An assembly concept for the consecutive introduction of unsymmetrical disulfide bonds: Synthesis of a releasable multidrug conjugate of folic acid

Article abstract of DOI:10.1021/jo070411z

(Chemical Equation Presented) We describe the development of methodology which allows for the introduction of a second disulfide bond into a molecular framework with a pre-existing disulfide linker system. Compounds which contain an S-9-fluorenylmethyl-pr

Full text of DOI:10.1021/jo070411z

An Assembly Concept for the Consecutive Introduction of
Unsymmetrical Disulfide Bonds: Synthesis of a Releasable
Multidrug Conjugate of Folic Acid
Iontcho R. Vlahov,* Hari Krishna R. Santhapuram, Yu Wang, Paul J. Kleindl, Fei You,
Stephen J. Howard, Elaine Westrick, Joseph A. Reddy, and Christopher P. Leamon
Endocyte Inc., 3000 Kent AVenue, Suite A1-100, West Lafayette, Indiana 47906
iVlahoV@endocyte.com
ReceiVed February 28, 2007
We describe the development of methodology which allows for the introduction of a second disulfide
bond into a molecular framework with a pre-existing disulfide linker system. Compounds which contain
an S-9-fluorenylmethyl-protected thiol and an additional disulfide linkage are deprotected in situ and
trapped with an activated thiophile. This methodology allowed for the synthesis of the first molecule
possessing two different biologically active agents covalently attached to a folate receptor targeting ligand
unit Via two disulfide-based release systems.
Herein, we describe a novel synthetic strategy which allows
for the introduction of a second reductively labile disulfide linker
system (or simply a disulfide bond) into a molecular framework
with a pre-existing disulfide linker system. Such methodology
allows for the delivery and release of multiple cargos (parent
drugs, reporter molecules, etc.) in targeted cells.
two different biologically active agents: dAc-VLB-mH and the
highly chemically sensitive alkylating agent mitomycin C
(MMC). Both cargos are covalently attached to a FA-Spacer
unit Via two disulfide-based release systems.
The selection of synthons with suitable protecting and leaving
groups, orthogonally stable toward multiple functional groups,
and their application in an environment of highly charged
molecules was of crucial importance to the synthetic design.
As indicated in the retrosynthetic analysis (Scheme 1), the key
precursor to the final construct I (simplified representation of
dual drug conjugate 1) is the thiol-containing disulfide conjugate
III. Molecules such as III, which possess a disulfide bond as
well as a free thiol functionality, are highly reactive, unstable,
and prone to rearrangement (VI) and intramolecular cyclization
(VII), thereby producing multiple side products. An elegant
solution to this challenging problem involves a synthetic route
in which III is generated in situ from the stable S-protected
monoconjugate IV. Trapping the thiol group of III with a Drug
2-containing, disulfide-forming synthon II should result in the
desired dual drug conjugate I. Monoconjugate IV would be
synthesized using conjugation techniques which we have
previously described.1
Previously we reported1 a disulfide-based folate conjugate
of the potent antimitotic agent desacetyl vinblastine monohy-
drazide (dAc-VLB-mH) which is composed of a single cytotoxic
molecule attached through a spacer and one releasable disulfide
linker to folic acid (Drug-Linker-Spacer-FA). This conjugate
exploits the folate receptor (FR), which is overexpressed in
several forms of cancer, as a molecular “Trojan horse” for
targeted delivery.2 To improve upon the encouraging biological
activity we saw with this compound,3 we wished to synthesize
a new folate conjugate with two releasable drug cargos with
disparate mechanisms of action. Our efforts have resulted in
the synthesis of 1 (Figure 1). 1 is the first molecule possessing
* Author to whom correspondence should be addressed. Phone: 765-807-
0619. Fax: 765-463-9271.
(1) Vlahov, I. R.; Santhapuram, H. K. R.; Kleindl, P. J.; Howard, S. J.;
Stanford, K. M.; Leamon, C. P. Bioorg. Med. Chem. Lett. 2006, 16, 5093.
