Synthesis of peptides containing DOPA (3,4-dihydroxyphenylalanine)

Article abstract of DOI:10.1016/S0040-4020(01)00601-9

Proteins from coral reefs structures, eggshells, and marine mollusk adhesives all contain the amino acid 3,4-dihydroxyphenylalanine (DOPA). The insoluble nature of these materials has hampered characterization and turned our efforts toward work with small peptide mimics. In this paper, we present the syntheses of various DOPA derivatives: Boc-DOPA, Fmoc-DOPA, DOPA(TBDMS)₂, DOPA(TBDPS)₂, Boc-DOPA(TBDPS)₂, Fmoc-DOPA(TBDMS)₂, and Fmoc-DOPA(TBDPS)₂ (where Boc=tert-butyloxycarbonyl, Fmoc=9-fluorenylmethyloxycarbonyl, TBDMS=tert-butyldimethylsilyl, and TBDPS=tert-butyldiphenylsilyl). These DOPA compounds were used to prepare peptides of various sequences. The synthetic procedure described provides an efficient route to DOPA-containing peptides in which sidechain deprotection and cleavage from resin can be accomplished in one step.

Full text of DOI:10.1016/S0040-4020(01)00601-9

TETRAHEDRON
Pergamon
Tetrahedron 57 (2001) 6139±6146
Synthesis of peptides containing DOPA
(3,4-dihydroxyphenylalanine)
Mary J. Sever and Jonathan J. Wilkerp
Department of Chemistry, 1393 H.C. Brown Laboratories, Purdue University, West Lafayette, IN 47907-1393, USA
Received 5 February 2001; accepted 30 May 2001
AbstractÐProteins from coral reefs structures, eggshells, and marine mollusk adhesives all contain the amino acid 3,4-dihydroxypheny-
lalanine (DOPA). The insoluble nature of these materials has hampered characterization and turned our efforts toward work with small
peptide mimics. In this paper, we present the syntheses of various DOPA derivatives: Boc-DOPA, Fmoc-DOPA, DOPA(TBDMS)2,
DOPA(TBDPS)2, Boc-DOPA(TBDPS)2, Fmoc-DOPA(TBDMS)2, and Fmoc-DOPA(TBDPS)2 (where Bocˆtert-butyloxycarbonyl,
Fmocˆ9-¯uorenylmethyloxycarbonyl, TBDMSˆtert-butyldimethylsilyl, and TBDPSˆtert-butyldiphenylsilyl). These DOPA compounds
were used to prepare peptides of various sequences. The synthetic procedure described provides an ef®cient route to DOPA-containing
peptides in which sidechain deprotection and cleavage from resin can be accomplished in one step. q 2001 Elsevier Science Ltd. All rights
reserved.
1. Introduction
reactions. Additional deprotection steps relying upon rather
harsh reagents were required speci®cally for DOPA.
The ubiquitous nature of 3,4-dihydroxyphenylalanine
(DOPA) touches biological ®elds ranging from medicine
to engineering. Most commonly known for pharmacological
value, l-DOPA has been the treatment of choice to alleviate
Parkinson's Disease symptoms since the late 1960s.1 DOPA
is also found as an amino acid residue in the proteins of
diverse animal species. For the most part, these proteins
play structural roles in the synthesis of biological materials.
Examples of substances based upon DOPA-containing
proteins include coral reef structures,2 eggshells,3,4
seashells,4 and adhesives produced by mollusks.4±7 In
these proteins, DOPA is derived from posttranslational
oxidation of tyrosine. Subsequent cross-linking of these
soluble proteins creates the requisite, hardened matrices of
which these materials are comprised.3,4 Such cross-linked
structures tend to be insoluble and, consequently, dif®cult to
study.
Our goal was to synthesize peptides containing the DOPA
residue employing one step deprotection and cleavage with
standard reagents. In this paper, we report the preparation of
various DOPA derivatives and their applications to peptide
synthesis. The success of our procedure is demonstrated by
the preparation of peptides with sequences derived from an
adhesive precursor protein from the common blue mussel,
Mytilus edulis.
2. Results
Solid phase peptide synthesis conditions require protection
of both the amine and side chain catecholic oxygens of
DOPA.19 Amine protections are largely reliant upon two
groups: tert-butyloxycarbonyl (Boc) and 9-¯uorenylmethyl-
oxycarbonyl (Fmoc).19 An equimolar reaction of DOPA and
di-tert-butyl dicarbonate afforded Boc-DOPA. Likewise,
Fmoc-DOPA was prepared by the 1:1 reaction of DOPA
and 9-¯uorenylmethyloxycarbonyl chloride (Fmoc-Cl).
We chose to focus our synthetic efforts upon Fmoc over
Boc chemistry owing to cleaner syntheses and no need to
work with gaseous hydro¯uoric acid.20,21
In order to understand the chemistry of these fascinating
biological materials, we have turned our attention to work
with small, soluble peptides containing the DOPA amino
acid residue. Previous reports have shown the successful
preparation of peptides containing DOPA.8±18 In most of
these methods, however, protecting groups for the
catecholic oxygens of the DOPA sidechain remained intact
after standard peptide deprotection and resin cleavage
The ®nal step of Fmoc-based solid phase peptide syntheses
involves treatment with tri¯uoroacetic acid (TFA) to effect
deprotection of all residue sidechains and cleavage from
the resin. Consequently, we discuss the use of acid
labile protecting groups for the DOPA catecholic oxygens.
Two acid labile groups employed commonly in catechol
Keywords: amino acids and derivatives; marine metabolites; peptides and
polypeptides; solid-phase synthesis.
p
Corresponding author. Tel.: 1765-496-3382; fax: 1765-494-0239;
e-mail: wilker@purdue.edu
0040±4020/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(01)00601-9

6140
M. J. Sever, J. J. Wilker / Tetrahedron 57 (2001) 6139±6146
TBDPS-Cl, and DBU in acetonitrile. The Boc group was
removed by stirring Boc-DOPA(TBDPS)2 for 24 h in ethyl
acetate saturated with gaseous HCl. Final amine protection
was accomplished by reaction of DOPA(TBDPS)2 with
Fmoc-Cl. Scheme 3 summarizes the synthetic route to
Fmoc-DOPA(TBDPS)2.
