U.S. patent application number 15/003362 was filed with the patent office on 2016-08-11 for large scale enzymatic synthesis of oligosaccharides.
The applicant listed for this patent is Academia Sinica. Invention is credited to Tsung-I TSAI, Chi-Huey WONG, Chung-Yi WU.
Application Number | 20160230201 15/003362 |
Document ID | / |
Family ID | 50100297 |
Filed Date | 2016-08-11 |
United States Patent
Application |
20160230201 |
Kind Code |
A1 |
WONG; Chi-Huey ; et
al. |
August 11, 2016 |
LARGE SCALE ENZYMATIC SYNTHESIS OF OLIGOSACCHARIDES
Abstract
A novel UDP-Gal regeneration process and its combined use with a
galactosyltransferase to add galactose to a suitable acceptor
substrate. Also described herein are synthetic methods for
generating Globo-series oligosaccharides in large scale, wherein
the methods may involve the combination of a glycosyltransferase
reaction and a nucleotide sugar regeneration process.
Inventors: |
WONG; Chi-Huey; (Rancho
Santa Fe, CA) ; TSAI; Tsung-I; (Taipei, TW) ;
WU; Chung-Yi; (New Taipei City, TW) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Academia Sinica |
Taipei |
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TW |
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|
Family ID: |
50100297 |
Appl. No.: |
15/003362 |
Filed: |
January 21, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13971353 |
Aug 20, 2013 |
9340812 |
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15003362 |
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61684974 |
Aug 20, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12P 19/18 20130101;
C12Y 207/0701 20130101; C12P 19/04 20130101 |
International
Class: |
C12P 19/18 20060101
C12P019/18; C12P 19/04 20060101 C12P019/04 |
Claims
1-34. (canceled)
35. A method for enzymatically synthesizing an oligosaccharide,
comprising: (i) producing UDP-GalNAc from GalNAc in the presence of
a set of UDP-GalNAc regeneration enzymes, wherein the set of
UDP-GalNAc regeneration enzymes comprises an N-acetylhexosamine
1-kinase, an N-acetylglucosamine 1-phosphate uridyltransferase, a
pyruvate kinase, and optionally, a pyrophosphatase, and (ii)
converting Gb3-OR.sup.1A into Gb4-OR.sup.1A in the presence of the
UDP-GalNAc and a beta-1,3-N-acetylgalactosaminyltransferase,
wherein R.sup.1A is hydrogen, substituted or unsubstituted alkyl,
substituted or unsubstituted alkenyl, substituted or unsubstituted
alkynyl, substituted or unsubstituted carbocyclyl, substituted or
unsubstituted heterocyclyl, substituted or unsubstituted aryl,
substituted or unsubstituted heteroaryl, or an oxygen protecting
group.
36. The method of claim 35, wherein (i) and (ii) occur in a
Gb4-synthesis reaction mixture comprising GalNAc, PEP, ATP, UTP,
the Gb3-OR.sup.1A, the beta-1,3-N-acetylgalactosaminyltransferase,
and the set of UDP-GalNAc regeneration enzymes.
37. The method of claim 35, wherein the
beta-1,3-N-acetylgalactosaminyltransferase is LgtD from H.
influenza, the N-acetylhexosamine 1-kinase is from B. longum, the
N-acetylglucosamine 1-phosphate uridyltransferase is from E. coli,
the pyruvate kinase is from E. coli, or the pyrophosphatase is from
E. coli.
38. The method of claim 35, wherein the R.sup.1A is hydrogen,
allyl, substituted alkyl, biotin, or a ceramide.
39. The method of claim 35, further comprising isolating the
Gb4-OR.sup.1A.
40. The method of claim 35, further comprising: (iii) converting
the Gb4-OR.sup.1A into Gb5-OR.sup.1A in the presence of UDP-Gal and
a beta-1,3-galactosyltransferase.
41. The method of claim 40, further comprising: (iv) producing the
UDP-Gal from galactose in the presence of a set of UDP-Gal
regeneration enzymes, wherein the set of UDP-Gal regeneration
enzymes comprises a galactokinase, an UDP pyrophosphorylase, a
pyruvate kinase, and optionally, a pyrophosphatase.
42. The method of claim 41, wherein (iii) and (iv) occur in a
Gb5-synthesis reaction mixture comprising galactose, PEP, ATP, UTP,
the Gb4-OR.sup.1A, the beta-1,3-galactosyltransferase, and the set
of UDP-Gal regeneration enzymes.
43. The method of claim 40, wherein the
beta-1,3-galactosyltransferase is LgtD from H. influenza; the
galactokinase is from E. coli, the UDP-sugar pyrophosphorylase is
from A. thaliana, the pyruvate kinase is from E. coli, or the
pyrophosphatase is from E. coli.
44. The method of claim 40, further comprising isolating the
Gb5-OR.sup.1A.
45. The method of claim 40, further comprising: (v) converting the
Gb5-OR.sup.1A into Fucosyl-Gb5-OR.sup.1A in the presence of GDPFuc
and an alpha-1,2-fucosyltransferase.
46. The method of claim 45, further comprising: (vi) producing the
GDP-Fuc from fucose in the presence of a set of GDP-Fuc
regeneration enzymes, wherein the set of GDP-Fuc regeneration
enzymes comprises an L-fucokinase/GDP-fucose pyrophosphorylase, a
pyruvate kinase, and optionally, a pyrophosphatase.
47. The method of claim 46, wherein (v) and (vi) occur in a
Fucosyl-Gb5-synthesis reaction mixture comprising fucose, ATP, GTP,
PEP, the Gb5-OR.sup.1A, the alpha-1,2-fucosyltransferase, and the
set of GDP-Fuc regeneration enzymes.
48. The method of claim 47, wherein the Fucosyl-Gb5-synthesis
reaction mixture is prepared by mixing the Gb5-synthesis reaction
mixture with at least fucose, GTP, the
alpha-1,2-fucosyltransferase, and the L-fucokinase/GDP-fucose
pyrophosphorylase.
49. The method of claim 45, wherein the L-fucokinase/GDP-fucose
pyrophosphorylase is from B. fragilis, or the
alpha-1,2-fucosyltransferase is from H. pylori.
50. The method of claim 45, further comprising isolating the
Fucosyl-Gb5-OR.sup.1A.
51. The method of claim 40, further comprising: (vii) converting
the Gb5-OR.sup.1A into Sialyl-Gb5-OR.sup.1A in the presence of
CMP-Neu5Ac and an alpha-2,3-sialyltransferase.
52. The method of claim 51, further comprising: (viii) producing
the CMP-Neu5Ac from Neu5Ac in the presence of a set of CMP-Neu5Ac
regeneration enzymes, wherein the set of CMP-Neu5Ac regeneration
enzymes comprises a cytidine monophosphate kinase, a CMP-sialic
acid synthetase, a pyruvate kinase, and optionally, a
pyrophosphatase.
53. The method of claim 52, wherein (vii) and (viii) occur in a
Sialyl-Gb5-synthesis reaction mixture comprising Neu5Ac, CTP, PEP,
the Gb5-OR.sup.1A, the alpha-2,3-sialyltransferase, and the set of
CMP-Neu5Ac regeneration enzymes.
54. The method of claim 53, wherein the Sialyl-Gb5-synthesis
reaction mixture is prepared by mixing the Gb5-synthesis reaction
mixture with at least Neu5Ac, CTP, the alpha-2,3-sialyltransferase,
the cytidine monophosphate kinase, and the CMP-sialic acid
synthetase.
55. The method of claim 51, wherein the alpha-2,3-sialyltransferase
is from M. bacteria, the cytidine monophosphate kinase is from E.
coli, or the CMP-sialic acid synthetase is from P. Multocida.
56. The method of claim 51, further comprising isolating the
Sialyl-Gb5-OR.sup.1A.
57-99. (canceled)
Description
RELATED APPLICATIONS
[0001] The present application claims priority under 35 U.S.C.
.sctn.119(e) to U.S. Provisional Patent Application No. 61/684,974,
filed Aug. 20, 2012, which is herein incorporated by reference in
its entirety.
BACKGROUND
[0002] Globopentaose (Gb5), fucosyl-Gb5 (Globo H), and sialyl-Gb5
(SSEA4) are globo-series glycosphingolipid and were first
discovered in 1983 in cultured human teratocarcinoma cell
line.sup.[1] and subsequently found in several malignant
cancers..sup.[2],[3] Report showed Globo H overexpression in up to
61%, Gb5 overexpression in 77.5% and SSEA4 overpression in 95% in
breast cancer patients..sup.[4] On the other hand, HER2 gene, the
target for therapeutic monoclonal antibody Trastuzumab (Herceptin)
that interferes with the HER2/neu receptor, is overexpressed in
only 25% breast cancer patients.sup.[5]. The comparison clearly
demonstrated that the glycosphingolipid antigens (Gb5 and its
derivative, Globo H and SSEA4) are better candidates to be
developed into cancer vaccine. Hence, Globo H has been conjugated
to the keyhole limpet hemocyanin (KLH) as a cancer vaccine, and is
under phase II clinical trial in some country now..sup.[6]
[0003] There are several disadvantages of current methods used for
the synthesis of Gb5, Globo H and SSEA4. The traditional chemical
synthesis is tedious and labor-consuming, and several protection
and de-protection steps are necessary to achieve high purity and
correct stereotype and always lead to the very low total yields.
Till now there are many reports for the chemical synthesis of Globo
H..sup.[7] [8] [9] [10] [11] [12] [13] [14] However, only two
reports have been published for SSEA4 synthesis. Hsu et al reported
a one-pot chemical synthesis approach to assembled the glycan part
of SSEA-4 in 24% yield.sup.[15] Zhen et al. reported the use of a
chemoenzymatic method to synthesize SSEA-4 in milligram
scale..sup.[16] On the other hand, the enzymatic synthesis of Globo
H based on Leloir-type glycosyltransferase only requires the active
nucleotide sugar as donor to catalyze the glycosylation reaction.
Nonetheless, the nucleotide sugar is too expensive to synthesize in
large scale. Moreover, the by-product pyrophosphate and nucleoside
diphosphate inhibit the nucleotide sugar formation of
pyrophosphorylase.sup.[15] and Leloir-type glycosyltransferase;
therefore, how to develop a regeneration strategy is necessary to
overcome the limitation and to recharge the nucleotide to achieve
constitute nucleotide sugar product in order to continue the
reaction. During the past several years, many groups worked to
tackle the major problem of nucleotide sugar regeneration and most
of the sugar nucleotide regeneration have been solved. However,
there is still some space to improve the technology of sugar
nucleotide regeneration, especially the UDP-Gal regenerate is much
difficult. For example, UDP-Gal regeneration was first proposed in
1982 by Wong and Whiteside via UDP-Glc C4 epimerase to
interconverse UDP-Glc and UDP-Gal (.sup.[17]). Ten years later, our
group developed the secondary UDP-Gal regeneration method. Instead
of using UDP-Glc C4 epimerase, Glc-1-phosphate uridylyltransferase
located in galactose operon in E. coli was used to interchange
Gal-1-phosphate and UDP-Glc to Glc-1-phosphate and
UDP-Gal..sup.[18] However, the final pathway to directly condense
UTP and Gal-1-phosphate to form UDP-Gal was not established due to
the absence of suitable enzyme. Because the target compounds Gb5,
Globo H and SSEA4 ae Gal-related molecules, how to overcome the
major difficult of UDP-Gal regeneration and increase its efficiency
will be the key point for large scale enzymatic synthesis of Gb5,
Globo H and SSEA4.
[0004] In summary, there are several limitations to current methods
of large scale synthesizing Gb5, Globo H and SSEA4 in the art.
Thus, there is a need for new synthetic procedures that produce
Gb5, Globo H, SSEA4, and intermediates thereto in an efficient
manner.
SUMMARY OF THE INVENTION
[0005] The present disclosure is based on the development of new
nucleotide sugar regeneration processes and their applications in
sugar synthesis. Such sugar synthesis methods, involving the
combination of at least one nucleotide sugar regeneration system
(e.g., the UDP-Gal regeneration system described herein) and at
least one glycosyltransferase (e.g., galactosyltransferase), were
used in synthesizing various oligosaccharides (tailed), including
allyl-tailed Gb3, Gb4, Gb5 (also known as SSEA3), Fucosyl-Gb5 (also
known as Globo H), and Sialyl-Gb5 (also known as SSEA4), with
unexpectedly high efficiency and yields. More specifically, the
synthetic approaches described herein unexpectedly allow chain
reactions to produce final products, such as Globo H and SSEA4,
without the need to purify intermediates.
[0006] Accordingly, one aspect of the present disclosure relates to
methods for adding a galactose residue to a substrate via the
action of a galactosyltransferase coupled with a UDP-Gal
regeneration process. The method comprises: (i) producing UDP-Gal
from galactose in the presence of a set of UDP-Gal regeneration
enzymes, wherein the set of UDP-Gal regeneration enzymes comprises
a galactokinase, an UDP-sugar pyrophosphorylase, a pyruvate kinase,
and optionally, a pyrophosphatase; (ii) reacting the UDP-Gal with a
substrate molecule (e.g., a polysaccharide, an oligosaccharide, a
glycoprotein, a glycolipid, or an aglycone) via action of a
galactosyltransferase (e.g., an alpha1,4-galactosyltransferase, a
beta1,4-galactosyltransferase, an alpha1,3-galactosyltransferase,
or a beta1,3-galactosyltransferase) to add a galactose residue to
the substrate molecule; and, optionally, (iii) isolating the
galactosylated product thus produced. Steps (i) and (ii) can take
place in a reaction mixture comprising the set of UDP-Gal
regeneration enzymes, the galactosyltransferase, the substrate
molecule, galactose, ATP, and UTP. In some examples, the substrate
molecule is a ceramide or a glycosphingolipid.
[0007] Another aspect of the present disclosure relates to methods
for synthesizing oligosaccharides involving at least one nucleotide
sugar regeneration process (e.g., UDP-Gal regeneration) and at
least one reaction of adding a monosaccaride, e.g., galactose
(Gal), N-acetylgalatocoamine (GalNAc), fucose (Fuc), and sialic
acid (Neu5Ac), onto a suitable acceptor via action of a
glycosyltransferase, e.g., galactosyltransferase,
fucosyltransferase, sialyltransferase, and
N-acetylgalactosaminyltransferase.
[0008] In some embodiments, the method described herein for
enzymatically synthesizing an oligosaccharide, uses lactose (e.g.,
tailed) as the starting material. The method comprises: (i)
producing UDP-Gal from galactose in the presence of a set of
UDP-Gal regeneration enzymes, wherein the set of UDP-Gal
regeneration enzymes comprises a galactokinase (e.g., from E.
coli), an UDP-sugar pyrophosphorylase (e.g., from A. thaliana), a
pyruvate kinase (e.g., from E. coli), and optionally, a
pyrophosphatase (e.g., from E. coli); (ii) converting Lac-OR.sup.1A
into Gb3-OR.sup.1A in the presence of the UDP-Gal and an alpha-1,4
galactosyltransferase (e.g., a LgtC such as that from N.
meningitides), wherein R.sup.1A is hydrogen, substituted or
unsubstituted alkyl, substituted or unsubstituted alkenyl,
substituted or unsubstituted alkynyl, substituted or unsubstituted
carbocyclyl, substituted or unsubstituted heterocyclyl, substituted
or unsubstituted aryl, substituted or unsubstituted heteroaryl, or
an oxygen protecting group. Lac-OR.sup.1A refers to lactose
(.beta.-D-galactopyranosyl-(1.fwdarw.4)-D-glucose) (e.g., also
encompassed by Formula (I), wherein each of R.sup.2A, R.sup.3A,
R.sup.5A, R.sup.2B, R.sup.3B, and R.sup.5B is hydrogen) wherein the
group attached to the anomeric carbon of lactose is an --OR.sup.1A
group, and wherein R.sup.1A is as defined herein.
[0009] Examples of R.sup.1A include, but are not limited to
hydrogen, allyl, biotin, a ceramide, or a non-hydrogen group (e.g.,
alkyl) which is further substituted with a substituted or
unsubstituted thio, substituted or unsubstituted amino, carbonyl
(e.g., carboxylic acid), azido, alkenyl (e.g., allyl), alkynyl
(e.g., propargyl), biotin, or a ceramide group. In certain
embodiments, R.sup.1A is hydrogen, allyl, substituted alkyl,
biotin, or a ceramide.
[0010] When necessary, Gb3-OR.sup.1A can be isolated from the
reaction mixture.
[0011] Steps (i) and (ii) can occur in a Gb3-synthesis reaction
mixture comprising galactose, PEP, ATP, UTP, the Lac-OR.sup.1A, the
alpha-1,4-galactosyltransferase, and the set of UDP-Gal
regeneration enzymes. In one example, the molar ratio of the
Lac-OR.sup.1A and galactose in the Gb3-synthesis reaction mixture
is 1:1 before occurrence of any enzymatic reactions.
[0012] Any of the methods described above can further comprise:
(iii) converting the Gb3-OR.sup.1A into Gb4-OR.sup.1A in the
presence of UDP-GalNAc and a
beta1,3-N-acetylgalactosaminyltransferase (e.g., a LgtD from a
suitable organism such as H. influenza), which can be coupled with
(iv) producing the UDP-GalNAc from GalNAc in the presence of a set
of UDP-GalNAc regeneration enzymes, wherein the set of UDP-GalNAc
regeneration enzymes comprises an N-acetylhexosamine 1-kinase
(e.g., from B. longum), an N-acetylglucosamine 1-phosphate
uridyltransferase (e.g., from E. coli), and a pyruvate kinase
(e.g., from E. coli), and optionally, a pyrophosphatase (e.g., from
E. coli). Steps (iii) and (iv) can be carried out in a
Gb4-synthesis reaction mixture comprising GalNAc, PEP, ATP, UTP,
the Gb3-OR.sup.1A, the beta1,3-N-acetylgalactosaminyltransferase,
and the set of UDP-GalNAc regeneration enzymes. In one example, the
Gb4-synthesis reaction mixture is prepared by mixing the
Gb3-synthesis reaction mixture with at least GalNAc, the
beta1,3-N-acetylgalactosaminyltransferase, the N-acetylhexosamine
1-kinase, and the N-acetylglucosamine 1-phosphate
uridyltransferase. When necessary, Gb4-OR.sup.1A can be isolated
from the reaction mixture.
[0013] After synthesis of Gb4-OR.sup.1A, the method as described
above can further comprise: (v) converting the Gb4-OR.sup.1A into
Gb5-OR.sup.1A in the presence of UDP-Gal and a
beta1,3-galactosyltransferase (e.g., a LgtD such as that from H.
influenza), which can be coupled with (vi) producing the UDP-Gal
from galactose in the presence of the set of UDP-Gal regeneration
enzymes described herein. In one example, (v) and (vi) take place
in a Gb5-synthesis reaction mixture comprising galactose, PEP, ATP,
UTP, the Gb4-OR.sup.1A, the beta1,3-galactosyltransferase, and the
set of UDP-Gal regeneration enzymes. The resultant Gb5-OR.sup.1A
can be isolated from the reaction mixture.
[0014] The above method can further comprise steps for converting
the Gb5-OR.sup.1A thus obtained into Fucosyl-Gb5-OR.sup.1A (Globo
H) or into Sialyl-Gb5-OR.sup.1A (SSEA4).
[0015] For Globo H synthesis, the method can further comprise:
(vii) converting the Gb5-OR.sup.1A into Fucosyl-Gb5-OR.sup.1A in
the presence of GDP-Fuc and an alpha1,2-fucosyltransferase (e.g.,
from H. pylori), which can be coupled with (viii) producing the
GDP-Fuc from fucose in the presence of a set of GDP-Fuc
regeneration enzymes, wherein the set of GDP-Fuc regeneration
enzymes comprises a L-fucokinase/GDP-fucose pyrophosphorylase
(e.g., B. fragilis), a pyruvate kinase (e.g., from E. coli), and a
pyrophosphatase (e.g., from E. coli). In one example, steps (vii)
and (viii) occur in a Fucosyl-Gb5-synthesis reaction mixture
comprising fucose, ATP, GTP, PEP, the Gb5-OR, the
alpha1,2-fucosyltransferase, and the set of GDP-Fuc regeneration
enzymes. The Fucosyl-Gb5-synthesis reaction mixture can be prepared
by mixing the Gb5-synthesis reaction mixture with at least fucose,
GTP, the alpha1,2-fucosyltransferase, and the
L-fucokinase/GDP-fucose pyrophosphorylase. When necessary, the
resultant Fucosyl-Gb5-OR.sup.1A can be isolated from the reaction
mixture.
[0016] For SSEA4 synthesis, the method can further comprise: (ix)
converting the Gb5-OR.sup.1A into Sialyl-Gb5-OR.sup.1A in the
presence of CMP-Neu5Ac and an alpha2,3-sialyltransferase (e.g.,
from M. bacteria), and (x) producing the CMP-Neu5Ac from Neu5Ac in
the presence of a set of CMP-Neu5Ac regeneration enzymes, wherein
the set of CMP-Neu5Ac regeneration enzymes comprises a cytidine
monophosphate kinase (e.g., from E. coli), a CMP-sialic acid
synthetase (e.g., from P. Multocida), a pyruvate kinase (e.g., from
E. coli), and optionally a pyrophosphatase (e.g., from E. coli).
Steps (ix) and (x) can occur in a Sialyl-Gb5-synthesis reaction
mixture comprising Neu5Ac, CTP, PEP, the Gb5-OR.sup.1A, the alpha
2,3-sialyltransferase, and the set of CMP-Neu5Ac regeneration
enzymes. The Sialyl-Gb5-synthesis reaction mixture is prepared by
mixing the Gb5-synthesis reaction mixture with at least Neu5Ac,
CTP, the alpha 2,3-sialyltransferase, the cytidine monophosphate
kinase, and the CMP-sialic acid synthetase. The
Sialyl-Gb5-OR.sup.1A can then be isolated from the reaction
mixture.
[0017] In one example, a method for synthesizing Globo H can be
performed as follows: (i) producing UDP-Gal from galactose in the
presence of the UDP-Gal regeneration enzymes as described herein,
(ii) converting Lac-OR.sup.1A as described herein into
Gb3-OR.sup.1A in a Gb3-synthesis reaction mixture comprising at
least the UDP-Gal, an alpha-1,4 galactosyltransferase, and the
UDP-Gal regeneration enzymes, (iii) mixing the Gb3-synthesis
reaction mixture with at least GalNAc, the
beta1,3-N-acetylgalactosaminyltransferase, the N-acetylhexosamine
1-kinase, and the N-acetylglucosamine 1-phosphate uridyltransferase
to form a Gb4-synthesis reaction mixture, (iv) incubating the
Gb4-synthesis reaction mixture under conditions allowing conversion
of Gb3-OR.sup.1A to Gb4-OR.sup.1A, (v) further incubating the
Gb4-synthesis reaction mixture in the presence of a
.beta.-1,3-galactosyltransferase under conditions allowing
conversion of the Gb4-OR.sup.1A to Gb5-OR.sup.1A, (vi) mixing the
Gb5-OR.sup.1A-containing reaction mixture with at least fucose,
GTP, the alpha1,2-fucosyltransferase, and the
L-fucokinase/GDP-fucose pyrophosphorylase to form a
Fucosyl-Gb5-OR.sup.1A reaction mixture; (vii) incubating the
Fucosyl-Gb5-OR.sup.1A reaction mixture under conditions allowing
conversion of the Gb5-OR.sup.1A to Fucosyl-Gb5-OR.sup.1A, and
optionally, (viii) isolating the Fucosyl-Gb5-OR.sup.1A.
[0018] In another example, a method for synthesizing Globo H can be
performed as follows: (i) producing UDP-Gal from galactose in the
presence of the UDP-Gal regeneration enzymes as described herein,
(ii) converting Lac-OR.sup.1A as described herein into
Gb3-OR.sup.1A in a Gb3-synthesis reaction mixture comprising at
least the UDP-Gal, an alpha-1,4 galactosyltransferase, and the
UDP-Gal regeneration enzymes, (iii) mixing the Gb3-synthesis
reaction mixture with at least GalNAc, the
beta1,3-N-acetylgalactosaminyltransferase, the N-acetylhexosamine
1-kinase, and the N-acetylglucosamine 1-phosphate uridyltransferase
to form a Gb4-synthesis reaction mixture, (iv) incubating the
Gb4-synthesis reaction mixture under conditions allowing conversion
of Gb3-OR.sup.1A to Gb4-OR.sup.1A; (v) isolating the Gb4-OR.sup.1A;
(vi) mixing the Gb4-OR.sup.1A with a beta1,3-galactosyltransferase
and the set of UDP-Gal regeneration enzymes to form a Gb5-synthesis
reaction mixture; (vii) incubating the Gb5-synthesis reaction
mixture under conditions allowing conversion of the Gb4-OR.sup.AI
to Gb5-OR.sup.1A, (viii) mixing the Gb5-synthesis reaction mixture
with at least at least fucose, GTP, the
alpha1,2-fucosyltransferase, and the L-fucokinase/GDP-fucose
pyrophosphorylase to form a Fucosyl-Gb5-OR.sup.1A reaction mixture;
(ix) incubating the Fucosyl-Gb5-OR.sup.1A reaction mixture under
conditions allowing conversion of the Gb5-OR.sup.1A to
Fucosyl-Gb5-OR.sup.1A; and optionally, (x) isolating the
Fucosyl-Gb5-OR.sup.1A.
[0019] A method for synthesizing SSEA4 can be performed as follows:
(i) producing UDP-Gal from galactose in the presence of the UDP-Gal
regeneration enzymes as described herein, (ii) converting
Lac-OR.sup.1A as described herein into Gb3-OR.sup.1A in a
Gb3-synthesis reaction mixture comprising at least the UDP-Gal, an
alpha-1,4 galactosyltransferase, and the UDP-Gal regeneration
enzymes, (iii) mixing the Gb3-synthesis reaction mixture with at
least GalNAc, the beta1,3-N-acetylgalactosaminyltransferase, the
N-acetylhexosamine 1-kinase, and the N-acetylglucosamine
1-phosphate uridyltransferase to form a Gb4-synthesis reaction
mixture, (iv) incubating the Gb4-synthesis reaction mixture under
conditions allowing conversion of Gb3-OR.sup.1A to Gb4-OR.sup.1A,
(v) further incubating the Gb4-synthesis reaction mixture in the
presence of a .beta.-1,3-galactosyltransferase under conditions
allowing conversion of the Gb4-OR.sup.1A to Gb5-OR.sup.1A, (vi)
mixing the Gb4-synthesis reaction mixture with at least at least
Neu5Ac, CTP, the alpha2,3-sialyltransferase, the cytidine
monophosphate kinase, and the CMP-sialic acid synthetase to form a
Sialyl-Gb5-OR.sup.1A reaction mixture; (vii) incubating the
Sialyl-Gb5-OR.sup.1A reaction mixture under conditions allowing
conversion of the Gb5-OR.sup.1A to Sialyl-Gb5-OR.sup.1A; and
optionally, (viii) isolating the Sialyl-Gb5-OR.sup.1A.
[0020] Alternatively, a method for synthesizing SSEA4 can be
performed as follows: (i) producing UDP-Gal from galactose in the
presence of the UDP-Gal regeneration enzymes as described herein,
(ii) converting Lac-OR.sup.1A as described herein into
Gb3-OR.sup.1A in a Gb3-synthesis reaction mixture comprising at
least the UDP-Gal, an alpha-1,4 galactosyltransferase, and the
UDP-Gal regeneration enzymes, (iii) mixing the Gb3-synthesis
reaction mixture with at least GalNAc, the
beta1,3-N-acetylgalactosaminyltransferase, the N-acetylhexosamine
1-kinase, and the N-acetylglucosamine 1-phosphate uridyltransferase
to form a Gb4-synthesis reaction mixture, (iv) incubating the
Gb4-synthesis reaction mixture under conditions allowing conversion
of Gb3-OR.sup.1A to Gb4-OR.sup.1A; (v) isolating the Gb4-OR.sup.1A;
(vi) mixing the Gb4-OR.sup.1A with a beta1,3-galactosyltransferase
and the set of UDP-Gal regeneration enzymes to form a Gb5-synthesis
reaction mixture; (vii) incubating the Gb5-synthesis reaction
mixture under conditions allowing conversion of the Gb4-OR.sup.1A
to Gb5-OR.sup.1A; (viii) mixing the Gb5-OR.sup.1A with an
alpha2,3sialyltransferase and a set of CMP-Neu5Ac regeneration
enzymes to form a Sialyl-Gb5-synthesis reaction mixture, wherein
the set of CMP-Neu5Ac regeneration enzymes comprises a cytidine
monophosphate kinase, a CMP-sialic acid synthetase, a pyruvate
kinase, and a pyrophosphatase; (ix) incubating the
Sialyl-Gb5-synthesis reaction mixture under conditions allowing
conversion of the Gb4-OR.sup.1A to Sialyl-Gb5-OR.sup.1A; and
optionally, (x) isolating the Sialyl-Gb5-OR.sup.1A.
