Synthesis of cyclophellitol, cyclophellitol aziridine, and their tagged derivatives

Article abstract of DOI:10.1002/ejoc.201402588

Cyclitol epoxides and aziridines are potent and selective irreversible inhibitors of retaining glycosidases. We have previously reported on our studies on the use of activity-based probes derived from cyclophellitol and from its aziridine analogue for activity-based profiling of retaining β-glucosidases in vitro, in situ, and in some examples also in vivo. In this work we disclose full details of the synthesis, purification, and analysis of a comprehensive panel of cyclophellitol analogues, all featuring the β-glucose configuration and designed as tools for selective inhibition and/or imaging of human acid glucosylceramidase (epoxides) or as broad-spectrum probes for retaining β-glucosidases (aziridines).

Full text of DOI:10.1002/ejoc.201402588

FULL PAPER
DOI: 10.1002/ejoc.201402588
Synthesis of Cyclophellitol, Cyclophellitol Aziridine, and Their Tagged
Derivatives
Kah-Yee Li,[a][‡] Jianbing Jiang,[a][‡] Martin D. Witte,[b] Wouter W. Kallemeijn,[c]
Hans van den Elst,[a] Chung-Sing Wong,[a] Sharina D. Chander,[a] Sascha Hoogendoorn,[a]
Thomas J. M. Beenakker,[a] Jeroen D. C. Codée,[a] Johannes M. F. G. Aerts,[c]
Gijs A. van der Marel,[a] and Herman S. Overkleeft*[a]
Keywords: Cyclitols / Enzyme inhibitors / Aziridines / Azides / Epoxides / Fluorescent probes
Cyclitol epoxides and aziridines are potent and selective
irreversible inhibitors of retaining glycosidases. We have pre- and analysis of a comprehensive panel of cyclophellitol ana-
viously reported on our studies on the use of activity-based logues, all featuring the β-glucose configuration and de-
probes derived from cyclophellitol and from its aziridine ana- signed as tools for selective inhibition and/or imaging of hu-
work we disclose full details of the synthesis, purification,
logue for activity-based profiling of retaining β-glucosidases
man acid glucosylceramidase (epoxides) or as broad-spec-
in vitro, in situ, and in some examples also in vivo. In this
trum probes for retaining β-glucosidases (aziridines).
mechanism, first proposed by Koshland,[3] with (+)-cyclo-
phellitol (1) results in the formation of a stable adduct (see
Figure 1) and thus in mechanism-based enzyme inacti-
vation.[4]
Introduction
(+)-Cyclophellitol (1, Figure 1), isolated and charac-
terized by Umezama and co-workers almost 25 years ago,[1]
is a potent and selective mechanism-based inhibitor of re-
taining β-glucosidases of various biological origins. It is a
close analogue of conduritol β-epoxide (CBE), a classical
glucosidase inhibitor first described by Günther Legler.[2]
(+)-Cyclophellitol (1) differs from CBE only in its C5-sub-
stituent (cyclophellitol numbering): whereas CBE features a
hydroxy group, (+)-cyclophellitol (1) possesses a hydroxy-
methylene moiety, which adds considerably to both potency
and specificity towards retaining β-exoglucosidases of this
natural product. The absolute stereochemistry of the sub-
stituents at C1 to C5 in 1 matches that of a β-glucopyrano-
side moiety, which explains tight binding of the cyclitol to
the active sites of retaining glucosidases. Upon binding, the
epoxide is optimally positioned to undergo acid-catalyzed
ring-opening by the catalytic diad consisting of two carbox-
Figure 1. (+)-Cyclophellitol (1) and cyclophellitol aziridine (2) are
mechanism-based inhibitors of retaining β-glucosidases.
Substitution of the epoxide moiety in 1 for an aziridine
ylic acid residues, positioned some 6–7 Å apart. Treatment yields cyclophellitol aziridine (2),[5] which inhibits retaining
of retaining β-glucosidases that hydrolyze β-glucosidic link- β-glucosidases in a similar fashion.[6]
ages through a formal two-step double displacement
We have previously reported on our work on activity-
based protein profiling of human retaining β-exoglucosid-
ases.[7–9] In those studies we capitalized on the remarkable
ability of compounds 1 and 2 to select and inactivate these
glycosidases in complex biological samples, ranging from
cell extracts to cells and animal models. Grafting a reporter
group at C8 of cyclophellitol, through an azide group
installed for this purpose at C8 [as in 8-deoxy-8-azido-
cyclophellitol (4), see Figure 2], allowed us to detect and
image human acid glucosylceramidase, or GBA, with high
selectivity and efficiency in the context of healthy and
[a] Leiden Institute of Chemistry, Leiden University,
Einsteinweg 55, 2300 RA Leiden, The Netherlands
E-mail: h.s.overkleeft@chem.leidenunv.nl
http://biosyn.lic.leidenuniv.nl
[b] Stratingh Institute of Chemistry, University of Groningen,
Nijenborgh 7, 9747 AG Groningen, The Netherlands
[c] Department of Medical Biochemistry, Academic Medical
Centre, University of Amsterdam,
Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands
[‡] K.-Y. Li and J. Jiang contributed equally to this work
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201402588.
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Cyclophellitol, Cyclophellitol Aziridine, and Tagged Derivatives
(Gaucher) diseased tissue.[7,8] Placing a reporter group on
the cyclophellitol aziridine nitrogen, through its acyl conge-
ner 5, enabled us to achieve broad profiling of all human
retaining β-glucosidase activities: in addition to GBA, non-
lysosomal β-glucosidase (GBA2), cytosolic (broad-specific-
ity) β-glucosidase (GBA3), and the β-glucosidase activity
presented by the intestinal lactase-phlorizin hydrolase
(LPH).[9]
Results and Discussion
Access both to epoxides and to aziridines bearing a vari-
ety of reporter moieties required easy access to key interme-
diate 3. For this purpose we revisited the work of Madsen
and co-workers,[10] and this led (see Scheme 1) to a modi-
fied, and in places optimized, synthetic route. Consistently
with the literature procedure,[11] selective triphenylphos-
phine-mediated substitution of the primary hydroxy group
in methyl xylofuranoside (6, α/β mixture, prepared by ki-
netic Fischer glycosylation of d-xylose in methanolic HCl;
see Exp. Sect.) with iodine provided iodides 7. Ensuing
benzylation of the two secondary alcohols in 7 proceeded
with complete conversion. However, this transformation
was accompanied by rearrangement of the benzylating
agent – benzyl trichloroacetimidate (which we needed to
add in excess) – to the corresponding benzyl trichloroacet-
amide, which we found difficult to separate from the desired
product 8. Subsequent reductive Vasella fragmentation of 8
contaminated with this acetamide proved suboptimal (39%
yield) and we thus turned to a somewhat more lengthy, but
ultimately higher-yielding route.
Figure 2. Key intermediate 3, targets cyclophellitol (1) and cyclo-
phellitol aziridine (2), and their azide derivatives 4 and 5, respec-
tively, that are the subjects of the synthetic studies presented here.
In this sequence, the primary hydroxy groups in methyl
xylofuranosides 6 were temporarily protected as trityl
The synthesis of key intermediates 4 and 5 was based on ethers. Next, the secondary hydroxy groups were benzylated
cyclohexene derivative 3 (Figure 2, Scheme 1), previously under basic conditions (BnBr, NaH, DMF), after which the
described by Madsen and co-workers in their paper on the primary hydroxy groups were liberated and substituted for
synthesis of (+)-cyclophellitol (1).[10] Here we report in de- iodine under the conditions described above. In this vein,
tail on the synthesis of compounds 1–5, on the difficulties iodoxylosides 8 were obtained in high purity in 47% yield
we encountered, both in the synthesis and in the purifica- over the four steps, with the Vasella fragmentation (zinc
tion steps, and on the optimized routes that emerged from dust, sonication) proceeding in 96% yield.
tackling these. We describe a palette of activity-based
The transformation of aldehyde 10 into key intermediate
probes varying in the reporter moiety (biotin, fluorophore, 3 proceeded by essentially the route published[10] by
combination of both) that we derived from azide derivatives Madsen and co-workers. The key step in this sequence of
4 and 5.
events is the lanthanum-triflate-catalyzed Barbier ad-
Scheme 1. Reagents and conditions: (a) I2, PPh3, imidazole, THF, 70 °C, 7:76%, 8: 88%; (b) BnOC(=NH)CCl3, TfOH, dioxane, 75%;
(c) (i) TrCl, Et3N, DMAP, DMF, (ii) NaH, BnBr, TBAI, DMF, 0 °C to room temp., (iii) p-TosOH, MeOH, CH2Cl2, 53% over four steps;
(d) zinc dust, THF/H2O (9:1, v/v), ultrasound, 40 °C, 96%; (e) indium powder, La(OTf)3, H2O, ultrasound, 80%; (f) second-generation
Grubbs catalyst, CH2Cl2, 40 °C, 89%; (g) (i) DIBAL-H, THF, 0 °C to room temperature, (ii) NaBH4, H2O, EtOAc, 94%.
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dition[12] of the indium Grignard reagent generated in situ gas afforded (+)-cyclophellitol (1) in good yield and pu-
from ethyl 4-bromocrotonate (11) to aldehyde 10. When this rity.[15]
reaction was performed on a small scale and the prescribed
Alternatively, selective tosylation of the primary hydroxy
indium reagent (60 mesh, 99.999% pure) was used, the de- group in 3, followed by azide substitution, afforded azide
sired diene 12 was obtained in good yield and stereoselecti- derivative 15. Because the azide functional group, installed
vity (13:1 diastereomeric ratio with respect to the newly for modification with reporter tags as demonstrated below,
generated chiral center, 12 obtained after purification in is not compatible with palladium-catalyzed hydrogenation
81% yield). The process could be performed on a large scale reactions, we decided to change the protective group
with equal efficiency (2 mmol vs. 40 mmol), but the cost pattern at this stage. In a one-pot, two-step procedure, the
of expensive 60 mesh indium powder became a significant benzyl ethers in 15 were removed by treatment with anhy-
factor. When the reaction vessel was placed in an ultra- drous BCl3 in dichloromethane. Upon completion of the
sound bath we found that the Barbier addition was com- reaction the mixture was concentrated and co-evaporated
plete overnight. Moreover, in this approach we found that from toluene to remove traces of the Lewis acid and the
the considerably less expensive indium powder containing formed benzyl chloride. The crude mixture was then treated
1% magnesium as anticaking agent performed equally well, with benzoyl chloride and pyridine to afford fully protected
and diene 12 could be prepared in 80% yield on a 44 mmol cyclohexene derivative 16 in 70% yield over the two steps.
scale.
Compound 16 lacks a (homo)allylic alcohol to guide the
Diene 12 was converted into cyclohexene 13 by treatment stereoselective epoxidation (as in the transformation of 3 to
with the second-generation Grubbs metathesis catalyst[13] in 14), and mCPBA-mediated epoxidation of 16 proceeded
dichloromethane at 40 °C. Optimal results were obtained with poor yield and resulted in the predominant formation
when 2–5 mol-% of the catalyst were added. Use of less cat- of 1,6-epi-cyclophellitol derivative 17. The desired protected
alyst or of the first-generation Grubbs catalyst[14] led to 8-deoxy-8-azidocyclophellitol isomer 18 could be obtained
lower yields and the formation of an unidentified re- in 20% yield by use of methyl(trifluoro)-dioxirane (formed
gioisomer. Reduction of the ethyl ester in 13 was ac- in situ from trifluoroacetone and oxone) as the oxidizing
complished in two steps by initial transformation into the agent, though also in this case the 1,6-epi-isomer 17 was
aldehyde (DIBAL-H) and subsequent reduction to the pri- formed as the major isomer (49%). Removal of the benzoyl
mary alcohol (NaBH4, H2O, EtOAc). Overall, key interme- esters finally proceeded uneventfully under Zemplén condi-
diate 3 can be produced on a 30 mmol scale in 30% overall tions to give target compound 4. Consistently with the
yield over five steps starting from 6.
study by Ogawa and co-workers, no methoxide attack on
Scheme 2 depicts the transformation of cyclohexene 3 the epoxide functionality was observed.[16]
into (+)-cyclophellitol (1) and 8-deoxy-8-azidocyclo-
phellitol (4).
Scheme 3 depicts an alternative and more efficient syn-
thesis of 8-deoxy-8-azidocyclophellitol (4). By starting from
Epoxidation of the homoallylic alcohol in 3 with meta- (+)-cyclophellitol (1), which we could prepare in sufficient
chloroperbenzoic acid went according to the literature pro- quantities by the procedures outlined in Schemes 1 and 2,
cedure in a stereoselective fashion. Optimal results were ob- orthogonally protected derivative 19 was readily prepared
tained when the epoxidation was carried out in a buffered in two steps by initial installation of the 4,8-di-tert-butylsil-
biphasic solvent system (1 m aqueous Na2HPO4, 1 m aque- ylidene protective group and subsequent acetylation of the
ous NaH2PO4, DCE) at 50 °C. Performing the reaction in two remaining secondary alcohol groups. HF/pyridine-
a non-buffered solution, with or without an inorganic base mediated removal of the silylidene protective group and se-
(NaHCO3, K2HPO4, or Na2HPO4) at ambient temperature lective triflation of the primary alcohol in 20 gave the corre-
led to a lower yield and a prolonged reaction time. sponding triflate, and azide substitution followed by basic
Hydrogenolysis with Pearlman’s catalyst and dihydrogen methanolysis of the acetate protective groups afforded 8-
Scheme 2. Reagents and conditions: (a) mCPBA, Na2HPO4 (aq., 1 m), NaH2PO4 (aq., 1 m), dichloroethane, 50 °C, 55%; (b) H2, Pd(OH)2,
MeOH, 88%; (c) (i) p-TosCl, Et3N, CH2Cl2, (ii) NaN3, DMF, 60 °C, 71%; (d) (i) BCl3, CH2Cl2, –78 °C, (ii) BzCl, pyridine, 70%; (e) oxone,
CF3OCH3, NaHCO3, acetonitrile, Na2EDTA (4 mm), 0 °C, 17, 49%, 18, 20%; (f) NaOMe, MeOH, 75%.
