Stabilizing unusual conformations in small peptides and glucopeptides using a hydroxylated cyclobutane amino acid

Article abstract of DOI:10.1039/b907091p

The synthesis and the conformational study in the solid state and in aqueous solution of a peptide and a glucopeptide containing the non-natural (1S,2S)-1-amino-2-hydroxycyclobutanecarboxylic acid (c₄Ser) residue are reported. This is the first

Full text of DOI:10.1039/b907091p

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Stabilizing unusual conformations in small peptides and glucopeptides using a
hydroxylated cyclobutane amino acid†
Alberto Ferna´ndez-Tejada, Francisco Corzana,* Jesu´s H. Busto, Alberto Avenoza and Jesu´s M. Peregrina*
Received 7th April 2009, Accepted 1st May 2009
First published as an Advance Article on the web 8th June 2009
DOI: 10.1039/b907091p
The synthesis and the conformational study in the solid state and in aqueous solution of a peptide and
a glucopeptide containing the non-natural (1S,2S)-1-amino-2-hydroxycyclobutanecarboxylic acid
(c4Ser) residue are reported. This is the first example of a glycopeptide containing a carbohydrate
moiety linked to an underlying non-natural amino acid residue. The conformational analysis in
solution combines NOEs and coupling constants data with Molecular Dynamics (MD) simulations
with time-averaged restraints. The study reveals that the c4Ser-Ala-Ala diamide peptide shows a
conformation of two consecutive b-turn type III structures (the basic structural element of a 310 helix).
However, none of the turns observed in the peptide are present in the derived glucopeptide. The
influence of the carbohydrate moiety on the peptide backbone can be explained by means of the
existence of two simultaneous hydrogen bonds, between the endocyclic oxygen of the glucose and two
amidic protons of the peptide. In addition, the non-natural residue favors the existence of an unusual
high energy conformation for the glycosidic linkage, the so-called anti-f conformation.
acids,8 show that these molecules can stabilize conformations
Introduction
present in naturally occurring molecules or exhibit some atypical
Small natural peptides are in general conformationally flexible and
conformations. This feature could be a powerful tool to design and
are therefore not suitable to study or control secondary peptide
modulate the conformational space of the different synthesized
structures. Taking into account that one attractive approach
glycopeptides.
to restrict peptide conformation involves the incorporation of
quaternary a-amino acids in the peptidic chain,1 the synthesis
With the intention of expanding our novel small systems,
we herein report the synthesis and the conformational anal-
of a,a-disubstituted a-amino acids has been the concern of many
synthetic organic chemists.2 Therefore, with the aim of increasing
the possibilities in terms of producing modified peptides with new
properties or functions, the synthesis and conformational study
of peptides containing non-natural amino acids is still undergoing
continual advancement and optimization. It is well-known, for
instance, that the Ca tetrasubstitution in small homopeptides
favours the stabilization of 310 or a-helixes.3 Additionally, in
short peptides, a,a-disubstituted a-amino acids stabilize turn
structures,4 which have received particular attention because they
play an important role in proteins from both structural and
functional points of view.5 On this basis, and in order to modify
the active conformation of a peptide, several artificial turns have
been reported in the past.6
ysis in aqueous solution of peptide 1 and its corresponding
b-O-glucopeptide 2 (Fig. 1). In addition, the structures found
in solution are compared to that obtained in the solid state for
peptide 1. It is important to emphasize that, to the best of our
knowledge, this is the first synthesized glycopeptide reported to
date that incorporates a non-natural amino acid at the underlying
residue.
To go one step further, and taking into account the important
role that glycopeptides play in fundamental biological processes,7
we are working on the design of novel glycopeptides that incorpo-
rate non-natural amino acids in their structures. Our preliminary
results, carried out on the simplest non-natural glycosylated amino
Departamento de Qu´ımica, Universidad de La Rioja, Grupo de S´ıntesis
Qu´ımica de La Rioja, UA-CSIC, 26006, Logron˜o, Spain. E-mail:
jesusmanuel.peregrina@unirioja.es; Fax: +34 941 299621
† Electronic supplementary information (ESI) available: NMR spectra for
n
all new compounds, full assignment of protons and JH,H couplings for
compound 2, sections of the 800 ms 2D NOESY spectra (400 MHz) in
H2O/D2O (9:1) for compound 2 and X-ray diffraction structure for com-
pound 9. CCDC reference number 716958. For ESI and crystallographic
data in CIF or other electronic format see DOI: 10.1039/b907091p
Fig. 1 Compounds studied in this work.
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MeONa/MeOH to give the glycopeptide 2 with a moderate overall
yield (Scheme 2).
