An Improved Protocol for the Synthesis and Purification of Yariv Reagents

Article abstract of DOI:10.1021/acs.joc.0c01812

Yariv reagents are glycoconjugate tris-azo dyes widely used in plant biology. These reagents are synthesized by diazo coupling between phloroglucinol and a para-diazophenyl glycoside. Despite their synthetic accessibility, well-defined protocols for obtai

Full text of DOI:10.1021/acs.joc.0c01812

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An Improved Protocol for the Synthesis and Purification of Yariv
Reagents
Raghuraj Hoshing, Michael Saladino, Helene Kuhn, David Caianiello, Robert F. Lusi, and Amit Basu*
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ABSTRACT: Yariv reagents are glycoconjugate tris-azo dyes
widely used in plant biology. These reagents are synthesized by
diazo coupling between phloroglucinol and a para-diazophenyl
glycoside. Despite their synthetic accessibility, well-defined
protocols for obtaining pure Yariv reagents, and their complete
compound characterization data, have not been reported. We
report here optimized protocols used to synthesize, purify, and
characterize a panel of six Yariv reagents and suggest approaches
that could be valuable for the purification and characterization of
other glycoconjugates as well.
lycoconjugates are synthetic presentations of carbohy-
drates widely used in studies of carbohydrate recognition
compounds 1 and 2, when we encountered variations in the
product purity and challenges in the product characterization. In
this report we describe a detailed set of protocols for the
purification and characterization of the Yariv reagents. We
developed these protocols for the two canonical Yariv reagents,
the β-D-glucosyl (1) and β-D-galactosyl (2) derivatives. These
optimized protocols were used to synthesize and purify several
other Yariv reagents (3−6). We also report the first complete
spectral characterization data (1H, 13C, COSY, and HSQC) for
these Yariv reagents. An initial version of this work was deposited
in ChemRxiv on June 24, 2020.25
Multiple batches of the β-D-glucosyl Yariv reagent (1) were
synthesized using a slight modification of a literature protocol.23
The1HNMRspectraofthecrudeproductsoffourrepresentative
batches are displayed in Figure 3 (for complete spectra, see
Figure S1), and exhibit wide batch-to-batch variations in the
purity and hydration of the crude materials. Resonances at 7.16
and 7.66 ppm corresponding to the protons of the p-azophenoxy
moiety were observed in each spectrum, but these resonances
were occasionally poorly resolved (e.g., batch 2). Several spectra
exhibit additional peaks in the aromatic region, indicating the
presence of impurities in these batches. The mass spectrometric
analysis of the crude product of batch 2 indicated a signal at m/z
691.1, corresponding to the bis-azo homologue. Also observed in
Figure 3 are variations in the peak shapes of the sugar −OH
resonances between 4.5 and 5.5 ppm, with many, but not all, of
G
inbiologicalsystems.1−5 Oneoftheearliestexamplesofsynthetic
glycoconjugatesare theYariv reagents(Figure1), asetoftris-aryl
glycosideazodyesreportedin1962.6 Severaloftheseeponymous
reagentsareusefultoolsinplantbiology.Theβ-D-glucopyranosyl
and β-D-galactopyranosyl derivatives 1 and 2 are used to identify
andisolatearabinogalactanproteins(AGPs). AGPsarebranched
proteoglycans found in plant cell walls that perform various cell
signaling and structural support roles.7−9 Yariv reagents are
primarily used for the histochemical staining,10−12 quantifica-
tion,13 and purification of AGPs.14,15 1 has also been used as a
probe to elucidate the roles of AGPs in cellular physiology.16,17
Biophysical studies indicate that the Yariv reagents aggregate in
solution and bind to the β-1,3 oligogalactan backbone of
AGPs.18−20
The Yariv reagents are synthesized by the diazotization of the
corresponding p-aminophenyl glycosides, followed by the
coupling of the diazonium salt with phloroglucinol (Figure
2).6,21−24 In Yariv’s original report, the dye was isolated from the
coupling reaction by precipitation from ethanol.6 Several
subsequent publications have reported additional synthetic
details, along with some minor modifications, but they provide
limited details on the further purification of the crude
product.23,24 Our own work as well as previous reports indicate
that the isolated crude product can vary in its purity.20
Furthermore, none of the literature reports of the synthesis of
the Yariv reagents provide complete NMR characterization data;
an incomplete list of 1H NMR resonances has only been reported
for compounds 1 and 2.23,24 We speculate that the challenges in
purification may have hampered a more thorough character-
ization of these important reagents.
Special Issue: A New Era of Discovery in Carbohy-
drate Chemistry
Received: July 28, 2020
The lack of detail in the purification protocols and the
characterization became very apparent during our syntheses of
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Figure 1. The Yariv reagents.
Figure 2. General synthetic scheme for Yariv reagents.
