KAHA ligations that form aspartyl aldehyde residues as synthetic handles for protein modification and purification

Article abstract of DOI:10.1021/ja511231f

Aldehydes are widely recognized as valuable synthetic handles for the chemoselective manipulation of peptides and proteins. In this report, we show that peptides and small proteins containing the aspartic acid semialdehyde (Asa) side chain can be easily p

Full text of DOI:10.1021/ja511231f

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Article
KAHA Ligations that form Aspartyl Aldehyde Residues as
Synthetic Handles for Protein Modification and Purification
Claudia E. Murar, Frederic Thuaud, and Jeffrey W. Bode
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/ja511231f • Publication Date (Web): 04 Dec 2014
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Journal of the American Chemical Society
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KAHA Ligations that form Aspartyl Aldehyde Residues as
Synthetic Handles for Protein Modification and Purification
† ‡
Claudia E. Murar†, Frédéric Thuaud† ‡ and Jeffrey W. Bode*,
,
,
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†Laboratorium für Organische Chemie, Department of Chemistry and Applied Biosciences, ETH–Zürich, 8093 Zürich, Switzerland
‡Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Chikusa, Nagoya 464-8602, Japan
Supporting Information Placeholder
ABSTRACT: Aldehydes are widely recognized as valuable synthetic handles for the chemoselective manipulation of peptides and proteins.
In this report, we show that peptides and small proteins containing the aspartic acid semi-aldehyde (Asa) side-chain can be easily prepared by
a chemoselective amide-forming ligation that results in the formation of the Asa residue at the ligation site. This strategy employs the α-keto-
acid–hydroxylamine (KAHA) ligation in combination with a new isoxazolidine monomer that forms a side-chain aldehyde upon ligation.
This monomer is easily prepared on a preparative scale by a catalytic, enantioselective approach and is readily introduced onto the N-
terminus of a peptide segment by solid phase peptide synthesis. The ligated product can be further functionalized by bioorthogonal reactions
between the aldehyde residue and alkoxyamines or hydrazides. We demonstrated that glucagon aldehyde, an unprotected 29-mer peptide
prepared by KAHA ligation, can be site-specifically and chemoselectively modified with biotin, dyes, aliphatic oximes and hydroxylamines.
We further describe a simple and high recovery one-step purification process based on the capture of a 29-mer glucagon aldehyde and a 76-
mer ubiquitin aldehyde by an alkoxyamine-functionalized polyethyleneglycol resin. The peptide or protein was released from the resin by
addition of a hydroxylamine to provide the corresponding oximes.
.
INTRODUCTION
The basis for this work is our recent identification of 5-
oxaproline as a chemically stable, yet highly reactive partner for
the KAHA ligation of large, unprotected peptide segments.
Aldehydes present in biological molecules including peptides,
proteins, and carbohydrates provide a convenient handle for site-
specific conjugation. By undergoing rapid, chemoselective reac-
15
1,2
Protein synthesis by KAHA ligation with 5-oxaproline offers the
advantages of easily prepared starting materials and ligations
under acidic aqueous conditions that are well-suited for
solubilizing peptide segments. Its primary disadvantage is the
formation of a non-canonical homoserine residue at the ligation
site. This is normally an innocuous mutation of most amino acids
in terms of tertiary structure and biochemical activity. The use of
the 5-oxaproline residue also leads to the formation of esters as
the initial ligation products; base induced rearrangements form
tions with
a
variety of functional groups, especially
hydroxylamines and hydrazines, aldehydes are ideal for the
attachment of dyes and tags. Their unique chemical reactivity
and ability to undergo reversible reactions also makes them well-
suited for affinity chromatography and catch-and-release
purification processes. Due to their high reactivity, however,
aldehydes must be introduced at a late stage of a chemical
synthesis by post-assembly chemical modification, either by a
3,4
16
the native peptide bond.
specific chemical reaction or by unmasking of a protected
5,6,7,8,9
form.
Several studies have been devoted to the synthesis of
Scheme 1. Ketoacid–hydroxylamine ligation to form
the peptide aspartic semi-aldehyde (Asa) residues
10
peptide aldehydes in solid phase peptide synthesis (SPPS), or to
the use of N-terminal peptide aldehydes.
