Hydrate of 2,3:4,5-di-O-isopropylidene-β-D-arabino-hexos-2-ulo-2,6-pyranose as new crystalline reagent in the preparation of Amadori derived peptides

Article abstract of DOI:10.2478/s11532-009-0123-y

We established, that crystalline hydrate of 2,3:4,5-di-O-isopropylidene-β-D-arabino-hexos-2-ulo-2,6-pyranose is a new, convenient and stable reagent for solid phase synthesis of peptide derived Amadori products. The structure of the title compound was studied by X-ray analysis, NMR spectroscopy, and high resolution ESI-MS. The crystal structure indicated the existence of two symmetry-independent molecules that were not connected with hydrogen bonds. A comparison with previously reported 2,3:4,5-di-O-isopropylidene-β-D-fructopyranose revealed, that these two compounds are isostructural. Versita Warsaw and Springer-Verlag Berlin Heidelberg.

Full text of DOI:10.2478/s11532-009-0123-y

Cent. Eur. J. Chem. • 8(1) • 2010 • 28–33
DOI: 10.2478/s11532-009-0123-y
Central European Journal of Chemistry
Hydrate of 2,3:4,5-di-O-isopropylidene-ß-D-
arabino-hexos-2-ulo-2,6-pyranose
as new crystalline reagent in the preparation of
Amadori derived peptides
Reserch Article
Piotr Stefanowicz*, Joanna Batorska, Monika Kijewska, Hubert Bartosz-Bechowski,
Zbigniew Szewczuk, Tadeusz Lis.
FacultyꢀofꢀChemistry,ꢀUniversityꢀofꢀWrocław,
50-383ꢀWrocław,ꢀPoland
Received 20 March 2009; Accepted 23 June 2009
Abstract: We established, that crystalline hydrate of 2,3:4,5-di-O-isopropylidene-ß-D-arabino-hexos-2-ulo-2,6-pyranose is a new, convenient and
stable reagent for solid phase synthesis of peptide derived Amadori products. The structure of the title compound was studied by X-ray
analysis, NMR spectroscopy, and high resolution ESI-MS. The crystal structure indicated the existence of two symmetry-independent
molecules that were not connected with hydrogen bonds. A comparison with previously reported 2,3:4,5-di-O-isopropylidene-ß-D-
fructopyranose revealed, that these two compounds are isostructural.
Keywords: ꢀ2,3:4,5-di-O-isopropylidene-ß-D-arabino-hexos-2-ulo-2,6-pyranose•ꢀꢀsolidꢀphaseꢀsynthesis•ꢀꢀX-rayꢀstudies•ꢀꢀAmadoriꢀproducts
© Versita Warsaw and Springer-Verlag Berlin Heidelberg.
1. Introduction
the studies of the biochemical consequences of Maillard
reaction as well as a search for biomarkers of diabetes
Reducing sugars react nonenzymatically with free amino requires the development of a fast, convenient, and
groups of proteins to form Amadori products, which general method of glycated peptide synthesis.
undergo a series of complex reactions leading to the
The 2, 3:4, 5 -di-O-isopropylidene-β-D-arabino-
formation of advanced glycation end products (AGEs) hexos-2-ulo-2,6-pyranose (I) is a common reagent
involved in aging, diabetes, and other pathological used for solid [5-8] and solution [9,10] phase synthesis
processes [1,2]. These reactions also occur during of peptide derived Amadori products by reductive
food processing or cooking, affecting flavors, color, alkylation of the ε-amino groups of the lysine residue or
and organoleptic properties of foods. Peptide-derived N-terminal α–amino groups in the presence of sodium
Amadori products are a subject of interest in clinical cyanoborohydride.Previouslythesamereagentwasalso
chemistry as markers of diabetes mellitus. Recent applied for synthesis of natural products, talatomycins
reports suggest a significant influence of the glycation A, B and 1-deoxy-6-castanospermine [11,12]. This
process on the function of relatively short-lived hormones compound was also an intermediate in the synthesis of
and regulatory peptides [3,4]. The dynamic progress in 3-heptuloses [13]. Unfortunately, I is an amorphous, oily
* E-mail: stp@wchuwr.pl
28

