A fluorous capping strategy for Fmoc-based automated and manual solid-phase peptide synthesis

Article abstract of DOI:10.1002/ejoc.200500958

Just add water: Peptides synthesized by the use of standardized Fmoc protocols with commercial automated synthesizers can be purified from deletion products by simple centrifugation of aqueous solutions. The deletion products are capped with fluorous triv

Full text of DOI:10.1002/ejoc.200500958

SHORT COMMUNICATION
DOI: 10.1002/ejoc.200500958
A Fluorous Capping Strategy for Fmoc-Based Automated and Manual Solid-
Phase Peptide Synthesis
Vittorio Montanari[a] and Krishna Kumar*[a,b]
Keywords: Protein design / Peptide synthesis / Organic methodology / Fluorous chemistry / Automated peptide synthesis /
Protein engineering
Just add water: Peptides synthesized by the use of standard-
ized Fmoc protocols with commercial automated synthesizers
can be purified from deletion products by simple centrifuga-
tion of aqueous solutions. The deletion products are capped
with fluorous trivalent iodonium salts. At the end of the syn-
thesis, the crude peptide is dissolved in water and centri-
fuged, and the deletion products precipitate leaving only the
full length peptide in solution. Protocols for generalized use
of this strategy are reported.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2006)
Automated solid-phase synthesis has revolutionized the by use of fluorous technology comprised treating the solid
construction of oligonucleotides[1] and peptides.[2] Indeed, support with reagent 1, which caps all unconsumed residual
the availability of reasonably priced, “off-the-shelf” oligos terminal amines resulting from incomplete reaction that
and peptides has become the central pillar on which mod- may participate in further couplings steps, giving rise to un-
ern molecular and cell biology have thrived. Within these desired deletion products.[7] The fluoroalkyl tag (RfCH2–)
two classes of ubiquitous biopolymers, solid-phase oligonu- appended in this manner renders amines unreactive in all
cleotide synthesis is clearly more efficient, where optimized subsequent steps of peptide synthesis. At the termination of
protocols allow the preparation of ca. 100mers routinely. the synthesis, the crude peptide can then be purified by sim-
However, the relative diversity of chemical functionality in ple centrifugation or by fluorous flash chromatography,
amino acid side chains and protecting groups in peptides which results in the removal of all tagged deletions (Fig-
makes the synthetic effort more demanding, limiting the ure 1).
length to ca. 50 residues. Furthermore, accumulation of de-
The trivalent iodonium salts 1 and 2 are extremely ag-
letion products, especially those missing a single residue (n – gressive electrophiles that react rapidly and quantitatively
1 products), are frequent, thus complicating the purification with amines. Indeed, in the absence of a primary amine, the
of the desired full-length product.[3] Nevertheless, several hindered base (2,4,6-collidine), commonly employed under
strategies have been devised to solve this general problem our capping conditions to neutralize the acid [bis(trifluoro-
with varying outcomes in the ease of implementation. The methylsulfonyl)imide], is itself consumed in less than 3 min-
more promising of these approaches have been the intro- utes.[46] Capping of unreacted amines during the automated
duction of handles that provide an avenue for the facile re- synthesis can be troublesome as secondary structure forma-
moval of unwanted products, mainly those due to failed tion or resin occlusion of amines may make these terminal
couplings.[4–6]
reactive groups inaccessible. The potent reactivity of 1 and
We and others have demonstrated the use of fluorous 2 makes the reagents attractive for complete fluorous tag-
capping during solid-phase synthesis of peptides,[7–9] oligo- ging of recalcitrant amines, which fail to react during the
nucleotides,[10] and carbohydrates.[11–15] In addition, fluo- coupling step.
rous compounds and solvents have found use in protein de-
Peptide synthesis protocols have embraced two major
sign,[16–27] reaction acceleration,[28] catalysis,[29–34] combina- protecting group strategies – using either tert-butoxycar-
torial chemistry,[35] proteomics[36] and organic separation bonyl (tBoc) or 9-fluorenylmethoxycarbonyl (Fmoc) func-
methodology.[34,37–45] Our approach for peptide purification tionalities. In automated solid-phase peptide synthesis
(SPPS), Fmoc chemistry predominates, because the depro-
[a] Department of Chemistry, Tufts University,
tection step is performed with base (piperidine in DMF)
rather than with corrosive trifluoroacetic acid. Further-
more, in tBoc-based synthesis, final cleavage of the peptide
from the resin is frequently accomplished with neat anhy-
drous HF, which requires specialized equipment and safety
protocols. In a recent report, we have shown the versatility
Medford, MA 02155-5813, USA
[b] Cancer Center, Tufts-New England Medical Center,
Boston, MA 02110, USA
Fax: +1-617-627-3443
E-mail: krishna.kumar@tufts.edu
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
874
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2006, 874–877

