Inhibition of human gastric and pancreatic lipases by chiral alkylphosphonates. A kinetic study with 1,2-didecanoyl-sn-glycerol monolayer

Chemistry and Physics of Lipids

100 (1999) 3–31

www.elsevier.com/locate/chemphyslip

Inhibition of human gastric and pancreatic lipases by chiral

alkylphosphonates. A kinetic study with

1,2-didecanoyl-sn-glycerol monolayer

Jean-Franc¸ois Cavalier ^a, Ste´phane Ransac ^b, Robert Verger ^b, Ge´rard Buono ^a,*

^aENSSPICAM, UMR 6516, ‘‘Synthe`se, Catalyse et Chiralite´’’, a6enue Escadrille Normandie-Niemen, F-13397 Marseille,

Cedex 20, France

^bLaboratoire de Lipolyse Enzymatique, UPR 9025, IFR 1 du CNRS, 31, Chemin Joseph Aiguier, F-13402 Marseille,

Cedex 20, France

Received 20 July 1998; received in revised form 18 January 1999; accepted 21 January 1999

Abstract

Enantiomerically pure alkylphosphonate compounds RR%P(O)PNP (R=C_nH_2n+1, R%=OY with Y=C_n%H_2n%+1

with n=n% or n"n%; PNP=p-nitrophenoxy) noted (RY), mimicking the transition state occurring during the

carboxyester hydrolysis were synthesized and investigated as potential inhibitors of human gastric lipase (HGL) and

human pancreatic lipase (HPL). The inhibitory properties of each enantiomer have been tested with the monomolec-

ular ﬁlms technique in addition to an enyzme linked immunosorbent assay (ELISA) in order to estimate simulta-

neously the residual enzymatic activity as well as the interfacial lipase binding. With both lipases, no obvious

correlation between the inhibitor molar fraction (h₅₀) leading to half inhibition, and the chain length, R or Y was

observed. (R₁₁Y₁₆)s were the best inhibitor of HPL and (R₁₀Y₁₁)s were the best inhibitors of HGL. We observed a

highly enantioselective discrimination, both with the pure enantiomeric alkylphosphonate inhibitors as well as a

scalemic mixture. We also showed, for the ﬁrst time, that this enantioselective recognition can occur either during the

catalytic step or during the initial interfacial adsorption step of the lipases. These experimental results were analyzed

with two kinetic models of covalent as well as pseudo-competitive inhibition of lipolytic enzymes by two enantiomeric

Keywords: Lipase enantioselective recognition; Chiral alkylphosphonates; Human gastric and pancreatic lipases;

Monomolecular ﬁlm technique

* Corresponding author. Fax: +33-491-027776.

E-mail addresses: verger@ir2cbm.cnrs-mrs.fr (R. Verger), buono@spi-chim.u-3mrs.fr (G. Buono)

PII: S 0 0 0 9 - 3 0 8 4 ( 9 9 ) 0 0 0 2 8 - 6

4

J.-F. Ca6alier et al. / Chemistry and Physics of Lipids 100 (1999) 3–31

1. Introduction

phonate as inhibitors of Candida rugosa lipase,

Cygler et al. have provided a structural basis of

the chiral preferences of lipases (Cygler et al.,

1994). It is worth noting that both phosphonate

groups were covalently bound to the Og oxygen

of the serine 209, and presented a (S_p) stereo-

chemistry, assuming that only the (S_p)-1R-Cl and

(S_p)-1S-Cl diastereomers in the original mixture

of (S_p, R_p) diastereomers reacted.

Acylglycerol analogues, in which one carbonyl

of the hydrolysable ester bonds was replaced by a

phosphonate group including a good leaving

group, were synthesized as lipase inhibitors (Man-

nesse et al., 1995; Stadler et al., 1996). In all the

cases, the lipases used exhibited a great stereose-

lectivity towards the chirality of these compounds

at the phosphorus and/or the glycerol sites. With

respect to HPL and HGL, Marguet et al. studied

their inhibition by the monomolecular ﬁlms tech-

nique coupled with ELISA tests, using mixed

ﬁlms of chiral pure sn-1,2- and sn-2,3-O-dide-

Lipases have potential applications in chemistry

(Rogalska et al., 1997; Schmid and Verger, 1998),

biotechnology (Vulfson, 1994; Ransac et al., 1996)

and medicine. In the latter domain, for instance,

conventional weight-reducing strategies have fo-

cused largely on controlling the energy intake, but

there is a doubt as to the long-term efﬁcacy of

these approaches. Reducing the adsorption of di-

etary fat by prescribing digestive lipases inhibitors

holds great promise as an anti-obesity strategy

(Gu¨zelhan et al., 1991; Drent and Vanderveen,

1993; Gu¨zelhan et al., 1994; Drent and Van-

derveen, 1995; Drent et al., 1995). Furthermore,

designing and synthesizing speciﬁc inhibitors is of

fundamental value for understanding the molecu-

lar mechanisms involved in the interfacial adsorp-

tion step as well as the catalytic activity of lipases.

Among the range of esterase inhibitors, phos-

phonate compounds present a fundamental inter-

est to understand the mechanisms of catalysis.

These compounds RR%P(O)PNP, noted (RY),

mimicking in both their charge distribution and

geometry the ﬁrst transition states occurring dur-

ing carboxyester hydrolysis were synthesized and

investigated as potential inhibitors of human gas-

tric (HGL) and pancreatic (HPL) lipases (Mar-

guet et al., 1994; Gargouri et al., 1997). Their

efﬁciency was studied on the basis of the alkyl

chain length, the nature of the leaving group and

the inﬂuence of the ester substituent (Marguet et

al., 1994). The released p-nitrophenol to enzyme

ratio indicates the formation of a 1:1 complex. In

the absence of substrate, the most powerful in-

hibitor was O-methyl-O-(p-nitrophenyl) n-

pentylphosphonate, (R₅Y₁), which possessed a

short alkyl chain (R=C₅H₁₁), a small methoxy

ester substituent (Y=CH₃) and a good leaving

group.

canoylglycerophosphonates

and

1,2-dicaprin

(Egloff et al., 1995b). With HPL, the four

stereoisomers exhibited a rather weak inhibitory

power and no signiﬁcant differences were ob-

served among them. With HGL, however, the

inhibition depended much more strongly on the

chirality at the sn-2 carbon of the glycerol back-

bone, while the chirality at the phosphorus atom

had no inﬂuence. Moreover, a clear correlation

was observed between the HGL surface concen-

tration and the inhibitor molar fraction (h₅₀) lead-

ing to half inhibition, the greatest enzymatic

inhibition was observed with ﬁlms containing the

enantiomeric inhibitor to which the HGL was

best adsorbed (Egloff et al., 1995b). Recently, the

three dimensional structure of HPL-colipase com-

plexes covalently bound to each enantiomer of a

chiral phosphonate inhibitor, the O-methyl-O-(p-

nitrophenyl) n-undecylphosphonate (R₁₁Y₁), was

resolved by X-ray diffraction (Egloff et al.,

1995a). The crystal structure of a racemic mixture

of (R₁₁Y₁) showed that the phosphorus atom of

each enantiomer were covalently bound to the O_g

oxygen of the active serine 152. Furthermore, the

use of each enantiomer separately in the crystal-

lization medium, revealed that the absolute

conﬁguration of the phosphorus atom of the most

Enantioselective inhibition was described for

the ﬁrst time by Patkar and Bjo¨rkling using Can-

dida antartica and Rhizomucor miehei lipases with

pure enantiomers of O-ethyl-O-(p-nitrophenyl) n-

hexylphosphonate (R₆Y₂) as inhibitors (Bjo¨rkling

et al., 1994; Patkar and Bjo¨rkling, 1994). Further-

more, using both diastereomers (S_p, R_p)-1R-Cl or

(S_p, R_p)-1S-Cl of O-menthyl n-hexyl chlorophos-

J.-F. Ca6alier et al. / Chemistry and Physics of Lipids 100 (1999) 3–31

5

reactive enantiomer, bound to the catalytic serine,

was (R_p). The C11 alkyl chain of this ﬁrst enan-

tiomer ﬁtted into a hydrophobic groove, mimick-

ing the interactions with the leaving acyl chain of

a glycerol substrate. The alkyl chain of the second

enantiomer (the less reactive bound to the cata-

lytic serine, (S_p) conﬁguration) possessed an elon-

gated conformation and interacted with

hydrophobic patches on the surface of the open

conformation of the amphipathic lid. This was

suggestive of the location of a second acyl chain

of a glycerol substrate. Finally, the alkyl chains of

the two enantiomers superimposed well with the

two fatty acyl chains of the phospholipid ob-

served in the ternary phosphatidylcholine-HPL-

colipase complex (van Tilbeurgh et al., 1993).

On the basis of these crystallographic studies,

and in an attempt to further characterize the

catalytic mechanism and in order to improve the

molecular ﬁt in the two hydrophobic clefts of the

HPL active site, we designed and synthesized new

chiral phosphonate inhibitors (RY). Their efﬁcacy

was studied by varying successively the alkyl or

ester chain length, R and Y respectively, from one

to 16 carbon atoms with p-nitrophenoxy as leav-

ing group (Scheme 1). In the present paper, we

wish to report the synthesis of these alkylphos-

phonate compounds, and the results of their inhi-

bition studies on HPL and HGL at the air–water

interface using the monomolecular ﬁlms technique

coupled with ELISA tests.

polyclonal antibodies against either native HGL

or native HPL were prepared and characterized as

previously described (Ivanova et al., 1993;

Aoubala et al., 1993, 1994).

2.3. General methods of synthesis

All reactions were carried out under scrupu-

lously dry conditions. All solvents and reagents

were puriﬁed according to usual methods (Perrin

et al., 1980). For TLC, precoated aluminium

sheets Merck silica gel 60 F₂₅₄were employed,

and the following detection methods were used:

UV lamp (254 nm); PMA, dipped into a solution

containing 5% phosphomolibdic acid in absolute

ethanol, and heated on a hot plate. All silica

chromatographic columns were performed as de-

scribed by Still et al. (1978) using Merck silica gel

60. Separation of the different enantiomers was

performed on a Water HPLC system using a

Daicel Chiralpack AS column 250×250 mm, and

a Waters UV detector at u=234 nm. The HPLC

analysis were carried out at 25°C using n-hexane–

2. Materials and methods

2.1. Materials

1,2-Didecanoyl-sn-glycerol (1,2-dicaprin) was

purchased from Serdary Research Laboratory

(Ont., Canada); streptavidin peroxydase and O-

phenilenediamine dihydrochloride were purchased

from Sigma.

2.2. Lipases

HGL and HPL were puriﬁed at the laboratory

using previously described procedures (De Caro et

al., 1977; Moreau et al., 1992). Monoclonal and

Scheme 1. Structure of alkylphosphonates (RY).

6

J.-F. Ca6alier et al. / Chemistry and Physics of Lipids 000 (1999) 3–31

2-propanol as eluent; ﬂow rate 4.5 ml min⁻¹

(see supplementary material for details). ³¹P

NMR experiments were all carried out with a

Brucker AC100 FT NMR spectrometer using an

³¹P NMR: l 42.9; ¹H NMR: l 2.55 (d, 3H,

J

_PH=16,3 Hz); ¹³C NMR: l 29.98 (d, P–CH₃,

¹J_PC=104,9 Hz).

1

85% H₃PO₄external reference. H and ¹³C NMR

2.4.3. Typical experimental procedure for the

preparation of O-alkyl O-(p-nitrophenyl)

n-undecylphosphonates (R₁₁Y_1–16) from 2-R₁₁,

described for compound (R₁₁Y₄)

experiments were all carried out with a Brucker

AC100 or AC200 FT NMR spectrometer.

CDCl₃was used as solvent and internal refer-

ence. Rotatory powers were all carried out with

a Perkin–Elmer P341 polarimeter. Elemental

analyses were performed at the Service Commun

de Microanalyse, University of Aix, Marseille

III. All products were fully characterised by ³¹P,

¹H, ¹³C NMR, elemental analyses and [h]²_D⁰.

Under nitrogen, to a solution of 1.0 g (3.66

mmol) n-undecylphosphonic acid dichloride 2-

R₁₁in CH₂Cl₂(5 ml) freed of ethanol, a solution

of 271.5 mg (3.66 mmol) of n-butanol 3-Y₄,

300.5 mg (3.66 mmol, 1 eq.) of N-methylimida-

zole (NMI) in CH₂Cl₂(5 ml) freed of ethanol

was added slowly dropwise. After the ﬁrst addi-

tion, a solution of 509.2 mg (3.66 mmol) of p-ni-

trophenol, 300.5 mg (3.66 mmol, 1 eq.) of NMI

in CH₂Cl₂(5 ml) freed of ethanol was then

added slowly dropwise. 15 min later, the solvent

was removed in vacuum, ether (10 ml) was

added and the salts were ﬁltered. The solution

was washed seven times with 5% K₂CO₃(7×5

ml), once with saturated NH₄Cl (3 ml) and twice

with brine. The organic layer was dried

(MgSO₄), ﬁltered and concentrated in vacuum.

The crude product was puriﬁed on silica with

petroleum ether–ether 7:3 as eluent (R_f=0.23,

PMA) to yield (R₁₁Y₄) (681 mg, 45%).

