Synthesis of α- And β-galactopyranose-configured isomers of cyclophellitol and cyclophellitol aziridine

Article abstract of DOI:10.1002/ejoc.201402589

Cyclophellitol and cyclophellitol aziridine are potent and irreversible mechanism-based inhibitors of retaining β-glucosidases. Alterations in the configuration of these compounds can lead to irreversible inhibition of different classes of retaining glycosidases. We have recently reported on the design of a set of α-galactopyranose-configured cyclophellitol and cyclophellitol aziridine derivatives that inhibit human retaining α-galactosidases. Moreover, we have shown that fluorescently labeled derivatives enable the activity-based profiling of these enzymes in vitro. In this report we describe in detail the synthetic strategies that were used to obtain these epoxide- and aziridine-based probes. In addition, we describe the parallel synthesis of a set of β-galactopyranose-configured cyclophellitol isomers as putative inhibitors of retaining β-galactosidases.

Full text of DOI:10.1002/ejoc.201402589

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FULL PAPER
DOI: 10.1002/ejoc.201402589
Synthesis of α- and β-Galactopyranose-Configured Isomers of Cyclophellitol
and Cyclophellitol Aziridine
Lianne I. Willems,[a] Thomas J. M. Beenakker,[a] Benjamin Murray,[a] Berend Gagestein,[a]
Hans van den Elst,[a] Erwin R. van Rijssel,[a] Jeroen D. C. Codée,[a] Wouter W. Kallemeijn,[b]
Johannes M. F. G. Aerts,[b] Gijsbert A. van der Marel,[a] and Herman S. Overkleeft*[a]
Keywords: Cyclitols / Enzyme inhibitors / Irreversible inhibitors / Aziridines / Epoxides / Fluorescent probes
Cyclophellitol and cyclophellitol aziridine are potent and
irreversible mechanism-based inhibitors of retaining β-gluc-
osidases. Alterations in the configuration of these compounds
can lead to irreversible inhibition of different classes of re-
taining glycosidases. We have recently reported on
the design of a set of α-galactopyranose-configured cyclo-
phellitol and cyclophellitol aziridine derivatives that inhibit
human retaining α-galactosidases. Moreover, we have shown
that fluorescently labeled derivatives enable the activity-
based profiling of these enzymes in vitro. In this report we
describe in detail the synthetic strategies that were used to
obtain these epoxide- and aziridine-based probes. In ad-
dition, we describe the parallel synthesis of a set of β-galacto-
pyranose-configured cyclophellitol isomers as putative inhib-
itors of retaining β-galactosidases.
resulting in the formation of a covalent and stable linkage
between the inhibitor and the enzyme.
Introduction
Cyclophellitol (1) is a naturally occurring β-glucosidase
inhibitor that was first isolated from the mushroom Phel-
linus sp. in 1990 (Figure 1).[1,2] Follow-up studies demon-
strated that this compound is a selective and potent mecha-
nism-based irreversible inhibitor of several retaining β-
exoglucosidases.[3] As first postulated by Koshland,[4] most
retaining glycosidases, including retaining β-glucosidases,
hydrolyze the glycosidic bond in their substrates by em-
ploying a double displacement mechanism that results in a
net retention of anomeric stereochemistry.[4] This two-step
process involves two acidic amino acid residues that act as
a catalytic nucleophile and a general acid/base. During hy-
Figure 1. Structures of cyclophellitol (1), conduritol B epoxide (2),
cyclophellitol aziridine (3), and conduritol B aziridine (4).
Conduritol B epoxide (2, Figure 1), which is structurally
similar to cyclophellitol except for the substituent at C5
(carbohydrate numbering), is a less potent and also less se-
lective retaining glucosidase inhibitor.[5,6] As a consequence
of the symmetry in this molecule, it inhibits not only retain-
ing β-glucosidases but also various retaining α-glucosidases,
though generally with lower efficiency. The aziridine deriva-
tives of these epoxide-based inhibitors – cyclophellitol azir-
idine (3)[7] and conduritol B aziridine (4)[8] – are irreversible
inhibitors of the same enzyme classes.
drolysis,
a covalent enzyme–substrate intermediate is
formed, and this is the cause of the irreversible inhibition
by cyclophellitol. This β-glucopyranose-configured inhibi-
tor binds with high affinity in the active site of a target
enzyme by mimicking a terminal β-linked glucoside sub-
strate. The epoxide is positioned in such a way that it can
be protonated by the general acid/base residue and at the
same time attacked by the catalytically active nucleophile,
An interesting application of potent and selective
irreversible mechanism-based inhibitors such as cyclophelli-
tol is the design of labeled derivatives, also known as ac-
tivity-based probes (ABPs), that enable the selective moni-
toring of enzymatic activity in a biological sample. Using
the structure of cyclophellitol and cyclophellitol aziridine
as a scaffold, we have previously reported on the design
and synthesis of several fluorescently labeled retaining β-
glucosidase ABPs.[9,10] In these studies, reporter groups
[a] Leiden Institute of Chemistry and the Netherlands Proteomics
Centre, Leiden University,
P. O. Box 9502, 2300 RA Leiden, The Netherlands
E-mail: h.s.overkleeft@chem.leidenuniv.nl
http://biosyn.lic.leidenuniv.nl
[b] Department of Medical Biochemistry, Academic Medical
Center,
Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201402589.
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H. S. Overkleeft et al.
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Figure 2. Structures of α-galactopyranose-configured cyclophellitol and cyclophellitol aziridine derivatives 5–10 and β-galactopyranose-
configured isomers 11–16.
were incorporated by means of an azide moiety installed at
the C6 position of the epoxide inhibitor or through acyl-
Results and Discussion
ation of the aziridine nitrogen with various tagged substitu-
ents. The resulting probes enabled the sensitive detection of ratory for the synthesis of cyclophellitol, cyclophellitol azir-
retaining β-exoglucosidase activities in situ and in vivo. idine, and their derivatives are based on key intermediate
The synthetic strategies previously developed in our labo-
The remarkable specificities and potencies of these com- 17, described by Madsen and co-workers (Figure 3).[14]
pounds, in combination with the highly conserved catalytic With all hydroxy substituents in the correct configuration,
mechanism of retaining glycosidases, opens up the potential this cyclohexene gave access to both epoxide- and aziridine-
to develop selective inhibitors and ABPs for different based β-glucosidase probes.[9,10] We reasoned that the sim-
classes of retaining glycosidases by modifying their configu- ilar dibenzylated compound 18, recently reported by Lleba-
rations to that of the natural substrate of an enzyme of ria et al.,[15] with the only difference being the configuration
interest. Several cyclophellitol isomers have been described of the hydroxy substituent at C4 (carbohydrate numbering),
in the literature. The α-glucopyranose-configured isomer of would provide an ideal starting point for both α- and β-
inhibitor 1, 1,6-epi-cyclophellitol, is an irreversible inhibitor galactopyranose-configured epoxides and aziridines (Fig-
of retaining α-glucosidases.[11] An α-mannopyranose-con- ure 3). The aziridine-based probes can be modified with re-
figured cyclophellitol isomer was demonstrated to be a re- porter entities by acylation of the aziridine nitrogen. In the
taining α-mannosidase inhibitor,[7] whereas its β-isomer has case of the epoxide inhibitors, an azide installed at C6 en-
been synthesized as a putative retaining β-mannosidase in- ables modification with alkyne-modified tags through cop-
hibitor.[12] Recently we have reported on the synthesis of per(I)-catalyzed “click” reactions.[16]
α-galactopyranose-configured epoxide 5 (Figure 2), which
Cyclohexene derivative 18 was synthesized in three steps
inhibits both retaining α- and β-galactosidases.[13] More- from aldehyde 20[14] and oxazolidinone 19[17] essentially as
over, we have also synthesized the various derivatives 6–8, described by Llebaria and co-workers (Scheme 1).[15] The
functionalized at C6 with reporter entities, as well as the key step in this procedure is the dibutylboryl-triflate-cata-
acylated aziridine analogue 9 and its fluorescently labeled lyzed stereoselective aldol condensation of 19 and 20. When
derivative 10 (Figure 2). We have demonstrated that the az- this reaction was performed as prescribed, by consecutive
iridine-based ABPs are very potent and selective inhibitors addition of all reagents at a temperature of –78 °C, followed
of the human retaining α-galactosidases, α-galactosidase A, by reaction at –20 °C to –15 °C for 3.5 h, compound 21
and α-N-acetylgalactosaminidase, and that Bodipy-tagged was obtained stereoselectively but with a considerably lower
probe 10 enables the labeling of the endogenous activity of yield (44%) than reported (83%). Because a considerable
these enzymes in cell extracts. In this report we give a de- amount of unreacted aldehyde could repeatedly be isolated
tailed overview of the synthetic strategies that we have used after the reaction, we attempted to improve the yield by
to generate the α-galactopyranose-configured probes, in- using freshly distilled Et3N and by drying solutions of 19
cluding potential pitfalls and difficulties that we encoun- and 20 in dichloromethane over activated molecular sieves
tered during the synthesis and purification of these com- (4 Å) for at least 1 h prior to use. Furthermore, we per-
pounds. In addition, we describe the parallel synthesis of formed the reaction at –20 °C overnight. With this pro-
compounds 11–16, a new series of epoxide- and aziridine- cedure, aldehyde 20 was completely consumed, and the de-
based probes with β-galactopyranose configuration.
sired product 21 could be isolated in 80% yield. Next, the
2
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α- and β-galacto-Configured Cyclophellitols
anhydrous BCl3 in dichloromethane, after which the crude
product was treated with benzoyl chloride in pyridine to
afford fully benzoylated product 23. Epoxidation was then
performed with m-chloroperoxybenzoic acid, which gave a
mixture of α- and β-epoxides 24 in a ratio of approximately
1:1. Separation of the two stereoisomers proved impossible
at this stage, and also after removal of the benzoyl esters by
methanolysis under basic conditions to give 25. However,
acetylation of 25 with acetic anhydride in pyridine gave a
separable mixture of fully acetylated α-epoxide 26, triacetyl-
ated β-epoxide 27, and a small amount of fully acetylated
β-epoxide 28. These results indicate that acetylation at the
C4 hydroxy group is sterically hindered by the β-epoxide.
After separation of the isomers by column chromatography,
deprotection by basic methanolysis yielded the enantiomer-
ically pure α- and β-epoxides 5 and 11, respectively.
Figure 3. Key intermediate 17, previously used for the synthesis of
β-glucopyranose-configured probes, and key intermediate 18 used
in this work for the synthesis of galactopyranose-configured inhibi-
tors and probes. R1 = OH, N3, or tag; R2 = N3- or reporter-group-
functionalized spacer.
Evans template was removed by reduction with LiBH4, and
the resulting alcohol 22 was subjected to ring-closing me-
tathesis in the presence of the second-generation Grubbs
catalyst to provide cyclohexene 18.
Scheme 2. Reagents and conditions. (a) (i) BCl3, CH2Cl2, –78 °C,
4 h, (ii) BzCl, pyridine, room temp., overnight, 76%; (b) mCPBA,
CH2Cl2, room temp., 4–8 d, 62%; (c) NaOMe, MeOH, room temp.,
1.5 h, 76%; (d) Ac2O, pyridine, room temp., 4 h, 26 (38%) + 27
(20%) + 28 (2%); (e) NaOMe, MeOH, room temp., 2 h, 5 (94%),
11 (84%).
The diastereomeric configuration of epoxides 5 and 11
was determined by 1H NMR analysis and further con-
firmed by DFT calculations. The optimized structures ob-
tained by these calculations reveal that both epoxides adopt
half-chair conformations (see the Supporting Information).
The calculated coupling constants agree well with the corre-
sponding experimentally measured values (Table 1), sup-
porting the experimentally assigned configurations. Evi-
dence is provided by the absence of an observable coupling
constant between H1 and H2 of the β-epoxide (calculated
value J1,2 = 0.04 Hz), in comparison with J1,2 = 2.5 Hz for
the α-epoxide, and the absence of an observable coupling
constant between H5 and H7 of the α-epoxide (calculated
value J5,7 = 0.04 Hz) in comparison with J5,7 = 1.7 Hz for
the β-epoxide. Characteristic proton peaks for α-epoxide 5
Scheme 1. Reagents and conditions: a) Bu2BSO3CF3, Et3N,
CH2Cl2, –78 °C to –20 °C, overnight, 80%; b) LiBH4, THF/H2O,
0 °CǞroom temp., 2 h, 85%; c) second-generation Grubbs cata-
lyst, CH2Cl2, 40 °C, overnight, 78%.
The synthetic route to α- and β-epoxides[18] 5 and 11 is are double doublets at δ = 3.3 ppm (JH1 = 4.0, 2.5 Hz) and
depicted in Scheme 2. We decided to change the protecting δ = 3.1 ppm (JH7 = 4.0, 1.8 Hz). For β-epoxide 11, the cor-
group pattern prior to epoxidation in order to enable facile responding peaks are a multiplet at δ = 3.3 ppm (H7) and
deprotection of the hydroxy groups in the presence of the a doublet at δ = 3.2 ppm (JH1 = 3.7 Hz). Very similar pat-
epoxide. Hence, the benzyl groups in 18 were removed with terns of epoxide proton peaks were observed for the acetyl-
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H. S. Overkleeft et al.
