Efficient syntheses of a flavin and an 8-hydroxy-5-deazaflavin amino acid and their incorporation into oligopeptides

Article abstract of DOI:10.1021/jo980643l

We report a convenient synthesis for the cofactor amino acids (S)-3 and (S)-4 in which the C₅-ribose chain of the original riboflavin and ribo-5- deazaflavin cofactors is replaced by a C₅-amino acid side chain. Both cofactor amino acids are available in enantiomerically pure form in gram quantities and can be incorporated into oligopeptides using a standard Fmoc- based solid-phase peptide synthesis protocol. The benzyl-protecting group of the 8-hydroxy-5-deazaflavin can be cleaved by hydrogenolysis directly on the peptide. This allows the investigation of the properties of the peptide bound redox active OH- and the deprotonated O^- form of the deazaflavin. Due to the electron- and energy-transfer properties of both cofactors, applications of both amino acid in the preparation of peptide- and protein-based biosensors, of catalytically active peptides, or as chemical rulers for distance measurements in biopolymers based on the fluorescence resonance energy- transfer technology can be envisaged.

Full text of DOI:10.1021/jo980643l

J . Org. Chem. 1998, 63, 8741-8747
8741
Efficien t Syn th eses of a F la vin a n d a n 8-Hyd r oxy-5-d ea za fla vin
Am in o Acid a n d Th eir In cor p or a tion in to Oligop ep tid es
Thomas Carell,*,† Holger Schmid,‡ and Markus Reinhard†
Departments of Organic Chemistry and Biochemistry, Swiss Federal Institute of Technology,
ETH-Zentrum, Universita¨tstrasse 16, 8092 Zu¨rich, Switzerland
Received April 7, 1998
We report a convenient synthesis for the cofactor amino acids (S)-3 and (S)-4 in which the C5-
ribose chain of the original riboflavin and ribo-5-deazaflavin cofactors is replaced by a C5-amino
acid side chain. Both cofactor amino acids are available in enantiomerically pure form in gram
quantities and can be incorporated into oligopeptides using a standard Fmoc-based solid-phase
peptide synthesis protocol. The benzyl-protecting group of the 8-hydroxy-5-deazaflavin can be
cleaved by hydrogenolysis directly on the peptide. This allows the investigation of the properties
of the peptide bound redox active OH- and the deprotonated O--form of the deazaflavin. Due to
the electron- and energy-transfer properties of both cofactors, applications of both amino acid in
the preparation of peptide- and protein-based biosensors, of catalytically active peptides, or as
chemical rulers for distance measurements in biopolymers based on the fluorescence resonance
energy-transfer technology can be envisaged.
In tr od u ction
The variety of organic reactions that can be catalyzed
by proteins is restricted by the side-chain functionalities
of the 21 encoded amino acids (including selenocysteine).
In several enzymes this limitation is overcome by the
incorporation of cofactors, which are bound to the protein
in special cofactor-binding pockets.1 The protein provides
a suitably shaped cofactor binding site and delivers the
appropriate side-chain functionalities required for the
catalytic process. The activity of the cofactor is further
modulated by the protein through the control of (1) the
polarity of the cofactor binding pocket, (2) H-bonds or salt
bridges to specific H-bond donor or acceptor groups of the
cofactor,2 and (3) the protonation or deprotonation of the
cofactors in various redox states.3 The desire to learn how
cofactor-dependent enzymes modulate cofactor activity
and the wish to prepare functionalized, and possibly
catalytically active, oligopeptides and proteins as artifi-
cial enzymes4-7 and biosensors8-10 have motivated the
preparation of cofactor-peptide chimeras.11-13 Here, the
incorporation of cofactor amino acids into oligopeptides
F igu r e 1. Depiction of the riboflavin 1 and the ribo-5-
deazaflavin 2 cofactors and of the two C5-chain flavin (S)-3
and 5-deazaflavin (S)-4 amino acid. R ) CH2C6H6.
† Department of Organic Chemistry.
‡ Department of Biochemistry.
(1) Bugg, T. An Introduction to Enzyme and Coenzyme Chemistry;
Blackwell Science: Oxford, 1997.
(2) Kirby, J . P.; Roberts, J . A.; Nocera, D. G. J . Am. Chem. Soc. 1997,
119, 9230-9236.
(3) Roberts, D. L.; Frerman, F. E.; Kim, J .-J . P. Proc. Natl. Acad.
Sci. U.S.A. 1996, 93, 14355-14360.
(4) Kaiser, E. T.; Lawrence, D. S. Science 1984, 226, 505-511.
(5) J ames, W.; Bryson, S. F. B.; Lu, H. S.; Suich, D. J .; Zhou, H. X.;
O’Neil, K. T.; DeGrado, W. F. Science 1995, 270, 935-941.
(6) Tuchscherer, G.; Dumy, P.; Mutter, M. Chimia 1996, 50, 644-
648.
(7) Dahiyat, B. I.; Mayo, S. L. Science 1997, 278, 82-87.
(8) Walkup, G. K.; Imperiali, B. J . Am. Chem. Soc. 1996, 118, 3053-
3054.
using convenient solid phase peptide synthesis (SPPS)
methodology was particularly successful and has recently
enabled the synthesis of pyridoxalphosphate-, thia-
mine-, and nicotinamide-cofactor-containing oligopep-
tides.13
Riboflavin (1) (Figure 1) is one of the most versatile
cofactors used in nature.14-16 It participates in reactions
such as amino acid deamination, activation of molecular
oxygen for oxidation reactions, one- and two-electron-
transfer reactions, and in a photoinduced electron-
(9) Torrado, A.; Walkup, G. K.; Imperiali, B. J . Am. Chem. Soc. 1998,
120, 609-610.
(13) Imperiali, B.; Roy, R. S.; Walkup, G. K.; Wang, L. In Molecular
Design and Bioorganic Catalysis; Wilcox, C. S., Hamilton, A. D., Eds.;
Kluwer Academic Publishers: Netherlands, 1996.
(10) Marvin, J . S.; Hellinga, H. W. J . Am. Chem. Soc. 1998, 120,
7-11.
