Synthesis, trehalase hydrolytic resistance and inhibition properties of 4- and 6-substituted trehalose derivatives

Article abstract of DOI:10.1080/14756366.2020.1837125

Although trehalose has recently gained interest because of its pharmaceutical potential, its clinical use is hampered due to its low bioavailability. Hence, hydrolysis-resistant trehalose analogues retaining biological activity could be of interest. In this study, 34 4- and 6-O-substituted trehalose derivatives were synthesised using an ether- or carbamate-type linkage. Their hydrolysis susceptibility and inhibitory properties were determined against two trehalases, i.e. porcine kidney and Mycobacterium smegmatis. With the exception of three weakly hydrolysable 6-O-alkyl derivatives, the compounds generally showed to be completely resistant. Moreover, a number of derivatives was shown to be an inhibitor of one or both of these trehalases. For the strongest inhibitors of porcine kidney trehalase IC₅₀ values of around 10 mM could be determined, whereas several compounds displayed sub-mM IC₅₀ against M. smegmatis trehalase. Dockings studies were performed to explain the observed influence of the substitution pattern on the inhibitory activity towards porcine kidney trehalase.

Full text of DOI:10.1080/14756366.2020.1837125

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Synthesis, trehalase hydrolytic resistance and
inhibition properties of 4- and 6-substituted
trehalose derivatives
Shari Dhaene , Johan Van der Eycken , Koen Beerens , Jorick Franceus , Tom
Desmet & Jurgen Caroen
To cite this article: Shari Dhaene , Johan Van der Eycken , Koen Beerens , Jorick Franceus , Tom
Desmet & Jurgen Caroen (2020) Synthesis, trehalase hydrolytic resistance and inhibition properties
of 4- and 6-substituted trehalose derivatives, Journal of Enzyme Inhibition and Medicinal Chemistry,
35:1, 1964-1989, DOI: 10.1080/14756366.2020.1837125
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2020, VOL. 35, NO. 1, 1964–1989
https://doi.org/10.1080/14756366.2020.1837125
RESEARCH PAPER
Synthesis, trehalase hydrolytic resistance and inhibition properties of 4- and
6-substituted trehalose derivatives
Shari Dhaenea , Johan Van der Eyckenb , Koen Beerensa , Jorick Franceusa , Tom Desmeta
Jurgen Caroenb
and
b
aDepartment of Biotechnology, Centre for Synthetic Biology, Ghent University, Ghent, Belgium; Department of Organic and Macromolecular
Chemistry, Laboratory for Organic and Bio-Organic Synthesis (LOBOS), Ghent University, Ghent, Belgium
ABSTRACT
ARTICLE HISTORY
Received 7 September 2020
Revised 5 October 2020
Accepted 9 October 2020
Although trehalose has recently gained interest because of its pharmaceutical potential, its clinical use is
hampered due to its low bioavailability. Hence, hydrolysis-resistant trehalose analogues retaining bio-
logical activity could be of interest. In this study, 34 4- and 6-O-substituted trehalose derivatives were syn-
thesised using an ether- or carbamate-type linkage. Their hydrolysis susceptibility and inhibitory properties
were determined against two trehalases, i.e. porcine kidney and Mycobacterium smegmatis. With the
exception of three weakly hydrolysable 6-O-alkyl derivatives, the compounds generally showed to be com-
pletely resistant. Moreover, a number of derivatives was shown to be an inhibitor of one or both of these
trehalases. For the strongest inhibitors of porcine kidney trehalase IC50 values of around 10 mM could be
determined, whereas several compounds displayed sub-mM IC50 against M. smegmatis trehalase. Dockings
studies were performed to explain the observed influence of the substitution pattern on the inhibitory
activity towards porcine kidney trehalase.
KEYWORDS
Trehalose derivatives;
trehalase; inhibition;
hydrolytic resistance;
molecular dockings
Introduction
degraded into glucose by the trehalase (EC 3.2.1.28) present in
our small intestine23. Trehalose analogues and derivatives that
show resistance against this human intestinal enzyme could,
therefore, be of great interest for pharmaceutical formulations and
for drug discovery due to their increased residence time in the
human body3,23. Recently, a trehalose analogue substituted on the
4-hydroxy group, lentztrehalose A 2 (Figure 1), was isolated from
the actinomycete Lentzea sp. and was shown to be only weakly
hydrolysed by porcine kidney trehalase24. Furthermore, it also
exhibited antitumor activity24. Subsequently, Wada and co-workers
isolated two additional natural analogues, lentztrehalose B 3 and
C 4 (Figure 1), which were shown to be possible inducers of
autophagy25. Importantly, these lentztrehaloses were found not to
be digested in a range of microbes and cancer cell lines, and their
in vivo bioavailability and stability was confirmed after oral admin-
istration to mice26.
In this study, an exploration of 34 trehalose derivatives (includ-
ing 33 new compounds next to lentztrehalose A 2) was estab-
lished. Inspired by the lentztrehaloses A-C 2–4, a series of 4-O-
substituted trehalose derivatives was synthesised using an ether-
or carbamate-type linkage. In addition, the analogous 6-O-substi-
tuted series was explored while some double substituted deriva-
tives were also investigated. Their biological relevance was
assessed by measuring their hydrolysis (as substrate) and binding
(as inhibitor) with two relevant trehalases, i.e. porcine kidney tre-
halase (91% similarity with human trehalase) and Mycobacterium
smegmatis trehalase (93% similarity with M. tuberculosis trehalase).
These enzymes are classified in family GH37 and GH15, respect-
Carbohydrates are the most diverse and abundant biomolecules
on earth, displaying a wide variety of functions1. During the last
decades, interest arose in trehalose 1 (Figure 1), a disaccharide
consisting of two D-glucose units linked via an a-1,1-a-bond,
resulting in a molecule with unique stabilising properties2,3. The
stability of this non-reducing glucobiose is reflected by several
interesting physicochemical properties like broad pH stability4,
high glass transition and melting temperatures5,6, low hydrolysis
rate5. During heating and processing of food products acrylamide
formation can be suppressed by trehalose as it interacts with glu-
cose and thus reduces the glucose-Asn reaction leading to the
toxic compound4,6. In nature, trehalose fulfils a biological role in
various organisms including bacteria, yeast, fungi, insects, inverte-
brates and plants, although it is not found in mammals6,7. It can
serve as energy and carbon source6,7, signalling molecule6,8,9 and
cell wall building block6,10. Furthermore, it can protect organisms
and compounds under stress conditions like desiccation7,11,12
dehydration7,13–15, heat7,13, cold7 and oxidation7,14
,
.
Since the establishment of the Hayashibara process, the avail-
ability of trehalose has drastically increased, leading to its wide-
spread use in the food, cosmetic and pharmaceutical industry6.
Moreover, due to its safeguarding capabilities, trehalose has
received attention as a chemical chaperone16,17 and autophagy
inducer16 in the treatment of neurodegenerative diseases like
Alzheimer’s18,19
,
Huntington’s20
and
Parkinson’s16,21,22
.
Unfortunately, the therapeutic use of trehalose is hampered due
to its low bioavailability. Indeed, this disaccharide is rapidly ively, of the CAZy-classification. Although they adopt a similar
CONTACT Tom Desmet
tom.desmet@ugent.be
Department of Biotechnology, Centre for Synthetic Biology (CSB), Ghent University, Ghent, Belgium
Supplemental data for this article is available online at here.
ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1965
Figure 1. Structure of trehalose 1 and lentztrehalose A-C 2–4.
overall fold (i.e. an (a/a)6-barrel) and follow the same general reac- 128.0 ppm, DMSO-d6: 2.50 and 39.43 ppm, acetone-d6: 2.05 and
29.84 ppm). 13 C-NMR Spectra recorded in D2O were referenced to
tion mechanism (i.e. inversion of the anomeric configuration
through single displacement), they are otherwise unrelated (about
20% similarity). Computational studies were performed to clarify
the structural determinants behind the activities and selectivity
profiles of the studied trehalose derivatives towards porcine kid-
ney trehalase.
the signal (30.89 ppm) of acetone (1 drop added). Coupling con-
stants, J, are reported in hertz (Hz). Infra-red spectra were
recorded on a Perkin-Elmer 1000 FT-IR infra-red spectrometer
(horizontal attenuated total reflection (HATR)). Optical rotation
was measured on a Perkin Elmer 241 Polarimeter.
Material and methods
General procedure A: O-alkylation
To a solution of an OH-containing starting material in anhydrous
DMF (0.1 M concentration) was added NaH (60% in mineral oil, 2.5
eq for each hydroxyl group). After stirring for 15 min, the alkyl halo-
genide (4 eq for each hydroxyl group) was added and the reaction
mixture was stirred overnight. Methanol (5 ml per mmol starting
material) was added dropwise (caution: gas formation) and the mix-
ture was transferred to a separation funnel using EtOAc (50 ml per
mmol starting material). The organic layer is washed with brine (5ꢁ,
50 ml per mmol starting material) and concentrated under reduced
pressure. The residue was purified by column chromatography.
Chemistry
General remarks
All reactions, unless otherwise stated, were carried out under
argon atmosphere in dry solvents. Dichloromethane and triethyl-
amine were freshly distilled from CaH2. Toluene was freshly dis-
tilled from Na. Tetrahydrofuran was freshly distilled from Na/
benzophenone. Other solvents and reagents were obtained from
commercial sources and were used as received without further
purification. Flash chromatography was carried out with Rocc sili-
cagel 60 Å, 40ꢀ63 mm. Precoated silica gel plates (Macherey-Nagel
SIL G-25 UV254) were used for TLC employing UV-absorption at
254 nm and Mo7O24/Ce(SO4)2/aq.H2SO4 staining for visualisation.
Electrospray mass spectra were recorded on an Agilent 1100 series
single quadrupole MS detector type VL with an APCI source and
an API-ES source, provided with a Phenomenex Luna C18 (2),
5 mm 250 mm ꢁ 4.60 mm column. High resolution mass spectrom-
etry (HRMS) was performed on an Agilent 1100 series connected
to a 6220 A TOF-MS detector equipped with an APCI-ESI multi-
mode source. 1H-NMR and 13 C-NMR spectra (see Supplementary
information) were recorded on a Bruker Avance 300, Bruker
Avance 400 or a Bruker AM 500 spectrometer as indicated with
chemical shifts reported in parts per million, referenced to the
residual solvent signals (CDCl3: 7.26 and 77.00 ppm, D2O:
General procedure B: carbamate formation
To a solution of an OH-containing starting material in CH2Cl2
(0.1 M concentration) was added DMAP (0.2 eq for each hydroxyl
group) and the isocyanate (3 eq for each hydroxyl group). The
reaction mixture was stirred at room temperature until the con-
sumption of starting material (TLC monitoring, 24–120 h). The mix-
ture was concentrated under reduced pressure and the residue
was purified by column chromatography.
General procedure C: hydrogenolysis
To a solution of a benzyl/benzylidene-protected starting material
4.75 ppm, CD3OD: 3.31 and 49.15 ppm, benzene-d6: 7.16 and in EtOAc/MeOH (1/1, 0.05 M concentration), Pd/C (10% w/w Pd,

1966
S. DHAENE ET AL.
0.05 eq) was added. A H2 balloon was placed and the reaction 2,3,4,6,2’,3’-Hexa-O-benzyl-4’,6’-O-benzylidene-a,a-D-trehalose (11)
mixture was stirred overnight after which it was filtered over cel-
ite. The filtrate was concentrated under reduced pressure and the
residue was purified by column chromatography.
Method 1: via benzylation of 7. To a solution of 7 (414 mg,
0.470 mmol) in anhydrous DMF (4.7 ml), NaH (60% in mineral oil,
78 mg, 1.17 mmol, 2.5 eq) was added slowly resulting in gas for-
mation. The grey suspension was cooled to 0 ꢂC and BnBr (113 mL,
0.94 mmol,
2 eq) was added dropwise. Next, TBAI (12 mg,
2,3,4,2’,3’-Penta-O-benzyl-4’,6’-O-benzylidene-a,a-D-trehalose (7)
To a cooled (ꢀ18 ꢂC) solution of 629 (5.576 g, 6.35 mmol, 1 eq) in
toluene (56 ml), DIBAL-H (supplied as 1.0 M solution in toluene,
31.9 ml, 31.9 mmol, 5 eq) was added dropwise. The cooling bath
was removed and the reaction mixture was stirred at room tem-
perature for 90 min. The solution was cooled to 0 ꢂC and the reac-
tion was quenched by dropwise addition of MeOH (11.5 ml) and
aqueous KOH (10% w/v, 3.75 ml). The resulting suspension was
transferred to a separation funnel using CH2Cl2 (400 ml) and H2O
(400 ml). The organic layer was separated and the aqueous phase
was extracted using CH2Cl2 (3 ꢁ 400 ml). The combined organic
layers were dried on MgSO4, the drying agent was filtered and
the filtrate was concentred under reduced pressure. The residue
was purified by flash column chromatography (gradient elution:
hexane/EtOAc 8/2 to 1/1) to obtain the title compound 7 as a
white foam with a yield of 80% (4.443 g; 5.05 mmol). Spectral data
were in agreement with literature27.
0.032 mmol, 0.07 eq) was added and the ice bath was removed.
After stirring overnight, the reaction mixture was cooled to 0 ꢂC
and carefully quenched (gas formation) with MeOH (2 ml). After
15 min, the mixture was diluted with ethyl acetate (35 ml) and
transferred to a separation funnel. The organic phase was washed
with brine (3 ꢁ 30 ml), and dried on MgSO4. The drying agent was
filtered and the filtrate was concentrated under reduced pressure.
The crude product was purified by flash column chromatography
(gradient elution: hexane/EtOAc 9/1 to 8/2) giving the title com-
pound 11 as a white solid (yield: 73%, 328 mg). Spectral data
were in agreement with literature29.
Method 2: alternative route via 14. To a suspension of anhyd-
rous trehalose (15 g, 43.82 mmol) and camphorsulfonic acid
(0.70 g, 2.19 mmol, 0.05 eq) in DMF (90 ml) benzaldehyde dimethyl
acetal (6.57 ml, 43.82 mmol, 1 eq) was added and the mixture was
heated to 90 ꢂC for 15 min. To the resulting colourless solution,
triethylamine (6.90 ml, 49.5 mmol, 1.1 eq) was added and the mix-
ture was concentrated under reduced pressure. The crude 4,6-O-
benzylidene-a,a-D-trehalose (13) (21.17 g) was dissolved in pyri-
dine/acetic anhydride (1/1, 80 ml) and the reaction mixture was
stirred for 24 h after which the volatiles are removed under
reduced pressure. The crude product was purified by column
chromatography (gradient eluent ion hexane/EtOAc 8/2 to 6/4)
giving 2,3,4,6,20,30-hexa-O-acetyl-40,60-O-benzylidene-a,a-D-trehal-
ose (14) as a white solid with a yield of 34% over two steps
(10.17 g, 14.90 mmol).
2,3,6,2’,3’-Penta-O-benzyl-4’,6’-O-benzylidene-a,a-D-trehalose
and 2,3,6,2’,3’,6’-hexa-O-benzyl-a,a-D-trehalose (9)
(8)
To a cooled (0 ꢂC) solution of 627 (2.000 g, 2.28 mmol, 1 eq) in
CH2Cl2 (2.0 ml), DIBAL-H (1.0 M solution in CH2Cl2, 11.4 ml,
11.4 mmol, 5 eq) was added dropwise. After stirring for 90 min at
0 ꢂC, the clear colourless reaction mixture was diluted with CH2Cl2
(20 ml) and the reaction was quenched by dropwise addition of
MeOH (6 ml). Subsequently, aqueous KOH (10% w/v, 6 ml) was
added and the resulting suspension was transferred to a separ-
ation funnel using CH2Cl2 (250 ml) and H2O (250 ml). The organic
layer was separated and the aqueous phase was extracted with
CH2Cl2 (2 ꢁ 250 ml). The combined organic layers were dried on
MgSO4, the drying agent was filtered and the filtrate was concen-
trated under reduced pressure. The residue was purified using col-
umn chromatography (gradient elution: hexane/EtOAc 8/2 to 6/4).
Following products were isolated (in order of elution): recovered
starting material 6 (200 mg, 0.228 mmol, 10%), compound 8 (4-
OH, 1.107 g, 1.256 mmol, 55%), compound 9 (4/40-bis-OH, 179 mg,
0.203 mmol, 9%) and compound 7 (6-OH, 380 mg, 0.431 mmol,
19%). Rf values in hexane/EtOAc 7/3: Rf 0.36 (6), Rf 0.31 (8), Rf
0.25 (9), Rf 0.11 (7). Spectral data of compounds 828 and 929 were
in agreement with the literature.
14: Rf 0.28 in hexane/EtOAc 1/1. 1H NMR (400 MHz, acetone-
d6): d 7.48–7.42 (m, 2H), 7.38–7.33 (m, 3H), 5.67 (s, 1H), 5.60 (t,
J ¼ 9.9 Hz, 1H), 5.53 (t, J ¼ 9.5 Hz, 1H), 5.40 (d, J ¼ 3.9 Hz, 1H), 5.34
(d, J ¼ 3.8 Hz, 1H), 5.10–5.00 (m, 3H), 4.28–4.15 (m, 3H), 4.11–4-03
(m, 2H), 3.94 (t, J ¼ 9.6 Hz, 1H), 3.86 (t, J ¼ 10.3 Hz, 1H), 2.12 (s, 3H),
2.11 (s, 3H), 2.02 (s, 6H), 2.01 (s, 3H), 1.98 (s, 3H) ppm. 13 C NMR
(100 MHz, acetone-d6): d 170.7 (C), 170.5 (C), 170.4 (C), 170.3 (C),
170.1 (C), 170.1 (C), 138.5 (C), 129.8 (CH), 128.9 (CH), 127.2 (CH),
102.4 (CH), 94.0 (CH), 92.9 (CH), 79.4 (CH), 71.4 (CH), 70.9 (CH),
70.6 (CH), 69.6 (CH), 69.4 (CH), 69.4 (CH), 69.0 (CH2) 64.5 (CH), 62.9
(CH2), 20.7 (CH3), 20.6 (CH3), 20.6 (CH3), 20.6 (CH3) ppm. IR (HATR):
2964 (w), 1741 (s), 1374 (m), 1229 (s), 1214 (s), 1160 (w), 1134 (m),
1062 (m), 1035 (m), 997 (m), 982 (m), 956 (m), 920 (w), 907 (m),
803 (w), 767 (w), 729 (w), 702 (w), 654 (w) cmꢀ1. ESMS [m/z (frag-
ment, intensity), positive mode]: 700.2 (M þ NH4þ, 100). HRMS
[M þ NH4]þ 700.2447;
þ
(ESI-TOF): calcd. for C31H42NO17
found 700.2444.
2,3,4,2’,3’,4’-Hexa-O-benzyl-a,a-D-trehalose (10)
To a cooled solution (0 ꢂC) of 7 (620 mg, 0.704 mmol, 1 eq) in tolu-
ene (6.32 ml), DIBAL-H (1.0 M in toluene, 3.52 ml, 3.52 mmol, 5 eq)
was added dropwise. After 15 min, the ice bath was removed and
the reaction mixture was stirred for 24 h. After cooling to 0 ꢂC,
MeOH (1.3 ml) and 10% aqueous KOH (0.4 ml) were added, and
the resulting mixture was transferred to a separation funnel using
CH2Cl2 (40 ml) and H2O (40 ml). The organic layer was separated
and the aqueous layer was extracted with CH2Cl2 (2 ꢁ 40 ml). The
combined organic layers were washed with brine (50 ml) and con-
centrated under reduced pressure. The residue was purified via
column chromatography (gradient elution: hexane/EtOAc 7/3 to 4/
6), affording the title compound 10 as a white foam (491 mg,
0.556 mmol, 79%). Rf 0.14 in hexane/EtOAc 1/1. Spectral data were
in agreement with the literature28.
To a suspension of 14 (1 g, 1.465 mmol) in MeOH (15 ml),
NaOMe (0.950 g, 17.58 mmol, 12 eq) was added. The reaction mix-
ture was stirred for 1 h after which silica (1 g) was added. The sus-
pension was filtered and the filtrate was concentrated under
reduced pressure. Purification by flash column chromatography
(eluent CH2Cl2/MeOH 8/2 to 6/4) gave 4,6-O-benzylidene-a,a-D-tre-
halose (13) as a white solid (831 mg), but still containing an
impurity according to TLC.
13 (purified sample): Rf 0.60 in acetonitrile/water 8/2. 1H NMR
(400 MHz, CD3OD): d 7.51–7.43 (m, 2H), 7.37–7.26 (m, 3H), 5.56 (s,
1H), 5.16 (d, J ¼ 4.0 Hz, 1H), 5.08 (d, J ¼ 3.8 Hz, 1H), 4.21 (dd,
J ¼ 9.8/4.9 Hz, 1H), 4.13–4.03 (m, 1H), 4.00 (t, J ¼ 9.4 Hz, 1H),
3.90–3.64 (m, 5H), 3.60 (dd, J ¼ 9.4/4.0 Hz, 1H), 3.49 (dd, J ¼ 9.8/
3.6 Hz, 1H), 3.47 (t, J ¼ 9.6 Hz, 1H), 3.37–3.30 (m, 2H) ppm.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1967
13 C NMR (100 MHz, CD3OD): d 139.2 (C), 129.9 (CH), 129.0 (CH), J ¼ 10.7 Hz, 1H), 4.39 (d, J ¼ 12.1 Hz, 1H), 4.20–4.13 (m, 1H),
127.5 (CH), 103.0 (CH), 95.9 (CH), 95.5 (CH), 83.1 (CH), 74.5 (CH),
73.9 (CH), 73.8 (CH), 73.2 (CH), 71.8 (CH), 71.5 (CH), 70.0 (CH2), 64.1
4.10–3.98 (m, 2H), 3.89 (app t, J ¼ 9.3 Hz, 1H), 3.75–3.47 (m, 7H),
3.40 (dd, J ¼ 10.7/1.9 Hz, 1H) ppm. 13 C NMR (100 MHz, CDCl3): d
(CH), 62.6 (CH2) ppm. IR (HATR): 3318 (m, br), 2982 (w), 2930 (w),
2868 (w), 1592 (m), 1455 (w), 1375 (m), 1345 (m), 1148 (m), 1122
(m), 1111 (m), 1071 (m), 1047 (m), 1024 (m), 1004 (m), 981 (s), 927
(w), 838 (w), 800 (w), 760 (w), 702 (w) cmꢀ1. ESMS [m/z (fragment,
intensity), API-ES negative modeþ]: 429.1 (M-Hþ, 100). HRMS (ESI-
138.8 (C), 138.7 (C), 138.3 (C), 138.0 (C), 137.9 (C), 137.8 (C), 128.5
(CH), 128.4 (CH), 128.4 (CH), 128.4 (CH), 128.3 (CH), 128.0 (CH),
127.9 (CH), 127.9 (CH), 127.8 (CH), 127.8 (CH), 127.7 (CH), 127.7
(CH), 127.6 (CH), 127.6 (CH), 127.3 (CH), 94.2 (CH), 94.0 (CH), 81.8
(CH), 80.9 (CH), 79.3 (CH), 79.2 (CH), 77.7 (CH), 75.6 (CH2), 75.2
(CH2), 75.0 (CH2), 73.5 (CH2), 72.9 (CH2), 72.5 (CH2), 71.2 (CH), 70.7
(CH), 70.5 (CH), 68.2 (CH2), 62.2 (CH2) ppm. IR (HATR): 3451 (br, m),
3059 (w), 3030 (w), 2926 (w), 2868 (w), 1496 (w), 1453 (m), 1360
(m), 1326 (w), 1272 (w), 1208 (w), 1153 (m), 1091 (m), 1054 (s),
1026 (m), 991 (s), 918 (w), 849 (w), 799 (w), 733 (s), 695 (s), 638
(w) cmꢀ1. ESMS [m/z (fragment, intensity), API-ES positive modeþ]:
900.3 (M þ NH4þ, 100). HRMS (ESI-TOF): calcd. for C54H62NO11
[M þ NH4]þ 900.4317; found 900.4321.
TOF): calcd. for C19H26NaO11
[M þ Na]þ 453.1367; found
453.1374. Literature data are available29.
To a solution of crude 13 in anhydrous DMF (12 ml), NaH (60%
in mineral oil, 700 mg, 17.4 mmol) was added slowly (gas forma-
tion). After stirring for 15 min, the resulting suspension was cooled
to 0 ꢂC and BnBr (1.67 ml, 13.9 mmol) was added dropwise, fol-
lowed by TBAI (129 mg, 0.348 mmol). The ice bath was removed
and the reaction mixture was stirred overnight. After cooling to
0 ꢂC, MeOH (5 ml) was added (gas formation is observed) and the
mixture is stirred for 10 min after which it is diluted with ethyl
acetate (40 ml). The organic layer was washed with brine
(5 ꢁ 40 ml), and dried on MgSO4. The drying agent was filtered
and the filtrate was concentrated under reduced pressure. The
crude product was purified by flash column chromatography (gra-
dient elution: hexane/EtOAc 95/5 to 8/2) giving 11 as a white
solid with a yield of 51% over two steps (725 mg, 0.747 mmol).
2,3,4,2’,3’-Penta-O-benzyl-4’,6’-O-benzylidene-6-O-methyl-a,a-D-tre-
halose (16-a)
General procedure
A
was applied (use of iodomethane).
Purification via column chromatography (gradient elution: hexane/
EtOAc 9/1 to 8/2) afforded the title compound 16-a as a white
solid (203 mg, 0.227 mmol, 80%).
1
Rf 0.27 in hexane/EtOAc 7/3. H NMR (400 MHz, benzene-d6): d
7.64–7.56 (m, 2H), 7.43–7.26 (m, 10H), 7.24–7.02 (m, 18H), 5.41 (d,
J ¼ 3.8 Hz, 1H), 5.39 (s, 1H), 5.33 (d, J ¼ 3.7 Hz, 1H), 5.00–4.90 (m,
3H), 4.84–4.63 (m, 5H), 4.58–4.46 (m, 4H), 4.41 (app t, J ¼ 9.4 Hz,
1H), 4.36 (app t, J ¼ 9.7 Hz, 1H), 4.19 (dd, J ¼ 10.1/4.9 Hz, 1H), 3.80
(t, J ¼ 10.1/9.2 Hz, 1H), 3.67–3.48 (m, 6H), 3.17 (s, 3H) ppm. 13 C
NMR (100 MHz, benzene-d6): d 140.2 (C), 140.1 (C), 139.8 (C), 139.3
(C), 139.0 (C), 138.8 (C), 129.3 (CH), 129.1 (CH), 129.0 (CH), 128.9
(CH), 128.8 (CH), 128.7 (CH), 128.3 (CH), 128.2 (CH), 128.2 (CH),
128.0 (CH), 127.8 (CH), 127.1 (CH), 102.0 (CH), 94.8 (CH), 94.0 (CH),
83.4 (CH), 82.7 (CH), 80.7 (CH), 79.7 (CH), 79.7 (CH), 78.9 (CH), 75.8
(CH2), 75.6 (CH2), 75.3 (CH2), 74.2 (CH2), 74.0 (CH2), 72.2 (CH2), 72.1
(CH), 69.7 (CH), 63.8 (CH), 59.4 (CH) ppm. IR (HATR): 3064 (w),
3029 (w), 2923 (w), 2868 (w), 1496 (w), 1453 (m), 1367 (m), 1330
(w), 1208 (w), 1149 (m), 1136 (m), 1085 (s), 1071 (s), 1049 (m),
1027 (m), 985 (s), 915 (w), 854 (w), 797 (w), 734 (m), 695 (s), 658
(w), 633 (w) cmꢀ1. ESMS [þm/z (fragment, intensity), API-ES positive
2,3,4,6,2’,3’,6’-Hepta-O-benzyl-a,a-D-trehalose (12)
To a solution of 11 (5.055 g; 5.205 mmol) in anhydrous THF
(52 ml), molecular sieves (4 Å, 5.00 g) were added. After stirring for
30 min, Me3N-BH3 (4.556 g; 62.46 mmol, 12 eq) was added. The
resulting mixture was cooled to 0 ꢂC and AlCl3 (8.328 g,
65.46 mmol, 12 eq) was added. The cooling bath was removed
and the reaction mixture was stirred overnight. The mixture was
diluted with EtOAc (1.6 L) and transferred to a flask containing a
solution of cold 1% aq H2SO4 (800 ml, containing ice). After stir-
ring for 30 min, the mixture was transferred to a separation funnel,
the organic layer was separated and the aqueous phase was
extracted with EtOAc (3 ꢁ 750 ml). The combined organic layers
were washed with saturated NaHCO3 solution (3 ꢁ 2.4 L) and brine
(3 ꢁ 2.4 L). The organic phase was dried on MgSO4, the drying
agent was filtered, and the filtrate was concentrated under
reduced pressure. The residue was purified by flash column chro-
matography (100% toluene, followed by gradient elution: hexane/
EtOAc 9/1 to 75/25), giving the title compound 12 as a colourless
oil (49%, 2.45 g, 2.52 mmol). Spectral data were in agreement with
literature30. In addition, the corresponding 6-OH regioisomer
(2,3,4,6,20,30,40-hepta-O-benzyl-a,a-D-trehalose) was isolated (1.90 g,
1.95 mmol, 38%). Spectral data were in agreement with
literature31.
mode]: 91þ2.3 (M þ NH4
, 100). HRMS (ESI-TOF): calcd. for
C55H62NO11 [M þ NH4]þ 912.4317; found 912.4343.
2,3,4,2’,3’-Penta-O-benzyl-4’,6’-O-benzylidene-6-O-ethyl-a,a-D-tre-
halose (16-b)
General procedure A was applied (use of iodoethane). Purification
via column chromatography (gradient elution: hexane/EtOAc 9/1
to 8/2) afforded the title compound as a white solid (476 mg,
0.524 mmol, 92%).
2,3,4,6,2’,3’-Hexa-O-benzyl-a,a-D-trehalose (15)
1
Rf 0.32 in hexane/EtOAc 7/3. H NMR (400 MHz, benzene-d6): d
To a solution of 11 (50 mg, 0.051 mmol) in CH2Cl2/MeOH (1/3,
1.2 ml) is added trifluoroacetic acid (0.1 ml) and the resulting reac-
tion mixture is stirred for 5 days. The mixture is diluted with
CH3CN and concentrated under reduced pressure. The residue is
purified via column chromatography (gradient elution: hexane/
EtOAc 1/1 to 4/6) to give the title compound 15 as a colourless
glass (34 mg, 0.039 mmol, 76%).
7.64–7.56 (m, 2H), 7.42–7.27 (m, 10H), 7.23–7.02 (m, 18H), 5.42 (d,
J ¼ 3.7 Hz, 1H), 5.38 (s, 1H), 5.33 (d, J ¼ 3.5 Hz, 1H), 5.02–4.91 (m,
3H), 4.84–4.66 (m, 5H), 4.58–4.46 (m, 4H), 4.40 (t, J ¼ 9.2 Hz, 1H),
4.36 (t, J ¼ 9.2 Hz, 1H), 4.18 (dd, J ¼ 10.2/4.9 Hz, 1H), 3.86 (t,
J ¼ 9.5 Hz, 1H), 3.68–3.49 (m, 6H), 3.45–3.15 (m, 1H), 3.35–3.24 (m,
1H), 1.11 (t, J ¼ 7.0 Hz, 3H) ppm. 13 C NMR (100 MHz, benzene-d6):
d 139.8 (C), 139.7 (C), 139.5 (C), 138.9 (C), 138.6 (C), 138.5 (C),
128.9 (CH), 128.7 (CH), 128.6 (CH), 128.4 (CH), 128.4 (CH), 128.3
(CH), 128.0 (CH), 127.9 (CH), 127.8 (CH), 127.6 (CH), 127.4 (CH),
126.8 (CH), 101.7 (CH), 94.4 (CH), 93.7 (CH), 83.0 (CH), 82.4 (CH),
Rf 0.34 in hexane/EtOAc 4/6. 1H NMR (400 MHz, CDCl3): d
7.43–7.20 (m, 28H), 7.18–7.11 (m, 2H), 5.24 (d, J ¼ 3.5 Hz, 1H), 5.21
(d, J ¼ 3.5 Hz, 1H), 5.03 (d, J ¼ 11.5 Hz, 1H), 5.01 (d, J ¼ 10.9 Hz, 1H),
4.89 (d, J ¼ 10.9 Hz, 1H), 4.84 (d, J ¼ 10.7 Hz, 1H), 4.78 (d,
J ¼ 11.5 Hz, 1H), 4.76–4.63 (m, 4H), 4.55 (d, J ¼ 12.1 Hz, 1H), 4.48 (d, 80.3 (CH), 79.3 (CH), 78.5 (CH), 75.5 (CH2), 75.2 (CH2), 74.9 (CH2),

