U.S. patent application number 10/797169 was filed with the patent office on 2004-09-16 for substrate analog for murg, methods of making same and assays using same.
This patent application is currently assigned to The Trustees Of Princeton University. Invention is credited to Ge, Min, Kahne, Suzanne Walker, Men, Hongbin, Park, Peter.
Application Number | 20040180813 10/797169 |
Document ID | / |
Family ID | 22113335 |
Filed Date | 2004-09-16 |
United States Patent
Application |
20040180813 |
Kind Code |
A1 |
Kahne, Suzanne Walker ; et
al. |
September 16, 2004 |
Substrate analog for murg, methods of making same and assays using
same
Abstract
General methods for monitoring the activity of MurG, a GlcNAc
transferase involved in bacterial cell wall biosynthesis, is
disclosed. More particularly, the synthesis of simplified substrate
analogs of Lipid I (the natural substrate for MurG), which function
as acceptors for UDP-GlcNAc in an enzymatic reaction catalyzed by
MurG, is described. Assays using the substrate analogs of the
invention are further disclosed, which are useful for identifying a
variety of other substrates, including inhibitors of MurG activity,
for facilitating mechanistic and/or structural studies of the
enzyme and for other uses. High throughput assays are also
described.
Inventors: |
Kahne, Suzanne Walker;
(Princeton, NJ) ; Men, Hongbin; (Princeton,
NJ) ; Park, Peter; (Wheeling, IL) ; Ge,
Min; (Princeton, NJ) |
Correspondence
Address: |
WOODCOCK WASHBURN LLP
ONE LIBERTY PLACE, 46TH FLOOR
1650 MARKET STREET
PHILADELPHIA
PA
19103
US
|
Assignee: |
The Trustees Of Princeton
University
|
Family ID: |
22113335 |
Appl. No.: |
10/797169 |
Filed: |
March 9, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10797169 |
Mar 9, 2004 |
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10127639 |
Apr 22, 2002 |
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6703213 |
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10127639 |
Apr 22, 2002 |
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09241862 |
Feb 2, 1999 |
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6413732 |
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60073376 |
Feb 2, 1998 |
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Current U.S.
Class: |
435/15 ; 514/2.4;
514/20.9; 530/322; 536/53 |
Current CPC
Class: |
Y10S 530/812 20130101;
C12N 9/1288 20130101; C07H 11/04 20130101; A61K 38/00 20130101 |
Class at
Publication: |
514/008 ;
530/322; 536/053 |
International
Class: |
A61K 038/14; C07K
009/00 |
Claims
What is claimed is:
1. A substance comprising the chemical moiety of the formula: 11in
which "R" is an acyl group comprising 2 or more carbon atoms,
"R.sub.1" is a substituted or unsubstituted alkyl group comprising
1 or more carbon atoms, "R.sub.2" is a hydrogen or a substituted or
unsubstituted alkyl group comprising 1 or more carbon atoms, "A" is
a substituted or unsubstituted amino acid residue or a peptide
comprising 2 or more substituted or unsubstituted amino acid
residues, "R.sub.3" is a substituted or unsubstituted alkyl group
comprising 5 or more carbon atoms, said substance exhibiting a
binding affinity for at least wild type MurG enzyme and provided
that said substance is not Lipid I, the natural substrate of wild
type MurG enzyme.
2. The substance of claim 1 which serves as an acceptor for the
GlcNAc transferase activity of at least wild type MurG enzyme.
3. The substance of claim 1 which inhibits the GlcNAc transferase
activity of at least wild type MurG enzyme or its homologs.
4. The substance of claim 1 in which "R" is an acetyl group.
5. The substance of claim 1 in which "R.sub.1" is a methyl
group.
6. The substance of claim 1 in which "R.sub.2" is a hydrogen.
7. The substance of claim 1 in which "R.sub.3" is citronellol.
8. The substance of claim 1 in which "A" is a pentapeptide.
9. The substance of claim 8 in which the amino acid residue
attached to the lactic acid moiety of the substance of the formula
(1) is Ala.
10. The substance of claim 9 in which the amino acid residue
attached to said Ala is Glu.
11. The substance of claim 10 in which the amino acid residue
attached to said Glu is Lys.
12. The substance of claim 8 in which said pentapeptide has the
sequence Ala-Glu-Lys-Ala-Ala, the amino terminal end of which is
attached to the lactic acid moiety of the substance of the formula
(I) via an amide bond.
13. The substance of claim 1 in which "A" is conjugated to a biotin
moiety.
14. The substance of claim 13 in which said biotin moiety is
attached covalently to an amino group of an amino acid residue
either directly or via a linker moiety.
15. The substance of claim 1 in which "R.sub.3" is bound to a solid
support.
16. A method of detecting GlcNAc transferase activity in a sample
suspected of containing a protein or an active fragment thereof
exhibiting GlcNAc transferase activity comprising: (a) providing a
sample suspected of containing a protein or an active fragment
thereof exhibiting GlcNAc transferase activity; (b) contacting the
sample with effective amounts of labeled UDP-GlcNAc substrate and a
substance comprising the chemical moiety of the formula: 12in which
"R" is an acyl group comprising 2 or more carbon atoms, "R.sub.1"
is a substituted or unsubstituted alkyl group comprising 1 or more
carbon atoms, "R.sub.2" is a hydrogen or a substituted or
unsubstituted alkyl group comprising 1 or more carbon atoms, "A" is
a substituted or unsubstituted amino acid residue or a peptide
comprising 2 or more substituted or unsubstituted amino acid
residues, "R.sub.3" is a substituted or unsubstituted alkyl group
comprising 5 or more carbon atoms, provided that said substance is
not Lipid I, the natural substrate of wild type MurG enzyme, under
conditions effective to provide a labeled coupling product
comprising labeled GlcNAc coupled to said substance via a
glycosidic bond in the presence of a protein or an active fragment
thereof exhibiting GlcNAc transferase activity; (c) detecting the
formation or presence of said labeled coupling product, which is
indicative of GlcNAc transferase activity in said sample.
17. The method of claim 16 in which said labeled GlcNAc substrate
is labeled UDP-GlcNAc.
18. The method of claim 16 in which at least a portion of said
sample comprises a portion of a lysed bacterial culture, a portion
of a supernatant thereof, a portion of a membrane fraction thereof,
a portion of a protein fraction thereof, a purified enzyme,
purified or synthesized lipid or mixtures of same.
19. The method of claim 16 in which the detection step comprises
separation of labeled coupling product from labeled UDP-GlcNAc
substrate.
20. An assay for detecting GlcNAc transferase activity in a sample
suspected of containing a protein or an active fragment thereof
exhibiting GlcNAc transferase activity comprising a compound of the
formula: 13in which "R" is an acyl group comprising 2 or more
carbon atoms, "R.sub.1" is a substituted or unsubstituted alkyl
group comprising 1 or more carbon atoms, "R.sub.2" is a hydrogen or
a substituted or unsubstituted alkyl group comprising 1 or more
carbon atoms, "A" is a substituted or unsubstituted amino acid
residue or a peptide comprising 2 or more substituted or
unsubstituted amino acid residues, "R.sub.3" is a substituted or
unsubstituted alkyl group comprising 5 or more carbon atoms, said
substance able to form a coupling product with a GlcNAc substrate
in the presence of a protein or an active fragment thereof
exhibiting GlcNAc transferase activity, provided that said
substance is not Lipid I, the natural substrate of wild type MurG
enzyme.
21. The assay of claim 20 which further comprises a labeled GlcNAc
substrate.
22. A screen for compounds exhibiting potential antibacterial
activity comprising (i) a protein or an active fragment thereof
exhibiting GlcNAc transferase activity, (ii) a substance comprising
the chemical moiety of the formula: 14in which "R" is an acyl group
comprising 2 or more carbon atoms, "R.sub.1" is a substituted or
unsubstituted alkyl group comprising 1 or more carbon atoms,
"R.sub.2" is a hydrogen or a substituted or unsubstituted alkyl
group comprising 1 or more carbon atoms, "A" is a substituted or
unsubstituted amino acid residue or a peptide comprising 2 or more
substituted or unsubstituted amino acid residues, "R.sub.3" is a
substituted or unsubstituted alkyl group comprising 5 or more
carbon atoms, said substance able to form a coupling product with a
GlcNAc substrate in the presence of a protein or an active fragment
thereof exhibiting GlcNAc transferase activity, provided that said
substance is not Lipid I, the natural substrate of wild type MurG
enzyme, and (iii) a labeled GlcNAc substrate.
23. A substrate analog of Lipid 1 (i) having a structure that is
accepted by at least wild type MurG enzyme such that a labeled
coupling product is produced by the GlcNAc transferase activity of
the enzyme in the presence of said substrate analog and labeled
UDP-GlcNAc, and (ii) having structural features that facilitate the
separation of labeled UDP-GlcNAc from said labeled coupling
product.
24. A substance comprising the chemical moiety of the formula: 15in
which "R" is an acyl group comprising 2 or more carbon atoms,
"R.sub.1" is a substituted or unsubstituted alkyl group comprising
1 or more carbon atoms, "R.sub.2" is a hydrogen or a substituted or
unsubstituted alkyl group comprising 1 or more carbon atoms, "A" is
a substituted or unsubstituted amino acid residue or a peptide
comprising 2 or more substituted or unsubstituted amino acid
residues, "R.sub.3" may be selected from H, an aliphatic group
comprising 1 to about 50 carbon atoms, an aromatic or
heteroaromatic group comprising 3 to about 55 carbon atoms,
pyrophosphate protecting groups and pharmaceutically acceptable
salts thereof, said substance exhibiting a binding affinity for at
least a soluble type MurG enzyme and provided that said substance
is not Lipid 1, the natural substrate of wild type MurG enzyme.
25. The substance of claim 24 in which "A"or "R.sub.3" is bound to
a solid support.
26. The substance of claim 25 in which said solid support is an
avidin or strepavidin coated resin and said "A" or "R.sub.3" are
conjugated to a biotin moiety.
27. The substance of claim 26 in which said biotin moiety is
attached to "A" or "R.sub.3" either directly or via a linker
moiety.
28. A pharmaceutical composition comprising the substance of claim
24 and a pharmaceutically acceptable carrier.
29. A method of detecting GlcNAc transferase activity in a sample
suspected of containing a protein or an active fragment thereof
exhibiting GlcNAc transferase activity comprising: (a) providing a
sample suspected of containing a protein or an active fragment
thereof exhibiting GlcNAc transferase activity; (b) contacting the
sample with effective amounts of labeled UDP-GlcNAc substrate and a
substance comprising the chemical moiety of the formula: 16in which
"R" is an acyl group comprising 2 or more carbon atoms, "R.sub.1"
is a substituted or unsubstituted alkyl group comprising 1 or more
carbon atoms, "R.sub.2" is a hydrogen or a substituted or
unsubstituted alkyl group comprising 1 or more carbon atoms, "A" is
a substituted or unsubstituted amino acid residue or a peptide
comprising 2 or more substituted or unsubstituted amino acid
residues, "R.sub.3" may be selected from H, an aliphatic group
comprising 1 to about 50 carbon atoms, an aromatic or
heteroaromatic group comprising 3 to about 55 carbon atoms,
pyrophosphate protecting groups and pharmaceutically acceptable
salts thereof, provided that said substance is not Lipid I, the
natural substrate of wild type MurG enzyme, under conditions
effective to provide a labeled coupling product comprising labeled
GlcNAc coupled to said substance via a glycosidic bond in the
presence of a protein or an active fragment thereof exhibiting
GlcNAc transferase activity; (c) detecting the formation or
presence of said labeled coupling product, which is indicative of
GlcNAc transferase activity in said sample.
30. The method of claim 29 in which said labeled GlcNAc substrate
is labeled UDP-GlcNAc.
31. The method of claim 30 in which at least a portion of said
sample comprises a portion of a lysed bacterial culture, a portion
of a supernatant thereof, a portion of a membrane fraction thereof,
a portion of a protein fraction thereof, a purified enzyme, a
soluble enzyme, purified or synthesized lipid or mixtures of
same.
32. The method of claim 29 in which the detection step comprises
separation of labeled coupling product from labeled UDP-GlcNAc
substrate.
33. The method of claim 29 in which said detection step comprises
binding said "A" or "R.sub.3" to a solid support via a biotin tag,
wherein said solid support includes an avidin or streptavidin
coated resin.
34. The method of claim 33 wherein said detection step provide a
continuous monitoring of product formation via the use of
scintillation proximity assay.
35. The method of claim 16 wherein said substance is a
biotin-labeled substance and said separation involves filtration
through an avidin-coated resin.
36. A method of identifying compounds with the ability to inhibit
GlcNAc transferase activity comprising: (a) providing a sample
containing a protein or active fragment exhibiting GlcNAc
transferase activity; (b) contacting the sample with the potential
inhibitor and effective amounts of labeled UDP-GlcNAc and a
substance of formula: 17(c) detecting the formation or presence of
coupled product and comparing the amount of product to that
obtained in the absence of any potential inhibitor.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to substrate analogs of a
UDP-GlcNAc:muramyl pentapeptide pyrophosphoryl,
N-acetylglucosaminyltrans- ferase (GlcNAc transferase, MurG, or its
homologs), an enzyme involved in bacterial cell wall biosynthesis.
The substrate analogs of the invention are useful as functional
substitutes of Lipid I, the membrane bound, natural substrate of
MurG. In particular, the substrate analogs of the present invention
can be used advantageously in an assay for the enzymatic activity
catalyzed by MurG, in methods for identifying other substrate
analogs of MurG, as well as inhibitors of enzymatic activity or
cell wall biosynthesis (i.e., potential antibacterial drugs), and
in the isolation/purification of MurG, including studies of its
active protein/peptide fragments.
BACKGROUND OF THE INVENTION
[0002] 2.1. Bacterial Enzymology
[0003] The emergence of resistance to existing antibiotics has
rejuvenated interest in bacterial enzymology. It is hoped that
detailed mechanistic and structural information about bacterial
enzymes involved in critical biosynthetic pathways could lead to
the development of new antibacterial agents. Because interference
with peptidoglycan biosynthesis is a proven strategy for treating
bacterial infections, all of the enzymes involved in peptidoglycan
biosynthesis are potential targets for the development of new
antibiotics. While some detailed structural and mechanistic
information on some of the early enzymes in the pathway is now
available, most of the downstream enzymes have proven very
difficult to study.
[0004] There are two main reasons for this difficulty: First, the
downstream enzymes are membrane-associated, making them
intrinsically hard to handle; secondly, discrete substrates for
most of the downstream enzymes are either not available or not
readily so. In some cases monomeric substrates are difficult to
obtain in large quantities from natural sources. In other cases
substrates, which may be available in large quantities from natural
sources, are intractable polymeric substances. In the absence of
readily available discrete substrates, it has been impossible to
develop enzyme assays that can be used to measure the activity of
the downstream enzymes reliably and under a well-defined set of
reaction conditions. This unfulfilled need has thwarted attempts to
purify many of the downstream enzymes in an active form suitable
for structural characterization, much less permitted attempts to
obtain detailed mechanistic information on such enzymes.
[0005] Some of the best antibiotics function by interfering with
the biosynthesis of the peptidoglycan polymer that surrounds
bacterial cells. With the emergence of bacterial pathogens that are
resistant to common antibiotics it has become imperative to learn
more about the enzymes involved in peptidoglycan biosynthesis.
Although remarkable progress has been made in characterizing some
of the early enzymes in the biosynthetic pathway (See, e.g., (a)
Fan, C.; Moews, P. C.; Walsh, C. T.; Knox, J. R. Science 1994, 266,
439; (b) Benson, T. E.; Filman, D. J.; Walsh, C. T.; Hogle, J. M.
Nat. Struct. Biol. 1995, 2, 644; (c) Jin, H. Y.; Emanuele, J. J.;
Fairman, R.; Robertson, J. G.; Hail, M. E.; Ho, H. T.; Falk, P.;
Villafranca, J. J. Biochemistry 1996, 35, 1423; (d) Skarzynski, T.;
Mistry, A.; Wonacott, A.; Hutchinson, S. E.; Kelly, V. A.; Duncan,
K. Structure 1996, 4, 1465; (e) Schonbrunn, E.; Sack, S.;
Eschenburg, S.; Perrakis, A.; Krekel, F.; Amrhein, N.; Mandelkow,
E. Structure 1996, 4, 1065. (f) Benson, T. E.; Walsh, C. T.; Hogle,
J. M. Biochemistry 1997, 36, 806.), the downstream enzymes have
proven exceedingly difficult to study. Part of the difficulty stems
from the fact that such downstream enzymes are membrane-associated
(See, e.g., (a) Gittins, J. R; Phoenix, D. A.; Pratt, J. M. FEMS
Microbiol. Rev. 1994, 13, 1; (b) Bupp, K; van Heijenoort, J. 1993,
175, 1841.), making them intrinsically hard to handle, and partly
because substrates for many of the enzymes are not readily
available. (See, e.g., (a) Pless, D. D.; Neuhaus, F. C. J. Biol.
