A water-soluble ruthenium glycosylated porphyrin catalyst for carbenoid transfer reactions in aqueous media with applications in bioconjugation reactions

Article abstract of DOI:10.1021/ja9077254

Water-soluble [Ru^II(4-Glc-TPP)(CO)] (1, 4-Glc-TPP ) = meso-tetrakis(4-(β-D-glucosyl)phenyl)porphyrinato dianion) is an active catalyst for the following carbenoid transfer reactions in aqueous media with good selectivities and up to 100% conversions: intermolecular cyclopropanation of styrenes (up to 76% yield), intramolecular cyclopropanation of an allylic diazoacetate (68% yield), intramolecular ammonium/ sulfonium ylide formation/[2,3]-sigmatroptic rearrangement reactions (up to 91% yield), and intermolecular carbenoid insertion into N-H bonds of primary arylamines (up to 83% yield). This ruthenium glycosylated porphyrin complex can selectively catalyze alkylation of the N-terminus of peptides (8 examples) and mediate N-terminal modification of proteins (four examples) using a fluorescent-tethered diazo compound (15). A fluorescent group was conjugated to ubiquitin via 1-catalyzed alkene cyclopropanation with 15 in aqueous solution in two steps: (1) incorporation of an alkenic group by the reaction of N-hydroxysuccinimide ester 19 with ubiquitin and (2) cyclopropanation of the alkene-tethered Lys ⁶ ubiquitin (23) with the fluorescentlabeled diazoacetate 15 in the presence of a catalytic amount of 1. The corresponding cyclopropanation product (24) was obtained with -55% conversion based on MALDI-TOF mass spectrometry. The products 23, 24, and the N-terminal modified peptides and proteins were characterized by LC-MS/MS and/or SDS-PAGE analyses.

Full text of DOI:10.1021/ja9077254

Published on Web 01/20/2010
A Water-Soluble Ruthenium Glycosylated Porphyrin Catalyst
for Carbenoid Transfer Reactions in Aqueous Media with
Applications in Bioconjugation Reactions
Chi-Ming Ho,† Jun-Long Zhang,‡ Cong-Ying Zhou,† On-Yee Chan,†
Jessie Jing Yan,† Fu-Yi Zhang,† Jie-Sheng Huang,† and Chi-Ming Che*,†
Department of Chemistry and Open Laboratory of Chemical Biology of the Institute of
Molecular Technology for Drug DiscoVery and Synthesis, The UniVersity of Hong Kong,
Pokfulam Road, Hong Kong, P. R. China, and Department of Chemistry, College of Chemistry
and Molecular Engineering, Peking UniVersity, Beijing 100871, P. R. China
Received September 11, 2009; E-mail: cmche@hku.hk
Abstract: Water-soluble [RuII(4-Glc-TPP)(CO)] (1, 4-Glc-TPP ) meso-tetrakis(4-(ꢀ-D-glucosyl)phenyl)por-
phyrinato dianion) is an active catalyst for the following carbenoid transfer reactions in aqueous media
with good selectivities and up to 100% conversions: intermolecular cyclopropanation of styrenes (up to
76% yield), intramolecular cyclopropanation of an allylic diazoacetate (68% yield), intramolecular ammonium/
sulfonium ylide formation/[2,3]-sigmatroptic rearrangement reactions (up to 91% yield), and intermolecular
carbenoid insertion into N-H bonds of primary arylamines (up to 83% yield). This ruthenium glycosylated
porphyrin complex can selectively catalyze alkylation of the N-terminus of peptides (8 examples) and mediate
N-terminal modification of proteins (four examples) using a fluorescent-tethered diazo compound (15). A
fluorescent group was conjugated to ubiquitin via 1-catalyzed alkene cyclopropanation with 15 in aqueous
solution in two steps: (1) incorporation of an alkenic group by the reaction of N-hydroxysuccinimide ester
19 with ubiquitin and (2) cyclopropanation of the alkene-tethered Lys6 ubiquitin (23) with the fluorescent-
labeled diazoacetate 15 in the presence of a catalytic amount of 1. The corresponding cyclopropanation
product (24) was obtained with ∼55% conversion based on MALDI-TOF mass spectrometry. The products
23, 24, and the N-terminal modified peptides and proteins were characterized by LC-MS/MS and/or SDS-
PAGE analyses.
effect.3 However, a serious problem likely to be encountered is
Introduction
the facile reaction of metal carbenoids with water to give O-H
insertion products4 and/or the poor solubility/stability of the
metal catalysts in aqueous solution. While metal-catalyzed
carbenoid transfer reactions in aqueous or aqueous biphasic
media are not unprecedented,5-13 such reactions, since the
studies by Nishiyama and co-workers,6a remain sparse in the
literature. And the reported reactions include the following:
Metal-catalyzed carbenoid transfer reactions, such as alkene
cyclopropanation, ylide formation/[2,3]-sigmatropic rearrange-
ment, and carbenoid insertion into X-H (X ) C, N, S) bonds,
have emerged to become a useful means for the construction
of organic molecules with complexity.1 These reactions are
usually performed in organic solvents, which are less favored
than in aqueous media in the context of green chemistry.2
Besides the environmentally benign nature, an organic reaction
in aqueous media could also benefit from the hydrophobic
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† The University of Hong Kong.
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Commun. 2001, 59. (b) Iwasa, S.; Tsushima, S.; Nishiyama, K.;
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2003, 47, 855.
‡ Peking University.
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1886 J. AM. CHEM. SOC. 2010, 132, 1886–1894
10.1021/ja9077254  2010 American Chemical Society

Water-Soluble Ru Glycosylated Porphyrin Catalyst
A R T I C L E S
alkene cyclopropanation catalyzed by the complexes of
ruthenium,6,7,12 cobalt,7 dirhodium,7,10 and iron;12 C-H insertion8,9
and sulfonium ylide formation/[2,3]-sigmatropic rearrangement11
catalyzed by dirhodium complexes; N-H insertion catalyzed
by complexes of dirhodium8 and iron.13
the first example of metalloporphyrin-catalyzed carbenoid
transfer for bioconjugation of proteins.
