Highlight: GM3 Synthase
Deficiency
Joshua Wesalo ’13
Advisor: Ken Hess
Tuesday, July 23, 13
“
Better living through
chemistry
Tuesday, July 23, 13
’’
Collaborations—The GM3 Team
Dr. Steve Withers
Drug Development Consultants
Dr. Steve Roth
Dr. David Zopf
Tuesday, July 23, 13
Dr. Matt Kremer
•
•
•
•
•
•
Dr. Ken Hess
Dr. Scott Brewer
Dr. Ed Fenlon
Dr. Rob Jinks
Dr. Scott Van Arman
Dr. Christine Piro
1
Tuesday, July 23, 13
What is GM3?
2
Synthetic Strategy
3
Results so far
4
Future Directions
GM3 Synthase
Deficiency
Founder effect in Amish leads to...
Blindness
Epilepsy
Deafness
Developmental Stagnation
•
•
•
•
Poor Feeding
Normal Vomiting
Birth
Tuesday, July 23, 13
3 mo.
1 yr.
Can’t walk, talk, reach, or sit
Nonpurposeful movements
Muscular weakness
Brain atrophy
3 yrs.
Teens
Patients
AMERICAN JOURNAL OF MEDICAL GENETICS PART A
AMERICAN JOURNAL OF MEDICAL GENETICS PART A
C
1. Cutaneous dyspigmentation in patients with ganglioside GM3 synthase deficiency. A: multiple freckle-like hyperpigmented macules on the right
of an 18-year-old female (S-1); (B) multiple freckle-like hyperpigmented macules on both legs of a 12-year-old female (M-1); (C) multiple
gmented macules with several hyperpigmented macules on the right arm of a 12-year-old female (M-1); (D) large depigmented patches on the
of a 25-year-old female (H-1); (E) sharply delineated depigmented patches on the right hand of a 24-year-old male (H-2); and (F) the forehead
25-year-old female (H-1) with white hairs and depigmented skin.
with ganglioside GM3 synthase deficiency. A: multiple freckle-like hyperpigmented macules on the right
ple freckle-likein
hyperpigmented
macules
on both legs of a 12-year-old
female (M-1);
multiple
described
this study are
disease-specific
in the context
of (C)American
patients was not found in any of the eight patients in
nted macules on the right arm of a 12-year-old female (M-1); (D) large depigmented patches on the
Cutaneous
dyspigmentation
inand
patients
with
ganglioside
GM3
synthase
deficiency.
A: multiple freckle-like
hyperpigmented
arly-onset
neurological
may serve
useful
cohort
in whom
electrocardiograms
were performed
(data not macules on the right
delineated
depigmented
patches on disease
the right hand
of a 24-year-old
maleas
(H-2);
and (F) theour
forehead
identifying
morefemale
patients
with(B)ganglioside
metabolic hyperpigmented
shown).
and
depigmented
skin.
an
18-year-old
(S-1);
multiple freckle-like
macules on both legs of a 12-year-old female (M-1); (C) multiple
sented
in themacules
future.
recent linkage study
iden-on theThe
for the development
with several ahyperpigmented
macules
rightmechanism
arm of a 12-year-old
female (M-1);of(D)dyspigmentation
large depigmented patches on the
A Interestingly,
homozygous
c.994 G>A
in the delineated
ST3GAL5 gene
remainspatches
unclear. on
Thethe
only
cellhand
from GM3
synthase deficient
patients
a 25-year-oldmutation
female (H-1);
(E) sharply
depigmented
right
of a 24-year-old
male (H-2);
and (F) the forehead
o GM3 synthase
3 patients
and studied
-year-old
female deficiency
(H-1) withinwhite
hairs with
and ‘‘Salt
depigmented
skin. to date has been the fibroblast, which demonstrates almost
syndrome’’
described
in
an
African-American
family
in
complete
of cellular gangliosides, suppressed epidermal
ecific in the context of American patients was not found in any of the eight
patients depletion
in
serve
as useful
our et
cohort
in whomThe
electrocardiograms
were performed
(datafactor
not receptor activation, and defective cell proliferation
ulmay
et al.,
1983;
Schwartz
al., 2011].
cutaneous changes
growth
shown).