(11) Imperiali, B.; Roy, R. S. J . Am. Chem. Soc. 1994, 116, 12083-
12084.
(12) Imperiali, B.; Roy, R. S. J . Org. Chem. 1995, 60, 1891-1894.
(14) Bruice, T. C. Acc. Chem. Res. 1980, 13, 256-262.
(15) Walsh, C. Acc. Chem. Res. 1980, 13, 148-155.
(16) Ghisla, S.; Massey, V. Eur. J . Biochem. 1989, 181, 1-17.
10.1021/jo980643l CCC: $15.00 © 1998 American Chemical Society
Published on Web 11/06/1998

8742 J . Org. Chem., Vol. 63, No. 24, 1998
Carell et al.
Sch em e 1. Syn th esis of th e F m oc-P r otected
F la vin Am in o Acid (S)-5
F igu r e 2. Representation of the 5-deazaflavin cofactor in its
redox competent “protonated” (8-OH-form) and its rather redox
incompetent deprotonated form (8-O--form).
transfer process required for the repair of pyrimidine
dimer DNA lesions by the enzyme DNA-photolyase.17 The
structurally related ribo-5-deazaflavin (2) (Figure 1)
functions in its “protonated” “8-OH-form” (Figure 2) as
a nicotinamide in “flavin clothing”,18 as a low potential
hydride-transfer agent involved in methane metabolism.
The deprotonated “8-O--cofactor form” (pKa ≈ 6) partici-
pates as a light-gathering antenna unit in the repair of
pyrimidine dimer DNA lesions.19,20 For the preparation
of defined redox active and potentially catalytically active
oligopeptides, it would therefore be highly desirable to
possess both cofactors as cofactor amino acid building
blocks, amenable to SPPS. Furthermore, since both the
flavin and the deazaflavin chromophore are known to be
highly fluorescent and undergo light-induced electron-
and energy-transfer reactions,21 applications of flavin and
deazaflavin amino acids in the synthesis of peptide- and
protein-based biosensor materials8,22,23 or as probes for
distance measurements in biopolymers based on the
fluoresecence resonance energy-transfer methodology can
be envisioned.
group, using catalytic hydrogenation and raising of the
pH (pH > 7), converts the deazaflavin into its deproto-
nated form.
The synthesis of the (S)-2-amino-NR-(9-fluorenylmethyl-
oxycarbonyl)-6-(7′,8′-dimethylisoalloxazin-10′-yl)hexanoic
acid [(S)-5] and of the (S)-2-amino-NR-(9-fluorenylmeth-
yloxycarbonyl)-6-(8′-O-benzyl-5′-carbaisoalloxazin-10′-yl)hexa-
noic acid [(S)-6] are outlined in Schemes 1 and 2. The
key step in the preparation of the Fmoc-protected flavin
amino acid (S)-5 involves an ipso-substitution reaction
between 4,5-dimethyl-1,2-dinitrobenzene (7) and the NR-
Boc-protected (S)-lysine derivative (S)-8. The 1,2-dini-
trobenzene starting material 7 was prepared from 4,5-
dimethyl-2-nitroaniline (9) using a known two-step
oxidation protocol, first involving oxidation of 9 to the
nitroso-nitro compound with Caros’ acid and then further
oxidation of this intermediate to the dinitro compound 7
with hydrogen peroxide and catalytic nitric acid.25,26 The
ipso-substitution was found to proceed without racem-
ization of the reaction product (S)-10 in an optimal
fashion either in a solvent mixture of n-butanol, water,
and ethanol at 80 °C with potassium acetate as the base
or in pure pyridine at 100 °C. The intensively red-colored
reaction product (S)-10 was readily separated from excess
dinitro compound 7 even on larger scale by filtration
through a silica gel plug eluting first with chloroform/
TEA to remove 7 and then with chloroform/methanol/
Resu lts a n d Discu ssion
In this paper, we report a convenient synthetic strategy
for the preparation of the flavin24 and the 5-deazaflavin
amino acids (S)-3 and (S)-4 in which the C5-ribose chain
of the original riboflavin and ribo-5-deazaflavin cofactors
is replaced by a C5-amino acid side chain, which is
derived retrosynthetically from a lysine building block.
Both cofactor amino acids are available in enantiomeri-
cally pure form in gram quantities as required for
efficient SPPS. To study both, the redox active OH- and
the deprotonated O--forms of the deazaflavin, we pre-
pared the 8-O-benzyl-protected 5-deazaflavin amino acid
(S)-4 (R ) Bn), which mimics the deazaflavin cofactor in
its “protonated” status. Deprotection of the 8-O-benzyl
(17) Sancar, A. Biochemistry 1994, 33, 2-9.
(18) Walsh, C. Acc. Chem. Res. 1986, 19, 216-221.
(19) Malhotra, K.; Kim, S.-T.; Walsh, C.; Sancar, A. J . Biol. Chem.
1992, 267, 15406-15411.
(20) J orns, M. S.; Ramsey, A. J . Biochemistry 1992, 31, 8437-8441.
(21) Epple, R.; Carell, T. Angew. Chem., Int. Ed. Engl. 1998, 37,
938-941. For a review, see: Carell, T.; Epple, R. Eur. J . Org. Chem.
1998, 1245-1258.
(22) Walkup, G. K.; Imperiali, B. J . Am. Chem. Soc. 1997, 119,
3443-3450.
(23) Elbaum, D.; Nair, S. K.; Patchan, M. W.; Thompson, R. B.;
Christianson, D. W. J . Am. Chem. Soc. 1996, 118, 8381-8387.
(24) Carell, T.; Butenandt, J . Angew. Chem., Int. Ed. Engl. 1997,
36, 1461-1464.
(25) Bamberger, E.; Hu¨bner, R. Ber. Dtsch. Chem. Ges. 1903, 36,
3803-3823.
(26) Kuhn, R.; v. Klaveren, W. Ber. Dtsch. Chem. Ges. 1938, 71, 779-
780.

