Synthesis of per- and poly-substituted trehalose derivatives: Studies of properties relevant to their use as excipients for controlled drug release

Article abstract of DOI:10.1080/07328300902887672

Per- and poly-substituted oligosaccharide derivatives, with trehalose cores, have been prepared and assessed for their potential for use as excipients in controlled-release formulations. The synthesized compounds, generally with acyl and amido substituent

Full text of DOI:10.1080/07328300902887672

Journal of Carbohydrate Chemistry, 28:198–221, 2009
ꢀC
Copyright Taylor & Francis Group, LLC
ISSN: 0732-8303 print / 1532-2327 online
DOI: 10.1080/07328300902887672
Synthesis of Per- and
Poly-Substituted Trehalose
Derivatives: Studies of
Properties Relevant to Their Use
as Excipients for Controlled
Drug Release
Thomas C. Baddeley1 and James L. Wardell2
1Department of Chemistry, University of Aberdeen, Meston Walk, Old Aberdeen, AB24
3UE, Scotland
2Department of Chemistry, University of Aberdeen, Meston Walk, Old Aberdeen,
AB24 3UE, Scotland; and Centro de Desenvolvimento Tecnolo´gico em Sau´de (CDTS),
Fundac¸a˜o Oswaldo Cruz (FIOCRUZ), Casa Amarela, Campus de Manguinhos, Av.
Brasil 4365, 21040-900, Rio de Janeiro, RJ, Brazil
Per- and poly-substituted oligosaccharide derivatives, with trehalose cores, have been
prepared and assessed for their potential for use as excipients in controlled-release for-
mulations. The synthesized compounds, generally with acyl and amido substituents, in-
cluded 6,6ꢀ-N, Nꢀ-diamido-6,6ꢀ-dideoxy-α,α-trehalose derivatives, 6,6ꢀ-bis(1,2,3,4-tetra-
O-acetyl-β-D-glucopyranuronyl)-α, α-trehalose derivatives, 2,2ꢀ,3,3ꢀ-tetra-O-acetyl-6,6ꢀ-
bis-(1,2,3,4-tetra-O-acetyl-β-D-glucopyranuronyl)-4,4ꢀ-di-O-acyl-α,α-trehalose, 2, 2ꢀ, 3,
3ꢀ-tetra-O-acetyl-6-(1,2,3,4-tetra-O-acetyl-β-D-glucopyranuronyl)-4,4ꢀ,6ꢀ-tri-O-acyl-α,α-
trehalose, and 2,2ꢀ,3,3ꢀ,4,4ꢀ-hexa-O-acetyl-6,6ꢀ-bis-(1,2,3,4-tetra-O-acetyl-6-O-succinyl-
β-D-glucopyranuronyl)-α,α-trehalose. Compounds were characterized by NMR, IR,
MS and optical rotations; elemental analyses; or HRMS. The compounds formed
amorphous materials either on fast quenching of melts or on spray drying. Properties,
used in the initial assessment of the potential as controlled-release excipients, were
log10 P and glass transition, Tg, values.
Keywords Trehalose derivatives; Glass transition temperatures; Controlled release
Received July 19, 2008; accepted March 11, 2009.
Address correspondence to Thomas C. Baddeley, Department of Chemistry, Uni-
versity of Aberdeen, Meston Walk, Old Aberdeen, AB24 3UE, Scotland. E-mail:
che523@abdn.ac.uk
198

Synthesis of Per- and Poly-Substituted Trehalose Derivatives
199
INTRODUCTION
The controlled release of actives, such as drugs, is of great interest in modern
therapeutic drug delivery, due to benefits arising from single-dose treatments,
increased pharmacologic efficacy, extended periods of action, greater flexibility
in administration, and improved targeting of specific sites.[1] Among the mate-
rials considered for use in controlled-release systems are polymers, both man-
made and natural systems,[2–9] glasses,[10,11] and materials based on carbo-
hydrates (e.g., cyclodextrin).[12–15] Other carbohydrate-based systems include
trehalose-hydroxyethylcellulose microspheres[16] and derivatives of di- and
other oligomeric saccharides, especially trehalose derivatives.[17,18] Many car-
bohydrates form reasonably stable amorphous phases, including glass phases,
by rapid cooling from melts. Such phases are suitable for use as matrices for
hosting, stabilization, and release of bioactive materials.[19,20] The melt tem-
peratures of these derivatives are sufficiently low to safely incorporate differ-
ent bioactives. In addition, spray drying techniques can also be employed to
generate encapsulated materials from solutions.
We have prepared a number of di-, tri-, and tetra-saccharide derivatives,
with trehalose cores, and have investigated selected properties that we con-
sider relevant to their use as excipients. For successful use in controlled-
release systems in aqueous media, an ideal excipient is considered to have
a suitable partition between water and lipids, have stability in aqueous me-
dia, and form an amorphous phase having a glass transition temperature, Tg,
above ambient and body temperatures. Too rapid dissolution in aqueous media
and interactions with water leading to plasticization and devitrification will all
limit the utility. Too low a glass transition temperature would allow undesir-
able devitrification of the host excipient to occur, usually leading to more rapid
release of its guests. By suitable derivatization, oligosaccharides can be ob-
tained having suitable Tg and log10 P values. The log10 P value, the ability to
partition between water and octanol, is a useful measure of the balance be-
tween the hydrophilicity and hydrophobicity of a material: values between 0
and 4 are taken as being indesirable.
Important considerations for complex saccharide excipients are their syn-
thesis and purification, especially with regard to the problem of mixed anomer
products. The choice of trehalose derivatives was made due to the symmetry
of the parent disaccharide, α,α-trehalose; the nonreducing properties of the
trehalose system; and the results of a published study.[17] The latter study re-
ported the viable use of per-esterified trehalose derivatives (see Fig. 1) in the
form of microparticles for the delivery of insulin via inhalation. We wish to re-
port the preparation and study of properties of other per- and poly-substituted
trehalose derivatives in order to establish a larger number of potentially useful
compounds for use in drug delivery systems generally, and not exclusively for
delivery via inhalation.

T. C. Baddeley & J. L. Wardell
200
Figure 1: Trehalose derivatives used by Davidson et al17
.
We now wish to report our results.
RESULTS AND DISCUSSION
Synthesis
The reported study[17] involved octa-acylated trehalose derivatives
including two hexa-O-acyl-(1,2,3,4-tetra-O-acetyl-β-D-glucopyranuronyl)-
α,α-trehalose derivatives (Fig. 1). In our study, we have extended the
types of derivatives studied to more carbon-rich compounds and to poly-
substituted compounds, with free hydroxyl groups, rather than exclusively to
per-substituted compounds. Compounds studied include 6,6ꢀ-N, Nꢀ-diamido-
6,6ꢀ-dideoxy-α,α-trehalose derivatives, 6,6ꢀ-bis(1,2,3,4-tetra-O-acetyl-β-D-
glucopyranuronyl)-α,α-trehalose derivatives, and pseudo-tetrasaccaharides
such
as
2,2ꢀ,3,3ꢀ,4,4ꢀ-hexa-O-acetyl-6,6ꢀ-bis-(1,2,3,4-tetra-O-acetyl-6-O-
succinyl-β- D-glucopyranuronyl)-α,α-trehalose.
Formation of 6,6ꢀ-N,Nꢀ-diamido-6,6ꢀ-dideoxy-α,α-trehalose derivatives
Amido-trehalose derivatives, as a group, had not been targeted previously
as potential drug excipients. 2,2ꢀ,3,3ꢀ,4,4ꢀ-Hexa-O-acetyl-6,6ꢀ-diamino-6,6ꢀ-
dideoxy-α,α-trehalose, 1, had been prepared by us[21] by the reduction of
2,2ꢀ,3,3ꢀ,4,4ꢀ-hexa-O-acetyl-6,6ꢀ-di-azido-6,6ꢀ-dideoxy-α,α-trehalose, using tri-
phenylphosphine in wet reagent-grade THF, in
a
Staudinger-type
reaction.[22–25] The use of PPh3 in THF gave good yields of 1, in contrast
to the literature reports of either poor yields or its in situ conversion to
6,6ꢀ-di-N-acetyl-2,2ꢀ,3,3ꢀ-tetra-O-acetyl-6,6ꢀ-dideoxy-α,α-trehalose, 2, on using
hydrogen and Pd/C.[22,26] Direct precipitation of 1 from the THF solution facili-
tated its separation and 1 simply purified by washing with 1,2-dichloroethane:
yields greater than 90% were obtained when reaction times greater than 4
days were employed.
Rearrangement of 1 to 2 was most readily achieved in pyridine solution,
although aqueous or methanolic solutions could also be used (see Sch. 1). The
acetyl migration could be followed using the acetamido methyl protons in the
1H NMR spectrum: of interest, additional peaks for an unsymmetric interme-
diate species could be detected during the reaction. Even after long reaction
1
times, the solution H NMR spectrum indicated sets of peaks for both 1 and

Synthesis of Per- and Poly-Substituted Trehalose Derivatives
201
Scheme 1. Reagents: (i) py, 3 h, rt; (ii) Ac2O, py; (iii) Pri COCl or cyclo-C6H11COCl, py, 4 h; (iv)
NaOMe, HOMe.
2, but on work-up, only product 2, showing complete migration, was isolated.
Thus, in solution the reaction apparently only proceeds to an equilibrium posi-
tion, but that work-up drives it to completion.[27] Of interest, the conditions
for the acetyl migration in 1 to 2 (a migration from O to N) occurs under
much milder conditions than required for the more frequently observed O to
O acetyl migrations. For example, under the same conditions used to trans-
form 1 to 2, no acetyl migration occurred with 2,2ꢀ, 3,3ꢀ,4,4ꢀ-hexa-O-acetyl-α,
α-trehalose.
Treatment of 2 with acetic anhydride in pyridine provided 3, an octa-
acylated compound. In contrast, the more reactive acylating agents, isobuty-
royl and cyclohexanoyl chlorides, furnished the per-(deca-)acylated derivatives,
4 and 5. Compound 3 could also be obtained directly from 1 on reaction with
excess acetic anhydride. Attempts to couple 1 with 1,2,3,4-tetra-O-acetyl-β-
D-glucuronic acid,[28] in the presence of 1,3-dicyclohexylcarbodiimide (DCC),
or the polymer-supported coupling agent, 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide on Merrifield resin, failed to provide 4,4ꢀ-hexa-O-acetyl-
6,6ꢀ-bis(1,2,3,4-tetra-O-acetyl-β-D-glucuronamido)-1,1ꢀ-dideoxy-α,α-trehalose.
Instead 2 was isolated (i.e., acetyl migration had resulted): this further indi-
cated that the free hydroxyls at the 4,4ꢀ sites in 2 are insufficiently reactive to
couple with 1,2,3,4-tetra-O-acetyl-β-D-glucuronic acid.

