Effective methods for the biotinylation of azamacrocycles

Article abstract of DOI:10.1021/jo071175v

(Chemical Equation Presented) The biotin-(strept)avidin interaction remains a gold standard of model biological recognition events. The biotinylation of azamacrocycles permits the investigation of signal transduction between this recognition event and the

Full text of DOI:10.1021/jo071175v

Effective Methods for the Biotinylation of Azamacrocycles
Sara J. Krivickas,† Emiliano Tamanini,† Matthew H. Todd,*,‡ and Michael Watkinson*,†
School of Biological and Chemical Sciences, Queen Mary, UniVersity of London, Mile End Road, London,
E1 4NS, United Kingdom, and School of Chemistry, UniVersity of Sydney,
New South Wales 2006, Australia
m.todd@chem.usyd.edu.au; m.watkinson@qmul.ac.uk
ReceiVed June 7, 2007
The biotin-(strept)avidin interaction remains a gold standard of model biological recognition events. The
biotinylation of azamacrocycles permits the investigation of signal transduction between this recognition
event and the metal center of an azamacrocycle complex, of wide potential interest in biosensing. There
are no generally applicable procedures in the literature for such functionalizations. We report here a
comprehensive investigation into the attachment of biotin to TACN, cyclen, and cyclam. Effective methods
have been found for each ring. The efficacy of the functionalization is critically dependent on the nature
of the azamacrocycle.
Introduction
fact that a number of biotinylated metal complexes has recently
been shown to bind to (strept)avidin,7 indicating that the
presence of a cationic center does not affect the binding unduly.
Exploration of the metal’s ability to report such binding
requires methodology for the synthesis of biotinylated azamac-
rocycles. Archibald et al.8 have recently reported an elegant
strategy for the biotinylation of a cross-bridged cyclam deriva-
tive, although binding of the biotinylated copper complex to
(strept)avidin was not demonstrated. Moreover, as we were keen
to establish the possibility of using generic synthetic methods
Azamacrocylic ligands continue to receive considerable
attention, and their metal complexes find widespread utility as
a consequence of their robust and well-defined coordination
chemistry.1 This broad ligand class is perhaps most commonly
associated with applications in the general area of radiophar-
maceuticals2 but has also been applied in many other areas such
as catalysis,3 biomimetics,4 and sensor devices.5
We recently became interested in the regioselective N-
functionalization of azamacrocycles with biologically relevant
recognition motifs with the aim of utilizing the metal coordinated
to the macrocycle to sense a subsequent binding event. We
elected to employ the biotin-(strept)avidin system as our model
because of its well-established high binding affinity6 and the
(5) See, for example: (a) Mizukami, S.; Nagano, T.; Urano, Y.; Odani,
A.; Kikuchi, K. J. Am. Chem. Soc. 2002, 124, 3920-3925. (b) Aoki, S.;
Kagata, D.; Shiro, M.; Takeda, K.; Kimura, E. J. Am. Chem. Soc. 2004,
126, 13377-13390. (c) Fabbrizzi, F.; Marcotte, N.; Stomeo, F.; Taglietti,
A. Angew. Chem., Int. Ed. 2002, 41, 3811-3814. (d) Aoki, S.; Sakurama,
K.; Matsuo, N.; Yamada, Y.; Takasawa, R.; Tanuma, S.-I.; Shiro, M.;
Takeda, K.; Kimura, K. Chem.sEur. J. 2006, 12, 9066-9080. (e) Cable,
M. L.; Kirkby, J. P.; Sorasaenee, K.; Gray, H. B.; Ponce, A. J. Am. Chem.
Soc. 2007, 129, 1474-1475.
† University of London.
‡ University of Sydney.
(1) Lindoy, L. F. The Chemistry of Macrocyclic Ligand Complexes;
Cambridge University Press: Cambridge, 1989.
(6) Tamanini, E.; Rigby, S. E. J.; Motevalli, M.; Todd, M. H.; Watkinson,
M., submitted.
(2) Merbach, A. E.; Toth, E. The Chemistry of Contrast Agents in Medical
Magnetic Resonance Imaging; Wiley: New York, 2001.
(3) Chae, P. S.; Kim, M.-S.; Jeung, C.-S.; Lee, S. D.; Park, H.; Lee, S.;
Suh, J. J. Am. Chem. Soc. 2005, 127, 2396-2397.
(4) See, for example: (a) Yin, G.; Danby, A. M.; Kitko, D.; Carter, J.
D.; Scheper, W. M.; Busch, D. H. Inorg. Chem. 2007, 46, 2173-2180. (b)
Yin, G.; Danby, A. M.; Kitko, D.; Carter, J. D.; Scheper, W. M.; Busch,
D. H. J. Am. Chem. Soc. 2007, 129, 1512-1513. (c) Belousoff, M. J.;
Duriska, M. B.; Graham, B.; Batten, S. R.; Moubaraki, B.; Murray, K. S.;
Spiccia, L. Inorg. Chem. 2006, 45, 3746-3755.
(7) For recent examples, see: (a) Skander, M.; Humbert, N.; Collot, J.;
Gradinaru, J.; Klein, G.; Loosli, A.; Sauser, J.; Zocchi, A.; Gilardoni, F.;
Ward, T. J. Am. Chem. Soc. 2004, 126, 14411-14418. (b) Eckermann, A.
L.; Barker, K. D.; Hartings, M. R.; Ratner, M. A.; Meade, T. J. J. Am.
Chem. Soc. 2005, 127, 11880-11881. (c) Lo, K. K-W.; Hui, W-K. Inorg.
Chem. 2005, 44, 1992-2002. (d) Letonder, C.; Pordea, A.; Humbert, N.;
Ivanova, A.; Mazurek, S.; Novic, M.; Ward, T. R. J. Am. Chem. Soc. 2006,
128, 8320-8328.
(8) Lewis, E. A.; Boylehave, R. W.; Archibald, S. J. Chem. Commun.
2004, 2212-2213.
10.1021/jo071175v CCC: $37.00 © 2007 American Chemical Society
Published on Web 09/29/2007
8280
J. Org. Chem. 2007, 72, 8280-8289

EffectiVe Methods for Biotinylation of Azamacrocycles
SCHEME 1a
SCHEME 2a
a Reagents and conditions: (a) ClCH2CN, K2CO3, MeCN, ∆, 24 h, N2
and (b) acrylonitrile, ∆, N2.
for the N-functionalization of a range of azamacrocycles, we
did not view this as a viable approach. Several strategies toward
our target ligands could be conceived, using the array of reported
methods for the functionalization of azamacrocycles. However,
it quickly became clear that it would not be possible to develop
generic methods as each azamacrocycle displays synthetic
idiosyncrasies and the synthetic strategy employed must be
tailored to the individual azamacrocycle. Herein, we report our
findings in the functionalization of TACN, 1, cyclen, 2, dimethyl
cyclam, 3, and cyclam, 4; of the azamacrocycles studied, cyclam
has proven to be the easiest to derivatize with biotin.
a Reagents and conditions: (a) K2CO3, MeCN, ∆, 24 h, N2; (b) 13,
K2CO3, MeCN, ∆; and (c) 20% TFA, DCM.
reported.10-13 We were, however, concerned that this methodol-
ogy would not be suitable for all of the ligands in which we
are interested. Our first concern with using an amide linker was
that Lewis acid activated amide hydrolysis could occur. While
Alsfasser has reported that such amide linkers are stable in
buffered solution at pH 6.8, hydrolysis was found to occur in
neutral solution and at higher pHs.14 Thus, if we were to apply
these macrocyclic conjugates in vivo, the loss of the biological
receptor was a real danger; these concerns were subsequently
justified (vide infra). Our other concern with an amide linker
centered on the unpredictable nature of its interaction with a
metal center. Not only are structurally authenticated Werner
complexes of tertiary amides extremely rare,15,16 but the mode
of the tertiary amide nitrogen-metal interaction is difficult to
predict, even in closely related systems,16,17 which could reduce
the stability and/or change the properties of the coordination
complex, thus limiting our ability to detect a binding event of
our macrocycle-biotin conjugate in a predictable and reproduc-
ible manner. We thus sought to introduce hydrolytically robust
alkyl pendant arms. R-,ω-Bromoamines have been utilized in
related systems as a means of introducing a primary amine
pendant arm by simple nucleophilic substitution of the alkyl
bromide when the primary amine is Z-protected.18 We preferred
to utilize the sulfonate esters 10 instead in the first instance
and found that the target macrocycle 11 could be isolated in
moderate yield (Scheme 2). We also found that sulfonamide
protection of the macrocyclic amine nitrogen atoms, as in 9,
significantly improved yields, suggesting that carbamate protect-
ing groups for the azamacrocycle are less compatible with this
Results and Discussion
Alkylation of TACN. Our first attempts to functionalize an
azamacrocyclic ligand with biotin centered on 1. Our aim was
to find a means of introducing a primary amine group to the
macrocycle via N-alkylation to which we could then couple the
carboxylic acid of biotin. We also wished to have sufficient
synthetic flexibility to allow the spacer unit between the primary
amine and the azamacrocycle to be varied to investigate whether
or not the spacer influenced the effect the binding of avidin
had upon the metal center. Thus, Schro¨der’s procedure for the
N-alkylation of 5 with electrophilic nitriles appeared to be a
particularly attractive strategy (Scheme 1),9 as this gave us a
means of introducing both primary amine and carboxylic acid
functional groups that could be used in a variety of ways to
incorporate biotin; indeed, if an unsymmetrical coupling could
be effected, the remaining amine or carboxylic acid could be
further utilized (e.g., to mount the material on a solid support
or surface). The synthesis of 6 and 7 from 5 proceeded smoothly,
although we found that 6 formed in higher yield when K2CO3
was used as the base rather than the reported triethylamine.
Nitrile hydrolysis could be easily achieved, but in our hands,
nitrile reduction proved capricious, so we decided to investigate
alternative strategies for the introduction of amines. We thus
turned to the di-Boc-protected analogue 8 (Scheme 2), which
we were able to prepare using the reported methods.10
(11) Ritter, S. C.; Eiblmaier, M.; Michlova, V.; Ko¨nig, B. Tetrahedron
2005, 61, 5241-5251.
(12) Ko¨nig, B.; Pelka, M.; Zieg, H.; Ritter, T.; Bouas-Laurent, H.;
Bonneau, R.; Desvergne, J.-P. J. Am. Chem. Soc. 1999, 121, 1681-1687.
(13) Wiest, O.; Harrison, C. B.; Saettel, N. J.; Cibulka, R.; Sax, M.;
Ko¨nig, B. J. Org. Chem. 2004, 69, 8183-8185.
(14) Geisser, B.; Ko¨nig, B.; Alsfasser, R. Eur. J. Inorg. Chem. 2001,
1543-1549.
