Synthetic, structural, and biosynthetic studies of an unusual phospho-glycopeptide derived from α-dystroglycan

Article abstract of DOI:10.1021/ja205473q

Aberrant glycosylation of α-dystroglycan (α-DG) results in loss of interactions with the extracellular matrix and is central to the pathogenesis of several disorders. To examine protein glycosylation of α-DG, a facile synthetic approach has been developed for the preparation of unusual phosphorylated O-mannosyl glycopeptides derived from α-DG by a strategy in which properly protected phospho-mannosides are coupled with a Fmoc protected threonine derivative, followed by the use of the resulting derivatives in automated solid-phase glycopeptide synthesis using hyper-acid-sensitive Sieber amide resin. Synthetic efforts also provided a reduced phospho-trisaccharide, and the NMR data of this derivative confirmed the proper structural assignment of the unusual phospho-glycan structure. The glycopeptides made it possible to explore factors that regulate the elaboration of critical glycans. It was established that a glycopeptide having a 6-phospho-O-mannosyl residue is not an acceptor for action by the enzyme POMGnT1, which attaches β(1,2)-GlcNAc to O-mannosyl moietes, whereas the unphosphorylated derivate was readily extended by the enzyme. This finding implies a specific sequence of events in determining the structural fate of the O-glycan. It has also been found that the activity of POMGnT1 is dependent on the location of the acceptor site in the context of the underlying polypeptide/glycopeptide sequence. Conformational analysis by NMR has shown that the O-mannosyl modification does not exert major conformational effect on the peptide backbone. It is, however, proposed that these residues, introduced at the early stages of glycoprotein glycosylation, have an ability to regulate the loci of subsequent O-GalNAc additions, which do exert conformational effects. The studies show that through access to discrete glycopeptide structures, it is possible to reveal complex regulation of O-glycan processing on α-DG that has significant implications both for its normal post-translational maturation, and the mechanisms of the pathologies associated with hypoglycosylated α-DG.

Full text of DOI:10.1021/ja205473q

ARTICLE
pubs.acs.org/JACS
Synthetic, Structural, and Biosynthetic Studies of an Unusual
Phospho-Glycopeptide Derived from r-Dystroglycan
Kai-For Mo, Tao Fang, Stephanie H. Stalnaker, Pamela S. Kirby, Mian Liu, Lance Wells, Michael Pierce,
David H. Live, and Geert-Jan Boons*
Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Road, Athens, Georgia 30602, United States
S Supporting Information
b
ABSTRACT: Aberrant glycosylation of R-dystroglycan (R-DG)
results in loss of interactions with the extracellular matrix and is
central to the pathogenesis of several disorders. To examine
protein glycosylation of R-DG, a facile synthetic approach has
been developed for the preparation of unusual phosphorylated O-
mannosyl glycopeptides derived from R-DG by a strategy in which
properly protected phospho-mannosides are coupled with a Fmoc
protected threonine derivative, followed by the use of the resulting
derivatives in automated solid-phase glycopeptide synthesis using hyper-acid-sensitive Sieber amide resin. Synthetic efforts also
provided a reduced phospho-trisaccharide, and the NMR data of this derivative confirmed the proper structural assignment of the
unusual phospho-glycan structure. The glycopeptides made it possible to explore factors that regulate the elaboration of critical
glycans. It was established that a glycopeptide having a 6-phospho-O-mannosyl residue is not an acceptor for action by the enzyme
POMGnT1, which attaches β(1,2)-GlcNAc to O-mannosyl moietes, whereas the unphosphorylated derivate was readily extended
by the enzyme. This finding implies a specific sequence of events in determining the structural fate of the O-glycan. It has also been
found that the activity of POMGnT1 is dependent on the location of the acceptor site in the context of the underlying polypeptide/
glycopeptide sequence. Conformational analysis by NMR has shown that the O-mannosyl modification does not exert major
conformational effect on the peptide backbone. It is, however, proposed that these residues, introduced at the early stages of
glycoprotein glycosylation, have an ability to regulate the loci of subsequent O-GalNAc additions, which do exert conformational
effects. The studies show that through access to discrete glycopeptide structures, it is possible to reveal complex regulation of O-
glycan processing on R-DG that has significant implications both for its normal post-translational maturation, and the mechanisms
of the pathologies associated with hypoglycosylated R-DG.
’ INTRODUCTION
be modified by R-O-mannosyl moieties (O-Man).12À14 This
type of protein modification was first described in yeast; however,
it has been established that it is widespread in nature and has, for
example, been found in plants, insects, and mammals. While
there is evidence for the existence of other mammalian glyco-
proteins modified with O-Man,15,16 R-DG is the only one
specifically identified as having this modification. The amino
acid sequence for the mucin-like region of R-DG is highly
conserved across vertebrates, reinforcing its significance. The
sites of O-glycosylation on R-DG have been mapped in unpre-
cedented detail,12,13 but the full characterization of this glyco-
protein remains a considerable challenge because of the intrinsic
heterogeneity associated with its glycosylation and the novel
structures of the glycans that continue to emerge.
The glycoprotein R-dystroglycan1 (R-DG) is a component of
the dystrophin glycoprotein complex found prominently in the
cell membranes of skeletal muscle, nerves, and brain.2,3 It has
received considerable attention because of its critical involve-
ment in linking the cytoskeleton to the extracellular matrix.
Although no mutations in the dystroglycan gene have been
identified, defects in O-glycan modification of the central mucin-
like region of R-DG have devastating effects on muscle fiber
integrity and neuronal migration and result in muscular dystro-
phies (dystroglycanopathies).2,4,5 R-DG is also the cellular
receptor for arena viruses6 and Mycobacterium leprae7 and
perturbations in R-DG processing have been associated with
cancer progression.8,9 There is evidence suggesting that O-
glycans of R-DG are ligands for extracellular proteins2,10,11 such
as laminin, agrin, and perlecan in muscle and neurexin in brain.
Aberrant glycosylation of R-DG results in loss of these interac-
tions that is causal to the pathogenesis of several disorders.2,3,5
The O-linked glycans of R-DG are structurally diverse.12,13 In
addition to the common mucin modification initiated with N-
acetyl-R-D-galactosamine (O-GalNAc) on the side chains of
serine (Ser) or threonine (Thr), these side chains can, instead,
The mannosyltransferases POMT1 and POMT2, which are
localized in the endoplasmic reticulum compartment, initiate the
attachment of a mannosyl residue to Ser/Thr by employing
dolichol phosphate-activated mannose (Dol-P-Man) as the
glycosyl donor.2,3 It is only after the O-mannosylated glycoprotein
Received: June 13, 2011
Published: August 03, 2011
r
2011 American Chemical Society
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Although the biological importance of the phosphorylated O-
mannosyl glycopeptide is evident, there are major gaps in our
understanding of its biosynthesis, the chemical nature of the
phosphodiester extension, and regulation of the formation of the
various classes of O-mannosylated structures. In this respect,
compound 2, isolated after tryptic digestion of R-DG, is the first
identified mammalian structure that has a GlcNAcβ (1À4)Man
motif in a non-N-linked glycan. The enzyme that adds the
β(1À4)GlcNAc residue has not been identified nor has the
process that controls differential extension of an O-mannosyl
residue with a 1,2- vs 1,4-linkage been uncovered. Furthermore,
compound 2 is the first vertebrate instance of a nonglycosylpho-
sphatidylinositol-anchored glycoprotein that is modified by a
phosphodiester linkage. The molecular weights from gel mobility
of R-DG produced either in the absence of POMGnT1 activity or
after chemical removal of the distal phosphoester component are
similar.21 Furthermore, POMGnT1 knockout brain proteins lack
extended O-Man structures and are not IIH6 reactive16 providing
definitive evidence for a regulatory role of POMGnT1 in the full
elaboration of the laminin binding component. The identity of
the distal ester component remains to be elucidated.
We anticipate that well-defined synthetic glycopeptides will
offer critical probes for uncovering the biosynthetic pathway and
enzymes involved in the assembly of compounds such as 2, which
includes the site where the novel laminin binding glycan is
attached, as well as proximal threonine residues on which O-
Man tetrasaccharides are found. To this end, we report here, for
the first time, a facile synthetic approach for the preparation of
phosphorylated O-mannosyl glycopeptides. Furthermore, rele-
vant R-DG biosynthetic glycopeptides intermediates were pre-
pared modified both with O-Man and O-Man-6-Phosphate
(Man-6-P). These compounds made it possible to explore factors
that regulate the elaboration of these critical glycans. The
synthetic effort has also provided the reduced trisaccharide 4
and the NMR data of this synthetic compound corresponds the
spectral features and assignments of the isolated compound
earlier reported,21 confirming the proper structural assignment
of the unusual phospho-glycan.
Figure 1. O-Mannosyl glycan 1 is a common structural motif of
glycoproteins of muscle and brain tissue. The unusual phosphoglyco-
peptide 2 is derived from the mucin-like domain of R-DG. Phospho-
diester extension of this compound is required for laminin binding.
The identity of this extension (R1) is unknown. The saccharide moiety
of 3 was identified by NMR analysis of 4, which was obtained by β-
elimination followed by reduction of the anomeric center. Glycopeptide
3 can have additional O-mannosyl sites as indicated by T*.
undergoes translocation to the Golgi that it can be elongated by
POMGnT1,17,18 which attaches a β(1,2)-linked GlcNAc residue.
The GlcNAc-β(1,2)-Man disaccharide can be further extended
by mammalian glycosyltransferases, and the resulting structures
are often variations of the tetrasaccharide NeuAcR(2À3)-Galβ-
(1À4)-GlcNAcβ(1À2)-ManR1-Ser/Thr (compound 1, Figure 1)
with different lengths and content (e.g., asialo and fucosides
R1,3-linked to GlcNAc). Small quantities of branched O-man-
nosylated structures have been found in brain and testes tissue,
and the biosynthesis of such compounds is mediated by
β(1,6)-N-acetylglucosaminyltransferase Vb (GlcNAcT-Vb or
GlcNAcT-IX) which adds a β(1,6)-linked GlcNAc residue
to O-linked mannosides that also express β(1,2)-linked
GlcNAc.19,20
Muscle-eye-brain disease (MEB), a form of muscular dystro-
phy, results when POMGnT1 activity is absent or compromised,
which also abrogates IIH6 antibody binding that serves as a
marker for functional R-DG.2 This observation suggests that
extension of the O-Man modification by a C2-linked GlcNAc is
an important element for proper R-DG function. The specific
mechanisms and glycan structures that impart functionality have
yet to be elucidated.
’ RESULTS AND DISCUSSION
Chemical Synthesis of Glycopeptides. In general, glyco-
peptides are chemically synthesized by a strategy in which
preformed glycosylated amino acids are used in stepwise solid-
phase peptide synthesis (SPPS).22 This approach requires,
however, special care in the selection of protecting groups
because O-glycosidic bonds can be hydrolyzed under acid con-
ditions and furthermore can undergo β-elimination23 upon
treatment with moderately strong bases. It has been established
that these problems can be circumvented by the use of acetyl
esters for hydroxyl protection combined with NR-9-fluorenyl-
methyloxycarbonyl (Fmoc) protected amino acids. Further-
more, solid-phase synthesis of phospho-peptides has been
accomplished by employing the Fmoc-strategy and amino acid
derivatives bearing a protected phospho-ester function. Thus, we
envisaged that solid-phase synthesis using trisaccharide threo-
nine 18 modified with stable phosphotriester and standard Fmoc
protected amino acids in combination with appropriate activa-
tion reagents would provide a facile synthetic route to phos-
phorylated O-mannosyl peptide 5 (Scheme 1).
Recently, Campbell and co-workers21 isolated a highly unu-
sual phosphorylated O-mannosyl glycopeptide from the mucin-
like domain of R-DG (compound 2, Figure 1). It was shown that
a phosphodiester modification of this compound is required for
laminin binding. Furthermore, patients with muscle-eye-brain
disease and Fukuyama congenital muscular dystrophy, as well as
mice with myodystrophy, were shown to have defects in post-
phosphoryl modification that prevent mannosyl phosphatedie-
ster (3, R = H) formation.
We designed a concise synthetic route for glycosylated amino
acid 18 based on the following strategic considerations.