T. C. Baddeley & J. L. Wardell
202
We found a slightly higher m.p. and larger [α]D value for 3 than that re-
ported [22]; this we assume is due to us having a purer product.
Acetyl migration in 1 can also be brought about under basic conditions.
Treatment of 1 with sodium methoxide in methanol[29] led to formation of 6,6ꢀ-
di-N-acetyl-6,6ꢀ-dideoxy-α,α-trehalose, 6 (i.e., both acetyl migration from the 4
to 6 sites and deacetylation at the remaining positions had occurred).
Preparations of 6,6ꢀ-bis(1,2,3,4-tetra-O-acetyl-β-D-glucopyranuronyl)-α,
α-trehalose derivatives
6,6ꢀ-Bis-(1,2,3,4-tetra-O-acetyl-β-D-glucopyranuronyl)-2,2ꢀ,3,3ꢀ,4,4ꢀ-hexa-
O-benzyl-α,α-trehalose, 7, and 6,6ꢀ-bis-(1,2,3,4-tetra-O-acetyl-β-D-glucopy-
ranuronyl)- α,α-trehalose, 8, were prepared, using as the starting compound
the known 2,2ꢀ,3,3ꢀ,4,4ꢀ-hexa-O-benzyl-α,α-trehalose, 9,[22,30] (see Sch. 2).
Reaction of 9 with 1,2,3,4-tetra-O-acetyl-β-D-glucopyranuronic acid, in the
presence of p-(dimethylamino)pyridine (DMAP) and DCC, in acetonitrile pro-
duced 6,6-bis-(1,2,3,4-tetra-O-acetyl-β-D-glucopyranuronyl)-2,2ꢀ,3,3ꢀ,4,4ꢀ-hexa-
O-benzyl-α,α-trehalose, 7, albeit in a low yield after extensive purifica-
tion. Debenzylation of 7 by H2/Pd/C gave 6,6ꢀ-bis-(1,2,3,4-tetra-O-acetyl-β-D-
glucopyranuronyl)-α,α-trehalose, 8.
The compounds, 2,2ꢀ,3,3ꢀ-tetra-O-acetyl-6,6ꢀ-bis-(1,2,3,4-tetra-O-acetyl-β-
D-glucopyranuronyl)-α,α-trehalose, 10; 2,2ꢀ,3,3ꢀ-tetra-O-acetyl-6,6ꢀ-bis-(1,2,3,
4-tetra-O-acetyl-β-D-glucopyranuronyl)-4,4ꢀ-di-O-isobutyryl-α,α-trehalose, 11;
and 2,2ꢀ,3,3ꢀ-tetra-O-acetyl-6,6ꢀ-bis-(1,2,3,4-tetra-O-acetyl-β-D-glucopyranuro-
nyl)-4,4ꢀ-di-O-cyclohexanecarbonyl-α,α-trehalose, 12, were obtained as shown
in Scheme 3.
2,2ꢀ,3,3ꢀ-Tetra-O-acetyl-α,α-trehalose, 13, was obtained from trehalose via
the following sequence: (i) benzylidation, using PhCH(OMe)2/p-toluenesulfonyl
chloride/DMF, (ii) acetylation, using acetyl anhydride/pyridine, and (iii) deben-
zylidation, using acetic acid at 45◦C. Reaction of 13 with 1,2,3,4-tetra-O-
acetyl-β-D-glucopyranuronic acid (1:2 mole ratio), in the presence of DCC
(two equivalents) and DMP in MeCN, gave a mixture of 10 and the prod-
uct of a mono-reaction, 2,2ꢀ,3,3ꢀ-tetra-O-acetyl-1-(1,2,3,4-tetra-O-acetyl-β-D-
glucopyranuronyl)-α,α-trehalose, 14. As expected, reactions with 13 only
occurred at the less hindered primary alcohol groups. We subsequently estab-
lished that DCC is partially consumed, even in the presence of DMAP, as an
adduct with 1,2,3,4-tetra-O-acetyl-β-D-glucopyranuronic acid. Separation of 10
and 14 could be readily made using column chromatography.
Reaction of the separated compounds with acyl chlorides led to the
expected products. However, it was more convenient and efficient to
treat the unseparated mixture of 10 and 14 with acyl chlorides and
then to separate products using column chromatography. Thus, reaction
with isobutyryl chloride, 10 and 14, gave a readily separable mixture of

Synthesis of Per- and Poly-Substituted Trehalose Derivatives
203
Scheme 2. Reagents: (i) DCC, DMAP, MeCN, 4 h; (ii) H2, Pd/C, MeOH, 3 d.
2,2ꢀ,3,3ꢀ-tetra-O-acetyl-6,6ꢀ-bis-(1,2,3,4-tetra-O-acetyl-β-D-glucopyranuronyl)-
4,4ꢀ-di-O-isobutyryl-α,α-trehalose, 11, and 2,2ꢀ,3,3ꢀ-tetra-O-acetyl-1-(1,2,3,4-
tetra-O-acetyl-β-D-glucopyranuronyl)-4,4ꢀ,1ꢀ-tri-O-isobutyryl-α,α-trehalose,
15. Similarly, 10 and 14 produced with cyclohexanoyl chloride a readily
separated mixture of 2,2ꢀ,3,3ꢀ-tetra-O-acetyl-6,6ꢀ-bis-(1,2,3,4-tetra-O-acetyl-
β-D-glucopyranuronyl)-4,4ꢀ-di-O-cyclohexanecarbonyl-α,α-trehalose, 12, and
2,2ꢀ,3,3ꢀ-tetra-O-acetyl-1-(1,2,3,4-tetra-O-acetyl-β-D-glucopyranuronyl)-4,4ꢀ,1ꢀ-
tri-O-cyclohexanecarbonyl-α,α-trehalose, 16.
Preparation of 2,2ꢀ,3,3ꢀ,4,4ꢀ-hexa-O-acetyl-6,6ꢀ-bis-(1,2,3,4-tetra-O-acetyl-6-O-
succinyl-β-D-glucopyranuronyl)-α,α-trehalose
The succinic acid ester, 17, a pseudo-tetrasaccharide, derived from
two different carbohydrate residues was obtained by an initial reaction of

T. C. Baddeley & J. L. Wardell
204
Scheme 3. Reagents: (i) PhCH(OMe)2, p-TsCl, DMF, 50◦C, 40 min; (ii) Ac2O, py, rt, 8 h; (iii)
AcOH, 45◦C, H2O, 20 min; (iv) DCC, DMAP, MeCN, RT, 8 h; (v) RCOCl (R = Pri or cyclohexyl),
py, rt, 8 h.
succinic anhydride with 2,2ꢀ,3,3ꢀ,4,4ꢀ-hexa-O-acetyl -α,α-trehalose, 18, to give
19, which was then coupled with 1,2,3,4-tetra-O-acetyl-β-D-glucopyranose
(see Sch. 4).
Our prepared sample of 18 had a considerably larger αD value (253◦) than
that quoted in the literature[36] (158.8◦): as our m.p. was higher and sharper
(102–103◦C vs. 93–96◦C), we assume our sample was the purer. However, the
m.p. of the intermediate, 6,6ꢀ-di-O-trityl-α,α-trehalose, used to obtain 18, was
much lower than that quoted in the literature[26] (191–193◦C compared to 278–
281◦C). NMR spectra were as expected for the compound, but a small amount
of water was clearly present.
Screening of Compounds Based on Tg and log Pow Value for
Potential Use as Excipients
Glass transition temperatures, Tg, and partition coefficients, log10 P val-
ues, were determined for the oligosaccharide derivatives (see Table 1). Melting
points, Tg values, and remelts of investigated compounds were obtained us-
ing heat, cool, and reheat programs in DSC experiments. Melting points are

