Determination of the substituent distribution in O-sulfonylbutyl-(1→4)-glucans

Article abstract of DOI:10.1016/S0008-6215(00)00056-2

A method has been developed to determine the distribution of substituents in the glucose units of sulfonylbutylethers of cyclomaltoheptaose (β-cyclodextrin). This method involves hydrolysis of the glucosidic linkages, permethylation, formation of sulfonylchlorides and subsequent transformation to the permethylated sulfonylfluoride derivatives. The latter were thermostable and could be analyzed by GLC and identified by EI and CIMS. For confirmation, the 2-, 3-, and 6-O-substituted standard compounds were independently synthesized and characterized by NMR and GLC-MS. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0008-6215(00)00056-2

www.elsevier.nl/locate/carres
Carbohydrate Research 327 (2000) 275–285
Determination of the substituent distribution in
O-sulfonylbutyl-(1“4)-glucans
Nikola Rogmann a, Peter Jones b, Petra Mischnick c,*
a Uni6ersita¨t Hamburg, Institut fu¨r Organische Chemie, Martin-Luther-King-Platz 6, D-20146 Hamburg, Germany
b Pfizer Central Research, Ramsgate Road, Sandwich, Kent CT13 9NJ, UK
c Technische Uni6ersita¨t Braunschweig, Institut fu¨r Lebensmittelchemie, Schleinitzstrasse 20,
D-38106 Braunschweig, Germany
Received 17 June 1999; received in revised form 14 February 2000; accepted 17 February 2000
Abstract
A method has been developed to determine the distribution of substituents in the glucose units of sulfonyl-
butylethers of cyclomaltoheptaose (b-cyclodextrin). This method involves hydrolysis of the glucosidic linkages,
permethylation, formation of sulfonylchlorides and subsequent transformation to the permethylated sulfonylfluoride
derivatives. The latter were thermostable and could be analyzed by GLC and identified by EI and CIMS. For
confirmation, the 2-, 3-, and 6-O-substituted standard compounds were independently synthesized and characterized
by NMR and GLC–MS. © 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Sulfonylbutyl cyclomaltoheptaoses; Substitution pattern; Synthesis of (4%-fluorosulfonyl)butylethers of glucose
1. Introduction
An advantage of the negative charge of the
sulfonylbutyl substituents is presumably its
behaviour against bile salts that are expected
to be repelled from the SBE substituent so
that the excretion of these salts should be
lessened [1]. The derivatization of CDs can
occur at all three positions on each glucopyran-
ose unit so that a mixture of multiply substi-
tuted CDs is generated. For pharmaceutical
applications, these products must be thor-
oughly characterized. Luna et al. determined
the distribution of sulfonylbutyl groups over
cyclodextrin molecules by capillary elec-
trophoresis [2]. Isolated monosubstituted CDs
were characterized by NMR spectroscopy [3]
of mixtures as well by mass spectrometry [2,4].
Other anionic (1“4)-glucan ethers, as sul-
fonylethyl- or carboxymethyl celluloses, have
been analyzed by means of high pH-anion
exchange chromatography with pulsed amper-
In connection with cyclomaltooligosaccha-
rides (cyclodextrins, CDs) the term ‘host–
guest interaction’ becomes more and more
important today, especially as among a broad
range of guest molecules even hydrophobic
drugs can readily be complexed by CDs. As
particularly the b-CD is poorly water soluble,
CD derivatives are synthesized for use as drug
carriers. Although a number of modified CDs
have been prepared, only three types of
derivatives can be used as pharmaceutical ex-
cipients: methyl-, hydroxypropyl- (HP) and
sulfonylbutylethers (SBE). Both the HP- and
the SBE-b-CDs show excellent safety profiles.
* Corresponding author. Tel.: +49-531-3917201; fax: +
49-531-3917230.
E-mail address: p.mischnick@tu-bs.de (P. Mischnick).
0008-6215/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(00)00056-2

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N. Rogmann et al. / Carbohydrate Research 327 (2000) 275–285
pling with mass spectrometry that helps to
identify the structure of the expected compo-
nents of complex mixtures, as well as unex-
pected side products. However, sufficient
volatility of the analytes is required. We there-
fore started to synthesize appropriate deriva-
tives of the sulfonic acid residues, as we had
done already with cationic starch ethers
[10,11]. First, ethyl esters were prepared via
the reaction sequence methanolysis, sodium–
trialkylammonium exchange, permethylation,
sodium–proton exchange, and esterification
with triethylorthoformate [12]. However, it
turned out that these compounds are not ther-
mostable. Therefore we prepared the sul-
fonylchlorides and subsequently the even
more volatile and stable sulfonylfluorides [13].
This procedure, the ‘sulfofluoride route’ com-
prises the following steps (see Scheme 1): (a)
hydrolysis with perchloric acid, followed by
neutralization with KOH; (b) cation-ex-
change: Et3HN+ for K+; (c) permethylation
with NaH and CH3I in DMF, followed by
purification by size-exclusion chromatography
on Sephadex LH20 with methanol; (d) once
more cation-exchange: Et3HN+ for Na+; (e)
sulfonylchlorination with sulfuryl chloride and
triphenyl phosphine in dichloromethane ac-
cording to Huang et al. [14]; (f) sulfonylfluori-
ometric detection (HPAEC-PAD) [5,6]. We
now report on the development of a method
for the analysis of the regioselectivity of the
sulfonylbutylation reaction in the glucose unit.
2. Results and discussion
The SBE7-b-CD (DS 0.73/glucose unit) was
prepared by addition of 1,4-butane sultone to
b-CD in aqueous sodium hydroxide [7] and
obtained as the sodium salt. The DS (degree
of substitution) was calculated from the sulfur
content.
HPAEC-PAD.—Due to the anionic char-
acter of the substituent, we first applied
HPAEC-PAD to the hydrolysate of the SBE-
b-CD. Group separation was achieved, but
the regioisomers of the mono- and disubsti-
tuted glucoses were only partially or not sepa-
rated. A drawback of pulsed amperometric
detection is the strong decrease of the re-
sponse with increasing number of substituents
[5,6,8]. Therefore, it was investigated whether
the permethylated SBE-glucoses gave better
separation and at the same time would give a
more uniform response. However, separation
was not significantly improved, and the effi-
ciency of the CarboPac column decreased with
each injection. The material of this column
also contains a resin with sulfonic acid groups
and, via ionic interaction, smaller latex parti-
cles with trialkylammonium residues are
bound on the surface of the anionic core [9].