Results and discussion
Synthesis
The synthesis of peptide 1 was started from the commercially
available N-Boc-Ala derivative 3. This compound was treated
with MeNH2·HCl and N,N,N¢,N¢-tetramethyl-O-(benzotriazol-
1-yl)uronium tetrafluoroborate (TBTU) as a coupling agent in
the presence of diisopropylethylamine (DIEA) as a base to give
the amide 4. Removal of the Boc group and the subsequent
treatment of the resulting amine 5 with an additional equivalent
of compound 3 gave the dipeptide 6. This compound was
firstly treated with trifluoroacetic acid (TFA) and then with the
racemic mixture of cyclobutane derivative 8, which was previously
synthesized by our group,2 to give a mixture of protected
peptides 9 and 10. The purification of compound 9 by column
chromatography on silica gel and hydrogenolysis of the benzyl
group with hydrogen in the presence of Pd/C as a catalyst gave
the desired peptide 1 (Scheme 1). Single crystals suitable for
X-ray analysis were obtained for compound 1, which allowed us
to determine the relative stereochemistry. Knowing the absolute S
configuration of two of the stereogenic centers allowed the con-
figuration of stereogenic centers in the cyclobutane ring also to be
deduced as S.
Solid state structure of peptide 1
The atom numeration and the labels of the different torsional
angles of compounds 1 and 2 are shown in Fig. 2.
Some important conclusions can be drawn with respect to
the solid state (Fig. 3 and Supporting Information†). Firstly,
a conformation consisting of two consecutive b-turns type III,
slightly deviating from an ideal 310 helix structure, was obtained in
the solid state for compound 1. The torsion angles (f,y) shown in
Fig. 3 resemble the typical values for a b-turn type III (-60◦, -30◦
and -60◦, -30◦). The structure is stabilized by two intramolecular
˚
hydrogen bonds (with N–H ◊ ◊ ◊ O distances of 2.02 and 2.06 A,
respectively and N–H ◊ ◊ ◊ O angles of 166◦ and 161◦, respectively).
A similar structure has been recently found for a tripeptide
containing a tetrahydrofuran Ca-tetrasubstituted amino acid.4
Secondly, the cyclobutane ring adopts only one conformation,
with a pucker angle q equal to -28.3◦. In addition, the substituent
at carbon Cb occupies an equatorial position. This result is in
accordance with the crystal structure of different cyclobutane
derivatives previously studied in the solid state.10
The corresponding model glycopeptide 2 was obtained follow-
ing the modified conditions of the Koenigs–Knorr glycosylation.9
The treatment of peptide 1 with 2,3,4,6-tetra-O-benzoyl-a-D-
glucopyranosyl bromide in the presence of AgTfO gave derivative
11. The hydroxyl groups of this compound were deprotected using
Conformational preferences of peptide 1 in aqueous solution
The conformational preferences of the tripeptide 1 were examined
with experimentally restrained MD simulations using a protocol11
Scheme 1 Synthesis of peptide diamide 1.
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Scheme 2 Synthesis of glucopeptide diamide 2.
Fig. 2 Molecular structure of glucopeptide 2, showing the atom numer-
ation and the most relevant torsional angles. Letter ‘s’ makes reference
to the carbohydrate moiety. The blue and green numbers make reference
to the numeration of the NH and methyl groups, respectively. The same
numbering and labels were used for peptide 1 (a). The pucker angle q
of the cyclobutane ring is defined as the acute angle between the planes
Ca-Cb-Cd and Cb-Cg-Cd (b).
Fig. 3 Some geometrical features of the crystal structure of peptide 1.
Heteroatoms are shown in color; blue for the nitrogen atom and red for
oxygen atom. Thermal ellipsoids drawn at the 50% probability level.
and NH3–NH4 NOEs (characteristic of folded ones), shown in
Fig. 4, suggests the coexistence of both conformations in aqueous
solution for the two alanine residues of peptide 1. On the other
hand, the medium NOE observed between NH1 and NH2 suggests
an important population of a helix-like conformation for the
cyclobutane residue.12
similar to that recently applied by our group on different model
glycopeptides.
NMR data. In a first step, full assignment of the protons of
compounds 1 and 2 was carried out using COSY and HSQC
experiments and it is given in Table 1 and in the Supporting
Information (Table S1†). Selective 1D-NOESY experiments in
D2O (25 ◦C, pH = 5.2) and 2D-NOESY experiments in H2O/D2O
(9/1) (25 ◦C, pH = 4.84) were then carried out (see Experimental
Section).