danglioside
in these metabolic
patients were
very similar with our findings here. and migration [Liu et al., 2008]. Interestingly, glucosylceramide
nt linkage study idenThe mechanism for the development of dyspigmentation
lor
of
skin
in
our
patients
is
much
lighter,
the
depigmented
synthase-deficient
melanoma cells, which have depletion of downn the ST3GAL5 gene remains unclear. The only cell from GM3 synthase deficient patients
are
missed
theytoare
comparison
with
streamalmost
ganglioside GM3, fail to produce pigment because of the
tientsoften
with ‘‘Salt
andsince
studied
datesubtle
has beeninthe
fibroblast, which
demonstrates
-American family
depletion
of cellularHowever,
gangliosides,the
suppressed
epidermal
t-colored
skin inof complete
the Amish
patients.
abnormal
intracellular sorting of tyrosinase in the Golgi complex
he
cutaneous
growthare
factor
receptor
defective
cellrather
proliferation
scribed
inchanges
this study
disease-specific
in
the context
of
American patients
not Further
foundstudies
in any
of the eight patients
cific
conduction
abnormality’’
foundactivation,
in theseand
Africanthan melanosomes
[Sprong etwas
al., 2001].
with
with our findings here. and migration [Liu et al., 2008]. Interestingly, glucosylceramide
ly-onset neurological disease and may serve as useful our cohort in whom
hter, the depigmented synthase-deficient melanoma cells, which have depletion of downe in comparison with
stream
gangliosidewith
GM3, fail
to produce pigment
because of theshown).
dentifying
patients
ganglioside
metabolic
B more
synthase
deficiency.
A: multiple
freckle-like
hyperpigmented
macules
on the right
ients. However,
the abnormal
intracellular
sorting
of tyrosinase in the
Golgi complex
n the
future.
a recent
linkage
study
The mechanism
nd
in these
African-Interestingly,
rather
than
melanosomes
[Sprong
etfemale
al., 2001].
Furtheridenstudies
with
pigmented
macules
on
both
legs
of a 12-year-old
(M-1);
(C)
multiple
in
electrocardiograms were performed (data not
for the development of dyspigmentation
mozygous
mutation
c.994
G>A
in the
ST3GAL5
geneon the
remains unclear. The only cell from GM3 synthase deficient patients
ght
arm of a 12-year-old
female
(M-1);
(D) large
depigmented
patches
synthase
deficiency
in 3 patients
withand‘‘Salt
studied to date has been the fibroblast, which demonstrates almost
edGM3
patches
on the right
hand of a 24-year-old
male (H-2);
(F) theand
forehead
n.
ndrome’’
described in an African-American family in complete depletion of cellular gangliosides, suppressed epidermal
et al., 1983; Schwartz et al., 2011]. The cutaneous changes growth factor receptor activation, and defective cell proliferation
n these patients were very similar with our findings here. and migration [Liu et al., 2008]. Interestingly, glucosylceramide
r of skin in our patients is much lighter, the depigmented synthase-deficient melanoma cells, which have depletion of downe often missed since they are subtle in comparison with stream ganglioside GM3, fail to produce pigment because of the
of American patients was not found in any of the eight patients in
colored
skin of the Amish patients. However, the abnormal intracellular sorting of tyrosinase in the Golgi complex
ul our cohort in whom electrocardiograms were performed (data not
fic conduction abnormality’’ found in these African- rather than melanosomes [Sprong et al., 2001]. Further studies with
lic
nne
nd
in
es
re.
ed
th
he
n-
shown).
The mechanism for the development of dyspigmentation
remains unclear. The only cell from GM3 synthase deficient patients
studied to date has been the fibroblast, which demonstrates almost
complete depletion of cellular gangliosides, suppressed epidermal
growth factor receptor activation, and defective cell proliferation
and migration [Liu et al., 2008]. Interestingly, glucosylceramide
synthase-deficient melanoma cells, which have depletion of downstream ganglioside GM3, fail to produce pigment because of the
abnormal intracellular sorting of tyrosinase in the Golgi complex
rather than melanosomes [Sprong et al., 2001]. Further studies with
Tuesday, July 23, 13
1 in 100 are carriers
Genetics & Disease Mechanism
Wild-Type
Nonsense mutation in GM3
Carrier
Synthase (SIAT9) gene
(694C→T, translating to R232X)
Affected
Tuesday, July 23, 13
Genetics & Disease Mechanism
GM3
Synthase
Nonsense mutation in GM3
Synthase (SIAT9) gene
(694C→T, translating to R232X)
Adapted from Rao FV et. al.. Nature Structural & Molecular Biology. 2009; 16: 1186–1188.