Results and Discussion
Synthesis of Water-Soluble Ruthenium Glycosylated
Porphyrin [RuII(4-Glc-TPP)(CO)] (1). Despite the reports of a
large variety of water-soluble porphyrin free bases,19 only a few
examples of water-soluble ruthenium porphyrins have been
known in literature, including poly(ethylene glycol)-supported
ruthenium porphyrins such as [RuII(4-Cl-TPP)(CO)]-PEG (2)20a
reported in our previous work;20 [RuII(TMPyP)(CO)]4+ (3),
[RuII(4-COOH-TPP)(CO)] (4), and [RuII(TPPS)(CO)]4- reported
earlier by Meunier and co-workers;21 and [RuII(D4-PorS*)-
(CO)]4- reported recently by Simonneaux and co-workers12
(Chart S1 in Supporting Information). These water-soluble
ruthenium porphyrins, except the PEG-supported ones, contain
peripheral pyridinium, carboxyl, or sulfonato functional groups;
such groups have been widely used to render metalloporphyrins
soluble in aqueous media.19
A formidable challenge is the application of metal-catalyzed
carbenoid transfer in bioconjugation reactions such as site
selective modification of proteins.14,15 As an appealing strategy
for modifying proteins, metal-catalyzed approaches can selec-
tively target the natural amino acid side chains that are hardly
modifiable by conventional methods and can also realize highly
selective protein modification upon incorporating suitable un-
natural functional groups into the amino acid side chains.15 The
challenges lie in that the bioconjugation of proteins not only
has to be performed in aqueous media without being complicated
by reactions with water but also should meet a number of
requirements: (i) high efficiency at the protein concentration
level (<100 µM); (ii) mild reaction conditions that allow proteins
to retain their activity or structure features; (iii) selective
modification of a single group out of many similar or the same
groups in different sites, with tolerance to various functional
groups present in the surface of the proteins.14,15 Of the
previously reported metal-catalyzed carbenoid transfer reactions
in aqueous media,6-13 [Rh2(OAc)4]-catalyzed carbenoid transfer
reaction has been applied to site-selective modification of
proteins,8,16 which targets the indole group of internal tryptophan
residues of proteins, as described in the work by Francis and
Antos.8
A different approach to water-soluble ruthenium porphyrin
was employed in this work, i.e. by covalently attaching sugar
moieties to the meso-aryl rings of a porphyrin ligand. We
envision that the sugar moieties, such as ꢀ-D-glucosyl (Glc),
not only can enhance the solubility of ruthenium porphyrins in
aqueous media but also might be able to direct the ruthenium
catalyst to approach a specific site of biomolecules via hydrogen-
bonding and dipole-dipole interactions. In literature, acetylated
sugar moieties, including 2,3,4,6-tetraacetyl-ꢀ-D-glucosyl
(Ac4Glc), have been incorporated into the porphyrin ligands in
a few iron and manganese catalysts for hydrocarbon oxidation
In this paper, we report the site-selective modification of the
N-terminus of proteins via metal-mediated carbenoid N-H
insertion, together with the modification, via metal-catalyzed
alkene cyclopropanation, of a protein prefunctionalized with a
styrene moiety, using a water-soluble metalloporphyrin catalyst
[RuII(4-Glc-TPP)(CO)] (1), wherein 4-Glc-TPP is the dianion
of a previously reported glycosylated porphyrin ligand meso-
tetrakis(4-(ꢀ-D-glucosyl)phenyl)porphyrin.17 Also reported herein
are 1-catalyzed cyclopropanation of styrenes and allylic diaz-
oacetate, ammonium/sulfonium ylide formation/[2,3]-sigmat-
ropic rearrangement of diazoketones, and carbenoid N-H
insertion reactions of primary arylamines in aqueous media.
Prior to the present work, a myriad of metal-catalyzed alkene
cyclopropanation reactions in organic solvents have been
reported in literature.1a-c Metalloporphyrin-catalyzed carbenoid
transfer reactions have been extensively studied in organic
solvents,18 examples of which performed in aqueous media are
rare. During the preparation of this manuscript, Simonneaux
and co-workers reported the intermolecular cyclopropanation
of styrene in water catalyzed by water-soluble iron and
ruthenium porphyrins;12 Gross and Aviv reported intermolecular
N-H insertion of anilines in THF/water catalyzed by myoglo-
bin.13 To the best of our knowledge, the present work contributes
(18) Selected examples: (a) Maxwell, J. L.; Brown, K. C.; Bartley, D. W.;
Kodadek, T. Science 1992, 256, 1544. (b) Brown, K. C.; Kodadek, T.
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9
J. AM. CHEM. SOC. VOL. 132, NO. 6, 2010 1887

A R T I C L E S
Ho et al.
Scheme 1
in organic solvents.22,23 However, concerning metal catalysts
for organic transformations, we could find no literature ex-
amples, apart from 1, that bear a water-soluble porphyrin ligand
functionalized with sugar moieties (such as Glc).
The preparation of 1 did not require isolation of the porphyrin
free base H2(4-Glc-TPP), which could be obtained by treatment
of H2(4-(Ac4Glc)-TPP) (meso-tetrakis(4-(2,3,4,6-tetraacetyl-ꢀ-
D-glucosyl)phenyl)porphyrin) with NaOMe.17 Reaction of H2(4-
(Ac4Glc)-TPP) with Ru3(CO)12 in refluxing decalin afforded
[RuII(4-(Ac4Glc)-TPP)(CO)]. Deprotection of the acetyl groups
of the porphyrin ligand in [RuII(4-(Ac4Glc)-TPP)(CO)] with a
catalytic amount of NaOMe in methanol gave 1 in 50% yield
(Scheme 1).
Complex 1 shows a UV-vis spectrum with Soret and ꢀ bands
at 412 and 527 nm, respectively, and an IR spectrum with an
intense ν(CO) band at 1940 cm-1, along with two sets of ortho
proton resonances of the meso-phenyl groups (δ 8.05, 7.95 ppm).
