Preparation of isotopically labelled benzophenone-containing lipid analogues

Article abstract of DOI:10.1002/jlcr.996

Methods have been developed for isotopic labelling of the benzophenone-containing cholesterol analogue 1 and the fatty acid analogue 2 by use of deuteriated sodium borohydride derivatives. These procedures avoid the commercial catalytic tritiation which h

Full text of DOI:10.1002/jlcr.996

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2005; 48: 781–788.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.996
Research Article
Preparation of isotopically labelled benzophenone-
containing lipid analogues
Pingzhen Wang and Thomas A. Spencer*
Department of Chemistry, Dartmouth College, Hanover, NH 03755, USA
Summary
Methods have been developed for isotopic labelling of the benzophenone-containing
cholesterol analogue 1 and the fatty acid analogue 2 by use of deuteriated sodium
borohydride derivatives. These procedures avoid the commercial catalytic tritiation
3
3
which had previously been used to obtain H-1 and H-2. Copyright # 2005 John
Wiley & Sons, Ltd.
Key Words: benzophenones; cholesterol; fatty acids; deuteriation; borohydrides
Introduction
We have been preparing benzophenone-containing analogues of sterols1 and
phospholipids2 for use as photoaffinity labelling agents in studies of cellular
cholesterol efflux and HDL formation.3–5 In particular, the cholesterol
analogue 1, known as FCBP,3 and the fatty acid analogue 2 (Figure 1),2
were required in radioactive form for use as biochemical research tools. This
has been accomplished to date by commercial catalytic tritiation of the
brominated i-steroid derivative 31 and unsaturated acid 42, respectively.
However, these catalytic tritiations have been somewhat capricious, requiring
carefully controlled conditions to avoid undesired reduction of the benzophe-
none carbonyl group; they are also expensive. Therefore, we wished to
developed alternative procedures for isotopic labelling of 1 and 2 that could be
conducted in our own laboratories. This paper describes such procedures,
employing deuteriated borohydride derivatives.
Results and discussion
The method adopted for labelling of FCBP (1) was an adaptation of that of
Malik et al.6 for tritiation of cholesterol via mild pyridinium chlorochromate
*Correspondence to: T. A. Spencer, Department of Chemistry, Dartmouth College, Hanover, NH 03755,
USA. E-mail: taspen@dartmouth.edu
Contract/grant sponsor: USPHS NIH; contract/grant number: HL 67294
Copyright # 2005 John Wiley & Sons, Ltd.
Received 14 June 2005
Accepted 23 June 2005

782
P. WANG AND T.A. SPENCER
O
O
Br
O
O
Br
HO
1
3
OCH3
O
O
COOH
4
COOH
2
Figure 1. Structures of photoactivable lipid analogues and tritiatable derivatives
(PCC) oxidation to 5-cholesten-3-one followed by reduction with tritiated
sodium borohydride (NaB3H4). In applying this approach to 1, of course, it
was necessary initially to protect the benzophenone carbonyl group, which was
accomplished by treatment of 1 with acidic ethylene glycol to afford ethylene
ketal 5 (Scheme 1). Oxidation of 5 with PCC according to the procedure of
Parish et al.7 then afforded unconjugated ketone 6. In order to establish the
validity of this approach for introduction of isotopic labelling, NaB2H4 was
used to reduce 6 back to 3a-2H-5 (7), which was converted without purification
1
to 3a-2H-1 (8) by hydrolysis in aqueous acidic acetone. The H NMR spectra
of 1 and the 8 thus produced are shown in Figure 2, clearly demonstrating
essentially complete absence of 1H at the 3a position of 8. The single
2
absorbance at d 3.55 in the H NMR spectrum of 8 and the essentially
1
complete disappearance of a peak at d 72.0 in the H-decoupled 13C NMR
spectrum of 8 confirmed its 3a deuteriation. The appearance of 13C NMR
signals for carbon atoms bonded to H depends on experimental conditions
2
such as spin-lattice relaxation time, and can vary from a distinct multiplet to
no detectable signal; e.g. see, Cunnane et al.8
As an alternative to commercial catalytic tritiation of unsaturated acid 4 for
radiolabelling of 2, we decided to investigate benzylic bromination followed by
chemical reduction. This approach was initially explored with the 2:1 mixture
of monobromo 9 and dibromo 10 produced by treatment of ester 112 with 1.5
equivalents of NBS in CCl4 in the presence of benzoyl peroxide (Scheme 2),
since we were ultimately interested in radiolabelling phosphatidylcholine
analogues containing acid 2 esterified in the C1 or C2 acyl chain.2 When the
Copyright # 2005 John Wiley & Sons, Ltd.
