Chemoenzymatic synthesis of hydrophobic glycoprotein: Synthesis of saposin c carrying complex-type carbohydrate

Article abstract of DOI:10.1021/jo3010155

The complex-type N-linked octasaccharide oxazoline having LacNAc as the nonreducing end sugar was efficiently synthesized using the benzyl-protected LacNAc, mannose, and β-mannosyl GlcNAc units as key building blocks. To achieve a highly β-selective glycosylation with the LacNAc unit, the N-trichloroacetyl group was used for the protection of the amino group in the LacNAc unit. After complete assembly of these units and deprotection, the obtained free sugar was successfully derivatized into the corresponding sugar oxazoline. On the other hand, the N-acetylglucosaminylated saposin C, a hydrophobic lipid-binding protein, was chemically synthesized by the native chemical ligation reaction. On the basis of the previous results related to the synthesis of the nonglycosylated saposin C, the O-acyl isopeptide structure was introduced to the N-terminal peptide thioester carrying GlcNAc to improve its solubility toward aqueous organic solvents. The ligation reaction efficiently proceeded with the simultaneous O- to N-acyl shift at the O-acyl isopeptide moiety. After the removal of the cysteine-protecting group and folding, saposin C carrying GlcNAc was successfully obtained. The synthetic sugar oxazoline was then transferred to this glycoprotein using the mutant of endo-β-N- acetylglucosaminidase from Mucor hiemalis (Endo-M) (glycosynthase), and the saposin C carrying the complex-type nonasaccharide was successfully obtained.

Full text of DOI:10.1021/jo3010155

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pubs.acs.org/joc
Chemoenzymatic Synthesis of Hydrophobic Glycoprotein: Synthesis
of Saposin C Carrying Complex-Type Carbohydrate
Hironobu Hojo,†,* Hiromasa Tanaka,† Masashi Hagiwara,† Yuya Asahina,† Akiharu Ueki,†
Hidekazu Katayama,† Yuko Nakahara,† Azusa Yoneshige,‡ Junko Matsuda,‡ Yukishige Ito,§
and Yoshiaki Nakahara*,†
‡
†Department of Applied Biochemistry and Institute of Glycoscience, Tokai University, 4-1-1 Kitakaname, Hiratsuka, Kanagawa
259-1292, Japan
§RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako, Saitama 351- 0198, Japan
S
* Supporting Information
ABSTRACT: The complex-type N-linked octasaccharide
oxazoline having LacNAc as the nonreducing end sugar was
efficiently synthesized using the benzyl-protected LacNAc,
mannose, and β-mannosyl GlcNAc units as key building
blocks. To achieve a highly β-selective glycosylation with the
LacNAc unit, the N-trichloroacetyl group was used for the
protection of the amino group in the LacNAc unit. After
complete assembly of these units and deprotection, the
obtained free sugar was successfully derivatized into the
corresponding sugar oxazoline. On the other hand, the N-
acetylglucosaminylated saposin C, a hydrophobic lipid-binding
protein, was chemically synthesized by the native chemical ligation reaction. On the basis of the previous results related to the
synthesis of the nonglycosylated saposin C, the O-acyl isopeptide structure was introduced to the N-terminal peptide thioester
carrying GlcNAc to improve its solubility toward aqueous organic solvents. The ligation reaction efficiently proceeded with the
simultaneous O- to N-acyl shift at the O-acyl isopeptide moiety. After the removal of the cysteine-protecting group and folding,
saposin C carrying GlcNAc was successfully obtained. The synthetic sugar oxazoline was then transferred to this glycoprotein
using the mutant of endo-β-N-acetylglucosaminidase from Mucor hiemalis (Endo-M) (glycosynthase), and the saposin C carrying
the complex-type nonasaccharide was successfully obtained.
technique for the peptide chain assembly. Using this strategy,
we have succeeded in the synthesis of various glycoproteins,5
such as the extracellular first immunoglobulin domain of
emmprin carrying the N-linked core pentasaccharide5a and 23
kDa mucin model carrying multiple O-GalNAcs.5b
To extend our strategy for the synthesis of N-glycoproteins
carrying larger carbohydrates, we recently established an
efficient route for the synthesis of 9-fluorenylmethoxycarbonyl
(Fmoc)-Asn carrying a complex-type nonasaccharide suitable
for the solid-phase peptide synthesis (SPPS) of a glycoprotein.6
The key to this synthesis was the combination of the armed
benzyl-protected sugar with the N-trichloroacetyl (TCA) group
for the protection of the amino group of the LacNAc moiety.
This protecting group showed a powerful stereocontrolling
ability as well as a high reactivity, enabling the stereoselective
formation of the β-LacNAc glycosidic linkage during the final
stage of the synthesis.7
INTRODUCTION
■
Glycosylation is a common post-translational modification of
proteins. More than half of natural proteins are supposed to be
glycosylated at the appropriate hydroxy groups of the Ser/Thr
residues and/or amido groups of Asn located at the Asn-Xaa-
Ser/Thr consensus motif.1 However, the structure of these
sugars is usually highly heterogeneous, making the functional
and structural studies of glycoproteins a difficult task. On the
basis of this fact, challenges toward the efficient preparation of
homogeneous glycoproteins by chemical, chemoenzymatic, or
semisynthetic methodologies have been performed by various
researchers.2 However, one of the problems of these methods is
that the synthesis of a sufficient amount of the sugars is still far
from routine, although the preparation of fairly large sugars,
such as the biantennary complex-type N-glycan, have been
reported.3 N-Linked carbohydrates can also be isolated from
natural sources; however, their structures are limited to the
natural forms.4 An efficient method for the glycan synthesis,
which is applicable to various glycoforms, is still required.
We have been developing a facile method for the
glycoprotein synthesis based on our benzyl-protection strategy
for the carbohydrate portion combined with a ligation
On the other hand, the chemoenzymatic synthesis of the N-
glycosylated protein, which uses the transglycosylation ability of
Received: May 17, 2012
© XXXX American Chemical Society
A
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endo-β-N-acetylglucosaminidases (Endo A and M) to the
GlcNAc residue on the polypeptide,8 has attracted much
attention, and the synthesis of glycopeptides as well as the
conversion of natural glycoproteins carrying a heterogeneous
sugar to the one with a homogeneous sugar has been
demonstrated.9 The major drawback of this method is that
the yield of the product is low due to the hydrolysis of the
product. This problem was recently solved by the development
of the Endo A and M mutant,10 although glycosyl oxazolines
lacking the reducing end GlcNAc has to be prepared as glycosyl
donors. This chemoenzymatic method has a great potential,
since a single glycoprotein can be derivatized into various
glycoforms, if we can prepare various glycosyl donors. Thus, we
intended to apply our synthetic procedure to the preparation of
the complex-type sugar oxazoline. Due to its highly convergent
nature, our synthetic methodology could be easily used for the
synthesis of the sugar oxazoline.
chemoenzymatic synthesis of glycosylated saposin C 1 (Figure
1) using the synthetic complex-type sugar oxazoline.
RESULTS AND DISCUSSION
Synthesis of Octasaccharide Oxazoline. Based on the
previous synthesis of N-glycoasparagine 2 (Figure 2),6 the
■
Saposins A−D are activators of glycosidases, which are
engaged in the degradation of sphingolipids in lysosomes.
Among them, saposin C helps in the degradation of
glucosylceramide (GlcCer) by GlcCer-β-glucosidase (GCase)
(Figure 1).11 Saposin C deficiency in humans showed the
Figure 2. Structure of Asn-linked nonasaccharide 2.
convergent route for the synthesis of oxazoline 3 was designed
(see Scheme 1). The target was divided into the LacNAc unit 4,
Scheme 1. Retrosynthetic Pathway to Sugar Oxazoline 3
mannosyl donor 5, and β-mannosyl GlcNAc 6. In the previous
synthesis of the complex type nonasaccharide unit 2, we first
attempted the glycosylation of the LacNAc-mannosyl donor
(h−g−f and e−d−c in Figure 2) to the β-mannosyl chitobiose
unit (b−a−a′). However, we could not achieve a highly
stereoselective di-α-glycosylation. Thus, the mannose and the
LacNAc units were sequentially glycosylated with the β-
mannosyl chitobiose unit to obtain unit 2. This sequential
glycosylation was also used in this synthesis. The phthaloyl
group previously used for the amino protection of the
chitobiose moiety was changed to the trichloroacetyl (TCA)
group, which was also used for the amino protection at the
LacNAc moieties. This change realizes the formation of all
acetamido groups in one step by the zinc in acetic acid
treatment. The 3-OH group of the Gal in the LacNAc unit was
protected by an allyl group so that the same LacNAc unit can
be used to construct the poly LacNAc structure or to achieve
sialylation of the 3-OH group in the future studies. The
obtained octasaccharide unit was completely deprotected and
then derivatized into the sugar oxazoline 3.
