Enzymatic synthesis of peptides on a solid support

Article abstract of DOI:10.1039/b816847d

We have previously shown that dipeptides can be synthesised in high yields from amino acids using protease catalysis in aqueous media, if the amino component is immobilised on porous PEGA resin (a copolymer of polyethylene glycol and polyacrylamide). Here

Full text of DOI:10.1039/b816847d

View Article Online / Journal Homepage / Table of Contents for this issue
PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Enzymatic synthesis of peptides on a solid support
Rose Haddoub, Martin Dauner, Fiona A. Stefanowicz, Valeria Barattini, Nicolas Laurent and Sabine L. Flitsch*
Received 25th September 2008, Accepted 5th November 2008
First published as an Advance Article on the web 9th December 2008
DOI: 10.1039/b816847d
We have previously shown that dipeptides can be synthesised in high yields from amino acids using
protease catalysis in aqueous media, if the amino component is immobilised on porous PEGA resin (a
copolymer of polyethylene glycol and polyacrylamide). Here we explore the scope of this methodology
for using protected and glycosylated amino acids as well as the synthesis of longer peptides on resin and
show that such a method can also be applied on non-porous surfaces, in particular on gold.
variety of peptide synthesis reactions from acyl donors with
1. Introduction
different functionalities.6 Thus, thermolysin was found to catalyse
Proteases represent a large class of enzymes involved in many
peptide-bond formation between hydrophobic Fmoc-protected
physiological functions and are important therapeutic targets1
for various diseases as well as being used as catalysts for
amino acids and a phenylalanine immobilised on PEGA1900
(poly-(acrylamide)-ethylene glycol) resin via the Wang (HMPA,
biotechnological applications.2 In water, the equilibrium of amide
bond formation and hydrolysis (Fig. 1) is generally shifted towards
hydrolysis, and thus proteases naturally hydrolyse amide bonds.
However, under low water conditions the equilibrium can shift
towards amide synthesis and proteases have shown to be effective
catalysts for peptide bond formation in a highly stereospecific
manner under mild conditions and without need for activation.3
We have shown previously that such equilibrium shift is also
observed when using a resin-bound amino substrate and a soluble
acyl donor even if the reaction is conducted in bulk aqueous
solution (Fig. 1).4
Thermolysin is a protease widely employed for peptide synthesis
and is well-known for the industrial synthesis of aspartame.5 While
the specificities and selectivities of the proteases usually limit
their synthetic application, thermolysin with its broad specificity
for carboxylic acid substrate can be employed to catalyse a
hydroxymethylphenoxyacetyl) linker in bulk aqueous media.4a The
aim of the present study was to apply these findings to the synthesis
of larger peptides and to investigate which components can be used
as soluble substrates to create a library of peptides with varying
size and functionalities.
2. Results and discussion
Enzymatic synthesis on porous support
Our initial studies reported the formation of dipeptide sequences
using the protease thermolysin from Bacillus thermoproteolyticus
Rokko.4a,b Yields for hydrophobic amino acid couplings (R1 in
Fig. 1) were generally high, whereas lower yields were observed
for polar and charged amino acids. From these studies the
question arose whether this enzymatic method would be limited to
hydrophobic dipeptides, or whether it could be applied to a wider
range of amino acids and to the synthesis of longer peptides.
We sought to overcome the low yields obtained with polar and
charged amino acids by using hydrophobic protecting groups,
Manchester Interdisciplinary Biocentre, The University of Manchester and
School of Chemistry, 131 Princess Street, Manchester, UK M1 7DN.
E-mail: sabine.flitsch@manchester.ac.uk
Fig. 1 Protease-catalysed synthesis/hydrolysis of dipeptides in solution and on resin. Whereas in aqueous solution hydrolysis is favoured, the reaction
equilibrium on PEGA resin favours dipeptide synthesis (Pg: protecting group).