These spectral features are similar to those of other carbonyl-
ruthenium(II) meso-tetraarylporphyrins reported in literature.24
The positive-ion mass spectrum of 1 shows a cluster peak at
m/z 1455.2 attributed to the parent ion M+.
Carbenoid Transfer Reactions Catalyzed by [RuII(4-Glc-
TPP)(CO)] (1) in Aqueous Media. The chemistry of ruthenium
porphyrin-catalyzed reactions is dominated by the catalysts
bearing hydrophobic porphyrin ligands such as TPP (meso-
tetraphenylporphyrinato dianion) and its derivatives,25 which
are difficult to be applied to reactions in aqueous media. We
and others have reported a considerable number of metallopor-
phyrin-catalyzed carbenoid transfer reactions in organic solvents,
including alkene cyclopropanation,18a-j,l-p carbenoid X-H
(X ) C, N, S) insertions,13,18q,r,26 and ammonium/sulfonium
ylide formation/[2,3]-sigmatropic rearrangements.27 Ruthenium
porphyrin-catalyzed ammonium ylide formation/[2,3]-sigmat-
ropic rearrangement reaction in CH2Cl2 has been applied to the
synthesis of alkaloid (()-platynecine.27b Carbonylruthenium(II)
porphyrins [RuII(Por)(CO)] are well-documented to catalyze
these types of carbenoid transfer reactions.18f-i,26a-c,27 Of
particular interest is the air and moisture stability of isolated
ruthenium porphyrin carbene complexes,18i,28 quite a few of
which have been structurally characterized by X-ray single
crystal analysis.18i,28c,e-j Thus, water-soluble [RuII(Por)(CO)]
catalysts, such as 1, are likely to be converted to a ruthenium
porphyrin carbene intermediate that has a sufficiently long
lifetime to allow for carbenoid transfer to organic substrates
in aqueous media (see also experimental results described
in ref 29).
Indeed, complex 1 catalyzed the following carbenoid transfer
reactions in aqueous media (Scheme 2; detailed results are
depicted in Tables S1-S5 in Supporting Information): (i)
intermolecular cyclopropanation of styrenes (5) with EDA (ethyl
diazoacetate) to give 6 (R ) H, Me, Cl; 70-76% yields; trans/
cis (4-5):1), (ii) intramolecular cyclopropanation of allylic
diazoacetates (7) to form 8 (Rt ) Rc ) Me; 68% yield), (iii)
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metallics 1998, 17, 565. (d) Simonneaux, G.; De Montigny, F.; Paul-
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Water-Soluble Ru Glycosylated Porphyrin Catalyst
A R T I C L E S
Scheme 2
intramolecular ammonium ylide formation/[2,3]-sigmatropic
rearrangement of diazoketones 9 to afford 10 (R ) Bn, Me,
CH2dCHCH2; n ) 1, 2; 83-89% yields), (iv) intramolecular
sulfonium ylide formation/[2,3]-sigmatropic rearrangement of
diazoketones 11 to produce 12 (n ) 1, 2; 90-91% yields), and
(v) intermolecular carbenoid N-H insertion of primary ary-
lamines 13 with EDA to afford 14 (R ) H, Me, Br, OMe;
76-83% yields). The substrate conversions were 85-92% for
the cyclopropanation of 5 and 100% for the other reactions.
as a Doyle-Kirmse reaction in the sulfonium case, are syntheti-
cally useful transformations for the construction of C-C
bonds1b,c,30 and have been widely studied in organic solvents
using catalysts such as [Rh2(OAc)4] and [Cu(acac)2].1b,c,30
Carbenoid insertion of diazo compounds into N-H bond is an
attractive method for the synthesis of R-amino carboxylic
derivatives.31 For the carbenoid N-H insertion reactions
catalyzed by 1, a simultaneous addition of amine and EDA to
avoid catalyst poisoning is not necessary, unlike those catalyzed
by [RuII(TMP)(CO)] (TMP ) meso-tetramesitylporphyrinato
dianion) in benzene.26a,b The reaction of 9 (R ) Bn; n ) 1)
catalyzed by [Rh2(OAc)4] or [Cu(acac)2] in aqueous media gave
10 in 21% and 31% yields, respectively (Table S3 in Supporting
Information), and the main product (isolated in 62% yield) in
the [Rh2(OAc)4]-catalyzed reaction resulted from carbenoid inser-
tion into the O-H bond of water. In contrast, no such O-H
insertion product was detected in the same reaction catalyzed by
1. Wang and Liao recently reported intermolecular [Rh2(OAc)4]-
Metal-catalyzed intramolecular ammonium/sulfonium ylide
formation/[2,3]-sigmatropic rearrangement reactions, also known
(29) Taking into account the well-documented preparation of carbene
complexes [Ru(Por)(CR1R2)] from [RuII(Por)(CO)] and diazo com-
pounds N2CR1R2 in organic solvents,18i,28c-j we made attempts to
prepare and isolate a ruthenium porphyrin carbene complex by treating
1 (0.1 mM) with N2CHCO2Et (EDA) or N2CPh2 (100 equiv) in
aqueous media at room temperature but have not been successful.
Probably, the corresponding carbene complex generated in water is
not sufficiently stable for isolation. The formation of a metal carbene
intermediate from the reaction of 1 with EDA in water is inferred by
the treatment of the reaction mixture with PhNH2 (100 equiv), which
gave PhNHCH2CO2Et in 53% yield (detected by GC, conversion of
EDA: 100%). This metal carbene species, however, escaped detection
by spectroscopic means including MALDI-TOF MS. By treating
[RuII(4-(Ac4Glc)-TPP)(CO)] (the acetylated form of 1) with N2CPh2
in CH2Cl2, we observed the formation of a new species that showed
a MALDI-TOF MS peak at m/z 2265 attributable to the carbene
complex [Ru(4-(Ac4Glc)-TPP)(CPh2)]+, along with a UV-vis spec-
trum featuring two Soret bands at 397 and 412 nm and a ꢀ band at
534 nm, similar to that of [Ru(D4-Por*)(CPh2)] reported in our previous
work (Soret: 397 and 432 nm, ꢀ: 536 nm).18i
(30) For reviews, see: (a) Trost, B. M.; Melvin, L. S., Jr. Sulfur Ylides;
Academic Press: New York, 1975; Chapter 7. (b) Vedejs, E. Acc.