We assume that competing interaction of
highly sulfonylbutylated analytes with this
core structure for the cationic resin particles
altered the shape of the stationary phase. The
sulfonylethylethers investigated by Kragten et
al. [5,6] had a much lower DS and did not
contain trisubstituted residues. Besides this
problem, peak broadening due to hydropho-
bic interactions of the butyl chain with the
stationary phase reduced separation efficiency.
Preparation of 6olatile sulfonic acid deri6a-
ti6es.—GLC is widely used for the analysis of
complex mixtures as they are obtained from
cleavage of poly- or oligosaccharide deriva-
tives. Separation efficiency is high and usually
superior to liquid chromatographic systems. A
further advantage is the well established cou-
nation
with
KF-18-crown-6–acetonitrile
complex in acetonitrile according to Bianchi et
al. [15], and subsequent purification by
column-chromatorgraphy on silica gel with
petrol 1:3 ether–ethyl acetate. Hydrolysis with
perchloric acid was preferred to methanolysis
or hydrolysis catalyzed by trifluoroacetic acid
since the solution could be concentrated under
neutral conditions after precipitating the acid
with KOH. In contrast, evaporation of the
solvent under acidic conditions at room tem-
perature caused significant formation of rever-
sion products, which might even have
enhanced the problems with the stability of
the CarboPac anion exchange column men-
tioned above. Transformation of the sul-
fonylchloride to the sulfonylfluoride derivative
was quantitative when phase transfer condi-
tions were applied. The use of an ion exchange
resin in the fluoride form according to Borders
et al. [16] was less effective.

N. Rogmann et al. / Carbohydrate Research 327 (2000) 275–285
277
Scheme 1. (a) HClO4 (70%), rt/10 min; dilution to 7% HClO4, 100 °C/16 h; neutralization with KOH (6 M), M+=K+h; (b)
M
+=K+“Et3HN+ (Amberlite IR-120, NEt3H+, H2O); (c) CH3I–NaH; DMF/rt/12 h, purification by SEC (Sephadex LH20;
methanol), “M+=Na+; (d) M+=Na+“Et3HN+ (Amberlite IR-120, NEt3H+, H2O); (e) Ph3P–SO2Cl2; CH2Cl2/rt/12 h; (f)
KF/18-crown-6–MeCN complex; MeCN/rt/12 h; Silica Gel 60; 1:3 petrol ether–EtOAc).
GLC
and
GLC–MS
analysis.—Sul-
fonylchloride derivatives were partially de-
graded when injected in a hot split injector
(250 °C). 4%-Chlorobutyl- and 3%-butenylethers
of glucose could be identified by GLC–MS.
However, the sulfonylfluoride form was com-
pletely stable. Fig. 1 shows the gas chro-
matogram of the components obtained from
SBE7-b-CD by the sulfonylfluoride route. The
position of substitution could be deduced
from the mass spectra of the mono- and di-
substituted methyl glucosides. The trisubsti-
tuted compound that has been detected by
HPAEC was not observed. The order of elu-
tion on a 60 m DB5 column was 6b\2b\
3b\6a\2a\3a and 2.3b\2.3a\3.6b\
2.6b\2.6a\3.6a. However, 3.6b and 2.6b as
well as 2.6a and 3.6a were only partially sepa-
rated. As a consequence, the integration of
these peaks is less reproducible and reliable.
Characteristic fragment ions of diagnostic
value were m/z 212 and 225 that correspond
to m/z 88 and 101 in permethylated methyl
glucopyranosides [17]. The mass difference of
methyl to (4%-fluorosulfonyl)butyl-(SFB) ethers
is 124. The C2 fragment ion with m/z 212 can
be formed from C-1+C-2, C-2+C-3, and
C-3+C-4 and therefore occurs in the mass
spectra of the 2- and 3-mono-O-substituted
regioisomers, as well as in the 2,6- and the
3,6-di-O-substituted methyl glucosides. For
Fig. 1. Gas chromatogram of the permethylated sulfonylfluo-
ride derivatives (SFB-Glc) obtained from b-cyclodextrinsul-
fonylbutylether SBE7-b-CD. Peaks are assigned according to
their mass spectral data and in some cases confirmed by
comparison with synthesized standard compounds. (a) Mono-
substituted methyl O-methyl- -glucopyranosides; (1) 6-SFB-b
D
(4b); (2) 2-SFB-b (9b); (3) 3-SFB-b (7b); (4) 6-SFB-a (4a); (5)
2-SFB-a (9a); (6) 3-SFB-a (7a); (x) probably methyl 2-O-(4%-
fluorosulfonyl)butyl-3,5,6-tri-O-methy-
D
-glucofuranoside; (b)
disubstituted methyl O-methyl-
D-glucopyranosides; (1) 2,3-
SFB-b; (2) 2,3-SFB-a; (3) 3,6-SFB-b; (4) 2,6-SFB-b; (5): 2,6-
SFB-a; (6) 3,6-SFB-a.

278
N. Rogmann et al. / Carbohydrate Research 327 (2000) 275–285
OH groups free for sulfonylbutylation. Methyl
2,3,4-tri-O-methyl-a- -glucopyranside (1a)
was obtained via tritylation, permethylation,
and subsequent detritylation of methyl a-
glucopyranoside. Methyl 2,4,6-tri-O-methyl-
a,b- -glucopyranoside (5) was accessible from
the 2,3-SFB isomer this fragment is partially
shifted to m/z 336. The ion at m/z 225 is
present for all 2-O-substituted-SFB-glucosides
since it contains C-2, C-3, and C-4 with the
original pattern at C-2 and C-4. The 6-O-
monosubstituted methyl glucosides show in-
tensive peaks at m/z 88 and 101. 3-SFB
derivatives additionally show m/z 199 corre-
sponding to m/z 75 in permethylated hexo-
pyranosides, formed by rearrangement of the
3-O-substituent to C-1 of the methyl glu-
coside. The molecular masses of 374 (mono-
SFB) and 498 (di-SFB) could be deduced from
the CI mass spectra with ammonia as reactant
gas. One further peak (assigned as component
x in Fig. 1), which was detected in this group,
showed the same molecular mass as the mono-
substituted derivatives and a mass spectrum
that was very similar to 2-O-SFB-Glc. Since
this peak increased with increasing 2-O-substi-
tution and showed a fragment ion typical for
methyl furanosides, it is assumed that this
peak belongs to the methyl 3,5,6-tri-O-methyl-
2-O-SFB-glucofuranoside. A second anomer
with the same mass spectrum could be de-
tected when complete separation of all eight
isomers was achieved on a special column
(Optima-d3, 60 m). In Fig. 1 it co-elutes with
the 2-SFB-a-glucoside. The relative amount of
component x was about 1.5%. Usually fura-
nosides play no significant role in the equi-
librium mixture of methyl glucosides obtained
under acidic conditions. However, 2-O-sul-
fonylbutylation may favour the formation of
furanosides. The analysis of an isolated mono-
2-O-SBE-b-CD [2] supported this interpreta-
tion as component x with its characteristic
mass spectrum was formed as a by-product of
this regioisomer. The anomer of component x
co-elutes with 2-SFB-a-Glc and therefore does
not interfere with quantification.
