High arenophlicity and water tolerance in direct derivatization of peptides and proteins by metal Π-coordination [10]

Full text of DOI:10.1021/ja982602c

11814
J. Am. Chem. Soc. 1998, 120, 11814-11815
High Arenophilicity and Water Tolerance in Direct
Derivatization of Peptides and Proteins by Metal
π-Coordination
Douglas B. Grotjahn,*,†,‡ Camil Joubran,‡,§ David Combs,† and
Daniel C. Brune‡
Department of Chemistry, San Diego State UniVersity
5500 Campanile DriVe, San Diego, California 92182-1030
Department of Chemistry and Biochemistry
Box 871604, Arizona State UniVersity
concentrations demonstrate high arenophilicity and water tolerance
in rapid room-temperature reactions, promising conditions for
applications to proteins.
Tempe, Arizona 85287-1604
Scheme 1 outlines the preparation of homologous chelates 1a,b.
Without purification of intermediates, thallium salts 2 were formed
in 56-60% overall yield from salts Br(CH2)nNH3Br by monoalky-
lation of CpLi (2 equiv),6d-8 protection of the amino nitrogen,
and deprotonation of the resulting cyclopentadiene mixture using
TlOEt.7b Metathesis between 2 and [Cl(µ-Cl)Ru(η6-arene)]2 and
exchange to the noncoordinating PF6- anion5a provided sandwich
complexes 3, which were deprotected by hydrogenolysis.5e,9
Photolysis of 4 in CH3CN produced 1. The rate of reaction was
greater for the longer side chain (n ) 3) and for the smaller
arene.10a Conversely, arene complexation to 1 in CD3NO2 is about
2 times faster for the shorter side chain, comparisons which may
indicate that the chelate ring in 1a is more strained.10b
Arenes (Scheme 2) of widely disparate steric demand, such as
1,3-dimethylbenzene (5a) and 1,4-di-tert-butylbenzene (5b) readily
opened chelate 1a under similar conditions (CD3NO2 or CD3-
OD, 60 °C, 1-6 h), to give sandwich complexes 6. Amino acid
derivatives 5c-h could also be used, to afford 6c-h. These
results and the ones below show that all functional groups present
in proteins except for thiols are tolerated.11 As seen for other
η6-arene complexes, the coordination of the metal to the arene
shifted the arene proton resonances upfield by approximately 1
ppm and shifted the C5H4R proton resonances downfield by about
1 ppm.8 The η6-arene complexes could be analyzed by MALDI-
MS, showing the robust nature of the Ru-arene linkage even on
irradiation by the laser.12
ReceiVed July 23, 1998
Site-specific modification of proteins leads to invaluable
structural and reactivity information for biochemical and medicinal
studies and to new biotherapeutics.1 Modification frequently
depends on reaction of electrophilic reagents with nucleophilic
atoms of the protein.1 Classic examples include reaction of BrCN
at S,2a RNCS at N,2b and electrophilic halogens at the tryptophan
π-system.2c The organic reagents which react with tryptophan
or tyrosine react much more slowly or not at all with the less
electron-rich phenylalanine. Moreover, the same electrophilic or
oxidizing reagents also enter into unwanted side reactions with
nucleophilic S- or N-donors.1 Here we show that metal π-com-
plexation to the arene-containing amino acid phenylalanine can
succeed even in the face of S- and N-donor ligands found in
methionine, cystine, or histidine, leaving these residues un-
changed. Metal π-complexation to amino acids and dipeptides
has been studied in nonaqueous solvents,3 and metals have been
covalently attached to proteins indirectly, by using organic ligands
which are attached to mercapto or amino sites.4 In contrast, here
we report the attachment of a metal directly to an arene ring in
a protein in water. Our strategy for reversible derivatization of
aryl amino acids exploits the capability of CpRu+ and Cp*Ru+
fragments to bind to arenes,3,5 providing air-stable products from
which arene subsequently can be released on photolysis. We have
made chelates 1, which unlike CpRu+ and Cp*Ru+ derivatives
are new compounds featuring a bound amino ligand,6 a nucleo-
phile or water-solubilizing group locked by coordination until
release by an incoming aryl amino acid. A series of experiments
Recognition of arene rings in proteins will succeed only if
coordination of the metal to other strong ligands such as amines
(5) (a) Gill, T. P.; Mann, K. R. Organometallics 1982, 1, 485-488. (b)
Fagan, P. J.; Ward, M. D.; Caspar, J. V.; Calabrese, J. C.; Krusic, P. J. J. Am.
