Syntheses of [14C]-detergents: Octaethyleneglycol-[1-14C]-dodecylether, [1-14C]-dodecylβ-D-maltoside and dibromo-analogues

Article abstract of DOI:10.1002/jlcr.485

Octaethylene-glycol-dodecylether and dodecyl-β-D-maltoside are two widely used detergents in membrane protein studies. We describe here the synthesis of the ¹⁴C-labelled brominated analogues, and of the ¹⁴C-labelled forms. [1-¹⁴C]5,6-Dibromo-dodecylether was prepared by coupling [1-¹⁴C]-(Z)-1-bromododec-5-ene with octaethylene glycol followed by bromination. [1-¹⁴C]-5,6-Dibromo-dodecyl-β-D-maltoside was synthesised from [1-¹⁴C]-(Z)-dodec-5-en-1-ol via a coupling with α-bromohepta-O-acetyl-maltose followed by a deprotection step and bromination. Following similar methods, octaethyleneglycol-[1-¹⁴C]-dodecylether and [1-¹⁴C]-dodecyl-β-D-maltoside were also obtained. Copyright

Full text of DOI:10.1002/jlcr.485

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Labelled Cpd Radiopharm 2001; 44: 575–585.
DOI: 10.1002/jlcr.485
Syntheses of [14C]-detergents: octaethylene-
glycol-[1-14C]-dodecylether, [1-14C]-dodecyl-
b-d-maltoside and dibromo-analogues
2
1
Dominique Georgin1,*, Marcle Maire and Jean Pierre Noel
.
1 CEA/Saclay, Service des Mole!cules Marque!es, Ba#t. 547,
F-91191 Gif sur Yvette Cedex, France
2
!
CEA/Saclay, Section de Biophysique des Proteines et des Membranes, CNRS,
#
URA 2096, Bat. 528, F-91191 Gif sur Yvette Cedex, France
Summary
Octaethylene-glycol-dodecylether and dodecyl-b-d-maltoside are two widely
used detergents in membrane protein studies. We describe here the synthesis of
the 14C-labelled brominated analogues, and of the 14C-labelled forms. [1-14C]-
5,6-Dibromo-dodecylether was prepared by coupling [1-14C]-(Z)-1-bromo-
dodec-5-ene with octaethylene glycol followed by bromination. [1-14C]-5,6-
Dibromo-dodecyl-b-d-maltoside was synthesised from [1-14C]-(Z)-dodec-5-en-
l-ol via a coupling with a-bromohepta-O-acetyl-maltose followed by a
deprotection step and bromination. Following similar methods, octaethylene-
glycol-[1-14C]-dodecylether and [1-14C]-dodecyl-b-d-maltoside were also obtai-
ned. Copyright # 2001 John Wiley & Sons, Ltd.
Key Words: Barton’s bromodecarboxylation; bromination; protein detergents;
14C-detergents
Introduction
Detergents like dodecyl-b-d-maltoside or octaethylene-glycol-dodecyl
ether provide a means of delineating the structure of biological
membranes in order to purify and characterise their membrane protein
!
*Correspondence to: D. Georgin, CEA/Saclay, Service des Molecules Marquees, Bat. 547, F-91191
Gif sur Yvette Cedex, France. E-mail: dominique.georgin@cea.fr
!
#
Copyright # 2001 John Wiley & Sons, Ltd.
Received 23 March 2001
Revised 5 April 2001
Accepted 11 April 2001
Published online \ \ \ \