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ated epoxides 26–28 (although, as might be expected, the
exact chemical shift values depend on the substitution of
the hydroxy groups), as well as for the C6-modified ana-
logues 6–8 and 12–14 and the benzylated epoxide 36 de-
scribed below.
Table 1. Comparison of experimentally measured [1H NMR analy-
sis (“J exp.”)] and calculated [from DFT calculations (“J calcd.”)]
coupling constants for epoxides 5 and 11.
α-Epoxide 5 J exp. J calcd. β-Epoxide 11 J exp.
J calcd.
[Hz]
[Hz]
[Hz]
[Hz]
H1,H7
H1,H2
H2,H3
H3,H4
H4,H5
H5,H7
H4,H7
4.0
2.5
8.6
1.9
3.6
n.d.
1.8
3.7
2.2
6.9
1.8
4.0
0.04
1.1
H1,H7
H1,H2
H2,H3
H3,H4
H4,H5
H5,H7
H4,H7
3.7
n.d.
8.6
n.d.
4.0
1.7
3.4
0.04
6.6
2.7
3.5
1.5
0.8
[a]
[b]
[a]
Scheme 3. Reagents and conditions: (a) (i) pTsCl, Et3N, CH2Cl2,
room temp., 4 d, (ii) NaN3, DMF, 60 °C, overnight, 63%;
(b) (i) BCl3, CH2Cl2, –78 °C, 4 h, (ii) BzCl, pyridine, room temp.,
overnight, 85%; (c) mCPBA, CH2Cl2, room temp., 4–8 d, 53%;
(d) NaOMe, MeOH, room temp., 1.5 h, 6 (42%) + 12 (8%).
[a]
n.d.
[a] Values not determined (n.d.) because of very small coupling
constants (J Ͻ 1 Hz). [b] Values not determined because of peak
overlap.
The synthesis of azide-functionalized epoxides 6 and 12 biotin-tagged α-epoxide ABPs 7 and 8 were prepared from
from 18 commenced with the installment of the azide moi- azide-functionalized epoxide 6 by treatment either with
ety at C6 by selective tosylation of the primary alcohol, Bodipy-alkyne 32[19] or with biotin-alkyne 33[20] in the pres-
followed by substitution with sodium azide to give 29 ence of copper(II) sulfate and sodium ascorbate overnight
(Scheme 3). Next, the same strategy of deprotection, benzo- (Scheme 4). Similarly, the Bodipy- and biotin-tagged β-ep-
ylation, and epoxidation as used for the non-tagged epoxide oxides 13 and 14 were synthesized from azido-epoxide 12.
inhibitors was employed to synthesize 31, which was ob- These reactions proceeded smoothly to afford the fluores-
tained as a 4:1 mixture of α- and β-epoxides. The α- and β- cently labeled and biotinylated epoxide probes in good
epoxides were not separable at this stage. However, after a yield.
final deprotection step the two isomers could be separated
The synthetic strategy directed towards the β-aziridine
by column chromatography to give the enantiomerically ABPs 15 and 16 is based on a procedure previously devel-
pure azide-functionalized α- and β-epoxides 6 and 12, oped in our laboratory for the corresponding β-gluco-
respectively.
pyranose-configured probes.[10] The approach relies on the
The azide groups in compounds 6 and 12 provide a stereocontrolled formation of a β-aziridine with the aid of
means to install reporter groups through copper(I)-cata- the primary alcohol at C6 in cyclohexene 18 (Scheme 5). By
lyzed azide–alkyne Huisgen [2+3] cycloaddition (“click” re- the reported tandem one-pot procedure, the primary
actions)[16] with alkyne-derivatized tags. The Bodipy- and hydroxy group in 18 was first selectively transformed into a
Scheme 4. Reagents and conditions: Bodipy-alkyne 32 or biotin-alkyne 33, CuSO4·5H2O, sodium ascorbate, DMF, room temp., overnight,
7 (88%), 8 (68%), 13 (90%), 14 (68%).
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α- and β-galacto-Configured Cyclophellitols
trichloroacetimidate, and this was then subjected to iodocy- possible to synthesize β-aziridine 34 by the same strategy,
clization by treatment with iodine under basic conditions. with no product being formed by attempted opening of the
Acidic hydrolysis of the intermediate imidate was followed benzylated α-epoxide isomer of 36 with sodium azide and
by the addition of base to effect intramolecular nucleophilic lithium perchlorate. Attempts to open the α-epoxide with
displacement of the iodine by the amine to provide partially sodium azide under acidic “nonchelating” conditions
protected aziridine 34, which can be used for subsequent [MeOH/1.2 m NH4Cl (4:1), 80 °C][21] or with use of phase-
functionalization with a reporter group.
transfer conditions (Bu4NCl, MeCN/H2O, 80 °C) were also
unsuccessful.
The final stage in the synthetic route to the aziridine-
based ABPs involves removal of the benzyl groups in 34
and 38 and acylation of the aziridine nitrogen with azide-
or Bodipy-functionalized spacers 39 and 40, respectively
(Scheme 6). Removal of the benzyl groups was achieved by
Birch reduction, after which acylation in the presence of 2-
ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) as
the coupling reagent at 0 °C yields the N-acylated prod-
uct.[10] As we have reported previously, these steps are com-
plicated by the lability of the acylated aziridine under either
basic or acidic conditions, with opening of the aziridine
during reversed-phase HPLC purification and/or ensuing
lyophilization being a major problem. Therefore, it is essen-
tial that the acylation step and the purification of the final
product are carried out under neutral conditions, including
acetonitrile/water gradients for HPLC purification and the
use of MilliQ for workup. In the case of α-aziridine 38, this
procedure uneventfully provided the azide- and Bodipy-
tagged products 9 and 10.
Scheme 5. Reagents and conditions: (a) (i) CCl3CN, DBU, CH2Cl2,
0 °C, 1.5 h, (ii) I2, NaHCO3, H2O, room temp., overnight, (iii) HCl,
MeOH, room temp., 3.5 h, (iv) HCl, dioxane, 60 °C, 1 h,
(v) NaHCO3, MeOH, room temp., 4 d, 52%; (b) NaH, BnBr,
TBAI, DMF, room temp., overnight, 85%; (c) mCPBA, CH2Cl2,
room temp., overnight, 63%; (d) NaN3, LiClO4, MeCN, 80 °C,
overnight, 73%; (e) Ph3P, MeCN, 80 °C, 2 h, 26%.
In the cases of the α-aziridine ABPs 9 and 10, the appli-
cation of a similar procedure would entail the formation of
a trichloroacetimidate with the hydroxy group at C2 to
achieve iodocyclization below the plane of the cyclohexane
ring. Such a strategy would also require several additional
protective group manipulations in order to obtain this
hydroxy group selectively unmasked. With the aim of find-
ing a shorter synthetic route to the α-aziridine probes, we
decided to use an alternative approach in which the α-azir-
idine moiety is generated from a β-epoxide. As depicted in
Scheme 5, this strategy commenced with the formation of
fully benzylated compound 35. Epoxidation under the same
conditions as described above yielded a mixture of α- and
β-epoxides. Fortunately, the more prevalent product was the
desired β-epoxide 36, together with only a minor amount
of the corresponding α-epoxide (ratio 6:1). Moreover, the
two isomers could easily be separated by column
chromatography. Next, the β-epoxide was treated according
to literature procedures with sodium azide and lithium per-
chlorate as a Lewis acid to give a mixture of trans-azido-
alcohols 37.[21] Subsequent treatment with triphenylphos-
phine led to the formation of α-aziridine 38 through an in-
tramolecular Staudinger-like ring closure.[22] The yield of
this reaction was rather low (26%), partly due to the fact
that it proved difficult to remove the triphenylphosphine
oxide byproduct. In addition, the intramolecular rearrange-
Scheme 6. Reagents and conditions: (a) Bodipy-alkyne 32,
CuSO4·5H2O, sodium ascorbate, DMF, 80 °C, 4.5 h, 87%; (b) Li,
NH3 (l), THF, –60 °C, 45 min; (c) 39 or 40, EEDQ, DMF, 0 °C,
90 min, 9 (14%), 10 (15%).
The synthesis of the β-aziridine ABPs, however, proved
ment of the imino-phosphorane intermediate is likely hin- more problematic. After debenzylation of β-aziridine 34
1
dered either by steric factors or by ring strain. Nonetheless, (Scheme 6), H NMR analysis of the crude product 42 re-
the α-aziridine 38 could be obtained in enantiomerically vealed a small amount of byproduct formation. Attempts
pure form in sufficient quantities. Notably, it proved im- to acylate 42 with azido-spacer 39 followed by reversed-
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phase HPLC purification and lyophilization resulted in sig- as mixtures of α- and β-isomers, stereoselective synthesis
nificant degradation of the aziridine. LC/MS analysis re- is also possible through alteration of the protective group
vealed the major product to be an H2O adduct, presumably patterns and/or by application of different epoxidation pro-
resulting from opening of the aziridine. In some cases an cedures, as was reported previously for the synthesis of β-
HCl adduct was also observed, despite the fact that no glucopyranose-configured epoxide ABPs.[9] In the case of
acids and no chloride salts were used during purification. the aziridine-based probes, two separate routes were re-
Further analysis by 1H NMR indicated almost complete quired to obtain the α- and β-configured isomers. The
disappearance of the aziridine proton peaks. Hence, it ap- β-configured aziridine 34 was synthesized stereoselectively
pears that the N-acylated β-aziridine moiety is rather labile by an intramolecular iodocyclization procedure, whereas
and prone to decomposition, even under our optimized α-aziridine 38 was obtained by azidolysis of the correspond-
conditions. As a result, ABPs 15 and 16 could not be syn- ing β-epoxide. The ability of the α-galactosidase probes
thesized.
presented here to inhibit recombinant human retaining
In the synthetic strategies described here, the formation α-galactosidases and to label the endogenous activities of
of both α- and β-epoxides by m-chloroperoxybenzoic acid- these enzymes in vitro has already been established.[13]
mediated epoxidation is favorable, as long as the two iso- Evaluation of the inhibitory potencies of the β-galacto-
mers can be separated at some point in the route of synthe- pyranose-configured epoxides will be the subject of future
sis. In other cases, however, it might be desirable to obtain research.
only one isomer, and for that purpose other oxidation meth-
ods should be evaluated. It is worth noting that epoxidation
of the perbenzoylated compound 23 gives a 1:1 ratio of α-
Experimental Section
and β-epoxides whereas an α/β ratio of 4:1 was obtained
General Methods: All reagents were commercial grade and were
with the C6-azide-functionalized analogue 30. Full benz-
used as received unless stated otherwise. Dichloromethane
ylation, as in compound 35, on the other hand, leads to
(CH2Cl2), dichloroethane (DCE), dimethylformamide (DMF), and
tetrahydrofuran (THF) (Biosolve) were of analytical grade and,
when used under anhydrous conditions, stored over flame-dried
molecular sieves (3 Å). Ethyl acetate (EtOAc, Riedel-de Haën) used
for column chromatography was of technical grade and distilled
more β-selectivity during epoxidation (α/β ratio of 1:6).
These results indicate that formation of the β-epoxide is fa-
vored in the absence of chelating effects and that the pres-
ence of a benzoyl group at either the allylic (C2) or the
homoallylic (C6) position can enhance α- or β-selectivity, before use. Reactions were monitored by TLC analysis (DC-
alufolien, Merck, Kieselgel 60, F254) with detection by UV absorp-
tion (254/366 nm), by spraying with H2SO4 in ethanol (20%) or a
solution of (NH4)6Mo7O24·4H2O (25 gL–1) and (NH4)4Ce(SO4)4·
2H2O (10 gL–1) in 10% aqueous sulfuric acid, followed by charring
at ca. 150 °C, or by spraying with an aqueous solution of KMnO4
(7%) and K2CO3 (2%). Column chromatography was performed
on silica gel (Screening Devices BV, 0.040–0.063 mm, 60 Å). LC/
MS analysis was performed with an LCQ Adventage Max (Thermo
Finnigan) ion-trap spectrometer (ESI+) coupled to a Surveyor
HPLC system (Thermo Finnigan) equipped with a C18 column
respectively. As expected, epoxidation of unprotected cyclo-
hexene derivatives, obtained by debenzylation either
of 18 or of 29, led mainly to the formation of α-epoxides
(α/β Ͼ 15:1), due to stereoselective control by the allylic
hydroxy group, which leads to syn epoxidation.
Conclusions
We describe the parallel synthesis of α- and β-galacto- (Gemini, 4.6 mmϫ50 mm, 5 μm particle size, Phenomenex). The
applied buffers were A: H2O, B: MeCN, and C: aqueous TFA (1%).
Reported gradients represent the percentage of buffer B in buffer A
with 10% buffer C. Alternatively, where indicated, LC/MS analysis
was performed with an API 3000 ESI (Q1) mass spectrometer (Ap-
plied Biosystems) coupled to a Jasco (900 series) HPLC system.
