Title: Glycorandomization and the production of novel erythronolide and coumarin analogs
Abstract: The present invention provides combinatorial methods for rapidly generating a diverse library of glycorandomized structures, comprising incubating one or more aglycons and a pool of NDP-sugars in the presence of a glycosyltransferase. The glycosyltransferase may be one that is associated with or involved in production of natural secondary metabolites, or one which is putatively associated with or involved in production of natural secondary metabolites. The glycosyltransferase may show significant flexibility with respect to its NDP-sugar donors and/or its aglycons. NDP-sugar donors may be commercially available, or may be produced by utilizing mutant or wild type nucleotidyltransferases significant flexibility with respect to their substrates.
Patent Number: 6,884,604 Issued on 04/26/2005 to Thorson
| Inventors:
|
Thorson; Jon S. (Madison, WI)
|
| Assignee:
|
Sloan-Kettering Institute for Cancer Research (New York, NY)
|
| Appl. No.:
|
109672 |
| Filed:
|
April 1, 2002 |
| Current U.S. Class: |
435/89; 435/97; 435/76; 435/72; 435/74; 435/75 |
| Intern'l Class: |
C12P 019//18; C12P 019//30 |
| Field of Search: |
435/97,89,76,72,74,75
|
References Cited [Referenced By]
U.S. Patent Documents
| 5998194 | Dec., 1999 | Summers et al.
| |
| 2003/0055235 | Mar., 2003 | Thorson et al.
| |
| Foreign Patent Documents |
| WO 9931224 | Jun., 1999 | WO.
| |
| WO 0248331 | Jun., 2002 | WO.
| |
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|
Primary Examiner: Prats; Francisco
Attorney, Agent or Firm: Godfrey & Kahn, S.C.
Goverment Interests
GOVERNMENT SUPPORT
This work was supported in part by grants from the National Cancer Institute
(NCI Core Grant 08748). The Government may have certain rights in this invention.
Parent Case Text
PRIORITY
The present application claims priority to U.S. Provisional Patent Application
No. 60/279,682, filed Jun. 30, 2000 which is also incorporated herein in its entirety.
Claims
1. A method of preparing a glycosylated compound comprising steps of:
(a) preparing a nucleotide sugar by combining an NTP and at least one sugar phosphate
in the presence of at least one nucleotidyltransferase Ep mutated at one or more
amino acids selected from the group consisting of V173, G147, W224, N112, G175,
D111, E162, T201, I200, E199, R195, L89, L89T, L109, Y146 and Y177;
(b) combining the nucleotide sugar prepared in step (a) with a glycosyltransferase
and a moiety capable of being glycosylated, thereby producing at least one glycosylated
compound; and
(c) recovering the glycosylated compound.
2. The method of claim 1, further wherein the method is carried out in vitro.
3. The method of claim 1, further wherein the nucleotide sugars is a nucleotide
diphospho sugar.
4. The method of claim 1, further wherein the nucleotide sugars is selected from
the group consisting of Thymidine 5′-(α-D-glucopyranosyl diphosphate);
Uridine 5′-(α-D-glucopyranosyl diphosphate); Thymidine 5′-(2-deoxy-α-D-glucopyranosyl
diphosphate); Uridine 5′-(2-deoxy-α-D-glucopyranosyl diphosphate);
Thymidine 5′-(3-deoxy-α-D-glucopyranosyl diphosphate); Uridine 5′-(3-deoxy-α-D-glucopyranosyl
diphosphate); Thymidine 5′-(4-deoxy-α-D-glucopyranosyl diphosphate);
Uridine 5′-(4-deoxy-α-D-glucopyranosyl diphosphate); Thymidine 5′-(6-deoxy-α-D-glucopyranosyl
diphosphate); Uridine 5′-(6-deoxy-α-D-glucopyranosyl diphosphate);
Thymidine 5′-(α-D-mannopyranosyl diphosphate); Uridine 5′-(α-D-mannopyranosyl
diphosphate); Thymidine 5′-(α-D-galactopyranosyl diphosphate); Uridine
5′-(α-D-galactopyranosyl diphosphate); Thymidine 5′-(α-D-allopyranosyl
diphosphate); Thymidine 5′-(α-D-altropyranosyl diphosphate); Uridine
5′-(α-D-allopyrano-syl diphosphate); Uridine 5′-(α-D-altropyranosyl
diphosphate); Thymidine 5′-(α-D-gulopyranosyl diphosphate); Uridine
5′-(α-D-gulopyranosyl diphosphate); Thymidine 5′-(α-D-idopyranosyl
diphosphate); Uridine 5′-(α-D-idopyranos-yl diphosphate); Thymidine
5′-(α-D-talopyranosyl diphosphate); Uridine 5′-(α-D-talopyranosyl
diphosphate); Thymidine 5′-(6-amino-6-deoxy-α-D-glucopyranosyl diphosphate);
Uridine 5′-(6-amino-6-deoxy-α-D-glucopyranosyl diphosphate); Thymidine
5′-(4-amino-4-deoxy-α-D-glucopyranosyl diphosphate); Uridine 5′-(4-amino-4-deoxy-α-D-glucopyranosyl
diphosphate); Thymidine 5′-(3-amino-3-deoxy-α-D-glucopyranosyl diphosphate);
Uridine 5′-(3-amino-3-deoxy-α-D-glucopyranosyl diphosphate); Thymidine
5′-(2-amino-2-deoxy-α-D-glucopyranosyl diphosphate); Uridine 5′-(2-amino-2-deoxy-α-D-glucopyranosyl
diphosphate); Thymidine 5′-(6-acetamido-6-deoxy-α-D-glucopyranosyl
diphosphate); Uridine 5′-(6-acetamido-6-deoxy-α-D-glucopyranosyl diphosphate);
Thymidine 5′-(4-acetamido-4-deoxy-α-D-glucopyranosyl diphosphate);
Uridine 5′-(4-acetamido-4-deoxy-α-D-glucopyranosyl diphosphate); Thymidine
5′-(3-acetamido-3-deoxy-α-D-glucopyranosyl diphosphate); Uridine 5′-(3-acetamido-3-deoxy-α-D-glucopyranosyl
diphosphate); Thymidine 5′-(2-acetamido-2-deoxy-α-D-glucopyranosyl
diphosphate); Uridine 5′-(2-acetamido-2-deoxy-α-D-glucopyranosyl diphosphate);
Thymidine 5′-(4-amino-4,6-dideoxy-α-D-glucopyranosyl diphosphate);
Uridine 5′-(4-amino-4,6-dideoxy-α-D-glucopyranosyl diphosphate); Thymidine
5′-(α-D-glucopyran-6-uronic acid diphosphate); Uridine 5′-(α-D-glucopyran-6-uronic
acid diphosphate); Thymidine 5′-(α-D-arabinopyranosyl diphosphate);
Uridine 5′-(α-D-arabinopyranosyl diphosphate); and
##STR15##
##STR16##
##STR17##
##STR18##
##STR19##
5. The method of claim 1, further wherein more than one nucleotide sugar is incubated
with the moiety capable of being glycosylated in the presence of the glycosyltransferase.