Figure 1. Structure of saposin C carrying complex-type non-
asaccharide.
phenotype similar to Gaucher disease with the accumulation of
GlcCer, which is due to a GCase deficiency. Saposin C-deficient
mice cause neurodegenerative diseases with the patterned loss
of cerebellar Purkinje cells and widespread axonal spheroids.
Although saposin C is N-glycosylated at Asn,21 its significance is
still controversial. Recently, we completed the synthesis of the
nonglycosylated saposin C.12 In spite of the relatively small size
of this protein, its synthesis by the native chemical ligation
(NCL) method13 was difficult due to the extremely low
solubility of the N-terminal segment in organic and aqueous
organic solvents. This fact might be due to the hydrophobic
nature of saposin C, which binds to sphingolipid. Thus, we
attempted to improve the solubility of the N-terminal segment
by introducing the O-acyl isopeptide structure14 and succeeded
in its synthesis using the NCL method. Based on the synthetic
route for the nonglycosylated saposin C, we now report the
The synthesis was started with the construction of β-
mannosyl GlcNAc unit 6 following Crich’s method15 as shown
in Scheme 2. The mannosyl thioglycoside 7 was activated with
1-benzenesulfinyl piperidine (BSP), 2,4,6-tri-tert-butylpyrimi-
dine (TTBP), and triflic anhydride (Tf2O) at −60 °C and
reacted overnight with GlcNAc unit 8 at −78 °C. The product
B
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a
by microwave irradiation and the desired product 14 was
obtained in 79% yield.7 The tert-butyldiphenylsilyl (TBDPS)
group was then removed by tetrabutylammonium fluoride
(TBAF) in the presence of AcOH in 86% yield to give
compound 15. Finally, the Bn groups were removed by catalytic
hydrogenation to give a completely free octasaccharide 16. The
conversion of this sugar to its oxazoline derivative 3 was
successfully achieved by reacting with 2-chloro-1,3-dimethyli-
midozolium chloride (DMC) according to Shoda’s method in
64% yield.18 Overall, this synthetic route efficiently gave the
desired product 3 with a highstereoselectivity. This route can be
easily extended to the synthesis of other N-linked carbohy-
drates.
Scheme 2. Synthesis of Tetrasaccharide 11
a
Reagents and conditions: (a) BSP, TTBP, Tf2O, MS4A, CH2Cl2, −78
°C, 1 d, 74%; (b) (1) Et3SiH, PhBCl2, MS4A, CH2Cl2, −78 °C, 0.5 h;
(2) 90% TFA aq, CH2Cl2, −10 °C, 0.5 h, 87% (two steps); (c)
Cp2ZrCl2, AgClO4, MS4A, CH2Cl2, −40 to −15 °C, 1.5 h, 85%; (d) 1
M NaOMe, MeOH−THF (9:1), rt, 1 d, 88%.
Synthesis of Saposin C carrying a Complex-Type
Sugar. In the previous synthesis of the nonglycosylated saposin
C 22 (Scheme 4), the SPPS of the N-terminal thioester as well
Scheme 4. Synthetic Route for Nonglycosylated Saposin C
2212
was obtained as a mixture of stereoisomers (β/α = 5/1) in 87%
yield. The β-isomer 9 (74% yield) was isolated and treated with
Et3SiH and PhBCl216 to regioselectively reduce the benzylidene
acetal. The MPM group was then removed by aqueous TFA to
afford 6 in 87% yield (two steps). As in the previous synthesis,
the mannosylation of 6 with mannosyl fluoride 517 efficiently
produced the α,α-dimannosylated tetrasaccharide 10 in 85%
yield, which was readily deacetylated to the diol 11 in a yield of
88%.
The glycosylation of 11 with 47b (3.5 equiv) was promoted
using a catalytic amount of trimethylsilyl triflate (TMSOTf) at
−78 °C for 1 h and then at −40 °C for 1 h to give the desired
octasaccharide 12 as a single isomer in 86% yield (Scheme 3).
The protected octasaccharide was then deallylated by the Ir-
complex treatment followed by the oxymercuration reaction to
give compound 13 in 78% yield (two steps). The TCA groups
were converted to Ac groups by Zn treatment in acetic acid and
ethyl acetate (EtOAc). This reaction was efficiently promoted
a
Scheme 3. Synthesis of Sugar Oxazoline 3
as the NCL reaction was hampered by the extreme low
solubility of the thioester.12 Sohma et al. reported that a peptide
with the isopeptide structure retains a significantly higher
solubility compared to the corresponding native peptide, which
is due to the inhibition of the aggregation caused by the
intermolecular β-sheet formation.14 Thus, we applied this
method to the synthesis of the N-terminal segment of saposin
C using the azido group as an amino protecting group at the
isopeptide site.12 However, the desired peptide thioester 17 was
not obtained by the Fmoc method, as the azido-protected
isopeptide moiety was unexpectedly decomposed during the
piperidine treatment. The use of the benzyloxylcarbonyl (Z)
group instead of the azido group also failed to obtain the
desired peptide having the Z-protected-O-acyl isopeptide
structure. Thus, the synthesis of 17 was accomplished by the
t-butoxylcarbonyl (Boc) strategy, which does not use a
nucleophilic base during the synthesis. The NCL reaction
with the C-terminal peptide 18 efficiently proceeded in the
absence of tris(2-carboxyethyl)phosphine (TCEP) and the
product carrying the azido-protected O-acyl isopeptide moieties
19 were successfully obtained (Scheme 4, method A).
However, the ligation also efficiently proceeded even in the
a
Reagents and conditions: (a) TMSOTf, MS AW-300, CH2Cl2, −78
to −40 °C, 2 h, 86%; (b) (1) Ir(COD)(PMe2Ph)2PF6, THF, t, 1 h;
(2) HgCl2, HgO, 90% acetone aq, rt, 1 h, 78% (two steps); (c) Zn,
AcOH, EtOAc, microwave (150 w), reflux, 1 h, 79%; (d) TBAF,
AcOH, THF, 0 °C to rt, 1 d, 86%; (e) (1) H2, Pd(OH)2, 70% THF aq,
rt, 2.5 d, 16: 96%, (2) DMC, Et3N, H2O, 0 °C, 6 h, 64%.
C
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presence of TCEP, which quickly reduces the azido group and
allows the O- to N-acyl shift reaction during the ligation
(method B). This result was somewhat unexpected, as the acyl
shift reaction would cause aggregation of the N-terminal
segment, which would lower the efficiency of the ligation. This
result might indicate that once the peptide is completely
dissolved in a buffer, it remains soluble even after the O-acyl
isopeptide structure is lost. Based on this speculation, we
expected that the azido protection at the isopeptide moiety is
not necessary. After removal of the Acm groups, the disulfide
bond was formed by DMSO oxidation in Tris buffer to obtain
the nonglycosylated saposin C 22.
strategy. During the synthesis, Boc-Thr(Fmoc-Ala)-OH and
Fmoc-Asn(GlcNAcBn3)-OH were introduced. After the
completion of the chain assembly, the peptide was cleaved
from the resin by the TFA cocktail, and the benzyl group of the
GlcNAc moiety was removed by the low-TfOH treatment.21
The peptide was then treated with 3-mercaptopropionic acid
(MPA) to perform the thioesterification by the NAC method.
The desired peptide thioester 23 was successfully obtained in
3.8% yield with a purity comparable to the one obtained by the
Boc strategy as shown in Figure 3. It seems that under the
The glycosylated saposin C 1 was chemoenzymatically
prepared based on the synthesis of the nonglycosylated one.
