Microreactor synthesis of β-peptides

Article abstract of DOI:10.1002/anie.200602167

Taming the insoluble: The reaction parameters of a β-peptide synthesis were optimized using only small amounts of reagents in a silicon microreactor (see picture). The use of unusually high temperatures (up to 120°C) reduces the reaction times. The insert

Full text of DOI:10.1002/anie.200602167

Communications
of b-peptides.[2,3] Owing to the formation of b-peptidic
secondary structures of short chains when synthesized on
Merrifield beads, the solid-phase assembly of b-peptides may
totally fail, especially when hair-pin turn structures can form.
In this case, one has to resort to fragment coupling.[2b,c]
Microreactors (i.e. miniaturized chemical reactors) are
attracting attention as an alternative to traditional synthesis in
round-bottom flasks.[4] However, in contrast to widely used
microanalysis systems, the development of microreactors as a
tool for organic synthesis has been slow. A range of chemical
transformations has been carried out by using microreactor
technologies, whereby most of these reactions only served as a
proof of principle.[5]
Herein we describe the first application of a silicon
continuous flow microreactor (Figure 1) to the assembly of
peptides. The microreactor not only allows for quick “scan-
ning” of reaction conditions, but also for the procurement of
synthetically useful amounts of peptides.[6] Our report is also
the first demonstration of 1) peptide couplings with tert-
butyloxycarbonyl (Boc)- and 9-fluorenylmethoxycarbonyl
(Fmoc)-protected amino acids in 1–5 minutes at temperatures
as high as 1208C, 2) the use of b2- and b3-homoamino acid
fluorides for b-peptide couplings, and 3) the advantageous
application of a C10H4F17-substituted benzylic ester protecting
group in solution-phase peptide synthesis.[7–9]
Microreactors
DOI: 10.1002/anie.200602167
Microreactor Synthesis of b-Peptides**
Microstructured reaction devices hold several advantages
for organic synthesis: only small amounts of valuable reagents
are required, reaction parameters, such as reaction time and
temperature, can be accurately controlled, and various
reaction conditions can be scanned in a continuous, time-
efficient fashion. The continuous-flow microreactor that we
used for our studies is designed to be compatible with a wide
range of organic solvents and can be operated over a broad
temperature range (À808C to +1508C) in a simple laboratory
setting (Figure 1).[10] The reaction volume of the microreactor
(78.3 mL) allows microscale reaction scanning (mmol of
reagents) and the production of several grams of target
material per day.
Oliver Flögel, Jeroen D. C. CodØe, Dieter Seebach, and
Peter H. Seeberger*
b-Amino acid oligomers (b-peptides) are a unique class of
peptides. In contrast to their natural a-amino acid counter-
parts, b-peptides require few residues to form secondary
structures such as turns, helices, or sheets.[1] As a result of
these structures and their metabolic stability, b-peptides have
attracted considerable interest. However, the secondary
structures can also severely hamper the synthesis of these
oligomers.[2] Small- to medium-length b-peptides are gener-
ally synthesized in solution[1b] or on a solid support,[1c] whereas
longer peptides can be prepared by chemical-ligation techni-
ques.[1e] Microwave-assisted solid-phase-peptide-synthesis
(SPPS) protocols have also been applied in the preparation
Initially, we explored the coupling of Fmoc-b3-homo-
phenylalanine fluoride (Fmoc-b3hPhe-F, 1) and H-b3hPhe-
OBn (2) in the microreactor (Scheme 1a). Acid fluorides
were selected as activated forms of the amino acids as 1) they
are readily available from the parent amino acids, 2) they are
powerful acylating agents, and 3) the use of a tertiary amine
base leads to an ammonium fluoride as the sole soluble by-
product.[11] The reaction was monitored at different temper-
atures (258C, 608C, and 908C) and different reaction times (1,
2, 5, and 10 min) by using Fmoc-b3hPhe-OBn (5) as the
internal standard for liquid chromatography LC-MS analysis.
Comparing the reaction progress at different temperatures
revealed that the maximum yield was obtained after
3 minutes at 908C (Scheme 1b). Interestingly, when the
coupling of 1 and 2 was conducted at a higher temperature
and/or for a prolonged period of time, the amount of
dipeptide diminished, as revealed in a second reaction scan
(Scheme 1c).[12] Under these conditions, tripeptide 4 was also,
not surprisingly, formed owing to Fmoc-cleavage and sub-
sequent peptide coupling with excess acid fluoride.[13] Follow-
ing reaction screening, dipeptide 3 was prepared on 0.5-mmol
[*] Dr. O. Flögel,[+] Dr. J. D. C. CodØe,[+] Prof. Dr. D. Seebach,
Prof. Dr. P. H. Seeberger
Laboratory ofOrganic Chemistry
Swiss Federal Institute ofTechnology (ETH) Zürich
Wolfgang-Pauli-Strasse 10, 8093 Zürich (Switzerland)
Fax: (+41)44-633-1235
E-mail: seeberger@org.chem.ethz.ch
[+] Both authors contributed equally to this work
[**] We thank the ETH Zürich, the Deutsche Forschungsgemeinschaft,
Swiss National Science Foundation, Novartis Pharma AG Basel, and
the Netherlands Organization for Scientific Research (NWO) for
financial support. Prof. Dr. K. F. Jensen and E. R. Murphy (Depart-
ment ofChemical Engineering, Massachusetts Institute ofTech-
nology, Cambridge) are gratefully acknowledged for providing the
microreactor used in this work.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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Scheme 2. Synthesis ofthe b3-dipeptides 7 and 9. 2-ClZ=2-chloroben-
zyloxycarbonyl.
