A highly efficient method for site-specific modification of unprotected peptides after chemical synthesis

Article abstract of DOI:10.1021/ja000217t

We have developed a highly efficient method for the site-specific attachment of biophysical probes to unprotected peptides after chemical synthesis. This methodology takes advantage of the selective reactivity of an N-methylaminooxy amino acid that is app

Full text of DOI:10.1021/ja000217t

VOLUME 122, NUMBER 15
APRIL 19, 2000
© Copyright 2000 by the
American Chemical Society
A Highly Efficient Method for Site-Specific Modification of
Unprotected Peptides after Chemical Synthesis
Steven J. Bark, Sandra Schmid, and Klaus M. Hahn*
Contribution from the Department of Cell Biology, The Scripps Research Institute,
10550 North Torrey Pines Road, BCC-162, La Jolla, California 92037
ReceiVed January 18, 2000
Abstract: We have developed a highly efficient method for the site-specific attachment of biophysical probes
to unprotected peptides after chemical synthesis. This methodology takes advantage of the selective reactivity
of an N-methylaminooxy amino acid that is appropriately protected for direct incorporation during solid-phase
peptide synthesis. The functional N-methylaminooxy group is unmasked using normal peptide cleavage
conditions and is capable of selective reaction with activated N-hydroxysuccinimide esters in the presence of
cysteine, lysine and the amino terminus, as demonstrated in model peptides and test proteins. Selective labeling
can be accomplished after synthesis using commercially available or chemically sensitive probes. This technology
is compatible with the synthesis of CR-thioester-containing peptides and amide-forming ligations, required
steps for the synthesis of proteins by either total chemical synthesis or expressed protein ligation. The
N-methylaminooxy amino acid can be introduced into different sites by parallel peptide synthesis to generate
a polypeptide analogue family with each member possessing a single specifically labeled site. This enables
the synthesis of optimized biosensors through combinatorial screening of different attachment sites for maximal
response and minimal perturbation of biological activity.
Introduction
An exciting recent development within this field has been the
construction of peptide and protein biosensors exhibiting altered
fluorescence properties in response to changes in their structure
or ligand binding.2-5 Appropriately labeled fluorescent biomol-
ecules allow spatial and temporal detection of biochemical
reactions inside living cells.4,5
Modified peptides and proteins are valuable biophysical tools
for studying biological processes, both in vitro and in vivo.1-5
In particular, quantitative live cell imaging of fluorescent
proteins and peptides is revolutionizing the study of cell biology.
* To whom correspondence should be addressed.
(1) Giuliano, K. A.; Post, P. L.; Hahn, K. M.; Taylor, D. L. Annu. ReV.
Biophys. Biomol. Struct. 1995, 24, 405-434.
The major obstacles in the development of fluorescent
biosensors have been: (1) The difficulty in site-specific place-
ment of the dye in the polypeptide and (2) determining exactly
which site is optimal for dye placement.1 Solvent-sensitive dyes
must be placed precisely for optimal response to changes in
protein structure without interference with biological activity.
Also, the need for site-specific incorporation of two dyes without
impairment of biological activity has proven a serious limitation
for utilization of FRET within a single protein. A potential
solution to these problems can be found in a recently developed
(2) (a) Day, R. N. Mol. Endocrinol. 1998, 12, 1410-9. (b) Adams, S.
R.; Harootunian, A. T.; Buechler, Y. J.; Taylor, S. S.; Tsien, R. Y. Nature
1991, 349, 694.
(3) Miyawaski, A.; Llopis, J.; Heim, R.; McCaffery, J. M.; Adams, J.
A.; Ikura, M.; Tsien, R. Y. Nature 1997, 388, 882-7.
(4) (a) Hahn, K.; DeBiasio, R.; Taylor, D. L. Nature 1992, 359, 736. (b)
Hahn, K. M.; Waggoner, A. S.; Taylor, D. L. J. Biol. Chem. 1990, 265,
20335.
(5) Richieri, G. V.; Ogata, R. T.; Kleinfeld, A. M. Mol. Cell. Biochem.
1999, 192, 87-94.
10.1021/ja000217t CCC: $19.00 © 2000 American Chemical Society
Published on Web 03/31/2000

3568 J. Am. Chem. Soc., Vol. 122, No. 15, 2000
Bark et al.
Scheme 1. Synthesis of
N-(2-Cl-Benzyloxycarbonyl)-N-methylaminooxy Acetic Acid
(3) and N-(2-Cl-Benzyloxycarbonyl)-N-
methylaminooxyacetyl-NR-Boc-R,â-diaminopropionic Acid
[(SA)Dapa-OH] (4)a
Figure 1. General strategy for site-specific labeling of polypeptides.
(Top) The protected aminooxy group is incorporated during solid-phase
peptide synthesis (synthesis on thioester-linker resin is shown). (Middle)
Cleavage from the resin generates a peptide possessing unprotected
side chains, an aminooxy group and a C-terminal thioester. (Bottom)
Ligation and subsequent site-specific labeling produces the full-length
polypeptide with a single dye attached at the aminooxy nitrogen. Rd
H or CH3.
technology, total chemical synthesis of proteins.6-9 However,
many biophysical probes suitable for fluorescent biosensors or
other purposes are not stable to the various conditions used for
peptide synthesis, and site-specific incorporation after synthesis
is difficult to achieve. The incorporation of an appropriately
protected unnatural amino acid during solid-phase peptide
synthesis, which can be selectively modified postsynthetically,
would resolve these problems.
We report here a simple and efficient methodology for site-
specific labeling of peptides after synthesis that satisfies the
requirements of high yield, selectivity, and compatibility with
both solid-phase peptide synthesis and CR-thioester peptides.
The applicability of this method has been tested using model
peptides and control proteins.
a N-(2-chlorobenzyloxycarbonyl)succinimide was reacted with excess
N-methyl hydroxylamine at pH 6-7 to generate the 2-Cl-Z hydroxamic
acid (1). Compound 1 was reacted with diisopropylethylamine and
bromoacetate tert-butyl ester to produce N-(2-Cl-benzyloxycarbonyl)-
N-methylaminooxy acetic acid tert-butyl ester (2), which was directly
converted to the free acid (3) by treatment with a 1:1 mixture of
trifluoroacetic acid and dichloromethane. Compound 3 was used for
the synthesis of SA-test peptide and the synthesis of (SA)Dapa-OH
(4) by condensation with NR-Boc-R,â-diaminopropionic acid. In each
case, compound 3 was activated as the N-hydroxysuccinimide ester.
peptide was achieved, extensive attempst to utilize the primary
aminooxy group during synthesis failed. Even when the primary
aminooxy group was protected as the 2-chlorobenzyloxycar-
bonyl carbamate, deprotonation allowed rapid acylation, so that
it could not be readily incorporated during peptide synthesis.