Synthesis of Per- and Poly-Substituted Trehalose Derivatives
205
Scheme 4. Reagents: (i) Ph3CCl, Ac2O, py, 2 h; (ii) IR 70 resin, H2O, MeCN, 5◦C, 20 h; (iii)
succinic anhydride, py, 8 h; (iv) DMAP, MeCN, rt, 8 h.
quoted as a single temperature as they were taken as the maximum of the
melting peak in the DSC trace. Octanol-water partition coefficients (log Pow
)
were determined using an HPLC method.[31] Being a partition coefficient, log10
P, which is defined as the partition coefficient between 1-octanol and water, is
not an absolute measure of hydrophobicity or solubility in water. However, it is
assumed that a high value of log10 P indicates a hydrophobic substance having
a low solubility in water.
A useful excipient is considered to have a Tg value at least 20◦C above
temperature of its use and a log Pow value between 0 and 4 for release into
aqueous media. Above Tg, devitrification will occur, one consequence of which
is the creation of pores or channels, which would allow incipients to be released
at increasing rates as crystallization proceeds.
On the basis of the stated criteria for a potentially useful excipient, com-
pounds 10, 11, 12, 14, 15, and17 are worthy of further study. However, com-
pounds 7 (too low a Tg value), 3 (too low a log10 P value), and 5 and 16 (log10
P

T. C. Baddeley & J. L. Wardell
206
Table 1: Properties of oligosaccharide derivatives
Compound
m.p.◦C
T◦gC
Log10
P
Mol. Weight
3
110
183
125
53
72
118
105
26
–1.19
4.26
676
872
4
5
8.36
1032
1570
1030
1198
1338
1418
854
a
7
8
124
220
96
61
1.30
2.26
3.30
3.88
1.06
3.50
4.93
3.04
0.97
10
111
85
11
12
91
205
106
88
14
129
15
99
1062
1182
1456
592
b
16
17
113
91
95
51
octa-O-acetyl-α,
α-trehalose
a Not determined as Tg is too low.
bNot determined as log10P is too low.
too high) can be immediately eliminated as potentially useful excipients, while
compound 4 is marginal.
It is, however, important to keep in mind that any compound in-
corporated into the excipient could lead to a reduction of the Tg value
of the excipient. This was realized with compound 3, formulated by
spray drying with (2S,3S)-(+)-cis-3-acetoxy-5-(2-dimethylaminoethanol)-2,3-
dihydro-2-(4-methoxyphenyl)-1,5-benzothiazepin-4(5H)-one hydrochloride (Di-
tiazem HCl) or pregna-1,4-diene-3,20-dione-6,7,21-trihydroxy-prednisolone
(prednisolone) at 10% loadings. In both cases, there were reductions in the Tg
value of pure 3 by 15◦C. However, the same drugs incorporated into 4 produced
much lower reductions (<5◦C).
As well as Tg and log10P values, other factors must be evaluated. For exam-
ple, the effects of humidity and water on excipients/formulations, which could
lead to crystallization and other unwanted results, must be considered: such
effects are not necessarily related to log10P values. These and other aspects
will be discussed in future articles.
The glass transition and melting point data, listed in Table 1, clearly il-
lustrate that melting points and glass transition temperatures do not cor-
relate with molecular weights even for related per-esterified saccharides,
which are free of such strong intermolecular associations as H-O—H hydrogen
bonds. Where strong H-bonding can occur as in compounds with free hydroxyl
groups, significant differences in Tgvalues are expected. This is exemplified
by the Tg/m.p. values for trehalose, 6,6ꢀ-yghdiamino-6,6ꢀ-dideoxy-α,α-trehalose,
and 6,6ꢀ-diacetamido-6,6ꢀ-dideoxy-α,α-trehalose of 70/202◦C, 10/222◦C, and
22/84◦C, respectively.[31]

Synthesis of Per- and Poly-Substituted Trehalose Derivatives
207
Conclusion
While further study is necessary, the Tg and log10P data for compounds 10,
11, 12, 14, 15, and 17 indicate their potential as excipients. These compounds,
having a range of Tg and log10P values within the recommended limits, can fur-
ther extend the utility of trehalose and trehalose derivatives, such as trehalose
octaacetate, already found to be useful in drug delivery systems.
EXPERIMENTAL
General
TLC was carried out on silica using 10% sulphuric acid, iodine, or ninhy-
drin to develop spots. Melting points were obtained on a Kofler hotstage or
a Perkin-Elmer DSC7 Differential Scanning Calorimeter and are uncorrected.
IR spectra were recorded in KBr discs using an Atti Mattson FTIR instrument;
1
UV-visible spectra on a Cary 300 UV spectrometer; H and 13C NMR spectra
on 250, 300, or 500 MHz Bruker instruments; or mass spectra on a Finnigan
Navigator Mass Spectrometer using ES+ ionization (low resolution); or by the
EPSRC mass spectrometer service at the University of Swansea (high resolu-
tion) and optical rotations (deg.cm2.g−1 and concentration, c, in g.100 mL−1
)
on a Bellingham + Stanley Ltd P20 polarimeter. C, H, and N analyses were
performed on a Perkin–Elmer 2400 apparatus. A Hewlett Packard HP 1100
instrument was used for HPLC measurements.
Materials characterization
Thermal analysis was carried out on 10-mg samples on a Perkin-Elmer
DSC7 Differential Scanning Calorimeter, using heat, cool, and reheat cycles
with temperature gradients of 10◦C per minute. Heating was taken to at least
20◦C above melting point, Tm, and cooling to at least 20◦C below the glass tran-
sition temperature, Tg. Thermogravimetric analysis was carried on a Perkin
Elmer TGA 7 instrument: Pyris 1 software was used.
Octanol-water partition coefficients (log Pow) were determined using an
HPLC method.[32] Values of log Pow for test compounds were calculated from
the linear correlation of log Pow and capacity factor, K, for the reference ma-
terials (benzyl alcohol, methyl benzoate, biphenyl, and bibenzyl), where the
capacity factor, K, is defined as K = (rtx– rto)/rto,where rtx is the retention time
for the compound, and rto is the retention time for acetonitrile, under a given
set of conditions. The retention times of the new saccharides and reference
compounds of known log Pow were measured on a Phenomenex Jupiter (C18
250 mm × 4.6 mm, 5 µm) column with 80:20 acetonitrile:water as the mobile
phase, with UV detection at 209 nm.

T. C. Baddeley & J. L. Wardell
208
Syntheses of Compounds
6,6ꢀ-Bis-(N-acetylamino)-2,2ꢀ,3,3ꢀ,4,4ꢀ-hexa-O-acetyl-6,6ꢀ-dideoxy-α,α-
trehalose, 3
To a solution of 2ꢀ,3,3ꢀ,4,4ꢀ-hexa-O-acetyl-6,6ꢀ-diamino-6,6ꢀ-dideoxy-α,α-
trehalose, 1[9] (2.0 g, 3.4 mmol), in methanol (30 mL) and pyridine (5 mL) was
added excess acetic anhydride (2 mL, 14 mmol). After leaving overnight, the
solvent was removed under reduced pressure to leave an oil. Diethyl ether was
added and the resulting sticky solid was recrystallized from CH2Cl2/Et2O at
–22◦C; yield 1.5 g (65.7%); m.p. 109–110◦C, lit. m.p. (ethyl acetate/ hexane)
92–98◦C[22]; [α]2D5 (c = 4, CHCl3) 170.4; lit [22] [α]D (CHCl3) 155.
1H NMR (400 MHz, CDCl3): δ: 1.94 (s, 3H, Me), 1.97 (s, 3H, Me), 2.02 (s, 3H,
Me), 2.03 (s, 3H, Me), 3.18 (m, 1H, H-6ꢀ), 3.51 (m, 1H, H-5), 3.76 (m, 1H,
H-6), 4.83 (t, 1H, J = 9.9 Hz, H-4), 4.85 (dd, 1H, J = 4.0, 9.9, H-2), 5.27
(d, 1H, J = 4.0 Hz, H-1), 5.42 (t, 1H, J = 9.9 Hz, H-3), 5.84 (t, 1H, J = 6.0,
NHCOCH3).
13C NMR (100 MHz, CDCl3): δ: 20.5, 20.6, 20.6, 23.0, 38.9, 69.0, 69.3, 69.5,
70.2, 91.4, 169.8, 169.9, 170.2, 170.3.
IR (KBr, cm−1): v: 3554–3411, 1753, 1655, 1543, 1372, 1225, 1040.
MS (FAB): 168.0 (100%), 677.0 (47%, M + H), 227.9 (28%), 698.9 (24%, M +
Na).
MS (ES+, Acc. Mass, M + H): calculated for C28H40N2O17: 677.2405, obtained:
677.2402.
6,6ꢀ-Bis-(N-acetylamino)-2,2ꢀ,3,3ꢀ-tetra-O-acetyl-6,6ꢀ-dideoxy-α,α-trehalose, 2
Diamine, 1 (0.5 g, 0.9 mmol), was dissolved either in pyridine (10 mL),
methanol (10 mL), or water (10 mL). The reaction mixtures were left overnight.
The aqueous or methanolic solution reaction mixtures were rotary evaporated
to leave a white solid, whereas ether was added to the pyridine reaction mix-
ture to precipitate a solid, m.p. 204◦C; [α]D25 (c = 3, MeOH) 123.2; lit[22] m.p.
(from ethanol/ether) 197–200◦C; lit.[22] [α]D (methanol/water, 3:1) 130.
1H NMR (250 MHz, CDCl3): δ: 1.80 (s, 3H, Me), 1.98 (s, 3H, Me), 2.02 (s, 3H,
Me), 2.91 (m, 1H, H-6), 3.38 (t, 1H, J = 9.2 Hz, H-4), 3.54 (m, 1H, H-5), 3.70
(m, 1H, H-6ꢀ), 4.79 (dd, 1H, J = 3.7 & 10.37 Hz, H-2), 5.18 (m, 2H, H-1 and
H-3), 7.81 (t, J = 4.3 Hz, NHCOCH3).
13C NMR (63 MHz, CDCl3): δ: 20.3, 20.8, 22.4, 69.0, 69.8, 71.4, 71.7, 90.6, 169.7,
169.9.

