U.S. patent application number 10/307490 was filed with the patent office on 2003-10-23 for method for releasing a product comprising chemical oxidation, method for detecting said product and uses thereof.
This patent application is currently assigned to Proteus. Invention is credited to Badalassi, Frabrizzio, Nguyen, Hong-Khanh, Reymond, Jean-Louis, Wahler, Denis.
Application Number | 20030199017 10/307490 |
Document ID | / |
Family ID | 26212439 |
Filed Date | 2003-10-23 |
United States Patent
Application |
20030199017 |
Kind Code |
A1 |
Reymond, Jean-Louis ; et
al. |
October 23, 2003 |
Method for releasing a product comprising chemical oxidation,
method for detecting said product and uses thereof
Abstract
This invention has as its object a method for releasing a
product by subjecting a compound of Formula (II'):
R'.sub.7R'.sub.8(HX)C.sub.1-C.sub- .2(YH)R'.sub.9R'.sub.10 to a
chemical oxidation that cleaves the bond C.sub.1-C.sub.2 to obtain
the product. In the compound of Formula (II'): R'.sub.7 to
R'.sub.10, which are identical or different, correspond to a
hydrogen atom, a substituted or unsubstituted alkyl group, or a
substituted or unsubstituted functional group; X and Y, which are
identical or different, are an oxygen atom, a sulfur atom, or an
amine of Formula -NR.sub.11R.sub.12, wherein R.sub.11 is a hydrogen
atom, an alkyl group, or a substituted or unsubstituted aryl group,
and R.sub.12 is not a hydrogen atom. The invention also has as its
object a method for releasing a product that comprises, before the
chemical oxidation stage, a first step for preparing the compound
of Formula (II'). The released product can be a volatile molecule
or an active substance or else a specific product. The invention
also relates to a method for detecting the released product as well
as its applications, in particular for detecting catalytic or
enzymatic activities.
Inventors: |
Reymond, Jean-Louis; (Berne,
CH) ; Wahler, Denis; (Berne, CH) ; Badalassi,
Frabrizzio; (Venturina, IT) ; Nguyen, Hong-Khanh;
(Nimes, FR) |
Correspondence
Address: |
HUNTON & WILLIAMS
INTELLECTUAL PROPERTY DEPARTMENT
1900 K STREET, N.W.
SUITE 1200
WASHINGTON
DC
20006-1109
US
|
Assignee: |
Proteus
|
Family ID: |
26212439 |
Appl. No.: |
10/307490 |
Filed: |
December 2, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10307490 |
Dec 2, 2002 |
|
|
|
PCT/FR01/01686 |
May 30, 2001 |
|
|
|
Current U.S.
Class: |
435/25 ; 435/189;
556/464; 558/73 |
Current CPC
Class: |
C07D 407/12 20130101;
C07D 311/16 20130101; C12Q 1/37 20130101; C12Q 1/26 20130101; C12Q
1/34 20130101; C12Q 2326/00 20130101; C12Q 2334/20 20130101; C12Q
2334/00 20130101; G01N 2333/918 20130101 |
Class at
Publication: |
435/25 ; 435/189;
556/464; 558/73 |
International
Class: |
C12Q 001/26; C12N
009/02; C07F 007/08; C07F 009/02 |
Foreign Application Data
Date |
Code |
Application Number |
May 30, 2001 |
FR |
00/06952 |
Oct 20, 2000 |
FR |
00/13487 |
Claims
What is claimed is:
1. A method for releasing a product, comprising subjecting a
compound of Formula (II'): 178to a chemical oxidation that cleaves
the bond C1-C2 to obtain a released product, wherein in the
compound of Formula (II'): R'.sub.7 to R'.sub.10, which are
identical or different, correspond to a hydrogen atom, an
optionally substituted alkyl group, or an optionally substituted
functional group, X and Y, which are identical or different, are an
oxygen atom, a sulfur atom, or an amine of Formula
-NR.sub.11R.sub.12; R.sub.11 is a hydrogen atom, an alkyl group, or
an optionally substituted aryl group, and R.sub.12 is not a
hydrogen atom.
2. The method of release according to claim 1, wherein the released
product corresponds to Formula R'.sub.7R'.sub.8C.sub.1=X and/or
R'.sub.9R'.sub.10C.sub.2=Y, in which R'.sub.7 to R'.sub.10, X, and
Y have the same meanings as above.
3. The method of release according to claim 1, wherein the released
product corresponds to the product of at least one secondary
reaction that is carried out on at least one compound of Formula
R'.sub.7R'.sub.8C.sub.1=X or R'.sub.9R'.sub.10C.sub.2=Y in which
R'.sub.7 to R'.sub.10, X, and Y have the same meanings as
above.
4. The method according to claim 1, wherein said released product
comprises a volatile molecule, an active substance, or a specific
compound.
5. The method according to claim 1, comprising carrying out said
chemical oxidation reaction with an oxidizing chemical agent.
6. The method according to claim 1, wherein the oxidizing chemical
agent is H.sub.5I.sub.6, RuO.sub.2, OSO.sub.4,
(CH.sub.3CH.sub.2CH.sub.2).sub.4- N(RuO.sub.4), NaClO.sub.4,
NaIO.sub.4, Na.sub.3H.sub.2IO.sub.6, NaMnO.sub.4, K.sub.2OsO.sub.4,
KIO.sub.4, KMnO.sub.4, KRuO.sub.4, K.sub.2RuO.sub.4, LiOCl, lead
acetate, tetrapropyl ammonium periodate, chromic acid or salts of
the latter, NaBiO.sub.3, Ph.sub.3BiCO.sub.3, Ca(OCl).sub.2,
reagents of Ce(IV), Cr(VI), salts of Co (II), IOAc, I(OAc).sub.3,
N-iodosuccinimide, VO(OAc), Pb(OAc).sub.4, MnO.sub.2,
H.sub.2O.sub.2, or a mixture of reagents H.sub.2O.sub.2,
Na.sub.2WO.sub.4, and H.sub.3PO.sub.4.
7. The method according to claim 5, wherein the oxidizing chemical
agent is a periodate salt.
8. The method according to claim 1, further comprising the step of
preliminarily preparing the compound of Formula (II').
9. The method according to claim 8, wherein the step of preparing
the compound of Formula (II') comprises synthesizing of a reduced
form of the ketone or aldehyde group of at least one product of
Formula R'.sub.9R'.sub.10C=Y and R'.sub.7R'.sub.8C.sub.1=X in which
R'.sub.7, R'.sub.8, R'.sub.9, R'.sub.10, X and Y have the same
meanings as above.
10. The method according to claim 8, wherein the compound of
Formula (II') is a compound of Formula (II) 179in which, X and Y,
which are identical or different, are an oxygen atom, a sulfur
atom, or an amine of Formula -NR.sub.11R.sub.12, and R.sub.11 is a
hydrogen atom, an alkyl group, or an optionally substituted aryl
group and R.sub.12 is not a hydrogen atom. R.sub.7 to R.sub.10,
which are identical or different, are a hydrogen atom, an
optionally substituted alkyl group, or an optionally substituted
functional group, and at least one of groups R.sub.7 to R.sub.10 is
a group Di, which is insensitive to [the stage (b)?] oxidation
reaction and which has properties that can be detected after the
cleavage of the bond C.sub.1-C.sub.2.
11. The method according to claim 10, comprising a first chemical
transformation of a substrate to obtain a compound of Formula (II)
then a chemical oxidation of said compound of Formula (II) to
directly or indirectly obtain a detectable product.
12. The method according to claims 10 or 11, comprising: a)
carrying out a chemical transformation of a substrate of Formula
(I) in which the bond C1-C2 is insensitive to a cleavage by a
chemical oxidation reaction: 180into a compound of Formula (II) in
which the bond C1-C2 is sensitive to a cleavage by a chemical
oxidation reaction: 181and (b) carrying out a chemical oxidation of
the compound of Formula (II) that is obtained in stage (a) that
cleaves the bond C1-C2 to directly or indirectly obtain a
detectable product, and wherein in the compounds of Formulas (I)
and (II): at least one of groups R.sub.1 to R.sub.6 is a group Di
as defined in claim 10, groups R.sub.1 to R.sub.6, which are
identical or different, are a hydrogen atom, a substituted or
unsubstituted alkyl group, a substituted or unsubstituted
functional group, and in the case of a functional group of Formulas
--OR12, --SR12, and -NR .sub.11R.sub.12, R.sub.11 is a hydrogen
atom, an alkyl group, or a substituted or unsubstituted aryl group,
and R.sub.12 is not a hydrogen atom, group or groups R.sub.1 to
R.sub.6 that after stage (a) form the groups of Formulas -XH and
-YH are insensitive to stage (b) oxidation, or may be sensitive to
this oxidation if the compound of corresponding Formula (I) does
not produce the compound of Formula (II) during an oxidation of the
type of that of stage (b), X and Y, which are identical or
different, are an oxygen atom, a sulfur atom, or an amine of
Formula -NR.sub.11R.sub.12, and R.sub.11 is a hydrogen atom, an
alkyl group, or a substituted or unsubstituted aryl group, and
R.sub.12 is not a hydrogen atom, R.sub.7 to R.sub.10, which are
identical or different, are either identical to at most four of
groups R.sub.1 to R.sub.6, or, because of the transformation of one
or more of groups R.sub.1 to R.sub.6, during the reaction of stage
(a), are a hydrogen atom, a substituted or unsubstituted alkyl
group, or a substituted or unsubstituted functional group, and at
least one of groups R7 to R10 is a group Di as defined in claim
10.
13. The method according to claim 8, comprising the following
stages: (a) transforming a substrate of Formula (I') in which the
bond C.sub.1-C.sub.2 is insensitive to a cleavage by a chemical
oxidation reaction: 182into a compound of Formula (II') in which
the bond C.sub.1-C.sub.2 is sensitive to a cleavage by a chemical
oxidation reaction: 183and, (b) the chemical oxidation of the
compound of Formula (II') that is obtained in stage (a) in the
presence of a compound of Formula II, which cleaves the bond
C.sub.1-C.sub.2 to obtain at least one detectable product directly
or indirectly, wherein in the compound of Formula (I'): groups
R'.sub.1 to R'.sub.6, which are identical or different, are a
hydrogen atom, a substituted or unsubstituted alkyl group, or a
substituted or unsubstituted functional group, and in the case of a
functional group of Formulas --OR.sub.12, --SR.sub.12, and
-NR.sub.11R.sub.12, R.sub.11 is a hydrogen atom, an alkyl group, or
a substituted or unsubstituted aryl group, and R.sub.12 is not a
hydrogen atom, group or groups R'.sub.1 to R'.sub.6 that after
stage (a) form the groups of Formulas -XH and -YH are insensitive
to stage (b) oxidation, or may be sensitive to this oxidation if
the corresponding compound of Formula (I') does not produce the
compound of Formula (II') during an oxidation of the type of that
of stage (b), wherein in the compound of Formula (II): R.sub.7 to
R.sub.10, which are identical or different, correspond to a
hydrogen atom, a substituted or unsubstituted alkyl group, or a
substituted or unsubstituted functional group, and at least one of
groups R.sub.7 to R.sub.10 is group Di as defined in claim 10, X
and Y, which are identical or different, are an oxygen atom, a
sulfur atom, an amine of Formula -NR.sub.11R.sub.12, and R.sub.11
is a hydrogen atom, an alkyl group, or a substituted or
unsubstituted aryl group, and R.sub.12 is not a hydrogen atom,
wherein in the compound of Formula (II'): X and Y, which are
identical or different, are an oxygen atom, a sulfur atom, or an
amine of Formula -NR.sub.11R.sub.12, and R.sub.11 is a hydrogen
atom, an alkyl group, or a substituted or unsubstituted aryl group,
and R.sub.12 is not a hydrogen atom, R'.sub.7 to R'.sub.10, which
are identical or different, are either identical to at most four of
groups R.sub.1 to R.sub.6, or, because of the transformation of one
or more of groups R.sub.1 to R.sub.6, during the reaction of stage
(a), are a hydrogen atom, a substituted or unsubstituted alkyl
group or a substituted or unsubstituted functional group.