After synthesis of Fmoc-DOPA(TBDMS)2, we wished to
ensure that chirality was maintained from the l-DOPA start-
ing material. The Fmoc protecting group was removed by
reaction of Fmoc-DOPA(TBDMS)2 with diethylamine to
yield DOPA(TBDMS)2. Subsequent action of tetrabutyl-
ammonium ¯uoride (TBAF) provided the desired DOPA.
This DOPA was then reacted with N-a-(5-¯uoro-2,4-dini-
trophenyl)-l-valine amide, FDNP-Val-NH2, a common
reagent for chiral resolution by high pressure liquid
chromatography, HPLC.31 FDNP-Val-NH2 was also reacted
with l-DOPA and d,l-DOPA. Fig. 1 shows HPLC traces of
each of the three DOPA sources after reaction with FDNP-
Val-NH2. HPLC of the l-DOPA reaction product shows
many peaks. The analogous trace from d,l-DOPA shows
all the same peaks as l-DOPA along with the addition of
four new species observed at 35.03, 45.43, 48.25, and
52.02 min. HPLC analysis of DOPA prepared from Fmoc-
DOPA(TBDMS)2 showed only those peaks attributable to
l-DOPA. These results indicate that the chirality of Fmoc-
DOPA(TBDMS)2 remains intact.
Scheme 1.
protection are the acetonide22,23 and the diphenylmethylene
ketal (Scheme 1).24±27 Surprisingly, we were unable to
prepare acetonide or diphenylmethylene ketal derivatives
of DOPA. In our hands, however, comparable protection
of catechol worked as presented in the literature.22,24 We
attempted the preparation of DOPA derivatives containing
acetonide or diphenylmethylene ketal groups using each of
DOPA, Boc-DOPA, and Fmoc-DOPA for starting materials.
Reactions of acetone with these DOPA compounds under
various conditions of temperature, solvent (toluene,
benzene, or THF), and reagents (P2O5 or para-toluenesulfo-
nic acid) failed to provide the acetonide in acceptable yield.
The reagent H3C±CBr2±CH3 also did not afford a DOPA-
acetonide species. Likewise, protection of DOPA,
Boc-DOPA, or Fmoc-DOPA by reaction with Cl2C(C6H5)2
failed to afford the appropriate diphenylmethylene ketal to
any appreciable extent.24±27
Successful preparations of Fmoc-DOPA(TBDMS)2 and
Fmoc-DOPA(TBDPS)2 enabled synthesis of DOPA-contain-
ing peptides. Manual, standard solid phase methodologies
were used to prepare the peptides.32 Brie¯y, amide forma-
tion was effected from the amino acid, and 1,3-dicyclo-
hexylcarbodiimide (DCC). In the case of asparagine,
1-hydroxybenzotriazole (HOBT) was used for coupling in
addition to DCC. Fmoc removal was accomplished with a
50% solution of piperidine in N,N-dimethylformamide
(DMF). The Kaiser test33 was used to assess coupling
ef®ciency.
The tert-butyldimethylsilyl protecting group, TBDMS, is
acid labile28 and has been installed on DOPA successfully.29
After preparation of DOPA(TBDMS)2, we effected amine
protection in a straightforward manner by reaction with
Fmoc-Cl (Scheme 2). Puri®ed Fmoc-DOPA(TBDMS)2 is
then suitable for use in solid phase peptide synthesis (vide
infra).
The analogous group tert-butyldiphenylsilyl, TBDPS, is
more stable with regard to hydrolysis relative to TBDMS
and preferable for long term storage.30 Preparation of
DOPA(TBDPS)2, however, did not proceed as well as that
found for DOPA(TBDMS)2. Reaction of DOPA, TBDPS-
Cl, and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) failed to
yield DOPA(TBDPS)2 to any appreciable extent. Reaction
of TBDPS-Cl with Fmoc-DOPA was also unsuccessful
owing to Fmoc deprotection by the DBU used for silylation.
Successful installation of TBDPS onto DOPA required
using Boc-DOPA for starting material. Intermediate Boc-
DOPA(TBDPS)2 was prepared by reaction of Boc-DOPA,
Using Fmoc-DOPA(TBDMS)2, we prepared peptides of
sequences Pro-DOPA-Val, Asn-DOPA-Arg-Gly (Scheme
4), and Gly-DOPA-Arg-Gly-Asn-DOPA. Amide formation
involving the acids of either DOPA or arginine required
excess coupling reagents and reactions times. Excellent
coupling ef®ciencies, however, did result. We chose to
cap the N-terminus by acetylation with acetic anhydride.
Cleavage from the Rink resin and deprotection of the side
chains was accomplished in one step by the use of a solution
containing 90% TFA, 5% thioanisol, 3% ethanedithiol, and
2% anisol. In order to optimize time of the deprotection/
Scheme 2.

M. J. Sever, J. J. Wilker / Tetrahedron 57 (2001) 6139±6146
6141
Scheme 3.
cleavage reaction, we performed a kinetic experiment using
HPLC on the 4-mer peptide, Asn-DOPA-Arg-Gly. As can
be seen in Fig. 2, treating the resin with the TFA solution for
4 h resulted in three major peaks. Mass spectral analysis of
the isolated peaks provided assignments of the completely
deprotected peptide at ,9 min retention time (mass calcd:
[M1H]ˆ566.2687. Found: 566.2709) and mono-TBDMS
peptides at ,22 and ,26 min (mass calcd: [M1H]ˆ681.4.
