Green synthesis of α,β- and β,β-dipeptides under solvent-free conditions

Article abstract of DOI:10.1021/jo101159a

The reactivity of N-tert-butyloxycarbonyl-N-carboxyanhydrides derived from β-alanine, (S)-β³-homophenylglycine, and (S)-β³-carboxyhomoglycine with different α- and β-amino ester hydrochlorides was examined under ball-milling activati

Full text of DOI:10.1021/jo101159a

pubs.acs.org/joc
Green Synthesis of r,β- and β,β-Dipeptides under Solvent-Free
Conditions
ꢀ
Jose G. Hernandez and Eusebio Juaristi*
ꢀ
´
ꢀ
Departamento de Quımica, Centro de Investigacion y de Estudios Avanzados del Instituto Politecnico
ꢀ
Nacional, Apartado Postal 14-740, 07000, Meꢀxico, D.F., Meꢀxico
ejuarist@cinvestav.mx
Received June 19, 2010
The reactivity of N-tert-butyloxycarbonyl-N-carboxyanhydrides derived from β-alanine, (S )-β3-
homophenylglycine, and (S )-β3-carboxyhomoglycine with different R- and β-amino ester hydro-
chlorides was examined under ball-milling activation. In particular, good to excellent yields of several
relevant R,β- and β,β-dipeptides were obtained. An illustrative application of this methodology
consisted in the high-yield synthesis of the mammalian R,β-dipeptide N-Boc-L-carnosine-OMe.
Introduction
reactions,3d Michael additions,3c functionalization of
fullerenes,3e synthesis of nitrones,3f and others.
In the past decade, interest in Green Chemistry has ex-
panded, and it now encompasses wide areas of the chemical
enterprise. Of particular interest are developments with poten-
tial impact on industry and laboratory research as a means to
continue chemical development in a more sustainable manner.
A way to evaluate how “green” a process is consists of deter-
mining how closely it fulfills the guidelines suggested by the so-
called principles of Green Chemistry. In particular, within the
“twelve principles” of Green Chemistry proposed by Anastas
and Warner in the mid-1990s,1 both the use of safer solvents and
the design of more energy-efficient processes are key concepts.
High-speed ball-milling (HSBM) is a sustainable mechano-
chemical technique, which has commonly been used for
milling minerals into fine particles, as well as in the synthesis
and modificationof inorganic solids and other organometallic
materials.2 Furthermore, in the area of synthetic organic chem-
istry, this technique has been successfully used to promote
several solvent-free reactions. Reported applications include:
Heck-type cross-couplings,3a asymmetric aldol reactions,3b
Knoevenagel condensation reactions,3c Baylis-Hillman
Recently, Lamaty and co-workers reported a novel strategy
for the synthesis of R-peptides under solvent-free conditions by
means of ball-milling activation.4 The methodology of Lamaty
and co-workers requires no solvent, and it is based on mechano-
chemical mixing of the starting amino acids, thus fulfilling the
aforementioned principles for a green synthesis.
As part of our current interest in the chemistry of β-amino
acids and β-peptides,5 we deemed it of interest to apply
Lamaty’s strategy to the synthesis of R,β- and β,β-dipeptides.
(3) (a) Tullberg, E.; Peters, D.; Frejd, T. J. Organomet. Chem. 2004, 689,
3778–3781. (b) Rodriguez, B.; Bruckmann, A.; Bolm, C. Chem.;Eur. J.
2007, 13, 4710–4722. (c) Kaupp, G.; Naimi-Jamal, M.; Schmeyers, J.
Tetrahedron 2003, 59, 3753–3760. (d) Mack, J.; Shumba, M. Green Chem.
2007, 9, 328–330. (e) Komatsu, K. Top. Curr. Chem. 2005, 254, 185–206. (f )
Colacino, E.; Nun, P.; Colacino, F. M.; Martinez, J; Lamaty, F. Tetrahedron
2008, 64, 5569–5576.
(4) Lamaty, F.; Martinez, J.; Nun, P.; Declerck, V. Angew. Chem., Int. Ed.
2009, 48, 9318–9321.
ꢀ
(5) (a) Anaya de Parrodi, C.; Clara-Sosa, A.; Quintero, L.; Perez, L.;
~ꢀ
~
Maranon, V.; Toscano, R. A.; Avina, J. A.; Rojas-Lima, S.; Juaristi, E.
Tetrahedron: Asymmetry 2001, 12, 69–79. (b) Juaristi, E.; Avina, J. Pure
~
Appl. Chem. 2005, 77, 1235–1241. (c) Avila-Ortiz, C.; Reyes-Rangel, G.;
Juaristi, E. Tetrahedron 2005, 61, 8372–8381. (d) Zubrzak, P.; Williams, H.;
Coast, G. M.; Isaac, R.-E.; Reyes-Rangel, G.; Juaristi, E.; Zabrocki, J.;
Nachman, R. J. Biopolymers: Pept. Sci. 2007, 88, 76–82. (e) Reyes-Rangel,
(1) Anastas, P. T.; Warner, J. C. Green Chemistry: Theory and Practice;
Oxford University Press: New York, 1998.
ꢀ ꢀ
G.; Jimenez-Gonzalez, E.; Olivares-Romero, J.; Juaristi, E. Tetrahedron:
ꢀ
Asymmetry 2008, 19, 2839–2849. (f ) Bandala, Y.; Rivero, I.; Gonzalez, T.;
~
Avina, J.; Juaristi, E. J. Phys. Org. Chem. 2008, 21, 349–358.