14. The method according to claim 12, wherein R.sub.1 to R.sub.6 or
R'.sub.1 to R'.sub.6 are such that the bond C.sub.1-C.sub.2 is part
of at least one cycle.
15. The method according to claim 12, wherein at least one pair of
groups R.sub.1 to R.sub.6 or R'.sub.1 to R'.sub.6 together form an
oxygen atom, a sulfur atom, or a group of Formula
-NR.sub.11R.sub.12, and R.sub.11 is a hydrogen atom, an alkyl
group, or a substituted or unsubstituted aryl group, and R.sub.12
is not a hydrogen atom.
16. The method according to claim 12, wherein at most one pair of
groups R.sub.1 to R.sub.6, from which one is R.sub.1, R.sub.2 or
R.sub.6 or R'.sub.1, R'.sub.2 and R'.sub.6 respectively, and the
other is R.sub.3, R.sub.4 or R.sub.5 or R'.sub.3, R'.sub.4 and
R'.sub.5 respectively, form a double bond between carbons C.sub.1
and C.sub.2.
17. The method according to claim 12, wherein at most two pairs of
groups R.sub.1 to R.sub.6 or R'.sub.1 to R'.sub.6 respectively,
from which one is R.sub.1, R.sub.2 or R.sub.6 or R'.sub.1, R'.sub.2
or R'.sub.6 respectively, and the other is R.sub.3, R.sub.4 or
R.sub.5 or R'.sub.3, R'.sub.4 or R'.sub.5 respectively, form a
triple bond.
18. The method according to claim 12, wherein groups R.sub.1 to
R.sub.6 or R'.sub.1 to R'.sub.6 are sensitive or insensitive to
stage (b) oxidation and wherein stages (a) and (b) are not
simultaneous.
19. The method according to claim 12, wherein group or groups
R.sub.1 to R.sub.6 or R'.sub.1 to R'.sub.6 are insensitive to stage
(b) oxidation and wherein stages (a) and (b) are simultaneous.
20. The method according to claim 12, wherein the chemical
transformation is a catalytic reaction.
21. The method according to claim 20, wherein the chemical
transformation is an enzymatic reaction.
22. The method according to claim 12, wherein groups R.sub.1 to
R.sub.10 or R'.sub.1 to R'.sub.10 are stable in a reaction medium
comprising an aqueous medium, an organic medium or a two-phase
medium or a solid medium.
23. The method according to claim 12, wherein one or the other or
both stages (a) and (b) of the method of the invention are carried
out in a reaction medium comprising an aqueous medium, an organic
medium, a two-phase medium or a solid medium.
24. The method according to claim 12, wherein the substrate that is
used in the process of the invention comprises one or more chiral
centers.
25. The method according to claim 12, wherein the substrate
corresponds to Formula (V) below: 184in which: Di, R.sub.1,
R.sub.2, R.sub.3, X and Y have the same meaning as in claim 12, and
at most one of groups P.sub.1 and P.sub.2 is a hydrogen atom, or
P.sub.1 and P.sub.2, which are identical or different, are an acyl
group that is substituted by an aryl or alkyl or peptidyl group, a
phosphate group, a phosphate ester group, a phosphonate group, a
carbamyl group that is substituted by an aryl or acyl or peptidyl
group, a glycosyl group or a sulfate group.
26. The method according to claim 12, wherein the substrate
corresponds to Formula (VI) below: 185in which: Di R.sub.1,
R.sub.2, R.sub.3, X and Y have the same meaning as in claim 12, and
P.sub.3 is a carbonyl group, a group --PO.sub.2R.sub.11 or a group
--OR.sub.11PO--, where R.sub.11 has the same meaning as in claim
12, a group --SO.sub.2, a group --CHOR.sub.13 where R.sub.13
represents an aryl, alkyl or glycosyl group, a group
SiR.sub.14R.sub.15 where R.sub.14 and R.sub.15, which are identical
or different, represent an aryl, alkyl, aryloxy or alkoxy group,
and a group ASO.sub.2H--.
27. The method according to claim 12, wherein the substrate
corresponds to Formula (VII) below: 186in which: Di, R.sub.1,
R.sub.2 and R.sub.3 have the same meaning as in claim 12, and G
represents an oxygen atom, a sulfur atom or an amine group of
Formula NR.sub.11R.sub.12 where R.sub.11 and R.sub.12 have the same
meaning as in claim 12.
28. The method according to claim 12, wherein the substrate
corresponds to Formula (VIII) below: 187in which: Di, R.sub.1,
R.sub.2 and R.sub.3, have the same meanings as in claim 12.
29. The method according to claim 12, wherein the substrate
corresponds to Formula (IX) below: 188in which Di, R.sub.1,
R.sub.3, X and Y have the same meaning as in claim 12.
30. The method according to claim 12, wherein the product that is
obtained from the transformation of stage (a) of the substrate
corresponds to Formula (X) below: 189in which Di, R.sub.8, R.sub.9,
R.sub.10, X and Y have the same meaning as in claim 12.
31. The method according to claim 12, wherein the product that is
obtained from the transformation of stage (a) corresponds to
Formula (XV) below: 190in which X, Y and Di have the same meaning
as in claim 12 and in which the chain that carries Di is attached
to any of positions 1, 2 or 3 of the glycerol-containing
derivative.
32. The method according to claim 1, wherein the oxidation reaction
is carried out in an organic medium.
33. The method according to claim 12, wherein the chemical
oxidation of the compound of Formula (II) produces the directly
detectable product of Formula (XI) below: 191in which Di, R.sub.9
and Y have the same meanings as above.
34. The method according to claim 33, wherein the product of
Formula (XI) is a group that comprises an aromatic ketone, an
aldehyde or a pheromone.
35. The method according to claim 12, wherein the chemical
oxidation of the compound of Formula (II) is followed by a
beta-elimination reaction and produces the detectable product of
Formula ZH.
36. The method according to claim 30 or 35, wherein group Di of the
product of Formula (II) or Formula (X) corresponds to Formula
(XIII) below: 192in which: R.sub.16, R.sub.17 and R.sub.18, which
are identical or different, represent a hydrogen atom, a
substituted or unsubstituted alkyl group or a substituted or
unsubstituted functional group, Z is a precursor of a detectable
product ZH.
37. The method according to claim 36, wherein detectable product ZH
is a derivative of an aromatic alcohol, a heteroaromatic alcohol, a
heteroaromatic amine, a halogen atom or a phosphoric ester.
38. The method according to claim 37, wherein ZH is fluorescein,
phenolphthalein, phenol red, p-nitrophenol, o-nitrophenol,
2,4-dinitrophenol, 6-hydroxynaphthoic acid, 8-hydroxy-pyrene
1,3,6-trisulfonic acid, tyrosine, luciferin,
5-bromo-4-chloro-indolyl, indolyl, quinolinium, nitro-anilinium or
pyridoxamine.
39. The method according to claim 12, wherein: the compound that is
obtained from the transformation of the substrate after stage (a)
corresponds to Formula (XIV) below: 193in which Di, R.sub.8,
R.sub.9, X and Y have the same meaning as above, it comprises a
pair of detectable groups Di, which are identical or different, one
attached to carbon C.sub.1, the second attached to carbon C.sub.2,
and the two detectable groups interact with one another.
40. The method according to claim 12, wherein the degree of
specificity of the substrate is provided by the structure of group
or groups R.sub.1 to R.sub.6 of Formula (I) or R'.sub.1 to R'.sub.6
of Formula (I').
41. The method for detecting a chemical transformation of a
substrate, comprising subjecting said substrate to a release method
according to claim 11, and detecting the released product.
42. A substrate capable of being used in the method according to
claim 12, corresponding to Formula (V) below: 194in which: Di,
R.sub.1, R.sub.2, R.sub.3, X and Y have the same meaning as in
claim 12, and at most one of groups P.sub.1 and P.sub.2 is a
hydrogen atom or P.sub.1, P.sub.2, which are identical or
different, are an acyl group that is substituted by an aryl or
alkyl or peptidyl group, a phosphate group, a phosphate ester
group, a phosphonate group, a carbamyl group that is substituted by
an aryl or alkyl or peptidyl group, a glycosyl group or a sulfate
group.
43. A substrate that is capable of being used in the method
according to claim 12, that corresponds to Formula (VI) below:
195in which: Di R.sub.1, R.sub.2, R.sub.3, X and Y have the same
meaning as in claim 12, and P.sub.3 is a carbonyl group, a group
--PO.sub.2R.sub.11 or a group R.sub.11PO--, where R.sub.1 has the
same meaning as in claim 12, a group --SO.sub.2, a group
--CHOR.sub.13 where R.sub.13 represents an aryl, alkyl or glycosyl
group, a group SiR.sub.14R.sub.15 where R.sub.14 and R.sub.15,
which are identical or different, represent an aryl, alkyl, aryloxy
or alkoxy group and a group AsO.sub.2H--.
44. A substrate that is capable of being used in the method
according to claim 12, that corresponds to Formula (VII) below:
196in which: Di, R.sub.1, R.sub.2 and R.sub.3 have the same meaning
as in claim 12, and G represents an oxygen atom, a sulfur atom or
an amine group of Formula NR.sub.11R.sub.12 where R.sub.11R.sub.12
have the same meaning as in claim 12.
45. A substrate that is capable of being used in the method
according to claim 12, that corresponds to Formula (VIII) below:
197in which: Di, R.sub.1, R.sub.2 and R.sub.3 have the same meaning
as in claim 12.
46. A substrate that is capable of being used in a method according
to claim 12, that corresponds to Formula (IX) below: 198in which,
Di, R.sub.1, R.sub.3, X and Y have the same meaning as in claim
12.
47. A substrate according to claim 42, wherein groups R1 to R3 are
stable in a reaction medium.
48. The substrate according to claim 47, wherein said reaction
medium is an aqueous, organic, two-phase or solid medium.
49. A chemical composition that comprises at least one compound of
Formula (II').
50. The composition according to claim 49, further comprising an
oxidizing agent.
51. A method for the identification of a new catalyst or new
activity of catalysts that are known to be able to transform a
substrate of Formula (I) or of Formula (I') respectively into a
compound of Formula (II) or Formula (II') comprising detecting a
chemical transformation according to claim 41.
52. A method for detecting said substrate of Formula (I) or of
Formula (I') that is produced during a chemical reaction comprising
detecting a chemical transformation according to claim 41.
53. The method according to claim 51, wherein the catalyst is
contained in a biological sample and wherein a substrate of Formula
(I) or Formula (I') is added to said sample.
54. A catalyst that can transform a substrate of Formula (I) or
Formula (I') respectively into a compound of Formula (II) or
Formula (II'), whereby said transformation can be detected by a
method according to claim 41.
55. An enzyme that is able to transform a substrate of Formula (I)
or Formula (I') respectively into a compound of Formula (II) or
Formula (II'), whereby said transformation can be detected by a
method according to claim 41.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] The present application is a continuation of international
application number PCT/FR01/01686 (corresponding to publication
number WO 01/92563), the disclosure of which is hereby incorporated
by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This present invention relates to a method for detecting a
chemical transformation of a substrate such that said chemical
transformation of this substrate releases a product, whereby said
released product is then a detectable product.