Chem. Res. 1984, 17, 358. (c) Pawda, A.; Hornbuckle, S. F. Chem.
ReV. 1991, 91, 263. (d) Marko´, I. E. In ComprehensiVe Organic
Synthesis; Pergamon: Oxford, 1991; Vol. 3,Chapter 3.10, p 913. (e)
Li, A.-H.; Dai, L.-X.; Aggarwal, V. K. Chem. ReV. 1997, 97, 2341.
(f) Hodgson, O. M.; Pierard, F. Y. T. M.; Stupple, P. A. Chem. Soc.
ReV. 2001, 30, 50. (g) Clark, J. S. In Nitrogen, Oxygen and Sulfur
Ylides Chemistry. A Practical Approach in Chemistry; Clark, J. S.,
Ed.; Oxford University Press: Oxford, 2002. (h) Sweeney, J. B. Chem.
Soc. ReV. 2009, 38, 1027.
9
J. AM. CHEM. SOC. VOL. 132, NO. 6, 2010 1889

A R T I C L E S
Ho et al.
Scheme 3
tography-tandem mass spectrometry (LC-MS/MS) analysis
(Figure S1 in Supporting Information). Only a trace amount of
18a (Scheme 3) was detected in the reaction mixture, with a
17a/18a ratio of >100:1 (entry 2 in Table 1). Neither other N-H
insertion products nor O-H insertion products were detected
by LC-MS/MS analysis of the reaction mixture. In the absence
of catalyst 1, no substrate conversion was observed (entry 1 in
Table 1). Changing the catalyst from 1 to 2-4 all resulted in a
< 5% substrate conversion (entries 10-12 in Table 1). The same
reaction catalyzed by [RuII(Por)(CO)] (Por ) TPP, TTP, TMP,
3,4,5-MeO-TPP) in PBS solution containing 1% dioxane led
to 0% or <10% substrate conversion (entries 13-16 in Table
1). When [Rh2(OAc)4] was used as catalyst, no modified peptide
product was observed (entry 17 in Table 1), and an O-H
insertion product HOCH2CO2RDns was detected by mass spec-
trometry (m/z 352).
catalyzed Doyle-Kirmse reactions in water.11 To the best of our
knowledge, the 1-catalyzed reactions of 9 and 11 are the first
examples of the following types of metal-catalyzed reactions in
aqueous media: (i) intramolecular Doyle-Kirmse reaction and
(ii) ammonium ylide formation/[2,3]-sigmatropic rearrangement
reaction.
To compare the catalytic properties of 1 with those of
previously reported water-soluble ruthenium porphyrins, we
examined the above carbenoid transfer reactions using catalysts
2-4 (the results are included in Tables S1-S5 in Supporting
Information). A comparison of the catalytic properties among
1-4, [RuII(D4-PorS*)(CO)]4-,12 and other related metal catalysts
is described in the Supporting Information.
With 1 as the catalyst, slightly raising the temperature to 37
°C increased the substrate conversion to >99%, and no 18a was
detected (entry 3 in Table 1). Upon reducing the loading of 1
from 10 mol % to 5 mol %, the substrate conversion decreased
to 52% (entry 4 in Table 1), with a product turnover of ∼10. A
marked decrease in substrate conversion was also found by
lowering the pH value of the reaction mixture from 7.4 to 6.4
and 5.0; further reduction of the pH value to 4.0 resulted in no
substrate conversion (entries 5-7 in Table 1). When the pH
value was increased from 7.4 to 8.4, the substrate conversion
slightly decreased to 97% (entry 8 in Table 1).
Peptide Modification through Carbenoid Transfer Reactions
Catalyzed by [RuII(4-Glc-TPP)(CO)] (1) in Aqueous Media. Metal-
catalyzed carbenoid insertion into X-H (X ) C, N, O, S) bonds
is possibly applicable for the modification of peptides that
contain amino acid residues such as tyrosine (Y), tryptophan
(W), lysine (K), cysteine (C), aspartic acid (D), glutamic acid
(E), threonine (T), and serine (S). A challenge for this type of
catalytic bioconjugation reactions is to achieve high site
selectivity and chemoselectivity for the reaction of a diazo
compound with peptides that contain multiple reactive X-H
(X ) C, N, O, S) sites. We prepared diazo compound
N2CHCO2RDns (15, RDns ) dansylaminoethyl), which contains
a dansyl fluorophore, by treating dansyl chloride with 2-ami-
noethanol followed by sequential reactions with diketene, MsN3,
and LiOH (Scheme S1 in Supporting Information). YTSSSKN-
VVR (16a) was chosen as a model peptide substrate for catalyst
screening, since it has various types of O-H (primary, second-
ary, and phenolic) and N-H (N-terminal, ε-, and guanidinyl)
sites possibly available for carbenoid insertion, allowing ex-
amination of site selectivity.
Table 1. Modification of Peptide YTSSSKNVVR (16a) by
1-Catalyzed Carbenoid Transfer Reactions under Various
Conditionsa (Results Using Catalysts 2-4 and Other Metal
Complexes Are Included for Comparison)
temp
(°C)
selectivity
(17a/18a)b
entry
pH
metal catalyst
conv (%)
1
2
7.4 RT
7.4 RT
7.4 37
7.4 37
4.0 37
5.0 37
6.4 37
8.4 37
7.4 37
7.4 RT
7.4 RT
7.4 RT
-
0
93
>99
52
0
19
46
97
81
<5
<5
<5
<5
<5
0
-
1
1
1
1
1
1
1
1
2
3
4
>100:1
17a only
100:1
-
17a only
17a only
3
4c
5
(i) N-H Insertion. Reaction of 16a (100 µM) with 15 (1.1
equiv) in a solution of phosphate-buffered saline (PBS, pH 7.4)
containing a catalytic amount of 1 (10 mol %) at room
temperature for 1 h afforded 17a (Scheme 3) with 93% substrate
conversion (entry 2 in Table 1), as revealed by liquid chroma-
6
7
8
100:1
9d
10
11
12
17a′ onlyd
-
-
-
-
-
-
-
-
13e 7.4 RT
14e 7.4 RT
15e 7.4 RT
16e 7.4 RT
[RuII(TPP)(CO)]
(31) Selected examples: (a) Cama, L. D.; Christensen, B. G. Tetrahedron
Lett. 1978, 19, 4233. (b) Aller, E.; Buck, R. T.; Drrysdale, M. J.; Ferris,
L.; Haigh, D.; Moody, C. J.; Pearson, N. D.; Sanghera, J. B. J. Chem.