2.4. Synthesis of organophosphorus compounds

2.4.1. Typical experimental procedure for the

synthesis of alkylphosphonic acid dichloride

2-R_4–16from O-,O-dimethyl alkylphosphonates

1-R_4–16, described for compound 2-R₄

5.2 g (31 mmol) of 1-R₄was added dropwise

to a mixture of 11.9 g (87 mol, 3 eq.) of freshly

distilled oxalyl chloride in ether (5 ml), and then

reﬂuxed for 5 days. After cooling the solution,

the excess of reagent was freed in vacuum and

the crude product was distilled in vacuum to

yield 2-R₄(4.75 g, 79%). Eb_0.023=50°C. NMR

1

³¹P: l 50.22; H: l 2.55 (m, 2H), 1.65 (m, 4H),

HPLC: eluent n-hexane–2-propanol 98:2, t_R1

14.04 min., [a]²_D⁰= +2.37 (c 3.63, CH₂Cl₂); t_R2

15.52 min., [a]²_D⁰= −2.37 (c 3.63, CH₂Cl₂). ³¹P

1

0.9 (s, 3H); ¹³C: l 42.21 (d,P–CH₂, J_PC=96.4

Hz), 24.44 (d, P–CH₂–CH₂, ²J_PC=6.7 Hz),

22.30 (d, P–CH₂–CH₂–CH₂, ³J_PC=22.2 Hz),

12.96 (CH₃).

1

3

NMR: l 29.64; H NMR: l 8.2 (d, 2H_ar, J=

3

9.4 Hz), 7.4 (d, 2H_ar, J=10.4 Hz), 4.2 (m, 2H),

1,9 (m, 2H), 1.7 (m, 2H), 1.3 (m, 20H), 0.9 (t,

2

2.4.2. Synthesis of methylphosphonic acid

6H); ¹³C NMR: l 155.72 (d, O–C_ar, J_PC=7.7

dichloride 2-R₁

Hz), 144.39 (C_ar–NO₂), 125.52 (O–C_ar–C_ar–C_ar),

120.86 (d, O–C_ar–C_ar, J_PC=4.8 Hz), 66.49 (d,

3

5 g (40 mmol) of 1-R₁was added dropwise to

a solution of 10 ml (2.5 eq.) of HCl 33%, and

then reﬂuxed for 3 h. The water and methanol

formed were then distilled during the reaction.

The product was dried under vacuum and P₂O₅.

3.84 g (40 mmol, 96%) of methylphosphonic acid

thus prepared, were treated by 16.66 g (80 mol,

2 eq.) of phosphorus pentachloride at reﬂux for

14 h. Phosphorus trichloride was ﬁrst removed

by fractional distillation. Next the crude product

was distilled in a vacuum to yield 2-R₁(4.3 g,

81%) as white solid. Eb_0.008=40°C; M_p=32°C.

O–CH₂, ²J_PC=7.24 Hz), 32.31 (d, O–CH₂–

CH₂, ³J_PC=5.9 Hz), 31.79 (P–(CH₂)₈–CH₂),

30.31 (d, P–CH₂–CH₂–CH₂, ³J_PC=16.45 Hz),

29.22–29.46 (P–(CH₂)₄–(CH₂)₄), 28.91 (P–

(CH₂)₃–CH₂), 25.81 (d, P–CH₂, ¹J_PC=139.6

Hz), 22.58 (P–(CH₂)₉–CH₂), 22.12 (d, P–CH₂–

2

CH₂, J_PC=5.7 Hz), 18.57 (O–CH₂–CH₂–CH₂),

13.97 (P–(CH₂)₁₀–CH₃); 13.39 (O–(CH₂)₃–

CH₃); Anal. Calc. for C₂₁H₃₆NO₅P (413.49): C

61.0, H 8.8, N 3.4, P 7.5%; found, C 62.1, H

8.5, N 3.8, P 7.9%.

J.-F. Ca6alier et al. / Chemistry and Physics of Lipids 100 (1999) 3–31

7

2.4.4. Typical experimental procedure for the prepa-

ration of O-(n-undecyl) O-(p-nitrophenyl)

alkylphosphonates (R_1–16Y₁₁) from 2-R_1–16, de-

scribed for compound (R₁Y₁₁)

or 10 mM sodium acetate–HCl, pH 5, 100 mM

NaCl, 21 mM CaCl₂and 1 mM EDTA for HGL

assays. Buffers were prepared with double-distilled

water and ﬁltered through a 0.45 mM Millipore

membrane. Residual surface-active impurities were

removed before each assay by sweeping and suction

of the surface (Verger and de Haas, 1973).

Under nitrogen, to a solution of 1.09 g (8.2

mmol) methylphosphonic acid dichloride 2-R₁in

CH₂Cl₂(5 ml) freed of ethanol, a solution of 1.41

mg (8.2 mmol) of n-undecanol 3-Y₁₁, 674 mg (8.2

mmol, 1 eq.) of NMI in CH₂Cl₂(5 ml) freed of

ethanol was added slowly dropwise. After the ﬁrst

addition, a solution of 1.14 mg (8.2 mmol) of

p-nitrophenol, 674 mg (8.2 mmol, 1 eq.) of NMI

in CH₂Cl₂(5 ml) freed of ethanol was then added

slowly dropwise. 15 min later, the solvent was

removed in vacuum, ether (10 ml) was added and

the salts were ﬁltered. The solution was washed

seven times with 5% K₂CO₃(7×5 ml), once with

saturated NH₄Cl (3 ml) and twice with brine. The

organic layer was dried (MgSO₄), ﬁltered and

concentrated in vacuum. The crude product was

puriﬁed on silica with petroleum ether–ether 1:1 as

eluent (R_f=0.20, PMA) to yield (R₁Y₁₁) (1.83 g,

60%).

2.6. Enzymes kinetics experiments

Measurements were performed with the KSV

2200 Barostat equipment (KSV-Helsinki). The

principle of the method has been described previ-

ously by Verger et al. (Verger and de Haas, 1973).

It involved the use of a ‘zero-order through’ with

two compartments: a reaction compartment and a

reservoir compartment, which were connected to

each other by a small surface channel. Enzyme

solution was injected into the subphase of the

reaction compartment only, whereas the lipid ﬁlm

covered both of them. A mobile barrier, automat-

ically driven by the barostat, moved back and forth

over the reservoir to keep the surface pressure (y)

constant, thus compensating for the substrate

molecules removed from the ﬁlm by the enzyme

hydrolysis. The surface pressure was measured on

the reservoir compartment with a Wilhelmy plate

(perimeter 3.94 cm) attached to an electromicrobal-

ance, connected in turn to a microprocessor pro-

grammed to regulate the mobile-barrier movement.

The reaction compartment was stirred at 250 rpm

by two 2.5 cm magnetic bars. The surface of the

reaction compartment was 100 cm²and its volume

100 ml. The reservoir compartment was 148 mm

wide and 249 long. Mixed ﬁlms of substrate–in-

hibitor were spread from a chloroform solution

(about 1 mg ml⁻¹), over the surface of the reaction

compartment only, whereas the reservoir was cov-

ered with a ﬁlm of pure substrate (1,2-dicaprin)

(Pie´roni and Verger, 1979, 1983; Gargouri et al.,

1987). The kinetics of hydrolysis were recorded for

20–25 mins, and the kinetics data were analyzed as

in the case of a previous model (Ransac et al., 1990,

1991).

HPLC: eluent n-hexane–2-propanol 99.5:0.5,

the two enantiomers were not separated. ³¹P NMR:

l 27.40; ¹H NMR: l 8.2 (d, 2H_ar, ³J=9.2 Hz), 7.4

(d, 2H_ar, ³J=9.2Hz), 4.1 (m, 2H), 1.6 (d, 3H,

¹J=17.65 Hz), 1.3 (m, 18H), 0.9 (t, 3H); ¹³C NMR:

2

l 155.49 (d, O–C_ar, J_PC=8.7 Hz), 144.69 (C_ar–

NO₂), 125.65 (O–C_ar–C_ar–C_ar), 120.91 (d, O–C_ar–

3

2

C_ar, J_PC=4.6 Hz), 67.05 (d, O–CH₂, J_PC=7.2

Hz), 31.84 (d, O–(CH₂)₈–CH₂), 30.32 (d, O–CH₂–

CH₂, ³J_PC=6.8 Hz), 29.03–29.64 (O–(CH₂)₃–

(CH₂)₅), 25.36 (O–CH₂–CH₂–CH₂), 22.63

(O–(CH₂)₉–CH₂), 14.05 (O–(CH₂)₁₀–CH₃), 11.53

(d, P–CH₃, ¹J_PC=145.5 Hz); Anal. calc. for

C₁₈H₃₀NO₅P (371.41): C 58.2, H 8.1, N 3.8, P 8.3%;

found, C 59.1, H 8.7, N 4.3, P 7.9%.

2.5. Monomolecular ﬁlm experiments

Before each utilization, the Teﬂon trough was

cleaned with tap water, then gently brushed in the

presence of distilled ethanol, before being washed

again with tap water and ﬁnally rinsed with double-

distilled water. The aqueous subphase was com-

posed of 10 mM Tris–HCl, pH 8, 100 mM NaCl,

21 mM CaCl₂and 1 mM EDTA for HPL assays

2.7. ELISA tests

The kinetics of hydrolysis were recorded as

8

J.-F. Ca6alier et al. / Chemistry and Physics of Lipids 100 (1999) 3–31

described above for 15 min, and the remaining ﬁlm

was aspirated as previously described (Aoubala et

al., 1995). The ﬁlm was recovered into a glass tube

(0.3–1 ml) by placing a barrier across the surface

channel and by sweeping it over the reaction

compartment and aspiring the ﬁlm thus collected.

After completely recovering the ﬁlm, an equal

volume of the sub-phase was sampled and placed

in another tube. The difference between the total

amounts of proteins between these two samples

was attributed to the surface excess of protein

molecules bound to the lipid ﬁlm.

nm using a micro ELISA reader (MR 5000, Dynat-

ech).

2.8. Determination of the amount of protein

adsorbed to the lipid monolayer

A reference curve was drawn up for each test and

was used to determine the amount of protein

adsorbed to the lipid monolayer. For this purpose,

proteins (HGL or HPL) at known concentrations

in 50 ml of buffer B were incubated in the wells of

a micro-titration plate previously coated with a

speciﬁc polyclonal antibody. An ELISA was per-

formed as described above. The optical density

values at 490 nm were plotted as a function of the

concentration of protein. Each assay was carried

out in duplicate. The obtained reference curve was

used to calculate the concentration of protein in the

aspirated samples recovered from the monomolec-

ular experiments as described above. In order to

calculate the amount of protein bound to the

monomolecular lipid ﬁlm, the volume occupied by

the lipid ﬁlm was not taken into account since it

was negligible with respect to the aspirated sub-

phase. We used the following equation:

All the ELISA tests were performed in 96-wells

poly(vinylchloride) (PVC) microplates (Maxisorb,

Nunc). The wells were coated with 250 ng of a

speciﬁc polyclonal antibody (anti-HGL or anti-

HPL for the titration of HGL and HPL respec-

tively) solubilized in 50 ml of 10 mM phosphate

buffer (pH 7.5) containing 137 mM NaCl, 2.7 mM

KCl and 8 mM Na₂HPO₄, 12 H₂O (buffer A) and

incubated overnight at 4°C. The wells were then

washed three times, 5 min for each, with 300 ml/well

of buffer A, and the excess protein binding sites

were saturated by incubation with 200 ml/well of

blocking agent (3% w/v Re´gilait skimmed milk

powders in buffer A) for 2 h at 37°C. The plates

were then washed three times with buffer B (buffer

A containing 0.05% v/v Tween 20), and dry the

excess buffer by aspiration. Fill each well with 50

ml of sample solution in diluted buffer A, and

incubate 1 h at 37°C. Wash the plates three times

for 5 min with buffer B, and ﬁll each well with 50

ml of speciﬁc biotinylated monoclonal anti-HGL or

anti-HPL (detector antibody) in buffer B, and keep

at 37°C for 1 h. Wash three times for 5 min with

buffer B, and ﬁll each well with 50 ml of Strep-

tavidine peroxydase diluted with buffer B at 1/

1000. Keep at 37°C for 45 min. After washing the

plates three times for 5 min with buffer B, and

drying the excess buffer by aspiration, 50 ml of

peroxydase substrate solution (O-phenylenedi-

amine dihydrochloride 0.4 g l⁻¹in 100 mM sodium

phosphate–150 mM sodium citrate (pH 5) contain-

ing 0.04% of fresh hydrogen peroxide) was added

to each well and incubated for 15 min in the dark

at room temperature. The enzyme reaction was

stopped by adding 50 ml of 0.5 M H₂SO₄to each

well. The optical density (OD) was measured at 490

[F+B]−[B]

Y=

· V_a

S

where Y is the surface excess of protein bound to

the lipid monolayer, expressed in pg cm⁻². [F+B]

is the concentration of protein present in the

aspirated ﬁlm with the aspirated bulk sub-phase, as

determined by ELISA test. [B] is the concentration

of protein in the bulk sample, also determined by

ELISA. V_ais the aspirated volume (ranging from

0.5 to 1 ml), and S is the area of the reactional

compartment of the trough (31 cm²).

2.9. Reliability of the sandwich ELISA for HGL

and HPL adsorbed lipid monolayers

During the monolayers experiments, the validity

of the sandwich ELISA for HGL and HPL was

tested in the presence of mixed ﬁlms of inhibitor–

1,2-dicaprin. The recovery levels of protein injected

under lipid monomolecular ﬁlms were determined

after each experiment as:

J.-F. Ca6alier et al. / Chemistry and Physics of Lipids 100 (1999) 3–31

9

Scheme 2. Synthetic pathway of various alkylphosphonate compounds (RY).

[B] · V_t+Y · S

Total recovery (%)=

· 100

nates (R_1–16Y₁₁), with chemical yields ranging

from 40 to 70%.