The applied buffers were A: H2O, B: MeCN, and C: NH4OAc in
H2O (100 mm). Reported gradients represent the percentage of
buffer B in buffer A with 10% buffer C. HRMS analysis was per-
formed with an LTQ Orbitrap (Thermo Finnigan) mass spectrome-
ter equipped with an electronspray ion source in positive mode
pyranose-configured cyclophellitol and cyclophellitol azir-
idine isomers as (putative) inhibitors and ABPs for retain-
ing α- and β-galactosidases, respectively. We present the
synthesis of non-tagged epoxide inhibitors 5 and 11, as well
as derivatives of these functionalized at C6 with an azide, a
Bodipy fluorophore, or a biotin tag (compounds 6–8 and
12–14). As well as the epoxide-based ABPs, α-galacto-
pyranose-configured aziridine-based probes in which an
azide or Bodipy tag is installed through N-acylation of the
aziridine were also synthesized (compounds 9 and 10). Un- (source voltage 3.5 kV, sheath gas flow 10 mLmin–1, capillary tem-
perature 250 °C) with resolution R = 60000 at m/z 400 (mass range
m/z = 150–2000) and dioctyl phthalate (m/z = 391.28428) as a “lock
mass”. The high-resolution mass spectrometer was calibrated prior
to measurements with a calibration mixture (Thermo Finnigan).
For reversed-phase HPLC purification an automated HPLC
system equipped with a C18 semiprep column (Gemini C18,
250 mmϫ10 mm, 5 μm particle size, 110 Å pore size, Phenomenex)
was used. 1H- and 13C-APT-NMR spectra were recorded with a
Bruker AV 400 (400/100 MHz) or a Bruker DMX 600 (600/
150 MHz) instrument and a cryoprobe. All NMR spectra were re-
fortunately, it proved impossible to isolate the acylated β-
aziridine isomers, due to severe lability of the aziridine dur-
ing purification procedures. Efforts to synthesize differently
substituted aziridine-based β-galactosidase ABPs are cur-
rently being undertaken in our laboratory.
The synthetic strategies directed towards both α- and β-
galactopyranose-configured probes, epoxides as well as
aziridines, are based on key cyclohexene intermediate 18,
from which all compounds were synthesized in a maximum
of six steps. Although our goal was to obtain the epoxides corded at room temperature (18–22 °C). Chemical shifts are given
6
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© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 0000, 0–0

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α- and β-galacto-Configured Cyclophellitols
in ppm (δ) relative to the solvent peak or to tetramethylsilane as
internal standard. Coupling constants (J) are given in Hz. All pre-
sented 13C-APT spectra are proton-decoupled. Peak assignments
alcohol 22 as a white solid (4.3 g, 12 mmol, 85%). [α]2D0 = +8 (c =
0.1, CHCl3). 1H NMR (400 MHz, CDCl3): δ = 7.45–7.21 (m, 10
H, CH arom.), 6.14–5.94 (m, 2 H, 2ϫCH=CH2), 5.49–5.33 (m, 2
H, 2ϫCH=CHH), 5.26 (dd, J = 10.4, 2.1 Hz, 1 H, CH=CHH),
5.13 (dd, J = 17.5, 2.2 Hz, 1 H, CH=CHH), 4.67 (d, J = 11.8 Hz,
1 H, CHH Bn), 4.57 (d, J = 11.1 Hz, 1 H, CHH Bn), 4.48 (d, J =
11.1 Hz, 1 H, CHH Bn), 4.39 (d, J = 11.8 Hz, 1 H, CHH Bn), 4.21
(dd, J = 7.0, 3.8 Hz, 1 H, CH-OH), 4.05 (d, J = 9.1 Hz, 1 H, CH-
OBn), 3.77–3.64 (m, 2 H, CH2-OH), 3.51 (dd, J = 9.1, 3.7 Hz, 1
H, CH-OBn), 3.39 (br. s, 1 H, OH), 2.58 (ddt, J = 7.9, 5.7, 2.9 Hz,
1 H, CH-CH=CH2), 2.20 (br. s, 1 H, OH) ppm. 13C NMR
(100 MHz, CDCl3): δ = 137.75, 137.26 (Cq arom.), 135.28, 133.85
(2ϫCH=CH2), 128.55, 128.36, 127.93, 127.86, 127.80 (CH arom.),
119.51, 119.05 (2ϫCH=CH2), 79.58, 78.97 (2ϫCH-OBn), 73.08
(CH2 Bn), 72.53 (CH-OH), 70.97 (CH2 Bn), 65.58 (CH2-OH),
47.33 (CH-CH=CH2) ppm. HRMS: calcd. for [C23H28O4Na]+
391.18798; found 391.18897.
1
are based on 2D H-COSY and 13C-HSQC NMR experiments.
Note: Numbering of proton peaks in cyclohexene and cyclohexane
epoxide derivatives is according to the numbering in Figure 3.
(S)-3-[(2S,3S,4S,5S)-4,5-Bis(benzyloxy)-3-hydroxy-2-vinylhept-6-
enoyl]-4-isopropyloxazolidin-2-one (21): Note: Solutions of aldehyde
20[14] and oxazolidinone 19[17] in CH2Cl2 were put under argon and
dried with activated molecular sieves (4 Å) for at least 1 h prior to
use. Et3N was freshly distilled from CaH2 and stored on activated
molecular sieves (4 Å) under argon prior to use.
A solution of oxazolidinone 19 (12 mmol, 2.4 g, 1.15 equiv.) in
CH2Cl2 under argon was cooled to –78 °C with the aid of a cryost-
ate before addition of Bu2BSO3CF3 (12 mmol, 12 mL 1 m in
CH2Cl2, 1.15 equiv.) and Et3N (14 mmol, 1.9 mL, 1.3 equiv.). The
reaction mixture was stirred at –78 °C for 1 h, then at 0 °C (ice
bath) for 15 min, and subsequently cooled back to –78 °C. A solu-
tion of aldehyde 20 (11 mmol, 3.2 g, 1.0 equiv.) in CH2Cl2 under
argon was added by cannula, after which the reaction mixture was
stirred at –20 °C overnight. Next, the mixture was removed from
the cold bath and put in an ice bath (–5 °C). After quenching with
PBS (20 mL), H2O2 (30% aqueous solution) was added dropwise
while the temperature of the mixture was kept below 5 °C, until no
more rise in temperature was observed. At this point the mixture
was stirred for another 45 min while being allowed to warm slowly
to room temperature, after which aqueous saturated NaHCO3 was
added and the aqueous layer was extracted with CH2Cl2 (3ϫ). The
combined organic layers were dried with MgSO4, filtered, and con-
centrated in vacuo. The crude product was purified by column
chromatography (pentaneǞ10% EtOAc in pentane) to afford the
aldol adduct 21 (4.2 g, 8.6 mmol, 80%). [α]2D0 = –48 (c = 0.1,
CHCl3). 1H NMR (400 MHz, CDCl3): δ = 7.39–7.25 (m, 10 H, CH
arom.), 6.06 (ddd, J = 18.5, 9.7, 7.1 Hz, 1 H, CH=CH2), 5.92 (ddd,
J = 17.3, 10.1, 8.8 Hz, 1 H, CH=CH2), 5.45–5.37 (m, 3 H,
CH=CH2, CH=CHH), 5.29 (dd, J = 10.2, 1.6 Hz, 1 H, CH=CHH),
5.02 (dd, J = 9.0, 7.2 Hz, 1 H, CH-CH=CH2), 4.69 (dd, J = 11.5,
4.0 Hz, 2 H, CH2 Bn), 4.55–4.40 (m, 3 H, CH2 Bn, CH-OH), 4.29
(dd, J = 7.3, 3.9 Hz, 1 H, CH-OBn), 4.03 (dt, J = 8.6, 3.5 Hz, 1 H,
CH oxazolidinone), 3.84 (dd, J = 9.0, 3.0 Hz, 1 H, CHH oxazolidi-
none), 3.58 (dd, J = 8.3, 3.9 Hz, 1 H, CH-OBn), 3.39 (br. s, 1 H,
OH), 3.26 (t, J = 8.8 Hz, 1 H, CHH oxazolidinone), 2.26–2.14
[m, 1 H, CH-(CH3 )2 ], 0.75 (dd, J = 18.8, 7.0 Hz, 6 H,
2ϫCH3) ppm. 13C NMR (100 MHz, CDCl3): δ = 172.53 (C=O),
153.53 (C=O), 138.01, 137.76 (Cq arom.), 134.55, 133.56
(2ϫCH=CH2), 128.17, 128.00, 127.64, 127.53, 127.32, 127.09 (CH
arom.), 120.43, 119.14 (2ϫCH=CH2), 81.78, 79.90 (2ϫCH-OBn),
73.04, 71.12 (2ϫCH2 Bn), 70.66 (CH-OH), 62.38 (CH2 oxazolidi-
none), 57.94 (CH oxazolidinone), 50.06 (CH-CH=CH2), 27.96
[CH-(CH3)2], 17.75, 14.41 (2 ϫ CH3) ppm. HRMS: calcd. for
[C29H36NO6]+ 494.25371; found 494.25349. HRMS: calcd. for
[C29H35NO6Na]+ 516.23566; found 516.23543.
Cyclohexene 18: The second-generation Grubbs catalyst (0.4 mmol,
0.34 g, 5 mol-%) was added under argon to a solution of 22
(7.8 mmol, 2.9 g) in CH2Cl2. The reaction mixture was stirred at
40 °C in the dark for 20 h, DMSO (40 mmol, 2.8 mL, 100 equiv.
with respect to catalyst) was added, and the mixture was stirred
overnight at room temperature before being concentrated in vacuo.
Purification by column chromatography [pentane Ǟ pentane/
EtOAc 1:1 (v/v)] gave cyclohexene 18 (7.2 mmol, 2.5 g, 92 %).
[α]2D0 = +104 (c = 0.1, CHCl3). H NMR (400 MHz, CDCl3): δ =
1
7.38–7.28 (m, 10 H, CH arom.), 5.84 (d, J = 10.4 Hz, 1 H,
CH=CH), 5.55 (d, J = 10.1 Hz, 1 H, CH=CH), 4.71 (d, J = 8.5 Hz,
4 H, 2ϫCH2 Bn), 4.33–4.31 (m, 2 H, CH-2, CH-4), 3.82–3.80 (m,
2 H, CH2-OH), 3.65 (d, J = 7.4 Hz, 1 H, CH-3), 2.95 (br. s, 2 H,
2 ϫ OH), 2.45 (d, J = 6.1 Hz, 1 H, CH-5) ppm. 13C NMR
(100 MHz, CDCl3): δ = 138.45, 137.93 (Cq arom.), 128.45, 128.32,
127.85, 127.75, 127.57, 127.43, 126.75 (CH arom., 2ϫCH=CH),
81.80 (CH-4), 77.31, 76.58 (CH-2, CH-3), 72.15 (CH2-OH), 41.87
(CH-5) ppm. HRMS: calcd. for [C21H25O4]+ 341.17474; found
341.17519. HRMS: calcd. for [C21H24O4Na]+ 363.15668; found
363.15714.
Benzoyl-cyclohexene 23: A solution of cyclohexene 18 (0.5 mmol,
0.17 g) in CH2Cl2 under argon was cooled to –78 °C, after which
BCl3 was added (5 mmol, 5 mL 1 m in CH2Cl2, 10 equiv.). The reac-
tion mixture was stirred under argon at –78 °C for 4 h and then
quenched with MeOH, concentrated in vacuo, and coevaporated
with toluene (3ϫ). Purification by column chromatography (10%
MeOH in EtOAcǞ20% MeOH in EtOAc) afforded the fully de-
protected compound, which was redissolved in pyridine and cooled
to 0 °C. Next, benzoyl chloride was added (5 mmol, 0.6 mL,
10 equiv.) and the mixture was stirred overnight at room tempera-
ture before being quenched with a saturated aqueous NaHCO3
solution. The aqueous layer was extracted with CH2Cl2 (3ϫ), and
the combined organic layers were dried with MgSO4, filtered, and
concentrated in vacuo. Purification by column chromatography
(pentaneǞ10% EtOAc in pentane) yielded the fully benzoylated
product 23 (0.22 g, 0.38 mmol, 76% over two steps). [α]2D0 = +250
1
(2R,3S,4S,5S)-4,5-Bis(benzyloxy)-2-vinylhept-6-ene-1,3-diol (22): (c = 0.1, CHCl3). H NMR (400 MHz, CDCl3): δ = 8.00–7.88 (m,
Oxazolidinone 21 (13.8 mmol, 6.8 g) was dissolved in THF
(100 mL) at 0 °C, after which H2O (8 mL) and LiBH4 (35 mmol,
17.5 mL 2 m in THF, 2.5 equiv.) were added. The reaction mixture
was stirred at 0 °C for 1 h and then at room temperature for 1 h,
before being quenched with NaOH (2 m). The resulting mixture
was extracted with Et2O (2ϫ), and the combined organic layers
were washed with aqueous saturated NaHCO3 and brine, dried
with MgSO4, and concentrated in vacuo. Purification by column
chromatography (pentane Ǟ 40 % EtOAc in pentane) yielded
8 H, CH arom.), 7.62–7.30 (m, 12 H, CH arom.), 6.23–6.18 (m, 2
H, CH-3, CH-4), 6.09–6.06 (m, 1 H, CH=CH), 5.88 (dd, J = 1.2,
10.4 Hz, 1 H, CH=CH), 5.78 (dd, J = 2.2, 8.6 Hz, 1 H, CH-2), 4.59
(dd, J = 6.4, 10.8 Hz, 1 H, CHH), 4.32 (dd, J = 8.8, 10.8 Hz, 1 H,
CHH), 3.44–3.40 (m, 1 H, CH-5) ppm. 13C NMR (100 MHz,
CDCl3): δ = 166.23, 166.14, 165.87, 165.51 (4ϫ C=O), 133.71,
133.42, 133.26, 133.20, 133.13, 129.80, 129.77, 129.74, 129.68,
129.51, 129.35, 129.24, 128.60, 128.47, 128.39, 128.31, 126.86,
126.81 (CH and Cq arom., 2ϫCH=CH), 73.10, 70.60, 69.30 (CH-
Eur. J. Org. Chem. 0000, 0–0
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Pages: 14
H. S. Overkleeft et al.
FULL PAPER
2, CH-3, CH-4), 63.05 (CH2), 39.47 (CH-5) ppm. LC/MS analysis:
Rt 10.1 min (linear gradient 10Ǟ90% B in 15 min), m/z 599.1 [M
+ Na]+, 1174.9 [2M + Na]+. HRMS: calcd. for [C35H29O8]+
577.18569; found 577.18602. HRMS: calcd. for [C35H28O8Na]+
599.16764; found 599.16727.
