Gas-phase structure and conformation of the glycosidase and ceramide glucosyltransferase inhibitor N-benzyl deoxynojirimycin

Article abstract of DOI:10.1039/b413672a

The deoxynojirimycin (DNJ) family of imino sugars are glucose analogues with an NH group replacing the oxygen atom in the pyranose ring. They are powerful inhibitors of glycosidases and ceramide glucosyltransferases. The conformation of N-benzyl-DNJ, isol

Full text of DOI:10.1039/b413672a

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R E S E A R C H P A P E R
Gas-phase structure and conformation of the glycosidase and
ceramide glucosyltransferase inhibitor N-benzyl deoxynojirimycinw
a
a
b
c
a
R. A. Jockusch, F. O. Talbot,z N. Asano, G. W. Fleet and J. P. Simons*
a
Department of Chemistry, Physical and Theoretical Chemistry Laboratory, South Parks Road,
Oxford, UK OX1 3QZ. E-mail: john.simons@chem.ox.ac.uk; Fax: þ44 1865 257410;
Tel: þ44 1865 275973
b
c
Faculty of Pharmaceutical Sciences, Hokuriku University, Kanazawa, 920-11, Japan
Department of Chemistry, Chemistry Research Laboratory, Mansfield Road, Oxford,
UK OX1 3TA
Received 15th September 2004, Accepted 14th October 2004
First published as an Advance Article on the web 22nd October 2004
The deoxynojirimycin (DNJ) family of imino sugars are glucose analogues with an NH group replacing the
oxygen atom in the pyranose ring. They are powerful inhibitors of glycosidases and ceramide
glucosyltransferases. The conformation of N-benzyl-DNJ, isolated in the gas phase, is studied using a
combination of resonant two-photon ionization (R2PI), UV/UV hole-burn, and IR ion-dip spectroscopies in
conjunction with electronic structure theory calculations. Three distinct conformers, one major and two minor,
are present and all three are assigned to structures in which the exocyclic hydroxymethyl group is axial to the
piperidine ring (gauche- to the ring nitrogen). This contrasts with the preferred equatorial (gaucheþ) orientation
observed for simple glucosides and may well contribute to the stronger binding of some enzymes to DNJ-based
inhibitors compared to their natural glucoside substrates.
4
ring, which is in a (distorted) C
1
chair conformation. Inter-
1
. Introduction
estingly, both also show ordered water molecules within the
active site interacting with the ring nitrogen. N-alkylation
could serve to displace the bound water molecules, represent-
ing an entropic advantage for binding these inhibitors (an
advantage also suggested for the increased potency of iso-
N-Benzyl deoxynojirimycin (N-Bn-DNJ) 1, the N-benzylated
derivative of deoxynojirimycin (DNJ) 2, is a mimic of glucose 3
(Scheme 1). DNJ, a member of an important family of imino
sugars that function as inhibitors of glycosidases, is an inhi-
bitor of a- and b-glucosidases. Enzymes other than glycosi-
dases are also inhibited by imino sugars; for example,
derivatives of DNJ are powerful inhibitors of ceramide-specific
glucosyltransferase whereas DNJ itself is not an effective
5
inhibitor of this enzyme. X-ray diffraction and NMR studies
of DNJ and its N-alkyl derivatives have revealed differing
conformational preferences. In DNJ the hydroxymethyl O(6)H
group adopts an equatorial orientation with respect to the
piperidine ring, possibly favoured by a weak, intramolecular
1
,2
11
10
fagomine 5 over DNJ by Zechel and coworkers ); no com-
plexes containing bound N-alkylated DNJ are available in the
protein data bank at this time. The only available structure
containing bound castanospermine shows the piperidine ring
distorted into a twisted boat conformation which is suggested
to be a precursor to the transition state for the cleavage of the
3
,4
6
,7
12
glucoside.
