Facile approach towards phosphorylated azasugars as potential glycosyl phosphate mimics

Article abstract of DOI:10.1016/S0957-4166(99)00322-5

A sequence of two chemoselective reductions enables the stereoselective synthesis of phosphorylated azasugars starting from products of dihydroxyacetonephosphate-dependent aldolases.

Full text of DOI:10.1016/S0957-4166(99)00322-5

Pergamon
Tetrahedron: Asymmetry 10 (1999) 3139–3145
Facile approach towards phosphorylated azasugars as potential
glycosyl phosphate mimics
Matthias Schuster ∗ and Siegfried Blechert
Institut für Organische Chemie, Sekr. C3, Technische Universität Berlin, Straße des 17. Juni 135, D-10623 Berlin, Germany
Received 8 June 1999; accepted 6 August 1999
Abstract
A sequence of two chemoselective reductions enables the stereoselective synthesis of phosphorylated azasugars
starting from products of dihydroxyacetonephosphate-dependentaldolases. © 1999 Elsevier Science Ltd. All rights
reserved.
1. Introduction
As apparent transition state analogs of glycoside-processing enzymes both polyhydroxylated piperi-
dines and pyrrolidines represent important bioactive compounds.1 One of the most versatile approaches
towards such azasugars combines enantioselective aldolase-catalyzed C–C bond formations with dia-
stereoselective reductive amination processes.2 Aldolase products have been reductively transformed
into three different types of cyclic products (A–C in Scheme 1) depending on the chemistry employed.
Enzyme-catalyzed removal of the phosphate group followed by catalytic hydrogenation gives rise to
cyclic amines A containing a hydroxymethyl group, whereas methyl-substituted cyclic amines B are
obtained upon direct hydrogenation of aldol products due to accompanying reductive dephosphorylation.
Recently,3 it has been demonstrated that cyclic imines C are accessible when the reduction of the
dephosphorylated aldolase products is performed at low pH (Fig. 1).
Scheme 1. Azasugars resulting from reductive treatment of products of DHAP-depending aldolases
∗
Corresponding author.
0957-4166/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(99)00322-5

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M. Schuster, S. Blechert / Tetrahedron: Asymmetry 10 (1999) 3139–3145
Figure 1.
Herein, we present a facile approach for the conversion of aldolase products into phosphorylated cyclic
amines and imines of type D and E, respectively. Both five- and six-membered cycles are accessible. The
products can be regarded as nitrogen-containing non-isosteric analogs of glycosyl phosphates, which are
key intermediates in the biosynthesis of complex carbohydrates and glycoconjugates.
2. Results and discussion
Scheme 2 illustrates the synthesis of phosphorylazasugar 5. Upon reaction of 2.5 equivalents of
azidoaldehyde 1 with DHAP in the presence of rabbit muscle aldolase (RAMA) under thermodynamic
control 2 is obtained as the exclusive product.4 When 2 is hydrogenated on Pd–C in 0.5 M HCl,
ammonium hemiketal 3 can be trapped in high yield. When the pH of the aqueous solution of 3 is raised,
an equilibrium between 3 and cyclic phosphorylimine 4 is established. At pH >8, 4 is the predominating
isomer as judged from the chemical shift of C-2 (178.6 ppm for 4 vs. 97.6 ppm for 3). In contrast to
hemiketal 3 the imine is unstable as indicated by 1H NMR. Whereas further catalytic hydrogenation of
3 results in the formation of a cyclic amine of type B due to reductive dephosphorylation, we found
that the phosphoryl function is retained when NaBH3CN is employed as the reducing agent, thus,
yielding phosphorylazasugar 5. The reductive amination proceeds with high diastereoselectivity. The
1
identity of 5 was unambigiously established by enzymatic dephosphorylation. H and 13C NMR spectra
of the resulting type A imino sugar are indistinguishable from those reported by Wong and co-workers.4
Notably, both reduction using NaBH3CN and catalytic hydrogenation on Pd–C led to products with the
2R configuration.
