An efficient synthesis of 5-aminolaevulinic acid (ALA)-containing peptides for use in photodynamic therapy

Article abstract of DOI:10.1016/j.tet.2005.05.036

An efficient and cost-effective procedure has been devised for the preparation of urethane-protected 5-aminolaevulinic acid (5-ALA) dipeptide ester derivatives which avoids problems associated with the instability of 5-ALA under basic conditions. The procedure is also applicable to the direct synthesis of N-(α)-acetyl amino acid-ALA dipeptides in high enantiomeric purity as potential novel prodrugs for photodynamic therapy (PDT).

Full text of DOI:10.1016/j.tet.2005.05.036

Tetrahedron 61 (2005) 6951–6958
An efficient synthesis of 5-aminolaevulinic acid (ALA)-containing
peptides for use in photodynamic therapy
Louis M.-A. Rogers,a Philip G. McGivern,b Anthony R. Butler,c Alexander J. MacRobertd
and Ian M. Egglestonb,
*
aCentre for Biomolecular Sciences, University of St. Andrews, North Haugh, St. Andrews, KY16 9ST Fife, Scotland, UK
bDivision of Biological Chemistry and Molecular Microbiology, School of Life Sciences, Carnelley Building, University of Dundee,
Dundee DD1 4HN, UK
cBute Medical School, University of St. Andrews, St. Andrews KY16 9TS, UK
dNational Medical Laser Centre, Division of Surgical Specialities, Royal Free and University College Medical School,
University College London, Charles Bell House, 67-73 Riding House Street, London W1P 7PN, UK
Received 25 February 2005; revised 14 April 2005; accepted 12 May 2005
Abstract—An efficient and cost-effective procedure has been devised for the preparation of urethane-protected 5-aminolaevulinic acid
(5-ALA) dipeptide ester derivatives which avoids problems associated with the instability of 5-ALA under basic conditions. The procedure is
also applicable to the direct synthesis of N-(a)-acetyl amino acid-ALA dipeptides in high enantiomeric purity as potential novel prodrugs for
photodynamic therapy (PDT).
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
Photodynamic therapy (PDT) is a non-thermal technique for
inducing tumour destruction with light following
administration of a light-activated photosensitising drug.1,2
Provided that the drug may be selectively introduced and
retained in cancerous cells relative to normal adjacent
tissue, necrosis is selective. A promising approach in PDT
involves the exogenous administration of 5-aminolaevulinic
acid (ALA), a naturally occurring compound present in
mammalian cells which can be metabolised to a porphyrin
photosensitiser, protoporphyrin IX (PpIX) via the haem
biosynthetic pathway (Fig. 1).3 Following accumulation of
Figure 1.
A significant drawback to ALA-PDT is the fact that ALA is
PpIX within the affected tissue, PDT treatment is then
a zwitterion at physiological pH resulting in low lipid
carried out using red laser light, activating PpIX and leading
solubility and limiting passage through biological barriers
to the production of cytotoxic reactive oxygen species. The
such as cellular membranes. To overcome this problem,
main clinical application of ALA-PDT at present is for the
various lipophilic ALA ester derivatives or other novel
treatment of skin cancers via topical application of ALA at
the appropriate place on the skin,4 but the technique is also
prodrugs and formulations have been investigated. In this
regard, we5 and others6 have conjectured that incorporation
particularly suited to the visualization and treatment of early
of 5-ALA into a short peptide derivative, would provide a
tumours in hollow organs where damage to underlying
suitable means of both facilitating transdermal delivery and
muscle must be minimized.
also improved targeting into cancerous cells. Release of
ALA once incorporated would then be mediated by
intracellular peptidase and esterase activities.
Keywords: Aminolaevulinic acid; Peptide; Prodrug; Photodynamic therapy.
*
Corresponding author. Tel.: C44 1382 344319; fax: C44 1382 345517;
e-mail: i.m.eggleston@dundee.ac.uk
We report herein an efficient route for the synthesis of a
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.05.036

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L. M.-A. Rogers et al. / Tetrahedron 61 (2005) 6951–6958
Figure 2. Possible degradation products of 5-ALA derivatives at neutral or basic pH.
range of 5-ALA peptide prodrug derivatives, suitable for
evaluation in skin explant and cellular assays.
components or a 5-ALA derivative. In the first case,6a
1 equiv of a Boc-protected amino acid was preactivated
with 5 equiv each of N-(3-dimethylaminopropyl)-N-ethyl-
carbodiimide hydrochloride (EDC$HCl) and HOBt and
coupled with 5 equiv of an ester derivative of 5-ALA.
Although apparently effective, the expense of 5-ALA makes
this approach less attractive for large scale preparations of
such 5-ALA-containing dipeptides. In a more recent
report,6b 5-ALA containing peptides were obtained via
reaction of in situ formed symmetrical anhydrides of di- or
tripeptide derivatives, necessitating the use of 2 equiv of the
latter relative to the 5-ALA component.
2. Results and discussion
Although Bertozzi et al.7 have reported the solid phase
synthesis of peptides containing 2-ALA, the preparation of
5-ALA-containing peptides is non-trivial. Acylation of
5-ALA derivatives is fraught with potential difficulties,
chiefly associated with the instability of ALA in solution
above pH 4.8 It has been shown that around pH 7, 5-ALA
dimerises to give pyrazine derivatives,9 while at higher pH,
pseudo-porphobilinogen may be formed.10 Conversion to
ester derivatives appears to exacerbate these problems of
instability, introducing the potential for formation of
lactam-type derivatives (Fig. 2).
For our studies in this area, we have developed an
alternative approach that provides access to various
orthogonally protected ALA dipeptides, derivatisable at
either N- or C-terminus, once the key ALA pseudopeptide
bond is formed, but crucially employing only 1 equiv of
both 5-ALA and the activated amino acid derivative. To
achieve this, and avoid competing decomposition of
5-ALA, we chose to react the latter, in the form of its
hydrochloride salt, with a urethane-protected amino acid
active ester in THF solution (in which 5-ALA hydrochloride
is insoluble). Initiation of the coupling reaction is then
effected by slow addition of base (DIPEA) such that any
5-ALA released into solution is immediately intercepted by
the acylating agent (in excess) before competing side
reactions can intervene (Scheme 1). This general strategy of
simultaneous deprotection and coupling has been utilized
previously in peptide chemistry to overcome the problem of
All these processes are typically associated with a
significant darkening of the ALA solution, and indeed we
have found that using conventional peptide synthesis
methodology, coupling of equimolar quantities of N-(a)-
protected amino acids to esters of ALA via carbodiimide/
1-hydroxybenzotriazole (HOBt) activation generally leads
to complex mixtures and uniformly low yields of the desired
peptides.11
Berger et al.6 have reported two separate approaches for the
preparation of 5-ALA-containing peptides, however both
employ either an excess of the activated amino acid
Scheme 1.