4.24–4.07 (m, 2 H, CH2), 3.59 (dd, J = 4.0, 2.4 Hz, 1 H, CH-1),
3.10 (dd, J = 4.0, 1.6 Hz, 1 H, CH-7), 2.68 (td, J = 8.1, 3.7 Hz, 1
H, CH-5), 2.13 (s, 3 H, CH3), 2.10 (s, 3 H, CH3), 2.06 (s, 3 H,
1
CH3), 2.00 (s, 3 H, CH3) ppm. H NMR (400 MHz, CDCl3) 28: δ
= 5.52–5.45 (m, 1 H, CH-4), 5.26 (d, J = 9.4 Hz, 1 H, CH-2), 4.95
(dd, J = 9.6, 2.0 Hz, 1 H, CH-3), 4.35–4.20 (m, 2 H, CH2), 3.26–
3.17 (m, 2 H, CH-1, CH-7), 2.78–2.74 (m, 1 H, CH-5), 2.12 (s, 3
H, CH3), 2.09 (s, 3 H, CH3), 2.05 (s, 3 H, CH3), 1.96 (s, 3 H,
CH3) ppm. 1H NMR (400 MHz, CDCl3) 27: δ = 5.35 (d, J =
9.1 Hz, 1 H, CH-2), 4.90 (dd, J = 9.1, 2.6 Hz, 1 H, CH-3), 4.46–
4.38 (m, 2 H, CH2), 3.99 (d, J = 9.3 Hz, 1 H CH-4), 3.44–3.37 (m,
1 H, CH-7), 3.30 (d, J = 3.6 Hz, 1 H, CH-1), 2.54 (tdd, J = 7.6,
3.5, 1.3 Hz, 1 H, CH-5), 2.46 (d, J = 11.0 Hz, 1 H, OH), 2.13 (s, 3
H, CH3), 2.12 (s, 3 H, CH3), 2.10 (s, 3 H, CH3) ppm. The β-isomers
27 and 28 were then combined and deprotected separately from 26
by treatment with sodium methoxide (30 μmol, 5 μL 5.6 m in
MeOH, 0.3 equiv. for 26 and 17 μmol, 3 μL 5.6 m in MeOH,
0.3 equiv. for 27/28) in MeOH at room temperature for 2 h. The
mixtures were neutralized with Amberlite-H+ IR-200, filtered, and
concentrated in vacuo. The crude products were finally purified by
column chromatography (10% MeOH in CH2Cl2 Ǟ15% MeOH in
CH2Cl2) to give α-epoxide 5 (17 mg, 94 μmol, 94%) and β-epoxide
11 (8.5 mg, 48 μmol, 84%).
Benzoyl-epoxide 24: 3-Chloroperoxybenzoic acid (1.1 mmol, 0.24 g,
2.5 equiv.) was added to a solution of 23 (0.42 mmol, 0.24 g) in
CH2Cl2 and the reaction mixture was stirred for 4 d at room tem-
perature. Then, an additional 1 equiv. of 3-chloroperoxybenzoic
acid was added and the reaction mixture was stirred again for 4 d
at room temperature, before being concentrated in vacuo. Purifica-
tion by column chromatography (pentaneǞ15% EtOAc in pent-
ane) yielded a mixture of the α- and β-epoxides 24 in a 1:1 ratio
1
(0.15 g, 0.26 mmol, 62%). H NMR (400 MHz, CDCl3): δ = 8.19–
7.76 (m, 8 H, 8ϫCH arom. α, 8ϫCH arom. β), 7.68–7.20 (m, 12
H, 12ϫCH arom. α, 12ϫCH arom. β), 6.14–5.98 (m, 1.5 H, CH-
2 α, CH-4 α, CH-4 β), 5.78 (d, J = 9.4 Hz, 0.5 H, CH-2 β), 5.68
(dd, J = 1.8, 9.2 Hz, 0.5 H, CH-3 α), 5.61 (dd, J = 2.6, 9.4 Hz, 0.5
H, CH-3 β), 4.78 (dd, J = 6.7, 11.1 Hz, 0.5 H, CHH β), 4.70–4.62
(m, 0.5 H, CHH α), 4.52–4.46 (m, 1 H, CHH α, CHH β), 3.92 (dd,
J = 2.3, 3.7 Hz, 0.5 H, CH-1 α), 3.50 (s, 1 H, CH-1 β, CH-7 β),
3.43 (dd, J = 1.5, 3.8 Hz, 0.5 H, CH-7 α), 3.21–3.01 (m, 1 H, CH-5
α, CH-5 β) ppm. 13C NMR (100 MHz, CDCl3): δ = 166.28, 165.69,
165.58 (C=O of α- and β-isomers), 133.59, 133.50, 133.44, 133.25,
α-Isomer 5: [α]2D0 = +85 (c = 0.2, MeOH). 1H NMR (600 MHz,
133.20, 133.16, 130.00, 129.86, 129.77, 129.68, 129.12, 129.08, MeOD): δ = 4.09 (dd, J = 8.5, 2.5 Hz, 1 H, CH-2), 3.85 (ddd, J =
129.01, 128.68, 128.58, 128.47, 128.40, 128.28 (CH and Cq arom. 3.6, 1.8, 1.8 Hz, 1 H, CH-4), 3.78 (dd, J = 11.0, 6.6 Hz, 1 H, CHH),
of α- and β-isomers), 72.13, 70.85, 70.30, 69.87, 68.71, 66.25 (CH- 3.74 (dd, J = 11.0, 8.0 Hz, 1 H, CHH), 3.41 (dd, J = 8.6, 1.9 Hz,
2, CH-3 and CH-4 of α- and β-isomers), 61.92, 61.79 (CH2 of α-
and-β isomers), 54.17, 53.98, 53.42, 51.73 (CH-1 and CH-7 of α-
and β-isomers), 38.59, 37.75 (CH-5 of α- and β-isomers) ppm. LC/
MS analysis: Rt 9.7 min (linear gradient 10Ǟ90% B in 15 min),
m/z 593.1 [M + H]+, 1206.9 [2M + Na]+. HRMS: calcd. for
[C35H29O9]+ 593.18061; found 593.18073. HRMS: calcd. for
[C35H28O9Na]+ 615.16255; found 615.16184.
1 H, CH-3), 3.33 (dd, J = 4.0, 2.5 Hz, 1 H, CH-1), 3.10 (dd, J =
4.0, 1.7 Hz, 1 H, CH-7), 2.04 (ddd, J = 8.0, 6.6, 3.5 Hz, 1 H, CH-
5) ppm. 13C NMR (150 MHz, MeOD): δ = 73.76 (CH-3), 72.77
(CH-4), 70.79 (CH-2), 62.28 (CH2), 58.10 (CH-1), 55.46 (CH-7),
44.34 (CH-5) ppm.
β-Isomer 11: [α]2D0 = +67 (c = 0.2, MeOH). 1H NMR (600 MHz,
MeOD): δ = 3.93 (d, J = 8.6 Hz, 1 H, CH-2), 3.88–3.82 (m, 3 H,
CH-4, CH2), 3.33–3.31 (m, 2 H, CH-3, CH-7), 3.17 (d, J = 3.7 Hz,
1 H, CH-1), 2.18 (tdd, J = 7.3, 4.0, 1.7 Hz, 1 H, CH-5) ppm. 13C
NMR (150 MHz, MeOD): δ = 76.72 (CH-3), 70.76 (CH-4), 69.96
Mixture of α- and β-Epoxides 25: A solution of tetrabenzoylated
epoxides 24 (α- and β-epoxides in 1:1 ratio, 0.30 mmol, 180 mg) in
MeOH was treated with sodium methoxide (0.12 mmol, 21 μL
5.6 m in MeOH, 0.4 equiv.) at room temperature for 1.5 h. After (CH-2), 61.87 (CH2), 57.76 (CH-1), 55.36 (CH-7), 42.82 (CH-
neutralization with Amberlite-H+ IR-200 the reaction mixture was
filtered and concentrated in vacuo. Purification by column
chromatography (10 % MeOH in CH2Cl2 Ǟ 15 % MeOH in
CH2Cl2) yielded the fully deprotected product 25 (α- and β-epox-
5) ppm.
Azido-cyclohexene 29: A solution of cyclohexene 18 (0.76 mmol,
0.26 g) in CH2Cl2 under argon was cooled to 0 °C, after which
pTsCl (0.84 mmol, 0.16 g, 1.1 equiv.) and Et3N (1.4 mmol,
0.19 mL, 1.8 equiv.) were added. The reaction mixture was stirred
overnight at room temperature, after which TLC analysis indicated
that the reaction was not complete. Therefore, another 0.5 equiv.
of pTsCl (75 mg) and 1.8 equiv. of Et3N (0.19 mL) were added after
24 h and again after 48 h. The mixture was stirred for a total of
4 d, after which aqueous HCl (1 m) was added and the aqueous
layer was extracted with Et2O (3ϫ). The combined organic layers
were dried with MgSO4, filtered, and concentrated in vacuo. The
crude tosylated product was then redissolved in DMF under argon,
and NaN3 (7.6 mmol, 0.50 g, 10 equiv.) was added. The reaction
mixture was heated to 60 °C, stirred overnight, and concentrated
in vacuo. The residue was then redissolved in EtOAc and washed
with aqueous HCl (1 m, 1ϫ), saturated aqueous NaHCO3 (1ϫ),
1
ides in 1.5:1 ratio) (40 mg, 0.23 mmol, 76%). H NMR (400 MHz,
CDCl3): δ = 4.07 (dd, J = 2.4, 8.4 Hz, 0.6 H, CH-2 α), 3.91 (d, J
= 8.4 Hz, 0.4 H, CH-2 β), 3.87–3.82 (m, 3 H, CH-4 α, CH-4 β,
CH2 α, CH2 β), 3.38 (dd, J = 1.6, 8.4 Hz, 0.6 H, CH-3 α), 3.32–
3.29 (m, 1.4 H, CH-1 α, CH-7 β, CH-3 β), 3.15 (d, J = 4.0 Hz, 0.4
H, CH-1 β), 3.08 (dd, J = 1.6, 4.0 Hz, 0.6 H, CH-7 α), 2.18–2.16
(m, 0.4 H, CH-5 β), 2.05–2.00 (m, 0.6 H, CH-5 α) ppm.
Separation of α- and β-Epoxides by Acetylation/Deacetylation (5,
11): An α/β mixture of deprotected epoxides 25 (0.26 mmol, 45 mg)
was coevaporated with toluene before being dissolved in pyridine
under argon. After addition of acetic anhydride (10 mmol, 0.9 mL,
38 equiv.) the mixture was stirred at room temperature for 4 h
and was then concentrated in vacuo and coevaporated with
toluene (3ϫ). After purification by column chromatography and brine (1ϫ), dried with MgSO4, filtered, and concentrated in
(CH2Cl2 Ǟ0.6% MeOH in CH2Cl2 in steps of 0.1%) three different
products were isolated: the tetraacetylated form of the α-isomer 26
(0.10 mmol, 35 mg, 38%), a small amount of tetraacetylated β-iso-
mer 28 (4.4 μmol, 1.5 mg, 2%), and triacetylated β-isomer 27
(53 μmol, 16 mg, 20%). 1H NMR (400 MHz, CDCl3) 26: δ = 5.52–
vacuo. Purification by column chromatography (pentaneǞ12%
EtOAc in pentane in steps of 2%) gave the dibenzylated azide 29
(0.17 g, 0.48 mmol, 63% over two steps). [α]2D0 = +118 (c = 0.1,
CHCl3). 1H NMR (400 MHz, CDCl3): δ = 7.46–7.31 (m, 10 H, CH
arom.), 5.86 (dt, J = 10.2, 2.6 Hz, 1 H, CH=CH), 5.48 (dd, J =
5.41 (m, 2 H, CH-2, CH-4), 5.00 (dd, J = 9.6, 1.8 Hz, 1 H, CH-3), 9.6, 1.6 Hz, 1 H, CH=CH), 4.81–4.73 (m, 4 H, 2ϫCH2 Bn), 4.40–
8
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α- and β-galacto-Configured Cyclophellitols
4.27 (m, 2 H, CH-2, CH-4), 3.70 (dd, J = 7.8, 2.2 Hz, 1 H, CH-3), 514.16066. HRMS: calcd. for [C28H23O7N3Na]+ 536.14282; found
3.60 (dd, J = 11.9, 9.3 Hz, 1 H, CHH-N3), 3.40 (dd, J = 11.9, 536.14242.
