Synthesis, analysis and application of specifically deuterated lipids

Prog. Lipid Res. Vol. 22, pp. 235-256, 1983 Printed in Great Britain.

0163-7827/83 $0.00 + .50

SYNTHESIS, ANALYSIS AND APPLICATION OF SPECIFICALLY DEUTERATED LIPIDS* A. P. TULLOCH Prairie Regional Laboratory, National Research Council of Canada, Saskatoon, Saskatchewan, Canada S 7N OW9

CONTENTS I. II.

INTRODUCTION DEUTERIUMINTRODUCTION BY EXCHANGE A. Exchange of enolizable protons 1. Exchange in aldehydes and ketones 2. Exchange in methyl esters and carboxylic acids B. Exchange of nonenolizable protons III. DEUTERIUMINTRODUCTION BY REDUCTION A. Reduction with metal deuterides 1. Lithium aluminum deuteride 2. Sodium borodeuteride 3. Sodium cyanoborodeuteride B. Reduction with Zn-D20 C. Catalytic reduction 1. Double bonds 2. Triple bonds D. [2H4]Hydrazine reduction IV. SYNTHESISFROM DEUTERATED INTERMEDIATES A. Synthesis from other acids B. Malonate as coupling unit C. Chain extension with enamines D. Anodic synthesis E. Acetylene as a coupling unit F. Coupling by the Wittig reaction G. Copper catalyzed coupling H. Tables of deuterated acids V. LIPID SYNTHESIS A. Lipids not containing phosphorus 1. Triacylglycerols and cholesteryl esters B. Deuterated phosphatidylcholines 1. Phosphatidylcholines labeled in choline or glycerol 2. Deuteration in both acyl chains 3. Deuteration in one acyl chain only C. Other deuterated phospholipids VI. ANALYSIS A. Analysis by mass spectrometry B. Analysis by nuclear magnetic resonance spectroscopy I. ~H NMR spectroscopy 2. 2H NMR spectroscopy 3. ~3C NMR spectroscopy C. Analysis by GC and HPLC VII. APPLICATIONSOF DEUTERATED LIPIDS A. ~H NMR spectroscopy B. Estimation of conformation and order in lipid bilayers by 2H NMR, FTIR and Raman spectroscopy 1. 2H NMR spectroscopy 2. FTIR and Raman spectroscopy C. Interpretation of MS, ~3C NMR and other spectral applications 1. MS 2. ~3C NMR spectra 3. Circular dichroism VIII. REFERENCES

236 236 236 236 237 237 237 237 237 238 239 239 239 239 240 240 240 240 241 241 242 243 244 245 245 246 246 246 246 247 247 248 248 249 249 249 249 250 250 250 250 250 251 251 251 252 252 252 254 254

Abbreviations--The abbreviations used are as follows. DMSO, dimethylsulphoxide; DME, dimethoxyethane; DMF, dimethylformamide; HMPA, hexamethylphosphoramide; MeOD, [O-2H]methanol; DHP, dihydropyran; THP, tetrahydropyranyl; Ms, methanesulphonyl; Ts, p-toluenesulphonyl; LAD, lithium aluminium deuteride; LAH, lithium aluminium hydride. *NRCC No. 21333. JPLR

22/4 A

235

236

A.P. Tulloch I. I N T R O D U C T I O N

Specifically deuterated fatty acids and derived lipids have been employed as nonradioactive tracers in investigations of biological processes and in studies of fragmentation patterns in mass spectrometry (MS) since quite soon after deuterium first became available. More recently there has been a great increase in the application of these compounds in other types of spectroscopy, particularly deuterium magnetic resonance of lipid membranes. These investigations have been reviewed. 1°°,1°4'1°6 There have been several previous reviews of synthesis of deuterated compounds and a book on deuterium labeling in organic chemistry has given a comprehensive account of the methods for deuterium incorporation up to 1970, ~'7 but specific syntheses of long chain compounds were not mentioned. A later review covered deuterated lipid synthesis to 1977. ~26 There have also been two reviews which dealt with deuterated lipids more briefly and also with 3H, 13C and ~4C labeled lipids. 42"43A recent review, to 1981, has described many of the experimental procedures employed, s~ The present review will extend our earlier one ~26will concentrate on results reported during the five years 1977-1982, and 92 references which were not mentioned before will be included. Since most deuterated lipids contain deuterium in the fatty acid portion, synthesis of deuterated acids will be discussed first; synthesis of lipids with deuterium in the glycerol, choline, ethanolamine or serine portions will be described later. Deuterium is introduced into an organic molecule essentially by exchange or by reduction ~7 but there are many different ways of effecting these two processes and also of incorporating a deuterated portion into a long chain: an account of these procedures will form a large part of this review. 11. D E U T E R I U M I N T R O D U C T I O N BY E X C H A N G E

The principal advantage of exchange processes is that, in many cases, they can be repeated with a fresh deuterium source until the required isotopic purity is attained. A list of exchange processes is given in Table 1.

A. Exchange of Enolizable Protons 1. Exchange in Aldehydes and Ketones Exchanges of protons ~ to ketonic or aldehydic carbonyls are readily catalyzed by weak acids or bases; pyridine is often used to catalyze exchange of e protons of aldehydes. 33-~2° Since c~ deuterated aldehydes and ketones are obtained under very mild conditions, precautions have to be taken against back exchange with moisture. It is best to convert the c~-deuterated products by further reaction to compounds in which the deuterium is stable: for example, to an aldehyde by the Wittig reaction ~3 or to an alcohol by reduction. 54 TABLE 1. Deuterium Introduction by Exchange Reactions Precursor

Reagents

--CH2CO2Na

NaOD/D20

--CH2CO2Me --CH2CHO --CH2COCH 2 -

NaOMe/MeOD D20/pyridine NaOD/MeOD

--CH2COCOCH2

CH3CO2D/DC1/D20

CH3(CH2)nCO2H CH3(CH2),CO2H

Pt/D2/D20/Na20 -, Pd/D 2

Products (after further reaction) --CD2CO2H (--CD2CH3) --CD2CO2Me --CD2CHO --CD2COCD2 (--CDzCH2CD2--) (--CD2CD2CD2--) (--CD2CF2CD2) --CD2COCOCD2 (--CD2CH=CHCD2) CD3(CD2).CO2H CD~(CD2),CO2H

References 9,62,75,122, 125.127.130 130 1.25,81,133 32,22,120 54,86 54 113 80,86 22 22 39 57