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Cyclophellitol, Cyclophellitol Aziridine, and Tagged Derivatives
deoxy-8-azidocyclophellitol (4). In this fashion, target com-
The procedure presented in Scheme 4 allows for the prep-
pound 4 was prepared in six steps and 49% overall yield aration of sufficient quantities of cyclophellitol aziridine (2)
from (+)-cyclophellitol (1) with complete stereo- and regi- for further modification. Acylation of the aziridine nitrogen
ochemical control.
with a functionalized (azide or alkyne) carboxylic acid,
however, proved much more complicated than we had ini-
tially foreseen. Condensation of 2 with hept-6-ynoic acid
under the agency of N,NЈ-diisopropylcarbodiimide (DIC)
proved abortive in that no acylated product could be iso-
lated. Treatment of 2 with the acyl chloride of hept-6-ynoic
acid did result in N-acylation, but the N-acylaziridine
proved highly susceptible to nucleophilic attack by the
chloride ion generated in situ, to yield a mixture of trans-
chlorocyclitolamine. Attempts to acylate with the N-hy-
droxysuccinimide ester of hept-6-ynoic acid proved abortive
and returned the starting material. From these initial stud-
ies we identified two complications. To start with, the azir-
idine nitrogen is less nucleophilic than “normal” aliphatic
amines, and, once acylated, the aziridine carbon atoms are
rather susceptible to nucleophilic attack.
In view of these factors we turned our attention to the
use of 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline
(EEDQ), an activating agent that upon reaction yields a non-
nucleophilic quinoline derivative as byproduct.[17] In a first
experiment, treatment of hept-6-ynoic acid with EEDQ fol-
lowed by addition of substoichiometric amounts of 2 af-
forded a mixture of N- and O-acylated products, with the
N-acyl aziridine moiety intact and acylation of the primary
hydroxy in 2 as the predominant side reaction. This side re-
action could be prevented by executing the reaction at 0 °C
and quenching by addition of methanol prior to workup.
By the same procedure as described for the synthesis of
alkyne 24, azide-modified aziridine 5 was prepared with
equal efficiency (Scheme 5). Both compounds 5 and 24
could be obtained in high purity after preparative HPLC.
In order to avoid opening of the acyl aziridine ring at this
stage we searched for optimal conditions. It turned out that
the standard conditions we normally use for reversed-phase
HPLC purification (water/acetonitrile gradient with tri-
fluoroacetic acid or formic acid) are detrimental to the acyl
aziridine, which opens either during the HPLC run or dur-
ing ensuing lyophilization. The acyl aziridine systems in 5
and 24 remained intact when either a neutral water/aceto-
nitrile gradient or a gradient including 25–50 mm aqueous
NH4HCO3 was used. On concentration of the NH4HCO3-
containing fractions the pH was kept between 7–8 to pre-
vent liberation of the acyl linker (see the Exp. Sect. for de-
tails). At all times after N-acylation we found that the
major decomposition routes involved chloride attack on the
acyl aziridine. These side products arose on HPLC purifica-
tion even though no chloride salts had been present during
the acylation steps. We found that this side reaction can
be suppressed when workup after the Birch reduction steps
(Scheme 4) was performed with chloride-free milliQ water.
In the end, compounds 5 and 24 were obtained in sufficient
quantities to allow elaboration to tagged activity-based
probes, as demonstrated below.
Scheme 3. Reagents and conditions: (a) (i) (tBu)2SiOTf2, pyridine,
DMF, –40 °C to 0 °C, (ii) Ac2O, pyridine, 60%; (b) HF/pyridine,
THF, 0 °C, quant; (c) (i) Tf2O, pyridine, CH2Cl2, –25 °C, (ii) NaN3,
15-crown-5, THF, –25 °C to 0 °C, 90%; (d) NaOMe, MeOH, 90%.
Scheme 4 depicts our optimized route towards cyclo-
phellitol aziridine (2). Introduction of the trichloroacet-
imidate functionality at the primary alcohol in 3 set the
stage for a stereospecific iodocyclization. Thus, treatment
of 3 with trichloroacetonitrile under basic conditions, fol-
lowed by treatment of the intermediate trichloroacetimidate
with iodine, afforded iodide 22 with complete stereocontrol.
Acidic hydrolysis of the trichloroacetimidate group in 23
was followed by base-induced intramolecular substitution
of the iodine to give partially protected cyclophellitol azir-
idine derivative 23. Removal of the two benzyl ether groups
in 23 under palladium-catalyzed hydrogenolysis conditions
[H2, Pd(OH)2] resulted in partial reduction of the aziridine
moiety. Lewis-acid-mediated debenzylation (BCl3, CH2Cl2)
proved abortive as well, and we therefore turned our atten-
tion to Birch reduction conditions. Treatment of 23 with
lithium in liquid ammonia afforded target compound 2 in
70% yield. Removal of the lithium hydroxide salts formed
during the Birch reduction was achieved by cation-exchange
chromatography first with an Amberlite H+ and next with
+
an Amberlite NH4 resin. Overall, cyclophellitol aziridine
(2) was prepared from cyclohexene 3 in 42% overall yield
over the five steps.
Scheme 4. Reagents and conditions: (a) (i) Cl3CN, DBU, CH2Cl2,
0 °C, (ii) I2, NaHCO3, H2O; (b) (i) 37% HCl, dioxane, 60 °C,
(ii) NaHCO3, MeOH, 60% over four steps; (c) Li, NH3, THF,
–60 °C, 70%.
With the azide- and alkyne-modified cyclophellitol and
cyclophellitol aziridine derivatives 4, 5, and 24 available in
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Scheme 5. Reagents and conditions: (a) (i) hept-6-ynoic acid, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ), DMF, 0 °C,
(ii) HPLC, gradient MeCN/H2O, 20%; (b) (i) 8-azidooctanoic acid, EEDQ, DMF, 0 °C, (ii) HPLC gradient MECN/H2O, 19 %.
sufficient quantities, a number of fluorescent and/or bio- alkynes 25–33 (see Exp. Sect. for their exact structures[19]
)
tinylated tags were installed. In each case the conjugation and the primary azide group in cyclophellitol derivative 4.
proceeded through copper(I)-catalyzed azide–alkyne In this fashion, by using symmetrical BODIPY-alkynes that
Huisgen [3+2] cycloaddition with the appropriate we had reported previously,[19a] we prepared the two red-
BODIPY/biotin alkyne or azide.[18] The click ligations pro- fluorescent and green-fluorescent cyclophellitol derivatives
ceeded uneventfully, with their purification presenting the 34 and 35 that we had used previously for the in situ moni-
most important obstacle, especially when dealing with the toring of GBA in the context of Gaucher disease. In the
acylaziridines, which are labile under both acidic and basic same fashion, biotin-cyclophellitol 36 was prepared. To
conditions as noted before.
demonstrate the viability of the strategy further we pre-
Scheme 6 shows the panel of cyclophellitol-derived ac- pared the cyclophellitol-derived activity-based retaining
tivity-based probes that we prepared through copper(I)-cat- glucosidase probes 37–40, bearing pH-responsive BODIPY
alyzed azide–alkyne click reactions between a number of dyes,[19c] varying from non-responsive (or permanently fluo-
Scheme 6. Reagents and conditions: (a) alkyne tag 25–32 (see Exp. Sect.), CuSO4, sodium ascorbate, toluene/tBuOH/H2O, 80 °C.
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Cyclophellitol, Cyclophellitol Aziridine, and Tagged Derivatives
rescent, compound 37) to responsive at various pH values phellitol aziridine 5 (Scheme 8). By the synthesis and purifi-
(4.5 to 6, 38–40). As a final example, doubly tagged[19b] cy- cation strategy outlined above for the cyclophellitol deriva-
clophellitol 41, equipped with both a BODIPY dye for visu- tives 33–35, the corresponding cyclophellitol aziridine de-
alization and a biotin for pull-down of potential reactive rivatives 44 and 45, bearing the same BODIPY-green and
glucosidases, was prepared.
BODIPY-red tag, respectively, and biotin-Ahx-cyclo-
The synthesis of BODIPY-green-cyclophellitol aziridine phellitol aziridine derivative 46 were obtained.
43 starting from alkyne 24 and azide 42 is depicted in
Scheme 7 and follows essentially the same conditions as
outlined in Scheme 6. As before, HPLC purification of acyl-
Conclusions
aziridine 43 was not straightforward, but use of the opti-
mized conditions based on a neutral gradient readily af-
forded the target compound after lyophilization.
In conclusion, we describe in this paper the full details
of the synthesis of a broad panel of inhibitors and activity-
based probes for retaining β-glucosidases, based on the
known inhibitors (+)-cyclophellitol (1) and cyclophellitol
aziridine (2). The synthetic strategy followed for both com-
pound classes is based on advanced intermediate 3 pre-
viously reported by Madsen and co-workers,[10] the synthe-
sis of which we have optimized by following the general
route as published and carefully optimizing the individual
steps, as well as the order in which some of the transforma-
tions are executed. We believe that the collective versatility
of these routes of synthesis should allow adaptation di-
rected towards a number of cyclophellitol-type mechanism-
based glycosidase inhibitors and activity-based probes. Re-
sults from this work provide guidelines for the synthesis of
derivatives of 1,6-epicyclophellitol and of 1,6-epicyclophel-
litol aziridine for the construction of retaining α-gluco-
sidase inhibitors and activity-based probes bearing reporter
tags similar to those demonstrated here. Adaptation of the
Scheme 7. Reagents and conditions: (a) (i) BODIPY-N3 42, CuSO4,
sodium ascorbate, DMF, 45%, (ii) HPLC (gradient MeCN/H2O).
By starting from 24 and employing copper(I)-catalyzed starting materials might lead to cyclophellitol derivatives
azide–alkyne cycloaddition, a diverse series of probes de- that emulate other carbohydrates in absolute stereochemis-
rived from cyclophellitol aziridine can, on paper, be synthe- try and that are able, by virtue of this, to target retaining
sized. However, because we have a number of fluorescent glycosidases that employ a Koshland-type double displace-
and/or biotinylated alkynes 25–26 and 33[18] (see Exp. Sect.) ment mechanism and have evolved to recognize other sub-
available, we switched to the use of azide-modified cyclo- strate glycosides.
Scheme 8. Reagents and conditions: (a) (i) tag alkyne 25–26 or 33 (see Exp. Sect.), CuSO4, sodium ascorbate, DMF, (ii) HPLC (gradient
MeCN/H2O).
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Note: NMR signal assignments and stereochemical descriptors
were made according to the atom numbering shown in the formulae
below.
Experimental Section
General Methods and Materials: All reagents and solvents were of
commercial grade and used as received unless stated otherwise.
THF and dichloromethane were stored over flamed-dried 3 Å mo-
lecular sieves. All reactions were performed under inert atmosphere
unless stated otherwise. Solvents used for flash chromatography
were of pro analysi quality. Reactions were monitored by TLC
analysis on aluminum sheets pre-coated with silica gel 60, with de-
tection by UV absorption (254 nm) and by spraying with a solution
of (NH4)6Mo7O24·H2O (25 gL–1) and (NH4)4Ce(SO4)4·H2O
(10 gL–1) in sulfuric acid (10%), followed by charring at ca. 150 °C,
or by spraying with sulfuric acid in ethanol (20%), followed by
charring at ca. 150 °C. Column chromatography was performed
with Screening Device silica gel in the indicated solvents. High-
resolution mass spectra were recorded with a LTQ Orbitrap instru-
ment (Thermo Finnigan). HPLC-MS purifications were performed
with an Agilent Technologies 1200 series automated HPLC system
and a Quadropole MS 6130 fitted with a semi-preparative Gem-
ini C18 column (Phenomenex, 250ϫ10, 5μ, particle size). 1H
NMR, 13C NMR, COSY, and HSQC spectra were recorded with
General HPLC Purification of Acylated Cyclophellitol Aziridines:
Purification of acylated cyclophellitol aziridines 5, 24, 42–45 by
HPLC can be performed either under neutral conditions (water/
acetonitrile) as described below or under basic conditions (25–
50 mm NH4HCO3/acetonitrile). When purifying under basic condi-
tions, the pH during the HPLC run, collection, and lyophilization
from tBuOH/water/acetonitrile is carefully kept between pH 7–8.