1968
S. DHAENE ET AL.
þ
73.8 (CH2), 73.6 (CH2), 71.7 (CH), 69.7 (CH2), 69.3 (CH2), 66.9 (CH2), mode]: 95þ4.4 (M þ NH4
, 100). HRMS (ESI-TOF): calcd. for
C58H68NO11 [M þ NH4]þ 954.4787; found 954.4778.
63.4 (CH), 15.5 (CH3) ppm. IR (HATR): 3064 (w), 3029 (w), 2973 (w),
2914 (w), 2862 (w), 1496 (w), 1454 (m), 1368 (m), 1328 (w), 1209
(w), 1135 (m), 1086 (s), 1071 (s), 984 (s), 915 (m), 797 (w), 734 (m),
695 (s), 659 (w) cmꢀ1. ESMS [m/z (fragment, intensity), API-ES posi-
tive mode]: 926.4 (M þ NH4þ, 100). HRMS (ESI-TOF): calcd. for
2,3,4,2’,3’-Penta-O-benzyl-4’,6’-O-benzylidene-6-O-(2-methylallyl)-
a,a-D-trehalose (16-e)
General procedure A was applied (use of 3-bromo-2-methylpro-
pene, “methallyl bromide”). Purification via column chromatog-
raphy (gradient elution: hexane/EtOAc 9/1 to 8/2) afforded the
title compound 16-e as a white solid (247 mg, 0.264 mmol, 93%).
þ
C56H64NO11 [M þ NH4]þ 926.4474; found 926.4485.
6-O-Allyl-2,3,4,2’,3’-penta-O-benzyl-4’,6’-O-benzylidene-a,a-D-tre-
halose (16-c)
1
Rf 0.64 in hexane/EtOAc 1/1. H NMR (400 MHz, benzene-d6): d
7.63–7.57 (m, 2H), 7.45–7.26 (m, 10H), 7.24–7.02 (m, 18H), 5.41 (d,
J ¼ 3.7 Hz, 1H), 5.38 (s, 1H), 5.33 (d, J ¼ 3.5 Hz, 1H), 5.11–5.05 (m,
1H), 5.03–4.90 (m, 3H), 4.89–4.84 (m, 1H), 4.57–4.46 (m, 4H), 4.40
(app t, J ¼ 9.3 Hz, 1H), 4.36 (app t, J ¼ 9.3 Hz, 1H), 4.19 (dd,
J ¼ 10.1/4.9 Hz, 1H), 3.91–3.74 (m, 3H), 3.70–3.47 (m, 6H), 1.67 (s,
3H) ppm. 13 C NMR (100 MHz, benzene-d6): d 142.7 (C), 139.7 (C),
139.7 (C), 139.4 (C), 138.9 (C), 138.6 (C), 138.5 (C), 128.9 (CH), 128.7
(CH), 128.6 (CH), 128.4 (CH), 128.4 (CH), 128.3 (CH), 128.0 (CH),
127.9 (CH), 127.9 (CH), 127.8 (CH), 127.6 (CH), 127.4 (CH), 126.7
(CH), 112.0 (CH2), 101.6 (CH), 94.3 (CH), 93.6 (CH), 83.0 (CH), 82.4
(CH), 80.4 (CH), 79.3 (CH), 79.3 (CH), 78.6 (CH), 75.5 (CH2), 75.5
(CH2), 75.2 (CH2), 75.0 (CH2), 73.9 (CH2), 73.6 (CH2), 71.7 (CH), 69.3
(CH2), 63.4 (CH), 19.6 (CH3) ppm. IR (HATR): 3088 (w), 3060 (w),
3030 (w), 2925 (w), 2861 (w), 1496 (w), 1453 (w), 1388 (w), 1367
(w), 1329 (w), 1279 (w), 1237 (w), 1209 (w), 1153 (m), 1136 (m),
1086 (s), 1072 (s), 1025 (s), 986 (s), 908 (w), 877 (w), 846 (w), 796
(w), 745 (m), 734 (m), 695 (s), 656 (w), 634 (w) cmꢀ1. ESMS [mþ/z
General procedure
A was applied (use of allyl bromide).
Purification via column chromatography (gradient elution: hexane/
EtOAc 9/1 to 8/2) afforded the title compound 16-c as a white
solid (460 mg, 0.499 mmol, 88%).
1
Rf 0.34 in hexane/EtOAc 7/3. H NMR (400 MHz, benzene-d6): d
7.62–7.57 (m, 2H), 7.41–7.26 (m, 10H), 7.23–7.02 (m, 18H), 5.84
(app ddt, J ¼ 17.2/10.4/5.3 Hz, 1H), 5.41 (d, J ¼ 3.8 Hz, 1H), 5.38 (s,
1H), 5.33 (d, J ¼ 3.5 Hz, 1H), 5.25 (app dq, J ¼ 17.3/1.8 Hz, 1H), 5.04
(app dq, J ¼ 10.4/1.5 Hz, 1H), 5.00–4.91 (m, 3H), 4.83–4.65 (m, 5H),
4.58–4.47 (m, 4H), 4.40 (app t, J ¼ 9.2 Hz, 1H), 4.36 (app t,
J ¼ 9.3 Hz, 1H), 4.19 (dd, J ¼ 10.1/4.9 Hz, 1H), 3.91 (app ddt,
J ¼ 13.1/5.3/1.5 Hz, 1H), 3.88–3.80 (m, 2H), 3.68–3.49 (m, 6H) ppm.
13 C NMR (100 MHz, benzene-d6): d 139.8 (C), 139.7 (C), 139.4 (C),
138.9 (C), 138.6 (C), 138.5 (C), 135.5 (CH), 128.9 (CH), 128.7 (CH),
128.6 (CH), 128.4 (CH), 128.4 (CH), 128.3 (CH), 127.9 (CH), 127.8
(CH), 127.6 (CH), 127.4 (CH), 126.8 (CH), 116.3 (CH2), 101.7 (CH),
94.4 (CH), 93.6 (CH), 83.0 (CH), 82.4 (CH), 80.3 (CH), 79.3 (CH), 79.3
(CH), 78.5 (CH), 75.5 (CH2), 75.2 (CH2), 75.0 (CH2), 73.9 (CH2), 73.6
(CH2), 72.5 (CH2), 71.7 (CH), 69.4 (CH2), 63.4 (CH) ppm. IR (HATR):
3064 (w), 3029 (w), 2923 (w), 2864 (w), 1496 (w), 1453 (m), 1366
(m), 1331 (w), 1209 (w), 1151 (m), 1135 (m), 1086 (s), 1071 (s), 985
(s), 928 (m), 734 (m), 695 (s), 656 (m) cmꢀ1. ESMS [m/z (fragment,
(fragment, intensity), API-ES positive mode]: 952.4 (M þ NH4
,
100). HRMS (ESI-TOF): calcd. for C58H66NO11 [M þ H]þ 952.4630;
þ
found 952.4641.
2,3,4,2’,3’-Penta-O-benzyl-4’,6’-O-benzylidene-6-O-(3,3-dimethy-
lallyl)-a,a-D-trehalose (16-f)
General procedure A was applied (use of 1-bromo-3-methyl-2-
butene, “prenyl bromide”). Purification via column chromatog-
raphy (hexane/EtOAc 8/2) afforded the partially purified title com-
pound 16-f as a white solid which was used as such in the
next step.
intensity), API-ES positive mode]: 938.4 (M þ NH4þ, 100). HRMS
[M þ NH4]þ 938.4474;
þ
(ESI-TOF): calcd. for C57H64NO11
found 938.4509.
2,3,4,2’,3’-Penta-O-benzyl-4’,6’-O-benzylidene-6-O-n-butyl-a,a-D-tre-
Rf 0.58 in hexane/EtOAc 6/4.
halose (16-d)
General procedure
A was applied (use of 1-iodobutane).
Purification via column chromatography (gradient elution: hexane/
EtOAc 9/1 to 7/3) afforded the title compound 16-d as a white
solid (445 mg, 0.475 mmol, 84%).
2,3,4,2’,3’-Penta-O-benzyl-4’,6’-O-benzylidene-6-O-n-decyl-a,a-D-tre-
halose (16-g)
1
General procedure
A was applied (use of 1-bromodecane).
Rf 0.54 in hexane/EtOAc 6/4. H NMR (400 MHz, benzene-d6): d
Purification via column chromatography (gradient elution: hexane/
EtOAc 9/1 to 8/2) afforded the partially purified title compound
16-g as a white solid (320 mg), which was used as such in the
next step.
7.64–7.56 (m, 2H), 7.43–7.27 (m, 10H), 7.23–7.02 (m, 18H), 5.43 (d,
J ¼ 3.8 Hz, 1H), 5.38 (s, 1H), 5.34 (d, J ¼ 3.5 Hz, 1H), 5.02 (d,
J ¼ 11.4 Hz, 1H), 4.95 (d, J ¼ 11.6 Hz, 1H), 4.94 (d, J ¼ 11.4 Hz, 1H),
4.84–4.66 (m, 5H), 4.58–4.46 (m, 4H), 4.45–4.33 (m, 2H), 4.20 (dd,
J ¼ 10.1/4.9 Hz, 1H), 3.95–3.85 (m, 1H), 3.72–3.48 (m, 6H), 3.46–3.38
(m, 1H), 3.35–3.26 (m, 1H), 1.63–1.27 (m, 4H), 0.85 (t, J ¼ 7.3 Hz,
3H) ppm. 13 C NMR (100 MHz, benzene-d6): d 139.8 (C), 139.7 (C),
139.5 (C), 138.9 (C), 138.6 (C), 138.5 (C), 128.9 (CH), 128.7 (CH),
128.6 (CH), 128.5 (CH), 128.4 (CH), 128.3 (CH), 127.9 (CH), 127.9
(CH), 127.9 (CH), 127.6 (CH), 127.4 (CH), 126.8 (CH), 101.7 (CH),
94.4 (CH), 93.7 (CH), 83.0 (CH), 82.4 (CH), 80.4 (CH), 79.4 (CH), 79.3
(CH), 78.6 (CH), 75.5 (CH2), 75.2 (CH2), 75.0 (CH2), 73.9 (CH2), 73.6
(CH2), 71.7 (CH), 71.5 (CH2), 70.0 (CH2), 69.3 (CH2), 63.4 (CH), 32.3
(CH2), 19.8 (CH2), 14.1 (CH3) ppm. IR (HATR): 3064 (w), 3027 (w),
2932 (w), 2862 (w), 1496 (w), 1454 (m), 1367 (m), 1328 (w), 1210
(w), 1154 (m), 1086 (s), 1072 (s), 985 (s), 915 (m), 733 (m), 695 (s),
Rf 0.67 in hexane/EtOAc 1/1. ESMS [m/z (fragment, intensity),
API-ES positive mode]: 1038.5 (M þ NH4þ, 100). HRMS (ESI-TOF):
þ
calcd. for C64H80NO11 [M þ NH4]þ 1038.5726; found 1038.5742.
6-O-Methyl-a,a-D-trehalose (17-a)
General procedure C was applied. Purification via column chroma-
tography (gradient elution: CH2Cl2/MeOH 8/2 to 6/4) afforded the
title compound 17-a as a white solid (77.9 mg, 0.219 mmol, 96%).
1
Rf 0.35 in CH2Cl2/MeOH 6/4. H NMR (400 MHz, CD3OD): d 5.09
(d, J ¼ 3.4 Hz, 1H), 5.08 (d, J ¼ 3.5 Hz, 1H), 3.94 (ddd, J ¼ 10.0/4.9/
2.6 Hz, 1H), 3.85–3.73 (m, 3H), 3.70–3.53 (m, 5H), 3.48 (d, J ¼ 3.8 Hz,
1H), 3.45 (d, J ¼ 3.8 Hz, 1H), 3.37 (s, 3H), 3.35–3.27 (m, 2H) ppm.
657 (w) cmꢀ1. ESMS [m/z (fragment, intensity), API-ES positive 13 C NMR (100 MHz, CD3OD): d 95.2 (CH), 74.6 (CH), 74.5 (CH), 73.8

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1969
(CH), 73.2 (CH), 73.1 (CH), 73.1 (CH2), 72.4 (CH), 72.0 (CH), 71.9 1146 (m), 1101 (m), 1077 (m), 1029 (m), 985 (s), 941 (m), 912 (w),
(CH), 62.6 (CH2), 59.5 (CH3) ppm. IR (HATR): 3292 (br, m), 2927 (w), 844 (w), 805 (w), 706 (w) cmꢀ1. [a]D þ271ꢂ (c 0.12, MeOH). ESMS
22
þ
1652 (w), 1637 (w), 1455 (w), 1438 (w), 1419 (w), 1363 (w), 1337 [m/z (fragment, intensity), API-ES positive mode]: 416.2 (M þ NH4
,
(w), 1265 (w), 1197 (w), 1146 (m), 1100 (m), 1079 (m), 1032 (m), 100). HRMS (ESI-TOF): calcd. for C16H30NaO11 [M þ Na]þ 421.1680;
þ
986 (s), 943 (m), 912 (w), 852 (w), 805 (w), 705 (w), 667 (w), 608 found 421.1676.
(w) cmꢀ1. [a]D þ198ꢂ (c 0.12, MeOH). ESMS [m/z (fragment,
22
intensity), API-ES positive mode]: 374.1 (M þ NH4þ, 100). HRMS
þ
6-O-Isobutyl-a,a-D-trehalose (17-e)
(ESI-TOF): calcd. for C13H28NO11
found 374.1661.
[M þ NH4]þ 374.1657;
General procedure C was applied. Purification via column chroma-
tography (gradient elution: CH2Cl2/MeOH 8/2 to 6/4) afforded the
title compound as a white solid (71.0 mg, 0.178 mmol, 79%).
1
6-O-Ethyl-a,a-D-trehalose (17-b)
Rf 0.32 in CH2Cl2/MeOH 6/4. H NMR (400 MHz, CD3OD): d 5.09
General procedure C was applied. Purification via column chroma- (app d, J ¼ 3.7 Hz, 2H), 3.92 (ddd, J ¼ 10.0/5.0/2.4 Hz, 1H), 3.84–3.74
tography (gradient elution: CH2Cl2/MeOH 8/2 to 6/4) afforded the (m, 4H), 3.70–3.58 (m, 3H), 3.48 (app t, J ¼ 3.8 Hz, 1H), 3.45 (app t,
title compound 17-b as a white solid (71.9 mg, 0.194 mmol, 98%).
J ¼ 3.8 Hz, 1H), 3.37–3.27 (m, 3H), 3.21 (dd, J ¼ 9.3/6.8 Hz, 1H), 1.85
Rf 0.29 in CH3CN/H2O 8/2, 0.41 in CH2Cl2/MeOH 6/4. 1H NMR (nonaplet, J ¼ 6.7 Hz, 1H), 0.90 (d, J ¼ 6.7 Hz, 3H), 0.90 (d, J ¼ 6.7 Hz,
(400 MHz, CD3OD): d 5.09 (d, J ¼ 3.4 Hz, 1H), 5.08 (d, J ¼ 3.5 Hz, 1H), 3H) ppm. 13 C NMR (100 MHz, CD3OD): d 95.0 (CH), 94.9 (CH), 79.5
3.92 (ddd, J ¼ 9.9/5.2/2.1 Hz, 1H), 3.85–3.72 (m, 4H), 3.72–3.41 (m, (CH2), 74.7 (CH), 74.6 (CH), 73.8 (CH), 73.2 (CH), 73.2 (CH), 72.6
7H), 3.38–3.28 (m, 2H), 1.18 (t, J ¼ 7.0 Hz, 3H) ppm. 13 C NMR (CH), 72.1 (CH), 71.9 (CH), 71.3 (CH2), 62.6 (CH2), 29.5 (CH), 19.7
(100 MHz, CD3OD): d 95.1 (CH), 95.0 (CH), 74.6 (CH), 74.5 (CH), 73.8 (CH3) ppm. IR (HATR): 3306 (br, m), 2953 (w), 2928 (w), 2875 (w),
(CH), 73.2 (CH), 73.0 (CH), 72.5 (CH), 72.1 (CH), 71.9 (CH), 70.9 1652 (w), 1637 (w), 1456 (w), 1436 (w), 1419 (w), 1388 (w), 1365
(CH2), 67.9 (CH2), 62.6 (CH2), 15.4 (CH3) ppm. IR (HATR): 3305 (br, (w), 1335 (w), 1270 (w), 1209 (w), 1147 (m), 1102 (m), 1077 (m),
m), 2976 (w), 2926 (w), 1652 (w), 1455 (w), 1418 (w), 1375 (w), 1031 (m), 985 (s), 941 (m), 913 (w), 846 (w), 804 (w), 706 (w), 642
22
1360 (w), 1335 (w), 1271 (w), 1206 (w), 1146 (m), 1100 (m), 1075 (w), 612 (w) cmꢀ1. [a]D þ165ꢂ (c 0.10, MeOH). ESMS [m/z (frag-
(m), 1028 (m), 985 (s), 941 (m), 913 (w), 853 (w), 804 (w), 734 (w), ment, intensity), API-ES negative mode]: 416.2 (M þ NH4þ, 100).
702 (w), 642 (w), 611 (w) cmꢀ1. [a]D þ213ꢂ (c 0.14, MeOH). ESMS HRMS (ESI-TOF): calcd. for C16H34NO11 [M þ NH4]þ 416.2126;
22
þ
þ
[m/z (fragment, intensity), API-ES positive mode]: 388.0 (M þ NH4
,
found 416.2138.
þ
100). HRMS (ESI-TOF): calcd. for C14H30NO11 [M þ NH4]þ 388.1813;
found 388.1827.
6-O-Isopentyl-a,a-D-trehalose (17-f)
General procedure C was applied. Purification via column chroma-
tography (gradient elution: CH2Cl2/MeOH 9/1 to 7/3) afforded the
6-O-n-Propyl-a,a-D-trehalose (17-c)
General procedure C was applied. Purification via column chroma- title compound 17-f as a white solid (40 mg, 0.097 mmol, 17%
tography (gradient elution: CH2Cl2/MeOH 9/1 to 65/35) afforded over 2 steps).
the title compound as a white solid (125 mg, 0.338 mmol, 71%).
Rf 0.29 in CH3CN/H2O 8/2, 0.49 in CH2Cl2/MeOH 6/4. 1H NMR
Rf 0.34 in CH3CN/H2O 8/2, 0.27 in CH2Cl2/MeOH 7/3. 1H NMR (400 MHz, CD3OD): d 5.10 (d, J ¼ 3.3 Hz, 1H), 5.09 (d, J ¼ 3.3 Hz, 1H),
(400 MHz, CD3OD): d 5.09 (d, J ¼ 3.3 Hz, 1H), 5.09 (d, J ¼ 3.5 Hz, 1H), 3.91 (ddd, J ¼ 9.9/4.9/2.2 Hz, 1H), 3.85–3.73 (m, 4H), 3.71–3.41 (m,
3.91 (ddd, J ¼ 10.0/5.2/2.1 Hz, 1H), 3.87–3.73 (m, 4H), 3.71–3.56 (m, 7H), 3.38–3.26 (m, 2H), 1.70 (heptaplet, J ¼ 6.7 Hz, 1H), 1.46 (q,
3H), 3.54–3.37 (m, 4H), 3.37–3.27 (m, 2H), 1.58 (app hexaplet, J ¼ 6.8 Hz, 2H), 0.91 (d, J ¼ 6.6 Hz, 6H) ppm. 13 C NMR (100 MHz,
J ¼ 7.1 Hz, 2H), 0.92 (t, J ¼ 7.4 Hz, 3H), ppm. 13 C NMR (100 MHz, CD3OD): d 95.1 (CH), 95.0 (CH), 74.6 (CH), 74.5 (CH), 73.8 (CH), 73.2
CD3OD): d 95.1 (CH), 95.0 (CH), 74.6 (CH), 74.5 (CH), 74.3 (CH2), (CH), 73.1 (CH), 72.6 (CH), 72.1 (CH), 71.9 (CH), 71.2 (CH2), 71.1
73.8 (CH), 73.2 (CH), 73.2 (CH), 72.6 (CH), 72.1 (CH), 71.9 (CH), 71.1 (CH2), 62.6 (CH2), 39.6 (CH2), 26.2 (CH), 23.1 (CH3), 23.0 (CH3) ppm.
(CH2), 62.6 (CH2), 23.8 (CH2), 10.8 (CH3) ppm. IR (HATR): 3312 (m, IR (HATR): 3308 (m, br), 2951 (w), 2926 (w), 2867 (w), 1455 (w),
br), 2928 (w), 2872 (w), 1435 (w), 1372 (w), 1334 (w), 1266 (w), 1423 (w), 1366 (w), 1337 (w), 1266 (w), 1204 (w), 1145 (m), 1102
1204 (w), 1146 (m), 1101 (m), 1075 (m), 1033 (m), 987 (s), 943 (m), (m), 1076 (m), 1032 (m), 987 (s), 942 (m), 914 (w), 847 (w), 806 (w)
912 (w), 853 (w), 845 (w), 804 (w), 700 (w) cmꢀ1. [a]D þ231ꢂ (c cmꢀ1. [a]D þ183ꢂ (c 0.06, MeOH). ESMS [m/z (fragment, inten-
22
22
0.10, MeOH). ESMS [m/z (fragment, intensity), API-ES negative sity), API-ES negative mode]: 430.2 (M þ NH4þ, 100). HRMS (ESI-
þ
mode]: 402.2 (M þ NH4
,
100). HRMS (ESI-TOF): calcd. for TOF): calcd. for C17H32NaO11 [M þ Na]þ 435.1837; found 435.1833.
C15H28NaO11 [M þ Na]þ 407.1524; found 407.1513.
6-O-n-Decyl-a,a-D-trehalose (17-g)
6-O-n-Butyl-a,a-D-trehalose (17-d)
General procedure C was applied. Purification via column chroma-
General procedure C was applied. Purification via column chroma- tography (gradient elution: CH2Cl2/MeOH 8/2 to 6/4) afforded the
tography (CH2Cl2/MeOH 7/3) afforded the title compound 17-d as title compound 17-g as a white solid (88.0 mg, 0.182 mmol, 64%
a white solid (145 mg, 0.364 mmol, 81%).
over two steps).
Rf 0.61 in CH2Cl2/MeOH 6/4. H NMR (400 MHz, CD3OD): d 5.10
1
Rf 0.21 in CH3CN/H2O 8/2, 0.24 in CH2Cl2/MeOH 7/3. 1H NMR
(400 MHz, CD3OD): d 5.13–5.06 (m, 2H), 3.92 (ddd, J ¼ 9.9/4.9/ (d, J ¼ 3.2 Hz, 1H), 5.09 (d, J ¼ 3.3 Hz, 1H), 3.92 (ddd, J ¼ 10.0/5.1/
2.1 Hz, 1H), 3.86–3.72 (m, 4H), 3.71–3.41 (m, 7H), 3.37–3.27 (m, 2H), 2.2 Hz, 1H), 3.85–3.74 (m, 4H), 3.70–3.58 (m, 3H), 3.56–3.41 (m, 4H),
1.62–1.48 (m, 2H), 1.38 (app hexaplet, J ¼ 7.5 Hz, 2H), 0.92 (t, 3.37–3.29 (m, 1H), 1.56 (quintet, J ¼ 6.9 Hz, 2H), 1.42–1.20 (m, 14H),
J ¼ 7.4 Hz, 3H) ppm. 13 C NMR (100 MHz, CD3OD): d 95.1 (CH), 95.0 0.89 (t, J ¼ 6.9 Hz, 3H) ppm. 13 C NMR (100 MHz, CD3OD): d 95.0
(CH), 74.6 (CH), 74.5 (CH), 73.8 (CH), 73.2 (CH), 73.2 (CH), 72.6 (CH), (CH), 94.9 (CH), 79.5 (CH2), 74.6 (CH), 74.5 (CH), 73.8 (CH), 73.2
72.4 (CH2), 72.1 (CH), 71.9 (CH), 71.2 (CH2), 62.6 (CH2), 32.8 (CH2), (CH), 73.1 (CH), 72.8 (CH), 72.6 (CH), 72.1 (CH), 71.9 (CH), 71.1
20.3 (CH2), 14.3 (CH) ppm. IR (HATR): 3301 (m, br), 2929 (w), 2872 (CH2), 62.6 (CH2), 33.1 (CH2), 30.7 (CH2), 30.7 (CH2), 30.6 (CH2), 30.5
(w), 1644 (w), 1455 (w), 1430 (w), 1372 (w), 1334 (w), 1263 (w), (CH2), 27.2 (CH2), 23.7 (CH2), 14.4 (CH3) ppm. IR (HATR): 3310 (m,

