From the
In the preceding paper, preliminary analysis revealed a new type
of O-linked oligosaccharide of 1244 Da at each of two proposed
glycosylation sites on several proteins secreted by the Gram-negative
bacterium Flavobacterium meningosepticum (Plummer, T. H., Jr.,
Tarentino, A. L., and Hauer, C. R.(1995) J. Biol. Chem. 270,
13192-13196). In this report we detail the linkage, sequence, and
branching of this unusual heptasaccharide by electrospray (ES)
ionization mass spectrometry (MS), and collision-induced dissociation
(CID). The proposed structure was supported by a combination of
isotopic labeling, composition and methylation analysis, and the
preparation of several chemical analogs and derivatives with each
product evaluated by MS and CID. The singly branched structure
contained seven residues, including three different uronyl analogs: a
methylated rhamnose and mannose, a glucose, and a reducing terminal
mannose. Only pyranose ring forms were detected
((2-OMe)Man1-4GlcNAcU1-4GlcU1-4Glc1-4(2-OMe)GlcU-4[(2-OMe)Rham1-2]Man).
In the course of studies on the hydrolases produced by
Flavobacterium meningosepticum, eight major proteins have been
purified, two of which were cloned and expressed in Escherichia
coli, (endo-
Collision of the doubly charged ion, m/z 1004.1
Nonreducing terminal losses from the focused precursor, m/z 1004
As an
example, the methylated alditol exhibited a major ion at m/z 649 with additional ions at m/z 1330 and 1071, all
shifted up by 16 Da, whereas the fragments m/z 954, 532, and
273 were invariant (Fig. S6). Additional ions were observed that
could be accounted for as internal cleavage products (m/z 459,
GlcUNAc-Glc, and m/z 718, GlcUNAc-GlcU-Glc). Thus, basic
treatment of either glycan (aldose or alditol) appeared to yield the
degradation products expected of internally linked,
4-O-hexuronyl residues, with six principle fragments each,
three nonreducing (invariant) termini and three reducing terminal
residues (Fig. S6).
The glycosidic loss of the uronyl residue from each trisaccharide
analog (m/z 449 (A), m/z 433 (B),
m/z 448 (C and D), ) and the
corresponding mass shifts attributed to cross-ring fragmentation
(m/z 505, 489, 504, and 504) identify the position of
glucuronyl O-methyl substitution as carbon 2. In a similar
manner, the cross-ring fragments (m/z 561, 545, 563, and 560),
eliminate position 4 O-methyl substitution on methyl rhamnose,
but fail to differentiate carbon 2 from 3. This was determined by
periodate oxidation (below).
These data were again compatible with a
branched trisaccharide at the reducing terminus. Since hexose analysis
had identified mannitol following reductive elimination, the doubly
linked hexose in this trisaccharide is of the manno configuration.
ORM chemistry also modified the
trisaccharide terminus and all fragments possessing this branched
residue (). The glycose product (G-ORM, m/z 651,
1105, 1364, and 1570, ) showed a 2-Da increase, whereas
the alditol product (A-ORM, ) was shifted down by 42 Da
(e.g.m/z 607, 1061, 1320, and 1542). This latter
difference may be considered the sum of a 44-Da decrement and a 2-Da
increment. Collision analysis of both products indicated no change in
the glucuronyl moiety (4-O-linkage and 2-O-methyl
ether blocking periodate oxidation), whereas the methylrhamnose was
responsible for the 2-Da increment.
The 2-Da methylrhamnose
increment fixes natural O-methylation to carbon 2, since CID
of the trisaccharide (, C or D) had
identified either carbon 2 or 3, and the latter product would be
resistant to oxidation. Of considerable interest was the 44-Da decrease
attributed to the mannitol analog following ORM. This mass loss is
consistent only with a combined 2-,4-branching pattern, since all other
combinations would result in multiple carbon-carbon cleavages yielding
different mass shifts (Fig. S9).
There have been few other well characterized secreted
prokaryotic glycans. The cellulolytic bacteria Clostridium
thermocellum and Bacteroides cellulosolvens produce an
extracellular complex containing O-linked glycans
(20) .