T

All chiral phosphonates were synthesized in a

racemic form. They were puriﬁed by silica-gel

chromatography. Each enantiomer was then sepa-

rated by performing chiral liquid chromatogra-

phy, as described in the experimental section. We

decided to deﬁne each enantiomer of the same

compound by its retention time on chiral HPLC,

noted f for ‘fast’, and s for ‘slow’. In the case of

(R₁Y₁₁) and (R₄Y₁₁), since the two respective

enantiomers were not separated by chiral HPLC,

these two alkylphosphonates were used in a

racemic form in the inhibition studies. It also

Where [B] is the concentration of HGL or HPL in

the sub-phase; V_tis the total volume of the reac-

tional compartment measured after each mono-

layer experiment (5091.5 ml); YS is the total

amount of protein adsorbed to the monomolecu-

lar ﬁlm; and T is the total amount of HGL (14.85

mg) or HPL (1 mg) injected under the monomolec-

ular ﬁlm.

3. Results and discussion

must be noted that for compounds (R₁₁Y_1–16

)

3.1. Synthesis of 6arious alkylphosphonates (RY)

there was a sign inversion of the rotatory power

between the two enantiomers from (R₁₁Y₁₀) to

(R₁₁Y₁₆).

Alkylphosphonic acid dichloride 2-R_4–16syn-

thesis has been easily performed from correspond-

ing O-O-dimethyl alkylphosphonates 1-R_4–16with

oxalyl chloride, as described by Marguet et al.

(1994), except for compound 2-R₁which has been

prepared using classical methods of successive

hydrolysis and chlorinating (Scheme 2) (Maier,

1973; Quast et al., 1974). Next, successive and

selective substitution of the chlorine atoms of

2-R_1–16by various alcohols 3-Y_1–16and p-nitro-

phenol in the presence of N-methylimidazole

(NMI) led to the formation of the desired

alkylphosphonates (RY), as depicted in Scheme 2.

We thus obtained various O-alkyl-O-(p-nitro-

phenyl) n-undecylphosphonates (R₁₁Y_1–16) and

3.2. Forces/area cur6es of alkylphosphonates

(RY)

In order to study the effects of the phospho-

nates (RY) on the human gastric and pancreatic

lipases, we ﬁrst determined the ﬁlm stability of all

the above compounds and their interfacial proper-

ties at the air–water interface. Experiments were

performed in the reservoir compartment of the

‘zero-order’ trough, as described in the experi-

mental section. With all the compounds tested,

sharp collapse points are indicative of a high

degree of purity. Moreover, the surface-pressure

O-undecyl-O-(p-nitrophenyl)

n-alkylphospho-

10

J.-F. Ca6alier et al. / Chemistry and Physics of Lipids 000 (1999) 3–31

tion from ideality when mixing 1,2-dicaprin and

phosphonate inhibitors (Andelman, 1989; Arnett

et al., 1989; Pathirana et al., 1992). In each case

the deviation from an ideal behavior was less

pronounced with the ‘fast’ enantiomers than with

the ‘slow’ enantiomers. This expansion effect was

approximately 11% in a 25:75 1,2-dicaprin–‘fast’

enantiomer mixture, compared with approxi-

mately 22% in a 75:25 1,2-dicaprin–’slow’ enan-

tiomer mixture. Furthermore, at various inhibitor

molar fractions (h): 0.25, 0.50, 0.75, we found

(data not shown) collapse pressure values (in mN

curves as a function of the molecular area were

found to be identical for the two enantiomers of a

same compound (Fig. 1). All the ﬁlms consisting

of the previous phosphonates, including (R₁Y₁₁)

and (R₁₁Y₁), in the pure state or mixed with

1,2-dicaprin, were stable with time, i.e. no signiﬁ-

cant decrease in the surface pressure was observed

within 1 h. For the kinetic studies, we selected a

surface pressure of y=20 mN m⁻¹for mixed

inhibitor–1,2-dicaprin ﬁlms. At this value, which

was below the collapse pressure of all the

alkylphosphonate (RY) tested, HPL and HGL are

active and characterised by linear kinetics when

using pure substrate. The covalent inhibition stud-

ies of HGL and HPL were then carried out using

the monomolecular ﬁlms technique (Verger and

de Haas, 1973; Pie´roni and Verger, 1979, 1983;

Gargouri et al., 1987).

m

⁻¹) of: 27.2, 23.4, 21.9 for (R₁₆Y₁₁)f and 23.6,

23.1, 21.6 for (R₁₆Y₁₁)s, respectively. The corre-

sponding collapse pressure values (in mN m⁻¹

)

were found to be (data not shown): 28.7, 25.4,

22.6 for (R₁₀Y₁₁)f and 24.4, 23.4, 22.9 for

(R₁₀Y₁₁)s, respectively. One can notice that at the

working surface pressure of 20 mN m⁻¹all the

above mentioned mixed ﬁlms, consisting of 1,2-di-

caprin and phosphonate inhibitors, are stable and

thus amenable to be studied by the monomolecu-

lar ﬁlm technique.

Fig. 2 shows the variation of the mean area per

molecule in mixtures of 1,2-dicaprin with each

enantiomer of compounds (R₁₀Y₁₁) (Fig. 2, panel

A) and (R₁₆Y₁₁) (Fig. 2, panel B) at a surface

pressure of 18 mN m⁻¹. A clear expansion effect

on the area per molecule was apparent from the

non linear relationship, reﬂecting a strong devia-

Arnett et al. have clearly demonstrated that the

chiral recognition in monolayers depends strongly

Fig. 1. Surface pressure versus molecular area of monomolecular ﬁlms of the two enantiomers, ‘fast’ (ꢀ) and ‘slow’ (), of the

n-decyl p-nitrophenyl n-undecylphosphonate (R₁₁Y₁₀).

J.-F. Ca6alier et al. / Chemistry and Physics of Lipids 000 (1999) 3–31

11

Fig. 2. Mean area per molecule in ﬁlms formed by mixtures of 1,2-dicaprin with each enantiomer ‘fast’ () and ‘slow’ (ꢀ) of

compounds (R₁₀Y₁₁) and (R₁₆Y₁₁) at a surface pressure of 18 mN m⁻¹. The straight dotted lines join the data for the pure

compounds indicating ideal mixing.

on the ability of the interacted molecules to bring

their chiral centers into favorable stereospeciﬁc

interactions (Arnett et al., 1989). Such intermolec-

ular effects have been theoretically estimated on

simple models by Andelman (1989). Recently,

Vodyanoy et al. have detected different

diastereomeric interactions of the odorants (+)

and (−)-carvone with

glycero-3-phosphocholine (

(Pathirana et al., 1992). The surface pressure-area

isotherms with each enantiomeric carvone show a

clear chiral discrimination effect attributed to dif-

L

−a-1,2-dipalmitoyl-sn-

-DPPC) monolayers

L

ferent packing arrangements of

layers exposed to different odorants.

L-DPPC in mono-

12

J.-F. Ca6alier et al. / Chemistry and Physics of Lipids 100 (1999) 3–31

In the case of the two enantiomers of com-

and ester chains length from four to eight carbon

atoms. This result conﬁrms the known preference

of HGL for medium and short lipid chains (Ro-

galska et al., 1990, 1995).

HPL and HGL stereoprefence for compounds

(RY) was then studied on the basis of a new

parameter, the stereoselectivity index (S.I.) (39,

40) as deﬁned in Eq. (1):

pounds (R₁₀Y₁₁) and (R₁₆Y₁₁), the differences ob-

served in the mean area per molecule at a

constant surface pressure (Fig. 2), disclose

diastereomeric interactions of the ‘fast’ or the

‘slow’ enantiomer with 1,2-dicaprin in mixed

ﬁlms, regardless of the alkyl chains length (C₁₀H₂₁

and C₁₆H₃₃).

However, during our investigations, the inhibi-

tion studies on HPL and HGL were performed

with low molar fraction of compounds (RY)

which were found to be very efﬁcient inhibitors

(vide infra). When using very low concentrations

of ‘fast’ or ‘slow’ alkylphosphonate compound in

mixed ﬁlms with 1,2-dicaprin, the molar fraction

of the inhibitor is very close to the relative surface

occupied by each component. In these conditions,

the alkylphosphonate enantiomers will not deviate

too much from the additivity rule of their respec-

tive area per molecule. Their inhibitory power can

then be expressed as the function of the molar

fractions of the inhibitor relative to the inhibitor

plus 1,2-dicaprin.

h f−h s

h₅₀f+h₅₀s

) )

50

S.I. (%)=

· 100

(1)

where h₅₀f and h₅₀s are the molar fraction of the

‘fast’ and ‘slow’ enantiomers of the same com-

pound, respectively. Fig. 4 shows the variation of

S.I. values as a function of the dissymmetry be-

tween the alkyl and the ester chain length, for

compounds (R₁₁Y_n) and (R_nY₁₁), with n ranging

from six to 16 carbon atoms. This new parameter

called the dissymmetry index (D.I.) was calculated

as follows:

D.I.=(n−11) with n varying from six to 16

carbon atoms

(2)

3.3. HGL and HPL acti6ities on mixed ﬁlms

containing alkylphosphonates

With HPL (Fig. 4, panel A), all compounds

presented S.I. values higher than 40%, indicating

a signiﬁcant chiral discrimination between two

enantiomers. Moreover, the highest values (ꢀ

87%) were reached for D.I. values equal to +1

and +5; showing a preference of HPL for com-

pounds (R₁₁Y₁₂) and (R₁₆Y₁₁), respectively. In

contrast to HPL, HGL (Fig. 4, panel B) shows a

strong dependency of S.I. values as a function of

D.I. values. The greatest stereoselectivity is ob-

served for a D.I. value equal to −1, correspond-

ing to the compounds (R₁₁Y₁₀) and (R₁₀Y₁₁).

From the data presented in Fig. 4, one can also

notice that there is a negligible chemoselective

discrimination in the lipases active site between

the isosteric methylene group (–CH₂–) of an

alkylphosphonate chain and the alkoxy group

(–OCH₂–) of an ester phosphonic chain; since a

permutation between R and Y chains does not

strongly affect the S.I. values. A molecular inter-

pretation of the above data has to wait for the

resolution of the three dimensional structures of

lipase-phosphonate complexes.

Lipase activities were measured as a function of

the inhibitor molar fraction (h). With both lipases

tested, the hydrolysis of 1,2-dicaprin decreased

sharply as the molar fraction of inhibitors (RY)

increased. Fig. 3 showed the variation, as a func-

tion of the alkyl R or ester Y chain length, of the

inhibitor molar fraction (h₅₀), which reduced en-

zyme activity to 50% of its initial value on pure

1,2-dicaprin.

With the two lipases, there is no obvious corre-

lation between h₅₀and the chain length, R or Y.

In the case of HPL (Fig. 3, panel A), compounds

(R₁₁Y_1–16)s and (R_1–16Y₁₁)s, with higher retention

times on chiral HPLC, are the most potent in-

hibitors. Moreover, between the above two series

of ‘slow’ enantiomers, the best inhibitors of HPL

are (R₁₁Y₁₆)s and (R₁₆Y₁₁)s. On the contrary, in

the case of HGL (Fig. 3, panel B), no clear

distinction could be made between the ‘slow’ or

the ‘fast’ enantiomers. The best inhibition of

HGL is obtained with compounds including alkyl

J.-F. Ca6alier et al. / Chemistry and Physics of Lipids 100 (1999) 3–31

13

Further experiments were carried out with the

best inhibitors: compounds (R₁₁Y₁₆) and

(R₁₆Y₁₁) on HPL, and compounds (R₁₁Y₁₀) and

(R₁₀Y₁₁) on HGL. The ‘slow’ enantiomers of

these alkylphosphonates were in fact the best

inhibitors of the digestive lipases (Fig. 3 and

Table 1). Moreover, these compounds displayed

the highest S.I. values (see Fig. 4).

We ﬁrst determined the inﬂuence of the enan-

tiomeric excess (ee) on the inhibitory power

(h₅₀) (Fig. 5). In three cases, an hyperbolic rela-

tionship was observed. However, no signiﬁcant

Fig. 3. Variation of the inhibitor molar fraction h₅₀(%), reducing HPL (panel A) or HGL (panel B) activity to 50% of its initial

value on 1,2-dicaprin during monolayer experiments at y=20 mN m⁻¹, as a function of the alkyl (R) or ester (Y) chain length,

for compounds (R₁₁Y_1–16) and (R_1–16Y₁₁).

14

J.-F. Ca6alier et al. / Chemistry and Physics of Lipids 000 (1999) 3–31

Fig. 4. Variation of the S.I. (%), as a function of the dissymmetry index (D.I.), with alkylphosphonates (R₁₁Y_1–16) and (R_1–16Y₁₁),

on HPL (panel A) or HGL (panel B). The lipases enantioselectivity for these compounds was studied on the basis of new

parameters, the S.I. deﬁne by analogy with the enantiomeric excess, and the D.I. showing the dissymmetry between the alkyl and

the ester chains length:

h

f−h s

50

) )

50

S.I. (%)=

· 100

h₅₀f+h₅₀

s

and D.I.=(n−11) with n ranging from six to 16 carbon atoms.

variation of h₅₀was observed for ee values above

−50%.