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Scheme 1a
a Reagents and conditions: (a) NIS, TMSOTf, DCM, 0 °C (91%); (b) TBAF, THF/AcOH; (83%); (c) Cl3CCN, Cs2CO3, DCM (86%); (d) TMSOTf,
DCM, À35 °C (86%); (e) NIS, AgOTf, DCM, À20 °C (72%); (f) H2NNH2, HOAc, DCM/MeOH; (g) N,N-diethyl-1,5-dihyro-2,4,3-benzodioxapho-
sphepin-3-amine, 1H-tetrazole, DCM followed by mCPBA oxidation at À20 °C; (71%, 3 steps); (h) DDQ, DCM/H2O then Zn, AcOH then Ac2O, Py
(56%, 3 steps); (i) Pd(PPh3)4, THF/H2O (92%).
Trisaccharide 13, which was assembled from building blocks 6,24
7, and 8, was coupled with properly protected threonine 925 to
give amino acid modified trisaccharide 14. We expected that the
Lev ester26 of 14 could be removed without affecting the other
protecting groups providing an opportunity to install the phos-
photriester. The late stage introduction of the anomeric amino
acid and phosphotriester was expected to avoid difficulties due to
the chemical labilities of these functionalities. Benzyl ethers were
avoided as permanent protecting groups for the carbohydrate
moiety because they may be difficult to remove after glycopep-
tide assembly,27 and furthermore, catalytic hydrogenation to
remove benzyl ethers of an Fmoc protected glycosylated amino
acid is problematic because it will result in loss of the Fmoc
protecting group. Therefore, we explored 2-methylnaphthyl
(Nap) ethers28 and acetals29 as permanent protecting groups.
These protecting groups are significantly more stable to acidic
conditions compared to a p-methoxybenzyl ether and acetal but
can readily be removed by oxidation with 2,3-dichloro-5,6-
dicyano-1,4-benzoquinone (DDQ).30,31
Coupling of the thiogalactoside 6 with glycosyl acceptor 7 in
the presence of NIS/TMSOTf32 as the promoter provided
the disaccharide 10 in a yield of 91% as only the β-anomer
(Scheme 1). Removal of the anomeric dimethylthexylsilyl (TDS)
ether of 10 was easily accomplished by treatment with tetra-n-
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ARTICLE
Scheme 2a
a Reagents and conditions: (a) standard Fmoc solid-phase peptide synthesis, HBTU/HOBt, DIPEA, DMF and manual coupling of 18, HATU, DIPEA,
DMF; (b) 1% TFA/DCM; (c) TFA/TIPS/DCM (5/5/90) followed by Pd/C, H2, MeOH/DCM then 5% H2NNH2/MeOH.
butylammonium fluoride (TBAF) buffered with AcOH in THF,
and the resulting hemiacetal 11 was immediately converted into
the corresponding trichloroacetimidate 12 by reaction with
trichloroacetonitrile in the presence of Cs2CO3. A TMSOTf-
promoted glycosylation of trichloroacetimidate 12 with thio-
mannosyl acceptor 8 gave trisaccharide 13 in an excellent yield of
86%. In this coupling reaction, no aglycon transfer33À35 of the
thioethyl moiety of 8 was observed, and furthermore, only the β-
anomer was formed due to neighboring group participation by
the 2,2,2-trichloroethoxycarbamate (Troc) function. Compound
13 could be used as a glycosyl donor without a need for any
manipulations and coupling with threonine glycosyl acceptor 9 in
the presence of NIS/AgOTf36 as the activator furnished glyco-
sylated amino acid 14 in 72% yield. Phosphorylation of 14 was
achieved by a three-step procedure involving selective removal of
the Lev ester using hydrazine acetate to give alcohol 15, which
was reacted with N,N-diethyl-1,5-dihyro-2,4,3-benzodioxapho-
sphepin-3-amine37 in the presence of 1H-tetrazole to give an
intermediate phosphite ester which was oxidized in situ with
meta-chloroperoxybenzoic acid (mCPBA) to give the phospho-
triester 16. Next, oxidative removal of the Nap ether and
naphthalene acetal was easily accomplished by treatment with
excess of DDQ to give an intermediate triol which was used
without purification in the subsequent step which involved
reductive removal of two Troc moieties using Zn/AcOH fol-
lowed by acetylation of the amines and alcohols using acetic
anhydride in pyridine provided compound 17. Finally, deal-
lylation of 17 using Pd(PPh3)4 as the catalyst furnished the
corresponding glycosylated threonine 18, which was employed
for the assembly of the glycopeptide 5. A full analysis of the 1H
and 13C NMR (HMQC and HMBC) spectra of this glycan was
carried out confirming the desired structure and linkages.
Hyper-acid-sensitive Sieber amide resin38 was selected as the
solid support for glycopeptides assembly, since release of the
compound can be accomplished under mild conditions using 1%
TFA in DCM. It was expected that these conditions would not
interfere with the rather labile phosphotriester function. The
assembly of the target glycopeptide 5 was performed on an
automatic solid-phase peptide synthesizer using a standard
Fmoc-based protocol using 2-(1H-benzotriazole-1-yl)-oxy-
1,1,3,3-tetramethyl-uronium hexafluorophosphate (HBTU)
and 1-hydroxybenzotriazole (HOBt)39 as the activator system
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Scheme 3a
a Reagents and conditions: (a) BnOH, NIS, TMSOTf, DCM, À20 °C (58%); (b) H2NNH2, HOAc, DCM/MeOH; (c) N,N-diethyl-1,5-dihyro-2,4,3-
benzodioxaphosphepin-3-amine, 1H-tetrazole, DCM followed by mCPBA, À20 °C; (65%, 3 steps); (d) Zn, Ac2O/AcOH/THF, CuSO4(aq) (71%);
(e) Pd(OH)2/C, H2, MeOH/DCM (78%); (f) NaOMe/MeOH/DCM followed by NaBH4, H2O (75%, 2 steps).
(Scheme 2). The glycosylated threonine 18 was introduced
manually using 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetra-
methyluronium hexafluorophosphate (HATU)40 as the activator
in the presence of the N,N-diisopropylethylamine (DIPEA) in
DMF. Under these conditions, only low equivalents (2 equiv) of
glycosylated amino acid were required and it was possible to
recover some of the excess starting material (∼0.7 equiv). The
fully assembled glycopeptide 19 was released from the resin by
treatment in 1% TFA in DCM to give, after purification by size
exclusion column chromatography (LH-20), the fully protected
phosphoglycopeptide 20. The trityl protecting group of the
glutamine moiety was removed by treatment with a cocktail of
trifluoroacetic acid (TFA)/ triisopropylsilane (TIPS)/DCM
(5/5/90, v/v/v) and subsequent hydrogenolysis using Pd/C as
the catalyst in a mixture of MeOH and DCM resulted in the removal
of the phosphate and threonine side chain protecting groups and
finally treatment with anhydrous hydrazine in MeOH resulted in the
cleavage of the acetyl esters to give the target glycopeptide 5.
Previously, the structure of the carbohydrate moiety of phos-
phoglycopeptide 3 was determined by NMR analysis of com-
pound 4 (Figure 1), which was obtained by treatment of
R-DG with aqueous NaOH (1 M) to release oligosaccharides
followed by reduction with NaBH4 and purification.21 As out-
lined above, the structure of 3 is highly unusual and the presence
of a β-GlcNAc moiety linked to the C-4 hydroxyl of a mannosyl
threonine residue had never been observed before. In this
respect, all previously identified mammalian O-mannosylated
glycoproteins are modified by a GlcNAc moiety linked to C-2 of
mannosyl serine or threonine. Thus, it was prudent to ascertain
the chemical structure of 3 by chemically synthesizing compound
4 and comparing its spectroscopic characteristics with that of
isolated material. For this purpose, thioglycoside 13 was con-
verted into benzyl glycoside 21 by a NIS/TMSOTf mediated
glycosylation with benzyl alcohol. Next, the Lev ester of 21 was
removed under standard conditions, and the resulting alcohol
phosphorylated by a two-step procedure entailing reaction with
N,N-diethyl-1,5-dihyro-2,4,3-benzodioxaphosphepin-3-amine in
the presence of 1H-tetrazole followed by in situ oxidation with
mCPBA to give the phospho-trisaccharide 23. The Troc moieties
of 23 were converted into acetamido residues by treatment with
Zn in a mixture of aqueous CuSO4, Ac2O, AcOH, and THF41
to give phospho-trisaccharide 24, which was subjected to
hydrogenolysis over Pd(OH)2/C in MeOH/DCM followed by
deacetylation and finally reduction of the anomeric center with
NaBH4 to furnish the target compound 4 (Scheme 3) whose
characterization by NMR is described below.
NMR Characterization of Trisaccharide 4 and Glycopep-
tide 5. NMR provides a powerful tool to characterize carbohy-
drate structures with several of its spectral parameters being very
sensitive to features of the glycosidic linkage. In view of the
novelty of the phospho-trisaccharide, we compared NMR param-
eters of the synthetic trisaccharide 4 with reported data to
confirm the proposed structural identity.
The assignment of saccharide resonances of 4 and 5 started at
the anomeric center of each monosaccharide residue as the
coordinates of their proton and carbon shifts occur in distinct
13
region of the 2-dimensional 1HÀ C correlation spectrum.
Homonuclear COSY and TOCSY experiments42 were used to
identify the proton-coupled spin systems of each of the mono-
13
saccharide residues. 1HÀ C HMQC experiments,42 which
provide correlations between directly bonded protons and
carbons, were employed to assign carbon resonances, similar to
the strategy followed for the material isolated from natural
sources.21 Vicinal proton coupling constants between the H1
and H2 protons of each monosaccharide residue, 8.4 Hz for
GalNAc and 8.3 Hz for GlcNAc, were used to establish the β
anomeric configurations. HMBC experiments,42 which can re-
veal long-range through-bond correlation, permitted identifica-
tion of connections across glycosidic bonds. In this respect, cross
peaks correlating C1 of GalNAc to H3 of GlcNAc and C3 of
GlcNAc to H1 of GalNAc, as well as C4 of the Man-ol to H1 of
GlcNAc and H4 of Man-ol to C1 of GlcNAc, were observed.
The resulting assignments of synthetic compounds 4 and 5
and the reported data for the reduced trisaccharide isolated from
R-DG are summarized in Table 1. As can be seen, there is excellent
agreement in chemical shifts of 4 and the previously isolated
material confirming that R-DG is modified with a trisaccharide
having an unusual GlcNAc-(1À4)-Man-6-P moiety. The small
variations in chemical shifts of the A5 and A6 sites of 4 between
those reported previously and here are likely due to small
differences in the pH of the samples, which could have an impact
on chemical shifts through minor changes in the average ioniza-
tion state of the phosphate group. It was observed that the
chemical shifts of the GlcNAc and GalNAc residues of the
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Table 1. Chemical Shifts of Carbon and Proton Signals of
Carbohydrate Moieties of Compound 4, the Corresponding
Isolated Derivative, and Glycopeptide 5a
synthetic trisaccharide 4 Yoshida-Moriguchi et al. glycopeptide 5
H1
C13
H1
C13
H1
C13
A1 3.61, 3.86
A2 3.90
A3 3.67
A4 4.00
A5 3.82
A6 3.87, 4.01
B1 4.68
B2 3.83
B3 3.71
B4 3.47
B5 3.55
B6 3.69, 3.92
C1 4.48
C2 3.83
C3 3.72
C4 3.90
C5 3.69
65.85
73.18
71.93
78.76
71.90
67.66
103.53
57.14
83.68
71.87
77.47
63.79
104.29
55.23
73.18
70.39
77.70
63.64
3.63, 3.87
3.92
65.80 4.95
73.10 3.85
71.90 3.82
78.70 3.90
71.70 3.81
68.30 3.98
103.50 4.61
57.11 3.86
83.60 3.80
71.80 3.52
77.50 3.57
104.15
72.37
71.71
79.17
73.15
65.32
103.96
56.98
83.50
73.38
77.98
3.68
4.02
3.89
3.92, 4.06
4.64
3.85
Figure 2. 1H NMR spectra taken in 90% H2O/10% D2O of the amide
protons of (A) Ac-GAIIQTPTLG-NH2 (26), (B) this peptide with the
phophorylated trisaccharide attached at T6 (5), and (C) this peptide
with an R-linked GalNAc at T6 (27). Peak numbering by residue
starting from N-termini, chemical shifts relative to DSS.
