Native chemical ligation with aspartic and glutamic acids as C-terminal residues: Scope and limitations

Article abstract of DOI:10.1002/ejoc.200300032

A new side reaction during Native Chemical Ligation (NCL) at aspartic and glutamic acid/cysteine ligation sites is reported. To exploit NCL at these sites, test peptides having either a C-terminal unprotected aspartyl or glutamyl thioester were ligated with a peptide containing an N-terminal free cysteine generating, in each case, two major compounds having equal masses but different retention times by HPLC analysis. Comparison of these ligation products with reference peptides of identical sequence synthesized by total SPPS (solid phase peptide synthesis) with both natural and unnatural backbones at the cysteinyl residue is in agreement with the hypothesis of migration of the thioester moiety on the side chain carboxyl group of both C-terminal Asp and Glu residues. Enzymatic digestion of the purified ligation products with a V8-protease further confirmed the nature of the side reaction, with a total resistance of the non-natural backbone peptide to the protease. To overcome this problem the use of a protecting group stable to both HF cleavage and ligation conditions, but removable after ligation, became a necessity. A series of side-chain protected derivatives were selected, specifically 9-fluorenylmethyl ester (OFm), (phenylsulfonyl)ethyl ester (OPse), 2,2,2-trichloroethyl ester (OTce), and phenacyl esters (OPac). For the glutamic acid, OPse gave the best results, generating the correct peptide and avoiding the isomerization at the 7-carboxyl group. In the case of the aspartic residue, all the protecting groups were removed under basic conditions, worsening the problem and generating, for example, 70% yield of the β-isomer after OFm deprotection. This prompted us to propose a protecting group derived from the phenacyl ester 1-methyl-2-oxo-2-phenylethyl ester (OMop). This protecting group showed greater stability during the ligation reaction than OPac and OTce, and was easily removed by Zn/acetic acid reduction after the ligation step. Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003.

Full text of DOI:10.1002/ejoc.200300032

FULL PAPER
Native Chemical Ligation with Aspartic and Glutamic Acids as C-Terminal
Residues: Scope and Limitations
Matteo Villain,[a] Hubert Gaertner,[a] and Paolo Botti*[a]
Keywords: Peptides / Protecting groups / Native chemical ligation / Isomerization
A new side reaction during Native Chemical Ligation (NCL)
ligation conditions, but removable after ligation, became a
at aspartic and glutamic acid/cysteine ligation sites is re- necessity. A series of side-chain protected derivatives were
ported. To exploit NCL at these sites, test peptides having
either a C-terminal unprotected aspartyl or glutamyl thioes-
ter were ligated with a peptide containing an N-terminal free
cysteine generating, in each case, two major compounds
having equal masses but different retention times by HPLC
selected, specifically 9-fluorenylmethyl ester (OFm), (phenyl-
sulfonyl)ethyl ester (OPse), 2,2,2-trichloroethyl ester (OTce),
and phenacyl esters (OPac). For the glutamic acid, OPse gave
the best results, generating the correct peptide and avoiding
the isomerization at the γ-carboxyl group. In the case of the
analysis. Comparison of these ligation products with refer- aspartic residue, all the protecting groups were removed un-
ence peptides of identical sequence synthesized by total
SPPS (solid phase peptide synthesis) with both natural and
unnatural backbones at the cysteinyl residue is in agreement
with the hypothesis of migration of the thioester moiety on
the side chain carboxyl group of both C-terminal Asp and
Glu residues. Enzymatic digestion of the purified ligation
products with a V8-protease further confirmed the nature of
the side reaction, with a total resistance of the non-natural
der basic conditions, worsening the problem and generating,
for example, 70% yield of the β-isomer after OFm deprotec-
tion. This prompted us to propose a protecting group derived
from the phenacyl ester 1-methyl-2-oxo-2-phenylethyl ester
(OMop). This protecting group showed greater stability dur-
ing the ligation reaction than OPac and OTce, and was easily
removed by Zn/acetic acid reduction after the ligation step.
backbone peptide to the protease. To overcome this problem ( Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
the use of a protecting group stable to both HF cleavage and
Germany, 2003)
Introduction
boxylic acid side chain. In this case the consequence would
be the formation of the unnatural β (for the aspartic acid)
Native chemical ligation[1] is emerging as a powerful tool or γ (for the glutamic acid) amide bond between the Asp/
in protein chemistry to prepare proteins of small to medium Glu and the cysteine as side product. To test our hypothesis
size by chemical synthesis. The key aspect of this technique model peptide Cα thioesters of Asp and Glu with unprotec-
is the possibility to ligate two unprotected polypeptide frag- ted side chains (Scheme 1) were synthesized and ligated un-
ments in aqueous solution when the C-terminal fragment der standard conditions with an N-terminal Cys peptide.