[0021] In some embodiments, the method described herein for
enzymatically synthesizing an oligosaccharide uses Gb3 (e.g.,
tailed) as the starting material. The method comprises: (i)
producing UDP-GalNAc from GalNAc in the presence of the set of
UDP-GalNAc regeneration enzymes as described above, and converting
Gb3-OR.sup.1A into Gb4-OR.sup.1A in the presence of the UDP-GalNAc
and a beta1,3-N-acetylgalactosaminyltransferase, wherein R.sup.1A
is hydrogen, substituted or unsubstituted alkyl, substituted or
unsubstituted alkenyl, substituted or unsubstituted alkynyl,
substituted or unsubstituted carbocyclyl, substituted or
unsubstituted heterocyclyl, substituted or unsubstituted aryl,
substituted or unsubstituted heteroaryl, or an oxygen protecting
group. Examples of R.sup.1A include, but are not limited to,
hydrogen, allyl, biotin, a ceramide, or a non-hydrogen group (e.g.,
alkyl) which is further substituted with a substituted or
unsubstituted thio, substituted or unsubstituted amino, carbonyl
(e.g., carboxylic acid), azido, alkenyl (e.g., allyl), alkynyl
(e.g., propargyl), biotin, or a ceramide group. In certain
embodiments, R.sup.1A is hydrogen, allyl, substituted alkyl,
biotin, or a ceramide. Steps (i) and (ii) can occur in a
Gb4-synthesis reaction mixture comprising GalNAc, PEP, ATP, UTP,
the Gb3-OR.sup.1A, the beta1,3-N-acetylgalactosaminyltransferase,
and the set of UDP-GalNAc regeneration enzymes. The Gb4-OR.sup.1A
can be isolated if necessary.
[0022] The above method can further comprise: (iii) converting the
Gb4-OR.sup.1A into Gb5-OR.sup.1A in the presence of UDP-Gal and a
beta1,3-galactosyltransferase, which can be coupled with (iv)
producing the UDP-Gal from galactose in the presence of the set of
UDP-Gal regeneration enzymes as described herein. (iii) and (iv)
can take place in a Gb5-synthesis reaction mixture comprising
galactose, PEP, ATP, UTP, the Gb4-OR.sup.1A, the
beta1,3-galactosyltransferase, and the set of UDP-Gal regeneration
enzymes. The resultant Gb5-OR.sup.1A can be isolated from the
reaction mixture.
[0023] In one example, the Gb5-OR.sup.1A is then converted into
Fucosyl-Gb5-OR.sup.1A as follows: (v) converting the Gb5-OR.sup.1A
into Fucosyl-Gb5-OR.sup.1A in the presence of GDP-Fuc and an
alpha1,2-fucosyltransferase, which can be coupled with (vi)
producing the GDP-Fuc from fucose in the presence of the set of
GDP-Fuc regeneration enzymes described herein. Steps (v) and (vi)
can be carried out in a Fucosyl-Gb5-synthesis reaction mixture
comprising fucose, ATP, GTP, PEP, the Gb5-OR.sup.1A, the
alpha1,2-fucosyltransferase, and the set of GDP-Fuc regeneration
enzymes. When desired, the Fucosyl-Gb5-synthesis reaction mixture
is prepared by mixing the Gb5-synthesis reaction mixture with at
least fucose, GTP, the alpha1,2-fucosyltransferase, and the
L-fucokinase/GDP-fucose pyrophosphorylase. The method can further
comprise isolating the Fucosyl-Gb5-OR.sup.1A.
[0024] In another example, the Gb5-OR.sup.1A is then converted into
Sialyl-Gb5-OR.sup.1A as follows: (vii) converting the Gb5-OR.sup.1A
into Sialyl-Gb5-OR.sup.1A in the presence of CMP-Neu5Ac and an
alpha 2,3-sialyltransferase, which can be coupled with (viii)
producing the CMP-Neu5Ac from Neu5Ac in the presence of the set of
CMP-Neu5Ac regeneration enzymes described herein. Steps (vii) and
(viii) can occur in a Sialyl-Gb5-synthesis reaction mixture
comprising Neu5Ac, CTP, PEP, the Gb5-OR.sup.1A, the alpha
2,3-sialyltransferase, and the set of CMP-Neu5Ac regeneration
enzymes. In some instances, the Sialyl-Gb5-synthesis reaction
mixture is prepared by mixing the Gb5-synthesis reaction mixture
with at least Neu5Ac, CTP, the alpha 2,3-sialyltransferase, the
cytidine monophosphate kinase, and the CMP-sialic acid synthetase.
The resultant Sialyl-Gb5-OR.sup.1A can be isolated from the
reaction mixture.
[0025] In yet other embodiments, the methods described herein
relate to synthesizing oligosaccharides, using Gb4 (e.g., tailed)
as a starting material. Such a method comprises: (i) producing
UDP-Gal from galactose in the presence of the set of UDP-Gal
regeneration enzymes described herein, and (ii) converting
Gb4-OR.sup.1A into Gb5-OR.sup.1A in the presence of UDP-Gal and a
beta1,3-galactosyltransferase, wherein R.sup.1A is hydrogen,
substituted or unsubstituted alkyl, substituted or unsubstituted
alkenyl, substituted or unsubstituted alkynyl, substituted or
unsubstituted carbocyclyl, substituted or unsubstituted
heterocyclyl, substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, or an oxygen protecting group. Examples
of R.sup.1A include, but are not limited, to hydrogen, allyl,
biotin, a ceramide, or a non-hydrogen group (e.g., alkyl) which is
further substituted with a substituted or unsubstituted thio,
substituted or unsubstituted amino, carbonyl (e.g., carboxylic
acid), azido, alkenyl (e.g., allyl), alkynyl (e.g., propargyl),
biotin, or a ceramide group. In certain embodiments, R.sup.1A is
hydrogen, allyl, substituted alkyl, biotin, or a ceramide. In this
method, steps (i) and (ii) can occur in a Gb5-synthesis reaction
mixture comprising galactose, PEP, ATP, UTP, the Gb4-OR.sup.1A, the
beta1,3-galactosyltransferase, and the set of UDP-Gal regeneration
enzymes. Alternatively or in addition, the Gb5-OR.sup.1A thus
produced can be isolated.
[0026] The above method can further comprise: (iii) converting the
Gb5-OR.sup.1A into Fucosyl-Gb5-OR.sup.1A in the presence of GDP-Fuc
and an alpha1,2-fucosyltransferase, which can be coupled with (iv)
producing the GDP-Fuc from fucose in the presence of the set of
GDP-Fuc regeneration enzymes, which is also described herein. Steps
(iii) and (iv) can take place in a Fucosyl-Gb5-synthesis reaction
mixture comprising fucose, ATP, GTP, PEP, the Gb5-OR.sup.1A, the
alpha1,2-fucosyltransferase, and the set of GDP-Fuc regeneration
enzymes. The Fucosyl-Gb5-synthesis reaction mixture is prepared by
mixing the Gb5-synthesis reaction mixture with at least fucose,
GTP, the alpha1,2-fucosyltransferase, and the
L-fucokinase/GDP-fucose pyrophosphorylase. The resultant
Fucosyl-Gb5-OR.sup.1A can be isolated from the reaction
mixture.
[0027] Alternatively, the above method can further comprise: (v)
converting the Gb5-OR.sup.1A into Sialyl-Gb5-OR.sup.1A in the
presence of CMP-Neu5Ac and an alpha 2,3-sialyltransferase, which
can be coupled with (v) producing the CMP-Neu5Ac from Neu5Ac in the
presence of the set of CMP-Neu5Ac regeneration enzymes described
herein. Steps (v) and (vi) can occur in a Sialyl-Gb5-synthesis
reaction mixture comprising Neu5Ac, CTP, PEP, the Gb5-OR.sup.1A,
the alpha 2,3-sialyltransferase, and the set of CMP-Neu5Ac
regeneration enzymes. The Sialyl-Gb5-synthesis reaction mixture is
prepared by mixing the Gb5-synthesis reaction mixture with at least
Neu5Ac, CTP, the alpha 2,3-sialyltransferase, the cytidine
monophosphate kinase, and the CMP-sialic acid synthetase. The
Sialyl-Gb5-OR.sup.1A produced in this method can be isolated from
the reaction mixture.
[0028] In some other embodiments, the methods described herein
relate to synthesis of a Fucosyl-Gb5 oligosaccharide (Globo H) from
Gb5. The method comprising: (i) producing GDP-Fuc from fucose in
the presence of the set of GDP-Fuc regeneration enzymes described
herein, (ii) converting Gb5-OR.sup.1A into Fucosyl-Gb5-OR.sup.1A in
the presence of the GDP-Fuc and an alpha1,2-fucosyltransferase,
and, optionally, (iii) isolating the Fucosyl-Gb5-OR.sup.1A Steps
(i) and (ii) can occur in a Fucosyl-Gb5-synthesis reaction mixture
comprising fucose, ATP, GTP, PEP, the Gb5-OR.sup.1A, the
alpha1,2-fucosyltransferase, and the set of GDP-Fuc regeneration
enzymes.
[0029] In some other embodiments, the methods described herein
relate to synthesis of a Sialyl-Gb5 oligosaccharide (Globo H) from
Gb5. The method comprises: (i) producing CMP-Neu5Ac from Neu5Ac in
the presence of the set of CMP-Neu5Ac regeneration enzymes
described herein, (ii) converting Gb5-OR.sup.1A into
Sialyl-Gb5-OR.sup.1A in the presence of CMP-Neu5Ac and an alpha
2,3-sialyltransferase, and, optionally, (iii) isolating the
Sialyl-Gb5-OR.sup.1A. Steps (i) and (ii) can take place in a
Sialyl-Gb5-synthesis reaction mixture comprising Neu5Ac, CTP, PEP,
the Gb5-OR, the alpha 2,3-sialyltransferase, and the set of
CMP-Neu5Ac regeneration enzymes.
[0030] In any of the synthesis methods described herein, either at
least one of the involved enzymes or at least one of the substrates
of each reaction (e.g., lactose, Gb3, Gb4, or Gb5) can be
immobilized on a support member.
[0031] Another aspect of the present disclosure features enzymatic
reactors for synthesizing oligosaccharides using the methods
described herein. Such an enzymatic reactor can comprise one or
more of the following reaction chambers:
[0032] (a) a reaction chamber for synthesizing Gb3-OR.sup.1A,
wherein the chamber comprises an alpha1,4-galactosyltransferase,
and a set of UDP-Gal regeneration enzymes, which comprises a
galactokinase, a UDP-sugar pyrophosphorylase, a pyruvate kinase,
and optionally a pyrophosphatase;
[0033] (b) a reaction chamber for synthesizing Gb4-OR.sup.1A,
wherein the chamber comprises a
beta1,3-N-acetylgalactosaminyltransferase and a set of UDP-GalNAc
regeneration enzymes, which comprises an N-acetylhexosamine
1-kinase, an N-acetylglucosamine 1-phosphate uridylyltransferase, a
pyruvate kinase, and optionally a pyrophosphatase;
[0034] (c) a reaction chamber for synthesizing Gb5-OR.sup.1A,
wherein the chamber comprises a beta1,3-galactosyltransferase, and
the set of UDP-Gal regeneration enzymes;
[0035] (d) a reaction chamber for synthesizing
Fucosyl-Gb5-OR.sup.1A, wherein the chamber comprises an
alpha1,2-fucosyltransferase and a set of GDP-Fuc regeneration
enzymes, which comprises an L-fucokinase/GDP-fucose
pyrophosphorylase, a pyruvate kinase, and optionally a
pyrophosphatase; and
[0036] (e) a reaction chamber for synthesizing
Sialyl-Gb5-OR.sup.1A, wherein the chamber comprises an
alpha2,3-sialyltransferase and a set of CMP-Neu5Ac regeneration
enzymes, which comprises a cytidine monophosphate kinase, a
CMP-sialic acid synthetase, a pyruvate kinase, and optionally a
pyrophosphatase.
[0037] In some examples, the enzymatic reactor comprises reaction
chambers: (a) and (b); (a), (b), and (c); (a), (b), (c), and (d);
(a), (b), (c), and (e); (b) and (c); (b), (c), and (d); (b), (c),
and (e); (c) and (d); or (c) and (e).
[0038] In another example, the enzymatic reactor described herein
comprises a reaction chamber that comprises a galactosyltransferase
(e.g., an alpha1,4-galactosyltransferase, a
beta1,4-galactosyltransferase, an alpha1,3-galactosyltransferase,
or a beta1,3-galactosyltransferase) and a set of UDP-Gal
regeneration enzymes as described herein, which may comprise a
galactokinase, an UDP pyrophosphorylase, a pyruvate kinase, and
optionally a pyrophosphatase.
[0039] In any of the reaction chambers, one or more of the enzymes
can be immobilized on a support member. In some examples, one or
more of the set of UDP-Gal regeneration enzymes, the set of
UDP-GalNAc regeneration enzymes, the set of GDP-Fuc regeneration
enzymes, and the set of CMP-Neu5Ac regeneration enzymes are each
immobilized on a support member. In other examples, all of the
enzymes in a reaction chamber are immobilized on a support
member.
[0040] Also within the scope of the present disclosure are
oligosaccharides obtained from any of the synthesis methods
described herein.
[0041] The details of one or more embodiments of the invention are
set forth in the Detailed Description of Certain Embodiments, as
described below. Other features, objects, and advantages of the
invention will be apparent from the Definitions, Drawings,
Examples, and Claims.
CHEMICAL DEFINITIONS
[0042] Definitions of specific functional groups and chemical terms
are described in more detail below. The chemical elements are
identified in accordance with the Periodic Table of the Elements,
CAS version, Handbook of Chemistry and Physics, 75.sup.th Ed.,
inside cover, and specific functional groups are generally defined
as described therein. Additionally, general principles of organic
chemistry, as well as specific functional moieties and reactivity,
are described in Organic Chemistry, Thomas Sorrell, University
Science Books, Sausalito, 1999; Smith and March March's Advanced
Organic Chemistry, 5.sup.th Edition, John Wiley & Sons, Inc.,
New York, 2001; Larock, Comprehensive Organic Transformations, VCH
Publishers, Inc., New York, 1989; and Carruthers, Some Modern
Methods of Organic Synthesis, 3.sup.rd Edition, Cambridge
University Press, Cambridge, 1987.
[0043] Compounds described herein can comprise one or more
asymmetric centers, and thus can exist in various stereoisomeric
forms, e.g., enantiomers and/or diastereomers. For example, the
compounds described herein can be in the form of an individual
enantiomer, diastereomer or geometric isomer, or can be in the form
of a mixture of stereoisomers, including racemic mixtures and
mixtures enriched in one or more stereoisomer. Isomers can be
isolated from mixtures by methods known to those skilled in the
art, including chiral high pressure liquid chromatography (HPLC)
and the formation and crystallization of chiral salts; or preferred
isomers can be prepared by asymmetric syntheses. See, for example,
Jacques et al., Enantiomers, Racemates and Resolutions (Wiley
Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725
(1977); Eliel, E. L. Stereochemistry of Carbon Compounds
(McGraw-Hill, NY, 1962); and Wilen, S. H. Tables of Resolving
Agents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ. of
Notre Dame Press, Notre Dame, Ind. 1972). The invention
additionally encompasses compounds as individual isomers
substantially free of other isomers, and alternatively, as mixtures
of various isomers.
[0044] When a range of values is listed, it is intended to
encompass each value and sub-range within the range. For example
"C.sub.1-6 alkyl" is intended to encompass, C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, C.sub.6, C.sub.1-6, C.sub.1-5,
C.sub.1-4, C.sub.1-3, C.sub.1-2, C.sub.2-6, C.sub.2-5, C.sub.2-4,
C.sub.2-3, C.sub.3-6, C.sub.3-5, C.sub.3-4, C.sub.4-6, C.sub.4-5,
and C.sub.5-6 alkyl.
[0045] As used herein, "alkyl" refers to a radical of a
straight-chain or branched saturated hydrocarbon group having from
1 to 30 carbon atoms ("C.sub.1-30 alkyl"). In some embodiments, an
alkyl group has 1 to 20 carbon atoms ("C.sub.1-20 alkyl"). In some
embodiments, an alkyl group has 1 to 10 carbon atoms ("C.sub.1-10
alkyl"). In some embodiments, an alkyl group has 1 to 9 carbon
atoms ("C.sub.1-9 alkyl"). In some embodiments, an alkyl group has
1 to 8 carbon atoms ("C.sub.1-8 alkyl"). In some embodiments, an
alkyl group has 1 to 7 carbon atoms ("C.sub.1-7 alkyl"). In some
embodiments, an alkyl group has 1 to 6 carbon atoms ("C.sub.1-6
alkyl"). In some embodiments, an alkyl group has 1 to 5 carbon
atoms ("C.sub.1-5 alkyl"). In some embodiments, an alkyl group has
1 to 4 carbon atoms ("C.sub.1-4 alkyl"). In some embodiments, an
alkyl group has 1 to 3 carbon atoms ("C.sub.1-3 alkyl"). In some
embodiments, an alkyl group has 1 to 2 carbon atoms ("C.sub.1-2
alkyl"). In some embodiments, an alkyl group has 1 carbon atom
("C.sub.1 alkyl"). In some embodiments, an alkyl group has 2 to 6
carbon atoms ("C.sub.2-6alkyl"). Examples of C.sub.1-6 alkyl groups
include methyl (C.sub.1), ethyl (C.sub.2), n-propyl (C.sub.3),
isopropyl (C3), n-butyl (C.sub.4), tert-butyl (C.sub.4), sec-butyl
(C.sub.4), iso-butyl (C.sub.4), n-pentyl (C.sub.5), 3-pentanyl
(C.sub.5), amyl (C.sub.5), neopentyl (C.sub.5), 3-methyl-2-butanyl
(C.sub.5), tertiary amyl (C.sub.5), and n-hexyl (C.sub.6).
Additional examples of alkyl groups include n-heptyl (C.sub.7),
n-octyl (C.sub.8) and the like. Unless otherwise specified, each
instance of an alkyl group is independently unsubstituted (an
"unsubstituted alkyl") or substituted (a "substituted alkyl") with
one or more substituents. In certain embodiments, the alkyl group
is an unsubstituted C.sub.1-10 alkyl (e.g., --CH.sub.3). In certain
embodiments, the alkyl group is a substituted C.sub.1-10 alkyl.
[0046] As used herein, "alkenyl" or "alkene" refers to a radical of
a straight-chain or branched hydrocarbon group having from 2 to 30
carbon atoms and one or more double bonds (e.g., 1, 2, 3, or 4
double bonds). In some embodiments, an alkenyl group has 2 to 20
carbon atoms ("C.sub.2-20 alkenyl"). In some embodiments, an
alkenyl group has 2 to 10 carbon atoms ("C.sub.2-10 alkenyl"). In
some embodiments, an alkenyl group has 2 to 9 carbon atoms
("C.sub.2-9 alkenyl"). In some embodiments, an alkenyl group has 2
to 8 carbon atoms ("C.sub.2-8 alkenyl"). In some embodiments, an
alkenyl group has 2 to 7 carbon atoms ("C.sub.2-7alkenyl"). In some
embodiments, an alkenyl group has 2 to 6 carbon atoms ("C.sub.2-6
alkenyl"). In some embodiments, an alkenyl group has 2 to 5 carbon
atoms ("C.sub.2-5 alkenyl"). In some embodiments, an alkenyl group
has 2 to 4 carbon atoms ("C.sub.2-4 alkenyl"). In some embodiments,
an alkenyl group has 2 to 3 carbon atoms ("C.sub.2-3 alkenyl"). In
some embodiments, an alkenyl group has 2 carbon atoms ("C.sub.2
alkenyl"). The one or more carbon-carbon double bonds can be
internal (such as in 2-butenyl) or terminal (such as in 1-butenyl).
Examples of C.sub.2-4 alkenyl groups include ethenyl (C.sub.2),
1-propenyl ("allyl", C.sub.3), 2-propenyl (C.sub.3), 1-butenyl
(C.sub.4), 2-butenyl (C.sub.4), butadienyl (C.sub.4), and the like.
Examples of C.sub.2-6 alkenyl groups include the aforementioned
C.sub.2-4 alkenyl groups as well as pentenyl (C.sub.5), pentadienyl
(C.sub.5), hexenyl (C.sub.6), and the like. Additional examples of
alkenyl include heptenyl (C.sub.7), octenyl (C.sub.8), octatrienyl
(C.sub.6), and the like. Unless otherwise specified, each instance
of an alkenyl group is independently unsubstituted (an
"unsubstituted alkenyl") or substituted (a "substituted alkenyl")
with one or more substituents. In certain embodiments, the alkenyl
group is an unsubstituted C.sub.2-10 alkenyl. In certain
embodiments, the alkenyl group is a substituted C.sub.2-10
alkenyl.
[0047] As used herein, "alkynyl" or "alkyne" refers to a radical of
a straight-chain or branched hydrocarbon group having from 2 to 30
carbon atoms and one or more triple bonds (e.g., 1, 2, 3, or 4
triple bonds) ("C.sub.2-10 alkynyl"). In some embodiments, an
alkynyl group has 2 to 20 carbon atoms ("C.sub.2-20 alkynyl"). In
some embodiments, an alkynyl group has 2 to 10 carbon atoms
("C.sub.2-10 alkynyl"). In some embodiments, an alkynyl group has 2
to 9 carbon atoms ("C.sub.2-9 alkynyl"). In some embodiments, an
alkynyl group has 2 to 8 carbon atoms ("C.sub.2-8 alkynyl"). In
some embodiments, an alkynyl group has 2 to 7 carbon atoms
("C.sub.2-7 alkynyl"). In some embodiments, an alkynyl group has 2
to 6 carbon atoms ("C.sub.2-6 alkynyl"). In some embodiments, an
alkynyl group has 2 to 5 carbon atoms ("C.sub.2-5 alkynyl"). In
some embodiments, an alkynyl group has 2 to 4 carbon atoms
("C.sub.2-4 alkynyl"). In some embodiments, an alkynyl group has 2
to 3 carbon atoms ("C.sub.2-3 alkynyl"). In some embodiments, an
alkynyl group has 2 carbon atoms ("C.sub.2 alkynyl"). The one or
more carbon-carbon triple bonds can be internal (such as in
2-butynyl) or terminal (such as in 1-butynyl). Examples of
C.sub.2-4 alkynyl groups include, without limitation, ethynyl
(C.sub.2), 1-propynyl (C.sub.3), 2-propynyl (C.sub.3), 1-butynyl
(C.sub.4), 2-butynyl (C.sub.4), and the like. Examples of C2,
alkenyl groups include the aforementioned C.sub.2-4 alkynyl groups
as well as pentynyl (C.sub.5), hexynyl (C.sub.6), and the like.
Additional examples of alkynyl include heptynyl (C.sub.7), octynyl
(C.sub.8), and the like. Unless otherwise specified, each instance
of an alkynyl group is independently unsubstituted (an
"unsubstituted alkynyl") or substituted (a "substituted alkynyl")
with one or more substituents. In certain embodiments, the alkynyl
group is an unsubstituted C.sub.2-10 alkynyl. In certain
embodiments, the alkynyl group is a substituted C.sub.2-10
alkynyl.
[0048] As used herein, "carbocyclyl" refers to a radical of a
non-aromatic cyclic hydrocarbon group having from 3 to 10 ring
carbon atoms ("C.sub.3-10 carbocyclyl") and zero heteroatoms in the
non-aromatic ring system. In some embodiments, a carbocyclyl group
has 3 to 8 ring carbon atoms ("C.sub.3-8 carbocyclyl"). In some
embodiments, a carbocyclyl group has 3 to 7 ring carbon atoms
("C.sub.3-7 carbocyclyl"). In some embodiments, a carbocyclyl group
has 3 to 6 ring carbon atoms ("C.sub.3-6 carbocyclyl"). In some
embodiments, a carbocyclyl group has 5 to 10 ring carbon atoms
("C.sub.5-10 carbocyclyl"). Exemplary C.sub.3-6 carbocyclyl groups
include, without limitation, cyclopropyl (C.sub.3), cyclopropenyl
(C.sub.3), cyclobutyl (C.sub.4), cyclobutenyl (C.sub.4),
cyclopentyl (C.sub.5), cyclopentenyl (C.sub.5), cyclohexyl
(C.sub.6), cyclohexenyl (C.sub.6), cyclohexadienyl (C.sub.6), and
the like. Exemplary C.sub.3-8 carbocyclyl groups include, without
limitation, the aforementioned C.sub.3-6 carbocyclyl groups as well
as cycloheptyl (C.sub.7), cycloheptenyl (C.sub.7), cycloheptadienyl
(C.sub.7), cycloheptatrienyl (C.sub.7), cyclooctyl (C.sub.5),
cyclooctenyl (C.sub.5), bicyclo[2.2.1]heptanyl (C.sub.7),
bicyclo[2.2.2]octanyl (C.sub.8), and the like. Exemplary C.sub.3-10
carbocyclyl groups include, without limitation, the aforementioned
C.sub.3-8 carbocyclyl groups as well as cyclononyl (C.sub.9),
cyclononenyl (C.sub.9), cyclodecyl (C.sub.10), cyclodecenyl
(C.sub.10), octahydro-1H-indenyl (C.sub.9), decahydronaphthalenyl
(C.sub.10), spiro[4.5]decanyl (C.sub.10), and the like. As the
foregoing examples illustrate, in certain embodiments, the
carbocyclyl group is either monocyclic ("monocyclic carbocyclyl")
or polycyclic (e.g., containing a fused, bridged or spiro ring
system such as a bicyclic system ("bicyclic carbocyclyl") or
tricyclic system ("tricyclic carbocyclyl")) and can be saturated or
can contain one or more carbon-carbon double or triple bonds.
"Carbocyclyl" also includes ring systems wherein the carbocyclyl
ring, as defined above, is fused with one or more aryl or
heteroaryl groups wherein the point of attachment is on the
carbocyclyl ring, and in such instances, the number of carbons
continue to designate the number of carbons in the carbocyclic ring
system. Unless otherwise specified, each instance of a carbocyclyl
group is independently unsubstituted (an "unsubstituted
carbocyclyl") or substituted (a "substituted carbocyclyl") with one
or more substituents. In certain embodiments, the carbocyclyl group
is an unsubstituted C.sub.3-10 carbocyclyl. In certain embodiments,
the carbocyclyl group is a substituted C.sub.3-10 carbocyclyl.
[0049] In some embodiments, "carbocyclyl" is a monocyclic,
saturated carbocyclyl group having from 3 to 10 ring carbon atoms
("C.sub.3-10 cycloalkyl"). In some embodiments, a cycloalkyl group
has 3 to 8 ring carbon atoms ("C.sub.3-8 cycloalkyl"). In some
embodiments, a cycloalkyl group has 3 to 6 ring carbon atoms
("C.sub.3-6 cycloalkyl"). In some embodiments, a cycloalkyl group
has 5 to 6 ring carbon atoms ("C.sub.5-6 cycloalkyl"). In some
embodiments, a cycloalkyl group has 5 to 10 ring carbon atoms
("C.sub.5-10 cycloalkyl"). Examples of C.sub.5-6 cycloalkyl groups
include cyclopentyl (C.sub.5) and cyclohexyl (C.sub.5). Examples of
C.sub.3-6 cycloalkyl groups include the aforementioned C.sub.5-6
cycloalkyl groups as well as cyclopropyl (C.sub.3) and cyclobutyl
(C.sub.4). Examples of C.sub.3-8 cycloalkyl groups include the
aforementioned C.sub.3-6 cycloalkyl groups as well as cycloheptyl
(C.sub.7) and cyclooctyl (C.sub.8). Unless otherwise specified,
each instance of a cycloalkyl group is independently unsubstituted
(an "unsubstituted cycloalkyl") or substituted (a "substituted
cycloalkyl") with one or more substituents. In certain embodiments,
the cycloalkyl group is an unsubstituted C.sub.3-10 cycloalkyl. In
certain embodiments, the cycloalkyl group is a substituted
C.sub.3-10 cycloalkyl.
[0050] As used herein, "heterocyclyl" refers to a radical of a 3-
to 14-membered non-aromatic ring system having ring carbon atoms
and 1 to 4 ring heteroatoms, wherein each heteroatom is
independently selected from nitrogen, oxygen, and sulfur ("3-14
membered heterocyclyl"). In heterocyclyl groups that contain one or
more nitrogen atoms, the point of attachment can be a carbon or
nitrogen atom, as valency permits. A heterocyclyl group can either
be monocyclic ("monocyclic heterocyclyl") or polycyclic (e.g., a
fused, bridged or spiro ring system such as a bicyclic system
("bicyclic heterocyclyl") or tricyclic system ("tricyclic
heterocyclyl")), and can be saturated or can contain one or more
carbon-carbon double or triple bonds. Heterocyclyl polycyclic ring
systems can include one or more heteroatoms in one or both rings.
"Heterocyclyl" also includes ring systems wherein the heterocyclyl
ring, as defined above, is fused with one or more carbocyclyl
groups wherein the point of attachment is either on the carbocyclyl
or heterocyclyl ring, or ring systems wherein the heterocyclyl
ring, as defined above, is fused with one or more aryl or
heteroaryl groups, wherein the point of attachment is on the
heterocyclyl ring, and in such instances, the number of ring
members continue to designate the number of ring members in the
heterocyclyl ring system. Unless otherwise specified, each instance
of heterocyclyl is independently unsubstituted (an "unsubstituted
heterocyclyl") or substituted (a "substituted heterocyclyl") with
one or more substituents. In certain embodiments, the heterocyclyl
group is an unsubstituted 3-14 membered heterocyclyl. In certain
embodiments, the heterocyclyl group is a substituted 3-14 membered
heterocyclyl.
[0051] In some embodiments, a heterocyclyl group is a 5-10 membered
non-aromatic ring system having ring carbon atoms and 1-4 ring
heteroatoms, wherein each heteroatom is independently selected from
nitrogen, oxygen, and sulfur ("5-10 membered heterocyclyl"). In
some embodiments, a heterocyclyl group is a 5-8 membered
non-aromatic ring system having ring carbon atoms and 1-4 ring
heteroatoms, wherein each heteroatom is independently selected from
nitrogen, oxygen, and sulfur ("5-8 membered heterocyclyl"). In some
embodiments, a heterocyclyl group is a 5-6 membered non-aromatic
ring system having ring carbon atoms and 1-4 ring heteroatoms,
wherein each heteroatom is independently selected from nitrogen,
oxygen, and sulfur ("5-6 membered heterocyclyl"). In some
embodiments, the 5-6 membered heterocyclyl has 1-3 ring heteroatoms
selected from nitrogen, oxygen, and sulfur. In some embodiments,
the 5-6 membered heterocyclyl has 1-2 ring heteroatoms selected
from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6
membered heterocyclyl has 1 ring heteroatom selected from nitrogen,
oxygen, and sulfur.