Furthermore, HPL and HGL superﬁcial con-

centrations at the lipidic interface were then

quantiﬁed by means of ELISA tests (Aoubala et

al., 1995). The latter method was developed at our

laboratory in order to measure the surface density

of lipases in the picogram range (Aoubala et al.,

1995). The recovery levels of each lipase injected

under the lipid monomolecular ﬁlms were as high

as 90% for HPL and 80% for HGL (see supple-

mentary material for details). As the monolayer

J.-F. Ca6alier et al. / Chemistry and Physics of Lipids 100 (1999) 3–31

15

system is characterised by a low speciﬁc surface

(around 1 cm²cm⁻³), only a small fraction of the

total amount of enzyme is actually bound to the

monomolecular ﬁlm. In the particular cases of

HPL and HGL, using the four above cited in-

hibitors, less than 0.7% (HPL) and 0.2% (HGL) of

the total amount of injected lipase, were recovered

after ﬁlm aspiration (see supplementary material

for details). Judging from this percentage of ﬁlm-

bound enzyme, the effective interfacial stoichiome-

try at the h₅₀inhibitor molar fraction can be

calculated and their values are reported in Table 2:

one molecule of HGL, bound to a mixed

monomolecular ﬁlm, could statistically interact

with 1.4×10⁴substrate molecules, and was inhib-

ited at 50% of its initial rate on 1,2-dicaprin by 110

molecules of inhibitor (R₁₀Y₁₁)s. In the case of

HPL, one molecule of this enzyme could statisti-

cally interact with 5.9×10⁴substrate molecules,

and was inhibited at 50% of its initial rate on

1,2-dicaprin by 153 molecules of inhibitor

(R₁₁Y₁₆)s. Concerning THL, its inhibitory power

(h₅₀) on HPL is 1.5×10⁻⁴%, which is 2000 times

higher than the best alkylphosphonate enantiomer

(data not shown). With HGL (Ransac et al., 1991),

the value of THL inhibitory power (0.25%) is quite

close to the value obtained in our study with

compound (R₁₀Y₁₁)s.

atically observed as the molar fraction of inhibitors

increased. In the case of HPL, the binding curves

obtained with each enantiomer were comparable.

Moreover, we showed that at a given inhibitor

molar fraction (h=15%), the variation of the

superﬁcial concentration of HPL (Y=37.1, 35.9,

36.7, 39.5, 40.3, 37.8, 38.2%) did not depend on the

enantiomeric excess (ee= −100, −96, −80, −

50, 0, +50, +100%, respectively) of inhibitor

(R₁₆Y₁₁).

With compounds (R₁₁Y₁₆)f, (R₁₁Y₁₆)s and

(R₁₆Y₁₁)s on HPL (Fig. 6) and compounds

(R₁₁Y₁₀)s and (R₁₀Y₁₁)s on HGL (Fig. 7), we

observed a clear difference between the inhibition

and the binding curves as a function of h. On the

contrary, with compound (R₁₆Y₁₁)f on HPL (Fig.

6) and compounds (R₁₁Y₁₀)f and (R₁₀Y₁₁)f on

HGL (Fig. 7), the inhibition and binding curves are

nearly superimposed.

These differences between the inhibition and

interfacial behavior are well illustrated by the ratio

between the molar fraction leading to half lipase

binding (Y₅₀), to the h₅₀inhibitor molar fraction

(Table 1). The highest values of this ratio, Y₅₀/h₅₀

correspond to the most potent enantiomeric

alkylphosphonate inhibitors.

3.4. Kinetic model of the co6alent inhibition of a

lipolytic enzyme at a lipid–water interface by two

enantiomeric compounds

Fig. 6 (HPL) and Fig. 7 (HGL) show the

variations of the residual activity (%) and the

superﬁcial concentration, Y (%), as a function of

the inhibitor molar fraction (h). In each case, a

decrease in the enzyme surface density was system-

The kinetic model presented in Fig. 8 has been

developed to describe the covalent inhibition of

Table 1

Values of the inhibitory power, h₅₀(%), lipases superﬁcial concentration, Y_h(%) (expressed as the ratio of the amount of lipase

adsorbed on each pure enantiomeric inhibitor at the h₅₀molar fraction, to the ⁵t⁰otal amount of lipase adsorbed on 1,2-dicaprin ﬁlms),

and inhibitor molar fraction, Y₅₀, leading to half lipase adsorption^a

HPL

HGL

(R₁₁Y₁₀

Fast

(R₁₁Y₁₆

Fast

)

(R₁₆Y₁₁

Fast

)

(R₁₀Y₁₁

)

Slow

Fast

Slow

h₅₀

Y_h

1.2

90.0

8.9

0.3

99.8

12.3

41.0

4.9

58.7

5.0

0.4

99.4

12.3

30.8

7.8

43.7

4.9

1.4

80.0

4.2

3

9.2

36.7

4.1

0.8

93.1

31.0

38.8

50

k₅₀

k_50/h₅₀

7.4

1.0

0.6

0.4

^aThese data were determined during inhibition studies and ELISA tests with mixed ﬁlms inhibitor–1,2-dicaprin at y=20 mN

m⁻¹for compounds (R₁₁Y₁₆) and (R₁₆Y₁₁) on HPL at pH 8, and compounds (R₁₁Y₁₀) and (R₁₀Y₁₁) on HGL at pH 5.

16

J.-F. Ca6alier et al. / Chemistry and Physics of Lipids 100 (1999) 3–31

Fig. 5. Variation of the inhibitory power h₅₀(%) as a function of the enantiomeric excess ee (%) of compound (R₁₁Y₁₆) or (R₁₆Y₁₁

)

on HPL (panel A) and compound (R₁₁Y₁₀) or (R₁₀Y₁₁) on HGL (Panel B), in mixed ﬁlms inhibitor–1,2-dicaprin at y=20 mN m⁻¹

using the monomolecular ﬁlms technique.

J.-F. Ca6alier et al. / Chemistry and Physics of Lipids 100 (1999) 3–31

17

Table 2

the formation of two different forms of cova-

lently inhibited enzyme (E*i_R) and (E*i_S). In this

model, we assume that the enzyme binding step

at the lipidic interface (E X E*) is slow as com-

pared to the three other reversible equilibria, in-

volving the substrate or the inhibitors. The

kinetic treatment of this new model was devel-

oped by analogy with the previous analysis by

Ransac et al. (1990, 1991). In the above kinetic

treatment, the inhibitor was assumed to be in

large molar excess as compared to the adsorbed

enzyme. This assumption was experimentally

checked to be fulﬁlled (see Table 2).

Interfacial stoichiometry values, at y=20 mN m⁻¹, by mole

of enzyme HPL or HGL, at the h₅₀inhibitor molar fraction,

for each enantiomer of compounds (R₁₁Y₁₆) and (R₁₆Y11)

with HPL, and (R₁₁Y₁₀) and (R₁₀Y₁₁) with HGL

Per HGL molecule

Fast

Slow

Fast

Slow

1,2-dicaprin

1.4×10⁴

5.9×10⁴

(R₁₁Y₁₀

(R₁₀Y₁₁

(R₁₁Y₁₆

(R₁₆Y₁₁

)

1080

1200

–

162

110

–

616

2771

–

153

210

–

The sigmoidal expression of the time depen-

dence of product released (P), normalized with

the enantiomeric excess (ee) and the inhibitor

molar fraction (h) is:

lipolytic enzymes at a lipid–water interface, by

two enantiomeric inhibitors (I_R) and (I_S), with

Fig. 6. Variation of HPL residual activity (%) (—) and superﬁcial concentration (%) (----) as a function of inhibitors molar fraction,

(R₁₁Y₁₆) or (R₁₆Y₁₁). HPL ﬁnal concentration 19.4 ng ml⁻¹in the sub-phase at pH 8, surface pressure 20 mN m⁻¹

.

18

J.-F. Ca6alier et al. / Chemistry and Physics of Lipids 100 (1999) 3–31

Fig. 7. Variation of HGL residual activity (%) (—) and Superﬁcial Concentration (%) (----) as a function of inhibitors molar

fraction, (R₁₁Y₁₀) or (R₁₀Y₁₁). HGL ﬁnal concentration 288.4 ng ml⁻¹in the sub-phase at pH 5, surface pressure 20 mN m⁻¹

.

Ks

Kf

k

K*

I

S

ꢁ ꢂ ꢁ ꢂ

1−h

h

2

i

S

more,

=

·

characterizes the ratio

P=

·

k_i

R

Ks · (1+ee)+Kf · (1−ee)

I

S

t

~

~₁· e^{− /}−~₂· e

of the speciﬁcity constants of the two enan-

tiomeric inhibitors (I_R) and (I_S).

~₁and ~₂are complex parameters (see supple-

mentary material for details) including k_d, k_p, ki_S,

− /

~

1

2

ꢀ

n

1−

(3)

~₁−~₂

with

ki_R, K* , K*I_S, K*I_Rand represent the time con-

k_iK*

K* k_cat

k_i

K*

M

s

R

E₀· Ks=

·

,

E₀· Kf=

·

,

stants of the combined partial reversible reactions

K* k_cat

I

R

I

s

(E X E*)

I_SX E*I_S) (E*+I_RX E*I_R), as well as the irre-

versible reactions (E*I_SE*i_S) and

(E*I_RE*i_R).

The corresponding equation of the enzymatic

velocity is:

(E*+S X E*S X E*S)

(E*+

I_R+I_S

I_S−I_R

I_R+I_S

h=

,

ee=

I_R+I_S+S

E₀Ks (for the ‘slow’ enantiomer) and E₀Kf (for

the ‘fast’ enantiomer) represent the ratio of the

speciﬁcity constant k_i/K* of each enantiomeric

I

inhibitor to the speciﬁcity constant of the sub-

dP 1−h

2

6=

=

·

strate k_cat/K* . The kinetic signiﬁcance of these

M

dt

h

Ks · (1+ee)+Kf · (1−ee)

t

~

constants, E₀Ks and E₀Kf, is in fact the relative

reactivity as well as the relative afﬁnity of one

inhibitor as compared to the substrate. Further-

− /

e^{− /}−e

~

1

2

(4)

~₁−~₂

J.-F. Ca6alier et al. / Chemistry and Physics of Lipids 100 (1999) 3–31

19

As can be seen from Eq. (4), the enzyme velocity

ln ~₁−ln ~

~₁−~₂

t_inﬂexion

=

²· ~₁· ~₂

(6)

possesses a maximal value, 6_inﬂexion(Eq. (5)) at a

given time t_inﬂexion(Eq. (6)) which correspond to

the inﬂexion point of the experimental kinetic

curve of the product release as a function of time.

On the basis of this new model, and starting

from Eq. (3), we performed curve ﬁtting on 20

experimental kinetics obtained with HPL, using

each of the following enantiomers (R₁₁Y₁₀),

(R₁₁Y₁₆) and (R₁₆Y₁₁). In a ﬁrst step, we ﬁtted

each kinetic experiment, P=f(t), and in a second

step we calculated by an iteration method the

values of Kf, Ks, ~₁, ~₂as a function of ee, using

MicroMath Scientist^®for Windows™ v. 2.01

software.

dP 1−h

6_inﬂexion

=

·

~

dt

h

2

~ −~

2

1

~

ꢁ ꢂ

2

1

2

·

(5)

Ks · (1+ee)+Kf · (1−ee) ~₁

~

1

It must be noted that ~₁and ~₂depend on both

substrate (S), inhibitor (I) concentrations and on

the enantiomeric excess (ee). In order to ﬁt our

experimental curves, P=f(t), we intentionally de-

cided to keep constant the two parameters, ~₁and

~₂for a given compound.

The classiﬁcation of the inhibitors according to

the theoretical values of E₀Ks and E₀Kf obtained

(Table 3) is in agreement with the classiﬁcation of

the inhibitory power (h₅₀), previously established

with HPL (Fig. 3, panel A):

(R₁₁Y₁₆)s\(R₁₆Y₁₁)s\(R₁₁Y₁₀)s\(R₁₁Y₁₆)f

\(R₁₁Y₁₀)f\(R₁₆Y₁₁)f

Fig. 8. Kinetic model illustrating the covalent inhibition of a

lipolytic enzyme at a lipid–water interface. Symbols and ab-

breviations are as follows: A, total interfacial area (surface); V,

total volume (volume); E₀, total enzyme concentration

(molecule/volume); E, free enzyme concentration (molecule/

volume); E*, interfacial enzyme concentration (molecule/sur-

Starting from the calculated values of the

parameters listed in Table 3, in the case of com-

pounds (R₁₁Y₁₆) and (R₁₆Y₁₁) with HPL (see sup-

plementary material for details), we found an

hyperbolic dependency of the h₅₀values as a

function of the enantiomeric excess, in good

agreement with the experimental data presented in

Fig. 5.

face);

S,

interfacial

concentration

of

substrate

(molecule/surface); I_R, interfacial concentration of inhibitor-

(R) (molecule/surface); I_S, interfacial concentration of in-

hibitor-(S) (molecule/surface); P, product concentration

(molecule/volume); E*S, interfacial enzyme-substrate complex

concentration (molecule /surface); E*I_R, interfacial enzyme-in-

hibitor-(R) complex concentration (molecule/surface); E*I_S,

interfacial enzyme–inhibitor-(S) complex concentration

(molecule/surface); E*i_R, interfacial enzyme covalently inhib-

ited by inhibitor-(R) concentration (molecule/surface); E*i_S,

interfacial enzyme covalently inhibited by inhibitor-(S) con-

centration (molecule/surface); k_d, desorption rate constant

(time⁻¹); k_p, penetration rate constant (volume surface⁻¹

3.5. Kinetic model of the pseudo-competiti6e

inhibition of a lipolytic enzyme at a lipid–water

interface by two enantiomeric compounds

In order to simplify the kinetic treatment of the

general covalent inhibition model presented in

Fig. 8, we can assume as a ﬁrst approximation

that HGL and HPL inhibition with alkylphospho-

nates (RY), is pseudo-competitive; i.e. the rate

constants (k_i, k_i) are slow and negligible as

time⁻¹); k₁, rate constant (surface molecule⁻¹time⁻¹); k₋₁

,

rate constant (time⁻¹); k_cat, catalytic rate constant (time⁻¹);

k_i, inhibition rate constant for the enzyme–inhibitor-(R)

R

complex (time⁻¹); k_i, inhibition rate constant for the en-

S

zyme–inhibitor-(S) complex (time⁻¹); K* , interfacial

M

Michae¨lis–Menten constant (molecule/surface); K* , interfa-

I

R

cial dissociation constant for the enzyme-inhibitor-(R) com-

R

S

compared to k_cat. This approximation is based on

the experimental observation that after the inﬂex-

plex (molecule/surface); K* , interfacial dissociation constant

I

for the enzyme-inhibitor-(S^S) complex (molecule/surface).