The next step was to experimentally determine some proton-
proton distances relevant to the conformational analysis. The
distances involving NH protons were semi-quantitatively deter-
mined by integrating the volume of the corresponding cross-peaks
in the 2D NOESY spectrum. Additionally, some proton-proton
distances were obtained from the corresponding NOE build-
up curves13 (see Supporting Information†). On the other hand,
3JNH,Ha coupling constants were measured from the splitting of the
resonance signals in the 1D spectrum. All these experimentally
The medium-strong Ha2–NH3, Ha3–NH4 (typical of extended
conformations), Ha2–NH2, and Ha3–NH3, as well as NH2–NH3
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n
Table 1 Full assignment of protons and JH,H couplings for com-
Table 2 Comparison of the experimental and MD-tar simulations
derived distances, q angle and 3J coupling constants for compounds 1
and 2a
pound 1a
d (in ppm) proton
splittingb (# protons) nJH,H (in Hz)
Compound 1
Compound 2
1.10–1.27
1.39
1.63–1.78
1.83
2.11
2.47
2.56
4.05
4.18
Me2,Me3 m (6H)
—
—
—
—
—
—
—
Exptl
MD-tar
Exptl
MD-tar
Hda
Hga
Me1
Hge
Hde
Me4
Ha3
Ha2
Hb
m (1H)
m (1H)
s (3H)
m (1H)
m (1H)
s (3H)
q (1H)
q (1H)
‘t’ (1H)
m (1H)c
d (1H)c
d (1H)c
s (1H)c
dNH4,Ha3
dNH3,Ha3
dNH3-Ha2
dNH2,Ha2
dNH1,Hda
dHb,NH1
dNH4,NH3
dNH3,NH2
dNH2,NH1
dHb,Hda
2.4
2.5
2.4
2.4
2.7
2.2
3.0
2.5
2.6
2.7
2.4
—
2.4
2.8
2.6
2.7
2.7
2.1
2.8
2.3
2.8
2.9
2.3
—
2.4
2.5
2.6
2.6
3.1
2.3
3.0
2.8
2.7
2.7
2.4
2.4
2.9
—
2.3
2.8
2.3
2.8
3.0
2.5
2.9
2.6
2.6
2.8
2.5
2.4
2.8
-23.4
6.0
6.7
3JHa3,Me3= 7.1
3JHa2,Me2= 7.1
3
4.26
3JHb,Hga = JHb,Hge = 7.1
7.49–7.57c NH4c
—
7.67c
8.08c
8.69c
NH3c
NH2c
NH1c
3JNH3,Ha3 = 6.2c
3JNH2,Ha2 = 5.8c
—
dHb,Hge
dHb,H1s
a Data extracted from 1D 1H NMR experiment carried out in D2O (25 ◦C,
dHb,H2s
—
—
5.8
6.2
—
pH = 5.2) at 400 MHz. b s = singlet, d = doublet, q = quartet, m =
<q >
-18.8
6.0
6.4
3JNH2,Ha2
5.6
6.5
multiplet. c Data extracted from 1D H NMR experiment carried out in
1
H2O/D2O (9/1) (25 ◦C, pH = 4.84) at 400 MHz.
3JNH3,Ha3
a
3
˚
Distances are given in A, J in Hz, and q in degrees.
The fi/yi (i = 1, 2 or 3) distribution obtained from the MD-
tar simulations for peptide 1 is shown in Fig. 5a. As can be seen,
the cyclobutane residue presents f1/y1 typical values for helix-
like conformations, such as the right and left handed a-helix (aD,
85%; aL, 13%), with a clear preference for the aD. It is important to
mention that these two major conformers lie at the local minima
previously found for a model peptide previously studied by our
group.8 Concerning residue Ala2, it exhibited dispersed values of
f2/y2, being the most populated helix-like conformation (about
53% of the total trajectory time). On the contrary, the extended
conformations (b, 23% and PPII, 50%) were the most populated
ones for Ala3. From these data, it can be stated that the peptide
1 is rather flexible in aqueous solution. Indeed, only the b-turn
exhibited in the crystalline structure between the carbonyl of the
N-acetyl group and the NH3 was observed in aqueous solution.
According to our MD-tar simulations, this b-turn is present about
30% of the total trajectory time.
=
The second b-turn (between C2 O2 and NH4) was only present
about 7% of the total time. Fig. 6a shows a superimposition
of 10 conformers of peptide 1, randomly collected from the
MD-tar simulations, together with the solid state conformation.