These differ from the acidic heptasaccharide produced by F.
meningosepticum in two ways: 1) they are attached to the protein
in Thr/Pro-rich regions but not at precise consensus sites, and 2) they
are composed of short di-, tri-, and tetrasaccharides containing more
typical components, 3-O-methyl-N-acetylglucosamine
and galactose, with linkage via galactose. Other glycoproteins
containing mannosyl-linked oligosaccharides have been described in
eukaryotes but are structurally altogether different from the
Flavobacterium acidic heptasaccharide. Yeast glycoproteins
contain O-linked glycans with chains ranging from Man1 to Man5
that are attached in Ser/Thr-rich regions, but again with no obvious
consensus site
(21) . Mannose has also been reported as the
linking sugar in keratin sulfate polysaccharides in the chondroitin
sulfate proteolytic glycan of brain
(22) , as well as in
glucuronyl
In summary the acidic
heptasaccharide produced by F. meningosepticum is unique among
both prokaryotes and eukaryotes. The function of this unusual acid
heptasaccharide is unknown and is under investigation.
A
= CH
Glc = glucose; Man = mannose; Rham =
rhamnose; GlcU = glucuronic acid; GlcUNAc =
2-acetamido-2-deoxy glucuronic acid; m = 2-O-methylated
residue, e.g. mMan = 2-O-methylmannose; G-M
= glycose-methylated; G-ORM = glycose,
periodate-oxidized, -reduced, and -methylated; A-ORM = alditol:
oxidized, reduced, and methylated.
The countless and precise chemical modifications
carried out in this study were performed by Ms. Shui-Yung Chan for
which we are most grateful.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-N-acetylglucosaminidases F
and F
). When compared on SDS-polyacrylamide gel
electrophoresis, the native enzymes were slightly larger than their
cloned counterparts and mass spectrometry (MS)
(
)
confirmed the products to be modified with carbohydrate
during secretion. In the preceding paper
(1) , these
O-linked carbohydrates on P40 and
endo-
-N-acetylglucosaminidases F
and F
appeared to be identical, and preliminary characterization
revealed an oligosaccharide of 1244 Da at each of the proposed
glycosylation sites. Collision-induced dissociation (CID) indicated an
identical product ion series and compositional analysis revealed
hexoses, methylated hexoses, and uronic acid derivatives
(1) .
Because of its unusual sugar composition, which included three
different uronic acid derivatives, initial attempts at structural
analysis of the Flavobacterium oligosaccharide by high
resolution
H
NMR spectroscopy were unsuccessful
in spite of the relative homogeneity (>80%) of the oligosaccharide
chain. The complex spectrum of this unusual oligosaccharide was
difficult to interpret in the absence of a suitable reference
oligosaccharide. In addition, the well known resistance of uronic
acid-containing polymers to acid hydrolysis and the instability of the
amine-containing uronic acid greatly complicated the stoichiometric
analysis of this oligosaccharide. An alternative approach using
electrospray-MS (ES-MS) and CID provided a detailed structure for this
O-linked heptasaccharide, including sequence, linkage, and
branching. The structural assignments were supported by a combination
of isotopic labeling, methylation, and composition analysis and the
preparation of several chemical analogs and derivatives.
P40 Glycopeptides
F. meningosepticum P40 and its tryptic glycopeptide
were isolated and purified as outlined in the previous
paper
(1) . Intact oligosaccharide was released with concomitant
reduction in 1 M NaBH in 50 mM NaOH at 45
°C for 16 h
(2) .
Carbohydrate Analysis
Methylglycoside-trimethylsilyl Ethers and
Acetates
Monosaccharide compositions from the glycopeptide,
carboxyl-reduced glycopeptide, and previously released glycan were
determined by gas-liquid chromatography following methanolysis and
trimethylsilylation
(3) and acetylation. All analyses were
spiked with an internal standard (C-16 hydrocarbon), and retention
times compared with commercial or synthetically prepared standards.
Alditol Acetates
Carbohydrate composition was also
determined as alditol acetates prepared from the glycopeptide,
carboxyl-reduced glycopeptide, and released glycan by gas-liquid
chromatography following hydrolysis with 2.5 M trifluoroacetic
acid at 120 °C for 2 h. The sample was neutralized with 0.1
M NaOH and reduced by the direct addition of 5 mg of solid
NaBD, where D is deuterium (which remained at room
temperature for an additional 16 h). Excess reducing agent was
destroyed by the addition of 5 µl of acetic acid and the solution
dried in a vacuum centrifuge. Borate was removed as its ester by
repeated addition and drying with methanol. The sample was
vacuum-desiccated overnight, and the dried residue was acetylated by
the consecutive addition of 100 µl of pyridine and acetic anhydride
and heated for 2 h at 75 °C. Gas-chromatography was performed on a
Hewlett-Packard 5980 (Avondale, PA) using a 30 min
0.25-mm DB-5
column (J & W Corp.).
