The design and synthesis of an α-Gal trisaccharide epitope that provides a highly specific anti-Gal immune response

Article abstract of DOI:10.1039/c7ob00448f

Carbohydrate antigens displaying Galα(1,3)Gal epitopes are recognized by naturally occurring antibodies in humans. These anti-Gal antibodies comprise up to 1% of serum IgG and have been viewed as detrimental as they are responsible for hyperacute organ rejections. In order to model this condition, α(1,3)galactosyltransferase-knockout mice are inoculated against the Galα(1,3)Gal epitope. In our study, two α-Gal trisaccharide epitopes composed of either Galα(1,3)Galβ(1,4)GlcNAc or Galα(1,3)Galβ(1,4)Glc linked to a squaric acid ester moiety were examined for their ability to elicit immune responses in KO mice. Both target epitopes were synthesized using a two-component enzymatic system using modified disaccharide substrates containing a linker moiety for coupling. While both glycoconjugate vaccines induced the required high anti-Gal IgG antibody titers, it was found that this response had exquisite specificity for the Galα(1,3)Galβ(1,4)GlcNAc hapten used, with little cross reactivity with the Galα(1,3)Galβ(1,4)Glc hapten. Our findings indicate that while homogenous glycoconjugate vaccines provide high IgG titers, the carrier and adjuvanting factors can deviate the specificity to an antigenic determinant outside the purview of interest.

Full text of DOI:10.1039/c7ob00448f

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The design and synthesis of an α-Gal trisaccharide epitope that
provides a highly specific anti-Gal immune response
+
ab
+ac
+a
a
a
Kensaku Anraku , Shun Sato , Nicholas T. Jacob , Lisa M. Eubanks , Beverly A. Ellis and Kim D.
Janda*
Received 00th January 20xx,
Accepted 00th January 20xx
a
DOI: 10.1039/x0xx00000x
Carbohydrate antigens displaying Galα(1,3)Gal epitopes are recognized by naturally occurring antibodies in humans. These
anti-Gal antibodies comprise up to 1% of serum IgG and has been viewed as detrimental as they are responsible for
hyperacute organ rejections. In order to model this condition, α(1,3)galactosyltransferase-knockout mice are innoculated
agaisnt the Galα(1,3)Gal epitope. In our study, two α-Gal trisaccharide epitopes composed of either
Galα(1,3)Galβ(1,4)GlcNAc or Galα(1,3)Galβ(1,4)Glc linked to a squaric acid ester moiety were examined for their ability to
elicit immune response in KO mice. Both target epitopes were synthesized using a two-component enzymatic system using
modified dissacharide substrates containing a linker moiety for coupling. While both glycoconjugate vaccines induced the
required high anti-Gal IgG antibody titers, it was found that this response had exquisite specificity for the
Galα(1,3)Galβ(1,4)GlcNAc hapten used, with little cross reactivity with the Galα(1,3)Galβ(1,4)Glc hapten. Our findings
indicate that while homogenous glycoconjugate vaccines provide high IgG titers, the carrier and adjuvanting factors can
www.rsc.org/
deviate
the
specificty
to
an
antigenic
determinant
outside
the
purview
of
interest.
method often results in a high production of anti-Gal IgM, with IgG
1
1,12
Introduction
production lower than in humans.
Our initial attempts to utilize
RRBC ghosts to immunize KO mice also resulted in a primarily IgM
response that failed to produce memory (Supp. Fig 1).
The α-Gal epitope (Galα1-3Gal) is a common type-2 terminus
abundantly synthesized on lipids and proteins of non-primate
mammals and New World monkeys through glycosylation via
It has been established that the α-Gal dissacharide exhibits a
1
3
1,2
high degree of conformational flexibility, but the addition of even
α1,3galactosyltransferase (α1,3GalT). Interestingly, this epitope is
absent in humans and Old World monkeys because the α1,3GalT
gene was inactivated in ancestral Old World primates millions of
14
a single sugar unit severely impacts this flexibility. Inhibition
studies indicate that the preferred target antigen in the anti-Gal
15,16
2
,3,4
response is Galα(1,3)Galβ(1,4)GlcNAc.
However, structural
years ago.
The lack of this epitope results in high levels of
insights suggest that there is no conformational difference between
naturally-occurring anti-Gal antibodies (Abs), which constitute ~1%
1
4
5
Galα(1-3)Galβ(1-4)Glc and Galα(1-3)Galβ(1-4)GlcNAc (Fig. 1), and
of circulating immunoglobulins. The presence of large amounts of
as a result are often used interchangeably when designing a hapten
anti-Gal Abs is a key primary factor in the acute and hyperacute
1
3,14,17,18,19
6
,7,8,9
for a glycoconjugate vaccine.
rejection of xenotransplants.
Typically, in order to recapitulate the physiologic condition of α-
1
0
Gal Abs in an animal model, α1,3GalT knockout (KO) mice are
inoculated with the α-Gal epitope via vaccination with rabbit red
blood cells (RRBCs) or pig kidney membranes (PKM) homogenates
having a high concentration of α-Gal epitopes. However, this
Figure 1. Structures of αGal trisaccharide epitope
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Figure 2. Synthetic strategy of the Galα(1,3)Galβ(1,4)Glc and Galα(1,3)Galβ(1,4)GlcNAc epitope.
Syntheses of the Galα(1,3)Galβ(1,4)Glc epitopes
During the course of our investigations using haptens based on
both the Galα(1-3)Galβ(1-4)GlcNAc and Galα(1-3)Galβ(1-4)Glc The syntheses of the Galα(1,3)Galβ(1,4)Glc epitopes were
epitopes, we discovered exquisite discrimination of the immune performed as shown in Scheme 1. Coupling of lactose peracetate 7
3 2
response between these two. Our findings demonstrated that while with 2-chloroethanol in the presence of BF -Et O to give 8
2
5
cross-reactivity is dominant with xenoreactive materials (RRBCs, proceeded in 52% yield. The reaction of peracetate 7 and 2-(2-
PKM homogenate), the immune response can be tailored to a chloroethoxy)ethanol was, however, inefficient (< 10% yield).
singular carbohydrate epitope if glycoside-pure materials are Therefore, we utilized the chloroacetimidate strategy to append the
evaluated in the vaccination process, and that care must be taken in 2-(2-chloroethoxy)ethyl and 2-[2-(2-chloroethoxy)ethoxy]ethyl
2
6
achieving a relevant approximation of the human condition of the linker moieties. This was accomplished by treatment of 7 with
anti-Gal response.
hydrazine acetate to selectively remove the anomeric acetyl group,
followed by nucleophilic addition of trichloroacetonitrile using DBU
to give the α-isomer of compound 9. The activated
chloroacetimidate 9 readily reacted with 2-(2-chloroethoxy)ethanol
Results
Design of α-Gal epitopes
or 2-[2-(2-chloroethoxy)ethoxy]ethanol in the presence of BF
3 2
-Et O
to give β-isomer of compounds 10 and 11 in 58% and 44% yield,
We designed α-Gal trisaccharide epitopes composed of either respectively. Subsequent azidation and global deacetylation of
Galα(1,3)Galβ(1,4)Glc or Galα(1,3)Galβ(1,4)GlcNAc linked to a compounds 8, 10, and 11 gave the azido-disaccharide
squaric acid ester moiety via 2-aminoethyl, 2-(2-aminoethoxy)ethyl, intermediates, which were then used as substrates for the
and 2-[2-(2-aminoethoxy)ethoxy]ethyl spacers (compounds 1−6, α1,3GalT-mediated incorporation of the terminal galactose.
Fig. 2). We varied the lengths of the spacer in an effort to determine Enzymatic reactions were carried out in 50 mM Tris buffer (pH 7.0)
o
its influence in inducing anti-Gal Abs. The squaric acid ester containing 10 mM MnCl
2
at 37 C (see Experimental Procedure) to
functionality serves as a handle that can be easily conjugated to give the α(1,3)-linked trisaccharides 15−17 in 34−54% yield. A
1
carrier proteins via condensation with lysine residues of the unique H-NMR peak was observed as a doublet at δ5.15−5.18 ppm
2
0,21
protein.
2
We note that synthetic methods for the preparation of (in D O solvent), which corresponds to the proton at the 1-position
2
7
α-Gal trisaccharides have been reported, however, these involved a of an α(1,3)-linked galactose. Reduction of azido compounds
2
2,23
need to control selectivity during the glycosylation step.
We 15−17 to their corresponding primary amines followed by squarate
devised a strategy utilizing a chemoenzymatic reaction mediated by coupling gave the target epitopes 1−3 in 39−66% yield.
α(1,3)galactosyltransferase (α1,3GalT) for incorporating the
terminal galactose to disaccharides, which resulted in the facile
2
4
preparation of our targeted α-Gal epitopes.
2
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Scheme 1. Reagents and conditions: (i) 2-chloroethanol, BF
DBU, Cl CCN, CH Cl
CH Cl , 0°C then rt, overnight, 58% (n=2), 44% (n=3); (iv) NaN , DMF, 80°C, 48 h, 52-84%. (v) (a) MeONa, MeOH, rt, 5 h; (b) 4mM
3
-Et
2
O, CH
2
2
Cl , 0°C then rt, overnight, 52%; (ii) (a)N
2
H
4
-OAc, DMF, rt, 4 h; (b)
3
2
2 3 2
, 0°C then rt, 2h, 58% for 2 steps; (iii) 2-(2-chlroethoxy)ethanol or 2-[2-(2-chloroethoxy)ethoxy]ethanol, BF -Et O,
2
2
3
substrates, 20 μg/mL α1,3GalT, 5 mM UDP-galactose, 10 mM MnCl
C, MeOH, rt, 24 h; (b) Squaric acid ethylester, Et N, 50% aq. EtOH, rt, 24h, 39-54%.
2 2
, 10 mM Tris-HCl (pH 7.0), 37°C for 72-168 h, 34-54%; (vi) (a) H /Pd-
3
Syntheses of the Galα(1,3)Galβ(1,4)GlcNAc epitopes
access α(1,3)-linked trisaccharides is by exploiting two enzymatic
systems [galactose 4-epimerase (GalE) and α1,3GalT] using the
The starting material N-acetyllactosamine 18 was prepared using inexpensive UDP-glucose as the substrate. In this case, UDP-glucose
2
8,29
the method of Wrodnigg et al.
Peracetylation of 18 followed by is first catalytically converted in situ by GalE to UDP-galactose,
reaction with TMSOTf gave the oxazoline intermediate 20, which which is then utilized as the substrate of α1,3GalT (Fig. 3). Such
was then coupled to azido-ethanol, 2-(2-azidoethoxy)ethanol or 2- two-component enzyme system worked to afford compounds 25
[
2-(2-azidoethoxy)ethoxy]ethanol under acidic conditions to yield and 26 in 52 and 57% yield, respectively. Hydrogenation of 24−26 to
3
0
compounds 21−23 (Scheme 2).