1970
S. DHAENE ET AL.
br), 2923 (m), 2854 (m), 1456 (w), 1428 (w), 1377 (w), 1337 (w), 2,3,4,2’,3’-Penta-O-benzyl-4’,6’-O-benzylidene-6-O-(N-cyclohexylcar-
1275 (w), 1202 (w), 1146 (m), 1104 (m), 1078 (m), 1034 (m), 987
bamoyl)-a,a-D-trehalose (18-c)
(s), 941 (m), 912 (w), 848 (w), 805 (w), 720 (w), 708 (w) cmꢀ1
.
General procedure B was applied (use of cyclohexyl isocyanate).
Purification via column chromatography (gradient elution: hexane/
EtOAc 8/2 to 7/3) afforded the title compound 18-c as a white
foam (411 mg, 0.408 mmol, 81%).
22
[a]D þ182ꢂ (c 0.10, MeOH). ESMS [m/z (fragment, intensity), API-
ES positive mode]: 500.3 (M þ NH4þ, 100). HRMS (ESI-TOF): calcd.
þ
for C22H42NaO11 [M þ Na]þ 505.2619; found 505.2616.
1
Rf 0.29 in hexane/EtOAc 6/4. H NMR (400 MHz, benzene-d6): d
7.63–7.58 (m, 2H), 7.39–7.28 (m, 10H), 7.23–7.02 (m, 18H), 5.39 (s,
1H), 5.36 (d, J ¼ 3.7 Hz,1H), 5.33 (d, J ¼ 3.4 Hz, 1H), 5.01–4.92 (m,
3H), 4.79–4.69 (m, 4H), 4.66–4.45 (m, 7H), 4.40 (app t, J ¼ 9.3 Hz,
1H), 4.37 (app t, J ¼ 9.3 Hz, 1H), 4.26–4.18 (m, 1H), 4.20 (dd, J ¼ 10.
2/4.8 Hz, 1H), 3.75 (app br t, J ¼ 9.2 Hz, 1H), 3.70–3.47 (m, 5H), 1.75
(app br t, J ¼ 12.8 Hz, 2H), 1.44–1.24 (m, 3H), 1.10–0.70 (m, 5H)
ppm. 13 C NMR (100 MHz, benzene-d6): d 155.4 (C), 139.6 (C), 138.9
2,3,4,2’,3’-Penta-O-benzyl-4’,6’-O-benzylidene-6-O-(N-n-propylcar-
bamoyl)-a,a-D-trehalose (18-a)
General procedure B was applied (use of n-propyl isocyanate).
Purification via column chromatography (gradient elution: hexane/
EtOAc 8/2 to 7/3) afforded the title compound 18-a as a white
foam (187 mg, 0.194 mmol, 85%).
1
Rf 0.55 in hexane/EtOAc 1/1. H NMR (400 MHz, benzene-d6): d (C), 138.7 (C), 138.4 (C), 138.4 (C), 128.9 (CH), 128.8 (CH), 128.7
7.63–7.00 (m, 30H), 5.38 (s, 1H), 5.34 (d, J ¼ 4.0 Hz, 1H), 5.31 (d, (CH), 128.5 (CH), 128.5 (CH), 128.4 (CH), 127.9 (CH), 127.8 (CH),
127.5 (CH), 127.5 (CH), 126.7 (CH), 101.7 (CH), 94.6 (CH), 93.6 (CH),
J ¼ 3.3 Hz, 1H), 5.03–4.90 (m, 3H), 4.81–4.03 (m, 16H), 3.79–3.44 (m,
82.9 (CH), 82.3 (CH), 80.2 (CH), 79.4 (CH), 79.2 (CH), 78.2 (CH), 75.5
(CH2), 75.3 (CH2), 75.1 (CH2), 74.1 (CH2), 73.5 (CH2), 70.4 (CH), 69.3
5H), 3.12 (s, 1H), 2.88 (q, J ¼ 6.6 Hz, 2H), 1.12 (app sextet,
J ¼ 7.3 Hz, 2H), 0.59 (t, J ¼ 7.4 Hz, 3H) ppm. 13 C NMR (100 MHz,
(CH2), 63.5 (CH), 49.9 (CH), 33.4 (CH2), 33.3 (CH2), 25.7 (CH2), 24.9
(CH2) ppm. IR (HATR): 3032 (w), 2930 (w), 2854 (w), 1719 (m), 1496
(m), 1453 (m), 1365 (w), 1331 (w), 1315 (w), 1272 (w), 1251 (w),
1211 (m), 1151 (m), 1086 (s), 1072 (s), 985 (s), 735 (m), 696 (s), 658
(w) cmꢀ1. ESMS [m/z (fragment, intensity), API-ES positive modeþ]:
1023.4 (M þ NH4þ, 100). HRMS (ESI-TOF): calcd. for C61H71N2O12
[M þ NH4]þ 1023.5002; found 1023.5013.
benzene-d6): d 156.2 (C), 139.6 (C), 138.9 (C), 138.7 (C), 138.43 (C),
138.4 (C), 128.9 (CH), 128.8 (CH), 128.7 (CH), 128.5 (CH), 128.5 (CH),
128.4 (CH), 128.4 (CH), 128.3 (CH), 127.9 (CH), 127.9 (CH), 127.8
(CH), 127.5 (CH), 126.7 (CH), 102.8 (CH), 101.7 (CH), 94.6 (CH), 93.6
(CH), 82.9 (CH), 82.3 (CH), 80.1 (CH), 79.4 (CH), 79.2 (CH), 78.2 (CH),
75.5 (CH2), 75.3 (CH2), 75.0 (CH2), 74.1 (CH2), 73.5 (CH2), 70.3 (CH),
69.3 (CH2), 63.5 (CH2), 63.5 (CH2), 51.9 (CH), 42.9 (CH2), 23.3 (CH2),
11.1 (CH3) ppm. IR (HATR): 3060 (w), 3031 (w), 2932 (w), 2869 (w),
1722 (m), 1517 (w), 1496 (w), 1454 (m), 1388 (w), 1368 (s), 1330
(w), 1279 (w), 1259 (w), 1234 (w), 1212 (w), 1153 (m), 1134 (m),
1085 (s), 1072 (s), 1005 (s), 981 (s), 930 (m), 874 (w), 846 (w), 798
(w), 747 (m), 734 (m), 695 (s), 654 (w), 631 (w) cmꢀ1. ESMS [mþ/z
2,3,4,2’,3’-Penta-O-benzyl-4’,6’-O-benzylidene-6-O-(N-phenylcarba-
moyl)-a,a-D-trehalose (18-d)
General procedure B was applied (use of phenyl isocyanate).
Purification via column chromatography (gradient elution: hexane/
EtOAc 9/1 to 7/3) afforded the title compound 18-d as a white
foam (184 mg, 0.185 mmol, 81%).
(fragment, intensity), API-ES positive mode]: 983.4 (M þ NH4
,
þ
100). HRMS (ESI-TOF): calcd. for C58H67N2O12
983.4689; found 983.4661.
[M þ NH4]þ
1
Rf 0.25 in hexane/EtOAc 8/2. H NMR (400 MHz, benzene-d6): d
7.64–7.58 (m, 2H), 7.39–7.25 (m, 12H), 7.24–6.98 (m, 20H), 6.82 (tt,
J ¼ 7.4/1.1 Hz, 1H), 6.14 (s, 1H), 5.39 (s, 1H), 5.33 (d, J ¼ 3.8 Hz, 1H),
5.31 (d, J ¼ 3.6 Hz, 1H), 5.02–4.88 (m, 3H), 4.82–4.63 (m, 4H),
4.60–4.32 (m, 9H), 4.21 (dd, J ¼ 10.1/4.9 Hz, 1H), 3.77–3.45 (m, 5H)
ppm. 13 C NMR (100 MHz, benzene-d6): d 153.1 (C), 139.6 (C), 139.5
(C), 138.8 (C), 138.7 (C), 138.6 (C), 138.4 (C), 138.4 (C), 129.1 (CH),
129.0 (CH), 128.8 (CH), 128.7 (CH), 128.5 (CH), 128.5 (CH), 128.5
(CH), 128.4 (CH), 128.3 (CH), 128.0 (CH), 128.0 (CH), 128.0 (CH),
127.9 (CH), 127.9 (CH), 127.8 (CH), 127.6 (CH), 127.5 (CH), 126.7
(CH), 123.3 (CH), 118.6 (CH), 101.7 (CH), 94.8 (CH), 93.7 (CH), 82.9
(CH), 82.3 (CH), 80.1 (CH), 79.4 (CH), 79.2 (CH), 77.6 (CH), 75.5
(CH2), 75.3 (CH2), 74.9 (CH2), 74.2 (CH2), 73.4 (CH2), 70.0 (CH), 69.3
(CH2), 63.8 (CH2), 63.5 (CH) ppm. IR (HATR): 3391 (w), 3318 (w),
3060 (w), 3031 (w), 2931 (w), 2866 (w), 1732 (m), 1715 (m), 1601
(w), 1525 (m), 1497 (w), 1454 (m), 1444 (m), 1385 (w), 1367 (w),
1313 (w), 1210 (m), 1156 (w), 1137 (w), 1086 (s), 1071 (s), 985 (s),
912 (w), 877 (w), 846 (w), 798 (w), 748 (m), 734 (m), 694 (s), 656
(w) cmꢀ1. ESMS [m/z (fragment, intensity), API-ES positive modeþ]:
1017.3 (M þ NHþ, 100). HRMS (ESI-TOF): calcd. for C61H65N2O12
[M þ NH4]þ 1017.4532; found 1017.4524.
2,3,4,2’,3’-Penta-O-benzyl-4’,6’-O-benzylidene-6-O-(N-isopropylcar-
bamoyl)-a,a-D-trehalose (18-b)
General procedure B was applied (use of isopropyl isocyanate).
Purification via column chromatography (hexane/EtOAc 8/2)
afforded the title compound 18-b as a white foam (174 mg,
0.180 mmol, 79%).
1
Rf 0.64 in hexane/EtOAc 1/1. H NMR (400 MHz, benzene-d6): d
7.63–7.58 (m, 2H), 7.38–7.27 (m, 10H), 7.25–6.92 (m, 38H), 5.38 (s,
1H), 5.35 (d, J ¼ 3.8 Hz, 1H), 5.32 (d, J ¼ 3.5 Hz, 1H), 5.00–4.92 (m,
3H), 4.80–4.62 (m, 4H), 4.62–4.31 (m, 9H), 4.19 (dd, J ¼ 10.1/4.9 Hz,
1H), 4.11–4.00 (m, 1H), 3.81–3.43 (m, 6H), 0.79 (d, J ¼ 6.6 Hz, 3H),
0.76 (d, J ¼ 6.6 Hz, 3H) ppm. 13 C NMR (100 MHz, benzene-d6): d
153.4 (C), 139.6 (C), 138.9 (C), 138.7 (C), 138.4 (C), 128.9 (CH), 128.8
(CH), 128.8 (CH), 128.5 (CH), 128.5 (CH), 128.4 (CH), 128.4 (CH),
128.3 (CH), 128.0 (CH), 127.9 (CH), 127.8 (CH), 127.5 (CH), 127.5
(CH), 126.7 (CH), 101.6 (CH), 94.6 (CH), 93.6 (CH), 82.9 (CH), 82.3
(CH), 80.2 (CH), 79.4 (CH), 79.2 (CH), 78.2 (CH), 75.5 (CH2), 75.2
(CH2), 75.0 (CH2), 74.1 (CH2), 73.5 (CH2), 70.3 (CH), 69.3 (CH2), 63.5
(CH), 63.4 (CH2), 43.0 (CH), 22.7 (CH3), 22.6 (CH3) ppm. IR (HATR):
3060 (w), 3031 (w), 2970 (w), 2932 (w), 2868 (w), 1718 (m), 1505
(w), 1497 (w), 1454 (m), 1385 (w), 1367 (w), 1321 (w), 1234 (w), 2,3,4,2’,3’-Penta-O-benzyl-4’,6’-O-benzylidene-6-O-(N-benzylcarba-
1212 (w), 1153 (w), 1134 (m), 1086 (s), 1071 (s), 1025 (m), 984 (s),
913 (w), 874 (m), 846 (w), 798 (w), 748 (m), 734 (m), 695 (s), 656
moyl)-a,a-D-trehalose (18-e)
General procedure B was applied (use of benzyl isocyanate).
Purification via column chromatography (gradient elution: hexane/
EtOAc 8/2 to 6/4) afforded the title compound 18-e as a white
foam (457 mg, 0.451 mmol, 79%).
(w), 631 (w) cmꢀ1. ESMS [m/z (fragment, intensity), API-ES positive
þ
mode]: 983þ.4 (M þ NH4
, 100). HRMS (ESI-TOF): calcd. for
C58H67N2O12 [M þ NH4]þ 983.4689; found 983.4666.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1971
1
Rf 0.27 in hexane/EtOAc 6/4. H NMR (400 MHz, benzene-d6): d 6-O-(N-n-Propylcarbamoyl)-a,a-D-trehalose (19-a)
General procedure C was applied. Purification via column chroma-
tography (gradient elution: CH2Cl2/MeOH 8/2 to 7/3) afforded the
title compound 19-a as a white solid (64.0 mg, 0.150 mmol, 93%).
Rf 0.56 in CH3CN/H2O 8/2, 0.11 in CH2Cl2/MeOH 9/1. 1H NMR
(400 MHz, CD3OD): d 5.08 (d, J ¼ 2.8 Hz, 1H), 5.07 (d, J ¼ 3.2 Hz, 1H),
4.25 (dd, J ¼ 11.6/1.7 Hz, 1H), 4.19 (dd, J ¼ 11.7/4.9 Hz, 1H), 3.98 (br
ddd, J ¼ 10.0/2.6/2.0 Hz, 1H), 3.85–3.73 (m, 4H), 3.66 (dd, J ¼ 12.0/
5.5 Hz, 1H), 3.55–3.31 (m, 4H), 3.05 (t, J ¼ 7.0 Hz, 2H), 1.49 (app sex-
tet, J ¼ 7.3 Hz, 2H), 0.90 (t, J ¼ 7.4 Hz, 3H) ppm. 13 C NMR (100 MHz,
CD3OD): d 159.2 (C), 95.2 (CH), 95.1 (CH), 74.6 (CH), 74.4 (CH), 73.9
(CH), 73.2 (CH), 73.2 (CH), 71.9 (CH), 71.8 (CH), 64.8 (CH2), 62.6
(CH2), 43.6 (CH2), 24.1 (CH2), 11.6 (CH3) ppm. IR (HATR): 3304 (br,
m), 2964 (w), 2933 (w), 1684 (m), 1540 (w), 1456 (w), 1419 (w),
1363 (w), 1337 (w), 1265 (m), 1147 (m), 1103 (m), 1076 (m), 1030
(m), 984 (s), 941 (w), 913 (w), 845 (w), 805 (w), 776 (w), 614 (w)
7.64–7.57 (m, 2H), 7.39–7.27 (m, 10H), 7.24–6.94 (m, 23H), 5.39 (s,
1H), 5.33 (br d, J ¼ 3.4 Hz, 1H), 5.31 (br d, J ¼ 2.9 Hz, 1H), 5.02–4.87
(m, 3H), 4.80–4.30 (m, 14H), 4.21 (dd, J ¼ 10.1/4.9 Hz, 1H),
4.15–4.01 (m, 2H), 3.76–3.49 (m, 5H) ppm. 13 C NMR (100 MHz, ben-
zene-d6): d 156.3 (C), 139.6 (C), 138.9 (C), 138.6 (C), 138.4 (C), 128.9
(CH), 128.8 (CH), 128.7 (CH), 128.6 (CH), 128.5 (CH), 128.5 (CH),
128.4 (CH), 128.1 (CH), 127.9 (CH), 127.5 (CH), 127.5 (CH), 127.4
(CH), 126.7 (CH), 101.7 (CH), 94.6 (CH), 93.6 (CH), 82.9 (CH), 82.2
(CH), 80.1 (CH), 79.4 (CH), 79.3 (CH), 78.1 (CH), 75.5 (CH2), 75.3
(CH2), 75.0 (CH2), 74.1 (CH2), 73.4 (CH2), 70.2 (CH), 69.3 (CH2), 63.7
(CH2), 63.5 (CH), 45.2 (CH2) ppm. IR (HATR): 3064 (w), 3030 (w),
2923 (w), 2871 (w), 1723 (m), 1513 (w), 1496 (m), 1453 (m), 1366
(m), 1331 (w), 1235 (m), 1210 (m), 1154 (m), 1138 (m), 1086 (s),
1073 (s), 985 (s), 915 (w), 735 (m), 696 (s), 652 (w) cmꢀ1. ESMS [m/
þ
z (fragment, intensity), API-ES positive mode]: 1031.4 (M þ NH4
,
22
cmꢀ1. [a]D þ150ꢂ (c 0.10, MeOH). ESMS [m/z (fragment, inten-
þ
100). HRMS (ESI-TOF): calcd. for C62H67N2O12
1031.4689; found 1031.4681.
[M þ NH4]þ
sity), API-ES negative mode]: 486.2 (M þ OAc-, 100). HRMS (ESI-
TOF): calcd. for C16H30NO12 [M þ H]þ 428.1763; found 428.1771.
þ
6-O-(N-Isopropylcarbamoyl)-a,a-D-trehalose (19-b)
2,3,4,2’,3’-Penta-O-benzyl-4’,6’-O-benzylidene-6-O-[N-(2-phenethyl)-
carbamoyl]-a,a-D-trehalose (18-f)
General procedure C was applied. Purification via column chroma-
tography (gradient elution: CH2Cl2/MeOH 8/2 to 6/4) afforded the
title compound 19-b as a white solid (207 mg, 0.484 mmol, 93%).
Rf 0.55 in CH3CN/H2O 8/2, 0.11 in CH2Cl2/MeOH 9/1. 1H NMR
(400 MHz, CD3OD): d 5.13–5.02 (m, 2H), 4.40–4.13 (m, 2H), 3.98 (br
d, J ¼ 8.9 Hz, 1H), 3.85–3.63 (m, 6H), 3.46 (app dd, J ¼ 9.8/3.7 Hz,
2H), 3.37–3.26 (m, 2H), 1.12 (d, J ¼ 6.5 Hz, 6H) ppm. 13 C NMR
(100 MHz, CD3OD): d 158.3 (C), 95.2 (CH), 95.1 (CH), 74.6 (CH), 74.4
(CH), 73.9 (CH), 73.2 (CH), 73.2 (CH), 71.9 (CH), 71.8 (CH), 64.7
(CH2), 62.6 (CH2), 44.0 (CH), 22.9 (CH3) ppm. IR (HATR): 3303 (br,
m), 2970 (w), 2932 (w), 1684 (m), 1540 (w), 1456 (w), 1436 (w),
1419 (w), 1388 (w), 1368 (w), 1349 (w), 1324 (w), 1254 (m), 1147
(w), 1103 (m), 1076 (m), 1042 (m), 1024 (m), 984 (s), 941 (m), 845
General procedure B was applied (use of phenethyl isocyanate).
Purification via column chromatography (gradient elution: hexane/
EtOAc 8/2 to 7/3) afforded the title compound 18-f as a white
foam (220 mg, 0.214 mmol, 94%).
1
Rf 0.13 in hexane/EtOAc 7/3. H NMR (400 MHz, benzene-d6): d
7.64–7.57 (m, 2H), 7.40–7.27 (m, 10H), 7.26–6.85 (m, 23H), 5.39 (s,
1H), 5.34 (d, J ¼ 3.8 Hz, 1H), 5.31 (d, J ¼ 3.5 Hz, 1H), 4.97 (d,
J ¼ 11.6 Hz, 1H), 4.95 (d, J ¼ 11.2 Hz, 1H), 4.93 (d, J ¼ 11.0 Hz, 1H),
4.80–4.10 (m, 15H), 3.77–3.47 (m, 5H), 3.26–3.07 (m, 2H), 2.53–2.38
(m, 2H) ppm. 13 C NMR (100 MHz, benzene-d6): d 156.1 (C), 139.6
(C), 139.3 (C), 138.9 (C), 138.6 (C), 138.4 (C), 138.4 (C), 129.0 (CH),
128.9 (CH), 128.8 (CH), 128.7 (CH), 128.7 (CH), 128.5 (CH), 128.5
(CH), 128.4 (CH), 128.4 (CH), 128.3 (CH), 128.0 (CH), 128.0 (CH),
128.0 (CH), 127.8 (CH), 127.5 (CH), 127.5 (CH), 126.7 (CH), 126.6
(CH), 101.7 (CH), 94.6 (CH), 93.6 (CH), 82.9 (CH), 82.3 (CH), 80.1
(CH), 79.4 (CH), 79.3 (CH), 78.1 (CH), 75.5 (CH2), 75.3 (CH2), 75.0
(CH2), 74.1 (CH2), 73.4 (CH2), 70.3 (CH), 69.3 (CH2), 63.5 (CH2), 63.5
(CH), 42.5 (CH2), 36.3 (CH2) ppm. IR (HATR): 3420 (w), 3060 (w),
3030 (w), 2932 (w), 2866 (w), 1722 (m), 1514 (w), 1496 (w), 1454
(m), 1391 (w), 1367 (w), 1329 (w), 1237 (w), 1210 (w), 1150 (w),
1137 (w), 1086 (s), 1072 (s), 984 (s), 913 (w), 874 (w), 849 (w), 824
(w), 798 (m), 734 (w), 695 (s), 656 (w) cmꢀ1. ESMS [m/z (fragment,
(w), 806 (w), 775 (w), 715 (w), 608 (w) cmꢀ1. [a]D þ222ꢂ (c 0.10,
22
MeOH). ESMS [m/z (fragment, intensity), API-ES negative modeþ]:
486.2 (M þ OAcꢀ, 100). HRMS (ESI-TOF): calcd. for C16H30NO12
[M þ H]þ 428.1763; found 428.1772.
6-O-(N-Cycloheylcarbamoyl)-a,a-D-trehalose (19-c)
General procedure C was applied. Purification via column chroma-
tography (gradient elution: CH2Cl2/MeOH 9/1 to 6/4) afforded the
title compound 19-c as a white solid (170 mg, 0.364 mmol, 88%).
Rf 0.15 in CH2Cl2/MeOH 8/2. 1H NMR (400 MHz, CD3OD): d
5.12–5.03 (m, 2H), 4.40–4.12 (m, 2H), 3.98 (br d, J ¼ 9.2 Hz, 1H),
3.85–3.73 (m, 4H), 3.66 (dd, J ¼ 12.0/5.6 Hz, 1H), 3.46 (dd, J ¼ 9.8/
3.7 Hz, 2H), 3.40–3.26 (m, 3H), 1.86 (app br d, J ¼ 11.0 Hz, 2H),
1.80–1.68 (m, 2H), 1.66–1.56 (m, 1H), 1.33 (app qt, J ¼ 12.5/3.1 Hz,
2H), 1.25–1.09 (m, 3H), ppm. 13 C NMR (100 MHz, CD3OD): d 158.3
(C), 95.2 (CH), 95.1 (CH), 74.6 (CH), 74.4 (CH), 73.9 (CH), 73.2 (CH),
73.2 (CH), 71.9 (CH), 71.8 (CH), 64.8 (CH2), 62.6 (CH2), 51.3 (CH),
34.2 (CH2), 26.6 (CH2), 26.2 (CH2) ppm. IR (HATR): 3293 (br, m),
2928 (m), 2858 (w), 1694 (m), 1538 (m), 1453 (m), 1418 (w), 1368
(w), 1317 (m), 1275 (m), 1253 (m), 1237 (m), 1145 (m), 1101 (m),
1076 (m), 1030 (s), 987 (s), 941 (m), 807 (w), 777 (w), 725 (m)
intensity), API-ES positive mode]: 1045.4 (M þ NH4þ, 100). HRMS
þ
(ESI-TOF): calcd. for C63H69N2O12
found 1045.4829.
[M þ NH4]þ 1045.4845;
2,3,4,2’,3’-Penta-O-benzyl-4’,6’-O-benzylidene-6-O-(N-n-octylcarba-
moyl)-a,a-D-trehalose (18-g)
General procedure B was applied (use of n-octyl isocyanate).
Purification via column chromatography (gradient elution: hexane/
EtOAc 8/2 to 7/3) afforded the partially purified title compound
18-g as a white foam (494 mg), which was used as such in the
next step.
cmꢀ1. [a]D þ166ꢂ (c 0.10, MeOH). ESMS [m/z (fragment, inten-
22
sity), API-ES negative mode]: 933.3 (2 M-Hþ, 44), 526.2 (M þ OAcꢀ,
þ
Rf 0.52 in hexane/EtOAc 6/4. HRMS (ESI-TOF): calcd. for 100). HRMS (ESI-TOF): calcd. for C19H33NNaO12
[M þ Na]þ
þ
C63H77N2O12 [M þ NH4]þ 1053.5471; found 1053.5472.
490.1895; found 490.1889.