Interestingly, the new peptide 1 presents a completely different
conformational behavior when it is compared with, i.e. the natural
derived tripeptide Z-Thr-Ala-Ala-CO2Me.15 The studies carried
out on the naturally derived molecule state that this molecule
takes mainly extended conformations in DMSO.
Finally, as in the solid state, the cyclobutane ring adopts mainly
(ca. 95% of the total trajectory time) one conformation in aqueous
solution, with <q > = -18.8◦. This result is in good agreement
Fig. 4 Sections of the 800 ms 2D NOESY spectra (400 MHz) in
H2O/D2O (9:1) at 25 ◦C and pH = 4.84 of compound 1.
˚
with the short distance (2.7 A) shown between the atoms Hb and
Hda.
determined distances, as well as the measured 3JNH,Ha, are shown
in Table 2. All these data were used as restraints in MD-tar
(MD with time-averaged restraints) simulations14 with the aim
of obtaining a distribution of conformers in aqueous solution able
to quantitatively reproduce the NMR data.
Conformational preferences of glycopeptide 2 in aqueous solution
The protocol above described for peptide 1 was also used
to quantitatively determine the conformers present in aqueous
solution for glucopeptide 2 (see Supporting Information and
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Fig. 5 fi/yi distributions obtained from the MD-tar simulations for compounds 1 (a) and 2 (b). The red dots correspond to the values of f and y
dihedrals observed in the solid state.
showed a clear preference for a helix-like conformation, in the
glucopeptide the extended conformers were the most populated
ones. As a consequence, the b-turns observed in the crystal
structure of compound 1 were present less than a 4% of the total
trajectory time in water solution for the glycopeptide 2. As can be
seen in Fig. 6b, the glucopeptide adopts an extended conformation
when compared with its parent peptide (Fig. 6a). Therefore, it can
be concluded that glycosylation of the peptide 1 results in a distinct
conformational change of the glycopeptide. This conformational
influence of glycosylation on the underlying peptide has been
frequently observed in natural glycopeptides.16
Concerning the glycosidic linkage, the values of the fs dihe-
dral angle are mainly determined by the exo-anomeric effect.17
Consequently, the glycosidic linkage adopts mostly the syn
conformation in aqueous solution [fs(O5s-C1s-O1s-Cb)= -60◦
for b-O-linkages], which corresponds to the minimum energy.
In the case of glucopeptide 2, we observe not only a strong-
medium NOE between Hb-H1s, which is representative of a syn
conformation of fs dihedral, but also a medium one between
Hb-H2s (Fig. 7a). These NOEs can never be explained without
assuming the existence of the anti-f population (the Hb-H2s
8
˚
distance for a syn conformation is longer than 4.5 A). The MD-
tar simulations suggest a population of ca. 20% for this high
energy conformation (Fig. 7). This feature is important since
although the existence of this higher energy conformation has
been experimentally confirmed for some oligosaccharides,18 to the
best of our knowledge this is the first time that this exceptionally
large percentage of anti-f conformation is experimentally (NOE)
observed in a glycopeptide.
Fig. 6 Calculated ensembles obtained from the MD-tar simulations
for compounds 1 (a) and 2 (b), showing the RMSDs for heavy atoms
superimposition. In the case of compound 1, the crystal structure is also
shown.
In order to gain insights into the structural changes induced by
the b-O-glycosylation on both the peptide backbone of 2 and the fs
dihedral angle, we decided to study the possible hydrogen bonds in
the glycopeptide. As a consequence, the relatively large proportion
of the anti-f conformers in aqueous solution could be stabilized by
the formation of intramolecular hydrogen bonds (Fig. 8). In fact,
Table 2). As can be seen in Fig. 5b, the cyclobutane residue adopts
almost exclusively the helix-like conformation (97% of the total
trajectory time). This result is similar to that observed for its parent
peptide. The major difference shown between compounds 1 and 2
was concerning Ala of residue 2. While in the former, this residue
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account that glycosylation of peptides, especially those containing
b-O-glycosidic linkages, results generally in induction of turn
conformations,16 the example shown in the present work is an
interesting contrast that emphasizes the use of non-natural amino
acids as a useful tool to obtain new compounds with novel
conformational behaviour.
Conclusions
Herein we report the first example of a glycopeptide containing
a non-natural amino acid residue. Our study reveals that the
c4Ser-Ala-Ala diamide peptide shows a conformation of two
consecutive b-turn type III structures in the solid state, the basic
structural element of a 310 helix. Remarkably, only the first b-turn
is partially present in solution. However, the b-O-glucosylation
notably modifies the conformation of the peptide backbone.