Subsequent deacetylation their corresponding primary amines followed by condensation with
followed by α1,3GalT-mediated appendage of galactose gave the squaric acid ethyl ester furnished the target epitopes 4−6.
α(1,3)-linked trisaccharides 24 and 25. An alternative strategy to
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Scheme 2. Reagents and conditions: (i) Ac O, DMAP, pyridine, rt, overnight, 83%; (ii) TMSOTf, ClCH CH Cl, 50°C, 24h, 68%; (iii) 2-
2
2
2
2 2
azidoethanol, 2-(2-azidoethoxy)ethanol or 2-[2-(2-azidoethoxy)ethoxy]ethanol, PPTS, ClCH CH Cl, 70°C, 24h, 40-74%; (iv) (a) MeONa,
MeOH, rt, 5 h; (b) 4 mM substrates, 20 μg/mL α1,3GalT, 5 mM UDP-galactose, MnCl , 10 mM Tris-HCl (pH 7.0), 37°C for 72-168 h, 32-
2
6
2%; (v) (a) MeONa, MeOH, rt, 5 h; (b) 4 mM substrates, 20 μg/mL α1,3GalT, 20 μg/mL GalE, 5 mM UDP-glucose, MnCl
HCl (pH 7.0), 37°C for 72-168 h, 52-57% (vi) (a) H /Pd-C, MeOH, rt, 24 h; (b) Squaric acid ethylester, Et N, 50% aq. EtOH, rt, 24h, 47-74%.
2
, 10 mM Tris-
2
3
Hapten
determinationTargeted α-Gal epitopes 1−6 were conjugated to knockout mice (50 μg α-Gal conjugate/mouse).
either immunogenic carrier protein keyhole limpet hemocyanin schedule of 1-, 2-, and 3-conjugated KLH were 2-week interval
KLH) or tetanus toxoid (TT) via amidation of the squaric acid injections for a total of 3 administrations (see experimental
conjugation,
vaccination
and
α-Gal
titer conjugate vaccines were determined by vaccination of α1,3GalT-
1
0,33
The vaccine
(
2
1
moieties under basic borate buffer conditions. In addition to procedure). Anti-Gal antibody titers of mouse serum samples were
31
alum, preliminary results utilizing a Galα(1-3)Galβ(1-4)GlcNAc determined by ELISA using compound 3-BSA as the coating antigen
glycoconjugate as well as RRBC ghosts indicated that the (Fig. 4). Titers at 42 days were similar over the course of vaccination
formulation benefited from the use of the glycolipid adjuvant C34 schedule in IgG (Midpoint IC ; 1:30,866, 2:30,556, 3:28,753) but
5
0
(
Supp. Fig 1). C34 has been shown to generally induce a class switch
3
2
from IgM to IgG in mice. The efficacies of the synthetic α-Gal
4
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Figure 3. Two enzymatic system of synthesis for Galα(1,3)Galβ(1,4)GlcNAc type epitope
Figure 4. Midpoint anti-Gal antibody titers from compound 1- (n=2), 2- (n=4), and 3-KLH (n=4) vaccinated mice, and compound 4- (n=7),
- (n=7), and 6-TT (n=7) vaccinated mice. (a) Anti-3 IgG titers and (b) anti-3 IgM (37 days) by ELISA using 3-BSA as a coating antigen. (c)
5
Anti-6 IgG titers and (d) anti-6 IgM (51 days) by ELISA using 6-BSA as a coating antigen.
different for IgM (Midpoint IC50; 1:7,000, 2:2,386, 3:6,169). KLH, days were different over the course of vaccination schedule in IgG
while an efficient T-cell epitope, presents difficulty in quantifying (Midpoint IC50; 4:31,203, 5:60,748, 6:43,568) and in IgM (Midpoint
the conjugation efficiency. Thus, we changed to tetanus toxoid (TT) IC ; 4:193, 5:2,084, 6:2,000). The low IgM titers for these mice was
5
0
conjugated to a Galα(1,3)Galβ(1,4)GlcNAc type trisaccharide. In this preferable as it indicated an efficient memory IgG response was
series, 4-, 5-, and 6-TT glycoconjugates were administered at 0, 4, engendered. Furthermore, compound having the 2-(2-
5
and 8 weeks in an effort to increase the class-switching to IgG and aminoethoxy)ethyl linker trisaccharide results in the highest titer
stimulate a memory response (see experimental procedure). values of all six haptens, indicating this linker length is preferable to
Antibody titers of mouse serum samples were determined by ELISA maximize the IgG titres than that of other linker lengths
using compound 6-BSA as the coating antigen (Fig. 4). Titers at 51
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Figure 5. The bar graph for the competitive interaction between anti-5 antibody sera and compound 6-BSA conjugate with compound
(a) and compound 5 (b) as competitors, which are presented at concentrations of 0, 0.6, 6.0, and 60 ꢀM.
2
(
compounds
4
and 6). Sham-vaccinated mice showed no
1
4
Galβ(1-4)GlcNAc portion of the trisaccharide. This has been
attributed to the relative inflexibility of the Galβ(1-4)GlcNAc
glycosidic linkage.
appreciable titer.
α-Gal antibody specificity
We prepared two highly homologous α-Gal epitopes,
Galα(1,3)Galβ(1,4)GlcNAc and Galα(1,3)Galβ(1,4)Glc, both
predicated upon a chemoenzymatic reaction with α1,3GalT.
Using dissacharide substrates functionalized with an ethylene
glycol linker, a two-component enzymatic synthesis provided
rapid access to these two haptens. In addition, this synthetic
Among the three TT-conjugates, 5-TT which contains the 2-(2-
minoethoxy)ethyl linker, elicited Abs with the highest titers as
determined by ELISA. In order to assess the specificity of the α-Gal
antibodies induced by 5-TT vaccination, we performed competitive
2
2
ELISA in the presence of varying concentrations (0, 0.6, 6.0, and 60 scheme was greatly enhanced by utilizing GalE. This allowed
μM)
of
either
the
Galα(1,3)Galβ(1,4)Glu
2
or the preparation of the very expensive but essential
Galα(1,3)Galβ(1,4)GlcNAc
5
epitope. As depicted in Fig. 5, intermediate, UDP-galactose substrate, which was obtained
Galα(1,3)Galβ(1,4)Glc 2 showed no inhibitory effect up to 60 μM from the inexpensive UDP-glucose.
(
Fig. 5a) whereas the Galα(1,3)Galβ(1,4)GlcNAc 5 epitope displayed
a concentration-dependent inhibition (with approximate IC50 of
6.0 μM; Fig. 5b) of the Ab-antigen binding. Moreover, the squaric
With facile routes to Galα(1,3)Galβ(1,4)GlcNAc and
Galα(1,3)Galβ(1,4)Glc epitopes, antibody selectivity could be
evaluated and thus vaccination of these carbohydrate epitopes
~
was initiated. To enhance the vaccination process,
a
acid ethylester attached to the 2-(2-minoethoxy)ethyl spacer (but
devoid of the trisaccharide unit) also showed no inhibition of
antigen binding, indicating that the Abs specifically recognize the α-
Gal epitope but not the linker moiety (data not shown). Our results
exemplify the exquisite specificity of 5-TT-elicited Abs for the
Galα(1,3)Galβ(1,4)GlcNAc-type epitope over Galα(1,3)Galβ(1,4)Glc.
glycosphingolipid bearing an α-galactosyl headgroup and acyl
chain terminated with a phenyl moiety, termed C34, was also
3
2
added to the vaccine cocktail. C34 has been shown to induce
a class switch from IgM to IgG in mice. Utilizing this vaccine,
high levels of anti-Gal IgG were achieved (Fig. 4), but it is clear
that this response has a specificity for the reducing end of the
glycone.
α-Gal antibodies in humans readily cross-react with a
variety of xenoantigens bearing the α-Gal terminus, the lack of
Discussion
The role of carbohydrates as key biological ligands is well antibody cross-reactivity pattern seen between the
established. Yet, in stark comparison to peptides and proteins, Galα(1,3)Galβ(1,4)GlcNAc and Galα(1,3)Galβ(1,4)Glc epitope-
carbohydrates have been notoriously difficult structures to vaccines we have prepared (See Fig. 5) demonstrates an
develop effective vaccines against. This low immunogenicity exquisite discrimination between these two epiopes. This
has been attributed to the flexibility and high solvation of result clearly indicates that the N-acetyl moiety is the key
3
4
glycans. In order to improve the response, it is often element in antigen recognition by these antibodies generated
necessary to engender greater T-cell help via conjugation of by both vaccines. We hypothesize this is due to the
the desired carbohydrate epitope to a T-cell epitope carrier.
3
4
conformational stability imparted by the additional residue at
We employed this strategy in order to achieve high levels of the reducing end of the oligosaccharide. This stability may
IgG titers in mice. Studies often use the Galα(1-3)Galβ(1- provide a higher stochastic exposure of this antigenic
4)GlcNAc and Galα(1-3)Galβ(1-4)Glc epitopes interchangeably, determinant during synapse formation, thus driving specificity
as the natural antibody response is primarily directed against to this end and outcompeting the response to the terminus.
1
3,14
the α-Gal terminus.
However, evidence suggests that part While this subtle change in a single functional group drastically
of the natural Ab response to αGal is directed against the alters antibody recognition, cases of antibody discrimination
6
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between these two functional groups has been documented with analysis on a plate reader (Molecular Devices SpectraMax
3
6
and thus highlight the subtle importance of steric and 250) at 562 nm , and determination of copy number of
electronic effects in determining antibody-carbohydrate protein conjugates was carried out using comparative MALDI-
epitope recognition. Similar discrimination has been reported MS, using sinapinic acid as the matrix on an Applied
3
5
37
in the isolation of anti-Gal monoclonal Abs, but not in a Biosystems VoyagerDE STR MS .
polyclonal response. Our results demonstrate the ability of this
glycoconjugate formulation to discriminate these epitopes Enzyme Expression
almost entirely in the polyclonal response.
The vectors pET15b-GalE and pET15b-
α
1,3galT encoding the
(GalE), and α1,3-
Galactosyltransferase (α1,3GalT), respectively, were
enzymes
Galactose-4-epimerase
a
Conclusion
generous gift from Prof. Peng George Wang. Proteins were
In summary a highly efficient, cost-effective chemoenzymatic
route has been developed to achieve glycoconjugate vaccines
for two common α-Gal haptens. Anti-Gal Abs represent a
significant portion of the natural antibody repertoire in
humans, and are contentious agents in xenotransplantation.
