Langmuir 1995,11, 3644-3647
3644
Synthesis and Structural Analysis of Long Chain N-Acetyl-N-alkyllactosylamines, a New Series of Surfactants Derived from Unprotected Lactose Florence Costes, Mostafa El Ghoul, Maryse Bon, Isabelle Rico-Lattes," and Armand Lattes Laboratoire des IMRCP, UA CNRS No. 470, Universitk Paul Sabatier, 118 route de Narbonne, 31062 Toulouse Ceder., France Received October 11, 1994. I n Final Form: January 25, 199P Surfactants based on sugars are veryimportant for extraction ofproteins. We describe here a new series easily prepared in a two-stage route starting from of surfactants, the N-acetyl-N-alkyllactosylamines, unprotected lactose. These new compounds have comparable surfactant activity to existing sugar-based homologs currently employed in the extraction of proteins from biological samples. Moreover, a complete structural analysis ofthese new nonionic amphiphiles by lH and 13CNMR showed a p anomeric conformation suitable for chiral induction in micelles.
Introduction There is increasing interest in surfactants derived from sugars in view of their pharmacological and biological properties. Since they are considerably less denaturing than conventional detergents, they are also widely employed in the extraction and purification of amphiphilic membrane proteins from biological samples, as essential step in the determination of the structure and function of this important class of biological macrom~lecules.~-~ However, methods usually described for their preparation require prior long and costly synthetic schemes. We have recently developed a new family of surfactants derived in two steps from unprotected lactose, the 1-(N-alkylaminol1-deoxylactitols,which have been used successfully in the extraction of opiate receptors.'O We present here a new family of surfactants derived from lactose, the N-acetyl-N-alkyllactosylaminesof general formula CHpOH
Scheme 1 'U: CH20H
CH.OH
CHzOH
OH
OH
HoQo*R .
OH
COW3 OH
The surfactant activity of these compounds was compared to that of homologs widely employed for the extraction of membrane proteins from biological samples. Of added interest is the finding that the /3 anomers of surfactants derived from sugars are stronger inducers of @
Abstract published inAdvance ACSAbstracts, August 15,1995.
(1)Stubbs, G.W.; Smith, H. G., Jr.; Litman, B. J. Biochim. Biophys. Acta 1976, 425, 46.
(2) Lin, J. T.; Riedel, S.;Kinne, R. Biochim. Biophys. Acta 1979,557, 179. (3) De Foresta, B.; Le Maire, M.; Orlowski, S.;Chameil, P.; Lund, S.; Moller, J. V.; Michelangeli, F.; Lee, A. G. Biochemistry 1989,28,2558. (4) Plusquellec, D.; Chevalier, G.; Talibart, R.; Wroblewski, H. Anal. Biochem. 1989, 179, 145. (5)Abram, D.; Boucher, F.; Hamanaka, T.; Hiraki, K.; Kito, Y . ; Koyama, K.; Leblanc, R. M.; Machida, H.; Munger, G.; Seidou, M.; Tessier, M. J. J . Colloid Interface Sci. 1989, 128, 230. (6)Ringsdorf, H.; Schlarb, B.; Wenzmer, J. Angew. Chem. Int. Ed. Engl. 1988, 27, 113. (7) Katoh, T.; Mimuro, M.; Takaichi, S.Biochim. Biophys.Acta 1989, 976. 233. (8) Mollereau, C.; Pascaud, A.; Baillat, G.; Mazarguil, H.; Puget, A,; Meunier, J. C. Eur. J . Pharm. 1988, 150, 75. (9) Helenius, A.; Simons, K. Biochim. Biophys. Acta 1975,424, 29. (10)Garelli-Calvet,R.; Latge, P.; Rico, I.; Lattes, A. Biochim. Biophys. Acta 1992, 1109, 55.
CHzOH
chirality in micelles than the a an0mers.l' With a view to using surfactants of this type for asymmetric induction in micellar media, we carried out a 'H and 13CNMR study together to determine the conformation of these new surfactants at the site of attachment of the acetyl €TOUP.
Experimental Section The lH and I3C NMR spectra were recorded on a Bruker AM 300wB instrument, and the chemical shifts were expressed with respect to TMS. Mass spectra were recorded on a Nermag R1010 spectrometer in the DCI/NHs mode. Microanalyses were carried out at the microanalysis center of the Ecole Nationale Superieure de Chimie de Toulouse (France). The compounds crystallize in a hydrated form (1.5 mol of HzO to 1 mol of substrate), and water content was determinedby the Karl Fischer method. Surface tension was measured at 25 "C on a Prolabo (TensimatNo. 3) apparatus usingthe stirrup detachmentmethod. The conformational calculations were carried out on a Silicon (11)Focher, B.; Savelli, G.; Toni, G.; Vecchio, G.; McKenzie, D. C.; Nicoli, D. F.; Bunton, C. A. Chem. Phys. Lett. 1989, 158 (61,491.