Chem. Soc. 1988, 110, 2981-2983. (c) Fagan, P. J.; Ward, M. D.; Calabrese,
J. C. J. Am. Chem. Soc. 1989, 111, 1698-1719. (d) Nolan, S. P.; Martin, K.
L.; Stevens, E. D.; Fagan, P. J. Organometallics 1992, 11, 3947-3953. (e)
Vichard, D.; Gruselle, M.; El Amouri, H.; Jaouen, G.; Vaissermann, J.
Organometallics 1992, 11, 976-979 and 2952-2956. (f) Lomenzo, S. A.;
Nolan, S. P.; Trudell, M. L. Organometallics 1994, 13, 676-681. (g)
Glatzhofer, D. T.; Liang, Y.; Funkhouser, G. P.; Khan, M. A. Organometallics
1994, 13, 315-321. (h) Dembek, A. A.; Fagan, P. J. Organometallics 1995,
14, 3741-3745. (i) Lead reference to related reactions of (arene)Ru2+: Ganja,
E. A.; Rauchfuss, T. B.; Stern, C. L. Organometallics 1991, 10, 270-275.
(6) Review of η5,η1-C5R4(CH2)2NR2 ligands: Jutzi, P.; Redeker, T. Eur.
J. Inorg. Chem. 1998, 663-674.
(7) (a) Keana, J. F. W.; Ogan, M. D. J. Am. Chem. Soc. 1986, 108, 7951-
7957. (b) Rausch, M. D.; Edwards, B. H.; Rogers, R. D.; Atwood, J. L. J.
Am. Chem. Soc. 1983, 105, 3882-3886.
(8) Full preparative details and characterization of new compounds,
including NMR and mass spectra for secretin complexation experiments appear
in Supporting Information.
culminating with reaction of the small protein secretin at 10-4
M
* Correspondence author. Current address: San Diego State University.
E-mail: grotjahn@chemistry.sdsu.edu.
† San Diego State University.
‡Arizona State University.
§ Current address: Astra Research Center Boston, 128 Sidney Street,
Cambridge, MA 02139.
(1) (a) Overview: Lundblad, R. L. Techniques in Protein Modification;
CRC Press: Boca Raton, Florida, 1995; pp 1-14. (b) The Protein Protocols
Handbook; Walker, J. M., Ed.; Humana Press: Totowa, NJ, 1996. (c)
Copeland, R. A. Methods for Protein Analysis; Chapman-Hall: New York,
1994.
(2) Reference 1a: (a) pp 51-61. (b) pp 35-50. (c) pp 187-208.
(3) (a) Moriarty, R. M.; Ku, Y.-Y.; Gill, U. S. J. Organomet. Chem. 1989,
362, 187-191. (b) Sheldrick, W. S.; Gleichmann, A. J. J. Organomet. Chem.
1994, 470, 183-187. (c) Gleichmann, A. J.; Wolff, J. M.; Sheldrick, W. S. J.
Chem. Soc., Dalton Trans. 1995, 1549-1554. (d) Wolff, J. M.; Sheldrick,
W. S. Chem. Ber.-Receuil 1997, 130, 981-988. (e) Kra¨mer, R. Angew. Chem.,
Int. Ed. Engl. 1996, 35, 1197-1199.