Figure 3. NMR spectra of the crude product from multiple batches of β-D-glucosyl Yariv reagent (1) syntheses. Spectra were obtained in DMSO-d6.
these spectra exhibiting peak broadening from the exchange with
water.
reprecipitated from ethanol to provide a material that we
designate 1-R1X. The NMR spectrum of 1-R1X indicates a
significant reduction in the amount of impurities (Figure 4).
A second round of redissolution and reprecipitation to
generate batch R2X resulted in a significant sharpening of the
sugar −OH peaks, and the coupling to the backbone C−H
protons was observed (Figures 4 and S2). DOSY NMR
experiments were carried out using two different samples of 1,
one exhibiting broad −OH resonances and the other exhibiting
sharp peaks. The diffusion coefficient of the sugar −OH
resonances was greater (logD = −5.9) in the samples containing
broad peaks compared to the sharp −OH peak samples (logD =
−6.0), indicating that the former peaks are diffusing faster
(Figures 5 and S3). The logD values for the nonexchangeable
aromatic protons of both samples displayed the same diffusion
constants (logD = −6.1). The differences in the diffusion
Our attempts at removing the impurities focused on using the
redissolution and reprecipitation protocol mentioned by Woods
et al., who were using analytical ultracentrifugation to character-
ize Yariv reagent aggregation in water.20 Upon the repeated
redissolution of 1 in water, followed by precipitation from
methanol, increases in the molecular weight distribution of the
Yariv aggregates were observed. They suggested that the crude
products contained impurities derived from incomplete diazo
coupling, i.e., bis- and mono-azo phloroglucinol derivatives that
were removed after this purification. Our observation of the bis-
azo species in the mass spectrum of the crude product
corroborates this suggestion. To evaluate if redissolution−
reprecipitation is effective in removing these impurities, the
crude product of 1 (batch 2) was dissolved in water and
B
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Figure4. Purificationoftheβ-D-glucosylYarivreagent(1)batch2crudeshowing, frombottomtotop, thecrude, thereprecipitatedonce(R1X), andthe
reprecipitated twice (R2X) spectra. Spectra were obtained in DMSO-d6.
Figure5. Comparisonof1HNMRandDOSYNMRspectraandofaβ-D-glucosylYariv reagent(1)batch2samplecontaining(a) broad−OHpeaksvsa
sample containing (b) sharp −OH peaks.
coefficient values of the sugar −OH resonances between the two
samples are consistent with an increased exchange of these
protons with water. The ROESY spectrum of 1 (Figures S4 and
S5) exhibits exchange crosspeaks between the sugar −OH
protons and the water resonance, corroborating the observations
from the DOSY experiment. An examination of the level of trace
water observed in the DMSO-d6 sample before and after the
dissolution of the Yariv reagent indicates an increase after analyte
addition (Figure S6), establishing that the excess water observed
in the NMR spectra originates from water associated with the
Yariv reagent and not solely from adventitious moisture in the
NMR solvent. This was corroborated by the observation of a
smaller diffusion constant for water in the sample exhibiting
broad Yariv −OH peaks (Table S1). Conventional methods for
the removal of this extraneous water, such as drying over P2O5 or
Drierite under high vacuum, were not successful.
Occasionally, additional processing beyond redissolution and
reprecipitation was required to observe sharp −OH peaks. For
example,asseeninFigure6,thecrudeproductfrom1batch3was
subject to two rounds of redissolution and reprecipitation to give
C
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1
Figure 6. H NMR spectra of, from bottom to top, the β-D-glucosyl Yariv reagent (1) batch 3 crude, the first reprecipitation (R1X), the second
reprecipitation (R2X), and the triturated material (R2XM).
R1X and R2X. This procedure successfully resolved individual
−OH proton resonances that were not visible in the crude
product, but these resonances remained slightly broadened even
in the R2X sample. It was also frequently observed that trace
amounts of ethanol would persist in these samples despite
prolonged drying attempts (Figure S7). We surmised that the
addition of methanol might help remove both the residual water
and ethanol and might itself be easier to remove due to its lower
boiling point. The trituration of 1 (batch 3) with boiling
methanol, followed by filtration and subsequent drying in a
vacuum oven, gave a methanol-processed batch, referred to here
as R2XM. This procedure successfully sharpened the −OH
resonances (Figure 6). Besides sharpening the −OH peaks, the
trituration process also effectively removed ethanol from the
sample (Figures S7 and S8), with mass recoveries of 78% and
95% in the final steps for batches 3 and 4, respectively.
To highlight the utility of improved protocols, several other
Yariv reagents (2−6) were synthesized and purified using these
optimized protocols. The diazotization of the cognate para-
aminophenyl glycoside and the coupling to phloroglucinol were
carriedoutinthesamemannerasthosefortheglucosylderivative
1. Several of the resulting crude products exhibited significant
−OH broadening (Figures S9−S13). In each case, redissolution
and reprecipitation effectively sharpened the −OH resonances.