Methylation
Vacuum-desiccated glycopeptide, carboxyl-reduced
glycopeptide, and released glycan were dissolved in 100 µl of a
NaOH/MeSO suspension, prepared by vortexing
Me
SO and powdered sodium hydroxide
(4) . After 1 h at
room temperature, 35 µl of methyl iodide was added and the
suspension set for 1 h at room temperature with occasional
vortexing
(5) . The methylated product was extracted into
chloroform and back-washed with water until neutral. Methylation may be
repeated to ensure complete derivatization.
Linkage Analysis as Methylated Alditol Acetates
Linkage analyses were performed following the established
procedures of Lindberg et
al.(6, 7, 8) , as modified by Geyer et
al.(9, 10) . 16 µg of the methylated glycan
(see above) was hydrolyzed with 160 µl of 2.5 M
trifluoroacetic acid at 120 °C for 2 h. The product was directly
dried by vacuum centrifugation and the residue reduced with 2.5 mg of
NaBH in 125 µl of 0.1 M NaOH. This was heated
to 65 °C for 1.5 h. The reaction was terminated with the addition
of acetic acid and dried by vacuum centrifugation. Borate salts were
removed as methyl esters by multiple addition of methanol and drying.
This residue was acetylated by addition of 100 µl of pyridine and
acetic anhydride and heated for 2 h at 75 °C. The products were
again dried by vacuum centrifugation and extracted into chloroform. The
products were identified and linkages determined by GS-MS.
Uronic Acid Esterification and Reduction
Esterification of carboxylate groups in polysaccharides
(11) and proteins
(12) have been described previously as
well as subsequent reduction to the corresponding alcohol
(11) .
These procedures have been adapted for this glycopeptide by using 20
µg of lyophilized P40 dissolved in 125 µl of 1.8 N HCl
in MeOH and letting set at room temperature for 1.5 h and then drying.
The esterified residue was dissolved in 75 µl of methanol and
reduced by the addition of 3 mg of NaBH and heated at 45
°C for 1.5 h. Excess reagent was decomposed by the addition of 10%
acetic acid. Borate was removed as its ester by repeated addition and
drying with methanol.
Periodate Oxidation and Reduction
Periodate oxidation
(5, 13) was performed
using a 9 mM solution of NaIO buffered with 0.1
M sodium acetate at pH 5.5 in a dark cold room (4 °C) for
3 days. The reaction was quenched with 3 µl of ethylene glycol and
set overnight under the same conditions. The product was neutralized
with 0.1 M NaOH and reduced by the direct addition of 2 mg of
solid NaBH
(which remained at room temperature for an
additional 16 h). Excess reducing agent was destroyed by the addition
of 5 µl of acetic acid and dried. Borate was removed as its ester
by repeated addition and drying with methanol. The sample was
vacuum-desiccated overnight prior to methylation.
Electrospray Ionization Mass Spectrometry
The instrument used in this study was a Finnigan-MAT TSQ-700
(Finnigan-MAT Corp., San Jose, CA) equipped with an electrospray ion
source. Methylated samples were dissolved in methanol:water solutions
(6:4, v/v) containing 0.25 mM sodium hydroxide and analyzed by
syringe pump flow injected at a rate of 0.75 µl/min directly into
the electrospray chamber through a stainless steel hypodermic needle.
The voltage difference between the needle tip and the source electrode
was -3.5 kV. For CID studies, multiply charged precursor ions
were selectively transmitted by the first mass analyzer and directed
into the collision cell containing argon at roughly 2 millitorr with
acceleration voltages of 30-40 V, hence kinetic energies of
60-80 eV.
Composition and Linkage Analysis
Composition analysis of the glycopeptide and reductively
eliminated glycan had shown the presence of glucose, mannose (mannitol
in reductively eliminated glycan samples), methylmannose,
methyldeoxyhexose, and glucuronic acid. Further indication of uronyl
residues was supported by a 42-Da increment to the glycan following
methyl esterification, suggesting three carboxyl groups
(1) .