Found: 681.6). Presumably, these two peaks arise from
monosilylation in the 3- and 4-positions. The di-TBDMS
peptide remains soluble in the ether precipitating solution
according to ultraviolet±visible spectroscopic data (not
shown). Reaction of the peptide with TFA solution beyond
the initial 4 h showed decreasing amounts of silyl protected
peptide and an increased yield of target 4-mer. Peak inte-
gration provided the following yields of 4-mer normalized
to the 24 h trace: 4 hˆ2.9%, 8 hˆ12%, 12 hˆ42%, and
24 hˆ84%. Selective nitration34 of this 4-mer demonstrated
the presence of DOPA in the peptide (data not shown). We
also prepared the 4-mer Asn-DOPA-Arg-Gly using Fmoc-
DOPA(TBDPS)2, but found deprotection/cleavage to be
impractical using only this TFA solution for deprotection.
The HPLC traces of Fig. 2 show that the above method
provides deprotected peptide after 24 h reaction in the
Figure 1. HPLC traces showing absorbance at 340 nm for reaction products
of FDNP-Val-NH2 with A) l-DOPA, B) d,l-DOPA C) fully deprotected
Fmoc-DOPA(TBDMS)2. The species attributable to d-DOPA are noted
with an asterick.
Scheme 4.

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M. J. Sever, J. J. Wilker / Tetrahedron 57 (2001) 6139±6146
Figure 3. HPLC trace monitored at 214 nm for the peptide Asn-DOPA-
Arg-Gly after synthesis and complete deprotection employing ¯uoride ion.
The peptide peak appears at ,9 min. The broad peak at ,3 min is from
acetic acid in which the peptide is stored in order to maintain stability of the
DOPA residue.
Figure 2. HPLC traces monitored at 280 nm for the deprotection and clea-
vage reaction of Asn-DOPA-Arg-Gly. Traces shown are for varied reaction
times of the resin-bound, protected peptide with a 90% TFA solution. The
label Asn-DOPA(Si)-Arg-Gly indicates the mono-TBDMS protected
species.
THF with addition of TBAF to the TFA cleavage/deprotec-
tion solution. In a typical deprotection and cleavage proce-
dure, the resin-bound peptide is reacted with TBAF in THF
for 5 min. The resin is then placed in the TFA solution
containing TBAF. Peptide is obtained by precipitation in
diethyl ether (see Section 4). Analogous reactions with
peptide synthesized from Fmoc-DOPA(TBDPS)2 exhibited
decomposition (absorption ,380 nm) upon deprotection
using these conditions. Fig. 3 shows an HPLC trace of
the 4-mer TBDMS peptide deprotected with TBAF accord-
ing to the above-mentioned procedure. We prepared and
deprotected the 3-mer Pro-DOPA-Val and 6-mer Gly-
DOPA-Arg-Gly-Asn-DOPA peptides by analogous methods.
TFA solution. This long TFA treatment, however, is both of
a cumbersome length and may not be suitable for use with
hydrolytically sensitive peptide sequences. Consequently,
we sought deprotection times to minimize exposure to
TFA. An excellent means of removing silyl groups is by
use of the ¯uoride anion.35
Reactions of TBAF with the silylated 4-mer DOPA peptide,
both on the resin and during cleavage reactions, were
examined. These studies exhibited a signi®cant dependence
upon solvent. Dichloromethane reactions of TBAF and
resin-bound peptide always resulted in decomposition of
the DOPA peptide after cleavage from resin, as evidenced
by absorption at ,380 nm (data not shown). Presumably,
this decomposition arises from the basic nature of ¯uoride36
and the known sensitivity of DOPA to base.37 A range of
reaction conditions in CH2Cl2 were examined, all of which
exhibited DOPA decomposition: 1±10 equiv. TBAF per
peptide (i.e. 0.5±5 equiv. per silyl group) and reaction
times of 1±30 min. Reactions in DMF exhibited slow depro-
tection and partial decomposition of DOPA. For DMF,
conditions were varied amongst 2±5 equiv. TBAF per
peptide and times of 1±225 min, none of which were satis-
factory. Use of tetrahydrofuran (THF) proved to be the most
successful. Reaction conditions examined included 2±
50 equiv. of TBAF per peptide and times of 5±150 min.
Longer reaction times (.5 min) or greater quantities of
TBAF (.6 equiv.) tended toward decomposition of
DOPA. We found THF reactions with 5 equiv. of TBAF
per peptide for 5 min to work best in terms of product
yield and lack of decomposition.
3. Discussion
We sought compounds and procedures to enable preparation
of DOPA-containing peptides with no necessary deviations
from standard protocols. Our chemistry is based upon Fmoc
amine protection in order to avoid the use of gaseous HF and
obtain cleaner peptide products relative to Boc methods.20,21
Consequently, we protected the DOPA sidechain catecholic
oxygens with acid labile groups. Surprisingly, we could not
prepare the acetonide or diphenylmethylene ketal deriva-
tives of DOPA. Analogous reactions with catechol did
yield the desired products. Apparently, the organic chemis-
try of DOPA is distinct enough from catechol to create these
differences in reactivity. Addition of two TBDMS protect-
ing groups to DOPA, by contrast, was straightforward and
useful in preparation of our ®nal reagent for peptide
synthesis, Fmoc-DOPA(TBDMS)2 (Scheme 2). This
compound was prepared in high purity and free from
common dipeptide side products.38 Full deprotection of
Fmoc-DOPA(TBDMS)2 and reaction with FDNP-Val-NH2
proved that the chiral integrity of starting l-DOPA persisted
throughout synthesis and deprotection.
We also examined deprotection of the silylated 4-mer
DOPA peptide by reactions with TBAF added to the TFA
cleavage/deprotection solution. Again, a range of conditions
were studied, including 2±125 equiv. TBAF per peptide and
total reaction times of 1±3 h. In the interest of minimizing
deprotection times and exposure of the peptide to TFA, we
chose 50 equiv. of TBAF per peptide and a time of 1 h. For
®nal optimization of our synthetic method in terms of
removing TBDMS groups (determined by HPLC) and
total yield of peptide (determined by UV±vis), we
combined a TBAF reaction of the resin-bound peptide in
Application of the TBDPS group to DOPA protection was
more complicated than TBDMS. An ef®cient route to
Fmoc-DOPA(TBDPS)2 was found using the intermediates
Boc-DOPA(TBDPS)2 and DOPA(TBDPS)2 (Scheme 3).
