Synthesis of a model for phycocyanin with respect to the acid-base chemistry between protein and chromophore

Article abstract of DOI:10.1007/s007060070065

Earlier investigations have shown that the cysteine adduct of phycocyanobilin dimethylester qualifies for intramolecular protonation [1]. In this context, the -Cys-Ala-Phe-Asp-tetrapeptide adduct of phycocyanobilin dimethylester was synthesized and examin

Full text of DOI:10.1007/s007060070065

Monatshefte fuÈr Chemie 131, 879±888 (2000)
Synthesis of a Model for Phycocyanin
with Respect to the Acid-Base Chemistry
between Protein and Chromophore
Michael Stanek1;2;Ã and Karl Grubmayr1
1
È
Institut fuÈr Chemie, Johannes-Kepler-Universitat, A-4040 Linz, Osterreich
È
DSM Fine Chemicals Austria GmbH, St.-Peter-Strasse 25, A-4021 Linz, Osterreich
È
2
Summary. Earlier investigations have shown that the cysteine adduct of phycocyanobilin dimethylester
quali®es for intramolecular protonation [1]. In this context, the -Cys-Ala-Phe-Asp-tetrapeptide adduct
of phycocyanobilin dimethylester was synthesized and examined for its protonation properties with
respect to the zwitterionic interaction between chromophore and protein.
Keywords. Phycocyanobilin; Tetrapeptide; Protonation; Zwitterions; Chromophores.
Introduction
Open chain 2,3-dihydrobilindiones such as phycocyanobilin [2, 3] or phytochromo-
bilin [4, 5] serve as prosthetic groups in antenna pigments of cyanobacteria or as
photoreceptors in the photomorphogenesis in higher plants. From crystal structures
of phycocyanin [6] as well as of other phycobiliproteins [7] such as allophycocyanin
[8], phycoerythrocyanin [9], and phycoerythrin [10, 11], a common principle becomes
evident: all chromophores are covalently linked to the apoprotein by thioethers and
adopt a stretched (allZ, 5anti, 10syn, 14anti)-geometry adjacent to their ꢀ-helical
protein segment due to the formation of various salt bridges (Fig. 1). Generally, these
stretched conformations are responsible for their distinct biologically important
photophysical and photochemical properties [12±18]. However, 2,3-dihydrobilin-
dione chromophores prefer a helical (allZ, 5anti, 10syn, 14anti)-geometry in solution
except when hexamethylphosphoric triamide (HMPT) is used as solvent [19].
The protonation of 2,3-dihydrobilindion chromophores, intermolecular as well as
intramolecular, always takes place at the nitrogen atom of the azafulvenic ring B
moiety [1]. Considering phycocyanin, it is exclusively caused by the highly
conserved aspartic acid residue. Protonation of bilindiones is indicated best by
signi®cant hyperchromic and bathochromic shifts of the long-wavelength absorption
maxima and can easily be detected by measuring their UV/Vis-spectra [20±22].
As shown recently by the synthesis of two diastereomers of a cysteine adduct of
phycocyanobilin dimethylester [1, 23], intramolecular protonation is entropically
Ã
Corresponding author

880
M. Stanek and K. Grubmayr
Fig. 1. (a) Extended (5anti, 10syn, 14anti) conformation of the protein bound phycocyanobilin
chromophore; (b) surroundings of the ꢀ-helical peptide segment -Cys84-Val-85-Arg86-Asp87- of
phycocyanin from Fremyella Diplosiphon [6] (stereo view of the wire frame model)
controlled and proceeds quantitatively at low temperatures, even if the protonated
chromophore is slightly more acidic ( pKa ˆ 4.6) [20, 21] than the carboxylic acid of
cysteine which can be compared to acetic acid ( pKa ˆ 4.76). Here we report on the
synthesis, the structure, and the spectroscopic properties of the model compound 8,
which is in equilibrium with its zwitterionic species 8Æ, representing the adduct of
the tetrapeptide of N-benzyloxycarbonyl-cysteinyl-alanyl-phenylalanyl-(ꢀ-methyl)-
aspartate (Z-Cys-Ala-Phe-Asp-OMe) and phycocyanoblin dimethylester (PCBDME)
in its (2R,3R,30R,CysR,AlaS,AspS)-con®guration. The tetrapeptide corresponds to
the consensus sequence of phycocyanines (-Cys-Xxx-Arg-Asp-) [24] with the
exception of phenylalanine and alanine, whereas alanine has been found in many
phycobiliprotein subunits at this position as well. Arginine was replaced by
phenylalanine due to the dif®culty of handling the propionic acid residues of
phycocyanobilin separately, one of which would probably support the formation of a

Acid-Base Chemistry of Phycocyanin
881
short ꢀ-helix by the arrangement of a salt bridge between arginine and the propionic
acid in position 12 of phycocyanobilin (Fig. 1). Concerning the ꢀ-helix propensity of
amino acids, phenylalanine might show a tendency towards the formation of ꢀ-
helices, as was deduced from statistical analysis of protein X-ray structures [25]. On
the other hand, re®ned statistical analysis on position-speci®c preferences clearly
indicated that the conformational propensity of amino acids are strongly position-
dependent [26]. Nevertheless, 8 is able to mimic the acid-base chemistry between
protein and chromophore under certain conditions due to the possibility of an
entropically controlled intramolecular protonation. However, a change of the helical
conformation of the chromophore to the stretched (5anti, 10syn, 14anti)-arrangement
was not observed. The formation of a salt bridge between the nitrogen atom of the
azafulvenic ring B moiety and the aspartic acid residue of the tetrapeptide seems to
provide insuf®cient driving force for changing the overall geometry.