6. The method of claim 1, further wherein more than one glycosylated compound
is produced in a single reaction vessel.
7. The method of claim 1, further wherein a diverse population of glycosylated
compounds is produced.
8. The method of claim 1, further wherein the moiety capable of being glycosylated
is selected from the group consisting of natural and synthetic metabolites, pyran
rings, furan rings, enediynes, anthracyclines, angucyclines, aureolic acids, orthosomycins,
macrolides, aminoglycosides, non-ribosomal peptides, polyenes, steroids, lipids,
indolocarbazoles, bleomycins, amicetins, benzoisochromanequinones coumarins, polyketides,
pluramycins, aminoglycosides, oligosaccharides, peptides, and proteins.
9. The method of claim 1, further wherein the moiety capable of being glycosylated
is selected from the group consisting of aglycons of bioactive anthracyclines,
angucyclines, nonribosomal peptides, macrolides, enediynes, indolocarbazoles, pluramycins,
aurelolic acids, orthosomycins, aminoglycosides, coumarins, bleomycins, amicetins,
polyenes, benzoisochromanequinones, and angucyclines.
10. The method of claim 1, further wherein the moiety capable of being glycosylated
is selected from the group consisting of enediynes, anthracyclines, angucyclines,
aureolic acids, orthosomycins, macrolides, aminoglycosides, non-ribosomal peptides,
polyenes, steroids, lipids, indolocarbazoles, bleomycins, amicetins, benzoisochromanequinones
coumarins, polyketides, pluramycins, aminoglycosides, oligosaccharides, peptides,
and proteins.
11. The method of claim 1, further wherein the moiety capable of being glycosylated
is selected from the group consisting of aglycons of bioactive anthracyclines,
angucyclines, nonribosomal peptides, macrolides, enediynes, indolocarbazoles, pluramycins,
aurelolic acids, orthosomycins, aminoglycosides, coumarins, bleomycins, amicetins,
polyenes, benzoisochromanequinones, and angucyclines.
12. The method of claim 1, further wherein more than one moiety capable of being
glycosylated is incubated with the at least one first nucleotide sugar in the presence
of the at least one first glycosyltransferase.
13. The method of claim 1, further wherein at least one of the at least one moieties
capable of being glycosylated comprises at least one glycosyl group.
14. The method of claim 1, further wherein at least one of the at least one first
glycosyltransferase is selected from the group consisting of CalB, CalE, CalN,
CalU, Gra orfl4, Gra orf5, LanGT1, LanGT2, LanGT3, LanGT4, MtmGI, MtmGII, MtmGTIII,
MtmGTIV, NovM, RhlB, Rif orf 7, SnogD, SnogE, SnogZ, UrdGT1a, UrdGT1b, UrdGT1c,
UrdGT2, AknK, AknS, DesVII, DnrS, OleG1, OleG2, TylCV, TylMII, TylN, DauH, DnrH,
EryBV, EryCIII, Ngt, BgtA, BgtB, BgtC, GftA, GftB, GftC, GftD, GftE, Gp1-1, Gp1-2,
RtfA, AveBI, BlmE, BlmF, MgtA, NysD1, OleD, OleI, SpcF, SpcG, StrH, Ugt51B1, Ugt51C1,
UGT52, UgtA, UgtB, UgtC, UgtD and homologs thereof.
15. The method of claim 1, further wherein the at least one moiety capable of
being glycosylated is incubated with the at least one novel nucleotide sugar in
the presence more than one glycosyltransferase.
16. A method comprising incubating at least one glycosylated compound produced
by the method of claim 1 that is capable of being glycosylated with and at least
one second nucleotide sugar in the presence of at least one second glycosyltransferase
to produce at least one twice-glycosylated compound having at least a first and
a second glycosyl attachment.
17. The method of claim 16, further wherein the first and second glycosyl attachments
are the same.
18. The method of claim 16, further wherein the first and second glycosyl attachments
are different.
19. The method of claim 16, further wherein the both the first and the second
glycosyl attachments are attached to the moiety capable of being glycosylated.
20. The method of claim 16, further wherein the second glycosyl attachment is
attached to the first glycosyl attachment.
21. The method of claim 16, further wherein the first and second glycosyl transferases
are the same.
22. The method of claim 16, further wherein the first and second glycosyl transferases
are different.
23. The method of claim 16, further wherein the at least one second nucleotide
sugar is the same as the at least one first nucleotide sugar.
24. The method of claim 16, further wherein the at least one second nucleotide
sugar is different than the at least one first nucleotide sugar.
Description
FIELD OF THE INVENTION
The present invention is directed to glycosyltransferases and methods for their
use. The present invention is also directed to methods of synthesizing novel glycosylated compounds.
BACKGROUND
A recent estimate suggests roughly 70% of current lead compounds in modern drug
discovery derive directly from the natural products, many of which are glycosylated
bacterial metabolites. Potier, P.
Actual. Chim. 11: 9 (1999). Thus, bacterial
glycosyltransferases and their corresponding sugar substrates contribute significantly
to the diversity of pharmaceutically important metabolites. A glycosylated metabolite
is one that is comprised of both a central core structure (often called the "aglycon")
and various sugar (or "glycosyl") attachments.
Carbohydrates are able to exhibit target specificity and often the
affinity of carbohydrate ligands for their target are defined by the structure
and length of the sugar chain carried by the aglycon. Traditionally, carbohydrate
ligands of bioactive agents have been implicated in the control of drug pharmacokinetics
such as absorption, distribution, metabolism and/or excretion. However, recent
growing evidence has led to a change in this dogmatic view.