While our work was in progress, a report appeared in
which the activated penta¯uorophenolate ester of Fmoc-
DOPA(TBDMS)2 was prepared.39 Although this paper did
not focus upon synthesis, the authors' use of the TBDMS
protecting group is consistent with our ®ndings of compat-
ibility with peptide preparations.

M. J. Sever, J. J. Wilker / Tetrahedron 57 (2001) 6139±6146
6143
Construction of peptides with the DOPA residue using
Fmoc-DOPA(TBDMS)2 has proven to be simple and ef®-
cient. Our ®rst synthetic target, Asn-DOPA-Arg-Gly, was
based upon sequence data from the adhesive protein Mefp-3
of the common blue mussel Mytilus edulis (Scheme 4).40 In
keeping with our goal of developing a synthetic procedure
using standard solid phase synthetic methodologies, we
found a reaction time of 24 h to be best suited for cleavage
from the solid support and sidechain deprotection. Although
this procedure worked well, exposure to TFA for such long
periods may diminish the utility of this method for peptides
of hydrolytically sensitive compositions. Consequently, we
explored reactions with ¯uoride ion to facilitate desilylation
of the DOPA residue. Previous reports demonstrated
compatibility of ¯uoride with derivatives of all twenty
standard amino acids used in peptide synthesis.41±43 In our
®nal procedure, we pretreated the peptide with ¯uoride
while remaining bound to the resin. Subsequently, we
completed desilylation by addition of ¯uoride to the TFA
cleavage/deprotection solution.
N-a-Fmoc-l-asparagine (Fmoc-Asn-OH), N-a-Fmoc-
NG-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-l-
arginine
(Fmoc-Arg(Pbf)-OH),
N-a-Fmoc-l-glycine
(Fmoc-Gly-OH), and Rink Amide resin. Piperidine and
diethylamine were purchased from Fisher Scienti®c.
4.2. High pressure liquid chromatography
Data were obtained using a Waters HPLC system composed
of a model 600 controller, a Delta 600E multisolvent
delivery system pump, a model 2487 dual wavelength
absorbance detector, and a Symmetry C 18 5 mm
(3.9£150 mm) column. Optical detection was monitored
at 214 and 280 nm. All samples were run for 30 min with
gradient proportioning between 0.1% TFA in water and
0.1% TFA in acetonitrile. The mobile phase began at
100% of 0.1% TFA in water and was ramped to 70% of
0.1% TFA in water and 30% of 0.1% TFA in acetonitrile
over 15 min. The eluent was continued at this 70/30 mixture
for an additional 15 min. Data were collected on a personal
computer using ChromPerfecte software (Justice Innova-
tions).
After preparation of Asn-DOPA-Arg-Gly, we examined the
generality of this synthetic method for DOPA-containing
peptides. The 6-mer Gly-DOPA-Arg-Gly-Asn-DOPA was
prepared successfully. Proline and valine are, typically,
two of the more dif®cult amino acids to include in
peptides.21 Thus, we sought a peptide containing proline,
valine, and DOPA. Synthesis of the 3-mer Pro-DOPA-Val
proceeded smoothly.
4.3. Synthesis
4.3.1. N-(tert-Butyloxycarbonyl)-3,4-dihydroxy-l-phenyl-
alanine (Boc-DOPA). This compound was prepared by
modi®cation of a general literature procedure for Boc
protection of amines.32 Dioxane (50 mL), water (25 mL),
1 M NaOH (25.4 mL, 25.4 mmol), and l-DOPA (5.00 g,
25.4 mmol) were stirred together for 10 min. After addition
of tert-butyl dicarbonate (5.55 g, 25.4 mmol), the reaction
was stirred for 20 h. Concentration of the solution in vacuo
to approximately 85 mL was followed by cooling on an ice
bath. Ethyl acetate (75 mL) was added and the mixture
acidi®ed to pH<2.5 with a dilute solution of KHSO4 (10 g
in 100 mL). This mixture was added to a separatory funnel
to divide the aqueous and organic layers. The aqueous layer
was washed with ethyl acetate (2£30 mL) and all organic
fractions were combined. The organic layer was washed
with water (3£30 mL), brine (3£30 mL), and dried over
anhydrous Na2SO4. A brown, foamy solid was obtained
after solvent removal in vacuo (6.20 g, 82.0% crude yield).
In conclusion, we have presented a series of compounds and
procedures for incorporating DOPA into peptides. This
chemistry requires no signi®cant deviation from standard,
solid phase peptide synthesis protocols. Such an easy route
to DOPA-containing peptides is sure to facilitate studies of
fascinating biological materials such as coral reefs, shells,
and molluskan adhesives.
4. Experimental
4.1. General procedures
All syntheses were carried out under an argon atmosphere
using standard Schlenk techniques. NMR spectra were
recorded on a Varian INOVA 300 spectrometer. Silica gel
for column chromatography was 230±400 mesh. TLC plates
were stained with aqueous solutions of Fe(NO3)3 as well as
I2. The following reagents were purchased from Acros
Organics: tert-butyldimethylsilyl chloride (TBDMS-Cl),
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 1,3-dicyclo-
hexylcarbodiimide (DCC), and l-3,4-dihydroxyphenyly-
alanine (l-DOPA). The following were purchased from
Advanced Chemtech: acetic anhydride, N,N-diisopropyl-
ethylamine (DIEA), 9-¯uorenylmethyloxycarbonyl chloride
(Fmoc-Cl), N-a-Fmoc-l-proline (Fmoc-Pro-OH), and N-a-
Fmoc-l-valine (Fmoc-Val-OH). The following were
purchased from Aldrich: tetrabutylammonium ¯uoride
hydrate (TBAF), tert-butyldiphenylsilyl chloride (TBDPS-
Cl), di-tert-butyl dicarbonate, l-3,4-dihydroxyphenyly-
alanine (l-DOPA), d,l-3,4-dihydroxyphenylalanine (d,l-
DOPA), and 1-hydroxybenzotriazole hydrate (HOBT).
