA), analog of phosphatidic acid; isobutyl phosphoethanolamine (IPE), analog of phosphatidylethanolamine; and isobutyl phosphocholine (lPCh), analog of phosphatidylcholine --- were synthesized for use in mixed crystals as initial material for modeling biomembrane fragments, lsobutyl oxophosphodichloride served as a common precursor yielding IPA when hydrolyzed. Cyclic compounds of the former with ethanolamine and ethylene glycol were used to synthesize IPE and IPCh. The structure of the obtained compounds was identified by means of NMR. For all three compounds the following signals were registered (ppm): 0.9, d, 6H, (CH3)2CH-; 1.9, m, 1H, (CHa)2CH-: 3.7, t, 2 H , - P O CH2-CH(CH3)2:which can be attributed to the isobutyl radical. In addition, the following signals were registered for IPE: 3.3, t, 2H, P O-CH2-CHz-NH2; 4.1, m, 2H, P - O - C H 2 CHz-NH2; and for IPCh: 3.2, s, 9H, -CH2 CH2-N+(CH3)3:4.2, m, 2H, P - O CH2-CHz-N+(CH3)3. X-ray analysis of the crystals is in progress as part of model studies of the biomembrane organization. K~'ywords: Biomembrane fragment modelling; Isobutyl analogs; NMR identification: Phospholipids: Synthesis

1. Introduction

The investigation and modeling of biological m e m b r a n e s is a problem of p a r a m o u n t importance for molecular biology. Physical reconstitution of a fragment of the zone-block model of biomembranes as undertaken in Refs. [1 3] can be considered as an a p p r o a c h to solve this problem. As shown in Fig. 1 the model implies that the bipolar groups of phospholipids (PLs) are directed inside the structure and form paired zones (zones I and II J. The so-called conjugated i o n i c - h y d r o g e n b o n d * Corresponding author. 0927-7757/96/$15.00 ~ 1996 Elsevier Science B.V. All rights reserved PII S0927-7757 (96 ~03609-6

systems (CIHBs) capable of carrying out charge transfer (electron transport) from donors ( H D ) to acceptors I l i A ) make up one zone (zone II. Amino groups of phosphatidylethanolamine (P[~Zt and phosphate groups of other PLs m a y contribute to the formation of such zones. Choline groups of another P L - - phosphatidylcholine (PCh) provide insulation of C I H B s (zone lit. Diglyceride radicals of PLs, according to the model, are buried in the protein layer of the m e m b r a n e so that O C = O groups of these radicals can participate together with proteins in the formation of two other zones of C I H B s (zones III and IV). The occurrence of an interzonal space between zones 1

84

V.A. Karasev et al./'Colloids"Surfaces A." Physicochem. Eng. Aspects 115 (1996) 83 8 7

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analogs of PLs were synthesized on the basis of one scheme and their structures were identified by means of NMR spectroscopy. The three isobutyl analogs were: isobutyl phosphoric acid (IPA), analog of phosphatidic acid (PA); isobutyl-2-aminoethyl phosphate (isobutyl phosphoethanolamine; IPE), analog of PE; and isobutyl-2-(trimethylammonia)-ethyl phosphate (isobutyl phosphocholine; IPCh), analog of PCh.

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2. Experimental

Fig. 1. Central area of the zone-block model of biomembranes. Filled circles represent carbon atoms with different numbers of hydrogen atoms.

2.1. Synthesis of PL analogs The synthesis was carried out in several steps with cyclic phosphorous compounds and oxoazaphospholan and dioxophospholan as intermediate products in the syntheses of IPE and IPCh respectively. The methods for such a synthesis based on cyclic phosphorous derivatives were proposed earlier [-4,5]. In the present study they were modified and adjusted for the synthesis of particular isobutyl derivatives. Basically, the following scheme was used for the synthesis:

and III as well as between zones II and IV is presumed, which can include terpenoid (T) molecules (cholesterol, carotenoids, etc.). To perform the physical reconstruction of the proposed model isobutyl analogs of PLs were chosen [3]. They retain the structural peculiarities of PL molecules necessary for modeling the central zones of CIHBs but do not contain bulky diglyceride radicals. In the present work three isobutyl

l.

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(CH3)2CHCH2 ~ P OH IPA jO CH2 / /

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isobutyl oxoazaphospholan 3.