D
D-
D
the mainly b-(1“3)-linked glucan laminaran
via permethylation and methanolysis. For the
2-O-SFB-standard (9), 3-O-methyl-glucose
was transformed to the methyl glucoside, pro-
tected as 4,6-O-benzylidene acetal and then
reacted with the 1,4-butane sultone in aqueous
sodium hydroxide to give the corresponding
2-O-(4%-sulfonyl)butyl derivative (8), which
was permethylated after cleavage of the ben-
zylidene group. Since sulfonylbutylation was
performed on the methyl glucopyranoside, no
furanoside (x) was observed. The a and b
anomers of the sulfonylfluoride derivative (9a
and 9b) could be separated and characterized
by NMR spectroscopy. For the synthesis of
the disubstituted standard compound, methyl
3,4-di-O-methyl-a,b- -glucopyranoside was
D
obtained from the (1“2)-linked disaccharide
sophorose via tert-butyldimethylsilylation of
the primary positions, followed by permethyl-
ation and methanolysis. The partially methyl-
ated compounds were transformed to the sul-
fonylbutylethers (sodium salt). From these the
SFB derivatives 4a, 7a and 9a,b were obtained
as described above following the sulfofluoride
route. The 2,6-SFB-Glc was only obtained as
a component of a crude product, but was
sufficient for comparison of GLC and MS
data (Table 1).
NMR spectra of sulfonylbutyl deri6ati6es.—
All standard compounds were investigated by
1H and 13C NMR spectroscopy as well as
two-dimensional NMR techniques (1H–1H
1
and H–13C correlation spectroscopy: COSY
13
and in addition for C: DEPT measurement).
Synthesis of standard compounds.—All peak
assignments of the mono-SFB glucosides and
that one of the 2,6-di-SFB glucoside could be
confirmed by independent synthesis of the
standard compounds. In addition, the order of
elution was found to be b before a anomers,
as has been observed for other methyl glu-
coside derivatives before. The strategy of syn-
thesis was aimed at the formation of partially
methylated methyl glucosides with one or two
NMR data of known intermediates were in
agreement with literature data.
Quantification.—The relative molar compo-
sition of the mono- and disubstituted glucosyl
residues could be determined from the peak
areas in the gas chromatogram. Since not all
steps of the sulfofluoride route are quantita-
tive, discrimination of higher substituted com-
pounds must be taken into consideration. The
unsubstituted fraction is also not obtained in a

N. Rogmann et al. / Carbohydrate Research 327 (2000) 275–285
279
Table 1
a
Compound
R1
R2
R3
R4
R6
H
1
2
3
4
5
6
7
8
9
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
benzylidene
CH3
(CH2)4SO−3 Na+
(CH2)4SO2Cl
(CH2)4SO2F
CH3
H
(CH2)4SO−3 Na+
(CH2)4SO2F
CH3
CH3
CH3
(CH2)4SO−3 Na+
(CH2)4SO2F
CH3
CH3
a a and b were added to the compound number to assign the a and b anomers (see text).
3-SFB-Glc : b/a of 6-SFB-Glc : b/a of 2-SFB-
Glc) was quite constant 2.4 : (1.1–1.7) : 1.0.
The reason for the wide range for 6-SFB-Glc
is not known. With the help of the relative
b/a ratios the composition of the monosubsti-
tuted fraction could even be calculated when
6b and 3a were not separated as was the case
on a 25 m CPSil8CB column. These values
were in excellent agreement with the data
obtained by complete separation on a 60 m
column.
representative ratio due to the non ionic char-
acter of these constituents. Therefore, only
the relative composition within the mono-
and disubstituted groups was determined.
Surprisingly, preferred 3-O-sulfonylbutylation
was found for SBE7-b-CD, followed by 2-
and 6-O-substitution as is obvious from the
gas chromatogram (Fig. 1). The disubstitution
is in full agreement with this regioselectivity
as 2,3-SBE is the main pattern, followed by
3,6 and 2,6. This unexpected behaviour was
further investigated. The results are reported
and discussed together with the quantitative
analysis of a number of further SBE-b-CDs
with different DS values according to the
method described in this paper [18].
For the determination of the molar frac-
tions of un-, mono- di- and trisubstituted glu-
cosyl units, capillary electrophoresis (CE) is
under investigation. Although group separa-
tion was achieved by HPAEC, no quantita-
tive evaluation is possible due to the different
response factors of regioisomers in the PAD
[5,6,8].
3. Conclusions
A method is presented that allows the de-
termination of the regioisomer composition in
the mono- and disubstituted fractions of sul-
fonylbutylethers of (1“4)-glucans. The per-
methylated (4%-fluorosulfonyl)butylethers of
methyl glucosides were prepared as volatile
and thermostable compounds that could be
analyzed by GLC and GLC–MS. HPAEC-
PAD was not applicable since hydrophobic
interactions of the resin with the butyl chains
prevented regioisomer separation, and the co-
operative effect of multiply SBE-substituted
analyte residues presumably impaired the sta-
tionary phase. Peak identity deduced from the
mass spectra could be confirmed by indepen-
dent synthesis of standard compounds.