20

J.-F. Ca6alier et al. / Chemistry and Physics of Lipids 100 (1999) 3–31

Table 3

Results of the curve ﬁtting on 20 experimental kinetic curves registered on HPL, with each enantiomer of the compounds (R₁₁Y₁₀),

(R₁₁Y₁₆) and (R₁₆Y₁₁), using MicroMath Scientist^®for Windows™ v. 2.01 software

E₀Kf (×10¹⁴

)

E₀Ks (×10¹⁴

)

~

(min)

~ (min)

2

K_s

K_f

k

K*

I

R

ꢁ ꢂ ꢁ ꢂ

i

s

1

=

·

K_I

k_i

R

s

(R₁₁Y₁₀

(R₁₁Y₁₆

(R₁₆Y₁₁

)

0.28

1.26

0.21

2.61

2.80

2.73

1.1

2.0

1.0

35.6

56.1

84.2

9.3

2.2

13.0

ion point, the decline with time of the recorded

kinetic curves is rather slow, reﬂecting qualita-

The relationship between h₅₀and ee (Eq. (9))

was deduced from Eq. (8) by analogy with the

kinetic treatment of Ransac et al. in the case of

competitive kinetic model with two inhibitors

(Ransac et al., 1990).

tively

a

slow irreversible covalent reaction

process.

Eq. (7) describes the steady-state velocity un-

der monolayer conditions in the case of this

pseudo-competitive model.

A

h₅₀=

(9)

B · ee+(B+2 · A−1)

dP

6= =k_cat· E₀·

dt

This hyperbolic dependency is in agreement

with the experiments presented in Fig. 5. In all

S

I_S

(7)

cases, the value of, A=K* · A_S· (1+k_d/(k_p· A/

I_R

k_d

ꢁ

ꢂ

M

K* 1+

M

+

+S

K* k_p· ^A/_V

I

S

V))+1 was found equal to 1, indicative of a

very low amount of adsorbed enzyme in the

lipid monolayer (k_d/(k_p· A/V)), as well as a low

K*

I

R

Using this latter equation we deﬁned R_vas the

ratio of steady-state velocities:

value of K* as compared to the interfacial sub-

M

strate concentration (1/A_S) . Unfortunately, the

calculated values of B can not be simply inter-

preted since they reﬂect the difference between

the ratio of the interfacial Michae¨lis–Menten

constant to the inhibitor dissociation constants

(Eq. (8)).

In order to simplify the interpretation of the

calculated constants, we consider the case of one

inhibitor (I). The rearrangement of Eq. (7),

gives the following hyperbolic expression of the

lipase residual activity:

R_v= (velocity in the presence of an inhibitor

with K*=K* )/(velocity in the presence of an

I

M

inhibitor with K*"K* ).

I

M

Starting from Eq. (7), the relationship be-

tween R_wand the inhibitor molar fraction (h)

and the enantiomeric excess (ee), can be de-

scribed as follows:

B · (ee+1)−1

R_v=1+h ·

(8)

A

6

Lipase residual activity (%)=

k_cat· E₀

With

1−h

h · B%+A

=

(10)

k

ꢁ ꢂ

d

A=K* · A_S· 1+

M

+1

With

k_p· ^A/_V

1

B= ·

2

K*

M

ꢀ n

M

k

ꢁ ꢂ

d

−

A=K* · A_S· 1+

M

+1,

k_p· ^A/_V

K* K*

I

S

R

J.-F. Ca6alier et al. / Chemistry and Physics of Lipids 100 (1999) 3–31

21

K*

ꢁ ꢂ

^M−1

K*

[E*] · [S]

[E*S]

[E*I]

[E*] · [I]

ꢁ ꢂ ꢁ ꢂ

B%=

M

=

·

(11)

K*

I

K*

I

The curve ﬁtting of the experimental data pre-

sented on Figs. 6 and 7, using Eq. (10), allows us

to estimate the value of the ratio K* /K* for each

enantiomer of the compounds (R₁₁Y₁₆) and

(R₁₆Y₁₁) with HPL, and the compounds (R₁₁Y₁₀)

and (R₁₀Y₁₁) with HGL (Table 4). In the case of

the ‘fast’ enantiomers, the small values of the ratio,

K* /K*, conﬁrm their low inhibitory power (see

Fig. 3). Contrary to compounds (R₁₁Y₁₆)s and

(R₁₆Y₁₁)s which display differential inhibitory

powers with HPL and HGL, compounds (R₁₁Y₁₀)s

and (R₁₀Y₁₁)s have similar values of K* /K* with

If we assume that the enzyme partitioning equi-

librium (E X E*), is not affected by the presence

of the inhibitor (see Table 1 and Figs. 6 and 7), at

the h₅₀molar fraction we have [E*S]=[E*I], and

Eq. (11) can be rewritten as Eq. (12):

M

I

M

I

K* [S]

M

=

(12)

K* [I]

I

M

I

One can notice that this simpliﬁed expression of

K* /K* is comparable to the reverse value of h₅₀,

M

both lipases.

I

When we are dealing with powerful inhibitors,

i.e. with low h₅₀values, minimal changes in the

interfacial excess of enzyme is to be expected in the

presence or absence of the inhibitor, if one assumes

that the global ‘interfacial quality’ is not signiﬁ-

cantly affected by small proportions of inhibitors.

Indeed, in the case of HPL and HGL, the superﬁ-

that is to say: (S+I/I).

In the case of the ‘slow’ enantiomers, the inter-

facial stoichiometry (Table 2) has been calculated.

We can thus deduce the values of the ratio, K* /

M

K* , by simply dividing the amount of substrate by

I

S

the amount of the ‘slow’ enantiomer, at the h₅₀

molar fraction.

cial concentrations (Y_a) at the h₅₀molar fractions

With HPL this ratio was then estimated as 386

and 281 for (R₁₁Y₁₆)s and (R₁₆Y₁₁)s, respectively.

In the case of HGL, this ratio was estimated to be

86 and 127 for (R₁₁Y₁₀)s and (R₁₀Y₁₁)s, respec-

tively (see Table 4). The obtained values with the

‘slow’ enantiomers are in good agreement with the

values calculated from Eq. (10) (see Table 4),

corroborating thus the assumption that the enzyme

partitioning equilibrium is not affected by the

presence of these inhibitors.

50

of inhibitors (R₁₁Y )s and (R₁₆Y₁₁)s, and

16

(R₁₁Y₁₀)s and (R₁₀Y₁₁)s is close to 100% (Table 1).

By deﬁnition, the ratio of the two interfacial

kinetic constants (K* /K*) can be expressed as:

M

I

Table 4

Values of the ratio K* /K* after curve ﬁtting with Eq. (10) of

M

I

the experimental data (Figs. 6 and 7) describing the lipase

residual activity as a function of the inhibitor molar fraction

of each enantiomer of compounds (R₁₁Y₁₆) and (R₁₆Y₁₁) on

HPL, and (R₁₁Y₁₀) and (R₁₀Y₁₁) on HGL^a

If we assume that k_catis small as compared to

k₋₁(Fig. 8), we can as usual assimilate K* as the

M

interfacial dissociation constant of the (E*S) com-

plex. With this assumption, the ratio K* /K* can

M

I

S

Ratio of K* /K*

be taken as the ratio of the relative interfacial

afﬁnities of the enzyme between the inhibitor and

the substrate.

M

I

HGL

Fast

HPL

Fast

Slow

(R₁₁Y₁₀

(R₁₀Y₁₁

(R₁₁Y₁₆

(R₁₆Y₁₁

)

14

12

95

26

101 (86)

133 (127)

99

22

28

72

22

102

141

393 (386)

277 (281)

4. Conclusion

The kinetic treatment of the experimentally

data were analyzed using both the covalent and

the pseudo-competitive inhibition models. For the

ﬁrst time, the relative interfacial afﬁnity, K* /K*,

17

^aValues in parenthesis indicate the ratio of K* /K* based on

the interfacial stoichiometry using Eq. (12).

M

I

M

I

22

J.-F. Ca6alier et al. / Chemistry and Physics of Lipids 100 (1999) 3–31

1

of HPL and HGL between each enantiomeric

alkylphosphonate inhibitor and 1,2-dicaprin has

been quantiﬁed.

P–CH₂, J_PC=96.1 Hz), 30.98 (P–(CH₂)₃–CH₂),

29.15 (d, P–CH₂–CH₂–CH₂, ³J_PC=21.4 Hz),

2

22.82 (d, P–CH₂–CH₂, J_PC=6.7 Hz), 22.20 (P–

From some of the experimental kinetic curves

(Figs. 6 and 7), we can conclude that the ob-

served inhibition can be correlated with a de-

crease in interfacial lipase binding to

monomolecular ﬁlms containing the enantiomeric

inhibitors. In other words, the inhibition can be

attributed to both interfacial binding as well as a

chiral interaction of the inhibitor with the lipase

active site. The chiral recognition of HPL and

HGL may then occur not only at the active site

but also during the initial adsorption step, assum-

ing the interfacial lipase binding being controlled

by a supramolecular chiral recognition process.

However, up to now, the experimentally deter-

mined interfacial excess of lipase molecules in-

cludes all the enzymatic species, (E*), (E*S) and

(E*I). The proportions of each of these enzy-

matic species can not be experimentally deter-

mined. The present study supports the interfacial

chiral recognition hypothesis in the case of mixed

chiral ﬁlms spread at a surface pressure of 20 mN

(CH₂)₄–CH₂), 13.87 (CH₃).

5.1.1.2. n-Octylphosphonic acid dichloride: 2-R₈.

Reﬂux 20 h. Eb_0.023=100°C. Yield=81%. ³¹P

1

NMR: l 50.17; H NMRNMR: l 2.6 (m, 2H),

1.84 (m, 2H), 1.4 (m, 10H), 0.9 (t, 3H); ¹³C

1

NMR: l 42.81 (d, P–CH₂, J_PC=96.5 Hz), 31.49

(P–(CH₂)₅–CH₂), 29.38 (d, P–CH₂–CH₂–CH₂,

³J_PC=20.8 Hz), 28.66 (P–(CH₂)₃–(CH₂)₂), 22.72

(d, P–CH₂–CH₂, ²J_PC=7.16 Hz), 22.36 (P–

(CH₂)₆–CH₂), 13.84 (CH₃).

5.1.1.3. n-Decylphosphonic acid dichloride: 2-R₁₀.

Reﬂux 20 h. Eb_0.008=102°C. Yield=77%. ³¹P

1

NMR: l 50.18; H NMRNMR: l 2.5 (m, 2H),

1.84 (m, 2H), 1.0–2.1 (m, 16H), 0.9 (t, 3H); ¹³C

1

NMR: l 42.84 (d, P–CH₂, J_PC=96.3 Hz), 33.22

(d, P–CH₂–CH₂–CH₂, ³J_PC=26.5 Hz), 31.69

(P–(CH₂)₇–CH₂), 28.77 (P–(CH₂)₃–CH₂), 29.0–

29.86 (P–(CH₂)₄–(CH₂)₃), 22.76 (d, P–CH₂–

CH₂, ²J_PC=6.8 Hz), 22.50 (P–(CH₂)₈–CH₂),

13.94 (CH₃).

m

⁻¹, but further experiments are required to

substantiate this theory.

5.1.1.4. n-Undecyphosphonic acid dichloride: 2-

R₁₁. Reﬂux 72 h. Eb_0.008=170°C. Yield=95%.

5. Supplementary material

1

³¹P NMR: l 50.30; H NMRNMR: l 2.4–2.8 (m,

³¹P, H, ¹³C NMR, elemental analyses and [h]²_D⁰

2H), 0.8–2.1 (m, 21H); ¹³C NMR: l 43.0 (d,

1

experimental data, HPLC separation methods of

the phosphorus compounds 1, 2, and (RY). Data

from the ELISA tests and details of covalent

kinetic model are provided.

P–CH₂, J_PC=96.2 Hz), 31.90 (P–(CH₂)₈–CH₂),

29.60 (d, P–CH₂–CH₂–CH₂, ³J_PC=21.5 Hz),

29.30–29.50 (P–(CH₂)₄–(CH₂)₄), 28.91 (P–

2

(CH₂)₃–CH₂), 22.89 (d, P–CH₂–CH₂, J_PC=6.9

Hz), 22.67 (P–(CH₂)₉–CH₂), 14.10 (CH₃).

5.1. Experimental and physical data of

organophosphorus compounds

5.1.1.5. n-Dodecyphosphonic acid dichloride: 2-

R₁₂. Reﬂux 20 h. Eb_0.003=120°C. Yield=82%.

