An efficient route towards a new branched tetrahydrofurane δ-sugar amino acid from a pyrolysis product of cellulose

Article abstract of DOI:10.1007/s00726-010-0690-4

(1R,5S)-1-Hydroxy-3,6-dioxa-bicyclo[3.2.1]octan-2-one, is a bicyclic lactone obtained in gram-scale by catalytic pyrolysis of the renewable source cellulose. Now it has been used as a chiral building block in the preparation of the new δ-sugar amino acid,

Full text of DOI:10.1007/s00726-010-0690-4

Amino Acids (2011) 40:633–640
DOI 10.1007/s00726-010-0690-4
ORIGINAL ARTICLE
An efficient route towards a new branched tetrahydrofurane
d-sugar amino acid from a pyrolysis product of cellulose
•
Andrea Defant Ines Mancini Cristian Torri
•
•
•
Danilo Malferrari Daniele Fabbri
Received: 8 June 2010 / Accepted: 6 July 2010 / Published online: 24 July 2010
ꢀ Springer-Verlag 2010
Abstract (1R,5S)-1-Hydroxy-3,6-dioxa-bicyclo[3.2.1]
octan-2-one, is a bicyclic lactone obtained in gram-scale by
catalytic pyrolysis of the renewable source cellulose. Now
it has been used as a chiral building block in the prepara-
tion of the new d-sugar amino acid, (3R,5S)-5-(amino-
ethyl)-3-hydroxytetrahydrofurane-3-carboxylic acid, by an
efficient synthesis in five steps with a 67% overall yield.
The structure of this tetrahydrofurane amino acid, isolated
in protonated form, was assigned by extensive mono- and
Introduction
Sugar amino acids (SAAs) are carbohydrate derivatives
widely used as building blocks, allowing to mimic nature in
organic synthesis. In recent years they have been involved in
the development of glycomimetics and peptidomimetics
(Schweizer 2002; Chakraborty et al. 2004), with application
also in drug design (Gruner et al. 2002). The advantages are
linked to their high density of functional groups present with
a well-defined stereochemistry, making them peculiar chiral
synthones able to introduce the suitable hydrophobic or
hydrophilic substitution patterns. A relevant example is
given by the potential of creating combinatorial amide
libraries of compounds in order to emulate the diversity of
biomolecules (Edwards et al. 2004). In addition, the incor-
poration at a specific location in a peptide sequence can
induce a conformational restriction responsible for defined
folding in peptides (Lohof et al. 2000).
1
bidimensional H- and 13C-NMR analysis and mass spec-
trometry, including measurements by electrospray and
matrix-assisted laser desorption ionization techniques, the
latter one for high-resolution experiments. This amino acid
is an isoster of dipeptide glycine-alanine (H-Gly-Ala-OH),
with a potential use in the access of new peptidomimetics
with conformationally restricted structures due to the
presence of tetrahydrofurane ring. As a preliminary study
in order to disclose this effect, density functional theory
calculation performed in water using polar continuum model
was applied to the new amino acid and H-Gly-Ala-OH
dipeptide, so that to evaluate and compare the relative
torsional angles for the energy-minimized structures.
The structures of sugar amino acids include variations on
the relative position of amino and carboxylic groups (a, b,…)
and on the oxygen heterocycle (with oxirane, oxetane, tetra-
hydrofurane and tetrahydropyrane as the major types)
(Risseeuw et al. 2007). In particular, d-SAAs are isosters of
scissible dipeptide bonds (Trabocchi et al. 2008) and for this
reason they have been thoroughly investigated in drug design
owing to the expected greater stability towards enzymatic
proteolysis with respect to natural peptides. Moreover, the
d-tetrahydrofurane series is characterized by a relative
rigidity able to introduce structural constraints and making
these SAAs attractive candidates as scaffolds in peptidomi-
metics, as well as by a propensity to induce conformational
preferences in relatively short sequences (Smith et al. 1998;
Simone et al. 2008). Most of these compounds consist of a
linear chain of carbon atoms in accordance with their origin
from simple carbohydrates (Risseeuw et al. 2007). Starting
Keywords Sugar amino acids ꢀ Chiral building block ꢀ
Dipeptide isoster ꢀ DFT calculation ꢀ Hydroxylactone ꢀ
Cellulose pyrolysis
A. Defant ꢀ I. Mancini (&)
Bioorganic Chemistry Laboratory, Department of Physics,
University of Trento, Povo-Trento, Italy
e-mail: mancini@science.unitn.it
C. Torri ꢀ D. Malferrari ꢀ D. Fabbri
Laboratory of Chemistry, Interdepartmental Research
Centre for Environmental Sciences (CIRSA),
University of Bologna, Ravenna, Italy
123

