Rapid synthesis of partially O-methylated alditol acetate standards for GC-MS: Some relative activities of hydroxyl groups of methyl glycopyranosides on Purdie methylation

Article abstract of DOI:10.1016/j.carres.2005.01.020

Mixtures containing the majority of partially O-methylated alditol acetates (PMAAs), necessary for the GC-MS based identification of glycosidic linkages in oligo- and polymeric structures were prepared. Rha, Fuc, Rib, Ara, Xyl, Man, Gal, and Glc were converted to their Me glycosides, and the products were progressively O-methylated using the Purdie reagent at 25°C. Resulting PMGs were assayed by TLC and at times that were optimum for formation of mono-O-methyl derivatives and later for higher degrees of methylation; they were converted to PMAAs, in a process incorporating NaB²H₄ reduction. The majority of these can be used as standards for simultaneous identification of pyranosides and some furanosyl units particularly in heteropolysaccharides. The relative reactivities of OH-groups were determined by GC-MS as: Me α- and β-Glcp, HO-2 > HO-4 > HO-3 > HO-6, Me α- and β-Galp, HO-3 > HO-2 > HO-4 > HO-6, Me α-Manp, HO-3 > HO-2 > HO-4 > HO-6, Me β-Manp, HO-3 > HO-4 ≥ HO-6 > HO-2, Me α-Rhap, OH-3 > OH-2 > OH-4; Me αβ-Fucp, OH-2 > OH-3 > OH-4, and Me αβ-Xylp, OH-2 > OH-4 > OH-3. The results differ from those obtained with Haworth, Hakomori, and Ciucanu methylation techniques, although some similarities occurred with the more rapid Kuhn method.

Full text of DOI:10.1016/j.carres.2005.01.020

Carbohydrate
RESEARCH
Carbohydrate Research 340 (2005) 731–739
Rapid synthesis of partially O-methylated alditol acetate
standards for GC–MS: some relative activities of hydroxyl groups
of methyl glycopyranosides on Purdie methylation
*
Guilherme L.Sassaki, Philip A.J.Gorin, Lauro M.Souza,
Phelipe A.Czelusniak and Marcello Iacomini
´ ´
Departamento de Bioquımica e Biologia Molecular, Universidade Federal do Parana, CP 19046, CEP 81531-990, Curitiba, PR, Brazil
Received 20 October 2004; accepted 17 January 2005
Abstract—Mixtures containing the majority of partially O-methylated alditol acetates (PMAAs), necessary for the GC–MS based
identification of glycosidic linkages in oligo- and polymeric structures were prepared.Rha, Fuc, Rib, Ara, Xyl, Man, Gal, and Glc
were converted to their Me glycosides, and the products were progressively O-methylated using the Purdie reagent at 25 ꢀC.Result-
ing PMGs were assayed by TLC and at times that were optimum for formation of mono-O-methyl derivatives and later for higher
degrees of methylation; they were converted to PMAAs, in a process incorporating NaB2H4 reduction.The majority of these can be
used as standards for simultaneous identification of pyranosides and some furanosyl units particularly in heteropolysaccharides.The
relative reactivities of OH-groups were determined by GC–MS as: Me a- and b-Glcp, HO-2 > HO-4 > HO-3 > HO-6, Me a- and
b-Galp, HO-3 > HO-2 > HO-4 > HO-6, Me a-Manp, HO-3 > HO-2 > HO-4 > HO-6, Me b-Manp, HO-3 > HO-4 P HO-6 > HO-2,
Me a-Rhap, OH-3 > OH-2 > OH-4; Me ab-Fucp, OH-2 > OH-3 > OH-4, and Me ab-Xylp, OH-2 > OH-4 > OH-3.The results dif-
fer from those obtained with Haworth, Hakomori, and Ciucanu methylation techniques, although some similarities occurred with
the more rapid Kuhn method.
ꢁ 2005 Elsevier Ltd. All rights reserved.
Keywords: Partially O-methylated alditol acetates; GC–MS standards; Purdie methylation; OH reactivity
1. Introduction
(PMAAs) via successive hydrolysis, reduction with so-
dium borohydride, and acetylation, and which can be
identified by GC–MS using their characteristic GC
retention times and EIMS fingerprints (the method is
extremely sensitive, requiring P50 lg samples).5 This
method has been refined by Carpita and Shea,6 who
used sodium borodeuteride in the reduction step and
avoided the problem of formation of identical, symmet-
rical derivatives from different partially O-methylated
aldoses (example: 2,3-Me2xylitol ꢀ 3,4-Me2xylitol).
However, for the analysis of polysaccharides with more
than one monosaccharide component, it is necessary to
compare the retention times of the PMAAs.Such syn-
theses can be time consuming, if done individually, so
we have now carried out partial O-methylation of
methyl glycosides to simultaneously provide a wide
range of products (PMGs), whose degree of methylation
was assayed by TLC, and whose structure was
Methylation analysis, in conjunction with NMR deter-
mination, is the most utilized method for determination
of carbohydrate structure, providing the structure of
monosaccharide units in oligo-, and polysaccharides
and in glycoconjugates.Several methods have been de-
scribed to give fully methylated carbohydrates, such as
those of Haworth,1 Kuhn et al.,2 Hakomori,3 and the
most used frequently nowadays being that of Ciucanu
and Kerek.4
These methods are sometimes used sequentially to
finally form fully O-methylated products that can be
converted to partially O-methylated alditol acetates
*
Corresponding author.Tel:. +55 41 361 1577; fax: +55 41 266 2042;
e-mail: sassaki@ufpr.br
0008-6215/$ - see front matter ꢁ 2005 Elsevier Ltd.All rights reserved.
doi:10.1016/j.carres.2005.01.020