Ratios of a/b anomers were calculated
from the gas chromatograms and were found
to depend strongly on the position of substi-
tution. Obviously, the b configuration is al-
ways the most preferred for the 3-O-SFB
methyl glucoside (up to 84%). While the abso-
lute b/a values vary, the relative ratio (b/a of

280
N. Rogmann et al. / Carbohydrate Research 327 (2000) 275–285
NMR spectroscopy.—Proton NMR spectra
4. Experimental
were measured with a Bruker WM 400 instru-
ment (1H: 400 MHz, 13C: 100.62 MHz); the
solvents D2O (internal standard: acetone, l
2.18) and CDCl3 (internal standard: TMS)
were purchased from E. Merck/Aldrich.
Column chromatography.—For purification
Silica Gel 60 (63–200 mm, E. Merck) was
used.
General.—The SBE7-b-CD (sodium form)
was obtained from Cydex, Kansas, USA. For
preparation, see Refs. [7,18]. All reagents were
of highest purity available and purchased
from Fluka, Aldrich or E. Merck.
GLC.—GLC separations were carried out
on a Carlo Erba GC 6000 Vega Series 2
instrument equipped with an on-column injec-
tor, a flame ionization detector (FID), a 25 m
capillary column CPSil 8CB (Chrompack)
connected with a retention gap (2 m), and a E.
Merck Hitachi D-2500 integrator. Hydrogen
was used as carrier gas (80 kPa). Complete
separation of all monosubstituted SBE-Glc
derivatives was obtained with a 60 m DB5,
i.d. 0.25 mm, helium as carrier gas at 2.0 kPa,
splitless injection (40 s) and the following tem-
perature program: 100 °C (1 min), with 7 °C/
min to 240 (10 min isotherm), with 10 °C/min
to 290 °C (hold). For quantification of the
disubstituted fraction the following program
was used: 120 °C (1 min), with 15 °C/min to
260 °C, 20 min hold, then with 10 °C/min to
290 °C (hold). Data from the FID were col-
lected with a Shimadzu Techlab C-R6A Chro-
matopac integrator.
GLC–MS.—EI (70 eV) mass spectra were
recorded on a VG Analytical VG/70-250S in-
strument. For CI, ammonia was used as a
reactant gas. Separation on an Optima
column-d3 (Machery and Nagel, 60 m) was
followed with an Ion Trap detector (ITD,
Finnigan MAT).
HR-FABMS.—High resolution FAB-mass
spectra (positive ion mode) were recorded on
a VG Analytical VG/70-250S instrument with
a xenon-gun and m-nitrobenzylalcohol as ma-
trix. Calibration was performed with
polyethyleneglycol. Precise masses were ob-
tained by linear interpolation between two
reference masses after electrical scanning of
the mass range [19]. Resolution: 6000 (for
compound 7 and 9), 4000 (for compound 4).
10–20 spectra were accumulated.
Size-exclusion chromatography.—For size-
exclusion chromatography Sephadex LH20
(Pharmacia) in MeOH was used.
HPAEC.—For the ion-exchange chro-
matography a Dionex 300 system equipped
with
a
semipreparative Carbo-Pac PA1
column (250×9 mm) and a PE-detector was
used. The pulse potentials were 0.05 V (E1),
0.6 V (E2) and 0.6 V (E3). The response time
was 1 s, the pulse times were 480, 120 and 60
ms. The following elution programme was
used (eluent A: 0.1 M NaOH; eluent B: 0.1 M
NaOAc): from 0 to 5 min: 95% A and 5% B;
at 30 min: 75% A and 25% B and from 45 to
60 min: 0% A and 100% B. The flow was 4
mL/min.
General procedures — ion-exchange.—The
ion-exchange resin (Amberlite IR-120, H+, E.
Merck) was treated with an aq satd triethyl-
amine solution until the pH was alkaline. The
excess of triethylamine was washed out with
distilled water. For regeneration the resin was
washed with 1 M HCl (volume: 20 fold vol-
ume of that of the resin). To transform the
sodium form of the sulfonic acids to the tri-
ethylammonium salts the samples were treated
with the Et3HN+ loaded resin and freeze-
dried afterwards.
HClO4 hydrolysis [5].—The SBE-b-CD
sample (2–5 mg) was stirred in a 1 mL V-vial
with HClO4 (70%, 100 mL) at room tempera-
ture (rt) for 10 min. The solution was diluted
with distilled water (900 mL) and stirred for 16
h at 100 °C. For neutralisation, a solution of 6
M KOH (about 300 mL) was added. The
precipitated KClO4 was removed by decanta-
tion and the supernatant, which contained the
products, was dried in a stream of nitrogen.
TFA hydrolysis.—The SBE-b-CD sample
(2–5 mg) was stirred in a 1 mL V-vial with 2
M trifluoroacetic acid (1 mL) at 120 °C for 3
h. Afterwards the acid was removed in a
ESIMS.—Electrospray mass spectra (posi-
tive mode) were recorded on a LC Esquire
instrument (Bruker/HP). The sample was dis-
solved in 1:1 MeOH–CH2Cl2 and introduced
directly via a syringe.

N. Rogmann et al. / Carbohydrate Research 327 (2000) 275–285
281
was added and the solution was stirred for 0.5
h at rt. Then 1,4-butane sultone (2 molar
equiv/OH) was added and the reaction mix-
ture was stirred overnight at rt. A further
amount of NaOH and butane sultone (2 mo-
lar equiv/OH) was added and the system was
stirred for 2 days at rt. To stop the reaction
and to destroy the unreacted sultone, more
NaOH (7 molar equiv per mol of glucoside)
and water were added and the mixture was
heated to 100 °C for 2.5 h. The solution was
neutralised with dilute aq HCl, the solvents
were removed by distillation, and the products
were purified by column chromatography.
Esterification of the sulfonylbutylethers
[12].—The methyl O-methyl-O-sulfonylbutyl-
stream of nitrogen. Residues of the acid were
removed by co-distillation with toluene (three
times).
Methanolysis.—The SBE-b-CD sample (2–
5 mg) was stirred in a 1 mL V-vial with 1.5 M
methanolic HCl (1 mL) at 100 °C for 2 h. The
acid and alcohol then were removed in a
stream of nitrogen.