Chem. 1973, 248, 1568; (b) van Heijenoort, Y.; Gomez, M.; Derrien,
M.; Ayala, J.; van Heijenoort, J. J. Bacteriol. 1992, 174, 3549.)
These problems have impeded the development of activity assays
suitable for detailed mechanistic investigations of the downstream
enzymes. For a fluorescent assay to monitor MraY activity, see:
Brandish, P. E.; Burnham, M. K.; Lonsdale, J. T.; Southgate, R;
Inukai, M.; Bugg, T. D. H. J. Biol. Chem. 1996, 271, 7609.
[0006] 2.2. MurG
[0007] One such downstream enzyme is MurG, which is involved in
peptidoglycan biosynthesis. MurG catalyzes the last intracellular
step in the biosynthetic pathway of peptidoglycan biosynthesis,
i.e., the transfer of UDP-N-acetylglucosamine (UDP-GlcNAc) to the
lipid-linked N-acetylmuramylpentapeptide substrate, Lipid I. (See,
Scheme 1, below.)
[0008] Although the murG gene is first identified in E. coli in
1980 and is sequenced independently by two groups in the early
1990's, very little is known about the MurG enzyme. There are no
mammalian homologs, and no direct assays for MurG activity have
been developed, in part because the lipid-linked substrate (Lipid
I, Scheme 1) is extremely difficult to isolate. This lipid-linked
substrate is present only in minute quantities in bacterial cells.
Although it is possible to increase the quantities of lipid-linked
substrate by using bacterial cells engineered to overexpress
enzymes involved in the synthesis of the lipid-linked substrate,
isolation remains very difficult. Moreover, the isolated substrate
is hard to handle.
[0009] Consequently, MurG activity is currently assessed using
crude membrane preparations by monitoring the incorporation of
radiolabel from radiolabeled UDP-GlcNAc donor group into
lipid-linked acceptor components in the membrane. To increase the
signal, the membranes are often prepared from bacterial cultures
that overexpress MraY and/or MurG. MraY is the enzyme that
catalyzes the reaction that attaches the MraY substrate,
UDP-N-acetyl muramic acid pentapeptide, to a lipid phosphate moiety
to provide Lipid I, which is the substrate for MurG. Typically, the
membrane preparations are supplemented with exogenous UDP-N-acetyl
muramic acid pentapeptide for conversion to Lipid I. This MraY
substrate can be readily isolated in large quantities from
bacterial cultures. Although this "coupled" enzyme assay is
manageable for screening of potential inhibitors of the MurG
enzyme, it is not suitable for detailed mechanistic investigations,
and it cannot be used to follow MurG activity during
purification.
[0010] More specifically, MurG is a cytoplasmic membrane-associated
enzyme which catalyzes the transfer of UDP-N-acetylglucosamine
(UDP-GlcNAc) to the C4 hydroxyl of an undecaprenyl pyrophosphate
N-acetylmuramyl pentapeptide substrate (Lipid I), resulting in the
assembly of the disaccharide-pentapeptide building block (Lipid II,
Scheme 1), which is incorporated into polymeric peptidoglycan. See,
e.g., (a) Bugg, T. D. H.; Walsh, C. T. Nat. Prod. Rep. 1992, 199;
(b) Mengin-Lecreulx, D.; Flouret, B.; van Heijenoort, J. J.
Bacteriol. 1982, 151, 1109. As already mentioned, the muramyl
pentapeptide substrate is unique to bacteria Hence, the MurG enzyme
is a potential target for the discovery or design of specific MurG
inhibitors.
[0011] Despite decades of effort spent characterizing MurG
activity, there is virtually no structural or mechanistic
information on the enzyme. See, e.g., (a) Anderson, J. S.;
Matsuhashi, M.; Haskin, M. A.; Strominger, J. L. Proc. Natl. Acad.
Sci. USA 1965, 53, 881; (b) Anderson, J. S.; Matsuhashi, M.;
Haskin, M. A.; Strominger, J. L. J. Biol. Chem. 1967, 242, 180; (c)
Taku, A.; Fan, D. P. J. Biol. Chem. 1976, 251, 6154; (d)
Mengin-Lecreulx, D.; Texier, L.; van Heijenoort, J. Nucl. Acid.
Res. 1990, 18, 2810; (e) Ikeda, M.; Wachi, M.; Jung, H. K.; Ishino,
F.; Matsuhashi, M. Nuci. Acid Res. 1990, 18, 4014; (f)
Mengin-Lecreulx, D.; Texier, L.; Rousseau, M.; van Heijenoort, J.
J. Bacteriol 1991, 173, 4652; (g) Miyao, A.; Yoshimura, A.; Sato,
T.; Yamamoto, T.;Theeragool, T.; Kobayashi, Y. Gene, 1992, 118,
147; (h) Ikeda, M.; Wachi, M.; Matshuhashi, M. J. Gen. Appl.
Microbiol., 1992, 38, 53. Difficulties isolating Lipid I have
prevented the development of a simple, direct assay for MurG
activity. Consequently, it has not been possible to purify MurG in
a quantifiably active form or to determine the minimal functional
length; nor has it been possible to carry out any detailed
mechanistic studies, or to determine the substrate
requirements.
[0012] Therefore, there exists a need for a direct enzyme assay
that can be used both for effective screening of enzyme inhibitors
and for the purification, characterization and identification of
MurG, its various mutants and active fragments thereof. 1
3. SUMMARY OF THE INVENTION
[0013] Substrate analogs for MurG enzyme, a GlcNAc transferase, are
disclosed. For the first time, a substrate analog of Lipid I, as
shown above in Scheme 1, (i) having a structure that is accepted by
at least wild type MurG enzyme such that a labeled coupling product
is produced by the GlcNAc transferase activity of the enzyme in the
presence of the substrate analog and labeled UDP-GlcNAc, and (ii)
having structural features that facilitate the separation of
labeled UDP-GlcNAc from the labeled coupling product.
[0014] In particular, a substance is described herein, which
comprises the chemical moiety of the formula: 2
[0015] in which "R" is an acyl group comprising 2 or more carbon
atoms, "R.sub.1" is a substituted or unsubstituted alkyl group
comprising 1 or more carbon atoms, "R.sub.2" is a hydrogen or a
substituted or unsubstituted alkyl group comprising 1 or more
carbon atoms, "A" is a substituted or unsubstituted amino acid
residue or a peptide comprising 2 or more substituted or
unsubstituted amino acid residues, "R.sub.3" is a substituted or
unsubstituted alkyl group comprising 5 or more carbon atoms, the
substance exhibiting a binding affinity for at least wild type MurG
enzyme and provided that the substance is not Lipid I, the natural
substrate of wild type MurG enzyme. More particularly, the
substance of the invention serves as an acceptor for the GlcNAc
transferase activity of at least wild type MurG enzyme or its
homologs.
[0016] Also disclosed is a method of detecting GlcNAc transferase
activity in a sample suspected of containing a protein or an active
fragment thereof exhibiting GlcNAc transferase activity. Preferably
the method comprises (a) providing a sample suspected of containing
a protein or an active fragment thereof exhibiting GlcNAc
transferase activity; (b) contacting the sample with effective
amounts of labeled GlcNAc substrate and a substance comprising the
chemical moiety of the formula (I), above, under conditions
effective to provide a labeled coupling product comprising labeled
GlcNAc coupled to the substance via a glycosidic bond in the
presence of a protein or an active fragment thereof exhibiting
GlcNAc transferase activity; and (c) detecting the formation or
presence of the labeled coupling product, which is indicative of
GlcNAc transferase activity in the sample.
[0017] It is also an objective of the present invention to provide
an assay for detecting GlcNAc transferase activity in a sample
suspected of containing a protein or an active fragment thereof
exhibiting GlcNAc transferase activity comprising a compound of the
formula (I), above. A screen and methods of utilizing same are also
contemplated by the present invention. In particular, a screen is
provided for compounds exhibiting potential antibacterial activity
comprising (i) a protein or an active fragment thereof exhibiting
GlcNAc transferase activity, (ii) a substance comprising the
chemical moiety of the formula (I), above, and (iii) a labeled
GlcNAc substrate.
[0018] Additionally, the method of this invention provides a
detection step comprising binding the "A" or "R.sub.3" groups of
formula I to a solid support via a biotin tag, wherein said solid
support includes an avidin or streptavidin coated resin. This step
provides a continuous monitoring of product formation via the use
of scintillation proximity assay. Furthermore, the separation of
biotin-labeled substance involves filtration through an
avidin-coated resin.
[0019] In a preferred embodiment of the invention "R.sub.3" may be
selected from H, an aliphatic group comprising 1 to about 50 carbon
atoms, an aromatic or heteroaromatic group comprising 3 to about 55
carbon atoms, pyrophosphate protecting groups and pharmaceutically
acceptable salts thereof.
[0020] Additionally, a method detection step comprises binding said
"A" or "R.sub.3" to a solid support via a biotin tag, wherein said
solid support includes an avidin or streptavidin coated resin
[0021] Hence, substrate analogs are prepared, which are used in an
enzyme assay for MurG or MurG-like activity. A direct assay for
MurG activity is thus provided.
[0022] These and other objects of the invention are described
further, below, along with the preferred embodiments of the
invention.
4. BRIEF DESCRIPTION OF THE FIGURE
[0023] FIG. 1. Plot of GlcNAc transfer as a function of the
concentration of substrate analog 5b and concentration of active
MurG enzyme. All reactions are run in 100 mM Tris-HCl, pH 7.6, 1 mM
MgCl.sub.2, with 0.5-1.0 .mu.g total protein and 9.4 .mu.M
14C-UDP-GlcNAc (265 mCi/mmol). Reactions for curves A, B, C, and D
are carried out using a cell lysate from a transformed
BL21(DE3)pLysS strain that overexpresses MurG: A)-.box-solid.7.1
.mu.M 5b; B)-.diamond-solid.3.5 .mu.M 5b; C)-.circle-solid.0.71
.mu.M 5b; D)-.largecircle. 7.1 .mu.M 5b+heat treated cell lysate
(65.degree. C., 5 min.). Reactions for curve E are carried out
using a BL21(DE3)pLysS cell lysate expressing only endogenous
levels of MurG: E)-.gradient. 7.1 .mu.M 5b.
[0024] FIG. 2(a). Double reciprocal plots of the initial rate data
with UDP-GicNAc as the varied substrate. Initial rates are measured
at fixed acceptor 1b concentrations of 7;.mu.M (.diamond.), 10
.mu.M (.gradient.), 15 .mu.M (.box-solid.), 30 .mu.M (+), 100 .mu.M
(.circle-solid.). 0.08 .mu.M of purified MurG is used for each
reaction.
[0025] FIG. 2(b). Secondary plots of the slope.
[0026] FIG. 2(c). Intercept versus [1b].sup.-1. Analysis of the
data assuming a rapid equilibrium sequential mechanism yields the
following kinetic parameters: K.sub.UDP-GLCNAC=110.+-.30 .mu.M,
K.sub.1b=60.+-.15 .mu.M.,
[0027] FIG. 3. IC50 measurements for compound 12a and UDP. All the
assays are performed under the same conditions with 18 .mu.M 1b and
34.3 .mu.M UDP-G1CNAc. Each IC.sub.50 value is determined by
fitting five or six data points to equation:
[0028] value is determined by fitting five or six data points to
equation: 1 v i v o = 1 1 + ( 1 ) IC 50
[0029] where v.sub.I is the initial rate in the presence of
inhibitor at concentration (1), and v.sub.o is the initial rate
without inhibitor.
[0030] FIG. 4. Structure of Lipid I and analogs (1a, 1b).
[0031] FIG. 5. Substrate-based inhibitors of MurG activity.
[0032] FIG. 6; Alternative acceptors for MurG.
[0033] FIG. 7. Synthesis of lipid I analogs (1a, 1b). Reagents and
conditions: (a) CCl.sub.3CH.sub.2OH, DCC/DMAP, THF, rt, 4 h, 80%;
(b) 1. H.sub.2/Pd, EtOAc, Mt, 0.5 h; 2. PhCH(OCH.sub.3).sub.2, cat
TsOH, DMF, rt, 10 h, 81%, 2 steps; (c) iPr.sub.2NP(OBn).sub.2,
.sup.1H-tetrazole, CH.sub.2Cl.sub.2, -20.degree. C.->0.degree.
C., 0.5 h, then mCPBA, -40.degree. C.->25.degree. C., 2 h, 70%;
(d) Zn dust, 90% AcOH/H.sub.2O, rt, 1 h, 91%; (e) HOBt, PyBop,
DIEA, DMF, 0.degree. C., 30 min, 87%; (f) 1. H.sub.2/Pd,
CH.sub.3OH, rt, 30 min, then py; 2.
(R)-(+)-.beta.-Citronellol-OPO.sub.3PO(OPh).sub.2, py,
CH.sub.2Cl.sub.2, rt, 18 h, 68%; (g) TBAF, DMF, rt, 24 h, 93%; (h)
6-((biotinoyl)amino)hexa- noic acid succinimide ester, NaHCO.sub.3,
H.sub.2O/dioxane, rt, 2 h, 80%.
[0034] FIG. 8. Synthesis of disaccharide by MurG.
5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0035] 5.1. General Aspects of the Invention
[0036] The present invention contemplates a substance comprising
the chemical moiety of the formula: 3
[0037] in which "R" is an acyl group comprising 2 or more carbon
atoms, "R.sub.1 " is a substituted or unsubstituted alkyl group
comprising 1 or more carbon atoms, "R.sub.2" is a hydrogen or a
substituted or unsubstituted alkyl group comprising 1 or more
carbon atoms, "A" is a substituted or unsubstituted amino acid
residue or a peptide comprising 2 or more substituted or
unsubstituted amino acid residues and "R.sub.3" is a substituted or
unsubstituted alkyl group comprising 5 or more carbon atoms.
Preferably, the substance of the invention (sometimes referred to
herein as a substrate analog or, simply, compound) exhibits a
binding affinity for at least wild type MurG enzyme. More
preferably, the substance of the invention serves as an acceptor
for the GlcNAc transferase activity of at least wild type MurG
enzyme. It is important to note that the substance of the invention
is not so broadly defined as to encompass Lipid I, the natural
substrate of wild type MurG enzyme.
[0038] It should be evident to one of ordinary skill that the
substance disclosed and described herein can also possess
inhibitory activity against the GlcNAc transferase activity of at
least wild type MurG enzyme, its homologs and, possibly, certain
mutant forms thereof, depending in part on the strength of its
binding affinity with the protein or its active fragments. That is,
a substrate analog of the present invention, by binding tenaciously
to the protein or active fragment thereof, can potentially inhibit
the ability of MurG or a MurG-like enzyme to catalyze the
glycosylation reaction that results in the transfer of GlcNAc to
the C4 hydroxyl position of the N-acetylmuramic acid moiety of
Lipid I. Of course, MurG and its homologs are derived from E. coli
and other gram-negative bacteria Gram-positive bacteria, such as B.
subtilis, E. faecalis, E. hirae, as well as M tuberculosis, are
also known to harbor homologs of MurG.
[0039] Accordingly, in a preferred embodiment of the invention, "R"
is an acyl group including, but not limited to, acetyl, proprionyl,
butanoyl, pentanoyl, hexanoyl and the like. The group "R.sub.1" is
a substituted or unsubstituted alkyl group including, but not
limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, phenyl,
benzyl, tolueyl, anthracyl and the like. The group "R.sub.2" is a
hydrogen or a substituted or unsubstituted alkyl group including,
but not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl,
phenyl, benzyl, tolueyl, anthracyl and the like.
[0040] Hence, the term "alkyl" group can encompass an aliphatic or
an aromatic group, and the term "substituted" means that the
particular alkyl group can have substituents including, but not
limited to, additional alkyl groups, heteroatoms or functional
groups containing heteroatoms, including, but not limited to,
alcohols, ethers, carboxylic acids, esters, amides, amines,
alkylamines, thiols, sulfides, sulfates, sulfoxides, sulfonic
acids, phosphoric acids, phosphate esters, phosphides,
phosphonates, phosphoramidates and the like. Any acyl group can
have 2 or more carbon atoms, and any alkyl group can have 1 or more
carbon atoms. Each group can have as many as 25 carbon atoms,
preferably up to 20 carbon atoms, more preferably up to 15, most
preferably up to 10 carbon atoms.
[0041] In one embodiment of the invention, the group "R.sub.3" may
be a substituted or unsubstituted alkyl group including, but not
limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, phenyl,
benzyl, tolueyl, naphthyl, anthracyl and the like. More
particularly, the group "R.sub.3" comprises a mimic of the
55-carbon hydrocarbon anchor found in the natural MurG substrate,
Lipid I. Such mimics include, but are not limited to, citronellol,
other polyprenol derivative, or an aromatic group. In addition, the
group "R.sub.3" can be bound to a solid support, such as a
synthetic resin or bead.