0743-746319512411-3644$09.00/00 1995 American Chemical Society
N-Acetyl-N-alkyllactosylamines as Surfactants
Langmuir, Vol. 11, No. 10, 1995 3645
Table 1. Cmc of N-Acetyl-N-alkyllactosylamines2 at 25 "C in Water
C1oH21
N-dodecyldeoxy-1-lactitolamine12
2.4 x 10-4 2.6 x 10-3
/?-1-n-OctylD-glucopyranoside.6* /?-1-n-DodecylD-maltoside.6
Scheme 2 6
CH,OH
OH
2.c
Table 2. Characteristic lH and lSC NMR Data for the mol 1-l) Two B Conformers of Compound 2c (C= 1 x 8 (ppm) C1 lactose C1N chain CH3CO C'1 lactose DzO C = 0.1 M 13C 'H 13C 1H 13C 13C 'H 13C 'H Form 1 87.42 5.33 45.75 3.15 174.79 22.06 2.19 103.5 4.45 3.35 Form 2 83.93 4.95 41.90 3.15 174.57 22.06 2.15 103.5 4.43 3.35
Graphics work station running the MAD software. All solvents and reagents were obtained from Aldrich and were used without h r t h e r purification. The solvents were preserved with molecular sieves (4 A). The synthesis. of N-alkyllactosylamine 1 is described elsewherei2 and the general method is indicated below. (i) Synthesis of N-Alkyllactosylamines 1. A solution of the alkylamine (50 mmol) in 2-propanol (100 mL) is added to a solution oflactose monohydrate (30 mmol) in water (60 mL). The reaction mixture is agitated for 24 h at ambient temperature and then heated at 60 "C for 30 min. The solvent is evaporated under reduced pressure. The residue is taken up in ethanol and reevaporated in the presence of toluene to eliminate residual water. The solid product is recrystallized from ethanol and then freeze-dried. (ii) Synthesis ofN-Acetyl-N-alkyllactosylamines 2 from N-Alkyllactosylamines 1. General Method. Triethylamine (0.02 mol) is added to a solution of alkyllactosylamine (0.02 mol) in 120 mL of DMF under argon. Aconcentrated solution of acetic anhydride (0.02 mol) in DMF is then added to this clear solution, and the mixture is stirred a t room temperature. The reaction is followed by TLC using CHCldCH30WNH40H (20/15/2, v/v/v) as eluent. The solvent is then evaporated under vacuum and the residue purified on a silica column using the eluent described above to give compound 2. N-Acetyl-N-octyllactosylamine 2a. NMR I3C (DMSO-ds): glucose moiety 81.65, 86.78 (Ci), 80.19 (C4), 76.91 (C3), 75.65 (C51, 70.30 (Cz), 60.33 (CS);galactose moiety 103.67 (CI,),75.50 (C5,), 73.26 (C3,),70.54 (Cy), 68.10 (C4,),60.57 (CS.);acetyl group 21.80 (CH3),170.66,170.90(C=O); alkyl chain 40.68,43.35 (C1),31.14 (CS), 28.66 (C4),28.60 (Cz),28.56 (C5),26.70 (c3), 21.95 (c7),13.81 (Ce). Mass: MH+ = 496, H1&NH2+COCH3 = 172. Elemental
PPm Figure 1. 2D-homonuclear ('H-lH) correlation (COSY) for compound 2c.
Costes et al.
3646 Langmuir, Vol. 11, No. 10, 1995
I
100
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'
'90'
'
'80
'
. 'io
'
. '60 . '50 '
'
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.
