Keratin protein-catalyzed nitroaldol (henry) reaction and comparison with other biopolymers

Article abstract of DOI:10.3390/molecules21091122

Here we describe a preliminary investigation on the ability of natural keratin to catalyze the nitroaldol (Henry) reaction between aldehydes and nitroalkanes. Both aromatic and heteroaromatic aldehydes bearing strong or moderate electron-withdrawing group

Full text of DOI:10.3390/molecules21091122

molecules
Article
Keratin Protein-Catalyzed Nitroaldol (Henry)
Reaction and Comparison with Other Biopolymers
Marleen Häring 1,2, Asja Pettignano 1,2, Françoise Quignard 2, Nathalie Tanchoux 2
and David Díaz Díaz 1,3,
1
*
Institute of Organic Chemistry, University of Regensburg, Universitätsstr 31, Regensburg 93053, Germany;
Marleen.Haering@chemie.uni-regensburg.de (M.H.); asja.pettignano@enscm.fr (A.P.)
Institute Charles Gerhardt Montpellier-UMR 5253 CNRS/UM/ENSCM, Matériaux Avancés pour la
Catalyse et la Santé, 8 rue de l'École Normale, Cedex 5, Montpellier 34296, France;
francoise.quignard@enscm.fr (F.Q.); nathalie.tanchoux@enscm.fr (N.T.)
2
3
IQAC-CSIC, Jordi Girona 18–26, Barcelona 08034, Spain
*
Correspondence: David.Diaz@chemie.uni-regensburg.de; Tel.: +49-941-943-4373; Fax: +49-941-943-4121
Academic Editor: Christophe Len
Received: 15 July 2016; Accepted: 2 August 2016; Published: 25 August 2016
Abstract: Here we describe a preliminary investigation on the ability of natural keratin to
catalyze the nitroaldol (Henry) reaction between aldehydes and nitroalkanes. Both aromatic and
heteroaromatic aldehydes bearing strong or moderate electron-withdrawing groups were converted
into the corresponding
β-nitroalcohol products in both DMSO and in water in the presence of
tetrabutylammonium bromide (TBAB) as a phase transfer catalyst. Negligible background reactions
(i.e., negative control experiment in the absence of keratin protein) were observed in these solvent
systems. Aromatic aldehydes bearing electron-donating groups and aliphatic aldehydes showed
poor or no conversion, respectively. In general, the reactions in water/TBAB required twice the
amount of time than in DMSO to achieve similar conversions. Moreover, comparison of the kinetics
of the keratin-mediated nitroaldol (Henry) reaction with other biopolymers revealed slower rates for
the former and the possibility of fine-tuning the kinetics by appropriate selection of the biopolymer
and solvent.
Keywords: keratin; biopolymer; C-C bond formation; nitroaldol reaction; Henry reaction
1. Introduction
Keratin is one of the most abundant non-food proteins and represents a group of insoluble,
cysteine-rich and stable filament-forming materials derived primarily from wool, hair, feathers, beaks,
hooves, horns and nails [1]. Depending on their sulfur content, keratins can be classified as soft or hard
keratins. Soft keratins, with a cysteine content up to 2% and found in outer layers of the epidermis
and hair core, have a weaker mechanical and chemical stability. Hard keratins with a cysteine content
of 10%–14% in hair, wool and skin, exhibit a higher resistance to thermal and chemical influences
due to higher content of disulfide bridges [
four segments in -helical conformation that are separated by three short linker segments in
conformation [ ]. The complete amino acid sequence of keratins obtained from different sources has
been reported in several publications [ ], making the protein a suitable candidate to be tested as a
2]. Keratins contain a central ~310 residue domain with
α
β-turn
3
4–7
catalyst. Recently, keratinous materials have attracted increasing attention as an important source of
renewable biomaterials, especially since keratin wastes have been estimated to be more than 5 million
tons per year [8], being used after degradation for different applications including scaffolds for tissue
engineering [9,10] and support matrices for catalytic metal nanoparticles [11], among others [12].