Table 1. Urethane-protected 5-ALA dipeptide derivatives
R
R1
Yield/%
Name
1a
1b
1c
1d
1e
1f
But
But
CH3
CH2CH(CH3)2
(CH2)4NHZ
CH2Ph
CH(CH3)2
H
65
87
85
79
32
80
Boc-Ala-ALA-OCH3
Boc-Leu-ALA-OCH3
Boc-Lys(Z)-ALA-OCH3
Boc-Phe-ALA-OCH3
Boc-Val-ALA-OCH3
Z-Gly-ALA-OCH3
But
But
But
CH2Ph

L. M.-A. Rogers et al. / Tetrahedron 61 (2005) 6951–6958
6953
Scheme 2.
diketopiperazine formation on coupling to an Xaa-Pro-OMe
dipeptide12 and has also been exploited more recently as
part of a highly efficient tandem deprotection-coupling
methodology using N-(a)-allyloxycarbonyl protected
amino acid derivatives.13
5-ALA peptides was observed under these conditions with
the key acylation step having already been performed.
To confirm that suitably protected 5-ALA dipeptides may be
further elaborated by standard peptide chemistry, the lysine-
containing derivative 1c was transformed into the
corresponding pseudotripeptides 3a and 3b as shown in
Scheme 3. Once again, no degradation of the 5-ALA unit
was observed either during acidolytic cleavage of N-(a)-
Boc protection or subsequent acylation reactions in the
presence of tertiary amine. The dipeptide fragment Ac-Phe-
Lys has previously been employed to create lysosomally-
cleavable prodrugs of the anticancer agent doxorubicin15
showing the potential value of expedient access to building
blocks such as 1a–f for the preparation of peptide prodrugs
that are susceptible to cleavage by specific proteases or
targeted to particular cellular transporters.16
As shown in Table 1, when a variety of amino acid
succinimidyl ester derivatives, were coupled with 5-ALA as
described above, the corresponding dipeptides were isolated
in generally very good yields, via conversion to the methyl
esters by treatment with ethereal diazomethane to facilitate
isolation. The sole exception was the Boc-Val derivative
which coupled rather slowly and ultimately gave a modest
yield of 32%. These results compare very favourably with
those reported by Berger et al.6 for the preparation of similar
dipeptides on the same scale of synthesis (e.g., 1a, 1d6a) and
it should be noted again that they provide a significant
economy in terms of the use of 5-ALA. All the dipeptides
were obtained in analytically pure form following isolation
by chromatography or crystallization.
In view of the effectiveness of our protocol for the coupling
of Boc or Z-protected amino acids to 5-ALA we were
interested to explore the possibility of the direct synthesis of
N-(a)-acetylated derivatives by this method in order to
facilitate rapid biochemical screening of such prodrugs.
When the N-(a)-acetyl succinimidyl ester derivatives of
Gly, D- or -L-Ala, D- or L-Phe, and L-Val were reacted with
5-ALA hydrochloride as before, followed by esterification
(Scheme 4), the desired dipeptides were obtained in
moderate yields (26–43%, Table 2, entries 1, 2, 4, 8–10)
and with surprisingly good optical purity.
We have found that the most appropriate 5-ALA dipeptide
substrates for PDT studies are those in which 5-ALA is
coupled to an N-(a)-acetyl amino acid derivative, since they
provide a useful balance of lipophilicity and water
solubility.14 As expected, Boc-protected derivatives such
as 1b and 1e, were readily transformable into novel prodrug
entities as illustrated in Scheme 2. Treatment of 1b or 1e
with 4 M HCl–dioxane provided the corresponding hydro-
chloride salts which were then treated with acetic anhydride
in the presence of DIPEA to give the corresponding
acetylated peptides in good yield. No degradation of the
Coupling with activated N-(a)-acetyl amino acids is rarely
used in peptide synthesis since the former are highly prone
Scheme 3.
Table 2. Direct preparation of Ac-Xaa-ALA-OCH3 derivatives
Entry
L or D
R1
Dilute base
eea/%
Yield/%
2a
2b
1
2
3
—
L
H
CH3
CH3
CH3
N
N
Y
N
Y
N
Y
N
N
N
Y
—
46
O98
41
O98
0
90
O98
O98
64
32
26
51
47
73
37
47
43
41
33
38
L
2c
4
D
D
L
5
CH3
CH2CH(CH3)2
2d
6b
7
L
2e
2f
2g
8
9
10
11
L
CH2Ph
CH2Ph
CH(CH3)2
D
L
L
O98
a ee determined by 1H NMR with the chiral shift reagent Eu(hfc)3 (hfc, 3-(heptafluoropropylhydroxy-methylene)-(C)-camphorate).
b Coupling via the pentafluorophenyl ester.

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L. M.-A. Rogers et al. / Tetrahedron 61 (2005) 6951–6958
to racemisation via oxazolone formation in the presence of
base,17 but for the Phe derivatives 2e and 2f (entries 8 and 9)
the desired peptide was obtained essentially as a single
enantiomer. Enantiomeric purities were assessed using
3. Conclusion
We have developed highly efficient and economic prep-
arations of 5-ALA peptides that overcome the known
instability of the amino acid under basic conditions. The
derivatives obtained may be elaborated into a wide range of
5-ALA prodrug derivatives using conventional peptide
chemistry for use as novel PDT agents. Details of these
studies will be reported shortly.14
1
chiral shift H NMR with Eu(hfc)3; addition of the reagent
led to a splitting of the resonance corresponding to the ester
methyl group, presumably via diastereoisomeric chelate
formation involving the ester and keto carbonyls of the ALA
residue. Integration of these signals then gave a direct
estimation of the enantiomeric purity of the peptide.