First, the entire polypeptide chain of saposin C carrying
GlcNAc was assembled by the NCL reaction, and then the
enzymatic transfer of the sugar oxazoline was carried out to the
reducing end GlcNAc on the polypeptide chain. It is noted that
the N-terminal Asn in the nonglycosylated saposin C 22 was
trimmed in this synthesis following a recent report.11e To
achieve this strategy, the N-terminal peptide thioester carrying
GlcNAc was synthesized as shown in Scheme 5. As the azido
Scheme 5. Synthesis of Peptide Thioester Carrying GlcNAc
23
Figure 3. RPHPLC profile of the thioesterification reaction: (a) T = 0,
(b) T = 2 d. Elution conditions: a column for protein purification (4.6
× 150 mm) at the flow rate of 1 mL/min and 50 °C; eluent, A, 0.1%
aqueous TFA, B, 0.1% TFA in acetonitrile. The asterisked peaks were
derived from the incomplete introduction of Asn12 during SPPS by the
synthesizer.
conditions of the Fmoc method, the O-acyl isopeptide structure
is stabilized by the Boc-protection, but not by the azido- or Z-
protection, the reason of which is not yet clear. The synthesis of
the C-terminal segment 25 was achieved by the same procedure
as described for the synthesis of the nonglycosylated saposin
C.12
Prior to the NCL reaction, the N- and C-terminal segments
were mixed in an equimolar amount in aqueous acetonitrile
containing 0.1% TFA and a part of the mixture was analyzed by
HPLC as shown in Figure 4 (a). After lyophilization, the
ligation was carried out in a sodium phosphate buffer
containing 6 M guanidium hydrochloride, 2% TCEP, and 2%
4-mercaptophenylacetic acid (MPAA)22 at pH 8.5 as shown in
Scheme 6. Even immediately after the peptides were dissolved
in the buffer, the O-acyl isopeptide thioester 23 was converted
to N-acyl peptide thioester 26 as shown by the comparison of
the HPLC of the acidic mixture (a) and the T = 0 of the
ligation reaction (b) in Figure 4. However, as we expected, the
ligation efficiently proceeded and was almost complete within 3
h. The isolated yield of the product 27 was 30%. The Acm
groups were removed by Ag+ treatment in aq DMSO in the
presence of N, N-diisopropylethylamine (DIEA). Finally, the
disulfide bond was formed by DMSO oxidation in Tris buffer
(pH 8.5). As in the case of the nonglycosylated saposin C,12 the
oxidized final product 29 was more hydrophobic than the
reduced form 28, which might be derived from the lipid-
binding character of this glycoprotein (Figure 4).
protection at the acyl isopeptide moiety was omitted on the
basis of the previous result, we returned to the use of the Fmoc
method, which was routinely used for the synthesis of peptides
having the O-acyl isopeptide structure.14 To synthesize it as a
thioester, our N-alkylcysteine (NAC) thioesterification meth-
od19 was applied. This method realizes the efficient post-SPPS
thioesterification reaction using the NAC introduced at the C-
terminus of the peptide as an N- to S-acyl migration device.
The NAC method is fully compatible with the conventional
Fmoc method and produces the peptide thioester in excellent
yield without any significant epimerization.20
For the synthesis of the N-terminal segment, the NAC
moiety was introduced as a dipeptide with the C-terminal
amino acid using Fmoc-Ala-(Et)Cys(Trt)-OH to the [Arg-
(Pbf)2]-Rink amide MBHA-resin using diisopropylcarbodii-
mide (DIC)-1-hydroxybenzotriazole (HOBt) method in
CH2Cl2. The peptide chain was then elongated by the Fmoc
Transglycosylation Reaction to the N-Acetylglucosa-
minylated Saposin C. According to the method of Huang et
al.,10b the transglycosylation reaction was carried out (Scheme
D
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Scheme 7. Synthesis of Saposin C Carrying Nonasaccharide
1
Figure 4. RPHPLC profile of the synthesis of saposin C carrying
GlcNAc 29: (a) acidic mixture of peptides 23 and 25, (b) T = 0 h of
the ligation mixture, (c) T = 3 h of the ligation mixture, (d) purified
glycoprotein 27, (e) reaction mixture after the removal of Acm groups,
(f) reaction mixture after DMSO oxidation. The asterisked peaks in (f)
are nonpeptidic components. Elution conditions are the same as those
of Figure 3.
1, indicating a successful reaction. The conversion ratio of
GlcNAc to the nonasaccaride was 64% based on a comparison
of the HPLC peak areas of both glycopeptides. By further
optimization, the amount of the sugar oxazoline used in this
reaction would be decreased. However, it should be pointed out
that the total amount of the sugar oxazoline was only a small
portion of the total amount of the synthetic sugar.
Scheme 6. Synthesis of Saposin C Carrying GlcNAc 29
CD Spectrum Measurement of Saposin C. The nona-
and monoglycosylated saposin C 1, 29, and nonglycosylated
saposin C 30 (see the Experimental Section for its synthesis)
were dissolved in 5 mM sodium phosphate containing 50 mM
NaCl (pH 4.5), and the CD spectrum was measured. As shown
in Figure 5, all of the saposin C's showed a very similar
spectrum rich in an α-helical structure, which is in good
agreement with those previously reported by other research-
ers.23 On the basis of these spectra, the carbohydrates on Asn,21
7). Saposin C carrying GlcNAc 29 and the sugar oxazoline 3
(10 equiv to 29) were dissolved in phosphate buffer containing
20% DMSO (pH 7.0), and then glycosynthase was added. After
the solution was kept at room temperature for 1 h, an
additional 10 equiv of oxazoline 3 (10 equiv) was added, and
the reaction was continued for another 3 h. As shown in Figure
S1 (Supporting Information), HPLC analysis of the reaction
mixture showed the appearance of a slightly hydrophilic peak.
The matrix assisted-laser desorption ionization-time-of-flight
(MALDI-TOF) mass analysis of this peak showed a value that
well corresponds to the theoretical one of the desired product
Figure 5. CD spectrum of the synthetic saposin C's in 5 mM sodium
phosphate buffer containing 50 mM NaCl at pH 4.5.
E
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unless noted otherwise. The H and 13C NMR spectra were recorded
by a spectrometer (1H at 400 MHz and 13C at 100 MHz). Chemical
shifts are expressed in ppm downfield from the signal for the internal
Me4Si for solutions in CDCl3. For a description of the NMR data, each
sugar residue in the oligosaccharide is indicated by an alphabetical
mark as shown in Scheme 1. Peak assignments were performed by H−
H and C−H COSY measurements. The MALDI TOF mass spectra
were obtained using a spectrometer using 2,5-dihydroxybenzoic acid as
a matrix. HPLC was performed in ODS columns (4.6 × 150 mm for
analysis, 10 × 250 mm and 20 × 250 mm for preparation). The
glycopeptide yield was determined by an amino acid analysis after the
samples were hydrolyzed in a sealed tube with 20% HCl and 0.5%
phenol at 150 °C for 2 h. The amino acids were analyzed by an amino
acid analyzer. Glycosynthase was purchased from a commercial
supplier.
1
regardless of their size, did not have specific effects on the
tertiary structure of saposin C. However, we noticed that the
nonglycosylated saposin C tends to form aggregates when
purified lyophilized powder was dissolved in an aqueous buffer.
Thus, one of the significant features of the glycosylation in
saposin C would be the increase in its solubility.
Analysis of GCase Activity. Activation of GCase by the
synthetic saposin C was analyzed using 4-methylumbelliferyl-β-
D-glucopyranoside (4MU-Glc) as the substrate in a detergent-
free system with the negatively charged lipid, L-α-phosphati-
dylserine (PS), as previously described with minor modifica-
tions.24 The activities of GCase increased 30- to 40-fold in the
presence of both PS and the saposin C (nonglycosylated
saposin C: 37-fold, monoglycosylated saposin C: 29-fold,
nonaglycosylated saposin C: 32-fold) compared to those in the
presence of only PS (Table 1). These activations of GCase
tert-Butyldiphenylsilyl 2-O-Benzyl-4,6-O-benzylidene-3-O-
(4-methoxyphenyl)methyl-β-D-mannopyranosyl-(1→4)-3,6-di-
O-benzyl-2-deoxy-2-trichloroacetamido-β-D-glucopyranoside
(9). A stirred mixture of compound 7 (154 mg, 0.27 mmol), BSP (85
mg, 0.41 mmol), TTBP (134 mg, 0.54 mmol), and dried molecular
sieves (MS) 4A (1.1 g) in anhydrous CH2Cl2 (2.5 mL) was cooled at
−60 °C for 30 min. The mixture was stirred for a further 20 min after
the addition of Tf2O (51 μL, 0.30 mmol). The solution was cooled at
−78 °C, and a solution of acceptor 8 (100 mg, 0.13 mmol) in
anhydrous CH2Cl2 (2.5 mL) was slowly added to the mixture through
a cannula. The mixture was then stirred overnight at the same
temperature before the reaction was quenched by addition of saturated
NaHCO3 aq. The mixture was diluted with EtOAc and filtered
through Celite. The organic layer of the combined filtrate was
successively washed with water and brine, dried over anhydrous
Na2SO4, and concentrated in vacuo. The residue was chromato-
graphed on silica gel with toluene−EtOAc (49:1) to give the
Table 1. Effect of Chemically Synthesized Saposin C on
GCase Activity
a
GCase activity (nmol/h/ng GCase)
PS only
0.0704 0.007
b
PS+nonglycosylated saposin C 30
PS+monoglycosylated saposin C 29
PS+nona-glycosylated saposin C 1
2.59 0.03
b
2.06 0.04
b
2.23 0.02
a
b
Values (nmol/h/ng GCase) are presented as mean SE (n = 3). P
< 0.05.
