Enzymatic fragment condensation of side chain-protected peptides using subtilisin A in anhydrous organic solvents: A general strategy for industrial peptide synthesis

Article abstract of DOI:10.1002/adsc.201200694

Herein, the enzymatic condensation of side chain-protected peptide fragments using subtilisin A in anhydrous organic solvents is described. A screening with dipeptide Cbz-Val-Xxx carboxamidomethyl esters with H-Xxx-Val-NH₂ nucleophiles was performed, wherein Xxx stands for every (side chain-protected) amino acid residue. Finally, it was demonstrated that it is feasible to enzymatically condense larger peptide fragments (up to the 10-mer level) bearing multiple side chain-protecting groups with very high conversion.

Full text of DOI:10.1002/adsc.201200694

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DOI: 10.1002/adsc.201200694
Enzymatic Fragment Condensation of Side Chain-Protected
Peptides using Subtilisin A in Anhydrous Organic Solvents:
A General Strategy for Industrial Peptide Synthesis
Timo Nuijens,a Annette H. M. Schepers,a Claudia Cusan,a John A. W. Kruijtzer,b
Dirk T. S. Rijkers,b Rob M. J. Liskamp,b and Peter J. L. M. Quaedfliega,*
a
DSM Innovative Synthesis B.V., P.O. Box 18, NL-6160 MD Geleen, The Netherlands
E-mail: peter.quaedflieg@dsm.com
Medicinal Chemistry and Chemical Biology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, P.O. Box
b
80082, NL-3508 TB Utrecht, The Netherlands
Received: August 3, 2012; Published online: January 4, 2013
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201200694.
Abstract: Herein, the enzymatic condensation of
side chain-protected peptide fragments using subti-
lisin A in anhydrous organic solvents is described.
A screening with dipeptide Cbz-Val-Xxx carboxam-
efficiently synthesized in one run on the solid phase.
Furthermore, during synthesis, long side chain-pro-
tected peptides tend to form tertiary structures (a
process which is called “hydrophobic collapse”)[4]
making peptide elongation very troublesome so that
a large excess of reagents and amino acid building
blocks is needed. Additionally the purification of the
final product is often very difficult due to the pres-
ence of significant amounts of truncated peptides and
peptides containing deletions.[5]
Taking into account the drawbacks of both SPPS
and solution phase peptide synthesis, it is generally
impossible to synthesize, for instance, a 30-mer pep-
tide solely by only one of these two strategies. To
remedy these limitations, a hybrid approach can be
used wherein protected peptide fragments are synthe-
sized by means of SPPS and subsequently chemically
coupled in solution. On paper, the “ideal” approach
for the synthesis of a 30-mer peptide would be a fully
ACHTUNGTRENNUNGidomethyl esters with H-Xxx-Val-NH2 nucleophiles
was performed, wherein Xxx stands for every (side
chain-protected) amino acid residue. Finally, it was
demonstrated that it is feasible to enzymatically
condense larger peptide fragments (up to the 10-
mer level) bearing multiple side chain-protecting
groups with very high conversion.
