Just add water: A new fluorous capping reagent for facile purification of peptides synthesized on the solid phase

Article abstract of DOI:10.1021/ja0479033

A novel fluorous capping reagent is introduced to facilitate purification during solid-phase peptide synthesis (SPPS). Reagent 1 is a trivalent iodonium salt that reacts vigorously with free amines to deliver a long-chain fluoroalkyl group. It has been us

Full text of DOI:10.1021/ja0479033

Published on Web 07/14/2004
Just Add Water: A New Fluorous Capping Reagent for Facile Purification of
Peptides Synthesized on the Solid Phase
Vittorio Montanari and Krishna Kumar*
Department of Chemistry, Tufts UniVersity, Medford, Massachusetts 02155
Received April 12, 2004; E-mail: krishna.kumar@tufts.edu
Scheme 1. Reactivity and Structure of Fluorous Capping Reagent
1, Model Compounds (2 and 3), and Sequences of Peptides P1,
P2, and P3
Solid-phase peptide synthesis (SPPS) has become a mainstay
method for the synthesis of small- to medium-length peptides and
proteins.1 With the advent of native chemical ligation methods, the
lengths achievable by SPPS have been significantly extended.2-6
However, the tedious aspect of SPPS remains the removal of shorter
peptide products, resulting from the failure of one or more of the
chain extension steps (deletion sequences), from the desired full-
length product. To facilitate purification, various new reagents have
been developed with the intention of tagging deletion sequences
or, alternatively, the full-length product, so that they may have
different elution profiles during chromatographic purification.
Several purification handles have been described that take advantage
of selective conditions under which retention time on ion-exchange,
hydrophobic interaction, affinity, and other types of chromato-
graphic columns could be varied.7-12 Still, sequences short of just
one residue (n - 1 products) are notoriously difficult to separate.
Recently, fluorous compounds have been employed in protein
design,13-21 reaction acceleration,22,23 catalysis,24-26 combinatorial
chemistry, and organic separation methodology.27-29 Among the
canonical amino acid side chain functionalities, none are expected
to extensively interact with fluorous chromatographic materials.
Thus, an appendage that is highly fluorinated should be unique in
its physical properties, compared to most peptide products.30 In this
report, we introduce a versatile new fluorous reagent that is useful
as a handle for the efficient removal of deletion products formed
during SPPS.
Automated peptide synthesizers typically use a capping step to
terminate chains resulting from incomplete coupling steps. One
approach is to make the capping reagent orthogonal in its properties
to aid purification. The following design elements were central to
our development of a new capping reagent. First, the reagent should
be aggressive in reactivity toward free amines of R-amino acids to
deliver a fluoroalkyl chain. Furthermore, the reactivity of the amine
must be eliminated with respect to further peptide coupling steps.
In addition, the new tag should be stable to subsequent peptide
coupling reactions, the deprotection steps during synthesis, and the
final cleavage of the peptide chain from the resin. We report here
that trivalent iodonium compound 1 possesses all these relevant
properties and functions as a useful tag for t-Boc-based SPPS.31 It
is compatible with the solvent systems of peptide synthesis and
efficient at tagging free amines with an Rf (C7F15-) group. Once
free amines have been alkylated in this manner, they are unreactive
in further peptide coupling steps and stable to both deprotection in
neat trifluoroacetic acid and to the final cleavage step in anhydrous
HF.
tyrosine 2, 2 equiv of compound 1 in the presence of collidine/
CH2Cl2 resulted in quantitative monoalkylation of the amino group
in 10 min or less. The purified alkylated product, when treated with
reagent 1 for an additional 30 min under identical conditions, did
not react further. The deactivated amine is also inert to peptide
coupling conditions. Upon reaction with 4 equiv of Boc-L-alanine,
3.6 equiv of HBTU as a coupling reagent, and 6 equiv of
diisopropylethylamine in DMF for 30 min, the monoalkylated
product 3 was recovered unchanged. In summary, the monofluo-
roalkylated R-amino acids survive all reactions that they would be
subject to during peptide synthesis using t-Boc chemistry.
To demonstrate the versatility and utility of reagent 1, we
employed it in capping amines from intentionally incomplete
couplings during SPPS. We used this approach on peptides of length
10 (P1), 21 (P2), and 14 residues (P3), which are variants of the
acyl carrier peptide 65-74, adrenocorticotropic hormone 18-39,
and insulin-like growth factor 28-41, respectively (Scheme 1).
Peptides were synthesized using the in situ neutralization protocol
using t-Boc chemistry on MBHA (methylbenzhydrylamine) resin.36
To ensure incomplete coupling at selected sites, we used 0.8 equiv
of amino acid and 0.72 equiv of HBTU and ran the coupling step
for 20 min. This was followed by a 1 min flow wash with DMF.
The resin was then washed thoroughly with CH2Cl2 using a
combination of shake and flow washes. The capping step was
performed with a solution containing 2 equiv of 1 in CH2Cl2 with
collidine as a base for 15 min and was repeated once with 1 equiv
of 1. The resin was thoroughly washed with CH2Cl2, and the cycle
of deprotection and coupling steps was then resumed in a normal
manner. Capping steps were performed only at sites where couplings
were incomplete.
The trivalent iodonium compound 1 has reactivity analogous to
earlier reagents that were mainly employed in a two-phase organic-
water solvent system with sodium bicarbonate as the base.32-34
Compound 1 is a fluorous trivalent iodonium salt, and its reactivity
has been fine-tuned to be compatible with peptide synthesis.35 In
model reactions, for example with the tert-butyl carboxyl ester of
The capped peptides were removed in one of two ways. In the
case of the smaller peptide P1, the crude product obtained from
the cleavage reaction was simply dissolved in 1% AcOH solution
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J. AM. CHEM. SOC. 2004, 126, 9528-9529
10.1021/ja0479033 CCC: $27.50 © 2004 American Chemical Society

C O M M U N I C A T I O N S
Studies along these lines are currently being pursued in our
laboratories and will be reported shortly.
Acknowledgment. This work was supported in part by NIH
(GM65500) and NSF (CHE-0236846 and CHE-0320783). K.K. is
a DuPont Young Professor. We thank A. Kennan for help with
ESI-MS experiments and M. d’Alarcao for helpful discussions.
Supporting Information Available: Experimental procedures and
accompanying analytical data (PDF). This material is available free of
charge via the Internet at http://pubs.acs.org.
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Figure 1. Fluorous tagging-aided purification. Counterclockwise from top
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