Recently, others have demonstrated a method for deprotection
Results and Discussion
of base labile Fmoc groups in the presence of thioesters:12
a
Site-Specific Labeling of the Secondary Aminooxy Group.
Under controlled pH conditions, the low pKa and enhanced
nucleophilicity of an aminooxy group relative to those of other
nucleophilic side chains found in peptides suggested the
possibility of site-specific reaction with standard electrophiles
such as succimidyl esters (Figure 1).10,11 While selective labeling
of a primary aminooxy group in the context of an unprotected
process which could be utilized to place an aminooxy substituent
on the side chain of a lysine within a peptide. However, we
alleviated this problem by utilizing a secondary N-methylami-
nooxy group.
We synthesized a test peptide containing both a secondary
aminooxy group and nucleophilic amino acids that were most
likely to interfere with selective labeling at the aminooxy
nitrogen (lysine, cysteine, and the amino terminus). NH2-
AKAARAAAAK*AARACA-CO2H, here designated SA-test
peptide, was synthesized by incorporation and deprotection of
N-(2-Cl-benzyloxycarbonyl)-N-methylaminooxy acetic acid (3)
during solid-phase peptide synthesis (Scheme 1 and Figure 2).
The reactivity of the protected secondary aminooxy group was
sufficiently attenuated to remain unreactive during Boc solid-
(6) (a) Wilken, J.; Kent, S. B. H. Curr. Opin. Biotechnol. 1998, 9, 412.
(b) Kent, S. B. H. Annu. ReV. Biochem. 1988, 57, 957-989.
(7) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H. Science
1994, 266, 776-779.
(8) Muir, T. W.; Sondhi, D.; Cole, P. A. Proc. Natl. Acad. Sci. 1998,
95, 6705-6710.
(9) Cotton, G. J.; Ayers, B.; Xu, R.; Muir, T. W. J. Am. Chem. Soc.
1999, 121, 1100-1101.
(10) (a) Jencks, W. P.; Carriuolo, J. J. Am. Chem. Soc. 1960, 82, 675.
(b) Jencks, W. P. J. Am. Chem. Soc. 1958, 80, 4581, 4585.
(11) Canne, L. E.; Bark, S. J.; Kent, S. B. H. J. Am. Chem. Soc. 1996,
118, 5891.
(12) Li, X.; Kawakami, T.; Aimoto, S. Tetrahedron Lett. 1998, 39, 8669-
8672.

Site-Specific Modification of Unprotected Peptides
J. Am. Chem. Soc., Vol. 122, No. 15, 2000 3569
Figure 2. Synthesis of SA-test peptide. For precursor peptide on the
resin, the ꢀ-amino group of lysine 10 is protected as the Fmoc
carbamate. Treatment with piperidine selectively removes the Fmoc-
protecting group. Either 2-Cl-Z aminoxy acetic acid or 2-Cl-Z N-
methylaminooxy acetic acid is then coupled on the lysine side chain
to generate, upon standard HF cleavage, PA-test peptide (RdH) or SA-
test peptide (RdCH3) in deprotected form.
Figure 3. Site-specific labeling and reductive cleavage of SA-test
peptide. (Top) HPLC analysis of purified SA-test peptide. (Bottom)
HPLC analysis of crude reaction products from optimized labeling
conditions. Smaller early eluting peak is unreacted SA-test peptide.
Main peak is SA-test peptide with single dye attached to secondary
aminooxy nitrogen. Smaller later eluting peaks are SA-test peptide with
two dyes attached and an acetylation side-product. Zinc/acetic acid
reduction of the singly labeled SA-test peptide yielded a new peak at
earlier retention time with a mass corresponding to cleavage of the
aminooxy N-O bond. Absorbance was monitored at 214 nm.
Table 1: Labeling of SA-Test Peptidea
buffers
percentage
dye
selec-
reaction gel filtration TCEP pHb equiv SM 1Dye 2Dye tivity
selectivity, as defined by the ratio of the peak areas of desired
single-labeled product over double-labeled products, was 6/1
(Table 1).
acetate
acetate
acetate
acetate
acetate
citrate
(NH4)HCO3 no
(NH4)HCO3 no
(NH4)HCO3 no
(NH4)HCO3 no
(NH4)HCO3 no
4.7 1.0 32
4.7 1.5 14
56
63
78
7
13
13
17
23
8:1
4.5:1
6:1
4.5:1
3:1
4.7 2.0
4.7 2.4
4.7 3.0
0
0. 76
71
4.3 89.6
yes 9.0 0.99 13 85
We found that many of the side reactions occurred during
size exclusion chromatography in the ammonium bicarbonate
solvent used to separate reaction products. Using an acidic
solvent system, 0.1% TFA, virtually eliminated multiply labeled
side products leading to considerable improvements in both
selectivity and yield. Including a mild reducing agent, tris(2-
carboxyethyl)phosphine (TCEP), in the reaction buffer also
significantly curtailed several minor side reactions revealed by
HPLC, especially disulfide formation. Labeling and gel filtration
under these optimized conditions produced a 70% recovered
yield of labeled SA-test peptide (90% yield based on HPLC
quantitation), 90% of which was labeled with only a single dye
at the aminooxy amine. The labeling selectivity was increased
to 22/1 (Table 1).
0
0.1% TFA
yes 4.7 4.3
4.1 22:1
0
carbonate 0.1% TFA
51:1c
a Conditions and outcome of labeling reactions using SA-test peptide
and tetramethylrhodamine N-hydroxysuccimidyl ester. Buffers: acetate
) 5% v/v acetic acid buffer (0.9 M) adjusted to pH ) 4.7 with NaOH;
citrate ) 100 mM sodium citrate, pH ) 4.7; carbonate ) 100 mM
sodium bicarbonate, pH ) 9.0. All buffers were used in labeling
reactions with 50% DMSO. TCEP was used at 5 mM. Equivalents were
based on the quantity of SA-test peptide in the reaction. Percentages
were determined by HPLC integration: SM ) starting SA-test peptide,
1Dye ) SA-test peptide with single dye on secondary aminooxy group;
2Dye ) doubly labeled SA-test peptide. Selectivity was determined as
the ratio of 1Dye/2Dye. b pH of the aqueous buffer used in the labeling
reaction solvent (50% aqueous buffer, 50% DMSO). c Selectivity based
on SA-test peptide product with a single dye on the secondary aminooxy
nitrogen divided by quantity of SA-test peptide with single dye at
alternate sites.
