Mimicking the function of eggshell matrix proteins: The role of multiplets of charged amino acid residues and self-assembly of peptides in biomineralization

Article abstract of DOI:10.1002/anie.200500261

(Figure Presented) An eggshell finish: Designed peptides can be used as a tool to unravel the structure-activity relationships of eggshell matrix proteins. The ordered arrangement of doublets of charged residues on the peptide and its self-assembling characteristics play a key role in the biomimetic nucleation of polycrystalline calcite crystal aggregates (see picture), which models that initiated by the goose eggshell matrix protein, ansocalcin.

Full text of DOI:10.1002/anie.200500261

Communications
Biomineralization
such proteins during in vitro experiments.[6c] Thus, to inves-
tigate the role of self-assembly and multiplets of charged
amino acids in calcium carbonate mineralization, we designed
a series of peptides (Table 1) and studied their structure–
activity relationships. The first two groups (I, II) of peptides
have an ordered arrangement of doublets of charged residues
which resembles the primary structure of avian eggshell
matrix proteins (a full sequence comparison is given in the
Supporting Information).[7] The significant structural differ-
ences between the peptides involve replacing the arginine
residue with lysine—a basic flexible residue with a primary
amino group and hydrophobic side chain—and the incorpo-
ration of a proline residue in the middle of the peptides in
group II. The proline residue is expected to limit the
conformational flexibility,[6c] and organisms use such domains
to form three-dimensional supramolecular structures of
proteins.[2a,8,9] Among the three peptides in group III,
K2E2W2D2G-17 and K2AE2AW2AD2P-23 have an ordered
arrangement of doublets of charged residues. How-
ever, the incorporation of one glycine residue in the
former and six alanine residues in the latter peptide is
expected to disrupt the ordered, stable secondary struc-
ture of the peptides and impart flexibility to the peptide
backbone. The peptide (KE)2(WD)2-16, with alternate acidic
and basic residues, is designed as a control peptide for
confirming the perceived critical role of multiplets of charged
residues.
To understand the role of these designed peptides in
calcite growth and habit modification, we carried out in vitro
mineralization of calcium carbonate by slow diffusion of CO2
and NH3 gas to the peptide dissolved in CaCl2 solution
(7.5 mm).[10] The first two groups of peptides showed signifi-
cant influence on the calcite crystal morphology and aggre-
gation as compared with the group III peptides (compare
images 2–5 with images 6–8 in Figure 1 and Supporting
Information).
The CaCO3 crystals formed in presence of group I and II
peptides showed—similarly to those formed in the presence
of ansocalcin[8]—concentration-dependent changes in crystal
morphology (Figure 1 and Supporting Information). Poly-
crystalline aggregates of calcite crystals were formed as the
concentration was increased to 2mgmL ꢀ1, and the size of the
crystal aggregates obtained with K2E2W2D2-16 ( ꢁ 41 mm) and
K2E2W2D2P-17 ( ꢁ 35 mm) was almost double that of crystals
obtained with R2E2W2D2-16 ( ꢁ 20 mm) and R2E2W2D2P-17
( ꢁ 21 mm). The observed superstructures are composed of
smaller rudimentary calcite crystals assembled into spherical
structures. The crystal lattice was further characterized by X-
ray diffraction studies. In addition, the nucleation density
(that is, the number of crystals per unit area) also increased
with an increase in the peptide concentration. Group III
peptides did not show significant influence on the calcite
crystal nucleation, growth, or morphology. This finding
indicates that not only the primary structural characteristics
but also control of the spatial disposition of the functional
sites on the peptide is important for the nucleation of crystal
aggregates. Thus, the designed peptides R2E2W2D2-16,
DOI: 10.1002/anie.200500261
Mimicking the Function of Eggshell Matrix
Proteins: The Role of Multiplets of Charged
Amino Acid Residues and Self-Assemblyof
Peptides in Biomineralization**
Parayil Kumaran Ajikumar,
Subramanian Vivekanandan,
Rajamani Lakshminarayanan, Seetharama D. S. Jois,
R. Manjunatha Kini, and Suresh Valiyaveettil*
The molecular-level understanding and rationalization of
natureꢀs clues on how to produce complex, organized
structures such as bone, teeth, and seashells at ambient
temperature and in aqueous environments has resulted in the