The naturally occurring α-Gal antibody repertoire binds with
several modes to α-Gal termini. However, utilizing two
chemically pure Galα(1,3)Gal epitopes, we were able to
generate a highly honed immune response able to discriminate
the Galα(1,3)Galβ(1,4)GlcNAc and Galα(1,3)Galβ(1,4)Glc
epitopes. The importance of an N-acetyl for a hydroxyl group
substitution between these two epitopes was critical in
polyclonal selectivity seen. The information and materials
garnered from this campaign are expected to facilitate future
vaccine and monoclonal antibody development for both
cancer-related as well as drugs of abuse treatments.
overexpressed in the E. coli expression strain BL21(DE3)
(
Invitrogen) and purified using TALON® metal affinity resin
Clontech). For GalE expression, 50 mL LB/Ampicillin (100
(
ug/mL) culture was inoculated with an overnight culture and
grown at 37°C with shaking (250 rpm) to an OD600 ~ 0.6
followed by the addition of isopropyl-1-thio-β-D-galactoside at
a final concentration of 0.5 mM. Cells were grown for an
additional 3 h at 37°C with shaking and subsequently,
harvested by centrifugation at 5837 rpm (~4000 rcf) and 4°.
The cell pellet yield was ~ 0.22g per 50 mL culture. For
α1,3GalT expression, cell growth was conducted at 20°C and
continued for an additional 18 h after IPTG induction.
Approximately 0.30 g of cell paste was obtained for α1,3GalT.
Cell pellets were stored at -80°C until further processing
Purification of Enzymes
Cell pellets were thawed on ice and then lysed with 5 mL of 0.2
g/L lysozyme (Sigma) in 20 mM Sodium phosphate buffer (pH
Materials and methods
7.0) containing 1% Triton X-100. Cells were disrupted by gentle
General Methods
pipetting followed by incubation at 37°C on rotator for 20 min.
1
13
H and C NMR spectra were obtained using a Bruker 500 or Subsequently, a 5 uL aliquot of a 2,000 unit/mL DNase I stock
00 MHz instrument unless otherwise noted. Chemical shifts (New England Biolab) was added to the solution, followed by
were reported in parts per million (ppm, ) referenced to the further incubation for 20 min at 37°C on rotator. The soluble
residual H resonance of the solvent (CDCl , 7.26 ppm) or fraction of the cell lysate was obtained by centrifugation at
OD, 3.31 ppm). C spectra were referenced to the 13,200 rpm (~16,000 rcf) and 4°C for 20 min and was added to
6
δ
1
3
1
3
(
CD
residual C resonance of the solvent (CDCl
CD OD, 49.0 ppm). Splitting patterns were designated as Sodium phosphate buffer (pH 7.0) containing 0.3 M NaCl. The
3
1
3
3
, 77.0 ppm) or 2 mL of TALON resin (Clonetech) pre-equilibrated with 20 mM
(
3
follows: s, singlet; br, broad; d, doublet; dd, doublet of column was washed with 10 mL of 20 mM Sodium phosphate
doublets; t, triplet; q, quartet; m, multiplet). Electrospray buffer (pH 7.0) containing 0.3 M NaCl and the protein was
Ionization (ESI) mass were obtained on a ThermoFinnigan LTQ eluted using a step gradient with increasing concentrations of
Ion Trap. Analytical thin layer chromatography (TLC) was imidazole (20, 50, 100, and 200 mM) in 20 mM Sodium
performed on Merck precoated analytical plates, 0.25 mm phosphate buffer (pH 7.0) containing 0.3 M NaCl. Protein
thick, silica gel 60 F254. Preparative TLC (PTLC) separations purity in each 5mL fraction was visualized by SDS-PAGE
were performed on Merck analytical plates (0.50 mm thick) (supplementary information 2). Fractions containing pure
precoated with silica gel 60 F254. Flash chromatography enzyme were pooled and concentrated to ~0.3-0.5 mL using an
separations were performed on Aldrich silica gel (60 Å pore Amicon 4 centrifugal filter (MWCO 10-kDa) followed by dialysis
size, 40-63 μm particle size, 230-400 mesh). The LC/MS against PBS. The final enzyme solution was added to equal
analysis was performed using an Agilent G-1956D single volume of glycerol to make a 50% glycerol solution for storage
quadrupole mass spectrometer equipped with an 1100 series at -20°C. Protein concentration was determined by BCA
LC system from Agilent Technologies. HPLC separations were method. Overall protein yield from a 50 mL culture was ~1.5
performed on a Vydac 218TP C18 reversed phase preparative mg for GalE and ~1.4 mg for α1,3GalT.
(
10−15
water. High resolution mass spectra were obtained in the Synthesis of 1-position modified
Scripps Center for Mass Spectrometry. Protein concentration
-D-galactopyranosyl-(1,4)- -D-glucopyranoside)-2-
was determined by BCA assay (Pierce BCA Protein Assay Kit) ethoxycyclobutene-3,4-dione derivatives
ꢀm) HPLC column using a gradient of acetonitrile and
β
-D-galactopyranosyl-(1,3)-
β
β
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
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Organic & Biomolecular Chemistry
Page 8 of 14
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DOI: 10.1039/C7OB00448F
ARTICLE
-Chloroethyl
Journal Name
2
2,3,4,6-tetra-O-acetyl-
β
-D-galactopyranosyl- To a mixture of chloroimidate
8
(0.244 g, 0.312 mmol) and 2-
(
1,4)-2,3,6-tri-O-acetyl-
β
-D-glucopyranoside (8)
(5.0 g, 7.36 mmol) and 2-
Cl (50 ml) was
(2-chlroethoxy)ethanol (0.037 ml, 0.343 mmol) in CH
2 2
Cl (50
ml) was added by dropwise addition of BF -Et O (1.20 ml, 9.58
3
2
To a mixture of Lactose peracetate
chlroethanol (0.494 ml, 7.36 mmol) in CH
added by dropwise addition of BF -Et O (1.20 ml, 9.58 mmol)
at 0 C over a time course of 20 min. The mixture was diluted
with CH Cl and washed with ice H O and saturated aqueous
NaHCO (two times), dried over Na SO and then
concentrated under reduced pressure. The residue was
purified by silica gel column chromatography
Pentane:AcOEt=1:1) to afford (2.69 g, 52%) as a yellow oil.
H NMR (500 MHz, Chloroform-d) δ 5.33 (dd, J = 3.5, 1.2 Hz,
7
o
mmol) at -20 C over a time course of 30 min and stirred at
room temperature for 3 hrs. The mixture was neutralized with
2
2
3
2
o
Et
residue was purified by preparative Thin layer chromatography
CH Cl :AcOEt=1:1) to afford 10 (0.134 g, 58%) as a yellow oil.
H NMR (500 MHz, Chloroform-d) δ5.34 (dd, J = 3.5, 1.2 Hz, 1H,
3
N (0.056 ml, 0.4 mmol) and concentrated in vacuo. The
2
2
2
(
1
2
2
3
2
4
,
H-4’), 5.19 (t, J = 9.3 Hz, 1H, H-3), 5.10 (dd, J = 10.4, 7.9 Hz, 1H,
H-2’), 4.95 (dd, J = 10.4, 3.5 Hz, 1H, H-3’), 4.89 (dd, J = 9.5, 7.9
Hz, 1H, H-2), 4.56 (d, J = 7.8 Hz, 1H, H-1’), 4.52 – 4.46 (m, 2H,
H-1, H-6a), 4.17 – 4.04 (m, 3H, H-6b, H-6a’, H-6b’), 3.91 (ddd, J
(
1
8
1
1
7
2
3
–
1
H, H-4’), 5.19 (t, J = 9.3 Hz, 1H, H-3), 5.09 (dd, J = 10.4, 7.9 Hz,
H, H-2’), 4.94 (dd, J = 10.4, 3.4 Hz, 1H, H-3’), 4.90 (dd, J = 9.5,
.9 Hz, 1H, H-2), 4.53 (d, J = 7.9 Hz, 1H, H-1’), 4.51 – 4.45 (m,
H, H-1, H-6a), 4.21 – 3.97 (m, 4H, H-6b, H-6a’, H-6b’, OCHH),
.88 – 3.83 (m, 1H, H-5’), 3.82 – 3.68 (m, 2H, H-4, OCHH), 3.67
=
1
2
11.0, 4.8, 3.6 Hz, 1H, OCHH), 3.86 (ddd, J = 7.4, 6.3, 1.2 Hz,
H, H-5’), 3.79-3.56 (m, 8H, OCHH, OCH , CH O, CH Cl, H-5),
.14, 2.11, 2.07-2.00, 1.96 (7 x s, 21H, COCH ). C NMR (126
) δ 170.49, 170.48, 170.27, 170.19, 169.89, 169.80,
69.20 (7 x COCH ), 101.20, 100.71, 76.37, 72.94, 72.81, 71.80,
71.55, 71.13, 70.83, 70.46, 69.26, 69.19, 66.75, 62.10, 60.93,
2.9, 21.01, 20.95, 20.87, 20.77, 20.64. MS (ESI), m/z 765.1978
2
2
2
13
3
MHz, CDCl
3
1
3
3.52 (m, 3H, H-5, CH
2
Cl), 2.14, 2.11, 2.05, 2.04, 2.03, 2.03,
1
). C NMR (500 MHz, Chloroform-d)
3
.95 (7 x s, 21H, 7 x COCH
3
4
δ 170.70, 170.49, 170.41, 170.08, 170.07, 169.42, 101.44,
+
(M + Na) (C30
f
H43ClO19 requires 765.1979). TLC; R 0.71
1
01.24, 76.54, 73.14, 72.97, 71.82, 71.35, 71.09, 70.31, 69.50,
(
CH Cl :AcOEt=1:1).
2
2
6
6.99, 62.24, 61.19, 42.79, 21.22, 21.17, 21.08, 21.01, 20.88.
+
MS (ESI), m/z 699.1900 (M + H) (C28
H39ClO18 requires
2
-[2-(2-Chloroethoxy)ethoxy]ethyl 2,3,4,6-tetra-O-acetyl-
galactopyranosyl-(1,4)-2,3,6-tri-O-acetyl- -D-glucopyranoside
11)
β-D-
6
f
99.1898). TLC; R 0.29 (Pentane:AcOEt=1:1).
β
(
2
,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl-(1,4)-2,3,6-tri-O-
acetyl-
α
-D-glucopyranose trichloracetimidate (9)
Chloroimidate 9 (0.276 g, 0.353 mmol) and 2-[2-(2-
chloroethoxy)ethoxy]ethanol (0.041 ml, 0.282 mmol) were
allowed to react under the same condition as described for the
preparation of 10 to give 11 (0.096 g, 44%) as a colorless oil.
