Solution phase synthesis of β-peptides using micro reactors

Article abstract of DOI:10.1016/S0040-4020(02)00513-6

The synthesis of β-peptides has been successfully performed using a borosilicate glass micro reactor, in which a network of channels has been produced using a photolithographic and wet etching method. The reagents were mobilised by electroosmotic flow (EOF). The micro reactor was initially evaluated using a carbodiimide coupling reaction to form a dipeptide. The methodology has been extended such that the peptides may also be produced via the pentafluorophenyl ester derivatives of amino acids. It was found that performing the pentafluorophenyl ester reactions in the micro reactor resulted in an increase in the reaction efficiency over the traditional batch method. We postulate that the enhancement in rate of reaction is an electrochemical phenomenon, due to the reaction being performed in an electric field, which is unique to micro reactor systems. It has also been demonstrated that selective deprotection of the resultant dipeptides can be achieved. This approach has been used in the synthesis of a tripeptide.

Full text of DOI:10.1016/S0040-4020(02)00513-6

TETRAHEDRON
Pergamon
Tetrahedron 58 (2002) 5427±5439
Solution phase synthesis of b-peptides using micro reactors
Paul Watts,a Charlotte Wiles,a Stephen J. Haswella,p and Esteban Pombo-Villarb
aDepartment of Chemistry, Faculty of Science and the Environment, University of Hull, Cottingham Road, Hull HU6 7RX, UK
bNervous System Research, WSJ-386.07.15, Novartis Pharma Ltd, CH4002 Basel, Switzerland
Received 19March 2002; revised 22 April 2002; accepted 16 May 2002
AbstractÐThe synthesis of b-peptides has been successfully performed using a borosilicate glass micro reactor, in which a network of
channels has been produced using a photolithographic and wet etching method. The reagents were mobilised by electroosmotic ¯ow (EOF).
The micro reactor was initially evaluated using a carbodiimide coupling reaction to form a dipeptide. The methodology has been extended
such that the peptides may also be produced via the penta¯uorophenyl ester derivatives of amino acids. It was found that performing the
penta¯uorophenyl ester reactions in the micro reactor resulted in an increase in the reaction ef®ciency over the traditional batch method. We
postulate that the enhancement in rate of reaction is an electrochemical phenomenon, due to the reaction being performed in an electric ®eld,
which is unique to micro reactor systems. It has also been demonstrated that selective deprotection of the resultant dipeptides can be
achieved. This approach has been used in the synthesis of a tripeptide. q 2002 Elsevier Science Ltd. All rights reserved.
1. Introduction
micro reactor manifold, electrophoretic separation would
enable isolation of pure products.21
A micro reactor is generally de®ned as a series of inter-
connecting channels (10±300 mm in diameter), formed in
a planar surface, in which small quantities of reagents are
manipulated. Thus reagents can be brought together in a
speci®c sequence, mixed and allowed to react for a speci®ed
time in a controlled region of the reactor. During the past 10
years, there has been a rapid growth in the development of
micro-Total Analytical Systems (mTAS)1±8 which exploit
electroosmotic ¯ow (EOF).9±12 The development of micro
reactor devices for chemical synthesis based on complimen-
tary technology is less common. However, recent research
has shown that Suzuki,13 Wittig,14,15 diazo coupling reac-
tions16 and enamines17 may be performed using micro
chemical systems. In addition, we have demonstrated that
peptide bonds may be prepared using such technology.18
The reactions performed in micro reactors have recently
been reviewed by Haswell and co-workers.19
To illustrate the principles of EOF, one can consider a micro
channel fabricated from a material having negatively
charged functional groups on the surface. If a liquid,
displaying some degree of dissociation, is brought into
contact with the surface, positive counter ions will form a
double layer such that the positively charged ions are
attracted to the negatively charged surface. Application of
an electric ®eld causes this layer to move towards the
negative electrode, thus causing the bulk liquid to move
within the channel (Fig. 1). Electroosmotic ¯ow is not just
restricted to aqueous systems and a range of organic
solvents can be mobilised using the technique. A major
advantage of EOF is that it gives a high degree of spacial
and temporal control to reactants mixing under a diffusive
regime. This high level of ¯uidic control and mixing has
been attributed to the reactions13±19 reported in the litera-
ture. Electroosmotic ¯ow also has the advantage that the
direction and magnitude of the ¯ow may be readily changed
by altering the applied voltage, where the ¯ow rates increase
Micro reactors are advantageous because they allow the
rapid optimisation of reactions and reduce exposure to
hazardous chemicals. They also have the ability to generate
reagents in situ, allowing the preparation and subsequent
reaction of highly reactive or toxic intermediates. The
ability to scale out the synthesis allows large quantities of
chemicals to be prepared and it has been calculated that 1 kg
of material could be produced in 24 h using 1000 micro
reactors in parallel.20 In addition, because EOF may be
used to mobilise the solvents and reagents around the
Keywords: b-peptides; electroosmotic ¯ow; dipeptides.
Corresponding author. Tel.: 144-1482-465469; fax: 144-1482-466416;
e-mail: s.j.haswell@hull.ac.uk
p
Figure 1. Principle of electroosmotic ¯ow.
0040±4020/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(02)00513-6

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P. Watts et al. / Tetrahedron 58 (2002) 5427±5439
phase peptide synthesis however, has the disadvantage that
an expensive polymer support is required. Extra steps are
also added to the synthesis as a result of initially linking the
amino acid to the support and ®nally having to remove the
peptide from the polymer. In this paper, a micro reactor
has been used to prepare peptides using solution phase
chemistry and the results are compared with traditional
solid phase procedures.
2. Fabrication and use of the micro reactors
A number of materials such as silicon, quartz, glass, metals
and polymers have been used to construct micro reactors.
Glass and certain polymers have been particularly useful
because of their physical properties and chemical inertness.
These substances allow the mobilisation of organic solvents
and reagents using a number of pumping mechanisms such
as hydrodynamic pumping and electroosmotic ¯ow. A range
of fabrication methods such as photolithography, hot
embossing, powder blasting, injection moulding and laser
micro forming alone, or in combination, have been reported
in the literature.19,27,28
The micro reactor devices used in this work were prepared
using the popular technique of photolithography and wet
etching to produce channels in a glass micro reactor (Fig.
2).29 Firstly, a thin layer of metal, such as chromium, is
deposited onto the surface of a borosilicate glass plate.
This controls the degree of undercutting during the wet
etching process. A layer of positive photoresist is then
spin coated on top of the chromium to a depth of
0.5±2.0 mm. The pattern of the required network of inter-
connecting channels is subsequently transferred to the
photoresist layer using photolithography. After exposure,
the photoresist is developed and removed, together with
the chromium layer, to reveal the areas of glass to be etched.
The plate is then heated to allow the volatiles to evaporate,
before performing the chemical etch. The channels are
etched using a mixture of 1% HF and 5% NH4F in water
at 658C, resulting in an etch rate of 0.3±0.5 mm min21. A
glass top block, with pre-drilled holes that act as reservoirs
for reagents, is then aligned with the channel geometry and
thermally bonded to the base plate, producing an all glass
device (Fig. 3). Microporous silica frits30 were placed in the
channels to prevent hydrodynamic ¯ow occurring.
Figure 2. Sequence of processes in photolithographic fabrication.
with voltage. The ¯ow rates, which are in the order
of ml min21, are reproducible using EOF. In addition, the
pumping technique has no mechanical or moving parts,
hence it is highly suitable for operation in miniaturised
systems. An automated power supply with multiple elec-
trodes can support several pumping channels. Importantly,
when using EOF, plugs of ¯uid are transported without
signi®cant hydrodynamic dispersion.
Following the discovery by Merri®eld in 1963,22 peptides
have been commonly prepared via solid supported
techniques. Solid phase peptide synthesis (SPPS) is based
on the addition of a protected amino acid residue to an
insoluble polymeric support. The acid-labile Boc group23
and base-labile Fmoc group24 have been commonly used
for N-protection. After removal of the protecting group
the next protected amino acid may be added using either a
coupling reagent or a pre-activated amino acid derivative. If
this dipeptide is the desired product, it may be cleaved from
the polymer support.25,26 If a longer peptide is required
additional amino acids can be added by repeating further
coupling reactions. Solid phase peptide synthesis is
advantageous because of the ease of product puri®cation.
In addition the process is readily automated and has become
common in the combinatorial synthesis of peptides. Solid
3. Results and discussion
We selected to demonstrate the multi-step synthesis of
peptides derived from b-amino acids for two reasons.
Firstly, the simplest b-amino acids lack chiral centres,
hence we avoid potential problems with racemisation and
the generation of diastereomeric mixtures, which would
complicate the analysis of the products in our initial studies.
In addition, b-peptides have attracted much interest due to
their structural31,32 and biological properties.33 In particular,
their stability to degradation by peptidases34,35 makes them
potentially superior to the drugs derived from a-amino
acids. Both solution and solid phase synthetic methods for
these have been developed which would serve to benchmark
the micro reactor synthesis.36,37
Figure 3. A borosilicate glass micro reactor.