Specifically deuterated lipids

237

In ketones, ~ exchange introduces 4 deuterons and subsequent reduction yields the CD2CH2CD2 group. Exchange of the ct protons of a ketone followed by fluorination of the carbonyl group gave the CD2CF2CD2 group. 86During Clemmensen reduction with Zn/DC1, both exchange and carbonyl reduction took place and a CD2CD2CD2 group was produced. 113Employing a a diketone, ~ exchange followed by reduction and elimination gave [8,11-2H4]oleic acid.22 2. Exchange in Methyl Esters and Carboxylic Acids Deuterium is introduced c~to the carboxyl group by exchange catalysed by stronger bases than those required for aldehydes. Reflux of methyl esters in MeOD, 0.7-1.0 M in NaOMe, gave almost quantitative incorporation of two deuterons. ~'8~Exchange of sodium salts in D20 containing 0.5~o NaOD at 150°C was first reported for C3 and C 4 acids and some dibasic acids. 9 Exchange of long chain acids was very slow at 150°C but at 200°C (0.1 M RCO2Na in D20 containing 1~o NaOD for 72 hr) 97-99~ dideuterated acids were obtained, m Monoalkenoic acids were dideuterated in the same way without double bond migration but alkynoic acids decomposed during this procedure and extensive introduction of deuterium into the chain occurred due to triple bond migration. ~27 Heating of a sodium alkynoate in D20, without excess NaOD, at 200°C, however, effected 97-98~o dideuteration without migration. ~3° An exchange of all the enolizable protons in an oxo ester to give 8-oxo [2,7,9-2H6]tetradecanoate, which was then fluorinated to the 8,8-difluoro ester, has been described. 8° A different exchange method for preparation of • monodeuterated acids, in which the dianion of an acid, prepared by treatment with two moles of lithium diisopropylamide was quenched with D20, has been outlined. 85 Repetition of the process could give dideuterated acid. 85 B. Exchange of Nonenolizable Protons These exchanges require vigorous conditions, high temperatures (200°C) and deuterium in the presence of Pd or Pt catalysts and are consequently not applicable to unsaturated acids. The method has particularly been applied to hydrocarbons, ~ but C6-C~4 acids were also exchanged with D2/Pd-C at 190°C.57 A modified process, applied to long chain acids, avoided continuous passage of D2 gas and consisted of heating an Na alkanoate in D20, with D2, reduced Adams catalyst, NaOD and Na20 2 at 240-270°C. 36'39Some decarboxylation to hydrocarbons with one less carbon and degradation to shorter chain acids and hydrocarbons occurred and became more pronounced at higher temperatures. 36 Another procedure, for partial exchange, is of interest because it showed considerable preference for the CH 3 group. Carboxylic acid was heated at 100°C with AcOD, D20, HCIO4, K2PtCI4 and pyrene; preferential exchange of CH 3 was pronounced with nonanoic acid but less so with octadecanoic acid where the preponderance of CH2 groups was probably too great. 26 III. DEUTERIUM INTRODUCTION BY REDUCTION In comparison with deuteration by exchange, reduction procedures have the disadvantage that they must give a product of high isotopic purity in one step. Various reduction methods and products are listed in Table 2. A. Reduction with Metal Deuterides 1. Lithium Aluminum Deuteride This reagent reduces any group reduced by LAH, in particular, esters and ketones, are rapidly reduced to alcohols. Solvents, such as Et20, THF and DME (depending on solubility requirements), which are unable to contribute protons to the reaction, are

A.P. Tulloch

238

TABLE 2. Deuterium Introduction by Reduction Procedures

Precursor

Products (after further reaction)

Reagents

. --CO,ME

LiAID 4

References

--CD,OH

"3

,~9,50.76, l 0,:'~

(CD,H, CDd

108,121,125 127.130

--COC1 --CO--

NaBD4/dioxane NaBD4/MeOD

--CD2OH --CDOH (CDH. CD,)

135 122,125,136

--C-~-N NHTs --CHOTs CHBr(CI) --CDOTs(MS) --CDOTs --CD~OTs(Ms)

NaBD3CN/DMF LiA 1D 4 LiAID4/LiD LiAID 4 NaBDsCN/HMPA LiAID4

--CHD --CHD --CH D --CD: --CD 2 --CD~

136 128 54,128 54,122 136 12 I, 125,130

--CD21, --CDI --CH--CHCO2H

NaBD3CN/HMPA NaBD 4

--CD~, - - C D : --CHOHCHDCO,H

87,135 72

O --CCI, --CC13 --C---~C--

Zn/D20 Zn/D20 Lindlar Pd/D~

--CD: --CD~ --CD=CD--

60 60 7.22,41.73. 76,98

--CH~CH---C~C--

P 2 Ni/D_, (Ph~PERhCI,'D:

--CDzCD---CD2CD_~--

97.115 2 5.32 34, 92

--CH=CH---CH=CH--

(Ph3P)xRhCI/D -,

--CHDCHD---CHDCHD--

t~0 8.113

\ /

N2D 4

employed. Tosylates and mesylates are also reduced by L A D though an excess of more than one mole of reagent is required. ~2~'m T w o successive reductions convert an ester or carbonyl group to CD3 or CD~ as shown below: L2~ LAD

H2C~CH(CH2)14CO2Me --

, H 2C~---CH(CH 2)I4CD:OH

MsC1

LAD

:-~-~ H?C~CH(CH2)I4CD2OMs

_ _

--~ H 2 C ~ ( C H 2 ) I 4 C D 3

Contrary to what is sometimes believed, L A D does not reduce primary and secondary aliphatic chlorides and bromides in the absence of lithium deuteride. 54'~2s This limitation was employed to advantage in a preparation of [8-2H3]octyl bromide, which is an intermediate in a synthesis of [18-2H3]oleic acid, 125 as shown below: LAD

Br(CH2)7CO2Me

. . . . .

, Br(CH2bCD2OH

MsC[

-- - - *

LAD

Br(CH2)TCD2OMs

-

-*Br(CH2)TCD~

The principal disadvantage o f L A D is lack of selectivity so that when, in synthesis of a deuterated ester, a substituent group such as mesylate is reduced, the ester group is also reduced. The resulting alcohol has to be oxidized back to the acid with consequent reduction in yield. 122'12s For this reason, milder reduction procedures have been examined. 2. Sodium Borodeuteride Esters are not affected by NaBD4 but ketones and aldehydes are reduced to deutero alcohols. Unlike L A D reduction, the solvent, whether protic or aprotic, has a considerable

Specifically deuterated lipids

239

influence on deuterium content of the product. Reduction of ketones with NaBD4 in MeOH appeared to give alcohols with isotopic purity of >95~/o .54'122'136 More recently, however, isotopic purities of only about 90~o were obtained with this system ~29but use of MeOD, in place of MeOH, gave 98~ purity.l~9 With isopropyl alcohol as solvent, 99~o isotopic purity was reported. 118 Attempts to avoid contamination by solvent protons by using aprotic solvents such as DME have led to other difficulties since reaction is apparently very slow in these solvents and some reduction is thought to occur when water or acid is added during work up. 95318 DMSO is not recommended as solvent either, because the CH 3 groups exchange slowly under basic conditions. H7 Reduction of an acid chloride to CD2OH with NaBD 4 in dioxane, with DzO decomposition, is however, a useful application. 135

3. Sodium Cyanoborodeuteride Sodium cyanoborodeuteride is a newer reagent and was introduced because, unlike NaBD4, it reduces tosylates and iodides, in suitable aprotic solvents, but still does not reduce esters. This is illustrated in the synthesis of methyl [14-2H3]tetradecanoate m as shown below:

MeO2C(CH2)~2COC1 NaBD4;D20,M e O 2 C ( C H 2 ) 1 2 C D 2 0

H

dioxane (PhO)3PMeI ~

,,-x ~ , - ~ .

x

~

.

) IVIe L/21..~( L. 1--12) 121..5LI21

HMPA

NaBD3CN ) MeO2C(CH2)12CD

3

HMPA

HMPA is the solvent in these reductions and is a potential proton source; however, it has been found that protons of HMPA exchange less than 2500 times as fast as those of DMSO 55 under the influence of base. Though very strong bases, such as butyl lithium and lithium cyclohexylamide, have effected partial exchange of protons of HMPA, TM exchange should not take place under milder conditions,v7 Sodium cyanoborodeuteride also reduces tosylhydrazones of ketones but it is important to notice that only one deuteron is introduced and CHD is obtained. ~z6This agrees with much earlier reductions of tosylhydrazones with LAD and NaBD4 which also introduced only one deuteron. 47 On the other hand, the one step reaction in which a tosylhydrazone of a ketone, with both ~ carbons deuterated, is formed and then reduced with sodium cyanoborohydride is not satisfactory because deuterium loss occurs, probably during tosylhydrazone formation. ~29 Apart from the difficulties mentioned above, reductions with metal deuterides give products with 97-99~o isotopic purity.