11,12,13
Bertozzi has
H2N
HN
SRRAQDFVQWLMNT
COOH
NH2
NH
exploited the formylglycine-generating enzyme (FGE) consensus
as a genetically encoded aldehyde tag for site- specific protein
HN
N
OH
COOH
O
O
H
N
O
H
N
HSQGTFTSDYSKY
OH
14
H3N
N
H
modification. The enzyme oxidizes a cysteine residue within a
O
OH
CONH2
NH3
Me
O
OH
∼13 amino acid consensus sequence to form an aldehyde-bearing
formylglycine residue.
EtO
Me
1 equiv
1.3 equiv
In this report, we introduce a new isoxazolidine monomer for
the α-ketoacid–hydroxylamine (KAHA) amide forming ligation
that reveals an aspartic semi-aldehyde side-chain upon reaction of
two unprotected peptide segments. The ligation product
possesses a single aldehyde functional group that can be readily
modified by standard oxime or hydrazone formation,
transformed to hydroxylamines for subsequent KAHA ligations,
or used for catch-and-release purification of the ligation products
from the reaction mixtures without preparative HPLC.
7:3 DMSO:H2O, 0.1 M oxalic acid
20 mM, 60 ˚C, 8 h
>100mg scale
61% after prep HPLC
H2N
HN
SRRAQDFVQWLMNT
COOH OH
NH2
Me
Me
HN
NH
N
OH
O
COOH
H
N
O
HSQGTFTSDYSKY
H3N
N
H
N
H
OH
O
H
CONH2
NH3
O
Chemoselective conjugations
Catch and release purification
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As part of ongoing efforts to design new stable
Scheme 2. Synthesis of the enantiopure (S)-
isoxazolidine
1
2
3
4
5
6
7
8
hydroxylamines that afford other unnatural or natural residues at
the ligation site, we considered the possibility of using KAHA
ligation to reveal an aldehyde upon N–O bond cleavage. In the
context of the synthesis of short β3-peptides, we have described
OTBS
Ph
Ph
N
H
Boc
OTBS
H
N
3 (20 mol%)
17
Boc
OTBS
isoxazolidine monomers that form ketones upon ligation, but it
N
H
EtO2C
O
EtO2C
O
CHCl3
was not clear if a corresponding design for an aldehyde-forming
substrate would be sufficiently stable to peptide cleavage, HPLC
purification and ligation. Furthermore, the increased steric
hindrance could interfere with the ligation of longer peptides. As
documented below, these fears proved unfounded and a suitable
monomer could be readily prepared, incorporated into peptide
segments, and used for KAHA ligation of unprotected segments
to give peptides bearing a single aspartic acid semi-aldehyde
residue (Asa) at the ligation site.
1
2
55%
4 (98% ee)
a
pTsOH
9
EtOH
10
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12
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14
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60
70%
Boc
Boc
LiBr, Et3N
N
O
N
O
EtO2C
OEt
98:2 CH3CN:H2O
HO2C
OEt
95%
5
6
a pTsOH = para-toluenesulfonic acid
a) concept and mechanism for Asa-forming ligations
HO2C
The key step in this route was the intramolecular
acetalization of aldehyde 4 in acidic ethanol to give cyclic adduct
5. Although the reaction was complete in several hours, some Boc
deprotection occurred and the reaction progress needed to be
monitored carefully. Ester hydrolysis of 55 with triethylamine and
lithium bromide provided (S)-isoxazolidine 6, ready for use in
SPPS. The relative and absolute configuration of 6 was assigned
by X-ray crystallographic analysis of an amide derivative (Figure
2). No epimerization was observed in the cyclization or the
hydrolysis step (See Supporting Information for epimerization
study).
OH
N
O
O
O
H
N
R
R
N
H
N
H
OH
O
O
O
EtO
EtO
O
O
O
H
N
H
N
R
R
N
N
H
H
H
OEt
O
O
H
OH
aspartic acid semi-aldehyde
residue (Asa)
b) potential monomers for Asa-forming KAHA ligations:
Me
Me
Br
Boc
Boc
Boc
Me
O
O
O
O
N
N
N
O
O
O
O
O
N
N
H
OH
OH
OH
EtO
NC
MeO
O
O
Figure 1. Monomer design
Me
18
Previous work on (S)-aspartic semi-aldehyde residues was
7
19
based on the introduction of a masked aldehyde linker, or the
incorporation of a Weinreb amide as a precursor of an aldehyde
Figure 2. X-Ray crystallographic analysis of 77
20
on the side-chain of aspartyl residues. There has also been
evidence of Asa in natural proteins arising from post-translational
21
Solid Phase Peptide Synthesis and Asa-Forming
Ligations. In order to demonstrate the utility of this monomer
for peptide aldehyde formation, we applied it to the chemical
synthesis of glucagon, a 29 amino acid peptide hormone. We
chose Leu (residue 14) and Asp (residue 15) as the ligation site.