634
A. Defant et al.
Fig. 1 Structure of the reported azido ester 1 as scaffold of the
branched d-tetrahydrofurane SAA 2
Fig. 2 Structures of the cellulose pyrolytic product 3, used as the
chiral building block for the synthesis of the new branched
d-tetrahydrofurane sugar amino acid 4
from a protected ribonolactone affordable from D-ribose the
methyl (3R,4R,5S)-5-azidomethyl 3,4-dihydroxy-tetra-
hydrofuran-3-carboxylate (1; Fig. 1) and the corresponding
epimer at 5 position were recently synthesized as the first
d-SAA scaffolds containing a branched carbon chain. The
sequence included a long series of steps among which the
reduction of the lactone, a function required in order to insert
the carbon branching by means of an aldol condensation with
formaldehyde. The lactone was successively regenerated by
oxidation followed by cyclisation of the ribonolactone
derivative affording the tetrahydrofurane ring (Simone et al.
2008). Previously the same authors had reported that azido
ester 1 was unable to give the methyl ester of amino acid 2
(Fig. 1) by palladium catalytic hydrogenation, due to the
spontaneous cyclization to bicylic lactame (Simone et al.
2005).
production of 3 were significantly increased in the presence
of commercial nanopowder metal oxides, with evidence that
nano-sized feature of aluminium titanate (Al Ti) oxides
resulted a determinant factor for its activity (Fabbri et al.
2007a). A preparative process was defined to give bio-oils
enriched of hydroxylactone 3. It proved that the behaviour of
the Al Ti nanopowder catalyst, which could be recycled in its
use quite effectively, resulted uniquely in promoting both the
formation of the main anhydromonosaccharide product 3 in
gram scale and in favoring its isolation from the pyrolytic
liquid (Fabbri et al. 2007b). Later, the efficiency of the cat-
alyst was verified also in the analytical pyrolysis of a series of
carbohydrates, with levoglucosan and its 4-O-acetyl deriv-
ative producing 3 in yields comparable to those of starch and
cellulose (Torri et al. 2009a). More recently, mesoporous
materials doped with different metals were also investigated,
with the best production of 3 was achieved with Sn-MCM-41
system (Torri et al. 2009b). These conditions open the route
to its potential use as chiral building block, never scrutinized
before probably due to the low production of 3. Recent
applications were done obtaining a suitable amide by the
effective and eco-friendly microwave assisted methodology
(Fabbri et al. 2007b), and later with the application as a
monomer in the synthesis of polyesters by ring-opening
polymerisation with L-lactide (Dobrzynski et al. 2009).
We report here on a simple and efficient route to the
novel amino acid 4 (Fig. 2), showing the methyleneamino
and the carboxylic acid components in 2,4-position of the
tetrahydrofurane ring with a cis relative configuration. The
synthesis employs 3 as chiral reagent, which enables to
overcome the need of oxidation/reduction steps of the lac-
tone moiety to insert the ramification and its ring closure
into tetrahydrofurane. It is the first production of a d-tetra-
hydrofurane SAA containing a branched carbon chain
obtained in a free form, after the report of the first branched
d-SAA scaffold derived from D-ribose (Simone et al. 2008).
Biomass treatment includes the conversion to biofuels
able to meet the energy demand under investigation as
alternative fuel source with environmental respect, but also
to produce chemicals, including building blocks to be
employed in the synthesis of useful compounds. Cellulose, a
polysaccharide found in plant materials, is the most abun-
dant biosynthesized organic substance on earth, and there-
fore represents a key biomass component. The relevance and
interest of this process is linked to the use of renewable
source of energy, due the employment of pure cellulose and
of cellulosic materials of recovery (i.e. wood). Cellulose can
be converted into a large series of compounds by several
processes and among them pyrolysis, which is a one-step
thermal degradation under non-oxidative conditions leading
to a liquid fraction (bio-oil). It contains low molecular
weight compounds, including polyfunctional dehydrated C6
monomers named anhydro-monosaccharides, peculiar for
retaining the structural record of the original stereochemis-
try in glucose units of cellulose and thus existing in pure
enantiomeric form, which accounts for their employment as
a chiral building block.