Synthesis of Per- and Poly-Substituted Trehalose Derivatives
209
IR (KBr): v: 3615–3096, 2867, 1739, 1643, 1571, 1437, 1378, 1236, 1141, 1047,
1017.
MS (ES+): 615.3 (100%, M + Na), 573.3 (6%, M-Ac + Na), 1207.7 (2%, 2M +
Na), 531.3 (1%, M-2Ac + Na).
Anal. Calcd for C24H36N2O15: C, 48.65; H, 6.12; N, 4.72. Found: C, 49.01; H,
6.31; N, 4.89.
2,2ꢀ,3,3ꢀ-Tetra-O-acetyl-6,6ꢀ-bis-(N-acetyl-N-isobutyroylamino)-6,6ꢀ-dideoxy-
4,4ꢀ-di-O-isobutyryl-α,α-trehalose, 4
To a solution of 3 (2.0 g, 3.4 mmol) in pyridine (20 mL) was added isobu-
tyroyl chloride (3 mL, 10.3 mmol). After stirring overnight, the solvent was
removed at reduced pressure to leave an oil, which was poured into water (500
mL). The resulting white precipitate was collected, dried, and recrystallized
from ethanol. Further recrystallization was achieved from aqueous methanol.
Yield of 4 was 0.9 g (30%); m.p. 183◦C; [α]D23 (c = 4, CHCl3) 132.6.
1H NMR (250 MHz, CDCl3): δ: 1.10 (t, 3H, J = 6.4 Hz, isoBut), 1.15 (t, 3H, J =
6.4 Hz, isoBut), 2.0 (s, 3H, Me), 2.1 (s, 3H, Me), 2.3 (s, 3H, Me), 2.6 (qn/spt,
1H, J = 7.0 Hz, COCHMe2), 3.15 (qn/spt, 1H, J = 6.4 Hz, COCHMe2), 3.35
(dd, 1H, J = 10.1 & 14.6 Hz, H-6), 3.75 (m, 1H, H-5), 4.01 (d(d), 1H, J = 14.6
Hz, H-6ꢀ), 4.85 (m, 2H, J = 4.0 & 10.1 Hz, H-1 and H-4), 5.0 (dd, 1H, J = 4.0
& 10.1 Hz, H-2), 5.5 (t, 1H, J = 10.1 Hz, H-3).
13C NMR (100 MHz, CDCl3): δ: 18.3, 18.7, 18.8, 19.6, 20.5, 20.6, 25.6, 33.9,
34.0, 45.1, 69.2, 69.3, 69.6, 70.1, 89.8, 169.4, 170.1, 173.7, 176.3, 181.8.
IR (KBr, cm−1): v: 2977, 2934, 2876, 1749, 1712, 1472, 1434, 1371, 1318, 1234,
1151, 1070, 1021, 983, 805, 477.
MS (FAB): 168.0 (100%), 428.1 (85%), 358.0 (68%), 238.0 (31%), 873.2 (12%, M
+ H), 895.2 (8%, M + Na).
MS (ES+, Acc. Mass, M + H) Calcd for C40H60N2O19: 873.3869. Obtained:
873.3878.
2,2ꢀ,3,3ꢀ-Tetra-O-acetyl-6,6ꢀ-bis-(N-acetyl,N-cyclohexanecarbonylamino)-4,4ꢀ-
di-O-cyclohexanecarbonoyl-6,6ꢀdideoxy-α,α-trehalose, 5
To a solution of 1 (5.0 g, 8.5 mmol) in pyridine (40 mL) that had been left
standing for 1 day was added excess cyclohexanecarbonyl chloride (10 mL, 68
mmol) and DMAP (5 mg). The reaction mixture was left for 4 h with stirring
and poured into water (200 mL), and the product extracted into CH2Cl2 twice
(100 mL). The CH2Cl2 solution was dried and concentrated and the residue
crystallized from methanol; yield 1.85 g; m.p. 124–125◦C; [α]D23 (c = 1, CHCl3)
148.1.

T. C. Baddeley & J. L. Wardell
210
1H NMR (400 MHz, CDCl3): δ: 1.3 (m, 12H, C6H11), 1.7 (m, 12H, C6H11), 1.95
(s, 3H, Me), 2.10 (s, 3H, Me), 2.30 (s, 3H, Me), 3.29 (dd, 1H, J = 9.9 & 14.7
Hz, H-6), 3.70 (ddd, 1H, J = 2.1, 9.9 & 10.3 Hz, H-5), 3.99 (dd, H1, J = 2.1 &
14.7 Hz, H-6ꢀ), 4.78 (d, 1H, J = 3.8 Hz, H-1), 4.85 (t, 1H, J = 10.3 Hz, H-4),
5.00 (dd, 1H, J = 3.8 & 10.3 Hz, H-2), 5.40 (t, 1H, J = 10.3 Hz, H-3).
13C NMR (63 MHz, CDCl3): δ:20.7, 25.2, 25.3, 25.5, 25.6, 25.8, 26.0, 28.2, 28.7,
28.9, 30.0, 31.0, 43.0, 43.6, 45.1, 69.1, 69.3, 69.7, 69.9, 90.0, 169.5, 170.2,
173.7, 175.4, 180.8.
IR (KBr, cm−1): v: 29.36, 28.58, 1759, 1715, 1694, 1452, 1165, 1140, 1074, 1024,
983.
MS (FAB): 398.1 (100%), 168.0 (96%), 111.0 (84%), 508.2 (37%), 1033.5 (7%,
M + H), 1055.4 (6%, M + Na).
MS (ES+, Acc. Mass, M + H) Calcd for C52H76N2O19: 1033.5121. Found:
1033.5116.
6,6ꢀ-Bis-(N-acetylamino)-6,6ꢀ-dideoxy-α,α-trehalose, 6
To a solution of 1 (2.0 g, 3.4 mmol) in methanol was added sodium
methoxide (18.5 mg, 3.4 mmol) and left stirring for 4 h. TLC using ethyl ac-
etate:methanol (2:1) as eluent showed complete conversion to a baseline prod-
uct. The solution was neutralized by stirring with DOWEX 50W X-8 resin and
filtered and the resin washed with methanol. The filtrates and washings were
combined, the solvent was removed at reduced pressure, and the residue was
recrystallized from acetone/ethanol and obtained as an acetone solvate. After
filtration, the crystals desolvated on standing to give a liquid, which was so-
lidified to a white solid by rotary evaporation; yield of title product was 0.95 g
(66.4%); m.p. 132–134◦C; [α]D23 (c = 1, CH3OH) 67.05.
1H NMR (250 MHz, CDCl3): δ: 1.83 (s, 3H, Me), 2.96 (t, 1H, J = 9.6 Hz, H-4),
3.24 (m, 3H, H-2,6,6ꢀ), 3.52 (t, 1H, J = 9.6 Hz, H-3), 3.69 (ddd, 1H, J = 3.1,
5.5, & 9.6 Hz, H-5), 4.86 (d, 1H, J = 3.8 Hz, H-1), 7.73 (brs, 1H, NHCOCH3).
13C NMR (63 MHz, CDCl3): δ: 22.5, 70.5, 71.3, 71.6, 72.3, 93.8, 170.1.
IR (KBr, cm−1): v: 3582–3183, 2919, 1638, 1561, 1439, 1382, 1150, 1109, 1043,
991.
Anal. Calcd for C16H28N2O11: C, 45.20; H, 6.64; N, 6.58. Found: C, 45.49;
H,6.48; N, 6.70.
6,6-Bis-(1,2,3,4-tetra-O-acetyl-β-D-glucopyranuronyl)-2,2ꢀ,3,3ꢀ,4,4ꢀ-hexa-O-
benzyl-α,α-trehalose, 7
To a stirred solution of 2,2ꢀ,3,3ꢀ,4,4ꢀ-hexa-O-benzyl-α,α-trehalose, 9[5,8]
(6.00 g, 6.8 mmol), 1,2,3,4-tetra-O-acetyl-β-D-glucopyranuronic acid (5.40 g,