[0042] The group "A" is broadly contemplated to encompass any
substituted or unsubstituted amino acid residue or any peptide
comprising 2 or more substituted or unsubstituted amino acid
residues. The group "A" can have as few as one, two, or three amino
acid residues, or many as 10 or more amino acid residues,
preferably no more than ten, more preferably no more than eight,
most preferably no more than five (e.g., a pentapeptide). Other
chemical moieties may be associated with the group "A," preferably
covalently attached, including but not limited to linker groups,
labeling groups (such as radiolabeled groups, fluorescent groups
and the like), affinity groups (such as biotin, avidin,
streptavidin and the like or haptens, such as dinitrophenol,
digoxegenin and the like), hydrophobic groups, hydrophilic groups,
and the like. In one embodiment of the invention biotin is
conjugated to the group "A", in which the biotin moiety is attached
covalently (e.g., to an amino group of an amino acid residue)
either directly or via a linker moiety.
[0043] In a preferred embodiment, the amino acid residue attached
to the lactic acid moiety of the substance of the formula (I) is
Ala A D-.gamma.-linked glutamic acid residue is preferably attached
next to this fist alanine residue. A lysine residue (L-Lys) is
preferably attached next to this glutamic acid residue,
particularly for gram-positive bacteria For gram-negative bacteria,
this third residue is preferably meso-diaminopimelate or "m-DAP."
Other residues at this position include, but are not limited to,
L-alanine, L-homoserine, L-diaminobutyric acid, L-glutamic acid,
L-ornithine, LL-DAP, as well as the meso-form, referred to, above.
Still others may include L-Orn, LL-Dpm, m-HyDpm, L-Dab, L-HyLys,
N.sup..gamma.-Acetyl-L-Dab, L-Hsr, L-Ala, or L-Glu. A preferred
amino acid sequence for a pentapeptide is
L-Ala-D-.gamma.-Glu-L-Lys-D-Ala-D-Ala, the amino terminal end of
which is attached to the lactic acid moiety of the substance of the
formula (I) via an amide bond. Yet another suitable amino acid
sequence may be L-Ala-D-.gamma.-Glu-meso-DAP-D-Ala-D-Ala A
tripeptide sequence of potential advantage is
L-Ala-D-.gamma.-Glu-L-Lys, optionally substituted at the L-Lys
amino acid residue with an affinity "handle," such as biotin,
avidin, streptavidin, an immunoglobulin, Protein A, and the like or
fragments thereof, or haptens, such as dinitrophenol, digoxegenin
and the like. Still possible is a dipeptide arrangement, including
but not limited to L-Ala-D-Lys, once again optionally substituted
at the D-Lys amino acid residue.
[0044] In a method of the present invention GlcNAc transferase
activity is detected in a sample suspected of containing a protein
or an active fragment thereof exhibiting GlcNAc transferase
activity. The method includes the steps of: (a) providing a sample
suspected of containing a protein or an active fragment thereof
exhibiting GlcNAc transferase activity; (b) contacting the sample
with effective amounts of labeled UDP-GlcNAc substrate and a
substance comprising the chemical moiety of the formula (I), above,
provided that the substance is not Lipid I, the natural substrate
of wild type MurG enzyme, under conditions effective to provide a
labeled coupling product comprising labeled GlcNAc coupled to the
substance via a glycosidic bond in the presence of a protein or an
active fragment thereof exhibiting GlcNAc transferase activity; and
(c) detecting the formation or presence of the labeled coupling
product, which is indicative of GlcNAc transferase activity in the
sample. Preferably, the labeled GlcNAc substrate is labeled
UDP-GlcNAc.
[0045] In the inventive method at least a portion of the sample may
comprise a portion of a lysed bacterial culture, a portion of a
supernatant thereof, a portion of a membrane fraction thereof, a
portion of a protein fraction thereof, a purified enzyme, purified
or synthesized lipid or mixtures of same.
[0046] Detection of the formation or presence of the labeled
coupling product can be effected in a number of ways, apparent to
those of ordinary skill. For example, the detection step may
comprise separation of the labeled coupling product from labeled
UDP-GlcNAc substrate. As discussed elsewhere in this disclosure,
separation of the labeled species can be accomplished using a
variety of approaches, including but not limited to, hydrophobic
capture, affinity chromatography, or other solid phase separation
techniques. Quantification of the labeled coupling product can then
follow depending on the nature of the label utilized.
[0047] Consistent with the objectives of the present invention an
assay is provided for detecting GlcNAc transferase activity in a
sample suspected of containing a protein or an active fragment
thereof exhibiting GlcNAc transferase activity. An assay of the
invention comprises a compound of the formula: 4
[0048] in which "R" is an acyl group comprising 2 or more carbon
atoms, "R.sub.1" is a substituted or unsubstituted alkyl group
comprising 1 or more carbon atoms, "R.sub.2" is a hydrogen or a
substituted or unsubstituted alkyl group comprising 1 or more
carbon atoms, "A" is a substituted or unsubstituted amino acid
residue or a peptide comprising 2 or more substituted or
unsubstituted amino acid residues, "R.sub.3" is a substituted or
unsubstituted alkyl group comprising 5 or more carbon atoms, the
substance able to form a coupling product with a GlcNAc substrate
in the presence of a protein or an active fragment thereof
exhibiting GlcNAc transferase activity, provided that the substance
is not Lipid I, the natural substrate of wild type MurG enzyme. The
assay further comprises a labeled GlcNAc substrate.
[0049] A screen for compounds exhibiting potential antibacterial
activity is also contemplated. Such a screen comprises: (i) a
protein or an active fragment thereof exhibiting GlcNAc transferase
activity, (ii) a substance comprising the chemical moiety of the
formula (I), above, the substance able to form a coupling product
with a GlcNAc substrate in the presence of a protein or an active
fragment thereof exhibiting GlcNAc transferase activity, provided
that the substance is not Lipid I, the natural substrate of wild
type MurG enzyme, and (iii) a labeled GlcNAc substrate.
[0050] Thus, a screen including the enzyme MurG, or an active
fragment thereof, is brought into contact with a substrate analog,
such as a substance of the formula (I), in the presence of labeled
GlcNAc substrate. The enzyme, of course, would catalyze the
coupling of the labeled GlcNAc (e.g., from C-14 labeled UDP-GlcNAc)
to the C4-hydroxyl group of the muramic acid moiety of the
substrate analog. The formation of labeled coupling product is then
monitored over time to produce a graph, such as that presented in
FIG. 1. (The coupling product may first have to be separated from
labeled GlcNAc substrate, e.g., by column chromatography, HPLC,
filtration (if the reaction is conducted in the solid phase) and
the like.) A potential inhibitory compound (or compounds) is then
added to the mixture, such as the control mixture described above,
and the decrease in the production of labeled coupling product is
monitored, preferably as a function of the concentration of the
potential inhibitory compound.
[0051] 5.2. The Preparation Of A Substrate Analog
[0052] Our first synthetic target, 8 (above, and Scheme 2, below),
differs from Lipid I in that the 55 carbon undecaprenol chain has
been replaced by the ten carbon chain of citronellol. A shorter
lipid chain is chosen because long chain lipids are difficult to
handle; a lipid containing a saturated isoprenol unit is further
chosen because allylic pyrophosphates are unstable. Although MurG
is a membrane-associated enzyme, which recognizes a lipid-linked
substrate, the chemistry takes place on the C4 hydroxyl of the
lipid-linked substrate, which is far removed from the lipid anchor.
Therefore, it is hoped that alteration of the lipid can be
accomplished without destroying substrate recognition. 5
[0053] To make 8 (See, Scheme 2, below), muramic acid derivative 1
(available from Sigma) is converted to the anomeric dibenzyl
phosphate 5 in 5 steps and coupled to the protected pentapeptide
13. Chen, J.; Dorman, G.; Prestwich, G. J. Org. Chem. 1996, 61,
393. The silyl protecting groups on the Lys and Glu are preferred
for facile deprotection under mild conditions. Hence, the
C-terminus of the peptide can be a methyl ester, as shown, or a
trimethylsilyl ethyl ester.
[0054] The protected pentapeptide is synthesized on a D-Ala-FMOC
Sasrin resin (available from Bachem Biosciences) in 11 steps in an
overall yield of 15% (See, Method 4, below). Experimental details
are provided in the Examples Section, below. Hydrogenolytic
deprotection produces the anomeric phosphate, which is treated with
diphenyl citronellol pyrophosphate to produce 7 (See, Scheme 2).
Diphenyl citronellol pyrophosphate (10, Method 1, below) is
generated in situ by treating citronellol phosphate with diphenyl
chlorophosphate (See, Example Section, below; see, also: Warren, C.
D.; Jeanloz, R W. Meth. Enzymol. 1978, 50, 122.) For other methods
to form glycosyl pyrophosphates, see: (a) Imperiali, B.; Zimmerman,
J. W. Tet Lett. 1990, 45, 6485; (b) Wittmann, V.; Wong, C.-H. J.
Org. Chem. 1997, 62, 2144. The pyrophosphate exchange reaction
takes place readily in the presence of the unprotected sugar
hydroxyls. Finally, the side chain protecting groups on the peptide
are removed with TBAF, which also hydrolyzes the C-terminal methyl
ester to give the desired product 8. It should be noted that 8 is
both acid- and base-sensitive. The synthesis minimizes exposure, to
acid and base, while providing for a convergent approach that
allows independent modification of all three building blocks, the
peptide, the carbohydrate, and the lipid.
[0055] Thus, using the same general scheme described above and
further illustrated below (in which TMSE is trimethylsilylethyl,
TEOC is trimethylsilylethyloxycarbonyl and N-linker is
6-aminohexanoic acid), one can prepare a variety of compounds to
define the requirements for substrate binding. 6
[0056] 5.3. GlcNAc Transferase Assay
[0057] Initial attempts to use substrate 8 in MurG activity assay
reveals some difficulties in separating radiolabeled product from
excess labeled UDP-GlcNAc, using relatively crude separation
methods like paper chromatography or thin layer chromatography.
Hence, in certain applications, it may be preferable to adjust the
length of the lipid chain to facilitate removal of excess labeled
UDP-GlcNAc. For instance, a longer lipid chain (e.g., ca.
C.sub.15-C.sub.40) may facilitate a separation method using a
hydrophobic resin or suitable filter to take advantage of
non-specific lipid-lipid interactions. What is more, a tether to a
solid phase resin may be more preferable in a commercial embodiment
of the invention. Still another alternative comprises an affinity
group, such as biotin, an IgG binding domain, or a hapten, such as
dinitrophenol, digoxegenin and the like, which is attached to the
substrate analog to facilitate separation by affinity
chromatography using an affinity resin comprising
avidin/streptavidin or Protein A, respectively.
[0058] The evidence suggests that MurG is relatively insensitive to
the identity of the third amino acid residue in the peptide chain.
E. coli strains (e.g., BL21) make a muramyl pentapeptide substrate
with meso-diaminopimelic acid (m-DAP) rather than L-lysine. E. coli
MurG accepts these lysine analogs. Fluorescently labeled analogs
are also accepted by some strains: Weppner, W. A.; Neuhaus, F. C.
J. Biol. Chem. 1978, 253, 472; White, D. Physiology and
Biochemistry of Prokaryotes Oxford Univ. Press:New York, 1995, pp
212-223. Accordingly, the third amino acid residue makes a
convenient location for attaching substituents onto the amino
acid/peptide moiety. In a preferred embodiment of the invention, an
affinity label substituted L-Lys is used as the third amino acid
residue of the peptide chain. More preferably a biotin moiety is
linked to the free amino group of lysine via a tether comprising a
bifunctional aliphatic agent, such as 6-aminohexanoic acid,
although shorter or longer tethers can be used. Tethers of various
lengths, which are attached to certain molecules of interest, such
as biotin, chromophores, fluorophores and the like, are
commercially available.
[0059] In this manner, biotin is attached (Scheme 2)
(6-{(biotinoyl)amino}hexanoic acid succinimide ester can be
purchased from Molecular Probes, Inc.) to the .epsilon. amino group
of the lysine residue via the carboxylic acid group of the
6-aminohexanoic acid linker so that radiolabeled product can be
readily separated from other radioactive components in the reaction
mixture using an avidin-derivatized resin (Tetralink.TM. Tetrameric
Avidin Resin, Promega). The ability of MurG to recognize the
biotin-labeled substrate 9 is evaluated by counting the
radioactivity that binds to the resin after incubation of various
crude membrane preparations with 9 and .sup.14C-UDP-GlcNAc. (See,
e.g., Baker, C. A.; Poorman, R. A.; Kezdy, F. J.; Staples, D. J.;
Smith, C. W.; Elhammer, A. P. Anal. Biochem. 1996, 239, 20.) The
reaction is rapid and efficient with a bacterial culture that
overexpresses MurG but barely detectable with a culture expressing
only endogenous levels of MurG (FIG. 1; compare curves A and
E).
[0060] The murG gene can be obtained from the pUG18 plasmid
available from Prof. W. D. Donachie (Univ. of Edinburgh). The E.
coli murG gene sequence is described by Mengin-Lecreulx, D. et al.,
in Nucleic Acids Res. 1990, 18, 2810 and Ikeda, M. et al., in Ibid.
1990, 18, 4014. Gene amplification by polymerase chain reaction
using the pUG 18 plasmid as the template is performed. The pT7BlueT
PCR cloning vector, which is available from Novagen, is used for
this purpose. The DNA fragment that contains murG is cleaved from
pT7BlueT plasmid by restriction enzymes NdeI and BamHI, and the
fragment is purified by gel electrophoresis. The purified fragment
is then inserted into the NdeI/BamHI cloning site of the pET15b
expression vector, also available from Novagen.
[0061] The murG gene is subcloned from pET15b into a pET3a plasmid
(Novagen). MurG is overexpressed in the IPTG-inducible
BL21(DE3)pLysS strain (Novagen). See: Studier, F. W.; Rosenberg, A.
H.; Dunn, J. J.; Dubendorff, J. W. Meth. Enzymol. 1990, 185, 60.
Heat treating the overexpressing cell lysate prior to adding it to
the substrates prevents the reaction from proceeding (See, FIG. 1;
compare A and D). Hence, the reaction depends on the presence of
active MurG. Furthermore, both the initial reaction rate and
conversion to coupled product increases with the concentration of 9
(See, FIG. 1; compare A, B, and C).
[0062] Therefore, the synthetic substrate analog functions
efficiently in a direct assay for MurG activity despite having a
different, and dramatically shorter, lipid chain. This synthetic
substrate can be used to evaluate enzyme activity in overexpressing
cell lysates, following structural modifications to the murG gene
which produce amino acid truncations, additions, deletions,
substitutions, or other mutations. The synthetic substrate analog
can also be used to assay for enzyme activity during purification,
as well as for detailed mechanistic studies on wholly or partially
purified enzyme. Thus, a high resolution structural analysis of
MurG is now possible. In addition, by evaluating the ability of
other synthetic substrates to compete with 9 for
.sup.14C-UDP-GlcNAc, it is possible to identify simpler acceptors
for use in direct screens for MurG inhibition.
[0063] As a further illustration of the invention, the following
examples are provided.
6. EXAMPLES
[0064] The following procedures are provided making specific
reference to Scheme 2, above, and Methods 1-4, below.
[0065] 6.1. Preparation of Compound 2
[0066] Compound 1 (482 mg, 1.022 mmol, Sigma) and
4-dimethylaminopyridine (10 mg, 0.080 mmol) are premixed, dried
three times by azeotropic distillation with toluene and then
dissolved in 8 mL of tetrahydrofuran (THF). Trichloroethanol (0.23
mL, 2.405 mmol) is added to the reaction vessel followed by
1,3-dicyclohexylcarbodiimide (248 mg, 1.203 mmol). After stirring
at room temperature for 4 h, the reaction solution is filtered
through a cotton plug and rinsed with ethyl acetate (EtOAc). The
filtrate is concentrated and purified by flash chromatography (15%
EtOAc/CH.sub.2Cl.sub.2) to give 453 mg (80%) of a white powder.
R.sub.f 0.39 (15% EtOAc/CH.sub.2Cl.sub.2); .sup.1H NMR (CDCl.sub.3,
270 MHz) .delta. 7.43-7.25 (m, 10H), 7.67 (d, 3=6.0 Hz, 1H), 5.59
(s, 1H), 5.34 (d, J=3.2 Hz, 1H), 4.98 (d, J=11.9 Hz, 1H), 4.71-3.70
(m, 10H), 2.04 (s, 3H), 1.50 (d, J=7.0 Hz, 3H); Mass spec.
[M+H].sup.+, 603.5.
[0067] 6.2. Preparation of Compound 3
[0068] Compound 2 (360 mg, 0.599 mmol) is dissolved in 30 mL of
EtOAc, and 900 mg of 20% Pd-C is added. The reaction vessel is
filled with hydrogen and stirred at room temperature. After 30 min,
the catalyst is filtered off and washed with methanol. The filtrate
is concentrated to give a fully hydrogenated product, which is used
in the next reaction without further purification.