576
D. GEORGIN ET AL.
components.1–3 14C-labelling and gel equilibrium chromatography can
be used to estimate the binding of the detergent to the protein and may
provide a measure of the hydrophobicsurface area of the membrane
protein.4
The bromination of the aliphaticchain of these detergents does not
seem to affect their physical properties, and it has been shown that such
brominated detergents quench the fluorescence of tryptophan octyl
ester,5 a hydrophobicmodel of tryptophan in proteins.
Spectroscopic quenching measurements can therefore provide some
topological information, such as the location of the tryptophan residues
in the transmembrane protein.6
The use of fluorescence quenching and brominated detergents points
to a new method of measuring protein binding. This method can be
validated by using 14C-labelled detergents both brominated and
otherwise to correlate the binding (thanks to radioactivity) with
fluorescence quenching.
Results and discussion
As the two syntheses were highly similar and presented the same kinds
of difficulties, we shall only describe the formation of the brominated
form and the last steps of the ordinary detergent. The two brominated
detergents we synthesised were characterised by a lateral chain
containing 12 carbons. [1-14C]-(Z)-dodec-5-enoic acid was therefore
chosen as the key molecule. Its double bond allows the introduction
of molecular bromine at the last step. Its carboxylic group can be
simply reduced to the corresponding alcohol, which is also easily
transformed to its bromo derivative by dibromotriphenylphospho-
rane.
To obtain [1-14C]-(Z)-dodec-5-enoic acid 4, we used a method
consisting of Barton’s bromodecarboxylation7,8 of commercially avail-
able 1, followed by nucleophilic substitution of the bromine by K14CN
in 81% radioactive yield. The hydrolysis of the cyano group afforded
the expected [1-14C]-(Z)-dodec-5-enoic acid 4 in 63% radioactive yield.
[1-14C]-(Z)-dodec-5-en-l-ol 5 was obtained by the reduction of the acid
with lithium aluminium hydride9 in 69% radioactive yield and
dibromotriphenylphosphorane permitted the substitution of the alkoxy
group by bromine on the labelled carbon10 in 57% radioactive yield
(Scheme 1).
Copyright # 2001 John Wiley & Sons, Ltd.
J Labelled Cpd Radiopharm 2001; 44: 575–585

SYNTHESIS OF [14C]-DETERGENTS
577
Scheme 1.
Scheme 2.
To obtain the octaethylene-glycol-[1-14C]-5-6-dibromododecylether 8,
we adapted a common method of coupling between a bromo derivative
and a polyethylene glycol using sodium hydride11 (Scheme 2). The
expected product also reacted with octaethylene glycol giving poly-
condensation products, which lowered the yield to 12%.
The similar reaction described in the reference afforded a better yield
(32.5%) as two equivalents of polyethylene glycol were used. Some
attempts using an unlabelled 1-bromo-dodec-5-ene were conducted, but
owing to the excess of octaethylene glycol the purification step was
unsuccessful.
The same chemical sequence (Scheme 3) was applied to dodecanoic
acid to form [1-14C]-dodecan-1-ol 9 (40% yield). 9 was then submitted
to chlorination, instead of bromination, with thionyl chloride to
synthesise [1-14C]-l-chlorododecane 10 in 80% yield with the aim of
increasing the coupling yield. This saturated chlorinated chain was even
less reactive with octaethylene-glycol than its unsaturated brominated
analogue and octaethylene-glycol-[1-14C]-dodecylether 11 was obtained
in only 6% yield.
Copyright # 2001 John Wiley & Sons, Ltd.
J Labelled Cpd Radiopharm 2001; 44: 575–585

578
D. GEORGIN ET AL.
Scheme 3.
Scheme 4.
[1-14C]-5,6-dibromododecyl-b-d-maltoside 14 was prepared from
[1-14C]-(Z)-dodec-5-en-1-ol 5 and a-bromohepta-O-acetyl-maltose by a
nucleophilic substitution procedure using silver carbonate.12,13 The low
yield (24%) of this step was balanced by a good selectivity, which
afforded only the b form of the expected product. After a simple
bromination (76% yield) and a deprotection step (58% yield), the
expected labelled detergent was obtained as detailed in Scheme 4.
Copyright # 2001 John Wiley & Sons, Ltd.
J Labelled Cpd Radiopharm 2001; 44: 575–585

