N-terminal protein modification through a biomimetic transamination reaction

Article abstract of DOI:10.1002/anie.200600368

(Chemical Equation Presented) One hit wonder: A biomimetic transamination reaction has been developed that employs pyridoxal-5-phosphate to modify the N terminus of proteins and peptides under mild conditions. This technique introduces a uniquely reactive

Full text of DOI:10.1002/anie.200600368

Angewandte
Chemie
Bioorganic Chemistry
DOI: 10.1002/anie.200600368
N-Terminal Protein Modification through a
Biomimetic Transamination Reaction**
Joshua M. Gilmore, Rebecca A. Scheck,
Aaron P. Esser-Kahn, Neel S. Joshi, and
Matthew B. Francis*
An important challenge in the field of protein bioconjugation
is the development of strategies that can modify a protein
once at a single site. Although many bioconjugation reactions
can functionalize specific amino acids in aqueous solution,[1]
most proteins display multiple copies of the targeted residue
on their surface. This commonly results in product mixtures
that present the newfunctionality in multiple locations on the
protein surface. As it is one of the rarest amino acids,[2]
cysteine is the most commonly targeted residue when site-
selective modification is required, but there remain many
situations in which the modification of a unique copy of this
residue is inconvenient or impossible. To address these
limitations, several chemoselective techniques have been
developed to target surface-accessible aromatic residues.[3]
To complement these methods further, we report herein a
biomimetic transamination reaction that can modify the
N terminus of proteins and peptides under mild conditions.
This technique introduces a uniquely reactive ketone or
aldehyde group in a single location, thus allowing further
modification through oxime or hydrazone formation. This
simple strategy does not require the use of site-directed
mutagenesis, and therefore has the potential to introduce
virtually any functional group on a wide range of protein
substrates.
The unique reactive properties of the N terminus have
resulted in several strategies targeting this location to achieve
site-selective protein modification. To a limited extent, the
lower pKa value of N-terminal amino groups (relative to
lysine side chains) can be used to direct acylation reactions to
[*] J. M. Gilmore,[+] R. A. Scheck,[+] A. P. Esser-Kahn, N. S. Joshi,
Prof. M. B. Francis
Department of Chemistry
University of California
Berkeley and Materials Sciences Division
Lawrence Berkeley National Laboratory
Berkeley, CA 94720 (USA)
Fax: (+1)510-643-3079
E-mail: francis@cchem.berkeley.edu
[+] These authors contributed equally to this work.
[**] The authors thank the DOE Nanoscale Science and Engineering
Technology (NSET) program for financial support, as well as the
Department of Chemistry at the University of California, Berkeley,
and the Berkeley Chemical Biology Graduate Program. N.S.J.
gratefully acknowledges the Saegebarth family for generous fellow-
ship support.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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Table 1: Modification of angiotensin I (1) using aldehyde reagents.[a]
this site through careful control of
the reaction pH.[4] However, the
large number of competing lysine
residues (10–40 on most proteins)
limits the selectivity of this reaction
in most cases. Alternative strat-
egies have targeted the N terminus
in combination with specific amino
acid side chains. In particular, the
reaction of N-terminal cysteine
residues with thioesters has been
used with great success, and affords
fusion proteins through native
chemical ligations.[5] N-terminal
cysteine residues have also been
modified through thiazolidine for-
Aldehyde
3 [%]
4a–h [%]
Aldehyde
3 [%]
4a–h [%]
2a
2b
ca. 10
trace
2e
2 f
0
25
14
65
0
17
0
10
50
>90
40
2c
2d
2g
2h
trace
20
0
mation
by
using
aldehyde
reagents.[6] Reactive aldehydes
can be formed through periodate
oxidation of N-terminal serine and
threonine residues,[7] and N-termi-
[a] Conditions: 1 mm 1, 10 mm aldehyde, 50 mm phosphate buffer (pH 6.5), 378C, 2 h. Product
distributions were determined by ESI-MS.