[0004] The invention also relates to substrates that can be used in
this method. The invention also relates to the use of this method
in a process for detection and optionally quantification of a
chemical transformation in a sample.
[0005] 2. Description of Related Art
[0006] After reaction, many substrates release a product that
provides an easily identifiable signal. It is possible to cite the
well known examples of glycosides, esters and phosphates of phenols
whose hydrolysis directly releases phenol, which is detected in
general by its color (chromogenic substrate) or its fluorescence
(fluorogenic substrate)) (D. C. Demirjian, P. C. Shah, F.
Moris-Varas, Top. Curr. Chem. 1999, 200, 1; M. T. Reetz, K.-E.
Jaeger, Top. Curr. Chem. 1999, 200, 31).
[0007] It is also possible to cite 2-methoxy-1-naphthalene
methanol, whose oxidation provides fluorescent
2-methoxy-1-naphthaldehyde (B. L. Vallee, U.S. Pat. No. 5,162,203;
B. List, C. F. Barbas, R. A. Lerner, Proc. Natl. Acad. Sci. USA
1998, 95, 15351 for use in aldolases).
[0008] These types of substrate have a major disadvantage: the
chromogenic or fluorogenic phenol is a strongly activated group,
which makes these substrates unstable and susceptible to
non-specific reactions. In the case of 2-methoxy-1-naphthalene
methanol, the problem is similar since the benzylic position is
sensitive to non-specific oxidation.
[0009] A second class of substrate leads to the revealing of a
product that is obtained after enzymatic secondary reaction and/or
spontaneous secondary reaction. Several examples exist, such as
penicillinase substrates (U.S. Pat. No. 5,583,217), alcohol
dehydrogenase (ADH) substrates (G. Klein, J.-L. Reymond, Bioorg.
Med. Chem. Lett. 1998, 8, 1113) and aldolase substrates (N.
Jourdain, R. Prez Carln, J.-L. Reymond, Tetrahedron Lett. 1998, 39,
9415).
[0010] It is also possible to note the existence of a substrate for
measuring the NADH (C. A. Roeschlaub, N. L. Maidwell, M. R. Rezai,
P. G. Sammes, Chem. Commun. 1999, 1637).
[0011] Finally, it is possible to cite substrates whose detected
product of the secondary reaction is obtained by the action of the
beta-galactosidase (K. L. Matta, C. F. Piskorz, J. J. Barlow,
Carbohydr. Res. 1981, 90, C1-C3) or by ADH (G. Klein, J.-L.
Reymond, Helv. Chim. Acta 1999, 82, 400).
[0012] This second class of substrate is more stable. This second
class of substrate, however, is limited to particular uses, because
the fact of using an enzyme for the secondary reaction represents a
drawback in terms of cost and flexibility.
[0013] The research work carried out within the scope of this
invention consisted in particular in developing a method for
detecting a chemical transformation. Thus, this invention proposes
solving the problems reported above by using a method for measuring
a chemical transformation that is reliable, sensitive and
reproducible, using a stable substrate within the environment in
which the chemical reaction occurs, and such that the successive
transformations release a detectable product.
SUMMARY AND OBJECTS OF THE INVENTION
[0014] This invention has as its object a method for releasing at
least one product that can be detected, characterized in that a
compound of Formula (II') below: 1
[0015] is subjected
[0016] To a chemical oxidation that cleaves the bond
C.sub.1-C.sub.2 to obtain the product, in the compound of Formula
(II'):
[0017] R'.sub.7 to R'.sub.10, which are identical or different,
correspond to a hydrogen atom, a substituted or unsubstituted alkyl
group, or a substituted or unsubstituted functional group,
[0018] X and Y, which are identical or different, are selected from
among an oxygen atom, a sulfur atom, an amine of Formula
-NR.sub.11R.sub.12, and R.sub.11 is selected from among: a hydrogen
atom, an alkyl group, and s substituted or unsubstituted aryl
group, and R.sub.12 is not a hydrogen atom.
[0019] This chemical oxidation reaction is carried out with an
oxidizing chemical agent that can correspond advantageously and in
a non-limiting way to one or more of the following reagents:
H.sub.5IO.sub.6, RuO.sub.2, OSO.sub.4,
(CH.sub.3CH.sub.2CH.sub.2).sub.4N(RuO.sub.4), NaClO.sub.4,
NaIO.sub.4, Na.sub.3H.sub.2IO.sub.6, NaMnO.sub.4,
K.sub.2O.sub.sO.sub.4, KIO.sub.4, KMnO.sub.4, KRuO.sub.4,
K.sub.2RuO.sub.4, LiOCl, lead acetate, tetrapropylammonium
periodate, chromic acid or salts of the latter, NaBiO3,Ph3BiCO3,
Ca(OCl).sub.2, the reagents Ce(IV), Cr(VI), the salts of Co (II),
IOAc, I(OAc)3, N-iodosuccinimide, VO(OAc), Pb(OAc).sub.4,
MnO.sub.2, H.sub.2O.sub.2 or the mixture of the reagents
[H.sub.2O.sub.2, Na.sub.2WO.sub.4, H.sub.3PO.sub.4].
[0020] Quite preferably the oxidizing chemical agent is a periodate
salt.
[0021] Said product, released directly or indirectly, can be a
volatile molecule or an active substance that can modulate a
function such as in particular a pharmacological substance or else
a detectable compound.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0022] Within the meaning of this invention there fall the
following definitions:
[0023] Volatile molecule: one or more molecules by themselves or in
combination that have the property of being volatile, in particular
any aldehyde-type or volatile ketone-type molecule, such as, for
example, numerous perfumes or numerous bioactive molecules such as
the pheromones.
[0024] Active substance: one or more molecules by themselves or in
combination that can modulate a function such as in particular a
pharmaceutical substance that can modulate, for example, a hormonal
action, a physiological action (blood pressure, mood, waking
state), or a malignant development (tumor). The active substances
can also correspond to molecules that have properties that can cure
or prevent a pathology. The active substances can also have an
antibiotic or insecticide effect or exhibit specific properties of
odor or taste.
[0025] Specific compound: one or more compounds that can exhibit by
themselves or in combination a variation of their biophysical,
biological or chemical properties before and after the chemical
oxidation stage, such as in particular a spectral-type variation or
a variation of the solubility.
[0026] Chemical oxidation: any chemical reaction that is carried
out in the presence of a chemical reagent that can oxidize the
product of Formula (II') and that induces a cleavage of the bond
between carbons C1 and C2.
[0027] Functional group: any chemical group that belongs to a class
of organic compounds that is characterized by chemical properties.
By way of example of a functional group, it is possible to cite:
amides, acyls, alkoxy, nitrites, aryls, heteroaryls, alkenyls,
carbonyls, thiocarbonyls, carboxyls, thiocarboxyls, carbamyls,
thiocarbamyls, thiocarbamides, alcohols, thiols, or amines that may
or may not be substituted.
[0028] Direct release: the release of product that is obtained
immediately after said chemical oxidation reaction.
[0029] Indirect release: the release of the product that is
obtained after one or more secondary reactions, in particular a
chemical reaction, after said chemical oxidation reaction. The
secondary reaction can correspond, by way of example, to a
beta-elimination reaction. Indirect releases are also those that
result from the exposure of a product that is obtained immediately
after said chemical oxidation reaction to irradiation or to the
action of enzymes that are more or less specific. These secondary
reactions can allow in particular the monitoring of the release of
said product.
[0030] In the case of a direct release, the released product
corresponds to the Formula
R'.sub.7R'.sub.8C.sub.1=X and/or R'.sub.9R'.sub.10C.sub.2=Y,
[0031] in which R'7 to R'10, X, and Y have the same meanings as
above.
[0032] In the case of an indirect release, the released product
corresponds to the product of at least one secondary reaction that
is carried out on at least one compound of Formula
R'.sub.7R'.sub.8C.sub.1=X or R'.sub.9R'10C.sub.2=Y,
[0033] in which R'.sub.7 to R'.sub.10, X, and Y have the same
meanings as above.
[0034] The product that is released in a direct or indirect manner
can be detected; mention is then made of detectable product that
corresponds to a detectable volatile molecule, to a detectable
active substance or else to a detectable specific compound.
[0035] In the case of the direct or indirect release of a volatile
molecule, an active substance or a specific compound, the compound
of Formula (II') makes it possible to release, after the chemical
oxidation, respectively at least one volatile molecule, at least
one active substance or at least one specific compound.
[0036] It is known that, for example, the pinacol groups or the
vicinal diol groups, just like the amino-alcohol groups, are not
sensitive to the oxidation of air and are not volatile. They can
therefore be used as stable derivatives of the volatile molecule,
which will be released in particular in a controlled manner by
exposure of said derivatives to an oxidizing agent, such as
periodate.
[0037] Thus, in the case where the released product corresponds to
a volatile molecule, the method of the invention can be used to
implement an atomizer of a volatile molecule, and said volatile
molecule will be released gradually from a substrate by a chemical
oxidation of the latter.
[0038] In the case where the product corresponds to an active
substance, the method of the invention can be used to release a
galenical form of a medication.
[0039] The invention therefore also has as its object a composition
that comprises a compound of Formula (II') from which it is
possible to release a product directly or indirectly by the method
of the invention.
[0040] Such a composition can be useful, by way of example, for the
preparation of an atomizer of a volatile molecule, comprising a
mixture of at least one compound of Formula (II') and an oxidizing
agent such as periodate.
[0041] According to a preferred implementation, the composition of
the invention comprises an inert solid matrix or a galenical form
of a medication.
[0042] By way of example, the volatile molecule can be a perfume or
a bioactive molecule such as a pheromone. Respectively a perfume
atomizer or a mosquito-repellant sprayer is then considered.
[0043] More particularly, the volatile molecule is selected from
among the group that comprises: benzaldehyde (artificial almond),
butanal, citronellal, anisaldehyde, menthone, cuminaldehyde,
salicylaldehyde (used in perfumery), vanillin, phenylacetaldehyde
(hyacinth) or isovaleraldehyde (lemon, mint, eucalyptus). 2
[0044] Advantageously, the active substance as defined in the
invention is selected from among the group that comprises:
testosterone, estrone or nicotine.
[0045] The method for releasing the product according to the
invention can also comprise a preliminary stage for preparing the
compound of Formula (II') by any technique that is known to one
skilled in the art.
[0046] It is, for example, the preparation of a stable derivative
of a volatile molecule or an active substance or a specific
compound.
[0047] It can be, for example, the preparation of a vicinal diol or
an amino alcohol of said product by any technique that is known to
one skilled in the art.
[0048] According to a particular implementation, the invention has
as its object a method for releasing at least one product that
comprises the following stages:
[0049] a) the preparation of a compound of Formula (II') 3
[0050] in which the bond C.sub.1-C.sub.2 is sensitive to a cleavage
by a chemical oxidation reaction, and R'.sub.7, R'.sub.8, R'.sub.9,
R'.sub.10, X and Y have the same meaning as above,
[0051] b) the chemical oxidation of the compound of Formula (II')
that is obtained at stage (a) that cleaves the bond C.sub.1-C.sub.2
to obtain said product.
[0052] Preparation stage (a) can comprise at least one chemical
transformation and/or at least one enzymatic transformation.
[0053] The preparation of the compound of Formula (II') can be
done, for example, by obtaining the reduced form of the ketone or
aldehyde function of at least one of said products of Formula
R'.sub.9R'.sub.10C.sub.2=Y and/or R'.sub.7R'.sub.8C.sub.1=X.
[0054] Thus, stage (a) can be, for example, a reaction for
dimerization of at least one volatile molecule, in particular an
aldehyde or a ketone.
[0055] Advantageously, the reaction for dimerization of stage (a)
of the method for releasing a product of the invention can be
carried out by biosynthesis of vicinal diols.