were somewhat greater than the reported value which showed a
12-fold activation of the GCase activity by the 8 mM
concentration of the nonglycosylated recombinant saposin
C.24b In this assay system with 4MU-Glc as the substrate, the
presence or absence of N-glycans on saposin C did not affect
the degree of GCase activation. Thus, in this in vitro assay
system, we could not find a significance of the glycan moiety of
saposin C, which was reported by the comparison of the activity
between the native and recombinant saposin Cs.24b We are now
doing further studies to clarify the biological role of saposin C
as well as its carbohydrate using our synthetic saposin C's.
disaccharide 9 (141 mg, 0.12 mmol, 87%, α/β = 17:83). [α]D
=
−23.0 (c 1.0). Rf = 0.63 (7:1 hexane−EtOAc). 1H NMR: δ 7.62−7.56
(m, 4H, Ar), 7.40−7.02 (m, 28H, Ar), 6.83 (d, 1H, J = 8.3 Hz, −NH),
6.74−6.71 (m, 2H, Ar), 5.41 (s, 1H, PhCH<), 4.97 (d, 1H, J = 10.7
Hz, −CH2Ph), 4.88 (d, 1H, J = 7.8 Hz, H-1a), 4.68 (d, 1H, J = 11.7
Hz, −CH2Ph), 4.63 (d, 1H, J=12.2 Hz, −CH2Ph), 4.55 (d, 1H, J =
11.7 Hz, −CH2Ph), 4.44 (brs, 1H, H-1b), 4.41 (d, 1H, J = 11.2 Hz,
−CH2Ph), 4.40 (d, 1H, J = 10.7 Hz, −CH2Ph), 4.32 (d, 1H, J = 12.2
Hz, −CH2Ph), 4.11 (d, 1H, J = 12.2 Hz, −CH2Ph), 3.98−3.88 (m, 3H,
H-4a, H-4b, H-6b), 3.77 (brt, 1H, J = 9.0 Hz, H-3a), 3.68−3.60 (m,
5H, H-2a, H-2b, −COCH3 × 3), 3.39 (t, 1H, J = 10.2 Hz, H-6b), 3.34
(dd, 1H, J = 3.2, 9.5 Hz, H-3b), 3.25 (dd, 1H, J = 2.9, 11.2 Hz, H-6a),
3.18 (dd, 1H, J = 2.4, 11.2 Hz, H-6a), 3.02 (ddd, 1H, J= 4.9, 9.8, 14.2
Hz, H-5b), 2.92 (m, 1H, H-5a), 0.98 (s, 9H, But). 13C NMR: δ 101.5
(1JCH = 159.3 Hz, C-1b), 101.2 (PhCH<), 94.8 (1JCH = 163.0 Hz, C-
1a), 92.5 (−CCl3). Anal. Calcd for C66H70Cl3NO12Si: C, 65.86; H,
5.86; N, 1.16. Found: C, 65.88; H, 5.96; N, 1.24.
CONCLUSION
■
Our convergent approach was successfully applied to the
synthesis of the N-linked complex-type sugar oxazoline. On the
other hand, the N-acetylglucosaminylated saposin C was
prepared by the NCL reaction incorporating the O-acyl
isopeptide structure as a solubility enhancing element. The
sugar oxazoline was then transferred to the saposin C carrying
GlcNAc by the tranglycosylation reaction using the Endo M
mutant (glycosythase) and the saposin C carrying the N-linked
complex-type nonasaccharide was successfully obtained. This
chemoenzymatic approach could be useful for the synthesis of
other hydrophobic glycoproteins. The synthetic saposins all
showed very similar α-helical structures in their CD spectra,
which indicates that the glycosylation does not have a specific
effect on the conformation of saposin C. We also did not
observe a significant difference in the GCase activation activity
among the synthetic saposin Cs. Further studies on the
significance of the glycosylation in saposin C is currently
underway.
tert-Butyldiphenylsilyl 2,4-Di-O-benzyl-β-D-mannopyrano-
syl-(1→4)-3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-β-D-
glucopyranoside 6. To a stirred mixture of 9 (2.3 g, 1.9 mmol) and
dried MS 4A (7.2 g) in anhydrous CH2Cl2 (170 mL) were successively
added Et3SiH (0.92 mL, 5.7 mmol) and PhBCl2 (0.87 mL, 6.7 mmol)
at −78 °C. The mixture was stirred for 30 min at that temperature.
The reaction was quenched by addition of Et3N and MeOH. The
resulting mixture was diluted with EtOAc and then filtered through
Celite. The combined filtrate and washings were successively washed
with saturated NaHCO3 aq, water, and brine, dried over anhydrous
Na2SO4, and concentrated in vacuo. The residue was dissolved in
CH2Cl2 (65 mL) and stirred with 90% aq TFA (65 mL) at −10 °C for
45 min. The acidic mixture was then carefully neutralized with
saturated NaHCO3 aq and extracted with EtOAc. The organic layer
was successively washed with saturated NaHCO3 aq, water, and brine,
dried over anhydrous Na2SO4, and concentrated in vacuo. The residue
was chromatographed on silica gel with toluene-EtOAc (7:1) followed
by gel-permeation chromatography (Bio-Beads S-X1, toluene) to
afford 6 (1.8 g, 1.7 mmol, 87% in two steps). [α]D = −22.3 (c 1.0). Rf
EXPERIMENTAL SECTION
General Experimental Methods. The optical rotation values
were determined using a polarimeter for the solutions in CHCl3,
■
1
= 0.39 (7:1 hexane−EtOAc). H NMR: δ 7.67 (m, 2H, Ar), 7.60 (m,
2H, Ar), 6.93 (d, 1H, J = 8.3 Hz, -NH), 5.01 (d, 1H, J = 10.7 Hz,
F
dx.doi.org/10.1021/jo3010155 | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Featured Article
−CH2Ph), 4.92 (d, 1H, J = 7.8 Hz, H-1a), 4.87 (d, 1H, J = 11.7 Hz,
−CH2Ph), 4.76 (d, 1H, J = 11.2 Hz, −CH2Ph), 4.52−4.46 (m, 4H,
−CH2Ph × 3, H-1b), 4.43 (d, 1H, J = 12.2 Hz, −CH2Ph), 4.22 (d, 1H,
J = 11.7 Hz, −CH2Ph), 3.95 (brt, 1H, J = 9.0 Hz, H-4a), 3.87 (brt, 1H,
J = 9.3 Hz, H-3a), 3.69 (m, 1H, H-2a), 2.01 (brs, −OH × 2), 1.01 (s,
9H, But). 13C NMR: δ 100.9 (C-1b), 94.8 (C-1a), 92.5 (−CCl3). Anal.