[0069] To a solution of the triol in 6 mL of DMF is added
benzylaldehyde dimethyl acetal (0.9 mL, 6 mmol) and
ptoluenesulfonic acid (11.4 mg, 0.06 mmol). The reaction mixture is
stirred at room temperature for 10 h, neutralized with saturated
NaHCO.sub.3, extracted with CH.sub.2Cl.sub.2 (3.times.20 mL), dried
over Na.sub.2SO.sub.4, filtered, concentrated, and purified by
flash chromatography (90% EtOAc/petroleum ether) to give 248 mg
(81%) of a mixture of .alpha., .beta. anomers.
[0070] 6.3. Preparation of Compound 4
[0071] Compound 3 (202 mg, 0.395 mmol) and 1H-tetrazole are
premixed and dried by azeotropic distillation with toluene, then
dissolved in 10 mL of CH.sub.2Cl.sub.2, and cooled to -30.degree.
C. To the solution is added dibenzyl N,N-diisopropylphosphamide
(0.266 mL, 0.791 mmol). The mixture is stirred at room temperature
for 1 h and cooled to 40.degree. C., m-CPBA (560 mg, 2 mmol) is
added, and the reaction is stirred for 30 min at 0.degree. C. and
then 30 min at room temperature. The mixture is diluted with
CH.sub.2Cl.sub.2, washed with 10% aqueous Na.sub.2SO.sub.3,
saturated NaHCO.sub.3, and water; then dried over Na.sub.2SO.sub.4,
filtered, concentrated, and purified by flash chromatography (65%
EtOAc/petroleum ether) to give 200 mg (70%) of white solid. R.sub.f
0.24 (70% EtOAc/petroleum ether); .sup.1H NMR (CDCl.sub.3, 270 MHz)
.delta. 7.44-7.33 (m, 15H), 7.20 (d, J=6.0 Hz, 1H), 6.10 (m, 1H),
5.56 (s, 1H), 5.05 (m, 6H), 4.61 (q, J=7.0 Hz, 2H), 4.10-3.61 (m,
6H), 1.86 (s, 3H), 1.48 (d, J=7.0 Hz, 3H).
[0072] 6.4. Preparation of Compound 5
[0073] Zinc dust is added to a solution of compound 4 (58 mg,
0.0752 mmol) in 5 mL of 90% AcOH/H.sub.2O. The mixture is stirred
vigorously at room temperature. After 1 h, the catalyst is filtered
off, the filtrate is concentrated and purified by flash
chromatography (10% MeOH/CHCl.sub.3, 0.1% AcOH) to give 44 mg (91%)
of product. R.sub.f 0.19 (5% MeOH/CHCl.sub.3, 0.1% AcOH); .sup.1H
NMR (CD.sub.3OD, 500 MHz) .delta. 7.44-7.25 (m, 15H), 6.11 (m, 1H),
5.55 (s, 1H), 5.02 (m, 4H), 4.33 (q, J=7.0 Hz, 1H), 3.96 (m, 1H),
3.77 (m, 1H), 3.73-3.66 (m, 4H), 1.94 (s, 3H), 1.32 (d, J=7.0 Hz,
3H).
[0074] 6.5. Preparation of Compound 6
[0075] Compound 5 (45 mg, 0.0704 mmol) and
NH.sub.2-L-Ala-.gamma.-D-Glu(O--
TMSE)-L-Lys(N-TEOC)-D-Ala-D-Ala-OCH.sub.3 (35 mg, 0.0469 mmol) are
premixed and dried by azeotropic distillation with toluene three
times, then dissolved in 0.9 mL of DMF, then cooled to 0.degree. C.
Diisopropylethylamine (41 .mu.L, 0.235 mmol) is added to reaction
vessel followed by HOBt (12.7 mg, 0.0938 mmol) and pyBOP (49 mg,
0.0938 mmol). After stirring for 30 min at room temperature, the
solution is diluted with 10 mL of EtOAc, washed with 0.01 N aqueous
HCl, and water. The solution is then concentrated and purified by
flash chromatography (5% MeOH/CHCl.sub.3) to give 59 mg (92%) of
compound 6. R.sub.f 0.16 (5% MeOH/CHCl.sub.3); .sup.1H NMR
(CD.sub.3OD, 500 MHz) .delta. 7.50-7.30 (m, 15H), 5.87 (m, 1H),
5.63 (s, 1H), 5.13 (m, 4H), 4.41 (m, 1H), 4.40 (m, 1H), 4.36 (m,
1H), 4.35 (m, 1H), 4.30 (m, 1H), 4.21 (m, 1H), 4.19 (m, 2H), 4.14
(m, 2H), 4.13 (m, 1H), 4.05 (m, 1H), 3.84 (m, 1H), 3.79 (m, 2H),
3.76 (m, 1H), 3.66 (s, 3H), 3.09 (t, J=8.8 Hz, 2H), 2.28 (t, J=8.8
Hz, 2H), 2.18 (m, 1H), 1.91 (m, 1H), 1.86 (s, 3H), 1.77 (m, 1H),
1.67 (m, 1H), 1.51 (m, 2H), 1.43-1.35 (m, 18H), 1.01-0.97 (m, 4H),
0.05-0.02 (s, s, 18H); Mass spec [M+H].sup.+1394.
[0076] 6.6. Preparation of Compound 7
[0077] Compound 6 (15 mg, 0.011 mmol) is dissolved in 1 mL of MeOH
and 20 mg of 20% Pd-C is added. The reaction vessel is filled with
hydrogen and stirred at room temperature. A drop of
diisopropylethylamine is added after 30 min, then the solution is
diluted in 5 mL of MeOH and stirred for 20 min. The mixture is
filtered, concentrated to give the hydrogenated, debenzylated
product (7a), which is used in the next reaction without further
purification. R.sub.f 0.28 (CHCl.sub.3:MeOH:H.sub.2O=3:2:0.5).
[0078] Citronellol phosphate (diisopropylethylammonium, 18 mg,
0.053 mmol) is dried three times by azeotropic distillation with
toluene, then dissolved in 1 mL of CH.sub.2Cl.sub.2.
Diisopropylethylamine (18.5 .mu.L, 0.106 mmol) is added. The
solution is cooled to -20.degree. C., and
diphenylphosphorochloridate (11.5 .mu.L, 0.080 mmol) is added. The
reaction vessel is allowed to warm up to room temperature and
stirred for 1 h at room temperature. After the addition of methanol
(0.1 mL), the reaction is stirred for a further 1 h at room
temperature, then the solvents are evaporated, and the residue is
dried twice by azeotropic distillation with toluene and dissolved
in 0.2 mL of DMF.
[0079] Compound 7a from above is dried three times by azeotropic
distillation with toluene and dissolved in 0.1 mL of DMF.
Diisopropylethylamine (3.9 .mu.L, 0.022 mmol) is added. 0.1 mL of
the citronellol diphenylpyrophosphate solution is transferred to
the solution containing compound 7a. The reaction mixture is
stirred for 48 h at room temperature, then loaded directly to a C18
reverse phase column (8 mm.times.80 mm, particle size 40 .mu.m,
pore size 60 .ANG., from J. T. Baker) and eluted with
CH.sub.3CN/H.sub.2O (0, 5%, 10%, 15%, 20%, 25%, 30%, 35% of 10 mL
each) with 0.1% triethylamine. The fractions containing the pure
compound are combined and concentrated to give 4.6 mg (28%) of
white powder. R.sub.f 0.36 (CHCl.sub.3:MeOH:H.sub.2O=3:2:0.5);
.sup.1H NMR (DMSO, 500 MHz) .delta. 8.36 (d, 3=7.2 Hz, 1H), 8.21
(d, 3=8.0 Hz, 1H), 8.19 (d, J=8.2 Hz, 1H), 8.10 (d, J=6.0 Hz, 1H),
7.32 (d, =7.5 Hz, 1H), 6.95 (t, J=5.0 Hz, 1H), 5.26 (d, J=6.0 Hz,
1H), 5.07 (t, J=7.0 Hz, 1H), 4.30 (m, 1H), 4.27 (m, 1H), 4.23 (m,
1H), 4.13 (m, 1H), 4.12 (m, 2H), 3.87 (m, 1H), 3.77 (m, 2H), 3.62
(m, 1H), 3.60 (s, 3H), 3.51 (m, 1H), 3.33 (m, 1H), 2.91 (m, 2H),
2.17 (m, 2H), 1.94 (m, 2H), 1.91 (m, 1H), 1.80 (s, 3H), 1.62 (s,
3H), 1.58 (s, 3H), 1.51 (m, 3H), 1.50 (m, 1H), 1.49 (m, 1H), 1.35
(m, 2H), 1.29 (d, 3=7.2 Hz, 3H), 1.27 (m, 2H), 1.25 (d, J=6.8 Hz,
3H), 1.24 (d, J=5.5 Hz, 3H), 1.23 (m, 2H), 1.19 (d, J=7.4 Hz, 3H),
1.11 (m, 1H), 0.84 (d, J=6.5 Hz, 3H), 0.02-0.01 (s, s, 18H); Mass
spec. [M+H].sup.+ 1321.
[0080] 6.7. Preparation of Compound 8
[0081] To a solution of compound 7 (5 mg, 0.0033 mmol) in 50 .mu.L
of DMF is added tetrabutylammonium fluoride (1 M in THF, 0.3 mL).
The reaction mixture is stirred for 24 h at room temperature, then
loaded directly to a C18 reverse phase column (8 mm.times.80 mm,
particle size 40 .mu.m, pore size 60 .ANG., from J. T. Baker) and
eluted with CH.sub.3CN/0.1% NH.sub.4HCO.sub.3 aqueous solution (0,
5%, 10%, 15%, 20%, 25%, 30% of 10 mL each). The fractions
containing the pure compound are combined, concentrated, and
lyophilized to remove salts. A white powder (2 mg, 57%) is
obtained. R.sub.f 0.18 (CHCl.sub.3:MeOH:H.sub.2O=3:3:1); .sup.1H
NMR (CD.sub.3OD, 500 MHz) .delta. 5.58 (m, 1H), 5.11 (t, J=6.5 Hz,
1H), 4.50-3.56 (m, 12H), 2.94 (m, 2H), 2.34 (m, 2H), 2.10 (s, 3H),
2.00 (m, 1H), 1.98 (m, 2H), 1.92 (m, 1H), 1.74 (m, 2H), 1.67 (s,
3H), 1.62 (m, 1H), 1.60 (s, 3H), 1.50-1.39 (m, 12H), 1.23 (m, 2H),
0.93 (d, J=6.5 Hz, 3H); Mass spec. [M+H].sup.+ 1062.
[0082] 6.8. Preparation of Compound 9
[0083] To a solution of compound 8 (2 mg, 0.0019 mmol) in 0.1 mL of
H.sub.2O/dioxane(1:1) is added NaHCO.sub.3 (3.2 mg, 0.038 mmol),
followed by 6-((biotinoyl)amino)hexanoic acid succinimide ester (2
mg, 0.0044 mmol). The reaction mixture is stirred for 2 h at room
temperature, then loaded directly to a C18 reverse phase column (8
mm.times.80 mm, particle size 40 .mu.m, pore size 60 .ANG., from J.
T. Baker) and eluted with CH.sub.3CN/0.1% NH.sub.4HCO.sub.3 aqueous
solution (0, 5%, 10%, 15%, 20%, 25%, 30% of 10 mL each). The
fractions containing the pure compound are combined, concentrated,
and lyophilized to remove salts. A white powder (2 mg, 76%) is
obtained. R.sub.f 0.40 (CHCl.sub.3:MeOH:H.sub.2O=3:3:1); .sup.1H
NMR (CD.sub.3OD, 500 MHz) .delta. 5.52 (d, J=4.5 Hz, 1H), 5.12 (t,
J=7.0 Hz, 1H), 4.50 (m, 1H), 4.39-4.19 (m, 8H), 4.00-3.72 (m, 4H),
3.51 (m, 1H), 3.22 (m, 1H), 3.18 (m, 2H), 2.95 (dd, J=12.5, 5.0 Hz,
1H), 2.71 (d, J=12.5 Hz, 1H), 2.27 (m, 2H), 2.02 (s, 3H), 2.01 (m,
2H), 1.85 (m, 2H), 1.67 (m, 2H), 1.67 (s, 3H), 1.62 (m, 1H), 1.61
(s, 3H), 1.53 (m, 2H), 1.45-1.37 (m, 12H), 1.38 (m, 1H), 1.17 (m,
1H), 0.94 (d, J=6.8 Hz, 3H); Mass spec. [M+H].sup.+ 1402.
[0084] 6.9. Preparation of Compound 10 (Method 1, Below)
[0085] (R)-(+)-.beta.-citronellol (330 mg, 2.111 mmol) is dried
three times by azeotropic distillation with toluene, then dissolved
in 21 mL of dry hexane. In another dry flask, phosphorus
oxychloride (0.98 mL, 10.56 mmol) and triethylamine (1.47 mL, 10.56
mmol) are dissolved in 10 mL of dry hexane and stirred at room
temperature. The citronellol solution is then added slowly (over 1
h) to the phosphorus oxychloride solution after which stirring is
continued for 30 min. A mixture of 70 mL
acetone/water/triethylamine (88:10:2) is added to the reaction,
which is allowed to stir for 18 h at room temperature to convert
citronellol phosphate dichloride to citronellol phosphate. The
solvent is evaporated in vacuo to give an aqueous residue, which is
loaded to a C18 reverse phase column (50 mm.times.12 cm, particle
size 40 .mu.m, pore size 60 .ANG., from J. T. Baker) and eluted
with CH.sub.3CN/H.sub.2O (0, 10%, 20%, 30%, 40%, 50%, 60% of 100 mL
each). The fractions containing the pure compound are combined and
concentrated to give 566 mg (62%) of oily residue. R.sub.f 0.42
(CHCl.sub.3:MeOH:H.sub.2O=3:2:0.5); .sup.1H NMR (CD.sub.3OD, 500
MHz) .delta.5.09 (t, J=5.0 Hz, 1H), 3.90 (m, 2H), 1.99 (m, 2H),
1.67 (m, 1H), 1.65 (s, 3H), 1.62 (m, 1H), 1.59 (s, 3H), 1.41 (m,
1H), 1.34 (m, 1H), 1.16 (m, 1H), 0.91 (d, J=6.5 Hz, 3H); .sup.13C
NMR (CD.sub.3OD, 500 MHz) .delta. 132.08, 125.96, 65.11, 39.00,
38.94, 30.45, 26.61, 26.10, 19.93, 17.92. 7
[0086] (a) 5 eq. POCl3, TEA, hexane, rt, 1 hr; then add
acetone/H2O/TEA (85:10:5), 10 hrs, 70%. 8
[0087] (a) 50% TFA/CH.sub.2Cl.sub.2, rt, 20 mins, 100%; (b) 1.2 eq.
2-(Trimethylsilyl)ethyl p-nitrophenyl carbonate, DIEA, DMF,
50.degree. C., 2 hrs, 95%. 9
[0088] (a) 1.2 eq. 9-Fluorenylmethyl chloroformate, 3 eq.
NaHCO.sub.3, H.sub.2O/Dioxane(1:1), ft, 1 hr. 93%; (b) 2 eq.
trimethylsilyl ethanol, DCC/DMAP, EtOAc, rt 2 hrs, 82%; (c) H2/Pd,
MeOH, rt, 10 mins, 90%. 10
[0089] (a) 55% piperidine/NMP, rt, 30 mins; (b) 4 eq.
HO-D-Ala-Fmoc, HOBT/HBTU, DIEA, NMP, rt, 2 hrs; identical coupling
and deprotection conditions for HOL-L-Lys(N-TEOC)-Fmoc,
HO-D-.gamma.-Glu(O-TMSE), and HO-L-Ala-Fmoc except for variations
in the amount of amino acid used; (c) 1% TFA/CH.sub.2Cl.sub.2, rt,
5.times.2 mins; (d) KHCO.sub.3, 50 eq. CH.sub.3l, DMF, rt, 2 hrs
overall yield 15%.
[0090] Sasrin resin-OOC-D-Ala-Fmoc, an acid-sensitive resin, is
available from BACHEM Bioscience Inc.
[0091] 6.10. Preparation of Compound 11 (Method 2)
[0092] To a solution of OH-L-Lys(N-BOC)-NHFmoc (607 mg, 1.295 mmol)
in 10 mL of CH.sub.2Cl.sub.2 is added 10 mL of trifluoroacetic
acid. The mixture is stirred for 20 min at room temperature, then
concentrated and lyophilized. The residue is dissolved in 10 mL of
DMF, then diisopropylethylamine (1.13 mL, 6.475 mmol) is added.
2-(Trimethylsilyl)ethyl p-nitrophenyl carbonate (440 mg, 1.554
mmol) is dissolved in 3 mL of DMF and transferred into the L-Lys
solution. The mixture is stirred for 2 h at room temperature. The
DMF solvent is evaporated in vacuo; the residue is purified by
flash chromatography (EtOAc, followed by 10% MeOH/CHCl.sub.3 with
0.1% AcOH) to give 635 mg (95%) of a white solid. R.sub.f 0.25 (10%
MeOH/CHCl.sub.3).