Pyran (or furan) ring rigidity in conjunction with glycosidic bond flexibility
lends itself to preorganization while deoxygenated and/or functionalized sugars
also provide unusual hydrophobic and hydrophilic domains. Furthermore, there exist
many examples in which removal of these critical ligands leaves barren aglycons
with little or no biological activity. Thus, carbohydrates provide great functional
diversity to secondary metabolite activity. Thorson, J. S. et al. "Nature's Carbohydrate
Chemists: The Enzymatic Glycosylation of Bioactive Bacterial Metabolites,"
Curr.
Org. Chem. 5: 139-167 (2001); Weymouth-Wilson, A. C. "The Role of Carbohydrates
in Biologically Active Natural Products,"
Nat. Prod. Rep. 14: 99-110 (1997).
Carbohydrate ligands often determine the specificity and affinity with
which bioactive metabolites bind to DNA. One of the best characterized glycoconjugates
is calicheamicin γ
1I (FIG. 1, 1), a member of the enediyne
family of antitumor antibiotics isolated from
Micromonospora echinospora. Thorson,
J. S. et al "Enediyne Biosynthesis and Self Resistance: A Progress Report,"
Bioorgan.
Chem. 27: 172-188 (1999) and references therein; Thorson, J. S. et al. "Understanding
and Exploiting Nature's Chemical Arsenal: The Past, Present and Future of Calicheamicin
Research,"
Curr. Pharm. Des. 6: 1841-1879 (2000) and references therein.
The aryltetrasaccharide of calicheamicin defines both the DNA binding specificity
and the high affinity (estimated to be 10
6-10
8) of calicheamicin.
In the related enediyne neocarzinostatin (FIG. 1, 2), the carbohydrate ligand
is 2,6-dideoxy-2-methylamino-α-D-galacto-hexopyranose (2-N-methyl-α-D-fucosamine)
and, in contrast to most minor groove-binding aminoglycosyl ligands, the neocarzinostatin
pyranose acts as an anchor, through numerous intermolecular contacts, and defines
how deep neocarzinostatin can penetrate the major groove. This locks the molecule
into position and thus, ultimately defines the specific sites of DNA-cleavage as
well as enhances (possibly as an internal base) the efficiency of cleavage. Stassinopoulos,
A. et al. "Solution Structure of a Two-Base DNA Bulge Complexed with an Enediyne
Cleaving Analog,"
Science 272: 1943-1946 (1996); Myers, A. G. et al. "A
Comparison of DNA Cleavage by Neocarzinostatin Chromophore and Its Aglycon: Evaluating
the Role of the Carbohydrate Residue,"
J. Am. Chem. Soc. 119: 2965-2972 (1997).
Like the sugar ligands of calicheamicin γ
1I and
neocarzinostatin, the carbohydrate ligands of anthracyclines (e.g. daunorubicin,
5, among the most potent and widely used anticancer agents) are known to contribute
directly to DNA binding, via intermolecular contacts, and to retard the activity
of polymerases in some cases. Also, a direct correlation between increased glycosylation
and lower toxicity has been demonstrated. Kirschning, A. et al. "Chemical and Biochemical
Aspects of Dexoysugars and Deoxysugar Oligosaccharides,"
Top. Curr. Chem. 188:
1-84 (1997). Similar roles for the carbohydrates in DNA minor groove binding of
the pluramycin antitumor antibiotics (e.g. altromycin B, a DNA alkylator, FIG.
1, 3), the antimicrobial aureolic acids (e.g. chromomycin A
3, an inhibitor
of replication/translation, FIG. 1, 8), and various other angucyclines, have been
observed. Hansen, M. et al. "Threads the DNA Helix Interacting with Both the Major
and Minor Grooves to Position Itself for Site-Directed Alkylation of Guanine N7
,"
J. Am. Chem. Soc. 117: 2421-2429 (1995); Pavlopoulos, S. et al. "Structural
Characterization of the 1:1 Adduct Formed between the Antitumor Antibiotic Hedamycin
and the Oligonucleotide Duplex d(CACGTG)2 by 2D NMR Spectroscopy."
Biochem.
35: 9314-9324 (1996); Pavlopoulos, S. et al. "Characterization of the Sequential
Non-covalent and Covalent Interactions of the Antitumor Antibiotic Hedamycin with
Double Stranded DNA by NMR Spectroscopy,"
J. Mol. Recognition 12: 346-354
(1999); Johnson, D. A. et al. "Mechanisms and Pathways from Recent Deoxysugar Biosynthesis
Research,"
Curr. Opin. Chem. Biol. 2: 642-649 (1998); Keniry, M. A. et al
"The Three-Dimensional Structure of the 4:1 Mithramycin:d(ACCCGGGT)2 Complex: Evidence
for an Interaction between the E Saccharides,"
Biopolymers 54: 104-114 (2000).
Saccharides of secondary metabolites are also responsible for interaction
with RNA. Examples include the orthosomycins such as the antibiotic evernimicin
(FIG. 1, 11), which specifically binds to the 50S ribosomal subunits of
E. coli
and
S. aureus and ultimately inhibits protein synthesis. McNicholas,
P. M. et al "Evernimicin Binds Exclusively to the 50S Ribosomal Subunit and Inhibits
Translation in Cell-Free Systems Derived from both Gram-Positive and Gram-Negative
Bacteria,"
Antimicrob. Agents &
Chemotherapy 44: 1121-1126 (2000).
Other examples include the macrolides (described further herein), such as erythromycin
D (FIG. 2
b, 18), which generally inhibit protein synthesis by inhibiting
the 50S ribosome via carbohydrate ligand-mediated binding with the 23S ribosomal
subunit and various proteins. Fish, S. A. et al. "Structure-Activity Studies of
Tylosin-related Macrolides,"
J. Antibiot. 49: 1044-1048 (1996). Extensive
work has established the critical importance of the macrolide carbohydrate ligands
in bioactivity. Kurihara, K. et al. "Analogues of Sixteen-Membered Macrolide Antibiotics.
I. Synthesis of 4-O-Alkyl-L-cladinose Analogues via Glycosylation,"
J. Antibiot.
49: 582-592 (1996). Likewise, the classical aminoglycosides, (e.g. streptomycin,
FIG. 1, 6) interact with the small (30S) subunit of eubacteria-type ribosomes which
generally leads to translational misreading.
Carbohydrate ligands also play a role in metabolites which interact
with cell walls/membranes. For example, the non-ribosomal peptide antibiotic vancomycin
(FIG. 1, 7) kills cells by binding to the N-acyl-D-Ala-D-Ala termini of uncrosslinked
lipid-PP-disaccharide-pentapeptides. Goldman, R. C. et al.,
Curr. Med. Chem.