nal tryptophan residues can be modified through Pictet–
Subsequent optimization experiments determined that a
1 mm peptide solution could be modified by using 10 mm PLP
in 50 mm phosphate buffer at pH 6.5 and 378C (Table 2,
entry 1). Consistent with other reactions involving carbonyl
condensations,[10] reactions at pH 6.5 afforded the optimal
Spengler reactions.[8]
An alternative strategy that does not depend on the
identity of the amino acid side chain can be envisioned
through the oxidation of the N-terminal amino group to an
imine, followed by hydrolysis to afford a ketone or an
aldehyde. This reaction pathway has indeed been demon-
strated for the chemospecific functionalization of N termini
by using glyoxylic acid, copper(II) salts, 1m pyridine, and 1m
HOAc.[9] However, these conditions are too harsh to maintain
the folded structure of most proteins, and are therefore more
appropriate for sequence-analysis applications. In contrast to
these reports, however, initial studies by our research group
found that the N-terminal aspartic acid residue of angioten-
sin I (1) unexpectedly decarboxylated to a small extent
(ca. 10%) when the peptide was simply treated with glyoxylic
acid (2a; Scheme 1). The product of the reaction was
identified as pyruvamide 3 through subsequent reaction
with O-benzylhydroxylamine (5) to form oxime 6. Screening
experiments indicated that the structure of the aldehyde
substitutent strongly influenced the reaction efficiency
(Table 1) and that the decarboxylated product was often
accompanied by various amounts of imines 4a–h. Interest-
ingly, pyridoxal-5-phosphate (PLP, 2c) emerged as the most
effective aldehyde, thus affording a 65% conversion of 1 into
ketone 3 in 2 h at 378C, with no observed imine formation.
Table 2: Peptide modification using 2c.[a]
Entry Peptide Sequence
pH T [8C] t [h] Conv [%]
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
7
8
9
DRYVIHPFHL
DRYVIHPFHL
DRYVIHPFHL
DRYVIHPFHL
DRYVIHPFHL
DRYVIHPFHL
DRYVIHPFHL
DNALRAFKYM
MYKFARLAND
6.5 37
5.5 37
7.5 37
8.5 37
6.5 65
6.5 22
6.5 22
6.5 37
6.5 37
1
1
1
1
2
2
18
2
2
2
65
29
46
43
>95
55
88
90
71
43
10
GLELEEYWKWGDKNYDG 6.5 37
[a] Conditions for peptides: 1 mm peptide, 10 mm 2c, 50 mm phosphate
buffer. Product distributions were determined by ESI-MS after oxime
formation with 5.
amount of product, although slight variations were still
tolerated (Table 2, entries 2–4). An increase in temperature
to 658C resulted in rapid conversion of 1 into 3 (> 95% in 2 h;
Table 2, entry 5), although excellent conversion could also be
obtained after 24 h at room temperature (Table 2, entry 7).
The reaction was found to proceed readily in 2-[4-(2-
hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES),
3-(N-morpholine)propanesulfonic acid (MOPS), and phos-
phate buffers, and, contrary to previous transamination
reports,[11] did not require the presence of divalent cations
or denaturing organic cosolvents. A variety of Lewis acids
were screened to confirm this, and little change in rate or
conversion was observed.[12] Moreover, experiments per-
formed in the presence of 10 mm ethylenediaminetetraacetate
(EDTA) to sequester any metal ions in the reaction solution
resulted in equivalent product yields.
Scheme 1. Formation of oxime 6 from the N-terminal asparticacid
residue of angiotensin I (1). Bn=benzyl.
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The most useful feature of this reaction is its
selectivity for the N terminus of the peptide. Under
the optimized conditions, peptide 7 afforded 90%
conversion into the ketone product (Table 2,
entry 8). These screening experiments also revealed
that the N-terminal residue does not need to be
aspartic acid, as peptides bearing N-terminal
methionine (8) and glycine (9) residues still afforded
good yields of the desired products. These peptides
also provided the singly modified conjugate, despite
the presence of internal lysine residues.
In a biological context, PLP is a cofactor that
effects a variety of metabolic transformations under
enzymatic control, including racemization, elimina-
tion, decarboxylation, and transamination.[13] Typi-
cally, PLP-mediated reactions first involve conden-
sation of the aldehyde with amino groups on a
peptide or protein, thus leading to the formation of
imine 4c (Scheme 2). In the absence of an enzyme,
this process could occur with all the amino groups on
a
particular biomolecule. However, the imine
formed with the N terminus has an a proton with a
much lower pKa value, which allows tautomerization
to occur uniquely at this site. Hydrolysis of the
resulting glyoxyl imine 10 (accompanied by decar-
boxylation in the case of aspartic acid) yields an
aldehyde or ketone specifically at the N terminus.