This journal is
The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 665–670 | 665
©

View Article Online
Table 1 Yields of dipeptides obtained by thermolysin catalysed bond
formation on Leu-HMPA-PEGA as shown in Fig. 2
Table 2 Thermolysin-catalysed coupling of N-protected di and tripep-
tides to Leu-HMPA-PEGA
Entry
Acyl donor
% Fmoc-AA-Leua
Entry
Acyl donor
Product (ratio)
1
2
3
4
5
6
7
8
9
Fmoc-Gly
Fmoc-Leu
Fmoc-Phe
Fmoc-Trp
Fmoc-Ala
Fmoc-Pro
Fmoc-Met
95
96
94
96
52
13
22
8
38
95
98
4
71
65
29
14
22
91
85
35
76
33
25
43
<1
49
5
1
2
3
4
FmocPhePhe
FmocTyrAla
FmocPheAsp
FmocPheAsp(tBu)
FmocPhePheLeu (4.5)
FmocPheLeu (95.5)
FmocTyrAlaLeu (25)
FmocTyrLeu (75)
FmocPheAspLeu (21)
FmocPheLeu (79)
FmocPheAsp(tBu)Leu (18)
FmocPheLeu (82)
FmocPheSerLeu
FmocPheSer(Bn)Leu (21)
FmocPheLeu (79)
FmocPheCys(Bn)Leu (77)
FmocPheLeu (23)
FmocPheLeu (traces)
FmocGlyGlyLeu (90)
FmocGlyLeu (10)
FmocPheGlyLeu (37)
FmocPheLeu (63)
ZAlaTyrLeu
Fmoc-Cys(Trt)
Fmoc-Cys(tBu)
Fmoc-Tyr
Fmoc-Tyr(tBu)
Fmoc-Ser
Fmoc-Ser(tBu)
Fmoc-Ser(Bn)
Fmoc-Thr
Fmoc-Thr(Bn)
Fmoc-Asp
Fmoc-Asp(tBu)
Fmoc-Asp(Bn)
Fmoc-Glu
Fmoc-Glu(tBu)
Fmoc-Asn
Fmoc-Asn(Trt)
Fmoc-Gln
Fmoc-Arg
Fmoc-Arg(Pbf)
Fmoc-His
Fmoc-His(Trt)
Fmoc-His(Bn)
Fmoc-Lys
Fmoc-Lys(Boc)
Fmoc-Lys(Mtt)
Fmoc-Ser(a-D-Man(OH)4)
Fmoc-Ser(a-D-Man(OAc)4)
Fmoc-Ser(b-D-Gal(OAc)4)
5
6
FmocPheSer
FmocPheSer(Bn)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
7
FmocPheCys(Bn)
8
9
FmocPheArg
FmocGlyGly
10
FmocPheGly
11
12
13
14
15
16
17
ZAlaTyr
ZGluTyr
ZLeuTyr
ZPhePhe
ZLeuLeu
ZTrpLeu
FmocGlyGlyGly
ZGluTyrLeu
ZLeuTyrLeu
Library
Library
Library
FmocGlyGlyGlyLeu (22)
FmocGlyLeu (88)
FmocPhePhePheLeu (traces)
FmocPheLeu
FmocLeuTyrPheLeu (58)
FmocLeuPheLeu (20)
FmocLeuTyrLeu (11)
FmocLeuTyrPhePheLeu (11)
FmocPheLeuOH (92)
FmocPhePheLeuOH (8)
18
19
FmocPhePhePhe
FmocLeuTyrPhe
33
41
0
96
4
0
78
70
20
FmocPheLeuPhe
a Yields determined by LC-MS.
31) and Fmoc-Lys(Mtt) (entry 32) suggested that apart from the
hydrophobicity, the size of the protecting group may also have a
considerable effect on the coupling efficiency. Comparable results
to those presented in Table 1 were obtained using an immobilised
phenylalanine instead of leucine as the C-terminal amino acid
(data not shown).
similar to those employed in chemical solid phase peptide synthesis
(SPPS). To evaluate the effect of protecting groups, experiments
were performed by adding a wide range of Fmoc-protected amino
acids having either a protected or non-protected side-chain residue
(Fig. 2) to leucine anchored via a Wang linker to PEGA resin (Leu-
HMPA-PEGA). The reactions were carried out in a suspension
of 0.025 mmol Fmoc-amino acid in 1 mL potassium phosphate
buffer (KPi) 0.1 M at pH 7.5 with 2 mg of thermolysin and
5 mg of resin-bound leucine (0.5 mmol). Yields were determined
using LC-MS after cleavage from the linker by integrating the UV
traces at 301 nm.4a,4b As expected, the best yields were obtained
with hydrophobic amino acids, which gave nearly quantitative
coupling in agreement with previous results4a (Table 1, entries
1–6, 10). Hydrophilic amino acids gave very low yields (Table 1,
entries 12, 15, 17, 20, 22, 24, 25, 27, 30), whereas their side-chain
protected counterparts resulted in increased product formation.