Results and Discussion
Synthesis
The synthesis of the cysteine adduct of phycocyanobilin dimethylester with
(2R,3R,30R,CysR)-con®guration was performed according to Ref. [1]. Since
Carpino's report on peptide coupling with 9-¯uorenylmethyloxycarbonyl (Fmoc)
amino acid halogenides occuring without racemization, a convenient method of
peptide synthesis in a two-layer system is at hand [27, 28]. Compound 6 was
synthesized following this approach (Fig. 2).
The ®nal coupling step towards the tetrapeptide 7 was accomplished using the
uronium salt O-(7-azabenzotriazole-1-yl)-N,N,N0,N0,-tetramethyluronium-hexa¯uoro-
Fig. 2. (a) The phycocyanobilin dimethyl ester chromophore in its helical (5syn, 10syn, 14syn)-
conformation can be interpreted as a protecting group for cysteine; the compound is drawn in its
zwitterionic form and exists at room temperature in equilibrium with its neutral form (Ref. [1]); (b)
reaction scheme for thesynthesis of model compound7: I)CHCl3, 40 min, II), CHCl3, 4-(aminomethyl)-
piperidine, 90 min, HATU, HOAt, DIPEA, DMF

882
M. Stanek and K. Grubmayr
phosphate (HATU), the peptide coupling additive 1-hydroxy-7-azabenzotriazole
(HOAt) and diisopropylethylamine (DIPEA) in DMF [29, 30]. After deprotection of
the trimethylsilylethyl (Tmse) protecting group with tetra-n-butylammonium ¯uoride
(TBAF) in THF and dilution with CH2Cl2 followed by treatment with aqueous
NaHCO3, aqueous HCl, H2O (3Â), and column chromatography on silica gel,
compound 8 with (4Z,9Z,15Z,2R,3R,30R,CysR,AlaS,PheS,AspS)-con®guration was
obtained. As a mater of fact, intermolecular protonated 2,3-dihydrobilindione
chromophores dissolved in CH2Cl2 or CHCl3 are changed to their neutral form by
vigorous shaking of the corresponding solution with H2O; it can therefore be
deduced with certainty that the aspartic acid residue in 8 prevails as carboxylic acid
and not as carboxylate.
Intramolecular protonation
By addition of the stoichiometric amount of acetic acid to the Tmse protected com-
pound 7 dissolved in chloroform, intermolecular protonation was not observed due to
the 2,3-dihydroblindione chromophore which is slightly more acidic than acetic acid
(see above). However, addition of a 4900-fold molar excess of acetic acid and cooling
to 54ꢀC led to a minor change of the long-wavelength absorption band (Fig. 3a).
The model compound 8 dissolved in chloroform only shows a small shoulder
around 670 nm in the UV/Vis spectra which might result from the intramolecular
protonation between the aspartic acid residue and the nitrogen atom of the
azafulvenic ring B moiety. This may be inferred from the disappearance of the
shoulder upon addition of DIPEA and, therefore, the formation of the diisopropyl
ethylammonium carboxylate of 8. In order to intesify and to prove this characteristic
± especially since no impressive change of the long-wavelength absorption band
could be detected by cooling or heating ±, a 2800-fold molar excess of acetic acid
was added. The use of acetic acid should not be interpreted as an initiation of
intermolecular protonation of the chromophore in this case, but is meant to change
the solvent polarity, inducing a slight change of the arbitrary peptide conformation
and effecting a closer distance beetween the aspartic acid residue and the nitrogen
atom of the azafulvenic ring B moiety. Actually, a batho- and hyperchromic shift of
the long-wavelength absorption band is observed at room temperature, which can
reversibly be extended to a ratio of approximately 1:1 between the long and short-
wavelength absorption bands by cooling to 54ꢀC (Fig. 3b). Assuming that the pKa
values of acetic acid and the carboxyl group of aspartic acid are more or less equal,
and knowing that the protection of a carboxyl group, like that of 7, leads to a
neglectable hyperchromicity of the long-wavelength absorption band upon addition
of acetic acid, the proton transfer in 8 must proceed intramolecularly, resulting in the
formation of the zwitterion 8Æ. Unfortunately, the equilibrium is not quantitatively
shifted towards 8Æ at 54ꢀC in chloroform as deduced from a slight shift of the
absorption maximum upon further cooling.
The desired change of the helical conformation of the chromophore to the
stretched (5anti, 10syn, 14anti)-arrangement which would also presuppose a short
ꢀ-helix formation of the peptide, was not observed. This change would easily have
been detected by a multiple enhancement of the long-wavelength absorption band
compared to its short-wavelength counterpart.

Acid-Base Chemistry of Phycocyanin
883
Fig. 3. (a) UV/Vis spectrum of 7 (dashed line) in CHCl3 (c ˆ 2:4 Á 10 5M); spectra with solid lines
recorded at different temperatures after the addition of a 4900-fold molar excess of acetic acid; (b)
UV/Vis specta showing the temperature dependence of the proton transfer equilibrium of 8 and 8Æ in
CHCl3 (c ˆ 2:7 Á 10 5M): spectra with solid lines recorded at different temperatures after the
addition of a 2800-fold molar excess of acetic acid, spectrum with dashed line: measured without
acetic acid; all values of " are corrected to room temperature
Although the overall change of the geometry does not happen, a conclusion
may be drawn for phycobiliproteins: intramolecular protonation by aspartic acid
residues is favoured by the highly ordered arrangement of consensus sequences
enabling the correct geometry necessary for effective ion-pair formation.