Hydroxylactone 3 (Fig. 2), namely (1R,5S)-1-hydroxy-
3,6-dioxa-bicyclo[3.2.1]octan-2-one, is an anhydro mono-
saccharide first isolated in a 1 mg amount from Lewis
acid-catalysed pyrolysis of cellulose (Furneaux et al. 1988).
Recently, some of us reported an analytical study on the
effect of different catalysts on the pyrolytic behaviour of
cellulose. In particular, it was shown that the levels of
Experimental
Materials and methods
The reagents and solvents were used in chemical reactions
without purification. High purity microgranular cellulose
123

An efficient route towards a new branched tetrahydrofurane d-sugar amino acid
635
was from Fluka. Mesophase of the 41 type doped with tin
(Sn-MCM-41) was prepared as previously described (Torri
et al. 2009b). All evaporations were carried out at room
temperature at reduced pressure. The reaction yields were
calculated on the products after chromatographic purifica-
tion. Thin layer chromatography (TLC) was carried out on
Merck Kieselgel 60 PF254 and flash-chromatography (FC)
on Merck silica gel 60 (15–25 lm), preparative thin layer
chromatography was realized on 20 9 20 cm Merck
Kieselgel 60 F254 0.5 mm plates. Infrared spectra were
recorded by using a FT-IR Tensor 27 Bruker spectrometer
(Attenuated Transmitter Reflection, ATR configuration)
at 1 cm-1 resolution in the absorption region Dm* 4,000–
1,000 cm–1. A thin solid layer is obtained by evaporation
of methanol solution of the sample. The instrument was
purged with a constant dry air flux and clean ATR crystal
as background was used. Spectra processing was made
using Opus software package. Melting points were deter-
mined on Reichert Thermovapor microscope and the data
are uncorrected. Polarimetric data were obtained with a
Bellingham & Stanley Limited ADP 440 apparatus,
reporting [a]D in dm-1 deg ml g-1. NMR spectra were
recorded on a Bruker-Avance 400 spectrometer by using
a 5 mm BBI probe with 90ꢁ proton pulse length of 8 ls at
directly applied onto the stainless-steel spectrometer plate
as 1 ll droplets, followed by the addition of 1 ll of DHB-
matrix solution (0.5 M of 2,5-DHB in methanol). Every
mass spectrum represents the average of about 100 single
laser shoots. Calibration for high resolution experiments
was performed on DHB peaks at m/z 177 ([M ? Na]?),
154 ([M]?ꢀ) and 137([M ? H–H2O]?).
Quantum chemical calculations were performed on a
Pentium IV/3.6 GHz personal computer using the Gaussian
03W revision E.01 package program set (Frisch et al.
2004). Restricted DFT was used, applied for geometry
optimization in water using Polar Continuum Model
(PCM) and invoking gradient geometry optimisation. The
basis set of choice resulted 6–31 G (d) for all the atoms.
The gradient-corrected DFT with the three-parameter
hybrid functional (B3) (Becke 1993) for the exchange part
and the Lee–Yang–Parr (LYP) correlation function (Lee
et al. 1988) were utilized.
Production of (1R,5S)-1-hydroxy-3,6-dioxa-bicyclo[3.2.1]
octan-2-one (3) from cellulose Cellulose (10 g) was
mixed with 10 g of mesoporous phase Sn-MCM-41, syn-
thesized as reported (Torri et al. 2009b). Four batches were
pyrolysed at 500ꢁC for 5 min under nitrogen. The bio-oil
(around 50% yield, containing 12% of 3) was obtained by
trapping the evolved products with cold acetonitrile. Col-
umn chromatography on silica gel eluting with 9:1 cyclo-
hexane/ethyl acetate and then pentane/dichloromethane
gradient elution, following vacuum distillation and filtra-
tion over charcoal furnished 3 with 99% purity by GC
analysis and 65% recovery from bio-oil, showing super-
imposable NMR and MS data to the ones previously
reported (Fabbri et al. 2007b).
1
a transmission power of 0 db; H at 400 MHz and 13C at
100 MHz in CDCl3 (by previous treatment on basic alu-
mina to avoid acidic traces), d values in ppm, in CDCl3
relative to the solvent residual signals dH = 7.25 and
dC = 77.00 ppm, or in D2O relative to dH = 4.70, J values
1
in Hz. Structural assignments are from H,1H-COSY, het-
eronuclear single quantum correlation (HSQC) and heter-
onuclear multiple bond correlation (HMBC) experiments.