The spectra of several of the Yariv reagents still exhibited the
presence of significant amounts of ethanol even after this
purification, as seen in the 1H NMR spectra of compounds 2−6
aftereachstageofpurification(FiguresS9−S13).Toremovethe
additional residual ethanol, the β-D-xylosyl analog 3 was
triturated with methanol and subsequently rotary evaporated
from methanol (“methovaped”), a process that we have found to
be as effective as trituration with higher sample recovery when
working at a smaller scale. Both L-fucosyl compounds 5 and 6
were also effectively purified via methovaping. Purified
compounds were obtained in good yields. Yields of the crude
product ranged from 72 to 93%, and mass balances of the final
product after redissolution−reprecipitation and methanol
processing ranged from 41 to 99% (Table S2). Finally, the
improved purification protocols allowed the complete character-
ization of these Yariv reagents by 1H and 13C NMR.
In summary, we have addressed several outstanding issues
associated with the synthesis of Yariv reagents, such as the
presence of impurities arising from incomplete azo coupling and
the broadening of sugar −OH NMR peaks due to the exchange
with water. We demonstrate that the Yariv reagents can be
purifiedbyasimpleredissolutionandreprecipitationprocessthat
provides a material of greater purity than is often obtained from
the original report of their precipitation from ethanol.6 We also
show that the water−sugar −OH exchange and the presence of
residual ethanol can be reduced by various treatments with
methanol. An additional benefit of the protocols reported here is
that they can potentially be used with glycoconjugates besides
just the Yariv reagents. It is worth noting here that many other
amphiphilic glycoconjugates exhibit similar challenges with
respect to resolving −OH resonances clearly. In several cases
where glycoconjugates were isolated from methanol, well
resolved −OH resonances were observed, indicating the utility
of processing glycoconjugates with methanol during purifica-
tion.26−29 Finally, several additional Yariv reagents were
synthesized, isolated, purified, and fully characterized using the
optimized procedures.
EXPERIMENTAL SECTION
■
Thin layer chromatography was carried out on Merck silica gel 60 F254
precoatedglassplates. CompoundswerevisualizedunderaUVlamp. All
NMR spectra were recorded on a Bruker Avance III HD Ascend 600
MHzinstrumentusingDMSO-d6 asthesolvent. 1HNMRand13CNMR
spectra were referenced to residual solvent peaks.30 Coupling constants
are given in hertz (Hz), and chemical shifts are given in parts per million
(ppm). Structural assignments were made with additional information
from gCOSY, gHSQC, and gHMBC experiments. Electrospray
ionization (ESI) mass spectra were obtained using a Thermo LCQ
Deca XP Max ion trap mass spectrometer. Purified water was obtained
from an EMD Millipore Direct-Q 3 Tap to Pure and Ultrapure Water
Purificationsystem.Avacuumovensetat75°CwasusedtodrytheYariv
reagents after the final purification.
Synthesis of theβ-D-Glucosyl Yariv reagent (1), Batch 4. A Parr
shaker bottle was charged with 4-nitrophenyl β-D-glucopyranoside (2.5
g, 8.3 mmol) and methanol (250 mL), and a stir bar was added to the
D
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bottle. The mixture was stirred while sparging for 20 min, with N2
introduced via a needle. To the Parr bottle was added 10% Pd/C (40
mg), which was put on a Parr shaker and shaken under 40 psi H2 for 7 h.
TLC showed complete conversion (2:1 dichloromethane/isopropyl
alcohol). The reaction mixture was filtered through Celite into a 1000
mL flask and then concentrated in vacuo to about 100 mL. The solution
was transferred to a 250 mL preweighed flask, concentrated in vacuo,
andplacedunderhighvacuum.Theaminosugar31 wasobtainedasapale
yellow-clear crystalline solid (2.20 g, 8.1 mmol, 97%). This material was
directlyused inthenext step:1HNMR (600 MHz, DMSO-d6)δ 6.77(d,
J= 8.8 Hz, 2H), 6.49 (d, J = 8.5Hz, 2H), 5.23(s, 1H), 5.07(d, J = 4.6Hz,
1H), 5.00 (d, J = 4.9 Hz, 1H), 4.69 (s, 2H), 4.57 (d, J = 7.7 Hz, 1H), 4.55
(t, J = 5.8 Hz, 1H, 1H), 3.71−3.65 (m, 1H), 3.46 (dt, J = 11.5, 5.4 Hz,
1H), 3.25−3.09 (m, 4H).