design and synthesis of novel materials.[1,2] The properties of
the crystals such as size, shape, stability, polymorph selectivity,
and crystallographic orientation are controlled by biomacro-
molecules, which are classified as “framework macromole-
cules” and “control macromolecules”.[2,3] The complexities of
the natural biomineralization process have resulted in
research efforts being focused on “tailor-made” low-molec-
ular-weight compounds and macromolecular additives for
structure–function investigations by in vitro mineralization
experiments.[4] So far, research efforts have been focused on
auxiliary oligopeptides to probe the role of functional motifs
in the kinetics, habit modifications, and polymorph selectivity
during the crystal growth process.[5,6]
The structure–function sequence analysis of eggshell-
specific proteins revealed that the presence of multiplets of
charged amino acid residues and characteristic self-aggrega-
tion in solution influence the formation of biominerals.[7,8] We
anticipate that the design and testing of peptides with
multiplets of charged residues could mimic the functions of
[*] Dr. P. K. Ajikumar, Dr. R. Lakshminarayanan, Prof. S. Valiyaveettil
Department of Chemistryand Singapore–MIT Alliance
National Universityof Singapore
3 Science Drive 3, Singapore 117543 (Singapore)
Fax: (+65)6779-1669
E-mail: chmsv@nus.edu.sg
Dr. S. D. S. Jois
Department of Pharmacy
National Universityof Singapore
Singapore 117543 (Singapore)
Dr. S. Vivekanandan, Prof. R. M. Kini
Department of Biological Sciences
National Universityof Singapore
Singapore 117543 (Singapore)
[**] This work was supported bythe Singapore–MIT Alliance. R.L.
acknowledges Singapore Millennium Foundation for the award of a
fellowship. The authors acknowledge technical support from the
Departments of Chemistry, Biological Sciences, and Materials
Sciences at National Universityof Singapore.
R2E2W2D2P-17,
K2E2W2D2-16,
and
K2E2W2D2P-17
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
(2mgmL ꢀ1, ꢁ 0.8 mm) induce the nucleation and formation
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Table 1: Details of the amino acid sequences and structure–activityrelationships of the model peptides.
Group Model peptide
Primarystructure
Results
Activityin crystal growth
Secondarystructure
I
R2E2W2D2-16 (I)
RREEWWDDRREEWWDD
KKEEWWDDKKEEWWDD
RREEWWDDPRREEWWDD
KKEEWWDDPKKEEWWDD
KKEEWWDDGKKEEWWDD
b sheet
b sheet
b sheet with turn
b sheet with turn
unordered
polycrystalline calcite aggregate formation
K2E2W2D2-16 (III)
R2E2W2D2P-17 (II)
K2E2W2D2P-17 (IV)
K2E2W2D2G-17 (V)
II
III
no influence on calcite growth or habit modification
K2AE2AW2AD2P-23 (VI) KKAEEAWWADDPKKAEEAWWADD
(KE)2(WD)2-16 (VII) KEKEWDWDKEKEWDWD
toward decreasing the conformational flexibility and enhanc-
ing the oligomerization of peptides into large aggregates. The
observed particle sizes for the group III peptides K2AE2-
AW2AD2P-23 (VI, 2.2 nm) and (KE)2(WD)2-16 (VII, 1.8 nm)
were low. Interestingly, alterations of the peptide sequences
result in the enhancement of the mean diffusion coefficient
(hDi, Figure 2). In addition, the observed mean diffusion
coefficient for the group I and II peptides is almost compa-
rable to that of monomeric forms of ansocalcin and lithosta-
thine.[8,11]
Figure 1. Crystal habit modification in the presence of the protein
ansocalcin (500 mgmLꢀ1) and designed peptides (2 mgmLꢀ1). The
scale bars represent 50 mm. Magnified images are given in the insets.
The study of the microenvironment around the trypto-
phan residues gives an insight into the self-aggregation
tendency of the peptides. All the peptides showed distinct
emission characteristics (see Supporting Information) and
changes in intensity of the emission maximum with change in
peptide concentration. The lysine-incorporated peptides
K2E2W2D2-16 and K2E2W2D2P-17 showed weaker emission
intensities than the peptides R2E2W2D2-16 and R2E2W2D2P-
17. Notably, the intensity increased linearly with concentra-
tion up to 0.25 mgmLꢀ1, followed by an approximately 85%
decrease in intensity for groups I and II peptides at a
concentration of 2mgmL ꢀ1. This result might be a conse-
quence of the quenching of the tryptophan fluorescence by
the polar functional groups (CO2H), which are brought into
close proximity through aggregation of the peptide mole-
cules.[12] However, in the case of the group III peptides, the
emission intensity increased linearly with concentration up to
of polycrystalline calcite crystal aggregates similar to the
parent protein ansocalcin.