P. Stefanowicz et al.
compound, what makes its purification, characterization hydroxyl H atoms) were generated in idealized positions
and handling inconvenient. In the following paper we and refined using the riding model (C—H bond length
have described the preparation and the structure of of 0.95 Å for H atoms from the methyl group, 0.99 Å
crystalline
2,3:4,5-di-O-isopropylidene-β-D-arabino- for H atoms from the methylene group). The hydroxyl
hexos-2-ulo-2,6-pyranose hydrate (II).
H atoms were found on the difference Fourier map
and kept using a distance restraint (0.82 Å for O—H
bond length; the displacement parameter: 1.5 Ueq(O)).
Absolute configuration was assumed based on the
starting compound’s known configuration (as Mo Kα
radiation was used in the X-ray diffraction experiment,
all Friedel pairs were merged).
2. Experimental procedure
2.1. Preparation 2,3:4,5-di-O-isopropylidene-
β-D-arabino-hexos-2-ulo-2,6-pyranose hydrate
(II)
2.3. Solid phase synthesis of glycoconjugate
Lys(Fru)-Ala-Ala-Phe
2,3:4,5-di-isopropylidene-β–D-fructopyranose has been
obtained by the reaction of β–D-fructopyranose with
acetone in the presence of sulfuric acid by the procedure
described by Brady [14]. The product was subsequently
oxidized with the pyridinium chlorochromate (VI),
following Cubero et al. [11]. The obtained oily product
was purified on silicagel column using diethyl ether as
an eluent. Two fold molar excess of water was added
to the purified oil and overnight the colorless crystals
of the title compound appeared. Obtained product was
washed with the mixture of diethyl ether – hexane (1:5)
and dried in the air. Yield of obtained hydrate 40%; mp.
The solid phase synthesis of glycoconjugate Lys(Fru)-
Ala-Ala-Phe(IV)wasperformedaccordingtoapreviously
reported procedure [5], replacing the 2,3:4,5-di-O-
isopropylidene-β-D-arabino-hexos-2-ulo-2,6-pyranose
with its hydrate II. Yield of crude product IV: 75%; purity
according to HPLC (C18 column Vydac (250×4.6 mm)
gradient0-80%BinA;A:water+0.1%TFA,B:acetonitrile
+ 0.1% TFA) 79% Retention time (min): 14.20; ESI-
MS (MicrOTOF-Q Bruker, Germany; solvent CH3CN/
H2O, positive ions [M+H]+) m/z measured: 598.3080;
calculated for C27H43N5O10 598.3088; ESI-MS/MS for
parent ion 598.3: 580.3 (-H2O); 562.3(-2H2O); 544.3
(-3H2O) 514.2 (-3H2O and –CH2O); 436.2 (y4-glucose)
308.1 (y3); 237.1 (y2); 166.1 (y1); Nomenclature for
the peptide fragment ions according to Roepstroff and
Fohlmann [16].
= 71-72oC; [α]D26= -31.01 (c=0.5, CHCl3); H NMR 500
1
1
MHz, DMSO-d6 H NMR, ppm; 5.70 (1H, J=6.8Hz, d),
5.63 (1H, J=6.4Hz, d), 4.67 (1H, J=6.8 Hz, J=6.4, dd),
4.53 (1H, J=2.1 Hz, J=8.0, dd), 4.32 (1H, J=2.2 Hz, d),
4.2 (1H, J=7.9 Hz, d), 3.71(1H, J=13.0 Hz, d), 3.56(1H,
J=13.0 Hz, d), 1.41 (3H, s), 1.35 (3H, s), 1.35 (3H, s),
1.27 (3H, s).
ESI-MS (MicrOTOF-Q Bruker, Germany; solvent
CH3CN/H2O, positive ions [M+Na]+) measured: 299.11;
calculated for C12H20O7Na 299.12); ESI-MS/MS for
parent ion 299.1: 259.1; 201.1; 143.0; 125.0; 115.0;
97.0; GC-MS (Hewlett–Packard 5890 II coupled with
Hewlett Packard HP-5971 A mass selective detector,
column: Elite 5 MS)): one chromatographic peak at 7.15
min, fragments: 243, 229, 201, 171, 143, 113, 85, 59, 43;
Anal. Calcd. for C12H20O7 C, 52.17% H, 7.30% Found: C,
52.41%, H, 7.11.
2.4. Synthesis novel lysine derivative: Fmoc-
Lys(i,i-Fru,BOC)-OH (III)
The synthesis of Fmoc-Lys(i,i-Fru,BOC)-OH, which
is a building block useful for the synthesis of post-
translationally modified glycated peptides, was
performed according to a previously reported procedure
[7]. Our procedure was modified by replacing the
2,3:4,5-di-O-isopropylidene-β-D-arabino-hexos-2-ulo-
2,6-pyranose with its hydrate II.
Yield of compound III: 46%, mp 80.5° - 85.0°C;
-14.50 (c 1.0, MeCN); HPLC: retention time (min):
1
2.2. Crystal structure determination
25
D
[a ]
42.83; H NMR (CDCl3), δ (ppm) = 1.32 – 1.52 (12H,
m), 1.45 (4H, m), 1.48 (9H, s), 1.74 (1H, m), 1.92 (1H,
m), 3.35 (2H, m), 3.50 (2H, s), 3.72 (1H, m), 3.85 (1H,
m), 4.19 – 4.24 ( 1H, 1H, m two signals overlap), 4.39
(1H, 2H, m two signals overlap), 4.57 (1H, m), 5.41 (1H,
m), 5.58 (1H, m), 7.19 – 7.77 (8H, m); HR-MS: Found:
711.3487 calculated for (C38H50N2O11 + H)+ 711.3492;
The crystallographic measurements were performed
on κ-geometry Kuma KM4CCD automated four-circle
difractometer with graphite monochromatized Mo Kα
radiation. The data for crystals were collected at 100 K
using the Oxford Cryosystems cooler. The structure was
solved by direct methods with SHELXS97 and refined
using SHELXL97 [15]. All H atoms (except for the
29
29