[0052] Exemplary 3-membered heterocyclyl groups containing 1
heteroatom include, without limitation, azirdinyl, oxiranyl,
thiorenyl. Exemplary 4-membered heterocyclyl groups containing 1
heteroatom include, without limitation, azetidinyl, oxetanyl and
thietanyl. Exemplary 5-membered heterocyclyl groups containing 1
heteroatom include, without limitation, tetrahydrofuranyl,
dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl,
pyrrolidinyl, dihydropyrrolyl and pyrrolyl-2,5-dione. Exemplary
5-membered heterocyclyl groups containing 2 heteroatoms include,
without limitation, dioxolanyl, oxathiolanyl and dithiolanyl.
Exemplary 5-membered heterocyclyl groups containing 3 heteroatoms
include, without limitation, triazolinyl, oxadiazolinyl, and
thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing
1 heteroatom include, without limitation, piperidinyl,
tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary
6-membered heterocyclyl groups containing 2 heteroatoms include,
without limitation, piperazinyl, morpholinyl, dithianyl, dioxanyl.
Exemplary 6-membered heterocyclyl groups containing 2 heteroatoms
include, without limitation, triazinanyl. Exemplary 7-membered
heterocyclyl groups containing 1 heteroatom include, without
limitation, azepanyl, oxepanyl and thiepanyl. Exemplary 8-membered
heterocyclyl groups containing 1 heteroatom include, without
limitation, azocanyl, oxecanyl and thiocanyl. Exemplary bicyclic
heterocyclyl groups include, without limitation, indolinyl,
isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl,
tetrahydrobenzothienyl, tetrahydrobenzofuranyl, tetrahydroindolyl,
tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl,
decahydroisoquinolinyl, octahydrochromenyl, octahydroisochromenyl,
decahydronaphthyridinyl, decahydro-1,8-naphthyridinyl,
octahydropyrrolo[3,2-b]pyrrole, indolinyl, phthalimidyl,
naphthalimidyl, chromanyl, chromenyl, 1H-benzo[e][1,4]diazepinyl,
1,4,5,7-tetrahydropyrano[3,4-b]pyrrolyl,
5,6-dihydro-4H-furo[3,2-b]pyrrolyl,
6,7-dihydro-5H-furo[3,2-b]pyranyl,
5,7-dihydro-4H-thieno[2,3-c]pyranyl,
2,3-dihydro-1H-pyrrolo[2,3-b]pyridinyl,
2,3-dihydrofuro[2,3-b]pyridinyl,
4,5,6,7-tetrahydro-1H-pyrrolo[2,3-b]pyridinyl,
4,5,6,7-tetrahydrofuro[3,2-c]pyridinyl,
4,5,6,7-tetrahydrothieno[3,2-b]pyridinyl,
1,2,3,4-tetrahydro-1,6-naphthyridinyl, and the like.
[0053] As used herein, "aryl" refers to a radical of a monocyclic
or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring
system (e.g., having 6, 10, or 14 .pi. electrons shared in a cyclic
array) having 6-14 ring carbon atoms and zero heteroatoms provided
in the aromatic ring system ("C.sub.6-14 aryl"). In some
embodiments, an aryl group has 6 ring carbon atoms ("C.sub.6 aryl";
e.g., phenyl). In some embodiments, an aryl group has 10 ring
carbon atoms ("C.sub.10 aryl"; e.g., naphthyl such as 1-naphthyl
and 2-naphthyl). In some embodiments, an aryl group has 14 ring
carbon atoms ("C.sub.14 aryl"; e.g., anthracyl). "Aryl" also
includes ring systems wherein the aryl ring, as defined above, is
fused with one or more carbocyclyl or heterocyclyl groups wherein
the radical or point of attachment is on the aryl ring, and in such
instances, the number of carbon atoms continue to designate the
number of carbon atoms in the aryl ring system. Unless otherwise
specified, each instance of an aryl group is independently
unsubstituted (an "unsubstituted aryl") or substituted (a
"substituted aryl") with one or more substituents. In certain
embodiments, the aryl group is an unsubstituted C.sub.6-14 aryl. In
certain embodiments, the aryl group is a substituted C.sub.6-14
aryl.
[0054] As used herein, "heteroaryl" refers to a radical of a 5-14
membered monocyclic or polycyclic (e.g., bicyclic, tricyclic) 4n+2
aromatic ring system (e.g., having 6, 10, or 14 .pi. electrons
shared in a cyclic array) having ring carbon atoms and 1-4 ring
heteroatoms provided in the aromatic ring system, wherein each
heteroatom is independently selected from nitrogen, oxygen and
sulfur ("5-14 membered heteroaryl"). In heteroaryl groups that
contain one or more nitrogen atoms, the point of attachment can be
a carbon or nitrogen atom, as valency permits. Heteroaryl
polycyclic ring systems can include one or more heteroatoms in one
or both rings. "Heteroaryl" includes ring systems wherein the
heteroaryl ring, as defined above, is fused with one or more
carbocyclyl or heterocyclyl groups wherein the point of attachment
is on the heteroaryl ring, and in such instances, the number of
ring members continue to designate the number of ring members in
the heteroaryl ring system. "Heteroaryl" also includes ring systems
wherein the heteroaryl ring, as defined above, is fused with one or
more aryl groups wherein the point of attachment is either on the
aryl or heteroaryl ring, and in such instances, the number of ring
members designates the number of ring members in the fused
polycyclic (aryl/heteroaryl) ring system. Polycyclic heteroaryl
groups wherein one ring does not contain a heteroatom (e.g.,
indolyl, quinolinyl, carbazolyl, and the like) the point of
attachment can be on either ring, i.e., either the ring bearing a
heteroatom (e.g., 2-indolyl) or the ring that does not contain a
heteroatom (e.g., 5-indolyl).
[0055] In some embodiments, a heteroaryl group is a 5-10 membered
aromatic ring system having ring carbon atoms and 1-4 ring
heteroatoms provided in the aromatic ring system, wherein each
heteroatom is independently selected from nitrogen, oxygen, and
sulfur ("5-10 membered heteroaryl"). In some embodiments, a
heteroaryl group is a 5-8 membered aromatic ring system having ring
carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring
system, wherein each heteroatom is independently selected from
nitrogen, oxygen, and sulfur ("5-8 membered heteroaryl"). In some
embodiments, a heteroaryl group is a 5-6 membered aromatic ring
system having ring carbon atoms and 1-4 ring heteroatoms provided
in the aromatic ring system, wherein each heteroatom is
independently selected from nitrogen, oxygen, and sulfur ("5-6
membered heteroaryl"). In some embodiments, the 5-6 membered
heteroaryl has 1-3 ring heteroatoms selected from nitrogen, oxygen,
and sulfur. In some embodiments, the 5-6 membered heteroaryl has
1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In
some embodiments, the 5-6 membered heteroaryl has 1 ring heteroatom
selected from nitrogen, oxygen, and sulfur. Unless otherwise
specified, each instance of a heteroaryl group is independently
unsubstituted (an "unsubstituted heteroaryl") or substituted (a
"substituted heteroaryl") with one or more substituents. In certain
embodiments, the heteroaryl group is an unsubstituted 5-14 membered
heteroaryl. In certain embodiments, the heteroaryl group is a
substituted 5-14 membered heteroaryl.
[0056] Exemplary 5-membered heteroaryl groups containing 1
heteroatom include, without limitation, pyrrolyl, furanyl and
thiophenyl. Exemplary 5-membered heteroaryl groups containing 2
heteroatoms include, without limitation, imidazolyl, pyrazolyl,
oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary
5-membered heteroaryl groups containing 3 heteroatoms include,
without limitation, triazolyl, oxadiazolyl, and thiadiazolyl.
Exemplary 5-membered heteroaryl groups containing 4 heteroatoms
include, without limitation, tetrazolyl. Exemplary 6-membered
heteroaryl groups containing 1 heteroatom include, without
limitation, pyridinyl. Exemplary 6-membered heteroaryl groups
containing 2 heteroatoms include, without limitation, pyridazinyl,
pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups
containing 3 or 4 heteroatoms include, without limitation,
triazinyl and tetrazinyl, respectively. Exemplary 7-membered
heteroaryl groups containing 1 heteroatom include, without
limitation, azepinyl, oxepinyl, and thiepinyl. Exemplary
5,6-bicyclic heteroaryl groups include, without limitation,
indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl,
isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl,
benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl,
benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl.
Exemplary 6,6-bicyclic heteroaryl groups include, without
limitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl,
cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplary
tricyclic heteroaryl groups include, without limitation,
phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl,
phenothiazinyl, phenoxazinyl and phenazinyl.
[0057] As used herein, the term "partially unsaturated" refers to a
ring moiety that includes at least one double or triple bond. The
term "partially unsaturated" is intended to encompass rings having
multiple sites of unsaturation, but is not intended to include
aromatic groups (e.g., aryl or heteroaryl moieties) as herein
defined.
[0058] As used herein, the term "saturated" refers to a ring moiety
that does not contain a double or triple bond, i.e., the ring
contains all single bonds.
[0059] Alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,
heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl
groups, as defined herein, are optionally substituted (e.g.,
"substituted" or "unsubstituted" alkyl, "substituted" or
"unsubstituted" alkenyl, "substituted" or "unsubstituted" alkynyl,
"substituted" or "unsubstituted" heteroalkyl, "substituted" or
"unsubstituted" heteroalkenyl, "substituted" or "unsubstituted"
heteroalkynyl, "substituted" or "unsubstituted" carbocyclyl,
"substituted" or "unsubstituted" heterocyclyl, "substituted" or
"unsubstituted" aryl or "substituted" or "unsubstituted" heteroaryl
group). In general, the term "substituted", whether preceded by the
term "optionally" or not, means that at least one hydrogen present
on a group (e.g., a carbon or nitrogen atom) is replaced with a
permissible substituent, e.g., a substituent which upon
substitution results in a stable compound, e.g., a compound which
does not spontaneously undergo transformation such as by
rearrangement, cyclization, elimination, or other reaction. Unless
otherwise indicated, a "substituted" group has a substituent at one
or more substitutable positions of the group, and when more than
one position in any given structure is substituted, the substituent
is either the same or different at each position. The term
"substituted" is contemplated to include substitution with all
permissible substituents of organic compounds, any of the
substituents described herein that results in the formation of a
stable compound. The present invention contemplates any and all
such combinations in order to arrive at a stable compound. For
purposes of this invention, heteroatoms such as nitrogen may have
hydrogen substituents and/or any suitable substituent as described
herein which satisfy the valencies of the heteroatoms and results
in the formation of a stable moiety.
[0060] Exemplary carbon atom substituents include, but are not
limited to, halogen, --CN, --NO.sub.2, --N.sub.3, --SO.sub.2H,
--SO.sub.3H, --OH, --OR.sup.aa, --ON(R.sup.bb).sub.2,
--N(R.sup.bb).sub.2, --N(R.sup.bb).sub.3.sup.+X.sup.-,
--N(OR.sup.cc)R.sup.bb, --SH, --SR.sup.aa, --SSR.sup.cc,
--C(.dbd.O)R.sup.aa, --CO.sub.2H, --CHO, --C(OR.sup.cc).sub.2,
--CO.sub.2R.sup.aa, --OC(.dbd.O)R.sup.aa, --OCO.sub.2R.sup.aa,
--C(.dbd.O)N(R.sup.bb).sub.2, --OC(.dbd.O)N(R.sup.bb).sub.2,
--NR.sup.bbC(.dbd.O)R.sup.aa, --NR.sup.bbCO.sub.2R.sup.aa,
--NR.sup.bbC(.dbd.O)N(R.sup.bb).sub.2, --C(.dbd.NR.sup.bb)R.sup.aa,
--C(.dbd.NR.sup.bb)OR.sup.aa, --OC(.dbd.NR.sup.bb)R.sup.aa,
--OC(.dbd.NR.sup.bb)OR.sup.aa,
--C(.dbd.NR.sup.bb)N(R.sup.bb).sub.2,
--OC(.dbd.NR.sup.bb)N(R.sup.bb).sub.2,
--NR.sup.bbC(.dbd.NR.sup.bb)N(R.sup.bb),
--C(.dbd.O)NR.sup.bbSO.sub.2R.sup.aa, --NR.sup.bbSO.sub.2R.sup.aa,
--SO.sub.2N(R.sup.bb).sub.2, --SO.sub.2R.sup.aa,
--SO.sub.2OR.sup.aa, --OSO.sub.2R.sup.aa, --S(.dbd.O)R.sup.aa,
--OS(.dbd.O)R.sup.aa, --Si(R.sup.aa).sub.3,
--OSi(R.sup.aa).sub.3--C(.dbd.S)N(R.sup.bb).sub.2,
--C(.dbd.O)SR.sup.aa, --C(.dbd.S)SR.sup.aa, --SC(.dbd.S)SR.sup.aa,
--SC(.dbd.O)SR.sup.aa, --OC(.dbd.O)SR.sup.aa,
--SC(.dbd.O)OR.sup.aa, --SC(.dbd.O)R.sup.aa,
--P(.dbd.O).sub.2R.sup.aa, --OP(.dbd.O).sub.2R.sup.aa,
--P(.dbd.O)(R.sup.aa).sub.2, --OP(.dbd.O)(R.sup.aa).sub.2,
--OP(.dbd.O)(OR.sup.aa).sub.2, --P(.dbd.O).sub.2N(R.sup.bb).sub.2,
--OP(.dbd.O).sub.2N(R.sup.aa).sub.2, --P(.dbd.O)(NR.sup.bb).sub.2,
--OP(.dbd.O)(NR.sup.bb).sub.2,
--NR.sup.bbP(.dbd.O)(OR.sup.cc).sub.2,
--NR.sup.bbP(.dbd.O)(NR.sup.bb).sub.2, --P(R.sup.cc).sub.2,
--P(R.sup.cc).sub.3, --OP(R.sup.cc).sub.2, --OP(R.sup.aa).sub.3,
--B(R.sup.aa).sub.2, --B(OR.sup.cc).sub.2, --BR(OR.sup.cc),
C.sub.1-10 alkyl, C.sub.1-10 perhaloalkyl, C.sub.2-10 alkenyl,
C.sub.2-10 alkynyl, C.sub.1-10 heteroalkyl, C.sub.2-10
heteroalkenyl, C.sub.2-10 heteroalkynyl, C.sub.3-14 carbocyclyl,
3-14 membered heterocyclyl, C.sub.6-14 aryl, and 5-14 membered
heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl,
heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and
heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5
R.sup.dd groups;
[0061] or two geminal hydrogens on a carbon atom are replaced with
the group .dbd.O, .dbd.S, .dbd.NN(R.sup.bb).sub.2,
.dbd.NNR.sup.bbC(.dbd.O)R.sup.aa,
.dbd.NNR.sup.bbC(.dbd.O)OR.sup.aa,
.dbd.NNR.sup.bbS(.dbd.O).sub.2R.sup.aa, .dbd.NR.sup.bb, or
.dbd.NOR.sup.cc;
[0062] each instance of R.sup.aa is, independently, selected from
C.sub.1-10 alkyl, C.sub.1-10 perhaloalkyl, C.sub.2-10 alkenyl,
C.sub.2-10 alkynyl, C.sub.1-10 heteroalkyl, C.sub.2-10
heteroalkenyl, C.sub.2-10 heteroalkynyl, C.sub.3-10 carbocyclyl,
3-14 membered heterocyclyl, C.sub.6-14 aryl, and 5-14 membered
heteroaryl, or two R.sup.aa groups are joined to form a 3-14
membered heterocyclyl or 5-14 membered heteroaryl ring, wherein
each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,
heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is
independently substituted with 0, 1, 2, 3, 4, or 5 R.sup.dd
groups;
[0063] each instance of R.sup.bb is, independently, selected from
hydrogen, --OH, --OR.sup.aa, --N(R.sup.cc).sub.2, --CN,
--C(.dbd.O)R.sup.aa, --C(.dbd.O)N(R.sup.cc).sub.2,
--CO.sub.2R.sup.aa, --SO.sub.2R, --C(.dbd.NR)OR.sup.aa,
--C(.dbd.NR.sup.aa)N(R.sup.cc).sub.2, --SO.sub.2N(R.sup.cc).sub.2,
--SO.sub.2R.sup.aa, --SO.sub.2OR.sup.aa, --SOR.sup.aa,
--C(.dbd.S)N(R.sup.cc).sub.2, --C(.dbd.O)SR.sup.cc,
--C(.dbd.S)SR.sup.cc, --P(.dbd.O).sub.2R.sup.aa,
--P(.dbd.O)(R.sup.aa).sub.2, --P(.dbd.O)N(R.sup.cc).sub.2,
--P(.dbd.O)(NR.sup.aa).sub.2, C.sub.1-10 alkyl, C.sub.1-10
perhaloalkyl, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl, C.sub.1-10
heteroalkyl, C.sub.2-10 heteroalkenyl, C.sub.2-10 heteroalkynyl,
C.sub.3-10 carbocyclyl, 3-14 membered heterocyclyl, C.sub.6-14
aryl, and 5-14 membered heteroaryl, or two R.sup.bb groups are
joined to form a 3-14 membered heterocyclyl or 5-14 membered
heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl,
heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and
heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5
R.sup.dd groups;
[0064] each instance of R.sup.cc is, independently, selected from
hydrogen, C.sub.1-10 alkyl, C.sub.1-10 perhaloalkyl, C.sub.2-10
alkenyl, C.sub.2-10 alkynyl, C.sub.1-10 heteroalkyl, C.sub.2-10
heteroalkenyl, C.sub.2-10 heteroalkynyl, C.sub.3-10 carbocyclyl,
3-14 membered heterocyclyl, C.sub.6-14 aryl, and 5-14 membered
heteroaryl, or two R.sup.cc groups are joined to form a 3-14
membered heterocyclyl or 5-14 membered heteroaryl ring, wherein
each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,
heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is
independently substituted with 0, 1, 2, 3, 4, or 5 R.sup.dd
groups;
[0065] each instance of R.sup.dd is, independently, selected from
halogen, --CN, --NO.sub.2, --N.sub.3, --SO.sub.2H, --SO.sub.3H,
--OH, --OR.sup.ee, --ON(R.sup.ff).sub.2, --N(R.sup.ff).sub.2,
--N(R.sup.ff).sub.3.sup.+X.sup.-, --N(OR.sup.ee)R.sup.ff, --SH,
--SR.sup.ee, --SSR.sup.ee, --C(.dbd.O)R.sup.ee, --CO.sub.2H,
--CO.sub.2R.sup.ee, --OC(.dbd.O)R.sup.ee, --OCO.sub.2R.sup.ee,
--C(.dbd.O)N(R.sup.ff).sub.2, --OC(.dbd.O)N(R.sup.ff).sub.2,
--NR.sup.ffC(.dbd.O)R.sup.ee, --NR.sup.ffCO.sub.2R.sup.ee,
--NR.sup.ffC(.dbd.O)N(R.sup.ff).sub.2, --C(.dbd.NR)OR.sup.ee,
--OC(.dbd.NR.sup.ff)R.sup.ee, --OC(.dbd.NR.sup.ff)OR.sup.ee,
--C(.dbd.NR.sup.ff)N(R.sup.ff).sub.2,
--OC(.dbd.NR.sup.ff)N(R.sup.ff).sub.2,
--NR.sup.ffC(.dbd.NR.sup.ff)N(R.sup.ff).sub.2,
--NR.sup.ffSO.sub.2R.sup.ee, --SO.sub.2N(R.sup.ff).sub.2,
--SO.sub.2R.sup.ee, --SO.sub.2OR.sup.ee, --OSO.sub.2R.sup.ee,
--S(.dbd.O)R.sup.ee, --Si(R.sup.ee).sub.3, --OSi(R.sup.ee).sub.3,
--C(.dbd.S)N(R.sup.ff).sub.2, --C(.dbd.O)SR.sup.ee,
--C(.dbd.S)SR.sup.ee, --SC(.dbd.S)SR.sup.ee,
--P(.dbd.O).sub.2R.sup.ee, --P(.dbd.O)(R.sup.ee).sub.2,
--OP(.dbd.O)(R.sup.ee).sub.2, --OP(.dbd.O)(OR.sup.ee).sub.2,
C.sub.1-6 alkyl, C.sub.1-6 perhaloalkyl, C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, C.sub.1-6 heteroalkyl, C.sub.2-6 heteroalkenyl,
C.sub.2-6heteroalkynyl, C.sub.3-10 carbocyclyl, 3-10 membered
heterocyclyl, C.sub.6-10 aryl, 5-10 membered heteroaryl, wherein
each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,
heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is
independently substituted with 0, 1, 2, 3, 4, or 5 R.sup.gg groups,
or two geminal R.sup.dd substituents can be joined to form .dbd.O
or .dbd.S;
[0066] each instance of R.sup.ee is, independently, selected from
C.sub.1-6 alkyl, C.sub.1-6 perhaloalkyl, C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, C.sub.1-6 heteroalkyl, C.sub.2-6 heteroalkenyl,
C.sub.2-6heteroalkynyl, C.sub.3-10 carbocyclyl, C.sub.6-10 aryl,
3-10 membered heterocyclyl, and 3-10 membered heteroaryl, wherein
each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,
heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is
independently substituted with 0, 1, 2, 3, 4, or 5 R.sup.u
groups;
[0067] each instance of R.sup.ff is, independently, selected from
hydrogen, C.sub.1-6 alkyl, C.sub.1-6 perhaloalkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 heteroalkyl, C.sub.2-6
heteroalkenyl, C.sub.2-6heteroalkynyl, C.sub.3-10 carbocyclyl, 3-10
membered heterocyclyl, C.sub.6-10 aryl and 5-10 membered
heteroaryl, or two R.sup.ff groups are joined to form a 3-14
membered heterocyclyl or 5-14 membered heteroaryl ring, wherein
each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,
heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is
independently substituted with 0, 1, 2, 3, 4, or 5 R.sup.gg groups;
and
[0068] each instance of R.sup.gg is, independently, halogen, --CN,
--NO.sub.2, --N.sub.3, --SO.sub.2H, --SO.sub.3H, --OH, --OC.sub.1-6
alkyl, --ON(C.sub.1-6alkyl).sub.2, --N(C.sub.1-6 alkyl).sub.2,
--N(C.sub.1-6alkyl).sub.3.sup.4X.sup.-, --NH(C.sub.1-6
alkyl).sub.2+X.sup.-, --NH.sub.2(C.sub.1-6 alkyl).sup.+X.sup.-,
--NH.sub.3.sup.+X.sup.-, --N(OC.sub.1-6 alkyl)(C.sub.1-6 alkyl),
--N(OH)(C.sub.1-6 alkyl), --NH(OH), --SH, --SC.sub.1-6 alkyl,
--SS(C.sub.1-6 alkyl), --C(.dbd.O)(C.sub.1-6alkyl), --CO.sub.2H,
--CO.sub.2(C.sub.1-6 alkyl), --OC(.dbd.O)(C.sub.1-6 alkyl),
--OCO.sub.2(C.sub.1-6alkyl), --C(.dbd.O)NH.sub.2,
--C(.dbd.O)N(C.sub.1-6 alkyl).sub.2, --OC(.dbd.O)NH(C.sub.1-6
alkyl), --NHC(.dbd.O)(C.sub.1-6 alkyl), --N(C.sub.1-6
alkyl)C(.dbd.O)(C.sub.1-6 alkyl), --NHCO.sub.2(C.sub.1-6 alkyl),
--NHC(.dbd.O)N(C.sub.1-6 alkyl).sub.2, --NHC(.dbd.O)NH(C.sub.1-6
alkyl), --NHC(.dbd.O)NH.sub.2, --C(.dbd.NH)O(C.sub.1-6alkyl),
--OC(.dbd.NH)(C.sub.1-6 alkyl), --OC(.dbd.NH)OC.sub.1-6 alkyl,
--C(.dbd.NH)N(C.sub.1-6 alkyl).sub.2, --C(.dbd.NH)NH(C.sub.1-6
alkyl), --C(.dbd.NH)NH.sub.2, --OC(.dbd.NH)N(C.sub.1-6
alkyl).sub.2, --OC(NH)NH(C.sub.1-6alkyl), --OC(NH)NH.sub.2,
--NHC(NH)N(C.sub.1-6 alkyl).sub.2, --NHC(.dbd.NH)NH.sub.2,
--NHSO.sub.2(C.sub.1-6 alkyl), --SO.sub.2N(C.sub.1-6 alkyl).sub.2,
--SO.sub.2NH(C.sub.1-6 alkyl), --SO.sub.2NH.sub.2,
--SO.sub.2C.sub.1-6 alkyl, --SO.sub.2OC.sub.1-4 alkyl,
--OSO.sub.2C.sub.1-6 alkyl, --SOC.sub.1-6 alkyl, --Si(C.sub.1-6
alkyl).sub.3, --OSi(C.sub.1-6 alkyl).sub.3-C(.dbd.S)N(C.sub.1-6
alkyl).sub.2, C(.dbd.S)NH(C.sub.1-6 alkyl), C(.dbd.S)NH.sub.2,
--C(.dbd.O)S(C.sub.1-6 alkyl), --C(.dbd.S)SC.sub.1-6 alkyl,
--SC(.dbd.S)SC.sub.1-6 alkyl, --P(.dbd.O).sub.2(C.sub.1-6 alkyl),
--P(.dbd.O)(C.sub.1-6alkyl).sub.2, --OP(.dbd.O)(C.sub.1-6
alkyl).sub.2, --OP(.dbd.O)(OC.sub.1-6alkyl).sub.2, C.sub.2-6 alkyl,
C.sub.1-6 perhaloalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl,
C.sub.1-6heteroalkyl, C.sub.2-6 heteroalkenyl,
C.sub.2-6heteroalkynyl, C.sub.3-10 carbocyclyl, C.sub.6-10 aryl,
3-10 membered heterocyclyl, 5-10 membered heteroaryl; or two
geminal R.sup.gg substituents can be joined to form .dbd.O or
.dbd.S; wherein X.sup.- is a counterion.
[0069] As used herein, the term "halo" or "halogen" refers to
fluorine (fluoro, --F), chlorine (chloro, --Cl), bromine (bromo,
--Br), or iodine (iodo, --I).
[0070] As used herein, a "counterion" is a negatively charged group
associated with a positively charged quarternary amine in order to
maintain electronic neutrality. Exemplary counterions include
halide ions (e.g., F.sup.-, Cl.sup.-, Br.sup.-, I.sup.-),
NO.sub.3.sup.-, ClO.sub.4.sup.-, OH.sup.-, H.sub.2PO.sub.4.sup.-,
HSO.sub.4.sup.-, sulfonate ions (e.g., methanesulfonate,
trifluoromethanesulfonate, p-toluenesulfonate, benzenesulfonate,
10-camphor sulfonate, naphthalene-2-sulfonate,
naphthalene-1-sulfonic acid-5-sulfonate, ethan-1-sulfonic
acid-2-sulfonate, and the like), and carboxylate ions (e.g.,
acetate, ethanoate, propanoate, benzoate, glycerate, lactate,
tartrate, glycolate, and the like).
[0071] As used herein, the term "carbonyl" refers a group wherein
the carbon directly attached to the parent molecule is sp.sup.2
hybridized, and is substituted with an oxygen, nitrogen or sulfur
atom, e.g., a group selected from ketones (.dbd.C(.dbd.O)R.sup.aa),
carboxylic acids (.dbd.CO.sub.2H), aldehydes (.dbd.CHO), esters
(.dbd.CO.sub.2R.sup.aa, --C(.dbd.O)SR.sup.aa,
--C(.dbd.S)SR.sup.aa), amides (.dbd.C(.dbd.O)N(R.sup.bb).sub.2,
--C(.dbd.--O)NR.sup.bbSO.sub.2R.sup.aa,
--C(.dbd.S)N(R.sup.bb).sub.2), and imines
(.dbd.--C(.dbd.NR.sup.bb)R.sup.aa, --C(.dbd.NR.sup.bb)OR.sup.aa),
--C(.dbd.NR.sup.bb)N(R.sup.bb).sub.2), wherein R.sup.aa and
R.sup.bb are as defined herein.
[0072] As used herein, "azide" or "azido" refers to the group
--N.sub.3.
As used herein, the term "thiol" or "thio" refers to the group
--SH. The term "substituted thiol" or "substituted thio," by
extension, refers to a thiol group wherein the sulfur atom directly
attached to the parent molecule is substituted with a group other
than hydrogen, and includes groups selected from --SR.sup.aa,
--S.dbd.SR.sup.aa, --SC(.dbd.S)SR.sup.aa, --SC(.dbd.O)SR.sup.aa,
--SC(.dbd.O)OR.sup.aa, and --SC(.dbd.O)R.sup.aa, wherein R.sup.aa
and R.sup.cc are as defined herein.
[0073] As used herein, the term, "amino" or "amine" refers to the
group --NH.sub.2. The term "substituted" amino or amine, by
extension, refers to a monosubstituted amino, a disubstituted
amino, or a trisubstituted amino, as defined herein. In certain
embodiments, the "substituted amino" is a monosubstituted amino or
a disubstituted amino group.
