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Chemo-enzymatic transformations of sensitive systems. Preparation of digoxigenin haptens via regioselective lipase mediated hydrolysis
Tetrahedron Letters. Vol. 36, No. 39, pp. 6987-6990, 1995
Pergamon
Elsevier Science Lid Printed in Great Britain 0040-4039/95 $9.50+0.00
0040-4039(95)01446-2
Chemo-Enzymati¢ Transformations of Sensitive Systems. Preparation of Digoxigenin Haptens via Regioselective Lipase Mediated Hydrolysis. Maciej Adamczyk,* John C. Gebler, and Jonathan Grote Depm'tmentof Cbemistry(DgNM), Al~xgtDiagnosticsDivision.AlCottLalxx'alm'i~,100 Abhor Park Road, AldmuPro-k,IL 60064-3500, USA
Abstract: We investigatedthe use of iipase for the preparationof a series of digoxigeninhaptens containing a 3-positioncarboxylicacid functionality.While synthesisof the requiredester derivativeswas straightforward, enzymatictransformationof the terminalesters to the correspondingacids was regioselective,but shown to be dependenton the length of the linkingarm. Thistransformationwas achievedwhile ineservingother sensitivefunctionality.
Digoxin (1) is a steroidal glycoside produced by the plant family Digitalis which is commonly used in the ueatment of various cardiac diseases. 1 Due to the toxicity of digoxin at higher concentrations, it is necessary to measure serum concentrations of the drug to maintain proper dosing. 2 Even though a simple, practical method is via irmnunoassay, monitoring by immunoassay is complicated by the cross-reactivities of the structurally similar metabolites produced by the extensive metabolism which occurs in man.2, 3 In fact, an ongoing controversy exists about whether digoxin immunoassays should correlate with parent drug concentration or reflect the pharmacological activities of digoxin arid its metabolites.4 We were interested in developing new immunogens which would trigger the production of antibodies showing a whole spectrum of reactivity to the steroid backbone and an intact lacmne ring. 5 For recognition of the steroidal portion of digoxin only, we required a digoxigenin immunogen which could be linked at the 3-position. Such an immunogen would orient putative antibodies particularly toward the lactone portion of the steroid. Selective functionalization at the 3-position of digoxigenin is complicated by side reaction at the free 12-hydroxyl. However, treatment of digoxin with acetic anhydride in pyridine acetylates all hydroxy groups on digoxin except the tertiary hydroxyl, and subsequent treatment of the crude product with aqueous HCI fully deglycosylates the steroid, producing selectively 12-acetylated digoxigenin (1). 6 From our experience, we reasoned that an antibody formed from an immunogen using such a hapten would have a binding pocket large enough m sterically ~ t e
the steroidal part of digoxin derivatives.
6987
6988
Utilization of this template allows for prelmafion of a series of alkyl diesters in straightforward fashion by r e a s o n with dim~id derivatives7 as shown below. 0
0
0
0
AcO
= -,o,°.
H
0
,
0
,, F Y
CH2CI2 R ~ ~ ~ ~ r 1
Y'o.
2a-f: R = CH 3 3a-f: R = B n a: n =
[ lipase
I
¢:n=3 d:n=4 *:n=5
~
"~
J
.