(4) (a) Rana, T. M.; Meares, C. F. J. Am. Chem. Soc. 1990, 112, 2457-
2458. (b) Hoyer, D.; Cho, H.; Schultz, P. G. J. Am. Chem. Soc. 1990, 112,
3249-3250. (c) Noncovalent attachment using enzyme-substrate affinity:
Schepartz, A.; Cuenoud, B. J. Am. Chem. Soc. 1990, 112, 3247-3249. (d) S-
or N-coordination of Pd(II) or Co(III) directs amide hydrolysis: Zhu, L.;
Kostic, N. J. Am. Chem. Soc. 1993, 115, 4566-4570. Sutton, P. A.;
Buckingham, D. A. Acc. Chem. Res. 1987, 20, 357-364. (e) Jaouen, G.;
Vessie´res, A.; Buther, I. S. Acc. Chem. Res. 1993, 26, 361-369. El
Mouatassim, B.; El Amouri, H.; Salmain, M.; Jaouen, G. J. Organomet. Chem.
1994, 479, C18-C20. (f) Attachment of N-acylated amino acids to CpRe-
(CO)3 via an oxygen of the carboxylate group: Minutolo, F.; Katzenellenbogen,
J. A. J. Am. Chem. Soc. 1998, 120, 4514-4515.
(9) Amino protection and deprotection were necessary: the Tl salt
corresponding to 2a could be made, but metathesis with [(arene)RuCl(µ-Cl)]2
was much slower, and other species, perhaps involving N-coordination by
Tl, were present.
(10) (a) Times for completion: 1a from 4a-η6-benzene, 8 h; from 4a-
η6-mesitylene, > 4 d; 1b from 4b-η6-mesitylene, 2 d. (b) We have not yet
been able to grow crystals of 1a,b suitable for X-ray diffraction.
(11) Presumably, thiol interference could be avoided by S-alkylation or
oxidization prior to complexation; see for example ref 1a, pp 63-90.
(12) (a) The MALDI-TOF laser used produces pulses of 337-nm light, and
the reported λmax for CpRu(η6-benzene)PF6 is 325 nm.12b In some experiments,
small losses of the Ru fragment may have occurred; this did not occur at all
while acquiring electrospray-MS data on secretin complex 6k. (b) McNair,
A. M.; Schrenk, J. L.; Mann, K. R. Inorg. Chem. 1984, 23, 2633-2640.
10.1021/ja982602c CCC: $15.00 © 1998 American Chemical Society
Published on Web 10/30/1998

Communications to the Editor
J. Am. Chem. Soc., Vol. 120, No. 45, 1998 11815
Scheme 1a
decompose.5f In contrast, warming the mixture containing 7 at
60 °C for 1 h led to its disappearance and formation of 6e,
revealed especially well by downfield shifts of the C5H4R proton
resonances (δ 5.29 and 5.39 ppm, each t, J ) 2 Hz, 2H) and
upfield shifts of the arene proton resonances (δ 6.05-6.22 ppm,
m, 5H). BOC-Met-Phe-OMe and BOC-Phe-His-OMe behaved
similarly, initially giving η1-complexes such as 8 at 25 °C, which
converted to η6-arene complexes (e.g, 6f) within 6 h at 60 °C.
While being a significant demonstration of the arenophilicity of
the Ru fragment, these conversions may only serve as models
for proteins in which the strong S- or N-donor is close to Phe,
and intramolecular migration of the Ru fragment is feasible. That
an S-donor need not be connected to Phe was shown by mixing
1a, BOC-Met-OMe, and BOC-Phe-OMe in a ratio of 1:1:1.
Within minutes at 25 °C, an η1-S complex formed; warming at
60 °C for 1 h led to η6-arene complex 6c. Tolerance of disulfides
was shown by similar results from a mixture of 1a and unprotected
cystine and Phe (molar ratio 1:1:1).
a (a) 2 CpLi, THF, 5 °C, 20 h; (b) Cbz-Cl, N-methylmorpholine, Et2O,
25 °C, 1.5 h; (c) TlOEt, Et2O, 25 °C, 0.5 d; (d) 2 + 0.5 [Cl(µ-Cl)Ru(η6-
arene)]2 (arene ) benzene or mesitylene), CH3CN, 25 °C, 5 h; (e) KPF6,
H2O; (f) H2 (40 psig), 10% Pd/C, 3-8 h; (g) Pyrex-filtered UV light,
CH3CN, time indicated in ref 10a.