P. Watts et al. / Tetrahedron 58 (2002) 5427±5439
5429
reservoir B and a solution of amine 3 (50 ml, 0.1 M) in DMF
was placed in reservoir C (Fig. 4). Anhydrous DMF (40 ml)
was placed in reservoir D, which was used to collect the
products of the reaction. Platinum electrodes were placed in
each of the reservoirs (A, B and C positive, D ground) and
an external voltage was applied to the channels inducing
electroosmotic ¯ow of the reagents. The reactions were
conducted at room temperature for a period of 20 min, in
order to acquire suf®cient volume of product to determine
the conversion of the reaction by HPLC. Product con-
versions were based on the amount of carboxylic acid 4
remaining in the sample.
Scheme 1. Synthesis of a standard dipeptide derivative.
In the ®rst instance, a one-step reaction was considered in
which an N-protected b-amino acid was reacted with an
O-protected b-amino acid, to prepare the protected b-dipep-
tide. To enable the methodology to be applicable to the
synthesis of more complex peptides, the use of orthogonal
protecting groups was clearly required. As in SPPS, the
base-labile Fmoc protecting group24 was selected for
N-protection while the Dmab ester38 was chosen for protec-
tion of the carboxylic acid. Importantly, both protecting
groups may be removed under mild conditions, since
electroosmotic ¯ow is retarded if the pH of the reaction is
less than 3.9±12 We have already reported the application of
micro reactors in the formation of peptide bonds,18 but here
we give full experimental details for all procedures and also
exemplify more complex examples.
When stoichiometric quantities of the reagents were used
only ca. 10% conversion to dipeptide 5 was achieved when a
voltage of 700 V was applied to each of the reagents (A, B
and C). By lowering the applied voltages to 500 V, hence
reducing the ¯ow rates and increasing the residence time of
the reaction, no signi®cant increase in conversion was
observed. However, by using 2 equiv. of EDCI (0.2 M
solution) an increase in conversion to ca. 20% was
observed. By applying a stopped ¯ow technique (2.5 s injec-
tion length with stopped ¯ow for 5 s) thus further increasing
the residence time of the reaction, the conversion was
further increased to approximately 50%. Since the ef®ciency
of the reaction appeared to be greatly dependent on the
number of equivalents of EDCI used, we wished to further
investigate the effect of carbodiimide concentration on the
reaction, however we found that EDCI was insoluble in
DMF above 0.2 M concentrations. In further experiments
dicyclohexylcarbodiimide (DCC) was used as the coupling
reagent as it was considerably more soluble in DMF. Using
5 equiv. of DCC (0.5 M solution in reservoir B) a con-
version of 93% to dipeptide 5 was obtained, using the
stopped ¯ow regime described above. This represented a
typical yield for a classic carbodiimide coupling reaction
in SPPS.
Commercially available Boc-b-alanine 1 was protected as
the Dmab ester using 1-(3-dimethylaminopropyl)-3-ethyl-
carbodiimide hydrochloride (EDCI) and 4-dimethylamino
pyridine (DMAP), to give the ester 2 in 92% yield (Scheme
1). Treatment of 2 with tri¯uoroacetic acid furnished the
desired amine 3 in 92% yield, which was subsequently
reacted with Fmoc-b-alanine 4 via a carbodiimide coupling
reaction, to give a synthetic sample of the target dipeptide 5
in 50% yield. Addition of a catalytic amount of DMAP
resulted in appreciable quantities of Fmoc deprotection,
hence DMAP was omitted in subsequent coupling reactions
when the Fmoc protecting group was present.
We wished to demonstrate that alternative methods used in
peptide synthesis, could also be applied to the synthesis
within the micro reactors. Another common method utilised
in peptide bond formation involves the reaction of a pre-
activated amino acid derivative, such as a penta¯uorophenyl
ester, with an amine.39,40 Fmoc-b-alanine 4 was activated as
penta¯uorophenyl ester 6 via an EDCI coupling reaction, to
give the product in 90% yield (Scheme 2). Subsequent treat-
ment of ester 6 with amine 3 afforded dipeptide 5 in 46%
yield in a bulk reaction, using DMF as solvent. The yield did
not increase even when prolonged reaction times were
employed.
Having prepared dipeptide 5, it represented a synthetic
target for preparation within the micro reactor. Prior to
synthesis, the micro reactor channels were primed with
anhydrous N,N-dimethylformamide (DMF) to remove any
air and moisture from the channels and the microporous
silica frits. A standard solution of Fmoc-b-alanine 4
(50 ml, 0.1 M) in anhydrous DMF was added to reservoir
A, a solution of EDCI (50 ml, 0.1 M) in DMF was placed in
Figure 4. Schematic of the micro reactor used in carbodiimide coupling reactions.

5430
P. Watts et al. / Tetrahedron 58 (2002) 5427±5439
Scheme 2. Preparation and reaction of penta¯uorophenyl esters of Fmoc
protected amino acids.
Scheme 4. Reaction of PFP ester of Fmoc-l-b-homophenylalanine.
Having prepared dipeptide 5 via the alternative pre-acti-
vated strategy, we wished to investigate if the reaction
could be performed within a micro reactor. A standard solu-
tion of the penta¯uorophenyl ester of Fmoc-b-alanine 6
(50 ml, 0.1 M) in anhydrous DMF was added to reservoir
A and a solution of amine 3 (50 ml, 0.1 M) was placed in
reservoir B. Anhydrous DMF (40 ml) was placed in reser-
voir D, which was used to collect the products of the reac-
tion. It was found that using continuous ¯ow of both
reagents, where the ester 6 was maintained at 700 V and
the amine 3 was maintained at 600 V, dipeptide 5 was
produced quantitatively in 20 min. Product conversions
were based on the amount of penta¯uorophenyl ester 6
remaining in the sample. This represented a signi®cant
increase in yield compared with solution phase synthesis,
where lengthy reaction times are also required.
different electroosmotic mobilities within the micro reactor
channels.
In order to demonstrate the generality of the technique we
wished to demonstrate that a selection of peptides could be
prepared. Thus, commercially available Fmoc-l-b-homo-
phenylalanine 10 was converted into penta¯uorophenyl
ester 11 in 86% yield, under standard conditions. This was
reacted with amine 3 to prepare a synthetic sample of
dipeptide 12 in 35% yield (Scheme 4).
The reaction was subsequently investigated in the micro
reactor. A solution of the penta¯uorophenyl ester 11 in
anhydrous DMF was added to reservoir A, a solution of
amine 3 was placed in reservoir B and anhydrous DMF
was placed in reservoir D. It was found that using con-
tinuous ¯ow of both reagents, where the ester 11 was main-
tained at 900 V and the amine 3 was maintained at 600 V,
dipeptide 12 was produced quantitatively in 20 min. As
before this represented a signi®cant increase in yield
compared with bulk reaction.
We were also interested to determine the effect of the
protecting group on the reaction. Thus, Boc-b-alanine 7
was activated as penta¯uorophenyl ester 8 via an EDCI
coupling reaction, to give the product in 79% yield. The
penta¯uorophenyl ester 8 was reacted with amine 3 in
DMF as solvent to give the dipeptide 9 in 57% yield
(Scheme 3). Subsequently, the reaction between the penta-
¯uorophenyl ester 8 of Boc-b-alanine and amine 3 was also
investigated in the micro reactor. In this case, when the
reagents were mixed using continuous ¯ow, with both
reagents maintained at 700 V, again quantitative conversion
to dipeptide 9 was observed. The fact that penta¯uorophenyl
ester 8 needed to be maintained at a higher potential, in
order to obtain a quantitative conversion, demonstrates
that the different penta¯uorophenyl esters 6 and 8 have
Similarly l-b-homo-p-chlorophenylalanine 13 was reacted
with 9-¯uorenylmethyl chloroformate41 to effect N-protec-
tion affording carboxylic acid 14, followed by an EDCI
coupling reaction with penta¯uorophenol to give the corre-
sponding penta¯uorophenyl ester 15. The ester 15 was
reacted with amine 3 in DMF, to prepare a synthetic sample
of dipeptide 16 in 36% yield (Scheme 5).
Scheme 5. Reaction of penta¯uorophenyl ester of Fmoc-l-b-homo-p-
chlorophenylalanine.
Scheme 3. Reaction of penta¯uorophenyl ester of Boc-b-alanine.

P. Watts et al. / Tetrahedron 58 (2002) 5427±5439
5431
Scheme 6. Synthesis of peptides containing lysine.
The reaction was then transferred to the micro reactor. As in
the previous example, it was found that using continuous
¯ow of both reagents, where the ester 15 was maintained at
900 V and the amine 3 was maintained at 600 V, dipeptide
16 was produced quantitatively in 20 min.
Scheme 8. Preparation of peptides containing glycine.
We were interested to observe that all reactions between
penta¯uorophenyl esters and amine 3 appeared to be faster
in the micro reactor than when performed in a bulk reaction.
However, bulk reactions were generally performed at much
higher concentrations than micro reactions. In order to make
comparison between rates we monitored the conversion of
penta¯uorophenyl ester 11 into peptide 12 at 0.05 M
concentration, the same as used in the micro reaction studies
(Scheme 8). Fig. 5 shows how conversion increases with
time, the graph shows that after 20 min, the length of a
micro reaction, only about 5% conversion to dipeptide
was observed in the bulk reaction. Even after 400 h (ca.
16 days) only 70% conversion was attained. This result
clearly shows that the rate of reactions is considerably
enhanced when the reactions are conducted in a micro
reactor. We postulate that the enhancement in rate of
reaction is an electrochemical phenomenon, due to the
reaction being performed in an electric ®eld.
Having demonstrated the success of the technique using
simple b-amino acids, we wished to perform the synthesis
of more complex peptides. We selected to prepare peptides
containing lysine, which contains two amine functionalities,
which require orthogonal protection. Thus, commercially
available N1-Boc-l-lysine 17 was converted into penta-
¯uorophenyl ester 19, under standard conditions (Scheme
6). This was reacted with amine 3 to prepare a synthetic
sample of dipeptide 20 in a disappointing 9% isolated yield.
The reaction was further investigated in the micro reactor. A
solution of the penta¯uorophenyl ester 19 in anhydrous
DMF was added to reservoir A, a solution of amine 3 was
placed in reservoir B and anhydrous DMF was placed in
reservoir D. It was found that using continuous ¯ow of both
reagents, where the ester 19 was maintained at 1000 V and
the amine 3 was maintained at 600 V, dipeptide 20 was
produced quantitatively in 20 min.
Having prepared numerous examples of peptides prepared
from the Dmab ester of b-alanine 3, we wished to demon-
strate that other amines may be used in the synthesis of
peptides. Thus, commercially available Boc-glycine 24
was protected as the Dmab ester via an EDCI/DMAP
coupling reaction to give the ester 25 in 100% yield in a
bulk reaction (Scheme 8). Treatment of 25 with tri¯uoro-
acetic acid furnished the desired amine 26 in 78% yield.
This was subsequently reacted with penta¯uorophenyl
ester 6 to give a synthetic sample of the dipeptide 27 in
35% yield.
Similarly Na-Boc-l-lysine 21 was converted into penta-
¯uorophenyl ester 22, in 59% yield (Scheme 7). This was
reacted with amine 3 to prepare a synthetic sample of dipep-
tide 23 in 50% yield. The reaction was then transferred to
the micro reactor. As in the previous example, it was found
that using continuous ¯ow of the reagents, where the ester 22
was maintained at 1000 V and the amine 3 was maintained
at 600 V, dipeptide 23 was produced quantitatively. As
expected, this result demonstrates that both penta¯uoro-
phenyl esters of lysine 19 and 22 have the same electro-
phoretic mobility within the channels of the micro
reactor. Fmoc deprotection of the peptides 20 and 23
would prepare different amines, which could be used in
further reactions.
The reaction was subsequently investigated in the micro
reactor. A solution of the penta¯uorophenyl ester 6 in
anhydrous DMF was added to reservoir A, a solution of
amine 26 was placed in reservoir B and anhydrous DMF
was placed in reservoir D, which was used to collect the
Figure 5. Conversion of penta¯uorophenyl ester 11 to dipeptide 12 in a
bulk reaction.
Scheme 7. Synthesis of peptides containing lysine.