Indeed, none of the turns observed in the peptide remain in the
glucopeptide derivative. In this case, although the c4Ser residue
retains the helix-like conformations, the two alanine residues
prefer extended conformations. This result differs from previous
studies with glycopeptides containing natural amino acids. The
influence of the carbohydrate moiety on the peptide backbone
can be explained by means of the existence of two simultaneous
hydrogen bonds between endocyclic oxygen of the glucose and the
amidic protons H-N1 and H-N2 of the peptide. Moreover, the non-
natural residue favors the existence of high energy conformations
for the glycosidic linkage, such as the anti-f conformation.
Fig. 7 NOE build-up curve corresponding to Hb of compound 2 (a).
Schematic representation of the characteristic NOEs of syn- and anti-f
conformations in 2 (b). (c) fs/ys distribution obtained from the MD-tar
simulations for glucopeptide 2e (c).
Experimental
General synthetic procedures
Melting points are uncorrected. All manipulations with air-
sensitive reagents were carried out under a dry argon atmosphere
using standard Schlenk techniques. Solvents were purified accord-
ing to standard procedures. The chemical reagents were purchased
from Aldrich Chemical Co. Analytical TLC was performed using
Polychrom SI F254 plates. Column chromatography was performed
using Kieselgel 60 (230–400 mesh). Organic solutions were dried
over anhydrous Na2SO4 and, when necessary, concentrated under
reduced pressure using a rotary evaporator. NMR spectra were
recorded at 300 or 400 MHz (1H) and at 75 or 100 MHz (13C) and
signals are reported in ppm downfield from TMS. Microanalyses
were carried out on a CE Instruments EA-1110 analyser and are
in good agreement with the calculated values.
Synthesis of compound 4. A solution of 3 (1.00 g, 5.29 mmol) in
acetonitrile (40 mL) was treated with DIEA (4.4 mL, 26.4 mmol),
methylamine hydrochloride (714 mg, 10.60 mmol) and TBTU
(2.21 g, 6.88 mmol). The reaction mixture was stirred at 25 ◦C
for 10 h, the solvent was then removed and the mixture was
dissolved in ethyl acetate (40 mL). The organic layer was washed
with aqueous 1M HCl (20 mL) and aqueous 5% NaHCO3 (20 mL),
dried over Na2SO4, filtered, and evaporated to give a residue that
was purified by silica gel column chromatography, eluting with
dichloromethane/MeOH (15:1) to give 4 (730 mg), as a white
solid, in 68% yield. Mp 106–108 ◦C. [a]D25: -26.1 (c 1.18, CHCl3).
dH (400 MHz, CDCl3) 1.36 (d, 3H, J = 7.0 Hz), 1.44 (s, 9H), 2.81
(d, 3H, J = 4.7 Hz), 4.08–4.26 (m, 1H), 5.20 (d, 1H, J = 7.2 Hz),
6.36–6.57 (m, 1H). dC (100 MHz, CDCl3) 18.6, 26.2, 28.3, 50.0,
Fig. 8 Time series monitoring the fs dihedral angle and distances
N1 ◊ ◊ ◊ O5s and N2 ◊ ◊ ◊ O5s in the MD-tar simulation for compound 2.
there is a strong correlation between the anti-f conformation and
the existence of two simultaneous hydrogen bonds, one between
the N1 and O5s and another one between N2 and O5s.
These hydrogen bonds are also responsible for the absence,
in glycopeptide 2, of the first b-turn in the peptide backbone
observed in peptide 1, which explains the effect of the carbohydrate
moiety on the peptide conformation. Therefore, and taking into
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80.0, 155.5, 173.4. Anal. calcd for C9H18N2O3: C, 53.45; H, 8.97;
N, 13.85. Found: C, 53.61; H, 8.99; N, 13.80%.
1.78 (m, 1H), 1.83 (s, 3H), 2.05–2.18 (m, 1H), 2.41–2.52 (m, 1H),
2.56 (s, 3H), 4.05 (q, J = 7.1 Hz, 1H), 4.18 (q, J = 7.1 Hz, 1H),
4.26 (‘t’, J = 7.1 Hz, 1H). dH (400 MHz, H2O/D2O): d 7.49–7.57
(m, 1H, NH4), 7.67 (d, J = 6.2 Hz, 1H, NH3), 8.08 (d, J = 5.8 Hz,
1H, NH2), 8.69 (s, 1H, NH1). dC (100 MHz, D2O): 16.1, 16.4, 21.8,
23.7, 25.6, 25.9, 49.8, 50.2, 65.8, 70.8, 172.9, 174.5, 175.1, 175.3.
Anal. calcd for C14H24N4O5: C, 51.21; H, 7.37; N, 17.06. Found:
C, 51.01; H, 7.36; N, 17.02%.
