Imines that react with phenols in water over a wide pH range

Article abstract of DOI:10.1021/jo8017389

(Chemical Equation Presented) Cyclic imine derivatives that react with phenols, including tyrosine residues of peptides, have been developed. Reactions of the imines with phenols proceeded in water over a wide pH range (pH 2-10) at room temperature to 37°C and afforded Mannich products without the need of additional catalysts.

Full text of DOI:10.1021/jo8017389

ligations.1e Here we report the development of phenol-reacting
molecules, cyclic imine derivatives, that can be used for
Mannich-type reactions of phenols in water over a wide pH
range at room temperature to 37 °C without the need of
additional catalysts.
Imines that React with Phenols in Water over a
Wide pH Range
Maki Minakawa, Hai-Ming Guo, and Fujie Tanaka*
Department of Molecular Biology, The Scripps Research
Institute, 10550 North Torrey Pines Road,
La Jolla, California 92037
The phenol functionality is contained in tyrosine, a natural
building block of proteins. Tyrosine residues are observed less
often on the surface of folded proteins than lysine or carboxylic
acid-containing residues, which are commonly used as labeling
sites; thus accessible tyrosine residues are attractive covalent
labeling sites.2,3 For proteins that do not have tyrosine on the
surface, the phenol functionality is orthogonal; these proteins
should not be affected by reactions with phenols. Therefore,
phenol-reacting molecules in aqueous media can be used for
both protein labeling at tyrosine and coupling reactions with
phenol-bearing molecules in the presence of biomolecules that
are not affected by the phenol-reacting molecules.
ftanaka@scripps.edu
ReceiVed August 5, 2008
Covalent bond-forming reactions at tyrosine residues have
been performed with imines prepared from formaldehyde and
aniline derivatives in situ;2 however, the optimal pH range for
this reaction is 5.5-6.5 and the reaction does not proceed above
pH 8.2a Other tyrosine or phenol labeling reactions include Pd-
catalyzed-O-alkylation, azonium coupling, and nitration with
tetranitromethane.3 Although these were pioneering bond-
forming reactions with phenols in water, molecules that react
with phenols over a wide pH range without additional catalysts
or multistep conversions after the first reaction step on phenols
are needed. When reactions occur over a wide pH range, reaction
conditions, including pH, can be chosen depending on proteins
present and on functionalities within molecules used as reactants.
Cyclic imine derivatives that react with phenols, including
tyrosine residues of peptides, have been developed. Reactions
of the imines with phenols proceeded in water over a wide
pH range (pH 2-10) at room temperature to 37 °C and
afforded Mannich products without the need of additional
catalysts.
We reasoned that imines should become useful phenol-
reacting molecules that can be used across a wide pH range
when reactivity and stability of the imines are tuned. First,
molecules should show some stability in water or aqueous
buffers; hydrolysis of imines is often a problem for reactions
of imines in water-containing solvents.4 We reasoned that cyclic
imines should be more resistant to hydrolysis than acyclic imines
or should easily reform the imines after hydrolysis (if ap-
plicable). Second, the cyclic imines should provide enough
electrophilicity for reactions with phenols. Nucleophilic addition
reactions to imines readily occur when the imines are protonated.
Protonation of the imines by the phenolic hydroxyl group of
phenols or by water of buffers may be key for the bond-forming
reaction of the imines with phenols in some pH ranges.2,5 In
addition, imines conjugated with electron-withdrawing groups
should be more reactive than imines without electron-withdraw-
ing groups. However, highly reactive imines may react non-
specifically and handling of these imine molecules may be
difficult. Third, the cyclic imines should not isomerize to
enamines, which may cause dimerization and lower the con-
centration of the imines.6
Bond-forming chemical transformations that can be performed
under biomolecule-compatible conditions (such as in water or
aqueous buffers at 4-40 °C) are necessary for covalent
attachment of small molecules to biomolecules and for coupling
of small molecule modules in the presence of biomolecules.1-3
Known transformations include reactions of thiol groups with
maleimides, reactions of primary amino groups with succinim-
idyl esters,1a azide-alkyne cycloadditions,1b-d and Staudinger
(1) (a) Antos, J. M.; Francis, M. B. Curr. Opin. Chem. Biol. 2006, 10, 253.