Keywords: anhydrous conditions; fragment conden-
sation; organic solvents; peptide synthesis; subtilisin
A
Although a few pharmaceutically relevant peptides symmetric (convergent) fragment condensation strat-
can be produced relatively cost-efficiently on a large egy wherein the length of the protected peptides syn-
scale by fermentation, whereas most pharmaceutical thesized by SPPS does not exceed 10 amino acids,
peptides on the large scale for short peptide sequen- e.g., a 10+10+10 strategy (Figure 1, A). However, if
ces are synthesized by chemical means.[1] Solution the desired peptide does not contain Gly or Pro resi-
phase stepwise chemical peptide synthesis is only fea- dues at the C-terminal coupling position of the frag-
sible for medium-sized and long peptides (containing ments, racemization is inevitable. Although with care-
10–50 amino acid residues), solid phase peptide syn- fully selected coupling positions and reagents or con-
thesis (SPPS) is most commonly applied.[2] The main densation techniques such as the azide method,[6] rac-
disadvantage of SPPS is that extremely high yields emization can be minimized, it can never be fully
should be realized for each coupling and deprotection eliminated. Especially in the case of pharmaceutical
cycle.[3] For instance, when per step (coupling, Fmoc- peptides, even a few tenths of percent of diastereoiso-
deprotection and final cleavage) a yield of 95% is re- mers in the final product is unacceptable. Therefore,
alized in the synthesis of a decamer, an overall yield a convergent approach is most often not feasible and
of only 34% of the crude peptide is obtained, which one has to adapt the peptide fragment length to the
should be purified by a laborious and cost-inefficient positions of the Gly and Pro residues, if they are pres-
HPLC method. Therefore, on the industrial scale, ent at all. This leads to the SPPS synthesis of undesir-
peptides longer than 10–15 amino acids are not cost- ably short (<5 amino acids) or long (>10 amino
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Figure 1. The hybrid peptide synthesis approach using solid phase synthesis of protected peptide fragments and condensation
of these fragments in solution.
acids) protected peptide fragments, which are often acyl donor. Specific esters have been designed, so-
very poorly soluble in organic solvents (Figure 1, B). called substrate mimetics, which have such a high af-
Alternatively, other coupling positions are chosen and finity for the enzyme so that side reactions could be
racemization is taken for granted. As a consequence, virtually eliminated.[11] However, peptides containing
a difficult preparative HPLC purification is required such a C-terminal ester moiety are notoriously diffi-
due to the similar retention times of the two diaste- cult to synthesize chemically in high stereoisomeric
reoisomers leading to (partial) overlap of peaks. purity. In fact, the coupling reagents required to syn-
Mixed fractions have to undergo an additional round thesize these substrate mimetics are often identical to
of preparative HPLC purification leading to much those used for chemical peptide bond formation.[12]
larger expenses and a lower product yield. few solid-phase methodologies have been reported
A
In contrast to chemical fragment condensation, the for the synthesis of peptide substrate mimetics, but
enzymatic coupling of peptide fragments is totally these strategies are complicated, low yielding and re-
free of racemization.[7] Therefore, the 10+10+10 quire the use of resins which are not suitable on an in-
fragment condensation strategy for the synthesis of dustrial scale.[13] Recently, we reported the SPPS of
a 30-mer peptide is at least in theory feasible when peptide carboxamidomethyl (Cam) esters, which are
using enzymes. Although many enzymes, i.e., proteas- highly active in subtilisin A-catalyzed peptide synthe-
es and lipases, have been applied to the synthesis of sis, using industrially applicable resins.[14] There are
short peptide sequences in aqueous buffered solution, several examples from the literature wherein subtili-
only very few examples of enzymatic peptide frag- sin is used for peptide fragment condensation with
ment condensations have been reported.[8] The major esters as the acyl donor.[15] However, there is always
drawback is that the presence of water, which is es- 1–5% of water present leading to partial hydrolysis of
sential for enzyme activity and stability, leads to unde- the acyl donor ester, a relatively large excess of amine
sired hydrolysis of the peptide backbone.[9] For in- is used and peptide fragments never exceed three
stance, when Wong et al. compared several commer- amino acids. Recently, we described the use of subtili-
cially available proteases for peptide synthesis, partial sin A for dipeptide synthesis in anhydrous organic
hydrolysis proved inevitable for all enzymes, even solvents. Molecular sieves were used to fully eliminate
when a 2.5 molar excess of nucleophile was used.[10] the hydrolytic side reactions and only 1.1 equivalents
Especially when longer fragments are used, with cost of nucleophile were needed for full conversion of the
prices up to E100,000 per kg for a 10-mer peptide, acyl donor ester.[16] Herein, we report the fragment
partial hydrolysis is unacceptable. To ensure a fast condensation of oligopeptide Cam esters with oligo-
coupling reaction and thereby reducing proteolysis, peptide amines under anhydrous reaction conditions
a peptide C-terminal activated ester is required as the with subtilisin A. To our surprise, peptide fragments
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Enzymatic Fragment Condensation of Side Chain-Protected Peptides using Subtilisin A
Table 1. Peptide fragment condensations using Subtilisin A in anhydrous organic solvent.