Figure 1. a) Microreactor setup. b) Schematic representation ofthe
microreactor showing the three inlets (acid fluoride (A), amino acid
benzyl ester (B) and N-methylmorpholine (NMM; C)), the quench port
(TFA and the internal standard (D)), the outlet, and the dimensions of
the channels (total prequench reactor volume=78.3 mL).
the purification procedure by fluorous solid-phase extraction
(FSPE).[7,8,14] Thus, the assembly of the tetrapeptide 17
3
3
À
commenced with the construction of the first b b peptidic
bond by applying the previously established conditions. A
reaction time of 3 min at 908C provided the Boc-protected
dipeptide 12b in 91% yield after FSPE. Notably, the product
precipitated in the collection flask, which was kept at ambient
temperature, indicating the poor solubility of this class of
compound.[15]
scale (3 min reaction time at 908C, 92% yield after column
chromatography). Identical conditions were used for the
construction of the b3-dipeptides 7 (0.5-mmol scale) and 9
(0.3 mmol; Scheme 2).
After having established the utility of our microreactor
system for the synthesis of b3-dipeptides, we set out to
assemble tetrapeptide 17 by using the Boc strategy
(Scheme 3). The tetrapeptide contains all possible b-peptide
3
2
À
b
The formation of the sterically more demanding b
peptide bond required higher temperatures and/or longer
residence times as revealed by a reaction scan at 908C and
1208C. Maximum conversion was obtained at 1208C and
5 min reaction time[16] to give tripeptide 14b in 93% yield on a
3
3
3
2
2
3
3
À
À
À
bonds, (b b )-, (b b )-, and (b b ) as well as a b h(R)Ala-
b2h(R)Val turn-inducing segment.[1f,2b] A fluorous benzyl
group was incorporated in the first amino acid to facilitate
0.4-mmol scale.[17] The final b b coupling was executed at
2
3
À
Scheme 1. a) Synthesis ofdipeptide 3 in the microreactor. b) LC–MS analysis ofthe of rmation of 3 as a function of time and temperature.
c) LC–MS analysis ofthe of rmation of 3 and 4 at higher temperatures and longer reaction times. Bn=benzyl, TFAÀ =trifluoroacetate.
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1208C and a reaction scan revealed the
coupling to be complete after 1.5 min.
Tetramer 16b was obtained in 81% yield.
Cleavage of the fluorous tag and removal
of the benzyloxycarbonyl function pro-
vided the zwitterionic target compound 17.
To compare the microreactor method-
ology with traditional assembly strategies,
tetrapeptide 17 was also synthesized on a
solid support by using the Fmoc strategy
and
HATU–DIPEA-mediated
cou-
plings,[18] as well as in solution by using
conventional laboratory glassware. For the
solution-phase assembly, two different
strategies were investigated: in the first
approach we used the above-mentioned
fluorous tag in combination with acid
fluorides, in the second approach we
started from H-b3hPhe-OBn and used
HATU–DIPEA coupling conditions. The
results of the four assembly methods are
collected in Table 1. All procedures pro-
vided the tetramer in comparable overall
yields. However, purification of the fluo-
rous peptides was much easier when com-
pared with the purification of the non-
fluorous peptides. The nonfluorinated
tetramer 16a was particularly difficult to
handle owing to its poor solubility. In
addition, the microreactor system allowed
for precise control over unconventionally
high reaction temperatures. This control
significantly reduces reaction times and
prevents product precipitation during the
reaction. Precipitation led to inhomoge-
neous gel-like reaction mixtures when
using traditional laboratory glassware in
the solution-phase synthesis.
Scheme 3. a) Synthesis ofthe tetrapeptides 16 and 17. b) Formation of 14b at different temperatures.
c) Formation of 16b at 1208C. Cbz=benzyloxycarbonyl, DCM=dichloromethane.
Table 1: Synthesis ofthe tetrapeptides 16a/b and 17 by microreactor technology, by solution-phase couplings, and by solid-phase assembly.[19]
Method
12a/b
14a/b
16a/b
17
Conditions (Yield)
Conditions (Yield)
Conditions (Yield)
Yield
microreactor
11b (2 equiv); NMM (4 equiv),
908C, 3 min
(91%)
13b (2 equiv); NMM (4 equiv),
1208C, 5 min
(93%)
15b (2 equiv); NMM (4 equiv),
1208C, 1.5 min
(81%)
>99%
>99%
79%
solution
phase, F tag
11b (2 equiv); NMM (4 equiv),
RT, 3 h.