(2) Leamon, C. P.; Reddy, J. A. AdV. Drug DeliVery ReV. 2004, 56, 1127.
(3) Reddy, J. A.; Dorton, R.; Westrick, E.; Dawson, A.; Smith, T.; Xu,
L. C.; Vetzel, M.; Kleindl, P. J.; Vlahov, I. R.; Leamon, C. P. Cancer Res.
2007, 67, 4434.
Proper selection of the protecting group for the thiol
functionality is key to the successful implementation of this
synthetic strategy. An ideal protecting group should be (i) easy
10.1021/jo070411z CCC: $37.00 © 2007 American Chemical Society
Published on Web 06/29/2007
5968
J. Org. Chem. 2007, 72, 5968-5972

ConsecutiVe Introduction of Unsymmetrical Disulfide Bonds
SCHEME 1
aminopropionic acid moiety prior to resin cleavage. Assembly
of monoconjugate 5 was performed by the addition of a solution
of the activated vinblastine disulfide 4 in THF to a pH-adjusted
(∼6.9) solution of the FA-Spacer 3 in water.1 5 was isolated
in excellent yield (75%) after preparative HPLC.
With monoconjugate 5 in hand, we attempted to remove the
S-Fm group in the presence of the known disulfide forming
MMC derivative 6.6 Our attempts to utilize the two common
deprotection protocols were of only limited success. First, the
removal of the Fm moiety using piperidine7 was slow and
generated multiple side products. Second, applying 2-tert-butyl-
1,1,3,3-tetramethylguanidine8 (TBTMG) in lieu of piperidine,
as suggested by the work of Corey et al., resulted in a complex
mixture of the desired conjugate 1, cyclic disulfide 8, and
2-pyridyl-disulfide 9 (Scheme 3 and Figure 2). One of our
original ideas for the deprotection of the Fm group was to use
DBU as a strong non-nucleophilic base. In the presence of 6,
these novel reaction conditions provided in less than 5 min a
mixture of 1 and 9 as a major side product. We concluded that
after the formation of the desired bis-disulfide 1, the released
2-thiopyridine acted as a thiophile and attacked the newly
generated disulfide bond in 1 to form 9. A concise solution to
this synthetic problem was developed using the known MMC
derivative 7.6 The presence of the strong electron-withdrawing
nitro group on the C-3-atom in the 2-thiopyridine moiety in 7
dramatically reduced the thiophilic and/or nucleophilic power
of the leaving group. While fine-tuning the reaction conditions
of the final conjugation step, we found that the addition of i-Pr2-
NEt to a DMF solution of the fully protonated form of 5 and 7,
followed by DBU, resulted in a clean and fast conversion to
the desired product. According to the HPLC profile (Figure 3),
the reaction was complete in 5 min. HPLC purification gave
the dual drug conjugate 1 in 88% yield.
We have found this methodology for introducing a second
disulfide bond to be superior to methods in which the second
disulfide linker-drug moiety is constructed separately and then
appended to the peptidic backbone of a monoconjugate. For
example, acylation of the C-3-amino group in the 2,3-di-
aminopropionic acid moiety in 10 using MMC-(CH2)2SS(CH2)2-
CO2H was hampered by poor, irreproducible yields (Scheme
4, first reaction). The extreme instability of this MMC derivative
and its tendency, after activation of the carboxy group, toward
self-condensation through the aziridine group was the likely
cause of this difficulty. We have also attempted to apply the
Huisgen reaction in a “click” chemistry protocol as described
by Sharpless et al.9 to synthesize a number of other dual drug
conjugates (Scheme 4, second reaction). A propargylglycine
moiety was incorporated into the peptidic backbone of a
monoconjugate (11), and a (O)CO(CH2)2SS(CH2)2N3 releasable
linker moiety was added to an appropriate nucleophilic site of
a second drug (12), to provide the alkyne and azide for the
dipolar [2 + 3] cycloaddition reaction. Unfortunately, only
moderate yields of 1,2,3-triazole regioisomeric dual drug
conjugates were obtained.10
to introduce in a solid-phase peptide synthesis (SPPS) protocol
on a suitable resin, (ii) stable under the strong acidic conditions
applied for the removal of the peptidic moiety from the resin
with simultaneous removal of all other protecting groups, and
(iii) possessing sufficient chemoselectivity to ensure a fast and
regioselective in situ generation and concomitant trapping of
the thiol group by an external thiophile in the presence of a
existing disulfide moiety. The rarely used S-9-fluorenylmethyl
thioether (Fm-SR)4 satisfies all of these criteria.