To a mixture of Lactose peracetate 7 (0.5 g, 0.74 mmol) in DMF
(5 ml) was added hydrazine acetate (0.73 g, 0.81 mmol). Upon
completion of anomeric deacetylation by TLC, 1 ml of water
was added to the mixture and concentrated in vacuo. The
residue was diluted with AcOEt (20 ml) and washed with 1%
1
H NMR (400 MHz, Chloroform-d) δ 5.30 (dd, J = 3.4, 1.1 Hz,
1
1
7
2
3
2
H, H-4’), 5.15 (t, J = 9.3 Hz, 1H, H-3), 5.06 (dd, J = 10.4, 7.9 Hz,
H, H-2’), 4.91 (dd, J = 10.4, 3.4 Hz, 1H, H-3’), 4.85 (dd, J = 9.6,
.9 Hz, 1H, H-2), 4.53 (d, J = 7.9 Hz, 1H, H-1’), 4.49 – 4.37 (m,
H, H-1, H-6a), 4.13 – 4.00 (m, 3H, H-6b, H-6a’, H-6b’), 3.92 –
HCl (20 ml), saturated NaHCO
3
(20 ml), and Brine (20 ml), and
SO . The organic phase
the organic phase was dried over Na
2
4
was filtered and concentrated in vacuo to give the crude
glycosyl hemiacetal. The dried crude glycosyl hemiacetal
.52 (m, 15H, H-4, H-5’, 3 x OCH
.08, 2.02, 2.00, 1.92 (7 x s, 21H, COCH
) δ 170.39, 170.37, 170.17, 170.08, 169.79, 169.68,
169.09, 101.11, 100.65, 76.31, 72.85, 72.68, 71.70, 71.41,
2
, 2 x CH
2
O, CH
2
Cl, H-5), 2.11,
13
3
). C NMR (101 MHz,
compound in CH
2 2
Cl (10 ml) was treated with DBU (0.044 ml,
CDCl
3
0
.30 mmol) and to the solution was added trichloroacetonitrile
o
(
0.22 ml, 2.2 mmol) at 0 C and stirred at room temperature for
hrs. The mixture was concentrated and purified by silica gel
column chromatography (Hexane:AcOEt=1:1) to give
compound as yellow oil (0.337 g, 58%).
The spectrum for compound was the same as reported
isomer . H NMR (500 MHz, Chloroform-
7
4
1.03, 70.71, 70.70, 70.42, 69.15, 69.10, 66.67, 62.06, 60.86,
3
2.84, 20.92, 20.86, 20.77, 20.69, 20.68, 20.67, 20.55. MS (ESI),
+
m/z 787.2419 (M + H) (C32
H47ClO20 requires 787.2422). TLC; R
f
9
0
.55 (CH
2
Cl
2
:AcOEt=1:1).
9
2
4 1
previously for the
α
2
-Azidoethyl
2,3,4,6-tetra-O-acetyl-
β
-D-galactopyranosyl-
d) δ8.65 (s, 1H, NH), 6.47 (d, J = 3.8 Hz, 1H, H-1), 5.54 (t, J = 9.7
Hz, 1H, H-3), 5.34 (d, J = 3.4 Hz, 1H, H-4’), 5.19 – 4.99 (m, 2H,
(
1,4)-2,3,6-tri-O-acetyl-
β
-D-glucopyranoside (12)
H-2, H-2’), 4.94 (dd, J = 10.4, 3.4 Hz, 2H, H-3’), 4.61 – 4.36 (m, To a solution of
8
(0.0841 g, 0.12 mmol) in DMF (4 ml) was
2
3
1
H, H-6a, H-1’), 4.20 – 4.01 (m, 4H, H-5, H-6b, H-6a’, H-6b’), added sodium azide (78.2 mg, 1.20 mmol). The mixture was
o
.97 – 3.75 (m, 2H, H-4, H-5’), 2.14, 2.09, 2.08 – 2.01, 1.99, stirred at 60 C for 48 h. The solvent was removed under
.95 (s, 21H, 7 x COCH
3
). TLC; R
f
0.34 (Pentane:AcOEt=1:1).
reduced pressure, and the residue was diluted with AcOEt and
washed with H O, dried over Na SO , and then concentrated
2,3,4,6-tetra-O-acetyl-β-D- under reduced pressure. The residue was purified by
2
2
4
2-(2-Chloroethoxy)ethyl
galactopyranosyl-(1,4)-2,3,6-tri-O-acetyl-
β
-D-glucopyranoside preparative thin layer chromatography (Pentane:AcOEt=1:1) to
(10)
afford 12 (0.0652 g, 77%) as a colorless oil.
1
H NMR (500 MHz, Chloroform-d) δ 5.34 (dd, J = 3.4, 1.2 Hz,
1
H, H-4’), 5.19 (t, J = 9.2 Hz, 1H, H-3), 5.10 (dd, J = 10.4, 7.9 Hz,
8
| J. Name., 2012, 00, 1-3
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Page 9 of 14
Journal Name
Orga Pn l iec a s&e Bd oi o nmo to al ed cj uu sl ta mr Ca rhg ei nms istry
View Article Online
DOI: 10.1039/C7OB00448F
ARTICLE
+
H, H-2’), 4.97 – 4.88 (m, 2H, H-3’, H-2), 4.55 (d, J = 7.9 Hz, 1H, MS (ESI), m/z 794.2828 (M + H) (C32
1
47 3
H N O20 requires
H-1’), 4.52 (dd, J = 12.0, 2.2 Hz, 1H, H-6a), 4.49 (d, J = 7.8 Hz, 794.2826). TLC; R 0.51 (CH Cl :AcOEt=1:1).
2
f
2
1H, H-1), 4.17 – 4.03 (m, 3H, H-6b, H-6a’, H-6b’), 3.98 (ddd, J =
10.6, 5.0, 3.4 Hz, 1H, OCHH), 3.86 (ddd, J = 7.5, 6.3, 1.3 Hz, 1H, 2-Azidoethyl
α
-D-galactopyranosyl-(1,3)-
β-D-
H-5’), 3.84 – 3.77 (m, 1H, H-4), 3.67 (ddd, J = 10.6, 8.2, 3.3 Hz, galactopyranosyl-(1,4)-β-D-glucopyranoside (15)
1
=
1
H, OCHH), 3.61 (ddd, J = 9.9, 5.0, 2.2 Hz, 1H, H-5), 3.46 (ddd, J
13.3, 8.2, 3.4 Hz, 1H, CHHN ), 3.26 (ddd, J = 13.4, 5.0, 3.3 Hz,
H, CHH N ), 2.14, 2.11, 2.05, 2.04, 1.95 (s, 21H, 7 x COCH ).
C NMR (126 MHz, CDCl ) δ170.45, 170.25, 170.17, 169.86,
To a solution of 12 (0.044 g, 0.0569 mmol) in dry MeOH (10
ml) was added 0.5 M NaOMe (0.224 ml, 0.112 mmol). The
mixture was stirred at room temperature for 5 h, and then
3
3
3
1
3
3
3
neutralized with CH COOH (0.0064 ml, 0.112 mmol). The
1
7
2
69.79, 169.18, 101.22, 100.58, 76.28, 72.95, 72.87, 71.63,
mixture was concentrated in vacuo, and the residue was
partially purified by silica gel column chromatography
1.10, 70.83, 69.26, 68.77, 66.74, 61.92, 60.92, 50.65, 20.97,
+
0.92, 20.84, 20.76, 20.63. MS (ESI), m/z 728.2121 (M + H)
2 2
(CH Cl :MeOH=3:1 to 1:1) to afford deacetylate crude
(C
28
H
39
N
3
O
18
requires
728.2102).
TLC;
R
f
0.34
compound, and an aliquot of this compound was used for
enzyme reaction without further purification. To a solution of
(Pentane:AcOEt=1:1).
5
mMUDP-Galactose and 4mM deacylated compound (0.0123
g, 0.03 mmol) in 50 mM Tris-HCl (pH 7.0) supplemented with
0 mM MnCl was added 1,3 GalT (20 μg/mL) to a final
volume of 7.5 ml. The solution was incubated at 37°C with
2
-(2-Azidoethoxy)ethyl
galactopyranosyl-(1,4)-2,3,6-tri-O-acetyl-
13)
2,3,4,6-tetra-O-acetyl-
β-D-
β
-D-glucopyranoside
1
2
α
(
Compound 10 (0.061 g, 0.0775 mmol) was allowed to react agitation and reaction progress was monitored by TLC. When
under the same condition as described for the preparation of the deacetylated compound was consumed, α1,3GalT was
1
2
to give 13 (0.030 g, 52%) as a colorless oil.
H NMR (500 MHz, Chloroform-d) δ 5.33 (dd, J = 3.5, 1.2 Hz, (MWCO 10-kDa) by centrifugation at 7,830 rpm and room
H, H-4’), 5.19 (t, J = 9.3 Hz, 1H, H-3), 5.09 (dd, J = 10.4, 7.9 Hz, temperature for 15 min. Filtrates were concentrated in vacuo,
H, H-2’), 4.94 (dd, J = 10.4, 3.4 Hz, 1H, H-3’), 4.89 (dd, J = 9.5, and the residue was dissolved in H O and then passed through
removed from reaction mixture using an Amicon 4 filter
1
1
1
7
2
2
-
.9 Hz, 1H, H-2), 4.56 (d, J = 7.9 Hz, 1H, H-1’), 4.52 – 4.45 (m, a chloride anion-exchange column [Dowex 1 (Cl )]. The eluate
H, H-1, H-6a), 4.15 – 4.03 (m, 3H, H-6b, H-6a’, H-6b’), 3.91 (dt, was concentrated and the residue was purified on a VYDAC C18
J = 11.1, 4.4 Hz, 1H, OCHH), 3.86 (ddd, J = 7.5, 6.3, 1.2 Hz, 1H, reversed phase semipreparative (250 mm × 22 mm, 10
H-5’), 3.79 (t, J = 9.4 Hz, 1H, H-4), 3.72 (ddd, J = 10.9, 6.6, 4.1 HPLC column using H O and 0.1% TFA in CH CN mobile phases.