Calcd for C58H64Cl3NO11Si: C, 64.17; H, 5.94; N, 1.29. Found: C,
64.08; H, 5.95; N, 1.25.
do-β-D-glucopyranosyl-(1→2)-3,4,6-tri-O-benzyl-α-D-manno-
pyranosyl-(1→6)]-2,4-di-O- benzyl-β-D-mannopyranosyl-(1→
4)-3,6-di-O-benzyl-2-deoxy-2- trichloroacetamido-β-D-gluco-
pyranoside (12). A stirred mixture of 4 (285 mg, 0.25 mmol), 11
(139 mg, 71 μmol), and dried MS AW-300 (0.71 g) in anhydrous
CH2Cl2 (3.6 mL) was cooled at −78 °C. TMSOTf (2.3 μL, 13 μmol)
was then added to the mixture. The stirring was continued for 1 h at
−78 °C and for another 1 h at −40 °C. After the reaction was
quenched by addition of saturated NaHCO3 aq, the mixture was
diluted with EtOAc and filtered through Celite. The organic layer was
successively washed with saturated NaHCO3 aq, water, and brine,
dried over anhydrous Na2SO4, and concentrated in vacuo. The crude
product was chromatographed on Bio-Beads S-X1 with toluene and
then on silica gel with toluene-EtOAc (19:1) to afford unit 12 (236
mg, 61 μmol, 86%). [α]D = +8.3 (c 1.0). Rf = 0.42 (2:1 hexane−
EtOAc). 1H NMR: δ 7.65 (d, 2H, J = 7.8 Hz, Ar), 7.61 (d, 2H, J = 7.8
Hz, Ar), 6.73 (d, 1H, J = 7.3 Hz, −NH), 6.68 (d, 1H, J = 7.3 Hz,
−NH), 5.96−5.87 (m, 2H, −CHCH2 × 2), 5.35−5.29 (m, 2H,
−CHCH2), 5.18 (d, 1H, J = 10.7 Hz, −CHCH2), 5.17 (d, 1H, J =
10.2 Hz, −CHCH2), 5.02 (s, 1H, H-1c), 4.84 (d, 1H, J = 7.8 Hz, H-
1a), 4.70 (s, 1H, H-1d), 2.83 (d, 1H, J = 9.3 Hz, H-5a), 1.02 (s, 9H,
But). 13C NMR: δ 102.8 and 102.7 (1JCH = 161.8 Hz, C-1e and 1 h),
100.8 (1JCH = 158.1 Hz, C-1b), 100.1 (1JCH = 169.6 Hz, C-1c or 1f),
98.8 (1JCH = 171.3 Hz, C-1c or 1f), 98.1 (1JCH = 163.8 Hz, C-1d or 1
g), 97.3 (C-1d or 1 g), 94.8 (1JCH = 163.8 Hz, C-1a), 92.6, 92.5, and
92.4 (−CCl3 × 3). Anal. Calcd for C216H228Cl9N3O41Si: C, 67.05; H,
5.94; N, 1.09. Found: C, 67.00; H, 5.98; N, 1.06.
tert-Butyldiphenylsilyl 2-O-Acetyl-3,4,6-tri-O-benzyl-α-D-
mannopyranosyl-(1→3)-[2-O-acetyl-3,4,6-tri-O-benzyl-α-D-
mannopyranosyl-(1→6)]-2,4-di-O-benzyl-β-D-mannopyrano-
syl-(1→4)-3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-β-D-
glucopyranoside (10). A mixture of Cp2ZrCl2 (153 mg, 0.52 mmol),
AgClO4 (218 mg, 1.1 mmol), and dried MS 4A (1.1 g) in anhydrous
CH2Cl2 (3.5 mL) was stirred under Ar at room temperature for 30
min and then cooled at −40 °C. To the stirred mixture was added a
mixture of 5 (261 mg, 0.53 mmol) and 6 (191 mg, 0.18 mmol) in
anhydrous CH2Cl2 (7.0 mL) through a cannula. The resulting mixture
was stirred at −15 °C for 1.5 h before the reaction was quenched by
the addition of saturated NaHCO3 aq. The mixture was diluted with
EtOAc and filtered through Celite. The organic layer was successively
washed with saturated NaHCO3 aq, water, and brine, dried over
anhydrous Na2SO4, and concentrated in vacuo. The crude product was
chromatographed on Bio-Beads S-X1 with toluene and then on silica
gel with toluene−EtOAc (14:1) to afford 10 (303 mg, 0.15 mmol,
85%). [α]D = +13.0 (c 1.0). Rf = 0.32 (7:1 toluene−EtOAc). 1H NMR:
δ 7.61 (d, 2H, J = 6.8 Hz, Ar), 7.54 (d, 2H, J = 6.8 Hz, Ar), 6.72 (d,
1H, J = 7.8 Hz, -NH), 5.37 (brs, 1H, H-2c), 5.26 (brs, 1H, H-2d), 5.03
(brs, 1H, H-1c), 4.94 (d, 1H, J = 10.7 Hz, −CH2Ph), 4.83 (d, 1H, J =
7.8 Hz, H-1a), 4.80 (d, 1H, J = 12.2 Hz, −CH2Ph), 4.76 (d, 1H, J =
10.7 Hz, −CH2Ph), 4.71 (d, 1H, J = 11.2 Hz, −CH2Ph), 4.68 (brs, 1H,
H-1d), 4.63 (d, 1H, J = 11.2 Hz, −CH2Ph), 4.59 (d, 1H, J = 12.2 Hz,
−CH2Ph), 4.51 (brs, 1H, H-1b), 4.51 (d, 1H, J = 10.7 Hz, −CH2Ph),
4.46 (d, 1H, J = 12.2 Hz, −CH2Ph), 4.44 (d, 1H, J = 11.7 Hz,
−CH2Ph), 4.24 (d, 1H, J = 12.2 Hz, −CH2Ph), 4.14 (d, 1H, J = 10.2
Hz, −CH2Ph), 4.11 (d, 1H, J=11.7 Hz, −CH2Ph), 3.91 (t, 1H, J = 8.8
Hz, H-4a), 3.85 (dd, 1H, J = 3.2, 9.0 Hz, H-3c), 3.10 (brd, 1H, J = 10.2
Hz, H-6a), 2.82 (brd, 1H, J = 9.3 Hz, H-5a), 1.99 (s, 3H, −CH3 × 3),
tert-Butyldiphenylsilyl 2,4,6-Tri-O-benzyl-β-D-galactopyra-
nosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-β-
D-glucopyranosyl-(1→2)-3,4,6-tri-O-benzyl-α-D-mannopyrano-
syl-(1→3)-[2,4,6-tri-O-benzyl-β-D-galactopyranosyl-(1→4)-3,6-
di-O-benzyl-2-deoxy-2-trichloroacetamido-β-D-glucopyrano-
syl-(1→2)-3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1→6)]-2,4-
di-O-benzyl-β-D-mannopyranosyl-(1→4)-3,6-di-O-benzyl-2-
deoxy-2-trichloroacetamido-β-D-glucopyranoside (13). A mix-
ture of Ir(COD)(PMe2Ph)2PF6 (58 mg, 70 μmol) in freshly distilled
THF (0.70 mL) was stirred at room temperature for 15 min under H2,
and the atmosphere was replaced by Ar. To the mixture of the
activated Ir complex in THF was added a solution of 12 (338 mg, 87
μmol) in THF (0.70 mL) under Ar. After the mixture was stirred for 1
h, it was evaporated under vacuo. The residue was dissolved in 90% aq
acetone (1.0 mL), and HgCl2 (113 mg, 0.42 mmol) and HgO (11 mg,
51 μmol) were then added. The mixture was stirred for 1.5 h, diluted
with EtOAc, and filtered through Celite. The combined filtrate and
washings were successively washed with aq potassium iodide, water,
and brine, dried over anhydrous Na2SO4, and concentrated in vacuo.
The residue was chromatographed on silica gel with toluene−EtOAc
(6:1) to afford 13 (256 mg, 68 μmol, 78%). [α]D = +11.8 (c 0.5). Rf =
1.93 (s, 3H, −CH3 × 3), 0.96 (s, 9H, But). 13C NMR: δ 101.0 (1JCH
=
157.2 Hz, C-1b), 99.7 (1JCH = 172.1 Hz, C-1c), 97.5 (1JCH = 171.3 Hz,
C-1d), 94.9 (1JCH = 166.3 Hz, C-1a), 92.4 (−CCl3 × 3). Anal. Calcd
for C116H124Cl3NO23Si: C, 68.48; H, 6.14; N, 0.69. Found: C, 68.22;
H, 6.23; N, 0.66.
tert-Butyldiphenylsilyl 3,4,6-Tri-O-benzyl-α-D-mannopyra-
nosyl-(1→3)-[3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1→6)]-
2,4-di-O-benzyl-β-D-mannopyranosyl-(1→4)-3,6-di-O-benzyl-
2-deoxy-2-trichloroacetamido-β-D-glucopyranoside (11). Com-
pound 10 (226 mg, 0.11 mmol) was dissolved in MeOH−THF (9:1,
1.1 mL), and 1 M NaOMe/MeOH (11 μL) was slowly added with
stirring. After the stirring was continued overnight, the solution was
neutralized by adding Amberlyst, filtered, and concentrated in vacuo.