Methyl 2,3-Di-O-benzyl-α-D-xylofuranoside (9): d-Xylose (15.0 g,
100 mmol) was added to a solution of acetyl chloride (3 mL,
42 mmol) in MeOH (300 mL). The reaction mixture was stirred for
5 h, after which it was neutralized with NaHCO3. The solution
was filtered and concentrated under reduced pressure. The resulting
product was taken up in a solution of MeOH/EtOAc (1:4, v/v,
500 mL), filtered, and concentrated again in vacuo in order to re-
move the remaining salts. The crude methyl xylofuranoside was
used without further purification. The crude product was dissolved
in DMF (500 mL), and trityl chloride (56 g, 200 mmol), Et3N
(22.3 mL, 160 mmol), and DMAP (610 mg, 0.05 mmol) were
added. The reaction mixture was stirred for 18 h and concentrated
under reduced pressure. The resulting oil was subsequently redis-
solved in Et2O, washed with water, dried with MgSO4, filtered, and
concentrated. The resulting oil was again taken up in DMF
(500 mL), and BnBr (29.7 mL, 250 mmol) and a catalytic amount
of TBAI (1.85 g, 5 mmol) were added, after which the solution was
cooled to 0 °C. Sodium hydride (60% dispersion in mineral oil,
17.6 g, 440 mmol) was added, and the reaction mixture was stirred
at ambient temperature. After 18 h, when TLC showed full con-
sumption of the starting material, the reaction mixture was cooled
to 0 °C and quenched by the addition of MeOH. The solution was
further diluted with water and subsequently washed with Et2O,
dried with MgSO4, filtered, and concentrated in vacuo. The re-
sulting product was redissolved in CH2Cl2 (250 mL) and MeOH
(250 mL), and p-toluenesulfonic acid was added until pH 2 was
reached. The reaction mixture was stirred at ambient temperature
for 18 h, after which it was quenched with saturated NaHCO3. The
layers were separated, and the water layer was extracted with
EtOAc. The combined organic layers were dried with MgSO4, fil-
tered, and concentrated under reduced pressure. Purification by sil-
ica column chromatography (pentane/EtOAc 70:30Ǟ60:40) gave
the partly deprotected methyl xylofuranoside (18.1 g, 52.5 mmol,
53%) as an anomeric mixture (α/β ratio 2:1). 1H NMR (400 MHz,
CDCl3): δ = 7.37–7.21 (m, 20 H, HArBn), 4.89 (d, J = 1.6 Hz, 1 H,
H-1α), 4.79 (d, J = 4.4 Hz, H-1β), 4.70 (d, J = 12.0 Hz, 1 H,
CH2Bn), 4.64–4.45 (m, 7 H, CH2Bn), 4.24 (t, J = 6.8 Hz, 1 H, H-
2β), 4.32–4.28 (m, 1 H, H-2α), 4.21–4.19 (m, 1 H, H-4α), 4.15 (dd,
J = 3.6, 6.8 Hz, 1 H, H-4β), 4.09–4.07 (m, 1 H, H-3α), 4.04 (dd, J
= 4.4, 6.4 Hz, 1 H, H-3β), 3.79–3.73 (m, 1 H, H-5α-β), 3.73 (s, 3
H, OMe-α), 3.36 (s, 3 H, OMe-β) ppm. 13C NMR (100 MHz,
CDCl3): δ = 137.4, 137.3, 128.3, 128.2, 127.9, 127.8, 127.7, 127.6,
127.5, 127.5, 107.7, 99.9, 86.8, 84.2, 82.4, 81.9, 80.4, 76.1, 72.4,
72.2, 72.1, 71.9, 62.0, 61.9, 55.4, 54.8 ppm. HRMS calcd. for
[C20H24O5 + Na] [M + H]+ 367.39154; found 367.39162.
a
Bruker DMX 400 (400/100 MHz), a Bruker AV 400 (400/
100 MHz), and/or a Bruker AV 600 (600/150 MHz) spectrometer
in the given solvent. Chemical shifts are reported as δ values in
ppm relative to the chloroform residual solvent peak or tetrameth-
ylsilane (TMS) as internal standard or the deuterated solvent signal
for CD3OD. Coupling constants are given in Hz. All given 13C
spectra are proton-decoupled.
Alkyne Tags 25–33 Used in this Study:
6036
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© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 6030–6043

Cyclophellitol, Cyclophellitol Aziridine, and Tagged Derivatives
Methyl 2,3-Di-O-benzyl-5-deoxy-5-iodo-α- -xylofuranoside (8):
Alcohol 9 (18.1 g, 52.5 mmol) was coevaporated with toluene, after
which it was dissolved in anhydrous THF (200 mL). Triphenylphos-
D
J = 7.0 Hz, 2 H, CH2 ester), 3.98 (d, J = 9.2 Hz, 1 H, 4-H), 3.54
(d, J = 7.6 Hz, 1 H, 3-H), 3.28 (d, J = 9.6 Hz, 1 H, 5-H), 1.22 (t,
J = 7.2 Hz, 3 H, CH3 ester) ppm. 13C NMR (100 MHz, CDCl3): δ
phine (20.7 g, 78.8 mmol) and imidazole (7.15 g, 105 mmol) were = 172.4, 138.4, 138.3, 134.8, 132.8, 128.3, 128.3, 127.9, 127.8, 127.7,
added to the solution, which was heated to reflux. A solution of
iodine (20.8 g, 78.8 mmol) in anhydrous THF (60 mL) was added,
and the reaction mixture was heated at reflux until TLC analysis
revealed full conversion of the starting material (3.5 h). The reac-
tion mixture was successively concentrated under reduced pressure,
redissolved in EtOAc, and washed with sodium thiosulfate (10%).
The organic layer was dried with MgSO4 and concentrated in
vacuo. Purification by silica column chromatography (pentane/
EtOAc 98:2 Ǟ80:20) afforded iodine 8 (α/β ratio 4:1, 21.0 g,
127.5, 119.9, 119.9, 82.8, 79.3, 74.5, 72.0, 70.7, 60.8, 55.1,
14.0 ppm. HRMS: calcd. for [C25H31O5] [M + H]+ 411.21660;
found 411.21653.
Ester 13: The second-generation Grubbs catalyst (753 mg,
0.89 mmol, 2.5 mol-%) was added to a solution of 12 (14.6 g,
35.5 mmol) in dichloromethane (180 mL). The reaction mixture
was heated at reflux in the dark for 18 h under a continuous flow
of argon at 40 °C, after which an additional quantity of the second-
generation Grubbs catalyst (603 mg, 0.71 mmol, 2.0 mol-%) was
added. The reaction mixture was again heated at reflux in the dark
under a continuous flow of argon until TLC showed complete con-
sumption of the starting material (6 h). The reaction mixture was
then concentrated and purified by silica column chromatography
(pentane/EtOAc 92:8Ǟ84:16) to afford 13 (12.2 g, 32.0 mmol,
89%) as an oil. 1H NMR (400 MHz, CDCl3): δ = 7.34–7.23 (m, 10
H, HArBn), 5.79 (dt, J = 2.4, 10.0 Hz, 1 H, 6-H), 5.66 (dt, J = 2.0,
10.4 Hz, 1 H, 1-H), 4.94 (d, J = 11.2 Hz, 1 H, CH2Bn), 4.79 (d, J
= 11.2 Hz, 1 H, CH2 Bn), 4.66 (dd, J = 11.6, 15.6 Hz, 2 H, CH2
Bn), 4.23–4.11 (m, 4 H, 3-H, 4-H, CH2 ester), 3.64 (dd, J = 7.6,
9.6 Hz, 1 H, 2-H), 3.26–3.23 (m, 1 H, 5-H), 3.04 (br., 1 H, OH),
1.26 (t, J = 8.4 Hz, CH3 ester) ppm. 13C NMR (100 MHz, CDCl3):
δ = 171.8, 138.3, 137.9, 128.4, 128.1, 127.8, 127.8, 127.7, 127.7,
123.9, 82.4, 79.1, 74.7, 71.8, 70.3, 61.2, 50.0, 14.0 ppm. HRMS:
calcd. for [C23H27O5] [M + H]+ 383.18530; found 381.18548.
1
46.2 mmol, 88%) as a yellow oil. H NMR (400 MHz, CDCl3): δ
= 7.32–7.26 (m, 20 H, HArBn), 4.91 (s, 1 H, H-1α), 4.82 (d, J =
3.6 Hz, H-1β), 4.61–4.36 (m, 8 H, H-2α-β, H-4α-β, 2ϫCH2Bn α-
β), 4.00–3.96 (m, 2 H, H-3α-β), 3.40–3.30 (m, 7 H, OMe-α-β, H-
5α), 3.19–3.15 (m, 1 H, H-5β) ppm. 13C NMR (100 MHz, CDCl3):
δ = 137.6, 137.4, 137.3, 137.2, 128.4, 128.3, 128.3, 128.0, 127.8,
127.8, 127.6, 127.6, 108.4, 100.6, 86.5, 83.6, 81.9, 817, 81.5, 77.5,
72.5, 72.4, 72.3, 71.9, 55.8, 55.3, 4.72, 3.11 ppm. HRMS: calcd. for
[C20H23IO4 +Na] [M + Na]+ 477.05332; found 477.05332.
(2R,3S)-2,3-Bis(benzyloxy)pent-4-enal (10): Zinc dust was activated
and dried immediately before use: it (54.4 g, 832 mmol) was added
slowly to a vigorously stirred HCl solution (3 m, 480 mL). The mix-
ture was stirred for 20 min at ambient temperature, filtered, and
subsequently rinsed with water and Et2O, after which it was dried
under high vacuum at elevated temperature. A solution of iodide 8
(21.0 g, 46.2 mmol) in THF/H2O (9:1, v/v, 520 mL) was sonicated
under an argon stream at 40 °C, after which the activated zinc dust
(45.3 g, 693 mmol) was added. The reaction mixture was flushed
again with argon and sonicated for 2.5 h at 40 °C, followed by fil-
tration through a pad of Celite and removal of THF in vacuo. The
resulting mixture was diluted with Et2O, washed with H2O, dried
with MgSO4, filtered, and concentrated under reduced pressure.
Purification by silica column chromatography (pentane/Et2O
98:2Ǟ90:10) afforded the relatively unstable aldehyde 10* (13.1 g,
Cyclohexene 3: Ester 13 (12.2 g, 32.0 mmol) was coevaporated with
toluene (3ϫ), after which it was dissolved in THF (320 mL).
DIBAL-H (1 m in hexane, 192 mL, 192 mmol) was added to the
solution at 0 °C. The reaction mixture was stirred for 30 min at
0 °C, followed by stirring for 2 h at ambient temperature. It was
then quenched with EtOAc (65 mL) at 0 °C, followed by addition
of H2O (75 mL) and sodium borohydride (7.87 g, 208 mmol). The
reaction mixture was stirred for 18 h at ambient temperature, after
which it was concentrated in vacuo. The crude product was poured
into EtOAc and washed with HCl solution (1 m). The organic layer
was dried with MgSO4 and concentrated in vacuo. Purification by
1
44.4 mmol, 96%) as a clear oil. H NMR (400 MHz, CDCl3): δ =
9.64 (s, 1 H, CHO), 7.30–7.17 (m, 10 H, HArBn), 5.96–5.87 (m, 1
H, CH=), 5.34–5.28 (m, 2 H, CH2=), 4.70 (d, J = 12.0 Hz, 1 H, silica column chromatography (pentane/EtOAc 60:40Ǟ40:60)
1
CH2Bn), 4.58 (t, J = 12.4 Hz, 2 H, CH2Bn), 4.32 (d, J = 12.0 Hz,
1 H, CH2Bn), 4.14 (dd, J = 4.0, 7.2 Hz, 1 H, CHCH=), 3.80 (d, J
yielded diol 3 (10.2 g, 30 mmol, 94%) as a colorless oil. H NMR
(400 MHz, CDCl3): δ = 7.30–7.20 (m, 10 H, HArBn), 5.73 (dt, J =
= 3.2 Hz, 1 H, CHCHO) ppm. 13C NMR (100 MHz, CDCl3): δ = 2.4, 10.0 Hz, 1 H, 6-H), 5.48 (dt, J = 2.0, 10.0 Hz, 1 H, 1-H), 4.98
202.4, 137.3, 136.9, 133.7, 128.2, 128.1, 127.9, 127.8, 127.4, 127.5,
119.5, 84.9, 79.6, 73.4, 70.6 ppm. HRMS: calcd. for [C19H20O3] [M
+ H]+ 297.14852; found 297.14922.
* Note: Aldehyde 10 is stable at –24 °C for up to several months,
whereas it decomposes at room temperature after 1 day.
(d, J = 11.2 Hz, 1 H, CH2Bn), 4.73 (d, J = 11.2 Hz, 1 H, CH2Bn),
4.64 (d, J = 9.2 Hz, 1 H, CH2Bn), 4.60 (d, J = 11.6 Hz, 1 H,
CH2Bn), 4.17–4.15 (m, 1 H, 2-H), 3.71–3.60 (m, 4 H, 3-H, 4-H, 8-
H), 3.28 (br., 1 H, OH), 2.99 (br., 1 H, OH), 2.44–2.43 (m, 1 H, 5-
H) ppm. 13C NMR (100 MHz, CDCl3): δ = 138.3, 137.9, 128.4,
128.3, 127.8, 127.7, 127.7, 127.6, 127.5, 127.2, 83.3, 80.1, 74.7, 72.1,
71.3, 64.6, 45.1 ppm. HRMS: calcd. for [C21H25IO4] [M + H]+
341.17474; found 341.17501.
Alkene 12: Ethyl 4-bromocrotonate (11, 26 mL, 142 mmol), La-
(OTf)3 (54.7 g, 93.2 mmol), and indium powder (11.7 g, 102 mmol)
were added to a solution of aldehyde 10 (13.1 g, 44.4 mmol) in H2O
(210 mL). After sonication at 40 °C for 18 h, the reaction mixture
was filtered through a plug of celite, extracted with Et2O, dried with
MgSO4, and concentrated under reduced pressure. Purification by
silica column chromatography (pentane/EtOAc 94:6Ǟ88:12)
yielded 12 (14.6 g, 35.5 mmol, 80%) as a clear oil. 1H NMR
(400 MHz, CDCl3): δ = 7.50–7.25 (m, 10 H, HArBn), 5.87–5.78 (m,
1 H, 1-H), 5.71 (dt, J = 9.6, 17.2 Hz, 1 H, 6-H), 5.42 (d, J =
12.0 Hz, 1 H, CH2=CHCHOBn), 5.38 (d, J = 4.8 Hz, 1 H,
CH2=CHCHOBn), 5.16 (d, J = 10.4 Hz, 1 H, CH2=CHCHC=O),
5.06 (d, J = 17.2 Hz, 1 H, CH2=CHCHC=O), 5.01 (d, J = 11.2 Hz,
1 H, CH2Bn), 4.60 (dd, J = 12.2, 14.4 Hz, 2 H, CH2Bn), 4.40 (d,
2,3-Di-O-benzylcyclophellitol (14): A mixture of alkene 3 (3.40 g,
10 mmol) and mCPBA (4.44 g, 18 mmol) in DCE (166 mL),
NaH2PO4 (1 m, 100 mL), and NaH2PO4 (1 m, 100 mL) was vigor-
ously stirred at 50 °C for 18 h. The layers were separated, and the
water layer was extracted with EtOAc. The combined organic layers
were dried with MgSO4 and concentrated under reduced pressure.