3.76
3.49
3.54
3.71, 3.94
4.49
63.80 3.69, 3.76 63.44
104.20 4.50
55.21 3.84
73.20 3.73
70.40 3.91
77.70 3.68
63.80 3.75
104.20
55.26
73.25
70.45
77.69
63.70
3.84
3.74
Table 2. Kinetic Parameters for the Modification of Glyco-
peptides 28À33 by POMGnT1
3.91
3.69
glycopeptide
Km (mM)
Vmax (nmol/h)
C6 3.74
13
3.77
a 1HÀ C HMQC NMR correlation spectra of compound 4 and the
corresponding isolated derivative are presented in the Supporting
Information (Figure SI 1). Letters refer to the sugar residues proceeding
from the reducing end, A = Man, B = GlcNAc, and C = GalNAc, with the
associated numbers referring to the carbon atom involved.
GAIIQT(Man)PTLG (28)
GAIIQT(Man-6-P)PTLG (29)
GAIIQTPT(Man)LG (30)
GAIIQT(Man)PT(Man)LG (31)
YVEPT(Man)AV (32)
1.7
1.03
no reaction
no reaction
-
-
2.3
4.0
no reaction
1.17
0.52
-
RIRTT(Man)TSGVPR (33)
reduced trisaccharide 4 and the glycopeptide 5 were very similar,
while the structural change associated with the cyclization of the
mannitol in 4 to a mannosyl in 5 is, as expected, accompanied by
some larger chemical shift changes. The insensitivity of the shifts
of the peripheral sugars between the trisaccharide and the
glycopeptide suggests at most limited interactions between these
carbohydrate residues and the peptide backbone.
Electron micrograph images of the mucin-like central region of
R-DG, which contains multiple O-Man and O-GalNAc
residues,43 indicate that it has an extended global conformation.
This type of organization has been recognized for sites with O-
GalNAc substitutions, and can be rationalized in terms of
intramolecular interactions between the proximal sugar residue
and the polypeptide backbone.44 To establish whether an O-
Man-6-P residue can affect the organization of the peptide
backbone, the patterns of peptide amide shifts, which are known
to be sensitive to backbone conformation, were analyzed for
glycopeptide 5, the similar unmodified peptide 26 (Ac-
GAIIQTPTLG-NH2), and O-GalNAc derivatized glycopeptide
27 (Ac-GAIIQT(R-GalNAc)PTLG-NH2).
Amide proton resonances of the peptide and glycopeptides
were observed in 90% H2O/10% D2O solutions. TOCSY
spectra42 with correlations originating from the amides were
used to identify the type of amino acid associated with the
individual amides. This experiment was complemented by
ROESY42 spectra that provide through-space correlations be-
tween the NH and the R proton of the preceding residue to
establish the identity of the neighboring residue, thereby resol-
ving ambiguities where there are multiple instances of the same
amino acid. As can be seen in Figure 2, the patterns of peptide
amide shifts of phosphoglycopeptide 5 (B) and peptide 26 (A)
are very similar. The largest difference due to the presence of the
O-Man-6-P modification was a downfield shifts of ∼0.04 ppm for
amides of residue 6 (saccharide modified T) and residue 9,
indicating the absence of significant peptide conformational
differences between two compounds. Glycopeptide 27 (C), which
is modified by an O-GalNAc moiety, exhibited more pronounced
changes, and in particular, the amide proton of glycosylated
T6 was shifted downfield by 0.35 ppm shift and T8 upfield by
∼0.07 ppm. These observations are consistent with structural
stabilization of 27 that is expected to arise from hydrogen bond-
ing interactions between the O-GalNAc amide proton and the
peptide backbone as well as interactions between the methyl
group of the acetamide moiety of O-GalNAc and the side chain of
the amino acid in the i+2 position of mucin structures.44
The data are consistent with a model in which mannosyl
modification does not exert major conformational effect on the
peptide backbone.45 These residues are, however, introduced at
the early stages of glycoprotein glycosylation (ER-based process)
and therefore have an ability to regulate the loci of subsequent O-
GalNAc additions through the action of polypeptide O-GalNAc
glycotransferases (Golgi-based process). Thus, by influencing
the patterning of O-GalNAc moieties on R-DG, O-mannosyla-
tion will indirectly influence the protein backbone conforma-
tion, which in turn is expected to impact specific molecular
recognition.
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Figure 3. MS/MS fragmentation of 31 following incubation with recombinant POMGnT1 (m/z 1560) yielded a glycopeptide mixture containing one
nonextended mannose and one GlcNAc-extended mannose as defined by accurate mass measurement in which one glycopeptide was extended at
Thr379 while the other isobaric glycopeptide was extended at Thr381. The generated b and y fragment ions allowed for confident identification and
assignment of the extended Thr residues. The b and y fragment ions shown in blue support the extension of Thr 379, while the b and y ions shown in red
support the extension of O-mannosyl residue on Thr381. In particular, the y3+Hex-HexNAc defines Thr381 as a site of extension while b6+Hex-
HexNAc define Thr-379 as being extended.
Enzymatic Studies. Recent studies have shown that, in
addition of β-D-GlcNAc-(1À2)-R-D-Man-Thr motifs, R-DG is
modified with an unusual β-D-GlcNAc-(1À4)-6-P-R-D-Man-Thr
moiety in the core of a glycan that is functionally relevant.21
Biosynthetic mechanisms that control elaboration R-D-Man-Thr
into various classes of extended structures have yet to be defined.
So far, the phosphorylated trisaccharide β-D-GalNAc-(1À3)-β-
D-GlcNAc-(1À4)-6-O-P-R-D-Man has only been associated with
a specific site of R-DG, namely, Thr379, though other loci have
been suggested,21 and it appears that this site is not modified by
the more common tetrasaccharide structure (compound 1,
Figure 1). Thus, a specific mechanism must control 1,2- vs 1,4-
extension of O-mannosyl residues in a site-specific manner. It is
possible that phosphorylation of an O-mannosyl residue blocks
the action of POMGnT1 thereby preventing the formation of
(1À2)-linked oligosaccharides. Furthermore, it is conceivable
that the underlying peptide influences the activity of POMGnT1,
directing activity to specific positions.
bis-O-mannosylated at T-6 and T-8. In addition, compounds 32
and 33 were prepared which are derived from residues 411À416
and 479À489 of R-DG and have an O-Man moiety at one of the
threonine residues. The apparent kinetic parameters for the
addition of a β(1À2)-O-GlcNAc moiety to the glycopeptide
acceptors 28À33 by recombinant POMGnT1 were determined
following earlier reported assays.17,18 As can be seen in Table 2,
the mannosyl moiety of glycopeptide 28 was readily modified by
POMGnT1, whereas the phophorylated derivative 29 was not an
acceptor for the enzyme. Interestingly, the mannosyl moiety at
T8 of glycopeptide 30 was also not an acceptor for POMGnT1,
while the bis-O-mannosylated glycopeptide 31 exhibited similar
kinetic parameters compared to monomannosylated derivative
28. The transformed product of 31 was further examined by
tandem mass spectrometry. The resulting MS/MS spectra
revealed a mixture of two structurally similar glycopeptides in
which either Thr379 or Thr381 was observed to be extended
with a O-GlcNAc residue following incubation with the recom-
binant POMGnT1 enzyme (Figure 3). Based on the b and y
fragment ions generated from the CID of m/z 1560, a confident
assignment can be made confirming that one or the other of the
O-mannosyl residues were extended with a GlcNAc. For exam-
ple, the observation of b6 + Hex-HexNAc and y4 + Hex indicates
that Thr379 is extended with GlcNAc in one glycoform. Like-
wise, the observation of b7 + Hex and y3 + Hex-HexNAc in the
To test these hypotheses, glycopeptides 28À31, based on the
sequence GAIIQTPTLG (amino acids 374À383 of full-length
R-DG) were prepared, including those modified with the unusual
phospho-trisaccharide (see Figure 1, compounds 2 and 3). Thus,
compounds 28 and 29 have an O-Man or O-Man-6-P on
T6 (corresponding to Thr379 of R-DG), respectively. Com-
pound 30 carries an O-Man residue on T8 and derivative 31 is
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same MS/MS spectra indicated that Thr381 is extended with a
GlcNAc residue in the other. Although we were able to observe
signature b and y ions indicating the presence of two peptides
with a single Thr extended, we were unable to observe an
instance in which both Thr residues were extended simulta-
neously. Compound 32 (411À416 of R-DG) exhibited glycosyl-
accepting properties for POMGnT1; however, the Km was less
favorable compared to those of compound 28, and finally,
compound 33 (479À489 of R-DG) was not modified by the
enzyme.
Collectively, the data suggest a model in which regulation of
modification of O-mannosyl residues by either a β(1,2)- or
β(1,4)-O-GlcNAc moiety is controlled by phosphorylation of
the C-6 position of an O-mannosyl residue at an early stage of the
biosynthetic transformation. Although the more common O-
mannosyl structures having a β-D-GlcNAc-(1À2)-R-D-Man link-
age have not been observed at T379 of R-DG, experiments with
model peptides 28 and 31 indicate that this site can readily be
modified by POMGnT1. It is well-established that mannosyla-
tion of Ser and Thr moieties of R-DG by POMT1 and POMT2
takes place in the ER, whereas POMGnT1 appears to be located
in the Golgi apparatus. Thus, it is conceivable that a site-specific
phosphorylation of O-Man can direct its modification to a β-D-
GlcNAc-(1À4)-6-P-R-D-Man moiety. Future identification of
the kinase and GlcNAc-transferase responsible for the biosynth-
esis of the unusual phospho-trisaccharide will make it possible to
confirm the model.
The data presented in Table 2 also indicate that the modifica-
tion preference of O-Man residues by POMGnT1 is modulated
by the peptide sequence and glycosylation status of neighboring
sites. A particularly surprising finding was that compound 30,
which has an O-Man residue at T-8, was not modified by
POMGnT1, whereas compound 31, which has an O-Man at
T-6 and T-8, was glycosylated by this enzyme at either site, but
not both. While this complicates the quantitative analysis of the
enzyme kinetic results, it points out that proximal glycosylation
as well as peptide sequence can affect the locus of POMGnT1
activity. Further studies are required to provide a rationale for this
observation.
The enzyme POMGnT1 was previously assumed to comprise
the obligatory activity in extending the initial O-Man residue with
a β(1À2)-GlcNAc residue. The recent elucidation of a novel O-
mannosyl modification having a β(1À4) linked O-GlcNAc, as
well as a 6-phosphate, suggests the existence of previously
unappreciated enzymatic activities. While several questions
remain to be answered, the observations reported here shed
light on the biosynthesis of O-mannosylated structures of R-DG
and the impact of POMGnT1 on determining the structural fate
of the glycan. First, the chemical synthesis of an alditol analogue
of the phospho-glycan confirmed the proper structural elucida-
tion of the novel form of glycosylation. Evaluation of several
glycopeptides indicates that when the relevant O-Man on residue
T379 does not have a 6-phosphoryl modification; it is an
appropriate substrate for POMGnT1, which, if modified by this
enzyme, would direct the glycan to a different biosynthetic path.
Furthermore, previous glycan mapping of R-DG suggests that
residue T379 is not modified by the more common tetrasacchar-
ide structure. These observations suggest that regulation by a
temporal separation needs to exist between the activities asso-
ciated with generating the Man-6-P structures and exposure to
POMGnT1, and suggests that the former shows considerable
specificity to this site in the sequence. The use of several possible
O-man glycopeptide substrates for POMGnT1 also demon-
strated that its activity is modulated by the underlying polypep-
tide sequence and the presence of glycans in the vicinity of a
potential acceptor site. Conformational analysis by NMR of
several glycopeptides showed that the O-mannosyl modification
does not exert major conformational effect on the peptide
backbone. These residues are, however, introduced at the early
stages of glycoprotein glycosylation, and therefore have an ability
to regulate the loci of subsequent O-GalNAc additions, which do
exert conformational effects. Collectively, the results reveal
additional levels of complexity in the regulation of O-glycan
processing on R-DG and provide important new clues in
searching for the enzyme activities associated with critical steps
in the processing of R-DG and spatial localization of glycans. The
synthetic phosphoglycopeptides presented here will provide
further opportunities to explore the biosynthesis and biological
properties of glycans of R-DG. In particular, we anticipate that
they will make it possible to identify enzymes associated with
phosphorylation and β(1À4)GlcNAcT activity and the chemical
nature of the phosphodiester moiety.