This disconnection required two peptide segments of around 15
residues each. In the ligated peptide, Asp (residue 15) would be
mutated to aspartic semi-aldehyde (Asp15Asa).
enzymatic reduction of aspartic acid. However, to the best of
our knowledge, there is no viable approach to the incorporation
of Asa into longer peptides or proteins by either synthetic or
enzymatic methods. Our study describes the first method for the
late stage introduction of the aldehyde function in the γ-position
(Asa), eliminating the risk of epimerization.
RESULTS AND DISCUSSION
The isoxazolidine segment of glucagon (15–29) 11 was
prepared on a resin bearing a Wang linker by automated Fmoc
SPPS, followed by manual coupling of the isoxazolidine
monomer 6 onto protected peptide 9. Side-chain deprotection
and cleavage of the peptide from the resin and purification by
HPLC proceeded without difficulty to yield glucagon (15–29)
11 (Scheme 3). The glucagon (1–14)-α-ketoacid segment 10
was synthesized in good yield by using one of our established
Monomer synthesis. Substituted isoxazolidine 66 was
readily prepared in an enantiomerically pure form through a
22
short, optimized sequence. Based on the work of MacMillan
and Cordova
23
on organocatalytic asymmetric conjugate
additions to α,β-unsaturated aldehydes, we established an
operationally simple route to the synthesis of the enantioenriched
isoxazolidine 6 (Scheme 2) on a gram scale. An alternative route
using an L-gulose derived chiral auxiliary for nitrone cyclization
26
procedures for the Fmoc SPPS of peptide α-ketoacids.
24,25
with ethyl vinyl ether was also feasible.
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Scheme 3. Preparation of glucagon aldehyde by Asa- from trapping of the putative nitrilium intermediate were
16
1
2
3
4
5
6
7
8
forming KAHA ligation.
observed.
NH
PbfNH
H2N
NH
N
BocN
OH COOH
O
Me
H2N
HN
SRRAQDFVQWLMNT
COOH
OH
NH2
HN
+
H3N
H
N
HN
a)
NH
HSQGTFTSDYSKY
CN
O
O
Me
COOH
SRRAQDFVQWLMNT
N
OH
O
S
NH3
Me
CONH2
H
N
O
COOtBu OtBu
O
HSQGTFTSDYSKY
CONH2
H3N
N
H
N
H
H
Me
CONH2
9
8
OH
O
NH3
1) 6, EDCI, HOBt, 2 h
2) 97:2:1 TFA:TIPS:H2O
Oxone (100 equiv)
1:1 CH3CN:H2O
3 min
HO OH
9
aldehyde hydrate
+ H2O - H2O
H2N
+
10
11
12
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14
15
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H2N
HN
SRRAQDFVQWLMNT
NH2
NH
HN
N
OH
COOH
O
O
H
N
+
H3N
O
OH
H
HSQGTFTSDYSKY
OH
N
N
O
H
CONH2
NH3
Me
O
Me
NH2
COOH
OH
HN
NH
EtO
Me
COOH
HN
10 (1 equiv)
glucagon (1–14)
11 (1.3 equiv)
glucagon (15–29)
Me
N
OH
O
H
N
N
H
O
SRRAQDFVQWLMNT
COOH
OH
HSQGTFTSDYSKY
H3N
N
H
OH
O
H
CONH2
NH3
7:3 DMSO:H2O, 0.1 M oxalic acid,
60 ˚C, 8 h, 20 mM
61% after prep HPLC
O
aldehyde
+
H2N
HN
SRRAQDFVQWLMNT
COOH
OH
NH2
Me
HN
NH
Me
N
OH
O
COOH
H
N
O
+
H3N
HSQGTFTSDYSKY
N
H
N
H
H
OH
O
CONH2
Me
NH3
H2N
NH2
NH
HN
O
Me
COOH
N
HN
OH
12 glucagon Asp15Asa peptide
O
H
N
O
HSQGTFTSDYSKY
H3N
N
H
SRRAQDFVQWLMNT
N
O
OH
CONH2
H
NH3
COOH
OH
HO
hemiaminal
a) Synthesis of glucagon α-ketoacid (1–14) 110 and glucagon
(15–29) 11 and their ligation to form glucagon Asp15Asa peptide.
b) Analytical RP-HPLC traces of the glucagon aldehyde forming
ligation. c) HRMS of glucagon aldehyde 112.