884
M. Stanek and K. Grubmayr
Experimental
All chemicals were of reagent grade. Solvents were generally distilled prior to use, THF was distilled
from sodium benzophenone ketyl. Column chromatography was performed on silica gel (E. Merck,
silica gel 60, 0.63±0.200 mm). NMR spectra were recorded either on a Bruker Avance DRX-500 or
on a Bruker WM-360 instrument. The assignment of 1H and 13C signals is based on gradient-
enhanced HMQC, gradient-enhanced phase sensitive DQF-COSY, and ROESY experiments. UV/Vis
and CD spectra were recorded on a Hitachi U-3210 spectrometer and a Jobin-Yvon Mark V circular
dichograph. The electrospray mass spectrum of 8 was measured on a Hewlett Packard MS-Engine
5989 API.
(S)-N-Benzyloxycarbonyl-ꢁ-(2-trimethylsilylethyl)-ꢀ-methyl-aspartate (1; C18H27NO6Si)
According to Ref. [31], 1.4 cm3 of pyridine and 1.45 cm3 of 2-trimethylsilyl-ethanol were added to a
solution of (S)-N-benzyloxycarbonyl-ꢀ-methyl-aspartate (2.43 g, 8.6 mmol) in 8.6 cm3 acetonitrile.
After cooling in an ice bath, 0.3 g 1-hydroxybenzotriazole (HOBt) and 1.9 g dicyclohexyl-
carbodiimide (DCC) were added. This solution was stirred at 0ꢀC for 2 h and kept in the refrigerator
over night.
Oxalic acid (0.5 cm3 5 M in DMF) was added to the reaction mixture which was stirred for
another 40 min and then ®ltered by suction. The ®ltrate was diluted with 50 cm3 of CH2Cl2, washed
with 100 cm3 of 0.1 N aqueous HCl, 100 cm3 of 0.2 M aqueous NaHCO3, and dried over Na2SO4.
The organic phase was evaporated to give 72% of 1 as a clear oil.
1H NMR (360 MHz, CDCl3): ꢂ ˆ 7:35 (m, 5H, phenyl-H), 5.78 (broad d, 1H, J(NH, ꢀCH) ˆ
8.3 Hz, Asp-NH), 5.12 (s, 2H, benzyl-CH2), 4.62 (ddd, 1H, Asp-ꢀCH), 4.17 (t, 2H, -O-CH2-), 3.75 (s,
3H, -O-CH3), 3.01 (dd, 1H, J(ꢁCH(1),ꢀCH) ˆ 4.4 Hz, J(ꢁCH(1),ꢁCH(2)) ˆ 17.1 Hz, Asp-ꢁCH(1)),
2.82 (dd, 1H, J(ꢁCH(2),ꢀCH) ˆ 4.5 Hz, J(ꢁCH(2),ꢁCH(1)) ˆ 17.1 Hz, Asp-ꢁCH(2)), 0.97 (t, 2H,
-CH2-Si-), 0.03 (s, 9H, -Si(CH3)3) ppm.
(S)-ꢁ-(2-Trimethylsilylethyl)-ꢀ-methyl-aspartate (2; C10H21NO4Si)
According to Ref. [32], 1 (1.1 g, 2.8 mmol) was dissolved in 5 cm3 of isopropanol. Palladium on
activated carbon (0.1 g) was added to the stirred solution, and the amino acid was decarboxylated by
hydrogenation in a ¯ask with a hydrogen-®lled balloon. The catalyst was ®ltered off, and the alcohol
was evaporated in vacuum to yield 2 quantitatively as a clear oil.
1H NMR (360 MHz, CDCl3): ꢂ ˆ 4:20 (t, 2H, -O-CH2-), 4.62 (m, 1H, Asp-ꢀCH), 3.79 (S, 3H,
-O-CH3) 3.01 (dd, 1H, J(ꢁCH(1), ꢀCH) ˆ 4 Hz, J(ꢁCH(1),ꢁCH(2))ˆ 17.1 Hz, Asp-ꢁCH(1)), 2.89 (dd,
1H, J(ꢁCH(2),ꢀCH) ˆ 6.3 Hz, J(ꢁCH(2),ꢁCH(1))ˆ 17.1Hz, Asp-ꢁCH(2)), 0.99 (t, 2H, -CH2-Si-), 0.04
(s, 9H, -Si(CH3)3) ppm.
(PheS, AspS)-N-9-Fluorenylmethyloxycarbonyl-phenylalanyl-ꢁ-(2-trimethylsilylethyl)-
ꢀ-methyl-aspartate (3; C34H40N2O7Si)
According to Refs [27, 28], to 0.68 g (2.75mmol) of 2 in 27 cm3 CHCl3 a solution of N-9-¯uorenyl-
methyloxycarbonyl-phenylalanine acid chloride [27] (Fmoc-Phe-Cl) (1.22 g, 3.03mmol) in 25 cm3 of
CHCl3 and 27cm3 of aqueous Na2CO3 (5%) were added. The two-phase mixture was stirred
vigorously for 40 min. The reaction mixture was diluted with 100 cm3 of CH2Cl2 and 60cm3 of H2O,
adjusted to pH 2±3 by addition of aqueous HCl (0.1 N), washed with 100 cm3 of H2O, and dried over
Na2SO4. After evaporation of the solvent the residue was puri®ed by column chromatography (silica
gel, CH2Cl2/EtOAcˆ 18/1) yielding 3 (1g, 60%) as a slightly yellowish oil.