(b) Lewis, W. G,; Green, L. G.; Grynszpan, F.; Radic, Z.; Carlier, P. R.; Taylor,
P.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2002, 41, 1053. (c)
Wang, Q.; Chan, T. R.; Hilgraf, R.; Fokin, V. V.; Sharpless, K. B.; Finn, M. G.
J. Am. Chem. Soc. 2003, 125, 3192. (d) Agard, N. J.; Prescher, J. A.; Bertozzi,
C. R. J. Am. Chem. Soc. 2004, 126, 15046. (e) Saxon, E.; Bertozzi, C. R. Science
2000, 287, 2007. (f) Dirksen, A.; Hackeng, T. M.; Dawson, P. E. Angew. Chem.,
Int. Ed. 2006, 45, 7581. (g) Crich, D.; Banerjee, A. J. Am. Chem. Soc. 2007,
129, 10064. (h) Tanaka, K.; Masuyama, T.; Hasegawa, K.; Tahara, T.; Mizuma,
H.; Wada, Y.; Watanabe, Y.; Fukase, K. Angew. Chem., Int. Ed. 2008, 47, 102.
(i) Prescher, J. A.; Bertozzi, C. R. Nat. Chem. Biol 2005, 1–13, See also. (j)
Maruoka, K.; Tayama, E.; Ooi, T. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 5824.
(k) Hadley, E. B.; Witek, A. M.; Freire, F.; Peoples, A. J.; Gellman, S. H. Angew.
Chem., Int. Ed. 2007, 46, 7056.
(2) (a) Joshi, N. S.; Whitaker, L. R.; Francis, M. B. A. J. Am. Chem. Soc.
2004, 126, 15942. (b) McFarland, J. M.; Joshi, N. S.; Francis, M. B. J. Am.
Chem. Soc. 2008, 130, 7639.
(3) (a) Tilley, S. D.; Francis, M. B. J. Am. Chem. Soc. 2006, 128, 1080. (b)
Hooker, J. M.; Kovacs, E. W.; Francis, M. B. J. Am. Chem. Soc. 2004, 126,
3718. (c) Kenner, R. A.; Neurath, H. Biochemistry 1971, 10, 551. (d) Hass,
J. A.; Frederick, M. A.; Fox, B. G. Protein Expr. Purif. 2000, 20, 274. (e) Koshi,
Y.; Nakata, E.; Miyagawa, M; Tsukiji, S.; Ogawa, T.; Hamachi, I. J. Am. Chem.
Soc. 2008, 130, 245. (f) Guo, H.-M.; Minakawa, M.; Tanaka, F. J. Org. Chem.
2008, 73, 3964.
(4) (a) Kobayashi, S.; Hamada, T.; Manabe, K. J. Am. Chem. Soc. 2002,
124, 5640. (b) Hamada, T.; Manabe, K.; Kobayashi, S. Chem.-Eur. J. 2006,
12, 1205.
(5) Miyano, S.; Abe, N. Tetrahedron Lett. 1970, 11, 1909.
(6) (a) Gravel, E.; Poupon, E.; Hocquemiller, R. Org. Lett. 2005, 7, 2497.
(b) Wanner, M.; Koomen, G.-J. J. Org. Chem. 1995, 60, 5634.