For a detailed investigation of the structure–activity
relationship, we investigated the self-assembly of the peptides
in solution by dynamic light scattering (DLS), tryptophan
fluorescence, circular dichroism (CD), NMR spectroscopy,
and molecular modeling studies. The DLS and fluorescence
analysis showed a concentration-dependent aggregation of
group I and II peptides in CaCl2 solution (Figure 2). The
observed hydrodynamic radii of the group I peptides (hRHi =
3.3 and 4.1 nm) are smaller than those of the corresponding
proline-incorporated group II peptides (hRHi = 6.4 and
8.1 nm). However, a fourfold reduction in the size of the
aggregate was observed when proline in K2E2W2D2P-17 (IV,
8.1 nm) was replaced with glycine in K2E2W2D2G-17 (V,
2.2 nm). These results highlight the expected role of proline
1 mgmLꢀ1 followed by a small intensity drop at 2mgmL ꢀ1
.
The CD spectra of the peptides were recorded in a
concentration range of 0.05 to 2mgmL ꢀ1 at 258C in CaCl2
solution (Figure 3 and Supporting Information). It is clear
that the secondary structure of the peptides R2E2W2D2-16 and
K2E2W2D2-16 in solution is an extended b-sheet conformation
Figure 2. A) Mean hydrodynamic radius (hRHi) and diffusion coefficient
(hDi) of synthetic peptides I–VII. The y axis represents the hRHi in
nanometers or hDi”10ꢀ7 in cm2 sꢀ1. B) Intensity( Fmax) versus concen-
Figure 3. Circular dichroism spectra of group I and II peptides in CaCl2
(7.5 mm) at various concentrations: 1) 50 mgmLꢀ1, 2) 100 mgmLꢀ1
,
^
tration profile of fluorescence emission of synthetic peptides I ( ), II
3) 250 mgmLꢀ1, 4) 500 mgmLꢀ1, 5) 1 mgmLꢀ1, and 6) 2 mgmLꢀ1. The
instrument settings used were: scan range, 190 to 260 nm; scan rate,
50 nmminꢀ1; sensitivity, 10 millidegrees; response time, 1 s.
~
&
*
( ), III ( ), IV ( ), V ( ), VI (+), and VII (”) in CaCl solution
*
2
(7.5 mm).
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at high peptide concentrations (2mgmL ꢀ1). However, the
The calculated d values for residues E4-D7 and R/K10-
D17 in the group II peptides are greater than random coil
values by 0.1 ppm, which indicates that both peptides adopt a
similar nonextended conformation with a turn structure for
the backbone. The calculated ratio of daN(i,i+1):dNN(i,i+1) is
greater than 1.4, which suggests an ordered peptide con-
incorporation of the proline in the middle of the sequences
(R2E2W2D2P-17 and K2E2W2D2P-17) disrupts the extended b-
sheet structure. The CD spectra showed the appearance of an
amide n–p* negative-transition band at 225 and 227 nm for
the peptides, which corresponds to a b-turn conformation.[13]
The replacement of proline with glycine in K2E2W2D2G-17
resulted in an unordered structure with turns (negative
minimum at 201 and 223 nm).[14] Similarly, the CD spectra
of the peptides K2AE2AW2AD2P-23 (negative minimum at
199 and 224 nm) and (KE)2(WD)2-16 (negative minimum at
199 nm and a weak positive maximum at 216 nm) also
indicated unordered structures in solution.
formation. A long-range NOE cross-relaxation transfer (daN
-
(i,i+2)) was observed between the residues P9/R11 and E12/
W14 for R2E2W2D2P-17 and D8/K10, P9/K11, and E12/W14
for K2E2W2D2P-17 (see Supporting Information). However,
in the case of the glycine-containing peptide (K2E2W2D2G-
17), no dNN(i,i+1) NOE connectivities were observed. Thus,
from the above observations we conclude that the peptides
R2E2W2D2P-17 and K2E2W2D2P-17 adopt a nonextended
structure with a turn or kink on the backbone.