Synthesis of Per- and Poly-Substituted Trehalose Derivatives
211
14.3 mmol), and p-(dimethylamino)pyridine (17.40 mg, 0.14 mmol) in acetoni-
trile (20 mL) was added a solution of DCC (2.95 g, 14.3 mmol) in acetonitrile
(10 mL) drop-wise over 10 min. After 4 h, the solution was filtered and the
filtrate reduced. The syrupy residue was added with stirring to hot diethyl
ether and filtered, and the white solid, (1,3-dicyclohexylcarbodiimide-1,2,3,4-
tetra-O-acetyl-β-D-glucopyranuronic acid adduct) discarded. The filtrate was
chromatographed using a Chromatotron, with ethyl acetate as eluent. The
third fraction was collected; evaporated to leave a residue, which was re-
dissolved in diethyl ether; and evaporated to give a white foam. The foam
was shown to be a mixture of two compounds, which were separated using
TLC with ethyl acetate:petrol (1:1). The less mobile compound, the title com-
pound, was collected as a foam; yield 1.6 g (15%); m.p. 53–56◦C; [α]D24 (c = 4,
CHCl3) 54.4.
1H NMR (400 MHz, CDCl3): δ: 1.97 (s, 3H, Me), 1.98 (s, 3H, Me), 2.00 (s, 3H,
Me), 2.01 (s, 3H, Me), 3.41 (m, 1H, H-5 [T]), 3.54 (m, 2H, H-6, 6ꢀ [T]), 4.00
(m, 2H, [G]), 4.16 (m, 2H, [T]), 4.47 (d, 1H, J = 10.9, CH2Ph), 4.66 (m, 2H,
CH2Ph), 4.83 (m, 2H, CH2Ph), 4.96 (dd, 1H, CH2Ph), 5.06 (m, 2H, [G, T]),
5.18 (m, 2H, [G, T]), 5.66 (d, 1H, J = 7.5, H-1 [G]), 7.2–7.4 (m, 15H, Ar) (G =
glucose; T = trehalose).
13C NMR (100 MHz, CDCl3): δ: 21.5, 21.6, 21.6, 21.7, 69.9, 70.0, 71.2, 71.2,
73.1, 74.1, 74.1, 74.2, 74.2, 76.1, 76.6, 82.5, 92.4, 128.4–129.6, 139.0, 139.3,
139.6, 167.1, 167.7, 170.2, 170.2, 171.0.
IR (KBr, cm−1): ν: 2929, 1752, 1670, 1511, 1365, 1245, 1216, 1067, 1035.
MS(FAB): 243.1 (100%), 1593.6 (7%, M + Na), 333.2 (5%).
Anal. Calcd for C82H90O31: C, 62.65; H, 5.77. Found: C, 62.46; H, 5.93.
6,6ꢀ-Bis-(1,2,3,4-tetra-O-acetyl-β-D-glucopyranuronyl)-α,α-trehalose, 8
A
solution of 6,6-bis-(1,2,3,4-tetra-O-acetyl-β-D-glucopyranuronyl)-
2,2ꢀ,3,3ꢀ,4,4ꢀ-hexa-O-benzyl-α,α-trehalose, 7 (2.00 g, 1.3 mmol), in ethanol
(25 mL) was hydrogenated over palladium on charcoal (1.05 g, 10%). After
3 days, the reaction mixture was filtered and the filtrate was evaporated
at reduced pressure to leave a colorless oil, which was crystallized from
ethanol/acetone at –22◦C. This solid liquified at rt. Recrystallization from
ethanol and diethyl ether produced a hydroscopic solid. Yield of the white
solid was 1.25 g (95.3%); m.p. 124◦C.
The 1H NMR spectrum was not resolved.
13C NMR (100 MHz, DMSO): δ: 21.0, 21.1, 21.2, 21.3, 21.4, 62.6, 62.7, 71.3,
71.7, 71.8, 73.1, 73.7, 73.7, 74.5, 94.9, 95.2, 171.2.

T. C. Baddeley & J. L. Wardell
212
MS (ES+): 709.6 (100%, M-35 + Na), 399.8 (35%), 1053.4 (30%, M + Na), 365.8
(32%), 650.9 (31%), 881.6 (15%).
Anal. Calcd for C40H54O31: C, 46.60; H, 5.28. Found: C, 46.87; H,5.41.
4,6:4ꢀ,6ꢀ-Di-O-benzylidene-α,α-trehalose
DMF (60 mL) was dried by refluxing with toluene (60 mL) and rotary evap-
oration at 60◦C to remove toluene and any residue water. p-Toluenesulfonic
acid monohydrate (0.24 g, 1.4 mmol) was dehydrated by rotary evaporation of
a suspension in toluene (60 mL). α,α-Trehalose dihydrate (10.00 g, 26.5 mmol)
was dried by refluxing in ethanol (60 mL) for 1 h and drying overnight at 60◦C.
Subsequent weight of anhydrous trehalose was 8.97 g (99%). To a suspension
of the anhydrous trehalose in dried DMF (30 mL) was added a solution of p-
toluenesulfonic acid and (dimethoxymethyl)benzene (4 mL, 26.5 mmol) in dry
DMF (30 mL). The mixture was heated at 100◦C for 10 min and concentrated at
60◦C. More (dimethoxymethyl)benzene (4 mL, 26.5 mmol) was added and the
heating procedure repeated. A further amount of (dimethoxymethyl)benzene
(1 mL, 6.6 mmol) was added, and the mixture was heated on a steam bath for
10 min to give a colorless solution. The solvent was removed at reduced pres-
sure and the residue crystallized on shaking with toluene and water. Yield of
title product was 12.36 g (91%); m.p. 140–145◦C (DSC); [α]D25 (c = 3, CH3OH)
165.2; lit.[33] m.p. 197–198◦C (from ethanol); lit[33][α]D (CHCl3) 93 and (MeOH)
81.3.[34]
1H NMR (400 MHz, CDCl3): δ: 3.45 (t, 1H, J = 9.Hz, H-3), 3.59 (dd, 1H, J = 3.9
& 9.3 Hz, H-2), 3.68 (t, 1H, J = 9.9 Hz, H-6), 3.99 (t, 1H, J = 9.3 Hz, H-4),
4.07 (dt, 1H, J = 4.8 & 9.3 Hz, H-5), 4.18 (dd, 1H, J = 4.8 & 9.9 Hz, H-6ꢀ),
4.54 (brs, 2H, OH), 5.08 (d, 1H, J = 3.9 Hz, H-1), 5.23 (s, 1H, CHAr).
13C NMR (100 MHz, CDCl3): δ: 63.8 (C-5), 69.6 (C-6), 71.1 (C-4), 73.4 (C-2),
82.6 (C-3), 96.0 (C-1), 102.7 (CHAr), 127.2, 128.7, 128.9, 129.6, 138.8.
MS (ES+): 541.0 (100%, M + Na), 119.0 (40%), 160.0 (30%), 371.0 (30%), 573.1
(15%, M + Na + MeOH), 557.1 (5%, M + K).
2,2ꢀ,3,3ꢀ-Tetra-O-acetyl-4,6:4ꢀ,6ꢀ-di-O-benzylidene-α,α-trehalose
To a solution of 4,6:4ꢀ,6ꢀ-di-O-benzylidene-α,α-trehalose (66.0 g, 12.7 mmol)
in pyridine (100 mL) and acetonitrile (100 mL) was added acetic anhydride
(80 mL, 0.78 mmol). After stirring for 18 h, the solvent was removed un-
der reduced pressure. The oily residue was precipitated on addition to wa-
ter with vigorous stirring. The precipitate was filtered and dried; yield of ti-
tle compound 63.7 g (73%); m.p. 267–268◦C (DSC), lit m.p. 246–247◦C (from
CH2Cl2/diethyl ether)[35]; [α]D24 (c = 4, CH3OH) 188.5, lit [α]D (CHCl3) value
124.[35]

Synthesis of Per- and Poly-Substituted Trehalose Derivatives
213
1H NMR (250 MHz, CDCl3): δ: 2.06 (s, 3H, Me), 2.12 (s, 3H, Me), 3.67 (t, 1H,
J = 9.8 Hz, H-4), 3.73 (dd, 1H, J = 9.8 & 10.4 Hz, H-6), 3.98 (ddd, 1H, J =
4.9, 9.8, & 9.8 Hz, H-5), 4.15 (dd, 1H, J = 4.9 & 10.4 Hz, H-6ꢀ), 4.99 (dd, 1H,
J = 4.0 & 9.8 Hz, H-2), 5.33 (d, 1H, J = 4.0 Hz, H-1), 5.48 (s, 1H, CHAr),
5.61 (t, 1H, J = 9.8 Hz, H-3), 7.33 (m, 3H, Ar), 7.41 (m, 2H, Ar).
13C NMR (63 MHz, CDCl3): δ: 20.7, 20.9, 63.1, 68.6, 69.0, 70.9, 79.0, 93.1, 101.8,
126.2, 128.3, 128.4, 129.2, 136.7, 169.7, 170.4.
IR (KBr, cm−1): ν: 2976, 2939, 2866, 1753, 1374, 1233, 1137, 1097, 1062, 1002,
982.
MS (ES+): 709.1 (100%, M + Na), 725.1 (12%, M + K).
2,2ꢀ,3,3ꢀ-Tetra-O-acetyl-α,α-trehalose, 13
2,2ꢀ,3,3ꢀ-Tetra-O-acetyl-4,6:4ꢀ,6ꢀ-di-O-benzylidene-α,α-trehalose (30.0 g,
43.7 mmol) was dissolved in acetic acid (540 mL) and heated to 95◦C, and
water (360 mL) was slowly added to the stirred solution. After 20 min at 95◦C,
the solvent was removed under reduced pressure. Toluene (200 mL) was added
to the residue, the solvent was removed under reduced pressure, and toluene
(200 mL) again was added, followed by diethyl ether. The mixture was rotary
evaporated. The procedure was repeated until the residue became a foam.
Crystallization from ethyl acetate yielded 17.05 g (78%) of the title product;
m.p. 180–183◦C (DSC); [α]D23 (c = 3, CH3OH) 303.7.
1H NMR (400 MHz, CDCl3): δ: 2.03 (s, 3H, Me), 2.04 (s, 3H, Me), 3.55 (t, 1H,
J = 9.6 Hz, H-4), 3.66 (dd, 1H, J = 5.1 & 12.0 Hz, H-6), 3.73 (dd, 1H, J = 2.4
& 12.0 Hz, H-6ꢀ), 3.85 (ddd, 1H, J = 2.4, 5.1, & 9.6 Hz, H-5), 4.84 (dd, 1H,
J = 3.8 & 10.3 Hz, H-2), 5.26 (d, 1H, J = 3.8 Hz, H-1), 5.36 (dd, 1H, J = 9.6
& 10.3 Hz, H-3).
13C NMR (63 MHz, CDCl3): δ: 20.9, 21.0, 62.2, 69.8, 72.1, 74.1, 74.4, 94.3, 171.9,
172.3.
IR (KBr, cm−1): ν: 3564–3341, 2937, 1746, 1438, 1371, 1249, 1133, 1051, 999,
942.
MS (ES+): 533.0 (100%, M + Na), 491.0 (10%, M-Ac + Na), 549.0 (4%, M + K).
Anal. Calcd for C20H30O15: C, 46.87; H, 5.90. Found: C, 46.59; H, 6.04.
2,2ꢀ,3,3ꢀ-Tetra-O-acetyl-6,6ꢀ-bis-(1,2,3,4-tetra-O-acetyl-β-D-glucopyranuronyl)-
α,α-trehalose, 10, and 2,2ꢀ,3,3ꢀ-tetra-O-acetyl-6-(1,2,3,4-tetra-O-acetyl-β-D-
glucopyranuronyl)-α,α-trehalose, 14
A solution of 1,2,3,4-tetra-O-acetyl-β-D-glucopyranuronic acid (3.28 g, 8.6
mmol) in CH2Cl2 (20 mL), was dried over anhydrous calcium chloride. After