’ CONCLUSIONS
Although it has been evident that the glycan structures on R-
DG, particularly those associated with O-mannosyl linkages, are
key elements in critical interaction with components of the
extracellular matrix (ECM), only recently have structural features
of compounds been revealed that directly interact with the ECM.
The core structure has features distinct from the more abundant
O-mannosyl glycan, which, although apparently not directly a
ligand for the ECM, plays a role in the ultimate function of R-DG.
The modification of the initiating O-mannosyl residues by a
β(1À2)-GlcNAc or a β(1À4)-GlcNAc moiety raises intriguing
questions regarding the regulation of biosynthetic pathways
associated with these structures. This issue is all the more
significant in light of the pathologies associated with aberrations
in either of these glycans. Faced with the myriad difficulties of
fully elucidating these aspects using cell-based studies, we have
exploited chemical synthesis to provide appropriate well-defined
models of intermediate forms of the post-translational pathway.
The availability of these reagents has allowed us to pursue the
experiments described, which has provided insights into glycan
modifications of this glycoprotein.
’ EXPERIMENTAL PROCEDURES
Reagents and General Procedures. Reagents were obtained
from commercial sources and used as purchased. Dichloromethane
(DCM) was freshly distilled using standard procedures. Other organic
solvents were purchased anhydrous and used without further purifica-
tion. Unless otherwise noted, all reactions were carried out at room
temperature in oven-dried glassware with magnetic stirring. Molecular
sieves were flame-dried under high vacuum prior to use. Organic
solutions were concentrated under diminished pressure with bath
temperatures <40 °C. Flash column chromatography was carried out
on silica gel G60 (Silicycle, 60À200 μm, 60 Å). Thin-layer chromatog-
raphy (TLC) was carried out on Silica gel 60 F254 (EMD Chemicals Inc.)
with detection by UV absorption (254 nm) were applicable, and
by spraying with 20% sulfuric acid in ethanol followed by charring
at ∼150 °C or by spraying with a solution of (NH4)6Mo7O24.H2O
(25 g/L) in 10% sulfuric acid in ethanol followed by charring
1
at ∼150 °C. H and 13C NMR spectra were recorded on a Varian
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Inova-300 (300/75 MHz), a Varian Inova-500 (500/125 MHz), and a
Varian Inova-600 (600/150 MHz) spectrometer equipped with Sun
workstations. Multiplicities are quoted as singlet (s), broad singlet (br s),
doublet (d), doublet of doublets (dd), triplet (t), or multiplet (m).
Spectra were assigned using COSY, DEPT, and HSQC experiments.
Signals marked with a superscript Roman numeral I were the reducing
end, whereas II and III were the second sugar from the reducing end and
the nonreducing end, respectively. All chemical shifts are quoted on the
δ-scale in parts per million (ppm). Residual solvent signals were used as
an internal reference. Reverse-phase HPLC was performed on an Aglient
1200 series system equipped with an autosampler, fraction-collector,
UV-detector, and eclipse XDB-C18 column (5 μm, 4.6 Â 250 mm or
9.4 Â 250 mm) at a flow rate of 1.5 mL/min. Mass spectra were recorded
on an Applied Biosystems 4800 MALDI-TOF proteomics analyzer. The
matrix used was 2,5-dihydroxybenzoic acid (DHB) and Ultamark 1621
as the internal standard.
General Methods for Glycopeptide Synthesis. Glycopep-
tides derived from the GAIIQTPTLG sequence were synthesized by an
automatic solid-phase peptide synthesizer (Applied Biosystems, ABI
433A) on Rink amide resin (79 mg, 0.05 mmol, loading: 0.63 mmol/g)
or Sieber amide resin (70.4 mg, 0.05 mmol, loading: 0.71 mmol/g) via
NR-Fmoc-based approach. 2-(1H-Benzotriazole-1-yl)-oxy-1,1,3,3-tetra-
methyl-uronium hexafluorophosphate (HBTU)/1-hydroxybenzotria-
zole (HOBt) were employed as the coupling reagents. The coupling
of glycosylated amino acids was performed manually using 2-(7-aza-1H-
R-D-glucopyranosyl Trichloroacetimidate (12). To a stirred and
cooled (0 °C) solution of 10 (524 mg, 0.478 mmol) in a mixture of THF
and AcOH (20/1, v/v, 7.2 mL) was added 1 M TBAF (1.43 mL, 1.43
mmol). After stirring at room temperature for 24 h, the reaction mixture
was diluted with DCM (180 mL) and washed with saturated NaHCO3
(2 Â 170 mL) and brine (120 mL). The organic layer was dried
(MgSO4) and filtered, and the filtrate was concentrated under reduced
pressure. The resulting yellow oil was purified by flash chromatography
over silica gel (EtOAc/hexanes, 1/1, v/v) to give 11 as an inseparable
mixture of R- and β-anomer (378 mg, 83%) as an amorphous white
solid: Rf = 0.34 (EtOAc/hexanes, 1/1, v/v). HR MALDI-TOF MS m/z:
calcd for C35H38Cl6N2O16 [M+Na]+, 975.0250; found 975.0255. To a
stirred and cooled (0 °C) solution of R- and β-hemiacetals 11 (350 mg,
0.368 mmol) in DCM (5.5 mL) was added trichloroacetonitrile
(3.68 mL, 3.68 mmol) followed by Cs2CO3 (120 mg, 0.368 mmol)
under Ar. After stirring at room temperature for 2 h, the reaction mixture
was filtered, and the filtrate was concentrated under reduced pressure.
The resulting yellow oil was purified by flash chromatography over silica
gel (EtOAc/hexanes, 1/2, v/v) to give 11 (347 mg, 86%) as an
amorphous white solid: Rf = 0.19 (EtOAc/hexanes, 1/2, v/v). 1H
NMR (300 MHz, CDCl3): δ 8.73 (s, 1H, Cl3CCdNH), 7.95À7.45
(m, 7H), 6.60 (br s, 1H, H-1I), 5.74 (s, 1H, >CHNap), 5.71 (d, 1H, J =
4.5 Hz, NHTrocI), 5.45 (dd, J = 3 and 11.4 Hz, 1H, H-3II), 5.29 (d, J =
2.7 Hz, 1H, H-4II), 5.16 (d, J = 9 Hz, 1H, H-1II), 5.07 (br s, 1H,
NHTrocII), 4.84À4.54 (m, 4H, OCH2CCl3I and II), 4.38 (dd, 1H, J = 4.7
and 10.4 Hz, H-6eI), 4.29 (t, J = 9.8 Hz, 1H, H-3I), 4.13À3.83 (m, 7H,
H-2I, H-4I, H-5I, H-6aI, H-5II and H-6aII and H-6eII), 3.51À3.42 (m, 1H,
H-2II), 21.5 (s, 3H), 1.88 (s, 3H), 1.76 (s, 3H). 13C NMR (75 MHz,
CDCl3): δ 170.13, 169.97, 169.91, 160.48, 154.51, 153.85, 134.02,
133.80, 133.00, 128.81, 128.45, 127.90, 127.02, 126.67, 125.86, 123.33,
102.45, 98.35 (C-1II), 95.56 (C-1I), 95.50, 95.06, 90.99, 80.07 (C-4I),
74.68, 74.28, 72.19 (C-3I), 71.01 (C-5II), 66.73 (C-6I), 68.31 (C-3II),
66.35 (C-4II), 65.85 (C-5I), 60.68 (C-6II), 54.73 (C-2I), 52.40 (C-2II),
20.81, 20.60, 20.36.
Ethyl O-[3,4,6-Tri-O-acetyl-2-deoxy-2-(2,2,2-trichloroethoxy)-
carbonylamino-β-D-galacto-pyranosyl]-(1f3)-O-[2-deoxy-
4,6-O-(2-naphthylidene)-2-(2,2,2-trichloroethoxy)carbonyl-
amino-β-D-glucopyranosyl]-(1f4)-2-O-acetyl-6-O-levulinoyl-
3-O-(2-methylnaphthyl)-1-thio-R-D-mannopyranoside (13). A
mixture of the thioglycosyl acceptor 8 (250 mg, 0.50 mmol) and
trichloroacetimidate 12 (367 mg, 0.33 mmol) and 4 Å molecular sieves
(900 mg) in DCM (9 mL) was stirred under Ar for 1 h. The reaction was
cooled (À35 °C) and TMSOTf (12 μL, 0.066 mmol) was added. After
stirring at À30 °C for 1.5 h, the reaction was quenched by the addition of
Et3N (0.1 mL). The reaction mixture was filtered, and the filtrate was
concentratedunder reducedpressure. The resulting yellow oil waspurified
by flash chromatography over silica gel (EtOAc/hexanes, 1/1, v/v) to give
13 (372 mg, 86%) as an amorphous white solid: Rf = 0.26 (EtOAc/
hexanes, 1/1, v/v). 1H NMR (500 MHz, CDCl3): δ 7.85À7.42 (m, 14H),
5.73 (br s, 1H, NHTrocII), 5.52 (s, 1H, >CHNap), 5.39 (s, 1H, H-2I);
5.24À5.13 (m, 4H, H-1I, H-3III, H-4III and NHTrocIII), 4.94À4.69
(m, 8H, H-1II, H-1III, OCH2Nap and OCH2CCl3II and III), 4.57 (br s,
1H, H-6aI), 4.25À4.16 (m, 3H, H-5I, H-6eI and H-3II), 4.03À4.00
(m, 3H, H-4I, H-6eII and H-6eIII), 3.86 (dd, 1H, J = 3.5 and 9 Hz,
H-3I), 3.81 (br s, 1H, H-6aIII), 3.70À3.63 (m, 4H, H-2II, H-4II, H-2III and
H-5III), 3.49 (t, 1H, J = 10.3 Hz, H-6aII), 3.41 (br s, 1H, H-5II), 2.93À2.52
(m, 6H), 2.25 (s, 3H), 2.15 (s, 3H), 2.07 (s, 3H), 1.85 (s, 3H), 1.77
(br s, 3H), 1.25 (t, 1H, J = 7.3 Hz). 13C NMR (75 MHz, CDCl3): δ
207.87, 172.92, 170.25, 170.22, 154.45, 154.05, 135.77, 134.58, 133.82,
133.37, 133.09, 132.92, 128.43, 128.34, 128.21, 128.01, 127.94, 127.87,
126.66, 126.39, 126.33, 126.01, 125.65, 125.55, 123.65, 101.54, 101.07
(C-1II and III), 96.01, 95.85, 82.78 (C-1I), 80.22 (C-4II), 78.42 (C-3II),
77.44 (C-3I), 76.81 (C-4I), 76.00, 74.51, 72.44, 71.15 (C-2I), 70.77
(C-5III), 69.68 (C-5I and C-3III), 68.78 (C-6II), 66.48 (C-4III), 66.07
benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate
(HATU)/N,N-diisopropylethylamine (DIPEA) as the activating reagents.
Upon completion of the manual coupling reaction as determined by
the Kaiser test, resins were resubjected to the automatic synthesizer and
completed the elongation.