The following were purchased from Novabiochem:
In a typical puri®cation, crude Boc-DOPA (0.753 g,
2.53 mmol) was loaded onto
a silica gel column
(2.5£30 cm) and eluted with dichloromethane (500 mL),
1.5% methanol in dichloromethane (4 L), 3.0% methanol
in dichloromethane (1 L), and 5.0% methanol in dichloro-
methane (1 L). Final yield of the colorless solid after puri-
1
®cation was 40.3%. H NMR (DMSO-d6) d 1.32 (s, 9H),
2.55±2.86 (m, 2H), 3.90±4.01 (m, 1H), 6.42±6.96 (m, 4H),
8.57±8.82 (br, 2H). 13C{1H} NMR (DMSO-d6) d 28.3,
36.01, 55.6, 78.1, 115.4, 116.6, 119.9, 128.8, 143.8, 144.9,
155.5, 173.9. Mass spec. Calcd for C14H19NO6:
[M1H]ˆ298.1291. Found: 298.1299.
4.3.2. N-(9-Fluorenylmethyloxycarbonyl)-3,4-dihydroxy-
l-phenylalanine (Fmoc-DOPA). Dioxane (36.8 mL), a
10% Na2CO3 solution (36.8 mL, 31.8 mmol), and l-
DOPA (2.50 g, 12.7 mmol) were stirred on an ice bath
10 min prior to addition of Fmoc-Cl (3.29 g, 12.7 mmol)
over 50 min. The reaction was stirred 4 h on an ice bath

6144
M. J. Sever, J. J. Wilker / Tetrahedron 57 (2001) 6139±6146
and 25 h at room temperature. The cream colored suspen-
sion was added to a separatory funnel and washed with ether
(3£100 mL). The aqueous layer was cooled on an ice bath
and acidi®ed to pH<3 using 6 M HCl. This aqueous solu-
tion was added to a separatory funnel and extracted with
ethyl acetate (3£100 mL). Ethyl acetate layers were
combined, washed with brine (3£100 mL), and dried over
anhydrous Na2SO4. Solvent was removed in vacuo to yield
3.12 g, 58.6% of a light brown, foamy solid.
(3£75 mL). After drying over anhydrous Na2SO4, solvent
was removed in vacuo to yield 13.0 g, 79.2% of light brown
foam.
In a typical puri®cation, crude Fmoc-DOPA(TBDMS)2
(6.57 g, 10.1 mmol) was loaded onto a silica gel column
(2.5£30 cm) and eluted with dichloromethane (2 L),
0.75% methanol in dichloromethane (2.5 L), 1.5% methanol
in dichloromethane (1.5 L), and 3% methanol in dichloro-
methane (5 L). The resulting colorless solid was obtained in
a ®nal, puri®ed yield of 52.0%. 1H NMR (DMSO-d6) d 0.14
(s, 6H), 0.16 (s, 6H), 0.94 (s, 9H), 0.95 (s, 9H), 2.66±3.10
(m, 2H), 4.00±4.36 (m, 3H), 6.78 (m, 3H), 7.31 (m, 2H),
7.42 (m, 2H), 7.68 (m, 2H), 7.90 (d, 2H), 12.3±13.1 (br,
1H). 13C{1H} NMR (DMSO-d6) d 24.3, 18.0, 25.6, 25.7,
35.8, 46.6, 53.4, 55.7, 65.7, 66.4, 120.0, 120.3, 121.7, 122.3,
125.2, 125.3, 127.0, 127.5, 131.3, 140.7, 143.7, 143.8,
144.6, 145.7, 156.0, 173.5. Mass spec. Calcd for
C36H49NO6Si2: [M1H]ˆ648.3177. Found: 648.3165.
Anal. Calcd for C36H49NO6Si2: C, 66.73; H, 7.62; N, 2.16.
Found: C, 66.56; H, 7.68; N, 2.42.
In a typical puri®cation, crude Fmoc-DOPA (0.760 g,
1.81 mmol) was loaded onto
a silica gel column
(2.5£30 cm) and eluted with dichloromethane (500 mL)
and 1.5% methanol in dichloromethane (4 L). Final yield
of the colorless solid after puri®cation was 64.6%. 1H
NMR (DMSO-d6) d 2.59±2.96 (m, 2H), 6.46±6.70 (m,
3H), 7.30 (q, 2H), 7.39 (m, 2H), 7.64 (m, 3H), 7.87 (d,
2H), 8.68 (s, 1H), 8.72 (s, 1H), 12.3±13.0 (br, 2H).
13C{1H} NMR (DMSO-d6) d 36.8, 56.7, 66.4, 116.1,
117.2, 120.6, 120.8, 126.0, 126.1, 127.8, 128.4, 129.4,
141.4, 144.5, 144.6, 145.7, 156.7, 174.3. Mass spec. Calcd
for C24H21NO6: [M1H]ˆ420.1447. Found: 420.1434.
4.3.5. 3,4-Bis(tert-butyldiphenylsilyloxy)-N-tert-butyloxy-
carbonyl-l-phenylalanine (Boc-DOPA(TBDPS)2). Ace-
tonitrile (7.5 mL), Boc-DOPA (4) (1.01 g, 3.41 mmol),
and TBDPS-Cl (2.57 mL, 9.88 mmol) were combined and
cooled on an ice bath. After 10 min stirring, DBU (1.54 mL,
10.2 mmol) was added over 15 min. Stirring continued on
the ice bath for 4 h and then at room temperature for 40 h.
The light brown solution was poured into a separatory
funnel with water (50 mL) and chloroform (50 mL). The
aqueous layer was washed with chloroform (3£50 mL)
and all organic fractions combined. After washing with
water (3£50 mL), brine (3£50 mL), and drying over
anhydrous Na2SO4, the solvent was removed in vacuo to
yield 2.93 g, 110.9% of crude, light brown foam.