Molecular mechanics (MM3) and low-level ab initio (HF/6-
31G) calculations for DNJ and castanospermine have sup-
ported the assignment of the two structures (established by
˚
NH - O6 hydrogen-bond [r(Nꢀ ꢀ ꢀO) ¼ 2.86 A, +NH–O ¼
6
1
041] but in the N-alkyl derivatives, where this H-bonding is
diffraction and NMR measurements in the condensed phase) to
7
no longer possible, its orientation switches to axial. It has been
suggested that this axial orientation is an important structural
requirement for potent a-glucosidase inhibition and the con-
sequent interference in the biosynthesis of N-linked glycopro-
8
teins (and anti-viral activity). The enhanced potency of some
a-glucosidases by castanospermine 4, where the O(6)H is
forced into an axial conformation by the fused five-membered
6
ring, has lent support to this view.
Despite the potential therapeutic uses of imino sugars, only
two crystal structures of biological complexes containing DNJ
as a tightly-bound inhibitor have been reported, one to an a-
9
,10
and one to a b-glucosidase.
In both, the O(6)H interacts
with an acidic residue and is oriented axially to the piperidine
w Electronic supplementary information (ESI) available: Synthetic
procedure for N-benzyl deoxynojirimycin and calculated Cartesian
coordinates of its low-energy conformers. See http://www.rsc.org/
suppdata/cp/b4/b413672a/
z Present address: Spectrometrie Ion & Mol Lab, 43 Bd 11 Novembre,
F-69622, Villeurbanne, France.
Scheme 1
T h i s j o u r n a l i s & T h e O w n e r S o c i e t i e s 2 0 0 4
P h y s . C h e m . C h e m . P h y s . , 2 0 0 4 , 6 , 5 2 8 3 – 5 2 8 7
5283

View Article Online
the global minimum conformations predicted for the two
6
molecules isolated in the gas phase. To date however, there
3
. Spectroscopy and structure
The lowest lying conformers of N-Bn-DNJ computed ab initio
are shown in Fig. 1w The global minimum structure is calculated
to lie at an energy 4.5 kJ mol below its nearest neighbour, see
have been no experimental determinations of their conforma-
tional landscapes under isolated conditions free of environ-
mental perturbations to compare with (higher-level) theoretical
results, or of the ease of populating alternative conformers.
Our recent investigations of the conformational structures of
the model sugars O-phenyl b-D-gluco- or D-galacto-pyranoside,
vaporised by heating or laser ablation and cooled in a free-jet
argon expansion, probed via UV mass-selected resonant two-
photon ionization (R2PI), resolved through ‘hole-burning’
techniques, and assigned through analysis of their resonant,
ꢁ
1
Table 1. All the conformers are associated with a gauche (Gꢁ)
[O6–C6–C5–N] conformation, creating an axially oriented
O(6)H group (regardless of the orientation of the N-benzyl
substituent): the lowest lying equatorially oriented structures,
gauche (Gþ) or trans (T), all lie at relative energies Z 9 kJ
ꢁ
1
mol above the global minimum. The six most stable confor-
mers differ primarily by the orientation of the benzyl ring (I vs.
II vs. III) and the orientation of the co-operative ‘daisy chain’
sequence of hydrogen bonds formed by the hydroxyls on the
ring carbons [(a) vs. (b): in conformers (a) they adopt a gauche
orientation with respect to the Cn11 carbon, while conformers of
type (b) present a trans orientation]. Conformers IIa,b and
IIIa,b are stabilized by a hydrogen bond between OH6 and
1
3,14
mid-IR ion-dip spectra, have prompted their extension to
enable the first investigation of an imino sugar in the gas phase.
2
. Methods
2
.1. Spectroscopy
˚
the ring nitrogen [r(OH6ꢀ ꢀ ꢀN) B 2.2–2.4 A, +OH–N B1151],
Gas-phase N-Bn-DNJ (synthetic details provided as electronic
supplementary information (ESI)w) was produced by placing
the sample in an oven attached to the expansion side of a
pulsed nozzle (General Valve, 0.8 mm diameter, 4 bar argon
backing pressure, 10 Hz pulse rate). The molecules, vaporized
at 175 1C, were thus entrained and cooled in the pulsed
expanding argon jet. The beam was skimmed and then inter-
sected by infrared (IR) and/or ultraviolet (UV) laser beam(s)
tuned to the desired wavelength(s) for excitation and ionization
in the acceleration region of a time-of-flight mass spectrometer
an interaction which is considerably weaker in the conformers
˚
Ia,b [r(OH6ꢀ ꢀ ꢀN) B 2.8–3.0 A, +OH–N B951] because of the
position of the benzyl ring; on the other hand conformer Ia, the
global minimum structure, displays the longest ‘daisy chain’
sequence of hydroxyl H-bonds.