0
0---(/
AcO ~)
f'n=6
H
H 4d-f While attempting regioselective transformations in sensitive systems such as rapamycin where traditional organic reactions had failed, 10 we previously recognized the utility of enzymes. We thus decided to investigate the use of lipase for selective ester hydrolysis of the digoxigenin subswates. The lipase (Amano LPL-80) was selected for its purity, and was confirmed to be >95% by SDS-PAGE. The purity of the lipase is important, since we wanted to avoid the effects of other contaminating proteins frequently present in less pure enzyme preparations. Hydrolysis of the digoxigenin methyl ester derivatives 2a-f was first attempted on a small scale (~1 mg at 300 ug/mL). When each of the methyl esters was incubated in a lipase free mauix for several days (as a control), no hydrolysis took place. In the presence of lipase, regioselective hydrolysis of the terminal methyl ester of the linking arm was observed only for the esters containing longer linking arms (n = 5 and 6, 2e and 2f, Table 1). No other products resulting from cleavage of the internal 3-position ester, lactone, or 12-acetoxy group were observed. In the previous work with rapamycin, we had observed that the susceptibility of the substrate toward lipase-mediated hydrolysis could be manipulated by changing the lipophilicity of the terminal ester. 10 We
6989
synthesized the corresponding series of benzyl esters 3a-f, and subjected them to the conurol matrix and lipase containing n~ctions. While no hydrolysis took place in the lipase free matrix, terminal benzyl esters 3¢-f were smoothly transformed to the corresponding acids by lipase-mediated deprotection (Table 1). Also,
Table 1. Hydrolysis of digoxigenin methyl esters 2a.f and benzyl esters 3a.f after 12 hrs.11
Ester
HPLC Retention Time 11 E,ster Acid
% Conversion
2a 2b 2c 2d 2e 2f
5.5 rain 6.3 rain 7.5 rain 9.1 rain 12.0 rain 16.4 rain
--------4.3 rain 4.9 rain
<1% <1% <1% <1% >99% >99%
3a 3b 3c 3d 3e 3f
8.1 rain 9.3 rain 11.5 min 13.1 rain 17.7 rain 24.1 rain
--3.0 rain 3.1 rain 3.5 rain 3.7 rain 4.2 rain
<1% 12% 59% >99% >99% >99%
in this case, no products resulting from the cleavage of the internal 3-position ester, lactone, or the 12acetoxy group were observed. We believe that the increased lipophilic nature of a benzyl ester as compared to a methyl ester is indeed the reason for the greater susceptibility of the terminal benzyl esters with shorter linking arms (3c and 3d) to lipase hydrolysis. This ehemo-enzyrnatie procedure was effectively used to hydrolyze each of the reactive benzyl esters (n = 4-6) on a larger scale (>100 mg),12 resulting in yields of acids 4d-f of -70%. It should be noted that this transformation was selective for terminal esters, and was achieved in a molecule containing two other esters and a laetone. Additionally, this lipase-mediated transformation served as an attractive, mild method for the cleavage of a benzyl ester, an alternative to benzyl ester deprotection via reduction.
References 1. Soldin, S.J. Clin Chem. 1986, 32, 5-12. 2. Stone, J.A.; Soldin, S.J. Clin. Chem. 1989, 35, 1326-1331. 3. For a recent new metabolite synthesis, see Adamczyk, M.; Grote, J. Tetrahedron Lett. 1995, 36, 63-66. 4. Miller, J.J,; Straub, R.W.; Valdes, R., Jr. Clin. Chem. 1994, 40, 1898-1903.
6990
5. Humber, D.C.; Jones, P.S.; Phillips, G,H. Steroids 1983, 42, 171-188. 6. Hanser, E.; Boffo, U.; Meister, L. 8awlewiez, L.; Linde, H.; Meyer, K. Heir. Chim. Acta. 1973, 56, 2782-2795. 7. The monomethyl diacid chlorides used to homologate the 12-acetyl-digoxigenin were ~ i a l l y available, while the mmotg~zyl diacid chlorides were prepared by treatment of corresponding benzyl half-est~ 8 with oxalyl chiot'ide.9 8. English, A. R.; Girard, D.; Jasys, V. J.; Martingano, R. J.; Kellogg, M.S.J. Med. Chem. 1990, 33, 344347. 9. Bosshard, H. H.; Mory, R.; Schmid, M. Helv. Chim. Acta. 1959, 42, 1653-1658. 10. Adamczyk, M., Gebler, J. C., and Mattingly, P. G. Tetrahedron Lett. 1994, 35, 1019-1022. 