Synthesis of compound 6. TFA (5 mL) was added to a solution
of 4 (226 mg, 1.12 mmol) in CH2Cl2 (10 mL) at 0 ◦C. The reaction
was maintained at 0 ◦C for 30 min, at 25 ◦C for 3 h, and then
concentrated to give derivative 5 (1.09 mmol). This compound
was then dissolved at 25 ◦C in CH3CN (15 mL) and treated with
3 (247 mg, 1.30 mmol), DIEA (0.9 mL, 5.44 mmol) and TBTU
(454 mg, 1.41 mmol), following the same protocol described for the
synthesis of derivative 4, to give compound 6 (238 mg), as a white
solid, in 80% yield. Mp 140–142 ◦C. [a]D25: -39.5 (c 1.07, CH3OH).
dH (400 MHz, CDCl3): d 1.37 (d, 3H, J = 4.3 Hz), 1.40 (d, 3H, J =
4.3 Hz), 1.46 (s, 9H), 2.80 (d, 3H, J = 4.8 Hz), 4.02–4.18 (m, 1H),
4.38–4.54 (m, 1H), 4.89–4.99 (m, 1H), 6.48 (br s, 1H), 6.58 (d, 1H,
J = 7.3 Hz). dC (100 MHz, CDCl3): d 18.0, 26.3, 28.3, 48.8, 50.8,
80.7, 155.8, 172.5. Anal. calcd for C12H23N3O4: C, 52.73; H, 8.48;
N, 15.37. Found: C, 52.75; H, 8.39; N, 15.40%.
Synthesis of compound 11. Silver triflate (60 mg, 0.25 mmol)
was added to a suspension of 1 (50 mg, 0.15 mmol) and powdered
˚
molecular sieves (4 A, 20 mg) in CH2Cl2 (4 mL), under an inert
atmosphere. The mixture was stirred at -30 ◦C and 2,3,4,6-tetra-
O-benzoyl-a-D-glucopyranosyl bromide (570 mg, 0.86 mmol) in
CH2Cl2 (4 mL) was then added. The mixture was stirred at this
temperature for 1 h and was then warmed to 25 ◦C and stirred for
additional 14 h. The crude was filtered, concentrated, and purified
by silica gel column chromatography, eluting with CH2Cl2/MeOH
(95:5), to give 11 (39 mg, 30% yield) as a colourless oil. [a]25: +11.4
(c 0.98, CH3OH). dH (400 MHz, CDCl3): d 1.22 (d, J =D7.3 Hz,
3H), 1.35 (d, J = 7.4 Hz, 3H), 1.51–1.62 (m, 1H), 1.83 (s, 3H),
2.10–2.19 (m, 1H), 2.22–2.33 (m, 1H), 2.42–2.54 (m, 1H), 2.70
(d, J = 4.6 Hz, 3H), 4.17–4.44 (m, 4H), 4.56 (dd, J1 = 12.3 Hz,
J2 = 5.3, 1H), 4.70 (dd, J1 = 12.4 Hz, J2 = 2.6, 1H), 5.07 (d, J =
7.8 Hz, 1H), 5.50–5.58 (m, 1H), 5.68 (t, J = 9.7 Hz, 1H), 5.93 (t,
J = 9.7 Hz, 1H), 6.73 (s, 1H), 6.81 (d, J = 4.7 Hz, 1H), 7.17–7.82
(m, 14H), 7.88–8.07 (m, 8H). dC (100 MHz, CDCl3): 16.5, 16.9,
22.6, 25.2, 26.0, 26.3, 49.2, 51.1, 62.9, 64.4, 69.3, 71.3, 72.4, 73.0,
77.9, 100.1, 128.4, 128.5, 128.5, 128.6, 128.7, 129.4, 129.6, 129.7,
129.8, 129.9, 133.4, 133.6, 133.7, 133.8, 165.2, 165.4, 165.7, 166.3,
171.1, 171.9, 172.3, 173.4. Anal. calcd for C48H50N4O14: C, 63.57;
H, 5.56; N, 6.18. Found: C, 63.70; H, 5.65; N, 6.10%.