The synthesis of 1 began with the construction of monocon-
jugate 5 from peptide-based derivative 3 (Scheme 2). Peptide-
based derivative 3 is a novel FA-Spacer unit designed to
introduce a discrete number of charged amino acids (Asp). As
such, 3 tailors the water-solubility and suppresses the passive
diffusion of the FA-Spacer-Linker-Drug system across the
lipophilic cell membrane, thereby decreasing undesirable de-
livery to cells not expressing the folate receptor. Pteroic acid5
served as the N-terminus, whereas the thiol group of cysteine
provided attachment site for one of the cleavable linkers. A
second attachment site was achieved by the addition of S-Fm-
3-thiopropionic acid to the â-amino functionality of the di-
The biological activity of 1 was evaluated by treating mice
bearing established, subcutaneous human KB tumor xenografts
(6) Vyas, D. M.; Chiang, Y.; Doyle, T. W. Ger Pat. 3413489, 1984.
(7) Ruiiz-Gayo, M.; Albericio, F.; Pedroso, E.; Eiralt, E. J. Chem. Soc.,
Chem. Commun. 1986, 20, 1501.
(4) Bodanszky, M.; Bednarek, M. A. Int J. Pept. Protein Res. 1982, 20,
434.
(5) Xu, L.; Vlahov, I. R.; Leamon, C. P.; Santhapuram, H. K. R.; Li, C.
PTC Int. Appl. WO2006/101845, 2006.
(8) Corey, E. J.; Gin, D. Y.; Kania, R. S. J. Am. Chem. Soc. 1996, 118,
9202.
(9) (a) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int.
Ed. 2001, 40, 2004.
J. Org. Chem, Vol. 72, No. 16, 2007 5969

Vlahov et al.
FIGURE 1. The dAc-VLB-mH- and MMC-containing folate conjugate.
FIGURE 2. Major byproducts resulting from the addition of MMC derivative 6 to monoconjugate 5.
SCHEME 2
(a) 2% NH2NH2 in DMF, 5 min, rt. (b) FmS(CH2)2CO2H, PyBoP, i-Pr2NEt, DMF. (c) TFA, H2O, TIPS, EDT, 65% overall yield from step a. (d) THF/
H2O (1:1), 75%. (e) NH2NH2, MeOH, ∆, 15 h, 78%. (f) PySS(CH2)2OC(O)OBt, TEA, DCM, 83%.
intravenously with 1 following the schedule indicated (Figure
4). 1 convincingly displayed potent antitumor activity, where
five out of five of treated animals were determined to be tumor
free by Day-26. Importantly, animals did not lose any weight
at any time during or after therapy, demonstrating a significant
reduction in toxicity to normal cells. The activity of 1 is superior
to that of its monodrug-folate counterparts (folate-MMC and
folate-dAc-VLB-mH) at equimolar concentrations and when
the monodrug-folate conjugates are used in combination, as
all of these treatment protocols failed to produce any complete
responses (n ) 5 in each study). These results, as well as others
which will be reported simultaneously in an appropriate
scientific journal, have led to the advancement of 1 to Phase 1
clinical trials.