7: 801 (2000). While it is known that the carbohydrate portion of vancomycin
is not directly involved in this binding event, deglycosylation or N-alkylation
of the terminal vancosamine sugar of vancomycin shows remarkably different antibacterial
profiles, while analogs with synthetically modified carbohydrates were found to
operate via a mechanism distinct from that of vancomycin. Solenberg, P. J. et al.
"Production of Hybrid Glycopeptide Antibiotics in vitro and in
Streptomyces
toyocaensis," Chem. Biol. 4: 195-202 (1997); Ge, M. et al. "Reconstruction
of Vancomycin by Chemical Glycosylation of the Pseudoaglycon,"
J. Am. Chem.
Soc. 120: 11014-11015 (1998); Thompson, C. et al "Synthesis of Vancomycin from
the Aglycon,"
J. Am. Chem. Soc. 121: 1237 (1999); Ge, M. et al. "Vancomycin
Derivatives that Inhibit Peptidoglycan Biosynthesis without Binding D-Ala-D-Ala,"
Science 284: 507-511(1999).
As another example, the polyenes, such as amphotericin B (FIG. 1, 9), bind selectively
to ergosterol in the cell membrane of susceptible fungi, inducing changes in permeability
that ultimately lead to cell death. Georgopapadakou, N. H., "Antifungals: Mechanism
of Action and Resistance, Established and Novel Drugs,"
Curr. Opin. Microbiol.
1: 547-557 (1998); Abusalah, K. M.,
Brit. J. Biomed. Sci. 53: 122 (1996).
In the amphotericin B-cholesterol aggregate cylindrical complex in the plasma membrane,
critical hydrogen-bonding contacts between the polyene sugar and sterol contribute
specificity for ergosterol over cholesterol.
Carbohydrate ligands often influence or determine interactions between
bioactive metabolites and proteins. In this regard, the indolocarbazoles are an
interesting class of metabolite. Prudhomme, M.,
Curr. Pharm. Des. 3: 265
(1997); Qu, X. G. et al. "A DNA Binding Indolocarbazole Disaccharide Derivative
Remains Highly Cytotoxic without Inhibiting Topoisomerase I,"
Anti-
Cancer
Drug Des. 14: 433-442 (1999); Bailly, C. et al. "Enhanced Binding to DNA and
Topoisomerase I Inhibition by an Analog of the Antitumor Antibiotic Rebeccamycin
Containing an Amino Sugar Residue,"
Mol. Pharmacol. 55: 377-385 (1999);
Bailly, C. et al. "Recognition of Specific Sequences in DNA by a Topoisomerase
I Inhibitor Derived from the Antitumor Drug Rebeccamycin,"
Mol. Pharmacol. 53:
77-87 (1998); Goossens, J. F. et al. "Cellular Uptake and Interaction with Purified
Membranes of Rebeccamycin Derivatives,"
Eur. J. Pharmacol. 389: 141-146
(2000). The indolocarbazoles, can be subdivided into two subgroups depending on
the nature of the linkage between the carbohydrate residue and the heterocyclic
chromophore. Compounds with the sugar attached to the two indole nitrogens (e.g.
staurosporine, FIG. 1, 12) have little or no interaction with nucleic acids but
strongly inhibit different protein kinases. In contrast, the second subgroup consists
of indolocarbazole derivatives in which the carbohydrate moiety is attached to
only one indole nitrogen, (e.g. rebeccamycin, 10) which does not inhibit PKC but
instead its activity is attributed to the ability to induce topoisomerase-I-dependent
DNA-strand breaks. These incredibly different activities attest to the critical
role of the saccharide ligand.
As another example, novobiocin (FIG. 1, 4, discussed further herein) is a naturally-occurring
coumarin which targets DNA gyrase, the bacterial type II topoisomerase which can
introduce negative supercoils into DNA using the free energy of ATP hydrolysis.
Structural analyses reveal a significant overlap of the novobiocin sugar constituent
and the binding site of the ATP adenine ring. Kampranis, S. C. et al. "Probing
the Binding of Coumarins and Cyclothialidines to DNA Gyrase,"
Biochem. 28:
1967-1976 (1999).
Macrolide antibiotics and coumarin antibiotics are clinically important
examples of biologically active glycosylated secondary metabolites. The macrolides
are a critical group of compounds due to their potent activity against Gram-positive
bacteria. These compounds are generally classified by ring size of the aglycon
lactone which contains either 12, 14, or 16 residues. Of these, the 14-membered
ring and 16-membered ring families have been extensively studied from which erythromycin
A
1, oleandromycin, spiramycin, josamycin and midecamycin are used clinically.
In general, these metabolites inhibit protein synthesis by inhibiting the 50S ribosome
via specific binding with the 23S ribosomal subunit and various proteins. Fish,
S. A. et al. (1996).
The 16-member macrolides are generally found to bind 23S rRNA and inhibit peptidyltransferase
activity while the 14-member macrolides generally inhibit the translocation of
peptidyl-tRNA. Extensive work has established the critical importance of the carbohydrate
ligands in bioactivity. Weymouth-Wilson, A. C. (1997); Kurihara, K. et al. (1996);
Bertho, G. et al. "Conformational Analysis of Ketolide, Conformations of RU 004
in Solution and Bound to Bacterial Ribosomes,"
J. Med. Chem. 41: 3373-3386
(1998); Bertho, G. et al. "Solution Conformation of Methylated Macrolide Antibiotics
Roxithromycin and Erythromycin Using NMR and Molecular Modeling. Ribosome-bound
Conformation Determined by TRNOE and Formation of Cytochrome P450-metbolite Complex,"
Internatl. J. Biol. Macromol. 22: 103-127 (1998); Bertho, G. et al. "Transferred
Nuclear Overhauser Effect Study of Macrolide-Ribosome Interactions: Correlation
between Antibiotic Activities and Bound Conformations,"
Biorg. &
Med.
Chem. 6: 209-221 (1998); Gharbi-Benarous, J. et al.
J. Chem. Soc. Per. Trans.
II 529 (1999); Verdier, L. et al.
Biorgan. &
Med. Chem. 8: 1225 (2000).
Katz and coworkers have demonstrated the biosynthesis of the megalomicins (e.g.