Figure 3. a) pH dependent hydration and hemiaminal formation
of a peptide containing aspartic semi-aldehyde side-chain. b) H
1
NMR spectra (600 MHz with presaturation, 300 K, KH2PO4/H3PO4
buffer, H2O/D2O 95:5 for pH 3 and 4.5. The pH was raised by
addition of solid Na2CO3). The asterisks * mark the aldehyde peak
resulting from the linear aldehyde glucagon-CHO. The measurement
at acidic pH showed the presence of the aldehyde peak along with the
hydrate form. Subsequent raising of the pH to 6.6 and 9.0 showed
almost no aldehyde signal left (marked by black arrow).
With the two peptide segments in hand, we proceeded with
milligram scale reactions using the standard ligation conditions
established with the 5-oxaproline substrates. No deviation from
15
our standard conditions for KAHA ligation was necessary and
the reactions proceeded cleanly to give a single ligation product.
Purification of the ligation mixture by preparative HPLC afforded
glucagon Asp15Asa 12 in 61% yield. This demonstrates that the
substituted isoxazolidine monomer is stable towards the ligation
conditions, tolerates acidic deprotection and cleavage conditions,
and that both the starting peptide segment and the Asa-
containing ligation products, can be purified by preparative
HPLC. The rate of the ligation was comparable to an experiment
performed using the glucagon segment bearing a 5-oxaproline
residue. In contrast to 5-oxaproline, we observed only the
expected amide products; no esters or other products arising
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Characterization of the Asa residue. High-resolution We also incorporated an affinity tag by the use of biotin
1
2
3
4
5
6
7
8
mass spectrometry (MALDI-HRMS) measurement confirmed
the exact mass of the glucagon peptide with the Asp15Asa
mutation. It appears likely that (S)-Asa residue exists primarily as
hydrazide. The reaction is known to proceed slowly at acidic pH
due to protonation of the hydrazide moiety, but to provide the
hydrazone in good yield at pH > 6. Hydrazide reagents are
known to have modest reaction kinetics because of the presence
of electron-withdrawing groups adjacent to the nucleophilic
nitrogen moiety, accounting for the moderate yield of our
conjugation reaction (Table 1, entry 2).
32
27
the hydrate in solution. Reversible cyclization of peptide
aldehydes into hemiaminals as a pH-dependant equilibrium has
1
28
already been documented and studied by H NMR by Geyer.
29
The work of Omura also described the formation of a five-
membered ring hemiaminal by the side-chain of an L-aspartic
semi-aldehyde with the backbone amide.
We also sought to generate hydrolytically stable reaction
products based on the recently reported Pictet-Spengler ligation
9
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10
11
12
13
14
15
16
17
18
19
20
21
22
23
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25
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60
To establish whether or not the Asa side-chain in our
synthetic glucagon was present in the aldehyde form or as a
hemiaminal with a backbone amide bond or another side-chain
functional group, we performed NMR studies over a range of pH
values between 3 and 9. These measurements showed that a
significant proportion of the hydrate form of the aldehyde was
present under acidic conditions. Under basic conditions, the
with aminoxy nucleophiles. We designed two reaction partners
for this transformation, aminoxy-functionalized indoles 17 and
18. We found that they reacted smoothly with glucagon aldehyde
to yield the stable conjugates at a slightly acidic pH in high yield
(Table 1, entry 3) when a ten-fold excess of reagent was
employed. With this result in hand, we aimed for generating a
stable product near neutral pH, by using the hydrazino-Pictet-
1
34
hemiaminal was predominant (Figure 3). These H NMR studies
Spengler (HIPS) ligation. Hydrazine-Pictet-Spengler products
28
are consistent with the observations by Geyer.
are known to exhibit a good stability and reactivity near neutral
pH compared to the oxime-linked conjugate. We prepared indole
21 that generated a hydrolytically stable reaction product upon
ligation with glucagon Asp15Asa 12 (Table 1, entry 4).