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LABELLED PHOTOACTIVABLE LIPID ANALOGUES
783
O
O
HO
R
1, R = H
8, R = D
a
d
O
O
O
HO
R
5, R = H
7, R = D
b
c
O
O
O
O
6
˚
Scheme 1. (a) HOCH2CH2OH, PTSA, PhH, D; (b) PCC, CaCO3, 4 A mole-
cular sieves, CH2Cl2, rt, 30 min; (c) NaB2H4, CH3OH, rt, 1 h; and (d) 9:1
acetone:H2O, PPTS, D
mixture of 9 plus 10 was treated with NaB2H3CN in HMPA at 908C for 48 h,
1
according to the procedure of Hutchins et al.,9 the H NMR spectrum of the
product showed complete disappearance of the peaks at d 4.98 and 4.54
characteristic of the protons on the brominated positions of 9 and 10, and the
2H NMR spectrum of the product showed peaks at d 2.69 and 2.46, indicating
clearly the formation of a mixture of 12 and 13. This conclusion was confirmed
by comparison of the 13C NMR spectra of 12 plus 13 vs 11, which showed that
the singlets at d 36.2 and 31.4 in 11, had, in 12 plus 13, essentially disappeared
and diminished in intensity, respectively.
The reaction of acid 2 itself with NBS under the same conditions likewise
produced a ca 2:1 mixture of 14 and 15. Debromination of 14 plus 15 was
initially, but unrewardingly, attempted with zinc in acetic acid.10 Reduction of
14 plus 15 with NaB2H3CN, however, again proved successful, resulting in a
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P. WANG AND T.A. SPENCER
1
Figure 2. H NMR spectra of: (a) FCBP (1) and (b) 3a-2H-1 (8) showing
disappearance of peak for 3a-1H at d 3.55
O
R1
COOR3
R2
11, R1 = R2 = H, R3 = CH3
2, R1 = R2 = R3 = H
12, R1 = H, R2 = D, R3 = CH3
13, R1 = R2 = D, R3 = CH3
16, R1 = H, R2 = D, R3 = H
17, R1 = R2 = D, R3 = H
a
b
O
R1
COOR2
Br
9, R1 = H, R2 = CH3
10, R1 = Br, R2 = CH3
14, R1 = R2 = H
15, R1 = Br, R2 = H
Scheme 2. (a) NBS, CCl4, (PhCO)2O2; (b) NaB2H3CN, HMPA, 908C, 48 h
moderate yield of a clean mixture of 16 and 17, which showed changes in its
1H, 2H, and 13C NMR spectra completely analogous to those observed for 12
plus 13.
Copyright # 2005 John Wiley & Sons, Ltd.
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LABELLED PHOTOACTIVABLE LIPID ANALOGUES
785
Experimental
General methods
1H NMR and 13C NMR spectra were taken at 300 MHz in CDCl3 unless
otherwise indicated. Flash column chromatography was carried out on EM
Reagent silica gel 60 (230–400 mesh). Thin layer chromatography (TLC) was
performed on polyester sheets precoated with silica gel 60 F-254. All plates
were visualized by a UV254 light source or staining with 5% phosphomolybdic
acid in ethanol. Na2SO4 was used as a drying agent unless otherwise noted.
Methylene chloride was distilled from CaH2. Reagents were obtained from
Aldrich, and were used without further purification unless otherwise noted.