The synthesis of 10 followed the same procedures. RAMA-catalyzed reaction of enantiomerically
pure aldehyde 6 with DHAP resulted in the formation of 7,5 which was catalytically hydrogenated under
acidic conditions. In contrast to 3, the lacking hydroxyl group in 8 prevents stabilization of the carbonyl
function via intramolecular hemiketal formation. Nevertheless, 8 was succesfully trapped in more than
80% yield, thus, demonstrating that selective azide reduction is not limited to aldol products of type 2.
When the pH is raised to 7.0, cyclic imine 9 is the predominating isomer as judged from the chemical
shift of C-2 (177.8 ppm for 9 vs. 208.3 ppm for 8). Reductive amination using NaBH3CN at pH 6–7
diastereoselectively yields phosphorylazasugar 10. The absolute configuration of 10 was again confirmed
by enzymatic dephosphorylation to the corresponding type A imino sugar.5
The synthesis of two six-membered phosphorylazasugars starting from an N-formyl-containing aldol
product is depicted in Scheme 3. The RAMA-catalyzed synthesis of 11 yielded a mixture of two
diastereomers.6 The N-formyl group was efficiently removed by treatment with 1 M HCl at 35°C for
24 h. Subsequent reductive amination (NaBH3CN, pH 6–7) yielded a mixture of phosphorylazasugars
12 and 13, which were separated by semipreparative HPLC. The stereochemistry of both isomers was
assigned based on their vicinal coupling constants assuming chair conformation.

M. Schuster, S. Blechert / Tetrahedron: Asymmetry 10 (1999) 3139–3145
3141
Scheme 2. Chemical-enzymatic synthesis of five-membered phosphorylazasugars 5 and 10. Reagents and conditions: (a)
H2/Pd–C, 0.5 M HCl, 15 min; (b) 2 equiv. NaBH3CN, pH 6–7, 4°C to 25°C, 12 h
Scheme 3. Synthesis of six-membered phosphorylazasugars 12 and 13. Reagents and conditions: (a) 0.55 M HCl, 12 h, 35°C;
(b) 2 equiv. NaBH3CN, pH 6–7, 4°C to 25°C, 12 h
3. Conclusion
In summary, a procedure for the conversion of phosphoryl-containing aldolase products into phos-
phorylazasugars has been developed. Its application to the synthesis of various compounds of the
pyrrolidine and piperidine type, respectively, has been demonstrated. In contrast to comparable aldolase
applications the dihydroxyacetonephosphate moiety is almost completely retained in the final products.
Notably, a wide range of stereoisomers should be accessible by choosing aldolases with the appropriate
stereoselectivities.2 Until recently, only very few reports on phosphorylated azasugars have been given
to the best of our knowledge,8–10 although these compounds may be regarded as nitrogen-containing
analogs of glycosyl phosphates, which are ubiquitous intermediates in the biosynthesis of complex car-
bohydrates or partial structures thereof. We are currently exploring the potential of phosphorylazasugars
as inhibitors of glycosyl transferring enzymes.
4. Experimental
All chemicals and solvents used were of the highest available purity. The following stationary phases
were used for chromatography. TLC: aluminum foils Merck 60 F 254 (elution with isopropanol:25%

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ammonium hydroxide:water 6:3:2), flash chromatography: 0.040–0.063 mm Merck silica. Semiprepara-
tive HPLC on a LiChrospher®-diol phase (8×250 mm) under elution with mixtures of MeOH and MTBE
containing 0.1% of trifluoroacetic acid in conjunction with a light scattering detector. The following
spectrometers were used to record physical data. NMR: Bruker DRX 500 (1H: 500.1 MHz, 13C: 125.8
MHz, 31P: 202.5 MHz) at 25°C against TMS as external standard (1H, 13C NMR). Data of proton-
decoupled 31P spectra were referenced against 85% phosphoric acid, which served as internal standard.