SYNTHESIS OF [14C]-DETERGENTS
579
Scheme 5.
The same method was applied to form [1-14C]-dodecyl-b-d-maltoside
16 from [1-14C]-dodecan-1-ol 9 (Scheme 5).
Conclusion
The relatively rapid synthesis of [1-14C]-(Z)-dodec-5-enoic acid and
[1-14C]-dodecanoic acid via Barton’s bromodecarboxylation of the
unlabelled forms yielded the four expected products in only 18 steps.
Furthermore, the selectivity of the chosen pathway permitted us, in the
case of [1-14C]-5,6-dibromododecyl-b-d-maltoside and [1-14C]-dodecyl-
b-d-maltoside, to selectively obtain the b epimer.
Experimental
K14CN was from Amersham Pharmacia Biotech. Other starting
materials and chemical reagents were from Aldrich. Sodium hydride
was obtained by 60% dispersion in mineral oil after washing with
Copyright # 2001 John Wiley & Sons, Ltd.
J Labelled Cpd Radiopharm 2001; 44: 575–585

580
D. GEORGIN ET AL.
tetrahydrofuran in a dry glove box. Flash chromatography was
performed using silica gel 60 (0.040–0.063 mm) from Merck. All
(
solvents were from SDS. Toluene was dried over 4 A molecular sieves,
diethyl ether and tetrahydrofuran were distilled from sodium benzo-
phenone ketyl and dichloromethane was dried by distillation over
calcium hydride. Analytical TLC was performed with silica gel 60F254
(Merck) and visualisation was carried out with iodine and the
radioactivity detected using a Berthold’s LB 2880-1 automatic TLC-
linear analyser fitted with an LB 511-chromatography data system.
NMR spectra were recorded on a Bruker AC 300 spectrometer (7.05 T;
300.13 MHz (1H)). Specific activity was determined by mass spectro-
metry (DCI/NH3) on a Finnigan instrument (Model 4600). GC/MS
analyses were effected using an HP 6890 Series gas chromatograph
system coupled to an HP 5973 mass selective detector with the following
operating conditions: column: HP-5MS (30 m ꢀ 0.25 mm ꢀ 0.25 mm).
Column temperature: 708C (5 min) to 3008C (5 min) at 158C/min.
Carrier gas: He (0.9 ml/min). Injector temperature: 2508C.
(Z)-1-Bromo-undec-4-ene (2)
Oxalyl chloride (10 g, 78.8 mmol) was added to a mixture of 1 (5 g,
25.2 mmol) in 40 ml of dry toluene stirred under argon. The mixture was
stirred at room temperature for 15 h under an argon atmosphere,
concentrated under vacuum and diluted three times with 30 ml of dry
toluene before concentration to remove oxalyl chloride residue. The
product was used without further purification. 2-Mercaptopyridine-N-
oxide, sodium salt hydrate (4.9 g, 33.3 mmol) and 4-dimethylaminopyr-
idine (348 mg, 2.8 mmol) were dried under vacuum overnight and
introduced with 75 ml of bromotrichloromethane into a three-necked
flask in a dry glove box. Under the ventilated hood, the flask was fitted
with a reflux condenser with a system providing a slow flow of argon.
The slurry was stirred and heated under reflux (1058C) for 30 min.
In a dry glove box the raw acid chloride was diluted with 25 ml of
bromotrichloromethane and stored in a syringe. This mixture was
added through a septum to the reaction flask over 30 min. Bubbles and
an orange colouration were observed and the reaction was stirred under
reflux for 2 h.
A brown product appeared as a side product of the reaction. After
cooling at room temperature the reaction mixture was filtered in order
to eliminate the brown residue. The crude product was then
Copyright # 2001 John Wiley & Sons, Ltd.
J Labelled Cpd Radiopharm 2001; 44: 575–585

