Aldehyde capture ligation for synthesis of native peptide bonds

Article abstract of DOI:10.1021/jacs.5b03538

Chemoselective reactions for amide bond formation have transformed the ability to access synthetic proteins and other bioconjugates through ligation of fragments. In these ligations, amide bond formation is accelerated by transient enforcement of an intramolecular reaction between the carboxyl and the amine termini of two fragments. Building on this principle, we introduce an aldehyde capture ligation that parlays the high chemoselective reactivity of aldehydes and amines to enforce amide bond formation between amino acid residues and peptides that are difficult to ligate by existing technologies.

Full text of DOI:10.1021/jacs.5b03538

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Article
Aldehyde Capture Ligation for Synthesis of Native Peptide Bonds
Monika Raj, Huabin Wu, Sarah L. Blosser, Marc A. Vittoria, and Paramjit S. Arora
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b03538 • Publication Date (Web): 12 May 2015
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Aldehyde Capture Ligation for Synthesis of Native Peptide
Bonds
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Monika Raj,‡,† Huabin Wu,‡ Sarah L. Blosser, Marc A. Vittoria, and Paramjit S. Arora*
Department of Chemistry, New York University, 100 Washington Square East, New York, NY 10003
Supporting Information Placeholder
ABSTRACT: Chemoselective reactions for amide bond formation have transformed the ability to access synthetic proteins and other bio-
conjugates through ligation of fragments. In these ligations, amide bond formation is accelerated by transient enforcement of an intramolecu-
lar reaction between the carboxyl and the amine termini of two fragments. Building on this principle, we introduce an aldehyde capture liga-
tion that parlays the high chemoselective reactivity of aldehydes and amines to enforce amide bond formation between amino acid residues
and peptides that are difficult to ligate by existing technologies.
the carboxyl and the amine partners (Figure 1b).29 The o-
INTRODUCTION
benzaldehyde ester (I) could condense with the amine functionali-
Methods for chemoselective formation of amide bonds between
unprotected peptide fragments have provided valuable access to
ty to form the hemiaminal (II), hemiaminal ester (III) or imine
(IV) intermediate.32 Subsequent rearrangement of the hemiami-
recombinant proteins and have become reliable methods for the
nal/imine/hemiaminal ester intermediate through a cyclic transi-
synthesis of bioconjugates.1-9 These advances have made total syn-
tion state would yield the amide product (V) and the ortho-
benzaldehyde auxiliary (VI). The ortho-aldehyde group in the
auxiliary is designed to reversibly capture the amine and intramo-
thesis of therapeutic peptides and proteins, hormones, and modi-
fied antibodies a credible objective.10-17 The premise underlying
peptide ligation approaches is that the amide bond formation step
can be accelerated by capturing the carboxyl and the amine func-
lecularly deliver it to an activated acid. The esters may also directly
condense with the amine;35-38 although our studies suggest that this
tionalities and enforcing intramolecular bond formation. Various
is not a major pathway. The mechanistic intermediates have been
ligation technologies18-24 including native chemical ligation
postulated by Kemp33 (Figure 2a) and also evaluated by Ito.34 As
(NCL)25 and the Staudinger ligation,26,27 as well as auxiliary based
the earlier studies by Kemp suggest that the imine is a nonproduc-
tive intermediate, we postulated that dehydration of the initially
formed hemiaminal II would need to be slower than the X!N shift
to yield the desired amide in high yield. If the hemiaminal is indeed
methods,5,28,29 utilize this concept and are widely used. Here we
describe an approach termed the aldehyde capture ligation, or
ACL, that exploits a similar fundamental logic to the auxiliary based
methods first conceived three decades ago by Kemp and others.28-34
the functional intermediate rather than an imine, we hypothesized
Kemp and Vellaccio postulated that an amine nucleophile may
that secondary amines may also undergo aldehyde capture ligation.
be captured by an aldehyde group in proximity of a carboxylic acid
Figure 2 compares ACL with three other aldehyde capture ap-
masked as an ester to drive its aminolysis.32 Reactions of 8-acetoxy-
proaches, including Kemp’s, that enforce amide bond formation.29
1-naphthaldehyde or 2-acetoxybenzaldehyde with benzylamine
Tam and coworkers utilized α-acyloxyacetaldehyde auxiliary to
provided a mixture of the desired amide as well as imine byproducts
activate C-terminal carboxylic acid groups (Figure 2b). Condensa-
in a solvent dependent manner.29 We postulated that the amount
tion of the activated acid with peptides bearing N-terminal cysteine,
of the dehydrated imine byproduct could be reduced if the carbox-
serine or threonine residues yield psuedoproline-linked
ylic acid was masked as a more reactive thio or seleno ester.34 If
fragments.8,39,40 Li et al. have recently demonstrated that O-
successful, the method will, in principle, be applicable to any N-
terminal amino acid residue for amide bond formation. For exam-
salicylaldehyde ester enables coupling of N-terminal ser-
ine/threonine fragments (Figure 2c).30,31 Serine-threonine ligation
ple, NCL requires the presence of a cysteine group as the N-
and ACL (Figure 2d) utilize a similar benzaldehyde auxiliary; how-
terminal residue but ACL should work for any amine because the
ever, we envisioned broader substrate scope for the ACL if the
capture step is changed from thioester exchange, as in NCL, to
initially formed hemiaminal could be acylated directly.
aldehyde–amine condensation (Figure 1a).