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G. L. Sassaki et al. / Carbohydrate Research 340 (2005) 731–739
confirmed by GC–MS examination of derived PMAAs.
The graded methylation method of choice was that of
Purdie (Ag2O–MeI).7 That method was selected as it is
relatively slow and easier to handle than that of Doares
et al.8 and it provides a wide spectrum of substitution, as
shown by Gagnaire and Odier.9 They found that Me
b-Glcp had relative reactivities of HO-2 > HO-6 > HO-
3 > HO-4, compared with the slow Haworth method,
which provided a large excess of 6-O- and very little
3-O-substitution, in the order HO-6 > HO-2 > HO-
4 > HO-3.10 The Purdie methylation appears analogous
to that of Kuhn et al.,2 which is more rapid however,
having DMF as an additional component.In the case
of Me a-Manp, the relative activities were HO-
2 > HO-3 > HO-4 P HO-6.11 We have now prepared
partially PMAAs, starting from methyl glycosides of
Glc, Man, Gal, Ara, Xyl, Fuc, and Rha, and have iden-
tified them by EIMS profiles and retention times.5,6
Figure 1. Progressive formation of PMGs on Purdie methylation of glycosides at 3 h intervals up to 12 h: Me Rha (A), Me Fuc (B), Me Ara (C), Me
Xyl (D), Me Man (E), Me Gal (F), and Me Glc (G).