The residue was purified by silica gel column chromatography
(pentane/Et2O 12:88Ǟ8:92) to provide β-epoxide 14 (1.94 g,
5.45 mmol, 55%) and its epidiastereoisomer (178 mg, 0.5 mmol,
5%) as a white solid.
1
β-Epoxide 14: H NMR (400 MHz, CDCl3): δ = 7.40–7.15 (m, 10
J = 11.6 Hz, 1 H, CH2Bn), 4.19 (t, J = 7.6 Hz, 1 H, 2-H), 4.10 (q, H, HArBn), 4.92 (d, J = 11.6 Hz, 2 H, CH2Bn), 4.79 (d, J =
Eur. J. Org. Chem. 2014, 6030–6043
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
6037

H. S. Overkleeft et al.
FULL PAPER
11.2 Hz, 2 H, CH2Bn), 4.65 (d, J = 11.2 Hz, 2 H, CH2Bn), 3.98
(dd, J = 6.4, 10.8 Hz, 1 H, 8-H), 3.85 (dd, J = 5.6, 10.8 Hz, 1 H,
MeOH at –78 °C. The solution was concentrated in vacuo to give
the triol intermediate, which was immediately used for benzo-
8-H), 3.79 (d, J = 7.6 Hz, 1 H, 2-H), 3.46 (t, J = 9.2 Hz, 1 H, 4- ylation. The crude product was coevaporated several times with
H), 3.37 (dd, J = 8.0, 10.0 Hz, 1 H, 3-H), 3.26 (d, J = 2.8 Hz, 1 H, anhydrous toluene, after which it was dissolved in pyridine (10 mL).
6-H), 3.14 (d, J = 4.0 Hz, 1 H, 1-H), 3.01 (br. s, 1 H, 1ϫOH), Benzoyl chloride (2.6 mL, 21.1 mmol) was added at 0 °C, and the
2.91 (br. s, 1 H, 1ϫOH), 2.15–2.06 (m, 1 H, 5-H) ppm. 13C NMR
(100 MHz, CDCl3): δ = 138.1 137.3, 128.5, 128.4, 128.0, 127.9,
127.8, 127.6, 83.4, 70.2, 74.8, 72.5, 68.3, 63.6, 54.8, 52.9, 43.3 ppm.
HRMS: calcd. for [C21H25O5] [M
357.16987.
reaction mixture was stirred for 18 h at ambient temperature. The
mixture was quenched with saturated aqueous NaHCO3, extracted
with EtOAc, dried with MgSO4, and concentrated in vacuo. Purifi-
cation by silica column chromatography (petroleum ether/EtOAc
96:4Ǟ94:6) afforded 16 (701.8 mg, 1.46 mmol, 70%) as a yellow
oil. 1H NMR (400 MHz, CDCl3): δ = 7.99 (d, J = 7.2 Hz, 2 H,
+
H]+ 357.16965; found
α-Epoxide: 1H NMR (400 MHz, CDCl3): δ = 7.41–7.17 (m,
HArBn), 4.97 (d, J = 11.2 Hz, CH2Bn), 4.79 (dd, J = 12.0, 21.2 Hz,
2 H, CH2Bn), 4.64 (d, J = 11.2 Hz, 1 H, CH2Bn), 3.86–3.77 (m, 3
H, 3-H, 8-H), 3.56 (t, J = 10.0 Hz, 1 H, 3-H), 3.41 (t, J = 10.0 Hz,
1 H, 4-H), 3.34–3.33 (m, 1 H, 6-H), 3.10 (d, J = 3.6 Hz, 1 H,
1-H), 2.88 (s, 1 H, OH), 2.54 (br., 1 H, OH), 2.15–2.10 (m, 1 H, 5-
H) ppm. 13C NMR (100 MHz, CDCl3): δ = 138.2, 137.9, 128.5,
128.4, 128.0, 127.9, 127.9, 125.5, 80.8, 79.4, 75.4, 72.5, 70.2, 62.5,
54.4, 54.0, 43.5 ppm. HRMS: calcd. for [C21H25O5] [M + H]+
357.16965; found 357.16989.
HArBz), 7.92 (d, J = 7.2 Hz, 2 H, HArBz), 7.84 (d, J = 7.2 Hz, 2
H, HArBz), 7.53–7.46 (m, 3 H, HArBz), 7.40–7.30 (m, 5 H, HArBz),
7.26–7.18 (m, 2 H, HArBz), 6.00–5.93 (m, 3 H, 2-H, 3-H, 6-H), 5.86
(d, J = 10.0 Hz, 1 H, 1-H), 5.72 (t, J = 9.2 Hz, 1 H, 4-H), 3.64 (dd,
J = 4.0, 12.4 Hz, 1 H, 8-H), 3.46 (dd, J = 6.4, 12.4 Hz, 1 H, 8-H),
2.99–2.97 (m, 1 H, 5-H) ppm. 13C NMR (100 MHz, CDCl3): δ =
166.0, 165.9, 133.3, 133.2, 133.1, 129.8, 129.7, 129.6, 129.4, 129.0,
128.9, 128.5, 128.4, 128.3, 126.2, 127.0, 72.7, 72.6, 70.3, 52.0,
42.5 ppm. HRMS: calcd. for [C28H25N3O6Na] [M
520.14791; found 520.14724.
+
Na]+
(+)-Cyclophellitol (1): A catalytic amount of Pd(OH)2 was added
to a solution of 14 (71.3 mg, 0.2 mmol) in MeOH (1 mL). The solu-
tion was stirred under H2 for 18 h, after which it was filtered
through a small pad of celite. Purification by silica column
chromatography (CH2Cl2/MeOH 84:16Ǟ80:20) afforded (+)-cyclo-
phellitol (1, 31 mg, 0.18 mmol, 88%) as a white solid. 1H NMR
(400 MHz, MeOD): δ = 4.00 (dd, J = 4.4, 10.8 Hz, 1 H, 8-H), 3.68
(dd, J = 9.2, 10.4 Hz, 1 H, 8-H), 3.64 (d, J = 8.0 Hz, 1 H, 2-H),
3.42 (d, J = 2.4 Hz, 1 H, 6-H), 3.21 (dd, J = 8.4, 10.0 Hz, 1 H, 3-
H), 3.13–3.08 (m, 2 H, 1-H, 4-H), 1.97 (ddt, J = 1.6, 3.6, 9.4 Hz,
1 H, 5-H) ppm. 13C NMR (100 MHz, MeOD): δ = 78.5, 72.8, 68.8,
62.5, 57.4, 56.0, 45.9 ppm. HRMS: calcd. for [C7H13O5] [M + H]+
177.07575; found 177.07576.
8-Azido-2,3,4-tri-O-benzoyl-8-deoxycyclophellitol (17 and 18): A
solution of Na2EDTA solution in H2O (0.4 mm, 3.1 mL) and tri-
fluoroacetone (1.34 mL, 15 mmol) were added to 16 (497 mg,
1.0 mmol) in acetonitrile (6.7 mL). A mixture of oxone (3.07 g,
5.0 mmol) and NaHCO3 (588 mg, 7.0 mmol) was added to the
solution over a period of 15 min. After the system had been stirred
at 4 °C for 4 h, an additional quantity of Na2EDTA in H2O
(0.4 mm, 1.5 mL), trifluoroacetone (0.7 mL, 7.5 mmol), and a mix-
ture of oxone (1.5 mg, 2.5 mmol) and NaHCO3 (290 mg, 3.5 mmol)
were added to the reaction mixture over a period of 15 min. The
reaction mixture was stirred at 0 °C for 30 min, after which it was
diluted with H2O. After extraction of the water layer with EtOAc,
the combined organic layers were dried with MgSO4 and concen-
trated in vacuo. Purification by silica column chromatography (pe-
troleum ether/Et2O 92:8Ǟ90:10 and 84:16Ǟ82:16) afforded 17
(254 mg, 0.49 mmol, 49%) and 18 (104 mg, 0.20 mmol, 20%) as
white crystals.
Azido-cyclohexene 15: p-Toluenesulfonyl chloride (1.04 g,
5.48 mmol) and triethylamine (0.90 mL, 6.57 mmol) were added to
a solution of 3 (1.24 g, 3.65 mmol) in dichloromethane (26 mL).
The solution was stirred at 40 °C for 5 h, after which it was poured
into HCl solution (1 m). The mixture was extracted with Et2O, and
the organic layer was dried with MgSO4, after which it was concen-
trated in vacuo to yield the crude tosylate, which was immediately
subjected to azidation. Sodium azide (2.40 g, 36.7 mmol) was
Compound 17: 1H NMR (400 MHz, CDCl3): δ = 8.02 (d, J =
7.2 Hz, 2 H, HArBz), 7.89 (d, J = 7.2 Hz, 2 H, HArBz), 7.78 (d, J
= 7.2 Hz, 2 H, HArBz), 7.53–7.19 (m, 5 H, HArBz), 5.96 (t, J =
9.6 Hz, 1 H, 3-H), 5.77 (d, J = 8.8 Hz, 1 H, 2-H), 5.55 (t, J =
9.6 Hz, 1 H, 4-H), 3.77–3.74 (m, 2 H, 6-H, 8-H), 3.64 (dd, J = 4.0,
12.8 Hz, 1 H, 8-H), 3.32 (s, 1 H, 1-H), 2.68 (ddd, J = 3.8, 5.2,
9.2 Hz, 1 H, 5-H) ppm. 13C NMR (100 MHz, CDCl3): δ = 166.0,
165.9, 165.6, 133.4, 133.0, 129.9, 129.7, 129.5, 129.0, 128.9, 128.6,
128.4, 128.3, 128.1, 72.1, 70.0, 69.9, 54.6, 53.8, 50.8, 40.9 ppm.
HRMS: calcd. for [C28H23N3O7] [M + H]+ 514.16017; found
514.16088.
added to
a solution of the tosylated intermediate (1.75 g,
3.65 mmol) in DMF (35 mL). The solution was stirred for 24 h at
60 °C, after which it was concentrated in vacuo. The crude product
was diluted with Et2O and successively washed with HCl (1 m),
saturated aqueous NaHCO3, and brine. The combined organic lay-
ers were dried with MgSO4 and concentrated under reduced pres-
sure. Purification by silica column chromatography (petroleum
ether/EtOAc 92:8Ǟ84:16) afforded 15 (900 mg, 2.46 mmol, 71%)
1
as a solid. H NMR (400 MHz, CDCl3): δ = 7.33–7.26 (m, 10 H,
Compound 18: 1H NMR (400 MHz, CDCl3): δ = 8.03 (d, J =
HArBn), 5.79 (dt, J = 2.4, 10.4 Hz, 1 H, 6-H), 5.58 (dt, J = 1.6, 7.4 Hz, 2 H, HArBz), 7.88 (d, J = 7.6 Hz, 2 H, HArBz), 7.79 (d, J
10.4 Hz, 1 H, 1-H), 5.02 (d, J = 11.2 Hz, 1 H, CH2Bn), 4.72–4.59
(m, 3 H, CH2 Bn), 4.18–4.16 (m, 1 H, 2-H), 3.61–3.53 (m, 3 H, 3-
= 7.6 Hz, 2 H, HArBz), 7.56 (t, J = 7.2 Hz, 1 H, HArBz), 7.46–7.36
(m, 5 H, HArBz), 7.32 (t, J = 7.6 Hz, 2 H, HArBz), 7.24 (t, J =
H, 4-H, 8-H), 3.42 (dd, J = 6.0, 12.0 Hz, 1 H, 8-H), 2.83 (s, 1 H, 7.2 Hz, 2 H, HArBz), 5.84 (t, J = 9.2 Hz, 1 H, 3-H), 5.56 (d, J =
OH), 2.48 (br., 1 H, 5-H) ppm. 13C NMR (100 MHz, CDCl3): δ = 8.4 Hz, 1 H, 2-H), 5.43 (t, J = 10.0 Hz, 1 H, 4-H), 3.67–3.62 (m, 4
138.3, 137.9, 128.9, 128.4, 128.3, 127.8, 127.7, 127.7, 127.5, 127.4,
83.3, 80.0, 74.7, 71.3, 69.9, 52.5, 43.6 ppm. HRMS: calcd. for
[C21H25NO3] [M + 3 H+ – N2] 340.19072; found 340.19080.
H, 6-H, 8-H), 3.44 (s, 1 H, 1-H), 2.72–2.68 (m, 1 H, 5-H) ppm. 13
C
NMR (100 MHz, CDCl3): δ = 165.7, 165.6, 165.4, 133.5, 133.3,
133.1, 129.8, 129.6, 129.5, 128.9, 128.7, 128.6, 128.4, 128.3, 128.1,
72.2, 71.4, 67.8, 54.7, 54.2, 50.5, 40.8 ppm. HRMS: calcd. for
[C28H23N3O7] [M + H]+ 514.16007; found 514.16088.