Electrospray ionization (ESI)-MS mass spectra were taken
with a Bruker Esquire-LC spectrometer with an electro-
spray ion source used in positive or negative ion mode by
direct infusion of a methanolic solution of the sample,
under the following conditions: source temperature 300ꢁC,
drying gas N2, 4 l/min, positive ion mode, ISV 4 kV, OV
38.3 V, scan range 100–1,000 m/z. Matrix assisted laser
desorption ionization-time of flight (MALDI-TOF) mea-
surements were performed on Bruker Daltonics Ultraflex
MALDI-TOF-TOF mass spectrometer equipped with a
reflectron unit. The acceleration voltage was set at 20 kV.
For desorption of the components, a nitrogen laser beam
(k = 337 nm) was focused on the template. The laser
power level was adjusted to obtain high signal-to-noise
ratios, while ensuring minimal fragmentation of the parent
ions. All measurements were carried out in the delayed
extraction mode, allowing the determination of monoiso-
topic mass values (m/z; mass-to-charge ratio). After crys-
tallization at ambient conditions, positive ion spectra were
acquired in the reflectron mode, giving mainly singly
protonated molecular ions ([M ? H]?). Samples were
(3R,5S)-Methyl 3-hydroxy-5-(hydroxymethyl)tetrahydro-
furan-3-carboxylate (5) To a solution of 3 (288 mg,
2.0 mmol.) in anhydrous methanol (5 ml) triethylamine
(0.28 ml, 2.0 mmol) was added. The resulted mixture was
stirred for 12 h at room temperature. After evaporation of
the solvent in vacuo, the residue was subjected to flash
chromatography on silica gel with dichloromethane/meth-
anol gradient elution, to give the ester 5 as viscous clear oil
(334 mg, 95%).
[a]2D0 = ?10.2ꢁ (c 0.5 in MeOH). Optical activity, NMR
and MS data are in accordance with those reported
(Furneaux et al. 1988; Fabbri et al. 2007b).
(3R,5S)-Methyl 3-hydroxy-5-(tosyloxymethyl)tetrahydrofu-
ran-3-carboxylate (6) To a solution of 5 (334 mg,
1.90 mmol) in 3 ml of anhydrous pyridine at 0ꢁC was
added slowly a solution of tosyl chloride (400 mg,
2.1 mmol) in anhydrous pyridine (2 ml). The solution was
stirred at room temperature for 18 h and then poured in a
beaker containing ice, the mixture was partitioned between
123

636
A. Defant et al.
water and dichloromethane, the organic phases were sep-
arated (93), combined and washed in sequence with 0.5 M
aq. HCl, saturated solution of NaHCO3 and water, dried on
anhydrous Na2SO4 and concentrated in vacuo. The residue
was subjected to flash-chromatography on silica gel with
hexane/EtOAc gradient elution, to give tosylate 6 as
a viscous yellowish oil (552 mg, 88%).
of Eu(tfc)3 in CDCl3 was added by 1–30 ll portions,
observing proton shifts without splitting of the signals.
After the addition of 0.30 mol equiv of shift-reagent Dd
for the signals of the protons were: 2.79 (OH), 0.79 (H-5),
0.75 and 0.73 (2H-4), 0.67 and 0.65 (2H-2), 0.59 and 0.37
(2H-6), 0.42 ppm (OMe).
(3R,5S)-5-(Azidomethyl)-3-hydroxytetrahydrofuran-3-car-
boxylic acid (8) To a solution of 7 (146 mg, 0.725 mmol)
in THF (5 ml) was added NaOH 0.5 M (1.5 ml,
0.363 mmol) and stirred for 3 h. The mixture was passed
through a column of Amberlyst 15 (H-form), washing with
acetonitrile and water. The eluate, having a pH value of 4,
was evaporated in vacuo to give the acid 8 (135 mg, 99%),
pure enough to be used in the next step.