Permethylation.—For permethylation, the
sugar derivatives were dissolved in DMF (30
mg/mL) and NaH (5 molar equivalents/OH
group) was added at 0 °C. The mixture was
stirred for 0.5 h and then CH3I was added (5
molar equiv/OH group). The reaction mixture
was stirred overnight at rt. After quenching
with MeOH–water, the reaction was neutral-
ized with dilute aq HCl, and afterwards the
solvents were evaporated. The crude product
was purified by size-exclusion chromato-
graphy with MeOH as eluent.
a,b- -glucosides (free acid form) obtained by
D
methanolysis, ion exchange, permethylation
and once again ion exchange of SBE-b-CD
were treated with triethyl orthoformate at
50 °C for 2 days. Thin-layer chromatography
(TLC) control (1:1 petrol ether–EtOAc)
showed complete reaction. The excess of or-
thoformate was removed by distillation.
Sulfonylchlorination [14].—For the sul-
fonylchlorination of the sulfonic acid (triethyl-
ammonium form, 1 molar equiv), triphenyl
phosphine (1.8 molar equiv) was dissolved in
CH2Cl2 (0.22 mmol/mL). At 0 °C freshly dis-
tilled SO2Cl2 (2 molar equiv) was added. The
educt — dissolved in CH2Cl2 (0.12 mmol/mL)
— was added dropwise at rt. The mixture was
stirred overnight at rt and the reaction was
stopped by adding an excess of 1:1 petrol
ether–diethyl ether. Triphenylphosphine oxide
precipitated and the supernatant was de-
canted. The solvents were removed by distilla-
tion and the crude sulfonyl chloride mixture
was used for the sulfonylfluorination reaction.
Synthesis of standard compounds.—Besides
the permethylated 6-O-substituted standard
compound (6-SFB-Glc, 4) the intermediates
formed on the way to it on the analytical
pathway were isolated and characterized by
NMR and MS (the sulfonic acid derivative 2,
the sulfonylchloride derivative 3). As the sul-
fonylchloride derivative is a reactive interme-
diate, the work-up procedure (column
chromatography) could cause losses. For that
reason the other standard compounds were
only isolated and characterized in their
sodium sulfonate or the sulfonylfluoride form.
Methyl 6-O-(4%-fluorosulfonyl)butyl-2,3,4-
Sulfonylfluorination
[15].—The
sul-
fonylchlorides of the permethylated glucosides
were dissolved in acetonitrile (0.12 mmol/mL)
and KF in a molar excess of 1.2 was added.
After adding a catalytic amount of the 18-
crown-6–acetonitrile complex that was freshly
prepared according to Gokel and Cram [20],
the mixture was stirred overnight at rt. The
products were purified by column chro-
matograhpy (3:4 petrol ether–1:3 EtOAc; Rf:
disubstituted methyl glucosides: 0.25-mono-
substituted methyl glucosides: 0.49).
tri-O-methyl-h-
D
-glucopyranoside (6-SFB-Glc,
-gluco-
4a).—Methyl 2,3,4-tri-O-methyl-a-
D
pyranoside (1a) was obtained via 6-O-trityla-
tion, permethylation and detritylation of com-
mercial methyl a- -glucopyranoside. NMR
D
data were in accordance with the literature
[22]. Intermediate 1a (154.4 mg, 0.65 mmol)
was sulfonylbutylated according to the general
method (see above). The crude product (2a)
was purified by column chromatography (elu-
ent: 5:1 CHCl3 –MeOH; Rf 0.21). Yield: 148.5
Sulfonylbutylation
[21].—The
partially
methylated methyl glucoside was dissolved in
a system of 10:1 isopropanol–water, pow-
dered NaOH (excess of 1.8 molar equiv/OH)
1
mg (0.38 mmol, 58%). H NMR (D2O, acet-
one as internal standard): l 4.90 (d, 1 H, H-1,

282
N. Rogmann et al. / Carbohydrate Research 327 (2000) 275–285
(C-4); 70.60 (C-1%); 69.90 (C-5); 69.80 (C-6);
60.90 (ꢀCH3); 60.50 (ꢀCH3); 59.00 (ꢀCH3);
55.20 (ꢀCH3); 27.90 (C-2%); 21.20 (C-3%).
GLC–MS: m/zB102 (5%); m/z 102–173 (\
1%); m/z 173–343 (\0.1%); m/z 41 (10); 45
(19); 55 (23); 71 (11); 73 (12); 75 (27); 88 (100);
89 (8); 101 (50); 111 (2); 117 (1.1); 127 (1.5);
131 (2); 139 (6); 145 (1.9); 187 (0.1); 195 (0.2);
225 (0.3); 251 (0.2); 279 (0.1); 283 (0.3); 311
(1.3); 312 (0.2). CIMS (ammonia): m/z 375
[M+1], 392 [M+18]. ESIMS (positive
mode): m/z 397 [M+Na], 413 [M+K]. HR-
FABMS (positive mode): m/z 343.1260 [M−
J1,2 3.56 Hz); 3.62 (m, 2 H, H-6); 3.60 (m, 1 H,
H-5); 3.52 (m, 2 H, H-1%); 3.50 (s, 3 H, ꢀCH3);
3.47 (s, 3 H, ꢀCH3); 3.41 (dd, 1 H, H-3, J3,4
3.56 Hz); 3.38 (s, 3 H, ꢀCH3); 3.31 (s, 3 H,
ꢀCH3); 3.27 (dd, 1 H, H-2); 3.22 (dd, 1 H,
H-4, J4,3 3.56 Hz); 2.88 (m, 2 H, H-4%); 1.71
13
(m, 2 H, H-3%); 1.66 (m, 2 H, H-2%). C NMR
(D2O): l 96.90 (C-1); 82.60 (C-3); 80.20 (C-2);
79.30 (C-4); 71.10 (C-1%); 69.70 (C-5); 68.90
(C-6); 60.40 (ꢀCH3); 60.30 (ꢀCH3); 58.20
(ꢀCH3); 55.30 (ꢀCH3); 51.10 (C-4%); 27.60 (C-
2%); 21.40 (C-3%). From 2a, first the methyl
(4%-chlorosulfonyl)butyl 2,3,4-tri-O-methyl-a-
D-glucoside (3a) was synthesized according to
MeOH+H]+,
(C14H27FO8S−MeOH+H)
the general method (see above) (94.66 mg,
0.24 mmol). The sulfonylchloride derivative
was purified by column chromatography (1:3
petrol ether–EtOAc; Rf 0.35). Yield: 81.4 mg
requires 343.1226); m/z 311.0906 [M−
2MeOH+H]+, (C14H27FO8S−2MeOH+H
requires 311.0965).