Azido-cyclohexene 16: Boron trichloride (21 mL, 21.1 mmol) was
added at –78 °C to a solution of 15 (777 mg, 2.11 mmol) in anhy-
drous dichloromethane (10 mL). The reaction mixture was kept be-
tween –78 °C and –60 °C for 6 h, after which it was quenched with
8-Deoxy-8-azidocyclophellitol (4): A catalytic amount of NaOMe
was added to a solution of 18 (104 mg, 0.20 mmol) in MeOH
6038
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© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 6030–6043

Cyclophellitol, Cyclophellitol Aziridine, and Tagged Derivatives
(1.0 mL), and the mixture was stirred for 1 h at ambient tempera-
ture. It was then neutralized with Amberlite IR-120 H+, filtered,
and concentrated in vacuo. Purification by silica column
chromatography (dichloromethane/MeOH 94:6Ǟ92:8) provided 4
(30.0 mg, 0.15 mmol, 75%) as a white solid. H NMR (400 MHz,
MeOD): δ = 3.84 (dd, J = 3.6, 8.4 Hz, 1 H, 8-H), 3.67 (d, J =
brine, dried with MgSO4, filtered, and concentrated in vacuo
(30 °C, 100 mbar). The triflated intermediate was co-evaporated
once with toluene and used in the next step without further purifi-
cation. The triflated product was dissolved in THF (2.5 mL) and
cooled to –25 °C under argon. NaN3 (53 mg, 0.75 mmol) and 15-
crown-5 (46 μL, 0.25 mmol) were added to the cooled solution, and
1
8.0 Hz, 1 H, 2-H), 3.51 (dd, J = 8.8, 12.0 Hz, 1 H, 8-H), 3.36 (d, J the reaction mixture was allowed to warm gradually to 0 °C. After
= 3.2 Hz, 1 H, 6-H), 3.23 (dd, J = 8.4, 10.0 Hz, 1 H, 3-H), 3.13–
3.08 (m, 2 H, 1-H, 4-H), 2.07 (ddt, J = 1.6, 3.6, 9.4 Hz, 1 H, 5-
H) ppm. 13C NMR (100 MHz, MeOD): δ = 78.3, 72.7, 68.6, 57.6,
20 min the reaction was complete, and the reaction mixture was
diluted with Et2O, silica gel was added, and the solvents were
removed under reduced pressure. The silica immobilized product
was purified by column chromatography (pentane/EtOAc
80:20Ǟ70:30), yielding 2,3-acetylated azidocyclophellitol 21 as a
colorless amorphous solid (64 mg, 0.23 mmol, 90%). 1H NMR
(400 MHz, CDCl3): δ = 5.07 (d, J = 8.4 Hz, 1 H, 2-H), 4.96 (dd, J
= 8.4, 10.4 Hz, 1 H, 3-H), 3.88 (dd, J = 4.0, 12.0 Hz, 1 H, 8-H),
3.59 (dd, J = 8.4, 12.0 Hz, 1 H, 8-H), 3.51 (t, J = 10.0 Hz, 1 H, 4-
H), 3.41 (d, J = 3.6 Hz, 1 H, 6-H), 3.14 (d, J = 3.6 Hz, 1 H, 1-H),
2.27–2.21 (m, 1 H, 5-H), 2.10 (d, J = 3.6 Hz, 6 H, 2ϫCH3
OAc) ppm. 13C NMR (100 MHz, CDCl3): δ = 75.2, 70.7, 66.3,
54.7, 53.9, 50.8, 42.1, 20.9, 20.8 ppm. HRMS: calcd. for
[C11H15N3O6] [M + H]+ 286.10336; found 286.10344.
56.1, 52.4, 43.9 ppm. HRMS: calcd. for [C7H14NO4] [M + 3 H+
N2]+ 176.09173; found 176.09179.
–
2,3-Di-O-acetyl-4,8-O-(tert-butylsilanediyl)cyclophellitol (19): Pyr-
idine (0.2 mL, 2.5 mmol) was added to a solution of (+)-cyclophel-
litol (1, 47.1 mg, 0.25 mmol) in DMF (2.3 mL), and the solution
was cooled to –40 °C. (tBu)2Si(OTf)2 (102 μL, 0.31 mmol) was
added to the cooled solution, and the reaction mixture was allowed
to warm gradually to 0 °C. At 0 °C the mixture was diluted with
EtOAc and washed with H2O. The aqueous phase was extracted
with EtOAc, and the combined organic layers were washed with
H2O and brine, dried with MgSO4, filtered, and concentrated in
vacuo. The resulting crude product was dissolved in pyridine
(2.5 mL) and cooled to 0 °C. Ac2O (0.25 mL) was added dropwise
to the cooled solution, and the reaction mixture was allowed to
warm to ambient temperature. After complete conversion the reac-
tion mixture was cooled to 0 °C and quenched with MeOH. The
reaction mixture was concentrated in vacuo, and traces of pyridine
were removed by co-evaporation with toluene. Purification by col-
umn chromatography (pentane/EtOAc 92.5:7.5Ǟ90:10) yielded
protected cyclophellitol 19 as a colorless oil (57.7 mg, 0.15 mmol,
8-Azido-8-deoxycyclophellitol (4): NaOMe solution in MeOH
(0.02 m, 0.5 mL, 0.01 mmol) was added to a solution of 2,3-acetyl-
ated azido cyclophellitol 21 (28.5 mg, 0.1 mmol) in MeOH
(0.5 mL) The reaction mixture was stirred overnight at room tem-
perature, and the reaction was quenched with Amberlite IR-120
H+ until pH-neutral. Amberlite was filtered off over a plug of cot-
ton wool, rinsed with MeOH. Removal of the solvent under re-
duced pressure yielded azido cyclophellitol 4 without further purifi-
cation as a colorless amorphous solid (17.9 mg, 0.089 mmol, 89%).
Analytical data are in accordance with those described previously
(see above).
1
60%). H NMR (400 MHz, CDCl3): δ = 5.06 (m, 2 H, 2-H, 3-H),
4.09–4.01 (m, H, 8-H), 3.99–3.91 (m, 1 H, 8-H), 3.66–3.61 (m, 1
H, 4-H), 3.41 (s, 1 H, 6-H), 3.10 (d, J = 4.8 Hz, 1 H, 1-H), 2.27–
2.21 (m, 1 H, 5-H), 2.10 (d, J = 3.6 Hz, 6 H, 2ϫCH3OAc) ppm.
13C NMR (100 MHz, CDCl3): δ = 170.4, 170.1, 74.4, 70.8, 70.3,
66.2, 53.9, 53.0, 42.1, 27.4, 26.9, 22.7, 21.0, 20.9, 19.9 ppm. HRMS:
calcd. for [C19H33O7Si] [M + H]+ 401.19901; found 401.19883.
2,3-Di-O-benzylcyclophellitol Aziridine Compound 23: Diol
3
(765 mg, 2.0 mmol) was co-evaporated thrice with toluene and sub-
sequently dissolved in anhydrous CH2Cl2 (50 mL). The solution
was cooled to 0 °C, and trichloroacetonitrile (200 μL, 2.0 mmol)
and 1,8-diazobicyclo[5.4.0]undec-7-ene (14 μL, 0.2 mmol) were
added. After the system had been stirred for 2 h at 0 °C, TLC
analysis revealed complete conversion to a higher-running product.
H2O (6.2 mL), sodium hydrogencarbonate (1.75 g, 20.8 mmol), and
iodine (1.57 g, 6.2 mmol) were added. The resulting mixture was
stirred overnight at room temperature, after which it was quenched
with Na2S2O3 (10%) and extracted with EtOAc. The organic layer
was dried with MgSO4 and concentrated under reduced pressure.
The resulting crude iminal 22 was dissolved in dioxane (18 mL),
after which it was cooled to 0 °C. Subsequently, concentrated hy-
drochloric acid (6.2 mL, 37% in H2O) was added, and the reaction
mixture was stirred at 60 °C for 1 h. The solution was concentrated
in vacuo and redissolved in MeOH (49 mL). Sodium hydrogencarb-
onate (3.36 g, 40 mmol) was added. After stirring at room tempera-
ture for 1 d, the reaction mixture was diluted with H2O and ex-
tracted with EtOAc, and the organic fraction was dried with
MgSO4 and concentrated in vacuo. Purification by silica gel col-
umn chromatography (dichloromethane/MeOH 96:4Ǟ90:10) af-
forded 23 (427 mg, 1.20 mmol, 60%) as a white solid. 1H NMR
(400 MHz, CDCl3): δ = 7.39–7.26 (m, 10 H, HArBn), 4.96 (d, J =
11.6 Hz, 1 H, CH2 Bn), 4.78 (d, J = 11.6 Hz, 1 H, CH2 Bn), 4.65
(d, J = 11.6 Hz, 2 H, CH2 Bn), 3.97 (dd, J = 5.6, 10.4 Hz, 1 H, 8-
H), 3.89 (dd, J = 4.4, 10.8 Hz, 1 H, 8-H), 3.74 (d, J = 8.0 Hz, 1 H,
2-H), 3.52 (t, J = 10.0 Hz, 1 H, 4-H), 3.37 (t, J = 9.6 Hz, 1 H, 3-
H), 2.42 (dd, J = 3.2, 6.0 Hz, 1 H, 6-H), 2.27 (d, J = 6.0 Hz, 1 H, 1-
H), 2.07 (td, J = 5.1, 8.8 Hz, 1 H, 5-H) ppm. 13C NMR (100 MHz,
CDCl3): δ = 138.4, 137.8, 128.5, 128.5, 127.9, 127.8, 127.8, 84.3,
2,3-Di-O-acetylcyclophellitol (20): Pyridine (0.25 mL) was added to
a
solution of silylene-protected cyclophellitol 19 (38.4 mg,
0.1 mmol) in THF (0.5 mL), and the mixture was cooled to 0 °C.
HF·pyridine (1 m, 0.25 mL, 0.25 mmol) was added to the cooled
reaction mixture, which was stirred for 1 h at 0 °C. The reaction
was quenched with NaHCO3 (s). Excess solids were filtered off over
a plug of cotton wool, rinsed with acetone, and the combined fil-
trate fractions were concentrated in vacuo. Purification by column
chromatography (dichloromethane/acetone 75:25) yielded 2,3-O-
acetylated cyclophellitol 20 as a colorless amorphous solid (27 mg,
0.1 mmol, quant.). 1H NMR (400 MHz, CDCl3): δ = 5.06 (m, 2 H,
2-H, 3-H), 4.09–4.01 (m, H, 8-H), 3.99–3.91 (m, 1 H, 8-H), 3.66–
3.61 (m, 1 H, 4-H), 3.36 (t, J = 7.6 Hz, OH), 3.38–3.34 (m, 2 H,
6-H, OH), 3.10 (d, J = 4.8 Hz, 1 H, 1-H), 2.27–2.21 (m, 1 H, 5-H),
2.10 (d, J = 3.6 Hz, 6 H, 2ϫCH3 OAc) ppm. 13C NMR (100 MHz,
CDCl3): δ = 171.3, 170.3, 75.1, 71.0, 67.0, 63.0, 55.2, 53.3, 43.7,
21.0, 20.9 ppm. HRMS: calcd. for [C11H16O7] [M + H]+ 261.09688;
found 261.09695.
2,3-Di-O-acetyl-8-azido-8-deoxycyclophellitol
(21):
Pyridine
(0.2 mL, 2.5 mmol) was added to a solution of 2,3-acetylated cyclo-
phellitol 20 (65 mg, 0.25 mmol) in CH2Cl2 (9.8 mL) and the solu-
tion was cooled to –25 °C. Triflic anhydride (52.5 μL, 0.36 mmol)
was added, and the reaction mixture was stirred for 1 h at –25 °C.
It was then diluted with EtOAc and successively washed with HCl
(aq., 0.1 m, 5 mL, pHՅ 6), satd. NaHCO3 (aq., pHՆ 6), H2O, and
Eur. J. Org. Chem. 2014, 6030–6043
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
6039

H. S. Overkleeft et al.
FULL PAPER
81.2, 74.7, 72.2, 68.2, 64.5, 42.5, 32.9, 31.4 ppm. HRMS: calcd. for
[C21H26NO4] [M + H]+ 356.18514; found 356.18563.
B: acetonitrile) afforded 5 (20 mg, 0.058 mmol, 25%) as a white
powder. 1H NMR (400 MHz, MeOD): δ = 4.05 (dd, J = 4.4,
10.4 Hz, 1 H, 8-H), 3.69–3.65 (m, 2 H, 2-H, 8-H), 3.30–3.25 (m, 2
H, CH2N3 and MeOD solvent signals), 3.19 (dd, J = 8.4, 10.0 Hz,
1 H, 3-H), 3.06 (t, J = 9.6 Hz, 1 H, 4-H), 3.01 (dd, J = 2.8, 6.0 Hz,
1 H, 6-H), 2.72 (d, J = 6.0 Hz, 1 H, 1-H), 2.48 (t, J = 7.2 Hz, 2 H,
NC=OCH2), 1.99–1.93 (m, 1 H, 5-H), 1.63–1.54 (m, 5 H, CH2
alkyl), 1.36 (s, 7 H, CH2 alkyl) ppm. 13C NMR (100 MHz, MeOD):
δ = 188.5, 79.0, 73.4, 69.3, 63.7, 52.4, 45.2, 42.4, 41.0, 36.8, 30.1,
29.8, 27.6 25.9 ppm. HRMS: calcd. for [C15H27N4O5] [M + H]+
343.19760; found 343.19761.
Cyclophellitol Aziridine Compound 2: Ammonia (20 mL) was con-
densed at –60 °C. Lithium (200 mg) was added, and the mixture
was stirred until the lithium was completely dissolved. A solution
of aziridine 23 (427 mg, 1.20 mmol) in THF (27 mL) was added.
The reaction mixture was stirred for 30 min at –60 °C and sub-
sequently quenched with milliQ-H2O (8 mL). The solution was al-
lowed to come to room temperature and stirred until all ammonia
had evolved. Next, the solution was concentrated in vacuo, redis-
solved in milliQ-H2O, and neutralized with Amberlite IR-120 H+.