22-(p-2-Phenyl-[1,3]dioxolan-2-ylphenoxy)-23,24-bisnorcholan-5-en-3b-ol (5)
To a solution of 200 mg (0.39 mmol) of 1 and 0.11 ml (1.95 mmol) of ethylene
glycol in 5 ml of dry benzene was added 4 mg (0.02 mmol) of p-toluenesul-
phonic acid (PTSA). The mixture was heated at reflux overnight, cooled,
diluted with 80 ml of EtOAc, washed with 2 ꢀ 15 ml of saturated NaHCO3 and
2 ꢀ 10 ml of brine, dried, filtered, and evaporated to give 243 mg of residue
which was chromatographed on 8 g of silica gel with 9:1 to 3:1 hexane:EtOAc
1
to give 150 mg (69%) of 5 as a colourless oil: H NMR (300 MHz) d 7.53 (m,
2H), 7.36 (m, 5H), 6.84 (m, 2H), 5.38 (s, 1H), 4.06 (m, 4H), 3.92 (dd, J ¼ 9:0,
3.0 Hz, 1H), 3.65 (dd, J ¼ 9:0, 7.0 Hz, 1H), 3.55 (m, 1H), 2.27-0.91 (m, 22H),
1.12 (d, J ¼ 6:6 Hz, 3H), 1.04 (s, 3H), 0.74 (s, 3H); 13C NMR (75 MHz) d
159.5, 142.5, 141.1, 134.2, 132.9, 128.4, 128.3, 126.5, 121.8, 114.3, 109.7, 73.2,
71.9, 65.1, 56.7, 53.0, 50.4, 42.8, 42.5, 39.9, 37.6, 36.8, 36.7, 32.2, 32.1, 31.9,
28.1, 24.7, 21.4, 19.7, 17.7, 12.2. Analytically Calculated for C37H48O4: C,
79.82; H, 8.69. Found: C, 79.60; H, 8.63.
22-(p-2-Phenyl-[1,3]dioxolan-2-ylphenoxy)-23,24-bisnorcholan-5-en-3b-one (6)
According to a procedure by Parish et al.,7 a mixture of 22.7 mg (0.045 mmol)
of 5, 19 mg (0.15 mmol) of CaCO3 and 4 mg of 4 A molecular sieves in 4 ml of
dry CH2Cl2 was stirred at room temperature for 15 min. Then 33 mg
(0.15 mmol) of PCC was added. The mixture was stirred at room temperature
for 30 min, diluted with 3 ml of brine, and extracted with 3 ꢀ 40 ml of ether.
The combined organic layers were passed through a short pad of MgSO4, and
evaporation of the filtrate gave 32 mg of residue which was chromatographed
on 3 g of silica gel with 15:1 to 4:1 hexane:EtOAc to give 17 mg (75%) of 6 as a
1
wax-like solid, plus 4.5 mg of overoxidized product: H NMR (500 MHz) d
7.52 (m, 2H), 7.40 (m, 2H), 7.32 (m, 3H), 6.84 (m, 2H), 5.36 (br, 1H), 4.10 (m,
4H), 3.92 (dd, J ¼ 9:0, 3.0 Hz, 1H), 3.66 (dd, J ¼ 9:0, 7.0 Hz, 1H), 3.30 (dd,
J ¼ 16:5, 2.5 Hz, 1H), 2.85 (dd, J ¼ 16:5, 2.5 Hz, 1H), 2.50 (m, 1H), 2.34
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P. WANG AND T.A. SPENCER
(m, 1H), 2.08 (m, 3H), 1.88 (m, 1H), 1.65-0.86 (m, 13H), 1.22 (s, 3H), 1.15 (d,
J ¼ 6:6 Hz, 3H), 0.77 (s, 3H); 13C NMR (125 MHz) d 210.6, 159.5, 142.5,
138.8, 134.2, 128.3, 128.2, 127.8, 126.5, 123.1, 114.2, 109.7, 73.2, 65.1, 56.5,
52.9, 49.4, 48.6, 42.8, 39.7, 37.9, 37.1, 36.7, 32.2, 32.0, 29.9, 28.0, 24.6, 21.6,
19.4, 17.7, 12.2.