10.1021/jo8017389 CCC: $40.75
Published on Web 10/10/2008
2008 American Chemical Society
J. Org. Chem. 2008, 73, 8669–8672 8669

TABLE 1. Reaction of Imine 1 with p-Cresola
SCHEME 1. Synthesis of Imines 1 and 2
entry
aqueous buffer (aqueous component)
200 mM NaH2PO4, pH 5
yieldb (%)
55
3a:3bc
Based on the considerations described above, we designed
and synthesized cyclic imines 1 and 2 as phenol-reacting
molecules. Cyclic imine 1 was readily obtained by mixing (()-
trans-1,2-diaminocyclohexane with ethyl glyoxylate (polymer
form) in 2-PrOH at room temperature (23 °C) (Scheme 1). The
reaction in other solvents, including toluene, ethyl acetate, and
water, also afforded 1. Cyclic imine 2 was synthesized using
cis-1,2-diaminocyclohexane by the same procedure used for the
synthesis of 1, although formation of 2 was slower than
formation of 1 (Scheme 1). Note that 1 and 2 were selectively
obtained; formation of imidazolidine derivatives7 was not
observed.
1
2
3
1.3:1
1.2:1
2.0:1
3.3:1
2.5:1
2.5:1
2.8:1
3.9:1
2.3:1
3.2:1
2.8:1
0.8:1
2.7:1
2.1:1
2.1:1
200 mM NaH2PO4-Na2HPO4, pH 6.0 36 (20)
200 mM NaH2PO4-Na2HPO4, pH 7.0 44
200 mM NaH2PO4-Na2HPO4, pH 8.0 46
200 mM NaH2PO4-Na2HPO4, pH 8.0 50
200 mM NaH2PO4-Na2HPO4, pH 8.0 52
200 mM NaH2PO4-Na2HPO4, pH 8.0 53
4
5d
6e
7f
8
9
200 mM Na2HPO4, pH 9
44
41
41
50
24
36
49
29
PBS, pH 7.4g
10
11
12
13
14
15
16h
17h
18h
19h
20j
100 mM Tris, pH 8.0g
25 mM HEPES, pH 7.5g
10% CH3COOH, pH 2
H2O
1
200 mM Na2CO3, pH 10
200 mM Et3N, pH 10
Imines 1 and 2 were soluble in water at 100 mM. H NMR
analysis of a solution of 1 in D2O indicated that the imine moiety
of 1 was partly hydrated.8 When a solution of 1 or 2 in water
was kept at 23 °C for several days and was extracted with
CH2Cl2, the imine was recovered quantitatively without sign
of decomposition. When acetic acid (1 equiv) was added to a
solution of imine 1 in D2O, the area of the signal of the imine
proton of 1 decreased. However, when this mixture was
neutralized by a solution of NaOH in D2O, the imine proton
signal area increased. After extraction of this mixture with
CH2Cl2, imine 1 was quantitatively recovered. Imine 1 was also
quantitatively recovered after treatment with aqueous NaH2PO4
(pH 5) or Na2HPO4 (pH 9). These results indicate that there is
an equilibrium between the imine and the hydrated form in water
or aqueous solutions and that re-formation of the imine from
the hydrated form occurs readily without hydrolysis to amine
and aldehyde.
Reactions of the imines were evaluated using a simple phenol
derivative, p-cresol (Tables 1 and 2). First, reactions of imine
1 (95 mM) with p-cresol (25 mM) were performed in aqueous
media (pH 5-9) at 23 °C (Table 1, entries 1-4 and 8).
Formation of product 3 was analyzed after 24 h. Product 3 was
formed without requirement of additional catalysts. Imine 1 and
p-cresol were soluble in the aqueous media used for the reactions
and the reaction mixtures were initially homogeneous, clear
solutions. Neither micelles9 nor biphasic mixtures10 were
observed. However, product 3 often precipitated with p-cresol;
this may have caused the moderate yield of the product. With
lower concentrations of imine 1 and p-cresol, product 3 was
also obtained (entries 5-7). Reaction between 1 and p-cresol
100 mM NaH2PO4, pH 5
100 mM NaH2PO4-Na2HPO4, pH 6
100 mM NaH2PO4-Na2HPO4, pH 7
100 mM Na2HPO4, pH 9
100 mM NaH2PO4-Na2HPO4, pH 7
23 (60) [143] 1.3:1
31 (48) [127] 1.5:1
39 (27) [93]
[76]
2.0:1
ndi
32 (45)
2.0:1
a Conditions: Imine 1 (95 mM) and p-cresol (25 mM) in 5% DMSO/
indicated aqueous buffer (or aqueous component) at 23 °C for 24 h.