6.5 Hz, 1 H, CHH-N3), 2.56 (dt, J = 6.3, 3.1 Hz, 1 H, CH-5), 2.50–
α-Epoxide 6 and β-Epoxide 12: Epoxide 31 (0.19 mmol, 96 mg) was
dissolved in MeOH (2.5 mL) and deprotected with NaOMe
(38 μmol, 6.8 μL 5.6 m in MeOH, 20 mol-%) over 1.5 h at room
2.47 (br. s, 1 H, OH) ppm. 13C NMR (100 MHz, CDCl3): δ =
138.51, 138.03 (Cq arom.), 128.56, 128.44, 127.96, 127.92, 127.86,
127.70 (CH arom., CH=CH), 125.50 (CH=CH), 82.12 (CH-3),
temperature. The reaction mixture was then quenched by addition
76.80 (CH-2), 72.42 (CH2 Bn), 72.26 (CH2 Bn), 67.58 (CH-4), 51.77
of Amberlite-H+ IR-200, filtered, and concentrated in vacuo. The
(CH2-N3), 40.63 (CH-5) ppm. HRMS: calcd. for [C21H24O3N3]+
product was purified by multiple rounds of column chromatog-
raphy (CH2Cl2 Ǟ3% MeOH in CH2Cl2 in steps of 0.5%) to sepa-
rate the α-epoxide 6 (16 mg, 80 μmol, 42%) and β-epoxide 12
366.18122; found 366.18143. HRMS: calcd. for [C21H23O3N3Na]+
388.16316; found 388.16322.
(3.0 mg, 15 μmol, 8%) (additional yield of mixed α/β product
7.0 mg, 35 μmol, 18%).
Azido-cyclohexene 30:
A solution of dibenzylated azide 29
(0.47 mmol, 0.17 g) in CH2Cl2 was cooled to –78 °C under argon
before addition of BCl3 (4.8 mmol, 4.8 mL 1 m in CH2Cl2,
10 equiv.). The reaction mixture was stirred at –78 °C for 4 h,
quenched with MeOH, concentrated in vacuo, and coevaporated
with toluene (3ϫ). The deprotected product was then dissolved in
pyridine, BzCl (4.7 mmol, 0.55 mL, 10 equiv.) was added, and the
mixture was stirred overnight at room temperature. After quench-
ing with aqueous saturated NaHCO3, the mixture was extracted
with CH2Cl2 (3ϫ), and the combined organic layers were dried
with MgSO4, filtered, and concentrated in vacuo. The product was
purified by column chromatography (pentaneǞ7.5% EtOAc in
pentane) to yield the tribenzoylated product 30 (0.20 g, 0.40 mmol,
85% over two steps). [α]2D0 = +265 (c = 0.2, CHCl3). 1H NMR
(400 MHz, CDCl3): δ = 8.19–7.86 (m, 6 H, CH arom.), 7.66–7.28
(m, 9 H, CH arom.), 6.21–6.12 (m, 1 H, CH-2), 6.12–6.07 (m, 1 H,
CH-4), 6.03 (dt, J = 10.3, 2.7 Hz, 1 H, CH=CH), 5.89–5.82 (m, 1
H, CH=CH), 5.71 (dd, J = 8.6, 2.3 Hz, 1 H, CH-3), 3.52 (dd, J =
12.1, 7.7 Hz, 1 H, CHH), 3.42 (dd, J = 12.1, 7.8 Hz, 1 H, CHH),
3.10–3.08 (m, 1 H, CH-5) ppm. 13C NMR (100 MHz, CDCl3): δ =
166.11, 165.84, 165.51 (3ϫC=O), 133.75, 133.55, 133.28, 133.21,
130.17, 129.78, 129.76, 129.72, 129.46, 129.20, 129.17, 128.66,
128.46, 128.40, 128.31 (CH and Cq arom.), 127.48, 126.55
(2ϫCH=CH), 73.14 (CH-3), 70.49 (CH-2), 69.85 (CH-4), 51.46
(CH2), 39.87 (CH-5) ppm. HRMS: calcd. for [C28H23O6N3Na]+
520.14791; found 520.14724.
α-Epoxide 6: [α]2D0 = +81 (c = 0.3, MeOH). 1H NMR (600 MHz,
MeOD): δ = 4.07 (dd, J = 8.6, 2.5 Hz, 1 H, CH-2), 3.78 (dd, J =
3.4, 1.8 Hz, 1 H, CH-4), 3.59 (dd, J = 12.2, 8.0 Hz, 1 H, CHH),
3.48 (dd, J = 12.3, 8.3 Hz, 1 H, CHH), 3.39 (dd, J = 8.5, 1.9 Hz,
1 H, CH-3), 3.32 (dd, J = 3.9, 2.4 Hz, 1 H, CH-1), 2.97 (dd, J =
3.9, 1.8 Hz, 1 H, CH-7), 2.09 (td, J = 8.2, 3.5 Hz, 1 H, CH-5) ppm.
13C NMR (150 MHz, MeOD): δ = 73.56 (CH-3), 72.15 (CH-4),
70.53 (CH-2), 58.08 (CH-1), 55.29 (CH-7), 51.73 (CH2), 41.83 (CH-
5) ppm.
1
β-Epoxide 12: [α]2D0 = +64 (c = 0.1, MeOH). H NMR (600 MHz,
MeOD): δ = 3.92 (dd, J = 8.7, 0.7 Hz, 1 H, CH-2), 3.82 (ddd, J =
4.1, 2.5, 1.3 Hz, 1 H, CH-4), 3.70 (dd, J = 12.1, 8.2 Hz, 1 H, CHH),
3.63 (dd, J = 12.1, 7.2 Hz, 1 H, CHH), 3.33 (dd, J = 8.6, 2.6 Hz,
1 H, CH-3), 3.26–3.22 (m, 1 H, CH-7), 3.17 (d, J = 3.7 Hz, 1 H,
CH-1), 2.28 (dddd, J = 8.2, 7.2, 4.1, 1.8 Hz, 1 H, CH-5) ppm. 13C
NMR (150 MHz, MeOD): δ = 76.39 (CH-3), 70.36 (CH-4), 69.50
(CH-2), 58.03 (CH-1), 55.21 (CH-7), 51.58 (CH2), 40.43 (CH-
5) ppm.
Bodipy-α-epoxide 7: Azido-epoxide 16 (25 μmol, 5.0 mg) and Bod-
ipy-alkyne 32[19] (25 μmol, 12 mg, 1.0 equiv.) were dissolved in
DMF (0.5 mL) under argon. After addition of copper(II)-
sulfate pentahydrate (5 μmol, 5 μL 1 m in H2O, 0.2 equiv.) and so-
dium ascorbate (10 μmol, 10 μL 1 m in H2O, 0.4 equiv.) the reaction
mixture was stirred at room temperature overnight. The mixture
was then concentrated in vacuo and purified by column chromatog-
raphy (CH2Cl2 Ǟ3% MeOH in CH2Cl2) to yield Bodipy-epoxide
7 as a purple solid (15 mg, 22 μmol, 88%). 1H NMR (400 MHz,
MeOD): δ = 7.89–7.82 (m, 4 H, CH arom.), 7.78 (s, 1 H, CH tri-
azole), 7.43 (d, J = 4.4 Hz, 2 H, 2ϫCH pyrrole), 7.01–6.94 (m, 4
H, CH arom.), 6.70 (d, J = 4.3 Hz, 2 H, 2ϫCH pyrrole), 4.60–
4.53 (m, 2 H, CH2-6), 4.13 (dd, J = 8.5, 2.4 Hz, 1 H, CH-2), 3.85
(s, 6 H, 2ϫOCH3), 3.66–3.61 (m, 1 H, CH-4), 3.42 (dd, J = 8.5,
1.8 Hz, 1 H, CH-3), 3.37–3.34 (m, 1 H, CH-1), 3.07–3.02 (m, 2 H,
CH2), 2.97 (dd, J = 3.9, 1.8 Hz, 1 H, CH-7), 2.79–2.75 (m, 2 H,
CH2), 2.55 (td, J = 8.2, 3.5 Hz, 1 H, CH-5), 1.88–1.83 (m, 4 H,
2ϫCH2) ppm. 13C NMR (100 MHz, MeOD): δ = 160.77 (Cq
arom.), 157.39 (Cq arom.), 145.31 (Cq arom.), 136.08 (Cq arom.),
130.71 (CH arom.), 127.03 (CH pyrrole), 125.10 (Cq arom.), 123.38
(CH triazole), 119.66 (CH pyrrole), 113.22 (CH arom.), 72.03 (CH-
3), 70.53 (CH-4), 69.06 (CH-2), 56.67 (CH-1), 54.41 (2ϫOCH3),
53.30 (CH-7), 49.13 (CH2-6), 41.27 (CH-5), 32.86, 29.58, 29.00,
24.48 (4ϫCH2) ppm. LC/MS analysis: Rt 10.4 min (linear gradient
10Ǟ50% B in 15 min), m/z 666.3 [M – F]+, 685.9 [M + H]+, 1371.0
[2M + H]+. HRMS: calcd. for [C36H39O6N5BF2]+ 686.29560; found
686.29599. HRMS: calcd. for [C36H38O6N5BF2Na]+ 708.27754;
found 708.27719.
Azido-epoxide 31: Tribenzoylated azide 30 (0.40 mmol, 0.20 g) was
dissolved in CH2Cl2 under argon, and 3-chloroperoxybenzoic acid
(1.2 mmol, 0.27 g, 3.0 equiv.) was added. The reaction mixture was
stirred at room temperature for a total of 4 d, concentrated in
vacuo, and purified by column chromatography (pentaneǞ10%
EtOAc in pentane), giving the azido-epoxide 31 as a mixture of
α- and β-isomers (ratio 4:1, 0.11 g, 0.21 mmol, 53%). 1H NMR
(400 MHz, CDCl3): δ = 8.12–7.81 (m, 6 H, 6ϫCH arom. α, 6ϫCH
arom. β), 7.68–7.25 (m, 9 H, 9ϫCH arom. α, 9ϫCH arom. β),
6.04 (dd, J = 9.3, 2.3 Hz, 0.8 H, CH-2 α), 5.93–5.86 (m, 1 H, CH-
4 α, CH-4 β), 5.75 (d, J = 9.4 Hz, 0.2 H, CH-2 β), 5.60 (dd, J =
9.3, 1.9 Hz, 0.8 H, CH-3 α), 5.53 (dd, J = 9.3, 2.6 Hz, 0.2 H, CH-
3 β), 3.88 (dd, J = 3.9, 2.3 Hz, 0.8 H, CH-1 α), 3.68–3.42 (m, 2.4
H, CH2 α, CH2 β, CH-1 β, CH-7 β), 3.35 (dd, J = 3.9, 1.7 Hz, 0.8
H, CH-7 α), 2.80–2.74 (m, 1 H, CH-5 α, CH-5 β) ppm. 13C NMR
(100 MHz, CDCl3): δ = 166.25, 165.68, 165.65, 165.54, 165.47,
165.26 (C=O of α- and β-isomers), 134.59, 133.66, 133.62, 133.53,
133.48, 133.36, 133.30, 130.88, 129.80, 129.79, 129.76, 129.73,
129.71, 129.64, 129.01, 128.96, 128.92, 128.89, 128.83, 128.67,
128.64, 128.58, 128.56, 128.49, 128.43, 128.33, 128.32 (CH and Cq
arom. of α- and β-isomers), 72.17, 70.85 (CH-3 of α- and β-iso-
mers), 70.32, 70.18 (CH-2 α, CH-4 α), 68.47 (CH-2 β), 66.77 (CH-
4 β), 54.44 (CH-1 or CH-7 β), 54.14 (CH-1 α), 53.67 (CH-7 α),
52.04 (CH-1 or CH-7 β), 49.77, 49.75 (CH2 of α- and β-isomers), Biotin-α-epoxide 8: Azido-epoxide 6 (25 μmol, 5.0 mg) and biotin-
39.01 (CH-5 α), 38.27 (CH-5 β) ppm. LC/MS analysis: Rt 9.3 min
(linear gradient 10Ǟ90% B in 15 min), m/z 514.1 [M + H]+, 1026.7
[2M + H]+. HRMS: calcd. for [C28H24O7N3]+ 514.16088; found
alkyne 33[20] (25 μmol, 7.0 mg, 1.0 equiv.) were dissolved in DMF
(0.5 mL) under argon. After addition of copper(II)sulfate pentahy-
drate (5 μmol, 5 μL 1 m in H2O, 0.2 equiv.) and sodium ascorbate
Eur. J. Org. Chem. 0000, 0–0
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Date: 12-08-14 18:46:12
Pages: 14
H. S. Overkleeft et al.
FULL PAPER
(10 μmol, 10 μL 1 m in H2O, 0.4 equiv.) the reaction mixture was
stirred at room temperature overnight. The mixture was then con-
centrated in vacuo and purified by column chromatography
(CH2Cl2 Ǟ30% MeOH in CH2Cl2) to yield biotin-epoxide 8 as a
white solid (8.0 mg, 17 μmol, 68%). [α]2D0 = +48 (c = 0.1, MeOH).