11. Analytical I-IPLC was performned on a 250 x 3.2 nun C18 (Phenongnex Primesphere 5 it) column: flowrate 0.5 mIJmin, 70:30 acetonitrile:50 mM ammonium acetate, 55 "C, with detection at 220 nm. 12. Ester 3e (381 rag, 0.57 ~ n o l ) was dissolved in 100 ml acetonitrile, and added to 230 ml ofpH 6.6 H 4 O A c buffer containing 230 mg lipase. After stirring for 8 hrs, the reaction was judged complete by HPLC. Most of the acetonitrile was evaporated in a stream of nitrogen, and the remaining solution lyophilized to dryness. The residue was extracted 3 x 50 ml with EtOAc. The combined extracts were dried over MgSO4 and concentrated to a thick oil. The oil was dissolved in methanol, and purified by preparative HPLC, eluting with 65% methanol/35% 40 mm NH4OAc. The fractions were combined, and lyophilized to dryness, to provide 229 mg (71%) of acid 4e as a white solid: 1H NMR (CDC13) 5 5.85 (1H, s), 5.08 (1H, s), 4.89 (1H, dd, J = 18.0 and 1.5 Hz), 4.77 (1H, dd, J = 18.0 and 1.7 Hz), 4.62 (1H, dd, J = 11.9 and 4.0 Hz), 2.90 (1H, dd, J = 9.0 and 6.3 Hz), 2.37-2.28 (4H, m), 2.19-2.11 (1H, m), 2.10 (3H, s), 2.06-1.20 (6H, m), 0.96 (3H, s), 0.89 (3H, s); 13C NMR (CDC13) 5 178.8, 174.4, 173.5, 173.0, 117.9, 85.7, 77.2, 73.3, 69.9, 53.9, 45.9, 41.2, 36.8, 35.1, 34.2, 33.7, 33.1, 33.0, 32.2, 30.4, 30.3, 28.4, 27.1, 26.4, 26.2, 25.0, 24.6, 24.3, 23.5, 21.4,21.2, 10.3; MS 575 (M + H)+, 592 (M + NH4)+. Anal. Calcd for C32I-I4609: C 66.88, H 8.07. Found: C, 66.66; H, 8.16.
(Received in USA 9 June 1995; revised 24 July 1995; accepted 27 July 1995)
Pergamon
Elsevier Science Lid Printed in Great Britain 0040-4039/95 $9.50+0.00
0040-4039(95)01446-2
Chemo-Enzymati¢ Transformations of Sensitive Systems. Preparation of Digoxigenin Haptens via Regioselective Lipase Mediated Hydrolysis. Maciej Adamczyk,* John C. Gebler, and Jonathan Grote Depm'tmentof Cbemistry(DgNM), Al~xgtDiagnosticsDivision.AlCottLalxx'alm'i~,100 Abhor Park Road, AldmuPro-k,IL 60064-3500, USA
Abstract: We investigatedthe use of iipase for the preparationof a series of digoxigeninhaptens containing a 3-positioncarboxylicacid functionality.While synthesisof the requiredester derivativeswas straightforward, enzymatictransformationof the terminalesters to the correspondingacids was regioselective,but shown to be dependenton the length of the linkingarm. Thistransformationwas achievedwhile ineservingother sensitivefunctionality.
Digoxin (1) is a steroidal glycoside produced by the plant family Digitalis which is commonly used in the ueatment of various cardiac diseases. 1 Due to the toxicity of digoxin at higher concentrations, it is necessary to measure serum concentrations of the drug to maintain proper dosing. 2 Even though a simple, practical method is via irmnunoassay, monitoring by immunoassay is complicated by the cross-reactivities of the structurally similar metabolites produced by the extensive metabolism which occurs in man.2, 3 In fact, an ongoing controversy exists about whether digoxin immunoassays should correlate with parent drug concentration or reflect the pharmacological activities of digoxin arid its metabolites.4 We were interested in developing new immunogens which would trigger the production of antibodies showing a whole spectrum of reactivity to the steroid backbone and an intact lacmne ring. 5 For recognition of the steroidal portion of digoxin only, we required a digoxigenin immunogen which could be linked at the 3-position. Such an immunogen would orient putative antibodies particularly toward the lactone portion of the steroid. Selective functionalization at the 3-position of digoxigenin is complicated by side reaction at the free 12-hydroxyl. However, treatment of digoxin with acetic anhydride in pyridine acetylates all hydroxy groups on digoxin except the tertiary hydroxyl, and subsequent treatment of the crude product with aqueous HCI fully deglycosylates the steroid, producing selectively 12-acetylated digoxigenin (1). 6 From our experience, we reasoned that an antibody formed from an immunogen using such a hapten would have a binding pocket large enough m sterically ~ t e
the steroidal part of digoxin derivatives.