b Sum of 3a and 3b; yield of 3c is indicated in parenthesis; determined
by 1H NMR of the extracted mixture. For entries 1-15, yield of 3c was
<10% except where noted. Percentage of modified ortho positions of
the phenolic OH of p-cresol is indicated in bracket; complete conversion
to 3c ) 200% in bracket. c Determined by 1H NMR of the extracted
mixture. d Imine 1 (95 mM) and p-cresol (5 mM). e Imine 1 (25 mM)
and p-cresol (500 µM). f Imine 1 (5 mM) and p-cresol (500 µM). g PBS
) phosphate buffered saline. Tris ) tris(hydroxymethyl)aminomethane.
HEPES ) 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. h Imine 1
(50 mM) and p-cresol (10 mM) in indicated buffer prepared with D2O
(no DMSO was added) at 37 °C for 24 h. Yield was determined by 1H
NMR of the reaction mixture. i Not determined. j Imine 1 (50 mM) and
p-cresol (10 mM) in the absence of DMSO at 37 °C.
in water and in other aqueous solutions also afforded product 3
(entries 9-15). Whereas imine 1 reacted with p-cresol at pH
range from 2 to 10 as shown in Table 1, no formation of 3 was
observed in 5% DMSO/aqueous 10% citric acid or in 5%
DMSO/aqueous 1% trifluoroacetic acid (TFA) at 23 °C after 1
day; the imine and p-cresol were recovered. In organic solvents
tested, including toluene, CH2Cl2, DMSO, DMF, and 2-PrOH,
imine 1 did not react with p-cresol or formation of 3 was very
slow (<5% after 1 day) at 23 °C. For the formation of 3 in
organic solvents at room temperature, addition of an acid, such
as TFA, was required.11
(7) Halland, N.; Hazell, R. G.; Jorgensen, K. A. J. Org. Chem. 2002, 67,
8331.
(8) (a) Struve, C.; Christophersen, C. Heterocycles 2003, 60, 1907. (b)
Calveras, J.; Bujons, J.; Parella, T.; Crehuet, R.; Espelt, L.; Joglar, J.; Clapes, P.
Tetrahedron 2006, 62, 2648.
(9) Itoh, J.; Fuchibe, K.; Akiyama, T. Synthesis 2006, 4075.
(10) Narayan, S.; Muldoon, J.; Finn, M. G.; Fokin, V. V.; Kolb, H. C.;
Sharpless, B. Angew. Chem., Int. Ed. 2005, 44, 3275.
Next, the reaction was performed at 37 °C in buffer prepared
with D2O and the product formation was directly analyzed by
1H NMR (entries 16-19). All compounds including products
were soluble under these conditions. Combined yields of 3a,
3b, and 3c after 24 h were 66-83% for the reactions at pH
5-7 (entries 16-18). Yields after extraction (entry 20) were
8670 J. Org. Chem. Vol. 73, No. 21, 2008

TABLE 2. Reaction of Imine 2 with p-Cresola
entry
aqueous buffer
yieldb (%)
FIGURE 1. Products 5-10 obtained by reactions with 1.
1
2
3
4
5
6
7c
200 mM NaH2PO4, pH 5
37
35
32
26
22
25
[68]
TABLE 3. Addition Reactions of Tyrosine-Containing Peptides to
Imines 1 and 11a
200 mM NaH2PO4-Na2HPO4, pH 6.0
200 mM NaH2PO4-Na2HPO4, pH 7.0
200 mM NaH2PO4-Na2HPO4, pH 8.0
200 mM Na2HPO4, pH 9
100 mM Tris, pH 8.0
100 mM NaH2PO4-Na2HPO4, pH 7
entry
imine
peptide
reaction pH
yieldb (%)
1
2
3
4
1
1
1
1
11
11
11
GlyTyr
GlyTyr
GlyTyr
AlaTyrAla
GlyTyr
pH 5
pH 7
pH 8.5
pH 7
pH 7
pH 7
pH 6
25c (54)d [133]e
34c (38)d[110]e
[91]e
[109]e
a Imine 2 (95 mM) and p-cresol (25 mM) at 23 °C for 24 h.