Experimental Section
Synthesis of Folate-Peptide Linker. Pte-γGlu-Asp-Asp-Asp-
Dap(S-Fm-3-thiopropionyl)-Cys-OH 3. In a peptide synthesis
vessel H-Cys(4-methoxytrityl)-2-chlorotrityl-resin (1.0 g, 0.56 mM)
was loaded and washed with i-PrOH (3 × 10 mL) followed by
DMF (3 × 10 mL). To the vessel were then introduced the amino
acid solution (2.0 equiv) in DMF, i-Pr2NEt (4.0 equiv), and PyBOP
(10) Unpublished results.
5970 J. Org. Chem., Vol. 72, No. 16, 2007

ConsecutiVe Introduction of Unsymmetrical Disulfide Bonds
SCHEME 3
FIGURE 3. HPLC profile of the addition of 5 and 7 under DBU
conditions at t ) 5 min. The major peak is the desired product 1.
(2.0 equiv). Argon was bubbled for 1 h, the coupling solution was
drained, and the resin was washed with DMF (3 × 10 mL) and
i-PrOH (3 × 10 mL). Kaiser tests were performed to assess reaction
completion. Fmoc deprotection was carried out using 20% piperi-
dine in DMF (3 × 10 mL), before each amino acid coupling. The
above sequence was repeated to complete six coupling steps. At
the end, the resin was washed with 2% hydrazine in DMF 3 × 10
mL (5 min) to cleave the trifluoroacetyl protecting group on pteroic
acid and the IvDde protecting group on diaminopropionic acid.
Finally, S-Fm-3-thiopropionic acid (1.25 equiv) in DMF using i-Pr2-
NEt (2.5 equiv) and PyBop (1.25 equiv) was coupled. Argon was
bubbled for 1 h, the coupling solution was drained, and the resin
was washed with DMF (3 × 10 mL) and i-PrOH (3 × 10 mL).
The resin was dried under argon for 30 min. Folate-peptide was
cleaved from the resin using a cleavage mixture consisting of 92.5%
CF3CO2H, 2.5% H2O, 2.5% triisopropylsilane, and 2.5% ethanedi-
thiol. A 25 mL amount of the cleavage mixture was introduced,
and argon was bubbled for 1.5 h. The cleavage mixture was drained
into a clean flask. The resin was washed three times with more
cleavage mixture. The combined mixture was concentrated under
reduced pressure to a smaller volume (∼ 5 mL) and precipitated
in ethyl ether. The precipitate was collected by centrifugation,
washed with ethyl ether (3 times), and dried under high vacuum.
The crude product was purified by preparative HPLC (mobile phase
A ) 10 mM ammonium acetate, pH ) 5; organic phase B )
FIGURE 4. Effect of 1 on KB tumor volume in mice. Treatment began
9 days after tumor implantation.
acetonitrile; method: 1% B to 20% B in 40 min at 15 mL/min)
and furnished folate-peptide spacer 3 (∼450 mg, 65%). 1H NMR
(300 MHz, DMSO-d6 with D2O): δ 8.60 (s, 1H), 7.78 (d, J ) 7.5
Hz, 2H), 7.71-7.67 (m, 2H), 7.56 (d, J ) 8.7 Hz, 2H), 7.31-7.25
(m, 4H), 6.61 (d, J ) 8.7 Hz, 2H), 4.51-4.38 (m, 5H), 4.35 (t, J
) 6.9 Hz, 1H), 4.19-4.09 (m, 3H), 3.28-3.04 (m, 4H), 2.84-
2.44 (m, 12H), 2.27-1.94 (m, 4H). 13C NMR (75.5 MHz, DMSO-
d6): 173.8, 172.0 (4C), 171.5, 171.1 (2C), 170.9, 170.3, 169.6,
166.4, 161.0, 156.5, 153.8, 150.8, 148.6 (2C), 146.0 (2C), 140.5
(2C), 129.1 (2C), 128.0, 127.4 (2C), 127.0 (2C), 125.0 (2C), 121.3,
119.9 (2C), 111.2 (2C), 54.6, 52.7, 52.2, 49.9, 49.8, 49.6, 46.3,
45.9, 36.1, 36.0, 35.9, 35.7, 35.1 (2C), 32.0, 27.9, 26.6, 25.4. HRMS
(MALDI): (M + H)+ ) Calculated for C54H60N13O18S2,1242.3518;
found 1242.3319.