Methyl
2,4,6-tri-O-methyl-h- -glucoside
D
1
(0.21 mmol; 88.0%). H NMR (CDCl3, TMS
(5a).—Laminaran from Laminaria digitata
(Sigma, L9634), a mainly (95%) b-(1“3)-
as internal standard): l 4.82 (d, 1 H, H-1, J1,2
3.56 Hz); 3.81 (dd, 2 H, H-4%); 3.63 (m, 2 H,
H-6); 3.62 (m, 4 H, H-5/ꢀCH3); 3.60 (m, 2 H,
H-1%); 3.54 (s, 3 H, ꢀCH3); 3.51 (s, 3 H, ꢀCH3);
3.50 (vt, 1 H, H-3); 3.41 (s, 3 H, ꢀCH3); 3.20
(dd, 1 H, H-2); 3.14 (dd, 1 H, H-4); 2.17 (m, 2
H, H-3%); 1.82 (m, 2 H, H-2%). 13C NMR
(CDCl3): l 97.50 (C-1); 83.50 (C-3); 81.80
(C-2); 79.50 (C-4); 70.70 (C-1%); 69.90 (C-5);
69.80 (C-6); 65.40 (C-4%); 60.90 (ꢀCH3); 60.50
(ꢀCH3); 59.00 (ꢀCH3); 55.20 (ꢀCH3); 27.50
(C-2%); 22.20 (C-3%). The methyl (4%-chlorosul-
linked
D-glucan (206.1 mg, 1.27 mmol AGU),
was dissolved in Me2SO (2 mL) and powdered
NaOH (440.0 mg, 11.0 mmol) was added. The
suspension was stirred for 0.5 h at rt and then
methyl iodide (510 mL, 8.2 mmol) was added.
The suspension was stirred overnight and, to
prevent undermethylation, permethylation
was performed twice. The permethylated lami-
naran was dialysed for 2 days against tap
water and for another 2 days against distilled
water (batchwise) in a cellulose membrane
from Spectra Por with a MWCO of 3500, and
freeze dried. Yield: 234.4 mg (1.15 mmol,
fonyl)butyl 2,3,4-tri-O-methyl-a- -glucoside
D
(81.4 mg, 0.21 mmol) was dissolved in MeCN
and transformed to the sulfonylfluoride
derivative by means of an anion exchange
resin (F− form, MeCN) prepared according to
Ref. [16]. Sulfonylchloride 3a was filtered
twice through the resin in a column to achieve
complete transformation. The solvent was
evaporated and the crude product purified by
column chromatography (2:1 EtOAc–EtOH;
Rf 0.69). Yield of 4a: 34.0 mg (0.09 mmol,
1
90%). H NMR (CDCl3): l 4.72 (d, 1 H, H-1,
J1,2 7.63 Hz); 3.77 (vt, 1 H, H-3, J2,3 8.6 Hz);
3.62 (m, 1 H, H-6); 3.6 (s, 3 H, ꢀCH3); 3.53 (s,
3 H, ꢀCH3); 3.41 (m, 4 H, H-6, ꢀCH3); 3.31
(m, 1 H, H-5); 3.21 (m, 1 H, H-4); 3.05 (vt, 1
H, H-2). For analytical control the permethyl-
ated laminaran (2 mg, 9.8 mmol) was submit-
ted to methanolysis, and the products were
trimethylsilylated. GLC analysis (60 °C (1
min), with 25 °C/min to 130 °C, with 4 °C/min
to 290 °C) yielded 81.7% of the permethylated
3-O-TMS-glucoside, 13.3% di-O-TMS deriva-
tives and 4.5% of permethylated methyl glu-
coside. The permethylated laminaran (234.4
mg, 1.15 mmol AGU) was degraded to
monomers by methanolysis (see Section 4.10).
1
43%). H NMR (CDCl3): l 4.81 (d, 1 H, H-1,
J1,2 3.56 Hz); 3.62 (m, 4 H, H-6/ꢀCH3); 3.61
(m, 1 H, H-5); 3.58 (m, 2 H, H-1%); 3.56 (m, 1
H, H-6); 3.54 (s, 3 H, ꢀCH3); 3.51 (s, 3 H,
ꢀCH3); 3.53 (m, 1 H, H-3); 3.40 (s, 3 H,
ꢀCH3); 3.39 (m, 2 H, H-4%); 3.19 (m, 1 H,
H-4); 3.13 (m, 1 H, H-2); 2.10 (m, 2 H, H-3%);
1.82 (m, 2 H, H-2%). 13C NMR (CDCl3): l
97.50 (C-1); 83.50 (C-3); 81.80 (C-2); 79.60
Methyl 2,4,6-tri-O-methyl-a-
D
-glucoside (5a)
was isolated by column chromatography (1:9

N. Rogmann et al. / Carbohydrate Research 327 (2000) 275–285
283
375.1439 [M+H]+ (C14H27FO8S+H requires
375.1489), 373.1346 (C14H27FO8S+2 H+H+
requires 373.1332), 343.1149 ([M−MeOH+
H]+ C14H27FO8S+2 MeOH+H requires
343.1226); 311.1013 [2 MeOH+H]+ C14H27-
FO8S+2 MeOH+H requires 311.0965).
Methyl 2-O-(4%-fluorosulfonyl)butyl-3,4,6-
petrol ether–EtOAc; Rf 0.22). Yield:
105.7 mg (0.45 mmol, 39%).13C NMR
(CDCl3): l C-1 (97.22); 81.74 (C-2); 79.47
(C-4); 73.57 (C-3); 71.52 (C-6); 70.06 (C-5);
60.89 (ꢀCH3); 59.65 (ꢀCH3); 58.82 (ꢀCH3);
55.66 (ꢀCH3). Analytical data were in accor-
dance with the literature [23].
Methyl 3-O-(4%-fluorosulfonyl)butyl-2,4,6-
tri-O-methyl-h,i-
Glc, 9).—Commercially available 3-O-
methyl-a,b- -glucopyranose (251.2 mg, 1.3
D
-glucopyranoside (2-SFB-
tri-O-methyl-h-
7a).—The methyl 2,4,6-tri-O-methyl-a-
D
-glucopyranoside (3-SFB-Glc,
D-glu-
D
coside (5a) was sulfonylbutylated following
the general procedure. The crude product was
purified by column chromatography (5:1
CHCl3–MeOH; Rf 0.21). Yield of 6a: 51.6 mg
mmol) was transformed into the methyl glu-
coside by a methanolysis (see Section 4.10).
