A versatile and selective chemo-enzymatic synthesis of β-protected aspartic and γ-protected glutamic acid derivatives

Article abstract of DOI:10.1016/j.tetlet.2009.03.130

Two versatile, high yielding, and efficient chemo-enzymatic methods for the synthesis of β-protected Asp and γ-protected Glu derivatives using Alcalase are described. The first method is based on the α-selective enzymatic hydrolysis of symmetrical aspartyl and glutamyl diesters. The second method involving mixed diesters comprises a three-step protocol using (i) α-selective enzymatic methyl-esterification, (ii) chemical β-esterification, and finally (iii) α-selective enzymatic methyl ester hydrolysis. The yields of the purified β- and γ-esters range from 77% to 91%.

Full text of DOI:10.1016/j.tetlet.2009.03.130

Tetrahedron Letters 50 (2009) 2719–2721
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
A versatile and selective chemo-enzymatic synthesis of b-protected aspartic
and c-protected glutamic acid derivatives
Timo Nuijens a,b, John A. W. Kruijtzer b, Claudia Cusan a, Dirk T. S. Rijkers b, Rob M. J. Liskamp b,
,
*
Peter J. L. M. Quaedflieg a,
*
a DSM Pharmaceutical Products, Innovative Synthesis and Catalysis, PO Box 18, NL-6160 MD Geleen, The Netherlands
b Medicinal Chemistry and Chemical Biology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, PO Box 80082, NL-3508 TB Utrecht, The Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
Two versatile, high yielding, and efficient chemo-enzymatic methods for the synthesis of b-protected Asp
Received 19 December 2008
Revised 6 March 2009
Accepted 17 March 2009
Available online 21 March 2009
and
enzymatic hydrolysis of symmetrical aspartyl and glutamyl diesters. The second method involving mixed
diesters comprises a three-step protocol using (i) -selective enzymatic methyl-esterification, (ii) chem-
ical b-esterification, and finally (iii) -selective enzymatic methyl ester hydrolysis. The yields of the puri-
fied b- and -esters range from 77% to 91%.
c-protected Glu derivatives using Alcalase are described. The first method is based on the a-selective
a
a
c
Keywords:
Enzymes
Ó 2009 Elsevier Ltd. All rights reserved.
Amino acids
Hydrolysis
Esters
Protecting groups
Protecting groups play a pivotal role in organic chemistry and
especially in peptide, carbohydrate, or nucleic acid synthesis. In
peptide synthesis, amine functionalities are usually protected as
carbamates, and carboxylic acid moieties are most often protected
as esters. A vast variety of esters have been developed to protect C-
terminal and side-chain carboxylic acids for different applications.
Orthogonality of these protecting groups and resistance to cou-
pling and deprotection reaction conditions are key factors for high
yields and for easy workup. For instance, allyl (All) esters, cleavable
with Pd(0),1 and trimethylsilylethyl (TMSE) esters, cleavable with
TBAF,2 are orthogonal to other commonly used protecting groups
such as tert-butyl (tBu) or methyl (Me) esters allowing selective
deprotection and modification of certain carboxylic acids during
peptide synthesis. Of special interest are selectively protected
aspartic acid (Asp) or glutamic acid (Glu) building blocks, which
A number of synthetic strategies have been disclosed for
(semi-)selective b- and
c-protection of Asp and Glu derivatives,
respectively.7 The most commonly used methods rely on the intra-
molecular anhydride formation of N-protected Asp/Glu derivatives
using a condensing reagent followed by moderately selective ring
opening with a nucleophile.8 Whereas with Glu residues a rela-
tively good selectivity of 9/1 of
c- over a-ester can be obtained,
selective b-protection of Asp residues remains difficult and is often
low yielding.
Only a few chemo-enzymatic approaches toward aspartyl b-es-
ters have been reported in the literature. These approaches are based
on the a-selective hydrolysis of Asp diesters. However, the reported
a
-selective hydrolysis with porcine liver esterase is only applicable
for N-terminal unprotected aspartic acid derivatives.9 Most prote-
ases proved to be unselective when N-unprotected aspartyl diesters
were used. Hydrolysis of N-protected aspartyl diesters with
papain,10 is limited to Cbz-Asp(OAll)-OAll. The very common N-ter-
are protected either at their
a-carboxylic acid functionality or at
their b-(Asp) or -(Glu) carboxylic acid moiety. These esters find
c
widespread application in on-resin synthesis of head-to-tail cyclic
peptides3 (using both Fmoc/tBu- and Boc/Bzl SPPS approaches),
side-chain lactam peptides,4 and branched peptides.5 Recently,
minal Fmoc/Boc-protected b-esters of Asp and
not been prepared enzymatically. Herein we demonstrate that the
cheap industrial protease Alcalase11 can catalyze the
-selective
c-esters of Glu have
a
we described the selective
a
-carboxylic acid esterification of
hydrolysis of a wide range of N-protected Asp and Glu symmetrical
diesters. In addition, we report a versatile and high yielding three-
step protocol for the synthesis of N-protected b-aspartyl esters.