The direct acylation of 5-ALA with N-(a)-acetyl derivatives
offers some advantages in terms of speed and economy,
particularly for cases such as Val, where coupling of the
Boc-protected derivative proceeds in a somewhat lower
yield (see Table 1). As anticipated, when the rate of addition
of base was carefully controlled (in dilute THF solution),
not only the yield but also the optical purity of the peptides
(Table 2, entries 3, 5, 7, 11) was significantly improved,
since controlled base addition limits the concentration of
free 5-ALA, the species required for coupling which may
also participate in polymerization or dimerisation reactions
etc (see above),8–10 but moreover minimises the amount of
base available to promote oxazolone formation and
racemisation. Preliminary studies with other active esters
such as pentafluorophenyl (entry 6) and 4-nitrophenyl
(not shown), did not suggest any significant advantage in
terms of yield relative to the readily available succinimidyl
esters which formed the basis of our study, however
racemisation appeared to be much more significant with
these species.
4. Experimental
4.1. General
Melting points were recorded on an Electrothermal IA9000
series digital melting point apparatus and are quoted
uncorrected. NMR spectra were recorded on a Varian
Gemini 300 (1H, 300 MHz; 13C, 75.4 MHz;) or Bruker
Avance DPX 300 FT-spectrometers. Chemical shifts (d) are
expressed in ppm and coupling constants (J) are given in
Hz. Mass spectra were recorded on a VG AUTOSPEC mass
spectrometer in electron impact (EI) mode, a VG Quattro
triple quadrupole instrument in positive electrospray (ES)
mode, or a TofSpec 2E instrument (MALDI-TOF).
Elemental analyses were performed in the Department of
Chemistry, University of St. Andrews, or by MEDAC Ltd,
Brunel Science Centre, Surrey UK. Optical rotations were
measured at 20 8C in a 10 cm path length cell using a Perkin
Elmer 343 polarimeter and are quoted in 10K1 deg. cm2 gK1
.
Flash chromotography was performed according to the
method of Still et al.,19 on columns of silica gel (Fluka Silica
Gel 60; 35–70 mm mesh, or Fluorochem; 40–60 mm mesh).
Ethereal diazomethane was prepared from Diazaldw.
Petroleum ether bp 40–60 8C was distilled through a
vigreux column prior to use. All other solvents were of
Analar quality or were dried using standard procedures.
Once again, derivatives such as 2a were readily transform-
able into other potential prodrugs by standard chemistry
with the key Xaa-ALA peptide bond now in place. For
example (Scheme 5), saponification of 2a with aq LiOH
proceeded in quantitative yield whereupon the dipeptide
acid was converted to the corresponding hexyl ester in 65%
yield by DMAP-catalysed esterification18 with DCC. This
further emphasizes the potential of derivatives such as 1a–f
and 2a–g as synthons for the preparation of more elaborate
5-ALA-containing peptides and also highlights the
possibility of employing other chemistries in the esterifica-
tion stage of our general procedure once the critical
acylation has been achieved.
Amino acid active esters. Urethane and acetyl-protected
amino acid N-hydroxysuccinimidyl ester derivatives were
either commercially available (Calbiochem or
SigmaAldrich), or were prepared by standard DCC-
mediated esterifications.20,21 Ac-L-Leu-OPfp was prepared
by DCC-mediated esterification of Ac-L-Leu with
pentafluorophenol.
Scheme 4.
Scheme 5.

L. M.-A. Rogers et al. / Tetrahedron 61 (2005) 6951–6958
6955
4.2. Preparation of ALA ‘dipeptides’
[MCNa]C); (Found: C, 53.07; H, 7.29; N, 8.75.
C14H24N2O6 requires: C, 53.15; H, 7.65; N, 8.85%).
Urethane derivatives (typical procedure). A suspension of
the amino acid active ester (1.5 mmol) and 5-ALA$HCl
(0.25 g, 1.5 mmol) in dry THF (20 mL) was cooled to
K5 8C under argon. A solution of DIPEA (0.26 mL,
1.5 mmol) in dry THF (10 mL) was slowly added over
120 min, then the reaction mixture was stirred overnight
under cooling. The solvent was evaporated and an excess of
freshly prepared ethereal diazomethane (40 mL) was added
with cooling in an ice bath. The reaction mixture was stirred
at room temperature for 2 h, then the solvent was carefully
evaporated. The crude product was redissolved in EtOAc
(40 mL) and was washed with 5% aq citric acid, 5% aq
NaHCO3, H2O, and saturated aq NaCl (40 mL each). The
organics were dried (MgSO4) and the solvent was
evaporated to give the crude product which was purified
by column chromatography (EtOAc/40–60 petrol or
MeOH/CH2Cl2 gradient) to give white solid or colourless
oils. For 1f, the product was purified by recrystallisation.
4.3.2. Boc-L-Leu-ALA-OMe (1b). 2.98 mmol scale. Yield
87% (colourless oil). [a]D K17.0 (cZ1.62 CHCl3); dH
(CDCl3) 0.95–0.98 (6H, m, CH3-Leu), 1.47 (9H, s, But),
1.55–1.73 (3H, m, CHCH2(CH3)2, CHCH2(CH3)2) 2.66–
2.70 (2H, m, COCH2CH2), 2.75–2.79 (2H, m, COCH2CH2),
3.70 (3H, s, CO2CH3), 4.21 (2H, d, JZ4.7 Hz, NHCH2CO),
4.13–4.29 (1H, m, CHCH2CH(CH3)2), 4.90 (1H, br,
urethane NH), 6.80 (1H, br, amide NH); dc 22.1, 23.4
(CH3-Leu), 25.1 (CHCH2(CH3)2), 27.9 (COCH2CH2), 28.7
(C(CH3)3), 34.9 (COCH2CH2), 41.8 (CHCH2CH(CH3)2),
49.4 (NHCH2CO), 52.3 (CO2CH3), 53.4 (CHCH2-
CH(CH3)2), 80.5 (C(CH3)3), 156.1 (urethane C]O),
173.3 (amide C]O), 173.4 (ester C]O), 204.1 (ketone
C]O); m/z (ES) 359 (100%, [MCH]C), 381 (16, [MC
Na]C); (Found: C, 56.63; H, 8.13; N, 8.10. C17H30N2O6
requires C, 56.97; H, 8.44; N, 7.82%).