b Determined by 1H NMR of the extracted mixture. For entries 1-6,
yield of diaddition product was <5%. Percentage of modified ortho
positions of the phenolic OH of p-cresol is indicated in bracket;
complete conversion to the diaddition product ) 200% in bracket.
c Imine 2 (50 mM) and p-cresol (10 mM) in the buffer prepared with
D2O (no DMSO was added) at 37 °C for 24 h.
5
[91]e
6
AlaTyrAla
AlaTyrAla
[74]e [99]e, f
[58]e [96]e, f
7g
a Conditions: Imine (50 mM) and peptide (10 mM) in 100 mM
NaH2PO4-Na2HPO4 (pH 7), 100 mM NaH2PO4 (for pH 5), or 100 mM
Na2HPO4 (for pH 8.5) prepared with D2O at 37 °C for 24 h.
b Determined by 1H NMR of the reaction mixture. c Monoaddition
product. d Diaddition product (diaddition at tyrosine). e Percentage of
modified ortho positions of the tyrosine phenolic OH; complete
similar to those obtained upon direct analysis of the reaction
mixture (entry 18).12
conversion to the diaddition product
)
200%. f Data after 48 h.
As reactions proceeded, yield of diaddition product 3c
increased before complete consumption of p-cresol. Bivalent
molecules often have the advantage of binding because of the
avidity.13 Use of derivatives of imine 1 in modification of
proteins and peptides at a single tyrosine residue under certain
conditions may furnish a concise route to useful bivalent
molecules.
g Reaction in D2O.
5, or 6). Mannich products 5, 6, and 7 were obtained in 41%,
41%, and 14% yield, respectively, after 24 h at pH 7.5;
formation of 8 was negligible (2% yield) after 24 h under the
same conditions.
Imine 2 also reacted with p-cresol and afforded corresponding
addition product 4 (Table 2). Although the reaction of 2 was
slower than the reaction of 1 under the same conditions,
importantly, cyclic imines 1 and 2 both reacted with the phenol
derivative in water over a wide pH range.
Reactions of imine 1 with potential nucleophiles were also
examined. Imine 1 did not react with imidazole, 2-PrOH,
n-butylamine, 3-methylindole, or p-anisidine in 5% DMSO/200
mM sodium phosphate (pH 7.5) at 23 °C. Tris, which possesses
amine and alcohol functionalities, did not react with imine 1,
as the reaction affording 3 occurred in Tris buffer. These results
suggest that tyrosine on a protein selectively reacts with imine
1, while histidine, lysine, N-terminal amino group, serine,
threonine, and tryptophan do not. An exception was thiol:
reaction of 1-dodecanethoil with imine 1 afforded addition
product 9. When both p-cresol and 1-dodecanethoil were present
(each at 25 mM) in a reaction mixture with 1 (95 mM) at pH
8.0, both 3 and 9 were formed after 1 day. However, 9 was
decomposed in aqueous 10% citric acid or in aqueous 1% TFA
at 23 °C and the thiol was recovered (imine 1 was not re-formed
by this decomposition and was not recovered). Note that product
3 was stable under these conditions as described above. As an
orthogonal functionality to natural proteins, indole (3-nonsub-
stituted indole) reacted with imine 1 and formed 10 (87% yield
after 24 h) in 5% DMSO/200 mM sodium phosphate (pH 7.5)
at 23 °C.