Incorporation of Flavins into Oligopeptides
J . Org. Chem., Vol. 63, No. 24, 1998 8743
Sch em e 2. Syn th esis of th e F m oc-P r otected
Subsequent reaction of (S)-15 with the bisbenzyl-protect-
ed 2,4-dihydroxybenzaldehyde 16, following the general
deazaflavin synthesis protocol of F. Yoneda and co-
workers31 afforded the NR-Boc-protected deazaflavin amino
acid (S)-17. This was treated with trifluoroacetic acid
to cleave both tert-butyl-containing protection groups to
afford (S)-4. Reaction of the unprotected deazaflavin
amino acid (S)-4 with Fmoc-OSu and sodium hydrogen
carbonate then yielded the NR-Fmoc-protected deazafla-
vin amino acid (S)-6. The final Fmoc-protected deazafla-
vin amino acid (S)-6 was obtained as a light yellow
powder in 15% overall yield, ready for its incorporation
into oligopeptides using SPPS. Despite the only moder-
ate yields obtained in the ipso-substitution reaction
between 7 and (S)-8, and in the condensation reaction of
(S)-11 with alloxan and of (S)-15 with the benzaldehyde
derivative 16 to give the isoalloxazine and 5-carbaisoal-
loxazine heterocycles (S)-12 and (S)-17, both cofactor
amino acids (S)-5 and (S)-6 are readily available in gram
quantities using the above-described procedures.
Dea za fla vin Am in o Acid (S)-6
To determine the enantiomeric purity of the cofactor
amino acids (S)-3 and (S)-4, we prepared the correspond-
ing (R)-configured compounds (R)-3 and (R)-4 using the
same procedures and reaction conditions but the (R)-
configured lysine starting materials (R)-8 and (R)-14. The
racemates (rac)-3 and (rac)-4 were obtained by mixing
the corresponding enantiomers. Separation of the enan-
tiomers was accomplished by thin layer, ligand exchange
chromatography on reverse phase CHIRALPLATES.32
The solvent mixture acetonitrile/water/methanol (4:1:1)
allowed the rapid (20 min) optical resolution of the
racemates (rac)-18, (rac)-3, and (rac)-4. Separation of the
racemate of the deprotected compound derived from (rac)-
15 was not possible using the CHIRALPLATE technology
due to low solubility. A dilution experiment, in which
decreasing amounts of the (R)-isomers (R)-18, (R)-3, and
(R)-4 were added to the corresponding (S)-forms and
subsequent scanning of the obtained TLC plates estab-
lished that amounts of the (R)-isomers down to 2-4% are
detectable. Since the final compounds derived from the
(S)-configured starting material (S)-3 and (S)-4 yield only
one spot on the CHIRALPLATES, as depicted in Figure
3 for the amino acids (R/ S)-18 and (R/ S)-3, we conclude
that the final synthetic cofactor amino acids (S)-5 and
(S)-6 possess an enantiomeric purity of ee > 95%.
Experiments aimed at finding suitable conditions for
the incorporation of the flavin and the deazaflavin amino
acids (S)-5 and (S)-6 into oligopeptides by SPPS revealed
that both cofactor amino acids are fully amendable to a
standard Fmoc-based SPPS protocol.33 Activation of the
cofactor amino acids was performed with a mixture of
HOBT and TBTU for 5 min prior to their coupling. The
coupling reaction was performed either in NMP or in
DMF using a Rink resin. Brief capping after each
coupling was performed with acetic anhydride and DIEA.
Deprotection of the Fmoc-group was achieved with 20%
piperidine in DMF. This causes no decomposition of the
otherwise nucleophile-sensitive isoalloxazin heterocycle.
Cleavage of the side-chain protection groups and of the
acetic acid (5:1:1%) to obtain the desired product (S)-10.
Hydrogenolytic reduction of the nitro group in (S)-10 and
subsequent filtration of the reaction product (S)-11
through Celite into a suspension of alloxan and boric acid
in acetic acid, following the general flavin synthesis
protocol of R. Kuhn and co-workers27,28 yielded the NR-
Boc-protected flavin amino acid (S)-12. Cleavage of the
Boc-protection group with trifluoroacetic acid afforded the
unprotected flavin amino acid (S)-3 which was reacted
with Fmoc-OSu in sodium hydrogen carbonate solution
to yield the desired NR-Fmoc-protected flavin amino acid
(S)-5 in 16% overall yield as an intensively yellow-colored
powder, ready for the incorporation into peptides using
SPPS.
For the preparation of the deazaflavin amino acid (S)-
6, the 6-chloro uracil 13 was first reacted with the NR-
Boc-protected (S)-lysine-tert-butyl ester building block
(S)-1429,30 in n-butanol at 80 °C to yield the uracil-lysine
compound (S)-15. Protection of the amino and carboxylic
acid functionalities in (S)-14 as the tert-butyl carbamate
and the tert-butyl ester, respectively, was required to
increase the solubility and allow purification of the
lysine-uracil reaction product (S)-15 by chromatography.
(31) Kimachi, T.; Tanaka, K.; Yoneda, F. J . Heter. Chem. 1991, 28,
439-443.
(27) Kuhn, R.; Weygand, F. Ber. Dtsch. Chem. Ges. 1934, 67, 1409-
1413.
(32) CHIRALPLATES are available from Macherey-Nagel GmbH
& Co. KG, D-52313 Du¨ren (Germany). See, for examples: Gu¨nther,
K. J . Chromatogr. 1988, 448, 11-30. Beck, A. K.; Seebach D. Chimia
1988, 42, 142-144.
(28) Kuhn, R.; Weygand, F. Ber. Dtsch. Chem. Ges. 1935, 68, 1282-
1288.
(29) Feldmann, P. L. Tetrahedron Lett. 1991, 32, 875-878.
(30) Maguire, M. P.; Feldmann, P. L.; Rapoport, H. J . Org. Chem.
1990, 55, 948-955.
(33) Grant, G. A. Synthetic Peptides A User’s Guide; W. H. Freeman
and Company: New York, 1992.