1H NMR (400 MHz, MeOD): δ = 7.97 (s, 1 H, CH triazole), 4.65–
4.5 Hz, 1 H, CH-NH), 3.98 (d, J = 8.5 Hz, 1 H, CH-2), 3.63 (ddd,
J = 4.1, 2.7, 1.2 Hz, 1 H, CH-4), 3.38–3.36 (m, 1 H, CH-3), 3.25–
3.22 (m, 1 H, CH-S), 3.21 (d, J = 3.8 Hz, 1 H, CH-1), 3.14–3.08
(m, 1 H, CH-7), 2.96 (dd, J = 12.7, 5.0 Hz, 1 H, CHH-S), 2.78
(ddt, J = 7.7, 5.4, 2.7 Hz, 1 H, CH-5), 2.73 (d, J = 12.8 Hz, 1 H,
CHH-S), 2.27 (t, J = 7.3 Hz, 2 H, CH2), 1.81–1.55 (m, 4 H,
4.60 (m, 2 H, CH2-6), 4.52 (ddd, J = 8.0, 5.0, 1.0 Hz, 1 H, CH- 2ϫCH2), 1.50–1.38 (m, 2 H, CH2) ppm. 13C NMR (100 MHz,
NH), 4.47 (s, 2 H, CH2-NH), 4.32 (dd, J = 7.9, 4.5 Hz, 1 H, CH-
NH), 4.14 (dd, J = 8.6, 2.4 Hz, 1 H, CH-2), 3.66 (dt, J = 3.7,
1.8 Hz, 1 H, CH-4), 3.43 (dd, J = 8.6, 1.8 Hz, 1 H, CH-3), 3.37–
MeOD): δ = 174.62 (C=O), 144.82 (C=O), 123.89 (CH triazole),
74.73 (CH-3), 68.46 (CH-4), 67.88 (CH-2), 61.95, 60.23 (2ϫCH-
NH), 56.79 (CH-1), 55.59 (CH-S), 53.25 (CH-7), 48.86 (CH2-6),
3.36 (m, 1 H, CH-1), 3.22 (ddd, J = 8.9, 5.9, 4.4 Hz, 1 H, CH-S), 39.87 (CH-5), 39.65 (CH2-S), 35.14, 34.22, 28.29, 28.04, 25.30
3.02 (dd, J = 4.0, 1.8 Hz, 1 H, CH-7), 2.96 (dd, J = 12.8, 5.0 Hz, (5ϫCH2) ppm. LC/MS analysis: Rt 3.7 min (linear gradient
1 H, CHH-S), 2.74 (d, J = 12.7 Hz, 1 H, CHH-S), 2.58 (td, J =
8.1, 3.5 Hz, 1 H, CH-5), 2.27 (t, J = 7.3 Hz, 2 H, CH2), 1.78–
1.57 (m, 4 H, 2ϫCH2), 1.48–1.42 (m, 2 H, CH2) ppm. 13C NMR
(100 MHz, MeOD): δ = 174.63 (C=O), 145.05 (C=O), 123.62 (CH
triazole), 72.00 (CH-3), 70.51 (CH-4), 69.03 (CH-2), 61.95, 60.23
(2ϫCH-NH), 56.70 (CH-1), 55.59 (CH-S), 53.32 (CH-7), 49.23
(CH2-6), 41.34 (CH-5), 39.66 (CH2-S), 35.14, 34.20, 28.28, 28.05,
25.29 (5ϫCH2) ppm. LC/MS analysis: Rt 3.6 min (linear gradient
10Ǟ50% B in 15 min), m/z 483.3 [M + H]+, 965.1 [2M + H]+,
1446.9 [3M + H]+. HRMS: calcd. for [C20H31O6N6S]+ 483.20203;
found 483.20176. HRMS: calcd. for [C20H30O6N6SNa]+ 505.18397;
found 505.18347.
10Ǟ50% B in 15 min), m/z 483.3 [M + H]+, 965.0 [2M + H]+,
1447.1 [3M + H]+. HRMS: calcd. for [C20H31O6N6S]+ 483.20203;
found 483.20179. HRMS: calcd. for [C20H30O6N6SNa]+ 505.18397;
found 505.18357.
Aziridine 34: Cyclohexene 18 (0.50 mmol, 0.17 g) was coevaporated
with toluene before being dissolved in CH2Cl2 (10 mL) at 0 °C un-
der argon. Next, CCl3CCN (0.50 mmol, 51 μL, 1.0 equiv.) and
DBU (25 μmol, 2.0 μL, 0.05 equiv.) were added and the reaction
mixture was stirred at 0 °C for 1.5 h. The mixture was removed
from the ice bath, after which H2O (1.5 mL), NaHCO3 (5.0 mmol,
0.40 g, 10 equiv.), and I2 (1.5 mmol, 0.38 g, 3.0 equiv.) were added.
After stirring at room temperature overnight, the reaction mixture
Bodipy-β-epoxide 13: Azido-epoxide 12 (25 μmol, 5.0 mg) and Bod- was quenched with aqueous Na2S2O3 (10%), and the aqueous layer
ipy-alkyne 32[19] (25 μmol, 12 mg, 1.0 equiv.) were dissolved in
DMF (0.5 mL) under argon. After addition of copper(II)sulfate
pentahydrate (5 μmol, 5 μL 1 m in H2O, 0.2 equiv.) and sodium as-
corbate (10 μmol, 10 μL 1 m in H2O, 0.4 equiv.) the reaction mix-
ture was stirred at room temperature overnight. The mixture was
then concentrated in vacuo and purified by column chromatog-
raphy (CH2Cl2 Ǟ3% MeOH in CH2Cl2) to yield Bodipy-epoxide
was extracted with EtOAc (2ϫ). The combined organics were dried
with MgSO4, filtered, and concentrated in vacuo. The residue was
redissolved in MeOH and cooled to 0 °C, and concentrated HCl
(6.3 mmol, 0.6 mL, 13 equiv.) was added, after which the reaction
mixture was stirred at room temperature for 3.5 h. Next, the mix-
ture was concentrated in vacuo, the residue was redissolved in diox-
ane, and concentrated HCl (16 mmol, 1.5 mL, 32 equiv.) was again
added. The mixture was stirred at 60 °C for 1 h, concentrated in
vacuo, and coevaporated with toluene (3ϫ). The residue was redis-
solved in MeOH, NaHCO3 (10 mmol, 0.8 g, 20 equiv.) was added,
and the reaction mixture was stirred at room temperature for 4 d.
1
13 as a purple solid (15 mg, 22 μmol, 90%). H NMR (400 MHz,
MeOD): δ = 1.91–7.82 (m, 4 H, CH arom.), 7.77 (s, 1 H, CH tri-
azole), 7.44 (d, J = 4.4 Hz, 2 H, 2ϫCH pyrrole), 7.03–6.93 (m, 4
H, CH arom.), 6.70 (d, J = 4.3 Hz, 2 H, 2ϫCH pyrrole), 4.70 (d,
J = 7.8 Hz, 2 H, CH2-6), 3.97 (d, J = 8.5 Hz, 1 H, CH-2), 3.86 (s, After addition of H2O, the aqueous layer was extracted with
6 H, 2ϫOCH3), 3.66–3.58 (m, 1 H, CH-4), 3.42–3.40 (m, 1 H, CH- EtOAc, and the organic layer was dried with MgSO4, filtered, and
3), 3.18 (d, J = 3.7 Hz, 1 H, CH-1), 3.11–2.97 (m, 3 H, CH-7,
CH2), 2.86–2.76 (m, 2 H, CH2), 2.73 (ddt, J = 7.6, 4.3, 2.2 Hz, 1
concentrated in vacuo. The crude product was purified by column
chromatography (CH2Cl2 Ǟ6% MeOH in CH2Cl2) to afford di-
H, CH-5), 1.91–1.82 (m, 4 H, 2ϫCH2) ppm. 13C NMR (100 MHz, benzylated β-aziridine 34 (92 mg, 0.26 mmol, 52%). 1H NMR
MeOD): δ = 160.78 (Cq arom.), 157.38 (Cq arom.), 144.42 (Cq (400 MHz, CDCl3): δ = 7.46–7.22 (m, 10 H, CH arom.), 4.83–4.61
arom.), 136.09 (Cq arom.), 130.79 (CH arom.), 127.03 (CH pyr-
role), 125.11 (Cq arom.), 123.06 (CH triazole), 119.63 (CH pyrrole),
(m, 4 H, 2ϫCH2 Bn), 4.13–4.04 (m, 2 H, CH-2, CH-4), 3.99 (dd,
J = 10.9, 7.5 Hz, 1 H, CHH-6), 3.90 (dd, J = 10.9, 6.2 Hz, 1 H,
113.22 (CH arom.), 74.73 (CH-3), 68.51 (CH-4), 67.89 (CH-2), CHH-6), 3.39 (dd, J = 7.9, 2.3 Hz, 1 H, CH-3), 2.50–2.32 (m, 2 H,
56.77 (CH-1), 54.41 (2ϫOCH3), 53.27 (CH-7), 48.74 (CH2-6), CH-1, CH-7), 2.06–1.96 (m,
1
H, CH-5) ppm. 13C NMR
39.84 (CH-5), 32.86, 29.59, 29.02, 24.46 (4ϫCH2) ppm. LC/MS
analysis: Rt 10.5 min (linear gradient 10Ǟ50% B in 15 min), m/z
666.3 [M – F]+, 686.0 [M + H]+, 1371.0 [2M + H]+. HRMS: calcd.
for [C36H39O6N5BF2]+ 686.29560; found 686.29615. HRMS: calcd.
for [C36H38O6N5BF2Na]+ 708.27754; found 708.27725.
(100 MHz, CDCl3): δ = 138.31, 138.17 (Cq arom.), 128.62, 128.58,
128.54, 128.49, 128.46, 128.43, 128.35, 128.29, 128.17, 128.15,
128.02, 127.96, 127.93, 127.90, 127.87, 127.85, 127.80, 127.78,
127.63 (CH arom.), 83.29 (CH-3), 78.57 (CH-2), 73.10, 71.69
(2ϫCH2 Bn), 67.42 (CH-4), 62.40 (CH2-6), 39.47 (CH-5), 32.63,
32.53 (CH-1, CH-7) ppm. LC/MS analysis: Rt 5.4 min (linear gra-
dient 10Ǟ90% B in 15 min), m/z 356.1 [M + H]+, 711.1 [2M +
H]+.
Biotin-β-epoxide 14: Azido-epoxide 12 (25 μmol, 5.0 mg) and bio-
tin-alkyne 33[20] (25 μmol, 7.0 mg, 1.0 equiv.) were dissolved in
DMF (0.5 mL) under argon. After addition of copper(II)sulfate
pentahydrate (5 μmol, 5 μL 1 m in H2O, 0.2 equiv.) and sodium as-
corbate (10 μmol, 10 μL 1 m in H2O, 0.4 equiv.) the reaction mix-
Benzyl-cyclohexene 35: A solution of cyclohexene 18 (3.5 mmol,
1.2 g) in DMF under argon was cooled to 0 °C, after which tetra-
ture was stirred at room temperature overnight. The mixture was butylammonium iodide (35 μmol, 13 mg, 1 mol-%), benzyl bromide
then concentrated in vacuo and purified by column chromatog-
raphy (CH2Cl2 Ǟ30% MeOH in CH2Cl2) to yield biotin-epoxide
14 as a white solid (8.0 mg, 17 μmol, 68%). [α]2D0 = +36 (c = 0.1,
MeOH). H NMR (400 MHz, MeOD): δ = 7.93 (s, 1 H, CH tri-
azole), 4.75 (d, J = 7.7 Hz, 2 H, CH2-6), 4.52 (ddd, J = 8.0, 5.0,
(8.4 mmol, 1.0 mL, 2.4 equiv.), and sodium hydride [8.4 mmol,
0.33 g (60% in mineral oil), 2.4 equiv.] were added. The reaction
mixture was stirred overnight at room temperature and sub-
sequently quenched with MeOH. Next, H2O was added and the
resulting mixture was extracted with EtOAc (3ϫ). The combined
1
0.9 Hz, 1 H, CH-NH), 4.46 (s, 2 H, CH2-NH), 4.32 (dd, J = 7.9, organic layers were washed with H2O (1ϫ) and brine (1ϫ), dried
10
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Eur. J. Org. Chem. 0000, 0–0

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α- and β-galacto-Configured Cyclophellitols
with MgSO4, filtered, and concentrated in vacuo, yielding a mix-
ture of products containing three or four benzyl groups. These were
separated by column chromatography (pentaneǞ5% EtOAc in
pentaneǞ20% EtOAc in pentane), after which the tribenzylated
products were again subjected to the benzylation reaction as before.