[a]2D0 = ?19.7 (c 0.04, CH2Cl2). 1H NMR (CDCl3):
d 7.80 (2H, d, J = 8.4 Hz, H-20 and H-60), 7.33 (2H, d,
J = 8.4 Hz, H-30 and H-50), 4.39 (1H, m, H-5), 4.15 (1H, dd,
J = 10.6, 3.8 Hz, H-6), 4.10 (1H, dd, J = 10.6, 4.8 Hz,
H-6), 4.09 (1H, d, J = 9.9 Hz, H-2), 3.81 (3H, s, OMe), 3.77
(1H, d, J = 9.9 Hz, H-2), 3.38 (1H, br. s, OH), 2.44 (3H, s,
Me-40), 2.23 (1H, dd, J = 12.8, 9.9 Hz, H-4), 2.09 (1 H, dd,
J = 12.8, 6.0 Hz, H-4). 13C NMR (CDCl3): d 174.3 (COO),
146.7 (C-10), 144.9 (C-40), 129.8 (C-30and C-50), 127.9 (C-20
and C-40), 81.0 (C-3), 78.2 (C-5), 76.6 (C-2), 70.1 (C-6),
53.4 (OMe), 41.5 (C-4), 21.6 (q, Me-40). Significant HMBC
correlations: d 7.80 ppm (H-20 and H-60) with C-10; 7.33
(H-30 and H-50) with Me-40; 4.15 (H-6) with C-4, 4.09 (H-2)
with COO, 3.81 (OMe) with C-3 and COO, 3.77 (H-2) with
COO, 2.09 (H-4) with C-3 and C-5. ESIMS (positive mode):
m/z 353 ([M ? Na]?), 331 ([M ? H]?); ESI–MS/MS
(331): 159([M ? H - TsO]?).
White powder. [a]2D0 = ?87.4 (c 0.2, EtOH). H NMR
1
(CDCl3): d 4.45 (1H, m, H-5), 4.30 (1H, d, J = 9.6 Hz,
H-2), 3.90 (1H, d, J = 9.6 Hz, H-2), 3.85 (1H, s, HO-3),
3.55 (1H, dd, J = 12.8, 3.5 Hz, H-6), 3.36 (1H, dd, J =
12.8, 5.6 Hz, H-6), 2.37 (1H, dd, J = 12.9, 9.8 Hz, H-4),
2.16 (1H, dd, J = 12.9, 5.8 Hz, H-4).13C NMR (CDCl3):
d 177.5 (COOH), 83.3 (C-3), 78.8 (C-2), 78.3 (C-5), 53.1
(C-6), 42.2 (C-4). Significant HMBC correlations:
d 4.30 ppm (H-2) with C-4, C-5 and COO; dH 3.90
(H-2) with C-4, C-5 and COO; 3.55 and 3.36 (2H-6) with
C-4 and C-5; 2.37 (H-4) with C-2, C-6 and COO. ESIMS
(negative mode): m/z 186 ([M–H]-).
(3R,5S)-Methyl 5-(azidomethyl)-3-hydroxytetrahydrofuran-
3-carboxylate (7) A mixture of tosylate 6 (552 mg,
1.67 mmol), sodium azide (163 mg, 2.5 mmol) and
Aliquatꢂ 336 (101 mg, 0.25 mmol) in anhydrous acetonitrile
(20 ml) was refluxed for 17 h. After cooling and filtration,
the solvent was removed in vacuo and the residue was
subjected to flash-chromatography on silica gel with
hexane/EtOAc gradient elution, to give the azide 7 as
viscous clear oil (292 mg, 87%).
(1R,5S)-1-hydroxy-6-oxa-3-azabicyclo[3.2.1]octan-2-one(9)
A solution of 7 (73 mg, 0.36 mmol) in 5 ml of ethanol was
hydrogenated for 3 h at room temperature and atmospheric
pressure in presence of 10% Pd/C. After filtration, the
solvent was evaporated in vacuo to obtain compound 9
(38 mg, 73%).
1H NMR (CDCl3): d 5.50 (1H, br. s, NH), 4.56 (1H, br.
s, H-5), 4.00 (1H, d, J = 8.0 Hz, H-2), 3.73 (1H, d,
J = 8.0 Hz, H-2), 3.46 (1H, d, J = 12.6 Hz, H-6), 3.36
(1H, d, J = 12.6, H-6), 2.50 (1H, dd, J = 11.9, 5.7 Hz,
H-4), 2.06 (1H, br. d, J = 11.8 Hz, H-4).13C NMR
(CDCl3): d 175.0 (CONH), 85.0 (C-3), 74.5 (C-5), 73.3
(C-2), 45.2 (C-6), 54.0 (C-4). Significant HMBC correla-
tions: d 4.00 and 3.73 ppm (2H-2) with C-5 and C=O; 2.06
(H-4) with C=O. ESIMS (positive mode): m/z 144
([M ? H]?), 166 ([M ? Na]?).