Benniston et al. have recently utilized 8 in a series of peptide
couplings to attach pendant arms to the 1,4,7-triazacyclononane
framework via amide linkages, and a number of Zn(II)
complexes of such amide-containing azamacrocycles have been
(15) Sibbons, K. F.; Al-Hashimi, M.; Motevalli, M.; Wolowska, J.;
Watkinson, M. Dalton Trans. 2004, 3163-3165.
(16) Schickaneder, C.; Heinemann, F. W.; Alsfasser, R. Eur. J. Inorg.
Chem. 2006, 2357-2363.
(17) Mondal, A.; Klein, E. L.; Khan, M. A.; Houser, R. P. Inorg. Chem.
2003, 42, 5462-5464.
(9) Blake, A. J.; Danks, J. P.; Li, W-S.; Lippolis, V.; Schro¨der, M. J.
Chem. Soc., Dalton Trans. 2000, 3034-3040.
(10) Benniston, A. C.; Gunning, P.; Peacock, R. D. J. Org. Chem. 2005,
70, 115-123.
(18) Boseggia, E.; Gatos, M.; Lucatello, L.; Mancin, F.; Moro, S.;
Palumbo, M.; Sissi, C.; Tecilla, P.; Tonellato, U.; Zagotto, G. J. Am. Chem.
Soc. 2004, 126, 4543-4549.
J. Org. Chem, Vol. 72, No. 22, 2007 8281

Krivickas et al.
SCHEME 3a
16 and 20c21 proceeded smoothly to give 21c in 77% yield,
our attempt to extend the length of the chain by using 20a saw
a dramatic reduction in the yield of 18a to 64% (Scheme 4).
Furthermore, we were unable to prepare 20b (n ) 2) due to
competing intramolecular cyclization of the aldehyde. Nonethe-
less, the primary amine in 22 could be readily revealed by
hydrogenation under standard conditions,19 and the primary
amine effectively coupled to biotin using HATU,22 a reagent
that we have generally found to be the best agent for the
coupling of biotin to azamacrocycles (vide infra). The free
amine, 24, was isolated as its TFA salt by simple deprotection
of 23.19
Acylation of Cyclen. We turned to the use of peptide
couplings for the functionalization of cyclen, despite our
concerns over amide stability in the presence of Lewis acids,
as our efforts to systematically vary the length of the spacer
between the macrocycle and the biotin had met with somewhat
limited success. We were keen to utilize Fmoc-protected amino
acids in the coupling reactions so that the methodology could
ultimately be applied to the development of solid phase
combinatorial libraries of azamacrocycles derivatized with short
oligopeptides.23 Unfortunately, adaptation of an effective pro-
cedure for the coupling of Z-protected glycine to 1610 was
unsuccessful for the Fmoc-protected glycine, 25. Fortunately,
adaptation of the procedure reported by Ritter et al.11 proceeded
smoothly for a number of Fmoc-protected amino acids, giving
the desired amides in good to excellent yield (28 ) 86% and
29 ) 64%) (Scheme 5).
We were readily able to remove the Fmoc and Boc protecting
groups19 yielding 34-36 and to show that a complex of amide
34, prepared from copper(II) chloride in aqueous solution
buffered at pH 9.8, appeared to be stable in methanolic solution,
as judged by a combination of IR and UV-vis spectroscopies
and ES MS. Encouraged, we therefore elaborated the synthesis
further and found that biotin could be coupled to 28, following
Fmoc group removal, in good yield to ultimately give the TFA
salt of 38. The biotinylated macrocycle could be metalated in
the same manner as 34, which to the best of our knowledge,
represents only the second example of a metal complex of a
biotinylated azamacrocyclic ligand8 (see Supporting Informa-
tion). Unfortunately, closer investigation of the coordination
chemistry of 34-36 caused us some concern as the coordination
mode of the ligand appeared to be critically dependent on the
anion used. With chloride, clear evidence of a shift in the
carbonyl band from 1678 to 1589 cm-1 was observed, consistent
with amide oxygen coordination, as reported by Alsfasser for
related cyclam complexes;16 however, a second carbonyl band
was also observed at 1686 cm-1 (cf. 1678 cm-1 in the free
ligand), which is indicative of the presence of another amide
mode (i.e., the presence of different coordination modes of the
ligand). Furthermore, when we investigated the use of other
anions, we found that the perchlorate complex appeared to be
unstable in methanolic solution, with changes occurring in its
UV-vis spectrum after only a few hours at room temperature.
Given that the ultimate application we envisage for the
functionalized metal complexes in sensors requires both well-
defined and robust coordination chemistry, we feel that this
a Reagents and conditions: (a) 10 or 17, K2CO3, MeCN, ∆, 4 days.
chemistry. The use of 9 instead of 8 introduces other issues
associated with sulfonamide deprotection, which we felt would
be problematic in the presence of biologically relevant recogni-
tion motifs. Therefore, we attempted to effect the global
deprotection of 11 using conventional methods19 and then couple
the product to biotin in the hope that the increased reactivity of
the primary amine (derived from the Z-carbamate in 11) might
give some regiocontrol. Unfortunately, intractable mixtures
resulted.
In an attempt to avoid this problem, we decided to try to
incorporate biotin in the electrophile prior to the coupling
reaction. Gratifyingly, reaction of 8 with 13 (see Supporting
Information for the synthesis of 13) proceeded smoothly in 61%
yield to give the biotinylated ligand 14. Removal of the Boc
groups could easily be effected with TFA to give the free amine
15 as its TFA salt quantitatively.
Alkylation of Cyclen. Confident that we had established an
effective method for the biotinylation of 1, we attempted to apply
the same conditions to the biotinylation of 2. It quickly became
clear that finding a generic functionalization strategy was
unlikely to be achieved. Although tri-Boc-protected cyclen 16
could be reliably prepared following literature procedures,20 its
reaction with 13 under the same conditions that had led to the
formation of 14 consistently resulted in the quantitative reiso-
lation of starting materials (Scheme 3). We then investigated
the efficacy of electrophiles 10 in the alkylation of 16 and again
saw a distinct difference in behavior between the different
macrocyclic ligands. This time, the sulfonate esters gave a very
poor yield of the target macrocycles 18 with either starting
materials recovered (10a, 56% recovered) or significant quanti-
ties of cyclic carbamate 19b (from 10b) being formed. We
therefore investigated the efficacy to R-,ω-bromides 17, which
although superior to 10, were still unsatisfactory. We attribute
this to the alkylation of 16 being much slower than it is for 8.
There is no obvious explanation for this other than that 16 adopts
a conformation that retards its alkylation. We elected not to
investigate the efficacy of sulfonamide protection of the
macrocyclic nitrogen atoms in this case given the perennial
problems already encountered with the removal of this group.
As an alternative strategy, we decided to utilize reductive
aminations that have previously been reported to occur with 16
by Chae et al.3 While the reported reductive amination between
(21) Delcross, J.-G.; Tomasi, S.; Carrington, S.; Martin, B.; Renault, J.;
Blagbrough, I. S.; Uriac, P. J. Med. Chem. 2002, 45, 5098-5111.
(22) Carpino, L. A. J. Am. Chem. Soc. 1993, 115, 4397-4398.
(23) (a) Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149-2154. (b)
Carpino, L. A. J. Am. Chem. Soc. 1970, 92, 5748-5749.
(19) Greene, T. W.; Wuts, P. G. M. ProtectiVe Groups in Organic
Synthesis, 3rd ed.; Wiley-Interscience: New York, 1999.
(20) Kimura, E.; Aoki, S.; Koike, T.; Shiro, M. J. Am. Chem. Soc. 1997,
119, 3068-3076.
8282 J. Org. Chem., Vol. 72, No. 22, 2007

EffectiVe Methods for Biotinylation of Azamacrocycles
SCHEME 4a
a Reagents and conditions: (a) 16, NaBH(OAc)3, THF; (b) Pd/C, H2, EtOH; (c) HATU, HOBT, DMAP, biotin, DMF/DCM; and (d) 20% TFA, DCM.
SCHEME 5a
a Reagents and conditions: (a) 16, HATU, HOBt, DMAP, DCM/DMF, 40 °C, 2 days; (b) TBAF, MeCN; (c) 20% TFA, DCM; (d) biotin, HATU, HOBt,
DCM/DMF, 40 °C, 2 days; and (e) 20% TFA, DCM.
behavior, while interesting, will preclude the use of this linker
in the devices.
have also observed amide cleavage with simpler groups attached
to the ring nitrogen (e.g., single amino acids).24
Alkylation of Cyclam. The hydrolytic instability of these
cyclam conjugates led us to investigate the alkylation of cyclam.
We returned to the reductive amination strategy described
previously for cyclen. Again, the reductive amination of 39 with
20c proceeded smoothly (Scheme 7). Elaboration of the mac-
rocyclic scaffold in the usual way lead to biotinylated cyclam
48c in acceptable yield, and we were able to prepare an
authenticated copper(II) complex of it and demonstrate its
binding to avidin.6 In contrast, the same methodology was
completely ineffective for 3 with the reduced aldehyde being
recovered quantitatively. Interestingly, the reactivity of 3
generally appears to be lower than that of the other macrocycles
investigated. For example, its alkylation with 10 and 17 was
ineffective, and only its alkylation with the highly reactive
acrylonitrile proceeded in satisfactory yield.9
In view of our inability to effectively vary the linker length
in 48c between the amine and the macrocycle using the reductive
amination strategy, we turned to the use of ethyl R-bromoethyl
acetate, which has been used extensively in the functionalization
of cyclen derivatized macrocycles,25 but which appears to have
been little utilized in cyclam chemistry. Our initial reluctance
to explore this methodology lay in the necessity to elaborate
the carboxylic acid in 49 with a singly protected diamine such
Acylation of Cyclam. As discussed, cyclam-amide conju-
gates are known to undergo Lewis acid-catalyzed hydrolysis,
and we have noted that the solution stability of some of the
cyclen amide complexes is also poor. Nevertheless, we felt it
worthwhile to investigate whether the methods shown in Scheme
5 could also be applied to cyclam. To our surprise, the Fmoc-
amino acid-protected coupling strategy that had been so effective
for 1611 proved to be ineffective for the equivalent cyclam, 39,
with the poor-yielding couplings that occurred further hampered
by complex reaction mixtures being formed. We therefore turned
to using Z-protected glycine 40,10 which gave 41 in quantitative
yield using DCC as the coupling agent. Simple palladium-
catalyzed hydrogenation19 gave the required coupling precursor,
42 (Scheme 6). We then found that the efficient coupling of
biotin to 42 was particularly sensitive to the coupling agent used,
with HATU being by far the most effective. Our efforts to effect
couplings of biotin using other methods were consistently lower
yielding (e.g., HBTU/DIPEA gave 43 in only 30% yield).
Deprotection of 43 was again easily achieved with TFA to give
44, again as its TFA salt. We then attempted to introduce copper-
(II) into the cyclam ring by carefully neutralizing the TFA salt
of 44 in a methanol/water mixture to pH 8 with 1 N NaOH
prior to the addition of Cu(ClO4)2‚6H2O. Unfortunately, inex-
pedient hydrolytic cleavage of the amide occurred, which was
confirmed by the formation of crystals of [Cu(4)(ClO4)2]. We
(24) Ramana, A. V.; Todd, M. H., unpublished results.