After workup and column chromatography purification (pent-
aneǞ5% EtOAc in pentane) the fully benzylated product 35 was
obtained (total yield 1.5 g, 3.0 mmol, 85%). [α]2D0 = +105 (c = 0.1,
CHCl3). 1H NMR (400 MHz, CDCl3): δ = 7.39–7.23 (m, 20 H, CH
arom.), 5.80–5.76 (m, 1 H, CH=CH), 5.48 (d, J = 10.4 Hz, 1 H,
CH=CH), 4.93 (d, J = 11.6 Hz, 1 H, CHH Bn), 4.78–4.68 (m, 4
H, 2ϫCH2 Bn), 4.64 (d, J = 11.6 Hz, 1 H, CHH Bn), 4.50–4.43
(m, 3 H, CH-2, CH2 Bn), 4.20 (dd, J = 1.6, 1.6 Hz, 1 H, CH-4),
3.75 (dd, J = 1.6, 8.0 Hz, 1 H, CH-3), 3.62–3.46 (m, 2 H, CH2-6),
2.72–2.68 (m, 1 H, CH-5) ppm. 13C NMR (100 MHz, CDCl3): δ =
139.03, 138.77, 138.70, 138.15 (Cq arom.), 128.40, 128.32, 128.30,
128.17, 127.87, 127.81, 127.79, 127.67, 127.49, 127.47, 127.44,
127.38, 126.66 (CH arom., 2ϫCH=CH), 83.20 (CH-3), 77.66 (CH-
2), 75.12 (CH-4), 74.03, 73.28, 72.37, 72.16 (4ϫCH2 Bn), 70.17
(CH2-6), 41.92 (CH-5) ppm. LC/MS analysis: Rt 10.8 min (linear
gradient 10Ǟ90% B in 15 min), m/z 521.1 [M + H]+, 543.3 [M +
Na]+. HRMS: calcd. for [C35H37O4]+ 521.26864; found 521.26864.
HRMS: calcd. for [C35H36O4Na]+ 543.25058; found 543.24988.
(CH-7), 40.19 (CH-5) ppm. LC/MS analysis: Rt 10.3 min (linear
gradient 10Ǟ90% B in 15 min), m/z 537.1 [M + H]+, 559.3 [M +
Na]+. HRMS: calcd. for [C35H37O5]+ 537.26355; found 537.26391.
HRMS: calcd. for [C35H36O5Na]+ 559.24550; found 559.24491.
trans-Azido Alcohols 37: A solution of β-epoxide 36 (0.48 mmol,
0.26 g) in MeCN was put under argon, sodium azide (24 mmol,
1.6 g, 50 equiv.) and lithium perchlorate (4.8 mmol, 0.51 g,
10 equiv.) were added, and the mixture was heated to 80 °C. After
stirring under reflux overnight, the reaction mixture was allowed
to cool to room temperature, quenched with H2O, and extracted
with CH2Cl2 (3ϫ). The combined organic layers were dried with
MgSO4, filtered, and concentrated in vacuo. Purification by col-
umn chromatography (pentaneǞ10% EtOAc in pentane) yielded
a mixture of the two trans-azido alcohols 37 in a ratio of 1:1.3
1
(0.20 g, 0.35 mmol, 73%). H NMR (400 MHz, CDCl3): δ = 7.39–
7.18 (m, 20 H, 20ϫCH arom. isomer 1, 20ϫCH arom. isomer 2),
5.02–4.67 (m, 5 H, 2ϫCH2 Bn isomer 1, CHH Bn isomer 1,
2ϫCH2 Bn isomer 2, CHH Bn isomer 2), 4.51–4.40 (m, 3 H, CH2
Bn isomer 1, CHH Bn isomer 1, CH2 Bn isomer 2, CHH Bn iso-
mer 2), 4.30–4.26 (m, 1.57 H, CH-2 isomer 1, CH-4 isomer 1, CH-
4 isomer 2), 4.11–4.09 (m, 0.57 H, CH-1 isomer 1), 3.91–3.87 (m,
1 H, OH isomer 1, CH-2 isomer 2), 3.79–3.77 (m, 1.57 H, CH-3
isomer 1, CH-7 isomer 1, OH isomer 2), 3.69–3.41 (m, 3.29 H,
CH2-6 isomer 1, CH2-6 isomer 2, CH-3 isomer 2, CH-1 isomer 2,
CH-7 isomer 2), 2.05–2.02 (m, 0.57 H, CH-5 isomer 1), 1.68–1.62
(m, 0.43 H, CH-5 isomer 2) ppm. 13C NMR (100 MHz, CDCl3): δ
= 138.14, 138.04, 137.99, 137.85, 137.80 (Cq arom.), 128.59, 128.45,
128.43, 128.41, 128.35, 128.23, 128.05, 127.94, 127.90, 127.81,
127.78, 127.75, 127.70, 127.66, 127.59, 127.51, 127.44, 127.42 (CH
arom.), 83.58 (CH-3 isomer 2), 81.21 (CH-2 isomer 2), 80.68 (CH-
3 isomer 1), 78.25 (CH-2 isomer 1), 76.93 (CH-1 isomer 2), 76.60
(CH-4 isomer 1), 75.86, 75.59, 75.02, 73.54 (4ϫCH2 Bn), 73.43
(CH-4 isomer 2), 73.39, 73.32, 73.28, 72.50 (4ϫCH2 Bn), 71.55
(CH-7 isomer 1), 67.71 (CH2-6 isomer 1, CH2-6 isomer 2), 64.75
(CH-1 isomer 1), 61.58 (CH-7 isomer 2), 42.31 (CH-5 isomer 2),
38.59 (CH-5 isomer 1) ppm. LC/MS analysis: Rt 10.4 min (linear
gradient 10Ǟ90% B in 15 min), m/z 579.9 [M + H]+; and Rt
10.6 min, m/z 580.0 [M + H]+. HRMS: calcd. for [C35H38N3O5]+
580.28060; found 580.28078. HRMS: calcd. for [C35H37N3O5Na]+
602.26254; found 602.26211.
β-Epoxide 36: 3-Chloroperoxybenzoic acid (1.7 mmol, 0.30 g,
2.5 equiv.) was added under argon to a solution of 35 (0.70 mmol,
0.36 g) in CH2Cl2. After stirring overnight, the reaction mixture
was quenched with saturated aqueous Na2SO3 and extracted with
CH2Cl2 (2ϫ). The combined organic layers were washed with satu-
rated aqueous NaHCO3 (1ϫ), dried with MgSO4, filtered, and
concentrated in vacuo. The crude products were purified and sepa-
rated by column chromatography (pentaneǞ10% EtOAc in pent-
ane) to afford β-epoxide 36 (0.24 g, 0.43 mmol, 63%) and its α-
epoxide isomer (37 mg, 70 μmol, 10%).
α-Epoxide: [α]2D0 = +30 (c = 0.1, CHCl3). 1H NMR (400 MHz,
CDCl3): δ = 7.41–7.22 (m, 20 H, CH arom.), 4.90–4.79 (m, 3 H,
CHH Bn, CH2 Bn), 4.70 (dd, J = 11.8, 17.8 Hz, 2 H, CH2 Bn),
4.53–4.45 (m, 3 H, CHH Bn, CH2 Bn), 4.25 (dd, J = 2.4, 8.4 Hz,
1 H, CH-2), 3.93–3.92 (m, 1 H, CH-4), 3.62 (dd, J = 8.6, 1.0 Hz,
1 H, CH-3), 3.58 (d, J = 8.0 Hz, 2 H, CH2-6), 3.37 (dd, J = 2.4,
4.0 Hz, 1 H, CH-1), 2.96 (dd, J = 1.4, 4.0 Hz, 1 H, CH-7), 2.32–
2.28 (m, 1 H, CH-5) ppm. 13C NMR (100 MHz, CDCl3) α-epoxide:
δ = 138.68, 138.60, 138.58, 137.89 (Cq arom.), 128.40, 128.31,
128.26, 128.21, 128.10, 127.97, 127.88, 127.85, 127.75, 127.73,
127.67, 127.52, 127.45 (CH arom.), 81.10 (CH-3), 76.73 (CH-2),
75.68 (CH-4), 74.23, 73.68, 73.35, 73.04 (4ϫCH2 Bn), 68.53 (CH2-
6), 55.20 (CH-1), 54.35 (CH-7), 40.89 (CH-5) ppm. LC/MS analy-
sis: Rt 10.3 min (linear gradient 10Ǟ90% B in 15 min), m/z 537.0
[M + H]+, 559.3 [M + Na]+. HRMS: calcd. for [C35H37O5]+
537.26355; found 537.26385. HRMS: calcd. for [C35H36O5Na]+
559.24550; found 559.24515.
Benzyl-aziridine 38:
A mixture of trans-azido alcohols 37
(0.50 mmol, 0.29 g) was dissolved in MeCN under argon. After ad-
dition of triphenylphosphine (0.65 mmol, 0.17 g, 1.3 equiv.), the re-
action mixture was heated to 80 °C and stirred under reflux for 2 h.
The mixture was then allowed to cool to room temperature and
concentrated in vacuo. The crude product was purified by column
chromatography (CH2Cl2 Ǟ0.8% MeOH in CH2Cl2) to give
α-aziridine 38 (pure yield 70 mg, 0.13 mmol, 26%, together with an
additional 77 mg product containing triphenylphosphine oxide).
[α]2D0 = +56 (c = 0.1, MeOH). 1H NMR (400 MHz, CDCl3): δ =
7.47–7.22 (m, 20 H, CH arom.), 4.89–4.79 (m, 3 H, CH2 Bn, CHH
Bn), 4.70 (dd, J = 11.6, 22.8 Hz, 2 H, CH2 Bn), 4.55–4.43 (m, 3 H,
CH2 Bn, CHH Bn), 4.23 (dd, J = 4.0, 8.4 Hz, 1 H, CH-2), 3.88–
3.87 (m, 1 H, CH-4), 3.61–3.55 (m, 3 H, CH-3, CH2-6), 2.55–2.52
(m, 1 H, CH-1), 2.19–2.15 (m, 1 H, CH-5), 2.02 (d, J = 6.0 Hz, 1
H, CH-7) ppm. 13C NMR (100 MHz, CDCl3): δ = 139.09, 138.92,
1
β-Epoxide 36: [α]2D0 = +66 (c = 0.1, CHCl3). H NMR (400 MHz,
CDCl3): δ = 7.38–7.20 (m, 20 H, CH arom.), 4.86 (d, J = 12.0 Hz,
1 H, CHH Bn), 4.81–4.66 (m, 4 H, 2ϫCH2 Bn), 4.56 (d, J =
12.0 Hz, 1 H, CHH Bn), 4.49–4.42 (m, 2 H, CH2 Bn), 4.14 (d, J =
8.8 Hz, 1 H, CH-2), 3.94 (dd, J = 4.0, 2.0 Hz, 1 H, CH-4), 3.73–
3.62 (m, 2 H, CH2-6), 3.45 (dd, J = 2.0, 8.8 Hz, 1 H, CH-3), 3.23
(d, J = 4.0 Hz, 1 H, CH-1), 3.16–3.15 (m, 1 H, CH-7), 2.32–2.30 138.84, 138.06 (Cq arom.), 132.03, 131.93, 128.47, 128.35, 128.32,
(m, 1 H, CH-5) ppm. 13C NMR (100 MHz, CDCl3): δ = 138.80,
128.25, 128.20, 128.13, 128.11, 127.85, 127.79, 127.72, 127.65,
138.44, 137.96 (Cq arom.), 128.36, 128.33, 128.25, 128.05, 127.81, 127.60, 127.36, 127.31, 127.27 (CH arom.), 81.88 (CH-3), 76.87
127.77, 127.71, 127.68, 127.63, 127.44, 127.42, 127.21 (CH arom.),
82.47 (CH-3), 76.11 (CH-2), 74.27, 73.38, 73.15 (3ϫCH2 Bn),
72.79 (CH-4), 72.62 (CH2 Bn), 68.79 (CH2-6), 54.37 (CH-1), 52.38
(CH-2), 76.25 (CH-4), 73.99, 73.10, 72.91, 72.35 (4ϫCH2 Bn),
69.81 (CH2-6), 41.89 (CH-5), 34.15 (CH-1), 32.08 (CH-7) ppm.
LC/MS analysis: Rt 7.7 min (linear gradient 10Ǟ90% B in
Eur. J. Org. Chem. 0000, 0–0
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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11

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/KAP1
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Pages: 14
H. S. Overkleeft et al.
FULL PAPER
15 min), m/z 536.1 [M + H]+, 1071.2 [2M + H]+. HRMS: calcd. H, 3ϫCH2) ppm. 13C NMR (150 MHz, MeOD): δ = 188.72
for [C35H38NO4]+ 536.27954; found 536.27943.
(C=O), 74.36 (CH-3), 73.13 (CH-4), 69.15 (CH-2), 62.84 (CH2-6),
52.44 (CH2-N3), 44.54 (CH-5), 43.27 (CH-1), 39.44 (CH-7), 37.12
(CH2-C=O), 30.17, 29.94, 27.68, 27.66, 25.90 (5ϫCH2) ppm. LC/
MS analysis (ammonium acetate): Rt 7.5 min (linear gradient
0Ǟ50% B in 15 min), m/z 343.4 [M + H]+, 360.5 [M + NH4]+.
HRMS: calcd. for [C15H27N4O5]+ 343.19760; found 343.19757.
HRMS: calcd. for [C15H27N4O5Na]+ 365.17954; found 365.17939.