Acid-Base Chemistry of Phycocyanin
885
Rf ˆ 0.46 (silica gel, CH2Cl2/EtOAc ˆ 12/1); 1H NMR (360 MHz, CDCl3): ꢂ ˆ 7:36 (m, 2H, 4,5-
¯uorenyl-H), 7.54 (m, 2H, 1,8-¯uorenyl-H), 7.40 (m, 2H, 3,6-¯uorenyl-H), 7.30 (m, 2H, 2,7-
¯uorenyl-H) 7.29 7.15 (m, 5H, phenyl-H), 6.92 (broad d, 1H, J(NH,ꢀCH) ˆ 7.1 Hz,Asp-NH), 5.46
(broad d, 1H, J(NH, ꢀCH ˆ 6.7 Hz, Phe-NH), 4.80 (ddd, 1H, Asp-ꢀCH), 4.41 (dd, 1H, ¯uorenyl-
CHH(1)), 4.32 (m, 1H, 9-¯uorenyl-H), 4.19 (dd, 1H, ¯uorenyl-CH(2)H), 4.12 (t, 2H, -O-CH2-), 3.71
(s, 3H, -O-CH3), 3.10 (m, 2H, Phe-ꢁCH2), 2.98 (dd, 1H, J(ꢁCH(1),ꢀCH) ˆ 4.2 Hz, J(ꢁCH(1),
ꢁCH(2)) ˆ 17.3 Hz, Asp-ꢁCH(1)), 2.77 (dd, 1H, J(ꢁCH(2),ꢀ(CH) ˆ 4.5 Hz, J(ꢁCH(2),
ꢁ(CH)(1)) ˆ 17.3 Asp-ꢁCH(2)), 0.94 (t, 2H, -CH2-Si-), 0.02 (s, 9H, -Si(CH3)3) ppm. 13C NMR
(90 MHz, CDCl3): ꢂ ˆ 170:87, 170.63, 170.57 (C=O), 155.70 (C=O), 143.75 143.67, 141.22, 136.03
(quaternary carbons), 129.23 (3-, 5-phenyl-CH), 128.90 (2-, 6-phenyl-CH), 127.65 (3-, 6-¯uorenyl-
CH), 127.00 (2-, 7-¯uorenyl-CH and 4-phenyl-CH), 125.00 (1-, 8-¯uorenyl-CH), 119.91 (4-, 5-
¯uorenyl-CH), 67.08 (¯uorenyl-CH2), 63.38 (-O-CH2-), 55.79 (Phe-ꢀCH), 52.67 (-O-CH3), 48.55
(Asp-ꢀCH), 47.04 (9-¯uorenyl-CH), 38.49 (Phe-ꢁCH2), 36.20 (Asp-ꢁCH2), 17.19 (-CH2-Si-), -1.62
(-Si(CH3)3) ppm.
(PheS,AspS)-Phenylalanyl-ꢁ-(2-trimethylsilylethyl)-ꢀ-methyl-aspartate (4; C19H30N2O5Si)
According to Refs. [27, 28], 3 (0.89 g, 1.4 mmol) was dissolved in 25 cm3 of CHCl3 and treated with
5 cm3 of 4-(aminomethyl)-piperidine. After 90 min, 70 cm3 of CHCl3 were added, and the organic phase
was washed with two 100 cm3 portions of saturated NaCl solution and with three 100 cm3 portions of
phosphate buffer of pH ˆ 5.5 (prepared from 90 g of KH2PO4 Á H2O and 32.7 g of Na2HPO4 in 500 ml
of deionized water). The organic phase was dried over Na2SO4 and evaporated to give 0.57 g of a
mixture of 4 and the corresponding diketo-piperazine by-product ((3S,6S)-trimethylsilylethyl-(3-(2,5-
dioxo-6-benzyl)-piperazinly)-ethanoat; the by-product was isolated by column chromatography after
preparation of 5).
ꢀ
1H NMR (360 MHz, CDCl3): ꢂ ˆ 8:15 (broad d, 1H, J(NH, CH) ˆ 8.5 Hz, Asp-NH), 7.35-7.15
(m, 5H, Phenyl-H), 4.86 (ddd, 1H, Asp-ꢀCH), 4.16 (m, 2H, -O-CH2-), 3.74 (s, 3H, -O-CH3), 3.66 (dd,
1H, J(ꢀCH,ꢁCH(1)) ˆ 3.8 Hz, J(ꢀCH,ꢁCH(2)) ˆ 4.21 Hz, Phe-ꢀCH), 3.22 (dd, 1H, J(ꢁCH(1),
ꢀCH) ˆ 3.8 Hz, J(ꢁCH(1),ꢁCH(2))ˆ 13.7Hz, Phe-ꢁCH(1)), 2.99 (dd, 1H, J(ꢁCH(1),ꢀCH) ˆ 4.7 Hz,
J(ꢁCH(1),ꢁCH(2)) ˆ 17.1Hz, Asp-ꢁCH(1)), 2.76 (dd, 1H, J(ꢁCH(2),ꢀCH) ˆ 4.6 Hz, J(ꢁCH(2),ꢁCH(1)
ˆ 17:1 Asp-ꢁCH(2)), 2.72 (dd, 1H, J(ꢁCH(2)) ˆ 4:2 Hz, J(ꢁCH(2),ꢁCH(1)) ˆ 13:7 Hz, Phe-ꢁCH(2)),
0.97 (t, 2H, -CH2-Si-), 0.04 (s, 9H, -Si(CH3)3) ppm.