Entry[a] Acyl donor Cam ester
Nucleophile
Conversion to
product [%][b]
1
2
3
4
5
6
7
8
Cbz-Phe-Leu-Ala-OCam
Cbz-Ala-Leu-Phe-OCam
Cbz-Gly-Ile-Ala-OCam
Cbz-Ala-Pro-Leu-OCam
Cbz-Ala-Pro-Leu-OCam
Cbz-Gly-Ile-Ala-OCam
Cbz-Ala-Pro-Leu-OCam
H-Ala-Leu-Phe-NH2
H-Leu-Phe-NH2
H-Gly-Phe-NH2
H-Gly-Phe-NH2
H-Gly-Leu-Met-NH2
hexapeptide, quant.
pentapeptide, 95
pentapeptide, quant.
pentapeptide, 87
hexapeptide, quant.
hexapeptide, 95
H-Ser
H-Trp
(Trt)-Met-OCam H-Leu-Phe-NH2
ACHTUNGTRENNUNG
G
ACHTUNGTRENNUNG
Ac-Leu-Ser
(t-Bu)-Lys
C
N
heptapeptide, 96
[a]
Conditions: Alcalase-CLEA-OM, DMF/THF (1/9, v/v), 1.5 equiv. nucleophile, 3ꢁ molecular sieves, 378C, 16 h (Proce-
dure A).
[b]
Conversions estimated using HPLC-MS by integration of the acyl donor starting material and the product peaks, assum-
ing identical response factors.
carrying the bulky protective groups on the side chain 400 possible coupling reactions were investigated as
functionalities are also well accepted by the enzyme. shown in Scheme 1 and Figure 2.
To the best of our knowledge, enzymatic fragment
condensation of fully protected peptides has never of a quite large number of dipeptide coupling combi-
been described before. nations proceed smoothly and high conversions were
As is clear from Figure 2, A, the coupling reactions
Initially, it was investigated whether Cam esters observed despite the fact the sterically demanding Val
could also be used to condense peptide fragments residues are located at the penultimate positions.
longer than dipeptides. As shown in Table 1,
With respect to the Cbz-Val-Xxx-OCam library
a number of peptide fragments without any function- with protected side chain functionalities, there is
alized side chain could be successfully coupled by sub- a clear preference for hydrophobic amino acids. How-
tilisin A (Alcalase) in anhydrous organic solvent (en- ever, also amino acids containing sterically demanding
tries 1–5). Excellent conversions were observed by an- protecting groups, such as GlnACHTUNTRGENNUG(Trt) and GluACHTUNGTERN(NUGN O-t-Bu),
alytical HPLC with only 1.5 equiv. of nucleophile. are very well accepted by the enzyme. It must be
After work-up and purification, a yield of 73% could noted that, when reaction times were extended and/or
be obtained for Cbz-Phe-Leu-Ala-Ala-Leu-Phe-NH2. more enzyme was applied, almost all reactions could
Unfortunately, peptide fragments containing amino be brought to complete conversion.
acids with unprotected functional groups in the side
chain, are very poorly soluble in organic solvents. cally demanding protecting groups proved difficult to
Therefore, it was investigated whether peptides con- incorporate, such as Cbz-Val-Ser(t-Bu)-OCam and
taining amino acids with protected side chain func- Cbz-Val-Thr(t-Bu)-OCam. Therefore, a second Cbz-
Some acyl donors containing amino acids with steri-
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
tionalities, which are better soluble in organic sol-
vents, were also accepted by subtilisin A. As shown in
Table 1 (entries 6–8), side chain-protected peptide
fragments, up to the pentamer level, were well accept-
ed by subtilisin A and high conversions to the desired
products were obtained, according to HPLC-MS anal-
ysis. It is rather surprising that large, sterically de-
manding protecting groups still fit in the substrate
binding pockets of the enzyme.
