Key words glycosylation - natural products - phenols - sulfoxides - trichloroacetimidates
Introduction
The glycosylation of proteins and natural products is an efficient means for organisms
to add an additional level of molecular complexity to the initial gene products.[
1
] In the case of ribosomally encoded proteins, N-linked glycosylation of asparagine
residues and O-glycosylation of serine and threonine side chains are the most abundant
structural motifs. Phenol glycosylation of tyrosine side chains, on the other hand,
is far less common, and has mainly been identified in glycogenin, S-layer proteins,[
2
] and most recently in amyloid precursor proteins and β-peptides connected to Alzheimer’s
disease.[
3
]
In contrast, microorganisms produce a number of secondary metabolites that carry a
carbohydrate moiety on the phenolic hydroxy group of tyrosine or hydroxyphenylglycine
derivatives, respectively. The glycosylation pattern of these metabolites is structurally
more diverse than on the protein level and features mono-, di-, and oligosaccharides
composed of a variety of α- or β-linked l - and d -sugars attached to the phenolic side chain. Prominent examples with interesting biological
activity are different glycopeptide antibiotics, e.g. vancomycin, teicoplanin,[
4
] and ramoplanin,[
5
] and the recently discovered group of anti-HIV cyclopeptides, the mirabamides.[
6
]
Our group is specifically interested in the chemical synthesis of the mannopeptimycins,
a group of glycopeptide antibiotics with excellent activity against resistant bacterial
strains (methicillin resistant Staphylococcus aureus , MRSA, and vancomycin resistant enterococci, VRE).
Figure 1 Structure of mannopeptimycin ε, the most active member of the naturally occurring
mannopeptimycin antibiotics; βhEnd = β-hydroxyenduracididine; βmePhe = β-methylphenylalanine
The mannopeptimycins contain an α-(1→4)-linked dimannoside that is attached to a
d -tyrosine side chain (Figure [1 ]).[
7
] Like the 4′-O -isovaleroyl derivative mannopeptimycin ε, the most active member of the group, all
naturally occurring mannopeptimycins with antibiotic activity contain a dimannoside
that carries the small hydrophobic isovaleroyl group at the terminal mannose unit,
whereas the unusual N-linked d -mannose unit attached to the cyclic guanidine of d -β-hydroxyenduracididine (d -βhEnd) is not essential for activity.[
8
]
While no total synthesis of the mannopeptimycins has been accomplished so far, efforts
towards glycosylated fragments and structural analogues have been reported. A group
at Wyeth described the synthesis of a mannopeptimycin derivative that contains a structurally
simplified cyclopeptide portion.[
9
] Because an unmodified dimannoside was the initial synthetic target, acyl groups
(Ac and Bz) could be used as temporary protecting groups for the glycosyl donors that
were required for the generation of an α-(1→4)-dimannosyl tyrosine building block.[
10
] As a consequence, this synthetic design is not generally applicable for esterified
derivatives like mannopeptimycin ε because of difficulties associated with the selective
removal of such acyl groups in the presence of other lipid esters. Following a completely
different approach, the O’Doherty group has used α-pyranone building blocks for the
iterative construction of a mannopeptimycin ε derived glycosylated d -tyrosine unit as well as a small set of structural analogues.[
11
] Although efficient, this ingenious strategy will be limited in terms of introducing
additional structural variations in the carbohydrate portion. Finally, the synthesis
of a mannopeptimycin ε derived dimannosyl N -phenyl trifluoroacetimidate has been reported, but this donor was so far only used
for the glycosylation of the side chain of a β-lactam acceptor.[
12
]
Because of the limitations described above, we were looking for an efficient and flexible
methodology to reliably attach different sugars to the tyrosine side chain via an
α-linkage. Published synthetic studies on phenol and tyrosine glycosylation, however,
have so far mostly been directed at the synthesis of β-glycosides of carbohydrates
with an equatorial 2-OH group, e.g. glucose and galactose. Accordingly, in these cases
glycosyl donors with 2-O -acyl groups were used, which ensured good β-selectivities by taking advantage of
neighboring group participation.[13 ]
[14 ]
[15 ]
[16 ] In principle, the same strategy can be used for the syntheses of α-linked mannosides,
because, in this case, neighboring group participation of the now axially oriented
2-O -acyl blocks the β-side. As described above, however, the use of 2-O -acylated donors is problematic for the synthesis of mannopeptimycin-type dimannosides
with a 4′-O -acyl group.
The formation of α-glycosylated tyrosine derivatives by using donors that carry non-participating
ether protecting groups at O2, on the other hand, has not been studied thoroughly,
and only a few examples in which surprisingly low glycosylation yields were obtained,
have been published.[14a ]
[17 ] Nevertheless, this second approach would not only avoid the tedious search for a
compatible 2-O -acyl group but also simplify the synthesis of the required mannopeptimycin building
blocks (vide infra ). Accordingly, we surveyed a number of perbenzylated glycosyl donors regarding their
performance in the α-mannosylation of different tyrosine acceptors. Here, we present
the results of these studies that concluded in the identification of reliable glycosylation
conditions for structurally complex donors and acceptors.
Results and Discussion
Model glycosylations : In our efforts towards the efficient and reliable synthesis of α-mannosylated tyrosine
units, we evaluated various ‘classical’ mannosyl donors. These donors were uniformly
protected with benzyl ethers, i.e. protecting groups that are orthogonal to the isovaleroyl
ester present in the mannopeptimycin dimannosyl unit. Initially, the perbenzylated
sulfoxide donor 1 was used, as glycosyl sulfoxides are known to be efficient donors for the glycosylation
of phenols.[
18
] Glycosylation of carbamate (Cbz) protected d -tyrosine methyl ester 3a with sulfoxide 1 under triflic anhydride activation led to the α-mannosyl amino acid 4a , albeit in only a moderate yield (45% at best). The corresponding Alloc derivative
gave similar results, whereas Boc- and Teoc-protected derivatives performed even worse
(yields <10%). Interestingly, the use of acceptors 3b and 3c
[
19
] in which the amines are protected as amide groups (trifluoroacetyl) or masked as
an azido group, led to much higher yields of the mannosylation products 4b and 4c (80% and 75%, respectively).
The same dependence on the N-protecting group was observed when the corresponding
trichloroacetimidate donor 2 was activated with trimethylsilyl triflate. Again, the carbamate-protected tyrosine
3a could be mannosylated in only 31% yield, whereas trifluoroacetamide derivate 3b led to mannoside 4b in 76% yield. Similar results were obtained with other glycosyl donors, e.g. the
corresponding glucosyl and galactosyl derivatives. We were also able to show that
the low yield for carbamate-containing acceptors was directly related to the phenolic
glycosylation site and not to the instability of the carbamate group under the glycosylation
conditions, because sulfoxide 1 could be used for the efficient glycosylation of the primary alcohol of Cbz-l -Ser-OMe.
The incompatibility of carbamate protecting groups with standard glycosyl donors for
the generation of α-glycosylated phenols would constitute a severe limitation for
the synthesis of structurally complex carbohydrate-containing compounds because of
the great utility of carbamates as N-protecting group in general.[
20
] A solution to this problem was ultimately found after screening additional activation
conditions for trichloroacetimidate 2 . The best results were obtained when donor 2 was activated with boron trifluoride–diethyl ether complex instead of trimethylsilyl
triflate, which led to vastly improved yields for carbamate-containing acceptors.
As exemplified for Cbz-protected tyrosine 3a , the yield of mannoside 4a increased from 31% to 76% when mannosyl trichloroacetimidate 2 was activated with 0.6 equivalents of boron trifluoride–diethyl ether complex (Scheme
[1 ]) at –30 °C. A similar increase was obtained for the mannosylation of the corresponding
N -Alloc derivative. Not surprisingly, the activation method had no influence on the
outcome of glycosylation reactions when acceptors without carbamate protecting groups
were used, e.g. trifluoroacetamide 3b or azide 3c . In conclusion, our experiments showed that the glycosylation of phenolic acceptors
with highly reactive glycosyl donors like sulfoxide 1 (activated by Tf2 O) or trichloroacetimidate 2 (in combination with TMSOTf) was only successful if no carbamate protecting groups
were present in the molecule. The activation of trichloroacetimidates with boron
trifluoride, on the other hand, proved to be compatible with carbamates and afforded
the glycosylated tyrosine derivatives in good yields.
Scheme 1 Glycosylation of different d -tyrosine acceptors 3a –c with mannosyl sulfoxide 1 and trichloroacetimidate donor 2 . Reagents and conditions : (a) Tf2 O (1 equiv), DTBP, CH2 Cl2 , –60 °C to –45 °C; (b) TMSOTf (0.8 equiv), CH2 Cl2 , –20 °C to 0 °C; (c) BF3 ·OEt2 (0.6 equiv), CH2 Cl2 , –30 °C to 0 °C. DTBP = 2,6-di-tert -butylpyridine, TFA = trifluoroacetyl.
Efficient glycosylation of mannopeptimycin precursor peptides : The results of our model studies were confirmed during the glycosylation of different
mannopeptimycin precursor peptides. Towards this end, we synthesized the dimannosyl
trichloroacetimidate donor 12 (Scheme [2 ]) starting from the known mannoside 9 .[
21
] In addition to serving as the glycosyl acceptor, alcohol 9 can also be converted into an appropriate sulfoxide donor 10 by acylation (i ValCl, DMAP, Et3 N, CH2 Cl2 ) to the 4-O -isovaleroyl derivative and subsequent oxidation with 3-chloroperoxybenzoic acid (MCPBA;
76% over 2 steps). Glycosylation of the thioglycoside acceptor 9 with sulfoxide 10 under triflic anhydride activation was rendered possible by the addition of 4-allyl-1,2-dimethoxybenzene
to the reaction mixture,[
22
] which afforded dimannoside 11 in acceptable yields.[
23
]
Scheme 2 Synthesis of dimannosyl building block 12 . Reagents and conditions: (a) i ValCl, DMAP, Et3 N, CH2 Cl2 , 0 °C; (b) MCPBA, CH2 Cl2 , –70 °C to –20 °C, 76% (2 steps); (c) Tf2 O, DTBP, 4-allyl-1,2-dimethoxybenzene, CH2 Cl2 , –70 °C to –25 °C, 52%; (d) NBS, acetone–H2 O, 0 °C, 81%; (f) Cl3 CCN, DBU, CH2 Cl2 , 0 °C, 79%.
Finally, thioglycoside 11 was transformed to the corresponding trichloroacetimidate donor 12 by hydrolysis of the thioglycoside (NBS, acetone–H2 O) and treatment of the resulting lactol with trichloroacetonitrile/DBU in good overall
yield (66%, 2 steps). Donor 12 was obtained as an anomeric mixture inseparable by flash chromatography and was used
directly in the glycosylation reactions.