(25) Sengupta, D.; Blasko´, A.; Bruice, T. C. Bioorg. Med. Chem. 1996,
4, 803-813.
J. Org. Chem, Vol. 72, No. 22, 2007 8283

Krivickas et al.
SCHEME 6a
a Reagents and conditions: (a) DCC, DMAP, DCM; (b) Pd/C, H2, MeOH; (c) HATU, DIPEA, DMAP, biotin, DMF; and (d) 20% TFA, DCM.
SCHEME 7a
a Reagents and conditions: (a) NaBH(OAc)3; (b) Pd/C, H2, MeOH; (c) HATU, DIPEA, DMAP, biotin, DMF; and (d) 20% TFA, DCM.
SCHEME 8a
a Reagents and conditions: (a) DCC, DMAP, DCM; (b) Pd/C, H2, MeOH; (c) HATU, DIPEA, DMAP, biotin, DMF; and (d) 20% TFA.
as 50. While ethylene diamine can be readily desymmetrized,
this is a more difficult procedure for longer diamines.19 In
addition, we felt that this method added unnecessary protection/
deprotection steps. Despite these reservations, the required
carboxylic acid was readily prepared by alkylation of 39 with
ethyl R-bromoethyl acetate, which was then saponified. The
carboxylic acid in 49 was effectively coupled to amine 50 with
deprotection of the Z-carbamate proceeding smoothly (Scheme
8). Coupling of 52 to biotin was again found to proceed
smoothly using HATU to give 53, which was readily converted
to 54 in the usual way.
method that can be applied to all of the macrocycles investigated,
we have been able to find effective routes to biotinylate 1, 2,
and 4; in contrast, 3 has proven to be considerably less reactive.
It is difficult, prima facie, to rationalize these observations, and
further studies are required into the conformational preferences
of these protected macrocycles. Reports relating to the variation
in reactivity in such systems are sparse, and we have been unable
to find any directly relevant analogues. Sherry and Kovacs have
exploited the variation in pKa of azamacrocyclic ligands to effect
a variety of selective functionalization strategies.26,27 Presum-
ably, this is a conformational effect, and this has been exploited
in host-guest chemistry where variations in anion binding of
azamacrocycles has been directly related to pKa.28 Moreover, it
has been noted that the effectiveness of polyazamacrocycle
Conclusion
A broad spectrum of methods has been investigated for the
selective functionalization of azamacrocycles with biotin.
Although we have found that there is apparently no generic
(26) Kovacs, Z.; Sherry, A. D. Synthesis 1996, 759-763.
(27) Kovacs, Z.; Sherry, A. D. Chem. Commun. 1995, 185-186.
(28) Schmidtchen, F. P.; Berger, M. Chem. ReV. 1997, 97, 1609-1646.
8284 J. Org. Chem., Vol. 72, No. 22, 2007

EffectiVe Methods for Biotinylation of Azamacrocycles
alkylation is dependent on the size of the ligand.29 Finally, Alder
et al. have correlated hydrogen bonding in bridged aminal
analogues with conformational effects.30 In contrast to the
present study, in none of these examples were the nitrogen atoms
of the macrocycles protected as Boc-carbamates, and thus, a
direct analogy can only be speculative, and investigations to
this end are currently ongoing. Most of the successful methods
we describe allow some variation in the nature of the macro-
cycle-biotin linker so that fine-tuning of the effect that (strept)-
avidin binding has on the metal center might be achieved. In
addition, many of the intermediates provide a means by which
other biological markers might be introduced to the macrocyclic
framework.
according to the previous procedure for 11. Compound 9 (500 mg,
1.14 mmol), 10b (560 mg, 1.37 mmol), and K2CO3 (316 mg, 2.29
mmol) in dry acetonitrile (20 mL) afforded 12b as a colorless oil
(646 mg, 88%): νmax (CH2Cl2)/cm-1 3403, 2936, 1716, 1643, 1597,
1524; 1H NMR (CDCl3, 270 MHz): δ ) 7.65 (d, J ) 8.2 Hz, 4H),
7.38-7.22 (m, 9H), 5.14-5.01 (m, 3H), 3.50-3.39 (m, 4H), 3.28-
3.12 (m, 6H), 2.91-2.79 (m, 4H), 2.56 (t, J ) 6.3 Hz, 2H), 2.43(s,
6H), 1.65-1.41 (m, 4H); 13C NMR (CDCl3, 67.5 MHz): δ ) 156.6,
143.6, 136.8, 135.3, 129.9, 128.5, 128.0, 127.2, 66.5, 56.7, 55.7,
52.9, 51.7, 27.5, 25.1, 21.6; MS (ESI) m/z: 666 [(M + Na)+], 643
[(M + H)+]; HRMS (ESI) calcd for C32H43N4O6S2 [(M + H)+]
643.2619, found 643.2620.
Toluene-4-sulfonic Acid 4-[5-(2-Oxo-hexahydro-thieno[3,4-
d]imidazol-4-yl)-pentanoylamino]-butyl Ester (13). A stirred
suspension of C (see Supporting Information; 120 mg, 0.382 mmol),
p-toluene sulfonyl chloride (146 mg, 0.742 mmol), triethylamine
(0.12 mL, 0.841 mmol), and DMAP (5 mg, 0.038 mmol) in
dichloromethane (5 mL) was stirred for 3 days at room temperature.
The solvent was removed in vacuo, and the crude material was
purified by silica gel column chromatography (1:10 methanol/
Experimental Section
The following compounds were prepared according to the
reported procedures: 5-7,9 8,10 9,9 10a,18 10b,25 N-benzyloxycar-
bonyl-4-amino-1-butanol, N-benzyloxycarbonyl-6-aminohexanol,
17,18 16,10 20c,21 and 39.31 For compounds that have been previously
reported but were prepared by different methods, see the Supporting
Information.
dichloromethane) to give 13 (31 mg, 20%) as a pale yellow oil:
1
ν
max(Nujol)/cm-1 1666; H NMR (CD3OD, 270 MHz): δ ) 7.78
(d, J ) 8.8 Hz, 2H), 7.43 (d, J ) 8.8 Hz, 2H), 4.48 (dd, J ) 7.7,
4.7 Hz, 1H), 4.29 (dd, J ) 7.9, 4.5 Hz, 1H), 4.04 (t, J ) 6.2 Hz,
2H), 3.24-3.15 (m, 1H), 3.12(t, J ) 4.4 Hz, 2H), 2.92 (dd, J )
12.9, 4.9 Hz, 1H), 2.69 (d, J ) 12.9 Hz, 1H), 2.45 (s, 3H), 2.17 (t,
J ) 7.2 Hz, 2H), 1.79-1.32 (m, 10H); 13C NMR (CD3OD, 67.5
MHz): δ ) 174.7, 164.8, 145.2, 133.2, 129.6, 127.8, 70.3, 62.0,
60.3, 55.7, 39.7, 38.2, 35.5, 28.4, 28.2, 26.1, 25.5, 25.2, 20.3; MS
(ESI) m/z: 492 [(M + Na)+], 470 [(M + H)+], 298, 226; HRMS
(ESI) calcd for C21H32N3O5S2 [(M + H)+] 470.1778, found
470.1780.
7-(6-Benzyloxycarbonylamino-hexyl)-1,4,7triazonane-1,4-dicar-
boxylic Acid Di-t-butyl Ester (11). To a stirred solution of 8 (14
mg, 0.043 mmol) and K2CO3 (12 mg, 0.085 mmol) in dry
acetonitrile (5 mL) was added 10a (26 mg, 0.064 mmol), and the
solution was refluxed overnight under a nitrogen atmosphere. The
reaction was cooled, acetonitrile was removed in vacuo, the residue
was taken up in dichloromethane, washed with water, dried over
MgSO4, and the volatiles were removed in vacuo. The crude residue
was purified by silica gel chromatography (1:1, ethyl acetate/
petroleum spirits) to afford 11 as a colorless oil (10 mg, 42%):
7-{4-[5-(2-Oxo-hexahydro-thieno[3,4-d]imidazol-6-yl)-pen-
tanoylamino]-butyl}-1,4,7triazonane-1,4-dicarboxylic Acid Di-t-
butyl Ester (14). A solution of 8 (31 mg, 0.094 mmol), 13 (52
mg, 0.114 mmol), and K2CO3 (35 mg, 0.251 mmol) in anhydrous
acetonitrile (5 mL) was refluxed under a nitrogen atmosphere for
4 days. After cooling, the solvent was removed in vacuo, and the
crude material was purified by silica gel column chromatography
(10% methanol/dichloromethane) to give 14 (36 mg, 61%) as a
colorless oil: νmax(CH2Cl2)/cm-1 1686, 1555, 1461, 1416, 1366;
1H NMR (CDCl3, 270 MHz): δ ) 6.62-6.46 (bm, 1H, NH), 6.14-
5.98 (bm, 1H, NH), 4.56-4.46 (m, 1H), 4.36-4.26 (m, 1H), 3.52-
3.10 (m, 11H), 2.91 (dd, J ) 12.6, 4.9 Hz, 1H), 2.74 (d, J ) 12.9
Hz, 1H), 2.66-2.42 (m, 6H), 2.28-2.14 (m, 2H), 2.05-1.58 (m,
6H), 1.54-1.34 (m, 22H); 13C NMR, not reported due to the
presence of rotamers, it was not possible to interpret the spectrum;
MS (ESI) m/z: 649.5 [(M + Na)+], 627.5 [(M + H)+], 528.4, 527.4,
428.4, 427.4; HRMS (ESI) calcd for C30H54N6O6S [(M + H)+]
627.3898, found 627.3888.
5-(2-Oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pentanoic Acid
(4-1,4,7Triazonan-1-yl-butyl)-amide-dihydrate-trifluoroacetate (15).
To a solution of 14 (10 mg, 16 µmol) in DCM (1 mL), TFA (42
equiv, 6.2 mmol, 50 µL) was added, and the resulting solution was
stirred at room temperature overnight. The solvent was then
evaporated in vacuo to give 15 as a pale yellow solid: νmax(neat)/
cm-1 1688, 1455. MS (ESI) m/z: 427.4 [(M + H)+]. HRMS (ESI)
calcd for C20H38N6O2S [(M + H)+], 427.2850, found 427.2858.
10-(6-Benzyloxycarbonylamino-hexyl)-1,4,7,10-tetraaza-cy-
clododecane-1,4,7-tricarboxylic Acid Tri-t-butyl Ester (18a). A
stirred solution of 16 (180 mg, 0.381 mmol), K2CO3 (105 mg, 0.763
mmol), and 10a (248 mg, 0.611 mmol) in dry acetonitrile (10 mL)
was refluxed overnight under a nitrogen atmosphere. The solution
was allowed to cool, and the acetonitrile was removed in vacuo.