³¹P NMR: l 50.27; ¹H NMRNMR: l 2.5 (m,

2H), 1.0–2.1 (m, 20H), 0.9 (t, 3H); ¹³C NMR: l

5.1.1. Synthesis of alkylphosphonic acid dichloride

2-R_6–16was performed as described for compound

2-R₄

1

42.92 (d, P–CH₂, J_PC=96.26 Hz), 33.28 (d, P–

CH₂–CH₂–CH₂, ³J_PC=26.6 Hz),31.81 (P–

(CH₂)₉–CH₂), 29.10–29.90 (P–(CH₂)₄–(CH₂)₅),

28.82 (P–(CH₂)₃–CH₂), 22.82 (d, P–CH₂–CH₂,

²J_PC=6.7 Hz), 22.60 (P–(CH₂)₁₀–CH₂), 14.0

(CH₃).

5.1.1.1. n-Hexylphosphonic acid dichloride: 2-R₆.

Reﬂux 60 h. Eb_0.011=75°C. Yield=84%. ³¹P

1

NMR: l 50.1; H NMRNMR: l 2.6 (m, 2H), 1.6

(m, 8H), 0.9 (t, 3H); ¹³C NMR: l 42.90 (d,

J.-F. Ca6alier et al. / Chemistry and Physics of Lipids 100 (1999) 3–31

23

5.1.1.6. n-Tetradecyphosphonic acid dichloride: 2-

R₁₄. Reﬂux 20 h. Eb_0.008=130°C. Yield=57%.

³¹P NMR: l 50.3; ¹H NMRNMR: l 2.55 (m, 2H),

1.0–2.1 (m, 24H), 0.9 (t, 3H); ¹³C NMR: l 42.96

155.45 (d, O–C_ar, ²J_PC=8.4 Hz), 144.28 (C_ar–

NO₂), 125.38 (O–C_ar–C_ar–C_ar), 120.79 (d, O–

C_ar–C_ar, ³J_PC=4.5 Hz), 66.84 (d, O–CH₂,

²J_PC=7.25 Hz), 31.85 (P–(CH₂)₈–CH₂), 31.18

1

(d, P–CH₂, J_PC=96.3 Hz), 33.31 (d, P–CH₂–

(O–(CH₂)₃–CH₂), 30.33 (d, P–CH₂–CH₂–CH₂,

CH₂–CH₂, ³J_PC=25.7 Hz), 31.87 (P–(CH₂)₁₁–

CH₂), 28.7–29.90 (P–(CH₂)₃–(CH₂)₈), 22.86 (d,

³J_PC=16.9 Hz), 30.31 (d, O–CH₂–CH₂, J_PC

=

3

5.6 Hz), 29.28–29.52 (P–(CH₂)₄–(CH₂)₄, O–

(CH₂)₃–CH₂), 28.98 (P–(CH₂)₃–CH₂), 25.72 (d,

2

P–CH₂–CH₂, J_PC=6.8 Hz), 22.64 (P–(CH₂)₁₂–

1

CH₂), 14.04 (CH₃).

P–CH₂, J_PC=139.83 Hz), 25.05 (O–CH₂–CH₂–

CH₂), 22.45–22.63 (P–(CH₂)₉–CH₂, O–(CH₂)₄–

CH₂), 22.14 (d, P–CH₂–CH₂, ²J_PC=5.4 Hz),

14.03 (P–(CH₂)₁₀–CH₃); 13.86 (O–(CH₂)₅–CH₃);

Anal. Calc. for C₂₃H₄₀NO₅P (441.54): C 62.6, H

9.1, N 3.2, P 7.0%; found, C 60.9, H 8.8, N 3.5, P

7.4%.

5.1.1.7. n-Hexadecyphosphonic acid dichloride: 2-

R₁₆. Reﬂux 20 h. Eb_0.008=150°C. Yield=57%.

³¹P NMR: l 50.26; ¹H NMRNMR: l 2.6 (m, 2H),

1.0–2.1 (m, 28H), 0.9 (t, 3H); ¹³C NMR: l 42.93

1

(d, P–CH₂, J_PC=96.3 Hz), 33.07 (d, P–CH₂–

CH₂–CH₂, ³J_PC=24.3 Hz), 31.84 (P–(CH₂)₁₃–

CH₂), 29.13–29.95 (P–(CH₂)₃–(CH₂)₁₀), 22.83 (d,

5.1.2.3. O-Octyl O-(p-nitrophenyl) n-undecylphos-

2

P–CH₂–CH₂, J_PC=6.8 Hz), 22.61 (P–(CH₂)₁₄–

phonate:

(R₁₁Y₈).

Flash

chromatography:

CH₂), 14.02 (CH₃).

petroleum ether–ether 7:3 (R_f=0.20, PMA).

Yield 36%. ³¹P NMR: l 29.86; ¹H NMR: l 8.2 (d,

3

5.1.2. Synthesis of O-alkyl O-(p-nitrophenyl)

n-undecylphosphoates (R₁₁Y_1–16) was performed

as described for compound (R₁₁Y₄)

2H_ar, J=9.1 Hz), 7.4 (d, 2H_ar, J=9.2 Hz), 4.2

(m, 2H), 1.6 (m, 32H), 0.9 (t, 6H); ¹³C NMR: l

2

155.54 (d, O–C_ar, J_PC=8.56 Hz), 144.28 (C_ar–

NO₂), 125.68 (O–C_ar–C_ar–C_ar), 120.02 (d, O–

C_ar–C_ar, ³J_PC=4.6 Hz), 67.24 (d, O–CH₂,

²J_PC=7.3 Hz), 31.89 (P–(CH₂)₈–CH₂), 31.73

5.1.2.1. O-Methyl O-(p-nitrophenyl) n-unde-

cylphosphonate: (R₁₁Y₁). Flash chromatography:

petroleum ether–ether 8:2 (R_f=0.48, PMA).

(O–(CH₂)₅–CH₂), 30.40 (d, P–CH₂–CH₂–CH₂,

1

3

Yield 53%. ³¹P NMR: l 30.9; H NMR: l 8.3 (d,

³J_PC=16.9 Hz), 30.38 (d, O–CH₂–CH₂, J_PC

=

3

2H_ar, J=9.0 Hz), 7.4 (d, 2H_ar, J=9.0 Hz), 3.8

6.1 Hz), 29.02–29.57 (P–(CH₂)₄–(CH₂)₄, O–

(CH₂)₃–(CH₂)₂), 29.14 (P–(CH₂)₃–CH₂), 25.74

(d, P–CH₂, ¹J_PC=139.8 Hz), 25.42 (O–CH₂–

CH₂–CH₂), 22.67 (P–(CH₂)₉–CH₂, O–(CH₂)₆–

CH₂), 22.16 (d, P–CH₂–CH₂, ²J_PC=5.67 Hz),

14.06 (P–(CH₂)₁₀–CH₃, O–(CH₂)₈–CH₃); Anal.

Calc. for C₂₅H₄₄NO₅P (469.60): C 63.9, H 9.4, N

3.0, P 6.6%; found, C 62.3, H 10.1, N 3.8, P 7.2%.

(d, 3H, ³J=11.0 Hz), 0.7–2.2 (m, 23H); ¹³C

NMR: l 155.77 (d, O–C_ar, J_PC=8.3 Hz), 144.48

2

(C_ar–NO₂), 125.65 (O–C_ar–C_ar–C_ar), 120.90 (d,

O–C_ar–C_ar, ³J_PC=4.8 Hz), 53.04 (d, O–CH₃,

²J_PC=7.3 Hz), 31.86 (P–(CH₂)₈–CH₂), 30.40 (d,

3

P–CH₂–CH₂–CH₂, J_PC=17.4 Hz), 29.29–29.53

(P–(CH₂)₄–(CH₂)₄), 28.98 (P–(CH₂)₃–CH₂),

25.52 (d, P–CH₂, ¹J_PC=139.8 Hz), 22.63 (P–

2

(CH₂)₉–CH₂), 22.14 (d, P–CH₂–CH₂, J_PC=5.5

5.1.2.4. O-Decyl O-(p-nitrophenyl) n-undecylphos-

Hz), 14.07 (P–(CH₂)₁₀–CH₃); Anal. Calc. for

C₁₈H₃₀NO₅P (371.41): C 58.2, H 8.1, N 3.8, P

8.3%; found, C 59.7, H 8.8, N 3.4, P 8.6%.

phonate:

(R₁₁Y₁₀).

Flash

chromatography:

petroleum ether–ether 7:3 (R_f=0.30, PMA).

Yield 60%. ³¹P NMR: l 29.64; ¹H NMR: l 8.2 (d,

3

2H_ar, J=9.1 Hz), 7.4 (d, 2H_ar, J=9.2 Hz), 4.1

5.1.2.2. O-Hexyl O-(p-nitrophenyl) n-undecylphos-

(m, 2H), 1.5–2.1 (m, 6H), 1.2 (m, 30H), 0.8 (t,

2

phonate:

(R₁₁Y₆).

Flash

chromatography:

6H); ¹³C NMR: l 155.72 (d, O–C_ar, J_PC=7.25

petroleum ether–ether 7:3 (R_f=0.20, PMA).

Hz), 144.32 (C_ar–NO₂), 125.45 (O–C_ar–C_ar–C_ar),

3

Yield 53%. ³¹P NMR: l 29.71; ¹H NMR: l 8.1 (d,

120.83 (d, O–C_ar–C_ar, J_PC=4.25 Hz), 66.75 (d,

3

2H_ar, J=9.1 Hz), 7.3 (d, 2H_ar, J=9.1 Hz), 4.0

(m, 2H), 1.7 (m, 28H), 0.8 (t, 6H); ¹³C NMR: l

O–CH₂, ²J_PC=7.2 Hz), 31.76 (P–(CH₂)₈–CH₂,

3

O–(CH₂)₇–CH₂), 30.30 (d, O–CH₂–CH₂, J_PC

=

24

J.-F. Ca6alier et al. / Chemistry and Physics of Lipids 100 (1999) 3–31

3

6.39 Hz), 30.28 (d, P–CH₂–CH₂–CH₂, J_PC

=

CH₂–CH₂–CH₂), 22.56 (P–(CH₂)₉–CH₂, O–

(CH₂)₁₂–CH₂), 22.12 (d, P–CH₂–CH₂, J_PC=5.7

Hz), 13.95 (P–(CH₂)₁₀–CH₃, O–(CH₂)₁₃–CH₃);

Anal. Calc. for C₃₁H₅₆NO₅P (553.76): C 67.2, H

10.2, N 2.5, P 5.6%; found, C 66.5, H 11.1, N 2.9,

P 6.0%.

2

16.97 Hz), 29.19–29.45 (P–(CH₂)₄–(CH₂)₄, O–

(CH₂)₃–(CH₂)₄), 28.95 (P–(CH₂)₃–CH₂), 25.79

(d, P–CH₂, ¹J_PC=139.35 Hz), 25.32 (O–CH₂–

CH₂–CH₂), 22.54 (P–(CH₂)₉–CH₂, O–(CH₂)₈–

CH₂), 22.11 (d, P–CH₂–CH₂, ²J_PC=5.66 Hz),

13.94 (P–(CH₂)₁₀–CH₃, O–(CH₂)₉–CH₃); Anal.

Calc. for C₂₇H₄₈NO₅P (497.65): C 65.2, H 9.7, N

2.8, P 6.2%; found, C 66.3, H 9.4, N 3.3, P 5.8%.

5.1.2.7. O-Hexadecyl O-(p-nitrophenyl) n-unde-

cylphosphonate: (R₁₁Y₁₆). Flash chromatography:

petroleum ether–ether 7:3 (R_f=0.27, PMA).

Yield 66%. ³¹P NMR: l 29.28; ¹H NMR: l 8.2 (d,

5.1.2.5. O-Dodecyl O-(p-nitrophenyl) n-unde-

cylphosphonate: (R₁₁Y₁₂). Flash chromatography:

petroleum ether–ether 7:3 (R_f=0.23, PMA).

Yield 54%. ³¹P NMR: l 29.45; ¹H NMR: l 8.2 (d,

2H_ar, J=9.1 Hz), 7.4 (d, 2H_ar, J=8.9 Hz), 4.0

(m, 2H), 1.8 (m, 4H), 1.2 (m, 36H), 0.7 (t, 6H);

¹³C NMR: l 155.57 (d, O–C_ar, ²J_PC=8.3 Hz),

144.20 (C_ar–NO₂), 125.28 (O–C_ar–C_ar–C_ar),

3

2H_ar, J=7.8 Hz), 7.4 (d, 2H_ar, J=9.2 Hz), 4.1

(m, 2H), 1.9 (m, 1H), 1.7 (m, 3H), 1.2 (m, 44H),

0.8 (t, 6H); ¹³C NMR: l 155.63 (d, O–C_ar, ²J_PC

=

8.6 Hz), 144.29 (C_ar–NO₂), 125.39 (O–C_ar–C_ar–

3

C_ar), 120.77 (d, O–C_ar–C_ar, J_PC=4.5 Hz), 66.71

(d, O–CH₂, ²J_PC=7.2 Hz), 31.81 (P–(CH₂)₈–

CH₂, O–(CH₂)₁₃–CH₂), 30.32 (d, O–CH₂–CH₂,

³J_PC=6.0 Hz), 30.31 (d, P–CH₂–CH₂–CH₂,

³J_PC=16.2 Hz), 29.23–29.60 (P–(CH₂)₄–(CH₂)₄,

O–(CH₂)₃–(CH₂)₁₀), 28.98 (P–(CH₂)₃–CH₂),

3

120.69 (d, O–C_ar–C_ar, J_PC=5.0 Hz), 66.60 (d,

O–CH₂, ²J_PC=7.2 Hz), 31.63 (P–(CH₂)₈–CH₂,

3

O–(CH₂)₉–CH₂), 30.16 (d, O–CH₂–CH₂, J_PC

=

1

25.82 (d, P–CH₂, J_PC=139.66 Hz), 25.35 (O–

3

5.9 Hz), 30.13 (d, P–CH₂–CH₂–CH₂, J_PC=17.0

Hz), 29.06–29.30 (P–(CH₂)₄–(CH₂)₄, O–(CH₂)₃–

(CH₂)₆), 28.79 (P–(CH₂)₃–CH₂), 25.64 (d, P–

CH₂–CH₂–CH₂), 22.58 (P–(CH₂)₉–CH₂, O–

2

(CH₂)₁₄–CH₂), 22.12 (d, P–CH₂–CH₂, J_PC=5.7

Hz), 13.98 (P–(CH₂)₁₀–CH₃, O–(CH₂)₁₅–CH₃);

Anal. Calc. for C₃₃H₆₀NO₅P (581.81): C 68.1, H

10.4, N 2.4, P 5.3%; found, C 69.2, H 10.7, N 2.9,

P 6.0%.