B. Reduction with Zn-D20 Dehalogenations of bromo and chloro acids have frequently been carried out with zinc dust. Treatment of methyl 12-trichlorododecanoate with Zn-D20 in dioxane at 90°C gave a good yield of [12-2H3]dodecanoate and 2-dichlorododecanoate, with Zn and AcOD, gave [2-2Hz]dodecanoate. 6° Deuterium has also been introduced by reduction of a halo compound with Zn-Cu couple in dioxane-D20. ~°9 No long chain compounds were examined but monodeuteration of bromo ketones occurred even though dideuteration by exchange might have been expected.

C. Catalytic Reduction 1. Double Bonds Reduction of double bonds with deuterium and common hydrogenation catalysts, such as Pd/charcoal, cannot be employed to prepare isotopically pure dideuterated acids

240

A.P. Tulloch

because appreciable deuterium scattering occurs. ~6 Reduction with the soluble catalyst, tris (triphenylphospine) rhodium chloride, however, gave a saturated product with only slight isotope scattering. 5'33~°

2. Triple Bonds Using the soluble catalyst system referred to above, triple bonds are reduced to the CD2CD2 group. 44'92There is usually some isotope scrambling with these reductions, about 5~o per bond reduced so that a product containing six deuterons has an isotopic purity of about 85~/,,.44The procedure is a convenient one step introduction of 4 deuterons, though exchange of an acid followed by LAD reduction should yield a similar product with better isotopic purity; a few more steps would be required to incorporate the CD~CD, group into a product. The selective Lindlar catalyst, which reduces only the more active triple bond to a double bond, effects semideuteration to the dideuterated olefinic product: isotopic purity is 96'! 0- :: or better and the product may consist of 98-99~i of the Z isomer. To avoid proton introduction, an aprotic solvent, either hexane 22 or dioxane, 7-~' is used. Triple bonds separated by methylene groups have also been reduced, either t w ¢ s or four as in several preparations of octadeuteroarachidonic acid. 4~'13v The opportunity for incomplete deuteration and double bond isomerization is much greater in reduction of an alkatetraynoic acid; use of P-2 nickel catalyst gave a purer product though only 58",, isotopic purity was attained.~5

D. [2Ha]Hydrazine Reduction As an alternative to homogeneous catalytic reduction, saturated cic-dideuterated acids have been obtained by reduction with deuterated hydrazine. ~5 Isotopic purity tbr introduction of 2 deuterons was 94~,, and of 8 deuterons, by reduction of arachidonic acid, was 80~o. ~3 More recently the complete series of 16 vic-dideuterated acids from [2,3-2H2] to [17,18-2H2] octadecanoic acids was synthesized by this method in yields of 40-90°~0, isotopic purity was 70-80°J;. ~

|V. SYNTHESIS FROM DEUTERATED INTERMEDIATES The preceding two sections have discussed methods of deuterium introduction and while, in some cases these lead directly to the required deuteroacid, frequently only a deuterated intermediate results which requires further steps to convert it to the end product. A classification of methods of fatty acid synthesis 53 has been adapted here in listing the procedures applied in synthesis of the final product.

A. Synthesis from Other Acids It is common practice in lipid chemistry to use naturally occurring fatty acids, which are often readily available, as precursors for required acids. Malonate chain extension of such acids is an example which proceeds in high yield so that two or more extensions can be carried out as in the synthesis of [5-2H2]hexadecanoic acid ~°2'129shown in Fig. 1. [3--'H2]. [5-2H2] and [3,5-2H4]tetracosanoic acids I°s and [4-2H2]-(Z)-ll-eicosenoic acid ([¥om [2-2H2]oleic acid 122) were prepared by this reaction. Other naturally occurring acids, and the products obtained in more elaborate syntheses, are listed in Table 3. Some acids were utilized without alteration of chain length but others. such as undecylenic and vernolic acids, were extensively modified. The double bond of these acids may be regarded as a potential carboxyl ~2~ or carbonyP ~ group which can be obtained by oxidative cleavage. Ricinoleic acid, a possible precursor of [12-2H2]oleic acid. could be converted to this acid only in very low yield. 125

Specifically deuterated lipids

241

LAD CH3(CH2)IoCO2Me

2, CH3(CH2)IoCD20H

MsCl CH3(CH2)IoCD2OMs NaCH(CO2Et)2 ; NaOH,H20 ; decarboxylation

CH3(CH2)IoCD2CH2C02H

LAH ; MsCI ; malonate extension > CH3(CH2)IoCD2(CH2)3C02 H Fig. 1. Malonate chain extension: Synthesis of [5-2Hz]hexadecanoic acid.

B. Malonate as Coupling Unit A number of gem-dideutero oxohexadecanoic and octadecanoic acids, in which the dideuterated carbon was separated from the oxo group by two CH2 groups, was prepared by reaction between tetrahydropyranyl sodio alkylmalonate and a half ester acid chloride. 122'129Synthesis of methyl 9-oxo-[12-2H2]hexadecanoate 129and its conversion to methyl [12-2HE]hexadecanoate is shown in Fig. 2. When an oxo acid with a dideuterated carbon in the chain between the oxo and carboxyl groups was required, a ketone was synthesized from a 2,2-dideutero unsaturated acid chloride and an alkyl malonate and the carboxyl group introduced by cleavage of the double bond. In this way, 8-oxo-[5-2H2], 1 l-oxo-[8-2H2] and 12-oxo-[9-2HE]OCtadecanoic acids were prepared. 122 C. Chain Extension with Enamines An acid chloride allowed to react with a morpholinocycloalkene (either -cyclopentene, -cyclohexene or -cyclododecene) yields, after removal of the morpholino group, a fl-diketone which on alkaline cleavage gives a 6-, 7- or 13-oxo acid. The chain length of an acid is thus extended by 5, 6 or 12 carbons. The oxo acids obtained have been converted to the corresponding dideuterated acids by metal deuteride reduction as described earlier j22 but this enamine reaction can also give oxo acids with deuterium on a carbon separated from the oxo group by two CH 2 groups. Synthesis of 13-oxo-[16-2HE]OCtadecanoic acid 122 is shown in Fig. 3. Deuterium was introduced by ct exchange, and two carbons added by malonate extension because the deuterated carbon must be separated from the carbonyl

TABLE 3. Deuterated Acids Synthesized from Readily Available or Naturally Occurring Acids Acid Octanedioic Nonanedioic 10-Undecenoic (2 R)-2-Hydroxyhexadecanoic 16-Hydroxyhexadecanoic (Z)-6-Octadecenoic (Z)-9-Octadecenoic 12-Hydroxy-(Z)-9-octadecenoic (Z)-9-Octadecen- 12-ynoic

12,13-Epoxy-(Z)-9-octadecenoic 17-Hydroxy-(Z)-9-octadecenoic 17-Hydroxyoctadecanoic 18-Hydroxyoctadecanoic

Deuterated acid [8-2HE]-(Z)-9-octadecenoic [7-2Hzl-(Z)-9-octadecenoic [18-2H3]-(Z)-9-octadecenoic [16-2H3]hexadecanoic [9-2Hz]octadecanoic [2S-2-2H] and [2R-2-ZH]hexadecanoic [16-2H3]hexadecanoic [ 15-2H2]hexadecanoic [5-2H2]octadecanoic [8-2H2]octadecanoic [ 12-2HJoctadecanoic [12,13-2Ha]-(Z)-9-octadecenoic [9,10,15,15,16,16-2Hj-(Z) - 12-octadecenoic [17-2Hz]-(Z)-9-octadecenoic [ 17-2H2]octadecanoic [ 18-2H3]octadecanoic

References 127 127 125 121 122 128 130 130 122 122 122 45 89 125 122 54

242

A.P. Tulloch

CH3(CH2)3CH2CO2Na LAH

NaOD,D20 ~- CH3(CH2)3CD2C02H

> CH3(CH2)3CD2CH20H

MsCl ; NaCH(CO2Et)2 ; >

NaOH,,H20 >CH3(CH2)3CD2CH2CH(CO2H)2 DHP; Na,CIOC(CH2)7CO2Me

> CH3(CH2)3CD2(CH2)2CO(CH2)7CO2Me

TsHNNH2,NaBH3CN > CH3(CH2)3CD2(CH2)IoCO2Me DMF Fig. 2. Malonate as a coupling unit: Synthesis of methyl [12-2H2]hexadecanoate. formed in the reaction to avoid back exchange. Reduction of the tosylhydrazone as before gave methyl [16-2H2]octadecanoate.~-'2 [18-2H3]Octadecanoic and [18-2H3]oleic acids have also been obtained using enamine synthesis with morpholinocyclohexene;66 in these preparations, the carbonyl group was removed by Wolf-Kishner reduction. Yields in this enamine reaction range from 40 to 70°,~,.