The purity of the product was confirmed using RP-HPLC,
mass spectroscopy, further chemical reaction, and isolation of
purposefully overlabeled products. The labeled peptide eluted
as a single peak under all HPLC conditions tested with a mass
consistent with that predicted for singly labeled SA-test peptide
(mass ) 1970). To determine that the labeling site was indeed
at the N-methylaminooxy group, we utilized a selective zinc/
acetic acid reduction procedure to cleave the N-O bond (Figure
3).11 HPLC of the reduction reaction showed >98% conversion
of the starting material and a new earlier eluting peak. The mass
of this peak (1530 amu) corresponded to the predicted mass of
the unlabeled SA-test peptide cleaved at the aminooxy N-O
bond. The residual zinc was washed several times with a
saturated solution of EDTA in water, which demonstrated that
the reduction reaction was complete.
phase peptide synthesis on thioester-linker resins. The 2-Cl-Z
protection for the N-methylaminooxy amino acid was efficiently
removed by standard HF cleavage procedures.
Conditions for selectively labeling the secondary aminooxy
group were determined by varying the reaction pH and dye
stoichiometry. Labeling with the succinimide ester of tetrameth-
ylrhodamine (TMR-OSu) was determined to be optimal in a
solvent system consisting of 50% DMSO/50% aqueous acetate
buffer at pH of 4.7 with 2 equiv of dye per mol of peptide (Table
1). The crude reaction products were separated from unreacted
dye and characterized by RP-HPLC and ESI-MS. Under these
conditions, a single molecule of dye was incorporated on the
aminooxy group with a 78% yield, based on HPLC quantifica-
tion. However, side reaction products were also isolated and
determined to be either SA-test peptide labeled with two dye
molecules (∼13%) or acetylated peptide products (∼5%). The
To eliminate the unlikely possibility that the HPLC peak
containing isolated single-labeled SA-test peptide product was
a mixture of two labeled species, SA-test peptide was reacted
under higher pH conditions (pH 9.0) to label all reactive sites.

3570 J. Am. Chem. Soc., Vol. 122, No. 15, 2000
Bark et al.
SA-test peptide contained three nucleophilic labeling sites which
would be irreversibly labeled: aminooxy, lysine, and N-terminal
amine. Dye labeling at high pH generated a mixture of peptides
labeled at all possible combinations of sites with one, two, or
three dyes. HPLC analysis of this reaction mixture showed eight
peaks, indicated by ESI-MS to correspond to unreacted SA-
test peptide, SA-test peptide single-labeled at the aminooxy
nitrogen, and six additional peaks corresponding to two single-
labeled peptides, three peptides bearing two dyes, and a single
triply labeled peptide species. This experiment revealed the
HPLC retention times of all of these products, none of which
coeluted with the peak identified as SA-test peptide labeled with
a single dye on the secondary aminooxy nitrogen.
resin, as described in Materials and Methods. (MPAL is the
C-terminal mercaptopropionyl-leucine group generated by cleav-
age of a peptide from TAMPAL resin.13)
Ligation of the LY-(SAOD)-AG-MPAL thioester peptide with
the peptide, CRANK-NH2,was tested by means of standard
procedures employing phosphate buffer with 6 M guanidine
hydrochloride at neutral pH in the presence of 2-3% thiophenol
by volume.7 The ligation proceeded over 24 h and generated
the desired ligation product, LY-(SAOD)-AGCRANK-NH2, at
∼85% yield. The major side product was attributable to
modification of unligated CRANK-NH2 peptide under the
ligation conditions (mass ) 714.5, data not shown) and was
not related to the presence of the aminooxy group. There was
also a single time-dependent side reaction, which generated a
product of 14 mass units lower than the desired. Using high
concentrations of reacting peptides and isolating the ligation
product after 24 h reduced this side reaction to acceptable levels
(<5%).
We also tested the ligation of a peptide containing multiple
potentially reactive functional groups, including a hexahistidine
tag useful for affinity chromatography. Coupling CEYRIDRVR-
LFVDKLDNIAQVPRVGAA-HHHHHH to LY-(SAOD)-AG-
MPAL thioester proceeded to completion in 5 h with minimal
side reactions. In both ligation reactions, there was less than
1% LY-(SAOD)-AG-MPAL self-condensation product, indicat-
ing that the aminooxy group and thioester do not appreciably
react with one another under the ligation conditions. These
results demonstrate that the inclusion of an unprotected ami-
nooxy group in the peptide chain is compatible with native
chemical ligation.
Labeling of the two ligation products using tetramethyl-
rhodamine succinimide ester proceeded with selectivity similar
to that for the SA-test peptide. HPLC integration indicated that
the product of the LY-(SAOD)-AG-MPAL ligation with CRANK-
NH2 was labeled with greater that 95% efficiency and with a
selectivity of 34:1. Mass spectral analysis and zinc reduction
demonstrated labeling at only the aminooxy group. For the
longer hexahistidine-containing polypeptide ligation product,
selectivity for the aminooxy group was greater than 10:1, but it
was difficult to achieve high yields. The histidines could
potentially have been affecting yield and selectivity by cata-
lyzing nucleophilic attack on the succinimide ester of the
reactive dye. Inclusion of guanidine hydrochloride in the reaction
solvent increased the yield to approximately 50%, indicating
that folding or poor solubility of the peptide was a factor in
preventing access of the reactive dye to the aminooxy group.
Selectivity was also improved, presumably because of the
availability of the reactive secondary aminooxy group. Single-
site labeling at the aminooxy group was proven by mass spectral
analysis of trypsin and R-chymotrypsin digests of the labeled
polypeptide product.
Site-specific labeling of the aminooxy group could even be
achieved at basic pH (Table 1). Using pH 9.0 carbonate buffer
in our solvent system, addition of 0.5 equiv of dye produced,
after 3 h, ∼50% conversion of the starting SA-test peptide to a
single peak with the elution time of the desired singly labeled
product. After addition of another 0.5 equiv of TMR-OSu and
an additional 3 h of reaction time, HPLC showed ∼85%
conversion to a peak with the retention time of the desired
product. Two minor peaks (∼2-3% of total peak area), were
also apparent and corresponded to the two other single-labeled
SA-test peptide species identified in the multiple labeling
experiment above. The N-hydroxysuccinimide ester of rhodamine
clearly showed selective reactivity with the aminooxy group.