Synthesis of compound 9. TFA (1 mL) was added to a solution
of 6 (52 mg, 0.19 mmol) in CH2Cl2 (2 mL) at 0 ◦C. The reaction
was maintained at 0 ◦C for 30 min, at 25 ◦C for 3 h, and then
concentrated to give derivative 7 (0.18 mmol). This compound
was then dissolved at 25 ◦C in DMF (3 mL) and treated with
racemic cyclobutane derivative 8 (48 mg, 0.18 mmol), DIEA
(0.2 mL, 0.90 mmol) and TBTU (75 mg, 0.23 mmol), following
a similar protocol to described for the synthesis of derivative
4, to give a mixture of compounds 9 and 10. The mixture
was purified by silica gel column chromatography, eluting with
dichloromethane/MeOH (15:1) to give pure compounds 9 (30 mg,
40%) and 10 (27 mg, 36%) as white solids. Compound 9: Mp 173–
◦
175 C. [a]2D5: +21.2 (c 1.22, CH3OH). dH (400 MHz, CDCl3): d
1.27 (d, 3H, J = 7.4 Hz), 1.43 (d, 3H, J = 7.3 Hz), 1.69 (‘q’, 1H,
J = 11.0 Hz), 1.94 (s, 3H), 1.97–2.13 (m, 1H), 2.14–2.32 (m, 1H),
2.49 (‘t’, 1H, J = 10.6 Hz), 2.74 (d, 3H, J = 4.6 Hz), 4.13–4.30 (m,
2H), 4.39 (‘t’, 1H, J = 7.7 Hz), 4.52 (d, 1H, J = 11.0 Hz), 4.63 (d,
1H, J = 11.0 Hz), 7.03–7.15 (m, 1H), 7.31 (br s, 5H), 7.71 (d, 1H,
J = 8.2 Hz), 8.01 (s, 1H), 8.56 (d, 1H, J = 5.2 Hz). dC (100 MHz,
CDCl3): d 16.8, 17.2, 22.5, 24.5, 24.7, 26.2, 49.7, 51.0, 63.7, 71.6,
79.6, 127.8, 128.1, 128.4, 137.0, 171.4, 173.0, 173.2, 173.8. Anal.
calcd for C21H30N4O5: C, 60.27; H, 7.23; N, 13.39. Found: C, 59.98;
H, 7.21; N, 13.37%. Compound 10: dH (400 MHz, CDCl3): d 1.06
(d, 3H, J = 7.3 Hz), 1.44 (d, 3H, J = 7.3 Hz), 1.45–1.51 (m,
1H), 1.72–1.82 (m, 1H), 2.05 (s, 3H), 2.17–2.25 (m, 1H), 2.70–2.75
(m, 1H), 2.76 (d, 3H, J = 4.6 Hz), 4.08–4.23 (m, 2H), 4.29–4.39
(m, 1H), 4.67 (d, 1H, J = 11.9 Hz), 4.74 (d, 1H, J = 11.9 Hz),
7.02–7.10 (m, 1H), 7.22 (d, 1H, J = 8.2 Hz), 7.33 (br s, 5H), 8.31
(d, 1H, J = 4.8 Hz). dC (100 MHz, CDCl3): d 17.0, 17.1, 22.3,
23.2, 25.8, 26.3, 48.9, 51.4, 66.1, 73.0, 80.1, 128.2, 128.3, 128.6,
137.0, 171.3, 171.4, 172.3, 173.1. Anal. calcd for C21H30N4O5:
C, 60.27; H, 7.23; N, 13.39. Found: C, 60.08; H, 7.29; N,
13.35%.
Synthesis of compound 2. A solution of 11 (30 mg, 0.04 mmol)
in MeOH (5 mL) was treated with MeONa/MeOH (0.5M) to
pH = 9. After stirring for 3 h at 25 ◦C, the mixture was neutralized
with Dowex 50-X8, filtered, and concentrated. Purification of the
residue with C18 reverse-phase sep-pak cartridge gave 15 mg of
2, as a colourless oil in 85% yield. [a]2D5: -12.5 (c 1.05, H2O). dH
(400 MHz, D2O): d 1.28–1.35 (m, 6H), 1.57 (dd, J1 = 20.8 Hz,
J2 = 11.2 Hz, 1H), 1.95 (s, 3H), 1.95–2.06 (m, 1H), 2.25 (dd, J1 =
19.0, Hz, J2 = 9.8 Hz, 1H), 2.54–2.63 (m, 1H), 2.67 (s, 3H), 3.20–
3.32 (m, 2H), 3.35–3.44 (m, 2H), 3.59–3.67 (m, 1H), 3.84–3.90 (m,
1H), 4.17 (q, J = 7.3 Hz, 1H), 4.24 (q, J = 7.2 Hz, 1H), 4.53 (d,
1
J = 7.9 Hz, 1H), 4.61 (t, J = 8.8 Hz, 1H). H NMR (400 MHz,
H2O/D2O): d 7.52–7.59 (m, 1H, NH4), 7.72 (d, J = 6.7 Hz, 1H,
NH3), 8.37 (d, J = 5.6 Hz, 1H, NH2), 8.87 (s, 1H, NH1). dC
(100 MHz, D2O): d 15.8, 16.4, 21.8, 24.3, 24.6, 25.9, 49.8, 50.6,
60.9, 65.1, 69.7, 72.3, 75.0, 75.7, 76.3, 100.5, 172.8, 174.2, 175.3,
175.3. Anal. calcd for C20H34N4O10: C, 48.97; H, 6.99; N, 11.42.
Found: C, 48.88; H, 6.93; N, 11.38%.