In parallel, we synthesized the Alloc-protected l -βmePhe-d -Tyr dipeptide 13 . The mannopeptimycin fragment 13 was assembled from a (2S ,3S )-β-methylphenylalanine derivative that was obtained conveniently through a newly
developed route that features a diastereoselective Strecker reaction (see Supporting
Information). Dipeptide 13 was efficiently glycosylated with trichloroacetimidate 12 to afford dimannosyl dipeptide 14 (60%) when the donor was activated with boron trifluoride (0.6 equiv; Scheme [3 ]). It should be noted that glycosylation of the carbamate containing 13 with trichloroacetimidates under trimethylsilyl triflate activation or sulfoxides
under triflic anhydride activation led to a significant decrease in the yields of
the glycosylated dipeptide, similar to the results of our model studies.
Scheme 3 Glycosylation of dipeptide 13 with dimannosyl donor 12
Scheme 4 Glycoslation of hexapeptide 15 and conversion into cyclopeptide 17
In addition to this dipeptide fragment, we also synthesized hexapeptide 15 (see Supporting Information), a linear hexapeptide precursor to the mannopeptimycins
(Scheme [4 ]). This acceptor contains two β-hydroxyenduracididine analogues in which N -Boc-protected N ,O -acetals serve as surrogates for the cyclic guanidine portion of these unusual amino
acids.[
24
] Gratifyingly, the structurally complex peptide 15 could also be efficiently glycosylated to afford 16 in 62% yield (76% brsm) employing only a slight excess of trichloroacetimidate donor
12 (1.4 equiv). In comparison to previous reactions, it was imperative to raise the
amount of boron trifluoride to 3.5 equivalents, presumably because of the larger number
of carbonyl groups present in this acceptor, which might sequester some part of the
Lewis acid through coordination. Following this successful glycosylation, the linear
hexapeptide 16 could be deprotected at the C-and N-termini and cyclized to mannopeptimycin ε precursor
cyclopeptide 17 by using 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H )-one (DEPBT) for activation of the carboxylic acid.
Glycosyl carbamates : Even after the successful identification of generally applicable glycosylation conditions,
the question remained why the trimethylsilyl triflate activation of trichloroacetimidates
and sulfoxide activation with triflic anhydride, respectively, is incompatible with
carbamate groups when very reactive perbenzylated donors are combined with less reactive
phenolic acceptors. In our model studies, the glycosylation products of carbamate-protected
acceptors were only isolated in yields below 50% for such combinations, and the only
major additional reaction products following workup were the mannosyl lactol and the
recovered acceptor. Other products of the glycosylation reaction remained elusive,
e.g. β-anomers or C-glycosides[
25
] as possible alternate glycosylation products, glycals resulting from elimination
within the activated donor,[
26
] or phenol triflates as a result of acceptor triflation. Therefore, the detection
of an additional product that was formed in small amounts during the sulfoxide glycosylation
of Boc- or Teoc-protected tyrosine methyl ester, the double glycosylation product
18 [Scheme [5 ] (A)], was extremely helpful for the interpretation of the observed reaction outcome.
Obviously, in addition to the phenolic hydroxy group, the carbamate can also react
with the activated glycosyl donor, which, after elimination of the tert -butyl or 2-(trimethylsilyl)ethyl group, respectively, led to the formation of a glycosyl
carbamate.
Scheme 5 Identification of glycosyl carbamates as reaction products. A: Side product 18 was identified by MS and 1 H NMR of crude product mixtures when Teoc-protected tyrosine acceptor was used and
was isolated in 25% yield for the glycosylation of Boc-protected acceptor. B: Glycosyl
carbamates 19 , 22 , and 23 were isolated as main products when Boc-containing compounds without any nucleophilic
hydroxy groups were reacted with sulfoxide 1 under Tf2 O activation.
Figure 2 The single crystal X-ray structure of glycocarbamate 19
[
27
] proves the α-configuration (O2) of the mannosyl carbamate. The thermal ellipsoids
correspond to 50% probability.
To further investigate these findings, we submitted Boc-l -Phe-OMe to exactly the same glycosylation conditions [Scheme [5 ] (B)]. This carbamate-protected amino acid is missing a hydroxy group (alcohol or
phenol) as a possible glycosylation site, and was converted in good yields into the
carbamoyl-O -glycosylated product 19 [75% after optimization of the reaction conditions: donor 1 (1.8 equiv), –25 °C, 18 h]. The presence of a glycosyl carbamate in 19 was unambiguously proven by a crystal structure (Figure [2 ]),[
27
] which also confirmed the α-linkage of the mannose residue already assigned based
on the 3
J
1,2 and 1
J
C,H coupling constants[
28
] (1.5 Hz and 177 Hz, respectively) in the 1 H and HMBC spectra. Moreover, the fully protected 19 could be converted into the carboxylic acid 20 by treatment with aqueous lithium hydroxide solution in 1,4-dioxane, thereby furnishing
a potential coupling partner for ligation to an amino group. We were also able to
remove the benzyl protecting groups of 19 by hydrogenation, which led to carbamate 21 with an unprotected mannose moiety.
To explore the scope of the reaction conditions, we tested additional amino acid substrates.
First, we could show that carbamate-protected secondary amines are also glycosylated.
In the event, mannosylation of Boc-protected l -proline methyl ester with sulfoxide 1 led to glycosyl carbamate 22 with similar efficiency (79%).[
29
]
Next, a chemoselective carbamate glycosylation was explored. In our model glycosylation
reactions, glycosyl carbamates could only be detected when Boc and Teoc derivatives
were used as acceptors. Because both groups contain reasonably good cationic leaving
groups [the tert -butyl and 2-(trimethylsilyl)ethyl cation, respectively], we reasoned that other carbamates,
e.g. Fmoc, that contain less-stabilized groups, could very well be tolerated. Indeed,
we found that a Fmoc- and Boc-containing dipeptide [Fmoc-l -Lys(NHBoc)-l -Phe-OMe] could be converted into the monomannosyl carbamate 23 , i.e. glycosylation was only observed in the lysine side chain, whereas the Fmoc
group remained intact.[
30
]
Glycosyl carbamates have previously been prepared by different methods, most notably
by the reaction of lactols with isocyanates,[
31
] the reaction of amines with activated sugar carbamates[
32
] or carbonates,[
33
] and by the acid-catalyzed glycosylation of carbamate-protected amines with either
glycosyl trichloroacetimidates[
34
] or glycosyl orthoesters.[
35
] In the latter cases, the formation of glycosyl carbamates was also observed serendipitously
as a byproduct of an attempted O-glycosylation of a Boc-protected amino acid acceptor.
This outcome was proposed to evolve through the intermediacy of a carbamic acid generated
via acid-catalyzed removal of the tert -butyl group, which subsequently serves as a glycosyl acceptor.[
35
] Here, we observed this alternate reaction pathway for the first time under basic
glycosylation conditions (sulfoxide activation with triflic anhydride in the presence
of a pyridine base), which should exclude the formation of carbamic acids as reaction
intermediates. Instead, the direct attack of the activated donor at the carbamate
carbonyl group is most probably involved, which is then followed by elimination of
the tert -butyl group. This reaction pathway is similar to the conversion of Boc-carbamates
into trialkylsilyl carbamates when treated with trialkylsilyl triflates.[
36
]
Scheme 6 Glycosylated carbamates of type A and their elimination products B could sequester the activated glycosyl donor. Upon hydrolysis, the observed reaction
mixtures are formed depending on the relative stability of the two possible leaving
groups (R vs. sugar).
Following this reasoning, such an attack of the activated glycosyl donor could also
account for the three reaction outcomes that were observed when tyrosine acceptors
are used. Firstly, in amide-protected derivatives, e.g. 3b , the carbonyl oxygen of the amide group is much less nucleophilic than the phenolic
group, whereas in azide 3c , a possible second glycosylation site is missing completely. Accordingly, such acceptors
are efficiently glycosylated at the phenolic hydroxy group with a variety of highly
reactive glycosyl donors, e.g. 1 and 2 , under different activation conditions. Secondly, in carbamate protected acceptors,
e.g. 3a , the carbonyl oxygen of the carbamate group is only slightly less nucleophilic than
the phenolic hydroxy group. As a result, a significant part of such acceptors is glycosylated
at the carbamate group instead of the phenol when sulfoxide donors (Tf2 O activation) and trichloroacetimidate donors (TMSOTf activation) are used, i.e. under
conditions that generate glycosyl triflates. In addition to the desired phenol glycoside
4a , this would lead to intermediates of type A or their neutral elimination products B (Scheme [6 ]). These intermediates, which effectively sequester the activated donor and prevent
it from further glycosylating the phenolic group, are then hydrolyzed during the aqueous
workup, either to the original carbamate (R = Cbz or Alloc) or towards glycosyl carbamates
(R = t -Bu or Teoc), i.e. depending on the stability of the cationic leaving groups liberated
from the different carbamate groups. This interpretation would explain the low yields
for the tyrosine glycosylation irrespective of the type of carbamate group employed,
as well as for formation of the minor side product 18 only in cases where reasonably good cationic leaving groups are present. So far,
we have never observed glycosyl carbamates in the reaction mixture prior to aqueous
workup, which also supports this view. Thirdly, and most importantly from a synthetic
point of view, the activation of trichloroacetimidates with boron trifluoride is able
to revert the reaction outcome towards predominant phenol glycosylation, irrespective
of the N-protecting group. The activated donor that is formed in the presence of boron
trifluoride preferentially reacts at the phenolic hydroxy group, possibly because
this species is less reactive than the corresponding anomeric triflate generated by
trimethylsilyl triflate activation. Further studies to provide additional experimental
data concerning this mechanistic proposal are currently under way.
Conclusion
The glycosylation of phenolic hydroxy groups is widespread among natural products
with interesting biological activity. The chemical glycosylation of such rather unreactive
acceptors is still a challenge, because subtle changes of reaction conditions can
have a profound impact on the efficiency of these transformations.
During our synthetic studies, we found that carbamate groups within the acceptor molecule
have a negative impact on the glycosylation yield when two of the most common glycosyl
donors are used, namely glycosyl trichloroacetimidates and sulfoxides. The diminished
yields were traced back to a sequestration of the activated donor by the carbamate
group, which in some cases led to glycosyl carbamates as side products. We were able
to determine conditions that circumvent this problem, specifically the use of trichloroacetimidate
donors that are activated by boron trifluoride. This methodology allows the efficient
glycosylation of structurally complex peptides that contain multiple carbamate groups
with mono- and disaccharide donors, thus highlighting the reliability of the presented
approach.[37 ]
[38 ] These results are of importance for the glycosylation of phenolic acceptors in general
and should be helpful for the proper choice of coupling partners and protecting group
patterns used in future total synthesis efforts aimed at the generation of compounds
containing glycosylated phenols.
In addition, the efficient and chemoselective generation of glycosyl carbamates under
basic conditions using glycosyl sulfoxides and Boc-protected amino acids developed
here nicely complements the known acid-mediated protocols for the formation of these
glycoconjugates.
All reactions were carried out under an argon atmosphere. All chemicals were of reagent
grade and were used as purchased. Solvents were dried according to established procedures.