T. C. Baddeley & J. L. Wardell
214
removal of the drying agent, toluene (50 mL) was added and the solvent was
removed at reduced pressure to leave a dried sample of solid 1,2,3,4-tetra-O-
acetyl-β-D-glucopyranuronic acid. To a solution of this solid with 2,2ꢀ,3,3ꢀ-tetra-
O-acetyl-α,α-trehalose, 13 (2.00 g, 3.9 mmol), and p-(dimethylamino)pyridine
(82 µg) in acetonitrile (30 mL) was slowly added a solution of DCC (1.78 g, 8.6
mmol) in acetonitrile (10 mL). After stirring overnight, the solid product, the
DCC adduct, was collected by filtration. More DCC adduct was obtained from
the filtrate on addition of methanol (10 mL). After removal of this by-product,
the solvent was removed at reduced pressure to leave a residue, which was
crystallized from a mixture of CH2Cl2 and diethyl ether; yield 3.10 g, m.p.
129–133◦C. TLC with ethyl acetate eluent indicated the presence of two prod-
ucts, which were separated by column chromatography using ethyl acetate as
eluent. From 400 mg of the mixed product, pseudo-tetrasaccharide (10, 170
mg) and pseudo-trisaccharide (14, 90 mg) were obtained in pure states; m.p. of
10 was 219–220◦C and 14 was 203–205◦C.
MS (ES+) of the unseparated products: 119.0 (100%), 150.9 (35%), 877.1 (15%,
14 + Na), 1221.2 (7%, 10 + Na).
1H NMR of 10 (400 MHz, CDCl3): δ: 1.97 (s, 6H, Me), 1.98 (s, 6H, Me), 2.05
(s, 3H, Me), 2.08 (s, 3H, Me), 3.51 (t, 1H, H-4 [T]), 3.86 (m, 1H, H-5 [T]),
4.22 (m, 2H, H-5 [G], H-6 [T]), 4.39 (dd, 1H, H-6ꢀ [T]), 4.93 (dd, 1H, H-2 [T]),
5.13–5.28 (m, 5H, H-2,3,4 [G], H-3,1 [T]), 5.73 (d, 1H, J = 7.9, H-1 [G]).
13C NMR of 10 (100 MHz, CDCl3): δ: 20.4, 20.5, 20.7, 20.9, 61.9, 64.2, 68.7,
69.1, 69.8, 70.1, 70.3, 71.9, 72.8, 72.9, 91.4, 94.4, 166.5, 168.8, 169.2, 169.7,
169.9, 170.0, 172.0.
Acc. Mass (M + NH4): Calcd for C48H66NO35: 1216.3415. Found 1216.3418.
1H NMR of 14 (400 MHz, CDCl3): δ: 1.98 (s, 3H, Me), 1.99 (s, 3H, Me), 2.00 (s,
3H, Me), 2.03 (s, 3H, Me), 2.06 (s, 3H, Me), 2.07 (s, 3H, Me), 2.08 (s, 3H, Me),
2.09 (s, 3H, Me), 3.52 (t, 1H, J = 9.9 Hz, H-4 [T2]), 3.64 (t, 1H, J = 9.6 Hz,
H-4a [T1]), 3.69–3.80 (m, 3H, H-5, 6, 6ꢀ, [T1]), 3.92 (m, 1H, H-5, [T2]), 4.23
(d, 1H, J = 9.2 Hz, H-5 [G]), 4.25 (dd, 1H, J = 5.4 & 11.0 Hz, H-6 [T2]), 4.38
(dd, 1H, J = 2.4 & 11.0 Hz, H-6ꢀ [T2]), 4.89 (dd, 2H, J = 4.0 & 7.2 Hz, H-2
[T1orT2]), 4.90 (dd, 2H, J = 3.6 & 7.2 Hz, H-2 [T1orT2]), 5.12 (t, 1H, J = 7.8
Hz, H-2 [G]), 5.18 (d, 1H, J = 4.0 Hz, H-1 [T1orT2]), 5.19 (d, 1H, J = 4.0 Hz,
H-1 [T1orT2]), 5.22–5.30 (m, 4H, H-3,4 [G], H-3 [T1&T2]), 5.74 (d, 1H, J =
7.8 Hz, H-1 [G]).
13C NMR of 14 (100 MHz, CDCl3): δ: 20.4, 20.5, 20.6, 20.7, 20.9, 62.0 (C-6 [T1]),
64.2 (C-6 [T2]), 68.8, 69.9, 70.1, 70.3, 71.9, 72.5, 72.8, 73.4, 91.5 (C-1 [G]),
92.4 (C-1 [T]), 94.4 (C-1 [T]), 166.5, 168.8, 169.2, 169.7, 169.9, 170.2, 172.0,
172.1.

Synthesis of Per- and Poly-Substituted Trehalose Derivatives
215
MS (ES+) of 14: 876.5 (100%, M + Na), 871.5 (76%, M + NH3), 794.5 (52%),
674.5 (38%), 428.5 (24%).
Anal. Calcd for C34H46O25: C, 47.78; H, 5.43. Found: C, 48.05; H, 5.64.
2,2ꢀ,3,3ꢀ-Tetra-O-acetyl-6,6ꢀ-bis-(1,2,3,4-tetra-O-acetyl-β-D-glucopyranuronyl)-
4,4ꢀ-di-O-isobutyryl-α,α-trehalose, 11, and 2,2ꢀ,3,3ꢀ-tetra-O-acetyl-6-
(1,2,3,4-tetra-O-acetyl-β-D-glucopyranuronyl)-4,4ꢀ,6ꢀ-tri-O-isobutyryl-α,α-
trehalose, 15
To a solution of the unseparated products of the above reaction, com-
pounds 10 and 14 (1.00 g) in pyridine (4 mL) and acetonitrile (4 mL), was
added isobutoyl chloride (2.5 mL, large excess). After stirring for 18 h the sol-
vent was removed under reduced pressure to leave an oil, which was poured
into water (30 mL), extracted into CH2Cl2 (10 mL), dried, filtered over acti-
vated charcoal, and crystallized from CH2Cl2and petroleum ether (40–60◦C).
Yield of the combined products was 0.23 g (20.6%), m.p. 109–113◦C. Com-
pounds 11 and 15 (55 mg) were separated using column chromatography, using
ethyl acetate and petrol ether 40–60◦C (2:1) as the eluent. The lower run-
ning spot was dissolved in ethyl acetate and crystallized on addition of di-
ethyl ether; yield of compound 11 (24 mg); m.p. (ethyl acetate/diethyl ether)
93–94◦C.
1H NMR of 11 (400 MHz, CDCl3): δ: 1.07 (d, 3H, J = 6.8 Hz, O2CCH(CH3)2),
1.09 (d, 3H, J = 2.7 Hz, O2CCH(CH3)2), 2.48 (m, 1H, J = 2.7 & 6.8Hz,
O2CCH(CH3)2), 3.93 (m, 2H, H-5, H-6 [T]), 4.11 (d(d), 1H, J = 10.3 Hz, H-6ꢀ
[T]), 4.22 (d, 1H, J = 9.2 Hz, H-5 [G]), 4.88 (t, 1H, J = 9.9Hz, H-4 [T]), 4.97
(dd, 1H, J = 4.1 & 9.9 Hz, H-2 [T]), 5.15 (t, 1H, J = 8.0 Hz, H-2 [G]), 5.20–
5.27 (m, 3H, H-3, H-4 [G], H-1 [T]), 5.47 (t, 1H, J = 9.9 Hz, H-3 [T]), 5.74 (d,
1H, J = 8.0 Hz, H-1 [G]).
13C NMR of 11 (100 MHz, CDCl3): δ: 18.6, 18.7, 20.4, 20.4, 20.5, 20.5, 20.6, 20.7,
33.8, 63.6 (C-6 [T]), 68.1 (C-4/5 [T]), 68.2 (C-4/5 [T]), 68.6 (C-3/4 [G]), 69.5
(C-3 [T]), 69.9 (C-2 [T]), 70.1 (C-2 [G]), 71.9 (C-3/4 [G]), 72.7 (C-5 [G]), 90.7
(C-1 [T]), 91.5 (C-1 [G]), 166.1 (C-6 [G]), 168.8, 169.1, 169.4, 169.6, 169.9,
169.9, 175.6.
MS (ES+) of 14: 119.0 (100%), 151.1 (45%), 1087.3 (40%), 1361.2 (20%, M +
Na).
Acc. Mass (M + NH4) of 14: Calcd 1356.4253. Found 1356.4284
1H NMR of 15 (400 MHz, CDCl3): δ: 1.07–1.12 (m, 18H, CH(CH3)2), 1.97 (s, 9H,
3 × Me), 1.98 (s, 3H, Me), 2.00 (s, 3H, Me), 2.03 (s, 3H, Me), 2.04 (s, 3H, Me),
2.07 (s, 3H, Me), 2.46–2.54 (m, 3H, CH(CH3)2), 3.93–4.02 (m, 4H), 4.11 (m,
2H), 4.24 (d, 1H, J = 9.6 Hz, H-5 [G]), 4.91 (t, 1H, J = 9.6 Hz), 4.95–5.04 (m,