Chemoselective conjugations to the Asa side-
chain. With the aldehyde group installed, we evaluated
chemoselective conjugations with aminooxy- or hydrazide-
functionalized molecules for site-specific modification. Reaction
partners included fluorescent imaging probe BODIPY-
hydroxylamine, affinity probes such as biotin hydrazide, and
conjugations with aliphatic hydroxylamines (Table 1). The
conjugation reactions with aminoxy-functionalized molecules
were carried out in aqueous buffers under slightly acidic pH, as
formation of oximes suffers from slow reaction kinetics at a pH >
Finally, we also used simple hydroxylamines to form oximes
(Table 1, entry 5), which were easily prepared and stable to
HPLC analysis and purification.
Conversion of aldehydes to hydroxylamines and
further ligation. The oxime products prepared from glucagon
aldehyde 12 and the simple hydroxylamines shown in Table 1,
entry
5 could be easily reduced to the corresponding
hydroxylamines with NaCNBH3. The hydroxylamine side-chain
was suitable for KAHA ligations with α-ketoacids (Scheme 4) in
30
6. Benzyl derived hydroxylamine proved to be more reactive
35
than hydroxylamine or O-methylhydroxylamine (Table 1, entry
good yield, provided an excess of the ketoacid was employed.
31
1,5). The use of aniline based catalysts (aniline, m-phenylene
We also found that the more stable, easily handled O-Me
diamine, 3,5-diaminobenzoic acid) only slightly improved the
outcome in our case. BODIPY-hydroxylamine was successfully
conjugated with glucagon Asp15Asa in 12 hours at 37 ºC,
affording fluorescent product 114 in good yield. The fluorescence
spectra of 14 with excitation and emission wavelengths is shown
in Figure 4.
alkoxyamine could be ligated with N-methyliminodiacetyl
(MIDA) acyl boronates (Scheme 4).
36
Although only simple α-ketoacids and MIDA acyl boronates
were employed in this study, the high reactivity and
chemoselectivity of these ligations offer a promising route to the
late-stage functionalization of unprotected peptides to form
4
37
stable, biocompatible amide bonds. Given the known instability
of oximes and hydrazines, this two-step approach may prove to be
better suited for many peptide modifications.
Figure 4. Absorbance and excitation spectrum of BODIPY-dye-
functionalized peptide 114
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Table 1. Examples of aldehyde conjugations and conditions a
1
2
3
Aldehyde
modifier
Reagent
concentration
Structure
Product
Conditions
4
5
6
7
8
12 (5 mM), 5:1 sodium acetate
buffer:CH3CN, pH 4.5, 37 ˚C, 6 h
1
2
BODIPY
50 mM
78% after prep HPLC (2.6 mg product
obtained)
hydroxylamine dye
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
12 (2.5 mM), 5:1 sodium phosphate
buffer:CH3CN, pH 6.6, 40 ˚C, 8 h
Biotin hydrazide
50 mM
47% after prep HPLC (0.5 mg product
obtained)
12 (2.5 mM), 5:1 sodium acetate
buffer:CH3CN, pH 4.5, 40 ˚C, 6 h
3
Aminoxy-indole
25 mM
70% after prep HPLC (~1 mg of each
product obtained)
12 (2.5 mM), 5:1 citric acid-sodium
dihydrogen phosphate buffer:CH3CN,
pH 6, 40 ˚C, 6 h
4
5
Hydrazino-indole
25 mM
70% after prep HPLC (~0.8 mg of
product obtained)
12 (1.5–2.5 mM), 5:1 sodium acetate
buffer:CH3CN, pH 4.5, 37 ˚C, 6–14 h
30-50 mM
Other
hydroxylamines
45–77% after prep HPLC (1–2 mg of
products obtained)
a
Conjugation experiments were conducted on 1–3 mg of glucagon Asp15Asa 112. Yields refer to isolated yields of pure product following
preparative HPLC.
Catch and release purification of ligation
products. Reversible, covalent reactions of aldehydes also
makes possible affinity based purification strategies. key
advantage of the Asa-forming ligations is the ability to easily
separate the ligation products from any unreacted starting
materials by affinity purification. The aspartic semi-aldehyde
residue provides
hydroxylamines or other covalent capture methods. We designed
a solid supported alkoxyamine that would selectively “catch” the
peptide aldehyde and would allow removal of the impurities, side-
products and unreacted starting materials. To achieve this we
required a resin compatible with organic solvents, but which also
possessed excellent swelling properties in an aqueous
environment under which the unprotected peptide would be
handled. In order to “release” the trapped peptide, a small
aminoxy group would be used to exchange the bound oxime for a
soluble one.