SYNTHESIS OF [14C]-DETERGENTS
581
concentrated under vacuum and flash chromatographed on silica gel
with hexane as eluent to give 2 (4.92 g, 14.77 mmol, 84% yield). The
purified product contained a little (1.5%) (Z)-1-chloro-undec-4-ene. No
further purification was attempted as this side product reacted with
[14C]-potassium cyanide in the same manner as 2.
GC/MS: (Z)-1-chloro-undec-4-ene (1.5%; rt=11.17 min; m=z ¼188–
190 M+), 2 (98%; rt=11.99 min; m=z ¼ 232–234 M+).
(Z)-Dodec-5-enenitrile (3)
In a 25ml-flask, 93 mg of 2 (0.4 mmol), 23.3 mg of [14C]-potassium
cyanide (55mCi/mmol, 0.375mmol, 20 mCi) and 10ml of dimethyl
sulphoxide were stirred for 5 h at 808C. After cooling, 100ml of water was
added and the aqueous layer extracted two times with 100ml of diethyl
ether. The mixture was dried under vacuum and purified using flash-
chromatography on silica gel with hexane/ethyl acetate (90/10) as eluent.
16.3 mCi (55 mCi/mmol, 0.30mmol) of 3 was obtained (81% yield).
TLC:hexane/ethyl acetate (95/5). Rf=0.24.
[1-14C]-(Z)-dodec-5-enoic acid (4)
In a 25 ml-flask, 16.3 mCi of 3 (0.30 mmol), 10 ml of a 30% aqueous
potassium hydroxide solution and 10 ml of absolute ethanol were stirred
for 14 h at 808C. The reaction was followed by TLC on silica gel with
hexane/diethyl ether/acetic acid (85/15/1). The mixture was then
acidified using 20 ml of 5 N HCl and extracted with 100 ml of diethyl
ether. The purification by flash chromatography using the same system
as the TLC afforded 10.2 mCi (55 mCi/mmol, 0.18 mmol) of pure 4
(63% yield).
TLC:hexane/diethyl ether/acetic acid (85/15/1). Rf=0.22.
The labelled product was diluted with 189 mg of pure unlabelled
compound and the specific activity of the mixture was measured by
mass spectrometry.
MS (DCI/NH3): m=z(%): 216(100), 217(20), 218(18). Specific activity
=9 mCi/mmol.
[1-14C]-(Z)-Dodec-5-en-1-ol (5)
In a three-necked flask under argon equipped with a magnetic stirrer,
224 mg (9 mCi/mmol, 10.2 mCi, 1.13 mmol) of 4 and 10 ml of anhydrous
Copyright # 2001 John Wiley & Sons, Ltd.
J Labelled Cpd Radiopharm 2001; 44: 575–585

582
D. GEORGIN ET AL.
diethyl ether were cooled in an ice bath. Gradual addition of 110 mg
(2.89 mmol, 2.5 eq.) of lithium aluminium hydride powder produced a
bubbling of the mixture which was then stirred overnight at room
temperature. Excess reducing agent was eliminated by slow addition of
ethyl acetate and solvents were evaporated. The crude product was
purified on silica gel with hexane/diethyl ether (66/33) giving 7.07 mCi of
5 (0.69 mg, 69% yield).
GC/MS: rt=12.17 min; m=z ¼ 166(M-18).
TLC:hexane/diethyl ether (70/30). Rf=0.23.
[1-14C]-(Z)-1-Bromo-dodec-5-ene (6)
Dibromotriphenylphosphorane (295 mg, 0.7 mmol, l eq.) and 5 ml of
dried dichloromethane were placed in an ice-cooled three-necked flask
fitted with a rubber septum. 7.07 mCi of 5 was diluted in 5 ml of dried
dichloromethane, stored in a syringe and slowly added, with magnetic
stirring, to the reaction mixture. After addition, the mixture was stirred
at 08C for 15 min and then 20 ml of water was added. The aqueous layer
was extracted twice with 20 ml of dichloromethane and the combined
organic layers were concentrated under vacuum. The residue was
purified by chromatography on silica gel with hexane/diethyl ether (70/
30) giving 2.2 mCi of 6 and 3.9 mCi of 5. The recycling of the unreacted
alcohol afforded 1.85 mCi of the expected product. 57% total yield.
GC/MS: rt=15.39 min; m=z ¼246–248 M+.
TLC:hexane/diethyl ether (70/30). Rf=0.30.
Octaethylene-glycol-[1-14C]-(Z)-dodec-5-ene (7)
In an oven-dried three-necked flask, a mixture of octaethylene-glycol
(164.5 mg, 0.44 mmol, l eq), sodium hydride (18 mg, 0.74 mmol, 1.7 eq.)
and 10 ml of tetrahydrofuran, was stirred under argon at room
temperature. After 1 h, 6 (4 mCi, 0.44 mmol) dissolved in 2 ml of
tetrahydrofuran and stored in a syringe was gradually added through a
rubber septum to the reaction flask. After 24 h at room temperature the
solution was dilulted with 50 ml of water and extracted four times with
20 ml of diethyl ether. The organic solution was concentrated under
vacuum and the crude material was flash-chromatographed, eluting
with pure acetone. After combination and evaporation of the collected
fractions, 476 mCi (0.05 mmol, 12% yield) of 7 was obtained.
TLC:hexane/acetone (60/40). Rf=0.15.
Copyright # 2001 John Wiley & Sons, Ltd.
J Labelled Cpd Radiopharm 2001; 44: 575–585