Cannot produce GM3
Cannot produce higher
gangliosides
Problems in cellular adhesion1,
cell signaling1, and axon-glial
interactions2
1-Simpson, Michael A., et. al.. Nature Genetics. 2004; 36(11): 1225–1229.
2-Yamashita, Tadashi, et. al.. PNAS. 2005; 102(8): 2725–2730.
Tuesday, July 23, 13
Genetics & Disease Mechanism
Simpson, Michael A., et. al.. Nature Genetics. 2004;
36(11): 1225–1229.
Tuesday, July 23, 13
Genetics & Disease Mechanism
se
tha
3
GM
n
Sy
a-series gangliosides
Tuesday, July 23, 13
b-series gangliosides
pKa 2.2–3.0
GM3
Neu5Ac Galactose Glucose
Fatty acid
Sphingosine
Neu5Ac(α2→3)Gal(β1→4)Glc(β1)Cer
10-7 M > CMC > 10-9 M (in aqueous solution)
Tuesday, July 23, 13
Neurodevelopmental milestones and concurrent changes in GSL expression.
Yu R K et al. J. Lipid Res. 2009;50:S440-S445
©2009 by American Society for Biochemistry and Molecular Biology
Tuesday, July 23, 13
TOTAL GANGLIOSIDES
3500
3000
u)
@
%
2500
@
@
@@
#
@ @
@
@
o
oo @
@
Q 2000
@
1500
1ooo
@
500
g
n
3
i
5
PRENATAL AGE. m o n t h s
t
l
l
It0
210
2
5
POSTNATAL AGE. years
i
50
t
100
Svennerholm, L.; Boström, K.; Fredman, P.; Månsson, J. E.; Rosengren, B.; Rynmark, B. M. Biochimica et Biophysica Acta, 1005 (1989): 109–117.
Fig. 1. The ganglioside concentration of human frontal cortex ex-
Tuesday, July 23, 13
GANGLIOSIDE
GM 1
t000
m 800
60G
@
@
@@ @
400
@
@
200
@ @ 0
@
~o
e
@ @Oo
O
00
@
@
0,,%, ,, @
,
3
5
PRENATAL AGE. m o n t h s
t
t
5t
,'o
2'0
50I
t~)0
POSTNATAL AGE. years
Svennerholm, L.; Boström, K.; Fredman, P.; Månsson, J. E.; Rosengren, B.; Rynmark, B. M. Biochimica et Biophysica Acta, 1005 (1989): 109–117.
Fig. 2. The concentration of ganglioside GM! expressed as #mol sialic
Tuesday, July 23, 13
GANGLIOSIDE GD I b
1000
==
I-. 800
o
<o
600
o
_o
@
.J
@
400
00
00
o
Q@
@
@
o
@
®
200
8e ®~
e~
I
I
t
3
5
PRENATAL AGE, m o n t h s
I
I
I
I
2
5
10
20
POSTNATAL AGE. years
I
50
I
100
Svennerholm, L.; Boström, K.; Fredman, P.; Månsson, J. E.; Rosengren, B.; Rynmark, B. M. Biochimica et Biophysica Acta, 1005 (1989): 109–117.
Fig. 4. Tile concentration of ganglioside GDI b expressed as /~mol
Tuesday, July 23, 13
Two Goals
1
Obtain the
Drug
Tuesday, July 23, 13
2
Tool to
Monitor
Treatment
GM3 from Natural
Sources
Tuesday, July 23, 13
Ganglioside 500 Buttermilk
•
•
•
•
•
•
0.56% GM3
0.6% GD3
34.0% other lipids
3.2% moisture
56.0% lactose
5.0% ash
Tuesday, July 23, 13
Other Lipids
Lactose
GD3
GM3
Ash
Moisture
Moisture
Other Lipids
GM3
Lactose
GD3
Ash
1
2
GM3 from Natural
Sources
Tuesday, July 23, 13
3
Difficulty
≠
1
2
GM3 from Natural
Sources
Tuesday, July 23, 13
3
Difficulty
≠
1
2
GM3 from Natural
Sources
Tuesday, July 23, 13
3
Difficulty
≠
GM3 Made in the Lab
GM3 from Natural
Sources
Tuesday, July 23, 13
Human vs. Cow GM3
≠
Tuesday, July 23, 13
≠
What happens after
.
o
3m
Tuesday, July 23, 13
of
?