FIG. 2, 19) proceeds from erythronolide B (16) in a stepwise manner (FIG. 2
b)
and interestingly, the conversion of erythromycin D (18) to megalomycin A (19),
via oxidation and the addition of a single sugar 2,3,4,6-tetradeoxy-3-dimethylamino-β-D-threo-hexopyranose
(megosamine), changes the molecule's activity from an antibiotic (erythromycin
D) to an antiparasitic/antiviral agent (megalomycin A). Volchegursky, Y. et al.
"Biosynthesis of the Anti-Parasitic Agent Megalomicin: Transformation of Erythromycin
to Megalomicin in
Saccharopolyspora erythraea," Mol. Microbiol. 37: 752-762 (2000).
Novobiocin (FIG. 1, 4) is a naturally-occurring coumarin from
Streptomyces
spheroides which targets DNA gyrase. DNA gyrase from
E. coli is an A
2B
2
complex in which each polypeptide displays distinct functional domains and the
coumarins specifically inhibit the ATPase reaction of GyrB in a competitive manner.
The complexes of the 24 kDa GyrB fragment with novobiocin and a related coumarin,
chlorbiocin, show the binding sites for ATP and coumarins partially overlap. Tsai,
F. T. F et al.
Proteins 28: 41 (1997); Lewis, R. J. et al.
EMBO J. 15:
1412 (1996). In particular, these high resolution structures reveal a significant
overlap of the drug sugar constituent (3-O-aminocarbonyl)-6-deoxy-5-C-methyl-4-O-methyl-β-D-lyxo-hexopyranose,
also known as β-D-noviose, in novobiocin) and the binding site of the ATP
adenine ring with specific sugar-protein hydrogen-bonding interactions between
the sugar C-2 and Asn 46, the sugar C-3 amide carbonyl with Thr 165 and amine with
Asp 73/Val 43 main chain atoms. Site directed mutagenesis of these GyrB amino acids
supports the structural assignments. Kampranis, S. C. et al.
Biochem. 28:
1967 (1999). Interestingly, while these interactions are critical, the replacement
of D-noviose with L-rhamnose has recently provided analogs with similar activity
and potency. Ferroud, D. et al.
Biorgan. &
Med. Chem. Lett. 9: 2881
(1999). Furthermore, replacement of the C-3 acylamino substituent with reversed
isosteres also provided highly potent analogs. Laurin, P. et al.
Biorgan. &
Med. Chem. Lett. 9: 2079 (1999). Recent studies also demonstrate a unique
interaction of novobiocin with heat shock protein 90 (Hsp90), which shares homology
with the a typical ATP-binding domaining of
E. coli GyrB and stabilizes
several oncogenic protein kinases. Marcu, M. G.
J. Nat. Cancer Inst. 92:
242 (2000).
The gene cluster from
S. spheroides which encodes for novobiocin biosynthesis
and self resistance was recently cloned and a single glycosyltransferase gene (novM,
accession AAF67506) was identified. Steffensky, M. et al.
Antimicrob. Agents
Chemotherap. 44: 1214 (2000). Given novobiocin contains a single saccharide,
it is presumed novM encodes for the transfer of D-noviose from the activated dTDP-D-noviose
to the aglycon novobiocic acid (FIG. 4, 20). The coumarins, while much more potent
inhibitors of DNA gyrase in vitro than the clinically utilized quinolones, have
failed clinically due to poor cell penetration, low solubility and toxicity in
eukaryotes (perhaps due to this Hsp90 interaction). Thus, as an example of an area
where engineering of secondary metabolites will be useful, glycosylated metabolites
based on the coumarin aglycon but having altered carbohydrate moities may produce
clinically useful compounds.
Both glycosyltransferases and nucleotidyltransferases play critical roles in
the formation of glycosylated secondary metabolites. The first step in metabolite
glycosylation is the reversible conversion of an α-D-hexose-1-phosphate to
the corresponding nucleotide diphospho (NDP) hexose. Enzymes that catalyze this
type of reaction (known as α-D-hexose-1-phosphate nucleotidyltransferases)
are prevalent in nature and, regardless of their origins, are generally allosterically
controlled with catalysis proceeding via an ordered bi-bi mechanism. Liu, H.-w.
et al. "Pathways and Mechanisms in the Biogenesis of Novel Deoxysugars by Bacteria,"
Annu. Rev. Microbiol. 48: 223-256 (1994).
The culminating attachment of a carbohydrate to a secondary metabolite aglycon
(or growing saccharide chain) is catalyzed by the family of enzymes known as glycosyltransferases.
These enzymes transfer a sugar, from its activated form (a nucleotide diphospho-sugar
or NDP-sugar), to an acceptor nucleophile to form a glycosidic bond and NDP. These
enzymes can catalyze transfer with retention (with respect to the NDP-sugar) or
inversion of anomeric stereochemistry. Drawing from the glycosidase analogy, the
current belief is "retaining" glycosyltransferases proceed via a double displacement
mechanism, which utilizes an enzyme-glycoside covalent intermediate, while the
"inverting" transferases proceed via a single displacement mechanism. Sinnott,
M. L. "Mechanisms of Glycosyl Hydrolysis and Transfer,"
Chem. Rev. 90: 1171-1202-1265
(1990). Based upon the known glycosylated metabolites, the majority of glycosyltransferases
in secondary metabolism are "inverting" enzymes and the acceptor nucleophile is
most often an aglycon or carbohydrate-derived heteroatom (O, N or S).
There are currently more than 70 putative secondary metabolite glycosyltransferase
genes in the public database and these can be divided into three major families
based upon sequence alignments. Thorson J. S. et al. (2001). Class I is the largest
family and contains glycosyltransferases from both aromatic and macrolide metabolite
pathways, Class II is predominately comprised of transferases associated with non-ribosomal
peptides and glycolipids, while the majority of Class III enzymes are involved
in metabolite inactivation. The number of known and putative secondary metabolite
glycosyltransferase genes in the public database is growing rapidly, as this is
an active area of research.
A number of genetic in vivo experiments have demonstrated that the glycosyltransferases
of secondary metabolism (which include those for anthracyclines, angucyclines,
nonribosomal peptides, macrolides and enediynes) are promiscuous with respect to
the NDP-sugar donor. Thorson J. S. et al. (2001); Hutchinson, C. R. "Combinatorial
Biosynthesis for New Drug Discovery,"
Curr. Opin. Microbiol. 1: 319-329
(1998). While these in vivo experiments have provided novel metabolites, the newly
formed metabolites, in most cases, were inactivated via host-catalyzed modification
to prevent killing the host producing organism. Thus, in biosynthetically altering
glycosylation, an in vitro scheme is desirable to eliminate this interference by
host inactivation mechanisms.