[0056] Such reactions for dimerization of a product to obtain a
stable derivative are, for example, those described in FIG. 7,
attached, in which:
[0057] a first dimerization reaction according to stage (a) is a
reducing dimerization of aldehyde 1 or ketone 2 of the released
product in the presence of zinc, so as to obtain a pinacol
group.
[0058] A second dimerization reaction according to stage (a) is
carried out in two stages, by olefination of carbonyl in the
presence of a phosphorated ylide, followed by a dihydroxylation by
osmium tetroxide. This second approach leads to a terminal diol
that, after oxidation by the sodium periodate, releases 1
equivalent of formaldehyde.
[0059] By way of examples of synthesis of a compound of Formula
(II'), in the case of volatile molecules, it is possible to cite:
4
[0060] Particular implementations of the method for releasing a
volatile molecule carried out by the applicant within the scope of
this invention are provided in detail in the examples below in
which diols are used:
[0061] 1 - A symmetric diol, hydrobenzoin, obtained by reduction by
sodium borohydride from benzoin, makes it possible to obtain
benzaldehyde, with the odor of almond, by chemical oxidation.
[0062] 2 - The oxidation of 1,2-pentanediol is also illustrated; it
releases an aliphatic aldehyde, butanal, that has an odor of
butter. 5
[0063] Particular implementations of the method for releasing an
active substance within the scope of this invention are provided in
detail in the examples below.
[0064] The first two examples correspond to direct releases of the
active substance and the last example to an indirect release in
which the oxidation reaction is followed by a beta elimination.
6
[0065] The method of the invention is noteworthy in that the
released product is a detectable product; it can then be used to
detect a chemical transformation.
[0066] Advantageously, when the method of the invention has as its
object the release of a detectable product, the compound of Formula
(II') is a compound of Formula (II) 7
[0067] in which,
[0068] X and Y, which are identical or different, are selected from
among an oxygen atom, a sulfur atom, an amine of Formula
-NR.sub.11R.sub.12, and R.sub.11 is selected from among: a hydrogen
atom, an alkyl group, an aryl group that may or may not be
substituted, and R.sub.12 is not a hydrogen atom,
[0069] R.sub.7 to R.sub.10, which are identical or different, are
selected from among a hydrogen atom, an alkyl group that may or may
not be substituted, or a functional group that may or may not be
substituted, and at least one of groups R.sub.7 to R.sub.10 is a
group Di, which is insensitive to the stage (b) chemical oxidation
reaction and which exhibits detectable properties, directly or
indirectly, alone or in combination, after the cleavage of the bond
C.sub.1-C.sub.2.
[0070] These properties are characteristic of the detectable
product.
[0071] By way of example, these properties can be biophysical,
biological or chemical, such as in particular a spectral-type
variation or a solubility variation.
[0072] The compound of Formula (II) corresponds to a particular
implementation of the compound of Formula (II'), thus the
statements made regarding the compound of Formula (II') are valid
for the compound of Formula (II).
[0073] The detectable product can then be released after having
carried out a first chemical transformation stage of a substrate to
obtain a compound of Formula (II) then a second chemical oxidation
stage of said compound of Formula (II).
[0074] The detection of this transformation is obtained by using
the method for releasing a detectable product or by carrying out a
chemical transformation of a substrate that produces the compound
of Formula (II), then by chemically oxidizing the compound of
Formula (II) to release the product that can be detected. In this
case, the release of the detectable product comprises the following
stages:
[0075] a) the chemical transformation of a substrate of Formula (I)
in which the bond C1-C2 is insensitive to a cleavage by a chemical
oxidation reaction: 8
[0076] into a compound of Formula (II) in which the bond
C.sub.1-C.sub.2 is sensitive to a cleavage by a chemical oxidation
reaction: 9
[0077] and
[0078] b) the chemical oxidation of the compound of Formula (II),
obtained in stage (a), that cleaves the bond C.sub.1-C.sub.2 to
obtain a detectable product directly or indirectly,
[0079] and in that in the compounds of Formulas (I) and (II):
[0080] At least one of groups R.sub.1 to R.sub.6 is a group Di as
defined above,
[0081] Groups R.sub.1 to R.sub.6, which are identical or different,
are selected from among: a hydrogen atom, an alkyl group that may
or may not be substituted, a functional group that may or may not
be substituted, and in the case of a functional group of Formulas
OR.sub.12, --SR.sub.12, and -NR.sub.11R.sub.12, R.sub.11 is
selected from among: a hydrogen atom, an alkyl group, an aryl group
that may or may not be substituted, and R.sub.12 is not a hydrogen
atom,
[0082] Group or groups R.sub.1 to R.sub.6 that form after stage (a)
the groups of Formulas -XH and -YH are insensitive to stage (b)
oxidation or can be sensitive to this oxidation if the
corresponding compound of Formula (I) does not produce the compound
of Formula (II) during an oxidation of the type of that of stage
(b),
[0083] X and Y, which are identical or different, are selected from
among an oxygen atom, a sulfur atom, or an amine of Formula
-NR.sub.11R.sub.12,
[0084] R.sub.7 to R.sub.10, which are identical or different, are
either identical to at most four of the groups R.sub.1 to R.sub.6,
or, because of the transformation of one or more of groups R.sub.1
to R.sub.6, during the reaction of stage (a), selected from among a
hydrogen atom, an alkyl group that may or may not be substituted,
or a functional group that may or may not be substituted, and at
least one of groups R.sub.7 to R.sub.10 is a group Di as defined
above.
[0085] After stage (b), the method of the invention obviously
comprises a stage for direct or indirect detection of the
detectable product to carry out, for example, the detection of the
chemical transformation of stage (a).
[0086] The method of the invention accepts several preferred
meanings of groups R.sub.1 to R.sub.6, among which the following
cases are considered more particularly:
[0087] R.sub.1 to R.sub.6 are selected so that the bond
C.sub.1-C.sub.2 is part of at least one cycle,
[0088] At least one pair of groups R.sub.1 to R.sub.6 together form
an oxygen atom, a sulfur atom, or a group of Formula
-NR.sub.11R.sub.12,
[0089] At most one pair of groups R.sub.1 to R.sub.6, one of which
is selected from among R.sub.1, R.sub.2, and R.sub.6, and the other
is selected from among R.sub.3, R.sub.4 and R.sub.5 form a double
bond between carbons C.sub.1 and C.sub.2,
[0090] At most two pairs of groups R.sub.1 to R.sub.6, one of which
is selected from among R.sub.1, R.sub.2 and R.sub.6, and the other
is selected from among R.sub.3, R.sub.4 and R.sub.5, form a triple
bond.
[0091] A diagram example for detecting a chemical transformation of
a substrate according to the invention is shown in FIG. 1,
attached, whereby the compounds of Formulas (III) and (IV) are
directly or indirectly detectable.
[0092] An advantageous implementation of the method for releasing a
detectable product comprises the following stages:
[0093] (a) the transformation of a substrate of Formula (I') in
which the bond C.sub.1-C.sub.2 is insensitive to a cleavage by a
chemical oxidation reaction: 10
[0094] into a compound of Formula (II') in which the bond
C.sub.1-C.sub.2 is sensitive to a cleavage by a chemical oxidation
reaction: 11
[0095] and
[0096] (b) the chemical oxidation of the compound of Formula (II')
that is obtained in stage (a) in the presence of a compound of
Formula II that cleaves the bond C.sub.1-C.sub.2 to obtain directly
or indirectly at least one detectable product,
[0097] where, in the compound of Formula (I'):
[0098] Groups R'.sub.1, to R'.sub.6, which are identical or
different, are selected from among: a hydrogen atom, an alkyl group
that may or may not be substituted, a functional group that may or
may not be substituted, and in the case of a functional group of
Formulas --OR.sub.12, --SR.sub.12, and -NR.sub.11R.sub.12, R.sub.11
is selected from among: a hydrogen atom, an alkyl group, an aryl
group, that may or may not be substituted, and R.sub.12 is not a
hydrogen atom,
[0099] Group or groups R'.sub.1 to R'.sub.6 that form after stage
(a) the groups of Formulas -XH and -YH are insensitive to stage (b)
oxidation or can be sensitive to this oxidation if the
corresponding compound of Formula (I') does not produce the
compound of Formula (II') during an oxidation of the type of that
of stage (b),
[0100] in the compound of Formula (II'):
[0101] X and Y, which are identical or different, are selected from
among an oxygen atom, a sulfur atom, an amine of Formula
-NR.sub.11R.sub.12, and R.sub.11 is selected from among: a hydrogen
atom, an alkyl group, an aryl group, that may or may not be
substituted, and R.sub.12 is not a hydrogen atom,
[0102] R'.sub.7 to R'.sub.10, which are identical or different, are
either identical to at most four of groups R'.sub.1 to R'.sub.6,
or, because of the transformation of one or more of groups R.sub.1
to R.sub.6, during the reaction of stage (a), selected from among a
hydrogen atom, an alkyl group that may or may not be substituted,
or a functional group that may or may not be substituted, and in
the compound of Formula (II):
[0103] R.sub.7 to R.sub.10, which are identical or different,
correspond to a hydrogen atom, an alkyl group that may or may not
be substituted, a functional group that may or may not be
substituted, and at least one of groups R.sub.7 to R.sub.10 is a
group Di as defined above,
[0104] X and Y, which are identical or different, are selected from
among an oxygen atom, a sulfur atom, an amine of Formula
-NR.sub.11R.sub.12, and R.sub.11 is selected from among: a hydrogen
atom, an alkyl group, an aryl group, that may or may not be
substituted, and R.sub.12 is not a hydrogen atom.
[0105] This advantageous implementation puts the compound of
Formula (II'), obtained from the chemical transformation of the
compound of Formula (I'), into competition with the compound of
Formula (II) during oxidation stage (b). The oxidation speed of the
compound of Formula (IT) will therefore decrease proportionately to
the amount of compound of Formula II' that is formed. The signal
that is associated with the transformation of the compound of
Formula (II), detectable thanks to the presence of a group Di that
is a precursor of a detectable product, will therefore also
decrease proportionately to the amount of compound of Formula (II')
that is produced.
[0106] In this manner, it is possible to detect via the compound of
Formula (II) the chemical reaction that transforms the compound of
Formula (I') into a compound of Formula (II').
[0107] In this advantageous implementation, the substrate of
Formula (I') can correspond to the non-modified specific substrate
of the chemical transformation such as, for example, the
non-modified specific substrate of an enzyme.
[0108] The method of the invention also accepts a modification of
one of groups R.sub.7 to R.sub.10 except for Di, by the chemical
oxidizing agent of stage (b), provided that this disturbs the
cleavage of the bond C.sub.1-C.sub.2 neither during stage (b) nor
during the subsequent detection.
[0109] In a particular implementation of the process of the
invention where stages (a) and (b) are simultaneous, group or
groups R.sub.1 to R.sub.6 are insensitive to stage (b)
oxidation.
[0110] Under these conditions, the reaction of chemical
transformation and chemical oxidation take place under the same
experimental conditions. When stages (a) and (b) are carried out
simultaneously, the profile of the signal that is obtained can give
information about the kinetic course of the transformation if the
latter is limiting in the process, i.e., slower than the
oxidation.
[0111] In another implementation, stages (a) and (b) are not
simultaneous. Groups R.sub.1 to R.sub.6 then may or may not be
sensitive to the oxidation of stage (b). Transformation reaction
(a) can take place under experimental conditions that may or may
not be similar to the oxidation reaction (b). When stages (a) and
(b) are not simultaneous, a reaction speed that is proportional to
the amount of accumulated product will be obtained.
[0112] The method of the invention can be used with a substrate
that comprises several chemical groups that can be transformed in
stage (a) and several chemical groups that are precursors of
detectable products.