On the other hand, the nitroaldol (Henry) reaction is a highly versatile C-C bond-forming
reaction between an aldehyde or ketone and a nitroalkane to yield
β-nitroalcohols, which can be
Molecules 2016, 21, 1122; doi:10.3390/molecules21091122
www.mdpi.com/journal/molecules

Molecules 2016, 21, 1122
2 of 9
easily modified into valuable building blocks [13
–17]. As part of one of our research programs devoted
to understanding the potential catalytic role of different biopolymers [18–21], we report here for the
first time the catalytic properties of keratin protein towards the nitroaldol (Henry) reaction as well as a
critical comparison with the performance of other natural polymers in the same process.
2. Results and Discussion
The nitroaldol (Henry) reaction between 4-nitrobenzaldehyde (1a, 0.1 mmol) and nitromethane
(
2a, 1.0 mmol) was chosen as a model reaction to demonstrate the potential ability of keratin to promote
C-C bond formation under mild conditions. Preliminary experiments using 10 mg of keratin in different
solvents showed no formation of the desired -nitroalcohol 3a in toluene, THF or CH3CN (Table 1,
β
entries 1–3), and very low yields of 3a (ca. 10%) were obtained in CH2Cl2, CHCl3 or H2O (Table 1,
entries 4, 5 and 7). However, DMSO and water containing tetrabutylammonium bromide (TBAB) as
phase-transfer catalyst were found to be appropriate solvents allowing the selective formation of 3a
in modest yields at room temperature with no or very low background reaction (Table 1, entry 6 and
entry 8). Thus, formation of 3a could be unequivocally ascribed to the intrinsic catalytic activity of the
protein. These results are similar to those obtained with other biopolymers [19]. We found that the
keratin loading could be reduced to 2 mg without detriment in the product yield (data not shown).
However, 10 mg were used in most experiments for practical reasons. In addition, the reaction can be
scaled up (i.e., 1 mmol of 1a) without detriment on the product yield.
Table 1. Initial solvent screening for the nitroaldol (Henry) reaction between 1a and 2a a.
Entry
Solvent
Yield c of 3a (%)
1
2
3
4
5
6
7
8
Toluene
THF
CH3CN
CH2Cl2
CHCl3
0
0
0
11
10
66 (0) d
11
DMSO
H2O
H2O/TBAB b
57 (3) d
a
Reaction conditions: 1a (0.1 mmol), 2a (1.0 mmol), keratin (10 mg), solvent (0.5 mL), RT, orbital shaking
(150 rpm), 24 h; b TBAB (0.1 mmol). The addition of the phase transfer catalyst did not change the pH of the
solution; c 1H-NMR yields of crude product obtained in the presence of dimethyl acetamide (DMA) as internal
standard; d Background reaction without keratin.
With these preliminary results in hand, we examined the substrate scope of the reaction using
different aldehydes in combination with nitromethane or nitroethane both in DMSO (Table 2) and
H2O/TBAB (Table 3) at room temperature. Aromatic aldehydes bearing strong electron-withdrawing
substituents were easily converted in DMSO into the corresponding
yields regardless the position of the substituent (ortho-, meta-, para-) (Table 2, entries 1–6). Weaker
acceptor substituents afforded the corresponding product in moderate yields (Table 2, entries 7, 8,
β-nitroalcohols 3a–f in excellent
3
10, 12, 15). Interestingly, here the meta-position (Table 2, entry 9) of the substituent seemed to be more
favored, giving rise to 3i in good yield and twice as much than the para-substituted substrate (Table 2,
entry 8). In contrast, benzaldehyde or aromatic aldehydes bearing electron-donating substituents
yielded only trace amounts of the expected product (Table 2, entries 11, 13,14). Although we did not

Molecules 2016, 21, 1122
3 of 9
perform these experiments under longer reaction times and/or higher temperatures, it is expected that
these factors could enhance to some extent the yields as we have observed with other biopolymers [19].
Heteroaromatic aldehydes such as 2-pyridinecarboxaldehyde (Table 2, entry 16) lead to the desired
product in excellent yield, whereas furfural (Table 2, entry 18) and 1-naphthaldehyde (Table 2, entry 19)
proceeded only with low yields.