Aldehyde capture ligation (ACL),28,30-32 envisions an o-
benzaldehyde ester to enforce an intramolecular reaction between
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Figure 1. Rationale for the aldehyde capture ligation (ACL). (a) Comparison of a phenylthioester-initiated native chemical ligation
(NCL) and ACL. ACL seeks to accelerate amide bond formation through transient condensation of the amine partner with an appended
aldehyde group and enforcement of a cyclic intermediate. NCL enhances the effective molarity between reacting partners through a tran-
sient thioester bond formation with the cysteine side chain. (b) Several discreet mechanisms may be postulated for ACL.
Figure 2. Mechanistic approaches that utilize aldehyde capture for amide bond formation. Approaches outlined by (a) Kemp, (b) Tam,
and (c) Li are compared with (d) ACL.
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phenol group. Importance of ortho-aldehyde group for ACL is
RESULTS AND DISCUSSION
1
also evident in the thioester series 1d-1f, as 1d, with an ortho-
aldehyde group, reacts more efficiently than the unsubstituted
thiophenyl and para-thiobenzaldehyde esters, 1e and 1f, respec-
tively (Table 1). Extensive analysis of aminolysis of thioesters
suggests that N-acylations also proceed through tetrahedral in-
termediates as oxoesters.44,45 (The thiobenzaldehyde ester deriva-
tives undergo a side reaction; this reaction can be suppressed by
converting the benzaldehyde to the corresponding benzophe-
none. Details on this benzophenone-analog are included in the
Supporting Information, Figure S1).
Evaluation of the reactivity of oxo-, thio-, and
seleno-benzaldehydesters for amide bond for-
mation
2
3
4
We began the ACL auxiliary development investigations by con-
densing salicylaldehyde-derived benzylester 1a with benzylamine
in DMF (Table 1).28,32 Complete conversion of the ester to am-
ide 2 was observed in less than five hours as monitored by LCMS.
Presence of the ortho-aldehyde group is critical for the ACL
mechanism as phenyl and para-benzaldehyde esters, 1b and 1c,
respectively, react at much slower rates. Compound 1c also
serves as a control for evaluating potential reactivity increases due
to the presence of an electron-withdrawing group at the ortho or
para position of the aromatic ring. Aminolysis reactions are not,
considered to be sensitive to minor pKb differences in the leaving
phenolate ions, as the formation of the tetrahedral transition state
is the rate limiting step; although, analysis is complicated by pro-
ton transfers.41-44 The pKa’s of the ortho-formylphenol is slightly
lower than the para substituted analog while both pKa’s are much
lower than that of the basic amine nucleophile. The para-
benzaldehyde ester analog, 1c, undergoes amide bond formation
much slower than the ortho analog 1a reflecting the faster rate of
aldehyde-amine union than direct acylation of the substituted
phenylesters. While the transient hemiaminal/imine in the ortho
analog is rapidly captured for acylation, a similar step is not possi-
ble for the para analog.
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To further accelerate the ACL reaction, we changed the leaving
group from a thiophenol moiety to a selenophenol derivative 1g
(Table 1).35,37,38 As expected, substitution with selenol provided a
additional boost to the reaction rate with complete conversion to
amide 2 occurring faster than could be measured under the reac-
tion conditions. The above reactions were performed using 50
mM of the oxo/thio/selenoesters and 2.5 eq of benzylamine. To
evaluate the difference in reactivity of different selenoester deriva-
tives, we utilized a lower concentration of selenoester (2 mM). A
2.5 eq excess of benzylamine was uniformly maintained in all
experiments (Supporting Information, Figure S2). (Under our
HPLC conditions designed for qualitative assessment of reaction
rates, we could not accurately measure the rates of reactions that
completed in less than 2 minutes; further lowering of substrate
concentrations to reduce reaction rates also affects accurate de-
tection of starting materials and products.) Under these condi-
tions, we find selenoester 1g with the o-benzaldehyde group un-
dergoes complete conversion to the amide in less than 2 min,
with rate substantially faster than the para and unsubstituted
benzylselenoester analogs 1h (> 60 h) and 1i (30 min), respec-
tively. The exceptionally slow rate of amide formation with the para-
selenoester analog 1h suggests that condensation of aldehydes with
amines is faster than aminolysis of selenoesters. We postulate that
the para-aldehyde group scavenges the amine and lowers the
concentration of the free amine impacting the rate of amide bond
formation; however, because we used an excess of the amine, the
amide bond should have formed at a faster rate if the rate of direct
acylation (with aldehyde substituted benzyl esters) were compet-
itive with aldehyde/amine condensation.
Table 1. Model studies for the development of ACL
R2
R3
O
O
DMF
HN
Ph
+
Ph
N
Ph
Ph
X
R3
R1
2
Ester
1aa
1ba
1ca
1da
1ea
1fa
X
R1
R2
H
H
CHO
H
R3
H
H
H
H
H
H
H
H
H
H
Timec
5 h
>40 h
O
O
O
S
S
S
Se
Se
Se
Se
Se
Se
Se
Se
CHO
H
H
>40 h
CHO
H
10 min
60 min
10 h
We also investigated if secondary amines are able to utilize the
ACL mechanism. A similar trend in reaction rates was observed
with the N-methylbenzylamine as above: the ortho-aldehyde ana-
log 1g undergoes selenoester aminolysis much faster than the
para-aldehyde (1h) or the unsubstituted selenoester (1i). Reac-
tivity of secondary amines result suggests that hemiaminal inter-
mediate is engaging the ester, as the imine formed from a second-
ary amine would be expected to be less nucleophilic.