To explore the scope and limitations of this peptide
fragment condensation strategy, two libraries of di-
peptides were synthesized, i.e., a Cbz-Val-Xxx-OCam
and an H-Yyy-Val-NH2 library, in which Xxx and Yyy
represent all proteinogenic amino acids with protect-
ed side chain functionalities. It was decided to take
a Val residue at both penultimate positions because
this usually results in lower coupling rates, so that the
differences between the Xxx and Yyy residues
Scheme 1. Coupling reactions between Cbz-Val-Xxx-OCam
become more clearly visible. The efficiencies of all and H-Yyy-Val-NH2.
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Figure 2. Comparison of coupling efficiencies (HPLC conversion to the desired tetramers) between Cbz-Val-Xxx-OCam and
H-Yyy-Val-NH2 dipeptides in which A: all functional side chains were protected and B: all functional side chains of the
Cbz-Val-Xxx-OCam were unprotected, and all functional side chains of the H-Yyy-Val-NH2 were protected.
Val-Xxx-OCam library was synthesized containing no ening the scope of the enzymatic peptide fragment
protecting groups on the side chain functionalities. As condensation strategy. Another large difference was
evident from Figure 2, B, the efficiency of several cou- observed for an unprotected Gln, which was found to
pling reactions in the case of acyl donors without pro- be one of the most efficient acyl donors.
tected side chain functionalities was increased com-
To show the versatility of this fragment condensa-
pared to the fully protected acyl donors. For instance, tion strategy, wherein only the C-terminal amino acid
t-Bu-protected Ser and Thr residues gave poor cou- residue of the Cam ester was unprotected, two antimi-
pling efficiencies, while the unprotected analogues crobial peptides containing ten amino acid residues
were readily accepted by subtilisin A, thereby broad- were synthesized, as shown in Table 2.[17,18] These pep-
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Enzymatic Fragment Condensation of Side Chain-Protected Peptides using Subtilisin A
Table 2. Synthesis of two antimicrobial peptides using an acyl donor with an unprotected C-terminal residue.
Entry[a] Acyl donor Cam ester
Nucleophile
Conversion to Decapeptide product after
product [%]
Isolated
full deprotection[b]
yield [%][c]
1
2
Boc-Leu-Asp
Ser(t-Bu)-Gln-OCam
Boc-His(Trt)-Lys(Boc)-Thr
Bu)-Asp(O-t-Bu)-Ser-OCam
G
R
G
H-Leu-Asp-Gln-Ser-Gln-
Phe-Val-Gly-Ser-Arg-NH2
H-His-Lys-Thr-Asp-Ser-Phe- 55
Val-Gly-Leu-Met-NH2
63
G
ACHTUNGTRENNUNG
G
(t-
ACHTUNGTRENNUNG
[a]
[b]
[c]
Conditions: Alcalase-CLEA-OM, DMF/MTBE (1/9, v/v), 3ꢁ molecular sieves, 378C, 16 h.
Conditions: TFA/TIS/H2O (95/2.5/2.5, v/v/v).
Isolated yield based on acyl donor starting material.
tides show activity against a number of Gram-positive to longer fragment condensations (entries 3 and 4)
and Gram-negative bacteria from the skin, the oral, due to the limited solubility of the peptides. However,
the respiratory and the gastrointestinal tracts.