The crude product was dissolved in DMF and
benzaldehyde dimethylacetal (1.1 equiv/equiv
glucose) and a catalytic amount of p-toluene
sulfonic acid was added. The reaction mixture
was stirred for 3 h at 60 °C under reduced
pressure (30 mbar). The temperature was in-
creased to 85 °C to remove the DMF, and the
crude methyl 4,6-O-benzylidene-3-O-methyl-
1
(0.13 mmol, 29%). H NMR (CDCl3): l 4.11
(d, 1 H, H-1, J1,2 8.14 Hz); 3.77 (m, 2 H,
H-1%); 3.62–3.07 sugar protons; 3.48–3.53 (2
s, 6 H, 2×ꢀCH3); 3.39 (s, 3 H, ꢀCH3); 3.40 (s,
3 H, ꢀCH3); 2.98 (m, 2 H, H-4%); 1.86 (m, 2 H,
H-3%); 1.67 (m, 2 H, H-2%). The 3-SBE-Glc (6a)
was transformed to the sulfonylfluoride
derivative following the analytical pathway
(see Section 4.10). The sulfonylfluoride deriva-
tive was purified by column chromatography
(1:3 petrol ether–EtOAc; Rf: 0.38). Yield of
a,b- -glucoside was purified by column chro-
D
matography (1:1 petrol ether–EtOAc; Rf
13
0.18). Yield: 209.7 mg (0.7 mmol, 54.5%). C
NMR (CDCl3):
l
128.99/128.25/126.06/
126.03/101.29 (bn); 99.83 (C-1); 82.04/80.66
(C-3/C-4); 72.32 (C-2); 69.03 (C-6); 62.56 (C-
5). This intermediate was sulfonylbutylated
following the general procedure. The methyl
4,6-O-benzylidene-3-O-methyl-2-O-(4%-sulfo-
1
7a: 32.3 mg (0.09 mmol, 66.2%). H NMR
(CDCl3): l 4.86 (d, 1 H, H-1, J1,2 3.56); 3.81
(m, 2 H, H-4%); 3.58 (vd, 2 H, H-6); 3.55 (m, 2
H, H-3/H-5); 3.41 (s, 3 H, ꢀCH3); 3.48 (m, 2
H, H-1%); 3.47 (s, 3 H, ꢀCH3); 3.42 (s, 3 H,
ꢀCH3); 3.41 (s, 3 H, ꢀCH3); 3.25 (m, 1 H,
H-4); 3.20 (dd, 1 H, H-2, J2,1 3.56); 2.09 (m, 2
H, H-3%); 1.77 (m, 2 H, H-2%). 13C NMR
(CDCl3): l 97.56 (C-1); 82.12 (C-2); 81.82
(C-3); 79.82 (C-4); 72.07 (C-4%); 71.38 (C-6);
70.37 (C-5); 60.93 (ꢀCH3); 59.61 (ꢀCH3); 58.86
(ꢀCH3); 55.61 (ꢀCH3); 51.10 (C-1%); 28.69 (C-
3%); 21.19 (C-2%). GLC–EIMSMS: m/zB102
(\5%); m/z 102–173 (\1%); m/z 173–343
(\0.5%); m/z 39 (6); 41 (17); 43 (7); 45 (66);
55 (100); 59 (6); 71 (39); 72 (5); 73 (11); 74
(64); 75 (35); 87 (16); 88 (24); 89 (8); 101 (73);
102 (12); 103 (1.7); 111 (3.5); 113 (1.8); 114
(1.8); 115 (1.2); 117 (2.6); 123 (1.1); 127 (5);
139 (69); 140 (4); 141 (4); 143 (1.7); 145 (4);
159 (1.2); 187 (4); 199 (26); 200 (2); 201 (1.4);
212 (84); 231 (8); 214 (5); 225 (6); 226 (0.7);
255 (0.9); 311 (0.8). CIMS (ammonia): m/z
375 [M+1]; m/z 392 [M+18]+. ESIMS (pos-
itive mode): m/z 397 [M+Na]+, 413 [M+
K]+. HRFABMS (positive mode): m/z
nyl)butyl-a,b- -glucoside was purified by
D
column chromatography (3:1 CHCl3–MeOH;
Rf 0.28). Yield of 8: 94.4 mg (0.22 mmol,
1
32%). H NMR (CDCl3): l 7.46 (m, 2 H, Ph);
7.33 (m, 3 H, Ph); 5.47 (s, 1 H, CHPh); 4.78
(d, 1 H, H-1, J1,2 3.56 Hz); 4.25 (m, 1 H,
sugar); 3.30–3.76 (m, 5 H, sugar); 3.70 (m, 2
H, H-1%); 3.58 (s, 3 H, OMe); 3.39 (s, 3 H,
OMe); 2.97 (m, 2 H, H-4%); 1.86 (m, 2 H,
H-2%); 1.70 (m, 2 H, H-3%). The benzylidene
group of 8 was cleaved according to Hann et
al. [24]. Compound 8 (94.4 mg, 0.22 mmol)
was dissolved in 0.01 M H2SO4 and heated for
3 h to 100 °C. The reaction mixture was neu-
tralised with diluted NaOH and the solvent
evaporated. After exchange of Na+ for
Et3HN+ (see Section 4.10) 69.9 mg (0.16
mmol, 77%) of methyl 3-O-methyl-2-O-(4%-
sulfonyl)butyl-a,b-
-glucoside (NEt3H+ form)
D
was obtained. After permethylation (see Sec-
tion 4.10) it was transformed into the sulfonyl-

284
N. Rogmann et al. / Carbohydrate Research 327 (2000) 275–285
quires
(C14H27FO8S)+H]+,
375.1489),
fluoride derivative following the analytical
pathway (see Section 4.10). The a and b stan-
dard compounds could be separated by
column chromatography (1:3 petrol ether–
EtOAc; Rf a: 0.5; Rf b 0.39). The yield of the
2-SFB-a-Glc (9a) was 6.7 mg (0.018 mmol)
and of the 2-SFB-b-Glc (9b) 15.5 mg (0.041
mmol). In addition a fraction of the a and b
anomer mixture (9a,b) could be isolated (13.9
mg, 0.037 mmol). The total yield of 9 was
36.2 mg (0.096 mmol, 60%). 1H NMR
(CDCl3); 9a, l 4.80 (d, 1 H, H-1, J1,2 3.56);
3.67 (m, 2 H, H-1%); 3.61 (s, 3 H, ꢀCH3);
3.575 (m, 3 H, 2 H-6/H-5); 3.53 (s, 3 H,
ꢀCH3); 3.49 (m, 2 H, H-4%); 3.46 (vt, 1 H,
H-3); 3.42 (3s, H, ꢀCH3); 3.40 (s, 3 H,
ꢀCH3); 3.268 (dd, 1 H, H-2, J2,1 3.56); 2.09
(m, 2 H, H-3%); 1.79 (m, 2 H, H-2%). 13C
NMR (CDCl3); 9a, l 98.01 (C-1); 83.50 (C-
3); 80.74 (C-2); 80.06 (C-4); 71.36 (C-6);
70.37 (C-5); 70.32 (C-1%); 61.35 (ꢀCH3); 60.83
(ꢀCH3); 59.63 (ꢀCH3); 55.53 (ꢀCH3); 51.11/
373.1346 [M−2 H+H]+, C14H27FO8S−2
MeOH+H requires 373.1332), 343.1136
[M−MeOH+H]+, (C14H27FO8S+MeOH+
H
requires 343.1226); 311.1000 [M−2
MeOH+H]+, C14H27FO8S−2 MeOH+H
requires 311.0965).