8744 J . Org. Chem., Vol. 63, No. 24, 1998
Carell et al.
parable to the reference Ala-containing peptide 21 (HPLC
trace not shown). The successful preparation of the 24-
and 27-mer oligopeptides 23 and 22 demonstrates that
even a large number of coupling cycles can be performed
after the incorporation of the cofactor amino acids
without significant cofactor destruction. The purification
of the cofactor peptides was finally performed by pre-
parative HPLC with a water (0.1% TFA)/acetonitrile
gradient. Here the strong absorption of both heterocycles
at 450 nm (flavin) and 400 nm (deazaflavin) allowed their
easy detection and facilitated the HPLC purification
procedures.
To cleave the 8-O-benzyl-protection group of the dea-
zaflavin and to convert the deazaflavin into its deproto-
nated form, deazaflavin-containing peptides such as 24
were dissolved in an acetic acid/water mixture (1:1) and
a very small amount of Pd/BaSO4 catalyst was added.21
This solution was gently stirred in an H2 atmosphere for
approximately 6 h. During this time, the fluorescence
of the sample (excitation at 366 nm) was carefully
monitored. It proved to be essential to avoid complete
reduction of the deazaflavin chromophore, which is
accompanied by a diminishment of the characteristic blue
fluorescence. After approximately 80% conversion, isola-
tion of the debenzylated peptide 25 was achieved by
preparative HPLC.
F igu r e 3. Depiction of the optical resolution of the racemates
(rac)-18 and (rac)-3 with CHIRALPLATES32 (solvent system
of acetonitrile, water, methanol (4:1:1)).
Lyophilization of the purified peptides yielded all
cofactor peptides in the form of intensively orange or
yellow powders which were characterized by HPLC,
electrospray or MALDI-TOF mass spectrometry, and
amino acid sequencing.
The UV and fluorescence spectra of the purified pep-
tides 19 and 23-25 in water at pH ) 8 are depicted as
typical examples in Figure 5. They prove the presence
of the cofactors, all stably incorporated into the oligopep-
tides. The benzylated deazaflavin-containing peptides
(such as 24) feature the expected absorption maxima
around 400 nm for the OH-form deazaflavin. The flavin
peptides 19 and 23 exhibit absorption maxima at 370 and
450 nm. The intense fluorescence at 430 nm (24) and
520 nm (23) shows the successful incorporation of the
intact heterocycles. UV/vis and fluorescence spectra
underline the successful debenzylation of the deazaflavin
cofactor inside the oligopeptide. The measured UV/vis
and fluorescence spectrum of the peptide 25, containing
the debenzylated deazaflavin, features under acidic
conditions the absorption and fluorescence characteristics
of the benzylated deazaflavin unit. Addition of triethyl-
amine, or measuring in water, buffered at pH 8, however,
caused deprotonation of the 8-OH group (pKa ) 6) as
evidenced by the increased absorption, the shift of the
absorption maximum from approximately 400 to 420 nm
and by the fluorescence maximum which appears now
at 470 nm, as expected for the deprotonated 8-hydroxy-
5-carbaisoalloxazine chromophore. Both values are in
full agreement with the spectroscopic data of the A.
nidulans DNA-photolyase, which contains the F420
cofactor as a light-gathering antenna chromophore in its
deprotonated form. Investigation of the fluorecence
properties of the flavin amino acid incorporated into the
tyrosine-rich peptide 19 shows that the flavin fluores-
cence is strongly reduced (Figure 5B). This experiment
confirms that the flavin environment critically influences
the spectroscopic properties of the cofactor and supports
F igu r e 4. HPLC traces of the crude peptides 19 (A) and 20
(B) obtained directly after the acid-mediated cleavage from the
solid support. Reverse phase HPLC. Gradient 100% A to 80%
B in 70 min (A, water with 0.1% TFA; B, acetonitrile).
peptide from the resin was achieved with 95% trifluoro-
acetic acid, 2.5% triisopropylsilane, and 2.5% water over
5 h. The crude peptide solution was evaporated in vacuo
and precipitated with diethyl ether. Figure 4 shows the
RP-HPLC traces obtained from the crude flavin- and
deazaflavin-containing peptides 19 and 20 (Table 1)
directly after acid-mediated cleavage from the solid
support. The cofactor peptides 19 and 20 are the main
reaction products and are obtained with a purity com-

Incorporation of Flavins into Oligopeptides
J . Org. Chem., Vol. 63, No. 24, 1998 8745
Ta ble 1. Sequ en ce of th e P r ep a r ed Cofa ctor P ep tid e Ch im er a s a n d Th eir Ca lcu la ted a n d Deter m in ed Molecu la r
Weigh ts
no.
sequence
calcd [M+]
obsd [M+] (M+ + Na)
21
19
20
24
25
22
23
AYA-A-EYYLE-OH
AYA-Fl-EYYLE-OH
AYA-dFl(OBn)-EYYLE-OH
AQDKA-dFl(OBn)-SKA-NH2
AQDKA-dFl-SKA-NH2
1092
1375
1452
1248
1158
3374
3012
(1118)a
1375b (1397)b
1451b (1474)b
1247b
1157b
PAAL-Fl-RARNTEAARRSRARKLQRLKEC-NH2
Ac-ADRRKAATERERRR-Fl-SKVNEAGGC-NH2
3375a
3011a
a
b
Maldi-TOF mass spectrometry. Electrospray mass spectrometry. Fl ) Flavin, dFl ) deazaflavin, dFl(OBn) ) 8-O-benzyl-deazaflavin.
A: Alanine. C: Cysteine. D: Aspartic acid. E: Glutamic acid. G: Glycine. K: Lysine. L: Leucine. N: Asparagine. P: Proline. Q: Glutamine.