FAB-MS: Finnigan MAT 95 SQ using a glycerol matrix.
Notably, 5, 10, 12 and 13 retain small amounts (<10%) of inorganic material even after extensive
purification.
4.1. Aldolase reactions
Aldolase-catalyzed syntheses of 2,4 75 and 116 were performed according to published procedures
using rabbit muscle aldolase (Fluka, 16–25 U/mg) and DHAP obtained by acid hydrolysis of dihydroxy-
acetone phosphate dimer bis(ethylketal) (Fluka).7
2: 54% Yield; 13C NMR (D2O): δ 96.96 (d, J=7.8 Hz, Cq), 71.76 (CH), 70.19 (CH), 65.78 (d, J=4.8
Hz, CH2), 60.52 (CH), 59.57 (CH2).
7: 52% Yield; 13C NMR (D2O): δ 210.6 (d, J=6.8 Hz, C_O), 75.49 (CH), 74.87 (CH), 68.51 (d, J=4.5
Hz, CH2), 59.41 (CH), 15.17 (CH3).
11: 1:1 mixture of diastereomers (54% combined yield); 13C NMR (D2O): δ 164.46, 164.38, 97.01
(d, J=3.5 Hz Cq), 71.52 (CH), 70.17 (CH), 69.22 (CH), 67.52 (CH), 63.61 (d, J=6.9 Hz, CH2), 61.05
(CH2), 59.31 (CH2), 42.46 (CH), 41.05 (CH), 35.82 (CH2), 33.26 (CH2).
4.2. Phosphoric acid mono-(5S-amino-2R,3S,4R-trihydroxy-tetrahydro-pyran-2-ylmethyl) ester 3
A solution of aldolase product 2 (142 mg, 0.5 mmol) in 5 mL of 0.5 M HCl was hydrogenated under
vigorous stirring in the presence of 50 mg of 10% Pd on coal for 15 min. At this time all starting material
was consumed as indicated by TLC. The catalyst was removed by centrifugation. Although the resulting
product solution is stable for at least three days at room temperature, rapid decomposition was observed
during concentration and chromatography. Therefore, 0.6 mL of the acidic solution was mixed with 0.05
mL of acetone-d6 and directly subjected to spectroscopic analysis. The use of 0.01 mL acetonitrile as an
internal standard revealed a conversion of >80%. 1H NMR (H2O/acetone-d6): δ 3.9 (dd, 1H, J=10.5, 6.0
Hz, H-1), 3.79 (d, 2H, J=9.5 Hz, H-6ax,eq), 3.77 (dd, 1H, J=9.5, 8.5 Hz, H-4), 3.73 (dd, 1H, J=10.5, 5.5
Hz, H-1), 3.46 (d, J=9.5 Hz, H-3), 3.19 (dt, J=8.5, 9.5 Hz, H-5); 13C NMR (H2O/acetone-d6): δ 97.58 (d,
J
C-2,P=9.5 Hz, C-2), 70.89 (C-3), 70.06 (C-4), 66.68 (d, JC-1,P=5.0 Hz, C-1), 58.80 (C-6), 51.73 (C-5);
31P (H2O/acetone-d6): δ -0.34.
4.3. Phosphoric acid mono-(3R,4R-dihydroxy-5S-hydroxymethyl-4,5-dihydro-3H-pyrrol-2-ylmethyl)
ester 4
To an NMR tube containing a solution of 3 in a mixture of water and acetone-d6 (see above) was
carefully added 3 M NaOH until the pH had reached 8.5. NMR analysis revealed the formation of 4
as the predominating species. Decomposition takes place in the range of several hours as indicated by
1
comparison of appropriate signals with the methyl group of the internal standard acetonitrile. H NMR
(H2O/acetone-d6): δ 4.62 (d, 1H, J =6.0 Hz, H-3), 4.20 (dd, 1H, J=6.0, 5.5 Hz, H-4), 4.01–3.96 (m, 1H,

M. Schuster, S. Blechert / Tetrahedron: Asymmetry 10 (1999) 3139–3145
3143
H-5), 3.70–3.60 (m, 2H, H-6); 13C NMR (H2O/acetone-d6): δ 178.61 (d, JC-2,P =7.0 Hz, C-2), 80.86
(C-3), 77.12 (C-4), 71.26 (C-5), 61.65 (d, JC-1,P =3.5 Hz, C-1); 31P (H2O/acetone-d6): δ −4.55.