CH₂,

¹J_PC=140.1

Hz),

25.17

(O–CH₂–CH₂–CH₂), 22.40 (P–(CH₂)₉–CH₂, O–

(CH₂)₁₀–CH₂), 21.96 (d, P–CH₂–CH₂, J_PC=5.6

Hz), 13.79 (P–(CH₂)₁₀–CH₃, O–(CH₂)₁₁–CH₃);

Anal. Calc. for C₂₉H₅₂NO₅P (525.70): C 66.3, H

10.0, N 2.7, P 5.9%; found, C 67.9, H 11.3, N 3.1,

P 6.1%.

2

5.1.3. Synthesis of O-(n-undecyl)

O-(p-nitrophenyl) alkylphosphoates (R_1–16Y₁₁)

was performed as described for compound (R₁Y₁₁)

5.1.2.6. O-Tetradecyl O-(p-nitrophenyl) n-unde-

cylphosphonate: (R₁₁Y₁₄). Flash chromatography:

petroleum ether–ether 7:3 (R_f=0.25, PMA).

Yield 63%. ³¹P NMR: l 29.49; ¹H NMR: l 8.1 (d,

5.1.3.1.

O-(n-Undecyl)

O-(p-nitrophenyl)

butylphosphonate: (R₄Y₁₁). Flash chromatography:

petroleum ether–ether 7:3 (R_f=0.25, PMA).

Yield 66%. ³¹P NMR: l 29.62; ¹H NMR: l 8.2 (d,

3

2H_ar, J=9.0 Hz), 7.3 (d, 2H_ar, J=8.4 Hz), 4.0

2H_ar, J=9.07 Hz), 7.4 (d, 2H_ar, J=10.27 Hz),

4.2 (m, 2H), 1.1–2.2 (m, 24H), 0.9 (t, 6H); ¹³C

NMR: l 155.61 (d, O–C_ar, ²J_PC=8.16 Hz),

144.21 (C_ar–NO₂), 125.40 (O–C_ar–C_ar–C_ar),

(m, 2H), 1.9 (m, 1H), 1.6 (m, 4H), 1.2 (m, 39H),

0.8 (t, 6H); ¹³C NMR: l 155.70 (d, O–C_ar, ²J_PC

=

8.6 Hz), 144.36 (C_ar–NO₂), 125.45 (O–C_ar–C_ar–

3

C_ar), 120.84 (d, O–C_ar–C_ar, J_PC=4.4 Hz), 66.78

120.74 (d, O–C_ar–C_ar, J_PC=4.8 Hz), 66.66 (d,

O–CH₂, J_PC=7.3 Hz), 31.67 (O–(CH₂)₈–CH₂),

30.18 (d, O–CH₂–CH₂, J_PC=5.87 Hz), 29.10–

29.30 (O–(CH₂)₄–(CH₂)₄), 28.83 (O–(CH₂)₃–

(d, O–CH₂, ²J_PC=7.2 Hz), 31.83 (P–(CH₂)₈–

CH₂, O–(CH₂)₁₁–CH₂), 30.32 (d, O–CH₂–CH₂,

³J_PC=5.9 Hz), 30.29 (d, P–CH₂–CH₂–CH₂,

³J_PC=17.2 Hz), 29.23–29.56 (P–(CH₂)₄–(CH₂)₄,

O–(CH₂)₃–(CH₂)₈), 28.96 (P–(CH₂)₃–CH₂),

25.80 (d, P–CH₂, ¹J_PC=140.4 Hz), 25.32 (O–

2

3

1

CH₂), 25.41 (d, P–CH₂, J_PC=140.4 Hz), 25.18

(O–CH₂–CH₂–CH₂), 24.04 (d, P–CH₂–CH₂,

²J_PC=5.6 Hz), 22.45 (O–(CH₂)₉–CH₂), 23.94 (d,

J.-F. Ca6alier et al. / Chemistry and Physics of Lipids 100 (1999) 3–31

25

P–CH₂–CH₂–CH₂, ³J_PC=17.8 Hz), 13.86 (O–

(CH₂)₁₀–CH₃), 13.29 (P–(CH₂)₃–CH₃); Anal.

Calc. for C₂₁H₃₆NO₅P (413.49): C 61.0, H 8.8, N

3.4, P 7.5%; found, C 62.1, H 9.6, N 3.7, P 8.1%.

5.1.3.4. O-(n-Undecyl) O-(p-nitrophenyl) de-

cylphosphonate: (R₁₀Y₁₁). Flash chromatography:

petroleum ether–ether 7:3 (R_f=0.25, PMA).

Yield 71%. ³¹P NMR: l 29.55; ¹H NMR: l 8.2 (d,

3

2H_ar, J=9.2 Hz), 7.4 (d, 2H_ar, J=9.95 Hz), 4.2

(m, 2H), 1.0–2.1 (m, 36H), 0.9 (t, 6H); ¹³C NMR:

2

5.1.3.2.

O-(n-Undecyl)

O-(p-nitrophenyl)

l 155.80 (d, O–C_ar, J_PC=8.26 Hz), 144.40 (C_ar–

hexylphosphonate: (R₆Y₁₁). Flash chromatogra-

phy: petroleum ether–ether 7:3 (R_f=0.25, PMA).

Yield 67%. ³¹P NMR: l 29.54; ¹H NMR: l 8.2 (d,

NO₂), 125.55 (O–C_ar–C_ar–C_ar), 120.92 (d, O–

C_ar–C_ar, ³J_PC=4.8 Hz), 66.81 (d, O–CH₂,

²J_PC=7.27 Hz), 31.88 (P–(CH₂)₇–CH₂, O–

3

2H_ar, J=9.2 Hz), 7.4 (d, 2H_ar, J=10.02 Hz),

(CH₂)₈–CH₂), 30.41 (d, O–CH₂–CH₂, J_PC=6.1

4.2 (m, 2H), 1.1–2.2 (m, 28H), 0.9 (t, 6H); ¹³C

Hz), 30.40 (d, P–CH₂–CH₂–CH₂, ³J_PC=16.77

Hz), 28.30–29.50 (P–(CH₂)₄–(CH₂)₃, O–(CH₂)₃–

(CH₂)₅), 28.69 (P–(CH₂)₃–CH₂), 25.91 (d, P–

2

NMR: l 155.60 (d, O–C_ar, J_PC=8.4 Hz), 144.18

(C_ar–NO₂), 125.34 (O–C_ar–C_ar–C_ar), 120.72 (d,

O–C_ar–C_ar, ³J_PC=4.53 Hz), 66.63 (d, O–CH₂,

CH₂,

¹J_PC=140.0

Hz),

25.42

²J_PC=7.27 Hz), 31.66 (O–(CH₂)₈–CH₂), 30.95

(O–CH₂–CH₂–CH₂), 22.86 (P–(CH₂)₈–CH₂, O–

3

2

(P–(CH₂)₃–CH₂), 30.18 (d, O–CH₂–CH₂, J_PC

=

(CH₂)₉–CH₂), 22.22 (d, P–CH₂–CH₂, J_PC=5.46

3

5.9 Hz), 29.85 (d, P–CH₂–CH₂–CH₂, J_PC

Hz), 14.06 (P–(CH₂)₉–CH₃, O–(CH₂)₁₀–CH₃);

Anal. Calc. for C₂₇H₄₈NO₅P (497.65): C 65.2, H

9.7, N 2.8, P 6.2%; found, C 64.8, H 10.5, N 3.6,

P 7.1%.

17.05 Hz), 28.83–29.30 (O–(CH₂)₃–(CH₂)₅),

25.67 (d, P–CH₂, ¹J_PC=140.2 Hz), 25.19 (O–

CH₂–CH₂–CH₂), 22.44 (P–(CH₂)₄–CH₂, O–

(CH₂)₉–CH₂), 22.0 (d, P–CH₂–CH₂, ²J_PC=6.8

Hz), 13.78 (P–(CH₂)₅–CH₃, O–(CH₂)₁₀–CH₃);

Anal. Calc. for C₂₃H₄₀NO₅P (441.54): C 62.6, H

9.1, N 3.2, P 7.0%; found, C 61.2, H 9.5, N 2.9, P

6.5%.

5.1.3.5. O-(n-Undecyl) O-(p-nitrophenyl) dode-

cylphosphonate: (R₁₂Y₁₁). Flash chromatography:

petroleum ether–ether 7:3 (R_f=0.25, PMA).

1

Yield 70%. ³¹P NMR: l 29.60; H NMR: l 8.23

3

(d, 2H_ar, J=9.2 Hz), 7.4 (d, 2H_ar, J=9.98 Hz),

4.14 (m, 2H), 1.1–2.2 (m, 40H), 0.9 (t, 6H); ¹³C

NMR: l 155.56 (d, O–C_ar, ²J_PC=8.16 Hz),

144.30 (C_ar–NO₂), 125.40 (O–C_ar–C_ar–C_ar),

5.1.3.3. O-(n-Undecyl) O-(p-nitrophenyl) octy-

lphosphonate: (R₈Y₁₁). Flash chromatography:

petroleum ether–ether 7:3 (R_f=0.25, PMA).

3

120.80 (d, O–C_ar–C_ar, J_PC=4.6 Hz), 66.77 (d,

1

2

Yield 79%. ³¹P NMR: l 29.5; H NMR: l 8.2 (d,

O–CH₂, J_PC=7.33 Hz), 31.75 (P–(CH₂)₉–CH₂,

3

2H_ar, J=9.16 Hz), 7.4 (d, 2H_ar, J=10.16 Hz),

O–(CH₂)₈–CH₂), 30.25 (d, O–CH₂–CH₂, J_PC

=

4.2 (m, 2H), 1.1–2.2 (m, 32H), 0.9 (t, 6H); ¹³C

NMR: l 155.84 (d, O–C_ar, J_PC=8.6 Hz), 144.41

5.97 Hz), 30.23 (d, P–CH₂–CH₂–CH₂, J_PC

3

2

16.92 Hz), 28.90–29.44 (P–(CH₂)₃–(CH₂)₆,

1

(C_ar–NO₂), 125.56 (O–C_ar–C_ar–C_ar), 120.93 (d,

O–C_ar–C_ar, ³J_PC=4.66 Hz), 66.82 (d, O–CH₂,

²J_PC=7.33 Hz), 31.82 (P–(CH₂)₅–CH₂, O–

O–(CH₂)₃–(CH₂)₅), 25.68 (d, P–CH₂, J_PC=

139.88 Hz), 25.27 (O–CH₂–CH₂–CH₂), 22.52

(P–(CH₂)₁₀–CH₂, O–(CH₂)₉–CH₂), 22.05 (d, P–

CH₂–CH₂, ²J_PC=5.62 Hz), 13.91 (P–(CH₂)₁₁–

CH₃, O–(CH₂)₁₀–CH₃); Anal. Calc. for

C₂₉H₅₂NO₅P (525.70): C 66.3, H 10.0, N 2.7, P

5.9%; found, C 67.1, H 9.5, N 3.1, P 5.4%.

(CH₂)₈–CH₂), 30.41 (d, P–CH₂–CH₂–CH₂,

3

³J_PC=16.85 Hz), 30.40 (d, O–CH₂–CH₂, J_PC

=

6.1 Hz), 28.90–29.50 (P–(CH₂)₃–(CH₂)₃, O–

(CH₂)₃–(CH₂)₅), 25.91 (d, P–CH₂, ¹J_PC=140.0

Hz), 25.55 (O–CH₂–CH₂–CH₂), 22.60 (P–

(CH₂)₆–CH₂, O–(CH₂)₉–CH₂), 22.22 (d, P–

5.1.3.6. O-(n-Undecyl) O-(p-nitrophenyl) tetrade-

cylphosphonate: (R₁₄Y₁₁). Flash chromatography:

petroleum ether–ether 7:3 (R_f=0.25, PMA).

CH₂–CH₂,

²J_PC=5.64

Hz),

14.04

(P–(CH₂)₇–CH₃, O–(CH₂)₁₀–CH₃); Anal. Calc.

for C₂₅H₄₄NO₅P (469.60): C 63.9, H 9.4, N 3.0, P

6.6%; found, C 64.3, H 9.9, N 3.7, P 5.8%.