[0093] 6.11. Preparation of Compound 12 (Method 3)
[0094] To a solution of D-Glu(benzyl) (1.046 g, 4.41 mmol) in 40 mL
of water/dioxane (1:1) is added a solution of NaHCO.sub.3 (1.1 g,
13.2 mmol) in 10 mL of water. The mixture is stirred for 20 min.
Then, 9-Fluoenylmethyl chloroformate (1.37 g, 5.29 mmol) is
dissolved in 10 mL of dioxane and added slowly (over 1 h) into the
D-Glu solution after which stirring is continued for 10 min. The
mixture is loaded directly to a silica gel column and eluted by 5%
MeOH/CHCl.sub.3 with 0.1% AcOH. Fractions containing product are
combined, concentrated, and purified again by flash chromatography
(EtOAc, followed by 5% MeOH/CHCl.sub.3 with 0.1% AcOH) to give 1.88
g (93%) of a white powder. R.sub.f 0.27 (5% MeOH/CHCl.sub.3 with
0.1% AcOH).
[0095] Fmoc-D-Glu(benzyl)-OH (350 mg, 0.762 mmol) and
4-dimethylaminopyridine (9.3 mg, 0.0762 mmol) are premixed and
dried three times by azeotropic distillation with toluene, and
dissolved in 8 mL of EtOAc. Trimethylsilyl ethanol (0.328 mL, 2.287
mmol) is added to the reaction vessel followed by
1,3-dicyclohexylcarbodiimide (314 mg, 1.525 mmol). After stirring
the mixture for 2 h at room temperature, the reaction solution is
filtered and washed with EtOAc. The filtration is concentrated and
purified by flash chromatography (15% EtOAc/petroleum ether) to
give 350 mg (82%) of a white powder. R.sub.f 0.33 (15%
EtOAc/petroleum ether).
[0096] Fmoc-D-Glu(benzyl) 2-(trimethylsilyl)ethyl ester (270 mg,
0.483 mmol) is dissolved in 11 mL of methanol and 500 mg of 20%
Pd-C is added. The reaction vessel is filled with hydrogen and
stirred at room temperature. After 10 min, the mixture is filtered,
concentrated, and purified by flash chromatography (10%
MeOH/CHCl.sub.3) to give 203 mg (90%) of a white powder. R.sub.f
0.43 (10% MeOH/CHCl.sub.3).
[0097] 6.12. Preparation of Compound 13 (Method 4)
[0098] Sasrin resin-OOC-D-Ala-NHFmoc (800 mg, 0.56 mmol) is put in
reaction vessel and washed successively by the following solvents
(20 mL each): CH.sub.2Cl.sub.2 (2.times.3 min), N-methylpyrrolidone
(NMP, 2.times.3 min), 20% piperidine/NMP (30 min), NMP (2.times.3
min), 50% dioxane/water (2.times.5 min), NMP (3.times.5 min),
CH.sub.2Cl.sub.2 (3.times.3 min), NMP (1.times.3 min).
OH-D-Ala-NHFmoc (701 mg, 2.24 mmol), diisopropylethylamine (0.59
mL, 3.36 mmol), HOBt/HBTU (0.45 M in DMF, 2.5 mL), and 10 mL of NMP
are added to the vessel and mixed thoroughly. The reaction vessel
is shaken for 2 h at room temperature, then washed successively
with the following solvents (20 mL each): NMP (5.times.8 min),
i-PrOH (5.times.8 min), CH.sub.2Cl.sub.2 (4.times.3 min) and NMP
(2.times.3 min).
[0099] The same procedure is used for the other 3 amino acids
except that the Fmoc group is not cleaved for the last amino acid
L-Ala-Fmoc.
[0100] After all of the amino acids are coupled, the pentapeptide
is cleaved off of the resin by ishing with 1% TFA/CH.sub.2Cl.sub.2
(5.times.2 min, 15 mL each) with slight agitation. The cleavage
solution is transferred via cannula into a vessel containing 2 mL
of pyridine and 20 mL of methanol. The filtration is concentrated
and purified three times by flash chromatography (5%
MeOH/CHCl.sub.3 with 1% AcOH) to give 300 mg (56%) of product.
R.sub.f 0.34 (10% MeOH/CHCl.sub.3).
[0101] KHCO.sub.3 (28.4 mg, 0.284 mmol) is ground to a fine powder
and mixed with
Fmoc-L-Ala-D-.gamma.-Glu(O-TMSE)-L-Lys(N-TEOC)-D-Ala-D-Ala-OH
(135.4 mg, 0.142 mmol). The mixture is dissolved in 2 mL of DMF,
CH.sub.3I (0.44 mL, 7.1 mmol) is added. The mixture is stirred for
2 h at room temperature and purified by flash chromatography (90%
EtOAc/petroleum ether) to give 47 mg (34%) of a white powder.
R.sub.f 0.40 (100% EtOAc);
[0102] .sup.1H NMR (DMSO-d.sub.6, 500 MHz) .delta. 8.24 (d, J=7.4
Hz, 1H), 8.18 (d, J=7.2 Hz, 1H), 8.17 (d, J=8.4 Hz, 1H), 8.01 (d,
J=7.1 Hz, 1H), 7.89 (d, J=7.4 Hz, 2H), 7.72 (dd, J=7.4, 7.4 Hz,
2H), 7.47 (d, J=7.8 Hz, 1H), 7.41 (dd, J=7.4, 7.4 Hz, 2H), 7.32
(dd, J=7.4, 7.4 Hz, 2H),6.94 (t, J=5.2 Hz, 1H), 4.29-4.12 (m, 8H),
4.11 (t, J=8.8 Hz, 2H), 4.00 (t, J=8.2 Hz, 2H), 3.59 (s, 3H), 2.91
(m, 2H), 2.17 (m, 2H), 1.92 (m, 1H), 1.79 (m, 1H), 1.56 (m, 1H),
1.48 (m, 1H), 1.35 (m, 2H), 1.28 (d, J=7.3 Hz, 3H), 1.23 (m, 5H),
1.19 (d, J=7.1 Hz, 3H), 0.93 (t, J=8.8 Hz, 2H), 0.89 (t, J=8.2 Hz,
2H), 0.02-0.01 (s, s, 18H); Mass spec [M+Na].sup.+ 992.
[0103]
Fmoc-L-Ala-D-.gamma.-Glu(O-TMSE)-L-Lys(N-TEOC)-D-Ala-D-Ala-OCH.sub.-
3 (100 mg, 0.104 mmol) is dissolved in 2 mL of 20% piperidine/DMF
and stirred for 30 min at room temperature. Solvent is evaporated
in vacuo, and the residue is purified by flash chromatography
(EtOAc, followed by 10% MeOH/CHCl.sub.3) to give 60 mg (78%) of the
desired product. R.sub.f 0.23 (10% MeOH/CHCl.sub.3).
[0104] 6.13. MurG Activity Assay Procedure
[0105] 6.13.1. Protein Preparation
[0106] The wild type murG gene is cloned into the pET3a plasmid and
transformed into the high-stringency expression host BL21(DE3)pLysS
(Novagen). The lysogenized cells are grown at 37.degree. C. in
2.times.YT media supplemented with 20 .mu.g/mL ampicillin and 34
.mu.g/mL chloramphenicol to an O.D..sub.600nm=0.7; overexpression
of the murG protein is achieved by induction for 1.25 h with 1 mM
IPTG. SDS/PAGE analysis shows production of a single new band
migrating at .about.38,000 MW. Several hundred aliquots of the
induced cell culture are prepared by centrifuging 1.0 mL samples at
5000 rpm for 10 min at 4.degree. C. The supernatant is removed, and
the pellet frozen at -20.degree. C. Frozen pellet stocks of
non-transformed BL21(DE3)pLysS culture are also prepared as a
negative control. Protein quantitation using a precipitated Lowry
assay (Sigma) with a BSA reference on the entire pellet shows total
protein concentration to be 11 and 17 .mu.g/pellet for the
BL21(DE3)pLysS and overexpressed cell cultures, respectively.
Immediately prior to reactions, pellets are thawed on ice and
resuspended in 100 .mu.L 1.times.Rxn buffer.
[0107] 6.13.2. Reaction Conditions
[0108] Biotinylated lipid substrate is aliquoted in autoclaved,
sterile, deionized H.sub.2O into 0.5 mL autoclaved Eppendorf tubes
containing Rxn buffer (1.times.:100 mM Tris-Cl pH 7.6, 1 mM
MgCl.sub.2). The ethanol is removed from an ethanol:water solution
of .sup.14C-UDP-GlcNAc (NEN Dupont) using an unheated SpeedVAC and
then added to the substrate mixture (1.1.times.10.sup.5 DPM; rxn
concentration of 9.4 .mu.M). Finally, 5 .mu.L iced crude cell
lysate containing 0.5-1.0 .mu.g protein are added to a total volume
of 20 .mu.L. All reactions are performed at 24.degree. C. Reactions
are quenched by the addition of 10 .mu.L 1% (w/v) SDS.
[0109] 6.13.3. Transferase Activity Determination
[0110] A molar excess of biotin-binding TetraLink Tetrameric Avidin
Resin (Promega) and deionized H.sub.2O are added to each quenched
reaction tube to a final volume of 350 .mu.L. The suspension is
incubated at room temperature for 10 min with frequent vortexing
and transferred to an empty 1.5 mL microcolumn tube with a 30 .mu.m
frit (Bio-Rad). The resin is washed (5.times.0.5 mL) using
deionized H.sub.2O. Washed resin is transferred using 1.0 mL
sterile, deionized H.sub.2O to 10 mL Ecolite (ICN) and vortexed.
Samples are counted immediately.
[0111] The results of various experiments are graphically depicted
in FIG. 1.
[0112] 6.14. Purification of Wild Type E. coli MurG
[0113] BL21(DE3)pLysS cells (Novagen) overexpressing wild type E.
coli MurG from a pET3a vector (Novagen) are grown in 8 L 2.times.YT
medium supplemented with 100 .mu.g/mL ampicillin and 34 .mu.g/mL
chloramphenicol. When the OD.sub.600nm reached 0.6, IPTG is added
to a final concentration of 1 mM. The induced cell culture is grown
for another 3.5 hours and then the cells are spun down in 500 mL
batches at 5000 rpm (Beckman RC5B centrifuge) for 10 minutes and
the supernatant is decanted. Each cell pellet is resuspended in 5
mL 25 mM MES (pH 6.0), 4 mM DTT and 3% Triton X-100, and the
suspensions are combined for a total of 80 mL, and then frozen at
-70.degree. C. The suspension is thawed at 4.degree. C., and to it
is added MgCl.sub.2 to a final concentration of 5 mM and DNAse to a
final concentration of 20 .mu.g/mL. After shaking for 1 hour at
4.degree. C., the debris is spun down at 15,000 rpm for 35 minutes.
The supernatant is decanted, diluted 6-fold with Buffer A (25 mM
NES pH 6.0, 4 mM DTT), and applied to a SP-Sepharose column
(Pharmacia Biotech) equilibrated with Buffer A. After washing for
40 minutes with 40% Buffer B (20 mM Tris pH 8.0, 1M NaCl, 4 mM
DTT)/Buffer A, the bound enzyme is eluted using a linear salt
gradient starting with 40% Buffer B and ending with 100% Buffer B
over 120 minutes. The eluted enzyme is concentrated to 7 mg/mL and
applied to a Superdex 200 HR 10/30 column (Pharmacia Biotech) at a
flow rate of 0.5 mL/min of TBSE buffer (100 mM NaCl, 20 mM Tris pH
8.0, 10 mM EDTA and 4 mM DTI). The protein eluted as a single,
symmetric peak at an estimated molecular weight of 72 kD. The
purity of the enzyme is estimated to be greater than 98% from a
Coomassie Blue-stained SDS-polyacrylamide gel. The yield of
purified enzyme is approximately 1.3 mg/L of bacterial culture. The
purified enzyme is stored at 4.degree. C., and is stable for at
least one month.
[0114] The following examples are best related to FIGS. 2-8 of the
specification.
[0115] 6.15. Initial Rate Assays With Purified, Soluble Enzyme
[0116] The following solutions are prepared prior to the assays: 1)
177.3 .mu.M [.sup.14C]-UDP-GlcNAc in H.sub.2O (0.05 mCi/mL); 2) 1.5
mM UDP-GlcNAc in H.sub.2O; 3) biotinylated Lipid I analog (1b) at
0.5 .mu.g/.mu.L; 4) 10.times. reaction buffer containing 50 mM
HEPES (pH 7.9) and 5 mM MgCl.sub.2. The enzyme stock is prepared by
diluting the purified enzyme with TBSE to a final concentration of
0.04 .mu.g/.mu.L in a 0.5 mL tube and storing at 4.degree. C. for
two days prior to running the assays.
[0117] Thirty reactions are prepared by individually mixing 2 mL of
10.times. reaction buffer with an appropriate amount of
biotinylated Lipid I analog (1b), radioactive UDP-GlcNAc,
nonradioactive UDP-GlcNAc, and H.sub.2O to a final volume of 18
.mu.l. The final concentrations for the Lipid I analog (1b) are 7
.mu.M, 10 .mu.M, 15 .mu.M, 30 .mu.M, 100 .mu.M, and for UDP-GlcNAc
11 .mu.M, 15 .mu.M, 20 .mu.M, 40 .mu.M, 100 .mu.M, 200 .mu.M.
Reactions are initiated by adding 2 .mu.L of the enzyme stock and
are run for 4 minutes at 24.degree. C. Reactions are stopped by
adding 10 .mu.l of 1% (w/v) SDS.
[0118] Radiolabeled product is separated from radiolabeled starting
material by incubating a 3-fold molar excess of biotin-binding
TetraLink Tetrameric Avidin Resin (Promega) to each tube. Deionized
H.sub.2O is added to each tube to a final volume of approximately
250 .mu.L and the suspension is transferred to a 1.0 .mu.m pore
size 96-well filter plate fitted to a vacuum-line fitted
MultiScreen Assay System (Millipore). The resin is washed 15 times
with 0.2 mL deionized H.sub.2O. Washed resin is transferred to a
scintillation vial containing 10 mL Ecolite and vortexed. Samples
are counted immediately on a Beckman LS5000 scintillation
counter.
[0119] 6.16. IC.sub.50 Measurements
[0120] The IC.sub.50 assays are performed the same way as the
initial rate assays except that the Lipid I analog (1b) and
UDP-GlcNAc concentrations are fixed at 18 .mu.M and 34.3 .mu.M,
respectively. Each set of assays is carried out at five or six
different concentrations of one of the inhibitory compounds. The
IC.sub.50 is taken as the concentration at which the reaction rate
(counts incorporated in a given time) decreased by 50%.
[0121] 6.17. General Methods
[0122] All amino acids are purchased from BAChem. Unless otherwise
stated, all chemicals are purchased from Aldrich or Sigma and used
without further purification. Dichloromethane, toluene, benzene,
pyridine, diisopropylethylamine and triethylamine are distilled
from calcium hydride under dry argon. Diethyl ether and
tetrahydrofuran are distilled from potassium benzophenone under dry
argon. DMF, ethyl acetate and methanol are dried over activated
molecular sieves.
[0123] Analytical thin layer chromatography (TLC) is performed on
silica gel 60 F.sub.254 plates (0.25 mm thickness) precoated with a
fluorescent indicator. The developed plates are examined under
short wave UV light and stained with anisaldehyde or Mo (Vaughn)
stain. Flash chromatography is performed using silica gel 60
(230-400 mesh) from EM Science.
[0124] NMR spectra are recorded on a JEOL GSX-270 NMR spectrometer
or a Varian Inova 500/VNMR spectrometer. Chemical shifts (8) are
reported in parts per million (ppm) downfield from
tetramethylsilane. Coupling constants (J) are reported in Hertz
(Hz). Multiplicities are abbreviated as follows: singlet (s),
doublet (d), triplet (t), quartet (q), multiplet (m), double of
doublets (dd), apparent triplet (apt), broad singlet (bs), pentet
(p), and octet (o).
[0125] High-resolution mass spectra (FAB) are obtained by Dr. Ron
New at the University of California at Riverside Department of
Chemistry Mass Spectrometry Facility. Low-resolution mass spectra
(ESI) are obtained by Dr. Dorothy Little at the Princeton
University Department of Chemistry.
[0126] 6.17.1. Compound 3
[0127] To a solution of compound 2 (482 mg, 1.02 mmol; see, FIG. 7)
and 4-dimethylaminopyridine (10 mg, 0.08 mmol) in 8 mL of THF is
added trichloroethanol (0.23 mL, 2.40 mmol) followed by
1,3-dicyclohexylcarbodi- imide (248 mg, 1.20 mmol). After stirring
at room temperature for 4 hours, the reaction solution is filtered
through cotton plug and the precipitate is rinsed with EtOAc. The
filtrate is concentrated and purified by flash chromatography (15%
EtOAc/CH.sub.2Cl.sub.2) to give 453 mg (80%) of 3 as a white
powder. R.sub.f 0.39 (15% EtOAc/CH.sub.2Cl.sub.2); .sup.1H NMR
(CDCl.sub.3, 500 MHz) .delta. 7.43-7.25 (m, 10H), 7.07 (d, J=6.0
Hz, 1H), 5.59 (s, 1H), 5.34 (d, J=3.2 Hz, 1H), 4.98 (d, J=11.9 Hz,
1H), 4.68 (d, J=12.0 Hz, 1H), 4.66 (q, J=7.0 Hz, 1H), 4.60 (d,
J=11.9 Hz, 1 H), 4.51 (d, J=12.0 Hz, 1H), 4.21 (dd, J=10.5, 4.8 Hz,
1H), 4.00 (m, 1H), 3.85 (m, 2H), 3.75 (m, 2H), 2.04 (s, 3H), 1.50
(d, J=7.0 Hz, 3H); .sup.13C NMR (CDCl.sub.3, 500 MHz) .delta.