4.4. Phosphoric acid mono-(3R,4R-dihydroxy-5S-hydroxymethyl-pyrrolidin-2R-ylmethyl) ester 5
The solution of 3 (2.5 mL) was cooled to 4°C and the pH was adjusted to 6–7 by addition of 3 M
NaOH. NaBH3CN (31 mg, 0.5 mmol) was added and the resulting solution was stirred at 4°C for 3 h
maintaining a pH of 6–7. It was then allowed to stand at room temperature overnight. Excess NaBH3CN
was destroyed by acidification to pH 2–3 using 6 M HCl. After neutralization with 3 M NaOH 1 g of
coarse silica gel (0.063–0.2 mm, Merck) was added and the solvent was evaporated at 45°C in vacuo.
The adsorbed material was subjected to silica gel chromatography under elution with isopropanol:25%
NH4OH:H2O (10:3:2–3:3:2) giving 34 mg (0.14 mmol) of 5 (57% yield from 2). [α]25 +9.9 (c=0.2,
D
H2O); 1H NMR (D2O): δ 4.24 (dd, 1H, J=4.0, 2.5 Hz, H-4), 4.15 (dd, 1H, J=4.0, 3.0Hz, H-3), 4.09–3.99
(m, 2H, H-1), 3.94 (dd, 1H, J=12.0, 5.0 Hz, H-6), 3.87 (dd, 1H, J=12.0, 6.0 Hz, H-6), 3.79–3.74 (m, 1H,
H-5), 3.62–3.57 (m, 1H, H-2); 13C NMR (D2O): δ 76.2 (C-3), 75.4 (C-4), 65.79 (d, JC-2,P=6.1 Hz, C-2),
62.60 (C-5), 61.79 (d, JC-1,P=3.6 Hz, C-1), 57.95 (C-6); 31P (D2O): δ −4.92; IR (KBr) ν 3194 (vs, broad),
2517 (m), 1634 (m), 1458 (m), 1402 (s), 1179 (s) 1117 (vs, broad), 778 (m) cm−1; HRMS (C6H15NPO7,
MH+): calcd 244.0586, obsd 244.0586.
4.5. Phosphoric acid mono-(5S-amino-3S,4R-dihydroxy-2-oxo-hexyl) ester 8
A solution of aldolase product 7 (135 mg, 0.5 mmol) in 5 mL of 0.5 M HCl was hydrogenated under
vigorous stirring in the presence of 50 mg of 10% Pd on coal for 15 min. At this time all starting material
was consumed as indicated by TLC. The catalyst was removed by centrifugation. Since the product was
found to be similarily unstable as 3 (see above), 0.6 mL of the acidic solution was mixed with 0.05
mL of acetone-d6 and directly subjected to spectroscopic analysis. The use of 0.01 mL acetonitrile as
internal standard revealed a conversion of >80%. 13C NMR (H2O/acetone-d6): δ 208.05 (d, JC-2,P=7.5
Hz, C-2), 75.96 (CH-O), 71.61 (CH-O), 69.15 (d, JC-1,P=4.0 Hz, C-1), 50.02 (C-5), 14.94 (C-6); 31P
(H2O/acetone-d6): δ −0.467.