3-(4-Desacetylvinblastinyl)hydrazinecarboxylic Acid (2′-Py-
ridyldithio)ethyl Ester 4. To a solution of 4-desacetyl-3-vinblastine
monohydrazide (200 mg, 0.260 mmol) and 2-[benzotriazole-1-yl-
SCHEME 4
J. Org. Chem, Vol. 72, No. 16, 2007 5971

Vlahov et al.
(oxycarbonyloxy)-ethyldisulfanyl]-pyridine (110 mg, 0.286 mmol)
in CH2Cl2 (5 mL) was added TEA (0.087 mL, 0.62 mmol). The
reaction was allowed to stir for 2 h. TLC (15% MeOH in CH2Cl2)
indicated that the reaction was complete. The reaction mixture was
diluted with CH2Cl2 and washed with 10% aqueous K2CO3. The
organic layer was dried over MgSO4 and concentrated to a small
volume. This material was then loaded onto a SiO2 column and
chromatographed (10% MeOH in CH2Cl2) to yield pure 3-(4-
desacetylvinblastinyl)hydrazinecarboxylic acid (2′-pyridyldithio)-
ethyl ester 4 (204 mg, 83%). 1H NMR (300 MHz, CD2Cl2): δ 8.44
(d, J ) 5.0 Hz, 1H), 8.15 (s, br, 1H), 7.78-7.70 (m, overlapped,
2H), 7.49 (d, J ) 7.6 Hz, 1H), 7.14 (m, 2H), 7.06 (m, 1H), 6.61 (s,
1H), 6.14 (s, 1H), 5.84 (dd, J ) 10.5, 3.8 Hz, 1H), 5.70 (d, J )
10.5 Hz, 1H), 4.37 (t, J ) 6.3 Hz, 2H), 4.01 (s, 1H), 3.93 (d, J )
12.3 Hz, 1H), 3.79 (s, 3H), 3.60 (s, 3H), 3.55 (s, 1H), 3.41 (d, J )
13.0 Hz, 1H), 3.33-3.12 (m, 2H), 3.08 (t, J ) 6.4 Hz, 2H), 2.84-
2.80 (overlapped, CH3 + 2H), 2.65 (s, 1H), 2.47-2.39 (m, 2H),
2.22 (dd, J ) 15.5, 3.2 Hz, 1H), 2.08-1.98 (m, 1H), 1.81-1.62
(m, 2H), 1.52-1.22 (m, 4H), 0.95-0.87 (overlapped, CH3+ CH3).
LCMS (ESI): (M + H)+ ) Calculated for C51H64N7O9S2, 982.41;
found 982.20
Pte-γGlu-Asp-Asp-Asp-Dap(S-Fm-3-thiopropionyl)-Cys(S-
ethyl-3-(4-desacetylvinblastinyl)hydrazinecarboxylate) 5. In a
polypropylene centrifuge bottle, Pte-γGlu-Asp-Asp-Asp-Dap(S-
Fm-3-thiopropionyl)-Cys-OH 3 (112 mg, 0.09 mmol) was dissolved
in 7.5 mL of water and bubbled with argon for 10 min. In another
flask, a 0.1 N NaHCO3 solution was bubbled with argon for 10
min, and the pH of the folate linker solution was carefully adjusted
to 6.9 using the 0.1 N NaHCO3 solution. The 3-(4-desacetylvin-
blastinyl)hydrazinecarboxylic acid 2-pyridyldithioethyl ester 4 (88
mg, 0.09 mM) in 7.5 mL of tetrahydrofuran (THF) was added to
the above solution. The resulting clear solution was stirred under
argon for 15 min to 1 h. Progress of the reaction was monitored by
analytical HPLC (10 mM ammonium acetate, pH ) 7.0 and
acetonitrile). THF was removed under reduced pressure, and the
aqueous solution was filtered and injected on a prep-HPLC column.