CH2Cl2 was found to be a very good alternative, since
Clearly, an almost complete conversion was ob- protected peptides containing more than 5–7 amino
tained for the peptide fragment condensation reac- acid residues tend to be highly soluble in this solvent.
tions. The semi-protected acyl donor with a C-termi- In fact, when CH2Cl2 was used as the solvent, nearly
nal unprotected Ser residue could be prepared by quantitative conversion was obtained for a number of
using a very acid labile protecting group, i.e., Trt, different peptide fragment condensations. When
which was simultaneously deprotected during the longer reaction times were applied, even an enzymat-
acidic cleavage of the peptide from the solid support. ic 9+10-mer side chain-protected peptide fragment
During this mild acidic cleavage, only partial depro- condensation proved well feasible and an excellent
tection of HisACHTUNGTRENNUNG(Trt) was observed, the other side chain conversion to the 19-mer product was observed
protecting groups were left intact. After enzymatic (Table 3, entry 4). It was surprising that such large
fragment condensation, full deprotection of the de- peptides, bearing bulky side chain protecting groups,
capeptides was performed to obtain the natural anti- were still accepted by the enzyme and that the cou-
microbial peptides in good yield (63, 55% respective- pling proceeded smoothly, despite the anhydrous re-
ly).
action conditions. This racemization-free enzymatic
After these successful 5+5 amino acid fragment side chain protected peptide fragment condensation
condensations, it was investigated whether even strategy opens new doors for the (industrial) synthesis
longer peptide fragments could be condensed. As of long peptides.
demonstrated in Table 3, the solvent mixture suitable
In conclusion, we have identified a novel enzymatic
for the 5+5-mer (entries 1 and 2) fragment condensa- fragment condensation strategy of side chain protect-
tion, i.e., DMF/MTBE (1/9, v/v), was not applicable ed peptides up to the coupling of 10+9-mer frag-
Table 3. Fragment condensation of side chain protected peptides of different lengths in DMF/MTBE (1/9, v/v) and CH2Cl2.
Entry[a] Acyl donor Cam ester
Nucleophile
Conversion in Conversion
DMF/MTBE in CH2Cl2
[%][b]
[%][b]
1
2
3
Ac-Asp
Gln-OCam (5-mer)
Ac-Thr(t-Bu)-Ser(t-Bu)-Asp
Ser(t-Bu)-Lys(Boc)-Gln-OCam (7-mer)
Ac-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(t-Bu)-
Asp(O-t-Bu)-Leu-Ser(t-Bu)-Lys(Boc)-Gln-
OCam (9-mer)
Ac-Thr(t-Bu)-Phe-Thr
Asp(O-t-Bu)-Leu-Ser(t-Bu)-Lys
OCam (9-mer)
G
N
G
G
G
quant.
quant.
AHCTUNGTRENNUNG
G
62
98
78
R
ACHTUNGTRENNUNG
R
not soluble
G
ACHTUNGTRENNUNG
4
A
not soluble
95[c]
E
N
AHCTUNGTRENNUNG
[a]
[b]
[c]
Conditions: Alcalase-CLEA-OM, DMF/MTBE (1/9, v/v) or CH2Cl2, 3ꢁ molecular sieves, 378C, 16 h (Procedures A and
C, respectively).
Conversions estimated using HPLC by integration of the acyl donor starting material and the product peaks, assuming
identical response factors.
After 1 week.
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mixture was shaken at 378C with 200 rpm for 16 h, and the
progress of the condensation reaction was monitored with
LC-MS.
ments in neat organic solvent using subtilisin A. It
was shown that the strategy is broadly applicable, es-
pecially when the side chain functionality of the C-ter-
minal amino acid Cam ester is left unprotected.