4.6. Phosphoric acid mono-(3R,4R-dihydroxy-5S-methyl-4,5-dihydro-3H-pyrrol-2-ylmethyl) ester 9
To an NMR tube containing a solution of 8 in a mixture of water and acetone-d6 (see above) was
carefully added 3 M NaOH until the pH had reached 7.0. NMR analysis revealed the formation of 9 as
the predominating species. 13C NMR (H2O/acetone-d6, 288 K): δ 177.8 (d, JC-2,P=7.0 Hz, C-2), 80.871
(CH-O), 77.56 (CH-O), 66.54 (C-5), 62.18 (d, JC-1,P=4.0 Hz, C-1), 13.64 (C-6); 31P (H2O/acetone-d6):
δ −1.25.
4.7. Phosphoric acid mono-(3R,4R-dihydroxy-5S-methyl-pyrrolidin-2R-ylmethyl) ester 10
The solution of 8 (2.5 mL) was treated with 31 mg (0.5 mmol) NaBH3CN exactly following the
procedure used for the synthesis of 5. The reaction product was adsorbed onto coarse silica as described
above. Silica gel chromatography using isopropanol:25% NH4OH:H2O (12:3:2–6:3:2) afforded 29 mg
(0.13 mmol) of 10 (51% yield from 2). [α]25 +29.2 (c=0.5, H2O); H NMR (D2O): δ 4.20–4.10 (m,
1
D
3H, H-3, H-4, H-1), 4.03 (dtbr, 1H, 12.0, 8.0 Hz, H-1), 3.90 (dq, 1H, J=4.0, 6.0 Hz, H-5), 3.70 (dt, 1H,
9.0, 4.0 Hz, H-2), 1.42 (d, 3H, J=6.0 Hz, H-6); 13C NMR (D2O): δ 76.08 (C-3), 75.57 (C-4), 65.94 (d,

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M. Schuster, S. Blechert / Tetrahedron: Asymmetry 10 (1999) 3139–3145
JC-2,P=6.0 Hz, C-2), 61.54 (d, JC-1,P=4.5 Hz, C-1), 57.69 (C-5), 10.33 (C-6); 31P (D2O): δ −3.89; IR
(KBr) ν 3190 (vs, broad), 2505 (m), 1633 (m), 1452 (m), 1401 (s), 1176 (s) 1105 (vs, broad), 801 (m)
cm−1; HRMS (C6H15NPO6, MH+): calcd 228.0637, obsd 228.0631.
4.8. Phosphoric acid mono-(3R,4R-dihydroxy-5R-hydroxymethyl-piperidin-2R-ylmethyl) ester 12 and
phosphoric acid mono-(3R,4R-dihydroxy-5S-hydroxymethyl-piperidin-2R-ylmethyl) ester 13
A 1:1 diastereomeric mixture of 11 (144 mg, 0.5 mmol) was dissolved in 5 mL of 0.55 M HCl and
maintained at 35°C overnight. TLC indicated complete removal of the N-formyl group. The solution
was then cooled to 4°C and the pH was adjusted to 6–7. Under stirring 62 mg (1 mmol) of NaBH3CN
was added. Keeping the pH constant the solution was stirred at 4°C for 3 h and at room temperature
overnight. The product mixture was treated as described in the synthesis of 5. Chromatography on silica
using isopropanol:25% NH4OH:H2O (12:3:2–6:3:2) as the eluent afforded 76 mg of a mixture of 11
1
and 12 (59% combined yield) as judged from the H NMR spectrum. 10 mg of each diastereomer was
isolated by semipreparative HPLC and spectroscopically characterized; 12: [α]25 +33.7 (c=0.15, H2O);