possesses an N-terminal cysteine, and the N-terminal frag-
ment is synthesized as a C-terminal thioester. The proposed
mechanism of this reaction involves two steps, a trans-thio-
esterification between the C-terminal thioester moiety with
the thiol of the N-terminal cysteine and a subsequent S-to-
N internal acyl shift through a five-membered ring inter-
mediate that generates a natural amide bond. Although an
extensive study[2] showed that any of the 20 naturally occur-
ring amino acids was compatible in a Xaa-Cys ligation, no
further investigation was carried out to identify potential
Scheme 1
side reactions at the C-terminal thioester residue. A possible
case study involves aspartic and glutamic acids, since the
proximity of the β- or γ-carboxyl to the thioester could
cause a migration of the thioester moiety onto the car-
Results and Discussion
Ligation with Asp/Glu Unprotected Side-Chain Thioesters
[a]
GeneProt Inc., Geneva Branch, Protein Synthesis
To investigate the compatibility of the native chemical
ligation strategy with Asp-Cys and Glu-Cys ligation sites,
the thioester (sr) peptides, H-LYRAD-sr (1), H-LYRAE-sr
´
2 Rue Pre de la Fontaine, 1217 Meyrin 2, Switzerland
Fax: (internat.) ϩ41-22-7198999
E-mail: Paolo.Botti@geneprot.com
Eur. J. Org. Chem. 2003, 3267Ϫ3272
DOI: 10.1002/ejoc.200300032
 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3267

M. Villain, H. Gaertner, P. Botti
FULL PAPER
relative ratio between the two ligation products is about 1:2.
Furthermore, during the ligation, 40% of the thioester pep-
tide was hydrolysed. A similar result was obtained in the
ligation involving the glutamyl thioester peptide, although
the two peaks corresponding to the ligated material were
present in a relative ratio of 1:4. Neither modification of
the pH of the ligation medium between pH 6.2 and 7.0 nor
reduction of the thiol concentration by lowering the con-
centration of thiophenol as low as 0.1% had any effect on
the final ratio between the two peaks in both ligations. To
correctly define the nature of the two entities with different
HPLC elution times, the expected ligation products H-LY-
RADCSYRFL-OH (4), H-LYRAECSYRFL-OH (5), and
the suspected ligation products — the unnatural isomers H-
LYRAD(β)CSYRFL-OH (6) and H-LYRAE(γ)CSYRFL-
OH (7) — were synthesized by SPPS. Comparing the Rt of
these standards with the ligation products we were able to
confirm that for both the Asp and Glu ligations the first
eluted peak (the most hydrophilic compound), which is pre-
sent in the lowest amount, corresponds to the unnatural
backbone isomers, supporting the idea of a migration of
the thioester to the side chain carboxyl (Scheme 1). This
was further confirmed by V8-protease digestion of purified
ligation products. This proteolytic enzyme, selective for
Asp-Xxx and Glu-Xxx, readily digested the second eluted
(more hydrophobic) ligation product for both Glu and Asp,
while it was ineffective on the first eluted compounds. The
same results were obtained with the fully synthetic prod-
ucts, both compounds 6 and 7 being totally resistant to the
protease. These experiments together confirm that ligation
at a Glu- or Asp-Cys site with the unprotected carboxyl
side chain results in 20Ϫ30% isomerization of the resulting
peptidic chain at the ligation site and a significant amount
of hydrolysed Asp-thioester.
Scheme 2. Protecting groups tested for Glu or Asp side chain pro-
tection: OFm (a), OPse (b), OTce (c), OPac (d), OMop (e), and
OdiMeOPac (f)
(2) and the N-terminal free Cys peptide, H-CSYRFL-OH
(3) were synthesized. Short peptides were retained for this
investigation to facilitate the detection by HPLC of side re-
action products. Analysis of the thioester peptides after HF
cleavage shows a single component for H-LYRAE-sr (2),
while H-LYRAD-sr (1) showed about 20% of thioester hy-
drolysis. After purification and lyophilization, peptide 1 was
found to be partially unstable, generating after eight weeks
storage at ϩ4 °C, a 30% yield of the hydrolysed product.