R: Arginine. S: Serine. T: Threonine. V: Valine. Y: Tyrosine.
scopic properties of a flavin and a deazaflavin which
illustrates their stable incorporation. Debenzylation of
the benzyl-protected 8-OH-deazaflavin group was achieved
using catalytic hydrogenolysis on the peptide. Although
this deprotection requires special care, it allows the study
of both forms (8-OH and 8-O-) of the deazaflavin cofactor
using the same building block for SPPS. We com-
municated recently that flavin-containing oligopeptides,
which possess the sequence of DNA-binding transcription
factors, are able to repair thymine dimer DNA-lesions
in a DNA single strand.24 We believe the presented
procedures now enable the preparation of flavin and
deazaflavin peptides as artificial DNA-repair enzymes
and of oligopeptides with novel energy- and electron-
transfer properties.35 Due to the ability of deazaflavins
to transfer excitation energy to the flavin,21 both cofactor
can also be used as new tools for fluorescence energy-
transfer experiments in biopolymers.
Exp er im en ta l Section
Gen er a l. Reagents and solvents were purchased reagent
grade and used without further purification. Anhydrous
MgSO4 was used as the drying agent after aqueous workup.
Evaporation and concentration in vacuo was done at H2O-
aspirator pressure. Column chromatography: Silica gel-H or
-S from Fluka. TLC: glass or aluminum sheets covered with
silica gel 60 F254 from Merck; visualization by UV light.
Melting points are uncorrected and measured in open capillary
tubes. IR spectra (cm-1) are measured as KBr pellets.
Fluorescence spectra were measured on a Spex 1680, 0.22m
double grating spectrometer, 450 Hg/Xe-lamp, in 1 cm quartz
1
cuvettes at RT. For H and 13C NMR, solvent peaks (2.49 ppm
for 1H and 39.7 ppm for 13C NMR, respectively) are used as
reference. MS (m/z): FAB measured in a 3-nitrobenzyl alcohol
matrix. R-Values were measured at 24 °C. HPLC was
F igu r e 5. UV/Vis spectra (A) and fluorescence spectra (B) of
performed with a flow of 1 mL/min using a Nucleosil RP18
the flavin and deazaflavin containing peptides measured at
(240 mm × 4 mm, 100 Å/5 µm) column. For analytical
oligopeptide HPLC, a linear gradient (H2O (0.1%TFA) to
acetonitrile) was used. Preparative HPLC was performed with
a Nucleoprep RP C18 column (250 mm × 21 mm, 100 Å/10
µm). Solvent system: A ) 0.1% TFA in H2O, B ) acetonitrile;
A to B in 120 min.
4,5-Dim eth yl-1,2-d in itr oben zen e (7). Compound 9 (36 g,
0.22 mol) was dissolved in ice water (500 mL). Potassium
persulfate (120 g) was added into concentrated sulfuric acid
(200 mL), and the mixture was stirred until a clear solution
was obtained. This solution was added into the suspension
of 9 in ice water. The reaction mixture was stirred at RT for
20 h, and then another batch of potassium persulfate (60 g)
in concentrated sulfuric acid (200 mL) was added until all
starting material had disappeared. The intermediate nitroso
product (41 g) was filtered off and dried in vacuo at 50 °C.
The product was subsequently dissolved in concentrated acetic
10-5 M in water (0.05 M Tris‚HCl, pH ) 8). A: (s) 19 and 23.
(- - -) 24. (‚‚‚) 25. B: (s) 19. (-‚‚) 23. (- - -) 24. (‚‚‚) 25.
the observation of McCormick and co-workers of reduced
flavin fluorescence in a tyrosine or tryptophan environ-
ment.34
Con clu sion s
In this paper, we describe an efficient and convenient
synthesis for the flavin and the deazaflavin amino acid
(S)-3 and (S)-4. Both cofactor amino acids can be
incorporated into oligopeptides using a rather standard
Fmoc-based solid-phase peptide synthesis protocol. The
incorporated cofactors show all of the expected spectro-
(34) Fo¨ry, W.; MacKenzie, R. E.; McCormick, D. B. J . Heterocyl.
Chem. 1968, 5, 625-630.
(35) Balzani, V.; Credi, A.; Venturi, M. Curr. Opin. Chem. Biol. 1998,
4, 506-513.

8746 J . Org. Chem., Vol. 63, No. 24, 1998
Carell et al.
acid (550 mL) at 100 °C. Nitric acid (70 mL, 65%) was added.
A mixture of H2O2 (35%, 500 mL) in concentrated acetic acid
(200 mL) was then added to the hot solution over 1.5 h. The
clear orange reaction solution was stirred for another 2 h at
100 °C. After complete reaction (TLC toluene/ethyl acetate
10:1), the reaction mixture was cooled to RT, and the solid
product (21.4 g, 50%) 7 was filtered off and dried in vacuo at
50 °C. For elemental analysis, 7 was recrystallized from
EtOH. For 7: mp 111-112 °C; IR (KBr) 3100, 3044, 2922,
2367, 1761, 1583, 1551, 1528, 1483, 1444, 1379, 1338, 1256,
1237, 1146, 1024, 1002, 894, 883, 859, 807, 751, 660, 584, 548,
obtained as a yellow powder after reprecipitation from water/
ethanol (1.2 g, 95%): [R]D ) 6.0 (c ) 1.0% in DMSO); mp >
215 °C; IR (KBr) 3446, 2360, 1687, 1578, 1544, 1461, 1350,
1267, 1205, 1139, 839, 800, 722, 667, 606; 1H NMR (400 MHz)
δ 1.45-1.65 (m, 2 H), 1.70-1.78 (m, 2H), 1.80-1.95 (m, 2H),
2.38 (s, 3H.43 (s, 3H), 3.91 (t, J ) 8 Hz, 1H), 4.58 (m, 2H),
7.78 (s, 1H), 7.88 (s, 1H), 8.15-8.40 (br.s., 1H), 11.30 (s, 1H);
13C NMR (100 MHz) δ 18.7, 20.6, 21.5, 25.9, 29.6, 43.6, 51.9,
116.1, 130.6, 131.0, 133.8, 135.8, 137.1, 146.7, 150.1, 155.8,
159.9, 171.0; MS (FAB+) m/z (%) 372.2 (30) [MH+]; HRMS calcd
for C18H21N5O4 [MH+] 372.1672, found 372.1683.