Disubstituted methyl glucosides.—Methyl
2,6-di-O-(4%-fluorosulfonyl)butyl-3,4-di-O-
methyl-a,b-
was prepared as follows. From sophorose
methyl 3,4-di-O-methyl-a,b- -glucopyrano-
D
-glucopyranoside (2,6-SFB-Glc)
D
side was obtained via 6,6%-di-O-tert-
butyldimethylsilylation, permethylation, and
methanolysis as one component of the disac-
charide. Sulfonylbutylation and transforma-
tion to the sulfonylfluoride yielded two
components, which co-eluted with the corre-
sponding components obtained from SBE7-b-
CD by the analytical procedure and which
had been identified as the 2,6-SFB-Glc deriva-
tives from their EI-mass spectra: EIMS: (m/
zB102 (\5%), m/zB300 (\1%) m/z 41
(16), 42 (7), 43 (6), 45 (20), 55 (100), 56 (5), 59
(5), 64 (8), 71 (17), 72 (7), 73 (6), 74 (34), 75
(45), 87 (13), 88 (21), 89 (5), 101 (8), 117 (2),
139 (61), 140 (3), 141 (3), 212 (25) 225 (39),
226 (3). CIMS (ammonia): m/z 499 [M+1]+,
516 [M+18]+. ESIMS (positive mode): m/z
521 [M+Na]+, 537 [M+K]+. The substitu-
tion pattern of the other disubstituted regio-
isomers were deduced from their mass spectra
(for interpretation of the mass spectra,
see text): GLC–EIMS (ion trap) of methyl
2,3-di-O-(4%-fluorosulfonyl)butyl-4,6-di-O-
1
50.94 (C-4%); 28.27 (C-2%); 21.42 (C-3%). H
NMR (9b): l 4.12 (d, 1 H, H-1, J1,2 7.63);
3.82/3.70 (m, 2 H, H-1%); 3.64 (dd, 1 H, H-6,
J6,6 10.68, J6,5 2.03); 3.61 (s, 3 H, ꢀCH3); 3.56
(dd, 1 H, H-6, J6,6 10.68, J6,5 4.58); 3.53 (s, 3
H, ꢀCH3); 3.51 (s, 3 H, ꢀCH3); 3.47 (m, 2 H,
H-4%); 3.26 (m, 1 H, H-5, J5,6 2.03, J5,6 4.58);
3.15 (m, 2 H, H-3/H-4, J3,2 3.56, J3,4 2.03);
3.03 (m, 1 H, H-2); 2.09 (m, 2 H, H-3%); 1.74
(m, 2 H, H-2%). 13C NMR (9b): l 104.56
(C-1); 86.77 (C-3); 82.42 (C-2); 80.13 (C-5);
75.05 (C-4); 71.60 (C-6)/(C-1%); 61.37 (CH3);
60.76 (CH3); 59.77 (CH3); 57.34 (CH3); 51.04/
50.88 (C-4%); 28.42 (C-2%); 21.10 (C-3%). GLC–
MS: (m/zB102 (\5%); m/z 102–173
(\1%); m/z 173–343 (\0.5%); m/z 39 (5);
41 (15); 43 (6); 45 (59); 55 (100); 59 (6); 59
(7); 71 (32); 73 (9); 74 (65); 75 (97); 87 (18);
88 (41); 89 (13); 101 (27); 102 (8); 111 (3);
113 (2); 114 (1.8); 115 (2); 117 (4); 123 (1.2);
127 (2.5); 139 (62); 140 (3); 141 (3); 143 (1.7);
149 (1.6); 187 (0.9); 199 (0.7); 212 (52); 213
(4.5); 214 (3); 225 (65); 226 (6); 227 (3.5); 235
(0.9); 251 (0.7); 255 (0.9); 269 (0.7); 300 (2.7);
311 (4). CIMS (ammonia): m/z 375 [M+1]+,
392 [M+18]+. ESIMS (positive mode): m/z
397 [M+Na]+, 413 [M+K]+. HRFABMS
(positive mode): m/z 375.1478 [M+H+ re-
methyl-a,b- -glucopyranoside (2,3-SFB-Glc):
D
m/z 55 (100), 71 (14), 101 (5), 139 (77), 199
(3), 212 (3), 225 (17), 336 (19). CIMS (ammo-
nia): m/z 499 [M+1], 516 [M+18]. Methyl
3,6-di-O-(4%-fluorosulfonyl)butyl-2,4-di-O-
methyl-a,b- -glucopyranoside (3,6-SFB-Glc).
D
EIMS (ion trap): m/z 55 (100), 71 (16), 74
(24), 75 (16), 101 (42), 111 (3), 139 (76), 199
(3), 212 (36), 225 (5), 435 (2). CIMS (ammo-
nia): m/z 499 [M+1]+, 516 [M+18]+.
Acknowledgements
We gratefully acknowledge financial sup-
port from Pfizer, Sandwich, UK.

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