Alcalase is often used for the hydrolysis and/or resolution of
amino acid esters.12 Recently,6 we identified a versatile synthetic
method for various N-protected amino acid esters (i.e., methyl,
ethyl, benzyl, tert-butyl, allyl, and trimethylsilylethyl) using
N-protected amino acids, including Asp and Glu residues, using
the industrial enzyme Alcalase.6 Selective synthesis of b-protected
Asp and c-protected Glu derivatives however, remains a challenge.
* Corresponding authors. Tel.: +31 6 52610991; fax: +31 46 4767604 (P.J.L.M.Q.).
E-mail address: peter.quaedflieg@dsm.com (P.J.L.M. Quaedflieg).
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.03.130

2720
T. Nuijens et al. / Tetrahedron Letters 50 (2009) 2719–2721
Alcalase-cross-linked-enzyme-aggregates (CLEA)13 in dry organic
solvents. The hydrolysis of these esters proved to be easy and
quantitative. Encouraged by these results, we decided to focus on
Table 1
HPLC and isolated yields of the synthesized b- and c-esters
Compound
Product
HPLC yield (%)
Isolated yield (%)
the
a-selective hydrolysis of N-protected aspartyl and glutamyl
1
2
3
4
5
6
7
8
Cbz-Asp(OAll)-OH
Cbz-Glu(OAll)-OH
Boc-Asp(OAll)-OH
Boc-Glu(OAll)-OH
Fmoc-Asp(OAll)-OH
Fmoc-Glu(OAll)-OH
Cbz-Asp-(OTMSE)-OH
Fmoc-Asp-(OPTMSE)-OH
96
97
98
98
95
97
95
96
87
85
87
86
77
78
85
n.d.
diesters (Scheme 1).
N-protected aspartyl (n = 1) and glutamyl (n = 2) diesters 1–8
were synthesized using EDC and the appropriate alcohol.14 These
symmetrical diesters were hydrolyzed using Alcalase-CLEA in
water at pH 7.5 using tBuOH or 1,4-dioxane as a co-solvent for dis-
solving the starting materials.15
Gratifyingly, all the hydrolytic reactions were completely
a
-selective, that is, no simultaneous b- or
observed by HPLC. As shown in Table 1, very high yields of b-aspar-
tyl and -glutamyl esters were obtained using a variety of
c-ester hydrolysis was
O
O
c
H
N
H
N
a
N-protecting groups. To our surprise, even sterically hindered di-
Cbz
OH
Cbz
OCH3
(2-phenyl-2-trimethylsilyl)ethyl (PTMSE) esters16 of Asp were
O
O
easily and
a-selectively hydrolyzed by Alcalase-CLEA. The newly
synthesized esters were analyzed by NMR17 and were found to
be identical to those reported in the literature18 or to commercially
OH
9
OH
available samples,19 which clearly proved the selective
sis of all the diesters used in this study.
a-hydroly-
b
However, as also disclosed by others,20 the di-tBu-ester deriva-
tives of Asp and Glu were not easily hydrolyzed enzymatically. We
O
O
H
N
H
N
envisioned that, by combining
Asp esters with chemical b-esterification followed by an
a
-selective synthesis of N-protected
-selec-
c
Cbz
OH
Cbz
O
OCH3
a
O
tive hydrolysis of the resulting diesters, we could obtain a feasible
approach toward b-protected Asp derivatives. Additionally, this
alternative strategy avoids the use of 2 equiv of an expensive
(e.g., TMSE-OH or PTMSE-OH) alcohol by preparation of a cheap
O
11
O
10
R
R
R = All or tBu
methyl ester as a temporary protecting group of the
acid followed by chemical esterification of the b- or
moiety (Scheme 2 and Table 2).
a
c
-carboxylic
-carboxylic
Scheme 2. Reaction conditions: (a) Alcalase-CLEA, 3 Å MS, MBTE/MeOH (14/1, v/v),
50 °C, 40 h; (b) EDC, DIPEA, AllOH, rt, 24 h or Boc2O, pyridine, DMAP, CH3CN/tBuOH
(2/1, v/v), rt, 24 h; (c) Alcalase-CLEA, pH 7.5 phosphate buffer/tBuOH or 1.4-dioxane
(1/1, v/v), 37 °C, 16 h.