As used herein, the term "monosubstituted amino" or
"monosubstituted amine" refers to an amino group wherein the
nitrogen atom directly attached to the parent molecule is
substituted with one hydrogen and one group other than hydrogen,
and includes groups selected from --NH(R.sup.bb),
--NHC(.dbd.O)R.sup.aa, --NHCO.sub.2R.sup.aa,
--NHC(.dbd.O)N(R.sup.bb).sub.2,
--NHC(.dbd.NR.sup.bb)N(R.sup.bb).sub.2, --NHSO.sub.2R.sup.aa,
--NHP(.dbd.O)(OR.sup.cc).sub.2, and --NHP(.dbd.O)(NR.sup.bb).sub.2,
wherein R.sup.aa, R.sup.bb and R.sup.cc are as defined herein, and
wherein R.sup.bb of the group --NH(R.sup.bb) is not hydrogen.
[0074] As used herein, the term "disubstituted amino" or
"disubstituted amine" refers to an amino group wherein the nitrogen
atom directly attached to the parent molecule is substituted with
two groups other than hydrogen, and includes groups selected from
--N(R.sup.bb).sub.2, --NR.sup.bb C(.dbd.O)R.sup.aa,
--NR.sup.bbCO.sub.2R.sup.aa, --NR.sup.bbC(.dbd.O)N(R.sup.bb).sub.2,
--NR.sup.bbC(.dbd.NR.sup.bb)N(R.sup.bb).sub.2,
--NR.sup.bbSO.sub.2R.sup.aa, --NR.sup.bbP(.dbd.O)(OR.sup.cc).sub.2,
and --NR.sup.bbP(.dbd.O)(NR.sup.bb).sub.2, wherein R.sup.aa,
R.sup.bb, and R.sup.cc are as defined herein, with the proviso that
the nitrogen atom directly attached to the parent molecule is not
substituted with hydrogen.
[0075] As used herein, the term "trisubstituted amino" or
"trisubstituted amine" refers to an amino group wherein the
nitrogen atom directly attached to the parent molecule is
substituted with three groups, and includes groups selected from
--N(R.sup.bb).sub.3 and --N(R.sup.bb).sub.3.sup.+X.sup.-, wherein
R.sup.bb and X.sup.- are as defined herein.
[0076] As used herein, "biotin", e.g., as an exemplary R.sup.1A
group, comprises the structure:
##STR00001##
[0077] As used herein, a "ceramide", e.g., as an exemplary R.sup.1A
group, comprises the structure:
##STR00002##
wherein R' is an optionally substituted C.sub.6-30 alkyl (e.g.,
C.sub.6, C.sub.7, C.sub.8, C.sub.9, C.sub.10, C.sub.11, C.sub.12,
C.sub.13, C.sub.14, C.sub.15, C.sub.16, C.sub.17, C.sub.18,
C.sub.19, C.sub.20, C.sub.21, C.sub.22, C.sub.23, C.sub.24,
C.sub.25, C.sub.26, C.sub.27, C.sub.28, C.sub.29, or C.sub.30
alkyl), optionally substituted C.sub.6-C.sub.30alkenyl (e.g.,
C.sub.6, C.sub.7, C.sub.8, C.sub.9, C.sub.10, C.sub.11, C.sub.12,
C.sub.13, C.sub.14, C.sub.16, C.sub.17, C.sub.18, C.sub.19,
C.sub.20, C.sub.21, C.sub.22, C.sub.23, C.sub.24, C.sub.25,
C.sub.26, C.sub.27, C.sub.28, C.sub.29, or C.sub.30 alkenyl), or
optionally substituted C.sub.6-C.sub.30alkynyl (e.g., C.sub.6,
C.sub.7, C.sub.8, C.sub.9, C.sub.10, C.sub.11, C.sub.12, C.sub.13,
C.sub.14, C.sub.15, C.sub.16, C.sub.17, C.sub.18, C.sub.19,
C.sub.20, C.sub.21, C.sub.22, C.sub.23, C.sub.24, C.sub.25,
C.sub.26, C.sub.27, C.sub.28, C.sub.29, or C.sub.30 alkynyl)
group.
[0078] Nitrogen atoms can be substituted or unsubstituted as
valency permits, and include primary, secondary, tertiary, and
quarternary nitrogen atoms. Exemplary nitrogen atom substitutents
include, but are not limited to, hydrogen, --OH, --OR.sup.aa,
--N(R.sup.cc).sub.2, --CN, --C(.dbd.O)R.sup.aa,
--C(.dbd.O)N(R.sup.cc).sub.2, --CO.sub.2R.sup.aa,
--SO.sub.2R.sup.aa, --C(.dbd.NR.sup.bb)R.sup.aa,
--C(.dbd.NR.sup.aa)OR.sup.aa, --C(.dbd.NR.sup.cc)N(R.sup.cc).sub.2,
--SO.sub.2N(R.sup.cc).sub.2, --SO.sub.2R.sup.cc,
--SO.sub.2OR.sup.cc, --SOR.sup.aa, --C(.dbd.S)N(R.sup.cc).sub.2,
--C(.dbd.O)SR.sup.aa, --C(.dbd.S)SR.sup.aa,
--P(.dbd.).sub.2R.sup.aa, --P(.dbd.O)(R.sup.aa).sub.2,
--P(.dbd.O).sub.2N(R.sup.cc).sub.2, --P(.dbd.O)(NR.sup.aa).sub.2,
C.sub.1-10 alkyl, C.sub.1-10 perhaloalkyl, C.sub.2-10 alkenyl,
C.sub.2-10 alkynyl, C.sub.1-10 heteroalkyl, C.sub.2-10
heteroalkenyl, C.sub.2-10 heteroalkynyl, C.sub.3-10 carbocyclyl,
3-14 membered heterocyclyl, C.sub.6-14 aryl, and 5-14 membered
heteroaryl, or two R.sup.cc groups attached to an N atom are joined
to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl
ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl,
heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and
heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5
R.sup.dd groups, and wherein R.sup.aa, R.sup.bb, R.sup.cc and
R.sup.dd are as defined above.
[0079] In certain embodiments, the substituent present on the
nitrogen atom is an nitrogen protecting group (also referred to
herein as an "amino protecting group"). Nitrogen protecting groups
include, but are not limited to, --OH, --OR.sup.aa,
--N(R.sup.cc).sub.2, --C(.dbd.O)R.sup.aa,
--C(.dbd.O)N(R.sup.cc).sub.2, --CO.sub.2R.sup.aa,
--SO.sub.2R.sup.aa, --C(.dbd.NR.sup.cc)R.sup.aa,
--C(.dbd.NR.sup.aa)OR.sup.aa, --C(.dbd.NR.sup.cc)N(R.sup.cc).sub.2,
--SO.sub.2N(R.sup.cc).sub.2, --SO.sub.2R.sup.cc,
--SO.sub.2OR.sup.cc, --SOR.sup.aa, --C(.dbd.S)N(R.sup.cc).sub.2,
--C(.dbd.O)SR.sup.cc, --C(.dbd.S)SR.sup.cc, C.sub.1-10 alkyl (e.g.,
aralkyl, heteroaralkyl), C.sub.2-10 alkenyl, C.sub.2-10 alkynyl,
C.sub.1-10 heteroalkyl, C.sub.2-10 heteroalkenyl, C.sub.2-10
heteroalkynyl, C.sub.3-10 carbocyclyl, 3-14 membered heterocyclyl,
C.sub.6-14 aryl, and 5-14 membered heteroaryl groups, wherein each
alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,
carbocyclyl, heterocyclyl, aralkyl, aryl, and heteroaryl is
independently substituted with 0, 1, 2, 3, 4, or 5 R.sup.dd groups,
and wherein R.sup.aa, R.sup.bb, R.sup.cc and R.sup.dd are as
defined herein. Nitrogen protecting groups are well known in the
art and include those described in detail in Protecting Groups in
Organic Synthesis, T. W. Greene and P. G. M. Wuts. 3.sup.rd
edition, John Wiley & Sons, 1999, incorporated herein by
reference.
[0080] For example, nitrogen protecting groups such as amide groups
(e.g., --C(.dbd.O)R.sup.aa) include, but are not limited to,
formamide, acetamide, chloroacetamide, trichloroacetamide,
trifluoroacetamide, phenylacetamide, 3-phenylpropanamide,
picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl
derivative, benzamide, p-phenylbenzamide, o-nitophenylacetamide,
o-nitrophenoxyacetamide, acetoacetamide,
(N'-dithiobenzyloxyacylamino)acetamide,
3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide,
2-methyl-2-(o-nitrophenoxy)propanamide,
2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide,
3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine
derivative, o-nitrobenzamide and o-(benzoyloxymethyl)benzamide.
[0081] Nitrogen protecting groups such as carbamate groups (e.g.,
--C(.dbd.O)OR.sup.aa) include, but are not limited to, methyl
carbamate, ethyl carbamante, 9-fluorenylmethyl carbamate (Fmoc),
9-(2-sulfo)fluorenylmethyl carbamate,
9-(2,7-dibromo)fluoroenylmethyl carbamate,
2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl
carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc),
2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl
carbamate (Teoc), 2-phenylethyl carbamate (hZ),
1-(1-adamantyl)-1-methylethyl carbamate (Adpoc), 1,
l-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl
carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate
(TCBOC), 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc),
1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2'-
and 4'-pyridyl)ethyl carbamate (Pyoc),
2-(N,N-dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate
(BOC), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl
carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), cinnamyl
carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl
carbamate, N-hydroxypiperidinyl carbamate, alkyldithio carbamate,
benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz),
p-nitobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzyl
carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl
carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl
carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl
carbamate, 2-(p-toluenesulfonyl)ethyl carbamate,
[2-(1,3-dithianyl)]methyl carbamate (Dmoc), 4-methylthiophenyl
carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc),
2-phosphonioethyl carbamate (Peoc), 2-triphenylphosphonioisopropyl
carbamate (Ppoc), 1,1-dimethyl-2-cyanoethyl carbamate,
m-chloro-p-acyloxybenzyl carbamate, p-(dihydroxyboryl)benzyl
carbamate, 5-benzisoxazolylmethyl carbamate,
2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc),
m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate,
o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate,
phenyl(o-nitrophenyl)methyl carbamate, r-amyl carbamate, S-benzyl
thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate,
cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl
carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxyacylvinyl
carbamate, o-(N,N-dimethylcarboxamido)benzyl carbamate,
1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate,
1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate,
2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl
carbamate, isobutyl carbamate, isonicotinyl carbamate,
p-(p'-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl
carbamate, 1-methylcyclohexyl carbamate,
1-methyl-1-cyclopropylmethyl carbamate,
1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate,
1-methyl-1-(p-phenylazophenyl)ethyl carbamate,
1-methyl-1-phenylethyl carbamate, 1-methyl-1-(4-pyridyl)ethyl
carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate,
2,4,6-tri-t-butylphenyl carbamate, 4-(trimethylammonium)benzyl
carbamate, and 2,4,6-trimethylbenzyl carbamate.
[0082] Nitrogen protecting groups such as sulfonamide groups (e.g.,
--S(.dbd.O).sub.2R.sup.aa) include, but are not limited to,
p-toluenesulfonamide (Ts), benzenesulfonamide,
2,3,6,-trimethyl-4-methoxybenzenesulfonamide (Mtr),
2,4,6-trimethoxybenzenesulfonamide (Mtb),
2,6-dimethyl-4-methoxybenzenesulfonamide (Pme),
2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte),
4-methoxybenzenesulfonamide (Mbs),
2,4,6-trimethylbenzenesulfonamide (Mts),
2,6-dimethoxy-4-methylbenzenesulfonamide (iMds),
2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc),
methanesulfonamide (Ms), 1-trimethylsilylethanesulfonamide (SES),
9-anthracenesulfonamide,
4-(4',8'-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS),
benzylsulfonamide, trifluoromethylsulfonamide, and
phenacylsulfonamide.
[0083] Other nitrogen protecting groups include, but are not
limited to, phenothiazinyl-(10)-acyl derivative,
N'-p-toluenesulfonylaminoacyl derivative, N'-phenylaminothioacyl
derivative, N-benzoylphenylalanyl derivative, N-acetylmethionine
derivative, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide,
N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide,
N-2,5-dimethylpyrrole, N-1,1,4,4-tetramethyldisilylazacyclopentane
adduct (STABASE), 5-substituted
1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted
1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted
3,5-dinitro-4-pyridone, N-methylamine, N-allylamine,
N-[2-(trimethylsilyl)ethoxy]methylamine (SEM),
N-3-acetoxypropylamine,
N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary
ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methylamine,
N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr),
N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr),
N-9-phenylfluorenylamine (PhF),
N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino
(Fcm), N-2-picolylamino N'-oxide, N-1,1-dimethylthiomethyleneamine,
N-benzylideneamine, N-p-methoxybenzylideneamine,
N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine,
N--(N',N'-dimethylaminomethylene)amine, N,N'-isopropylidenediamine,
N-p-nitrobenzylideneamine, N-salicylideneamine,
N-5-chlorosalicylideneamine,
N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine,
N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine,
N-borane derivative, N-diphenylborinic acid derivative,
N-[phenyl(pentaacylchromium- or tungsten)acyl]amine, N-copper
chelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine
N-oxide, diphenylphosphinamide (Dpp), dimethylthiophosphinamide
(Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates,
dibenzyl phosphoramidate, diphenyl phosphoramidate,
benzenesulfenamide, o-nitrobenzenesulfenamide (Nps),
2,4-dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide,
2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide,
and 3-nitropyridinesulfenamide (Npys).
[0084] In certain embodiments, the substituent present on an oxygen
atom is an oxygen protecting group (also referred to herein as an
"hydroxyl protecting group"). Oxygen protecting groups include, but
are not limited to, --R.sup.aa, --N(R.sup.bb).sub.2,
--C(.dbd.O)SR.sup.aa, --C(.dbd.O)R.sup.aa, --CO.sub.2R.sup.aa,
--C(.dbd.O)N(R.sup.bb).sub.2, --C(.dbd.NR.sup.bb)R.sup.aa,
--C(.dbd.NR.sup.bb)OR.sup.aa, --C(.dbd.NR.sup.bb)N(R.sup.bb).sub.2,
--S(.dbd.O)R.sup.aa, --SO.sub.2R.sup.aa, --Si(R.sup.aa).sub.3,
--P(R.sup.cc).sub.Z, --P(R.sup.cc).sub.3,
--P(.dbd.O).sub.2R.sup.aa, --P(.dbd.O)(R.sup.aa).sub.2,
--P(.dbd.O)(OR.sup.cc).sub.2, --P(.dbd.O).sub.2N(R.sup.bb).sub.2,
and --P(.dbd.O)(NR.sup.bb).sub.2, wherein R.sup.aa, R.sup.bb, and
R.sup.cc are as defined herein. Oxygen protecting groups are well
known in the art and include those described in detail in
Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M.
Wuts, 3.sup.rd edition, John Wiley & Sons, 1999, incorporated
herein by reference.
[0085] Exemplary oxygen protecting groups include, but are not
limited to, methyl, methoxylmethyl (MOM), methylthiomethyl (MTM),
t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM),
benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM),
(4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM),
t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl,
2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl,
bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR),
tetrahydropyranyl (THP), 3-bromotetrahydropyranyl,
tetrahydrothiopyranyl, 1-methoxycyclohexyl,
4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl,
4-methoxytetrahydrothiopyranyl S,S-dioxide,
1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP),
1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl,
2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl,
1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl,
1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl,
2,2,2-trichloroethyl, 2-trimethylsilylethyl,
2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl,
p-methoxyphenyl, 2,4-dinitrophenyl, benzyl (Bn), p-methoxybenzyl,
3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl,
2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl,
4-picolyl, 3-methyl-2-picolyl N-oxido, diphenylmethyl,
p,p'-dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl,
.alpha.-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl,
di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl,
4-(4'-bromophenacyloxyphenyl)diphenylmethyl,
4,4',4''-tris(4,5-dichlorophthalimidophenyl)methyl,
4,4',4''-tris(levulinoyloxyphenyl)methyl,
4,4',4''-tris(benzoyloxyphenyl)methyl,
3-(imidazol-1-yl)bis(4',4''-dimethoxyphenyl)methyl,
1,1-bis(4-methoxyphenyl)-1'-pyrenylmethyl, 9-anthryl,
9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl,
1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido,
trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl
(TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl
(DEIPS), dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS),
t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl,
triphenylsilyl, diphenylmethylsilyl (DPMS),
t-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate,
acetate, chloroacetate, dichloroacetate, trichloroacetate,
trifluoroacetate, methoxyacetate, triphenylmethoxyacetate,
phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate,
4-oxopentanoate (levulinate), 4,4-(ethylenedithio)pentanoate
(levulinoyldithioacetal), pivaloate, adamantoate, crotonate,
4-methoxycrotonate, benzoate, p-phenylbenzoate,
2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate,
9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl
2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl
carbonate (TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec),
2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutyl
carbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkyl
p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl
p-methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate,
alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl
S-benzyl thiocarbonate, 4-ethoxy-1-naphthyl carbonate, methyl
dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate,
4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate,
2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl,
4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate,
2,6-dichloro-4-methylphenoxyacetate,
2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,
2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate,
isobutyrate, monosuccinate, (E)-2-methyl-2-butenoate,
o-(methoxyacyl)benzoate, .alpha.-naphthoate, nitrate, alkyl
N,N,N',N'-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate,
borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate,
sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate
(Ts).
[0086] These and other exemplary substituents are described in more
detail in the Detailed Description, Examples, and claims. The
invention is not intended to be limited in any manner by the above
exemplary listing of substituents.
[0087] As used herein, the term "salt" refers to any and all
salts.
[0088] The term "pharmaceutically acceptable salt" refers to those
salts which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of humans and lower
animals without undue toxicity, irritation, allergic response and
the like, and are commensurate with a reasonable benefit/risk
ratio. Pharmaceutically acceptable salts are well known in the art.
For example, Berge et al., describes pharmaceutically acceptable
salts in detail in J. Pharmaceutical Sciences (1977) 66:1-19.
Pharmaceutically acceptable salts of the compounds of this
invention include those derived from suitable inorganic and organic
acids and bases. Examples of pharmaceutically acceptable, nontoxic
acid addition salts are salts of an amino group formed with
inorganic acids such as hydrochloric acid, hydrobromic acid,
phosphoric acid, sulfuric acid and perchloric acid or with organic
acids such as acetic acid, oxalic acid, maleic acid, tartaric acid,
citric acid, succinic acid or malonic acid or by using other
methods used in the art such as ion exchange. Other
pharmaceutically acceptable salts include adipate, alginate,
ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,
borate, butyrate, camphorate, camphorsulfonate, citrate,
cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, formate, fumarate, glucoheptonate,
glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate,
hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate,
laurate, lauryl sulfate, malate, maleate, malonate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,
oleate, oxalate, palmitate, pamoate, pectinate, persulfate,
3-phenylpropionate, phosphate, picrate, pivalate, propionate,
stearate, succinate, sulfate, tartrate, thiocyanate,
p-toluenesulfonate, undecanoate, valerate salts, and the like.
Pharmaceutically acceptable salts derived from appropriate bases
include alkali metal, alkaline earth metal, ammonium and
N.sup.+(C.sub.1-4alkyl).sub.4 salts. Representative alkali or
alkaline earth metal salts include sodium, lithium, potassium,
calcium, magnesium, and the like. Further pharmaceutically
acceptable salts include, when appropriate, nontoxic ammonium,
quaternary ammonium, and amine cations formed using counterions
such as halide, hydroxide, carboxylate, sulfate, phosphate,
nitrate, lower alkyl sulfonate, and aryl sulfonate.
BRIEF DESCRIPTION OF THE DRAWINGS
[0089] FIG. 1 depicts the chemical structures of allyl-tailed Gb3,
Gb4, Gb5, Globo H, and SSEA4.
[0090] FIG. 2 depicts glycosylation reactions combined with
nucleotide sugar regeneration and synthesis results monitored by
TLC. A: Combined galactosylation with UDP-Gal regeneration for
synthesizing, e.g., allyl-Gb3. B: Combined acetylgalactosamination
with UDP-GalNAc regeneration for synthesizing, e.g., allyl-Gb4. C:
Combined galactosylation with UDP-Gal regeneration for
synthesizing, e.g., allyl-Gb5. D: Combined fucosylation with
GDP-Fuc regeneration for synthesizing, e.g., allyl-Globo H. E:
Combined sialylation with CMP-Neu5Ac regeneration for synthesizing,
e.g., allyl-SSEA4.
[0091] FIG. 3 depicts the biosynthetic pathway of
glycosphingolipids, involving addition of galactose residues, which
can be calayzed by a galactosyltransferase coupled with the UDP-Gal
regeneration process described herein.
[0092] FIG. 4 depicts the enzymatic synthetic strategy in the
manufacture of Globo H via the Lac.fwdarw.Gb3.fwdarw.Gb4.fwdarw.Gb5
pathway using a nucleotide sugar regeneration system.
[0093] FIG. 5 depicts the enzymatic synthetic strategy in the
manufacture of SSEA4 via the Lac.fwdarw.Gb3.fwdarw.Gb4.fwdarw.Gb5
pathway using a nucleotide sugar regeneration system.
[0094] FIG. 6 depicts the enzymatic synthetic strategy in the
manufacture of allyl-Globo H via the
allyl-Lac.fwdarw.allyl-Gb3.fwdarw.allyl-Gb4.fwdarw.allyl-Gb5
pathway using a nucleotide sugar regeneration system.
[0095] FIG. 7 depicts the enzymatic synthetic strategy in the
manufacture of allyl-SSEA4 via the
allyl-Lac.fwdarw.allyl-Gb3.fwdarw.allyl-Gb4.fwdarw.allyl-Gb5
pathway using a nucleotide sugar regeneration system.
[0096] FIG. 8 depicts the high purity obtained in the biosynthesis
of intermediates allyl-Gb3, allyl-Gb4, and allyl-Gb5.
[0097] FIG. 9 depicts the high purity obtained in the biosynthesis
of allyl-Globo H from allyl-Gb5 using unmodified and modified
FutC.
[0098] FIG. 10 depicts the high purity obtained in the biosynthesis
of allyl-SSEA4 from allyl-Gb5 using JT-FAJ-16.
DETAILED DESCRIPTION OF THE INVENTION
[0099] Described herein are newly developed nucleotide sugar
regeneration processes and their uses in adding sugar residues to
suitable acceptors via the action of a suitable
glycosyltransferase. These approaches allow chain reactions for
synthesizing glycosylated molecules, such as oligosaccharides
(e.g., Gb3, Gb4, Gb5, Globo H, and SSEA4) without the need to
purify intermediates, resulting in unexpectedly rapid production of
the glycosylated products with unexpectedly high yields. In
addition, the synthesis methods described herein can be used for
large scale production of desired oligosaccharides and
glycoconjugates.
UDP-Gal Regeneration System and its Use in Galactosylation
[0100] The UDP-Gal regeneration system is exemplified in FIG. 2A,
involving the enzymes listed in Table 1 below:
TABLE-US-00001 TABLE 1 Enzymes Used in UDP-Gal Regeneration System
Enzyme Activity Examples Galactokinase (GalK) Catalyzes the
phosphorylation of E. coli (e.g., GenBank accession
alpha-D-galactose to produce no. AP012306.1 galactose-1-phosphate
(Gal-1-P) H. sapiens (e.g., GenBank in the presence of ATP
accession no. NP_000145) M. hydrothermalis (e.g., GenBank accession
no. YP_004368991) S. sputigena (e.g., GenBank accession no.
AEC00832) H. hydrossis (e.g., GenBank accession no. YP_004451189)
UDP-sugar Catalyzes the conversion of Gal- A. thaliana (e.g.,
GenBank pyrophosphorylase 1-P to UDP-Gal in the presence accession
no. AF360236.1 (USP) of UTP L. major (e.g., GenBank accession no.
ABY79093) T. cruzi (e.g., GenBank accession no. ADD10758) L.
donovani (e.g., GenBank accession no. XP_00385998) G. max (e.g.,
GenBank accession no. NP_001237434) Pyruvate kinase Catalyzes the
transfer of a E. coli (e.g., GenBank accession (PykF) phosphate
group from no. U00096.2) phosphoenolpyruvate (PEP) to N.
hamburgensis (e.g., GenBank ADP, producing pyruvate and accession
no. YP_576506) ATP or UTP R. palustris (e.g., GenBank accession no.
YP_7830161) M. ruestringensis (e.g., GenBank accession no.
YP_004787669) H. hydrossis (e.g., GenBank accession no.
YP_004450514) S. coccoides (e.g., GenBank accession no.
YP_00441096) Pyrophosphatase Acid anhydride hydrolase that E. coli
(e.g., GenBank accession (PPA) (Optional) acts upon diphosphate
bonds no. U00096.2 G. theta (e.g., GenBank accession no. CAI77906)
C. butyricum (e.g., GenBank accession no. ZP_04525837) L. plantarum
(e.g., GenBank accession no. EFK28054) L. suebicus (e.g., GenBan
accession no. ZP_09451344)
[0101] The enzymes to be used in the UDP-Gal regeneration system
described herein can be a wild-type enzyme. As used herein, a
wild-type enzyme is a naturally occurring enzyme found in a
suitable species. In some examples, the GalK, USP, PykF, and PPA
enzymes can be from E. coli, A. thaliana, E. coli, and E. coli,
respectively. Examples of the enzymes from these species are listed
in Table 1 above. Others can be readily identified by those skilled
in the art, e.g., search a publicly available gene database, such
as GenBank. In other examples, these enzymes are homologs of those
from the just-noted species, which are within the knowledge of
those skilled in the art. For example, such homologs can be
identified by searching GenBank using the amino acid sequence or
the coding nucleotide sequence of an exemplary enzyme as a search
query.
[0102] Alternatively, the enzymes involved in the UDP-Gal
regeneration system can be a functional variant of a wild-type
counterpart. As used herein, a functional variant of a wild-type
enzyme possesses the same enzymatic activity as the wild-part
counterpart and typically shares a high amino acid sequence
homology, e.g., at least 80%, 85%, 90%, 95, or 98% identical to the
amino acid sequence of the wild-type counterpart. The "percent
identity" of two amino acid sequences is determined using the
algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA
87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl.
Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated
into the NBLAST and XBLAST programs (version 2.0) of Altschul, et
al J. Mol. Biol. 215:403-10, 1990. BLAST protein searches can be
performed with the XBLAST program, score=50, wordlength=3 to obtain
amino acid sequences homologous to the protein molecules of
interest. Where gaps exist between two sequences, Gapped BLAST can
be utilized as described in Altschul et al., Nucleic Acids Res.
25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST
programs, the default parameters of the respective programs (e.g.,
XBLAST and NBLAST) can be used.
[0103] A functional variant can have various mutations, including
addition, deletion, or substitution of one or more amino acid
residues. Such a variant often contain mutations in regions that
are not essential to the enzymatic activity of the wild-type enzyme
and may contain no mutations in functional domains or contain only
conservative amino acid substitutions. The skilled artisan will
realize that conservative amino acid substitutions may be made in
lipoic acid ligase mutants to provide functionally equivalent
variants, i.e., the variants retain the functional capabilities of
the particular lipoic acid ligase mutant. As used herein, a
"conservative amino acid substitution" refers to an amino acid
substitution that does not alter the relative charge or size
characteristics of the protein in which the amino acid substitution
is made. Variants can be prepared according to methods for altering
polypeptide sequence known to one of ordinary skill in the art such
as are found in references which compile such methods, e.g.
Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds.,
Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989, or Current Protocols in Molecular Biology, F.
M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York.
Conservative substitutions of amino acids include substitutions
made amongst amino acids within the following groups: (a) M, I, L,
V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g)
E, D.
[0104] Any of the enzymes involved in the UDP-Gal regeneration
system can be prepared via routine technology. In one example, the
enzyme is isolated form a natural source. In other examples, the
enzyme is prepared by routine recombinant technology. When
necessary, the coding sequence of a target enzyme can be subjected
to coden optimization based on the host cell used for producing the
enzyme. For example, when E. coli cells are used as the host for
producing an enzyme via recombinant technology, the gene encoding
that enzyme can be modified such that it contains codons commonly
used in E. coli.
[0105] As illustrated in FIG. 2A, the UDP-Gal regeneration system
can be used in conjunction with a galactosylation reaction via the
activity of a galactosyltransferase to add a galactose residue to a
suitable substrate. Examples of galactosyltransferases are listed
in Table 2 below:
TABLE-US-00002 TABLE 2 Galactosyltransferases Galactosyltransferase
Enzymatic Activity Examples Beta-1,4-Galactosyltransferase
Catalyzes the transfer of Homo sapiens [e.g., GI: (B4GALT),
including isoforms galactose from UDP-Gal to 903740] 1-7 a suitable
acceptor, such as Rattus norvegicus [e.g., GI:
(Beta-1,4-galactosyltransferase a glycoprotein or glycolipid
3258653] 1-7) acceptor having a terminal Zobellia galactanivorans
[e.g., 2-acetamido-2-deoxy-D- GI: 340619721] glucosyl-group, in an
Clostridium perfringens [e.g., beta1,4-linkage GI: 18309463]
Beta-1,3-Galactosyltransferase Catalyzes the transfer of Culex
quinquefasciatus [e.g., (B3GALNT) galactose from UDP-Gal to GI:
167873909] a suitable acceptor, such as Zea mays [e.g., GI:
195643406] a glycoprotein or glycolipid Brachyspira pilosicoli
[e.g., GI: acceptor having a terminal 300871377]
2-acetamido-2-deoxy-D- Enterococcus faecium [e.g., GI:
glucosyl-group, or a 257822935] GalNAc residue, in an LgtD, from,
e.g., Haemophilus beta1,3-linkage influenza [L42023.1] Alpha-1,4-
Catalyzes the transfer of a Homo sapiens [e.g.,
Galactosyltransferase galactose from UDP-Gal to GI: 55956926]
(A4GALT) a suitable acceptor such as a Mustela putorius furo [e.g.,
GI: e.g.: glycoprotein or a glycolipid 355666115] Lactosylceramide
4-alpha- having, e.g., a terminal Mus musculus [e.g., GI:
galactosyltransferase galactose residue or a 51921295] GlcNAc
residue in an alpha Rattus norvegicus [e.g., GI: 1,4-linkage
67677925] LgtC from, e.g., Neisseria meningitides [e.g.,
AF355193.1] Alpha-1,3- Catalyzes the transfer of a Mus musculus
[e.g., Galactosyltransferase galactose from UDP-Gal to GI:
224922807] (A3GALT) a suitable acceptor such as a Mustela putorius
furo [e.g., GI: e.g.: glycoprotein or a glycolipid 355690122]
Alpha-1,3- having, e.g., a terminal Cebus paella [e.g., GI:
Galactosyltransferase 1 galactose residue or a 19698748] Alpha-1,3-
GlcNAc residue in an alpha Rattus norvegicus [e.g., GI:
Galactosyltransferase 2 1,3-linkage 28625949]
[0106] Both wild-type galactosyltransferases and functional
variants, as described above, are within the scope of this
description. Such glycosyltransferases can be prepared via any
routine method.