Soc., Perkin Trans. 1 1996, 2879. (c) Bolm, C.; Kasyan, A.; Drauz,
K.; Gu¨nther, K.; Raabe, G. Angew. Chem., Int. Ed. 2000, 39, 2288.
(d) Morilla, M. E.; D´ıaz-Requejo, M. M.; Belderrain, T. R.; Nicasio,
M. C.; Trofimenko, S.; Pe´rez, P. J. Chem. Commun. 2002, 2998. (e)
Matsushita, H.; Lee, S.-H.; Yoshida, K.; Clapham, B.; Koch, G.;
Zimmermann, J.; Janda, K. D. Org. Lett. 2004, 6, 4627. (f) Davies,
J. R.; Kane, P. D.; Moody, C. J. J. Org. Chem. 2005, 70, 7305. (g)
Aviv, I.; Gross, Z. Chem. Commun. 2006, 4477. (h) Liu, B.; Zhu,
S.-F.; Zhang, W.; Chen, C.; Zhou, Q.-L. J. Am. Chem. Soc. 2007,
129, 5834. (i) Lee, E. C.; Fu, G. C. J. Am. Chem. Soc. 2007, 129,
12066. (j) Taber, D. F.; Berry, J. F.; Martin, T. J. J. Org. Chem. 2008,
73, 9334.
[RuII(TTP)(CO)]f
[RuII(TMP)(CO)]
[RuII(3,4,5-MeO-TPP)(CO)]g
[Rh2(OAc)4]
9
0
17
7.4 RT
a Reaction conditions: 16a (100 µM), 15 (110 µM), catalyst (10 µM),
PBS (pH 7.4, 100 mM NaCl), 1 h. Product and substrate conversion
were determined by LC-MS/MS. b Based on ion count of the peaks in
LC-MS. c Metal catalyst (5 µM). d EDA (instead of 15) was used; 17a′
differs from 17a in that the former bears an Et group, instead of an RDns
group. e PBS (pH 7.4, 100 mM NaCl) containing 1% dioxane. f TTP )
meso-tetrakis(p-tolyl)porphyrinato dianion. g 3,4,5-MeO-TPP ) meso-
tetrakis(3,4,5-trimethoxyphenyl)porphyrinato dianion.
9
1890 J. AM. CHEM. SOC. VOL. 132, NO. 6, 2010

Water-Soluble Ru Glycosylated Porphyrin Catalyst
A R T I C L E S
Scheme 4
Table 2. Modification of Peptides (16b-h) by 1-Catalyzed
Carbenoid Transfer Reactionsa
The results depicted in Table 1 reveal that the ruthenium
glycosylated porphyrin 1 is a highly selective catalyst for
modification of the N-terminus of 16a in an aqueous medium
via carbenoid insertion into the N-H bond. In a previous work,
we demonstrated a selective modification of the N-terminus of
peptides (including 16a) by a different type of metal-catalyzed
reaction, namely, N-terminal acylation with alkynes in aqueous
NaHCO3 buffer using the “[Mn(2,6-Cl2TPP)Cl] + H2O2”
oxidation system (2,6-Cl2TPP ) meso-tetrakis(2,6-dichlorophe-
nyl)porphyrinato dianion).32
entry
peptide
conv (%)b
1
2
3
4
5
6
7
YLSGANLNL (16b)
PPGFSPFR (16c)
GGG (16d)
ALILTLVS (16e)
HDMNKVLDL (16f)
TYGPVFMSL (16g)
DRVYIHPFHL (16h)
95
93
∼80
46
34
21
5
a Conditions: peptide (100 µM), 15 (110 µM), 1 (10 µM), PBS (pH
7.4, 100 mM NaCl), 37 °C, 1 h. b Determined by LC-MS/MS.
Interestingly, the 1-catalyzed carbenoid N-H insertion reac-
tion of 16a occurred predominately at the N-terminal amine
(pKa 7.6-8.014) but not at the more basic internal lysine ε-amine
(pKa 9.3-9.514); this contrasts with the modification of the same
peptide under similar conditions (37 °C, PBS, pH 7.4) using
N-hydroxysuccinimide (NHS) ester 19 (Scheme 4; NHS ester
is a widely used acylation agent for amines), in which case the
major product was the lysine modified peptide 20 (Figure S2
in Supporting Information), instead of the corresponding N-
terminal modified peptide. A rationalization is that most of the
lysine ε-amine groups are protonated at pH 7.4-8.4 in aqueous
media, rendering these amine groups not available for 1-cata-
lyzed carbenoid N-H insertion under these pH conditions. This
is supported by a dramatic decrease in substrate conversion upon
lowering the pH from 7.4 to 5.0 (Table 1), as most of the
N-terminal amines would also be protonated at pH 5.0 (no
reaction was observed when the pH was lowered to 4.0). The
reason for the absence of the O-H insertion product in the
1-catalyzed reaction might be due to a lower nucleophilicity of
the OH groups compared with the N-terminal amine group.
The 1-catalyzed carbenoid insertion reaction is useful for
selective modification of peptides not only at N-terminal tyrosine
(in 16a) but also at other N-terminal amino acids such as glycine,
alanine, and histidine. Under the optimum conditions (37 °C,
pH 7.4), a variety of peptides YLSGANLNL (16b), PPGFSPFR
(16c), GGG (16d), ALILTLVS (16e), HDMNKVLDL (16f),
TYGPVFMSL (16g), and DRVYIHPFHL (16h) reacted with
15 in the presence of catalyst 1, resulting in modification of
the N-terminus of 16b-g (Figures S3-S8 in Supporting
Information) with 21-95% substrate conversions (Table 2).