1
417; H NMR (200 MHz, CDCl3) δ 2.44 (s, 6 H), 7.70 (s, 2 H);
(S)-2-Am in o-Nr-(9-flu or en ylm eth yloxycar bon yl)-6-(7′,8′-
d im eth ylisoa lloxa zin -10′-yl)h exa n oic Acid ((S)-5). (S)-2-
Amino-6-(7′,8′-dimethylisoalloxazin-10′-yl)hexanoic acid ((S)-
3) (1.0 g, 2.7 mmol) was dissolved in an aqueous sodium
hydrogen carbonate solution (150 mL, 10%). The suspension
was cooled to 0 °C, and Fmoc-OSu (1.1 g, 3.3 mmol) dissolved
in DMF (15 mL) was added. The resulting thick slurry was
shaken for another 30 min and diluted with water, and the
aqueous phase was extracted with diethyl ether. The aqueous
phase was collected, acidified with citric acid (pH ) 4), and
extracted three times with CHCl3. The combined organic
phases were dried with MgSO4, filtered, and evaporated in
vacuo. The product (S)-5 was precipitated through the addi-
tion of diethyl ether and dried in vacuo. Compound (S)-5 was
MS (EI) m/z (%) 196 (75) [M+], 108, 91, 77, 65, 51, 39. Anal.
Calcd for C8H8N2O4 (196.16): C, 48.93; H, 4.11; N, 14.28.
Found C, 48.89; H, 3.90; N, 14.37.
(S)-2-Am in o-Nr-(ter t-bu tyloxycar bon yl)-6-(4′,5′-dim eth yl-
2′-n itr oa n ilin -N-yl)h exa n oic Acid ((S)-10). Compound 7
(2.0 g, 10.2 mmol) and NR-(tert-butyloxycarbonyl)-(S)-lysine
(S)-8 (4.0 g, 16.2 mmol) were suspended in pyridine (250 mL),
and the mixture was heated at reflux for 72 h. The dark red
reaction solution was diluted with CHCl3 (400 mL) and
extracted three times with citric acid solution (10% in water).
The organic phase was finally washed with water, dried with
MgSO4, and, after the addition of 50 g silica gel, evaporated
to dryness. The resulting red powder was added on top of a
silica gel column. Unreacted 7 was separated by eluting the
column with CHCl3 (1% TEA). The product (S)-10 was
separated with CHCl3/MeOH (5:1, 1% acetic acid) as the
eluent. After evaporation of the solvent in vacuo, (S)-10 was
obtained as a red oil that crystallized at 4 °C (3.0 g, 74%): [R]D
) 2.0 (c ) 1.0% in DMSO); mp 74-76 °C; IR (KBr) 3378, 2978,
2922, 2867, 2356, 1700, 1678, 1633, 1572, 1506, 1406, 1367,
1306, 1239, 1161, 1056, 1022, 861, 761, 667; 1H NMR (500
MHz) δ 1.35 (s, 9 H), 1.58 (m, 4H), 1.73 (m, 2H), 2.11 (s, 3H),
2.23 (s, 3H), 3.27 (m, 2H), 3.76 (br.s., 1H), 6.29 (br.s., 1H), 6.81
(s, 1H), 7.78 (s, 1H), 7.97 (t, J ) 5.4 Hz, 1H); 13C NMR (125.8
MHz) δ 17.9, 20.1, 22.7, 28.2, 28.3, 32.3, 42.3, 54.5, 77.4, 114.4,
123.9, 125.3, 128.5, 143.9, 147.5, 154.9, 166.9; MS (FAB-) m/z
(%) 394 (40) [M- - H], 306 (100); HRMS calcd for C19H29N3O6
[M- - H] ) 394.1978, found 394.1982.
20
obtained as a yellow powder (1.2 g, 75%): [R]D ) -13.1 (c )
1.0% in DMSO); mp > 90-95 °C (dec); IR (KBr) 3419, 2360,
1716, 1667, 1579, 1543, 1450, 1400, 1350, 1261, 1172, 741, 667;
1H NMR (400 MHz) δ 1.48-1.60 (m, 2 H), 1.70-1.80 (m, 4H),
2.36 (s, 3H), 2.48 (s, 3H), 3.95-3.99 (m, 1H), 4.20-4.25 (m,
1H), 4.25-4.33 (m, 2H), 4.50-4.65 (m, 2H), 7.32 (dd, J ) 14.
Hz, 6.7 Hz, 2H), 7.41 (t, J ) 7.4 Hz, 2H), 7.71 (m, 4H), 7.89
(m, 3H, Ar-H), 11.30 (s, 1H); 13C NMR (100 MHz) δ 15.1, 18.7,
22.9, 25.9, 30.5, 43.9, 46.6, 53.6, 65.5, 115.9, 120.0, 125.2, 126.9,
127.5, 130.6, 130.9, 133.7, 135.6, 137.0, 140.6, 143.7, 146.5,
149.9, 155.6, 156.1, 159.8, 173.7; MS (FAB-) m/z (%) 593.4
(100) [M•-]; HRMS calcd for C33H31N5O6 [M•-] 593.2274, found
593.2269. Anal. Calcd for C33H31N5O6 + 1.5 H2O (620.68):
C, 63.85; H, 5.52; N, 11.28. Found: C, 64.06; H, 5.61; N, 10.93.