The residue was taken up in dichloromethane, washed with water,
and dried over MgSO4, and the volatiles were removed. The crude
material was purified by column chromatography (silica gel, 1:1
ethyl acetate/petroleum spirits) to give 18a (39 mg, 15%) as a
colorless oil: νmax(CH2Cl2)/cm-1 1686, 1460, 1416; 1H NMR (270
ν
max (CH2Cl2)/cm-1 1685; 1H NMR (400 MHz, CDCl3): δ ) 7.38-
7.28 (m, 5H), 5.05 (s, 2H), 4.75 (bs, 1H), 3.52-3.40 (m, 4H), 3.30-
3.14 (m, 6H), 2.66-2.55 (m, 4H), 2.51-2.41 (m, 2H), 1.66-1.21
(m, 30H); 13C NMR (100 MHz, CDCl3): δ ) 155.4, 154.6, 154.4,
135.7, 127.5, 127.1, 65.5, 55.8, 53.2, 53.1, 52.9, 52.6, 50.0, 49.62,
49.55, 49.3, 48.6, 40.0, 28.7, 27.6, 26.8, 26.0, 25.7; MS (ESI) m/z:
563.5 [(M + H)+], 455.4, 429.4, 330.3; HRMS (ES) calcd for
C30H51N4O6 [(M + H)+] 563.3803, found 563.3801.
{6-[4,7-Bis-(toluene-4-sulfonyl)-1,4,7triazonan-1-yl]-hexyl}-car-
bamic Acid Benzyl Ester (12a). To a stirred solution of 9 (200
mg, 0.46 mmol) and K2CO3 (130 mg, 0.92 mmol) in dry acetonitrile
(10 mL) was added 10a (220 mg, 0.55 mmol), and the solution
was refluxed overnight under a nitrogen atmosphere. The reaction
was cooled, acetonitrile was removed in vacuo, the residue was
taken up in dichloromethane, washed with water, and dried over
MgSO4, and the volatiles were removed. The crude residue was
purified by silica gel chromatography (10:1, dichloromethane/
methanol) to afford 12a as a colorless oil (242 mg, 75%): νmax
(CH2Cl2)/cm-1 3391, 2931, 2858, 1717, 1597, 1527, 1454; 1H NMR
(CDCl3, 270 MHz): δ ) 7.66 (d, J ) 8.4 Hz, 4H), 7.39-7.22 (m,
9H), 5.08 (s, 2H), 4.85 (bs, 1H), 3.55-3.44 (m, 4H), 3.24-3.05
(m, 6H), 2.91-2.79 (m, 4H), 2.52 (t, J ) 7.0 Hz, 2H), 2.37 (s,
6H), 1.58-1.20 (m, 8H); 13C NMR (CDCl3, 67.5 MHz): δ ) 156.6,
143.5, 136.8, 135.4, 129.8, 128.5, 128.1, 127.3, 66.6, 57.4, 55.9,
52.6, 51.5, 41.1, 29.9, 27.7, 27.1, 26.7, 21.7, 21.4; MS (ESI) m/z:
671 [(M + H)+], 141, 90; HRMS (ESI) calcd for C34H47N4O6S2
[(M + H)+] 671.2932, found 671.2937.
{4-[4,7-Bis-(toluene-4-sulfonyl)-1,4,7triazonan-1-yl]-butyl}-car-
bamic Acid Benzyl Ester (12b). Compound 12b was made
(29) Kruper, W. J., Jr.; Rudolf, P. R.; Langhoff, C. A. J. Org. Chem.
1993, 58, 3869-3876.
(30) Alder, R. W.; Carniero, T. M. G.; Mowlam, R. W.; Orpen, A. G.;
Petillo, P. A.; Vachon, D. J.; Weisman, G. R.; White, J. M. J. Chem. Soc.,
Perkin Trans 2 1999, 589-599.
(31) Mishra, A. K.; Draillard, K.; Faiver-Chauvet, A.; Gestin, J. F.; Curtet,
C.; Chatal, J.-F. Tetrahedron Lett. 1996, 37, 7515-7518.
J. Org. Chem, Vol. 72, No. 22, 2007 8285

Krivickas et al.
MHz, CDCl3): δ ) 7.47-7.29 (m, 5H), 5.05 (s, 2H), 4.84 (bs,
1H), 3.61-3.11 (m, 14H), 2.71-2.44 (m, 6H), 1.60-1.15 (m, 35H);
13C NMR (CDCl3, 67.5 MHz): δ ) 156.5, 156.2, 155.8, 155.5,
136.7, 128.6, 128.2, 79.6, 79.4, 66.7, 54.9, 53.7, 52.3, 50.1, 48.0,
47.7, 41.1, 30.0, 28.8, 28.6, 27.5, 26.7, 23.7; MS (ESI) m/z: 706
[(M + H)+], 473; HRMS (ESI) calcd for C37H64N5O8 [(M + H)+]
706.4749, found 706.4745.
10-(6-Benzyloxycarbonylamino-hexyl)-1,4,7,10-tetraaza-cy-
clododecane-1,4,7-tricarboxylic Acid Tri-t-butyl Ester (18a).
Alternative procedure: a solution of 16 (187 mg, 0.396 mmol),
triacetoxy sodium borohydride (191 mg, 0.902 mmol), and 20a (235
mg, 0.451 mmol) in THF (10 mL) was stirred overnight at room
temperature. The solution was basified with saturated NaHCO3,
the volatiles were removed in vacuo, and the aqueous layer was
extracted with dichloromethane. The combined organics were dried
over MgSO4, and the volatiles were removed in vacuo. The crude
material was purified by silica gel chromatography (1:1 ethyl
acetate/petroleum spirits) to afford 18a (177 mg, 64%) as a colorless
oil.
10-(4-Benzyloxycarbonylamino-butyl)-1,4,7,10-tetraaza-cy-
clododecane-1,4,7-tricarboxylic Acid Tri-t-butyl Ester (18b). A
stirred solution of 16 (100 mg, 0.212 mmol), K2CO3 (58 mg, 0.424
mmol), and 10b (133 mg, 0.424 mmol) in dry acetonitrile (10 mL)
was refluxed overnight under a nitrogen atmosphere. The solution
was allowed to cool, and the acetonitrile was removed in vacuo.
The residue was taken up in dichloromethane, washed with water,
and dried over MgSO4, and the volatiles were removed. The crude
material was purified by silica gel chromatography (1:1, ethyl
acetate/petroleum spirits) to give 18b as a colorless oil (12 mg,
8%); νmax(CH2Cl2)/cm-1 1662, 1538, 1456, 1416; 1H NMR (CDCl3,
270 MHz): δ ) 7.33-7.22 (m, 5H), 5.07(s, 2H), 3.53-3.10 (m,
16H), 2.63-2.40 (m, 4H), 1.48-1.50 (m, 4H), 1.39 (s, 9H), 1.37
(s, 18H); 13C NMR (CDCl3, 67.5 MHz): δ ) 156.5, 156.1, 155.8,
155.4, 136.7, 128.5, 128.2, 128.1, 79.6, 79.5, 79.3, 66.6, 55.0, 53.4,
51.8, 50.0, 48.1, 47.8, 47.4, 40.9, 40.6, 28.5, 28.0, 21.1; MS (ESI)
m/z: 701 [(M + Na)+], 678 [(M + H)+]; HRMS (ESI) calcd for
C35H60N5O8 [(M + H)+] 678.4436, found 678.4438.
128.1, 79.7, 79.5, 66.4, 55.2, 54.4, 49.9, 48.9, 47.6, 38.9, 28.7, 28.5;
MS (ESI) m/z: 664[(M + H)+]; HRMS (ESI) calcd for C34H58N5O8
[(M + H)+] 664.4280, found 664.4280.
10-(3-Amino-hexyl)-1,4,7,10-tetraazacyclododecane-1,4,7-tri-
carboxylic Acid Tri-t-butyl Ester (22a). A solution of 21a
(384 mg, 0.56 mmol), Pd/C (17 mg), and acetic acid (0.04 mL,
0.61 mmol) in methanol (7 mL) under a H2 atmosphere was stirred
overnight at room temperature. The solution was filtered, and the
solvent was removed under reduced pressure to give 22a as a
1
colorless oil (272 mg, 88%); νmax(CHCl3)/cm-1 1686; H NMR
(CDCl3, 270 MHz): δ ) 3.54-3.06 (m, 12H), 2.75-2.30 (m, 8H),
1.64-1.09 (m, 35H); 13C NMR (CDCl3, 67.5 MHz): δ ) 156.1,
155.7, 155.4, 79.4, 79.2, 55.0, 53.7, 52.6, 50.0, 48.0, 47.6, 42.2,
28.7, 28.5, 27.7, 26.9; MS (ESI) m/z: 572[(M + H)+]; HRMS (ESI)
calcd for C29H58N5O6 [(M + H)+] 572.4382, found 572.4378.
10-{6-[5-(2-Oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pen-
tanoylamino]-hexyl}-1,4,7,10-tetraazacyclododecane-1,4,7-tri-
carboxylic Acid Tri-t-butyl Ester (23a). To a stirred solution of
22a (99 mg, 0.177 mmol) in 1:1 dichloromethane/dimethylforma-
mide (2 mL) was added biotin (47 mg, 0.196 mmol), HATU (81
mg, 0.213 mmol), HOBt (33 mg, 0.213 mmol), and DMAP (26
mg, 0.213 mmol). The yellow solution was stirred at 40 °C for 2
days. The volatiles were removed in vacuo, and the residue was
purified by silica gel chromatography (1:10 methanol/dichlo-
romethane) to afford 23a as a colorless oil (70 mg, 50%): νmax
-
(CHCl3)/cm-1 1690, 1545; 1H NMR (CDCl3, 270 MHz): δ ) 6.19
(s, 1H), 6.13-6.02 (m, 1H), 5.44 (s, 1H), 4.54-4.43 (m, 1H), 4.32-
4.22 (m, 1H), 3.59-3.07 (m, 15H), 3.01-2.82 (m, 3H), 2.77-
2.41 (m, 7H), 2.22-2.11 (m, 2H), 1.80-1.55 (m, 6H), 1.55-1.15
(m, 35H); 13C NMR (CDCl3, 67.5 MHz): δ ) 173.3 (C), 163.9
(C), 156.2 (C), 155.8 (C), 155.5 (C), 79.5 (C), 79.3 (C), 61.9 (CH),
60.3 (CH), 55.7 (CH), 54.9 (CH2), 53.6 (CH2), 52.2 (CH2), 50.1
(CH2), 47.9 (CH2), 47.6 (CH2), 40.6 (CH2), 40.1 (CH2), 39.5 (CH2),
36.1 (CH2), 29.6 (CH2), 28.8 (CH3), 28.6 (CH3), 28.3 (CH3), 28.2
(CH2), 27.5 (CH), 26.9 (CH2), 25.8 (CH2), 23.5 (CH2). MS (ESI)
m/z: 820[(M + Na)+], 798[(M + H)+]; HRMS (ESI) calcd for
C39H71N7O8SNa [(M + Na)+] 798.5158, found 798.5156.