G. L. Sassaki et al. / Carbohydrate Research 340 (2005) 731–739
733
2. Experimental
solvent: 4:1 n-PrOH–H2O) for 15 min, which were devel-
oped using orcinol–H2SO4 spray at 100 ꢀC for 5 min.12
Quantification was carried out at 3 h intervals using
TLC (solvent: 9:1 CHCl3–EtOH; spray: as above).Prod-
ucts with increasing degrees of O-methylation were
examined using the Scion Image Program (Scion Corpo-
ration, Maryland, USA), based on perception of the im-
age of each spot, after obtaining a slope of displacement
by intensity.A relation between the area under the
peaks of intensity and their perceived concentration
was obtained.The graphics were prepared using Micro-
cal Origin v.50. software.
2.1. Acquisition and preparation of methyl glycosides
Gal, Glc, Man, Fuc, Rha, Rib, Ara, Xyl, Me a-Glcp,
Me b-Glcp, Me a-Galp, Me b-Galp, Me a-Manp, and
Me b-Manp were obtained from Sigma–Aldrich (MO,
USA).
Methyl glycosidation of Gal, Glc, Man, Rha, Fuc,
Rib, Ara, and Xyl (10 mg each) was carried out by
refluxing in 3% MeOH–HCl (1.0 mL) for 2 h, followed
by neutralization (AgCO3), filtration, and evaporation.
In the case of Rha and Man, the a-pyranosides were
formed almost exclusively.
2.4. Preparation of PMAAs and GC–MS examination
2.2. Purdie methylation of methyl glycosides
Another 0.2 mL aliquot of the PMG mixture was evapo-
rated, and the residue was hydrolyzed with M H2SO4 for
8 h at 100 ꢀC.The solution was neutralized (BaCO 3), and
the mixture containing partially O-methylated aldoses
was reduced with NaB2H4 (5 mg) for 4 h at room temper-
ature.The solution was neutralized with 50 lL glacial
HOAc, dried under reduced pressure, and co-distilled
with 100 lL of MeOH at 50 ꢀC.This step was repeated
The glycosides were submitted to shaking in CH3I
(1.5 mL), containing Ag2O (250 mg) at 25 ꢀC over a per-
iod of 12 h.They gradually dissolved in the suspension,
and the degree of methylation was monitored at 3 h
intervals using aliquots, which were removed on decan-
tation, and resulting PMGs were detected by TLC and
converted to PMAAs for GC–MS examination.
thrice.The product was acetylated with 1:1 Ac O–pyr-
2
idine (500 lL), overnight at room temperature.The
PMAAs were extracted with CHCl3 and washed with
2% aq CuSO4 solution, and the organic layer containing
PMAAs was dried at room temperature, and the residue
was dissolved in acetone before GC–MS analysis.
2.3. TLC determination of degree of O-methylation
TLC of a portion (0.2-mL aliquot) of each PMG mix-
ture was performed on silica gel plates (10 · 7 cm;
Table 1. Partially O-methylated alditol acetates obtained in syntheses following Purdie methylation for 12 h of methyl glycosides of Rha, Fuc, Rib,
Ara, Xyl, Gal, Glc, and Mana
b
tR
c
%
OMe-Alditol
acetatesb
OMe-Alditol
acetates
tR
%
OMe-Alditol acetates tR
%
OMe-Alditol acetates tR
%
2,3,5-Me3-Rha
3,4-Me2-Rha
2,3-Me2-Rha
2-Me-Rha
7.9
8.5 2,3,5-Me3-Ara
7.8 11.6 2,3,6,-Me3-Man
8.4 30.0 2,3,4,-Me3-Man
13.5 35.1 2,4-Me2-Gal
14.2 5.2 2,3-Me2-Gal
22.3
21.2
23.0
1.2
9.7 16.1 2,3,4-Me3-Ara
9.9 42.0 3,5-Me2-Ara
2.8
0.8
9.4
9.7
1.1 4,6-Me2-Man
7.6 2,6-Me2-Man
16.5 tr
16.6 tr
3,4-Me2-Gal
2-Me-Gal
4.1 3-Me-Gal
11.7
12.1
3.8 2,5-Me2-Ara
2.2 2,3-Me2-Ara
25.8 tr
30.4 tr
4-Me-Rha
3-Me-Rha
10.5 25.1 3,6-Me2-Man
10.6 13.1 2,3-Me2-Man
18.3
19.1
20.5
20.9
24.4
28.1
12.6 25.6 2,4-Me2-Ara
8.3 5.8 3,4-Me2-Ara
7.3 2,3,5,6-Me4-Glc
1.6 2,3,4,6-Me4-Glc
0.7 3,4,6-/2,3,4-Me3-Glcf
0.2 2,3,6,-Me3-Glc
0.8 2,6-Me2-Glc
9.8
10.1
2.4
7.3
2,3,5-Me3-Fuc
2,3,4-Me3-Fuc
2,3-Me2-Fuc
3,4-Me2-Fuc
2-Me-Fuc
11.0
11.4
13.1
13.7
8.0
0.3 3,4-Me2-Man
1.3 2,4-Me2-Man
6.1 2-Me-Man
8.5 27.9 5-Me-Ara
10.3 47.9 2-Me-Ara
13.0 16.7
14.4 36.5
10.6
11.8 10.2 2,3,5-Me3-Xyl
12.6 trd
2,3,4-Me3-Xyl
12.9 3.7 2,5-Me2-Xyl
7.6 17.4 2,4-Me2-Xyl
3.6 3-Me-Ara
3.1 3-/4-Me-Man
1.2 2,3,5,6-Me4-Gal
17.6
18.5
20.2
2.8
2.3
5.8
10.4 10.4 3,6-Me2-Glc
10.7 22.6 2,4-Me2-Glc
4-Me-Fuc
3-Me-Fuc
8.5 27.9 2,3,4,6-Me4-Gal
10.1 2.0 2,5,6,-Me3-Gal
10.5 31.6 2,4,6,-Me3-Gal
13.2
13.7
9.9 2,3-Me2-Glc
4.9 3,4-Me2-Glc
20.6 14.1
20.8 10.3
26.2 tr
2,3,5-Me3-Rib
2,3,4-Me3-Rib
2,5-Me2-Rib
2,4-Me2-Rib
2,3-/3,4-Me2-Rib
2-/3-Me-Rib
7.7 16.8 2,3-/3,4-Me2-Xyl 10.9 27.6 2,3,6,- Me3-Gal
14.0 22.2 2-Me-Glc
15.8 13.4 3-Me-Glc
9.5
9.8
5.7 2-/3-/4-Me-Xyle
7.3 2,3,4,6-Me4-Man 10.0 28.9 2,3,4-Me2-Gal
14.1
9.4 2,3,5,-Me3-Gal
28.2
30.9 tr
1.7
16.1
17.0
18.6
3.9 4-Me-Glc
9.9 42.3 3,4,6,-Me3-Man
12.7 10.1 2,4,6,-Me3-Man
12.9 15.6 2,6-Me2-Gal
13.3 0.4 3,6-Me2-Gal
6.6
1.3
a DB-225 at 215 ꢀC.
b Retention time in minutes.
c Relative percentage.
d Trace.
e The 2-/4-mixture was resolved from the 3-derivative using DB-210.
f Resolved at 200 ꢀC with DB-225.