5432
P. Watts et al. / Tetrahedron 58 (2002) 5427±5439
with the penta¯uorophenyl ester. This was con®rmed from
a batch reaction, in which piperidine was reacted with the
penta¯uorophenyl ester of Boc-b-alanine 8 (Scheme 10).
The only product isolated from the reaction was amide 29,
resulting from the nucleophilic attack of piperidine on the
ester.
As a result, an alternative method of Fmoc deprotection was
required that would not cause the aforementioned problem.
Using the micro reactor, the Dmab ester of Fmoc-b-alanine
28 was reacted with 1 equiv. of 1,8-diazabicyclo[5.4.0]-
undec-7-ene (DBU)44,45 to give the free amine 3 which
was then reacted with the penta¯uorophenylester of
Boc-b-alanine 8, in an attempt to form the dipeptide 9.
Scheme 9. Multi-step peptide synthesis.
products of the reaction. It was found that using continuous
¯ow of reagents, where both were maintained at 800 V,
dipeptide 27 was produced quantitatively in 20 min.
In this case, when the reagents were mixed using continuous
¯ow, with the reagents maintained at 700 V, product 9 was
observed in typically 25% yield. Surprisingly, when the
reagents were mobilised using a stopped ¯ow technique,
the yield of product was actually reduced. By comparing
the ¯ows of each reagent at this stage we were able to
optimise the reaction. The Dmab ester of Fmoc-b-alanine
28 was maintained at 750 V while reacted with DBU at
800 V. The deprotected amine was further reacted, using
continuous ¯ow, with the penta¯uorophenyl ester of Boc-
b-alanine 8, maintained at 700 V, to give a conversion of
96%, based on the amount of Dmab ester 28 present at the
end of the reaction. Importantly we have shown that depro-
tection may be achieved in micro reactions using just
1 equiv. of DBU, compared with 2% DBU used in
SPPS.44,45
Having extensively demonstrated that peptide bonds could
be successfully formed when using a micro reactor, we
wished to show that we could extend the methodology to
the preparation of longer chain peptides. Consequently, we
needed to be able to conduct deprotection reactions and
subsequently perform further peptide bond forming reac-
tions. Fmoc-b-alanine 4 was converted into the Dmab
ester 28, in a bulk reaction, using standard conditions
(Scheme 9). It was proposed to convert ester 28 into
amine 3 by deprotection of the Fmoc group and subse-
quently react the amine `in situ' within the micro reactor
with penta¯uorophenyl ester 8, to give the dipeptide 9.
Several methods for the deprotection of the Fmoc group are
reported in the literature, the most common method being
treatment with 20% piperidine in DMF.42,43 The advantage
of using piperidine in the reaction, is that the piperidine
subsequently reacts with the dibenzofulvene, to form the
DMF soluble piperidine adduct. This is advantageous, as
precipitation or polymerisation of the dibenzofulvene within
the micro reactor could cause blockage of the channels.
Preliminary experiments revealed that treatment of 28,
with 10 equiv. of piperidine in DMF using continuous
¯ow within the micro reactor, resulted in 60±70% deprotec-
tion over a 20 min period, to give amine 3.
Having shown that more complex peptides could be
produced by removal of the N-protecting group we wished
to determine if we could remove the Dmab protecting group
using hydrazine.38 Hence, a solution of the Dmab ester of
Fmoc-b-alanine 28 (50 ml, 0.1 M) in anhydrous DMF was
added to reservoir A, a solution of hydrazine (50 ml, 0.1 M)
was placed in reservoir B and anhydrous DMF (40 ml) was
placed in reservoir D. Using continuous ¯ow of both
reagents maintained at 700 V, quantitative deprotection
was observed to give carboxylic acid 4 (Scheme 11).
Again deprotection was achieved in the micro reactions
using just 1 equiv. of hydrazine, compared with 2% solu-
tions used in SPPS.38 This suggests that reactions conducted
within micro reactors are more molecule ef®cient.
Subsequently, a standard solution of the Dmab ester of
Fmoc-b-alanine 28 (50 ml, 0.1 M) in anhydrous DMF was
added to reservoir A, a solution of piperidine (50 ml, 1.0 M)
was placed in reservoir B and a solution of penta¯uoro-
phenyl ester 8 (50 ml, 0.1 M) was placed in reservoir C, in
an attempt to prepare dipeptide 9 using this multi-step
approach. Anhydrous DMF (40 ml) was placed in reservoir
D, to collect the products of the reaction. The HPLC of the
reaction mixture showed that Fmoc deprotection had
occurred, however no dipeptide 9 was evident.
Scheme 11. Removal of the Dmab protecting group.
Having successfully demonstrated that peptide bonds could
be formed and that the protecting groups could be selec-
tively removed, we wished to show that we could use the
methodology in the preparation of tripeptides within micro
reactor devices. Our initial target was the tripeptide of
b-alanine 31. We selected to initially investigate the syn-
thesis of dipeptide 9 followed by the selective deprotection
of the Fmoc group and subsequent reaction of amine 30 with
another equivalent of penta¯uorophenyl ester 6 (Scheme
12).
It was postulated that the excess piperidine was reacting
Scheme 10. Reaction of piperidine with penta¯uorophenyl esters.

P. Watts et al. / Tetrahedron 58 (2002) 5427±5439
5433
performed in an electric ®eld. It has also been demonstrated
that selective deprotection of the resultant dipeptides can be
achieved. The Fmoc group was successfully deprotected
using 1 equiv. of DBU while the Dmab ester was removed
by treatment with 1 equiv. of hydrazine. After deprotection
further peptide bond forming reactions were performed,
resulting in the synthesis of longer chain peptides. We
have successfully used this approach in the synthesis of
tripeptide 31. Further studies are currently underway in
our laboratories to increase the overall conversion of the
multi-step reactions and to purify the peptides by electro-
phoretic separation.
Scheme 12. Tripeptide synthesis.
5. Experimental
5.1. Micro reactions
A standard solution of the penta¯uorophenyl ester of Fmoc-
b-alanine 6 (50 ml, 0.1 M) in anhydrous DMF was added to
reservoir A and a solution of amine 3 (50 ml, 0.1 M) was
placed in reservoir B, such that dipeptide 9 could be
prepared in the channel of the micro reactor (Fig. 6). A
solution of DBU (50 ml, 0.1 M) was placed in reservoir C
to effect Fmoc deprotection and a second equivalent
of penta¯uorophenyl ester 6 was added to reservoir D.
Anhydrous DMF (40 ml) was placed in reservoir E, which
was used to collect the products of the reaction. Using
continuous ¯ow of all reagents where reservoir A was
maintained at 1000 V, reservoir B was at 1000 V, reservoir
C was at 400 V and reservoir D was at 700 V, tripeptide 31
was prepared in 30% overall conversion, for the three step
synthesis. Further work is currently underway to purify the
tripeptide by electrophoretic separation.
Platinum electrodes were placed in each of the reservoirs of
the micro reactor and an external voltage was applied to the
channels inducing electroosmotic ¯ow of the reagents. The
power supply was manufactured by King®eld Electronics
(Shef®eld, UK) and was controlled using LabVIEWe
software written by Dr X. Zhang (University of Hull).
The reactions were conducted at room temperature for a
period of 20 min, in order to acquire suf®cient volume of
product to determine the conversion of the reaction. Reac-
tion products were determined by HPLC via comparison
with retention times and spectra with those obtained from
synthetic standards. Analysis was achieved by high per-
formance liquid chromatography (Jupiter C18 10 mm,
4.6£250 mm, mobile phase composition: 0.1% tri¯uoro-
acetic acid in water and 0.1% tri¯uoroacetic acid in aceto-
nitrile, using a gradient system of 30% aqueous to 70%
aqueous over 20 min, with a ¯ow rate of 2.5 ml min21 at
room temperature). Product conversions were based on
the amount of carboxylic acid or penta¯uorophenyl ester
remaining in the sample.
4. Conclusion
We have demonstrated the application of micro reactors to
perform multi-step synthesis, potentially allowing the high
throughput synthesis of peptides. It has been demonstrated
that peptide bonds may be prepared in high yield by either
a carbodiimide coupling reaction or from pre-activated
derivatives such as penta¯uorophenyl esters. It was found
that performing these reactions in the micro reactor resulted
in an increase in the reaction ef®ciency over the traditional
batch method. We propose that this enhancement in rate is
an electrochemical phenomenon, due to the reaction being
5.2. Bulk reactions
All solvents were purchased as anhydrous solutions over
molecular sieves from Fluka. Reagents were purchased
from Aldrich and used as supplied. Amino acids were
obtained from Bachem (UK) and Peptech. Column chroma-
tography was carried out using Fluka silica gel 60 as the
solid support. Compounds were eluted using various
mixtures of ethyl acetate, hexane and methanol. Thin
layer chromatography was performed using Merck Kiesel-
gel 60 HF254 aluminium backed TLC plates with various
mixtures of ethyl acetate and hexane as the eluent. Visuali-
sation of the plates was carried out by either exposure to
short wave ultra violet light or by development in aqueous
potassium permanganate (0.5%) and sodium hydrogen
carbonate (2.5%) solution, followed by heating with a hot
air gun.
Nuclear magnetic resonance (NMR) spectra were recorded
as solutions in deuteriochloroform, unless otherwise stated,
using tetramethylsilane (TMS) as internal standard. The
spectra were recorded on Jeol GX270 or GX400 spectro-
meters. The chemical shift values for all spectra are given in
Figure 6. Schematic of the micro reactor used to prepare tripeptide.