6987
6988
Utilization of this template allows for prelmafion of a series of alkyl diesters in straightforward fashion by r e a s o n with dim~id derivatives7 as shown below. 0
0
0
0
AcO
= -,o,°.
H
0
,
0
,, F Y
CH2CI2 R ~ ~ ~ ~ r 1
Y'o.
2a-f: R = CH 3 3a-f: R = B n a: n =
[ lipase
I
¢:n=3 d:n=4 *:n=5
~
"~
J
.
0
0---(/
AcO ~)
f'n=6
H
H 4d-f While attempting regioselective transformations in sensitive systems such as rapamycin where traditional organic reactions had failed, 10 we previously recognized the utility of enzymes. We thus decided to investigate the use of lipase for selective ester hydrolysis of the digoxigenin subswates. The lipase (Amano LPL-80) was selected for its purity, and was confirmed to be >95% by SDS-PAGE. The purity of the lipase is important, since we wanted to avoid the effects of other contaminating proteins frequently present in less pure enzyme preparations. Hydrolysis of the digoxigenin methyl ester derivatives 2a-f was first attempted on a small scale (~1 mg at 300 ug/mL). When each of the methyl esters was incubated in a lipase free mauix for several days (as a control), no hydrolysis took place. In the presence of lipase, regioselective hydrolysis of the terminal methyl ester of the linking arm was observed only for the esters containing longer linking arms (n = 5 and 6, 2e and 2f, Table 1). No other products resulting from cleavage of the internal 3-position ester, lactone, or 12-acetoxy group were observed. In the previous work with rapamycin, we had observed that the susceptibility of the substrate toward lipase-mediated hydrolysis could be manipulated by changing the lipophilicity of the terminal ester. 10 We
6989
synthesized the corresponding series of benzyl esters 3a-f, and subjected them to the conurol matrix and lipase containing n~ctions. While no hydrolysis took place in the lipase free matrix, terminal benzyl esters 3¢-f were smoothly transformed to the corresponding acids by lipase-mediated deprotection (Table 1). Also,
Table 1. Hydrolysis of digoxigenin methyl esters 2a.f and benzyl esters 3a.f after 12 hrs.11
Ester
HPLC Retention Time 11 E,ster Acid
% Conversion
2a 2b 2c 2d 2e 2f
5.5 rain 6.3 rain 7.5 rain 9.1 rain 12.0 rain 16.4 rain
--------4.3 rain 4.9 rain
<1% <1% <1% <1% >99% >99%
3a 3b 3c 3d 3e 3f
8.1 rain 9.3 rain 11.5 min 13.1 rain 17.7 rain 24.1 rain
--3.0 rain 3.1 rain 3.5 rain 3.7 rain 4.2 rain
<1% 12% 59% >99% >99% >99%
in this case, no products resulting from the cleavage of the internal 3-position ester, lactone, or the 12acetoxy group were observed. We believe that the increased lipophilic nature of a benzyl ester as compared to a methyl ester is indeed the reason for the greater susceptibility of the terminal benzyl esters with shorter linking arms (3c and 3d) to lipase hydrolysis. This ehemo-enzyrnatie procedure was effectively used to hydrolyze each of the reactive benzyl esters (n = 4-6) on a larger scale (>100 mg),12 resulting in yields of acids 4d-f of -70%. It should be noted that this transformation was selective for terminal esters, and was achieved in a molecule containing two other esters and a laetone. Additionally, this lipase-mediated transformation served as an attractive, mild method for the cleavage of a benzyl ester, an alternative to benzyl ester deprotection via reduction.