When the aspartyl thioester peptide 1 was involved in a
ligation reaction with the C-terminal peptide 3 under stand-
ard NCL conditions (pH 6.5, 1% thiophenol and 1%
benzylmercaptan) the presence of two ligated products was
observed after 2 h incubation, both compounds presenting
the expected mass for the correct product (Figure 1). The
To clarify the mechanism of this reaction, H-LYRAD-sr
was incubated for 1 h at pH 6.5 without any thiol and no
N-terminal Cys peptide. Mass spectrometry of fractions ob-
tained by HPLC analysis of the product mixture after 30
minutes provided evidence of the presence of 50% thioester
hydrolysed product, 18% of a product hydrolysed and dehy-
drated consistent with the generation of succinic anhydride
at the C-terminus, and 20% of the original thioester peptide.
The presence of the succinic anhydride product could sup-
port the idea that this intermediate in the ligation reacts
either with thiophenol or with the Cys-containing peptide
on the β-carbonyl, generating the β-isomer. Furthermore,
due to its high reactivity, the succinic anhydride intermedi-
ate is highly unstable in aqueous solutions and rapidly un-
dergoes ring opening, generating the thioester hydrolysed
product.
Ligation with Side-Chain-Protected Glutamyl Thioesters
It is clear from previous results that a protecting group
strategy has to be taken into consideration for the synthesis
of Asp/Glu-thioester peptides. Such a protecting group
should have the following characteristics: i. be stable to HF
cleavage, ii. be stable during the ligation reaction, and iii.
be easily removable after the ligation reaction without jeop-
Figure 1. HPLC analysis of ligation products using different H-
LYRAE(X)-sr peptides: (a) ligation between 2 and 3; (b) ligation
between substrates 2 and 3 co-injected with compound 7; (c) lig-
ation product between substrates 8 and 3 after basic treatment; (d)
ligation product between substrates 9 and 3 after basic treatment
3268
 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2003, 3267Ϫ3272

Native Chemical Ligation with Aspartic and Glutamic Acids
FULL PAPER
ardizing the integrity of a fully unprotected polypeptide. (OFm)CSYRFL-OH (13), only one compound was gener-
Most of the available protecting groups do not match the ated with the correct α-backbone, and there was no trace
first requirement. We were focusing at this stage on com- of any side reaction.
mercially available Nα-Boc-Asp/Glu with side chain pro-
tecting groups, and we selected for our experiments the 9-
fluorenylmethyl ester (OFm)[3] and the (phenylsulfonyl)-
ethyl ester (OPse)[4] (Scheme 2). Synthesis by SPPS of H-
LYRAE(OFm)-sr (8) and H-LYRAE(OPse)-sr (9) gave, in
both cases, predominantly the correct peptides, but with
significantly less side products with the last protecting
group. The major identified side products with the OFm
protecting group were the loss of the OFm group (ഠ 20%
of the crude material)[5] and the alkylation of the OFm
group by Br-Z (ഠ 25% of the crude material). Obviously
this problem is limited to peptides containing a tyrosine,
but this side reaction has also been reported in the case of
the benzyl protecting group.[6]
When these peptides were tested in an NCL reaction at
pH 6.2, both peptides generated the expected ligation prod-
uct within two hours. It appears that the protected glutamic
acid undergoes ligation at a similar rate to the unprotected
residue, which is considered itself as a favorable site for lig-
ation among the 20 amino acids.[2] The stability of these
protecting groups under ligation conditions at different pH
values was investigated. The OPse group was shown to be
less stable than the OFm group, especially at higher pH
values. If the OPse protecting group is employed, the lig-
ation has to be carried out at pH 6.0 and over a short per-
iod of time, and hence with a higher excess of N-terminal
Cys peptide in order to generate the ligation product before
any significant loss of the protecting group. The OFm
group is, on the other hand, completely stable in the pH
range analysed.
Ligation with Aspartyl Thioesters Protected with
β-Removable Protecting Groups
The ligation with the aspartyl thioester peptide proved to
be a more difficult case than the glutamic acid thioester.
Introduction of the previously described OPse group on the
Asp side chain [H-LYRAD(OPse)-sr (14)] for the ligation
reaction results in the formation of 50% of the unprotected
aspartimide derivative of the ligation product as well as the
expected protected ligation product. Furthermore, depro-
tection of the first compound under the conditions de-
scribed for Glu(OPse) results in the formation of 70% of
the β-form and 30% of the natural α-product (Figure 2e).