1972
S. DHAENE ET AL.
þ
6-O-(N-Phenylcarbamoyl)-a,a-D-trehalose (19-d)
General procedure C was applied. Purification via column chroma- 490.1919; found 490.1922.
tography (gradient elution: CH2Cl2/MeOH 8/2 to 7/3) afforded the
(M þ OAc-, 100). HRMS (ESI-TOF): calcd. for C21H32NO12 [M þ H]þ
title compound 19-d as a white solid (41.0 mg, 0.089 mmol, 57%).
Rf 0.59 in CH3CN/H2O 8/2, 0.15 in CH2Cl2/MeOH 9/1. 1H NMR
(400 MHz, CD3OD): d 7.41 (d, J ¼ 7.9 Hz, 2H), 7.25 (t, J ¼ 8.0 Hz, 2H),
7.00 (t, J ¼ 7.4 Hz, 1H), 5.12 (app d, J ¼ 3.8 Hz, 2H), 4.37 (dd,
J ¼ 11.7/1.9 Hz, 1H), 4.31 (dd, J ¼ 11.7/4.9 Hz, 1H), 4.06 (ddd,
J ¼ 10.0/4.8/2.3 Hz, 1H), 3.87–3.72 (m, 4H), 3.66 (dd, J ¼ 11.8/5.3 Hz,
1H), 3.54–3.28 (m, 6H) ppm. 13 C NMR (100 MHz, CD3OD): d 156.1
(C), 140.1 (C), 129.8 (CH), 124.1 (CH), 119.9 (CH), 95.3 (CH), 95.2
(CH), 74.6 (CH), 74.4 (CH), 73.9 (CH), 73.2 (CH), 73.2 (CH), 71.9 (CH),
71.8 (CH), 71.7 (CH), 65.0 (CH2), 62.6 (CH2) ppm. IR (HATR): 3302
6-O-(N-n-Octylcarbamoyl)-a,a-D-trehalose (19-g)
General procedure C was applied. Purification via column chroma-
tography (gradient elution: CH2Cl2/MeOH 8/2 to 7/3) afforded the
title compound 19-g as a white solid (191 mg, 0.179 mmol, 84%
over 2 steps).
Rf 0.50 in CH2Cl2/MeOH 6/4. 1H NMR (400 MHz, CD3OD): d
5.14–5.03 (m, 2H), 4.30–4.14 (m, 2H), 3.99 (br d, J ¼ 7.8 Hz, 1H),
3.87–3.74 (m, 4H), 3.67 (br dd, J ¼ 11.8/5.2 Hz, 1H), 3.55–3.43 (m,
2H), 3.39–3.26 (m, 2H), 3.07 (app br t, J ¼ 6.7 Hz, 2H), 1.55–1.20 (m,
(br, m), 2970 (w), 2932 (w), 1694 (m), 1684 (m), 1540 (w), 1455 (w), 12H), 0.95–0.83 (m, 3H) ppm. 13 C NMR (100 MHz, CD3OD): d 159.1
1447 (w), 1419 (w), 1388 (w), 1368 (w), 1322 (w), 1251 (w), 1147
(w), 1100 (m), 1078 (m), 1039 (m), 1025 (m), 985 (s), 941 (w), 844
(C), 95.2 (CH), 95.1 (CH), 74.5 (CH), 74.3 (CH), 73.8 (CH), 73.2 (CH),
73.1 (CH), 71.9 (CH), 71.7 (CH), 64.8 (CH2), 62.6 (CH2), 41.8 (CH2),
33.0 (CH2), 30.9 (CH2), 30.4 (CH2), 30.4 (CH2), 27.9 (CH2), 23.7 (CH2),
14.4 (CH3) ppm. IR (HATR): 3314 (m, br), 2924 (m), 2857 (w), 1693
(m), 1539 (m), 1456 (w), 1371 (w), 1334 (w), 1260 (m), 1147 (m),
1106 (m), 1076 (m), 1025 (m), 986 (s), 941 (m), 912 (w), 853 (w),
(w), 806 (w), 774 (w), 751 (w), 720 (w), 695 (w) cmꢀ1. [a]D þ152ꢂ
22
(c 0.10, MeOH). ESMS [m/z (fragment, intensity), API-ES negativþe
mode]: 460.2 (M-Hþ, 100). HRMS (ESI-TOF): calcd. for C19H31N2O12
[M þ NH4]þ 479.1872; found 479.1867.
22
805 (w), 777 (w) cmꢀ1. [a]D þ171ꢂ (c 0.10, MeOH). ESMS [m/z
(fragment, intensity), API-ES negative mode]: 993.4 (2 M-Hþ, 45þ),
556.2 (M þ OAc-, 100). HRMS (ESI-TOF): calcd. for C21H39NNaO12
[M þ Na]þ 520.2365; found 520.2355.
6-O-(N-Benzylcarbamoyl)-a,a-D-trehalose (19-e)
General procedure C was applied. Purification via column chroma-
tography (gradient elution: CH2Cl2/MeOH 8/2 to 6/4) afforded the
title compound 19-e as a white solid (200 mg, 0.421 mmol, 74%
over 2 steps).
2,3,6,2’,3’-Penta-O-benzyl-4’,6’-O-benzylidene-4-O-methyl-a,a-D-tre-
Rf 0.33 in CH3CN/H2O 8/2, 0.09 in CH2Cl2/MeOH 8/2. 1H NMR
(400 MHz, CD3OD): d 7.36–7.17 (m, 5H), 5.09 (d, J ¼ 3.4 Hz, 1H),
5.07 (d, J ¼ 3.4 Hz, 1H), 4.30 (br dd, J ¼ 12.0/1.7 Hz, 1H), 4.28 (s,
2H), 4.22 (dd, J ¼ 11.7/5.0 Hz, 1H), 4.00 (br ddd, J ¼ 9.9/4.8/1.9 Hz,
1H), 3.85–3.72 (m, 4H), 3.66 (dd, J ¼ 12.0/5.6 Hz, 1H), 3.51–3.41 (m,
2H), 3.38–3.27 (m, 2H) ppm. 13 C NMR (100 MHz, CD3OD): d 159.3
(C), 140.6 (C), 129.5 (CH), 128.3 (CH), 128.1 (CH), 95.2 (CH), 95.1
(CH), 74.6 (CH), 74.4 (CH), 73.9 (CH), 73.2 (CH), 73.2 (CH), 71.9 (CH),
71.8 (CH), 65.1 (CH2), 62.6 (CH2), 45.5 (CH2) ppm. IR (HATR): 3311
(m, br), 2930 (w), 1688 (m), 1540 (m), 1454 (w), 1433 (w), 1364 (w),
1338 (w), 1258 (m), 1147 (m), 1102 (m), 1076 (m), 1026 (m), 985
(s), 941 (m), 848 (w), 805 (w), 777 (w), 736 (w), 696 (w), 610 (w)
halose (20-a)
General procedure
Purification via column chromatography (gradient elution: hexane/
EtOAc 9/1 to 8/2 containing 1% Et3N) afforded the title compound
20-a as a colourless glass (194 mg, 0.217 mmol, 95%).
A
was applied (use of iodomethane).
1
Rf 0.39 in hexane/EtOAc 7/3. H NMR (400 MHz, benzene-d6): d
7.63–7.56 (m, 2H), 7.39–7.28 (m, 10H), 7.24–7.02 (m, 18H), 5.39 (d,
J ¼ 3.8 Hz, 1H), 5.37 (s, 1H), 5.32 (d, J ¼ 3.5 Hz, 1H), 4.94 (d,
J ¼ 11.6 Hz, 1H), 4.92 (d, J ¼ 11.4 Hz, 1H), 4.80 (d, J ¼ 11.6 Hz, 1H),
4.76 (d, J ¼ 11.3 Hz, 1H), 4.70 (d, J ¼ 11.6 Hz, 1H), 4.69 (d,
J ¼ 12.1 Hz, 1H), 4.59–4.43 (m, 6H), 4.37 (app t, J ¼ 9.3 Hz, 1H), 4.28
(app t, J ¼ 9.3 Hz, 1H), 4.18 (dd, J ¼ 10.1/4.9 Hz, 1H), 3.72–3.48 (m,
7H), 3.45 (s, 3H) ppm. 13 C NMR (100 MHz, benzene-d6): d 140.2 (C),
140.1 (C), 139.5 (C), 139.3 (C), 139.1 (C), 138.9 (C), 129.3 (CH), 129.1
(CH), 129.0 (CH), 128.9 (CH), 128.8 (CH), 128.7 (CH), 127.8 (CH),
127.8 (CH), 127.1 (CH), 102.0 (CH), 94.8 (CH), 94.1 (CH), 83.3 (CH),
82.7 (CH), 80.6 (CH), 80.6 (CH), 79.7 (CH), 79.6 (CH), 75.7 (CH2), 75.6
(CH2), 74.1 (CH2), 74.0 (CH2), 74.0 (CH2), 72.1 (CH), 70.0 (CH2), 69.6
(CH2), 63.8 (CH), 60.7 (CH) ppm. IR (HATR): 3032 (w), 2921 (m),
2859 (w), 1728 (w), 1496 (w), 1453 (m), 1369 (m), 1331 (w), 1262
(w), 1208 (w), 1154 (m), 1138 (m), 1086 (s), 1049 (m), 986 (s), 913
(w), 797 (w), 733 (m), 695 (s), 656 (w) cmꢀ1. ESMS [m/z (fragment,
22
cmꢀ1. [a]D þ140ꢂ (c 0.10, MeOH). ESMS [m/z (fragment, inten-
sity), API-ES negative mode]: 949.2 (2 M-Hþ, 47), 534.2 (M þ OAc-,
þ
100). HRMS (ESI-TOF): calcd. for C20H29NNaO12
498.1582; found 498.1579.
[M þ Na]þ
6-O-[N-(2-Phenethyl)carbamoyl]-a,a-D-trehalose (19-f)
General procedure C was applied. Purification via column chroma-
tography (gradient elution: CH2Cl2/MeOH 8/2 to 7/3) afforded the
title compound 19-f as a white solid (85.8 mg, 0.175 mmol, 90%).
Rf 0.47 in CH3CN/H2O 8/2, 0.35 in CH2Cl2/MeOH 7/3. 1H NMR
(400 MHz, CD3OD): d 7.31–7.13 (m, 5H), 5.08 (d, J ¼ 3.9 Hz, 1H),
5.07 (d, J ¼ 3.9 Hz, 1H), 4.27 (dd, J ¼ 11.7/1.7 Hz, 1H), 4.20 (dd,
J ¼ 11.8/4.9 Hz, 1H), 3.98 (ddd, J ¼ 9.9/4.5/1.8 Hz, 1H), 3.86–3.73 (m,
4H), 3.67 (dd, J ¼ 12.1/5.6 Hz, 1H), 3.51–3.42 (m, 2H), 338–3.24 (m,
4H), 2.77 (t, J ¼ 7.4 Hz, 2H) ppm. 13 C NMR (100 MHz, CD3OD): d
159.1 (C), 140.6 (C), 129.8 (CH), 129.5 (CH), 127.3 (CH), 95.2 (CH),
95.1 (CH), 74.6 (CH), 74.4 (CH), 73.9 (CH), 73.2 (CH), 73.2 (CH), 71.9
(CH), 71.8 (CH), 71.8 (CH), 64.8 (CH2), 62.6 (CH2), 43.5 (CH2), 37.1
(CH2) ppm. IR (HATR): 3306 (br, m), 3026 (w), 2931 (w), 1694 (m),
1540 (w), 1497 (w), 1454 (w), 1436 (w), 1419 (w), 1363 (w), 1332
(w), 1254 (m), 1204 (w), 1147 (m), 1103 (m), 1075 (m), 1047 (m),
1026 (m), 985 (s), 941 (m), 847 (w), 806 (w), 773 (w), 748 (w), 698
intensity), API-ES positive mode]: 912.3 (M þ NH4þ, 100). HRMS
þ
(ESI-TOF): calcd. for C55H62NO11
found 912.4333.
[M þ NH4]þ 912.4317;
2,3,6,2’,3’-Penta-O-benzyl-4’,6’-O-benzylidene-4-O-ethyl-a,a-D-tre-
halose (20-b)
General procedure A was applied (use of iodoethane). Purification
via column chromatography (gradient elution: hexane/EtOAc 9/1
to 8/2 containing 1% Et3N) afforded the title compound 20-b as a
colourless glass (195 mg, 0.215 mmol, 83%).
Rf 0.35 in hexane/EtOAc7/3. 1H NMR (400 MHz, benzene-d6): d
7.62–7.56 (m, 2H), 7.39–7.27 (m, 10H), 7.23–7.01 (m, 18H), 5.40 (d,
(w), 667 (w) cmꢀ1. [a]D þ152ꢂ (c 0.10, MeOH). ESMS [m/z (frag-
22
ment, intensity), API-ES negative mode]: 977.3 (2 M-Hþ, 60), 548.2 J ¼ 3.7 Hz, 1H), 5.37 (s, 1H), 5.32 (d, J ¼ 3.7 Hz, 1H), 4.97–4.89 (m,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1973
2H), 4.81 (d, J ¼ 11.4 Hz, 1H), 4.76 (d, J ¼ 12.1 Hz, 1H), 4.70 (d, J ¼ 6.2 Hz, 1H), 3.89 (app t, J ¼ 6.2 Hz, 1H), 3.75–3.48 (m, 8H),
J ¼ 11.6 Hz, 1H), 4.68 (d, J ¼ 12.1 Hz, 1H), 4.61–4.40 (m, 7H), 4.37 1.58–1.20 (m, 2H), 0.84 (t, J ¼ 7.3 Hz, 3H) ppm. 13 C NMR (100 MHz,
(app t, J ¼ 9.2 Hz, 1H), 4.28 (app t, J ¼ 9.3 Hz, 1H), 4.18 (dd, CDCl3): d 139.9 (C), 139.8 (C), 139.1 (C), 139.0 (C), 138.7 (C), 138.5
J ¼ 10.3/4.9 Hz, 1H), 3.94–3.82 (m, 1H), 3.73–3.48 (m, 8H), 1.08 (t, (C), 128.9 (CH), 128.7 (CH), 128.6 (CH), 128.6 (CH), 128.4 (CH), 128.3
J ¼ 7.0 Hz, 3H) ppm. 13 C NMR (100 MHz, benzene-d6): d 140.3 (C), (CH), 128.0 (CH), 127.9 (CH), 127.7 (CH), 127.4 (CH), 127.4 (CH),
140.2 (C), 139.5 (C), 139.4 (C), 139.1 (C), 138.9 (C), 129.3 (CH), 129.1 126.8 (CH), 101.6 (CH), 94.4 (CH), 93.7 (CH), 83.0 (CH), 82.4 (CH),
(CH), 129.0 (CH), 128.9 (CH), 128.8 (CH), 128.7 (CH), 128.3 (CH), 80.3 (CH), 79.3 (CH), 78.6 (CH), 75.2 (CH2), 73.8 (CH2), 73.7 (CH2),
128.3 (CH), 128.1 (CH), 127.8 (CH), 127.1 (CH), 102.0 (CH), 94.8 72.8 (CH2), 71.9 (CH), 69.5 (CH2), 69.3 (CH2), 63.4 (CH), 33.0 (CH2),
(CH), 94.1 (CH), 83.3 (CH), 82.7 (CH), 80.6 (CH), 79.7 (CH), 79.6 (CH), 19.7 (CH2), 14.2 (CH) ppm. IR (HATR): 3064 (w), 3030 (w), 2930 (w),
79.0 (CH), 75.8 CH2), 75.6 (CH2), 74.1 (CH2), 74.1 (CH2), 74.0 (CH2), 2862 (w), 1496 (w), 1453 (m), 1366 (m), 1329 (w), 1208 (w), 1135
72.2 (CH), 69.9 (CH2), 68.7 (CH2), 63.8 (CH), 16.5 (CH3) ppm. IR (m), 1086 (s), 1049 (m), 985 (s), 913 (m), 796 (w), 733 (m), 695 (s),
(HATR): 3064 (w), 3030 (w), 2968 (w), 2923 (w), 2863 (w), 1496 (w), 657 (m) cmꢀ1. ESMS [mþ/z (fragment, intensity), API-ES positive
1453 (m), 1370 (m), 1329 (w), 1208 (w), 1151 (m), 1135 (m), 1085 mode]: 95þ4.4 (M þ NH4
, 100). HRMS (ESI-TOF): calcd. for
(s), 1076 (s), 1049 (m), 1013 (m), 984 (s), 915 (w), 877 (w), 796 (w), C58H68NO11 [M þ NH4]þ 954.4787; found 954.4788.
733 (m), 695 (s), 656 (w), 632 (w) cmꢀ1. ESMS [m/z (fragment,
intensity), API-ES positive mode]: 926.4 (M þ NH4þ, 100). HRMS
þ
2,3,6,2’,3’-Penta-O-benzyl-4’,6’-O-benzylidene-4-O-(2-methylallyl)-
a,a-D-trehalose (20-e)
(ESI-TOF): calcd. for C56H64NO11
found 926.4491.
[M þ NH4]þ 926.4474;
General procedure A was applied (use of 3-bromo-2-methylpro-
pene, “methallyl bromide”). Purification via column chromatog-
raphy (gradient elution: hexane/EtOAc 9/1 to 8/2 containing 1%
Et3N) afforded the title compound 20-e as a colourless glass
4-O-Allyl-2,3,6,2’,3’-penta-O-benzyl-4’,6’-O-benzylidene-a,a-D-tre-
halose (20-c)
General procedure
Purification via column chromatography (gradient elution: hexane/
EtOAc 9/1 to 75/25 containing 1% Et3N) afforded the title com-
A was applied (use of allyl bromide). (203 mg, 0.217 mmol, 88%).
1
Rf 0.07 in hexane/EtOAc 9/1. H NMR (400 MHz, benzene-d6): d
7.64–7.56 (m, 2H), 7.43–7.27 (m, 10H), 7.24–7.02 (m, 18H), 5.41 (d,
J ¼ 3.8 Hz, 1H), 5.38 (s, 1H), 5.33 (d, J ¼ 3.7 Hz, 1H), 5.10–5.03 (m,
pound 20-c as a colourless glass (179 mg, 0.194 mmol, 93%).
1
Rf 0.41 in hexane/EtOAc 7/3. H NMR (400 MHz, benzene-d6): d 1H), 4.94 (d, J ¼ 11.7 Hz, 1H), 4.93 (d, J ¼ 11.2 Hz, 1H), 4.86–4.81 (m,
7.63–7.57 (m, 2H), 7.40–7.27 (m, 10H), 7.24–7.01 (m, 18H), 5.85 1H), 4.78 (d, J ¼ 11.4 Hz, 1H), 4.77 (d, J ¼ 12.0 Hz, 1H), 4.70 (d,
(app ddt, J ¼ 17.2/10.5/5.3 Hz, 1H), 5.39 (d, J ¼ 3.7 Hz, 1H), 5.37 (s, J ¼ 11.7 Hz, 1H), 4.66 (d, J ¼ 12.0 Hz, 1H), 4.59–4.26 (m, 9H), 4.19
1H), 5.32 (d, J ¼ 3.7 Hz, 1H), 5.21 (app dq, J ¼ 17.2/1.7 Hz, 1H), 5.01 (dd, J ¼ 10.1/4.9 Hz, 1H), 4.03 (d, J ¼ 12.1 Hz, 1H), 3.78–3.48 (m, 7H),
(app dq, J ¼ 10.5/1.4 Hz, 1H), 4.94 (d, J ¼ 11.5 Hz, 1H), 4.92 (d, 1.61 (s, 3H) ppm. 13 C NMR (100 MHz, benzene-d6): d 143.0 (C),
J ¼ 11.1 Hz, 1H), 4.79 (d, J ¼ 11.2 Hz, 1H), 4.77 (d, J ¼ 12.1 Hz, 1H), 139.8 (C), 139.7 (C), 139.0 (C), 139.0 (C), 138.6 (C), 138.5 (C), 128.9
4.74–4.64 (m, 2H), 4.59–4.34 (m, 8H), 4.30 (app t, J ¼ 9.3 Hz, 1H), (CH), 128.7 (CH), 128.6 (CH), 128.4 (CH), 128.3 (CH), 128.0 (CH),
4.18 (dd, J ¼ 10.3/4.9 Hz, 1H), 4.14 (app ddt, J ¼ 12.9/5.3/1.5 Hz, 127.9 (CH), 127.8 (CH), 127.7 (CH), 127.4 (CH), 127.4 (CH), 126.8
1H), 3.78–3.47 (m, 7H) ppm. 13 C NMR (100 MHz, benzene-d6): d (CH), 111.4 (CH2), 101.6 (CH), 94.4 (CH), 93.7 (CH), 83.0 (CH), 82.4
139.8 (C), 139.7 (C), 139.0 (C), 139.0 (C), 138.7 (C), 138.5 (C), 135.8 (CH), 80.3 (CH), 79.4 (CH), 79.3 (CH), 78.5 (CH), 76.8 (CH2), 75.4
(CH), 128.9 (CH), 128.7 (CH), 128.6 (CH), 128.4 (CH), 128.3 (CH), (CH2), 75.2 (CH2), 73.9 (CH2), 73.6 (CH2), 71.7 (CH), 69.5 (CH2), 69.3
128.0 (CH), 127.9 (CH), 127.9 (CH), 127.9 (CH), 127.8 (CH), 127.7 (CH2), 63.4 (CH), 19.7 (CH3) ppm. IR (HATR): 3085 (w), 3065 (w),
(CH), 127.4 (CH), 126.8 (CH), 115.7 (CH2), 101.6 (CH), 94.4 (CH), 93.7 3030 (w), 2930 (w), 2862 (w), 1496 (w), 1453 (m), 1366 (m), 1328
(CH), 83.0 (CH), 82.3 (CH), 80.2 (CH), 79.3 (CH), 78.3 (CH), 75.4 (w), 1208 (w), 1155 (m), 1137 (m), 1085 (s), 1075 (s), 1049 (m), 984
(CH2), 75.2 (CH2), 73.8 (CH2), 73.7 (CH2), 73.7 (CH2), 73.6 (CH2), 71.7 (s), 907 (m), 796 (w), 733 (m), 695 (s), 656 (w), 631 (w) cmꢀ1. ESMS
þ
(CH), 69.5 (CH2), 69.3 (CH2), 63.4 (CH) ppm. IR (HATR): 3064 (w), [m/z (fragment, intensity), API-ES positive mode]: 952.4 (M þ NH4
,
3032 (w), 2927 (w), 2864 (w), 1496 (w), 1453 (m), 1367 (m), 1328 100). HRMS (ESI-TOF): calcd. for C58H66NO11 [M þ NH4]þ 952.4630;
(w), 1208 (w), 1135 (m), 1083 (s), 1072 (s), 985 (s), 927 (m), 875 found 952.4623.
(w), 796 (w), 733 (m), 695 (s), 656 (w) cmꢀ1. ESMS [m/z (fragment,
þ
intensity), API-ES positive mode]: 938.4 (M þ NH4þ, 100). HRMS
þ
[M þ H]þ
938.4474;
2,3,6,2’,3’-Penta-O-benzyl-4’,6’-O-benzylidene-4-O-(3,3-dimethy-
lallyl)-a,a-D-trehalose (20-f)
(ESI-TOF):
found 938.4488.
calcd.
for
C57H64NO11
General procedure A was applied (use of 1-bromo-3-methyl-2-
butene, “prenyl bromide”). Purification via column chromatog-
raphy (gradient elution: hexane/EtOAc 9/1 to 85/15 containing 1%
Et3N) afforded the title compound 20-f as a colourless glass
2,3,6,2’,3’-Penta-O-benzyl-4’,6’-O-benzylidene-4-O-n-butyl-a,a-D-tre-
halose (20-d)
General procedure A was applied (use of iodobutane). Purification (665 mg, 0.701 mmol, 78%).
1
Rf 0.38 in hexane/EtOAc 7/3. H NMR (400 MHz, benzene-d6): d
7.63–7.54 (m, 2H), 7.42–7.26 (m, 10H), 7.23–7.01 (m, 18H),
5.53–5.46 (m, 1H), 5.40 (d, J ¼ 3.8 Hz, 1H), 5.37 (s), 1H), 5.34 (d,
via column chromatography (gradient elution: hexane/EtOAc 9/1
to 8/2) afforded the title compound 20-d as a colourless glass
(205 mg, 0.219 mmol, 96%).
1
Rf 0.17 in hexane/EtOAc 8/2. H NMR (400 MHz, benzene-d6): d J ¼ 3.7 Hz, 1H), 5.00–4.83 (m, 3H), 4.80–4.66 (m, 3H), 4.59–4.44 (m,
7.63–7.56 (m, 2H), 7.40–7.28 (m, 10H), 7.24–7.00 (m, 18H), 5.41 (d, 7H), 4.42–4.25 (m, 3H), 4.18 (dd, J ¼ 10.2/4.9 Hz, 1H), 3.80–3.69 (m,
J ¼ 3.8 Hz, 1H), 5.38 (s, 1H), 5.34 (d, J ¼ 3.5 Hz, 1H), 4.95 (d, 3H), 3.62 (dd, J ¼ 7.3/3.7 Hz, 1H), 3.60 (dd, J ¼ 7.6/3.7 Hz, 1H), 3.54
J ¼ 11.6 Hz, 1H), 4.93 (d, J ¼ 11.7 Hz, 1H), 4.81 (d, J ¼ 11.4 Hz, 1H), (app t, J ¼ 9.5 Hz, 1H), 3.52 (app t, J ¼ 10.4 Hz, 1H), 1.59 (s, 3H),
4.76 (d, J ¼ 12.0 Hz, 1H), 4.69 (d, J ¼ 11.6 Hz, 1H), 4.68 (d, 1.45 (s, 3H) ppm. 13 C NMR (100 MHz, benzene-d6): d 140.3 (C),
J ¼ 12.1 Hz, 1H), 4.59–4.44 (m, 6H), 4.38 (app t, J ¼ 9.3 Hz, 1H), 4.28 140.2 (C), 139.5 (C), 139.4 (C), 139.1 (C), 138.9 (C), 135.5 (C), 129.3
(app t, J ¼ 9.3 Hz, 1H), 4.19 (dd, J ¼ 10.2/4.9 Hz, 1H), 3.92 (app t, (CH), 129.1 (CH), 129.0 (CH), 128.8 (CH), 128.7 (CH), 128.4 (CH),

