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Microbiological aldolisations. Synthesis of 2-keto-3-deoxy-D-gluconate
Tetmhedrm:Asynm?#ry Vol. 4. No. 6.p~.
09574166/93 $6.MW.C@ PRgamonRess~
1165-1168.1993
Primted inGreatBritain
Microbiological Aldolisations. Synthesis of Z-Keto-3-DeoxyPGluconate Claudine Aug@ and Vhnique
Debt
(Received 15 Februury 1993)
Ahtract : An cfllclmt syothesisof2-keto-3dcoxy-D-gh1~0oate (KDG)wasachievedvi0
staeospecific aldolisation using the fimgus hpergillw
niger. Whenas incubation of D-
glycefaM&y&aadpyruvatewitheazymakextnctledtoadUawme&mixnaeofthtW ~~~inthc13:87ratio.ifictlbationwithnaingcellstumedont~oenhance ratioupto4:%infwrdKDG.
-yG =
oft
Kc-o.33 nlM . .
OH
-mycls
+ 0 3
1
+a 0 2
We wish to report here, initial studies in the investigatioo of this -on of aldolisalioa fa organic synthesis, caked out with enqmatic extracts and resting ails ss well. Aspergillus nip was Browa 011D1165
1166
C. AUGEand V. DELE~T
gluconate,the tnycelium was recove& after 3 days then either used like that or ground with the French Press. From the crude extract, the enxyme was partially purified by heat treatment and prucipiuuion with acetone accouGrg to the published procedure (specific activity : 0.32 U/mg).4 Alternatively Aspergillus nign was gnnvncmDgalaaonate. Results atu summsked in the table.
Table. Diasta#wtrie compositionsof the pducts resultingfrom aldol condensauon of 2 and 3.8
SourceofCarbon
glyamldehyde
Buymade
Myeelium
kuatedyield %
lko&dlm ratio
w-
D
+
73
13 : 87
Dghlmnate Pghlauate Dghtastate
L DJ,
+
74 36
40:60 46:54
65 88
4:% 9:91
Dgf&XolW
D D
f + +
adependingupmlbree~:the-ofCerboa~inthcarlmn.tbeenantiomaic.fomrdgl~~tbeincubation
Surprisingly condensation of D-glyceraldehyde 2 with pyruvatc 3 in the presence of the enzymatic exwmdidnotleadaasinglecompoundbnttoa~~oftwodiastenomrs spectroscopy,the major ccmpouud, 3decxy-D-q&o-2-hexulosonate
inthe13:87ratio.~FmmNMR (KDG ) was identified as a mixture of
pyranose ln and fnranose lb, and the minor one, 3-deoxy-D-zhreo-2-hexulosonate4, as the pyranose form.6 IIte enantictnericpurity of the startingmate&l, D-glycemldehyde,wss asecmkd by derivatizationwith (+) utnetJtyCbenzylatnineinto the Schist base, followedby teduckr and peracetylation? Therefote, we must admit that the enzymatic prepamdon contained two distinct aldolase!Jwith compleWnary facial selectivity. W two different degrading systems co-exist in D-gluconate-grown Aspergillus tiger, the one degrading Dglucottate, the other degrading L-araLxunueand involving KDA aldolase.*compound 4 exhibited the ssme sterecchemisttyon C-4 than 2-Item-3-deoxy-L-srahonate(IDA 51, the intemdhte degradationpmduct of Larabonsteinto pyruvate and glycolaldehyde~ Enxyrnaticcondensation ofL-glyceraldehyde with 3 Ied to a mhtttne of threo/erytbrocompoundsin the ratio 40 : 60 in 73% yield. The condensation products6 and 7 arc the enantiomers of 1 and 4, and in that case this is the L-three compound7 that results from the action of KDG aldolase.Thus RDA aldolase wss supposed to have higher affinity for L-glyceraldehydethan KDG aldolsse. Moreover we observed that the use of resting cells instead of enzymatic extract was of major interest. Indeed in that csse, the diastereomericratio wss euhanced up to 96% in favor of the erythro.roThe reason for this increase is still not clear; the three compound might be metabolized more quickly than the erythto. FurthermoreRDG synthesiswas much more efficientby incubation with the myceliun~than with the enxymatic extracu ten millimoles of KDG could be easily prepared with the amount of mycelium obtained from 1 L of cukure. KDG has just been discoveredas a componentof bacterial polysacc~,lu it is also supposedto play a role as an inducer in the synthesis of the pectate lyase, an enzyme involved in the cleavage of
1167
OH -0oc
OH
7
Growing Aspergillus n&r on D-gal-ate instead of D-glncona* did not make great difference in the diaswe4nnuic mixture obtained by inc&ation with myceliuxq the erythm compound was -wdY formed. Work is now in progress to better &&late possibilities
these microbiological
for the synthesis of analogs. Preliminary ex@ments
akkdisations
and
to explon
the
let us suppose that these aldolases exhibit
broadspecificitytowatdstheacceptor. -ical
experiment with enzymatic extract : 0.2 M solution of D or L 01:D,L-glyce~ddehydet~ in 0.02 M
potassium phosphate buffer pH 8.0 (5 mLJ was incubated with 1.1 equiv. of pyruvate in the preswce of 2 U ~~~~~~f~8h. Typical experiment with mycelium
: 0.05 M solution of D-glyceraldchyde
in 0.02 M potassium
phosphate buffer pH 8.0 (20 mL) was gently stirnd at 27’ for 24 h, with 1.1 cquiv. of pyruvate and mycelium obtain4 from 40 aiL ofculture.