Excellent substrate conversions of 95% and 93% were obtained
for 16b and 16c, respectively (entries 1, 2 in Table 2).
5 and Figure S9 in Supporting Information) with complete substrate
conversion, indicating that a S-H insertion reaction is more rapid
than a N-H insertion reaction. The thioether group of methionine
in the peptide remained intact throughout the reaction. Under the
same conditions, 1 catalyzed the reaction of 15 with SSCSSC-
PLSSK (16j), a peptide containing a disulfide group and both
N-terminal and internal amine groups, to give the N-terminal N-H
insertion product 17j only, with 73% substrate conversion (Scheme
5 and Figure S10 in Supporting Information), and no cleavage of
the disulfide bond was observed.
Modification of N-Terminus of Proteins through Carbenoid
Transfer Reactions Mediated by [RuII(4-Glc-TPP)(CO)] (1)
in Aqueous Media. Bioconjugation of proteins is a useful tool in
biological studies. There is an increasing need to develop new
synthetic technologies for the bioconjugation reaction of proteins,14
and metal-catalyzed site-selective modification of proteins has
attracted considerable interest in recent years.15 Despite the wide
applications of metal-catalyzed carbenoid transfer reactions in
synthetic organic chemistry,1 modification of proteins by this type
of metal catalysis has previously been confined to [Rh2(OAc)4]-
catalyzed carbenoid transfer to the indole group of internal
tryptophan residues of proteins including myoglobin,8a ꢀ-lacto-
globulin,16 subtilisin,8b lysozyme,8b and mutants of FKBP.8b In
contrast to the [Rh2(OAc)4]-catalyzed modification of proteins under
denaturing conditions (at pH ∼3.5 in the presence of HONH2 ·HCl
additive8a or at pH 6-7 in the presence of BuNHOH at 75-95
t
°C8b), the protein modification reactions mediated by 1 can be
conducted at 37 °C in a pH range of 5.0-8.4 in the absence of an
additive, without causing the proteins to denature.
(ii) Competition between N-H and S-H Insertion. The
competition between N-H and S-H bonds for a carbenoid
insertion reaction is commonly encountered in peptide modification.
Treatment of peptide AYEMWCFHQK (16i), which contains both
N-terminal amine and internal SH groups, with 15 in PBS (pH
7.4) in the presence of catalyst 1 (10 mol %) at room temperature
for 1 h afforded the S-H insertion product 21 exclusively (Scheme
Reaction of an N-terminal lysine-containing protein, RNase A
(10 µM), with 15 (1.1 equiv) in PBS (pH 7.4) solution in the
presence of 1 (1 equiv) at 37 °C for 1 h increased the mass of
RNase A by ∼334 amu on the basis of liquid chromatography-
mass spectrometry (LC-MS) analysis (Figure S11 in Supporting
Information), indicating that one dansyl carbene unit CHCO2RDns
(mass: 334 amu) had been incorporated into this protein. The
substrate conversion based on MS analysis was 65% (entry 1 in
Table 3). LC-MS/MS analysis of the modified protein, after
(32) Chan, W.-K.; Ho, C.-M.; Wong, M.-K.; Che, C.-M. J. Am. Chem.
Soc. 2006, 128, 14796.
9
J. AM. CHEM. SOC. VOL. 132, NO. 6, 2010 1891

A R T I C L E S
Ho et al.
Scheme 5
Francis and co-workers recently reported N-terminal protein
modification by introducing a ketone or aldehyde group.34 Note
that the 1-mediated N-H insertion reaction would not be compat-
ible with the free SH group of cysteine, as both thiolate (RS-) and
free SH are stronger nucleophiles than NH, which would render
the SH group a more favorable site for carbenoid insertion (see
Scheme 5). This limitation on N-H insertion could actually suggest
another potential application of the 1-mediated carbenoid transfer
reactions, i.e. selective modification of proteins via S-H insertion.
Table 3. Alkylation of N-Terminus of Proteins by 1-Catalyzed
Carbenoid Transfer Reactionsa
entry
protein
N-terminal amino acid
conv (%)
1
2
RNase A
insulin
lysine
65
glycine (A-chain) and
phenylalanine (B-chain)
glycine
54b
3
4
myoglobin
ubiquitin
trace
trace
methionine
a Conditions: protein (10 µM), 15 (11 µM), 1 (10 µM), PBS (pH 7.4,
NaCl, 100 mM), 37 °C, 1 h. b Total conversion (singly modified 48%,
doubly modified 6%).
Modification of Alkene-Tethered Protein through Carbenoid
Transfer Reaction Mediated by [RuII(4-Glc-TPP)(CO)] (1) in
Aqueous Media. As direct modification of ubiquitin by 1-mediated
N-H insertion was not effective, we performed a site-selective
modification of this protein in two steps. The first step is to
covalently tether an alkene group to the lysine residue Lys6 of
ubiquitin by treatment of this protein with 19 (Scheme 7) in PBS
solution (pH 7.4), analogous to the previously reported reaction of
ubiquitin with sulfosuccinimidobiotin to give Lys6-biotinylated
ubiquitin.35 The alkene-tethered ubiquitin (23) purified by HPLC
exhibited a peak at m/z 8710 (Figure S16 in Supporting Informa-
tion) in matrix-assisted laser desorption/ionization time-of-flight
mass spectrometry (MALDI-TOF MS). After tryptic digestion of
23, the resulting peptides were analyzed by LC-MS/MS, and the
results, as compared with those of ubiquitin, revealed attachment
of the alkenic moiety (C10H8O, 144 amu) to the Met1-Lys11 peptide
(MQIFVKTLTGK) only (the details are given in Table S6 of
Supporting Information). A doubly charged ion peak at m/z 705.4
attributable to this alkene-tethered peptide was observed by LC-
MS for the trypsin-digested 23. MS/MS analysis confirmed that
the alkene was specifically incorporated at Lys6.
digestion with trypsin, revealed that only the N-terminal lysine of
RNase A was modified (Figure S12 in Supporting Information),
consistent with the formation of 22 (Scheme 6). This “15 + 1”
protocol was also effective for insulin, giving singly modified
insulin with 48% substrate conversion; a doubly modified insulin
was also detected, with a low substrate conversion of 6% (Figure
S13 in Supporting Information and entry 2 in Table 3). The doubly
modified insulin species is probably due to acylation of both
N-termini of A- and B-chains of the insulin molecule. However,
attempts to apply this protocol to myoglobin and ubiquitin, which
contain 17 and 7 lysine residues, respectively, besides N-terminal
glycine or methionine, were not successful (see Figures S14 and
S15 in Supporting Information).