4.3.3. Boc-L-Lys(Z)-ALA-OMe (1c). 1.50 mmol scale.
Yield: 85% (white solid). Mp: 85–87 8C (from CH2Cl2/
40–60 petrol); [a]D K9.6 (cZ0.87 CHCl3); dH (CDCl3)
1.32–1.70 (4H, m, CHCH2CH2CH2CH2NH), 1.72–1.88
(2H, m, CHCH2(CH2)3NH), 1.45 (9H, s, But), 2.64–2.68
(2H, m, COCH2CH2), 2.72–2.76 (2H, m, COCH2CH2),
3.18–3.24 (2H, m, CH2NH), 3.69 (3H, s, CO2CH3), 4.14–
4.25 (1H, m, CH(CH2)4NH), 4.19 (2H, d, JZ4.8 Hz,
NHCH2CO), 5.00 (1H, br, CH2NH), 5.11 (2H, s,
OCH2Ph), 5.11–5.19 (1H, m, ButOCONH), 6.85 (1H, br,
amide NH), 7.30–7.38 (5H, m, Ph); dc 22.8 (CHCH2CH2-
(CH2)2NH), 27.9 (COCH2CH2), 28.7 (C(CH3)3), 29.8
(CH(CH2)2CH2CH2NH), 32.4 (CHCH2(CH2)3NH), 34.9
(COCH2CH2), 40.8 (CH(CH2)3CH2NH), 49.4 (NHCH2CO),
52.3 (CO2CH3), 54.7 (CH(CH2)4NH), 67.0 (CH2Ph), 128.5,
128.9, 137.0 (Ph), 156.1, 157.0 (urethane C]O), 172.7
(amide C]O), 173.3 (ester C]O), 204.1 (ketone C]O);
m/z (ES) 508 (100%, [MCH]C), 530 (36, [MCNa]C), 546
(21, [MCK]C); (Found: C, 59.13; H, 7.49; N, 8.23.
C25H37N3O8 requires C, 59.16; H, 7.35; N, 8.27%).
Acetyl derivatives (Method A). The coupling reaction and
esterification were performed as for the urethane-protected
derivatives, except that the base was added directly rather
than in THF solution. The crude products were pre-absorbed
onto silica using MeOH as solvent and purified by column
chromatography using MeOH/CH2Cl2 (CH2Cl2 to 10%
MeOH/CH2Cl2) as eluent, followed by recrystallisation
from EtOAc/40–60 petrol to give the dipeptides as white
solids.
Method B. The coupling reaction and esterification were
performed exactly as for the urethane-protected derivatives.
The crude products were pre-absorbed onto silica using
MeOH as solvent and purified by column chromatography
using MeOH/CH2Cl2 (CH2Cl2 to 10% MeOH/CH2Cl2) as
eluent. If required, the dipeptides were further purified by
recrystallisation from EtOAc/40–60 petrol as previously.
1
4.3. H NMR chiral shift experiments with N-(a)-
acetylated dipeptides
4.3.4. Boc-L-Phe-ALA-OMe (1d). 1.10 mmol scale. Yield:
79% (colourless glass, Lit.,6a Mp: 83–85 8C); [a]D K1.4
(cZ0.89 CHCl3); dH (CDCl3) 1.33 (9H, s, But), 2.57–2.65
(4H, m, COCH2CH2), 3.01–3.12 (2H, m, CH2Ph), 3.60 (3H,
s, CO2CH3), 3.98–4.17 (2H, m, NHCH2CO), 4.35 (1H, br,
CHCH2Ph), 4.85 (1H, br, urethane NH), 6.50 (1H, br, amide
NH), 7.11–7.25 (5H, m, Ph); dc 28.0 (COCH2CH2), 28.6
(C(CH3)3), 34.9 (COCH2CH2), 38.8 (CHCH2Ph), 49.5
(NHCH2CO), 52.3 (CO2CH3), 56.1 (CHCH2Ph), 80.7
(C(CH3)3), 127.4, 129.1, 129.7, 136.9 (Ph), 155.8 (urethane
C]O), 171.8 (amide C]O), 173.2 (ester C]O), 203.6
(ketone C]O); m/z (ES) 393 (100%, [MCH]C), 415 (26,
[MCNa]C), 431 (13, [MCK]C); (Found: C, 61.07; H,
7.26; N, 7.17. C20H28N2O6 requires C, 61.21; H. 7.19; N.
7.14%).
Optimum resolution was obtained when a molar ratio of
Eu(hfc)3 versus 5-ALA ‘dipeptide’ of approximately 1:4
was employed. In a typical experiment, a solution of the
5-ALA ‘dipeptide’ (10 mg, ca. 30 mmol) in dry CDCl3
(0.7 mL) was treated with Eu(hfc)3 (10 mg, ca. 8 mmol). The
method was verified by examining mixtures of Ac-D-Phe-
ALA-OMe and Ac-L-Phe-ALA-OMe. Integral ratios for
each enantiomer were found to be consistent with the ratio
of each in solution.