Product bound to the resin was washed with water and sub-
sequently eluted with NH4OH solution (1 m) and evaporated under
reduced pressure. The resulting solid was again purified on Am-
General Click Procedure for Epoxides 34–36 and 41: Azido-
cyclophellitol (1 equiv.) and the desired BODIPY-alkyne (1 equiv.)
were dissolved in tBuOH/toluene/H2O (1–2 mL, 1:1:1, v/v/v).
CuSO4 (0.1 equiv., 100 mm in H2O) and sodium ascorbate
(0.1 equiv., 100 mm in H2O) were added. Subsequently, the reaction
mixture was heated to 80 °C and stirred overnight. The solution
was diluted with CH2Cl2, washed with H2O, dried with MgSO4,
concentrated under reduced pressure, and purified by silica column
chromatography.
+
berlite IR-120 NH4 with milliQ-water as eluent until the eluate
was neutral. Concentration of the combined eluate under reduced
pressure gave the fully deprotected aziridine 3 (147 mg, 0.84 mmol,
1
70%). H NMR (400 MHz, D2O): δ = 3.61 (dd, J = 4.4, 10.8 Hz,
1 H, 8-H), 3.34–3.25 (m, 2 H, 8-H, 2-H), 2.89 (t, J = 9.6 Hz, 1 H,
3-H), 2.68 (t, J = 9.6 Hz, 1 H, 4-H), 2.22 (dd, J = 3.2, 6.0 Hz, 6-
H), 1.93 (d, J = 6.4 Hz, 1 H, 1-H), 1.68–1.64 (m, 1 H, 5-H) ppm.
13C NMR (100 MHz, D2O): δ = 78.8, 73.3, 69.1, 62.9, 44.2, 36.0,
33.4 ppm. HRMS: calcd. for [C7H14NO4] [M + H]+ 176.09173;
found 176.09170.
BODIPY-Cyclophellitol Derivative 34: Silica column chromatog-
raphy (dichloromethane/MeOH 100:0Ǟ95:5) furnished 34 as a pur-
ple powder (14.3 mg, 20.8 mmol, 77%). 1H NMR (400 MHz,
MeOD): δ = 7.83 (d, J = 7.2 Hz, 4 H, HArPh), 7.58 (s, 1 H, CHtrz),
7.32 (d, J = 4.4 Hz, 2 H, 2ϫHAr), 6.95 (d, J = 8.8 Hz, 4 H, HArPh),
6.63 (d, J = 4.4 Hz, 2 H, 2ϫHAr), 4.80 (dd, J = 3.6, 13.6 Hz, 1 H,
8-H), 4.57 (dd, J = 8.8, 14.0 Hz, 1 H, 8-H), 3.85 (s, 6 H,
2ϫCH3OMe), 3.70 (d, J = 8.0 Hz, 1 H, 2-H), 3.27 (dd, J = 8.4,
10.0 Hz, 1 H, 3-H), 3.17 (t, J = 9.6 Hz, 1 H, 4-H), 3.09 (d, J =
4.0 Hz, 1 H, 6-H), 3.04–3.01 (m, 3 H, 1-H, CH2 alkyl), 2.80–2.79
(m, 2 H, CH2 alkyl), 2.41 (td, J = 2.0, 3.6, 8.8 Hz, 1 H, 5-H), 1.89
(br. s, 4 H, 2ϫCH2 alkyl) ppm. 13C NMR (100 MHz, MeOD): δ
= 162.2, 158.8, 148.7, 137.5, 132.2, 128.4, 126.5, 124.4, 121.0, 114.6,
78.3, 72.5, 68.6, 57.8, 55.5, 50.7, 49.6, 44.7, 34.2, 31.0, 30.5,
Cyclophellitol N-(Hept-6-ynoyl)aziridine Compound 24: A preacti-
vated mixed anhydride solution (1 m) was prepared by dissolving
EEDQ (209 mg, 0.85 mmol) and hept-6-ynoic acid (0.11 mL,
0.85 mmol) in DMF (0.85 mL), and the reaction mixture was
stirred at room temperature for 2 h before use. Cyclophellitol azir-
idine (3, 38 mg, 0.22 mmol) was dissolved in DMF (1.3 mL), and
the solution was cooled to 0 °C, after which the preactivated mixed
anhydride solution (1 m, 0.11 mL, 0.11 mmol) was added. The reac-
tion mixture was stirred at 0 °C for 30 min, and additional preacti-
vated solution (0.11 mL, 0.11 mmol) was added. After stirring for
45 min at 0 °C, the reaction mixture was quenched with MeOH
and concentrated under reduced pressure. Purification by semipre-
parative reversed-phase HPLC (linear gradient: 13%Ǟ16%, 3CV,
solutions used: A: H2O, B: acetonitrile) afforded compound 24
25.8 ppm. HRMS: calcd. for [C36H39BF2N5O4] [M
686.29560; found 686.29559.
+
H]+
BODIPY-Cyclophellitol Derivative 35: Silica column chromatog-
raphy (dichloromethane/MeOH 100:0Ǟ95:5) afforded 35 as an
orange powder (12.5 mg, 23.6 mmol, 56%). 1H NMR (400 MHz,
MeOD): δ = 7.77 (s, 1 H, CHtrz), 6.11 (s, 2 H, 2ϫHAr), 4.68 (d, J
= 4.0, 14.0 Hz, 1 H, 8-H), 4.60 (dd, J = 7.2, 13.6 Hz, 1 H, 8-H),
3.58 (d, J = 8.0 Hz, 1 H, 2-H), 3.21 (t, J = 10.0 Hz, 1 H, 3-H), 3.12
(t, J = 10.0 Hz, 1 H, 4-H), 3.03–2.99 (m, 2 H, CH2 alkyl), 2.96 (s,
2 H, 1-H, 6-H), 2.79 (t, J = 6.8 Hz, 2 H, CH2 alkyl), 2.66 (s, 1 H,
CH2 alkyl), 2.44 (s, 6 H, 2ϫCH3Me), 2.38 (s, 6 H, 2ϫCH3Me),
1.86 (td, J = 7.6, 7.6, 15.0 Hz, 1 H, 5-H), 1.92–1.87 (m, 3 H, CH2
alkyl), 1.69–1.62 (m, 2 H, CH2 alkyl) ppm. 13C NMR (100 MHz,
MeOD): δ = 154.9, 149.4, 148.5, 147.9, 142.2, 132.6, 124.5, 122.6,
78.3, 72.5, 68.6, 57.4, 55.4, 50.6, 44.7, 32.2, 31.1, 30.8, 29.0, 25.9,
16.5, 14.4 ppm. HRMS: calcd. for [C26H35BF2N5O4] [M + H]+
530.27447; found 530.27454.
1
(12.2 mg, 0.04 mmol, 20%) as a white solid. H NMR (400 MHz,
MeOD): δ = 4.06 (dd, J = 4.4, 10.4 Hz, 1 H, 8-H), 3.71–3.66 (m,
2 H, 2-H, 8-H), 3.20 (dd, J = 8.0, 10.0 Hz, 1 H, 3-H), 3.07 (t, J =
10.0 Hz, 1 H, 4-H), 3.02 (dd, J = 3.2, 6.0 Hz, 1 H, 6-H), 2.72 (d, J
= 6.0 Hz, 1 H, 1-H), 2.51 (td, J = 1.6, 8.8 Hz, 2 H, CH2C=O),
2.25–2.15 (m, 3 H, CH2Cϵ, CϵCH), 1.99–1.93 (m, 1 H, 5-H),
1.76–1.70 (m,
2 H, C=OCH2CH2), 1.57–1.50 (m, 2 H,
CH2CH2Cϵ) ppm. 13C NMR (100 MHz, MeOD): δ = 188.2, 84.6,
79.1, 73.4, 69.8, 69.3, 63.5, 45.3, 42.5, 41.1, 36.3, 29.1, 25.2,
18.8 ppm. HRMS: calcd. for [C14H22NO4] [M + H]+ 284.14925;
found 284.14952.
Cyclophellitol N-(8-Azidooctanoyl)aziridine 5: A preactivated mixed
anhydride solution (1 m) was prepared by dissolving EEDQ (74 mg,
0.30 mmol) and 8-azidooctanoic acid (56 mg, 0.30 mmol) in DMF
(0.3 mL), and the reaction mixture was stirred at room temperature
for 2 h before use. Crude deprotected aziridine (40 mg, 0.23 mmol)
was dissolved in DMF (1.3 mL), and the solution was cooled to
0 °C, after which preactivated mixed anhydride solution (1 m,
0.15 mL, 0.15 mmol) was added. The reaction mixture was stirred
at 0 °C for 30 min, and additional preactivated solution (0.15 mL,
0.15 mmol) was added. After stirring for 45 min at 0 °C, the reac-
tion mixture was quenched with MeOH and concentrated under
reduced pressure. Purification by semipreparative reversed-phase
HPLC (linear gradient: 25%Ǟ31%, 3CV, solutions used: A: H2O,
Biotin-Cyclophellitol Derivative 36: Silica column chromatography
(dichloromethane/MeOH 100:0Ǟ80:20) furnished 36 as a white
1
powder (12.3 mg, 12.6 μmol, 90%). H NMR (600 MHz, DMSO):
δ = 7.81 (s, 1 H, CHtrz), 4.57 (dd, J = 3.6, 13.8 Hz, 1 H, 8-H),
4.36 (dd, J = 10.8, 13.2 Hz, 1 H, 8-H), 4.13–4.23 (m, 3 H, 6-H,
CH2NHC=O), 4.13–4.11 (m, 1 H, 1-H), 3.62 (t, J = 3.0 Hz, 1 H,
CH biotin), 3.09–3.09 (m, CH biotin), 2.82 (dd, J = 4.8, 12.6 Hz,
1 H, CH2 biotin), 2.11–2.05 (m, 3 H, CH2 alkyl), 1.64–1.58 (m, 1
H, CH2 alkyl), 1.54–1.37 (m, 3 H, CH2 alkyl), 1.31–1.29 (m, 3 H,
CH2 alkyl) ppm. 13C NMR (151 MHz, DMSO): δ = 172.0, 162.7,
144.7, 144.6, 123.3, 74.7, 72.2, 70.6, 70.4, 67.7, 61.0, 59.2, 55.4,
6040
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© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 6030–6043

Cyclophellitol, Cyclophellitol Aziridine, and Tagged Derivatives
47.6, 42.9, 34.9, 34.1, 28.2, 28.0, 25.2, 20.4 ppm. HRMS: calcd. for
[C20H30N6O6S] [M + H]+ 483.20203; found 483.20170.
15.1, 14.8, 14.6, 11.2 ppm. HRMS: calcd. for [C39H44BF2N6O5] [M
+ H]+ 725.34305; found 725.34288.
General Procedure for the Synthesis of pH-Activatable Cyclophellitol
Probes 37–40: Azidocyclophellitol (1 equiv.) and the desired BOD-
IPY-alkyne (1 equiv.) were dissolved in tBuOH/toluene/H2O (1–
2 mL, 1:1:1, v/v/v) and sonicated for 30 min under argon. CuSO4
(0.1 equiv., 100 mm in H2O) and sodium ascorbate (0.1 equiv.,
100 mm in H2O) were added, and the mixture was heated to 80 °C.
After 2 h an additional amount of CuSO4 (0.1 equiv.) and sodium
ascorbate (0.15 equiv.) were added, and the mixture was stirred at
80 °C for 2 h. The mixture was concentrated under reduced pres-
sure and co-evaporated with toluene. HPLC-MS purification and
lyophilization from tBuOH/H2O resulted in 37–40 as purple pow-
ders.
Diethylaminophenyl-BODIPY-Cyclophellitol Derivative 40: Yield
3.46 mg, 4.1 μmol, 14%. 1H NMR (600 MHz, MeOD): δ = 8.13 (s,
1 H, CHtrz), 7.55 (d, J = 8.7 Hz, 2 H, 2ϫHAr Ph), 7.49 (d, J =
16.3 Hz, 1 H, CH=), 7.31 (d, J = 16.3 Hz, 1 H, CH=), 7.06 (dd, J
= 3.2, 8.7 Hz, 4 H, 4ϫHAr Ph), 6.85 (d, J = 8.7 Hz, 2 H, 2ϫHAr
Ph), 6.72 (s, 1 H, HAr), 6.06 (s, 1 H, HAr), 5.23 (s, 2 H, OCH2),
4.67 (dd, J = 8.6, 13.9 Hz, 2 H, 8-H), 3.62 (d, J = 8.1 Hz, 1 H, 2-
H), 3.45 (q, J = 7.0 Hz, 4 H, 2ϫCH2 ethyl), 3.24 (dd, J = 8.2,
9.0 Hz, 1 H, 3-H), 3.15 (t, J = 9.8 Hz, 1 H, 4-H), 3.04 (d, J =
3.7 Hz, 2 H, 1-H, 6-H), 2.52 (s, 3 H, CH3 Me), 2.42 (td, J = 4.4,
8.6, 9.1 Hz, 1 H, 5-H), 1.60 (s, 3 H, CH3 Me), 1.56 (s, 3 H, CH3
Me), 1.20 (t, J = 7.0 Hz, 6 H, 2ϫCH3 ethyl) ppm. 13C NMR
(150 MHz, MeOD): δ = 160.5, 155.2, 154.0, 149.8, 144.9, 144.2,
144.0, 143.6, 136.6, 134.7, 133.7, 131.5, 130.3, 129.8, 126.4, 122.3,
121.8, 118.3, 116.4, 113.2, 78.2, 72.5, 68.7, 62.5, 57.6, 55.5, 51.0,
45.4, 44.7, 31.1, 15.1, 14.9, 14.6, 12.7 ppm. HRMS: calcd. for
[C40H46BF2N6O5] [M + H]+ 739.35853; found 739.35866.