D. Anodic Synthesis When a mixture of an acid and a dicarboxylic acid half methyl ester is electrolyzed, the anions decarboxylate giving radicals which couple randomly. The desired product results from cross coupling of dissimilar radicals; alkane and dicarboxylic acid are by-products from coupling of similar radicals and may be partly suppressed by using an excess of the less valuable acid. Anodic coupling was one of the first reactions to be applied to synthesis of deuterated acids 35 and synthesis of methyl [10-2H2]tetradecanoate sj is shown as an example in Fig. 4. [2-2Hz]Hexanoic acid was electrolyzed with methyl hydrogen sebacate in the presence of 2~o of the NaOMe required to neutralize the acids; most of the gem-dideuterotetradecanoic

CH3CH2CH2CO2Na

NaOD,D20 > CH3CH2CD2CO2H

LAH ; MsCl ; malonate extension ~CH3CH2CD2(CH2)2CO2H

(COCl)2. CH3CH2CD2(CH2)2COCI + Et3N ; H+

~

N

__/O

F~----~O CO(CH2)2CD2CH2CH3

NaOH ; MeOH,H+ CH3CH2CD2(CH2)2CO(CH2)ICO2Me TsHNNH2,NaBH3CN > CH3CH2CD2(CH2)I4CO2Me DMF Fig. 3. Enamine chain extension: Synthesis of methyl [16-2H2]octadecanoate.

Specifically deuterated lipids

CH3(CH2)3CH2COzMe

MeOD,NaOMe

243

CH3(CH2)3CD2CO2Me

repeated three times

D20,MeOD,NaOMe ~, CH3(CH2)3CD2CO2H HO2C(CH2)8CO2Me > CH3(CH2)3CD2(CH2)sCO2Me MeOH,NaOMe,electrolysis Fig. 4. Anodic synthesis: Preparation of methyl [10-2Hz]tetradecanoate. acids were prepared by this reation, sl Electrolysis of perdeuterated stearic and palmitic acids with methyl hydrogen malonate gave perdeuterated acids with two protons on C-2, only one carbon was added to the chain length. 38 Though the reaction is simple and contains essentially only two steps, yields are only 20-40~o and often lower when unsaturated acids are involved. One of the most convenient uses is preparation of terminally trideuterated acids, such as [18-2H3]octadecanoic acid, by electrolysis of commercially available [2H4]acetic acid with methyl hydrogen octa-

decanedioate.35'54 E. Acetylene as a Coupling Unit

Olefinic acids were often prepared from acetylenic intermediates before application of this route to synthesis of deuterated analogs) 3 Availability of lithium acetylide (ethylene diamine complex) and of co-halo alcohols and development of better methods of alkylating triple bonds have much improved this method. Synthesis of methyl [14-2H2]-oleate is shown in Fig. 5. Deuterium was introduced by ~t exchange of hexanoic acid, and the chain lengthened by malonate extension to [4:H2]octanoic acid which was then converted to

CH3(CH2)3CH2C02H as in Fig.2; ,~decarboxylation

HOCH2(CH2)6CH20H

CH3(CH2)3CD2(CH2)2CO2H LAH; Ph3PBr2

BrCH2(CH2)6CH2OH

I I

HBr

DHP; LiC~ CH.H2N(CH2)2NH2

~

HC~C(CH2)7CH2OTHP BuLi,DME; MeOH,TsOH

CH3(CH2)3CD2(CH2)3Br

CH3(CH2)3CD2(CH2)3C~C(CH2)7CH20H CrO3,CH3COCH3;

I

MeOH,H+

CH3(CH2)3CD2(CH2)3C~C(CH2)7CO2Me

I

H2,Lindlar catalyst

CH3(CH2)3CD2(CH2)3CH=CH(CH2)7C02 Me Fig. 5. Acetyleneas a coupling unit: Synthesis of methyl [14-2H~-(Z)-9-octadecenoate. JPLR 22/4

a

244

A.P. Tulloch

[4-2H2]octyl bromide. The bromide was allowed to react with the lithium derivative of the THP ether of 9-decynol, prepared from octanediol, to yield [14-2Hz]-9-octadecynol. Oxidation to the octadecynoic acid and Lindlar reduction then gave the required product. The overall yield was about 30°Jl,.t25t2~ Most of the gem-dideuterated oleic acids were obtained by this route, a common intermediate was employed for some; thus, LAD reduction of 5-tetradecynoic acid and extension led to [5-2H2]oleic acid or conversion to [2-2H2]-(Z)5-tetradecenoic by double bond reduction and exchange yielded [6-2H2]oleic acid by extension? 27 In general, it was best to retain the triple bond to the last step because acetylenic intermediates were crystalline and not liable to isomerize. Though long, the method has the advantage that the product has high isotopic purity and usually consists of 98-99%o of the Z isomer. Acetylenic coupling was also involved in a synthesis of [1 1-2H2]linoleic acid in which the deuterated carbon was derived from [2H2]formaldehyde.~'9 There was no loss of deuterium from carbons next to the unsaturation, during Lindlar reduction or in earlier stages of these reactions. ~19,127

F. Coupling by the Wittig Reaction This reaction, in which a phosphorane derived from a triphenylalkyl phosphonium halide is allowed to react with an aldehyde to yield an olefin directly, is an obvious choice for synthesis of unsaturated compounds. In combination with homogeneous catalytic deuterium reduction, its application has yielded about 12 tetra- or hexadeutero octadecenoic and octadecadienoic acids. 44 Synthesis of methyl [9,10,15,15,16,16-2H6]-(Z) - 12-octadecenoate, in Fig. 6, illustrates the method. Reduction of 3-hexynol introduced four deuterons and two further steps gave the phosphonium iodide. Treatment of the latter with sodium methoxide gave a phosphorane which, on reaction with a vic-dideuterated aldehyde, gave hexadeuterooctadecenoate, s9 The aldehyde was prepared by lead tetraacetate cleavage of the dideuterated diol derived from vernolic acid. 89 The product contained 88~o Z isomer and 12% E isomer which were separated on a silver resin column. ~ Deuterium scattering has occurred in Wittig reactions, carried out under these conditions, when the aldehyde was labeled in the c~-position but use of t-butyl lithium as base

CH3CH2C~ CCH2CH20H I DHP,H

CH3CH2C~CCH2CH2OTHP I D2,(Ph3P)3RhCI CH3CH2CD2CD2CH2CH2OTHP

Vernoniaanthelminticaseedoil I AcOH;KOH,MeOH;

MeOH,H+ CH3(CH2)4CHOHCHOHCH2CH=CH(CH2)7CO2CH 3 I D2,(Ph3P)3RhCI

I NaI,H3PO

CH3CH2CD2CD2CH2CH2I

CH3(CH2)4CHOHCHOHCH2(CHD)2(CH2)7CO2CH 3

I Ph3P

CH3CH2CD2CD2CH2CH2PPh31

I Pb304,AcOH

OCHCH2(CHD)2(CH2)7CO2C3H NaOMe,DMF

CH3CH2CD2CD2CH2CH=CHCH2CHDCHD(C7C02Me H2) Fig. 6. Coupling by the Wittig reaction: Synthesis of methyl [9,10~15,15,16,16-2H6]-(Z)-I2octadecenoate.