Dimethylthexylsilyl O-[3,4,6-tri-O-Acetyl-2-deoxy-(2,2,2-
trichloroethoxy)carbonylamino-β-D-galactopyranosyl]-
(1f3)-2-deoxy-4,6-O-(2-naphthylidene)-2-(2,2,2-trichloro-
ethoxy)-carbonylamino-β-D-glucopyranoside (10). A mixture
of the glucosyl acceptor 7 (0.45 g, 0.66 mmol) and galactosyl donor 6
(0.38 g, 0.79 mmol) and 4 Å molecular sieves (1.3 g) in DCM (15 mL)
was stirred under Ar for 1 h. The reaction was cooled (0 °C) and NIS
(267 mg, 1.19 mmol) was added followed by the addition of TMSOTf
(29 μL, 0.16 mmol). After stirring for 30 min, the reaction was quenched
by the addition of Et3N (0.5 mL). The mixture was filtered, and the
filtrate (100 mL) was washed with 10% Na2S2O3 (120 mL) and brine
(100 mL). The organic phase was dried (MgSO4) and filtered, and the
filtrate was concentrated under reduced pressure. The resulting yellow
oil was purified by flash chromatography over silica gel (EtOAc/hexanes,
2/5, v/v) to give 10 (0.66 g, 91%) as an amorphous white solid: Rf = 0.21
(EtOAc/hexanes, 1/3, v/v). 1H NMR (500 MHz, CDCl3): δ 7.92À7.46
(m, 7H), 5.69 (s, 1H, >CHNap), 5.42 (br s, 1H, NHTrocI), 5.25 (s, 1H,
H-4II), 5.13À5.03 (m, 3H, H-1I, H-3II and NHTrocII), 4.79À4.56 (m,
5H, H-1II and OCH2CCl3I and II), 4.38 (br s, 1H, H-3I), 4.32 (dd, 1H, J =
4.8 and 10.3 Hz, H-6eI), 4.09 (br s, 1H, H-6eII), 3.85À3.71 (m, 5H, H-4I,
H-6aI, H-2II, H-5II and H-6aII), 3.51 (br s, 1H, H-5I), 3.19 (br s, 1H,
H-2I), 2.09 (s, 3H), 1.88 (s, 3H), 1.82 (br s, 3H), 1.64À1.59 (m, 1H),
0.87À0.83 (m, 12H), 0.14 (s, 3H), 0.12 (s, 3H). 13C NMR (75 MHz,
CDCl3): δ 170.24, 154.00, 153.88, 134.60, 133.82, 132.93, 128.45,
128.39, 127.90, 126.67, 126.41, 125.71, 123.73, 101.64, 101.22 (C-1II),
95.71, 95.56 (C-1I), 95.45, 80.07 (C-4I), 77.76 (C-3I), 74.63, 74.43,
70.72 (C-5II), 69.58 (C-3II), 68.86 (C-6I), 66.47 (C-4II), 66.34 (C-5I),
61.02 (C-6II), 60.50 (C-2I), 53.16 (C-2II), 34.03, 24.91, 20.77, 20.60,
20.54, 20.14, 20.10, 18.68, 18.65, À1.79, À3.22. HR MALDI-TOF MS
m/z: calcd for C43H56Cl6N2O16Si [M+Na]+, 1117.1428; found
1117.1441.
O-[3,4,6-Tri-O-acetyl-2-deoxy-(2,2,2-trichloroethoxy)-
carbonylamino-β-D-galactopyranosyl]-(1f3)-2-deoxy-4,6-O-
(2-naphthylidene)-2-(2,2,2-trichloroethoxy)-carbonylamino-
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(C-5II), 63.70 (C-6I), 60.88 (C-6III), 58.28 (C-2II), 53.31 (C-2III), 38.10,
30.19, 28.10, 25.69, 21.29, 20.77, 20.55, 20.52, 15.02. HR MALDI-TOF
MS m/z: calcd for C61H68Cl6N2O23S [M+Na]+, 1461.1962; found
1461.1974.
min, the reaction mixture was diluted with DCM (80 mL) and washed
with saturated NaHCO3 (2 Â 70 mL) and brine (60 mL). The organic
layer was dried (MgSO4) and filtered and the filtrate was concentrated
under reduced pressure. The resulting yellow oil was purified by flash
chromatography over silica gel (EtOAc/hexanes, 2/3f1/1, v/v) to give
16 (182 mg, 71% over 3 steps) as an amorphous white solid: Rf = 0.41
(EtOAc/hexanes, 1/1, v/v). 1H NMR (500 MHz, CDCl3): 7.85À7.77
(m, 26H), 7.18 (d, J = 8.5 Hz, 1H, NHTrocII), 5.89À5.81 (m, 1H,
OCH2CHdCH2), 5.54 (s, 1H, >CHNap), 5.42À5.06 (m, 12H, H-2I,
H-3III, H-4III, NHFmoc, NHTrocIII, OCH2CHdCH2, two POCH2 and
NR-(9-Fluorenylmethyloxycarbonyl)-O-{[3,4,6-tri-O-acetyl-
2-deoxy-2-(2,2,2-trichloro-ethoxy)carbonylamino-β-D-gala-
ctopyranosyl]-(1f3)-O-[2-deoxy-4,6-O-(2-naphthylidene)-
2-(2,2,2-trichloroethoxy)carbonylamino-β-D-glucopyranosyl]-
(1f4)-2-O-acetyl-6-O-levulinoyl-3-O-(2-methylnaphthyl)-R-
D-mannopyranosyl}-L-threonine allyl Ester (14). A mixture of
the threonine acceptor 9 (168 mg, 0.44 mmol) and glycosyl donor 13
(320 mg, 0.22 mmol) and 4 Å molecular sieves (700 mg) in DCM
(7 mL) was stirred under Ar for 1 h. The reaction was cooled (À25 °C)
and NIS (74 mg, 0.33 mmol) was added, followed by the addition of
AgOTf (11 mg, 0.044 mmol) in toluene (0.44 mL). After stirring at
À20 °C for 2 h, the reaction was quenched by the addition of pyridine
(0.5 mL). The reaction mixture was filtered, and the filtrate (∼100 mL)
was washed with 10% Na2S2O3 (120 mL) and brine (100 mL). The
organic phase was dried (MgSO4) and filtered and the filtrate was
concentrated under reduced pressure. The resulting yellow oil was
purified by flash chromatography over silica gel (EtOAc/hexanes, 2/
3f1/1, v/v) to give 14 (279 mg, 72%) as an amorphous white solid: Rf =
0.31 (EtOAc/hexanes, 1/1, v/v). 1H NMR (500 MHz, CDCl3):
7.86À7.31 (m, 22H), 5.91À5.83 (m, 1H, OCH2CHdCH2), 5.77 (br
s, 1H, NHTrocII), 5.53 (s, 1H, >CHNap), 5.41 (d, J = 10 Hz, 1H,
NHFmoc), 5.33À5.11 (m, 6H, H-2I, H-3III, H-4III, NHTrocIII and
OCH2CHdCH2), 4.89À4.43 (m, 15H, H-1I, H-6aI, H-1II, H-1III,
OCH2Nap, OCH2CCl3II and III, OCH2CHdCH2, CH2CHFmoc and
CHCOOAll), 4.33À4.14 (m, 4H, H-6eI, H-3II, OCHCH3 and
CH2CHFmoc), 4.01À3.91 (m, 6H, H-3I, H-4I, H-5I, H-6eII H-6aIII
and H-6eIII), 3.68À3.65 (m, 4H, H-2II, H-4II, H-2III and H-5III), 3.50 (t,
1H, J = 9.8 Hz, H-6aII), 3.37 (br s, 1H, H-5II), 2.86À2.54 (m, 4H), 2.24
(s, 3H), 2.12 (s, 3H), 2.07 (s, 3H), 1.84 (s, 3H), 1.76 (br s, 3H), 1.26 (d,
3H, J = 6 Hz, OCHCH3). 13C NMR (75 MHz, CDCl3): δ 207.87,
172.89, 170.23, 170.08, 169.92, 156.79, 154.51, 154.05, 144.02, 143.94,
141.49, 135.86, 134.53, 133.87, 133.43, 133.13, 132.95, 131.58, 128.46,
128.40, 128.29, 128.04, 127.98, 127.95, 127.90, 127.33, 126.72, 126.43,
126.08, 125.86, 125.69, 125.42, 125.38, 125.34, 123.66, 120.20, 119.70,
101.63, 101.07 (C-1I), 99.60 (C-1II and III), 96.07, 95.81, 80.28 (C-4II),
78.31 (C-3II), 77.43, 76.18 (C-3I), 75.98 (C-4I), 74.54, 72.36, 70.80 (C-
5III), 69.74 (C-5I), 69.57 (C-3III or C-4III), 69.30 (C-3III or C-4III), 68.78
(C-6II), 67.62, 66.73, 66.50 (C-2I), 66.17 (C-5II), 63.64 (C-6I), 60.85
(C-6III), 58.90, 58.42 (C-2II), 53.34 (C-2III), 47.35, 38.11, 30.22, 29.86,
28.08, 21.20, 20.79, 20.56, 20.55, 18.34. HR MALDI-TOF MS m/z:
calcd for C81H85Cl6N3O28 [M+Na]+, 1780.3348; found 1780.3366.
N-(9-Fluorenylmethyloxycarbonyl)-O-{[3,4,6-tri-O-acetyl-
2-deoxy-2-(2,2,2-trichloro-ethoxy)carbonylamino-β-D-ga-
lactopyranosyl]-(1f3)-O-[2-deoxy-4,6-O-(2-naphthy-
lidene)-2-(2,2,2-trichloroethoxy)carbonylamino-β-D-gluco-
pyranosyl]-(1f4)-2-O-acetyl-6-O-(1,5-dihydro-3-oxo-3λ5-3H-
2,4,3-benzodioxaphosphepin-3yl)-3-O-(2-methylnaphthyl)-
R-D-mannopyranosyl}-L-threonine allyl ester (16). To a stirred
solution of 14 (245 mg, 0.139 mmol) in a mixture of MeOH and DCM
(5 mL, 1/8, v/v) was added hydrazine acetate (26 mg, 0.278 mmol).
After stirring for 1.5 h, the reaction mixture was diluted with DCM
(50 mL) and washed with saturated NaHCO3 (2 Â 45 mL) and brine
(40 mL). The organic phase was dried (MgSO4) and filtered and the
filtrate was concentrated under reduced pressure to afford crude 15 as an
amorphous white solid. Compound 15 was dissolved in DCM (6 mL)
and placed under an atmosphere of Ar, and subsequently N,N-diethyl-
1,5-dihyro-2,4,3-benzodioxaphosphepin-3-amine (67 mg, 0.278 mmol)
and 1H-tetrazole (3 wt % solution in CH3CN, 1.6 mL, 0.556 mmol)
were added. After stirring for 2 h, the reaction was cooled (À20 °C) and
m-CPBA (77%, 124 mg, 0.556 mmol) was added. After stirring for 30
one proton from OCH2CCl3II or III), 4.87À4.53 (m, 11H, H-1I, H-6aI,
II and III
H-1II, H-1III, OCH2Nap and three protons from OCH2CCl3
,
OCH2CHdCH2), 4.44À4.41 (m, CH2CHFmoc and CHCOOAll),
4.31À4.23 (m, H-6eI, OCHCH3 and CH2CHFmoc), 4.11À3.65 (m,
10H, H-3I, H-4I, H-5I, H-2II, H-3II, H-4II, H-6eII, H-2III, H-6aIII and
H-6eIII), 3.57 (br s, 1H, H-5III), 3.46 (t, 1H, J = 10 Hz, H-6aII), 3.37 (br s,
1H, H-5II), 2.11 (s, 3H), 2.05 (s, 3H), 1.84 (s, 3H), 1.74 (br s, 3H), 1.23
(d, 3H, J = 6 Hz, OCHCH3). 13C NMR (75 MHz, CDCl3): δ 170.29,
170.14, 169.97, 169.77, 156.68, 154.70, 154.17, 143.89, 143.81, 141.38,
135.97, 135.05, 134.86, 134.51, 133.77, 133.32, 133.01, 132.85, 131.48,
129.68, 129.54, 129.32, 129.18, 129.11, 128.38, 128.34 128.07, 127.93,
127.87, 127.82, 127.25, 126.60, 126.32, 126.29, 125.92, 125.84, 125.66,
125.44, 125.28, 125.24, 123.64, 120.11, 119.53, 102.72 (C-1I), 101.62,
101.32 (C-1II), 99.34 (C-1III), 96.43, 95.88, 80.46 (C-4II), 79.05 (C-4I),
77.44, 76.28 (C-3II), 75.68 (C-3I), 74.40, 74.27, 72.89, 70.68, 70.58 (C-
5I), 69.81 (C-5III), 69.33 (C-3III or C-4III), 69.24 (C-3III or C-4III),
68.83, 68.74 (C-6II), 67.54, 66.62 (C-2I), 66.44 (C-5II), 66.37 (C-6I),
65.93, 60.93 (C-6III), 58.65, 57.72 (C-2II), 53.06 (C-2III), 47.22, 29.75,
21.11, 20.69, 20.52, 20.44, 18.28. HR MALDI-TOF MS m/z: calcd for
C84H86Cl6N3O29P [M+Na]+, 1864.3113; found 1864.3128.
N-(9-Fluorenylmethyloxycarbonyl)-O-[(2-acetamido-3,4,6-
tri-O-acetyl-2-deoxy-β-D-galactopyranosyl)-(1f3)-O-(2-acet-
amido-4,6-di-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1f4)-
2,3-di-O-acetyl-6-O-(1,5-dihydro-3-oxo-3λ5-3H-2,4,3-ben-
zodioxaphosphepin-3yl)-R-D-mannopyranosyl]-L-threonine
Allyl Ester (17). To a stirred solution of 16 (100 mg, 0.054 mmol) in
DCM (3 mL) and water (0.3 mL) was added 2,3-dichloro-5,6-dicyano-
1,4-benzoquinone (DDQ) (123 mg, 0.54 mmol). After stirring vigor-
ously in the dark for 16 h, the reaction mixture was diluted with DCM
(60 mL) and washed with saturated NaHCO3 (2 Â 60 mL) and brine
(45 mL). The organic phase was dried (MgSO4) and filtered and the
filtrate was concentrated under reduced pressure to afford a yellow oil.