1974
S. DHAENE ET AL.
128.3 (CH), 128.3 (CH), 128.2 (CH), 128.1 (CH), 127.8 (CH), 127.8
(CH), 127.1 (CH), 123.3 (CH), 102.0 (CH), 94.8 (CH), 94.1 (CH), 83.3
(CH), 82.9 (CH), 80.7 (CH), 79.7 (CH), 79.6 (CH), 78.3 (CH), 75.8
(CH2), 75.6 (CH2), 74.1 (CH2), 72.3 (CH), 70.0 (CH2), 69.7 (CH2), 63.8
(CH), 26.1 (CH3), 18.3 (CH3) ppm. IR (HATR): 3085 (w), 3059 (w),
3030 (w), 2930 (w), 2861 (w), 1496 (w), 1453 (m), 1366 (m), 1328
(w), 1279 (vw), 1208 (w), 1153 (m), 1137 (m), 1070 (s), 984 (s), 913
(m), 874 (vw), 840 (w), 796 (w), 732 (s), 695 (s), 656 (m), 631 (w)
cmꢀ1. ESMS [m/z (fragment, intensity), API-ES positive modeþ]:
966.4 (M þ NH4þ, 100). HRMS (ESI-TOF): calcd. for C59H68NO11
[M þ NH4]þ 966.4787; found 966.4791.
Rf 0.50 in hexane/EtOAc 1/1. 1H NMR (300 MHz, CDCl3): d
7.54–7.22 (m, 30H), 5.55 (s, 1H), 5.18–5.11 (m, 2H), 5.05–4.60 (m,
8H), 4.52 (app d, J ¼ 12.1 Hz, 1H, A-part of AB-system), 4.41 (app d,
J ¼ 12.1 Hz, 1H, B-part of AB-system), 4.31–3.98 (m, 4H), 3.92 (app
t, J ¼ 9.3 Hz, 1H), 3.83 (app dd, J ¼ 9.5/2.2 Hz, 1H), 3.72–3.28 (m,
9H), 2.94 (br s, OH), 2.29 (br s, OH), 1.10 (app br s, 3H), 1.02 (s,
ꢃ0.4H, minor diastereoisomer), 0.98 (s, ꢃ2.6H, major diastereoiso-
mer) ppm. 13 C NMR (75 MHz, CDCl3, only major diastereoisomer
visible): d 138.8 (C), 138.4 (C), 138.2 (C), 137.8 (C), 137.6 (C), 137.5
(C), 128.9 (CH), 128.5 (CH), 128.4 (CH), 128.3 (CH), 128.2 (CH), 128.0
(CH), 128.0 (CH), 127.9 (CH), 127.8 (CH), 127.8 (CH), 127.7 (CH),
127.6 (CH), 127.5 (CH), 126.1 (CH), 101.2 (CH), 95.0 (CH), 94.2 (CH),
82.4 (CH), 81.2 (CH), 79.5 (CH), 78.9 (CH), 78.7 (CH), 78.0 (CH), 76.2
2,3,6,2’,3’-Penta-O-benzyl-4’,6’-O-benzylidene-4-O-n-decyl-a,a-D-tre- (CH), 75.6 (CH2), 75.2 (CH2), 73.7 (CH2), 73.6 (CH2), 73.6 (CH2), 73.0
(CH2), 71.2 (C), 70.5 (CH), 69.0 (CH2), 68.4 (CH2), 63.0 (CH), 26.5
(CH3), 24.5 (CH3) ppm. IR (HATR): 3480 (w, br), 3086 (w), 3064 (w),
3027 (w), 2927 (w), 2863 (w), 1496 (w), 1453 (m), 1368 (m), 1331
(w), 1209 (w), 1135 (m), 1086 (s), 1072 (s), 1047 (m), 1011 (m), 985
(s), 915 (m), 877 (w), 797 (w), 734 (m), 695 (s), 656 (w) cmꢀ1. ESMS
[m/z (fragment, intensity), API-ES positive mode]: 1000.3
(M þ NH4þ, 100). ESMS [m/z (fragment, intensity), API-ES negative
mode]: 1041.3 (M þ OAc-, 100). HRMS (ESI-TOF): calcd. for
C59H70NO13 [M þ NH4]þ 1000.4842; found 1000.4865.
halose (20-g)
General procedure
Purification via column chromatography (gradient elution: hexane/
EtOAc 10/0 to 8/2 containing 1% Et3N) afforded the title com-
pound 20-g as a colourless glass (236 mg, 0.231 mmol, 93%).
A was applied (use of bromodecane).
1
Rf 0.07 in hexane/EtOAc 7/3. H NMR (400 MHz, benzene-d6): d
7.63–7.55 (m, 2H), 7.43–7.27 (m, 10H), 7.26–7.02 (m, 18H), 5.41 (d,
J ¼ 3.8 Hz, 1H), 5.37 (s, 1H), 5.34 (d, J ¼ 3.7 Hz, 1H), 4.97 (d,
J ¼ 11.5 Hz, 1H), 4.93 (d, J ¼ 11.6 Hz, 1H), 4.85 (d, J ¼ 11.5 Hz, 1H),
4.76 (d, J ¼ 12.0 Hz, 1H), 4.68 (app d, J ¼ 11.5 Hz, 2H), 4.60–4.44 (m,
6H), 4.38 (app t, J ¼ 9.3 Hz, 1H), 4.30 (app t, J ¼ 9.3 Hz, 1H), 4.18
(dd, J ¼ 10.1/4.9 Hz, 1H), 4.00–3.90 (m, 1H), 3.80–3.48 (m, 8H),
1.64–1.47 (m, 2H), 1.43–1.18 (m, 14H), 0.92 (t, J ¼ 6.8 Hz, 3H) ppm.
13 C NMR (100 MHz, benzene-d6): d 139.9 (C), 139.8 (C), 139.1 (C),
139.0 (C), 138.7 (C), 138.5 (C), 128.9 (CH), 128.7 (CH), 128.6 (CH),
128.6 (CH), 128.4 (CH), 128.3 (CH), 127.9 (CH), 127.9 (CH), 127.7
(CH), 127.6 (CH), 127.4 (CH), 127.4 (CH), 101.6 (CH), 94.4 (CH), 93.7
(CH), 83.0 (CH), 82.4 (CH), 80.3 (CH), 79.3 (CH), 79.3 (CH), 78.6 (CH),
75.4 (CH2), 75.2 (CH2), 73.7 (CH2), 73.7 (CH2), 73.7 (CH2), 73.2 (CH2),
71.9 (CH), 69.6 (CH2), 69.3 (CH2), 63.4 (CH), 32.3 (CH2), 31.0 (CH2),
30.1 (CH2), 30.1 (CH2), 30.0 (CH2), 29.8 (CH2), 26.7 (CH2), 23.1 (CH2),
14.3 (CH3) ppm. IR (HATR): 3085 (w), 3059 (w), 3030 (w), 2924 (m),
2854 (m), 1496 (w), 1453 (m), 1366 (m), 1328 (w), 1208 (w), 1153
(m), 1137 (m), 1087 (s), 1049 (m), 986 (s), 913 (w), 874 (w), 796
(w), 732 (m), 695 (s), 656 (w), 634 (w) cmꢀ1. ESMS [m/z (fragment,
4-O-Methyl-a,a-D-trehalose (21-a)
General procedure C was applied. As the product was poorly sol-
uble in MeOH, the celite layer was additionally washed with water
and the filtrate was lyophilised. The resulting solid was washed
with MeOH to afford the title compound 21-a as a white solid
(46.7 mg, 0.131 mmol, 80%).
Rf 0.30 in CH3CN/H2O 8/2, 0.35 in CH2Cl2/MeOH 6/4. 1H NMR
(400 MHz, D2O): d 5.14 (d, J ¼ 3.8 Hz, 1H), 5.14 (d, J ¼ 3.8 Hz, 1H),
3.89 (app t, J ¼ 9.6 Hz, 1H), 3.85–3.68 (m, 7H), 3.62 (dd, J ¼ 9.8/
3.9 Hz, 1H), 3.60 (dd, J ¼ 9.8/3.9 Hz, 1H), 3.54 (s, 3H), 3.40 (app t,
J ¼ 9.4 Hz, 1H), 3.22 (app t, J ¼ 9.4 Hz, 1H) ppm. 13 C NMR
(100 MHz, D2O): d 93.2 (CH), 93.1 (CH), 79.4 (CH), 72.5 (CH), 72.3
(CH), 72.1 (CH), 71.1 (CH), 71.0 (CH), 71.0 (CH), 69.7 (CH), 60.5
(CH2), 60.2 (CH2), 60.1 (CH3) ppm. IR (HATR): 3488 (w), 3478 (w),
3400 (br, m), 3300 (w), 3160 (w), 3012 (w), 2976 (w), 2938 (w),
2920 (w), 2874 (w), 1654 (w), 1594 (w), 1456 (m), 1414 (w), 1380
(w), 1354 (w), 1304 (w), 1288 (w), 1250 (w), 1206 (w), 1190 (w),
1148 (m), 1132 (m), 1116 (m), 1098 (m), 1072 (m), 1044 (m), 1022
(m), 980 (s), 964 (s), 938 (m), 904 (m), 868 (w), 854 (w), 828 (w),
intensity), API-ES positive mode]: 1038.5 (M þ NH4þ, 100). HRMS
[M þ NH4]þ 1038.5726;
þ
(ESI-TOF): calcd. for C64H80NO11
found 1038.5713.
804 (w), 770 (w), 726 (w), 710 (w), 698 (w), 654 (w) cmꢀ1. ESMS
2,3,6,2’,3’-Penta-O-benzyl-4’,6’-O-benzylidene-4-O-[(2S)-2,3-dihy-
droxy-3-methylbut-1-yl]-a,a-D-trehalose (20-h)
þ
[m/z (fragment, intensity), API-ES positive mode]: 374.1 (M þ NH4
,
100), 339.1 (M-H2O þ Hþ, 40), 177.1 (M-glucose þ Hþ, 85). HRMS
In a sealed tube AD-mix a (1.21 g), MeSO2NH2 (106 mg, 1.11 mmol,
2.5 eq) and (DHQ)2PHAL (95 mg, 0.122 mmol, 0.28 eq) were dis-
solved in H2O/tert-butanol (1/1, 18.4 ml) and the solution was
stirred for 30 min. The obtained yellow solution was cooled to
0 ꢂC and stirred for 10 min at 0 ꢂC. To this, a cooled (0 ꢂC) solution
of 20-f (418 mg, 0.440 mmol, 1 eq) in acetone (8.3 ml) was added
and the resulting mixture was stirred for 10 d at 0–5 ꢂC. An aque-
ous Na2SO3 solution (10% w/v, 3 ml) was added and the resulting
mixture was stirred for 2 h at 0 ꢂC after which it was transferred to
a separation funnel using H2O (10 ml) and EtOAc (30 ml). After
separation of the organic layer, the aqueous phase was extracted
with EtOAc (2 ꢁ 30 ml). The combined organic layers were washed
with brine (100 ml) and concentrated under reduced pressure. The
residue was purified using column chromatography (gradient elu-
tion: hexane/EtOAc 75/25 to 6/4 containing 1% Et3N) to give the
title compound 20-h as a colourless glass (240 mg, 0.244 mmol,
(ESI-TOF): calcd. for C13H24NaO11
found 379.1225.
[M þ Na]þ 379.1211;
þ
4-O-Ethyl-a,a-D-trehalose (21-b)
General procedure C was applied. Purification via column chroma-
tography (gradient elution: CH2Cl2/MeOH 8/2 to 7/3) afforded the
title compound 21-b as a white solid (59.9 mg, 0.162 mmol, 90%).
Rf 0.27 in CH3CN/H2O 8/2, 0.22 in CH2Cl2/MeOH 7/3. 1H NMR
(400 MHz, CD3OD): d 5.10 (d, J ¼ 3.8 Hz, 1H), 5.08 (d, J ¼ 3.7 Hz, 1H),
3.95–3.72 (m, 7H), 3.71–3.60 (m, 3H), 3.51–3.42 (m, 2H), 3.33–3.27
(m, 1H), 3.21 (dd, J ¼ 9.7/9.2 Hz, 1H), 1.17 (t, J ¼ 7.0 Hz, 3H) ppm.
13 C NMR (100 MHz, CD3OD): d 95.1 (CH), 95.0 (CH), 79.6 (CH), 74.6
(CH), 74.5 (CH), 73.8 (CH), 73.3 (CH), 73.2 (CH), 72.9 (CH), 71.9 (CH),
69.1 (CH2), 62.6 (CH2), 62.1 (CH2), 16.0 (CH3) ppm. IR (HATR): 3308
(br, m), 2970 (w), 2929 (w), 1440 (w), 1413 (w), 1374 (w), 1332 (w),
1248 (w), 1204 (w), 1145 (m), 1103 (m), 1030 (m), 984 (s), 941 (m),
1
55%, 73% de based on H NMR analysis).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1975
25
877 (w), 843 (w), 799 (w), 706 (w) cmꢀ1. [a]D þ164ꢂ (c 0.310, 4-O-Isopentyl-a,a-D-trehalose (21-f)
MeOH). ESMS [m/z (fragment, intensity), API-ES positive modeþ]: General procedure C was applied. Purification via column chroma-
388.1 (M þ Naþ, 100). HRMS (ESI-TOF): calcd. for C14H26NaO11
tography (gradient elution: CH2Cl2/MeOH 8/2 to 7/3) afforded the
[M þ Na]þ 393.1367; found 393.1365.
title compound 21-f as a white solid (27.2 mg, 0.066 mmol, 39%).
Rf 0.46 in CH3CN/H2O 8/2, 0.36 in CH2Cl2/MeOH 7/3. 1H NMR
(400 MHz, CD3OD): d 5.10 (d, J ¼ 3.8 Hz, 1H), 5.08 (d, J ¼ 3.8 Hz, 1H),
4-O-n-Propyl-a,a-D-trehalose (21-c)
General procedure C was applied. Purification via column chroma-
tography (gradient elution: CH2Cl2/MeOH 8/2 to 7/3) afforded the
3.95–3.71 (m, 7H), 3.71–3.57 (m, 3H), 3.51–3.42 (m, 2H), 3.36–3.27
(m, 1H), 3.19 (dd, J ¼ 9.8/9.1 Hz, 1H), 1.70 (app nonaplet, J ¼ 6.7 Hz,
1H), 1.56–1.35 (m, 2H), 0.91 (d, J ¼ 6.7 Hz, 3H), 0.90 (d, J ¼ 6.7 Hz,
3H) ppm. 13 C NMR (100 MHz, CD3OD): d 95.1 (CH), 95.0 (CH), 79.6
title compound 21-c as a white solid (57.3 mg, 0.149 mmol, 90%).
Rf 0.30 in CH3CN/H2O 8/2, 0.30 in CH2Cl2/MeOH 7/3. 1H NMR
(CH), 74.7 (CH), 74.5 (CH), 73.8 (CH), 73.4 (CH), 73.2 (CH), 72.9 (CH),
72.2 (CH2), 71.9 (CH), 62.6 (CH2), 62.2 (CH2), 40.4 (CH2), 26.0 (CH),
(400 MHz, CD3OD): d 5.10 (d, J ¼ 3.9 Hz, 1H), 5.09 (d, J ¼ 3.9 Hz, 1H),
3.92–3.72 (m, 7H), 3.71–3.62 (m, 2H), 3.58–3.43 (m, 3H), 3.36–3.28
23.2 (CH3), 22.9 (CH3) ppm. IR (HATR): 3327 (br, m), 2953 (w), 2927
(m, 1H), 3.20 (dd, J ¼ 9.8/9.2 Hz, 1H), 1.67–1.48 (m, 2H), 0.92 (t,
(w), 2875 (w), 1461 (w), 1419 (w), 1385 (w), 1366 (w), 1335 (w),
J ¼ 7.4 Hz, 3H) ppm. 13 C NMR (100 MHz, CD3OD): d 95.0 (CH), 79.6
1240 (w), 1204 (w), 1145 (w), 1103 (m), 1069 (m), 1030 (m), 986
(CH), 75.5 (CH2), 74.6 (CH), 74.5 (CH), 73.7 (CH), 73.4 (CH), 73.2
(s), 843 (w), 800 (w), 706 (w), 614 (w) cmꢀ1. [a]D þ177ꢂ (c 0.09,
25
(CH), 72.9 (CH), 71.8 (CH), 62.6 (CH2), 62.1 (CH2), 24.5 (CH2), 10.9
MeOH). ESMS [m/z (fragment, intensity), API-ES positive modeþ]:
(CH3) ppm. IR (HATR): 3326 (br, m), 2934 (w), 2880 (w), 1455 (w),
430.2 (M þ NH4þ, 100). HRMS (ESI-TOF): calcd. for C17H32NaO11
1416 (w), 1363 (w), 1257 (w), 1204 (w), 1145 (m), 1103 (m), 1072
[M þ Na]þ 435.1837; found 435.1841.
(m), 1030 (m), 987 (s), 843 (w), 802 (w), 706 (w), 617 (w) cmꢀ1
.
25
[a]D þ183ꢂ (c 0.557, MeOH). ESMS [m/z (fragment, intensity),
API-ES positive mode]: 402.2 (M þ NH4þ, 100). HRMS (ESI-TOF):
calcd. for C15H28NaO11 [M þ Na]þ 407.1524; found 407.1520.
þ
4-O-n-Decyl-a,a-D-trehalose (21-g)
General procedure C was applied. Purification via column chroma-
tography (gradient elution: CH2Cl2/MeOH 9/1 to 7/3) afforded the
4-O-n-Butyl-a,a-D-trehalose (21-d)
title compound 21-g as
a
colourless glass (98 mg,
General procedure C was applied. Purification via column chroma-
tography (gradient elution: CH2Cl2/MeOH 9/1 to 6/4) afforded the
title compound 21-d as a white solid (75 mg, 0.188 mmol, 100%).
Rf 0.40 in CH3CN/H2O 8/2, 0.14 in CH2Cl2/MeOH 8/2. 1H NMR
(300 MHz, CD3OD): d 5.09 (d, J ¼ 4.1 Hz, 1H), 5.08 (d, J ¼ 4.0 Hz, 1H),
3.94–3.50 (m, 10H), 3.50–3.40 (m, 2H), 3.35–3.25 (m, 1H), 3.19 (app
t, J ¼ 9.9 Hz, 1H), 1.64–1.26 (m, 4H), 0.92 (t, J ¼ 7.3 Hz, 3H) ppm.
13 C NMR (75 MHz, CD3OD): d 95.1 (CH), 95.0 (CH), 79.7 (CH), 74.7
(CH), 74.5 (CH), 73.8 (CH), 73.6 (CH2), 73.4 (CH), 73.3 (CH), 72.9
(CH), 71.9 (CH), 62.6 (CH2), 62.2 (CH2), 33.6 (CH2), 20.3 (CH2), 14.3
(CH3) ppm. IR (HATR): 3338 (m, br), 2934 (w), 2872 (w), 1455 (w,
br), 1361 (w, br), 1258 (w), 1236 (w), 1209 (w), 1145 (m), 1104 (m),
1074 (m), 1040 (m), 990 (s), 939 (w), 909 (w), 842 (w), 801 (w)
0.203 mmol, 98%).
1
Rf 0.23 in CH2Cl2/MeOH 8/2. H NMR (400 MHz, CD3OD): d 5.10
(d, J ¼ 3.8 Hz, 1H), 5.09 (d, J ¼ 3.8 Hz, 1H), 3.92–3.72 (m, 7H),
3.71–3.52 (m, 3H), 3.51–3.43 (m, 2H), 3.33–3.28 (m, 1H), 3.19 (dd,
J ¼ 9.8/9.0 Hz, 1H), 1.65–1.47 (m, 2H), 1.43–1.20 (m, 14H), 0.89 (t,
J ¼ 6.9 Hz, 3H) ppm. 13 C NMR (100 MHz, CD3OD): d 95.0 (CH), 94.9
(CH), 79.6 (CH), 74.6 (CH), 74.5 (CH), 73.9 (CH2), 73.8 (CH), 73.4
(CH), 73.2 (CH), 72.9 (CH), 71.9 (CH), 62.6 (CH2), 62.2 (CH2), 33.0
(CH2), 31.4 (CH2), 30.8 (CH2), 30.7 (CH2), 30.7 (CH2), 30.4 (CH2), 27.2
(CH2), 23.7 (CH2), 14.4 (CH3) ppm. IR (HATR): 3320 (m, br), 2922
(m), 2854 (w), 1650 (w), 1455 (w), 1370 (w), 1337 (w), 1145 (m),
1101 (m), 1076 (m), 1030 (s), 988 (s), 940 (w), 912 (w), 840 (w), 803
25
(w), 720 (w), 620 (w) cmꢀ1. [a]D þ142ꢂ (c 0.750, MeOH). ESMS
cmꢀ1. [a]D þ182ꢂ (c 0.10, MeOH). ESMS [m/z (fragment, inten-
22
þ
[m/z (fragment, intensity), API-ES positive mode]: 500.3 (M þ NH4
,
sity), API-ES positive mode]: 416.2 (M þ NH4þ, 100). HRMS (ESI-
TOF): calcd. for C16H30NaO11 [M þ Na]þ 421.1680; found 421.1677.
þ
100). HRMS (ESI-TOF): calcd. for C22H42NaO11 [M þ Na]þ 505.2619;
found 505.2620.
4-O-Isobutyl-a,a-D-trehalose (21-e)
General procedure C was applied. Purification via column chroma-
tography (gradient elution: CH2Cl2/MeOH 85/15 to 7/3) afforded
the title compound 21-e as
0.193 mmol, 100%).
Lentztrehalose A (2)
General procedure C was applied. Purification via column chroma-
tography (gradient elution: CH2Cl2/MeOH 8/2 to 6/4) afforded the
title compound 2 as a white solid (68 mg, 0.153 mmol, 100%).
Literature data is available
a
white solid (77 mg,
24,30
Rf 0.16 in CH2Cl2/MeOH 8/2. 1H NMR (400 MHz, CD3OD): d
5.12–5.07 (m, 2H), 3.93–3.73 (m, 6H), 3.73–3.63 (m, 3H), 3.51–3.43
(m, 2H), 3.35–3.28 (m, 2H), 3.19 (dd, J ¼ 9.9/9.0 Hz, 1H), 1.83 (app
nonaplet, J ¼ 6.6 Hz, 1H), 0.91 (d, J ¼ 7.0 Hz, 3H), 0.89 (d, J ¼ 7.0 Hz,
3H) ppm. 13 C NMR (100 MHz, CD3OD): d 95.0 (CH), 94.9 (CH), 80.7
(CH2), 79.6 (CH), 74.7 (CH), 74.5 (CH), 73.7 (CH), 73.4 (CH), 73.2
(CH), 72.9 (CH), 71.8 (CH), 62.6 (CH2), 62.2 (CH2), 30.2 (CH), 19.9
(CH3), 19.7 (CH3) ppm. IR (HATR): 3326 (br, m), 2951 (w), 2925 (m),
2873 (w), 1458 (w), 1414 (w), 1380 (w), 1365 (w), 1336 (w), 1259
(w), 1204 (w), 1145 (m), 1104 (m), 1031 (m), 983 (s), 951 (m), 841
.
Rf 0.17 in CH3CN/H2O 8/2, 0.32 in CH2Cl2/MeOH 6/4. 1H NMR
(300 MHz, CDCl3, two diastereoisomers): d 5.10 (app d, J ¼ 4.0 Hz,
1H), 5.08 (app d, J ¼ 3.8 Hz, 1H), 4.05–3.60 (m, 10H), 3.57–3.41 (m,
3H), 3.35–3.22 (m, 2H), 1.21–1.13 (m, 6H) ppm. 13 C NMR (75 MHz,
CDCl3, only major diastereoisomer visible): d 95.1 (CH), 95.0 (CH),
80.4 (CH), 78.2 (CH), 74.7 (CH2), 74.5 (CH), 74.4 (CH), 73.8 (CH), 73.4
(CH), 73.2 (CH), 72.8 (CH), 72.8 (C), 71.9 (CH), 62.6 (CH2), 62.2 (CH2),
26.5 (CH3), 25.4 (CH3) ppm. IR (HATR): 3309 (m, br), 2923 (w), 1362
(w, br), 1229 (w), 1145 (m), 1074 (m), 1033 (m), 985 (s), 934 (m),
855 (w), 843 (w), 800 (w) cmꢀ1. [a]D þ228ꢂ (c 0.10, MeOH). ESMS
22
25
(w), 802 (w), 732 (w), 706 (w), 613 (w) cmꢀ1. [a]D þ151ꢂ (c 0.77,
þ
[m/z (fragment, intensity), API-ES positive mode]: 462.2 (M þ NH4
,
MeOH). ESMS [m/z (fragment, intensity), API-ES positive modeþ]:
416.2 (M þ NH4þ, 100). HRMS (ESI-TOF): calcd. for C16H30NaO11
[M þ Na]þ 421.1680; found 421.1676.
100). HRMS (ESI-TOF): calcd. for C17H32NaO13 [M þ Na]þ 467.1735;
found 467.1734.