Small GM3 levelC18via placental transfer
C18
C16
30125
30125 C16
0 months
0
M
0M
RT: 7.01 - 10.03
8.03
RT:100
7.01 - 10.03
8.03
100
90
7.75
7.75
90
80
Relative
Relative
Abundance
Abundance
80
70
70
60
60
50
50
40
C20
8.32
C20
40
30
30
20
20
10
7.13
7.27
7.13
7.27
10
0
0
7.43
7.437.47
7.47
7.2
7.4
7.2
7.4
RT: 6.66 - 9.64
RT: 6.66 - 9.64
100
100
90
90
80
80
70
70
60
60
50
50
40
40
30
30
6.78
20
6.78
20
10
10
0
6.8
0
6.8
Relative
Relative
Abundance
Abundance
C22
C22
8.60
8.32
7.89
7.59
7.89
7.71
7.59
7.71
7.6
7.6
30953
30953
3
3M
M
8.18
8.46
8.18
8.46
7.8
8.0
8.2
7.8
8.0
8.2
C26
9.18
C26
8.86
8.74
8.74
8.4
8.6
8.4 Time (min)
8.6
8.92
C16
7.39
C16
8.8
8.92 9.02
9.02
9.0
9.2
8.8
9.0
9.2
9.49
9.30
9.49
9.61
9.79
9.93
9.4
9.61
9.6
9.79
9.8
9.93
9.4
9.6
9.8
(Same
affected
individual)
10.0
10.0
7.67
3 months
C24
C24
8.51
8.51
C22
C23
C22
8.24
C23
8.24 8.37
8.37
C26
C26
8.81
7.96
8.81
7.03
7.03 7.11 7.19
7.11 7.19
7.2
7.2
9.30
G500
G500
C18
C18
7.67
7.39
C20
7.96
C20
7.0
7.0
9.18
Time (min)
6.92
6.92
1300
1300
Tuesday, July 23, 13
8.60
C24
C24
8.86
7.53
7.53
7.4
7.4
7.6
7.6
7.82
7.82
7.8
7.8
8.67
8.67
8.10
8.10
8.0
8.2
8.0 Time (min)
8.2
Time (min)
1400
1400
8.4
8.4
8.6
8.6
8.8
8.8
8.99 9.11
8.99 9.11
9.0
9.2
9.0
9.2
1500
1500
9.33 9.40 9.56
9.33 9.40 9.56
9.4
9.6
9.4
9.6
1600
1600
Plasma Glycosphingolipid Levels
Tuesday, July 23, 13
90
Non&Amish&Cont.
Sph&HexCer
80
70
Relative Abundance
1010.783
z=1
#4
100
863.727
z=1
60
50
40
918.805
z=1
837.712
z=1
1120.896
z=1
30
1038.814
z=1
938.590
z=1
20
1094.879
z=?
10
0
800
900
1000
1242.922
z=1
1100
1200
1325.002
z=1
1300
1400.001
z=1
1482.082
z=1
1400
m/z
1010.784
z=1
#31
100
1371.969
z=1
1214.890
z=1
1570.137
z=1
1500
1600
1733.155
z=1
1700
1800
1900
2000
LacCer
90
80
Relative Abundance
70
899.510
z=1
60
50
837.713
z=1
40
Amish&Cont.
918.806
z=1
30
1120.897
z=1
939.410
z=1
20
10
0
800
900
1000
90
1100
1200
1350.080
z=2
1300
1456.067
z=1
1400.002
z=1
1400
m/z
1010.783
z=1
#23
100
1298.987
z=1
1139.782
z=1
1054.775
z=1
1371.970
z=1
1214.891
z=1
1514.115
z=1
1500
1600
1700
1800
1900
2000
80
Relative Abundance
70
Amish&Carrier
60
50
40
899.508
z=1
837.712
z=1
30
20
918.804
z=1
1120.895
z=1
938.590
z=1
10
0
800
900
1000
1100
1300
1120.896
z=1
70
Relative Abundance
1200
60
40
1094.880
z=1
916.790
z=1
837.713
z=1
1066.848
z=1
938.590
z=1
10
0
800
1400
m/z
1500
1570.137
z=1
1600
1733.154
z=?
1700
No&
GM3
863.728
z=1
50
1460.023
z=1
1484.096
z=1
1821.208
z=1
1800
1900
2000
LacCer&carrying&C24:0&and&C24:1&as&FA&components.