SYNTHESIS OF [14C]-DETERGENTS
583
14C]-5,6-dibromododecylether (8)
Octaethylene-glycol-[1-
To a cooled (08C) mixture of 7 (476 mCi, 0.05 mmol) in 10 ml of dry
dichloromethane, a solution of bromine in dichlormethane (1 mg/ml)
was added dropwise over a 15-min period until a yellow colour
persisted. The reaction mixture was then concentrated under reduced
pressure and the residue purified by flash chromatography on silica gel
with pure acetone giving 287 mCi (0.03 mmol, 60% yield) of pure 8.
TLC:reversed phase (KC18F, Whatman), methanol/water (90/10).
Rf=0.32.
HPLC: column: Zorbax SB C18 (250 ꢀ 4.6 mm). Solvent system:
acetonitrile/water (70/30).
Flow rate: 1.5 ml/min. Rt=8.98 min. Chemical and radiochemical
purity >99%.
MS (DCI/NH3): m=z (%): 714 (100), 715 (67), 716 (43), 717 (15).
[1-14C]-(Z)-Dodec-5-ene-b-d-acetyl-maltoside (12)
5 (3 mCi, 0.24 mmol, specific activity=12.8 mCi/mmol, new synthesis)
was dissolved in 5 ml of dry dichloromethane and introduced, under
argon, in an oven-dried three-necked flask protected from light with an
aluminium film. a-Bromohepta-O-acetyl-maltose (168 mg, 0.24 mmol,
(
1 eq.) was added and after 5 min 60 mg of powered, freshly activated 4 A
molecular sieves were added to the mixture with magnetic stirring. Silver
carbonate (92.6 mg, 0.336 mmol, 1.4 eq.) was introduced after 30 min
and the progress of the reaction was followed by TLC. After 12 h, only
40% of the starting material had been consumed and one more
equivalent of both a-bromohepta-O-acetyl-maltose and silver carbonate
were added. After 36 h no side products appeared and 10% more
product had been formed. One more equivalent of each reactant was
charged and after 72 h the reaction was stopped by filtration as the TLC
control showed a 60% conversion into the expected product. The
organiclayer was first washed with a saturated solution of sodium
hydrogen carbonate and then washed with a saturated solution of
sodium chloride.
After evaporation of the solvent, the crude product was flash
chromatographed on silica gel with diethyl ether/hexane (80/20), giving
400 mCi of the starting alcohol and 711 mCi (0.05 mmol, 24% yield) of
the expected product 12.
TLC:diethyl ether/hexane (80/20). Rf=0.20.
Copyright # 2001 John Wiley & Sons, Ltd.
J Labelled Cpd Radiopharm 2001; 44: 575–585