Regarding the nature of the nucleophile, the use of nitroethane instead of nitromethane provided
slightly higher yields of the desired product albeit with no or little diastereoselectivity favoring the
syn diastereomer (Table 2, entries 2, 6, 17 vs. 1, 5, 16, respectively). Slight anti-diastereoselectivy was
observed only with 3-nitrobenzaldehyde (Table 2, entry 4). Relative configurations were assigned by
comparison with 1H-NMR data reported in the literature. For instance, in the model reaction between
1a and 2b, the anti diastereomer is characterized by a doublet at 4.85 ppm (J = 8.3 Hz), whereas the syn
diastereomer shows the doublet at 5.41 ppm (J = 2.4 Hz). These results are similar to those obtained
with other biopolymers such as gelatin [19] and suggest that the acidity of the nitroalkane (nitroethane
pKa = 8.6; nitromethane pKa = 10.2) constitutes in most cases here a more important factor than steric
effects [22]. It is also worth mentioning that aliphatic aldehydes such as isobutyraldehyde were not
converted (data not shown). Moreover, despite the α-helical chiral structure of the keratin, HPLC
analysis of the reaction mixtures showed no enantioselection, as we have previously observed with
other biocatalysts in the same reaction [19–21].
Table 2. Keratin-catalyzed nitroaldol (Henry) reaction in DMSO a.
0
Yield b of 3 (%)
dr d (anti/syn)
Entry
R (1)
R (2)
3
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
(4-NO2)-C6H4
(4-NO2)-C6H4
(3-NO2)-C6H4
(3-NO2)-C6H4
(2-NO2)-C6H4
(2-NO2)-C6H4
(4-F)-C6H4
(4-Cl)-C6H4
(3-Cl)-C6H4
(4-Br)-C6H4
(4-OH)-C6H4
(4-CN)-C6H4
(4-Me)-C6H4
C6H4
H
CH3
H
CH3
H
CH3
H
H
H
H
H
H
H
H
H
H
CH3
H
H
84
96
93
98
94
96
10
25
48
25
5
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
-
1:1
-
1:0.8
-
1:1.3
-
-
-
-
-
-
-
-
-
30
7
9
(4-CHO)-C6H4
Pyrid-2-yl
Pyrid-2-yl
Furfur-2-yl
Naphtha-1-yl
65 c
90
96
3
-
1:1.7
-
-
13
3s
a Reaction conditions:
24 h; b Yield determined by 1H-NMR in the presence of DMA as internal standard; c Yield of monosubstituted
-nitroalcohol; d Diastereomeric ratio anti/syn determined by 1H-NMR analyses. “Not applicable” is represented
1 (0.1 mmol), 2 (1.0 mmol), keratin (10 mg), DMSO (0.5 mL), RT, orbital shaking (150 rpm),
β
by a dash (-).
Although the desired β-nitroalcohols 3 were also obtained in H2O/TBAB, the reactions took
two times longer than in DMSO to achieve similar conversions. In H2O/TBAB the substituent

Molecules 2016, 21, 1122
4 of 9
position in the aldehyde
1
was found to have higher influence than in DMSO. For example, strong
electron-withdrawing substituents in ortho- and para-position favored the reaction (Table 3, entries 1
and 5), whereas a significant decrease of the yield was observed with the same substituent placed in
meta-position (Table 3, entry 3). The reasons for the differences observed in H2O/TBAB vs. DMSO
may be related to the solubilization of the reactants and/or involve important variations in the
mechanistic pathways, which remain unclear and will be investigated in future research. Furthermore,
the expected products were obtained in good yields also with 4-cyanobenzaldehyde (Table 3, entry 12)
and 2-pyridine carboxaldehyde (Table 3, entry 17). Other aldehydes were converted less efficiently.
Furthermore, the use of nitroethane instead of nitromethane provided significant higher yields of the
desired product (Table 3, entries 2, 6, 13, 18 vs. 1, 5, 12, 17, respectively) albeit with almost negligible
diastereoselectivity (Table 3, entries 6, 18). It is worth to mention that the replacement of TBAB by
other ionic materials such as 1-butyl-3-methylimidazolium hexaflorophosphate (BMIM-PF6) afforded
the desired compound albeit in lower yield even at longer reaction time (Table 3, entry 1).
Table 3. Keratin-catalyzed nitroaldol (Henry) reaction in H2O/tetrabutylammonium bromide (TBAB) a.
