Enzymatic C-terminal amidation of amino acids and peptides

Article abstract of DOI:10.1016/j.tetlet.2012.05.039

Herein, we describe two versatile and high yielding enzymatic approaches for the conversion of semi-protected amino acid and peptidyl C-terminal α-carboxylic acids into their corresponding amides. In the first approach, the lipase Candida antarctica lipase-B (Cal-B), and in the second approach, the protease Subtilisin A, are used, respectively. We found that by using the ammonium salt of the α-carboxylic acid instead of separate ammonia sources, the enzymatic amidation reactions proceeded much faster without side reactions and gave near to quantitative yields of products.

Full text of DOI:10.1016/j.tetlet.2012.05.039

Tetrahedron Letters 53 (2012) 3777–3779
Contents lists available at SciVerse ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Enzymatic C-terminal amidation of amino acids and peptides
Timo Nuijens a,b, Elena Piva a, John A. W. Kruijtzer b, Dirk T. S. Rijkers b, Rob M. J. Liskamp b,
Peter J. L. M. Quaedflieg a,
⇑
a DSM Innovative Synthesis B.V., 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:
Herein, we describe two versatile and high yielding enzymatic approaches for the conversion of semi-
Received 9 March 2012
Revised 2 May 2012
Accepted 10 May 2012
Available online 15 May 2012
protected amino acid and peptidyl C-terminal
the first approach, the lipase Candida antarctica lipase-B (Cal-B), and in the second approach, the protease
Subtilisin A, are used, respectively. We found that by using the ammonium salt of the -carboxylic acid
instead of separate ammonia sources, the enzymatic amidation reactions proceeded much faster without
side reactions and gave near to quantitative yields of products.
Ó 2012 Elsevier Ltd. All rights reserved.
a-carboxylic acids into their corresponding amides. In
a
Keywords:
Enzymes
Amino acids
Peptides
Amidation
Many peptide hormones and pharmaceutically relevant pep-
tides contain a C-terminal primary amide functionality, which is
not only important for their biological activity, but also increases
their stability against exoproteases.1 Furthermore, the primary
amide functionality is often used as a (temporary) protecting group
in chemical and enzymatic peptide synthesis; its cleavage can be
achieved by using peptide amidases such as the peptidase from
the flavedo of oranges.2 Solution phase synthesis of peptides bear-
ing a C-terminal primary amide functionality usually starts from
the C-terminal amino acid carboxamide and the desired peptide
sequence is assembled stepwise in the C?N direction using N-car-
bamate protected amino acid building blocks to minimize racemi-
zation. However, the generally limited solubility of amino acid and
peptide C-terminal amides makes the synthesis of longer peptide
amides rather laborious by the solution phase methodology. There-
fore, peptide amides are mostly synthesized by solid phase peptide
synthesis (SPPS) using, among others, Rink Amide resin or 4-meth-
ylbenzhydrylamine (MBHA) resin in Fmoc- or Boc-SPPS, respec-
tively.3 However, these resins are expensive and not attractive
for large scale use. On the other hand the solid-phase synthesis
of peptide C-terminal carboxylic acids is much more cost-efficient
since cheap and industrially available resins can be used (such as
the 2-chlorotritylchloride resin). Alternatively, several peptide C-
terminal carboxylic acids are accessible via fermentation routes,
while their C-terminal amides are the desired end product. There-
fore, several chemical approaches have been developed to convert
peptide C-terminal carboxylic acids into the corresponding amides.
Unfortunately, these methods lack selectivity toward the C-termi-
nus and usually cause racemization or prove incompatible with la-
bile substrates or other protecting groups.4
In contrast to chemical approaches for modification of amino
acids and peptides, enzymatic conversions proceed under mild
conditions and are often chemo-, regio-, and stereoselective.
Several enzymes are candidates to be used for the amidation of
amino acids and peptidyl C-terminal carboxylic acids. For instance,
a few peptide amidases have been described in the literature, but
they tend to be selective for a limited number of C-terminal amino
acids and cannot be produced on large scale.2 Lipases, such as
Candida antarctica lipase-B (Cal-B), have been used to perform
esterification and subsequent amidation of aliphatic carboxylic
acids, but amidation of peptides by Cal-B has so far not been
reported.5 The C-terminal amidation of amino acid esters and
peptidyl esters has been reported using the protease Subtilisin
A.6 Subtilisin A is a serine endoprotease produced from Bacillus
licheniformis and commercially available as Alcalase in which it is
the main constituent. This amidation reaction has recently been
optimized using C-terminal peptide methyl esters and ammonium
carbamate as the amine source.7 However, the chemical synthesis
of the C-terminal peptide (methyl) esters that are required for the
enzymatic reaction encounters the same disadvantages as the
direct chemical amidation, that is lack of C-terminal selectivity,
racemization issues, and incompatibility with labile substrates or
other protecting groups. Herein, we describe, for the first time,
the direct and C-terminally selective enzymatic amidation of
side-chain protected as well as unprotected amino acids and
⇑
Corresponding author. Tel.: +31 46 4761592.