The C-C bond formed by the reaction was stable even under
the conditions where the Mannich-type reaction of p-cresol with
the imine did not occur: When 3a was kept in 5% DMSO/200
mM sodium phosphate (pH 8.0), 5% DMSO/200 mM NaH2PO4
(pH 5), 5% DMSO/10% citric acid, or 5% DMSO/1% TFA at
23 °C for 1 day, neither decomposition of 3a (as would be
expected from retro-Mannich or retro-Michael14 reaction) nor
isomerization of 3a to 3b was detected. The same experiments
with 3b (3b:3a >99:1) showed that isomerization of 3b to 3a
occurred slowly (3b:3a ) 97:3 at pH 5 and 94:6 at pH 8.0 after
1 day), but no decomposition of 3b was detected. Addition
product 4 was also stable under the conditions used for the
experiments of 3a and 3b.
To provide further information about the reactivity of the
imines, reactions of imine 1 with a series of phenol derivatives
affording 5-8 (Figure 1) in aqueous buffers were examined.
Reactions of phenols with electron-withdrawing groups (forma-
tion of 7 and 8) were slower than that without (formation of 3,
Imine 1 covalently reacted with tyrosine-containing peptides
as shown in Table 3. 1H NMR analyses of the reaction mixtures
showed that bond-formation occurred at tyrosine. Formation of
the Mannich products was also confirmed by high-resolution
mass analysis. A 24-mer peptide, YKLLKELLAKLKWLL-
RKLLGPTSL,15 also covalently reacted with 1 in sodium
phosphate (pH 7.0), in Tris (pH 8.0), and in HEPES (pH 7.5)
as confirmed by mass analysis. Imine 1 also reacted with
(11) (a) Tohma, S.; Rikimaru, K.; Endo, A.; Shimamoto, K.; Kan, T.;
Fukuyama, T. Synthesis 2004, 909. (b) Chen, Y.-J.; Lei, F.; Liu, L.; Wang, D.
Tetrahedron 2003, 59, 7609. (c) Rondot, C.; Zhu, J. Org. Lett. 2005, 7, 1641.
(12) Reactions in buffer prepared with D2O were slightly slower than the
reactions in buffer prepared with H2O under identical conditions; this may be
due to the solvent isotope effect.
(13) Tanaka, F.; Kinoshita, K.; Tanimura, R.; Fujii, I. J. Am. Chem. Soc.
1996, 118, 2332.
(14) Wang, P.; Liu, R.; Wu, X.; Ma, H.; Cao, X.; Zhou, P.; Zhang, J.; Weng,
X.; Zhang, X.-L.; Qi, J.; Zho, X.; Weng, L. J. Am. Chem. Soc. 2003, 125, 1116.
(15) Tanaka, F.; Fuller, R.; Barbas, C. F., III Biochemistry 2005, 44, 7583.
J. Org. Chem. Vol. 73, No. 21, 2008 8671

SCHEME 2. Synthesis of Imine 11
CDCl3): δ 1.37-1.45 (m, 1H), 1.46-1.55 (m, 3H), 1.62-1.68 (m,
1H), 1.70-1.77 (m, 2H), 1.86-1.92 (m, 1H), 3.70 (m, 1H),
3.85-3.87 (m, 1H), 5.79 (brs, 1H), 7.75 (t, J ) 2.3 Hz, 1H). 13C
NMR (125 MHz, CDCl3): δ 20.7, 22.8, 28.2, 28.8, 48.5, 57.7, 155.9,
157.8. HRMS: calcd for C8H13N2O (MH+) 153.1022, found
153.1026.
Reactions of Imine 1 with p-Cresol (Table 1, entries 1-4
and 8-15). A 100 mM solution of imine 1 in 200 mM sodium
phosphate buffer (or indicated aqueous component) was prepared
immediately before starting reaction. To the 100 mM solution of 1
(950 µL), a 500 mM solution of p-cresol in DMSO (50 µL) was
added at rt and the mixture was stirred for 24 h. After pH of the
reaction mixture was adjusted to pH 7-8 with 200 mM Na2HPO4
or with 200 mM NaH2PO4, the mixture was extracted with CH2Cl2.