The residue was purified by silica gel with toluene−EtOAc (4:1) to
give 11 (190 mg, 98 μmol, 88%). [α]D = +11.8 (c 1.0). Rf = 0.32 (3:1
1
0.31 (4:1 toluene−EtOAc). H NMR: δ 7.57 (d, 2H, J = 8.3 Hz, Ar),
7.53 (d, 2H, J = 7.8 Hz, Ar), 6.72 (d, 1H, J = 7.3 Hz, −NH), 6.61 (d,
1H, J = 7.8 Hz, −NH), 2.77 (d, 1H, J = 9.8 Hz, H-5a), 0.95 (s, 9H,
But). 13C NMR: δ 102.7 and 102.6 (C-1e and 1 h), 100.9 (C-1b),
100.1 (C-1c or 1f), 98.9 (C-1c or 1f), 97.8 (C-1d or 1 g), 97.2 (C-1d
or 1 g), 94.9 (C-1a), 92.6, 92.5, and 92.4 (−CCl3 × 3).
1
toluene−EtOAc). H NMR: δ 7.69 (d, 2H, J = 6.8 Hz, Ar), 7.63 (d,
2H, J = 6.8 Hz, Ar), 6.81 (d, 1H, J = 7.3 Hz, -NH), 5.13 (brs, 1H, H-1c
or -1d), 5.09 (d, 1H, J = 11.2 Hz, −CH2Ph), 4.89 (d, 1H, J = 11.7 Hz,
−CH2Ph), 4.88 (d, 1H, J = 7.8 Hz, H-1a), 4.81 (brs, 1H, H-1b), 4.80
(d, 1H, J = 10.7 Hz, −CH2Ph), 4.77 (d, 1H, J = 11.7 Hz, −CH2Ph),
4.67 (d, 1H, J = 12.2 Hz, −CH2Ph), 4.62 (d, 1H, J = 11.2 Hz,
−CH2Ph), 4.34 (d, 1H, J = 12.2 Hz, −CH2Ph), 4.21 (d, 1H, J = 12.2
Hz, −CH2Ph), 4.03 (t, 1H, J = 8.8 Hz, H-4a), 3.93 (brs, 1H, H-2c or
2d), 3.32 (dd, 1H, J=2.4 11.7 Hz, H-6a), 2.88 (brd, 1H, J = 9.3 Hz, H-
5a), 1.91 (brs, 2H, -OH × 2), 1.05 (s, 9H, But). 13C NMR: δ 101.4 (C-
1c or 1d), 100.9 (C-1c or 1d), 99.6 (C-1b), 94.9 (C-1a), 92.4
(−CCl3). Anal. Calcd for C112H120Cl3NO21Si: C, 68.96; H, 6.20; N,
0.72. Found: C, 68.82; H, 6.25; N, 0.72.
tert-Butyldiphenylsilyl 2,4,6-Tri-O-benzyl-β-D-galactopyra-
nosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-2-acetamido-β-D-gluco-
pyranosyl-(1→2)-3,4,6-tri-O-benzyl-α-D- mannopyranosyl-(1→
3)-[2,4,6-tri-O-benzyl-β-D-galactopyranosyl-(1→4)-3,6-di-O-
benzyl-2-deoxy-2-acetamido-β-D-glucopyranosyl-(1→2)-3,4,6-
tri-O-benzyl-α-D-mannopyranosyl-(1→6)]-2,4-di-O-benzyl-β-D-
mannopyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-2-acetami-
do-β-D-glucopyranoside (14). A mixture of 13 (109 mg, 28 μmol),
powdered Zn (1.4 g), and AcOH (1.4 mL) in EtOAc (14 mL) was
stirred under microwave irradiation at 150 W for 1 h. The microwave
unit was controlled to allow gentle refluxing during this reaction
period. After cooling, the insoluble materials were removed by
filtration through Celite, and the filtrate was concentrated in vacuo.
The residual crude product was purified by chromatography on silica
gel with toluene−EtOAc (2:1−5:3) to afford 14 (78 mg, 22 μmol,
79%). [α]D = +12.1 (c 0.5). Rf = 0.39 (3:2 toluene-EtOAc). 1H NMR:
δ 7.57 (d, 2H, J = 7.3 Hz, Ar), 7.51 (d, 2H, J = 7.3 Hz, Ar), 5.58 (d,
tert-Butyldiphenylsilyl 3-O-Allyl-2,4,6-tri-O-benzyl-β-D-gal-
actopyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-2-trichloroace-
tamido-β-D-glucopyranosyl-(1→2)-3,4,6-tri-O-benzyl-α-D-man-
nopyranosyl-(1→3)-[3-O-allyl-2,4,6-tri-O-benzyl-β-D- galacto-
pyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-2-trichloroacetami-
G
dx.doi.org/10.1021/jo3010155 | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Featured Article
1H, J = 6.3 Hz, -NH), 5.18 (d, 1H, J = 7.8 Hz, -NH), 5.08 (d, 1H, J =
7.3 Hz, -NH), 1.62 (s, 3H, −COCH3 × 3), 1.58 (s, 3H, −COCH3 ×
3), 1.41 (s, 3H, −COCH3 × 3), 0.96 (s, 9H, But). 13C NMR: δ 102.8
and 102.6 (C-1e and 1 h), 100.7 (C-1b), 99.6 (C-1c or 1f), 98.1, 98.0,
and 97.9 (C-1c or 1f, d and g), 94.9 (C-1a). HRMS: calcd for
C210H229N3O41Si [M + 2Na]2+, m/z 1762.2772 (100), found
1762.2761.
activated by DIC (0.077 mL, 0.50 mmol) and HOBt (62 mg, 0.45
mmol) in dichloromethane for 30 min at room temperature, was
added, and the mixture was shaken for 3 h at room temperature. The
Fmoc group was removed by 20% piperidine/NMP treatment for 5
min, followed by a 15-min treatment using the new reagent. Fmoc-
Asn(GlcNAcBn3)-OH (0.26 g, 0.31 mmol), activated by DCC (93 mg,
0.45 mmol) and HOBt (62 mg, 0.45 mmol) in NMP for 30 min at
room temperature, was added, and the mixture was shaken overnight
at room temperature. The obtained resin was again subjected to
automated synthesis to produce Boc-Thr[H-Val-Ile-Leu-Cys(Acm)-
Gln(Trt)-Thr(But)-Cys(Acm)-Gln(Trt)-Phe-Val-Met-Asn(Trt)-Lys-
(Boc)-Phe-Ser(But)-Glu(OBut)-Leu-Ile-Val-Asn(Trt)-Asn-
(GlcNAcBn3)-Ala]-Glu(OBut)-Glu(OBut)-Leu-Leu-Val-Lys(Boc)-
Gly-Leu-Ser(But)-Asn(Trt)-Ala-(Et)Cys(Trt)-Arg(Pbf)-Arg(Pbf)-
NH-resin (2.1 g). A part of the resin (0.50 g) was treated with TFA
containing 2.5% TIS and 2.5% H2O (5.0 mL) at room temperature for
1 h. After the reaction mixture was filtered, the TFA was removed by a
nitrogen stream, and the crude peptide was precipitated by diethyl
ether and dried in vacuo. The product was then treated with the low-
TfOH (TfOH/TFA/dimethyl sulfide/m-cresol, 10:50:30:10, 2 mL) at
−15 °C for 2 h. The product was precipitated by cold diethyl ether,
washed two times with the same solvent, and dried in vacuo. The
residue was dissolved in 50% aqueous acetonitrile containing 6 M urea
and 5% MPA (10 mL), and an aliquot of the solution was analyzed by
RPHPLC. Peptide 24 was identified by MALDI-TOF mass analysis
(found: m/z 4558.4 (average) (M + H)+, calcd for (M + H)+ 4559.4
(average)). The solution was stored overnight at 40 °C and loaded on
a RPHPLC column (Mightysil 5C18, 10 × 250 mm). The fraction
containing the product was isolated and lyophilized to give the desired
peptide thioester 23 (2.3 μmol, 3.8% based on the C-terminal Ala
residue on the initial resin). MALDI-TOF mass: found m/z 4204.5
(average), calcd for (M + H)+ 4204.9 (average). Amino acid analysis:
2,4,6-Tri-O-benzyl-β-D-galactopyranosyl-(1→4)-3,6-di-O-
benzyl-2-deoxy-2- acetamido-β-D-glucopyranosyl-(1→2)-3,4,6-
tri-O-benzyl-α-D-mannopyranosyl-(1→3)-[2,4,6-tri-O-benzyl-β-
D-galactopyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-2-acetami-
do-β-D-glucopyranosyl-(1→2)-3,4,6-tri-O-benzyl-α-D-manno-
pyranosyl-(1→6)]-2,4-di-O-benzyl-β-D-mannopyranosyl-(1→4)-
3,6-di-O-benzyl-2-deoxy-2-acetamido- β-D-glucopyranose
(15). To a stirred mixture of 14 (77 mg, 22 μmol) and AcOH (13
μL, 0.23 mmol) in freshly distilled THF (0.40 mL) was added 1 M
tetra-n-butylammonium fluoride (TBAF)/THF (88 μL, 88 μmol) at 0
°C. The mixture was stirred at room temperature for 20 h. The THF
was evaporated in vacuo, and the product was extracted with EtOAc.