E-mail address: peter.quaedflieg@dsm.com (P.J.L.M. Quaedflieg).
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.05.039

3778
T. Nuijens et al. / Tetrahedron Letters 53 (2012) 3777–3779
Table 1
Table 2
C-terminal amidation of amino acids and peptides with Cal-B using ammonium
C-terminal amidation of peptide ammonium salts using Alcalase-CLEA in tBuOH/DMF
(82.5/17.5, v/v)a,13
benzoate or NH3 gas in toluenea,9
Entry Amide product
Conversion (%) into
amide using
Conversion (%) into
amide using
Entry
Amide product
Conversion
into amide (%)
Transamidation (%)
ammonium benzoate ammonia gas
1
2
3
4
5
6
7
8
Cbz-Phe-Leu-Ala-NH2
Cbz-Gly-Gly-Ala-NH2
Cbz-Val-Ala-NH2
94
92
9114
89
89
87
87
86
85
85
81
81
81
80
79
78
77
68
53
46
30
1
1
2
3
4
5
6
7
8
9
Cbz-Pro-NH2
Cbz-Ala-NH2
Cbz-Gly-NH2
Cbz-Met-NH2
Cbz-Val-Pro-Pro-NH2
Cbz-Gly-Leu-Ala-NH2
99
95
80
60
90
65
ndb
nd
nd
nd
nd
nd
82
85
56
—
—
—
1
10
Cbz-Ile-Met-NH2
Cbz-Gly-Leu-Ala-NH2
Cbz-Ala-Leu-NH2
Cbz-Ala-Phe-NH2
Cbz-Ala-Ala-NH2
—
—
—
—
—
—
—
—
—
—
—
—
—
4
Cbz-Gly-Phe-Ala-NH2 60
Cbz-Gly-Ile-Ala-NH2
Cbz-Val-Val-Pro-NH2
52
12
9
Cbz-Val-Met-NH2
Cbz-Gly-Pro-Ala-NH2
Cbz-Ala-Met-NH2
Cbz-Tyr-Leu-NH2
Cbz-Gly-Phe-NH2
Cbz-Val-Phe-NH2
Cbz-Leu-Phe-NH2
Cbz-Gly-Ile-Ala-NH2
Cbz-Phe-Leu-NH2
Cbz-Ala-Pro-Leu-NH2
Cbz-Pro-Leu-Gly-NH2
Cbz-Gly-Tyr-NH2
Cbz-Val-Gly-NH2
10
11
12
13
14
15
16
17
18
19
20
21
a
Conversions were determined by HPLC relative to the carboxylic acid starting
material. The amide products were identified by LC-MS analysis and compared with
commercially available reference compounds.
b
Experiment not done.
peptides in high yield starting from the corresponding carboxylic
acids using Cal-B and Subtilisin A.
—
—
The direct amidation of N-protected amino acids and peptides is
energetically unfavorable and should therefore be conducted in dry
organic solvents with removal of the formed water to drive the
equilibrium to the amide product. Additionally, the activation en-
ergy of the amidation reaction starting from the carboxylic acids
is much higher than starting from the ester congeners. Therefore,
longer reaction times are required and side reactions, such as
hydrolysis or transamidation of the peptide backbone, can be ex-
pected. In fact, when we applied the reported optimized amidation
conditions with Alcalase7 on Cbz-Phe-Leu-Ala-OH as the substrate,
we observed 81% conversion of the starting material into the de-
sired product, but also formation of a significant amount (15%) of
side products caused by transamidation and hydrolysis.8 Therefore,
we first tested the versatility of lipases for the direct amidation,
since these enzymes virtually lack the ability to hydrolyze or tran-
samidate peptide bonds. Cal-B, a commercially available lipase ac-
tive in organic solvents, proved to be the most promising candidate
(data of lipase screening not shown). Gratifyingly, in the case of
Cal-B we did not observe any side products during the direct ami-
dation of a number of amino acid and peptide acids, as shown in
Table 1.