Hz, 1H, OCHH), 3.66 – 3.56 (m, 5H, OCH , CH O, H-5), 3.38 – A linear gradient was employed (95:5 H O/0.1%TFA in CH CN
), 2.14, 2.11, 2.05, 2.03, 1.95 (s, 21H, 5:95 H O/0.1%TFA in CH CN) over a period of 40 min at a
). C NMR (101 MHz, CDCl ) δ 170.49, 170.47, 170.27, flow rate of 10 mL/min. Fractions containing the desired
−15 ꢀm)
2
3
2
2
2
3
3
.32 (m, 2H, CH
2
N
3
→
2
3
1
3
COCH
3
3
1
7
6
2
7
70.19, 169.88, 169.81, 169.19, 101.18, 100.74, 76.37, 72.91, product were collected, frozen, and lyophilized to give
2.77, 71.81, 71.12, 70.80, 70.49, 70.31, 69.23, 69.20, 66.73, compound 15 (5.9 mg, 34%) as a white solid.
1
2.09, 60.92, 50.88, 21.00, 20.95, 20.84, 20.78, 20.77, 20.76,
+
H NMR (500 MHz, Deuterium Oxide) δ 5.14 (d, J = 3.7 Hz, 1H,
0.65. MS (ESI), m/z 750.2563 (M + H) (C30
50.2562). TLC; R 0.19 (Pentane:AcOEt=1:1).
H
43
N
3
O
19 requires H-1’’), 4.53 (t, J = 7.4 Hz, 2H, H-1’, H-1), 4.19 (d, J = 6.0 Hz, 2H,
f
H-4’, H-5’’), 4.08 – 3.91 (m, 3H, H-6a, H-4’’, H-3’’), 3.88 – 3.51
1
m, 16H), 3.40 – 3.30 (m, 1H, H-2). C NMR (500 MHz,
3
(
2-[2-(2-azidoethoxy)ethoxy]ethyl 2,3,4,6-tetra-O-acetyl-β-D- Deuterium Oxide) δ 102.33, 101.66, 94.92, 78.05, 76.68, 74.54,
galactopyranosyl-(1,4)-2,3,6-tri-O-acetyl-
β
-D-glucopyranoside 74.27, 73.89, 72.23, 70.32, 69.06, 68.77, 68.61, 68.03, 67.69,
(
14)
64.29, 60.48, 60.41 59.61, 50.02. MS (ESI), m/z 574.2090 (M +
+
H) (C20
H
35
N
3
O16 requires 574.2090). TLC;
R
f
0.56 (2-
Compound 11 (0.3 g, 0.381 mmol) was allowed to react under
the same condition as described for the preparation of 13 to
give 14 (0.241 g, 84%) as a colorless oil.
propanol:AcOEt:H
2
O=2:2:1).
1
2-(2-Azidoethoxy)ethyl
galactopyranosyl-(1,4)-
α
-D-glucopyranoside (16)
-D-galactopyranosyl-(1,3)-
β-D-
H NMR (400 MHz, Chloroform-d) δ 5.34 (dd, J = 3.5, 1.2 Hz,
β
1
1
7
2
3
3
H, H-4’), 5.19 (t, J = 9.3 Hz, 1H, H-3), 5.10 (dd, J = 10.4, 7.9 Hz,
H, H-2’), 4.94 (dd, J = 10.4, 3.4 Hz, 1H, H-3’), 4.89 (dd, J = 9.6, Compound 13 (0.030 g, 0.0400 mmol) was allowed to react
.9 Hz, 1H, H-2), 4.56 (d, J = 7.9 Hz, 1H, H-1’), 4.52 – 4.41 (m, under the same condition as described for the preparation of
H, H-1, H-6a), 4.18 – 4.02 (m, 3H, H-6b, H-6a’, H-6b), 3.95 – deacetylated compound from compound 12 and the aliquot
.84 (m, 2H, OCHH, H-5’), 3.78 (t, J = 9.5 Hz, 1H, H-4), 3.75 – (0.0137 g, 0.03 mmol) of this compound was used for enzyme
.54 (m, 10H, H-5, OCHH, 2 x OCH
2 2
, 2 x CH O), 3.39 (t, J = 5.0 reaction to give compound 16 (6.7 mg, 36%) as a white solid.
1
Hz, 2H, CH
2
N
3
), 2.14, 2.11, 2.05, 2.05 – 2.00 , 1.96 (s, 21H,
H NMR (500 MHz, Deuterium Oxide) δ 5.16 (d, J = 3.9 Hz, 1H,
1
) . C NMR (126 MHz, CDCl
3
COCH
3
3
) δ 170.72, 170.69, 170.49, H-1’’), 4.54 (dd, J = 7.9, 5.1 Hz, 2H, H-1’, H-1), 4.27 – 4.16 (m,
1
7
6
70.40, 170.12, 170.00, 169.42, 101.45, 100.99, 76.65, 73.19, 2H, H-4’, H-5’’), 4.15 – 3.92 (m, 4H), 3.92 – 3.57 (m, 17H), 3.57
1
3
3.00, 72.04, 71.36, 71.08, 71.06, 70.76, 70.42, 69.49, 69.41, – 3.48 (m, 2H, CH
2 3
N ), 3.41 – 3.31 (m, 1H, H-2). C NMR (151
6.98, 62.39, 61.17, 51.05, 21.23, 21.18, 21.07, 21.00, 20.87. MHz, D O) δ 102.36, 101.61, 94.93, 78.10, 76.69, 74.55, 74.23,
2
7
3.88, 72.28, 70.33, 69.17, 69.08, 68.79, 68.66, 68.62, 68.23,
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 9
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Organic & Biomolecular Chemistry
Page 10 of 14
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DOI: 10.1039/C7OB00448F
ARTICLE
Journal Name
6
6
0
7.70, 64.30, 60.49, 60.42, 59.64, 49.67. MS (ESI), m/z 1-[2-(2-aminoethoxy)]ethyl
α
-D-galactopyranosyl-(1,3)-
-D-glucopyranoside)-2-
ethoxycyclobutene-3,4-dione (2)
β-D-
+
18.2350 (M + H) (C22
H
39
N
3
O17 requires 618.2352). TLC; R
f
galactopyranosyl-(1,4)-β
.50 (2-propanol:AcOEt:H
2
O=2:2:1).
Compound 16 (0.0067 g, 0.0108 mmol) was allowed to react
under the same condition as described for the preparation of
to give (0.0051 g, 66% from compound 16) as a colorless oil.
H NMR (600 MHz, Deuterium Oxide) δ 5.16 (d, J = 3.7 Hz, 1H,
under the same condition as described for the preparation of H-1’’), 4.77 – 4.70 (m, 2H, OCH CH ), 4.52 (td, J = 9.1, 8.6, 4.1
deacetylated compound from compound 12 and the aliquot Hz, 2H, H-1’, H-1), 4.23 – 4.15 (m, 2H, H-4’, H-5’’), 4.11 – 3.52
2
-[2-(2-azidoethoxy)ethoxy]ethyl
α
-D-galactopyranosyl-(1,3)-
1
β
-D-galactopyranosyl-(1,4)-
β
-D-glucopyranoside (17)
2
1
Compound 14 (0.059 g, 0.0743 mmol) was allowed to react
2
3
(
0.015 g, 0.03 mmol) of this compound was used for enzyme (m, 23H), 3.34 (d, J = 8.3 Hz, 1H, H-2), 1.58 – 1.37 (m, 3H,
3
1
reaction to give compound 17 (14.3 mg, 68%) as a white solid.
1
H NMR (500 MHz, Deuterium Oxide) δ H NMR (500 MHz, 176.91, 176.63, 173.27, 102.38, 101.65, 94.93, 78.16, 76.69,
Deuterium Oxide) δ 5.17 (d, J = 3.9 Hz, 1H, H-1’’), 4.55 (dd, J = 74.87, 74.56, 74.29, 73.96, 72.29, 72.03, 70.46, 70.34, 70.17,
2 3 2
OCH CH ). C NMR (151 MHz, D O) δ 188.43, 182.94, 182.85,
1
7.9, 2.7 Hz, 2H, H-1’, H-1), 4.29 – 4.15 (m, 2H, H-4’, H-5’’), 69.54, 69.24, 69.09, 68.96, 68.80, 68.63, 68.31, 68.24, 67.72,
4
.12– 3.93 (m, 4H), 3.93 – 3.58 (m, 21H), 3.56 – 3.49 (m, 2H, 64.31, 60.50, 60.42, 59.64, 43.47, 42.72, 14.51. MS (ESI), m/z
1
3
+
), 3.37 (td, J = 7.7, 2.4 Hz, 1H, H-2). C NMR (126 MHz, 716.2609 (M + H) (C28
CH
2
N
3
f
H45NO20 requires 716.2608). TLC; R 0.26
D
2
O) δ 103.25, 102.49, 95.82, 79.01, 77.60, 75.44, 75.14, (2-propanol:AcOEt:H
4.77, 73.19, 71.23, 70.08, 69.98, 69.89, 69.68, 69.59, 69.52,
9.14, 68.60, 65.21, 61.37, 61.31, 60.55, 50.53. MS (ESI), m/z 1-[2-{2-(2-aminoethoxy)]ethoxy]ethyl
2
O=2:2:1).
7
6
6
0
α
-D-galactopyranosyl-
+
62.2613 (M + H) (C24
H
43
N
3
O18 requires 662.2614). TLC; R
f
(1,3)-β-D-galactopyranosyl-(1,4)-β-D-glucopyranoside)-2-
ethoxycyclobutene-3,4-dione (3)
.47 (2-propanol:AcOEt:H
2
O=2:2:1).
Compound 17 (0.0089 g, 0.014 mmol) was allowed to react
under the same condition as described for the preparation of
to give (0.0058 g, 54% from compound 17) as a colorless oil.
H NMR (600 MHz, Deuterium Oxide) δ 5.15 (dd, J = 3.8, 1.7
To a solution of 15 (0.0074 g, 0.0129 mmol) in MeOH (5 ml) Hz, 1H, H-1’’), 4.78 – 4.68 (m, 2H, OCH CH ), 4.57 – 4.47 (m,
was added 5% Pd-C (5.0 mg). The mixture was stirred under 2H, H-1’, H-1), 4.26 – 4.14 (m, 2H, H-4’, H-5’’), 4.10 – 3.53 (m,
hydrogen atmosphere at room temperature for 24 h. The 27H), 3.42 – 3.28 (m, 1H, H-2), 1.54 – 1.36 (m, 3H, OCH CH ).