Comparison of the yields obtained with Fmoc-Lys(Boc) (entry
Our group has
a strong interest in the synthesis of
glycopeptides.7 To explore the breadth of the present methodology,
we therefore tested N-Fmoc protected glycosylated amino acids,
such as galactosylated and mannosylated serine. Interestingly,
glycosyl amino acid with an unprotected glycosyl moiety (entry 33)
resulted in no detectable amount of coupling product. However,
using per-acetylated mannosyl or galactosyl serine, high yields
were obtained (entries 34–35), therefore showing that thermolysin
was able to tolerate highly diverse structural modifications of the
acyl donors.
Given that thermolysin is an endopeptidase, the amide coupling
reaction described here was tested for the coupling of peptide
fragments, adding di- and tripeptides instead of single amino
acids as acyl donors to the reaction (Fig. 3). For example when
Fig. 2 Thermolysin catalysed formation of dipeptides on resin.
666 | Org. Biomol. Chem., 2009, 7, 665–670
Fig. 3 Thermolysin catalysed coupling of dipeptides.
This journal is
The Royal Society of Chemistry 2009
©

View Article Online
FmocPheSer was mixed with thermolysin and Leu-HMPA-
PEGA, LC-MS analysis of the product showed that the enzyme
was able to achieve coupling to yield the corresponding tripep-
tide FmocPheSerLeu (Table 2, entry 5). However, when Fmoc-
protected dipeptides that are potential substrates for thermolysin
were chosen as acyl donors, mixtures of di- and tripeptides were
formed (Table 2). These dipeptide side-products appeared to be
formed from Fmoc-protected amino acids released from the dipep-
tide substrate in solution followed by coupling to the solid phase
substrate. Overall, the yields of coupling were moderate (30–40%).
Similarly, Z-protected dipeptides 11–13 yielded pure tripeptides,
whereas 14–16 were more prompt to hydrolysis and thus, libraries
of various size peptides were obtained. When even longer peptides
were investigated as acyl donors, mixtures of peptides were
obtained, which appeared to be the results of a series of hydroly-
sis/synthesis steps leading to some degree of scrambling of peptide
sequence (Table 2, entries 17–20). The best result of tetrapeptide
was obtained when using FmocLeuTyrPhe as substrate (Table 2,
entry 19).
An alternative way of generating larger peptides on the solid
phase using this biocatalytic method is by stepwise elongation
using a series of repeated coupling and Fmoc-deprotection steps
similar to a chemical SPPS protocol.8 The advantage of such a
method is that coupling is mediated by biocatalysis and thus no
activation of amino acid is needed. Furthermore, when using
a racemic mixture of D/L amino acid, selective formation of
L/L dipeptide can be achieved by thermolysin.8 This approach
might be particularly useful for non-natural L-amino acids that
are not readily available because the resolution of enantiomers
from racemic mixtures and the coupling is achieved in one step.
First, the most simple case of synthesis of polyleucine peptides
was studied by repeated cycles of enzymatic coupling at 30 ◦C and
chemical Fmoc-deprotection (Fig. 4). Each step was monitored by
LC-MS and it was found that clean coupling could be achieved up
to the tetramer Fmoc-Leu4-HMPA-PEGA (Fig. 4A) without any
visible side-products. Beyond tetraleucine, hydrolysis of peptide
started to compete with synthesis and a mixture of products were
obtained. The maximal achievable length was Fmoc-Leu6-HMPA-
PEGA, which was part of a mixture comprising 3 to 6 Leu residues
(Fig. 4B). For other amino acids such as poly-Phe and poly-
Tyr, formation of mixtures occurred already during the second
coupling step towards Fmoc-Phe3-HMPA-PEGA and Fmoc-Tyr3-
HMPA-PEGA respectively. The Fmoc tetrapeptides Phe4 and
Tyr4 were the maximal achievable length. It was also found
that the library distribution was dependent on the temperature
of enzymatic reaction, as mixtures of poly-Leu peptides were
obtained already during the third coupling step when performed
at higher temperatures of 37 ◦C. On the contrary, synthesis of
a PheLeuPheLeu tetrapeptide (with alternating Phe and Leu
residues) gave only traces of shorter peptides. Therefore, it is
likely that the smaller peptides arise from hydrolysis of the
resin bound peptide by thermolysin, and that hydrolysis appears
to be dependent on the amino acid sequence of the substrate.