[a]2D0 = ?90.3 (c 0.06, MeOH). 1H NMR (CDCl3):
d 4.40 (1H, m, H-5), 4.23 (1H, d, J = 9.5 Hz, H-2), 3.85
(3H, s, OMe), 3.83 (1H, d, J = 9.5 Hz, H-2), 3.54 (1H, dd,
J = 12.8, 3.6 Hz, H-6), 3.40 (1H, br. s, OH), 3.31 (1H, dd,
J = 12.8, 5.6 Hz, H-6), 2.28 (1H, dd, J = 12.8, 9.9 Hz,
H-4), 2.08 (1H, dd, J = 12.8, 5.4 Hz, H-4).13C NMR
(CDCl3): d 174.4 (COO), 77.8 (C-5), 81.6 (C-3), 78.2
(C-2), 53.3 (OMe), 53.2 (C-6), 53.3 (OMe), 42.3 (C-4).
Significant HMBC correlations: d 3.85 ppm (H-2) with
C-4; 4.23 (H-2) with C-4, C-5 and C-4 COO, 3.85 (OMe)
with COO, 4.40 (H-5) with C-2, C-6 and C-4, 3.54 (H-6)
with C-4 and C-5; 3.31 (H-6) with C-2 and C-4; 2.28 (H-4)
with C-6, C-2 and COO; 2.08 (H-4) with C-2. Significant
HMBC correlations: d 4.23 ppm (H-2) with C-5 and COO;
3.83 (H-2) with C-3, C-5 and COO; 3.54 (H-6) with C-4,
C-5; 2.28 (H-4) with C-2, C-6,COO; 2.08 (H-4) with C-3.
ESIMS (positive mode): m/z 224 ([M ? Na]?).
((2S,4R)-4carboxy-4-hydroxytetrahydrofuran-2-yl) methana-
minium salt A solution of 8 (135 mg, 0.72 mmol) in
10 ml of ethanol was hydrogenated for 3 h at room tem-
perature and atmospheric pressure in presence of 10% Pd/
C. After filtration, the solvent was evaporated in vacuo to
obtain 4, as protonated form due to the acid condition of
the starting reagent (110 mg, 95%).
White crystalline solid, m.p. 192–194ꢁC. [a]2D0 = ?24.3
(c 0.15, H2O). IR mmax: 2,910 (v br) 1,716 (vs), 1,591 (s),
Chiral shift-reagent study with 7 To compound 7
(2.0 mg, 0.01 mmol) in 0.5 ml of CDCl3 a 0.11 M solution
123

An efficient route towards a new branched tetrahydrofurane d-sugar amino acid
637
1,502, 1,394, 1,225 (s), 1,134, 1,024 (vs), 812 and
1
(3R, 5S) stereochemistry. By conversion of the primary
hydroxyl group through the reaction with 1.2 molar
equivalents of p-toluenesulfonyl chloride in pyridine by
stirring at room temperature overnight, tosylate 6 was
obtained in 88% yield. The sterically HO-3 did not require
time demanding protection/deprotection steps, but it is
noteworthy that an excess of tosyl chloride was able to
produce also the ditosylate derivative by conversion of the
tertiary hydroxyl function.
710 cm-1. H NMR (D2O): d 4.40 (1H, m, H-5), 4.11
(1H, d, J = 9.8 Hz, H-2), 3.80 (1H, d, J = 9.8 Hz, H-2),
3.17 (1H, dd, J = 13.0, 3.1 Hz, H-6), 3.07 (1H, dd,
J = 13.0, 8.7 Hz, H-6), 2.20 (2H, m, H-4).13C NMR
(D2O): d 176.8 (COOH), 81.4 (C-3), 76.8 (C-2), 75.1 (C-5),
41.5 (C-4), 41.3 (C-6). Significant HMBC correlations:
d 4.40 ppm (H-5) with C-2; 4.11 and 3.80 (2H-2) with
C-3, C-4, C-5 and COO; 3.17 and 3.06 (2H-6) with C-4,
C-5; 2.20 (2H-4) with C-3, C-6 and COO. ESIMS (posi-
tive mode): m/z 162 ([M ? H]?); ESI–MS/MS (162):
145 ([M ? H - NH3]?), 144 ([M ? H - H2O]?), 117
([M ? H - CO2]?), 116 ([M ? H - HCOOH]?), 99
([C5H7O2]?). HR-MALDI-TOF/TOF MS (positive mode):
m/z 162.0751 0.005, calculated for C6H12NO4
([M ? H]?) 162.0766.