80
20
1371.968
z=1
1139.780
z=1
90
30
1325.002
z=1
1010.783
z=1
#30
100
1038.814
z=1
1214.889
z=1
900
Tuesday, July 23, 13
1000
1100
1214.890
z=1
1200
1325.003
z=1
1298.986
z=1
1300
Amish&Affected
1460.025
z=1
1400
m/z
1500
1570.138
z=1
1600
1700
1800
1900
2000
70
60
50
40
Non Amish Cont
30
Amish Cont
20
M/+
10
M/M
Tuesday, July 23, 13
G
D
3
G
M
1b
3
M
G
G
b4
G
b3
er
La
cC
M
Gl
cC
C er
H
0
70
60
50
40
Non Amish Cont
30
Amish Cont
20
M/+
10
M/M
G
D
3
G
M
1b
M
3
G
G
b4
G
b3
er
cC
La
Gl
cC
C er
M
H
0
1
0.8
0.6
0.4
0.2
0
GD3
Tuesday, July 23, 13
GM1b
nmol/ml&plasma
30"
25"
20"
15"
Non&Amish&Cont.
10"
5"
0"
CMH"
LacCer"
CTH"
CQH"
GM3"
GD3"
GM1"
30"
25"
20"
15"
Amish&Cont.
10"
5"
0"
CMH"
LacCer"
CTH"
CQH"
GM3"
GD3"
GM1"
30"
25"
20"
15"
Amish&Carrier
10"
5"
0"
CMH"
30"
LacCer"
CTH"
CQH"
GM3"
GD3"
GM1"
max
q3
median
q1
25"
20"
15"
Amish&Affected
min
10"
5"
0"
CMH"
Tuesday, July 23, 13
LacCer"
CTH"
CQH"
GM3"
GD3"
GM1"
Rel.&%
45"
40"
35"
31%
30"
25"
Amish&Cont.
20"
15"
10"
5"
0"
GlcCer
CMH"
LacCer"
CTH"
CQH"
GM3"
GD3"
GM1"
80"
70"
Amish&Affected
60%
60"
50"
40"
30"
20"
10"
—None—
0"
CMH"
GlcCer
Tuesday, July 23, 13
LacCer"
CTH"
CQH"
GM3"
GD3"
GM1"
26
1
2
Tuesday, July 23, 13
What is GM3?
The Plan: Synthetic Strategy
3
Results so far
4
Future Directions
Convergence
Build this up...
O
OH
OH
OH
OH
O
O
O
OH
O
OH
O
HO
Build this up...
HN
OH
O
OH
NH
O
O
HO
HO
HO
OH
Couple together
OH
OH
OH
OH
OH
O
HO
OH
HO
O
O
O
O
O
O
NH
OH
O
HO
HO
HN
HO
OH
Tuesday, July 23, 13
Synthetic Strategies
Chemical
Semisynthesis
Chemoenzymatic
Semisynthesis
Coupling
Tuesday, July 23, 13
Chemical
Semisynthesis
D-erythro-Sphingosine
Preparing the pole
NH22
NH
OH
OH
Phytosphingosine (S. cerevisae)
HO
OH
OH
NH22
HO
OH
OH
D-erythro-sphingosine
van den Berg, R. J. B. H. N. et al. J. Org. Chem., 2004, 69, 5699–5704.
Tuesday, July 23, 13
Chemical
Semisynthesis
O
NH
D-erythro-Sphingosine
Ph
95%
HCl
NH2
OH
OH
OH
Phytosphingosine
D-ribo-phytosphingosine
(Side-product doubly tosylated)
76%
OH
N
(CH2)12CH3
Ph
O
(CH2)12CH3
CH2Cl2
reflux (40 ºC)
48 h.
OTs
N
TsCl
3:2 pyr:CH2Cl2
O
(CH2)12CH3
N
OH
DBN
THF
0 ºC
1 h.
N
Oxazoline
5
4
Tosylate
6
OH
NaH
Ph
Ph
OK
NH2
N
D-erythro-sphingosine
(CH2)12CH31. 2 M HCl, THF, rt, 8 h. O
OH
OH
(CH2)12CH3 DBN
2. NaOH, MeOH, 100 ºC, 2 h.
2
Sphingosine
N
TMSI
OTMS
8
MeCN
40 86 ºC
O
(CH2)12CH3
O
7 Epoxide
Silyl Ether
van den Berg, R. J. B. H. N. et al. J. Org. Chem., 2004, 69, 5699–5704.