The selectivity observed at higher pH cannot be explained
by the nucleophicity of the aminooxy group alone. In fact, others
have shown that, in an uncatalyzed reaction with phenyl acetate
at high pH, amines are more reactive than O-alkyl aminooxy
groups.10 Therefore, we suggest that kinetic factors are con-
tributing to the selective reactivity of the N-methylaminooxy
group, even when competing groups are not protonated. Possible
reasons for this include: (1) the aminooxy oxygen localizes the
nitrogen near the activated ester via formation of a hydrogen
bonded “bridged” intermediate, or (2) a base-catalyzed reaction
pathway under the conditions of our reaction.10 This exceptional
reactivity has important practical implications, as it can allow
the selective labeling of acid-labile polypeptides and synthetic
proteins under physiological or basic conditions.
The Secondary Aminooxy Group Is Compatible with Cr-
Thioesters and Amide-Forming Ligation. Preparation of
proteins by total chemical synthesis often requires the ligation
of large polypeptides prepared by solid-phase peptide synthesis
on thioester-linker resins.6,9 The most generally applicable
methods available for ligations are native chemical ligation7 and
expressed protein ligation.8,9 These processes utilize the same
basic chemistry to join two peptides, one with an N-terminal
cysteine and the other with a C-terminal thioester, through a
regiospecific and site-specific reaction to generate a larger
polypeptide. The application of aminooxy-labeling chemistry
to the synthesis of large polypeptides and proteins requires
compatibility with these solid-phase peptide synthesis and
ligation chemistries.
Specificity of Labeling in Protein Domains Containing
Aminooxy Amino Acids. As a control to establish the selectiv-
ity of labeling for aminooxy amino acid, we attempted to label
native â-Lactoglobulin with tetramethylrhodamine N-hydroxy-
succinimide ester. Under our conditions, nonspecific labeling
of this 162-amino acid (aa) protein containing15 lysines and 4
cysteines was minimal (<1%) even after 6 h.
Finally, we synthesized the GTPase binding domain of p21
activated kinase (45 aa, 4 Lys, 1 Cys) with a secondary
aminooxy amino acid incorporated at the amino-terminus
The optimal approach for utilizing aminooxy-labeling chem-
istry in the chemical synthesis of proteins is direct incorporation
of the aminooxy group as part of an amino acid used during
standard solid-phase peptide synthesis. For this purpose, we
generated a suitably protected N-methylaminooxy amino acid,
R-Boc-â-[N-(2-chlorobenzyloxycarbonyl)-N-methylaminooxy
acetyl]-R,â-diaminopropionic acid [Boc-2-Cl-Z-(SA)Dapa-OH]
(4), as shown in Scheme 1. This amino acid, referred to as
SAOD, was incorporated into the peptide sequence LY-(SAOD)-
AG-MPAL thioester by synthesis on TAMPAL thioester-linker
(13) (a) Hojo, H.; Kwon, Y.; Kakuta, Y.; Tsuda, I.; Hikichi, K.; Aimoto,
S. Bull. Chem. Soc. Jpn. 1993, 66, 2700-2706. (b) Hackeng, T. M.; Griffin,
J. M.; Dawson, P. E. Proc. Natl. Acad. Sci. U.S.A., in press.

Site-Specific Modification of Unprotected Peptides
J. Am. Chem. Soc., Vol. 122, No. 15, 2000 3571
(SAOD-PBD). Previous experiments have demonstrated that
PBD domains labeled with fluorescent reporter dyes at this
terminus could be used as biosensors of GTPase activation.14
Labeling of PBD using the new methodology would enable the
production of sufficient quantities to apply the biosensors in
vivo and in pharmaceutical screening applications and would
allow incorporation of sensitive dyes enabling applications
within living cells.
SAOD-PBD was readily labeled with Alexa-532 N-hydroxy-
succinimide ester by titration addition of dye at pH 4.7 over 72
h. The labeling efficiency was commensurate with that reported
for the longest model peptide (∼50% yield by HPLC quanti-
tation) and there was no indication of multiple labeling. In this
case, isolation of labeled SAOD-PBD by RP-HPLC proved
difficult. Separation to baseline resolution was not achieved,
but small quantities of unalabeled PBD in the labeled product
do not preclude the use of the labeled material in biosensor
applications. Previous reports indicate that separation of labeled
product from starting polypeptide is highly dependent on the
specific peptide and the attached dye.
of ketone amino acids through synthetic procedures21-25 or
molecular biology,26 and expressed protein ligation.8,9 While
each method has utility for producing a particular class of
biosensor, all have limitations that restrict their general use.
Labeling of natural amino acid side chains in solution is often
impractical because of the existence of many other competing
nucleophiles. To use unnatural amino acids, such as those
bearing ketones for selective labeling, requires the synthesis of
dye constructs or amino acids that are difficult to make and not
available commercially. Peptide labeling on the resin limits
usable fluorophores to those that can survive the harsh conditions
used in synthesis and cleavage of the labeled polypeptide. The
procedure we have developed resolved many of these issues
and should be generally applicable to a wide range of polypep-
tides and biophysical probes.
Materials and Methods
General. For column chromatography, silica gel (230-400 mesh)
was used in standard glass columns with gravity or air pressure.
Reversed-phase high performance liquid chromatography (RP-HPLC)
was performed on a Waters HPLC system with UV detection at 214
nm using either a Vydac C-18 analytical column (5 µm, 0.46 × 25
cm), a Waters RCM 8 × 10 module equipped with a semipreperative
Delta Pack C-18 Radial Pack cartridge column (15 µm, 8 × 100 mm)
from Millipore, or a Vydac C-18 preparative scale column (15 µm,
1.0 × 25 cm). Linear gradients of solvent B (0.09% TFA in 90%
acetonitrile/10% water) in solvent A (0.1% TFA in water) were used
for all HPLC chromatographic separations.6b
In summary, these results clearly demonstrate that the
optimized site-specific labeling chemistry reported here is
compatible with the steps required for the preparation of proteins
by total chemical synthesis.