(AlaS,PheS,AspS)-N-9-Fluorenylmethyloxycarbonyl-alanyl-phenylalanyl-ꢁ-(2-trimethylsilylethyl)-
ꢀ-methyl-aspartate (5; C37H45N3O8Si)
5 was synthesized according to the protocol described for the synthesis of 3 employing N±9-
¯uorenylmethyloxycarbonyl-alanine acid chloride [27] (Fmoc-Ala-Cl). The residue was puri®ed by
column chromatography (silica gel, CH2Cl2/MeOH ˆ 20/1) yielding 5 (0.44 g, 44%) as a yellowish oil.
Rf ˆ 0.75 (silica gel, CH2Cl2/MeOH ˆ 15/1); 1H NMR (360MHz, CDCl3): ꢂ ˆ 7:76 (m, 2H, 4,5-
¯uorenyl-H), 7.58 (m, 2H, 1,8-¯uorenyl-H), 7.40 (m, 2H, 3,6-¯uorenyl-H), 7.31 (m, 2H, 2,7-¯uorenyl-
H) 7.28-7.16 (m, 5H, phenyl-H), 6.95 (broad) d, 1H, J(NH,ꢀCH, ˆ 7.6Hz,Asp-NH), 6.72 (broad d, 1H,
J(NH,ꢀCHˆ 6.8 Hz, Phe-NH), 5.41 (broad d, 1H, J(NH,ꢀCH) ˆ 6.7 Hz, Ala-NH), 4.79 (ddd, 1H,
Asp-ꢀCH), 4.73 (ddd, 1H, Phe-ꢀCH), 4.37 (m, 2H, ¯uorenyl-CHH(1) and Ala-ꢀ(CH), 4.24 (m, 1H, 9-
¯uorenyl-H), 4.19 (dd, 1H, ¯uorenyl-CH(2)H), 4.13 (t, 2H, -O-CH2-), 3.71 (s, 3H, -O-CH3), 3.14 (dd,
1H, J(ꢁCH(1),ꢀCH) ˆ 6.5 Hz, J(ꢁCH(1)ꢁCH(2)) ˆ 14 Hz, Phe-ꢁCH(1)), 3.06 (dd, 1H,
J(ꢁCH(2),ꢀCH) ˆ 6.6 Hz, J(ꢁCH(2),ꢁCH(1)) ˆ 14 Hz, Phe-ꢁCH(2)), 2.93 (dd, 1H, J(ꢁCH(1),
ꢀ(CH) ˆ 4.4 Hz, J(ꢁCH(1),ꢁCH(2)) ˆ 17.2 Hz,Asp-ꢁCH(1)), 2.74 (dd, 2H, J(ꢁ(CH(2),ꢁCH(1)) ˆ
17.2Hz, Asp-ꢁCH(2)), 1.33 (d, 3H, J(ꢁCH3,ꢀCH) ˆ 6.5 Hz Ala-ꢁCH3)), 0.94 (t, 2H, -CH2-Si-), 0.02
(s, 9H, -Si(CH3)3) ppm; 13C NMR (90 MHz, CDCl3): ꢂ ˆ 172:11, 170.81, 170.67, 170.31 (C=O),

886
M. Stanek and K. Grubmayr
155.89 (C=O), 143.79, 141.25, 136.03, (quaternary carbons atoms), 129.30 (3-, 5-phenyl-CH), 128.48
(2-,6-phenyl-CH), 127.68 (3-, 6-¯uorenyl-CH), 127.03 (2-, 7-¯uorenyl-CH) 126.92 (4-phenyl-CH),
125.06 (1-, 8-¯uorenyl-CH), 119.93 (4-, 5-¯uorenyl-CH), 67.10 (¯uorenyl-CH2), 63.37 (-O-CH2-),
54.08 (Phe-ꢀCH), 52.66 (-O-CH3), 50.47 (9-¯uorenyl-CH), 48.49 (Asp-ꢀCH), 47.08 (Ala-ꢀCH), 38.19
(Phe-ꢁCH2), 36.22 (Asp-ꢁCH2), 18.68 (Ala-ꢁCH3), 17.22 (-CH2-Si-), 1.59 (-Si(CH3)3) ppm.
(AlaS,PheS,AspS)-Alanyl-phenylalanyl-ꢁ-(2-trimethylsilylethyl)-ꢀ-methyl-aspartate
(6; C22H35N3O6Si)
6 was synthesised according to the protocol described for the synthesis of 4 in a yield of 98%.