Fig. 2 shows the one-colour R2PI spectrum of jet-cooled N-
Bn-DNJ (top trace) and below it three distinct UV hole-burn
spectra, labeled a, b and b ; they account for all of the intense
peaks observed in the R2PI spectrum and correspond to three
different conformers of the molecule. The UV contour and
vibronic spacings displayed by the principal conformer, a, are
0
(
RM Jordan) [using a resonant two-photon ionization (R2PI)
scheme] enabling mass selective detection of the ions thus
produced. Tuneable UV laser radiation for the R2PI experi-
ments was generated by frequency doubling the output of a
Nd:YAG-pumped pulsed dye laser [GCR-170/Laser Analytical
Systems (LAS)] operated with a mixture of Coumarin 153 and
quite distinct from the (nearly identical) spectra displayed by
0
the two minor conformers, b and b . In addition to their S ’
1
ꢁ1
S
0
origin bands, centred around 37 280 cm (a) and 37 500
ꢁ1
0
cm (b/b ), all three hole-burn spectra display a series of more
intense vibronic bands located B530 cm higher in energy
ꢁ1
ꢁ1
Coumarin 500 dye (B0.4–1 mJ pulse ). This laser was
employed as the scanned, ‘burn’ laser in UV/UV hole-burn
experiments: the fixed ‘probe’ pulse, fired B200 ns after the
burn laser, was generated by a second, excimer-pumped pulsed
dye laser (Lambda Physik FL3002), tuned to successive fea-
tures of interest in the R2PI spectrum. Resonant IR ion dip
scheme spectra were recorded in an analogous manner with the
burn laser generating tuneable pulsed IR radiation (B3000–
(
associated with the excitation of a benzyl ring mode, a feature
commonly seen in one colour R2PI spectra of compounds
17
containing this chromophore ). Each band accommodates a
series of much lower frequency vibronic progressions with
ꢁ1
spacings of B37.5 and 41 cm (conformer a) and B21 and
ꢁ1
0
2
4 cm (conformers b and b ). These spectra suggest that
0
conformers b and b are extremely similar to each other while
conformer a is somewhat different.
The mass of N-Bn-DNJ is 253 u but in all three conformers
more than 90% of the observed ion signal is at mass 222, a loss
of 31 u. Spectra measured in the fragment and parent ion mass
ꢁ1
ꢁ1
ꢁ1
4
difference frequency mixing the output of a Nd:YAG laser
000 cm , 0.4 cm bandwidth, 4.5 mJ pulse ), through
(
(
Powerlite Precision 8000) and that of a pumped dye laser
ND6000, Continuum) in a LiNbO3 crystal: the UV probe
0
channels show the same features, though conformers b and b
pulse was provided by the LAS laser system, fired B150 ns
after the IR pulse.
have a greater ratio of fragment to parent ions than conformer a.
The fragmentation, in all probability, corresponds to the
cleavage of the C5–C6 bond and loss of the hydroxymethyl
2
.2. Computation
Starting structures for geometry optimization were generated
using molecular mechanics (MMFF94c and MM3 forcefields)
and conformation searching with the Monte Carlo multiple
minimization method implemented in the MacroModel soft-
1
5
¨
ware (MacroModel v.8.5, Schrodinger, LLC); 1000 struc-
tures were generated. A total of 40 conformers were selected
for subsequent electronic structure theory calculations; these
ꢁ
1
included all structures within 15 kJ mol of the most stable in
the conformational search, as well as other assorted structures,
4
such as ones with the benzyl group axial to the ( C
1
) piperidine
1
4
ring, C conformers and boat conformers. The selected con-
formers were further optimized using B3LYP hybrid-method
density functional theory and the 6-31þG(d) basis set. Analy-
tical frequency and thermochemistry calculations were per-
formed on the B3LYP geometry-optimized conformations, as
were single-point energy calculations, using perturbation the-
ory (MP2) and the 6-311þG(d,p) basis set. All electronic
structure theory calculations were performed using the GAUS-
Fig. 1 Six lowest-energy conformers of N-benzyl deoxynojirimycin
(
N-Bn-DNJ) optimized at B3LYP/6-31þG(d). Relative energies are
ꢁ1
given in parenthesis in kJ mol at the MP2/6-311þG(d,p)//B3LYP/
1
6
SIAN software.