A
a perfect handle for immobilization with
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Scheme 4. Chemoselective reactions for hydroxylamine side-chain
Page 6 of 11
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60
We began by evaluating resins compatible with a mixture of
H2O/CH3CN. Initially, we tested amino PEGA resin, but found it
was difficult to handle the wet beads. The use of an amino
NovaPEG resin improved the outcome due to its superior
Scheme 5. Preparation of the hydroxylamine “Catch”
resin
mechanical properties; the resin beads are free flowing in the dry
OH
OPhtal
38
state. We synthesized the Wang-type linker 33 via
a
39
Mitsunobu reaction of alcohol 31 and N-hydroxyphtalimide,
followed by allyl deprotection (Scheme 5). Loading 33 onto
NovaPEG resin via amide coupling, followed by hydrazinolysis
gave stable, storable resin 334.
Pd(PPh3)4,
Morpholine
N-hydroxyphtalimide,
DIAD, PPh3
O
O
65%
85%
CO2Allyl
CO2Allyl
To demonstrate the efficiency and utility of the catch-and-
32
31
40
NH2
O
release strategy, we examined its application to the synthesis of
OPhtal
glucagon aldehyde from the two corresponding peptide
segments. For this purpose, we performed a ligation between the
two peptide segments of glucagon and, after aldehyde product
formation, incubated the ligation mixture with the resin under
1)
P
NH2,
HCTU, NMM
O
O
2) NH2NH2 . H2O
º
gentle shaking at 45 C (Scheme 6). We were pleased to see that
P
CO2H
the hydroxylamine resin quantitatively and selectively trapped the
glucagon aldehyde over 12 hours at slightly acidic pH. Small
amounts of decomposition products or unreacted peptide
aldehyde were observed, but all traces were removed by washing
the resin containing the bound peptide aldehyde. The peptide
was released from the resin by treatment with a solution of O-
N
H
O
33
34
refers to NovaPEG polymer support
P
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Scheme 6. Monitoring of the catch-and -release purification of Asp15Asa glucagon peptide
3
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methylhydroxylamine or hydroxylamine at pH 3 for 4 hours to
afford, after filtration, oximes 224 or 25. Additionally, the peptide
could be recovered as oxime 225 by cleavage with a standard
TFA:TIPS:H2O cocktail. However, after this treatment the
hydroxylamine resin cannot be recovered or reused.
hydroxylamines can be further used in chemoselective reactions,
such as those shown in Scheme 4.
To further demonstrate the selectivity and applicability to
otherwise extremely challenging purification tasks, we mixed
glucagon (1-29) acid (no Asa modification, no mutation) 35
with glucagon Asp15Asa 12. Using the same conditions as
previously stated, we incubated this mixture for 8 h together with
the hydroxylamine resin and evaluated the specificity of our
purification method. As expected, only glucagon aldehyde was
captured on the resin, while the second peptide remained in
solution. The degree of purification achieved by this method is
The solution of the released peptide can either be lyophilized
to afford the corresponding oximes without further purification
or reduced by NaCNBH3 in the same pot. Reduction was
achieved in several hours, providing the desired hydroxylamine
peptides in high purity with a good overall yield after preparative
HPLC (Scheme 6). It is noteworthy that the freshly generated
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significant (Figure 5), as from our knowledge mixtures of this Synthesis and catch-and-release purification of
1
2
3
4
type are nearly impossible to separate by HPLC.
ubiquitin Ala46Asa. The Asa-forming ligations are also
suitable for improving synthetic access and purification of protein
targets. We successfully incorporated the aldehyde functional
41
group into ubiquitin Ala46Asa. We chose a ligation site between
5
Phe (residue 45) and Ala (residue 46, mutated to Asa).
6
7
8
9
The isoxazolidine segment of ubiquitin (46–76) 37 was
prepared by automated Fmoc SPPS followed by manual coupling
of the isoxazolidine monomer 6. Side-chain deprotection,
cleavage from resin and purification provided ubiquitin (46–76)
37 (Scheme 7). The ubiquitin (2–45)-α-ketoacid segment 36
was synthesized in a good yield by using a linker for preparing α-
10
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15
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42
ketoacids. With the two peptide segments in hand, the
milligram scale ligation proceeded cleanly to the desired product
38. Purification of the ligation mixture by preparative HPLC
afforded ubiquitin Ala46Asa in 58% yield. This demonstrated
that our isoxazolidine monomer could be applied to a small
protein and the aspartic semi-aldehyde residue could be
introduced successfully into ubiquitin.