O) δ 191.76, 188.40, 182.90, 176.85,
1
-(2-aminoethyl)
α
-D-galactopyranosyl-(1,3)-
-D-glucopyranoside)-2-
ethoxycyclobutene-3,4-dione (1)
β
-D-
1
galactopyranosyl-(1,4)-
β
3
1
2
3
2
3
1
3
solution was filtered through a pad of celite, and then
C NMR (151 MHz, D
2
concentrated under reduced pressure to afford azide reduced 176.61, 173.30, 102.36, 101.59, 94.92, 78.12, 76.70, 74.56,
crude compound, and this compound was used for next 74.26, 73.90, 72.29, 70.34, 70.16, 69.23, 69.18, 69.09, 68.97,
reaction for further purification. To a solution of amine 68.90, 68.79, 68.63, 68.27, 67.71, 64.31, 60.49, 60.42, 59.65,
+
compound in 50% aqueous EtOH (2ml) was added Diethyl 43.47, 43.35, 14.52. MS (ESI), m/z 760.2867 (M + H)
squarate (0.0094 ml, 0.0636 mmol) and Et
.0518 mmol), and stirred at room temperature for 24 hrs. The propanol:AcOEt:H
elute was concentrated and the residue was purified on a
VYDAC C18 reversed phase semipreparative (250 mm × 22 mm, Synthesis of 1-position modified
15 m) HPLC column using water and CH CN mobile -D-galactopyranosyl-(1,4)-O-2-acetamido-2-deoxy-
phases. A linear gradient was employed (95:5 H O/CH CN glucopyranoside derivatives.
:95 H O/CH CN) over a period of 40 min at a flow rate of 10
3
N (0.0072 ml, (C30
H
49NO21 requires 760.2870). TLC;
R
f
0.33 (2-
0
2
O=2:2:1).
α
-D-galactopyranosyl-(1,3)-
-D-
10
−
ꢀ
3
β
β
2
3
→
5
2
3
2
-Acetoamido-1,3,6-tri-O-acetyl-2-deoxy-4-O-(2,3,4,6-tetra-O-
mL/min. Fractions containing the desired product were
acetyl- -D-galactopyranosyl)-D-glucopyranose (19)
β
collected, frozen, and lyophilized to give compound
1 (0.0034
mg, 34% from compound 15) as a colorless oil.
1
To a solution of N-acetyllactosamine 18 (0.5 g, 1.3 mmol) in
H NMR (600 MHz, Deuterium Oxide) δ 5.15 (t, J = 3.0 Hz, 1H, pyridine (5 ml) was added acetic anhydride (2.84 ml, 30 mmol)
CH ), 4.52 (ddt, J = 6.6, 4.9, 1.8 Hz, and 4-dimethylaminopyridine (0.061 g, 1.3 mmol), and the
H, H-1’, H-1), 4.20 (q, J = 5.0, 3.2 Hz, 2H, H-4’, H-5’’), 4.08 – mixture was stirred at room temperature for overnight. The
.55 (m, 19H), 3.33 (s, 1H, H-2), 1.55 – 1.34 (m, 3H, CH CH ). mixture was concentrated in vacuo and the resulting
H-1’’), 4.75 (ddd, 2H, OCH
2
3
2
3
2
3
1
3
C NMR (600 MHz, Deuterium Oxide) δ 191.76, 190.35, residuepurified by silica gel column chromatography
88.52, 188.46, 183.00, 182.92, 176.96, 176.76, 173.28, (Toluene:AcOEt=1:9) to give compound 5 as yellow oil (0.73 g,
02.36, 101.70, 94.93, 78.11, 76.70, 74.87, 74.57, 74.30, 74.27, 83%). The spectrum for compound 19 was the same as
1
1
7
6
5
6
3
8
3.96, 73.91, 72.31, 72.03, 70.46, 70.35, 70.15, 70.11, 69.24, reported previously for the
α
isomer.
H NMR (600 MHz, Chloroform-d) δ 6.09 (d, J = 3.6 Hz, 1H, H-
9.65, 43.71, 43.64, 14.57, 14.53, 14.48. MS (ESI), m/z 1), 5.74 (d, J = 9.2 Hz, 1H, NH), 5.36 (dd, J = 3.5, 1.2 Hz, 1H, H-
1
9.09, 68.90, 68.79, 68.63, 68.38, 67.71, 64.31, 60.50, 60.43,
+
72.2343 (M + H) (C26
H41NO19 requires 672.2345). TLC; R
f
0.13 4’), 5.23 (dd, J = 11.1, 8.4 Hz, 1H, H-3), 5.11 (dd, J = 10.5, 7.9
Hz, 1H, H-2’), 4.97 (dd, J = 10.4, 3.4 Hz, 1H, H-3’), 4.53 (d, J =
(
2-propanol:AcOEt:H
2
O=2:2:1).
7.9 Hz, 1H, H-1’), 4.44 – 4.32 (m, 2H, H-2, H-6a), 4.19 – 4.05 (m,
1
0 | J. Name., 2012, 00, 1-3
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Journal Name
Orga Pn l iec a s&e Bd oi o nmo to al ed cj uu sl ta mr Ca rhg ei nms istry
View Article Online
DOI: 10.1039/C7OB00448F
ARTICLE
3
H’, H-6b, H-6a’, H-6b’), 3.97 – 3.81 (m, 3H, H-5, H-4, H-5), Oxazoline 20 (0.12 g, 0.194 mmol) and 2-(2-
.18, 2.15, 2.11, 2.09, 2.06, 1.96, 1.93 (7 x s, 21H, 7 x COCH ). azidoethoxy)ethanol (0.254 ml, 1.94 mmol) were allowed to
2
3
+
MS (ESI), m/z 678.2240 (M + H) (C28
H39NO18 requires react under the same condition as described for the
678.224). TLC; R
f
0.20 (AcOEt:Toluene=6:1).
preparation of 21 to give 22 (0.108 g, 74%) as a white solid.
1
H NMR (600 MHz, Chloroform-d) δ 5.99 (d, J = 9.5 Hz, 1H, NH),
2
-Methyl-{3,6-di-O-acetyl-1,2-dideoxy-4-O-(2,3,4,6-tetra-O-
5.35 (dd, J = 3.4, 1.2 Hz, 1H, H-4’), 5.08 (ddd, J = 26.6, 10.1, 8.1
Hz, 2H, H-2’, H-3), 4.96 (dd, J = 10.5, 3.4 Hz, 1H, H-3’), 4.58 (d, J
= 7.9 Hz, 1H, H-1’), 4.52 – 4.40 (m, 2H, H-1, H-6a), 4.21 – 3.99
acetyl-β
oxazoline (20)
-D-galactopyranosyl)-D-glucopyrano}-[2,1-d]-
(
m, 3H, H-6b, H-6a’, H-6b’), 3.95 – 3.83 (m, 2H, H-2, OCHH),
.83 –3.63 (m, 8H, H-5’, H-4, H-5, OCHH, 2 x OCH ), 3.52 – 3.23
(m, 2H, CH ), 2.14, 2.11, 2.07, 2.05, 1.98 – 1.93 (7 x s, 21H, 7
). C NMR (151 MHz, CDCl ) δ 170.62, 170.50, 170.42,
70.27, 170.20, 170.11, 169.37, 101.38, 101.08, 75.95, 72.76,
To a solution of peracetate 19 (0.49 g, 0.723 mmol) in 1,2-
3
2
dichlroethane was added TMSOTf (0.161 ml, 0.889 mmol), and
o
the resulting mixture was stirred at 50 C for 5hrs. To the
2 3
N
13
x COCH
3
3
mixture was added Et
3
N (0.145 ml, 1.04 mmol) and
1
7
5
concentrated in vacuo. The residue was purified by silica gel
2.75, 70.98, 70.76, 70.06, 69.17, 68.66, 66.70, 62.40, 60.87,
3
column chromatography (Toluene:AcOEt:Et N=100:200:1) to
3.36, 51.02, 23.27, 20.96, 20.93, 20.72, 20.59. MS (ESI), m/z
+
afford 20 (0.304 g, 68%) as a yellow oil.
1
H NMR (600 MHz, Chloroform-d) δ 5.88 (d, J = 7.3 Hz, 1H, H-
749.2726 (M + H) (C30
H
4
N
4
O18 requires 749.2723). TLC; R
f
0.40
(n-hexanes:acetone=1:1).
1
), 5.61 (dd, J = 2.6, 1.1 Hz, 1H, H-3), 5.34 (dd, J = 3.6, 1.2 Hz,
H, H-4’), 5.14 (dd, J = 10.4, 8.0 Hz, 1H, H-2’), 4.97 (dd, J = 10.4,
.5 Hz, 1H, H-3’), 4.62 (d, J = 8.0 Hz, 1H, H-1’), 4.18 (dd, J =
2.0, 2.4 Hz, 1H, H-6a), 4.15 – 4.06 (m, 3H, H-2, H-6a’, H-6b’),
.04 (dd, J = 12.0, 5.9 Hz, 1H, H-6b), 3.92 (ddd, J = 7.6, 6.4, 1.2
1
3
1
4
2-[2-(2-Azidoethoxy)ethoxy]ethyl (2,3,4,6-tetra-O-acetyl-β-D-
galactopyranosyl)-(1,4)-O-2-acetamido-3,6-di-O-acetyl-2-
deoxy-β-D-glucopyranoside (23)
Hz, 1H, H-5’), 3.63 (dt, J = 9.5, 1.4 Hz, 1H, H-5), 3.46 (ddd, J = Oxazoline 20 (0.079 g, 0.128 mmol) and 2-[2-(2-
9
.5, 5.7, 2.3 Hz, 1H, H-4), 2.13, 2.08, 2.07, 2.02, 2.01, 2.00, azidoethoxy)ethoxy]ethanol (0.224 mg, 1.28 mmol) were
+
1
.94 (7 x s, 21H, 7 x COCH
requires
:MeOH=20:1).
3
). MS (ESI), m/z 618.2031 (M + H)
allowed to react under the same condition as described for the
(C
26
H
35NO16
618.2029).
TLC;
R
f
0.32 preparation of 21 to give 23 (0.040 g, 40%) as a colorless oil.
1
H NMR (600 MHz, Chloroform-d) δ 6.28 (d, J = 9.6 Hz, 1H, NH),
(CHCl
3
5.33 (dd, J = 3.5, 1.2 Hz, 1H, H-4’), 5.09 (dd, J = 10.5, 7.9 Hz, 1H,
2-Azidoethyl (2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)- H-2’), 5.01 (dd, J = 10.2, 8.7 Hz, 1H, H-3), 4.94 (dd, J = 10.4, 3.4
(1,4)-O-2-acetamido-3,6-di-O-acetyl-2-deoxy-
β
-D-
Hz, 1H, H-3’), 4.64 (d, J = 8.3 Hz, 1H, H-1), 4.52 – 4.41 (m, 2H,
glucopyranoside (21)
H-1’, H-6a), 4.14 – 4.04 (m, 4H, H-2, H-6b, H-6a’, H-6b’), 3.89 –
3
.54 (m, 15H, H-5, H-4, H-5’, OCH
.3, 4.4, 0.9 Hz, 2H, CH ), 2.12, 2.10, 2.06 – 2.01, 1.95 (7 x s,
). C NMR (151 MHz, CDCl ) δ 170.60, 170.57,
70.52, 170.44, 170.23, 170.14, 169.35, 102.04, 101.23, 76.33,
2 2
x 3, CH O x 2), 3.50 (ddd, J =
To a solution of 20 (0.1 g, 0.162 mmol) and 2-azidoethanol
0.141 mg, 1.62 mmol) in 1,2-dichlroethane(10 ml) was added
5
2 3
N
(
13
o
21H, 7 x COCH
3
3
Pyridinium p-TsOH (0.005 g, 0.0199 mmol) and stirred at 70 C
for overnight. The mixture was cooled and neutralized with
pyridine (0.25 ml, 3.1 mmol), and concentrated in vacuo. The
residue was purified by silica gel column chromatography (n-
hexanes:AcOEt=1:1) to afford 21 (0.067 g, 59%) as a colorless
oil.