(2) (a) Balema, V. P.; Dennis, K. W.; Pecharsky, V. K. Chem. Commun.
2000, 1665–1666. (b) Braga, D.; Giaffreda, S. L.; Grepioni, F.; Pettersen, A.;
Maini, L.; Curzi, M.; Polito, M. Dalton Trans. 2006, 1249–1263.
DOI: 10.1021/jo101159a
r
Published on Web 09/27/2010
J. Org. Chem. 2010, 75, 7107–7111 7107
2010 American Chemical Society

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Hernandez and Juaristi
JOCArticle
SCHEME 1. Synthesis of R,β- and β,β-Dipeptides under Solvent-
Free Conditions
FIGURE 1. X-ray structure of crystal 1a and 1b.
1a and 1b were determined by single-crystal X-ray diffraction
analysis (Figure 1). Particularly interesting is the axial
orientation of the phenyl group in 1b as a consequence of
the allylic A1,3 strain.7 Complete spectroscopic analysis and
X-ray diffraction crystal structure determination of 1a,b are
presented in the Supporting Information.
Following the preparation of the required β-UNCAs, we
carried out the coupling reaction between 1a and R-amino
ester hydrochlorides 2a-f, in the presence of NaHCO3 (1.5
equiv) and using the high-energy ball-milling process. The
capsule containing the mixture of solid substrates was
shaken at a frequency of 3800 rpm. The change in mass of
the capsule was quantified during the shaking process until
the recorded difference in mass was constant. This indicated
that the reaction had ended; i.e., no more CO2 was being
liberated. In general, the required reaction time was 2 h.
The reaction mixture was removed from the capsule,
dissolved in EtOAc, washed with brine, and dried to afford
the desired R,β-dipeptides as white solids, except for 3f (oil)
in good yields (Table 1, entries 1-6).
All reactions were repeated at least twice to confirm the
outcome of the procedure. Furthermore, in some assays the
reaction time was varied, observing that with less time of
milling the yield of the produced dipeptide decreased sig-
nificantly. On the other hand, longer times did not lead to
increased yields. The physical and spectroscopic properties
of the isolated products 3a-e are in agreement with previous
reports of their synthesis in liquid phase.8
This required the coupling of urethane-protected β-amino
acid N-carboxyanhydride (UNCA) derivatives with hydro-
chloride salts derived from R- and β-amino esters. In this
regard, the scientific literature records the preparation and
reactivity (in liquid phase) of UNCAs derived from β2- and
β3-amino acids.6a-c
Extension of Lamaty’s strategy to β-amino acid substrates
is not trivial since the reactivity of β-UNCAs is usually
limited relative to the R analogues. Indeed, Roumestant
and co-workers6a examined the reaction between N-Boc-R-
methyl-β-alanine N-carboxyanhydride and 1a with benzyl-
amine, benzyl alcohol, and the lithium enolate of ethyl
acetate as nucleophiles. Although these reactions proceeded
rapidly, yields were only modest (54-79%). By contrast, the
same reaction with R-amino acid N-carboxyanhydrides af-
forded better yields of the expected products.6d Further-
more, the reactivity of methyl glycinate hydrochloride with
N-Boc-R-methyl-β-alanine-NCA in CH2Cl2, in the presence
of NEt3, was evaluated, obtaining the expected dipeptide in
80% yield.6c Nevertheless, the synthesis of R,β- and β,β-
dipeptides from β-UNCAs in solvent-free conditions has not
been reported. In view of this, we proceeded to study the
coupling reaction of N-tert-butyloxycarbonyl N-carboxyan-
hydrides derived from β-alanine 1a, (S )-β3-homophenyl-
glycine 1b, and (S )-β3-carboxyhomoglycine 1c with various
R- or β-aminoester hydrochlorides 2a-h (Scheme 1).
The coupling of Boc-β-Ala-NCA 1a and Boc-(S )-β3-
hPhg-NCA 1b with β-aminoester hydrochlorides 2g-h was
also carried out under the described conditions affording the
four β,β-dipeptides 3g-j in good yield (Table 1, entries
7-10). Interesting R,β- and β,β-dipeptides 3k-l were ob-
tained by coupling the R-amino ester hydrochlorides 2a and
2g with the novel β-UNCA 1c (Table 1, entries 11 and 12).
Thus, all R,β- and β,β-dipeptides were obtained with yields
that ranged in from 79% to 96% (Table 1). The best yields
were obtained for the coupling reaction between 1a-c and
the β-amino ester hydrochlorides 2g-h.