1

Yield 40%. ³¹P NMR: l 29.68; H NMR: l 8.24

3

(d, 2H_ar, J=8.9 Hz), 7.4 (d, 2H_ar, J=9.27 Hz),

26

J.-F. Ca6alier et al. / Chemistry and Physics of Lipids 100 (1999) 3–31

Table 5

Eluent of separation, retention time (t_R) and rotatory power of each enantiomer of compounds (R₁₁Y_1–16

)

(R₁₁Y_1–16

)

Eluent:hexane–2-propanol

t_R(min)

[h]²_D⁰

1

CH₃

90:10

8.50

12.80

14.04

17.52

10.26

16.34

21.70

28.90

14.74

19.55

20.41

25.34

17.01

20.70

14.36

16.98

+9.0

−9.0

+2.37

−2.37

+0.93

−0.93

+0.44

−0.44

−2.25

2.25

−0.85

0.85

−0.47

0.47

−0.52

0.52

(c 0.2, CH₂Cl₂)

(c 3.63, CH₂Cl₂)

(c 3.34, CH₂Cl₂)

(c 3.66, CH₂Cl₂)

(c 1.56, CH₂Cl₂)

(c 1.53, CH₂Cl₂)

(c 2.8, CH₂Cl₂)

(c 5.56, CH₂Cl₂)

4

C₄H₉

98:2

6

C₆H₁₁

C₈H₁₇

C₁₀H₂₁

C₁₂H₂₅

C₁₄H₂₉

C₁₆H₃₃

95:5

8

99:1

10

12

14

16

99.5:0.5

4.12 (m, 2H), 1.0–2.2 (m, 47H), 0.9 (t, 6H); ¹³C

NMR: l 155.54 (d, O–C_ar, ²J_PC=8.32 Hz),

144.32 (C_ar–NO₂), 125.59 (O–C_ar–C_ar–C_ar),

5.95 Hz), 28.90–29.60 (P–(CH₂)₃–(CH₂)₁₁, O–

(CH₂)₃–(CH₂)₅), 25.70 (d, P–CH₂, J_PC=140.29

1

Hz), 25.30 (O–CH₂–CH₂–CH₂), 22.56 (P–

(CH₂)₁₄–CH₂, O–(CH₂)₉–CH₂), 22.06 (d, P–

3

120.89 (d, O–C_ar–C_ar, J_PC=4.83 Hz), 66.93 (d,

2

O–CH₂, J_PC=7.1 Hz), 31.85 (P–(CH₂)₁₁–CH₂,

CH₂–CH₂,

²J_PC=5.62

Hz),

13.96

3

O–(CH₂)₈–CH₂), 30.36 (d, O–CH₂–CH₂, J_PC

=

(P–(CH₂)₁₅–CH₃, O–(CH₂)₁₀–CH₃); Anal. Calc.

for C₃₃H₆₀NO₅P (581.81): C 68.1, H 10.4, N 2.4, P

5.3%; found, C 67.5, H 11.8, N 3.1, P 5.9%.

The separation of each enantiomer alkylphos-

phonate was performed on a Water HPLC system

using a Daicel Chiralpack AS column 250×250

mm, and a Waters UV detector at u=254 nm.

The HPLC analysis were carried out at 25°C

using n-hexane–2-propanol as eluent; ﬂow rate

4.5 ml min⁻¹. Ten injections of 100 ml (100 mg

ml⁻¹) sample phosphonates gave each enan-

tiomer in 99% ee. Tables 5 and 6 gave the differ-

ent proportions of n-hexane and 2-propanol used

in the HPLC separation, the retention time and

the rotatory powers of each enantiomer of

alkylphosphonates (RY).

3

6.13 Hz), 30.35 (d, P–CH₂–CH₂–CH₂, J_PC

16.34 Hz), 29.0–29.50 (P–(CH₂)₃–(CH₂)₈,

1

O–(CH₂)₃–(CH₂)₅), 25.84 (d, P–CH₂, J_PC

=

139.92 Hz), 25.40 (O–CH₂–CH₂–CH₂), 22.37

(P–(CH₂)₁₂–CH₂, O–(CH₂)₉–CH₂), 22.12 (d, P–

CH₂–CH₂, ²J_PC=5.0 Hz), 14.04 (P–(CH₂)₁₃–

CH₃, O–(CH₂)₁₀–CH₃); Anal. Calc. for

C₃₁H₅₆NO₅P (553.76): C 67.2, H 10.2, N 2.5, P

5.6%; found, C 68.5, H 9.6, N 2.1, P 6.3%.

5.1.3.7. O-(n-Undecyl) O-(p-nitrophenyl) hexade-

cylphosphonate: (R₁₆Y₁₁). Flash chromatography:

petroleum ether–ether 7:3 (R_f=0.25, PMA).

1

Yield 60%. ³¹P NMR: l 29.71; H NMR: l 8.23

(d, 2H_ar, ³J=9.14 Hz), 7.4 (d, 2H_ar, ³J=9.19

Hz), 4.13 (m, 2H), 1.0–2.1 (m, 48H), 0.9 (t, 6H);

2

¹³C NMR: l 155.52 (d, O–C_ar, J_PC=8.48 Hz),

5.2. Co6alent kinetic model

144.39 (C_ar–NO₂), 125.49 (O–C_ar–C_ar–C_ar),

120.83 (d, O–C_ar–C_ar, J_PC=4.7 Hz), 66.90 (d,

O–CH₂, J_PC=7.33 Hz), 31.79 (P–(CH₂)₁₃–CH₂,

3

2

5.2.1. Theoretical expressions

Sigmoidal expression of the time dependence of

product released:

O–(CH₂)₈–CH₂), 30.27 (d, P–CH₂–CH₂–CH₂,

³J_PC=16.9 Hz), 30.26 (d, O–CH₂–CH₂, J_PC

=

3

J.-F. Ca6alier et al. / Chemistry and Physics of Lipids 100 (1999) 3–31

27

S

P=k_cat· E₀·

·

and

K*

M

k_p· ^A/

S

I

k_i

I

ꢁ ꢂ

-t/~

^V· 1+

+

·

1

2

1

~₁· (1−e^-t/~)−~₂· (1−e

)

X=1+

·

k_d

K* K*

M

k_dK*

I

k_i

~₁−~₂

I_R· ^R+I_S·

S

k

₋₁+k_cat

K*

I

K*

I

S

K* =

M

R

k₁

The corresponding equation of the enzymatic

velocity is:

5.2.2. Recalculation, in the case of compounds

(R₁₁Y₁₆) and (R₁₆Y₁₁), of the h₅₀6alues from Ks,

Kf, ~₁and ~₂theoretical data

dP

S

6= =k_cat· E₀·

dt

·

Ransac et al. (1990, 1991) deﬁned a velocities

ratio as: R_v=(velocity in the presence of an in-

hibitor with K*=K* )/(velocity in the presence of

K*

M

t

~

− /

~

1

2

1

e^{− /}−e

·

I

M

k_i

K_i

~₁−~₂

I_R· ^R+I_S·

S

an inhibitor with K*"K* ).

I M

K*

I

K*

I

S

This ratio varies linearly with the inhibitor mo-

R

lar fraction (h) and can be rewritten as:

with

v

ꢁ ꢂ

1,2−dicaprin

R_v=(1−h) ·

=1+h · Z

v_inﬂexion

~₁=

2

k_d

S

I

ꢁ ꢂ

where 6_1,2-dicaprinis the maximal value of the en-

zyme velocity in the presence of a ﬁlm of 1,2-di-

caprin; and 6_inﬂexionis the maximal value of the

enzyme velocity in the presence of a mixed ﬁlm of

inhibitor–1,2-dicaprin.

· 1+

+

K* K*

M I

k_p· ^A/

k_i

I

S

I

'

ꢁ ꢂ

X− X²−4 ·

·

^V· 1+

+

k_dK*

k_d

K* K*

M I

I

~₂=

The molar fraction of inhibitor which reduces

the enzyme activity to 50% of its initial value, h₅₀,

can then be written as:

2

k_d

k_i

S

I

ꢁ ꢂ

· 1+

+

K* K*

M I

I

k_p· ^A/

S

I

'

ꢁ ꢂ

1

h₅₀=

X+ X²−4 ·

·

^V· 1+

+

k_dK*

k_d

K* K*

M I

I

2+Z

Table 6

Eluent of separation, retention time (t_R) and rotatory power of each enantiomer of compounds (R_1–16Y₁₁

)

(R_1–16Y₁₁

)

Eluent:hexane–2-propanol

t_R

[h]²_D⁰

1

4

6

CH₃

C₄H₉

C₆H₁₁

99.5:0.5 (not separated)

99.5:0.5

42.59

48.04

30.82

36.29

25.90

30.86

21.19

24.08

19.29

24.06

16.14

19.95

13.27

15.98

–

−1.16

+1.16

−0.96

+0.96

−0.62

+0.62

−0.48

+0.48

−0.39

+0.39

−0.27

+0.27

(c 4.32, CH₂Cl₂)

8

C₈H₁₇

99:1

(c 3.43, CH₂Cl₂)

(c 4.53, CH₂Cl₂)

(c 4.78, CH₂Cl₂)

(c 3.82, CH₂Cl₂)

(c 5.56, CH₂Cl₂)

10

12

14

16

C₁₀H₂₁

C₁₂H₂₅

C₁₄H₂₉

C₁₆H₃₃

99.5:0.5

Table 7

HPL superﬁcial concentration, Y (pg cm⁻²), determined by an ELISA with monomolecular ﬁlms of mixed inhibitor (R₁₆Y₁₁)–1,2-dicaprin at y=20 mN m⁻¹after

15 min of incubation^a

Films

(%)

HPL concentration in recovered ﬁlm HPL concentration in bulk

Total recovery

(%)

HPL superﬁcial concentration Y Film adsorbed li-

(ng ml⁻¹

)

phase (ng ml⁻¹

)

(pg cm⁻²

)

pase (%)

1,2-dicaprin

24.7/23.8

19.3/19.1

19.2/18.4

20.5/16.0

16.0/14.9

15.9/16.7

19.3/21.6

10.2/10.3

19.5/19.3

19.6/18.7

20.9/16.3

9.7/12.0

54/52

97/96

98/93

100/84

50/62

64/52

74/85

224.6/207.0

0.0/0.0

0.69/0.65

0.0/0.0

Fast

100

/

60

30

15

10

5

0.0/0.0

83.0/79.2

96.9/88.0

115.5/136.1

0.26/0.25

0.30/0.27

0.36/0.42

12.4/10.1

14.7/16.4

Slow

100

60

30

15

10

5

14.4/16.0

15.5/17.8

14.0/13.4

17.2/15.3

18.7/17.4

17.0/18.8

14.6/16.2

15.8/18.1

12.6/9.1

14.4/12.4

12.0/10.6

10.9/12.6

73/81

80/90

65/46

74/64

62/55

55/66

0.0/0.0

42.7/67.8

79.3/86.2

119.5/122.7

197.5/200.0

0.0/0.0

0.13/0.21

0.25/0.27

0.37/0.38

0.61/0.62

100

^aWe have, however, measured the interfacial binding of HPL on pure ﬁlms of (R₁₆Y₁₁)s and (R₁₆Y₁₁)f as a function of surface pressure. At 15 and 20 mN m_,⁻¹

no signiﬁcant surface excess of HPL could be detected. In contrast, at 10 mN m⁻¹a substantial interfacial binding was measured: 41.6 and 107.1 pg cm⁻²of HPL

in the case of (R₁₆Y₁₁)s and (R₁₆Y₁₁)f, respectively. Furthermore, this surface excess values increase by about 1.7-fold when working on pure ﬁlms of the same

phosphonate compounds spread at 5 mN m⁻¹(data not shown).

(

Table 8

HGL superﬁcial concentration, Y (pg cm⁻²), determined by an ELISA with monomolecular ﬁlms of mixed inhibitor (R₁₀Y₁₁)–1,2-dicaprin at y=20 mN m⁻¹after

15 min incubation

Films

(%)

HGL concentration in recovered ﬁlm HGL concentration in bulk

Total recovery

(%)

HGL superﬁcial concentration Y Film adsorbed li-

(ng ml⁻¹

)

phase (ng ml⁻¹

)

(pg cm⁻²

)

pase (%)

/

1,2-dicaprin

206.8/210.5

183.2/181.7

64/63

911.8/1111.9

0.193/0.236

Fast

100

206.9/201.6

239.7/237.4

240.6/238.3

221.0/216.4

222.8/223.6

227.2/229.3

198.1/192.8

232.3/230.9

232.8/230.6

204.6/199.3

212.5/213.3

210.7/214.0

69/67

81/80

71/69

74/74

73/74

146.9/147.5

187.8/167.0

211.0/210.0

301.8/313.1

359.1/356.4

485.1/452.5

0.03/0.03

0.04/0.03

0.05/0.04

0.06/0.07

0.08/0.08

0.10/0.10

60

30

15

10

5

Slow

100

60

30

227.2/232.2

207.2/215.7

272.4/274.3

210.7/212.8

184.1/188.9

253.5/260.5

73/74

64/66

90/91

378.1/422.7

424.9/493.7

568.7/565.8

0.08/0.09

0.09/0.10

0.12/0.12

100

(

30

J.-F. Ca6alier et al. / Chemistry and Physics of Lipids 100 (1999) 3–31

Example: in the case of compound (R₁₆Y₁₁

_1,2-dicaprin=8.78 mm min⁻¹

From Eq. (4) and data presented in Table 3, we

)

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Article Doi

Inhibition of human gastric and pancreatic lipases by chiral alkylphosphonates. A kinetic study with 1,2-didecanoyl-sn-glycerol monolayer

DOI: 10.1016/S0009-3084(99)00028-6

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Article abstract of DOI:10.1016/S0009-3084(99)00028-6

Full text of DOI:10.1016/S0009-3084(99)00028-6

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