A solution of cold freshly prepared 1.2 M HCl (17 mL, 2.5 equiv) was
added to 4-aminophenyl β-D-glucopyranoside31 (2.2 g, 8.11 mmol, 1
equiv) in a 250 mL round-bottom flask. This solution was cooled in an
ice bath while stirring. NaNO2 (616 mg, 8.9 mmol, 1.1 equiv) was
dissolved in 1 mL of cold water in an Eppendorf tube. The NaNO2
solution was added to a 5 mL syringe with a 4 in. × 22 gauge needle that
was clamped above the reaction flask, leading to a gravity-assisted
dropwise addition. The reaction was stirred at 0 °C for 3 h.
Phloroglucinol (348 mg, 2.8 mmol, 0.34 equiv) was dissolved in a
minimal amount of cold 5 M NaOH (1−2 mL) and added dropwise to
the reaction mixture. To the mixture was added 5 M NaOH until the pH
was basic as indicated by a pH paper, at which point the ice bath was
removed. The pH was maintained at ∼11 (dark blue on the pH strip) by
theadditionof5MNaOHasnecessary.Thereactionmixturewasstirred
overnight. Cold 100% ethanol (200 mL) was added to the reaction
mixture, which was neutral at this point as determined by the pH paper.
To the mixture was added 1.2 M HCl until the solution was acidic as
indicated by a pH paper. The reaction flask was placed in a freezer for at
least 24 h until a dark red-brown precipitate was observed. The
precipitate was filtered through a Hirsch funnel to provide the crude
Yariv reagent 1 as a glossy red sticky powder, which clumped together.
This solid was air-dried for a few hours, at which point it hardened to a
solid mass that became a powder when crushed. The powder was
transferred to a preweighed scintillation vial and dried in a vacuum oven
at 75 °C for 24 h. This crude product (2.31 g, 2.4 mmol) was obtained in
an 86% yield as a dark red powder. Some of this material was further
purified twice using the redissolution−reprecipitation protocol,
followed by trituration.
Synthesis of the β-D-Galactosyl Yariv Reagent (2). 4-Nitro-
phenyl β-D-galactopyranoside (0.7 g, 2.4 mmol) and 10% Pd/C (42 mg)
were used to prepare the aniline using the same procedure used for 4-
aminophenyl β-D-glucopyranoside. TLC showed complete conversion
(2:1 dichloromethane/methanol). The product was dried in vacuo
overnight to give the aniline31 as a pale-yellow powder (0.6 g, 2.2 mmol,
91%). This material was directly used in the next step: 1H NMR (600
MHz, DMSO-d6)6.76(d,J=9Hz,2H),6.49(d,J=9.1Hz,2H), 5.07(d,
J = 5.1 Hz, 1H), 4.79 (d, J = 6.2 Hz, 1H), 4.78 (s, 2H), 4.62 (t, J = 5.4 Hz,
1H), 4.54 (d, J = 7.7 Hz, 1H), 4.45 (d, J = 4.6 Hz, 1H), 3.67 (t, J = 3.9 Hz,
1H), 3.55 (dt, J = 10.3, 5.4 Hz, 1H), 3.47 (m, 3H), 3.37 (m, 1H).
Using largely the same procedure as that used for 1, 1.2 M HCl(5 mL,
2.5 equiv) and 4-aminophenyl β-D-galactopyranoside31 (608 mg, 2.24
mmol, 1 equiv) were used to synthesize the Yariv reagent 2. However,
the basificationofthe reactionwasdone using2MNaOHinsteadof5M
NaOH. The crude product (0.8 g, 0.76 mmol) was obtained in a 100%
yield. Some of this material was further purified using the redissolution−
reprecipitation protocol twice, followed by methovaping; 0.66 g of the
crude product was reprecipitated from 20 mL of water and 130 mL of
ethanol to give 0.4 g of the β-D-galactosyl Yariv reagent (2) R1X product
(60%). The second redissolution−reprecipitation with R1X (300 mg)
from 20 mL of water and 130 mL of ethanol provided 0.23 g of the β-D-
galactosyl Yariv reagent (2) R2X (77%), which was was purified by
rotary evaporation from methanol (methovaping).