C. AUCE and V. DELFZ.T
1168
After incubation the products were purifkd by anion exchange c-by on AG l-X8 resin (HC!O3*,1Of&200m&Q and isolated as their ammonium salts xuxding to the f&llowingprocodurr.: elution with a O-O.4 M ammonium bicarbonate linear gradient, freeze-drying, deionization with Dowex SOH+, neutrakation with dilute ammonia andagainkez&ying. We thank Ptvfessor A. Lubbkeaufor his encouragements. Referencea
and Notes
1.
C. AugC, C. Gautheron. S. David, A. Malkon, 201.
B. Cavayt and B. Bouxom, Tetrahedron, 1990,46.
2.
N-C!.Floyd, MH. Liebster and NJ. Tmner, J. Chem. Sec. Perkin Trans., 2.1992,1085.
3.
S.T. Allen, G.R. Hemkelman and E-J. Toone, 1. Org. Ckm.. 1992,57,426.
4.
A.M. Allam, MM. Hassan, T.A. Elzainy, J. Bacterial., 1975.1128 ; the enzymetic assay for KDG
5.
aldolase was carried out by determining pyruvate liberated from KDG. KDG has been obtained by putida,seeref.3. enqmatic synthesis with KDPG aldolase fmm PseUifo~ The amount of dueo compound was estimated fkom tH NMR spectrum by the intensity of the peak at 1.77 ppm attributed to H-3ax and from t3c NMR by comparkg the intensity of the peak at 39.00 and 33. 94, corresponding respectively to the C-3 signal of three and erythro in the p pyranose form. Cormction wasmade~~~co~occlnmainlyin~conformation,whareasthepercenemgeof~
in
this conformation is only 49%. Similar results were found from both estimations. 6.
Compound 1: tH and *SCNMR identical to the literam
: R. Plant&Royon. F. Mona,
Condemine, W. Nasser and J. Robert-Baudouy, J. C~b&y&.
Gem., 1991,10,787
D. Anker, G.
; compound 4: lH
NMR (250 MHz, m0, HOD= 4.80 ppm) 6 : 1.77 (dd, J3ax,3eq=13Hz, J3aq4=11.5Hz, H-3ax), 2.22 (dd, J% 4=%2, H&q), t3C NMR (62.9MHz, extemal l&dioxanc refemnce, 6 66.64) 8 : 39.00 C-3, 62.94 C-i, 68.93 C-4, 70.00 C-5, 96.60 C-2, 176.29 C-l. These values are very close to the ones observed for H-3ax and H-feq in 3-deoxy-D&zero-D-galncto-nonulosonic
acid (KDN) which exhibits
the ssme steraochcmistry on C-4 and C-5 ss compound 4: C. Au& and C. Gautheron, J. C&m. Sue., CheRI. Commun., 1987,859. 7 8.
R Oshima and J. Kumanotani, Chem. fen., 1981,943.
9.
T.A. Elzainy, M.M. Hassan and A.M. Elshafei, Ann. Microbial.. 1981,31,9.
10.
[a]*= -26 (c ,2, water at pH 7) ; &era[a$& Carbohyd?. Res., 1%8,8, 193. F. Fern&i, L. Kenne, B. Lindberg and W. Niich,
11. 12. 13.
MM. Hassan, A.M. Allam, A.M. Elshafei and T.A. Elzainy. Egypt. J. Bat.. 1985.28.165. -31.6 fc , 1.67, water at pH 6) : D. Portsmouth, Carbohydr. Res., 1991,210,255.
R. Ramage, A.M. MacLeod and G.W. Rose, Tetrahedron, 1991,47,5625. D-glycaaldchydehyde was either purchased from Fluka or prepared from D-fructose by oxidation with lead tetraacetate : A.S. Perk, Methods in Curb&&. Ckm. , 1962,1,61.