The glycosyltransferases of secondary metabolism rely almost exclusively upon
pyrimidine (uridine or thymidine) diphosphosugars, yet, in vitro studies in this
area are severely lacking due to the inability to access the appropriate NDP-sugar
substrates. Easy access to UDP- or dTDP-sugars would revolutionize the biochemical
characterization and exploitation of these critical glycosyltransferases.
Surprisingly, a three dimensional structure for any enzyme from this
important class of enzymes is lacking and of the many nucleotidyltransferases studied,
the dTDP-α-D-glucose forming thymidylyltransferases have received the least
attention. The best characterized thymidylyltransferase (rmlA-encoded E
p)
is from
Salmonella, which catalyzes the reaction shown in FIG. 2
a.
Lindquist, L. et al. "Purification, Characterization and HPLC Assay of Salmonella
Glucose-1-phosphate Thymidylyltransferase from the Cloned rfbA Gene,"
Eur. J.
Biochem. 211: 763-770 (1993). Preliminary E
p substrate specificity
studies, limited to only a few commercially available hexopyranosyl phosphates
and NTPs, revealed E
p could utilize both dTTP and UTP as well as α-D-glucosamine-1-phosphate
as a substitute for natural substrate (α-D-glucose-1-phosphate). Kinetic
analysis revealed a ping-pong mechanism with K
m values for the forward
direction for dTTP and α-D-glucose-1-phosphate of 0.02 mM and 0.11 mM, respectively.
In the reverse reaction the K
m values for dTDP-α-D-glucose and
diphosphate were 0.083 mM and 0.15 mM, respectively. Lindquist, L. et al. (1993).
The above examples illustrate that carbohydrate ligands often define the biological
activity of a particular secondary metabolite and suggest alteration of saccharide
ligands should lead to new compounds which may display novel biological activity.
However, the complex structure of most glycosylated natural products preclude the
ability to synthetically exchange their sugar ligands.
Further, while in vivo experiments have provided novel metabolites, the
newly formed metabolites, in most cases, were inactivated via host-catalyzed modification
to prevent killing the host producing organism. As the organisms producing the
novel metabolites are killed, it is not feasible to produce sufficient amounts
of novel metabolites for analysis or therapeutic use in in vivo systems. Additionally,
producing novel metabolites in vivo requires the use of recombinant DNA technology
to alter gene expression. Such methods are too time consuming for rapid production
of numerous novel metabolites for testing as drug candidates. Further still, the
production of these new agents was also severely limited by the host's biosynthetic
machinery so that the number and diversity of compounds that may be produced by
such methods is likewise severely limited.
Thus, for biosynthetically altering glycosylation, an in vitro scheme is needed
to eliminate the problems associated with in vivo manipulation. Further, a scheme
that allows such manipulation despite the complexities of biologically active secondary
metabolites is needed.
SUMMARY OF THE INVENTION
The present invention provides combinatorial methods for rapidly generating a
diverse library of glycorandomized structures, comprising incubating one or more
aglycons and a pool of NDP-sugars in the presence of a glycosyltransferase. The
glycosyltransferase may be one that is associated with or involved in production
of natural secondary metabolites, or one which is putatively associated with or
involved in production of natural secondary metabolites. The glycosyltransferase
may show significant flexibility with respect to its NDP-sugar donors and/or its
aglycons. NDP-sugar donors may be commercially available, or may be produced by
utilizing mutant or wild type nucleotidyltransferases significant flexibility with
respect to their substrates.
The present invention provides a novel method of chemo-enzymatic synthesis of
glycosylated entities. The present invention provides a simple and efficient method
to bypass the severe barriers to synthesis posed by both the complexities of biologically
active secondary metabolites and the difficulties and limitations of in vivo manipulation,
for the first time providing the ability to construct large libraries of diverse
macrolides with varied carbohydrate attachments as therapeutic candidates and for
use in, e.g., biomedical processes, production of downstream compounds, and biomedical
and chemical research.
The present invention enables the rapid synthesis of compounds (typically based
upon natural products) too complex for chemical synthesis but not accessible by biosynthesis.
The present invention enables the rapid generation of libraries of novel chemical
entities not available through synthesis or biosynthesis. Since these compounds
are generally based on biologically active natural products and the carbohydrate
ligands being randomized are generally critical to this activity, the potential
for compounds with novel activities is great.
The present invention provides methods of glycorandomization and methods for
producing novel compounds through the use of glycorandomization.
The present invention provides methods for producing novel glycosylated entities.
The present invention provides chemo-enzymatic methods for altering any given glycosylated
entity or entity capable of being glycosylated to produce novel entities. In a
preferred embodiment of the present invention, novel entities with enhanced or
unique biological activities are produced. Entities which may be altered include,
but are not limited to, natural and synthetic aglycons, natural product metabolites,
enediynes, anthracyclines, angucyclines, aureolic acids, orthosomycins, macrolides,
aminoglycosides, non-ribosomal peptides, polyenes, steroids, lipids, indolocarbazoles,
bleomycins, amicetins, benzoisochromanequinones coumarins, polyketides, pluramycins,
aminoglycosides, oligosaccharides, peptides, proteins, numerous other classes of
bioactive compounds, and hybrids consisting of one or more these components.
In one embodiment, a method of the present invention comprises incubating a pool
of entities capable of being glycosylated with a glycosyltransferase (which may
also be referred to herein as glycosyltransferases) and a pool of nucleotidyl sugars
to produce a glycosylated entity.
In certain embodiments, the pool of sugars consists of a single sugar. In other
embodiments, the pool of sugars comprises different sugars. In one such embodiment,
the pool of sugars comprises a population of sugars that is highly diverse. In
certain embodiments, the pool of sugars comprises known nucleotidyl sugars and/or
novel nucleotidyl sugars.
In certain embodiments, the pool of NDP-sugar donors comprises naturally occurring
sugars. In certain embodiments, the pool of NDP-sugar donors comprises novel or
"unnatural" sugars. In certain embodiments the pool of NDP-sugar donors comprises
or is selected from a library or libraries of NDP-sugars catalyzed by utilizing
the promiscuity of wild type and/or engineered
Salmonella enterica LT2 α-D-glucopyranosyl
phosphate thymidylyltransferase (Ep).