Comparable results were obtained when the protected lig-
ation product was first isolated and then deprotected under
basic conditions.
H-LYRAD(OFm)-sr (15), on the other hand, proved to
be completely stable during the ligation reaction, generating
a single product. During the deprotection reaction at pH
12, however, two isoforms were generated, with the β-form
representing 70% of the final product (Figure 2, c). This
cannot be related to the presence of the free thiol of the
neighboring Cys since OFm deprotection of the S-aceta-
mide-alkylated H-LYRAD(OFm)C(Cam)SYRFL-OH pep-
tide results in the formation of the two isoforms in the
same ratio.
Aspartic Acid Protection Removed by Reduction
It was clear at this stage that the basic conditions em-
The OPse group has the advantage of allowing the use of ployed for the OFm and OPse removal were the major ob-
much milder conditions for the removal procedure, and this stacle to obtain the native product. This led us to investigate
reaction can be carried out directly in the ligation medium another class of protecting groups that are stable to HF but
in order to reduce the number of purification steps. As can be promptly removed under reductive acidic conditions,
[7]
shown in Table 1, the OPse group required less drastic con- which includes the phenacyl ester (OPac)
and the 2,2,2-
ditions giving complete removal in 0.1  Na2CO3, 10% trichloroethyl (OTce) esters[8] (Scheme 2). Since the Asp
BME, pH 9 at 37 °C in 2 h, while the OFm group was protected with these groups is not commercially available,
cleaved in 15 minutes in the presence of 10% BME, 20% these protecting groups were first incorporated into a series
DMF, 20% piperidine at pH12Ϫ13. It is interesting to no- of test peptides with the aim of investigating the stability
tice that β-mercaptoethanol heavily affected the rate of de- of such moieties in the ligation conditions (0.2  sodium
protection. When these conditions were applied to phosphate, 6  guanidine, 0.1% thiophenol, pH 6.5 at room
H-LYRAE(OPse)CSYRFL-OH (12) and H-LYRAE- temperature). The Ac-D(X)YAKYAKL-OH peptide was
Table 1. Conditions tested for OFm and OPse removal
Buffer conditions
OPse extent of deprotection
(%/time)
OFm extent of deprotection
(%/time)
NaHCO3 0.2 , pH ϭ 9
Na2CO3 0.2 , pH ϭ 12
50%/two days
38%/two days
Ϫ
Ϫ
NaHCO3 0.2 , 10% BME, pH ϭ 9
Na2CO3 0.2 , 10% BME, pH ϭ 12
20% pip., 20% DMF, pH ϭ 13
20% pip., 20% DMF, 10% BME, pH ϭ 13
200 equiv. DEA, 10% BME, pH ϭ 11
100%/2 h
100%/2 h
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
70%/2 h
100%/15 min
100%/2 h
Eur. J. Org. Chem. 2003, 3267Ϫ3272
www.eurjoc.org
 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3269

M. Villain, H. Gaertner, P. Botti
FULL PAPER
Ligation with Asp(OMop) Thioester Peptide
Synthesis of Boc-Asp(OMop)-OH was carried out with a
slight modification of a procedure described for Boc-
Asp(Pac)-OH.[9] The synthesis of H-LYRAD(OMop)-sr
(16) did not give any problems. HF cleavage of the peptide
was conducted at Ϫ5 °C since at 4 °C we observed the pres-
ence of 10% of the deprotected peptide. At this level it was
not possible to detect the two different diastereoisomers de-
rived from the OMop protection. Ligation with H-
CSYRFL-OH was completed in 1 h at pH 6.5, with genera-
tion of a unique ligated product with the OMop protection
and 5% already deprotected peptide. Zn/Acetic Acid depro-
tection of isolated H-LYRAD(OMop)CSYRFL-OH for 30
minutes resulted in only the H-LYRAD(α)CSYRFL-OH,
confirming that the methyl group does not affect the rate
of reduction (Figure 2, f).