Yield b of 3 (%)
Entry
R (1)
R’ (2)
3
dr e (anti/syn)
1
2
3
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
(4-NO2)-C6H4
(4-NO2)-C6H4
(3-NO2)-C6H4
(2-NO2)-C6H4
(2-NO2)-C6H4
(4-F)-C6H4
(4-Cl)-C6H4
(3-Cl)-C6H4
(4-Br)-C6H4
(4-OH)-C6H4
(4-CN)-C6H4
(4-CN)-C6H4
(4-Me)-C6H4
C6H4
H
CH3
H
H
CH3
H
H
H
H
H
H
CH3
H
H
H
H
CH3
H
73 (50) c
94
21
70
98
9
3a
3b
3c
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
-
n.d.
-
-
1:0.8
-
-
-
-
-
-
n.d.
-
-
-
18
34
14
1
66
89
4
8
19 d
72
85
11
0
(4-CHO)-C6H4
Pyrid-2-yl
Pyrid-2-yl
Furfur-2-yl
Naphtha-1-yl
-
1:0.9
-
-
3s
3t
H
a
Reaction conditions:
1 (0.1 mmol), 2 (1.0 mmol), TBAB (0.1 mmol), keratin (10 mg), H2O (0.5 mL), RT, orbital
shaking (150 rpm), 48 h; b Yield determined by 1H-NMR in the presence of DMA as internal standard; c Yield
of the reaction carried out in the presence of BMIM-PF6 (0.1 mmol) instead of TBAB. Reaction time = 72 h;
d
Yield of monosubstituted β
-nitroalcohol; e Diastereomeric ratio anti/syn determined by 1H-NMR analyses.
Abbreviation: n.d. = not determined. “Not applicable” is represented by a dash (-).
The heterogeneous nature of the protein catalyst allowed its recovery from the reaction mixture
and its reuse for further cycles. However, a gradual deactivation of the catalyst could be observed in
both organic and aqueous media, although the reduction of the catalytic activity was clearly more
pronounced in DMSO (Figure 1). Such behavior has also been observed with other biopolymers [19–21]
and could be associated to loss of catalyst loading (e.g., inefficient isolation by filtration) after each
cycle and/or formation of intermediate linear or cyclic aminals that could block catalytic sites of the

Molecules 2016, 21, 1122
5 of 9
protein. These potential competitive processes are somehow more critical in DMSO than in aqueous
solution; however, the reasons behind these differences remain unclear.
Figure 1. Typical recycling experiments for keratin-catalyzed nitroaldol (Henry) reaction in DMSO
and H2O/TBAB. Reaction conditions: 4-Nitrobenzaldehyde (1a, 15.1 mg, 0.1 mmol), nitromethane
(
2a, 54 µL, 1.0 mmol), solvent (0.5 mL), keratin powder (10 mg), room temperature, 24–48 h.
Yields correspond to 1H-NMR values obtained from at least two experiments. The error bars represent
the standard deviation of the measurements. For the experiments in H2O/TBAB, addition of new
TBAB after each cycle (i.e., 32.2 mg, 0.1 mmol) was necessary in order to ensure a constant concentration
during the reactions as confirmed by NMR analyses of the reaction mixtures.
At this point, we carried out kinetic analyses of the model reaction between 1a and 2a catalyzed
by keratin in both DMSO and H2O/TBAB under the described conditions. The results were compared
with the performance of other biopolymers as biocatalysts for the same reaction that we have
previously studied in our group and others (i.e., chitosan [18], gelatin [19], collagen [19], bovine
serum albumin (BSA) [19,23], silk fibroin [20] and alginate [21]) under optimized conditions. Figure 2
shows the first-order kinetics corresponding to each biopolymer within the first hours of reaction. In
general, powdered keratin displayed rate constants in the range of aerogel calcium alginate [21] and
significantly below the other biopolymers demonstrating the possibility of fine-tuning the kinetics of
the nitroaldol (Henry) reaction by appropriate selection of the biopolymer and solvent system. It is
worth mentioning that although these biopolymers are not superior to standard base catalysts such
as tetramethylethylenediamine [16], the former avoided the formation of byproducts and allowed
to work under ecofriendly and heterogeneous conditions. However, far beyond the interest of these
materials as standard catalyst, their mechanism of action to promote C-C bond formation reactions
may be more relevant within the context of biological evolution.

Molecules 2016, 21, 1122
6 of 9
Figure 2. First-order kinetics plots for the nitroaldol (Henry) reaction between 1a and 2a catalyzed
by different biopolymers in powder form. Unless otherwise indicated, reactions were made in
DMSO as described in the text and experimental section. Apparent rate constants are given in
units of h−1. Each data point represents the average of at least two independent measurements.