References 1. Soldin, S.J. Clin Chem. 1986, 32, 5-12. 2. Stone, J.A.; Soldin, S.J. Clin. Chem. 1989, 35, 1326-1331. 3. For a recent new metabolite synthesis, see Adamczyk, M.; Grote, J. Tetrahedron Lett. 1995, 36, 63-66. 4. Miller, J.J,; Straub, R.W.; Valdes, R., Jr. Clin. Chem. 1994, 40, 1898-1903.
6990
5. Humber, D.C.; Jones, P.S.; Phillips, G,H. Steroids 1983, 42, 171-188. 6. Hanser, E.; Boffo, U.; Meister, L. 8awlewiez, L.; Linde, H.; Meyer, K. Heir. Chim. Acta. 1973, 56, 2782-2795. 7. The monomethyl diacid chlorides used to homologate the 12-acetyl-digoxigenin were ~ i a l l y available, while the mmotg~zyl diacid chlorides were prepared by treatment of corresponding benzyl half-est~ 8 with oxalyl chiot'ide.9 8. English, A. R.; Girard, D.; Jasys, V. J.; Martingano, R. J.; Kellogg, M.S.J. Med. Chem. 1990, 33, 344347. 9. Bosshard, H. H.; Mory, R.; Schmid, M. Helv. Chim. Acta. 1959, 42, 1653-1658. 10. Adamczyk, M., Gebler, J. C., and Mattingly, P. G. Tetrahedron Lett. 1994, 35, 1019-1022. 11. Analytical I-IPLC was performned on a 250 x 3.2 nun C18 (Phenongnex Primesphere 5 it) column: flowrate 0.5 mIJmin, 70:30 acetonitrile:50 mM ammonium acetate, 55 "C, with detection at 220 nm. 12. Ester 3e (381 rag, 0.57 ~ n o l ) was dissolved in 100 ml acetonitrile, and added to 230 ml ofpH 6.6 H 4 O A c buffer containing 230 mg lipase. After stirring for 8 hrs, the reaction was judged complete by HPLC. Most of the acetonitrile was evaporated in a stream of nitrogen, and the remaining solution lyophilized to dryness. The residue was extracted 3 x 50 ml with EtOAc. The combined extracts were dried over MgSO4 and concentrated to a thick oil. The oil was dissolved in methanol, and purified by preparative HPLC, eluting with 65% methanol/35% 40 mm NH4OAc. The fractions were combined, and lyophilized to dryness, to provide 229 mg (71%) of acid 4e as a white solid: 1H NMR (CDC13) 5 5.85 (1H, s), 5.08 (1H, s), 4.89 (1H, dd, J = 18.0 and 1.5 Hz), 4.77 (1H, dd, J = 18.0 and 1.7 Hz), 4.62 (1H, dd, J = 11.9 and 4.0 Hz), 2.90 (1H, dd, J = 9.0 and 6.3 Hz), 2.37-2.28 (4H, m), 2.19-2.11 (1H, m), 2.10 (3H, s), 2.06-1.20 (6H, m), 0.96 (3H, s), 0.89 (3H, s); 13C NMR (CDC13) 5 178.8, 174.4, 173.5, 173.0, 117.9, 85.7, 77.2, 73.3, 69.9, 53.9, 45.9, 41.2, 36.8, 35.1, 34.2, 33.7, 33.1, 33.0, 32.2, 30.4, 30.3, 28.4, 27.1, 26.4, 26.2, 25.0, 24.6, 24.3, 23.5, 21.4,21.2, 10.3; MS 575 (M + H)+, 592 (M + NH4)+. Anal. Calcd for C32I-I4609: C 66.88, H 8.07. Found: C, 66.66; H, 8.16.
(Received in USA 9 June 1995; revised 24 July 1995; accepted 27 July 1995)