These compositions were confirmed by methanolysis of the
glycopeptide
(3) , but the sum of the peak areas was well below
the anticipated mass of the glycan. To check for incomplete
methanolysis, the reaction mixture was analyzed by ES-MS following
acetylation (data not shown). The spectrum exhibited masses for the
expected neutral monosaccharides (A-E) but, in addition,
showed the presence of uronyl-containing disaccharides
(F-H), a trisaccharide (I) (Fig. S1) and
several unassigned higher mass ions. Interestingly, a methyluronyl
residue was detected only in the disaccharide F. Thus, the poor
quantification observed following composition analysis may be explained
by the presence of uronyl residues that are relatively
acid-stable
(14) . To a lesser degree, 2-acetamidohexoses share
the same properties so quantification of these components was greatly
compromised.
Figure S1:
To offset the influence of uronyl residues, the
carboxyl groups in the glycopeptide were esterified and reduced to the
corresponding alcohol. Carbohydrate composition and linkage analysis of
this carboxyl-reduced product identified two new components,
2-deoxy-2-acetamidoglucose and 2-O-methylglucose, indicating
all three uronyl precursors to be glucuronyl analogs (glucuronic,
2-deoxy-2-acetamidoglucuronic, and 2-O-methylglucuronic). From
these results, the glycan composition analysis suggested three hexoses
(mannose, glucose, and 2-O-methylglucose), one deoxyhexose,
(2-O-methylrhamnose),(
)
and the three
glucuronyl analogs for a total of seven unique structures. A repeat of
the composition analysis following reductive elimination from the
peptide detected the unique presence of mannitol and an absence of
mannose providing evidence for O-linkage to the peptide.
Linkage analysis, aided by CD
-methylation (where D is
deuterium), indicated two termini (2-O-methylmannose,
2-O-methylrhamnose) and a 4-O-linked glucosyl
residue. Interestingly, no dibranched structures were detected (see
below).
ES-MS-CID-MS of the Glycopeptide
Tryptic digestion of Flavobacterium P40 followed by
gel filtration provided a single early eluting peak when analyzed by
phenol-HSO
and absorbance at 230 nm. Amino acid
analysis of this glycopeptide fraction yielded the sequence
S-I-L-D-S*-T-K, and this material served as the major source for
subsequent experiments
(1) . Analysis of this fraction by ES-MS
produced prominent ions at m/z 1003.9 and 669.7, which was
consistent with a doubly and triply protonated glycopeptide of 2006.8
Da. The peptide sequence (762.8 Da) leaves 1244 Da as the residue
weight for the oligosaccharide, concordant with earlier
results
(1) .
, provided the spectrum in Fig. 1,
and ion intervals correspond to the residues identified in the
composition analysis (e.g.m/z 162, 176, 190, 217 Da,
hexose, methyl hexose or hexuronic acid, methyl uronic, and
N-acetyl hexuronic acid, respectively). From this spectrum
three series of glycosidic cleavage ions can be observed that suggest
glycan sequence. One series (Fig. S2), can be followed as
oligosaccharide increments to the protonated peptide (m/z 763)
and the other two as decrements from the focused precursor
(Fig. S3). Fragments from the oligosaccharide nonreducing end
appear incomplete, although ions at m/z 394 and 732 do
indicate some reducing end sequence information. Internal double
cleavage ions, m/z 218 (GlcU) and m/z 556
(GlcUNAc-GlcU-Glc) were also detected and observed in the CID spectrum
of the glycan alditol
(1) . Increasing the collision energy
resulted in some peptide fragmentation, but their intensities at all
voltages were considerably less than those attributed to glycosidic
cleavage.
Figure 1:
Collision spectrum (CID)
obtained from F. meningosepticum P40 glycopeptide. Focused
precursor ion, m/z 1004.1, provided all
singly charged products with the exception of m/z 807.4
, 835.7
,
916.0
, and 925.1
, representing
nonreducing end losses (Schemes 2, 3, and
4).