5434
P. Watts et al. / Tetrahedron 58 (2002) 5427±5439
parts per million with coupling constants in Hertz. Mass
spectra were recorded on a Thermo-Finnigan LCQ, using
electrospray ionisation. Infrared spectra were recorded in
the range 4000±600 cm21 using a Perkin Elmer Paragon
1000 FT-IR spectrometer and peaks are reported (nmax) in
wavenumbers (cm21). Liquid samples were obtained as thin
®lms and solid samples were dissolved in chloroform, on
sodium chloride discs. Melting points were measured on a
Gallenkamp melting point apparatus using open capillary
tubes and are uncorrected. Elemental combustion analyses
were measured using a Fisons Carlo Erba EA1108 analyser.
53.8, 66.1, 107.8, 126.7, 129.2, 134.8, 137.0, 171.0, 176.4,
196.3 and 200.2; m/z 401 (M111).
5.2.3. Fmoc-b-alanine-b-alanine-ODmab 5. A solution of
EDCI (91 mg, 0.47 mmol) in DCM (10 ml) was added to a
stirred solution of Fmoc-b-alanine 4 (94 mg, 0.30 mmol)
and amine 3 (130 mg, 0.33 mmol) in DCM (10 ml) at
room temperature under nitrogen. After 24 h, the solvent
was washed with dilute hydrochloric acid (20 ml) and the
aqueous layer was further extracted with DCM (2£50 ml).
The combined organic extracts were dried over magnesium
sulphate and concentrated in vacuo to give an oil which was
puri®ed by column chromatography. Elution with ethyl
acetate gave Fmoc-b-alanine-b-alanine-ODmab 5 (105
mg, 50%) as a colourless gum; dH (399.6 MHz) 0.77 (6H,
d, Jˆ6.8 Hz, CH(CH3)2), 1.08 (6H, s, C(CH3)2), 1.84 (1H,
m, CH(CH3)2), 2.39(2H, m, CH 2CO2), 2.40 (2H, s,
CH2C(CH3)2), 2.49(2H, s, C H2C(CH3)2), 2.61 (2H, t, Jˆ
6.4 Hz, CH2CO), 3.00 (2H, m, CH2CH(CH3)2), 3.48 (2H, m,
NHCH2), 3.56 (2H, m, NHCH2), 4.20 (1H, t, Jˆ6.8 Hz,
CHCH2O), 4.37 (2H, d, Jˆ6.8 Hz, CHCH2O), 5.30 (2H, s,
CO2CH2), 5.52 (1H, m, NH), 6.17 (1H, br t, NH), 7.12 (2H,
d, Jˆ8.0 Hz, ArH), 7.30 (2H, t, Jˆ7.6 Hz, ArH), 7.39(2H, t,
Jˆ7.1 Hz, ArH), 7.40 (2H, d, Jˆ8.0 Hz, ArH), 7.59(2H, d,
Jˆ7.8 Hz, ArH), 7.76 (2H, d, Jˆ7.8 Hz, ArH) and 15.29
(1H, s, ArNH); dC (100.4 MHz) 22.6, 28.2, 29.6, 30.0,
31.9, 34.0, 34.9, 38.4, 47.2, 52.3, 53.7, 64.5, 65.8, 66.7,
107.8, 120.0, 125.1, 126.7, 127.1, 127.7, 129.1, 135.1,
137.0, 141.3, 143.9, 155.9, 172.1, 172.3, 176.5, 196.5 and
200.3; m/z 694 (M111).
5.2.1. Boc-b-alanine-ODmab 2. A solution of EDCI
(1.27 g, 6.62 mmol) and DMAP (72 mg, 0.59mmol) in
DCM (100 ml) was added to a stirred solution of Boc-b-
alanine 1 (1.10 g, 5.81 mmol) and DmabOH (1.92 g,
5.83 mmol) in DCM (100 ml) at room temperature under
nitrogen. After 24 h, the solvent was washed with dilute
hydrochloric acid (50 ml) and the aqueous layer was further
extracted with DCM (2£50 ml). The combined organic
extracts were dried over magnesium sulphate and concen-
trated in vacuo to give a solid, which was puri®ed by column
chromatography. Elution with 20% ethyl acetate in hexane
gave Boc-b-alanine-ODmab 2 (2.67 g, 92%) as a white
solid; mp 78±808C (from DCM/hexane); (Found C, 67.03;
H, 7.79; N, 5.36. C28H40N2O6 requires C, 67.20; H, 8.00; N,
5.60%); nmax (cm21) 3453, 3360, 2940, 1734, 1708, 1641,
1454 and 754; dH (399.6 MHz) 0.78 (6H, d, Jˆ6.6 Hz,
CH(CH3)2), 1.08 (6H, s, C(CH3)2), 1.43 (9H, s, C(CH3)3),
1.84 (1H, m, CH(CH3)2), 2.40 (2H, s, CH2C(CH3)2), 2.49
(2H, s, CH2C(CH3)2), 2.61 (2H, t, Jˆ6.1 Hz, CH2CO2), 3.01
(2H, br d, Jˆ6.1 Hz, CH2CH(CH3)2), 3.43 (2H, dt, Jˆ6.1
and 6.1 Hz, NHCH2), 5.01 (1H, m, NHCH2), 5.17 (2H, s,
CO2CH2), 7.14 (2H, d, Jˆ8.4 Hz, ArH), 7.41 (2H, d, Jˆ
8.4 Hz, ArH) and 15.30 (1H, s, ArNH); dC (100.4 MHz)
22.6, 28.3, 29.5, 30.0, 34.6, 36.1, 38.3, 52.3, 53.8, 65.5,
79.4, 100.5, 107.8, 126.7, 129.0, 135.3, 136.9, 155.7,
172.2, 176.4, 196.3 and 200.2; m/z 501 (M111).
5.2.4. Fmoc-b-alanine-OPFP 6. A solution of EDCI
(1.32 g, 6.88 mmol) in DCM (50 ml) was added to a stirred
solution of Fmoc-b-alanine 4 (2.06 g, 6.62 mmol) and
penta¯uorophenol (1.22 g, 6.63 mmol) in DCM (100 ml)
at room temperature under nitrogen. After 24 h, the solvent
was washed with dilute hydrochloric acid (50 ml) and the
aqueous layer was further extracted with DCM (2£50 ml).
The combined organic extracts were dried over magnesium
sulphate and concentrated in vacuo to give a solid which
was which was puri®ed by column chromatography. Elution
with 20% ethyl acetate in gave Fmoc-b-alanine-OPFP 6
(2.85 g, 90%) as a white solid; mp 112±1148C (from
EtOAc/hexane); (Found C, 60.06; H, 3.09; N, 2.67.
C24H16NO4F5 requires C, 60.38; H, 3.35; N, 2.94%); nmax
(cm21) 3422, 1787, 1696, 1518, 1002 and 758; dH
(399.6 MHz) 2.94 (2H, t, Jˆ6.1 Hz, CH2CO2), 3.60 (2H,
dt, Jˆ5.8 and 6.1 Hz, NHCH2), 4.22 (1H, t, Jˆ6.8 Hz,
CHCH2O), 4.42 (2H, d, Jˆ6.8 Hz, CHCH2O), 5.22 (1H,
m, NH), 7.31 (2H, t, Jˆ7.3 Hz, ArH), 7.40 (2H, t, Jˆ
7.3 Hz, ArH), 7.58 (2H, d, Jˆ6.8 Hz, ArH) and 7.76 (2H,
d, Jˆ7.5 Hz, ArH); dC (100.4 MHz) 33.8, 36.4, 47.2, 66.9,
120.0, 125.0, 127.0, 127.7, 136.6 (m, CF), 138.4 (m, CF),
139.1 (m, CF), 139.8 (m, CF), 140.8 (m, CF), 141.3, 142.2
(m, CF), 143.7, 156.3 and 168.3; m/z 478 (M111).
5.2.2. b-Alanine-ODmab 3. Tri¯uoroacetic acid (20 ml)
was added to a solution of Boc-b-alanine-ODmab 2
(1.28 g, 2.56 mmol) in DCM (5 ml). After stirring for
30 min, the reaction mixture was concentrated in vacuo to
remove the excess tri¯uoroacetic acid. The residue was
dissolved in DCM (50 ml) and washed with saturated
sodium hydrogen carbonate solution (50 ml). The aqueous
solution was further extracted with DCM (2£50 ml) and the
combined organic extracts were dried over magnesium
sulphate and concentrated in vacuo to give a solid which
was recrystallised using DCM/Et2O/hexane to give
b-alanine-ODmab (as the TFA salt) 3 (1.21 g, 92%) as a
pale yellow solid; mp 162±1648C; (Found C, 58.39; H, 6.50;
N, 5.29. C25H33N2O6F3 requires C, 58.37; H, 6.42; N,
5.45%); nmax (cm21) 3451, 3251, 2960, 1730, 1672, 1648,
1450, 1195 and 755; dH (399.6 MHz) 0.77 (6H, d,
Jˆ6.6 Hz, CH(CH3)2), 1.08 (6H, s, C(CH3)2), 1.84 (1H,
m, CH(CH3)2), 2.40 (2H, s, CH2C(CH3)2), 2.49(2H, s,
CH2C(CH3)2), 2.86 (2H, t, Jˆ 6.4 Hz, CH2CO2), 3.00 (2H,
br d, Jˆ5.1 Hz, CH2CH(CH3)2), 3.26 (2H, t, Jˆ6.6 Hz,
NH2CH2), 5.01 (1H, m, NHCH2), 5.17 (2H, s, CO2CH2),
7.14 (2H, d, Jˆ8.4 Hz, ArH), 7.41 (2H, d, Jˆ8.4 Hz,
ArH), 8.42 (2H, br s, NH2) and 15.31 (1H, s, ArNH); dC
(100.4 MHz) 22.6, 28.2, 29.5, 30.0, 31.3, 35.3, 38.3, 52.3,
5.2.5. Fmoc-b-alanine-b-alanine-ODmab 5. A solution of
penta¯uorophenyl ester 6 (181 mg, 0.38 mmol) and amine 3
(161 mg, 0.40 mmol) in DMF (2 ml) was stirred for 24 h.
The DMF was removed in vacuo, the residue was dissolved
in DCM (50 ml) and washed with dilute hydrochloric acid
(20 ml). The aqueous layer was further extracted with DCM
(2£50 ml) and the combined organic extracts were dried