[0113] According to the chemical transformation that is being
studied and/or the structure of the substrate that is used, the
method of the invention can be carried out in a suitable reaction
medium that is selected from among an aqueous, organic, two-phase
or solid medium.
[0114] In this case,
[0115] chemical transformation or chemical reaction means any
transformation of a substrate that may or may not be spontaneous
and that can call for particular experimental conditions such as
heat, UV, etc. In the case of a non-spontaneous chemical
transformation, the reaction can call for a chemical reagent or a
catalyst such as an enzyme. This chemical transformation of stage
(a) can be carried out with several sequential reactions that may
or may not be simultaneous.
[0116] Groups R.sub.1 to R.sub.10, regardless of their meaning, can
consist of or comprise an isotope, such as, for example, deuterium.
In addition, advantageously, groups R.sub.1 to R.sub.10 are stable
in a reaction medium, in particular an aqueous, organic, two-phase,
or solid medium, etc.
[0117] Advantageously, the substrate that is used in the process of
the invention comprises one or more chiral centers. The substrate
that is used in the process of the invention can therefore consist
of a mixture of enantiomers, or diastereomers or enantiomers that
are pure.
[0118] Preferred substrates that correspond to Formula I are
described below.
[0119] A first class of substrate (I) corresponds to Formulas (V)
to (IX) below: 12
[0120] in which:
[0121] Di is a group as defined above
[0122] R.sub.1 to R.sub.3 and X and Y have the same meanings as
above,
[0123] At most one of groups P.sub.1 and P.sub.2 is a hydrogen
atom. P.sub.1 and P.sub.2, which are identical or different, are
selected from among an acyl group that is substituted by an aryl or
alkyl or peptidyl group, a phosphate group, a phosphate ester
group, a phosphonate group, a carbamyl group that is substituted by
an aryl or acyl or peptidyl group, a glycosyl group and a sulfate
group,
[0124] P.sub.3 is selected from among a carbonyl group, a group
--PO.sub.2R.sub.11 or a group R.sub.11PO--, where R.sub.11 has the
same meaning as above, a group --SO.sub.2, a group --CHOR.sub.13
where R.sub.13 represents an aryl, alkyl or glycosyl group, a group
SiR.sub.14R.sub.15 where R.sub.14 and R.sub.15, which are identical
or different, represent an aryl, alkyl, aryloxy or alkoxy group,
and a group AsO.sub.2H--,
[0125] G is selected from among an oxygen atom, a sulfur atom, or
an amine group of Formula NR.sub.11R.sub.12 where R.sub.11 has the
same meaning as above.
[0126] The product of the substrate that is obtained after the
first stage of the method of the invention from substrates of
Formulas (V), (VI), (VII), (VI) and (IX) corresponds to Formula
(II) or advantageously to Formula (X) below: 13
[0127] in which Di, X, Y, R.sub.8, R.sub.9 and R.sub.10 have the
same meaning as above.
[0128] In a particular case, the production of the substrate that
is obtained after first chemical transformation stage (a) of the
invention from substrates of Formulas (I) can also correspond to
Formula (XV) below: 14
[0129] in which X, Y and Di have the same meaning as above, whereby
the chain that carries Di can be in 1-, 2- or 3-position of the
glycerol-containing derivative.
[0130] Entirely preferably, groups X and Y of the compound of
Formula (XV) are hydroxyl groups.
[0131] The detection of the chemical transformation according to
the invention is used either by a direct revelation or by an
indirect revelation of the compound that is obtained after the
chemical oxidation of the compound of Formulas (II) or (X) carried
out with an oxidizing chemical agent according to stage (b).
[0132] Tetrapropyl ammonium periodate, soluble in organic phase, is
advantageously used when the method of the invention is carried out
in an organic medium.
[0133] According to a type of preferred revelation named direct
revelation below, according to which the compound that corresponds
to Formula (XI) obtained after the chemical oxidation reaction that
is carried out on the derivative that corresponds to Formulas (II)
or (X) after stage (b) is directly detectable according to the
diagram below. 15
[0134] An embodiment of the invention with a direct revealing uses
the direct detection of a property of the compound of Formula (XI),
not present in the compounds of Formulas (I) or (II) or (X).
[0135] In a type of direct revelation or direct releasing of a
detectable product:
[0136] Di in the compound of Formula (X) is a precursor of a
detectable product, and
[0137] Di in the compound of Formula (XI) has directly detectable
properties.
[0138] By way of non-limiting examples, it is possible to cite
among these properties of compound (XI): a physical property, such
as solubility; a physicochemical property, such as a spectral
property; or a biological property, such as the activation of an
enzyme, an odor, or the action of a pheromone.
[0139] The molecule that corresponds to Formula (XI) can correspond
to aromatic ketones, for example a beta-aromatic ketone that is
detected by a spectral variation, an aldehyde such as benzaldehyde
or citronellal that is detected by odor, or to a pheromone that is
detected by the attraction of insects. (Suzuki et al., (1980).
Agric. Biol. Chem. 44, 2519 and Millar et al. (1996). Bioorg. Med.
Chem. 3, 331-340).
[0140] In a second type of preferred revelation, named indirect
revelation below, compound (XII), obtained after chemical oxidation
stage (b) of the compound of Formulas (II) or (X), undergoes a
beta-elimination reaction that produces detectable product ZH,
according to the diagram below. 16
[0141] The group Di that corresponds to Formula (XIII) below:
17
[0142] in which:
[0143] R.sub.16, R.sub.17 and R.sub.18, which are identical or
different, represent a hydrogen atom, an alkyl group that may or
may not be substituted or a functional group that may or may not be
substituted, and
[0144] Z is a precursor of detectable product ZH.
[0145] In this type of indirect revelation or indirect release of a
detectable product:
[0146] Di in the compounds of Formulas (X) and (XII) is the
precursor of said detectable product that will be released after a
secondary beta-elimination reaction.
[0147] In this embodiment of the invention with an indirect
revelation, the detection of a property of product ZH that is
obtained by a beta-elimination reaction that is carried out on the
compound of Formula (XII) that is obtained after chemical oxidation
stage (b) is carried out. This beta-elimination reaction,
advantageously spontaneous, is preferably carried out in the
presence of a base called B that can correspond to bovine serum
albumin (BSA).
[0148] Among the properties of compound ZH, it is possible to cite
as non-limiting examples a physical property such as solubility; a
physicochemical property such as a spectral property; or a
biological property such as the induction of bacterial growth.
[0149] Compound ZH is selected from among an aromatic alcohol, a
heteroaromatic alcohol, a heteroaromatic amine, a halogen atom, or
a phosphoric ester. By way of non-limiting examples, there can be
cited: fluorescein, phenolphthalein, phenol red, p-nitrophenol,
o-nitrophenol, 2,4-dinitrophenol, 6-hydroxynaphthoic acid,
8-hydroxy-pyrene 1,3,6-trisulfonic acid, tyrosine, luciferin,
indolyl, 5-bromo-4-chloro-indolyl, quinolinium, nitro-anilinium or
pyridoxamine.
[0150] In a third type of preferred revelation, the compound that
is obtained after stage (a) for chemical transformation of the
substrate corresponds to Formula XIV below: 18
[0151] in which:
[0152] Di, R8, R9, X and Y have the same meanings as above,
and,
[0153] The two groups Di, which are identical or different, one
attached to carbon C.sub.1, the second attached to carbon C.sub.2,
interact with one another.
[0154] In this case, the cleavage of the bond C.sub.1-C.sub.2 to
this compound (XIV) causes a detectable spectral variation.
[0155] A non-limiting example of interaction between the two groups
Di is a FRET-type energy transfer.
[0156] A diagram that shows this revelation using a FRET-type
energy transfer is shown in FIG. 2, attached.
[0157] Examples of substrates that allow the implementation of the
invention with a direct revelation are compounds of Formula (V) in
which groups X, Y, R.sub.1, R.sub.2, R.sub.3 and Di are selected
from among those that are described in the following table.
[0158] For all of the compounds that are described in the following
tables that illustrate particular examples of substrates according
to the invention, the stereochemistry is always carried out in all
of the possible variants.
1TABLE I No. X Y R.sub.1 R.sub.2 R.sub.3 Di 6 NH O H H H
6'-Methoxy-2'-naphthyl 7 NH O COOH H H 6'-Methoxy-2'-naphthyl 8 NH
O CONH- H H 6'-Methoxy-2'-naphthyl peptide 17 O O H H H
6'-Methoxy-2'-naphthyl
[0159] The group 6'-methoxy-2'-naphthyl corresponds to the formula:
19
[0160] Examples of substrates that allow the implementation of the
invention with an indirect detection are compounds of Formula (V)
in which groups X, Y, R.sub.11, R.sub.12, R.sub.13 as well as
groups R.sub.1, R.sub.2, R.sub.3 and ZH are selected from among
those that are described in Table II below.
2 TABLE II Di No. X Y R.sub.1 R.sub.2 R.sub.3 R.sub.16 R.sub.17
R.sub.18 Z 1 NH O H H H H H H Coum*/NP** 2 NH O CH.sub.2 H H H
CH.sub.2 H Coum*/NP** 3 NH O COOH H H H H H Coum*/NP** 4 NH O CONH-
H H H H H Coum*/NP** peptide 5 NH O H H Me H H H Coum*/NP** 9 O O H
H H H H H Coum*/NP** 10 O O Me Me H H H H Coum*/NP** 11 O O H
CH.sub.2 H H CH.sub.2 H Coum*/NP** 12 O O (MeO)CHO H H O-- H H
Coum*/NP** CH(OMe) 13 O O H H Me H H H Coum*/NP** 14 O O Et H H H H
H Coum*/NP** 15 O O (MeO)CHO H H O-- H H Coum*/NP** CH(OMe) 16 O O
(Ade)CHO H H O-- H H Coum*/NP** CH(Ade) 18 NH N H H H H H H
Coum*/NP** H *Coum = 7-oxycoumarin 20 **NP = paranitro-phenoxy
21
[0161] The structures that correspond to substrates of Table II are
shown below:
[0162] Amino alcohols 2223
[0163] The diamines can be mono- or disubstituted, whereby groups
P.sub.1 and P.sub.2 cannot both correspond to H.
[0164] P.sub.1 and/or P.sub.2 can correspond to: 2425
[0165] The diols can be mono- or disubstituted.
[0166] In the particular case of a disubstitution, the two hydroxyl
radicals can be substituted identically as follows: 26
[0167] In the case of a mono-substitution, by way of a particular
example, the hydroxyl radicals can be monofunctionalized as above,
and in addition as follows: 27
[0168] R'''''=5' end of a DNA or RNA chain
[0169] By way of a particular implementation example of the
invention, the revelation of stage (b) is carried out sequentially
by using substrates that are at least disubstituted such as the
disubstituted substrate of Formula XVII below: 28
[0170] in which: Di, R.sub.1, R.sub.2, R.sub.6, P.sub.1, P.sub.2, X
and Y have the same meanings as above.
[0171] A preferred embodiment of the method of the invention that
uses a substrate that corresponds to Formula (V) above comprises
the transformation of mono- or diester derivatives of diols (1 a/b)
by a lipase or an esterase to produce compound (2) according to the
diagram below. 29
[0172] Another preferred embodiment of the method of the invention
that uses a substrate that belongs to the second class above
comprises the transformation of mono- or diester derivatives of
diols (3 a/b) by a lipase or an esterase to produce compound (4)
according to the diagram below. 30
[0173] By way of example of substrates of Formula (VI), it is
possible to cite more particularly those that correspond to the
formulas below: 31
[0174] By way of example of substrates of Formula (VII) that is
used in the method of the invention with a direct revelation, it is
possible to cite more particularly those that are described in
Table III below:
3 TABLE III Di No. R.sub.1 R.sub.2 R.sub.3 R.sub.16 R.sub.17
R.sub.18 Z 19 H H H H H H coum/NP 20 H H Me H H H coum/NP 21 Me Me
H H H H coum/NP 22 Et H H H H H coum/NP 23 CH.sub.2-- H H H
CH.sub.2-- H coum/NP
[0175] Coum and NP have the same meanings as above.