[0107] The combination of the UDP-Gal regeneration system and one
or more galactosyltransferases can be used for adding a galactose
residue to a suitable substrate (an acceptor) with high yields.
Substrates for galactosyltransferase, e.g., described in Table 2
above, are well known to those skilled in the art. Preferably, the
substrate has a terminal sugar residue (e.g., Gal, GalNAc, or
GlcNAc) to which the galactose residue can be added. In some
examples, the substrate is a polysaccharide (having >50
monosaccharide units), an oligosaccharide (having 2-50
monosaccharide units), a glycoprotein or glycopeptide, or a
glycolipid. The type of a galactosyltransferase to be used in the
galactosylation methods descried herein depends on the end product
of interest and the substrate for synthesizing the end product,
which is well within the knowledge of a skilled artisan. The
combined UDP-Gal regeneration system/galactosyltransferase approach
described herein can be used to synthesize glycosphingolipids.
Examples are illustrated in FIG. 3.
[0108] In other examples, the combined UDP-Gal generation
system/galactosyltransferase approach can be used for synthesizing
Globo-series oligosaccharides, such as synthesis of Gb3 from
lactose or synthesis of Gb5 from Gb4. FIGS. 2A and 2C. See also
descriptions below.
UDP-GalNAc Regeneration System and its Use in
N-Acetylgalactosamination
[0109] A UDP-GalNAc regeneration system can be co-used with an
N-acetylgalactosaminyltransferase (GalNAcT), such as a
beta1,3-N-acetylgalactosaminyltransferase, for addition of a GalNAc
residue onto a suitable acceptor.
[0110] Enzymes involved in an exemplary UDP-GalNAc regeneration
system are shown in Table 3 below:
TABLE-US-00003 TABLE 3 Enzymes Used in UDP-GalNAc Regeneration
System Enzyme Activity Examples N-Acetylhexosamine 1- Acts by a
sequential two NahK from B. longum (e.g., Kinase (GalNAcK)
substrates-two products GenBank accession no. mechanism to convert
ATP and CP000246.1 N-acetylhexosamine into ADP B. breve (e.g.,
GenBank and N-acetyl-alpha-D- accession no. ZP_06596651) hexosamine
1-phosphate. A. haemolyticum (e.g., GenBank accession no.
YP_003696399 B. bifidum (e.g., GenBank accession no. YP_003938776)
N-acetylglucosamine 1- Catalyzes the conversion of GlmU from E.
coli (e.g., phosphate UTP and N-acetyl-alpha-D- GenBank accession
no. uridylyltransferase (GlmU) glucosamine 1-phosphate into
U00096.2 diphosphate and UDP-N- A. thaliana (e.g., GenBank
acetyl-D-glucosamine accession no. AEE31311) G. bemidjiensis (e.g.,
GenBank accession no. ACH37122) H. pylori (e.g., GenBank accession
no. YP_003728906) Pyruvate kinase (PykF) Catalyzes the transfer of
a E. coli (e.g., GenBank phosphate group from accession no.
U00096.2) phosphoenolpyruvate (PEP) to N. hamburgensis (e.g., ADP,
producing pyruvate and GenBank accession no. ATP or UTP YP_576506)
R. palustris (e.g., GenBank accession no. YP_7830161) M.
ruestringensis (e.g., GenBank accession no. YP_004787669) H.
hydrossis (e.g., GenBank accession no. YP_004450514) S. coccoides
(e.g., GenBank accession no. YP_00441096) Pyrophosphatase (PPA)
Acid anhydride hydrolase that E. coli (e.g., GenBank (Optional)
acts upon diphosphate bonds accession no. U00096.2 G. theta (e.g.,
GenBank accession no. CAI77906) C. butyricum (e.g., GenBank
accession no. ZP_04525837) L. plantarum (e.g., GenBank accession
no. EFK28054) L. suebicus (e.g., GenBan accession no.
ZP_09451344)
[0111] N-acetylgalactosaminyltransferase (e.g., beta-1,3-GalNAcT or
beta-1,4-GalNAcT) is an enzyme that catalyzes the reaction in which
a GAlNAc residue is added onto a suitable acceptor, such as a
peptide or an oligosaccharide. Examples include LgtD from H.
influenza, (GenBank accession no. L42023.1. Other examples include,
but are not limited to, LgtD of B. garinii (e.g., GenBank accession
no. AEW68905), LgtD of N. lactamica (e.g., GenBank accession no.
AAN08512), and LgtD of R. felis (e.g., GenBank accession no.
YP_246702).
[0112] Any of the enzymes used in the combined UDP-GalNAc
regeneration system/GalNAcT approach can be either a wild-type
enzyme or a functional variant thereof, as described herein. Any
conventional method can be used for preparing such enzyme. In one
example, this approach is applied for synthesizing Gb4 from Gb3.
See, e.g., FIG. 2B.
GDP-Fuc Regeneration System and its Use in Fucosylation
[0113] An GDP-Fuc regeneration system can be co-used with a
fucosyltransferase (e.g., an alpha1,2-fucosyltransferase, an
alpha1,3-fucosyltransferase, or an alpha2,6-fucosyltransferase) to
add a fucose residue to a suitable acceptor, such as an
oligosaccharide, which can be conjugated to another molecule such
as a lipid or a polypeptide.
[0114] Enzymes involved in an exemplary GDP-Fuc regeneration system
are shown in Table 4 below:
TABLE-US-00004 TABLE 4 Enzymes Used in GDP-Fuc Regeneration System
Enzyme Activity Examples L-fucokinase/GDP-fucose A biofunctional
enzyme that B. fragilis (e.g., GenBank pyrophosphorylase (FKP)
generates Fuc-1-P and GDP- accession no. CR626927.1 Fuc from fucose
and ATP H. sapiens (e.g., GenBank accession no. NP_003829) R.
norvegicus (e.g., GenBank accession no. NP_955788) Pyruvate kinase
(PykF) Catalyzes the transfer of a E. coli (e.g., GenBank accession
phosphate group from no. U00096.2) phosphoenolpyruvate (PEP) to N.
hamburgensis (e.g., GenBank ADP, producing pyruvate and accession
no. YP_576506) ATP or UTP R. palustris (e.g., GenBank accession no.
YP_7830161) M. ruestringensis (e.g., GenBank accession no.
YP_004787669) H. hydrossis (e.g., GenBank accession no.
YP_004450514) S. coccoides (e.g., GenBank accession no.
YP_00441096) Pyrophosphatase (PPA) Acid anhydride hydrolase that E.
coli (e.g., GenBank accession (Optional) acts upon diphosphate
bonds no. U00096.2 G. theta (e.g., GenBank accession no. CAI77906)
C. butyricum (e.g., GenBank accession no. ZP_04525837) L. plantarum
(e.g., GenBank accession no. EFK28054) L. suebicus (e.g., GenBan
accession no. ZP_09451344)
[0115] A fucosyltransferase transfers an L-fucose sugar from a
GDP-fucose (guanosine diphosphate-fucose) donor substrate to an
acceptor substrate, which can be another sugar. Fucosyltransferase
can add the fucose residue to a core GlcNAc (N-acetylglucosamine)
sugar as in the case of N-linked glycosylation, or to a protein, as
in the case of O-linked glycosylation. Fucosyltransferases include
alpha1,3-fucosyltransferase, alpha1,2-fucosyltransferase, and
alpha1,6-fucosyltransferase. Examples include
alpha1,2-fucosyltransferase from E. coli (e.g., GenBank accession
no. U00096.2), alpha 1,3-fucosyltransferase from B. fragilis (e.g.,
GenBank accession no. YP_213404) and from X. laevis (e.g., GenBank
accession no. NP_001083664), alpha 1,6-fucosyltransferase from X.
Any of the enzymes used in the combined GDP-Fuc regeneration
system/FucT approach can be either a wild-type enzyme or a
functional variant thereof, as described herein. Any conventional
method can be used for preparing such enzyme. In one example, this
approach is applied for synthesizing Gb4 from Gb3. See, e.g., FIG.
2D.
CMP-Neu5Ac Regeneration System and its Use in Sialylation
[0116] An CMP-Neu5Ac regeneration system can be coupled with a
sialyltransferase, such as an alpha 2,3-sialyltransferase, to add a
sialic acid residue (Neu5Ac) to a suitable acceptor substrate, such
as an oligosaccharide.
[0117] Enzymes involved in an exemplary CMP-Neu5Ac regeneration
system are shown in Table 5 below:
TABLE-US-00005 TABLE 5 Enzymes Used in CMP-Neu5Ac Regeneration
System Enzyme Activity Examples Cytidine monophosphate Catalyzes
phosphorylation of E. coli (e.g., GenBank accession kinase (CMK)
CMP to produce CDP no. U00096.2 B. amyloliquefaciens (e.g., GenBank
accession no. ABS74466) M. leprae (e.g., GenBank accession no.
CAB08279) M. avium (e.g., GenBank accession no. AAS03731) B.
garinii (e.g., GenBank accession no. AEW68468) CMP-sialic acid
Catalyzes the synthesis of P. multocida (e.g., GenBank synthetase
(Css) CMP sialic acid from CTP accession no. AE004439.1 and sialic
acid. N. meningitidis (e.g., GenBank accession no. AAB60780) O.
mykiss (e.g., GenBank accession no. BAB47150) I. ioihiensis (e.g.,
GenBank accession no. AAV81361) C. jejuni (e.g., GenBank accession
no. ABI32334) Pyruvate kinase (PykF) Catalyzes the transfer of a E.
coli (e.g., GenBank accession phosphate group from no. U00096.2)
phosphoenolpyruvate (PEP) to N. hamburgensis (e.g., GenBank ADP,
producing pyruvate and accession no. YP_576506) ATP or UTP R.
palustris (e.g., GenBank accession no. YP_7830161) M.
ruestringensis (e.g., GenBank accession no. YP_004787669) H.
hydrossis (e.g., GenBank accession no. YP_004450514) S. coccoides
(e.g., GenBank accession no. YP_00441096) Pyrophosphatase (PPA)
Acid anhydride hydrolase that E. coli (e.g., GenBank accession
(Optional) acts upon diphosphate bonds no. U00096.2 G. theta (e.g.,
GenBank accession no. CAI77906) C. butyricum (e.g., GenBank
accession no. ZP_04525837) L. plantarum (e.g., GenBank accession
no. EFK28054) L. suebicus (e.g., GenBan accession no.
ZP_09451344)
[0118] Sialyltransferases are enzymes that transfer sialic acid to
nascent oligosaccharide. This family of enzymes adds sialic acid to
the terminal portions of sialylated glycolipids (gangliosides) or
to the N- or O-linked sugar chains of glycoproteins. There are
about twenty different sialyltransferases, including
sialyltransferases that add sialic acid with an alpha 2,3 linkage
to galactose (e.g., alpha 2,3-sialyltransferase), and
sialyltransferases that add sialic acid with an alpha 2,6 linkage
to galactose or N-acetylgalactosamine (e.g., alpha
2,6-sialyltransferase). Examples include alpha
2,3-sialyltransferase from, e.g., M. bacteria (GenBank accession
no. AB308042.1), M. musculus (e.g., GenBank accession no.
BAA06068), or P. multocida (e.g., GenBank accession no. AET17056);
and alpha 2,6-sialyltransferase from, e.g., B. taurus (e.g.,
GenBank accession no. NP_001008668), C. griseus (e.g., GenBank
accession no. NP_001233744), or R. norvegicus (e.g., GenBank
accession no. AAC42086).
[0119] Any of the enzymes used in the combined CMP-Neu5Ac
regeneration system/sialyltransferase approach can be either a
wild-type enzyme or a functional variant thereof, as described
herein. Any conventional method can be used for preparing such
enzyme. In one example, this approach is applied for synthesizing
Gb4 from Gb3. FIG. 2E.
Synthesis of Globo-Series Oligosaccharides
[0120] The above-described combined approaches involving UDP-Gal
regeneration/galactosyltransferase, UDP-GalNAc
regeneration/GalNAcT, GDP-Fuc regeneration/fucosyltransferase, and
CMP-Neu5Ac regeneration/sialyltransferase can be applied, either
independently, or in combination, to synthesize Globo-series
oligosaccharides, including Gb3. Gb4, Gb5, Globo H (fucosyl-Gb5),
and SSEA4 (sialyl-Gb5). As discussed in greater detail below, all
of these Globo-series oligosaccharides can be either substituted or
unsubstituted.
Step S-1
[0121] The first step in the biosynthetic approach (S-1) involves
enzymatic conversion of a compound of Formula (I), or salt thereof,
to a compound of Formula (II), or salt thereof:
##STR00003##
wherein R.sup.1A is hydrogen, substituted or unsubstituted alkyl,
substituted or unsubstituted alkenyl, substituted or unsubstituted
alkynyl, substituted or unsubstituted carbocyclyl, substituted or
unsubstituted heterocyclyl, substituted or unsubstituted aryl,
substituted or unsubstituted heteroaryl, or an oxygen protecting
group; and each instance of R.sup.2, R.sup.3A, R.sup.5A, R.sup.2B,
R.sup.3B, R.sup.5B, R.sup.2C, R.sup.4C, and R.sup.5C is
independently hydrogen, substituted or unsubstituted C.sub.1-6
alkyl, or an oxygen protecting group.
[0122] Thus, in one aspect, provided is a method of enzymatically
synthesizing a compound of Formula (II), or salt thereof, from a
compound of Formula (I), or salt thereof, comprising converting a
compound of Formula (I) to a compound of Formula (H), or salt
thereof, in the presence of uridine diphosphate-Gal (UDP-Gal) and
an alpha-1,4 galactosyltransferase, and regenerating UDP-Gal from
galactose in the presence of the set of enzymes listed in Table 1
above. See, e.g., FIG. 2A. To perform this enzymatic reaction,
necessary components, such as galactose, galactosyltransferase, the
set of UDP-Gal regeneration enzymes, ATP, UTP, and others (e.g.,
Mg.sup.++), can be mix to form a reaction mixture, which can be
incubated under suitable conditions allowing production of Formula
(II) compounds. Such conditions are well known to those skilled in
the art. See also Examples below.
[0123] The R.sup.1A group can serve as a functional group allowing
conjugation of the Globo-series oligosaccharides to another
molecule, such as a protein or a lipid. Alternative, it can serve
as a protecting group.
[0124] In certain embodiments, R.sup.1A is hydrogen.
[0125] In other embodiments, R.sup.1A is substituted or
unsubstituted alkyl, e.g., substituted or unsubstituted
C.sub.1-6alkyl, substituted or unsubstituted C.sub.2-6alkyl,
substituted or unsubstituted C.sub.3-6alkyl, substituted or
unsubstituted C.sub.4-6alkyl, substituted or unsubstituted
C.sub.5-6alkyl, substituted or unsubstituted C.sub.2-5alkyl,
substituted or unsubstituted C.sub.2-4alkyl, substituted or
unsubstituted C.sub.2-3alkyl, substituted or unsubstituted
C.sub.1alkyl, substituted or unsubstituted C.sub.2alkyl,
substituted or unsubstituted C.sub.3alkyl, substituted or
unsubstituted C.sub.4alkyl, substituted or unsubstituted
C.sub.5alkyl, or substituted or unsubstituted C.sub.6alkyl. Biotin
and a ceramide, as defined herein, are encompassed by substituted
alkyl. In certain embodiments, R.sup.1A is an unsubstituted alkyl,
e.g., in certain embodiments, R.sup.1A is methyl, ethyl, propyl,
isopropyl, sec-butyl, iso-butyl, or tert-butyl. Alternatively, in
certain embodiments, R.sup.1A is a substituted alkyl. In certain
embodiments, R.sup.1A is alkyl which is further substituted with a
substituted or unsubstituted thio, substituted or unsubstituted
amino, carbonyl (e.g., carboxylic acid), azido, alkenyl (e.g.,
allyl), alkynyl (e.g., propargyl), biotin, or a ceramide group. In
certain embodiments, such substituents are substituted at the
terminal position (last carbon atom) of the alkyl group. In certain
embodiments, R.sup.1A is alkyl substituted with one or more amino
(--NH.sub.2) groups. In certain embodiments, R.sup.1A is alkyl
substituted at the terminal position (last carbon atom) with an
amino (.dbd.NH.sub.2) group. In certain embodiments, R.sup.1A is
--(CH.sub.2).sub.n--NH.sub.2 wherein n is 1, 2, 3, 4, 5, or 6. In
certain embodiments, R.sup.1A is 5-pentylamino
(--(CH.sub.2).sub.5--NH.sub.2).
[0126] In certain embodiments, R.sup.1A is substituted or
unsubstituted alkenyl, e.g., substituted or unsubstituted
C.sub.2-6alkenyl, substituted or unsubstituted C.sub.3-6alkenyl,
substituted or unsubstituted C.sub.4-6alkenyl, substituted or
unsubstituted C.sub.5-6alkenyl, substituted or unsubstituted
C.sub.2-5alkenyl, substituted or unsubstituted C.sub.2-4alkenyl,
substituted or unsubstituted C.sub.2-3alkenyl, substituted or
unsubstituted C.sub.2alkenyl, substituted or unsubstituted
C.sub.3alkenyl, substituted or unsubstituted C.sub.4alkenyl,
substituted or unsubstituted C.sub.5alkenyl, or substituted or
unsubstituted C.sub.6alkenyl. In certain embodiments, R.sup.1A is
--(CH.sub.2).sub.m--CH.dbd.CH.sub.2, wherein n is 1, 2, or 3. In
certain embodiments, R.sup.1A is allyl (--CH.sub.2CH.dbd.CH.sub.2).
In certain embodiments, R.sup.1A is alkenyl which is further
substituted with a substituted or unsubstituted thio, substituted
or unsubstituted amino, carbonyl (e.g., carboxylic acid), azido,
alkenyl (e.g., allyl), alkynyl (e.g., propargyl), biotin, or a
ceramide group. In certain embodiments, such substituents are
substituted at the terminal position (last carbon atom) of the
alkenyl group
[0127] In certain embodiments, R.sup.1A is substituted or
unsubstituted alkynyl, e.g., substituted or unsubstituted
C.sub.2-6alkynyl, substituted or unsubstituted C.sub.3-6alkynyl,
substituted or unsubstituted C.sub.4-6alkynyl, substituted or
unsubstituted C.sub.5-6alkynyl, substituted or unsubstituted
C.sub.2-5alkynyl, substituted or unsubstituted C.sub.2-4alkynyl,
substituted or unsubstituted C.sub.2-3alkynyl, substituted or
unsubstituted C.sub.2alkynyl, substituted or unsubstituted
C.sub.3alkynyl, substituted or unsubstituted C.sub.4alkynyl,
substituted or unsubstituted C.sub.5alkynyl, or substituted or
unsubstituted C.sub.6alkynyl. In certain embodiments, R.sup.1A is
alkynyl which is further substituted with a substituted or
unsubstituted thio, substituted or unsubstituted amino, carbonyl
(e.g., carboxylic acid), azido, alkenyl (e.g., allyl), alkynyl
(e.g., propargyl), biotin, or a ceramide group. In certain
embodiments, such substituents are substituted at the terminal
position (last carbon atom) of the alkynyl group.
[0128] In certain embodiments, R.sup.1A is substituted or
unsubstituted heterocyclyl, e.g., substituted or unsubstituted 5-
to 8-membered heterocyclyl, substituted or unsubstituted 5- to
7-membered heterocyclyl, substituted or unsubstituted 5- to
6-membered heterocyclyl, substituted or unsubstituted 5-membered
heterocyclyl, substituted or unsubstituted 6-membered heterocyclyl,
substituted or unsubstituted 7-membered heterocyclyl, or
substituted or unsubstituted 8-membered heterocyclyl. In certain
embodiments, R.sup.1A is heterocyclyl which is further substituted
with a substituted or unsubstituted thio, substituted or
unsubstituted amino, carbonyl (e.g., carboxylic acid), azido,
alkenyl (e.g., allyl), alkynyl (e.g., propargyl), biotin, or a
ceramide group.
[0129] In certain embodiments, R.sup.1A is substituted or
unsubstituted carbocyclyl, e.g., substituted or unsubstituted
C.sub.3-6 carbocyclyl, substituted or unsubstituted C.sub.3-5
carbocyclyl, substituted or unsubstituted C.sub.3-4 carbocyclyl,
substituted or unsubstituted C.sub.3 carbocyclyl, substituted or
unsubstituted C.sub.4 carbocyclyl, substituted or unsubstituted
C.sub.5 carbocyclyl, or substituted or unsubstituted C.sub.6
carbocyclyl. In certain embodiments, R.sup.1A is carbocyclyl which
is further substituted with a substituted or unsubstituted thio,
substituted or unsubstituted amino, carbonyl (e.g., carboxylic
acid), azido, alkenyl (e.g., allyl), alkynyl (e.g., propargyl),
biotin, or a ceramide group.
[0130] In certain embodiments, R.sup.1A is substituted or
unsubstituted aryl, e.g., substituted or unsubstituted C.sub.6 aryl
(phenyl) or substituted or unsubstituted C.sub.10 aryl (naphthyl).
In certain embodiments, R.sup.1A is aryl which is further
substituted with a substituted or unsubstituted thio, substituted
or unsubstituted amino, carbonyl (e.g., carboxylic acid), azido,
alkenyl (e.g., allyl), alkynyl (e.g., propargyl), biotin, or a
ceramide group.
[0131] In certain embodiments, R.sup.1A is substituted or
unsubstituted heteroaryl, e.g., substituted or unsubstituted
5-membered heteroaryl or substituted or unsubstituted 6-membered
heteroaryl. In certain embodiments, R.sup.1A is heteroaryl which is
further substituted with a substituted or unsubstituted thio,
substituted or unsubstituted amino, carbonyl (e.g., carboxylic
acid), azido, alkenyl (e.g., allyl), alkynyl (e.g., propargyl),
biotin, or a ceramide group.
[0132] In certain embodiments, R.sup.1A is hydrogen, allyl,
substituted alkyl, biotin, or a ceramide.
[0133] It is further contemplated herein that R.sup.1A can be a
mixture of any of the above recited non-hydrogen groups, e.g.,
substituted or unsubstituted alkyl, substituted or unsubstituted
alkenyl, substituted or unsubstituted alkynyl, substituted or
unsubstituted carbocyclyl, substituted or unsubstituted
heterocyclyl, substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, to provide a linker group comprising 1,
2, 3, 4, 5, 6, 7, 8, 9, or 10 different combinations of groups. As
a non-limiting example, R.sup.1A may be a linker group comprising
alkyl and aryl combination of groups, e.g., such as
alkyl-aryl-alkyl, and which may optionally be further substituted
at any position on the linker group (e.g., the terminal position)
with a substituted or unsubstituted thio, substituted or
unsubstituted amino, carbonyl (e.g., carboxylic acid), azido,
alkenyl (e.g., allyl), alkynyl (e.g., propargyl), biotin, or a
ceramide group.
[0134] In certain embodiments, R.sup.1A is an oxygen protecting
group, as defined herein.
[0135] In certain embodiments, R.sup.2A is hydrogen. In certain
embodiments, R.sup.2A is substituted or unsubstituted C.sub.1-6
alkyl, e.g., substituted or unsubstituted C.sub.1alkyl, substituted
or unsubstituted C.sub.2alkyl, substituted or unsubstituted
C.sub.3alkyl, substituted or unsubstituted C.sub.4alkyl,
substituted or unsubstituted C.sub.5alkyl, or substituted or
unsubstituted C.sub.6alkyl. In certain embodiments, R.sup.2A is an
oxygen protecting group.
[0136] In certain embodiments, R.sup.3A is hydrogen. In certain
embodiments, R.sup.3A is substituted or unsubstituted C.sub.1-6
alkyl, e.g., substituted or unsubstituted C.sub.1alkyl, substituted
or unsubstituted C.sub.2alkyl, substituted or unsubstituted
C.sub.3alkyl, substituted or unsubstituted C4alkyl, substituted or
unsubstituted C.sub.5alkyl, or substituted or unsubstituted
C.sub.6alkyl. In certain embodiments, R.sup.3A is an oxygen
protecting group.
[0137] In certain embodiments, R.sup.5A is hydrogen. In certain
embodiments, R.sup.5A is substituted or unsubstituted C.sub.1-6
alkyl, e.g., substituted or unsubstituted C.sub.1alkyl, substituted
or unsubstituted C.sub.2alkyl, substituted or unsubstituted
C.sub.3alkyl, substituted or unsubstituted C.sub.4alkyl,
substituted or unsubstituted C.sub.5alkyl, or substituted or
unsubstituted C.sub.6alkyl. In certain embodiments, R.sup.SA is an
oxygen protecting group.
[0138] In certain embodiments, R.sup.2B is hydrogen. In certain
embodiments, R.sup.2B is substituted or unsubstituted
C.sub.1-6alkyl, e.g., substituted or unsubstituted C.sub.1alkyl,
substituted or unsubstituted C.sub.2alkyl, substituted or
unsubstituted C.sub.3alkyl, substituted or unsubstituted
C.sub.4alkyl, substituted or unsubstituted C.sub.5alkyl, or
substituted or unsubstituted C.sub.6alkyl. In certain embodiments,
R.sup.2B is an oxygen protecting group.
[0139] In certain embodiments, R.sup.3B is hydrogen. In certain
embodiments, R.sup.3B is substituted or unsubstituted C.sub.1-6
alkyl, e.g., substituted or unsubstituted C.sub.1alkyl, substituted
or unsubstituted C.sub.2alkyl, substituted or unsubstituted
C.sub.3alkyl, substituted or unsubstituted C.sub.4alkyl,
substituted or unsubstituted C.sub.5alkyl, or substituted or
unsubstituted C.sub.6alkyl. In certain embodiments, R.sup.3B is an
oxygen protecting group.
[0140] In certain embodiments, R.sup.5B is hydrogen. In certain
embodiments, R.sup.5B is substituted or unsubstituted C.sub.1-6
alkyl, e.g., substituted or unsubstituted C.sub.1alkyl, substituted
or unsubstituted C.sub.2alkyl, substituted or unsubstituted
C.sub.3alkyl, substituted or unsubstituted C.sub.4alkyl,
substituted or unsubstituted C.sub.5alkyl, or substituted or
unsubstituted C.sub.6alkyl. In certain embodiments, R.sup.5B is an
oxygen protecting group.
[0141] In certain embodiments, R.sup.2C is hydrogen. In certain
embodiments, R.sup.2C is substituted or unsubstituted
C.sub.1-6alkyl, e.g., substituted or unsubstituted C.sub.1alkyl,
substituted or unsubstituted C.sub.2alkyl, substituted or
unsubstituted C.sub.3alkyl, substituted or unsubstituted
C.sub.4alkyl, substituted or unsubstituted C.sub.5alkyl, or
substituted or unsubstituted C.sub.6alkyl. In certain embodiments,
R.sup.2C is an oxygen protecting group.
[0142] In certain embodiments, R.sup.4C is hydrogen. In certain
embodiments, R.sup.4C is substituted or unsubstituted C.sub.1-6
alkyl, e.g., substituted or unsubstituted C.sub.1alkyl, substituted
or unsubstituted C.sub.2alkyl, substituted or unsubstituted
C.sub.3alkyl, substituted or unsubstituted C.sub.4alkyl,
substituted or unsubstituted C.sub.5alkyl, or substituted or
unsubstituted C.sub.6alkyl. In certain embodiments, R.sup.2C is an
oxygen protecting group.
[0143] In certain embodiments, R.sup.4C is hydrogen. In certain
embodiments, R.sup.4C is substituted or unsubstituted C.sub.1-6
alkyl, e.g., substituted or unsubstituted C.sub.1alkyl, substituted
or unsubstituted C.sub.2alkyl, substituted or unsubstituted
C.sub.3alkyl, substituted or unsubstituted C.sub.4alkyl,
substituted or unsubstituted C.sub.5alkyl, or substituted or
unsubstituted C.sub.6alkyl. In certain embodiments, R.sup.4C is an
oxygen protecting group.
[0144] In certain embodiments, each instance of R.sup.2A, R.sup.3A,
R.sup.5A, R.sup.2B, R.sup.3B, R.sup.5B, R.sup.2C, R.sup.4C, and
R.sup.5C is independently hydrogen. In certain embodiments,
R.sup.1A is substituted or unsubstituted alkenyl, and each instance
of R.sup.2A, R.sup.3A, R.sup.5A, R.sup.2B, R.sup.3B, R.sup.5B,
R.sup.2C, R.sup.4C, and R.sup.5C is independently hydrogen. In
certain embodiments, R.sup.1A is substituted or unsubstituted
alkyl, and each instance of R.sup.2A, R.sup.3A, R.sup.5A, R.sup.2B,
R.sup.3B, R.sup.5B, R.sup.2C, R.sup.4C, and R.sup.5C is
independently hydrogen.