Although in principle this reaction sequence could
take place on the N termini of peptides and proteins
in living cells, this pathway has not been reported to
date. This is likely due to the lowconcentration of
unbound PLP in the cytoplasm.
Figure 1. Site-selective protein modification with PLP. a) Myoglobin was treated with 2c at
378C for 24 h. After removal of small molecules by gel filtration, oxime 14 was formed after
reaction with 13. b) ESI-LCMS studies indicated that only one modification had occurred.
c) UV/Vis spectra of 12 subjected to the reaction conditions in the absence (dotted red)
and presence (dashed blue) of 2c. d) Product 14 was digested with trypsin, and the
resulting fragments were analyzed by MALDI-TOF MS. The only modified fragment
observed contained the N-terminal residue. e) Predicted (left) and observed (right) isotope
patterns for the modified myoglobin peptide fragment shown in (d). All MS values agree
with those predicted within 0.01%.
dence that the tertiary structure of the protein was still intact
following the modification procedure.
We were able to confirm the site-specificity of this
modification after subjecting 14 to a proteolytic digest with
trypsin. Analysis of the peptide fragments by MALDI-TOF-
MS showed that the reaction had occurred on the N-terminal
fragment of the protein (Figure 1d). The bromine atom of 13
provided a useful analysis tag, as the characteristic isotope
pattern was specific to the modified fragment and could be
observed clearly (Figure 1e). Despite the 17 lysine residues
on the protein, no other modified peptides were observed, a
result that is consistent with the observation that these
Scheme 2. Formation of pyruvamide 3.
The development of the reaction for protein targets was
initiated with horse heart myoglobin (12), which has an N-
terminal glycine residue. Optimal conditions for myoglobin
modification were found to be 50 mm protein and 10 mm PLP
for 20 h at 378C in 25 mm phosphate buffer (pH 6.5;
Figure 1a). Following treatment of the mixture with p-
bromobenzyloxyamine (13), it was determined by ESI-MS
that a single product was obtained that corresponded to the
expected oxime 14 (Figure 1b). The overall conversion of the
reaction was determined to be greater than 75% by ESI-MS
and 69% using sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE analysis techniques, see below).
Following removal of excess PLP, the absorbance of the heme
component was found to be unchanged over the course of the
reaction (Figure 1c). This observation provides good evi-
residues do not participate in this reaction.
The oxime linkage is well suited for use as a probe or
linker in biochemical systems, as it is selective for ketone and
aldehyde functionalities, forms in high yield, and is stable
under physiological conditions.[14] As an example, the N ter-
minus of myoglobin was fluorescently labeled with chromo-
phore 15. SDS-PAGE analysis revealed selective labeling of
the protein after activation with PLP (Figure 2b, lane 1),
whereas no labeling was observed for unactivated myoglobin
(lane 2).
This technique can also be used to label proteins when
chemistry appropriate for cysteine groups is not available. For
example, cysteine 70 in the green fluorescent protein[15]
(EGFP, 16a) is reactive toward maleimide reagents and this
protein (in our hands) cannot be expressed in fluorescent
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Figure 3. Site-selective attachment of PEG polymers to proteins.
a) PEG-alkoxyamine 17 was synthesized and coupled to N-terminal
carbonyl groups introduced using 2c. b) Analysis by SDS-PAGE with
Coomassie staining confirmed single-site labeling of the proteins
(lanes 1–3, 6–11). The red arrows indicate the shift in the position on
the gel caused by the attached PEG polymer. PEG-NHS ester 18 was
used to label lysine residues of 12 nonspecifically (lanes 4 and 5). A
small portion of thioredoxin (20) reacts with the PEG-alkoxyamine
without treatment with 2c (lane 10). This background reaction gives
rise to the small portion of doubly labeled product shown in lane 11.
Protein 19 is RNase A.