H
H
CHO
H
1ga
1gb
1hb
1ib
CHO
CHO
H
<2 mind
<2 mind
>60 h
30 min
<2 mind
>60 h
H
CHO
H
H
1gb
1hb
1ib
CHO
H
H
CH3
CH3
CH3
H
CHO
H
Lastly, we tested the potential of a different electron withdraw-
ing group in place of the aldehyde group to mediate coupling of
selenoesters with benzylamine through direct aminolysis.34,46
Compound 1j, which features an ortho-nitrophenyl selenoester,
undergoes aminolysis on par with ortho-benzaldehyde selenoes-
ter; although accurate comparisons are not possible under our
HPLC conditions. The pKa of ortho-nitrophenol (7.23) falls
between that of ortho-formylphenol (6.79) and para-
formylphenol (7.66) thus the rate of aminolysis for the nitro de-
rivative may not be readily explained by the leaving group ability
of the particular selenols, assuming the pKa trend from phenols
translates to selenol derivatives. This observation is consistent
H
30 min
1jb
NO2
H
<2 mind
aReaction conditions: esters (50 µmol) and amines (125 µmol)
in 1mL DMF. bReaction conditions: esters (2 µmol) and amines
(5 µmol) in 1mL DMF. Time for >95 % conversion of starting
ester to the amide product at room temperature as determined
by LCMS. These rapid reaction rates could not be accurately
c
d
measured with the HPLC assay.
Next, we sought to enhance the efficiency of the ACL reaction
by converting the leaving group from a phenol moiety to a thio-
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with literature reports that the formation and not the breakdown We determined the compatibility of ACL for synthesis of pep-
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of the tetrahedral transition state is the rate limiting step in ester
aminolysis reactions, as discussed above.41-44 Further studies,
vide infra, with the benzaldehyde selenoesters and nitrophenyl
selenoesters indicate that subtle electronic effects may be modu-
lating the reactivity of the benzaldehyde selenoesters. Overall,
although, we cannot rule out direct acylation of amines as a con-
tributor to amide bond formation, our model studies support the
aldehyde capture mechanism for ortho-selenobenzaldehyde de-
rivatives with a hemiaminal as the major pathway.
tides in aqueous buffers (with DMF as a co-solvent) for potential
applications aimed at synthesis of large peptides and proteins or
other bioconjugates by fragment coupling. The formation and
reactions of hemiaminals and imines are known to be sensitive to
buffer pH;47 however, hydrolysis of selenoesters is also pH de-
pendent. Our results show that initial rates of dipeptide for-
mation were higher at basic pH, with pH 8.5 proving to be opti-
mal (Figure 3 and Figure S4).
9
In these exploratory studies designed to determine the scope of
the aldehyde capture ligation, we initially prepared amino acid
and peptide thio- and seleno-benzaldehyde esters by coupling
activated carboxylic acids and o-benzaldehyde disulfide (6) or
diselenide (7) in the presence of a reducing agent, such as TCEP
(Figure 4). The diselenide, itself, is available in one step from 2-
bromobenzaldehyde (Supplementary Information).48 (We have
also explored solid phase synthesis of peptide selenoesters as
described below.)
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Application of ACL to amino acid derivatives:
Evaluation of potential epimerization
We next sought to determine if amino acid derived selenoben-
zaldehyde esters can provide the desired dipeptides in a chemose-
lective and efficient manner. The ACL reaction between alanine-
derived selenobenzaldehyde ester, 3, and tryptophan methyl ester
follows a second order rate constant, (FmocAla-COSe-o-
PhCHO, k = 1.65 0.06 M-1S-1) (Table 2 and Supporting Infor-
mation, Figure S3). This reaction undergoes complete conver-
sion to the dipeptide roughly 100-times faster as compared to the
selenoester analog without the o-aldehyde group (FmocAla-
COSePh, k = 0.015 0.001 M-1S-1).
O
H
O
H
X
R
OH
R
DCC, TCEP
X
X
H
THF
55 - 70%
O
O
R = AA
or
Table 2. Kinetics of the aldehyde capture ligation for dipeptide
O
dipeptide
formationa.
6: X = S
7: X = Se
8: X = S
9: X = Se
R
Figure 4. Synthesis of seleno- and thio-o-benzaldehyde esters
from Fmoc amino acids (AA) or peptides.
Se
Trp-OMe
DMF
FmocHN
Fmoc-Ala-Trp-OMe
O
5
Selenoester
R
k [M-1S-1]
Relative Rates
a)
b)
3
4
CHO
H
1.653 0.0587
0.015 0.0011
110
1
aConditions: FmocAla-seleno-ester, 3 or 4 (2 µmol), HCl⋅NH2-
Trp-OMe (2 µmol) and Et3N (2 µmol) in 1mL DMF.
c)
d)
CHO
Se
Trp-OMe
DMF
Fmoc-Ala-Trp-OMe
FmocHN
0.45
3 O
5
Figure 5. HPLC studies to determine potential epimerization
of the amino acid selenoester: (a) Analytical HPLC trace of a
1:1 mixture of diastereoisomers, FmocV-
A-AQ. (b) Analytical HPLC trace of a 1:4 mixture of diastereo-
isomers, FmocV- A-AQ and Fmoc-V- A-AQ. (c) Analytical
HPLC trace of a 1:49 mixture of diastereoisomers, FmocV- A-
AQ and FmocV- A-AQ (d) Crude HPLC trace of ACL reaction
mixture FmocVal- Ala-seleno-ortho-benzaldehyde ester (10
DA-AQ and Fmoc-V-
L
D
L
D
0
L
DMF
6.5
7.0
8.5
9.3
7.5
L
PBS buffer (pH): DMF
µmol), TFA⋅Ala-Gln-CONH2 (20 µmol) and Et3N (20 µmol)
in 1 mL DMF. HPLC Conditions: 0.1 % TFA (v/v) in water
(solvent A): acetonitrile (solvent B); gradient 35-65% in 60
min, flow rate = 0.5 mL/min.