6-Oxo-hexyl-carbamic Acid Benzyl Ester (20a). Oxalyl chlo-
ride (0.39 mL, 3.79 mmol) was dissolved in anhydrous dichlo-
romethane (20 mL) under a nitrogen atmosphere and cooled to -78
°C. Dimethyl sulfoxide (0.43 mL, 7.59 mmol) was added dropwise
over 5 min, and the reaction mixture was stirred at -78 °C for 10
min. N-Benzyloxycarbonyl-6-amino-1-hexanol (0.956 mg, 3.79
mmol) in dichloromethane (50 mL) was added slowly over 5 min.
The reaction mixture was then warmed to room temperature and
then cooled to -78 °C and stirred for a further 10 min. Triethy-
lamine (2.8 mL, 18.9 mmol) was added, and the reaction again
was warmed to room temperature. Water (50 mL) was added, the
organic layer was separated, and the aqueous layer was extracted
with dichloromethane. The combined organics were dried over
MgSO4 and filtered, and the volatiles were removed in vacuo. Silica
gel chromatography (2:3-1:1 ethyl acetate/petroleum spirits) af-
10-{3-[5-(2-Oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pen-
tanoylamino]-propyl}-1,4,7,10-tetraazacyclododecane-1,4,7-tri-
carboxylic Acid Tri-t-butyl Ester (23c). Prepared in an analogous
manner to 23a: 22c (133 mg, 0.251 mmol), biotin (67 mg, 0.277
mmol), HATU (115 mg, 0.302 mmol), HOBt (46 mg, 0.302 mmol),
and DMAP (37 mg, 0.302 mmol) in dichloromethane/dimethyl-
formamide (4 mL) gave 23c (74 mg, 38%): νmax(CHCl3)/cm-1
3304, 2974, 2930, 1686, 1545; 1H NMR (CDCl3, 270 MHz): δ )
7.09 (bs, 1H), 6.28 (bs, 1H), 5.62 (bs, 1H), 4.60-4.46 (m, 1H),
4.40-4.26 (m, 1H), 3.79-3.10 (m, 14H), 2.90 (dd, J ) 13.1, 4.9
Hz, 1H), 2.75 (d, J ) 12.6 Hz, 1H), 2.81-2.46 (m, 6H), 2.26 (t, J
) 7.2 Hz, 2H), 1.88-1.57 (m, 6H), 1.88-1.57 (m, 29H); 13C NMR
(CDCl3, 67.5 MHz): δ ) 173.7, 164.0, 156.3, 155.8, 155.3, 79.8,
79.7, 79.6, 61.9, 60.3, 55.7, 55.2, 54.6, 50.7, 50.3, 49.8, 48.5, 40.6,
37.1, 36.0, 28.7, 28.6, 28.4, 28.2, 25.9, 25.1; MS (ESI) m/z: 778-
[(M + Na)+], 756[(M + H)+]; HRMS (ESI) calcd for C36H65N7O8-
SNa [(M + Na)+] 756.4687, found 756.4688.
1
forded 20a as a colorless oil: νmax(CH2Cl2)/cm-1 1697, 1645; H
NMR (CDCl3, 270 MHz): δ ) 9.75 (s, 1H), 7.50-7.20 (m, 5H),
5.09 (s, 2H), 4.79 (bs, 1H), 3.21 (q, J ) 6.4 Hz, 2H), 2.44 (t, J )
6.4 Hz, 2H), 1.78-1.24 (m, 6H); 13C NMR (CDCl3, 67.5 MHz):
δ ) 202.5, 136.7, 128.6, 128.2, 66.7, 43.8, 40.9, 29.8, 26.3, 21.7;
MS (ESI) m/z: 250 [M + H+]+, 232 [M + MeOH]+, 267 [M +
NH4+]+.
10-(3-Benzyloxycarbonylamino-propyl)-1,4,7,10-tetraaza-cy-
clododecane-1,4,7-tricarboxylic Acid Tri-t-butyl Ester (21c).
Macrocycle 21c was prepared in an analogous manner to 18a: 16
(258 mg, 0.547 mmol), triacetoxy sodium borohydride (397 mg,
1.873 mmol), and 20c (180 mg, 0.938 mmol) in THF (10 mL)
afforded 21c (274 mg, 77%): νmax(CH2Cl2)/cm-1 1686, 1533, 1460,
1416; 1H NMR (270 MHz, CDCl3): δ ) 7.39-7.23 (m, 5H), 5.70
(bs, 1H), 5.05 (s, 2H), 3.64-3.05 (m, 14H), 2.70-2.41 (m, 6H),
1.70-1.52 (m, 2H), 1.41 (s, 9H), 1.38 (s, 18H); 13C NMR (67.5
MHz, CDCl3): δ ) 156.72, 156.3, 155.8, 155.4, 136.9, 128.4,
5-(2-Oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pentanoic Acid
[6-(1,4,7,10-Tetraazacyclododec-1-yl)-hexyl]amide-trihydro-tri-
fluoroacetate (24a). Carbamate 23a (70 mg, 0.09 mmol) was
dissolved in dichloromethane (3 mL) and treated with trifluoroacetic
acid (0.42 g, 3.69 mmol). The solution was stirred at room
temperature overnight and then evaporated to give 24a (72 mg,
98%):
ν
max(neat)/cm-1 1673, 1454, 1420; 1H NMR (D2O,
270 MHz): δ ) 4.64-4.54 (m, 1H), 4.42-4.36 (m, 1H), 3.38-
3.26 (m, 1H), 3.24-3.06 (m, 16H), 3.02-2.88 (m, 5H), 2.75 (d, J
) 13.1 Hz, 1H), 2.23 (t, J ) 6.7 Hz, 2H), 1.72-1.22 (m, 14H);
13C NMR (D2O, 100 MHz): δ ) 175.8, 164.5, 69.0, 61.26, 59.4,
56.3, 54.6, 53.5, 51.9, 48.0, 42.6, 41.7, 40.8, 38.8, 38.2, 34.7, 27.4,
27.0, 26.8, 25.4, 25.1, 25.0, 24.3, 22.2; MS (ESI) m/z: 498[(M +
H)+]; HRMS (ESI) calcd for C24H48N7O2S [(M + H)+] 498.3585,
found 498.3585.
8286 J. Org. Chem., Vol. 72, No. 22, 2007

EffectiVe Methods for Biotinylation of Azamacrocycles
5-(2-Oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pentanoic Acid
[3-(1,4,7,10-Tetraazacyclododec-1-yl)-propyl]-amide-trihydro-
trifluoroacetate (24c). Prepared in an identical manner to 24a: 23c
(74 mg, 0.09 mmol) and trifluoroacetic acid (0.42 g, 3.69 mmol)
in dichloromethane (3 mL) gave 24c (79 mg, 99%) as a colorless
oil: νmax(neat)/cm-1 3774, 2928, 2854, 1674, 1454, 1423; 1H NMR
(D2O, 270 MHz): δ ) 4.63-4.53 (m, 1H), 4.44-4.34 (m, 1H),
3.36-2.87 (m, 22H), 2.81-2.68 (m, 3H), 2.23 (t, J ) 6.9 Hz, 2H),
1.82-1.26 (m, 8H); 13C NMR (D2O, 100 MHz): δ ) 176.9, 165.4,
62.1, 60.3, 55.5, 49.9, 48.9, 48.0, 44.0, 41.9, 39.7, 37.1, 35.4, 27.9,
27.7, 25.1, 23.6; MS (ESI) m/z: 456 [(M + H)+], 228; HRMS
(ESI) calcd for C21H42N7O2S [(M + H)+] 456.3115, found
456.3114.
10-[2-(9H-Fluoren-9-ylmethoxycarbonylamino)-acetyl]-1,4,7,-
10-tetraazacyclododecane-1,4,7-tricarboxylic Acid Tri-t-butyl
Ester (28). To a stirred solution of 16 (145 mg, 0.307 mmol) in a
dichloromethane/dimethylformamide mixture (1:1, 2 mL) was added
25 (100 mg, 0.338 mmol), HATU (140 mg, 0.367 mmol), HOBt
(56 mg, 0.367 mmol), and DMAP (45 mg, 0.376 mmol). The yellow
solution was stirred at 40 °C for 2 days. The solvents were removed
in vacuo, and the residue was purified by silica gel chromatography
(2:1-1:1 petroleum spirits/ethyl acetate) to afford 28 as a colorless
oil (199 mg, 86%): νmax(CH2Cl2)/cm-1 3294, 2932, 1694, 1516,
1466; 1H NMR (270 MHz, CDCl3): δ ) 7.75 (d, J ) 7.4 Hz, 2H),
7.61 (d, J ) 7.4 Hz, 2H), 7.39 (t, J ) 7.1 Hz, 2H), 7.30 (t, J ) 6.9
Hz, 2H), 5.81 (bs, 1H), 4.38-4.30 (m, 2H), 4.28-4.16 (m, 1H),
4.08-4.00 (m, 2H), 3.62-3.32 (m, 16H), 1.54-1.38 (m, 27H);
13C NMR (67.5 MHz, CDCl3): δ ) 168.9, 157.1, 157.0, 156.2,
155.6, 144.0, 141.3, 127.7, 127.1, 125.2, 120.0, 80.6, 80.5, 67.2,
51.5, 49.9, 49.6, 47.2, 42.7, 28.5; MS (ESI) m/z: 774 [(M + Na)+],
752 [(M + H)+], 652, 552, 452; HRMS (ESI) calcd for C40H57N5O9-
Na [(M + Na)+] 774.4048, found 774.4055.
10-[2-(9H-Fluoren-9-ylmethoxycarbonylamino)-propionyl]-
1,4,7,10-tetraaza-cyclododecane-1,4,7-tricarboxylic Acid Tri-t-
butyl Ester (29). Prepared in an identical manner to 28: 16 (750
mg, 1.5 mmol), 26 (544 mg, 1.7 mmol), HOBt (292 mg, 0.1.9
mmol), HATU (725 mg, 1.9 mmol), and DMAP (233 mg, 1.9
mmol) in DMF (2 mL) and DCM (2 mL) afforded 29 (777 mg,
64%) as a colorless oil: νmax(CH2Cl2)/cm-1 1693, 1651, 1466, 1414;
1H NMR (270 MHz, CDCl3): δ ) 7.76 (d, J ) 7.4 Hz, 2H), 7.59
(d, J ) 7.4 Hz, 2H), 7.40 (t, J ) 7.4 Hz, 2H), 7.31 (t, J ) 7.7 Hz,
2H), 5.69 (d, J ) 7.9 Hz, 1H), 4.63 (apparent q, J ) 7.4 Hz, 1H),
4.31 (d, J ) 6.9 Hz, 2H), 4.22-4.14 (m 1H), 3.81-3.05 (m, 16H),
1.54-1.31 (m, 30H); 13C NMR (67.5 MHz, CDCl3): δ ) 173.3
(C), 157.4 (C), 157.0 (C), 155.6 (C), 155.4 (C), 144.0 (C), 143.9
(C), 141.3 (C), 127.7 (CH), 127.1 (CH), 125.2 (CH), 120.0 (CH),
80.6 (C), 67.0 (CH2), 50.3 (CH2), 50.0 (CH2), 49.7 (CH2), 47.2
(CH), 28.5 (CH3), 19.8 (CH3); MS (ESI) m/z: 788 [(M + Na)+],
766 [(M + H)+], 666, 610, 566, 466; HRMS (ESI) calcd for
C41H60N5O9 [(M + H)+] 766.4386, found 766.4393. [R]25 ) +21.8
(c ) 5.0, CH2Cl2).