By using the available NMR restraints, the conformations
of the peptides were calculated with the help of molecular-
dynamics-simulated annealing/minimization experiments to
determine a plausible lowest-energy conformational ensem-
ble (ten structures) that fits the target NMR data set. The
conformers with the fewest NOE violations were used to
represent the structure. Figures 4B,D show the backbones of
ten overlapped, energy-minimized structures of the peptides
R2E2W2D2P-17 and K2E2W2D2P-17, whereas the surface
structures (Figure 4C,E) represent the distribution of func-
tional groups. Analysis of the f,y distribution obtained for
the ten conformational ensembles revealed that the dihedral
pairs near proline consistently centered within the f,y regions
associated with a turn structure in polypeptides. Comparison
of the dihedral angles shows a type II turn structure near P9-
R10 residues of the peptide R2E2W2D2P-17 and a type I turn
structure in the region of P9-R10 and E12to E13 of
K2E2W2D2P-17.
The results obtained from CD spectrometry are supported
by data from NMR experiments. The 1D NMR spectra of
R2E2W2D2-16 and K2E2W2D2-16 showed a significant peak
broadening (Figure 4A) as a result of the formation of an
The observations from the CD and NMR spectra and
molecular modeling studies are well-correlated and consis-
tent. The peptides R2E2W2D2P-17 and K2E2W2D2P-17 exhibit
a b-sheet-type structure with a turn near the proline residue.
Such an unfolded conformation would minimize the electro-
static side-chain charge repulsion, orient the charged residues
(D, E, R, and K), and make them accessible for complexation
with calcium ions in solution.[16]
The observed extensive overlap of the NMR signals in the
amide region of group III peptides indicates the presence of
an unordered open structure with rapid conformational
exchanges. In addition, the low hydrodynamic radius (hRHi)
and high value of the mean diffusion coefficient (hDi)
obtained from the DLS experiments also corroborate the
findings from the CD and NMR studies. Moreover, the
fluorescence studies revealed the absence of concentration-
dependent self-assembly of group III peptides in solution.
The results from conformational studies of the peptides are
consistent with the observed mineralization behavior in
solution. Thus, in the nonaggregated form, the random
movement of the peptides resulted in a nonspecific inter-
action with the CaCO3 crystals, thereby inducing pits and
truncation of the calcite crystal surfaces.
Figure 4. A) 1D pulsed-field gradient 1H NMR spectra of the NH re-
gion of the model peptides; 300 K, in 90% H2O/10% D2O, pH 3.0.
Proton chemical shifts are referenced from internal [2,2,3,3-D4]TSP. B)–
E) Conformational ensembles and surface structures obtained from
molecular dynamics simulations of the peptides R2E2W2D2P-17 (B,C)
and K2E2W2D2P-17 (D,E).
extended b-sheet conformation in solution. In contrast, the
1D NMR spectra of the peptides R2E2W2D2P-17 and
K2E2W2D2P-17 showed sharp NH peaks within the range of
Dd = 1.3 ppm, which indicates an ordered structure in solu-
tion. The NMR spectrum of K2E2W2D2G-17 showed a strong
overlap of amide protons, thus indicating an unordered
structure. Similarly, the peptides (KE)2(WD)2-16 and
K2AE2AW2AD2P-23 also showed an extensive chemical
shift overlap, which indicated an unordered structure in
solution. The calculated proton chemical shift difference
measured with TSP references (TSP = 3-(trimethylsilyl)pro-
pionate) showed deviation from the random coil values for
the R2E2W2D2P-17 and K2E2W2D2P-17 peptides (see Sup-
porting Information).[15]
In conclusion, seven peptides were designed and fully
characterized to mimic the function of the goose eggshell
matrix protein, ansocalcin. The ordered arrangement of
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Chemie
ecules 2003, 4, 1321 – 1326; d) D. Volkmer, M. Fricke, T. Huber,
doublets of charged residues and the self-assembling charac-
teristics of the group I and II peptides generated a functional-
group-segregated surface which facilitates the nucleation,
growth, and aggregation of calcium carbonate crystal nuclei
to form polycrystalline calcite crystal aggregates. The group
III peptides showed an unordered structure in solution and no
activity toward mineralization. This finding is supported by
evidence from DLS, NMR, CD, and fluorescence spectros-
copy investigations.