Organic layers were combined, washed with brine, dried over
Na2SO4, and concentrated under reduced pressure. The crude
mixture was dissolved in CDCl3 and was analyzed by 1H NMR to
determine the yield.
lysozyme, which has tyrosine on the folded surface,2,16 as
confirmed by mass analysis. Myoglobin, which lacks surface-
accessible tyrosine,2,3a,16 did not react with imine 1 under the
conditions used for the reactions of lysozyme with 1.
To use the imines for protein labeling and module coupling,
the imines must be conjugated to labeling molecules (such as
fluorescent molecule or biotin) or modules. The imine conjugates
may be prepared using an ester or amide linkage on the
cyclohexane ring of the imines. To demonstrate the feasibility
of reactions of such imine conjugates, imine derivative 11 was
synthesized as a mixture of the regio- and diastereo-isomers
(Scheme 2)17 and reactions of 11 with tyrosine-containing
peptides were evaluated (Table 3). Formation of addition
products was observed, suggesting that derivatives of imines 1
and 2 bearing other moieties at the cyclohexane ring may also
react with phenols.
We have developed cyclic imine derivatives that can co-
valently modify phenols, including tyrosine residues, in water
across a wide pH range at room temperature to 37 °C without
the need of additional catalysts. These imines were relatively
stable but efficiently reacted with phenols. Derivatives of these
imines bearing fluorescent or other moieties should be useful
for modification of peptide and proteins at tyrosine and for
module coupling reactions in aqueous media.
Compound 3a. 1H NMR (500 MHz, CDCl3-CD3OD): δ
1.30-1.47 (m, 4H), 1.81-1.86 (m, 2H), 1.89-1.92 (m, 2H), 2.25
(s, 3H, CH3), 2.65 (ddd, J ) 3.5 Hz, 9.5 Hz, 11.0 Hz, 1H),
3.31-3.26 (m, 1H), 4.59 (s, 1H), 6.71 (d, J ) 8.0 Hz, 1H), 6.98
(dd, J ) 2.0 Hz, 8.0 Hz, 1H), 7.04 (d, J ) 2.0 Hz, 1H). 13C NMR
(125 MHz, CDCl3-CD3OD): δ 19.8, 23.4, 24.2, 30.0, 30.7, 57.2,
57.7, 62.3, 115.9, 123.1, 128.5, 129.6, 131.0, 152.7, 170.9. HRMS:
calcd for C15H21N2O2 (MH+) 261.1597, found 261.1606.
Compound 3b. 1H NMR (500 MHz, CDCl3): δ 1.18-1.36 (m,
4H), 1.75 (m 1H), 1.79-1.86 (m, 2H), 1.96 (dd, J ) 2.8, 12.6 Hz,
1H), 2.26 (s, 3H), 2.64 (ddd, J ) 3.5 Hz, 9.5 Hz, 11.5 Hz, 1H),
3.08 (dt, J ) 4.0 Hz, 10.5 Hz, 1H), 4.96 (s, 1H), 6.76 (brs, 1H),
6.80 (d, J ) 8.0 Hz, 1H), 7.00 (dd, J ) 2.0 Hz, 8.0 Hz, 1H), 7.06
(s, 1H) 10.8 (br, 1H). 13C NMR (125 MHz, CDCl3): δ 20.8, 23.7,
24.8, 30.6, 31.6, 53.4, 58.9, 59.4, 117.5, 122.1, 127.3, 128.7, 129.7,
154.7, 170.6. HRMS: calcd for C15H21N2O2 (MH+) 261.1597, found
261.1595.
Compound 3c. 1H NMR (500 MHz, CDCl3): δ 1.24-1.39 (m,
8H), 1.76-1.85 (m, 8H), 2.25 (s, 3H), 2.55-2.64 (m, 2H),
3.15-3.21 (m, 2H), 4.54 (s, 1H), 4.75 (s, 1H), 6.23 (s, 1H), 6.33
(s, 1H), 7.02 (s, 1H), 7.07 (s, 1H). 13C NMR (125 MHz, CDCl3):
δ 20.5, 20.6, 23.8, 24.6, 24.7, 30.50, 30.53, 31.3, 31.4, 57.8, 58.2,
58.4, 61.4, 62.9, 124.7, 125.2, 128.3, 128.7, 130.5, 131.6, 151.7,
170.6, 170.7. HRMS: calcd for C23H33N4O3 (MH+) 413.2547, found
413.2544.