[0176] By way of example of substrates of Formula (VII) that is
used in the method of the invention with an indirect revelation, it
is possible to cite more particularly those that are described in
Table IV below:
4 TABLE IV No. R.sub.1 R.sub.2 R.sub.3 Di 24 H H H naphth
[0177] The structures that correspond to the substrates of Tables
III and IV are shown below: 32
[0178] A preferred embodiment of the method of the invention that
uses a substrate that corresponds to Formula (VII) above comprises
the transformation of epoxide (7) by an epoxide esterase to produce
compound (2) according to the diagram below. 33
[0179] Another preferred embodiment of the method of the invention
that uses a substrate that corresponds to Formula (VII) above
comprises the transformation of epoxide (8) by an epoxide hydrolase
to produce compound (4) according to the diagram below. 34
[0180] By way of example of substrates of Formula (VIII) that is
used in the method of the invention with an indirect revelation, it
is possible to cite more particularly those that are described in
Table V below, where the groups coum and NP have the same meanings
as above.
5 TABLE V Di No. R.sub.1 R.sub.2 R.sub.3 R.sub.16 R.sub.17 R.sub.18
Z 25 H H H H H H coum/NP 26 H H Me H H H coum/NP 27 Me Me H H H H
coum/NP 28 Et H H H H H coum/NP 29 CH.sub.2-- H H H CH.sub.2-- H
coum/NP
[0181] By way of example of substrates of Formula (VIII) that are
used in the method of the invention with a direct revelation, it is
possible to cite more particularly those that are described in
Table VI below:
6 TABLE VI No. R.sub.1 R.sub.2 R.sub.3 Di 30 H H H naphth
[0182] The structures that correspond to the substrates of Tables V
and VI are shown below: 35
[0183] A preferred embodiment of stage (a) of the method of the
invention that uses a substrate according to Formula (VIII) above
comprises the transformation of olefin (9) by a dihydroxylase or a
chemical catalyst such as an alkaloid or an amino alcohol in the
presence of OsO4 to produce compound (2) according to the diagram
below: 36
[0184] Another preferred embodiment of stage (a) of the method of
the invention that uses a substrate according to Formula (VIII)
above comprises the transformation of olefin (10) by a
dihydroxylase to produce compound (4) according to the diagram
below. 37
[0185] By way of example of oxidation reaction, according to stage
(b), products that are obtained from stage (a), it is possible to
cite the two reactions below:
[0186] With a direct revelation method 38
[0187] With an indirect revelation method 39
[0188] The invention also relates to the substrates that correspond
to Formulas (V), (VI), (VII), (VIII) or (IX).
[0189] These substrates have a particular advantage, and in
particular, they are able to be used in the method that is
described above.
[0190] Advantageously, these substrates are stable in the reaction
medium, in particular in the very wide ranges of pH and
temperature, but also in very diverse solvents.
[0191] By way of example, substrates that can be used in the method
of the invention are shown in Table XI, attached.
[0192] The invention also has as its object a composition that
comprises at least one compound of Formula (II').
[0193] This compound of Formula (II') can correspond to a form that
is stabilized by a volatile molecule, an active substance or a
specific compound.
[0194] Advantageously, the composition according to the invention
comprises an oxidizing agent.
[0195] The method of the invention is noteworthy in that it makes
it possible to detect a chemical transformation that is optionally
present in a sample by selecting a substrate that is the most
suitable for the transformation that it is desired to analyze.
[0196] Advantageously, these substrates have various degrees of
specificity. These degrees of specificity are provided by the
structure of one or more groups R.sub.1 to R.sub.6 of Formula (I)
or R.sub.1 to R.sub.3, or G, or P.sub.1 to P.sub.3 of Formulas (V),
(VI), (VII), (VIII) or (IX).
[0197] By way of example, in the case of the detection of a
chemical transformation that is carried out by a lipase, group or
groups R.sub.1 to R.sub.6 are selected so that the substrates that
correspond to Formula (I) are close to the structure of the
specific substrate. In particular, they can correspond to a fatty
acid chain. One skilled in the art will know, based on the type of
chemical transformation to be detected, to adapt the selection of
group or groups R.sub.1 to R.sub.6. In the case of implementing the
method of the invention, where two compounds of Formula (II) are
put into competition at stage (2), the substrate of Formula (I) can
correspond to the non-modified specific substrate of the enzyme,
which represents an advantage in the case of use of the method of
the invention for the identification of new catalysts or of the new
activity of known catalysts that can transform a substrate of
Formula (I) or (I') respectively into a compound of Formula (II) or
(II').
[0198] The method of the invention makes it possible to identify an
enantioselective or stereoselective chemical transformation in a
sample.
[0199] Advantageously, the chemical transformation is carried out
with a catalyst and in particular an enzyme. By way of non-limiting
example, the method of the invention makes possible the detection
of the activity of an enzyme that is selected from among: lipase,
esterase, protease, glycosidase, glycosyl transferase, phosphatase,
kinase, mono- or dioxygenase, haloperoxidase, lignin peroxidase,
diarylpropane peroxidase, epoxide hydrolase, nitrile hydratase,
nitrilase, transaminase, amidase, acylase, dihydroxylase, phytase,
xylanase, nuclease, and reductase.
[0200] In the case of a chemical transformation that may or may not
be spontaneous, it is possible to cite in particular: a spontaneous
or thermal hydrolysis of an ester, an olefin dihydroxylation by
AD-mix reagents, and a hydrolysis of epoxides by chromium
complexes.
[0201] The invention therefore finally relates to a process for
detecting and/or quantifying a known chemical transformation in a
sample that consists of using the method for detecting a chemical
transformation described above in the presence of said sample and a
substrate that is suitable for the desired activity.
[0202] The invention also makes it possible to detect the substrate
of Formula (I) or (I') in the presence of a chemical or biochemical
catalyst reagent.
[0203] The method of the invention can be used for screening,
particularly at a high flow rate, a catalyst from an in vivo or in
vitro expression library. These libraries can be prepared, for
example, from microorganisms or microalgae that preferably have
extremophile properties.
[0204] The method of the invention can also be used to identify
catalysts that have a different activity relative to a starting
activity. These catalysts will be, for example, products of
directed mutagenesis or directed evolution.
[0205] A particular embodiment of the method of the invention
relates to the case where the substrate of Formula (I) or (I') is
itself the product of a first unknown chemical transformation,
whose activity is desired to be identified.
[0206] By way of example of this particular embodiment of the
process of the invention, it is possible to cite the epoxidation
reaction of olefins to transform them into epoxides of Formulas (I)
or (I') or (VII), and said epoxides then undergo an enzymatic
reaction to obtain the diols of Formulas (II) or (II') or (X),
which will be subjected to a chemical oxidation reaction by the
periodate.
[0207] The method of the invention is noteworthy in that it also
makes possible the identification and the isolation of new chemical
or biochemical catalysts that can transform a substrate of Formula
(I) or (I') respectively into a compound of Formula (II) or (II').
Actually, the method of the invention makes it possible to disclose
the presence of a catalyst that, thanks to chemical oxidation,
generates a signal that is directly or indirectly detectable.
[0208] Consequently, the invention also has as its object the use
of the method for detecting a chemical transformation, as described
above, for the identification of new catalysts, or the new activity
of a known catalyst. The invention also relates to a catalyst that
can be identified by the method of the invention.
[0209] These new catalysts are specific to the experimental
conditions used during their detection; they advantageously
correspond to enzymes.
[0210] These new enzymes are specific to the experimental
conditions that are used during their detection.
[0211] Other advantages and characteristics of the invention will
emerge from reading the examples below that are part of the
experimental works carried out by the applicant within the scope of
the invention and where reference is made to the accompanying
figures in which:
[0212] FIG. 3 represents the enzymatic activity of the Candida
antarctica lipase based on the temperature. The enzymatic reactions
were carried out in the PIPES buffer (0.1 M, pH 7) at 50, 60, 70,
80 and 95.degree. C. for 40 minutes by using
2-hydroxy-4-p-nitrophenyl-butyl-decanoate. (.box-solid.) Candida
antarctica lipase; () without enzyme.
[0213] FIG. 4 illustrates the enzymatic activity of the Candida
antarctica lipase based on the temperature. The enzymatic reactions
were carried out in the PIPES buffer (0.1 M, pH 7) at 50, 60, 70,
80 and 95.degree. C. for 40 minutes by using
para-nitrophenylbutyrate. (.box-solid.) Candida antarctica lipase;
() without enzyme.
[0214] FIG. 5 illustrates the activity of the Candida antarctica
lipase based on the pH of the solution. The tests were carried out
by using 2-hydroxy-4-p-nitrophenyl-butyl-decanoate.
[0215] FIG. 6 illustrates the activity of the Candida antarctica
lipase based on the pH of the solution. The tests were carried out
by using para-nitrophenolbutyrate.
[0216] I - Synthesis of the Substrates.
EXAMPLE 1:
Preparation of 4-(7-coumarinyloxy)-1-butene
[0217] 40
[0218] 4-(7-coumarinyloxy)-1-butene.
[0219] A suspension of 7-hydroxycoumarin (3 g, 19.6 mmol) in DMF
(20 ml) is treated with NaH (60% suspension in oil, 1.03 g, 25.9
mmol), then with 4-bromo-1-butene (2.65 g, 19.6 mmol). It is heated
to 60.degree. C. for 24 hours, then it is diluted with ethyl
acetate (400 ml), and it is washed with water (3.times.300 mL) then
with 1 M NaOH (3.times.200 ml). The residue is chromatographed with
the mixture (ethyl acetate-hexane) to obtain
4-(7-coumarinyloxy)-1-butene (2.7 g, 67%).
EXAMPLE 2:
Preparation of (S)-4-(7-coumarinyloxy)-1,2-butanediol.
[0220] 41
[0221] (S)-4-(7-coumarinyloxy)-1,2-butanediol (2)
[0222] An AD-alpha-mix suspension (4.2 g) is stirred in 30 ml of
t-BuOH/water 1:1 until a single phase is obtained (5 minutes). It
is then cooled to 0.degree. C., and olefin is added (0.648 g, 3
mmol). After 16 hours at 0.degree. C., it is quenched with
Na.sub.2S.sub.2O.sub.5 (4.5 g). After one hour at ambient
temperature, it is extracted with CH.sub.2Cl.sub.2 and
chromatographed (CH.sub.2Cl.sub.2/acetone elution 7:3) to obtain
0.6 g (80%) of pure diol (S).
[0223] Melting point 92-93.degree. C., [a].sub.D.sup.20=22.4 (c
0.46, CH.sub.3OH); .sup.1H-NMR (CD.sub.3OD).sub.--7.83 (d, 1H,
J=9.8 Hz), 7.47 (d, 1H, J=8.3 Hz), 6.85-6.92 (m, 2H), 6.21 (d, 1H,
J=9.3 Hz), 4.16-4.23 (m, 2H), 3.81-3.93 (m, 1H), 3.54 (d, 2H,
J=6.35 Hz), 1.97-2.13 (m, 1H), 1.74-1.91 (m, 1H).
[0224] The same procedure with the AD-beta-mix provides enantiomer
R.
EXAMPLE 3:
Preparation of (S)-4-(7-coumarinyloxy-1,2-epoxybutane.