1976
S. DHAENE ET AL.
2,3,6,2’,3’-Penta-O-benzyl-4’,6’-O-benzylidene-4-O-(N-n-propylcar-
2,3,6,2’,3’-Penta-O-benzyl-4’,6’-O-benzylidene-4-O-(N-cyclohexylcar-
bamoyl)-a,a-D-trehalose (22-c)
bamoyl)-a,a-D-trehalose (22-a)
General procedure B was applied (use of cyclohexyl isocyanate).
Purification via column chromatography (gradient elution: hexane/
EtOAc 9/1 to 75/25) afforded the title compound 22-c as a colour-
less oil (499 mg, 88%).
General procedure B was applied (use of n-propyl isocyanate).
Purification via column chromatography (gradient elution: tolu-
ene/acetone 99/1 to 95/5) afforded the title compound 22-a as a
colourless oil (372 mg, 0.352 mmol, 68%).
Rf 0.31 in hexane/EtOAc 7/3. 1H NMR (400 MHz, CDCl3): d
7.37–7.17 (m, 33H), 7.16–7.10 (m, 2H), 5.29 (d, J ¼ 3.5 Hz, 1H), 5.23
(d, J ¼ 3.5 Hz, 1H), 4.99 (d, J ¼ 10.9 Hz, 1H), 4.91–4.83 (m, 2H), 4.81
(d, J ¼ 10.7 Hz, 1H), 4.77–4.67 (m, 3H), 4.65 (m, 2H), 4.62 (m, 1H),
4.57 (s, 1H), 4.54 (s, 1H), 4.53–3.35 (m, 4H), 4.21–4.14 (m, 2H), 4.04
(t, J ¼ 9.3 Hz, 1H), 3.96 (t, J ¼ 9.5 Hz, 1H), 3.68 (t, J ¼ 9.6 Hz, 1H),
3.62 (dt, J ¼ 9.7/3.7 Hz, 2H), 3.55 (dd, J ¼ 10.7/3.2 Hz, 1H), 3.42 (dt,
J ¼ 10.6/2.3 Hz, 3H), 3.36 (dd, J ¼ 10.7/5.6 Hz, 1H), 1.95–1.80 (m,
2H), 1.75–1.60 (m, 3H), 1.42–1.26 (m, 2H), 1.22– 1.00 (m, 3H) ppm.
13 C NMR (100 MHz, CDCl3): d 154.4 (C), 138.9 (C), 138.7 (C), 138.4
(C), 138.2 (C), 138.1 (C), 138.0 (C), 137.8 (C), 128.3 (CH), 128.3 (CH),
128.2 (CH), 128.2 (CH), 128.2 (CH), 128.0 (CH), 127.9 (CH), 127.9
(CH), 127.8 (CH), 127.7 (CH), 127.7 (CH), 127.6 (CH), 127.5 (CH),
127.5 (CH), 127.4 (CH), 127.4 (CH), 93.6 (CH), 93.5 (CH), 81.1 (CH),
79.2 (CH), 79.1 (CH), 78.9 (CH), 77.7 (CH), 75.5 (CH2), 75.0 (CH2),
74.9 (CH2), 73.7 (CH2), 73.5 (CH2), 73.0 (CH2), 72.4 (CH2), 71.2 (CH),
70.6 (CH), 69.8 (CH), 69.1 (CH2), 68.1 (CH2), 49.8 (CH), 33.4 (CH2),
33.3 (CH2), 25.5 (CH2), 24.7 (CH2) ppm. IR (HATR): 3410 (w), 3060
(w), 3032 (w), 2928 (w), 2854 (w), 1723 (m), 1496 (m), 1453 (m),
1361 (w), 1315 (w), 1271 (w), 1252 (w), 1212 (m), 1155 (m), 1140
(m), 1097 (s), 1059 (s), 1027 (s), 994 (s), 892 (w), 799 (w), 733 (s),
695 (s) cmꢀ1. ESMS [m/z (fragment, intensity), API-ES positive
mode]: 1098.4 (M þ Hþ, 10), 558.2 (4-O-cyclohexylcarbamoyl-2-O,3-
O,6-O-tribenzyþlglucosyl cation, 100). HRMS (ESI-TOF): calcd. for
C68H75NNaO12 [M þ Na]þ 1120.5181; found 1120.5143.
Rf 0.34 in hexane/EtOAc 7/3, 0.19 in toluene/acetone 95/5. 1H
NMR (400 MHz, CDCl3): d 7.36–7.09 (m, 35H), 5.26 (d, J ¼ 3.5 Hz,
1H),5.21 (d, J ¼ 3.5 Hz, 1H), 4.98 (d, J ¼ 11.0 Hz, 1H), 4.90–4.35 (m,
15H), 3.41 (d, J ¼ 1.7 Hz, 1H), 3.45–3.37 (m, 2H),0.35 (dd, J ¼ 10.6/
5.5 Hz, 1H), 3.08 (q, J ¼ 6.5 Hz, 2H), 1.49–1.37 (m, 2H), 0.86 (t,
J ¼ 7.3 Hz, 3H) ppm. 13 C NMR (100 MHz, CDCl3): d 155.3 (C), 138.9
(C), 138.7 (C), 138.4 (C), 138.2 (C), 138.1, 138.0 (C), 137.8 (C), 128.3
(CH), 128.2 (CH), 128.1 (CH), 128.0 (CH), 127.9 (CH), 127.8 (CH),
127.7 (CH), 127.7 (CH), 127.6 (CH), 127.5 (CH), 127.5 (CH), 127.4
(CH), 127.4 (CH), 93.6 (CH), 93.5 (CH), 81.8 (CH), 79.2 (CH), 78.9
(CH), 77.7 (CH), 75.5 (CH2), 75.0 (CH2), 74.9 (CH2), 73.6 (CH2), 73.5
(CH2), 73.0 (CH2), 72.4 (CH2), 71.3 (CH), 70.6 (CH), 69.8 (CH), 69.1
(CH2), 68.1 (CH2), 42.8 (CH2), 23.1 (CH2), 11.2 (CH3) ppm. IR (HATR):
3419 (w), 3331 (w), 3060 (w), 3030 (w), 2926 (w), 2870 (w), 1723
(m), 1514 (w), 1496 (m), 1453 (m), 1361 (m), 1332 (w), 1312 (w),
1263 (m), 1231 (m), 1212 (m), 1140 (m), 1097 (s), 1067 (s), 1025
(m), 993 (s), 917 (w), 848 (w), 801 (w), 733 (s), 695 (s) cmꢀ1. ESMS
[m/z (fragment, intensity), API-ES positive mode]: 1075.4
(M þ NH4þ, 42), 518.2 (4-O-n-propylcarbamoyl-2-O,3-O,6-O-triben-
þ
zylglucosyl cation, 100). HRMS (ESI-TOF): calcd. for C65H71NNaO12
[M þ Na]þ 1080.4869; found 1080.4847.
2,3,6,2’,3’-Penta-O-benzyl-4’,6’-O-benzylidene-4-O-(N-isopropylcar-
bamoyl)-a,a-D-trehalose (22-b)
General procedure B was applied (use of isopropyl isocyanate).
Purification via column chromatography (gradient elution: hexane/
EtOAc 9/1 to 75/25) afforded the title compound 22-b as a colour-
less glass (447 mg, 0.422 mmol, 82%).
2,3,6,2’,3’-Penta-O-benzyl-4’,6’-O-benzylidene-4-O-(N-phenylcarba-
moyl)-a,a-D-trehalose (22-d)
General procedure B was applied (use of phenyl isocyanate).
Purification via column chromatography (gradient elution: tolu-
ene/acetone 99/1 to 95/5) afforded the partially purified title com-
pound 22-d as a colourless oil which was used as such in the
next step.
Rf 0.31 in hexane/EtOAc 7/3. 1H NMR (400 MHz, CDCl3): d
7.38–7.19 (m, 33H), 7.16–7.11 (m, 2H), 5.29 (d, J ¼ 3.3 Hz, 1H), 5.23
(d, J ¼ 3.5 Hz, 1H), 5.00 (d, J ¼ 11.0 Hz, 1 H), 4.87 (dd, J ¼ 11.0/
2.4 Hz, 2H), 4.83 (d, J ¼ 10.9 Hz, 1H),) , 4.73 (s, 3H), 4.72–4.61 (m,
4H), 4.56 (d, J ¼ 12.1 Hz, 1H), 4.53–4.36 (m, 2H), 4.50 (d, J ¼ 2.1 Hz,
1H), 4.47 (s, 1H), 4.22–4.14 (m, 2H), 4.05 (t, J ¼ 9.5 Hz, 1H), 3.96 (t,
J ¼ 9.5 Hz, 1H), 3.8 (q, J ¼ 6.0 Hz, 1H), 3.69 (t, J ¼ 9.5 Hz, 1H), 3.63
(dt, J ¼ 9.7/3.3 Hz, 2H), 3.55 (dd, J ¼ 10.8/3.2 Hz, 1H), 3.50–3.40 (m,
2H), 3.36 (dd, J ¼ 10.7/5.6 Hz, 1H) 11.12 (s, 3H)) 1.09 (s, 3H) ppm.
13 C NMR (100 MHz, CDCl3): d 154.4 (C), 138.9 (C), 138.7 (C), 138.4
(C), 138.2 (C), 139.1 (C), 138.0 (C), 137.8 (C), 128.3 (CH), 128.3 (CH),
128.2 (CH), 128.2 (CH), 128.2 (CH), 128.0 (CH),127.9 (CH), 127.8
(CH), 127.8 (CH), 127.7 (CH), 127.6 (CH), 127.6 (CH), 127.5 (CH),
127.5 (CH), 127.4 (CH), 127.4 (CH), 127.4 (CH), 93.6 (CH), 93.5 (CH),
81.8 (CH), 79.2 (CH), 79.1 (CH), 79.0 (CH), 77.7 (CH), 75.5 (CH2), 75.0
(CH2), 74.9 (CH2), 73.7 (CH2), 73.5 (CH2), 73.0 (CH2), 72.4 (CH2), 71.1
(CH), 70.6 (CH), 69.8 (CH), 69.1 (CH2), 68.1 (CH2), 43.1 (CH), 23.0
(CH3), 22.9 (CH3) ppm. IR (HATR): 3410 (w), 3327 (w), 3059 (w),
3030 (w), 2972 (w), 2923 (w), 2868 (w), 1723 (m), 1496 (m), 1453
(m), 1387 (w), 1362 (m), 1322 (w), 1269 (w), 1230 (m), 1156 (m),
1135 (m), 1097 (m), 1068 (s), 1026 (m), 995 (s), 952 (m), 852 (w),
797 (w), 733 (s), 695 (s), 641 (m) cmꢀ1. ESMS [m/z (fragment,
intensity), API-ES positive mode]: 1075.4 (M þ NH4þ, 42), 518.2 (4-
O-isopropylcarbamoyl-2-O,3-O,6-O-tribenzyþlglucosyl cation, 100).
HRMS (ESI-TOF): calcd. for C65H71NNaO12 [M þ Na]þ 1080.4869;
found 1080.4847.
Rf 0.38 in hexane/EtOAc 7/3.
2,3,6,2’,3’-Penta-O-benzyl-4’,6’-O-benzylidene-4-O-(N-benzylcarba-
moyl)-a,a-D-trehalose (22-e)
General procedure B was applied (use of benzyl isocyanate).
Purification via column chromatography (gradient elution: tolu-
ene/acetone 100/0 to 97/3) afforded the title compound 22-e as a
colourless oil (283 mg, 0.256 mmol, 50%).
Rf 0.33 in hexane/EtOAc 7/3. 1H NMR (400 MHz, CDCl3): d
7.36–7.10 (m, 40H), 5.28 (d, J ¼ 3.5 Hz, 1H), 5.23 (d, J ¼ 3.4 Hz, 1H),
5.02–4.91 (m, 2H), 4.86 (d, J ¼ 10.4 Hz, 2H), 4.81–4.75 (m, 2H),
4.75–4.60 (m, 5H), 4.56 (d, J ¼ 12.1 Hz, 1H), 4.48 (q, J ¼ 12.8 Hz, 1H)
4.47 (m, 2H), 4.41 (d, J ¼ 12.1 Hz, 1H), 4.32 (m, 2H), 4.21–4.14 (m,
2H), 4.03 (t, J ¼ 9.3 Hz, 1H), 3.96 (t, J ¼ 9.3 Hz, 1H), 3.69 (t,
J ¼ 9.7 Hz, 1H), 3.63 (dd, J ¼ 9.7/3.5, 1H), 3.61 (dd, J ¼ 8.3/3.4 Hz,
1H), 3.55 (dd, J ¼ 10.7/3.3 Hz, 1H), 3.47–3.40 (m, 2H), 3.36 (dd,
J ¼ 10.4/5.0 Hz, 1H) ppm. 13 C NMR (100 MHz, CDCl3): d 155.3 (C),
138.9 (C), 138.7 (C), 138.4 (C),138.3 (C), 138.2 (C), 138.1 (C), 138.0
(C), 137.8 (C), 128.7 (CH), 128.3 (CH), 128.2 (CH), 128.2 (CH), 128.0
(CH),127.9 (CH), 127.8 (CH), 127.7 (CH), 127.7 (CH), 127.6 (CH),
127.6 (CH), 127.5 (CH), 127.5 (CH), 127.5 (CH), 127.4 (CH), 127.4
(CH), 93.6 (CH), 93.5 (CH), 81.8 (CH), 79.2 (CH), 79.0 (CH), 78.9 (CH),

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1977
77.7 (CH), 75.5 (CH2), 75.0 (CH2), 74.9 (CH2), 73.7 (CH2), 73.5 (CH2), 74.5 (CH), 73.8 (CH),73.5 (CH), 73.4 (CH),73.1 (CH), 72.4 (CH), 72.1
(CH), 71.9 (CH), 62.6 (CH2), 62.4 (CH2), 43.7 (CH2), 24.0 (CH2), 11.5
(CH3) ppm. IR (HATR): 3336 (m, br), 2938 (w), 1716 (m), 1522 (m),
1442 (w), 1361 (w), 1317 (w), 1267 (m), 1230 (m), 1144 (m), 1104
(m), 1086 (m), 1047 (m), 1025 (s), 991 (s), 961 (m), 922 (w), 897
73.0 (CH2), 72.5 (CH2), 71.6 (CH), 70.6 (CH), 68.7 (CH), 69.0 (CH2),
68.1 (CH2), 45.2 (CH2) ppm. IR (HATR): 3410 (w), 3332 (w), 3059
(w), 3031 (w), 2920 (w), 2868 (w), 1724 (m), 1510 (w), 1496 (w),
1453 (w), 1362 (w), 1247 (w), 1208 (w), 1154 (m), 1138 (m), 1097
(m), 1068 (m), 1026 (m), 995 (m), 850 (w), 797 (w), 734 (w), 695 (s),
645 (w) cmꢀ1. ESMS [m/z (fragment, intensity), API-ES positive
mode]: 1123.4 (M þ NH4þ, 95), 566.2 (4-O-benzylcarbamoyl-2-O,3-
O,6-O-tribenzyþlglucosyl cation, 100). HRMS (ESI-TOF): calcd. for
C69H71NNaO12 [M þ Na]þ 1128.4868; found 1128.4828.
(w), 837 (w), 810 (w), 766 (w), 720 (w) cmꢀ1. [a]D þ162ꢂ (c 0.10,
22
MeOH). ESMS [m/z (fragment, intensity), API-ES positive mode]:
428.1 (M þ Hþ, 100).
4-O-(N-Isopropylcarbamoyl)-a,a-D-trehalose (23-b)
General procedure C was applied. Purification via column chroma-
tography (gradient elution: CH2Cl2/MeOH 9/1 to 6/4) afforded the
title compound 23-b as a white foam (129 mg, 0.302 mmol, 76%).
Rf 0.59 in CH3CN/H2O 8/2, 0.12 in CH2Cl2/MeOH 8/2. 1H NMR
(400 MHz, CD3OD): d 5.14 (d, J ¼ 3.8 Hz, 1H), 5.11 (d, J ¼ 3.8 Hz, 1H),
4.53 (dd, J ¼ 10.4/9.5 Hz, 1H), 3.97–3.89 (m, 2H), 3.85–3.78 (m, 1H),
3.77 (m, 1H), 3.76–3.62 (m, 2H), 3.60–3.53 (m, 2H), 3.48 (dd,
J ¼ 9.8/3.8 Hz, 1H), 3.31 (d, J ¼ 9.5 Hz, 1H), 11.14 (s, 3H), 1.14 (s, 3H)
ppm. 13 C NMR (100 MHz, CD3OD): d 157.9 (C), 95.0 (CH), 94.8 (CH),
74.5 (CH), 73.8 (CH), 73.4 (CH), 73.1 (CH), 72.4 (CH), 72.1 (CH), 71.9
(CH), 62.6 (CH2), 62.4 (CH2), 44.2 (CH), 22.8 (CH) ppm. IR (HATR):
3301 (m, br), 2936 (w), 1694 (m), 1540 (w), 1457 (w), 1325 (w),
1256 (m), 1138 (m), 1108 (m), 1074 (m), 1031 (s), 992 (s), 946 (m),
2,3,6,2’,3’-Penta-O-benzyl-4’,6’-O-benzylidene-4-O-[N-(2-phenethyl)-
carbamoyl]-a,a-D-trehalose (22-f)
General procedure B was applied (use of phenethyl isocyanate).
Purification via column chromatography (gradient elution: hexane/
EtOAc 9/1 to 75/25) afforded the title compound 22-f as a white
foam (130 mg, 0.116 mmol, 57%).
1
Rf 0.28 in hexane/EtOAc. H NMR (400 MHz, CDCl3): d 7.35–7.05
(m, 40H), 5.25 (d, J ¼ 3.3 Hz, 1H), 5.20 (d, J ¼ 3.4 Hz, 1H), 5.00–4.35
(m, 16H), 4.20–4.10 (d, J ¼ 9.9 Hz, 2H), 4.01 (t, J ¼ 7.8 Hz, 1H), 3.91
(t, J ¼ 9.4 Hz, 1H), 3.67 (t, J ¼ 9.5 Hz, 1H), 3.62–3.56 (m, 2H), 3.52
(dd, J ¼ 10.7/3.4 Hz, 1H), 3.43–3.36 (m, 3H), 3.35–3.30 (m, 1H),
2.79–2.63 (m, 2H) ppm. 13 C NMR (100 MHz, CDCl3): d 155.2 (C),
138.9 (C), 138.7 (C), 138.6 (C), 138.4 (C), 138.2 (C), 138.0 (C), 138.0
(C), 137.8 (C), 128.7 (CH), 128.6 (CH), 128.3 (CH), 128.2 (CH), 128.2
(CH), 128.2 (CH), 128.0 (CH), 127.9 (CH), 127.8 (CH), 127.7 (CH),
127.7 (CH), 127.6 (CH), 127.6 (CH), 127.5 (CH), 127.5 (CH), 127.4
(CH), 127.4 (CH), 127.4 (CH), 126.5 (CH), 93.6 (CH), 93.5 (CH), 81.7
(CH), 79.1 (CH), 78.9 (CH), 77.6 (CH), 75.5 (CH2), 75.0 (CH2), 74.9
(CH2), 73.6 (CH2), 73.5 (CH2), 73.0 (CH2), 72.4 (CH2), 69.0 (CH2), 68.1
(CH2), 42.1 (CH2), 36.0 (CH2) ppm. IR (HATR): 3414 (w), 3336 (w),
3064 (w), 3028 (w), 2922 (w), 2868 (w), 1726 (m), 1496 (m), 1453
(m), 1394 (w), 1362 (m), 1331 (w), 1241 (m), 1208 (w), 1154 (m),
1140 (m), 1098 (s), 1066 (s), 1026 (s), 995 (s), 920 (w), 852 (w), 802
(w), 733 (s), 695 (s), 646 (w) cmꢀ1. ESMS [m/z (fragment, intensity),
API-ES positive mode]: 1137.4 (M þ NH4þ, 28), 580.2 (4-O-phene-
914 (w), 841 (w), 806 (w) cmꢀ1. [a]D þ190ꢂ (c 0.10, MeOH). ESMS
22
[m/z (fragment, intensity), API-ES positive mode]: 428.2
(M þ Hþ, 100).
4-O-(N-Cyclohexylcarbamoyl)-a,a-D-trehalose (23-c)
General procedure C was applied. Purification via column chroma-
tography (gradient elution: CH2Cl2/MeOH 9/1 to 6/4) afforded the
title compound 23-c as a white foam (116 mg, 0.248 mmol, 58%).
Rf 0.54 in CH3CN/H2O 8/2, 0.59 in CH2Cl2/MeOH 6/4. 1H NMR
(300 MHz, D2O): d 5.20 (d, J ¼ 4.5 Hz, 1H), 5.16 (d, J ¼ 3.8 Hz, 1H),
4.56 (t, J ¼ 9.6 Hz, 1H), 3.99 (t, J ¼ 9.8 Hz, 1H), 3.86–3.55 (m, 9H),
3.42 (t, J ¼ 9.2 Hz, 1H), 3.4 (s, 1H), 1.86–1.76 (m, 2H), 1.73–1.64 (m,
2H), 1.60–1.50 (m, 1H), 1.35–1.05 (m, 5H) ppm. 13 C NMR (75 MHz,
D2O): d 157.6 (C), 94.0 (CH), 93.8 (CH), 73.2 (CH), 72.9 (CH), 72.1
(CH), 71.7 (CH), 71.5 (CH), 71.3 (CH), 71.1 (CH), 70.3 (CH), 61.2
(CH2), 61.0 (CH2), 50.8 (CH), 33.0 (CH2), 25.6 (CH2), 25.0 (CH2) ppm.
IR (HATR): 3244 (m, br), 2943 (m), 2857 (w), 1678 (s), 1465 (w),
1447 (w), 1409 (m), 1374 (w), 1347 (w), 1329 (w), 1265 (w), 1133
(m), 1102 (m), 1076 (m), 1046 (s), 1037 (s), 1018 (s), 993 (s), 953
(m), 929 (w), 890 (w), 868 (w), 805 (w), 789 (w), 720 (w), 703 (w)
thylcarbamoyl-2-O,3-O,6-O-tribenzylglucosyl cation, 100). HRMS
þ
(ESI-TOF): calcd. for C70H73NNaO12
found 1142.4978.
[M þ Na]þ 1142.5025;
2,3,6,2’,3’-Penta-O-benzyl-4’,6’-O-benzylidene-4-O-(N-n-octylcarba-
moyl)-a,a-D-trehalose (22-g)
General procedure B was applied (use of n-octyl isocyanate).
Purification via column chromatography (gradient elution: hexane/
EtOAc 95/5 to 75/25) afforded the partially purified title com-
pound 22-g as a colourless oil which was used as such in the
next step.
cmꢀ1. [a]D þ171ꢂ (c 0.10, MeOH). ESMS [m/z (fragment, inten-
22
sity), API-ES positive mode]: 468.2 (M þ Hþ, 55), 288.1 (M-
glucose þ Hþ, 100).
4-O-(N-Phenylcarbamoyl)-a,a-D-trehalose (23-d)
Rf 0.45 in hexane/EtOAc 7/3.
General procedure C was applied. Purification via column chroma-
tography (gradient elution: CH2Cl2/MeOH 10/0 to 7/3) afforded
the title compound 23-d as a white foam (55 mg, 0.119 mmol,
16% over two steps).
4-O-(N-n-Propylcarbamoyl)-a,a-D-trehalose (23-a)
General procedure C was applied. Purification via column chroma-
tography (gradient elution: CH2Cl2/MeOH 10/0 to 7/3) afforded
1
Rf 0.46 in CH3CN/H2O 8/2. H NMR (400 MHz, D2O): d 4.41–7.35
the title compound 23-a as
0.314 mmol, 89%).
a
white foam (134 mg,
(m, 4H), 7.20–7.14 (m, 1H), 5.22 (d, J ¼ 3.9 Hz, 1H), 5.18 (d,
J ¼ 3.8 Hz, 1H), 4.72 (d, J ¼ 9.9 Hz, 1H), 4.08 (t, J ¼ 9.8 Hz, 1H),
4.06–3.97 (m, 1H), 3.87–3.79 (m, 3H), 3.78–3.70 (m, 3H), 3.68– 3.60
(m, 2H), 3.43 (t, J ¼ 9.5 Hz, 1H) ppm. 13 C NMR (100 MHz, D2O): d ):
155.7 (C), 137.9 (C), 130.0 (CH), 125.1 (CH), 120.8 (CH), 94.1 (CH),
93.9 (CH), 73.2 (CH), 72.9 (CH), 72.4 (CH), 71.7 (CH), 71.5 (CH), 71.3
(CH), 70.9 (CH), 70.3 (CH), 61.2 (CH2), 60.9 (CH2) ppm. IR (HATR):
Rf 0.43 in CH3CN/H2O 8/2. 1H NMR (400 MHz, CD3OD): d 5.14
(d, J ¼ 3.8 Hz, 1H), 5.11 (d, J ¼ 3.8 Hz, 1H), 4.53 (t, J ¼ 9.6 Hz, 1H),
3.98–3.90 (m, 2H), 3.85–3.74 (m, 3H), 3.67 (dd, J ¼ 12.0/5.5 Hz, 1H),
3.62–3.50 (m, 3H), 3.48 (dd, J ¼ 9.8/3.8 Hz, 1H), 3.32 (m, 1H), 3.07
(dt, J ¼ 7.0/1.5 Hz, 2H), 1.56–1.45 (m, 2H), 0.91 (t, J ¼ 7.5 Hz, 3H)
ppm. 13 C NMR (100 MHz, CD3OD): d 158.8 (C), 95.1 (CH), 94.8 (CH), 3279 (m, br), 2932 (w), 1702 (m), 1601 (m), 1542 (m), 1501 (w),