The crude product was dissolved in glacial acetic acid (2 mL), and zinc
metal power (76 mg, 1.16 mmol) was added. After stirring for 1 h, the
reaction mixture was filtered, and the filtrate was concentrated under
reduced pressure to afford a yellow oil. The crude product was dissolved
in pyridine (1 mL), and acetic anhydride (0.2 mL, 2.12 mmol) was added.
After stirring for 12 h, the reaction mixture was diluted with DCM
(50 mL) and washed with saturated NaHCO3 (2 Â 50 mL) and brine
(40 mL). The organic phase was dried (MgSO4) and filtered, and the
filtrate was concentrated under reduced pressure. The resulting yellow oil
was purified by flash chromatography over silica (MeOH/DCM, 1/
40f1/30, v/v) to give 17 (43 mg, 56% over 3 steps) as an amorphous
white solid: Rf = 0.26 (MeOH/DCM, 1/20, v/v). 1H NMR (500 MHz,
CDCl3): 7.76À7.30 (m, 12H), 5.93À5.87 (m, 2H, NHAcIII and
OCH2CHdCH2), 5.62 (d, J = 9.5 Hz, NHFmoc), 5.43 (t, 1H, J = 12
Hz, one proton from POCH2), 5.34À5.10 (m, 9H, H-3I, H-3III, H-4III,
NHAcII, OCH2CHdCH2 and three protons from POCH2), 5.02 (s, 1H,
H-2I), 4.92 (t, 1H, J = 8 Hz, H-4II), 4.83À4.81 (m, 2H, H-1I and H-1III),
4.70À4.60 (m, 4H, H-1II, H-6aI and OCH2CHdCH2), 4.48À4.33 (m,
5H, H-6eIII, OCHCH3, CHCOOAll and CH2CHFmoc), 4.27À4.20 (m,
2H, H-6eI and CH2CHFmoc), 4.12À3.85 (m, 8H, H-4I, H-5I, H-2II,
H-3II, H-6aII, H-6eII, H-5III and H-6aIII), 3.74À3.71 (m, 1H, H-5II),
3.65À3.60 (m, 1H, H-2III), 2.10 (s, 3H), 2.09 (s, 3H), 2.07 (s, 3H), 2.05
14427
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Journal of the American Chemical Society
ARTICLE
(s, 3H), 2.01 (s, 3H), 1.99 (s, 3H), 1.96 (s, 3H), 1.94 (s, 3H), 1.28 (d, 3H,
J = 4.5 Hz, OCHCH3). 13C NMR (75 MHz, CDCl3): δ 171.37, 171.21,
171.00, 170.59, 170.40, 170.35, 169.86, 169.76, 169.70, 156.87, 144.03,
143.96, 141.46, 135.01, 134.83, 131.58, 130.00, 129.84, 129.52, 129.40,
127.93, 125.48, 120.17, 119.74, 101.74 (C-1II), 99.67 (C-1III), 99.06 (C-
1I), 78.23 (C-3II), 77.44, 74.13 (C-4I), 72.27 (C-5II), 70.83 (C-5III),
70.71(C-5I), 70.49 (C-2I), 70.05, 69.62 (C-4II), 69.54 (C-3III or C-4III),
68.94 (C-3III or C-4III), 67.84, 66.92, 66.83 (C-3I), 65.34 (C-6I), 62.76
(C-6III), 61.16 (C-6II), 58.69, 55.77 (C-2II), 52.27 (C-2III), 47.27, 29.89,
23.66, 23.52, 21.09, 21.03, 20.96, 20.86, 20.83, 18.31. HR MALDI-TOF
MS m/z: calcd for C66H80N3O30P [M+Na]+, 1448.4462; found
1448.4477.
mixture of the thioglycosyl 13 (108 mg, 0.0749 mmol) and benzyl
alcohol (32 mg, 0.300 mmol) and 4 Å molecular sieves (200 mg) in
DCM (2.5 mL) was stirred under an atmosphere of Ar for 1 h. The
reaction was cooled (À20 °C) and NIS (25 mg, 0.11 mmol) was added
followed by the addition of TMSOTf (2.7 μL, 0.015 mmol). After
stirring for 40 min, the reaction was quenched by the addition of Et3N
(0.1 mL). The reaction mixture was filtered, and the filtrate (70 mL) was
washed with 10% Na2S2O3 (80 mL) and brine (60 mL). The organic
phase was dried (MgSO4) and filtered and the filtrate was concentrated
under reduced pressure. The resulting yellow oil was purified by flash
chromatography over silica gel (EtOAc/hexanes, 1/1, v/v) to give 21
(65 mg, 58%) as an amorphous white solid: Rf = 0.22 (EtOAc/hexanes,
1/1, v/v). 1H NMR (500 MHz, CDCl3) δ 7.84À7.26 (m, 19H), 5.72 (br
s, 1H, NHTrocII), 5.51 (s, 1H, >CHNap), 5.36 (s, 1H, H-2I), 5.22
(s, 1H, H-4III), 5.15À5.13 (m, 2H, H-3III and NHTrocIII), 4.86À4.46
(m, 12H, H-1I, H-6aI, H-1II, H-1III, OCH2Ph, OCH2Nap and OCH2C-
Cl3II and III), 4.18À3.99 (m, 6H, H-3I, H-4I, H-6eI, H-3II, H-6eII and
H-6eIII), 3.88À3.81 (m, 2H, H-5I and H-6aIII), 3.67À3.63 (m, 4H,
H-2II, H-4II, H-2III and H-5III), 3.48 (t, 1H, J = 10 Hz, H-6aII), 3.37 (br s,
1H, H-5II), 2.87À2.58 (m, 4H), 2.25 (s, 3H), 2.13 (s, 3H), 2.07 (s, 3H),
1.84 (s, 3H), 1.77 (br s, 3H). 13C NMR (75 MHz, CDCl3) δ 207.91,
172.94, 170.32, 170.25, 154.47, 154.06, 136.60, 135.99, 134.59, 133.88,
133.43, 133.12, 132.96, 128.76, 128.48, 128.39, 128.33, 128.26, 128.05,
127.98, 127.91, 126.71, 126.43, 126.37, 126.04, 125.91, 125.70, 125.56,
123.70 101.62, 101.60 (C-1III), 97.32 (C-1I and II), 96.06, 80.22 (C-4I),
78.37 (C-3II), 76.32 (C-3I and C-4I), 75.80, 74.59, 72.33, 70.81 (C-5III),
70.00 (C-3III), 69.62 (C-2I), 69.42 (C-5I), 68.80 (C-6II), 66.52 (C-4III),
66.14 (C-5II), 63.71 (C-6I), 60.89 (C-6III), 58.45 (C-2II), 53.36 (C-2III),
38.17, 30.24, 29.89, 28.17, 21.30, 20.82, 20.60. HRMALDI-TOF MSm/z:
calcd for C66H70Cl6N2O24 [M+Na]+, 1507.2347; found 1507.2357.
Benzyl O-[3,4,6-Tri-O-acetyl-2-deoxy-2-(2,2,2-trichloroe-
thoxy)carbonylamino-β-D-galactopyranosyl]-(1f3)-O-[2-
deoxy-4,6-O-(2-naphthylidene)-2-(2,2,2-trichloroethoxy)-
carbonyl-amino-β-D-glucopyranosyl]-(1f4)-2-O-acetyl-6-
O-(1,5-dihydro-3-oxo-3λ5-3H-2,4,3-benzodioxaphosphe-
pin-3yl)-3-O-(2-methylnaphthyl)-R-D-mannopyranoside (23).
To a stirred solution of 21 (60 mg, 0.0404 mmol) in a mixture of
MeOH and DCM (2 mL, 1/8, v/v) was added hydrazine acetate (7.5
mg, 0.0808 mmol). After stirring for 1.5 h, the reaction mixture was
diluted with DCM (40 mL) and washed with saturated NaHCO3 (2 Â
30 mL). The organic layer was dried (MgSO4) and filtered, and the
filtrate was concentrated under reduced pressure to afford crude 22 as an
amorphous white solid. Compound 22 was dissolved in DCM (1.2 mL)
and placed under an atmosphere of Ar, and subsequently, N,N-diethyl-
1,5-dihyro-2,4,3-benzodioxaphosphepin-3-amine (19 mg, 0.0808
mmol) and 1H-tetrazole (3 wt % solution in CH3CN, 0.47 mL, 0.16
mmol) were added. After stirring for 3 h, the reaction was cooled
(À20 °C) and m-CPBA (77%, 28 mg, 0.16 mmol) was added. After
stirring for 1 h, the reaction mixture was diluted with DCM (40 mL) and
washed with saturated NaHCO3 (2 Â 30 mL) and brine (25 mL). The
organic phase was dried (MgSO4) and filtered, and the filtrate was
concentrated under reduced pressure. The resulting yellow oil was
purified by flash chromatography over silica gel (EtOAc/hexanes, 1/
1f3/2, v/v) to give 23 (41 mg, 65% over 3 steps) as an amorphous
white solid: Rf = 0.29 (EtOAc/hexanes, 3/2, v/v); 1H NMR (500 MHz,
CDCl3) δ 7.89À7.27 (m, 23H), 7.11 (d, 1H, J = 9 Hz, NHTrocII), 5.58
(s, 1H, >CHNap), 5.46 (t, 1H, J = 12.3 Hz, one proton from POCH2),
5.37À5.32 (m, 2H, H-2I and one proton from POCH2), 5.24À5.15 (m,
5H, H-3III, H-4III, two protons from POCH2, one proton from OCH2Ph),
N-(9-Fluorenylmethyloxycarbonyl)-O-[(2-acetamido-3,4,6-
tri-O-acetyl-2-deoxy-β-D-galactopyranosyl)-(1f3)-O-(2-
acetamido-4,6-di-O-acetyl-2-deoxy-β-D-glucopyranosyl)-
(1f4)-2,3-di-O-acetyl-6-O-(1,5-dihydro-3-oxo-3λ5-3H-2,4,
3-benzodioxaphosphepin-3yl)-R-D-mannopyranosyl]-L-threo-
nine (18). To a stirred solution of 17 (150 mg, 0.105 mmol) in
THF (7 mL) and water (0.7 mL) was added tetrakis(triphenyl-
phosphine)palladium(0) [Pd(PPh3)4] (61 mg, 0.0525 mmol) under
Ar. After stirring for 1 h, the reaction mixture was filtered through a
short pad of silica gel and the filtrate was concentrated under reduced
pressure. The resulting yellow oil was purified by flash chromatography
over silica gel (MeOH/DCM, 1/20, v/v with 0.1% acetic acid) to give 18
(134 mg, 92%) as an amorphous white solid: Rf = 0.22 (MeOH/DCM,
1
1/20/, v/v with 0.1% acetic acid). H NMR (500 MHz, acetone-d6):
7.73À7.20 (m, 12H), 7.05 (d, J = 9 Hz, NHFmoc), 6.80 (d, J = 8 Hz,
NHAcIII), 5.43À5.37 (m, 2H, one POCH2), 5.28À5.25 (m, 1H, H-3III),
5.17À4.99 (m, 6H, H-2I, H-3I, H-3III, H-4III and one POCH2), 4.93 (d,
J = 8 Hz, H-1III), 4.90 (s, 1H, H-1I), 4.73 (t, J = 9.5 Hz, H-4II), 4.64 (d, J =
8 Hz, H-1II), 4.43À4.36 (m, 3H, H-6aI, H-6eI and OCHCH3), 4.27 (br s,
1H, CHCOOAll), 4.23À3.82 (m, 11H, H-4I, H-5I, H-3II, H-6aII, H-6eII,
H-5III, H-6aIII, H-6eIII, CH2CHFmoc and CH2CHFmoc), 3.77À3.74
(m, 1H, H-2II), 3.63À3.61 (m, 1H, H-5II), 3.42À3.36 (m, 1H, H-2III),
1.95 (br s, 6H), 1.92 (s, 6H), 1.91 (s, 3H), 1.90 (s, 3H), 1.85 (br s, 6H),
1.83 (s, 3H), 1.29 (d, 3H, J = 6.5 Hz, OCHCH3). 13C NMR (75 MHz,
acetone-d6): δ 171.13, 171.01, 170.84, 170.69, 170.65, 170.28, 170.14,
170.07, 169.98, 157.74, 145.13, 145.05, 142.11, 137.27, 137.08, 130.33,
130.22, 128.56, 128.04, 126.27, 120.82, 102.41 (C-1II), 100.82 (C-1III),
99.78 (C-1I), 78.06 (C-4I), 77.71, 74.34 (C-3II), 72.51 (C-5II), 71.34,
71.24, 71.06 (C-5I), 70.61 (C-5III), 70.49 (C-2I), 70.16, 69.81 (C-3III),
69.64 (C-3I), 69.52 (C-4III), 69.43, 67.75 (C-4II), 67.44, 66.35 (C-6I),
63.27 (C-6II), 62.02 (C-6III), 59.53, 56.38 (C-2II), 52.89 (C-2III), 48.03,
32.65, 23.76, 23.44, 23.39, 23.35, 21.10, 20.96, 20.77, 20.69, 20.63, 18.71,
14.38. HR MALDI-TOF MS m/z: calcd for C63H76N3O30P [M+Na]+,
1408.4149; found 1408.4158.