P. Watts et al. / Tetrahedron 58 (2002) 5427±5439
5435
over magnesium sulphate and concentrated in vacuo to give
a yellow oil which was puri®ed by column chromatography.
Elution with ethyl acetate gave Fmoc-b-alanine-b-alanine-
ODmab 5 (121 mg, 46%) as a colourless oil. Spectroscopic
data as reported.
86%) as a white solid; mp 140±1428C (DCM/hexane);
nmax (cm21) 3326, 1782, 1691, 1518, 993 and 754; dH
(399.6 MHz) 2.83±3.05 (4H, m, CH2CO2 and CH2Ph),
4.20 (1H, t, Jˆ6.8 Hz, CHCH2O), 4.36±4.42 (3H, m,
CHCH2O and CHNH), 5.13 (1H, d, Jˆ8.8 Hz, NH), 7.19±
7.33 (7H, m, ArH), 7.40 (2H, t, Jˆ7.3 Hz, ArH), 7.55 (2H,
d, Jˆ7.3 Hz, ArH) and 7.76 (2H, d, Jˆ7.3 Hz, ArH); dC
(100.4 MHz) 36.8, 39.7, 47.1, 49.2, 66.8, 120.0, 125.0,
127.0, 127.1, 127.7, 128.8, 129.3, 136.6 (m, CF), 138.4
(m, CF), 136.8, 139.1 (m, CF), 139.8 (m, CF), 140.8 (m,
CF), 141.3, 142.2 (m, CF), 143.8, 155.5 and 167.5; m/z 567
(M111).
5.2.6. Boc-b-alanine-OPFP 8. A solution of EDCI (2.35 g,
12.26 mmol) and DMAP (60 mg, 0.49mmol) in DCM
(100 ml) was added to a stirred solution of Boc-b-alanine
7 (2.28 g, 12.05 mmol) and penta¯uorophenol (2.25 g,
12.22 mmol) in DCM (100 ml) at room temperature under
nitrogen. After 24 h, the solvent was washed with dilute
hydrochloric acid (50 ml) and the aqueous layer was further
extracted with DCM (2£50 ml). The combined organic
extracts were dried over magnesium sulphate and concen-
trated in vacuo to give a solid which was recrystallised using
DCM and hexane to give Boc-b-alanine-OPFP 8 (3.37 g,
79%) as a white solid; mp 56±578C; (Found C, 47.27; H,
3.86; N, 3.61. C14H14NO4F5 requires C, 47.32; H, 3.94; N,
3.94%); nmax (cm21) 3374, 2989, 1797, 1691, 1518, 1097,
988 and 756; dH (399.6 MHz) 1.45 (9H, s, (CH3)3CO), 2.93
(2H, t, Jˆ6.1 Hz, CH2CO2), 3.53 (2H, dt, Jˆ6.1 and 6.1 Hz,
NHCH2) and 4.98 (1H, m, NH); dC (100.4 MHz) 28.4, 34.0,
36.0, 79.9, 136.6 (m, CF), 138.3 (m, CF), 139.2 (m, CF),
139.8 (m, CF), 140.8 (m, CF), 142.3 (m, CF), 155.8 and
168.5; m/z 356 (M111).
5.2.9. Fmoc-l-b-homophenylalanine-b-alanine-ODmab
12. A solution of penta¯uorophenyl ester 11 (171 mg,
0.30 mmol) and amine 3 (125 mg, 0.31 mmol) in DMF
(2 ml) was stirred for 24 h. The DMF was removed in
vacuo, the residue was dissolved in DCM (50 ml) and
washed with dilute hydrochloric acid (20 ml). The aqueous
layer was further extracted with DCM (2£50 ml) and the
combined organic extracts were dried over magnesium
sulphate and concentrated in vacuo to give a yellow oil
which was puri®ed by column chromatography. Elution
with ethyl acetate gave Fmoc-l-b-homophenylalanine-b-
alanine-ODmab 12 (83 mg, 35%) as a cream solid; mp
116±1188C (DCM/hexane); nmax (cm21) 3432, 3316,
2951, 1734, 1686, 1556, 1445, 1166 and 756; dH (399.6
MHz) 0.76 (6H, d, Jˆ6.8 Hz, CH(CH3)2), 1.08 (6H,
s, C(CH3)2), 1.83 (1H, m, CH(CH3)2), 2.40 (2H, s,
CH2C(CH3)2), 2.49(2H, s, C H2C(CH3)2), 2.61 (2H, br t,
CH2CO2), 2.87±2.99 (6H, m, CH2CH(CH3)2, CH2CO2 and
CH2Ph), 3.54 (2H, m, CH2NH), 4.18 (5H, t, Jˆ7.0 Hz,
CHCH2O), 4.28±4.39(2H, m, C HCH2O and CHNH), 5.12
(2H, s, CO2CH2), 5.84 (1H, br d, Jˆ8.4 Hz, NH), 6.28 (1H,
m, NH), 7.09(2H, d, Jˆ7.3 Hz, ArH), 7.18±7.57 (13H, m,
ArH), 7.76 (2H, d, Jˆ7.3 Hz, ArH) and 15.28 (1H, s,
ArNH); dC (100.4 MHz) 22.6, 28.2, 29.5, 30.0, 34.0, 34.9,
36.5, 38.3, 40.2, 47.2, 52.3, 53.7, 65.7, 66.7, 107.8, 120.0,
125.1, 126.7, 127.1, 127.7, 128.6, 129.1, 129.3, 135.0,
136.8, 137.8, 141.3, 143.9, 155.9, 162.6, 170.9, 172.2,
176.4, 196.4 and 200.2; m/z 784 (M111).
5.2.7. Boc-b-alanine-b-alanine-ODmab 9. A solution of
penta¯uorophenyl ester 8 (142 mg, 0.40 mmol) and amine 3
(176 mg, 0.44 mmol) in DMF (2 ml) was stirred for 48 h.
The DMF was removed in vacuo, the residue was dissolved
in DCM (50 ml) and washed with dilute hydrochloric acid
(20 ml). The aqueous layer was further extracted with DCM
(2£30 ml) and the combined organic extracts were dried
over magnesium sulphate and concentrated in vacuo to
give a yellow oil which was puri®ed by column chromato-
graphy. Elution with ethyl acetate gave Boc-b-alanine-b-
alanine-ODmab 9 (129mg, 57%) as a colourless oil; dH
(399.6 MHz) 0.78 (6H, d, Jˆ6.8 Hz, CH(CH3)2), 1.08
(6H, s, C(CH3)2), 1.43 (9H, s, C(CH3)3), 1.85 (1H, m,
CH(CH3)2), 2.38 (2H, t, Jˆ6.1 Hz, CH2CO2), 2.45 (4H, s,
2£CH2C(CH3)2), 2.63 (2H, t, Jˆ6.1 Hz, CH2CO), 3.00 (2H,
br d, Jˆ6.1 Hz, CH2CH(CH3)2), 3.38 (2H, m, NHCH2), 3.56
(2H, dt, Jˆ6.1 and 6.1 Hz, NHCH2), 5.01 (1H, m, NHCH2),
5.14 (1H, br s, NH), 5.17 (2H, s, CO2CH2), 6.21 (1H, br s,
NH), 7.14 (2H, d, Jˆ8.3 Hz, ArH), 7.41 (2H, d, Jˆ8.3 Hz,
ArH) and 15.30 (1H, s, ArNH); dC (100.4 MHz) 22.6, 28.2,
28.4, 29.5, 30.0, 34.0, 34.8, 36.3, 38.4, 53.0, 54.9, 65.7,
79.4, 107.8, 126.7, 129.1, 135.1, 137.0, 156.0, 171.4,
172.2, 176.4, 196.3 and 200.2; m/z 572 (M111).
5.2.10. Fmoc-l-b-homo-p-chlorophenylalanine 14. A
solution of 9-¯uorenylmethyl chloroformate (0.37 g,
1.45 mmol) in dioxane (30 ml) was added to a stirred
solution of l-b-homo-p-chlorophenylalanine 13 (0.30 g,
1.22 mmol) in saturated aqueous sodium hydrogen carbo-
nate (50 ml). After stirring for 24 h the mixture was concen-
trated in vacuo and acidi®ed using dilute hydrochloric acid
(50 ml) and extracted using dichloromethane (3£50 ml).
The combined organic extracts were dried over magnesium
sulphate and concentrated in vacuo to give a solid which
was recrystallised from DCM/hexane to give Fmoc-l-b-
homo-p-chlorophenylalanine 14 (0.37 g, 70%) as a white
solid; mp 176±1788C; (Found C, 68.90; H, 5.16; N, 3.14.
C25H22NO4Cl requires C, 68.97; H, 5.06; N, 3.22%); nmax
(cm21) 3326, 1682, 1652, 1537 and 737; dH (DMSO,
399.6 MHz) 2.47 (2H, d, Jˆ6.1 Hz, CH2CO2), 2.84 (2H,
d, Jˆ6.6 Hz, CH2Ph), 4.12±4.36 (4H, m, CHCH2O and
NH), 4.42 (2H, d, Jˆ5.6 Hz, CHCH2O), 6.63 (1H, d, Jˆ
8.0 Hz, NH), 7.15 (2H, d, Jˆ8.1 Hz, ArH), 7.21 (2H, d,
Jˆ8.1 Hz, ArH), 7.31 (2H, dt, Jˆ7.3 and 3.7 Hz, ArH),
7.40 (2H, t, Jˆ7.3 Hz, ArH), 7.58 (2H, m, ArH) and 7.76
5.2.8. Fmoc-l-b-homophenylalanine-OPFP 11. A solu-
tion of EDCI (0.37 g, 1.96 mmol) in DCM (50 ml) was
added to a stirred solution of Fmoc-l-b-homophenylalanine
10 (0.43 g, 1.24 mmol) and penta¯uorophenol (0.35 g,
1.91 mmol) in DCM (50 ml) at room temperature under
nitrogen. After 24 h, the solvent was washed with dilute
hydrochloric acid (50 ml) and the aqueous layer was further
extracted with DCM (2£50 ml). The combined organic
extracts were dried over magnesium sulphate and concen-
trated in vacuo to give a solid which puri®ed by column
chromatography. Elution with 20% ethyl aceate in hexane
gave Fmoc-l-b-homophenylalanine-OPFP 11 (0.55 g,