The Cal-B catalyzed amidations proceeded smoothly in toluene
using 10 equiv of ammonium benzoate. Up to quantitative yields
were obtained in the presence of molecular sieves to absorb the
liberated water during amide formation (Table 1, entries 1–5).
Although no peptide-based side products were observed, the ami-
dation of benzoic acid turned out to be a serious problem, espe-
cially when more challenging peptide substrates were used
(Table 1, entries 6–9). Therefore, we investigated the possibility
of using a direct NH3 source (e.g., NH3 in 1,4-dioxane) or the
ammonium salt of the corresponding amino acid or peptide acid.
Gratifyingly, the amidation reaction with Cal-B in the presence of
only one equivalent of NH3 gas or NH3/1,4-dioxane (via the amino
acid or peptide ammonium salt) resulted in a much higher yield
and proceeded much faster than in the presence of ammonium
benzoate (Table 1, entries 7–9).9 However, Cal-B appeared rather
restricted in accepting bulky side-chains in its active site, so that
phenylalanine, tyrosine, tryptophan, or arginine and peptides con-
taining these amino acids at the C-terminus, were not converted
into the corresponding amides (data not shown).
a
Conversions were determined by HPLC relative to the carboxylic acid starting
material. Products were identified by LC-MS analysis and compared with com-
mercially available reference compounds when available.
Table 3
a
-Selective amidation of amino acid ammonium salts using Alcalase-CLEA in tBuOH/
DMF (82.5/17.5, v/v)a,13
Entry
Amide product
Conversion (%)
1
2
3
4
5
Cbz-Gln-NH2
Cbz-Ser-NH2
Cbz-Asn-NH2
96
87
70
67
57
b
Cbz-Glu-NH2
b
Cbz-Asp-NH2
a
Conversions were determined by HPLC relative to the carboxylic acid starting
material. The amide products were compared with commercially available refer-
ence compounds and identified by LC-MS analysis.
b
Using 2.1 equiv of NH3 in 1,4-dioxane.
and is also active in organic solvents. We applied this biocatalyst
in the form of cross-linked enzyme aggregates (Alcalase-CLEA,
from CLEA Technologies)12 to achieve maximum stability and
activity in the anhydrous organic solvent applied.
As shown in Table 2, a large number of peptide ammonium salts
could be converted into their corresponding amides using Alcalase.
Surprisingly, transamidation did not occur at all in most substrates,
or to a very limited extent in a few. High conversions were ob-
tained for almost all the substrates and by increasing the reaction
time or the amount of enzyme, even the amidation of more chal-
lenging substrates, such as Cbz-Val-Gly-NH2 (entry 21), could be
brought to full completion. To avoid any hydrolysis, the amidation
reactions were run in the presence of molecular sieves. To show
the versatility of the amidation reaction, and to demonstrate the
complete specificity toward
was also applied to amino acids containing reactive side-chain
functionalities, as shown in Table 3.
The amidation of amino acids turned out to be slower than the
amidation of peptide acids. Nevertheless, no side reactions oc-
a-carboxylic acids, Alcalase-CLEA
curred and full
a-selectivity was observed when b- or c-carboxylic
Hence, we investigated the use of Subtilisin A in the direct ami-
dation reaction of amino acid and peptide C-terminal carboxylic
ammonium salts under anhydrous conditions. It is known11 that
this serine endoprotease has a very broad substrate tolerance
acids were present (entries 4 and 5).
Finally, we applied the direct amidation approach to the tetra-
mer fragment Tyr-Met-Asp-Phe with several degrees of protection
(Table 4).

T. Nuijens et al. / Tetrahedron Letters 53 (2012) 3777–3779
3779
Table 4
Straathof, A. J. J.; Jongejan, J. A.; Heijnen, J. J. Tetrahedron 1999, 55, 12411–
12418.
6. Chen, S. T.; Jang, M. K.; Wang, K. T. Synthesis 1993, 858–860.
7. Boeriu, C. G.; Frissen, A. E.; Boer, E.; Kekem, K.; van Zoelen, D.-J.; Eggen, I. F. J.
Mol. Catal. B: Enzym. 2010, 66, 33–42.