173.8, 170.9, 137.5, 137.4, 129.3, 128.6, 128.5, 128.1, 128.0,
126.1, 101.6, 97.5, 94.6, 83.4, 75.2, 75.1, 74.3, 70.5, 69.2, 63.1,
54.2, 23.4, 18.9; HRMS(FAB) calcd for
C.sub.27H.sub.31NO.sub.8Cl.sub.3 [M+H.sup.+]: 602.1115, found:
602.1130.
[0128] 6.17.2. Compound 4
[0129] To a solution of compound 3 (360 mg, 0.60 mmol) in 30 mL of
EtOAc is added 500 mg of 20% Pd-C. The reaction vessel is filled
with hydrogen. After stirring at room temperature for 30 minutes,
the suspension is filtered and the catalyst is rinsed with
methanol. The filtrate is concentrated to give a clear oil which is
used in the next reaction without further purification.
[0130] To a solution of this clear oil in 6 mL of DMF is added
benzylaldehyde dimethyl acetal (0.9 mL, 6.0 mmol) followed by
ptoluenesulfonic acid (11.4 mg, 0.06 mmol). The reaction is stirred
at room temperature for 10 hours and neutralized with saturated
NaHCO.sub.3. Then the mixture is extracted with CH.sub.2Cl.sub.2
(3.times.20 mL). The CH.sub.2Cl.sub.2 layers are combined, dried
over anhydrous sodium sulfate, filtered, concentrated, and purified
by flash chromatography (90% EtOAc/petroleum ether) to give 248 mg
(81%) of 4 as a mixture of .alpha., .beta. anomers
(.alpha.:.beta.=4:1). R (.alpha. anomer) 0.33, R.sub.f (.beta.
anomer) 0.28 (90% EtOAc/petroleum ether); a anomer .sup.1H NMR
(CDCl.sub.3, 270 MHz) .delta. 7.50-7.35 (m, 5H), 5.66 (bs, 1H),
5.58 (s, 1H), 5.02 (d, J=12.0 Hz, 1H), 4.95 (m, 1H), 4.67 (m, 1H),
4.58 (d, J=12.0 Hz, 1H), 4.27 (dd, J=10.0, 5.0 Hz, 1H), 4.05 (m,
1H), 2.06 (s, 3H), 1.52 (d, 3=7.0 Hz, 3H); .sup.13C NMR
(CDCl.sub.3, 270 MHz) .delta. 174.2, 171.9, 137.6, 129.2, 128.5,
126.2, 101.5, 94.7, 91.4, 83.5, 75.5, 75.0, 74.6, 69.2, 62.9, 54.9,
23.4, 18.9; HRMS(FAB) calcd for C.sub.20H.sub.25NO.sub.8Cl.sub.3
[M+H.sup.+]; 512.0646, found: 512.0653.
[0131] 6.17.3. Compound 5
[0132] Compound 4 (202 mg, 0.40 mmol) and 1H-tetrazole are premixed
and co-evaporated with toluene and dissolved in 10 mL of
CH.sub.2Cl.sub.2. The reaction solution is cooled to -30.degree. C.
and dibenzyl N,N-diisopropylphosphamide (0.27 mL, 0.79 mmol) is
added. The reaction is warmed up to room temperature in 30 minutes
and stirred for another hour. Then the reaction is cooled to
-40.degree. C. and m-CPBA (560 mg, 2 mmol) is added. After stirring
for 30 minutes at 0.degree. C. and another 30 minutes at room
temperature, the reaction is diluted with 20 mL of
CH.sub.2Cl.sub.2, extracted with 10% aqueous Na.sub.2SO.sub.3
(2.times.20 mL), saturated NaHCO.sub.3 (2.times.20 mL), and water
(2.times.20 mL). The CH.sub.2Cl.sub.2 layer is dried over anhydrous
sodium sulfate, filtered, concentrated, and purified by flash
chromatography (65% EtOAc/petroleum ether) to give 200 mg (70%) of
5 as a white solid. R.sub.f 0.24 (70% EtOAc/petroleum ether);
.sup.1H NMR (CDCl.sub.3, 500 MHz) .delta. 7.44-7.33 (m, 15H), 7.20
(d, J=6.0 Hz, 1H), 6.10 (m, 1H), 5.56 (s, 1H), 5.07 (m, 4H), 5.02
(d, J=12.0 Hz, 1H), 4.64 (q, J=7.0 Hz, 2H), 4.59 (d, J=12.0 Hz,
1H), 4.09 (m, 1H), 4.03 (m, 1H), 3.95 (m, 1H), 3.83-3.68 (m, 3H),
1.86 (s, 3H), 1.48 (d, J=7.0 Hz, 3H); .sup.13C NMR (CDCl.sub.3, 500
MHz) .delta. 173.8, 171.2, 137.1, 129.4, 128.8, 128.5, 128.2,
128.0, 126.1, 101.7, 96.2, 96.1, 82.6, 75.3, 74.3, 74.2, 69.7,
68.6, 64.6, 54.2, 54.1, 23.0, 18.8;
[0133] HRMS(FAB) calcd for C.sub.34H.sub.37NO.sub.11Cl.sub.3PNa
[M+Na.sup.+]:794.1068, found 794.1095.
[0134] 6.17.4. Compound 6
[0135] To a solution of compound 5 (58 mg, 0.075 mmol) in 5 mL of
90% AcOH/H.sub.2O is added zinc dust (30 mg). The reaction is
stirred vigorously at room temperature for 1 hour. The suspension
is filtered and the precipitate is rinsed with methanol. The
filtrate is concentrated and purified by flash chromatography (10%
MeOH/CHCl.sub.3/0.1% AcOH) to give 44 mg (91%) of 6 as a white
solid. E 0.19 (5% MeOH/CHCl.sub.3, 0.1% AcOH); .sup.1H NMR
(CD.sub.3OD, 500 MHz) .delta. 7.44-7.25 (m, 15H), 6.11 (m, 1H),
5.55 (s, 1H), 5.02 (m, 4H), 4.33 (q, J=7.0 Hz, 1H), 3.96 (m, 1H),
3.77 (m, 1H), 3.7-3.66 (m, 4H), 1.94 (s, 3H), 1.32 (d, J=7.0 Hz,
3H); .sup.13C NMR (CD.sub.3OD, 500 MHz) .delta. 181.2, 174.2,
139.0, 137.1, 130.0, 129.9, 129.3, 129.2, 127.3, 102.8, 97.4, 83.2,
78.3, 75.0, 71.2, 69.2, 66.4, 56.2, 56.1, 22.8, 19.7; HRMS(FAB)
calcd for C.sub.32H.sub.36NO.sub.11PNa [M+Na.sup.+]: 664.1924,
found 664.1938.
[0136] 6.17.5. Compound 7
[0137] 6.17.5.1. Fmoc-L-Lys(N-TEOC)--OH
[0138] To a solution of Fmoc-L-Lys(N-BOC)-OH (607 mg, 1.30 mmol) in
10 mL of CH.sub.2Cl.sub.2 is added 10 mL of trifluoroacetic acid.
The mixture is stirred for 20 minutes at room temperature and
concentrated. The residue is dissolved in 10 mL of DMF.
Diisopropylethylamine (1.1 mL, 6.48 mmol) is added.
2-(Trimethylsilyl)ethyl p-nitrophenyl carbonate (440 mg, 1.55 mmol)
is dissolved in 3 mL of DMF and transferred into the reaction
solution. After stirring for 2 hours at room temperature, solvent
is removed under vacuum. The residue is purified by flash
chromatography (eluting first with EtOAc then with 10%
MeOH/CHCl.sub.3/0.1% AcOH) to give 635 mg (95%) of the desired
product as a white solid. R.sub.f 0.54 (10% MeOH/CHCl.sub.3).
[0139] 6.17.5.2. Z-D-Glu(OH)-OTMSE
[0140] To a solution of Z-D-Glu(O-bzl)-OH (1.1 g, 3.0 mmol) and
DMAP (37 mg, 0.3 mmol) in 30 mL of EtOAc is added DCC (0.7 g, 3.6
mmol) and 2-(Trimethylsilyl)ethanol (0.5 mL, 3.6 mmol). After
stirring for 20 minutes at room temperature, the reaction is
filtered. The filtrate is concentrated and purified by flash
chromatography (15% EtOAc/petroleum ether) to give 1.3 g (91%) of
Z-D-Glu(O-bzl)OTMSE as a white solid. R.sub.f 0.30 (15%
EtOAc/petroleum ether).
[0141] To a solution of Z-D-Glu(O-bzl)-OTMSE (1.2 g, 2.6 mmol) in
30 mL of MeOH is added 900 mg of 20% Pd-C. After stirring for 10
minutes at room temperature, the suspension is filtered. The
filtrate is concentrated and dissolved in 20 mL of H.sub.2O/dioxane
(1:1). To the solution is added NaHCO.sub.3 (0.44, 5.2 mmol). A
solution of Cbz-succinimide (0.8 g, 3.1 mmol) in 5 mL of dioxane is
added to the reaction over 30 minutes. Then 1 mL of AcOH is added.
Solvent is removed under vacuum. The residue is purified by flash
chromatography (eluting first with 10% EtOAc/CH.sub.2Cl.sub.2 then
with 10% MeOH/CHCl.sub.3/0.1% AcOH) to give 0.9 g (87%) of
Z-D-Glu(OH)-OTMSE as a white solid. R.sub.f 0.49 (10%
MeOH/CHCl.sub.3); .sup.1H NMR (CD.sub.3OD, 500 MHz) .delta.
7.24-7.15 (m, 5H), 4.96 (d, J=3.0 Hz, 1H), 4.10 (m, 4H), 2.28 (t,
J=7.6 Hz, 2H), 2.02 (m, 1H), 1.80 (m, 1H), 0.87 (t, J=8.6 Hz, 2H),
-0.08 (s, 9H); .sup.13C NMR (CD.sub.3OD, 500 MHz) .delta. 176.3,
173.9, 158.6, 138.2, 129.5, 129.1, 128.9, 67.7, 64.7, 55.0; 31.2,
27.8, 18.2, -1.3;
[0142] Peptide 7 is synthesized by standard HOBt/HBTU method with
Fmoc protected amino acids. R.sub.f 0.29 (10% MeOH/CHCl.sub.3);
.sup.1H NMR (DMSO, 500 MHz) .delta. 8.15 (d, J=5.0 Hz, 1H), 8.14
(d, J=5.0 Hz, 1H), 8.10 (d, J=8.0 Hz, 1H), 8.02 (d, J=5.0 Hz, 1H),
6.92 (t, J=5.0 Hz, 1H), 4.30 (m, 1H), 4.19 (m, 2H), 4.174.07 (m,
5H), 4.00 (t, J=8.5 Hz, 2H), 3.31 (q, J=8.5 Hz, 1H), 2.92 (m, 2H),
2.18 (m, 2H), 1.95 (m, 1H), 1.80 (m, 1H), 1.57 (m, 1H), 1.48 (m,
1H), 1.36 (m, 2H), 1.29 (d, J=8.5 Hz, 3H), 1.25 (m, 2H), 1.20 (d,
J=8.5 Hz, 3H), 1.13 (d, J=8.5 Hz, 3H), 0.92 (m, 6H), 0.02-0.00 (3s,
27H);
[0143] .sup.13C NMR (DMSO, 500 MHz) .delta. 175.8, 172.3, 172.0,
171.8, 171.5, 171.4, 156.2, 62.6, 62.4, 61.2, 52.9, 51.3, 50.1,
47.7, 47.6, 31.4, 31.2, 29.2, 27.2, 22.6, 21.4, 18.0, 17.4, 16.9,
16.8, 16.7, -1.4, -1.5, -1.6; HRMS(FAB) calcd for
C.sub.36H.sub.7N.sub.6O.sub.10Si.sub.3Na [M+Na.sup.+]: 855.4515,
found: 855.4564.
[0144] 6.17.6 Compound 8
[0145] To a solution of compound 6 (85 mg, 0.13 mmol) and
NH.sub.2-L-Ala-O-D-Glu(O-TMSE)-L-Lys(N-TEOC)-D-Ala-D-Ala-OTMSE (7)
(153 mg, 0.18 mmol) in 1.5 mL of DMF is added diisopropylethylamine
(116 .mu.L, 0.66 mmol) followed by HOBt (27 mg, 0.20 mmol) and
PyBOP (104 mg, 0.20 mmol). After stirring for 30 minutes at room
temperature, the solution is diluted in 10 mL of EtOAc and washed
with 0.01 N aqueous HCl (3.times.10 mL). The organic layer is
concentrated, dried over anhydrous sodium sulfate, and purified by
flash chromatography (5% MeOH/CHCl.sub.3) to give 168 mg (87%) of 8
as a white solid. R.sub.f 0.24 (5% MeOH/CHCl.sub.3); .sup.1H NMR
(CD.sub.3OD, 500 MHz) .delta. 7.52-7.37 (m, 15H), 5.88 (m, 1H),
5.65 (s, 1H), 5.13 (m, 4H), 4.41 (m, 2H), 4.35 (m, 3H), 4.17 (m,
8H), 4.06 (dd, J=9.5, 3.5 Hz, 1H), 3.84 (m, 3H), 3.77 (m, 1H), 3.10
(m, 2H), 2.29 (t, J=14.5 Hz, 2H), 2.19 (m, 1H), 1.90 (m, 1H), 1.88
(s, 3H), 1.77 (m, 1H), 1.67 (m, 1H), 1.51 (m, 2H), 1.43-1.35 (m,
14H), 1.01-0.97 (m, 6H), 0.06-0.04 (3s, 27H); .sup.13C
NMR(CDCl.sub.3, 500 MHz) .delta. 173.9, 172.8, 172.4, 171.8, 171.3,
157.1, 137.1, 135.5, 135.4, 129.2, 129.0, 128.9, 128.7, 128.4,
128.1, 126.1, 101.6, 97.1, 82.5, 81.0, 78.2, 76.7, 70.0, 69.6,
68.4, 64.8, 64.1, 63.8, 63.0, 53.9, 53.3, 51.4, 50.0, 49.1, 48.4,
40.4, 31.6, 31.5, 29.6, 27.9, 23.1, 22.7, 19.6, 18.0, 17.9, 17.8,
17.5, 17.4, -1.3, -1.4, -1.5; HRMS(FAB) calcd for
C.sub.68H.sub.106N.sub.7O.sub.20PSi.sub.3Na [M+Na.sup.+]:
1478.6436, found: 1478.6417.
[0146] 6.17.7 Compound 9
[0147] To a solution of compound 8 (87 mg, 0.06 mmol) in 5 mL of
MeOH is added 20 mg of 20% Pd-C. The reaction vessel is filled with
hydrogen and stirred at room temperature. 1 mL of pyridine is added
after 30 minutes. The solution is diluted with 15 mL of MeOH and
stirred for 30 minutes. The catalyst is filtered off. The filtrate
is concentrated to give product 9a which is used in the next
reaction without further purification. R.sub.f 0.28 (CHCl.sub.3:
MEOH:H.sub.2O=3:2:0.5).
[0148] Citronellol phosphate (25 mg, 0.11 mmol) [Ref: Warren, C.
D., Jeanloz, R. W., Biochem, 14, 412-419, 1975] is coevaporated
with toluene (3.times.1 mL) and dissolved in 2 mL of
CH.sub.2Cl.sub.2. Diisopropylethylamine (92 .mu.l, 0.53 mmol) is
added. The solution is cooled to -20.degree. C. and
diphenylphosphorochloridate (26 .mu.L, 0.13 mmol) is added. The
reaction is allowed to warm up to room temperature in 10 minutes
and stirred at room temperature. After 1 hour, methanol (1 mL) is
added and the reaction is stirred for another hour at room
temperature. Solvent is removed under vacuum. The residue is
coevaporated with toluene (3.times.1 mL) and dissolved in 0.5 mL of
CH.sub.2Cl.sub.2.