6-31þG(d) level of theory and include zero-point energy corrections.
5
284
P h y s . C h e m . C h e m . P h y s . , 2 0 0 4 , 6 , 5 2 8 3 – 5 2 8 7
T h i s j o u r n a l i s & T h e O w n e r S o c i e t i e s 2 0 0 4

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ꢁ1
Table 1 Relative energies (in kJ mol ) of low-energy conformers of N-Bn-DNJ calculated at the MP2/6-311þG(d,p)//B3LYP/6-31þG(d) level of
theory
a
Name
Conformation
U
E
0
TDS(175 1C)
DG(175 1C)
Ia
gggG-gþ_I
gggG-gþ_II
gggG-gþ_III
tttG-gþ_I
0.0
5.3
6.1
8.7
5.1
7.9
0.0
4.7
7.2
7.5
4.5
8.5
0.0
6.7
0.7
1.9
6.2
0.8
1.3
0.0
7.8
7.7
0.3
9.2
IIa
IIIa
Ib
IIb
IIIb
a
tttG-gþ_II
tttG-gþ_III
The naming system used follows that used in other reports on sugar molecule conformation (see, for example, refs. 13 and 14). The letters refer to
–O –C
dihedral which defines the position of the hydroxy
the position of the rotatable dihedral angles. The first three letters (all ttt or ggg here) indicate the trans or gauche orientations of the H
–C –C –O
n
n
n
–
C
n11, n ¼ 2–4 dihedral angles. The next (capital) letter indicates value of the O
6
6
5
5
methyl group (all conformers here are Gaucheꢁ: angles between 01 and 1201). The following letter indicates the C
gaucheþ: angles between 2401 and 3601). The final roman numerals refer to the orientation of the benzyl ring.
5 6 6 6
–C –O –H dihedral (all here are
radical. Although the benzyl ring is ionized in the R2PI scheme
used, the ionization energy of tertiary amines is B1 eV lower
groups appear at slightly higher wave number. All three
conformers of N-Bn-DNJ exhibit one relatively free O–H
mode, two bands consistent with a weakly hydrogen bonded
and one band associated with a more strongly bonded OH
1
8
than benzyl compounds and migration of the charge from the
benzyl ring chromophore to the amine will endow the mole-
cular ion with significant internal energy and promote its
fragmentation, further promoted by excitation of the ring
0
group. In conformers b and b , particularly, this latter band is
ꢁ
1
considerably shifted (B80 cm below the central doublet)
indicating a strong perturbation of one of the OH stretches in
both these conformers.
ꢁ
1
deformation mode at B530 cm . The difference in frag-
mentation ratios among the conformers may simply be due
to the larger excess energy in the vibrationally excited ions, but
it may also be due to differences in Franck–Condon overlap
between the excited and ionized states in the different con-
As the hydroxyl groups attached directly to the ring (in its
C chair conformation) are geometrically constrained and
1
4
0
formers a and b/b .
Ion-dip spectra of the three conformers, measured in the O–
H stretching region of the infrared are shown in Fig. 3. In each
case all four O–H modes are visible although the central pair lie
very close in energy and therefore appear as a partially resolved
doublet. The close similarity between the minor conformers,
0
b and b , is again apparent; each one presents a very similar
vibrational spectrum with the O–H band at lowest wavenum-
ber more strongly shifted than for conformer a.
The O–H stretching modes are very sensitive to the local
environment and provide good indicators of conformation. In
1
3,14
the monosaccharides
pyranoside, vibrational bands appearing in the region 3630–
3
O-phenyl b-D-gluco- and D-galacto-
ꢁ
1
650 cm are associated with a sequence of weakly bonded
hydroxyl groups arranged in an equatorial fashion around the
pyranoside sugar ring. Stronger H-bonded interactions shift
the O–H bands to lower wavenumber and broaden them; those
associated with relatively ‘free’ (not H-bond donating) OH
Fig. 3 Experimental and computed infrared ion-dip spectra of N-Bn-
DNJ in the O–H stretching region. Computed bands are labeled with
Fig. 2 Resonant two-photon ionization (R2PI) and UV/UV hole-
0
the (C
n
ꢁ) O–H stretching motion to which they correspond according
burn spectra of N-Bn-DNJ. Peaks marked with * burned with b .
to the normal coordinate analysis.