2D NMR experiments
Synthesis of compound 1. A solution of compound 9 (50 mg,
0.12 mmol) in MeOH (3 mL) was hydrogenolyzed, using 10 mg
of 10% Pd/C as a catalyst, at 25 ◦C for 4 h. The catalyst and
solvent were removed and further purification of the residue with
C18 reverse-phase sep-pak cartridge gave 1 (35 mg), as a white
solid in 90% yield. Mp 129–131 ◦C. [a]D25: +17.4 (c 1.03, H2O). dH
(400 MHz, D2O): d 1.10–1.27 (m, 6H), 1.31–1.47 (m, 1H), 1.63–
NMR experiments were recorded on a Bruker Avance 400 spec-
trometer at 298 K. Magnitude-mode ge-2D COSY spectra were
recorded with gradients and using the cosygpqf pulse program
with 90 degree pulse width. Phase-sensitive ge-2D HSQC spectra
were recorded using z-filter and selection before t1 removing the
decoupling during acquisition by use of invigpndph pulse program
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with CNST2 (JHC) = 145. 2D NOESY experiments were made
using phase-sensitive ge-2D NOESY for CDCl3 spectra and phase-
sensitive ge-2D NOESY with WATERGATE for H2O/D2O (9:1)
spectra. Selective ge-1D NOESY experiments were carried out
using the 1D-DPFGE NOE pulse sequence. NOEs intensities
were normalized with respect to the diagonal peak at zero mixing
time. Experimental NOEs were fitted to a double exponential
function, f(t) = p0(e-p1t)(1 - e-p2t) with p0, p1 and p2 being adjustable
parameters.13 The initial slope was determined from the first
derivative at time t = 0, f¢(0) = p0p2. From the initial slopes,
interproton distances were obtained by employing the isolated
spin pair approximation.
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MD simulations: MD-tar simulations
MD-tar simulations were performed with AMBER19 6.0 and
AMBER94 force field,20 which was implemented with GLYCAM
0421 and the General Amber Force Field (GAFF)22 parameters to
accurately simulate the conformational behavior of the carbohy-
drate moiety and the cyclobutane ring, respectively. NOE-derived
distances were included as time-averaged distance constraints, and
scalar coupling constants J as time-averaged coupling constraints.
A <r-6>-1/6 average was used for the distances and a linear average
was used for the coupling constants. Final trajectories were run
using an exponential decay constant of 8000 ps and a simulation
length of 80 ns with a dielectric constant e = 80.
X-Ray crystallography
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The diffraction data for the crystalline compounds were collected
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˚
using Mo Ka radiation (l = 0.71073 A). The structures were
solved by direct methods and refined by a full-matrix least-squares
procedure with the SHELXL suite of programs.23 Hydrogen atoms
were located from mixed methods (electron-density maps and
theoretical positions). Further details on the crystal structures of
the compound 1 are available on the CIF data of the Supporting
Information.‡
7 P. Van den Steen, P. M. Rudd, R. A. Dwek and G. Opdenakker, Crit.
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‡ Crystal data for compound 1: C14H28N4O7, Mw = 364.40, colorless prism
of 0.32 ¥ 0.25 ¥ 0.20 mm, T = 293(2) K, orthorhombic, space group
11 F. Corzana, J. H. Busto, G. Jime´nez-Ose´s, J. L. Asensio, J. Jime´nez-
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˚
˚
˚
˚
P212121, Z = 4, a = 7.8140(5) A, b = 14.5374(8) A, c = 16.0994(10) A,
3
-3
˚
V = 1828.72(19) A , dcalc = 1.309 g cm , F(000) = 768, l = 0.71073 A
(Mo, Ka), m = 0.105 mm-1, Nonius kappa CCD diffractometer, q range
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0.1125, wR2 = 0.1761 all data), goodness of fit = 1.019, residual electron
-3
˚
density between 0.238 and -0.336 e A . Hydrogen atoms were located
from mixed methods (electron-density maps and theoretical positions).
CCDC 716958.
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