Hydrate of 2,3:4,5-di-O-isopropylidene-ß-D-arabino-hexos-2-ulo-
2,6-pyranose as new crystalline reagent in the preparation of
Amadori derived peptides
MS/MS (parent 711.35): 369.2, 495.2, 535.2, 553.3, are more complex. The signals of geminal hydroxyl
611.3, 655.3.
groups are still present, but the strong, sharp singlet at
9.51 ppm indicated the presence of free, unhydrated
aldehyde in the solution. The content of free 2,3:4,5-di-
O-isopropylidene-β-D-arabino-hexos-2-ulo-2,6-
pyranose in solution 20 min after dissolving the sample
evaluated on the basis of integrated aldehyde peak is
21% in CD3CN and respectively 24% in CDCl3. These
experiments suggest that a partial dehydratation of
gem-diol II takes place spontaneously in the organic
solvents.
3. Results and discussion
Compound I, which can be obtained by oxidation of
2,3:4,5-di-O-isopropylidene-β-D-fructopyranose
in
presence of water, forms solid, crystalline derivative II
which has a gem-diol structure. The compound is stable
and can be stored at room temperature for many months
without any evidence of oxidation or decomposition.
Molecular mass of hydrate measured by ESI-MS for
[M+Na]+ ion is consistent with that calculated for the
formula C12H20O7Na. GC-MS measurement gave the
same retention time and fragmentation pattern as GC-
MS of the starting aldehyde. Hydrate II was analysed
The crystal structure of compound II shows that
it’s C1 atom is substituted by two OH groups, forming
gem-diol moiety (Table 1). Overall structure of the
investigated hydrate II is very similar to that reported
for
2,3:4,5-di-O-isopropylidene-β-D-fructopyranose
1
by H NMR. Spectra were measured in CDCl3, CD3CN
[17] III. The molecule III differs from the molecule II
by the absence of one of the hydroxyl groups on
carbon C1. Both crystal structures are isostructural.
In the unit cell two symmetry-independent molecules
could be distinguished, namely A and B. The atom
labeling system is adopted from Lis and Weichsel [17]
(Fig. 1) for comparison purposes. The H atoms from
hydroxyl groups bonded to C1 atoms are disordered
and DMSO-d6. Spectrum measured in DMSO is in a
good agreement with the structure proposed for the
hydrate. The doublets at 5.62 and 5.70 ppm confirmed
the presence of two geminal hydroxyl groups attached
to the carbon C1 while the signal corresponding to the
free aldehyde group is not present. On the other hand,
the spectra measured in acetonitrile and chloroform
Table 1. Selected geometrical parametrs characterizing the molecular environment of the carbon atom C1
Molecule A
Molecule B
Bond lengths for both molecules [Å]
O01A-C1A
O02A-C1A
C1A-C2A
1.411(2)
O01B-C1B
O02B-C1B
C1B-C2B
1.411(2)
1.412(2)
1.537(2)
1.413(2)
1.535(2)
Valence angles for both molecules [o]
O01A-C1A-O02A
O01A-C1A-C2A
O02A-C1A-C2A
111.1(2)
O02B-C1B-O01B
O02B-C1B-C2B
O01B-C1B-C2B
111.3(2)
109.7(2)
109.4(2)
109.9(2)
109.3(2)
Torsion angles for both molecules [o]
O01A-C1A-C2A-O6A
O02A-C1A-C2A-O6A
O01A-C1A-C2A-O2A
O02A-C1A-C2A-O2A
O01A-C1A-C2A-C3A
O02A-C1A-C2A-C3A
52.7(2)
O02B-C1B-C2B-O6B
O01B-C1B-C2B-O6B
O02B-C1B-C2B-O2B
O01B-C1B-C2B-O2B
O02B-C1B-C2B-C3B
O01B-C1B-C2B-C3B
175.3(1)
53.0(2)
54.9(2)
-67.4(2)
-60.4(2)
177.3(2)
174.9(2)
-67.4(2)
54.8(2)
177.3(2)
-60.5(2)
30