N. Sewald, Chem. Commun. 2004, 1872– 1873.
[7] a) R. Lakshminarayanan, S. Valiyaveettil, R. M. Kini, Proc. Natl.
Acad. Sci. USA 2002, 99, 5155 – 5159; b) R. Lakshminarayanan,
S. Valiyaveettil, V. S. Rao, R. M. Kini, J. Biol. Chem. 2003, 278,
2938 – 2946; c) R. Lakshminarayanan, J. S. Joseph, S. Valiya-
veettil, R. M. Kini, Biomacromolecules, 2005, 6, 741 – 751.
[8] a) K. Mann, F. Siedler, Biochim. Biophys. Acta Proteins Pro-
teom. 2004, 1696, 41 – 50; b) J. P. Reyes-Grajeda, A. Moreno, A.
Romero, J. Biol. Chem. 2004, 279, 40876 – 40881; c) K. Mann,
Br. Poult. Sci. 2004, 45, 483 – 490.
[9] a) M. Bergdoll, M. H. Remy, C. Cagnon, J. M. Masson, P.
Dumas, Structure 1997, 5, 391 – 398; b) M. W. MacArthur, J. M.
Thornton, J. Mol. Biol. 1991, 218, 397 – 412; c) S. C. Benson,
F. H. Wilt in Calcification in Biological Systems (Ed.: E.
Bonucci), CRC, Boca Raton, FL, 1992, pp. 157 – 178; d) J. P.
Gorski, Calcif. Tissue Int. 1992, 50, 391 – 396; e) J. Moradian-
Oldak, J. Tan, A. G. Fincham, Biopolymers 1998, 46, 225 – 238;
f) J. Moradian-Oldak, W. Leung, A. G. Fincham, J. Struct. Biol.
1998, 122, 320 – 327; g) C. E. Killian, F. C. Wilt, J. Biol. Chem.
1996, 271, 9150 – 9159.
[10] S. Albeck, J. Aizenberg, L. Addadi, S. Weiner, J. Am. Chem. Soc.
1993, 115, 11691 – 11697.
[11] C. Cerini, V. Peyrot, C. Garnier, L. Duplan, S. Veesler, J. P.
Le Caer, J. P. Bernard, H. Bouteille, R. Michel, A. Vazi, P.
Dupuy, B. Michel, Y. Berland, J. M. Verdier, J. Biol. Chem. 1999,
274, 22266 – 22274.
[12] a) A. P. Demchenko, Topics in Fluorescence Spectroscopy, Vol. 3
(Ed.: J. R. Lackowicz), Plenum, New York, 1992, pp. 77; b) L. W.
Ruddock, T. R. Hirst, R. B. Freedman, Biochem. J. 1996, 315,
1001 – 1005.
[13] a) F. J. Blanco, G. Rivas, L. Serrano, Nat. Struct. Biol. 1994, 1,
584 – 590; b) M. C. Mannin, M. Illangasekare, R. W. Woody,
Biophys. Chem. 1998, 31, 77 – 86.
[14] a) G. D. Fasman, Circular Dichroism and the Conformational
Analysis of Biomolecules, Plenum, New York, 1996; b) S. C.
Shankaramma, S. K. Singh, A. Sathyamurthy, P. Balaram, J. Am.
Chem. Soc. 1999, 121, 5360 – 5363.
[15] D. S. Wishart, B. D. Sykes, F. M. Richards, J. Mol. Biol. 1991, 222,
311 – 333.
Received: January 24, 2005
Revised: May 5, 2005
Published online: August 1, 2005
Keywords: biomineralization · eggshell · peptides · proteins ·
.
structure–activityrelationships
[1] a) Materials Synthesis Based on Biological Processes (Eds.: M.
Alper, P. D. Calvert, R. Frankel, P. C. Rieke, D. A. Tirrell),
Materials Research Society, Pittsburgh, PA, 1991; b) Biomimetic
Materials Chemistry (Ed.: S. Mann), VCH, New York, 1996; c) S.
Zhang, Nat. Biotechnol. 2003, 21, 1171 – 1178.