584
D. GEORGIN ET AL.
[1-14C]-5,6-Dibromo-dodecyl-b-d-acetyl-maltoside (13)
This product was obtained by following the same procedure as
described for 8. The final chromatographic purification was performed
on silica gel with diethyl ether/hexane (80/20). 545 mCi (0.04 mmol, 76%
yield) of the pure material 13 was recovered.
TLC:diethylether/hexane (80/20). Rf=0.15.
MS (DCI/NH3): m=z (%): 978 (50), 979 (72), 980 (100), 981 (77), 982
(84).
[1-14C]-5,6-Dibromo-dodecyl-b-d-maltoside (14)
A mixture of methanol/water/triethylamine (20/10/1) was prepared.
20 ml of this solution was inserted, with magneticstirring, into a flask
containing the previous protected product 13. After 12 h, the reaction
mixture was concentrated and flash chromotographed on silica gel with
pure acetone. 320 mCi (0.025 mmol, 58% yield) of [1-14C]-5,6-dibromo-
dodecyl-b-d-maltoside was obtained.
TLC:reversed phase (KC18F, Whatman), methanol/water (80/20).
Rf=0.25.
HPLC:column: Zorbax SB C18 (250 ꢀ 4.6 mm).
Solvent system: acetonitrile/water (75/25). Flow rate: 1.5 ml/min.
Rt=9 min. Chemical and radiochemical purity>99%.
MS (DCI/NH3): m=z (%): 687 (53), 689 (100), 690 (33), 691 (53).
[1-14C]-1-Chloro-dodecane (10)
9 (54 mCi) and 10 ml of thionyl chloride were placed in an oven-dried,
ice-cooled, one-necked flask equipped with a magnetic stirrer and a
reflux condenser. After 5 min of stirring at 08C, the mixture was heated at
1008C for 4 h. After washing with 30 ml of water, the aqueous layer was
extracted twice with 20 ml of diethyl ether and the combined organic
layers were concentrated under vacuum. The residue was purified on
silica gel with hexane/diethyl ether (60/40) giving 43 mCi of 10.80% yield.
TLC:hexane/diethyl ether (60/40). Rf=0.42.
Octaethylene-glycol-[1-14C]-dodecylether (11)
2.45 MCi (6% yield) of 11 was obtained from 10 as described for its
unsaturated analogue 7.
TLC:hexane/acetone (50/50). Rf=0.20.
Copyright # 2001 John Wiley & Sons, Ltd.
J Labelled Cpd Radiopharm 2001; 44: 575–585

SYNTHESIS OF [14C]-DETERGENTS
585
[1-14C]-dodecyl-b-d-acetyl-maltoside (15)
25 MCi (14% yield) of 15 was synthesised from 177 mCi of 9 following
the procedure described for 12.
TLC:diethyl ether/hexane (70/30). Rf=0.32.
[1-14C]-dodecyl-b-d-maltoside (16)
The same treatment as detailed for 14 afforded 22 mCi (88% yield) of
the expected product 16.
HPLC: column ODS 2 (250 ꢀ 4.6 mm). Solvent system: methanol/
water (85/15). Flow rate: 1.5 ml/min. Rt=5.39 min.
References
1. Krag-Hansen U, le Maire M, Mꢀller JV. Biophys J 1998; 75: 2932–2946.
2. Mꢀller JV, le Maire M, Andersen JP. Progress in Protein–Lipid Interaction
2. Elsevier Science Publishers BV (Biomedical Division): Amsterdam,
1986; 147–196.
3. le Maire M, Champeil P, Mꢀller JV. Biochim Biophys Acta (Biomembrane
Review) 2000; 1508(1–2): 86–111.
4. Mꢀller JV, le Maire M. J Biol Chem 1993; 268: 18 659–18 672.
5. de Foresta B, Gallay J, Sopkova J, Champeil P, Vincent M. Biophys J
1999; 77: 1–14.
!
6. Soulie S, de Foresta B, Mꢀller JV, Bloomberg GB, Groves JD, le Maire M.
Eur J Biochem 1998; 257: 216–227.
7. Barton DHR, Crich D, Motherwell WB. Tetrahedron Lett 1983; 24: 4979–
4982.
8. Channing MA, Simpson N. J Labelled Cpd Radiopharm 1993; 33: 541–546.
9. Jacobson M, Keiser I, Chambers DL, Miyashita DH, Harding C. J Med
Chem 1971; 14: 236–239.
10. Ohtani T, Keiichi K, Kamezawa M, et al. J Chem Soc Perkin Trans 1 1996;
10: 961–962.
11. Fujiwara K, Murata Y, Wan TSM, Komatsu K. Tetrahedron 1998; 54:
2049–2058.
12. Andrews CW, Rodebaugh R, Fraser-Reid B. J Org Chem 1996; 61: 5280–
5289.
13. Rosevear P, Van Aken T, Baxter J, Fergusen-Miller S. Biochemistry 1980;
19: 4108–4115.
Copyright # 2001 John Wiley & Sons, Ltd.
J Labelled Cpd Radiopharm 2001; 44: 575–585