In certain embodiments, at least one of the at least one nucleotide sugar is
selected
from the group consisting of Uridine 5′-(α-D-allopyranosyl diphosphate);
Uridine 5′-(α-D-altropyranosyl diphosphate); Thymidine 5′-(α-D-gulopyranosyl
diphosphate); Uridine 5′-(α-D-gulopyranosyl diphosphate); Thymidine
5′-(α-D-idopyranosyl diphosphate); Uridine 5′-(α-D-idopyranosyl
diphosphate); Thymidine 5′-(α-D-talopyranosyl diphosphate); Uridine
5′-(α-D-talopyranosyl diphosphate); Thymidine 5′-(6-amino-6-deoxy-α-D-glucopyranosyl
diphosphate); Uridine 5′-(6-amino-6-deoxy-α-D-glucopyranosyl diphosphate);
Thymidine 5′-(4-amino-4-deoxy-α-D-glucopyranosyl diphosphate); Uridine
5′-(4-amino-4-deoxy-α-D-glucopyranosyl diphosphate); Thymidine 5′-(3-amino-3-deoxy-α-D-glucopyranosyl
diphosphate); Uridine 5′-(3-amino-3-deoxy-α-D-glucopyranosyl diphosphate);
Thymidine 5′-(2-amino-2-deoxy-α-D-glucopyranosyl diphosphate); Uridine
5′-(2-amino-2-deoxy-α-D-glucopyranosyl diphosphate); Thymidine 5′-(6-acetamido-6-deoxy-α-D-glucopyranosyl
diphosphate); Uridine 5′-(6-acetamido-6-deoxy-α-D-glucopyranosyl diphosphate);
Thymidine 5′-(4-acetamido-4-deoxy-α-D-glucopyranosyl diphosphate);
Uridine 5′-(4-acetamido-4-deoxy-α-D-glucopyranosyl diphosphate); Thymidine
5′-(3-acetamido-3-deoxy-α-D-glucopyranosyl diphosphate); Uridine 5′-(3-acetamido-3-deoxy-α-D-glucopyranosyl
diphosphate); Thymidine 5′-(2-acetamido-2-deoxy-α-D-glucopyranosyl
diphosphate); Uridine 5′-(2-acetamido-2-deoxy-α-D-glucopyranosyl diphosphate);
Thymidine 5′-(4-amino-4,6-dideoxy-α-D-glucopyranosyl diphosphate);
Uridine 5′-(4-amino-4,6-dideoxy-α-D-glucopyranosyl diphosphate); Thymidine
5′-(α-D-glucopyran-6-uronic acid diphosphate); Uridine 5′-(α-D-glucopyran-6-uronic
acid diphosphate); Thymidine 5′-(α-D-arabinopyranosyl diphosphate);
Uridine 5′-(α-D-arabinopyranosyl diphosphate); and
##STR1##
##STR2##
##STR3##
##STR4##
##STR5##
##STR6##
In certain other embodiments, at least one of the at least one nucleotide sugar
is selected from the group consisting of Thymidine 5′-(α-D-glucopyranosyl
diphosphate); Uridine 5′-(α-D-glucopyranosyl diphosphate); Thymidine
5′-(2-deoxy-α-D-glucopyranosyl diphosphate); Uridine 5′-(2-deoxy-α-D-glucopyranosyl
diphosphate); Thymidine 5′-(3-deoxy-α-D-glucopyranosyl diphosphate);
Uridine 5′-(3-deoxy-α-D-glucopyranosyl diphosphate); Thymidine 5′-(4-deoxy-α-D-glucopyranosyl
diphosphate); Uridine 5′-(4-deoxy-α-D-glucopyranosyl diphosphate);
Thymidine 5′-(6-deoxy-α-D-glucopyranosyl diphosphate); Uridine 5′-(6-deoxy-α-D-glucopyranosyl
diphosphate); Thymidine 5′-(α-D-mannopyranosyl diphosphate); Uridine
5′-(α-D-mannopyranosyl diphosphate); Thymidine 5′-(α-D-galactopyranosyl
diphosphate); Uridine 5′-(α-D-galactopyranosyl diphosphate); Thymidine
5′-(α-D-allopyranosyl diphosphate); and Thymidine 5′-(α-D-altropyranosyl diphosphate).
The present invention provides a method for producing novel glycosylated compounds
comprising: combining at least one moiety capable of being glycosylated and at
least one first nucleotide sugar in the presence of at least one first glycosyltransferase,
wherein the method is carried out in vitro and at least one novel glycosylated
compound is produced.
The present invention provides a method comprising combining (a) at least one
moiety capable of being glycosylated and (b) at least one first nucleotide sugar
produced by combining nucleotide triphosphate (NTP) and at least one sugar phosphate
in the presence of at least one mutated nucleotidyltransferase; in the presence
of at least one first glycosyltransferase, wherein at least one glycosylated compound
is produced.
In certain embodiments, at least one of the at least one mutated nucleotidyltransferase
is E
p mutated at one or more amino acids selected from the group consisting
of V173, G147, W224, N112, G175, D111, E162, T201, I200, E199, R195, L89, L89T,
L109, Y146 and Y177. In certain embodiments, at least one of the at least one mutated
nucleotidyltransferase is E
p mutated at one or more amino acids in its
active site, its divalent cation binding site, and/or its auxiliary site.
Methods according to the present invention are preferably carried out in vitro.
In certain preferred embodiments, at least on of the at least one novel glycosylated
compounds produced has enhanced and/or unique biological activity as compared to
at least one of the at least one moieties capable of being glycosylated. In certain
other preferred embodiments, more than one type of glycosylated compound is produced
in a single reaction vessel and at least one of the at least one glycosylated compounds
produced is a novel glycosylated compound.
In certain other preferred embodiments, highly diverse population of glycosylated
compounds is produced and at least one of the at least one glycosylated compounds
produced is a novel glycosylated compound.
In certain embodiments, at least one of the at least one moiety capable of being
glycosylated is selected from the group consisting of natural and synthetic metabolites,
pyran rings, furan rings, enediynes, anthracyclines, angucyclines, aureolic acids,
orthosomycins, macrolides, aminoglycosides, non-ribosomal peptides, polyenes, steroids,
lipids, indolocarbazoles, bleomycins, amicetins, benzoisochromanequinones coumarins,
polyketides, pluramycins, aminoglycosides, oligosaccharides, peptides, proteins,
and hybrids thereof.