[2] a) H. A. Lowenstam, S. Weiner, On Biomineralization, Oxford
University Press, New York, 1989; b) S. Mann, Biomineraliza-
tion: Principles and Concepts in Bioinorganic Materials Chemis-
try, Oxford University Press, New York, 2001.
[3] a) C. M. Zaremba, A. M. Belcher, M. Fritz, Y. L. Li, S. Mann,
P. K. Hansma, D. E. Morse, J. S. Speck, G. D. Stucky, Chem.
Mater. 1996, 8, 679 – 690; b) H. C. Lichtenegger, T. Schoberl,
M. H. Bartl, H. Waite, G. D. Stucky, Science 2002, 298, 389 – 392;
c) L. Addadi, S. Weiner, Angew. Chem. 1992, 104, 159 – 176;
Angew. Chem. Int. Ed. Engl. 1992, 31, 153 – 169.
[4] a) A. P. Wheeler, C. S. Sikes in Chemical Aspects of Regulation
of Mineralization (Eds.: C. S. Sikes, A. P. Wheeler), University of
South Alabama, Mobile, AL, 1988, pp. 9 – 13; b) A. P. Wheeler,
C. S. Sikes in Material Synthesis Utilizing Biological Process
(Eds.: P. C. Rieke, P. D. Calvert, M. Alper), Materials Research
Society, Pittsburgh, PA, 1989, pp. 45 – 50; c) S. Mann, D. D.
Archibald, J. M. Didymus, T. Douglas, B. R. Heywood, F. C.
Meldrum, N. J. Reeves, Science 1993, 261, 1286 – 1292; d) J. M.
Didymus, P. Oliver, S. Mann, A. L. Devries, P. V. Hauschka, P.
Westbroek, J. Chem. Soc. Faraday Trans. 1993, 89, 2891 – 2900;
e) A. Wierzbicki, C. S. Sikes, J. D. Madura, B. Drake, Calcif.
Tissue Int. 1994, 54, 133 – 141; f) C. Geffroy, A. Foissy, J. Persello,
B. Cabane, J. Colloid Interface Sci. 1999, 211, 45 – 53; g) D. K.
Keum, K.-M. Kim, K. Naka, Y. Chujo, J. Mater. Chem. 2002, 12,
2449 – 2452; h) K. Naka, Top. Curr. Chem. 2003, 228, 141 – 158.
[5] a) Y. Levi, S. Albeck, A. Brack, S. Weiner, L. Addadi, Chem.
Eur. J. 1998, 4, 389 – 396; b) S. R. Whaley, D. S. English, E. L. Hu,
P. F. Barbara, A. M. Belcher, Nature 2000, 405, 665 – 668; c) C.
Li, G. D. Botsaris, D. L. Kaplan, Cryst. Growth Des. 2002, 2, 387 –
393; d) M. Gilbert, J. W. Shaw, J. R. Long, K. Nelson, G. P.
Drobny, C. M. Giachelli, P. S. Stayton, J. Biol. Chem. 2000, 275,
16213 – 16218; e) B. Zhang, B. A. Wustman, D. E. Morse, J. S.
Evans, Biopolymers 2002, 63, 358 – 372; f) B. Zhang, G. Z. Xu,
J. S. Evans, Biopolymers 2000, 54, 464 – 475; g) B. A. Wustman,
D. E. Morse, J. S. Evans, Langmuir 2002, 18, 9901 – 9906;
h) B. A. Wustman, J. C. Weaver, D. E. Morse, J. S. Evans,
Connect. Tissue Res. 2003, 44, 10 – 15; i) G. He, T. Dahl, A.
Veis, A. George, Nat. Mater. 2003, 2, 552– 558.
[16] a) J. S. Evans, Curr. Opin. Colloid Interface Sci. 2003, 8, 48 – 54;
b) B. A. Wustman, J. C. Weaver, D. E. Morse, J. S. Evans,
Langmuir 2003, 19, 9373 – 9381.
[6] a) D. B. DeOliveria, R. A. Laursen, J. Am. Chem. Soc. 1997, 119,
10627 – 10631; b) D. J. H. Gaskin, K. Starck, E. N. Vulfson,
Biotechnol. Lett. 2000, 22, 1211 – 1216; c) P. K. Ajikumar, R.
Lakshminarayanan, S. Valiyaveettil, R. M. Kini, Biomacromol-
Angew. Chem. Int. Ed. 2005, 44, 5476 –5479ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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