Phenyl-BODIPY-Cyclophellitol Derivative 37: Yield 2.74 mg,
4.1 μmol, 26%. 1H NMR (600 MHz, MeOD): δ = 8.13 (s, 1 H,
CHtrz), 7.57 (dd, J = 5.0, 7.4 Hz, 5 H, 5ϫHArPh), 7.50 (d, J =
16.2 Hz, 1 H, CH=), 7.30–7.38 (m, 3 H, CH=, 2ϫHArPh), 7.07 (d,
J = 8.8 Hz, 2 H, 2ϫHArPh), 6.75 (s, 1 H, HAr), 6.08 (s, 1 H, HAr),
5.24 (s, 1 H, OCH2), 4.67 (dd, J = 8.6, 13.9 Hz, 2 H, 8-H), 3.62 (d,
J = 8.2 Hz, 1 H, 2-H), 3.24 (dd, J = 8.2, 10.0 Hz, 1 H, 3-H), 3.15
(t, J = 9.8 Hz, 1 H, 4-H), 3.00–3.07 (m, 2 H, 1-H, 6-H), 2.53 (s, 3
H, CH3 Me), 2.42 (td, J = 4.5, 8.5, 9.2 Hz, 1 H, 5-H), 1.46 (s, 3 H,
CH3 Me), 1.42 (s, 3 H, CH3Me) ppm. 13C NMR (151 MHz,
MeOD): δ = 160.7, 155.9, 154.8, 144.8, 144.2, 143.8, 141.8, 137.3,
136.4, 133.9, 132.8, 131.3, 130.4, 130.3, 129.9, 120.9, 126.4, 122.1,
188.7, 118.1, 116.4, 116.3, 78.2, 72.5, 68.7, 62.5, 57.6, 55.4, 51.0,
44.7, 14.8, 14.6, 14.5 ppm. HRMS: calcd. for [C36H37BF2N5O5]
[M + H]+ 668.28503; found 668.28553.
Biotin-BODIPY-Cyclophellitol Derivative 41: Silica column
chromatography (dichloromethane/MeOH 100:0Ǟ80:20) furnished
41 as a purple powder (12.3 mg, 12.6 μmol, 90%). 1H NMR
(400 MHz, MeOD): δ = 7.84 (d, J = 8.8 Hz, 2 H, HAr), 7.73 (s, 1
H, CHtrz), 7.58 (s, 2 H, HAr Ph), 7.24 (s, 1 H, CHtrz), 7.01 (d, J =
4.0 Hz, 1 H, HAr), 6.92 (d, J = 8.8 Hz, HAr Ph), 6.55 (d, J = 4.0 Hz,
HAr), 3.31 (br. s, H2O solvent peak and 8-H), 4.58 (t, J = 6.8 Hz,
2 H, CH2O), 4.48–4.38 (m, 6 H, 8-H, 2ϫCH2NHC=O, CH biotin),
4.22 (dd, J = 4.4, 7.6 Hz, CH biotin), 4.02 (t, J = 5.6 Hz, 2 H, CH2
alkyl), 3.62 (d, J = 8.4 Hz, 1 H, 2-H), 3.21 (dd, J = 8.4, 10.0 Hz,
3-H), 3.12–3.06 (m, 2 H, 4-H, SCH), 3.00 (d, J = 3.6 Hz, 1 H, 6-
H), 2.96–2.95 (m, 1 H, 1-H), 2.86 (dd, J = 4.8, 12.8 Hz, 1 H, CH
biotin), 2.73 (t, J = 7.2 Hz, 1 H, CH2 alkyl), 2.67 (d, J = 12.4 Hz,
1 H, CH biotin), 2.48 (s, 3 H, CH3 Me), 2.42–2.30 (m, 5 H, 5-H,
CH2 alkyl), 2.19–2.16 (m, 5 H, CH3 Me, CH2 alkyl), 1.65–1.57 (m,
5 H, CH2 alkyl), 1.55–1.24 (m, 3 H, CH2 alkyl) ppm. 13C NMR
(150 MHz, MeOD): δ = 175.2, 173.8, 160.2, 160.0, 155.8, 141.2,
135.1, 131.3, 128.8, 126.6, 124.4, 123.9, 118.8, 77.7, 71.7, 68.0, 64.9,
62.6, 57.0, 56.3, 54.9, 50.3, 47.9, 43.8, 40.8, 36.2, 36.1, 35.2, 35.1,
30.3, 29.0, 28.7, 26.0, 13.3, 9.6 ppm. HRMS: calcd. for
[C45H56BF2N12O8S] [M + H]+ 987.42848; found 987.42855.
Dimethylaminophenyl-BODIPY-Cyclophellitol Derivative 38: Yield
1
5.44 mg, 7.66 μmol, 30%. H NMR (600 MHz, MeOD): δ = 8.15
(s, 1 H, CHtrz), 7.56 (d, J = 8.7 Hz, 2 H, 2ϫHAr), 7.49 (d, J =
16.3 Hz, 1 H, CH=), 7.33 (d, J = 16.4 Hz, 1 H, CH=), 7.11 (d, J
= 8.6 Hz, 2 H, 2ϫHAr Ph), 7.07 (d, J = 8.7 Hz, 2 H, 2ϫHAr Ph),
6.91 (d, J = 8.6 Hz, 2 H, 2ϫHAr Ph), 6.74 (s, 1 H, HAr), 6.07 (s, 1
H, HAr), 5.24 (s, 2 H, OCH2), 4.87 (dd, J = 3.8, 13.8 Hz, 1 H, 8-
H), 4.67 (dd, J = 8.6, 13.9 Hz, 1 H, 8-H), 3.62 (d, J = 8.2 Hz, 1 H,
2-H), 3.20–3.26 (m, 1 H, 3-H), 3.14 (t, J = 9.8 Hz, 1 H, 4-H), 3.03
(d, J = 6.3 Hz, 8 H, 1-H, 6-H, 2ϫCH3 NMe2), 2.52 (s, 3 H, CH3
Me), 2.42 (td, J = 2.5, 8.7, 9.2 Hz, 1 H, 5-H), 1.57 (s, 3 H, CH3
Me), 1.52 (s, 3 H, CH3) ppm. 13C NMR (150 MHz, MeOD): δ =
160.6, 155.3, 154.1, 152.6, 144.9, 144.2, 144.0, 143.4, 136.7, 136.4,
133.6, 131.5, 130.1, 129.8, 126.4, 123.4, 121.8, 118.3, 116.4, 113.7,
78.2, 72.5, 68.7, 62.5, 57.6, 55.5, 51.0, 44.7, 40.6, 15.1, 14.8,
BODIPY-Cyclophellitol Aziridine Derivative 43: Alkyne 24 (4.2 mg,
15 μmol) was dissolved in DMF (0.65 mL). BODIPY-azide 42
(6.2 mg, 17 μmol), CuSO4 (12 μL of 1M solution in H2O), and so-
dium ascorbate (13 μL of a 1 m solution in H2O) were added, and
the solution was stirred for 1 h at ambient temperature. The vola-
tiles were removed under reduced pressure, and compound 43 was
purified by semipreparative reversed-phase HPLC (linear gradient:
40%Ǟ50%, 3 CV, solutions used: A: H2O, B: acetonitrile) to yield
43 as an orange powder (4.43 mg, 6.75 μmol, 45%). 1H NMR
(400 MHz, MeOD): δ = 7.74 (s, 1 H, CHtrz), 6.13 (s, 2 H, HAr),
14.6 ppm. HRMS: calcd. for [C38H42BF2N6O5] [M
711.32723; found 711.32738.
+
H]+
N-Methyl-N-Ethylaminophenyl-BODIPY-Cyclophellitol Derivative
1
39: Yield 2.43 mg, 4.1 mmol, 18%. H NMR (600 MHz, MeOD):
δ = 8.15 (s, 1 H, CHtrz), 7.56 (d, J = 8.7 Hz, 2 H, 2ϫHAr Ph), 7.49
(d, J = 16.4 Hz, 1 H, CH=), 7.32 (d, J = 16.3 Hz, 1 H, CH=), 7.08
(dd, J = 8.7, 11.3 Hz, 4 H, 4ϫHAr Ph), 6.89 (d, J = 8.7 Hz, 2 H, 4.37 (t, J = 6.8 Hz, 2 H, NCH2), 4.05 (dd, J = 4.4, 10.0 Hz, 1 H,
2ϫHAr Ph), 6.74 (s, 1 H, HAr), 6.07 (s, 1 H, HAr), 5.24 (s, 2 H, 8-H), 3.67 (dd, J = 9.2, 10.4 Hz, 1 H, 8-H), 3.65 (d, J = 8.0 Hz, 1
OCH2), 4.87 (dd, J = 3.8, 13.9 Hz, 1 H, 8-H), 4.67 (dd, J = 8.6,
13.9 Hz, 1 H, 8-H), 3.62 (d, J = 8.2 Hz, 1 H, 2-H), 3.51 (q, J =
7.0 Hz, 2 H, CH2 ethyl), 3.24 (dd, J = 8.2, 9.9 Hz, 1 H, 3-H), 3.14
(t, J = 9.9 Hz, 1 H, 4-H), 3.04 (q, J = 3.8 Hz, 2 H, 1-H, 6-H), 2.99
H, 2-H), 3.19 (dd, J = 84, 10.4 Hz, 1 H, 3-H), 3.06 (t, J = 9.6 Hz,
1 H, 4-H), 3.01–2.97 (m, 3 H, 6-H, CH2C=), 2.72 (t, J = 7.6 Hz, 3
H, 1-H, CH2-alkyl), 2.54 (t, J = 7.2 Hz, 2 H, NC=OCH2), 2.43 (d,
J = 6.0 Hz, 12 H, 4ϫOMe), 1.99–1.93 (m, 3 H, 1-H, CH2 alkyl),
(s, 3 H, CH3NMe), 2.52 (s, 3 H, CH3 Me), 2.42 (td, J = 4.3, 9.8 Hz, 1.70–1.52 (m, 8 H, CH2 alkyl), 1.41–1.35 (m, 2 H, CH2 alkyl) ppm.
1 H, 5-H), 1.58 (s, 3 H, CH3 Me), 1.54 (s, 3 H, CH3 Me), 1.16 (t, 13C NMR (100 MHz, MeOD): δ = 188.2, 154.9, 148.8, 148.1, 142.2,
J = 7.0 Hz, 3 H, CH3 ethyl) ppm. 13C NMR (150 MHz, MeOD):
δ = 160.6, 155.3, 154.1, 151.1, 144.9, 144.2, 144.0, 143.5, 136.7,
135.7, 133.6, 131.5, 130.2, 129.8, 126.4, 122.9, 121.8, 118.3, 116.4,
113.6, 78.2, 72.5, 68.7, 62.5, 57.6, 55.5, 51.0, 47.6, 44.7, 37.7,
132.6, 123.2, 122.6, 79.1, 73.4, 69.3, 63.5, 51.1, 45.2, 42.5, 41.1,
36.5, 32.9, 31.2, 30.5, 29.9, 29.2, 27.2, 25.9, 25.3, 16.6, 14.4 ppm.
HRMS: calcd. for [C33H48BF2N6O5] [M + H]+ 657.37418; found
657.37464.
Eur. J. Org. Chem. 2014, 6030–6043
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
6041

H. S. Overkleeft et al.
FULL PAPER
BODIPY-Cyclophellitol Aziridine Derivative 44: BODIPY-alkyne
25 (9 mg, 26 μmol), CuSO4 (20 μL of 1M solution in H2O), and
sodium ascorbate (23 μL of 1 m solution in H2O) were added to a
solution of 5 (14 mg, 0.03 mmol) in DMF (1 mL). The reaction
2.84 (t, J = 8.0 Hz, 2 H, NC=OCH2), 2.25–2.18 (m, 4 H,
2ϫCH2CONH), 2.02–1.96 (m, 1 H, 5-H), 1.95–1.86 (m, 2 H,
CH2Ahϫ), 1.79–1.58 (m, 9 H, CH2 alkyl, CH2Ahx, COCH2CH2),
1.55–1.42 (m, 4 H, CH2CH2CHS, CH2CH2CHS), 1.38–1.30 (m, 10
mixture was stirred at ambient temperature for 2 h, after which it H, CH2 alkyl, CH2Ahx) ppm. 13C NMR (100 MHz, MeOD): δ =
was concentrated under reduced pressure. Purification by semipre-
parative reversed-phase HPLC [linear gradient: 52Ǟ58% B, 3CV
(solutions used A: H2O, B: acetonitrile)] afforded the title com-
pound (5.77 mg, 7.0 μmol, 27%) as a purple powder. 1H NMR
(600 MHz, MeOD): δ = 7.83 (d, J = 9.0 Hz, 4 H, HAr Ph), 7.67 (s,
1 H, CHtrz), 7.42 (d, J = 4.2 Hz, 2 H, CHAr), 6.96 (d, J = 9.0 Hz,
4 H, CHAr), 6.68 (d, J = 4.2 Hz, 2 H, CHAr), 4.31 (t, J = 7.2 Hz,
2 H, CH2Ntrz), 4.30 (dd, J = 4.2, 10.2 Hz, 1 H, 8-H), 3.84 (s, 6 H,
2ϫOMe), 3.68–3.65 (m, 2 H, 2-H, 8-H), 3.18 (dd, J = 8.4, 10.2 Hz,
1 H, 3-H), 3.09–3.03 (m, 3 H, 4-H, =CCH2), 2.97 (dd, J = 3.0,
6.0 Hz,1 H, 6-H), 2.77 (t, J = 6.6 Hz, 2 H CH2 alkyl), 2.69 (d, J =
6.0 Hz, 1 H, 1-H), 2.44–2.39 (m, 2 H, CH2 alkyl), 1.98–1.93 (m, 1
H, 5-H), 1.85–1.81 (m, 6 H, CH2 alkyl), 1.56–1.51 (m, 2 H, CH2
alkyl), 1.33–1.20 (m, 6 H, CH2 alkyl) ppm. 13C NMR (150 MHz,
MeOD): δ = 188.5, 162.2, 158.8, 148.6, 146.8, 137.5, 132.2, 132.2,
132.1, 128.4, 126.5, 123.2, 121.0, 114.7, 79.1, 73.4, 69.4, 63.6, 55.8,
51.2, 45.3, 42.4, 41.0, 36.8, 34.1, 31.1, 31.0, 30.3, 30.0, 29.6, 27.2,
25.8, 25.8 ppm. HRMS: calcd. for [C44H54BF2N6O5] [M + H]+
827.41172; found 827.41207.