TLC was performed on silica gel 60 F254 plates (Merck KGaA). Detection was carried out by fluorescence quenching under UV
light (λ = 254 nm) or by staining with 20% H2 SO4 or ninhydrin soln followed by heating to ca. 300 °C. Flash chromatography was performed
on silica gel 60 (0.040–0.063 mm) from Merck KGaA. The petroleum ether (PE) used was
of the fraction boiling in the 40–60 °C range. NMR spectra were recorded on Bruker
AV 300, Bruker DRX 400, AV 500, DRX 500, and DRX 600 spectrometers at the NMR facilities,
Philipps-Universität Marburg referenced to the solvent residual signal. All coupling
constants are J
H,H couplings unless indicated otherwise. All signals are described as they appear in
the 1D 1 H NMR spectra, i.e. the actual multiplicitiy and not the expected one is given and
the coupling constants of each signal are actual values for each signal and not averaged
ones. Signals were assigned with the aid of COSY, HMBC and HMQC spectra. Diastereotopic
protons are labeled according to their chemical shifts: δa < δb . In the NMR data listings, the β-hydroxyenduracididine precursor amino acids are
abbreviated l - and d -Hep, respectively. HRMS (ESI) were acquired with a LTQ-FT mass spectrometer (Thermo
Fischer Scientific); the resolution was set to 100.000. The optical rotation was measured
on a Perkin-Elmer 241 polarimeter.
Model glycosylations were performed according to General Procedure A or B. The acceptor
phenols were prepared according to established procedures. For the synthesis of tyrosine
derivatives 3a and 3b , d -Tyr-OMe was acylated with benzyl chloroformate or trifluoroacetic anhydride, respectively.
Azido-d -Tyr-OMe (3c ) was prepared from d -Tyr-OMe using imidazole-1-sulfonyl azide.[
19
] The perbenzylated sulfoxide 1 was prepared by MCPBA oxidation[
39
] of the corresponding phenyl thioglycoside.[
40
] The known trichloroacetimidate donor 2 was synthesized from the corresponding lactol (DBU, Cl3 CCN), which in turn was obtained from the phenyl thioglycoside by treatment with NBS/H2 O.[
40
] Mannoside 9 was prepared according to known procedures.[
21
]
Experimental details for the synthesis of (2S ,3S )-β-methylphenylalanine, dipeptide 13 , and hexapeptide 15 and copies of NMR spectra are given in the Supporting Information. Details of the
synthesis of tetrapeptide S8 (see Supporting Information for the structure) will be described in a separate account.
Mannosylation of Phenolic Acceptors with Sulfoxides; General Procedure A
Mannosylation of Phenolic Acceptors with Sulfoxides; General Procedure A
Sulfoxide 1
[39 ]
[40 ] (1.5 equiv) and DTBP (3.0 equiv) were coevaporated with toluene (2 ×) and dissolved
in CH2 Cl2 (5 mL/mmol sulfoxide) under argon. After the addition of molecular sieves (4 Å),
the soln was stirred at r.t. (30 min). The soln was cooled to –70 °C and Tf2 O (1.5 equiv) was added. The mixture was stirred for 5 min at –60 °C, a soln of the
acceptor 3 (1 equiv) [previously coevaporated with toluene (2 ×)] in CH2 Cl2 (5 mL/mmol) was added, and the mixture was stirred at –60 °C to –45 °C for 1 h. The
reaction was stopped by the addition of MeOH, diluted with CH2 Cl2 , and poured into sat. aq NaHCO3 –brine (1:1). The aqueous phase was extracted with CH2 Cl2 (3 ×) and the combined organic phases were dried (MgSO4 ), filtered, and evaporated. Purification of the residue by flash chromatography (PE–EtOAc,
2:1) gave the mannosylated phenol 4 .
Mannosylation of Phenolic Acceptors with Trichloroacetimidates; General Procedure
B
Mannosylation of Phenolic Acceptors with Trichloroacetimidates; General Procedure
B
To a soln of 2,3,4,6-tetra-O -benzyl-mannose[
40
] (1 equiv) and trichloroacetonitrile (4.5 equiv) in CH2 Cl2 (20 mL/mmol lactol) at 0 °C was added DBU (0.3 equiv). After 30 min at this temperature,
the mixture was evaporated and purified directly by flash chromatography (PE–EtOAc,
6:1 to 2:1), which gave trichloroacetimidate 2 as a colorless oil (85–90%). To a soln of trichloroacetimidate 2 (1.5 equiv) and the acceptor 3 (1 equiv) in CH2 Cl2 was added either TMSOTf (0.8 equiv based on donor 2 ) at –20 °C or BF3 ·OEt2 (0.6 equiv based on donor 2 ) at –30 °C. The mixture was warmed to 0 °C over 1 h and the reaction was stopped
by the addition of Et3 N. Evaporation of the mixture and purification of the residue by flash chromatography
(PE–EtOAc, 2:1) gave the mannosylated phenol 4 .
Benzyloxycarbonyl-d -Tyr-O -(2,3,4,6-tetra-O -benzyl-α-d -mannopyranosyl)-OMe (4a)
Benzyloxycarbonyl-d -Tyr-O -(2,3,4,6-tetra-O -benzyl-α-d -mannopyranosyl)-OMe (4a)
Mannoside 4a was prepared according to General Procedure A and B and was obtained as a colorless
oil; yields: see Scheme [1 ]; Rf
= 0.55 (PE–EtOAc, 2:1).
1 H NMR (300 MHz, DMSO-d
6 ): δ = 2.80 (dd, 2
J = 13.6, 3
J
α,βa = 10.0 Hz, 1 H, β-Ha
Tyr ), 2.97 (dd, 2
J = 13.6, 3
J
α,βb = 5.0 Hz, 1 H, β-Hb
Tyr ), 3.52–3.75 (m, 3 H, H5, 2 H6), 3.61 (s, 3 H, OCH3 ), 3.87 (t, 3
J
3,4 = 3
J
4,5 = 9.2 Hz, 1 H, H4), 3.98 (dd, 3
J
2,3 = 2.9, 3
J
3,4 = 9.3 Hz, 1 H, H3), 4.09 (dd, 3
J
1,2 = 2.1, 3
J
2,3 = 3.0 Hz, 1 H, H2), 4.19 (ddd, 3
J
α,βt = 5.2, 3
J
α,NH = 7.9, 3
J
α,βh = 10.2 Hz, 1 H, α-HTyr ), 4.35–4.81 (m, 8 H, 4 CH2
Bn ), 4.95 and 5.00 (2 d, 2
J = 12.6 Hz, each 1 H, CH2
Cbz ), 5.69 (d, 3
J
1,2 = 1.9 Hz, 1 H, H1), 6.93–7.04 (m, 2 H, o -Harom
Tyr ), 7.11–7.47 (m, 27 H, Harom ), 7.82 (d, 3
J
α,NH = 8.2 Hz, 1 H, NH).
13 C NMR (75 MHz, CDCl3 ): δ = 37.4 (C-βTyr ), 52.3 (OCH3 ), 55.6 (C-αTyr ), 65.4 (CH2
Cbz ), 68.8 (C6), 70.8, 71.9 (CH2
Bn ), 72.0 (C5), 72.2, 74.1 (3 CH2
Bn ), 74.2 (C2), 74.3 (C4), 79.4 (C3), 96.5 (C1), 116.7, 121.1, 127.4, 127.6, 127.6,
127.7, 127.8, 127.9, 127.9, 128.1, 128.2, 128.2, 128.3, 128.4, 128.5, 128.7, 130.3
(CHarom ), 138.2, 138.3, 138.5, 139.5 (Carom ), 150.4, 155.0 (Carom
Tyr , C=OCbz ), 169.6 (COOMe).
HRMS (ESI): m /z [M + Na]+ calcd for C52 H53 NNaO10 : 874.3567; found: 874.3579.
Trifluoroacetyl-d -Tyr-O -(2,3,4,6-tetra-O -benzyl-α-d -mannopyranosyl)-OMe (4b)
Trifluoroacetyl-d -Tyr-O -(2,3,4,6-tetra-O -benzyl-α-d -mannopyranosyl)-OMe (4b)
Mannoside 4b was prepared according to General Procedure A and B and was obtained as a colorless
oil; yields: see Scheme [1 ]; R
f = 0.50 (PE–EtOAc, 2:1).
1 H NMR (300 MHz, DMSO-d
6 ): δ= 2.96 (dd, 2
J = 13.8, 3
J
α,βa = 10.6 Hz, 1 H, β-Ha
Tyr ), 3.14 (dd, 2
J = 13.9, 3
J
α,βb = 4.9 Hz, 1 H, β-Hb
Tyr ), 3.48–3.57 (m, 1 H, H6a ), 3.60–3.71 (m, 5 H, H5, H6b , OCH3 ), 3.87 (t, 3
J
3,4 = 3
J
4,5 = 9.2 Hz, 1 H, H4), 3.98 (dd, 3
J
2,3 = 3.0, 3
J
3,4 = 9.2 Hz, 1 H, H3), 4.08–4.13 (m, 1 H, H2), 4.31–4.84 (m, 9 H, 4 CH2
Bn , α-HTyr ), 5.72 (d, 3
J
1,2 = 1.6 Hz, 1 H, H1), 7.01–7.44 (m, 24 H, Harom ), 9.92 (d, 3
J
α,NH = 8.0 Hz, 1 H, NH).
13 C NMR (75 MHz, DMSO-d
6 ): δ= 34.8 (C-βTyr ), 52.4 (OCH3 ), 54.1 (C-αTyr ), 68.7 (C6), 70.8, 71.9 (2 CH2
Bn ), 72.0 (C5), 72.2, 74.1 (2 CH2
Bn ), 74.1 (C2), 74.2 (C4), 78.9 (C3), 95.6 (C1), 116.7, 127.3, 127.4, 127.5, 127.5,
127.6, 127.7, 127.7, 128.1, 128.2, 128.2, 130.1 (CHarom ), 130.5, 138.2, 138.4, 138.5 (Carom ), 154.5 (OCarom ), 156.3 (q, 3
J
C,F = 36.8 Hz, C=OTFA ), 170.4 (COOMe).
HRMS (ESI): m /z [M + Na]+ calcd for C46 H46 F3 NNaO9 : 836.3022; found: 836.3020.
Azido-d -Tyr-O -(2,3,4,6-tetra-O -benzyl-α-d -mannopyranosyl)-OMe (4c)
Azido-d -Tyr-O -(2,3,4,6-tetra-O -benzyl-α-d -mannopyranosyl)-OMe (4c)
Mannoside 4c was prepared according to General Procedure A and B and was obtained as a colorless
oil; yields: see Scheme [1 ]; Rf
= 0.45 (PE–EtOAc, 4:1).