(94%)
13b (2 equiv); NMM (4 equiv),
RT, overnight
(93%)
15b (2 equiv); NMM (4 equiv),
RT, overnight
(93%)
solution
phase[a]
11a (2 equiv); HATU (1.8 equiv),
NMM (4 equiv),
RT, overnight
13a (2 equiv); HATU (1.8 equiv),
NMM (4 equiv),
RT, overnight
15a (2 equiv); HATU (1.8 equiv),
NMM (4 equiv)
RT, overnight
(85%)
(85%)
(87%)
solid phase[b] Fmoc-11a (3 equiv); HATU (2.8 equiv), Fmoc-13a (3 equiv); HATU
Fmoc-15a (3 equiv); HATU
(2.8 equiv), DIPEA (6 equiv)
RT, 1 h
55% (after
HPLC)[20]
(Wang Resin) DIPEA (6 equiv)
RT, 1.5 h
(2.8 equiv), DIPEA (6 equiv)
RT, 1 h
[a] HATU=2-(1H-7-azybenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate. [b] DIPEA=diisopropylethylamine.
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[8] For fluorous tagging in solid-phase a-peptide syntheses, see:
In summary, we have demonstrated the use of a silicon-
based microreactor for the effective synthesis of peptides. The
microstructured reaction device allowed for the detailed
investigation and optimization of reaction parameters by
using minimal amounts of reagents. Furthermore, it opened
the way for the use of unprecedented high reaction temper-
atures, leading to homogeneous reaction mixtures and of
significantly reduced reaction times. Synthesis efficiency was
further enhanced by the use of a fluorous benzyl tag that was
applied for the first time in the assembly of b-peptides and
proved to be particularly useful for the purification of poorly
soluble products.[8] Not only will the synthetic strategy
outlined herein find applications in the construction of
challenging peptides, it will also be applied to the assembly
of other biopolymers, including oligosaccharides and oligo-
nucleotides. In general, the continuous-flow process opens
many opportunities for multistep syntheses and automa-
tion.[21]
a) D. V. Filippov, D. J. van Zoelen, S. P. Oldfield, G. A. van der
Marel, H. S. Overkleeft, J. W. Drijfhout, J. H. van Boom, Tetra-
hedron Lett. 2002, 43, 7809 – 7812; b) P. C. de Visser, M. van
Helden, D. V. Filippov, G. A. van der Marel, J. W. Drijfhout,
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2003, 44, 9013 – 9016.
[9] For a-peptide synthesis on a fluorous support, see: M. Mizuno,
K. Goto, T. Miura, T. Matsuura, T. Inazu, Tetrahedron Lett. 2004,
45, 3425 – 3428.
[10] D. M. Ratner, E. R. Murphy, M. Jhunjhunwala, D. A. Snyder,
K. F. Jensen, P. H. Seeberger, Chem. Commun. 2005, 5, 578 – 580.
[11] L. A. Carpino, M. Beyermann, H. Wenschuh, M. Bienert, Acc.
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[12] Longer reaction times (30 min) led to an irregular flow as judged
from the large error margin in the relative LC–MS UV
absorbances of the products and the internal standard.
[13] Tetramers were also observed (data not shown).
[14] a) D. P. Curran, Synlett 2001, 1488 – 1496; b) W. Zhang, Chem.
Rev. 2004, 104, 2531 – 2556.
[15] The poor solubility dictated the use of a relatively large amount
of DMF (30 mgmLÀ1) to apply it to the fluorous silica column
(Fluorchrom, 20 g). The large amount of DMF did not adversely
affect the purification. Also see the Supporting information.
[16] The low solubility of the product prohibited lower temperatures
and longer reaction times in the microreactor.
[17] a) For this condensation, a ca. 93:7 mixture of enantiomers (R)-
13b/(S)-13b was used, see [17b]. LC–MS analysis of the product
revealed the same diastereomer ratio in the product, indicating
that no epimerization had occurred under these reaction
conditions. A more detailed epimerization study is currently
underway. b) For the synthesis of amino acid fluoride 13, a 93:7
diasteromeric mixture of Boc-b2-homovaline (R/S = 93:7) was
used: T. Hinterman; D. Seebach, Helv. Chim. Acta 1998, 81,
2093.
[18] Synthesis of Peptides and Peptidomimetics (Houben-Weyl Meth-
ods of Organic Synthesis), Vol. E22a (Hrsg.: M. Goodman, A.
Felix, L. Moroder, C. Toniolo), Georg Thieme, Stuttgart, 2002,
pp. 665 – 877.
Received: May 31, 2006
Published online: September 29, 2006
Keywords: b-peptides · fluorous tags · microreactors ·
.
synthetic methods
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[19] For detailed experimental conditions, see the Supporting
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