Cinfi = final concentration, at infinite time; Ct = concentration at given time t; C0 = initial concentration
at t = zero time.
3. Materials and Methods
3.1. Materials
Unless otherwise indicated, analytical grade solvents and reactants were commercially available
and used as received without further purification. Deionized water was used for experiments in
aqueous solutions. Aldehydes (purity by GC > 98%) were purchased from TCI Europe (TCI Europe,
Zwijndrecht, Belgium). Keratin extracted from wool (CAS 69430-36-0, Cat. Nr. AB 250197)
was purchased from ABCR. Tetrabutylammonium bromide (CAS 1643-19-2, Cat Nr. 86860) was
purchased from Fluka (Fluka Chemical Corp., Milwaukee, WI, USA). 1-Buthyl-3-methylimidazolium
hexafluorophosphate (BMIM-PF6) was purchased from TCI Europe (CAS 174501-64-5, Cat. Nr. B2320).
3.2. Methods
1H-NMR spe◦ctra were recorded on Avance 300 or Avance 400 spectrometers (Bruker, Billerica,
MA, USA) at 25 C. Chemical shifts for 1H-NMR were reported as
δ, parts per million, relative to
external standards. Yields were determined by 1H-NMR analyses of the crude product in CDCl3 using
dimethyl acetamide (0.1 mmol, 9.2 L) as internal standard after complete work-up of the reaction.
Enantiomeric excess was evaluated by chiral HPLC (Agilent Technologies, Santa Clara, CA, USA)
(column: Phenomenex (Phenomenex, Aschaffenburg, Germany) Lux Cellulose-1, 4.6 mm × 250 mm
m, eluents: n-heptane, i-propanol 70:30, flow: 0.5 mL/min). Relative configurations were assigned
µ
,
5
µ
1
by comparison with H-NMR data reported in the literature. For example, in the model reaction
between 1a and 2b, the anti diastereomer was identified by a doublet at 4.85 ppm (J = 8.3 Hz),
whereas the syn diastereomer showed the doublet at 5.41 ppm (J = 2.4 Hz). For kinetics calculations,
the 1H-NMR analyses of the reaction mixtures were performed in the presence of an internal standard
as above indicated. In general, given yield values correspond to the average of at least two independent
measurements with STDV
kinetics plots correspond to the best fit of the first-order model (e.g., (nitromethane) ≥ (aldehyde)).
±
2%–4%. Among various kinetics models, the straight lines shown in the

Molecules 2016, 21, 1122
7 of 9
3.3. General Procedure for Keratin-Catalyzed Nitroaldol (Henry) Reaction
Keratin (10 mg) was added in one portion to a mixture of 4-nitrobenzaldehyde (1a, 0.1 mmol,
15.1 mg), nitromethane (2a, 1.0 mmol, 54 L) and solvent (0.5 mL) placed into a 4-mL screw-capped vial.
µ
The resulting reaction mixture was gently shaked in an orbital shaker (150 rpm) for the appropriate
time at room temperature. After completion, water (1 mL) was added. The reaction mixture was
extracted with EtOAc (4
×
1.5 mL), dried over anhydrous sodium sulfate, filtrated and evaporated
1
under reduced pressure. Yield was determined by H-NMR of the crude product in CDCl3 using
dimethylacetamide (9.2 µL, 0.1 mmol) as internal standard. All β-nitroalcohol products are known
and the spectroscopic data obtained from NMR analysis of the reaction mixtures were in agreement
with those reported in the literature.
3.4. Typical Recycling Procedure
After reaction and extraction with EtOAc, the aqueous phase with remaining catalyst was
freeze-dried prior addition of the reaction substrates and solvent for the next run.