D
1H NMR (D2O): δ 4.28–4.20 (m, 2H, H-1), 3.85 (dd, 1H, J=12.0, 3.5 Hz, H-7), 3.77 (dd, 1H, J=12.0,
6.0 Hz, H-7), 3.70 (t, 1H, J=9.5 Hz, H-3), 3.59 (t, 1H, J=9.5 Hz, H-4), 3.55 (dd, 1H, J=12.5, 4.5 Hz, H-
6eq), 3.38–3.31 (m, 1H, H-2), 3.07 (t, 1H, J=12.5 Hz, H-6ax), 2.09–1.98 (m, 1H, H-5); 13C NMR (D2O):
δ 71.00 (C-4), 68.91 (C-3), 61.29 (d, JC-1,P=4.0 Hz, C-1), 59.03 (C-7), 58.83 (d, JC-2,P=8.0 Hz, C-2),
44.39 (C-6), 40.85 (C-5); 31P (D2O): δ −0.84; IR (KBr) ν 3176 (vs, broad), 2529 (m), 1650 (m), 1452
(m), 1400 (s), 1175 (s) 1118 (vs, broad), 931 (m) cm−1; HRMS (C7H17NPO7, MH+): calcd 258.0742,
obsd 258.0746; 13: [α]25 −4.5 (c=0.15, H2O); H NMR (D2O): δ 4.30 (dt, 1H, J=12.0, 6.0 Hz, H-1),
1
D
4.19–4.15 (ddd, 1H, J=12.0, 5.5, 3.0 Hz, H-1), 4.04 (t, 1H, J=8.0 Hz, H-3), 3.99 (dd, 1H, J=8.0, 5.0 Hz,
H-4), 3.94 (dd, 1H, J=11.0, 5.0 Hz, H-7), 3.86 (dd, 1H, J=11.0, 4.5 Hz, H-7), 3.59 (dd, 1H, J=13.0, 4.5
Hz, H-6), 3.46–3.41 (m, 1H, H-2), 3.31 (dd, 1H, J=13.0, 4.0 Hz, H-6), 2.41 (qbr, 1H, J=4,5 Hz, H-5); 13
C
NMR (D2O): δ 70.03 (C-4), 66.15 (C-3), 60.83 (d, JC-1,P=4.5 Hz, C-1), 59.00 (d, JC-2,P=8.5 Hz, C-2),
58.79 (C-7), 42.74 (C-6), 37.02 (C-5); 31P (D2O): δ −0.92; IR (KBr) ν 3174 (vs, broad), 2520 (m), 1645
(m), 1455 (m), 1402 (s), 1176 (s) 1107 (vs, broad), 925 (m) cm−1; HRMS (C7H17NPO7, MH+): calcd
258.0742, obsd 258.0748.
4.9. Enzymatic dephosphorylation reactions
A sample of 5 or 10 (20 mg) was dissolved in 0.5 mL of 0.1 M sodium acetate buffer (pH 4.8). After
adding 50 units of acid phosphatase (Sigma) the solution was stirred for 3 days at 37°C. The solvent was
evaporated in vacuo and the residue was purified by flash chromatography using mixtures of CH2Cl2,
methanol and aqueous NH4OH to give the following products.
Dephosphorylation product of 5:4 1H NMR (D2O): δ 3.93 (dd, 1H, J=5.0, 3.0 Hz), 3.68 (dd, 1H,
J=5.5, 3.0 Hz), 3.60 (dd, 1H, 11.5, 6.0 Hz), 3.55 (dd, 1H, J=12.0, 5.0 Hz), 3.50–3.43 (m, 2H), 3.13 (dt,
1H, J=5.0, 5.5 Hz), 2.82 (dt, 1H, J=5.5, 5.0 Hz); 13C NMR (D2O): δ 76.42, 74.93, 67.17, 63.47, 59.65,
57.28.
Dephosphorylation product of 10:5 1H NMR (CD3OD): δ 3.85 (dd, 1H, J=3.5, 1.5 Hz), 3.70–3.60
(m, 3H); 3.20 (dq, 1H, J=4.0, 6.5 Hz), 2.93 (dt, 1H, J=3.5, 4.0 Hz); 1.15 (d, 1H, 6.5 Hz); 13C NMR
(CD3OD): δ 81.00, 80.40, 70.45, 63.27, 59.65, 13.53.

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3145
Acknowledgements
Financial support from Fonds der Chemischen Industrie is gratefully acknowledged.
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