Figure 3. Effect of pH on reaction rates. Conditions: FmocAla-
COSe-o-PhCHO, 3 (2 µmol), HCl⋅NH2-Trp-OMe (4 µmol) and
Et3N (4 µmol) in 1mL DMF or 1:1, DMF:100 mM NaH2PO4
buffer (pH ~ 6.5-9.3). Plotted values are the average of two inde-
pendent experiments.
Epimerization of activated amino acids is a critical concern in
peptide synthesis. The selenobenzaldehyde-modified peptide
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may be expected to undergo epimerization during the course of As part of these studies, we compared the reactivity of amino
1
2
3
4
5
6
7
8
aldehyde capture ligation possibly through the formation of an
azalactone. To investigate this possibility and quantify the
amount of epimerized product, we prepared a tetrapeptide
FmocVal-LAla-Ala-Gln-NH2 and the expected epimerized analog,
FmocVal-DAla-Ala-Gln-NH2. These products would result from
ligation of FmocVal-LAla-COSe-o-PhCHO with NH2-Ala-Gln-
CONH2. An HPLC assay that allows detection of as low as 2%
epimerization was developed; careful analysis showed the ab-
sence of racemization under the reaction conditions (Figure 5
and Figure S5).
acid selenobenzaldehyde esters with standard activated esters
utilized for peptide synthesis. Performance of activated Fmoc-
valine esters derived from DCC/DMAP, HBTU, and DCC/NHS
(N-hydroxysuccinimide) was compared with FmocVal-
selenobenzaldehyde ester for condensation with tryptophan me-
thyl ester. Coupling using the classical DCC/DMAP conditions
requires 96 hours for 70% conversion. All reactions were per-
formed at 10 mM substrate concentrations in DMF (Supporting
Information, Table S1). Reaction with the preformed NHS-ester
is completed in 30 h while condensation with HBTU derived OBt
ester is completed in 30 mins. Reaction rates for the selenoben-
zaldehyde ester are slightly slower than for the HBTU-derived
OBt ester, a highly efficient but non-chemoselective and water
sensitive coupling agent. This analysis suggests that ACL may
potentially be useful for performing ligation reactions under
aqueous conditions on solid-phase matrices.
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Condensation of FmocVal-Ala selenobenzaldehyde with NH2-
Ala-Gln-CONH2 is a relatively fast reaction. To rule out the pos-
sibility that the epimerization is occurring but at a slower rate, we
tested condensation of FmocAla-Val-L-COSe-o-PhCHO with
NH2-Ala-Gln-CONH2 under the same reaction conditions. Am-
ide bond formation involving valine selenobenzaldehyde esters is
relatively slow (Table 3, entries 8 and 9). Roughly 2% epimeriza-
tion was observed for this slower ACL coupling (Figure S5).
Table 3. Scope of the aldehyde capture ligation for the synthesis
The low level of epimerization observed for the ACL reactions
was surprising because activated acids, especially in the context of
peptides where the azalactone formations are often observed as
an intermediate to epimerization.49 Above, we described aminol-
ysis experiments with the nitrophenyl selenoesters. We sought to
compare the epimerization levels between the benzaldehyde and
nitrophenyl derivatives; however we were unable to synthesize
the dipeptide nitophenyl selenoester presumably because of its
disposition to undergo azalactonization and form other side
products (Scheme 1). The azalactone side product was observed
by LCMS of crude reaction mixtures. We hypothesize that the
absence of epimerization with the benzaldehyde derivative and its
slower rate of aminolysis as compared to the nitrophenyl analog
suggests an electronic (n-π*) interaction between the carbonyl of
the formyl group and the selenoester that diminishes azalactone
formation.50-52 Further studies will be required to support this
postulate.
of peptides. Residues at the junction are underlined.a
Entry
1
Ligation product
Timeb
2 min
2 min
2 min
2 min
5 min
5 min
3 min
2 h
Fmoc-AW-COOMe
Fmoc-AV-COOMe
Fmoc-AR-COOMe
Fmoc-GC-COOMe
Fmoc-AS-COOBn
2
3
4
5
6c
(Fmoc-A)2K-COOH
Fmoc-FL- COOtBu
Fmoc-VW-COOMe
Fmoc-VV-COOMe
Fmoc-AibW-COOMe
Fmoc-AVDE-CONH2
Fmoc-AASY-CONH2
Fmoc-AASY-CONH2
Fmoc-AAAH-CONH2
Fmoc-AVDAFE-CONH2
Fmoc-FVDAFE-CONH2
Fmoc-VAVDAFE-CONH2
7
8
9
4 h
10
11
12
13d
14
15
16
17
2 h
5 min
2 min
2 min
2 min
5 min
5 min
5 min
Scheme 1
O
FmocNH
OH
N
H
O
O2N
Se Se
7, DCC, TCEP
NO2
O
aReaction conditions: Fmoc amino acid-selenobenzaldehyde
ester (10 µmol), N-terminal amino acid/peptide HCl or
CF3COOH salts (20 µmol) and Et3N (20 μmol) in 1 mL DMF.
b Time for > 95 % conversion to product at room temperature.