734
G. L. Sassaki et al. / Carbohydrate Research 340 (2005) 731–739
Each PMAA product was examined by GC–MS using
3. Results and discussion
a Varian model 3300 gas chromatograph linked to a
Finnigan ion trap, model 800R mass spectrometer.
PMAA mixtures were applied to a capillary column of
DB-225 (30 m · 0.25 mm i.d.). Conditions were: elec-
tron impact at 70 eV; injector temp: 250 ꢀC; initial temp:
50 ꢀC (1 min); temp program: 40 ꢀC minÀ1 to 215 ꢀC,
maintained for 40 min.In order to resolve 3,4,6- and
2,3,4-Me3Glc derivatives, the temp was reduced to
200 ꢀC.A DB-210 column at 180 ꢀC was used to resolve
the acetates of 2-/4- and 3-Me-xylitol.Post run analysis
of the partially PMAAs was performed after optimized
separation.
Ara, Xyl, Fuc, Rha, Gal, Glc, Man were each converted
to mixtures of methyl glycosides with MeOH–HCl.
Methylation of the products with the Purdie reagent over
a period of time gave rise to methyl O-methyl glycosides
(PMGs), whose increasing degree of methylation over a
period was determined by TLC (spray: orcinol–sulfuric
acid).The Rfs of the spots indicated the degree of meth-
ylation, and their areas were quantified by Scion Imaging
(Fig.1 ).The formation of the PMGs depended on the
dissolution of the methyl glycosides in iodomethane,
which was only gradual.But over a period of 12 h, those
Figure 2. EIMS patterns at m/z 80 to 270 of PMAA standards, deuterated at C-1: 2-MeAra (A1), 3-MeAra (A2), 4-MeAra (A3), 5-MeAra (A4), 2,3,-
Me2Ara (A5), 2,4-Me2Ara (A6), 2,5-Me2Ara (A7), 3,4-/2,3-Me2Xyl (A8), 3,5-Me3Ara (A9), 2,3,4-Me3-Ara (A10), 2,3,5-Me3Ara (A11), 2-MeGal
(B1), 3-MeGlc impure (B2), and 4-MeGlc (B3), 2,3-Me2Gal (B4), 2,4-Me2Gal (B5), 2,6-Me2Gal (B6), 3,4-Me2Gal (B7), 3,6-Me2Gal (B8),
2,3,4-Me3Gal (B9), 2,3,5-Me3Gal (B10), 2,3,6-Me3Gal (B11), 2,4,6-Me3Gal (B12), 2,5,6-Me3Gal (B13), 3,4,6-Me2Man (B14), 2,3,4,6-Me4Gal
(B15), 2,3,5,6-Me4Gal (B16), 2-MeFuc (C1), 3-MeFuc (C2), 4-MeFuc (C3), 2,3-Me2Fuc (C4), 2,4-Me2Fuc (C5), 3,4-Me2Fuc (C6), 2,3,4-Me3Fuc
(C7), 2,3,5-Me3Fuc (C8).