Conclusions
Our work has proved that the ligation at Asp-Cys and
Glu-Cys ligation sites, when conducted with an unprotected
residue at the ligation site, results in the generation of
20Ϫ30% non-natural backbone isoforms. Characterization
of this side reaction was easy on a small peptide but could
be challenging on a larger fragment. To overcome this prob-
lem we investigated commercially available protecting
groups for glutamic acid and proved that the use of OPse
side-chain protection, although not ideal, is the best solu-
tion since OPse removal can be achieved under milder con-
ditions than for OFm. Aspartic acid was more problematic,
since the use of the available side-chain protecting groups
enhanced the generation of the non-natural isoform. For
this amino acid (and probably also for glutamic acid), the
best solution lies in a protecting group amenable to removal
by reduction with Zn in acetic acid. Unfortunately, the
classical phenacyl ester proved to be unstable in the ligation
reaction. A sterically hindered version — 1-methyl-2-oxo-2-
phenylethyl ester — withstands longer reaction times, and
generates only the expected natural α-compound at the lig-
ation site. This study pointed out OMop protection as one
of the possible ways to overcome the isomerization reaction
when approaching a ligation at an aspartic acid site. Con-
sidering the advantage of avoiding basic conditions for the
deprotection step, we suggest further exploration of OMop
also for glutamic acid protection. A further possible devel-
opment of this research could come from the exploration
of novel phenacyl-based derivatives with additional electron
donating groups on the phenyl ring of OMop, to enhance
the ligation stability of this protecting group.
Figure 2. HPLC analysis of ligation products using different H-
LYRAD(X)-sr peptides: (a) ligation between substrates 1 and 3; (b)
ligation between substrates 1 and 3 co-injected with compound 6;
(c) ligation product between substrates 15 and 3 after basic treat-
ment; (d) ligation product between substrates 14 and 3; (e) ligation
product between substrates 14 and 3 after basic treatment; (f) lig-
ation product between substrates 16 and 3 after Zn° reduction
derivatized with four different protecting groups: OTce,
OPac, 1-methyl-2-oxo-2-phenylethyl ester (OMop) and
(2ЈЈ,4ЈЈ-dimethoxy)phenacyl (OdiMeOPac). Since there
have been controversial reports on OPac stability,[9,10] and
with the aim of reducing potential hydrolysis, we syn-
thesised the sterically hindered version of OPac
—
OMop — by introducing an extra methyl in position 1Ј.[9]
This approach has been successfully used to increase the
stability of the phenacyl group when employed as a handle
for solid-phase synthesis.[15,16] Furthermore, we introduced
two methoxyl groups in positions 2ЈЈ and 4ЈЈ of the phenyl
group to verify the incidence of the phenyl ring electron
donating effect, generating diMeOPac. As reported in
Table 2, the OMop group presented the highest stability,
with a half-life of 10 h under ligation conditions, which is
sufficient for a ligation at a favorable residue site.[2] All the
other compounds showed much shorter half-live.
Table 2. Stability of the different Asp protections tested at pH 6.5,
0.1% thiophenol
Experimental Section
Protection
Hydrolysis t1/2 (h)
OMop
OdiMeOPac
OTce
9.9
3.8
1.5
1.2
Abbreviations
ACN: acetonitrile; BME: 2-mercaptoethanol; Boc: tert-butoxycar-
bonyl; COSY: correlation spectroscopy; DAE: dimethylamine;
DCM: dichloromethane; DIEA: N,N-diisopropylethylamine;
OPac
3270
 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2003, 3267Ϫ3272

Native Chemical Ligation with Aspartic and Glutamic Acids
FULL PAPER
DMAP: 4-dimethylaminopyridine; DMF: dimethylformamide; gradient unit [Bruker BioSpin AG, Fällanden, Switzerland].
DMSO: dimethyl sulfoxide; HBTU: N-[(1H-benzotriazol-1-yl)(di- Chemical shift data are given in ppm relative to internal TMS. Ap-
methylamino)methylene]-N-methylmethanaminium
hexafluoro- parent scalar couplings are given in Hz. Assignment of 1H and 13
phosphate; HF: hydrogen fluoride; HMBC: heteronuclear multiple chemical shift data is ascertained by making use of the appropriate
C
bond correlation; HSQC: heteronuclear single-quantum corre-
lation; MPAL: β-mercaptopropionic acid-leucine; NCL: native
chemical ligation; NMM: N-methylmorpholine; OdiMeOPac:
(2ЈЈ,4ЈЈ-dimethoxy)phenacyl group; OFm: 9-fluorenylmethyl ester;
homonuclear or heteronuclear correlation experiments including
COSY, HSQC, and HMBC (heteronuclear multiple bond corre-
lation).[14] Boc-Asp(OMop)-OH is an inseparable diastereomeric
mixture (1:1) of [2S,1ЈR]- and [2S,1ЈS]-4-(1-methyl-2-oxo-2-pheny-
OMop: 1-methyl-2-oxo-2-phenylethyl ester; OPac: phenacyl esters; lethyl) 2-[(tert-butoxycarbonyl)amino]succinate.