T h i s j o u r n a l i s & T h e O w n e r S o c i e t i e s 2 0 0 4
P h y s . C h e m . C h e m . P h y s . , 2 0 0 4 , 6 , 5 2 8 3 – 5 2 8 7
5285

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0
cannot form strong interactions with each other, this implies a
strong interaction between the hydroxymethyl group and most
likely, the ring nitrogen in the two conformers, b and b .y
Infrared spectra calculated for each of the six lowest-lying
conformers (see Fig. 1) are also shown in Fig. 3. Conformers Ia
and Ib, differing in the orientation of the ring hydroxyl groups
importantly, both the major (a) and the two minor (b, b )
conformers are associated with structures in which the OH6
group is axial (Gꢁ) to the ring nitrogen.
0
The conserved Gꢁ orientation of the hydroxymethyl group
of N-Bn-DNJ differs from the preferred orientation in simple
glucosides. In 1-O-phenyl b-D-gluco-pyranoside, only B25%
of the observed population adopts a Gꢁ orientation; the most
(
note the effect on the relative positions of the alternately ‘free’
1
3
or hydrogen-bonded O–H stretch modes, s4 and s2, in each
one of the three ‘a,b’ conformer pairs) and conformer IIa all
present very similar IR spectra and each could provide a
reasonable match for the recorded spectrum of the principal
conformer a. On balance however, its assignment to the
structure Ia is strongly favoured for the following reasons:
populated conformation (B70%) is Gþ. In the correspond-
ing galacto-pyranoside, the preference for Gþ is even stronger
1
4
(B90%). This innate preference for the Gꢁ orientation in the
imino sugars may well contribute to their strong binding
compared to the natural glucoside substrate. Although the
present investigation has focused on a neutral N-alkylated
imino sugar, which is not thought to be protonated in its
anti-ceramide glucosyltransferase activity, the ring nitrogen
atom of DNJ-based inhibitors is probably protonated in the
(
i) Calculations at 0 K place conformer Ia at the global
ꢁ
1
minimum,z lying 4.5 kJ mol below the energy of conformer
ꢁ
1
IIa and 7.5 kJ mol below Ib.
ii) There is a very close correspondence between the spacings
in the vibronic progressions in its R2PI spectrum, B37.5 and
5
(
active sites of glycosidases. A study of the conformation of
protonated DNJ in the gas phase may provide further insight
into the mechanism of such inhibition.
ꢁ
1
41.0 cm (in the S
benzyl ring wag modes of conformer Ia calculated (in the S
state) at 37.6 and 42.3 cm . The equivalent lowest wavenumber
modes in conformer IIa are calculated at 20.6 and 24.1 cm
The assignment of the two minor conformers, b and b , is
less straightforward; the strong shift of the lowest wavenumber
OH band provides an important clue but each one of the
conformer pairs IIa/b and IIIa/b exhibits a hydrogen bond
between the hydroxymethyl group and the ring nitrogen which
significantly lowers the calculated wave number of the O–H6
band, s6. Nonetheless, one of the remaining conformers, b or
1
state) and the in-plane and out-of-plane
0
ꢁ
1
ꢁ
0
1
.
Acknowledgements
The authors thank Dr Mark Wormald for helpful discussions.
We appreciate the support provided by the EPSRC, the Royal
Society (R.A.J., USA Royal Society Fellowship), the Lever-
hulme Trust (Grant No. F/08788D), the CLRC Laser Loan
Pool and the Physical and Theoretical Chemistry Laboratory
at Oxford.