The absence of racemization or epimerization in the prepara-
tion of R,β-dipeptides using ball-milling activation was demon-
strated by Lamaty and co-workers.4 In this regard, we com-
pared the specific optical rotations of dipeptides 3d and 3m with
those reported in the literature. The recorded values showed
Results and Discussion
To do this, we synthesized β-UNCAs 1a-c according to
the method described by Roumestant,6a starting with com-
mercial β-alanine, the (S )-β3-homophenylglycine [(S )-β3-
hPhg], which was prepared according to the methodology
reported by Juaristi and co-workers,5f or natural L-aspartic
acid acting as β-amino acid, (S )-β3-carboxyhomoglycine
[(S )-β3-chg]. The structure and solid-state conformation of
(7) Seebach, D.; Lamatsch, B.; Beck, A. K.; Dobler, M.; Egli, M.; Fitzi, R.;
ꢀ
Meetzke, T.; Mourino, A.; Pfammatter, E.; Plattner, D. A.; Schickli, C.;
Gautschi, M.; Herradon, B.; Hidber, P. C.; Irwin, J. J.; Locher, R.; Maestro, M.;
~
(6) (a) Roumestant, M. L.; Fehrentz, J. A.; Huck, J.; McKiernan, M.;
Viallefont, P.; Martinez, J. J. Org. Chem. 2001, 66, 6541–6544. (b) Huck, J.;
Receveur, J. M.; Roumestant, M. L.; Martinez, J. Synlett 2001, 9, 1467–1469.
(c) Huck, J.; Roumestant, M. L.; Martinez, J. J. Pept. Res. 2003, 62, 233–237.
(d) Paris, M.; Fehrentz, J. A.; Heitz, A.; Loffet, A.; Martinez, J. Tetrahedron
Lett. 1996, 37, 8489–8492.
Schweiser, W. B.; Seifer, P.; Stucky, G.; Petter, W.; Escalante, J.; Juaristi, E.;
Quintana, D.; Miravitlles, C.; Molins, E. Helv. Chim. Acta 1992, 75, 913–934.
(8) (a) Guha, S.; Drew, M. G. B.; Banerjee, A. Chem. Mater. 2008, 20,
2282–2290. (b) Guha, S.; Drew, M. G. B.; Banerjee, A. Org. Lett. 2007, 9,
1347–1350.
7108 J. Org. Chem. Vol. 75, No. 21, 2010

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TABLE 1. R,β- and β,β-Dipeptides Prepared under Solvent-Free Conditions in This Work
entry
N-Boc-β-NCA
amino methyl ester hydrochloride
dipeptide N-Boc-aa-aa-OMe
yield (%)
1
2
3
4
5
6
7
8
9
β-ala
β-ala
β-ala
β-ala
β-ala
β-ala
β-ala
β-ala
Ala
Val
Leu
Phe
Ile
Gly
β-ala
(S)-β3-hPhg
β-ala
(S)-β3-hPhg
Ala
β-ala-Ala (3a)
β-ala-Val (3b)
β-ala-Leu (3c)
β-ala-Phe (3d)
β-ala-Ile (3e)
β-ala-Gly (3f)
β-ala-β-ala (3g)
88
82
87
83
80
88
96
91
93
94
79
91
β-ala-(S)-β3-hPhg (3h)
(S)-β3-hPhg-β-ala (3i)
(S)-β3-hPhg-(S)-β3-hPhg (3j)
(S)-β3-chg(CO2CH3)-Ala (3k)
(S)-β3-chg(CO2CH3)-β-ala (3l)
(S)-β3-hPhg
(S)-β3-hPhg
(S)-β3-chg-(CO2CH3)
(S)-β3-chg-(CO2CH3)
10
11
12
β-ala
SCHEME 2. Proposed Mechanism for the Synthesis of Dipep-
tides under Solvent-Free Conditions
FIGURE 2. Natural L-carnosine.
SCHEME 3. Synthesis of Dipeptide Boc-β-Ala-L-His-OMe
under Solvent-Free Conditions
total agreement with those reported, indicating that no racemi-
zation or epimerization took place.9
L-Carnosine has proven to be an antiglycating agent,
antioxidant-based on in vitro tests, and exhibits hydroxyl-
radical scavenger properties. As a result, L-carnosine is pre-
sently an active topic of study in chemistry.12
Given the importance of L-carnosine, and to demonstrate
the general applicability of the present synthetic procedure
for the preparation of R,β-dipeptides under solvent-free
conditions, we deemed it of interest to synthesize dipeptide
Boc-β-Ala-L-His-OMe 3m as an approach to the architecture
of L-carnosine (Scheme 3).
An efficient procedure for synthesizing peptides consists of
the ring-opening polymerization of R-amino acid-N-carboxy-
anhydrides (NCAs). The mechanism of the ring opening in this
process has been well estudied.10 On the basis of those studies,
we propose the mechanism depicted in Scheme 2 for the
formation of the dipeptide Boc-β-Ala-L-Ala-OMe 3a.
The importance of the incorporation of β-amino acids into
peptides is recognized not only in view of their increased
stability against proteolytic degradation in vitro and in vivo
but also because β-peptides offer the potential for the design
of drugs based on β-peptidic architecture.11
The aminoester hydrochloride 2i was prepared from com-
mercial L-histidine. Ball-milling of 1a, 2i, and NaHCO3 for 2 h
yielded the protected dipeptide 3m in excellent yield. It is worth
mentioning that the general procedure implemented in this
work usually involves the use of 1.5 equiv of NaHCO3 to ensure
the total liberation of the amino ester. However, when the
reaction of 1a and 2i was carried out by applying this stoichio-
metric relationship, the yield of 3m was low and the reac-
tion generated many byproducts. Thus, we used 2 equiv of
NaHCO3, finding that the coupling was clean and the yield was
91%. Under these conditions, the 2 equiv of NaHCO3 liberated
Although not many examples of R,β-dipeptides exist in
nature, L-carnosine, a mammalian dipeptide composed of
the amino acids β-alanine and L-histidine and found in
muscle and brain tissues, is one salient example (Figure 2).