The azide intermediate 7 was achieved by nucleophilic
substitution of tosylate 6 with sodium azide in a hetero-
geneous system containing methyltrioctylammonium
chloride (Aliquatꢂ 336). Although the use of this catalyst is
generally reported in water (Balo et al. 1998), in our case
acetonitrile resulted a better solvent giving 85% yield,
whereas in water we observed partial hydrolysis of the
tosylate group. Otherwise the standard method involving
the use of sodium azide in dimethylformamide (DMF) as
solvent, applied to tosylate 6 furnished a very low yield of
azide 7, due to the difficult isolation of the water-soluble
product. The conversion of azido ester 7 to amino acid 4
involved a first hydrolysis by aqueous sodium hydroxide in
tetrahydrofurane (THF) at room temperature for 3 h pro-
viding pure azido acid 8 in quantitative amount, which was
later subjected to Pd/C catalysed hydrogenation in ethanol
for 3 h with a 95% yield. Otherwise, the direct reduction to
amine of the azido function in compound 7 did not work,
but intramolecular cyclization to lactame 9 occurred, in
line with the compound 1 giving a corresponding lactame
(Simone et al. 2005), whose structure was firmly estab-
lished by X-ray crystallographic analysis (Punzo et al.
2004).
Results and discussion
Synthesis of sugar amino acid 4
Hydroxylactone 3, here obtained in higher yields by a
modified procedure involving Sn-MCM-41 system
(‘‘Experimental’’), was converted into the new amino acid
4 according to the sequence illustrated in Scheme 1. All the
products were purified by silica gel chromatography and
structurally characterized (‘‘Experimental’’).
In details, the lactone group of 3 could be easily opened
to the methyl ester derivative 5 by triethylamine treatment
in methanol at room temperature for 12 h. The product was
obtained in 95% yield and characterized by 1H- and
13C-NMR technique, observing superimposable data with
the ones already reported (Furneaux et al. 1988; Fabbri
et al. 2007b). The value of optical activity (‘‘Experimental’’)
was also in agreement with literature, supporting the
Amino acid was isolated in protonated form, due to the
acidic conditions present in the workup of the azido car-
boxylate when it was subjected to a filtration on a cationic
resin before reduction by Pd/C catalysed hydrogenation.
Scheme 1 Arbitrary numbering
is for convenience. Reaction
reagents and conditions:
a MeOH/Et3N (2.5 eq), r.t.,
12 h, 95%; b TsCl (1.2 eq)/Py,
r.t., overnight, 88%; c NaN3
(1.5 eq)/Aliquatꢂ336 (0.1 eq),
CH3CN, reflux 17 h, 85%;
d 0.5 M aq.NaOH (1 eq), THF,
r.t., 3 h, H?-form Amberlyst 15,
99%; e H2, p = 1 atm, Pd/C
10%; EtOH, 3 h, 95%; f H2,
p = 1 atm, Pd/C 10%; EtOH,
1 h, 73%
123

638
A. Defant et al.
Sugar amino acid 4 as H-Gly-Ala-OH isoster
Electrospray (ESI)-mass spectra showed the signal at m/z
162 corresponding to the molecular ion of the protonated
form, and fragmentation experiments on this selected mass
value gave signals for the loss of ammonia, water, carbon
dioxide and formic acid. High-resolution measurement by
matrix-assisted laser desorption ionization (MALDI)-MS
It has been reported that d-amino acids are able to affect
the structure of the peptides where they are involved
(Edwards et al. 2006). Amino acid 4 is comparable to
H-Gly-Ala-OH dipeptide, with the peculiarity of assuming
a restricted conformation due to the presence of the tetra-
hydrofurane ring, formally derived by closure between
oxygen atom of peptide bond and methyl group of alanine
residue. In particular 2,5-cis-tetrahydrofurane amino acid
provide building blocks for peptides, whose b-turn-type
structure is due to hydrogen bonds, as predicted by calcu-
lation in the gas phase (Jockusch et al. 2006). By using
density functional theory (DFT) method with polarizable
continuum model, stable conformers can be calculated in
water to simulate solvation effect (Iwaoka et al. 2002).