188.5, 176.0, 175.9, 166.1, 146.3, 124.1, 79.1, 73.4, 69.3, 63.6, 63.4,
61.6, 57.0, 51.3, 49.4, 45.3, 42.4, 41.1, 41.0, 40.2, 36.8, 36.7, 35.7,
31.2, 30.1, 29.9, 29.8, 29.6, 29.5, 27.5, 27.2, 26.9, 26.5, 25.9 ppm.
HRMS: calcd. for [C46H57BF2N12O8S] [M + H]+ 737.40201; found
737.40146.
BODIPY-cyclophellitol derivatives 34 and 35 have previously been
used as activity-based glucosidase probes under the names
MDW941 and MDW933, respectively (see ref.[7]). BODIPY-cyclo-
phellitol aziridine 43 and biotin-Ahx-cyclophellitol aziridine 46
have been reported previously under the names BODIPY-aziridine
cyclitol and biotin-Ahx-aziridine cyclitol (see ref.[9]).
Supporting Information (see footnote on the first page of this arti-
cle): 1H NMR and 13C APT NMR spectra for all new compounds.
Acknowledgments
The authors are grateful for financial support from the European
Research Council (ERC) (AdG, to H. S. O.), the Netherlands Or-
ganization of Scientific Research (NWO-CW) (grant to H. S. O.),
and the Chinese Scholarship Council (CSC) (grant to J.-B. J.).
BODIPY-Cyclophellitol Aziridine Derivative 45: BODIPY-alkyne
26 (10.4 mg, 0.03 mmol), CuSO4 (20 μL of a 1 m solution in H2O),
and sodium ascorbate (23 μL of a 1 m solution in H2O) were added
to a solution of 5 (10 mg, 0.03 mmol) in DMF (1 mL). The reaction
mixture was stirred at ambient temperature for 2 h, after which it
was concentrated under reduced pressure. Purification by semipre-
parative reversed-phase HPLC [linear gradient: 45Ǟ48% B, 3CV
(solutions used A: H2O, B: acetonitrile)] afforded the title com-
[1] a) S. Atsuma, K. Umezawa, H. Iinuma, H. Naganawa, H. Nak-
amura, Y. Iitaka, T. Takeuchi, J. Antibiot. 1990, 43, 49–53; for
some reviews on cyclophellitol chemistry and biochemistry, see:
b) J. Marco-Contelles, Eur. J. Org. Chem. 2001, 1607–1618; c)
B. P. Rempel, S. G. Withers, Glycobiology 2008, 18, 570–586.
[2] a) G. Legler, Hoppe-Seyler’s Z. Physiol. Chem. 1966, 345, 197–
214; b) G. Legler, Hoppe-Seyler’s Z. Physiol. Chem. 1968, 349,
767–774.
[3] a) D. Koshland, Biol. Rev. 1953, 28, 416–436; for some reviews
on the mechanism of glycosyl hydrolases, see: b) D. J. Vocadlo,
G. J. Davies, Curr. Opin. Chem. Biol. 2008, 12, 539–555; c) G.
Davies, B. Henrissat, Structure 1995, 15, 853–859; d) see
also the CAZypedia website on carbohydrate-active enzymes,
1
pound (6.53 mg, 9.7 μmol, 33%) was an orange powder. H NMR
(600 MHz, MeOD): δ = 7.74 (s, 1 H, CHtrz), 6.13 (s, 2 H, HAr),
4.36 (t, J = 7.2 Hz, 2 H, CH2Ntrz), 4.07 (dd, J = 4.2, 10.2 Hz, 1 H,
8-H), 3.71–3.67 (m, 2 H, 2-H, 8-H), 3.22 (dd, J = 8.4, 10.2 Hz, 1
H, 3-H), 3.09 (t, J = 10.2 Hz, 1 H, 4-H), 3.03–3.00 (m, 3 H, 6-H,
=CCH2), 2.79 (t, J = 7.2 Hz, 2 H, CH2 alkyl), 2.73 (d, J = 6.0 Hz,
1 H, 1-H), 2.48 (dd, J = 1.8, 7.2 Hz, 2 H, CH2NC=O), 2.45 (s, 6
H, 2ϫCH3), 2.39 (s, 6 H, 2ϫCH3), 2.00–1.97 (m, 1 H, CH2 alkyl),
1.92–1.86 (m, 4 H, CH2 alkyl), 1.69–1.64 (m, 2 H, CH2 alkyl), 1.60–
1.56 (m, 2 H, CH2 alkyl), 1.36–1.25 (m, 7 H, CH2 alkyl) ppm. 13C
NMR (150 MHz, MeOD): δ = 188.5, 154.9, 148.5, 147.9, 142.2,
132.6, 123.4, 122.8, 122.6, 79.1, 73.4, 69.4, 63.6, 51.2, 45.3, 42.4,
41.0, 36.8, 32.2, 31.1, 30.8, 29.9, 29.6, 29.1, 27.2, 25.9, 25.8, 16.5,
specifically:
hydrolases.
http://www.cazypedia.org/index.php/Glycoside_
[4] T. M. Gloster, R. Madsen, G. J. Davies, Org. Biomol. Chem.
2007, 5, 444–446.
[5] a) M. Nakata, C. Chong, Y. Nitawa, K. Toshima, K. Tatsuta,
J. Antibiot. 1993, 46, 1919–1924; b) for a mechanistic study on
the mode of action of the corresponding conduritol B aziridine,
see: G. Caron, S. G. Withers, Biochem. Biophys. Res. Commun.
1989, 163, 495–499.
14.4 ppm. HRMS: calcd. for [C34H49BF2N6O5] [M
671.39044; found 671.39033.
+
H]+
Biotin-Ahx-Cyclophellitol Aziridine Derivative 46: Azide 5 (20 mg,
58 μmol) was dissolved in DMF (2 mL). Biotin-Ahx-alkyne 33
(23 mg, 58 μmol), CuSO4 (0.14 mL of 100 mm solution in H2O),
and sodium ascorbate (0.14 mL of a 100 mm solution in H2O) were
added, and the solution was stirred at 80 °C for 18 h. The solution
was concentrated under reduced pressure and purified by semipre-
parative reversed-phase HPLC (linear gradient: 18%Ǟ27%, 3 CV,
solutions used: A: H2O, B: acetonitrile) to yield 46 as a white pow-
der (8.9 mg, 12 μmol, 17%). 1H NMR (400 MHz, MeOD): δ = 7.85
(s, 1 H, CHtrz), 4.50 (dd, J = 5.2, 8.0 Hz, 1 H, SCH2CHNH), 4.43
(s, 2 H, CqCH2NH), 4.37 (t, J = 6.8 Hz, 2 H, CH2CH2NH), 4.31
(dd, J = 4.4, 8.0 Hz, 1 H, CH2CHNH), 4.06 (dd, J = 4.4, 10.0 Hz,
1 H, 8-H), 3.72–3.65 (m, 2 H, 2-H, 8-H), 3.24–3.14 (m, 4 H, 3-H,
CH2Ntrz, CH biotin), 3.08 (t, J = 9.6 Hz, 1 H, 4-H), 3.03 (dd, J =
3.2, 6.0 Hz, 1 H, 6-H), 2.93 (dd, J = 4.6, 12.8 Hz, 1 H, SCH2CH),
2.73 (d, J = 5.6 Hz, 1 H, 1-H), 2.71 (d, J = 12.0 Hz, 1 H, SCH2CH),
[6] K. Tatsuta, Pure Appl. Chem. 1996, 68, 1341–1346.
[7] M. D. Witte, W. W. Kallemeijn, J. Aten, K.-Y. Li, A. Strijland,
W. E. Donker-Koopman, A. M. C. H. van den Nieuwendijk, B.
Bleijlevens, G. Kramer, B. I. Florea, B. Hooibrink, C. E. M.
Hollak, R. Ottenhoff, R. G. Boot, G. A. van der Marel, H. S.
Overkleeft, J. M. F. G. Aerts, Nat. Chem. Biol. 2010, 6, 907–
913.
[8] M. D. Witte, M. T. C. Walvoort, K.-Y. Li, W. W. Kallemeijn,
W. E. Donker-Koopman, R. G. Boot, J. M. F. G. Aerts, J. D. C.
Codée, G. A. van der Marel, H. S. Overkleeft, ChemBioChem
2011, 12, 1263–1269.
[9] W. W. Kallemeijn, K.-Y. Li, M. D. Witte, A. R. A. Marques, J.
Aten, S. Scheij, J. Jiang, L. I. Willems, T. M. Voorn-Brouwer,
C. P. A. A. van Roomen, R. Ottenhoff, R. G. Boot, H.
van den Elst, M. T. C. Walvoort, B. I. Florea, J. D. C. Codée,
G. A. van der Marel, J. M. F. G. Aerts, H. S. Overkleeft, An-
gew. Chem. Int. Ed. 2012, 51, 12529–12533.
6042
www.eurjoc.org
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 6030–6043

Cyclophellitol, Cyclophellitol Aziridine, and Tagged Derivatives
[10] F. G. Hansen, E. Bundgaard, R. Madsen, J. Org. Chem. 2005,
70, 10139–10142.
[17]
[18]
R. Vicik, M. Busemann, C. Gelhaus, N. Stiefl, J. Scheiber,
W. Schmitz, F. Schulz, M. Mladenovic, B. Engels, M. Leippe,
K. Baumann, T. Schirmeister, ChemMedChem 2006, 1, 1126–
1141.
a) Q. Wang, T. R. Chan, R. Hilgraf, V. V. Fokin, K. B.
Sharpless, M. G. Finn, J. Am. Chem. Soc. 2003, 125, 3192–
3193; b) C. W. Tornøe, C. Christensen, M. Meldal, J. Org.
Chem. 2002, 67, 3057–3064.
[11] P. R. Skaanderup, C. S. Paulsen, L. Hyldtoft, M. R. Jørgensen,
R. Madsen, Synthesis 2002, 1721–1727.
[12] T.-P. Loh, G.-Q. Cao, J. Pei, Tetrahedron Lett. 1998, 39, 1453–
1456.
[13] M. Scholl, S. Ding, C. W. Lee, R. H. Grubbs, Org. Lett. 1999,
1, 953–956.
[14] P. Schwab, R. H. Grubbs, J. W. Ziller, J. Am. Chem. Soc. 1996,
[19] For the synthesis of the BODIPY tags employed in the studies
presented here, see: a) M. Verdoes, U. Hillaert, B. I. Florea, M.
Sae-Heng, M. D. P. Risseeuw, D. V. Filippov, G. A.
van der Marel, H. S. Overkleeft, Bioorg. Med. Chem. Lett.
2007, 17, 6169–6171; b) M. Verdoes, B. I. Florea, U. Hillaert,
L. I. Willems, W. A. van der Linden, M. Sae-Heng, D. V. Filip-
pov, A. F. Kisselev, G. A. van der Marel, H. S. Overkleeft,
ChemBioChem 2008, 9, 1735–1738; c) S. Hoogendoorn, K. L.
Habets, S. Passemard, J. Kuiper, G. A. van der Marel, B. I. Flo-
rea, H. S. Overkleeft, Chem. Commun. 2011, 47, 9363–9365; d)
S. Hoogendoorn, A. E. M. Blom, L. I. Willems, G. A.
van der Marel, H. S. Overkleeft, Org. Lett. 2011, 13, 5656–
5659.
118, 100–110.
[15] For related studies describing the synthesis of the natural prod-
uct (+)-cyclophellitol, see: a) K. Tatsuta, Y. Niwata, K. Umez-
awa, K. Toshima, M. Nakata, Tetrahedron Lett. 1990, 31,
1171–1172; b) K. Sato, M. Bokura, H. Moriyama, T. Igarashi,
Chem. Lett. 1994, 37–40; c) R. H. Schlessinger, C. P. Berg-
strom, J. Org. Chem. 1995, 60, 16–17; d) M. E. Jung, S. W. T.
Choe, J. Org. Chem. 1995, 60, 3280–3281; e) F. E. Ziegler, Y. Z.
Wang, J. Org. Chem. 1998, 63, 426–427; f) B. M. Trost, E. J.
Hembre, Tetrahedron Lett. 1999, 40, 219–222; g) T. Ishikawa,
Y. Shimizu, T. Kudoh, S. Saito, Org. Lett. 2003, 5, 3879–3882;
h) A. S. Kireev, A. T. Breithaupt, W. Collins, O. N. Nadein, A.
Kornienko, J. Org. Chem. 2005, 70, 742–745; i) S. Mondal, A.
Prathap, K. M. Sureshan, J. Org. Chem. 2013, 78, 7690–7700.
[16] Y. Shibata, S. Ogawa, Carbohydr. Res. 1989, 189, 309–322.
Received: May 16, 2014
Published Online: August 14, 2014
Eur. J. Org. Chem. 2014, 6030–6043
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