These results also suggest that the enzyme-catalysed peptide
synthesis and hydrolysis reactions on the solid phase are reversible
and distribution of the peptide libraries depends on reaction
conditions. Further studies probing this reversibility are currently
under way.
Enzymatic synthesis on gold surfaces
A disadvantage of using porous polymer beads such as PEGA or
Tentagel is poor enzyme diffusion into the bead which limits their
application to proteins of lower than average molecular weight.9
Fig. 4 A: Stepwise elongation of peptides on solid phase. B: LC trace of a poly-leucine library.
The Royal Society of Chemistry 2009 Org. Biomol. Chem., 2009, 7, 665–670 | 667
This journal is
©

View Article Online
Fig. 5 Thermolysin catalysed amino acid transfer on a gold surface. Example of MALDI-ToF MS spectra before (top) and after (bottom) incubation
of an immobilised Phe with thermolysin and FmocLeu. m/z 1269 is the sodium adduct of the mixed disulfide formed between the hydroxyl-terminated
and the Phe-terminated alkanethiols. m/z 1620 is the potassium adduct of the product detected as a mixed disulfide.
Table 3 m/z detected after incubation of immobilized Phe with a
mixture of thermolysin and Fmoc amino acid (for clarity only
masses of the sodium and potassium adducts of the mixed disulfide
formed between the peptide and the tri(ethyleneglycol) alkanethiols are
shown)
Very recently, we have investigated the use of self-assembled mono-
layers (SAMs) on gold surfaces and have generally found excellent
enzyme accessibility.10 These surfaces are easy to prepare and
monitoring of enzymatic reactions can be achieved using label-free
analytical techniques such as MALDI-ToF MS (matrix-assisted
laser desorption ionization/time-of flight mass spectrometry).11
Noteworthy, analysis can be performed on-chip, therefore ruling
out the need for cleavage from the support. Therefore, the ability
of thermolysin to catalyse amino acid transfer on gold surfaces
was investigated (Fig. 5).
Entry Fmoc amino acid added
Product
m/z detected
1
2
3
FmocPhe
FmocLeu
FmocAla
FmocPhePhe
FmocLeuPhe
Phe
FmocAlaPhe
Phe
1654 (M + K)
1620 (M + K)
1285 (M + K)
1575 (M + K)
1269 (M + Na)
1269 (M + Na)
4
5
FmocGly
FmocSer(tBu)
To this end, Fmoc phenylalanine was immobilised onto a
disposable 64-well gold slide (Applied Biosystems) coated with
a 1 : 1 mixture of alkanethiols terminated either by a primary
amine or by a non-reactive hydroxyl group (providing a control of
the surface density by mixing the two linkers at different ratios).10b
After Fmoc-deprotection, the array was incubated with a mixture
of thermolysin and a saturated solution of Fmoc-amino acid in
Phe
6
FmocLys(Boc)
7
8
FmocAsp
FmocGlu(tBu)
FmocGlu(tBu)Phe 1676 (M + Na)
Phe 1269 (M + Na)
FmocTyr(tBu)Phe 1710 (M + Na)
9
FmocTyr(tBu)
◦
KPi buffer at 37 C. After 16 h, MALDI-ToF MS interrogation
10
11
FmocTyr
FmocGln(trt)
Phe
1285 (M + K)
1285 (M + K)
1861 (M + Na)
1877 (M + K)
of the array revealed that thermolysin was able to catalyse the
coupling of these amino acids to the immobilised Phe (Table 3). For
example, coupling of Fmoc-Leu (Fig. 5) resulted in the complete
disappearance of the starting material and formation of a single
species corresponding to the addition of the Fmoc-Leu. Other
Fmoc amino acids were tested, and MS data are shown in Table 3.
Overall, these results were in good agreement with those
obtained using PEGA-anchored peptides and demonstrated that
themolysin was able to react on a planar surface where surface
density can be tailored in order to maximize the enzyme’s
accessibility. Furthermore, parallel screening can be performed on
a single chip, and the use of MALDI-ToF MS analysis provided
a straightforward insight into the determination of thermolysin
activity.
Phe
FmocGln(trt)Phe
a wide range of peptides particularly by using side-chain protected
amino acids. The structure of such peptides is currently limited
by thermolysin specificity, and studies on using complementary
proteases are on the way. In a number of examples studied
here, mixtures of products were obtained, which appeared to
be the results of consecutive hydrolysis and synthesis steps,
suggesting a reversibility under the reaction conditions. Finally,
we have shown that the shift of equilibrium observed initially
only for PEGA resin, can also occur on other surfaces, such
as SAMs on gold. Such surfaces are increasingly used in solid
phase biocatalysis and should broaden further the scope of this
methodology.