As shown in Table 2, the
a
-methyl esterification21 proceeded
smoothly and the chemical b-esterification, by activation with
either EDC22 for the All-ester or with Boc2O23 for the tBu-ester, fur-
nished the desired esters in high yields. Much to our satisfaction,
Table 2
subsequent Alcalase-CLEA-mediated enzymatic hydrolysis of 10
Yields of intermediates and Asp b-ester products
t
(R = All or Bu) proceeded completely
a-selectively giving the de-
Compound
Product
Isolated yield (%)
sired b-protected Asp derivatives 11 in high yields.24 Although this
approach required three-reaction steps, the overall yields (76% for
Cbz-Asp(OAll)-OH) were considerably higher compared to the
overall yields obtained via hydrolysis of the diesters (57%) or those
obtained by chemical means (around 50%). Even more importantly,
this method allowed the synthesis of aspartyl b-esters which are
9
Cbz-Asp-OMe
93
94
92
91
89
10a (R = tBu)
10b (R = All)
11a (R = tBu)
11b (R = All)
Cbz-Asp(OtBu)-OMe
Cbz-Asp(OAll)-OMe
Cbz-Asp(OtBu)-OH
Cbz-Asp(OAll)-OH
not available via the diester hydrolysis method, for example, Cbz-
Asp(OtBu)-OH.
In conclusion, via two attractive approaches, we have demon-
strated that the complete a-selectivity of Alcalase-CLEA in the syn-
thesis and hydrolysis of various esters of N-protected Asp and Glu
derivatives, can be utilized to prepare b-esters of N-protected
aspartic acid or c-esters of N-protected glutamic acid in high yields
and purity. These derivatives are very useful for the synthesis of
O
O
O
H
N
H
N
H
N
R2
b
a
R1
O
OH
R2
R1
O
O
R1
O
OH
n
n
n
O
O
OH
R1
R2
R2
#
n
peptides and peptide derivatives.
1
2
3
4
5
6
7
8
Cbz
Cbz
Boc
Boc
Fmoc
Fmoc
Cbz
All
All
All
All
All
All
TMSE
PTMSE
1
2
1
2
1
2
1
1
References and notes
1. Willson, T. M.; Kocienski, P.; Jarowicki, K.; Isaac, K.; Faller, A.; Campbell, S. F.;
Bordner, J. Tetrahedron 1990, 46, 1757–1766.
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3. Lambert, J. N.; Mitchell, J. P.; Roberts, K. D. J. Chem. Soc., Perkin Trans. 1 2001,
471–484.
4. Taylor, J. W. Biopolymers 2002, 66, 49–75.
Fmoc
5. Delforge, D.; Dieu, M.; Delaive, E.; Art, M.; Gillon, B.; Devreese, B.; Raes, M.; Van
Beeumen, J.; Remacle, J. Lett. Pept. Sci. 1996, 3, 89–97.
6. Nuijens, T.; Cusan, C.; Kruijtzer, J. A. W.; Rijkers, D. T. S.; Liskamp, R. M. J.;
Quaedflieg, P. J. L. M. Synthesis 2009, 5, 809–814.
Scheme 1. Reaction conditions: (a) EDC, HOAt, DIPEA, CH2Cl2/R2OH (25/1, v/v), rt,
24 h. (b) Alcalase-CLEA, pH 7.5 phosphate buffer/tBuOH or 1,4-dioxane (1/1, v/v),
37 °C, 16 h.

T. Nuijens et al. / Tetrahedron Letters 50 (2009) 2719–2721
2721
7. (a) Benoiton, N. L.; Chen, F. M. F. Int. J. Pept. Protein Res. 1994, 43, 321–324; (b)
Belshaw, P. J.; Adamson, J. G.; Lajoie, G. A. Synth. Commun. 1992, 22, 1001–
1005; (c) Baldwin, J. E.; Moloney, M. G.; North, M. Tetrahedron 1989, 45, 6319–
6330; (d) Belshaw, P. J.; Mzengeza, S.; Lajoie, G. A. Synth. Commun. 1990, 20,
3157–3160; (e) Baldwin, J. E.; North, M.; Flinn, A.; Moloney, M. G. Tetrahedron
1989, 45, 1453–1464.