[0145] Exemplary compounds of Formula (I) include, but are not
limited to, and salts thereof.
[0146] Exemplary compounds of Formula (II) include, but are not
limited to,
##STR00004##
and salts thereof.
Step S-2
[0147] The second step in the biosynthetic approach (S-2) involves
enzymatic conversion of a compound of Formula (II), or salt
thereof, to a compound of Formula (III), or salt thereof:
##STR00005##
wherein R.sup.1A, R.sup.2A, R.sup.3A, R.sup.5A, R.sup.2B, R.sup.3B,
R.sup.5B, R.sup.2C, R.sup.4C, and R.sup.5C are as defined herein;
and each instance of R.sup.4D and R.sup.5D is independently
hydrogen, substituted or unsubstituted C.sub.1-6 alkyl, or an
oxygen protecting group.
[0148] In certain embodiments, R.sup.2D is hydrogen. In certain
embodiments, R.sup.2D is substituted or unsubstituted C.sub.1-6
alkyl, e.g., substituted or unsubstituted C.sub.1alkyl, substituted
or unsubstituted C.sub.2alkyl, substituted or unsubstituted
C.sub.3alkyl, substituted or unsubstituted C.sub.4alkyl,
substituted or unsubstituted C.sub.5alkyl, or substituted or
unsubstituted C.sub.6alkyl. In certain embodiments, R.sup.2D is a
nitrogen protecting group, e.g., acetyl (Ac,
--C.dbd.OCH.sub.3).
[0149] In certain embodiments, R.sup.4D is hydrogen. In certain
embodiments, R.sup.4D is substituted or unsubstituted C.sub.1-6
alkyl, e.g., substituted or unsubstituted C.sub.1alkyl, substituted
or unsubstituted C.sub.2alkyl, substituted or unsubstituted
C.sub.3alkyl, substituted or unsubstituted C.sub.4alkyl,
substituted or unsubstituted C.sub.5alkyl, or substituted or
unsubstituted C.sub.6alkyl. In certain embodiments, R.sup.4D is an
oxygen protecting group.
[0150] In certain embodiments, R.sup.5D is hydrogen. In certain
embodiments, R.sup.5D is substituted or unsubstituted C.sub.1-6
alkyl, e.g., substituted or unsubstituted C.sub.1alkyl, substituted
or unsubstituted C.sub.2alkyl, substituted or unsubstituted
C.sub.3alkyl, substituted or unsubstituted C.sub.4alkyl,
substituted or unsubstituted C.sub.5alkyl, or substituted or
unsubstituted C.sub.6alkyl. In certain embodiments, R.sup.5D is an
oxygen protecting group.
[0151] In certain embodiments, both of R.sup.4D and R.sup.5D are
hydrogen. In certain embodiments, R.sup.2D is a nitrogen protecting
group, e.g., acetyl (Ac, --C.dbd.OCH.sub.3), and R.sup.4D and
R.sup.5D are each hydrogen.
[0152] Exemplary compounds of Formula (III) include, but are not
limited to,
##STR00006##
and salts thereof.
[0153] In Step S-2, a method of enzymatically synthesizing a
compound of Formula (III), or salt thereof, from a compound of
Formula (II), or salt thereof, is performed under suitable
conditions. A substrate of Formula (II) can be prepared by any
method known in the art or disclosed herein. In some examples, the
Formula (II) compound is isolated from the reaction mixture
described in Step S-1 above. In other examples, the whole reaction
mixture of Step S-1 is used without purification of the Formula
(II) compound produced therein. The Formula (II) compound can be
incubated with UDP-GalNAc in the presence of a GalNAcT (e.g., a
beta1,3-GalNAcT) under conditions allowing conversion of the
Formula (II) compound to a Formula (III) compound. In some example,
this GalNAcT-catalyzed reaction is coupled with the UDP-GalNAc
regeneration process as described herein. FIG. 2B. See also
Examples below.
Step S-3
[0154] The third step in the biosynthetic approach (S-3) involves
enzymatic conversion of a compound of Formula (III), or salt
thereof, to a compound of Formula (IV), or salt thereof:
##STR00007##
wherein R.sup.1A, R.sup.2A, R.sup.3A, R.sup.5A, R.sup.2B, R.sup.3B,
R.sup.5B, R.sup.2C, R.sup.4C, R.sup.5C, R.sup.2D, R.sup.4D and
R.sup.5D are as defined herein; and each instance of R.sup.2E,
R.sup.3E, R.sup.4E, and R.sup.5E is independently hydrogen,
substituted or unsubstituted C.sub.1-6 alkyl, or an oxygen
protecting group.
[0155] In certain embodiments, R.sup.2E is hydrogen. In certain
embodiments, R.sup.2E is substituted or unsubstituted C.sub.1-6
alkyl, e.g., substituted or unsubstituted C.sub.1alkyl, substituted
or unsubstituted C.sub.2alkyl, substituted or unsubstituted
C.sub.3alkyl, substituted or unsubstituted C.sub.4alkyl,
substituted or unsubstituted C.sub.5alkyl, or substituted or
unsubstituted C.sub.6alkyl. In certain embodiments, R.sup.2E is an
oxygen protecting group.
[0156] In certain embodiments, R.sup.3E is hydrogen. In certain
embodiments, R.sup.3E is substituted or unsubstituted C.sub.1-6
alkyl, e.g., substituted or unsubstituted C.sub.1alkyl, substituted
or unsubstituted C.sub.2alkyl, substituted or unsubstituted
C.sub.3alkyl, substituted or unsubstituted C.sub.4alkyl,
substituted or unsubstituted C.sub.5alkyl, or substituted or
unsubstituted C.sub.6alkyl. In certain embodiments, R.sup.3E is an
oxygen protecting group.
[0157] In certain embodiments, R.sup.4E is hydrogen. In certain
embodiments, R.sup.4E is substituted or unsubstituted C.sub.1-6
alkyl, e.g., substituted or unsubstituted C.sub.1alkyl, substituted
or unsubstituted C.sub.2alkyl, substituted or unsubstituted
C.sub.3alkyl, substituted or unsubstituted C.sub.4alkyl,
substituted or unsubstituted C.sub.5alkyl, or substituted or
unsubstituted C.sub.6alkyl. In certain embodiments, R.sup.4E is an
oxygen protecting group.
[0158] In certain embodiments, R.sup.5E is hydrogen. In certain
embodiments, R.sup.5E is substituted or unsubstituted C.sub.1-6
alkyl, e.g., substituted or unsubstituted C.sub.1alkyl, substituted
or unsubstituted C.sub.2alkyl, substituted or unsubstituted
C.sub.3alkyl, substituted or unsubstituted C.sub.4alkyl,
substituted or unsubstituted C.sub.5alkyl, or substituted or
unsubstituted C.sub.6alkyl. In certain embodiments, R.sup.5E is an
oxygen protecting group.
[0159] In certain embodiments, R.sup.3E is hydrogen. In certain
embodiments, R.sup.3E is substituted or unsubstituted C.sub.1-6
alkyl, e.g., substituted or unsubstituted C.sub.1alkyl, substituted
or unsubstituted C.sub.2alkyl, substituted or unsubstituted
C.sub.3alkyl, substituted or unsubstituted C.sub.4alkyl,
substituted or unsubstituted C.sub.5alkyl, or substituted or
unsubstituted C.sub.6alkyl. In certain embodiments, R.sup.3E is an
oxygen protecting group.
[0160] In certain embodiments, each instance of R.sup.2E, R.sup.3E,
R.sup.4E, and R.sup.5E is hydrogen.
[0161] Exemplary compounds of Formula (IV) include, but are not
limited to,
##STR00008##
and salts thereof.
[0162] Step S-3 involves an enzymatic reaction via the activity of
a beta1,3-galactosyltransferase, which is performed under suitable
conditions known to those skilled in the art. A substrate of
Formula (III), such as Gb4, can be prepared by any method known in
the art or disclosed herein. In some examples, the Formula (m)
compound is isolated from the reaction mixture described in Step
S-2 above. In other examples, the whole reaction mixture of Step
S-2 is used without purification of the Formula (III) compound
produced therein. The Formula (III) compound can be incubated with
UDP-Gal in the presence of a beta1,3-galactosyltransferase under
conditions allowing conversion of the Formula (I) compound to a
Formula (IV) compound. In some example, this GalT-catalyzed
reaction is coupled with the UDP-Gal regeneration process as
described herein. FIG. 2A. See also Examples below.
[0163] In some embodiments, a beta1,3-GalNAcT/beta1,3-GalT
bifunctional enzyme, such as LgtD from, e.g., H. influenza, is used
in both Steps S-2 and S-3.
Step S-4
[0164] The compound of Formula (IV) may then be substituted at
various positions on the terminal Ring E. For example, in certain
embodiments of Formula (IV), wherein R.sup.2E is hydrogen, an
optional step in the biosynthetic approach (S-4) involves enzymatic
conversion of a compound of Formula (IV-a), or salt thereof, to a
compound of Formula (V), or salt thereof:
##STR00009##
wherein R.sup.1A, R.sup.2A, R.sup.3A, R.sup.5A, R.sup.2B, R.sup.3B,
R.sup.5B, R.sup.2C, R.sup.4C, R.sup.5C, R.sup.2D, R.sup.4D,
R.sup.5D, R.sup.3E, R.sup.4E, and R.sup.5E are as defined herein;
and each instance of R.sup.1F, R.sup.2F, and R.sup.3F is
independently hydrogen, substituted or unsubstituted
C.sub.1-6alkyl, or an oxygen protecting group.
[0165] In certain embodiments, R.sup.1F is hydrogen. In certain
embodiments, R.sup.1F is substituted or unsubstituted C.sub.1-6
alkyl, e.g., substituted or unsubstituted C.sub.1alkyl, substituted
or unsubstituted C.sub.2alkyl, substituted or unsubstituted
C.sub.3alkyl, substituted or unsubstituted C.sub.4alkyl,
substituted or unsubstituted C.sub.5alkyl, or substituted or
unsubstituted C.sub.6alkyl. In certain embodiments, R.sup.1F is an
oxygen protecting group.
[0166] In certain embodiments, R.sup.2F is hydrogen. In certain
embodiments, R.sup.2F is substituted or unsubstituted
C.sub.1-6alkyl, e.g., substituted or unsubstituted C.sub.1alkyl,
substituted or unsubstituted C.sub.2alkyl, substituted or
unsubstituted C.sub.3alkyl, substituted or unsubstituted
C.sub.4alkyl, substituted or unsubstituted C.sub.5alkyl, or
substituted or unsubstituted C.sub.6alkyl. In certain embodiments,
R.sup.2F is an oxygen protecting group.
[0167] In certain embodiments, R.sup.3F is hydrogen. In certain
embodiments, R.sup.3F is substituted or unsubstituted
C.sub.1-6alkyl, e.g., substituted or unsubstituted C.sub.1alkyl,
substituted or unsubstituted C.sub.2alkyl, substituted or
unsubstituted C.sub.5alkyl, substituted or unsubstituted
C.sub.4alkyl, substituted or unsubstituted C.sub.5alkyl, or
substituted or unsubstituted C.sub.6alkyl. In certain embodiments,
R.sup.3F is an oxygen protecting group.
[0168] In certain embodiments, each instance of R.sup.1F, R.sup.2F,
and R.sup.3F is hydrogen.
[0169] Exemplary compounds of Formula (V) include, but are not
limited to,
##STR00010##
and salts thereof.
[0170] Step S-4 involves an enzymatic reaction via the activity of
an alpha1,2-fucosyltransferase, which is performed under suitable
conditions known to those skilled in the art. A substrate of
Formula (IV), such as Gb5, can be prepared by any method known in
the art or disclosed herein. In some examples, the Formula (IV)
compound is isolated from the reaction mixture described in Step
S-3 above. In other examples, the whole reaction mixture of Step
S-3 is used without purification of the Formula (V) compound
produced therein. The Formula (IV) compound can be incubated with
GDP-Fuc in the presence of the fucosyltransferase under conditions
allowing conversion of the Formula (IV) compound to a Formula (V)
compound. In some example, this FucT-catalyzed reaction is coupled
with the GDP-Fuc regeneration process as described herein. FIG. 2D.
See also Examples below.
Step S-5
[0171] In other embodiments of Formula (IV), wherein R.sup.3E is
hydrogen, an optional step in the biosynthetic approach (S-5)
involves enzymatic conversion of a compound of Formula (IV-b), or
salt thereof, to a compound of Formula (VI), or salt thereof:
##STR00011##
wherein R.sup.1A, R.sup.2A, R.sup.3A, R.sup.5A, R.sup.2B, R.sup.3B,
R.sup.5B, R.sup.2C, R.sup.4C, R.sup.5C, R.sup.2D, R.sup.4D,
R.sup.5D, R.sup.2E, R.sup.4E, and R.sup.5E are as defined herein;
R.sup.3G is hydrogen, substituted or unsubstituted C.sub.1-6 alkyl,
or a nitrogen protecting group; and each instance of R.sup.6G,
R.sup.7G, R.sup.8G, and R.sup.9G, is independently hydrogen,
substituted or unsubstituted C.sub.1-6 alkyl, or an oxygen
protecting group.
[0172] In certain embodiments, R.sup.3G is hydrogen. In certain
embodiments, R.sup.3G is substituted or unsubstituted
C.sub.1-6alkyl, e.g., substituted or unsubstituted C.sub.1alkyl,
substituted or unsubstituted C.sub.2alkyl, substituted or
unsubstituted C.sub.3alkyl, substituted or unsubstituted
C.sub.4alkyl, substituted or unsubstituted C.sub.5alkyl, or
substituted or unsubstituted C.sub.6alkyl. In certain embodiments,
R.sup.3G is a nitrogen protecting group, e.g., acetyl (Ac,
--C.dbd.OCH.sub.3).
[0173] In certain embodiments, R.sup.6G is hydrogen. In certain
embodiments, R.sup.6G is substituted or unsubstituted C.sub.1-6
alkyl, e.g., substituted or unsubstituted C.sub.1alkyl, substituted
or unsubstituted C.sub.2alkyl, substituted or unsubstituted
C.sub.3alkyl, substituted or unsubstituted C.sub.4alkyl,
substituted or unsubstituted C.sub.5alkyl, or substituted or
unsubstituted C.sub.6alkyl. In certain embodiments, R.sup.6G is an
oxygen protecting group.
[0174] In certain embodiments, R.sup.7G is hydrogen. In certain
embodiments, R.sup.7G is substituted or unsubstituted C.sub.1-6
alkyl, e.g., substituted or unsubstituted C.sub.1alkyl, substituted
or unsubstituted C.sub.2alkyl, substituted or unsubstituted
C.sub.3alkyl, substituted or unsubstituted C.sub.4alkyl,
substituted or unsubstituted C.sub.5alkyl, or substituted or
unsubstituted C.sub.6alkyl. In certain embodiments, R.sup.7G is an
oxygen protecting group.
[0175] In certain embodiments, R.sup.8G is hydrogen. In certain
embodiments, R.sup.8G is substituted or unsubstituted C.sub.1-6
alkyl, e.g., substituted or unsubstituted C.sub.1alkyl, substituted
or unsubstituted C.sub.2alkyl, substituted or unsubstituted
C.sub.3alkyl, substituted or unsubstituted C.sub.4alkyl,
substituted or unsubstituted C.sub.5alkyl, or substituted or
unsubstituted C.sub.6alkyl. In certain embodiments. R.sup.8G is an
oxygen protecting group.
[0176] In certain embodiments, R.sup.9G is hydrogen. In certain
embodiments, R.sup.9G is substituted or unsubstituted C.sub.1-6
alkyl, e.g., substituted or unsubstituted C.sub.1alkyl, substituted
or unsubstituted C.sub.2alkyl, substituted or unsubstituted
C.sub.3alkyl, substituted or unsubstituted C.sub.4alkyl,
substituted or unsubstituted C.sub.5alkyl, or substituted or
unsubstituted C.sub.6alkyl. In certain embodiments, R.sup.9G is an
oxygen protecting group.
[0177] In certain embodiments, each instance of R.sup.6G, R.sup.7G,
R.sup.8G, and R.sup.9G is hydrogen. In certain embodiments,
R.sup.3G is a nitrogen protecting group, e.g., acetyl (Ac,
--C.dbd.OCH.sub.3), each instance of R.sup.6G, R.sup.7G, R.sup.8G,
and R.sup.9G is hydrogen.
[0178] Exemplary compounds of Formula (V) include, but are not
limited to,
##STR00012##
and salts thereof.
[0179] Step S-5 involves an enzymatic reaction via the activity of
an alpha2,3-sialyltransferase, which is performed under suitable
conditions known to those skilled in the art. A substrate of
Formula (IV), such as Gb5, can be prepared by any method known in
the art or disclosed herein. In some examples, the Formula (IV)
compound is isolated from the reaction mixture described in Step
S-3 above. In other examples, the whole reaction mixture of Step
S-3 is used without purification of the Formula (IV) compound
produced therein. The Formula (IV) compound can be incubated with
CMP-Neu5Ac in the presence of the sialyltransferase under
conditions allowing conversion of the Formula (IV) compound to a
Formula (V) compound. In some example, this
Sialyltransferase-catalyzed reaction is coupled with the CMP-Neu5Ac
regeneration process as described herein. FIG. 2E. See also
Examples below.
[0180] Each of the Steps S1-S5, as well as any combination of
consecutive steps as described above, is within the scope of this
disclosure. Also within the scope of the present disclosure are any
of the compounds produced in any of the synthesis methods described
herein, e.g., those described above.
[0181] In some embodiments, the present disclosure features methods
for synthesizing Globo H or SSEA.sub.4 from lactose via a chain
reaction comprising Steps S-1, S-2, S-3, and S-4 or Steps S-1, S-2,
S-3, or S-5 described above. The Globo H or SSEA4 can be either
untailed (R.sup.1A being hydrogen; see FIGS. 3 and 4), or tailed
(e.g., R.sup.1A being allyl; see FIGS. 5 and 6). In each step, the
glycosyltransferase reaction can be coupled with the corresponding
nucleotide sugar regeneration process. FIGS. 3-6. In one example,
the above-described method is performed in a one-pot manner, i.e.,
each prior reaction mixture is used directly for the next step
reaction without purifying the substrate produced in the prior
reaction. In other words, the one-pot approach is free of any step
for purifying any intermediate. Alternatively, Steps S-1 and S-2
are performed in a one-spot manner without purification of any
intermediate. After Step S-2, Gb4 is isolated from the reaction
mixture and the purified GB4 is used for the following Steps S3,
S4, and/or S5. No further purification step is performed for
isolating other intermediate.
[0182] The enzymes used in each reaction step can be dissolved in
each reaction mixture, or immobilized on one or more support
members. When necessary, additional enzymes can be added during the
chain reaction.
Enzymatic Reactors
[0183] A chain enzymatic reaction comprising any combination of two
or more consecutive steps as described above can be performed in an
enzymatic reactor, which comprises one or more reaction chambers.
Each reaction chamber is designed for perform one step of the chain
reaction. In particular, each reaction chamber comprises enzymes
involved in one step of the reaction, including each of Steps 1-S
to 5-S described above.
[0184] In some embodiments, one or more enzymes, or all of the
enzymes, in each reaction chamber are immobilized on a suitable
support member (e.g., a support membrane). When necessary, reaction
chambers for consecutive reaction steps can be connected such that,
after termination of the enzymatic reaction in a prior reaction
chamber, the resultant reaction mixture can flow into the following
reaction chamber to allow the next reaction step to occur. In some
examples, the product from the prior reaction is not purified and
the whole reaction mixture including the product is added into the
next reaction chamber to allow occurrence of the next enzymatic
reaction. See, e.g., FIGS. 3 and 4.
[0185] For example, the reaction of Step 1-S can be performed in a
first reaction chamber in the enzymatic reactor, wherein one or
enzymes involved in Step 1-S are immobilized on a support member.
After termination of Step 1-S, the reaction mixture (including the
Gb3 product) in the first reaction chamber is placed into a second
reaction chamber containing all enzymes and reagents necessary for
Step 2-S for synthesis of Gb4. In one example, the Gb4 is purified
and used for the next reaction step. In another example, the whole
reaction mixture in the second reaction chamber, including Gb4, is
placed into a third reaction chamber that contains enzymes and
reagents necessary for Step 3-S, in which Gb5 is synthesized.
Afterwards, the reaction mixture from the third reaction chamber
can be placed into a fourth reaction chamber containing enzymes and
reagents necessary for Step 4-S or placed into a fifth reaction
chamber containing enzymes and reagents necessary for Step 5-S.
[0186] In other embodiments, the enzymatic reactor contains one
reaction chamber including enzymes, reagents, and the suitable
substrate, necessary for one of the synthesis steps described
above. The substrate is immobilized on a support member. In one
example, a reaction chamber contains the enzymes and reagents
necessary for Step 1-S, in which the substrate, Lac-allyl, is
immobilized on a support member. After Step 1-S, in which Gb3-allyl
is synthesized, the reaction mixture in the reaction chamber is
replaced with a second reaction mixture containing enzymes and
reagents necessary for Step 2-S. After synthesis of Gb4-allyl in
Step 2-S, the second reaction mixture is replaced with a third
reaction mixture containing enzymes and reagents for Step 3-S, in
which Gb5-allyl is synthesized. Afterwards, the third reaction
mixture is replaced with either a fourth reaction mixture
containing the enzymes and reagents for Step 4-S (for synthesis of
Globo H-allyl) or a fifth reaction mixture containing the enzymes
and reagents for Step 5-S (for synthesis of SSEA.sub.4-allyl).
[0187] Without further elaboration, it is believed that one skilled
in the art can, based on the above description, utilize the present
invention to its fullest extent. The following specific embodiments
are, therefore, to be construed as merely illustrative, and not
limitative of the remainder of the disclosure in any way
whatsoever. All publications cited herein are incorporated by
reference for the purposes or subject matter referenced herein.
EXAMPLES
[0188] These and other aspects of the present invention will be
further appreciated upon consideration of the following Examples,
which are intended to illustrate certain particular embodiments of
the invention but are not intended to limit its scope, as defined
by the claims.
Example 1
Synthesis of Globo-Series Oligosaccharides
New Method for UDP-Gal Regeneration
[0189] In 2004, Kotake's group discovered an enzyme from Pea
Sprouts, UDP-Sugar Pyrophosphorylase, which has broad substrate
specificity toward different monosaccharide-1-phosphate to form
UDP-Sugar..sup.[19] Two years later, Kotake's and Somers' groups
independently published similar function enzyme, AtUSP, existed in
Arabidopsis..sup.[20],[21] Very recently, the homologous enzymes
also proved existing in parasites, Leishmania and
Trypanosoma..sup.[22],[23] The AtUSP enzyme is interesting because
of its intrinsic ability to condense UTP with not only
Glc-1-phosphate and Gal-1-phosphate but also other
monosaccharide-1-P, GlcA-1-phosphate, and Xyl-1-phosphate.
Therefore, we chose AtUSP to condense Gal-1-phosphate with UTP
directly to render the UDP-Gal regeneration and to fulfill the
third regeneration of UDP-Gal synthesis.
Synthesis of Allyl-Gb3
[0190] The reaction mixture (200 mL) contained 10 mmol of
allyl-lac, 10 mmol of galactose, 22 mmol of Phosphoenolpyruvic acid
(PEP), 0.05 mmol of ATP, 0.125 mmol of UTP with 10 mM MgCl.sub.2 in
100 mM Tris-HCl buffer (pH 7.0). The reaction was initiated by
addition 100 U of .alpha.-1,4-galactosyltransferase (LgtC), 50 U of
galactokinase (GalK), 150 U of UDP-sugar pyrophosphorylase (AtUSP),
200 U of pyruvate kinase (PK) and 200 U of pyrophosphatase (PPA).
The flask was incubated at 25.degree. C. and the reaction progress
was monitored by TLC, and stained by p-anisaldehyde. More enzymes
were added if any of the reaction was incomplete until the reaction
was complete, and the products were confirmed by TLC and
ESI-MS.
Synthesis of Allyl-Gb4
[0191] Following the allyl-Gb3 synthesis, additional components
were added, including 9.9 mmol of N-acetylgalactosamine (GalNAc),
22 mmol of PEP, 100 U of
.beta.-1,3-N-acetylgalactosaminyltransferase (.beta.1,3GalNAcT,
LgtD), 50 U of N-acetylhexosamine 1-kinase (NahK), 200 U of
N-acetylglucosamine 1-phosphate uridylyltransferase (GlmU), 100 U
of PK and 100 U of PPA, in 220 mL solution. The mixture was
incubated at 25.degree. C. and monitored by TLC and ESI-MS as
before until the reaction was complete. The product was further
purified by a C-18 gel column and characterized by NMR.
Synthesis of Allyl-Gb5
[0192] The reaction mixture (250 mL) contained 9 mmol of allyl-Gb4,
9 mmol of galactose, 22 mmol of PEP, 0.05 mmol of ATP, 0.125 mmol
of UTP with 10 mM MgCl.sub.2 in 100 mM Tris-HCl buffer (pH 7.0).
The reaction was initiated by addition of 200 U of
.beta.-1,3-galactosyltransferase (.beta.1,3GalT, LgtD), 50 U of
GalK, 150 U of AtUSP, 100 U of PK and 100 U of PPA and incubated at
25.degree. C., until completion.
Synthesis of Allyl-Globo H
[0193] A half amount of the reaction product of allyl-Gb5
(.about.4.5 mmol) without additional purification was used to
produce allyl-globo H directly. A solution containing 5 mmol of
fucose, 0.05 mmol of ATP, 0.5 mmol of GTP, 11 mmol PEP with 10 mM
MgCl.sub.2 in 100 mM Tris-HCl buffer (pH 7.0) was added 200 U of
L-fucokinase/GDP-fucose pyrophosphorylase (FKP), 200 U of PK, 200 U
of PPA and 200 U of .alpha.-1,2-fucosyltransferase (FutC) incubated
at 25.degree. C. until the reaction was complete, and the product
was purified by C-18 gel chromatography as before and
characterized.
Synthesis of Allyl-SSEA4
[0194] Another half of the allyl-Gb5 (4.5 mmol) reaction mixture
was used for the synthesis of allyl-SSEA4 by adding 5 mmol of
N-acetylneuraminic acid (Neu5Ac), 0.05 mmol of ATP, 0.25 mmol of
CTP, 11 mmol of PEP with 10 mM MgCl.sub.2 in 100 mM Tris-HCl buffer
(pH 8.0) followed by 50 U of Cytidine monophosphate kinase (CMK),
120 U of CMP-sialic acid synthetase (CSS), 100 U of PK, 100 U of
PPA and 150 U of .alpha.-2,3-sialyltransferase (JT-FAJ-16). The
progress was monitored by TLC and the product was purified and
characterized as described above.
Purification and Characterization of Oligosaccharides
[0195] Proteins in reaction mixture were removed by heating to
90.degree. C. for 30 minutes and followed by centrifugation (5000
rpm, 20 min). The filtrate was then purified by C-18 gel
chromatography and eluted by a gradient from 100% H.sub.2O to 10%
methanol in H.sub.2O. The fractions were collected and monitored by
TLC [butanol/ammonium hydroxide/water=5:3:2 (v/v/v)] and the
fractions with allyl-oligosaccharides were pooled and lyophilized.
More than 99% purity of product could be gathered by HPLC using
Cosmosil 5SL-II column in (H.sub.2O/Acetonitrile=19/81) in an
isocratic mode. The structure of allyl-Lac, allyl-Gb3, allyl-Gb4,
allyl-Gb5, allyl-Globo H and allyl-SSEA4 were analyzed by 1H NMR,
.sup.13C NMR, and mass spectrometry (Avance 600 and APEX-ultra 9.4
T FTICR-MS, Bruker Daltonics).
Cloning of Genes for Nucleotide Sugar Synthesis,
Glycosyltransferases and ATP Regeneration
[0196] All genes obtained via PCR from genomic DNA or cDNA library
by respective primer (Table 5), and PCR product were ligated into
the modified pET47b vector. After ATG, following are the His-tag,
AcTEV protease cutting site and ccdB positive selection gene
flanked by special restriction recognition enzymes, or pET28a in
C-terminal His-tag. In order to increase the gene expression level,
the four glycosyltransferases were synthesized by codon
optimization for E. coli. The plasmid with correct sequence was
transformed into ArcticExpress/RIL competent cell by chemical
transformation method. Picked single colony and inoculated into TB
medium with kanamycin antibiotics overnight, and refresh the cell
culture into fresh TB medium, then inducing target protein
expression by final concentration 0.1 mM IPTG when OD600 was
reaching 0.5. After that, allowed grown at 16.degree. C. for 24 h.
The E. coli cells were harvested and disrupted in a buffer
containing 50 mM sodium phosphate buffer, pH8.0, 300 mM sodium
chloride, and 10 mM imidazole by microfluidizer. Centrifuge the
cell in 10,000 rpm at 4.degree. C. for 30 minutes. Then, poured the
supernatant into the equilibrated Ni-NTA agarose and discard the
precipitate. The bound protein was eluted in the same buffer but
containing higher concentration imidazole (250 mM). The protein
concentration was determined by Qubit Protein Quantitation
(Invitrogen, CA), and purity was confirmed by SDS-PAGE.