10-(2-Aminopropionyl)-1,4,7,10-tetraazacyclododecane-1,4,7-
tricarboxylic Acid Tri-t-butyl Ester (32). Compound 29 (777 mg,
1.02 mmol) was dissolved in a solution of TBAF (641 mg, 2.03
mmol) in acetonitrile (0.05 M) and stirred at room temperature for
90 min, and the reaction was followed by TLC. The reaction was
stopped by the addition of dichloromethane (100 mL), the organics
were washed with water (2 × 100 mL), and the combined aqueous
layers were washed with dichloromethane (2 × 100 mL). The
combined organics were dried over MgSO4 and removed in vacuo.
The crude material was purified by silica gel chromatography (1:
10 methanol/dichloromethane) to give 32 as a colorless oil (408
mg, 74%): νmax(CH2Cl2)/cm-1 1689, 1639; 1H NMR (CDCl3, 270
MHz): δ ) 3.70-3.31 (m, 17H), 2.22 (bs, 2H), 1.48 (s, 9H), 1.46
(s, 18H), 1.20 (d, J ) 9.8 Hz, 3H); 13C NMR (67.5 MHz, CDCl3):
δ ) 176.5 (C), 157.2 (C), 157.0 (C), 155.5 (C), 80.6 (C), 80.5 (C),
80.4 (C), 51.6 (CH2), 50.4 (CH2), 49.7 (CH2), 49.6 (CH2), 47.1
(CH), 28.5 (CH3), 21.7 (CH3); MS (ESI) m/z: 544 [(M + H)+],
488, 444, 345, 245; HRMS (ESI) calcd for C26H50N5O7 [(M + H)+]
544.3705, found 544.3705. [R]25 ) +39.1 (c ) 5.0, CH2Cl2).
2-Amino-1-(1,4,7,10-tetraazacyclododec-1-yl)-ethanone-tet-
rahydro-trifluoroacetate (34). Prepared in an identical manner to
35: 31 (169 mg, 0.32 mmol) and trifluoroacetic acid (0.99 mL,
13.4 mmol) in dichloromethane (10 mL) gave 34 (178 mg, 98%)
1
as a colorless oil: νmax(neat)/cm-1 1670, 1427; H NMR (D2O,
270 MHz): δ ) 4.09 (s, 2H), 3.76-3.62 (m, 4H), 3.42-3.12 (m,
12H); 13C NMR (67.5 MHz, D2O): δ ) 169.2, 47.0, 46.4, 45.9,
45.8, 45.0, 44.3, 43.5, 43.4, 40.8; MS (ESI) m/z: 230 [(M + H)+];
HRMS (ESI) calcd for C10H24N5O [(M + H)+] 230.1975, found
230.1973.
2-Amino-1-(1,4,7,10-tetraazacyclododec-1-yl)-propan-1-one-
tetrahydro-trifluoroacetate (35). Carbamate 32 (408 mg, 0.75
mmol) was dissolved in dichloromethane (33 mL) and treated with
trifluoroacetic acid (3.6 g, 31.62 mmol), and the solution was stirred
at room temperature overnight and then evaporated to give 35 (404
1
mg, 98%): νmax(CH2Cl2)/cm-1 1674; H NMR (D2O, 270 MHz):
δ ) 4.47 (q, J ) 6.9 Hz, 1H), 4.05-3.05 (m, 16H), 1.52 (d, J )
6.9 Hz, 3H); 13C NMR (D2O, 67.5 MHz): δ ) 172.7(C), 47.9-
(CH), 47.4(CH2), 46.8(CH2), 46.4(CH2), 46.2(CH2), 44.9(CH2),
44.4(CH2), 43.6(CH2), 43.3(CH2), 15.7(CH3); MS (ESI) m/z: 244-
[(M + H)+]; HRMS (ESI) calcd for C11H26N5O [(M + H)+]
244.2132, found 244.2130. [R]25 ) +10.7 (c ) 1.64, MeOH).
2-Amino-1-phenyl-(1,4,7,10-tetraazacyclododec-1-yl)-propan-
1-one-tetrahydro-trifluoroacetate (36). Prepared in an identical
manner to 35: 33 (73 mg, 0.118 mmol) and TFA (0.3 mL, 4.96
mmol) in dichloromethane (5 mL) gave 36 (71 mg, 96%); νmax(CH2-
1
Cl2)/cm-1 1670; H NMR (D2O, 270 MHz): δ ) 7.23-7.10 (m,
3H), 7.08-6.99 (m, 2H), 4.48 (t, J ) 7.4 Hz, 1H), 3.86-3.72 (m,
1H), 3.40-2.62 (m, 17H); 13C NMR (D2O, 67.5 MHz): δ ) 170.9,
133.8, 129.3, 128.9, 127.9, 51.7, 46.4, 46.2, 46.0, 45.5, 45.3, 44.1,
43.6, 37.2; MS (ESI) m/z: 320 [(M + H)+], 242, 120, 102; HRMS
(ESI) calcd for C17H30N5O [(M + H)+] 320.2445, found 320.2444.
10-{2-[5-(2-Oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pen-
tanoylamino]-acetyl}-1,4,7,10-tetraazacyclododecane-1,4,7-tri-
carboxylic Acid Tri-t-butyl Ester (37). To a stirred solution of
28 (124 mg, 0.235 mmol) in 1:1 dichloromethane/dimethylforma-
mide (4 mL) was added biotin (63 mg, 0.258 mmol), HATU (107
mg, 0.282 mmol), HOBt (43 mg, 0.282 mmol), and DMAP (34
mg, 0.282 mmol). The yellow solution was stirred at 40 °C for 2
days. The solvents were removed in vacuo, and the residue was
purified by silica gel chromatography (1:10 methanol/dichlo-
romethane) to give 37 as a colorless oil. (121 mg, 68%): νmax(CH2-
1
Cl2)/cm-1 1688, 1470, 1413; H NMR (CDCl3, 270 MHz): δ )
6.96 (bs, 1H), 6.29 (bs, 1H), 5.35 (bs, 1H), 4.47 (m, 1H), 4.52-
4.43 (m, 1H), 4.37-4.25 (m, 2H), 4.15-3.94 (m, 2H), 3.62-3.25
(m, 16H), 3.18-3.08 (m, 1H), 2.90 (dd, J ) 12.6, 4.7 Hz, 1H),
2.73 (d, J ) 12.6 Hz, 1H), 2.31-2.18 (m, 2H), 1.82-1.55 (m,
4H), 1.55-1.31 (s, 29H); 13C NMR (CDCl3, 67.5 MHz): δ ) 173.4,
169.7, 164.2, 157.2, 156.9, 155.7, 80.7, 80.5, 61.8, 60.3, 55.7, 51.4,
50.2, 49.8, 40.1, 40.6, 35.8, 28.5, 28.3, 28.2, 25.7; MS (ESI) m/z:
778 [(M + Na)+], 628, 572, 528, 428; HRMS (ESI) calcd for
C35H61N7O9SNa [(M + Na)+] 778.4144, found 778.4144.
5-(2-Oxo-hexahydro-thieno[3,4-d-imidazol-4-yl)-pentanoic Acid
[2-Oxo-2-(1,4,7,10-tetraazayclododec-1-yl)-ethyl]-amide-trihy-
dro-trifluoroacetate (38). Carbamate 37 (55 mg, 0.07 mmol) was
dissolved in dichloromethane (5 mL) and treated with trifluoroacetic
acid (0.35 g, 3.07 mmol), and the solution was stirred at room
temperature overnight and then evaporated to give 38 (56 mg,
97%): νmax(neat)/cm-1 1682, 1455, 1420; 1H NMR (D2O, 270
MHz): δ ) 4.64-4.55 (m, 1H), 4.46-4.37 (m, 1H), 4.10 (s, 2H),
3.83-3.63 (m, 4H), 3.38-3.14 (m, 12H), 2.98 (dd, J ) 12.9, 4.7
Hz, 1H), 2.76 (d, J ) 13.1 Hz, 1H), 2.34 (t, J ) 7.2 Hz, 2H),
1.78-1.35 (m, 6H); 13C NMR (CDCl3, 270 MHz): δ ) 177.9,
173.0, 165.6, 62.5, 60.5, 55.5, 48.9, 47.0, 46.1, 45.5, 45.0, 44.2,
43.6, 42.8, 41.7, 39.8, 35.0, 27.9, 27.8, 25.1; MS (ESI) m/z: 479
[(M + Na)+], 456 [(M + H)+]. HRMS (ESI) calcd for C20H38N7O3S
[(M + H)+], 456.2751, found 456.2753.
J. Org. Chem, Vol. 72, No. 22, 2007 8287

Krivickas et al.
11-(2-Benzyloxycarbonylamino-acetyl)-1,4,8,11-tetraaza-cy-
clotetradecane-1,4,8-tricarboxylic Acid Tri-t-butyl ester (41). To
a solution of 39 (1 mmol, 500 mg) in DCM (30 mL) were added
40 (1 mmol, 209 mg), DCC (1 mmol, 207 mg), and DMAP (1
mmol, 122 mg). The reaction was stirred at room temperature for
3 days, and the white precipitate was filtered. The solution was
concentrated in vacuo, and the crude material was purified by flash
chromatography on silica gel (EtOAc) to give 41 as a white solid
(690 mg, 100%). mp 79-83 °C; νmax(CH2Cl2)/cm-1 3055, 2985,
removed in vacuo, and the residue was dissolved in DCM and
washed with NaHCO3 (5%) and water. The organic phase was dried
over MgSO4, filtered, and concentrated, and the crude material thus
obtained was purified by flash chromatography on silica gel
(EtOAc) to give 45c as a white solid (815 mg, 80%): mp 44-47
°C; νmax(CH2Cl2)/cm-1 3055, 2985, 1710, 1685; 1H NMR (CDCl3,
270 MHz): δ ) 7.36-7.28 (m, 5H), 5.56 (bs, 1H), 5.07 (s, 2H),
3.38-3.14 (m, 14H), 2.57-2.47 (m, 2H), 2.46-2.32 (m, 4H),
1.88-1.54 (m, 6H), 1.44 (s, 1H), 1.42 (s, 9H); 13C NMR (CDCl3,
67.5 MHz): δ ) 156.4, 155.6, 155.3, 136.8, 128.3, 127.9, 127.8,
79.3, 66.1, 53.4, 52.7, 51.5, 47.7, 47.3, 46.7, 45.9, 39.6, 28.4, 26.6;
HRMS (ES) calcd for [M + H]+, (C36H62N5O8) 692.4593, found
692.4599.