The extract was washed with water and brine, dried over anhydrous
Na2SO4, and concentrated in vacuo. The residue was chromato-
graphed on HPLC (JAYGEL 2H, 20 × 600 mm, CH2Cl2) to give
compound 15 (62 mg, 19 μmol, 86%). Rf = 0.36, 0.27 (29:1 CHCl3−
MeOH). 1H NMR: δ 5.70 (δ, 1H, J = 6.3 Hz, −NH), 5.32 (d, 1H, J =
7.3 Hz, −NH. HRMS: calcd for C194H211N3O41 [M + 2Na]2+, m/z
1643.2184 (100), found 1643.2173.
β-D-Galactopyranosyl-(1→4)-2-deoxy-2-acetamido-β-D-glu-
copyranosyl-(1→2)-α-D-mannopyranosyl-(1→3)-[β-D-galacto-
pyranosyl-(1→4)-2-deoxy-2-acetamido-β-D- glucopyranosyl-
(1→2)-α-D-mannopyranosyl-(1→6)]-β-D-mannopyranosyl-(1→
4)-2-deoxy-2-acetamido-β-D-glucopyranose (16). Compound 15
(56 mg, 17 μmol) was dissolved in 70% aq THF (8.5 mL), and
Pd(OH) 2 (56 mg) was added. After the resulting mixture was stirred
for 2.5 d under a H2 atmosphere, the product was extracted by THF−
H2O and filtered through Celite, and the filtrate was concentrated in
vacuo. The crude product was purified by RPHPLC using an ODS
column (10 × 250 mm) in distilled water to give compound 16 (24
mg, 17 μmol, 96%). MALDI-TOF mass, found m/z 1460.68, calcd for
(M + Na)+ 1460.50. The NMR spectrum of compound 16 was
identical to a previously reported one (see the Supporting
Information).9b MALDI-TOF MS: found m/z 1460.56 (M + Na)+,
calcd for (M + Na)+ 1460.50.
β-D-Galactopyranosyl-(1→4)-2-deoxy-2-acetamido-β-D-glu-
copyranosyl-(1→2)-α-D-mannopyranosyl-(1→3)-[β-D-galacto-
pyranosyl-(1→4)-2-deoxy-2-acetamido-β-D-glucopyranosyl-
(1→2)-α-D-mannopyranosyl-(1→6)]-β-D-mannopyranosyl-(1→
4)-1,2-dideoxy-β-D-glucopyrano-2,1-D-2-oxazoline (3). Com-
pound 16 (20 mg, 14 μmol) was dissolved in distilled water (0.40
mL) containing Et3N (84 μL, 0.61 mmol), and DMC (34 mg, 0.20
mmol) was added at 0 °C. After the reaction mixture was stored at 0
°C for 1 h, the solution was applied to an ODS column (10 × 250 mm
at a flow rate of 2.5 mL/min) using a linear gradient of acetonitrile
concentration in distilled water from 1% to 15% in 14 min, and the
fraction containing the desired product was collected. After 1 M
NaOH (0.2 equiv to the compound 16 used) was added, the solution
was lyophilized to obtain the product 3 (12 mg, 8.7 μmol, 64%). The
NMR spectrum of compound 3 was identical with a previously
reported one (see the Supporting Information).9b
Asp3.73Thr1.30Ser1.69Glu4.74Gly1.01Ala2Val2.97Met0.94Ile1.11Leu4.68Phe1.86
Lys2.02
-
.
[Cys(Acm)71,77]-Saposin C (35−80)-NH2 (25). Fmoc-Rink amide
MBHA resin (0.74 g, 0.25 mmol) was subjected to an automated
synthesis to produce H-Cys(Trt)-Gly-Val-Leu-Pro-Asp(OBut)-Pro-
Ala-Arg(Pbf)-Thr(But)-Lys(Boc)-Cys(Acm)-Gln(Trt)-Glu(OBut)-
Val-Val-Gly-Thr(But)-Phe-Gly-Pro-Ser(But)-Leu-Leu-Asp(OBut)-Ile-
Phe-Ile-His(Trt)-Glu(OBut)-Val-Asn(Trt)-Pro-Ser(But)-Ser(But)-
Leu-Cys(Acm)-Gly-Val-Ile-Gly-Leu-Cys(Acm)-Ala-Ala-Arg(Pbf)-NH-
resin (2.4 g). A part of the resin (0.94 g) was treated with Reagent K
(15 mL) for 2 h at room temperature. The TFA was removed by a
nitrogen stream, and the product was precipitated by ether, which was
washed twice with the same solvent and dried in vacuo. The powder
obtained was purified by RPHPLC to obtain 25 (14 μmol, 15%).
MALDI-TOF MS: found m/z 4994.9 (M + H)+, calcd for (M + H)+
4994.6. Amino acid analysis: Asp2.91Thr1.80Ser2.47Glu3.04Pro3.96Gly5-
Ala3.06Val4.27Ile2.54Leu5.02Phe2.06Lys1.02His0.91Arg1.97
.
[Asn(GlcNAc)21, Cys(Acm)4,7,46,71,77]-Saposin C (27). The peptide
thioester 23 and peptide 25 (240 nmol each) were dissolved in 0.1 M
sodium phosphate containing 2% TCEP, 2% MPAA, and 6 M
guanidine HCl (120 μL), and the solution was stored at room
temperature. At T = 0, an aliquot of the solution was analyzed by
RPHPLC and peptide thioester 26 was identified (found m/z 4225.9
(average) (M + H)+, calcd for (M + H)+ 4226.9 (average)). After 3 h,
the product was purified by gel filtration chromatography to obtain the
glycoprotein 27 (70 nmol, 30%). MALDI-TOF MS: found m/z 9094.1
(average) (M + H)+, calcd for (M + H)+ 9095.7 (average). Amino acid
Synthesis of Saposin C Carrying GlcNAc. [Asn(GlcNAc)21,
Cys(Acm)4,7]-Saposin C (1−34)-SCH2CH2COOH (23). Fmoc-Rink
amide MBHA resin (0.74 g, 0.25 mmol) was subjected to automated
synthesis by the FastMoc protocol to produce the H-Arg(Pbf)-
Arg(Pbf)-NH-resin. To this resin, Fmoc-alanyl N-ethyl-S-tritylcysteine
(0.30 g, 0.44 mmol), activated by DIC (0.10 mL, 0.66 mmol) and
HOBt (89 mg, 0.66 mmol) in dichloromethane for 30 min at room
temperature, was added. After the mixture was shaken overnight, the
peptide chain was elongated by a synthesizer using O-(benzotriazol-1-
yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU) as
an activator and H-Glu(OBut)-Glu(OBut)-Leu-Leu-Val-Lys(Boc)-Gly-
Leu-Ser(But)-Asn(Trt)-Ala-(Et)Cys(Trt)-Arg(Pbf)-Arg(Pbf)-NH-
resin was obtained. Boc-Thr(Fmoc-Ala)-OH (230 mg, 0.45 mmol),
analysis: Asp6.89Thr3.33Ser4.41Glu7.90Pro3.96Gly6.02Ala5Val6.86Met0.89
Ile3.21Leu9.84Phe3.78Lys3.20His0.79Arg2.03
-
.
[Asn(GlcNAc)21]-Saposin C (29). The peptide 27 (45 nmol) was
dissolved in DMSO (90 μL) containing DIEA (0.15 μL, 0.86 μmol),
and AgNO3 (1.1 mg, 6.5 μmol) dissolved in distilled water (15 μL)
was added. The reaction mixture was stored at 50 °C for 2 h in the
dark. After the silver ions were quenched by DTT (10 mg), the
product was isolated by gel filtration chromatography and lyophilized.