In certain other embodiments, at least one of the at least one moiety capable
of being glycosylated is selected from the group consisting of aglycons of bioactive
anthracyclines, angucyclines, nonribosomal peptides, macrolides, enediynes, indolocarbazoles,
pluramycins, aurelolic acids, orthosomycins, aminoglycosides, coumarins, bleomycins,
amicetins, polyenes, benzoisochromanequinones, angucyclines, and hybrids thereof.
In certain other embodiments, at least one of the at least one moiety capable
of being glycosylated is selected from the group consisting of enediynes, anthracyclines,
angucyclines, aureolic acids, orthosomycins, macrolides, aminoglycosides, non-ribosomal
peptides, polyenes, steroids, lipids, indolocarbazoles, bleomycins, amicetins,
benzoisochromanequinones coumarins, polyketides, pluramycins, aminoglycosides,
oligosaccharides, peptides, proteins, and hybrids consisting of one or more these components.
In certain embodiments, at least one of the at least one first glycosyltransferase
is selected from the group consisting of CalB, CalE, CalN, CalU, Gra orfl4, Gra
orf5, LanGT1, LanGT2, LanGT3, LanGT4, MtmGI, MtmGII, MtmGTIII, MtmGTIV, NovM, RhlB,
Rif orf7, SnogD, SnogE, SnogZ, UrdGT1a, UrdGT1b, UrdGT1c, UrdGT2, AknK, AknS, DesVII,
DnrS, OleG1, OleG2, TylCV, TylMII, TylN, DauH, DnrH, EryBV, EryCIII, Ngt, BgtA,
BgtB, BgtC, GftA, GftB, GftC, GftD, GftE, Gp1-1, Gp1-2, RtfA, AveBI, BlmE, BlmF,
MgtA, NysD1, OleD, OleI, SpcF, SpcG, StrH, Ugt51B1, Ugt51C1, UGT52, UgtA, UgtB,
UgtC, UgtD and homologs thereof; is selected from the group consisting of those
glycosyltransferases known to be involved in the synthesis of bioactive metabolites;
or is is produced by expressing the product of a putative glycosyltransferase gene.
In certain embodiments, more than one moiety capable of being glycosylated is
incubated with the at least one novel nucleotide sugar in the presence at least
one type of glycosyltransferase.
In certain embodiments, at least one moiety capable of being glycosylated is
incubated
with more than one novel nucleotide sugar in the presence more than one type of glycosyltransferase.
In certain embodiments, at least one moiety capable of being glycosylated is
incubated
with the at least one novel nucleotide sugar in the presence more than one type
of glycosyltransferase.
The present invention also provides a method comprising incubating at least one
glycosylated compound produced by the method of claim C that is capable of being
glycosylated with and at least one second nucleotide sugar in the presence of at
least one second glycosyltransferase to produce at least one twice-glycosylated
compound having at least a first and a second glycosyl attachment, wherein the
first and second may be of the same type or of different types and the second glycosyl
attachment may be attached to the original moiety capable of being glycosylated
or to the first glycosyl attachment.
The present invention provides a method comprising subjecting at least one glycosylated
compound produced according to the methods of the present invention to repeated
cycles of incubation with at least one nucleotide sugar in the presence of at least
one glycosyltransferase until a population multiply-glycosylated compounds of the
desired type and size is achieved.
The present invention also provides novel compounds produced by the methods of
the present invention. Non-limiting examples of the such novel compounds that are
provided by the present invention include two novel novobiocin (designated Nov-1
and Nov-2) derivatives and six novel erythromycin (designated Ery-1-Ery-6) analogs.
##STR7##
##STR8##
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 provides chemical structures of several bioactive metabolites.
FIG. 2(
a) provides chemical structures illustrating a portion
of the biosynthesis of megosamine. FIG. 2(
b) provides chemical structures
illustrating a portion of the biosynthesais of erythromycin D and Megalomicin A.
FIG. 3 provides chemical structures for new chemical entities (NCEs) Ery-1,
Ery-2, Ery-3, Ery-4, Ery-5, and Ery-6. FIG. 3 also provides chemical structures
illustrating a portion of the biosynthesis of these NCEs.
FIG. 4 provides chemical structures for new chemical entities (NCEs) Nov-1 and
Nov-2. FIG. 3 also provides chemical structures illustrating a portion of the biosynthesis
of these NCEs.
FIG. 5 provides a chemical structure for antitumor agent mithramycin.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a simple and efficient method to bypass the severe
barriers to synthesis posed by both the complexities of biologically active secondary
metabolites and the difficulties and limitations of in vivo manipulation, for the
first time providing the ability to construct large and diverse libraries macrolides
with varied carbohydrate attachments.
The present invention utilizes the promiscuity of nucleotidyltransferases and
glycosyltransferases for their respective substrates and donor molecules to provide
a method for producing libraries of glycosylated entities, which then may be screened
by methods known in the art for compounds useful in, e.g., clinical therapy, biomedical
research, and chemical synthesis of downstream products.
A number of genetic in vivo experiments have demonstrated that the glycosyltransferases
of secondary metabolism (which include those for anthracyclines, angucyclines,
nonribosomal peptides, macrolides and enediynes) are promiscuous with respect to
the NDP-sugar donor.
However, prior in vitro studies in this area were severely limited due to
the inability to access the appropriate NDP-sugar substrates.
The present inventors recently vastly increased the pool of UDP- and dTDP-sugar
substrates available by systematically re-examining the substrate specificity of
purified E
p, which revealed this enzyme can accommodate a wide array
of hexopyranosyl phosphates as a replacement for FIG. 2, 14 in this reaction. See,
e.g., Jiang J, et al., "Expanding the Pyrimidine Diphosphosugar Repertoire: The
Chemoenzymatic Synthesis of Amino- and Acetamidoglucopyranosyl Derivatives"
Angew
Chem Int Ed Engl 40(8): 1502-1505 (2001); Jiang J, et al., "A General Enzymatic
Method for the Synthesis of Natural and ‘Unnatural’ UDP- and TDP-Nucleotide
Sugars,"
Journal of the American Chemical Society 122(28): 6803-6804 (2000).
In comparison to the tedious chemical synthesis of nucleotide sugars, this one-step
E
p-catalyzed enzymatic conversion is a rapid and effective method to
construct libraries of both the desired UDP- and dTDP-nucleotide diphosphosugars
for