4.3.1. Boc-L-Ala-ALA-OMe (1a). 1.10 mmol scale. Yield:
65% (colourless oil). [a]D K17.3 (cZ0.54 CHCl3); dH
(CDCl3) 1.39 (3H, d, JZ7.1 Hz, CH3-Ala), 1.47 (9H, s,
But), 2.66–2.69 (2H, m, COCH2CH2), 2.75–2.79 (2H, m,
COCH2CH2), 3.70 (3H, s, CO2CH3), 4.21–4.29 (3H, m,
NHCH2CO, CHCH3), 5.01 (1H, br, urethane NH), 6.83 (1H,
br, amide NH). dc 18.8 (CHCH3), 28.0 (COCH2CH2), 28.7
(C(CH3)3), 34.9 (COCH2CH2), 49.5 (NHCH2CO), 50.5
(CHCH3), 52.3 (CO2CH3), 80.7 (C(CH3)3), 155.8 (urethane
C]O), 173.2 (2 signals, amide and ester C]O), 203.9
(ketone C]O); m/z (ES) 317 (100%, [MCH]C), 339.2 (23,
4.3.5. Boc-L-Val-ALA-OMe (1e). 1.50 mmol scale. Yield:
32% (white solid). Mp: 73.5–77 8C; [a]D K21.2 (cZ1.32
CHCl3); dH (CDCl3) 0.84 (3H, d, JZ6.8 Hz, CH3-Val), 0.90
(3H, d, JZ6.8 Hz, CH3-Val), 1.38 (9H, s, But), 2.05–2.16
(1H, m, CH(CH3)2), 2.56–2.61 (2H, m, COCH2CH2),
2.66–2.70 (2H, m, COCH2CH2), 3.61 (3H, s, CO2CH3),

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L. M.-A. Rogers et al. / Tetrahedron 61 (2005) 6951–6958
3.89–3.99 (1H, m, CHCH(CH3)3), 4.14 (2H, d, JZ4.8 Hz,
NHCH2CO), 4.95 (1H, br, urethane NH), 6.62 (1H, br,
amide NH); dc 17.95, 19.6 (CH3-Val), 27.9 (COCH2CH2),
28.7 (C(CH3)3), 31.2 (CH(CH3)2), 34.9 (COCH2CH2), 49.4
(NHCH2CO), 52.3 (CO2CH3), 60.2 (CHCH(CH3)2), 80.3
(C(CH3)3), 156.2 (urethane C]O), 172.2 (amide C]O),
173.2 (ester C]O), 204.0 (ketone C]O); m/z (ES) 345
(100%, [MCH]C), 367 (100, [MCNa]C), 348 (48, [MC
K]C); (Found: C, 55.45; H, 8.13; N, 8.10. C16H26N2O6
requires: C, 55.80; H, 8.20; N, 8.13%).
Ala); (Found: C, 51.05; H, 7.06; N, 10.76. C11H18N2O5
requires C, 51.19; H, 7.03; N, 10.85%).
Method B. 1.30 mmol scale. Yield 73%. dH (CDCl3/
Eu(hfc)3) single enantiomer.
4.3.10. Ac-L-Leu-ALA-OMe (2d). Method A (using the
pentafluorophenyl ester). 1.50 mmol scale. Yield: 37%. Mp:
74–76 8C. dH (CDCl3) 0.92–0.95 (6H, m, CH3-Leu), 1.50–
1.70 (3H, m, CHCH2(CH3)2, CHCH2(CH3)2), 2.01 (3H, s,
COCH3), 2.63–2.67 (2H, m, COCH2CH2), 2.72–2.76 (2H,
m, COCH2CH2), 3.68 (3H, s, CO2CH3), 4.08–4.24 (2H, m,
NHCH2CO), 4.51–4.56 (1H, m, CHCH2CH(CH3)2), 6.25
(1H, d, JZ5.2 Hz, CH3CONH), 7.00 (1H, br, NHCHCH2-
CH(CH3)2CO);); dH (CDCl3/Eu(hfc)3) 3.767 (1.5H, s,
CO2CH3-L-Leu), 3.813 (1.5H, s, CO2CH3-D-Leu); dc 22.0,
22.8 (CH3-Leu), 23.0 (COCH3) 24.7 (CHCH2(CH3)2), 27.4
(COCH2CH2), 34.4 (COCH2CH2), 41.2 (CHCH2-
CH(CH3)2), 49.0 (NHCH2CO), 51.5 (CO2CH3), 51.8
(CHCH2CH(CH3)2), 170.4, 172.6 (amide C]O), 173.0
(ester C]O), 203.7 (ketone C]O); m/z (EI) 300 (3%, MC),
269 (7, [MKOCH3]C), 156 (62, [MKALA-OCH3]C), 86
(100); (Found: C, 56.20; H, 7.97; N, 9.23. C14H24N2O5
requires C, 55.99; H, 8.05; N, 9.33%).
4.3.6. Z-Gly-ALA-OMe (1f). 1.10 mmol scale. Yield 80%
(white solid). Mp: 103–105 8C; dH (CDCl3) 2.66–2.70 (2H,
m, COCH2CH2), 2.74–2.79 (2H, m, COCH2CH2), 3.70 (3H,
s, CO2CH3), 3.94 (2H, d, JZ5.6 Hz, NHCH2CONH), 4.23
(2H, d, JZ4.6 Hz, NHCH2CO) 5.16 (2H, s, OCH2Ph), 5.51
(1H, br, urethane NH), 6.78 (1H, br, amide NH), 7.32–7.44
(5H, m, Ph); dc 27.9 (COCH2CH2), 34.9 (COCH2CH2), 44.7
(NHCH2CONH), 49.4 (NHCH2CO), 52.4 (CO2CH3), 67.6
(OCH2Ph), 128.5, 128.6, 128.9, 136.6 (Ph), 157.1 (urethane
C]O), 169.8 (amide C]O), 173.3 (ester C]O), 204.2
(ketone C]O); m/z (ES) 337 (100%, [MCH]C), 359 (35,
[MCNa]C), 375 (12, [MCK]C); (Found: C, 57.11; H,
6.00; N, 8.35. C16H20N2O6 requires C, 57.14; H, 5.99; N,
8.32%).
Method B (using the succinimidyl ester). 1.50 mmol scale.
Yield 47%. dH (CDCl3/Eu(hfc)3) 3.611 (2.85H, s, CO2CH3-
L-Leu), 3.6689 (0.15H, s, CO2CH3-D-Leu).
4.3.7. Ac-Gly-ALA-OMe (2a). Method A. 2.30 mmol scale.
Yield: 32%. Mp: 192–194 8C. dH (CDCl3) 2.06 (3H, s,
COCH3), 2.64–2.69 (2H, m, COCH2CH2), 2.73–2.78 (2H,
m, COCH2CH2), 3.67 (3H, s, CO2CH3), 3.99 (2H, d, JZ
5.0 Hz, NHCH2CONH), 4.22 (2H, d, JZ5.0 Hz, NHCH2-
CO), 6.33 (1H, br, CH3CONHCH2), 6.76 (1H, br, NHCH2-
COCH2CH2); dc 23.4 (COCH3), 28.1 (COCH2CH2), 35.0
(COCH2CH2), 43.5 (NHCH2CONH), 49.6 (NHCH2CO),
52.5 (CO2CH3), 169.7, 171.5 (amide C]O), 173.4 (ester
C]O), 204.1 (ketone C]O); m/z (EI) 244 (11%, MC), 213
(7, [MKOMe]C), 171 (8) 130 (45), 115 (78), 100 (100,
[MKALA-OCH3]C); (Found: C, 49.14; H, 6.60; N, 11.44.