Elution with 1 mM sodium phosphate (pH ) 7.0) buffer A and
acetonitrile B (method: 1% B to 80% B in 25 min at 25 mL/min)
resulted in pure fractions containing the product. Pure fractions were
pooled, acetonitrile was removed under reduced pressure at ambient
temperature, and pH was adjusted to 4.0 using 0.1 N HCl.
Vinblastine-folate conjugate 5 was isolated after freeze-drying for
48 h (157 mg, 75%). 1H NMR (300 MHz, DMSO-d6 with D2O) δ
8.60 (s, 1H), 7.78 (d, J ) 6.9 Hz, 2H), 7.68-7.65 (m, 2H), 7.58
(d, J ) 8.7 Hz, 2H), 7.46-7.22 (m, 6H), 7.07-6.95 (m, 2H), 6.61
(d, J ) 8.4 Hz, 2H), 6.36 (s, 1H), 6.19 (s, 1H), 5.68 (m, 1H), 5.58
(d, J ) 10.2 Hz, 1H), 4.56-4.09 (m, 11H), 3.83-1.85 (m, 50H),
1.56 (br s, 2H), 1.38-1.26 (m, 5H), 0.79 (t, J ) 6.9 Hz, 3H), 0.70
(t, J ) 7.2 Hz, 3H). 13C NMR (75.5 MHz, DMSO-d6): 174.5, 173.9,
172.9, 172.3, 172.1, 172.0, 171.8, 171.4, 171.2, 171.0, 170.8, 170.0,
169.7, 166.4, 161.3, 157.4, 155.78, 155.77, 154.2, 152.5, 150.8,
148.7, 148.5, 146.0 (2C), 140.5 (2C), 135.5, 131.9, 131.6, 129.0
(2C), 128.4, 127.8, 127.3 (2C), 127.0 (2C), 125.0 (2C), 123.4, 122.6,
122.5, 121.5, 121.4, 119.9 (2C), 118.8, 118.4, 117.8, 114.1, 111.7,
111.3 (2C), 92.9, 82.8, 80.5, 73.5, 67.0, 64.6, 62.4, 62.0, 56.1 (2C),
54.9 (2C), 52.8 (2C), 52.5 (2C), 52.2 (2C), 52.1 (2C), 50.0, 49.8,
49.4, 48.9, 46.2 (2C), 45.9, 45.5, 45.0, 42.0, 37.8, 37.0, 36.4, 35.9
(2C), 35.7, 35.1 (2C), 34.2, 31.9, 31.8, 27.8 (2C), 26.7, 8.1, 7.0.
HRMS (MALDI): (M + H)+ ) Calculated for C100H118N19O27S3,
2112.7500; found 2112.7701.
Pte-γGlu-Asp-Asp-Asp-Dap{S-[7-N-(2-mercaptoethyl)mito-
mycin C]-3-thiopropionyl}-Cys(S-ethyl-3-(4-desacetylvinblasti-
nyl)hydrazinecarboxylate) 1. Anhydrous DMF (4.5 mL) was
syringed into a mixture of Pte-γGlu-Asp-Asp-Asp-Dap(S-Fm-3-
thiopropionyl)-Cys(S-ethyl-3-(4-desacetylvinblastinyl)hydrazine-car-
boxylate) 5 (103 mg, 48.7 µmol) and N7-((3′-nitropyridyl-2′-
yl)dithioethyl)mitomycin C 7 (33.4 mg, 1.25 equiv) at room
temperature under argon. To the resulting solution were syringed
i-Pr2NEt (84.9 µL, 10 equiv) and DBU (72.9 µL, 10 equiv.) in
tandem. The reaction mixture was stirred at room temperature under
argon for 20 min. Analytical HPLC (mobile phase A ) 1.0 mM
phosphate buffer, pH 7.0; organic phase B ) acetonitrile; method:
5% B to 50% B in 10 min at 1 mL/min) of the reaction mixture
confirms the completion of the reaction. The reaction mixture was
transferred into stirring diethyl ether (50 mL). The resulting
suspension was centrifuged, and the precipitate was washed with
diethyl ether (15 mL × 2), dissolved in phosphate buffer (9 mL,
1.25 mM, pH 6.8), and subjected to preparative HPLC (mobile
phase A ) 1.0 mM phosphate buffer, pH 7.0; organic phase B )
acetonitrile; method: 10% B to 40% B in 25 min at 25 mL/min).