T. C. Baddeley & J. L. Wardell
216
3H), 5.17 (dd, J = 7.9 & 8.9 Hz), 5.20–5.29 (m, 4H), 5.49 (t, 2H, J = 9.2 Hz,
H-3 [T & Tꢀ]), 6.76 (d, 1H, J = 7.9 Hz, H-1 [G]).
13C NMR of 15 (100 MHz, CDCl3): δ: 18.7, 18.7, 18.8, 18.9, 20.5, 20.6, 20.6,
20.7, 29.7, 33.7, 33.8, 61.5, 63.6, 68.1, 68.2, 68.4, 68.6, 69.7, 69.8, 69.9, 70.2,
72.0, 72.7, 91.5, 166.1, 168.8, 169.2, 169.5, 169.6, 169.8, 169.9, 169.9, 175.5,
175.6, 176.6.
Anal. Calcd for C46H62O28: C, 51.97; H, 5.88. Found: C, 52.13; H, 5.93.
2,2ꢀ,3,3ꢀ-Tetra-O-acetyl-6,6ꢀ-bis-(1,2,3,4-tetra-O-acetyl-β-D-glucopyranuronyl)-
4,4ꢀ-di-O-cyclohexanecarbonyl-α,α-trehalose, 12, and 2,2ꢀ,3,3ꢀ-tetra-O-
acetyl-6-(1,2,3,4-tetra-O-acetyl-β-D-glucopyranuronyl)-4,4ꢀ,6ꢀ-tri-O-
cyclohexanecarbonyl-α,α-trehalose, 16
To a solution of the unseparated compounds 10 and 14 (1.00 g) in pyri-
dine (4 mL) and acetonitrile (5 mL) was added cyclohexanecarbonyl chloride
(2 mL, large excess). After stirring for 18 h, the solvent was removed under
reduced pressure to leave an oil, which was poured into water (20 mL), ex-
tracted into CH2Cl2 (10 mL), dried, and filtered over activated charcoal. The
solvent was removed under reduced pressure and the residue was crystallized
from diethyl ether and petroleum ether (40–60◦C). Yield of combined title prod-
ucts 0.73 g (61.7%). TLC with ethyl acetate and petroleum ether (40–60◦C)
eluent provided good separation of the products. Column chromatography, us-
ing the same eluent system, of the white solid (400 mg) yielded the pseudo-
trisaccharide (16, 47.3 mg) as the higher running TLC spot and the pseudo-
tetrasaccharide (12, 181.7 mg) as the lower running TLC spot.
1H NMR of 12 (400 MHz, CDCl3): δ: 1.16–1.34 (m, 5H, C6H11CO2), 1.59–1.80
(m, 6H, C6H11CO2), 1.96 (s, 3H, Me), 1.97 (s, 3H, Me), 1.98 (s, 3H, Me), 2.00
(s, 3H, Me), 2.03 (s, 3H, Me), 2.07 (s, 3H, Me), 3.94 (m, 2H, H-5, H-6 [T]),
4.12 (d(d), 1H, J = 10.6 Hz, H-6ꢀ [T]), 4.24 (d, 1H, J = 9.2 Hz, H-5 [G]), 4.90
(t, 1H, J = 9.6 Hz, H-4 [T]), 4.98 (dd, 1H, J = 4.1 Hz, H-2 [T]), 5.17 (t, 1H,
J = 8.2 Hz, H-2 [G]), 5.24 (m, 3H, H-3, H-4 [G], H-1 [T]), 5.48 (t, 1H, J = 9.9
Hz, H-3 [T]), 5.75 (d, 1H, J = 7.9 Hz, H-1 [G]).
13C NMR of 12 (63 MHz, CDCl3): δ: 20.5, 20.6, 20.7, 20.8, 25.3, 25,5, 28.7, 42.9,
63.6 (C-6 [T]), 68.1 (C-4 [T]), 68.2 (C-5 [T]), 68.7 (C-3/4 [G]), 69.5 (C-3 [T]),
70.0 (C-2 [T]), 70.1 (C-2 [G]), 72.0 (C-3/4 [G]), 72.7 (C-5 [G]), 91.0 (C-1 [T]),
91.5 (C-1 [G]), 166.1 (C-6 [G]), 168.9, 169.2, 169.5, 169.7, 169.9, 170.0, 174.6.
MS (ES+) of 12: 293.0 (100%), 261.0 (75%), 499.1 (35%), 539.2 (22%), 1441.3
(15%, M + Na).
Acc. Mass (M + NH4) of 12: Calcd for C62H82O37: 1436.4879. Found 1436.4885.

Synthesis of Per- and Poly-Substituted Trehalose Derivatives
217
1H NMR of 16 (400 MHz, CDCl3): δ: 1.16–1.40 (m, 15H), 1.52 (s, 3H), 1.60–1.81
(m, 15H), 1.97 (s, 3H, Me), 1.97 (s, 6H, 2 × Me), 1.99 (s, 3H, Me), 2.00 (s,
3H, Me), 2.04 (s, 3H, 2 × Me), 2.08 (s, 3H, Me), 3.91–4.01 (m, 4H, H-5, 6 [T
& Tꢀ]), 4.12 (m, 2H, H-6ꢀ [T & Tꢀ]), 4.25 (d, 1H, J = 9.2 Hz, H-5 [G]), 4.92
(t, 1H, J = 9.6, H-4 [T or Tꢀ]), 4.97–5.06 (m, 3H, H-2 [T&Tꢀ], H-4 [T or Tꢀ]),
5.18 (t, 1H, J = 7.8 Hz, H-2 [G]), 5.16–5.30 (m, 4H, H-3, 4 [G], H-1 [T & Tꢀ]),
5.45–5.50 (t, 2H, J = 9.3 Hz, H-3 [T & Tꢀ]), 5.77 (d, 1H, J = 7.5 Hz, H-1 [G]).
13C NMR of 16 (63 MHz, CDCl3): δ: 20.5, 20.6, 20.7, 20.8, 25.3, 25.4, 25.4, 25.5,
25.7, 28.8, 29.0, 42.9, 61.3, 63.6, 67.8, 68.1, 68.1, 68.4, 68.7, 69.7, 69.8, 70.0,
70.2, 72.1, 72.7, 91.5, 91.6, 91.8, 166.1, 168.9, 169.2, 169.5, 169.6, 169.7,
169.8, 169.9, 170.0, 174.5, 174.6, 175.6.
MS (ES+) of 16: 1124.7 (100%), 1201.7 (35%), 1206.6 (25%, M + Na).
Anal. Calcd for C55H74O28: C, 55.83; H, 6.30. Found: C, 55.69; H, 6.43.
6,6ꢀ-Di-O-trityl-α,α-trehalose
6,6ꢀ-Di-O-trityl-α,α-trehalose was obtained using a modification of the
method reported by Brederick.[36] Trityl chloride (17.0 g, 61 mmol) was slowly
added to a solution of anhydrous trehalose (10.5 g, 31 mmol) in pyridine (40
mL). The reaction mixture was stirred at 40◦C for 36 h and evaporated under
reduced pressure to 2 mL, and the residue poured into cold water with stir-
ring. The pale yellow precipitate was washed with water and dried at 70◦C.
Hot methanol was added to the solid and stirred for 5 min to form a slurry,
which was filtered and the solid dried at 70◦C for 12 h. Yield of product was
19.9 g (83%); m.p. 191–193◦C: lit.[26] value 278–281◦C; [α]2D4 (c = 2, DMSO)
+100.1: lit.[26] value [α]1D9 (pyridine) +62.8.
1H NMR (250 MHz, DMSO-d6): δ: 3.04 (m, 1H, H-6), 3.20 (m, 2H, H-4, 6ꢀ), 3.40
(dd, 1H, J = 3.1, 9.3, H-2), 6.63 (t, 1H, J = 9.3, H-3), 4.05 (m, 1H, H-5),
5.15 (d, 1H, J = 3.1, H-1), 7.23–7.35 (m, 9H, m, p−Ar), 7.45 (d, 6H, J = 7.3,
o−Ar).
13C NMR (63 MHz, DMSO-d6): δ: 63.5, 70.6, 71.1, 71.8, 73.5, 85.5, 126.8, 126.9,
127.0, 127.7, 127.9, 128.3, 128.4, 128.5, 128.6, 144.1.
2,2ꢀ,3,3ꢀ,4,4ꢀ-Hexa-O-acetyl-6,6ꢀ-di-O-trityl-α,α-trehalose
Acetic anhydride (20 mL, 196 mmol) was slowly added to a solution of 6,6ꢀ-
di-O-trityl-α,α-trehalose (19.86 g, 24 mmol) in pyridine (60 mL) to maintain
the temperature below 30◦C. The mixture was stirred for 4 h and evaporated
and the residue poured into ice water with vigorous stirring. The pale brown
precipitate was washed with water (150 mL) and stirred with methanol for 10
min at 60◦C. The solid obtained on cooling was collected and dried overnight