Conclusions
In this paper, we report a simple and efficient synthetic
methodology for site-specific labeling of peptides after synthesis
that satisfies the requirements of high yield, selectivity and
compatibility with both solid-phase peptide synthesis and CR-
thioester peptides. The approach and primary advantages can
be summarized as follows: (1) A protected N-methylaminooxy
amino acid which can be incorporated into peptides by means
of optimized solid-phase Boc synthesis procedures16 has been
synthesized. (2) Labeling procedures have been optimized to
yield highly efficient and specific modification of the N-
methylaminooxy nitrogen in the presence of unprotected
competing nucleophiles, including cysteine, lysine, and the
amino terminus. (3) The electrophile used in labeling, an
activated carboxylic ester, is readily available in the majority
of commercially available fluorescent dyes and labels.17a (4)
Labeling of the N-methylaminooxy group occurs after synthesis
and purification, thus enabling the use of chemically sensitive
fluorophores and labels that would otherwise not survive earlier
synthetic procedures. (5) This synthetic methodology is compat-
ible with the critical steps required for the synthesis of proteins
by total chemical synthesis or expressed protein ligation, namely
synthesis of CR-peptide thioesters and amide-forming ligations.6-9
(6) Combinatorial screening of both the dye used and its
placement18 could enable the rapid synthesis of optimally labeled
polypeptide-based biosensors.
Mass spectra of peptides were obtained either with a Sciex API-III
electrospray ionization (ESI) triple-quadrupole mass spectrometer (PE
Biosystems, Foster City), or matrix assisted laser desorption ionization
time-of-flight (MALDI-TOF) instruments from Thermo Bioanalysis
(Thermo Bioanalysis, Ltd., UK) or Kratos Analytical (Chestnut Ridge,
NY). For ESI-MS, the observed masses reported were derived from
the experimental m/z states for all observed charge states of a molecular
species using the program MacSpec (Sciex, version 2.4.1) for electro-
spray mass spectrometry. MALDI-MS observed masses were relative
to internal calibration using R-cyano-hydroxycinnammic acid or sini-
pinic acid matrixes. Calculated masses reported were derived from either
MacProMass (Terry Lee and Sunil Vemuri, Beckman Research Institute,
Duarte, CA) or PAWS (Version 8.1.1, ProteoMetrics) and reflect the
average isotope composition of the singly charged molecular ion. Proton
nuclear magnetic resonance spectrometry was recorded on a Bruker
AC-250 mass spectrometer and data were analyzed using WinNMR
(Bruker Instruments). Ultraviolet-visible spectroscopy was performed
on a Hewlett-Packard photodiode-array spectrophotometer.
Boc-L-amino acids were purchased form Novabiochem (La Jolla,
CA) or Bachem Bioscience, Inc. (King of Prussia, PA). [[4-(Hy-
droxymethyl)phenyl]-acetamido]methyl (-OCH2-Pam) resin was pur-
chased from PE Biosystems (Foster City, CA), and methylbenzhydryl-
amine (MBHA) resin was purchased from Peninsula Laboratories, Inc.
(San Carlos, CA). Solvents were synthesis grade or better and were
purchased from Fisher Scientific (Tustin, CA). Trifluoroacetic acid
(TFA) and anhydrous hydrogen fluoride were purchased from Halo-
carbon (New Jersey) and Matheson Gas (Rancho Cucamonga, CA).
Dyes were obtained from Molecular Probes (Eugene, OR). All other
reagents were analytical grade or better and were purchased from
Other procedures for site-specific modification of polypep-
tides have been described, including chemically selective
labeling in solution19 and on resin-bound peptides,20 introduction
(14) Kraynov, V.; Chamberlain, C.; Bokoch, G.; Balch, W.; Hahn, K.
M., submitted.
(15) Brune, M.; Hunter, J. L.; Corrie, J. E. T.; Webb, M. R. Biochemistry
1994, 33, 8262-8271.
(16) Schnolzer, M.; Alewood, P.; Jones, A.; Alewood, D.; Kent, S. B.
H. Int. J. Pept. Protein Res. 1992, 40, 180-193.
(20) Hackeng, T.; Yegneswanan, S.; Johnson, A. E.; Griffin, J. H. J.
Biol. Chem., submitted.
(21) Rose, K. J. Am. Chem. Soc. 1994, 116, 30-33.
(22) King, T. P.; Zhao, S. W.; Lam, T. Biochemistry 1986, 25, 5774-
5779.
(23) Rose, K.; Zeng, W.; Regamey, P.-O.; Chernushevich, I. V.; Standing,
K. G.; Gaertner, H. F. Bioconjugate Chem. 1996, 7, 552-556.
(24) Marcaurelle, L. A.; Bertozzi, C. R. Tetrahedron Lett. 1998, 39,
7279-7282.
(25) Wahl, F.; Mutter, M. Tetrahedron Lett. 1996, 37, 6861-6864.
(26) Cornish, V. W.; Hahn, K. M.; Schultz, P. G. J. Am. Chem. Soc.
1996, 118, 8150.
(17) (a) Haugland, R. P., Ed. Handbook of Fluorescent Probes and
Research Chemicals; Molecular Probes: Eugene, OR, 1992; pp 81-88.
(b) Waddel, W. J. J. Lab. Clin. Med. 1956, 48, 311-314.
(18) Dawson, P. E.; Fitzgerald, M. C.; Muir, T. W.; Kent, S. B. H. J.
Am. Chem. Soc. 1997, 119, 7917-7927.
(19) Brinkley, M. Bioconjugate Chem. 1992, 3, 2-13.

3572 J. Am. Chem. Soc., Vol. 122, No. 15, 2000
Bark et al.
Aldrich (Milwaukee, WI), Lancaster (Windham, NH), Peptides Inter-
national (Louisville, KY) or Richelieu Biotechnologies (Montreal,
Canada).
tic Acid (3).29 Compound 2 (1.1 g, 3.30 mmol) was dissolved in 4 mL
of DCM, and with rapid stirring, neat TFA (5 mL) was added dropwise
over 2 min at room temprature. After 1 h, the reaction was quenched
with 20 mL of water and extracted three times with DCM, and then
the combined DCM layers were dried over sodium sulfate. The DCM
was removed in vacuo to yield 0.9 g (3.28 mmol, 99%) of an off-
white solid. TLC Rf ) 0.2 (Hex/EtOAc/AcOH 80/20/1). 1H NMR: 3.23
(s, 3H), 4.50 (s, 2H), 5.32 (s, 2H), 7.28 (m, 2H), 7.40 (m, 2H).
HRMS: Expected Mass ) 274.0482, Observed Mass ) 274.0479.