Methovaping (Representative Protocol). β-D-Galactosyl (2) R2X
(0.055 g) was weighed out into a scintillation vial. To the vial was added
3 mL of methanol, which was then evaporated using a rotovap. The vial
was dried in a vacuum oven overnight at 75 °C overnight to afford 0.054
g (99%) of β-D-galactosyl R2XM as a mixture of dark red and black
pellets, which could be crushed into a powder. The vial containing the
product was stored in a desiccator containing Drierite for several days:
1HNMR(600MHz, DMSO)δ15.91and15.53−14.77(s,3H, coreAr−
OH), 7.60 (d, J = 8.4 Hz, 6H, ArH ortho to azo), 7.15 (d, J = 8.6 Hz, 6H,
ArH ortho to sugar), 5.27 (d, J = 5.1 Hz, 3H, 2-OH), 4.93 (d, J = 5.1 Hz,
3H, 3-OH), 4.89 (d, J = 7.5 Hz, 3H, H1), 4.76(t, J = 5.1 Hz, 3H, 6-OH),
4.59 (d, J = 4.5 Hz, 3H, 4-OH), 3.73 (t, J = 3.3 Hz, 3H, H4), 3.60 (m,
12H, H2, H5, H6), 3.45(s, 3H, H3);13C{1H}NMR(151MHz, DMSO-
d6) δ 177.9 (core Ar, C−O), 156.9(Ar, C−O−sugar), 136.5(Ar, C−N),
128.8(coreAr, C−N), 119.0(ArC−C−N), 117.9(ArC−C−O−sugar),
101.5 (C1), 76.1 (C2), 73.7 (C3), 70.8 (C5), 68.7 (C4), 60.9 (C6);
HRMS (ESI) m/z [M + H]+ calcd for C42H49N6O21 973.2951, found
973.2920.
Redissolution−Reprecipitation(RepresentativeProtocol). To0.6g
of the crude product (β-D-glucosyl Yariv reagent (1) batch 4) was added
20 mL of water, and the mixture was heated and vortexed until complete
dissolution. To the solution was added 130 mL of cold 100% ethanol.
ThemixturewasfilteredthroughaHirschfunnel, andtheprecipitatewas
allowed to air-dry and then transferred to a preweighed vial. The
precipitatewasthenallowedtodryinavacuumovenovernight,and0.5g
of material R1X (81%) was obtained. A second redissolution−
reprecipitation with (1) R1X (0.4 g) provided 0.35 g of β-D-glucocyl
(1) batch 4 R2X (85%).
Trituration (Representative Protocol). The β-D-glucosyl R2X Yariv
reagent (1) batch 4 (0.19 g) was weighed out into an Erlenmeyer flask.
The material was then rinsed with 10 mL of boiling methanol five times
and filtered through a Hirsch funnel. It was then dried in a vacuum oven
overnight to afford 0.18 g (95%) of β-D-glucosyl (1) batch 4 R2XM as a
mixture of dark red and black pellets, which were crushed into a powder:
1H NMR(600 MHz, DMSO-d6)δ 15.91, 15.86, 15.11, and 15.06 (s, 3H,
core Ar−OH), 7.66 (d, J = 8.6 Hz, 6H, ArH ortho to azo), 7.18 (d, J = 8.7
Hz, 6H, ArH ortho to sugar), 5.37 (d, J = 5.0 Hz, 3H, 2-OH), 5.11 (d, J =
4.7 Hz, 3H, 3-OH), 5.05 (d, J = 5.3 Hz, 3H, 4-OH), 4.94 (d, J = 7.4 Hz,
3H, H1), 4.61 (t, J = 5.8 Hz, 3H, 6-OH), 3.73 (ddd, J = 12.0, 5.3, 2.1 Hz,
3H, H6a), 3.50 (dt, J = 11.9, 6.0 Hz, 3H, H6b), 3.38 (ddd, J = 9.7, 5.7, 2.3
Hz, 3H, H5), 3.34−3.23 (m, 6H, H3, H2), 3.19 (td, J = 9.0, 4.9 Hz, 3H,
H4); 13C{1H} NMR (151 MHz, DMSO-d6) δ 178.1 (core Ar, C−O),
156.8 (Ar, C−O−sugar), 136.6 (Ar, C−N), 128.9 (core Ar, C−N),
119.0 (Ar, C−C−N), 118.0 (Ar, C−C−O−sugar), 101.0 (C1), 77.6
(C5), 77.1 (C3), 73.7 (C2), 70.2 (C4), 61.2 (C6); HRMS (ESI) m/z [M
+ H]+ calcd for C42H49N6O21 973.2951, found 973.2927.
Synthesis of the β-D-xylosyl Yariv Reagent(3). 4-Nitrophenyl β-
D-xylopyranoside(1.0g, 3.7mmol)and10%Pd/C(36mg)wereusedto
prepare the aniline using the same procedure that used for 4-
aminophenyl β-D-glucopyranoside. TLC showed complete conversion
(5:1 dichloromethane/methanol). The amino sugar32 was obtained as a
pale yellow-clear crystalline solid (0.9 g, 3.6 mmol, 97%). This material
was directly used in the next step: 1H NMR (600 MHz, DMSO-d6) δ
6.72 (d, J = 8.8 Hz, 2H), 6.49 (d, J = 8.7 Hz, 2H), 5.26 (s, 1H), 5.10 (s,
1H), 5.04 (s, 1H), 4.70 (s, 2H), 4.56 (d, J = 7.2 Hz, 1H), 3.70 (dd, J =
11.2, 5.3 Hz, 1H), 3,38 (m, 1H), 3.22−3.08 (m, 3H).