T. C. Baddeley & J. L. Wardell
218
at 60◦C; yield 22.11 g (86%); m.p. 248–250◦C: lit[36] value 245–250◦C; [α]2D3 (c =
6, CHCl3) 251.5; lit.[36] value [α] (CHCl3)+114.7.
1H NMR (250 MHz, CDCl3): δ: 1.74 (s, 6H), 1.89 (s, 6H), 1.99 (s, 6H), 3.10 (m,
2H, H-6,6ꢀ), 4.12 (m, 1H, H-5), 5.14 (t, 1H, J = 9.8 Hz, H-4), 5.19 (dd, 2H,
J = 3.7 Hz, H-2), 5.45 (t, 1H, J = 9.8 Hz, H-3), 5.46 (d, 1H, J = 3.7 Hz, H-1),
7.17–7.42 (m, 15H, 3 × Ph).
13C NMR (63 MHz, CDCl3): δ: 20.5, 20.6, 20.8, 61.8, 68.9, 69.7, 69.8, 70.6, 86.6,
92.8, 127.1, 127.3, 127.9, 128.6, 143.4, 169.2, 169.7, 170.2.
2,2ꢀ,3,3ꢀ,4,4ꢀ-Hexa-O-acetyl-α,α-trehalose, 18
To a solution of 2,2ꢀ,3,3ꢀ,4,4ꢀ-hexa-O-acetyl-6,6ꢀ-di-O-trityl-α,α-trehalose
(45 g, 42 mmol) in dichloromethane (200 mL) was added freshly ground ferric
chloride hexahydrate (30 g, 111 mmol) with stirring. After 1 h, water (200 mL)
was added, the mixture was stirred for 20 min, and the organic layer collected.
This was washed with water (2 × 150 mL), dried over anhydrous magnesium
sulphate, and evaporated. Fractional recrystallization of the residue initially
from methanol, to remove the trityl coproduct, and finally from methyl t-butyl
ether at 0◦C, gave the title product, 12 g (49%); m.p. 102◦C: lit.[36] value 93–
96◦C; [α]2D4 (c = 4, CHCl3) 253; lit[36] value [α]D19 (CHCl3)+158.8.
1H NMR (250 MHz, CDCl3): δ: 2.03 (s, 3H, Me), 2.07 (s, 3H, Me), 2.08 (s, 3H,
Me), 3.57 (m, 2H, H-6,6ꢀ), 3.94 (ddd, 1H, J = 3.1, 4.3, & 10.1 Hz, H-5), 4.97
(dd, 1H, J = 3.97 & 10.1 Hz, H-2), 5.00 (dd, 1H, J = 9.5 Hz, 10.1, H-4), 5.30
(d, 1H, J = 3.97 Hz, H-1), 5.52 (dd, 1H, J = 9.5 & 10.1 Hz, H-3).
13C NMR (63 MHz, CDCl3): δ: 20.7, 61.0, 60.8, 69.8, 70.2, 70.6, 93.2, 170.0,
170.2, 170.3.
IR (KBr): v: 3498, 2951, 1757, 1431, 1371, 1227, 1130, 1043, 984, 953.
MS (ES+): 617.1 (100%, M + Na), 131.2 (20%), 428.3 (15%), 1211.2 (8%, 2M +
Na).
Anal. Calcd for C24H34O17: C, 48.48; H, 5.76. Found: C, 48.65; H, 5.90.
2,2ꢀ,3,3ꢀ,4,4ꢀ-Hexa-O-acetyl-6,6ꢀ-di-O-succinyl-α,α-trehalose, 19
A solution of 2,2ꢀ,3,3ꢀ,4,4ꢀ-hexa-O-acetyl-α,α-trehalose, 18 (2.50 g, 4.2
mmol), and succinic anhydride (3.25 g, 32.5 mmol) in pyridine (20 ml) was
stirred for 24 h, poured into water (100 mL), and acidified with concen-
trated hydrochloric acid (10 mL). The aqueous solution was extracted with
dichloromethane (2 × 50 ml). The combined organic solutions were dried, the
solvent was removed under reduced pressure, and the residue recrystallized
from diethyl ether, yield 1.67 g (50%); m.p. 142–145◦C (DSC); [α]D24 (c = 2.5,
CHCl3) 131.3.

Synthesis of Per- and Poly-Substituted Trehalose Derivatives
219
1H NMR (250 MHz, CDCl3): δ: 2.00 (s, 3H, Me), 2.02 (s, 3H, Me), 2.10 (s, 3H,
Me), 2.35–2.85 (m, 4H, O2CCH2CH2CO2), 3.74 (dd, 1H, J = 1.8 & 11.9 Hz,
H-6ꢀ), 4.04 (ddd, 1H, J = 1.8, 8.9, & 9.7 Hz, H-5), 4.68 (dd, 1H, J = 8.9 &
11.9 Hz, H-6), 4.92 (t, 1H, J = 9.7 Hz, H-4) 5.01 (dd, 1H, J = 3.4 & 9.7 Hz,
H-2), 5.27 (d, 1H, J = 3.4 Hz, H=1), 5.48 (t, 1H, J = 9.7 Hz, H-3).
13C NMR (63 MHz, CDCl3): δ: 15.3, 20.6, 20.7, 28.4, 29.1, 43.5, 61.6, 65.9, 68.0,
69.7, 69.8, 90.9, 169.7, 170.0, 172.1, 179.4.
IR (KBr): ν: 3612-3347, 2964, 1761, 1740, 1718, 1436, 1371, 1245, 1168, 1042,
984, 956.
MS (ES+): 817.1 (100%, M + Na), 833.1 (10%, M + K).
Anal. Calcd for C32H42O23: C, 48.31; H, 5.33. Found: C, 48.51; H, 5.42.
2,2ꢀ,3,3ꢀ,4,4ꢀ-Hexa-O-acetyl-6,6ꢀ-bis-(1,2,3,4-tetra-O-acetyl-6-O-succinyl-β-D-
glucopyranuronyl)-α,α-trehalose, 17
2,2ꢀ,3,3ꢀ,4,4ꢀ-Hexa-O-acetyl-6,6ꢀ-di-O-succinyl-α,α-trehalose, 19 (1.00 g, 1.3
mmol), 1,2,3,4-tetra-O-acetyl-β-D-glucopyranose (0.99 g, 2.7 mmol), and p-
(dimethylamino)pyridine (4 mg, 0.03 mmol) were dissolved in acetonitrile (10
mL). To this mixture was slowly added a solution of DCC (0.55 g, 2.6 mmol) in
acetonitrile (10 mL) with stirring. After 18 h the solution was filtered to remove
the 1,3-dicyclohexylcarbodiimide adduct and the filtrate reduced. An impure
sample of the title compound, contaminated with one other compound, was
obtained on crystallization of the residue from aqueous methanol. Further pu-
rification was achieved by column chromatography using ethyl acetate:petrol
ether (40–60◦C) (2:1) as eluent. The title compound was the more mobile of the
two components in the mixture; yield 200 mg (11%); m.p. 113–115◦C.
1H NMR (250 MHz, CDCl3): δ: 1.99 (s, 3H, Me), 2.02 (s, 9H, 3 × Me), 2.03 (s,
3H, Me), 2.08 (s, 3H, Me), 2.11 (s, 3H, Me), 2.65 (br.s, 4H, O2CCH2CH2CO2),
3.85 (m, 1H, H-5 [G]), 4.03 (d, 1h, J = 12.8Hz, H-6ꢀ [T]), 4.17 (m, 1H, H-5
[T]), 4.17 (d(d), 1H, H-6ꢀ [G]), 4.24 (m, 2H, H-6 [G], H-6 [T]), 5.03 (m, 2H,
H-2, 4 [T]), 5.12 (t, 2H, J = 8.2 Hz, H-2, 4 [G]), 5.22 (t, 1H, J = 9.2 Hz, H-3
[G]), 5.29 (d, 1H, J = 3.7 Hz, H-1 [T]), 5.48 (dd, 1H, J = 8.24 & 10.1 Hz, H-3
[T]), 5.70 (d, 1H, J = 8.2 Hz, H-1 [G]).
13C NMR (63 MHz, CDCl3): δ: 20.4, 20.6, 20.8, 28.6, 28.7, 61.8 (C-6, [T]+[G]),
67.8 (C-4, [G]), 68.2 (C-5, [T]), 68.6 (C-4, [T]), 69.8 (C-2, [T]), 70.0 (C-3, [T]),
70.3 (C-2, [G]), 72.7 (C-5, [G]), 72.8 (C-3, [G]), 91.7 (C-1, [G]), 92.1 (C-1, [T]),
169.0, 169.2, 169.4, 169.6, 169.7, 170.0, 170.1, 171.8, 171.9.
MS (FAB): 269.1 (100%), 229.1 (52%), 209.0 (46%), 557.1 (34%), 335.1 (30%),
719.1 (25%), 431.1 (20%), 845.1 (16%), 1477.4 (15%, M + Na).
Anal. Calcd for C60H70O41: C, 49.55; H, 4.85. Found: C, 49.74; H, 4.77.

T. C. Baddeley & J. L. Wardell
220
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