TABLE-US-00006 TABLE 5 Primers used for sialidase expressions in E.
coli. Gene source SEQ ID Restriction from genome NO Primer.sup.a
Sequence (5'.fwdarw.3') enzyme site or cDNA pool SEQ ID galK-F
CTGTATTTTCAGGGAGCGATCGCTA AsiSI E. coli NO: 1
TGAGTCTGAAAGAAAAAACA.sup.b MG1655 SEQ ID galK-R
GCCTCGAGTCATTACGTTTAAACTC PmeI ATCC NO: 2 AGCACTGTCCTGCTCCTTG
700926 SEQ ID atusp-F CTGTATTTTCAGGGAGCGATCGCTA AsiSI cDNA NO: 3
TGGCTTCTACGGTTGATTC pool of SEQ ID atusp-R
GCCTCGAGTCATTACGTTTAAACTC PmeI Arabidopsis NO: 4
AATCTTCAACAGAAAATTTGC thaliana SEQ ID lgtC-F.sup.b
GATATACCATGGAAATGGACATCGT NcoI Gene NO: 5 TTTCGCGGCG optimization
SEQ ID lgtC-R.sup.b GTGGTGCTCGAGGTAGATTTTACGC XhoI NO: 6 AGGAAACG
SEQ ID nahK-F CTGTATTTTCAGGGAGCGATCGCTA AsiSI Bifidobacterium NO: 7
TGAACAAGACTTATGATTTTAAAAG longum SEQ ID nahK-R
GCCTCGAGTCATTACGTTTAAACTT PmeI Atcc NO: 8 AAATGTATGAATATACTATCTTC
15697 SEQ ID glmU-F CTGTATTTTCAGGGAGCGATCGCTA AsiSI E. coli NO: 9
TGTTGAATAATGCTATGAGC MG1655 SEQ ID glmU-R GCCTCGAGTCATTACGTTTAAACTC
PmeI ATCC NO: 10 ACTTTTTCTTTACCGGACG 700926 SEQ ID lgtD-F.sup.b
GATATACCATGGAAAACTGCCCGCT NcoI Gene NO: 11 GGTTTCT optimization SEQ
ID lgtD-R.sup.b GTGGTGCTCGAGGAAGATAACGTTG XhoI NO: 12 ATTTTACGG SEQ
ID fkp-F CAGGGAGCGATCGCTATGCAAAAAC AsiSI Bacteroides NO: 13
TACTATCTTTA fragilis 9343 SEQ ID fkp-R CATTACGTTTAAACTTATGATCGTG
PmeI ATCC NO: 14 ATACTTGGAA 25285 SEQ ID futC-F.sup.b
CTGTATTTTCAGGGAGCGATCGCTA AsiSI Gene NO: 15 TGGCGTTCAAAGTTGTTCAG
optimization SEQ ID futC-R.sup.b GCCTCGAGTCATTACGTTTAAACTT PmeI NO:
16 ACGCGTTGTATTTCTGAGAT SEQ ID cmk-F CAGGGAGCGATCGCTATGACGGCAA
AsiSI E. coli NO: 17 TTGCCCCGGTT MG1655 SEQ ID cmk-R
CATTACGTTTAAACTTATGCGAGAG PmeI ATCC NO: 18 CCAATTTCTG 700926 SEQ ID
CSS-F GATATACCATGGAAACAAATATTGC NcoI Pasteurella NO: 19 GATCATTCCTG
multocida SEQ ID CSS-R GTGGTGCTCGAGTTTATTGGATAAA XhoI ATCC BAA- NO:
20 ATTTCCGCGAG 1113 SEQ ID jt-faj- GATATACCATGGAAATGAACAACGA NcoI
Gene NO: 21 16-F.sup.b CAACTCTACC optimization SEQ ID jt-faj-
GTGGTGCTCGAGGATGTCAGAGATC XhoI NO: 22 16-R.sup.b AGTTTGATG SEQ ID
pykF-F CTGTATTTTCAGGGAGCGATCGCTA AsiSI E. coli NO: 23
TGAAAAAGACCAAAATTGTTTG MG1655 SEQ ID pykF-R
GCCTCGAGTCATTACGTTTAAACTT PmeI ATCC NO: 24 ACAGGACGTGAACAGATG
700926 SEQ ID ppa-F CAGGGAGCGATCGCTATGAGCTTAC AsiSI E. coli NO: 25
TCAACGTCCCT MG1655 SEQ ID ppa-R CATTACGTTTAAACTTATTTATTCT PmeI ATCC
NO: 26 TTGCGCGCTC 700926 .sup.aapair of primers for forward (F) and
reversed (R) PCR reactions to amplify the coding sequence of each
gene. .sup.bUnderline with bold means the site of restriction
enzyme recognition. .sup.cCodon optimization for E. coli. See,
e.g., Puigb et al., Nucleic Acids Research (2007)
35(S2):W126-W130
Enzyme Assay
[0197] In order to maintain constant assay conditions, all activity
was measured at 37.degree. C. with 10 mM MgCl.sub.2, 100 mM Tris,
and at a pH of 7.5.
(i) Measurement of the Galactokinase (GalK), N-Acetylhexosamine
Kinase (NahK), Fucokinase (FKP) and Cytidine Monophosphate Kinase
(CMK) Activity
[0198] The fluorometric assay method was based on monitor of ADP
production (ATP consumption) by using the pyruvate kinase/lactate
dehydrogenase coupled enzymatic assay for the NADH consumption.
See, e.g., Murray et al., "Mechanism of Human
.alpha.-1,3-Fucosyltransferase V: Glycosidic Cleavage Occurs Prior
to Nucleophilic Attack" Biochemistry (1997) 36:823-831; and
Gosselin et al., "A Continuous Spectrophotometric Assay for
Glycosyltransferases" Analytical Biochemistry (1994) 220:92-97.
Fluorescence property of NADH has an excitation wavelength of 340
nm and an emission wavelength of 450 nm. A 100 uL of reaction
mixture was prepared containing the coupling enzyme (5 units of
pyruvate kinase and 7 units of lactic dehydrogenase from rabbit
muscle) and substrates and cofactors (0.2 mM NADH, 0.8 mM PEP, 10
mM MgCl.sub.2) in 100 mM Tris (pH 7.5). Reactions were initiated by
the addition of the respective sugar. The kinetic parameters,
K.sub.cat and K.sub.m were calculated by curve fitting the
experimental data with the theoretical equation, using Grafit
version 7 (Erithacus Software, Middlesex, UK). One unit of sugar
kinase activity is defined as 1 umol of sugar-1-P formation per
minute at 25.degree. C.
(ii) Measurement of UDP-Sugar Pyrophosphorylase (AtUSP), N-Acetyl
Glucosamine-1-Phosphate Uridyltransferase (GlmU), GDP-L-Fucose
Pyrophosphorylase (FKP) and CMP-Sialic Acid Synthetases (CSS)
Activity
[0199] The production of pyrophosphate was measured using the
EnzCheck Pyrophosphate Assay Kit (Invitrogen, CA, USA). Assay
components including: 200 uM 2-amino-6-mercapto-7-methylpurine
ribonucleoside, 1 unit nucleoside phosphorylase, 0.03 unit
inorganic pyrophosphatase, 10 mM MgCl.sub.2, 50 mM Tris, pH 7.5 in
100 uL scale in UV-Star microplates (Greiner Bio One, Germany). All
components except FKP were mixed in the microplates and allowed to
equilibrate until a flat baseline was achieved. Reactions were
initiated by the addition of enzyme. One unit of enzyme activity is
defined as the producing 1 umol of nucleotide sugar from the
respective sugar-1-Ps per minute at 25.degree. C., except for
CMP-sialic acid synthetase, which is defined as 1 umol of
pyrophosphate formation per minute at 25.degree. C.
(iii) Measurement of glycosyltransferase:
.alpha.-1,4-galactosyltransferase (LgtC),
.beta.1,3-N-acetylgalactosaminyltransferase (.beta.1,3GalNAcT,
LgtD), .beta.-1,3-galactosyltransferase (LgtD),
.alpha.-1,2-fucosyltransferase (FutC) and
.alpha.-2,3-sialyltransferase (JT-FAJ-16).
[0200] The fluorometric assay method monitored UDP, GDP, or CDP
production using the pyruvate kinase/lactate dehydrogenase coupled
enzymatic assay for the NADH consumption. See, e.g., Murray et al.,
"Mechanism of Human .alpha.-1,3-Fucosyltransferase V: Glycosidic
Cleavage Occurs Prior to Nucleophilic Attack" Biochemistry (1997)
36:823-831; and Gosselin et al., "A Continuous Spectrophotometric
Assay for Glycosyltransferases" Analytical Biochemistry (1994)
220:92-97. The assay components except nucleotide sugar were
simultaneously incubated in the multiple plate fluorometer
(SpectraMax M2 Readers, Molecular Devices) at 25.degree. C.
Reactions were initiated by the addition of corresponding
nucleotide sugar. The kinetic parameters, K.sub.cat and K.sub.m
were calculated by curve fitting the experimental data with the
theoretical equation, using Grafit version 7 (Erithacus Software,
Middlesex, UK). One unit of activity is defined as the amount of
enzyme that catalyzes the transfer 1 umol sugar from respective
nucleotide sugar to acceptor per minute at 25.degree. C.
(iv) Measurement of Pyruvate Kinase (PyrK)
[0201] Pyruvate kinase assay was slightly modified from sugar
kinase measurement previous mentioned, also based on NADH
consumption. A 100 uL of reaction mixture is prepared containing
0.8 mM ADP, 0.8 mM PEP, 0.2 mM NADH, 10 mM MgCl.sub.2, and 5 units
of lactic dehydrogenase from rabbit muscle in 100 mM Tris (pH 7.5)
in black multiplate. NADH has an excitation wavelength at 340 nm
and an emission wavelength at 450 nm. Reaction is initiated by
adding a suitable amount of recombinant E. coli pyruvate kinase.
One unit of pyruvate kinase is defined as conversion of 1.0
.mu.mole of phospho(enol)pyruvate to pyruvate per minute at
25.degree. C.
(v) Measurement of Pyrophosphatase (PPA)
[0202] Pyrophosphatase assay is slightly modified from
pyrophorylase protocol from commercial kit EnzCheck Pyrophosphate
Assay Kit (Invitrogen, CA, USA). Assay components including: 1 mM
pyrophosphate, 200 uM 2-amino-6-mercapto-7-methylpurine
ribonucleoside, 1 unit nucleoside phosphorylase, 10 mM MgCl.sub.2,
50 mM Tris, at a pH of 7.5 in 100 uL scale in UV-Star microplates
(Greiner Bio One, Germany) with suitable amount of recombinant E.
coli pyrophosphatase. One unit of pyrophosphatase activity is
defined as liberation of 1.0 umole of inorganic pyrophosphate per
minute at 25.degree. C.
(vi) Measurement of Optimum pH
[0203] The optimum pH for enzyme activity was determined in the
standard enzyme assay mentioned above in the pH range 4.0-10.0,
including sodium acetate, MES, MOPS, HEPES, Tris-HCl, CHES buffer.
The pH of the buffer was adjusted at the temperature of incubation.
All reactions were performed in triplicate for statistical
evaluation.
(vii) Measurement of Optimum Divalent Metal Ion
[0204] The assay for metal requirement was conducted in standard
assay condition. Enzymes were mixed with metal ion (Mg.sup.2+,
Mn.sup.2+, Mg.sup.2+, Mn.sup.2+, Ca.sup.2+, Zn.sup.2+, Co.sup.2+,
or Ni.sup.2+) in a final concentration of 10 mM, in the presence
and absence of EDTA. All reactions were performed in triplicate for
statistical evaluation.
(viii) Measurement of Optimum Temperature
[0205] The effect of temperature on the activity of enzymes were
determined by incubating an appropriate amount of purified enzyme
in MOPS buffer (pH 7.0), 10 mM MgCl.sub.2 and respective
substrates. In order to keep the assay consist, all components were
mixed well and preheated at assay temperature for 5 min, and the
reaction was started by adding the enzyme and recorded by multimode
plate readers (SpectraMax MS, Molecular Devices) in constant
temperature. The temperature ranged from 20 to 60.degree. C. All
reactions were performed in triplicate for statistical
evaluation.
Enzyme Composition
UDP-Gal Regeneration/Galactosylation
[0206] 1. GalK: galactokinase, from E. coli [0207] 2. AtUSP:
UDP-sugar pyrophosphorylase from Arabidopsis thaliana [0208] 3.
LgtC: .alpha.1,4galactosyltransferase, from Neisseria meningitidis,
but codon optimization for E. coli [0209] 4. PykF: pyruvate kinase,
from E. coli [0210] 5. PPA: pyrophosphatase, from E. coli
[0211] The coding sequence of the coden-optimized LgtC enzyme is
provided below (SEQ ID NO: 27):
TABLE-US-00007 ATGGACATCGTTTTCGCGGCGGACGACAACTACGCGGCGTACCTGTGC
GTTGCGGCGAAATCTGTTGAAGCGGCGCACCCGGACACCGAAATCCGT
TTCCACGTTCTGGACGCGGGTATCTCTGAAGCGAACCGTGCGGCGGTT
GCGGCGAACCTGCGTGGTGGTGGTGGTAACATCCGTTTCATCGACGTT
AACCCGGAAGACTTCGCGGGTTTCCCGCTGAACATCCGTCACATCTCT
ATCACCACCTACGCGCGTCTGAAACTGGGTGAATACATCGCGGACTGC
GACAAAGTTCTGTACCTGGACATCGACGTTCTGGTTCGTGACTCTCTG
ACCCCGCTGTGGGACACCGACCTGGGTGACAACTGGCTGGGTGCGTGC
ATCGACCTGTTCGTTGAACGTCAGGAAGGTTACAAACAGAAAATCGGT
ATGGCGGACGGTGAATACTACTTCAACGCGGGTGTTCTGCTGATCAAC
CTGAAAAAATGGCGTCGTCACGACATCTTCAAAATGTCTTGCGAATGG
GTTGAACAGTACAAAGACGTTATGCAGTACCAGGACCAGGACATCCTG
AACGGTCTGTTCAAAGGTGGTGTTTGCTACGCGAACTCTCGTTTCAAC
TTCATGCCGACCAACTACGCGTTCATGGCGAACCGTTTCGCGTCTCGT
CACACCGACCCGCTGTACCGTGACCGTACCAACACCGTTATGCCGGTT
GCGGTTTCTCACTACTGCGGTCCGGCGAAACCGTGGCACCGTGACTGC
ACCGCGTGGGGTGCGGAACGTTTCACCGAACTGGCGGGTTCTCTGACC
ACCGTTCCGGAAGAATGGCGTGGTAAACTGGCGGTTCCGCACCGTATG
TTCTCTACCAAACGTATGCTGCAGCGTTGGCGTCGTAAACTGTCTGCG
CGTTTCCTGCGTAAAATCTACTGA
UDP-GalNAc Regeneration/Acetylgalactosamination
[0212] 1. GalNAcK: N-Acetylliexosarnine 1-Kinases, from B. longum
[0213] 2. GlmU: N-acetylglucosamine 1-phosphate uridylyltransferase
from E. coli [0214] 3. LgtD: .beta.1,3galactosyltransferase, from
Haemophilus influenza, but codon optimization for E. coli [0215] 4.
PykF: pyruvate kinase, from E. coli [0216] 5. PPA: pyrophosphatase,
from E. coli
[0217] The coding sequence of the coden-optimized LgtD enzyme is
provided below (SEQ ID) NO: 28):
TABLE-US-00008 ATGGAAAACTGCCCGCTGGTTTCTGTTATCGTTTGCGCGTACAACGCG
GAACAGTACATCGACGAATCTATCTCTTCTATCATCAACCAGACCTAC
GAAAACCTGGAAATCATCGTTATCAACGACGGTTCTACCGACCTGACC
CTGTCTCACCTGGAAGAAATCTCTAAACTGGACAAACGTATCAAAATC
ATCTCTAACAAATACAACCTGGGTTTCATCAACTCTCTGAACATCGGT
CTGGGTTGCTTCTCTGGTAAATACTTCGCGCGTATGGACGCGGACGAC
ATCGCGAAACCGTCTTGGATCGAAAAAATCGTTACCTACCTGGAAAAA
AACGACCACATCACCGCGATGGGTTCTTACCTGGAAATCATCGTTGAA
AAAGAATGCGGTATCATCGGTTCTCAGTACAAAACCGGTGACATCTGG
AAAAACCCGCTGCTGCACAACGACATCTGCGAAGCGATGCTGTTCTAC
AACCCGATCCACAACAACACCATGATCATGCGTGCGAACGTTTACCGT
GAACACAAACTGATCTTCAACAAAGACTACCCGTACGCGGAAGACTAC
AAATTCTGGTCTGAAGTTTCTCGTCTGGGTTGCCTGGCGAACTACCCG
GAAGCGCTGGTTAAATACCGTCTGCACGGTAACCAGACCTCTTCTGTT
TACAACCACGAACAGAACGAAACCGCGAAAAAAATCAAACGTGAAAAC
ATCACCTACTACCTGAACAAAATCGGTATCGACATCAAAGTTATCAAC
TCTGTTTCTCTGCTGGAAATCTACCACGTTGACAAATCTAACAAAGTT
CTGAAATCTATCCTGTACGAAATGTACATGTCTCTGGACAAATACACC
ATCACCTCTCTGCTGCACTTCATCAAATACCACCTGGAACTGTTCGAC
CTGAAACAGAACCTGAAAATCATCAAAAAATTCATCCGTAAAATCAAC GTTATCTTCTAG
GDP-FK Regeneration/Fucosylation
[0218] 1. FKP: L-fucokinase/GDP-fucose pyrophosphorylase, from
Bacteroides fragilis [0219] 2. FutC: .alpha.1,2fucosyltransferase,
from Helicobacter, pylor, but codon optimization for E. coli [0220]
3. PykF: pyruvate kinase, from E. coli [0221] 4. PPA:
pyrophosphatase, from E. coli
[0222] The coding sequence of the coden-optimized FutC enzyme is
provided below (SEQ ID NO: 29):
TABLE-US-00009 ATGGCGTTCAAAGTTGTTCAGATCTGCGGTGGTCTGGGTAACCAGATG
TTCCAGTACGCGTTCGCGAAATCTCTGCAGAAACACTCTAACACCCCG
GTTCTGCTGGACATCACCTCTTTCGACTGGTCTGACCGTAAAATGCAG
CTGGAACTGTTCCCGATCGACCTGCCGTACGCGTCTGCGAAAGAAATC
GCGATCGCGAAAATGCAGCACCTGCCGAAACTGGTTCGTGACGCGCTG
AAATGCATGGGTTTCGACCGTGTTTCTCAGGAAATCGTTTTCGAATAC
GAACCGAAACTGCTGAAACCGTCTCGTCTGACCTACTTCTTCGGTTAC
TTCCAGGACCCGCGTTACTTCGACGCGATCTCTCCGCTGATCAAACAG
ACCTTCACCCTGCCGCCGCCGCCGGAAAACAACAAAAACAACAACAAA
AAAGAAGAAGAATACCAGTGCAAACTGTCTCTGATCCTGGCGGCGAAA
AACTCTGTTTTCGTTCACATCCGTCGTGGTGACTACGTTGGTATCGGT
TGCCAGCTGGGTATCGACTACCAGAAAAAAGCGCTGGAATACATGGCG
AAACGTGTTCCGAACATGGAACTGTTCGTTTTCTGCGAAGACCTGGAA
TTCACCCAGAACCTGGACCTGGGTTACCCGTTCATGGACATGACCACC
CGTGACAAAGAAGAAGAAGCGTACTGGGACATGCTGCTGATGCAGTCT
TGCCAGCACGGTATCATCGCGAACTCTACCTACTCTTGGTGGGCGGCG
TACCTGATCGAAAACCCGGAAAAAATCATCATCGGTCCGAAACACTGG
CTGTTCGGTCACGAAAACATCCTGTGCAAAGAATGGGTTAAAATCGAA
TCTCACTTCGAAGTTAAATCTCAGAAATACAACGCGTAA
CMP-Neu5Ac Regeneration/Sialylation
[0223] 1. CMK: Cytidine monophosphate kinase, from E. coli [0224]
2. Css: CMP-sialic acid synthetase, from Pasteurella multocida
[0225] 3. JT-FAJ-16: .alpha.2,3sialyltransferase, from marine
bacteria, but codon optimization for E. coli [0226] 4. PykF:
pyruvate kinase, from E. coli [0227] 5. PPA: pyrophosphatase, from
E. coli
[0228] The coding sequence of the coden-optimized JT-FAJ-16 enzyme
is provided below (SEQ ID NO: 30):
Materials and Chemicals
TABLE-US-00010 [0229]
ATGAACAACGACAACTCTACCACCACCAACAACAACGCGATCGAAATC
TACGTTGACCGTGCGACCCTGCCGACCATCCAGCAGATGACCAAAATC
GTTTCTCAGAAAACCTCTAACAAAAAACTGATCTCTTGGTCTCGTTAC
CCGATCACCGACAAATCTCTGCTGAAAAAAATCAACGCGGAATTCTTC
AAAGAACAGTTCGAACTGACCGAATCTCTGAAAAACATCATCCTGTCT
GAAAACATCGACAACCTGATCATCCACGGTAACACCCTGTGGTCTATC
GACGTTGTTGACATCATCAAAGAAGTTAACCTGCTGGGTAAAAACATC
CCGATCGAACTGCACTTCTACGACGACGGTTCTGCGGAATACGTTCGT
ATCTACGAATTCTCTAAACTGCCGGAATCTGAACAGAAATACAAAACC
TCTCTGTCTAAAAACAACATCAAATTCTCTATCGACGGTACCGACTCT
TTCAAAAACACCATCGAAAACATCTACGGTTTCTCTCAGCTGTACCCG
ACCACCTACCACATGCTGCGTGCGGACATCTTCGACACCACCCTGAAA
ATCAACCCGCTGCGTGAACTGCTGTCTAACAACATCAAACAGATGAAA
TGGGACTACTTCAAAGACTTCAACTACAAACAGAAAGACATCTTCTAC
TCTCTGACCAACTTCAACCCGAAAGAAATCCAGGAAGACTTCAACAAA
AACTCTAACAAAAACTTCATCTTCATCGGTTCTAACTCTGCGACCGCG
ACCGCGGAAGAACAGATCAACATCATCTCTGAAGCGAAAAAAGAAAAC
TCTTCTATCATCACCAACTCTATCTCTGACTACGACCTGTTCTTCAAA
GGTCACCCGTCTGCGACCTTCAACGAACAGATCATCAACGCGCACGAC
ATGATCGAAATCAACAACAAAATCCCGTTCGAAGCGCTGATCATGACC
GGTATCCTGCCGGACGCGGTTGGTGGTATGGGTTCTTCTGTTTTCTTC
TCTATCCCGAAAGAAGTTAAAAACAAATTCGTTTTCTACAAATCTGGT
ACCGACATCGAAAACAACTCTCTGATCCAGGTTATGCTGAAACTGAAC
CTGATCAACCGTGACAACATCAAACTGATCTCTGACATCTAA
[0230] All nucleotide, sugar, nucleotide sugar and chemicals were
purchased from Sigma-Aldrich (St. Louis, Mo.). Restriction enzyme
and T4 DNA ligase acquired from NEB (Beverly, Mass.). Primer
ordered from Proligo Singapore Pte Ltd (Singapore). Ni-NTA Agarose
obtained from Qiagen (Santa Clarita, Calif.). Bio-Gel P2 gel was
purchase from Bio-Rad (Hercules, Calif.). Plasmid pET28a, pET47b
and precoated glass plates TLC covered in Silica Gel 60, F254 with
0.25 mm layer thickness was purchase from EMD Chemicals Inc
(Carlsbad, Calif.) were purchased from EMD Chemicals Inc (Carlsbad,
Calif.). ArcticExpress/RIL competent cell were obtained from
Agilent Genomics (La Jolla, Calif.). All other materials not
mentioned above were purchased as high quality as possible.
[0231] All reactions were monitored by thin-layer chromatography.
(mobile phase: Butanol:acetate:water=5:3:2). Staining the TLC by
p-Anisaldehyde.
Synthesis of Allyl-Lac
[0232] The synthesis of different lactose with linker was followed
by the literature reported method [Carbohydrate Research 2004, 339,
2415-2424.]. .sup.1H NMR (600 MHz, D2O) .delta. 6.01 (m, 1H),
5.40-5.37 (dd, J=17.3, 1.4 Hz, 1H), 5.30-5.28 (d, J=10.3 Hz, 1H),
4.54 (d, J=8.1 Hz, 1H), 4.46 (d, J=7.8 Hz, 1H), 4.41-4.38 (m, 1H),
4.25-4.22 (m, 1H), 4.00-3.97 (dd, J=12.3, 2.1 Hz, 1H), 3.93 (d,
J=3.3 Hz, 1H), 3.80-3.71 (m, 4H), 3.67-3.53 (m, 5H), 3.35-3.33 (m,
1H); .sup.13C NMR (150 MHz, D2O) .delta. 133.3, 118.7, 102.9,
101.0, 78.3, 75.3, 74.7, 74.4, 72.8, 72.5, 70.9, 70.6, 68.5, 60.9,
60.1; HRMS (ESI-TOF, MNa.sup.+) C.sub.15H.sub.26O.sub.11Na.sup.+
calcd for 405.1367. found 405.1346.
Large Scale Production of Gb3 with Linker
[0233] 5 mmol lactose with linker, 5 mmol galactose, 12 mmol
Phosphoenolpyruvic acid (PEP), 0.25 mmol ATP, 0.25 mmol UTP and 10
mM MgCl.sub.2 were added into 100 mM Tris-HCl buffer (pH 7.5)
solution. The reaction was initiated by addition suitable amount of
.alpha.-1,4-galactosyltransferase (LgtC), galactokinase (GalK),
UDP-sugar pyrophosphorylase (AtUSP), pyruvate kinase (PK) and
pyrophosphatase (PPA). The flask was placed into an incubator at
16-50.degree. C. with gentle shaking. The reaction was monitored by
TLC. More enzymes are added if the reaction stops. The reaction is
stopped when no more starting material is observed by TLC. The Gb3
product was isolated by .sup.18C reverse phase column in 99%
yield.
[0234] Allyl-Gb3: .sup.1H NMR (600 MHz, D.sub.2O) .delta. 6.00 (m,
1H), 5.42 (d, J=17.2 Hz, 1H), 5.32 (d, J=10.4 Hz, 1H), 4.97 (d,
J=3.3 Hz, 1H), 4.56 (d, J=7.9 Hz, 1H), 4.53 (d, J=7.7 Hz, 1H),
4.43-4.37 (m, 2H), 4.27-4.24 (m, 1H), 4.06-3.58 (m, 16H), 3.37-3.34
(t, J=8.0 Hz, 1H); .sup.13C NMR (150 MHz, D2O) .delta. 133.3,
118.7, 103.3, 100.9, 100.3, 78.6, 77.3, 75.4, 74.8, 74.5, 72.9,
72.2, 70.9, 70.8, 70.6, 69.1, 68.9, 68.5, 60.5, 60.4, 60.0; HRMS
(ESI-TOF, MNa.sup.+) C.sub.21H.sub.36O.sub.16Na.sup.+ calcd for
567.1896. found 567.1858.
##STR00013##
[0235] .sup.1H NMR (600 MHz, D.sub.2O) .delta. 4.97 (d, J=3.3 Hz,
1H), 4.56 (d, J=7.9 Hz, 1H), 4.54-4.50 (m, 2H), 4.37 (dd, J=6.0 Hz,
J=0.6 Hz, 1H), 4.27-4.24 (m, 1H), 4.06-3.59 (m, 18H), 3.35 (t,
J=8.0 Hz, 1H), 3.03 (t, J=7.4 Hz, 2H), 1.75-1.68 (m, 4H), 1.51-1.46
(m, 2H); .sup.13C NMR (150 MHz, D2O) .delta. 103.3, 101.9, 100.3,
78.7, 77.3, 75.4, 74.8, 74.5, 72.9, 72.2, 70.9, 70.8, 70.6, 69.1,
68.9, 68.5, 60.5, 60.4, 60.0, 39.3, 28.1, 26.4, 22.1.
Large Scale Production of Gb4 with Linker
[0236] 5 mmol Gb3 with linker, 5 mmol N-acetylgalactosamine
(GalNAc), 12 mmol Phosphoenolpyruvic acid (PEP), 0.25 mmol ATP,
0.25 mmol UTP and 10 mM MgCl.sub.2 were added into 100 mM Tris-HCl
buffer (pH 7.5) solution. The reaction was initiated by addition
suitable amount of .beta.-1,3-N-acetylgalactosaminyltransferase
(LgtD), N-acetylhexosamine 1-kinase (NahK), N-acetylglucosamine
1-phosphate uridylyltransferase (GlmU), pyruvate kinase (PK) and
pyrophosphatase (PPA). The flask was placed into an incubator at
16-50.degree. C. with gentle shaking. The reaction was monitored by
TLC. More enzymes are added if the reaction stops. The reaction is
stopped when no more starting material is observed by TLC. The Gb4
product was isolated by .sup.18C reverse phase column in 96%
yield.