1
7
6
2
3.51, 72.68, 71.79, 71.08, 70.75, 70.60, 69.95, 69.23, 68.78,
6.72, 62.51, 60.87, 53.58, 50.60, 23.11, 21.01, 21.00, 20.76,
+
0.74, 20.63. MS (ESI), m/z 793.2985 (M + H) (C32
H
48
N
4
O
19
requires 793.2974). TLC; R
f
0.50 (n-hexane:acetonet=1:1).
1
H NMR (600 MHz, Chloroform-d) δ 5.86 (d, J = 9.3 Hz, 1H, NH),
2
-Azidoethyl -D-galactopyranosyl-(1,3)−β-D-
α
5
.33 (dd, J = 3.5, 1.2 Hz, 1H, H-4’), 5.13 – 5.04 (m, 2H, H-2’, H-
), 4.95 (dd, J = 10.5, 3.4 Hz, 1H, H-3’), 4.56 (d, J = 7.7 Hz, 1H,
galactopyranosyl-(1,4)-O-2-acetamido-2-deoxy-β-D-
glucopyranoside (24)
3
H-1’), 4.53 – 4.46 (m, 2H, H-1, H-6a), 4.13 – 3.93 (m, 5H, H-2,
H-6b, H-6a’, H-6b’ OCHH), 3.86 (ddd, J = 7.5, 6.3, 1.3 Hz, 1H, H- To a solution of 21 (0.0187 g, 0.0265 mmol) in dry MeOH (10
5
3
5
’), 3.77 (t, J = 8.6 Hz, 1H, H-4), 3.67 – 3.59 (m, 2H, OCHH, H-5), ml) was added 0.5 M NaOMe (0.5 ml, 0.25 mmol). The mixture
.45 (ddd, J = 13.4, 8.3, 3.3 Hz, 1H, CHHN ), 3.24 (ddd, J = 13.4, was stirred at room temperature for 5 h, and then neutralized
.0, 3.2 Hz, 1H, CHHN ), 2.12, 2.09, 2.05, 2.04 – 2.00, 1.94 (7 x with CH COOH (0.017 ml, 0.3 mmol). The mixture was
) δ 170.68, concentrated in vacuo, and the residue was purified on a
70.48, 170.47, 170.44, 170.20, 170.12, 169.39, 101.11, VYDAC C18 reversed phase semi preparative (250 mm × 22 mm,
00.84, 75.88, 72.79, 72.44, 70.90, 70.78, 69.15, 68.26, 66.68, 10 15 m) HPLC column using H O and 0.1% TFA in CH CN
2.17, 60.85, 53.32, 50.67, 23.34, 20.96, 20.94, 20.73, 20.71, mobile phases. A linear gradient was employed (95:5
3
3
3
1
3
3 3
s, 21H, 7 x COCH ). C NMR (151 MHz, CDCl
1
1
6
2
7
−
ꢀ
2
3
+
0.60. MS (ESI), m/z 705.2462 (M + H) (C28
H
40
N
4
O17 requires
2 3 2 3
H O/0.1%TFA in CH CN → 5:95 H O/0.1%TFA in CH CN) over a
05.2461). TLC; R 0.35 (n-hexanes:acetone=1:1).
f
period of 40 min at a flow rate of 10 mL/min. Fractions
containing the desired product were collected, frozen, and
-D- lyophilized to give deacetylated compound quantitatively. To a
solution of 5 mM UDP-Galactose and 4 mM deacetylated
compound (0.0118 g, 0.0192 mmol) in 50 mM Tris-HCl (pH 7.0)
2-(2-Azidoethoxy)ethyl
(2,3,4,6-tetra-O-acetyl-β
galactopyranosyl)-(1,4)-O-2-acetamido-3,6-di-O-acetyl-2-
deoxy- -D-glucopyranoside (22)
β
This journal is © The Royal Society of Chemistry 20xx
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Organic & Biomolecular Chemistry
Page 12 of 14
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DOI: 10.1039/C7OB00448F
ARTICLE
Journal Name
supplemented with 10 mM MnCl
2
was added 1,3
α
GalT (20 compound (0.015 g, 0.03 mmol) in 50 mM Tris-HCl (pH 7.0)
was added 1,3 GalT (20
μg/mL) to a final volume of 7.5 ml. The solution was incubated supplemented with 10 mM MnCl α
at 37°C with agitation and reaction progress was monitored by μg/mL) and GalE (20 μg/mL) to a final volume of 7.5 ml. The
2
2
2
TLC. When the deacetylated compound was consumed, same work-up procedure as described for compound 24
1 αGalT was removed using an Amicon 4 filter (MWCO 10- afforded compound 25 (11.2 mg, 57%) as a white solid.
,3
kDa) by centrifugation at 7,830 rpm and room temperature for
5 min. Filtrates were then concentrated in vacuo and the 2-[2-(2-azidoethoxy)ethoxy]ethyl
residue was dissolved in H O and passed through a chloride -D-galactopyranosyl-(1,4)-O-2-acetamido-2-deoxy-β
1
α
-D-galactopyranosyl-(1,3)-
-D-
2
β
-
anion-exchange column [Dowex 1 (Cl )]. The eluate was glucopyranoside (26)
concentrated and the residue was also purified on a VYDAC C18
The deacylated compound from 23 was allowed to react under
the same conditions as described for the preparation of
compound 25 to give compound 26 (11 mg, 54% from
compound 23) as a white solid.
reversed phase semi preparative (250 mm × 22 mm, 10
m) HPLC column using H O and 0.1% TFA in CH CN mobile
phases. A linear gradient was employed (95:5 H O/0.1%TFA in
CH CN 5:95 H O/0.1%TFA in CH CN) over a period of 40 min
−15
ꢀ
2
3
2
3
→
2
3
1
H NMR (600 MHz, Deuterium Oxide) δ 5.15 (d, J = 3.9 Hz, 1H,
at a flow rate of 10 mL/min. Fractions containing the desired
product were pooled, frozen, and lyophilized to afford
compound 24 (5.1 mg, 32%) as a white solid.
H-1’’), 4.63 – 4.51 (m, 1H, H-1’), 4.48 (d, J = 7.9 Hz, 1H, H-1),
4
.25 – 4.16 (m, 1H, H-4’), 4.08 – 3.89 (m, 4H), 3.90 – 3.48 (m,
13
1
25H, CH
2
N
3
), 2.05 (s, 3H, COCH
3 2
). C NMR (151 MHz, D O) δ
H NMR (600 MHz, Deuterium Oxide) δ 5.07 (d, J = 3.9 Hz, 1H,
1
7
6
74.03, 102.32, 100.47, 94.94, 78.21, 76.70, 74.57, 74.24,
H-1’’), 4.53 (d, J = 8.3 Hz, 1H, H-1’), 4.47 (d, J = 7.8 Hz, 1H, H-1),
2.07, 70.48, 70.35, 69.15, 68.84, 68.64, 68.50, 67.71, 64.32,
4
1
=
7
.16 – 4.06 (m, 2H, H-4’, H-5’’), 3.98 (ddd, J = 11.5, 5.6, 3.0 Hz,
H), 3.94 (d, J = 3.6 Hz, 1H), 3.92 (d, J = 2.1 Hz, 1H), 3.87 (dd, J
10.4, 3.3 Hz, 1H), 3.81 – 3.50 (m, 14H), 3.41 (ddd, J = 13.9,
0.50, 59.65, 54.55, 49.74, 21.69. MS (ESI), m/z 703.2879 (M +
+
H) (C26
H
46
N
4
O18 requires 703.288).
.7, 3.0 Hz, 1H, CHHN
3
), 3.34 (ddd, J = 13.8, 5.8, 3.0 Hz, 1H,
1
3
1-(2-aminoethyl)
Dgalactopyranosyl-(1,4)-O-2-acetamido-2-deoxy-
glucopyranoside-2-ethoxycyclobutene-3,4-dione (4)
α
-D-galactopyranosyl-(1,3)-
β-
CHHN
3
3 2
), 1.97 (s, 3H, COCH ). C NMR (151 MHz, D O) δ
β
-D-
1
7
6
74.19, 102.32, 100.50, 94.94, 78.19, 76.70, 74.57, 74.30, 2.06,
1.56, 70.35, 69.11, 68.63, 68.27, 67.70, 64.32, 61.99, 60.50,
0.43, 59.62, 54.50, 49.87, 21.76. MS (ESI), m/z 615.2353 (M + To a solution of 24 (0.0038 g, 0.00647 mmol) in MeOH (5 ml)
+
H) (C22
H
38
N
4
O16 requires 615.2355). TLC;
R
f
0.22 (2- was added 5% Pd-C (5.0 mg). The mixture was stirred under
hydrogen atmosphere at room temperature for 24 h. The
solution was filtered through a pad of celite, and then
propanol:AcOEt:H O=2:2:1).
2
2
-(2-Azidoethoxy)ethyl
α
-D-galactopyranosyl-(1,3)-
β
-D- concentrated under reduced pressure to afford azide reduced
crude compound, and this compound was used for next
reaction for further purification. To a solution of amine
compound in 50% aqueous EtOH (2ml) was added Diethyl
galactopyranosyl-(1,4)-O-2-acetamido-2-deoxy-β-D-
glucopyranoside (25)
Compound 22 (0.079 g, 0.105 mmol) was allowed to react
3
squarate (0.0047 ml, 0.032 mmol) and Et N (0.0035 ml, 0.023
under the same condition as described for the preparation of
mmol), and stirred at room temperature for 24 hrs. The elute
was concentrated and the residue was purified on a VYDAC C18
compound 24
deacetylated compound from 22 gave compound 25 (12.2 mg,
2% from compound 22) as a white solid.