G. L. Sassaki et al. / Carbohydrate Research 340 (2005) 731–739
735
Figure 2. (continued)
of Fuc, Rha, and Ara dissolved completely after 4 h,
whereas those of Gal, Glc, and Man took 6–8 h.
standards, necessary for identification of Galf and Araf
units in oligomers and polymers, were also formed.Al-
most all of the PMAA structures were obtained in the
rapid syntheses, exceptions being those of 4-MeAra,
4-MeGal, 2,4-Fuc, and 2,4-Rha, 2,5- and 3,5-Me2Gal,
and 3,5,6-Me3Gal and 5-or 6-Me-Hex.The EIMS spec-
tra for each of the neutral monosaccharide classes are
represented in Figure 2.
As a corollary to the Purdie methylation of methyl
glycosides to give PMGs, some conclusions can be
reached concerning the relative reactivities of their hy-
droxyl groups.The most relevant results were obtained
by mono-methylation, since further methylation rates
could be increased when a vic-methoxyl group is present.
However, some conclusions on the relative reactivity
order of each hydroxyl group can be reached (Table
2).We found that in the case of both Me a- and b-Glcp,
PMGs aliquots were removed from the methylation
mixture after 2.5 h to obtain mainly mono-O-methyl
derivatives and at 12 h for samples with higher degrees
of methylation.Each batch was converted to PMAAs
via successive hydrolysis, reduction with sodium boro-
deuteride, and acetylation.The resulting PMAA mix-
tures could then be employed as GC–MS standards in
methylation analysis of polysaccharides, the majority
of which contain pyranosyl, as well some containing
Araf and Galf units (see Table 1).
The resolution of components of PMAAs of Gal, Glc,
Man, Ara, Xyl, Rib, Rha and Fuc, on GC with a DB-
225 capillary column at 215 ꢀC was excellent, although
it was sometimes necessary to reduce the temperature
to 200 ꢀC or to use a DB-210 column (Table 1).PMAA

736
G. L. Sassaki et al. / Carbohydrate Research 340 (2005) 731–739
Figure 2. (continued)
the rate was similar with an order of HO-2 > HO-
4 > HO-3 > HO-6, and that for Me a- and b-Galp are
identical in the order HO-3 > HO-2 > HO-4 > HO-6.
The two anomeric forms of Me Manp were of interest
since, after 3 h, Me b-Manp gave rise to 54% Me3Manp
and 4% Me Me4-Manp derivatives, whereas the
products from the a anomer were formed slowest of
all, with significant amounts of Me1-Manp (76%) and
24% of 2,3-Me2-Manp.There were also significant differ-
ences in the order of reactivity, as it was observed for the
b anomer that HO-3 > HO-4 P HO-6 > HO-2 and for
the a anomer that HO-3 > HO-2 > HO-4 > HO-6.As
previously observed for a Kuhn methylation of Me
a-Manp, the order was not greatly different, with

G. L. Sassaki et al. / Carbohydrate Research 340 (2005) 731–739
737
Figure 2. (continued)
HO-2 P HO-3 > HO-4 P HO-6.11 For the struct-
urally related Me a-Rhap, the most reactive group
was HO-3, with an order of OH-3 > OH-2 >
OH-4.
Other methyl glycosides that were methylated by the
Purdie reagent contained both a- and b-pyranosides
and sometimes furanosides.The mixture of Me ab-Fucp
had the reactivity sequence HO-3 > HO-2 > HO-4, sim-
ilar to that of Me a- and Me b-Galp.The order of Me
ab-Xylp was HO-2 > HO-4 > HO-3, which is identical
to that observed by Ovodov and Evtushenko for Me
b-Xylp.13 The methyl glycosides derived from Ara and

738
G. L. Sassaki et al. / Carbohydrate Research 340 (2005) 731–739
Figure 2. (continued)
Rib contained both furanosides and pyranosides, so
that the order of reactivity was difficult to assess.How-
ever, in both systems, the HO-2 was the most reactive.
Thus in practical terms, for methylation analysis of
carbohydrate-containing oligomers and polymers, par-
tially O-methylated alditol acetate standards (PMAAs)
have been simultaneously prepared for GC–MS in rapid
syntheses, after determination of the degree of
Purdie methylation of methyl glycoside precursors by
TLC.