Experimental Section
Synthesis of Imine 1. To a solution of trans-1,2-diaminocyclo-
hexane (228 mg, 2.00 mmol) in 2-PrOH (3.0 mL), a solution of
ethyl glyoxylate polymer form (45-50% in toluene, 0.21 mL, 1.00
mmol) in 2-PrOH (3.0 mL) was added dropwise over 15 min at
room temperature (23 °C). The mixture was stirred for 2 h,
concentrated under reduced pressure, and purified by flash column
chromatography (AcOEt) to afford 1 (151 mg, 99%) as a colorless
solid. 1H NMR (500 MHz, CDCl3): δ 1.28-1.37 (m, 1H),
1.39-1.49 (m, 3H), 1.80-1.84 (m, 1H), 1.88-1.92 (m, 1H),
1.94-1.98 (m, 1H), 2.35-2.41 (m, 1H), 3.06-3.12 (m, 1H), 3.17
(dt, J ) 3.9 Hz, 11.6 Hz, 1H), 7.07 (brs, 1H), 7.72 (t, J ) 2.8 Hz,
1H). 13C NMR (125 MHz, CDCl3): δ 23.6, 25.2, 31.0, 31.5, 54.1,
63.0, 156.3, 158.0. HRMS: calcd for C8H13N2O (MH+) 153.1022,
found 153.1019.
Synthesis of Imine 2. To a solution of cis-1,2-diaminocyclo-
hexane (228 mg, 2.00 mmol) in 2-PrOH (3.0 mL), a solution of
ethyl glyoxylate polymer form (45-50% in toluene, 0.21 mL, 1.00
mmol) in 2-PrOH (3.0 mL) was added dropwise over 15 min at rt.
The mixture was stirred for 17 h, concentrated under reduced
pressure, and purified by flash column chromatography (AcOEt)
to afford 2 (114 mg, 75%) as a colorless solid. 1H NMR (500 MHz,
1
Compound 4. H NMR (500 MHz, CDCl3): δ 1.33-1.39 (m,
1H), 1.40-1.46 (m, 1H), 1.55-1.59 (m, 3H), 1.63-1.69 (m, 1H),
1.72-1.78 (m, 1H), 1.88-1.95 (m, 1H), 2.25 (s, 3H), 3.26 (dt, J
) 7.5 Hz, 3.5 Hz, 1H), 3.64 (brd, J ) 3.5, 1H), 4.81 (s, 1H), 6.57
(brs, 1H), 6.77 (d, J ) 8.0 Hz, 1H), 6.99 (dd, J ) 2.0 Hz, 8.0 Hz,
1H), 7.06 (d, J ) 2.0 Hz, 1H). 13C NMR (125 MHz, CDCl3): δ
20.6, 21.3, 22.0, 27.4, 30.5, 48.6, 51.8, 58.1, 117.3, 122.1, 128.7,
129.0, 129.8, 154.5, 169.9. HRMS: calcd for C15H21N2O2S8 (MH+)
261.1597, found 261.1594.
Acknowledgment. This study was supported by NIH R21
GM078447.
Supporting Information Available: Additional experimental
procedures, synthesis and characterization of compounds, and
NMR spectra. This material is available free of charge via the
Internet at http://pubs.acs.org.
(16) Ly, T.; Julian, R. R. J. Am. Chem. Soc. 2008, 130, 351.
(17) Azides are potentially hazardous. For safety in handling of azides, see:
Brase, S.; Gil, C.; Knepper, K.; Zimmermann, V. Angew. Chem., Int. Ed. 2005,
44, 5188.
JO8017389
8672 J. Org. Chem. Vol. 73, No. 21, 2008