OPse: (phenylsulfonyl)ethyl ester; OTce: 2,2,2-trichloroethyl ester;
Pam: 4-hydroxymethylphenylacetic acid; PyBOP: benzotriazolylox-
ytris(pyrrolidinio)phosphonium hexafluorophosphate; RP-HPLC:
reversed-phase high performance liquid chromatography; Rt: reten-
tion time; SPPS: solid phase peptide synthesis; sr: β-mercaptopro-
pionic acid-leucine; TFA: trifluoroacetic acid; TMS: tetramethylsil-
ane; TIS: triisopropylsilane.
Reagents
Boc amino acids were used with the following side chain protec-
tion: Arg(Tos), Asp(OChx), Cys(pMeBzl), Glu(OChx), Lys(2ClZ),
Ser(Bzl) and Tyr(2BrZ). Boc amino acids were obtained from Or-
pegen Pharma (Heidelberg, Germany). HBTU was obtained from
Luxembourg Industries (Tel-Aviv, Israel). All the other chemicals
were from Fluka or Aldrich (Buchs, Switzerland) and used as re-
ceived. DIEA was from Applied Biosystems (Foster City, CA).
Boc-Asp(OPse)-OH.DCHA and Boc-Glu(OPse)-OH.DCHA were
from A&PEP Inc. (Bucheon, Korea). Boc-Asp(OFm)-OH, Boc-
Asp-OChx. DCHA, Boc-Asp-OtBu, Boc-Glu-OChx and Boc-Glu-
(OFm)-OH were from Bachem (Bubendorf, Switzerland). V8 pro-
tease (Type XVII-B) was from Sigma (Buchs, Switzerland).
Figure 3. Structure of Boc-Asp(OMop)-OH
If the diastereomers show clearly distinct resonances, two shift val-
ues are given in the following listings. All data refers to the domi-
nant rotamer of the N-Boc grouping (Ͼ 95%). 1H NMR
([D6]DMSO, 500 MHz): δ ϭ 1.36 (br. s, tBu), 1.41 [d, J ϭ 7 Hz,
H3C(3Ј)]; 2.65Ϫ2.90 (m, AB parts of two ABMX systems, H2C(3)];
4.28Ϫ4.35 (m, M parts of two ABMX systems, HC(2)]; 6.03/6.04
(two quart. J ϭ 7 Hz, HC(1Ј)]; 7.08/7.10 (2d, J ϭ 8.7 Hz, X-parts
of two ABMX systems, HN), 7.55 (arom. m-H), 7.68 (arom. p-H),
7.98 (arom. o-H), 12.7 (s, br. HO2C(1)] ppm. 13C NMR
([D6]DMSO, 125 MHz): δ ϭ 16.86/16.93 [H3C(3Ј)]; 28.14/28.46
(Me3 of tBu), 35.53/35.78 [H2C(3)]; 49.79/49.84 [HC(2)]; 71.70/
71.78 [HC(1Ј)]; 78.30 [C(6)]; 128.1/128.4 (arom. o-C), 129.0/129.1
(arom. m-C), 133.7 (arom. C(1ЈЈ), 133.8/133.9 (arom. p-C), 155.2
[C(5)]; 168.9/169.0 [C(4)]; 172.4/172.5 [C(1)]; 196.3/196.4 [C(2Ј)]
ppm.
Solid Phase Peptide Synthesis
All peptides were prepared by SPPS on a 0.2 mmolar scale, using
machine-assisted protocols on a custom-modified Applied Biosys-
tems model 433A peptide synthesizer, using in situ neutralization/
HBTU activation procedure for Boc chemistry as described pre-
viously.[11] C-terminal peptides were synthesized on the appropriate
Boc-aminoacyl-Pam preloaded resin. C-terminal Asp or Glu thio-
ester peptides were synthesized starting from the MPAL resin (β-
mercaptopropionic acid-leucine) following a published procedure
using PyBop as activator.[13,14] Boc-Asp(OPse)-OH and Boc-Glu-
(OPse)-OH were desalted according to standard protocols (Novabi-
ochem, Laüfelfingen, Switzerland) before activation with PyBop.