1H NMR (360 MHz, CDCl3): ꢂ ˆ 7:70 broad d, 1H, J(NH,ꢀCH, ˆ 8.3 Hz, Phe-NH), 7.36-7.19 (m,
5H, phenyl-H) 6.99 broad d, 1H, J(NH,ꢀCH ˆ 8.0Hz, Asp-NH), 4.78 (ddd, 1H, Asp-ꢀCH), 4.67 (ddd,
1H, Phe-ꢀCH), 4.13 (m, 2H, -O-CH2-), 3.71 (s, 3H, -O-CH3) 3.44 (dd. 1H, J(ꢀCH,ꢁCH3) ˆ 7 Hz,
AlaꢀCH),3.18 (dd, 1H, J(ꢁCH(1),ꢀCH) ˆ 6.2 Hz, J(ꢁCH(1)ꢁCH(2))ˆ 14Hz, Phe-ꢁCH(1)), 3.04 (dd,
1H, J(ꢁCH(2),ꢀCH) ˆ 7.6 Hz, J(ꢁCH(2),ꢁCH(1))ˆ 14Hz, Phe-ꢁCH(2)), 2.94 (dd, 1H, J(ꢁCH(1),
ꢀ(CH) ˆ 4.5 Hz, J(ꢁCH(1),ꢁ(CH)(2) ˆ 17.1Hz, Asp-ꢁCH(1)), 2.76 (dd, 1H, J(ꢁCH(2),ꢀCH(2) ˆ
4.8 Hz, J(ꢁ(CH(2),ꢁCH(1))ˆ 17.1Hz, Asp-ꢁCH(2)), 0.95 (t, 2H -CH2-Si-), 0.02 (s, 9H, -Si(CH3)3)
ppm; 13C NMR (90 MHz, CDCl3): ꢂ ˆ 175:73, 170.78, 170.73 (C=O), 136.43 (1-phenyl-C) 129.27 (3-
, 5-phenyl-CH), 128.38 (2-, 6-phenyl-CH), 126.76 (4-phenyl-CH), 63.32 (-O-CH2-), 53.57 (Phe-ꢀCH),
52.63 (-O-CH3), 50.58 (Ala-ꢀCH), 48.49 (Asp-ꢀCH), 37.81 (Phe-ꢁCH2), 36.22 (Asp-ꢁCH2), 21.31
(Ala-ꢁCH3) 17.17 (-CH2-Si-), 1.63 (-Si(CH3)3) ppm.
(4Z,9Z,15Z,2R,3R,30R,CysR,AlaS,PheS,AspS)-3-(1-(N-Benzyloxycarbonyl-cysteinyl-alanyl-
phenylalanyl-ꢁ-(2-trimethylsilylethyl)-ꢀ-methyl-aspartyl-S-yl)-ethyl)-18-ethyl-2,3-dihydro-
8,12-bis-(2-methoxycarbonylethyl)-2,7,13,17-tetramethyl-23H-bilin-1,19-(21H,24H)-dione
(7; C68H88N8O15SSi)
A solution of 6 (16 mg, 33 mmol) in 0.5 cm3 of DMF (0.5ml) and a solution of HATU (9 mg, 22 mmol),
HOAt (3 mg, 22 mmol), and DIPEA (4 ml 3 mg) in 0.5 cm3 of DMF were added to a stirred solution of
(4Z,9Z,15Z,2R,3R,30R,CysR)-3-(1-(N-benzyloxycarbonyl-cystein-S-yl)-ethyl)-18-ethyl-2,3-dihydro-
8,12-bis-(2-ethoxycarbonylethyl)-2,7,13,17-tetramethyl-23H-bilin-1,19-(21H,24H)-dione (1Æ in
Ref. [1]; 10 mg, 11.8 mmol) in 4 cm3 of DMF. The reaction mixture was stirred under Ar at 25ꢀC
for 2 h, diluted with 80 cm3 of CH2Cl2, washed with 0.5 N aqueous HCl and 2% aqueous NaHCO3,
and dried over Na2SO4. After evaporation of the solvent the residue was puri®ed by column
chromatography (silica gel, CH2Cl2/MeOH ˆ 30/1) to afford 14.6 mg (96%) of 7 as a blue solid.