Cbz-Phe-Leu-Ala-Ala-Leu-Phe-NH2
To a suspension of Alcalase-CLEA-OM (100 mg) and 3ꢁ
molecular sieves (100 mg) in THF (9 mL) was added a solu-
tion of Cbz-Phe-Leu-Ala-OCam (300 mmol, 162 mg) and H-
Ala-Leu-Phe-NH2 (450 mmol, 157 mg, 1.5 equiv.) in DMF
(1 mL). The obtained reaction mixture was shaken at 378C
with 200 rpm for 16 h. Afterwards, the reaction mixture was
filtered and the solid enzyme particles washed with CH2Cl2
(10 mLꢂ2) and DMF (5 mLꢂ2). The combined organic
phases were concentrated under vacuum to a volume of
10 mL and purified by preparative HPLC. The pure frac-
tions were pooled and lyophilized yielding Cbz-Phe-Leu-
Experimental Section
General Condtions
Before use, Alcalase-CLEA-OM was dried as follows: 3 g
Alcalase-CLEA-OM (CLEA technologies, 850 AGEU/g),
was suspended in 100 mL t-BuOH and crushed with a spatu-
la. After filtration, the enzyme was resuspended in 50 mL
MTBE followed by filtration and the solid was dried for
1 min at ambient temperature. Protected peptide nucleo-
philes were synthesized on a Sieber resin using standard
Fmoc SPPS protocols.[18] Protected peptide Cam esters were
synthesized using literature procedures.[12] The side chain-
protected Cbz-Val-Xxx-OCam library was deprotected using
5 mmol peptide in TFA/H2O (1 mL, 95/5, v/v) and stirred for
1 h at ambient temperature. Afterwards, the volatiles were
removed by a nitrogen flow and the residue was lyophilized
from CH3CN/H2O (3/1, v/v). The resulting lyophilized pow-
ders were dissolved in DMF (90 mL) containing piperidine
(10 mmol, 1 mL) and used as such for the coupling efficiency
assays described in general procedure B.
Ala-Ala-Leu-Phe-NH2 as
a white solid; yield: 176 mg
(73%). 1H NMR (DMSO-d6, 300 MHz): d=0.77–0.95 (m,
12H), 1.20 (dd, J=7.2 and 9.9 Hz, 6H), 1.30–1.67 (m, 6H),
2.68–2.85 (m, 2H), 2.98–3.04 (m, 2H), 4.13–4.44 (m, 6H),
4.94 (s, 2H), 7.09–7.32 (m, 17H), 7.48 (d, J=8.4 Hz, 1H),
7.72 (d, J=7.8 Hz, 1H), 7.83 (d, J=7.8 Hz, 1H), 7.93 (d, J=
7.2 Hz, 1H), 8.05–8.09 (m, 2H); 13C NMR (DMSO-d6,
75 MHz): d=17.7, 21.5, 22.8, 22.9, 23.9, 24.0, 37.4, 48.1, 51.0,
51.3, 53.3, 55.9, 65.1, 126.1, 127.3, 127.5, 127.9, 128.1, 129.0,
136.9, 137.6, 137.9, 155.7, 171.3, 171.4, 171.7, 171.9, 172.5;
LC-MS: Rt =22.07 min and m/z=814, [M + H]+, calcd. for
+
C44H60N7O8 : 814.
General Procedure A
H-Leu-Asp-Gln-Ser-Gln-Phe-Val-Gly-Ser-Arg-NH2
Enzymatic peptide coupling reactions in DMF/THF or
DMF/MTBE (1/9, v/v): to a suspension of Alcalase-CLEA-
OM (10 mg) and 3ꢁ molecular sieves (10 mg) in THF or
MTBE (900 mL), a solution of the acyl donor Cam ester
(30 mmol) and protected peptide nucleophile (45 mmol,
1.5 equiv.) in DMF (100 mL) was added. The obtained reac-
tion mixture was shaken at 378C with 200 rpm for 16 h and
subjected to LC-MS analysis.