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PPm Figure 2. 2D-heteronuclear (I3C-'H) correlations for compound 2c. were carried out usinga Bruker AM 300 MB instrument equipped Anal. Calcd for CzzH41011N.1.5HzO: C, 50.57; H, 8.42; N, 2.68. with a n Aspect 3000 computer. The following parameters were Found: C, 50.89; H, 8.48; N, 2.69. used: (1)lH spectra were recorded a t a nominal frequency of N-Acetyl-N-nonyllactosylamine2b. NMR I3C (DMSO-ds): 300.13 MHz with 5 mm i.d. tubes; and (2) 1D spectra were glucose moiety 81.43, 86.62 (Cd, 80.20 (Cd, 76.83 (Cd, 75.72 recorded in DzO at 298 K with a spectral width of 3000 Hz, an (C5), 70.23 (CZ),60.26 (c6);galactose moiety 103.68 (CY),75.59 aquisition time of 2.7 s, and a pulse angle of 35"; and (3) I3C (C5,),73.19 (Cy), 70.44 (CZ,),68.02 ( C ~ T60.41 ), (CS.);acetyl group spectra were recorded at a nominal frequency of 75.47 MHz with 21.76 (CH3), 170.68,170.92 (C-0); alkyl chain 40.68,43.23 (CA, 10 mm i.d. tubes, a spectral width of 3000 Hz, a n acquisition 31.20 (C7),28.59-28.92 ( C Z , C ~ , C ~ ,26.72 C ~ ) , (c3),22.00 (cs), 13.87 time of 1 s, and a pulse angle of 40". (Cs). Mass: MH+ = 510, MH+ - GalOH = 330, H&NHz+COCH3 = 186. Elemental Anal. Calcd for C Z ~ H ~ ~ O ~ ~ N * ~ . ~ HThe Z Opulse : sequences were programmed using the software C, 51.49; H, 8.58; N, 2.61. Found: C, 51.45; H, 8.81; N, 2.89. provided by Bruker: Jmodule heteronuclear and 13Cgated COSY45 homonuclear-2D (p/2, t,p/4, Acq); 2D heteronuclear correlation N-Acetyl-N-decyllactosylamine2c. NMR 13C(DMSO-ds): glucose moiety 81.61, 86.62 (Cl), 80.21 (C4), 76.84 (C3), 75.59 (C5), 13C-lH (CPD decoupling) using XH CORREL.AUR software. 70.24 (CZ),60.27 (c6); galactose moiety 103.69 (CY),75.59 (Cr), The spectral width for the homonuclear and heteronuclear 73.18 ( C ~ P70.45 ), (CZ.),68.04 (C4,),60.41 (c6,); acetyl group 21.87 correlations was 1445 Hz, and for the 13C spectra was 7040 Hz. (CH3),170.64,170.88 (C=O); alkyl chain 40.67,43.30 (Cd, 31.21 The 2D spectra in quadrature in two directions were obtained (cs), 28.46-28.99 ( C Z , C ~ , C ~ , C ~26.74 , C ~ )(c3), , 22.00 ( c g ) , 13.89 from 512 x 1024 point ('HI and 256 x 1024 point P3C) matrices, MH+ = 524, MH+ - GalOH = 344, H21CloNH2+( C ~ O )Mass: . zero-filled to 1024 x 2048 and 512 x 2048 points, respectively. COCH3 = 200. Elemental Anal. Calcd for C24H45011N.1.5HzO: The heteronuclear correlations were enhanced by mathematical C, 52.36; H, 8.72; N, 2.54. Found: C, 52.80; H, 8.66; N, 2.62. treatment (in both dimensions, fraction S "sine-bell window" N-Acetyl-N-dodecyllactosylamine 2d. NMR 13C (DMSO-dd: corresponding to a shift of p/3). glucose moiety 81.49, 86.67 (CA, 80.22 (C4), 76.86 (Cd, 75.61 (C5), 70.25 (CZ),60.29 (c6);galactose moiety 103.69 (CY), 75.61 Results and Discussion (CY), 73.21 (Cy), 70.47 (CZ,),68.05 (Cg), 60.46 (CS.); acetyl (1) Synthesis of N-Acetyl-N-alkyllactosylamines group 21.85 (CH3), 170.65, 170.90 (C=O); alkyl chain 40.65, 43.26 (ci),31.20 (cs), 28.47-28.97 ( c ~ , c 4 , c ~ , c ~ 26.74 ,c~), 2. The N-acetyl-N-alkyllactosylamines2 were prepared (C3),21.99 (Cg), 13.85 (Clo). Mass: MH+ = 552, MH+ GalOH in a two-stage reaction w i t h o u t prior protection of lactose = 372, Hz~CIZNHZ+COCH~ = 228. Elemental Anal. Calcd for (Scheme 1). C Z ~ H ~ ~ O ~ ~ N C, * ~53.97; . ~ H H, ZO 8.99; : N, 2.42. Found: C, 54.08; The first stage has been described in a previous H, 9.01; N, 2.31. publication12(see the Experimental Section). Compounds (iii) Complete Structural Analysis of N-Acetyl-N-all a and Id w e r e obtained in yields ranging from 60% to kyllactosylamines2 by lH and 13CNMR. All measurements 70%. The N-alkyllactosylamines 1 cannot be used directly as s u r f a c t a n t s since t h e y spontaneously hydrolyze in (12) Latge, P.; Rico, I.; Garelli, R.; Lattes, A. J.Disp. Sci. Technol. 1991,12 (3 & 41, 227. a q u e o u s solution t o give the starting amine and lactose.