Tuesday, July 23, 13
Chemical Activating Sugar
Semisynthesis
CCl3
OH
O
HO
O
OAc
~90%
O
AcO
OH
76%
OAc
HO
OH
O
OH
O
AcO
OAc
O
OAc
Pyridine
12 h.
OAc
OAc
N
H2NNH2 H2O
CH3CN
DBU, CH2Cl2, 1.5 h.
O
MeO
O
O
O
N
H
HO
OAc
AcO
Tuesday, July 23, 13
MeO
AcO
Protected
O
O
N
H
O
Per-O-Acetyl 3'-Sialyllactose Methyl Ester
Duclos R. I., Jr. Carbohydr. Res. 2000, 328, 489–507.
Bavaro, T. et al. Eur. J. Org. Chem. 2011, 6181-6185.
Khan, R. et al. Aust. J. Chem. 1996, 49, 293–298.
OAc
AcO
OAc
AcO
O
N
O
NH
3’-SL
OAc
N
OAc
3'-Sialyllactose
AcO
Cl3C
O
O
O
O
O
AcO
OH
O
OAc
AcO
AcO
OAc
HO
OAc
OAc
O
O
NH
O
O
O
O
2.
O
HO
1. CH3I, DMSO
O
O
OAc
OAc
OH
76%
O
O
OH
O
HN
OAc
OH
OAc
AcO
1-Alcohol
Regioselectively
Deprotected
Trichloroacetimidate
AcO
Activated
Chemical Ceramide Preparation
Semisynthesis
D-erythro-sphingosine
NH2
HO
OH
Acylation
O
O
(CH2)14CH3
HN
96%
(CH2)14CH3
HN
85%
TrCl
HO
(CH2)13CH3
N
OH
89%
Cl
TrO
(CH2)14CH3
HN
(CH2)13CH3
CH2Cl2
18 h.
O
O
O
N
OH
Et2O BF3
6:4 Toluene:MeOH
OH
4 h.
TrO
4-pyrrolidinopyridine
5 min.
(CH2)13CH3
(CH2)13CH3
Ph
Ph
O
O
O
O
Duclos R. I., Jr. Carbohydr. Res. 2000, 328, 489–507.
Rai, A. N.; Basu, A. Org. Lett. 2004, 6, 2861–2863.
Tuesday, July 23, 13
(CH2)14CH3
HN
OH
c
OA
(CH2)14CH3
HN
OH
%
HN
76
c
OA
h.
N
O
O
Ac
O
Ac
.5
N
C
,1
Cl 3
Cl 2
H2
N
,C
BU
O
N
H
c
OA
O
O
O
Me
(CH2)13CH3
CC
O
O
Ph
O
l3
c
OA
O
O
O
O
Ac
c
OA
c
OA
c
OA
O
O
N
H
c
OA
ti
ace
oro
chl
Tri
Couple together
mi
1. BF3 • Et2O
2. NaOCH3
3. Amberlite IRC-50
e
dat
O
OH
OH
OH
OH
O
OH
O
HO
OH
O
HN
O
O
O
O
NH
OH
O
HO
HO
OH
GM3
Duclos R. I., Jr. Carbohydr. Res. 2000, 328, 489–507.
Tuesday, July 23, 13
OH
1
Tuesday, July 23, 13
What is GM3?
2
Synthetic Strategy
3
Results so far
4
Future Directions
Model Coupling
Tuesday, July 23, 13
~90%
OH
HO
O
O
HO
AcO
OH
O
HO
O
O
HO
OH
OH
Lactose
O
I2
12 h.
OAc
O
AcO
AcO
O
OAc
AcO
OAc
O
H2NNH2 H2O
O
CH3CN
AcO
AcO
OAc
OAc
OAc
O
AcO
AcO
DBU, CH2Cl2, 1.5 h.
O
OAc
CCl3
N
AcO
H2
C
=
AcO
CH
)9
(CH2
O
1. BF3
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
Tuesday, July 23, 13
, HO
O
2
t
•E
OAc
OAc
O
O
AcO
AcO
O
OAc
84%
O
NH
CCl3
OH
8-O-acetyl lactose
HO
OH
O
OH
O
O
HO
HO
HO
Lactose
5.63 g
Tuesday, July 23, 13
O
O
OH
OH
OAc
AcO
O
I2
12 h.