(S)-2-Am in o-Nr-(ter t-bu tyloxyca r bon yl)-6-(6′-a m in op y-
r im id in e-2′,4′-d ion -N-yl)h exa n oic Acid ter t-Bu tyl Ester
((S)-15). 6-Chloro uracil 13 (3.0 g, 20 mmol) and NR-(tert-
butyloxycarbonyl)-(S)-lysine tert-butyl ester ((S)-14) (7.0 g, 23
mmol) were heated in n-butanol (50 mL) to 80 °C for 5 h. The
reaction solution was evaporated in vacuo, and the residual
material was dried in vacuo until a solid material was
obtained. The product (S)-15 was purified by chromatography
on silica gel with CHCl3/MeOH (20:1). Compound (S)-15 was
obtained as an off-white powder (4.1 g, 49%), which was used
without further purification: [R]D ) -9.9 (1.0% in DMSO); mp
) 173-176 °C; IR (KBr) 2878, 2922, 2356, 1713, 1615, 1517,
1450, 1389, 1368, 1289, 1244, 1155, 1044, 1022, 844, 781, 547;
1H NMR (400 MHz) δ 1.25-1.50 (m, 22 H), 1.50-1.70 (m, 2H),
3.00 (m, 2H), 3.78 (m, 1H), 4.40 (s, 1H), 6.05 (m, 1H), 7.05 (d,
J ) 7.7 Hz, 1H), 9.83 (s, 1H), 10.15 (s, 1H); 13C NMR (100
MHz) δ 22.8, 27.6, 27.7, 30.3, 41.0, 54.2, 72.4, 77.9, 80.1, 150.7,
153.9, 155.5, 164.1 (2C), 171.8; MS (FAB+) m/z (%) 413.4 (95)
[MH+]. Anal. Calcd for C19H32N4O6 (412.49): C, 55.33; H,
7.82; N, 13.58. Found C, 55.32; H, 7.72; N, 13.46.
(S)-2-Am in o-Nr-(ter t-bu tyloxyca r bon yl)-6-(7′,8′-d im eth -
ylisoa lloxa zin -10′-yl)h exa n oic Acid ((S)-12). (S)-2-Amino-
NR-(tert-butyloxycarbonyl)-6-(4′,5′-dimethyl-2′-nitroanilin-N-yl-
)hexanoic acid ((S)-10) (2.8 g, 7.1 mmol) was dissolved in acetic
acid (100 mL) and stirred after the addition of cat. Pd/C (10%)
for 24 h under an H2 atmosphere. The colorless reaction
solution was filtered through Celite, and subsequently alloxan
monohydrate (3.0 g) and boric acid (4.0 g) were added. The
mixture was stirred at RT in the dark for another 12 h. The
solution was diluted with CHCl3 (500 mL), and the organic
phase was washed three times with water. The organic phase
was separated, dried with MgSO4, and evaporated in vacuo.
Diethyl ether was added to the resulting oil in order to
precipitate the product. Compound (S)-12 was obtained as a
yellow powder, which was dried in vacuo (2.0 g, 60%): [R]D
)
-1.0 (c ) 1.0% in DMSO); mp > 300 °C; IR (KBr) 3411, 2966,
2367, 1678, 1578, 1544, 1461, 1400, 1367, 1330, 1167, 1056,
1017, 861, 827, 811, 772, 739, 678, 605, 583, 499, 470, 452; 1H
NMR (400 MHz) δ 1.38 (s, 9 H), 1.48-1.85 (m, 6H), 2.39 (s,
3H), 2.51 (s, 3H), 3.88 (br.s., 1H), 4.55 (m, 2H), 7.04 (d, J )
8.1 Hz), 7.76 (s, 1H), 7.89 (s, 1H), 11.28 (s, 1H), 12.50 (br.s.,
1H); 13C NMR (100 MHz) δ 18.7, 20.7, 22.8, 25.9, 28.1, 30.5,
43.9, 53.2, 64.8, 77.9, 115.0, 130.6, 131.0, 133.7, 135.5, 137.0,
146.5, 149.9, 155.6, 159.9, 174.0; MS (FAB-) m/z (%) 471.3 (45)
[M- - H]. Anal. Calcd for C23H29N5O6 + H2O (471.51 +
18.02): C, 56.43; H, 6.39; N, 14.31. Found: C, 56.56; H, 6.50;
N, 14.06.
(S)-2-Am in o-Nr-(ter t-bu tyloxyca r bon yl)-6-(8′-O-ben zyl-
5′-ca r ba isoa lloxa zin -10′-yl)h exa n oic Acid ter t-Bu tyl Es-
t er ((S)-17). (S)-2-Amino-NR-(tert-butyloxycarbonyl)-6-(6′-
aminopyrimidine-2′,4′-dion-N-yl)hexanoic acid tert-butyl ester
((S)-15) (1.5 g, 3.6 mmol) and 2,4-(dibenzyloxy)benzaldehyde
(16) (2.3 g, 7.2 mmol) were dissolved in DMF (50 mL) and
heated to 120 °C for 12 h. The solvent was evaporated in
vacuo, and diethyl ether was added to the solid residual
material yielding the product as a light yellow precipitate
which was filtered off, washed with diethyl ether twice, and
dried in vacuo. Compound (S)-17 was obtained as a light
yellow powder (900 mg, 41%): [R]D20 ) -10.4° (1.0% in DMSO);
mp ) 193-195 °C; IR (KBr) 3422, 3133, 2978, 2811, 2367,
1706, 1654, 1600, 1528, 1489, 1456, 1406, 1234, 1156, 1000,
850, 580, 444; 1H NMR (400 MHz) δ 1.30-1.50 (m, 20 H),
1.55-1.70 (m, 4H), 3.80 (m, 1H), 4.65 (m, 2H), 5.43 (s, 2H),
(S)-2-Am in o-6-(7′,8′-d im eth ylisoa lloxa zin -10′-yl)h exa -
n oic Acid ((S)-3). (S)-2-Amino-NR-(tert-butyloxycarbonyl)-6-
(7′,8′-dimethylisoalloxazine-10′-yl)hexanoic acid ((S)-12) (1.6
g, 3.4 mmol) was stirred in a mixture of CHCl3/trifluoroacetic
acid (1:1, 25 mL) for 1 h at RT under the exclusion of light.