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I I

i O CH 2

isobutyl dioxophospholan N(CH3) 3 OI + (CH3)2CHCH 2 O-P O CH2CH2N(CH3) 3 O IPCh

V.A. Karasev et al./Colloids Surfaces' A: Physicochem. Eng. Aspects 115 (1996) 83 87

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Fig. 2. ~H N M R spectra of isobutyl analogs of phospholipids in D:O: (a) IPA; (b) IPE: {c) IPC.

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86

V.A. Karasev et al./Colloids Surfaces A." Physicochem. Eng. Aspects 115 (1996) 83 87

In the first stage isobutyl oxophosphodichloride (compound I) was obtained from phosphorus chloroxide and isobutanol. Compound I was used as the precursor in the subsequent synthesis of all the analogs. The synthesis was carried out with a procedure similar to that used for the synthesis of butyl oxophosphodichloride [6]. After vacuum distillation water was added to compound I according to Ref. [7], which resulted in the formation of IPA in the second stage. In the synthesis of IPE, ethanolamine was added in the second stage to compound I dissolved in tetrahydrofuran in the presence of triethylamine as catalyst, according to Ref. [4], thus yielding isobutyl oxoazaphospholan. In the third stage the ring was opened by water as described in Ref. I-8]. Similarly, in the second stage of the synthesis of IPCh, which proceeded in the presence of triethylamine, ethylene glycol was added to compound ! dissolved in benzol with the formation of dioxophospholan [5]. In the third stage a trimethylammonium group was introduced and the ring was simultaneously opened, as proposed in Ref. [5]. The advantage of this scheme for the synthesis of PL analogs in comparison with other methods consists of a high, nearly quantitative, yield of products in the last two stages. Thus obtained, IPE and IPCh were recrystallized from 85% aqueous ethanol and from dimethylformamide containing water (2%) respectively. The products obtained were used for identification and in further experiments on growing crystals. 2.2. Identification o f P L analogs

The structure ascribed to the analogs of PL was confirmed by 1H NMR spectroscopy. The spectrum was registered in D20 (2% solution) by means of an AG-200 Bruker instrument, working frequency 200.13MHz, internal standard DSS (2,2-dimethyl-2-silanpentan-5-sulfoacid).

spectrum of IPA is characterized by three unambiguously interpreted signals relating to the isobutyl radical: a doublet (0.9 ppm, 6H) corresponding to two methyl groups, a CH proton septet (1.9 ppm, 1H), and a triplet (3.7 ppm, 2H) corresponding to CH 2 protons. The same group of signals with nearly the same values of chemical shifts can be traced distinctly in the spectra of the other PLs analogs: IPE and IPCh (Figs. 2b and 2c). Signals from the ethanolamine radicals are added in the spectrum of IPE. These are a triplet of protons from the - O - C H 2 group (3.3 ppm, 2H) and a multiplet from the second CH2 group of this radical, whose protons interact with the protons of the amino group (4.1 ppm, 2H). To confirm the presence of the amino group the 1PE spectrum was registered in deuterated dimethylsulfoxide. IPE is practically insoluble in this solvent; however, the addition of 10% LiC1, which facilitates the rupture of hydrogen bonds when chitin is being dissolved in dimethylformamide [9], proved efficient in our case. The spectrum remained practically unchanged, but a signal from the three N+H3 protons (8.7 ppm, 3H) appeared. A peculiar feature of the IPCh spectrum consists in the coincidence of the triplet signals from protons of both O-CH 2- groups (3.6 ppm), which are symmetrical with respect to the phosphorus atom. This is the reason why only one triplet of twice the intensity is observed (4H). In addition, the spectrum of IPCh also contains a singlet (3.2 ppm, 9H) belonging to the group N+(CH3)3, as well as an unresolved multiplet of protons from the CH2 group (4.2 ppm, 2H) in the vicinity of the trimethylammonia group.

4. Conclusion

The compounds obtained were identified quite reliably by means of NMR spectroscopy. X-ray analysis of the crystals of PL analogs and their complexes is in progress as part of model studies of biomembrane organization.

3. Results and discussion

The spectra of the three compounds IPA, IPE and IPC are presented in Fig. 2. From the presented data (Fig. 2a) it can be seen that the

References

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I . A. Karasev et al./Colloids Sur/ilces A." Physicochem. Eng. Aspects 115 ( 1996 ; 83 X7

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