C-terminal amidation of Tyr-Met-Asp-Phe using Alcalase-CLEA in tBuOH/DMF (82.5/
17.5, v/v)a,13
Entry
Amide product
Yield (%)
8. The reaction mixture was analyzed by HPLC-MS and all resulting peaks could
be identified. Area distribution: 4% Cbz-Phe-Leu-Ala-OH (starting material),
81% Cbz-Phe-Leu-Ala-NH2 (product), 9% Cbz-Phe-Leu-NH2, 4% Cbz-Phe-Leu-OH
and 2% Cbz-Phe-NH2.
1
2
3
Ac-Trp(Boc)-Met-Asp(tBu)-Phe-NH2
77
68
63
H-Trp(Boc)-Met-Asp(tBu)-Phe-NH2
b
H-Trp-Met-Asp-Phe-NH2
9. Using ammonium benzoate: Cal-B (100 mg) was added to a mixture of Cbz-Xaa-
OH (50 mg), ammonium benzoate (10 equiv) and 3 Å MS (200 mg) in toluene
(6 mL). The mixture was shaken at 50 °C, 150 rpm for 16 h.
a
Yield after purification by preparative HPLC.15
Using 2.1 equiv of NH3 in 1,4-dioxane.
b
Using NH3 gas: NH3 gas was bubbled through a solution of tripeptide (50 mg) in
toluene (6 mL) for 30 s. Subsequently, Cal-B (100 mg) and 3 Å MS (200 mg)
were added. The mixture was shaken at 50 °C, 150 rpm for 16 h.
10. After the enzymatic conversion of Cbz-Ala-OH the reaction mixture was
filtered and the Cal-B enzyme particles resuspended in MeOH followed by
filtration (3 Â 5 mL). The combined organic layers were concentrated in vacuo.
The crude product was purified by column chromatography on silica gel using
MeOH/CH2Cl2 (7/93 v/v) as the eluent. The pure fractions were combined,
concentrated in vacuo and the volatiles co-evaporated with toluene (2Â) and
CHCl3 (2Â). Cbz-Ala-NH2 was obtained in 87% yield.
Both diagnostic peptides Sincalide and Pentagastrine contain
16
the C-terminal sequence Tyr-Met-Asp-Phe-NH2 which is a gen-
eral motif of gastrin-related peptides. As a first approach we exam-
ined the amidation of the fully protected peptide (Table 4, entry 1).
Its carboxylic acid congener is easily accessible by SPPS and is
highly soluble in anhydrous organic solvents. Despite the presence
of two bulky side-chain protecting groups, the peptide acid was
well recognized by alcalase-CLEA and smoothly converted into its
corresponding amide. The same peptide sequence, with a free N-
terminal amine (entry 2), was also amidated without side reac-
tions. This result is important, since side-chain protected peptides
with a C-terminal primary amide and an unprotected N-terminus
are very useful building blocks for chemical peptide fragment
condensation, for example in the synthesis of the pharmaceutical
peptide products Fuzeon and Exenatide.17 Finally, the unprotected
tetrapeptide (entry 3) was also amidated in a good yield, without
amidation of the aspartic acid side-chain.
11. (a) Nuijens, T.; Cusan, C.; Kruijtzer, J. A. W.; Rijkers, D. T. S.; Liskamp, R. M. J.;
Quaedflieg, P. J. L. M. Synthesis 2009, 809–814; (b) Nuijens, T.; Kruijtzer, J. A.
W.; Cusan, C.; Rijkers, D. T. S.; Liskamp, R. M. J.; Quaedflieg, P. J. L. M.
Tetrahedron Lett. 2009, 50, 2719–2721; (c) Nuijens, T.; Cusan, C.; Kruijtzer, J. A.
W.; Rijkers, D. T. S.; Liskamp, R. M. J.; Quaedflieg, P. J. L. M. J. Org. Chem. 2009,
74, 5145–5150.
12. Sheldon, R. A. Biochem. Soc. Trans. 2007, 35, 1583–1587.
13. Alcalase-CLEA (50 mg, washed once with dry tBuOH and once with MTBE) was
added to
a mixture of peptide (0.10 mmol) and 2 N NH3 in 1,4-dioxane
(0.11 mmol) in tBuOH/DMF (5 mL, 82.5/17.5 v/v). The mixture was shaken at
50 °C, 150 rpm for 16 h.