Pure fractions were collected, and acetonitrile was removed under
reduced pressure and freeze-dried to afford 99.2 mg (88%) of dual
drug conjugate 1. 1H NMR (300 MHz, DMSO-d6 with D2O) δ 8.61
(s, 1H), 7.51 (d, J ) 8.7 Hz, 2H), 7.35 (d, J ) 7.8 Hz, 1H), 7.22
(d, J ) 8.1 Hz, 1H), 7.00 (t, J ) 7.8 Hz, 1 H), 6.95 (t, J ) 8.1 Hz,
1H), 6.61 (d, J ) 9.0 Hz, 2H), 6.38 (s, 1H), 6.18 (s, 1H), 5.71 (m,
1H), 5.57 (d, J ) 10.2 Hz, 1H), 4.54-4.39 (m, 5H), 4.30-4.19
(m, 5H), 4.06-3.96 (m, 4H), 3.77 (br s, 3H), 3.51 (s, 3H), 3.39-
3.34 (m, 4H), 3.24-3.20 (m, 3H), 3.08 (s, 6H), 2.96-2.83 (m,
8H), 2.74 (s, 3H), 2.70-2.28 (m, 21H), 2.08-1.69 (m, 10H), 1.54
(br s, 2H), 1.32-1.15 (m, 5H), 0.77 (t, J ) 7.2 Hz, 3H), 0.71 (t, J
) 7.5 Hz, 3H). 13C NMR (125.71 MHz, DMSO-d6/D2O): 178.7,
176.9, 176.5 (4C), 176.3, 175.1, 174.4, 173.5, 173.1 (2C), 173.0,
170.7, 167.5, 164.1, 158.5 (2C), 158.3, 157.4, 156.4, 155.9, 155.5,
153.5, 151.5, 150.3, 149.7, 148.7, 136.0, 131.9, 131.6, 129.8 (2C),
129.3, 128.4, 124.8, 124.2, 123.5, 123.2, 122.5, 119.9, 119.0, 116.1,
112.7 (2C), 112.3, 109.9, 106.8, 104.1, 94.1, 83.4, 81.3, 74.5, 73.0,
68.4, 65.9, 64.0, 63.5, 62.7, 62.5, 62.2, 57.0 (3C), 55.9, 55.3, 54.9,
54.1, 53.7 (2C), 53.4, 52.2 (3C), 51.5, 50.8, 50.5, 50.4 (3C), 50.0,
46.5, 45.7, 44.1 (2C), 43.7, 42.9, 42.2, 41.1 (2C), 38.3, 36.4, 35.9,
35.1, 34.4, 32.9 (3C), 29.3 (2C), 10.5, 9.1, 7.8. LCMS (ESI): (M
+ H)+ ) Calculated for C103H128N23O32S4, 2327.80; found 2327.93
Acknowledgment. Part of this work was supported by SBIR
grant #5R44CA096020-03. The Analytical Group and Biology
Department at Endocyte are gratefully acknowledged.
Supporting Information Available: Experimental procedures
and spectral data. This material is available free of charge via
Internet at http://pubs.acs.org.
JO070411Z
5972 J. Org. Chem., Vol. 72, No. 16, 2007