3.5. Kinetic Studies
Reaction conversions were unequivocally calculated by 1H-NMR analysis of the reaction mixtures
according to the integration of characteristic signals of the species in the reaction mixture in the
presence of a suitable internal standard. Each experimental point represents the average of at least
two experiments. Among various kinetics models, lines presented in the kinetic plots show best-fits of
the first-order model for each case (i.e., (NO2R)
reached 100% yield, data fitting was made according to the variation of ln((Ct
≥
(aldehyde)). Due to the fact that not all reactions
C∞ )/(C∞ C0)) with
−
−
time, where Ct is the concentration at a given time t; C∞ the final concentration (at infinite time) and
C0 the initial concentration (at t = zero time). For reaction conversions close to 100%, plots of ln(Ct/C0)
versus time provided consistent results (C∞ = 0). Under these considerations, minor differences were
observed between the exponential and linear fits. All errors reported for the rate constants k were
calculated by graphical analysis. Kinetics data for other biopolymers have been previously reported
by us and were used for comparison: Chitosan [18], gelatin [19], collagen [19], BSA [19], freeze-dried
silk fibroin [20] and calcium alginate aerogel [21]. Sample preparation and details of the reactions can
be found in the corresponding references.
4. Conclusions
In conclusion, we have demonstrated that keratin proteins are able to promote C-C bond
formation via the nitroaldol (Henry) reaction between various aldehydes and nitroalkanes. Appropriate
control experiments demonstrated the intrinsic catalytic activity of the keratin. Both aromatic and
heteroaromatic aldehydes having strong or moderate electron-withdrawing groups were converted
exclusively into the corresponding β-nitroalcohol products in both DMSO and in water in the presence
of TBAB as phase transfer catalyst. In contrast, aromatic aldehydes bearing electron-donating groups
and aliphatic aldehydes showed poor or no conversion, respectively. In general, the reactions in
water/TBAB required twice the amount of reaction time than in DMSO to achieve similar conversions.
Although the heterogeneous nature of the reaction allowed for the recovery and reuse of the keratin,
a gradual deactivation of the catalyst was observed after each cycle. Comparative kinetic studies with
other biopolymers revealed that the rate of the nitroaldol (Henry) reaction strongly depends on the
nature of the biopolymer. The effect of different forms of keratins on the ability to catalyze this and
other C-C bond forming reactions, as well as detailed mechanistic studies in both organic and aqueous
medium, including different ionic liquids, are currently under study in our laboratories and the results
will be reported in due course.

Molecules 2016, 21, 1122
8 of 9
Acknowledgments: D.D.D. thanks the Deutsche Forschungsgemeinschaft (DFG) for the Heisenberg Professorship
Award. A.P. thanks the SINCHEM program for her PhD Grant. SINCHEM is a Joint Doctorate program selected
under the Erasmus Mundus Action 1 Program—FPA 2013-0037.
Author Contributions: M.H. and A.P. performed the experiments. F.Q. and N.T. contributed to results discussion.
D.D.D. designed the experiments. M.H. and D.D.D. wrote the paper. All authors took part in data analysis
and discussion.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
4.
Rouse, J.G.; Van Dyke, M.E. A review of keratin-based biomaterials for biomedical applications. Materials
2010, 3, 999–1014. [CrossRef]
Kornillowics-Kowalska, T.; Bohacz, J. Biodegradation of keratin waste: Theory and practical aspects.
Waste Manag. 2011, 31, 1689–1701. [CrossRef] [PubMed]
Lee, C.H.; Kim, M.S.; Chung, B.M.; Leahy, D.J.; Coulombe, P.A. Structural basis for heteromeric assembly and
perinuclear organization of keratin filaments. Nat. Struct. Mol. Biol. 2012, 19, 707–715. [CrossRef] [PubMed]
Zhou, X.M.; Idler, W.W.; Steven, A.C.; Roop, D.R.; Steinert, P.M. The complete sequence of the human
intermediate filament chain keratin 10. Subdomainal divisions and model for folding of end domain
sequences. J. Biol. Chem. 1988, 263, 15584–15589. [PubMed]
0
5.
6.
7.
O Donnell, J. The complete amino acid sequence of a feather keratin from Emu (Dromaius Novae-Hollandiae).
Aust. J. Bioi. Sci. 1973, 26, 415–437. [CrossRef]
Arai, K.M.; Takahashi, R.; Yokote, Y.; Akahane, K. Amino-acid sequence of feather keratin from fowl.
Eur. J. Biochem. 1983, 132, 501–507. [CrossRef] [PubMed]
Steinert, P.M.; Rice, R.H.; Roop, D.R.; Trus, B.L.; Steven, A.C. Complete amino acid sequence of a mouse
epidermal keratin subunit and implications for the structure of intermediate filaments. Nature 1983, 302,
794–800. [CrossRef] [PubMed]
8.