4-Aminophenyl β-D-xylopyranoside32 (0.89 g, 3.7 mmol, 1 equiv)
used to synthesize the Yariv reagent 3 using the same procedure as that
used for 1. However, the basification of the reaction was done using 2 M
NaOHinsteadof5MNaOH. Thecrudeproduct(0.84g, 0.9mmol)was
obtained in a 77% yield. This material was further purified twice using
the redissolution−reprecipitation protocol, followed by methovaping
(see below);0.8 gof the crudeproduct wasreprecipitated from 30 mL of
water and 80 mLof ethanolto give 0.45 gof the β-D-xylosyl Yarivreagent
(3) R1X (53%). A second redissolution−reprecipitation with (3) R1X
(0.45 g) reprecipitated from 30 mL of water and 80 mL of ethanol
provided 0.36 g of the β-D-xylosly (3) R2X (82%). Then, 0.36 g of the β-
D-xylosly (3) R2X was triturated to afford 0.33 g of the β-D-xylosyl (3)
R2XM (92%). The β-D-xylosyl (3) R2XM sample (0.037 g) was
methovaped and dried in a vacuum oven overnight at 75 °C overnight to
afford 0.033 g (90%) of the β-D-xylosyl R2XM2 as a mixture of dark red
andblackpellets, whichwascrushedintoapowder:1HNMR(600MHz,
DMSO-d6) δ 15.91, 15.86, 15.09, and 15.05 (s, 3H, core Ar−OH), 7.64
(d, J=8.7Hz, 6H, ArHorthotoazo), 7.15(d,J=8.5Hz,6H, ArHorthoto
E
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sugar), 5.40 (d, J = 3.8 Hz, 3H, 2-OH), 5.14 (d, J = 3.2 Hz, 3H, 3-OH),
5.10 (d, J = 4.6 Hz, 3H, 4-OH), 4.94(d, J = 6.3Hz, 3H, H1), 3.79 (dd, J=
10.9, 5.1 Hz, 3H, H5a), 3.40 (dq, J = 9.1, 4.3 Hz, 3H, H4), 3.32 (d, J =
10.9Hz,3H, H5b),3.27(m, 6H,H3andH2);13C{1H}NMR(151MHz,
DMSO-d6)δ178.1(core Ar, C−O), 156.5(Ar, C−O−sugar), 136.6(Ar,
C−N), 128.9(core Ar, C−N), 119.0 (Ar C−C−N), 118.0 (Ar C−C−
O−sugar), 101.5 (C1), 76.9 (C3), 73.5 (C2), 69.8 (C4), 66.2 (C5);
HRMS (ESI) m/z [M + H]+ calcd for C39H43N6O18 883.2634, found
883.2644.
Synthesis of the β-D-Fucosyl Yariv Reagent (4). 4-Nitrophenyl
β-D-fucopyranoside(0.5g, 1.7mmol)and10%Pd/C(37mg)wereused
to prepare the aniline using the same procedure as that used for 4-
aminophenylβ-D-glucopyranoside. The aminosugar32 wasobtainedasa
pale yellow-clear crystalline solid (0.4 g, 1.6 mmol, 99%). This material
was directly used in the next step: 1H NMR (600 MHz, DMSO-d6) δ
6.72 (d, J = 8.6 Hz, 2H), 6.48 (d, J = 8.8 Hz, 2H), 5.06 (s, 1H), 4.85 (s,
1H), 4.67 (s, 2H), 4.55 (d, J = 7.6 Hz, 1H), 4.47 (s, 1H), 3.63 (q, J = 6.6
Hz, 1H), 3.49−3.40 (m, 2H), 3.40−3.34 (m, 1H), 1.13 (d, J = 6.4 Hz,
3H).
71.8 (C3), 69.9 (C4), 68.1 (C2), 67.9 (C5), 17.0 (C6); HRMS (ESI) m/
z [M + H]+ calcd for C42H48N6O18 925.3103, found 925.3092.
Synthesisoftheβ-L-FucosylYarivReagent(6). 4-Nitrophenylβ-
L-fucopyranoside (0.8 g, 2.82 mmol) and 10% Pd/C (35 mg) were used
to prepare the aniline using the same procedure as that used for 4-
aminophenyl β-D-glucopyranoside. The aminosugar34 wasobtained asa
pale yellow-clear crystalline solid (0.72 g, 2.8 mmol, 99%). This material
was directly used in the next step: 1H NMR (600 MHz, DMSO-d6) δ
6.73 (d, J = 8.7 Hz, 2H), 6.49 (d, J = 8.9 Hz, 2H), 5.03 (s, 1H), 4.76 (s,
1H), 4.67 (s, 2H), 4.55 (d, J=7.6Hz, 1H), 4.47 (s, 1H), 3.63(qd, J=6.4,
1.1Hz, 1H), 3.48−3.42(m, 2H), 3.40−3.34(m,1H), 1.13(d, J=6.4Hz,
3H).