1 H NMR (300 MHz, acetone-d
6 ): δ = 2.96 (dd, 2
J = 14.1, 3
J
α,βa = 8.5 Hz, 1 H, β-Ha
Tyr ), 3.12 (dd, 2
J = 14.1, 3
J
α,βb = 5.4 Hz, 1 H, β-Hb
Tyr ), 3.62–3.88 (m, 3 H, H5, 2 H6), 3.74 (s, 3 H, OCH3 ), 4.09 (t, 3
J
3,4 = 3
J
4,5 = 9.2 Hz, 1 H, H4), 4.09 (dd, 3
J
2,3 = 2.9, 3
J
3,4 = 9.3 Hz, 1 H, H3), 4.17 (dd, 3
J
1,2 = 2.1, 3
J
2,3 = 3.0 Hz, 1 H, H2), 4.29 (dd, 3
J
α,βb = 5.4, 3
J
α,βa = 8.5 Hz, 1 H, α-HTyr ), 4.46–4.98 (m, 8 H, 4 CH2
Bn ), 5.69 (d, 3
J
1,2 = 1.9 Hz, 1 H, H1), 7.04–7.11 (m, 2 H, CHarom ), 7.16–7.39 (m, 22 H, CHarom ).
13 C NMR (75 MHz, CDCl3 ): δ = 37.2 (C-βTyr ), 52.8 (OCH3 ), 63.8 (C-αTyr ), 70.3 (C6), 72.5, 73.5, 73.7 (3 CH2
Bn ), 73.7 (C5), 75.4 (CH2
Bn ), 75.7 (C4), 76.1 (C2), 80.8 (C3), 97.7 (C1), 116.7 (o -CHarom
Tyr ), 127.3, 127.4, 127.5, 127.5, 127.6, 127.7, 127.7, 128.1, 128.2, 128.2, 130.1 (CHarom ), 130.5, 138.2, 138.4, 138.5 (Carom ), 154.5 (OCarom ), 170.4 (COOMe).
HRMS (ESI): m /z [M + Na]+ calcd for C44 H45 N3 NaO8 : 766.3104; found: 766.3115.
Phenyl 2,3,6-Tri-O -benzyl-4-O -isovaleroyl-1-thio-α-d -mannopyranoside S -Oxide (10)
Phenyl 2,3,6-Tri-O -benzyl-4-O -isovaleroyl-1-thio-α-d -mannopyranoside S -Oxide (10)
Thioglycoside 9
[
21
] (2.16 g, 3.98 mmol) and Et3 N (2.49 mL, 17.8 mmol) were dissolved in CH2 Cl2 (50 mL). Isovaleroyl chloride (1.41 mL, 11.9 mmol) and DMAP (30 mg, cat.) were added
at 0 °C and the mixture was stirred for 6 h at 0 °C. The reaction was quenched by
the addition of MeOH (3 mL) and diluted with CH2 Cl2 (100 mL). The mixture was washed with 2 M HCl (50 mL) and sat. aq NaHCO3 (50 mL), and dried (MgSO4 ); the solvent was evaporated to afford the crude 4-O -isovaleoryl derivative (2.3 g).
The crude 4-O -isovaleoryl derivative was dissolved in CH2 Cl2 (250 mL) and was subsequently treated dropwise with a soln of MCPBA (70–75%, 1.03 g,
4.2–4.5 mmol) in CH2 Cl2 (100 mL) at –70 °C. The mixture was slowly warmed to –20 °C over a period of 6 h,
and then sat. aq NaHCO3 (100 mL) was added. After the addition of brine (300 mL), the phases were separated,
the aqueous phase was extracted with CH2 Cl2 (2 × 200 mL), and the combined organic phases were dried (MgSO4 ) and evaporated. Purification of the residue by flash chromatography (PE–EtOAc, 4:1)
afforded the sulfoxide 10 (3.08 g, 76% over 2 steps) as a colorless amorphous powder; Rf
= 0.35 (PE–EtOAc, 4:1).
1 H NMR (300 MHz, CDCl3 ): δ = 0.89 and 0.90 (2 d, 3
J = 6.3 Hz, each 3 H, 2 CH3
i Val ), 1.98–2.12 (m, 3 H, CH2
i Val , CH
i Val ), 3.48–3.62 (m, 2 H, 2 H6), 4.22 (dd, 3
J
2,3 = 3.0, 3
J
3,4 = 9.5 Hz, 1 H, H3), 4.23–4.32 (m, 1 H, H5), 4.33–4.36 (m, 1 H, H2), 4.42–4.56 (m,
6 H, 3 CH2
Bn ), 4.58 (d, 3
J
1,2 = 2.1 Hz, 1 H, H1), 5.39 (t, 3
J
3,4 = 3
J
4,5 = 9.5 Hz, 1 H, H4), 7.12–7.24 (m, 5 H, CHarom ), 7.27–7.50 (m, 13 H, CHarom ), 7.54–7.59 (m, 2 H, CHarom ).
13 C NMR (75 MHz, CDCl3 ): δ = 22.4 (2 CH3
i Val ), 25.5 (CH
i Val ), 43.3 (CH2
i Val ), 67.6 (C4), 70.0 (C6), 71.0 (C2), 72.6, 72.6, 73.5 (3 CH2
Bn ), 76.5 (C5), 76.6 (C3), 95.8 (C1), 124.3, 127.7, 127.8, 128.0, 128.3, 128.3, 129.2,
131.2 (CHarom ), 137.4, 137.8, 137.9, 141.6 (Carom ), 171.9 (C=O).
HRMS (ESI): m /z [M + Na]+ calcd for C38 H42 NaO7 S: 665.2543; found: 665.2561.
Phenyl 2,3,6-Tri-O -benzyl-4-O -(2′,3′,6′-tri-O -benzyl-4′-O -isovaleroyl-α-d -mannopyranosyl)-1-thio-α-d -mannopyranoside (11)
Phenyl 2,3,6-Tri-O -benzyl-4-O -(2′,3′,6′-tri-O -benzyl-4′-O -isovaleroyl-α-d -mannopyranosyl)-1-thio-α-d -mannopyranoside (11)
Acceptor 9 (422 mg, 0.78 mmol, 1.0 equiv), DTBP (0.52 mL, 2.33 mmol, 3.0 equiv), and 4-allyl-1,2-dimethoxybenzene
(0.32 mL, 1.87 mmol, 2.4 equiv) were coevaporated with toluene (2 × 10 mL). The residue
was dissolved in CH2 Cl2 (10 mL), molecular sieves (4 Å) were added, and the resulting mixture was stirred
at r.t. for 30 min under argon. The mixture was cooled to –70 °C and then Tf2 O (0.16 mL, 0.93 mmol, 1.2 equiv) was added dropwise, followed by a soln of sulfoxide
10 (600 mg, 0.93 mmol, 1.2 equiv) in CH2 Cl2 (4 mL). The mixture was slowly warmed to –25 °C over 1 h, quenched with Et2 NH (0.4 mL), diluted with CH2 Cl2 (100 mL), and poured into sat. aq NaHCO3 –brine–H2 O (1:1:2, 100 mL). The aqueous phase was extracted with CH2 Cl2 (2 × 50 mL) and the combined organic phases were dried (MgSO4 ) and evaporated. Purification of the residue by flash chromatography (PE–EtOAc, 10:1,
8:1, 6:1) afforded disaccharide 11 (427 mg, 52%) as a colorless syrup; Rf
= 0.45 (PE–EtOAc, 4:1).
1 H NMR (500 MHz, CDCl3 ): δ = 0.87 and 0.88 (2d, 3
J = 6.6 Hz, each 3 H, 2 CH3
i Val ), 1.97–2.10 (m, 3 H, CH
i Val , CH2
i Val ), 3.47 (dd, 2
J = 10.6, 3
J
5′,6a′ = 2.8 Hz, 1 H, H6′a ), 3.53 (dd, 2
J = 10.6, 3
J
5′,6b′ = 6.2 Hz, 1 H, H6′b ), 3.72 (dd, 3
J
1′,2′ = 2.5, 3
J
2′,3′ = 2.8 Hz, 1 H, H2′), 3.73 (dd, 3
J
2,3 = 2.9, 3
J
3,4 = 9.2 Hz, 1 H, H3), 3.83 (dd, 3
J
2′,3 = 2.9, 3
J
3′,4′ = 9.6 Hz, 1 H, H3′), 3.83 (dd, 2
J = 10.8, 3
J
5,6a = 6.9 Hz, 1 H, H6a ), 3.93 (ddd, 3
J
5′,6′a = 2.7, 3
J
5′,6′b = 6.2, 3
J
4′,5′ = 9.7 Hz, 1 H, H5′), 3.96 (dd, 2
J = 11.1, 3
J
5,6b = 1.9 Hz, 1 H, H6b ), 4.00 (dd, 3
J
1,2 = 2.0, 3
J
2,3 = 3.1 Hz, 1 H, H2), 4.12 (t, 3
J
3,4 = 3
J
4,5 = 9.3 Hz, 1 H, H4), 4.28–4.36 (m, 5 H, H5, 2 CH2
Bn ), 4.45–4.70 (m, 8 H, 4 CH2
Bn ), 5.31 (d, 3
J
1′,2′ = 2.2 Hz, 1 H, H1′), 5.37 (dd, 3
J
3′,4′ = 9.7, 3
J
4′,5′ = 9.8 Hz, 1 H, H4′), 5.61 (d, 3
J
1,2 = 1.9 Hz, 1 H, H1), 7.15–7.56 (m, 35 H, CHarom ).
13 C NMR (126 MHz, CDCl3 ): δ = 22.6 (2 CH3
i Val ), 25.6 (CH
i Val ), 43.6 (CH2
i Val ), 68.7 (C4′), 70.3 (C6′), 70.5 (C5′, C6), 71.2 (CH2
Bn ), 71.8 (C3′), 71.9, 72.3 (2 CH2
Bn ), 72.5 (C5), 73.1, 73.2, 73.7 (3 CH2
Bn ), 75.4, 75.4, 75.5 (C2, C4, C2′), 77.0 (C3′), 80.1 (C3), 85.6 (C1), 100.4 (C1′),
127.3, 127.5, 127.5, 127.6, 127.6, 127.6, 127.8, 127.9, 127.9, 128.1, 128.2, 128.3,
128.4, 128.4, 128.5, 128.7, 129.2, 132.2 (CHarom ), 134.2, 137.9, 137.9, 138.4, 138.6, 138.6, 138.9 (Carom ), 172.0 (C=O
i Val ).
HRMS (ESI): m /z [M + Na]+ calcd for C65 H70 NaO11 S: 1081.4537; found: 1081.4556.