Conversion of 6 to 8
To a solution of 35 mg (0.063 mmol) of 6 in 2 ml of MeOH was added 8 mg
(0.189 mmol) of NaB2H4. The mixture was stirred at room temperature for 1 h,
diluted with 2 ml of brine, extracted with 3 ꢀ 30 ml of CH2Cl2, washed with
2 ꢀ 5 ml of brine, dried over Na2SO4, filtered, evaporated to give 36 mg of
crude 7. This material was dissolved in 10 ml of 9:1 acetone:H2O solution and
treated with 46 mg (0.185 mmol) of pyridium toluenesulphonate (PPTS). The
resulting mixture was heated at reflux overnight, cooled, evaporated, diluted
with 50 ml of CH2Cl2, and washed with 2 ꢀ 5 ml of sat. NaHCO3 solution and
5 ml of brine. The organic layer was separated and evaporated to give 39 mg of
residue which was chromatographed on 3 g of silica gel with 4:1 hexane:EtOAc
to give 23 mg (72%) of 8 as a colourless solid: 1H NMR (500 MHz) d 7.84-7.76
(m, 4H), 7.57 (m, 1H), 7.49 (m, 2H), 6.95 (m, 2H), 5.38 (br, 1H), 4.01 (dd,
J ¼ 9:0, 3.0 Hz, 1H), 3.76 (dd, J ¼ 9:0, 7.0 Hz, 1H), 2.33-0.97 (m, 22H), 1.16
2
(d, J ¼ 6:5 Hz, 3H), 1.05 (s, 3H), 0.78 (s, 3H); H NMR d 3.55; 13C NMR d
195.8, 163.4, 141.0, 138.5, 132.8, 132.0, 130.0, 129.9, 128.3, 121.8, 114.2, 73.5,
56.6, 52.8, 50.3, 42.8, 42.5, 39.8, 37.5, 36.7, 36.6, 32.1, 32.1, 31.8, 28.0, 24.6,
21.3, 19.6, 17.6, 12.2.
Bromination of methyl 11-[4-(4-methylbenzoyl)-phenyl]-undecanoate (11) to 9
plus 10
To a solution of 316 mg (0.8 mmol) of 11 in 30 ml of CCl4 was added 216 mg
(1.21 mmol) of NBS and 37 mg (0.15 mmol) of benzoyl peroxide. The mixture
was heated at reflux for 14 h, cooled, filtered, and evaporated to give 820 mg of
residue which was chromatographed on 5 g of silica gel with 25:1 to 20:1
hexane:EtOAc to give 200 mg (53%) of 9 and 109 mg (25%) of 10 as colourless
oils. Compound 9: 1H NMR d 7.74 (m, 4H), 7.49 (m, 2H), 7.28 (m, 2H), 4.98
(t, J ¼ 7:5 Hz, 1H), 3.67 (s, 3H), 2.45 (s, 3H), 2.30 (t, J ¼ 7:5 Hz, 2H), 2.30 (m,
1H), 2.18 (m, 1H), 1.62 (m, 2H), 1.28 (br, 12H); 13C NMR d 196.0, 174.5,
146.6, 143.6, 137.9, 134.9, 130.6, 130.5, 130.0, 127.4, 54.6, 51.7, 40.0, 34.3,
29.5, 29.4, 29.3, 29.1, 28.4, 25.2, 21.9. Compound 10: 1H NMR d 7.79 (m, 4H),
7.51 (m, 4H), 4.98 (t, J ¼ 7:5 Hz, 1H), 4.54 (s, 2H), 3.67 (s, 3H), 2.30 (t,
J ¼ 7:5 Hz, 2H), 2.30 (m, 1H), 2.09 (m, 1H), 1.60 (m, 2H), 1.28 (br, 12H); 13
C
NMR d 195.5, 174.5, 147.1, 142.5, 137.5, 137.3, 130.8, 130.7, 129.2, 127.6,
54.4, 51.7, 40.0, 34.3, 32.5, 29.5, 29.4, 29.3, 29.1, 28.4, 25.2.
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LABELLED PHOTOACTIVABLE LIPID ANALOGUES
787
Reduction of 9 plus 10 to 12 plus 13
According to the procedure of Hutchins et al.,9 to 34 mg of a 2:1 mixture of 9
and 10 in 1 ml of dry HMPA was added 45 mg (0.72 mmol) of NaB2H3CN.