1978
S. DHAENE ET AL.
1445 (m), 1318 (m), 1231 (m), 1143 (w), 1108 (m), 1086 (m), 1027
(s), 992 (s), 939 (m), 914 (w), 838 (w), 805 (w), 755 (w), 723 (w),
690 (w) cmꢀ1. ESMS [m/z (fragment, intensity), API-ES positive
mode]: 282.1 (M-glucose þ Hþ, 100).
z
(fragment, intensity), API-ES negative mode]: 556.2
(M þ OAc-, 100).
2,3,4,2’,3’,4’-Hexa-O-benzyl-6,6’-di-O-methyl-a,a-D-trehalose (24)
General procedure
A
was applied (use of iodomethane).
Purification via column chromatography (eluent: hexane/EtOAc 7/
3) afforded the title compound 24 as a colourless glass (482 mg,
0.529 mmol, 99%).
4-O-(N-Benzylcarbamoyl)-a,a-D-trehalose (23-e)
General procedure C was applied. Purification via column chroma-
tography (gradient elution: CH2Cl2/MeOH 10/0 to 7/3) afforded
Rf 0.18 in hexane/EtOAc 7/3. 1H NMR (300 MHz, CDCl3): d
7.40–7.22 (m, 30H), 5.19 (d, J ¼ 3.6 Hz, 2H), 4.99 (d, J ¼ 10.9 Hz, 2H),
4.87 (d, J ¼ 11.1 Hz, 2H), 4.85 (d, J ¼ 10.9 Hz, 2H), 4.70 (d,
J ¼ 12.4 Hz, 2H, A-part of AB-system), 4.66 (d, J ¼ 12.2 Hz, 2H, B-
part of AB-system), 4.13 (app dt, J ¼ 10.0/2.1 Hz, 2H), 4.02 (app t,
J ¼ 9.3 Hz, 2H), 3.64 (app t, J ¼ 9.6 Hz, 2H), 3.56 (dd, J ¼ 9.7/3.7 Hz,
2H), 3.42 (dd, J ¼ 10.6/2.8 Hz, 2H), 3.32–3.24 (m, 2H), 3.26 (s, 6H)
ppm. 13 C NMR (100 MHz, CDCl3): d 138.9 (C), 138.4 (C), 138.2 (C),
128.4 (CH), 128.3 (CH), 128.0 (CH), 127.8 (CH), 127.7 (CH), 127.5
(CH), 127.5 (CH), 94.7 (CH), 81.7 (CH), 79.3 (CH), 77.6 (CH), 75.5
(CH2), 75.1 (CH2), 72.7 (CH2), 70.6 (CH2), 70.5 (CH), 59.1 (CH3) ppm.
IR (HATR): 3027 (w), 2918 (w), 2882 (w), 1727 (w), 1496 (w), 1453
(m), 1359 (m), 1326 (w), 1267 (w), 1198 (m), 1149 (m), 1135 (m),
1090 (s), 1067 (s), 1027 (m), 992 (s), 915 (m), 848 (w), 802 (w), 734
(m), 695 (s), 642 (w) cmꢀ1. ESMS [m/z (fragment, intensity), API-ES
positive mode]: 928.4 (M þ NH4þ, 100). HRMS (ESI-TOF): calcd. for
the title compound 23-e as
0.208 mmol, 87%).
a
white foam (99 mg,
Rf 0.44 in CH3CN/H2O 8/2. 1H NMR (400 MHz, CD3OD): d
7.32–7.27 (m, 4H), 7.25–7.19 (m, 1H), 5.14 (d, J ¼ 3.8 Hz, 1H), 5.11
(d, J ¼ 3.7 Hz, 1H), 4.58 (t, J ¼ 9.8 Hz, 1H),4.30 (s, 2H), 4.00–3.91 (m,
2H), 3.84–3.74 (m, 3H), 3.70–3.45 (m, 5H), 3.32 (m, 1H) ppm. 13 C
NMR (100 MHz, CD3OD): d 158.9 (C), 140.5 (C), 129.5 (CH), 128.3
(CH), 128.2 (CH), 95.1 (CH), 94.8 (CH), 74.5 (CH), 73.8 (CH), 73.8
(CH), 73.4 (CH), 73.1 (CH), 72.1 (CH), 71.9 (CH), 62.6 (CH2), 62.4
(CH2), 45.6 (CH2) ppm. IR (HATR): 3311 (m, br), 2931 (w), 1698 (m),
1539 (w), 1454 (w), 1339 (w), 1254 (m), 1207 (w), 1145 (m), 1104
(w), 1080 (m), 1028 (m), 989 (s), 944 (m), 841 (w), 802 (w), 747 (w),
22
720 (w), 698 (m) cmꢀ1. [a]D þ142ꢂ (c 0.10, MeOH). ESMS [m/z
(fragment, intensity), API-ES positive mode]: 476.2 (M þ Hþ, 100).
C56H66NO11 [M þ NH4]þ 928.4630; found 928.4626.
þ
4-O-[N-(2-Phenethyl)carbamoyl]-a,a-D-trehalose (23-f)
General procedure C was applied. Purification via column chroma-
tography (gradient elution: CH2Cl2/MeOH 9/1 to 7/3) afforded the
title compound 23-f as a white foam (112 mg, 0.229 mmol, 75%).
Rf 0.47 in CH3CN/H2O 8/2. 1H NMR (400 MHz, CD3OD): d
7.30–7.14 (m, 5H), 5.14 (d, J ¼ 3.8 Hz, 1H), 5.10 (d, J ¼ 3.8 Hz, 1H),
4.57 (s, 1H), 4.58 (t, J ¼ 9.6 Hz, 1H), 3.96–3.90 (m, 2H), 3.85–3.75 (m,
3H), 3.67 (dd, J ¼ 12.0/6.5 Hz, 1H), 3.56 (dd, J ¼ 9.9/3.8 Hz, 2H),
3.35–3.45 (m, 2H), 3.40–3.27 (m, 2H), 2.85–2.72 (m, 2H) ppm. 13 C
NMR (100 MHz, CD3OD): d 158.7 (C), 140.5 (C), 129.9 (CH), 129.5
(CH), 127.4 (CH), 95.1 (CH), 94.8 (CH), 74.5 (CH), 73.9 (CH), 73.6
(CH), 73.4 (CH), 73.1 (CH), 72.4 (CH), 72.0 (CH), 71.9 (CH), 62.6
(CH2), 62.4 (CH2), 43.5 (CH2), 37.0 (CH2) ppm. IR (HATR): 3320 (m,
br), 2931 (w), 1699 (m), 1540 (m), 1452 (w), 1339 (w), 1251 (m),
1202 (w), 1148 (m), 1106 (w), 1080 (m), 1030 (m), 989 (s), 846 (w),
6,6’-Di-O-methyl-a,a-D-trehalose (25)
General procedure C was applied. Purification via column chroma-
tography (gradient elution: CH2Cl2/MeOH 9/1 to 7/3) afforded the
title compound 25 as a white solid (164 mg, 0.443 mmol, 92%).
Rf 0.31 in CH3CN/H2O 8/2, 0.29 in CH2Cl2/MeOH 7/3. 1H NMR
(400 MHz, CD3OD): d 5.05 (d, J ¼ 3.7 Hz, 2H), 3.94 (ddd, J ¼ 10.0/
4.7/2.6 Hz, 2H), 3.76 (app t, J ¼ 9.3 Hz, 2H), 3.66–3.53 (m, 4H), 3.45
(dd, J ¼ 9.8/3.8 Hz, 2H), 3.37 (s, 6H), 3.34–3.27 (m, 2H) ppm. 13 C
NMR (100 MHz, CD3OD): d 95.3 (CH), 74.5 (CH), 73.1 (CH), 73.0
(CH2), 72.5 (CH), 72.0 (CH), 59.5 (CH3) ppm. IR (HATR): 3300 (m, br),
2929 (w), 1454 (w), 1379 (w), 1334 (w), 1275 (w), 1241 (w), 1195
(w), 1148 (m), 1108 (m), 1079 (m), 1038 (m), 1015 (m), 986 (s), 944
22
(m), 911 (m), 856 (w), 808 (w), 688 (w) cmꢀ1. [a]D þ190ꢂ (c 0.10,
806 (w), 748 (w), 698 (m) cmꢀ1. [a]D þ132ꢂ (c 0.10, MeOH).
22
MeOH). ESMS [m/z (fragment, intensity), API-ES positive modeþ]:
388.2 (M þ NH4þ, 100). HRMS (ESI-TOF): calcd. for C14H26NaO11
[M þ Na]þ 393.1367; found 393.1360.
ESMS [m/z (fragment, intensity), API-ES positive mode]: 490.2
(M þ Hþ, 20), 310.0 (M-glucose þ Hþ, 100).
4-O-(N-n-Octylcarbamoyl)-a,a-D-trehalose (23-g)
General procedure C was applied. Purification via column chroma-
tography (gradient elution: CH2Cl2/MeOH 10/0 to 7/3) afforded
2,3,4,2’,3’,4’-Hexa-O-benzyl-6,6’-di-O-[N-(2-phenethyl)carbamoyl]-
a,a-D-trehalose (26)
General procedure B was applied (use of phenethyl isocyanate).
Purification via column chromatography (gradient elution: hexane/
EtOAc 85/15 to 6/4) afforded the partially purified title compound
26 as a colourless glass which is used as such in the next step.
Rf 0.11 in hexane/EtOAc 7/3.
the title compound 23-g as
a
white foam (154 mg,
0.310 mmol, 74%).
Rf 0.27 in CH3CN/H2O 8/2. 1H NMR (400 MHz, CD3OD): d 5.14
(d, J ¼ 3.9 Hz, 1H), 5.10 (d, J ¼ 3.7 Hz, 1H), 4.53 (t, J ¼ 9.8 Hz, 1H),
3.98 (m, 2H), 3.84–3.74 (m, 2H), 3.66 (dd, J ¼ 12.0/5.5 Hz, 1H),
3.62–3.50 (m, 3H), 3.47 (dd, J ¼ 9.6/3.7 Hz, 1H), 3.31 (m, 1H), 3.10
(t, J ¼ 7.0 Hz, 2H), 3.09 (t, J ¼ 7.2 Hz, 2H), 1–55-1.45 (m, 2H),
1.35–1.25 (m, 10H), 0.89 (t, J ¼ 6.7 Hz, 3H) ppm. 13 C NMR
(100 MHz, CD3OD): d 158.8 (C), 95.1 (CH), 94.8 (CH), 74.5 (CH), 73.8
(CH), 73.5 (CH), 73.4 (CH), 73.1 (CH), 72.4 (CH), 72.1 (CH), 71.9 (CH),
62.6 (CH2), 62.4 (CH2), 41.9 (CH2), 33.0 (CH2), 30.9 (CH2), 30.4 (CH2),
30.4 (CH2), 27.9 (CH2), 23.7 (CH2), 14.4 (CH) ppm. IR (HATR): 3320
(m, br), 2928 (m), 2854 (w), 1697 (m), 1534 (m), 1456 (w), 1376
6,6’-Di-O-[N-(2-phenethyl)carbamoyl]-a,a-D-trehalose (27)
General procedure C was applied. Purification via column chroma-
tography (gradient elution: CH2Cl2/MeOH 9/1 to 6/4) afforded the
title compound 27 as a white solid (178 mg, 0.280 mmol, 65%
over 2 steps).
Rf 0.52 in CH3CN/H2O 8/2, 0.34 in CH2Cl2/MeOH 8/2. 1H NMR
(400 MHz, CD3OD): d 7.30–7.10 (m, 10H), 5.13–4.99 (m, 2H),
4.37–4.14 (m, 4H), 4.09–3.92 (m, 2H), 3.77 (t, J ¼ 9.3 Hz, 2H), 3.44
(w), 1257 (m), 1148 (m), 1104 (m), 1080 (m), 1027 (m), 992 (s), 944
22
(m), 846 (w), 804 (w) cmꢀ1. [a]D þ157ꢂ (c 0.12, MeOH). ESMS [m/ (br dd, J ¼ 9.6/3.4 Hz, 2H), 3.37–3.23 (m, 6H), 2.76 (t, J ¼ 7.4 Hz, 4H)

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1979
ppm. 13 C NMR (100 MHz, CD3OD): d 159.1 (C), 140.5 (C), 129.8 4H), 4.54 (d, J ¼ 12.1 Hz, 1H), 4.45 (d, J ¼ 10.8 Hz, 1H), 4.36 (d,
(CH), 129.5 (CH), 127.3 (CH), 95.3 (CH), 74.5 (CH), 73.2 (CH), 71.9 J ¼ 12.2 Hz, 1H), 4.16 (dt, J ¼ 9.9/2.2 Hz, 1H), 4.05 (dt, J ¼ 10.1/
(CH), 71.8 (CH), 64.9 (CH2), 43.5 (CH2), 37.1 (CH2) ppm. IR (HATR): 2.4 Hz, 1H), 4.02 (app t, J ¼ 9.5 Hz, 1H), 3.91 (app t, J ¼ 9.3 Hz, 1H),
3342 (m, br), 2934 (w), 1697 (s), 1520 (m), 1496 (w), 1454 (w), 3.68 (app br t, J ¼ 9.6 Hz, 1H), 3.59 (dd, J ¼ 9.6/3.5 Hz, 1H),
1347 (w), 1327 (w), 1240 (m), 1204 (w), 1148 (m), 1099 (m), 1074 3.54–3.28 (m, 6H), 3.53 (s, 3H), 3.32 (s, 3H) ppm. 13 C NMR
(100 MHz, CDCl3): d 139.0 (C), 138.9 (C), 138.4 (C), 138.3 (C), 138.2
(C), 137.8 (C), 128.4 (CH), 128.3 (CH), 128.3 (CH), 128.3 (CH), 128.0
(CH), 128.0 (CH), 127.9 (CH), 127.8 (CH), 127.7 (CH), 127.6 (CH),
127.5 (CH), 127.4 (CH), 127.4 (CH), 94.7 (CH), 94.7 (CH), 81.8 (CH),
81.5 (CH), 79.4 (CH), 79.3 (CH), 79.1 (CH), 77.6 (CH), 75.6 (CH2), 75.4
(CH2), 75.1 (CH2), 73.5 (CH2), 72.7 (CH2), 70.7 (CH2), 70.6 (CH), 70.5
(CH), 68.1 (CH2), 60.5 (CH3), 59.1 (CH3) ppm. IR (HATR): 3059 (w),
3028 (w), 2915 (w), 2868 (w), 1496 (w), 1453 (m), 1363 (m), 1328
(w), 1265 (w), 1199 (w), 1153 (m), 1140 (m), 1087 (s), 1069 (s),
1026 (m), 992 (s), 920 (m), 853 (w), 799 (w), 733 (s), 695 (s), 638
(w) cmꢀ1. ESMS [m/z (fragment, intensity), API-ES positive modeþ]:
928.4 (M þ NH4þ, 100). HRMS (ESI-TOF): calcd. for C56H66NO11
[M þ NH4]þ 928.4630; found 928.4645.
(m), 1040 (m), 1015 (m), 984 (s), 942 (m), 909 (w), 853 (w), 808 (w),
772 (w), 749 (w), 698 (m) cmꢀ1. [a]D þ110ꢂ (c 0.10, MeOH).
22
ESMS [m/z (fragment, intensity), API-ES negative mode]: 637.2
(M þ Hþ, 100). HRMS (ESI-TOF): calcd. for C30H40N2NaO13 [M þ Na]þ
659.2423; found 629.2414.
2,3,6,2’,3’,6’-Hexa-O-benzyl-4,4’-di-O-methyl-a,a-D-trehalose (28)
General procedure
A was applied (use of iodomethane).
Purification via column chromatography (eluent: hexane/EtOAc 8/
2) afforded the title compound 28 as a colourless glass (302 mg,
0.331 mmol, 98%).
Rf 0.21 in hexane/EtOAc 8/2. 1H NMR (400 MHz, CDCl3): d
7.43–7.22 (m, 30H), 5.19 (d, J ¼ 3.5 Hz, 2H), 4.97 (d, J ¼ 10.9 Hz, 2H),
4.86 (d, J ¼ 10.9 Hz, 2H), 4.69 (d, J ¼ 12.1 Hz, 2H, A-part of AB-sys-
tem), 4.67 (d, J ¼ 12.2 Hz, 2H, B-part of AB-system), 4.58 (d,
J ¼ 12.1 Hz, 2H), 4.43 (d, J ¼ 12.1 Hz, 2H), 4.09 (app dt, J ¼ 10.1/
2.3 Hz, 2H), 3.94 (app t, J ¼ 9.3 Hz, 2H), 3.57–3.36 (m, 8H), 3.47 (s,
6H) ppm. 13 C NMR (100 MHz, CDCl3): d 138.9 (C), 138.2 (C), 138.0
(C), 128.3 (CH), 128.3 (CH), 128.3 (CH), 127.9 (CH), 127.8 (CH), 127.6
(CH), 127.5 (CH), 127.4 (CH), 94.6 (CH), 81.7 (CH), 79.3 (CH), 79.2
(CH), 75.5 (CH2), 73.4 (CH2), 72.7 (CH2), 70.6 (CH), 68.2 (CH2), 60.6
(CH3) ppm. IR (HATR): 3064 (w), 3028 (w), 2930 (w), 2859 (w), 1496
(w), 1453 (m), 1364 (m), 1331 (w), 1207 (w), 1155 (m), 1135 (m),
1090 (s), 1024 (m), 993 (s), 913 (m), 854 (w), 797 (w), 732 (s), 695
(s), 636 (w) cmꢀ1. ESMS [þm/z (fragment, intensity), API-ES positive
4,6-Di-O-methyl-a,a-D-trehalose (31)
General procedure C was applied. Purification via column chroma-
tography (gradient elution: CH2Cl2/MeOH 9/1 to 7/3) afforded the
title compound 31 as a white glass (42.8 mg, 0.116 mmol, 87%).
Rf 0.29 in CH3CN/H2O 8/2, 0.16 in CH2Cl2/MeOH 8/2. 1H NMR
(400 MHz, CD3OD): d 5.07 (d, J ¼ 3.8 Hz, 1H), 5.05 (d, J ¼ 3.8 Hz, 1H),
3.91 (ddd, J ¼ 10.0/4.2/2.1 Hz, 1H), 3.85 (app t, J ¼ 9.4 Hz, 1H),
3.82–3.72 (m, 3H), 3.66 (dd, J ¼ 12.0/5.5 Hz, 1H), 3.62–3.52 (m, 2H),
3.53 (s, 3H), 3.47 (dd, J ¼ 9.8/3.8 Hz, 1H), 3.45 (dd, 9.8/3.7 Hz, 1H),
3.38 (s, 3H), 3.35–3.27 (m, 1H), 3.09 (dd, J ¼ 10.0/9.0 Hz, 1H) ppm.
13 C NMR (100 MHz, CD3OD): d 95.2 (CH), 95.0 (CH), 81.3 (CH), 74.5
(CH), 74.4 (CH), 73.8 (CH), 73.3 (CH), 73.2 (CH), 72.6 (CH2), 71.8
(CH), 71.5 (CH), 62.6 (CH2), 60.8 (CH3), 59.4 (CH3) ppm. IR (HATR):
3338 (m, br), 2922 (w), 1373 (m, br), 1192 (w), 1069 (m), 1032 (m),
mode]: 92þ8.4 (M þ NH4
, 100). HRMS (ESI-TOF): calcd. for
C56H66NO11 [M þ NH4]þ 928.4630; found 928.4639.
989 (s), 968 (m), 936 (m), 924 (m), 912 (m), 802 (m), 681 (w) cmꢀ1
.
22
[a]D þ203ꢂ (c 0.10, MeOH). ESMS [m/z (fragment, intensity), API-
ES positive mode]: 388.2 (M þ NH4þ, 100). HRMS (ESI-TOF): calcd.
for C14H26NaO11 [M þ Na]þ 393.1367; found 393.1359.
4,4’-Di-O-methyl-a,a-D-trehalose (29)
General procedure C was applied. Purification via column chroma-
tography (gradient elution: CH2Cl2/MeOH 9/1 to 7/3) afforded the
title compound 29 as a white solid (99 mg, 0.267 mmol, 86%).
Rf 0.28 in CH3CN/H2O 8/2, 0.31 in CH2Cl2/MeOH 7/3. 1H NMR
(400 MHz, CD3OD): d 5.06 (d, J ¼ 3.8 Hz, 2H), 3.85 (app t, J ¼ 9.4 Hz,
2H), 3.79 (ddd, J ¼ 10.0/4.3/2.0 Hz, 2H), 3.74 (dd, J ¼ 11.8/2.1 Hz,
2H), 3.65 (dd, J ¼ 11.9/4.5 Hz, 2H), 3.55 (s, 6H), 3.45 (dd, J ¼ 9.8/
3.8 Hz, 2H), 3.11 (dd, J ¼ 9.7/9.2 Hz, 2H) ppm. 13 C NMR (100 MHz,
CD3OD): d 95.0 (CH), 81.1 (CH), 74.5 (CH), 73.3 (CH), 72.8 (CH), 62.1
(CH2), 60.8 (CH3) ppm. IR (HATR): 3380 (m, br), 2932 (w), 1660 (w),
1472 (w), 1442 (w), 1420 (w), 1389 (w), 1339 (w), 1317 (w), 1244
(w), 1229 (w), 1209 (w), 1192 (w), 1156 (m), 1135 (m), 1101 (m),
1063 (s), 1029 (m), 992 (s), 963 (s), 928 (m), 854 (m), 804 (w), 702
2,3,4,2’,3’,4’-Hexa-O-benzyl-6-O-methyl-a,a-D-trehalose (32)
To a cooled (0 ꢂC) solution of 16-a (420 mg, 0.469 mmol, 1 eq) in
toluene (5 ml), DIBAL-H (supplied as 1.0 M solution in toluene,
2.35 ml, 2.35 mmol, 5 eq) was added dropwise. After 15 min, the
cooling bath was removed and the reaction mixture was stirred
for 48 h. The resulting solution was cooled to 0 ꢂC after which
MeOH (1.3 ml) and 10% aqueous KOH (0.4 ml) were added. The
mixture was transferred to a separation funnel using H2O (35 ml)
and dichloromethane (35 ml). The organic layer was separated and
the aqueous layer was extracted with dichloromethane (3 ꢁ 35 ml).
The combined organic layers were washed with brine (50 ml) and
concentrated under reduced pressure. The residue was purified
via column chromatography (gradient elution: hexane/EtOAc 7/3
to 6/4), giving the title compound 32 as a colourless glass in 76%
yield (277 mg, 0.3088 mmol) based on recovered starting mater-
ial (55 mg).
(w), 655 (w) cmꢀ1. [a]D þ220ꢂ (c 0.10, MeOH). ESMS [m/z (frag-
22
ment, intensity), API-ES positive mode]: 393.1 (M þ Naþ, 41), 388.2
þ
þ
(M þ NH4
, 100). HRMS (ESI-TOF): calcd. for C14H26NaO11
[M þ Na]þ 393.1367; found 393.1361.
2,3,2’,3’,4’,6’-Hexa-O-benzyl-4,6’-di-O-methyl-a,a-D-trehalose (30)
Rf 0.12 in hexane/EtOAc 6/4. 1H NMR (400 MHz, CDCl3): d
7.40–7.23 (m, 30H), 5.18 (d, J ¼ 3.5 Hz, 1H), 5.15 (d, J ¼ 3.7 Hz, 1H),
General procedure
A was applied (use of iodomethane).
Purification via column chromatography (gradient elution: hexane/
EtOAc 85/15 to 75/25) afforded the title compound 30 as a col- 5.00 (d, J ¼ 10.9 Hz, 1H), 4.99 (d, J ¼ 10.9 Hz, 1H), 4.92–4.83 (m, 4H),
ourless oil (131 mg, 0.144 mmol, 85%).
4.76–4.62 (m, 5H), 4.58 (d, J ¼ 11.0 Hz, 1H), 4.18–3.98 (m, 4H),
Rf 0.12 in hexane/EtOAc 8/2. 1H NMR (400 MHz, CDCl3): d 3.69–3.49 (m, 6H), 3.42 (br dd, J ¼ 10.6/3.1 Hz, 1H), 3.31 (br dd,
7.40–7.18 (m, 28H), 7.16–7.08 (m, 2H), 5.20 (d, J ¼ 3.5 Hz, 1H), 5.17 J ¼ 10.6/2.0 Hz, 1H), 3.27 (s, 3H), 1.46 (br t, J ¼ 6.0 Hz, 1H) ppm. 13 C
(d, J ¼ 3.7 Hz, 1H), 4.99 (d, J ¼ 11.0 Hz, 1H), 4.95 (d, J ¼ 11.0 Hz, 1H), NMR (100 MHz, CDCl3): d 138.9 (C), 138.8 (C), 138.4 (C), 138.2 (C),
4.85 (app d, J ¼ 10.9 Hz, 2H), 4.80 (d, J ¼ 10.7 Hz, 1H), 4.71–4.60 (m, 138.2 (C), 138.1 (C), 128.5 (CH), 128.4 (CH), 128.4 (CH), 128.4 (CH),

1980
S. DHAENE ET AL.
Scheme 1. Synthesis of strategic intermediates. Reagents and conditions: (a) 2.3 eq benzaldehyde dimethyl acetal, 0.05 eq camphorsulfonic acid, DMF, 95 ꢂC, 30 min;
(b) 10 eq NaH, 8 eq BnBr, 0.2 eq TBAI, DMF, 0 ꢂC-rt, 18 h; (c) 5 eq DIBAL-H, toluene, ꢀ18 ꢂC-rt, 2 h; (d) 5 eq DIBAL-H, CH2Cl2, 0 ꢂC-rt, 90 min; (e) 5 eq DIBAL-H, toluene,
48 h; (f) 2.5 eq NaH, 4 eq BnBr, DMF, 16 h; (g) 12 eq Me3N-BH3, 12 eq AlCl3, THF, molecular sieves (4 Å), 16h; (h) 1 eq benzaldehyde dimethyl acetal, 0.05 eq camphor-
sulfonic acid, DMF, 90 ꢂC, 15min; (i) Ac2O/pyridine 1/1, 24 h; (j) 12 eq NaOMe, MeOH, 1 h; (k) 15 eq NaH, 12 eq BnBr, 0.2 eq TBAI, 0 ꢂC-rt, 16h; (l) TFA/CH2Cl2/MeOH 1/
3/9, 120 h.
128.3 (CH), 128.1 (CH), 128.0 (CH), 127.9 (CH), 127.8 (CH), 127.7 (CH), 69.5 (CH), 63.1 (CH2), 59.1 (CH3), 42.1 (CH2), 36.0 (CH2) ppm.
(CH), 127.6 (CH), 127.6 (CH), 127.6 (CH), 127.5 (CH), 127.4 (CH),
94.4 (CH), 94.2 (CH), 81.7 (CH), 81.6 (CH), 79.5 (CH), 79.3 (CH), 77.6
(CH), 75.6 (CH2), 75.5 (CH2), 75.1 (CH2), 75.1 (CH2), 72.9 (CH2), 72.8
(CH2), 71.2 (CH), 70.7 (CH2), 70.6 (CH), 61.5 (CH2), 59.1 (CH3) ppm.
IR (HATR): 3479 (w, br), 3059 (w), 3029 (w), 2920 (w), 2873 (w),
1496 (w), 1453 (m), 1396 (w), 1359 (m), 1328 (w), 1208 (w), 1153
(m), 1133 (m), 1088 (s), 1068 (s), 1027 (m), 990 (s), 920 (m), 847
(w), 798 (w), 732 (s), 695 (s), 641 (w) cmꢀ1. ESMS [m/z (fragment,
intensity), API-ES positive mode]: 914.4 (M þ NH4þ, 100). HRMS
IR (HATR): 3410 (w), 3341 (w), 3086 (w), 3059 (w), 3026 (w), 2920
(w), 2877 (w), 1722 (m), 1514 (w), 1496 (m), 1454 (m), 1401 (w),
1359 (w), 1328 (w), 1242 (m), 1208 (w), 1153 (m), 1086 (m), 1068
(s), 1027 (m), 991 (s), 911 (w), 850 (w), 797 (w), 733 (s), 695 (s), 640
(w) cmꢀ1. ESMS [m/z (fragment, intensity), API-ES positive mode]:
1061.4 (M þ NH4þ, 100). HRMS (ESI-TOF): calcd. for C64H73N2O12
[M þ NH4]þ 1061.5158; found 1061.5179.
(ESI-TOF): calcd. for C55H64NO11 [M þ NH4]þ
914.4474;
found 914.4493.
6-O-Methyl-6’-O-[N-(2-Phenethyl)carbamoyl]-a,a-D-trehalose (34)
General procedure C was applied. Purification via column chroma-
tography (gradient elution: CH2Cl2/MeOH 9/1 to 7/3) afforded the
title compound 34 as a white glass (96 mg, 0.191 mmol, 83%).
Rf 0.49 in CH3CN/H2O 8/2, 0.17 in CH2Cl2/MeOH 8/2. 1H NMR
2,3,4,2’,3’,4’-Hexa-O-benzyl-6-O-methyl-6’-O-[N-(2-phenethyl)carba-
moyl]-a,a-D-trehalose (33)
General procedure B was applied (use of phenethyl isocyanate).
Purification via column chromatography (gradient elution: hexane/
EtOAc 8/2 to 6/4) afforded the title compound 33 as a colourless
glass (280 mg, 0.268 mmol, 98%).
(400 MHz, CD3OD):
d 7.32–7.13 (m, 5H), 5.14–4.99 (m, 2H),
4.36–4.14 (m, 2H), 4.08–3.88 (m, 2H), 3.83–3.71 (m, 2H), 3.66–3.52
(m, 2H), 3.51–3.40 (m, 2H), 3.37 (s, 3H), 3.35–3.26 (m, 4H), 2.77 (t,
J ¼ 7.4 Hz, 2H) ppm. 13 C NMR (100 MHz, CD3OD): d 159.1 (C), 140.5
(C), 129.8 (CH), 129.5 (CH), 127.3 (CH), 95.3 (CH), 74.5 (CH), 74.5
(CH), 73.1 (CH), 73.1 (CH2), 72.5 (CH), 72.0 (CH), 71.8 (CH), 64.9
(CH2), 59.5 (CH3), 43.5 (CH2), 37.1 (CH2) ppm. IR (HATR): 3308 (m,
br), 2926 (w), 1697 (m), 1526 (w), 1496 (w), 1455 (w), 1334 (w),
1252 (m), 1197 (w), 1146 (m), 1101 (m), 1074 (m), 1037 (m), 1018
(m), 987 (s), 943 (m), 909 (w), 856 (w), 807 (w), 772 (w), 750 (w),
Rf 0.13 in hexane/EtOAc 7/3. 1H NMR (400 MHz, CDCl3): d
7.40–7.10 (m, 35H), 5.19 (d, J ¼ 3.4 Hz, 1H), 5.15 (d, J ¼ 3.4 Hz, 1H),
5.01 (d, J ¼ 11.2 Hz, 1H), 4.98 (d, J ¼ 11.6 Hz, 1H), 4.92–4.77 (m, 4H),
4.73–4.49 (m, 7H), 4.31–3.95 (m, 6H), 3.68–3.25 (m, 8H), 3.27 (s,
3H), 2.75 (t, J ¼ 7.0 Hz, 2H) ppm. 13 C NMR (100 MHz, CDCl3): d
155.9 (C), 138.9 (C), 138.7 (C), 138.6 (C), 138.4 (C), 138.1 (C), 137.9
(C), 128.7 (CH), 128.6 (CH), 128.4 (CH), 128.4 (CH), 128.4 (CH), 128.4
(CH), 128.3 (CH), 128.0 (CH), 127.9 (CH), 127.8 (CH), 127.7 (CH),
127.7 (CH), 127.6 (CH), 127.5 (CH), 127.4 (CH), 126.5 (CH), 94.7
(CH), 94.3 (CH), 81.7 (CH), 81.6 (CH), 79.3 (CH), 79.1 (CH), 75.6
700 (w) cmꢀ1. [a]D þ132ꢂ (c 0.10, MeOH). ESMS [m/z (fragment,
22
intensity), API-ES positive mode]: 504.2 (M þ Hþ, 100). HRMS (ESI-
TOF):
calcd.
for
C22H33NNaO12
[M þ Na]þ
526.1895;
(CH2), 75.5 (CH2), 75.1 (CH2), 72.9 (CH2), 72.7 (CH2), 70.6 (CH2), 70.6 found 526.1879.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1981
Scheme 2. Synthesis of 6-O-substituted trehalose derivatives. Reagents and conditions: (a) 2.5 eq NaH, 4 eq R-Br or R-I, DMF, 16 h; (b) 0.05 eq Pd/C, EtOAc/MeOH 1/1,
16 h; (c) 3 eq R-N ¼ C ¼ O, 0.2 eq DMAP, CH2Cl2, 24–120 h.
Enzyme activity assays
was performed with AutoDock VINA using default parameters and
ligands were allowed to rotate freely during the simulation. The
conformer with an appropriate glucose in both the ꢀ1 and þ1
subsite was selected as the binding mode for further analysis.
Trehalose and each derivative (20 mM) were incubated with
0.01 mg mlꢀ1 trehalase from porcine kidney (Sigma Aldrich) or
Mycobacterium smegmatis (NZYtech) in 100 mM of sodium phos-
phate buffer (pH 7) at 37 ꢂC for 24 h. Mixtures with Mycobacterium
smegmatis trehalase also contained 6 mM of MgSO4, as the
enzyme requires Mg2þ for activity32. Samples were taken at regu-
lar time intervals and diluted with ultrapure water for analysis
with high performance anion-exchange chromatography with
pulsed amperometric detection (HPAEC-PAD, Dionex ICS-3000).
In order to evaluate their relative inhibition percentage, each
trehalose derivative (20 mM) was incubated with 0.01 mg mlꢀ1 of
above-mentioned trehalases with 3 or 20 mM of trehalose for por-
cine kidney and Mycobacterium smegmatis trehalases, respectively,
in 100 mM of sodium phosphate buffer (pH 7) at 37 ꢂC for 14 min.
Again, 6 mM of MgSO4 was added to the reaction mixture with
Mycobacterium smegmatis trehalase. Samples were taken every
2 min and diluted with ultrapure water for analysis with HPAEC-
PAD. The residual activity of the enzyme towards trehalose in
each mixture was determined by comparing the measured activity
to the activity determined in the absence of trehalose derivative.
For selected compounds 2, 17-a, 19-d, 19-e, 19-f, 21-a, 23-f,
and 31, IC50 values were determined by incubating different con-
centrations of the trehalose derivative (0.01–50 mM) with the
selected trehalases under the same conditions as mentioned
above. Samples were taken every 2 min and diluted for analysis
with HPAEC-PAD. The residual activity of the enzyme towards tre-
halose in each mixture was determined compared to a sample
without trehalose derivative. Plots and curve fits were obtained
via Sigmaplot 13. All reactions were performed in triplicate, values
are represented as the mean and standard deviations were in the
range of ꢄ15% of the reported value.
Results
Chemistry
Synthesis of strategic intermediates
Generally, synthesis of all final analogues was achieved from a
central set of partially protected strategic intermediates 7–10, 12
and 15 which could be obtained from trehalose 1 (Scheme 1).
After protection of both 4- and 6-hydroxyl groups in trehalose as
benzylidene acetal, the remaining OH functional groups were con-
verted to the corresponding benzyl ethers, leading to 627. In the
following crucial desymmetrisation step, regioselective reductive
opening of one of the acetal moieties to either the 4- or 6-mono-
alcohol can be achieved using diisobutylaluminium hydride
(DIBAL-H) in an appropriate solvent, according to existing litera-
ture27. Based on reported conditions, selective opening towards
strategic intermediate 7 containing a free 6-OH group was
achieved through reaction in toluene. The alternative 4-OH isomer
8 could be obtained via DIBAL-H reduction in dichloromethane: in
a typical experiment, starting material still remained at moment of
quenching (10% recovered), while in addition to the desired com-
pound 8 (55%), regio-isomer 7 (19%) and product 9 (9%, resulting
from overreduction) were also isolated through tedious chromato-
graphic separation. Compound 9 was found to be useful as an
additional strategic intermediate to access 4/40-bis-substituted
derivatives; the corresponding 6/60-bis-OH intermediate 10 was
obtained after a second DIBAL-H treatment of 7 in toluene. To
avoid the above-mentioned practical issues concerning synthesis
of 8, we decided to additionally synthesise alternative strategic
intermediate 12, also allowing the subsequent synthesis of 4-sub-
stituted derivatives. This was achieved via sequential O-benzyla-
tion of 7 and opening of the resulting mono-benzylidene acetal
11 using Me3N-BH3/AlCl334. Under the latter conditions, the
desired 4-OH isomer 12 could be isolated in 49% yield, while the
fraction of corresponding 6-OH regio-isomer amounted to 38%
(compound not shown). Alternatively, intermediate 11 could be
Molecular modelling
All manipulations were performed with the molecular modelling
program YASARA and the YASARA/WHATIF twinset and figures
were created with PyMOL 2.0. The ligand free and inhibitor bound
crystal structures of trehalase from Enterobacter cloacae (GH37
family) (PDB code 5Z6H and 5Z66, respectively)33 were used as
template for docking. Trehalose derivative structures were created obtained via the mono-benzylidene acetal-protected 13. Although
with YASARA Structure and subsequently minimised with the this was expected to involve a shorter reaction route from trehal-
AMBER03 force field. The grid box for docking had a dimension of ose, an extra acetylation/deacetylation sequence via 14 was found
25 ꢁ 25 ꢁ 25 Å and comprised the entire catalytic cavity. Docking to be necessary to facilitate intermediate purification, leading to