Specifically deuterated lipids

A. CH3(CH2)8CO2Me HBr;Mg

LAD

245

CH3(CH2)sCD20H

CH3(CH2)8CDzMgBr

,Br(CH2)sCO2MgCl >CH3(CH2)BCD2(CH2)5C02 H Li2CuCI4;H+ NaBD4

B. MeO2C(CH2)IICOCl ~-MeO2C(CH2)IICD20H dioxane TsCl Li(CH3)2Cu :~MeO2C(CH2)IICD2OTs -20 °

CH3CD2(CH2)IICO2Me Fig. 7. Copper catalyzed coupling: (A) synthesis of [7-2H2]hexadecanoic acid; (B) synthesis of methyl [13-2H2ltetradecanoate.

eliminated this difficulty. 32'33 Though it has not been done in fatty acid synthesis, other work with cyclohexane derivatives l° and acyclic terpenes lz has shown that it is possible to label just one of the double bond carbons with deuterium by using a [1-2H]aldehyde. The major disadvantage of the Wittig reaction in general is that the product contains both Z and E isomers which have to be separated. The conditions which have been reported for preparation of deuterated alkenoic acids are based on those of earlier syntheses of natural Z unsaturated acids ~4 and still yield about 10~ of E isomer. More recently, use of sodium 15 or potassium 1°7 bis(trimethylsilyl)amides has given good yields of olefins containing up to 98~o of Z isomer, in fact quite comparable with those obtainable by Lindlar reduction.

G. Copper Catalyzed Coupling There have been two syntheses of deuterated saturated acids by copper catalyzed coupling. One of these, preparation of [7-2H2]hexadecanoic acid, 29 is shown in Fig. 7a. Deuterium was introduced by LAD reduction of methyl decanoate and the Grignard reagent from the derived bromide was then coupled, by LiCuCI4 catalysis, with the magnesium chloride salt of 6-bromohexanoic acid. The yield in the coupling reaction was about 80~o. The other reaction ~36 is shown in Fig. 7b. The half ester acid chloride from tridecanedioic acid was reduced with NaBD4-dioxane as described above (Section III. A. 2,3) to the dideutero hydroxy ester. The tosylate was formed, and when coupled with lithium dimethyl cuprate gave the C13 ester dideuterated on the penultimate carbon. This type of coupling reaction has obvious advantages, for syntheses of saturated acids, when intermediate oxo acids are not required, and will probably be applied more frequcntly in the future.

H. Tables of Deuterated Acids Deuterated octadecanoic acids were listed in the previous review ~26and very few more have since been reported; a large number of deuterated tetradecanoic and hexadecanoic acids, however, have been synthesized and these are listed in Table 4. Deuterated unsaturated 18-carbon acids are shown in Table 5, most of which were not listed previously. ~26 Unsaturated deuterated acids of chain lengths other than 18 carbons are shown in Table 6; a number of deuterated eicosatetraenoic acids related to arachidonic acid and its metabolites have been reported recently.

246

A.P. Tulloch TABLE4. Deuterated Tetradecanoic and Hexadecanoic Acids* Chain length

Position of deuterons

14 14 14 14 14 14 14 14 14 14 14 14 14 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16

,~,~ ~ 2 2 7,7,9,9+ 3.3 4,4

16

AI1 D

5,5,7,7++ 6,6 8,8 10,10 12,12 13 13,13 14,14,14 All D (2S)-2 (2R)-2 "~-' 3,3 4.4 5,5 5,5,6,6 6,6 7,7 7,7,8,8 8,8 9,10 10,10

11,11,12,t2 12,12 13,13 14,14 15,15 16,16,16

References 1,50,75,81 80 76,81 81 86 81 81 81 81 136 136 81,135,136 57,63 128 128 ~ 9 "~~ .5.30,75,10~,1.. 50.76 1,50 102,129 92 129 29 92 129 4 74,129 92 129 122 129 130 92,121,130, 135 39,93

*Isotopic purity was not reported in many preparations but the methods suggest that for products with one or two deuterons it is at least 95'!,,. +Two ~F substituents at C-8. ++Two ~F substituents at C-6.

v. L I P I D S Y N T H E S I S P r e p a r a t i o n s o f a large n u m b e r o f lipids, r e p r e s e n t i n g at least six types, have b e e n d e s c r i b e d b u t m o s t p r e p a r a t i o n s are c o n c e r n e d w i t h p h o s p h a t i d y l c h o l i n e , d e u t e r a t e d in v a r i o u s p o s i t i o n s . In general, m e t h o d s p r e v i o u s l y e s t a b l i s h e d for synthesis o f u n l a b e l e d lipids h a v e b e e n used.

A. Lipids Not Containing Phosphorus 1. Triacylglycerols and Cholestervl Esters D i r e c t a c y t a t i o n o f glycerol ( h y d r o x y l p r o t o n s e x c h a n g e d first) with [2-2H2]tetra decanoic, [3-2H2]pentadecanoic, [4-2H2]hexadecanoic, [5-2H2]heptadecanoic and [6-2Hz]octadecanoic acids gave the triacylglycerols. ~ S y n t h e s e s o f t r i a c y l g l y c e r o l s f r o m [5-2H2], [8-2H2], [1 1-2H2], [16-2H2] a n d [18-2H3]oleic acids were r e p o r t e d . 5~ T r i a c y l g l y c e r o l s were also p r e p a r e d f r o m [12,13-2H4]oleic 45 a n d f r o m [9,10,15,15,16,16-2H6] - 1 2 - o c t a d e c e n o i c acids b y a c y l a t i o n o f g l y c e r o l ? 9 T r e a t m e n t o f glycerol with the acid c h l o r i d e o f [9,10-2H2] - 1 2 Z , 1 5 E - o c t a d e c a d i e n o i c acid yielded the triacyiglycerol.90 R e a c t i o n o f the a c y l i m i d a z o l e f r o m [2-2H2] a n d fully d e u t e r a t e d h e x a d e c a n o i c acids with cholesterol gave cholesteryl esters in g o o d yields. 52

Specifically deuterated lipids

247

TABLE 5. Deuterated 18-Carbon Unsaturated Acids Position and nature of unsaturation

Position of deuterons

2Z,E 6Z 6Z 8Z,E 8Z,E 9Z 9Z 9Z 9Z 9Z 9Z 9Z 9Z 9E 9Z 9Z 9Z 9Z 9Z 9Z 9Z 9Z,E 9Z 9Z 10Z,E 10Z,E 10Z,E

2 2,2 6,7 17,18 13,13,14,14 2,2 3,3 4,4 5,5 6,6 7,7 8,8 9,10 9,10 11,11 12,12 14,14 16,16 17,17 18,18,18 8,8,11,11 13,13,14,14 9,10,13,13,14,14 14,14,15,15,17,18 13,14 14,15 14,14,15,15

11Z

11,12

IIZ,E 11Z,E 1IZ,E 11Z,E 12E 12Z,E 12Z,E 13Z 13Z 9Z,12Z 9Z,12Z 12Z,15Z; 12E,15Z 12Z,15E; 12E,15E

15,16 15,15,16,16 10,10,15,15,16,16 14,14,15,15,17,18 9,10 15,15,16,16 9,10,15,15,16,16 17,18 17,17,18,18 11,11 16,16,17,17 9,10 9,10

Isotopic purity 91 97 ? 92 93 98 ? ? 97 98 98 97 96 87 98 95 96 98 96 97 79 87 87 84 89 76 85 84 89 83 71 86 85 89 80 93 87 98 96 87 85

References 99 122 7 5 5 51,75,122 127 127 127 127 127 127 7,22,76,129 45 120,127 125 125 125 125 66,125 22 4 4 2 34 34 34 7

33 33 33 34 89 89 89 5 5 119 3 90 90

B. Deuterated Phosphatidylchol&es D e u t e r a t e d f o r m s o f p h o s p h a t i d y l c h o l i n e with d e u t e r i u m either in the h e a d g r o u p , o r the glycerol o r one o r b o t h o f the acyl chains, have been synthesized. A p e r d e u t e r a t e d p h o s p h o l i p i d was also synthesized in which every p r o t o n in all p a r t s o f the m o l e c u l e was r e p l a c e d b y deuterium.