0
b , is most likely to be associated with the conformation IIb,
since this is the second most stable conformer at 0 K and its
References
calculated IR spectrum provides an excellent match to the
0
measured IR spectra of both b and b . Assignment of the
partner conformer to the structure IIIb (which differs from IIb
only in the orientation of the benzyl group) is tempting but at
1
2
N. Asano, Glycobiology, 2003, 13, 93R.
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ꢁ1
3
0
conformer IIa [and at the temperature of the oven IIa becomes
K it is calculated to lie B4 kJ mol higher in energy than
the lowest energy conformer while IIIb is the highest of the six
0
4
5
(
favoured by their similar low frequency vibration/internal
Table 1)]. Assignment of conformers b/b to the IIa/IIb pair is
rotation modes (calculated to be a twist of the benzyl group)
ꢁ1
lying at 24.1 and 20.6 cm , respectively (in the ground state,
S ); these match very well the measured (S ) vibronic progres-
6
7
8
A. Hempel, N. Camerman, D. Mastropaolo and A. Camerman,
J. Med. Chem., 1993, 36, 4082.
0
1
ꢁ
1
sions of 24 and 21 cm . In contrast, normal mode analysis of
the conformers IIIa/b show the lowest energy mode (also a
benzyl twist though with some benzyl wag coupled in) at 34.5
and 35.3 cm . Additional evidence favouring assignment to
the IIa/IIb pair is provided by the extreme similarity of their
R2PI spectra. The environment of the benzyl ring chromo-
phore of conformer IIb is closer to that of conformer IIa
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ꢁ
1
9
0
1
(
which differs in the orientation of the chain of hydroxyl
groups) than conformer IIIb (which differs in the orientation
of the benzyl ring). On balance, therefore the assignment of
11 V. H. Lillelund, H. Z. Liu, X. F. Liang, H. Sohoel and M. Bols,
Org. Biomol. Chem., 2003, 1, 282.
0
b and b to conformers IIa/IIb is the most favoured. Most
1
2
S. M. Cutfield, G. J. Davies, G. Murshudov, B. F. Anderson, P. C.
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294, 771.
y The hydroxymethyl group could also form a strong hydrogen bond
with O4, forming a gggTt structure. This would also result in a single
O–H mode which is shifted to lower wavenumbers, but IR ion-dip
spectra of the monosaccharide mannose (manuscript in preparation) in
13 F. O. Talbot and J. P. Simons, Phys. Chem. Chem. Phys., 2002,
4, 3562.
14 R. A. Jockusch, F. O. Talbot and J. P. Simons, Phys. Chem. Chem.
Phys., 2003, 5, 1502.
15 F. Mohamadi, N. G. J. Richards, W. C. Guida, R. Liskamp,
M. Lipton, C. Caufield, G. Chang, T. Hendrickson and W. C.
Still, J. Comput. Chem., 1990, 11, 440.
ꢁ1
which this conformer is seen, show this mode to be B35 cm higher in
energy than that measured for N-Bn-DNJ. Also, the most stable gggTt
ꢁ1
ꢁ1
conformer is calculated to be 4.5 kJ mol (about 390 cm ) higher in
energy than conformers IIa and b.
z Conformer Ia benefits from an extended chain of hydrogen bonds
encircling the piperidine ring; the stability conferred by this arrange-
ment has become a recurring theme in gas phase studies of analogous
sugar molecules [see refs. 13, 14]. The entropic constraint provided by
this arrangement disfavours conformation Ia at higher temperatures;
free energy calculations (harmonic approximation, treating internal
rotations as vibrations) place conformer Ia slightly higher in energy
16 M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A.
Robb, J. R. Cheeseman, J. J. A. Montgomery, T. Vreven, K. N.
Kudin, J. C. Burant, J. M. Millam, S. S. Iyengar, J. Tomasi, V.
Barone, B. Mennucci, M. Cossi, G. Scalmani, N. Rega, G. A.
Petersson, H. Nakatsuji, M. Hada, M. Ehara, K. Toyota, R.
Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O.
Kitao, H. Nakai, M. Klene, X. Li, J. E. Knox, H. P. Hratchian, J.
B. Cross, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann,
O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski,
ꢁ1
than conformer IIa (by 1.5 kJ mol at 448 K, the temperature of the
oven used to vaporize the sample).
5
286
P h y s . C h e m . C h e m . P h y s . , 2 0 0 4 , 6 , 5 2 8 3 – 5 2 8 7
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