Synthesis of N-(2-Cl-Benzyloxycarbonyl)-N-methylaminooxy-
acetyl-r-Boc-r,â-Diaminopropionic Acid [(SA)Dapa-OH](4).25,30
N-(2-Chlorobenzyloxycarbonyl)-N-methylaminooxy acetic acid (3) (2.5
g, 9.2 mmol) was activated with N-hydroxysuccinimide (2.11 g, 2 equiv)
and DIC (1.440 mL, 1.0 equiv) in 20 mL of DCM. This reaction was
rapidly stirred at room temperature for 2 h prior to the addition of NR-
Boc-R,â-diaminopropionic acid (2.3 g, 1.2 equiv) and DIEA (3.20 mL,
2 equiv). After 4 h, the DCM solvent was removed in vacuo, and 50
mL of ethyl acetate was added. The ethyl acetate layer was washed
twice with 0.5 M acetate buffer, pH ) 4.0 and then twice with 0.1 N
sulfuric acid. The combined acid washes were then washed with 50
mL of ethyl acetate. The combined ethyl acetate layers were dried over
sodium sulfate and then concentrated in vacuo to yield a viscous yellow
oily solid. This solid was subjected to three hexane precipitations from
diethyl ether to yield 2.16 g (51% yield) of an off-white solid. TLC
Rf ) 0.2-0.4 (Hex/EtOAc/AcOH 30/70/0.5). 1H NMR: 1.45 (s, 9H),
3.16 (s, 3H), 3.54 (d-of-t, 1H, J ) 14.3, 4.6 Hz), 3.93 (m, 1H, J )
14.3, 7.5, 4.6 Hz), 4.31 (s, 0.5H), 4.38 (s, 1H), 4.45 (s, 1H), 4.51 (s,
0.5H), 5.34 (s, 2H), 5.97 (broad-d, 1H, J ) 7.3 Hz), 7.30 (m, 2H),
7.43 (m, 2H), 8.50 (broad-s, 1H). HRMS: Expected Mass ) 460.1487,
Observed Mass ) 460.1480.
Synthesis of Secondary Aminooxy Test Peptide (SA-test peptide).
The SA-test peptide, NH2-AKAARAAAAK*AARACA-CO2H, was
synthesized with Lys 10 side chain Fmoc protection as described
previously.31 Incorporation of the secondary aminooxy group was
accomplished by coupling 2-Cl-Z-protected N-methylaminooxyacetic
(300 mgs, 1.09 mmol) activated with diisopropylcarbodiimide (157 µl,
1.00 mmol) and N-hydroxysuccinimide (140 mg, 1.22 mmol) in 2 mL
of DCM for 1-2 h and then diluted with 2 mL of DMF just prior to
coupling to the ꢀ-amino group of Lys 10. Optimized coupling, cleavage,
and purification protocols were utilized.6b,10 Amino acid analysis was
consistent with the desired peptide. Expected Mass ) 1560, Observed
Mass ) 1559.
Synthesis of LY-(SAOD)-AG-MPAL-Thioester. LY-(SAOD)-AG-
MPAL-thioester was synthesized using optimized in situ neutralization
protocols for Boc chemistry on TAMPAL resin.13,16 Coupling of the
NR-Boc-(SA)Dapa-OH amino acid was accomplished by reacting the
in situ activated N-hydroxysuccinimide ester to the deprotected amino-
terminal nitrogen of alanine.31 (SA)Dapa-OH (4) (230 mgs, 0.5 mmol)
was dissolved in 1 mL of DCM and N-hydroxysuccinimide (115.1 mgs,
1.0 mmol) and DIC (74.4 µL, 0.47 mmol) were added. The reaction
was mixed briefly and allowed to activate for 1-2 h at room
temperature prior to coupling to the deprotected N-terminus of the
peptide chain. After this coupling, no further modifications of the
synthetic protocols were required. Expected mass ) 797, Observed
mass ) 797.
Peptide Segment Synthesis. Synthesis of peptides was carried out
manually using optimized stepwise solid-phase synthesis methods with
in situ neutralization and HBTU activation procedures for Boc chemistry
on either -OCH2-Pam, MBHA, or Trt-protected mercaptopropionyl-
Leu (TAMPAL) resin.13,16 Standard Boc-protecting group strategies
were employed.6b,16 Coupling was monitored by quantitative ninhydrin
assay after 15 min coupling cycles.27 After chain assembly, standard
deprotection and cleavage from the resin support was carried out by
treatment at 0 °C for 1 h with anhydrous HF containing either 10%
p-cresol or anisole as scavenger. Purification was performed using RP-
HPLC.6b
Synthesis of TAMPAL Resin.13 MBHA resin (2.5 g, 0.865 mmol/
g, 2.16 mmol of amine) was swelled in DMF. Boc-Leu-OH (1.1 g, 4.4
mmol) was activated with HBTU (8 mL, 0.5M solution) and DIEA (2
mL) and then coupled to the MBHA resin until complete reaction by
ninhydrin assay. The NR-Boc group of the linked leucine was removed
with neat TFA, and then S-Trt-â-mercaptopropionic acid (1.5 g, 4.3
mmol), activated in the same manner as Boc-Leu-OH, was added to
the deprotected Leu-MBHA resin and allowed to couple until complete
reaction. The S-Trt-â-mercaptopropionyl-Leu-MBHA resin was washed
extensively with DMF and then with DCM/MeOH (1/1) and finally
dried in vacuo to yield 3.39 g of thioester resin. Substitution calculated
by weight gain yielded 0.549 mmol/gram.
Deprotection of TAMPAL Resin. S-trityl protection was removed
by two 5 min treatments with 95% TFA/5% triisopropylsilane.13 The
deprotected resin was extensively washed with DMF before coupling
the first amino acid, activated using optimized in situ neutralization
protocols.
Synthesis of N-(2-Cl-Benzyloxycarbonyl)-N-methylhydroxyl-
amine (1).10 N-methylhydroxylamine hydrochloride (0.95 g, 11.37
mmol) was dissolved in 3 mL of water with rapid stirring. The pH of
this solution was adjusted to 6-7 by dropwise addition of a saturated
solution of sodium bicarbonate. 2-Chlorobenzyloxycarbonyl-N-hydroxy-
succinimidyl carbonate (1.2 g, 4.23 mmol) was dissolved in 4 mL of
THF and added slowly to the rapidly stirring solution of neutralized
N-methylhydroxylamine. After stirring at room temperature for 14 h,
the reaction was quenched with 20 mL of saturated sodium bicarbonate
and extracted three times with 20 mL of ethyl acetate. The combined
ethyl acetate layers were washed once with saturated sodium bicarbonate
and dried over anhydrous sodium sulfate, and the solvent was removed
in vacuo to yield 0.77 g (3.77 mmol, 84%) of an off-white solid. TLC
Rf ) 0.2 (Hex/EtOAc/AcOH 80/20/1). 1H NMR: 3.23(s, 3H), 5.25 (s,
2H), 7.24 (m, 2H), 7.37 (m, 2H). HRMS: Expected ) 216.0427,
Observed ) 216.0425.