4.3.3. 3,4-Bis(tert-butyldimethylsilyloxy)-l-phenylalanine
(DOPA(TBDMS)2). To a stirred solution of TBDMS-Cl
(6.25 g, 41.5 mmol) in anhydrous acetonitrile (31.0 mL)
was added l-DOPA (2.72 g, 13.8 mmol). The colorless
suspension was cooled on an ice bath for 10 min prior to
addition of DBU (6.24 mL, 41.4 mmol) over 10 min. The
reaction was left stirring on an ice bath for 4 h, followed by
further stirring at room temperature for an additional 20 h.
Solvent was removed in vacuo and the colorless solid
transferred to a fritted glass funnel where it was washed
with cold chloroform (150 mL). The solid was dissolved
in methanol (150 mL) and ®ltered. Solvent was removed
in vacuo to yield a crude, colorless solid (5.96 g, 101%).
Alternatively, addition of cold (2208C) acetonitrile to the
reaction solution results in a colorless precipitate. Filtration
and washing with cold acetonitrile yields crude
DOPA(TBDMS)2.
In a typical puri®cation, crude Boc-DOPA(TBDPS)2
(0.754 g, 9.75 mmol) was loaded onto a silica gel column
(2.5£30 cm) and eluted with dichloromethane (500 mL) and
1.5% methanol in dichloromethane (1 L). Final yield of the
colorless solid after puri®cation was 43.8%. 1H NMR
(DMSO-d6) d 0.99 (s, 9H), 1.09 (s, 9H), 1.27 (s, 9H),
2.35±2.46 (m, 2H), 3.61±3.73 (m, 1H), 6.18±6.42 (m,
3H), 7.31±7.53 (m, 12H), 7.62±7.80 (m, 8H). 13C{1H}
NMR (DMSO-d6) d 18.8, 19.1, 26.3, 26.7, 28.1, 35.6,
54.6, 54.9, 58.0, 77.8, 119.2, 120.9, 121.5, 127.9, 128.0,
130.1, 130.4, 132.2, 132.3, 132.5, 135.0, 135.1, 144.0,
145.1, 152.2, 173.4. Mass spec. Calcd for C46H55NO6Si2:
[M1Na]ˆ796.3466. Found: 796.3488.
In a typical puri®cation, crude DOPA(TBDMS)2 (0.755 g,
1.78 mmol) was loaded onto
a silica gel column
(2.5 cm£30 cm) and eluted with methanol (1 L). Final
yield of the colorless solid after puri®cation was 44.3%.
1H NMR (CD3OD) d 0.22 (m, 13H), 1.01 (s, 9H), 1.01 (s,
9H), 2.80±3.28 (m, 2H), 3.70 (m, 1H), 6.74±6.91 (m, 3H).
13C{1H} NMR (CD3OD) d 23.7, 19.5, 26.6, 26.7, 37.8,
57.9, 122.6, 123.5, 123.7, 130.8, 141.6, 148.5, 174.0.
Mass spec. Calcd for C21H39NO4Si2: [M1H]ˆ426.2496.
Found: 426.2503.
4.3.6. 3,4-Bis(tert-butyldiphenylsilyloxy)-l-phenylalanine
(DOPA(TBDPS)2). Ethyl acetate saturated with HCl was
prepared by bubbling HCl gas through ethyl acetate (50 mL)
for approximately 10 min. After stirring crude Boc-
DOPA(TBDPS)2 (7.95 g, 11.8 mmol) in HCl-saturated
ethyl acetate (17.4 mL) for 18 h, the solution was poured
into a separatory funnel with water (50 mL). The aqueous
later was extracted with ethyl acetate (3£50 mL). Organic
layers were combined, washed with water (3£50 mL),
brine (3£50 mL), and dried over anhydrous Na2SO4.
Solvent was removed in vacuo to yield 6.40 g, 80.5%
of a colorless solid.
4.3.4. 3,4-Bis(tert-butyldimethylsilyloxy)-N-(9-¯uorenyl-
methyloxycarbonyl)-l-phenylalanine (Fmoc-DOPA
(TBDMS)2). On an ice bath, DOPA(TBDMS)2 (10.8 g,
25.3 mmol), dioxane (150 mL), and a 10% Na2CO3 solution
(53.8 mL, 63.2 mmol) were combined. The cream colored
suspension was stirred 10 min prior to adding Fmoc-Cl
(6.55 g, 25.3 mmol). The reaction was stirred 4 h on an
ice bath and then at room temperature for 18 h. The suspen-
sion was added to a separatory funnel with water (75 mL)
and extracted with chloroform (3£75 mL). Chloroform
layers were washed with water (3£75 mL) and brine

M. J. Sever, J. J. Wilker / Tetrahedron 57 (2001) 6139±6146
6145
In a typical puri®cation, crude DOPA(TBDPS)2 (1.14 g,
1.69 mmol) was loaded onto silica gel column
removed. The remaining colorless solid was washed three
times with THF and dried under reduced pressure.
a
(2.5 cm£30 cm) and eluted with chloroform (2 L), 1.0%
methanol in chloroform (1.5 L), 3% methanol in chloroform
(1 L), and 10% methanol in chloroform (1 L). Final yield of
the light brown solid after puri®cation was 46.7%. 1H NMR
(CDCl3) d 1.14 (s, 9H), 1.15, (s, 9H), 2.04 (m, 2H), 6.10±
6.41 (m, 3H), 7.14 (m, 4H), 7.26 (m, 2H), 7.30±7.44 (m,
6H), 7.62±7.81 (m, 8H). 13C{1H} NMR (CDCl3) d 19.6,
26.7, 26.8, 56.3, 76.8, 77.2, 77.4, 77.7, 120.8, 121.0,
121.7, 127.9, 128.0, 129.1, 130.0, 130.2, 132.7, 133.0,
133.1, 135.5, 135.6, 145.4, 146.4. Mass spec. Calcd for
C41H47NO4Si2: [M1H]ˆ674.3122. Found: 674.3136.