Figure 5. Analytical RP-HPLC traces at 220 nm of the
supernatant of mixed glucagon aldehyde and acid at different stages
within the same HPLC monitoring conditions. (a) Initial conditions
prior to capture; (b) 8 h capture; (c) reference peak of aldehyde.
We evaluated the efficiency of the catch-and-release
purification strategy for ubiquitin Ala46Asa (Scheme 7). The
hydroxylamine resin captured 65% of ubiquitin aldehyde when
the resin loading was 0.3 mmol/g. However, by decreasing the
loading of the hydroxylamine resin to ~0.1 mmol/g, the catch
efficiency increased (see Suporting Information for analytical
Scheme 7. Synthesis and Catch-and-Release Purification of ubiquitin Ala46Asa
aCatch monitoring of ubiquitin Ala46Asa is only 65% if using a resin loading of 0.3 mmol/g. When using a resin loading of ~0.1 mmol/g, the catch
efficiency increased (see Supporting Information)
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HPLC traces when using 0.1 mmol/g resin loading). The release
with O-methylhydroxylamine proceeded well, and the
supernatant was lyophilized to afford the ubiquitin protein in one
chemical ligation, without any HPLC purification.
1
2
3
4
5
6
7
8
(5) For a review on the synthesis of peptide aldehydes: Moulin, A.;
Martinez, J.; Fehrentz, J. A. J. Pept. Sci. 2007, 13, 1–15.
(6) Sorg, G.; Thern, B.; Mader, O.; Rademann, J.; Jung, G. J. Pept. Sci.
2005, 11, 142–152.
(7) Guichard, G.; Briand, J. P.; Friede, M. Peptide research 1993, 6,
121–124.
(8) Galeotti, N.; Giraud, M.; Jouin, P. Int. J. Pep. Res. Ther. 1997, 4,
437–440.
(9) Garrett, G. S.; Correa, P. E.; McPhail, S. J.; Tornheim, K.; Burton,
J. A.; Eickhoff, D. J.; Engerholm, G. G.; McIver, J. M. J. Pep. Res. 1998,
52, 60–71.
Finally, we successfully conjugated ubiquitin Ala46Asa with
BODIPY hydroxylamine dye following the same reaction
conditions as Table 1, entry 1 to give the desired product in 50%
isolated yield (see Supporting Information for experimental
details).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
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55
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CONCLUSION
(10) (a) Murphy, A. M.; Dagnino Jr, R.; Vallar, P. L.; Trippe, A. J.;
Sherman, S. L.; Lumpkin, R. H.; Tamura, S. Y.; Webb, T. R. J. Am. Chem.
Soc. 1992, 114, 3156–3157. (b) Ede, N. J.; Bray, A. M. Tet. Lett. 1997,
38, 7119–7122. For additional information see book: Chan, W.C.; White,
P.D. Fmoc Solid Phase Peptide Synthesis (book chapter: Peptide
aldehydes by solid phase synthesis, 153)
(11) Groth, T.; Meldal, M. J. Comb. Chem. 2001, 3, 34–44.
(12) Geoghegan, K. F.; Stroh, J. G. Bioconjugate chem. 1992, 3, 138–
146.
(13) (a) Gilmore, J. M.; Scheck, R. A.; Esser-Kahn, A. P.; Joshi, N. S.;
Francis, M. B. Angew. Chem., Int. Ed. 2006, 45, 5307–5311. (b) Scheck,
R. A.; Dedeo, M. T.; Iavarone, A. T.; Francis, M. B. J. Am. Chem. Soc.
2008, 130, 11762–11770.
(14) (a) Rush, J. S.; Bertozzi, C. R., J. Am. Chem. Soc. 2008, 130,
12240–12241. (b) Rabuka, D.; Rush, J. S.; Dehart, G. W.; Wu, P.;
Bertozzi, C. R. Nat. Protoc. 2012, 7, 1052–1067.
In summary, the facile generation of peptide aldehydes was
made possible by a chiral isoxazolidine monomer that undergoes
KAHA ligation with C-terminal peptide α-ketoacids to form the
aspartic semi-aldehyde (Asa) residue at the ligation site. We have
43
succeeded in installing an aldehyde motif into a therapeutically
relevant peptide and a small protein by KAHA ligation with easily
prepared segments. We successfully showed that we could
introduce site-selective modifications with a variety of modifiers
and hope that this new route to aldehyde-containing peptides
complement biochemical techniques to incorporate synthetic
handles into peptides. The ability to prepare a panel of site-
specific modifications of proteins will be useful in the preparation
44
of a wide array of post-translational modified proteins, or in the
(15) Pattabiraman, V. R.; Ogunkoya, A. O.; Bode, J. W. Angew.