M.p.: 81ꢀC; Rf ˆ 0.36 (silica gel, CH2Cl2/MeOH ˆ 30/1); 1H NMR (500 MHz, CDCl3; two
different conformations of 7 in a ratio of 68.32 were observed at 298 K; Bzl ˆ benzyl): ꢂ ˆ 10:41
broad s, 1H, 32% chromophore-NH), 8.74 (d, 1H, J(NH,ꢀCH, ˆ 4.5 Hz, 1st Cys-NH), 8.10 (d, 1H, J
(NH,ꢀCH) ˆ 3.5 Hz, 2nd Cys-NH), 6.88 (broad s, 1H, 32% chromophore-NH), 7.39-6.97 (m, 19H, 1st
Ala-NH, 1st and 2nd Phe-phenyl-H, 1st Asp-NH, 1st and 2nd 3,4,5-Bzl-H, 1st Phe-NH), 6.96-6.80 (m,
5H, 1st and 2nd/1,6-Bzl-H, 2nd Ala-NH), 6.72 (s, 1H, 1st 10-H), 6.69 (d, 1H, 2nd Asp-NH), 6.70 (s, 1H,
2 10-H), 6.56 (d, 1H, J(NH,ꢀCH) ˆ 7.5 Hz, 2nd/Phe-NH), 5.99 (s, 1H, 1st 15-H), 5.98 (s, 1H, 2nd 15-
H), 5.59 (s, 1H, 1st 5-H), 5.42 (s, 1H, 2nd 5-H), 4.98 (d, 1st part of the 1st AB-system, 1H,
J(CHH,CHH) ˆ 12.5 Hz, Bzl-CHH-), 4.81 (ddd, 1H, 1st Asp-ꢀCH), 4.75 (d, 1st part of the 2nd AB-
system, 1H, Bzl-CHH-), 4.67 (ddd, 1H, 2nd Asp-ꢀCH), 4.60 (ddd, 1H, 1st Phe-ꢀCH), 4.38 (m, 2nd part
of 1st and 2nd AB-system, 3H, 2ÂBzl-CHH- and 2nd Phe-ꢀCH), 4.28 (ddd, 1H, 1st Ala-ꢀCH), 4.24-
4.04 (m, 7H, 2nd Ala-ꢀCH, 1st and 2nd Cys-ꢀCH, 1st and 2nd -O-CH2-), 3.73-3.58 (6s, 18H, 2Â1st and
2
nd 8,12-COOCH3, 1st and 2nd Asp-O-CH3), 3.29 (dd, 2H, 1st and 2nd Phe-ꢁCH(1)), 3.12 (qd, 1H, 1st
3,-H), 3.04-2.40 (m, 31H, 2Â1st and 2nd 8,12-CH2-, 1st and 2nd Cys-ꢁCH2 2nd 3,-H, 1st and 2nd 3-H,
1st and 2nd Phe-ꢁCH(2), 1st and 2nd Asp-ꢁCH2, 1st and 2nd 2-H, 2Â1st and 2nd 8,12-CH2-COO-), 2.30

Acid-Base Chemistry of Phycocyanin
887
(m, 4H, 1st and 2nd 18-CH2-CH3) 2.16-2.03 (4s, 12H, 1st and 2nd 17-CH3, 1st and 2nd 13-CH3), 2.02,
1.80 (2s, 6H, 1st 7-CH3 and 2nd 7-CH3), 1.47 (d, 6H, 1st and 2nd 30-CH3), 1.30 (d, 3H,
J(ꢁCH3,ꢀCH) ˆ 5.1 Hz, 1st Ala-ꢁ-CH3), 1.22 (d, 3H, J(2-CH3,2-H) ˆ 5.4 Hz, 1st 2-CH3), 1.18 (d, 3H,
J(2-CH3,2-H) ˆ 5.4 Hz, 2nd 2-CH3), 1.13 (d, 3H, J(ꢁCH3,ꢀCH) ˆ 5.1 Hz, 2nd Ala-ꢁCH3), 1.06 (t, 6H,
1st and 2nd 18-CH2-CH3), 0.95 (m, 4H, 1st and 2ndCH2-Si-), 0.28 (2s, 18H, 1st and 2nd -Si(CH3)3)
ppm; 1H NMR (360 MHz, C6D5-CD3, T ˆ 358 K (above Tc)): ꢂ ˆ 7.40-6.80 (m, 14H, Asp-NH, Phe-
phenyl-H, Bzl-H Cys-NH, 10-H and Ala-NH), 6.73 (broad d, 1H, Phe-NH), 5.83 (s, 1H, 15-H), 5.49
(s, 1H, 5-H), 4.90 (broad, dd, 2H, Bzl-CH2), 4.81 (m, 1H, Asp-ꢀCH), 4.63 (m, 1H, Phe-ꢀCH), 4.40
(m, 1H, Cys-ꢀCH), 4.30 (m, 1H, Ala-ꢀCH), 4.15 (m, 2H, -O-CH2-) 3.40, 3.39 (2s, 9H, 2Â8,12-
COOCH3, Asp-COOCH3), 3.25-2.65 (m, 12H, Phe-ꢁCH230-H, Cys-ꢁCH2 2-H, Asp-ꢁCH2, 2Â8,12-
CH2-), 2.59 (broad s, 1H, 3-H), 2.51 (m, 4H, 2Â8,12-CH2-COO-) 2.32 (m, 3H, 18-CH2-CH3) 2.01 (s,
3H, 7-CH3), 1.94 (s, 3H, 13-CH3), 1.88 (s, 3H, 17-CH3), 1.25 (d, 3H, 2-CH3), 1.18, (m, 6H, 18-CH2-
CH3, Ala-ꢁCH3), 1.06 (d, 3H, J(3-CH3,30-H) ˆ 7.0 Hz, 30-CH3), 0.90 (m, 2H, -CH2-Si-), .043 (s,
9H -Si(CH3)3 ppm; IR (CHCl3): ꢃ~ ˆ 3425, 3330, 3027, 2875, 1734, 1669,1636, 1595 cm 1; UV/Vis
(298 K, ꢄmax (")): CHCl3: 592 (15100), 352 (34200), 275 (20700) nm; CHCl3 (TFA): 632 (38900),
352 (25600), 331 (26300), 276 (13400), 260 (13000) nm; CHCl3 (Zn2‡): 656 (17500), 382 (24700),
354 (25400) nm; CHCl3 (TBD), guanidine base): 762 (20600), 408 (25500), 364 (29500), 314
(18960) nm; CD (298 K, ꢄmax (Á")): CHCl3: 585 ( 77), 351 (133), 277 ( 47) nm; CHCl3 (TFA):
623 ( 30), 353(46), 275 ( 19) nm; CHCl3 (TBD): 760 ( 77), 413 (28), 379 (26), 354 (24) nm.