5436
P. Watts et al. / Tetrahedron 58 (2002) 5427±5439
(2H, d, Jˆ7.6 Hz, ArH); dC (100.4 MHz) 29.4, 38.4,
47.1, 49.4, 65.8, 119.8, 125.1, 127.0, 127.6, 128.2, 130.8,
131.7, 136.9, 141.0, 143.8, 155.6 and 173.0; m/z 436
(M111).
sodium hydrogen carbonate (50 ml). After stirring for 24 h
the mixture was concentrated in vacuo and acidi®ed using
dilute hydrochloric acid (100 ml) and extracted using
dichloromethane (3£100 ml). The combined organic
extracts were dried over magnesium sulphate and concen-
trated in vacuo to give a solid which was recrystallised from
DCM/hexane to afford N1-Boc-Na-Fmoc-l-lysine 18
(0.80 g, 93%) as a white solid; mp 44±468C (from DCM/
hexane); nmax (cm21) 3422, 3336, 2970, 1705, 1648, 1516
and 757; dH (DMSO, 399.6 MHz) 1.25±1.41 (4H, m,
CH2CH2), 1.36 (9H, s, (CH3)3CO), 1.51±1.68 (2H, m,
CH2), 2.89(2H, m, C H2NH), 3.89(1H, m, C HCO2H),
4.21 (1H, t, Jˆ6.1 Hz, CHCH2O), 4.27 (1H, d, Jˆ6.1 Hz,
CHCH2O), 6.77 (1H, t, Jˆ5.6 Hz, NHCH2), 7.31 (2H, t,
Jˆ7.3 Hz, ArH), 7.41 (2H, t, Jˆ7.3 Hz, ArH), 7.72 (2H,
d, Jˆ7.6 Hz, ArH), 7.88 (2H, d, Jˆ7.3 Hz, ArH) and
12.56 (1H, br s, CO2H); m/z 467 (M121).
5.2.11. Fmoc-l-b-homo-p-chlorophenylalanine-OPFP 15.
A solution of EDCI (0.20 g, 1.07 mmol) in DCM (20 ml)
was added to a stirred solution of Fmoc-l-b-homo-p-chloro-
phenylalanine 14 (0.31 g, 0.71 mmol) and penta¯uoro-
phenol (0.14 g, 0.75 mmol) in DCM (20 ml) at room
temperature under nitrogen. After 24 h, the solvent was
washed with dilute hydrochloric acid (50 ml) and the
aqueous layer was further extracted with DCM (2£50 ml).
The combined organic extracts were dried over magnesium
sulphate and concentrated in vacuo to give a solid which
was recrystallised using DCM and hexane to give Fmoc-l-
b-homo-p-chlorophenylalanine-OPFP 15 (0.40 g, 93%) as
a white solid; mp 160±1628C; nmax (cm21) 3322, 1777,
1687, 1520, 991, 758 and 741; dH (399.6 MHz) 2.86±3.01
(4H, m, CH2CO2 and CH2Ph), 4.20 (1H, t, Jˆ5.6 Hz,
CHCH2O), 4.30 (3H, m, CHNH), 4.42 (2H, d, Jˆ5.6 Hz,
CHCH2O), 5.09(1H, d, Jˆ9.0 Hz, NH), 7.12 (2H, d, Jˆ
7.6 Hz, ArH), 7.26±7.32 (4H, m, ArH), 7.40 (2H, t, Jˆ
7.3 Hz, ArH), 7.54 (2H, d, Jˆ7.3 Hz, ArH) and 7.76 (2H,
d, Jˆ7.6 Hz, ArH); dC (100.4 MHz) 36.8, 39.1, 47.2, 49.2,
66.8, 120.0, 125.0, 127.1, 127.8, 129.0, 130.6, 133.0, 135.3,
136.6 (m, CF), 138.4 (m, CF), 139.1 (m, CF), 139.8 (m, CF),
140.8 (m, CF), 141.4, 142.2 (m, CF), 143.8, 155.5 and
167.4; m/z 602 (M111).
5.2.14. N1-Boc-Na-Fmoc-l-lysine-OPFP 19. A solution
of EDCI (0.32 g, 1.68 mmol) in DCM (30 ml) was added
to a stirred solution of N1-Boc-Na-Fmoc-l-lysine 18
(0.74 g, 1.58 mmol) and penta¯uorophenol (0.29g, 1.60
mmol) in DCM (50 ml) at room temperature under nitrogen.
After 24 h, the solvent was washed with dilute hydrochloric
acid (50 ml) and the aqueous layer was further extracted
with DCM (2£50 ml). The combined organic extracts
were dried over magnesium sulphate and concentrated
in vacuo to give a solid, which was puri®ed by column
chromatography. Elution with 20% ethyl acetate in hexane
gave N1-Boc-Na-Fmoc-l-lysine-OPFP 19 (0.54 g, 54%) as
a white solid; mp 100±1028C (from DCM/hexane); (Found
C, 60.30; H, 4.81; N, 4.25. C32H31N2O6F5 requires C, 60.57;
H, 4.89; N, 4.42%); nmax (cm21) 3432, 3345, 2900, 1787,
1681, 1518, 993 and 753; dH (399.6 MHz) 1.28±1.59 (4H,
m, CH2CH2), 1.44 (9H, s, (CH3)3CO), 1.91±2.09 (2H, m,
CH2), 3.16 (2H, m, CH2NH), 4.24 (1H, t, Jˆ6.8 Hz,
CHCH2O), 4.40±4.72 (4H, m, CHCH2O, CHCO2 and
NH), 5.58 (1H, d, Jˆ7.8 Hz, NH), 7.31 (2H, t, Jˆ7.3 Hz,
ArH), 7.40 (2H, t, Jˆ7.3 Hz, ArH), 7.61 (2H, d, Jˆ7.3 Hz,
ArH) and 7.77 (2H, d, Jˆ7.6 Hz, ArH); dC (100.4 MHz)
22.1, 28.4, 29.7, 31.4, 39.6, 47.1, 53.7, 67.2, 79.4,
120.0, 125.0, 127.1, 127.7, 136.6 (m, CF), 138.4 (m,
CF), 139.1 (m, CF), 139.7 (m, CF), 141.0 (m, CF), 141.3,
142.3 (m, CF), 143.7, 155.9, 156.3 and 168.8; m/z 635
(M111).
5.2.12. Fmoc-l-b-homo-p-chlorophenylalanine-b-ala-
nine-ODmab 16. A solution of penta¯uorophenyl ester 15
(130 mg, 0.21 mmol) and amine 3 (102 mg, 0.26 mmol) in
DMF (2 ml) was stirred for 24 h. The DMF was removed in
vacuo, the residue was dissolved in DCM (50 ml) and
washed with dilute hydrochloric acid (20 ml). The aqueous
layer was further extracted with DCM (2£50 ml) and the
combined organic extracts were dried over magnesium
sulphate and concentrated in vacuo to give a yellow oil
which was puri®ed by column chromatography. Elution
with ethyl acetate gave Fmoc-l-b-homo-p-chlorophenyl-
alanine-b-alanine-ODmab 16 (64 mg, 36%) as a white
solid; mp 152±1568C (DCM/hexane); nmax (cm21) 3451,
3297, 2951, 1734, 1681 and 753; dH (399.6 MHz) 0.76
(6H, d, Jˆ6.7 Hz, CH(CH3)2), 1.08 (6H, s, C(CH3)2), 1.83
(1H, m, CH(CH3)2), 2.40 (2H, s, CH2C(CH3)2), 2.49(2H, s,
CH2C(CH3)2), 2.60 (2H, br t, CH2CO2), 2.78±3.00 (6H, m,
CH2CH(CH3)2, CH2CO2 and CH2Ph), 3.54 (2H, m,
CH2NH), 4.18 (5H, t, Jˆ6.7 Hz, CHCH2O), 4.30±4.39
(2H, m, CHCH2O and CHNH), 5.13 (2H, s, CO2CH2),
5.80 (1H, br d, Jˆ8.2 Hz, NH), 6.24 (1H, m, NH), 7.11
(2H, d, Jˆ7.6 Hz, ArH), 7.22±7.56 (12H, m, ArH), 7.76
(2H, d, Jˆ7.6 Hz, ArH) and 15.29(1H, s, ArNH); dC
(100.4 MHz) 22.6, 28.3, 29.6, 30.0, 34.0, 34.9, 36.5, 38.4,
39.4, 47.2, 52.3, 53.8, 65.8, 66.6, 107.8, 120.0, 125.1, 126.7,
127.1, 127.7, 128.7, 129.1, 130.6, 132.5, 135.0, 136.4,
137.0, 141.3, 143.9, 155.9, 162.6, 170.7, 172.2, 176.4,
196.4 and 200.3; m/z 818 (M111).
5.2.15. N1-Boc-Na-Fmoc-l-lysine-b-Aalanine-ODmab
20. A solution of carboxylic acid 18 (142 mg, 0.30 mmol),
amine 3 (131 mg, 0.33 mmol) and EDCI (95 mg, 0.50
mmol) in DCM (20 ml) was stirred under nitrogen. After
24 h, the solvent was washed with dilute hydrochloric acid
(50 ml) and the aqueous layer was further extracted with
DCM (2£50 ml). The combined organic extracts were
dried over magnesium sulphate and concentrated in vacuo
to give an oil which was puri®ed by column chroma-
tography. Elution with ethyl acetate in gave N1-Boc-Na-
Fmoc-l-lysine-b-alanine-ODmab 20 (23 mg, 9%) as a
colourless gum; nmax (cm21) 3432, 3336, 2951, 1667,
1551, 1450, 1166 and 748; dH (399.6 MHz) 0.77 (6H, d,
Jˆ6.4 Hz, CH(CH3)2), 1.08 (6H, s, C(CH3)2), 1.24±1.54
(6H, m, CH2CH2CH2), 1.43 (9H, s, (CH3)3CO), 1.83 (1H,
m, CH(CH3)2), 2.40 (2H, s, CH2C(CH3)2), 2.49(2H, s,
CH2C(CH3)2), 2.61 (2H, t, Jˆ5.6 Hz, CH2CO2), 2.99 (2H,
5.2.13. N1-Boc-Na-Fmoc-l-lysine 18. A solution of
9-¯uorenylmethyl chloroformate (0.52 g, 2.02 mmol) in
dioxane (50 ml) was added to a stirred solution of N1-
Boc-l-lysine 17 (0.45 g, 1.83 mmol) in saturated aqueous