(9) (a) Giordano, C.; Lucente, G.; Nalli, M.; Pagani-Zecchini, G.; Paglialunga-
Paradisi, M.; Varani, K.; Spisani, S. Il Farmaco. 2003, 58, 1121–1130. (b) Wiles, C.;
Watts, P. Synthesis 2007, 17, 2608–2610.
(10) (a) Idelson, M.; Blout, E. R. J. Am. Chem. Soc. 1958, 80, 2387–2393.
(b) Luximon-Bhaw, A.; Jhurry, D.; Belleney, J.; Goury, V. Macromolecules
2003, 36, 977–982. (c) Pickel, L. D.; Politakos, N.; Avgeropoulos, A.;
Messman, J. M. Macromolecules 2009, 42, 7781–7788.
(11) Leading references: (a) Seebach, D.; Beck, A. K.; Bierbaum, D. J.
Chem. Biodiversity 2004, 1, 1111–1239. (b) Horne, W. S.; Gellman, S. H. Acc.
Chem. Res. 2009, 41, 1399–1408. (c) Goodman, C. M.; Choi, S.; Shandler, S.;
DeGrado, W. F. Nat. Chem. Biol. 2007, 3, 252–262.
both amino functions of the amino ester. All spectra (1H, 13
C
NMR, COSY, HETCOR and HMBS spectroscopy) of 3m are
presented in the Supporting Information.
(12) (a) Hipkiss, R. A.; Brownson, C.; Carrier, J. M. Mech. Ageing Dev.
2001, 122 (13), 1431–1445. (b) Guiotto, A.; Calderan, A; Ruzza, P.; Borin, G.
Curr. Med. Chem. 2005, 12, 2293–2315. (c) Bauer, K. Neurochem. Res. 2005,
30, 1339–1345. (d) Chan, W. K. M.; Decker, E. A.; Lee, J. B.; Butterfield,
D. A. J. Agric. Food Chem. 1994, 42 (7), 1407–1410. (e) Heck, T.; Makam,
V. S.; Lutz, J.; Blank, M. L.; Schmid, A.; Seebach, D.; Kohler, H-P. E.;
Geueke, B. Adv. Synth. Catal. 2010, 352 (2), 407–415. (f ) Heyland, J.;
Antweiler, N.; Lutz, J.; Heck, T.; Geueke, B.; Kohler, H-P. E.; Blank,
L. M.; Schmid, A. Microb. Biotechnol. 2010, 3 (1), 74–83.
Conclusion
In summary, a “green” protocol for the high-yield synth-
esis of R,β-dipeptides and several novel β,β-dipeptides under
solvent-free conditions, starting with urethane-protected
β-amino acid N-carboxyanhydrides and R- or β-aminoesters,
is reported. As an illustrative application of this strategy,
J. Org. Chem. Vol. 75, No. 21, 2010 7109

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JOCArticle
N-Boc-L-Carnosine-OMe, a protected derivative of natural
R,β-dipeptide L-carnosine, was prepared in high yield.
Research toward the synthesis of other unnatural dipep-
tides and evaluation of its potential organocatalytic activity
is already in progress in our laboratory.
Boc-β-Ala-L-Val-OMe (3b). (25 mg, 82%) as a gummy solid.
[R]25D = þ12.0 (c 1.6, CHCl3). (FT-IR/ATR cm-1) νmax 3321,
1
2968, 2933, 1736, 1693, 1655. H NMR (500 MHz, CDCl3) δ
6.15 (1H, br, Val-NH), 5.18 (1H, br-s, β-Ala-NH), 4.53 (1H, m,
(CHCO2)), 3.73 (3H, s, OCH3), 3.40 (2H, m, CH2NBoc), 2.45
(2H, m, CH2CdO), 2.15 (1H, m, (CH)), 1.41 (9H, s, t-Bu), 0.92
(3H, d, CH3, J = 6.8 Hz), 0.89 (3H, d, CH3, J = 6.8 Hz) ppm.
13C NMR (125 MHz, CDCl3) δ 176.6, 171.8, 156.2, 79.3, 57.2,
52.2, 36.8, 36.2, 31.1, 28.4, 19.0, 17.9 ppm. HR-ESI-TOF
Calculated for C14H27N2O5 [M þ H]þ: 303.1914. Found:
303.1921 (2.1 ppm error).
Experimental Section
General Procedure for the Synthesis of the UNCAs Derived
from β-Amino Acids. To a cooled (-20 °C) solution of DMF
(1.18 mL, 15.3 mmol) in freshly distilled acetonitrile (7.5 mL)
was added dropwise oxalyl chloride (1.34 mL, 15.3 mmol). After
30 min of stirring at -20 °C, a cooled solution (-20 °C) of N-
Bis-Boc-β-alanine (740 mg, 2.56 mmol) and pyridine (207 μL,
2.56 mmol) in acetonitrile (4.5 mL) was added dropwise. The
solution was stirred at -20 °C for 2 h and after that allowed to
warm to room temperature (over 1 h) and stirred for a further
4 h. The reaction was quenched by pouring onto ice and the
product extracted into AcOEt (3 ꢀ 30 mL). The combined
organics were washed with NaHCO3 solution (pH 8) and brine
(50 mL). The organic phases were dried (MgSO4) and evapo-
rated under vacuum to give a yellowish oil; recrystallization
from ethyl acetate gave the respective UNCA 1a-c.