[0237] Allyl-Gb4: .sup.1H NMR (600 MHz, D.sub.2O) .delta. 6.01 (m,
1H), 5.40-5.38 (dd, J=17.3, 1.4 Hz, 1H), 5.30 (d, J=10.5 Hz, 1H),
4.92 (d, J=3.9 Hz, 1H), 4.64 (d, J=8.5 Hz, 1H), 4.54 (d, J=7.9 Hz,
1H), 4.53 (d, J=7.8 Hz, 1H), 4.42-4.38 (m, 2H), 4.26-4.22 (m, 2H),
4.05 (d, J=2.9 Hz, 1H), 4.01-3.99 (dd, J=12.3, 1.8 Hz, 1H),
3.98-3.89 (m, 5H), 3.86-3.74 (m, 7H), 3.72-3.57 (m, 7H), 3.37-3.34
(t, J=8.6 Hz, 1H), 2.05 (s, 3H); .sup.13C NMR (150 MHz, D2O)
.delta. 133.2, 118.7, 103.3, 103.2, 100.9, 100.4, 78.7, 78.6, 77.2,
75.4, 74.9, 74.8, 74.5, 72.9, 72.1, 70.9, 70.8, 70.6, 70.2, 68.9,
67.7, 67.6, 60.9, 60.5, 60.3, 60.2, 60.0, 52.6, 22.2; HRMS (MALDI,
MNa.sup.+) C.sub.29H.sub.49NO.sub.21Na.sup.+ calcd for 770.2689.
found 770.2654.
Large Scale Production of Gb5 with Linker
[0238] 5 mmol allyl-Gb4, 5 mmol galactose, 12 mmol
Phosphoenolpyruvic acid (PEP), 0.25 mmol ATP, 0.25 mmol UTP with 10
mM MgCl.sub.2 were added into 100 mM Tris-HCl buffer (pH 7.5). The
reaction was initiated by addition suitable amount of
.beta.-1,3-galactosyltransferase, galactokinase (GalK), UDP-sugar
pyrophosphorylase (AtUSP), pyruvate kinase (PK) and pyrophosphatase
(PPA). The flask was placed into an incubator at 16-50.degree. C.
with gentle shaking. The reaction was monitored by TLC. More
enzymes are added if the reaction stops. The reaction is stopped
when no more starting material is observed by TLC. The Gb5 product
was purified by .sup.18C reverse phase column in 95% yield.
[0239] Allyl-Gb5: .sup.1H NMR (600 MHz, D.sub.2O) .delta. 6.01 (m,
1H), 5.41-5.38 (dd, J=17.3, 1.4 Hz, 1H), 5.31 (d, J=10.6 Hz, 1H),
4.93 (d, J=4.0 Hz, 1H), 4.71 (d, J=8.5 Hz, 1H), 4.55 (d, J=8.1 Hz,
1H), 4.53 (d, J=7.8 Hz, 1H), 4.47 (d, J=7.7 Hz, 1H), 4.42-4.39 (m,
2H), 4.27-4.23 (m, 2H), 4.20 (d, J=3.2 Hz, 1H), 4.09-3.90 (m, 8H),
3.87-3.59 (m, 17H), 3.55-3.52 (m, 1H), 3.36-3.33 (t, J=8.6 Hz, 1H),
2.04 (s, 3H); .sup.13C NMR (150 MHz, D2O) .delta. 175.1, 133.2,
118.7, 104.8, 103.3, 102.9, 100.9, 100.4, 79.6, 78.7, 78.6, 77.2,
75.4, 74.9, 74.8, 74.6, 74.5, 72.9, 72.4, 72.1, 70.9, 70.6 (2C),
70.2, 68.9, 68.5, 67.9, 67.6, 60.9 (2C), 60.33, 60.28, 60.0, 51.5,
22.2; HRMS (ESI-TOF, MNa.sup.+) C.sub.35H.sub.59NO.sub.26Na.sup.+
calcd for 932.3218. found 932.3235.
##STR00014##
[0240] .sup.1H NMR (600 MHz, D.sub.2O), 4.47 (d, 1H, J=8.42 Hz),
4.30 (d, 1H, J=7.9 Hz), 4.28 (d, 1H, J=8.1 Hz), 4.24 (d, 1H, J=7.7
Hz), 4.19 (t, 1H J=7.0 Hz), 4.04 (d, 1H, J=2.8 Hz), 3.97 (d, 1H,
J=2.98 Hz), 3.87-3.35 (m, 32H), 3.30 (t, 1H, J=7.7 Hz), 3.09 (t,
1H, J=8.5 Hz), 2.79 (t, 2H, J=7.6 Hz), 1.82 (s, 3H), 1.51-1.43, (m,
4H), 1.28-1.21 (m, 2H) .sup.13C NMR (150 MHz, D.sub.2O), .delta.
175.0, 104.7, 103.1, 102.8, 101.8, 100.2, 79.4, 78.5, 78.4, 76.9,
75.3, 74.8, 74.7, 74.4, 74.3, 72.8, 72.2, 71.9, 70.6, 70.4, 70.0,
69.9, 68.7, 68.4, 67.8, 67.4, 60.82, 60.77, 60.13, 60.1, 59.8,
51.3, 39.1, 28.0, 26.3, 22.1, 21.9 MALDI-TOF:
C.sub.37H.sub.66N.sub.2O.sub.26 [M+H].sup.+ calculated 955.3904.
found 955.3972.
Large Scale Production of Globo H with Linker
[0241] 5 mmol Gb5 with linker, 5 mmol fucose, 12 mmol
Phosphoenolpyruvic acid (PEP), 0.25 mmol ATP, 0.25 mmol GTP with 10
mM MgCl.sub.2 were added into 100 mM Tris-HCl buffer (pH 7.5). The
reaction was initiated by addition suitable amount of
.alpha.-1,2-fucosyltransferase, L-fucokinase/GDP-fucose
pyrophosphorylase (FKP), pyruvate kinase (PK) and pyrophosphatase
(PPA). The flask was placed into an incubator at 16-50.degree. C.
with gentle shaking. The reaction was monitored by TLC. More
enzymes are added if the reaction stops. The reaction is stopped
when no more starting material is observed by TLC. The Globo H
product was purified by .sup.18C reverse phase column in 94%
yield.
[0242] Allyl-Globo H: .sup.1H NMR (600 MHz, D.sub.2O) .delta. 6.01
(m, 1H), 5.41-5.38 (dd, J=17.3, 1.4 Hz, 1H), 5.31 (d, J=10.7 Hz,
1H), 5.24 (d, J=4.0 Hz, 1H), 4.91 (d, J=3.9 Hz, 1H), 4.63 (d, J=7.7
Hz, 1H), 4.56-4.52 (m, 3H), 4.42-4.40 (m, 2H), 4.26-4.23 (m, 3H),
4.12 (d, J=2.2 Hz, 1H), 4.05 (d, J=3.0 Hz, 1H), 4.03-3.59 (m, 28H),
3.36-3.33 (t, J=8.2 Hz, 1H), 2.06 (s, 3H), 1.24 (d, J=6.5 Hz, 3H);
.sup.13C NMR (150 MHz, D2O) .delta. 174.3, 133.2, 118.7, 103.9,
103.2, 102.0, 100.9, 100.4, 99.3, 78.7, 78.3, 77.1, 76.3, 76.1,
75.5, 75.0, 74.8, 74.6, 74.5, 73.5, 72.9, 72.1, 71.8, 70.8, 70.6,
70.1, 69.5, 69.2, 69.1, 68.5, 68.0, 67.8, 66.8, 60.95, 60.93, 60.3
(2C), 60.0, 51.6, 22.2, 15.3; HRMS (MALDI, MNa+)
C.sub.41H.sub.70NO.sub.30Na.sup.+ calcd for 1079.3875. found
1078.4145.
##STR00015##
[0243] .sup.1H NMR (600 MHz, D.sub.2O) .delta. 5.12 (d, 1H, J=3.9
Hz), 4.78 (d, 1H, J=3.6 Hz), 4.50 (d, 1H, J=7.7 Hz), 4.43 (d, 1H,
J=7.5 Hz), 4.40 (d, 1H, J=7.7 Hz), 4.37 (d, 1H, J=8.0 Hz), 4.30 (t,
1H, J=6.2 Hz), 4.15-4.10 (m, 2H), 3.99 (d, 1H, J=1.8 Hz), 3.92 (d,
1H, J=2.2 Hz), 3.90-3.47 (m, 33H), 3.19 (t, 1H, J=8.3 Hz), 2.89 (t,
2H, J=7.5 Hz), 1.94 (s, 3H), 1.60-1.55 (m, 4H), 1.38-1.31 (m, 2H),
1.11 (d, 3H, J=6.4 Hz). .sup.13C NMR (150 MHz, D.sub.2O) .delta.
176.1, 105.7, 105.0, 103.74, 103.65, 102.1, 100.97, 80.5, 79.9,
78.8, 78.0, 77.8, 77.2, 76.76, 76.5, 76.3, 76.2, 75.3, 74.6, 73.8,
73.5, 72.5, 71.81, 71.78, 71.2, 71.1, 70.9, 70.8, 70.2, 69.7, 69.5,
68.5, 62.66, 62.64, 62.0, 61.7, 53.3, 41.0, 29.9, 28.1, 23.9, 23.8,
17.0 MALDI-TOF: C.sub.43H.sub.76N.sub.2O.sub.30 [M+Na].sup.+
calculated 1123.4381. found 1123.4385.
Large Scale Production of SSEA4 with Linker
[0244] 5 mmol Gb5 with linker, 5 mmol fucose, 12 mmol
phosphoenolpyruvic acid (PEP), 0.25 mmol ATP, 0.25 mmol CTP with 10
mM MgCl.sub.2 were added into 100 mM Tris-HCl buffer (pH 7.5). The
reaction was initiated by addition suitable amount of
.alpha.-2,3-sialyltransferase, cytidine monophosphate kinase (CMK),
CMP-sialic acid synthetase (CSS), pyruvate kinase (PK) and
pyrophosphatase (PPA). The flask was placed into an incubator at
16-50.degree. C. with gentle shaking. The reaction was monitored by
TLC. More enzymes are added if the reaction stops. The reaction is
stopped when no more starting material is observed by TLC. The
SSEA4 product was isolated by .sup.18C reverse phase column in 45%
yield.
[0245] Allyl-SSEA.sub.4: .sup.1H NMR (600 MHz, D.sub.2O) .delta.
6.00 (m, 1H), 5.40-5.37 (d, J=17.3 Hz, 1H), 5.30-5.28 (d, J=10.4
Hz, 1H), 4.92 (d, J=3.9 Hz, 1H), 4.70 (d, J=8.5 Hz, 1H), 4.54-4.51
(m, 3H), 4.40-4.38 (m, 2H), 4.25-4.18 (m, 3H), 4.10-3.52 (m, 34H),
3.35-3.32 (t, J=8.6 Hz, 1H), 2.77 (dd, J=12.5, 4.6 Hz, 1H), 2.03
(s, 6H), 1.80 (t, J=12.1 Hz, 1H); .sup.13C NMR (150 MHz, D2O)
.delta. 175.2, 175.1, 174.1, 133.4, 121.6, 118.9, 104.7, 103.4,
103.1, 101.1, 100.5, 99.8, 79.9, 78.9, 78.8, 77.3, 75.7, 75.5,
75.0, 74.7, 74.6, 73.0, 72.9, 72.2, 72.1, 71.9, 71.0, 70.8, 70.4,
69.1, 69.0, 68.5, 68.2, 68.0, 67.7, 67.5, 62.6, 61.1, 60.5, 60.4,
60.1, 51.7, 51.4, 39.8, 22.4, 22.1; HRMS (ESI-TOF, M-H)
C.sub.46H.sub.75N.sub.2O.sub.34O.sup.- calcd for 1199.4196. found
1199.4208.
##STR00016##
[0246] .sup.1H NMR (600 MHz, D.sub.2O) .delta. 4.94 (d, J=3.8 Hz,
1H), 4.72 (d, J=8.5 Hz, 1H), 4.54-4.50 (m, 3H), 4.40 (t, J=6.4 Hz,
1H), 4.27 (d, J=2.0 Hz, 1H), 4.20 (d, J=2.8 Hz, 1H), 4.10-3.54 (m,
37H), 3.34-3.31 (m, 1H), 3.02 (t, J=7.6 Hz, 2H), 2.78 (dd, J=12.4,
4.6 Hz, 1H), 2.05 (m, 6H), 1.80 (t, 12.2 Hz, 1H), 1.74-1.67 (m,
4H), 1.51-1.45 (m, 2H); .sup.13C NMR (150 MHz, D.sub.2O) .delta.
175.0, 174.9, 173.9, 104.5, 103.2, 102.9, 101.9, 100.3, 99.6, 79.7,
78.8, 78.7, 77.1, 75.5, 75.4, 74.8, 74.7, 74.6, 74.5, 72.9, 72.7,
72.1, 71.8, 70.8, 70.2, 70.0, 68.9, 68.9, 68.3, 68.0, 67.8, 67.5,
67.3, 62.4, 60.9, 60.3, 60.3, 60.0, 51.6, 51.3, 39.7, 39.3, 28.1,
26.5, 22.3, 22.0, 22.0; HRMS (ESI-TOF, MNa.sup.+) calcd for
C.sub.48H.sub.83N.sub.3O.sub.34Na 1268.4756. found 1268.4760.
TABLE-US-00011 TABLE 6 Basic composition of glycosphingolipids
Globoseries Gal Glc GalNAc GlcNAc Neu5Ac Fuc Globotetraose 2 1 1 0
0 0 (Gb4) Globopentaose 3 1 1 0 0 0 (Gb5) Globo H 3 1 1 0 0 1
(Fucosyl-Gb5) SSEA4 3 1 1 0 1 0 (Sialyl-Gb5) Isoglobotetraose 2 1 1
0 0 0 Neolactoseries 2 1 0 1 1 0 Lactoseries 2 1 0 1 1 0
Ganglioseries 2 1 1 0 2 0
TABLE-US-00012 TABLE 7 Yields of Each step of glycosylation with
regeneration Enzyme involvement Product Yield Step 1. GalK, AtUSP,
PykF, PPA, LgtC* allyl-Gb3 99% Step 2. NahK, GlmU, PykF, PPA, LgtD*
allyl-Gb4 96% Step 3. GalK, AtUSP, PykF, PPA, LgtD* allyl-Gb5 95%**
Step 4a. FKP, PykF, PPA, FutC* allyl- 94% Globo H Step 4b. CSS,
CMK, PykF, PPA, JT-FAJ-16* allyl-SSEA4 45% *DNA sequences were
optimized for E. coli expression. **When using pure allyl-Gb4 as an
acceptor.
Example 2
One-Step Synthesis of Allyl-Gb5(SSEA3) from Allyl-Lactose
[0247] Allyl-Gb5 was synthesized from allyl-lac via a one-step
chain reaction as illustrated in FIG. 6, without purifying any of
the intermediates.
[0248] 5 mmol Allyl-lac, 5 mmol galactose, 12 mmol PEP, 0.25 mmol
ATP, 0.25 mmol UTP with 10 mM MgCl.sub.2 in 100 mM Tris-HCl buffer
(pH 7.5) were mixed in a flask. Enzymatic reaction was initiated by
adding into the flask a suitable .alpha.1,4-galactosyltransferase
(LgtC), GalK, AtUSP, PK and PPA to synthesize allyl-Gb3. The flask
containing the reaction mixture was placed in a 16.about.50.degree.
C. incubator with gently shaking. TLC analysis was performed to
monitor the synthesis process. If no further synthesis of allyl-Gb3
is observed, additional enzymes were added.
[0249] After synthesis of allyl-Gb3, another set of components,
including 5 mmol of GalNAc, 12 mmol PEP, and a suitable amount of
N-acetylhexosamine 1-kinase (NahK-CP), N-acetylglucosamine
1-phosphate uridylyltransferase (GlmU), PK, PPA and .beta.
1,3-N-acetylgalactosaminyltransferase (LgtD), was added into the
flask. The reaction mixture thus formed was incubated under the
same conditions under which allyl-Gb3 was synthesis. If no further
synthesis of allyl-Gb4 is observed, additional amounts of the
enzymes can be added.
[0250] After synthesis of allyl-Gb4, 5 mmol galactose and 12 mmol
PEP were added into the flask without purifying the allyl-Gb4. The
next galactosylation reaction was initiated by adding suitable
.beta.1,3-galactosyltransferase (LgtD), GalK, AtUSP, PK and PPA to
synthesize allyl-Gb5. The flask containing the reaction mixture was
placed in a 16-50.degree. C. incubator with gently shaking. TLC was
performed to monitor the synthesis process. Additional amounts of
enzymes can be added if no further synthesis of allyl-Gb5 is
observed. The yield of this one-step synthesis of allyl-Gb5 from
allyl-lac is about 40%.
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Other Embodiments
[0295] In the claims articles such as "a," "an," and "the" may mean
one or more than one unless indicated to the contrary or otherwise
evident from the context. Claims or descriptions that include "or"
between one or more members of a group are considered satisfied if
one, more than one, or all of the group members are present in,
employed in, or otherwise relevant to a given product or process
unless indicated to the contrary or otherwise evident from the
context. The invention includes embodiments in which exactly one
member of the group is present in, employed in, or otherwise
relevant to a given product or process. The invention includes
embodiments in which more than one, or all of the group members are
present in, employed in, or otherwise relevant to a given product
or process.
[0296] Furthermore, the invention encompasses all variations,
combinations, and permutations in which one or more limitations,
elements, clauses, and descriptive terms from one or more of the
listed claims is introduced into another claim. For example, any
claim that is dependent on another claim can be modified to include
one or more limitations found in any other claim that is dependent
on the same base claim. Where elements are presented as lists,
e.g., in Markush group format, each subgroup of the elements is
also disclosed, and any element(s) can be removed from the group.
It should it be understood that, in general, where the invention,
or aspects of the invention, is/are referred to as comprising
particular elements and/or features, certain embodiments of the
invention or aspects of the invention consist, or consist
essentially of, such elements and/or features. For purposes of
simplicity, those embodiments have not been specifically set forth
in haec verba herein. It is also noted that the terms "comprising"
and "containing" are intended to be open and permits the inclusion
of additional elements or steps. Where ranges are given, endpoints
are included. Furthermore, unless otherwise indicated or otherwise
evident from the context and understanding of one of ordinary skill
in the art, values that are expressed as ranges can assume any
specific value or sub-range within the stated ranges in different
embodiments of the invention, to the tenth of the unit of the lower
limit of the range, unless the context clearly dictates
otherwise.
[0297] This application refers to various issued patents, published
patent applications, journal articles, and other publications, all
of which are incorporated herein by reference. If there is a
conflict between any of the incorporated references and the instant
specification, the specification shall control. In addition, any
particular embodiment of the present invention that falls within
the prior art may be explicitly excluded from any one or more of
the claims. Because such embodiments are deemed to be known to one
of ordinary skill in the art, they may be excluded even if the
exclusion is not set forth explicitly herein. Any particular
embodiment of the invention can be excluded from any claim, for any
reason, whether or not related to the existence of prior art.
[0298] Those skilled in the art will recognize or be able to
ascertain using no more than routine experimentation many
equivalents to the specific embodiments described herein. The scope
of the present embodiments described herein is not intended to be
limited to the above Description, but rather is as set forth in the
appended claims. Those of ordinary skill in the art will appreciate
that various changes and modifications to this description may be
made without departing from the spirit or scope of the present
invention, as defined in the following claims.
Sequence CWU 1
1
30146DNAArtificial SequenceSynthetic oligonucleotide 1ctgtattttc
agggagcgat cgctatgagt ctgaaagaaa aaacab 46244DNAArtificial
SequenceSynthetic oligonucleotide 2gcctcgagtc attacgttta aactcagcac
tgtcctgctc cttg 44344DNAArtificial SequenceSynthetic
oligonucleotide 3ctgtattttc agggagcgat cgctatggct tctacggttg attc
44446DNAArtificial SequenceSynthetic oligonucleotide 4gcctcgagtc
attacgttta aactcaatct tcaacagaaa atttgc 46535DNAArtificial
SequenceSynthetic oligonucleotide 5gatataccat ggaaatggac atcgttttcg
cggcg 35633DNAArtificial SequenceSynthetic oligonucleotide
6gtggtgctcg aggtagattt tacgcaggaa acg 33750DNAArtificial
SequenceSynthetic oligonucleotide 7ctgtattttc agggagcgat cgctatgaac
aagacttatg attttaaaag 50848DNAArtificial SequenceSynthetic
oligonucleotide 8gcctcgagtc attacgttta aacttaaatg tatgaatata
ctatcttc 48945DNAArtificial SequenceSynthetic oligonucleotide
9ctgtattttc agggagcgat cgctatgttg aataatgcta tgagc
451044DNAArtificial SequenceSynthetic oligonucleotide 10gcctcgagtc
attacgttta aactcacttt ttctttaccg gacg 441132DNAArtificial
SequenceSynthetic oligonucleotide 11gatataccat ggaaaactgc
ccgctggttt ct 321234DNAArtificial SequenceSynthetic oligonucleotide
12gtggtgctcg aggaagataa cgttgatttt acgg 341336DNAArtificial
SequenceSynthetic oligonucleotide 13cagggagcga tcgctatgca
aaaactacta tcttta 361435DNAArtificial SequenceSynthetic
oligonucleotide 14cattacgttt aaacttatga tcgtgatact tggaa
351545DNAArtificial SequenceSynthetic oligonucleotide 15ctgtattttc
agggagcgat cgctatggcg ttcaaagttg ttcag 451645DNAArtificial
SequenceSynthetic oligonucleotide 16gcctcgagtc attacgttta
aacttacgcg ttgtatttct gagat 451736DNAArtificial SequenceSynthetic
oligonucleotide 17cagggagcga tcgctatgac ggcaattgcc ccggtt
361835DNAArtificial SequenceSynthetic oligonucleotide 18cattacgttt
aaacttatgc gagagccaat ttctg 351936DNAArtificial SequenceSynthetic
oligonucleotide 19gatataccat ggaaacaaat attgcgatca ttcctg
362036DNAArtificial SequenceSynthetic oligonucleotide 20gtggtgctcg
agtttattgg ataaaatttc cgcgag 362135DNAArtificial SequenceSynthetic
oligonucleotide 21gatataccat ggaaatgaac aacgacaact ctacc
352234DNAArtificial SequenceSynthetic oligonucleotide 22gtggtgctcg
aggatgtcag agatcagttt gatg 342347DNAArtificial SequenceSynthetic
oligonucleotide 23ctgtattttc agggagcgat cgctatgaaa aagaccaaaa
ttgtttg 472443DNAArtificial SequenceSynthetic oligonucleotide
24gcctcgagtc attacgttta aacttacagg acgtgaacag atg
432536DNAArtificial SequenceSynthetic oligonucleotide 25cagggagcga
tcgctatgag cttactcaac gtccct 362635DNAArtificial SequenceSynthetic
oligonucleotide 26cattacgttt aaacttattt attctttgcg cgctc
3527936DNAEscherichia coli 27atggacatcg ttttcgcggc ggacgacaac
tacgcggcgt acctgtgcgt tgcggcgaaa 60tctgttgaag cggcgcaccc ggacaccgaa
atccgtttcc acgttctgga cgcgggtatc 120tctgaagcga accgtgcggc
ggttgcggcg aacctgcgtg gtggtggtgg taacatccgt 180ttcatcgacg
ttaacccgga agacttcgcg ggtttcccgc tgaacatccg tcacatctct
240atcaccacct acgcgcgtct gaaactgggt gaatacatcg cggactgcga
caaagttctg 300tacctggaca tcgacgttct ggttcgtgac tctctgaccc
cgctgtggga caccgacctg 360ggtgacaact ggctgggtgc gtgcatcgac
ctgttcgttg aacgtcagga aggttacaaa 420cagaaaatcg gtatggcgga
cggtgaatac tacttcaacg cgggtgttct gctgatcaac 480ctgaaaaaat
ggcgtcgtca cgacatcttc aaaatgtctt gcgaatgggt tgaacagtac
540aaagacgtta tgcagtacca ggaccaggac atcctgaacg gtctgttcaa
aggtggtgtt 600tgctacgcga actctcgttt caacttcatg ccgaccaact
acgcgttcat ggcgaaccgt 660ttcgcgtctc gtcacaccga cccgctgtac
cgtgaccgta ccaacaccgt tatgccggtt 720gcggtttctc actactgcgg
tccggcgaaa ccgtggcacc gtgactgcac cgcgtggggt 780gcggaacgtt
tcaccgaact ggcgggttct ctgaccaccg ttccggaaga atggcgtggt
840aaactggcgg ttccgcaccg tatgttctct accaaacgta tgctgcagcg
ttggcgtcgt 900aaactgtctg cgcgtttcct gcgtaaaatc tactga
93628972DNAEscherichia coli 28atggaaaact gcccgctggt ttctgttatc
gtttgcgcgt acaacgcgga acagtacatc 60gacgaatcta tctcttctat catcaaccag
acctacgaaa acctggaaat catcgttatc 120aacgacggtt ctaccgacct
gaccctgtct cacctggaag aaatctctaa actggacaaa 180cgtatcaaaa
tcatctctaa caaatacaac ctgggtttca tcaactctct gaacatcggt
240ctgggttgct tctctggtaa atacttcgcg cgtatggacg cggacgacat
cgcgaaaccg 300tcttggatcg aaaaaatcgt tacctacctg gaaaaaaacg
accacatcac cgcgatgggt 360tcttacctgg aaatcatcgt tgaaaaagaa
tgcggtatca tcggttctca gtacaaaacc 420ggtgacatct ggaaaaaccc
gctgctgcac aacgacatct gcgaagcgat gctgttctac 480aacccgatcc
acaacaacac catgatcatg cgtgcgaacg tttaccgtga acacaaactg
540atcttcaaca aagactaccc gtacgcggaa gactacaaat tctggtctga
agtttctcgt 600ctgggttgcc tggcgaacta cccggaagcg ctggttaaat
accgtctgca cggtaaccag 660acctcttctg tttacaacca cgaacagaac
gaaaccgcga aaaaaatcaa acgtgaaaac 720atcacctact acctgaacaa
aatcggtatc gacatcaaag ttatcaactc tgtttctctg 780ctggaaatct
accacgttga caaatctaac aaagttctga aatctatcct gtacgaaatg
840tacatgtctc tggacaaata caccatcacc tctctgctgc acttcatcaa
ataccacctg 900gaactgttcg acctgaaaca gaacctgaaa atcatcaaaa
aattcatccg taaaatcaac 960gttatcttct ag 97229903DNAEscherichia coli
29atggcgttca aagttgttca gatctgcggt ggtctgggta accagatgtt ccagtacgcg
60ttcgcgaaat ctctgcagaa acactctaac accccggttc tgctggacat cacctctttc
120gactggtctg accgtaaaat gcagctggaa ctgttcccga tcgacctgcc
gtacgcgtct 180gcgaaagaaa tcgcgatcgc gaaaatgcag cacctgccga
aactggttcg tgacgcgctg 240aaatgcatgg gtttcgaccg tgtttctcag
gaaatcgttt tcgaatacga accgaaactg 300ctgaaaccgt ctcgtctgac
ctacttcttc ggttacttcc aggacccgcg ttacttcgac 360gcgatctctc
cgctgatcaa acagaccttc accctgccgc cgccgccgga aaacaacaaa
420aacaacaaca aaaaagaaga agaataccag tgcaaactgt ctctgatcct
ggcggcgaaa 480aactctgttt tcgttcacat ccgtcgtggt gactacgttg
gtatcggttg ccagctgggt 540atcgactacc agaaaaaagc gctggaatac
atggcgaaac gtgttccgaa catggaactg 600ttcgttttct gcgaagacct
ggaattcacc cagaacctgg acctgggtta cccgttcatg 660gacatgacca
cccgtgacaa agaagaagaa gcgtactggg acatgctgct gatgcagtct
720tgccagcacg gtatcatcgc gaactctacc tactcttggt gggcggcgta
cctgatcgaa 780aacccggaaa aaatcatcat cggtccgaaa cactggctgt
tcggtcacga aaacatcctg 840tgcaaagaat gggttaaaat cgaatctcac
ttcgaagtta aatctcagaa atacaacgcg 900taa 903301146DNAEscherichia
coli 30atgaacaacg acaactctac caccaccaac aacaacgcga tcgaaatcta
cgttgaccgt 60gcgaccctgc cgaccatcca gcagatgacc aaaatcgttt ctcagaaaac
ctctaacaaa 120aaactgatct cttggtctcg ttacccgatc accgacaaat
ctctgctgaa aaaaatcaac 180gcggaattct tcaaagaaca gttcgaactg
accgaatctc tgaaaaacat catcctgtct 240gaaaacatcg acaacctgat
catccacggt aacaccctgt ggtctatcga cgttgttgac 300atcatcaaag
aagttaacct gctgggtaaa aacatcccga tcgaactgca cttctacgac
360gacggttctg cggaatacgt tcgtatctac gaattctcta aactgccgga
atctgaacag 420aaatacaaaa cctctctgtc taaaaacaac atcaaattct
ctatcgacgg taccgactct 480ttcaaaaaca ccatcgaaaa catctacggt
ttctctcagc tgtacccgac cacctaccac 540atgctgcgtg cggacatctt
cgacaccacc ctgaaaatca acccgctgcg tgaactgctg 600tctaacaaca
tcaaacagat gaaatgggac tacttcaaag acttcaacta caaacagaaa
660gacatcttct actctctgac caacttcaac ccgaaagaaa tccaggaaga
cttcaacaaa 720aactctaaca aaaacttcat cttcatcggt tctaactctg
cgaccgcgac cgcggaagaa 780cagatcaaca tcatctctga agcgaaaaaa
gaaaactctt ctatcatcac caactctatc 840tctgactacg acctgttctt
caaaggtcac ccgtctgcga ccttcaacga acagatcatc 900aacgcgcacg
acatgatcga aatcaacaac aaaatcccgt tcgaagcgct gatcatgacc
960ggtatcctgc cggacgcggt tggtggtatg ggttcttctg ttttcttctc
tatcccgaaa 1020gaagttaaaa acaaattcgt tttctacaaa tctggtaccg
acatcgaaaa caactctctg 1080atccaggtta tgctgaaact gaacctgatc
aaccgtgaca acatcaaact gatctctgac 1140atctaa 1146
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