Ac-Gly-Ala-Ile-Ile-Gln-Thr[GalNAc-β-(1À3)-GlcNAc-β-(1À4)-
Man(6-PO4)-r-O]-Pro-Thr-Leu-Gly-NH2 (5). According to the
general method for glycopeptide synthesis, compound 5 was prepared
using Sieber amide resin (70.4 mg, 0.05 mmol, loading: 0.71 mmol/g).
The synthetic procedure is similar to that of glycopeptide S8 except the
glycosylated trisaccharide threonine 18 (138 mg, 0.1 mmol) was
employed in the manual coupling step. The crude product was purified
by reversed-phase HPLC on an analytical C-18 column using a gradient
of 0f100% acetonitrile in H2O (0.1% TFA) over a 50 min period to
give, after lyophilization of the appropriate fractions, glycopeptide 5 (5.8
mg, 7%, based on initial loading of the resin) as an amorphous white
solid. HR MALDI-TOF MS m/z: calcd for C67H115N14O32P [M+Na]+,
1681.7437; found 1681.7452.
5.03 (br s, 1H, NHTrocIII), 4.94À4.68 (m, 9H, H-1I, H-6aI, H-1II, H-1III,
Benzyl O-[3,4,6-Tri-O-acetyl-2-deoxy-2-(2,2,2-trichloroethoxy)-
carbonylamino-β-D-galacto-pyranosyl]-(1f3)-O-[2-deoxy-
4,6-O-(2-naphthylidene)-2-(2,2,2-trichloroethoxy)carbonyl-
amino-β-D-glucopyranosyl]-(1f4)-2-O-acetyl-6-O-levuli-
noyl-3-O-(2-methylnaphthyl)-R-D-mannopyranoside (21). A
II and
one proton from OCH2Ph, and OCH2CCl3
III), 4.66À4.50
(m, 2H, OCH2Nap), 4.25À4.23 (m, 1H, H-6eI), 4.13À4.01 (m, 5H,
H-3I, H-4I, H-3II, H-6eII and H-6eIII), 3.87À3.72 (m, 5H, H-5I, H-2II,
H-4II, H-2III and H-6aIII), 3.62 (br s, 1H, H-5III), 3.50 (t, 1H, J = 10.3 Hz,
14428
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Journal of the American Chemical Society
ARTICLE
H-6aII), 3.40 (br s, 1H, H-5II), 2.16 (s, 3H), 2.09 (s, 3H), 1.88 (s, 3H),
1.79 (br s, 3H). 13C NMR (75 MHz, CDCl3) δ 170.35, 170.30, 170.21,
154.77, 154.14, 136.55, 136.14, 135.24, 135.01, 134.61, 133.91, 133.43,
133.11, 132.99, 129.75, 129.60, 129.41, 129.22, 128.78, 128.51, 128.47,
128.39, 128.18, 128.15, 128.05, 127.97, 127.92, 126.69, 126.42, 126.32,
125.98, 125.78, 125.64, 123.73, 102.69 (C-1II), 101.77, 101.24 (C-1III),
97.35 (C-1I), 96.45, 95.93, 80.50 (C-4I), 79.08 (C-3I or C-4I), 77.44
(C-3II), 76.34 (C-3I or C-4I), 75.98, 74.46, 72.90, 70.73, 70.47 (C-5III),
70.38(C-2I), 70.09 (C-5I), 69.92, 69.43(C-3III), 69.35, 68.86 (C-6II),
66.52 (C-5II), 66.47 (C-6I), 66.18 (C-4III), 60.89 (C-6III), 57.86 (C-2II),
53.23 (C-2III), 29.89, 21.31, 20.80, 20.60, 20.57. HR MALDI-TOF
MS m/z: calcd for C69H71Cl6N2O25P [M+Na]+, 1591.2112; found
1591.2131.
δ 101.96 (C-1II), 101.72 (C-1III), 91.70 (C-1I), 84.09, 77.45, 76.21,
72.64, 70.73, 70.62, 69.78, 69.64, 68.81, 67.59, 66.69, 63.06, 61.00, 57.73,
49.93, 19.27, 19.24, 19.17, 19.16.19.15. HR MALDI-TOF MS m/z: calcd
for C30H47N2O23P [M+Na]+, 857.2205; found 857.2200.
O-(2-Acetamido-2-deoxy-β-D-galactopyranosyl)-(1f3)-O-(2-
acetamido-2-deoxy-β-D-glucopyranosyl)-(1f4)-6-O-phos-
phate-D-mannitol (4). To a stirred solution of 25 (5.0 mg, 6.0 μmol)
in a mixture of MeOH and DCM (1/2, v/v, 1 mL) was added NaOMe in
a methanolic solution (10 μL, 1.0 M, 10 μmol) and the resulting reaction
mixture was stirred for 6 h. The reaction was then neutralized with 1%
AcOH in MeOH until pH ∼7. Removal of solvents under reduced
pressure afforded a white solid. The crude product was dissolved in H2O
(0.5 mL) and NaBH4 (1.1 mg, 30 μmol) was added. After stirring for 2 h,
the resulting mixture was concentrated under reduced pressure. The
resulting yellow solid was purified by Bio-Gel P2 (H2O) to give pure 4
(3.0 mg, 75% over 2 steps) as an amorphous white solid: 1H NMR (600
MHz, D2O) δ 4.68 (d, 1H, J = 8 Hz, H-1II), 4.48 (d, 1H, J = 8 Hz, H-1III),
4.01À3.92 (m, 3H), 3.90À3.74 (m, 9H), 3.72À3.59 (m, 6H),
3.56À3.52 (m, 1H), 3.50À3.45 (m, 1H), 2.02 (s, 3H), 1.91 (s, 3H).
13C NMR (HMQC, 150 MHz, D2O) δ 104.28 (C-1III), 103.53 C-1II),
83.68, 78.76, 77.70, 77.47, 73.18, 71.93, 71.90, 71.87, 70.39, 67.66, 65.85,
63.79, 63.64, 57.14, 55.23, 19.16.19.15. HR MALDI-TOF MS m/z: calcd
for C22H41N2O19P [M+Na]+, 691.1939; found 691.1947.
Structural and Conformational Analysis. NMR spectra were
recorded on a Varian NMRS 600 MHz spectrometer with a 3 mm cold
probe in D2O or H2O/D2O, 9/1, v/v. Pulse sequences for double
quantum filtered COSY (DQCOSY), total correlation spectroscopy
(TOCSY), gradient heteronuclear multiple quantum coherence
(gHMQC), and gradient heteronuclear multiple bond coherence
(gHMBC) experiments, as provided in the VnmrJ software, were used.
Data were processed and analyzed using the VnmrJ software
and nmrPipe. The reported shifts were based on HMQC peak positions
determined in nmrDraw. Shifts are referenced to the calculated position
of 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) with the approach
widely used in biomolecular NMR, using the position of the residual
HDO signal as a secondary reference.
Enzyme Kinetics. A recombinant form of the soluble catalytic
domain of POMGnT1 cloned into the plasmid pSecTAG2B was a gift of
Prof. Huaiyu Hu. POMGnT1 was obtained from a culture of HEK293
cells stably transfected with the plasmid encoding an epitope tagged
form of the human catalytic domain. The assays were carried out in
solution of 6.25 mM MnCl2, 80 mM MES buffer, pH 6.5, and 2 mM
UDP-GlcNAc (10 μL) with 105 counts of tritium/assay tube. The
glycopeptide concentration was varied up to 2 mM. The tubes were
incubated for 6 h at 37 °C, after which the reaction was quenched with
addition of water (1 mL). C18 SepPak material was prewash with ethanol
(10 mL) and water (30 mL). The column was then washed with of water
(60 mL), followed by elution of the glycopeptide with ethanol (1.5 mL).
The latter fraction was collected and radioactivity counted to determine
the amount of GlcNAc incorporated into the glycopeptides. Kinetic
parameters were extracted from the data after fitting to the MichaelisÀ
Menten equation using the enzyme kinetics module of the Sigma Plot
software.
Analysis of O-Man Glycopeptides Following Enzymatic
Extension by POMGnT1. Following separation by HPLC, the
synthetic O-mannosyl glycopeptides that were extended with a GlcNAc
residue following incubation with recombinant POMGnT1 were ana-
lyzed by direct infusion. Briefly, a solution of extended O-mannosyl glycopep-
tides (2 μL, 2 mM) was diluted into formic acid (20 μL, 0.1%). The
glycopeptides were directly infused into a linear ion trap mass spectrometer
(LTQ-XL, Thermo Scientific) at a flow rate of 0.4À0.6 μL/min and both
full MS and MS/MS fragmentation spectra were acquired. From the
acquired full MS scans, select m/z values were selected to undergo MS/
MS fragmentation by collision-induced dissociation (CID). The MS/
Benzyl O-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-gala-
ctopyranosyl)-(1f3)-O-[2-acetamido-2-deoxy-4,6-O-
(2-naphthylidene)-β-D-glucopyranosyl]-(1f4)-2-O-acetyl-
6-O-(1,5-dihydro-3-oxo-3λ5-3H-2,4,3-benzodioxaphosphe-
pin-3yl)-3-O-(2-methylnaphthyl)-R-D-mannopyranoside (24).
To a stirred solution of 23 (32 mg, 0.0204 mmol) in Ac2O/AcOH/
THF (4 mL, 1/2/3, v/v/v) was added zinc metal power (30 mg, 0.45
mmol) followed by saturated CuSO4 (0.2 mL). After stirring for 30 min,
the reaction mixture was filtered, and the filtrate was concentrated under
reduced pressure. The resulting yellow oil was purified by flash
chromatography over silica gel (MeOH/DCM, 1/25 v/v) to give 24
(20 mg, 71%) as an amorphous white solid: Rf = 0.30 (MeOH/DCM, 1/
15, v/v). 1H NMR (600 MHz, CDCl3) δ 7.85À7.25 (m, 23H), 5.74 (d,
1H, J = 7 Hz, NHAcIII), 5.49 (s, 1H, >CHNap), 5.38 (t, 1H, J = 10.5 Hz,
one proton from POCH2), 5.34À5.29 (m, 2H, H-2I and one proton
from POCH2), 5.23À5.12 (m, 4H, H-3III, H-4III, POCH2), 4.99 (d, 1H,
J = 7 Hz, H-1II), 4.87 (s, 1H, H-1I), 4.83 (d, 1H, J = 7 Hz, H-1III), 4.80 (s,
2H, OCH2Ph), 4.60À4.50 (m, 2H, OCH2Nap), 4.59 (t, 1H, J = 7 Hz,
H-6aI), 4.23À4.21 (m, 1H, H-6eI), 4.15 (t, 1H, J = 7.8 Hz, H-3II), 4.10 (t,
1H, J = 7.8 Hz, H-6aIII), 4.07À4.00 (m, 3H, H-3I, H-4I and H-6eIII),
3.95À3.83 (m, 5H, H-5I, H-2II, H-6eII, H-2III and H-6eIII), 3.67À3.64
(m, 2H, H-4II and H-5III), 3.45 (t, 1H, J = 10 Hz, H-6aII), 3.37À3.33 (m,
1H, H-5II), 2.08 (s, 6H), 2.06 (s, 3H), 1.90 (s, 3H), 1.87 (s, 3H), 1.82 (s,
3H). 13C NMR (150 MHz, CDCl3) δ 171.25, 170.97, 170.50, 170.44,
170.19, 136.78, 136.09, 135.27, 135.13, 134.82, 133.89, 133.50, 133.16,
133.04, 129.76, 129.67, 129.40, 129.27, 128.72, 128.49, 128.31, 128.22,
128.14, 128.07, 127.97, 127.90, 126.65, 126.39, 126.33, 125.97, 125.93,
125.82, 125.51, 123.82, 102.06 (C-1II), 101.64, 100.17 (C-1III), 97.51
(C-1I), 80.12 (C-4I), 77.68 (C-3II), 76.24 (C-3I or C-4I), 75.72 (C-3I or
C-4I), 72.61, 70.79 (C-5III), 70.60 (C-5I), 70.55, 70.52 (C-2I), 70.18,
69.75, 69.35 (C-3III), 69.30, 68.96, 68.92 (C-6II), 66.85 (C-4III), 66.21
(C-6I), 66.06 (C-5II), 61.36 (C-6III), 57.02(C-2III), 51.82 (C-2II), 29.87,
23.68, 23.53, 21.19, 20.80, 20.74, 20.63. HR MALDI-TOF MS m/z:
calcd for C67H73N2O23P [M+Na]+, 1327.4239; found 1327.4247.