8-(Bodipy-triazole)-octanoic Acid 40: 8-Azidooctanoic acid 39[23]
(0.50 mmol, 93 mg) and Bodipy-alkyne 32[19] (0.50 mmol, 0.24 g,
1.0 equiv.) were dissolved in DMF under argon. After addition of
copper(II)sulfate pentahydrate (100 μmol, 100 μL 1 m in H2O,
0.2 equiv.) and sodium ascorbate (200 μmol, 200 μL 1 m in H2O,
0.4 equiv.) the reaction mixture was heated to 80 °C and stirred for
4.5 h. The mixture was then concentrated in vacuo and purified by
column chromatography (CH2Cl2 Ǟ2% MeOH in CH2Cl2) to yield
Bodipy-acid 40 as a purple solid (0.29 g, 0.43 mmol, 87%). 1H
NMR (400 MHz, CDCl3): δ = 7.83 (d, J = 8.8 Hz, 4 H, CH arom.),
7.27–7.26 (m, 2 H, 2ϫCH pyrrole), 7.18 (s, 1 H, CH triazole), 6.92
(d, J = 8.8 Hz, 4 H, CH arom.), 6.58 (d, J = 3.2 Hz, 2 H, 2ϫCH
pyrrole), 4.30–4.28 (m, 2 H, CH2-N), 3.83 (s, 6 H, 2ϫOCH3), 2.96–
2.95 (m, 2 H, CH2-C=CH), 2.79–2.77 [m, 2 H, CH2-(CH2)3-
C=CH], 2.32 (t, J = 7.2 Hz, 2 H, CH2-CO2H), 1.90–1.83 (m, 6 H,
Bodipy-α-aziridine 10: Bodipy acid 40 (0.10 mmol, 67 mg) was pre-
activated with EEDQ (0.10 mmol, 25 mg, 1.0 equiv.) in DMF
(1 mL) under argon over 2 h at room temperature. Deprotected az-
iridine 41 (60 μmol) was dissolved in DMF (0.5 mL) under argon
and cooled to 0 °C, after which 0.3 mL of the activated ester solu-
tion was added (30 μmol, 0.5 equiv.) and the mixture was stirred at
0 °C for 30 min. Then another 0.5 equiv. of the activated ester was
added, and the reaction mixture was stirred at 0 °C for another
60 min. After quenching with MeOH, the mixture was concen-
trated in vacuo, purified by HPLC under neutral conditions with
use of a gradient of 48Ǟ51% MeCN in H2O in 12 min, and lyo-
philized to yield aziridine 10 as a purple powder (7.4 mg, 9.0 μmol,
3ϫCH2), 1.62–1.58 (m,
2 H, CH2), 1.32–1.25 (m, 6 H,
3ϫCH2) ppm. 13C NMR (100 MHz, CDCl3): δ = 178.12 (C=O),
160.29, 157.15 (Cq arom.), 144.67 (Cq triazole), 135.88 (Cq arom.),
130.71 (CH triazole), 126.73 (CH pyrrole), 124.89 (Cq arom.),
119.73 (CH pyrrole), 113.47 (2ϫCH arom.), 55.00 (2ϫOCH3),
53.33 (CH2-CO2H), 33.77, 32.72, 30.02, 29.78, 29.05, 28.49, 28.30,
25.95, 24.71, 24.31 (10ϫCH2) ppm.
1
15%). H NMR (400 MHz, MeOD): δ = 7.87 (d, J = 8.8 Hz, 4 H,
CH arom.), 7.72 (s, 1 H, CH triazole), 7.46 (d, J = 4.3 Hz, 2 H,
2ϫCH pyrrole), 7.00 (d, J = 9.2 Hz, 4 H, CH arom.), 6.72 (d, J =
4.3 Hz, 2 H, 2ϫCH pyrrole), 4.36 (t, J = 7.0 Hz, 2 H, CH2-N),
4.10 (dd, J = 8.6, 4.0 Hz, 1 H, CH-2), 3.88 (s, 6 H, 2ϫOCH3),
3.87–3.80 (m, 1 H, CH-4), 3.79 (dd, J = 7.4, 4.3 Hz, 2 H, CH2-6),
3.38 (dd, J = 8.6, 1.8 Hz, 1 H, CH-3), 3.12–3.06 (m, 2 H, CH2-
C=CH), 2.96 (dd, J = 6.0, 4.0 Hz, 1 H, CH-1), 2.82–2.78 [m, 2 H,
CH2-(CH2)3-C=CH], 2.62–2.58 (m, 1 H, CH-7), 2.55–2.35 (m, 2 H,
CH2-C=O), 2.03 (td, J = 7.8, 7.3, 3.6 Hz, 1 H, CH-5), 1.94–1.82
(m, 6 H, 3ϫCH2), 1.63–1.52 (m, 2 H, CH2), 1.32–1.21 (m, 6 H,
3ϫCH2) ppm. LC/MS analysis: Rt 7.4 min (linear gradient
10Ǟ90% B in 15 min), m/z 827.07 [M + H]+, 807.47 [M – F]+,
1653.40 [2M + H]+. Notably, under these acidic LC/MS conditions
the aziridine is partially opened to give a second peak at Rt 7.2 min
(linear gradient 10Ǟ90% B in 15 min), m/z 845.07 [(M + H2O) +
H]+, 825.40 [(M + H2O) – F]+, 1689.13 [2(M + H2O) + H]+.
HRMS: calcd. for [C44H54BF2N6O7]+ 827.41096; found 827.41180.
α-Aziridine 41: Ammonia was condensed at –60 °C (ca. 5 mL) un-
der argon flow with use of Birch equipment. Lithium (50 mg) was
added and the solution was stirred for 30 min at –60 °C, after which
a solution of α-aziridine 38 (0.10 mmol, 36 mg) in THF (5 mL) was
added. The reaction mixture was stirred at –60 °C under argon for
45 min, quenched with MilliQ (2 mL), and allowed to warm to
room temperature. After all ammonia had evaporated, the mixture
was further concentrated in vacuo and redissolved in MilliQ. Neu-
tralization with Amberlite-H+ resulted in resin-bound product,
which was filtered, washed with MilliQ, and then eluted with aque-
ous NH4OH solution (1 m, 10ϫ1 mL). After evaporation in vacuo
+
the product was redissolved in MilliQ, and Amberlite-NH4 was
added. Product was eluted from the resin by filtration and washing
with MilliQ until the pH of the eluted solution was neutral (pH 7).
The combined eluates were concentrated in vacuo to yield the crude
deprotected α-aziridine 41, which was used without further purifi-
β-Aziridine 42: The β-aziridine 34 (0.10 mmol, 36 mg) was depro-
tected by the same procedure as described for α-aziridine 41 to give
β-aziridine 42. 1H NMR (400 MHz, D2O): δ = 4.10 (d, J = 8.0 Hz,
1 H, CH-2), 3.87–3.78 (m, 3 H, CH2, CH-4), 3.43 (d, J = 8.0 Hz,
1 H, CH-3), 2.44 (br. s, 1 H, CH-1 or CH-7), 2.34–2.33 (m, 1 H,
CH-1 or CH-7), 2.04 (br. s, 1 H, CH-5) ppm.
1
cation. H NMR (400 MHz, D2O): δ = 4.01 (dd, J = 4.2, 9.0 Hz,
1 H, CH-2), 3.78 (s, 1 H, CH-4), 3.68–3.64 (m, 2 H, CH2), 3.28 (d,
J = 8.8 Hz, 1 H, CH-3), 2.54–2.51 (m, 1 H, CH-1 or CH-7), 2.08 (d,
J = 6.4 Hz, 1 H, CH-1 or CH-7), 1.90–1.86 (m, 1 H, CH-6) ppm.
Azido-β-aziridine 15: Deprotected β-aziridine 42 (0.10 mmol) was
acylated with azido-spacer 39[23] by the same procedure as de-
scribed for α-aziridine 9 to give β-aziridine 15. However, LC/MS
analysis (under basic conditions under which the aziridine should
be stable) revealed the major product to be an H2O adduct, pre-
sumably formed by opening of the aziridine. LC/MS analysis (am-
monium acetate): minor peak Rt 7.6 min (linear gradient 0Ǟ50%
B in 15 min), m/z 343.4 [M + H]+, 360.4 [M + NH4]+; major peak
Rt 7.0 min (linear gradient 0Ǟ50% B in 15 min), m/z 361.4 [(M +
H2O) + H]+, 378.7 [(M + H2O) + NH4]+. After HPLC purification
and lyophilization, NMR analysis revealed that no aziridine proton
peaks were present, indicating that the aziridine had completely
decomposed.
Azido-α-aziridine 9: 8-Azidooctanoic acid (39[23]
, 0.40 mmol,
78 mg) was preactivated with EEDQ (0.40 mmol, 99 mg, 1.0 equiv.)
in DMF (0.4 mL) under argon for 2 h at room temperature. Depro-
tected aziridine 41 (65 μmol) was dissolved in DMF (1 mL) under
argon and cooled to 0 °C, after which 33 μL of the activated ester
solution was added (33 μmol, 0.5 equiv.) and the mixture was
stirred at 0 °C for 30 min. Then another 0.5 equiv. of the activated
ester was added and the reaction mixture was stirred at 0 °C for
another 60 min. After quenching with MeOH, the mixture was
concentrated in vacuo, purified by HPLC under neutral conditions
with use of a gradient of 22Ǟ28% MeCN in H2O in 12 min, and
lyophilized to yield aziridine 9 as a white powder (3.0 mg, 9.0 μmol,
1
14%). H NMR (600 MHz, MeOD): δ = 4.08 (dd, J = 4.2, 9.0 Hz,
1 H, CH-2), 3.87–3.86 (m, 1 H, CH-4), 3.82–3.75 (m, 2 H, CH2- DFT Calculations: The calculated 1H NMR coupling constants
6), 3.37 (dd, J = 1.8, 8.4 Hz, 1 H, CH-3), 3.30–3.27 (m, 2 H, CH2-
N3), 2.97 (dd, J = 4.2, 6.0 Hz, 1 H, CH-1), 2.61 (d, J = 6.0 Hz, 1
H, CH-7), 2.56–2.44 (m, 2 H, CH2-C=O), 2.02 (td, J = 7.8, 7.2,
were obtained by first finding the lowest-energy conformations of
both epoxide isomers (5 and 11), for which a library of gas-phase
conformations was generated by use of the conformer distribution
3.6 Hz, 1 H, CH-5), 1.66–1.57 (m, 4 H, 2ϫCH2), 1.39–1.34 (m, 6 option included in the Spartan 04[24] program with employment of
12
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α- and β-galacto-Configured Cyclophellitols
[14] F. G. Hansen, E. Bundgaard, R. Madsen, J. Org. Chem. 2005,
70, 10139–10142.
DFT/B3LYP 6–31G(d). All conformers were further optimized by
use of Gaussian 03[25] at DFT/B3LYP 6–311G(d,p), their zero-
point energies were calculated, and the energies were corrected for
solvent by another optimization step with employment of a Polariz-
able Continuum Model set for water. The energies of these con-
formers, corrected for their zero-point energies, were compared,
and for the lowest energy-conformer an NMR calculation was per-
formed with the aid of the Gauge-Independent Atomic Orbital
(GIAO) method with added spin–spin coupling calculation.
[15] Y. Harrak, C. M. Barra, A. Delgado, A. R. Castaño, A. Lleba-
ria, J. Am. Chem. Soc. 2011, 133, 12079–12084.
[16] a) C. W. Tornøe, C. Christensen, M. Meldal, J. Org. Chem.
2002, 67, 3057–3064; b) V. V. Rostovtsev, L. G. Green, V. V.
Fokin, K. B. Sharpless, Angew. Chem. Int. Ed. 2002, 41, 2596–
2599; Angew. Chem. 2002, 114, 2708; c) Q. Wang, T. R. Chan,
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[17] D. A. Evans, E. B. Sjogren, J. Bartroli, R. L. Dow, Tetrahedron
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[18] Throughout this paper, the designation of the α-/β-stereochem-
istry of all epoxides and aziridines is based on α-/β-galacto-
pyranose nomenclature.
[19] M. Verdoes, U. Hillaert, B. I. Florea, M. Sae-Heng,
M. D. P. Risseeuw, D. V. Filippov, G. A. van der Marel, H. S.
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1735–1738.
Supporting Information (see footnote on the first page of this arti-
1
cle): H NMR and 13C-APT NMR spectra for all compounds and
optimized structures of epoxides 5 and 11 obtained by DFT calcu-
lations.
Acknowledgments
Funding from the Netherlands Organization for Scientific Research
(NWO-CW), the Netherlands Genomics Initiative (NGI), and the
European Research Council (ERC) is acknowledged.
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Received: May 16, 2014
Published Online: ᭿
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13

Job/Unit: O42589
/KAP1
Date: 12-08-14 18:46:12
Pages: 14
H. S. Overkleeft et al.
FULL PAPER
Cyclophellitol Derivatives
L. I. Willems, T. J. M. Beenakker,
B. Murray, B. Gagestein, H. van den Elst,
E. R. van Rijssel, J. D. C. Codée,
W. W. Kallemeijn, J. M. F. G. Aerts,
G. A. van der Marel,
H. S. Overkleeft* ............................ 1–14
The synthesis of α- and β-galactopyranose-
configured derivatives of cyclophellitol and
of cyclophellitol aziridine, either non-
tagged or functionalized with various
reporter entities, is described. All epoxide-
and aziridine-based compounds are synthe-
sized from a single cyclohexene precursor –
(3S,4S,5S,6R)-3,4-dibenzyloxy-5-hydroxy-
6-(hydroxymethyl)cyclohex-1-ene.
Synthesis of α- and β-Galactopyranose-
Configured Isomers of Cyclophellitol and
Cyclophellitol Aziridine
Keywords: Cyclitols / Enzyme inhibitors /
Irreversible inhibitors / Aziridines / Epox-
ides / Fluorescent probes
14
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Eur. J. Org. Chem. 0000, 0–0