[0149] Compound 9a (58 mg, 0.04 mmol) is coevaporated with toluene
(3.times.1 mL) and dissolved in 1 mL of CH.sub.2Cl.sub.2. 0.4 mL of
the citronellol diphenylpyrophosphate solution is added to the
reaction followed by pyridine (20 .mu.L, 0.24 mmol). The reaction
is stirred at room temperature for 18 hours. Solvent is removed
under vacuum and the residue is loaded to a C18 reverse phase
column (8 mm.times.80 mm, particle size 40 .mu.m, pore size 60
.ANG., from J. T. Baker) and eluted with CH.sub.3CN/0.1%
NH.sub.4HCO.sub.3 aqueous solution (0, 5%, 10%, 15%, 20%, 25%, 30%,
35% of 10 mL each). The fractions containing desired product are
combined and concentrated to give 34 mg (68%) of 9 as a white
powder. R.sub.f 0.21 (CHCl.sub.3: MeOH: H.sub.2O=4.5:1.5:0.2). This
product is used in the next reaction without further purification.
ESI-MS calcd for C.sub.57H.sub.109N.sub.7O.sub.23P.sub.2Si.sub.3Na
[M+Na.sup.+]: 1429, found: 1429.
[0150] 6.17.8 Compound 1a
[0151] To a solution of compound 9 (43 mg, 0.023 mmol) in 0.7 mL of
DMF is added tetrabutylammonium fluoride (1 M in THF, 0.7 mL). The
reaction is stirred at room temperature for 24 hours. Solvent is
removed under vacuum. The residue is loaded to a C18 reverse phase
column (8 mm.times.80 mm, particle size 40 .mu.m, pore size 60
.ANG., from J. T. Baker), and eluted with CH.sub.3CN/0.1%
NH.sub.4HCO.sub.3 aqueous solution (0, 5%, 10%, 15%, 20%, 25%, 30%
of 10 mL each). The fractions containing the desired product are
combined and concentrated. The crude product is further purified on
a diethylaminoethyl cellulose column (14 mm.times.80 mm, from
Whatman Labsales, Inc.), eluted with 250 mM NH.sub.4HCO.sub.3, to
give 24 mg of 1a (93%) as a white powder after lyophilization.
R.sub.f 0.18 (CHCl.sub.3: MeOH: H.sub.2O=3:3:1); .sup.1H NMR
(CD.sub.3OD, 500 MHz) .delta. 5.58 (m, 1H), 5.11 (t, J=6.5 Hz, 1H),
4.50-3.56 (m, 12H), 2.94 (m, 2H), 2.34 (m, 2H), 2.10 (s, 3H), 2.00
(m, 1H), 1.98 (m, 2H), 1.92 (m, 1H), 1.74 (m, 2H), 1.67 (s, 3H),
1.62 (m, 1H), 1.60 (s, 3H), 1.50-1.39 (m, 12H), 1.23 (m-, 2H), 0.93
(d, J=6.5 Hz, 3H);
[0152] .sup.13C NMR (D.sub.2O, 500 MHz) .delta. 178.2, 177.9,
176.7, 176.6, 176.5, 176.4, 176.3, 165.3, 135.5, 127.6, 97.0, 82.2,
80.3, 75.4, 74.1, 72.0, 71.9, 71.8, 70.4, 67.6, 62.7, 56.6, 55.8,
52.3, 51.9, 51.2, 41.5, 38.8, 34.0, 32.7, 31.0, 30.0, 28.6, 27.2,
27.1, 24.6, 24.4, 21.0, 19.2, 19.1, 18.8; ESI-MS calcd for
C.sub.41H.sub.74O.sub.21N.sub.7P.sub.2 [M+H.sup.+]: 1062, found:
1062.
[0153] 6.17.9 Compound 1b
[0154] To a solution of compound 1a (25 mg, 0.022 mmol) in 1.5 mL
of H.sub.2O/dioxane(1:1) is added NaHCO.sub.3 (23 mg, 0.4 mmol)
followed by 6-((biotinoyl)amino)hexanoic acid succinimide ester (12
mg, 0.027 mmol). The reaction is stirred at room temperature for 2
hours. Solvent is removed under vacuum. The residue is loaded on a
diethylaminoethyl cellulose column (14 mm.times.80 mm, from Whatman
Labsales, Inc.), eluted with 250 mM NH.sub.4HCO.sub.3 to give 16 mg
(80%) of 1b as a white powder after lyophilization. R.sub.f 0.40
(CHCl.sub.3: MeOH: H.sub.2O=3:3:1); .sup.1H NMR (CD.sub.3OD, 500
MHz) .delta. 5.49 (dd, J=3.0, 7.3 Hz, 1H), 5.11 (t, J=7.2 Hz, 1H),
4.50 (dd, J=4.8, 7.8 Hz, 1H), 4.37 (m, 2H), 4.31 (dd, J=4.3, 7.8
Hz, 1H), 4.29 (m, 1H), 4.24 (m, 3H), 4.16 (d, J=10.4 Hz, 1H), 4.02
(m, 2H), 3.99 (m, 1H), 3.90 (d, J=11.0 Hz, 1H), 3.74 (m, 1H), 3.70
(m, 1H), 3.49 (dd, J=9.5, 9.5 Hz, 1H), 3.21 (m, 1H), 3.17 (m, 4H),
2.94 (dd, J=4.8, 12.8 Hz, 1H), 2.71 (d, J=12.8 Hz, 1H), 2.31 (m,
1H), 2.28 (m, 2H), 2.25 (m, 1 H), 2.20 (m, 4H), 2.02 (s, 3H), 2.00
(m, 2H), 1.86 (m, 2H), 1.82 (m, 1H), 1.73 (m, 4H), 1.67 (s, 3H),
1.63 (m, 5H), 1.61 (s, 3H), 1.52 (m, 4H), 1.45 (m, 2H), 1.44 (d,
J=7.3 Hz, 3H), 1.43 (d, J=6.2 Hz, 3H), 1.41 (m, 2H), 1.38 (d, J=7.3
Hz, 3H), 1.37 (d, J=7.2 Hz, 3H), 1.35 (m, 2H), 1.17 (m, 1H), 0.93
(d, J=6.7 Hz, 3H); .sup.13C NMR (CD.sub.3OD, 500 MHz) .delta.
177.2, 176.5, 176.2, 176.1, 176.0, 175.6, 174.7, 174.6, 174.5,
174.2, 166.3, 132.1, 126.2, 96.4, 81.3, 78.8, 75.2, 71.0, 65.7,
63.6, 63.0, 61.8, 57.2, 55.7, 55.0, 54.2, 50.9, 50.7, 50.4, 41.2,
40.4, 40.2, 39.1, 39.0, 38.6, 37.2, 37.0, 33.0, 32.5, 30.6, 30.3,
30.2, 30.0, 29.6, 27.7, 27.1, 26.9, 26.7, 26.1, 24.5, 23.5, 20.0,
19.5, 18.4, 18.3, 18.0, 17.9; HRMS(FAB) calcd for
C.sub.57H.sub.95N.sub.10O.sub- .24P.sub.2SNa
[M-3H.sup.++2Na.sup.+]: 1443.5512, found: 1443.5494.
[0155] 6.17.10 Compound 10
[0156] Compound 10 is made following the same scheme as 1a except
that in step e, intermediate 6 is coupled to dipeptide
CH.sub.3NH-D-.gamma.-Glu(O- -TMSE)-L-Ala-NH.sub.2 instead of to 7.
R.sub.f 0.41 (CHCl.sub.3: MeOH: H.sub.2O=3:3:1); .sup.1H NMR
(CD.sub.3OD, 500 MHz) .delta. 5.49 (dd, J=3.0, 7.0 Hz, 1H), 5.11
(t, J=6.6 Hz, 1H), 4.33 (q, J=7.0 Hz, 1H), 4.27 (q, J=7.0 Hz, 1H),
4.24 (dd, J=3.8, 7.6 Hz, 1H), 4.16 (m, 1H), 4.04 (m, 2H), 4.00 (m,
1H), 3.90 (dd, J=1.8, 11.8 Hz, 1H), 3.75 (dd, J=9.6, 9.6 Hz, 1H),
3.70 (dd, J=5.7, 11.8 Hz, 1H), 3.48 (dd, J=9.6, 9.6 Hz, 1H), 2.64
(s, 3H), 2.18 (m, 2H), 2.16 (m, 1H), 2.02 (s, 3H), 1.98 (m, 2H),
1.92 (m, 1H), 1.72 (m, 1H), 1.67 (s, 3H), 1.62 (m, 1H), 1.61 (s,
3H), 1.47 (m, 1H), 1.43 (d, d, J=7.0 Hz, 6H), 1.37 (m, 1H), 1.18
(m, 1H), 0.94 (d, J=6.6 Hz, 3H); .sup.13C NMR (CD.sub.3OD, 500 MHz)
.delta. 177.2, 176.1, 176.0, 174.4, 174.2, 132.0, 126.1, 96.3,
81.1, 78.9, 75.2, 70.8, 65.7, 63.0, 55.1, 54.9, 51.0, 39.1, 38.6,
33.3, 30.6, 30.1, 26.7, 26.5, 26.1, 23.4, 19.9, 19.5, 18.2, 17.9;
HRMS(FAB) calcd for C.sub.30H.sub.53N.sub.4O.sub.17P.sub.2
[M-H.sup.+]: 803.2881, found: 803.2861.
[0157] 6.17.11 Compound 1a
[0158] Compound 11a is made following the same scheme as 1a except
that in step e, compound 6 is coupled to
TEOC-NHCH.sub.2CH.sub.2NH.sub.2 instead of to 7. The sily
protecting group is cleaved using TBAF, the same as in making 1a.
R.sub.f 0.20 (CHCl.sub.3: MeOH: H.sub.2O=3:2:0.5); .sup.1H NMR
(CD.sub.3OD, 500 MHz) .delta. 5.58 (bs, 1H), 5.11 (t, J=7.0 Hz,
1H), 4.30 (q, J=6.7 Hz, 1H), 4.21 (m, 1H), 4.04 (m, 3H), 3.72 (m,
1H), 3.78 (m, 1H), 3.73 (m, 1H), 3.64 (m, 1H), 3.50 (dd, J=9.4, 9.4
Hz, 1H), 3.40 (m, 1H), 3.13 (m, 2H), 2.03 (s, 3H), 2.00 (m, 2H),
1.73 (m, 1H), 1.67 (s, 3H), 1.63 (m, 1H), 1.61 (s, 3H), 1.46 (m,
1H), 1.39 (m, 1H), 1.38 (d, J=6.7 Hz, 3H), 1.18 (m, 1H), 0.94 (d,
J=6.7 Hz, 3H); .sup.13C NMR (CD.sub.3OD, 500 MHz) .delta. 176.2,
173.6, 131.2, 125.2, 95.6, 80.7, 78.1, 74.3, 70.3, 64.9, 62.0,
54.2, 39.7, 38.2, 37.8, 37.5, 29.8, 25.8, 25.2, 22.5, 19.1, 18.6,
17.0; HRMS(FAB) calcd for C.sub.23H.sub.43N.sub.3-
O.sub.13P.sub.2Na [M-2H.sup.++Na.sup.+]: 654.2169, found
654.2199.
[0159] 6.17.12 Compound 11b
[0160] Compound 11a (4 mg, 0.006 mmol) and 4-nitrophenyl acetate
(1.2 mg, 0.007 mmol) is dissolved in 0.4 mL of DMF. Large amount of
KHCO.sub.3 is added to increase PH. Equal amount of 4-nitrophenyl
acetate is added every 12 hours. After 3 days, the reaction is
completed. The solvent is removed and the residue is loaded to a
C18 reverse phase column (8 mm.times.80 mm, particle size 40 .mu.m
pore size 60 .ANG., from J. T. Baker) and eluted with
CH.sub.3CN/0.1% NH.sub.4HCO.sub.3 aqueous solution (0, 5%, 10%,
15%, 20%, 25%, 30%, 35% of 10 mL each). The fractions containing
desired product are combined and concentrated to give 3 mg (71%) of
11b as a white powder. R.sub.f 0.26 (CHCl.sub.3: MeOH:
H.sub.2O=3:2:0.5); .sup.1H NMR (CD.sub.3OD, 500 MHz) .delta. 5.50
(bs, 1H), 5.12 (t, J=7.0 Hz, 1H), 4.22 (q, J=7.0 Hz, 1H), 4.04 (m,
2H), 4.00 (m, 1H), 3.89 (d, J=12.2 Hz, 1H), 3.72 (m, 2H), 3.46 (dd,
J=9.5, 9.5 Hz, 1H), 3.36 (m, 2H), 3.28 (m, 2H), 2.04 (s, 3H), 2.02
(m, 2H), 1.98 (s, 3H), 1.73 (m, 1H), 1.68 (s, 3H), 1.63 (m, 1H),
1.62 (s, 3H), 1.46 (m, 1H), 1.40 (d, J=7.0 Hz, 3H), 1.38 (m, 1H),
1.18 (m, 1H), 0.94 (d, J=6.7 Hz, 3H); .sup.13C NMR (CD.sub.3OD, 500
MHz) .delta. 176.4, 174.5, 173.8, 132.0, 126.1, 96.4, 81.9, 79.2,
75.1, 71.0, 65.6, 62.9, 55.0, 40.2, 40.1, 39.0, 38.6, 30.6, 26.7,
26.0, 23.4, 22.8, 19.9, 19.5, 17.9; HRMS(FAB) calcd for
C.sub.25H.sub.46N.sub.3O.sub.14P.sub.2 [M-H.sup.+]: 674.2455, found
674.2488.
[0161] 6.17.13 Compound 11c
[0162] Compound 11e is made from 11a and
6-((biotinoyl)amino)hexanoic acid succinimide ester using the same
chemistry described in step h (scheme II). R.sub.f 0.30
(CHCl.sub.3: MeOH: H.sub.2O=3:2:0.5); .sup.1H NMR (CD.sub.3OD, 500
MHz) .delta. 5.49 (dd, J=2.7, 7.0 Hz, 1H), 5.12 (t, J=7.2 Hz, 1H),
4.50 (dd, J=5.0, 7.5 Hz, 1H), 4.32 (dd, J=4.4, 7.5 Hz, 1H), 4.20
(q, J=6.7 Hz, 1H), 4.16 (m, 1H), 4.03 (m, 2H), 3.98 (m, 1H), 3.90
(d, J=12.0 Hz, 1H), 3.71 (m, 1H), 3.70 (m, 1H), 3.45 (dd, J 9.4,
9.4 Hz, 1H), 3.23 (m, 1H), 3.18 (m, 6H), 2.94 (dd, J=5.0, 12.8 Hz,
1H), 2.72 (d, J=12.8 Hz, 1H), 2.24 (t, J=7.6 Hz, 2H), 2.21 (t,
J=7.6 Hz, 2H), 2.03 (s, 3H), 2.00 (m, 2H), 1.73 (m, 3H), 1.68 (s,
3H), 1.64 (m, 6H), 1.62 (s, 3H), 1.53 (m, 2H), 1.45 (m, 3H), 1.40
(d, J=6.7 Hz, 3H), 1.36 (m, 3H), 1.18 (m, 1H), 0.94 (d, J=6.7 Hz,
3H); .sup.13C NMR (CD.sub.3OD, 500 MHz) .delta. 176.5, 176.4,
176.1, 174.4, 166.3, 132.0, 126.1, 96.5, 81.9, 79.2, 75.2, 70.9,
65.7, 63.5, 62.9, 61.8, 57.1, 55.0, 41.2, 40.4, 40.1, 39.1, 39.0,
38.6, 37.2, 37.0, 30.6, 30.3, 30.0, 29.6, 27.8, 27.1, 26.8, 26.7,
26.1, 23.4, 20.0, 19.6, 18.0; HRMS(FAB) calcd for
C.sub.39H.sub.69N.sub.6O.sub.16P.sub.2S [M-H.sup.+]: 971.3966,
found: 971.3948.
[0163] 6.17.14 Compound 12a
[0164] The intermediate from hydrogenation of compound 8 is
deprotected with TBAF using the same method for making 1a. R.sub.f
0.16 (CHCl.sub.3: MeOH: H.sub.2O=3:4:1.5); .sup.1H NMR (CD.sub.3OD,
500 MHz) .delta. 5.34 (dd, J=3.0, 7.0 Hz, 1H), 4.24 (m, 3H),4.17
(dd, J=6.7, 6.7 Hz, 1H),4.08 (dd, J=4.6, 8.5 Hz, 1H),4.03 (q, J=7.0
Hz, 1H), 3.93 (m, 1H), 3.80 (m, 1H), 3.75 (m, 1H), 3.59 (dd, J=5.5,
11.6 Hz, 1H), 3.56 (m, 1H), 3.38 (dd, J=9.7, 9.7 Hz, 1H), 2.82 (t,
J=7.3 Hz, 2H), 2.22 (m, 2H), 2.15 (m, 1H), 1.86 (s, 3H), 1.70 (m,
4H), 1.58 (m, 2H), 1.40 (m, 1H), 1.31 (m, 6H), 1.25 (m, 6H);
.sup.13C NMR (CD.sub.3OD, 500 MHz) .delta. 179.4, 178.8, 178.0,
176.2, 175.9, 174.7, 174.0, 173.8, 95.3, 81.2, 78.7, 74.9, 71.2,
62.8, 55.5, 55.3, 55.0, 51.9, 51.1, 50.8, 40.5, 33.1, 32.5, 30.4,
28.4, 23.7, 23.4, 19.8, 19.4, 18.4, 18.0; HRMS(FAB) calcd for
C.sub.31H.sub.53N.sub.7O.sub.18P [M-H.sup.+]: 842.3185, found:
842.3212.