Boc-β-Ala-L-Leu-OMe (3c). (28 mg, 88%) as a white solid.
D
mp = 50-52 °C. [R]25 = þ5.5 (c 0.1, CHCl3). (FT-IR/ATR
1
cm-1) νmax 3347, 2960, 2932, 1752, 1691, 1648. H NMR (500
MHz, CDCl3) δ 6.35 (1H, br, Leu-NH), 5.24 (1H, br-s, β-Ala-
NH), 4.56 (1H, m, (CHCO2)), 3.68 (3H, s, OCH3), 3.36 (2H, m,
CH2NBoc), 2.41 (2H, m, CH2CdO), 1.65-1.55 (2H, m, CH2i-
Bu), 1.54-1.44 (1H, m, CHi-Bu), 1.38 (9H, s, t-Bu), 0.89 (6H, d,
CH3i-Bu, J = 6.0 Hz) ppm. 13C NMR (125 MHz, CDCl3) δ
173.5, 171.5, 156.1, 77.4, 52.3, 50.7, 41.3, 36.7, 36.1, 28.4, 24.9,
22.8, 21.9 ppm. HR-ESI-TOF Calculated for C15H29N2O5 [M þ
H]þ: 317.2070. Found: 317.2075 (1.2 ppm error).
Boc-β-Ala-L-Phe-OMe (3d). (29 mg, 83%) as a white solid.
mp = 89-91 °C. [R]25D = þ52.0 (c 1.0, CHCl3). (FT-IR/ATR
1
3-tert-Butyloxycarbonyl-4,5-dihydro-1,3-oxazine-2,6-dione
(N-Boc-β-alanine-N-carboxyanhydride), 1a. 320 mg (58%) of
white crystals, mp = 122-124 °C. (FT-IR/ATR cm-1) νmax
cm-1) νmax 3320, 2976, 2931, 1735, 1691, 1654. H NMR (500
MHz, CDCl3) δ 7.31-7.20 (3H, m, H-Ar), 7.11-7.08 (2H, d,
Horto-Ar, J = 7.0 Hz), 6.04 (1H, br, Phe-NH), 5.10 (1H, br-s, β-
Ala-NH), 4.88 (1H, m, CH), 3.74 (3H, s, OCH3), 3.36 (2H, m,
CH2NBoc), 3.16, (1H, dd, CH2Ph, J = 6.3, 13.8 Hz), 3.07 (1H, dd,
CH2Ph, J = 5.7, 13.8 Hz), 2.38 (2H, t, CH2CdO, J = 5.5 Hz), 1.44
(9H, s, t-Bu) ppm. 13C NMR (125 MHz, CDCl3) δ 172.0, 171.2,
156.1, 135.8, 129.3, 128.7, 127.3, 79.4, 53.2, 52.5, 38.0, 36.6, 36.2,
28.5 ppm. HR-ESI-TOF Calculated for C18H27N2O5 [M þ H]þ:
351.1914. Found: 351.1923 (2.4 ppm error).
1
2985, 2939, 1824, 1790, 1739. H NMR (500 MHz, CDCl3) δ
3.90 (2H, t, CH2N, J = 6.6 Hz), 2.87 (2H, m, CH2CO), 1.54 (9H,
s, t-Bu) ppm. 13C NMR (125 MHz, CDCl3) δ 163.7, 150.5, 144.5,
85.7, 39.2, 29.5, 27.7 ppm.
(S )-3-tert-Butyloxycarbonyl-4-phenyl-4,5-dihydro-1,3-ox-
azine-2,6-dione ((S )-N-Boc-β3-hPhg-N- carboxyanhydride) 1b.
208 mg (60%) of white crystals. 1H NMR (500 MHz, CDCl3) δ
7.41-7.33 (3H, m, ArH), 7.22-7.20 (2H, m, ArH), 5.62 (1H, m,
CH-Ph), 3.23 (1H, dd, J = 16.4, J = 6.4 Hz, CH2), 3.13 (1H,
dd, J = 16.4, J = 6.4 Hz, CH2), 1.48 (9H, s, t-Bu) ppm. 13C
NMR (125 MHz, CDCl3) δ 162.7, 150.3, 144.8, 137.1, 129.6,
129.1, 125.2, 86.0, 53.6, 36.8, 27.9 ppm.
Boc-β-Ala-L-Ile-OMe (3e). (25.2 mg, 80%) as a gummy solid.
[R]25 = þ5.0 (c 0.7, CHCl3). (FT-IR/ATR cm-1) νmax 3345,
D
2958, 1755, 1689, 1651. 1H NMR (500 MHz, CDCl3) δ 6.05 (1H,
br, Ile-NH), 5.17 (1H, br-s, β-Ala-NH), 4.58 (1H, m, NCH), 3.74
(3H, s, OCH3), 3.42-3.39 (2H, m, CH2NBoc), 2.47-2.44, (2H,
m, CH2CdO), 1.90-1.80 (1H, m, CH), 1.47-1.37 (1H, m,
CH2CH3), 1.22-1.12, (1H, m, CH2CH3), 1.43 (9H, s, t-Bu),
0.86-0.96 (6H, m, 2ꢀCH3) ppm. 13C NMR (125 MHz, CDCl3)
δ 172.5, 171.5, 156.2, 79.4, 56.5, 52.3, 38.0, 36.8, 36.4, 28.5, 25.4,
15.6, 11.7 ppm. HR-ESI-TOF Calculated for C15H29N2O5 [M þ
H]þ: 317.2070. Found: 317.2076 (1.6 ppm error).