P. Stefanowicz et al.
Figure 1. Molecule A with atom labeling scheme. The disordered part is indicated with dashed and solid line for major and minor components,
respectively
in two positions. In molecule A the occupancy of HΦ2 may convert spontaneously to the compound I.
position is 0.62 while the occupancy of HΦ3 position Therefore the hydrate can be used as a convenient and
is 0.38. For the B molecule the occupancy is 0.66 and stable precursor of the compound I. In order to test this
0.34 respectively. Molecules A and B are of similar possibility we used the crystalline hydrate for the solid
conformation (see crystallographic data, Cambridge phase synthesis of Amadori products on the ε-amino
Crystallographic Data Centre No. 651195). The group of lysine.
pyranosyl, 2,3-isopropylidene and 4,5-isopropylidene
Synthesis of the model compound Lys(Fru)-Ala-Ala-
rings conformations are essentially the same as in Phe was performed in conditions given in our previous
2,3:4,5-di-O-isopropylidene-β-D-fructopyranose [17]. In paper[5].ThecrudereactionproductwastestedbyHPLC
both cases pyranosyl ring forms 2S0 twisted boats (Table and ESI-MS methods. After purification by preparative
2). The 2,3-isopropylidene rings are of envelope (3E) HPLC, the identity of the product was confirmed by high
conformation while the 4,5-isopropylidene rings are close resolution ESI-MS and ESI-MS/MS. Yields and purity
5
to 1 T twist inAmolecule and to 1E envelope conformation of the obtained product was similar to that synthesized
in B molecule. In crystal structure of compound II the applying the free aldehyde [5]. Compound II was also
molecules A and B are not interconnected with hydrogen tested in synthesis of (Nα-9-fluorenylmethoxycarbonyl-
bonds. The same situation takes plase for the crystal of Nε-tert-butyloxycarbonyl-Nε-N-(2,3:4,5-di-O-
compound III. Similar motifs of hydrogen bonds forming a isopropyliden-1-deoxy-β-D-fructopyranose-1-ylo)lysine,
square about four-fold axis at (0,0,z) and (1/2,1/2,z+1/2) Fmoc-Lys(i,i-Fru,BOC)-OH (III), which is a building
could be distinguished for compounds II and III (Table block useful for incorporating the glycated lysine moiety
3). In these motifs both disordered components of both into the peptide chain.7,8 The product III was obtained by
hydroxyl groups bonded to C1 atom are involved and act reductive alkylation of Fmoc-Lys-OH trifluoroacetate
as hydrogen bond donors as well as acceptors.
in THF using compound II in the presence of sodium
Relatively high content of free aldehyde in solution of cyanoborohydride. After the glycation, the secondary
hydrate II in organic solvents revealed by NMR studies amino group of lysine was protected using Boc2O.
suggests that the crystalline hydrate of 2,3:4,5-di-O- The scheme of synthesis was presented in Scheme 1.
isopropylidene-β-D-arabino-hexos-2-ulo-2,6-pyranose The final product was purified on a silica-gel column.
31
31