(2,3,6-Tri-O -benzyl-4-O -chloroacetyl-α-d -mannopyranosyl)-(1→4)-2,3,6-tri-O -benzyl-α-d -mannopyranosyl Trichloroacetimidate (12)
(2,3,6-Tri-O -benzyl-4-O -chloroacetyl-α-d -mannopyranosyl)-(1→4)-2,3,6-tri-O -benzyl-α-d -mannopyranosyl Trichloroacetimidate (12)
Thioglycoside 11 (620 mg, 0.58 mmol) was dissolved in acetone–H2 O (19:1, 12 mL) and cooled to 0 °C. After the addition of NBS (313 mg, 1.76 mmol,
3 equiv), the mixture was stirred for 30 min at 0 °C. The reaction was stopped by
the addition of 10% aq Na2 S2 O3 soln (1 mL), diluted with EtOAc (150 mL), and washed with sat. aq NaHCO3 soln–brine (1:1, 50 mL). The organic phase was dried (MgSO4 ), evaporated, and the residue was purified by flash chromatography (PE–EtOAc, 3:1,
2:1) to afford the corresponding lactol (460 mg, 81%) as a colorless solid; Rf
= 0.45 (PE–EtOAc, 4:1).
1 H NMR (500 MHz, DMSO-d
6 ): δ= 0.81 and 0.82 (2 d, 3
J = 6.6 Hz, each 3 H, 2 CH3
i Val ), 1.88 (septet, 3
J = 6.7 Hz, 1 H, CH
i Val ), 2.06 (d, 3
J = 6.9 Hz, 2 H, CH2
i Val ), 3.28 (dd, 2
J = 10.9, 3
J
5′,6′a = 2.8 Hz, 1 H, H6′a ), 3.30–3.33 (m, 1 H, H6′b ), 3.62 (dd, 2
J = 10.8, 3
J
5,6a = 5.0 Hz, 1 H, H6a ), 3.67 (dd, 2
J = 10.7, 3
J
5,6b = 1.8 Hz, 1 H, H6b ), 3.72 (dd, 3
J
2′,3′ = 2.9, 3
J
3′,4′ = 9.8 Hz, 1 H, H3′), 3.75 (ddd, 3
J
5′,6′a = 3.1, 3
J
5′,6′b = 4.6, 3
J
4′,5′ = 10.0 Hz, 1 H, H5′), 3.84 (dd, 3
J
1′,2′ = 2.2, 3
J
2′,3′ = 2.9 Hz, 1 H, H2′), 3.85–3.90 (m, 3 H, H5, H2, H3), 3.93 (dd, 3
J
3,4 = 9.2, 3
J
4,5 = 9.5 Hz, 1 H, H4), 4.18–4.65 (m, 12 H, 6 CH2
Bn ), 5.17 (dd, 3
J
1,2 = 2.2, 3
J
1,OH = 4.1 Hz, 1 H, H1), 5.19 (dd, 3
J
3′,4′ = 9.9, 3
J
4′,5′ = 10.0 Hz, 1 H, H4′), 5.30 (d, 3
J
1′,2′ = 1.9 Hz, 1 H, H1′), 6.73 (d, 3
J
1,OH = 4.3 Hz, 1 H, 1-OH), 7.08–7.38 (m, 30 H, CHarom ).
13 C NMR (126 MHz, 300 K, DMSO-d
6 ): δ= 22.0, 22.0 (2 CH3
i Val ), 24.9 (CH
i Val ), 42.6 (CH2
i Val ), 67.4 (C4′), 68.9 (C6′), 69.9 (C6), 70.0 (CH2
Bn ), 70.0 (C5), 70.5 (C5′), 70.5, 71.5, 71.6, 72.2, 72.4 (5 CH2
Bn ), 73.6 (C4), 74.6 (C2), 74.8 (C2′), 76.4 (C3′), 79.4 (C3), 90.9 (C1), 98.7 (C1′),
127.0, 127.2, 127.3, 127.3, 127.3, 127.3, 127.4, 127.4, 127.4, 127.5, 127.5, 128.0,
128.1, 128.1, 128.1, 128.1, 128.2 (CHarom ), 138.1, 138.2, 138.3, 138.3, 138.4, 138.7 (Carom ), 171.1 (C=O
i Val ).
HRMS (ESI): m /z [M + Na]+ calcd for C59 H66 NaO12 : 989.4452; found: 989.4431.
The lactol (110 mg, 114 μmol) and trichloroacetonitrile (51 μL, 512 μmol, 4.5 equiv)
were dissolved in CH2 Cl2 (2 mL) under an atmosphere of argon. At 0 °C, DBU (5 μL, 34 μmol, 0.3 equiv) was
added. After stirring for 30 min, the solvent was evaporated and the crude product
was purified by flash chromatography (PE–EtOAc, 6:1, 4:1) to afford trichloroacetimidate
12 (100 mg, 79%) as a colorless oil, which was used directly in the glycosylation reactions;
Rf
= 0.48 (PE–EtOAc, 4:1).
HRMS (ESI): m /z [M + Na]+ calcd for C61 H66 Cl3 NNaO12 : 1132.3543; found: 1132.3522 (correct isotope pattern).
Alloc-l -(3S )-βmePhe-d -Tyr-{O -[2,3,6-tri-O -benzyl-4-O -(2′,3′,6′-tri-O -benzyl-4′-O -isovaleroyl-α-d -mannopyranosyl)-α-d -mannopyranosyl]}-OMe (14)
Alloc-l -(3S )-βmePhe-d -Tyr-{O -[2,3,6-tri-O -benzyl-4-O -(2′,3′,6′-tri-O -benzyl-4′-O -isovaleroyl-α-d -mannopyranosyl)-α-d -mannopyranosyl]}-OMe (14)
Trichloroacetimidate 12 (100 mg, 90 μmol, 1.3 equiv) and dipeptide 13 (30 mg, 68 μmol, 1 equiv) were dissolved in CH2 Cl2 (2 mL). The mixture was cooled to –30 °C and BF3 ·OEt2 (7 μL, 54 μmol, 0.6 equiv based on donor 12 ) was added. After 1 h at this temperature, the reaction was quenched by the addition
of Et3 N (0.2 mL) and purified directly by flash chromatography (PE–EtOAc, 2:1) to afford
the dimannosyl dipeptide 14 (57 mg, 60%) as a colorless amorphous solid; Rf
= 0.20 (PE–EtOAc, 2:1).
1 H NMR (500 MHz, acetone-d
6 ): δ = 0.86, 0.88 (2 d, 3
J = 6.7 Hz, each 3 H, 2 CH3
i Val ), 1.14 (d, 3
J
β,Me = 7.1 Hz, 3 H, CH3
mePhe ), 1.98 (septet, 3
J = 6.7 Hz, 1 H, CH
i Val ), 2.08–2.12 (m, 2 H, CH2
i Val ), 2.97 (dd, 2
J = 14.0, 3
J
α,βa = 9.2 Hz, 1 H, β-Ha
Tyr ), 3.09–3.17 (m, 1 H, β-HmePhe ), 3.16 (dd, 2
J = 14.2, 3
J
α,βb = 5.2 Hz, 1 H, β-Hb
Tyr ), 3.41–3.48 (m, 2 H, H6′), 3.62–3.71 (m, 2 H, H6), 3.66 (s, 3 H, OCH3 ), 3.79 (m, 1 H, H5), 3.87 (dd, 3
J
2′,3′ = 2.9, 3
J
3′,4′ = 9.8 Hz, 1 H, H3′), 3.90 (m, 1 H, H5′), 3.94 (dd, 3
J
1′,2′ = 2.1, 3
J
2′,3′ = 3.1 Hz, 1 H, H2′), 4.10 (dd, 3
J
2,3 = 3.1, 3
J
3,4 = 9.2 Hz, 1 H, H3), 4.19 (dd, 3
J
1,2 = 2.4, 3
J
2,3 = 3.1 Hz, 1 H, H2), 4.25 (t, 3
J
3,4 = 3
J
4,5 = 9.4 Hz, 1 H, H4), 4.30–4.80 (m, 14 H, 6 × CH2
Bn , CH2
All ), 4.32–4.38 (m, 1 H, α-HmePhe ), 4.64–4.75 (m, 1 H, α-HTyr ), 5.05–5.11, 5.13–5.20 (2 m, each 1 H, =CH2
All ), 5.38 (t, 3
J
3′,4′ = 3
J
4′,5′ = 9.9 Hz, 1 H, H4′), 5.40 (d, 3
J
1′,2′ = 1.7 Hz, 1 H, H1′), 5.69 (d, 3
J
1,2 = 1.7 Hz, 1 H, H1), 5.77–5.86 (m, 1 H, =CHAll ), 5.92 (br d, 3
J
α,NH = 8.9 Hz, 1 H, NHmePhe ), 6.96–7.51 (m, 39 H, Harom ), 7.66 (br d, 3
J
α,NH = 8.4 Hz, 1 H, NHTyr ).
13 C NMR (126 MHz, acetone-d
6 ): δ = 19.1 (CH3
mePhe ), 22.7 (2 CH3
i Val ), 26.1 (CH
i Val ), 37.6 (C-βTyr ), 42.9 (C-βmePhe ), 43.9 (CH2
i Val ), 52.3 (OCH3 ), 54.8 (C-αTyr ), 60.7 (C-αmePhe ), 65.7 (OCH2
All) , 69.1 (C4′), 70.6 (C6), 70.9 (C6′), 71.7, 72.1 (2 × CH2
Bn ), 72.2 (C5′), 73.0 (CH2
Bn ), 73.1 (C5), 73.3, 73.5, 73.9 (4 × CH2
Bn ), 75.0 (C4), 75.0 (C2), 76.4 (C2′), 78.0 (C3′), 80.7 (C3), 97.3 (C1′), 100.7 (C1),
117.1 (=CH2
All ), 117.8 (2 m -CHarom
Tyr ), 127.4, 128.0, 128.1, 128.1, 128.2, 128.2, 128.3, 128.3, 128.4, 128.5, 128.5, 128.6,
128.6, 128.6, 128.7, 128.8, 128.9, 128.9, 129.0, 129.0, 129.0, 129.1, 129.2, 129.3,
131.3, 131.3 (CHarom ), 131.3 (o -CHarom
Tyr ), 131.9 (i -Carom
Tyr ), 134.3 (=CHAll ), 139.4, 139.6, 139.6, 139.6, 139.8, 139.9, 143.4 (i -Carom ), 156.5 (p -Carom
Tyr , C=Ocarbamate ), 171.7 (C=Oamide ), 172.1 (C=O
i Val ), 172.5 (COOMe).
HRMS (ESI): m /z [M + H]+ calcd for C83 H93 N2 O17 : 1389.6474; found: 1389.6472.
Dimannosylated Linear Hexapeptide 16
Dimannosylated Linear Hexapeptide 16
Trichloroacetimidate 12 (160 mg, 144 μmol, 1.4 equiv) and linear hexapeptide 15 (126 mg, 101 μmol, 1 equiv) were dissolved in CH2 Cl2 (1.5 mL) and cooled to –30 °C. BF3 ·OEt2 (62 μL, 504 μmol, 3.5 equiv based on donor 12 ) was added dropwise, and the reaction was stirred at this temperature for 40 min.