4-Aminophenylβ-L-fucopyranoside34 (0.72g, 2.8mmol, 1equiv)was
used to synthesize the Yariv reagent 6 using largely the same procedure
as that used for 1. However, the basification of the reaction was done
using 2 M NaOH instead of 5 M NaOH. This (6) crude product (0.71g,
1 mmol)was obtainedin an80% yield. This materialwas furtherpurified
twice using the redissolution−reprecipitation protocol, followed by
methovaping;0.71gofthecrudeproductwasreprecipitatedfrom20mL
of water and 180 mL of ethanol to give 0.52 g of the β-L-fucosyl (6) R1X
product(73%).Thesecondredissolution−reprecipitationwith(6)R1X
(0.5g)from20mLofwaterand180mLofethanolprovided0.44gofthe
β-L-fucosyl (6) R2X (84%). Methovaping 31 mg of (6) R2X using 3 mL
of methanol gave 28 mg of (6)R2XM (90%) asamixture of dark red and
black pellets, which was crushed into a powder: 1H NMR (600 MHz,
DMSO-d6) δ 15.94, 15.88, 15.10, and 15.06 (s, 3H, core Ar−OH), 7.62
(d, J=8.7Hz, 6H, ArHorthotoazo), 7.14(d,J=8.5Hz,6H, ArHorthoto
sugar), 5.19 (d, J = 5.0 Hz, 3H, 2-OH), 4.90 (d, J = 7.6 Hz, 3H, H1), 4.84
(m, 3H, 3-OH), 4.57 (s, 3H, 4-OH), 3.80 (q, J = 6.5 Hz, 3H, H5), 3.57
(m, 3H, H2), 3.51 (s, 3H, H4), 3.45 (d, J = 10.2 Hz, 3H, H3), 1.18 (d, J =
6.5 Hz, 9H, H6); 13C{1H} NMR (151 MHz, DMSO-d6) δ 178.1 (core
Ar, C−O), 156.9 (Ar, C−O−sugar), 136.3 (Ar, C−N), 128.8 (core Ar,
C−N), 119.0 (Ar, C−C−N), 117.8 (Ar C−C−O−sugar), 101.2 (C1),
73.9 (C3), 71.4 (C4), 70.8 (C5), 70.4 (C2), 17.1 (C6); HRMS (ESI) m/
z [M + H]+ calcd for C42H48N6O18 925.3103, found 925.3068.
4-Aminophenyl β-D-fucopyranoside32 (0.4 g, 1.73 mmol, 1 equiv)
was used to synthesize the Yariv reagent 4 using the same procedure as
that used for 1. The crude product (0.39 g, 0.4 mmol) was obtained in a
72% yield. Some of this material was further purified twice using the
redissolution−reprecipitation protocol; 0.27 g of the crude product was
reprecipitated from 20 mL of water and 150 mL of ethanol to give 0.16 g
of the β-D-fucosyl Yariv reagent (4) R1X product (59%). The second
redissolution−reprecipitation with R1X (0.09 g) from 20 mL of water
and 150 mL of ethanol provided 0.035 g of the β-D-fucosyl Yariv reagent
1
(4) R2X (41%) to provide a dark red sticky powder: H NMR (600
MHz, DMSO-d6) δ 15.94,15.89, 15.11, and 15.08 (s, 3H, core Ar−OH),
7.65 (d, J = 9.0 Hz, 6H, ArH ortho to azo), 7.15 (d, J = 9.0 Hz, 6H, ArH
ortho to sugar), 5.18 (d, J = 5.2 Hz, 3H, 2-OH), 4.90 (d, J = 7.6 Hz, 3H,
H1), 4.84 (d, J=5.8Hz, 3H, 3-OH), 4.57(d, J= 4.9Hz, 3H, 4-OH), 3.81
(q, J = 6.6 Hz, 3H, H5), 3.56 (m, 3H, H2), 3.50 (t, J = 4.3 Hz, 3H, H4),
3.44 (m, 3H, H3), 1.18 (d, J = 6.4 Hz, 9H, H6); 13C{1H} NMR (151
MHz, DMSO-d6) δ 178.0 (core Ar, C−O), 156.8 (Ar, C−O−sugar),
136.3 (Ar, C−N), 128.8 (core Ar, C−N), 119.0 (Ar, C−C−N), 117.8
(Ar,C−C−O−sugar),101.1(C1),73.9(C3),71.4(C4),70.8(C5),70.4
(C2), 17.1 (C6); HRMS (ESI) m/z [M + H]+ calcd for C42H48N6O18
925.3103, found 925.3091.