Scheme 2a
The high arenophilicity of the complexes prompted binding
studies of 1a with the gastrointestinal hormone secretin13 (mass
3039 daltons). Secretin (5k) contains 27 residues, including a
unique Phe, along with N-terminal His, four Arg, one Asp, and
two Glu, all containing potentially interfering functional groups.
Secretin (0.1 or 0.25 mg) and an internal standard were dissolved
in deoxygenated CD3OD or D2O in a 5-mm NMR tube.8 After
acquisition of the 1H NMR spectrum, chelate 1a was added as a
solution in CD3OD.14 Optimum conditions for consumption of
secretin, monitored by the upfield shift of the Phe C6H5 proton
resonances, appeared to require only 3.3 to 10 equiv of 1a.15
Preliminary indications point to a remarkable and useful solvent
effect that is, whereas the reactions in CD3OD require mild heating
at 60 °C to be completed within 5 h, similar reactions in D2O are
completed within 8 h at room temperature, promising for
applications to protein chemistry. The resulting ruthenated protein
6k (yield 70-100% as determined by NMR integration8) could
be purified by reversed-phase HPLC, analyzed by MALDI-MS
or electrospray-MS, and subjected to automated Edman degrada-
tion, which by lack of an HPLC peak for the phenylthiohydantoin
of Phe further verified that the Phe residue had been covalently
modified. Finally, liberation of arene from sandwich complexes
6 by irradiation in water-acetonitrile mixtures is possible.16
In summation, others have studied either π-complexation to
small amino acid derivatives in nonaqueous solvents3 or attach-
ment of preformed π-complexes to biomolecules using organic
chemistry.4e In distinct contrast, we have shown that direct
π-complexation of a protein in aqueous medium is possible, even
in the presence of other coordinating groups. The implications
of enhanced reactivity of 1 in water and further applications
involving the amino-containing side chain are under investigation.
a Piv ) N-pivaloyl.
Scheme 3
Acknowledgment. We thank Minh Huynh for preparing a number
of peptides and John Nemmers of Finnigan Corporation for electrospray-
MS data on 6k. Engelhardt Corp. gave RuCl3‚xH2O.
Supporting Information Available: Full preparative details and
spectral data for new compounds, and representative NMR and MS spectra
for 6k (29 pages print/PDF). See any current masthead page for ordering
information and Web access instructions.
(e.g., lysine), thioethers and disulfides (methionine and cystine),
and histidine does not interfere. A series of control experiments
using 1a,b showed conclusively that η1-S or η1-N coordination
is kinetically favored but that arene coordination is thermo-
dynamically preferred. For example (Scheme 3), when 1a was
mixed with phenylethanamine (5e) in CD3NO2 at 25 °C, within
10 min 1a and the free amine had disappeared, and 1 mol of
CH3CN and a new complex, formulated as 7, were observed.
Particularly diagnostic spectral features for 7 include four slightly
broad one-proton multiplets between δ 3.70 and 4.12 ppm,
consistent with the maintenance of three σ-donor ligands on Ru
and the formation of a chiral complex in which all four C5H4R
protons are inequivalent. A complex similar to 7 from
[Cp*Ru(CH3CN)3]+TfO- and tryptamine had been seen to
JA982602C
(13) Physicians Desk Reference, 52nd edition; Medical Economics Co.:
Montvale, NJ, 1998; pp 3215-3216. Jorpes, E.; Mutt, V. Acta Chem. Scand.
1961, 15, 1790-1791.
(14) The amount of CD3OD thus added (25-75 µL) is small compared to
the amount of buffered D2O (1 mL). The triflate analogue 1a-OTf, showing
better solubility in CD3OD, was used in some of these experiments.8
(15) After complexation was complete and before HPLC purification,
3-mercaptopropionic acid (ca. 20 mol per mol of 1a used) was added to
scavenge any excess metal which might be loosely bound to the protein.8
(16) Pyrex-filtered light from a 450-W Hg lamp was used on (η6-
benzene)Ru(η5-C5H4CH2CH2NH2)+ Cl- in D2O-CD3CN (1:3) for 32 h to form
benzene (91% yield by NMR integration) and 1a (presumed to have the Cl-
counterion).8