09574166/93 $6.MW.C@ PRgamonRess~
1165-1168.1993
Primted inGreatBritain
Microbiological Aldolisations. Synthesis of Z-Keto-3-DeoxyPGluconate Claudine Aug@ and Vhnique
Debt
(Received 15 Februury 1993)
Ahtract : An cfllclmt syothesisof2-keto-3dcoxy-D-gh1~0oate (KDG)wasachievedvi0
staeospecific aldolisation using the fimgus hpergillw
niger. Whenas incubation of D-
glycefaM&y&aadpyruvatewitheazymakextnctledtoadUawme&mixnaeofthtW ~~~inthc13:87ratio.ifictlbationwithnaingcellstumedont~oenhance ratioupto4:%infwrdKDG.
-yG =
oft
Kc-o.33 nlM . .
OH
-mycls
+ 0 3
1
+a 0 2
We wish to report here, initial studies in the investigatioo of this -on of aldolisalioa fa organic synthesis, caked out with enqmatic extracts and resting ails ss well. Aspergillus nip was Browa 011D1165
1166
C. AUGEand V. DELE~T
gluconate,the tnycelium was recove& after 3 days then either used like that or ground with the French Press. From the crude extract, the enxyme was partially purified by heat treatment and prucipiuuion with acetone accouGrg to the published procedure (specific activity : 0.32 U/mg).4 Alternatively Aspergillus nign was gnnvncmDgalaaonate. Results atu summsked in the table.
Table. Diasta#wtrie compositionsof the pducts resultingfrom aldol condensauon of 2 and 3.8
SourceofCarbon
glyamldehyde
Buymade
Myeelium
kuatedyield %
lko&dlm ratio
w-
D
+
73
13 : 87
Dghlmnate Pghlauate Dghtastate
L DJ,
+
74 36
40:60 46:54
65 88
4:% 9:91
Dgf&XolW
D D
f + +
adependingupmlbree~:the-ofCerboa~inthcarlmn.tbeenantiomaic.fomrdgl~~tbeincubation
Surprisingly condensation of D-glyceraldehyde 2 with pyruvatc 3 in the presence of the enzymatic exwmdidnotleadaasinglecompoundbnttoa~~oftwodiastenomrs spectroscopy,the major ccmpouud, 3decxy-D-q&o-2-hexulosonate
inthe13:87ratio.~FmmNMR (KDG ) was identified as a mixture of
pyranose ln and fnranose lb, and the minor one, 3-deoxy-D-zhreo-2-hexulosonate4, as the pyranose form.6 IIte enantictnericpurity of the startingmate&l, D-glycemldehyde,wss asecmkd by derivatizationwith (+) utnetJtyCbenzylatnineinto the Schist base, followedby teduckr and peracetylation? Therefote, we must admit that the enzymatic prepamdon contained two distinct aldolase!Jwith compleWnary facial selectivity. W two different degrading systems co-exist in D-gluconate-grown Aspergillus tiger, the one degrading Dglucottate, the other degrading L-araLxunueand involving KDA aldolase.*compound 4 exhibited the ssme sterecchemisttyon C-4 than 2-Item-3-deoxy-L-srahonate(IDA 51, the intemdhte degradationpmduct of Larabonsteinto pyruvate and glycolaldehyde~ Enxyrnaticcondensation ofL-glyceraldehyde with 3 Ied to a mhtttne of threo/erytbrocompoundsin the ratio 40 : 60 in 73% yield. The condensation products6 and 7 arc the enantiomers of 1 and 4, and in that case this is the L-three compound7 that results from the action of KDG aldolase.Thus RDA aldolase wss supposed to have higher affinity for L-glyceraldehydethan KDG aldolsse. Moreover we observed that the use of resting cells instead of enzymatic extract was of major interest. Indeed in that csse, the diastereomericratio wss euhanced up to 96% in favor of the erythro.roThe reason for this increase is still not clear; the three compound might be metabolized more quickly than the erythto. FurthermoreRDG synthesiswas much more efficientby incubation with the myceliun~than with the enxymatic extracu ten millimoles of KDG could be easily prepared with the amount of mycelium obtained from 1 L of cukure. KDG has just been discoveredas a componentof bacterial polysacc~,lu it is also supposedto play a role as an inducer in the synthesis of the pectate lyase, an enzyme involved in the cleavage of
1167
OH -0oc
OH
7
Growing Aspergillus n&r on D-gal-ate instead of D-glncona* did not make great difference in the diaswe4nnuic mixture obtained by inc&ation with myceliuxq the erythm compound was -wdY formed. Work is now in progress to better &&late possibilities
these microbiological
for the synthesis of analogs. Preliminary ex@ments
akkdisations
and
to explon
the
let us suppose that these aldolases exhibit
broadspecificitytowatdstheacceptor. -ical
experiment with enzymatic extract : 0.2 M solution of D or L 01:D,L-glyce~ddehydet~ in 0.02 M
potassium phosphate buffer pH 8.0 (5 mLJ was incubated with 1.1 equiv. of pyruvate in the preswce of 2 U ~~~~~~f~8h. Typical experiment with mycelium
: 0.05 M solution of D-glyceraldchyde
in 0.02 M potassium
phosphate buffer pH 8.0 (20 mL) was gently stirnd at 27’ for 24 h, with 1.1 cquiv. of pyruvate and mycelium obtain4 from 40 aiL ofculture.