Analysis by HPLC traces of the crude reaction mixture. cDouble
acylation of lysine on the α- and ε-amine groups occurs.
dFmocAla-selenobenzaldehyde ester (20 µmol), N-terminal
CF3COOH·ASY-CONH2 (10 µmol) and Et3N (10 µmol) in 1
mL DMF; after 6 h only the amidation product is obtained.
DCC, TCEP
NO2
O
CHO
FmocNH
Se
FmocNH
Se
N
H
N
H
O
O
unstable
Stable
(not synthesized)
O
O
FmocNH
N
Potential of ACL for chemoselective ligation
The utility and attractiveness of native chemical ligation results
from the fact that it affords chemoselective ligation of peptides,
and other biomolecules, without need of protecting groups on
reactive side chain functionality. We explored the tolerance of
azalactone (observed by MS)
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ACL for unprotected side chain groups for a variety of amino acid
phase synthesis of thioesters (Figure 6).57 A model pentapeptide
1
2
3
4
5
6
7
8
partners including Arg, Asp, Cys, Glu, His, Leu, Phe, Ser, Trp,
Tyr and Val (Table 3). These amino acid residues underwent the
desired ligation suggesting that ACL is a chemoselective reaction.
Reactions of selenobenzaldehyde esters with N-terminal serine
and cysteine residues may provide the amide products through
the formation of respective pseudoproline intermediates as re-
ported for oxobenzaldehyde esters, 30,31,39,40 rather than through an
ACL mechanism. We investigated this possibility but did not
observe the formation of the pseudoproline derivatives with sele-
no-benzaldehyde esters in DMF or pyridine/acetic acid mixtures
(Figure S6). (The serine/threonine ligation has been shown to
be optimal in pyridine/acetic acid mixtures.31) N-terminal cyste-
ine may undergo condensation under an NCL or ACL mecha-
nism.
(FmocLYRAG) N-acyl-benzimidazolinone (Nbz) was synthe-
sized using the Dawson Nbz protocol. The peptide was cleaved as
the o-benzaldehydeselenoester by treatment of the resin with
diselenide 7 (Figure 6) and tributylphosphine. The crude yield of
the cleaved peptide was calculated to be 59% based on resin load-
ing using the Fmoc absorbance. The selenoester was character-
ized using LCMS (Figure 7a). (The HPLC analysis shows a peak
for the hydrolyzed peptide along with the desired selenoester.
We posit that the selenoester likely hydrolyzes during the HPLC
run under the acidic aqueous conditions utilized for the analysis
and not during the selenoester synthesis. The hydrolysed peptide
is not observed upon further reaction of the unpurified selenoes-
ter with an amine (Figure 7b), supporting this hypothesis).
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H
O
A potential limitation of ACL is that the aldehyde group on se-
Se
lenobenzaldehyde esters may react with any amine, i.e. ε-amino
of lysine would undergo an ACL as well as N-terminal amines
(Table 3, entry 6). Possible solutions to control reactions of ly-
sine side chain amines include standard protecting group strate-
gies or reactions with peptides and proteins under pH-controlled
conditions. The N-terminal amino group of proteins has a signifi-
Se
O
FmocAn--A3-A2-A1
O
O
7
H
O
N
O
PBu3, THF
N
H
cantly lower pKa value than the ε-amine of lysines.53 Prior studies
have shown that this pKa difference can be exploited to selectively
modify the N-terminal amine in the presence of lysine residues.54
We have explored the possibility of selectively modifying N-
terminal amines of proteins using ACL (vide infra).
O
Se
FmocAn--A3-A2-A1
CHO
Figure 6. Solid phase synthesis of peptide seleno-o-
benzaldehyde esters.
We investigated the possibility if side chain alcohol functionali-
ty of serine or tyrosine is acylated, especially with an excess of
selenobenzaldehyde esters. Tripeptide ASY-CONH2 was treated
with 2 or 0.5 eq of Fmoc-alanine selenobenzaldehyde ester (Ta-
ble 3, entries 12-13). Acylation of the side chain alcohol group
was not observed even after prolonged reaction periods. C-
terminal aspartic and glutamic acid thioesters can undergo reac-
tions with side chain carboxylates;55 this side reaction will likely
also preclude placement of Asp and Glu residues, along with oth-
er reactive side chains such as cysteine, at the C-terminus in pep-
tido selenobenzaldehydes, as in NCL.
Evaluation of ACL for difficult couplings
The efficiency of peptide fragment couplings by native chemi-
cal ligation, and by other coupling agents, is significantly reduced
for bulky amino acid substrates.56 We examined a range of sele-
nobenzaldehyde esters featuring bulky C-terminal residues in-
cluding valine and aminoisobutyric acid (Aib) to evaluate the
potential of ACL for these challenging couplings. The sterically
demanding Val and Aib selenobenzaldehyde esters require longer
ligation times (2-4 h) for >95 % conversion (Table 3, entries 8-
10) as compared to 2-5 minutes for Ala, Gly and Phe analogs
(Table 3, entries 1-7).56 Ligation of selenobenzaldehyde esters
with pentapeptide (VDAFE) underwent completion in just 5
minutes (Table 3, entries 15-17), suggesting that ACL with pep-
tides is as efficient as with single amino acid residues.