The reaction was stopped by the addition of Et3 N (0.2 mL), and the mixture was diluted with CH2 Cl2 (30 mL) and washed with sat. aq NH4 Cl–H2 O (1:1, 15 mL) and sat. aq NaHCO3 (15 mL). The organic phase was dried (MgSO4 ) and evaporated, and the residue was purified by flash chromatography (CH2 Cl2 –MeOH, 60:1, 40:1, 20:1). This afforded unreacted acceptor 15 (23 mg, 18%) and the glycosylated hexapeptide 16 (137 mg, 62%) as a colorless solid; Rf
= 0.50 (CH2 Cl2 –MeOH, 20:1).
[α]D
22 +33.1 (c 1.0, CHCl3 ).
1 H NMR (500 MHz, 350 K, DMSO-d
6 ): δ = 0.84, 0.85 (2 d, 3
J = 6.6 Hz, each 3 H, 2 CH3
i Val ), 0.92 (d, 3
J
β,Me = 7.1 Hz, 3 H, CH3
mePhe ), 1.38 (br s, 3 H, CH3
i -Pr ), 1.42 (br s, 9 H, CH3
Boc ), 1.44 (br s, 12 H, CH3
i -Pr , CH3
Boc ), 1.49 (br s, 3 H, CH3
i -Pr ), 1.51 (br s, 3 H, CH3
i -Pr ), 1.91 (septet, 3
J = 6.6 Hz, 1 H, CH
i Val ), 2.07 (d, 3
J = 6.8 Hz, 2 H, CH2
i Val ), 2.79 (dd, 2
J = 14.0, 3
J
α,βa = 9.7 Hz, 1 H, β-Ha
Tyr ), 2.90 (dq, 3
J
β,Me = 6.8, 3
J
α,β = 7.5 Hz, 1 H, β-HmePhe ), 3.11 (dd, 2
J = 14.1, 3
J
α,βb = 4.3 Hz, 1 H, β-Hb
Tyr ), 3.28–3.44 (m, 2 H, H6a , H6′a ), 3.51–3.60 (m, 2 H, H6b , H6′b ), 3.61 (s, 3 H, OCH3 ), 3.67–3.72 (m, 2 H, β-H2
Ser ), 3.71–3.75 (m, 1 H, H5), 3.75–3.80 (m, 3 H, H3′, H5′, δ-Ha
l -Hep ), 3.82–3.87 (m, 2 H, H2′, δ-Ha
d -Hep ), 3.87–3.94 (m, 4 H, γ-Ha
d -Hep , γ-Ha
l -Hep , CH2
Gly ), 3.97–4.03 (m, 2 H, H3, β-H
l -Hep ), 4.08 (t, 3
J
3,4 = 3
J
4,5 = 9.4 Hz, 1 H, H4), 4.10 (dd, 3
J
1,2 = 2.4, 3
J
2,3 = 3.0 Hz, 1 H, H2), 4.09–4.13 (m, 2 H, δ-Hb
d -Hep , δ-Hb
l -Hep ), 4.22 (t, 3
J = 9.0 Hz, 1 H, α-HmePhe ), 4.25–4.30 (m, 1 H, β-H
d -Hep ), 4.26–4.52 (m, 13 H, OCH2
All , 5.5 OCH2
Bn ), 4.33–4.37 (m, 1 H, α-H
d -Hep ), 4.45–4.48 (m, 1 H, α-H
l -Hep ), 4.59 (ddd, 3
J
α,βa = 5.7, 3
J
α,NH = 7.5, 3
J
α,βb = 8.0 Hz, 1 H, α-HSer ), 4.63–4.75 (m, 4 H, 1.5 OCH2
Bn , α-HTyr ), 4.89 (br d, 3
J = 5.7 Hz, 1 H, OH
l -Hep ), 4.91 (br s, 1 H, OH
d -Hep ), 5.04–5.16 (m, 2 H, =CH2
All ), 5.21 (t, 3
J
3′,4′ = 3
J
4′,5′ = 9.8 Hz, 1 H, H4′), 5.29 (d, 3
J
1′,2′ = 2.2 Hz, 1 H, H1′), 5.60 (d, 3
J
1,2 = 2.2 Hz, 1 H, H1), 5.73–5.79 (m, 1 H, =CHAll ), 6.42 (br s, 1 H, NHmePhe ), 6.97–7.40 (m, 44 H, Harom ), 7.98 (br s, 1 H, NHSer ), 7.99 (br d, 3
J
α,NH = 7.2 Hz, 1 H, NH
l -Hep ), 8.13 (br t, 3
J
CH2α,NH = 6.4 Hz, 1 H, NHGly ), 8.16 (br s, 1 H, NH
d -Hep ), 8.19 (br d, 3
J = 6.7 Hz, 1 H, NHTyr ).
13 C NMR: (126 MHz, 350 K, DMSO-d
6 ): δ = 18.3 (CH3
mePhe ), 21.6 (2 CH3
i Val ), 24.1 (2 CH3
i -Pr ), 24.5 (CH
i Val ), 26.1 (2 CH3
i -Pr ), 27.7 (CH3
Boc ), 27.8 (CH3
Boc ), 36.6 (C-βTyr ), 40.5 (CH2
Gly ), 41.2 (C-βmePhe ), 42.4 (CH2
i Val ), 51.1 (OCH3 ), 52.6 (C-αSer ), 54.0 (C-αTyr ), 54.9 (C-α
l -Hep ), 56.6 (C-α
d -Hep ), 58.1, 58.4 (C-γ
d -Hep , C-γ
l -Hep ), 59.6 (C-αmePhe ), 62.4 (C-δ
d -Hep ), 62.5 (C-δ
l -Hep ), 64.1 (OCH2
Allyl ), 67.5 (C4′), 69.0 (2 C, C6, C6′), 69.4 (C-βSer ), 69.4 (C-β
d -Hep ), 70.2 (CH2 Ph, C-β
l -Hep ), 70.4 (CH2 Ph), 70.5 (C5), 71.4, 71.4, 71.6, 71.9 (CH2 Ph), 72.5 (C4), 73.6 (C2), 74.9 (C2′), 76.2 (C5′, C3′), 78.7 (C3), 79.0, 79.1 (Cq
Boc ), 92.7, 92.9 (Cq
i -Pr ), 95.9 (C1′), 98.6 (C1), 116.4 (=CH2
All ), 116.4, 125.7, 126.7, 126.7, 126.8, 126.8, 126.9, 126.9, 127.0, 127.1, 127.1, 127.2,
127.2, 127.4, 127.6, 127.6, 127.7, 127.7, 129.7, 127.8, 131.4 (CHarom , Carom ), 132.9 (=CHAll ), 137.8, 137.9, 137.9, 137.9, 138.0, 138.1, 142.6 (Carom ), 151.3, 151.4 (C=OBoc ), 154.2 (C=OAlloc ), 154.4 (p -Carom
Tyr ), 169.3 (COOMe), 169.4, 170.3, 170.4 (C=Oamide ), 170.6 (C=O
i Val ).
HRMS (ESI): m /z [M + Na]+ calcd for C121 H150 N8 NaO30 : 2219.0389; found: 2219.0402.
Dimannosyl Cyclopeptide 17
Dimannosyl Cyclopeptide 17
To a soln of linear hexapeptide 16 (81 mg, 37 μmol, 1 equiv) in THF (degassed, 0.5 mL) was added morpholine (16 μL,
184 μmol, 5 equiv) and Pd(PPh3 )4 (9 mg, 8 μmol, 0.2 equiv), and the mixture was stirred for 30 min at r.t. After evaporation
of the solvent, the residue was directly purified by flash chromatography (CH2 Cl2 –MeOH, 40:1, 20:1, 10:1), which afforded the corresponding terminal amine (52 mg,
67%) as an amorphous solid; Rf
= 0.30 (CH2 Cl2 –MeOH, 20:1).
HRMS (ESI): m /z [M + H]+ calcd for C117 H147 N8 O28 : 2113.0358; found: 2113.0375.
The amine (52 mg, 25 μmol) was dissolved in 1,4-dioxane (0.5 mL) and treated with
aq 1 M LiOH (50 μL, 50 μmol, 2 equiv) at 8 °C. After stirring for 90 min, the reaction
was stopped by the addition of 2 M HCl (38 μL, 75 μmol, 3 equiv), and the solvents
were removed under reduced pressure to afford the N- and C-terminally deprotected
hexapeptide.
HRMS (ESI): m /z [M + H]+ calcd for C116 H145 N8 O28 : 2099.0202; found: 2099.0232.
The crude residue was dissolved in THF (25 mL) and cooled with an ice-water bath.
Following the addition of solid NaHCO3 (8 mg, 100 μmol, 4 equiv) and DEPBT (22 mg, 75 μmol, 3 equiv), the mixture was stirred
at r.t. for 4 d. After evaporation of the solvent, the residue was partitioned between
EtOAc (30 mL) and sat. aq NaHCO3 (20 mL). The organic phase was washed with sat. aq NaHCO3 (20 mL) and brine (20 mL), dried (MgSO4 ), and evaporated. Purification of the residue by flash chromatography (CH2 Cl2 –MeOH, 60:1, 40:1, 20:1) afforded cyclopeptide 17 (37 mg, 72%) as a colorless, amorphous solid; Rf
= 0.40 (CH2 Cl2 –MeOH, 20:1).
[α]D
22 +37.7 (c 1.0, CHCl3 ).