G. L. Sassaki et al. / Carbohydrate Research 340 (2005) 731–739
739
Table 2. Determination of the relative reactivities of the hydroxyl groups after 2.5 h of methylation and the influence of the anomeric configuration
on the PMAA formation using Purdie methylation
OMe-Alditol acetate
a
b
aba
OMe-Alditol acetate
a
b
2,3,4-Me3Rhap
3,4-Me2Rhap
2,3-Me2Rhap
2-Me-Rhap
6.3
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
3,6-Me2Manp
2,3-Me2Manp
3,4-Me2Manp
2,4-Me2Manp
2-MeManp
—
11.7
7.1
7.
7.0
65.0
3.2
1.1
16.6
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
24.0
—
—
—
9
6
—
2.
4-Me-Rhap
3-Me-Rhap
—
—
27.3
48.7
0.9
1.4
—
0.5
1.0
1.1
—
3-/4-MeManp
2,3,4,6-Me4Galp
2,4,6-Me3Galp
2,3,4-Me3Galp
2,3,6-Me3Galp
2,6-Me2Galp
4,6-Me2Galp
3,6-Me2Galp
2,4-Me2Galp
2,3-Me2Galp
3,4-Me2Galp
2-MeGalp
2,3,4-Me3Fucp
2,3-Me2Fucp
3,4-Me2Fucp
2-MeFucp
22.2
44.1
8.0
12.3
15.2
8.2
1.8
66.0
24.0
0.3
50.1
36.9
4.5
6.3
0.2
1.5
18.8
27.4
53.6
0.1
—
3.
3
6.2
8.0
1.5
3.9
4.0
8.5
11.4
11.0
19.4
5.4
4.1
1.8
3.2
3.2
4.7
22.4
0.1
23.1
11.7
20.1
—
6.2
8.6
1.4
3.9
3.7
8.0
11.0
11.2
18.7
6.7
6.1
1.3
4.9
1.2
10.2
39.5
1.6
12.1
7.9
8.3
—
3-MeFucp
2,3,4-Me3Ribfp
2,4-Me2Ribfp
2,3-Me2Ribfp
2-/4-MeRibfp
2,3,5-Me3Arafp
2,3,4-Me3Arafp
2,3-Me2Arafp
2,4-Me2Arafp
2-MeArafp
3-MeGalp
3,4,6-Me3Glcp
2,3,6-Me3Glcp
4,6-Me2Glcp
2,6-Me2Glcp
2,4-Me2Glcp
2,3-Me2Glcp
3,4-Me2Glcp
6-MeGlcp
4-MeArafp
3-MeArafp
2,3,4-Me3Xylp
2,4-Me2Xylp
2,3-MeXylp
2-/3-/4-MeXylpb
2,3,4,6-Me4Manp
3,4,6-Me3Manp
2,4,6-Me3Manp
2,3,6-Me3Manp
4,6-Me2Manp
3.9
2-MeGlcp
3-MeGlcp
23.7
9.5
—
—
4-MeGlcp
—
21.7
10.4
—
—
a Mixture of ab anomers obtained on methanolysis.
b The 2-/3- were resolved from the 3-derivative using DB-210.
5. Jansson, P.-E.; Kenne, L.; Liedgren, H.; Lindberg, B.;
Lo¨nngren, J. Chem. Commun. (Stockholm) 1970, 8, 1–
70.
6.Carpita, N.C;.Shea, E.M.Linkage structure of carbo-
hydrates by gas chromatography–mass spectrometry
(GC–MS) of partially methylated alditol acetates: analysis
of carbohydrates by GLC and MS.In Analysis of
Carbohydrates by GLC and MS; Biermann, C.J,. McGin-
nis, G. D., Eds.; CRC: Boca Raton, 1989; pp 157–216.
Acknowledgements
The authors wish to thank the Brazilian agencies FINEP
(PRONEX-CARBOIDRATOS, PADCT II/SBIO) and
´
Conselho Nacional de Desenvolvimento Cientıfico e
Tecnologico (CNPq) and Fundac¸a˜o Araucaria, State
´
´
´
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