Ligation Reaction
After chain assembly was completed, the peptides were deprotected
and cleaved from the resin by treatment with anhydrous HF for 1 h
at 0 °C with 5% p-cresol as scavenger. The peptides were precipi-
tated with diethyl ether, dissolved in aqueous acetonitrile, lyophil-
ized, and purified by preparative RP-HPLC on a Waters 600 HPLC
module, using a C18 Waters DeltaPak preparative column. Peptide
identity was confirmed by ESI-MS with a Bruker Esquire 3000 Ion
Trap (Bruker Daltonics, Bremen, DE).
The C-terminal unprotected peptide (1.5 equivalents) and the N-
terminal peptide (1 equivalent) were solubilized in freshly degassed
0.2  sodium phosphate buffer, pH 7.5, containing 6  guanidine
at a concentration of 2.5 m each. After addition of 1% thiophenol
and 1% benzylmercaptan the pH was adjusted to 6.5 (or different
thiols concentration or pH as reported in the text). Aliquots of this
solution were treated with equal volumes of BME for 5 minutes
and then with 10% TCEP for 10 minutes to completely hydrolyse
any thiol adduct before HPLC analysis. The ligation reaction was
monitored by HPLC using a gradient starting from either 10% of
B with a gradient slope of 1.66% of B per minute or from 20% of
B and with an increase of 0.33% B per minute when a baseline
resolution of the different isomerization products was required.
Analytical RP-HPLC of all the products was performed on a
Waters 2690 HPLC module with 214 nm UV detection, using a
Symmetry 300 C18 column, with a linear gradient of buffer B in
buffer A over 30 minutes at 1 mL/min. Buffer A ϭ 0.1% TFA in
water; buffer B ϭ 0.1% TFA in ACN. Data were recorded and
analysed using the software system Millennium 32.
Enzymatic Digestion
NMR Studies
The peptides were solubilized in 0.1  NH4HCO3, 0.2% BME at a
5 m concentration, and V8 protease added for a final enzyme/
peptide ratio of 1 unit/20 µg. Digestion was stopped after 24 h incu-
bation at 37 °C by acidification with an equal volume of 10%
High-resolution NMR spectra of Boc-Asp(OMop)-OH were re-
corded in [D6]DMSO with a Bruker DRX-500 NMR spectrometer
operating at a field of 11.74 T and equipped with a pulsed field-
Eur. J. Org. Chem. 2003, 3267Ϫ3272
www.eurjoc.org
 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3271

M. Villain, H. Gaertner, P. Botti
FULL PAPER
TCEP. The extent of digestion was evaluated by RP-HPLC and the Zn/Acetic Acid OMop Removal
fragments obtained were characterized by ESI-MS.
The purified peptide H-LYRAD(OMop)CSYRFL-OH (5 mg) ob-
tained via ligation, was diluted in 30% aqueous acetic acid, mixed
with activated zinc powder and stirred for 30 minutes. The aqueous
solution was separated and the final product was obtained by a
desalting step with no further purification. Zinc powder (1 g) was
previously activated as follows: 1  aqueous HCl (4 ϫ 4 mL,
3 min), H2O (4 ϫ 4 mL, 1 min), and stored in H2O until use (da-
ily prepared).
Synthesis of Protected Asp Peptides
Evaluation of different protecting groups for the β-carboxyl of Asp
was carried out by coupling the precursor Fmoc-Asp(tBu)-OH at
the N-terminus of the peptide H-YAKYAKL-OH still on the Pam
resin. The N-terminus was deprotected with piperidine, acetylated
and the tBu was then removed in order to add the different protect-
ing groups. For OPac, OMop and OdiMeOPac the resins were re-
acted with the α-bromo ketones phenacyl bromide, α-bromopropi-
ophenone and 2-bromo-2Ј,4Ј-dimethoxyacetophenone, respectively.
The same protocol was used for the three products, using a 20 
solution of bromo ketone over the resin-free carboxylic acid with
1 equivalent of DIEA in DMF overnight at room temperature. For
OTce the resin was treated with 2,2,2-trichloroethanol (40 equiv. of
the resin free carboxylic acid), DMAP (0.1 equiv.), NMM (1 equiv.)
and PyBop (1 equiv.) in DMF overnight at 37 °C. The final prod-
ucts were cleaved and deprotected by HF treatment. The peptides
were purified by preparative RP-HPLC. OMop generated two dia-
stereoisomers which were resolved by HPLC. Only the major prod-
uct was used. The overall esterification yield in this procedure was
about 50% and only the OTce derivative was obtained in a signifi-
cant amount (15%).