Figure 7. HPLC analysis of peptide-selenoester condensation.
(a) Crude analytical HPLC trace of side chain-deprotected
FmocLYRAG-Se-o-PhCHO synthesized from the correspond-
ing resin bound Nbz-peptide. The peptide was treated with
trifluoroacetic acid:TES:H2O (95:2.5:2.5) to remove the pro-
tecting groups prior to LCMS analysis. (b) Crude HPLC trace
of FmocLYRAGFRANG-CONH2 obtained from the condensa-
tion of unpurified FmocLYRAG-Se-o-PhCHO from part (a)
and FRANG-CONH2. HPLC Conditions: 0.1 % TFA (v/v) in
water (solvent A): acetonitrile (solvent B); gradient 5-95% B in
30 min, flow rate = 0.5 mL/min. m/z* = Masses of these peaks
do not correspond to the starting selenoester or identifiable side
products.
Solid phase synthesis of peptide selenobenzalde-
hyde esters: Evaluation of ACL for peptide liga-
tion
To extend the ACL technology to large peptides, we explored a
solid phase method for the synthesis of seleno-benzaldehyde
esters using the approach outlined by Dawson et al. for the solid
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We utilized the solid phase synthesis methodology to evaluate
CRANK-CONH2 needs more than 48 h for 60% completion.56
The ACL-mediated condensation of peptides in aqueous solu-
tions is slower than in DMF, requiring roughly three times longer
for completion. The HPLC analysis of crude reaction mixture
shows efficient conversion of the selenoester to the desired pep-
tide (Figure 7).
1
2
3
4
5
6
7
8
the applicability of ACL to the synthesis of model peptides in
aqueous solutions. For direct comparison with NCL, we pre-
pared peptide sequences that have previously been explored by
Table 4. Aldehyde capture ligation of peptide segments
Ligation producta
Solvent
DMF
10% DMF in H2O 90 mind
Timeb
Comparison of ACL and NCL for proline ligation
c
d
c
Fmoc-LYRAGFRANG-CONH2
Fmoc-LYRAGFRANG-CONH2
Fmoc-LYRAVFRANG-CONH2
30 min
Acylated prolines thioesters react slowly (<15% yield in >48 h)
under NCL conditions.56 The rates of these NCL reactions have
been reported to be independent of the number of residues at-
tached to the N-terminus of proline.58,59 This observation is con-
sistent with the postulated contribution of the n-π* interaction on
proline reactivity as only the carbonyl attached directly to the
proline residue perturbs the rate of the reaction.51 We synthe-
sized various acylated proline derivatives to gauge the potential of
proline ligations using the ACL approach (Table 5, entries 1 and
2), and find that ACL excels at difficult ligation of proline resi-
dues indiscriminant of whether it is a C-terminal or N-terminal
residue. Couplings of FmocVal, FmocPro, FmocAla-Pro and
FmocVal-Pro selenobenzaldehyde esters with proline methyl
ester proceed rapidly (Table 5, entries 1 and 3); whereas, analo-
gous reactions of FmocVal and FmocPro selenophenyl esters
(without the ortho-aldehyde group) require significantly longer
periods (Table 5, entries 2 and 4). These studies demonstrate
that preformed phenylselenoesters, which are typically consid-
ered to be highly reactive,35 are less efficient than the ACL auxilia-
ry for difficult couplings.
9
DMF 8 h
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aReaction progress analyzed by HPLC; residues at the junction
b
are underlined. Time required for 95 % conversion to product
at room temperature. cFmocLYRAX-Se-o-PhCHO (2 µmol),
FRANG-CONH2 (4 µmol) and Et3N (4 µmol) in 1 mL DMF.
dFmocLYRAG-Se-o-PhCHO (1 µmol), FRANG-CONH2 (2
µmol) and Et3N (2 µmol) in 0.5 mL of 100 mM NaH2PO4
buffer (pH-6.5):DMF (90:10).
Dawson et al.56 These coworkers noted that NCL rates of pen-
tapeptides AcLYRAX-SPh, where X is any residue, with CRANK-
CONH2 are largely dependent on the identity of X, with bulky
residues (i.e. valine) at this position leading to inefficient cou-
plings. We used analogs of these pentapeptide sequences, Fmoc-
LYRAG and FmocLYRAV selenobenzaldehyde esters, to probe
the effectiveness of ACL for peptide ligations with peptide
FRANG-CONH2 (Table 4). As expected, ACL with glycinyl
selenobenzaldehyde proceeds much faster than the analogous
valinyl selenobenzaldehyde, which requires 8 h. Yet, the ACL
reaction rate with FmocLYRAV-Se-o-PhCHO is remarkable
considering native chemical ligation between AcLYRAV-SPh and
Table 5. Coupling of Proline Residues with ACL.a
Entry
C-terminus
N-terminus
Ligation site
Conversion (%)b
> 95
Timec
CHO
Se
OMe
N
X
N
30 min
1
N
H
O
N
O
O
O
O
X = Fmoc, P1d, P2
OMe
OMe
Se
N
2e
3
~ 20
> 95
7 h
2 h
N
N
H
N
O
O
O
Fmoc
O
CHO
N
Se
N
H
FmocHN
N
H
O
O
O
OMe
N
Se
N
H
4
> 95
> 96 h
FmocHN
N
H
O
O
O
O
aReaction conditions: C-terminal seleno-benzaldehyde ester (10 µmol), N-terminal AA/peptide (20 µmol) and Et3N (20 µmol) in 1 mL DMF.
bAnalysis by HPLC traces of the crude reaction mixture. cRoom temperature. dReaction in buffer (pH-8.5) > 95 % conversion in 90 min. P1: Fmoc-A–
–, P2: Fmoc-V––. eAfter 10 h, unidentified side products are observed.