P. Watts et al. / Tetrahedron 58 (2002) 5427±5439
5437
br d, Jˆ5.3 Hz, CH2CH(CH3)2), 3.10 (2H, m, CH2NH), 3.55
(2H, m, CH2NH), 4.21 (2H, t, Jˆ6.8 Hz, CHCH2O), 4.39
(2H, m, CHCH2O and CHCH), 4.69(1H, m, NH), 5.09(2H,
s, CO2CH2), 5.56 (1H, m, NH), 6.70 (1H, m, NH), 7.09(2H,
d, Jˆ8.4 Hz, ArH), 7.31 (2H, dt, Jˆ7.3 and 1.1 Hz, ArH),
7.33 (2H, t, Jˆ8.4 Hz, ArH), 7.40 (2H, t, Jˆ7.3 Hz, ArH),
7.59(2H, d, Jˆ7.3 Hz, ArH), 7.76 (2H, d, Jˆ8.4 Hz, ArH)
and 15.28 (1H, s, ArNH); dC (100.4 MHz) 22.6, 28.3, 24.4,
29.6, 29.7, 30.0, 32.0, 33.9, 35.0, 38.4, 39.8, 47.2, 52.3,
53.8, 54.9, 60.4, 65.7, 67.1, 79.3, 107.8, 120.0, 125.1,
126.7, 127.1, 127.8, 129.1, 135.1, 136.9, 141.3, 143.8,
156.3, 171.8, 172.1, 176.5, 196.5 and 200.2; m/z 851 (M111).
CH2C(CH3)2), 2.61 (2H, t, Jˆ5.6 Hz, CH2CO2), 3.00 (2H,
br d, Jˆ5.3 Hz, CH2CH(CH3)2), 3.18 (2H, m, CH2NH), 3.55
(2H, m, CH2NH), 4.02 (1H, m, NH), 4.20 (1H, m,
CHCH2O), 4.39(2H, m, CHC H2O), 5.09(1H, m, NH),
5.18 (1H, m, NH), 5.30 (2H, s, CO2CH2), 6.75 (1H, m,
NH), 7.12 (2H, d, Jˆ8.1 Hz, ArH), 7.30 (2H, dt, Jˆ7.3
and 0.9Hz, ArH), 7.39(4H, t, Jˆ8.4 Hz, ArH), 7.59(2H,
d, Jˆ7.3 Hz, ArH), 7.76 (2H, d, Jˆ8.1 Hz, ArH) and 15.29
(1H, s, ArNH); dC (100.4 MHz) 22.6, 28.2, 24.3, 29.4, 29.5,
30.0, 31.9, 33.9, 34.9, 38.3, 40.3, 47.2, 52.3, 53.4, 53.7,
60.4, 65.6, 66.6, 80.1, 107.8, 119.9, 125.0, 126.7, 127.0,
127.7, 129.1, 135.2, 136.9, 141.3, 143.9, 156.6, 172.0,
172.2, 176.4, 196.4 and 200.2; m/z 851 (M111).
5.2.16. Na-Boc-N1-Fmoc-l-lysine-OPFP 22. A solution
of 9-¯uorenylmethyl chloroformate (0.62 g, 2.40 mmol) in
dioxane (30 ml) was added to a stirred solution of Na-Boc-
l-lysine 21 (0.50 g, 2.05 mmol) in saturated aqueous
sodium hydrogen carbonate (30 ml). After stirring for 24 h
the mixture was concentrated in vacuo and acidi®ed using
dilute hydrochloric acid and extracted using dichloro-
methane (3£50 ml). The combined organic extracts were
dried over magnesium sulphate and concentrated in vacuo
to give an oil which was then reacted with EDCI (0.55 g,
2.86 mmol) and penta¯uorophenol (0.41 g, 2.22 mmol) in
DCM (100 ml). After 24 h, the solvent was washed with
dilute hydrochloric acid (50 ml) and the aqueous layer
was further extracted with DCM (2£50 ml). The combined
organic extracts were dried over magnesium sulphate and
concentrated in vacuo to give a solid, which was puri®ed by
column chromatography. Elution with 20% ethyl acetate in
hexane gave Na-Boc-N1-Fmoc-l-lysine-OPFP 22 (0.76 g,
59%) as a white solid; mp 146±1488C (from DCM/hexane);
(Found C, 60.80; H, 5.19; N, 4.62. C32H31N2O6F5 requires
C, 60.57; H, 4.89; N, 4.42%); nmax (cm21) 3413, 3336, 1787,
1686, 1518, 998 and 758; dH (399.6 MHz) 1.25±1.65 (4H,
m, CH2CH2), 1.44 (9H, s, (CH3)3CO), 1.83±2.00 (2H, m,
CH2), 3.22 (2H, m, CH2NH), 4.20 (1H, t, Jˆ6.8 Hz,
CHCH2O), 4.37±4.83 (4H, m, CHCH2O, CHCO2 and
NH), 5.58 (1H, d, Jˆ7.8 Hz, NH), 7.30 (2H, t, Jˆ7.3 Hz,
ArH), 7.39(2H, t, Jˆ7.3 Hz, ArH), 7.58 (2H, d, Jˆ7.3 Hz,
ArH) and 7.76 (2H, d, Jˆ7.3 Hz, ArH); dC (100.4 MHz)
22.3, 28.3, 29.6, 31.8, 40.4, 47.4, 53.4, 66.7, 77.4, 120.0,
125.1, 127.1, 127.7, 136.7 (m, CF), 138.4 (m, CF), 139.2 (m,
CF), 139.9 (m, CF), 141.0 (m, CF), 141.4, 142.3 (m, CF),
144.0, 155.3, 156.7 and 169.2; m/z 635 (M111).
5.2.18. Boc-glycine-ODmab 25. A solution of EDCI
(0.95 g, 4.96 mmol) and DMAP (62 mg, 0.51 mmol) in
DCM (50 ml) was added to a stirred solution of Boc-glycine
24 (0.82 g, 4.68 mmol) and DmabOH (1.55 g, 4.70 mmol)
in DCM (100 ml) at room temperature under nitrogen. After
24 h, the solvent was washed with dilute hydrochloric acid
(50 ml) and the aqueous layer was further extracted with
DCM (2£50 ml). The combined organic extracts were
dried over magnesium sulphate and concentrated in vacuo
to give a solid which was puri®ed by column chroma-
tography. Elution with 20% ethyl acetate in hexane gave
Boc-glycine-ODmab 25 (2.30 g, 100%) as a white solid;
mp 50±528C (from DCM/hexane); (Found C, 66.52; H,
7.97; N, 5.49. C27H38N2O6 requires C, 66.67; H, 7.82; N,
5.76%); nmax (cm21) 3441, 3364, 2951, 1749, 1710, 1633,
1551 and 754; dH (399.6 MHz) 0.78 (6H, d, Jˆ6.6 Hz,
CH(CH3)2), 1.08 (6H, s, C(CH3)2), 1.45 (9H, s, C(CH3)3),
1.84 (1H, m, CH(CH3)2), 2.40 (2H, s, CH2C(CH3)2),
2.49(2H, s, C H2C(CH3)2), 3.00 (2H, br d, Jˆ6.1 Hz,
CH2CH(CH3)2), 3.99 (2H, d, Jˆ5.6 Hz, NHCH2), 5.07
(1H, m, NHCH2), 5.21 (2H, s, CO2CH2), 7.14 (2H, d, Jˆ
8.3 Hz, ArH), 7.41 (2H, d, Jˆ8.3 Hz, ArH) and 15.30 (1H, s,
ArNH); dC (100.4 MHz) 22.5, 28.2, 29.5, 30.0, 38.3, 42.4,
52.2, 53.7, 66.1, 80.1, 107.8, 126.7, 129.1, 134.9, 137.0,
155.7, 170.2, 176.4, 196.3 and 200.2; m/z 487 (M111).
5.2.19. Glycine-ODmab 26. Tri¯uoroacetic acid (20 ml)
was added to a solution of Boc-glycine-ODmab 25
(0.43 g, 0.89mmol) in DCM (10 ml). After stirring for
30 min, the reaction mixture was concentrated in vacuo to
remove the excess tri¯uoroacetic acid. The residue was
dissolved in DCM (50 ml) and washed with saturated
sodium hydrogen carbonate solution (50 ml). The aqueous
solution was further extracted with DCM (2£50 ml) and the
combined organic extracts were dried over magnesium
sulphate and concentrated in vacuo to give a solid which
was recrystallised using DCM and hexane to give glycine-
ODmab 26 (0.35 g, 78%) as a pale yellow solid; mp 132±
1368C; (Found C, 57.51; H, 6.49; N, 5.53. C24H31N2O6F3
requires C, 57.60; H, 6.20; N, 5.60%); nmax (cm21) 3430,
2958, 1755, 1669, 1558 and 754; dH (DMSO, 399.6 MHz)
0.68 (6H, d, Jˆ6.8 Hz, CH(CH3)2), 0.99 (6H, s, C(CH3)2),
1.70 (1H, m, CH(CH3)2), 2.38 (4H, s, 2£CH2C(CH3)2), 2.96
(2H, br d, Jˆ6.1 Hz, CH2CH(CH3)2), 3.92 (4H, br s,
NH2CH2), 5.28 (2H, s, CO2CH2), 7.33 (2H, d, Jˆ8.6 Hz,
ArH), 7.51 (2H, d, Jˆ8.6 Hz, ArH) and 15.28 (1H, s,
ArNH); dC (100.4 MHz) 22.3, 27.8, 28.8, 29.7, 37.3, 66.1,
107.1, 126.4, 129.1, 134.7, 136.5, 167.6, 175.0, 196.3 and
200.2; m/z 387 (M111).
5.2.17. Na-Boc-N1-Fmoc-l-lysine-b-alanine-ODmab 23.
A
solution of penta¯uorophenyl ester 22 (184 mg,
0.293 mmol) and amine 3 (139mg, 0.35 mmol) in DMF
(2 ml) was stirred for 24 h. The DMF was removed in
vacuo, the residue was dissolved in DCM (50 ml) and
washed with dilute hydrochloric acid (20 ml). The aqueous
layer was further extracted with DCM (2£50 ml) and the
combined organic extracts were dried over magnesium
sulphate and concentrated in vacuo to give a yellow oil
which was puri®ed by column chromatography. Elution
with ethyl acetate gave Na-Boc-N1-Fmoc-l-lysine-b-
alanine-ODmab 23 (125 mg, 50%) as a pale yellow oil;
nmax (cm21) 3393, 2951, 1710, 1648, 1547, 1243, 1162
and 757; dH (399.6 MHz) 0.77 (6H, d, Jˆ6.7 Hz,
CH(CH3)2), 1.07 (6H, s, C(CH3)2), 1.34±1.63 (6H, m,
CH2CH2CH2), 1.42 (9H, s, (CH3)3CO), 1.83 (1H, m,
CH(CH3)2), 2.40 (2H, s, CH2C(CH3)2), 2.49(2H, s,

5438
P. Watts et al. / Tetrahedron 58 (2002) 5427±5439
5.2.20. Fmoc-b-alanine-glycine-ODmab 27. A solution of
penta¯uorophenyl ester 8 (144 mg, 0.30 mmol) and amine 3
(50 mg, 0.10 mmol) in DMF (1 ml) was stirred for 48 h. The
DMF was removed in vacuo, the residue was dissolved in
DCM (50 ml) and washed with dilute hydrochloric acid
(20 ml). The aqueous layer was further extracted with
DCM (2£30 ml) and the combined organic extracts were
dried over magnesium sulphate and concentrated in vacuo
to give a yellow oil which was puri®ed by column chroma-
tography. Elution with ethyl acetate gave Fmoc-b-alanine-
glycine-ODmab 27 (24 mg, 35%) as a colourless oil; dH
(399.6 MHz) 0.69 (6H, d, Jˆ6.5 Hz, CH(CH3)2), 1.00
(6H, s, C(CH3)2), 1.76 (1H, m, CH(CH3)2), 2.32 (2H, m,
CH2CO), 2.41 (2H, s, CH2C(CH3)2), 2.50 (2H, s,
CH2C(CH3)2), 2.45 (2H, m, CH2CO), 2.89(2H, d, Jˆ
5.8 Hz, CH2CH(CH3)2), 3.42 (2H, m, NHCH2), 4.12 (1H,
t, Jˆ6.8 Hz, CHCH2O), 4.27 (2H, d, Jˆ6.8 Hz, CHCH2O),
5.30 (2H, s, CO2CH2), 5.47 (1H, m, NH), 5.70 (1H, br t,
NH), 7.04 (2H, d, Jˆ8.4 Hz, ArH), 7.32 (2H, t, Jˆ7.3 Hz,
ArH), 7.31 (2H, t, Jˆ7.3 Hz, ArH), 7.51 (2H, d, Jˆ7.3 Hz,
ArH), 7.67 (2H, d, Jˆ7.6 Hz, ArH), 7.93 (2H, s, ArH) and
15.21 (1H, s, ArNH); dC (100.4 MHz) 22.6, 28.3, 29.6, 30.1,
32.0, 34.3, 35.4, 38.4, 47.3, 52.3, 53.8, 66.3, 66.7, 107.8,
120.0, 125.2, 126.8, 127.1, 127.7, 129.3, 134.8, 137.1,
141.3, 144.0, 156.6, 171.6, 172.1, 176.5, 196.6 and 200.3;
m/z 680 (M111).
hydrochloric acid (20 ml). The DCM was dried over mag-
nesium sulphate and concentrated in vacuo to give Boc-b-
alanine-piperidine 29 (20 mg, 83%) as a yellow oil; dH
(399.6 MHz) 1.42 (9H, s, (CH3)3CO), 1.51±1.67 (6H, m,
CH2(CH2)3), 2.49(2H, t, Jˆ5.6 Hz, CH2CO), 3.36 (2H, t,
Jˆ5.6 Hz, CH2N), 3.42 (2H, dt, Jˆ5.8 and 5.6 Hz, NHCH2),
3.55 (2H, t, Jˆ5.6 Hz, CH2N) and 5.35 (1H, m, NH); dC
(100.4 MHz) 24.5, 25.6, 26.4, 28.4, 33.3, 36.4, 42.6, 46.4,
79.0, 156.1 and 169.8; m/z 257 (M111).
5.2.23. Fmoc-b-alanine-b-alanine-b-alanine-ODmab 31.
A solution of 9-¯uorenylmethyl chloroformate (0.39 g,
1.92 mmol) in dioxane (10 ml) was added to a stirred solu-
tion of b-ALA-b-ALA (175 mg, 1.09mmol) in saturated
aqueous sodium hydrogen carbonate (10 ml). After stirring
for 24 h the mixture was concentrated in vacuo and acidi®ed
using dilute hydrochloric acid and extracted using dichloro-
methane (3£50 ml). The combined organic extracts were
dried over magnesium sulphate and concentrated in vacuo
to give an oil which was then reacted with EDCI (0.41 g,
2.13 mmol) and penta¯uorophenol (0.42 g, 2.29mmol) in
DCM (50 ml). After 24 h, the solvent was washed with
dilute hydrochloric acid (50 ml) and the aqueous layer
was further extracted with DCM (2£50 ml). The combined
organic extracts were dried over magnesium sulphate and
concentrated in vacuo to give a solid, which was used with-
out further puri®cation. The penta¯uorophenyl ester was
then added to amine 3 (48 mg, 0.12 mmol) in DMF (1 ml)
and the reaction was stirred for 48 h. The DMF was
removed in vacuo, the residue was dissolved in DCM
(50 ml) and washed with dilute hydrochloric acid (20 ml).
The aqueous layer was further extracted with DCM
(2£30 ml) and the combined organic extracts were dried
over magnesium sulphate and concentrated in vacuo to
give a yellow oil which was puri®ed by column chroma-
tography. Elution with 5% methanol in ethyl acetate gave
Fmoc-b-alanine-b-alanine-b-alanine-ODmab 31 (21 mg,
3% over three step synthesis) as a pale yellow gum; dH
(399.6 MHz) 0.76 (6H, d, Jˆ6.7 Hz, CH(CH3)2), 1.07
(6H, s, C(CH3)2), 1.83 (1H, m, CH(CH3)2), 2.39(2H, t,
Jˆ5.9Hz, CH 2CO), 2.39(2H, s, C H2C(CH3)2), 2.49(4H,
br s, CH2C(CH3)2 and CH2CO), 2.56 (2H, t, Jˆ5.9Hz,
CH2CO), 2.99 (2H, d, Jˆ4.5 Hz, CH2CH(CH3)2), 3.43±
5.2.21. Fmoc-b-alanine-ODmab 28. A solution of EDCI
(0.41 g, 2.15 mmol) in DCM (20 ml) was added to a stirred
solution of Fmoc-b-alanine 4 (0.55 g, 1.78 mmol) and
DmabOH (0.59g, 1.80 mmol) in DCM (100 ml) at room
temperature under nitrogen. After 24 h, the solvent was
washed with dilute hydrochloric acid (50 ml) and the
aqueous layer was further extracted with DCM (2£50 ml).
The combined organic extracts were dried over magnesium
sulphate and concentrated in vacuo to give a solid which
was puri®ed by column chromatography. Elution with 20%
ethyl acetate in hexane gave Fmoc-b-alanine-ODmab 28
(1.05 g, 95%) as a white solid; mp 44±468C (from DCM/
hexane); (Found C, 73.61; H, 7.04; N, 4.33. C38H42N2O6
requires C, 73.31; H, 6.75; N, 4.50%); nmax (cm21) 3451,
3365, 2951, 1720, 1638, 1450 and 756; dH (399.6 MHz)
0.77 (6H, d, Jˆ6.1 Hz, CH(CH3)2), 1.08 (6H, s, C(CH3)2),
1.84 (1H, m, CH(CH3)2), 2.40 (2H, s, CH2C(CH3)2), 2.49
(2H, s, CH2C(CH3)2), 2.64 (2H, t, Jˆ6.1 Hz, CH2CO2), 3.00
(2H, br d, Jˆ5.9Hz, C H2CH(CH3)2), 3.51 (2H, dt, Jˆ6.1
and 5.6 Hz, NHCH2), 4.20 (1H, t, Jˆ7.1 Hz, CHCH2O),
4.40 (2H, d, Jˆ7.1 Hz, CHCH2O), 5.17 (2H, s, CO2CH2),
5.29(1H, m, NH), 7.12 (2H, d, Jˆ8.1 Hz, ArH), 7.30 (2H,
dt, Jˆ7.6 and 1.0 Hz, ArH), 7.39(2H, t, Jˆ7.1 Hz, ArH),
7.40 (2H, d, Jˆ8.1 Hz, ArH), 7.59(2H, d, Jˆ7.6 Hz, ArH),
7.76 (2H, d, Jˆ7.6 Hz, ArH) and 15.30 (1H, s, ArNH); dC
(100.4 MHz) 22.6, 28.3, 29.6, 30.2, 34.4, 36.5, 38.3, 47.2,
52.3, 53.8, 65.7, 66.7, 107.8, 120.0, 125.0, 126.7, 127.0,
127.7, 129.0, 135.2, 137.0, 141.3, 143.9, 156.3, 172.1,
176.4, 196.4 and 200.2; m/z 623 (M111).
3.52 (6H, m, 3£NHCH2), 4.19(1H, t,
Jˆ7.0 Hz,
CHCH2O), 4.35 (2H, d, Jˆ7.0 Hz, CHCH2O), 5.11 (2H, s,
CO2CH2), 5.72 (1H, br t, Jˆ5.6 Hz, NH), 6.50 (1H, m, NH),
6.70 (1H, br t, NH), 7.11 (2H, d, Jˆ8.2 Hz, ArH), 7.29±7.40
(6H, m, ArH), 7.58 (2H, d, Jˆ7.6 Hz, ArH), 7.75 (2H, d,
Jˆ7.6 Hz, ArH) and 15.27 (1H, s, ArNH); dC (100.4 MHz)
22.6, 28.2, 29.6, 30.0, 34.0, 35.0, 35.5, 36.1, 37.2, 38.4,
47.2, 52.3, 53.7, 60.4, 65.8, 66.8, 107.8, 120.0, 125.1,
126.7, 127.1, 127.7, 129.1, 135.1, 137.0, 141.3, 143.9,
156.7, 171.8, 172.4, 176.5, 196.5 and 200.3; m/z 765
(M111).
5.2.22. Boc-b-alanine-piperidine 29. Piperidine (20 ml,
0.20 mmol) was added to a stirred solution of Boc-b-
alanine-OPFP 8 (33 mg, 0.09mmol) in DMF (0.5 ml)
under nitrogen. After 10 min the reaction was concentrated
in vacuo and the residue was diluted with DCM (50 ml) and
washed with dilute sodium hydroxide (20 ml) and dilute
Acknowledgements
We wish to thank Novartis Pharmaceuticals (P. W. and C.
W.) for ®nancial support. We are grateful to Dr Tom
McCreedy (University of Hull) for help in fabricating the
micro reactor devices.