(S)-3-tert-Butyloxycarbonyl-4-carboxymethyl-4,5-dihydro-1,3-
oxazine-2,6-dione ((S )-N-Boc-β3-chg-(CO2CH3)-N-carboxyanhy-
dride), 1c. 250 mg (62%) as a gummy solid. 1H NMR (500 MHz,
CDCl3) δ 4.55 (1H, dd, J = 8.0, J = 2.6 Hz, CH-CO2CH3),
3.66 (3H, s, -OCH3), 3.20 (1H, dd, J = 19.4, J = 8.0 Hz, CH2),
2.92 (1H, dd, J = 19.4, J = 2.6 Hz, CH2), 1.53 (9H, s, t-Bu) ppm.
13C NMR (125 MHz, CDCl3) δ 169.2, 148.1, 147.1, 118.3, 85.9,
58.5, 53.0, 35.8, 28.0 ppm. (FT-IR/ATR cm-1) νmax 2982, 1833,
1805, 1738. HR-ESI-TOF: Calculated for C9H10N7O5Na
[MþNa]þ: 296.0737. Found: 296.0741 (1.0 ppm error).
General Procedure for the Coupling of UNCAs 1 with Ami-
noester Hydrochloride 2. A mixture of UNCA 1 (0.1 mmol),
aminoester hydrochloride 2 (0.1 mmol), and NaHCO3 (0.15 mmol)
was vigorously milled for 2 h at 3800 rpm in a digital Mixer/
Amalgamator used with a reactor made of Nylamid (cylinder, 25
mm long and with a diameter 10 mm) containing one stainless steel
ball with a 5 mm diameter. The solid residue (or the melted mixture)
was dissolved in AcOEt and washed with brine. The organic phase
was dried over MgSO4 and concentrated to yield the dipeptide.
Boc-β-Ala-L-Ala-OMe (3a). (24 mg, 88%) as a white solid. mp
Boc-β-Ala-Gly-OMe (3f). (23 mg, 88%) as a liquid. (FT-IR/
1
ATR cm-1) νmax 3316, 2957, 1743, 1688, 1656. H NMR (500
MHz, CDCl3) δ 6.49 (1H, br, gly-NH), 5.25 (1H, br-s, β-Ala-
NH), 4.01 (2H, d, CH2CO2, J = 5.37 Hz), 3.73 (3H, s, OCH3),
3.38 (2H, q, CH2NBoc, J = 6.0 Hz), 2.45 (2H, t, CH2CdO, J =
5.8 Hz), 1.39 (9H, s, t-Bu) ppm. 13C NMR (125 MHz, CDCl3) δ
172.0, 170.5, 156.2, 79.4, 52.5, 41.2, 36.6, 36.1, 28.5 ppm. HR-
ESI-TOF Calculated for C11H21N2O5 [M þ H]þ: 261.1444.
Found: 261.1447 (0.7 ppm error).
Boc-β-Ala-β-Ala-OMe (3g). (28.5 mg, 96%) as a white solid.
mp = 77-78 °C. (FT-IR/ATR cm-1) νmax 3357, 3288, 2972,
1732, 1683, 1643. 1H NMR (500 MHz, CDCl3) δ 6.19 (1H, br, β-
Ala-NH), 5.16 (1H, br-s, β-Ala-BocNH), 3.69 (3H, s, OCH3),
3.51 (2H, q, CH2NBoc, J = 6.1 Hz), 3.38 (2H, q, CH2CdO, J =
6.1 Hz), 2.53 (2H, t, CH2NHCO, J = 6.1 Hz), 2.36 (2H, t,
CH2CO2, J = 5.9 Hz), 1.41 (9H, s, t-Bu) ppm. 13C NMR (125
MHz, CDCl3) δ 173.0, 171.6, 156.2, 79.3, 51.9, 36.7, 36.2, 34.9,
33.9, 28.4 ppm. HR-ESI-TOF Calculated for C12H23N2O5 [M þ
H]þ: 275.1601. Found: 275.1609 (2.7 ppm error).
= 78-80 °C. [R]25 = þ55.0 (c 0.1, CHCl3). (FT-IR/ATR
D
1
cm-1) νmax 3356, 3321, 2983, 1745, 1682, 1648. H NMR (500
MHz, CDCl3) δ 6.30 (1H, br, Ala-NH), 5.19 (1H, br-s, β-Ala-
NH), 4.55 (1H, q, CH, J = 7.2 Hz), 3.73 (3H, s, OCH3), 3.38
(2H, m, CH2NBoc), 2.41 (2H, t, CH2CdO, J = 5.8 Hz), 1.40
(9H, s, t-Bu), 1.38 (3H, d, CH3, J = 7.1 Hz) ppm. 13C NMR (125
MHz, CDCl3) δ 173.5, 171.2, 156.1, 79.3, 52.9, 48.1, 36.6, 36.1,
28.4, 18.3 ppm. HR-ESI-TOF Calculated for C12H23N2O5 [M þ
H]þ: 275.1601. Found: 275.1609 (2.7 ppm error).