OAc
O
O
AcO
AcO
AcO
O
OAc
10.67 g
95.52% Yield
OAc
7-O-acetyl lactose
O
OAc
AcO
OAc
O
O
AcO
AcO
AcO
2.00 g
O
OAc
OAc
H2NNH2 OH
DMF, 1 h., 50 ºC
AcO
Zn(OAc)2
AcO
CH3OH, 12 h.
OAc
O
O
AcO
AcO
2O
H
·
2
H
N
N
e
l
H2
i
r
t
i
n
~4 g
Aceto
~75% Yield
Duclos R. I., Jr. Carbohydr. Res. 2000, 328, 489–507.
Kaya, E.; Sonmez, F.; Kucukislamoglu, M.; Nebioglu, M. Chem. Pap. 2012, 66, 312–315.
Khan, R. et al. Aust. J. Chem. 1996, 49, 293–298.
Tuesday, July 23, 13
OAc
O
OAc
OH
7OAc Lac 1-Trichloroacetimidate
AcO
OAc
AcO
OAc
O
O
AcO
AcO
CCl3
O
AcO
OAc
OH
N
OAc
OAc
O
O
DBU, CH2Cl2, 1.5 h.
N
N
AcO
AcO
AcO
OAc
Performed on 400 mg scale
Tried NaH instead of DBU (100 mg scale)
Duclos R. I., Jr. Carbohydr. Res. 2000, 328, 489–507.
Schmidt, R. R.; Michel, J. Angew. Chem. Int. Ed. Engl. 1980, 19, 731–732.
Tuesday, July 23, 13
NH
O
O
CCl3
Model Coupling
OAc
AcO
OAc
O
O
AcO
AcO
AcO
O
OAc
O
HO(CH2)9HC=CH2
NH
AcO
AcO
OAc
O
BF3· Et2O, CH2Cl2
CCl3
OAc
O
AcO
AcO
O
OAc
O
Performed on 300 mg scale
One-pot event after trichloroacetimidate formation
MS provides evidence of successful coupling
Tuesday, July 23, 13
1
Tuesday, July 23, 13
What is GM3?
2
Synthetic Strategy
3
Results so far
4
Future Directions
Finish Synthesis!
Tuesday, July 23, 13
Make the Sugar Here!
Tuesday, July 23, 13
Engineering a cell line to produce 3’-sialyllactose
Journal of Biotechnology 134 (2008) 261–265
Contents lists available at ScienceDirect
Journal of Biotechnology
journal homepage: www.elsevier.com/locate/jbiotec
Genetic engineering of Escherichia coli for the economical
production of sialylated oligosaccharides
Nicolas Fierfort, Eric Samain ∗
Centre de Recherches sur les Macromolécules Végétales (CERMAV – CNRS), affiliated with Joseph Fourier University and
member of the ICMG (Institut de Chimie Moléculaire de Grenoble), BP 53, 38041 Grenoble Cedex 9, France
a r t i c l e
i n f o
Article history:
Received 4 September 2007
Received in revised form 6 December 2007
Accepted 6 February 2008
Keywords:
Sialyllactose
Sialyltransferase
Oligosaccharide
Escherichia coli
Metabolic engineering
Tuesday, July 23, 13
a b s t r a c t
We have previously described a microbiological process for the conversion of lactose into 3" sialyllactose
and other ganglioside sugars by living Escherichia coli cells expressing the appropriate recombinant glycosyltransferase genes. In this system the activated sialic acid donor (CMP-Neu5Ac) was generated from
exogenous sialic acid, which was transported into the cells by the permease NanT. Since sialic acid is an
expensive compound, a more economical process has now been developed by genetically engineering
E. coli K12 to be capable of generating CMP-Neu5Ac using its own internal metabolism. Mutant strains
devoid of Neu5Ac aldolase and of ManNAc kinase were shown to efficiently produce 3" sialyllactose by
coexpressing the !-2,3-sialyltransferase gene from Neisseria meningitidis with the neuC, neuB and neuA
Campylobacter jejuni genes encoding N-acetylglucosamine-6-phosphate-epimerase, sialic acid synthase
and CMP-Neu5Ac synthetase, respectively. A sialyllactose concentration of 25 g l−1 was obtained in long-
1. Establish Safety
2. Proof of Principal
Make ~1–2 g
Use mouse model
Tuesday, July 23, 13
Clinical Trial
Tuesday, July 23, 13
Acknowledgements
•
•
•
•
•
Dr. Hess
Dr.Van Arman
Dr. Brewer
Dr. Fenlon
Dr. Piro
Funding
•F&M Chemistry Department
•Hackman Scholars Fund
•Dr. Eric Rackow
•HHMI
•Eyler Grant
Collaborators
•Dr. Rob Jinks
Jon Salandra
•Dr. Kevin Strauss
•Dr. D. Holmes Morton
•Dr. Stephen Roth
•Dr. Matthew Kremer
•Dr. Theresa Swenson
•Adam Heaps
•Dr. Stephen G. Withers
•Dr. Shawn DeFrees
Special Thanks to Lisa Mertzman and to Carol Strausser
Tuesday, July 23, 13
Synthetic Strategies
Chemical
Semisynthesis
Tuesday, July 23, 13
Chemoenzymatic
Semisynthesis
Sugar
Chemoenzymatic
Semisynthesis
~90%
OH
HO
O
O
O
HO
OH
OAc
OAc
O
O
HO
OH
Lactose
Ng, E. S. P. Master’s Thesis. U. British Columbia 2005.