1. Phosphatidylcholines Labeled in Choline or Glycerol [1-EH2] a n d [2-2H2]cholines as well as choline with o n l y one t r i d e u t e r o m e t h y l g r o u p were synthesized a n d i n c o r p o r a t e d into p h o s p h o l i p i d . 49 O n e c o m m o n p r o c e d u r e for p r e p a r a t i o n o f p h o s p h a t i d y l c h o l i n e labeled in the h e a d g r o u p is a l k y l a t i o n o f p h o s p h a t i d y l e t h a n o l a m i n e , either egg-derived o r synthetic, with t r i d e u t e r o m e t h y l iodide.74.111.114 One p r e p a r a t i o n investigated the m o s t effective m e t h o d o f c a r r y i n g o u t the m e t h y l a t i o n . 8z

2. Deuteration in Both Acyl Chains In these syntheses, g l y c e r o l p h o s p h o r y l choline was p r e p a r e d a n d acylated. F a t t y acids with v a r i o u s degrees o f d e u t e r a t i o n were e m p l o y e d ; in one e x a m p l e , the acids

248

A . P . Tulloch TABLE 6. Deuterated Unsaturated Acids with Chain lengths 11 to 22 ( a r b o n s (except 18 Carbons) Chain length I1 12 13 13 14 15 16 16 16 17 20 20 20 20 20 20 20 20 22

Position and nature of unsaturation 10 3Z,E 3Z.E 12 3 Z. E 3Z,5Z,TE,9Z 3Z, E 9Z 9Z

5Z.8Z, IOZ,E* 8Z 11Z 5Z,8Z, 11Z, 14Z 5Z,8Z,10Z,E,14Z* 5Z,SZ,11Z,14Z 5Z,8Z, IIZ,14Z 5Z,SZ,11Z,14Z 6E,gZ,11Z 14Zt 13Z

Position of deuterons ..~'~'~ 2 2 4,4 2 9,10 2 15,15 16.16,16 5,6,8,9 4,4 4,4 20.20,20 5,6,8,9

5,68,9,11,12.14,15 5,68.9 11 12,14,15 5.6,8,9,11,12,14,15

5.6,8,9,11,12,14,15 2,2

Isotopic purit 5 95 92 93 95 93 '.' 90 99 99 '., 97 9S '., 7 86 '.' 5~ 6(1 ?

References 122 ~)9 9~I 122 ~)9

99 130 130 9~ 122 t22 66 '~ 41 iv7 ! I~ % 75

*Hydroxyl substitution at C-12. tHydroxyl substitution at C-5.

were perdeuterated C~4,TM in others they were [16-2H3]hexadecanoic2" [2-2H2] or [5-2H2]hexadecanoic, l°z or [10-2H2]hexadecanoic. TM A synthesis of a phosphatidylcholine, fully deuterated in every position in the choline, glycerol and two tetradecanoyl chains, 72 in all, has been described. 6xThe glycerol portion was prepared by LAD reduction ofdiethyl oxomalonate and converted to the 3-phosphate by glycerokinase. Perdeuterated tetradecanoic acid was prepared by the usual exchange procedure 39 and, as the anhydride, used to acylate the glycerophosphate. Deuterated ethanolamine was obtained by LAD reduction of ethyl cyanoformate and alkylated with trideuteromethyl iodide. 3. Deuteration in One Ao, l Chain Onh' Both these types of phosphatidylcholines were synthesized by means of 2-1yso compounds, thus starting with a phosphatidylcholine with two normal chains and, replacing that at position 2, gave a product with label in the 2-chain. Starting with two labeled chains and replacing the 2-chain with an unlabeled acyl group gave a product with the chain at position 1 labeled. Thus, there were several syntheses of phosphatidylcholine with a [16-2H3]hexadecanoyl group at position 1 and the 2-acyl group was either (Z)-9-hexadecenoyl28,~'~or docosahexaenoyl. 3~ In another synthesis, either the 1- or 2-acyl group was perdeuterated hexadecanoyl. 4s Phosphatidylcholines have been prepared in which the labeled chain at position 2 was [2-2H2], [3-2H2] or [7,8-2H4] or [13-2H2] or [16-2H3]hexadecanoyP ~4 or various dideutero tetradecanoyl groups sl or dideutero (Z)- or (E)-9-octadecenoyl. "'s There have been two preparations of phosphatidylcholines with doubly labelled chains at position 2. In one, this chain was [2,2,7,7,9,9-2H6-8,8-tgF2]tetradecanoyl s° and in the other it was [1-13C]hexadecanoyl dideuterated at either C-4, C-8 or C-12. ~s C. Other Deuterated Phospholipids Deuterated phosphatidylethanolamines with 2 hexadecanoyl groups were synthesized with [1-ZH2], [2-2H2] and [l,2-2H4]ethanolamines from phosphatidylcholine using phospholipase D. lm Phosphatidylserines with [2-2H2] or [3-2H2]serine 2° and also with 2 hexa-

Specifically deuterated lipids

249

decanoyl or 2 tetradecanoyl groups have been prepared. 21 Preparations of phosphatidylserines with 2 hexadecanoyl chains, dideuterated at C-2 or C-3 or C-6 or C-10 or C-14, or with 2 [9,1 0-2H2]oleyl chains have also been described. 21Phosphatidylcholine with unnatural head groups, [1,3-2H2]propanediol and [l,2-2H4]ethanediol and with 2-tetradecanoyl chains have also been synthesized. 19

vI. ANALYSIS Any discussion of the synthesis of deuterated lipids should consider the isotopic purity of the product, that is the percentage of the product which is labeled to the required extent. Many preparations have been reported without any indication of isotopic purity or if a value is given the method of obtaining it is not described.

A. Analysis by Mass Spectrometry Most of the spectroscopic analyses of deuterated lipids which have been reported have been by mass spectrometry. In assessing accuracy of MS results, decisions have to be made about the type of mass fragmentation to be examined and assumptions have to be made about what the results mean. These considerations have been discussed for deuterated molecules in general 16,H7and for perdeuterated long-chain acids in particular. 67,134Usually only the molecular ion is examined and it is assumed that: (a) deuterated and nondeuterated compounds will ionize equally readily, (b) unexpected M - 1 or M + 1 ions will not appear in the spectrum of the deuterated compound and (c) there will not be preferential loss of deutero or protio component in the spectrometer, due perhaps to secondary isotope effects, or if GC-MS is employed in the GC due to partial fractionation. The M ion is preferred because fragment ions may not be formed equally readily, but difficulties arise when M is very small in the particular MS system used. Sometimes this is the case with polyunsaturated methyl esters such as arachidonate, and here the t-butyldimethylsilyl ester has been prepared and the M-57 peak examined. 41 TMS esters of acids, and ethers of the corresponding alcohol, give strong M- 15 fragments and isotopic purities obtained with these derivatives for [2-2H]hexadecanoic acid were very similar to those obtained using the M ion of the methyl ester. 128 In studies of MS of dideuterated octadecenoic acids, pyrrolidides sometimes gave more intense molecular ions. 6'7 The above assumptions seem reasonable for deuterated long chain compounds, particularly those which contain only a few deuterons and a large number of protons. The contribution of 13C to M + 1 peaks has to be allowed for in calculating isotopic purity for partially deuterated compounds.16 The particular case of this effect in perdeuterated esters has been discussed. TM If the product has high isotopic purity and contains only a few deuterons, then the contribution of 13C can be neglected since the fraction of the product with one less than the required number of deuterons is very small. More recently, analysis of deuterated products has been by GC-MS with multiple ion detection 64 where most of the data collecting ability of the instrument is concentrated on a few peaks with m/z close to that of M. Field desorption MS has also been applied to measurement of isotopic purity with good results since M is usually the base peak; it is particularly suited to MS of normally nonvolatile phospholipids. 65Analysis of a deuterated hexadecanoic acid, in which carbons 9-16 were fully deuterated, by MS was confirmed by the Raman spectrum. 7~ While MS estimations, under suitable experimental conditions, most probably give accurate results, it is preferable to have an alternative procedure which will give an independent estimate of isotopic purity. Moreover, MS is quite unreliable as an indication of deuterium position because of deuterium scattering in the spectrometer and because the relative intensities of the fragments are influenced by deuterium isotope effects on bond strength. 117