Synthesis of the α-L-Fucosyl Yariv Reagent (5). 4-Nitrophenyl
α-L-fucopyranoside (1 g, 3.38 mmol) and 10% Pd/C (36 mg) were used
to prepare the aniline using the same procedure as that used for 4-
aminophenylβ-D-glucopyranoside. The aminosugar33 wasobtainedasa
pale yellow-clear crystalline solid (0.9 g, 3.35 mmol, 99%). This material
was directly used in the next step: 1H NMR (400 MHz, DMSO-d6) δ
6.74(d, J=8.8Hz, 2H), 6.49(d, J=8.6Hz, 2H), 5.08(d, J=3.5Hz, 1H),
4.68(d, J=6.5Hz, 1H), 4.68(s,2H), 4.61(d, J=5.5Hz, 1H), 4.50(d,J=
4.4 Hz, 1H), 4.00−3.90 (m, 1H), 3.68 (m, 2H), 3.55 (s, 1H), 1.07 (d, J =
6.5 Hz, 3H).
ASSOCIATED CONTENT
* Supporting Information
The SupportingInformationisavailablefree ofchargeathttps://
pubs.acs.org/doi/10.1021/acs.joc.0c01812.
■
sı
1H NMR, 13C NMR, COSY, and HSQC spectra for 1−6;
DOSY and ROESY spectra of 1; and table of yields after
processing and purification (PDF)
FAIR data, including the primary NMR FID files, for
compounds 1−6 (ZIP)
AUTHOR INFORMATION
Corresponding Author
■
4-Aminophenyl α-L-fucopyranoside33 (0.85 g, 3.34 mmol, 1 equiv)
was used to synthesize that Yariv reagent 5 using the same procedure as
that used for 1. This crude product (0.97 g, 1 mmol) was obtained in a
93% yield. This material was further purified twice using the
redissolution−reprecipitation protocol, followed by methovaping;
0.97 g of the (5) crude product was reprecipitated from 20 mL of
water and 180 mL of ethanol to give 0.52 g of the α-L-fucosyl R1X (5)
product(59%). Thesecondredissolution−reprecipitationwith(5)R1X
(0.5g)from20mLofwaterand180mLofethanolprovided0.35gofthe
α-L-fucosyl (5) R2X (68%). Methovaping 33.8 mg of (5) R2X using 3
mL of methanol gave 31.1 mg of (5) R2XM (92%) as dark red pellets,
which were crushed into a powder: 1H NMR (600 MHz, DMSO-d6) δ
15.95, 15.90, 15.11, and 15.07 (s, 3H, core Ar−OH), 7.63 (d, J = 8.1 Hz,
6H, ArH ortho to azo), 7.18 (d, J = 8.1 Hz, 6H, ArH ortho to sugar), 5.46
(d, J = 3.2 Hz, 3H, H1), 4.90 (d, J = 5.7 Hz, 3H, 2-OH), 4.71 (d, J = 5.2
Hz, 3H, 4-OH), 4.60 (d, J = 4.4 Hz, 3H, 3-OH), 3.92 (q, J = 6.7 Hz, 3H,
H5), 3.80 (m, 6H, H4 and H2), 3.60 (t, J = 3.8 Hz, 3H, H3), 1.09 (d, J =
6.5 Hz, 9H, H6); 13C{1H} NMR (151 MHz, DMSO-d6) δ 178.1 (core
Ar, C−O), 156.7 (Ar, C−O−sugar), 136.3 (Ar, C−N), 128.8 (core Ar,
C−N), 119.0 (Ar, C−C−N), 118.4 (Ar, C−C−O−sugar), 98.8 (C1),
Amit Basu − Department of Chemistry, Brown University,
Providence, Rhode Island 02912, United States; orcid.org/
0000-0001-6312-675X; Email: abasu@brown.edu
Authors
Raghuraj Hoshing − Department of Chemistry, Brown University,
Providence, Rhode Island 02912, United States
Michael Saladino − Department of Chemistry, Brown University,
Providence, Rhode Island 02912, United States
Helene Kuhn − Department of Chemistry, Brown University,
Providence, Rhode Island 02912, United States
David Caianiello − Department of Chemistry, Brown University,
Providence, Rhode Island 02912, United States
Robert F. Lusi − Department of Chemistry, Brown University,
Providence, Rhode Island 02912, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.joc.0c01812
F
https://dx.doi.org/10.1021/acs.joc.0c01812
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
pubs.acs.org/joc
Note
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the National Science Foundation
(no. CHE1607554), and a Brown University Teaching and
Research Assistantship award to R.F.L. Drs. Tun-Li Shen and
Russell Hopson are thanked for their assistance with mass
spectrometry and NMR spectroscopy, respectively.
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