The mixture was heated at 908C for 48 h, cooled, and chromatographed
directly on 5 g of silica gel with 30:1 to 20:1 hexane:EtOAc to give 11 mg (42%)
1
of a colourless oily mixture of 12 and 13. H NMR d 7.73 (m, 4H), 7.28 (m,
4H), 3.68 (s, 3H), 2.69 (t, J ¼ 7:6 Hz, 1H), 2.45 (s, 2.7H), 2.31 (t, J ¼ 7:6 Hz,
2
2H), 1.65 (m, 4H), 1.30 (s, 12H); H NMR d 2.69, 2.46; 13C NMR d 196.6,
174.6, 148.1, 143.1, 135.6, 135.4, 130.4, 129.1, 128.5, 51.7, 34.3, 31.4, 29.72,
29.70, 29.67, 29.5, 29.49, 29.4, 25.2, 21.9.
Bromination of 11-[4-(4-methylbenzoyl)-phenyl]-undecanoic acid (2) to 14
plus 15
To a solution of 200 mg (0.53 mmol) of 2 in 20 ml of CCl4 was added 140 mg
(0.79 mmol) of NBS and 26 mg (0.11 mmol) of benzoyl peroxide. The mixture
was heated at reflux for 18 h, cooled, filtered, and evaporated to give 400 mg of
residue which was chromatographed on 5 g of silica gel with 4:1 to 2:1
hexane:EtOAc to give 244 mg of light-yellowish oily ca 2:1 mixture of 14 and
15. Rechromatography on 15 g of silica gel effected separation to give pure 14
1
and 15: Compound 14: H NMR d 7.76 (m, 4H), 7.49 (m, 2H), 7.28 (m, 2H),
4.98 (t, J ¼ 7:5 Hz, 1H), 2.45 (s, 3H), 2.33 (t, J ¼ 7:5 Hz, 2H), 2.29 (m, 1H),
2.18 (m, 1H), 1.63 (m, 2H), 1.28 (br, 12H); 13C NMR d 196.0, 180.1, 146.6,
143.7, 137.9, 134.9, 130.6, 130.5, 130.0, 127.4, 54.6, 40.0, 34.2, 29.5, 29.48,
29.4, 29.2, 29.1, 28.4, 24.9, 21.9. Compound 15: 1H NMR d 7.78 (m, 4H), 7.51
(m, 4H), 4.98 (t, J ¼ 7:5 Hz, 1H), 4.55 (s, 2H), 2.30 (t, J ¼ 7:5 Hz, 2H), 2.26
(m, 1H), 2.17 (m, 1H), 1.63 (m, 2H), 1.29 (br, 12H); 13C NMR d 195.5, 179.8,
147.1, 142.5, 137.5, 137.3, 130.8, 130.7, 129.2, 127.6, 54.4, 40.0, 34.2, 32.5,
29.5, 29.4, 29.3, 29.2, 29.1, 28.4, 25.2.
Reconversion of 14 and 15 to 16 and 17
To a mixture of 42 mg of 14 and 15 in 0.8 ml of dry HMPA was added 70 mg
(1.06 mmol) of NaB2H3CN. The mixture was heated at 808C for 48 h, cooled,
and chromatographed directly on 3 g of silica gel with 4:1 to 3:1 hexane:EtOAc
to give 15 mg (45%) of a colourless oily mixture of 15 and 16. 1H NMR d 7.74
(m, 4H), 7.28 (m, 4H), 2.69 (m, 1H), 2.45 (br, 2.7H), 2.37 (t, J ¼ 7:2 Hz, 2H),
1
1.65 (m, 4H), 1.32 (s, 12H); H NMR d 2.69, 2.45; 13C NMR d 196.6, 179.1,
148.1, 143.2, 135.6, 135.4, 130.5, 130.5, 129.1, 128.5, 34.1, 31.4, 29.7, 29.6,
29.6, 29.5, 29.4, 29.3, 24.9, 21.9.
Conclusion
Convenient, reliable procedures for isotopic labelling of photoactivable
cholesterol analogue 1 and fatty acid analogue 2 have been developed, using
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P. WANG AND T.A. SPENCER
NaB2H4 and NaB2H3CN, respectively, that will be useful as less expensive
alternatives to commercial catalytic tritiation for introduction of radiolabels
into these biochemical research tools.
Acknowledgements
This research was supported by USPHS NIH grant HL 67294.
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