The ineffective modification of myoglobin and ubiquitin by
1-mediated N-H insertion could be due to the relatively small
solvent-accessible areas of the nitrogen atoms of their N-terminal
amine groups, which were calculated to be 29.06 and 18.68 Å2,
respectively, based on the crystal structure data of horse heart
myoglobin (1YMB) and bovine ubiquitin (1AAR) (using GETAR-
EA 1.433), compared with the corresponding value of 40.20 Å2
calculated for RNase A (1FS3). We reason that a larger solvent-
accessible area favors the bioconjugation reaction, as the N-H
site at which the reaction takes place is not bounded by the protein
molecule. Insulin contains two N-termini: the one belonging to
phenylalanine has a solvent-accessible area of 31.19 Å2, larger than
that of 18.31 Å for the one belonging to glycine; thus, the former
is more exposed and more reactive, consistent with the observation
described above. However, such rationalization should be taken
with caution, as proteins have a dynamic structure in solution. Also,
the carbenoid transfer reaction is dependent on the pKa value of
amines, which may vary with the amino acids, salts, buffer,
temperature, and ionic strength. These factors may also affect the
reactivity of carbenoid group transfer and should not be neglected.
The water-soluble ruthenium porphyrin complex 1 could be used
to mediate the conjugation of a fluorescent group, through
carbenoid N-H insertion, to the N-terminus of proteins having
different classes of N-terminal amino acids such as proline,
threonine, tyrosine, alanine, histidine, glutamic acid, and glycine.
In the second step, reaction of 23 (100 µM) with 15 (1.1 equiv)
in PBS (pH 7.4) containing 1 (10 mol %) at 25 °C for 1 h afforded
the cyclopropanation product 24 (Scheme 7) with ∼55% substrate
conversion (∼5.5 product turnovers). Similar selectivity and product
yield were found for the reaction even when the concentration of
23 was lowered to 10 µM. MALDI-TOF MS analysis of the
product, upon purification by HPLC, revealed a peak at m/z 9044
(Figure S16 in Supporting Information) attributed to 24. This peak
has an m/z value 334 larger than that observed for 23, consistent
with the incorporation of one dansyl carbene unit CHCO2RDns (334
amu) into 23. Indeed, the dansyl-labeled protein 24 could be seen
as a yellow fluorescent band under UV-irradiation by SDS-PAGE
analysis (Figure S16 in Supporting Information). For the 1-cata-
lyzed reaction of 23 with 15, other forms of modified 23, such as
(34) Gilmore, J. M.; Scheck, R. A.; Esser-Kahn, A. P.; Joshi, N. S.; Francis,
M. B. Angew. Chem., Int. Ed. 2006, 45, 5307.
(35) Shang, F.; Deng, G.; Liu, Q.; Guo, W.; Haas, A. L.; Crosas, B.; Finley,
D.; Taylor, A. J. Biol. Chem. 2005, 280, 20365.
(33) Fraczhiewicz, R.; Braun, W. J. Comput. Chem. 1998, 19, 319.
9
1892 J. AM. CHEM. SOC. VOL. 132, NO. 6, 2010

Water-Soluble Ru Glycosylated Porphyrin Catalyst
A R T I C L E S
Scheme 6
Scheme 7
a doubly modified one, were not detected in the reaction mixture.
No modification of 23 was observed in the absence of catalyst 1.
aqueous media, including inter- and intramolecular alkene
cyclopropanation, intermolecular carbenoid N-H insertion, and
intramolecular ammonium/sulfonium ylide formation/[2,3]-
sigmatropic rearrangement but also can be applied to site-
selective modification of peptides and proteins. By using 1 as
a catalyst, the N-terminus of a number of peptides can be
modified through carbenoid N-H insertion in aqueous media
Conclusion
We have synthesized a water-soluble glycosylated ruthenium
porphyrin catalyst (1), which not only exhibits high activity and
selectivity toward a number of carbenoid transfer reactions in
9
J. AM. CHEM. SOC. VOL. 132, NO. 6, 2010 1893

A R T I C L E S
Ho et al.
determined by GC analysis using the internal standard method (1,4-
dichlorobenzene).
with high site selectivity and moderate to excellent substrate
conversions. Proteins RNase A and insulin can also be modified
at their N-terminus in a similar manner. The bioconjugation
reaction is selective to protein structure. Site-selective modifica-
tion of ubiquitin via metal catalysis has been achieved by
introducing an alkene group to the protein followed by 1-cat-
alyzed cyclopropanation of the alkene-tethered ubiquitin with
a fluorescent-labeled diazo compound in aqueous media.
Modification of proteins by the 1-mediated carbenoid transfer
can be performed at pH 5.0-8.4, an optimum pH range for
most proteins, with high chemoselectivity and without nonpro-
ductive binding to the protein surface. This ruthenium porphyrin-
mediated bioconjugation method could be applicable for proteins
containing various N-terminal amino acids. For peptides having
the SH group, the modification would be dominated by
carbenoid S-H insertion instead of N-H insertion reaction.
The selective conjugation of the fluorescent dansyl probe to
protein chains by the method reported in this work is potentially
useful for biological studies.