Synthesis of N-(2-Cl-Benzyloxycarbonyl)-N-methylaminooxy Ace-
tic Acid-tert-butyl ester (2).28 Compound 1 (0.96 g, 4.71 mmol) was
dissolved at room temperature in 10 mL of THF with rapid stirring.
To the solution was added bromoacetate tert-butyl ester (1.05 g, 5.38
mmol) and then sodium iodide (1.5 g, 10.01 mmol) followed by DIEA
(2.5 mL, 15.92 mmol). The reaction changed to an orange-yellow color
after addition of sodium iodide. The reaction was quenched with 30
mL of water after complete reaction (∼3 h) and extracted three times
with ethyl acetate. The combined ethyl acetate layers were dried over
sodium sulfate, and the ethyl acetate was removed in vacuo. The
resultant oily solid was purified by silica chromatography on 230-
400 mesh silica gel using hexanes/ethyl acetate/acetic acid (80/20/1)
to yield 1.40 g (4.29 mmol, 90%) of a pure yellow oil. TLC Rf ) 0.5
(Hex/EtOAc/AcOH 80/20/1). 1H NMR: 1.46 (s, 9H), 3.29 (s, 3H), 4.36
(s, 2H), 5.26 (s, 2H), 7.24 (m, 2H), 7.40 (m, 2H). HRMS: Expected
Mass ) 330.1108, Observed Mass ) 330.1104.
Ligation of LY-(SAOD)-AG-MPAL-Thioester with CRANK-NH2
Peptide. LY-(SAOD)-AG-MPAL-thioester (3 mg, 3.8 µmol) was
dissolved in 100 µL of 50 mM phosphate buffer containing 6 M
guanidine hydrochloride, pH ) 7.2. To this solution was added
CRANK-NH2 peptide dissolved in 100 µL of the same phosphate buffer
and 3 µL of thiophenol.7 The reaction was monitored by analytical
reversed-phase HPLC. After 24 h, the ligated product, LY-(SAOD)-
AGCRANK-NH2, was isolated by semi-preperative reversed-phase
HPLC (gradient ) 10-50% B over 60 min) and lyophilized to yield
Synthesis of N-(2-Cl-Benzyloxycarbonyl)-N-methylaminooxy Ace-
(27) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Anal.
Biochem. 1970, 34, 595.
(28) (a) March, J. AdVanced Organic Chemistry, 3rd ed.; John Wiley &
Sons: New York, 1989; pp 381. (b) Nyberg, D. D.; Christensen, B. E. J.
Am. Chem. Soc. 1957, 79, 1222. (c) Motorina, I. A.; Parly, F.; Grierson, D.
S. Synlett 1996, 389.
(29) Bryan, D. B.; Hall, R. F.; Holden, K. G.; Huffman, W. F.; Gleason,
J. G. J. Am. Chem. Soc. 1977, 99, 2353.
(30) Anderson, G. W.; Zimmerman, J. E.; Callahan, F. M. J. Am. Chem.
Soc. 1964, 86, 1839.
(31) Canne, L. E.; Ferre-D’Amare, Burley, S. K.; Kent, S. B. H. J. Am.
Chem. Soc. 1995, 117, 2998-3007.

Site-Specific Modification of Unprotected Peptides
J. Am. Chem. Soc., Vol. 122, No. 15, 2000 3573
a fluffy white solid. Amino acid analysis was consistent with the desired
product peptide. Expected Mass ) 1168, Observed Mass ) 1168.
Ligation of LY-(SAOD)-AG-MPAL-Thioester with CEYRIDRVR-
LFVDKLDNIAQ-VPRVGAA-HHHHHH. LY-(SAOD)-AG-MPAL
(0.3 mg, 0.37 mmol) and CEYRIDR-VRLPFVDKLDNIAQ-VPRV-
GAA-HHHHHH (1.5 mg, 3.8 mmol) were subjected to the same
ligation and purification conditions as those described above to yield
1.0 mg (58% yield) of a white fluffy solid. Expected Mass ) 4518,
Observed Mass ) 4517.
Synthesis of Amino-Terminal P21 Binding Domain (PBD) Pep-
tide Fragment, (SAOD)-KKKEKERPEISLPSDFEHTIHVGFDA-
MPAL Thioester. The amino-terminal PBD thioester containing a
secondary aminooxy group was synthesized as described above using
TAMPAL resin. HF cleavage utilizing p-cresol scavenger followed by
HPLC purification yielded (SAOD)-KKKEKERPEISLPSDFEHTIH-
VGFDA-MPAL containing two DNP groups protecting the histidines.
Mass Expected ) 3745, Mass Observed ) 3745.
Synthesis of Carboxy-Terminal of P21 Binding Domain (PBD)
Peptide Fragment, CTGEFTGMPEQWARLLQT. The native car-
boxy-terminal half of PBD was synthesized using standard FMOC
synthesis protocols by the Scripps Peptide and Protein Core Facility.
Mass Expected ) 2068, Mass Observed ) 2068.
Synthesis of SAOD-Modified PBD, SAOD-KKKEKERPEIS-
LPSDFEHTIHVGFDA-CTGEFTGMPEQWARLLQT. SAOD-K-
KKEKERPEISLPSDFEHTIHVG-FDA-MPAL (1.5 mg, 0.4 mmol) was
ligated to 1 mg (4.8 mmol) of carboxy-terminal fragment, CTGEFT-
GMPEQWARLLQT, as described above. After 48 h, the ligated PBD
proteins were isolated by RP-HPLC and lyophilized, 1.5 mg (70%
yield), Mass Expected ) 5262, Mass Observed ) 5262.