3
Conclusions
In summary, these results have shown that the initial coupling
reaction of two natural amino acids can be broadened to generate
668 | Org. Biomol. Chem., 2009, 7, 665–670
This journal is
The Royal Society of Chemistry 2009
©

View Article Online
slide was dipped into a 20% solution of piperidine in DMF for
10 min to ensure Fmoc removal, then rinsed and dried as above.
10 mg of Fmoc phenylalanine were dissolved in 0.25 mL of a
solution containing 52 mg/mL of PyBOP (benzotriazol-1-yl-
oxy-tris-pyrrolidino-phosphonium hexafluorophosphate) and
36 mL/mL of DIPEA (diisopropylethylamine) in dry DMF and
allowed to react for 5 min at room temperature. 0.4 mL of the
above solution was then spotted in each well of the gold slide,
and the slide was then placed at 37 ◦C in a sealed container. After
1 h, the surface was rinsed with DMF, ethanol and dried under a
stream of nitrogen. Terminal Fmoc removal was carried out as
above.
4
Experimental details
General methods
Liquid chromatography/mass spectrometry was performed on
an Agilent 1100 Series LC system, equipped with a 1100 series
MSD with a multimode ion source. Samples were analysed with a
Phenomenex Lunaꢀ 5 m C18 column (250 ¥ 2 mm) and a 0–85%
MeCN (0.1% TFA)/H2O (0.1% TFA) gradient with flow rate of
0.5 ml/min. The UV trace was recorded at 210 nm, 254 nm, 280 nm
and 301 nm. Data were processed with the Agilent Chemstation
Software.
R
Attachment of the first amino acid to HMPA-PEGA
Thermolysin-catalysed peptide-bond formation on gold
Fmoc protected amino acid (10 equivalents to resin) was dissolved
in dry DCM, DIC (diisopropylcarbodimide) (5 equivalents to
resin) was added dropwise and the solution was stirred under
nitrogen atmosphere at RT for 20 min. DCM was evaporated
and a small volume of dry DMF was added. The resulting
solution was added to HMPA-derivatized PEGA resin to form
a gel. DMPA (dimethylaminopyridine) (0.1 eq) was dissolved
in a minimum volume of dry DMF and added to the reaction
mixture, which was then agitated at RT for 2 h. The resin
was then filtered and extensively washed. The loading densities
were determined by Fmoc cleavage and were typically 0.06–
0.07 mmol/g.
Each well of the slide was incubated overnight at 37 ◦C with a
saturated solution of Fmoc amino acid (about 10 mg/mL) and
thermolysin (2 mg/mL) in KPi buffer 0.1 M with a pH 7.5. The
slide was then thoroughly rinsed with deionised water, DMF, and
ethanol and dried under a stream of nitrogen.
MALDI-ToF MS analysis
Each well of the surface was coated with a solution of THAP
(2,4,6-trihydroxyacetophenone, 10 mg/mL in acetone), and the
target was loaded into a Voyager-DE STR Biospectrometry
MALDI-ToF mass spectrometer (PerSeptive Biosystems) operat-
ing with a 337 nm nitrogen laser. Mass spectra were acquired using
reflector ToF, positive ion mode using an accelerating voltage of
20 kV and an extraction delay of 200 ns.
Thermolysin-catalysed peptide-bond formation on PEGA
5 mg Leu-HMPA-PEGA were treated with 0.025 mmol of the
acyl donor in the presence of 2 mg thermolysin in 1 mL 0.1 M
KPi buffer with pH = 7.5. After 16 h the resin was washed and the
reaction products were cleaved from the resin with 95% TFA/H2O
for 2 h. Analysis and quantification of reaction products were
achieved by LC-MS.
Acknowledgements
The authors would like to acknowledge funding from the EC,
BBSRC, Wellcome Trust and the Royal Society. Thanks are due
to DSM (Holland) for the generous gift of thermolysin.
Preparation of the amine-terminated self-assembled monolayers on
gold and immobilization of phenylalanine
A
disposable 64-well gold surface (Applied Biosystems)
was cleaned with a 5 : 1 solution of concentrated sulfuric
acid and hydrogen peroxide 35% (caution: very oxidizing
agent) and thoroughly rinsed with deionised water, ethanol
References
1 D. Leung, G. Abbenante and D. P. Fairlie, J. Med. Chem., 2000, 43,
305.