The present work, to the best of our knowledge, contributes
the first examples of (a) ruthenium porphyrin-catalyzed modi-
fication of peptides and proteins in aqueous media and (b) metal-
mediated carbenoid transfer for modification of the N-terminus
of proteins. The 1-catalyzed carbenoid transfer to alkene-tethered
protein in aqueous media confirmed by LC-MS and LC-MS/
MS could open up an entry to new bioconjugation reactions
for protein modifications using metalloporphyrins as catalysts.
Typical Procedure for 1-Catalyzed Intermolecular Carbenoid
N-H Insertion of Amines. To a vigorously stirred aqueous solution
of amine (1 mmol) and 1 (0.005 mmol) in water (5 mL) was slowly
added an aqueous solution of EDA (0.5 mmol) in water (5 mL)
via a syringe pump over 10 h at RT. The reaction mixture was
stirred until all the substrate was consumed, as monitored by TLC.
The aqueous phase was extracted with Et2O (15 mL × 3), and the
combined organic extracts were dried over anhydrous Na2SO4. Pure
products were obtained by flash chromatography.
Typical Procedure for Modification of Peptides by
1-Catalyzed Carbenoid Transfer Reactions. A PBS solution (pH
7.4) containing peptide (100 µM) and 1 (10 µM) in a 1.5 mL
eppendorf tube was treated with a solution of the diazo compound
15 (1.1 equiv) in dioxane (1 mg/mL). The solution was incubated
at 37 °C for 1 h. The resulting N-terminal modified peptide was
characterized by LC-MS/MS analysis.
Typical Procedure for Modification of N-Terminus of
Proteins by 1-Mediated Carbenoid Transfer Reactions. A PBS
solution (pH 7.4) containing protein (10 µM) and 1 (10 µM) in a
1.5 mL eppendorf tube was treated with a solution of the diazo
compound 15 (1.1 equiv) in dioxane (1 mg/mL). The solution was
incubated at 37 °C for 1 h. The resulting N-terminal modified
protein was characterized by LC-MS/MS analysis.
Modification of Alkene-Tethered Lys6 Ubiquitin (23) by
1-Catalyzed Carbenoid Transfer Reaction. 100 µL of alkene-
tethered Lys6 ubiquitin (100 µM) in PBS at pH 7.4 was added to
a 1.5 mL vial, followed by adding 10 µL of an aqueous solution of
15 (110 µM). 10 mol % of 1 in 10 µL of dioxane was added, and
the reaction mixture was generally mixed in a thermomixer
(Eppendrof Thermomixer Comfort) at 25 °C for 1 h. After removal
of small molecules by ultrafiltration (Millipore, Centricon, YM-3),
the purified protein sample 24 was resuspended in 100 µL of PBS
(pH 7.4) solution and then analyzed by SDS-PAGE and MALDI-
TOF MS. Upon digestion of 24 with trypsin, the resulting peptides
were analyzed by LC-MS/MS. A doubly charged peptide fragment at
873 m/z was identified. MS/MS analysis of this peptide fragment
showed a full y-ion series that is consistent with the cyclopropanation
of the alkene tethered to the Lys6 residue of ubiquitin (Figure S17 in
Supporting Information). The formulation of the cyclopropanation
product 24 was also based on the well-documented cyclopropanation
of alkenes with diazo compounds catalyzed by ruthenium porphyrins25a,c
and the catalytic activity of 1 toward cyclopropanation of alkenes with
diazoacetates (see, for example, Scheme 2).
Experimental Section
Synthesis of Glycosylated Ruthenium Porphyrin [RuII(4-Glc-
TPP)(CO)] (1). A mixture of Ru3(CO)12 (200 mg) and H2(4-
(Ac4Glc)-TPP) (200 mg) in decalin (50 mL) was refluxed under
nitrogen for 24 h. The resulting red solution was loaded on an
alumina column. Decalin and some impurities were removed with
hexane as the eluent. The brick red band containing the desired
product was eluted with hexane/EtOAc (1:2 v/v) and collected. After
removal of solvent, a red solid of [RuII(4-(Ac4Glc)-TPP)(CO)] was
obtained (yield: 50%. IR: ν(CO) 1940 cm-1). This complex was
refluxed in methanol (50 mL) containing NaOMe (50 mg) for 30
min. Treatment of the mixture with ion-exchange resin to pH ) 7
and concentration of the filtrate afforded the desired product 1 as
a red solid. Yield: 50%. UV-vis (MeOH): λmax (log ε) 412 (5.46),
527 nm (4.43); IR: ν(CO) 1940 cm-1; 1H NMR (CD3OD): 8.58 (s,
ꢀ-H, 8H), 8.05 (d, J ) 6.3 Hz, 4H), 7.95 (d, J ) 8.1 Hz, 4H), 7.41
(m, 8H), 5.18 (d, J ) 6.6 Hz, 4 H), 3.97 (d, J ) 10.8 Hz, 4H),
3.78 (m, 4H), 3.58 (m, 12H), 3.45 (m, 4H); ESI-MS: m/z 1455.2
(M+).
Typical Procedure for 1-Catalyzed Intermolecular Cyclopro-
panation of Styrenes. To a vigorously stirred aqueous solution of
1 (0.01 mmol) in water (10 mL) was added styrene (1 mmol). An
aqueous solution of EDA (1.2 mmol) in water (10 mL) was slowly
added to the reaction mixture via a syringe pump over 24 h at RT.
The reaction mixture was stirred at RT, with the reaction progress
monitored by TLC. The aqueous phase was extracted with Et2O
(15 mL × 3), and the combined organic extracts were dried over
anhydrous Na2SO4. Conversions, yields, and trans/cis ratios were
Acknowledgment. This work was supported by The University
of Hong Kong (University Development Fund), the University Grants
Council of HKSAR (the Area of Excellence Scheme: AoE 10/01P),
and the Hong Kong Research Grants Council (HKU 1/CRF/08).
Supporting Information Available: Experimental section
including general, product identification, procedures for prepara-
tion of 15, 19, and 23, Tables S1-S6, Chart S1, Scheme S1,
and Figures S1-S17. This material is available free of charge
via the Internet at http://pubs.acs.org.
JA9077254
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1894 J. AM. CHEM. SOC. VOL. 132, NO. 6, 2010