Selective Labeling of SA-Test Peptide with Tetramethyl-
rhodamine N-Hydroxysuccinimidyl ester. A solution of SA-test
peptide (3.396 µg/µL, 2.18 mM) in 5% acetate buffer, pH ) 4.7
incorporating 5 mM TCEP was utilized for labeling. A stock solution
of dye (5 µg/µL, 9.5 mM) was made by dissolving TMR-OSu in neat
DMSO. For each reaction, the dye stock was diluted so that the desired
number of dye equivalents could be added in 20 µL of DMSO. The
following equivalents of dye were tested: 1.2, 1.5, 1.8, 2.0, 2.4, 3.0,
and 4.3. With constant stirring, 20 µL of dye solution was added in
two 10 µL aliquots to 20 µL of peptide solution at room temperature.
The second aliquot of dye in DMSO was added 10 min after the initial
dye addition. After complete addition of dye, the reaction was briefly
vortexed and then incubated at room temperature. After 3 h, the labeled
reaction product(s) were separated from unreacted dye by gel filtration
on Sephadex G-10 or G-15 columns using either 100 µM ammonium
bicarbonate or, after optimization, 0.1% TFA in water. The individual
peptide product(s) were then separated by RP-HPLC and analyzed by
ESI-MS. Mass Expected ) 1971, Mass Observed ) 1970.
Non-Selective Labeling of SA-Test Peptide with Tetramethyl-
rhodamine N-Hydroxysuccinimide ester. SA-test peptide (3.396 µg/
µL, 2.18 mM) was dissolved 100 mM sodium carbonate, pH ) 9.01
containing 5 mM TCEP. 18 µL of neat DMSO was added to 20 µL of
peptide solution. With rapid mixing, 1 µL of stock dye solution in
DMSO was added to this mixture. Upon completion of the addition,
the reaction was vortexed briefly and then incubated at room temper-
ature. After 3 h, a 15 µL aliquot was removed and evaluated by RP-
HPLC and ESI-MS. This process was repeated until significant levels
of reaction were detected by the formation of single-labeled SA-lysine
test peptide products.
sample of 20 µL of LY-(SAOD)-AGCRANK-NH2 (2.5 µg/µL, 2.18
mM) in 200 mM citrate buffer, pH ) 4.7, 5 mM TCEP was labeled
using 4.3 equiv of dye in DMSO, purified, and analyzed. Expected
Mass ) 1580, Observed Mass ) 1580. A 20 µL sample of LY-(SAOD)-
AGCEYRIDRVRLFVDKLDNIAQVPRVGAA-HHHHHH (9.7 µg/µL,
2.12 mM) in 200mM citrate buffer, pH ) 4.7, containing 5 mM TCEP
and 3 M guanidine hydrochloride was labeled using a modified
procedure. Eighteen microliters of a solution of tetramethylrhodamine
N-hydroxysuccinimide (10 µg/µL) in DMSO was added in 6 µL aliquots
over 15 min with rapid mixing. The reaction was incubated at room
temperature for 5 h prior to gel filtration/RP-HPLC purification and
mass spectral analysis. Expected Mass ) 4930, Observed Mass ) 4929.
Labeling of â-Lactoglobulin with Tetramethylrhodamine N-
Hydroxysuccinimide Ester. A solution of tetramethylrhodamine
N-hydroxysuccinimide ester (10 µL, 10 mg/mL) in DMSO (9.5 equiv
compared to protein), was added to a solution containing 10 µL of
DMSO and 20 µL of a solution of â-lactoglobulin (20.3 mg/mL or 1.1
mM) in 2.8 M guanidine hydrochloride with 5 mM TCEP (pH 4.7).
After 3 h, protein was separated from unreacted dye by gel filtration,
and labeling was determined by analysis of the dye-to-protein ratio
(protein concentration was determined by the method of Waddel and
ꢀ for tetramethylrhodamine in phosphate buffer, pH ) 8.0 of 81 000).17
Labeling of PBD Proteins with Alexa-532 N-Hydroxysuccinimide
Ester. SAOD-modified PBD protein (150 µg) was dissolved in 105
µL of 200 mM sodium citrate buffer, pH ) 4.8, containing 5 mM TCEP
(protein concentration ∼0.28 mM). A solution of Alexa-532-OSu in
DMSO (dye concentration ∼10 mg/mL in DMSO) was titrated into
the protein solution in 5 µL aliquots over 72 h. Four hours after each
addition, the extent of labeling was determined by RP-HPLC and MS.
Labeling was continued until quantities of double-labeled PBD were
obtained (SAOD-modified PBD). Alexa-532 labeled SAOD-modified
PBD, Mass Expected ) 5871, Mass Observed ) 5870.
Zinc/Acetic Acid Reduction of the N-Methylaminooxy N-O
Bond in Peptides. Reductive cleavage of the N-O bond was performed
using zinc and aqueous acetic acid.11 Effervescence in the reaction was
evident after a few seconds and subsided after 60-120 min. After 14
h, the reaction supernatant was analyzed by RP-HPLC and ESI-MS.
Reduction of labeled SA-Test peptide, Expected Mass ) 1530,
Observed Mass ) 1530: Reduction of labeled (SAOD)-AGCRANK-
NH2: Expected Mass ) 1139, Observed Mass ) 1139.
Trypsin/Chymotrypsin Cleavage of Labeled LY-(SAOD)-AGCEY-
RIDRVRLFVDKLDN-IAQVPRVGAA-HHHHHH Peptide. A solu-
tion of either trypsin or R-chymotrypsin (10 µL, 0.05 mg/mL) in 25
mM ammonium carbonate (without pH adjustment) was added to 5
µL of a 10-20 µg/µL solution of pure tetramethylrhodamine-labeled
peptide in water (final concentration of protease is 0.033 mg/mL). The
reaction was incubated at room temperature for 24 h prior to analysis
of peptide fragments by MALDI-MS.
Acknowledgment. We gratefully acknowledge Dr. Wilna
Moree, Dr. Philip Dawson, and Dr. John Offer for helpful
discussions. We thank Dr. Ganga Beligere for providing a
sample of the His6-tagged peptide used in these studies. We
also thank Dr. Gary Siuzdak and the Scripps Mass Spectroscopy
Facility for the exact mass spectral analysis reported. Funding
for this research was obtained from the NSF (MCB-9812248)
and the National Institutes of Health (CA 58689 and GM
57464). S.J.B was supported by an NRSA Fellowship from the
National Institutes of Health.
Selective Labeling of LY-(SAOD)-AGCRANK-NH2 and LY-
(SAOD)-AGCEYRIDRVR-LFVDKLDNIAQVPRVGAA-HHHH-
HH with Tetramethylrhodamine N-Hydroxy-succinimidyl Ester. A
JA000217T