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Langmuir, Vol. 11, No. 10, 1995 3647
N-Acetyl-N-alkyllactosylamines as Surfactants
However, the N-acetyl-N-alkyllactosylamines2 obtained in the second stage are stable and can be employed as surfactants in aqueous solution. They were obtained after purification in yields ranging from 30% for long chain compounds to 55% for short chain compounds (see the Experimental Section). These yields, decreasing with increasing chain length, were attributed to the increased adsorption of the longer chain surfactants on the silica during the chromatographic purification. The compounds obtained were identified by mass spectrometry, NMR, and microanalysis. The mass spectrometric data (DCI/NH3) indicated that acetylation took place on the nitrogen atom: the fragmentation RN, H2+COCH3 was clear evidence for acetylation on the amino group. (2) Surfactant Properties of N-Acetyl-N-alkyllactosylamines2. The critical micelle concentrations (cmc) and the areas per head group of the N-acetyl-N-alkyllactosylamines 2 were determined by measurement of surface tension in aqueous solution at 25 "C. The values of cmc are listed in Table 1. Cmc of widely employed homologues are also shown for comparison. As shown in Table 1,the cmc of compounds 2 are decreasing as with classical nonionic surfactants. They are quite similar to those of commercially available homologous surfactants used in the extraction of membrane proteins. Moreover, the head group areas (0)of compounds 2 have been evaluated to be 0.55 nm2,which is also similar to the value obtained by Zemb and co-workersfor ,6-dodecylmaltoside (0.50 nm2).13To determine the structure of micelles, work is now on progress using X-ray and neutron scattering methods.l4 (3) Structural Analysis by lH and 13CNMR of the N-Acetyl-N-alkyllactosylamines 2. A previous 1D and 2D NMR (IH and 13C)study of the N-alkyllactosylamines 1 demonstrated that they existed in a and ,6 forms with the 6, form as the major species (=70%).15 We carried out a similar study of the N-acetyl-N-alkyllactosylamines2 to determine the conformation at the site of attachment of the acetyl group. A typical example is represented by compound 2c (Scheme 2). The lH and 13Csignals were assigned from 1D and 2D studies and by comparison with our previous results15 and a similar work with ,6-dodecyl maltoside13 using DzO as solvent. The experiments were carried out at different concentrations from 1.1x 10-1 to 2.5 x lov3M (above and mol L-l at 25 "C). below the cmc = 3 x In all cases, two forms in equal proportions were indicated both by 13C and lH NMR data (Table 2 and Figures 1 and 2). Moreover, the proton anomer H1 of lactose gave rise to two doublets with chemical shifts of 4.95 and 5.33 ppm with high coupling constants ( J = 8.5 Hz). These high J 1 - 2 coupling constants indicated that the protons were axial. Both forms are thus in the ,6 Conformation. This is in marked contrast to the results obtained with the N-alkyllactosylamines 1,which were observed in a and ,6 forms. These results are confirmed by the area per head group value (0.55 nm2), which is in agreement with the value obtained for the ,6 conformation of dodecyl maltoside.13 A molecular modeling study, based on the work of Zemb and ~ o - w o r k e r swas , ~ ~ thus carried out to determine the structure of these two stable ,6 conformers. The structures of the two most stable ,6 conformers obtained with the software MAD16 are represented in Figure 3. (13) Cecutti, C.; Focher, B.; Perly, B.; Zemb, T. Langmuir 1991, 7, 2580. (14)Auvray, X.; Petipas, C.; Costes, F.; Rico-Lattes, I.; Lattes, A. In press. (15) Latge, P.; Bon, M.; Rico, I.; Lattes, A. New J . Chem. 1992, 16, 387. (16)Molecular Advanced Design (MAD) distributed by Oxford Molecular Ltd. Oxford OX4 4GA, England.
Q\ l
0
a
U
Ez22.34 Kcal/mol
Q
W I
b
b
P
W
E=22,49Kcal/mol
Figure 3. Molecular models of the two stable p conformers of N-acetyl-N-decyllactosylamine2c.
-
To confirm the stability of these two ,6 conformers (E 22.5 kcal molp1) relative to that of an a conformer, we carried out a similar study of compound 2c in the a conformation. The most a stable form was found at a value of E = 55.55 kcal molp1, indicating that the two3!, conformers are the most favored energetically. This was attributed to steric hindrance between the acetyl group and the OH group on C2 in the a conformation.
Conclusion The preparation of a new family of surfactants, the N-acetyl-N-alkyllactosylaminesby a convenient two-step route from unprotected lactose is described. These amphiphiles were found to have similar surfactant properties to conventional sugar-based homologs, indicating that they would have value in biological app1i~ations.l~ Furthermore, complete structural analysis of these compounds by lH and 13C NMR demonstrated that they exist in a ,6 anomeric conformation suitable for micellar chiral induction.
Acknowledgment. We are grateful to CNRS and STEPAN-EUROPE for their financial support. LA9407929 (17) The compounds have been patented by STEPAN-EUROPE, European Patent, no. 924014038, May 22, 1992.