1 H NMR (500 MHz, 350 K, DMSO-d
6 ): δ = 0.72 (br s, 3 H, CH3
mePhe ), 0.84, 0.85 (2 d, 3
J = 6.7 Hz, each 3 H, 2 CH3
i Val ), 1.30 (br s, 3 H, CH3
i -Pr ), 1.41 (br s, 9 H, CH3
Boc ), 1.42 (br s, 3 H, CH3
i -Pr ), 1.48 (br s, 9 H, CH3
Boc ), 1.49 (br s, 3 H, CH3
i -Pr ), 1.56 (br s, 3 H, CH3
i -Pr ), 1.91 (septet, 3
J = 6.7 Hz, 1 H, CH
i Val ), 2.04–2.08 (m, 2 H, CH2
i Val ), 2.76 (dd, 2
J = 14.0, 3
J
α,βa = 10.2 Hz, 1 H, β-Ha
Tyr ), 2.90 (dq, 3
J
β,Me = 6.7, 3
J
α,β = 7.1 Hz, 1 H, β-HmePhe ), 3.22 (br d, 2
J = 13.8 Hz, 1 H, β-Hb
Tyr ), 3.34 (dd, 2
J = 11.0, 3
J
5,6 = 3.0 Hz, 1 H, H6′a ), 3.37 (d, 2
J = 11.0, 3
J
5,6 = 4.6 Hz, 1 H, H6′b ), 3.42 (br d, 2
J = 11.0 Hz, 1 H, CHa
Gly ), 3.57–3.63 (m, 2 H, 6H), 3.65 (dd, 2
J = 9.5, 3
J = 8.0 Hz, 1 H, β -CHa
Ser ), 3.73–3.80 (m, 5 H, CHb
Gly , β -Hb
Ser , H5, H3′, H5′), 3.80–3.87 (m, 5 H, δ-Ha
d -Hep , δ-Ha
l -Hep , γ-Ha
d -Hep , γ-Ha
l -Hep , H2′), 3.89 (br s, 1 H, β -H
l -Hep ), 3.97 (br s, 1 H, α-H
d -Hep ), 4.00 (dd, 3
J
2,3 = 3.1, 3
J
3,4 = 9.0 Hz, 1 H, H3), 4.07–4.14 (m, 4 H, δ-Hb
d -Hep , δ-Hb
l -Hep , H2, H4), 4.25 (br t, 3
J = 6.6 Hz, 1 H, β -H
d -Hep ), 4.28, 4.34 (2 d, each 1 H, OCH2
Bn ), 4.34–4.41 (m, 1 H, α-HmePhe ), 4.26–4.49 (m, 7 H, 3.5 OCH2
Bn ), 4.49–4.59 (m, 2 H, α-H
l -Hep , α-HSer ), 4.51, 4.54, 4.69, 4.70 (3 d and 1 s, 5 H, 2.5 OCH2
Bn ), 4.65–4.78 (m, 3 H, α-HTyr , OH
d -Hep , OH
l -Hep ), 5.20 (t, 3
J
3′,4′ = 3
J
4′,5′ = 9.6 Hz, 1 H, H4′), 5.29 (d, 3
J
1′,2′ = 2.0 Hz, 1 H, H1′), 5.56 (d, 3
J
1,2 = 2.1 Hz, 1 H, H1), 7.00–7.40 (m, 45 H, Harom , NH
l -Hep ), 7.76 (br s, 1 H, NHmePhe ), 7.83 (br s, 1 H, NHGly ), 8.26 (br s, 1 H, NHSer ), 8.42 (br d, 3
J = 8.4 Hz, 1 H, NHTyr ), 8.58 (br s, 1 H, NH
d -Hep ).
13 C NMR: (126 MHz, 350 K, DMSO-d
6 ): δ = 18.0 (CH3
mePhe ), 21.3 (2 CH3
i Val ), 23.5, 23.7 (2 CH3
i -Pr ), 23.9 (CH
i Val ), 25.3, 25.6 (2 CH3
i -Pr ), 27.4 (CH3
Boc ), 27.5 (CH3
Boc ), 34.6 (C-βTyr ), 39.7 (C-βmePhe ), 40.8 (CH2
Gly ), 42.0 (CH2
i Val ), 51.2 (C-αSer ), 53.1 (C-αTyr ), 54.1 (C-α
l -Hep ), 56.7 (C-αmePhe ), 57.4 (C-α
d -Hep ), 57.8 (C-γ
d -Hep , C-γ
l -Hep ), 62.0 (C-δ
d -Hep ), 62.5 (C-δ
l -Hep ), 67.2 (C4′), 68.0 (C-β
l -Hep ), 68.5 (C-βSer ), 68.6 (C6, C6′), 69.8, 70.1 (CH2 Ph), 70.3 (C-β
d -Hep ), 70.4 (C5), 71.2 (C5′), 71.3, 71.4, 71.6, 71.8, 71.9 (CH2 Ph), 72.8 (C4), 73.1 (C2), 74.3 (C2′), 75.8 (C3′), 78.5 (C3), 78.2, 79.2 (Cq
Boc ), 92.4, 92.7 (Cq
i -Pr ), 95.7 (C1′), 98.3 (C1), 116.1, 125.5–142.7 (Carom ), 154.3 (p -Carom
Tyr ), 170.5 (C=O
i Val ).
HRMS (ESI): m /z [M + H]+ calcd for C116 H143 N8 O27 : 2081.0096; found: 2081.0107.
Methyl N -(2,3,4,6-Tetra-O -benzyl-α-d -mannopyranosyloxycarbonyl)-l -phenylalaninate (19)
Methyl N -(2,3,4,6-Tetra-O -benzyl-α-d -mannopyranosyloxycarbonyl)-l -phenylalaninate (19)
Sulfoxide 1 (259 mg, 0.4 mmol, 1.8 equiv) and DTBP (0.15 mL, 0.69 mmol, 3.1 equiv) were coevaporated
with toluene (2 × 5 mL). The residue was dissolved in CH2 Cl2 (2 mL), molecular sieves (4 Å) were added, and the resulting mixture was stirred
at r.t. for 30 min under argon. After cooling the mixture to –70 °C, Tf2 O (52 μL, 0.31 mmol, 1.4 equiv) was added dropwise, followed by a soln of Boc-l -Phe-OMe (56 mg, 0.22 mmol, 1 equiv) in CH2 Cl2 (1 mL). After stirring for 18 h at –25 °C, the reaction was quenched by the addition
of MeOH (0.1 mL), diluted with CH2 Cl2 (30 mL), and poured into a mixture of sat. aq NaHCO3 –brine (1:1, 30 mL). The aqueous phase was extracted with CH2 Cl2 (3 × 20 mL) and the combined organic phases were dried (MgSO4 ) and evaporated. Purification of the residue by flash chromatography (PE–EtOAc, 4:1)
afforded glycocarbamate 19 (124 mg, 75%) as a colorless solid. Crystallization of an analytical sample (PE–THF–Et2 O) gave crystals suitable for X-ray analysis.[
27
] Rf
= 0.20 (PE–EtOAc, 4:1).
1 H NMR (400 MHz, acetone-d
6 ): δ = 3.00 (dd, 2
J = 13.8, 3
J
α,βa = 9.1 Hz, 1 H, β-Ha
Phe ), 3.17 (dd, J = 13.8, 3
J
α,βb = 5.3 Hz, 1 H, β-Hb
Phe ), 3.65 (s, 3 H, OCH3 ), 3.66–3.73 (m, 1 H, H6a ), 3.75–3.83 (m, 2 H, H6b , H5), 3.86–3.94 (m, 2 H, H2, H3), 4.03 (t, 3
J = 9.3 Hz, 1 H, H4), 4.43–4.55 (m, 1 H, α-HPhe ), 4.51–4.90 (m, 8 H, 4 OCH2
Bn ), 6.04 (d, 3
J
1,2 = 1.5 Hz, 1 H, H1), 6.94 (d, 3
J
NH,α = 8.8 Hz, 1 H, NH), 7.16–7.46 (m, 25 H, Harom ).
13 C NMR (100 MHz, acetone-d
6 ): δ = 38.2 (C-βPhe ), 52.4 (OCH3 ), 56.4 (C-αPhe ), 70.1 (C6), 72.4, 73.3, 73.8 (3 × CH2 Ph), 74.8 (C5), 75.3 (C2, C4), 75.5 (CH2 Ph), 80.7 (C3), 93.1 (C1), 127.6, 128.1, 128.2, 128.3, 128.5, 128.6, 128.6, 128.7,
129.0, 129.0, 129.1, 129.1, 129.2, 130.1 (CHarom ), 138.1, 139.7, 139.8, 139.8, 139.9 (Carom ), 154.8 (C=Ocarbamate ), 172.6 (COOMe).
HRMS (ESI): m /z [M + Na]+ calcd for C45 H47 NNaO9 : 745.3251; found: 745.3257.
N -(2,3,4,6-Tetra-O -benzyl-α-d -mannopyranosyloxycarbonyl)-l -phenylalanine (20)
N -(2,3,4,6-Tetra-O -benzyl-α-d -mannopyranosyloxycarbonyl)-l -phenylalanine (20)
To a soln of ester 19 (24 mg, 32 μmol) in 1,4-dioxane (0.4 mL) was added at 8 °C aq 1 M LiOH soln (64 μL,
64 μmol, 2 equiv). The mixture was stirred for 3 h at this temperature and then it
was diluted with EtOAc (20 mL), washed with sat. aq NH4 Cl–H2 O (1:1), and dried (MgSO4 ). Evaporation of the solvent gave acid 20 (21 mg, 89%) as a colorless solid; Rf
= 0.50 (CH2 Cl2 –MeOH, 9:1).
1 H NMR (400 MHz, acetone-d
6 ): δ = 2.96 (dd, 2
J = 14.0, 3
J
α,βa = 8.4 Hz, 1 H, β-Ha
Phe ), 3.19 (dd, 2
J = 14.0, 3
J
α,βb = 4.9 Hz, 1 H, β-Hb
Phe ), 3.64 (dd, 2
J = 11.0, 3
J
5,6a = 1.7 Hz, 1 H, H6a ), 3.70–3.76 (m, 2 H, H5, H6b ), 3.74 (dd, 3
J
1,2 = 2.1, 3
J
2,3 = 3.2 Hz, 1 H, H2), 3.80 (dd, 3
J
2,3 = 3.1, 3
J
3,4 = 9.5 Hz, 1 H, H3), 4.02 (t, 3
J
3,4 = 3
J
4,5 = 9.7 Hz, 1 H, H4), 4.44 (dd, 3
J
α,βa = 9.0, 3
J
α,βb = 5.4 Hz, 1 H, α-HPhe ), 4.44–4.84 (m, 8 H, 4 CH2
Bn ), 6.03 (d, 3
J
1,2 = 2.0 Hz, 1 H, H1), 7.10–7.39 (m, 25 H, Harom ).
13 C NMR (100 MHz, acetone-d
6 ): δ = 38.2 (C-βPhe ), 55.9 (C-αPhe ), 67.7 (C6), 69.3, 72.7, 73.1 (3 CH2
Bn ), 74.1 (C5), 74.5 (C2), 74.9 (C4), 75.8 (CH2
Bn ), 80.0 (C3), 92.9 (C1), 127.4, 128.2, 128.3, 128.4, 128.7, 128.7, 128.9, 129.0 (CHarom ), 129.9 (o -CHarom
Phe ), 137.6 138.5, 138.8 (Carom ), 155.1 (C=Ocarbamate ), 174.4 (COOH).
HRMS (ESI): m /z [M + Na]+ calcd for C44 H45 NNaO9 : 754.2987; found: 754.2975.
Methyl N -(α-d -Mannopyranosyloxycarbonyl)-l -phenylalaninate (21)
Methyl N -(α-d -Mannopyranosyloxycarbonyl)-l -phenylalaninate (21)
Glycocarbamate 19 (24 mg, 32 μmol) was dissolved in MeOH (0.5 mL), and 10% Pd/C was added (5 mg). The
mixture was stirred at r.t. under an atmosphere of H2 (1 bar) for 12 h, diluted with CH2 Cl2 (2 mL), and purified directly by flash chromatography (CH2 Cl2 –MeOH, 6:1), which afforded the tetraol 21 (8 mg, 64%) as a colorless solid; Rf
= 0.25 (CH2 Cl2 –MeOH, 9:1).