(4Z,9Z,15Z,2R,3R,30R,CysR,AlaS,PheS,AspS)-3-(1-(N-Benzyloxycarbonyl-cysteinyl-alanyl-
phenylalanyl-ꢀ-mehyl-aspartyl-S-yl)-ethyl)-18-ethyl-2,3-dihydro-8,12-bis-
(2-methoxycarbonylethyl)-2,7,13,17-tetramethyl-23H-bilin-1,19-(21H,24H)-dione
(8; C63H76N8O15S)
TBAF (4 mg, 12.6 mmol) was added to a solution of 7 (5 mg, 3.8 mmol) in 2 cm3 of THF. The reaction
mixture was stirred under Ar for 40 min, diluted with 50 cm3 of CH2Cl2, washed with 40 cm3 of
0.2 M aqueous NaHCO3, 40 cm3 0.1N aqueous HCl, 3Â100 cm3 H2O and dried over Na2SO4. After
evaporation of the solvent the residue was puri®ed by column chromatography (silica gel, CH2Cl2/
MeOH ˆ 8/1) to afford 8 (4.2 mg, 90%) as a blue solid.
M.p.: 152ꢀC Rf ˆ 0:38 (silica gel, CH2Cl2/MeOH ˆ 10/1); 1H NMR (500 MHz, DMSO-d6):
ꢂ ˆ 9:79 (broad s, 1H, chromophore-NH), 8.36 (d, 1H, Asp-NH), 8.14 (d, 1H, Ala-NH), 7.91 (d, 1H,
J(NH,ꢀCH) ˆ 7.4 Hz, Phe-NH), 7.54 (d, 1H, J(NH,ꢀCH) ˆ 8.4 Hz, Cys-NH), 7.37-7.10 (m, 10H,
Phe-phenyl-H, Bzl-H), 6.78 (s, 1H, 10-H), 5.96 (s, 1H, 15-H), 5.76 (s, 1H, 5-H), 4.96 (s, 2H, Bzl-
CH2-), 4.59 (ddd, 1H, Asp-ꢀCH), 4.49 (ddd, 1H, Phe-ꢀCH), 4.20 (ddd, 2H, Ala-ꢀCH and Cys-ꢀCH),
3.57 (s, 3H, Asp-O-CH3), 3.55 (s, 6H, 2Â8,12-COOCH3), 3.34 (m, 1H, 30-H), 3.10-2.35 (m, 15H,
Phe-ꢁCH2, 3-H, Cys-ꢁ CH2, 8,12-CH2-, 8,12-CH2-COO-, Asp-ꢁCH2), 2.28 (m, 1H, 2-H), 2.16 (q,
2H, 18-CH2-CH3), 2.06 (s, 3H, 17-CH3), 2.05 (s, 3H, 13-CH3), 1.94 (s, 3H, 7-CH3), 1.28 (d, 3H,
J(30-CH3,30-H) ˆ 6.6 Hz, 30CH3), 1.10 (m, 6H, 2-CH3 and Ala-ꢁCH3), 0.83 (t, 3H, J(18-CH2-CH3,18-
CH2-CH3) ˆ 6.7 Hz, 18-CH2-CH3) ppm; ROESY (500 MHz, DMSO-d6, 298 K): Asp-
NH$Phe-ꢀCH, Ala-NH$(Cys-ꢀCH, Ala-ꢁCH3), Phe-NH$(Ala-ꢀCH, Phe-ꢁCH), Bzl-2,6H$Cbz-
CH2-, 10-H$(8,12-CH2-, 8,12-CH2-COO-), 15-H$(13-CH3, 17-CH3), 5-H$(30-H,3-H, 7-CH3,
Cys-ꢀCH), Phe-ꢀCH$(Phe-phenyl-2,6H, Asp-NH), Cys-ꢀCH$(Ala-NH, 30-H), 30-H$(5-H,
Cys-ꢀCH, weak (2-H)), Phe-ꢁCH$Phe-phenyl-2,6H), 3-H$(5-H,30-CH3, 2-CH3), 12-CH2-$(10-
H, 13-CH3) 8-CH2-$(10-H, 7-CH3), 2-H$(30-CH3, weak (30-H)); UV/Vis (298 K, ꢄmax (")):
CHCl3: 601 (13600), 357 (28500), 276 (16600) nm; CHCl3 (TFA): 637 (35700), 333 (27600), 278
(10400) nm; CHCl3 (HOAc): 612 (17000), 356 (28300), 276 (14900) nm; CHCl3 (Zn2‡: 641
(13800), 378 (21900), 355 (21400), 283 (13200) nm; CD (298 K, ꢄmax (Á")): CHCl3: 578 ( 54), 353
(90), 277 ( 38) nm; CHCl3 (TFA): 631 (9), 389 ( 2), 345 (3), 283 ( 10) nm; CHCl3 (excess of
‡
HOAc): 587 ( 38), 353 (57), 277 ( 27) nm; MS (ESIp, TFA): m/z (%): 1217 [M‡H] (45), 609
(38), 242 (100).

888
M. Stanek and K. Grubmayr: Acid-Base Chemistry of Phycocyanin
Acknowledgements
We thank Dr. W. Ahrer for mass spectroscopic measurements. This work was supported by the
Austrian Science Foundation (P9166-CHE).
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Received March 15, 2000. Accepted March 21, 2000