[0165] 6.17.15 Compound 12b
[0166] Compound 12b is made from 12a and
6-((biotinoyl)amino)hexanoic acid succinimide ester using the same
chemistry described in step h (scheme II). R.sub.f 0.27
(CHCl.sub.3: MeOH: H.sub.2O=3:4:1.5); .sup.1H NMR (CD.sub.3OD, 500
MHz) .delta. 5.45 (dd, J=7.0, 3.0 Hz, 1H), 4.51 (dd, J=5.0, 7.5 Hz,
1H), 4.39 (m, 2H), 4.32 (m, 2H), 4.26 (m, 3H), 4.12 (m, 1H), 3.91
(m, 1H), 3.86 (d, J=11.6 Hz, 1H), 3.73 (dd, J=5.5, 11.6 Hz, 1H),
3.69 (m, 1H), 3.53 (m, 1H), 3.22 (m, 1H), 3.17 (m, 4H), 2.94 (dd,
J=5.0, 12.8 Hz, 1H), 2.72 (d, J=12.8 Hz, 1H), 2.30 (m, 4H), 2.21
(m, 4H), 1.99 (s, 3H), 1.89 (m, 1H), 1.82 (m, 1H), 1.74 (m, 2H),
1.63 (m, 4H), 1.53 (m, 4H), 1.46 (m, 2H), 1.44 (m, 6H), 1.39 (m,
6H), 1.35 (m, 4H); .sup.13C NMR (CD.sub.3OD, 500 MHz) .delta.
177.5, 177.4, 177.3, 176.5, 176.3, 174.8, 174.7, 174.6, 174.3,
174.2, 166.0, 94.3, 80.6, 78.6, 73.2, 68.9, 62.8, 61.1, 60.9, 56.1,
55.0, 54.3, 54.2, 51.6, 50.4, 50.1, 40.4, 39.8, 39.6, 36.4, 36.2,
32.5, 31.4, 28.8, 28.7, 28.6, 28.5, 28.4, 26.2, 25.9, 25.8, 23.2,
22.7; HRMS(FAB) calcd for C.sub.47H.sub.79N.sub.10O.sub-
.21P.sub.2S [M-H.sup.+]: 1181.4801, found: 1181.4769.
[0167] 6.17.16 Compound 13a
[0168] To a solution of compound 6 (12 mg, 0.019 mmol) in 1 mL of
methanol is added 10 mg of pearlman's catalyst. The reaction vessel
is filled with hydrogen. After stirring at room temperature for 30
min, a few drops of pyridine is added. The suspension is filtered
after stirring for another 30 min. The filtration is concentrated
to give a yellow oil which is purified on a diethylaminoethyl
cellulose column (14 mm.times.80 mm, from Whatman Labsales, Inc.),
eluted with 1M NH.sub.4HCO.sub.3, to give 7 mg (90%) of 13a as a
white powder. R.sub.f 0.29 (CHCl.sub.3: MeOH: H.sub.2O=3:4:1.5);
.sup.1H NMR (CD.sub.3OD, 500 MHz) .delta. 5.73 (d, J=7.3 Hz, 1H),
4.72 (q, J=6.7 Hz, 1H), 3.86 (m, 1H), 3.84 (d, J=11.6 Hz, 1H), 3.74
(m, 1H), 3.70 (m, 1H), 3.66 (dd, J=5.5, 11.6 Hz, 1H), 3.45 (dd,
J=9.8, 9.8 Hz, 1H), 2.0 (s, 3H), 1.83 (d, J=7.3 Hz, 3H); .sup.13C
NMR (CD.sub.3OD, 500 MHz) .delta. 180.4, 173.2, 93.7, 77.7, 77.4,
74.2, 71.8, 62.0, 54.5, 22.2, 19.2; HRMS(FAB) calcd for
C.sub.11H.sub.19NO.sub.11P[M-- H.sup.+]: 372.0696, found:
372.0711.
[0169] 6.17.17 Compound 13b
[0170] To a solution of 2 (20 mg, 0.042 mmol) in 1 mL of
CH.sub.2Cl.sub.2 is added DIEA (16 .mu.L, 0.924 mmol). The reaction
vessel is cooled to -30.degree. C., then MeOTf (5.2 .mu.L, 0.046
mmol) is added. The reaction is complete after stirring at room
temperature for 30 min. Saturated NaHCO.sub.3 is added. The mixture
is extracted with CH.sub.2Cl.sub.2 (3.times.5 mL). The organic
layers are combined, dried over anhydrous sodium sulfate, filtered,
concentrated and purified by flash chromatography (45%
EtOAc/petroleum ether) to give 18 mg (87%) of product as a white
powder. The following chemistry is the same as for 13a. R.sub.f
0.12 (CHCl.sub.3: MeOH: H.sub.2O=3:2:0.5); .sup.1H NMR (CD.sub.3OD,
500 MHz) .delta. 5.50 (dd, J=3.4, 7.3 Hz, 1H), 4.58 (q, J=6.7 Hz,
1H), 3.87 (m, 2H), 3.84 (m, 1H), 3.73 (3, 3H), 3.62 (m, 2H), 3.42
(dd, J=9.2, 9.2 Hz, 1H), 2.00 (s, 3H), 1.37 (d, J=7.0 Hz, 3H);
.sup.13C NMR (CD.sub.3OD, 500 MHz) .delta. 176.4, 173.8, 95.0,
80.7, 77.3, 74.8, 72.8, 62.9, 54.9, 52.6, 23.2, 19.4; ESI-MS calcd
for C.sub.12H.sub.23NO.sub.11P [M+H.sup.+]: 388, found: 388.
[0171] 6.17.18 Compound 14
[0172] Compound 14a-c are made by the same approach as 1a, except
by using R--OPO.sub.3PO(OPh).sub.2 instead of
(R)-(+)-.beta.-citronellol-OPO.sub.3- PO(OPh).sub.2. ESI-MS for 14a
C.sub.32H.sub.58N.sub.7O.sub.21P.sub.2 [M+H.sup.+]: 938; ESI-MS for
14b C.sub.33H.sub.60N.sub.7O.sub.21P.sub.2 [M+H.sup.+]: 952; ESI-MS
for 14c C.sub.34H.sub.60N.sub.7O.sub.21P.sub.2 [M+H.sup.+]:
964.
[0173] 6.17.19 Compound 15
[0174] To a microfuge tube containing 1 equivalent 1b (10 .mu.g)
and 3 equivalents .sup.14C-UDP-GlcNAc in 100 .mu.L HEPES reaction
buffer (25 mM HEPES, pH 7.9, and 2.5 mM MgCl2) is added 1 .mu.g
purified MurG. The reaction is terminated after 30 minutes by
heating MurG to 65.degree. C. for five minutes. The reaction is
evaluated by transferring a 10 .mu.L aliquot to a tube containing a
3-fold molar excess of TetraLink Tetrameric Avidin Resin (based on
the amount of 1b expected in one tenth of a volume of the reaction
mixture), diluting with H.sub.2O, transferring the suspension to a
96 well filter plate, and ishing to remove unbound radioactivity as
described in more detail under the experimental for the initial
rate assays. The resin is then transferred to a scintillation vial
containing Ecolite and counted. The conversion to disaccharide
product 15 is estimated to be greater than 90% based on the counts
incorporated into the resin. The mixture containing 15 is suitable
for evaluating transglycosylase activity.
[0175] .sup.1H NMR Assignments are Made From 1D and 2D Spectra
(COSY)
[0176] .sup.1H NMR (CD.sub.3OD, 500 MHz) .delta. ppm 5.09 (t, J=5.0
Hz, 1H, H-7), 3.90 (m, 2H, H-1), 1.99 (m, 2H, H-6), 1.67 (m, 1H,
H-2), 1.65 (s, 3H, H-9), 1.62 (m, 1H, H-3), 1.59 (s, 3H, H-10),
1.41 (m, 1H, H-2'), 1.34 (m, 1H, H-5), 1.16 (m, 1H, H-5'), 0.91 (d,
J=6.5 Hz, 3H, H-4).
[0177] .sup.1HNMR Assignments are Made From 1D and 2D Spectra
(COSY, ROESY)
[0178] .sup.1H NMR (DMSO, 500 MHz) .delta. ppm 8.24 (d, J=7.5 Hz,
1H, D-.gamma.-Glu-NH), 8.18 (d, J=8.5 Hz, 1H, D-Ala.sub.2-NH), 8.17
(d, J=7.0 Hz, 1H, D-Ala.sub.1-NH), 8.01 (d, J=7.5 Hz, 1H,
L-Lys-NH), 7.47 (d, J=7.5 Hz, 1H, L-Ala-NH), 6.94 (t, J=5.0 Hz, 1H,
L-Lys-NHCOOR), 4.29 (m, 1H, D-Ala.sub.1-H.alpha.), 4.24 (m, 1H,
D-Ala.sub.2-H.alpha.), 4.18 (m, 1H, D-.gamma.-Glu-H.alpha.), 4.14
(m, 1H, L-Lys-H.alpha.), 4.12 (m, 1H, L-Ala-H.alpha.), 3.58 (s, 3H,
D-Ala.sub.2--COOCH.sub.3), 2.91 (m, 2H, L-Lys-He), 2.17 (m, 2H,
D-.gamma.-Glu-H.gamma.), 1.92 (m, 1H, D-.gamma.-Glu-H.beta.), 1.79
(m, 1H, D-.gamma.-Glu-H.beta.'), 1.56 (m, 1H, L-Lys-H.beta.), 1.48
(m, 1H, L-Lys-H.beta.'), 1.35 (m, 2H, L-Lys-H.delta.), 1.29 (d,
J=7.0 Hz, 3H, D-Ala.sub.2-CH.sub.3), 1.23 (d, J=6.5 Hz, 3H,
L-Ala-CH.sub.3), 1.22 (m, 1H, L-Lys-H.gamma.), 1.19 (m, 1H,
L-Lys-H.gamma.'), 1.19 (d, J=7.0 Hz, 3H, D-Ala.sub.1-CH.sub.3),
0.01-0.00 (s, 9H; s, 9H, TMS-CH.sub.3).
[0179] .sup.1H NMR Assignments are Made From 1D and 2D Spectra
(COSY, NOESY)
[0180] .sup.1H NMR (DMSO, 500 MHz) .delta. ppm 8.36 (d, J=7.2 Hz,
1H, L-Lys-NH), 8.21 (d, J=8.0 Hz, 1H, D-Ala.sub.2-NH), 8.19 (d,
J=8.2 Hz, 1H, D-Ala.sub.1-NH), 8.10 (d, J=6.0 Hz, 1H,
D-.gamma.-Glu-NH), 7.32 (d, J=7.5 Hz, 1H, L-Ala.sub.2-NH), 6.95 (t,
J=5.0 Hz, 1H, L-Lys-NHCOOR), 5.26 (d, J=6.0 Hz, 1H, H-1'), 5.07 (t,
J=7.0 Hz, 1H, H-7), 4.30 (m, 1H, L-Ala-H.alpha.), 4.27 (m, 1H,
D-Ala.sub.2-H.alpha.), 4.23 (m, 1H, D-Ala.sub.1-H.alpha.), 4.13 (m,
1H, D-.gamma.-Glu-H.alpha.), 4.12 (m, 1H, L-Lys-H.alpha.), 4.12 (m,
1H, H-7'), 3.87 (m, 1H, H-2'), 3.77 (m, 2H, H-1), 3.62 (m, 1H,
H-5'), 3.60 (s, 3H, D-Ala.sub.2-COOCH.sub.3), 3.51 (m, 1H, H-3'),
3.33 (m, 1H, H-4'), 2.91 (m, 2H, L-Lys-H.epsilon.), 2.17 (m, 2H,
D-.gamma.-Glu-H.gamma.), 1.94 (m, 2H, H-6), 1.91 (m, 1H,
D-.gamma.-Glu-H.beta.), 1.51 (m, 1H, D-.gamma.-Glu-H.beta.), 1.80
(s, 3H, NHCOCH.sub.3-2'), 1.62 (s, 3H, CH.sub.3-9), 1.58 (s, 3H,
CH.sub.3-10), 1.50 (m, 1H, H-3), 1.51 (m, 1H, L-Lys-H.beta.), 1.49
(m, 1H, L-Lys-H.beta.), 1.35 (m, 2H, L-Lys-H.delta.), 1.51 (m, 1H,
H-2), 1.27 (m, 1H, H-2), 1.29 (d, J=7.2 Hz, 3H,
D-Ala.sub.2-CH.sub.3), 1.19 (d, J=7.4 Hz, 3H,
D-Ala.sub.2-CH.sub.3), 1.24 (d, J=5.5 Hz, 3H, CH.sub.3-8'), 1.27
(m, 1H, H-5), 1.11 (m, 1H, H-5), 1.25 (d, J=6.8 Hz, 3H,
L-Ala-CH.sub.3), 1.23 (m, 2H, L-Lys-H.gamma.), 0.84 (d, J=6.5 Hz,
3H, CH.sub.34), 0.02-0.01 (s, 9H; s, 9H, TMS-CH.sub.3).
[0181] .sup.1HNMR are Made From ID and 2D Spectra (COSY).
[0182] .sup.1H NMR (CD.sub.3OD, 500 MHz), .delta. ppm 5.58 (1H,
H-1'), 5.11 (t, J=6.5 Hz, 1H, H-7), 4.504.00 (L-Ala-H.alpha.,
D-.gamma.-Glu-H.alpha., L-Lys-H.alpha., D-Ala.sub.1,2-H.alpha.,
H-7'), 4.10 (m, 1H, H-2'), 3.98 (m, 1H, H-5'), 3.87 (m, 1H, H-6'),
3.80 (m, 1H, H-3'), 3.75 (m, 1H, H-6'), 3.56 (m, 1H, H4'), 2.94 (m,
2H, L-Lys-H.epsilon.), 2.34 (m, 2H, D-.gamma.-Glu-H.gamma.), 2.10
(s, 3H, NHCOCH.sub.3-2'), 2.00 (m, 1H, D-.gamma.-Glu-H.beta.), 1.92
(m, 1H, D-.gamma.-Glu-H.beta.), 1.98 (m, 2H, H-6), 1.74 (m, 2H,
L-Lys-H.delta.), 1.67 (s, 3H, CH.sub.3-9), 1.62 (m, 1H, H-3), 1.60
(s, 3H, CH.sub.3-10), 1.50-1.39 (12H, L-Ala-CH.sub.3,
D-Ala.sub.1,2-CH.sub.3, CH.sub.3-7'), 1.23 (m, 2H, L-Lys-H.gamma.),
0.93 (d, J=6.5 Hz, 3H, CH.sub.3-4).
[0183] .sup.1HNMR are Made From 1D and 2D Spectra (COSY).
[0184] .sup.1H NMR (CD.sub.3OD, 500 MHz) .delta. ppm 5.52 (d, J=4.5
Hz, 1H, H-1'), 5.12 (t, J=7.0 Hz, 1H, H-7), 4.50 (m, 1H, H-b1),
4.39-4.19 (L-Ala-H.alpha., D-.gamma.-Glu-H.alpha., L-Lys-H.alpha.,
D-Ala.sub.1,2-H.alpha., H-7'), 4.31 (m, 1H, H-b2), 4.20 (m, 1H,
H-2'), 4.00 (m, 1H, H--S'), 3.89 (m, 1H, H-6'), 3.76 (m, 1H, H-3'),
3.72 (m, 1H, H-6'), 3.51 (m, 1H, H4'), 3.22 (m, 1H, H-b4), 3.18 (m,
2H, H-b9), 2.95 (dd, J=12.5, 5.0 Hz, 1H, H-b3), 2.71 (d, J=12.5 Hz,
1H, H-b3'), 2.27 (m, 2H, D-.gamma.-Glu-H.gamma.), 2.02 (s, 3H,
NHCOCH.sub.3-2'), 2.01 (m, 2H, H-6), 1.85 (m, 2H,
D-.gamma.-Glu-H.beta.), 1.67 (m, 2H, H-b5), 1.67 (s, 3H,
CH.sub.3-9), 1.61 (s, 3H, CH.sub.3-10), 1.62 (m, 1H, H-3), 1.53 (m,
2H, H-b10), 1.45-1.37 (12H, L-Ala-CH.sub.3, D-Ala.sub.1,2-CH.sub.3,
CH.sub.3-8'), 1.38 (m, 1H, H-5), 1.17 (m, 1H, H-5), 0.94 (d, J=6.8
Hz, 3H, CH.sub.3-4).
[0185] The preceding examples are provided as a further
illustration of the present invention. The specific embodiments
described above are not to be construed to limit the invention in
any way, which invention broadly encompasses such embodiments, as
well as those embodiments that would be evident to those of
ordinary skill upon consideration of the disclosure herein
provided. The invention is limited solely by the claims, which
follow.
* * * * *