1 H NMR (400 MHz, CD3 OD): δ = 2.95 (dd, J = 13.8, 3
J
α,βa = 9.3 Hz, 1 H, β-Ha
Phe ), 3.16 (dd, J = 13.8, 3
J
α,βb = 5.2 Hz, 1 H, β-Hb
Phe ), 3.59–3.64 (m, 1 H, H5), 3.66–3.74 (m, 3 H, H4, H3, H6a ), 3.70 (s, 3 H, OCH3 ), 3.74 (dd, 3
J
1,2 = 1.7, 3
J
2,3 = 3.2 Hz, 1 H, H2), 3.79 (dd, 2
J = 12.1, 3
J
5,6b = 2.2 Hz, 1 H, H6b ), 4.41 (dd, 3
J
α,βa = 9.3, 3
J
α,βb = 5.3 Hz, 1 H, α-HPhe ), 5.80 (d, 3
J
1,2 = 1.9 Hz, 1 H, H1), 7.18–7.25 (m, 5 H, CHarom ).
13 C NMR (100 MHz, CD3 OD): δ = 38.5 (C-βPhe ), 52.7 (OCH3 ), 57.0 (C-αPhe ), 62.7 (C6), 68.0 (C4), 71.2 (C2), 72.2 (C3), 76.6 (C5), 96.1 (C1), 127.9, 129.5,
130.3, 138.3 (Carom ), 156.4 (C=Ocarbamate ), 173.6 (COOMe).
HRMS (ESI): m /z [M + Na]+ calcd for C17 H23 NNaO9 : 408.1265; found: 408.1259.
Methyl N -(2,3,4,6-Tetra-O -benzyl-α-d -mannopyranosyloxycarbonyl)-l -prolinate (22)
Methyl N -(2,3,4,6-Tetra-O -benzyl-α-d -mannopyranosyloxycarbonyl)-l -prolinate (22)
Boc-l -Pro-OMe (27 mg, 118 μmol, 1 equiv) and DTBP (98 μL, 425 μmol, 3.6 equiv) were coevaporated
with toluene (2 × 5 mL). The residue was dissolved in CH2 Cl2 (2 mL), molecular sieves (4 Å) were added, and the resulting mixture was stirred
at r.t. for 30 min under argon. After cooling the mixture to –70 °C, Tf2 O (35 μL, 212 μmol, 1.8 equiv) was added dropwise, followed by a soln of sulfoxide
1 (138 mg, 212 μmol, 1.8 equiv) in CH2 Cl2 (1 mL), and the mixture was slowly warmed to r.t. over 5 h. The reaction was quenched
by the addition of MeOH (0.1 mL), diluted with CH2 Cl2 (30 mL), and poured into a mixture of sat. aq NaHCO3 –brine (1:1, 30 mL). The aqueous phase was extracted with CH2 Cl2 (3 × 20 mL) and the combined organic phases were dried (MgSO4 ) and evaporated. Purification of the residue by flash chromatography (PE–EtOAc, 4:1,
3:1) afforded glycocarbamate 22 (65 mg, 79%) as a colorless oil; Rf
= 0.40 (PE–EtOAc, 2:1).
1 H NMR (400 MHz, DMSO-d
6 , mixture of cis - and trans -isomers, ratio 3:2): δ = 1.77–1.96 (m, 3 H, cis - + trans -βPro -Ha , 2 γPro -H), 2.13–2.30 (m, 1 H, cis - + trans -βPro -Hb ), 3.28–3.35 (m, 1.2 H, cis -δPro -CH2 ), 3.41 (t, 3
J
γ,δ = 7.0 Hz, 0.8 H, trans -δPro -CH2 ), 3.58 (s, 1.2 H, trans -OCH3 ), 3.60–3.64 (m, 1 H, cis - + trans -H6a ), 3.61 (s, 1.8 H, cis -OCH3 ), 3.65–3.74 (m, 2.4 H, trans -H3, cis - + trans -H4, cis - + trans -H6b ), 3.79–3.84 (m, 1 H, trans -H2, cis -H3), 3.84–3.93 (m, 1.6 H, cis -H2, cis - + trans -H5), 4.24 (dd, 3
J
α,β = 3.9, 3
J
α,β = 9.0 Hz, 0.6 H, cis -αPro -CH), 4.35 (dd, 3
J
α,β = 4.1, 3
J
α,β = 8.5 Hz, 0.4 H, trans -αPro -CH), 4.43–4.81 (m, 8 H, 4 CH2 Ph), 5.95 (d, 3
J
1,2 = 1.8 Hz, 0.6 H, cis -H1), 5.97 (d, 3
J
1,2 = 1.8 Hz, 0.4 H, trans -H1), 7.16–7.23, 7.24–7.39 (2 m, 2 H and 18 H, cis - + trans -βHarom ).
13 C NMR (100 MHz, DMSO-d
6 ): δ= 23.1 (trans -C-γPro ), 23.7 (cis -C-γPro ), 29.4 (cis -C-βPro ), 30.1 (trans -C-βPro ), 46.2 (cis -C-δPro ), 46.7 (trans -C-δPro ), 51.9 (cis -OCH3 ), 52.0 (trans -OCH3 ), 58.5 (trans -C-αPro ), 58.7 (cis -C-αPro ), 68.7 (C6), 70.7 (CH2 Ph), 71.0 (C5), 71.9, 71.9, 72.3, 72.3, 73.3 (C4), 73.4 (C2), 73.6 (CH2 Ph), 73.7, 73.8, 73.9, 74.2, 78.2 (cis -C3), 79.6 (trans -C3), 91.9 (C1), 127.3, 127.4, 127.4, 127.4, 127.5, 127.5, 127.5, 127.6, 127.6, 127.7,
127.7, 127.8, 128.1, 128.2, 128.2, 128.2, 139.8, 139.8, 139.9 (Carom ), 151.2 (trans -C=Ocarbamate ), 151.8 (cis -C=Ocarbamate ), 172.3 (cis -COOMe), 172.8 (trans -COOMe).
HRMS (ESI): m /z [M + Na]+ calcd for C41 H45 NNaO9 : 718.2992; found: 718.2975.
N
α -Fmoc-l -Lys-N
ε -(2,3,4,6-tetra-O -benzyl-α-d -mannopyranosyloxycarbonyl)-l -Phe-OMe (23)
N
α -Fmoc-l -Lys-N
ε -(2,3,4,6-tetra-O -benzyl-α-d -mannopyranosyloxycarbonyl)-l -Phe-OMe (23)
Perbenzylated sulfoxide 1 (129 mg, 0.2 mmol, 1.5 equiv) and DTBP (91 μL, 0.41 mmol, 3.1 equiv) were coevaporated
with toluene (2 × 5 mL). The residue was dissolved in CH2 Cl2 (4 mL), and molecular sieves (4 Å) were added. The resulting mixture was stirred
for 30 min under an argon atmosphere. The mixture was cooled –70 °C and Tf2 O (30 μL, 0.2 mmol, 1.5 equiv) was added dropwise followed by a soln of Fmoc-l -Lys-(N
ε -Boc)-l -Phe-OMe (83 mg, 131 μmol) in CH2 Cl2 (5 mL). The mixture was stirred at –25 °C for 18 h, and the reaction was stopped
by the addition of MeOH (0.3 mL), diluted with CH2 Cl2 (30 mL) and poured into sat. aq NaHCO3 –brine–H2 O (1:1:2, 30 mL). The aqueous phase was extracted with CH2 Cl2 (3 × 20 mL), and the combined organic phases were dried (MgSO4 ) and evaporated. The residue was purified by flash chromatography (CH2 Cl2 –MeOH, 100:1, then 80:1), which afforded mannosyl carbamate 23 (43 mg, 39 μmol, 44%) as a colorless solid; Rf
= 0.30 (CH2 Cl2 –MeOH, 40:1).
1 H NMR (500 MHz, acetone-d
6 ): δ = 1.32–1.46 (m, 2 H, 2 γ-HLys ), 1.47–1.57 (m, 2 H, 2 δ-HLys ), 1.59–1.70 (m, 1 H, β-Ha
Lys ), 1.74–1.85 (m, 1 H, β-Hb
Lys ), 3.02 (dd, J = 13.8, 3
J
α,βa = 7.8 Hz, 1 H, β-Ha
Phe ), 3.09–3.18 (m, 3 H, β-Hb
Phe , 2 ε-HLys ), 3.64 (s, 3 H, OCH3 ), 3.69 (d, 2
J = 10.7 Hz, 1 H, H6a ), 3.77 (dd, 2
J = 11.2, 3
J
5,6b = 4.7 Hz, 1 H, H6b ), 3.79–3.84 (m, 1 H, H5), 3.91 (dd, 3
J
2,3 = 3.0, 3
J
3,4 = 9.5 Hz, 1 H, H3), 3.98 (dd, 3
J
1,2 = 1.7, 3
J
2,3 = 3.0 Hz, 1 H, H2), 4.04 (t, 3
J
3,4 = 3
J
4,5 = 9.3 Hz, 1 H, H4), 4.15–4.21 (m, 1 H, α-HLys ), 4.22 (br t, 3
J = 7.3 Hz, 1 H, CHFmoc ), 4.26–4.39 (m, 2 H, CH2
Fmoc ), 4.51–4.68 (m, 5 H, 2 CH2
Bn , CHa
Bn ), 4.69–4.75 (m, 1 H, α-HPhe ), 4.76 (br s, 2 H, CH2
Bn ), 4.89 (d, 2
J = 11.0 Hz, 1 H, CHb
Bn ), 6.14 (d, 3
J
1,2 = 1.7 Hz, 1 H, H1), 6.52–6.63 (m, 2 H, α-NHLys , ε-NHLys ), 7.12–7.46 (m, 29 H, Harom ), 7.48 (d, 3
J
NH,α = 7.8 Hz, 1 H, NHPhe ), 7.65–7.73 (m, 2 H, Harom
Fmoc ), 7.82–7.88 (m, 2 H, Harom
Fmoc ).
13 C NMR (125 MHz, acetone-d
6 ): δ = 23.5 (γ-CLys ), 30.3 (δ-CLys ), 32.6 (β-CLys ), 38.4 (β-CPhe ), 41.4 (ε-CLys ), 48.1 (CFmoc ), 52.4 (OCH3 ), 54.2 (α-CPhe ), 56.2 (α-CLys ), 67.3 (CH2
Fmoc ), 70.1 (C6), 72.3, 73.3, 73.8, 75.4 (4 CH2 Ph), 74.7 (C5), 75.6 (C4), 75.7 (C2), 80.8 (C3), 92.6 (C1), 120.8 (CHarom
Fmoc ), 126.2, 127.6, 128.1, 128.2, 128.3, 128.5, 128.6, 128.6, 128.6, 129.0, 129.0, 129.1,
129.1, 129.2 (CHarom ), 130.1 (o -CHarom
Phe ), 136.8, 138.1, 139.7, 139.8, 139.8, 139.9 (Carom ), 142.1 (CHarom
Fmoc ), 145.0, 145.1 (Carom
Fmoc ), 155.1 (C=Ocarbamate
Man ), 157.0 (C=Ocarbamate
Fmoc ), 172.5 (COOMe).
HRMS (ESI): m /z [M + Na]+ calcd for C66 H69 N3 NaO12 : 1118.4779; found: 1118.4799.