Hydrate of 2,3:4,5-di-O-isopropylidene-ß-D-arabino-hexos-2-ulo-
2,6-pyranose as new crystalline reagent in the preparation of
Amadori derived peptides
Table 2. Hydrogen-bond lengths [Å] and angles[o]
D-H
D-H[Å]
0.82
H…A[Å]
2.06
D…A[Å]
2.876(2)
2.779(2)
2.880(2)
2.815(2)
2.779(2)
2.815(2)
D-H…A[O]
172
A
O01A-H01A
O02A-H03A
O01B-H01B
O02B-H03B
O02A-H02A
O02B-H02B
O01Ai
O02Aii
O01Biii
O02Biv
O02Ai
O02Biii
0.82
1.97
166
0.82
2.07
169
0.82
2.03
159
0.82
1.98
166
0.82
2.08
149
Symmetry operations : (i) y, -x, z; (ii) –y, x, z; (iii) 1-y, x, z; (iv) y, 1-x, z.
Table 3. Puckering parameters and conformations
Puckering parameters
Molecule
Q (Å)
θ (°)
θ (°)
Conformation
Pyranose ring
O6-C2-C3-C4-C5-C6
O6-C2-C3-C4-C5-C6
2,3-Isopropylidene ring
O2-C9-O3-C3-C2
A
B
0.675(1)
0.661(1)
95.54(9)
96.29(9)
150.0(1)
150.5(1)
2S0
2S0
A
B
0.325(1)
0.327(1)
80.8(2)
80.8(2)
3E
3E
O2-C9-O3-C3-C2
4,5-Isopropylidene ring
O4-C10-O5-C5-C4
A
B
0.249(1)
0.302(1)
157.3(2)
178.1(2)
51T
1E
O4-C10-O5-C5-C4
Scheme 1. The scheme of synthesis of Fmoc-Lys(i,i-Fru,BOC)-OH
32

P. Stefanowicz et al.
The purity and identity of obtained derivative (III) was
confirmed by analytical HPLC, TLC, ESI-MS, ESI-MS/
MS and NMR The obtained results were the same as
those reported for synthesis utilizing the free aldehyde I
[7]. The reactivity of the aldehyde (I) and the crystaline
hydrate of 2,3:4,5-di-O-isopropylidene-β-D-arabino-
hexos-2-ulo-2,6-pyranose (II) in applied conditions is
comparable which was confirmed by very similar yields
and purities obtained using reagents (I) and (II).
Acknowledgement
This work was supported by a Grant No. 3 T09A 029
28 from the State Committee for Scientific Research
(Poland). We thank MSc Marek Chojniak for GC-MS
measurements.
Supplementary data
4. Conclusion
Completecrystallographicdataforthestructuralanalysis
have been deposited with Cambridge Crystallographic
Data Centre No. 651195. Copies of this information
may be obtained free of charge from the Director,
Cambridge Crystallographic Data Centre, 12 Union
Road, Cambridge CB2 IEZ, UK. (fax +44-1223-336033,
e-mail deposit@ccdc.cam.ac.uk or via www.ccdc.cam.
ac.uk )
The stable and crystaline hydrate of 2,3:4,5-di-O-
isopropylidene-β-D-arabino-hexos-2-ulo-2,6-pyranose
(II) was obtained. The title compound has geminal
diol structure in solid state and in DMSO, however in
acetonitrile and chloroform spontaneously decomposes
to free aldehyde (I). Presented examples show that
compound (II) can be successfully used for the synthesis
of peptides and other derivatives containing glycated
lysine residue.
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