O-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galacto-
pyranosyl)-(1f3)-O-(2-acetamido-2-deoxy-β-D-gluco-
pyranosyl)-(1f4)-2-O-acetyl-6-O-phosphate-R/β-D-manno-
pyranose (25). Compound 24 (10 mg, 7.67 μmol) was dissolved in a
mixture of MeOH and DCM (1/1, v/v, 3 mL) and Pd(OH)2/C (40 mg,
20 wt %, Degussa type) was added. The resulting mixture was placed
under a hydrogen atmosphere (1 psi). After stirring for 24 h, the catalyst
was filtered off and washed thoroughly with MeOH and DCM. The
combined filtrates (∼50 mL) were concentrated under reduced pres-
sure. The resulting yellow solid was purified by Bio-Gel P2 (MeOH/
H2O, 1/4, v/v) to give 25 (5.0 mg, 78%) as an amorphous white solid:
1H NMR R-anomer (500 MHz, CD3OD) δ 5.28 (d, 1H, J = 3.5 Hz,
H-4III), 5.02 (dd, 1H, J = 3.6 and 11 Hz, H-3III), 4.96À4.94 (m, 2H, H-1I
and H-2I), 4.66À4.62 (m, H-1II and H-1III), 4.15À4.09 (m, 4H),
3.91À3.81 (m, 6H), 3.67À3.53 (m, 3H), 3.44À3.41 (m, 1H),
3.36À3.32 (m, 1H), 2.06 (s, 3H), 2.03 (s, 3H), 2.00 (s, 3H), 1.96 (s,
3H), 1.88 (s, 3H), 1.86 (s, 3H). 13C NMR (HSQC, 150 MHz, CD3OD)
14429
dx.doi.org/10.1021/ja205473q |J. Am. Chem. Soc. 2011, 133, 14418–14430

Journal of the American Chemical Society
ARTICLE
MS fragmentation spectra were generated by applying 40% collision
energy and collected over 30 s. The acquired MS/MS spectra were
manually interpreted to determine which O-mannosyl residue had been
extended with a GlcNAc residue. No signal was observed in the full MS
spectra or trapped MS/MS spectra for a glycopeptide extended with two
GlcNAc residues.
(17) Takahashi, S.; Sasaki, T.; Manya, H.; Chiba, Y.; Yoshida, A.;
Mizuno, M.; Ishida, H. K.; Ito, F.; Inazu, T.; Kotani, N.; Takasaki, S.;
Takeuchi, M.; Endo, T. Glycobiology 2001, 11, 37–45.
(18) Zhang, W. L.; Betel, C.; Schachter, H. Biochem. J. 2002,
361, 153–162.
(19) Inamori, K.; Endo, T.; Gu, J. G.; Matsuo, I.; Ito, Y.; Fujii, S.;
Iwasaki, H.; Narimatsu, H.; Miyoshi, E.; Honke, K.; Taniguchi, N. J. Biol.
Chem. 2004, 279, 2337–2340.
(20) Kaneko, M.; Alvarez-Manilla, G.; Kamar, M.; Lee, I.; Lee, J. K.;
Troupe, K.; Zhang, W. J.; Osawa, M.; Pierce, M. FEBS Lett. 2003,
554, 515–519.
(21) Yoshida-Moriguchi, T.; Yu, L. P.; Stalnaker, S. H.; Davis, S.;
Kunz, S.; Madson, M.; Oldstone, M. B. A.; Schachter, H.; Wells, L.;
Campbell, K. P. Science 2010, 327, 88–92.
’ ASSOCIATED CONTENT
Supporting Information. Complete ref 11, H and 13C
1
S
b
NMR spectra, and experimental procedures for the preparation
of compounds 7 and 8 and glycopeptides 28À33. This material is
available free of charge via the Internet at http://pubs.acs.org.
(22) Buskas, T.; Ingale, S.; Boons, G. J. Glycobiology 2006,
16, 113r–136r.
’ AUTHOR INFORMATION
(23) Sjolin, P.; Elofsson, M.; Kihlberg, J. J. Org. Chem. 1996,
61, 560–565.
Corresponding Author
E-mail: gjboons@ccrc.uga.edu
(24) Ellervik, U.; Magnusson, G. Carbohydr. Res. 1996, 280, 251–260.
(25) Ciommer, M.; Kunz, H. Synlett 1991, 593–595.
(26) Zhu, T.; Boons, G. J. Tetrahedron: Asymmetry 2000, 11, 199–205.
(27) Nakahara, Y.; Nakahara, Y.; Ito, Y.; Ogawa, T. Carbohydr. Res.
1998, 309, 287–296.
’ ACKNOWLEDGMENT
(28) Gaunt, M. J.; Yu, J. Q.; Spencer, J. B. J. Org. Chem. 1998,
63, 4172–4173.
This research was supported by grants from the National
Institute of General Medicine (NIGMS) of the National Insti-
tutes of Health (Grant No R01GM090269, GJB) and the Center
for Research Resources (P41RR018502, MP and LW), and
P41RR005351 (GJB, DL) and the National institute of Health
(R21AR056055, DL)
(29) Matsuoka, K.; Nishimura, S.-I.; Lee, Y. C. Tetrahedron: Asym-
metry 1994, 5, 2335–2338.
(30) Xia, J.; Abbas, S. A.; Locke, R. D.; Piskorz, C. F.; Alderfer, J. L.;
Matta, K. L. Tetrahedron Lett. 2000, 41, 169–173.
(31) Liptak, A.; Borbas, A.; Janossy, L.; Szilagyi, L. Tetrahedron Lett.
2000, 41, 4949–4953.
(32) Veeneman, G. H.; Vanleeuwen, S. H.; Vanboom, J. H. Tetra-
hedron Lett. 1990, 31, 1331–1334.
(33) Zhu, T.; Boons, G. J. Carbohydr. Res. 2000, 329, 709–715.
(34) Li, Z. T.; Gildersleeve, J. C. J. Am. Chem. Soc. 2006,
128, 11612–11619.
(35) Geurtsen, R.; Boons, G. J. Tetrahedron Lett. 2002, 43, 9429–9431.
(36) Konradsson, P.; Udodong, U. E.; Fraserreid, B. Tetrahedron
Lett. 1990, 31, 4313–4316.
(37) Watanabe, Y.; Komoda, Y.; Ebisuya, K.; Ozaki, S. Tetrahedron
Lett. 1990, 31, 255–256.
’ REFERENCES
(1) Ibraghimov-Beskrovnaya, O.; Ervasti, J. M.; Leveille, C. J.;
Slaughter, C. A.; Sernett, S. W.; Campbell, K. P. Nature 1992,
355, 696–702.
(2) Barresi, R.; Campbell, K. P. J. Cell Sci. 2006, 119, 199–207.
(3) Martin, P. T.; Freeze, H. H. Glycobiology 2003, 13, 67R–75R.
(4) Jimenez-Mallebrera, C.; Brown, S. C.; Sewry, C. A.; Muntoni, F.
Cell. Mol. Life Sci. 2005, 62, 809–823.
(38) Sieber, P. Tetrahedron Lett. 1987, 28, 6147–6150.
(39) Knorr, R.; Trzeciak, A.; Bannwarth, W.; Gillessen, D. Tetra-
hedron Lett. 1989, 30, 1927–1930.
(40) Carpino, L. A. J. Am. Chem. Soc. 1993, 115, 4397–4398.
(41) Winans, K. A.; King, D. S.; Rao, V. R.; Bertozzi, C. R.
Biochemistry 1999, 38, 11700–11710.
(42) van de Ven, F. J. M. Multidimensional NMR in Liquids; Wiley-
VCH: New York, 1995.
(43) Brancaccio, A.; Schulthess, T.; Gesemann, M.; Engel, J. FEBS
Lett. 1995, 368, 139–142.
(5) Moore, C.; Hewitt, J. Glycoconjugate J. 2009, 26, 349–357.
(6) Cao, W.; Henry, M. D.; Borrow, P.; Yamada, H.; Elder, J. H.;
Ravkov, E. V.; Nichol, S. T.; Compans, R. W.; Campbell, K. P.; Oldstone,
M. B. A. Science 1998, 282, 2079–2081.
(7) Rambukkana, A.; Yamada, H.; Zanazzi, G.; Mathus, T.; Salzer,
J. L.; Yurchenco, P. D.; Campbell, K. P.; Fischetti, V. A. Science 1998,
282, 2076–2079.
(8) Bao, X. F.; Kobayashi, M.; Hatakeyama, S.; Angata, K.; Gullberg,
D.; Nakayama, J.; Fukuda, M. N.; Fukuda, M. Proc. Natl. Acad. Sci. U. S.
A. 2009, 106, 12109–12114.
(44) Coltart, D. M.; Royyuru, A. K.; Williams, L. J.; Glunz, P. W.;
Sames, D.; Kuduk, S. D.; Schwarz, J. B.; Chen, X. T.; Danishefsky, S. J.;
Live, D. H. J. Am. Chem. Soc. 2002, 124, 9833–9844.
(45) Barb, A. W.; Borgert, A. J.; Liu, M. A.; Barany, G.; Live, D. In
Methods in Enzymology; Academic Press, San Diego, CA 2010; Vol. 478,
pp 365À388.
(9) de Bernabe, D. B. V.; Inamori, K.; Yoshida-Moriguchi, T.;
Weydert, C. J.; Harper, H. A.; Willer, T.; Henry, M. D.; Campbell,
K. P. J. Biol. Chem. 2009, 284, 11279–11284.
(10) Martin, P. T. Glycobiology 2003, 13, 55R–66R.
(11) Yoshida, A.; et al. Dev. Cell 2001, 1, 717–724.
(12) Stalnaker, S. H.; Hashmi, S.; Lim, J.-M.; Aoki, K.; Porterfield,
M.; Gutierez-Sanchez, G.; Wheeler, J.; Ervasti, J. M.; Bergmann, C.;
Tiemeyer, M.; Wells, L. J. Biol. Chem. 2010, 285, 24882–24891.
(13) Nilsson, J.; Nilsson, J.; Larson, G.; Grahn, A. Glycobiology 2010,
20, 1160–1169.
(14) Endo, T. Glycoconjugate J. 2004, 21, 3–7.
(15) Chai, W. G.; Yuen, C. T.; Kogelberg, H.; Carruthers, R. A.;
Margolis, R. U.; Feizi, T.; Lawson, A. M. Eur. J. Biochem. 1999,
263, 879–888.
(16) Stalnaker, S. H.; Aoki, K.; Lim, J. M.; Porterfield, M.; Liu, M.;
Satz, J. S.; Buskirk, S.; Xiong, Y.; Zhang, P.; Campbell, K. P.; Hu, H.; Live,
D.; Tiemeyer, M.; Wells, L. J. Biol. Chem. 2011, 286, 21180–21190.
14430
dx.doi.org/10.1021/ja205473q |J. Am. Chem. Soc. 2011, 133, 14418–14430