11-(3-Amino-propyl)-1,4,8,11-tetraaza-cyclotetradecane-1,4,8-
tricarboxylic Acid Tri-t-butyl Ester (46c). Pd/C (10 mol %, 121
mg) was added to 45c (1.13 mmol, 780 mg) dissolved in MeOH
(50 mL), and the resulting mixture was stirred under H2 (1 atm)
for 16 h at room temperature. The crude mixture was filtered
through a short plug of Celite, and the solvent was removed in
vacuo to give 46c as a white solid (628 mg, 100%), which was
used without further purification: mp 47-49 °C; νmax(CH2Cl2)/
cm-1 3055, 2985, 1686; 1H NMR (CDCl3, 270 MHz): δ ) 3.46-
3.12 (m, 12H), 2.82-2.30 (m, 10H), 1.94-1.54 (m, 6H), 1.44 (s,
18H), 1.43 (s, 9H); 13C NMR (CDCl3, 67.5 MHz): δ ) 155.7
(2×C), 155.5 (C), 79.5 (3×C), 53.4 (CH2), 52.9 (CH2), 51.4 (CH2),
48.2 (2×CH2), 47.4 (m, 4×CH2), 45.8 (CH2), 40.2 (CH2), 28.5
(9×CH3), 26.6 (m, 2×CH2); HRMS (ES) calcd for [M + H]+,
(C28H56N5O6) 558.4225, found 558.4228.
1
1701, 1685, 1654; H NMR (CDCl3, 270 MHz): δ ) 7.36-7.27
(m, 5H), 5.79 (bs, 1H), 5.10 (s, 2H), 4.01 (s, 2H), 3.54-3.18 (m,
16H), 1.85-1.64 (m, 4H), 1.44 (s, 18H), 1.42 (s, 9H); 13C NMR
(CDCl3, 67.5 MHz): δ ) 167.8, 156.2, 156.0, 155.5, 136.5, 128.5,
128.1, 128.0, 79.9, 66.8, 50.0, 48.9, 48.4, 47.5, 46.9, 46.5, 46.0,
42.7, 42.5, 28.5, 27.7; HRMS (ES) calcd for [M + H]+, (C35H58N5O9)
692.4229, found 692.4229.
11-(2-Amino-acetyl)-1,4,8,11-tetraaza-cyclotetradecane-1,4,8-
tricarboxylic Acid Tri-t-butyl Ester (42). Pd/C (10 mol %, 87
mg) was added to 41 (0.81 mmol, 560 mg) dissolved in MeOH
(40 mL), and the resulting mixture was stirred under H2 (1 atm)
for 16 h at room temperature. The reaction mixture was filtered
through a Celite pad, and the solvent was removed in vacuo to
give 42 as a white solid (436 mg, 96%). The product was used
without further purification: mp 68-71 °C; νmax(CH2Cl2)/cm-1
3433, 3055, 2985, 1685; 1H NMR (CDCl3, 270 MHz): δ ) 3.62-
3.21 (m, 18H), 1.88-1.62 (m, 4H), 1.44 (s, 27H); 13C NMR (CDCl3,
67.5 MHz): δ ) 155.8, 155.5, 155.3, 80.3, 80.0, 79.7, 49.4, 48.2,
47.3, 46.9, 46.5, 46.3, 45.9, 44.9, 41.9, 28.3, 27.5; HRMS (ES)
calcd for [M + H]+, (C27H52N5O7) 558.3861, found 558.3855.
11-{2-[5-(2-Oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pen-
tanoylamino]-acetyl}-1,4,8,11-tetraaza-cyclotetradecane-1,4,8-
tricarboxylic Acid Tri-t-butyl Ester (43). Biotin (0.37 mmol, 89
mg), HATU (0.37 mmol, 139 mg), DIPEA (0.38 mmol, 67 µL),
and DMAP (3 mg, 10 mol %) were added to a solution of 42 (0.24
mmol, 136 mg) in DMF (10 mL). The solution was stirred for 24
h at room temperature. The solvent was evaporated in vacuo, and
the crude material was purified by column chromatography on silica
gel with CHCl3/MeOH (95:5 to 90:10) to afford 43 (156 mg, 83%)
as a pale yellow solid: mp 83-85 °C; νmax(CH2Cl2)/cm-1 1693,
1643, 1461, 1265; 1H NMR (CDCl3, 270 MHz): δ ) 7.17 (broad,
1H, NH), 6.66 (broad, 1H, NH), 5.65 (broad, 1H, NH), 4.53-4.43
(m, 1H), 4.34-4.25 (m, 1H), 4.18-3.92 (m, 2H), 3.54-3.20 (m,
16H), 3.17-3.05 (m, 1H), 2.89 (dd, J ) 4.2, 12.7 Hz, 1H), 2.73
(d, J ) 12.7 Hz, 1H), 2.34-2.20 (m, 2H), 1.87-1.54 (m, 8H),
1.43 (s, 27H); 13C NMR (CDCl3, 67.5 MHz): δ ) 173.7, 168.8,
164.5, 155.9, 155.6, 80.6, 80.0, 79.9, 61.8, 60.3, 55.8, 50.0-45.0,
40.9, 40.5, 35.8, 28.5, 28.1, 25.7; HRMS (ES) calcd for [M +H]+,
(C37H66N7O9S) 784.4637, found 784.4642.
11-{3-[5-(2-Oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pen-
tanoylamino]-propyl}-1,4,8,11-tetraaza-cyclotetradecane-1,4,8-
tricarboxylic Acid Tri-t-butyl Ester (47c). Biotin (374 mg, 1.67
mmol), HATU (634 mg, 1.67 mmol), DIPEA (580 µL, 3.33 mmol),
and DMAP (14 mg, 10 mol %) were preactivated in DMF (10 mL)
for 10 min at room temperature. Then, 46c (620 mg, 1.11 mmol)
was added, and the yellow solution was stirred for 24 h at room
temperature. The solvent was evaporated in vacuo, and the crude
material was purified by column chromatography on silica gel
(CHCl3/MeOH, 90:10 to 85:15) to give 47c as a pale yellow solid
(450 mg, 52%): mp 109-111 °C; νmax(CH2Cl2)/cm-1 1685, 1465,
1
1265; H NMR (CDCl3, 270 MHz): δ ) 6.24 (broad, 1H, NH),
5.49 (broad, 1H, NH), 4.53-4.42 (m, 1H), 4.34-4.23 (m, 1H),
3.46-3.04 (m, 15H), 2.96-2.82 (m, 1H), 2.71 (d, J ) 12.8 Hz,
1H), 2.58-2.47 (m, 2H), 2.46-2.24 (m, 4H), 2.24-1.98 (m, 4H),
1.92-1.48 (m, 10H), 1.43 (s, 27H); 13C NMR (CDCl3, 67.5
MHz): δ ) 173.5, 164.1, 155.6, 79.8, 61.9, 60.3, 55.8, 51.8, 48.1-
45.5 (m, 7×CH2), 40.6, 37.5, 36.0, 28.6, 28.4, 28.1, 26.5 (m,
2×CH2), 25.8; HRMS (ES) calcd for [M + H]+, (C38H70N7O8S)
784.5001, found 784.5000.
11-{2-[5-(2-Oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pen-
tanoylamino]-acetyl}-1,4,8,11-tetraaza-cyclotetradecane-trihy-
dro-trifluoroacetate (44). To a solution of 43 (120 mg, 0.15 mmol)
in DCM (10 mL), TFA (42 equiv, 6.2 mmol, 460 µL) was added,
and the resulting solution was stirred at room temperature overnight.
The solvent was evaporated in vacuo to give 43 as a pale yellow
solid (112 mg, 91%): mp 45-47 °C; νmax(CH2Cl2)/cm-1 3340
(broad), 3055, 3985, 1681; 1H NMR (D2O, 270 MHz): δ ) 4.66-
4.56 (m, 1H), 4.48-4.38 (m, 1H), 4.09 (s, 2H), 3.90-3.18 (m,
18H), 3.00 (dd, J ) 5.2 and 13.3 Hz, 1H), 2.77 (d, J ) 13.3 Hz,
1H), 2.35 (t, J ) 7.1 Hz, 2H), 2.30-2.06 (m, 4H), 1.78-1.36 (m,
6H); 13C NMR (D2O, 67.5 MHz): δ ) 177.7, 172.4, 165.5, 163.5
(TFA), 116.5 (TFA), 62.2, 60.5, 55.5, 46.1, 44.6, 44.2, 44.0, 42.7,
42.3, 41.8, 41.4, 39.8, 38.8, 35.2, 27.9, 25.2, 24.0, 19.0; HRMS
(ES) calcd for [M + H]+, (C22H42N7O3S) 484.3064, found 484.3064.
11-(3-Benzyloxycarbonylamino-propyl)-1,4,8,11-tetraaza-cy-
clotetradecane-1,4,8-tricarboxylic Acid Tri-t-butyl Ester (45c).
Aldehyde 20c (305 mg, 1.47 mmol) dissolved in THF (5 mL) was
added to a solution of 39 (882 mg, 1.76 mmol) in THF (15 mL).
Then, NaBH(OAc)3 (934 mg, 4.41 mmol) was added, and the
mixture was stirred at room temperature for 16 h. The solvent was
11-{3-[5-(2-Oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pen-
tanoylamino]-propyl}-1,4,8,11-tetraaza-cyclotetradecane-trihy-
dro-trifluoroacetate (48c). To a solution of 47c (219 mg, 0.28
mmol) in DCM (10 mL), TFA (42 equiv, 11.7 mmol, 873 µL) was
added, and the resulting solution was stirred at room temperature
overnight. The solvent was then evaporated in vacuo to give 48c
as a pale yellow solid (229 mg, 100%): mp 41-43 °C; νmax(CH2-
Cl2)/cm-1 3294 (broad), 3055, 3985, 1681 (broad); 1H NMR (D2O,
270 MHz): δ ) 4.61 (dd, J ) 4.7 and 7.9 Hz, 1H), 4.41 (dd, J )
4.4 and 7.9 Hz, 1H), 3.66-3.16 (m, 20H), 3.10 (m, 1H), 3.00 (dd,
J ) 4.7 and 12.8 Hz, 1H), 2.77 (d, J ) 12.8 Hz, 1H), 2.28 (t, J )
6.9 Hz, 2H), 2.18-1.98 (m, 4H), 1.97-1.79 (m, 2H), 1.78-1.28
(m, 6H); 13C NMR (D2O, 100 MHz): δ ) 177.6, 165.7, 163.3
(TFA), 116.7 (TFA), 62.4, 60.6, 55.8, 51.9, 49.8, 47.4, 45.2-42.0
(m, 4×CH2), 40.7 (2×CH2), 40.0, 36.6, 35.7, 28.3, 28.0, 25.4, 23.9,
20.9, 19.7; HRMS (ES) calcd for [M + H]+, (C23H46N7O2S)
484.3428, found 484.3430.