Chem. Int. Ed. 2012, 51, 5114–5118.
formation of peptide-conjugated dendrimers for drug delivery
45
applications. In addition, we developed a simple purification
(16) Wucherpfennig, T. G.; Rohrbacher, F.; Pattabiraman, V. R.;
Bode, J. W. Angew. Chem. Int. Ed. 2014, 53, 12244–12247.
(17) Ishida, H.; Carrillo, N.; Bode, J. W. Tet. Lett. 2009, 50, 3258–
3260.
(18) For initial work on the synthesis of aspartic acid semi-aldehyde:
Tudor, D. W.; Lewis, T.; Robins, D. J. Synthesis 1993, 11, 1061–1062.
(19) (a) Spetzler, J. C.; Hoeg-Jensen, T. J. Pept Sci. 2001, 7, 537–
551. (b) Johannesson, P.; Erdélyi, M.; Lindeberg, G.; Frändberg, P. A.;
Nyberg, F.; Karlén, A.; Hallberg, A. J. Med. Chem. 2004, 47, 6009–6019.
(20) Paris, M.; Pothion, C.; Goulleux, L.; Heitz, A.; Martinez, J.;
Fehrentz, J. A. React. Funct. Polym. 1999, 41, 255–261.
(21) Anderson, L. B.; Ouellette, A. J. A.; Eaton-Rye, J.; Maderia, M.;
MacCoss, M. J.; Yates Iii, J. R.; Barry, B. A. J. Am. Chem. Soc. 2004, 126,
8399–8405.
(22) (a) Chen, Y. K.; Yoshida, M.; MacMillan, D. W. C. J. Am. Chem.
Soc. 2006, 128, 9328–9329. (b) Ouellet, S. G.; Tuttle, J. B.; MacMillan,
D. W. C. J. Am. Chem. Soc. 2005, 127, 32–33.
(23) (a) Vesely, J.; Ibrahem, I.; Rios, R.; Zhao, G. L.; Xu, Y.; Córdova,
A. Tet. Lett. 2007, 48, 2193–2198. (b) Ibrahem, I.; Rios, R.; Vesely, J.;
Zhao, G. L.; Córdova, A., Chem. Commun. (Cambridge, U. K.) 2007,
849–851.
(24) Vasella, A.; Voeffray, R. J. Chem. Soc., Chem. Commun. 1981,
97–98.
(25) Yu, S.; Ishida, H.; Juarez-Garcia, M. E.; Bode, J. W. Chem. Sci.
2010, 1, 637–641.
(26) (a) Ju, L.; Bode, J. W. Org. Biomol. Chem. 2009, 7, 2259–2264.
(b) Ju, L.; Lippert, A. R.; Bode, J. W., J. Am. Chem. Soc. 2008, 130,
4253–4255.
(27) Coulter, C. V.; Gerrard, J. A.; Kraunsoe, J. A. E.; Moore, D. J.;
Pratt, A. J. Tetrahedron 1996, 52, 7127–7136.
(28) Enck, S.; Kopp, F.; Marahiel, M. A.; Geyer, A. Org. Biomol.
Chem. 2010, 8, 559–563.
(29) Hirose, T.; Sunazuka, T.; Sugawara, A.; Endo, A.; Iguchi, K.;
Yamamoto, T.; Ui, H.; Shiomi, K.; Watanabe, T.; Sharpless, K. B.;
Ömura, S. J. Antibiot. 2009, 62, 277–282.
method for the aldehyde peptide through a catch-and-release
strategy. This method is inexpensive, effective and could be
highly attractive due to the facile elimination of impurities.
ASSOCIATED CONTENT
Supporting Information. Experimental procedures and spectros-
copic data for all new compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
Corresponding Author
bode@org.chem.ethz.ch
Funding Sources
This work was supported by ETH Zürich and the Swiss National
Science Foundation (200020_150073).
ACKNOWLEDGMENT
We thank the LOC MS Service for analyses, NMR Service for analy-
ses, Hidetoshi Noda and Safwan Aroua for helpful discussions.
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