Boc-β-Ala-(S )-β3-hPhg-OMe (3h). (32 mg, 91%) as a white
solid. mp = 82-84 °C. [R]25 = -47.0 (c 1.5, CHCl3). (FT-IR/
D
ATR cm-1) νmax 3369, 3336, 2976, 2927, 1736, 1678, 1645. 1HNMR
(500 MHz, CDCl3) δ 7.33-7.24 (5H, m, H-Ar), 6.80 (1H, br,
7110 J. Org. Chem. Vol. 75, No. 21, 2010

ꢀ
Hernandez and Juaristi
JOCArticle
β3-hPhg-NH), 5.42 (1H, m, CH), 5.22 (1H, br-s, β-Ala-NH), 3.62
(3H, s, OCH3), 3.38 (2H, dd, CH2NBoc, J = 5.8, J = 6.3 Hz), 2.88
(1H, dd, CH2CO2, J = 6.3, J = 15.9 Hz), 2.84 (1H, dd, CH2CO2,
J=6.3, J =15.9Hz), 2.43(2H, t, CH2CdO, J=6.3Hz), 1.43 (9H,
s, t-Bu) ppm. 13C NMR (125 MHz, CDCl3) δ 171.5, 170.8, 156.1,
140.4, 128.8, 127.7, 126.2, 79.3, 51.9, 49.7, 40.0, 36.7, 36.3, 28.4 ppm.
HR-ESI-TOF: Calculated for C18H27N2O5 [M þ H]þ: 351.1914.
Found: 351.1920 (1.5 ppm error).
CDCl3) δ 6.21 (1H, br-s, β-Ala-NH), 5.72 (1H, d, J = 8.1 Hz,
β3-chg-NH), 4.50 (1H, m, CH), 3.74 (3H, s, OCH3), 3.70 (3H, s,
OCH3), 3.51-3.48, (2H, m, CH2N), 2.86 (1H, dd, J = 15.9, J = 4.4
Hz, CH2CH), 2.68 (1H, dd, J = 15.9, J = 4.4 Hz, CH2CH), 2.53
(2H,t,CH2CO2,J=5.7Hz),1.43(9H,s,t-Bu) ppm. 13CNMR(125
MHz, CDCl3) δ172.9, 171.9, 169.8, 155.6, 79.9, 52.7, 51.8, 50.3, 37.9,
34.7, 33.6, 28.2 ppm. HR-ESI-TOF Calculated for C14H25N2O7 -
[M þ H]þ: 333.1656. Found: 333.1663 (2.0 ppm error).
Boc-(S )-β3-hPhg-β-Ala-OMe (3i). (16.3 mg, 93%, (0.05
mmol of 1b and 2g were used)) as a white solid. mp =
Boc-β-Ala-L-His-OMe (Boc-Carnosine-OMe) (3m). (62 mg,
91%, (0.2 mmol of 1a and 2i were used)) as a white solid. mp =
140-142 °C. [R]25D = -2.2 (c 0.3, CHCl3). (FT-IR/ATR cm-1
)
78-82 °C. [R]25 = 6.0 (c 0.3, CH3OH). (FT-IR/ATR cm-1
)
D
1
1
νmax 3346, 2961, 2925, 1734, 1680, 1645. H NMR (500 MHz,
CDCl3) δ 7.32-7.21 (5H, m, H-Ar), 6.18 (1H, br-s, β3-hPhg-
NH), 6.01 (1H, br-s, β-Ala-NH), 5.01 (1H, br-s, CH), 3.64 (3H,
s, OCH3), 3.64-3.28 (2H, m, CH2N), 2.71-2.55 (2H, m,
CH2CdO), 2.41-2.27 (2H, m, CH2CO2), 1.41 (9H, s, t-Bu)
ppm. 13C NMR (125 MHz, CDCl3) δ 173.0, 170.5, 155.4, 141.6,
128.7, 127.4, 126.2, 79.6, 51.9, 51.9, 43.0, 34.7, 34.7, 33.6, 28.5
ppm. HR-ESI-TOF Calculated for C18H27N2O5 [M þ H]þ:
351.1914. Found: 351.1915 (0.1 ppm error).
νmax 3283, 2977, 2931, 1738, 1686, 1655, 1523. H NMR (500
MHz, CDCl3) δ 7.55 (1H, s, H-2), 7.30 (1H, br-s, His-NHCO),
6.79 (1H, s, H-4), 5.61 (1H, t, β-Ala-NH, J = 5.7 Hz), 4.79 (1H,
m, CH), 3.70 (3H, 2, OCH3), 3.40 (2H, m, CH2NBoc), 3.10 (2H,
d, CH2, J = 5.1 Hz), 2.43 (2H, t, CH2CdO, J = 5.8 Hz), 1.43
(9H, s, t-Bu) ppm. 13C NMR (125 MHz, CDCl3) δ 171.9
(CO2CH3), 171.7 (CONH), 156.2 (CO-Carbamate), 135.3
(C-2), 134.0 (C-5), 116.0 (C-4), 79.4 (C-t-Bu), 52.7 (CH), 52.4
(OCH3), 36.9 (CH2NBoc), 36.4 (CH2CdO), 28.9 (CH2), 28.4
(CH3-t-Bu) ppm. COSY correlation [δH/δH]: 7.65/6.45 [2-H/4-
H], 7.30/4.78 [His-NH/CH], 6.45/7.65 [4-H/2-H], 5.60/3.40 [ β-
Ala-NH/CH2NBoc], 4.78/7.30/3.10 [CH/His-NH/CH2], 3.40/
5.60/2.43 [CH2NBoc/β-Ala-NH/CH2CdO], 3.10/4.78 [CH2/
CH], 2.43/3.40 [CH2CdO/CH2NBoc]. HETCOR correlation
[δH/δC]: 7.55/135.3 [H-2/C-2], 6.79/116.0 [H-4/C-4], 4.79/52.7
[CH/CH], 3.70/52.4 [OCH3/OCH3], 3.40/36.9 [CH2NBoc/
CH2NBoc], 3.10/28.9 [CH2/CH2], 2.43/36.4 [CH2CdO/
CH2CdO], 1.43/28.4 [CH3-t-Bu/CH3-t-Bu]. HMBC correlation
[δH/δC]: 7.55/116.0/134.0 [H-2/C-4/C-5], 6.79/135.3 [H-4/C-2],
4.79/28.9/134.0/171.9 [CH/CH2/C-5/CO2CH3], 3.70/171.9 [O
CH3/CO2CH3], 3.40/36.4/156.2/171.7 [CH2NBoc/CH2CdO/
CO-Carbamate/CONH], 3.10/52.7/116.0/134.0/171.9 [CH2/
CH/C-4/C-5/CO2CH3], 2.43/36.9/171.7 [CH2CdO/CH2NBoc/
CONH], 1.43/28.4/79.4[CH3-t-Bu/CH3-t-Bu/C-t-Bu]. HR-ESI-
TOF Calculated for C15H25N4O5 [M þ H]þ: 341.1823. Found:
341.1819 (1.0 ppm error).
Boc-(S )-β3-hPhg-(S )-β3-hPhg-OMe (3j). (40.0 mg, 94%) as a
white solid. mp = 154-156 °C. [R]25D = -77.1 (c 0.8, CHCl3).
(FT-IR/ATR cm-1) νmax 3308, 2929, 1736, 1683, 1650. 1H
NMR (500 MHz, CDCl3) δ 7.34-7.21 (10H, m, H-Ar), 6.50
(1H, d, NHCO, J = 8.5 Hz), 6.19 (1H, br-s, NHBoc), 5.27 (1H,
m, CHNHCO), 5.06 (1H, br-s, CHNHBoc), 3.54 (3H, s, OCH3),
2.80-2.50 (4H, m, 2xCH2), 1.40 (9H, s, t-Bu) ppm. 13C NMR
(125 MHz, CDCl3) δ 171.6, 169.7, 155.4, 141.7, 140.1, 128.8,
128.8, 127.8, 127.5, 126.3, 126.2, 79.7, 52.0, 51.9, 49.4, 43.0, 39.4,
28.5 ppm. HR-ESI-TOF Calculated for C24H31N2O5 [M þ H]þ:
427.2227. Found: 427.2226 (0.3 ppm error).
Boc-(S )-β3-chg-(CO2CH3)-L-Ala-OMe (3k). (28.8 mg, 79%)
as a gummy solid. [R]25D = 16.5 (c 0.37, CHCl3). (FT-IR/ATR
1
cm-1) νmax 3311, 2920, 1755, 1741, 1691, 1650. H NMR (500
MHz, CDCl3) δ 6.15 (1H, d, J = 6.6 Hz, Ala-NH), 5.70 (1H, br-
d, J = 6.8 Hz, β3-chg-NH), 4.58-4.52 (1H, m, CH-CO2CH3),
4.58-4.52 (1H, m, CH-CH3), 3.75 (6H, s, 2ꢀ-OCH3), 2.92
(1H, dd, J = 16.1, J = 4.5 Hz, CH2), 2.72 (1H, dd, J = 15.6, J =
4.4 Hz, CH2), 1.44 (9H, s, t-Bu), 1.39 (3H, d, J = 7.2 Hz, CH3)
ppm. 13C NMR (125 MHz, CDCl3) δ 173.1, 171.8, 169.4, 155.6,
80.0, 52.6, 52.5, 50.3, 48.1, 37.9, 28.3, 18.4 ppm. HR-ESI-TOF
Calculated for C14H25N2O7 [M þ H]þ: 333.1656. Found:
333.1656 (0.1 ppm error).
Acknowledgment. The authors are indebted to Conacyt,
ꢀ
Mexico, for financial support via grant 60366-Q. We thank
ꢀ
Conacyt, Mexico, for a doctoral fellowship (N° 22211).
Supporting Information Available: Copies of NMR spectra
of all compounds, X-ray crystal structure, and crystallographic
information files (CIFs) of 1a,b. This material is available free of
charge via the Internet at http://pubs.acs.org.
Boc-(S )-β3-chg-(CO2CH3)-β-Ala-OMe (3l). (33.2 mg, 91%)
as a gummy solid. [R]25D = þ2.3 (c 0.95, CHCl3). (FT-IR/ATR
1
cm-1) νmax 3357, 2929, 1734, 1711, 1653. H NMR (500 MHz,
J. Org. Chem. Vol. 75, No. 21, 2010 7111