11-Carboxymethyl-1,4,8,11-tetraaza-cyclotetradecane-1,4,8-
tricarboxylic Acid Tri-t-butyl Ester (49). To a solution of 39 (500
mg, 1 mmol) in CH3CN (20 mL) were added Na2CO3 (126 mg,
1.1 mmol) and ethyl bromoacetate (134 µL, 1.1 mmol). The mixture
8288 J. Org. Chem., Vol. 72, No. 22, 2007

EffectiVe Methods for Biotinylation of Azamacrocycles
was stirred at reflux for 16 h. The reaction was cooled to room
temperature, and the insoluble salts were removed by filtration. The
solution was concentrated in vacuo, and the crude material was
purified by flash chromatography on silica gel (EtOAc) to give the
intermediate ester as a gummy solid (550 mg, 94%): νmax(CH2-
MHz): δ ) 173.1, 155.7 (3×C), 79.8, 79.6 (2×C), 59.6, 54.2, 52.9,
47.7 (2×CH2), 47.3 (2×CH2), 46.6, 46.2, 40.5, 37.8, 28.6 (9×CH3),
26.3 (2×CH2); HRMS (ES) calcd for [M + H]+, (C29H57N6O7)
601.4283, found 601.4287.
11-({2-[5-(2-Oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pen-
tanoylamino]-ethylcarbamoyl}-methyl)-1,4,8,11-tetraaza-cy-
clotetradecane-1,4,8-tricarboxylic Acid Tri-t-butyl Ester (53).
To a solution of 52 (0.373 mmol, 223 mg) in DMF (5 mL) were
added biotin (136 mg, 0.559 mmol), HATU (212 mg 0.559 mmol),
DIPEA (130 µL, 0.746 mmol), and DMAP (5 mg, 10 mol %). The
resulting yellow solution was stirred at room temperature for 40 h.
The solvent was removed in vacuo, and the crude material was
purified by flash chromatography on silica gel (CHCl3/MeOH 9:1
ramping to 8:2) to give 53 as a pale yellow solid (181 mg, 59%):
1
Cl2)/cm-1 2977, 2931, 1732, 1678; H NMR (270 MHz, CDCl3):
δ ) 4.10 (q, J ) 6.9, 14. Hz, 2H), 3.40-3.16 (m, 14H), 2.84-
2.78 (m, 2H), 2.66-2.56 (m, 2H), 1.94-1.78 (m, 2H), 1.72-1.58
(m, 2H), 1.43 (s, 27H), 1.23 (t, J ) 6.9 Hz, 3H); 13C NMR (CDCl3,
67.5 MHz): δ ) 170.9, 155.7, 155.6, 155.5, 79.5 (3×C), 60.2,
55.4, 53.6, 52.8, 51.8, 48.4, 47.4, 47.1, 46.8, 45.3, 28.4 (9×CH3),
27.0 (2×CH2), 14.3; HRMS (ES) calcd for [M + H]+, (C29H55N4O8)
587.4014, found 587.4011. The ester (0.43 mmol, 250 mg) was
dissolved in MeOH (5 mL), and a 1 N solution of NaOH (3 mL)
added. The solution was stirred for 2 h at room temperature. MeOH
was removed in vacuo, and the pH of the remaining aqueous
solution was adjusted to 5 by adding a 5% aq solution of citric
acid. The aqueous phase was extracted with DCM (3 × 20 mL),
and the organic phases were combined, dried over MgSO4, and
concentrated in vacuo to give 49 as a white solid (221 mg, 91%):
1
mp 85-87 °C; νmax(CH2Cl2)/cm-1 1687, 1463, 1419, 1265; H
NMR (CDCl3, 270 MHz): δ ) 7.28 (bs, 1H, NH), 6.52 (bs, 1H,
NH), 5.60 (bs, 1H, NH), 4.53-4.47 (m, 1H), 4.33-4.28 (m, 1H),
3.52-3.20 (m, 16H), 3.18-2.97 (m, 3H), 2.90 (dd, J ) 4.9 and
12.8 Hz, 1H), 2.73 (d, J ) 12.8 Hz, 1H), 2.68-2.56 (m, 2H), 2.56-
2.44 (m, 2H), 2.19 (t, J ) 6.6 Hz, 2H), 1.96-1.54 (m, 10H), 1.44
(s, 18H), 1.42 (s, 9H); 13C NMR (CDCl3, 100 MHz): δ ) 173.0,
171.3, 163.3, 154.6 (3×C), 79.1, 78.9 (2×C), 60.7, 59.2, 58.4, 54.8,
52.1, 51.9, 46.9 (2×CH2), 46.6 (2×CH2), 45.8, 45.4, 39.6, 38.8
(2×CH2), 34.9, 27.8, 27.5 (9×CH3), 27.4, 27.2, 25.1 (2×CH2), 24.6;
HRMS (ES) calcd for [M + H]+, (C39H71N8O9S) 827.5059, found
827.5048.
1
mp 89-91 °C; νmax(CH2Cl2)/cm-1 3300-2700 (broad), 1685; H
NMR (270 MHz, CDCl3): δ ) 3.45-3.12 (m, 14H), 2.82-2.48
(m, 4H), 1.94-1.60 (m, 4H), 1.50-1.24 (m, 27H); 13C NMR
(CDCl3, 67.5 MHz): δ ) 172.1, 156.3, 155.3 (2×C), 80.5, 79.9
(2×C), 56.5, 53.9, 52.6, 47.7, 47.5 (2×CH2), 46.6, 46.0, 28.5
(9×CH3), 26.4 (2×CH2); HRMS (ES) calcd for [M + H]+,
(C27H51N4O8) 559.3701, found 559.3705.
11-({2-[5-(2-Oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pen-
tanoylamino]-ethylcarbamoyl}-methyl)-1,4,8,11-tetraaza-cy-
clotetradecane-trihydro-trifluoroacetate (54). To a solution of
53 (0.121 mmol, 100 mg) in DCM (3 mL), TFA (5.08 mmol,
502 µL) was added, and the solution was stirred overnight at room
temperature. The solvent was removed in vacuo to give 54 as a
pale yellow semisolid (97 mg, 92%): νmax(CH2Cl2)/cm-1 3312
11-[(2-Benzyloxycarbonylamino-ethylcarbamoyl)-methyl]-
1,4,8,11-tetraaza-cyclotetradecane-1,4,8-tricarboxylic Acid Tri-
t-butyl Ester (51). To a solution of 49 (0.423 mmol, 236 mg) and
50 (164 mg, 0.846 mmol) in DCM (30 mL), DCC (87 mg, 0.423
mmol) and DMAP (52 mg, 0.423 mmol) were added. The mixture
was stirred overnight at room temperature, the insoluble salts that
had formed were removed, and the resulting solution was washed
with brine. The organic phase was dried over MgSO4, filtered, and
concentrated in vacuo. The crude material was purified by flash
chromatography on silica gel (EtOAc/n-Hex 7:3 ramping to EtOAc
1
(broad), 1682 (broad); H NMR (D2O, 270 MHz): δ ) 4.64 (m,
1H), 4.48-4.40 (m, 1H), 3.46-2.92 (m, 21H), 2.90-2.74 (m, 3H),
2.28 (pseudo-t, J ) 6.4 Hz, 2H), 2.10-1.88 (m, 4H), 1.82-1.34
(m, 6H); 13C NMR (D2O, 100 MHz): δ ) 180.1, 176.3, 168.2,
165.8 (TFA), 119.1 (TFA), 65.0, 63.2, 58.3, 57.9, 57.2, 56.2, 50.1,
49.3, 48.4, 47.6, 45.9, 42.6, 42.1, 40.9, 38.4, 36.5, 30.9, 30.6, 28.0,
26.9, 25.5; HRMS (ES) calcd for [M + H]+, (C23H46N7O2S)
484.3428, found 484.3430.
100%) to give 51 as a white solid (248 mg, 84%): mp 81-83 °C;
1
ν
max(CH2Cl2)/cm-1 1705, 1686; H NMR (270 MHz, CDCl3): δ
) 7.37-7.25 (m, 5H), 5.88 (bs, 1H, NH), 5.06 (s, 2H), 3.48-3.18
(m, 18H), 3.05 (s, 2H), 2.66-2.56 (m, 2H), 2.56-2.46 (m, 2H),
1.88-1.60 (m, 4H), 1.44 (s, 18H), 1.40 (s, 9H); 13C NMR (CDCl3,
67.5 MHz): δ ) 172.0, 156.9, 155.7 (3×C), 136.7, 128.5, 128.0
(2×CH), 80.3, 80.0 (2×C), 66.6, 59.5, 54.2, 53.0, 48.3, 48.0, 47.5,
47.2, 46.9, 46.8, 41.4, 39.4, 28.5 (9×CH3), 26.6 (2×CH2); HRMS
(ES) calcd for [M + H]+, (C37H63N6O9) 735.4651, found 735.4648.
11-[(2-Amino-ethylcarbamoyl)-methyl]-1,4,8,11-tetraaza-cy-
clotetradecane-1,4,8-tricarboxylic Acid Tri-t-butyl Ester (52).
Pd/C (10 mol %, 34 mg) was added to 51 (0.313 mmol, 230 mg)
dissolved in MeOH (15 mL), and the resulting mixture was stirred
under H2 (1 atm) for 16 h at room temperature. The crude mixture
was filtered through a short plug of Celite, and the solvent was
removed in vacuo to give 52 as a white solid (169 mg, 90%) that
could be used without further purification: mp 49-51 °C; νmax(CH2-
Acknowledgment. We are grateful to the BBSRC (68/
E19752), EPSRC GR/T17014/01, and the University of Sydney
Cancer Research Fund for financial support. We also gratefully
acknowledge the EPSRC National Mass Spectrometry Service,
University of Wales Swansea.
Supporting Information Available: Synthesis and character-
ization data for 6, 10a, 10b, A-C, 19b, 22c, 30, 31, 33, and 50;
1H and 13C NMR data for 6, 10a,b, 11, 12, A-C, 13, 14, 18-24,
28-32, 34-38, and 41-54; 2-D NMR data for 23a; mass spectral
data for copper complexes of ligands 35, 36, and 38; and UV-vis
data for copper complexes of ligand 35. This material is available
free of charge via the Internet at http://pubs.acs.org.
1
Cl2)/cm-1 3680, 3433, 1686; H NMR (CDCl3, 270 MHz): δ )
3.66-3.50 (m, 2H), 3.46-3.04 (m, 16H), 2.74-2.48 (m, 4H),
1.90-1.54 (m, 4H), 1.50-1.30 (m, 27H); 13C NMR (CDCl3, 67.5
JO071175V
J. Org. Chem, Vol. 72, No. 22, 2007 8289