[0225] 42
[0226] (S)-4-(7-coumarinyloxy)-1,2-epoxybutane (7)
[0227] Trimethyl orthoacetate (1 ml), then pyridinium paratoluene
sulfonate (1 mg) are added to a solution of
(S)-4-(7-coumarinyloxy)-1,2-b- utanediol (0.15 g, 0.6 mmol) in
CH.sub.2Cl.sub.2. After 40 minutes, it is evaporated in its
entirety, the residue is dissolved in CH.sub.2Cl.sub.2 (1 ml), and
it is treated with Me.sub.3SiCl (0.1 ml, 0.78 mmol). After one
hour, dry methanol (1 ml) and potassium carbonate (0.2 g, 1.5 mmol)
are added. It is left for 2 hours at 20.degree. C., then it is
filtered, and the filtrate (hexane/ethyl acetate 6:4) is
chromatographed to obtain the epoxide (S) (0.097 g, yield 70%).
[0228] Melting point 61-64.degree. C., [.alpha.].sub.D.sup.20=23.0
(c 0.3, CHCl.sub.3); .sup.1H-NMR (CDCl.sub.3): 7.58 (d, 1H, J=9.3
Hz), 7.31 (d, 1H, J=8.3 Hz), 6.75-6.81 (m, 2H), 6.18 (d, 1H, J=9.8
Hz), 4.03-4.19 (m, 2H), 3.05-3.14 (m, 1H), 2.78 (t, 1H, J=4.9 Hz),
2.53 (dd, 1H, J=4.9 and 2.4 Hz), 2.04-2.20 (m, 1H), 1.79-1.97 (m,
1H).
[0229] The same procedure with
(R)-4-(7-coumarinyloxy)-1,2-butanediol provides enantiomer R.
EXAMPLE 4:
Preparation of (S)-1,2-Diacetoxy-4-(7-coumarinyloxy)-butane.
[0230] 43
[0231] (S)-1,2 Diacetoxy-4-(7-coumarinyloxy)-butane
[0232] 2 ml of acetic anhydride is added to a solution of
(S)-4-(7-coumarinyloxy)-1,2-butanediol (0.1 g, 0.4 mmol) in 4 ml of
anhydrous pyridine at 0.degree. C. After 18 hours at 20.degree. C.,
it is evaporated with toluene, and the residue is chromatographed
to obtain diacetate S (quantitative yield).
[0233] Melting point 74-76.degree. C., [.alpha.].sub.D.sup.20=9.8
(c 0.5, CHCl.sub.3); .sup.1H-NMR (CDCl.sub.3): 7.61 (d, 1H, J=9.8
Hz), 7.34 (d, 1H, J=8.8 Hz), 6.74-6.82 (m, 2H), 6.22 (d, 1H, J=9.3
Hz), 5.24-5.35 (m, 1H), 4.32 (dd, 1H, J=11.7 and 3.4 Hz), 3.97-4.15
(m, 2H), 4.10 (dd, 1H, J=11.7 and 5.9 Hz), 2.05-2.16 (m, 2H), 2.05
(s, 6H).
[0234] The same procedure with
(R)-4-(7-coumarinyloxy)-1,2-butanediol provides enantiomer R.
EXAMPLE 5
Preparation of (S)-1-Acetaxy-4-(7-coumarinyloxy)-2butanol.
[0235] 44
[0236] (S)-1-Acetoxy-4-(7-coumarinyloxy)-2-butanol
[0237] Acetyl chloride (0.36 mmol, 0.024 ml) is added to a solution
of (S)-4-(7-coumarinyloxy)-1,2-butanediol (0.090 g, 0.36 mmol) and
triethylamine (0.1 ml, 0.72 mmol) in 8 ml of dry CH.sub.2Cl.sub.2.
After 40 minutes at 0.degree. C., it is diluted with
CH.sub.2Cl.sub.2, and it is washed with an aqueous solution of
NaHCO.sub.3. The chromatography of the residue after evaporation
provides monoacetate S (0.058 g, 0.22 mmol, 60%).
[0238] [.alpha.].sub.D.sup.20=9.6 (c 0.31, CHCl.sub.3); .sup.1H-NMR
(CDCl.sub.3): 7.61 (d, 1H, J=9.8 Hz), 7.33 (d, 1H, J=7.8 Hz),
6.78-6.83 (m, 2H), 6.21 (d, 1H, J=8.8 Hz), 4.01-4.29 (m, 5H), 2.09
(s, 3H), 1.91-2.00 (m, 2H).
[0239] The same procedure with
(R)-4-(7-coumarinyloxy)-1,2-butanediol provides enantiomer R.
EXAMPLE 6:
Preparation of 1-Amino -4-(7-coumarinyloxy)-2-butanol.
[0240] 45
[0241] 1-Amino-4-(7-coumarinyloxy)-2-butanol
[0242] 5 ml of 30% aqueous ammonia and Gd (OTf.sub.3) (0.039 g,
0.065 mmol) are added to a solution of racemic
4-(7-coumarinyloxy)-1,2-epoxybut- ane (0.150 g, 0.65 mmol) in 2 ml
of ethanol. It is heated at 65.degree. C. for 15 hours, the solvent
is evaporated, the residue is diluted in ethyl acetate, and it is
washed with an NaCl-saturated solution. The residue provides the
crude amine (0.081 g, yield 50% ) that is used without purification
for the next stage.
EXAMPLE 7:
Preparation of 1-Phenylacetamido-4-(7coumarinyloxy)-2-butanol.
[0243] (5) 46
[0244] 1-Phenylacetamido-4-(7-coumarinyloxy)-2-butanol
[0245] Triethylamine (1.3 mmol, 0.18 ml), then phenotype chloride
(0.65 mmol, 0.085 ml) are added to a solution of
1-amino-4-(7-coumarinyloxy)-2-- butanol (0.162 g, 0.65 mmol) in 3
ml of dry CH.sub.2Cl.sub.2 at 0.degree. C. It is stirred at
0.degree. C. for 2 hours, then it is diluted with CH.sub.2Cl.sub.2
and washed with an NaHCO.sub.3-saturated aqueous solution. The
crude product is chromatographed to obtain the amide (0.170 g, 0.48
mmol, 74 %).
EXAMPLE 8:
Preparation of 6-Methoxy-2-naphthaldehyde and
6-dimethylamino-2-naphthalde- hyde.
[0246] 47
[0247] 6-Methoxy-2-naphthaldehyde and
6-dimethylamino-2-naphthaldehyde.
[0248] 6-Methoxy-2-naphthaldehyde is prepared by successive
treatment of 2-bromo-6-methoxynaphthalene in solution in dry ether
with n-butyllithium and then dimethylformamide. (Literature: J.
Med. Chem. 1998, 1308-1312). 6-Dimethylamino-2-naphthaldehyde is
prepared according to the described procedure (Barbas et al., Proc.
Natl. Acad. Sci. USA 1998, 95, 15351) as follows: gaseous
dimethylamine is introduced into a mixture of 2.4 ml of dry benzene
and 2.4 ml of hexamethylphosphoramide (HMPA) until 750 mg (16.7
mmol) is dissolved. At 0.degree. C. and under inert atmosphere,
n-butyllithium (1.6 M in hexane, 16.7 mmol) is added, then after 15
minutes, 6-methoxy-naphthaldehyde (390 mg, 2.09 mmol) is added.
Stirring is continued for 14 hours at 20.degree. C., then it is
poured into aqueous phosphate buffer, pH 7.4, and it is extracted
with ether. Purification by chromatography provides
6-dimethylamino-2-naphthaldehyde (350 mg, 84%).
[0249] 6-Methoxy-2-naphthaldehyde: .sup.1H-NMR (CDCl.sub.3): 10.09
(s,1H), 8.26 (s,1H), 7.78-7.95 (m, 3H), 7.17-7.26 (m, 2H), 3.96 (s,
3H).
EXAMPLE 9:
Preparation of 6-Methoxy and
6-Dimethylamino-2-vinylnaphthalene.
[0250] 48
[0251] 6-Methoxy and 6-dimethylamino-2-vinylnaphthalene.
[0252] 6-Methoxy-naphthaldehyde (432 mg, 2.35 mmol) is added to a
solution of instantaneous ylide
(Ph.sub.3P.sup.+Br.sup.-+NaNH.sub.2, 1.48 g, 2.4 mmol) in anhydrous
THF (5 ml). After one hour of stirring at ambient temperature, it
is diluted with ether, it is filtered on Celite, and it is
chromatographed (hexane/AcOEt 5:1) to obtain
6-methoxy-2-vinyl-naphtha- lene (390 mg, 90%).
[0253] 6-Methoxy-2-vinyl-naphthaldehyde: .sup.1H-NMR (CDCl.sub.3):
7.59-7.74 (m, 4H), 7.11-7.16 (mn, 2H), 6.86 (dd, 1H, J=17.3 and
10.9 Hz), 5.82 (d, 1H, J=17.6 Hz), 5.28 (d, 1H, J=10.7 Hz), 3.93
(s, 3H).
EXAMPLE 10:
Preparation of (R) and (S)
6-Methoxy-2-(1',2'-dihydroxyethyl)naphthalene.
[0254] 49
[0255] (R) and (S)
6-Methoxy-2-(1',2'-dihydroxyethyl)naphthalene.
[0256] These products, in the form of enantiomers R (>99% ee)
and S (>99% ee), were prepared by Shamnisens asymmetric
dihydroxylation of the corresponding olefin as described above for
coumarin derivatives.
[0257] Diol: .sup.1H-NMR (Acetone-d6): 7.71-7.84 (m, 3H), 7.52 (dd,
1H, J=8.5 and 1.5 Hz), 7.25-7.16 (m, 1H), 7.16 (dd, 1H, J=8.9 and
2.5), 4.87 (dd, 1H, J=7.5 and 4.2 Hz), 3.94 (s, 3H), 3.50-3.80 (m,
4H, CH.sub.2O+2OH).
EXAMPLE 11:
Preparation of (R)- and (S)-2-(6-Methoxy-2-naphthyl)oxyran.
[0258] 50
[0259] (R)- and (S)-2-(6-Methoxy-2-naphthyl)oxyran.
[0260] These products were prepared from corresponding diols
according to the method that is described above for the coumarin
derivatives and obtained optically pure (ee >99% according to
chiral HPLC).
[0261] .sup.1H NMR (CDCl3): 7.70-7.75 (m, 3H), 7.26-7.32 (m, 1H),
7.13-7.19 (m, 2H), 4.00 (dd, 1H, J=4.1 and 2.4 Hz), 3.92 (s, 3H),
3.22 (dd, 1H, J=5.4 and 4.0 Hz), 2.91 (dd, 1H, J=5.4 and 2.4
Hz).
[0262] II - Oxidation by Periodate.
[0263] The oxidation of 1,2 diols and amino alcohols by periodate
in an aqueous medium is a reaction that is well known to one
skilled in the art. This reaction provides a purely chemical
alternative for the conversion of alcohols into carbonyl groups in
aqueous medium with a high chemoselectivity in the presence of
other functional groups. The applicant now found that,
advantageously, hydrolytic enzymes that release diols or amino
alcohols from substrates that are resistant to oxidation by
periodate can be detected by fluorescence in the presence of
periodate and bovine serum albumin (BSA).
[0264] Diesters (R)- (S) as well as monoesters (R)- and
(S)-represented by Formula (I) (compounds 1 a/b, 3 a/b) constitute
excellent substrates for lipases and esterases given their
structural similarity with glycerides. The amides that are shown by
Formula I (compound 5) react with the amidases and peptidases.