4.4. Chirality determination
Stock solutions of 200 mM amino acid in 1 M HCl were
prepared for l-DOPA, d,l-DOPA, and DOPA deprotected
from FmocDOPA(TBDMS)2. A 100 mM solution of FDNP-
Val-NH2 in acetone was also prepared. For derivitization of
each amino acid sample, 25 mL of the 200 mM DOPA solu-
tion, 25 mL of 1 M NaHCO3, 50 mL of 100 mM FDNP-Val-
NH2 in acetone, and an additional 40 mL of 1 M NaHCO3
were combined and heated for 1 h at 408C.31 After heating,
20 mL of 2 M HCl was then added and the samples lyophi-
lized overnight. The resulting solid was dissolved in 1.0 mL
of dimethyl sulfoxide (DMSO) and ®ltered (0.45 mm) for
analysis by HPLC. Optical detection was monitored at 280
and 340 nm. Samples were run for 60 min with gradient
proportioning between 0.1% TFA in water and 0.1% TFA
in acetonitrile. The mobile phase began at 100% of 0.1% TFA
in water and was ramped to 60% of 0.1% TFA in water and
40% of 0.1% TFA in acetonitrile over 50 min. The eluent was
continued at this 60/40 mixture for an additional 10 min.
4.3.7. 3,4-Bis(tert-butlydiphenlysilyloxy)-N-(9-¯uorenyl-
methyloxycarbonyl)-l-phenylalanine (Fmoc-DOPA
(TBDPS)2). Dioxane (15 mL), a 10% Na2CO3 solution
(21.6 mL, 18.6 mmol), and DOPA(TBDMS)2 (5.03 g,
7.47 mmol) were combined and stirred on an ice bath for
10 min prior to addition of Fmoc-Cl (1.94 g, 7.49 mmol).
The reaction was stirred 4 h on the ice bath and 25 h at room
temperature. After pouring the light brown solution into a
separatory funnel with water (50 mL) and chloroform
(50 mL), the aqueous layer was washed with chloroform
(3£50 mL). The combined organic layers were washed
with water (3£50 mL) and brine (3£50 mL) before drying
over anhydrous Na2SO4. Solvent was removed in vacuo to
yield 5.54 g, 82.8% of a light brown, ¯aky solid.
4.5. Peptide synthesis
Preparation of the 4-mer Asn-DOPA-Arg-Gly is typical and
follows: Rink amide resin (0.109 g, 0.0500 mmol) was
placed in a peptide synthesis vessel and washed with
CH2Cl2 (5 mL, 1 min), DMF (2£5 mL, 1 min each), 50%
piperidine in DMF (5 mL, 20 min), DMF (2£5 mL, 1 min
each), and CH2Cl2 (2£5 mL, 1 min each) by rotation of the
¯ask. At this point, the Kaiser test33 is performed to identify
the presence of free amine available for coupling. The resin
is further washed with ethanol (2£5 mL, 3 min each),
CH2Cl2 (2£5 mL, 1 min each), and DMF (2£5 mL, 1 min
each). In a separate, dried 100 mL round bottom ¯ask ®tted
with a drying tube, a coupling solution is prepared contain-
ing Fmoc-Gly-OH (0.0603 g, 0.200 mmol) and DMF
(2.17 mL). This mixture is stirred until all solid dissolves,
then CH2Cl2 (2.17 mL) is added and the solution cooled on
an ice bath. DCC (0.0380 g, 0.184 mmol) is added and the
solution stirred for a further 30 min on the ice bath. Addition
of this coupling solution to the peptide synthesis ¯ask
containing the resin is followed by a reaction time of
45 min. A solution of 10% DIEA in CH2Cl2 (,3 mL) is
added and the ¯ask rotated for an additional 15 min. The
resin is then washed with DMF (2£5 mL, 1 min each) and
CH2Cl2 (2£5 mL, 1 min each). The Kaiser test is performed
to identify the extent of coupling. Successive addition of
amino acids proceeds in a manner analogous to that
provided above for washing followed by coupling of glycine
to the resin. Couplings for arginine and DOPA required
doubling of reaction time (i.e. 90 min rather than 45 min)
and equivalents of the Fmoc-protected amino acid (i.e.
8 equiv. rather than 4). Addition of asparagine required
preparation of a slightly modi®ed coupling solution: In a
dried 100 mL round bottom ¯ask were combined Fmoc-
Asn-OH (0.0716 g, 0.200 mmol), HOBT (0.0278 g,
0.200 mmol), and DMF (2.17 mL). After stirring to dissolve
the solids, CH2Cl2 (2.17 mL) was added and the solution
chilled on an ice bath. DCC (0.0825 g, 0.400 mmol) was
added and this mixture poured into the peptide synthesis
In a typical puri®cation Fmoc-DOPA(TBDPS)2 (4.37 g,
4.88 mmol) was loaded onto
a silica gel column
(6.5 cm£35 cm) and eluted with dichloromethane (3.5 L)
and 0.75% methanol in dichloromethane (8 L). Final yield
of the colorless solid after puri®cation was 56.9%. 1H NMR
(DMSO-d6) d 0.99 (s, 9H), 1.08 (s, 9H), 2.26±2.47 (m, 2H),
3.58±3.74 (m, 1H), 6.19±6.39 (m, 3H), 7.23 (m, 3H), 7.39
(m, 16H), 7.57 (q, 2H), 7.71 (m, 8H), 7.84 (d, 2H), 12.2±
12.8 (br, 2H). 13C{1H} NMR (DMSO-d6) d 18.8, 19.1, 26.3,
26.6, 35.6, 46.7, 55.1, 65.5, 66.4, 119.4, 120.0, 120.9, 121.5,
125.2, 125.3, 127.0, 127.6, 127.9, 130.1, 130.4, 132.3,
132.4, 132.5, 135.0, 135.2, 140.7, 143.8, 144.2, 145.2,
155.7, 173.2. Mass spec. Calcd for C56H57NO6Si2:
[M1H]ˆ894.3. Found: 894.3. Anal. Calcd for
C56H57NO6Si2: C, 75.05; H, 6.41; N, 1.56. Found: C,
74.68; H, 6.42; N, 1.65.