Figure 2. Site-specific chromophore labeling. Proteins possessing N-
terminal carbonyl groups introduced by 2c were labeled with 15.
Reaction success was confirmed by SDS-PAGE analysis using Coomas-
sie staining (left) and fluorescence visualization (right).
form when this residue has been substituted. Thus, the
labeling of other positions on this protein is difficult to
achieve by standard mutagenesis techniques. We have found
that the N-terminal modification method can overcome this
limitation, and results in the attachment of 15 at the N-
terminal valine residue of 16a (Figure 2b, lanes 3 and 4) and
at the N-terminal glycine residue of mutant 16b. Subsequent
experiments have confirmed that the internal chromophore
retains its fluorescence after modification with PLP.[12]
The attachment of poly(ethylene glycol) (PEG) chains to
proteins has been shown to improve circulation lifetime,
lower immunogenicity, and confer proteolysis resistance.[16]
These conjugates are typically prepared through lysine
modification, which results in complex mixtures that exhibit
varying levels of efficacy. To avoid this, we have used the two-
step strategy outlined above to attach a single polymer chain
to the N terminus of proteins.[17,18] Specifically, the aldehyde
of PLP-activated myoglobin was treated with various equiv-
alents of the previously reported[19] PEG alkoxyamine 17. As
seen in Figure 3b (lanes 1–3), a single bioconjugate was
obtained even when using a large excess of 17. Using similar
conditions, this reaction strategy has also proven effective for
EGFP (16b, lanes 6 and 7), RNase A (19, lanes 8 and 9), and
thioredoxin (20, lanes 10 and 11).
Coomassie staining (Table 3). For myoglobin, 69% overall
conversion was achieved at 378C, and for EGFP (16b) 53%
conversion was obtained. High levels of modification could be
obtained for 16a and 16b at 558C. RNase A (19) was cleanly
modified in 50% conversion at 378C, as was thioredoxin (20)
under identical conditions. The large number of disulfide and
free thiol groups that proteins 19 and 20 possess render them
difficult to modify in a single location by other methods.
Finally, initial studies have found that protein G’ (21) can be
modified to 30% at 418C, thus indicating that proteins with
N-terminal methionine residues are compatible with this
technique. Studies to increase the conversion for this impor-
tant class of substrates are in progress, as are studies to
evaluate the compatibility of other N-terminal amino acids.
We anticipate that proteins possessing N-terminal serine,
threonine, cysteine, and tryptophan residues will be incom-
patible with this technique because of known side reactions
with aldehydes.[6,8] It is also presumed that N-terminal proline
residues will be unreactive. Outside of these limitations, this
reaction provides a generally applicable tool for the site-
selective labeling of protein targets. Our current efforts use
Table 3: Protein modification using 2c.[a]
For comparison, PEG-NHS ester
18 yielded a ladder of polymer
conjugates for the labeling of 12
(Figure 3b, lanes 4 and 5). Clean
conversion to a singly modified
protein was not observed under
any conditions using this approach.
The shift on the gel caused by
Entry
Protein
N-terminal amino acid
Conc. [mm]
T [8C]
Conv. [%]
1
2
3
4
myoglobin (12)
GFP-1V (16a)
GFP-1G (16b)
GFP-1G (16b)
RNase A(19)
glycine
valine
glycine
glycine
lysine
50
10
10
10
50
94
33
37
55
37
55
37
37
41
69
67
41
80
50
50
30
5
6
thioredoxin (20)
protein G’ (21)
glycine
methionine
7[b]
polymer attachment provides
a
useful method for the quantitation
of reaction conversion by optical
densitometry measurements after
[a] Conditions: 10 mm 2c, 25 mm phosphate buffer, pH 6.5, 18–20 h. Product yields were determined by
densitometry after derivitization with 17 and separation by SDS-PAGE (Coomassie stain, see Figure 3).
[b] Extent of protein modification was determined by ESI-MS after oxime formation with 5.
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this methodology for the site-specific placement of fluoro-
phores for sensing applications and the dual modification of
both protein termini for device integration.
Received: January 27, 2006
Revised: May 27, 2006
Published online: July 17, 2006
Keywords: bioconjugation · bioorganic chemistry · oximes ·
.
protein modifications
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