C10H16N2O5 requires C, 49.18; H, 6.60; N, 11.47%).
From Boc-L-Leu-OCH3. 1b (0.796 g, 2.22 mmol) was
treated with 4 M HCl in dioxane (14 mL) and the resulting
solution was stirred at room temperature for 40 min. The
solvent was evaporated and the crude hydrochloride salt was
dried thoroughly in vacuo. A suspension of the hydro-
chloride salt in CH2Cl2 (30 mL) was cooled in an ice bath
and was treated with DIPEA (0.46 mL, 2.64 mmol),
followed by acetic anhydride (0.42 mL, 4.45 mmol). The
reaction mixture was allowed to attain room temperature
overnight, then it was diluted with CH2Cl2 (30 mL) and was
washed with 5% aq NaHCO3, 5% aq citric acid and
saturated aq NaCl (30 mL each). The aqueous layers were
back-extracted with CH2Cl2 (4!30 mL) and the combined
organics were dried (MgSO4) and the solvent evaporated to
give a colourless oil which was purified by column
chromatography using MeOH/CH2Cl2 (CH2Cl2 to 7%
MeOH/CH2Cl2) as eluant. This gave a white solid
(0.508 g, 76%), indistinguishable (1H, 13C NMR) from the
samples prepared by Methods A and B.
4.3.8. Ac-L-Ala-ALA-OMe (2b). Method A. 1.50 mmol
scale. Yield: 26%. Mp: 102–106 8C. dH (CDCl3) 1.38 (3H,
d, JZ6.9 Hz, CH3-Ala), 2.01 (3H, s, COCH3), 2.63–2.67
(2H, m, COCH2CH2), 2.72–2.77 (2H, m, COCH2CH2), 3.70
(3H, s, CO2CH3), 4.18 (2H, d, JZ4.8 Hz, NHCH2CO),
4.50–4.60 (1H, m, CHCH3), 6.31 (1H, d, JZ6.6 Hz,
CH3CONH), 6.94 (1H, br, NHCHCH3CO); dH (CDCl3/
Eu(hfc)3) 3.709 (2.2H, s, CO2CH3-L-Ala), 3.742 (0.8H, s,
CO2CH3-D-Ala); dc 17.9 (CHCH3), 22.4 (CH3CO), 27.0
(COCH2CH2), 33.9 (COCH2CH2), 48.5 (NHCH2CO), 48.9
(CHCH3), 51.4 (CO2CH3), 170.2, 172.6 (amide C]O),
173.0 (ester C]O), 204.0 (ketone C]O); m/z (MALDI-
TOF) 281 (100%, [MCNa]C), 297 (23, [MCK]C);
(Found: C, 51.30; H, 6.87; N, 11.08. C11H18N2O5 requires
C, 51.19; H, 7.03; N, 10.85%).
4.3.11. Ac-L-Phe-ALA-OMe (2e). Method A. 1.38 mmol
scale. Yield: 43%. Mp: 129–130 8C. dH (CDCl3) 1.95 (3H, s,
COCH3), 2.60–2.70 (4H, m, COCH2CH2), 3.00–3.15 (2H,
m, CH2Ph), 3.68 (3H, s, CO2CH3), 4.00–4.20 (2H, m,
NHCH2CO), 4.70–4.80 (1H, br, CHCH2Ph), 6.20 (1H, br,
CH3CONH), 6.70 (1H, br, NHCHCH2Ph); dH (CDCl3/
Eu(hfc)3) single enantiomer; dc 23.1 (COCH3) 27.5
(COCH2CH2), 35.0 (COCH2CH2), 38.3 (CH2Ph), 49.0
(NHCH2CO), 52.0 (CO2CH3), 56.1 (CHCH2Ph), 127.0,
128.6, 129.2, 136.3 (Ph), 170.1, 170.9 (amide C]O), 172.8
(ester C]O), 203.2 (ketone C]O); m/z (EI) 334 (11%,
MC), 303 (3, [MKOCH3]C), 275 (15, [MKCO2CH3]C),
219 (5), 190 (20), 171 (8), 120 (100); (Found: C, 60.75; H,
Method B. 1.50 mmol scale. Yield: 51%. dH (CDCl3/
Eu(hfc)3) single enantiomer.
4.3.9. Ac-D-Ala-ALA-OMe (2c). Method A. 1.50 mmol
scale. Yield: 47%. Mp: 102–104 8C. dH (CDCl3/Eu(hfc)3)
3.709 (0.9H, s, CO2CH3-D-Ala), 3.742 (2.1H, s, CO2CH3-L-

L. M.-A. Rogers et al. / Tetrahedron 61 (2005) 6951–6958
6957
6.68; N, 8.15 C17H22N2O5 requires C, 61.07; H, 6.61; N,
8.38%).
(20 mL each). The organic layer was dried (MgSO4) and the
solvent was evaporated to give an off-white solid which was
purified by column chromatography using MeOH/CH2Cl2
(5–9% MeOH/CH2Cl2) as eluant. This gave a white solid
(0.163 g, 89%). Mp: 103.5–107.5 8C; [a]D K17.0 (cZ1.0
CHCl3); dH (CDCl3 0.83–1.54 (4H, m, CHCH2CH2CH2-
CH2NH), 1.31 (9H, s, But), 1.70–1.90 (2H, m, CHCH2-
(CH2)3), 2.55–2.57 (2H, m, COCH2CH2), 2.61–2.63 (2H, m,
COCH2CH2), 2.93–3.10 (4H, m, CH(CH2)3CH2NH,
CHCH2Ph), 3.59 (3H, s, CO2CH3), 4.01–4.10 (2H, m,
NHCH2CO), 4.32–4.37 (2H, m, CH(CH2)4NH, CHCH2Ph),
4.95–5.10 (4H, m, OCH2Ph, 2!urethane NH), 6.60–6.67
(2H, m, 2!amide NH), 7.10–7.34 (10H, m, 2! Ph); dc 22.6
(CHCH2CH2CH2CH2NH), 27.9 (CHCH2CH2CH2CH2NH),
28.6 (C(CH3)3), 29.7 (COCH2CH2), 31.8 (CHCH2(CH2)3-
NH), 34.6 (COCH2CH2), 38.4 (CHCH2Ph), 40.8
(CH(CH2)3CH2NH), 49.4 (NHCH2CO), 52.3 (CO2CH3),
53.3 (CH(CH2)4NH), 56.4 (CHCH2Ph), 67.0 (OCH2Ph),
80.9 (C(CH3)3), 127.4, 128.5, 128.9, 129.1, 136.7, 137.0
(Ph), 156.1, 157.0 (urethane C]O), 171.7, 172.0 (amide
C]O), 173.3 (ester C]O), 203.0 (ketone C]O); m/z (ES)
655 (57%, [MCH]C), 677.4 (16, [MCNa]C); (Found: C,
62.32; H. 7.26; N, 8.47. C34H46N4O8 requires C, 62.37; H,
7.08; N, 8.55%).