Epoxides (R)- and (S)- that are represented by Formula (II)
(compounds 7 and 8) react with the epoxy-hydrolase enzymes. The
applicant thus found that the transformation undergone by these
fluorogenic substrates transformed by sodium periodate, in the
presence of hydrolytic enzymes, provides a new method for detecting
said enzymes.
[0265] III - Stability of the Substrates
[0266] The substrates acetates 1, amide 5 and epoxide 6 are stable
in the presence of periodate and BSA without the aqueous media.
Under the same conditions, diol 2 and amino alcohol 6 release
umbelliferone. Under optimized reaction conditions using 100 .mu.m
of substrate, 1 mmol of sodium periodate, 2 mg/ml of BSA in a
borate buffer with a pH of 8.8, diol 2 is transformed into
umbelliferone with a yield of 72% without observable secondary
reactions. The oxidation yield is independent of the presence of
BSA, which indicates that these reagents do not interfere with one
another in a notorious manner. The oxidation of amino alcohol 6
takes place with a yield of 85% under the same conditions.
[0267] IV - Development of the Test.
[0268] Acetates 1, amide 5 and epoxides (R)-7 and (S)-7 were tested
in the presence of corresponding hydrolytic enzymes. The acetates
were tested compared to 25 different esterases and lipases. The
epoxides were tested compared to the epoxy-hydrolase extracted from
Aspergillus Niger (X-J. Chen, A. Archelas, R. Furstoss, J. Org.
Chem. 1993, 58, 5528) and Rhodotorula glutinis (C. A. G. M.
Weijers, A. L. Botes, M. S. van Dyk, J. A. M. de Bont, Tetrahedron
Asymm. 1998, 9, 467).
[0269] Phenylacetamide 5 was tested against penicillin G acylase,
chymotrypsin and papain. An increase of fluorescence that is
clearly dependent on time is observed when the enzymatic activity
is present. The analyses that are carried out by high-pressure
liquid chromatography (HPLC) confirm that the increase of
fluorescence is caused by the release of umbelliferone from
substrates.
[0270] In all of the cases, the results that are obtained are
similar at both pH 7.2 and pH 8.8.
[0271] All of the enzymes that showed activity against any
substrate preserve their activity regardless of whether
preincubation of the enzyme with the substrate is done before the
addition of periodate and BSA or during the reaction, in the
presence of all of the components. This clearly proves that the
addition of periodate and BSA as secondary agents does not affect
the enzymatic activity of the enzymes that should be analyzed. The
analyses by HPLC of the reaction with the active enzymes in the
absence of periodate and BSA show that diol 2 and amino alcohol 6
are actually released by the enzymes that were considered as active
in the fluorescence test.
[0272] V - Measirement of the Lipase Activity by Using Substrate
2-Hydroxy-4-p-nitrophenyl-butyl-decanonte, and
p-Nitro-phenylbutyrate
[0273] The hydrolytic activity of the Candida antarctica lipase was
tested on 2 substrates: 2-hydroxy-4-p-nitrophenyl-butyl-decanoate
and p-nitro-phenylbutyrate. The lipase activity is detected after
hydrolysis of ester functions of substrates that produce the
release (direct or indirect) of yellow-colored para-nitrophenol
that is measured by colorimetry at 414 nm. The tests were carried
out at different temperatures and pH.
[0274] The stock solutions of
2-hydroxy-4-p-nitrophenyl-butyl-decanoate and
para-nitrophenolbutyrate (SIGMA) are prepared at the concentration
of 20 mmol respectively in acetonitrile and isopropanol. The
concentration of lipase B that is used is 0.01 mg/ml in a phosphate
buffer (100 mmol, pH 7).
[0275] V.1 - Measurement of the Lipase Activity Based on
Temperature
[0276] V.1.1 - Measurement of Lipase Activity with
2-Hydroxy-4-p-nitrophen- yl-butyl-decanote.
[0277] 74 .mu.l of PIPES buffer (100 mmol, pH 7) and 10 .mu.l of a
lipase solution at 0.01 mg/ml are added to 13.6 .mu.mol of
2-hydroxy-4-p-nitrophenyl-butyl-decanoate. The reaction is
incubated in a water bath for 40 minutes at 50, 60, 70, 80 and
95.degree. C. The samples are then removed from the water bath.
Then, 40 .mu.l of NaIO.sub.4 (100 mmol) and 4 .mu.l of BSA (50
mg/ml) are added to the mixture. The pH of the solution is then
adjusted to 10 by adding 40 .mu.l of an Na.sub.2CO.sub.3 solution
at 0.2 M. The para-nitrophenol that is released is then measured
with a colorimeter at 414 nm.
[0278] A negative control without enzyme was carried out under the
same experimental conditions.
[0279] V.1.2 - Measurement of Lipase Activity with
p-Nitrophenylbutyrate
[0280] 74 .mu.l of PIPES buffer (100 mmol, pH 7) and 10 .mu.l of a
lipase solution at 0.01 mg/ml are added to 13.6 .mu.mol of
para-nitrophenylbutyrate. The reaction is incubated in a water bath
for 40 minutes at 50, 60, 70, 80 and 95.degree. C. The samples are
then removed from the water bath, and the pH of the solution is
adjusted to 10 with a 0.2 M Na.sub.2CO.sub.3 solution. The released
para-nitrophenol is then measured with a calorimeter at 414 nm.
[0281] A negative control without enzyme was carried out under the
same experimental conditions.
[0282] V.2. - Measurement of Lipase Activity Based on the pH of the
Solution
[0283] The enzymatic activity of the Candida antarctica lipase was
carried out at different pH levels by using buffers (100 mmol)
listed in Table VII below.
7 TABLE VII Buffer pH Formate 3 Formate 4 Acetate 5 MES 6.5 PIPES 7
Phosphate 8
[0284] V.2.1 - Measurement of Lipase Activity with
2-Hydroxy-4-p-nitrophen- yl-butyl-decanoate
[0285] 74 .mu.l of buffer at the desired pH (100 mmol) and 10 .mu.l
of a lipase solution at 0.01 mg/ml are added to 13.6 .mu.mol of
2-hydroxy-4-p-nitrophenyl-butyl-decanoate. The reaction is
incubated in a water bath for 40 minutes at 60.degree. C. The
samples are then removed from the water bath. Then, 40 .mu.l of
NaIO.sub.4 (100 mmol) and 4 .mu.l of BSA (50 mg/ml) are added to
the mixture. The pH of the solution is then adjusted to 10 by
adding 40 .mu.l of a solution of Na.sub.2CO.sub.3 at 0.2 M. The
para-nitrophenol that is released is then measured with a
calorimeter at 414 nm.
[0286] A negative control without enzyme was carried out under the
same experimental conditions.
[0287] V.2.2 Measurement of Lipase Activity with
p-Nitrophenylbutyrate
[0288] 74 .mu.l of buffer at the desired pH (100 mmol) and 10 .mu.l
of a lipase solution at 0.01 mg/ml are added to 13.6 .mu.mol of
para-nitrophenylbutyrate. The reaction is incubated in a water bath
for 40 minutes at 60.degree. C. The samples are then removed from
the water bath, and the pH of the solution is adjusted to 10 with a
0.2 M Na.sub.2CO.sub.3 solution. The para-nitrophenol that is
released is then measured with a colorimeter at 414 nm.
[0289] A negative control without enzyme was carried out under the
same experimental conditions.
[0290] V.3 - Results of the Detection of Lipase Activity.
[0291] The 2-hydroxy-4-p-nitrophenyl-butyl-decanoate and the
para-nitrophenylbutyrate commercial substrate were tested compared
to the Candida antarctica lipase. The appearance of a yellow
coloration linked to the release of para-nitrophenol is observed
when hydrolytic activity is present.
[0292] The activity of the lipase based on the incubation
temperature was measured by using
2-hydroxy-4-p-nitrophenyl-butyl-decanoate and the
para-nitrophenylbutyrate commercial substrate. Table VIII shows the
measurements of the activity of the Candida antarctica lipase that
are made by using 2-hydroxy-4-p-nitrophenyl-butyl-decanoate and
para-nitrophenylbutyrate. The activities are expressed in %. The
enzymatic reactions were carried out in the PIPES buffer (0.1 M, pH
7) at 50, 60, 70, 80 and 95.degree. C. for 40 minutes. A negative
control without enzyme was also carried out by using the same
experimental conditions.
8 TABLE VIII p- 2-Hydroxy-4-p-nitrophen- yl- Nitrophenylbutyrate
butyl-decanoate Without Temperature (.degree. C.) Lipase B Without
enzyme Lipase B enzyme 50 100 8.7 100 83.6 60 75.9 9.4 75.4 67.3 70
61.5 9.5 60.4 59.5 80 44.2 8.1 55.9 59.8 95 26.1 8.6 66.7 71.5
[0293] The hydrolytic activity of the lipase based on the pH of the
solution was also measured. Table IX below shows the activity of
the Candida antarctica lipase that is measured by using
2hydroxy-4-p-nitrophenyl-butyl-decanoate and
para-nitrophenylbutyrate. The activities are expressed in %. The
enzymatic reactions were incubated at 60.degree. C. for 40 minutes
at pH 3, 4, 5, 6.5, 7 and 8. A negative control without enzyme was
also carried out by using the same experimental conditions.
9 TABLE IX 2-Hydroxy-4-p-nitrophenyl- butyl- decanoate
p-Nitrophenylbutyrate pH Lipase B Without enzyme Lipase B Without
enzyme 3 58.9 3.9 94.2 54.9 4 80.9 2.7 73 63.6 5 96.1 2.6 100 65.6
6.5 100 2.9 79.5 69.2 7 72 3.4 60.8 51.4 8 40 2.8 103.9 103.9
[0294] VI. Detection of the Release of a Volatile Compound.
[0295] The first tests were conducted with three different
substrates: benzaldehyde, the volatile form, and hydrobenzoin and
1,2-pentanediol, the vicinal forms.
[0296] 10 mg of each product is mixed with 10 mg of NaIO4 and 1 g
of MgSO4, then is left just in air on a shelf.
[0297] The detection of aromas is carried out from time to time by
sampling a little powder and by adding water. The results that
correspond to the observations relative to the perception of
perfumes are summarized in Table X below.
10TABLE X Period Benzaldehyde Hydrobenzoin 1,2-Pentanediol day
Strong odor A simple stirring of A simple stirring the solid with
of the solid with ultrasound is ultrasound is suff- sufficient to
release icient to release a a perceptible odor perceptible odor 5
days No more odor 2 or 3 drops of 2 or 3 drops of water should be
water should be added to several added to several milligrams of
solid milligrams of solid 1 month idem 2 or 3 ml of water 2 or 3 ml
of water should be added to should be added to several milligrams
several milligrams of solid of solid 5 months idem idem A larger
amount of solid is necessary
[0298] The detection of the "almond" odor of benzaldehyde, the
volatile form, is stable for at least one day. By contrast, thanks
to the process of the invention, the oxidation of the vicinal diols
corresponding to benzaldehyde makes it possible to spray the
"almond" odor of benzaldehyde for several months.
11TABLE XI List of Substrates by Functional Groups Diols, Amino
Alcohols and Azido-alcohol 51 52 53 54 55 56 57 58 59 60 61 62 63
64 65 66 67 68 69
[0299]
12 Esters 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88
89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107
108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124
125 126 127 128 129
[0300]
13 Amides 130 131 132 133 134 135
[0301]
14 Alcohols 136 137
[0302]
15 Phosphates 138 139
[0303]
16 Triols and Derivatives 140 141 142 143 144 145 146 147
[0304]
17 Epoxides 148 149 150 151 152 153 154 155 156 157 158 159 160 161
162 163 164 165 166 167 168
[0305]
18 Carbonates 169 170 171 172 173 174 175 176 177
* * * * *