The obtained peptide 28 (found m/z 8739.5 (average) (M + H)+,
calcd for (M + H)+ 8738.2 (average)) was dissolved in 6 M guanidine
hydrochloride (100 μL), which was dropped in 50 mM Tris-HCl
H
dx.doi.org/10.1021/jo3010155 | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Featured Article
buffer (3.0 mL) containing 0.5 M guanidine hydrochloride and 10%
DMSO (pH 8.5). After the solution was kept at 5 °C for 2 d, the
product was isolated by RPHPLC to give [Asn(GlcNAc)21]-saposin C
(29) (13 nmol, 29%). MALDI-TOF MS: found m/z 8734.4 (average)
(M + H)+, calcd for (M + H)+ 8734.2 (average). Amino acid analysis:
Asp7.01Thr3.22Ser3.27Glu7.90Pro3.93Gly6Ala4.93Val8.30Met0.66Ile4.44
mixture was stored at 50 °C for 2 h in the dark. After the silver ions
were quenched by DTT (3.6 mg, 23 μmol), the product was isolated
by gel filtration chromatography and lyophilized. The obtained powder
was dissolved in 6 M guanidine hydrochloride (100 μL), which was
dropped in 50 mM Tris-HCl buffer (6.0 mL) containing 0.5 M
guanidine hydrochloride and 10% DMSO (pH 8.5). After the solution
was stored at 5 °C for 2 d, the product was isolated by RPHPLC to
give the protein 30 (8.8 nmol, 17%). MALDI-TOF MS: found m/z
8734.4 (average) (M + H)+, calcd for (M + H)+ 8734.2 (average).
Amino acid analysis: Asp6.77Thr3.44Ser4.56Glu7.86Pro4.16Gly6-
Leu9.95Phe3.94Lys3.00His0.96Arg2.00
.
Enzymatic Transfer of Complex-Type Sugar to Saposin C.
{Asn[(Gal-GlcNAc-Man)2Man-GlcNAc2]21}-Saposin C (1). [Asn-
(GlcNAc)21]-Saposin C 29 (12 nmol) was dissolved in 50 mM
sodium phosphate containing 20% DMSO (pH 7.0, 5.0 μL).
Octasaccharide oxazoline 3 (0.17 mg, 0.12 μmol) dissolved in distilled
water (3 μL), glycosynthase (5 munit) was added, and the resulting
solution was stored for 3 h at room temperature. Octasaccharide
oxazoline (0.17 mg, 0.12 μmol) and glycosynthase (3 munit) were
further added, and the solution was stored for another 1 h. The
product was purified by an RPHPLC column for protein purification
(4.6 × 150 mm at a flow rate of 1.0 mL/min and at 50 °C) using a
linear gradient of acetonitrile concentration in distilled water
containing 0.1% TFA from 40% to 50% in 20 min and the fraction
containing the desired product were collected. The glycoprotein 1 (4.9
nmol, 41%) was obtained as a powder after lyophilization. MALDI-
TOF MS: found m/z 10156.0 (average) (M + H)+, calcd for (M + H)+
Ala5.11Val6.65Met0.66Ile3.32Leu9.37 Phe3.76Lys2.89His0.72Arg1.91
.
CD Spectrum Measurements. The synthetic saposin C's were
dissolved in 5 mM sodium phosphate containing 50 mM NaCl (pH
4.5) at a concentration of 50−100 μg/mL. The CD spectrum was
measured between 190 nm and 260 nm by a spectropolarimeter at
room temperature using a 1 mm path-length cell. The concentration of
the samples was determined by the amino acid analysis of each sample.
GCase Activation Assay. Activation of GCase by the chemically
synthesized saposin C was analyzed with 4MU-Glc as the substrate in a
detergent-free system with PS. Imiglucerase was used as the GCase
source. The enzyme assay mixture contained the final concentrations
of 200 mM sodium citrate phosphate buffer (pH 5.5), 20 μg/mL PS, 1
nM imiglucerase, 2.5 mM 4MU-Glc, and 2 mM of each chemically
synthesized saposin C (1), 29, and 30 in a 100 μL volume. Prior to the
addition of 4MU-Glc, the imiglucerase was preincubated at pH 5.5
with PS vesicles and each saposin C for 30 min at room temperature to
facilitate the interaction of the enzyme, PS, and saposin C. 4MU-Glc
was then added to the assay mixture and incubated for 30 min at 37
°C. The GCase activities were determined by the amount of
fluorescence of the liberated 4MU with Ex/Em 460/515 nm. The
data were analyzed by Mann−Whitney’s U-test.
10154.5 (average). Amino acid analysis: Asp6.87Thr3.28Ser3.55Glu7.80
-
Pro3.79Gly6Ala4.88Val8.00Met0.70Ile4.24Leu9.81Phe3.91Lys2.95His0.97Arg1.94
.
Synthesis of Nonglycosylated Saposin C. [Cys(Acm)4,7]-
Saposin C (1−34)-SCH2CH2COOH (31). The Fmoc-Rink amide
MBHA resin (0.29 g, 0.10 mmol) was subjected to automated
synthesis by the FastMoc protocol to produce the H-Arg(Pbf)-
Arg(Pbf)-NH-resin. To this resin, Fmoc-alanyl N-ethyl-S-tritylcysteine
(0.10 g, 0.15 mmol), activated by DIC (50 μL, 0.30 mmol) and HOBt
(27 mg, 0.20 mmol) in dichloroethane (0.50 mL) for 30 min at room
temperature, was added. After the mixture was shaken overnight, the
peptide chain was elongated by an synthesizer and H-Glu(OBut)-
Glu(OBut)-Leu-Leu-Val-Lys(Boc)-Gly-Leu-Ser(But)-Asn(Trt)-Ala-
(Et)Cys(Trt)-Arg(Pbf)-Arg(Pbf)-NH-resin was obtained. After Boc-
Thr(Fmoc-Ala)-OH (77 mg, 0.15 mmol), activated by DIC (50 mL,
0.30 mmol) and HOBt (27 mg, 0.20 mmol) in dichloromethane for 30
min at room temperature, was added and the mixture was shaken for 1
h at 40 °C, the peptide chain was again elongated by the FastMoc
protocol to produce Boc-Thr[H-Val-Ile-Leu-Cys(Acm)-Gln(Trt)-Thr-
(But)-Cys(Acm)-Gln(Trt)-Phe-Val-Met-Asn(Trt)-Lys(Boc)-Phe-Ser-
(But)-Glu(OBut)-Leu-Ile-Val-Asn(Trt)-Asn(Trt)-Ala]-Glu(OBut)-
Glu(OBut)-Leu-Leu-Val-Lys(Boc)-Gly-Leu-Ser(But)-Asn(Trt)-Ala-
(Et)Cys(Trt)-Arg(Pbf)-Arg(Pbf)-NH-resin (720 mg). A part of the
resin (150 mg) was treated with TFA containing 2.5% TIS and 2.5%
H2O (2.0 mL) at room temperature for 2 h. After the reaction mixture
was filtered, TFA was removed by a nitrogen stream, and the crude
peptide was precipitated by diethyl ether and dried in vacuo. The
residue was dissolved in 50% aqueous acetonitrile containing 6 M urea
and 5% MPA (5.0 mL), then stored at 40 °C for 2 d. The solution was
loaded on a RPHPLC column (Mightysil 5C18, 10 × 250 mm) and
the fraction containing the product was isolated and lyophilized to give
the desired peptide thioester 31 (344 nmol, 1.7% based on the C-
terminal Ala residue in the initial resin). MALDI-TOF mass: found m/
z 4001.3 (average), calcd for (M + H)+ 4001.7 (average). Amino acid
ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR spectra of compounds 6, 9, 11−16, and 3
and HPLC profile of the transglycosylation reaction (Figure
S1). This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: hojo@keyaki.cc.u-tokai.ac.jp, yonak@sd.dcns.ne.jp.
■
ACKNOWLEDGMENTS
■
This work was supported by a grant-in-aid for scientific research
from the Ministry of Education, Sports, Science and
Technology of Japan (23380065). We thank Dr. H. Koshino
for the HRNMR, Dr. T. Nakamura for the HRMS, and the staff
of the analysis laboratory for the elemental analysis at Riken.
We also thank Tokai University for their support with a grant-
in-aid for high technology research as well as Dr. Yuji Matsuzaki
of Tokyo Chemical Industry for kindly providing the
glycosynthase.
analysis: Asp3.24Thr1.20Ser1.57Glu4.08Gly1.06Ala2Val2.54Met0.68Ile0.85
-
REFERENCES
Leu4.45Phe1.51Lys1.74
.
■
[Cys(Acm)4,7,46,71,77]-Saposin C (32). The peptide thioester 31 and
peptide 25 (220 nmol each) were dissolved in 0.1 M sodium
phosphate containing 2% TCEP, 2% MPAA and 6 M guanidine HCl
(100 μL), then the solution was stored at room temperature. After 6 h,
the product was purified by RPHPLC to obtain 32 (61 nmol, 27%).
MALDI-TOF MS: found m/z 8890.9 (average) (M + H)+, calcd for
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