4.3.12. Ac-D-Phe-ALA-OMe (2f). Method A. 1.38 mmol
scale. Yield: 41%. Mp: 126–128 8C. dH (CDCl3/Eu(hfc)3)
single enantiomer. (Found: C, 60.78; H, 6.63; N, 8.36.
C17H22N2O5 requires C, 61.07; H, 6.61; N, 8.38%).
4.3.13. Ac-L-Val-ALA-OMe (2g). Method A. 1.50 mmol
scale. Yield: 33%. Mp: 147–148 8C. dH (CDCl3) 0.94 (3H,
d, JZ4.6 Hz, CH3-Val), 0.96 (3H, d, JZ4.6 Hz, CH3-Val),
2.00–2.12 (4H, m, COCH3, CH(CH3)2), 2.62–2.67 (2H, m,
COCH2CH2), 2.72–2.77 (2H, m, COCH2CH2), 3.67 (3H, s,
CO2CH3), 4.10–4.28 (2H, m, NHCH2CO) 4.34–4.39 (1H,
m, CHCH(CH3)3), 6.35 (1H, br, CH3CONH), 6.70 (1H, br,
NHCH(CH3)2CO); dH (CDCl3/Eu(hfc)3) 3.723 (2.45H, s,
CO2CH3-L-Val), 3.775 (0.55H, s, CO2CH3-D-Val); dc 18.0,
19.0 (CH3-Val), 23.1 (COCH3), 27.4 (COCH2CH2), 31.0
(CH(CH3)2), 34.4 (COCH2CH2), 49.0 (NHCH2CO), 52.0
(CO2CH3), 58.3 (CHCH(CH3)2), 170.3, 172.2 (amide
C]O), 172.9 (ester C]O), 203.6 (ketone C]O); m/z
(EI) 286 (3%, MC), (5, M-OCH3), 142 (40, [MKALA-
OCH3]C), 114 (100), 72 (95); (Found: C, 54.86; H, 7.92; N,
9.71. C13H22N2O5 requires C, 54.53; H, 7.74; N, 9.78%).
Method B. 1.50 mmol scale. Yield: 38%. dH (CDCl3/
Eu(hfc)3) single enantiomer.
4.3.15. N-Acetyl-L-phenylalanyl-N3-(benzyloxycar-
bonyl)-L-lysinyl-5-aminolaevulinic acid methyl ester
(Ac-L-Phe-L-Lys(Z)-ALA-OMe) (3b). 3a (0.122 g,
0.186 mmol) was treated with 4 M HCl in dioxane (2 mL)
and the resulting solution was stirred at room temperature
for 1 h. The solvent was evaporated and the crude
hydrochloride salt was dried thoroughly in vacuo. A
suspension of the hydrochloride salt in CH2Cl2 (4 mL)
was cooled in an ice bath and was treated with DIPEA
(40 mL, 0.23 mmol), followed by acetic anhydride (35 mL,
0.37 mmol). The reaction mixture was allowed to attain
room temperature overnight at which point a gelatinous
white solid had separated out. The reaction mixture was
diluted with CH2Cl2 (25 mL) to dissolve the solid and was
washed with 5% aq NaHCO3, 5% aq citric acid and
saturated aq NaCl (25 mL each). Drying (MgSO4) and
evaporation of the solvent gave an off-white solid which
was precipitated from CH2Cl2/hexane to give a white
powder (95 mg, 85%). Mp: 144–146 8C; [a]D K6.9 (cZ1.0
CH3OH); dH (CDCl3) 1.20–1.91 (6H, m, CHCH2CH2CH2-
CH2NH), 1.87 (3H, s, COCH3), 2.55–2.58 (2H, m, COCH2-
CH2), 2.62–2.66 (2H, m, COCH2CH2), 2.92–3.10 (4H, m,
CH(CH2)3CH2NH, CHCH2Ph), 3.60 (3H, s, CO2CH3),
4.01–4.08 (2H, m, NHCH2CO), 4.30–4.40 (1H, m,
CH(CH2)4NH), 4.65 (1H, ABq, CHCH2Ph) 5.01–5.18
(4H, m, OCH2Ph, 2! urethane NH), 6.25 (1H, d, JZ
7.2 Hz, CH3CONH) 6.55–6.60 (1H, m, amide NH), 6.68
((1H, d, JZ7.7 Hz, amide NH), 7.12–7.32 (10H, m, 2!