8. (a) Jouln, P.; Castro, B.; Zeggaf, C.; Pantaloni, A. Tetrahedron Lett. 1987, 28,
1665–1668; (b) Cooper, J. W.; Waters, M. L. Org. Lett. 2005, 7, 3825–3828; (c)
Huang, X.; Luo, X.; Roupioz, Y.; Keillor, J. W. J. Org. Chem. 1997, 62, 8821–8825;
(d) Chen, F. M. F.; Benoiton, N. L. Int. J. Pept. Protein Res. 1992, 40, 13–18.
9. Stein, K. A.; Toogood, P. L. J. Org. Chem. 1995, 60, 8110–8112.
10. Xaus, N.; Clapés, P.; Bardaji, E.; Torres, X.; Horba, X.; Mata, J.; Valencia, G.
Tetrahedron 1989, 45, 7421–7426.
11. Obtained from Novozymes, 10 wt % Alcalase solution, 6.75 €/kg (a) Maurer, K.
H. Curr. Opin. Biotechnol. 2004, 15, 330; (b) Smith, E. L.; DeLange, R. J.; Evans, W.
H.; Landon, M.; Markland, F. S. J. Biol. Chem. 1968, 243, 2184; (c) Siezen, R. J.;
Leunissen, J. J. Protein Sci. 1997, 6, 501.
16. (a) Wagner, M.; Kunz, H. Synlett 2000, 3, 400–402; (b) Wagner, M.; Kunz, H. Z.
Naturforsch. 2002, 57b, 928–936.
17. Cbz-Glu(OAll)-OH: 1H NMR (300 MHz, CDCl3) d = 1.94–1.99 (m, 1H), 2.10–2.15
(m, 1H), 2.33–2.39 (m, 2H), 4.24–4.27 (m, 1H), 4.35 (d, J = 6.0 Hz, 2H), 5.01 (s,
2H), 5.13–5.25 (m, 2H), 5.75 (d, J = 7.9 Hz, 1H), 5.74–5.85 (m, 1H), 7.19–7.26
(m, 5H). 13C NMR (75 MHz, CDCl3) d = 27.2, 39.9, 53.4, 65.7, 67.5, 118.7, 128.2,
128.4, 128.7, 132.0, 136.1, 156.4, 177.4, 178.4; Cbz-Asp(OTMSE)-OH: 1H NMR
(300 MHz, CDCl3) d = 0.00 (s, 9H), 0.92–1.01 (m, 2H), 2.85 (dd, J = 4.4 and
17.2 Hz, 1H), 2.90 (dd, J = 4.0 and 16.8 Hz, 1H), 4.18–4.25 (m, 2H), 4.58–4.64
(m, 1H), 5.10 (s, 2H), 5.84 (d, J = 8.1 Hz, 1H), 7.30–7.39 (m, 5H). 13C NMR
(75 MHz, CDCl3) d = À1.5, 17.3, 36.7, 50.4, 53.5, 64.5, 136.2, 159.9, 170.9, 175.4.
18. Cbz-Asp(OAll)-OH: Ginisty, M.; Gravier-Pelletier, C.; Le Merrer, Y. Tetrahedron:
Asymmetry 2006, 17, 142; Boc-Asp(OAll)-OH, Fmoc-Asp(OAll)-OH and Fmoc-
Glu(OAll)-OH: Webster, K. L.; Maude, A. B.; O’Donnell, M. E.; Mehrotra, A. P.;
Gani, D. J. Chem. Soc., Perkin Trans. 1 2001, 1673–1695.
19. Boc-Glu(OAll)-OH: CAS 132286-79-4.
20. Eggen, I.F.; Boeriu, C.G. WO Pat. Appl. No. 2007 082890, CAS No. 147:210280
HCA.
12. Chen, S. T.; Wang, K. T.; Wong, C. H. J. Chem. Soc., Chem. Commun. 1986, 1514–1517.
13. Sheldon, R. A. Biochem. Soc. Trans. 2007, 35, 1583.
21. Alcalase-CLEA (3 g) was added to 0.5 g of Cbz-Asp-OH, 28.0 mL of MTBE, 2.0 mL
of MeOH, and 2.0 g of 3 Å molecular sieves. The mixture was shaken at 50 °C at
150 rpm for 16 h. After filtration, the enzyme was washed thoroughly with
aqueous HCl (pH 1, 3 Â 50 mL) and EtOAc (3 Â 50 mL) followed by filtration.