Rich, J. R.; Withers, S. G. Angew. Chem. Int. Ed. 2012, 51, 8640–8643.
Tuesday, July 23, 13
AcO
OH
O
HO
O
I2
12 h.
O
AcO
O
AcO
AcO
OAc
OAc
7:3
AcO
HF
N
OAc
O
O
1 h., 0 ºC
AcO
O
OH
HO
OAc
NaOCH3
O
CH3OH, 30 min
AcO
AcO
HO
OAc
F
OH
O
O
HO
HO
OH
Ng, E. S. P. Master’s Thesis. U. British Columbia 2005.
Rich, J. R.; Withers, S. G. Angew. Chem. Int. Ed. 2012, 51, 8640–8643.
Tuesday, July 23, 13
F
7:3
OAc
AcO
HF
N
O
O
1 h., 0 ºC
AcO
OH
HO
OAc
NaOCH3
O
O
CH3OH, 30 min
AcO
AcO
HO
OAc
OH
O
O
HO
HO
F
OH
F
NH2
NH2
N
N
O
O
O
HO
O
O P O
O
O
O-
OH
HO
OH
O
O
H
H
H
H
OH OH
O
HO
Ng, E. S. P. Master’s Thesis. U. British Columbia 2005.
Rich, J. R.; Withers, S. G. Angew. Chem. Int. Ed. 2012, 51, 8640–8643.
Tuesday, July 23, 13
O-
P
O
O
O-
HO
O
OH
H
H
H
OH
H
OH
OH
HO
OH
OH
NANA
N
O
O
CMP-Sialic Acid Synthetase
Bovine Alkaline Phosphatase
OH
HO
N
O
HO
CMP-NANA
O
7:3
OAc
AcO
HF
N
O
O
1 h., 0 ºC
AcO
OH
HO
OAc
NaOCH3
O
O
CH3OH, 30 min
AcO
AcO
HO
OAc
OH
O
O
HO
HO
F
OH
F
NH2
2
N
N
O
N
O
O
O-
P
O
O
O-
HO
O
OH
α(2,3)-sialyl
transferase
O
H
H
H
OH
H
OH
O
OH
OH
HO
OH
OH
OH
CMP-NANA
Ng, E. S. P. Master’s Thesis. U. British Columbia 2005.
Rich, J. R.; Withers, S. G. Angew. Chem. Int. Ed. 2012, 51, 8640–8643.
Tuesday, July 23, 13
OH
OH
O
HO
HO
OH
O
NH
O
O
OH
O
HO
HO
3’-SL Fluoride
O
OH
F
Chemoenzymatic
Semisynthesis: Coupling
3’-SL Fluoride
O
D-erythro-sphingosine
OH
OH
OH
OH
OH
NH2
O
HO
O
OH
O
O
O
OH
NH
O
HO
HO
O
OH
HO
ase
OH
OH
OH
OH
OH
O
HO
OH
OH
EGC
F
O
O
E35
1S
NH2
O
O
O
NH
OH
O
HO
HO
Vaughan, M. D.; Johnson, K.; Defrees, S.; Tang, X.; Warren, R. A. J.;
Withers, S. G. J. Am. Chem. Soc. 2006, 128, 6300–6301.
Tuesday, July 23, 13
OH
lyso-GM3
Acylation
OH
GM3