To a suspension of Alcalase-CLEA-OM (100 mg) and 3ꢁ
molecular sieves (100 mg) in THF or MTBE (9 mL), was
added
Bu)-Gln-OCam (0.3 mmol, 330 mg) and H-Phe-Val-Gly-Ser-
(t-Bu)-Arg(Pbf)-NH2 (0.45 mmol, 1.5 equiv., 393 mg) in
a solution of Boc-Leu-AspACHTUNGTRNENG(U OtBu)-GlnACHTUTGNRNENUG(Trt)-SerACHTUNGTRENNUNG(t-
A
ACHTUNGTRENNUNG
DMF (1 mL). The obtained reaction mixture was shaken at
378C with 200 rpm for 16 h. Afterwards, the reaction mix-
ture was filtered and the solid enzyme particles and molecu-
lar sieves washed with CH2Cl2 (100 mLꢂ5) and DMF
(5 mLꢂ2). The combined organic phases were concentrated
under vacuum and the residue dissolved in TFA/TIS/H2O
(10 mL, 95/2.5/2.5, v/v/v) and stirred at ambient temperature
for 60 min. The reaction mixture was added to MTBE/n-
heptanes (100 mL, 1/1, v/v) and the precipitates collected by
centrifugation (4000 rpm, 10 min). The residue was purified
by preparative HPLC, the pure fractions were pooled and
lyophilized affording H-Leu-Asp-Gln-Ser-Gln-Phe-Val-Gly-
Ser-Arg-NH2 as a white solid; yield: 214 mg (63%). LC-MS:
Rt =13.67 min and m/z=568 [M+2H]2+, calcd. for
C48H80N16O162+: 568.
General Procedure B
Coupling efficiencies between Cbz-Val-Xxx-OCam and H-
Yyy-Val-NH2 with protected side chain functionalities: frag-
ment condensations were performed in a 96-well format
with glass vial inserts. To a glass insert was added a solution
of Cbz-Val-Xxx-OCam (2.5 mmol) in DMF (45 mL) and a so-
lution of H-Yyy-Val-NH2 (5 mmol, 2 equiv.) in DMF (45 mL).
Subsequently, a suspension of crushed molecular sieves
(5 mg) and Alcalase-CLEA-OM (4.5 mg) in t-BuOH
(410 mL) were added. The plate was covered with an alumi-
na lid and shaken at 378C with 150 rpm for 1 h. Afterwards,
samples of the supernatant (300 mL) were taken and added
to DMSO (700 mL) and analyzed by HPLC. The identity of
the tetrapeptide products was confirmed by LC-MS analysis.
H-His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH2
To a suspension of Alcalase-CLEA-OM (100 mg) and 3ꢁ
molecular sieves (100 mg) in THF or MTBE (9 mL), was
General Procedure C
added a solution of Boc-His
ACHTUNGERTN(UNGN Trt)-LysAHCTUNRTEGNN(GUN Boc)-ThrACHTGUNTREN(NNGU t-Bu)-Asp-
Enzymatic peptide coupling reactions in CH2Cl2: to a suspen-
sion of Alcalase-CLEA-OM (10 mg) and 3ꢁ molecular
sieves (10 mg) in CH2Cl2 (500 mL), was added a solution of
the peptide Cam ester (3.0 mmol) and the peptide nucleo-
phile (4.5 mmol) in CH2Cl2 (500 mL). The obtained reaction
ACHTUNGTREN(NGNU O-t-Bu)-Ser-OCam (0.3 mmol, 313 mg) and H-Phe-Val-
Gly-Leu-Met-NH2 (0.45 mmol, 1.5 equiv., 251 mg) in DMF
(1 mL). The obtained reaction mixture was shaken at 378C
with 200 rpm for 16 h. Afterwards, the reaction mixture was
filtered and the solid enzyme particles and molecular sieves
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Enzymatic Fragment Condensation of Side Chain-Protected Peptides using Subtilisin A
washed with CH2Cl2 (100 mLꢂ5) and DMF (5 mLꢂ2). The
combined organic phases were concentrated under vacuum
and the residue dissolved in TFA/TIS/H2O (10 mL, 95/2.5/
2.5, v/v/v) and stirred at ambient temperature for 60 min.
The reaction mixture was added to MTBE/n-heptanes
(100 mL, 1/1, v/v) and the precipitates collected by centrifu-
gation (4000 rpm, 10 min). The residue was purified by prep-
arative HPLC, the pure fractions were pooled and lyophi-
lized affording H-His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-
Met-NH2 as a white solid; yield: 187 mg (55%). LC-MS:
Rt =10.23 min and m/z=379 [M+3H]3+, calcd. for
C50H83N14O14S3+: 379.
[9] B. Sauerbrei, T. Kappes, H. Waldmann, Top. Curr.
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