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Figure 8. (a) Ubiquitin (15N labelled) was treated with selenoester 10 in 10% DMF in 1X phosphate buffered saline (PBS), pH 7.0.
Mono-labeling of ubiquitin was observed as the exclusive product. (b) Ubiquitin contains seven lysine residues and provides a stringent
test for evaluation of the specificity of reaction at the N-terminus. (c) The reaction progress was evaluated by MALDI and LCMS; the
MALDI spectrum after 40 hours is shown. MS/MS analysis confirms that labelling is localized to the N-terminus (Figure S7). (d) MALDI
MS of the HPLC purified sample. The analytical HPLC trace is shown in the inset.
In summary, we have introduced a new method for peptide li-
gation, which builds on the fundamental principles utilized by
common ligation techniques. The key feature of aldehyde capture
ligation is that it employs the rapid association between an alde-
hyde group and an amine to enforce an intramolecular reaction
leading to the desired native amide bond formation. Because
primary and secondary amines react with the selenobenzaldehyde
esters readily, we postulate that the hemiaminal II (Figure 1) is
the active intermediate in the reaction, but contributions from the
imine as a reactive intermediate with primary amines or direct
acylation of selenoesters cannot be ruled out. We have demon-
strated the potential of this approach for ligating a variety of un-
protected amino acids and peptides, including difficult sequences.
Preliminary results indicating mono-labeling of ubiquitin, a mod-
el protein, highlight the potential of ACL as a strategy for protein
modification. Importantly, we outline a strategy for the facile
synthesis of peptide selenoesters on solid phase, which will fur-
ther enable evaluation of the ACL methodology for the synthesis
of more complex peptides.
Potential of ACL for protein modifications: N-
terminal modification of ubiquitin as
protein
a model
ACL adds to a growing list of methods that allow chemoselec-
tive formation of the amide bond in aqueous buffers. The attrac-
tiveness of ACL is that it potentially allows coupling of any set of
amino acid residues, without requiring specific N-terminal amino
acids such as cysteine. A potentially significant limitation is that
the aldehyde may capture any amine, i.e., it may be difficult to
selectively modify lysine side chains or the N-terminal amines.
Two solutions may be envisioned to address this potential limita-
tion: (a) in short peptides or other synthetic oligomers and small
molecules, the appropriate amine may be protected using stand-
ard approaches; (b) the pKa difference between the N-terminal
amino group and the ε-amine of lysine may be exploited to
achieve selective modification of the N-terminus.53,54,60
We evaluated the potential of ACL to specifically acylate the
N-terminus of ubiquitin. We conjectured that ubiquitin would
provide a stringent test to determine the specificity of reaction at
the N-terminus because it contains seven lysine residues (Figure
8). We used diazobenzene dye 10 as a model substrate for reac-
tion with ubiquitin. Five-fold excess of the dye selenoester was
incubated with the protein (100 µM) in 10% DMF in 1X PBS
buffer, pH 7.0. The reaction was monitored using mass spectros-
copy (MALDI and LCMS). After 40 h, roughly 70% of ubiquitin
was modified by the dye and only the peak corresponding to
mono-labeled ubiquitin (Ubm) was observed by Maldi and LCMS
(Figure 8). Further analysis by MS/MS showed that the modifi-
cation is localized to the N-terminal hexapeptide fragment of
ubiquitin (Figure S7). This result further highlights the attrac-
tiveness of the ligation strategy proposed herein, and suggests
potential uses of ACL as a strategy for N-terminal modification of
proteins.61-63
EXPERIMENTAL SECTION
General. Commercial-grade reagents and solvents were used
without further purification except as indicated. All reactions
were stirred magnetically or mechanically shaken; moisture-
sensitive reactions were performed under nitrogen or argon at-
mosphere. Reverse-phase HPLC experiments were conducted
with 0.1% aqueous trifluoroacetic acid and acetonitrile as eluents
with 4.6 mm × 150 mm (analytical scale) or 21.4 mm × 150 mm
(preparative scale) Waters C18 Sunfire columns using a Beckman
Coulter HPLC equipped with a System Gold 168 Diode array
detector. The typical flow rates for analytical and preparative
HPLC were 0.5-1 mL/min and 8 mL/min, respectively. ESI-MS
data were obtained on an Agilent 1100 series LC/MSD (XCT)
electrospray trap. Protein MALDI data was collected on Bruker
MALDI-TOF/TOF UltrafleXtreme Spectrometer. MS/MS
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(2) Davis, B. G. Science 2004, 303, 480.
(3) Nilsson, B. L.; Soellner, M. B.; Raines, R. T. Annu. Rev. Biophys.
Biomol. Struct. 2005, 34, 91.
(4) Pattabiraman, V. R.; Bode, J. W. Nature 2011, 480, 471.