14. When twice the amount of enzyme was used for the amidation of Cbz-Val-Ala-
OH, the HPLC conversion was 98%. The reaction mixture was filtered and the
solid enzyme particles were resuspended in MeOH (3 Â 5 mL) and in EtOAc
(25 mL) followed by filtration. The combined organic layers were washed with
saturated aqueous NaHCO3 (50 mL), brine (50 mL), dried over Na2SO4,
concentrated in vacuo and the volatiles co-evaporated with toluene
(2 Â 50 mL) and CHCl3 (2 Â 50 mL). Cbz-Val-Ala-NH2 was obtained in a yield
of 89%.
In conclusion, we have shown for the first time, that amino
acids and peptide acids can be enzymatically converted into their
C-terminal amide congeners directly using Cal-B or Subtilisin A.
The amidation is high yielding and competitive with other func-
tional groups and is independent of the presence of protecting
groups.
15. Ac-Trp(Boc)-Met-Asp(OtBu)-Phe-NH2: 1H NMR (DMSO-d6, 300 MHz): d = 1.35 (s,
9H), 1.62 (s, 9H), 1.77–1.94 (m, 5H), 2.01 (s, 3H), 2.28–2.45 (m, 2H), 2.61–2.68
(m, 1H), 2.79–3.09 (m, 4H), 4.26–4.41 (m, 2H), 4.50–4.64 (m, 2H), 7.14–7.33
(m, 9H), 7.53 (s, 1H), 7.67 (d, J = 7.8 Hz, 1H), 7.84 (d, J = 8.1 Hz, 1H), 8.00 (d,
J = 8.1 Hz, 1H), 8.17–8.24 (m, 3H); 13C NMR (DMSO-d6, 75 MHz): d = 14.5, 27.6,
29.2, 32.5, 37.2, 49.4, 51.6, 51.8, 53.7, 80.2, 83.6, 113.3, 114.7, 119.4, 122.3,
124.4, 125.6, 126.2, 127.9, 129.0, 129.8, 134.8, 137.6, 148.9, 168.0, 169.2, 169.7,
170.1, 172.4; TFA.H-Trp(Boc)-Met-Asp(OtBu)-Phe-NH2: 1H NMR (DMSO-d6,
300 MHz): d = 1.36 (s, 9H), 1.63 (s, 9H), 1.68–1.95 (m, 2H), 2.03 (s, 3H), 2.64–
2.81 (m, 2H), 2.91–3.05 (m, 2H), 3.11–3.21 (m, 1H), 4.10–4.21 (m, 1H), 4.36–
4.48 (m, 2H), 4.55–4.63 (m, 1H), 7.14–7.37 (m, 9H), 7.63 (s, 1H), 7.78 (d,
J = 7.8 Hz, 1H), 7.93 (d, J = 8.1 Hz, 1H), 8.00–8.10 (m, 5H), 8.40 (d, J = 7.8 Hz,
1H), 8.90 (d, J = 7.5 Hz, 1H); 13C NMR (DMSO-d6, 75 MHz): d = 14.5, 22.4, 26.9,
27.6, 29.3, 31.7, 37.3, 49.5, 52.0, 52.5, 53.8, 80.2, 83.4, 114.5, 116.6, 119.4,
122.3, 123.8, 124.2, 126.1, 128.0, 129.0, 130.2, 134.5, 137.7, 149.0, 169.3, 169.4,
169.7, 170.8, 171.5, 172.6; TFA.H-Trp-Met-Asp-Phe-NH2: 1H NMR (DMSO-d6,
300 MHz): d = 1.68–1.90 (m, 2H), 1.96 (s, 3H), 2.80–2.92 (m, 2H), 3.01–3.13 (m,
2H), 3.14–3.20 (m, 1H), 3.94–4.05 (m, 1H), 4.28–4.40 (m, 2H), 4.46–4.53 (m,
1H), 6.95–7.32 (m, 11H), 7.62 (d, J = 7.8 Hz, 1H), 7.88–7.94 (m, 4H), 8.32 (d,
J = 7.5 Hz, 1H), 8.75 (d, J = 8.1 Hz, 1H), 10.9 (s, 1H), 12.3 (s, 1H); 13C NMR
(DMSO-d6, 75 MHz): d = 14.5, 16.8, 27.3, 29.3, 32.3, 35.9, 36.4, 37.3, 49.5, 51.8,
52.4, 53.8, 106.7, 111.4, 118.4, 121.0, 125.0, 126.1, 127.0, 128.0, 129.0, 136.2,
137.7, 168.4, 167.0, 170.3, 171.9, 172.5.
Acknowledgements
We would like to thank Mr. Math Boesten B.Sc. for his analytical
support and Dr. Claudia Cusan for fruitful discussions.
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