1982
S. DHAENE ET AL.
Scheme 3. Synthesis of 4-O-substituted trehalose derivatives. Reagents and conditions: (a) 2.5 eq NaH, 4 eq R-Br or R-I, DMF, 16 h; (b) AD-mix a, 2.5 eq MeSO2NH2,
0.25 eq (DHQ)2PHAL, tBuOH/H2O:acetone 1/1/0.9, 240 h; (c) 0.05 eq Pd/C, EtOAc/MeOH 1/1, 16 h; (d) 3 eq R-N ¼ C ¼ O, 0.2 eq DMAP, CH2Cl2, 24–120 h.
an equal number of reaction steps but significantly lower overall Biological evaluation
yield. Finally, acid hydrolysis of the acetal moiety in 11 was used
Resistance to trehalase hydrolytic activity
to obtain 4/6-bis-OH strategic intermediate 15. With these stra-
The hydrolytic resistance of each trehalose derivative was eval-
tegic intermediates in hand, different sets of 4- or 6-monosubsti-
uated by incubating 20 mM of substrate with 0.01 mg mlꢀ1 of
tuted, and 4/40-, 6/60- and 4/6-double substituted analogues were
enzyme at pH 7 and 37 ꢂC for 24 h. No degradation was observed
accessible, via derivatisation of the remaining unprotected alco-
for the majority of derivatives, whereas trehalose was already bro-
hol moieties.
ken down completely after just 12 h (data not shown). The only
exceptions showing significant hydrolysis were derivatives 17-a (7
and 8% relative hydrolysis by the trehalase from porcine kidney
and M. smegmatis, respectively), 17-b (1 and 6%, respectively) and
Derivatisation of strategic intermediates to final compounds
First, using strategic intermediate 7, two series of 6-O-monosubsti-
17-c (1% and 1%, respectively), which all contain a small alkyl
tuted derivatives were prepared (Scheme 2). On one hand, O-alkyl-
group (i.e. methyl, ethyl and n-propyl, respectively) on the 6-
ation using alk(en)yl halogenides with increasing chain length
under standard conditions delivered intermediates 16-a–g, which
O-position.
were overall deprotected using catalytic hydrogenolysis to obtain
the intended 6-O-alkylated trehalose derivatives 17-a–g. On the
other hand, a series of 6-O-carbamoyl derivatives 19-a–g was pre-
pared via treatment of 7 with a set of isocyanates in presence of
DMAP, followed by hydrogenolysis.
Next, two analogous series of 4-O-substituted trehalose deriva-
tives were envisaged (Scheme 3). Indeed, alkylation of 8 using the
same set of alk(en)yl halogenides mentioned above gave the
expected ethers 20-a–g which were deprotected to the final
Inhibitory activity towards trehalase
The inhibitory activity of the compounds was evaluated at a con-
centration of 20 mM, using trehalose as substrate at a concentra-
tion equal to the respective Michaelis–Menten constant (3 mM
and 20 mM for porcine kidney and M. smegmatis trehalase,
respectively). Despite their resistance to hydrolysis, several of
these compounds were found to still bind quite tightly to these
trehalases, causing enzyme inhibition (Table 1).
Regarding porcine kidney trehalase, the most interesting inhibi-
tors are grouped within the series of 6-O-substituted derivatives,
as their counterparts with 4-O-substitutions are all poor inhibitors
(ꢄ35% inhibition at 20 mM). Only two derivatives, namely 17-a
derivatives 21-a–g. Additionally, lentztrehalose A 2 was synthes-
ised following reported conditions, via Sharpless asymmetric dihy-
droxylation of 4-O-prenylated intermediate 20-f and subsequent
1
hydrogenolysis of 20-h30. From H-NMR analysis, a diastereomeric
excess of 73% 20-h could be determined (Figure S1 in
Supplementary Information). Both diastereomers could not be
separated chromatographically and the resulting lentztrehalose A
2 was therefore used as such for trehalase evaluation. In analogy
to the 6-O-series 19-a–g, we also prepared the corresponding 4-
O-carbamates 23-a-g in two steps from strategic intermediate 12.
Finally, a selection of double-substituted analogues was pre-
pared (Scheme 4). To this end, the appropriate bis-OH strategic
intermediates 10, 9 and 15 were reacted with MeI and subse-
quently deprotected to obtain the 6/60-, 4/40- and 4/6-bis-O-
methyl derivatives 25, 29 and 31, respectively. Additionally, 6/60-
bis-phenethylcarbamoyl trehalose 27 was obtained from 10.
Lastly, mixed derivative 34 was obtained via methylation of inter-
mediate 16-a, followed by regioselective acetal opening using
DIBAL-H in toluene, conversion to the carbamate 33 and final
debenzylation.
and 19-f, exhibited complete inhibition. However, these com-
pounds bear very different substituents in both size and chemical
properties, i.e. methyl ether and phenethyl carbamate, respect-
ively. Compared to 17-a, extension of the alkyl ether substituent
resulted in a clear drop in inhibition. Similar to compound 19-f,
the other phenyl group-containing carbamates, 19-d and 19-e,
exhibited significant inhibitory effects (>80%). Compounds in the
carbamate series devoid of a phenyl moiety, i.e. 19-a, 19-b, 19-c
and 19-g, showed a clearly lowered inhibition, suggesting that
the aromatic group forms a crucial interaction with the enzyme.
Interestingly, compared to 17-a and 19-f, the additional introduc-
tion of either of their substituents on the 60-O-position, i.e. leading
to 25, 27 and 34, led to a lower rather than a higher affinity.
Likewise, the introduction of an additional methyl substituent on
the 4-O-position in 31 led to
a similar lowered inhib-
ition outcome.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1983
Scheme 4. Synthesis of double-substituted trehalose derivatives. Reagents and conditions: (a) 5 eq NaH, DMF, 0 ꢂC, 15 min; then: 8 eq MeI, DMF, 0 ꢂC-rt, 16 h; (b) 0.05
eq Pd/C, EtOAc/MeOH 1/1, 16 h; (c) 6 eq 2-phenethyl isocyanate, 0.4 eq DMAP, CH2Cl2, 48h; (d) 5 eq DIBAL-H, toluene, 48 h; (e) 3 eq 2-phenethyl isocyanate, 0.2 eq
DMAP, CH2Cl2, 72 h.
Table 1. Inhibition (%) of porcine kidney trehalase (3 mM trehalose) and M. smegmatis trehalase (20 mM trehalose) in the presence of 4- (left) and 6-O-substituted
(right) derivatives of trehalose (20 mM).
4-position
Porcine kidney (%)a
6-position
Porcine kidney (%)a
Substituent
Cpd
M. smegmatis (%)a
Cpd
M. smegmatis (%)a
-O(CH2)2OHCH(CH3)2OH
-OCH3
-OCH2CH3
-O(CH2)2CH3
-O(CH2)3CH3
-OCH2CH(CH3)2
-O(CH2)2CH(CH3)2
-O(CH2)9CH3
2
1
20
7
3
35
0
0
0
0
0
100 (93b)
–
–
–
21-a
21-b
21-c
21-d
21-e
21-f
21-g
23-a
23-b
23-c
23-d
23-e
23-f
23-g
29
90
82
88
31
40
10
0
24
17-a
17-b
17-c
17-d
17-e
17-f
17-g
19-a
19-b
19-c
19-d
19-e
19-f
19-g
–
100 (4b)
100 (76b)
45
47
36
24
7
28
23
17
94
82
84
95
96
0
83
67
78
-OC(¼O)NH(CH2)2CH3
-OC(¼O)NHCH2(CH3)2
-OC(¼O)NHCy
44
0
100 (38b)
5
83
-OC(¼O)NHPh
32
18
8
0
10
–
16
–
100 (51b)
100 (44b)
100 (47b)
100 (74b)
0
-OC(¼O)NHCH2Ph
-OC(¼O)NH(CH2)2Ph
-OC(¼O)NH(CH2)7CH3
4,40-bis-OCH3
87
93
90
100 (12b)
0
26
–
47
16
0
100 (43b)
–
6,60-bis-OCH3
–
31
–
–
–
25
31
27
34
91
4,6-bis-OCH3
100 (88b)
100 (88b)
100 (42b)
98
6,60-bis-OOCNH(CH2)2Ph
6-OCH3,60-OC(¼O)NH(CH2)2Ph
–
–
–
7
aMean from three different assays, analysis with HPAEC-PAD (errors were in the range of ꢄ15% of the reported value).
b% Inhibition at inhibitor concentration of 1 mM.
Towards M. smegmatis trehalase, contrarily, members of both corresponding 4-O-alkyl analogues), carbamate derivatives bearing
series of 4- and 6-O-substituted compounds generally demon- a cyclohexyl, phenyl, benzyl or phenethyl group on either the 4-
strated interesting inhibitory properties at a concentration of or 6-O-position (19-c-f, 23-c-f) all cause a high to complete inhib-
20 mM. Whereas high inhibition is observed for 6-O-alkyl deriva- ition. Furthermore, all 4,40-, 6,60- and 4,6-bis-substituted derivatives
tives (in contrast to the somewhat lower activity of the show an inhibition percentage that exceeds 90%. Compounds

1984
S. DHAENE ET AL.
Table 2. IC50 values of selected trehalose derivatives for porcine kidney and M.
smegmatis trehalase.
Compound 21-a (4-O-methyltrehalose) was picked as a 4-O-
substituted representative in order to find an explanation for their
low inhibitory activity and high resistance to hydrolysis. In the
open form, two docking poses were found, i.e. with the methyl
substituent positioned in either subsite ꢀ1 or þ1. However, nei-
ther of these fits match with the one of trehalose, and very little
interactions are formed between enzyme and ligand (Figure 3).
There obviously is little space around the 4-hydroxy position of
trehalose in both subsite ꢀ1 and þ1 (Figure 2), explaining why
the bulkier compounds cannot take on the same pose as trehal-
ose. Furthermore, no productive docking result could be obtained
in the closed structure.
IC50 (mM)
Compound
Porcine kidney trehalase
M. smegmatis trehalase
2
ꢅ20a
0.67
0.71
1.11
1.03
0.74
5.27
5.11
0.35
17-a
19-d
19-e
19-f
21-a
23-f
31
8.61
>20a
>20a
9.91
>20a
>20a
>20a
aValue not determined, based on observed weak inhibition at 20 mM (see
Table 1).
In contrast to their 4-O-substituted counterparts, 6-O-substi-
tuted derivatives show a far more interesting inhibitory profile. A
distinction can be made between the ether and carbamate deriva-
tives. For example, the 6-methoxy derivative 17-a triggered minor
hydrolytic activity as well as an attractive inhibitory effect.
Docking simulations confirmed our hypothesis that the derivative
could find a productive conformation in both the open and closed
structure (Figure 3). Interestingly, the substitution is accommo-
dated in subsite ꢀ1 rather than þ1, although the latter seems to
offer more space (Figure 2). In contrast, substitutions containing
four or more carbons are preferentially positioned in subsite þ1,
although their inhibitory potential is considerably lower. These
findings suggest that a greater affinity can be achieved when the
substituent is sufficiently small to be placed in subsite ꢀ1.
with longer (n-octyl/n-decyl) hydrophobic chains at the 4- or 6-O-
positions (17-g, 19-g, 21-g and 23-g), resulted in a lack of inhibi-
tory effect towards M. smegmatis trehalase.
To further rank the derivatives that caused complete inhibition,
the abovementioned experiments were repeated with a lower
inhibitor concentration, i.e. 1 mM instead of 20 mM (Table 1).
Additionally, eight trehalose derivatives were also subjected to
half maximal inhibitory concentration (IC50) experiments (Table 2).
This confirmed that compounds 17-a and 19-f are the strongest
inhibitors for porcine kidney trehalase, while derivatives 2, 17-a,
19-f and 31 displayed sub-mM IC50 values against M. smegmatis
trehalase. Interestingly, the IC50 of the strongest inhibitors of por-
cine kidney trehalase is about 10 mM whereas substantially lower
values (down to 0.35 mM) are found for the M. smegma-
tis trehalase.
A trend could be noticed within the 6-O-carbamate derivatives,
as the inhibitory potential among these compounds clearly is
highest when a phenyl group is present. For instance, complete
inhibition of porcine kidney trehalase without hydrolysis was
observed with compound 19-f, containing a N-phenethylcarba-
moyl group. This trehalose derivative was able to find a fit in the
open active site (Figure 3), while its positioning in the closed
structure was predicted to have a negative binding energy (i.e.
repulsion, Table 3). A strong stacking interaction is established
between the compound’s aromatic group and F154, and again, the
substituent was found to be accommodated in subsite ꢀ1.
Docking simulations were also performed with both other phenyl-
containing carbamates 19-d and 19-e and cyclohexyl carbamate
19-c (Figure 3). The positioning of the benzyl substituent of 19-e
resembles that of 19-f. Remarkably, the phenyl carbamate moiety
of 19-d is preferentially placed in the þ1 subsite instead, although
the interaction with F154 still is retained. Just as with the alkylated
derivatives, the positioning of the substituent in subsite þ1
appears to lower the compound’s inhibitory potential. In contrast,
the cyclohexyl carbamate 19-c exhibited a different behaviour: its
preferred docking poses do not match the one of trehalose, high-
lighting the importance of the aromatic group.
Molecular docking studies
To clarify the displayed activities of the new derivatives, molecular
docking studies were carried out. Unfortunately, no structural elu-
cidation was possible for the enzyme from M. smegmatis as no
crystal structure of a trehalase from family GH15 has yet been
determined. However, several crystal structures of trehalases from
family GH37 are described in literature, namely from Escherichia
coli, Saccharomyces cerevisiae and Enterobacter cloacae33,35–37
.
Interestingly, the latter has been crystallised in two different
forms, i.e. with and without the inhibitor validoxylamine A 36,
which resulted in structures that can be described as ‘closed’ and
‘open’, respectively. Indeed, the so-called lid loop (G506-G519) and
side loop (Y147-Y159) were found to undergo a significant conform-
ational change upon ligand binding (Figure 2)33. Docking simula-
tions were performed with these structures as representative
scenario for family GH37 trehalases, such as the one from porcine
kidney. The bacterial enzyme shares about 30% sequence identity
and 40% similarity with porcine and human trehalase, but all
active site residues within a distance of 4 Å from validoxylamine A
36 are fully conserved (Supplementary Figure S2). It was hypoth-
esised that a compound sensitive to hydrolysis should find a pro-
ductive docking pose in both the open and closed conformation,
whereas a hydrolysis-resistant inhibitor should only be accommo-
dated by the former.
After removal of the cocrystallised validoxylamine A 36, trehal-
ose 1 was docked in the active site pocket of the closed structure
and compared with the reference compound 36 (Figure 2, bot-
tom). A very good overlay was observed after superposition, with
most of the crucial interactions being conserved. A similar result
was obtained after docking of trehalose 1 and validoxylamine A
36 in the open structure (Figure 2, top). Subsequently, the binding
of various trehalose derivatives was simulated (Figure 3; Table 3).
Discussion
In our work, we have evaluated a series of 4- and/or 6-O-substi-
tuted trehalose derivatives towards trehalase degradation.
Generally, all compounds were resistant to hydrolysis for both
tested trehalases, with the exception of 6-O-alkylated derivatives
with short carbon chain length (C1-C3). For porcine kidney treha-
lase, as representative of GH37 trehalases, this trend was con-
firmed by docking studies: the hydrolysable 6-O-substituted
derivatives could find a productive fit in the closed (i.e. hydrolyti-
cally active) enzyme structure, whereas that was not the case for
analogues containing larger substituents at this position or for
any of the 4-substituted derivatives. This is consistent with the

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1985
Figure 2. Docking of trehalose 1 (left, orange) and validoxylamine 36 (right, green) in the active site pocket of the open enzyme conformation (upper) and the closed
conformation (lower) with surface view of active site pocket (catalytic residues D312 and E496, salmon; side loop Y147-Y159, raspberry; lid loop G506-G519, green).
findings of Asano et al., who pointed out that the enzyme is most 0.1–1 mM (Figure 4). In comparison, as substrate analogue inhibi-
tolerant towards substitutions at the 6-O-position38.
tors, the presented 6-substituted trehaloses 17-a and 19-f have
IC50 values around 9 mM. The observed inhibition could be
explained via docking experiments: binding of 17-a and 19-f was
shown to be possible in the open state of the enzyme via place-
ment of the side chain in the ꢀ1 subsite.
To evaluate whether the non-hydrolysable trehalose derivatives
are still able to bind to the enzyme, inhibition experiments were
performed with the trehalases from porcine kidney (family GH37)
and M. smegmatis (family GH15).
From a pharmaceutical perspective, inhibitors of intestinal gly-
cosidases, including trehalase (GH37 family), can be interesting
and may be orally administered in the treatment of diabetes (type
II) to regulate the absorption of carbohydrates39. A distinction can
be made between transition state analogues and substrate ana-
logues40. The former are inhibitors that optimally bind the transi-
Trehalases could also be attractive drug targets in the ongoing
battle against pathogenic microorganisms due to the essentiality
of trehalose and its metabolic derivatives for their survival23.
Trehalose is a building block of the cell wall in mycobacteria and
corynebacteria, as it is a basic component of their glycolipids6,10
.
For example, the cell wall of the pathogen Mycobacterium tubercu-
tion state of trehalase, by mimicking the glucosyl-oxocarbenium losis contains trehalose-6,60-dimycolate, a toxic lipid that has been
ion which is developing during enzymatic action35,40. Examples identified as the main virulence factor of tuberculosis and is
are validamycin A 35, validoxylamine A41 36 and trehazolin35 37, responsible for the low permeability of the cell wall, leading to
which have IC50 values in the nanomolar range for porcine kidney drug resistance10,45. Interestingly, Shleeva and co-workers investi-
trehalase (Figure 4). In contrast, substrate analogues like manno- gated the importance of trehalase activity on the resuscitation of
trehalose42,43 38, 5-thiotrehalose42 39 and 3,30-diketotrehalose44 dormant mycobacterial cells46. Validamycin A 35 (Figure 4), a tre-
40 are mildly potent inhibitors with IC50 values in the range of halase inhibitor that mimics the transition-state, had a negative

1986
S. DHAENE ET AL.
Figure 3. Docking of trehalose derivatives (blue) and validoxylamine 36 (green) in the active site pocket of the open (upper) and closed (bottom) enzyme conform-
ation. An overlay of trehalose (orange) was added for reference. Salmon rectangle: compounds docked in both open and closed conformations, grey rectangle: com-
pounds with aromatic group (catalytic residues D312 and E496, salmon; side loop Y147-Y159, raspberry; lid loop G506-G519, green).
effect on the resuscitation of dormant Mycobacterium smegmatis derivatives 2, 17-a, 19-d, 19-e, 19-f, 23-a, 23-c, 23-d, 27, 29 and
cells, hereby highlighting the importance of trehalose breakdown 31, exhibiting complete inhibition of M. smegmatis trehalase at an
for cell revival46. Based on our obtained results, trehalose inhibitor concentration of 20 mM, are suggested to be mildly

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1987
Table 3. Binding energy (kcal molꢀ1) and the possible presence of a productive conformation determined by simulated molecular docking experiments versus
experimentally observed hydrolysis and inhibition.
Cpd
Binding energy open
Productive conformation?
Inhibition?a
Binding energy closed
Productive conformation?
Hydrolysis?
1
2
6.88 0.79
7.39 0.47
7.21 0.75
8.33 0.48
9.02 0.55
8.85 0.51
8.80 0.54
6.76 0.68
7.30 0.71
YES
NO
YES
NO
YES
YES
YES
NO
–
3.36 3.61
ꢀ1.17 2.63
0.81 6.99
ꢀ2.06 1.24
ꢀ7.14 2.48
ꢀ5.53 1.06
ꢀ2.42 1.19
ꢀ0.17 4.64
2.84 4.59
YES
NO
YES
NO
NO
NO
NO
NO
YES
YES
NO
YES
NO
NO
NO
NO
NO
NOb
NO
YES
NO
YES
YES
YES
NO
YES
17-a
19-c
19-d
19-e
19-f
21-a
36
YES
a>80% inhibition percentage (see Table 1).
bNon-hydrolysable compound (no glycosidic linkage).
Figure 4. Structure of trehalose derivatives that are hydrolysis resistant and inhibitors of trehalases (IC50,PT and IC50,MT; half maximal inhibitory concentration of porcine
kidney and M. smegmatis trehalase, respectively).
potent inhibitors with IC50 values down to 0.35 mM for 31. To the substituted derivatives; several compounds (2, 21-a, 23-c, 23-d,
best of our knowledge, this is the first report of substrate ana- 23-f, 29, 31) show an interesting inhibition of M. smegmatis treha-
logues that inhibit the activity of M. smegmatis trehalase.
lase (>90% inhibition at 20 mM concentration) but lack inhibitory
effect towards porcine kidney trehalase. Notably, the trehalase
hydrolysis-resistant lentztrehalose A 2 inhibited the activity of M.
smegmatis trehalase completely, whereas it only had a poor effect
on porcine kidney trehalase activity (1% inhibition) at the same
concentration, the latter confirming previously reported data25.
This binding selectivity is also reflected in the IC50 values of these
Finally, clear differences can be noticed between the two
studied trehalases concerning interaction with our series of trehal-
ose derivatives; generally, M. smegmatis trehalase is more sensitive
to inhibitory effects in comparison to porcine kidney trehalase,
reflecting their classification in different families (GH15 vs. GH37,
respectively). Carrol and co-workers already pointed this out, as it
was found that the transition-state mimic trehazolin35,47, a known 4-O-substituted compounds, which are in the lower- to sub-mM
inhibitor of porcine kidney trehalase, had no inhibitory effect range (0.35–5.27 mM) for M. smegmatis trehalase, but could not
towards M. smegmatis trehalase32 (Figure 4). In our study, this be determined for porcine kidney trehalase (>20 mM). Due to this
effect is particularly noticeable in the performance of 4-O- selective inhibition profile and the hydrolytic stability, these 4-

1988
S. DHAENE ET AL.
substituted trehalose derivatives could be investigated as poten-
tial selective anti-pathogenic agents that exert a limited inhibitory
effect in the human and/or mammalian gastro-intestinal tract
without being easily degraded by intestinal trehalases.
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Koen Beerens
Jorick Franceus
Tom Desmet
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http://orcid.org/0000-0001-6608-0443
http://orcid.org/0000-0003-4377-5933
http://orcid.org/0000-0002-5788-3022
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