9.
Tonin, C.; Aluigi, A.; Zoccola, M. Characterisation of keratin biomass from butchery and wool industry
wastes. J. Mol. Struct. 2009, 938, 35–40.
Sierpinski, P.; Garrett, J.; Ma, J.; Apel, P.; Klorig, D.; Smith, T.; Koman, L.A.; Atala, A.; Van Dyke, M. The use
of keratin biomaterials derived from human hair for the promotion of rapid regeneration of peripheral
nerves. Biomaterials 2008, 29, 118–128. [CrossRef] [PubMed]
10. Saul, J.M.; Ellenburg, M.D.; de Guzman, R.C.; van Dyke, M. Keratin hydrogels support the sustained release
of bioactive ciprofloxacin. J. Biomed. Mater. Res. A 2011, 98A, 544–553. [CrossRef] [PubMed]
11. Hengchang, M.; Zhikang, B.; Guobin, H.; Ningning, Y.; Yufei, X.; Zengming, Y.; Wei, C.; Yuan, M.
Nanoparticulate palladium catalyst stabilized by supported on feather keratin for Suzuki coupling reaction.
Chin. J. Catal. 2013, 52, 578–584.
12. Karthikeyan, R.; Balaji, S.; Sehgal, P.K. Industrial applications of keratins—A review. J. Sci. Ind. Res. 2007, 52,
710–715.
13. Luzzio, F.A. The Henry reaction: Recent examples. Tetrahedron 2001, 57, 915–945. [CrossRef]
14. Palomo, C.; Oiarbide, M.; Laso, A. Recent advances in the catalytic asymmetric nitroaldol (Henry) reaction.
Eur. J. Org. Chem. 2007, 2007, 2561–2574. [CrossRef]
15. Boruwa, J.; Gogoi, N.; Saikia, P.P.; Barua, N.C. Catalytic asymmetric Henry reaction. Tetrahedron Asymmetry
2006, 17, 3315–3326. [CrossRef]
16. Sharma, K.K.; Biradar, A.V.; Asefa, T. Substituent- and catalyst-dependent selectivity to aldol or nitrostyrene
products in a heterogeneous base-catalyzed Henry reaction. ChemCatChem 2010, 2, 61–66. [CrossRef]
17. Ballini, R.; Gabrielli, S.; Palmieri, A.; Petrini, M. Nitroalkanes as key compounds for the synthesis of amino
derivatives. Curr. Org. Chem. 2011, 15, 1482–1506. [CrossRef]
18. Kühbeck, D.; Saidulu, G.; Reddy, K.R.; Díaz, D.D. Critical assessment of the efficiency of chitosan biohydrogel
beads as recyclable and heterogenous organocatalyst for C-C bond formation. Green Chem. 2012, 14, 378–392.
[CrossRef]
19. Kühbeck, D.; Dhar, B.B.; Schön, E.-M.; Cativiela, C.; Gotor-Fernández, V.; Díaz, D.D. C-C bond formation
catalyzed by natural gelatin and collagen proteins. Beilstein J. Org. Chem. 2013, 9, 1111–1118.

Molecules 2016, 21, 1122
9 of 9
20. Kühbeck, D.; Ghosh, M.; Gupta, S.S.; Díaz, D.D. Investigation of C-C Bond formation mediated by Bombyx
Mori silk fibroin materials. ACS Sustain. Chem. Eng. 2014, 2, 1510–1517. [CrossRef]
21. Kühbeck, D.; Mayr, J.; Häring, M.; Hofmann, M.; Quignard, F.; Díaz, D.D. Evaluation of the nitroaldol
reaction in the presence of metal ion-crosslinked alginates. New J. Chem. 2015, 39, 2306–2315.
22. Akutu, K.; Kabashima, H.; Seki, T.; Hattori, H. Nitroaldol reaction over solid base catalysts. Appl. Catal. A
2003, 247, 65–74. [CrossRef]
23. Busto, E.; Gotor-Fernández, V.; Gotor, V. Protein-mediated nitroaldol addition in aqueous media.
Catalytic promiscuity or unspecific catalysis? Org. Process Res. Dev. 2011, 15, 236–240. [CrossRef]
Sample Availability: Samples of the compounds are available from the authors.
©
2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).