250

A.P. Tulloch

B. Analysis by Nuclear Magnetic Resonance Spectroscopy 1. ~H NMR Spectroscopy Disappearance of signals of protons ~ to carbonyl groups is readily followed by ~H NMR.~2°'~22 Fairly quantitative measurements of isotopic purity can be made by comparing intensities of these ~H signals and of those due to CH2OH and CH2OMs, in derived compounds, with those due to OCH3 and terminal methyl, if suitable experimental conditions are employed. 54,~7,~2~ The accuracy of the method applied to [3-ZH2] and [2-2H2]methyl esters has been increased by using shift reagents so that the intensity of residual protons at C-3 can be compared to that of protons at C-2, for the former ester. and intensities of residual protons at C-2 can be compared to those of protons C-3, for the latter ester. 75'76 Use of Eu(fod) 3 displaced signals of protons on C-2, C-3 and C-4 downfield but, when deuterated olefinic acids were examined, Pr(fod)3 was added to displace the signals upfield and so avoid overlap with signals of double bond protons. ~57~ 2. 2H NMR Spectroscopy Though deuterium N M R has not been employed in investigations of deuterated products to any extent because of low sensitivity, it has some advantages. This is particularly so when incorporation is low and isotopic distribution is required. Due to low sensitivity, observations were made under FT conditions with full ~H decoupling (2H-2H coupling was not observed) and Eu(fod)3 was added to displace the signals of deuterium on carbons 2, 3, 4 and 5. 84 Incorporation of deuterium into the terminal methyl group was thus compared with that in the methylene groups, z6

3. ~3C NMR Spectroscopy Since the chemical shift range in ~3C N M R is much greater than in IH NMR, and even in long-chain compounds many carbons have separate shifts, investigation of deuterium labeling by 13C N M R has some advantages. The ~3C signal of a CDH group is a triplet, due to 13C-ZHcoupling, displaced upfield from the shift of a CH2 group; integration of this signal can show the extent of monodeuteration.~°9 Signals of carbons with 2 or 3 deuterons are not seen in normal spectra TM due to very long spin lattice relaxation times (T~, probably > 40 sec for CD3129)and because of extensive splitting due to ~3C-2H coupling. Deuteration could be estimated by the absence of particular signals, as in ~H NMR, but there seem to have been no reports of this procedure as a quantitative method. To avoid some of these difficulties, another method was employed 59'6~ in which 2H coupling, as well as ~H coupling, was removed by heteronuclear decoupling. Also Tt was made very short by addition of a paramagnetic relaxation agent, chromium acetylacetonate, which provided an alternative relaxation mechanism, hexafluorobenzene was added to give an internal 19F lock. Since each deuterium substituent caused an upfield displacement of 13C shift of 0.3-0.5 ppm, separate signals were seen for each deuterated species and, for the undeuterated carbon, integration gave an accurate measure of extent of deuteration. This procedure can probably only be applied to well separated signals, that is to those of C-2, C-3 and the last 3 carbons of a fatty acid chain. So far, it has been used to analyze biosynthetically labeled compounds $9'68 but it should be applicable to measurement of isotopic purity resulting from chemical labeling.

C. Analysis by GC and HPLC It is well known that during GC analysis the more highly deuterated components have shorter retention times than the less deuterated 37,67'83'~°8,H7A34and in theory this might be a way of estimating isotopic purity. In practice, however, it seems that complete, or even partial, resolution has not been achieved except of perdeutero from undeuterated esters, 3v even using capillary G C ? 3 Separation of partially deuterated methyl hexadecanoate from undeuterated ester by HPLC has been described but degree of deuteration was not reported. 27

Specificallydeuterated lipids

251

vii. APPLICATIONS OF DEUTERATED LIPIDS

A. 1H NMR Spectroscopy Since investigation of motion of small molecules by 1H NMR in a phospholipid bilayer is difficult against a background of phospholipid protons, a fully deuterated phosphatidylcholine was prepared so that the ~H signals could be seen. 63 Polyenoic compounds such as hydroxyeicosatetraenoic acids, have complex IH NMR spectra in which signals of protons on the double bond carbons are not well resolved but preparation of derivatives in which some of these protons have been replaced by deuterons considerably simplifies the spectra. 98

B. Estimation of Conformation and Order in Lipid Bilayers by ZH NMR, FTIR and Raman Spectroscopy 1. 2H NMR Spectroscopy Deuterium introduced into a molecule as a "spectroscopic probe" has several advantages over other groups. It is very small and causes little perturbation, the spectrum is simple and dipolar couplings are much reduced due to the smaller magnetic moment of the deuteron. Anisotropic motion is readily detected by 2H NMR, since though for small molecules rapid isotropic motion gives a one-line signal for a deuteron, for large molecules, like phospholipids which undergo anisotropic motion, the signal of each deuteron is a doublet due to the 2H quadrupole moment, l°° Determination of signal splitting for CD2 groups at a number of positions along an acyl chain in a phospholipid shows that splitting is greatest where the degree of order, or rigidity, is greatest, which is in the half of the chain closest to the phosphoryl glycerol head group. Greater motion is possible near the end of the chain and in consequence the splitting and degree of order are much smaller. 1°4 In earlier studies, specifically deuterated acids were examined in model membranes t'2 but later labeled palmitic acids "° and oleic acids9~ were biosynthetically incorporated into the membrane of the microorganism, Acholeplasma laidlawii. Results with natural membranes agreed with those obtained with models; the effects of temperature on liquid crystal and gel phases were also studied. 9~ It had been found that 2H NMR spectra of phospholipids dideuterated at C-2 of the sn-2 acyl chain usually contained two quadrupole couplings. ~°2,1°4Measurement of the spectrum of phosphatidylcholine in which the 2-acyl chain was [2R-2-ZH]hexadecanoyl showed that the two couplings arose from two nonequivalent deuterons.46 The influence of Z and E double bonds in the acyl chain of phosphatidylcholine was studied with oleic acid derivatives labeled at several positions. ~°3 The conformation of phosphatidylethanolamine in gel and liquid crystal phases has been examined by double labeling in the 2-hexadecanoyl chain, 13C in the carboxyl group and ZH at either C-4, C-8 or C-12. ~8 The effect of addition of cholesterol on the gel phase of this system was also determined.~7 Studies of phosphatidylcholine labeled with both ZH and 19F showed that the disturbing effect of the CFz group was intramolecular rather than intermolecular.8° Effects of temperature on micellar and crystalline phases in soap solution were examined using perdeuterated and ~-dideuterated potassium palmitate; at higher temperatures, there was an overall decrease in the order of the chain. 3°