Ph); dc 22.6 (CHCH2CH2CH2CH2NH), 23.3 (COCH3) 27.9
(CHCH2CH2CH2CH2NH), 29.7 (COCH2CH2), 32.4
(CHCH2(CH2)3NH), 34.9 (COCH2CH2), 38.8 (CHCH2Ph),
40.9 (CH(CH2)3CH2NH), 49.5 (NHCH2CO), 52.3
(CO2CH3), 53.3 (CH(CH2)4NH), 54.8 (CHCH2Ph), 66.9
(OCH2Ph), 127.3, 128.4, 128.9, 129.7, 136.9, 137.1 (Ph),
157.1 (urethane C]O), 170.9, 171.9 (2 signals) (amide
C]O), 173.3 (ester C]O), 204.3 (ketone C]O); m/z (ES)
597 (100%, [MCH]C), 619 (27, [MCNa]C); (Found: C,
From Boc-L-Val-OCH3. 1e (0.164 g, 0.476 mmol) was
treated with 4 M HCl in dioxane (2 mL) and the resulting
solution was stirred at room temperature for 1 h. The solvent
was evaporated and the crude hydrochloride salt was dried
thoroughly in vacuo. A suspension of the hydrochloride salt
in CH2Cl2 (4 mL) was cooled in an ice bath and was treated
with DIPEA (0.11 mL, 0.63 mmol), followed by acetic
anhydride (96 mL, 1.02 mmol). The reaction mixture was
allowed to attain room temperature overnight, then it was
diluted with CH2Cl2 (25 mL) and was washed with 5% aq
NaHCO3, 5% aq citric acid and saturated aq NaCl (25 mL
each). The aqueous layers were back-extracted with CH2Cl2
(4!25 mL) and the combined organics were dried (MgSO4)
and the solvent evaporated to give an off-white solid which
was recrystallised from CH2Cl2-hexane to give a white solid
(90 mg, 66%), indistinguishable (1H, 13C NMR) from the
samples prepared by Methods A and B.
4.3.14. N-(tert-Butoxycarbonyl)-L-phenylalanyl-N3-(ben-
zyloxycarbonyl)-L-lysinyl-5-aminolaevulinic acid methyl
ester (Boc-L-Phe-L-Lys(Z)-ALA-OMe) (3a). 1c (0.141 g,
0.278 mmol) was treated with 4 M HCl in dioxane (2.5 mL)
and the resulting solution was stirred at room temperature
for 30 min. The solvent was evaporated and the crude
hydrochloride salt was dried thoroughly in vacuo. A stirred
solution of Boc-L-Phe (74 mg, 0.279 mmol) and 1-hydroxy-
benzotriazole monohydrate (58 mg, 0.429 mmol) in CH2Cl2
(1.5 mL) and DMF (1.5 mL) was cooled in an ice bath and
EDC$HCl (53 mg, 0.276 mmol) was added. After 40 min, a
solution of the preceding hydrochloride salt in DMF (2 mL)
was added, followed by DIPEA (0.11 mL, 0.63 mmol) and
the reaction mixture was allowed to attain room temperature
overnight. After evaporation of the solvents, the residue was
dissolved in EtOAc (20 mL) and was washed with 5% aq
citric acid, 5% aq NaHCO3, H2O, and saturated aq NaCl

6958
L. M.-A. Rogers et al. / Tetrahedron 61 (2005) 6951–6958
R. Chemical Aspects of Photodynamic Therapy; Gordon and
Breach: Amsterdam, 2000.
62.02; H. 6.73; N, 9.31. C34H46N4O8 requires C, 62.40; H,
6.76; N, 9.39%).
2. Milgrom, L.; MacRobert, A. J. Chem. Brit. 1998, 45–50.
3. Peng, Q.; Warloe, T.; Berg, K.; Moan, J.; Kongshaug, M.;
Giercksky, K.-E.; Nesland, J. M. Cancer 1997, 79, 2282–2308.
4. (a) Casas, A.; Batlle, A. Curr. Med. Chem. 2002, 2, 465–475.
(b) Lopez, R. F. V.; Lange, N.; Guy, R.; Bentley, M. V. L. B.
Adv. Drug. Deliv. Rev. 2004, 56, 77–94.
4.3.16. N-Acetylglycyl-5-aminolaevulinic acid hexyl ester
(Ac-Gly-ALA-OHex) (4). A stirred solution of 2a (0.152 g,
0.53 mmol) in CH3OH (1.2 mL) and H2O (1.8 mL) was
treated with LiOH (16 mg, 0.66 mmol). The reaction
mixture was stirred at room temperature for 40 min, then
it was applied to a column of Amberlyst IR 120 resin (plus)
cation exchange resin which was eluted with 60% aq
CH3OH. Acidic fractions were collected and the solvent was
evaporated to give the crude acid as a white solid (0.182 g).
A solution of the acid (70 mg, 0.25 mmol) in CHCl3
(10 mL) was treated with DCC (58 mg, 0.28 mmol), DMAP
(5 mg, 40 mmol) and hexan-1-ol (40 mL, 0.32 mmol). The
reaction mixture was stirred at room temperature for 21 h,
then it was filtered to remove DCU and the solvent was
evaporated. The crude product was purified by column
chromatography using 5% MeOH/CH2Cl2 as eluant, then
recrystallised from CH3OH/diethyl ether to give a white
solid (52 mg, 65%); Mp: 124–125 8C; dH (CDCl3) 0.82 (3H,
t, JZ6.7 Hz, (CH2)5CH3), 1.10–1.35 (6H, m, (CH2)2-
CH2CH2CH2CH3), 1.40–1.70 (2H, m, CH2CH2(CH2)3-
CH3), 1.99 (3H, s, COCH3), 2.57–2.68 (4H, m,
COCH2CH2), 3.91 (2H, d, JZ5.2 Hz, NHCH2CONH),
4.16 (2H, d, JZ4.7 Hz, NHCH2CO), 6.10 (1H, br, CH3-
CONH), 6.50 (1H, br, NHCH2COCH2CH2); dc 14.0
((CH2)5CH3), 22.5, 25.5, 27.8, 28.5 (CH2)CH2CH2CH2-
CH3, COCH2CH2), 31.3 (CH2CH2(CH2)3CH3), 34.5
(COCH2CH2), 43.0 (NHCH2CONH), 49.1 (NHCH2CO),
65.1 (CH2(CH2)4CH3)), 169.1, 170.1 (amide C]O), 172.5
(ester C]O), 203.6 (ketone C]O); [Found: (EI)
314.18417, C15H26N2O5 requires 314.18351]; m/z (ES)
315 (70%, [MCH]C), 337 (100, [MCNa]C), 353 (10,
[MCNa]C).
5. Casas, A.; Battle, A. M. C.; Butler, A. R.; Robertson, D.;
Brown, E. H.; MacRobert, A.; Riley, P. A. Br. J. Cancer 1999,
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´
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References and notes
1. (a) Bonnett, R. Chem. Soc. Rev. 1995, 24, 19–33. (b) Bonnett,