Figure S2:
Scheme 2
Figure S3:
Scheme 3
Increments to the peptide ion, m/z 763
(Fig. 1) can be initiated by 162 Da for the fragment m/z 925, supporting a hexose moiety proximal to the peptide
(Fig. S2) and consistent with the mannitol detected in the
composition analysis of the reductively eliminated glycan. The
subsequent intervals for methylrhamnose, methyluronic, hexose, uronic,
N-acetyl uronic, and a methyl hexose residue suggest a linear
sequence. But, this simple interpretation was compromised by the
isomers of methylhexose and uronic acid and the inability to recognize
double cleavage fragments that identify polymer branching.
, provided two ion series initiated by a
differential loss of 160 Da (Fig. 2, m/z 916.0
, 925.1
). Considering
only one glycan has been identified, this suggests branching with a
terminating methylrhamnose and a methylhexose residue (or the isomeric
uronic acid).
Figure 2:
Collision
spectrum (CID) of the trisaccharide (B, Table I) obtained following
basic methylation representing the reducing terminus fragment, m/z 633.3 (Scheme 7).
Alternatively, the upper fragment series may be
explained by a secondary loss of 160 Da from each of the ions in the
lower series (double glycosidic cleavage). Since the intact
glycopeptide, and all ions of the lower branch, exhibit this loss, the
residue must reside on the hexose moiety linked to the protein and be a
6-deoxyhexose, the methylrhamnose identified in composition analysis.
Moreover, the lower branch (Fig. S3) intervals coincide with
increments to the peptide (Fig. S2), again suggesting branching
to be proximal to the peptide. Thus, the composition analysis combined
with ES-MS and CID indicate the O-linked glycan to be a
heptomer with a single branched methylrhamnose at the reducing end
(Fig. S4).
Figure S4:
Scheme 4
Polymer stability to acid hydrolysis, the lack of
selected methylated alditol acetate standards, and the absence of some
sequence ions in the CID spectra have compromised the above data. To
compensate for these structural shortcomings and corroborate the
proposed structure, further studies were initiated with the released
oligosaccharide. In unrelated work with methylated oligosaccharides,
CID has provided complete sequence, branching, and linkage information,
the latter by detection of unique cross-ring fragments of the reducing
side residue
(15, 16) . When contrasting glycoproteins,
glycopeptides, and methylated glycans, the latter CID spectra have
proven to be more comprehensive and complete
(17, 18) .
Unfortunately, monomers with substituent groups (i.e. carboxyl, acetamido, amino, acetyl) do not show linkage-specific
ions, and samples so substituted must be chemically modified.
ES-MS-CID-MS of the Glycan
Basic Deglycosylation
For glycan
characterization, two strategies have been utilized for peptide
deglycosylation, the classical base elimination methods of
Carlson
(2) , using hydride reduction to trap the alditol
product, and direct base methylation
(4) , as developed for
O-linked peptides
(3) . The former method provided the
glycan as the alditol which was methylated (m/z 1566), whereas
the latter procedure released and methylated a glycose analog in one
step (m/z 1550). For the Flavobacterium P40 sample,
both procedures resulted in extensive glycan degradation with the
product spectra dominated by a major trisaccharide and several
additional ions of lower abundance. The extensive degradation may be
attributed to uronyl base-labile C-5 protons which causes a tautomeric
shift in the -carboxyl group and
-elimination of any C-4
residues (Fig. S5)
(19) .
Figure S5:
Scheme 5
Since esterification and
sequence data had indicated the glycan to possess three internal uronyl
moieties, one would anticipate the generation of a new methylglycoside
and unsaturated uronyl terminus from each base-labile residue following
methylation (Fig. S5). These partially eliminated products were
observed when profiled by ES-MS and a comparison of the glycose and
alditol product spectra provided further identification of all
fragments possessing a reducing end (differ by 16 Da).
Figure S6:
Scheme 6
To clarify the positions of natural
O-methylation, the reducing end trisaccharide was studied in
greater detail with four chemical analogs. Differential methylation of
these products provided unique ways to label specific fragments for
structural identification. In addition to the alditol, three other
samples were prepared, CH- and CD
-methylated
glycose, and a carboxyl ester back-exchange of the
CD
-methylated glycan by during
CH
-remethylation. These four analogs produced the
corresponding trisaccharides, identified as A (m/z 649), B (m/z 633); C (m/z 654),
and D (m/z 651), respectively ().