2. FTIR and Raman Spectroscopy FTIR spectra can also provide information about physical properties of fatty acid derivatives. 1°5 Anomalous behavior was observed in the C-D stretching regions of spectra of specifically dideuterated hexadecanoic and octadecanoic acids when substitution was near the end of the chain? 6 Vibrational spectra were assigned for phosphatidylcholine in which the 2-hexanoyl chain was deuterated, t14 and IR spectra of multibilayers of this lipid indicated the strength of interchain interactions. 24 JPLR

22/4-

(-

252

A . P . Tulloch

2H N M R and Raman spectra were compared as methods of investigating fluidity and order in the regions of CH3 o r C D 3 groups of the acyl chains; when applied to phosphatidylcholines with either both chains [16-2H3]hexadecanoyl or [10-2H2]hexadecanoyl, the sensitivities of the methods were not identical. 69 Vibrational modes were assigned in Raman spectra of phosphatidylcholine with perdeuterated C~6 acyl chains and also N-CD 3 groups. 23 Raman spectra of phosphatidylcholines, with a perdeuterated chain either in position 1 or in position 2, showed that the conformations of the two chains were nonequivalent. 4s Other Raman spectral studies of chain conformation, in phosphatidylcholines with perdeuterated Cl6 chains, 7° have shown that it is much altered in the vicinity of protein. ~6 Variation in Raman spectra with CD~ position in the 2-tetradecanoyl chain of phosphatidylcholine has been reported. ~

C. Interpretation of MS, 13C N M R and Other Spectral Applications 1. M S One of the earliest applications of deuterated lipids to spectral interpretation was to MS: hydrogen atom rearrangements are common in MS of methyl esters and use of specifically dideuterated esters explained the formation of the prominent ions at m/z 74 and 87. ~ One major disadvantage of MS has been that it could not determine double bond position, but derived vic-dideutero esters gave MS in which the position of the C H D C H D grouping was clearly indicated. 6'35 Olefinic acids with deuterium on the double bond carbons were used in a study of double bond migration during MS, more informative spectra were obtained with pyrrolidides rather than methyl esters. 7 Earlier conclusions about MS of triacylglycerols were confirmed and formation of a number of unexpected ions, particularly M-18, was explained from MS of triacyglycerols with deuterium in the acyl chains and in the glycerol. 1 2. ~3C N M R Spectra The ~3C N M R spectra, determined at 25.2 MHz, of long chain methyl esters, particularly those of saturated esters, are characterized by a larger number of unresolved signals. In the spectrum of methyl octadecanoate, signals due to C-7 to C-14 are not resolved; of the other signals, those due to C-I to C-3 and C-16 to C-18 could be assigned from spectra of smaller molecules but those due to C-15 and C-4 to C-6, which are separated by only 0.3 ppm, could not be assigned without further investigation. TM It has been suggested that signals could be assigned by measurement of relaxation times, since these tend to increase towards the ends of the chain, but this can only be done if signals are sufficiently resolved; also use of deuterated decanoic acids has shown that some previous assignments based on Tls were incorrect. ~29 Use of specifically dideuterated esters has made possible unambiguous assignment of almost all signals in spectra of 18-carbon compounds. As mentioned above (Section VIB 3), a C D 2 carbon does not normally give an observable signal so that the deuterated carbon is assigned by the absence of signal. The C D 2 group also affects signals of the two carbons on each side, the isotope effect on the ~ carbons is - 0 . 2 0 to - 0 . 2 5 ppm (upfield) and on the/~ carbons is - 0.04 to - 0.07 ppm (upfield) and these signals are also broadened 2 to 3-fold by long range J3C to 2H coupling. TM From ~3C spectra of all the gem dideuterated octadecanoates, all the signals have been assigned in the spectrum of methyl octadecanoate and chemical shifts have been estimated for the unresolved signals, though those of carbons 11-13 could not be distinguished, k:3 The values are shown in Table 7. The shift of C-14 is of particular interest since it is upfield of those of C-9 to C-13. A spectrum of methyl octadecanoate at 90.5 MHz, which showed separate signals for each carbon so that relaxation times could be measured, confirmed the results in Table 7. j29 Most signals in spectra of all the isomeric oxo, hydroxy and acetoxy octadecanoates were assigned from the spectra of deuterated e s t e r s . L23'~24'132 Long range c~to 0 effects of the oxygen containing substituents were determined and can be used in structural studies on naturally occurring isomeric mixtures.

Specifically deuterated lipids

253

TABLE 7. 13C Chemical Shifts of Methyl Octadecanoate* Carbon

Chemical shift

Carbon

Chemical shift

1 2 3 4 5 6 7 8 9

174.21 34.14 25.01 29.21 29.31 29.50 29.64 29.69 29.72

10 11 12 13 14 15 16 17 18

29.73 29.76 29.76 29.76 29.71 29.41 31.97 22.73 14.11

*Measured at 25.2 MHz, chemical shifts are in ppm from tetramethylsilane.

All signals in spectra of methyl oleate and petroselinate were assigned from spectra of some gem dideuterated oleates so that long range effects of the double bonds could be calculated. 132The assignments were later confirmed by the spectra of oleates dideuterated at C-4 to C-8 and C-I 1, C-12, C-14 and C-16.125A27 Figure 8 shows the central portions of the spectra of methyl oleate and [7-2H2] and [14-2H2]oleates and illustrates the upfield displacement and line broadening effects of deuteration on the neighboring ~ and fl carbons.~29 Isotope effects of a deuterated carbon ~ to a double bond on the signals of the double bond carbons is slightly different from effects on signals of saturated carbons. In the spectrum of [8-2H2]oleate, the signal of C-9 was displaced upfield by only 0.1 1 ppm, or less

D4-2H2]OLEATE

~4,13, 15

15,15

[7- 2H2] OLEATE 4

15, 15 OLEATE 54

I

I

I

29,6

29.2

28,B

ppm

Fig. 8. 28.8-30.0 ppm region of 13C NMR spectra of methyl oleate, methyl [7-2Hz]oleate and methyl [14JH2]oleate. Spectra were measured at 25.2 MHz with a spectral width of 1000 Hz and horizontally expanded ten times.

A . P . Tulloch

254

than half the usual effect, but that of C-t0 experienced a downfield effect of 0.04 ppm and the line width of both signals approximately doubled: the same effects were seen in the spectrum [11--~H2]oleate. ~27 The result of these effects is that for [8-2H2]oleate the double bond carbon signals are 0.38 ppm apart but in [11-2H2]oleate they are only 0.09 ppm apart. The observation confirmed an earlier assignment of the double bond carbon signals o1" methyl oleate, t-~'~ Signals in spectra of the corresponding dideuterated 9-octadecynoates were also assigned from isotope effects and showed the long range effects of the triple bond. ~-7 Effects on signals of triple bond carbons were unusual, in spectra of [8-:H~] - and [1 l-2H2]-9-octadecynoates, that of the triple bond carbon ~ to CD, was displaced upfield only 0.06-0.09 ppm, though the line width increased three times, that of the other triple bond carbon was unaffected. There seem to have been no reports of confirmation of signal assignments in spectra of linoleate or linolenate using deuterated derivatives.

3. Circular Diehroism Specific rotations of c o m p o u n d s which owe their chirality to deuterium substitution arc very small and consequently optical purity, which depends on isotopic purity, cannot be estimated accurately. 54 When the chiral center was C-2 of an acid, circular dichroism was measured and the molecular ellipticity values gave a better estimation of optical purity. ~:~ Investigation of the circular dichroism of short ~-deuterated acids has shown that ~-deuterium substitution in a carboxylic acid makes a dissignate contribution to the Cotton effect 24

(Received 29 March 1983)

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