Under these conditions, the comparison of energy mini-
mized structures has been here achieved for H-Gly-Ala-OH
dipeptide and compound 4 (Fig. 3). Fleet and co-workers,
for example have shown that quite short oligomers of tet-
rahydrofurane dipeptide isosteres derived from carbohy-
drates adopt regular conformations in solution due to
stabilization by hydrogen bonds (Smith and Fleet 1999).
In order to disclose the effect given by 4 as a replace-
ment for the Gly-Ala residue in a peptide sequence, the
values of the its dihedral angles corresponding to the
rotations about OC–N bond (xi angle) and about N–CCO
bond in the following aminoacid residue (Ui?1 angle) as
reported in Fig. 3, have been taken into account. In detail,
the torsional angles were evaluated for the energy-
minimized structures of compound 4 and H-Gly-Ala-OH
1
confirmed the molecular composition C6H12NO4. H-and
13CNMR spectra recorded in D2O allowed to fully assign
the structure, supported by one bond and long range het-
erocorrelation (HSQC and HMBC, respectively). By
infrared spectrum in attenuated transmitter reflection
(ATR) configuration on a solid sample a strong peak at
1,716 cm-1 attributable to stretching of the CO group in
the carboxylic unit was evident, as well as a very broad and
intense signal centered at 2,910 cm-1 due to CH and OH
stretching. The OH groups are probably involved in inter-
and intra-molecular hydrogen bonds as suggested by the
asymmetric broadening of the band and the decreased in
wavenumber value if compared to the free group (Perrin
and Nielson 1997). Otherwise, in the structure of amino
acid 4, or in its protonated form, the number of hydrogen
bond donor/acceptor atoms is also higher than in
hydroxylactone 3, whose OH group was involved in
intramolecular hydrogen bonding with the vicinal carbonyl
group, as confirmed by FT-IR spectroscopic analysis in
solid state and in solution (Dobrzynski et al. 2009).
The enantiomeric purity of 4 is expected by the use of
hydroxylactone 3 as building block existing in pure enan-
tiomeric form due to its natural origin from cellulose,
combined to a sequence of reactions not affecting the
configuration of the asymmetric centres. It is in line with
the optical activity shown by the final aminoacid 4, as well
as for each intermediate of the synthesis. A further support
came from chiral shift reagent study, where 1HNMR
spectrum of azido ester 7 was investigated in the presence
of europium trifluorohydroxymethylene-D-camphorato,
Eu(tfc)3. The choice of the product 7 is imposed by its
solubility in CDCl3 and above all by the presence of a well-
detectable singlet NMR signal (the COOMe group) to be
monitored by addition of increasing amount of Eu-complex.
Otherwise Eu(tfc)3 was selected as the chiral shift reagent
for its ability to complex with tertiary a-hydroxyl methyl
esters (Fraser 1983). No splitting was observed for the
COOMe singlet, neither for each other signal; these results
speaking for the lack of diastereomeric complexes and
confirming the enantiomeric purity of the compound, but
shifts were observed for all the protons, in downfield as
typical when induced by europium complexes. The stron-
gest one was for OH proton, giving indication of the kind
of chelation with this functional group, whereas a pro-
nounced shift was obtained also for H-5 located in the same
side of the plane with OH, thus confirming their relative
syn position.
Fig. 3 Energy minimized structures by DFT-B3LYP 6–31 G (d)
calculation in water of Gly-Ala dipeptide, with indication of the
torsional angles (top), and of amino acid 4 (bottom)
123

An efficient route towards a new branched tetrahydrofurane d-sugar amino acid
639
peptidomimetics. It represents a Gly-Ala dipeptide isoster,
with the peculiarity of assuming a restricted conformation.
In order to evaluate the peculiarity of compound 4, DFT
calculation was performed and the values of torsional
angles were compared to the ones computed for H-Gly-
Ala-OH dipeptide and the known pyrrole amino acid 10.
These studies are under investigation.
Table 1 Torsional angles for the energy-minimized structures of
sugar aminoacid 4, H-Gly-Ala-OH and 5-(aminomethyl)pyrrole-2-
carboxylic acid (10) by DFT-B3LYP 6–31 G (d) calculation in water
Compound
xi
Ui?1
H-Gly-Ala-OH
?173
-98
-157
?178
?145
?179
Acknowledgments The authors thank the financial contribution of
Provincia di Ravenna supporting this research. They are grateful to
prof. L. Gentilucci, University of Bologna for the fruitful discussion
and to Mr. A. Sterni and Mr. M. Rossi, university of Trento for mass
spectra recording and technical assistance, respectively.
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