P. Watts et al. / Tetrahedron 58 (2002) 5427±5439
5439
21. Jacobson, S. C.; Hergenroder, R.; Koutny, L. B.; Ramsey, J. M.
Anal. Chem. 1994, 66, 1114.
References
22. Merri®eld, R. B. J. Am. Chem. Soc. 1963, 85, 2149.
23. Munster, P.; Steglich, W. Synthesis 1987, 223.
24. Penke, B.; Rivier, J. J. Org. Chem. 1987, 52, 1197.
25. Whitney, D. B.; Tam, J. P.; Merri®eld, R. B. Tetrahedron
1984, 40, 4237.
1. Haswell, S. J. Analyst 1997, 112, 1R.
2. Manz, A.; Harrison, D. J.; Verpoorte, E.; Fettinger, J. C.; Ludi,
H.; Widmer, H. M. Chimia 1991, 45, 103.
3. Manz, A.; Harrison, D. J.; Verpoorte, E.; Widmer, H. M. Adv.
Chromatogr. 1993, 33, 1.
26. Chan, W. C.; White, P. D. Fmoc Solid Phase Peptide
Synthesis; Oxford University Press: New York, 2000.
27. McCreedy, T. TrAC 2000, 19, 396.
4. Manz, A.; Effenhauser, C. S.; Burggraf, N.; Verpoorte, E.;
Raymond, D. E.; Widmer, H. M. Anal. Mag. 1994, 22, M25.
5. Proceedings of the Micro Total Analytical Systems 98' Work-
shop; Harrison, D. J., van den Berg, A., Eds.; Kluwer
Academic: Dordrecht, 1998.
28. Micro Total Analysis Systems 2001; Ramsey, J. M., van den
Berg, A., Eds.; Kluwer Academic: Dordretch, 2001.
29. McCreedy, T. Anal. Chim. Acta 2001, 427, 39 .
30. Christensen, P. D.; Johnson, S. W. P.; McCreedy, T.; Skelton,
V.; Wilson, N. G. Anal. Commun. 1998, 35, 341.
31. Daura, X.; Gademann, K.; Schaefer, H.; Jaun, B.; Seebach, D.;
van Gunsteren, W. F. J. Am. Chem. Soc. 2001, 123, 2393.
32. Seebach, D.; Schreiber, J. V.; Abele, S.; Daura, X.; van
Gunsteren, W. F. Helv. Chim. Acta 2000, 83, 34.
33. Dado, G. P.; Gellman, S. H. J. Am. Chem. Soc. 1994, 116,
1054.
6. van den Berg, A.; Lammerink, T. S. J. Top. Curr. Chem. 1998,
194, 21.
7. Harrison, D. J.; Fluri, K.; Seiler, K.; Fan, Z. H.; Effenhauser,
C. S.; Manz, A. Science 1993, 261, 895.
8. Jacobson, S. C.; Hergenroder, R.; Koutny, L. B.; Ramsey, J. M.
Anal. Chem. 1994, 66, 1114.
9. Spence, D. M.; Crouch, S. R. Anal. Chem. 1998, 358, 9 5.
10. Schoot, B. H.; Jeanneret, S.; Berg, A. Anal. Meth. Instrum.
1993, 1, 38.
34. Gademann, K.; Ernst, M.; Seebach, D.; Hoyer, D. Helv. Chim.
Acta 2000, 83, 16.
11. Das Gupta, P. K.; Lui, S. Anal. Chem. 1994, 66, 1792.
12. Fletcher, P. D. I.; Haswell, S. J.; Paunov, V. N. Analyst 1999,
124, 1273±1282.
35. Werder, M.; Hauser, H.; Abele, S.; Seebach, D. Helv. Chim.
Acta 1999, 82, 1774.
13. Greenway, G. M.; Haswell, S. J.; Morgan, D. O.; Skelton, V.;
Styring, P. Sens. Actuators, B 2000, 63, 153.
14. Skelton, V.; Greenway, G. M.; Haswell, S. J.; Styring, P.;
Morgan, D. O.; Warrington, B.; Wong, S. Y. F. Analyst
2001, 126, 7.
36. Arvidsson, P. I.; Rueping, M.; Seebach, D. Chem. Commun.
2001, 649.
37. Schreiber, J. V.; Seebach, D. Helv. Chim. Acta 2000, 83, 3139.
38. Chan, W. C.; Bycroft, B. W.; Evans, D. J.; White, P. D. Chem.
Commun. 1995, 2209.
15. Skelton, V.; Greenway, G. M.; Haswell, S. J.; Styring, P.;
Morgan, D. O.; Warrington, B.; Wong, S. Y. F. Analyst
2001, 126, 11.
39. Kisfaludy, L.; Schon, I. Synthesis 1983, 325.
40. Atherton, E.; Cameron, L. R.; Sheppard, R. C. Tetrahedron
1988, 44, 843.
16. Salimi-Moosavi, H.; Tang, T.; Harrison, D. J. J. Am. Chem.
Soc. 1997, 119, 8716.
41. Johnson, T.; Quibell, M.; Owen, D.; Sheppard, R. C. Chem.
Commun. 1993, 369.
17. Sands, M.; Haswell, S. J.; Kelly, S. M.; Skelton, V.; Morgan,
D. O.; Styring, P.; Warrington, B. H. Lab on a Chip 2001, 1,
64.
42. Carpino, L. A.; Han, G. Y. J. Org. Chem. 1972, 37, 3404.
43. Carpino, L. A.; Cohen, B. J.; Stephens, K. E.; Sadat-Aalaee,
S. J.; Tien, J.-H.; Langridge, D. E. J. Org. Chem. 1986, 51,
3732.
18. Watts, P.; Wiles, C.; Haswell, S. J.; Pombo-Villar, E.; Styring,
P. Chem. Commun. 2001, 990.
44. Wade, J. D.; Bedford, J.; Sheppard, C.; Tregear, G. W. Pept.
Res. 1991, 4, 194.
19. Haswell, S. J.; Middleton, R. J.; O'Sullivan, B.; Skelton, V.;
Watts, P.; Styring, P. Chem. Commun. 2001, 391.
20. Fletcher, P. D. I.; Haswell, S. J. Chem. Br. 1999, 35, 38.
45. Kates, S. A.; Nuria, A.; Sole, M.; Beyermann, M.; Barany, G.;
Albericio, F. Pept. Res. 1996, 9, 106.