. The aliquot (0.015 g, 0.03 mmol) of
reversed phase semi preparative (250 mm × 22 mm, 10
m) HPLC column using water in CH CN mobile phases. A
linear gradient was employed (95:5 H O/CH CN 5:95
O/CH CN) over a period of 40 min at a flow rate of 10
mL/min. Fractions containing the desired product were
collected, frozen, and lyophilized to give compound (2.5 mg,
4% from compound 24) as a colorless oil.
−15
6
1
ꢀ
3
H NMR (600 MHz, Deuterium Oxide) δ 5.11 (d, J = 3.9 Hz, 1H,
H-1’’), 4.55 (dd, J = 8.3, 1.6 Hz, 1H, H-1’), 4.51 (d, J = 7.9 Hz, 1H,
H-1), 4.19 – 4.11 (m, 2H, H-4’, H-5’’), 4.01 – 3.87 (m, 4H), 3.85
2
3
→
H
2
3
–
3.59 (m, 17H), 3.55 (d, J = 7.0 Hz, 1H), 3.49 – 3.43 (m, 2H,
1
3
4
2 3 3 2
CH N ), 2.01 (s, 3H, COCH ). C NMR (151 MHz, D O) δ 174.04,
5
1
1
6
4
02.30, 100.44, 94.93, 78.18, 76.69, 74.53, 74.21, 72.04, 70.32,
H NMR (600 MHz, Deuterium Oxide) δ 5.16 (d, J = 3.9 Hz, 1H,
H-1’’), 4.75 (ddd, 2H, OCH CH ), 4.61 – 4.48 (m, 2H, H-1’, H-1),
.26 – 4.16 (m, 2H, H-4’, H-5’’), 4.10 – 3.92 (m, 4H), 3.91 – 3.53
m, 16H), 2.00 (s, 3H, COCH ), 1.46 (q, J = 7.5 Hz, 3H, CH CH ).
MS (ESI), m/z 713.2607 (M + H) (C28 19 requires
O=1:1:1).
9.08, 68.80, 68.62, 67.69, 64.31, 60.48, 60.42, 59.62, 54.53,
+
9.71, 21.66. MS (ESI), m/z 659.2617 (M + H) (C24
2
3
H
42
N
4
O
17
4
requires 659.2618). TLC; R
f
0.31 (2-propanol:AcOEt:H
2
O=2:2:1).
(
3
2
3
+
44 2
H N O
2-(2-Azidoethoxy)ethyl
α
-D-galactopyranosyl-(1,3)-β
-D-
7
f 2
13.2611). TLC; R 0.32 (2-propanol:AcOEt:H
galactopyranosyl-(1,4)-O-2-acetamido-2-deoxy-β-D-
glucopyranoside (25)
1
-[2-(2-aminoethoxy)]ethyl
α
-D-galactopyranosyl-(1,3)-
-D-
glucopyranoside-2-ethoxycyclobutene-3,4-dione (5)
β-D-
galactopyranosyl-(1,4)-O-2-acetamido-2-deoxy-
β
In an alternative method for the generation of compound 25
the deacylated compound from 22 was allowed to react in a
coupled enzyme system containing both 1,3 GalT and GalE. To
a solution of 5 mM UDP-Glucose, 4 mM deacetylated
,
α
1
2 | J. Name., 2012, 00, 1-3
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Journal Name
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DOI: 10.1039/C7OB00448F
ARTICLE
Compound 25 (0.0112 g, 0.0177 mmol) was allowed to react number (hapten density) for each
under the same condition as described for the preparation of samples were submitted for MALDI-TOF analysis and
to give (0.0063 g, 47%) as a colorless oil. compared MW of -BSA or -BSA and unmodified BSA,
H NMR (600 MHz, Deuterium Oxide) δ 5.16 (d, J = 3.9 Hz, 1H, respectively, as per the formula: copy number = (MW Gal
H-1’’), 4.77 – 4.67 (m, 2H, OCH CH ), 4.57 (dd, J = 13.2, 8.1 Hz, BSA – MW BSA) / (MW Gal – MW EtOH); MW Gal = 759
H, H-1’, H-1), 4.27 – 4.15 (m, 2H, H-4’, H-5’’), 4.08 – 3.90 (m, (compound ), 800 (compound ) Da, MW EtOH = 46 Da; MW
H), 3.91 – 3.47 (m, 20H), 2.04 (s, 3H, COCH ), 1.46 (q, J = 7.4 -BSA = 76,514 Da, MW -BSA = 78,102, MW BSA = 66,432 Da
O) δ 188.46, 173.92, (see supplementary information 2). The resulting protein
3-BSA and 6-BSA conjugates,
4
5
3
6
1
α
2
3
α
α
2
3
6
4
3
3
6
1
3
2 3 2
Hz, 3H, CH CH ). C NMR (151 MHz, D
1
7
6
5
73.17, 102.33, 100.53, 94.94, 78.23, 77.99, 76.69, 74.87, conjugates were dialyzed into PBS (pH 7.4) at 4 °C. KLH and TT
4.56, 74.29, 72.04, 71.98, 70.35, 70.17, 69.23, 69.10, 68.79, conjugates were used for immunization; BSA conjugates were
8.63, 68.53, 68.47, 68.06, 67.71, 64.31, 60.49, 60.42, 59.67, used for ELISA plate coating.
+
4.51, 43.50, 21.67, 14.51. MS (ESI), m/z 757.2873 (M + H)
(
C
30
H
48
N
2
O
20
requires 757.2875). TLC;
R
f
0.41 (2-
Vaccine formulation/administration and blood collection
propanol:AcOEt:H O=1:1:1).
2
On a per mouse basis, 50 µg of KLH-conjugate (or 50 µg of TT-
1
-[2-{2-(2-aminoethoxy)]ethoxy]ethyl
1,3)- -D-galactopyranosyl-(1,4)-O-2-acetamido-2-deoxy-
glucopyranoside-2-ethoxycyclobutene-3,4-dione (6)
α-D-galactopyranosyl- conjugate) in ~50 µL PBS pH 7.4 was combined with 50 µL of
(
β
β
-D- Alhydrogel® (10 mg/mL, Invivogen). The resulting suspension
was mixed and subsequently administered to mice
intraperitoneally on days 0, 14, and 28 for KLH-conjugates (or
Compound 26 (0.0114 g, 0.0168 mmol) was allowed to react
under the same condition as described for the preparation of
to give (0.01 g, 74%) as a colorless oil.
H NMR (600 MHz, Deuterium Oxide) δ 5.16 (d, J = 3.9 Hz, 1H,
H-1’’), 4.77 – 4.67 (m, 2H, OCH CH ), 4.57 (dd, J = 13.2, 8.1 Hz,
H, H-1’, H-1), 4.27 – 4.15 (m, 2H, H-4’, H-5’’), 4.08 – 3.90 (m,
H), 3.91 – 3.47 (m, 24H), 2.04 (s, 3H, COCH ), 1.46 (q, J = 7.4 ELISA
O) δ 188.38, 173.95,
0
, 28, 42 for TT-conjugate). Each mouse was bled (via retro-
4
orbital sinus) on days 28 and 42 for KLH-conjugates (or 37 and
51 for TT-conjugates). Blood samples were then centrifuged at
6
1
1
0,000 rpm for 10 min to collect sera.
2
3
2
4
3
1
3
2 3 2
Hz, 3H, CH CH ). C NMR (151 MHz, D
Production of anti-αGal IgG antibody was evaluated by ELISA.
1
7
6
1
8
02.41, 100.53, 94.94, 74.87, 74.57, 74.29, 72.07, 72.02, 70.48,
Microtiter plates (Costar 3690) were incubated with coating
antigen-BSA in PBS (4 μg/mL, 25 μL) (18 h, 37 °C). Then, 5%
nonfat milk in PBS (30 min, 37 °C) was added to block
nonspecific binding. Mouse sera in 2% BSA were serially
diluted across the plate before incubation in a moist chamber
0.35, 70.16, 69.30, 69.21, 69.16, 68.98, 68.80, 68.64, 68.56,
8.07, 67.71, 64.32, 60.50, 60.43, 59.58, 54.53, 43.48, 21.69,
+
4.53. MS (ESI), m/z 801.3131 (M + H) (C32
H
52
N
2
O21requires
01.3135). TLC; R
f
0.13 (2-propanol:AcOEt:H
2
O=1:1:1).
(
24 h, 4 °C). The plate was washed with dH
incubation with peroxidase-conjugated donkey anti-mouse IgG
(knockout) (SouthernBiotech) in a moist chamber (2 h, 25 °C). The plates
2
1,3galactosyltransferase genes and are referred to as were further washed with dH O before being developed with
2
O before
Animals
Mice
used
have
disrupted
16
α
knockout (KO) mice (6-10 week old). Mice were group-housed using TMB (Thermo Pierce) and the absorbance at 450 nm
in an AAALAC-accredited vivarium containing temperature- measured on a microplate reader (SpectraMax M2e Molecular
and humidity-controlled rooms, with mice kept on a reverse Devices). Titers were calculated as the dilution corresponding
light cycle (lights on: 9PM-9AM). All experiments were to 50% of the maximum absorbance from a plot of the
performed during the dark phase, generally between 1PM- absorbance versus log(dilution) using GraphPad Prism 6.
4PM. General health was monitored by both the scientists and Competitive ELISA was also performed in a similar manner but
veterinary staff at The Scripps Research Institute. All studies with an added step: serum was incubated with dilution of
were performed in compliance with the Scripps Institutional compounds 2 and 5 (0, 0.6, 6.0, 60
Animal Care and Use Committee, and were in accordance with plates for 24 h (4°C).
the National Institutes of Health Guide for the Care and Use of
ꢀM) in hapten-BSA coated
Laboratory Animals.
Acknowledgements
We thank Prof. George Peng Wang for his generous gift of
enzyme plasmids. Funding for this work was provided by the
NIH, NIDA R01DA008590 (to K.D.J) and NCATS CTSA Award UL1
TR001114 (to N.T.J). The authors acknowledge Dr. Major
Gooyit for helpful discussions.
Conjugation of Haptens
Dialyzed KLH, TT or BSA in 0.5M Borate buffer (pH 9.0) was
added to the hapten [ca. 447 (compound
), 393 (compound ) equiv. for KLH, 205 (compound
compound ), 180 (compound ) equiv. for TT, and 92
compound ), 87 (compound ) equiv. for BSA] to give a final
1
), 420 (compound
2
3
4), 193
(
(
5
6
3
6
concentration of 1.25 mg/mL in 0.5M Borate buffer (pH 9.0) Notes and References
and the solutions mixed (rt, 24 h). In order to quantify copy
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Organic & Biomolecular Chemistry
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DOI: 10.1039/C7OB00448F
ARTICLE
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