2 F. Bordusa, Chem. Rev., 2002, 102, 4817.
and dried under
a stream of nitrogen. Self-assembled
3 (a) M. Erbeldinger, X. Ni and P. J. Halling, Enzyme Microb. Technol.,
1998, 23, 141; (b) R. V. U. lijn, A. E. M. Janssen, B. D. Moore and P. J.
Halling, Chem.–Eur. J., 2001, 7, 2089; N. Laurent, R. Haddoub and
S. L. Flitsch, Trends Biotech., 2008, 26, 328.
4 (a) R. V. Ulijn, B. Baragana, P. J. Halling and S. L. Flitsch, J. Am.
Chem. Soc., 2002, 124, 10988; (b) R. V. Ulijn, N. Bisek, P. J. Halling
and S. L. Flitsch, Org. Biomol. Chem., 2003, 1, 1277; (c) P. J. Halling,
R. V. Ulijn and S. L. Flitsch, Curr. Opin. Biotechnol., 2005, 16, 385;
(d) C. E. Humphrey, N. J. Turner, M. Easson, S. L. Flitsch and R. V.
Ulijn, J. Am. Chem. Soc., 2003, 46, 13952; (e) R.H. P. Doeze, C. L.
Egan, R. V. Ulijn and S. L. Flitsch, Angew. Chem. Int. Ed., 2004, 43,
3138.
monolayers on gold surfaces were prepared by spotting a
DMSO solution (final concentration 0.1 mg/mL, 0.4 mL
per spot) of a 1 : 1 mixture of carboxylic acid-terminated
and tri(ethyleneglycol)-terminated alkanethiols (ProChimia,
chemical formulas HS(CH2)17(OCH2CH2)6CH2CO2H and
HS(CH2)17(OCH2CH2)3OH respectively) into each well and
left overnight to react before rinsing with DMSO, ethanol
and drying as above. The terminal carboxylic acid was then
activated for 1 h at room temperature by spotting 0.4 mL per
spot of a freshly prepared DMF solution of EDC (1-ethyl-
3-(3-dimethylaminopropyl)carbodiimide, 0.2 M) and NHS
(N-hydroxysuccinimide, 0.05 M). After rinsing and drying as
above, a DMF solution of N-Fmoc diaminobutane (Aldrich,
100 mM) was spotted (0.4 mL per spot) and left to react overnight
at room temperature. Following washing steps as above, the gold
5 H. D. Jakubke, P. Kuhl and A. Konnecke, Angew. Chem. Int. Ed., 1985,
24, 85.
6 R. L. Henrikson, Methods Enzymol., 47, 175.
7 (a) M. Bejugam and S. L. Flitsch, Org. Lett., 2004, 6, 4001; (b) B. A.
Maltman, M. Bejugam and S. L. Flitsch, Org. Biomol. Chem., 2005, 3,
2505; (c) M. Bejugam, B. A. Maltman and S. L. Flitsch, Tetrahedron:
Asymmetry, 2005, 16, 21.
This journal is
The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 665–670 | 669
©

View Article Online
8 R. V. Ulijn, N. Bisek and S. L. Flitsch, Org. Biomol. Chem., 2003, 1,
621.
9 A. Y. Bosma, R. V. Ulijn, G. McConnell, J. Girkin, P. J.
Halling and S. L. Flitsch, Chem. Commun., 2003, 22,
2790.
883; (b) Z. l. Zhi, N. Laurent, A. K. Powell, R. Karamanska, M. Fais,
J. Voglmeir, A. Wright, J. M. Blackburn, P. R. Crocker, D. A. Russell,
S. L. Flitsch, R. A. Field and J. E. Turnbull, ChemBioChem, 2008, 9,
568.
11 (a) M. Mrksich, ACS Nano, 2008, 2, 7; (b) J. Su and M. Mrksich, Angew.
Chem. Int. Ed., 2002, 41, 4715; (c) B. T. Housman and M. Mrksich,
Angew. Chem. Int. Ed., 1999, 38, 782.
10 (a) N. Laurent, J. Voglmeir, A. Wright, J. Blackburn, N. T. Pham,
S. C. C. Wong, S. J. Gaskell and S. L. Flitsch, ChemBioChem, 2008, 9,
670 | Org. Biomol. Chem., 2009, 7, 665–670
This journal is
The Royal Society of Chemistry 2009
©