The combined organic layers were washed with 100 mL of aqueous HCl (pH 1),
dried (Na2SO4), and concentrated in vacuo. The resulting oil was redissolved in
20 mL of CH2Cl2/MeOH/AcOH (89.9:10:0.1) followed by a filtration over silica
gel. The mixture was concentrated in vacuo and dried by co-evaporation with
50 mL of toluene (2Â) and 50 mL of CHCl3 (2Â). The NMR data were identical to
those of the commercially obtained compound.
14. Diallyl ester synthesis of N-protected Asp and Glu; general procedure: 2.0 g of
N-protected amino acid, 2.1 equiv of N-(3-dimethylaminopropyl)-N’-
ethylcarbodiimide. HCl (EDCÁHCl), 2.1 equiv of 7-aza-N-hydroxybenzotriazole
(HOAt), and 2.1 equiv of diisopropylethylamine (DIPEA) were dissolved in
100 mL of CH2Cl2 containing 4.0 mL of the appropriate alcohol. The mixture
was stirred at ambient temperature for 24 h and concentrated in vacuo. The
resulting oil was partitioned between 100 mL of EtOAc and 100 mL of saturated
aqueous NaHCO3. The organic phase was washed with 100 mL of saturated
aqueous NaHCO3, 100 mL of aqueous HCl (pH 1, 2Â), 100 mL of brine and
concentrated in vacuo. The resulting oil was purified by preparative HPLC
(using 15% of 0.1 mL/L formic acid in H2O and 85% of 0.1 mL/L formic acid in
acetonitrile as eluent).
22. Cbz-Asp-OMe (281 mg, 1.0 mmol) was dissolved in 50 mL of EtOAc.
Subsequently, 209 mg of EDCÁHCl (1.1 mmol), 192
lL of DIPEA (1.1 mmol),
and 2.0 mL of All-OH were added. The reaction mixture was stirred for 24 h at
rt. The mixture was washed with 50 mL of saturated aqueous NaHCO3, 50 mL
of aqueous HCl (pH 3, 2Â), 50 mL of brine, filtered over basic alumina, dried
(Na2SO4) and concentrated in vacuo. NMR data corresponded to those reported
by: Sears, P.; Schuster, M.; Wang, P.; Witte, K.; Wong, C. H. J. Am. Chem. Soc.
1994, 116, 6521–6530.
15. Enzymatic
a-hydrolysis of diallyl-protected Asp and Glu; general procedure:
3 g of Alcalase-CLEA (purchased from Codexis (Jülich, Germany) and used as
received) was added to 0.5 g of diallyl-protected amino acid, 15.0 mL of tBuOH
or 1,4-dioxane, and 15.0 mL of phosphate buffer (pH 7.5, 50 mM). The mixture
was shaken at 37 °C at 200 rpm for 16 h. After filtration, the enzyme was re-
suspended in 50 mL of saturated aqueous NaHCO3 followed by filtration. This
procedure was repeated twice with saturated aqueous NaHCO3 and twice with
50 mL of EtOAc. The combined organic phase was washed with saturated
aqueous NaHCO3. The combined aqueous phase was acidified to pH 1 with
aqueous HCl (0.1 N) followed by extraction with 100 mL of EtOAc (3Â). The
combined organic layers were washed with 250 mL of brine, dried over
Na2SO4, concentrated in vacuo, and dried by co-evaporation with 50 mL of
toluene (2Â) and 50 mL of CHCl3 (2Â). Additional column chromatography
with EtOAc/n-hexane mixtures was performed if necessary. The purity of the b-
23. Cbz-Asp-OMe (281 mg, 1.0 mmol) was dissolved in 60 mL of CH3CN/tBuOH (2/
1, v/v). Subsequently, 218 mg of Boc2O (1.0 mmol), 81 lL of pyridine
(1.0 mmol), and 5 mg of (N,N)-dimethylaminopyridine were added. The
reaction mixture was stirred at rt for 24 h and concentrated in vacuo. The
residue was dissolved in 50 mL of EtOAc and washed with 50 mL of saturated
aqueous NaHCO3, 50 mL of aqueous HCl (pH 3, 2Â), 50 mL of brine, filtered
over basic alumina, dried (Na2SO4) and concentrated in vacuo. NMR data
corresponded to those reported by David, C.; Bischoff, L.; Roques, B. P.; Fournie-
Zaluski, M. C. Tetrahedron 1999, 56, 209–215.
or
c
-ester of the N-protected Asp or Glu derivatives, respectively, was >98% as
24. NMR data corresponded with the commercially available reference compound,
determined by HPLC.
Cbz-Asp(OtBu)-OH: CAS. 5545-52-8.