Acknowledgments
We would like to thank Benoit Depresle, Nicolas Dafflon, Mari-
anne Paolini and Sylvain Raimondi for their excellent technical as-
sistance. We thank Dr. Keith Rose for the helpful discussion during
the project development and Prof. Ulrich Burger for assistance in
NMR assignments.
[1]
P. E. Dawson, T. W. Muir, I. Clark-Lewis, S. B. Kent, Science
1994, 266, 776Ϫ779.
T. M. Hackeng, J. H. Griffin, P. E. Dawson, Proc. Natl. Acad.
[2]
Sci. USA 1999, 96 (18), 10068Ϫ73.
F. al-Obeidi, D. G. Sanderson, V. J. Hruby, Int. J. Pept. Protein.
[3]
Res. 1990, 35(3), 215Ϫ218.
Y. S. Lee, H. J. Lee, P. I. Pozdnyakov, V. V. Samukov, H. J. Kim,
[4]
Synthesis of Boc-Asp(OMop)-OH
J. Pept. Res. 1999, 54(4), 328Ϫ335.
C. B. Xue, J. M. Becker, F. Naider, Int. J. Pept. Protein. Res.
[5]
The cesium salt of Boc-Asp-OtBu (1.38 mmol) was prepared under
standard conditions and reacted with α-bromopropiophenone
(1.3 mmol) in DMF (4 mL) by incubation overnight at room tem-
perature. The white precipitate was discarded after filtration and
the DMF solution was dried under vacuum. The resulting oil was
diluted with ethyl acetate, and the organic phase was washed with
1  NaHCO3 (4 times), then with saturated NaCl, dried with
MgSO4, and finally concentrated under vacuum.
1991, 37(6), 476Ϫ486.
J. Robles, E. Pedroso, A. Grandasy, Int. J. Pept. Protein Res.
[6]
1994, 43(4), 359Ϫ62.
[7]
G. C. Stalakatos, A. Paganou, L. Zervas, J. Chem. Soc. 1966,
11918.
[8]
R. B. Woodward, K. Heusler, J. Gosteli, P. Naegeli, W. Op-
polzer, R. Ramage, S. Ranganathan, H. Vorbrügger, J. Am.
Chem. Soc. 1966, 88, 852.
[9]
C. C. Yang, R. B. Merrifield, J. Am. Chem. Soc. 1976, 41,
The resulting Boc-Asp(OMop)-OtBu (single spot by TLC, Rt ϭ
0.95 chloroform/methanol/acetic acid, 90:8:2) was deprotected in
TFA/Tis/H2O (95:2.5:2.5,v/v) for 2 h. TFA was evaporated under
vacuum and the remaining TFA was removed by multiple cycles of
acetonitrile and ethanol evaporation. The resulting oil was solubil-
ized in DMSO (2.5 mL), Boc2O (2.76 mmol) and DIEA
(2.76 mmol) were added and the reaction mixture was stirred over-
night at room temperature. The solution was diluted with water
(10 mL) and acidified to pH 3.5 with KHSO4. The aqueous phase
was extracted with diethyl ether, the combined organic fractions
were washed with 1  KHSO4 and saturated NaCl, dried with
MgSO4 and finally concentrated at reduced pressure. The oily prod-
uct was used without further purification for coupling to MPAL
resin using PyBop as activator.
1032Ϫ1041.
[10]
M. Bodanszky, J. Martinez, J. Org. Chem. 1978, 43,
3071Ϫ3073.
M. Schnölzer, P. Alewood, A. Jones, D. Alewood, S. B. H.
[11]
Keny, Int. J. Pept. Protein Res. 1992, 40, 180Ϫ193.
[12]
H. Hojo, S. Aimoty, Bull. Chem. Soc., Jpn. 1991, 64, 111Ϫ117.
[13]
H. Hojo, Y. Kwon, Y. Kakuta, S. Tsuda, I. Anaka, K. Hikichi,
S. Aimoto, Bull. Chem. Soc., Jpn. 1993, 66, 2700Ϫ2706.
D. W. Timothy Claridge, in High-Resolution NMR Techniques
[14]
in Organic Chemistry, PergamonϪElsevier Science Ltd., Am-
sterdam, 1999.
[15]
D. Bellof, M. Mutter, Chimia 1985, 39, 317Ϫ320.
[16]
J. C. Sheehan, K. Umezawa, J. Org. Chem. 1973, 38,
3771Ϫ3774.
Received January 22, 2003
3272
 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2003, 3267Ϫ3272