A Fluorous Capping Strategy for Solid-Phase Peptide Synthesis
SHORT COMMUNICATION
Figure 1. Generalized fluorous capping strategy. Amino acids that fail to couple leave an unprotected terminal amino group that is capped
with reagent 1 (or 2) in standard peptide synthesis solvents. All products lacking one (or more) residues are therefore tagged with a
fluorous tag that is easily removed at the end of the synthesis by simple centrifugation or by fluorous flash chromatography.
of reagent 1 during manual tBoc-solid-phase peptide syn-
thesis.[7] This work was motivated by the need to make our
strategy general and establish the utility of trivalent iodon-
ium salts as robust capping reagents during routine Fmoc
synthesis.
Here, we describe two related reagents 1 and 2, that differ
only in the length of the perfluoroalkyl chains, that are equ-
ally efficient in automated Fmoc peptide synthesis. The cap-
ping protocol can be included as a programmable step in
commercially available synthesizers using standard solvents.
As a control experiment, the fluoroalkylated compound 3
(Figure 2) was stirred in piperidine/DMF (1:1) for 20 hours
at 20 °C and recovered in 91% yield after workup and
chromatography. Furthermore, when the fluoroalkylated ty-
rosine derivative 4 was treated with 4 equiv. of Fmoc--ala-
nine, 3.6 equiv. of HBTU as coupling agent and 6 equiv. of
DIEA in DMF for 45 min, it was recovered unchanged.
When 4 was treated with an excess of 2 under typical cap-
Figure 2. Sequences of peptides P1–P3; structures of reagents 1 and
2; and of model compounds 3 and 4. Sites of intentional incom-
plete couplings in P1–P3 are shown in bold and marked with aster-
isks.
ping conditions, it was again recovered intact. These prelim- identical conditions, and were either capped with Ac2O or
inary experiments established that the capping reagents and with fluorous capping reagent 2. To ensure identical levels
tagged products generated from them are compatible with of incomplete couplings, 20% of the resin by weight was
the solvents and reagents normally employed during Fmoc removed following deprotection of the Fmoc group, but
peptide synthesis. In particular, there were no problems prior to amino acid coupling. The incoming amino acid was
with elimination reactions which lead to reactive com- coupled to the remainder (80%) of the solid support. The
pounds that might result in undesired impurities.
resin was then washed, and the previously removed portion
We tested the capping reagents by both manual and lacking the terminal amino acid returned to the synthesis
automated Fmoc synthesis. Model peptides of length 10 vessels followed by the capping step. The capping step was
(P1), 14 (P2) and 10 (P3) residues, which are analogs of programmed as an additional step in the synthesizer, which
acyl carrier peptide 65–74, of insulin-like growth factor 28– involved prewashing of the resin (2×3 min DMF followed
41 and bombesin 5–14, respectively, were prepared, and in- by 2×3 min CH2Cl2), addition of 10 equiv. of reagent 1 or
complete couplings were intentionally introduced at se- 2 in CH2Cl2, followed by addition of 2,4,6-collidine in
lected positions (marked with asterisks and shown in bold CH2Cl2 which was stored separately. After 10 minutes of
in Figure 2). The peptides were prepared in tandem using shaking this step was repeated. After two washing cycles
Eur. J. Org. Chem. 2006, 874–877
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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875

V. Montanari, K. Kumar
SHORT COMMUNICATION
with DMF, standard cycles resumed. Peptides P1 and P2 which could be recovered by simple draining. The base col-
were synthesized in this manner using reagent 2 with cap- lidine was then added in a separate step. In this manner, the
ping carried out only at the sites of incomplete coupling. solution of iodonium salt could be recycled several times.
At the end of the synthesis and subsequent to cleavage and Because the salt and the base are employed in two consecu-
deprotection, crude peptides were simply dissolved in 1% tive steps, other bases besides collidine may in principle be
AcOH, centrifuged at 14000 rpm for 5 minutes and then an- used in this protocol. Successful capping and purification
alyzed by reversed-phase HPLC. Figure 3 shows that in by simple centrifugation gave Ͼ95% pure P3 (see support-
both cases, a dramatic improvement in the purity of the ing information; for supporting information see also the
crude peptide is observed. The fluorous-tagged deletion footnote on the first page of this article). We note that the
products tend to precipitate out of aqueous solutions, and reagents are cost-effective and competitive with Ac2O in
the supernatant contains mostly the desired product. We that each capping step adds ca. $ 6 to the overall materials
have noted previously, that for longer peptides passage cost resulting in substantial financial benefit in purification
through fluorous silica gel may sometimes be necessary[7] of peptides (Figure 4).
but for the shorter peptides employed here, mere centrifuga-
tion is enough to remove most unwanted products.
We note that nineteen of the twenty canonical amino ac-
ids are compatible with the capping protocol, however, the
Peptide P3 was synthesized by manual Fmoc synthesis relatively uncommon amino acid methionine is alkylated
with capping reagent 2. A new protocol was employed that rapidly by 1 or 2 to yield the corresponding sulfonium salt.
involved soaking the resin with the iodonium salt solution, Nevertheless, our protocol is still applicable to peptides
Figure 3. Analytical reverse-phase HPLC analysis of crude peptides after fluorous capping: (a) reverse-phase (Vydac C18) HPLC chroma-
togram of peptide P1 synthesized using an automated synthesizer with one incomplete coupling at position marked with an asterisk (also
shown in bold) as shown in Figure 2 and capping with Ac2O; (b) peptide P1 made under identical conditions as in (a) but with fluorous
capping reagent 2; (c) peptide P3 synthesized manually using Ac2O capping (see intentional deletions in Figure 2) and (d) using fluorous
capping reagent 2. Samples in (a)–(d) were dissolved in 1% AcOH and centrifuged at 14,000 rpm for 5 min and then the supernatant
injected. Chromatogram peaks marked with arrows are full length peptides and those marked with asterisks are unknown impurities. In
(c), peaks marked 1 and 2 are the acetyl capped products of increasing mass.
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Eur. J. Org. Chem. 2006, 874–877

A Fluorous Capping Strategy for Solid-Phase Peptide Synthesis
SHORT COMMUNICATION
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Figure 4. Photograph of precipitate resulting from centrifugation
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ported in part by National Institutes of Health Grants GM65500
and by National Science Foundation Grants CHE-0236846 and
CHE-0320783. K. K. is a DuPont Young Professor.
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Received: December 7, 2005
Published Online: January 3, 2006
Eur. J. Org. Chem. 2006, 874–877
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