C. AUCE and V. DELFZ.T
1168
After incubation the products were purifkd by anion exchange c-by on AG l-X8 resin (HC!O3*,1Of&200m&Q and isolated as their ammonium salts xuxding to the f&llowingprocodurr.: elution with a O-O.4 M ammonium bicarbonate linear gradient, freeze-drying, deionization with Dowex SOH+, neutrakation with dilute ammonia andagainkez&ying. We thank Ptvfessor A. Lubbkeaufor his encouragements. Referencea
and Notes
1.
C. AugC, C. Gautheron. S. David, A. Malkon, 201.
B. Cavayt and B. Bouxom, Tetrahedron, 1990,46.
2.
N-C!.Floyd, MH. Liebster and NJ. Tmner, J. Chem. Sec. Perkin Trans., 2.1992,1085.
3.
S.T. Allen, G.R. Hemkelman and E-J. Toone, 1. Org. Ckm.. 1992,57,426.
4.
A.M. Allam, MM. Hassan, T.A. Elzainy, J. Bacterial., 1975.1128 ; the enzymetic assay for KDG
5.
aldolase was carried out by determining pyruvate liberated from KDG. KDG has been obtained by putida,seeref.3. enqmatic synthesis with KDPG aldolase fmm PseUifo~ The amount of dueo compound was estimated fkom tH NMR spectrum by the intensity of the peak at 1.77 ppm attributed to H-3ax and from t3c NMR by comparkg the intensity of the peak at 39.00 and 33. 94, corresponding respectively to the C-3 signal of three and erythro in the p pyranose form. Cormction wasmade~~~co~occlnmainlyin~conformation,whareasthepercenemgeof~
in
this conformation is only 49%. Similar results were found from both estimations. 6.
Compound 1: tH and *SCNMR identical to the literam
: R. Plant&Royon. F. Mona,
Condemine, W. Nasser and J. Robert-Baudouy, J. C~b&y&.
Gem., 1991,10,787
D. Anker, G.
; compound 4: lH
NMR (250 MHz, m0, HOD= 4.80 ppm) 6 : 1.77 (dd, J3ax,3eq=13Hz, J3aq4=11.5Hz, H-3ax), 2.22 (dd, J% 4=%2, H&q), t3C NMR (62.9MHz, extemal l&dioxanc refemnce, 6 66.64) 8 : 39.00 C-3, 62.94 C-i, 68.93 C-4, 70.00 C-5, 96.60 C-2, 176.29 C-l. These values are very close to the ones observed for H-3ax and H-feq in 3-deoxy-D&zero-D-galncto-nonulosonic
acid (KDN) which exhibits
the ssme steraochcmistry on C-4 and C-5 ss compound 4: C. Au& and C. Gautheron, J. C&m. Sue., CheRI. Commun., 1987,859. 7 8.
R Oshima and J. Kumanotani, Chem. fen., 1981,943.
9.
T.A. Elzainy, M.M. Hassan and A.M. Elshafei, Ann. Microbial.. 1981,31,9.
10.
[a]*= -26 (c ,2, water at pH 7) ; &era[a$& Carbohyd?. Res., 1%8,8, 193. F. Fern&i, L. Kenne, B. Lindberg and W. Niich,
11. 12. 13.
MM. Hassan, A.M. Allam, A.M. Elshafei and T.A. Elzainy. Egypt. J. Bat.. 1985.28.165. -31.6 fc , 1.67, water at pH 6) : D. Portsmouth, Carbohydr. Res., 1991,210,255.
R. Ramage, A.M. MacLeod and G.W. Rose, Tetrahedron, 1991,47,5625. D-glycaaldchydehyde was either purchased from Fluka or prepared from D-fructose by oxidation with lead tetraacetate : A.S. Perk, Methods in Curb&&. Ckm. , 1962,1,61.