CD
-methylation identifies the number of methyl groups,
differentiates natural methylation, and CH
-remethylation
characterizes carboxyl ester fragments by a 3-Da decrement. The
collision spectra obtained from samples B and D are
presented in Fig. 2and 3, and the major fragments for all
samples are tabulated in . From these resulting mass
shifts, fragmentation patterns have been summarized in Schemes 7 and 8.
Glycan Periodate Oxidation, Reduction, and Methylation
(ORM)
To further strengthen the linkage and sequence
information, and localize indigenous methylation on each nonreducing
terminus, the released alditol and glycopeptide were periodate-oxidized
and reduced. Methylation again induced 4-O-elimination from
uronyl residues (Fig. S5) and eliminated the oxidized glycan from
the glycopeptide as the methylated glycose. The ES-MS profiles provided
information on linkage and natural methylation (). No mass
shifts were found on internal residues, whereas fragments containing a
nonreducing terminal methylmannose exhibited a 2-Da increase. This
increment would be compatible with natural methylation at position 2 or
4, since a 6-O-methyl group would cause a 44-Da decrement,
whereas a 3-O-methyl group would leave the mass invariant.
Indigenous methylation at position 2 was consistent with composition
and methylation analysis.
Figure S9:
Scheme 9
Monomer Ring Form
Furanose ring forms are uncommon
in most oligosaccharides, but are important structural differences not
frequently considered. In this bacterial glycan all internal residues
are 4-linked, and only the termini may be considered possible furanose
candidates. A terminal methylrhamnofuranose would be unmodified
following ORD, whereas a methylmannfuranose would show a 44-Da
decrement. Since each termini showed only a 2-Da increment following
ORM, they both must be pyrans. The peptide-linked mannose residue has
been considered to be 2,4-linked from ORM studies; however, a
3,5-linked furan structure would satisfy both the ORM and CID sequence
results, and this possibility has not been excluded.
Uronyl Ester Reduction
Reduction of uronyl esters
within the polymer avoids the problem of alkali instability and
provides a hexose analog for characterization and an oligosaccharide
product suitable for CID linkage analysis by cross-ring cleavage. Thus,
the Flavobacterium P40 glycopeptide was
esterified
(14) , reduced to the primary alcohol, and released by
the separate steps of reductive elimination and methylation. Each
sample provided a major doubly charged parent ion when analyzed by
ES-MS with an absence of base degradation. Collision analysis of this
analog released by methylation, m/z 765.6,
provided the spectrum in Fig. 4.
Figure 4:
Collision
spectrum (CID) of the F. meningosepticum P40 glycan which was
first prepared with uronyl carboxyl groups reduced and released by
basic methylation (m/z 1508.7). Details of fragmentation
defined in Scheme 10.
As expected, one major
glycosidic cleavage dominated the spectrum which occurred adjacent to
the acetamidohexose residue leaving a nonreducing terminal disaccharide
fragment, m/z 486.4, and its pentasaccharide counterpart,
m/z 1045.3. Smaller fragments define the sequence from both
termini and four cross-ring fragments identify 4-O-linkages
for each of four internal residues, m/z 329.1, 574.9, 778.4,
and 982.4 (Fig. S10). A similar fragment to identify the
penultimate GlcU-Man linkage was not expected or detected for the
terminally positioned residue.
Figure S10:
Scheme 10
ES-MS and CID supported by isotopic
labeling and methylation analysis provide a powerful approach to
structures not previously encountered in biological systems. In the
present study, a new class of oligosaccharide has been discovered on
several proteins secreted by the Gram-negative bacterium, F.
meningosepticum. This simple prokaryote has a complex
glycosyltransferase system which assembles a well defined highly acidic
heptasaccharide at specific Asp-Ser or Asp-Thr-Thr consensus sites in
several different secreted proteins. In this regard the system is
similar to, but possibly predates, the attachment of oligosaccharides
to consensus sites such as Asp-X-Ser/Thr sites in eukaryotic
proteins.
(1
6)mannosyl-O-threonine linkages in
cuticle collagen of the clam worm
(23) .
Table:
CID of trisaccharide analogs
-alditol; B = CH
-glycose; C
= CD
-glycoside; D = carboxylester back
exchange of C by CH
-methylation.
Table:
Direct methylation (M), alditol ORM, and
products
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.