The reaction mixture was evaporated in vacuo, and diethyl
ether was added to the resulting oil to precipitate the free
amino acid (S)-3. The product (S)-3 was filtered off and

Incorporation of Flavins into Oligopeptides
J . Org. Chem., Vol. 63, No. 24, 1998 8747
7.10 (d, J ) 7.7 Hz, 1H), 7.25 (m, 2H), 7.36 (m, 1H), 7.43 (t, J
) 7.1 Hz, 2H), 7.52 (d, J ) 7.1 Hz, 2H,), 8.11 (d, J ) 9.5 Hz,
1H), 8.89 (s, 1H), 10.90 (s, 1H); 13C NMR (100 MHz) δ 22.8,
24.5, 27.5, 27.9, 30.4, 43.9, 54.2, 70.1, 77.9, 80.1, 100.0, 111.9,
114.7, 115.8, 127.8, 128.1, 128.5, 133.7, 136.0, 141.0, 142.0,
145.2, 155.5, 156.5, 162.2, 164.0, 171.7; MS (FAB+) m/z (%)
627 (30) [M+ + Na], 605 (100) [M+ + H], 449 (95). Anal. Calcd
for C33H40N4O7 (604.70): C, 65.55; H, 6.67; N, 9.27. Found:
C, 65.49; H, 6.72; N, 9.29.
(S)-2-Am in o-6-(8′-O-ben zyl-5′-ca r ba isoa lloxa zin -10′-yl)-
h exa n oic Acid ((S)-4). (S)-2-Amino-NR-(tert-butyloxycarbo-
nyl)-6-(8′-O-benzyl-5′-carbaisoalloxazin-10′-yl)hexanoic acid tert-
butyl ester ((S)-17) (900 mg, 1.5 mmol) was stirred in
trifluoroacetic acid (15 mL) and water (0.5 mL) for 3 h at RT
under the exclusion of light. The reaction mixture was
evaporated in vacuo, and diethyl ether was added to the
residual oil to precipitate the trifluoroacetic acid salt of the
product (S)-4‚TFA as a light yellow powder (800 mg, 95%): [R]D
) -1.6 (1.0% in DMSO); mp ) 214-218 °C; IR (KBr): 3446,
2367, 1683, 1602, 1528, 1489, 1406, 1245, 1200, 1139, 1022,
833, 800; 1H NMR (400 MHz) δ 1.45-1.70 (m, 4 H), 1.70-
1.95 (m, 2H), 3.90 (m, 1H), 4.65 (m, 2H), 5.43 (s, 2H), 7.27 (m,
2H), 7.36 (m, 1H), 7.43 (t, J ) 7.3 Hz, 2H), 7.52 (d, J ) 7.4
Hz, 2H), 8.13 (d, J ) 9.3 Hz, 1H), 7.90-8.70 (br.s., 3H), 8.90
(s, 1H), 11.00 (s, 1H, NH); 13C NMR (100 MHz) δ 21.5, 25.9,
29.7, 43.6, 52.0, 70.1, 100.2, 112.0, 114.6, 115.9, 127.82, 128.2,
128.6, 133.7, 136.0, 141.1, 142.0, 156.8, 157.4, 162.3, 164.1,
171.0; HRMS calcd for C24H24N4O5 [MH+] 449.1834, found
449.1824.
suspension was shaken for 3 h at RT and aqueous 10% citric
acid solution was carefully added until pH ) 4 was reached.
The mixture was diluted with CHCl3 (200 mL), and the organic
phase was separated. The aqueous phase was once again
extracted with CHCl3. The combined organic phases were
washed with water, dried with MgSO4, and evaporated in
vacuo. Diethyl ether was added to the remaining oil in order
to precipitate the product (S)-6 as a light yellow powder.
Recrystallization from methanol yielded the Fmoc-protected
deazaflavin amino acid as a yellow powder (760 mg, 80%):
20
[R]D ) -16.8 (1.0% in DMSO); mp ) 135-138 °C; IR (KBr)
3433, 2367, 1700, 1603, 1561, 1529, 1444, 1400, 1239, 1161,
1083, 1028, 740; 1H NMR (400 MHz) δ 1.42-1.55 (m, 2 H),
1.55-1.75 (m, 2 H), 1.75-1.85 (m, 2H), 4.00 (m, 1H), 4.65 (dd,
J ) 7.4 Hz, 14.5 Hz, 1H), 4.25 (m, 2H), 4.65 (br.s., 2H), 5.42
(s, 2H), 7.20-7.45 (m, 9H), 7.52 (d, J ) 7.1 Hz, 2H), 7.64 (d, J
) 7.9 Hz, 1H), 7.70 (t, J ) 6.8 Hz, 2H), 7.88 (d, J ) 7.5, 2H),
8.08 (d, J ) 8.8 Hz, 1H), 8.87 (s, 1H), 10.95 (s, 1H), 12.5 (br.s.,
1H); 13C NMR (100 MHz) δ 23.0, 25.9, 30.5, 43.9, 46.53, 53.8,
65.5, 70.0, 100.0, 111.9, 114.7, 115.9, 120.0, 125.2, 126.9, 127.5,
127.7, 128.1, 128.5, 133.6, 135.9, 140.6, 141.0, 142.0, 143.7,
156.0, 156.6, 157.3, 162.2, 164.0, 173.7; MS (FAB+) m/z (%)
671 (100) [M+ + 1]. Anal. Calcd for C39H34N4O7‚2H2O (670.74
+ 2(18.02)): C, 66.27; H, 5.42; N, 7.93. Found C, 66.44; H,
5.33; N, 7.82.
Ack n ow led gm en t. We gratefully acknowledge the
financial support of the Swiss National Science Founda-
tion and the ETH Zurich. T.C. thanks particularly Prof.
F. Diederich for his generous support, Robert Epple,
J ens Butenandt, and Anja Schwo¨gler for many helpful
discussions and Liam Cox for the careful inspection of
the manuscript.
(S)-2-Am in o-Nr-(9-flu or en ylm eth yloxyca r bon yl)-6-(8′-
O-ben zyl-5′-ca r ba isoa lloxa zin -10′-yl)h exa n oic Acid ((S)-
6). (S)-2-Amino-6-(8′-O-benzyl-5′-carbaisoalloxazine-10′-yl)-
hexanoic acid trifluoroacetic acid salt ((S)-4‚TFA) (800 mg, 1.42
mmol) was dissolved in DMF (25 mL) and aqueous 10%
NaHCO3 solution. To the suspension was added Fmoc-OSu
(600 mg, 1.78 mmol), dissolved in DMF (2 mL). The reaction
J O980643L