(5) Hackenberger, C. P. R.; Schwarzer, D. Angew. Chem. Int. Ed.
2008, 47, 10030.
(6) Stephanopoulos, N.; Francis, M. B. Nat. Chem. Biol. 2011, 7,
876.
(7) Ogunkoya, A. O.; Pattabiraman, V. R.; Bode, J. W. Angew. Chem.
Int. Ed. 2012, 51, 9693.
(8) Tam, J. P.; Xu, J.; Eom, K. D. Peptide Science 2001, 60, 194.
(9) Mao, H.; Hart, S. A.; Schink, A.; Pollok, B. A. J. Am. Chem. Soc.
2004, 126, 2670.
(10) Wang, P.; Dong, S.; Shieh, J.-H.; Peguero, E.; Hendrickson, R.;
Moore, M. A. S.; Danishefsky, S. J. Science 2013, 342, 1357.
(11) Payne, R. J.; Wong, C.-H. Chem. Commun. 2010, 46, 21.
(12) Scheck, R. A.; Francis, M. B. ACS Chem. Biol. 2007, 2, 247.
(13) Gamblin, D. P.; Scanlan, E. M.; Davis, B. G. Chem. Rev. 2008,
109, 131.
(14) Chalker, J. M.; Bernardes, G. J. L.; Davis, B. G. Acc. Chem. Res.
2011, 44, 730.
(15) Kiessling, L. L.; Splain, R. A. Annu. Rev. Biochem 2010, 79, 619.
(16) Dawson, P. E.; Kent, S. B. H. Annu. Rev. Biochem 2000, 69, 923.
(17) Raibaut, L.; Ollivier, N.; Melnyk, O. Chem. Soc. Rev. 2012, 41,
7001.
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Engl. 2006, 45, 1248.
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13244.
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1027.
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5611.
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Chem. Int. Ed. 2012, 51, 5114.
(23) Aimoto, S. Peptide Sci. 1999, 51, 247.
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Chem. Int. Ed. 2008, 47, 4411.
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1994, 266, 776.
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2141.
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1939.
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1724.
(31) Zhang, Y.; Xu, C.; Lam, H. Y.; Lee, C. L.; Li, X. Proc. Natl. Acad.
Sci. U. S. A. 2013, 110, 6657.
(32) Kemp, D. S.; Vellaccio, F., Jr. J. Org. Chem. 1975, 40, 3003.
(33) Kemp, D. S.; Grattan, J. A.; Reczek, J. J. Org. Chem. 1975, 40,
3465.
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Lett. 2003, 44, 3187.
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12042.
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Soc. 1963, 85, 3458.
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analysis was performed on Agilent 1100 Series LCMSD VL MS
Spectrometer. Proton NMR spectra of selenoesters were record-
ed on a Bruker AVANCE 400 MHz spectrometer.
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3
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7
8
Peptide synthesis. Peptides were synthesized on a CEM Lib-
erty microwave peptide synthesizer using Fmoc solid-phase
chemistry on Rink amide resin and purified by reversed-phase
HPLC. The identity and the purity of the peptides were con-
firmed by ESI-MS.
General procedure for synthesis of thiobenzaldehyde and
selenobenzaldehyde esters: Amino acid (AA)/peptide (1
mmol) was dissolved in anhydrous THF (5 mL) in an oven-dried
round bottom flask under N2. The resulting solution was stirred
and cooled at 0 °C in ice-bath for 10 min. Then, DCC (1 mmol)
was added at 0 °C. The reaction was stirred at 0 °C for 30 min.
Then, di-thio (6) or di-seleno benzaldehyde (7) (0.5 mmol),
TCEP⋅HCl (0.6 mmol), Et3N (0.6 mmol) and 2 drops of water
were added. The resulting reaction mixture was stirred at room
temperature for 30 min. The reaction was then cooled in ice bath
and filtered to remove dicyclohexyl urea. The filtrate was concen-
trated under vacuum and purified by flash chromatography or
HPLC to give AA/peptide-thio-benzaldehyde or AA/peptide-
seleno-benzaldehyde ester, respectively as pale yellow solid (55-
70% yield).
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General procedure for the Aldehyde Capture Ligation: To
a solution of C-terminal amino acid or peptide-oxo, thio or seleno
ester (2-10 µmol) in 1 mL DMF, was added N-terminal amino
acid or peptide (2 eq) and Et3N (2 eq). The reaction was stirred
at 22 °C.
Analytical analysis of ACL reactions: For determination of
reaction rates, the reaction mixture was diluted 20-fold in acetoni-
trile and kept over dry-ice until HPLC analysis. HPLC (C-18
columns): 0.1% TFA (v/v) in water (solvent A): acetonitrile
(solvent B); gradient 45-85 % in 30 min, flow rate = 0.5 mL/min,
detection wavelength 280 nm.
ASSOCIATED CONTENT
Supporting Information.
Supporting Figures and experimental procedures and analytical data
for new compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
arora@nyu.edu
Present Addresses
†Department of Chemistry & Biochemistry, Seton Hall University,
South Orange, NJ 07079.
Author Contributions
‡These authors contributed equally to this work.
ACKNOWLEDGMENTS
We thank the US National Science Foundation (CHE-1506854) for
funding. P.S.A. thanks Anna Mapp, Laura Kiessling and Neville Kal-
lenbach for helpful suggestions.
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11
ACS Paragon Plus Environment