4.3.8. Deprotection of Fmoc-DOPA(TBDMS)2. Complete
deprotection of Fmoc-DOPA(TBDMS)2 was accomplished
in two steps. First, the Fmoc amine protecting group was
removed with base. In a typical procedure, Fmoc-
DOPA(TBDMS)2 (0.250 g, 0.386 mmol), dry acetonitrile
(2.5 mL), and diethylamine (28.8 mg, 1.931 mmol) were
placed in a 10 mL round bottom ¯ask and stirred for 1 h.
During this time, a colorless solid precipitated from solu-
tion. The resulting suspension was centrifuged and the
supernatant decanted. After several acetonitrile washes,
the colorless precipitate was dried under reduced pressure.
Second, the TBDMS groups were removed using TBAF in
order to bring about complete deprotection. To a 10 mL
round bottom ¯ask was added DOPA(TBDMS)2 (90.0 mg,
0.210 mmol), THF (2.0 mL) and TBAF (0.111 g,
0.420 mmol). Within 20 min, a colorless precipitate formed.
The suspension was centrifuged and the supernatant

6146
M. J. Sever, J. J. Wilker / Tetrahedron 57 (2001) 6139±6146
W. R.; Hane, D. L.; Sheppard, H. J. Med. Chem. 1974, 17,
422±426.
¯ask containing the resin. Coupling time was 2 h. After
addition of the ®nal amino acid (Asn), we removed the
remaining Fmoc and acetylated the N-terminus by the addi-
tion of CH2Cl2 (4.4 mL), DIEA (43.6 mL, 0.250 mmol), and
acetic anhydride (23.6 mL, 0.250 mmol). The reaction
proceeded for 1 h, was followed by a CH2Cl2 wash
(4£5 mL, 1 min each) and assayed by a Kaiser test.
11. Hu, B. H.; Messersmith, P. B. Tetrahedron Lett. 2000, 41,
5795±5798.
12. Nakonieczna, L.; Przychodzen, W.; Chimiak, A. Amino Acids
1995, 8, 109±112.
13. Taylor, C.; Weir, C. A. J. Org. Chem. 2000, 65, 1414±1421.
14. Wang, H. P.; Lee, J. S.; Tsai, M. C.; Lu, H. H.; Hsu, W.
Bioorg. Med. Chem. Lett. 1995, 5, 2195±2198.
15. Yamamoto, H.; Hayakawa, T. Biopolymers 1977, 16, 1593±
1607.
Deprotection of the sidechains and cleavage from the resin
for the 4-mer peptide, Asn-DOPA-Arg-Gly, was accom-
plished by reaction with a solution of 90% TFA, 5% thio-
anisol, 3% ethanedithiol, and 2% anisol. Reaction times for
deprotection/cleavage are discussed in Section 2. Deprotec-
tion using the ¯uoride ion also proved useful. In a typical
deprotection and cleavage procedure, the resin-bound
peptide (0.016 mmol) was reacted with TBAF
(0.077 mmol) in THF (5.0 mL) for 5 min, followed by
washing with THF (3£5.0 mL, 1 min each). After drying
under vacuum, the resin was placed in 4.0 mL of 90%
TFA, 5% thioanisol, 3% ethanedithiol, 2% anisol, and
TBAF (0.77 mmol). The peptide was then obtained by
precipitation in diethyl ether. This TFA/peptide solution
was added to 50 mL of cold ether and stored at 2208C for
4 h. The reaction mixture was centrifuged and the ether
supernatant decanted. The precipitated peptide was further
washed with 50 mL of cold ether followed by centrifugation
and removal of the supernatant. The solid was dried under
vacuum. After dissolution in water or 5% acetic acid (v/v),
the peptide was puri®ed by HPLC and analyzed by mass
spectrometry. Mass spec. Calcd for C23H36N9O8:
[M1H]ˆ566.2687. Found: 566.2709. Analogous procedures
were used to prepare the 3-mer peptide, Pro-DOPA-Val, and
6-mer peptide, Gly-DOPA-Arg-Gly-Asn-DOPA. Mass Spec.
3-mer peptide Calcd for C21H30N4O6: [M1Na]ˆ457.2063.
Found 457.602. Mass Spec. 6-mer peptide Calcd for
C34H48N11O12: [M1H]ˆ802.3484. Found 802.898.
16. Yamamoto, H. J. Chem. Soc. Perkin Trans. 1 1987, 613±618.
17. Yamamoto, H.; Kuno, S.; Naghai, A.; Nishida, A.; Yamauchi,
S.; Ikeda, K. Int. J. Biol. Macromol. 1990, 12, 305±310.
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161±214.
20. Bodanszky, M. Peptide Chemistry. A Practical Textbook;
Springer: New York, 1988.
21. Bodanszky, M. Principles of Peptide Synthesis; Springer: New
York, 1984.
22. Cole, E. R.; Crank, G.; Minh, H. T. H. Aust. J. Chem. 1980, 33,
675±680.
23. Iwagami, H.; Yatagal, M.; Nakazawa, M.; Orita, H.; Honda,
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28. Greene, T. W.; Wuts, P. G. Protective Groups in Organic
Synthesis; Wiley: New York, 1999 pp. 127±141.
29. Nakonieczna, L.; Przychodzen, W.; Chimiak, A. Leibigs Ann.
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Acknowledgements
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esis; Springer: New York, 1994.
We are grateful to Harry C. Wang, Jean A. Chmielewski,
and Mark A. Lipton for helpful discussions. This work was
supported generously with startup funds provided by Purdue
University and the National Science Foundation Faculty
Early Career Development (CAREER) Program.
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